WO2022200848A1 - Pterostilbene for use in the treatment or prevention of infertility - Google Patents

Pterostilbene for use in the treatment or prevention of infertility Download PDF

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Publication number
WO2022200848A1
WO2022200848A1 PCT/IB2021/058009 IB2021058009W WO2022200848A1 WO 2022200848 A1 WO2022200848 A1 WO 2022200848A1 IB 2021058009 W IB2021058009 W IB 2021058009W WO 2022200848 A1 WO2022200848 A1 WO 2022200848A1
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Prior art keywords
pterostilbene
infertility
decline
use according
individual
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PCT/IB2021/058009
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French (fr)
Inventor
Antonio La Marca
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Proxenia S.R.L.
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Publication of WO2022200848A1 publication Critical patent/WO2022200848A1/en

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    • 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
    • A61K31/075—Ethers or acetals
    • A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
    • A61K31/09—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages
    • 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

Definitions

  • the present invention relates to pterostilbene for use in the treatment or prevention of infertility in an individual who is affected by infertility or a decline in fertility or who is at risk of suffering from infertility or a decline in fertility.
  • Infertility is defined by the World Health Organization (WHO) as the failure to achieve a pregnancy after 12 months of regular unprotected sexual intercourse. This interval of time must be reduced to 6 months for women aged over 35 or in the presence of risk factors.
  • WHO World Health Organization
  • Pharmacological therapy for the treatment of male infertility aims to correct the alterations that have caused the infertility as there is no unique therapy for increasing the concentration of spermatozoa and correcting abnormal shapes.
  • certain hormones are successfully used for fighting hypogonadotropic hypogonadism or after varicocele surgery.
  • Antibiotics and anti-inflammatories are used for treating testicle and prostate inflammations and antioxidants are used to improve the quality of the spermatozoa.
  • infertility is connected with pathologies not susceptible to a medical approach, surgery intervenes; this is for the purpose of solving pathologies such as varicocele, obstruction of the genital ducts, cysts or malformations of the prostatic utricle.
  • a second aspect of the present invention relates to a composition comprising pterostilbene for the medical uses described above.
  • the composition comprises salts, buffers, excipients, carriers, preservatives and/or combinations thereof accepted for the preparation of pharmaceutical products.
  • a third aspect of the present invention relates to an in vitro method for increasing the percentage of success of in vitro or in vivo fertilisation, comprising at least one step of culturing a zygote or an embryo fertilised in vitro in a culture medium comprising pterostilbene.
  • a fourth aspect of the present invention relates to a method for the treatment or prevention of infertility in an individual who is affected by infertility or a decline in fertility or who is at risk of suffering from infertility or a decline in fertility.
  • Said method comprises at least one step of administering an effective quantity of pterostilbene or of a composition that contains it to an individual who needs it.
  • Figure 1 shows a graph with the dose-response effect of increasing concentrations of pterostilbene on decidualisation: a) expression of the prolactin marker (PRL); b) expression of the IGFBP-1 marker;
  • PRL prolactin marker
  • Figure 2 shows the expression of the genes PRL and IGFBP-1 evaluated over the course of the decidualisation process obtained with pterostilbene [1 mM], classic protocol or classic protocol + pterostilbene [1 mM];
  • Figure 3 shows a graph of the dose-response effect of increasing concentrations of pterostilbene on the expression of the LIF implant marker
  • Figure 4 shows the expression of LIF evaluated over the course of the decidualisation process obtained with pterostilbene [1mM], classic protocol or classic protocol + pterostilbene [1 mM];
  • Figure 5 shows: A) prolactin secreted into the medium quantified through ELISA assay; B) IGFBP-1 secreted into the medium quantified through ELISA assay;
  • Figure 6 shows LIF secreted into the medium quantified through ELISA assay
  • Figure 7 shows an immunoblot: the columns refer to: 1 ESC control; 2 ESC + pterostilbene [1mM]; 3 ESC + progesterone + cAMP; 4 ESC + progesterone + cAMP + pterostilbene [1mM]
  • the term “ultrasound antral follicle count” or “AFC” means the antral follicle count in an individual through transvaginal pelvic ultrasound study.
  • ovarian reserve means the number of oocytes i.e. the capacity of the ovary to produce ovarian follicles that can be fertilised to start a pregnancy.
  • the expression “reducing the rate of decline in serum anti-Mullerian hormone (AMH) levels” means reducing by at least 30% the normal decline in serum values of AMH in a female individual. In an individual aged between 18 and 40, the reduction in serum values of AMH is comprised between 0.1 and 0.15 ng/ml in 12 months.
  • infertility means a pathological condition in an individual characterised by the failure to achieve a pregnancy after 12 months or more of regular unprotected sexual intercourse.
  • a first aspect of the present invention relates to pterostilbene for use in the treatment or prevention of infertility in an individual who is affected by infertility or a decline in fertility or who is at risk of suffering from infertility or a decline in fertility.
  • said treatment or prevention of infertility comprises increasing fertility or reducing the rate of decline in fertility or restoring fertility in an individual.
  • the treatment or prevention of infertility comprises restoring or stimulating ovulation in an anovulatory female individual or improving the quality of the sperm in a male individual.
  • the treatment or prevention of infertility comprises reducing the rate of decline in the antral follicle count (AFC), or reducing the rate of decline in the serum levels of the anti-Mullerian hormone (AMH), or increasing the decidualisation of the endometrium, or decreasing the rate of decline in oocytes, or increasing the number of oocytes, or increasing the pregnancy success rate in a female subject who is suffering from a decline in fertility, or preventing gestational diabetes or pre-eclampsia.
  • AFC antral follicle count
  • AH anti-Mullerian hormone
  • the individual is affected by polycystic ovary syndrome (PCOS).
  • PCOS polycystic ovary syndrome
  • the individual has a normal ovarian reserve.
  • the individual has an AFC value greater than 10, more preferably greater than 12 and/or the individual has a serum AMH dose greater than 2 ng/ml, more preferably greater than 2.5 ng/ml.
  • the individual has a reduced ovarian reserve.
  • the individual has an AFC value less than 10, more preferably less than 8 and/or the individual has a serum AMH dose less than 2 ng/ml, more preferably less than 1.5 ng/ml.
  • treatment with pterostilbene reduces the rate of decline in serum AMH values and in AFC by 35-60% with respect to the expected AMH and AFC reduction values based on the age of the individual.
  • reducing the rate of decline in serum AMH levels comprises maintaining a reduction comprised between 0.05 and 0.1 ng/ml of AMH in 12 months, more preferably between 0.06 and 0.09 ng/m in 12 months.
  • reducing the rate of decline in AFC comprises maintaining a reduction comprised between 0.1 and 0.5 follicles in 12 months, more preferably between 0.2 and 0.4 follicles in 12 months.
  • increasing the decidualisation of the endometrium comprises increasing the gene and/or protein prolactin (PRL) expression levels, and/or insulin-like growth factor binding protein-7 (IGFBP-1) and/or leukaemia inhibiting factor (LIF) with respect to the gene and//or protein expression levels in an individual not treated with pterostilbene.
  • PRL protein prolactin
  • IGFBP-1 insulin-like growth factor binding protein-7
  • LIF leukaemia inhibiting factor
  • the pterostilbene is administered at least once a day, preferably at least twice a day.
  • the pterostilbene is administered for a period of time of at least 45 days, preferably for a period comprised between 50 and 365 days, more preferably comprised between 70 and 200 days.
  • the pterostilbene is administered for at least 60 days.
  • the pterostilbene is enterally administered, preferably orally administered.
  • the pterostilbene is administered in association or in combination with an infertility treatment, preferably in association or in combination with ovarian stimulation.
  • the pterostilbene is administered before the infertility treatment, preferably before ovarian stimulation.
  • pterostilbene acts on the reproductive apparatus of a female individual improving the decidualisation of the endometrium, restoring ovulation in anovulatory patients and reducing the rate of decline of the primordial follicles.
  • pterostilbene reduces the percentage of sperm DNA fragmentation with possible positive repercussions on fertilisation.
  • a second aspect of the present invention relates to a composition comprising pterostilbene for the medical uses described above.
  • the composition comprises salts, buffers, excipients, carriers, preservatives and/or combinations thereof accepted for the preparation of pharmaceutical products.
  • said composition is formulated for enteral administration, preferably for oral administration.
  • the composition is formulated in solid form, preferably in the form of pills, capsules, tablets, granular powder, hard gelatin capsules, buccal granules, sachets or lozenges.
  • a third aspect of the present invention relates to a method for increasing the percentage of success of in vitro fertilisation.
  • Said in vitro method comprises at least one step of culturing a zygote or an embryo fertilised in vitro in a culture medium comprising pterostilbene.
  • the gamete used for in vitro fertilisation is collected from an individual who has taken pterostilbene for at least 3 months prior to the collection of the gamete.
  • the individual has taken pterostilbene for at least 3 months at the dose of 125 mg per day prior to the collection of the gamete.
  • said treatment with pterostilbene is associated with an increase in oocyte recovery and an increase in the percentage of euploid embryos available for transfer to the uterus.
  • a fourth aspect of the present invention relates to a method for the treatment or prevention of infertility in an individual who is affected by infertility or a decline in fertility or who is at risk of suffering from infertility or a decline in fertility.
  • Said method comprises at least one step of administering an effective quantity of pterostilbene or of a composition that contains it to an individual who needs it.
  • the individual is affected by polycystic ovary syndrome (PCOS).
  • PCOS polycystic ovary syndrome
  • the individual has a normal ovarian reserve.
  • the individual has an AFC value greater than 10, more preferably greater than 12 and/or the individual has a serum AMH dose greater than 2 ng/ml, more preferably greater than 2.5 ng/ml.
  • the individual has a reduced ovarian reserve.
  • the individual has an AFC value less than 10, more preferably less than 8 and/or the individual has a serum AMFI dose less than 2 ng/ml, more preferably less than 1.5 ng/ml.
  • the method described above reduces the rate of decline in serum AMFI values and in AFC by 35-60% with respect to the expected AMFI and AFC reduction values based on the age of the individual.
  • reducing the rate of decline in serum AMFI levels comprises maintaining a reduction comprised between 0.05 and 0.1 ng/ml of AMFI in 12 months, more preferably between 0.06 and 0.09 ng/m in 12 months.
  • reducing the rate of decline in AFC comprises maintaining a reduction comprised between 0.1 and 0.5 follicles in 12 months, more preferably between 0.2 and 0.4 follicles in 12 months.
  • treatment or prevention of infertility comprises increasing the gene and protein prolactin (PRL) expression levels, and/or insulin-like growth factor binding protein-7 (IGFBP-1) and/or leukaemia inhibiting factor (LIF) with respect to the gene and//or protein expression levels in an individual not treated with pterostilbene.
  • PRL prolactin
  • IGFBP-1 insulin-like growth factor binding protein-7
  • LIF leukaemia inhibiting factor
  • the pterostilbene is administered at least once a day, preferably at least twice a day.
  • the pterostilbene is administered for a period of time of at least 45 days, preferably for a period comprised between 50 and 365 days, more preferably comprised between 70 and 200 days.
  • the pterostilbene is administered in association or in combination with an infertility treatment, preferably in association or in combination with ovarian stimulation.
  • the pterostilbene is administered before the infertility treatment, preferably before ovarian stimulation.
  • the pterostilbene is administered in association or in combination with at least one drug used for the infertility treatment.
  • at least one drug is chosen from among: FSH, hMG, clomiphene citrate, an antioestrogen, an FSFI analogue, a GnRFI analogue, a dietary supplement, vitamin complexes, probiotics, paraprobiotics, coenzyme Q10, carnitine, d-chiro-inositol, myo-inositol, resveratrol and combinations thereof.
  • PCOS polycystic ovary syndrome
  • Exclusion criteria were: presence of ovarian cysts, presence of endocrine/metabolic diseases.
  • the patients were subjected to the ovarian reserve study through the dosage of serum AMH and the ultrasound antral follicle count (AFC). The demographic characteristics of the patients are shown in Table I.
  • the primary aim of the study was to assess the effect of the treatment on the patients’ menstrual cycles.
  • the endometrium samples were collected from the uterus of 3 female patients with a regular menstrual cycle who were subjected to exploratory diagnostic hysteroscopy performed according to standard practice for patients referred for in vitro fertilisation due to infertility of the male of the couple.
  • the cell suspension was distributed in multi-well dishes at the concentration of 10 6 cells/well and incubated at 37°C with 5% CO2 per 24 hours. Treatment with collagenase enables a “pure” cell population of endometrial stromal cells to be obtained i.e. without the contamination of other cell types such as glandular epithelial cells.
  • the ESCs thus obtained were plated in 4 dishes and kept in culture until reaching 70% confluence according to the following outline:
  • RNA was retrotranscribed to cDNA (RTmaxima, Biorad, USA). 1 mI of cDNA deriving from each sample was used as a template for the qPCR reaction using the specific primers for every gene analysed and the enzyme SYBRgreen (Biorad).
  • the expression of the analysed genes PRL; IGFBP-1 , LIF was normalised using the constitutively expressed gene b-actin.
  • the PRL, IGFBP-1 and LIF proteins as secreted in the medium were measured in the supernatant as described in Rose et al., 1978.
  • the secretion of proteins was measured through ELISA assay (R&D) following the specific protocol for each protein supplied by the manufacturer.
  • the optical density generated in each well corresponding to each treatment was read at a wavelength of 450 nm using the MultiSkan FC (Thermo Fisher) plate reader.
  • the total proteins of the samples were extracted from the treated cells in culture or control at 4°C for 20 minutes, after the removal of the supernatant used for the ELISA assay, using the lysis buffer prepared as follows (50 mM Tris-CI, pH 7.8, containing 1% Nonidet P40, 140 mM NaCI, 0.1% SDS, 0.1% Na desoxicolate, 1 mM Na3V04, 1 X protease inhibition cocktail).
  • the following antibodies diluted 1 :1000 in TBS-T were incubated with the membranes overnight at 4°C in agitation: anti-PRL; anti-IGFBP-1 ; anti-LIF. All the antibodies were taken from rabbit (rabbit anti-human) and purchased from Santa Cruz.
  • the membrane was incubated with the following secondary antibody conjugated to horseradish peroxidase (FIRP): goat anti-rabbit IgG antibody (1 :10000) for 90 minutes at room temperature.
  • FIRP horseradish peroxidase
  • the membranes were treated in order to remove the primary antibody used (stripping of the membrane) in order to be able to enable further incubation with a different primary antibody i.e. anti-human b-tubulin (SIGMA) as an internal control over the amount of protein loaded.
  • SIGMA anti-human b-tubulin
  • the ESC control cell cultures treated were incubated with a solution of PBS-trypsin-EDTA (Life Technologies) for 15 minutes at 37°C to enable the detachment of the cells from the well.
  • the cell suspensions obtained were washed three times with PBS 1X and then transferred onto a slide through the use of cytospin (Shandon Cytospin4, Thermo Scientific) at 1000 rpm for 5 minutes and finally fixed with formalin (paraformaldehyde 4%) at 4°C for 1 hour and further washed with PBS 1X three times.
  • the fixed cells were used in immunofluorescence methods and analysed by two different investigators double blind.
  • the slides were incubated with the primary antibodies (the same ones used for the Immunoblot) at a concentration of 1 :25 in PBS 1X for an hour at room temperature. After washing with 1X PBS the samples were incubated for 1 hour at room temperature with the secondary antibody diluted 1 :200 in 1X PBS containing 3% BSA (goat anti-rabbit FITC conjugated). After washing in 1X PBS and one in water the samples were counter-stained with 1 pg/ml DAPI in H20 and mounted using an anti-fading medium (0.21 M DABCO and 90% glycerol in 0.02 M Tris, pH 8.0). The negative controls were incubated with the incubation mix without primary antibody.
  • BSA bovine serum albumin
  • the images were acquired under the optical microscope equipped with a Leica TCS SP2 AOBS fluorescence lamp.
  • the samples that bonded the DAPI (blue stain of the DNA contained in the nucleus) and the FITC bonded to the secondary antibody, were excited at the wavelength of -405 nm/25 mW line- generated by the laser blue diode and at the wavelength of 488-nm/20 mW generated by the Argon laser.
  • the excitation and detection of the fluorescence of the samples were performed in a sequential way avoiding any overlapping of signals.
  • the original images obtained in green under the confocal were converted into greyscale by applying a filter called a median filter.
  • An intensity value comprised between 0 (black) and 255 (white) was assigned to each pixel.
  • the background fluorescence (background noise) was subtracted from the analysis and the intensity of the immunofluorescence was calculated as the average intensity for each area selected.
  • the frequency of the positivity of positive cells for PRL was determined by observing more than 1000 nuclei for each experimental sample.
  • ESCs purified and placed in culture were incubated with increasing concentrations of pterostilbene (range 0, 0.5, 1 mM).
  • pterostilbene range 0, 0.5, 1 mM
  • 2 marker genes PRL and IGFBP-1
  • pterostilbene increases the expression of the decidualisation and endometrial receptivity marker genes with a dose-dependent trend.
  • the results of the immunohistochemistry staining are shown in Table III.
  • the results relate to the count of 1000 cells per slide per field and expressed as a percentage of the average positive cell count of the antibody anti-PRL.
  • the percentages of positive cells decidualised increases if as well as the decidualisation protocol the ESCs are co-incubated with pterostilbene [1mM]
  • Table III immunohistochemistry (percentage of positive cells to the antibody anti-prolactin: the columns relate to the following: 1 ESC control; 2 ESC + pterostilbene [1mM]; 3 ESC + progesterone + cAMP; 4 ESC + progesterone + cAMP + pterostilbene [1mM]
  • the ovarian stimulation was performed with recombinant FSH (rFSH) starting from the 2nd day of the period, choosing a personalised daily dose based on age: 150 IU of rFSFI for patients aged ⁇ 35, or 225 IU of rFSFI for patients > 35.
  • Pituitary suppression was obtained with GnRFI antagonist, from the day on which the largest follicle reached 14 mm average diameter until the day of the rhCG.
  • Follicular growth was monitored with transvaginal ultrasound on alternate days.
  • Recombinant hCG (rhCG) was administered to induce final oocyte maturation when 3 or more follicles had a diameter >16 mm, exactly 35.5 hours prior to the oocyte recovery (pick-up) operation. No cycle was interrupted due to inadequate or excessive ovarian response.
  • PPS Pre implantation genetic screening
  • the primary aim of the study was to compare the number of oocytes recovered among the patients who had performed a pre-treatment with pterostilbene and the control patients.
  • the patients were enrolled in an age range between 18 and 40. Exclusion criteria were: presence of known infertility factors (endocrine-ovulatory factor, reduced ovarian reserve, tubal factor, endometriosis), presence of ovarian cysts, presence of endocrine-metabolic diseases.
  • the demographic characteristics of the patients are shown in Table VI.
  • the 30 patients in the study took pterostilbene for 12 months at the dosage of 100 mg twice a day. They were subjected to the ovarian reserve study through the dosage of serum AMH and the ultrasound antral follicle count (AFC) at time 0 and at time 12 months.
  • AFC ultrasound antral follicle count
  • the ovarian reserve of these patients was compared with that of 300 control patients with similar demographic characteristics, who were not taking any therapy.
  • the data related to the ovarian reserve of the control patients were collected in a perspective way by measuring the ovarian reserve in every patient twice after 12 months. It emerged that in 12 months the AMH undergoes a 0.2 ng/ml decline, whereas the AFC decreases by 2 follicles. Aims of the study

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Abstract

The present invention relates to pterostilbene for use in the treatment or prevention of infertility in an individual who is affected by infertility or a decline in fertility or who is at risk of suffering from infertility or a decline in fertility. Preferably, said treatment or prevention of infertility comprises increasing fertility or reducing the rate of decline in fertility or restoring fertility in an individual.

Description

PTEROSTILBENE FOR USE IN THE TREATMENT OR PREVENTION OF INFERTILITY
DESCRIPTION
FIELD OF THE INVENTION
The present invention relates to pterostilbene for use in the treatment or prevention of infertility in an individual who is affected by infertility or a decline in fertility or who is at risk of suffering from infertility or a decline in fertility.
PRIOR ART
Infertility is defined by the World Health Organization (WHO) as the failure to achieve a pregnancy after 12 months of regular unprotected sexual intercourse. This interval of time must be reduced to 6 months for women aged over 35 or in the presence of risk factors.
It is estimated that about 15% of couples of childbearing age, throughout the world, have conception difficulties; therefore, we are talking about a relatively common condition which affects about 100 million people. According to more recent data, it appears that these numbers are constantly increasing; this is due to various factors such as incorrect lifestyles (smoking, alcohol and drugs), pollution and chemical agents that have a negative effect on female and male reproduction, obesity and infections. Furthermore, the causes of infertility may be connected with a pathological condition in women, men or both. The female factor seems to be responsible in about 30-40% of cases of infertility; the male factor is responsible in about 30-40% of cases, whereas in 10-20% of cases, together with the female factor, joint infertility takes part. Finally, in about 10-20% of cases, we talk about idiopathic infertility, i.e. when the diagnostic tests have not been able to identify any specific cause.
There are numerous cases of female infertility and of different kinds: infections, hormonal, immunological or reproductive apparatus alterations, congenital malformations, endometriosis, etc. Regardless of the cause, the reproductive capacity of the couple declines with age and this phenomenon occurs more significantly in women because of hormonal factors, an increase in miscarriages connected with age and, especially, because of a reduced ovarian reserve and the ageing of the oocytes. Fertility gradually drops with age, undergoing a significant drop after the age of 35 which becomes even more marked after the age of 40 (at 30 the possibility to conceive in a fertile cycle is around 30-40%, whereas the possibility drops to 10% at 40).
In the case of tubal alterations, and in particular obstructions, if they are slight it is possible to combine microsurgery techniques with the administration of drugs. If, instead, failure to fall pregnant is connected with polycystic ovary syndrome, the physician may decide to resort to a pharmacological treatment based on FSH and LH to support ovulation. Flowever, pharmacological therapy cannot produce significant effects and in these cases the most appropriate solution remains assisted conception. Fertile capacity in males is based on a secretory phase (production of spermatozoa) and a subsequent excretory phase (transport via the excretory ducts). Therefore, male infertility is both due to idiopathic causes (up to 60%) and known causes, i.e. connected with insufficient production of spermatozoa, abnormalities in the quality of the spermatozoa produced (due to reduced motility, altered morphology or damaged DNA) or to an obstacle to transport along the spermatic ducts.
Pharmacological therapy for the treatment of male infertility aims to correct the alterations that have caused the infertility as there is no unique therapy for increasing the concentration of spermatozoa and correcting abnormal shapes.
For example, certain hormones are successfully used for fighting hypogonadotropic hypogonadism or after varicocele surgery. Antibiotics and anti-inflammatories are used for treating testicle and prostate inflammations and antioxidants are used to improve the quality of the spermatozoa.
If the infertility is connected with pathologies not susceptible to a medical approach, surgery intervenes; this is for the purpose of solving pathologies such as varicocele, obstruction of the genital ducts, cysts or malformations of the prostatic utricle.
Therefore, the development of a therapy able to treat and/or prevent female and male infertility in a non-invasive way is of current interest. In particular, there is a strongly perceived need for a pharmacological therapy able to act on various contributing factors that can cause male and female infertility.
SUMMARY OF THE INVENTION
A first aspect of the present invention relates to pterostilbene for use in the treatment or prevention of infertility in an individual who is affected by infertility or a decline in fertility or who is at risk of suffering from infertility or a decline in fertility. Preferably, said treatment or prevention of infertility comprises increasing fertility or reducing the rate of decline in fertility or restoring fertility in an individual.
A second aspect of the present invention relates to a composition comprising pterostilbene for the medical uses described above. In one embodiment, the composition comprises salts, buffers, excipients, carriers, preservatives and/or combinations thereof accepted for the preparation of pharmaceutical products.
A third aspect of the present invention relates to an in vitro method for increasing the percentage of success of in vitro or in vivo fertilisation, comprising at least one step of culturing a zygote or an embryo fertilised in vitro in a culture medium comprising pterostilbene.
A fourth aspect of the present invention relates to a method for the treatment or prevention of infertility in an individual who is affected by infertility or a decline in fertility or who is at risk of suffering from infertility or a decline in fertility.
Said method comprises at least one step of administering an effective quantity of pterostilbene or of a composition that contains it to an individual who needs it.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows a graph with the dose-response effect of increasing concentrations of pterostilbene on decidualisation: a) expression of the prolactin marker (PRL); b) expression of the IGFBP-1 marker;
Figure 2 shows the expression of the genes PRL and IGFBP-1 evaluated over the course of the decidualisation process obtained with pterostilbene [1 mM], classic protocol or classic protocol + pterostilbene [1 mM];
Figure 3 shows a graph of the dose-response effect of increasing concentrations of pterostilbene on the expression of the LIF implant marker;
Figure 4 shows the expression of LIF evaluated over the course of the decidualisation process obtained with pterostilbene [1mM], classic protocol or classic protocol + pterostilbene [1 mM];
Figure 5 shows: A) prolactin secreted into the medium quantified through ELISA assay; B) IGFBP-1 secreted into the medium quantified through ELISA assay;
Figure 6 shows LIF secreted into the medium quantified through ELISA assay; and
Figure 7 shows an immunoblot: the columns refer to: 1 ESC control; 2 ESC + pterostilbene [1mM]; 3 ESC + progesterone + cAMP; 4 ESC + progesterone + cAMP + pterostilbene [1mM]
DEFINITIONS
In the context of the present invention, the term “ultrasound antral follicle count” or “AFC” means the antral follicle count in an individual through transvaginal pelvic ultrasound study. In the context of the present invention the term “ovarian reserve” means the number of oocytes i.e. the capacity of the ovary to produce ovarian follicles that can be fertilised to start a pregnancy.
In the context of the present invention the expression “reducing the rate of decline in serum anti-Mullerian hormone (AMH) levels” means reducing by at least 30% the normal decline in serum values of AMH in a female individual. In an individual aged between 18 and 40, the reduction in serum values of AMH is comprised between 0.1 and 0.15 ng/ml in 12 months.
In the context of the present invention the term “infertility” means a pathological condition in an individual characterised by the failure to achieve a pregnancy after 12 months or more of regular unprotected sexual intercourse.
In the context of the present invention the term “decline in fertility” means a sub-pathological condition characterised by conception difficulties and/or AMH serum values below normal standard values and/or with lower than normal AFC values.
DETAILED DESCRIPTION OF THE INVENTION
A first aspect of the present invention relates to pterostilbene for use in the treatment or prevention of infertility in an individual who is affected by infertility or a decline in fertility or who is at risk of suffering from infertility or a decline in fertility.
Preferably, said treatment or prevention of infertility comprises increasing fertility or reducing the rate of decline in fertility or restoring fertility in an individual.
In one embodiment, the treatment or prevention of infertility comprises restoring or stimulating ovulation in an anovulatory female individual or improving the quality of the sperm in a male individual.
Preferably, the treatment or prevention of infertility comprises reducing the rate of decline in the antral follicle count (AFC), or reducing the rate of decline in the serum levels of the anti-Mullerian hormone (AMH), or increasing the decidualisation of the endometrium, or decreasing the rate of decline in oocytes, or increasing the number of oocytes, or increasing the pregnancy success rate in a female subject who is suffering from a decline in fertility, or preventing gestational diabetes or pre-eclampsia.
In one embodiment of the invention, the individual is affected by polycystic ovary syndrome (PCOS).
In one embodiment, the treatment or prevention of infertility comprises increasing the sperm count, or increasing sperm motility, or reducing the percentage of sperm DNA fragmentation in a male individual who is suffering from a decline in fertility.
In one embodiment of the invention, the individual has a normal ovarian reserve. Preferably, the individual has an AFC value greater than 10, more preferably greater than 12 and/or the individual has a serum AMH dose greater than 2 ng/ml, more preferably greater than 2.5 ng/ml.
In one embodiment of the invention, the individual has a reduced ovarian reserve. Preferably, the individual has an AFC value less than 10, more preferably less than 8 and/or the individual has a serum AMH dose less than 2 ng/ml, more preferably less than 1.5 ng/ml.
In a preferred embodiment, treatment with pterostilbene reduces the rate of decline in serum AMH values and in AFC by 35-60% with respect to the expected AMH and AFC reduction values based on the age of the individual.
Preferably, reducing the rate of decline in serum AMH levels comprises maintaining a reduction comprised between 0.05 and 0.1 ng/ml of AMH in 12 months, more preferably between 0.06 and 0.09 ng/m in 12 months. Preferably, reducing the rate of decline in AFC comprises maintaining a reduction comprised between 0.1 and 0.5 follicles in 12 months, more preferably between 0.2 and 0.4 follicles in 12 months.
Preferably, increasing the decidualisation of the endometrium comprises increasing the gene and/or protein prolactin (PRL) expression levels, and/or insulin-like growth factor binding protein-7 (IGFBP-1) and/or leukaemia inhibiting factor (LIF) with respect to the gene and//or protein expression levels in an individual not treated with pterostilbene.
In one embodiment, said pterostilbene is administered, for the medical purposes reported above, in an amount comprised between 50 and 400 mg a day, more preferably between 70 and 250 mg a day, even more preferably comprised between 100 and 150 mg a day.
In one embodiment, the pterostilbene is administered at least once a day, preferably at least twice a day. For the medical purposes described above, the pterostilbene is administered for a period of time of at least 45 days, preferably for a period comprised between 50 and 365 days, more preferably comprised between 70 and 200 days.
In a preferred embodiment, the pterostilbene is administered for at least 60 days.
Preferably, the pterostilbene is enterally administered, preferably orally administered.
In one embodiment, the pterostilbene is administered in association or in combination with an infertility treatment, preferably in association or in combination with ovarian stimulation.
Preferably, the pterostilbene is administered before the infertility treatment, preferably before ovarian stimulation.
In one embodiment, the pterostilbene is administered in association or in combination with at least one drug used for the infertility treatment. Preferably said at least one drug is chosen from among: FSFI, hMG, clomiphene citrate, an antioestrogen, an FSFI analogue, a GnRFI analogue, a dietary supplement, vitamin complexes, probiotics, paraprobiotics, coenzyme Q10, carnitine, d-chiro-inositol, myo-inositol, resveratrol and combinations thereof.
In fact, the Applicant has demonstrated that pterostilbene acts on the reproductive apparatus of a female individual improving the decidualisation of the endometrium, restoring ovulation in anovulatory patients and reducing the rate of decline of the primordial follicles. In a male individual, pterostilbene reduces the percentage of sperm DNA fragmentation with possible positive repercussions on fertilisation.
A second aspect of the present invention relates to a composition comprising pterostilbene for the medical uses described above. In one embodiment, the composition comprises salts, buffers, excipients, carriers, preservatives and/or combinations thereof accepted for the preparation of pharmaceutical products.
In a preferred embodiment of the invention, the composition comprises pterostilbene as a single active ingredient, i.e. the composition does not comprise any further molecules with pharmacological activity on the reproductive apparatus of a male or female individual.
In one embodiment of the invention, said composition is formulated for enteral administration, preferably for oral administration. In particular, the composition is formulated in solid form, preferably in the form of pills, capsules, tablets, granular powder, hard gelatin capsules, buccal granules, sachets or lozenges.
A third aspect of the present invention relates to a method for increasing the percentage of success of in vitro fertilisation. Said in vitro method comprises at least one step of culturing a zygote or an embryo fertilised in vitro in a culture medium comprising pterostilbene. Preferably, the gamete used for in vitro fertilisation is collected from an individual who has taken pterostilbene for at least 3 months prior to the collection of the gamete. Preferably, the individual has taken pterostilbene for at least 3 months at the dose of 125 mg per day prior to the collection of the gamete. In fact, said treatment with pterostilbene is associated with an increase in oocyte recovery and an increase in the percentage of euploid embryos available for transfer to the uterus.
A fourth aspect of the present invention relates to a method for the treatment or prevention of infertility in an individual who is affected by infertility or a decline in fertility or who is at risk of suffering from infertility or a decline in fertility.
Said method comprises at least one step of administering an effective quantity of pterostilbene or of a composition that contains it to an individual who needs it.
In one embodiment of the invention, the individual is affected by polycystic ovary syndrome (PCOS).
In one embodiment of the invention, the individual has a normal ovarian reserve. Preferably, the individual has an AFC value greater than 10, more preferably greater than 12 and/or the individual has a serum AMH dose greater than 2 ng/ml, more preferably greater than 2.5 ng/ml.
In one embodiment of the invention, the individual has a reduced ovarian reserve. Preferably, the individual has an AFC value less than 10, more preferably less than 8 and/or the individual has a serum AMFI dose less than 2 ng/ml, more preferably less than 1.5 ng/ml.
In a preferred embodiment, the method described above reduces the rate of decline in serum AMFI values and in AFC by 35-60% with respect to the expected AMFI and AFC reduction values based on the age of the individual.
Preferably, reducing the rate of decline in serum AMFI levels comprises maintaining a reduction comprised between 0.05 and 0.1 ng/ml of AMFI in 12 months, more preferably between 0.06 and 0.09 ng/m in 12 months. Preferably, reducing the rate of decline in AFC comprises maintaining a reduction comprised between 0.1 and 0.5 follicles in 12 months, more preferably between 0.2 and 0.4 follicles in 12 months.
Preferably, treatment or prevention of infertility comprises increasing the gene and protein prolactin (PRL) expression levels, and/or insulin-like growth factor binding protein-7 (IGFBP-1) and/or leukaemia inhibiting factor (LIF) with respect to the gene and//or protein expression levels in an individual not treated with pterostilbene.
In one embodiment, said pterostilbene is administered, for the medical purposes reported above, in an amount comprised between 50 and 400 mg a day, more preferably between 70 and 250 mg a day, even more preferably comprised between 100 and 150 mg a day.
In one embodiment, the pterostilbene is administered at least once a day, preferably at least twice a day. For the medical purposes described above, the pterostilbene is administered for a period of time of at least 45 days, preferably for a period comprised between 50 and 365 days, more preferably comprised between 70 and 200 days.
In a preferred embodiment, the pterostilbene is administered for at least 60 days. Preferably, the pterostilbene is enterally administered, preferably orally administered.
In one embodiment, the pterostilbene is administered in association or in combination with an infertility treatment, preferably in association or in combination with ovarian stimulation.
Preferably, the pterostilbene is administered before the infertility treatment, preferably before ovarian stimulation.
In one embodiment, the pterostilbene is administered in association or in combination with at least one drug used for the infertility treatment. Preferably said at least one drug is chosen from among: FSH, hMG, clomiphene citrate, an antioestrogen, an FSFI analogue, a GnRFI analogue, a dietary supplement, vitamin complexes, probiotics, paraprobiotics, coenzyme Q10, carnitine, d-chiro-inositol, myo-inositol, resveratrol and combinations thereof.
EXAMPLE
Effects of 12-month treatment with pterostilbene in patients with polycystic ovary syndrome
A perspective pilot trial was performed on 13 patients affected by polycystic ovary syndrome (PCOS). The aim of the study was to investigate the effects on the menstrual cycle and ovarian reserve of a 12- month treatment with pterostilbene. The patients were enrolled in an age range between 18 and 40. The main inclusion criterion was compliance with the Rotterdam criteria for the diagnosis of PCOS (presence of oligo-anovulation and at least one from among: hyperandrogenism/hyperandrogenemia, or ovaries with a polycystic appearance at ultrasound).
Exclusion criteria were: presence of ovarian cysts, presence of endocrine/metabolic diseases. The patients were subjected to the ovarian reserve study through the dosage of serum AMH and the ultrasound antral follicle count (AFC). The demographic characteristics of the patients are shown in Table I.
All the patients took pterostilbene for 12 months at the dosage of 100 mg twice a day.
Aims of the study
The primary aim of the study was to assess the effect of the treatment on the patients’ menstrual cycles.
Secondary aims:
• To assess the effect of the treatment on weight loss
• Reduction of AMH in 12 months
• Reduction of AFC in 12 months
Results
From the study it emerged that, after treatment for 12 months with pterostilbene, 68% of patients had started to have regular periods again (25±7 days), in association with modest weight loss. The average time for the recovery of the menstrual cycle was 5.5 months. It was observed in particular that the patients who started to have regular menstrual cycles again were those with the lowest AMH and AFC at the baseline examination (Table II).
Some of the patients (6) were taking inositol as a dietary supplement. In these patients the average recovery time of the menstrual cycle was significantly shorter (3.8 months vs. 6.5; p<0.01). Furthermore, in all the patients treated with pterostilbene, the rate in reduction over time of AMH and AFC was 50% lower than expected on the basis of the nomograms that represent the decline in these markers with age (Table II).
Table I. Demographic characteristics of the patients
Figure imgf000013_0001
Table II. Effects of the treatment
Figure imgf000013_0002
Assessment of the activity of pterostilbene on decidualisation and on the expression of proteins of the implant in primary cultures of stromal endometrial cells
The effects of pterostilbene on the decidualisation of undifferentiated ESCs were analysed with a particular focus on the regulation of genes considered as decidualisation markers (prolactin, PRL; insulin-like growth factor binding protein-1 , IGFBP-1) and of the gene considered to be a marker of endometrial receptivity (leukaemia inhibiting factor, LIF) through quantitative PCR methods (qPCR). The translation of these genes into proteins was then analysed through enzyme-linked immunosorbent assays (ELISA), immunoblot and immunohistochemistry staining. The role of pterostilbene in inducing decidualisation was also compared with ESCs of the same patient treated by applying the direct decidualisation protocol (positive internal control).
The data obtained demonstrate that pterostilbene induces undifferentiated ESCs to assume a decidualised phenotype, with activation levels and times of the marker genes that are comparable with one another and with those obtained with decidualisation induced by progesterone and cAMP (direct). The results obtained at gene level were also confirmed at protein level demonstrating the similarity of the protocols applied in inducing decidualisation.
From this study it can also be deduced that, when progesterone and cAMP are added to the culture system as well as pterostilbene, according to the direct decidualisation protocol, the gene and protein expression levels produced are significantly increased, in fact anticipating the achievement of the decidualised state of the ESCs, as confirmed by the percentages of decidualised cells counted after immunohistochemistry analysis.
The possibility that pterostilbene can have a positive effect on the uterine micro-environment improving decidualisation and therefore receptivity during the implant window is of notable importance especially for the development of new treatments/investigations for increasing the possibility of conception both in vivo and in vitro (IVF).
Materials and methods Collection of endometrium samples
The endometrium samples were collected from the uterus of 3 female patients with a regular menstrual cycle who were subjected to exploratory diagnostic hysteroscopy performed according to standard practice for patients referred for in vitro fertilisation due to infertility of the male of the couple.
Informed consent was obtained from every patient prior to the surgical operation in order to use the tissues taken from the patient for this study. The patients used in this study had an average age of 42 ± 4 and had not followed any hormone therapy in the 6 months prior to the operation. Primary cell cultures of ESCs
The endometrium samples were obtained during the proliferative phase of the menstrual cycle.
The primary cultures were prepared following the protocol reported in Ciarmela et al., 2011 and described here in brief
1) 2 washes in 1X PBS
2) The individual samples were deposited on a sterile Petri dish with the addition of Collagenase Type II [0.1%, or 10 mg in 10 ml] or type VIII [10 mg in 12 ml] diluted in DMEM without FBS (serum);
3) After the removal of the thin outer layer the sample was chopped into the smallest possible pieces;
4) The tissue thus fragmented was placed in a 50 ml tube and covered with the collagenase solution up to 8 ml;
5) The samples were then incubated at 37°C until the complete digestion of the tissue (the process takes at least 4 hours);
6) The digested samples were filtered;
7) The lysates thus obtained were centrifuged at 1200 rpm for 12 minutes;
8) Removal of the supernatant;
9) Re-suspension of the cells accumulated on the bottom of the tube (about 1 ml) with the addition of 3 ml of FBS and then incubation at 37°C in a controlled atmosphere with 5% CO2 for 20 minutes;
10) Repetition of points 7, 8 and 9; and
11) The cell suspension was distributed in multi-well dishes at the concentration of 106 cells/well and incubated at 37°C with 5% CO2 per 24 hours. Treatment with collagenase enables a “pure” cell population of endometrial stromal cells to be obtained i.e. without the contamination of other cell types such as glandular epithelial cells.
The ESCs thus obtained were plated in 4 dishes and kept in culture until reaching 70% confluence according to the following outline:
Plate 1 : Control ESC (106 cells/well)
Plate 2: ESC (106 cells/well) + Pterostilbene [0, 0.5, 1 mM]
Plate 3: ESCs treated according to the decidualisation protocol reported in Dimitriadis et al., 2005, i.e. by dissolving 600 ng/ml of progesterone and 10 ng/ml of cAMP in the medium for 8-10 days.
Plate 4: ESCs treated according to the decidualisation protocol reported in Dimitriadis et al., 2005, i.e. by dissolving 600 ng/ml of progesterone and 10 ng/ml of cAMP in the medium for 8-10 days + pterostilbene [0, 0.5, 1 mM] To perform the subsequent analyses the samples were processed on a daily basis as described below.
Measurement of the mRNA for the quantification of gene activation The cells (control and treated) present in every individual well were directly lysed in the commercial product Tri-reagent for the extraction of total RNA according to the protocol provided by the company.
After the quantification and determination of the degree of purity of the RNA extracted, which was evaluated using the spectrophotometer, about 1 pg of total RNA was retrotranscribed to cDNA (RTmaxima, Biorad, USA). 1 mI of cDNA deriving from each sample was used as a template for the qPCR reaction using the specific primers for every gene analysed and the enzyme SYBRgreen (Biorad). The expression of the analysed genes (PRL; IGFBP-1 , LIF) was normalised using the constitutively expressed gene b-actin. The specificity of the amplified products thus obtained was checked both through the analysis of the melting curve and after the fractioning of the aforesaid amplified products in a 1% agarose gel run in tris-acetic acid-EDTA buffer (TAE) 1X. ELISA
The PRL, IGFBP-1 and LIF proteins as secreted in the medium were measured in the supernatant as described in Rose et al., 1978. The secretion of proteins was measured through ELISA assay (R&D) following the specific protocol for each protein supplied by the manufacturer. The optical density generated in each well corresponding to each treatment was read at a wavelength of 450 nm using the MultiSkan FC (Thermo Fisher) plate reader. The sensitivity and detection limit of the assay were calculated according to the instructions of the kit by comparing the optical density of the positive control with the more diluted standard used for obtaining the calibration curve (Sensitivity = 0.67 pmol/ml; Detection limit = 0.31 pmol/ml; Variation coefficient R2 = 0.9974) thanks to the computational tool MyAssay for calculating the concentrations of the proteins secreted into the medium.
Western blot analysis of PRL, IGFBP-1 and LIF
Only the samples related to the 6 days of incubation were used for analysis through the immunoblot technique. The total proteins of the samples were extracted from the treated cells in culture or control at 4°C for 20 minutes, after the removal of the supernatant used for the ELISA assay, using the lysis buffer prepared as follows (50 mM Tris-CI, pH 7.8, containing 1% Nonidet P40, 140 mM NaCI, 0.1% SDS, 0.1% Na desoxicolate, 1 mM Na3V04, 1 X protease inhibition cocktail). The lysates were then centrifuged for 15 minutes in a centrifuge refrigerated at maximum speed of 16000 x g and immediately boiled in sodium-dodecyl- sulphate (SDS) buffer for loading the samples. The extracted proteins were quantified with the Bradford method. About 50 pg of total proteins per sample were subjected to vertical electrophoresis under (SDS-PAGE) denaturing and reducing conditions and the proteins thus fractionated were transferred onto a nitrocellulose membrane. The protocol of this method called Immunoblot below is the one described in Sacchi et al. 2017. Once the transfer had taken place, the membrane was blocked with 5% full-fat powdered milk in TBS-Tween 20 (TBS-T) for an hour at room temperature. The following antibodies diluted 1 :1000 in TBS-T were incubated with the membranes overnight at 4°C in agitation: anti-PRL; anti-IGFBP-1 ; anti-LIF. All the antibodies were taken from rabbit (rabbit anti-human) and purchased from Santa Cruz. At the end of incubation with the primary antibodies, after 3 washes in TBS-T to remove the excess non-bonded primary antibody, the membrane was incubated with the following secondary antibody conjugated to horseradish peroxidase (FIRP): goat anti-rabbit IgG antibody (1 :10000) for 90 minutes at room temperature. The immunoreactive proteins were detected with ECL (Amersham). After a first acquisition the membranes were treated in order to remove the primary antibody used (stripping of the membrane) in order to be able to enable further incubation with a different primary antibody i.e. anti-human b-tubulin (SIGMA) as an internal control over the amount of protein loaded.
Immuno cytochemistry staining
The ESC control cell cultures treated were incubated with a solution of PBS-trypsin-EDTA (Life Technologies) for 15 minutes at 37°C to enable the detachment of the cells from the well. The cell suspensions obtained were washed three times with PBS 1X and then transferred onto a slide through the use of cytospin (Shandon Cytospin4, Thermo Scientific) at 1000 rpm for 5 minutes and finally fixed with formalin (paraformaldehyde 4%) at 4°C for 1 hour and further washed with PBS 1X three times. The fixed cells were used in immunofluorescence methods and analysed by two different investigators double blind.
After treatment with 3% purified bovine serum albumin (BSA) in PBS 1X for 30 minutes at room temperature, the slides were incubated with the primary antibodies (the same ones used for the Immunoblot) at a concentration of 1 :25 in PBS 1X for an hour at room temperature. After washing with 1X PBS the samples were incubated for 1 hour at room temperature with the secondary antibody diluted 1 :200 in 1X PBS containing 3% BSA (goat anti-rabbit FITC conjugated). After washing in 1X PBS and one in water the samples were counter-stained with 1 pg/ml DAPI in H20 and mounted using an anti-fading medium (0.21 M DABCO and 90% glycerol in 0.02 M Tris, pH 8.0). The negative controls were incubated with the incubation mix without primary antibody.
The images were acquired under the optical microscope equipped with a Leica TCS SP2 AOBS fluorescence lamp. The samples that bonded the DAPI (blue stain of the DNA contained in the nucleus) and the FITC bonded to the secondary antibody, were excited at the wavelength of -405 nm/25 mW line- generated by the laser blue diode and at the wavelength of 488-nm/20 mW generated by the Argon laser. The excitation and detection of the fluorescence of the samples were performed in a sequential way avoiding any overlapping of signals.
The original images obtained in green under the confocal were converted into greyscale by applying a filter called a median filter. An intensity value comprised between 0 (black) and 255 (white) was assigned to each pixel. The background fluorescence (background noise) was subtracted from the analysis and the intensity of the immunofluorescence was calculated as the average intensity for each area selected. The frequency of the positivity of positive cells for PRL was determined by observing more than 1000 nuclei for each experimental sample.
Statistical analysis
The statistical analysis was carried out with the Kruskal-Wallis test followed by the Bonferroni test with a probability P O.001 selected as the significance threshold. The statistical analysis on the secreted proteins analysed with the ELISA method was carried out using the computational tool specified in the kit. The relative expression of each gene analysed was obtained using the 2_DDa method [Livak and Schmittgen, 1992] and expressed as a ratio to the control samples placed arbitrarily at 1. Results
ESCs purified and placed in culture were incubated with increasing concentrations of pterostilbene (range 0, 0.5, 1 mM). To follow the effects of pterostilbene on the decidualisation process the expression of 2 marker genes (PRL and IGFBP-1) was analysed through qPCR. As shown in Figure 1 , pterostilbene increases the expression of the decidualisation and endometrial receptivity marker genes with a dose-dependent trend.
As control over the decidualisation capacity of the ESCs used in our experiment, we compared the PRL, IGFBP-1 expression profiles generated by the dose with the highest concentration of pterostilbene (1 mM) with the expression profile obtained in ESCs treated with the direct classic decidualisation profile. As can be deduced from Figure 2, pterostilbene induces gene expression profiles comparable with those induced by the classic decidualisation profile. Furthermore, the addition of pterostilbene to the ESC culture treated with the classic decidualisation profile increases the expression of all the genes taken into consideration at decisively higher levels with respect to the simple culture or to ESCs treated with the decidualisation protocol in the absence of pterostilbene. Therefore, pterostilbene promotes the reaching of the decidualised phenotype in a shorter time (Figure 2).
To understand whether, in addition to influencing decidualisation, pterostilbene could also influence endometrial receptivity, the expression of the LIF marker was carried out over the course of the dose-response treatment and analysed with qPCR. As can be deduced from the data reported in Figure 3 the treatment of the ESCs with an increasing dose of pterostilbene generates significantly higher expression levels of LIF than the controls. Furthermore, the levels of LIF induced by pterostilbene are comparable to those in ESC in decidualisation (classic protocol). Furthermore, the expression levels of LIF were significantly higher in the samples where pterostilbene was added to ESCs treated with the classic decidualisation protocol (Figure 4) confirming a possible beneficial role in improving the receptivity of the endometrium during decidualisation.
In order to confirm the results obtained on gene regulation with qPCR methods, the presence and effective production of the final proteins were confirmed with immunoenzymatic methods of the ELISA type. As can be deduced from Figure 5, the quantification of the two proteins secreted into the medium displays a general trend comparable to the one observed on gene regulation with ratios and production levels that can be superimposed in relation to simple co-culture and ESCs subjected to decidualisation protocol. A general positive contribution of pterostilbene to the decidualisation protocol is noted again. This positive contribution can also be found in the case of the LIF endometrial receptivity marker (Figure 6) although there are marginal discrepancies in terms of time both between the treatments and on the times which can probably be attributed to the protein transcription kinetic specifications.
In order to confirm the results obtained with the ELISA method, samples deriving from the different treatments were analysed with the Immunoblot method. Based on the results obtained with qPCR only the samples treated for 6 days with pterostilbene [1 mM] and their related controls were evaluated with this method. As can be deduced from Figure 7, all three proteins considered display a significant increase in expression following the treatments.
The results of the immunohistochemistry staining are shown in Table III. The results relate to the count of 1000 cells per slide per field and expressed as a percentage of the average positive cell count of the antibody anti-PRL. As can be deduced from the results summarised in the table the percentages of positive cells decidualised increases if as well as the decidualisation protocol the ESCs are co-incubated with pterostilbene [1mM] Table III: immunohistochemistry (percentage of positive cells to the antibody anti-prolactin: the columns relate to the following: 1 ESC control; 2 ESC + pterostilbene [1mM]; 3 ESC + progesterone + cAMP; 4 ESC + progesterone + cAMP + pterostilbene [1mM]
Figure imgf000022_0001
Evaluation of the effects of pterostilbene on the outcomes of an in vitro fertilisation cycle
A pilot trial of the perspective kind was carried out on 20 patients waiting to undergo an in vitro fertilisation cycle (IVF/ICSI) for the exclusive male factor. For 3 months before the cycle the patients took pterostilbene or they took a placebo for 3 months (control group).
In the patients treated with pterostilbene it was administered at the dose of 125 mg twice a day. The patients were enrolled by gradually choosing 10 cases and 10 controls of the same age, in an age range between 18 and 40. All the patients were similar in terms of demographic characteristics (Table IV) and had regular cycles and no known female infertility factor. Exclusion criteria were: irregular cycles, presence of ovarian cysts, presence of endocrine/metabolic diseases or stage lll/IV endometriosis. The in vitro fertilisation cycle was performed in all the patients according to common clinical practice. The ovarian stimulation was performed with recombinant FSH (rFSH) starting from the 2nd day of the period, choosing a personalised daily dose based on age: 150 IU of rFSFI for patients aged <35, or 225 IU of rFSFI for patients > 35. Pituitary suppression was obtained with GnRFI antagonist, from the day on which the largest follicle reached 14 mm average diameter until the day of the rhCG. Follicular growth was monitored with transvaginal ultrasound on alternate days. Recombinant hCG (rhCG) was administered to induce final oocyte maturation when 3 or more follicles had a diameter >16 mm, exactly 35.5 hours prior to the oocyte recovery (pick-up) operation. No cycle was interrupted due to inadequate or excessive ovarian response. Pre implantation genetic screening (PGS) were carried out on the embryos. The embryos were then frozen pending the result of the genetic analysis, for subsequent possible transfer into the uterus.
Aims of the study
The primary aim of the study was to compare the number of oocytes recovered among the patients who had performed a pre-treatment with pterostilbene and the control patients.
Secondary aims were: number of growing follicles (diameter >10 mm) per patient
% of mature oocytes Mil per patient
% of fertilisations per patient number of high quality embryos (A or B) per patient
% of euploid embryos per patient
Results of the study
From the study it emerged that, in the group of patients who had taken pterostilbene for 3 months, the number of oocytes recovered and the number of growing follicles was greater with respect to the control group. Furthermore, also the % of mature oocytes, the percentage of fertilisations, the number of high quality embryos and the percentage of euploid embryos was greater in the study group with respect to the control group (Table V). Table IV. Demographic characteristics of the patients
Figure imgf000024_0001
Table V. Outcomes of the IVF cycle
Figure imgf000024_0002
Figure imgf000025_0001
Evaluation of the effects of pterostilbene on the ovarian reserve in sub- fertile patients A perspective pilot trial was performed on 30 patients affected by idiopathic infertility. The aim of the study was to look at the effects on ovarian reserve of a 12-month treatment with the study composition pterostilbene.
The patients were enrolled in an age range between 18 and 40. Exclusion criteria were: presence of known infertility factors (endocrine-ovulatory factor, reduced ovarian reserve, tubal factor, endometriosis), presence of ovarian cysts, presence of endocrine-metabolic diseases. The demographic characteristics of the patients are shown in Table VI.
The 30 patients in the study took pterostilbene for 12 months at the dosage of 100 mg twice a day. They were subjected to the ovarian reserve study through the dosage of serum AMH and the ultrasound antral follicle count (AFC) at time 0 and at time 12 months.
The ovarian reserve of these patients was compared with that of 300 control patients with similar demographic characteristics, who were not taking any therapy. The data related to the ovarian reserve of the control patients were collected in a perspective way by measuring the ovarian reserve in every patient twice after 12 months. It emerged that in 12 months the AMH undergoes a 0.2 ng/ml decline, whereas the AFC decreases by 2 follicles. Aims of the study
The primary aim of the study was to evaluate in the study patients:
• Reduction of AMH in 12 months
• Reduction of AFC in 12 months The secondary aim was to compare the reduction in ovarian reserve markers between the study patients and the control patients.
Results of the study
It emerged from the study that, after a 12-month treatment with pterostilbene, in the study patients, the AMH and AFC at time 0 and at time 12 months were almost unvaried (Table VII).
The comparison with the control population, where a reduction in the aforesaid markers in 12 months had been pointed out, therefore confirmed the beneficial effects of treatment with pterostilbene. Table VI. Demographic characteristics of the patients
Figure imgf000026_0001
Table VII. Ovarian reserve markers at 12 months
Figure imgf000026_0002
Figure imgf000027_0001

Claims

1. Pterostilbene for use in the treatment or prevention of infertility in an individual who is affected by infertility or a decline in fertility or who is at risk of suffering from infertility or a decline in fertility.
2. Pterostilbene for use according to claim 1 , wherein the treatment or prevention of infertility comprises increasing fertility or reducing the rate of decline in fertility or restoring fertility in an individual.
3. Pterostilbene for use according to claim 1 or 2, wherein the individual is affected by polycystic ovary syndrome (PCOS).
4. Pterostilbene for use according to any one of claims 1-3, wherein the individual has a normal ovarian reserve defined as a serum AMH level greater than 2 ng/ml, preferably greater than 2.5 ng/ml and/or defined as an AFC greater than 10, more preferably greater than 12.
5. Pterostilbene for use according to claim 1 or 2, wherein the individual has a reduced ovarian reserve defined as a serum AMH level of less than 2 ng/ml, preferably less than 1.5 ng/ml and/or defined as an AFC of less than 10, preferably less than 8.
6. Pterostilbene for use according to any one of claims 1-5, wherein the treatment or prevention of infertility comprises restoring the normal menstrual and ovulatory cycle, reducing the rate of decline in the antral follicle count (AFC), or reducing the rate of decline in the serum levels of the anti-Mullerian hormone (AMH), or increasing the decidualisation of the endometrium, or decreasing the rate of decline in oocytes, or increasing the number of oocytes, or increasing the pregnancy success rate, or preventing gestational diabetes or pre-eclampsia in a female subject who is suffering from a decline in fertility, or increasing the sperm count, or increasing sperm motility, or reducing the percentage of sperm DNA fragmentation in a male individual who is suffering from a decline in fertility.
7. Pterostilbene for use according to claim 6, wherein reducing the rate of decline in serum AMH levels comprises maintaining a reduction comprised between 0.05 and 0.1 ng/ml of AMH in 12 months, more preferably between 0.06 and 0.09 ng/ml of AMH in 12 months.
8. Pterostilbene for use according to claim 6 or 7, wherein reducing the rate of decline in AFC comprises maintaining a reduction comprised between 0.1 and 0.5 follicles in 12 months, more preferably between 0.2 and 0.4 follicles in 12 months.
9. Pterostilbene for use according to any one of claims 1-8, wherein increasing the decidualisation of the endometrium comprises increasing the levels of gene and/or protein expression of prolactin (PRL), of insulin like growth factor binding protein-1 (IGFBP-1) and of leukaemia inhibitory factor (LIF).
10. Pterostilbene for use according to any one of claims 1-9, wherein said pterostilbene is enterally administered, preferably orally administered.
11. Pterostilbene for use according to any one of claims 1-10, wherein said pterostilbene is administered in an amount comprised between 50 and 400 mg a day, more preferably between 70 and 250 mg a day, even more preferably comprised between 100 and 150 mg a day.
12. Pterostilbene for use according to any one of claims 1-11 , wherein the pterostilbene is administered for a period of time of at least 45 days, preferably for a period comprised between 50 and 365 days, more preferably comprised between 70 and 200 days.
13. Pterostilbene for use according to any one of claims 1-12, wherein the pterostilbene is administered in association or in combination with an infertility treatment in an individual, preferably in association or in combination with ovarian stimulation.
14. Pterostilbene for use according to any one of claims 1-13, wherein the pterostilbene is administered before the infertility treatment, preferably before ovarian stimulation.
15. Pterostilbene for use according to any one of claims 1-14, wherein the pterostilbene is administered in association or in combination with at least one drug for the treatment of infertility selected from: FSH, hMG, clomiphene citrate, an antioestrogen, an FSH analogue, a GnRH analogue, a dietary supplement, vitamin complexes, probiotics, paraprobiotics, coenzyme Q10, carnitine, d-chiro-inositol, myo-inositol, resveratrol and combinations thereof.
16. A composition for use according to any one of claims 1-15, wherein the composition comprises pterostilbene as the sole active ingredient.
17. An in vitro method for increasing the percentage of success of in vitro fertilisation, comprising at least one step of culturing a zygote or an embryo fertilised in vitro in a culture medium comprising pterostilbene.
18. An in vitro method according to claim 17, wherein a gamete used for the in vitro fertilisation has been drawn from an individual to whom pterostilbene has been administered for at least 3 months before collection of the gamete; preferably, pterostilbene has been administered to the individual at a dosage of 125 mg per day for at least 3 months before collection of the gamete.
PCT/IB2021/058009 2021-03-25 2021-09-02 Pterostilbene for use in the treatment or prevention of infertility WO2022200848A1 (en)

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WO2018200357A1 (en) * 2017-04-26 2018-11-01 Elysium Health, Inc. Methods and compositions of improving fertility
US20190083510A1 (en) * 2017-09-15 2019-03-21 City Of Hope Methods and compositions for treating endometrial cancer

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