Oocyte and cumulus cell cooperativity and metabolic plasticity under the direction of oocyte paracrine factors.

Mammalian oocytes develop and mature in a mutually dependent relationship with surrounding cumulus cells. The oocyte actively regulates cumulus cell differentiation and function by secreting soluble paracrine oocyte-secreted factors (OSFs). We characterized the molecular mechanisms by which two model OSFs, cumulin and BMP15, regulate oocyte maturation and cumulus-oocyte cooperativity. Exposure to these OSFs during mouse oocyte maturation in vitro altered the proteomic and multispectral autofluorescence profiles of both the oocyte and cumulus cells. In oocytes, cumulin significantly upregulated proteins involved in nuclear function. In cumulus cells, both OSFs elicited marked upregulation of a variety of metabolic processes (mostly anabolic), including lipid, nucleotide, and carbohydrate metabolism, whereas mitochondrial metabolic processes were downregulated. The mitochondrial changes were validated by functional assays confirming altered mitochondrial morphology, respiration, and content while maintaining ATP homeostasis. Collectively, these data demonstrate that cumulin and BMP15 remodel cumulus cell metabolism, instructing them to upregulate their anabolic metabolic processes, while routine cellular functions are minimized in the oocyte during maturation, in preparation for ensuing embryonic development.NEW & NOTEWORTHY Oocyte-secreted factors (OSFs) promote oocyte and cumulus cell cooperativity by altering the molecular composition of both cell types. OSFs downregulate protein catabolic processes and upregulate processes associated with DNA binding, translation, and ribosome assembly in oocytes. In cumulus cells, OSFs alter mitochondrial number, morphology, and function, and enhance metabolic plasticity by upregulating anabolic pathways. Hence, the oocyte via OSFs, instructs cumulus cells to increase metabolic processes on its behalf, thereby subduing oocyte metabolism.

Reproductive Ageing: Metabolic contribution to age-related chromosome missegregation in mammalian oocytes.

In brief: Chromosome missegregation and declining energy metabolism are considered to be unrelated features of oocyte ageing that contribute to poor reproductive outcomes. Given the bioenergetic cost of chromosome segregation, we propose here that altered energy metabolism during ageing may be an underlying cause of age-related chromosome missegregation and aneuploidy. Abstract: Advanced reproductive age in women is a major cause of infertility, miscarriage and congenital abnormalities. This is principally caused by a decrease in oocyte quality and developmental competence with age. Oocyte ageing is characterised by an increase in chromosome missegregation and aneuploidy. However, the underlying mechanisms of age-related aneuploidy have not been fully elucidated and are still under active investigation. In addition to chromosome missegregation, oocyte ageing is also accompanied by metabolic dysfunction. In this review, we integrate old and new perspectives on oocyte ageing, chromosome segregation and metabolism in mammalian oocytes and make direct links between these processes. We consider age-related alterations to chromosome segregation machinery, including the loss of cohesion, microtubule stability and the integrity of the spindle assembly checkpoint. We focus on how metabolic dysfunction in the ageing oocyte disrupts chromosome segregation machinery to contribute to and exacerbate age-related aneuploidy. More specifically, we discuss how mitochondrial function, ATP production and the generation of free radicals are altered during ageing. We also explore recent developments in oocyte metabolic ageing, including altered redox reactions (NAD+ metabolism) and the interactions between oocytes and their somatic nurse cells. Throughout the review, we integrate the mechanisms by which changes in oocyte metabolism influence age-related chromosome missegregation.

Serum GDF9 and BMP15 as potential markers of ovarian function in women with and without polycystic ovary syndrome.

OBJECTIVE: Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are critical paracrine regulators of female fertility and are predominantly expressed by oocytes. However, it is unknown if serum concentrations reflect changes in ovarian function and/or reproductive endocrine disorders. This study aimed to determine if serum GDF9/BMP15 are associated with ovarian, pituitary, oestrogenic, androgenic and metabolic characteristics and the ovarian pathologies, polycystic ovarian morphology (PCOM) and polycystic ovary syndrome (PCOS). DESIGN: Women aged 21-45 years (n = 381) were included from a cross-sectional study at the National University Hospital, Singapore. PATIENTS: Participants were volunteers and patients with possible PCOS. MEASUREMENTS: Anthropometric measurements, transvaginal ultrasound scans and serum sampling were performed and a questionnairecompleted. Serum GDF9 and BMP15 concentrations were matched with menstrual cycle length, ovarian protein and steroid hormone production, pituitary hormone production and metabolic assessments in women with PCOM or PCOS and those with neither (control). RESULTS: Serum GDF9 and BMP15 were detectable in 40% and 41% of women, respectively and were positively correlated with each other (r = 0.08, p = 0.003). GDF9, but not BMP15, was positively correlated with ovarian volume (p = 0.02) and antral follicle count (AFC) (p = 0.004), but not with anti-Müllerian hormone (p = 0.05). However, serum GDF9 and BMP15 concentrations were not significantly different between control, PCOM and PCOS women, nor associated with androgenic or metabolic PCOS features. However, the relationship between GDF9 and AFC differed between control, PCOM and PCOS women (p = 0.02). CONCLUSIONS: Serum GDF9 and BMP15 concentrations somewhat reflect ovarian but not androgenic or metabolic characteristics of PCOS, with increased GDF9 reflecting high AFC as seen in PCOM/PCOS.

Androgen signaling in adipose tissue, but less likely skeletal muscle, mediates development of metabolic traits in a PCOS mouse model.

Polycystic ovary syndrome (PCOS) is a common, multifactorial disorder characterized by endocrine, reproductive, and metabolic dysfunction. As the etiology of PCOS is unknown, there is no cure and symptom-oriented treatments are suboptimal. Hyperandrogenism is a key diagnostic trait, and evidence suggests that androgen receptor (AR)-mediated actions are critical to PCOS pathogenesis. However, the key AR target sites involved remain to be fully defined. Adipocyte and muscle dysfunction are proposed as important sites involved in the manifestation of PCOS traits. We investigated the role of AR signaling in white adipose tissue (WAT), brown adipose tissue (BAT), and skeletal muscle in the development of PCOS in a hyperandrogenic PCOS mouse model. As expected, dihydrotestosterone (DHT) exposure induced key reproductive and metabolic PCOS traits in wild-type (WT) females. Transplantation of AR-insensitive (AR-/-) WAT or BAT from AR knockout females (ARKO) into DHT-treated WT mice ameliorated some metabolic PCOS features, including increased body weight, adiposity, and adipocyte hypertrophy, but not reproductive PCOS traits. In contrast, DHT-treated ARKO female mice transplanted with AR-responsive (AR+/+) WAT or BAT continued to resist developing PCOS traits. DHT-treated skeletal muscle-specific AR knockout females (SkMARKO) displayed a comparable phenotype with that of DHT-treated WT females, with full development of PCOS traits. Taken together, these findings infer that both WAT and BAT, but less likely skeletal muscle, are key sites of AR-mediated actions involved in the experimental pathogenesis of metabolic PCOS traits. These data further support targeting adipocyte AR-driven pathways in future research aimed at developing novel therapeutic interventions for PCOS.NEW & NOTEWORTHY Hyperandrogenism is a key feature in the pathogenesis of polycystic ovary syndrome (PCOS); however, the tissue sites of androgen receptor (AR) signaling are unclear. In this study, AR signaling in white and brown adipose tissue, but less likely in skeletal muscle, was found to be involved in the development of metabolic PCOS traits, highlighting the importance of androgen actions in adipose tissue and obesity in the manifestation of metabolic disturbances.

Approaches to oocyte meiotic arrest in vitro and impact on oocyte developmental competence.

Oocytes are maintained in a state of meiotic arrest following the first meiotic division until ovulation is triggered. Within the antral follicle, meiotic arrest is actively suppressed in a process facilitated by the cyclic nucleotides cGMP and cAMP. If removed from this inhibitory follicular environment and cultured in vitro, mammalian oocytes undergo spontaneous meiotic resumption in the absence of the usual stimulatory follicular stimuli, leading to asynchronicity with oocyte cytoplasmic maturation and lower developmental competence. For more than 50 years, pharmacological agents have been used to attenuate oocyte germinal vesicle (GV) breakdown in vitro. Agents that increase intra-oocyte cAMP or prevent its degradation have been predominantly used; however, agents such as kinase and protein synthesis inhibitors have also been trialed. Twenty years of research demonstrates that maintaining GV arrest for a period before in vitro maturation (IVM) improves oocyte developmental competence, and is likely attributed to maintenance of bidirectional communication with cumulus cells leading to improved oocyte metabolic function. However, outcomes are influenced by various factors including the mode of action of the modulators, dose, treatment duration, species, and the degree of hormonal priming of the oocyte donor. Cyclic GMP and/or cAMP modulation in a prematuration step (called pre-IVM) prior to IVM has shown the greatest consistency in improving oocyte developmental competence, whereas kinase and protein synthesis inhibitors have proven less effective at improving IVM outcomes. Such pre-IVM approaches have shown potential to alter current use of artificial reproductive technologies in medical and veterinary practice.

Multispectral autofluorescence characteristics of reproductive aging in old and young mouse oocytes.

Increasing age has a major detrimental impact on female fertility, which, with an ageing population, has major sociological implications. This impact is primarily mediated through deteriorating quality of the oocyte. Deteriorating oocyte quality with biological age is the greatest rate-limiting factor to female fertility. Here we have used label-free, non-invasive multi-spectral imaging to identify unique autofluorescence profiles of oocytes from young and aged animals. Discriminant analysis demonstrated that young oocytes have a distinct autofluorescent profile which accurately distinguishes them from aged oocytes. We recently showed that treatment with the nicotinamide adenine dinucleotide (NAD+) precursor nicotinamide mononucleotide (NMN) restored oocyte quality and fertility in aged animals, and when our analysis was applied to oocytes from aged animals treated with NMN, 85% of these oocytes were classified as having the autofluorescent signature of young animals. Spectral unmixing using the Robust Dependent Component Analysis (RoDECA) algorithm demonstrated that NMN treatment altered the metabolic profile of oocytes, increasing free NAD(P)H, protein bound NAD(P)H, redox ratio and the ratio of bound to free NAD(P)H. The frequency of oocytes with simultaneously high NAD(P)H and flavin content was also significantly increased in mice treated with NMN. Young and Aged + NMN oocytes had a smoother spectral distribution, with the distribution of NAD(P)H in young oocytes specifically differing from that of aged oocytes. Identifying the multispectral profile of oocyte autofluorescence during aging could have utility as a non-invasive and sensitive measure of oocyte quality.

Effect of cumulin and super-GDF9 in standard and biphasic mouse IVM.

PURPOSE: In vitro maturation (IVM) is a technology that generates mature oocytes following culture of immature cumulus-oocyte complexes (COC) in vitro. IVM is characterized by minimal patient stimulation, making it attractive for certain patient groups. Recently, a biphasic IVM system, capacitation (CAPA)-IVM, has shown improved clinical outcomes relative to standard IVM; however, it remains less efficient than IVF. This study assessed whether supplementation of CAPA-IVM culture media with the novel TGFß superfamily proteins cumulin and super-GDF9 improves subsequent mouse embryo development. METHODS: Immature mouse COCs were cultured by standard IVM or biphasic IVM ± cumulin or super-GDF9. RESULTS: Both cumulin and super-GDF9 in standard IVM significantly improved day-6 blastocyst rate (53.9% control, 73.6% cumulin, 70.4% super-GDF9; p = 0.006; n = 382-406 oocytes). Cumulin or super-GDF9 in CAPA-IVM did not alter embryo yield or blastocyst cell allocation in an unstimulated model. Moreover, cumulin did not alter these outcomes in a mild PMSG stimulation model. Cumulin in CAPA-IVM significantly increased cumulus cell expression of cumulus expansion genes (Ptgs2, Ptx3, Adamts1, Gfat2) and decreased Lhr expression relative to control. However, cumulin-induced mRNA expression of cumulus cell (Ptgs2, Ptx3) and oocyte genes (Gdf9, Bmp15, Oct4, Stella) in CAPA-IVM remained significantly lower than that of in vivo matured cells. CONCLUSION: Cumulin did not provide an additional beneficial effect in biphasic IVM in terms of blastocyst yield and cell allocation; however in standard IVM, cumulin and super-GDF9 significantly improve oocyte developmental competence.

A variant of human growth differentiation factor-9 that improves oocyte developmental competence.

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are co-expressed exclusively in oocytes throughout most of folliculogenesis and play central roles in controlling ovarian physiology. Although both growth factors exist as homodimers, recent evidence indicates that GDF9 and BMP15 can also heterodimerize to form the potent growth factor cumulin. Within the cumulin complex, BMP15 "activates" latent GDF9, enabling potent signaling in granulosa cells via type I receptors (i.e. activin receptor-like kinase-4/5 (ALK4/5)) and SMAD2/3 transcription factors. In the cumulin heterodimer, two distinct type I receptor interfaces are formed compared with homodimeric GDF9 and BMP15. Previous studies have highlighted the potential of cumulin to improve treatment of female infertility, but, as a noncovalent heterodimer, cumulin is difficult to produce and purify without contaminating GDF9 and BMP15 homodimers. In this study we addressed this challenge by focusing on the cumulin interface formed by the helix of the GDF9 chain and the fingers of the BMP15 chain. We demonstrate that unique BMP15 finger residues at this site (Arg301, Gly304, His307, and Met369) enable potent activation of the SMAD2/3 pathway. Incorporating these BMP15 residues into latent GDF9 generated a highly potent growth factor, called hereafter Super-GDF9. Super-GDF9 was >1000-fold more potent than WT human GDF9 and 4-fold more potent than cumulin in SMAD2/3-responsive transcriptional assays in granulosa cells. Our demonstration that Super-GDF9 can effectively promote mouse cumulus cell expansion and improve oocyte quality in vitro represents a potential solution to the current challenges of producing and purifying intact cumulin.

Impact of nicotinamide mononucleotide on transplanted mouse ovarian tissue.

Ovarian tissue cryopreservation and future transplantation is the only strategy to preserve the fertility of young female adolescent and prepubertal patients. The primary challenge to ovarian graft longevity is the substantial loss of primordial follicles during the period of ischaemia post-transplantation. Nicotinamide mononucleotide (NMN), a precursor of the essential metabolite NAD+, is known to reduce ischaemic damage. Therefore, the objective of the current study was to assess the impact of short- and long-term NMN administration on follicle number and health following ovarian tissue transplantation. Hemi-ovaries from C57Bl6 mice (n = 8-12/group) were transplanted under the kidney capsule of bilaterally ovariectomised severe combined immunodeficient (SCID) mice. Recipient mice were administered either normal drinking water or water supplemented with NMN (2 g/L) for either 14 or 56 days. At the end of each treatment period, ovarian transplants were collected. There was no effect of NMN on the resumption of oestrous or length of oestrous cycles. Transplantation significantly reduced the total number of follicles with the greatest impact observed at the primordial follicle stage. We report that NMN did not prevent this loss. While NMN did not significantly impact the proportion of apoptotic follicles, NMN normalised PCNA expression at the primordial and intermediate stages but not at later stages. In conclusion, NMN administration did not prevent ovarian follicle loss under the conditions of this study.

Perspectives on the development and future of oocyte IVM in clinical practice.

Oocyte in vitro maturation (IVM) is an assisted reproductive technology designed to obtain mature oocytes following culture of immature cumulus-oocyte complexes collected from antral follicles. Although IVM has been practiced for decades and is no longer considered experimental, the uptake of IVM in clinical practice is currently limited. The purpose of this review is to ensure reproductive medicine professionals understand the appropriate use of IVM drawn from the best available evidence supporting its clinical potential and safety in selected patient groups. This group of scientists and fertility specialists, with expertise in IVM in the ART laboratory and/or clinic, explore here the development of IVM towards acquisition of a non-experimental status and, in addition, critically appraise the current and future role of IVM in human ART.

In-vitro maturation of oocytes versus conventional IVF in women with infertility and a high antral follicle count: a randomized non-inferiority controlled trial.

STUDY QUESTION: Is one cycle of IVM non-inferior to one cycle of conventional in IVF with respect to live birth rates in women with high antral follicle counts (AFCs)? SUMMARY ANSWER: We could not demonstrate non-inferiority of IVM compared with IVF. WHAT IS KNOWN ALREADY: IVF with ovarian hyperstimulation has limitations in some subgroups of women at high risk of ovarian stimulation, such as those with polycystic ovary syndrome. IVM is an alternative ART for these women. IVM may be a feasible alternative to IVF in women with a high AFC, but there is a lack of data from randomized clinical trials comparing IVM with IVF in women at high risk of ovarian hyperstimulation syndrome. STUDY DESIGN, SIZE, DURATION: This single-center, randomized, controlled non-inferiority trial was conducted at an academic infertility center in Vietnam from January 2018 to April 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 546 women with an indication for ART and a high AFC (≥24 follicles in both ovaries) were randomized to the IVM (n = 273) group or the IVF (n = 273) group; each underwent one cycle of IVM with a prematuration step versus one cycle of IVF using a standard gonadotropin-releasing hormone antagonist protocol with gonadotropin-releasing hormone agonist triggering. The primary endpoint was live birth rate after the first embryo transfer. The non-inferiority margin for IVM versus IVF was -10%. MAIN RESULTS AND THE ROLE OF CHANCE: Live birth after the first embryo transfer occurred in 96 women (35.2%) in the IVM group and 118 women (43.2%) in the IVF group (absolute risk difference -8.1%; 95% confidence interval (CI) -16.6%, 0.5%). Cumulative ongoing pregnancy rates at 12 months after randomization were 44.0% in the IVM group and 62.6% in the IVF group (absolute risk difference -18.7%; 95% CI -27.3%, -10.1%). Ovarian hyperstimulation syndrome did not occur in the IVM group, versus two cases in the IVF group. There were no statistically significant differences between the IVM and IVF groups with respect to the occurrence of pregnancy complications, obstetric and perinatal complications, preterm delivery, birth weight and neonatal complications. LIMITATIONS, REASONS FOR CAUTION: The main limitation of the study was its open-label design. In addition, the findings are only applicable to IVM conducted using the prematuration step protocol used in this study. Finally, the single ethnicity population limits the external generalizability of the findings. WIDER IMPLICATIONS OF THE FINDINGS: Our randomized clinical trial compares live birth rates after IVM and IVF. Although IVM is a viable and safe alternative to IVF that may be suitable for some women seeking a mild ART approach, the current study findings approach inferiority for IVM compared with IVF when cumulative outcomes are considered. Future research should incorporate multiple cycles of IVM in the study design to estimate cumulative fertility outcomes and better inform clinical decision-making. STUDY FUNDING/COMPETING INTEREST(S): This work was partly supported by Ferring grant number 000323 and funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) and by the Fund for Research Flanders (FWO). LNV has received speaker and conference fees from Merck, grant, speaker and conference fees from Merck Sharpe and Dohme, and speaker, conference and scientific board fees from Ferring; TMH has received speaker fees from Merck, Merck Sharp and Dohme, and Ferring; RJN has received conference and scientific board fees from Ferring, is a minor shareholder in an IVF company, and receives grant funding from the National Health and Medical Research Council (NHMRC) of Australia; BWM has acted as a paid consultant to Merck, ObsEva and Guerbet, and is the recipient of grant money from an NHMRC Investigator Grant; RBG reports grants and fellowships from the NHMRC of Australia; JS reports lecture fees from Ferring Pharmaceuticals, Biomérieux, Besins Female Healthcare and Merck, grants from Fund for Research Flanders (FWO), and is co-inventor on granted patents on CAPA-IVM methodology in the US (US10392601B2) and Europe (EP3234112B1); TDP, VQD, VNAH, NHG, AHL, THP and RW have no financial relationships with any organizations that might have an interest in the submitted work in the previous three years, and no other relationships or activities that could appear to have influenced the submitted work. TRIAL REGISTRATION NUMBER: NCT03405701 (www.clinicaltrials.gov). TRIAL REGISTRATION DATE: 16 January 2018. DATE OF FIRST PATENT'S ENROLMENT: 25 January 2018.

Neuroendocrine androgen action is a key extraovarian mediator in the development of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a complex hormonal disorder characterized by reproductive, endocrine, and metabolic abnormalities. As the origins of PCOS remain unknown, mechanism-based treatments are not feasible and current management relies on treatment of symptoms. Hyperandrogenism is the most consistent PCOS characteristic; however, it is unclear whether androgen excess, which is treatable, is a cause or a consequence of PCOS. As androgens mediate their actions via the androgen receptor (AR), we combined a mouse model of dihydrotestosterone (DHT)-induced PCOS with global and cell-specific AR-resistant (ARKO) mice to investigate the locus of androgen actions that mediate the development of the PCOS phenotype. Global loss of the AR reveals that AR signaling is required for all DHT-induced features of PCOS. Neuron-specific AR signaling was required for the development of dysfunctional ovulation, classic polycystic ovaries, reduced large antral follicle health, and several metabolic traits including obesity and dyslipidemia. In addition, ovariectomized ARKO hosts with wild-type ovary transplants displayed normal estrous cycles and corpora lutea, despite DHT treatment, implying extraovarian and not intraovarian AR actions are key loci of androgen action in generating the PCOS phenotype. These findings provide strong evidence that neuroendocrine genomic AR signaling is an important extraovarian mediator in the development of PCOS traits. Thus, targeting AR-driven mechanisms that initiate PCOS is a promising strategy for the development of novel treatments for PCOS.

Live births after oocyte in vitro maturation with a prematuration step in women with polycystic ovary syndrome.

PURPOSE: Standard oocyte in vitro maturation (IVM) usually results in lower pregnancy rates than in vitro fertilization (IVF). IVM preceded by a prematuration step improves the acquisition of oocyte developmental competence and can enhance embryo quality (EQ). This study evaluated the effectiveness of a biphasic culture system incorporating prematuration and IVM steps (CAPA-IVM) versus standard IVM in women with polycystic ovarian morphology (PCOM). METHODS: Eighty women (age < 38 years, ≥ 25 follicles of 2-9 mm in both ovaries, no major uterine abnormalities) were randomized to undergo CAPA-IVM (n = 40) or standard IVM (n = 40). CAPA-IVM uses two steps: a 24-h prematuration step with C-type natriuretic peptide-supplemented medium, then 30 h of culture in IVM media supplemented with follicle-stimulating hormone and amphiregulin. Standard IVM was performed using routine protocols. RESULTS: A significantly higher proportion of oocytes reached metaphase II at 30 h after CAPA-IVM versus standard IVM (63.6 vs 49.0; p < 0.001) and the number of good quality embryos per cumulus-oocyte complex tended to be higher (18.9 vs 12.7; p = 0.11). Clinical pregnancy rate per embryo transfer was 63.2% in the CAPA-IVM versus 38.5% in the standard IVM group (p = 0.04). Live birth rate per embryo transfer was not statistically different between the CAPA-IVM and standard IVM groups (50.0 vs 33.3% [p = 0.17]). No malformations were reported and birth weight was similar in the two treatment groups. CONCLUSIONS: Use of the CAPA-IVM system significantly improved maturation and clinical pregnancy rates versus standard IVM in patients with PCOM. Furthermore, live births after CAPA-IVM are reported for the first time.

Biphasic in vitro maturation (CAPA-IVM) specifically improves the developmental capacity of oocytes from small antral follicles.

PURPOSE: To investigate the effectiveness of a biphasic IVM culture strategy at improving IVM outcomes in oocytes from small follicles (< 6 mm) compared with routine Standard IVM in patients with polycystic ovaries. METHODS: This prospective pilot study was performed in 40 women with polycystic ovaries whose oocytes were randomized to two IVM culture methods. Patients received a total stimulation dose of 450 IU rFSH. Cumulus-oocyte complexes (COCs) from follicles < 6 mm and ≥ 6 mm were retrieved and cultured separately in either a prematuration medium with c-type natriuretic peptide followed by IVM (CAPA-IVM), or STD-IVM. Primary outcomes were maturation rate, embryo quality, and the number of vitrified day 3 embryos per patient. RESULTS: Use of the CAPA-IVM system led to a significant improvement in oocyte maturation (p < 0.05), to a doubling in percentage of good and top-quality day 3 embryos per COC, and to an increased number of vitrified day 3 embryos (p < 0.001), compared to STD IVM. Oocytes from follicles < 6 mm benefited most from CAPA-IVM, showing a significant increase in the amount of good and top-quality embryos compared to STD IVM. CAPA-IVM yielded significantly (p < 0.0001) less GV-arrested oocytes and larger oocyte diameters (p < 0.05) than STD IVM. CONCLUSIONS: CAPA-IVM brings significant improvements in maturation and embryological outcomes, most notably to oocytes from small antral follicles (< 6 mm), which can be easily retrieved from patients with a minimal ovarian stimulation. The study demonstrates the robustness and transferability of the CAPA-IVM method across laboratories and populations.

In-vitro regulation of primordial follicle activation: challenges for fertility preservation strategies.

Ovarian tissue is increasingly being collected from cancer patients and cryopreserved for fertility preservation. While the only available option to restore fertility is autologous transplantation, this treatment is not appropriate for all patients due to the risk of reintroducing cancer cells and causing disease recurrence. Harnessing the full reproductive potential of this tissue to restore fertility requires the development of culture systems that support oocyte development from the primordial follicle stage. While this has been achieved in the mouse, the goal of obtaining oocytes of sufficient quality to support embryo development has not been reached in higher mammals despite decades of effort. In vivo, primordial follicles gradually exit the resting pool, whereas when primordial follicles are placed into culture, global activation of these follicles occurs. Therefore, the addition of a factor(s) that can regulate primordial follicle activation in vitro may be beneficial to the development of culture systems for ovarian tissue from cancer patients. Several factors have been observed to inhibit follicle activation, including anti-Müllerian hormone, stromal-derived factor 1 and members of the c-Jun-N-terminal kinase pathway. This review summarizes the findings from studies of these factors and discusses their potential integration into ovarian tissue culture strategies for fertility preservation.

Effect of pre-in vitro maturation with cAMP modulators on the acquisition of oocyte developmental competence in cattle.

The administration of follicle-stimulating hormone (FSH) prior to oocyte retrieval improves oocyte developmental competence. During bovine embryo production in vitro, however, oocytes are typically derived from FSH-unprimed animals. In the current study, we examined the effect of pre-in vitro maturation (IVM) with cAMP modulators, also known as the second messengers of FSH, on the developmental competence of oocytes derived from small antral follicles (2-4 mm) of FSH-unprimed animals. Pre-IVM with N6,2'-O-dibutyryladenosine 3',5'-cyclicmonophosphate (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX) for 2 h improved the blastocyst formation in oocytes stimulated by FSH or amphiregulin (AREG). Furthermore, pre-IVM enhanced the expression of the FSH- or AREG-stimulated extracellular matrix-related genes HAS2, TNFAIP6, and PTGS2, and epidermal growth factor (EGF)-like peptide-related genes AREG and EREG. Additionally, pre-IVM with dbcAMP and IBMX enhanced the expression of EGFR, and also increased and prolonged cumulus cell-oocyte gap junctional communication. The improved oocyte development observed using the pre-IVM protocol was ablated by an EGF receptor phosphorylation inhibitor. These results indicate that pre-IVM with cAMP modulators could contribute to the acquisition of developmental competence by bovine oocytes from small antral follicles through the modulation of EGF receptor signaling and oocyte-cumulus/cumulus-cumulus gap junctional communication.

The safety and efficacy of controlled ovarian hyperstimulation for fertility preservation in women with early breast cancer: a systematic review.

STUDY QUESTION: Can controlled ovarian hyperstimulation (COH) for fertility preservation be effectively conducted in women with breast cancer without worsening their prognosis? SUMMARY ANSWER: COH with co-administration of letrozole suppresses oestradiol levels without significantly impacting oocyte yield or decreasing disease-free survival rates. WHAT IS KNOWN ALREADY: Oestradiol has the capacity to stimulate the proliferation of breast cancer cells. COH can cause oestradiol levels to rise by an order of magnitude above physiological levels. Concern exists regarding the effect of supra-physiological oestradiol levels in women with a recent diagnosis of breast cancer. STUDY DESIGN, SIZE, DURATION: A systematic review of the literature was performed using MEDLINE (PubMed database), EMBASE and the Cochrane Library. The search was restricted to articles written in English. No restrictions regarding the date of publication were applied. Safety was assessed in terms of relapse rates and cancer-related mortality rates. Peak oestradiol concentrations were recorded for different stimulation protocols. Efficacy was measured in terms of the total number of oocytes collected and proportion of mature oocytes. The primary outcome was mortality/recurrence in premenopausal women with Stage I-IIIB breast cancer who underwent COH in the immediate post-operative period, prior to chemotherapy. PARTICIPANTS/MATERIALS, SETTING, METHODS: This is a systematic review of randomized control trials (RCTs), case control and cohort studies reporting on the primary outcome of mortality/recurrence after COH in women with early breast cancer, or secondary outcomes of oocyte yield and peak oestrogen concentration. Owing to the small number of RCTs available, other study types were included. The last electronic search was run in April 2016. Two prospective non-randomized studies reported relapse and breast cancer-related mortality rates in 397 women with breast cancer, of whom 149 underwent COH. Twelve studies reported the peak oestradiol concentrations of 882 women undergoing COH with letrozole co-administration. Four studies compared the oocyte yield of 248 women who underwent COH plus letrozole with 254 women who underwent standard COH. Two studies compared peak oestradiol concentrations and oocyte yield in 61 women who underwent COH with tamoxifen co-administration and 49 women who underwent COH without tamoxifen. One study compared letrozole and tamoxifen co-administration, and another study compared the co-administration of letrozole and anastrozole. MAIN RESULTS AND THE ROLE OF CHANCE: The search identified 1002 records of which 15 were included in the final analysis. There was no evidence of a decline in relapse-free survival rates in the two studies of women with breast cancer who received COH with letrozole co-administration compared with women who did not undergo fertility preservation procedures. The largest of these studies reported recurrences in 6/120 (5.0%) women who received COH plus letrozole compared with 12/217 (5.5%) women who did not undergo COH (mean follow-up 5.0 versus 6.9 years; hazard ratio for recurrence 0.77, 95%CI 0.28-2.13). Conclusions regarding women with breast cancer who received tamoxifen during COH could not be made due to insufficient data. Peak oestradiol concentrations (338-829 pg/ml) were suppressed by letrozole when commenced on Days 2-3, with no decrease in oocyte yield. Tamoxifen does not suppress oestradiol concentrations, but may convey protection via its inhibitory action on the oestrogen receptor. LIMITATIONS, REASONS FOR CAUTION: Any statements regarding the safety of COH in women with breast cancer are based on a limited number of observational studies. High quality evidence is unlikely to become available for ethical and practical reasons. Whilst available data do not indicate a decline in disease-free survival, a small effect cannot be excluded. Breast cancers are heterogeneous in their genetic profile and receptor status, making the results of studies difficult to generalize to individual cases. The implication of alterations in other hormone levels such as androgens, progestins or vascular endothelial growth factor secondary to COH in women with breast cancer has not been quantified. WIDER IMPLICATIONS OF THE FINDINGS: The co-administration of 5 mg of letrozole daily commencing on Day 2 and continuing throughout COH is recommended as it reduces peak oestradiol concentrations without significantly decreasing oocyte yield. The use of a GnRH agonist trigger is beneficial as oestradiol concentrations rapidly decrease post-administration and rates of ovarian hyperstimulation are lower than with an hCG trigger, without a corresponding reduction in clinical pregnancy or live birth rates in cryopreservation cycles. The protective effect of tamoxifen has not been evaluated although theoretically may be of benefit due to its action on the oestrogen receptor. STUDY FUNDING/COMPETING INTEREST(S): None. REGISTRATION NUMBER: None.

Failure to launch: aberrant cumulus gene expression during oocyte in vitro maturation.

In vitro maturation (IVM) offers significant benefits for human infertility treatment and animal breeding, but this potential is yet to be fully realised due to reduced oocyte developmental competence in comparison with in vivo matured oocytes. Cumulus cells occupy an essential position in determining oocyte developmental competence. Here we have examined the areas of deficient gene expression, as determined within microarrays primarily from cumulus cells of mouse COCs, but also other species, between in vivo matured and in vitro matured oocytes. By retrospectively analysing the literature, directed by focussing on downregulated genes, we provide an insight as to why the in vitro cumulus cells fail to support full oocyte potential and dissect molecular pathways that have important roles in oocyte competence. We conclude that the roles of epidermal growth factor signalling, the expanded extracellular matrix, cumulus cell metabolism and the immune system are critical deficiencies in cumulus cells of IVM COCs.

Promotion of EGF receptor signaling improves the quality of low developmental competence oocytes.

Oocytes acquire developmental competence with progressive folliculogenesis. Cumulus oocyte complexes (COCs) from small antral follicles have inherent low competence and are poorly responsive to amphiregulin (AREG) which normally mediates oocyte maturation and ovulation. Using low competence porcine COCs, in an in vitro AREG-induced oocyte maturation system, the combined exposure to N(6),2'-O-dibutyryladenosine 3':5' cyclic monophosphate (cAMP) and bone morphogenetic protein 15 (B15) and growth differentiation factor 9 (G9) was necessary to enhance the rate of oocyte meiotic maturation and blastocyst formation. Furthermore, the combination of cAMP+B15+G9 enabled AREG-stimulated cumulus expansion and increased expression of the matrix-related genes HAS2, TNFIPA6 and PTGS2. Additionally, the combination enhanced p-ERK1/2 which is downstream of the EGF receptor. The enhanced nuclear maturation and blastocyst formation rates with the combinational treatment were ablated by an EGF receptor phosphorylation inhibitor. These results indicate that cAMP and oocyte-secreted factors cooperate to promote EGF receptor functionality in developing COCs, representing a key component of the acquisition of oocyte developmental competence.