WO2013052995A2 - Improved developmental competence of oocytes - Google Patents

Improved developmental competence of oocytes Download PDF

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WO2013052995A2
WO2013052995A2 PCT/AU2012/001219 AU2012001219W WO2013052995A2 WO 2013052995 A2 WO2013052995 A2 WO 2013052995A2 AU 2012001219 W AU2012001219 W AU 2012001219W WO 2013052995 A2 WO2013052995 A2 WO 2013052995A2
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oocyte
inducer
endoplasmic reticulum
heat shock
shock proteins
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PCT/AU2012/001219
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French (fr)
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WO2013052995A3 (en
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Rebecca Louise ROBKER
Linda Linyan WU
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Adelaide Research & Innovation Pty Ltd
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Publication of WO2013052995A2 publication Critical patent/WO2013052995A2/en
Publication of WO2013052995A3 publication Critical patent/WO2013052995A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0609—Oocytes, oogonia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/07—Heat shock proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/10—Conditioning of cells for in vitro fecondation or nuclear transfer

Definitions

  • the present disclosure relates to methods and culture medium for improving the developmental competence of oocytes, and embryos arising from the oocytes, and also to pharmaceutical compositions and methods of treatment for improving fertility.
  • PCOS is characterised by multiple follicles which develop from primordial follicles, but the development is arrested at an early antral stage due to disturbed ovarian function. Obesity is believed to be the main contributing factor to PCOS, and the majority of patients with the condition have insulin resistance and/or are obese.
  • oocytes of obese women also often fail to develop to the blastocyst stage when fertilised in vitro, indicating that obesity disrupts sensitive aspects of oocyte maturation that influence subsequent embryo growth.
  • the present disclosure is based on the determination that the developmental competence of oocytes may be improved by exposing oocytes to an endoplasmic stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method of improving developmental competence of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method of improving fertility in a female subject, the method comprising administering to the subject an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method of treating reduced fertility in a female subject, the method comprising administering to the subject an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method of improving ovulation in a female subject, the method comprising administering to the subject an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method of assisted reproduction comprising an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method of improving cumulus cell expansion in a cumulus oocyte complex, the method comprising exposing an oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method of increasing the level of cumulus cell protein in a cumulus oocyte complex, the method comprising exposing an oocyte to an endoplasmic reticulum stress inhibitor, and/or an inducer of heat shock proteins
  • Certain embodiments of the present disclosure provide a method of in vitro maturation of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method of improving the developmental competence of an embryo produced by fertilisation of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments provide a method of improving blastocyst development of an embryo produced by fertilisation of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide an oocyte and/or embryo culture medium, the medium comprising an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • kits for improving developmental competence comprising:
  • an oocyte and/or embryo culture medium an oocyte and/or embryo culture medium
  • Certain embodiments provide a pharmaceutical composition for improving fertility in a female subject, the composition comprising an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method of preventing and/or treating reduced fertility in a female subject, the method comprising administering to the subject a therapeutically effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide use of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in the preparation of a medicament for preventing and/or treating reduced fertility in a female subject.
  • Certain embodiments of the present disclosure provide a method of increasing the likelihood of a female subject falling pregnant, the method comprising exposing the subject to an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method of treating reduced fertility in a female subject, the method comprising:
  • Certain embodiments of the present disclosure provide a method of preventing and/or treating a subject with reduced fertility, the method comprising administering to the subject a therapeutically effective amount of endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins; wherein the subject comprises one or more of the following characteristics: a body mass index of greater than 25 kg/m ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal to 30 kg/m 2 ; obesity; Polycystic ovary syndrome; reduced fertility; sub-fertility; infertility; ovarian dysfunction; anovulation; reduced ovulation rate; pre-diabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet.
  • Certain embodiments of the present disclosure provide a combination product comprising:
  • FIG. 1 shows that thapsigargin induces ER stress in COCs which can be reversed by Salubrinal.
  • Mouse COCs were matured in vitro for 16 hours in the presence of ⁇ thapsigargin (Thap) or ⁇ thapsigargin plus ⁇ Salubrinal (Thap+Sal), or ⁇ Salubrinal (Sal) alone, or in the absence of thapsigargin and salubrinal as control.
  • Ovulated COCs in vivo matured) from eCG, hCG 16 h treated mice were used for comparison to in vitro controls.
  • FIG. 2 shows that thapsigargin impairs cumulus expansion and protein secretion which can be reversed by Salubrinal.
  • COCs from eCG-treated mice were matured in vitro for 16 hours in the presence of ⁇ thapsigargin (Thap), ⁇ thapsigargin plus ⁇ Salubrinal (Thap+Sal), ⁇ Salubrinal (Sal) alone, or in the absence of thapsigargin and salubrinal as control.
  • Thap ⁇ thapsigargin
  • Thap+Sal ⁇ Salubrinal
  • Sal ⁇ Salubrinal
  • C Matrix protein pentraxin-3 (PTX3) (red- fluorescence) was reduced in cumulus matrix of thapsigargin (Thap) treated COCs by immunohistochemistry. DAPI nuclear stain is shown as blue fluorescence.
  • D Western blot analysis of PTX3 from cumulus matrix extracts, and PTX3, phospho-IREl and IRE1 proteins from cell pellets obtained from COCs matured for 8 or 16 hour.
  • FIG. 3 shows that ER stress reduces oocyte mitochondrial membrane potential which is reversed by Salubrinal.
  • Mitochondrial membrane potential was assessed by JC-1 staining (A) in oocytes matured in vitro for 16 hours in the presence of ⁇ thapsigargin (Thap) or ⁇ thapsigargin plus ⁇ Salubrinal (Thap+Sal), or ⁇ Salubrinal (Sal) alone, or in the absence of thapsigargin and salubrinal as control.
  • Ovulated COCs in vivo matured) from eCG, hCG 16 h treated mice were used as a comparison for the in vitro controls.
  • Red fluorescence indicates high mitochondrial membrane potential and green indicates low mitochondrial membrane potential.
  • FIG. 4 shows that COCs matured in the presence of thapsigargin have significantly impaired embryo developmental competence following IVF, which is reversible with Salubrinal.
  • COCs were matured in vitro for 16 hours in the presence of 500nM thapsigargin (Thap) or 500nM thapsigargin plus 200nM Salubrinal (Thap+Sal), or 200nM Salubrinal (Sal) alone, or in the absence of thapsigargin and salubrinal as control.
  • Ovulated COCs (in vivo matured) from eCG, hCG 16 h treated mice were used as a comparison to the in vitro controls. COCs were then fertilized in vitro and the rate of embryo development assessed.
  • FIG. 5 shows that palmitic acid dose-dependently induces ER stress in COCs.
  • COCs were matured in vitro for 16h in 1% FCS as control or plus 150, 275, 400 or 525 ⁇ palmitic acid.
  • FIG. 6 shows induction of ER stress markers by high dose (400 ⁇ ) palmitic acid is reversible by salubrinal.
  • Immature COCs from eCG-treated mice (0 hours) COCs were matured in vitro in 1% FCS as control or plus 150 ⁇ or 400 ⁇ palmitic acid or 400 ⁇ palmitic acid and 200nM salubrinal (Sal) for 8 hours or 16 hours.
  • Total RNA was extracted from COCs and subjected to RT-PCR for analysis of ER stress marker genes mRNA expression.
  • Figure 7 shows high dose (400 ⁇ ) palmitic acid reduces Ptx3 protein production in COCs that is normalized with salubrinal.
  • Immature COCs from eCG- treated mice (0 hour) COCs were matured in vitro in 1% FCS as control or plus 150 ⁇ or 400 ⁇ palmitic acid or 400 ⁇ palmitic acid and 200nM salubrinal (Sal) for 8 hours or 16 hours.
  • DAPI nuclear stain is shown as blue fluorescence.
  • (C) PTX3 mRNA expression levels were determined by RT-PCR. Values are mean + SEM expressed as fold change compared with calibrator sample; n 3 pools of cells per treatment group. Different letters indicate significant differences by one-way ANOVA within each timepoint, Bonferroni post hoc test.
  • FIG. 8 shows oocyte mitochondrial membrane potential is reduced in COCs treated with high dose palmitic acid and reversed by Salubrinal but not by L-Carnitine. Mitochondrial membrane potential was assessed by JC-1 staining in oocytes matured in vitro in the presence of 150 ⁇ or 400 ⁇ palmitic acid or without palmitic acid as control. High dose (400 ⁇ ) palmitic acid treated COCs were also treated with either salubrinal (Sal; 200nM) or L-carnitine (5mM) as indicated. In vivo matured oocytes from eCG, hCG 16h treated mice were used for comparison to in vitro controls (A, C).
  • FIG. 9 shows that COCs treated with high dose palmitic acid have significantly impaired embryo development that is reversed by salubrinal.
  • COCs from eCG-treated mice were matured in vitro for 16 hours in the presence of 150 ⁇ or 400 ⁇ palmitic acid and/or 200nM salubrinal (Sal) or without palmitic acid as control.
  • In vivo matured COCs from eCG hCG 16h treated mice were used for comparison to in vitro controls.
  • Figure 10 shows the effect of ER stress inhibitors TUDCA and PBA on COC morphology.
  • Figure 11 shows fertilisation of COCs matured in TUDCA (ImM) results in very poor embryo development, even compared to thapsigargen (TG) treatment.
  • Figure 12 shows the effect of salubrinal added to bovine oocytes during oocyte maturation in vitro, followed by IVF and assessments of embryo development.
  • FIG 13 shows that the obese ovarian environment directly affects oocytes.
  • Follicle fluid (FF) from either obese women or moderate weight women was added to mouse COCs during 16h of maturation; and compared to controls matured for 16h in vivo.
  • Figure 14 shows reduced ovulation rates in homocysteine (Hcy)-treated mice. In mice fed Hey for 2 weeks poor ovulation rate is normalised by treatment with salubrinal for 4 days.
  • Hcy homocysteine
  • FIG. 15 shows that that Blobby mice have poor oocyte quality compared to their wildtype or heterozygous littermates. Oocytes of Blobby mice when fertilized by IVF have reduced fertilization rates (2-cells) and poor development to the 4-cell and blastocyst stages. When the mice are treated with salubrinal (lmg/kg i.p. daily) for 4 days prior to ovulation, oocyte quality is restored to normal in the obese Blobby mice.
  • Figure 16 shows that ovulation in Blobby mice is improved upon administration of an ER stress inhibitor.
  • Figure 17 shows that treatment with an ER stress improves mitochondria in Blobby mice.
  • Figure 18A shows that treatment with an ER stress inhibitor restores fertilization rates in Blobby mice.
  • Figure 18B shows that treatment with an ER stress inhibitor improves rates of blastocyst formation on Blobby mice.
  • FIG 19 shows photographs of mouse cumulus-oocyte complexes (COCs) following in vitro culture with no supplement (control), 400 ⁇ palmitic acid (PA) or 400 ⁇ palmitic acid and the hydroximic acid derivatives BGP-15 or BGP-15M for 16 hours.
  • the oocyte can be seen at the center of each complex surrounded by many layers of cumulus cells and extracellular matrix.
  • PA palmitic acid.
  • Figure 20 shows mouse COCs exposed to high lipid (i.e. palmitic acid (PA) at 400 ⁇ ) have significantly impaired embryo development that is restored by co- treatment with BGP-15M (400mg/L).
  • COCs were exposed to the indicated treatment for 16 hours followed by IVF under identical conditions.
  • the percentage of oocytes that formed 2-cell embryos following IVF was assessed at 24 hours/ day 2 (A).
  • the percentage of 2-cell embryos developing to blastocysts was assessed on day 5 (B).
  • Figure 21 shows that treatment of mouse COCs with high lipid (400uM palmitic acid (PA)) during their maturation in vitro (IVM) impairs subsequent fertilisation rate, as well as blastocyst development rates (not shown).
  • PA 400uM palmitic acid
  • IVM maturation in vitro
  • Different letters indicate significant differences by one-way ANOVA, Bonferroni Post hoc test; P ⁇ 0.05.
  • the present disclosure relates to methods, culture medium and compositions for improving oocyte developmental competence.
  • the present disclosure also relates to methods and compositions for preventing and/or treating reduced fertility.
  • the present disclosure is based, at least in part, upon the recognition that the developmental competence of oocytes may be improved by exposure of the oocytes, in vitro and in vivo, to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure are directed to methods for improving developmental competence of oocytes, media for culturing oocytes and/or embryos, methods for treating reduced fertility, and pharmaceutical compositions for treating reduced fertility. Kits and combination products are also provided.
  • Certain embodiments of the present disclosure are directed to methods for improving developmental competence of oocytes, media for culturing oocytes and/or embryos, methods for treating reduced fertility, methods for improving fertility, and pharmaceutical compositions for treating reduced fertility, that have one or more advantages.
  • some of the advantages of the embodiments disclosed herein include one or more of the following: improving developmental competence of oocytes in female subjects (including, for example, obese women and/or women with PCOS); assist in the formulation of culture media for oocytes from female subjects (including, for example, obese women and/or women with PCOS); provide improved pharmaceutical compositions for improving fertility in female subjects (including, for example, obese women and/or women with PCOS); assist in prevention and/or treatment of reduced fertility in female subjects (including, for example, obese women and/or women with PCOS); improve the likelihood of female subjects (including, for example, obese women and/or women with PCOS) falling pregnant; improving fertility in female subjects (including, for example, obese women and/or women with PCOS); improve outcomes for assisted reproduction technologies involving oocytes in animals and humans; assist in the uptake of assisted reproduction technologies in animals and humans; to improve fertility; improve the rate of live births per IVF cycle; improve success rates for women seeking natural conceptions (including, for example, obese women and/or women with PCOS); improve
  • developmental competence includes one or more of: (i) the ability and/or likelihood of the oocyte to produce an embryo (for example, upon fertilization of an oocyte or by other mechanisms, such as parthenogenic activation); (ii) one or more of the ability, likelihood and rate of an oocyte to progress through blastocyst development upon formation of an embryo; and (iii) the quality of the embryo (for example, as determined by morphological and/or biochemical assessments) achieved upon the production of an embryo from the oocyte.
  • the developmental competence is improved.
  • the ability of an embryo produced from an oocyte exposed to an endoplasmic reticulum stress to progress through blastocyst development may be improved and/or has one or more of an increased ability to progress through blastocyst development, an increased fertilization rate, an increased likelihood of progression through blastocyst development, an increased rate of forming a two cell embryo, an increased rate of forming a four cell embryo, an increased rate of forming a blastocyst, an increased rate of progressing through blastocyst development, and an increased rate of blastocyst hatching.
  • the method may be used to increase fertilization rate, to increase blastocyst formation, and to increase blastocyst hatching.
  • Certain embodiments of the present disclosure provide a method of increasing fertilization rate of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • the fertilization rate is 95% or greater, 96% or greater, 97% or greater, or 98% or greater.
  • Certain embodiments of the present disclosure provide a method of increasing blastocyst formation of an embryo produced from an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method of increasing blastocyst hatching of an embryo produced from an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • the rate of blastocyst hatching is 95% or greater, 96% or greater, 97% or greater, or 98% or greater.
  • the oocyte is a part of a cumulus oocyte complex.
  • the oocyte is a denuded oocyte.
  • Methods for removing cumulus cells from a cumulus oocyte complex are known.
  • the term "oocyte" as used herein includes an oocyte alone or an oocyte in association with one or more other cells, such as an oocyte as part of a cumulus oocyte complex. Animal and human oocytes are contemplated.
  • the oocyte comprises an increased lipid content and/or an increased lipid level. In certain embodiments, the oocyte comprises an increased content and/or level of neutral lipids. In certain embodiments, the oocyte comprises an increased content and/or level of one or more toxic lipids. In certain embodiments, the oocyte comprises an oocyte exposed to an increased lipid environment and/or an oocyte exposed to one or more toxic lipids. In certain embodiments, the oocyte is a normal oocyte. In certain embodiments, the oocyte does not comprise an increased lipid content and/or an increased lipid level.
  • lipid droplet abundance and localization in an oocyte may be measured using a fluorescent stain, such as BODIPY 493/503 (Invitrogen) and comparison to oocytes with a normal lipid content.
  • a fluorescent stain such as BODIPY 493/503 (Invitrogen)
  • the oocyte is obtained from a female subject comprising one or more of the following characteristics: a body mass index of greater than 25 kg/m 2 ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal to 30 kg/m ; obesity; Polycystic Ovary Syndrome; reduced fertility; sub- fertility, infertility; ovarian dysfunction; anovulation; reduced ovulation rate; prediabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet.
  • the oocyte is obtained from a female subject comprising one or more of the following characteristics: a normal body mass index; a body mass index of equal to or less than 25 kg/m ; normal weight; non-obese; and normal fertility.
  • the endoplasmic reticulum stress inhibitor comprises one or more of an inhibitor of an inositol requiring protein- 1 (IREl) signal transduction pathway, an inhibitor of a protein kinase RNA-like endoplasmic reticulum kinase (PERK) signal transduction pathway, an inhibitor of a transcription factor (ATF6) signal transduction pathway, and an inducer of heat shock proteins.
  • IREl inositol requiring protein- 1
  • PERK protein kinase RNA-like endoplasmic reticulum kinase
  • ATF6 transcription factor
  • the endoplasmic reticulum stress inhibitor comprises an inducer of heat shock proteins, such as a derivative of a hydroximic acid.
  • the endoplasmic reticulum stress inhibitor comprises an inhibitor of eIF2a dephosphorylation.
  • Inhibitors of eIF2a dephosphorylation are known and available.
  • the eIF2a dephosphorylation is salubrinal and/or a derivative of salubrinal that is capable of inhibiting eIF2a dephosphorylation, such as a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate of salubrinal.
  • salubrinal refers to salubrinal and any of the aforementioned derivatives:
  • the endoplasmic reticulum stress inhibitor comprises tauroursodeoxycholate (TUDCA) and/or 4-phenyl butyric acid (PBA); and/or a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate of either these compounds.
  • TDCA tauroursodeoxycholate
  • PBA 4-phenyl butyric acid
  • a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate of either these compounds is used.
  • tauroursodeoxycholate refers to tauroursodeoxycholate and any of the aforementioned derivatives.
  • 4-phenyl butyric acid refers to 4-phenyl butyric acid and any of the aforementioned derivatives.
  • the inducer of heat shock proteins comprises a derivative of hydroximic acid.
  • the derivative of hydroximic acid comprises one or more of BGP-15, propanolol, bimoclomol, arimoclomal, NG-94, iroxanadine, and/or a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate of any of the aforementioned.
  • the structures of the aforementioned hydroximic acid derivatives are as follows:
  • the inducer of heat shock proteins comprises an inducer of Hsp72.
  • Methods for exposing oocytes to agents are known.
  • the oocyte is exposed to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in vitro.
  • the oocyte is exposed to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in vivo.
  • the oocyte is exposed to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in vitro and in vivo.
  • Examples of in vitro exposure include exposure of an oocyte to an agent in a liquid medium, exposure to a pre-drug that is metabolised to an active agent in the oocyte, introduction of a product into the oocyte directly, such as injection, introduction of a nucleic acid encoding the agent into an oocyte, such as by viral infection or transfection, and exposure of the oocyte to a compound that induces the expression of an agent in the oocyte.
  • the method comprises exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in vitro.
  • an oocyte may be cultured in a medium comprising an endoplasmic recticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Examples of in vivo exposure include various methods for the administration of an agent to a subject, such as a human or animal subject, are described herein.
  • the oocyte is exposed to an exogenous endoplasmic reticulum stress inhibitor, such as salubrinal. In certain embodiments, the oocyte is exposed to an endogenous endoplasmic reticulum stress inhibitor.
  • the oocyte is exposed to a concentration of the endoplasmic reticulum stress inhibitor of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1000 nM or greater, 10 uM or greater, 0.1 nM or less, 0.5 nM or less, 1 nM or less, 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, 1000 nM or less, 10 uM or less, 0.1 nM to 10 uM, 0.1 nM to 1000 nM, 0.1 nM to 500 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.1 nM to 1 nM, 0.5
  • the oocyte is exposed to a concentration of salubrinal of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1000 nM or greater, 10 uM or greater, 0.1 nM or less, 0.5 nM or less, 1 nM or less, 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, 1000 nM or less, 10 uM or less, 0.1 nM to 10 uM, 0.1 nM to 1000 nM, 0.1 nM to 500 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.1 nM to 1 nM, 0.5 nM to 10
  • the oocyte is exposed to a concentration of salubrinal in the range of 50 nM to 500 nM.
  • the oocyte is exposed to a concentration of tauroursodeoxycholate of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1000 nM or greater, 10 uM or greater, 0.1 nM or less, 0.5 nM or less, 1 nM or less, 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, 1000 nM or less, 10 uM or less, 0.1 nM to 10 uM, 0.1 nM to 1000 nM, 0.1 nM to 500 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.1 nM to 1 nM, 0.5 nM to 100 nM,
  • the oocyte is exposed to a concentration of tauroursodeoxycholate in the range of ⁇ 10 ⁇ .
  • the oocyte is exposed to a concentration of 4-phenyl butyric acid of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1000 nM or greater, 10 uM or greater, 0.1 nM or less, 0.5 nM or less, 1 nM or less, 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, 1000 nM or less, 10 uM or less, 0.1 nM to 10 uM, 0.1 nM to 1000 nM, 0.1 nM to 500 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.1 nM to 1 nM, 0.5 nM to 100 nM, 0.1
  • the oocyte is exposed to a concentration of 4-phenyl butyric acid in the range of ⁇ lnM.
  • the oocyte is exposed to an inducer of heat shock proteins, such as a derivative of hydroximic acid. In certain embodiments, the oocyte is exposed to an endogenous inducer of heat shock proteins. In certain embodiments, the oocyte is exposed to an exogenous inducer of heat shock proteins.
  • an inducer of heat shock proteins such as a derivative of hydroximic acid.
  • the oocyte is exposed to an endogenous inducer of heat shock proteins.
  • the oocyte is exposed to an exogenous inducer of heat shock proteins.
  • the oocyte is exposed to a concentration of a derivative of a hydroximic acid (for example BGP-15) at a concentration of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1000 nM or greater, 10 uM or greater, 0.1 nM or less, 0.5 nM or less, 1 nM or less, 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, 1000 nM or less, 10 uM or less, 0.1 nM to 10 uM, 0.1 nM to 1000 nM, 0.1 nM to 500 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.1 nM to 100 nM
  • the oocyte is exposed to a concentration of a derivative of a hyroximic acid (for example BGP-15) of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1000 nM or greater, 10 uM or greater, 0.1 nM or less, 0.5 nM or less, 1 nM or less, 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, 1000 nM or less, 10 uM or less, 0.1 nM to 10 uM, 0.1 nM to 1000 nM, 0.1 nM to 500 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.1 nM to 100 nM,
  • the oocyte is exposed to a concentration of a derivative of a hyroximic acid (for example BGP-15) in the range of 0.1 to 1000 mg/ml, 0.1 to 500 mg/ml, 0.1 to 400 mg/ml, 0.1 to 200 mg/ml, 0.1 to 100 mg/ml, 0.1 to 50 mg/ml, 0.1 to 20 mg/ml, 0.1 to 10 mg/ml, 0.1 to 5 mg/ml, 0.1 to 4 mg/ml, 0.1 to 2 mg/ml, 0.1 to 1.0 mg/ml, 0.1 to 0.5 mg/ml, 0.1 to 0.4 mg/ml, 0.1 to 0.2 mg/ml, 0.2 to 1000 mg/ml, 0.2 to 500 mg/ml, 0.2 to 400 mg/ml, 0.2 to 200 mg/ml, 0.2 to 100 mg/ml, 0.2 to 50 mg/ml, 0.2 to 20 mg/ml, 0.2 to 10 mg/ml,
  • Certain embodiments of the present disclosure provide a method of culturing an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, wherein exposure of the oocyte to the endoplasmic stress inhibitor improves the developmental competence of the oocyte. Culturing of oocytes from human, animals and other species are contemplated.
  • the oocyte is an immature oocyte. In certain embodiments, the oocyte is a mature oocyte. Methods for determining the maturation status of an oocyte are known.
  • the oocyte is an oocyte matured in vitro.
  • Methods for collecting/obtaining oocytes and performing in vitro maturation are known, including in humans and animals.
  • the method comprises obtaining an oocyte from a female subject. In certain embodiments, the method comprises obtaining an immature oocyte from a female subject. In certain embodiments, the method comprises obtaining an immature oocyte from a female subject and in vitro maturing the oocyte.
  • the oocyte is an oocyte matured in vivo.
  • Methods for collecting/obtaining mature oocytes from a female subject are known.
  • the method comprises obtaining a mature oocyte from a female subject.
  • the method comprises exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in a subject in vivo.
  • the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins are administered to a subject in order to expose an oocyte to the endoplasmic stress inhibitor.
  • Administration and delivery of the endoplasmic stress inhibitor and/or an inducer of heat shock proteins may be performed by a known method.
  • administration may be one or more of intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical route, and direct injection. The mode and route of administration in most cases will depend on the type of disease, condition or state being treated.
  • the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins are administered to the subject at a dose of 0.05 mg/kg or greater, 0.1 mg/kg or greater, 0.5 mg/kg or greater, 1 mg/kg or greater, 2 mg/kg or greater, 5 mg/kg or greater, 10 mg/kg or greater, 50 mg/kg or greater, 100 mg/kg or greater, 0.05 mg/kg or less, 0.1 mg/kg or less, 0.5 mg/kg or less, 1 mg/kg or less, 2 mg/kg or less, 5 mg/kg or less, 10 mg/kg or less, 50 mg/kg or less, 100 mg/kg or less, 0.05 to 100 mg/kg, 0.05 to 50 mg/kg, 0.05 to 10 mg/kg, 0.05 to 5 mg/kg, 0.05 to 1 mg/kg, 0.05 to 0.5 mg/kg, 0.05 to 0.1 mg/kg, 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 10 mg/kg, 0.1 to 100 mg/kg,
  • the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins are administered to the subject at a dose of 0.2 to 2 mg/kg.
  • the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins are administered to the subject at a dose 0.05 mg/kg/day or greater, 0.1 mg/kg/day or greater, 0.5 mg/kg/day or greater, 1 mg/kg/day or greater, 2 mg/kg/day or greater, 5 mg/kg/day or greater, 10 mg/kg/day or greater, 50 mg/kg/day or greater, 100 mg/kg/day or greater, 0.05 mg/kg/day or less, 0.1 mg/kg/day or less, 0.5 mg/kg/day or less, 1 mg/kg/day or less, 2 mg/kg/day or less, 5 mg/kg/day or less, 10 mg/kg/day or less, 50 mg/kg/day or less, 100 mg/kg/day or less, 0.05 to 100 mg/kg/day, 0.05 to 50 mg/kg/day, 0.05 to 10 mg/kg/day, 0.05 to 5 mg/kg/day, 0.05 to 1 mg/kg/kg/kg/day, 0.05
  • the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins are administered to the subject at a dose of 0.2-2 mg/kg/day.
  • the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins are administered to a human subject at a dose of 50 to 500 mg/day. In certain embodiments, the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins are administered to a human subject at a dose of 100 to 400 mg/day. For example, two 100 mg capsules by mouth in the morning; and two 100 mg capsules by mouth in the evening; or two 50 mg capsules by mouth in the morning and two 50 mg capsules by mouth in the evening.
  • the method further comprises exposing the oocyte to an inducer of lipid metabolism and/or to a Peroxisome Proliferator Activated Receptor (PPAR) agonist.
  • the Peroxisome Proliferator Activated Receptor (PPAR) agonist comprises a Peroxisome Proliferator Activated Receptor alpha agonist and/or a Peroxisome Proliferator Activated Receptor gamma agonist.
  • the method comprises exposing the oocyte to an inducer of lipid metabolism, such as L-carnitine, and/or a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate thereof.
  • an inducer of lipid metabolism such as L-carnitine
  • a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate thereof such as L-carnitine, and/or a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate thereof.
  • the method comprises exposing the oocyte to a Peroxisome Proliferator Activated Receptor alpha agonist and/or a Peroxisome Proliferator Activated Receptor gamma agonist.
  • the Peroxisome Proliferator Activated Receptor alpha agonist is fenofibrate.
  • the Peroxisome Proliferator Activated Receptor gamma agonist is rosiglitazone.
  • the method comprises exposing the oocyte to an inducer of lipid metabolism (such as L-carnitine) and a Peroxisome Proliferator Activated Receptor (PPAR) agonist.
  • an inducer of lipid metabolism such as L-carnitine
  • PPAR Peroxisome Proliferator Activated Receptor
  • the oocyte is a mammalian oocyte.
  • the oocyte is an oocyte from a human, a primate, a livestock animal (eg. a horse, a cow (eg bos taurus, bos indicus), a sheep, a pig, or a goat), a companion animal (eg. a dog, a cat), or a laboratory animal (eg. a mouse, a rat, a guinea pig, a rabbit, a bird, a frog).
  • the oocyte is a bovine oocyte, such as an oocyte from a dairy cow.
  • the oocyte is a human oocyte. Oocytes from other species are contemplated.
  • the oocyte is obtained from a female subject comprising one or more of the following characteristics: a body mass index of greater than 25 kg/m 2 ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal 30 kg/m ; obesity; Polycystic Ovary Syndrome; reduced fertility; infertility; sub-fertility, ovarian dysfunction; anovulation; reduced ovulation rate; pre-diabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet.
  • the method is used to improve maturation of an oocyte, to improve developmental competence of an embryo produced by fertilisation of the oocyte exposed to the endoplasmic reticulum stress inhibitor, to improve blastocyst development of an embryo produced by fertilisation of the oocyte exposed to the endoplasmic reticulum stress inhibitor, to improve cumulus cell expansion in a cumulus oocyte complex, to increase the level of cumulus cell protein in a cumulus oocyte complex; to improve assisted reproduction; to improve fertility; to treat sub-fertility, to treat infertility; to treat ovarian dysfunction; to treat anovulation; to improve ovulation rate, to improve fertility in a female subject, to treat reduced fertility in a female subject, and to improve ovulation in a female subject.
  • Certain embodiments of the present disclosure provide a non-human oocyte or a non-human embryo produced after exposure to the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins as described herein. Certain embodiments of the present disclosure provide a non-human animal produced from the oocyte or embryo. Methods for producing animals from oocytes are known, and include for example, in vitro fertilization. A female animal may also, for example, be treated with an endoplasmic stress inhibitor and/or an inducer of heat shock proteins, and subsequently fertilized. Examples of animals are described herein.
  • Certain embodiments of the present disclosure provide a method of assisted reproduction comprising an oocyte, the method comprising exposing the oocyte to an endoplasmic stress inhibitor and/or an inducer of heat shock proteins, as described herein.
  • Certain embodiments of the present disclosure provide a method of assisted reproduction comprising an oocyte, the method comprising:
  • Examples of assisted reproduction comprising an oocyte, or an embryo produced from an oocyte include a fertilization technique in humans and animals involving an isolated oocyte, including a technique using an oocyte in vitro (for example in vitro maturation of an oocyte), in vitro fertilization (IVF; aspiration of an oocyte, fertilization in the laboratory and transfer of the embryo into a recipient), gamete intrafallopian transfer (GIFT; placement of oocytes and sperm into the fallopian tube), zygote intrafallopian transfer (ZIFT; placement of fertilized oocytes into the fallopian tube), tubal embryo transfer (TET; the placement of cleaving embryos into the fallopian tube), peritoneal oocyte and sperm transfer (POST; the placement of oocytes and sperm into the pelvic cavity), intracytoplasmic sperm injection (ICSI), testicular sperm extraction (TESE), and microsurgical epididymal sperm aspiration (MES
  • the oocyte is first harvested or collected from an ovary of a subject.
  • Oocyte collection can be performed according to standard techniques, for example as described in Textbook of Assisted Reproduction: Laboratory and Clinical Perspectives (2003) Editors Gardner, D. K., Weissman, A., Howies, CM., Shoham, Z. Martin Dunits Ltd, London, UK; and Gordon, I. (2003) Laboratory Production of Cattle Embryos 2nd Edition CABI Publishing, Oxon, UK.
  • the assisted reproduction technology comprises in vitro fertilisation.
  • Certain embodiments provide a method of in vitro fertilisation, the method comprising exposing the oocyte to an endoplasmic stress inhibitor and/or an inducer of heat shock proteins, as described herein.
  • IVF relates to the fertilization of an oocyte in vitro, wherein the oocyte is isolated from the subject and typically incubated in liquid media to allow fertilization of the oocyte. It is contemplated that fertilisation of the oocyte will generally occur greater than 24 hours, but usually not later than 60 hours, after the oocyte collection step, such that maturity of the oocyte is at a sufficient stage to maximise the success of subsequent steps in the IVF procedure.
  • Certain embodiments of the present disclosure provide a method of in vitro fertilisation, the method comprising:
  • the assisted reproduction technology comprises in vitro maturation.
  • Methods of in vitro maturation of oocytes are known.
  • Certain embodiments of the present disclosure provide a method of in vitro maturation of an oocyte, the method comprising exposing the oocyte to an endoplasmic stress inhibitor and/or an inducer of heat shock proteins, as described herein.
  • Certain embodiments of the present disclosure provide a method of in vitro maturation of an oocyte, the method comprising:
  • Certain embodiments of the present disclosure provide a method of improving the developmental competence of an embryo produced by fertilisation of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, as described herein.
  • Methods for fertilising an oocyte include fertilization in vitro, artificial insemination and natural insemination.
  • Certain embodiments of the present disclosure provide a method of improving the developmental competence of an embryo produced by fertilisation of an oocyte, the method comprising:
  • Certain embodiments of the present disclosure provide a method of improving blastocyst development of an embryo produced by fertilisation of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, as described herein.
  • Certain embodiments of the present disclosure provide a method of improving blastocyst development of an embryo produced by fertilisation of an oocyte, the method comprising:
  • Certain embodiments of the present disclosure provide a method of improving cumulus cell expansion in a cumulus oocyte complex, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, as described herein.
  • Certain embodiments of the present disclosure provide a method of increasing the level of cumulus cell protein in a cumulus oocyte complex, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, as described herein.
  • Certain embodiments of the present disclosure provide a method of culturing an oocyte.
  • Certain embodiments of the present disclosure provide a method of culturing an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, as described herein.
  • Certain embodiments of the present disclosure provide a method of culturing an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, wherein exposure of the oocyte to the endoplasmic stress inhibitor and/or the inducer of heat shock proteins improves developmental competence of the oocyte.
  • Certain embodiments of the present disclosure provide a method of culturing an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, wherein exposure of the oocyte to the endoplasmic stress inhibitor and/or the inducer of heat shock proteins improves blastocyst development of an embryo produced from the oocyte.
  • kits for performing the methods as described herein comprises one or more reagents as described herein and/or instructions for performing the methods as described herein.
  • Certain embodiments of the present disclosure provide a combination product for performing the methods as described herein.
  • Certain embodiments of the present disclosure provide use of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins as described herein in the preparation of an oocyte and/or embryo culture medium.
  • Certain embodiments of the present disclosure provide an oocyte and/or embryo culture medium, the medium comprising an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins. Endoplasmic reticulum stress inhibitors and/or an inducer of heat shock proteins, and their use, are as described herein.
  • the oocyte is part of a cumulus oocyte complex. In certain embodiments, the oocyte is a denuded oocyte. In certain embodiments, the oocyte comprises an increased lipid content. Oocytes are as described herein.
  • the endoplasmic reticulum stress inhibitor comprises one or more of an inhibitor of an inositol requiring protein- 1 (IRE1) signal transduction pathway, an inhibitor of a protein kinase RNA-like endoplasmic reticulum kinase (PERK) signal transduction pathway, an inhibitor of a transcription factor (ATF6) signal transduction pathway, and an inducer of heat shock proteins.
  • IRE1 inositol requiring protein- 1
  • PERK protein kinase RNA-like endoplasmic reticulum kinase
  • ATF6 transcription factor
  • the endoplasmic reticulum stress inhibitor comprises an inhibitor of eIF2a dephosphorylation.
  • the culture medium comprises a concentration of the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1000 nM or greater, 10 uM or greater, 0.1 nM or less, 0.5 nM or less, 1 nM or less, 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, 1000 nM or less, 10 uM or less, 0.1 nM to 10 uM, 0.1 nM to 1000 nM, 0.1 nM to 500 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1
  • the culture medium comprises an endoplasmic stress inhibitor and/or an inducer of heat shock proteins at a concentration in the range of 0.1 to 1000 mg/ml, 0.1 to 500 mg/ml, 0.1 to 400 mg/ml, 0.1 to 200 mg/ml, 0.1 to 100 mg/ml, 0.1 to 50 mg/ml, 0.1 to 20 mg/ml, 0.1 to 10 mg/ml, 0.1 to 5 mg/ml, 0.1 to 4 mg/ml, 0.1 to 2 mg/ml, 0.1 to 1.0 mg/ml, 0.1 to 0.5 mg/ml, 0.1 to 0.4 mg/ml, 0.1 to 0.2 mg/ml, 0.2 to 1000 mg/ml, 0.2 to 500 mg/ml, 0.2 to 400 mg/ml, 0.2 to 200 mg/ml, 0.2 to 100 mg/ml, 0.2 to 50 mg/ml, 0.2 to 20 mg/ml, 0.2 to 10 mg/ml, 0.2 to 5 mg//
  • the endoplasmic reticulum stress inhibitor comprises one or more of salubrinal, tauroursodeoxycholate and 4-phenyl butyric acid and/or a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate of any of the aforementioned.
  • the medium comprises a concentration of salubrinal in the range of 50-500 nM.
  • the medium comprises a concentration of tauroursodeoxycholate in the range of ⁇ 10 ⁇ .
  • the medium comprises a concentration of 4-phenyl butyric acid in the range of ⁇ lnM.
  • the medium comprises an inducer of heat shock proteins, as described herein.
  • the medium comprises a derivative of a hydroximic acid, as described herein.
  • the concentration of a derivative of hydroximic acid - (for example BGP-15) is in the range of 4 to 4000 mg/ml.
  • the medium comprises a concentration of a derivative of hydroximic acid - (for example BGP-15) in the range of 4 to 4000 mg/ml. Other ranges are contemplated and described herein.
  • the oocyte is an immature oocyte. In certain embodiments, the oocyte comprises a mature oocyte. In certain embodiments, the oocyte is an oocyte matured in vitro. In certain embodiments, the oocyte is an oocyte matured in vivo.
  • the oocyte is a mammalian oocyte. In certain embodiments, the oocyte is a bovine oocyte. In certain embodiments, the oocyte is a human oocyte. In certain embodiments, the oocyte is an animal oocyte.
  • the oocyte is obtained from a female subject comprising one or more of the following characteristics: a body mass index of greater than 25 kg/m 2 ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal to 30 kg/m ; obesity; Polycystic Ovary Syndrome; reduced fertility; infertility; ovarian dysfunction; anovulation; reduced ovulation rate; pre-diabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet.
  • Culture media for oocytes and embryos are known and commercially available, including culture media for human oocytes and embryos and culture media for animal oocytes.
  • the culture medium is one or more of:
  • IVF media are known, and typically comprise the following components: Calcium Chloride; •Gentamicin sulphate; Glucose; Human Albumin Solution; Magnesium Sulfate; Potassium Chloride; Sodium Bicarbonate; Sodium Chloride; Sodium phosphate; Sodium Pyruvate; Synthetic Serum Replacement.
  • an IVF medium comprising the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins may be used as follows:
  • Embryos are prepared and transferred to the uterus in 20 to 30 ⁇ of pre- equilibrated IVF Medium;
  • In vitro maturation medium typically comprise the following the components: Adenine; Alanine; AMP; Arginine; Ascorbic acid; Aspartic Acid; ATP; Calcium Chloride; Calcium pantothenate; Cholesterol; Choline Chloride; Cysteine; Cystine; D-Biotin; Deoxy ribose; Folic Acid; Gentamicin sulphate; Glucose; Glutamic Acid; Glutamine; Glutathione; Glycine; Guanine Hydrochloride; Histidine; Hydroxy Proline; Hypoxanthine; Inositol; Iron(III) Nitrate; Isoleucine; Leucine; Lysine; Magnesium Sulfate; Menadione; Methionine; Niacin; Niacinamide; Para-aminobenzoic Acid; Phenylalanine; Potassium Chloride; Proline; Pyridoxal; Pyridoxine; Riboflavin
  • medium(s) for in vitro maturation comprising the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins may be used as follows:
  • Medium 1 Used for pre-incubation of immature oocytes, which may or may not comprise an endoplasmic reticulum stress inhibitor and/or an inducer of heat shick proteins. Typically comprises Synthetic Serum Replacement, Human serum albumin (HSA), Physiological salts, Glucose, Sodium pyruvate, Sodium bicarbonate, Gentamicin sulphate 10 ⁇ g/ml, Phenol Red
  • Medium 2 Used for maturing immature oocytes, comprising an endoplasmic reticulum stress inhibitor and/or an inducer of heat shick proteins. Typically further comprises Glucose, Sodium pyruvate, Physiological salts, Amino acids, Nucleotides, Vitamins, Sodium bicarbonate, Gentamicin sulphate lC ⁇ g/ml, Phenol Red.
  • hCG human chorionic gonadotropin
  • FSH human follicle stimulating hormone
  • the medium further comprises an inducer of lipid metabolism and/or a Peroxisome Proliferator Activated Receptor (PPAR) agonist.
  • the Peroxisome Proliferator Activated Receptor (PPAR) agonist comprises a Peroxisome Proliferator Activated Receptor alpha agonist and/or a Peroxisome Proliferator Activated Receptor gamma agonist.
  • the inducer of lipid metabolism is L-carnitine, and/or a derivative as described herein.
  • the medium comprises a Peroxisome Proliferator Activated Receptor alpha agonist and/or a Peroxisome Proliferator Activated Receptor gamma agonist.
  • the Peroxisome Proliferator Activated Receptor alpha agonist is fenofibrate.
  • the Peroxisome Proliferator Activated Receptor gamma agonist is rosiglitazone.
  • the medium comprises an inducer of lipid metabolism (such as L-carnitine) and a Peroxisome Proliferator Activated Receptor (PPAR) agonist.
  • an inducer of lipid metabolism such as L-carnitine
  • PPAR Peroxisome Proliferator Activated Receptor
  • Certain embodiments of the present disclosure provide a non-human oocyte or a non-human embryo exposed to the medium as described herein. Certain embodiments provide a non-human animal produced from the oocyte or embryo. Certain embodiments of the present disclosure provide an animal oocyte or an animal embryo exposed to the medium as described herein.
  • Certain embodiments of the present disclosure provide a method of assisted reproduction, the method comprising exposing an oocyte and/or an embryo to a medium as described herein.
  • the method of assisted reproduction comprises in vitro fertilization of an oocyte.
  • Certain embodiments of the present disclosure provide a method of assisted reproduction, the method comprising culturing an oocyte and/or an embryo in a medium as described herein.
  • kits for use in the methods as described herein comprises an oocyte and/or embryo culture medium as described herein.
  • the kit comprises instructions for culturing the oocyte and/or embryo in the culture medium.
  • kits for improving developmental competence of an oocyte and/or an embryo produced from the oocyte comprising:
  • an oocyte and/or embryo culture medium an oocyte and/or embryo culture medium
  • Certain embodiments of the present disclosure provide a combination product.
  • Certain embodiments of the present disclosure provide a combination product for use in the methods as described herein, the combination product comprising an oocyte and/or embryo culture medium as described herein. [00172] Certain embodiments of the present disclosure provide a combination product comprising:
  • an oocyte and/or embryo culture medium an oocyte and/or embryo culture medium
  • compositions for use in the methods and/or kits as described herein are provided.
  • the composition is a pharmaceutical composition.
  • Certain embodiments of the present disclosure provide a pharmaceutical composition.
  • Certain embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutically effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, as described herein.
  • Certain embodiments of the present disclosure provide a pharmaceutical composition for use in the relevant methods as described herein.
  • compositions for improving fertility in a female subject comprising an effective amount of endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • the endoplasmic reticulum stress inhibitor comprises one or more of an inhibitor of an inositol requiring protein- 1 (IREl) signal transduction pathway, an inhibitor of a protein kinase RNA-like endoplasmic reticulum kinase (PERK) signal transduction pathway, an inhibitor of a transcription factor (ATF6) signal transduction pathway, and an inducer of heat shock proteins.
  • IREl inositol requiring protein- 1
  • PERK protein kinase RNA-like endoplasmic reticulum kinase
  • ATF6 transcription factor
  • the endoplasmic reticulum stress inhibitor comprises an inhibitor of eIF2a dephosphorylation.
  • the endoplasmic reticulum stress inhibitor comprises one or more of salubrinal, tauroursodeoxycholate and 4-phenyl butyric acid and/or a pharmaceutically acceptable derivative, solvate, salt, tautomer, stereoisomer, or racemate of any of the aforementioned.
  • the inducer of heat shock proteins comprises a derivative of hydroximic acid.
  • the derivative of hydroximic acid comprises one or more of BGP-15, propanolol, bimoclomol, arimoclomal, NG-94, iroxanadine, and/or a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate of any of the aforementioned.
  • the pharmaceutical composition further comprises a Peroxisome Proliferator Activated Receptor (PPAR) agonist.
  • PPAR Peroxisome Proliferator Activated Receptor
  • the pharmaceutical composition further comprises a Peroxisome Proliferator Activated Receptor alpha agonist and/or a Peroxisome Proliferator Activated Receptor gamma agonist.
  • the Peroxisome Proliferator Activated Receptor alpha agonist is fenofibrate. In certain embodiments, the Peroxisome Proliferator Activated Receptor gamma agonist is rosiglitazone.
  • the pharmaceutical composition comprises the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins in an amount of 0.05 mg or greater, 0.1 mg or greater, 0.2 mg or greater, 0.5 mg or greater, 1 mg or greater, 2 mg or greater, 5 mg or greater, 10 mg or greater, 50 mg or greater, 100 mg or greater, 0.05 mg or less, 0.1 mg or less, 0.5 mg or less, 1 mg or less, 2 mg or less, 5 mg or less, 10 mg or less, 50 mg or less, 100 mg or less, 0.05 to 100 mg, 0.05 to 50 mg, 0.05 to 10 mg, 0.05 to 5 mg, 0.05 to 1 mg, 0.05 to 0.5 mg, 0.05 to 0.1 mg, 0.1 to 100 mg, 0.1 to 50 mg, 0.1 to 10 mg, 0.1 to 5 mg, 0.1 to 1 mg, 0.1 to 0.5 mg, 0.5 to 100 mg, 0.5 to 50 mg, 0.5 to 10 mg, 0.5 to 5 mg, 0.5 to 1 mg, 1 to 100 mg, 0.5 to 50 mg,
  • the pharmaceutical composition comprises the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins in an amount of 0.2 to 2 mg.
  • the term "therapeutically effective amount” includes the quantity which, when administered to a subject in need of treatment, improves the prognosis and/or state of the subject.
  • the amount to be administered to a subject will depend on the particular characteristics of the disease, condition or state in the subject, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, body weight and tolerance to drugs.
  • administration and delivery of the compositions may be for example by one or more of the intravenous, intraperitoneal, subcutaneous, intramuscular, oral, or topical route, by direct injection or any combination of these administration routes.
  • the mode and route of administration in most cases will depend on the type of disease, condition or state being treated, and/or the treatment of a human or non-human animal.
  • the administration of the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and any other agents may also include the use of one or more pharmaceutically acceptable additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients, preservatives and bulking agents, taking into consideration the particular physical, microbiological and chemical characteristics of the agents to be administered.
  • pharmaceutically acceptable additives including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients, preservatives and bulking agents, taking into consideration the particular physical, microbiological and chemical characteristics of the agents to be administered.
  • the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and/or the other agents can be prepared into a variety of pharmaceutically acceptable compositions in the form of, e.g., an aqueous solution, an oily preparation, a fatty emulsion, an emulsion, a lyophilised powder for reconstitution, etc. and can be administered as a sterile and pyrogen free intramuscular or subcutaneous injection or as injection to an organ, or as an embedded preparation or as a transmucosal preparation through nasal cavity, rectum, uterus, vagina, lung, etc.
  • the composition may be administered in the form of oral preparations (for example solid preparations such as tablets, caplets, capsules, granules or powders; liquid preparations such as syrup, emulsions, dispersions or suspensions).
  • compositions containing the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and/or any other agents may also contain one or more pharmaceutically acceptable preservatives, buffering agents, diluents, stabilisers, chelating agents, viscosity enhancing agents, dispersing agents, pH controllers, or isotonic agents.
  • Examples of suitable preservatives are benzoic acid esters of para- hydroxybenzoic acid, propylene glycol, phenols, phenylethyl alcohol or benzyl alcohol.
  • suitable buffers are sodium phosphate salts, citric acid, tartaric acid and the like.
  • suitable stabilisers are, antioxidants such as alpha-tocopherol acetate, alpha-thioglycerin, sodium metabisulphite, ascorbic acid, acetylcysteine, 8- hydroxyquinoline, chelating agents such as disodium edetate.
  • Suitable viscosity enhancing agents, suspending or dispersing agents are substituted cellulose ethers, substituted cellulose esters, polyvinyl alchohol, polyvinylpyrrolidone, polyethylene glycols, carbomer, polyoxypropylene glycols, sorbitan monooleate, sorbitan sesquioleate, polyoxy ethylene hydrogenated castor oil 60.
  • pH controllers examples include hydrochloric acid, sodium hydroxide and the like.
  • suitable isotonic agents are glucose, D-sorbitol or D-mannitol, sodium chloride.
  • a endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and/or the other agents may also be in the form of a composition containing a pharmaceutically acceptable carrier, diluent, excipient, suspending agent, lubricating agent, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, glidant, anti-adherant, binder, flavorant or sweetener, taking into account the physical, chemical and microbiological properties of the agents being administered.
  • the composition may be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, mucosally, transdermally, bucally, vaginally, intraventricularly, via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically-acceptable carriers, or by any other convenient dosage form.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.
  • compositions When administered parenterally, the compositions will normally be in a unit dosage, sterile, pyrogen free injectable form (solution, suspension or emulsion, which may have been reconstituted prior to use) which is generally isotonic with the blood of the recipient with a pharmaceutically acceptable carrier.
  • sterile injectable forms are sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable vehicles, dispersing or wetting agents and suspending agents.
  • the sterile injectable forms may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3-butanediol.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides, corn, cottonseed, peanut, and sesame oil.
  • Fatty acids such as ethyl oleate, isopropyl myristate, and oleic acid and its glyceride derivatives, including olive oil and castor oil, especially in their polyoxyethylated versions, are useful in the preparation of injectables.
  • These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants.
  • the carrier may contain minor amounts of additives, such as substances that enhance solubility, isotonicity, and chemical stability, for example anti- oxidants, buffers and preservatives.
  • additives such as substances that enhance solubility, isotonicity, and chemical stability, for example anti- oxidants, buffers and preservatives.
  • compositions may be in a form to be reconstituted prior to administration.
  • examples include lyophilisation, spray drying and the like to produce a suitable solid form for reconstitution with a pharmaceutically acceptable solvent prior to administration.
  • compositions may include one or more buffers, bulking agents, isotonic agents and cryoprotectants and lyoprotectants.
  • excipients include, phosphate salts, citric acid, non-reducing such as sucrose or trehalose, polyhydroxy alcohols, amino acids, methylamines, and lyo tropic salts which are usually used instead of reducing sugars such as maltose or lactose.
  • the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and/or the other agents will usually be formulated into unit dosage forms such as tablets, caplets, cachets, powder, granules, beads, chewable lozenges, capsules, liquids, aqueous suspensions or solutions, or similar dosage forms, using conventional equipment and techniques known in the art.
  • Such formulations typically include a solid, semisolid, or liquid carrier.
  • Exemplary carriers include excipients such as lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, substituted cellulose ethers, polyoxyethylene sorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and the like.
  • excipients such as lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, substituted cellulose ethers, polyoxyethylene sorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate,
  • a tablet may be made by compressing or molding the agent optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active, or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine, a mixture of the powdered active ingredient and a suitable carrier moistened with an inert liquid diluent.
  • the administration of the endoplasmic reticulum stress inhibitor and/or the other agents may also utilize controlled release technology.
  • the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and/or the other agents may also be administered as a sustained-release pharmaceutical composition.
  • the agent may be formulated with additional components such as vegetable oil (for example soybean oil, sesame oil, camellia oil, castor oil, peanut oil, rape seed oil); middle fatty acid triglycerides; fatty acid esters such as ethyl oleate; polysiloxane derivatives; alternatively, water-soluble high molecular weight compounds such as hyaluronic acid or salts thereof, carboxymethylcellulose sodium hydroxypropylcellulose ether, collagen polyethylene glycol polyethylene oxide, hydroxypropylmethylcellulosemethylcellulose, polyvinyl lalcohol, and polyvinylpyrrolidone.
  • vegetable oil for example soybean oil, sesame oil, camellia oil, castor oil, peanut oil, rape seed oil
  • middle fatty acid triglycerides fatty acid esters such as eth
  • the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and/or the other agents may be incorporated into a hydrophobic polymer matrix for controlled release over a period of days.
  • the agent may then be moulded into a solid implant, or externally applied patch, suitable for providing efficacious concentrations of the agents over a prolonged period of time without the need for frequent re-dosing.
  • Such controlled release films are well known to the art.
  • Other examples of polymers commonly employed for this purpose that may be used include nondegradable ethylene-vinyl acetate copolymer a degradable lactic acid- glycolic acid copolymers, which may be used externally or internally.
  • Certain hydrogels such as poly(hydroxyethylmethacrylate) or poly(vinylalcohol) also may be useful, but for shorter release cycles than the other polymer release systems, such as those mentioned above.
  • the carrier may also be a solid biodegradable polymer or mixture of biodegradable polymers with appropriate time -release characteristics and release kinetics.
  • the agent may then be moulded into a solid implant suitable for providing efficacious concentrations of the agents over a prolonged period of time without the need for frequent re-dosing.
  • the agent can be incorporated into the biodegradable polymer or polymer mixture in any suitable manner known to one of ordinary skill in the art and may form a homogeneous matrix with the biodegradable polymer, or may be encapsulated in some way within the polymer, or may be moulded into a solid implant.
  • the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and/or the other agents may be in the form of a solution, spray, lotion, cream (for example a non-ionic cream), gel, paste or ointment.
  • the composition may be delivered via a liposome, nanosome, rivosome, or nutri-diffuser vehicle.
  • agents are also contemplated, including the use of a nucleic acid encoding a polypeptide for delivering of such agents, if such agents are a RNA or a polypeptide.
  • an endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins as described herein may be used to improve fertility in a female subject, to increase the likelihood of a female subject falling pregnant, to improve the time to pregnancy of a female subject, to prevent and/or treat reduced fertility in a female subject, to prevent and/or treat reduced fertility in a female subject, to prevent and/or treat infertility in a female subject, to prevent and/or treat ovarian dysfunction in a female subject, to prevent and/or treat sub-fertility in a female subject, to prevent and/or treat anovulation in a female subject, and to prevent and/or treat reduced ovulation rate in a female subject.
  • Certain embodiments of the present disclosure provide a method and/or a pharmaceutical composition to: (i) improve fertility in a female subject, to increase the likelihood of a female subject falling pregnant, (ii) to improve the time to pregnancy of a female subject, (iii) to prevent and/or treat reduced fertility or sub-fertility in a female subject, (iv) to prevent and/or treat reduced fertility in a female subject, (v) to prevent and/or treat infertility in a female subject, (vi) to prevent and/or treat ovarian dysfunction in a female subject, (vii) to prevent and/or treat anovulation in a female subject, and (viii) to prevent and/or treat reduced ovulation rate in a female subject; the aforementioned methods comprising exposing an oocyte in vivo in the female subject to an endoplasmic stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method of improving fertility in a female subject, the method comprising exposing an oocyte in vivo in the female subject to an endoplasmic stress inhibitor and/or the inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method of improving fertility in a female subject, the method comprising administering to the female subject a therapeutically effective amount of an endoplasmic stress inhibitor.
  • Certain embodiments of the present disclosure provide use of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in the preparation of a medicament for any of the following: (i) improve fertility in a female subject, to increase the likelihood of a female subject falling pregnant, (ii) to improve the time to pregnancy of a female subject, (iii) to prevent and/or treat reduced fertility in a female subject, (iv) to prevent and/or treat reduced fertility or sub-fertility in a female subject, (v) to prevent and/or treat infertility in a female subject, (vi) to prevent and/or treat ovarian dysfunction in a female subject, (vii) to prevent and/or treat anovulation in a female subject, and (viii) to prevent and/or treat reduced ovulation rate in a female subject.
  • Certain embodiments of the present disclosure provide use of an endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins in the preparation of a medicament for improving fertility in a female subject.
  • Certain embodiments of the present disclosure provide a method of improving fertility of a female subject, the method comprising administering to the subject an endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method to (i) improve fertility in a female subject, to increase the likelihood of a female subject falling pregnant, (ii) to improve the time to pregnancy of a female subject, (iii) to prevent and/or treat reduced fertility in a female subject, (iv) to prevent and/or treat reduced fertility or sub-fertility in a female subject, (v) to prevent and/or treat infertility in a female subject, (vi) to prevent and/or treat ovarian dysfunction in a female subject, (vii) to prevent and/or treat anovulation in a female subject, and (viii) to prevent and/or treat reduced ovulation rate in a female subject; the aforementioned methods comprising administering to the subject an effective amount of a pharmaceutical composition as described herein.
  • Certain embodiments of the present disclosure provide a method of improving fertility of a female subject comprising administering to the subject an effective amount of a pharmaceutical composition as described herein.
  • the subject is administered the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins at a dose 0.05 mg/kg/day or greater, 0.1 mg/kg/day or greater, 0.5 mg/kg/day or greater, 1 mg/kg/day or greater, 2 mg/kg/day or greater, 5 mg/kg/day or greater, 10 mg/kg/day or greater, 50 mg/kg/day or greater, 100 mg/kg/day or greater, 0.05 mg/kg/day or less, 0.1 mg/kg/day or less, 0.5 mg/kg/day or less, 1 mg/kg/day or less, 2 mg/kg/day or less, 5 mg/kg/day or less, 10 mg/kg/day or less, 50 mg/kg/day or less, 100 mg/kg/day or less, 0.05 to 100 mg/kg/day, 0.05 to 50 mg/kg/day, 0.05 to 10 mg/kg/day, 0.05 to 5 mg/kg/day, 0.05 to 1 mg/kg/kg/day, 0.05 to 1 mg
  • the subject is administered the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins at a dose of 0.1- 8 mg/kg/day. In certain embodiments, the subject is administered the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins at a dose of 0.2- 4 mg/kg/day. In certain embodiments, the subject is administered the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins at a dose of 0.2- 2 mg/kg/day.
  • the female subject is a mammal.
  • the female subject is human, a primate, a livestock animal (eg. a horse, a cow, a sheep, a pig, or a goat), a companion animal (eg. a dog, a cat), or a laboratory animal (eg. a mouse, a rat, a guinea pig, a rabbit).
  • a livestock animal eg. a horse, a cow, a sheep, a pig, or a goat
  • a companion animal eg. a dog, a cat
  • a laboratory animal eg. a mouse, a rat, a guinea pig, a rabbit.
  • the female subject comprises one or more of the following characteristics: a body mass index of greater than 25 kg/m ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal to 30 kg/m 2 ; obesity; Polycystic ovary syndrome; reduced fertility; infertility; sub-fertility, ovarian dysfunction; anovulation; reduced ovulation rate; pre-diabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet.
  • Certain embodiments of the present disclosure provide a treatment regime for a female subject with reduced fertility, the treatment regime comprising administering to the subject an endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a treatment regime for a female subject with reduced fertility, the treatment regime comprising administering to the subject a pharmaceutical composition as described herein.
  • a suitable treatment regime may be designed by a person skilled in the art.
  • Certain embodiments of the present disclosure provide a method of preventing and/or treating a female subject with reduced fertility, the method comprising administering to the subject a therapeutically effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method of preventing and/or treating a female subject with reduced fertility, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition as described herein.
  • Certain embodiments of the present disclosure provide use of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in the preparation of a medicament for preventing and/or treating reduced fertility in a female subject.
  • Certain embodiments of the present disclosure provide a method of preventing and/or treating a subject with reduced fertility, the method comprising administering to the subject a therapeutically effective amount of endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins; wherein the subject comprises one or more of the following characteristics: a body mass index of greater than 25 kg/m ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal to 30 kg/m 2 ; obesity; Polycystic ovary syndrome; reduced fertility; sub-fertility, infertility; ovarian dysfunction; anovulation; reduced ovulation rate; pre-diabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet.
  • Certain embodiments of the present disclosure provide a method of increasing the likelihood of a female subject falling pregnant, the method comprising exposing the subject to an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
  • Certain embodiments of the present disclosure provide a method of increasing the likelihood of a female subject falling pregnant, the method comprising exposing the subject to an effective amount of a pharmaceutical composition as described herein.
  • exposure of an oocyte in vitro to an endoplasmic stress inhibitor may also be used in an assisted reproduction technology.
  • the exposure may be used to improve fertility in a female subject, to increase the likelihood of a female subject falling pregnant, to improve the time to pregnancy, and to prevent and/or treat reduced fertility in a female subject.
  • Certain embodiments of the present disclosure provide a method of treating reduced fertility in a female subject, the method comprising:
  • mice CBAxC57Bl/6 Fl were maintained on a 12-h light, 12-h dark cycle with rodent chow and water available ad libitum. All experiments were approved by the University of Sydney's Animal Ethics Committee and were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.
  • Immature unexpanded COCs were isolated from 23-day-old prepubertal female mice by puncturing the antral follicles of ovaries collected 44 h after i.p. injection of 5 IU equine chorionic gonadotropin (eCG, Professional Compounding Centre of Australia). Mature expanded COCs were obtained from oviducts by blunt dissection 44 h after eCG injection and 16 h after i.p. administration of 5 IU human chorionic gonadotropin (hCG/Pregnyl, Organon) to stimulate ovulation. All COCs were collected in HEPES -buffered a-MEM (Invitrogen) supplemented with 1% or 5% (vol/vol) fetal calf serum (FCS, Invitrogen) as indicated.
  • HEPES -buffered a-MEM Invitrogen
  • FCS fetal calf serum
  • Treatments consisted of control media supplemented with ER stress inducer ⁇ or 500nM thapsigargin (Merk), or ⁇ or 500nM ER stress inducer thapsigargin plus ⁇ or 200nM eIF-2a inhibitor Salubrinal (Merck), or ⁇ or 200nM Salubrinal alone (see Figures 1-4).
  • ER stress inducer ⁇ or 500nM thapsigargin Merk
  • 1% (vol/vol) FCS was used in the culture media.
  • Treatments were control media supplemented with 150, 275, 400, 525 ⁇ palmitic acid for dose response experiments (see Figure 5), or 400 ⁇ palmitic acid with 200nM salubrinal, or 200nM salubrinal alone (see Figures 6-9).
  • Real-time PCR was performed in triplicate using SYBR Green PCR Master Mix (Applied Biosystems) and a Rotor-Gene 6000 (Corbett) real-time rotary analyzer. Real-time RT-PCR data were analyzed using the 2-AACT method and expressed as the fold change relative to a calibrator sample which was included in each run.
  • the COCs were then incubated with the primary antibody at a 1: 100 dilution in blocking buffer overnight at 4°C.
  • Specific primary antibodies used were rabbit polyclonal anti-PTX3 (Santa Cruz) and goat polyclonal anti-TSG6 (Santa Cruz).
  • Following washing in PBS COCs were incubated with biotinylated goat-anti-rabbit (Millipore) or biotinylated donkey-anti-goat (Millipore) secondary antibodies for 1 h at room temperature.
  • COCs were washed in PBS and incubated with 1 ng/mL streptavidin-Alexa594 (Molecular Probes) and 1 mg/mL DAPI (Invitrogen) in PBS for 1 h at room temperature. COCs were visualised and images captured using a Leica TCS SP5 spectral scanning confocal microscope system.
  • Protein extracts from matrix fractions or cell pellet fractions were resolved by 10% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes (Millipore). Membranes were blocked in TBST (10 mM Tris, 150 mM NaCl and 0.05% Tween 20, pH 7.5) containing 3% (w/v) non-fat milk for 1 h at room temperature. Membranes were then incubated with primary antibodies for 2 h at room temperature in 3% milk/TBST.
  • COCs matured in vitro and COCs isolated from oviduct at 16 h post-hCG (in vivo, ovulated) were used for in vitro fertilization.
  • Sperm were collected from 8 week old CBAxC57Bl/6 Fl males from the vas deferens and caudal region of the epididymis.
  • Sperm were capacitated in bicarbonate -buffered a-MEM supplemented with 3 mg/ml of BSA (fatty acid-free) for 1 h at 37°C in an atmosphere of 5% C0 2 and 95% air.
  • BSA fatty acid-free
  • COCs were washed twice, and COCs and sperm (35 000 sperm/ml) were coincubated in ⁇ bicarbonate-buffered a-MEM supplemented with 3 mg/ml of BSA (fatty acid free) for 4 h at 37°C in an atmosphere of 5% C0 2 and 95% air. All cumulus cell-free oocytes were then transferred to embryo development culture medium (Vitro Cleave, COOK Australia). Twenty-four hours following in vitro fertilization (Day 2), the fertilization rate was assessed, and 2-cell embryos were transferred to a fresh, 20 ⁇ drop of culture medium.
  • BSA fatty acid free
  • Embryo morphology was assessed on Day 3 (44 h of embryo culture) and Day 5 (the end of the culture period, 96-100 hours post- fertilization). Embryos were classified as appropriately developed ('on- time') using the following criteria: on Day 2, embryos at the 2-cell stage; on Day 3, embryos at the 4- to 8-cell stage; and on Day 5, blastocyst or hatching blastocyst. The rate of development was assessed on day 2 as the percentage of embryos meeting the development criteria from the starting number of oocytes; while the rate of development was assessed on day 3 and 5 as the percentage of embryos meeting the development criteria from 2-cell embryos on day 2.
  • COCs were cultured in media containing both thapsigargin ( ⁇ ) and the ER stress eIF-2a inhibitor salubrinal ( ⁇ ).
  • ⁇ thapsigargin
  • ⁇ ER stress eIF-2a inhibitor
  • salubrinal normalized expression of each of the four ER stress markers to levels similar to those of control COCs matured either in vitro or in vivo.
  • the ER stress inhibitor salubrinal can reverse each of the thapsigargin-induced defects. ER stress is also tightly linked to mitochondrial dysfunction and thus to investigate whether ER stress inducer thapsigargin can cause mitochondrial damage in oocytes, mitochondrial membrane potential of oocytes was determined by staining with the inner membrane potential dye JC-1. In ovulated COCs (matured in vivo) oocytes exhibited red punctuate fluorescence localized to the pericortical region of oocytes indicating high MMP, while green fluorescence localized to the deeper cytoplasm of oocytes indicating low MMP (Figure 3A).
  • Oocytes from control COCs matured in vitro had a similar pattern although reduced in.
  • red fluorescence intensity and thus mitochondrial membrane potential was reduced in the pericortical region (Fig. 3A), indicating that ER stress inducer thapsigargin impairs mitochondrial function in oocytes.
  • red fluorescence intensity in the pericortical region of oocytes was similar to controls.
  • COCs matured in 150 ⁇ palmitic acid had similar mRNA expression levels to control COCs (Figure 5); however Xbpls mRNA was significantly increased at 275 ⁇ , ATF4 mRNA was significantly increased at 400 ⁇ , and ATF6 and GRP78 mRNA were significantly increased at 525 ⁇ palmitic acid.
  • COCs treated with high (400 ⁇ ) concentrations of palmitic acid were also co- treated with salubrinal (200nM) during maturation to determine whether salubrinal can reduce palmitic acid-induced ER stress.
  • COCs treated with salubrinal for 8 hours had dramatically increased expression of ATF4 and GRP78 mRNA compared to both untreated control COCs as well as high dose (400 ⁇ ) palmitic acid treated COCs, while expression levels of ATF6 and Xbpls were reduced to control levels.
  • All four genes were reduced to control groups' levels by salubrinal treatment (Figure 6).
  • intracellular PTX3 protein was relatively similar in all treatments.
  • the IRE1 reduction in extracellular PTX3 protein was associated with increased levels of phosphorylated IRE1 which was highest in COCs matured in high dose palmitic acid for 8h ( Figure 7B).
  • the reductions in PTX3 protein secretion were not reflective of changes in Ptx3 mRNA since all of the treatment groups had PTX3 mRNA levels that were identical to controls at 8h of maturation, and that were not decreased compared to controls at 16h of maturation (Figure 7C).
  • mitochondrial membrane potential of oocytes was determined by staining with the inner membrane potential dye JC-1. Oocytes matured in vitro have less red fluorescence intensity than in vivo matured oocytes, as expected. However, in oocytes maturing in high dose (400 ⁇ ) palmitic acid but not low dose (150 ⁇ ) palmitic acid, red fluorescence intensity was reduced in the pericortical region (Fig. 8A), indicating that high dose palmitic acid decreases mitochondrial activity in oocytes.
  • L-carnitine is a cellular metabolite that regulates fatty acid transportation from cytosol into mitochondria.
  • L-carnitine can improve mitochondrial activity assessed as increased JC-1 staining. It also found be able to protect cardiac mitochondrial structure damage and function reduction against 20 ⁇ palmitoyl CoA caused.
  • L-carnitine increases fatty acid metabolism in COCs and improves embryo development in vitro. Thus we investigated whether the mitochondrial dysfunction induced by high dose palmitic acid can be repaired by adding L-carnitine, similar to the effect of salubrinal.
  • L-carnitine treatment was not able to normalize oocyte mitochondrial activity (0.13+0.15) of COCs treated with high dose palmitic acid (0.11+0.08) ( Figure 8 C and D).
  • L-carnitine can improve mitochondrial activity of oocytes matured in vitro it cannot reverse the oocyte mitochondrial dysfunction seen in COCs exposed to high dose palmitic acid.
  • the fertilized oocytes from COCs treated with high dose palmitic acid were also slower to develop to 4 cells on day 3 (87.9%) and to blastocysts and hatching blastocysts on day 5 (50%), which was significantly lower than the in vitro matured control group on day 3 (100%).
  • Salubrinal treatment 200nM normalized the palmitic acid induced decrease in fertilization rates and day 3 development rates to control levels.
  • This study elucidates a cellular mechanism by which lipotoxicity contributes to female infertility. Specifically, during oocyte in vitro maturation, exposure to toxic lipids, such as the saturated fatty acid palmitic acid at concentrations mimicking physiological levels present in obese women, induces ER stress in COCs thereby impairing the secretion of cumulus cell protein, mitochondrial activity in oocytes, oocyte maturation and fertilisation.
  • toxic lipids such as the saturated fatty acid palmitic acid at concentrations mimicking physiological levels present in obese women
  • EXAMPLE 2 Effect of ER stress inhibitors: PBA and TUDCA during mouse COC maturation and fertilization
  • Immature COCs isolated from mice treated with eCG for 44-46 hours
  • Treatments consisted of Tauroursodeoxycholic acid (TUDCA, Calbiochem, Cat. 580549) or 4-Phenylbutyrate (PBA, Calbiochem, Cat 567616). TUDCA or PBA were added to IVM media alone (i.e. in the absence of ER stress inducer Thapsigargin) at the following concentrations:
  • TUDCA lOmM, 2mM, ImM , 900 ⁇ , 800 ⁇ , 700 ⁇ , 500 ⁇ , 100 ⁇ , and 10 ⁇
  • PBA 5mM, 2mM ImM, 100 ⁇ , 10 ⁇ , 1 ⁇ , ⁇ , ⁇ , InM
  • FIG 10 shows representative examples of COCs evaluated after the standard time of 16hours of rVM.
  • morphology was very poor, with cumulus cells falling from the complex and dispersed. COCs were found completely denuded in the higher concentrations of TUDCA and PBA.
  • FIG 11 shows the results of an experiment that investigated the effects of TUDCA on oocyte developmental competence.
  • COCs matured in vitro in fetal calf serum (FCS; the standard in vitro control condition) and COCs matured in vivo (ie isolated from the oviduct after ovulation) showed normal high rates of fertilization and blastocyst development.
  • Maturation of COCs in the ER stress inducer thapsigargen (TG; ⁇ ) resulted in reduced fertilization and blastocyst development as observed in other experiments.
  • Fertilisation of COCs matured in TUDCA (ImM) resulted in very poor embryo development, even compared to thapsigargen (TG) treatment.
  • Bovine oocytes are known to have higher intracellular lipid content than mouse or human oocytes.
  • bovine COCs were matured in vitro by treatment TUDCA (500 ⁇ ), PBA ( ⁇ ), or Salubrinal ( ⁇ ). COCs were then subjected to IVF under identical standard conditions. The cleavage rate was not different among treatments and controls (data not shown). TUDCA and PBA treatments resulted in the lowest blastocyst rates, while salubrinal tended to increase the blastocyst rate higher than control.
  • Blood-free follicle fluid was collected at oocyte pick-up from women of known Body Mass Index (BMI; kg/m ) undergoing IVF/ICSI treatment who had given written, informed consent to participate in a study approved by the Women's and Children's Hospital, Sydney, South Australia. Triglyceride and free fatty acid levels in follicular fluid were measured by automated Roche Hitachi 912 Chemistry Analyzer.
  • Immature COCs were isolated from 23 day old mice (CBAXC57B1/6F1) by puncturing antral follicles of ovaries collected 44h after i.p. injection of 5 IU equine chorionic gonadotropin (eCG/Gestyl, Professional Compounding Centre of Australia, Sydney, NSW, AU). Mature COCs were dissected from oviducts of mice following 44h eCG and 13h post i.p.
  • COCs were collected in Hepes-buffered a-MEM (Gibco, Invitrogen Australia Pty. Ltd., VIC, AU) and cultured in groups of 20 at 37°C in 6% C0 2 / 94% air in 100 ⁇ drops of bicarbonate-buffered a-MEM, supplemented with 50% (v/v) human follicle fluid and overlaid with sterile paraffin oil (Merck, Darmstadt, DE).
  • Maturation was stimulated by treatment with 50 mlU/ml recombinant human FSH and 10 ng/ml Epidermal Growth Factor (EGF). Cumulus expansion, germinal vesicle breakdown (GVBD) and first polar body extrusion were evaluated following 16h of culture.
  • EGF Epidermal Growth Factor
  • BODIPY 493/503 Lipophilic dye BODIPY 493/503 (Invitrogen) which stains intracellular neutral lipids, was used to localize lipid droplets in oocytes. Briefly, COCs were paraformaldehyde-fixed, stained in BODIPY 493/503 and imaged using a Leica SP5 spectral scanning confocal microscope and identical conditions throughout all experiments. Fluorescence in the oocyte was calculated by Analysis Pro Software (Olympus Australia Pty. Ltd., Mt Waverly, VIC, AU).
  • COCs Cumulus-oocyte complexes from mice were stimulated to mature for 16h in lipid-rich follicle fluid (high lipid FF) and compared to those matured in lipid- poor follicle fluid (low lipid FF) and those matured in vivo, i.e. isolated from oviducts following ovulation. COCs were stained with neutral lipid dye and lipid content within oocytes determined. Oocytes matured in lipid-rich follicle fluid contained significantly more neutral lipid than oocytes matured in lipid-poor follicle fluid or those matured in vivo (Fig. 13A).
  • perilipin-2 a lipid droplet protein expressed in the COC that coats lipid droplets in mouse oocytes, was also significantly higher in COCs matured in lipid-rich follicle fluid than in COCs matured in lipid-poor follicle fluid or in vivo (Fig. 13B).
  • Expression of ER stress markers Atf4, Atf6 and Grp78 were also significantly increased in COCs matured in lipid-rich follicle fluid compared to COCs matured in lipid-poor follicle fluid (Fig. 13 C-E), similar to COCs from mice with diet-induced obesity.
  • COC maturation was assessed by measuring cumulus expansion score, oocyte germinal vesicle breakdown (GVBD) and oocyte polar body extrusion. Cumulus expansion and GVBD were not affected by culture in either of the follicle fluids compared to in vivo matured COCs (data not shown). However, maturation in lipid-rich follicle fluid dramatically decreased oocyte maturation to Mil, assessed as polar body formation, to approximately 25% of in vivo rates (Fig. 13F).
  • EXAMPLE 5 Reduced ovulation rate and oocyte quality in two mouse ER stress models and restoration with salubrinal
  • Homocysteine (Hey) in drinking water is an established in vivo model of ER stress in mice and mimics clinical hyperhomocysteinemia, which occurs in women with PCOS and is correlated with poor embryo outcome.
  • mice were given 3.6g/L homocysteine in drinking water for 2 weeks to induce systemic ER stress. We are the first to show that these mice have reduced ovulation rate, and most importantly, that this can be reversed by treatment with salubrinal (lmg/kg) injected i.p. for just 4 days ( Figure 14).
  • mice represent a model of obesity-induced lipotoxicity. These mice have a mutation that causes severe hyperphagia and weight gain even when mice are fed a standard chow diet.
  • FIG. 15 shows that Blobby mice (which are obese) have poor oocyte quality compared to their wildtype or heterozygous littermates. Specifically the oocytes of Blobby mice when fertilized by IVF have reduced fertilization rates (2-cells) and poor development to the 4-cell and blastocyst stages. When we treat the mice with salubrinal (Sal; lmg/kg i.p. daily) for 4 days prior to ovulation we are able to restore oocyte quality to normal in the obese Blobby mice. Veh: vehicle-treated controls.
  • Figure 16 Values are mean + SEM expressed as number of ovulated oocytes and different letters indicate significant differences by one-way ANOVA, Bonferroni Post hoc test; P ⁇ 0.05.Thus ER stress responses in COCs contribute to ovulation deficiencies in obese Blobby mice since the ER stress inhibitor salubrinal treatment can restore ovulation capacity.
  • EXAMPLE 7 Improved mitochondria in Blobby mice upon treatment with an ER stress inhibitor
  • Figure 17 shows that treatment with an ER stress improves mitochondria in Blobby mice.
  • oocyte mitochondrial membrane potential ( ⁇ ) was reduced in ovulated oocytes from Blobby mice by staining with the inner membrane potential dye 5,5' ,6,6' -tetrachloro- 1 , 1 ' ,3,3 ' -tetraethylbenzimidazolylcarbocyanine iodide (JC-1).
  • JC-1 oocyte mitochondrial membrane potential
  • oocytes of ovulated COC exhibited red punctuate fluorescence localized to the pericortical region, indicating high ⁇ , whereas green fluorescence indicating low ⁇ localized to the deeper cytoplasm of oocytes ( Figure 17).
  • Figure 18A shows that treatment with an ER stress inhibitor restores fertilization rates in Blobby mice.
  • Figure 18B shows that treatment with an ER stress inhibitor improves rates of blastocyst formation on Blobby mice.
  • BGP-15 and BGP-15M are compounds that induce heat shock proteins, for example Hsp72.
  • COCs were treated with thapsigargen (100 nM) in the presence of BGP-15 or BGP-15M at doses of 50 mg/L, 100 mg/L or 200 mg/L. Additional cohorts were treated with BGP-15 (100 mg/L) alone or BGP-15M (100 mg/L) alone or were untreated controls. The COCs were then stimulated to undergo 'expansion', a process whereby they rapidly produce extracellular matrix in preparation for fertilization, and their responses were scored 16 hours later. Thapsigargen impairs cumulus expansion, an effect that can be reversed by salubrinal.
  • BGP-15 or BGP-15M alone at 400 mg/L did not impair cumulus expansion and some COCs exhibited more robust expansion than controls.
  • COCs exposed to thapsigargen exhibited very poor expansion, as expected, and co-treatment with either BGP-15 or BGP-15M at 400 mg/L restored cumulus expansion to control levels.
  • BGP-15M appeared to be slightly more effective at restoring cumulus expansion than BGP-15. This experiment was conducted twice with identical results.
  • BGP-15 is not detrimental to health and viability of mouse cumulus-oocyte complexes and that at 400 mg/L it can reverse the detrimental effect of the ER stressor thapsigargen on cumulus expansion. Therefore BGP-15 may be effective in reversing other types of ER stress in COCs, such as ER stress induced by dyslipidemia or obesity.
  • Control COCs exhibited normal oocyte mitochondrial activity, assessed by JC- 1 staining, with red-stained 'active' mitochondria in the periphery of the oocyte and primarily green-stained 'inactive' mitochondrial in the center of the oocyte. Palmitic acid treatment (400 uM) reduced mitochondrial activity as we have previously observed. BGP-15M (400 mg/L) co-treatment restored oocyte mitochondrial activity to normal in palmitic acid-treated COCs. BGP-15M alone (400 mg/L) also increased oocyte mitochondrial activity.
  • BGP-15M is able to improve mitochondrial activity in oocytes exposed to high lipid.
  • EXAMPLE 10 - ER stress inhibitors can improve oocyte developmental competence whether used during IVM or IVF.
  • Figure 21 shows that treatment of mouse COCs with high lipid (400uM palmitic acid (PA)) during their maturation in vitro (IVM) impairs subsequent fertilisation rate, as well as blastocyst development rates (not shown).
  • PA 400uM palmitic acid
  • IVM maturation in vitro
  • EXAMPLE 11 - Ovulation rate is significantly increased in adult female mice by in vivo treatment with heat shock inducer BGP-15M
  • EXAMPLE 12 Treatment of oocytes from obese mice with an ER stress inducer normalises fetal size and weight
  • alterations in the oocytes of obese Blobby mice lead to altered fetal (and perhaps plancental) growth even though the oocytes are fertilized with sperm from a non-obese male and transferred into the uterus of a non-obese female for gestation.
  • fetal size is normalized by a brief peri- conception treatment of the obese mothers.
  • Different letters indicate significant differences by one-way AN OVA, Bonferroni Post hoc test; P ⁇ 0.05.

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Abstract

The present disclosure relates to a method of improving developmental competence of an oocyte. The method comprises exposing the oocyte and/or an embryo to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.

Description

IMPROVED DEVELOPMENTAL COMPETENCE OF OOCYTES
PRIORITY CLAIM
[001] This application claims priority to Australian provisional patent application 2011904259 filed on 10 October 2011, the content of which is hereby incorporated by reference.
FIELD
[002] The present disclosure relates to methods and culture medium for improving the developmental competence of oocytes, and embryos arising from the oocytes, and also to pharmaceutical compositions and methods of treatment for improving fertility.
BACKGROUND
[003] Despite improvements in the understanding of the complex biology of embryo development and female fertility, female infertility in humans still remains a serious health problem.
[004] In addition, while assisted reproduction technologies are becoming increasing important in the animal sector, the rates of success of these technologies still remain relatively low and provide a significant economic barrier to the widespread adoption of these technologies.
[005] Advances in in vitro fertilisation (Γν ) techniques have assisted some human couples with fertility deficiencies, but the success rates of IVF have remained relatively static and in many instances this technique does not assist in improving fertility in some women.
[006] There is also an urgent need to develop new strategies to stop the impact of the obesity epidemic on female fertility and embryo development. Western countries in particular have a high obesity rate: in Australia, for example, over 50% of women are overweight or obese. A serious health consequence of increased body mass index is female infertility. For example, obesity is a risk factor for anovulation, including the increasingly common failure to respond to gonadotropin treatment. Further, even in women who are cycling regularly, obesity is associated with increased time-to- pregnancy and decreased chance of pregnancy. Obesity is also definitively and causally associated with the onset of Polycystic Ovary Syndrome (PCOS). PCOS is characterised by multiple follicles which develop from primordial follicles, but the development is arrested at an early antral stage due to disturbed ovarian function. Obesity is believed to be the main contributing factor to PCOS, and the majority of patients with the condition have insulin resistance and/or are obese.
[007] In humans, oocytes of obese women also often fail to develop to the blastocyst stage when fertilised in vitro, indicating that obesity disrupts sensitive aspects of oocyte maturation that influence subsequent embryo growth.
[008] Pharmacological interventions for improving fertility still have a number of disadvantages, including variable efficacy and undesired side effects. For example, currently obese women (including those with PCOS) are encouraged to lose weight or they are treated with drugs, such as clomiphene citrate, but this is often ineffective. This agent is used mainly for ovarian stimulation in female infertility due to anovulation, but has common side effects such as hot flashes, breast tenderness, mood swings, and nausea. In addition, treatment can also lead to multiple ovulations, hence increasing the chance of twins.
[009] As the prevalence of obesity rises, assisted reproduction technologies such as IVF, are increasingly required to achieve pregnancies. Unfortunately, even with these invasive technologies the oocytes of women with obesity and/or PCOS often fail to develop to the blastocyst stage.
[0010] Accordingly, there is a need for methods and compositions for improving assisted reproduction technologies, and methods and compositions for treating reduced fertility in women, including to address one or more problems in the art and/or to provide one or more advantages. SUMMARY
[0011] The present disclosure is based on the determination that the developmental competence of oocytes may be improved by exposing oocytes to an endoplasmic stress inhibitor and/or an inducer of heat shock proteins.
[0012] Certain embodiments of the present disclosure provide a method of improving developmental competence of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[0013] Certain embodiments of the present disclosure provide a method of improving fertility in a female subject, the method comprising administering to the subject an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[0014] Certain embodiments of the present disclosure provide a method of treating reduced fertility in a female subject, the method comprising administering to the subject an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[0015] Certain embodiments of the present disclosure provide a method of improving ovulation in a female subject, the method comprising administering to the subject an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[0016] Certain embodiments of the present disclosure provide a method of assisted reproduction comprising an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[0017] Certain embodiments of the present disclosure provide a method of improving cumulus cell expansion in a cumulus oocyte complex, the method comprising exposing an oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins. [0018] Certain embodiments of the present disclosure provide a method of increasing the level of cumulus cell protein in a cumulus oocyte complex, the method comprising exposing an oocyte to an endoplasmic reticulum stress inhibitor, and/or an inducer of heat shock proteins
[0019] Certain embodiments of the present disclosure provide a method of in vitro maturation of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[0020] Certain embodiments of the present disclosure provide a method of improving the developmental competence of an embryo produced by fertilisation of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[0021] Certain embodiments provide a method of improving blastocyst development of an embryo produced by fertilisation of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[0022] Certain embodiments of the present disclosure provide an oocyte and/or embryo culture medium, the medium comprising an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[0023] Certain embodiments of the present disclosure provide a kit for improving developmental competence, the kit comprising:
an oocyte and/or embryo culture medium;
an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins; and
optionally instructions for culturing the oocyte and/or embryo in the culture medium.
[0024] Certain embodiments provide a pharmaceutical composition for improving fertility in a female subject, the composition comprising an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins. [0025] Certain embodiments of the present disclosure provide a method of preventing and/or treating reduced fertility in a female subject, the method comprising administering to the subject a therapeutically effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[0026] Certain embodiments of the present disclosure provide use of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in the preparation of a medicament for preventing and/or treating reduced fertility in a female subject.
[0027] Certain embodiments of the present disclosure provide a method of increasing the likelihood of a female subject falling pregnant, the method comprising exposing the subject to an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[0028] Certain embodiments of the present disclosure provide a method of treating reduced fertility in a female subject, the method comprising:
exposing an oocyte in vitro to an effective amount of an endoplasmic stress inhibitor and/or an inducer of heat shock proteins; and
introducing the oocyte, or an embryo produced from the oocyte, into the female subject.
[0029] Certain embodiments of the present disclosure provide a method of preventing and/or treating a subject with reduced fertility, the method comprising administering to the subject a therapeutically effective amount of endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins; wherein the subject comprises one or more of the following characteristics: a body mass index of greater than 25 kg/m ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal to 30 kg/m 2 ; obesity; Polycystic ovary syndrome; reduced fertility; sub-fertility; infertility; ovarian dysfunction; anovulation; reduced ovulation rate; pre-diabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet. [0030] Certain embodiments of the present disclosure provide a combination product comprising:
an oocyte and/or embryo culture medium; and
an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[0031] Other certain embodiments are disclosed herein. BRIEF DESCRIPTION OF THE FIGURES
[0032] Certain embodiments are illustrated by the following figures. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the description.
[0033] Figure 1 shows that thapsigargin induces ER stress in COCs which can be reversed by Salubrinal. Mouse COCs were matured in vitro for 16 hours in the presence of ΙΟΟηΜ thapsigargin (Thap) or ΙΟΟηΜ thapsigargin plus ΙΟΟηΜ Salubrinal (Thap+Sal), or ΙΟΟηΜ Salubrinal (Sal) alone, or in the absence of thapsigargin and salubrinal as control. Ovulated COCs (in vivo matured) from eCG, hCG 16 h treated mice were used for comparison to in vitro controls. Total RNA was extracted from COCs and expression of ER stress marker genes ATF4 (A), ATF6 (B), Xbpls (C) and GRP78 (D) was determined by RT-PCR. Values are mean + SEM expressed as fold change compared with calibrator sample; n = 3 pools of COCs per treatment group. Different letters indicate significant differences by one-way ANOVA, Tukey post test, A: P =0.0016, B: P=0.0022, C: P<0.0001, D: P=0.0184.
[0034] Figure 2 shows that thapsigargin impairs cumulus expansion and protein secretion which can be reversed by Salubrinal. COCs from eCG-treated mice were matured in vitro for 16 hours in the presence of ΙΟΟηΜ thapsigargin (Thap), ΙΟΟηΜ thapsigargin plus ΙΟΟηΜ Salubrinal (Thap+Sal), ΙΟΟηΜ Salubrinal (Sal) alone, or in the absence of thapsigargin and salubrinal as control. (A) The morphology of COCs matured in vitro. (B) COC expansion assessed according to a qualitative scoring system, 0 to +4, and presented as the mean + SEM, n=3 experimental replicates with >25 COCs per treatment group. Different letters indicate significant differences by one-way ANOVA, Tukey post test P < 0.0001. (C) Matrix protein pentraxin-3 (PTX3) (red- fluorescence) was reduced in cumulus matrix of thapsigargin (Thap) treated COCs by immunohistochemistry. DAPI nuclear stain is shown as blue fluorescence. (D) Western blot analysis of PTX3 from cumulus matrix extracts, and PTX3, phospho-IREl and IRE1 proteins from cell pellets obtained from COCs matured for 8 or 16 hour.
[0035] Figure 3 shows that ER stress reduces oocyte mitochondrial membrane potential which is reversed by Salubrinal. Mitochondrial membrane potential was assessed by JC-1 staining (A) in oocytes matured in vitro for 16 hours in the presence of ΙΟΟηΜ thapsigargin (Thap) or ΙΟΟηΜ thapsigargin plus ΙΟΟηΜ Salubrinal (Thap+Sal), or ΙΟΟηΜ Salubrinal (Sal) alone, or in the absence of thapsigargin and salubrinal as control. Ovulated COCs (in vivo matured) from eCG, hCG 16 h treated mice were used as a comparison for the in vitro controls. Red fluorescence indicates high mitochondrial membrane potential and green indicates low mitochondrial membrane potential. (B) The ratio of red to green fluorescence was quantified as an indicator of mitochondrial activity. Data are presented as mean + SEM, n = 25-35 oocytes from three independent experiments. Different letters indicate significant differences by one-way ANOVA, Tukey post test; P<0.05.
[0036] Figure 4 shows that COCs matured in the presence of thapsigargin have significantly impaired embryo developmental competence following IVF, which is reversible with Salubrinal. COCs were matured in vitro for 16 hours in the presence of 500nM thapsigargin (Thap) or 500nM thapsigargin plus 200nM Salubrinal (Thap+Sal), or 200nM Salubrinal (Sal) alone, or in the absence of thapsigargin and salubrinal as control. Ovulated COCs (in vivo matured) from eCG, hCG 16 h treated mice were used as a comparison to the in vitro controls. COCs were then fertilized in vitro and the rate of embryo development assessed. Data presented as the mean % on time embryo development + SEM, n = 3 experimental replicates, representative of 90 oocytes per treatment. Day 3 and Day 5 development is from 2-cell embryos on Day2. Different letters indicate significant differences by one-way ANOVA within each developmental stage, Tukey post test; p<0.05.
[0037] Figure 5 shows that palmitic acid dose-dependently induces ER stress in COCs. COCs were matured in vitro for 16h in 1% FCS as control or plus 150, 275, 400 or 525 μΜ palmitic acid. Total RNA was extracted from COCs and expression of ER stress marker genes ATF4 (A), ATF6 (B), Xbpls (C) and GRP78 (D) was analyzed by RT- PCR. Values are mean + SEM expressed as fold change compared with calibrator sample; n = 3 pools of COCs per treatment group. Different letters indicate significant differences by one-way ANOVA, Tukey post hoc test; P<0.05.
[0038] Figure 6 shows induction of ER stress markers by high dose (400μΜ) palmitic acid is reversible by salubrinal. Immature COCs from eCG-treated mice (0 hours) COCs were matured in vitro in 1% FCS as control or plus 150 μΜ or 400μΜ palmitic acid or 400μΜ palmitic acid and 200nM salubrinal (Sal) for 8 hours or 16 hours. Total RNA was extracted from COCs and subjected to RT-PCR for analysis of ER stress marker genes mRNA expression. ATF4 (A), ATF6 (B), Xbpls (C) and GRP78 (D) mRNA expression levels were determined. Values are mean + SEM expressed as fold change compared with calibrator sample; n = 3 pools of cells per experiment. Different letters indicate significant differences by one-way ANOVA within each time course, Tukey post hoc test; p<0.05
[0039] Figure 7 shows high dose (400μΜ) palmitic acid reduces Ptx3 protein production in COCs that is normalized with salubrinal. Immature COCs from eCG- treated mice (0 hour) COCs were matured in vitro in 1% FCS as control or plus 150 μΜ or 400μΜ palmitic acid or 400μΜ palmitic acid and 200nM salubrinal (Sal) for 8 hours or 16 hours. (A) Cumulus matrix protein pentraxin-3 (PTX3; red- fluorescence) detected by immunocytochemistry was reduced in the cumulus matrix in 400μΜ palmitic acid treated COCs. DAPI nuclear stain is shown as blue fluorescence. (B) Western blot analysis of extracellular PTX3 from cumulus matrix extracts, and intracellular PTX3, phospho-IREl and IRE1 proteins from cell pellets obtained from COCs matured in vitro for 8 or 16 hours. (C) PTX3 mRNA expression levels were determined by RT-PCR. Values are mean + SEM expressed as fold change compared with calibrator sample; n = 3 pools of cells per treatment group. Different letters indicate significant differences by one-way ANOVA within each timepoint, Bonferroni post hoc test.
[0040] Figure 8 shows oocyte mitochondrial membrane potential is reduced in COCs treated with high dose palmitic acid and reversed by Salubrinal but not by L-Carnitine. Mitochondrial membrane potential was assessed by JC-1 staining in oocytes matured in vitro in the presence of 150μΜ or 400μΜ palmitic acid or without palmitic acid as control. High dose (400μΜ) palmitic acid treated COCs were also treated with either salubrinal (Sal; 200nM) or L-carnitine (5mM) as indicated. In vivo matured oocytes from eCG, hCG 16h treated mice were used for comparison to in vitro controls (A, C). The ratio of red to green fluorescence was quantified as an indicator of mitochondrial activity. Data are presented as mean + SEM, n = 30 -55 oocytes from three independent experiments. Different letters indicate significant differences by one-way ANOVA, Tukey post hoc test (B, D).
[0041] Figure 9 shows that COCs treated with high dose palmitic acid have significantly impaired embryo development that is reversed by salubrinal. COCs from eCG-treated mice were matured in vitro for 16 hours in the presence of 150 μΜ or 400μΜ palmitic acid and/or 200nM salubrinal (Sal) or without palmitic acid as control. In vivo matured COCs from eCG, hCG 16h treated mice were used for comparison to in vitro controls. COCs were fertilized in vitro and oocyte developmental competence was assessed by the rate of embryo development following in vitro fertilization. Data presented as the mean % on time embryo development + SEM, n= 3 independent experiments, representative of 90 oocytes per treatment. Day 3 and Day 5 development is from 2-cell embryos on Day 2. Different letters indicate significant differences by one-way ANOVA with Tukey post hoc test at each developmental stage.
[0042] Figure 10 shows the effect of ER stress inhibitors TUDCA and PBA on COC morphology.
[0043] Figure 11 shows fertilisation of COCs matured in TUDCA (ImM) results in very poor embryo development, even compared to thapsigargen (TG) treatment.
[0044] Figure 12 shows the effect of salubrinal added to bovine oocytes during oocyte maturation in vitro, followed by IVF and assessments of embryo development.
[0045] Figure 13 shows that the obese ovarian environment directly affects oocytes. Follicle fluid (FF) from either obese women or moderate weight women was added to mouse COCs during 16h of maturation; and compared to controls matured for 16h in vivo. [0046] Figure 14 shows reduced ovulation rates in homocysteine (Hcy)-treated mice. In mice fed Hey for 2 weeks poor ovulation rate is normalised by treatment with salubrinal for 4 days.
[0047] Figure 15 shows that that Blobby mice have poor oocyte quality compared to their wildtype or heterozygous littermates. Oocytes of Blobby mice when fertilized by IVF have reduced fertilization rates (2-cells) and poor development to the 4-cell and blastocyst stages. When the mice are treated with salubrinal (lmg/kg i.p. daily) for 4 days prior to ovulation, oocyte quality is restored to normal in the obese Blobby mice.
[0048] Figure 16 shows that ovulation in Blobby mice is improved upon administration of an ER stress inhibitor.
[0049] Figure 17 shows that treatment with an ER stress improves mitochondria in Blobby mice.
[0050] Figure 18A shows that treatment with an ER stress inhibitor restores fertilization rates in Blobby mice. Figure 18B shows that treatment with an ER stress inhibitor improves rates of blastocyst formation on Blobby mice.
[0051] Figure 19 shows photographs of mouse cumulus-oocyte complexes (COCs) following in vitro culture with no supplement (control), 400 μΜ palmitic acid (PA) or 400 μΜ palmitic acid and the hydroximic acid derivatives BGP-15 or BGP-15M for 16 hours. The oocyte can be seen at the center of each complex surrounded by many layers of cumulus cells and extracellular matrix. PA: palmitic acid.
[0052] Figure 20 shows mouse COCs exposed to high lipid (i.e. palmitic acid (PA) at 400 μΜ) have significantly impaired embryo development that is restored by co- treatment with BGP-15M (400mg/L). COCs were exposed to the indicated treatment for 16 hours followed by IVF under identical conditions. The percentage of oocytes that formed 2-cell embryos following IVF was assessed at 24 hours/ day 2 (A). The percentage of 2-cell embryos developing to blastocysts was assessed on day 5 (B). Data are presented as mean + SEM, n= 2 replicate experiments with 30 oocytes per treatment group. Different letters indicate significant differences by one-way ANOVA, Bonferroni Post hoc test; P<0.05.
[0053] Figure 21 shows that treatment of mouse COCs with high lipid (400uM palmitic acid (PA)) during their maturation in vitro (IVM) impairs subsequent fertilisation rate, as well as blastocyst development rates (not shown). The addition of salubrinal to either the IVM media OR the fertilisation media normalises fertilisation (and blastocyst) rates. Data is mean + SEM; n=3 independent experiments using >20 COCs per treatment group in each experiment. ***p<0.0001 compared to all other groups.
[0054] Figure 22 shows that ovulation rate is significantly increased in adult female mice by in vivo treatment with BGP-15M for 4 days (20mg/kg i.p. once per day). N=3 mice per treatment group. *p=0.015 by t-test.
[0055] Figure 23 shows fetal crown-rump length (A) and fetal weight (B) in animals at 14.5 days of gestation (6 blastocysts were transferred in each horn, wildtype, n=8 fetuses from 3 recipient horns; Blobby, n=7 fetuses from 2 recipient horns; and Blobby + 4 days salubrinal treatment (lmg/kg), n= 9 fetuses from 3 recipient horns). Different letters indicate significant differences by one-way ANOVA, Bonferroni Post hoc test; P<0.05.
DETAILED DESCRIPTION
[0056] The present disclosure relates to methods, culture medium and compositions for improving oocyte developmental competence. The present disclosure also relates to methods and compositions for preventing and/or treating reduced fertility.
[0057] The present disclosure is based, at least in part, upon the recognition that the developmental competence of oocytes may be improved by exposure of the oocytes, in vitro and in vivo, to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[0058] Certain embodiments of the present disclosure are directed to methods for improving developmental competence of oocytes, media for culturing oocytes and/or embryos, methods for treating reduced fertility, and pharmaceutical compositions for treating reduced fertility. Kits and combination products are also provided.
[0059] Certain embodiments of the present disclosure are directed to methods for improving developmental competence of oocytes, media for culturing oocytes and/or embryos, methods for treating reduced fertility, methods for improving fertility, and pharmaceutical compositions for treating reduced fertility, that have one or more advantages. For example, some of the advantages of the embodiments disclosed herein include one or more of the following: improving developmental competence of oocytes in female subjects (including, for example, obese women and/or women with PCOS); assist in the formulation of culture media for oocytes from female subjects (including, for example, obese women and/or women with PCOS); provide improved pharmaceutical compositions for improving fertility in female subjects (including, for example, obese women and/or women with PCOS); assist in prevention and/or treatment of reduced fertility in female subjects (including, for example, obese women and/or women with PCOS); improve the likelihood of female subjects (including, for example, obese women and/or women with PCOS) falling pregnant; improving fertility in female subjects (including, for example, obese women and/or women with PCOS); improve outcomes for assisted reproduction technologies involving oocytes in animals and humans; assist in the uptake of assisted reproduction technologies in animals and humans; to improve fertility; improve the rate of live births per IVF cycle; improve success rates for women seeking natural conceptions (including, for example, obese women and/or women with PCOS); improve ovulation rates in women (including, for example, obese women and/or women with PCOS); improve time to pregnancy in women (including, for example, obese women and/or women with PCOS); provide a pharmacological treatment with improved efficacy and/or reduced side effects; improve blastocyst development of embryos produced from oocytes (including, for example, from obese women and/or women with PCOS); improve the maturation of oocytes (including, for example, from obese women and/or women with PCOS). Other advantages of certain embodiments are disclosed herein.
[0060] Certain embodiments of the present disclosure provide a method of improving developmental competence of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins. [0061] The term "developmental competence" as used herein includes one or more of: (i) the ability and/or likelihood of the oocyte to produce an embryo (for example, upon fertilization of an oocyte or by other mechanisms, such as parthenogenic activation); (ii) one or more of the ability, likelihood and rate of an oocyte to progress through blastocyst development upon formation of an embryo; and (iii) the quality of the embryo (for example, as determined by morphological and/or biochemical assessments) achieved upon the production of an embryo from the oocyte.
[0062] Methods for determining the developmental competence of an oocyte are known.
[0063] In certain embodiments, the developmental competence is improved. For example, the ability of an embryo produced from an oocyte exposed to an endoplasmic reticulum stress to progress through blastocyst development may be improved and/or has one or more of an increased ability to progress through blastocyst development, an increased fertilization rate, an increased likelihood of progression through blastocyst development, an increased rate of forming a two cell embryo, an increased rate of forming a four cell embryo, an increased rate of forming a blastocyst, an increased rate of progressing through blastocyst development, and an increased rate of blastocyst hatching.
[0064] In certain embodiments, the method may be used to increase fertilization rate, to increase blastocyst formation, and to increase blastocyst hatching.
[0065] Certain embodiments of the present disclosure provide a method of increasing fertilization rate of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins. In certain embodiments, the fertilization rate is 95% or greater, 96% or greater, 97% or greater, or 98% or greater.
[0066] Certain embodiments of the present disclosure provide a method of increasing blastocyst formation of an embryo produced from an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[0067] Certain embodiments of the present disclosure provide a method of increasing blastocyst hatching of an embryo produced from an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins. In certain embodiments, the rate of blastocyst hatching is 95% or greater, 96% or greater, 97% or greater, or 98% or greater.
[0068] In certain embodiments, the oocyte is a part of a cumulus oocyte complex. In certain embodiments, the oocyte is a denuded oocyte. Methods for removing cumulus cells from a cumulus oocyte complex are known. The term "oocyte" as used herein includes an oocyte alone or an oocyte in association with one or more other cells, such as an oocyte as part of a cumulus oocyte complex. Animal and human oocytes are contemplated.
[0069] In certain embodiments, the oocyte comprises an increased lipid content and/or an increased lipid level. In certain embodiments, the oocyte comprises an increased content and/or level of neutral lipids. In certain embodiments, the oocyte comprises an increased content and/or level of one or more toxic lipids. In certain embodiments, the oocyte comprises an oocyte exposed to an increased lipid environment and/or an oocyte exposed to one or more toxic lipids. In certain embodiments, the oocyte is a normal oocyte. In certain embodiments, the oocyte does not comprise an increased lipid content and/or an increased lipid level.
[0070] Methods for determining the lipid content of oocytes are known. For example, lipid droplet abundance and localization in an oocyte may be measured using a fluorescent stain, such as BODIPY 493/503 (Invitrogen) and comparison to oocytes with a normal lipid content.
[0071] In certain embodiments, the oocyte is obtained from a female subject comprising one or more of the following characteristics: a body mass index of greater than 25 kg/m 2 ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal to 30 kg/m ; obesity; Polycystic Ovary Syndrome; reduced fertility; sub- fertility, infertility; ovarian dysfunction; anovulation; reduced ovulation rate; prediabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet.
[0072] In certain embodiments, the oocyte is obtained from a female subject comprising one or more of the following characteristics: a normal body mass index; a body mass index of equal to or less than 25 kg/m ; normal weight; non-obese; and normal fertility.
[0073] In certain embodiments, the endoplasmic reticulum stress inhibitor comprises one or more of an inhibitor of an inositol requiring protein- 1 (IREl) signal transduction pathway, an inhibitor of a protein kinase RNA-like endoplasmic reticulum kinase (PERK) signal transduction pathway, an inhibitor of a transcription factor (ATF6) signal transduction pathway, and an inducer of heat shock proteins.
[0074] In certain embodiments, the endoplasmic reticulum stress inhibitor comprises an inducer of heat shock proteins, such as a derivative of a hydroximic acid.
[0075] In certain embodiments, the endoplasmic reticulum stress inhibitor comprises an inhibitor of eIF2a dephosphorylation. Inhibitors of eIF2a dephosphorylation are known and available.
[0076] In certain embodiments, the eIF2a dephosphorylation is salubrinal and/or a derivative of salubrinal that is capable of inhibiting eIF2a dephosphorylation, such as a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate of salubrinal. The term "salubrinal" as used herein refers to salubrinal and any of the aforementioned derivatives:
Figure imgf000017_0001
[0077] An example of a derivative of salubrinal that is capable of inhibiting eIF2a dephosphorylation is as follows:
Figure imgf000018_0001
[0078] In certain embodiments, the endoplasmic reticulum stress inhibitor comprises tauroursodeoxycholate (TUDCA) and/or 4-phenyl butyric acid (PBA); and/or a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate of either these compounds. The term "tauroursodeoxycholate" as used herein refers to tauroursodeoxycholate and any of the aforementioned derivatives. The term "4-phenyl butyric acid" as used herein refers to 4-phenyl butyric acid and any of the aforementioned derivatives.
[0079] In certain embodiments, the inducer of heat shock proteins comprises a derivative of hydroximic acid. In certain embodiments, the derivative of hydroximic acid comprises one or more of BGP-15, propanolol, bimoclomol, arimoclomal, NG-94, iroxanadine, and/or a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate of any of the aforementioned. The structures of the aforementioned hydroximic acid derivatives are as follows:
Figure imgf000019_0001
Figure imgf000019_0002
[0080] In certain embodiments, the inducer of heat shock proteins comprises an inducer of Hsp72.
[0081] Methods for exposing oocytes to agents are known. In certain embodiments, the oocyte is exposed to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in vitro. In certain embodiments, the oocyte is exposed to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in vivo. In certain embodiments, the oocyte is exposed to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in vitro and in vivo.
[0082] Examples of in vitro exposure include exposure of an oocyte to an agent in a liquid medium, exposure to a pre-drug that is metabolised to an active agent in the oocyte, introduction of a product into the oocyte directly, such as injection, introduction of a nucleic acid encoding the agent into an oocyte, such as by viral infection or transfection, and exposure of the oocyte to a compound that induces the expression of an agent in the oocyte. [0083] In certain embodiments, the method comprises exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in vitro. For example, an oocyte may be cultured in a medium comprising an endoplasmic recticulum stress inhibitor and/or an inducer of heat shock proteins.
[0084] Examples of in vivo exposure include various methods for the administration of an agent to a subject, such as a human or animal subject, are described herein.
[0085] In certain embodiments, the oocyte is exposed to an exogenous endoplasmic reticulum stress inhibitor, such as salubrinal. In certain embodiments, the oocyte is exposed to an endogenous endoplasmic reticulum stress inhibitor.
[0086] In certain embodiments, the oocyte is exposed to a concentration of the endoplasmic reticulum stress inhibitor of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1000 nM or greater, 10 uM or greater, 0.1 nM or less, 0.5 nM or less, 1 nM or less, 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, 1000 nM or less, 10 uM or less, 0.1 nM to 10 uM, 0.1 nM to 1000 nM, 0.1 nM to 500 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.1 nM to 1 nM, 0.5 nM to 10 uM, 0.5 nM to 1000 nM, 0.5 nM to 500 nM, 0.5 nM to 100 nM, 0.5 nM to 50 nM, 0.5 nM to 10 nM, 0.5 nM to 1 nM; 1 nM to 10 uM, 1 nM to 1000 nM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 5 nM to 10 uM, 5 nM to 1000 nM, 5 nM to 500 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 10 nM, 10 nM to 10 uM, 10 nM to 1000 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 100 nm to 10 uM, 100 nM to 1000 nM, 100 nM to 500 nM, 500 nM to 10 uM, 500 nM to 1000 nM, 1000 nM to 10 uM, or 1 uM to 10 uM.
[0087] In certain embodiments, the oocyte is exposed to a concentration of salubrinal of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1000 nM or greater, 10 uM or greater, 0.1 nM or less, 0.5 nM or less, 1 nM or less, 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, 1000 nM or less, 10 uM or less, 0.1 nM to 10 uM, 0.1 nM to 1000 nM, 0.1 nM to 500 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.1 nM to 1 nM, 0.5 nM to 10 uM, 0.5 nM to 1000 nM, 0.5 nM to 500 nM, 0.5 nM to 100 nM, 0.5 nM to 50 nM, 0.5 nM to 10 nM, 0.5 nM to 1 nM; 1 nM to 10 uM, 1 nM to 1000 nM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 5 nM to 10 uM, 5 nM to 1000 nM, 5 nM to 500 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 10 nM, 10 nM to 10 uM, 10 nM to 1000 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 100 nm to 10 uM, 100 nM to 1000 nM, 100 nM to 500 nM, 500 nM to 10 uM, 500 nM to 1000 nM, 1000 nM to 10 uM, or 1 uM to 10 uM.
[0088] In certain embodiments, the oocyte is exposed to a concentration of salubrinal in the range of 50 nM to 500 nM.
[0089] In certain embodiments, the oocyte is exposed to a concentration of tauroursodeoxycholate of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1000 nM or greater, 10 uM or greater, 0.1 nM or less, 0.5 nM or less, 1 nM or less, 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, 1000 nM or less, 10 uM or less, 0.1 nM to 10 uM, 0.1 nM to 1000 nM, 0.1 nM to 500 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.1 nM to 1 nM, 0.5 nM to 10 uM, 0.5 nM to 1000 nM, 0.5 nM to 500 nM, 0.5 nM to 100 nM, 0.5 nM to 50 nM, 0.5 nM to 10 nM, 0.5 nM to 1 nM; 1 nM to 10 uM, 1 nM to 1000 nM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 5 nM to 10 uM, 5 nM to 1000 nM, 5 nM to 500 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 10 nM, 10 nM to 10 uM, 10 nM to 1000 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 100 nm to 10 uM, 100 nM to 1000 nM, 100 nM to 500 nM, 500 nM to 10 uM, 500 nM to 1000 nM, 1000 nM to 10 uM, or 1 uM to 10 uM.
[0090] In certain embodiments, the oocyte is exposed to a concentration of tauroursodeoxycholate in the range of <10μΜ.
[0091] In certain embodiments, the oocyte is exposed to a concentration of 4-phenyl butyric acid of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1000 nM or greater, 10 uM or greater, 0.1 nM or less, 0.5 nM or less, 1 nM or less, 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, 1000 nM or less, 10 uM or less, 0.1 nM to 10 uM, 0.1 nM to 1000 nM, 0.1 nM to 500 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.1 nM to 1 nM, 0.5 nM to 10 uM, 0.5 nM to 1000 nM, 0.5 nM to 500 nM, 0.5 nM to 100 nM, 0.5 nM to 50 nM, 0.5 nM to 10 nM, 0.5 nM to 1 nM; 1 nM to 10 uM, 1 nM to 1000 nM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 5 nM to 10 uM, 5 nM to 1000 nM, 5 nM to 500 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 10 nM, 10 nM to 10 uM, 10 nM to 1000 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 100 nm to 10 uM, 100 nM to 1000 nM, 100 nM to 500 nM, 500 nM to 10 uM, 500 nM to 1000 nM, 1000 nM to 10 uM, or 1 uM to 10 uM.
[0092] In certain embodiments, the oocyte is exposed to a concentration of 4-phenyl butyric acid in the range of <lnM.
[0093] In certain embodiments, the oocyte is exposed to an inducer of heat shock proteins, such as a derivative of hydroximic acid. In certain embodiments, the oocyte is exposed to an endogenous inducer of heat shock proteins. In certain embodiments, the oocyte is exposed to an exogenous inducer of heat shock proteins.
[0094] In certain embodiments, the oocyte is exposed to a concentration of a derivative of a hydroximic acid (for example BGP-15) at a concentration of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1000 nM or greater, 10 uM or greater, 0.1 nM or less, 0.5 nM or less, 1 nM or less, 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, 1000 nM or less, 10 uM or less, 0.1 nM to 10 uM, 0.1 nM to 1000 nM, 0.1 nM to 500 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.1 nM to 1 nM, 0.5 nM to 10 uM, 0.5 nM to 1000 nM, 0.5 nM to 500 nM, 0.5 nM to 100 nM, 0.5 nM to 50 nM, 0.5 nM to 10 nM, 0.5 nM to 1 nM; 1 nM to 10 uM, 1 nM to 1000 nM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 5 nM to 10 uM, 5 nM to 1000 nM, 5 nM to 500 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 10 nM, 10 nM to 10 uM, 10 nM to 1000 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 100 nm to 10 uM, 100 nM to 1000 nM, 100 nM to 500 nM, 500 nM to 10 uM, 500 nM to 1000 nM, 1000 nM to 10 uM, or 1 uM to 10 uM.
[0095] In certain embodiments, the oocyte is exposed to a concentration of a derivative of a hyroximic acid (for example BGP-15) of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1000 nM or greater, 10 uM or greater, 0.1 nM or less, 0.5 nM or less, 1 nM or less, 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, 1000 nM or less, 10 uM or less, 0.1 nM to 10 uM, 0.1 nM to 1000 nM, 0.1 nM to 500 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.1 nM to 1 nM, 0.5 nM to 10 uM, 0.5 nM to 1000 nM, 0.5 nM to 500 nM, 0.5 nM to 100 nM, 0.5 nM to 50 nM, 0.5 nM to 10 nM, 0.5 nM to 1 nM; 1 nM to 10 uM, 1 nM to 1000 nM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 5 nM to 10 uM, 5 nM to 1000 nM, 5 nM to 500 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 10 nM, 10 nM to 10 uM, 10 nM to 1000 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 100 nm to 10 uM, 100 nM to 1000 nM, 100 nM to 500 nM, 500 nM to 10 uM, 500 nM to 1000 nM, 1000 nM to 10 uM, or 1 uM to 10 uM.
[0096] In certain embodiments, the oocyte is exposed to a concentration of a derivative of a hyroximic acid (for example BGP-15) in the range of 0.1 to 1000 mg/ml, 0.1 to 500 mg/ml, 0.1 to 400 mg/ml, 0.1 to 200 mg/ml, 0.1 to 100 mg/ml, 0.1 to 50 mg/ml, 0.1 to 20 mg/ml, 0.1 to 10 mg/ml, 0.1 to 5 mg/ml, 0.1 to 4 mg/ml, 0.1 to 2 mg/ml, 0.1 to 1.0 mg/ml, 0.1 to 0.5 mg/ml, 0.1 to 0.4 mg/ml, 0.1 to 0.2 mg/ml, 0.2 to 1000 mg/ml, 0.2 to 500 mg/ml, 0.2 to 400 mg/ml, 0.2 to 200 mg/ml, 0.2 to 100 mg/ml, 0.2 to 50 mg/ml, 0.2 to 20 mg/ml, 0.2 to 10 mg/ml, 0.2 to 5 mg/ml, 0.2 to 4 mg/ml, 0.2 to 2 mg/ml, 0.2 to 1 mg/ml, 0.2 to 0.5 mg/ml, 0.2 to 0.4 mg/ml, 0.4 to 1000 mg/ml, 0.4 to 500 mg/ml, 0.4 to 400 mg/ml, 0.4 to 200 mg/ml, 0.4 to 100 mg/ml, 0.4 to 50 mg/ml, 0.4 to 20 mg/ml, 0.4 to 10 mg/ml, 0.4 to 5 mg/ml, 0.4 to 4 mg/ml, 0.4 to 2 mg/ml, 0.4 to 1 mg/ml, 0.4 to 0.5 mg/ml, 0.5 to 1000 mg/ml, 0.5 to 500 mg/ml, 0.5 to 400 mg/ml, 0.5 to 200 mg/ml, 0.5 to 100 mg/ml, 0.5 to 50 mg/ml, 0.5 to 20 mg/ml, 0.5 to 10 mg/ml, 0.5 to 5 mg/ml, 0.5 to 4 mg/ml, 0.5 to 2 mg/ml, 0.5 to 1.0 mg/ml, 1 to 1000 mg/ml, 1 to 500 mg/ml, 1 to 400 mg/ml, 1 to 200 mg/ml, 1 to 100 mg/ml, 1 to 50 mg/ml, 1 to 20 mg/ml,
1 to 10 mg/ml, 1 to 5 mg/ml, 1 to 4 mg/ml, 1 to 2 mg/ml, 2 to 1000 mg/ml, 2 to 500 mg/ml, 2 to 400 mg/ml, 2 to 200 mg/ml, 2 to 100 mg/ml, 2 to 50 mg/ml, 2 to 20 mg/ml,
2 to 10 mg/ml, 2 to 5 mg/ml, 2 to 4 mg/ml, 4 to 1000 mg/ml, 4 to 500 mg/ml, 4 to 400 mg/ml, 4 to 200 mg/ml, 4 to 100 mg/ml, 4 to 50 mg/ml, 4 to 20 mg/ml, 4 to 10 mg/ml, 4 to 5 mg/ml, 5 to 1000 mg/ml, 5 to 500 mg/ml, 5 to 400 mg/ml, 5 to 200 mg/ml, 5 to 100 mg/ml, 5 to 50 mg/ml, 5 to 20 mg/ml, 5 to 10 mg/ml, 10 to 1000 mg/ml, 10 to 500 mg/ml, 10 to 400 mg/ml, 10 to 200 mg/ml, 10 to 100 mg/ml, 10 to 50 mg/ml, 10 to 20 mg/ml, 20 to 1000 mg/ml, 20 to 500 mg/ml, 20 to 400 mg/ml, 20 to 200 mg/ml, 20 to 100 mg/ml, 20 to 50 mg/ml, 50 to 1000 mg/ml, 50 to 500 mg/ml, 50 to 400 mg/ml, 50 to 200 mg/ml, 50 to 100 mg/ml, 100 to 1000 mg/ml, 100 to 500 mg/ml, 100 to 400 mg/ml, 100 to 200 mg/ml, 200 to 1000 mg/ml, 200 to 500 mg/ml, 200 to 400 mg/ml, and 500 to 1000 mg/ml.
[0097] Certain embodiments of the present disclosure provide a method of culturing an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, wherein exposure of the oocyte to the endoplasmic stress inhibitor improves the developmental competence of the oocyte. Culturing of oocytes from human, animals and other species are contemplated.
[0098] In certain embodiments, the oocyte is an immature oocyte. In certain embodiments, the oocyte is a mature oocyte. Methods for determining the maturation status of an oocyte are known.
[0099] In certain embodiments, the oocyte is an oocyte matured in vitro. Methods for collecting/obtaining oocytes and performing in vitro maturation are known, including in humans and animals.
[00100] In certain embodiments, the method comprises obtaining an oocyte from a female subject. In certain embodiments, the method comprises obtaining an immature oocyte from a female subject. In certain embodiments, the method comprises obtaining an immature oocyte from a female subject and in vitro maturing the oocyte.
[00101] In certain embodiments, the oocyte is an oocyte matured in vivo. Methods for collecting/obtaining mature oocytes from a female subject are known. In certain embodiments, the method comprises obtaining a mature oocyte from a female subject.
[00102] In certain embodiments, the method comprises exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in a subject in vivo. In certain embodiments, the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins are administered to a subject in order to expose an oocyte to the endoplasmic stress inhibitor. [00103] Administration and delivery of the endoplasmic stress inhibitor and/or an inducer of heat shock proteins may be performed by a known method. For example, administration may be one or more of intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical route, and direct injection. The mode and route of administration in most cases will depend on the type of disease, condition or state being treated.
[00104] In certain embodiments, the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins are administered to the subject at a dose of 0.05 mg/kg or greater, 0.1 mg/kg or greater, 0.5 mg/kg or greater, 1 mg/kg or greater, 2 mg/kg or greater, 5 mg/kg or greater, 10 mg/kg or greater, 50 mg/kg or greater, 100 mg/kg or greater, 0.05 mg/kg or less, 0.1 mg/kg or less, 0.5 mg/kg or less, 1 mg/kg or less, 2 mg/kg or less, 5 mg/kg or less, 10 mg/kg or less, 50 mg/kg or less, 100 mg/kg or less, 0.05 to 100 mg/kg, 0.05 to 50 mg/kg, 0.05 to 10 mg/kg, 0.05 to 5 mg/kg, 0.05 to 1 mg/kg, 0.05 to 0.5 mg/kg, 0.05 to 0.1 mg/kg, 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 10 mg/kg, 0.1 to 5 mg/kg, 0.1 to 1 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 100 mg/kg, 0.5 to 50 mg/kg, 0.5 to 10 mg/kg, 0.5 to 5 mg/kg, 0.5 to 1 mg/kg, 1 to 100 mg/kg, 1 to 50 mg/kg, 1 to 10 mg/kg, 1 to 5 mg/kg, 5 to 100 mg/kg, 5 to 50 mg/kg, 5 to 10 mg/kg, 10 to 100 mg/kg, or 10 to 50 mg/kg.
[00105] In certain embodiments, the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins are administered to the subject at a dose of 0.2 to 2 mg/kg.
[00106] In certain embodiments, the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins are administered to the subject at a dose 0.05 mg/kg/day or greater, 0.1 mg/kg/day or greater, 0.5 mg/kg/day or greater, 1 mg/kg/day or greater, 2 mg/kg/day or greater, 5 mg/kg/day or greater, 10 mg/kg/day or greater, 50 mg/kg/day or greater, 100 mg/kg/day or greater, 0.05 mg/kg/day or less, 0.1 mg/kg/day or less, 0.5 mg/kg/day or less, 1 mg/kg/day or less, 2 mg/kg/day or less, 5 mg/kg/day or less, 10 mg/kg/day or less, 50 mg/kg/day or less, 100 mg/kg/day or less, 0.05 to 100 mg/kg/day, 0.05 to 50 mg/kg/day, 0.05 to 10 mg/kg/day, 0.05 to 5 mg/kg/day, 0.05 to 1 mg/kg/day, 0.05 to 0.5 mg/kg/day, 0.05 to 0.1 mg/kg/day, 0.1 to 100 mg/kg/day, 0.1 to 50 mg/kg/day, 0.1 to 10 mg/kg/day, 0.1 to 5 mg/kg/day, 0.1 to 1 mg/kg/day, 0.1 to 0.5 mg/kg/day, 0.5 to 100 mg/kg/day, 0.5 to 50 mg/kg/day, 0.5 to 10 mg/kg/day, 0.5 to 5 mg/kg/day, 0.5 to 1 mg/kg/day, 1 to 100 mg/kg/day, 1 to 50 mg/kg/day, 1 to 10 mg/kg/day, 1 to 5 mg/kg/day, 5 to 100 mg/kg/day, 5 to 50 mg/kg/day, 5 to 10 mg/kg/day, 10 to 100 mg/kg/day, or 10 to 50 mg/kg/day.
[00107] In certain embodiments, the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins are administered to the subject at a dose of 0.2-2 mg/kg/day.
[00108] In certain embodiments, the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins are administered to a human subject at a dose of 50 to 500 mg/day. In certain embodiments, the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins are administered to a human subject at a dose of 100 to 400 mg/day. For example, two 100 mg capsules by mouth in the morning; and two 100 mg capsules by mouth in the evening; or two 50 mg capsules by mouth in the morning and two 50 mg capsules by mouth in the evening.
[00109] In certain embodiments, the method further comprises exposing the oocyte to an inducer of lipid metabolism and/or to a Peroxisome Proliferator Activated Receptor (PPAR) agonist. In certain embodiments, the Peroxisome Proliferator Activated Receptor (PPAR) agonist comprises a Peroxisome Proliferator Activated Receptor alpha agonist and/or a Peroxisome Proliferator Activated Receptor gamma agonist.
[00110] In certain embodiments, the method comprises exposing the oocyte to an inducer of lipid metabolism, such as L-carnitine, and/or a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate thereof.
[00111] In certain embodiments, the method comprises exposing the oocyte to a Peroxisome Proliferator Activated Receptor alpha agonist and/or a Peroxisome Proliferator Activated Receptor gamma agonist. In certain embodiments, the Peroxisome Proliferator Activated Receptor alpha agonist is fenofibrate. In certain embodiments, the Peroxisome Proliferator Activated Receptor gamma agonist is rosiglitazone. [00112] In certain embodiments, the method comprises exposing the oocyte to an inducer of lipid metabolism (such as L-carnitine) and a Peroxisome Proliferator Activated Receptor (PPAR) agonist.
[00113] In certain embodiments, the oocyte is a mammalian oocyte. In certain embodiments, the oocyte is an oocyte from a human, a primate, a livestock animal (eg. a horse, a cow (eg bos taurus, bos indicus), a sheep, a pig, or a goat), a companion animal (eg. a dog, a cat), or a laboratory animal (eg. a mouse, a rat, a guinea pig, a rabbit, a bird, a frog). In certain embodiments, the oocyte is a bovine oocyte, such as an oocyte from a dairy cow. In certain embodiments, the oocyte is a human oocyte. Oocytes from other species are contemplated.
[00114] In certain embodiments, the oocyte is obtained from a female subject comprising one or more of the following characteristics: a body mass index of greater than 25 kg/m 2 ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal 30 kg/m ; obesity; Polycystic Ovary Syndrome; reduced fertility; infertility; sub-fertility, ovarian dysfunction; anovulation; reduced ovulation rate; pre-diabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet.
[00115] In certain embodiments, the method is used to improve maturation of an oocyte, to improve developmental competence of an embryo produced by fertilisation of the oocyte exposed to the endoplasmic reticulum stress inhibitor, to improve blastocyst development of an embryo produced by fertilisation of the oocyte exposed to the endoplasmic reticulum stress inhibitor, to improve cumulus cell expansion in a cumulus oocyte complex, to increase the level of cumulus cell protein in a cumulus oocyte complex; to improve assisted reproduction; to improve fertility; to treat sub-fertility, to treat infertility; to treat ovarian dysfunction; to treat anovulation; to improve ovulation rate, to improve fertility in a female subject, to treat reduced fertility in a female subject, and to improve ovulation in a female subject. [00116] Certain embodiments of the present disclosure provide a non-human oocyte or a non-human embryo produced after exposure to the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins as described herein. Certain embodiments of the present disclosure provide a non-human animal produced from the oocyte or embryo. Methods for producing animals from oocytes are known, and include for example, in vitro fertilization. A female animal may also, for example, be treated with an endoplasmic stress inhibitor and/or an inducer of heat shock proteins, and subsequently fertilized. Examples of animals are described herein.
[00117] Certain embodiments of the present disclosure provide a method of assisted reproduction comprising an oocyte, the method comprising exposing the oocyte to an endoplasmic stress inhibitor and/or an inducer of heat shock proteins, as described herein.
[00118] Certain embodiments of the present disclosure provide a method of assisted reproduction comprising an oocyte, the method comprising:
exposing an oocyte in vitro to an endoplasmic recticulum stress inhibitor and/or an inducer of heat shock proteins; and
using the oocyte in an assisted reproduction technology.
[00119] Examples of assisted reproduction comprising an oocyte, or an embryo produced from an oocyte, include a fertilization technique in humans and animals involving an isolated oocyte, including a technique using an oocyte in vitro (for example in vitro maturation of an oocyte), in vitro fertilization (IVF; aspiration of an oocyte, fertilization in the laboratory and transfer of the embryo into a recipient), gamete intrafallopian transfer (GIFT; placement of oocytes and sperm into the fallopian tube), zygote intrafallopian transfer (ZIFT; placement of fertilized oocytes into the fallopian tube), tubal embryo transfer (TET; the placement of cleaving embryos into the fallopian tube), peritoneal oocyte and sperm transfer (POST; the placement of oocytes and sperm into the pelvic cavity), intracytoplasmic sperm injection (ICSI), testicular sperm extraction (TESE), and microsurgical epididymal sperm aspiration (MESA); or any other in vitro technique for producing embryos in humans and/or animals comprising an oocyte, such as nuclear transfer and parthenogenic activation [00120] In certain embodiments, the assisted reproduction technology is used to produce a mammalian embryo. In certain embodiments, the assisted reproduction technology is used to produce a human embryo. In certain embodiments, the assisted reproduction technology is used to produce a bovine embryo. In certain embodiments, the assisted reproduction technology is used in an animal.
[00121] In certain embodiments, the oocyte is first harvested or collected from an ovary of a subject. Oocyte collection can be performed according to standard techniques, for example as described in Textbook of Assisted Reproduction: Laboratory and Clinical Perspectives (2003) Editors Gardner, D. K., Weissman, A., Howies, CM., Shoham, Z. Martin Dunits Ltd, London, UK; and Gordon, I. (2003) Laboratory Production of Cattle Embryos 2nd Edition CABI Publishing, Oxon, UK.
[00122] In certain embodiments, the assisted reproduction technology comprises in vitro fertilisation. Certain embodiments provide a method of in vitro fertilisation, the method comprising exposing the oocyte to an endoplasmic stress inhibitor and/or an inducer of heat shock proteins, as described herein.
[00123] IVF relates to the fertilization of an oocyte in vitro, wherein the oocyte is isolated from the subject and typically incubated in liquid media to allow fertilization of the oocyte. It is contemplated that fertilisation of the oocyte will generally occur greater than 24 hours, but usually not later than 60 hours, after the oocyte collection step, such that maturity of the oocyte is at a sufficient stage to maximise the success of subsequent steps in the IVF procedure.
[00124] Certain embodiments of the present disclosure provide a method of in vitro fertilisation, the method comprising:
exposing an oocyte in vitro and/or in vivo to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins; and
fertilising the oocyte in vitro.
[00125] In certain embodiments, the assisted reproduction technology comprises in vitro maturation. Methods of in vitro maturation of oocytes are known. [00126] Certain embodiments of the present disclosure provide a method of in vitro maturation of an oocyte, the method comprising exposing the oocyte to an endoplasmic stress inhibitor and/or an inducer of heat shock proteins, as described herein.
[00127] Certain embodiments of the present disclosure provide a method of in vitro maturation of an oocyte, the method comprising:
exposing the oocyte to an endoplasmic stress inhibitor and/or an inducer of heat shock proteins; and
maturing the oocyte in vitro.
[00128] Certain embodiments of the present disclosure provide a method of improving the developmental competence of an embryo produced by fertilisation of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, as described herein.
[00129] Methods for fertilising an oocyte are known, and include fertilization in vitro, artificial insemination and natural insemination.
[00130] Certain embodiments of the present disclosure provide a method of improving the developmental competence of an embryo produced by fertilisation of an oocyte, the method comprising:
exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in vitro and/or in vivo; and
fertilising the oocyte to produce an embryo.
[00131] Certain embodiments of the present disclosure provide a method of improving blastocyst development of an embryo produced by fertilisation of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, as described herein.
[00132] Certain embodiments of the present disclosure provide a method of improving blastocyst development of an embryo produced by fertilisation of an oocyte, the method comprising:
exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in vitro and/or in vivo; and fertilising the oocyte to produce an embryo.
[00133] Certain embodiments of the present disclosure provide a method of improving cumulus cell expansion in a cumulus oocyte complex, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, as described herein.
[00134] Certain embodiments of the present disclosure provide a method of increasing the level of cumulus cell protein in a cumulus oocyte complex, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, as described herein.
[00135] Certain embodiments of the present disclosure provide a method of culturing an oocyte.
[00136] Certain embodiments of the present disclosure provide a method of culturing an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, as described herein.
[00137] Certain embodiments of the present disclosure provide a method of culturing an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, wherein exposure of the oocyte to the endoplasmic stress inhibitor and/or the inducer of heat shock proteins improves developmental competence of the oocyte.
[00138] Certain embodiments of the present disclosure provide a method of culturing an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, wherein exposure of the oocyte to the endoplasmic stress inhibitor and/or the inducer of heat shock proteins improves blastocyst development of an embryo produced from the oocyte.
[00139] Certain embodiments provide a kit for performing the methods as described herein. In certain embodiments, the kit comprises one or more reagents as described herein and/or instructions for performing the methods as described herein.
[00140] Certain embodiments of the present disclosure provide a combination product for performing the methods as described herein.
[00141] Certain embodiments of the present disclosure provide use of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins as described herein in the preparation of an oocyte and/or embryo culture medium.
[00142] Certain embodiments of the present disclosure provide an oocyte and/or embryo culture medium, the medium comprising an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins. Endoplasmic reticulum stress inhibitors and/or an inducer of heat shock proteins, and their use, are as described herein.
[00143] In certain embodiments, the oocyte is part of a cumulus oocyte complex. In certain embodiments, the oocyte is a denuded oocyte. In certain embodiments, the oocyte comprises an increased lipid content. Oocytes are as described herein.
[00144] In certain embodiments, the endoplasmic reticulum stress inhibitor comprises one or more of an inhibitor of an inositol requiring protein- 1 (IRE1) signal transduction pathway, an inhibitor of a protein kinase RNA-like endoplasmic reticulum kinase (PERK) signal transduction pathway, an inhibitor of a transcription factor (ATF6) signal transduction pathway, and an inducer of heat shock proteins. In certain embodiments, the endoplasmic reticulum stress inhibitor comprises an inhibitor of eIF2a dephosphorylation.
[00145] In certain embodiments, the culture medium comprises a concentration of the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1000 nM or greater, 10 uM or greater, 0.1 nM or less, 0.5 nM or less, 1 nM or less, 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, 1000 nM or less, 10 uM or less, 0.1 nM to 10 uM, 0.1 nM to 1000 nM, 0.1 nM to 500 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.1 nM to 1 nM, 0.5 nM to 10 uM, 0.5 nM to 1000 nM, 0.5 nM to 500 nM, 0.5 nM to 100 nM, 0.5 nM to 50 nM, 0.5 nM to 10 nM, 0.5 nM to 1 nM; 1 nM to 10 uM, 1 nM to 1000 nM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 5 nM to 10 uM, 5 nM to 1000 nM, 5 nM to 500 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 10 nM, 10 nM to 10 uM, 10 nM to 1000 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 100 nm to 10 uM, 100 nM to 1000 nM, 100 nM to 500 nM, 500 nM to 10 uM, 500 nM to 1000 nM, 1000 nM to 10 uM, or 1 uM to 10 uM. Endoplasmic stress inhibitors and inducers of heat shock proteins are as described herein.
[00146] In certain embodiments, the culture medium comprises an endoplasmic stress inhibitor and/or an inducer of heat shock proteins at a concentration in the range of 0.1 to 1000 mg/ml, 0.1 to 500 mg/ml, 0.1 to 400 mg/ml, 0.1 to 200 mg/ml, 0.1 to 100 mg/ml, 0.1 to 50 mg/ml, 0.1 to 20 mg/ml, 0.1 to 10 mg/ml, 0.1 to 5 mg/ml, 0.1 to 4 mg/ml, 0.1 to 2 mg/ml, 0.1 to 1.0 mg/ml, 0.1 to 0.5 mg/ml, 0.1 to 0.4 mg/ml, 0.1 to 0.2 mg/ml, 0.2 to 1000 mg/ml, 0.2 to 500 mg/ml, 0.2 to 400 mg/ml, 0.2 to 200 mg/ml, 0.2 to 100 mg/ml, 0.2 to 50 mg/ml, 0.2 to 20 mg/ml, 0.2 to 10 mg/ml, 0.2 to 5 mg/ml, 0.2 to
4 mg/ml, 0.2 to 2 mg/ml, 0.2 to 1 mg/ml, 0.2 to 0.5 mg/ml, 0.2 to 0.4 mg/ml, 0.4 to 1000 mg/ml, 0.4 to 500 mg/ml, 0.4 to 400 mg/ml, 0.4 to 200 mg/ml, 0.4 to 100 mg/ml, 0.4 to 50 mg/ml, 0.4 to 20 mg/ml, 0.4 to 10 mg/ml, 0.4 to 5 mg/ml, 0.4 to 4 mg/ml, 0.4 to 2 mg/ml, 0.4 to 1 mg/ml, 0.4 to 0.5 mg/ml, 0.5 to 1000 mg/ml, 0.5 to 500 mg/ml, 0.5 to 400 mg/ml, 0.5 to 200 mg/ml, 0.5 to 100 mg/ml, 0.5 to 50 mg/ml, 0.5 to 20 mg/ml, 0.5 to 10 mg/ml, 0.5 to 5 mg/ml, 0.5 to 4 mg/ml, 0.5 to 2 mg/ml, 0.5 to 1.0 mg/ml, 1 to 1000 mg/ml, 1 to 500 mg/ml, 1 to 400 mg/ml, 1 to 200 mg/ml, 1 to 100 mg/ml, 1 to 50 mg/ml, 1 to 20 mg/ml, 1 to 10 mg/ml, 1 to 5 mg/ml, 1 to 4 mg/ml, 1 to 2 mg/ml, 2 to 1000 mg/ml, 2 to 500 mg/ml, 2 to 400 mg/ml, 2 to 200 mg/ml, 2 to 100 mg/ml, 2 to 50 mg/ml, 2 to 20 mg/ml, 2 to 10 mg/ml, 2 to 5 mg/ml, 2 to 4 mg/ml, 4 to 1000 mg/ml, 4 to 500 mg/ml, 4 to 400 mg/ml, 4 to 200 mg/ml, 4 to 100 mg/ml, 4 to 50 mg/ml, 4 to 20 mg/ml, 4 to 10 mg/ml, 4 to 5 mg/ml, 5 to 1000 mg/ml, 5 to 500 mg/ml, 5 to 400 mg/ml,
5 to 200 mg/ml, 5 to 100 mg/ml, 5 to 50 mg/ml, 5 to 20 mg/ml, 5 to 10 mg/ml, 10 to 1000 mg/ml, 10 to 500 mg/ml, 10 to 400 mg/ml, 10 to 200 mg/ml, 10 to 100 mg/ml, 10 to 50 mg/ml, 10 to 20 mg/ml, 20 to 1000 mg/ml, 20 to 500 mg/ml, 20 to 400 mg/ml, 20 to 200 mg/ml, 20 to 100 mg/ml, 20 to 50 mg/ml, 50 to 1000 mg/ml, 50 to 500 mg/ml, 50 to 400 mg/ml, 50 to 200 mg/ml, 50 to 100 mg/ml, 100 to 1000 mg/ml, 100 to 500 mg/ml, 100 to 400 mg/ml, 100 to 200 mg/ml, 200 to 1000 mg/ml, 200 to 500 mg/ml, 200 to 400 mg/ml, and 500 to 1000 mg/ml.
[00147] In certain embodiments, the endoplasmic reticulum stress inhibitor comprises one or more of salubrinal, tauroursodeoxycholate and 4-phenyl butyric acid and/or a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate of any of the aforementioned.
[00148] In certain embodiments, the medium comprises a concentration of salubrinal in the range of 50-500 nM.
[00149] In certain embodiments, the medium comprises a concentration of tauroursodeoxycholate in the range of <10μΜ.
[00150] In certain embodiments, the medium comprises a concentration of 4-phenyl butyric acid in the range of <lnM.
[00151] In certain embodiments, the medium comprises an inducer of heat shock proteins, as described herein. In certain embodiments, the medium comprises a derivative of a hydroximic acid, as described herein. In certain embodiments, the concentration of a derivative of hydroximic acid - (for example BGP-15) is in the range of 4 to 4000 mg/ml. In certain embodiments, the medium comprises a concentration of a derivative of hydroximic acid - (for example BGP-15) in the range of 4 to 4000 mg/ml. Other ranges are contemplated and described herein.
[00152] In certain embodiments, the oocyte is an immature oocyte. In certain embodiments, the oocyte comprises a mature oocyte. In certain embodiments, the oocyte is an oocyte matured in vitro. In certain embodiments, the oocyte is an oocyte matured in vivo.
[00153] In certain embodiments, the oocyte is a mammalian oocyte. In certain embodiments, the oocyte is a bovine oocyte. In certain embodiments, the oocyte is a human oocyte. In certain embodiments, the oocyte is an animal oocyte.
[00154] In certain embodiments, the oocyte is obtained from a female subject comprising one or more of the following characteristics: a body mass index of greater than 25 kg/m 2 ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal to 30 kg/m ; obesity; Polycystic Ovary Syndrome; reduced fertility; infertility; ovarian dysfunction; anovulation; reduced ovulation rate; pre-diabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet.
[00155] Culture media for oocytes and embryos are known and commercially available, including culture media for human oocytes and embryos and culture media for animal oocytes.
[00156] In certain embodiments, the culture medium is one or more of:
(i) a medium for any one or more of retrieval, holding and washing of an oocyte;
(ii) a medium for fertilization and culture, for example until the 2-8 cell stage;
(iii) a medium for one or more of fertilization, culture (eg until the 2-8 cell stage) and transfer;
(iv) a medium for culture from the 4-8 cell stage through to the blastocyst stage, and /or for embryo transfer;
(v) a medium for culturing until 2-8 cell stage;
(vi) a medium for transfer of embryos and blastocysts; and
(vii) a medium for preincubation and/or maturing of immature oocytes.
[00157] For example, IVF media are known, and typically comprise the following components: Calcium Chloride; •Gentamicin sulphate; Glucose; Human Albumin Solution; Magnesium Sulfate; Potassium Chloride; Sodium Bicarbonate; Sodium Chloride; Sodium phosphate; Sodium Pyruvate; Synthetic Serum Replacement.
[00158] For example, an IVF medium comprising the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins may be used as follows:
1. Equilibrate medium for a minimum of 2 hours in 5-6% C02 at 37°C prior to use; 2. Recover oocytes and prepare sperm;
3. Carry out fertilization (Day 0) in pre-equilibrated IVF medium.
4. At 16-20 hours (Day 1), check for formation of pronuclei, then carefully wash and transfer zygotes to fresh 50 μΐ microdrops or 0.5 ml wells/dishes of IVF medium covered with Liquid Paraffin. Embryos should be cultured singly or in multiples to a maximum of 4 per well.
Embryo transfer at Day 2 or Day 3:
5. Embryos are prepared and transferred to the uterus in 20 to 30 μΐ of pre- equilibrated IVF Medium;
6. Flush transfer catheter with chosen transfer medium prior to use.
[00159] In vitro maturation medium are known, and typically comprise the following the components: Adenine; Alanine; AMP; Arginine; Ascorbic acid; Aspartic Acid; ATP; Calcium Chloride; Calcium pantothenate; Cholesterol; Choline Chloride; Cysteine; Cystine; D-Biotin; Deoxy ribose; Folic Acid; Gentamicin sulphate; Glucose; Glutamic Acid; Glutamine; Glutathione; Glycine; Guanine Hydrochloride; Histidine; Hydroxy Proline; Hypoxanthine; Inositol; Iron(III) Nitrate; Isoleucine; Leucine; Lysine; Magnesium Sulfate; Menadione; Methionine; Niacin; Niacinamide; Para-aminobenzoic Acid; Phenylalanine; Potassium Chloride; Proline; Pyridoxal; Pyridoxine; Riboflavin; Ribose; Serine; Sodium acetate; Sodium Bicarbonate; Sodium Chloride; 'Sodium phosphate; Sodium Pyruvate; Thiamine; Threonine; Thymine; Tryptophan; Tween 80; Tyrosine; Uracile; Valine; Vitamin A; Vitamin D2; Vitamin E; and Xanthine.
[00160] For example, medium(s) for in vitro maturation comprising the endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins may be used as follows:
Medium 1: Used for pre-incubation of immature oocytes, which may or may not comprise an endoplasmic reticulum stress inhibitor and/or an inducer of heat shick proteins. Typically comprises Synthetic Serum Replacement, Human serum albumin (HSA), Physiological salts, Glucose, Sodium pyruvate, Sodium bicarbonate, Gentamicin sulphate 10μg/ml, Phenol Red
Medium 2: Used for maturing immature oocytes, comprising an endoplasmic reticulum stress inhibitor and/or an inducer of heat shick proteins. Typically further comprises Glucose, Sodium pyruvate, Physiological salts, Amino acids, Nucleotides, Vitamins, Sodium bicarbonate, Gentamicin sulphate lC^g/ml, Phenol Red.
1. Pre-equilibrate 3 ml Medium 1 and 10 ml of Medium 2 in C02 environment at 37 °C for a minimum of 12 hours.
2. After oocyte pick up store the immature oocytes in Medium lin C02 environment at 37°C for 2-3 hours prior to transfer to the Medium 2.
3. Preparation of final maturation medium:
9 ml Medium 1
1 ml patient's own serum
10 μΐ human chorionic gonadotropin (hCG) solution (lOOmlU/ml) 100 μΐ human follicle stimulating hormone (FSH) solutin (75mIU/ml).
4. Transfer the oocytes to the final maturation medium and incubate in C02 environment at 37°C for 28-32 hours.
[00161] In certain embodiments, the medium further comprises an inducer of lipid metabolism and/or a Peroxisome Proliferator Activated Receptor (PPAR) agonist. In certain embodiments, the Peroxisome Proliferator Activated Receptor (PPAR) agonist comprises a Peroxisome Proliferator Activated Receptor alpha agonist and/or a Peroxisome Proliferator Activated Receptor gamma agonist.
[00162] In certain embodiments, the inducer of lipid metabolism is L-carnitine, and/or a derivative as described herein.
[00163] In certain embodiments, the medium comprises a Peroxisome Proliferator Activated Receptor alpha agonist and/or a Peroxisome Proliferator Activated Receptor gamma agonist. In certain embodiments, the Peroxisome Proliferator Activated Receptor alpha agonist is fenofibrate. In certain embodiments, the Peroxisome Proliferator Activated Receptor gamma agonist is rosiglitazone.
[00164] In certain embodiments, the medium comprises an inducer of lipid metabolism (such as L-carnitine) and a Peroxisome Proliferator Activated Receptor (PPAR) agonist.
[00165] Certain embodiments of the present disclosure provide a non-human oocyte or a non-human embryo exposed to the medium as described herein. Certain embodiments provide a non-human animal produced from the oocyte or embryo. Certain embodiments of the present disclosure provide an animal oocyte or an animal embryo exposed to the medium as described herein.
[00166] Certain embodiments of the present disclosure provide a method of assisted reproduction, the method comprising exposing an oocyte and/or an embryo to a medium as described herein. In certain embodiments, the method of assisted reproduction comprises in vitro fertilization of an oocyte.
[00167] Certain embodiments of the present disclosure provide a method of assisted reproduction, the method comprising culturing an oocyte and/or an embryo in a medium as described herein.
[00168] Certain embodiments of the present disclosure provide a kit for use in the methods as described herein. In certain embodiments, the kit comprises an oocyte and/or embryo culture medium as described herein. In certain embodiments, the kit comprises instructions for culturing the oocyte and/or embryo in the culture medium.
[00169] Certain embodiments of the present disclosure provide a kit for improving developmental competence of an oocyte and/or an embryo produced from the oocyte, the kit comprising:
an oocyte and/or embryo culture medium;
an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins; and
optionally instructions for culturing the oocyte and/or embryo in the culture medium.
[00170] Certain embodiments of the present disclosure provide a combination product.
[00171] Certain embodiments of the present disclosure provide a combination product for use in the methods as described herein, the combination product comprising an oocyte and/or embryo culture medium as described herein. [00172] Certain embodiments of the present disclosure provide a combination product comprising:
an oocyte and/or embryo culture medium;
an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins; and
optionally instructions for culturing the oocyte and/or embryo in the culture medium.
[00173] Certain embodiments of the present disclosure provide a composition for use in the methods and/or kits as described herein. In certain embodiments, the composition is a pharmaceutical composition.
[00174] Certain embodiments of the present disclosure provide a pharmaceutical composition.
[00175] Certain embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutically effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, as described herein.
[00176] Certain embodiments of the present disclosure provide a pharmaceutical composition for use in the relevant methods as described herein.
[00177] Certain embodiments of the present disclosure provide a pharmaceutical composition for improving fertility in a female subject, the composition comprising an effective amount of endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[00178] In certain embodiments, the endoplasmic reticulum stress inhibitor comprises one or more of an inhibitor of an inositol requiring protein- 1 (IREl) signal transduction pathway, an inhibitor of a protein kinase RNA-like endoplasmic reticulum kinase (PERK) signal transduction pathway, an inhibitor of a transcription factor (ATF6) signal transduction pathway, and an inducer of heat shock proteins.
[00179] In certain embodiments, the endoplasmic reticulum stress inhibitor comprises an inhibitor of eIF2a dephosphorylation.
[00180] In certain embodiments, the endoplasmic reticulum stress inhibitor comprises one or more of salubrinal, tauroursodeoxycholate and 4-phenyl butyric acid and/or a pharmaceutically acceptable derivative, solvate, salt, tautomer, stereoisomer, or racemate of any of the aforementioned.
[00181] In certain embodiments, the inducer of heat shock proteins comprises a derivative of hydroximic acid. In certain embodiments, the derivative of hydroximic acid comprises one or more of BGP-15, propanolol, bimoclomol, arimoclomal, NG-94, iroxanadine, and/or a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate of any of the aforementioned.
[00182] In certain embodiments, the pharmaceutical composition further comprises a Peroxisome Proliferator Activated Receptor (PPAR) agonist.
[00183] In certain embodiments, the pharmaceutical composition further comprises a Peroxisome Proliferator Activated Receptor alpha agonist and/or a Peroxisome Proliferator Activated Receptor gamma agonist.
[00184] In certain embodiments, the Peroxisome Proliferator Activated Receptor alpha agonist is fenofibrate. In certain embodiments, the Peroxisome Proliferator Activated Receptor gamma agonist is rosiglitazone.
[00185] Methods for the preparation of pharmaceutical compositions are known, for example as described in Remington's Pharmaceutical Sciences, 18th ed., 1990, Mack Publishing Co., Easton, Pa. and U.S. Pharmacopeia: National Formulary, 1984, Mack Publishing Company, Easton, Pa.
[00186] In certain embodiments, the pharmaceutical composition comprises the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins in an amount of 0.05 mg or greater, 0.1 mg or greater, 0.2 mg or greater, 0.5 mg or greater, 1 mg or greater, 2 mg or greater, 5 mg or greater, 10 mg or greater, 50 mg or greater, 100 mg or greater, 0.05 mg or less, 0.1 mg or less, 0.5 mg or less, 1 mg or less, 2 mg or less, 5 mg or less, 10 mg or less, 50 mg or less, 100 mg or less, 0.05 to 100 mg, 0.05 to 50 mg, 0.05 to 10 mg, 0.05 to 5 mg, 0.05 to 1 mg, 0.05 to 0.5 mg, 0.05 to 0.1 mg, 0.1 to 100 mg, 0.1 to 50 mg, 0.1 to 10 mg, 0.1 to 5 mg, 0.1 to 1 mg, 0.1 to 0.5 mg, 0.5 to 100 mg, 0.5 to 50 mg, 0.5 to 10 mg, 0.5 to 5 mg, 0.5 to 1 mg, 1 to 100 mg, 1 to 50 mg, 1 to 10 mg, 1 to 5 mg, 5 to 100 mg, 5 to 50 mg, 5 to 10 mg, 10 to 100 mg, or 10 to 50 mg.
[00187] In certain embodiments, the pharmaceutical composition comprises the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins in an amount of 0.2 to 2 mg.
[00188] The term "therapeutically effective amount" includes the quantity which, when administered to a subject in need of treatment, improves the prognosis and/or state of the subject. The amount to be administered to a subject will depend on the particular characteristics of the disease, condition or state in the subject, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, body weight and tolerance to drugs.
[00189] As discussed previously herein, administration and delivery of the compositions may be for example by one or more of the intravenous, intraperitoneal, subcutaneous, intramuscular, oral, or topical route, by direct injection or any combination of these administration routes. The mode and route of administration in most cases will depend on the type of disease, condition or state being treated, and/or the treatment of a human or non-human animal.
[00190] The dosage form, frequency and will depend on the mode and route of administration.
[00191] As described above, the administration of the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and any other agents, may also include the use of one or more pharmaceutically acceptable additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients, preservatives and bulking agents, taking into consideration the particular physical, microbiological and chemical characteristics of the agents to be administered. [00192] For example, the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and/or the other agents, can be prepared into a variety of pharmaceutically acceptable compositions in the form of, e.g., an aqueous solution, an oily preparation, a fatty emulsion, an emulsion, a lyophilised powder for reconstitution, etc. and can be administered as a sterile and pyrogen free intramuscular or subcutaneous injection or as injection to an organ, or as an embedded preparation or as a transmucosal preparation through nasal cavity, rectum, uterus, vagina, lung, etc. The composition may be administered in the form of oral preparations (for example solid preparations such as tablets, caplets, capsules, granules or powders; liquid preparations such as syrup, emulsions, dispersions or suspensions).
[00193] Compositions containing the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and/or any other agents, may also contain one or more pharmaceutically acceptable preservatives, buffering agents, diluents, stabilisers, chelating agents, viscosity enhancing agents, dispersing agents, pH controllers, or isotonic agents.
[00194] Examples of suitable preservatives are benzoic acid esters of para- hydroxybenzoic acid, propylene glycol, phenols, phenylethyl alcohol or benzyl alcohol. Examples of suitable buffers are sodium phosphate salts, citric acid, tartaric acid and the like. Examples of suitable stabilisers are, antioxidants such as alpha-tocopherol acetate, alpha-thioglycerin, sodium metabisulphite, ascorbic acid, acetylcysteine, 8- hydroxyquinoline, chelating agents such as disodium edetate. Examples of suitable viscosity enhancing agents, suspending or dispersing agents are substituted cellulose ethers, substituted cellulose esters, polyvinyl alchohol, polyvinylpyrrolidone, polyethylene glycols, carbomer, polyoxypropylene glycols, sorbitan monooleate, sorbitan sesquioleate, polyoxy ethylene hydrogenated castor oil 60.
[00195] Examples of suitable pH controllers include hydrochloric acid, sodium hydroxide and the like. Examples of suitable isotonic agents are glucose, D-sorbitol or D-mannitol, sodium chloride.
[00196] The administration of a endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and/or the other agents, may also be in the form of a composition containing a pharmaceutically acceptable carrier, diluent, excipient, suspending agent, lubricating agent, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, glidant, anti-adherant, binder, flavorant or sweetener, taking into account the physical, chemical and microbiological properties of the agents being administered.
[00197] For these purposes, the composition may be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, mucosally, transdermally, bucally, vaginally, intraventricularly, via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically-acceptable carriers, or by any other convenient dosage form. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.
[00198] When administered parenterally, the compositions will normally be in a unit dosage, sterile, pyrogen free injectable form (solution, suspension or emulsion, which may have been reconstituted prior to use) which is generally isotonic with the blood of the recipient with a pharmaceutically acceptable carrier. Examples of such sterile injectable forms are sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable vehicles, dispersing or wetting agents and suspending agents. The sterile injectable forms may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Among the pharmaceutically acceptable vehicles and solvents that may be employed are water, ethanol, glycerol, saline, Ringer's solution, dextrose solution, isotonic sodium chloride solution, and Hanks' solution. In addition, sterile, fixed oils are conventionally employed as solvents or suspending mediums. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides, corn, cottonseed, peanut, and sesame oil. Fatty acids such as ethyl oleate, isopropyl myristate, and oleic acid and its glyceride derivatives, including olive oil and castor oil, especially in their polyoxyethylated versions, are useful in the preparation of injectables. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants.
[00199] The carrier may contain minor amounts of additives, such as substances that enhance solubility, isotonicity, and chemical stability, for example anti- oxidants, buffers and preservatives.
[00200] In addition, the compositions may be in a form to be reconstituted prior to administration. Examples include lyophilisation, spray drying and the like to produce a suitable solid form for reconstitution with a pharmaceutically acceptable solvent prior to administration.
[00201] Compositions may include one or more buffers, bulking agents, isotonic agents and cryoprotectants and lyoprotectants. Examples of excipients include, phosphate salts, citric acid, non-reducing such as sucrose or trehalose, polyhydroxy alcohols, amino acids, methylamines, and lyo tropic salts which are usually used instead of reducing sugars such as maltose or lactose.
[00202] When administered orally, the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and/or the other agents, will usually be formulated into unit dosage forms such as tablets, caplets, cachets, powder, granules, beads, chewable lozenges, capsules, liquids, aqueous suspensions or solutions, or similar dosage forms, using conventional equipment and techniques known in the art. Such formulations typically include a solid, semisolid, or liquid carrier. Exemplary carriers include excipients such as lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, substituted cellulose ethers, polyoxyethylene sorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and the like.
[00203] A tablet may be made by compressing or molding the agent optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active, or dispersing agent. Moulded tablets may be made by moulding in a suitable machine, a mixture of the powdered active ingredient and a suitable carrier moistened with an inert liquid diluent. [00204] The administration of the endoplasmic reticulum stress inhibitor and/or the other agents may also utilize controlled release technology.
[00205] The endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and/or the other agents, may also be administered as a sustained-release pharmaceutical composition. To further increase the sustained release effect, the agent may be formulated with additional components such as vegetable oil (for example soybean oil, sesame oil, camellia oil, castor oil, peanut oil, rape seed oil); middle fatty acid triglycerides; fatty acid esters such as ethyl oleate; polysiloxane derivatives; alternatively, water-soluble high molecular weight compounds such as hyaluronic acid or salts thereof, carboxymethylcellulose sodium hydroxypropylcellulose ether, collagen polyethylene glycol polyethylene oxide, hydroxypropylmethylcellulosemethylcellulose, polyvinyl lalcohol, and polyvinylpyrrolidone.
[00206] Alternatively, the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and/or the other agents, may be incorporated into a hydrophobic polymer matrix for controlled release over a period of days. The agent may then be moulded into a solid implant, or externally applied patch, suitable for providing efficacious concentrations of the agents over a prolonged period of time without the need for frequent re-dosing. Such controlled release films are well known to the art. Other examples of polymers commonly employed for this purpose that may be used include nondegradable ethylene-vinyl acetate copolymer a degradable lactic acid- glycolic acid copolymers, which may be used externally or internally. Certain hydrogels such as poly(hydroxyethylmethacrylate) or poly(vinylalcohol) also may be useful, but for shorter release cycles than the other polymer release systems, such as those mentioned above.
[00207] The carrier may also be a solid biodegradable polymer or mixture of biodegradable polymers with appropriate time -release characteristics and release kinetics. The agent may then be moulded into a solid implant suitable for providing efficacious concentrations of the agents over a prolonged period of time without the need for frequent re-dosing. The agent can be incorporated into the biodegradable polymer or polymer mixture in any suitable manner known to one of ordinary skill in the art and may form a homogeneous matrix with the biodegradable polymer, or may be encapsulated in some way within the polymer, or may be moulded into a solid implant.
[00208] For topical administration, the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins, and/or the other agents, may be in the form of a solution, spray, lotion, cream (for example a non-ionic cream), gel, paste or ointment. Alternatively, the composition may be delivered via a liposome, nanosome, rivosome, or nutri-diffuser vehicle.
[00209] It will be appreciated that other forms of administration of agents are also contemplated, including the use of a nucleic acid encoding a polypeptide for delivering of such agents, if such agents are a RNA or a polypeptide.
[00210] In certain embodiments, an endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins as described herein may be used to improve fertility in a female subject, to increase the likelihood of a female subject falling pregnant, to improve the time to pregnancy of a female subject, to prevent and/or treat reduced fertility in a female subject, to prevent and/or treat reduced fertility in a female subject, to prevent and/or treat infertility in a female subject, to prevent and/or treat ovarian dysfunction in a female subject, to prevent and/or treat sub-fertility in a female subject, to prevent and/or treat anovulation in a female subject, and to prevent and/or treat reduced ovulation rate in a female subject.
[00211] Certain embodiments of the present disclosure provide a method and/or a pharmaceutical composition to: (i) improve fertility in a female subject, to increase the likelihood of a female subject falling pregnant, (ii) to improve the time to pregnancy of a female subject, (iii) to prevent and/or treat reduced fertility or sub-fertility in a female subject, (iv) to prevent and/or treat reduced fertility in a female subject, (v) to prevent and/or treat infertility in a female subject, (vi) to prevent and/or treat ovarian dysfunction in a female subject, (vii) to prevent and/or treat anovulation in a female subject, and (viii) to prevent and/or treat reduced ovulation rate in a female subject; the aforementioned methods comprising exposing an oocyte in vivo in the female subject to an endoplasmic stress inhibitor and/or an inducer of heat shock proteins.
[00212] Certain embodiments of the present disclosure provide a method of improving fertility in a female subject, the method comprising exposing an oocyte in vivo in the female subject to an endoplasmic stress inhibitor and/or the inducer of heat shock proteins.
[00213] Certain embodiments of the present disclosure provide a method of improving fertility in a female subject, the method comprising administering to the female subject a therapeutically effective amount of an endoplasmic stress inhibitor.
[00214] Certain embodiments of the present disclosure provide use of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in the preparation of a medicament for any of the following: (i) improve fertility in a female subject, to increase the likelihood of a female subject falling pregnant, (ii) to improve the time to pregnancy of a female subject, (iii) to prevent and/or treat reduced fertility in a female subject, (iv) to prevent and/or treat reduced fertility or sub-fertility in a female subject, (v) to prevent and/or treat infertility in a female subject, (vi) to prevent and/or treat ovarian dysfunction in a female subject, (vii) to prevent and/or treat anovulation in a female subject, and (viii) to prevent and/or treat reduced ovulation rate in a female subject.
[00215] Certain embodiments of the present disclosure provide use of an endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins in the preparation of a medicament for improving fertility in a female subject.
[00216] Certain embodiments of the present disclosure provide a method of improving fertility of a female subject, the method comprising administering to the subject an endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins.
[00217] Certain embodiments of the present disclosure provide a method to (i) improve fertility in a female subject, to increase the likelihood of a female subject falling pregnant, (ii) to improve the time to pregnancy of a female subject, (iii) to prevent and/or treat reduced fertility in a female subject, (iv) to prevent and/or treat reduced fertility or sub-fertility in a female subject, (v) to prevent and/or treat infertility in a female subject, (vi) to prevent and/or treat ovarian dysfunction in a female subject, (vii) to prevent and/or treat anovulation in a female subject, and (viii) to prevent and/or treat reduced ovulation rate in a female subject; the aforementioned methods comprising administering to the subject an effective amount of a pharmaceutical composition as described herein.
[00218] Certain embodiments of the present disclosure provide a method of improving fertility of a female subject comprising administering to the subject an effective amount of a pharmaceutical composition as described herein.
[00219] In certain embodiments, the subject is administered the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins at a dose 0.05 mg/kg/day or greater, 0.1 mg/kg/day or greater, 0.5 mg/kg/day or greater, 1 mg/kg/day or greater, 2 mg/kg/day or greater, 5 mg/kg/day or greater, 10 mg/kg/day or greater, 50 mg/kg/day or greater, 100 mg/kg/day or greater, 0.05 mg/kg/day or less, 0.1 mg/kg/day or less, 0.5 mg/kg/day or less, 1 mg/kg/day or less, 2 mg/kg/day or less, 5 mg/kg/day or less, 10 mg/kg/day or less, 50 mg/kg/day or less, 100 mg/kg/day or less, 0.05 to 100 mg/kg/day, 0.05 to 50 mg/kg/day, 0.05 to 10 mg/kg/day, 0.05 to 5 mg/kg/day, 0.05 to 1 mg/kg/day, 0.05 to 0.5 mg/kg/day, 0.05 to 0.1 mg/kg/day, 0.1 to 100 mg/kg/day, 0.1 to 50 mg/kg/day, 0.1 to 10 mg/kg/day, 0.1 to 5 mg/kg/day, 0.1 to 1 mg/kg/day, 0.1 to 0.5 mg/kg/day, 0.5 to 100 mg/kg/day, 0.5 to 50 mg/kg/day, 0.5 to 10 mg/kg/day, 0.5 to 5 mg/kg/day, 0.5 to 1 mg/kg/day, 1 to 100 mg/kg/day, 1 to 50 mg/kg/day, 1 to 10 mg/kg/day, 1 to 5 mg/kg/day, 5 to 100 mg/kg/day, 5 to 50 mg/kg/day, 5 to 10 mg/kg/day, 10 to 100 mg/kg/day, or 10 to 50 mg/kg/day.
[00220] In certain embodiments, the subject is administered the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins at a dose of 0.1- 8 mg/kg/day. In certain embodiments, the subject is administered the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins at a dose of 0.2- 4 mg/kg/day. In certain embodiments, the subject is administered the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins at a dose of 0.2- 2 mg/kg/day.
[00221] In certain embodiments, the female subject is a mammal. In certain embodiments, the female subject is human, a primate, a livestock animal (eg. a horse, a cow, a sheep, a pig, or a goat), a companion animal (eg. a dog, a cat), or a laboratory animal (eg. a mouse, a rat, a guinea pig, a rabbit). [00222] In certain embodiments, the female subject comprises one or more of the following characteristics: a body mass index of greater than 25 kg/m ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal to 30 kg/m 2 ; obesity; Polycystic ovary syndrome; reduced fertility; infertility; sub-fertility, ovarian dysfunction; anovulation; reduced ovulation rate; pre-diabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet.
[00223] Certain embodiments of the present disclosure provide a treatment regime for a female subject with reduced fertility, the treatment regime comprising administering to the subject an endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins.
[00224] Certain embodiments of the present disclosure provide a treatment regime for a female subject with reduced fertility, the treatment regime comprising administering to the subject a pharmaceutical composition as described herein.
[00225] A suitable treatment regime may be designed by a person skilled in the art.
[00226] Certain embodiments of the present disclosure provide a method of preventing and/or treating a female subject with reduced fertility, the method comprising administering to the subject a therapeutically effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[00227] Certain embodiments of the present disclosure provide a method of preventing and/or treating a female subject with reduced fertility, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition as described herein.
[00228] Certain embodiments of the present disclosure provide use of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in the preparation of a medicament for preventing and/or treating reduced fertility in a female subject.
[00229] Certain embodiments of the present disclosure provide a method of preventing and/or treating a subject with reduced fertility, the method comprising administering to the subject a therapeutically effective amount of endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins; wherein the subject comprises one or more of the following characteristics: a body mass index of greater than 25 kg/m ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal to 30 kg/m 2 ; obesity; Polycystic ovary syndrome; reduced fertility; sub-fertility, infertility; ovarian dysfunction; anovulation; reduced ovulation rate; pre-diabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet.
[00230] Certain embodiments of the present disclosure provide a method of increasing the likelihood of a female subject falling pregnant, the method comprising exposing the subject to an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
[00231] Certain embodiments of the present disclosure provide a method of increasing the likelihood of a female subject falling pregnant, the method comprising exposing the subject to an effective amount of a pharmaceutical composition as described herein.
[00232] As described herein, exposure of an oocyte in vitro to an endoplasmic stress inhibitor may also be used in an assisted reproduction technology. In certain embodiments, the exposure may be used to improve fertility in a female subject, to increase the likelihood of a female subject falling pregnant, to improve the time to pregnancy, and to prevent and/or treat reduced fertility in a female subject.
[00233] Certain embodiments of the present disclosure provide a method of treating reduced fertility in a female subject, the method comprising:
exposing an oocyte in vitro to an effective amount of an endoplasmic stress inhibitor and/or an inducer of heat shock proteins; and
introducing the oocyte, or an embryo produced from the oocyte, into the female subject.
[00234] The present disclosure is further described by the following examples. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.
EXAMPLE 1 - ER stress contributes to female infertility [00235] Materials and Methods
[00236] (i) Animals
[00237] Mice (CBAxC57Bl/6 Fl) were maintained on a 12-h light, 12-h dark cycle with rodent chow and water available ad libitum. All experiments were approved by the University of Adelaide's Animal Ethics Committee and were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.
[00238] (ii) Isolation of mouse cumulus-oocyte complexes (COCs)
[00239] Immature unexpanded COCs were isolated from 23-day-old prepubertal female mice by puncturing the antral follicles of ovaries collected 44 h after i.p. injection of 5 IU equine chorionic gonadotropin (eCG, Professional Compounding Centre of Australia). Mature expanded COCs were obtained from oviducts by blunt dissection 44 h after eCG injection and 16 h after i.p. administration of 5 IU human chorionic gonadotropin (hCG/Pregnyl, Organon) to stimulate ovulation. All COCs were collected in HEPES -buffered a-MEM (Invitrogen) supplemented with 1% or 5% (vol/vol) fetal calf serum (FCS, Invitrogen) as indicated.
[00240] (iii) In vitro maturation of cumulus-oocyte complexes (COCs)
[00241] For in vitro maturation, immature COCs (isolated from mice treated with eCG for 44-46 hours) were cultured in groups of 30 in bicarbonate-buffered a-MEM supplemented with 5% (vol/vol) FCS, 50 mlU/ml recombinant human FSH (Sigma- Aldrich) and 10 ng/ml epidermal growth factor (EGF, Sigma- Aldrich Pty. Ltd) as control (Figures 1- 4), or with the addition of the indicated treatment, in drops of 100 μΐ overlaid with sterile mineral oil (Sigma-Aldrich) and incubated at 37°C in an atmosphere of 5% C02 and 95% air for 16 hours. Treatments consisted of control media supplemented with ER stress inducer ΙΟΟηΜ or 500nM thapsigargin (Merk), or ΙΟΟηΜ or 500nM ER stress inducer thapsigargin plus ΙΟΟηΜ or 200nM eIF-2a inhibitor Salubrinal (Merck), or ΙΟΟηΜ or 200nM Salubrinal alone (see Figures 1-4). In experiments assessing the effect of palmitic acid during in vitro maturation (see Figures 5- 9), 1% (vol/vol) FCS was used in the culture media. Treatments were control media supplemented with 150, 275, 400, 525 μΜ palmitic acid for dose response experiments (see Figure 5), or 400 μΜ palmitic acid with 200nM salubrinal, or 200nM salubrinal alone (see Figures 6-9).
[00242] (iv) Assessment of cumulus expansion
[00243] The degree of cumulus expansion of in vitro matured oocytes was assessed after 16h of IVM culture by independent blinded assessor. Briefly, a score of 0 indicates no expansion of cumulus cells; +1 the most outer layers of cumulus cells expanded; +2 expansion of the outer half of cumulus cells; +3 all layers expanded except the corona radiata; +4 expansion of all layers of cumulus cells. For each treatment group a mean cumulus expansion index (CEI) (0.0-4.0) was calculated.
[00244] (v) RNA isolation and real-time RT-PCR
[00245] Total RNA was isolated from COCs using RNeasy Micro Kit (Qiagen) as per manufacturer's instructions. RNA concentration and purity were quantified using a Nanodrop ND-1000 Spectrophotometer (Biolab) before reverse transcribing 600 ng RNA using random primers (Roche) and Superscript III Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. Ribosomal protein L19 (rpll9) was used as a validated internal control for every sample. All primers were Quantitect Primer Assays (Qiagen) and shown to have comparable amplification efficiency against the internal control. Real-time PCR was performed in triplicate using SYBR Green PCR Master Mix (Applied Biosystems) and a Rotor-Gene 6000 (Corbett) real-time rotary analyzer. Real-time RT-PCR data were analyzed using the 2-AACT method and expressed as the fold change relative to a calibrator sample which was included in each run.
[00246] (vi) Immunocytochemistry [00247] In each experiment, 10 COCs per treatment group were fixed as "whole mount" in 4% paraformaldehyde (w/v) in PBS with lmg/mL polyvinylpyrrolidone (PVP, Sigma) to prevent sticking and washed thoroughly in PBS with lmg/mL PVP. The COCs were incubated in blocking buffer containing 10% normal rabbit or goat serum (Vector Laboratories) in PBS (80mM Na2HP04, 20mM NaH2P04, lOOmM NaCl, pH7.5) for 1 h at room temperature. The COCs were then incubated with the primary antibody at a 1: 100 dilution in blocking buffer overnight at 4°C. Specific primary antibodies used were rabbit polyclonal anti-PTX3 (Santa Cruz) and goat polyclonal anti-TSG6 (Santa Cruz). Following washing in PBS COCs were incubated with biotinylated goat-anti-rabbit (Millipore) or biotinylated donkey-anti-goat (Millipore) secondary antibodies for 1 h at room temperature. Finally, COCs were washed in PBS and incubated with 1 ng/mL streptavidin-Alexa594 (Molecular Probes) and 1 mg/mL DAPI (Invitrogen) in PBS for 1 h at room temperature. COCs were visualised and images captured using a Leica TCS SP5 spectral scanning confocal microscope system.
[00248] (vii) Cumulus matrix extraction and Western Blot
[00249] For each experiment, 90 COCs from each control or treatment group were collected in 20μ1 culture media and incubated with 4μ1 lOOOIU/ml hyaluronidase (Invitrogen) and gentle rotation at room temperature for 1 minute followed by centrifugation at 5000g for 1 minute to separate the matrix extract (supernatant) and the cell pellet. Cell pellets were then resuspended in 24μ1 PBS. 6μ1 of 5x reducing Laemmli buffer (2% (w/v) sodium dodecyl sulphate (SDS) and 5% (v/v) β-mercaptoethanol) was added to each fraction, and the samples were boiled at 100°C for 10 minutes.
[00250] Protein extracts from matrix fractions or cell pellet fractions were resolved by 10% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes (Millipore). Membranes were blocked in TBST (10 mM Tris, 150 mM NaCl and 0.05% Tween 20, pH 7.5) containing 3% (w/v) non-fat milk for 1 h at room temperature. Membranes were then incubated with primary antibodies for 2 h at room temperature in 3% milk/TBST. Primary antibodies used were rabbit monoclonal IRE la antibody at 1: 1000 (Cell Signaling), rabbit phospho IRE la [Ser724] antibody at 1: 1000 (Novus Biologicals) and rat monoclonal PTX3 antibody at 1:2000 (Enzo, Life Sciences). Membranes were then washed in TBST and incubated with horseradish peroxidase- linked anti-rabbit immunoglobulin G at 1: 1000 (Millipore) or horseradish peroxidase- linked anti-rat immunoglobulin G at 1: 1000 (Millipore). Enhanced chemiluminescence detection (Amersham) was used as per manufacturer's instructions.
[00251] (viii) Analysis of inner mitochondrial membrane potential
[00252] Denuded oocytes were incubated with the inner mitochondrial membrane dye JC- 1 (5,5',6,6'-tetrachloro- 1 , l',3,3'-tetraethylbenzimidazolylcarbocyanine iodide, Invitrogen) at 1.5mM for 15 min at 37 °C in the dark. Oocytes were then imaged immediately in both green and red fluorescence channels using a Leica SP5 spectral scanning confocal microscope at identical magnification and gain settings throughout experiments. Using Analysis Pro software (Olympus), a square was placed to cover the oocyte image. Red or green fluorescence intensity was determined as the sum total of fluorescence in the boxed area.
[00253] (ix) Embryo Production and Assessment of Embryo Morphology and Development
[00254] COCs matured in vitro and COCs isolated from oviduct at 16 h post-hCG (in vivo, ovulated) were used for in vitro fertilization. Sperm were collected from 8 week old CBAxC57Bl/6 Fl males from the vas deferens and caudal region of the epididymis. Sperm were capacitated in bicarbonate -buffered a-MEM supplemented with 3 mg/ml of BSA (fatty acid-free) for 1 h at 37°C in an atmosphere of 5% C02 and 95% air. Following sperm capacitation COCs were washed twice, and COCs and sperm (35 000 sperm/ml) were coincubated in ΙΟΟμΙ bicarbonate-buffered a-MEM supplemented with 3 mg/ml of BSA (fatty acid free) for 4 h at 37°C in an atmosphere of 5% C02 and 95% air. All cumulus cell-free oocytes were then transferred to embryo development culture medium (Vitro Cleave, COOK Australia). Twenty-four hours following in vitro fertilization (Day 2), the fertilization rate was assessed, and 2-cell embryos were transferred to a fresh, 20 μΐ drop of culture medium. Embryo morphology was assessed on Day 3 (44 h of embryo culture) and Day 5 (the end of the culture period, 96-100 hours post- fertilization). Embryos were classified as appropriately developed ('on- time') using the following criteria: on Day 2, embryos at the 2-cell stage; on Day 3, embryos at the 4- to 8-cell stage; and on Day 5, blastocyst or hatching blastocyst. The rate of development was assessed on day 2 as the percentage of embryos meeting the development criteria from the starting number of oocytes; while the rate of development was assessed on day 3 and 5 as the percentage of embryos meeting the development criteria from 2-cell embryos on day 2.
[00255] (x) Statistical analysis
[00256] All measures are reported as mean + SEM. Statistical significance was determined as indicated; by t test or one-way ANOVA with Tukey post hoc tests, as appropriate, using Graph Pad Prism version 5.01 for Windows (GraphPad Software Inc., San Diego, CA). A P value of <0.05 was considered statistically significant.
[00257] Results
[00258] (i) ER stress inducer thapsigargin causes ER stress in mouse cumulus-oocyte complexes that is reversible with Salubrinal
[00259] The mRNA expression of classic ER stress marker genes (ATF4, ATF6, Xbpls and GRP78) was examined in maturing COCs that had been exposed to the ER stress inducer thapsigargin, the ER stress inhibitor salubrinal or both; and compared to expression in control COCs that had undergone maturation either in vitro or in vivo (Figure 1). COCs treated with thapsigargin expressed increased ATF4 (4.42-fold), ATF6 (5.28-fold), Xbpls (5.96-fold) and GRP78 (4.8-fold) compared to control COCs matured in vitro. To determine whether ER stress in maturing COCs can be reversed by the ER stress inhibitor salubrinal, COCs were cultured in media containing both thapsigargin (ΙΟΟηΜ) and the ER stress eIF-2a inhibitor salubrinal (ΙΟΟηΜ). Co- treatment with salubrinal normalized expression of each of the four ER stress markers to levels similar to those of control COCs matured either in vitro or in vivo. These results demonstrate that thapsigargin induces markers of ER stress in COCs and that this is reversible with co-treatment with the ER stress inhibitor salubrinal.
[00260] (ii) Thapsigargin impairs cumulus cell protein secretion, mitochondrial membrane potential and embryo development; defects which can be reversed by salubrinal
[00261] After 16 hours of maturation, COC morphology was evaluated and compared between control COCs and those treated with thapsigargin (ΙΟΟηΜ), thapsigargin plus salubrinal (ΙΟΟηΜ) and salubrinal (ΙΟΟηΜ) alone. Poor expansion was evident in COCs cultured in the presence of thapsigargin, with the outer layers of cumulus cells falling off (Figure 2A). The four groups of IVM COCs were scored by independent blinded assessor and the expansion scores were analysed by 3 independent experiments (Figure 2B).
[00262] We next sought to determine with the impaired COC expansion was associated with impaired production of cumulus matrix proteins such as PTX3. Control COCs exhibited clear expression of Ptx3 protein localized extracellularly to the cumulus matrix; however, COCs treated with thapsigargin displayed very little Ptx3 positive staining in cumulus matrix and the complexes were exceedingly fragile (Figure 2C). Thus PTX3 protein levels in cumulus matrix and cumulus cells were further examined by western blots. After 8 hours of maturation, COCs treated with thapsigargin or thapsigargin plus salubrinal had less PTX3 protein detected in the extracellular matrix than control COCs and those treated with salubrinal alone. Levels of PTX3 protein within cumulus cells was also less in COCs treated with thapsigargin compared to the other groups. After 16 hours of maturation, COCs treated with thapsigargin had very low levels of PTX3 protein in the extracellular matrix extracts; however co-treatment of thapsigargin plus salubrinal, restored extracellular PTX3 protein to similar levels as those in the controls. (Figure 2D). The thapsigargin-induced reduction in PTX3 protein appears to be a defect in secretion since PTX3 protein levels within cumulus cells were similar in all treatment groups. The reduction in PTX3 protein was associated with the induction of ER stress in cumulus cells. At both 8 and 16 hours of maturation, cumulus cells from thapsigargin and thapsigargin plus salubrinal COCs have high levels of phospho-IREl compared to control COCs or those treated with salubrinal alone. Total IRE1 protein however is similar in all treatment groups (Figure 2D). These results clearly show that thapsigargin-induced ER stress in mouse COCs results in poor cumulus expansion and dramatically reduced PTX3 protein secretion by cumulus cells.
[00263] The ER stress inhibitor salubrinal can reverse each of the thapsigargin-induced defects. ER stress is also tightly linked to mitochondrial dysfunction and thus to investigate whether ER stress inducer thapsigargin can cause mitochondrial damage in oocytes, mitochondrial membrane potential of oocytes was determined by staining with the inner membrane potential dye JC-1. In ovulated COCs (matured in vivo) oocytes exhibited red punctuate fluorescence localized to the pericortical region of oocytes indicating high MMP, while green fluorescence localized to the deeper cytoplasm of oocytes indicating low MMP (Figure 3A). Oocytes from control COCs matured in vitro had a similar pattern although reduced in. In COCs treated with thapsigargin, red fluorescence intensity and thus mitochondrial membrane potential was reduced in the pericortical region (Fig. 3A), indicating that ER stress inducer thapsigargin impairs mitochondrial function in oocytes. In COCs treated with thapsigargin and ER stress inhibitor salubrinal, red fluorescence intensity in the pericortical region of oocytes was similar to controls. The ratio of red/green fluorescence intensity provides an index of mitochondrial activity and further demonstrated that oocytes from COCs matured in thapsigargin had significantly decreased mitochondrial activity (0.11+0.188) compared to controls (0.26+0.049) (Figure 3B). Importantly the presence of salubrinal normalized mitochondrial activity (0.23+0.059) to levels similar to those of untreated controls matured in vitro. These results demonstrate that ER stress in COCs causes mitochondrial dysfunction in oocytes and that salubrinal can normalize this defect.
[00264] To determine the impact of ER stress on fertilization and oocyte developmental competence, COCs matured in thapsigargin, or thapsigargin plus salubrinal fertilized in vitro and compared to controls. Treatment of COCs with ΙΟΟηΜ thapsigargin during maturation had no significant effect on fertilization rate on day 2 or blastocyst development rates on day 5 (data not shown). An increased dose of thapsigargin (500nM) during COC maturation resulted in oocytes that were morphologically identical to controls but exhibited a significantly lower fertilization rate on day 2 (87.7%). Co-treatment with salubrinal (200nM) normalized fertilization rates back to (96.3%). The fertilized oocytes that had been treated with thapsigargin also exhibited reduced competence to form embryos with only 77.9% developed to 4 cells on day 3 and 70% developed to blastocysts and hatching blastocysts on day 5 which was significant lower on day 3 than the embryo development rates of the controls (in vitro controls: 98.3%; ovulated in vivo controls 100%). Importantly, co-treatment with salubrinal during maturation normalized embryo development rates to 98% on day3. [00265] (iii) Palmitic acid dose-dependently induces ER stress in COCs that is reversible with salubrinal.
[00266] Based on the previous results using thapsigargin, COCs were treated with a more physiological stimulator of ER stress, palmitic acid. Initial experiments determined whether palmitic acid induces ER stress and lipotoxicity responses by treating mouse COCs with increasing doses of palmitic acid (150, 275, 400 or 525μΜ) which is a range comparable to levels of FFAs in plasma from normal to obese patients. All 4 ER stress marker genes (ATF4, ATF6, Xbpls and GRP78) were dose-dependently increased by palmitic acid. COCs matured in 150μΜ palmitic acid had similar mRNA expression levels to control COCs (Figure 5); however Xbpls mRNA was significantly increased at 275μΜ, ATF4 mRNA was significantly increased at 400μΜ, and ATF6 and GRP78 mRNA were significantly increased at 525μΜ palmitic acid.
[00267] We next investigated whether the palmitic acid-induced ER stress response in COCs is temporally regulated and whether salubrinal can reverse the ER stress induced at high doses. Mouse COCs were matured in low (150μΜ) and high (400μΜ) concentrations of palmitic acid and ER stress marker genes were assessed at 8 and 16 hours later. A 400μΜ dose of palmitic acid, but not a 150μΜ dose induced a clear ER stress response within 8 hours with increased mRNA expression of each of the ER stress markers ATF4, ATF6, Xbpls and GRP78 (Figure 6). After 16 hours, ATF4 and Xbpls mRNA levels were still significantly higher in COCs treated with high dose palmitic acid compared to low dose or untreated controls.
[00268] COCs treated with high (400μΜ) concentrations of palmitic acid were also co- treated with salubrinal (200nM) during maturation to determine whether salubrinal can reduce palmitic acid-induced ER stress. Interestingly, COCs treated with salubrinal for 8 hours had dramatically increased expression of ATF4 and GRP78 mRNA compared to both untreated control COCs as well as high dose (400 μΜ) palmitic acid treated COCs, while expression levels of ATF6 and Xbpls were reduced to control levels. By 16 hours, all four genes were reduced to control groups' levels by salubrinal treatment (Figure 6). This data confirms that ER stress induced by high concentration of palmitic acid in maturing mouse COCs can be reversed by ER stress inhibitor salubrinal. By increasing eIF2alpha phosphorylation, salubrinal stimulates the production of key UPR components, such as ATF4 and GRP78 and appears to function efficiently within 8 hours of maturation; but salubrinal ultimately normalizes UPR pathways in maturing COCs treated with palmitic acid within 16 hours.
[00269] (iv) High dose palmitic acid impairs PTX3 secretion by cumulus cells, oocyte mitochondrial membrane potential and embryo development; defects that are reversible by salubrinal.
[00270] The previous experiments showed that thapsigargin induced ER stress and that this caused poor cumulus expansion and dramatically reduced matrix protein secretion to cumulus matrix; since the ER stress inhibitor salubrinal was able to completely reverse these defects. To investigate whether high dose of palmitic acid will also cause similar defects in COCs that are reversible by salubrinal, we again measured the levels and localization of cumulus matrix protein PTX3 by wholemount immunocytochemistry and western blot of both extracellular and intracellular fractions. Immunocytochemistry clearly showed that COCs matured in high dose palmitic acid had very little PTX3 protein in the cumulus matrix; while COCs matured in high dose palmitic acid plus salubrinal had strong PTX3 staining in cumulus matrix, similar to in vivo matured COCs and control COCs matured in vitro (Figure 7A). Western blot also clearly showed the amount of PTX3 protein found in the extracellular cumulus matrix was reduced in COCs treated with high dose palmitic acid for 8h, and that by 16h of maturation salubrinal treatment increased PTX3 secretion in high dose palmitic acide treated COCs (Figure 7B). In contrast, intracellular PTX3 protein was relatively similar in all treatments. The IRE1 reduction in extracellular PTX3 protein was associated with increased levels of phosphorylated IRE1 which was highest in COCs matured in high dose palmitic acid for 8h (Figure 7B). The reductions in PTX3 protein secretion were not reflective of changes in Ptx3 mRNA since all of the treatment groups had PTX3 mRNA levels that were identical to controls at 8h of maturation, and that were not decreased compared to controls at 16h of maturation (Figure 7C).
[00271] To investigate whether treatment of maturing COCs in high dose of palmitic acid would also reduce oocyte mitochondrial membrane potential due to the induction of ER stress, mitochondrial membrane potential of oocytes was determined by staining with the inner membrane potential dye JC-1. Oocytes matured in vitro have less red fluorescence intensity than in vivo matured oocytes, as expected. However, in oocytes maturing in high dose (400μΜ) palmitic acid but not low dose (150μΜ) palmitic acid, red fluorescence intensity was reduced in the pericortical region (Fig. 8A), indicating that high dose palmitic acid decreases mitochondrial activity in oocytes. In oocytes from high dose palmitic acid plus salubrinal, similar red fluorescence intensity was present in the pericortical region as control oocytes matured in vitro. Quantification of fluorescence intensities further verified that the mean ratio of red/green fluorescence intensity of oocytes matured in high dose palmitic acid was significantly decreased (0.11+0.08) compared to controls (0.22+0.19) or low dose palmitic acid treatment ( 0.23+0.19) (Figure 8B). Co-treatment with salubrinal normalized mitochondrial activity in COCs treated with high dose palmitic acid (0.27+0.06) , demonstrating that the mitochondrial dysfunction induced by high dose palmitic acid is due to ER stress.
[00272] L-carnitine is a cellular metabolite that regulates fatty acid transportation from cytosol into mitochondria. In models of mitochondrial dysfunction in neuroblastoma cell lines and platelet concentrates, L-carnitine can improve mitochondrial activity assessed as increased JC-1 staining. It also found be able to protect cardiac mitochondrial structure damage and function reduction against 20μΜ palmitoyl CoA caused. In addition we have also found that L-carnitine increases fatty acid metabolism in COCs and improves embryo development in vitro. Thus we investigated whether the mitochondrial dysfunction induced by high dose palmitic acid can be repaired by adding L-carnitine, similar to the effect of salubrinal. COCs treated with L-carnitine (5mM) alone had increased oocyte mitochondrial acitivity (0.33+0.17) compared to oocytes from control COCs (0.23+0.18), but still less than in vivo matured COCs (0.44+0.19). In contrast to the effects of salubrinal, L-carnitine treatment was not able to normalize oocyte mitochondrial activity (0.13+0.15) of COCs treated with high dose palmitic acid (0.11+0.08) (Figure 8 C and D). Thus although L-carnitine can improve mitochondrial activity of oocytes matured in vitro it cannot reverse the oocyte mitochondrial dysfunction seen in COCs exposed to high dose palmitic acid.
[00273] To determine the impact of palmitic acid induced ER stress on fertilization and embryo developmental competence, COCs matured in high dose (400μΜ) palmitic acid, and high dose palmitic acid plus salubrinal were fertilized in vitro and compared to controls and those treated with low dose palmitic acid (Figure 9). On day 2, the fertilization rate of oocytes matured in high dose palmitic acid was significantly lower (43.7%) than that of COCs matured in low dose palmitic acid or controls. The fertilized oocytes from COCs treated with high dose palmitic acid were also slower to develop to 4 cells on day 3 (87.9%) and to blastocysts and hatching blastocysts on day 5 (50%), which was significantly lower than the in vitro matured control group on day 3 (100%). Salubrinal treatment (200nM) normalized the palmitic acid induced decrease in fertilization rates and day 3 development rates to control levels.
[00274] Discussion
[00275] This study elucidates a cellular mechanism by which lipotoxicity contributes to female infertility. Specifically, during oocyte in vitro maturation, exposure to toxic lipids, such as the saturated fatty acid palmitic acid at concentrations mimicking physiological levels present in obese women, induces ER stress in COCs thereby impairing the secretion of cumulus cell protein, mitochondrial activity in oocytes, oocyte maturation and fertilisation. Identical results were observed when COCs were treated with a classical ER stress inducer thapsigargin, and most importantly, ER stress and all impairments in protein secretion, mitochondrial activity and oocyte developmental competence were normalized with an ER stress inhibitor salubrinal, demonstrating that ER stress is the key mechanism mediating these defects. This lipotoxicity mechanism is likely to be responsible for the impaired oocyte quality and reduced fertility that affects obese women.
EXAMPLE 2 - Effect of ER stress inhibitors: PBA and TUDCA during mouse COC maturation and fertilization
[00276] Additional ER stress inhibitors were tested for their effects on mouse COCs for comparison with salubrinal.
[00277] Immature COCs (isolated from mice treated with eCG for 44-46 hours) were matured in vitro by culturing in groups of 30 in bicarbonate-buffered a-MEM supplemented with 5 % (vol/vol) FCS, 50 mlU/ml recombinant human FSH (Sigma- Aldrich) and 10 ng/ml epidermal growth factor (EGF, Sigma- Aldrich Pty. Ltd) as control, or with the addition of the indicated treatment, in drops of 100 μΐ overlaid with sterile mineral oil (Sigma-Aldrich) and incubated at 37 °C in an atmosphere of 5 % C02 and 95 % air for 16 hours. Treatments consisted of Tauroursodeoxycholic acid (TUDCA, Calbiochem, Cat. 580549) or 4-Phenylbutyrate (PBA, Calbiochem, Cat 567616). TUDCA or PBA were added to IVM media alone (i.e. in the absence of ER stress inducer Thapsigargin) at the following concentrations:
[00278] TUDCA: lOmM, 2mM, ImM , 900μΜ, 800 μΜ, 700 μΜ, 500 μΜ, 100 μΜ, and 10 μΜ
[00279] PBA: 5mM, 2mM ImM, 100 μΜ, 10 μΜ, 1 μΜ, ΙΟΟηΜ, ΙΟηΜ, InM
[00280] Figure 10 shows representative examples of COCs evaluated after the standard time of 16hours of rVM. In COCs treated with either TUDCA or PBA, morphology was very poor, with cumulus cells falling from the complex and dispersed. COCs were found completely denuded in the higher concentrations of TUDCA and PBA.
[00281] Figure 11 shows the results of an experiment that investigated the effects of TUDCA on oocyte developmental competence. COCs matured in vitro in fetal calf serum (FCS; the standard in vitro control condition) and COCs matured in vivo (ie isolated from the oviduct after ovulation) showed normal high rates of fertilization and blastocyst development. Maturation of COCs in the ER stress inducer thapsigargen (TG; ΙΟΟηΜ) resulted in reduced fertilization and blastocyst development as observed in other experiments. Fertilisation of COCs matured in TUDCA (ImM) resulted in very poor embryo development, even compared to thapsigargen (TG) treatment.
[00282] EXAMPLE 3 - Salubrinal as an IVM/IVF additive in the bovine system
[00283] Bovine oocytes are known to have higher intracellular lipid content than mouse or human oocytes. To investigate whether ER stress inhibitor can improve bovine oocyte quality and developmental competence, bovine COCs were matured in vitro by treatment TUDCA (500μΜ), PBA (ΙΟΟηΜ), or Salubrinal (ΙΟΟηΜ). COCs were then subjected to IVF under identical standard conditions. The cleavage rate was not different among treatments and controls (data not shown). TUDCA and PBA treatments resulted in the lowest blastocyst rates, while salubrinal tended to increase the blastocyst rate higher than control. It should be noted that the dose of salubrinal used for these experiments (ΙΟΟηΜ) was lower than that used to reverse palmitic-acid induced lipotoxicity (200nM) above. Overall this bovine data also confirms that Salubrinal is an ER stress inhibitor that is beneficial for developmental competence when used during oocyte maturation.
[00284] The data is shown in Figure 12.
EXAMPLE 4 - Follicular fluid from obese women effects mouse oocytes
[00285] We have previously shown that the ovarian follicle environment is altered in obese women, specifically that the follicle fluid of obese women contains higher levels of triglycerides than follicle fluid of non-obese women. The impact that these high lipid levels in follicle fluid may have on peri-conception oocyte maturation and health is not known. We sought to determine whether the high lipid content of follicular fluid from obese women influences oocyte maturation.
[00286] Materials and Methods
[00287] (i) Ovarian Follicle Fluid
[00288] Blood-free follicle fluid was collected at oocyte pick-up from women of known Body Mass Index (BMI; kg/m ) undergoing IVF/ICSI treatment who had given written, informed consent to participate in a study approved by the Women's and Children's Hospital, Adelaide, South Australia. Triglyceride and free fatty acid levels in follicular fluid were measured by automated Roche Hitachi 912 Chemistry Analyzer.
[00289] (ii) Isolation and in vitro maturation of mouse cumulus-oocyte complexes (COCs)
[00290] Experiments were approved by the University of Adelaide Animal Ethics Committee and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Immature COCs were isolated from 23 day old mice (CBAXC57B1/6F1) by puncturing antral follicles of ovaries collected 44h after i.p. injection of 5 IU equine chorionic gonadotropin (eCG/Gestyl, Professional Compounding Centre of Australia, Sydney, NSW, AU). Mature COCs were dissected from oviducts of mice following 44h eCG and 13h post i.p. administration of 5 IU human chorionic gonadotropin (hCG/ Pregnyl, Organon, Sydney, NSW, AU). COCs were collected in Hepes-buffered a-MEM (Gibco, Invitrogen Australia Pty. Ltd., VIC, AU) and cultured in groups of 20 at 37°C in 6% C02/ 94% air in 100 μΐ drops of bicarbonate-buffered a-MEM, supplemented with 50% (v/v) human follicle fluid and overlaid with sterile paraffin oil (Merck, Darmstadt, DE). Maturation was stimulated by treatment with 50 mlU/ml recombinant human FSH and 10 ng/ml Epidermal Growth Factor (EGF). Cumulus expansion, germinal vesicle breakdown (GVBD) and first polar body extrusion were evaluated following 16h of culture.
[00291] (iii) Lipid droplet staining
[00292] Lipophilic dye BODIPY 493/503 (Invitrogen) which stains intracellular neutral lipids, was used to localize lipid droplets in oocytes. Briefly, COCs were paraformaldehyde-fixed, stained in BODIPY 493/503 and imaged using a Leica SP5 spectral scanning confocal microscope and identical conditions throughout all experiments. Fluorescence in the oocyte was calculated by Analysis Pro Software (Olympus Australia Pty. Ltd., Mt Waverly, VIC, AU).
[00293] (iv) RNA isolation and real time RT-PCR
[00294] Total RNA was isolated from COCs using RNeasy Micro Kit (Qiagen Pty Ltd, Doncaster VIC, AU) and 600ng reverse transcribed using random primers (Roche) and SuperscriptTM III Reverse Transcriptase (Invitrogen). Plin2, Atf4, Atf6, Grp78 and ribosomal protein L19 (internal control) primers were Quantitect Primer Assays (Qiagen). PCR was performed in triplicate using the Rotor-GeneTM 6000 (Corbett Research) analyzer with SYBR® Green PCR Master Mix (Applied Biosystems). Data was analyzed using the 2-AACT method, for quantification relative to a calibrator sample and was expressed as the relative fold change.
[00295] (v) Statistical analysis
[00296] Statistically significant correlations were detected by Spearman (two-tailed) correlation. Other measures are reported as mean + SEM and analyzed by unpaired two- tailed t-test or One-way ANOVA with Newman-Keuls Multiple Comparison Test as indicated. Analysis was performed using Graph Pad Prism version 5.01 (GraphPad Software Inc., San Diego, CA). A p-value of <0.05 was considered statistically significant.
[00297] Results
[00298] Increasing BMI in women (n=64) was positively correlated with increasing levels of triglyceride (r=0.44; p=0.0003) and free fatty acids (r=0.25; p=0.04) in ovarian follicle fluid. To examine how the increased lipid content of follicle fluid of obese women may impact oocyte maturation the most lipid-rich and lipid-poor follicle fluid samples were sought. Levels of triglyceride and free fatty acids in follicle fluid were positively correlated (p<0.001) and samples that were relatively high or relatively low in these two measures were identified. The lipid-rich samples (n=8) were from obese women (BMI 32.6 + 1.7) and, as expected, had significantly elevated levels of free fatty acids (0.38 + 0.04 meq/L) and triglycerides (0.24 + 0.015 mmol/L) compared to the lipid-poor samples (n=8) which were from non-obese women (BMI 24.8 + 1.0) and contained significantly lower levels of lipids (free fatty acids: 0.16 + 0.02 meq/L; p=0.0001; triglycerides: 0.09 + 0.004 mmol/L, p<0.0001 by t-test compared to lipid-rich samples). Equal volumes of 2 or 3 samples of each type were pooled.
[00299] Cumulus-oocyte complexes (COCs) from mice were stimulated to mature for 16h in lipid-rich follicle fluid (high lipid FF) and compared to those matured in lipid- poor follicle fluid (low lipid FF) and those matured in vivo, i.e. isolated from oviducts following ovulation. COCs were stained with neutral lipid dye and lipid content within oocytes determined. Oocytes matured in lipid-rich follicle fluid contained significantly more neutral lipid than oocytes matured in lipid-poor follicle fluid or those matured in vivo (Fig. 13A). Expression of perilipin-2 (Plin2/ADRP), a lipid droplet protein expressed in the COC that coats lipid droplets in mouse oocytes, was also significantly higher in COCs matured in lipid-rich follicle fluid than in COCs matured in lipid-poor follicle fluid or in vivo (Fig. 13B). Expression of ER stress markers Atf4, Atf6 and Grp78 were also significantly increased in COCs matured in lipid-rich follicle fluid compared to COCs matured in lipid-poor follicle fluid (Fig. 13 C-E), similar to COCs from mice with diet-induced obesity.
[00300] COC maturation was assessed by measuring cumulus expansion score, oocyte germinal vesicle breakdown (GVBD) and oocyte polar body extrusion. Cumulus expansion and GVBD were not affected by culture in either of the follicle fluids compared to in vivo matured COCs (data not shown). However, maturation in lipid-rich follicle fluid dramatically decreased oocyte maturation to Mil, assessed as polar body formation, to approximately 25% of in vivo rates (Fig. 13F).
[00301] These results show that the hyperlipidemia associated with obesity in women extends directly into the ovarian follicular microenvironment; with increasing BMI associated with markedly increased triglyceride and free fatty acid levels in follicle fluid. Maturation of COCs in this lipid-rich environment promoted lipid accumulation and increased lipid droplet protein mRNA expression in mouse COCs exposed to it in vitro. This disrupted physiological maturation environment resulted in the induction of endoplasmic reticulum stress in the COCs and was detrimental to oocytes, leading to impaired maturation.
EXAMPLE 5 - Reduced ovulation rate and oocyte quality in two mouse ER stress models and restoration with salubrinal
[00302] Homocysteine (Hey) in drinking water is an established in vivo model of ER stress in mice and mimics clinical hyperhomocysteinemia, which occurs in women with PCOS and is correlated with poor embryo outcome.
[00303] In this ER stress animal model, mice were given 3.6g/L homocysteine in drinking water for 2 weeks to induce systemic ER stress. We are the first to show that these mice have reduced ovulation rate, and most importantly, that this can be reversed by treatment with salubrinal (lmg/kg) injected i.p. for just 4 days (Figure 14).
[00304] In a second in vivo model, 'Blobby' mice represent a model of obesity-induced lipotoxicity. These mice have a mutation that causes severe hyperphagia and weight gain even when mice are fed a standard chow diet. Bodyweights of 12 week old female Blobby mice are typically 30.5 + 1.4g compared to 21.1 + 0.6g in wildtype littermates (n=8 of each; t-test p<0.0001). We are the first to show that these mice exhibit reduced ovulation rates that are tightly correlated to bodyweight (not shown) and that COCs from Blobby females exhibit significantly reduced embryo development following IVF, compared to COCs from normal littermates.
[00305] Figure 15 shows that Blobby mice (which are obese) have poor oocyte quality compared to their wildtype or heterozygous littermates. Specifically the oocytes of Blobby mice when fertilized by IVF have reduced fertilization rates (2-cells) and poor development to the 4-cell and blastocyst stages. When we treat the mice with salubrinal (Sal; lmg/kg i.p. daily) for 4 days prior to ovulation we are able to restore oocyte quality to normal in the obese Blobby mice. Veh: vehicle-treated controls.
[00306] These results show that salubrinal treatment in vivo for just 4 days improves reproductive function in female mice. Specifically salubrinal is able to reverse the anovulation that occurs with homocysteine-induced ER stress and salubrinal is able to normalise the poor oocyte quality and developmental competence that results from obesity.
EXAMPLE 6 - Improved ovulation in Blobby mice
[00307] Treatment with an ER stress inhibitor (Sal) helps restore ovulation rates. The results are presented in Figure 16.
[00308] To assess ovulatory function, 14 week old female Blobby mice and their wildtype and heterozygous littermates were treated with eCG and hCG and ovaries were analyzed histologically and numbers of COCs ovulated into the oviduct were counted at 16 hours post-hCG treatmen. Ovaries of Blobby mice had more unovulated follicles compared to their wildtype and heterozygous littermates (not shown). Ovulation rate (the number of ovulated COCs found in the oviduct) was negatively correlated with increased body weight (P=0.0212; not shown). Treatment of Blobby mice with the ER stress inhibitor Salubrinal (lmg/kg i.p. once daily) for 4 days significantly increased ovulation rate (Figure 16; Salubrinal-treated Blobby: 12.5+1.69, n=8, vs, Blobby: 5.8+1.241, n=5). Salubrinal-treated non-obese (wildtype and heterozygous) littermates had slightly increased ovulation rate (23.2+4.271, n=5) compared to untreated non- obese mice (18.4+2.909, n=5) but this was not significantly different. Figure 16: Values are mean + SEM expressed as number of ovulated oocytes and different letters indicate significant differences by one-way ANOVA, Bonferroni Post hoc test; P<0.05.Thus ER stress responses in COCs contribute to ovulation deficiencies in obese Blobby mice since the ER stress inhibitor salubrinal treatment can restore ovulation capacity.
EXAMPLE 7 - Improved mitochondria in Blobby mice upon treatment with an ER stress inhibitor
[00309] Figure 17 shows that treatment with an ER stress improves mitochondria in Blobby mice.
[00310] We determined whether oocyte mitochondrial membrane potential (ΔΨιη) is reduced in ovulated oocytes from Blobby mice by staining with the inner membrane potential dye 5,5' ,6,6' -tetrachloro- 1 , 1 ' ,3,3 ' -tetraethylbenzimidazolylcarbocyanine iodide (JC-1). As expected, oocytes of ovulated COC exhibited red punctuate fluorescence localized to the pericortical region, indicating high ΔΨιη, whereas green fluorescence indicating low ΔΨιη localized to the deeper cytoplasm of oocytes (Figure 17). In oocytes from obese Blobby mice, the red punctuate fluorescence in the pericortical region was visibly reduced compared to oocytes from non-obese wildtype/ heterozygous mice (Figure 17). Oocytes from Blobby mouse treated with ER stress inhibitor salubrinal (lmg/kg, i.p., once daily) for 4 days before ovulation (Figure 17) did not have reduced punctuate fluorescence in pericortical region. The ratio of red/green fluorescence intensity provides an index of mitochondrial activity and further demonstrated significantly decreased mitochondrial activity in ovulated oocytes from Blobby mice and that salubrinal treatment in vivo is able to reverse the decreased mitochondrial membrane potential ΔΨιη (Figure 17). Figure 17: data are presented as mean + SEM, n= 15-33 oocytes from 4 mice per group and different letters indicate significant differences by one-way ANOVA, Bonferroni Post hoc test;
EXAMPLE 8 - Restored oocyte developmental competence
[00311] Figure 18A shows that treatment with an ER stress inhibitor restores fertilization rates in Blobby mice. Figure 18B shows that treatment with an ER stress inhibitor improves rates of blastocyst formation on Blobby mice.
[00312] To determine the impact of obesity induced ER stress on fertilization and oocyte developmental potential, ovulated COCs isolated from oviducts of Blobby mice, or Blobby mice treated with salubrinal for 4 days prior were fertilized in vitro, and oocyte viability and embryo development were compared with COCs from oviducts of non-obese mice with and without salubrinal treatment. Four hours post-fertilization, some oocytes were undergoing fragmentation and deemed non-viable. The percentage of viable oocytes was significantly lower in Blobby mice (0.40+0.06%, n=14) than in non-obese mice (0.77+0.03%, n=25; n=25-60 oocytes from 3 independent experiments; P<0.05 by one-way ANOVA, Bonferroni Post hoc test) but salubrinal-treated Blobby mice did not exhibit this defect and had similar proportions of viable oocytes as the non- obese mice (0.82+0.03%, n=8). The morphologically viable COCs from Blobby mice exhibited a significantly lower fertilization rate on d2 (44.7% two-cell embryos) (Figure 18A) that was normalized in salubrinal-treated Blobby mice (70.9% two-cell embryos) and similar to rates in non-obese mice (77.4% two-cell embryos). The 2-cell embryos from oocytes of Blobby mice were also significantly delayed in developing to four cells on d3 (33.6% 4-cell embryo) and blastocysts on d5 (18.9%) (Figure 18B). Salubrinal treatment of Blobby mice normalized embryo development following IVF (2-cells: 70.9%; 4-cells: 68.1%; blastocysts: 61.6%) to rates that were similar to those of embryos from oocytes of non-obese mice (2-cells: 77.4%: 4-cells: 68.1%; blastocysts: 51.5%). Figure 18: Data presented as the mean % on time embryo development + SEM, n = 3 independent experiments, representative of 15-50 oocytes per group. Blastocyst development is from viable oocytes on dl. Different letters indicate significant differences by one-way ANOVA, Bonferroni Post hoc test; P<0.05, at each developmental stage.
EXAMPLE 9- Effect of BGP-15 on oocytes and embryos
[00313] BGP-15 and BGP-15M are compounds that induce heat shock proteins, for example Hsp72.
[00314] An initial dose response study tested the effects of BGP-15 and BGP-15M on the viability and morphology of mouse cumulus-oocyte complexes (COCs). COCs were treated with thapsigargen (100 nM) in the presence of BGP-15 or BGP-15M at doses of 50 mg/L, 100 mg/L or 200 mg/L. Additional cohorts were treated with BGP-15 (100 mg/L) alone or BGP-15M (100 mg/L) alone or were untreated controls. The COCs were then stimulated to undergo 'expansion', a process whereby they rapidly produce extracellular matrix in preparation for fertilization, and their responses were scored 16 hours later. Thapsigargen impairs cumulus expansion, an effect that can be reversed by salubrinal.
[00315] COCs exposed to BGP-15 or BGP-15M alone exhibited robust cumulus expansion, in fact better than controls, indicating that this compound does not detrimentally affect the health or viability of mouse COCs. BGP-15 at 50 mg/L did not completely reverse the effects of thapsigargen, however BGP-15 at 100 mg/L or 200 ng/L partially reversed the effects of thapsigargen. Identical results were observed with BGP-15M.
[00316] Subsequent experiments tested the effects of BGP-15 and BGP-15M at 400mg/L on restoring cumulus expansion in thapsigargen-treated mouse COCs.
[00317] BGP-15 or BGP-15M alone at 400 mg/L did not impair cumulus expansion and some COCs exhibited more robust expansion than controls. COCs exposed to thapsigargen exhibited very poor expansion, as expected, and co-treatment with either BGP-15 or BGP-15M at 400 mg/L restored cumulus expansion to control levels. At this dose level, BGP-15M appeared to be slightly more effective at restoring cumulus expansion than BGP-15. This experiment was conducted twice with identical results.
[00318] Cumulatively, these and the above results demonstrate that BGP-15 is not detrimental to health and viability of mouse cumulus-oocyte complexes and that at 400 mg/L it can reverse the detrimental effect of the ER stressor thapsigargen on cumulus expansion. Therefore BGP-15 may be effective in reversing other types of ER stress in COCs, such as ER stress induced by dyslipidemia or obesity.
[00319] To determine whether BGP-15 could reverse the effects of dyslipidemia on cumulus-oocyte complexes, the next experiments treated mouse COCs with high dose palmitic acid in vitro and investigated whether BGP-15M could reverse the detrimental effect on oocyte mitochondrial activity. At least 20 oocytes were used in each treatment group.
[00320] Control COCs exhibited normal oocyte mitochondrial activity, assessed by JC- 1 staining, with red-stained 'active' mitochondria in the periphery of the oocyte and primarily green-stained 'inactive' mitochondrial in the center of the oocyte. Palmitic acid treatment (400 uM) reduced mitochondrial activity as we have previously observed. BGP-15M (400 mg/L) co-treatment restored oocyte mitochondrial activity to normal in palmitic acid-treated COCs. BGP-15M alone (400 mg/L) also increased oocyte mitochondrial activity.
[00321] Thus, similar to our results with salubrinal, BGP-15M is able to improve mitochondrial activity in oocytes exposed to high lipid.
[00322] The final in vitro experiments tested the ability of BGP-15M to reverse the detrimental effect of high dose palmitic acid on embryo development. Mouse COCs were treated with high dose palmitic acid (400 μΜ), palmitic acid and BGP-15M (400 mg/L), BGP-15M alone or control conditions for 16 hours. COCs were then subjected to in vitro fertilization (IVF) and culture under identical conditions and embryo development monitored (Figure 19).
[00323] At 16 hours of treatment, palmitic acid-treated COCs exhibited poor expansion compared to controls.
[00324] BGP-15M alone resulted in robust expansion and BGP-15M in the presence of palmitic acid improved expansion. Following IVF under identical conditions, embryos were scored on day 2 (2-cell stage) and day 5 (blastocyst stage). As we have previously observed, palmitic-treated COCs exhibit markedly reduced rates of 2-cell embryo formation (Figure 20 A). Co-treatment of COCs with BGP-15M in the presence of palmitic acid was able to restore embryo formation to normal. The embryos that formed 2-cells were again scored for blastocyst development at day 5 (Figure 20B). Palmitic acid-treated COCs exhibited significantly reduced potential to form blastocysts; an effect that was reversed by co-treatment with BGP-15M. [00325] These results demonstrate that treatment of cumulus-oocyte complexes with BGP-15M can reverse the detrimental effects of high lipid exposure on subsequent embryo development.
EXAMPLE 10 - ER stress inhibitors can improve oocyte developmental competence whether used during IVM or IVF.
[00326] Figure 21 shows that treatment of mouse COCs with high lipid (400uM palmitic acid (PA)) during their maturation in vitro (IVM) impairs subsequent fertilisation rate, as well as blastocyst development rates (not shown). The addition of salubrinal to either the IVM media OR the fertilisation media normalises fertilisation (and blastocyst) rates. Data is mean + SEM; n=3 independent experiments using >20 COCs per treatment group in each experiment. ***p<0.0001 compared to all other groups. These results show that ER stress inhibitor can improve oocyte developmental competence whether used during IVM or IVF.
EXAMPLE 11 - Ovulation rate is significantly increased in adult female mice by in vivo treatment with heat shock inducer BGP-15M
[00327] Figure 22 shows that ovulation rate is significantly increased in adult female mice by in vivo treatment with BGP-15M for 4 days (20mg/kg i.p. once per day). N=3 mice per treatment group. *p=0.015 by t-test.
EXAMPLE 12 - Treatment of oocytes from obese mice with an ER stress inducer normalises fetal size and weight
[00328] Preimplantation embryos generated by IVF of oocytes from obese Blobby mice or non-obese wildtype littermates were transferred to the uterus of non-obese recipient mice. Fetuses that developed from embryos generated from Blobby oocytes were significantly bigger (crown rump length 10.94+0.2745 cm; fetal weight 207.4+13.29 mg, n=7 fetuses from 2 horns) than those developed from embryos generated from wildtype oocytes (crown rump length 10+0.2182 cm; fetal weight 156.0+9.532 mg, n=8 fetuses from 3 horns) (Figure 23A, B). In contrast, the fetuses developed from oocytes of salubrinal-treated Blobby mice were of similar sizes (crown rump length 10.11+0.20 cm; fetal weight 186.4+5.85 mg, n=9 fetuses from 3 horns) as fetuses from wildtype oocytes (Figure 23A and B). Thus alterations in the oocytes of obese Blobby mice lead to altered fetal (and perhaps plancental) growth even though the oocytes are fertilized with sperm from a non-obese male and transferred into the uterus of a non-obese female for gestation. Importantly, the alterations in fetal size are normalized by a brief peri- conception treatment of the obese mothers. Fetal crown -rump length (A) and fetal weight (B) in animals at 14.5 days of gestation (6 blastocysts were transferred in each horn, wildtype, n=8 fetuses from 3 recipient horns; Blobby, n=7 fetuses from 2 recipient horns; and Blobby + 4 days salubrinal treatment (lmg/kg), n= 9 fetuses from 3 recipient horns). Different letters indicate significant differences by one-way AN OVA, Bonferroni Post hoc test; P<0.05.
[00329] Although the present disclosure has been described with reference to particular embodiments, it will be appreciated that the disclosure may be embodied in many other forms. It will also be appreciated that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.
[00330] Also, it is to be noted that, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context already dictates otherwise.
[00331] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
[00332] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
[00333] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.
[00334] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.
[00335] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.
[00336] Future patent applications may be filed on the basis of the present application, for example by claiming priority from the present application, by claiming a divisional status and/or by claiming a continuation status. It is to be understood that the following claims are provided by way of example only, and are not intended to limit the scope of what may be claimed in any such future application. Nor should the claims be considered to limit the understanding of (or exclude other understandings of) the present disclosure. Features may be added to or omitted from the example claims at a later date.

Claims

1. A method of improving developmental competence of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
2. The method according to claim 1, wherein the oocyte is part of a cumulus oocyte complex.
3. The method according to claims 1 or 2, wherein the oocyte comprises an increased lipid content.
4. The method according to any one of claims 1 to 3, wherein the endoplasmic reticulum stress inhibitor comprises one or more of an inhibitor of an inositol requiring protein- 1 (IRE1) signal transduction pathway, an inhibitor of a protein kinase RNA-like endoplasmic reticulum kinase (PERK) signal transduction pathway, an inhibitor of a transcription factor (ATF6) signal transduction pathway, and an inducer of heat shock proteins.
5. The method according to any one of claims 1 to 4, wherein the endoplasmic reticulum stress inhibitor comprises an inhibitor of eIF2a dephosphorylation.
6. The method according to any one of claims 1 to 5, wherein the endoplasmic reticulum stress inhibitor comprises one or more of salubrinal, tauroursodeoxycholate and 4-phenyl butyric acid and/or a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate of any of the aforementioned.
7. The method according to claim 6, wherein the oocyte is exposed to a concentration of salubrinal in the range of 50nM to 500nM.
8. The method according to claims 6 or 7, wherein the oocyte is exposed to a concentration of tauroursodeoxycholate in the range of <10μΜ.
9. The method according to any one of claims 6 to 7, wherein the oocyte is exposed to a concentration of 4-phenyl butyric acid in the range of <lnM.
10. The method according to any one of claims 1 to 9, wherein the inducer of heat shock proteins comprises a derivative of hydroximic acid.
11. The method according to claim 10, wherein the derivative of hydroximic acid comprises one or more of BGP-15, propanolol, bimoclomol, arimoclomal, NG-94, iroxanadine, and/or a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate of any of the aforementioned.
12. The method according to claims 10 or 11, wherein the oocyte is exposed to a concentration of the derivative hydroximic acid in the range of 4 mg/ml to 4000 mg/ml.
13. The method according to any one of claims 1 to 12 wherein the oocyte is an immature oocyte.
14. The method according to any one of claims 1 to 13, wherein the oocyte is a mature oocyte.
15. The method according to claim 14, wherein the oocyte is an oocyte matured in vitro.
16. The method according to claim 14, wherein the oocyte is an oocyte matured in vivo.
17. The method according to any one of claims 1 to 16, wherein the method comprises exposing the oocyte to the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins in vitro.
18. The method according to any one of claims 1 to 16, wherein the method comprises exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins in a subject in vivo.
19. The method according to claim 18, wherein the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins are administered to the subject at a dose of 0.2- 2 mg/kg.
20. The method according to claims 18 or 19, wherein the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins are administered to the subject at a dose of 0.2-2 mg/kg/day.
21. The method according to any one of claims 1 to 20, wherein the oocyte is a mammalian oocyte.
22. The method according to any one of claims 1 to 21, wherein the oocyte is a bovine oocyte.
23. The method according to any one of claims 1 to 21, wherein the oocyte is a human oocyte.
24. The method according to claim 23, wherein the oocyte is obtained from a female subject comprising one or more of the following characteristics: a body mass index of greater than 25 kg/m 2 ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal to 30 kg/m ; obesity; Polycystic ovary syndrome; reduced fertility; sub-fertility; infertility; ovarian dysfunction; anovulation; reduced ovulation rate; pre-diabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet.
25. The method according to any one of claims 1 to 24, wherein the method is used to improve maturation of an oocyte, to improve developmental competence of an embryo produced by fertilisation of the oocyte exposed to the endoplasmic reticulum stress inhibitor, to improve blastocyst development of an embryo produced by fertilisation of the oocyte exposed to the endoplasmic reticulum stress inhibitor, to improve cumulus cell expansion in a cumulus oocyte complex, to increase the level of cumulus cell protein in a cumulus oocyte complex; to improve assisted reproduction, to improve fertility in a female subject, to treat reduced fertility in a female subject, and to improve ovulation in a female subject.
26. A method of improving fertility in a female subject, the method comprising administering to the subject an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
27. A method of treating reduced fertility in a female subject, the method comprising administering to the subject an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
28. A method of improving ovulation in a female subject, the method comprising administering to the subject an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
29. A method of assisted reproduction comprising an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
30. A method of improving cumulus cell expansion in a cumulus oocyte complex, the method comprising exposing an oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
31. A method of increasing the level of cumulus cell protein in a cumulus oocyte complex, the method comprising exposing an oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
32. A method of in vitro maturation of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
33. A method of improving the developmental competence of an embryo produced by fertilisation of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
34. A method of improving blastocyst development of an embryo produced by fertilisation of an oocyte, the method comprising exposing the oocyte to an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
35. A kit for performing the method of any one or more of claims 1 to 30.
36. An oocyte and/or embryo culture medium, the medium comprising an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
37. The medium according to claim 36, wherein the oocyte is part of a cumulus oocyte complex.
38. The medium according to claims 36 or 37, wherein the oocyte comprises an increased lipid content.
39. The medium according to any one of claims 36 to 38, wherein the oocyte is obtained from a female subject comprising one or more of the following characteristics: a body mass index of greater than 25 kg/m 2 ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal to 30 kg/m ; obesity; Polycystic ovary syndrome; reduced fertility; infertility; sub-fertility; ovarian dysfunction; anovulation; reduced ovulation rate; pre-diabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet.
40. The medium according to any one of claims 36 to 39, wherein the endoplasmic reticulum stress inhibitor comprises one or more of an inhibitor of an inositol requiring protein-1 (IREl) signal transduction pathway, an inhibitor of a protein kinase RNA-like endoplasmic reticulum kinase (PERK) signal transduction pathway, and an inhibitor of a transcription factor (ATF6) signal transduction pathway.
41. The medium according to any one of claims 36 to 40, wherein the endoplasmic reticulum stress inhibitor comprises an inhibitor of eIF2a depho sphorylation .
42. The medium according to any one of claims 36 to 41, wherein the endoplasmic reticulum stress inhibitor comprises one or more of salubrinal, tauroursodeoxycholate and 4-phenyl butyric acid and/or a pharmaceutically acceptable derivative, solvate, salt, tautomer, stereoisomer, or racemate of any of the aforementioned.
43. The medium according to claim 42, wherein the medium comprises a concentration of salubrinal in the range of 50-500 nM.
44. The medium according to claims 42 or 43, wherein the medium comprises a concentration of tauroursodeoxycholate in the range of <10μΜ.
45. The medium according to any one of claims 42 to 44, wherein the medium comprises a concentration of 4-phenyl butyric acid in the range of <lnM.
46. The medium according to any one of claims 36 to 45, wherein the inducer of heat shock proteins comprises a derivative of hydroximic acid.
47. The medium according to claim 46, wherein the derivative of hydroximic acid comprises one or more of BGP-15, propanolol, bimoclomol, arimoclomal, NG-94, iroxanadine, and/or a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate of any of the aforementioned.
48. The medium according to claims 46 or 47, wherein the oocyte is exposed to a concentration of the derivative hydroximic acid in the range of 4 mg/ml to 4000 mg/ml.
49. The medium according to any one of claims 36 to 48, wherein the oocyte is an immature oocyte.
50. The medium according to any one of claims 36 to 49, wherein the oocyte is a mature oocyte.
51. The medium according to claim 50, wherein the oocyte comprises an oocyte matured in vitro.
52. The medium according to claim 51, wherein the oocyte comprises an oocyte matured in vivo.
53. The medium according to any one of claims 36 to 52, wherein the oocyte is a mammalian oocyte.
54. The medium according to any one of claims 36 to 53, wherein the oocyte is a bovine oocyte.
55. The medium according to any one of claims 36 to 54, wherein the oocyte is a human oocyte.
56. The medium according to any of the claims 36 to 55, wherein the medium comprises an inducer of lipid metabolism and/or a Peroxisome Proliferator Activated Receptor (PPAR) agonist.
57. The medium according to claim 56, wherein the inducer of lipid metabolism is L-carnitine.
58. The medium according to claims 56 or 57, wherein the medium comprises a Peroxisome Proliferator Activated Receptor alpha agonist and/or a Peroxisome Proliferator Activated Receptor gamma agonist.
59. The medium according to claim 52, wherein the Peroxisome Proliferator Activated Receptor alpha agonist is fenofibrate.
60. The medium according to claim 58, wherein the Peroxisome Proliferator Activated Receptor gamma agonist is rosiglitazone.
61. A non-human oocyte or embryo exposed to the medium according to any one of claims 33 to 60, or a non-human animal produced from the oocyte or embryo.
62. Use of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in the preparation of an oocyte and/or embryo culture medium according to any one of claims 33 to 60.
63. A method of assisted reproduction, the method comprising exposing an oocyte and/or an embryo to a medium according to any one of claims 33 to 60.
64. The method according to claim 63, wherein the method of assisted reproduction comprises in vitro fertilization of an oocyte.
65. A kit for improving developmental competence, the kit comprising:
an oocyte and/or embryo culture medium;
an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins; and
optionally instructions for culturing the oocyte and/or embryo in the culture medium.
66. A pharmaceutical composition for improving fertility in a female subject, the composition comprising an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
67. The pharmaceutical composition according to claim 66, wherein the endoplasmic reticulum stress inhibitor comprises one or more of an inhibitor of an inositol requiring protein-1 (IREl) signal transduction pathway, an inhibitor of a protein kinase RNA-like endoplasmic reticulum kinase (PERK) signal transduction pathway, an inhibitor of a transcription factor (ATF6) signal transduction pathway, and an inducer of heat shock proteins.
68. The pharmaceutical composition according to claims 66 or 67, wherein the endoplasmic reticulum stress inhibitor comprises an inhibitor of eIF2a depho sphorylation .
69. The pharmaceutical composition according to any one of claims 66 to 68, wherein the endoplasmic reticulum stress inhibitor comprises one or more of salubrinal, tauroursodeoxycholate and 4-phenyl butyric acid and/or a pharmaceutically acceptable derivative, solvate, salt, tautomer, stereoisomer, or racemate of any of the aforementioned.
70. The pharmaceutical composition according to any one of claims 66 to 69, wherein the inducer of heat shock proteins comprises a derivative of hydroximic acid.
71. The pharmaceutical composition according to claim 70, wherein the derivative of hydroximic acid comprises one or more of BGP-15, propanolol, bimoclomol, arimoclomal, NG-94, iroxanadine, and/or a pharmaceutically acceptable derivative, prodrug, solvate, salt, tautomer, stereoisomer, or racemate of any of the aforementioned.
72. The pharmaceutical composition according to any one of claims 66 to 71, wherein the composition further comprises a Peroxisome Proliferator Activated Receptor (PPAR) agonist.
73. A method of improving fertility of a female subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition according to any one of claims 66 to 72.
74. The method according to claim 73, wherein the subject is administered the endoplasmic reticulum stress inhibitor and/or the inducer of heat shock proteins at a dose of 0.2- 2 mg/kg/day.
75. The method according to claims 73 or 74, wherein the subject is a mammal.
76. The method according to any one of claims 73 to 75, wherein the subject is a cow.
77. The method according to any one of claims 73 to 76, wherein the subject is a human.
78. The method according to claim 77, wherein the female subject comprises one or more of the following characteristics: a body mass index of greater than 25 kg/m 2 ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal to 30 kg/m ; obesity; Polycystic ovary syndrome; reduced fertility; sub-fertility; infertility; ovarian dysfunction; anovulation; reduced ovulation rate; pre-diabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet.
79. A treatment regime for a female subject with reduced fertility, the treatment regime comprising administering to the subject a pharmaceutical composition according to any one of claims 66 to 72.
80. A method of preventing and/or treating reduced fertility in a female subject, the method comprising administering to the subject a therapeutically effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
81. Use of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins in the preparation of a medicament for preventing and/or treating reduced fertility in a female subject.
82. A method of increasing the likelihood of a female subject falling pregnant, the method comprising exposing the subject to an effective amount of an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
83. A method of treating reduced fertility in a female subject, the method comprising:
exposing an oocyte in vitro to an effective amount of an endoplasmic stress inhibitor and/or an inducer of heat shock proteins; and
introducing the oocyte, or an embryo produced from the oocyte, into the female subject.
84. A method of preventing and/or treating a subject with reduced fertility, the method comprising administering to the subject a therapeutically effective amount of endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins, wherein the subject comprises one or more of the following characteristics: a body mass index of greater than 25 kg/m 2 ; a body mass index of 25-29.9 kg/m 2 ; a body mass index of greater than or equal to 30 kg/m ; obesity; Polycystic ovary syndrome; reduced fertility; sub-fertility; infertility; ovarian dysfunction; anovulation; reduced ovulation rate; prediabetes; diabetes; hyperandrogenism; insulin resistance; impaired glucose tolerance; hyperinsulinemia; dyslipidaemia; and exposure to a high fat diet.
85. A combination product comprising:
an oocyte and/or embryo culture medium; and
an endoplasmic reticulum stress inhibitor and/or an inducer of heat shock proteins.
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CN114917215B (en) * 2022-04-27 2024-01-23 中国科学院生物物理研究所 Use of active compounds for preventing or treating ovarian dysfunctional diseases
CN117143809A (en) * 2023-10-30 2023-12-01 云南省畜牧兽医科学院 Method for improving in-vitro maturation quality of porcine oocytes
CN117143809B (en) * 2023-10-30 2023-12-26 云南省畜牧兽医科学院 Method for improving in-vitro maturation quality of porcine oocytes

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