WO2017197425A1

WO2017197425A1 - A method of treatment

Info

Publication number: WO2017197425A1
Application number: PCT/AU2016/050372
Authority: WO
Inventors: Justin Charles ST JOHN
Original assignee: Hudson Institute of Medical Research
Priority date: 2016-05-18
Filing date: 2016-05-18
Publication date: 2017-11-23

Abstract

The present disclosure relates to a method for facilitating embryogenesis potential via manipulation of a female mammalian germ cell, in particular an oocyte. Taught herein is a method for enhancing the potential of an oocyte fertilized in vitro to develop to term following implantation.

Description

A METHOD OF TREATMENT

BACKGROUND FIELD

[0001] The present disclosure relates to a method for facilitating embryogenesis potential via manipulation of a female mammalian germ cell, in particular an oocyte. Taught herein is a method for enhancing the potential of an oocyte fertilized in vitro to develop to term following implantation.

DESCRIPTION OF RELATED ART

[0002] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

[0003] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgement or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0004] An increasing number of women fail to achieve pregnancy through assisted reproduction technology due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Meiotically-arrested germ cells, or more particularly oocytes, develop in female gonads of mammalian species including humans. The oocytes are enclosed in primordial follides and are released during each menstrual cycle (Morita and Tilly (1999) Dev Biol 273: 1-17). Loss of oocytes reduces the level of fertility. There are only limited treatment regimes available for preserving fertility and normal ovarian function, especially under adverse conditions. In addition, there is reduced availability of healthy receptive oocytes for use in assisted reproduction protocols, for example, for women of advanced age, women undergoing pre-menopause or menopause and women with polycystic ovary syndrome. Poor respiratory activity mediated by mitochondria has been suggested as one possible cause of reduced oocyte competency (Spikings et al. (2007) Biol Reprod 7(5:327-335; El Shourbagy et al. (2006) Reproduction 737:233-245; Murakoshi et al. (2013) J Assist Reprod Genet 30: 1367-1375; May-Panloup et al. (2005) Hum Reprod 20:593-597; Santos et al. (2006) Fertil Steril 55:584-591; Reynier et al. (2001) Mol Hum Reprod 7:425-429; Xu et al. (2015) Scientific Reports 5: 10779; Boucret et al. (2015) Hum Reprod 30: 1653- 1664). Hence, insufficient mitochondrial activity in ova can result in eggs failing to fertilize or, if they do fertilize, the resulting embryo can exhibit an arrested development.

[0005] Mitochondria are organelles present in the cytoplasm of cells. Their main function is to generate energy in the form of ATP. Mitochondria possess DNA referred to as mitochondrial DNA or mtDNA. All mtDNA is inherited from the copies present in an ovum at the time of fertilization. mtDNA is a double-stranded circular genome that is approximately 16.6 kb in size and is located in the mitochondrial matrix (Andrews et al. (1999) Nat Genet 23: 147). It encodes 13 of the 80+ subunits of the electron transfer chain (ETC), which generates the vast majority of cellular ATP through oxidative phosphorylation (OXPHOS) [Schon et al. (2012) Nature Reviews Genetics 73:878-890; Rich (2003) Nature ¥27:583]. The remaining OXPHOS subunits are encoded by the chromosomal genome. The mitochondrial genome also encodes tRNAs and rRNAs and has one non-coding region, the D-Loop, which is the site of interaction for the nuclear- encoded transcription and replication factors that translocate to the mitochondrion to first drive mtDNA transcription then replication (Falkenberg et al. (2007) Annual Review of Biochemistry 7(5:679-699). As indicated above, cells possess multiple copies of mtDNA, which are inherited solely from the population present in the oocyte at fertilization and passed from generation to generation through the female germline (Shoubridge and Wai (2007) Curr Top Dev Biol 77:87-11). These copies of mtDNA are effectively identical leading to a state of homoplasmy. [0006] Supplementing oocytes with mitochondria or mitochondrial DNA (mtDNA) to overcome mtDNA deficiency and enhance developmental competence has not hither before met with significant success. Cytoplasmic transfer has been used to introduce extra mitochondria into the ovum at the time of fertilization. However, the source of the mitochondria has been eggs from younger, unrelated subjects. Hence, embryos comprise two genetically distinct populations of mtDNA, referred to as heteroplasmy. This can lead to the health and wellbeing of offspring being compromised by physiological abnormalities (Brenner et al. (2000) Fertil Steril 74:573-578; Acton et al. (2007) Biol Reprod 77:569- 576; Sharpley et al. (2012) Cell 757:333-343). In addition, experimental reduction of mtDNA copy number has been shown not to impair implanted embryo development in mice (Wai et al. (2010) Biol. Reprod. 53:52-62).

[0007] mtDNA disorders (McFarland et al. (2007) Curr Top Dev Biol 77: 113-155), include mtDNA deficiency syndromes that manifest in somatic tissues and organs and primarily affect cells that are highly dependent on OXPHOS for the generation of ATP (McFarland et al. (2007) supra). Maturing mammalian oocytes and developing embryos are not highly dependent on OXPHOS. Their mitochondria are structurally and functionally quiescent, and they likely derive most of their energy through alternative pathways, such as the adenosine salvage pathway (Scantland et al. (2014) Biol Reprod 97:75). They are also involved in a number of cellular functions including the sequestration and release of intracellular calcium. Furthermore, women harboring severe mtDNA mutations retain the capacity to be fertile (Gorman et al. (2015) N Engl J Med 372:885-887) hence the persistence of mild and severe forms of mtDNA disease (McFarland et al. (2007) supra).

[0008] Oocytes can be selected by various expression markers which change over time depending on developmental competency. Glucose-6-phosphate dehydrogenase (G6PD) is one such marker [Roca et al. (1998) Reprod Fertil Dev 70:479-485]. G6PD shows progressive down-regulation during normal oocyte growth and developmental. To this extent, the level of G6PD has been used in various mammalian species to assess developmental competence (Roca et al. (1998) supra; Rodriguez-Gonzalez et al. (2002) Theriogenology 57: 1397-1409). In addition, developmentally competent pig oocytes contain significantly higher levels of mtDNA copy number compared to less competent oocytes (Spikings et al. (2007) supra; El Shourbagy et al. (2006) supra). Indeed, the pig is an excellent model of oocyte and embryo development as it is very similar to that of the human (Humpherson et al. (2005) Theriogenology 64: 1852-1866; Bode et al. (2010) J Pharmacol Toxiol Methods (52: 196-220). In addition, mtDNA replication and reduction events have been mapped in porcine oocytes and embryos (Spikings et al. (2007) supra; El Shourbagy et al. (2006) supra).

[0009] There is a need to ensure harvested oocytes are of sufficiently high quality for successful fertilization in vitro and subsequent culture to blastocyst stage followed by implantation and embryo development to term. This is particularly important for assisted reproduction in female mammals of advanced maternal age and/or subjects with poor ovarian reserve or subjects with physiological issues such as polycystic ovary syndrome. It is also important for assisted reproduction of non-human animals.

SUMMARY

[0010] Successful fertilization and subsequent embryo development is influenced by the level of oocyte mitochondrial DNA (mtDNA) investment. Such an investment provides sufficient mtDNA at fertilization prior to embryonic genome activation or EGA. As taught herein, supplementation of oocytes prior to, simultaneously with or following fertilization with autologous mtDNA or mitochondria leads to the induction of early mtDNA replication events prior to EGA and enables the development of blastocysts which have a greater number of cells and which are more robust than blastocysts at the same stage which are derived from non-supplemented oocytes. In addition, the gene expression profiles match those of cells in blastocysts derived from developmentally competent oocytes. The present invention is predicated, therefore, on the use of autologous transfer of mtDNA or mitochondria into oocytes.

[0011] Accordingly, taught herein is a method for enhancing oocyte quality whilst maintaining genetic identity by introducing to an isolated oocyte, autologous mtDNA or autologous mitochondria. The term "genetic identity" includes "genetic integrity". In an embodiment, the oocytes are selected on the basis of biomarkers, mtDNA deficiency, reduced cytoplasmic maturation and/or volume and/or lower developmental competence. Alternatively, generally developmentally competent oocytes are selected. By "mtDNA deficiency" includes low copy number and/or poorer respiratory capacity compared to healthy controls. "Mitochondrial deficiency" has the same meaning. Supplementation of oocytes at fertilization with autologous mtDNA or autologous mitochondria increases developmental rates to blastocyst stage in vitro including blastocysts with a comparatively greater number of cells, the final stage of preimplantation development and promotes mtDNA replication prior to embryonic genome activation. Blastocysts exhibit transcriptome profiles which closely resemble those of blastocysts from developmentally competent oocytes. In addition, mtDNA or mitochondrial supplementation reduce gene expression patterns associated with metabolic disorders identified in blastocysts from mtDNA deficient oocytes. The use of autologous mtDNA or mitochondria avoids the risk of heteroplasmy. In an embodiment, the autologous mtDNA or mitochondria are derived from mature oocytes or immature oocytes or pre-cursors to oocytes, including from the oogenia.

[0012] Further taught herein is an association between poor mtDNA regulation at the blastocyst stage and aneuploidy, a state where there is an abnormal number of chromosomes in a cell. Following supplementation of oocytes with mtDNA or mitochondria the resulting blastocyst have more cells. mtDNA must be more efficiently regulated since as there are more cells, the supplemented blastocyst have fewer copies of mtDNA per cell, based on the ratio of cells to copy number. Hence, poor mtDNA regulation, i.e. more mtDNA copies per cell, is likely to lead to a state of aneuploidy.

[0013] Enabled herein is a method for enhancing fe male mammalian oocyte fertilization and embryo development capacity, the method comprising isolating a biological sample comprising an oocyte from a female mammalian subject, selecting an oocyte and supplementing the oocyte with autologous mtDNA or whole mitochondria prior to, simultaneous with or following fertilization of the oocyte. By "selecting" includes selecting a potentially poorly competent oocyte. A potentially poorly competent oocyte may be detected in any number of ways. One indicator, for example, is an oocyte which converts brilliant cresyl blue (BCB) to a colorless product due to the presence of glucose- 6-phosphate dehydrogenase (G6PD). Generally, a competent oocyte exhibits reduced to zero levels of G6PD. This enzyme converts BCB to the colorless product making the oocyte BCB^". Another indicator is reduced cytoplasmic maturation or volume. Any indicator of oocyte competency may be used to screen for potentially competent or non- competent oocytes. Whilst the present invention exemplifies the use of mtDNA or mitochondrial supplementation for developmentally poorly competent oocytes, the subject invention extends to the supplementation of developmentally competent oocytes with mtDNA or mitochondria. Reference to "fertilization" includes in vitro fertilization and intracytoplasmic sperm injection (ICSI). It is proposed herein that mtDNA or mitochondrial supplementation promotes mtDNA replication at fertilization and prior to embryonic genome activation. This ensures the resultant blastocysts have increased cell numbers and gene expression profiles in cells that are associated with blastocysts from developmentally competent oocytes. Blastocysts from supplemented oocytes based on the ratio of cells to copy number of mtDNA may have lower overall copy number hence indicative of efficient mtDNA regulatory potential. As indicated above, autologous mtDNA or mitochondria may be derived from autologous mature oocytes, immature oocytes and/or oogenia. The mtDNA/mitochondria may be sourced from either developmentally competent or non-competent oocytes.

[0014] In an embodiment, the female mammalian subject is a human female. Alternatively, the female mammalian subject is a non-human female mammal. Conveniently, the mtDNA or mitochondria are introduced to the oocyte simultaneously with a sperm.

[0015] Generally, the fertilized oocyte is cultured in vitro for a time and under conditions for a blastocyst to develop which is then implanted into the uterus of the same female mammalian subject as the donor of the oocyte. Generally, the blastocysts have comparatively more cells compared to blastocysts at the same stage derived from a developmentally competent oocyte or a mtDNA/mitochondria deficient oocyte neither of which is subject to supplementation. The blastocyst may alternatively be implanted into a non-autologous female mammalian subject of the same species. Hence, aspects of the present invention extend to surrogacy. In a further alternative, a preimplantation embryo or up to and including the blastocyst is subject to freeze storage for subsequent use.

[0016] In an embodiment, the implanted blastocyst enables development of an embryo to term. Usefully, the mtDNA to be introduced to an oocyte is first screened for DNA mutations or copy number or donor cells comprising mitochondria or isolated mitochondria are first screened for respiration ability wherein mitochondria with normal to enhanced respiration capability compared to a control are selected for transfer to the oocyte.

[0017] Another aspect enabled herein is a method for facilitating fertilization and developmental competency of a mammalian oocyte, the method comprising supplementing an isolated oocyte with autologous mtDNA or autologous mitochondria to enable early mtDNA replication at fertilization with a sperm or following intracytoplasmic sperm injection (ICSI) and culturing the fertilized oocyte in vitro to blastocyst stage such that the blastocyst comprises cells with a gene expression profile associated with the gene expression profile of a developmentally competent oocyte and appropriate numbers of mtDNA copy and increased numbers of cells in the blastocysts stage embryo.

[0018] Further enabled herein is the use of autologous mtDNA or autologous mitochondria in combination with a sperm in the supplementation of an oocyte which is then capable of forming a blastocyst to be implanted to a female mammalian host. Contemplated herein is mtDNA or mitochondria and a sperm for use in fertilizing an oocyte capable of being cultured to blastocyst stage and then to be implanted to a female mammalian host. In an embodiment, the oocyte is selected on the basis of being potentially developmentally incompetent or poorly competent. In an embodiment, mtDNA or mitochondria are introduced to an oocyte to promote mtDNA replication at fertilization and prior to embryonic genome activation. In an embodiment, mtDNA replication leads to gene expression patterns at blastocyst stage associated with blastocysts from developmentally competent oocytes. In another embodiment, oocytes are supplemented with autologous mtDNA or autologous mitochondria regardless of their level of competency or non- competency.

[0019] The present invention represents an improvement to assisted reproduction technology. In the method of fertilization in vitro of an oocyte, culturing the fertilized oocyte to blastocyst stage and implanting the blastocyst in a female mammalian host, the improvement comprising supplementing the oocyte with autologous mtDNA or autologous mitochondria prior to, simultaneously with or following fertilization with a sperm. This allows mtDNA replication prior to embryonic genome activation and gene expression patterns at the blastocyst stage associated with healthy embryos. Blastocysts also have comparatively more cells than blastocysts at the same stage derived from developmentally competent oocytes or oocytes deficient in mtDNA or mitochondria neither of which is subject to mtDNA/mitochondrial supplementation. [0020] Abbreviations used herein are defined in Table 1.

[0021] Primer sequences are referred to by a sequence identifier number (SEQ ID NO). The SEQ ID NOs correspond numerically to the sequence identifiers <400>1 (SEQ ID NO: l), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 12. A sequence listing is provided after the claims.

BRIEF DESCRIPTION OF THE FIGURES

[0022] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0023] Figures la through lc are graphical representations showing mean (± SEM) mtDNA copy number in maturing and mature oocytes determined by real time PCR. (a) BCB⁺ and (b) BCB^" maturing (0 hour and 22 hours) and immature (44 hours) and mature (44 hours) oocytes (n=10 for each). Statistical analysis was performed using ANOVA. (c) Comparison between BCB⁺ and BCB^" Mil oocytes performed by t-test.

[0024] Figures 2a through 2d are photographic representations showing generation of mICSI-derived blastocysts, (a) Localization of mitochondria following injection into BCB^" Mil oocytes at 1 hour and 24 hours post insemination. Mitochondria endogenous to the oocyte are labeled with MitoTracker Deep Red (grey). The injected mitochondria are labeled with TMRM (red) and MitoTracker Green (green). The confocal z-stacks are displayed as maximum intensity projections (X40 magnification), (b) Transmission Electron Microscopy of Mil oocytes, ICSI-1 hour, ICSI-24 hours, mICSI-1 hour, and mICSI-24 hour at 2500X, 5000X and 25,000X magnification. Abbreviations include; LD=lipid droplet, V=vacuole, M=mitochondria. (c) DAPI staining of ICSI-BCB⁺ and mICSI-BCB^" blastocysts highlighting the presence of an inner cell mass, (d) Enlargement of a mICSI-BCB^" blastocyst.

[0025] Figures 3a through 3e are graphical representations of mtDNA copy number for preimplantation embryos, (a) Mean (± SEM) mtDNA copy number for BCB⁺ IVF; (b) ICSI-BCB⁺; (c) ICSI-BCB^"; and (d) mICSI-BCB^"-derived embryos determined by real time PCR (n = 5-10). (e) Mean (± SEM) mtDNA copy for each stage of development for embryos generated by IVF, ICSI and mICSI from BCB⁺ and BCB^" oocytes. [0026] Figures 4a through 4c are photographic representations of global gene expression analysis of single blastocysts following microarray. (a) Principal component analysis plot of microarray data from single blastocysts. Red, blue and brown points represent individual transcriptomes from ICSI-BCB⁺, ICSI-BCB^" and mICSI-BCB^" blastocysts, respectively, (b) Heat map of global gene expression following Pearson's correlation to determine hierarchical clustering between blastocysts of each group, (c) Venn diagram representing differentially expressed genes between ICSI-BCB⁺ and ICSI-BCB^"; mlCSI- BCB^" and ICSI-BCB^"; and mICSI-BCB^" and ICSI-BCB⁺ blastocysts following unpaired t- tests, with FC>2 (abs) and significance of p<0.01.

[0027] Figure 5 is a graphical representation of O₂ consumption rates for mitochondrial extracts isolated from BCB⁺ and BCB^" oocytes after 44 hours of IVM. Mitochondrial extracts were subjected to 5 mM succinate, followed by ADP and increased FCCP concentrations (50 mM increments). Respiration was abolished by addition of Antimycin A.

[0028] Figures 6a through 6c are photographic representations of shape analysis of mitochondrial clusters, a) BCB^" and BCB⁺ Mil oocytes are stained with MitoTracker Red. b) Region of interests (ROIs) were thresholded and binarized. Cluster area, perimeter and circularity were chosen as parameters for characterization, c) Cluster analysis was performed using two-tailed t-test.

[0029] Figures 7a through 7e are graphical representations of normalized microarray intensity values for genes associated with blastocyst development for ICSI-BCB⁺, ICSI- BCB^" and mICSI-BCB⁺ derived blastocysts, a) pluripotency; b) epigenetic reprogramming; c and d) energy metabolism; and e) microRNA genes.

[0030] Figures 8a through 8h are graphical representations of relative gene expression for early regulators of development in blastocysts derived from IVF, ICSI and mICSI. Real-time RT-PCR values were normalized to ACT-B values and compared to IVF- blastocysts as the reference point. IVF and ICSI-BCB⁺ values represent the mean (± SEM) of gene expression levels from 2 pools of 5 blastocysts each. Values for the mICSI-BCB^" group represent gene expression from one pool of 5 blastocysts each.

[0031] Figure 9 is a graphical representation of litter size following the generation of founder mice through mICSI. The founder offspring were generated using ovulated oocytes that were not selected for mtDNA deficiency. Data from the first generation consist of values for 5 parities from each of the individuals. The second and third generations were generated from the daughters of the first and second generations, respectively. Colony values represent litter size from 93 pregnancies from mice based in the same facility as the founders were generated and the pregnancies were established through natural matings. * = P<0.05, ** = P<0.01.

DETAILED DESCRIPTION

[0032] 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 method step or group of elements or integers or method steps but not the exclusion of any other element or integer or method steps or group of elements or integers or method steps.

[0033] As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a mitochondrion" includes a single mitochondrion, as well as two or more mitochondria; reference to "an oocyte" includes a single oocyte, as well as two or more oocytes; reference to "the disclosure" includes a single and multiple aspects taught by the disclosure; and so forth. Aspects taught and enabled herein are encompassed by the term "invention". All such aspects are enabled within the width of the present invention. Any variants and derivatives contemplated herein are encompassed by "forms" of the invention.

[0034] The present invention is predicated in part on the determination that autologous mitochondrial DNA supplementation or the equivalent in mitochondria of an oocyte remarkably improves the overall health of the oocyte leading to enhanced fertilization and subsequent developmental competence and blastocyst quality including blastocysts with more cells compared to blastocysts at the same stage derived from developmentally competent oocytes or mtDNA- or mitochondria-deficient oocytes neither of which is subject to supplementation. This in turn improves the likelihood of embryo development to term.

[0035] Whilst not intending to limit the present invention to any one theory or mode of action, it highlights the importance of providing a sufficient amount of mtDNA to induce early mtDNA replication events at fertilization, particularly but not exclusively in mtDNA deficient embryos, to thereby enhance embryo quality by stabilizing the embryo prior to embryonic genome activation (EGA). In particular, the supplementation of mtDNA or mitochondria enables early mtDNA replication at fertilization prior to EGA resulting in blastocysts having increased cell numbers and enhanced gene expression profiles or patterns that are associated with blastocysts and embryos from healthy, competent oocytes. Interestingly, as a ratio of cells to copy number of the mtDNA in the blastocyst, there is an overall lower copy number per cell. However, this is indicative of efficient mtDNA regulation.

[0036] Accordingly, taught herein is a method of enhancing the fertilization competency of an oocyte, the method comprising introducing into the oocyte, autologous mtDNA or autologous mitochondria. In a related embodiment, a method is enabled herein for enhancing the developmental competency of a fertilized oocyte or an oocyte to be fertilized, the method comprising introducing into the oocyte prior to, simultaneous with, or subsequent to fertilization, autologous mtDNA or autologous mitochondria. In a further related embodiment, the present specification contemplates a method for facilitating the developmental competency and blastocyst quality of a fertilized oocyte, the method comprising introducing into the oocyte autologous mtDNA or autologous mitochondria. Enabled herein is a method for enhancing female mammalian oocyte fertilization and embryo development capacity, the method comprising isolating a sample comprising an oocyte from a female mammalian subject, selecting an oocyte and supplementing the oocyte with autologous mtDNA or whole mitochondria prior to, simultaneous with or following fertilization of the oocyte. In an embodiment the oocyte is selected on the basis of being a potentially poorly competent oocyte. Alternatively, any oocyte, competent or otherwise, is subject to mtDNA or mitochondrial supplementation. In an embodiment, this leads to early mtDNA replication at fertilization and prior to EGA. In an embodiment, mtDNA or mitochondrial supplementation leads to gene expression profiles at the blastocyst stage which are associated with healthy blastocysts from developmentally competent oocytes. In an embodiment, mtDNA- or mitochondria-supplemented oocytes go on to develop blastocysts comprising more cells than blastocysts at the same stage derived from either developmentally competent oocytes or inferior or less competent oocytes neither of which is subject to mtDNA/mitochondrial supplementation. [0037] In an embodiment, supplementation of oocytes with mtDNA or mitochondria leads to a sufficient amount of mtDNA being provided. This in turn can enable early replication of mtDNA at fertilization.

[0038] The term "oocyte" refers to a cell in a mammalian ovary which may undergo meiotic division to form an ovum. It is a cell from which an egg or ovum develops by meiosis. The term "female gametocyte" may also be used to describe the oocyte. The oocyte disclosed herein may be derived from any mammalian source including a human, non-human primate, a laboratory test animal such as a mouse, rat, rabbit, guinea pig or hamster or a larger animal such as a pig, horse, sheep, cow or camel. A precursor of an oocyte may also be isolated and cultured in the presence of cytokines, hormones and/or growth factors for a time and under conditions sufficient for it to develop into an oocyte.

[0039] In an embodiment, the oocyte is of human origin. In another embodiment, the oocyte is of non-human origin. Hence, the method of enhancing oocyte fertilization and developmental competency enabled herein has application to fertilization in vitro and subsequent implantation of human embryos as well as non-human embryos in veterinary applications.

[0040] In an embodiment, the oocyte is selected on the basis of being deficient in mtDNA or mitochondria. This can be determined directly or indirectly via markers or indicators. Such an mtDNA deficient oocyte is then subject to autologous mtDNA or mitochondrial supplementation prior to, simultaneous with or following fertilization by a donor sperm. The fertilized mtDNA or mitochondrial supplemented oocyte is then implanted to permit blastocyst and subsequent embryo development. Supplemented blastocysts comprise more cells than blastocysts at the same stage from non-supplemented oocytes whether developmentally competent or non-competent. Reference to "stage" means days post- fertilization. Alternatively, the in vitro developed blastocyst is freeze stored for later use. Whilst the present invention includes the selection of mtDNA deficient oocytes, it does not exclude the use of oocytes which do not exhibit mtDNA deficiency. Hence, once an oocyte is isolated, whether or not it exhibits mtDNA deficiency, it may be supplemented with autologous mtDNA or autologous mitochondria prior to, simultaneous with or following fertilization. Critically, the supplementation step ensures or enables early mtDNA replication at fertilization and prior to EGA. In an embodiment, mtDNA or mitochondrial supplementation leads to gene expression profiles at blastocyst stage associated with blastocysts from developmentally competent oocytes.

[0041] The determination of mtDNA deficiency or otherwise may be conducted in a number of ways. Conveniently, an oocyte is tested with brilliant cresyl blue (BCB), a nontoxic dye that is reduced to a colorless compound by glucose-6-phosphate dehydrogenase (G6PD). During oocyte growth, G6PD is progressively down-regulated, indicative of oocyte competency. Hence, a developmentally competent oocyte stains BCB⁺. A developmentally incompetent oocyte is BCB^" as the G6PD has not been down-regulated. Reference to a "mtDNA deficient oocyte" is one which stains BCB^". Alternatively, an oocyte is screened for reduced cytoplasmic maturation or volume where a reduced maturation level or volume compared to a control is an indicator of non-competency or potential non-competency.

[0042] Enabled herein is a method for enhancing the fertilization and developmental competency of a mammalian oocyte, the method comprising isolating an oocyte from a mammal, ascertaining whether the oocyte is mtDNA deficient wherein if it is mtDNA deficient, introducing to the oocyte prior to, simultaneous with or following fertilization, an effective amount of autologous mtDNA or autologous mitochondria. As indicated above, if an oocyte is determined not to be mtDNA deficient, it may nevertheless still be supplemented with autologous mtDNA or autologous mitochondria. As indicated above, a mtDNA deficient oocyte includes one which is BCB^". A mtDNA competent oocyte is BCB⁺. Alternatively, another indicator of oocyte competency or non-competency is employed such as level of cytoplasmic maturation or volume. Yet in a further alternative, no screening step is employed and an isolated oocyte is supplemented with mtDNA or mitochondria. In an embodiment, the mammalian oocyte is a human oocyte. This is shown, for instance, in Example 8 (and Figure 9). In another embodiment, the oocyte is a non-human mammalian oocyte. The fertilized oocyte is then cultured in vitro to the blastocyst stage and then implanted into the same female mammal from which the oocyte was isolated or a surrogate female mammal of the same species. Alternatively, the blastocyst may be subject to freeze storage.

[0043] Still a further embodiment enabled herein is a method for facilitating fertilization and developmental competency of a mammalian oocyte, the method comprising supplementing an isolated oocyte with autologous mtDNA or autologous mitochondria to enable early mtDNA replication at fertilization with a sperm or following intracytoplasmic sperm injection (ICSI) and culturing the fertilized oocyte in vitro to blastocyst stage such that the blastocyst comprises cells with a gene expression profile associated with the gene expression profile of a developmentally competent oocyte. In an embodiment, mtDNA replication occurs prior to EGA. In an embodiment, the mammalian oocyte is a human oocyte. In an embodiment, it is a non-human oocyte.

[0044] The term "autologous" as used herein refers to mtDNA obtained from cells of the same female subject from which the oocyte is isolated. Hence, the mtDNA is autologous to the oocyte which, after fertilization in vitro and autologous mtDNA supplementation, is implanted into the female mammal, e.g. human or non-human mammal, from which it was isolated or into a surrogate female mammal. The term "autologous mitochondria" has in effect the same meaning as autologous mtDNA. Hence, the oocyte may be supplemented with either isolated mtDNA or mitochondria comprising same. Whilst a mature oocyte is a useful source for autologous mtDNA or mitochondria, the use of immature oocytes or precursors to oocytes (oogenia) obviates the need to use mature oocytes.

[0045] The term "isolated" refers to the oocyte, mtDNA or mitochondria which has been physically separated or removed from its natural biological environment. An isolated oocyte, mtDNA or mitochondria need not be purified. A sample comprising an oocyte is referred to as a "biological sample".

[0046] In an embodiment, the mtDNA or mitochondria sample is derived from another oocyte or a precursor to an oocyte (i.e. from oogenia) autologous to the fertilized oocyte which results in maintaining mtDNA genetic integrity and identity. The mtDNA or mitochondria is isolated from another oocyte or its precursor whether deemed genetically competent or not.

[0047] The female mammal may be referred to as a subject, patient, person, animal, individual or implantation recipient or other such term to indicate the mammalian source of the oocyte and to which the fertilized oocyte is to be implanted. The subject may be a human or non-human mammal. The subject may also be a surrogate mammal of the same species.

[0048] Reference to a "mitochondrion" or the plural form "mitochondria" encompasses a functional entity meaning that it can produce ATP. A functional mitochondrion or functional mitochondria means a respiring mitochondrion or mitochondria.

[0049] The present invention provides in an embodiment, a method of fertilization in vitro and subsequent in vitro culture to blastocyst stage and subsequently full embryo development in vivo. The method comprises in non-limiting order:

(i) selecting a mammalian female recipient;

(ii) isolating an oocyte from the mammalian female subject;

(iii) isolating of mitochondria or mtDNA from a cell autologous to the source of the oocyte;

(v) supplementing the oocyte with autologous mtDNA or autologous mitochondria prior to, simultaneous with or following fertilization and culturing the fertilized oocyte to blastocyst stage in vitro; and

(vi) implanting the fertilized and supplemented oocyte in the mammalian female recipient. [0050] In an embodiment, the female mammalian recipient is a human subject. [0051] Hence, taught herein is a method comprising in non-limiting order:

(i) selecting a human female recipient;

(ii) isolating an oocyte from the human female subject;

(vi) implanting the fertilized and supplemented oocyte in the human female recipient. [0052] In an embodiment, the female mammalian recipient is a non -human subject. [0053] Accordingly, contemplated herein is a method comprising in non-limiting order;

(i) selecting a non-human female recipient;

(ii) isolating an oocyte from the non-human female subject;

(vi) implanting the fertilized and supplemented oocyte in the non-human female recipient.

[0054] In an alternative to these aspects, following fertilization and in vitro culture the preimplantation embryo up to blastocyst stage is freeze stored. In these embodiments, the source of mtDNA or mitochondria may be from a mature, competent oocyte or a precursor cell to an oocyte which includes an immature oocyte and a developmentally non- competent oocyte. Reference to a "blastocyst" includes a blastocyst with more cells, including more robust, i.e. healthy cells, compared to a blastocyst at the same stage derived from a non-supplemented oocyte.

[0055] These aspects may additionally comprise a step ii(a) whereby an oocyte is screened for being potentially developmentally competent or non-competent. This may be achieved in any number of ways such as the BCB assay (where a BCB⁺ oocyte is deemed competent and a BCB^" oocyte is deemed developmentally non-competent) or by measuring the level of cytoplasmic maturation or volume (where an oocyte is compared to an oocyte having a level of cytoplasmic maturation or volume of a competent or non-competent oocyte). At step v, mtDNA or mitochondrial supplementation permits early mtDNA replication at fertilization and prior to EGA. At blastocyst stage, cells comprise a gene expression profile associated with a developmentally competent oocyte.

[0056] Oocytes can be isolated by any convenient means from adult ovarian tissues. Methods for the preparation and transfer of mtDNA or mitochondria and transfer of mtDNA or mitochondria are also standard in the art. See, for instance, the "methods" in the Example section herein or using comparable methods disclosed in Perez et al. (2007) Cell Death and Differentiation 3:524-533 and Perez et al. (2000) Nature 403:500-501. In an embodiment, oocytes are subject to sorting based on being BCB⁺ (competent) or BCB^" (non-competent). The oocytes are subject to fertilization in vitro including intracytoplasmic sperm injection (ICSI) generally, but not necessarily simultaneously with mtDNA or mitochondrial supplementation. mtDNA or mitochondria are in an embodiment, isolated from any autologous oocyte or its precursor form or from an in vitro matured metaphase II BCB⁺ oocyte. Alternatively, a developmentally competent or non- competent oocyte is determined based on cytoplasmic maturation or volume level or other indicator. Yet in a further alternative, the oocytes are supplemented with mtDNA or mitochondria regardless of their state or level of competency. The mtDNA or mitochondria are then introduced into the oocyte prior to, simultaneously with or following sperm fertilization or injection. This leads to early mtDNA replication at fertilization. Conveniently, the sperm and mtDN A/mitochondria are introduced simultaneously by injection. Fertilized oocytes are then cultured in vitro to blastocyst stage and comprise cells with a gene expression profile associated with a healthy oocyte. The blastocysts have comparatively more cells than blastocysts at the same stage derived from oocytes not subject to mtDNA or mitochondrial supplementation. Preimplantation stage embryos upto and including blastocyst stage may be cryopreserved. Otherwise, they are implanted at the appropriate age.

[0057] The oocytes may be retrieved by any of a number of techniques. The mammalian female subject may first be subject to ovarian hyperstimulation to generate multiple follicles of the ovaries. Factors affecting predicted response include age, overall health of the subject, antra follicle count and/or level of anti-Mullerian hormone. The hyperstimulation may also include a suppression step to prevent spontaneous ovulation such as using the GnRH agonist protocol or GnRH antagonist protocol. The oocytes may also first be cryopreserved (freeze stored). Upon reaching appropriate development of ovarian follicles, final oocyte maturation is induced such as by infection with chronic gonadotropin. Transvaginal oocyte retrieval is a convenient protocol to retrieve oocytes which uses an ultrasound-guided needle piercing the vaginal wall to reach the ovaries. Through this needle, follicles are aspirated. The oocytes are isolated and, in one embodiment, subject to BCB⁺/BCB^" determination. BCB⁺ oocytes can be used as a source of mtDNA or mitochondria. BCB^" oocytes are then used for mtDNA or mitochondria supplementation prior to, simultaneous with or following fertilization in vitro. As previously indicated, any test for oocyte competency or non-competency or potential competency or non-competency may be employed. Another test is cytoplasmic maturation or volume. Yet a further test is respiration levels. Potentially competent oocytes are one source of mtDNA or mitochondria. Potentially non-competent oocytes are selected for mtDN A/mitochondria supplementation. In an alternative embodiment, oocytes are not selected for either competency or non-competency and are nevertheless supplemented with autologous mtDNA or autologous mitochondria. In relation to the latter, sperm are collected and subject to sperm washing to remove seminal fluid and unwanted cellular material. The sperm may be pre-treated and also may be cryopreserved prior to use.

[0058] In an embodiment, the sperm and oocyte are co-incubated together. The ratio of sperm to oocyte may depend on mammalian species. For humans, a ratio of 75,000: 1 (sperm: oocyte) is generally employed. mtDNA or mitochondria are introduced at this time or prior to or following fertilization. A single sperm and mtDNA or mitochondria may also be introduced by intracytoplasmic sperm injection (ICSI). The fertilizing oocyte is then incubated for 24-72 hours (e.g. 58 hours). The oocyte is then allowed to develop to blastocyst stage as an embryo. Fertilized embryos may then be cryopreserved or transferred to the autologous host or a surrogate recipient. Oocyte competency may be determined by other means such as cytoplasmic maturation or volume. There is no requirement, however, to determine the state or level of competency in order to supplement oocytes with mtDNA or mitochondria.

[0059] Further enabled herein is a method for fertilization in vitro of a mammalian oocyte, the method comprising supplementing an isolated oocyte with autologous mtDNA or autologous mitochondria prior to, simultaneous with or following fertilization to enable early mtDNA replication and subsequent developmental in vitro to blastocyst stage wherein the blastocyst comprises cells having a gene expression pattern associated with a developmentally competent embryo. The blastocysts comprise comparatively more cells than blastocysts at the same stage derived from oocytes which have not been supplemented with mtDNA or mitochondria, whether they are competent or non-competent oocytes.

[0060] Enabled herein is a method for fertilization in vitro of a mammalian oocyte, the method comprising selecting an oocyte which is BCB^" and supplementing the BCB^" oocyte which autologous mtDNA or mitochondria prior to, simultaneous with or following fertilization with a sperm. Other indicators of potential oocyte competency or non- competency may also be employed (e.g. cytoplasmic maturation or volume or level of respiration). In an alternative method, taught here is a method for fertilization in vitro of a mammalian oocyte, the method comprising supplementing the oocyte which autologous mtDNA or mitochondria prior to, simultaneous with or following fertilization with a sperm. Reference to a "mammalian oocyte" includes a human oocyte or an oocyte or egg equivalent from a non-human mammal.

[0061] In an embodiment, the fertilized oocyte is cultured to blastocyst stage then either cryopreserved or implanted into a mammalian female recipient either of the same species or in the same subject from which the oocyte was isolated. Alternatively, preimplantation embryos to blastocyst stage may be cryopreserved. Blastocysts derived following mtDNA or mitochondrial supplementation have comparatively more cells than blastocysts at the same stage derived from less competent oocytes.

[0062] In an embodiment, the oocyte is a human oocyte. In an embodiment, the oocyte is a non-human oocyte.

[0063] Reference to "fertilization in vitro" includes standard in vitro fertilization as well as intracytoplasmic sperm injection (ICSI). Cytoplasmic transfer may also be carried out on the basis that an oocyte is supplemented with autologous mtDNA or mitochondria.

[0064] The present invention has capacity to improve successful pregnancy rates for human women especially those predisposed to generating a higher proportion of poorly competent oocytes such as pre-menopausal women, post-menopausal women, women at advance maternal age or women which polycystic ovary syndrome. The present invention also has application in veterinary fields in controlled or mass breeding of farm animals such as pigs, sheep, cattle or horses. [0065] Following isolation of mtDNA or mitochondria, integrity of the mtDNA (e.g. presence of mutations) or functionality of the mitochondria (e.g. ATP production or respiration potential) can be assessed by known methods (e.g. Duran et al. (2011) Fertility and Sterility 96(2):284-388; Chen et al. (2011) BMC Medical Genetics 72:8).

[0066] Mitochondria functionality can be assessed, for example, by respiratory analysis such as by high-resolution respirometery.

[0067] The percentage of mutations in the mtDNA from a population of mitochondria can be assessed by first determining the number of mitochondria present in a biological sample and next, determining the copy number of mtDNA present in the sample. Standard mutation analysis can be employed and compared to the number of mitochondria and copy number of mtDNA to calculate the percentage of mutations in the population of mitochondria.

[0068] The material to be injected (e.g., mtDNA or mitochondrial suspension) is transferred to a microinjection needle according to methods known in the art. Microinjection needles and holding pipettes can be made using a Sutter pipette (Sutter Instruments, Novato, Calif, USA) and a De Fonbrune Microforge (EB Sciences, East Granby, Conn., USA) or a pipette from The Pipette Company, Australia. The microinjection needles have inner diameters of about 5-6 μm with blunt tips. The material to be injected is aspirated into the needle by negative suction. Between 1 -10 pi of mitochondrial isolate is injected into the oocyte which includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 pl of mitochondrial isolate (e.g. 3 pi).

[0069] Methods for the preparation and transfer of nuclear-free cytoplasmic fractions are known in the art. For example, reference can be made to Cohen et al. (1998) Mol Hum Reprod 4:269-80. Briefly, in one method, approximately 4 hours after oocyte retrieval, recipient oocytes are exposed to 0.1% w/v hyaluronidase, and oocytes are selected for injection. All corona cells are removed with fine bore pipettes. After exposure to 0.1 % w/v hyaluronidase, zonae are opened mechanically using a microspear. Oocytes of various sizes are separated from the aspirate. Oocytes that survive the procedure are transferred for culture and optionally, assessment or cryopreservation prior to in vitro fertilization or intrauterine insemination.

[0070] Alternatively, conventional intracytoplasmic sperm injection (ICSI) methods can be employed in connection with the transfer of nuclear-free cytoplasmic fractions or isolated mitochondria. Reference may be made to Cohen et al. (1998) supra. As one example, the zonae of the recipient oocytes are opened mechanically over the polar body area using a microspear. The polar body is removed after re-positioning the oocyte on the holding pipette in such a way that the zona can be dissected using the closed microspear. The same position is used to insert the ooplast, which had contained the polar body. The zona is closed tight using the same tool. Spermatozoa are immobilized in 10% v/v polyvinylpyrrolidone (PVP) for ICSI. Methods of fertilization in vitro are well known in the art.

[0071] Individual oocytes can be evaluated morphologically and transferred to a petri dish containing culture media. A sample of sperm is provided and processed using a "swim up" procedure, whereby the most active, motile sperm will be obtained for insemination. If the female's oviducts are present, gamete intrafallopian transfer can be performed at this time. By this approach, oocyte-cumulus complexes surrounded by sperm are placed directly into the oviducts by laparoscopy. This procedure best simulates the normal sequences of events and permits fertilization to occur within the oviducts.

[0072] The present specification further contemplates kits for undertaking oocyte retrieval, mtDN A/mitochondria retrieval and for fertilization and mtDNA or mitochondrial supplementation in vitro. The kits may also be provided with instructions for use and/or with reagents to test the functionality of mitochondria or the integrity of mtDNA.

[0073] Further enabled herein is an ability to genetically modify a non-human mammal by genetically altering or selecting an oocyte or sperm, subjecting the oocyte to fertilization in vitro prior to, simultaneously with or following mtDNA or mitochondrial supplementation, culturing the fertilized oocyte in vitro to blastocyst stage, optionally screening the blastocyst for expression of a genetic trait and either implanting the resulting embryo for development to term or cryopreserving the embryo. Variations of this method may also be employed to genetically correct mutations in a human oocyte or sperm.

[0074] The present invention represents an improvement to assisted reproduction technology. Accordingly, in the method of fertilization in vitro of an oocyte by the steps of fertilizing the oocyte, culturing the fertilized oocyte to blastocyst stage and implanting the blastocyst in a female mammalian host, the improvement comprising supplementing the oocyte with autologous mtDNA or autologous mitochondria prior to, simultaneously with or following fertilization with a sperm. In an embodiment, the mammalian host is a human. In general, blastocysts derived following mtDNA or mitochondrial supplementation have either more cells than blastocysts at the same stage derived from oocytes not subject to mtDNA or mitochondrial supplementation.

EXAMPLES

[0075] Aspects disclosed herein are further described by the following non-limiting Examples. Some aspects disclosed herein are published in Cagnone et al. (2016) Scientific Reports (5:23229; doi: 10.1038/srep23229 (2016), the entire contents of which are incorporated herein by reference.

Methods

Chemicals

[0076] All chemicals were obtained from Sigma, unless states otherwise. BCB staining IVM and embryo culture

[0077] Pig ovaries were collected from a local abattoir. They were washed and maintained in PBS at 37-38°C. Cumulus-oocyte complexes (COCs) were aspirated from the ovaries using a syringe with an 18G needle (BD) containing around 1 ml warm flush medium (ViGRO, Bioniche Australia). After washing in pre-equilibrated in in vitro maturation (IVM) medium consisting of TCM 199, 0.1% v/v polyvinyl alcohol, 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 10 ng/mL EGF, 10 IU/mL LH (Chorulon, Intervet DE), 10 IU/mL FSH (Folligon, Intervet, DE) and 50 μg/mL penicillin/streptomycin, COCs were stained with 12 μΜ BCB in IVM medium for 60 mins at 39°C, 5% v/v C0₂ at maximum humidity. COCs were then washed in warm flush medium containing the supplements listed for IVM medium. BCB⁺ and BCB^" COCs were isolated using a stereomicroscope. After sorting, COCs were plated (50 per well) into pre- equilibrated IVM medium and incubated for 44 hours at 39°C, 5% v/v C0₂ at maximum humidity.

[0078] After IVM, COCs underwent one of IVF, ICSI or parthenogenic activation followed by in vitro culture in Porcine Zygote Medium (PZM; saline solution containing 0.20 mM sodium pyruvate, 2 mM calcium lactate, 1 mM L-glutamine, 5 mM hypotaurine, basal medium eagle and nonessential amino-acids, 0.05 mg/ml penicillin/streptomycin and 0.3% w/v BSA). For IVF, 50 COCs were plated in a 4-well plate containing pre- equilibrated mTBM solution (Tris Buffer Medium 20 with 5 mM sodium pyruvate, 0.02 mM fresh adenosine, 0.2 mM fresh L-glutathione and 0.1 % w/v BSA) and incubated with 90% w/v Percoll-pelleted spermatozoa (0.5x10⁶) for 4 hours at 39°C, 5% v/v CO₂ at maximum humidity.

[0079] For intracytoplasmic sperm injection (ICSI), two injection plates were prepared with a central 7μ1 drop of sperm catch (Nidacon SC-100), with a Ιμl arm of DPBS/FBS. This was surrounded by 4, 7μ1 drops of pre-equilibrated PZM and overlayed with pre- equilibrated mineral oil (Sage 4008). Plates were equilibrated at 38.5°C, 5% v/v CO₂, 5% v/v O₂ for at least 1 hour. Sperm was added to the PBS/FBS arm and allowed to swim into the sperm catch. Immediately prior to injection, a single oocyte was loaded into each of the PZM drops. ICSI was performed on a Nikon TE300 inverted microscope with a heated stage and fitted with Eppendorf micromanipulators. Injection and holding pipettes were supplied by The Pipette Company, Australia. Injection pipettes had an internal diameter of 6 μm. Individual sperm that had migrated into the sperm catch and were motile were immobilized by scoring the tail and aspirated into the injection pipette. The injection pipette was moved to a drop containing an oocyte. The oocyte was held with the polar body orientated at either 6 or 12 o'clock. The injection pipette with the sperm in the tip was introduced into the oocyte at 3 o'clock and the cytoplasm aspirated to ensure the oolemma had been ruptured before depositing the sperm. Post-injected oocytes were transferred to pre-equilibrated PZM for culture at 38.5°C, 5% v/v CO₂, 5% v/v O₂. mICSI was performed as for ICSI except that 3pl of mitochondrial isolate was collected into the injection pipette after the sperm and injected at the time of sperm injection.

[0080] After insemination/activation, zygotes were cultured in PZM for 7 days at 39°C, 5% v/v CO₂, 5% v/v O₂ at maximum humidity with media changes after 48 and 120 hours. Imaging of DAPI-stained nuclei in expanded blastocysts was undertaken using a multi- photon confocal microscope (Leica TCS SP5). Mitochondrial isolation and supplementation

[0081] Mitochondrial isolation from in vitro matured metaphase II BCB+ oocytes was performed using a drill-fitted Teflon pestle (Johnston et al. (2002) J Biol Chem 277:42197- 42204). Briefly, after hyaluronidase treatment, mechanical stripping and multiple PBS- washes to eliminate all cumulus cells, denuded oocytes were resuspended in 5 mL mitochondrial isolation buffer + 2mg/ml BSA (20 mM Hepes pH 7.6, 220 mM Mannitol, 70 mM sucrose, 1 mM EDTA) and homogenized by no more than 10 strokes of the pestle, at 4°C or on ice. The oocyte homogenate was centrifuged at 800 g for 10 minutes to remove cell debris and the supernatant was centrifuged at 10,000 g for 20 minutes to pellet the mitochondrial fraction. The pellet was resuspended in isolation buffer without BSA then centrifuged at 10,000 g for 20 minutes. The supernatant was removed and the mitochondrial pellet resuspended in isolation buffer without BSA. The mitochondrial suspension was further concentrated by transferring it to a sealed straw (Flexipet, Cook Australia) followed by centrifugation for 25 seconds at 10,000g. The mitochondrial suspension was dispensed onto an ICSI plate. At injection, a single sperm along with 3pl of mitochondrial isolate was drawn up into the injection pipette. The 3pl of mitochondrial isolate along with the sperm was injected into the oocyte.

[0082] The mitochondrial isolate was quantified for mtDNA copy number by injecting an identical volume to that injected into oocytes into 2 μl of H2O within a PCR tube, and analyzed in triplicate by real time PCR using mtDNA-specific primers, as described in 'Analysis of mtDNA copy number. Furthermore, the non-concentrated mitochondrial suspension (10 μl) underwent respiration analysis, as described in 'Mitochondrial respiration analysis'.

Mitochondrial respiration analysis

[0083] O₂ consumption rates for isolated mitochondria were determined by high-resolution respirometry (Oroboros Oxygraph-2K, Innsbruck, Austria). The Oxygraph was calibrated at 0% (5% v/v sodium hydrosulfide) and maximal (air) O₂ concentration in mitochondrial respiration buffer (225 mM D-mannitol, 75 mM sucrose, 10 mM KC1, 10 mM Tris-HCl, 5 mM KH2PO4). After 2 minute washes of H2O, EtOH 80% v/v, EtOH 100% v/v and H2O respectively, the chambers were filled with 2 ml respiration buffer maintained at 37°C and continuously stirred at 750 rpm. O₂ consumption was measured using the integrated software package Datlab (Version 3.1; Oroboros, Innsbruck, Austria), which presented respiration as O₂ flux, pmol O₂ per unit per second. Following O₂ flux stabilization, 25 μl of succinate (1 M) was added to the chamber as well as mitochondrial isolate and initial resting measurements were recorded. 10 μl of ADP (25 mM) followed by increasing doses of FCCP (carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone) were added at 10 minute intervals to determinate maximal uncoupled ETC respiratory capacity. Finally, 10 μl (5 mM) of the complex III inhibitor Antimycin A was used to abolish respiration.

Analysis of mtDNA copy number

[0084] Individual denuded oocytes and embryos were freeze-thawed in 50 μl of water. 2 μl was added to 10 μl SYBR green (Bioline, Australia), 6 μl of H2O and 0.5 μΜ of each primer. Table 2 shows primer sequences, product size, annealing temperature, and accession number. Reactions were run in a Rotorgene-3000 (Corbett Research, Cambridge, UK), according to the following conditions: 95°C for 5 minutes, 45 cycles of melting temperature for 30 seconds and acquisition temperature for 15 seconds, followed by 1 cycle at ramping temperature from 72°C to 95°C with continuous fluorescence acquisition. All samples were analyzed in triplicate. mtDNA copy number was extrapolated from standard curves (10-fold serial dilutions of 2 ng target PCR product) and calculated according to the target PCR product length. To determine the number of mtDNA copies introduced into oocytes by mICSI, the volume of mitochondrial isolate used to supplement the oocytes was directly added to a PCR tube and processed and analyzed, as described in 'Mitochondrial isolation and supplementation'.

Imaging of mitochondria in inseminated oocytes

[0085] Denuded metaphase II (Mil) oocytes were labeled with 100 nM MitoTracker Deep Red (Molecular Probes) in IVM medium for 45 minutes prior to injection. The mitochondrial isolate labeled with 200 nM MitoTracker Green FM (Molecular Probes) and 20 nM TMRM (Molecular Probes) was centrifuged at 10,000 g for 20 minutes. The mitochondrial pellet was washed to discard any unbound stain and concentrated as described in 'Mitochondrial isolation and supplementation'. Injection was performed as described in 'Mitochondrial isolation and supplementation'. Presumptive embryos were transferred into 8-well chamber slides on a coverslip (Sarstedt, product no. 94.6190.802).

[0086] Brightfield and MitoTracker Deep red images of presumptive embryos and fluorescent images of injected mitochondria were recorded on a Nikon CI confocal microscope using a 40x oil immersion objective. 488nm, 561nm, and 639nm lasers were used to specifically excite MitoTracker Green, TMRM and MitoTracker Deep Red in a sequential manner. Confocal stacks of 50 μm thickness with 5 μm increments between slices were recorded of the lower half of each oocyte.

Shape Analysis of Mitochondrial Clusters

[0087] Oocytes were stained with 100nΜ MitoTracker Deep Red (Molecular Probes) for 45 minutes and then transferred into 8-well chamber slides on a coverslip (Sarstedt, product no. 94.6190.802). Images of MitoTracker Deep Red were recorded on a Nikon CI confocal microscope using a 40x oil immersion objective. Confocal z-stacks of 50 μm thickness with 5 μm increments between slices were recorded of the lower half of each oocyte.

[0088] Image processing and analysis was performed using Fiji software. For shape analysis of mitochondrial clusters, a suitable region of interest (ROI) with even staining of mitochondria and excluding any oocyte edges (Figures la, c) was chosen for each data set. The ROIs were then thresholded and binarized (Figures lb, d). Particle analysis was then performed to detect and characterize clusters. Cluster area, perimeter and circularity were chosen as parameters for characterization. Clusters at ROI edges were excluded from the analysis. A total of 7 BCB^" and 9 BCB⁺ oocytes and 402 and 651 mitochondrial clusters, respectively, were analyzed.

Imaging of blastocysts to assess cell number

[0089] Day 7 blastocysts derived from ICSI-BCB⁺ and mICSI-BCB^" oocytes were fixed using 4% v/v paraformaldehyde, then permeabilized in 1% v/v TRITON X-100 and stained with DAPI. Image capture of DAPI stained blastocysts was performed by confocal microscopy using the multiphoton Leica SP8 (Leica Microsystems, Ontario Canada). Cell number represents the number of DAPI-stained nuclei per blastocyst.

Transmission Electron Microscopy

[0090] Porcine eggs were fixed for at least 48 hours at Mil, 1 hour and 24 hours post-ICSI and/or post-mlCSI in freshly made 2% v/v paraformaldehyde and 2.5% v/v glutaraldehyde in 0.1M sodium cacodylate buffer. Samples then underwent osmication and uranyl acetate staining, dehydration in alcohols and embedded in Taab 812 Resin (Marivac Ltd., Nova Scotia, Canada). Subsequent blocks were cut and sectioned with a Leica ultracut microtome, picked up on 100 mesh formvar/carbon coated Cu grids, stained with 0.2% w/v lead citrate, and imaged under the Phillips Technai BioTwin Spirt electron microscope.

RNA extraction, amplification and reverse transcription

[0091] RNA was extracted from single or pooled blastocysts using the Picopure RNA isolation Kit (Arcturus), according to the manufacturer's instructions. Extracted RNA was treated with DNAse I to remove genomic DNA. RNA was amplified by 2 rounds of in vitro transcription (Arcturus RiboAmp HS PLUS, Life Technologies) or reverse- transcribed (random primers) using Superscript III (Life Technologies), according to the manufacturer's instructions. After reverse-transcription, cDNA was diluted in 50 μl H₂O and real time PCR was conducted using a Rotorgene-3000. Relative differences in Ct values were compared to IVF BCB⁺ blastocysts and normalized against β-Actin (ACTB). Primer sequences, product size, annealing temperature, and accession numbers are provided in Table 2.

Microarray analysis

[0092] Highly diluted RNA Spike-Ins (Agilent #5188-5279, CA) were added at the time of RNA extraction to individual blastocysts (Zahurak et al. (2007) BMC Bioinformatics 5: 142; McCall and Irizarry (2008) Nucleic Acids Research 36:e108). Quality and concentration of extracted RNA were analyzed before and after T7 RNA amplification (Bio analyzer, Agilent). 1.8 μg amplified RNA was labeled with Cy3-ULS using the Agilent DNA ULS Fluorescent Labeling kit. The Degree of Labeling (DOL) was measured with the Nanodrop ND-1000 Spectrophotometer (Thermo Scientific). 1.65 μg of Cy3 labeled antisense RNA (aRNA; DOL 1-3.5%) was fragmented at 60°C for 30 minutes in a reaction volume of 25 μl containing lx Agilent fragmentation buffer and 4.4x Agilent blocking agent, according to the manufacturer's instructions. On completion, 55 μl of 2x Agilent gene expression hybridization buffer, 3 μl FLO and 27 μl CGH ULS block were added and 100 μl of the mixture was hybridized for 17 hours at 67°C in a rotating Agilent hybridization oven. After hybridization, EmbryoGENE Porcine - 4x 44K (ID 031068) microarrays (Agilent Technologies, CA) were washed for 1 minute at room temperature with GE wash buffer 1 (Agilent) and 1 minute with 37°C GE wash buffer 2 (Agilent, CA). Slides were scanned immediately after washing on the Agilent C DNA microarray scanner. The scanned images were analyzed using Feature Extraction Software 11.0.1.1 (Agilent). Ingenuity pathway analysis (IPA) was used to determine the signaling pathways underlying the transcriptomic differences (p<0.05, abs. FC>2) between blastocysts derived from ICSI-BCB⁺, ICSI-BCB^" and mICSI-BCB^" oocytes. Lists of DEGs from each direct comparison were prepared and imported into IPA by uploading lists of DEGs into separate experiments.

Gene expression analysis in IPA

[0093] DEGs were matched with their universal gene symbols, then compiled into canonical pathways as well as gene product interactions (networks) that are developed from information contained in Ingenuity's Knowledge Base. Canonical pathway analysis identified the pathways from the IPA library of canonical pathways that were most significant to the data set (enrichment score and probability). Networks of network-eligible molecules were algorithmically generated based on their connectivity. Green and red symbols represented genes respectively down- and upregulated. DEGs were also submitted to upstream regulator analysis, which is based on prior knowledge of expected effects between transcriptional regulators and their target genes stored in the Ingenuity (Registered Trade Mark) Knowledge Base. The analysis examines how many known targets of each transcription regulator are present in the dataset, and also compares their direction of change (i.e. expression in the experimental sample(s) relative to control) to what is expected from the literature in order to predict likely relevant transcriptional regulators. Each potential transcriptional regulator is given a p-value (probability to overlap with the data set) and an activation score (likelihood to be activated or inhibited with regard to the expression of target genes).

Statistics

[0094] An unpaired t-test was used to compare mtDNA copy number between Mil BCB⁺ and BCB^" oocytes and lysis rates. Direct comparison of global gene expression from microarray analysis was performed using an unpaired t-test corrected for false discovery rate (FDR) or filtered for absolute (abs.) fold-change >2 and p-value <0.01. One-way ANOVA was used to compare Mil rates and lysis rates; blastocyst rates; mtDNA replication profiles during either IVM, preimplantation development or between BCB staining/treatments; and differential gene expression in BCB blastocysts generated by ICSI or mICSI. ANOVA results for differential gene expression were corrected for FDR. Using Agilent's GeneSpring GX software, microarray data for each single blastocyst were subjected to principal component analysis and global gene expression was clustered by hierarchical Pearson's correlation. Ingenuity Pathway Analysis (IP A, QIAGEN Redwood City, www.qiagen.com/ingenuity) was used to determine significant enrichment of biological pathways associated with differentially expressed genes. Statistical significance is represented as *, **, ***, **** for p values of <0.05, 0.01, 0.001, 0.0001, respectively.

EXAMPLE 1

Nuclear and cytoplasmic maturation in BCB⁺ and BCB^' oocytes

[0095] In an ovary, typically 38.7 % ± 2.1 (mean ± SEM) oocytes stain negatively for BCB (BCB^"). To validate the use of BCB staining as a differential marker of oocyte maturation for oocytes that had not been synchronized to the S-phase of the cell cycle (Spikings et al. (2007) supra), aspirated BCB⁺ and BCB^" oocytes were assessed for Metaphase II (Mil; polar body extrusion) after 44 hr of in vitro maturation (IVM). Significantly more BCB⁺ oocytes (51.4%) developed to Mil than BCB^" oocytes (20.3%; PO.0001; whilst lysis was higher in the BCB^" population (51.2%; PO.0001).

[0096] Real-time PCR was performed on individual in vitro matured BCB⁺ and BCB^" oocytes at 0, 22 and 44 hr post-isolation from ovaries. BCB⁺ oocytes had high copy number at 0 hour, which decreased significantly at 22 hr (P<0.01) and returned to higher levels at 44 hours (Figure la). This was the case for oocytes that reached and failed to reach MIL BCB^" oocytes behaved in the opposite manner with lower copy number at 0 hour followed by an increase at 22 hours and a return to far lower levels at 44 hours (Figure lb), demonstrating their incapacity to mediate nucleo -cytoplasmic interactions. The few BCB^'oocytes reaching Mil had lower mtDNA copy number than immature oocytes at 44 hours. A direct comparison of Mil BCB⁺ and BCB^" oocytes at 44 hours showed that BCB^" oocytes had significantly fewer copies of mtDNA (P<0.01; Figure lc).

[0097] Furthermore, mitochondria isolated from BCB⁺ oocytes exhibited lower respiration rates than mitochondria from BCB^" oocytes when stimulated with succinate and ADP, but higher respiratory capacity when uncoupled with FCCP (Figure 5). BCB⁺ mitochondria were also in a more quiescent state than their BCB^" counterparts. In addition, there were distinct profiles for the distribution and clustering of mitochondria in BCB⁺ and BCB^" oocytes following staining with MitoTracker Deep Red (Figure 6). When cluster area, perimeter and circularity were chosen as parameters for characterization, there were significant differences between cluster area and perimeter for the two populations of oocytes. EXAMPLE 2

Supplementation of BCB^' oocytes with isolated mitochondria

[0098] Using cohorts of Mil BCB⁺ and BCB oocytes, a series of blastocyst stage embryos were generated through in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and mitochondrial supplementation in combination with ICSI (mICSI) [Table 3]. A mitochondrial isolate was generated from 5 to 10 BCB⁺ metaphase II oocytes for each ovary pair. The number of mtDNA copies delivered to each oocyte at 44 hours of IVM was 787.5 ± 409.3 with 44 hours deemed the most appropriate time as BCB^" oocytes showed large fluctuations in mtDNA copy number during the first 18 to 28 hours of IVM when compared to BCB⁺ oocytes. Indeed, supplemented mitochondria were tightly compact at 0 to 1 hour post-insemination and maintained their viability (Figure 2a). At 24 hours, they were still present and viable within the cytoplasm and were beginning to exhibit less of a clustered feature (Figure 2b). This was corroborated by transmission electron microscopy where quiescent mitochondrial morphology and dense organelles with translucent cristae were observed (Figure 2c). Some mitochondria were found with more defined and possibly more biologically active cristae in the BCB^" mISCI group at 1 hour post injection, but these mitochondria where no longer detectable at 24 hours.

[0099] BCB⁺ oocytes inseminated by ICSI had the highest fertilization rates (77.7%), as did BCB^" oocytes (59.9%) compared with IVF (58.4 % vs 38.1%; Table 3). Blastocyst rates were not significantly different following insemination by IVF, ICSI and mICSI for the BCB⁺ group. For the BCB^" group, mICSI led to significantly higher blastocyst rates (31.5%)) compared with IVF (7.6%>; P<0.05) and improved blastocyst rates compared with ICSI (23.9%o). Blastocyst rates for BCB^" oocytes inseminated by mICSI were very similar to BCB⁺ blastocysts generated by ICSI and mICSI. Furthermore, BCB^" mICSI-derived blastocysts had well-defined blastomeres with higher cell numbers (n= 51) than BCB⁺ ICSI-derived blastocysts (n= 33; Figures 2d and 2e).

EXAMPLE 3

mtDNA replication during preimplantation development

[0100] To determine whether mtDNA was differentially replicated during preimplantation development following each treatment, copy number was assessed from the 2-cell to the blastocyst stage. Although there was a slight turnover at the 4-cell stage in BCB⁺ IVF generated embryos, copy number decreased during preimplantation development (Figure 3a, P<0.001) until a significant increase at the expanded blastocyst stage (Figure 3a, P<0.05). In ICSI-BCB⁺ generated embryos, mtDNA copy number decreased at the 2-and 4-cell stages with significant expansion at the morula (Figure 3b; P<0.05) and expanded blastocyst stages (P<0.01). For BCB⁺ oocytes, there was a significant turnover of mtDNA copy number at the 4-cell stage after IVF but not ICSI (Figure 3e; P<0.05). At later stages (Figure 3e), there was significantly higher copy number in ICSI-generated compacting morulae than from IVF (P<0.001). The profile for the ICSI-derived BCB^" embryos was very different to the IVF- and ICSI generated BCB⁺ embryos (cf. Figures 3a, 3b and 3c; 3e).

[0101] BCB^" mICSI-generated embryos also exhibited very different profiles (Figure 3d). Supplementation led to significantly higher mtDNA copy number at the 2-cell stage compared to ICSI (p<0.01 ; Figures 3d; 3e). This represented a 4.4-fold increase (Table 4), whilst ICSI-generated 2 cell embryos retained only 27.8% (BCB⁺) and 50.1% (BCB^") of their initial mtDNA copy number. Likewise, there were significant differences in mICSI 8- cell embryos compared to the other treatments (Figure 3e). For day 7 expanded blastocysts, mtDNA copy number was > 200,000 copies following each treatment (Figure 3e). For mICSI-BCB^" blastocysts, this represented a 4.8-fold increase from the Mil oocyte stage (Table 4) whilst for BCB⁺ blastocysts generated through IVF and ICSI, the increases were 1.7-fold and 1.8-fold, respectively.

EXAMPLE 4

Global analysis of differential gene expression

[0102] To determine if mitochondrial transplantation modulated gene expression patterns in BCB^" derived blastocysts, global gene expression analysis was performed on single blastocysts generated from ICSI-BCB⁺ (n=4), ICSI-BCB^" (n=3) and mICSI-BCB^" (n=4) oocytes. Using a linear RNA amplification protocol and a global genome microarray, the expression of 28,944 probes was assessed across the different groups (Quantile normalization, 20- 100th percentile and detection flag filters). Principal component analysis (PCA) demonstrated the clustering of replicates to their respective groups, except for one mICSI-BCB- blastocyst outlier (Figure 4a). Heat maps for gene expression also showed good clustering and greater similarity between the mICSI-BCB^" and ICSI-BCB⁺ blastocysts than the ICSI-BCB^" blastocysts (Figure 4b). Indeed, the probe intensities examined for genes associated with blastocyst development showed consistent expression of genes involved in pluripotency (Figure 7a), epigenetic reprogramming (Figure 7b), energy metabolism (Figures 7c and d) and microRNAs (Figure 7e) for mICSI-BCB^" and ICSI- BCB⁺ blastocysts.

[0103] Amongst each group of blastocysts, direct unpaired t-test comparisons identified several differentially expressed genes (DEG) [False Discovery Rate (FDR) = 0.05, Table 5]. There were 5 DEGs between ICSI-BCB^" and ICSI-BCB⁺ blastocysts, 7 DEGs between mICSI-BCB^" and ICSI-BCB⁺ blastocysts, and 1 DEG between mICSI-BCB^" and ICSI- BCB^" blastocysts. Across the three groups of blastocysts, ANOVA determined significant differences for 6 genes (FDR=0.05, Table 6). One DEG (predicted Ras and Rab interactor 3, LOC100155709) showed a greater fold change for the mICSI-BCB^" group than for the ICSI-BCB^" group compared to ICSI-BCB⁺ blastocysts. However, the other 5 DEGs (the putative olfactory receptor 51Hl-like LOC100511338, the YKT6 v-SNARE homolog LOC100513964, the uncharacterized LOC100621956 and 2 novel genes) had greater fold change for ICSI-BCB^" than mICSI-BCB^" blastocysts compared to ICSI-BCB⁺ blastocysts. [0104] Analysis by ANOVA without FDR (pO.01, absolute fold-change ((FC)) > 2) produced a total of 309 DEGs with significant DEGs profiled according to relative fold change between the groups. This profiling showed greater fold-change differences in the ICSI-BCB^" blastocysts compared to ICSI-BCB⁺ or mICSI-BCB^" blastocysts. In particular, ICSI-BCB^" blastocysts had a greater number of upregulated DEGs with fold changes higher than 5 or 10, whilst mICSI-BCB^" blastocysts showed a greater number of downregulated DEGs.

[0105] As large numbers of DEGs are required to reach sufficient enrichment for in silico pathway analysis, pairs of groups without FDR but with FC >2 and significance of p<0.01 were compared (Figure 4c; and Table 7). Using Ingenuity Pathway Analysis, 276/378 DEGs were annotated for the comparison between ICSI-BCB^" vs ICSI-BCB⁺ blastocysts, which clustered into 12 networks (Table 8). The top three networks were cellular assembly and organization; cell morphology; and amino acid metabolism. For the cellular assembly and organization pathway, a large number of genes were upregulated in the ICSI-BCB^" blastocysts. Likewise, for the cell morphology and the amino acid metabolism networks, a higher proportion of genes were upregulated in the ICSI-BCB^" blastocysts than were down regulated. For the canonical pathways, PPAR signaling was the most affected. Of the predicted upstream regulators of the DEGs, CREBl, ERB2 and BMP2 were activated, as was the pathway that is modulated by the anti-Type 2 Diabetes pharmaceutical agent Troglitazone (Tables 9 and 10).

[0106] In contrast, 127/192 DEGs were annotated from the comparison between mlCSI- BCB^" and ICSI-BCB^" blastocysts, which clustered into seven networks (Table 11). The cellular movement, cellular development and cell morphology networks ranked highest. The majority of genes in these networks were downregulated in the mICSI-BCB^" cohorts. The PPAR canonical pathway was also affected in ICSI-BCB^" blastocysts. Likewise, three predicted upstream regulators of the DEGs, namely NFKB, ILS and URAS were significantly inhibited whilst the pathway modulated by the pharmaceutical agent resveratrol, which regulates Sirtuin activity and thus mitochondrial biogenesis (Sato (2014) PLoS One 9:e94488), was activated (Table 9). [0107] The comparison of mICSI-BCB^" and ICSI-BCB⁺ blastocysts resulted in 222/311 DEGs being annotated, which clustered into 10 networks. The highest ranked of these were cell cycle, cellular compromise and developmental disorders. For these networks, there was an improved balance between upregulated and downregulated DEGs compared to ICSI-BCB^" blastocysts and more DEGs showed no marked differences. Of the canonical pathways, the regulators of metabolism were most affected (Table 9) whilst MYC and STAT4 were predicted upstream regulators to be activated, as was the pathway modulating the anti-cancer pharmaceutical agent Streptozocin.

[0108] Genes were analyzed that were not differently expressed between ICSI-BCB⁺ and mICSI-BCB^" but were differently expressed in ICSI-BCB^" blastocysts. 90/168 annotated genes clustered into 5 networks. The top three networks were cell morphology, gene expression and protein synthesis, and cell to cell signaling and interaction and cellular assembly and association. Glycine biosynthesis I, methylmalonyl pathway, and 2- oxobutanoate degradation I were the top three affected canonical pathways. However, no upstream regulators were significantly activated or inhibited.

EXAMPLE 5

Analysis of patterns of gene expression specific to embryonic development

[0109] Cohorts of blastocysts (n= 5 to 10) were also analyzed to determine the patterns of expression for key regulatory genes. Each of the pluripotent genes, OCT4 (Figure 8a), SOX2 (Figure 8b) and REXl (Figure 8c) showed consistent levels of expression across the different groups, whilst NANOG expression was very low (Ct > 40) with no detection in mICSI-BCB⁺ blastocysts (Figure 8d), as expected for porcine embryos (Wolf et al. (2011) Dev Dyn 240:204-210). The trophectodermal marker, CDX2, was consistently expressed in IVF, ICSI and mICSI blastocysts (Figure 8e). Two mtDNA-encoded genes, ND1 (Figure 8f) and ATP6 (Figure 8g), and the nuclear-encoded mtDNA transcription factor TFAM (Figure 8h) were highly expressed and within similar range.

EXAMPLE 6

The role of mtDNA in oocyte development

[0110] The number of mtDNA copies present in developmentally competent Mil oocytes is an essential investment in oocyte development, as embryonic cells only initiate mtDNA replication post-gastrulation which is a large number of cell divisions post-fertilization. These are important developmental events as they establish the mtDNA set point from which all naive cells possess low numbers of mtDNA. These copies of mtDNA are then replicated in a cell specific manner in order that mature, specialized cells acquire their specified numbers of mtDNA copy to produce sufficient ATP through OXPHOS to perform their specialized functions.

[0111] Previously, it has been demonstrated that the supplementation of oocytes with genetically distinct populations of mtDNA, through cytoplasmic transfer, resulted in heteroplasmy (Brenner et al. (2000) supra; Acton et al. (2007) supra). In mice, the health and wellbeing of the offspring was compromised by a host of physiological abnormalities (Acton et al. (2007) supra). Other studies in heteroplasmic mice have indicated similar outcomes (Sharpley et al. (2012) supra). The transfer of donor cytoplasm to treat women whose embryos undergo repetitive developmental arrest prior to or at EGA (Cohen et al. (1997) Lancet 350: 186-187; Lanzendorf et al. (1999) Fertil Steril 77:575-577; Dale et al. (2001) Hum Reprod 76: 1469-1472; Huang et al. (1999) Fertil Steril 72:702-706) resulted in heteroplasmy at higher than anticipated levels even though the cytoplasmic extract was not a mitochondrial concentrate. Indeed, 40% of the offspring's total mtDNA originated from the donor cytoplasm (Brenner et al. (2000) supra; Barritt et al. (2001) Reprod Biomed Online 3:47-48), which supports the findings in the present Examples that mtDNA introduced at fertilization 'hitchhikes' on the pre-EGA mtDNA replication event to provide sufficient mtDNA investment for subsequent development. Nevertheless, this approach led to autism and Turner's syndrome (XO) but not in all cases (Dale et al. (2001) supra; Huang et al. (1999) supra; Barritt et al. (2001) supra). [0112] It is proposed herein that supplementation with autologous populations of mitochondrial isolate does not perturb the genetic identity of the offspring but generates a stabilization effect, which is reflected in the enhanced gene expression patterns observed at the blastocyst stage. The gene expression patterns for BCB^" supplemented oocytes (mlCSI- BCB^") are far more aligned to the ICSI-BCB⁺ cohort. The key gene networks positively affected in mICSI-BCB^" blastocysts included cellular movement, cellular development and cell morphology, which are essential to developmental outcomes. These networks closely resemble deficits observed in ICSI-BCB^" derived blastocysts that attempted to compensate by increasing gene expression associated with amino acid metabolism, which is essential to early development (Humpherson et al. (2005) supra).

[0113] From the DEG profiles, it appears that ICSI-BCB^" blastocysts had a higher number of upregulated genes compared to ICSI-BCB⁺ blastocysts, with significant enrichment in the PPAR signaling pathway. This up-regulation is also predicted to involve activation of transcription factors, such as CREBl (Table 5), an important metabolic regulator of blastocyst development (Jin and O'Neill (2007) Reproduction 134:661-615). As CREBl is also involved in mitochondrial maintenance prior to EGA and energy homeostasis (Rohas et al. (2007) Proceedings of the National Academy of Sciences of the United States of America 704:7933-7938), it would also communicate mtDNA deficiency to BCB^" embryos prior to EGA.

[0114] The upregulation of gene expression in BCB^" derived blastocysts was stabilized by mICSI and affected PPAR signaling, as well as other signalling pathways such as NKkB. Notably, mICSI down-regulated genes correlated with upstream activation by resveratrol (Table 5). Resveratrol is an exogenous compound beneficial to oocyte maturation and embryo development in mice (Liu et al. (2013) Hum Reprod 28:101-111), cattle (Wang et al. (2014) Fertility and sterility 707:577-586) and pigs (Sato et al. (2014) supra). The effects of resveratrol involves activation of Sirtl, which influences mitochondrial biogenesis as well as mtDNA copy number in porcine oocytes (Sato et al. (2014) supra). Consequently, the effect of mICSI on blastocyst gene expression would be to communicate via retrograde signaling the stabilizing effect of increased mtDNA copy number in early embryos. Collectively, these outcomes promote stabilized mtDNA replication under pluripotent gene control that is essential to the undifferentiated state, as observed in embryonic stem cells (Facucho-Oliveira et al. (2007) J Cell Sci 720:4025-4034; Facucho- Oliveira et al. (2009) Stem Cell Rev 5: 140-158; Kelly et al. (2013) Stem Cell Rev 9: 1-15). Likewise, the activation of MYC and STAT4 in mICSI-BCB^" blastocysts, when compared to ICSI-BCB⁺ blastocysts, is associated with increased cell proliferation in other cellular systems (Matikainen et al. (1999) Blood 93: 1980-1991), and, would account for the higher cell number observed in mICSI-BCB^" blastocysts.

[0115] Whilst oocyte and early embryonic mitochondria do not fully mature before the blastocyst stage, the Examples show here that the mtDNA content can be modulated throughout preimplantation development, and, particularly, prior to embryonic genome activation. In mtDNA deficient oocytes, this modulation does not occur which could account for lower blastocyst development. On the other hand, the increased mtDNA copy number in BCB^" oocytes following mICSI could counteract the homeostatic response to early mtDNA deficiency, and influence embryonic development by stabilizing communication between the mitochondrial and nuclear genomes, which fails in other cellular systems, such as tumours, where the failure to efficiently replicate mtDNA prevents differentiation from taking place (Dickinson et al. (2013) Cell Death Differ 20: 1644-1653). Whilst we argue that the increase in mtDNA copy number is proposed herein to induce a stabilising effect on the embryo prior to embryonic genome activation, others have proposed that metabolic quiescence in oocytes and early embryos serves to protect the integrity of the mitochondrial genome (de Paula et al. (2013) Philosophical Transactions of the Royal Society of London Series B, Biological Sciences 3(55:20120263). If mitochondrial supplementation is to be introduced into clinical treatment, it would be pertinent to evaluate the potential for DNA damage by determining the levels of double strand breaks through, for example gamma H2AX labeling, or the comet assay, and to test for aneuploidy. This would demonstrate that there are no detrimental effects from supplementation on genome integrity and that, indeed, mtDNA supplementation is not inducing a metabolic affect that harms the embryo but, rather, supports subsequent development. [0116] In conclusion, outcomes demonstrated herein include the importance of mtDNA to oocyte developmental competence, whereby supplementation of mtDNA deficient oocytes at fertilization enhances embryo development and blastocyst quality. It also highlights the importance of inducing early mtDNA replication events at fertilization in mtDNA deficient oocytes to enhance embryo quality by stabilizing the embryo prior to EGA. This mtDNA investment ensures that resultant blastocysts have increased cell numbers and enhanced gene expression profiles that are associated with blastocysts from developmentally competent oocytes. It is concluded herein that mitochondrial supplementation could significantly improve fertilization and development rates in mammals. In addition to advanced maternal age and poor ovarian reserve, this procedure is relevant to the rescue of in vitro matured human oocytes, notably in women with polycystic ovary syndrome.

EXAMPLE 7

Generation of live offspring through mitochondrial supplementation

[0117] This Example describes the transfer of autologous populations of mitochondria from egg (oocyte) precursor cells into mature eggs. No selection of eggs (oocytes) is made based on mtDNA or mitochondrial deficiency. The resulting supplementation of mitochondria at time of fertilization is not detrimental to the development of the embryo and results in the generation of healthy offspring. The use of precursor egg cells ensures that mature eggs are not sacrificed for this process and that as many eggs as possible are available to generate embryos.

[0118] It is proposed that the introduction of autologous populations of mitochondria from immature eggs enhances embryonic development resulting in healthy offspring.

[0119] This validates the autologous transfer of mitochondria to the oocytes of women who present with repeated failed fertilization or embryonic developmental arrest. This enables clinicians and embryologists to use this technique without concern for any unforeseen and undesirable outcomes. The outcomes of this invention are also directly applicable to animals, where valuable species, which have poor oocyte function and embryo development, can be preserved through autologous mitochondrial transfer. It is also useful for controlled breeding of non-human mammals.

[0120] The aim of this Example is to determine whether the transfer of autologous mitochondria affects: embryo development rates to blastocyst and blastocyst quality; live birth rates; and offspring viability. Experimental procedures:

[0121] Overview : The experiments here test whether the transfer of autologous populations of mitochondria by mICSI into oocytes at the time of fertilization results in the generation of healthy offspring. Oocytes are superovulated through hormonal stimulation, sperm injected with mitochondria simultaneously, embryos transferred to recipients that are pseudopregnant and offspring allowed to be born naturally. Animals are monitored over 3 to 4 months to determine that they develop normally.

Breed of mouse : Fl : C57BL/6xCB A.

Breed of mitochondrial donors: Fl : C57BL/6xCBA. The source of mtDNA is confirmed by genotyping prior to use to ensure that genetic identity is maintained.

Breed of foster females : CD1.

Sex:

Females:

1) Provide oocytes following superovulation.

2) Pseudopregnant females used as recipients for embryo transfer.

Age: 4 to 6 weeks to ensure sexual maturity has been reached. Males:

1) Mature males provide sperm from the epididymides for fertilization.

2) Vasectomized males were introduced to females (CD1) in natural oestrus to generate pseudopregnant females (foster females for embryo transfer).

Age: 8 to 10 weeks of age to ensure sexual maturity. [0122] Oocytes are supplemented with autologous populations of mitochondria as they undergo sperm injection (mICSI) to fertilize oocytes and produce embryos. The resultant embryos are cultured in vitro and transferred to pseudopregnant mice.

[0123] Oocyte collection: Oocytes are obtained from mice (n= 30 per treatment) following superovulation. Female mice of 4-6 weeks of age are superovulated using pregnant mare serum gonadotropin (PMSG), 5IU/animal, and followed 48 hr later by hCG, 5IU/animal. Females are killed 13-15 hrs post-hCG injection for oocyte collection.

[0124] Sperm collection and cryopreservation: Male mice (8-10 weeks old; n = 6 per treatment) are killed and their epididymides collected to obtain sperm by gentle expression of luminal contents into medium. The sperm are used immediately for mICSI or ICSI or are cryopreserved and stored for future use.

[0125] mICSI: Oocytes are supplemented with autologous populations of mitochondria, prepared from egg precursor cells. The purified mitochondria are introduced into the egg at the same time as it was fertilized by direct injection of either fresh or cryopreserved sperm collected from the epididymides of mature male mice.

[0126] Mitochondria are isolated from egg precursor cells and transferred at a dose equivalent to ~ 800 copies into mature eggs and fertilized at the same time (mICSI). The resultant embryos are cultured to the blastocyst stage. Embryos were then transferred to pseudopregnant mice to establish pregnancy. The offspring are delivered vaginally at term.

[0127] Embryo transfer: Embryos are transferred into the primed uteri of foster females for normal intrauterine development and offspring are delivered at term. This is a standard mouse embryo transfer procedure conducted under anaesthesia.

[0128] The oestrous cycle in the female mice selected for transfer is monitored by vaginal smears and, in the early luteal phase, the female is anaesthetized and the uteri exposed through lateral skin/body wall incisions anterior to the leg flexure on each side. Embryos loaded into a transfer pipette (3-6 embryos per transfer) in a 5-10μ1 droplet of medium are transferred. Females are allowed to give birth naturally.

[0129] Outcome of pregnancy: Outcome of pregnancy is assessed by recording the number of offspring born (litter size) for each treatment and compared to controls (natural matings from the colony populations) [Figure 9].

[0130] Matings to determine litter size and transgenerational effects: Female offspring (FO) are then mated with colony males to determine whether they are fertile over five parities. The offspring from the FO females (Fl) are then mated as are their daughters to determine if there is a transgenerational effect over three generations.

[0131] Monitoring of live offspring: Live offspring are monitored for 90 to 120 days, which include equivalent time points to early childhood, adolescence, onset of breeding age, and adulthood. They are assessed each day from days 1 to 14 for a range of developmental characteristics including skin color, skin density, milk spot, ear structure, pigmentation, colored fuzz, fur growth, nipple growth, activity, development of teeth, opening of eyes, uptake of solids, weight and size. Thereafter, they are monitored each week. They are assessed for weight from day 14 for 8 weeks at weekly intervals.

[0132] Methods of statistical analysis: Numerical analysis is run in duplicate and repeated three times. Parametric and non-parametric tests are chosen after assessing Gaussian distribution curves. Multiple variables within single sets of samples are analyzed by One- Way ANOVA. Multiple variables within multiple sets of samples are assessed by Two- Way ANOVA. Individual tests within multivariable sets are performed using, for example, Bonferroni correction (parametric) and Dunn's (non-parametric) post-hoc tests. Results:

Establishment of founders:

[0133] In total, 8 mICSI founders are generated. These result from 2 cell transfer embryos as the developmental progression to blastocyst is delayed resulting from mICSI and resulted in asynchrony with the female recipients. The offspring (FO) develop normally and their weights are within normal range for mice of the same breed.

[0134] The FO mice are mated naturally and their litter sizes are enhanced (1.2 offspring per litter over 5 parities; P<0.05; Figure 9; 1^st generation) when compared with naturally mated colony controls. When the subsequent offspring (Fls) were mated, litter size remained significantly higher (P<0.01 ; Figure 9; 2^nd generation), and for a further generation also (P<0.05; Figure 9; 3^rd generation). The results show that the autologous transfer of mitochondria has no adverse consequences in embryo development to blastocyst stage. Blastocysts are clearly of high quality in terms of success of the pregnancy. Live birth rates and offspring variability show no adverse consequences.

EXAMPLE 8

The role of mtDNA regulation

[0135] Following mtDNA/mitochondrial supplementation, the copy number at blastocyst stage is similar to blastocysts derived from other treatments. However, mtDNA copy number is more efficiently regulated since, whilst copy number is comparable, there are more cells in supplemented blastocysts. Therefore, the higher cell number means that supplemented blastocysts have fewer copies per cell. For example, ICSI-BCB⁺ = 263067/33 = 7972 mDNA copies per cell; mICSI-BCB^" = 214841/51 = 4213 mDNA copies per cell. In other words, developmentally non-competent oocytes supplemented with mtDNA have more cells at blastocyst stage but have slightly lower overall copy number. This is highlighted when viewed as the ratio of copy number to cell number. This indicates that supplemented oocytes can better regulate their mtDNA copy number and overcome the association between poor mtDNA regulation at the blastocyst stage and aneuploidy.

Table 12

Summary of sequence identifiers

[0136] Those skilled in the art will appreciate that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure contemplates all such variations and modifications. The disclosure also enables 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 or compositions or compounds.

Claims

CLAIMS:

1. A method for enhancing female mammalian oocyte fertilization and embryo development capacity, said method comprising isolating a sample comprising an oocyte from a female mammalian subject, selecting an oocyte and supplementing the oocyte with autologous mitochondrial DNA or autologous whole mitochondria prior to, simultaneous with or following fertilization of the oocyte.

2. The method of Claim 1 wherein the oocyte is selected as potentially poorly competent.

3. The method of Claim 2 wherein a potentially poorly competent oocyte is one which converts brilliant cresyl blue (BCB) to a colorless product (BCB^").

4. The method of Claim 2 wherein a potentially poorly competent oocyte is one which has a cytoplasmic volume associated with non-competency.

5. The method of Claim 1 wherein the oocyte is selected regardless of its level of potential developmental competency or non-competency.

6. The method of Claim 1 wherein fertilization is by fertilization in vitro.

7. The method of Claim 1 or 6 wherein fertilization is by intracytoplasmic sperm injection (ICSI).

8. The method of any one of Claims 1 to 7 wherein the female mammalian subject is a human female.

9. The method of Claim 1 wherein the female mammalian subject is a non-human female mammal.

10. The method of Claim 1 wherein the mtDNA or mitochondria are introduced to the oocyte simultaneously with a sperm.

11. The method of any one of Claims 1 to 10 wherein the fertilized oocyte is cultured in vitro for a time and under conditions for a blastocyst to develop.

12. The method of Claim 10 wherein the blastocyst comprises more cells than a blastocyst at the same stage derived from an oocyte not supplemented with mtDNA or mitochondria.

13. The method of Claim 1 wherein the blastocyst comprises cells with a copy number of mtDNA per cell lower than in a blastocyst at the same stage derived from an oocyte not supplemented with mtDNA or mitochondria.

14. The method of Claim 11 or 12 wherein the supplementation enables early mtDNA replication at fertilization and prior to embryonic genome activation.

15. The method of Claim 11 or 12 or 13 wherein the blastocyst comprises cells with a gene expression profile or pattern associated with a blastocyst from a developmentally competent, fertilized oocyte.

16. The method of any one of Claims 11 to 15 wherein the blastocyst is implanted into the same female mammalian subject as the donor of the oocyte.

17. The method of any one of Claims 11 to 15 wherein the blastocyst is implanted into a non-autologous female mammalian subject of the same species.

18. The method of Claim 16 or 17 wherein the implanted blastocyst develops to term.

19. The method of Claim 18 wherein in non-human mammalian subjects, litter sizes are increased compared to litters derived from oocytes not supplemented with mtDNA or mitochondria.

20. The method of Claim 1 wherein the mtDNA is first screened for DNA mutations or copy number.

21. The method of Claim 1 wherein the mitochondria are first screened for respiration ability wherein mitochondria with respiration capability are selected for transfer to the oocyte.

22. A method for facilitating fertilization and developmental competency of a mammalian oocyte, said method comprising supplementing an isolated oocyte with autologous mtDNA or autologous mitochondria to provide a sufficient amount of mtDNA at fertilization with a sperm or following intracytoplasmic sperm injection (ICSI) and culturing the fertilized oocyte in vitro to blastocyst stage such that the blastocyst comprises cells with a gene expression profile associated with the gene expression profile of a developmentally competent oocyte.

23. The method of Claim 22 wherein the supplemented mtDNA enables early mtDNA replication at fertilization.

24. The method of Claim 22 or 23 wherein the blastocyst comprises more cells than a blastocyst at the same stage derived from an oocyte not supplemented with mtDNA or mitochondria.

25. Use of autologous mtDNA or mitochondria in combination with a sperm in the supplementation of an oocyte which is then capable of forming a blastocyst to be implanted to a female mammalian host.

26. mtDNA or mitochondria and a sperm for use in fertilizing an oocyte capable of being cultured to blastocyst stage and then to be implanted to a female mammal host.

27. In the method of fertilization in vitro of an oocyte by the steps of fertilizing the oocyte, culturing the fertilized oocyte to blastocyst stage and implanting the blastocyst in a female mammalian host, the improvement comprising supplementing the oocyte with autologous mtDNA or mitochondria prior to, simultaneously with of following fertilization with a sperm.

28. The method of Claim 27 wherein the mammalian host is human.

29. The use of Claim 25, mtDNA or mitochondria of Claim 26 or the method of Claim 27 or 28 wherein the blastocyst comprises more cells than a blastocyst at the same stage derived from an oocyte not supplemented with mtDNA or mitochondria.