[Show abstract][Hide abstract] ABSTRACT: What are the consequences of polycystic ovary syndrome (PCOS) pathology and metformin-pretreatment in vivo in women with PCOS on the metabolism and steroid production of follicular phenotype- and long-term cultured-granulosa cells (GC)?
[Show abstract][Hide abstract] ABSTRACT: Is it possible to restore ovarian function and natural fertility following the cryopreservation and autotransplantation of whole ovaries, complete with vascular pedicle, in adult females from a large monovulatory animal model species (i.e. sheep)?
[Show abstract][Hide abstract] ABSTRACT: Mitochondria are responsible for the production of ATP which drives cellular metabolic and biosynthetic processes. This is the first study to quantify mtDNA copy number across all stages of oogenesis in a large monovulatory species, it includes assessment of the activity of mitochondria in GV and MII oocytes through JC1 staining. Primordial to early antral follicles (n=249) were isolated from sheep ovarian cortex following digestion at 37°C for 1 hr and all oocytes were disaggregated from their somatic cells. Germinal vesicle oocytes (n=133) were aspirated from 3-5 mm diameter antral follicles and mature MII oocytes (n=71) were generated following IVM. The mtDNA copy number in each oocyte was quantified using real-time PCR and showed a progressive, but variable increase in the amount of mtDNA in oocytes from primordial follicles (605±205, n=8) to mature MII oocytes (744,633±115,799, n=13; P<0.05). Mitochondrial activity (P>0.05) was not altered during meiotic progression from GV to MII during IVM. The observed increase in mtDNA copy number across oogenesis reflects the changing ATP demands needed to orchestrate cytoskeletal and cytoplasmic reorganisation during oocyte growth and maturation and the need to fuel the resumption of meiosis in mature oocytes following the preovulatory gonadotrophin surge.
Molecular Human Reproduction 03/2013; · 4.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: STUDY QUESTION: Can amino acid profiling differentiate between human oocytes with differing competence to mature to metaphase II (MII) in vitro? SUMMARY ANSWER: Oocytes which remained arrested at the germinal vesicle (GV) stage after 24 h of in vitro maturation (IVM) displayed differences in the depletion/appearance of amino acids compared with oocytes which progressed to MII and patient age, infertile diagnosis and ovarian stimulation regime significantly affected oocyte amino acid turnover during IVM. WHAT IS KNOWN ALREADY: Amino acid profiling has been proposed as a technique which can distinguish between human pronucleate zygotes and cleavage stage embryos with the potential to develop to the blastocyst stage and implant to produce a pregnancy and those that arrest. Most recently, the amino acid turnover by individual bovine oocytes has been shown to be predictive of oocyte developmental competence as indicated by the gamete's capacity to undergo fertilization and early cleavage divisions in vitro. STUDY DESIGN, SIZE, DURATION: The study was conducted between March 2005 and March 2010. A total of 216 oocytes which were at the GV or metaphase I (MI) stages at the time of ICSI were donated by 67 patients. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: The research was conducted in university research laboratories affiliated to a hospital-based infertility clinic. Oocytes were cultured for 24 h and the depletion/appearance of amino acids was measured during the final 6 h of IVM. Amino acid turnover was analysed in relation to oocyte meiotic progression, patient age, disease aetiology and controlled ovarian stimulation regime. MAIN RESULTS AND THE ROLE OF CHANCE: The depletion/appearance of key amino acids was linked to the maturation potential of human oocytes in vitro. Oocytes which arrested at the GV stage (n = 9) depleted significantly more valine and isoleucine than those which progressed to MI (n = 32) or MII (n = 107) (P < 0.05). Glutamate, glutamine, arginine and valine depletion or appearance differed in MII versus degenerating oocytes (n = 20) (P < 0.05). Glutamine, arginine, methionine, phenylalanine, total depletion and total turnover all differed in oocytes from patients aged < 35 years versus patients ≥35 years (P < 0.05). MII oocytes obtained following ovarian stimulation with recombinant FSH depleted more isoleucine (P < 0.05) and more alanine and lysine (P < 0.05) appeared than oocytes from hMG-stimulated cycles. MII oocytes from patients with a polycystic ovary (PCO) morphology (n = 33) depleted more serine (P < 0.05) than oocytes from women with normal ovaries (n = 61). LIMITATIONS, REASONS FOR CAUTION: Immature oocytes collected at the time of ICSI were used as the model for human oocyte maturation. These oocytes have therefore failed to respond to the ovulatory hCG trigger in vivo (they are meiotically incompetent), and have limited capacity to support embryo development in vitro. The lack of cumulus cells and stress of the conditions in vitro may have influenced turnover of amino acids, and owing to the small sample sizes further studies are required to confirm these findings. WIDER IMPLICATIONS OF THE FINDINGS: The findings provide support for the hypothesis that oocyte metabolism reflects oocyte quality. Longitudinal studies are required to link these functional metabolic indices of human oocyte quality with embryo developmental competence. Oocyte amino acid profiling may be a useful tool to quantify the impact of new assisted reproduction technologies (ART) on oocyte quality. STUDY FUNDING/COMPETING INTERESTS: This project was funded by the UK Biology and Biotechnology Research Council (BB/C007395/1) and the Medical Research Council (G 0800250). K.E.H was in receipt of a British Fertility Society/Merck Serono studentship. H.J.L. is a shareholder in Novocellus Ltd, a company which seeks to devise a non-invasive biochemical test of embryo health.
[Show abstract][Hide abstract] ABSTRACT: Fertility preservation by whole ovarian cryopreservation requires successful cryopreservation of both the ovary and its vascular supply. Previous work has indicated detrimental effects of both perfusion and cryopreservation on the ovarian vasculature. This study assessed the effects of blood perfusion, alone or in combination with cryopreservation, on functional effects in the follicle population and ovarian function in vivo following short term autotransplantation of the tissue after vascular reanastomosis and measured acute changes in endothelial cell related gene expression within the ovarian medulla and pedicle. Following autotransplantation for 7 days, primordial, transitional and primary follicle densities were significantly reduced (P<0.05) and stromal Ki67 and caspase-3 expression significantly increased (P<0.05) in cryopreserved but not fresh or perfused whole ovaries. There was evidence of clot formation and fluorescent microsphere (FMS) extravasation in the medulla of all cryopreserved ovaries, indicating vascular damage. Utilising a customised RT-PCR array or conventional RT-PCR, we found that perfusion alone resulted in downregulation in expression of CASP6 and THBS1 genes in the medulla. Following additional cryopreservation, eNOS, ET-1, EDNRA and Bcl-2 expression were significantly (P<0.05) downregulated. In the pedicle, both perfusion and cryopreservation caused a (P<0.05) downregulation of eNOS, and THBS1 and an upregulation in Bax expression. Perfusion also caused a downregulation of TNF and upregulation of EDN2 expression (P<0.05). In conclusion, this study has identified a number of endothelial cell related genes expressed in the medulla which are acutely affected by both cryopreservation and perfusion, supporting the hypothesis that both interventions have deleterious effects on endothelial cell function.
Molecular Human Reproduction 11/2012; · 4.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is evidence that expression and methylation of the imprinted paternally expressed gene 1/mesoderm-specific transcript homologue (PEG1/MEST) gene may be affected by assisted reproductive technologies (ARTs) and infertility. In this study, we sought to assess the imprinting status of the MEST gene in a large cohort of in vitro-derived human preimplantation embryos, in order to characterise potentially adverse effects of ART and infertility on this locus in early human development. Embryonic genomic DNA from morula or blastocyst stage embryos was screened for a transcribed AflIII polymorphism in MEST and imprinting analysis was then performed in cDNA libraries derived from these embryos. In 10 heterozygous embryos, MEST expression was monoallelic in seven embryos, predominantly monoallelic in two embryos, and biallelic in one embryo. Screening of cDNA derived from 61 additional human preimplantation embryos, for which DNA for genotyping was unavailable, identified eight embryos with expression originating from both alleles (biallelic or predominantly monoallelic). In some embryos, therefore, the onset of imprinted MEST expression occurs during late preimplantation development. Variability in MEST imprinting was observed in both in vitro fertilization and intracytoplasmic sperm injection-derived embryos. Biallelic or predominantly monoallelic MEST expression was not associated with any one cause of infertility. Characterisation of the main MEST isoforms revealed that isoform 2 was detected in early development and was itself variably imprinted between embryos. To our knowledge, this report constitutes the largest expression study to date of genomic imprinting in human preimplantation embryos and reveals that for some imprinted genes, contrasting imprinting states exist between embryos.European Journal of Human Genetics advance online publication, 4 July 2012; doi:10.1038/ejhg.2012.102.
European journal of human genetics: EJHG 07/2012; · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The response of Graafian follicles to pre-ovulatory surge levels of FSH and LH in vivo triggers the terminal differentiation of granulosa cells and oocyte maturation. In polyovular species, the LH-driven signalling uses the epidermal growth factor (EGF)-like ligands AREG, EREG and BTC to promote oocyte maturation and cumulus expansion. This experimental series used a physiologically relevant ovine in vitro maturation (IVM) system to evaluate the impact of exposure to pre-ovulatory levels (100 ng/ml) of LH and FSH on ovine cumulus cell expression of EGF-like ligands in vitro. The serum-free sheep IVM system supported high levels (91.4%) of gonadotrophin-induced maturation of cumulus-enclosed oocytes and embryo development to the blastocyst stage (34.5%). Results were equivalent to a serum-based IVM system (85.1% IVM, 25.8% blastocyst rate; P>0.05) but were significantly different (P<0.05) to serum-free medium without gonadotrophins (69.5% IVM; 8.0% blastocyst rate). Ovine BTC was cloned and sequenced. Gonadotrophin-induced AREG, EREG, BTC and EGFR expressions were quantified in cumulus and mural granulosa cells during IVM. A rapid induction of AREG expression was apparent in both cell types within 30 min of gonadotrophin exposure in vitro. LHCGR (LHR) was detected in mural cells and FSHR in both cumulus and mural granulosa cells. The data confirm the involvement of AREG and EGFR during gonadotrophin-induced cumulus expansion, oocyte maturation and the acquisition of developmental competence by sheep oocytes matured in vitro.
[Show abstract][Hide abstract] ABSTRACT: Amino acid profiling has been used to distinguish between human embryos of differing developmental competence. We sought to determine whether amino acid profiling could be used to distinguish between metaphase II (MII) bovine oocytes with different developmental capabilities in vitro. Amino acid turnover was assayed during the final 6 h of in vitro maturation prior to oocytes undergoing individual fertilization in vitro. Following insemination, zygotes were immobilized in groups of 16 on the base of a Petri dish using Cell-Tak tissue adhesive to enable the developmental progress of each to be tracked to the blastocyst stage. Spent droplets of in vitro maturation medium were analyzed by high performance liquid chromatography, which revealed glutamine, arginine, and asparagine were depleted in the greatest quantities. Incompetent MII oocytes that failed to cleave by 72 h postfertilization depleted significantly more glutamine from (P = 0.0006) and released more alanine (P = 0.0001) into the medium than oocytes that cleaved. When cutoff values were selected for the turnover of alanine, arginine, glutamine, leucine, and tryptophan and modeled to predict fertilization and cleavage potential, oocytes that did not exceed the cutoff values for ≥2 of these key amino acids were more likely to cleave. The sensitivity, specificity, accuracy, and positive predictive value of this model were 60.5%, 76.8%, 63.5%, and 92.0%, respectively. Significant differences (P ≤ 0.015) in the consumption/production of alanine and glutamine were also observed when comparing uncleaved oocytes with those that produced blastocysts. The data show that noninvasive amino acid profiling can be used to measure oocyte developmental competence.
Biology of Reproduction 02/2012; 86(5):165, 1-12. · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metabolic studies of mammalian embryos started with the development of in vitro culture systems more than 40 years ago. More recently, metabolic studies have begun to shed light on the requirements of growing oocytes/follicles from the earliest stages of folliculogenesis. While growing oocytes preferentially metabolise pyruvate over glucose, the somatic compartment of ovarian follicles is more glycolytic. The metabolic preferences of the oocyte are reflected in the early zygote, which becomes increasingly dependent on glycolytic energy production as development progresses to the blastocyst stage. Furthermore, the intricate metabolic relationship between each oocyte and its somatic surroundings is critical for oocyte growth and developmental competence. Measurements of amino acid turnover in bovine oocytes indicate that glutamine, arginine and leucine are consistently depleted, while alanine is produced, showing similarities with amino acid turnover in preimplantation embryos. Amino acid profiling is a good predictor of embryo quality and might also turn out to be a predictor of oocyte developmental competence. Finally, recent studies have uncovered lipid metabolism in oocytes and early embryos, suggesting that endogenous fatty acids might be used for energy production. Together, metabolic studies have revealed the multiplicity of energetic substrates used by oocytes and early embryos, and suggest that the versatility of the metabolic pathways available for energy production is key for high developmental potential. Metabolic studies of early embryos are now being applied to follicle culture, and the goal of describing the metabolome of the growing oocyte in its follicle is now very attainable.
The International journal of developmental biology 01/2012; 56(10-11-12):799-808. · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transient Receptor Potential Melastatin 3 (TRPM3) is a widely expressed calcium-permeable non-selective cation channel that is stimulated by high concentrations of nifedipine or by physiological steroids that include pregnenolone sulphate. Here we sought to identify steroids that inhibit TRPM3. Channel activity was studied using calcium-measurement and patch-clamp techniques. Progesterone (0.01-10μM) suppressed TRPM3 activity evoked by pregnenolone sulphate. Progesterone metabolites and 17β-oestradiol were also inhibitory but the effects were relatively small. Dihydrotestosterone was an inhibitor at concentrations higher than 1μM. Corticosteroids lacked effect. Overlay assays indicated that pregnenolone sulphate, progesterone and dihydrotestosterone bound to TRPM3. In contrast to dihydrotestosterone, progesterone inhibited nifedipine-evoked TRPM3 activity or activity in the absence of an exogenous activator, suggesting a pregnenolone sulphate-independent mechanism of action. Dihydrotestosterone, like a non-steroid look-alike compound, acted as a competitive antagonist at the pregnenolone sulphate binding site. Progesterone inhibited endogenous TRPM3 in vascular smooth muscle cells. Relevance of TRPM3 or the progesterone effect to ovarian cells, which have been suggested to express TRPM3, was not identified. The data further define a chemical framework for competition with pregnenolone sulphate at TRPM3 and expand knowledge of steroid interactions with TRPM3, suggesting direct steroid binding and pregnenolone sulphate-independent inhibition by progesterone.
[Show abstract][Hide abstract] ABSTRACT: Familial biparental hydatidiform mole (FBHM) is the only known pure maternal-effect recessive inherited disorder in humans. Affected women, although developmentally normal themselves, suffer repeated pregnancy loss because of the development of the conceptus into a complete hydatidiform mole in which extraembryonic trophoblastic tissue develops but the embryo itself suffers early demise. This developmental phenotype results from a genome-wide failure to correctly specify or maintain a maternal epigenotype at imprinted loci. Most cases of FBHM result from mutations of NLRP7, but genetic heterogeneity has been demonstrated. Here, we report biallelic mutations of C6orf221 in three families with FBHM. The previously described biological properties of their respective gene families suggest that NLRP7 and C6orf221 may interact as components of an oocyte complex that is directly or indirectly required for determination of epigenetic status on the oocyte genome.
The American Journal of Human Genetics 09/2011; 89(3):451-8. · 11.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report the first quantitative assessment of DNA methylation for any gene in the human preimplantation embryo to reveal that imprints exist at KvDMR1, RB1, SNRPN, and GRB10 in the human blastocyst. For comparison, in two human embryonic stem cell lines, imprints were also observed at KvDMR1, SNRPN, GRB10, and other imprinted loci, whereas RB1 and MEG3 were hypermethylated.
Fertility and sterility 05/2011; 95(8):2564-7.e1-8. · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Information on the ovarian follicle reserve in the African elephant (Loxodonta africana) is lacking. This study set out to determine the ratios of early preantral follicles and their relative dimensions in the ovaries of 16 African elephant aged 10-34 years. The ovaries were sectioned histologically. Follicles were counted and classified according to expansion of the pre-granulosa cells. Early primary follicles were the most common (75.8%±11.8%), followed by true primary follicles (23.8%±11.8%), whereas primordial follicles were the most rare (<2%). Measurements made on at least 100 early preantral follicles from each animal (n=1464) indicate that growth in oocyte and nuclear diameters started with transition to the true primary stage P<0.01. This, together with the observed ratios between the three types of early preantral follicles suggest that both classical primordial and early primary follicles contribute to the ovarian reserve in the African elephant.
[Show abstract][Hide abstract] ABSTRACT: Ovarian tissue cryopreservation, in combination with autotransplantation or long-term culture, has been proposed as a means of fertility preservation. However follicle density within ovarian cortex has a profound impact on the success of in vivo and in vitro systems designed to support follicle growth and restore fertility. The objective of this study was to investigate the dye neutral red (NR) as a tool to quantify follicle density in situ, without compromising follicle viability and developmental potential.
In the first experimental series thin slices of cryopreserved and fresh ovine cortical tissue were incubated in 50 μg/ml NR and assessed for the presence of red colouration. Slices were then used for follicular structure isolation and viability evaluation using 5-(and 6)-carboxyfluoresceindiacetate succinimidylester (CFDA-SE), or prepared histologically for follicle counting or evaluation of apoptosis via terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL). An additional subset of slices were cultured for 8 days, followed by re-evaluation of follicle viability. NR staining was further assessed in a pilot study using thin slices of cryopreserved human ovarian tissue donated by 17 patients undergoing laparoscopic sterilisation or elective Caesarean section.
In both ovine and human ovarian cortex NR concentrated in follicular structures within weakly stained stroma. NR colouration was observed in 41.7 ± 4.6% of cryopreserved and 49.3 ± 6.5% of the fresh ovine tissue slices, and NR staining was consistently predictive of the presence of follicles. Non-stained ovine slices contained highly apoptotic follicles, while lower levels of apoptosis were observed in NR positive slices, indicating preferential detection of viable follicles by NR. Following culture the majority of ovine slices re-stained with NR, no significant increases in the levels of apoptosis were observed and 94.6 ± 3.1% of follicles were viable by CFDA-SE. In the human study, NR identified follicles in 19.3 ± 3.7% of tissue slices, and follicle density tended to decrease with advancing patient age.
NR predicts viable follicle density in situ in slices of ovine and human ovarian cortex. Furthermore incubation of tissue in NR prior to culture does not compromise subsequent follicle survival in vitro, indicating the potential suitability of this approach in fertility preservation regimes.
Human Reproduction 10/2010; 25(10):2559-68. · 4.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polycystic ovary syndrome (PCOS) is associated with metabolic disturbances which include impaired insulin signalling and glucose metabolism in ovarian follicles. The oocyte is metabolically dependent upon its follicle environment during development, but it is unclear whether PCOS or polycystic ovarian (PCO) morphology alone affect oocyte metabolism and energy-demanding processes such as meiosis.
Immature human oocytes were donated by PCOS (n = 14), PCO (n = 14) and control (n = 46) patients attending the assisted conception programme at Leeds Teaching Hospitals NHS Trust. Oocytes were cultured individually and carbohydrate metabolism was assessed during overnight in vitro maturation (IVM). Meiotic status was assessed and oocyte intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H) content and mitochondria activity were measured prior to karyotype analysis by multifluor in situ hybridization.
Patient aetiology had no significant effect on oocyte maturation potential or incidence of numerical chromosome abnormalities (44%), although PCOS and PCO oocytes were more likely to suffer predivision. Group G chromosomes were most likely to be involved in non-disjunction and predivision. PCOS was associated with increased glucose consumption (2.06 +/- 0.43 and 0.54 +/- 0.12 pmol/h for PCOS and control oocytes, respectively) and increased pyruvate consumption (18.4 +/- 1.2 and 13.9 +/- 0.9 pmol/h for PCOS and control oocytes, respectively) during IVM. Prior prescription of metformin significantly attenuated pyruvate consumption by maturing oocytes (8.5 +/- 1.8 pmol/h) from PCOS patients. Oocytes from PCO patients had intermediate metabolism profiles. Higher pyruvate turnover was associated with abnormal oocyte karyotypes (13.4 +/- 1.9 and 19.9 +/- 2.1 pmol/h for normal versus abnormal oocytes, respectively). Similarly, oocyte NAD(P)H content was 1.35-fold higher in abnormal oocytes.
The chromosomal constitution of in vitro matured oocytes from PCOS is similar to that of controls, but aspects of oocyte metabolism are perturbed by PCOS. Elevated pyruvate consumption was associated with abnormal oocyte karyotype.
Human Reproduction 09/2010; 25(9):2305-15. · 4.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study investigated the relationship between human preimplantation embryo metabolism and aneuploidy rates during development in vitro. One hundred and eighty-eight fresh and cryopreserved embryos from 59 patients (33.9 +/- 0.6 years) were cultured for 2-5 days. The turnover of 18 amino acids was measured in spent media by high-performance liquid chromatography. Embryos were either fixed for interphase fluorescent in situ hybridization analysis of chromosomes 13, 18, 19, 21, X or Y, or were assayed for mitochondrial activity. Amino acid turnover was different (P < 0.05) between stage-matched fresh and cryopreserved embryos due to blastomere loss following warming. The proportion of embryos with aneuploid cells increased as cell division progressed from pronucleate- (23%) to late cleavage stages (50-70%). Asparagine, glycine and valine turnover was significantly different between uniformly genetically normal and uniformly abnormal embryos on Days 2-3 of culture. By Days 3-4, the profiles of serine, leucine and lysine differed between uniformly euploid versus aneuploid embryos. Gender significantly (P < 0.05) affected the metabolism of tryptophan, leucine and asparagine by cleavage-stage embryos. Pronucleate zygotes had a significantly higher proportion of active:inactive mitochondria compared with cleavage-stage embryos. Furthermore, mitochondrial activity was correlated (P < 0.05) with altered aspartate and glutamine turnover. These results demonstrate the association between the metabolism, cytogenetic composition and health of human embryos in vitro.
Molecular Human Reproduction 08/2010; 16(8):557-69. · 4.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND Female cancer patients are offered 'banking' of gametes before starting fertility-threatening cancer therapy. Transplants of fresh and frozen ovarian tissue between healthy fertile and infertile women have demonstrated the utility of the tissue banked for restoration of endocrine and fertility function. Additional methods, like follicle culture and isolated follicle transplantation, are in development. METHODS Specialist reproductive medicine scientists and clinicians with complementary expertise in ovarian tissue culture and transplantation presented relevant published literature in their field of expertise and also unpublished promising data for discussion. As the major aims were to identify the current gaps prohibiting advancement, to share technical experience and to orient new research, contributors were allowed to provide their opinioned expert views on future research. RESULTS Normal healthy children have been born in cancer survivors after orthotopic transplantation of their cryopreserved ovarian tissue. Longevity of the graft might be optimized by using new vitrification techniques and by promoting rapid revascularization of the graft. For the in vitro culture of follicles, a successive battery of culture methods including the use of defined media, growth factors and three-dimensional extracellular matrix support might overcome growth arrest of the follicles. Molecular methods and immunoassay can evaluate stage of maturation and guide adequate differentiation. Large animals, including non-human primates, are essential working models. CONCLUSIONS Experiments on ovarian tissue from non-human primate models and from consenting fertile and infertile patients benefit from a multidisciplinary approach. The new discipline of oncofertility requires professionalization, multidisciplinarity and mobilization of funding for basic and translational research.
Human Reproduction Update 01/2010; 16(4):395-414. · 9.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The development of technologies to grow oocytes from the most abundant primordial follicles to maturity in vitro holds many attractions for clinical practice, animal production technology and research. The production of fertile oocytes and live offspring has been achieved in mice following the long-term culture of oocytes in primordial follicles from both fresh and cryopreserved ovarian tissue. In contrast, in non-rodent species advances in follicle culture are centred on the growth of isolated preantral follicles. As a functional unit, mammalian preantral follicles are well-suited to culture but primordial and primary follicles do not grow well after isolation from the ovarian stroma. The current challenges for follicle culture are numerous and include: optimisation of culture media and the tailoring of culture environments to match the physiological needs of the cell in vivo; the maintenance of cell-cell communication and signalling during culture; and the evaluation of the epigenetic status, genetic health and fertility of in vitro derived mature oocytes. In large animals and humans, the complete in vitro growth and maturation of oocytes is only likely to be achieved following the development of a multistage strategy that closely mimics the ovary in vivo. In this approach, primordial follicle growth will be initiated in situ by the culture of ovarian cortex. Isolated preantral follicles will then be grown to antral stages before steroidogenic function is induced in the somatic cells. Finally, cytoplasmic and nuclear maturation will be induced in the in vitro derived oocytes with the production of fertile metaphase II gametes.