Non-invasive cumulus cell analysis can be applied for oocyte ranking and is useful for countries with legal restrictions on embryo generation or freezing.

RESEARCH QUESTION: Can a strategy for scoring oocyte quality, based on cumulus cell (CC) gene expression, prioritize oocytes with the highest implantation potential, while limiting the number of embryos to be processed in culture and the number of supernumerary embryos to be vitrified? DESIGN: An interventional, blinded, prospective cohort study was retrospectively analyzed. In the original study, patients underwent a fresh Day3 single embryo transfer with embryos ranked based on morphology and CC gene expression (Aurora Test). The additional ranking of the embryos with the Aurora Test resulted in significant higher clinical pregnancy and live birth rates. Now it is investigated if the Aurora Test ranking could be applied to select oocytes. The effect of an Aurora Test based restriction to 2 and 3 2PN or MII oocytes on clinical pregnancy and other outcomes, was analyzed in two subsets of patients with all 2PN (n = 83) or all MII oocytes (n = 45) ranked. RESULTS: Considering only the top three ranked 2PN oocytes, 95% of the patients would have received a fresh SET on Day3 resulting in 65% clinical pregnancies. This was not different from the pregnancy rate obtained in a strategy using all oocytes but significantly reduced the need for vitrification of supernumerary embryos by 3-fold. Considering only top-ranked MII oocytes gave similar results. CONCLUSIONS: In countries with legal restrictions on freezing of embryos, gene expression of CC can be used for the selective processing of oocytes and would thus decrease the twin pregnancy rate and workload, especially for embryo morphology scoring and transfers as the handling and processing of lower competence oocytes is prevented, while improving the ART outcome.

A fresh start for IVM: capacitating the oocyte for development using pre-IVM.

BACKGROUND: While oocyte IVM is practiced sporadically it has not achieved widespread clinical practice globally. However, recently there have been some seminal advances in our understanding of basic aspects of oocyte biology and ovulation from animal studies that have led to novel approaches to IVM. A significant recent advance in IVM technology is the use of biphasic IVM approaches. These involve the collection of immature oocytes from small antral follicles from minimally stimulated patients/animals (without hCG-priming) and an ∼24 h pre-culture of oocytes in an advanced culture system ('pre-IVM') prior to IVM, followed by routine IVF procedures. If safe and efficacious, this novel procedure may stand to make a significant impact on human ART practices. OBJECTIVE AND RATIONALE: The objectives of this review are to examine the major scientific advances in ovarian biology with a unique focus on the development of pre-IVM methodologies, to provide an insight into biphasic IVM procedures, and to report on outcomes from animal and clinical human data, including safety data. The potential future impact of biphasic IVM on ART practice is discussed. SEARCH METHODS: Peer review original and review articles were selected from PubMed and Web of Science searches for this narrative review. Searches were performed using the following keywords: oocyte IVM, pre-IVM, biphasic IVM, CAPA-IVM, hCG-triggered/primed IVM, natural cycle IVF/M, ex-vivo IVM, OTO-IVM, oocyte maturation, meiotic competence, oocyte developmental competence, oocyte capacitation, follicle size, cumulus cell (CC), granulosa cell, COC, gap-junction communication, trans-zonal process, cAMP and IVM, cGMP and IVM, CNP and IVM, EGF-like peptide and IVM, minimal stimulation ART, PCOS. OUTCOMES: Minimizing gonadotrophin use means IVM oocytes will be collected from small antral (pre-dominant) follicles containing oocytes that are still developing. Standard IVM yields suboptimal clinical outcomes using such oocytes, whereas pre-IVM aims to continue the oocyte's development ex vivo, prior to IVM. Pre-IVM achieves this by eliciting profound cellular changes in the oocyte's CCs, which continue to meet the oocyte's developmental needs during the pre-IVM phase. The literature contains 25 years of animal research on various pre-IVM and biphasic IVM procedures, which serves as a large knowledge base for new approaches to human IVM. A pre-IVM procedure based on c-type natriuretic peptide (named 'capacitation-IVM' (CAPA-IVM)) has undergone pre-clinical human safety and efficacy trials and its adoption into clinical practice resulted in healthy live birth rates not different from conventional IVF. WIDER IMPLICATIONS: Over many decades, improvements in clinical IVM have been gradual and incremental but there has likely been a turning of the tide in the past few years, with landmark discoveries in animal oocyte biology finally making their way into clinical practice leading to improved outcomes for patients. Demonstration of favorable clinical results with CAPA-IVM, as the first clinically tested biphasic IVM system, has led to renewed interest in IVM as an alternative, low-intervention, low-cost, safe, patient-friendly ART approach, and especially for patients with PCOS. The same new approach is being used as part of fertility preservation in patients with cancer and holds promise for social oocyte freezing.

Epigenetic variation in neonatal tissues in infants conceived using capacitation-in vitro maturation vs. in vitro fertilization.

OBJECTIVE: To investigate alterations of the global DNA methylation profile in placenta, cord blood, and neonatal buccal smears in infants conceived using in vitro maturation (IVM) with a prematuration step (capacitation-IVM [CAPA-IVM]) vs. in vitro fertilization (IVF). DESIGN: Analysis of data from the offspring of participants in a randomized controlled trial. SETTING: Private clinic. PATIENTS: Forty-six women with polycystic ovary syndrome and/or high antral follicle count and their offspring (58 newborns). INTERVENTION(S): Women with polycystic ovary syndrome and/or a high antral follicle count participating in the clinical trial were randomized to undergo CAPA-IVM or conventional IVF. MAIN OUTCOME MEASURE(S): At delivery, biological samples including cord blood, placental tissue, and a neonatal buccal smear were collected. Genome-wide DNA methylation was determined using the Illumina Infinium MethylationEPIC BeadChip. Variability in methylation was also considered, and mean variances for the two treatment categories were compared. RESULTS: In neonatal buccal smears, there were no significant differences between the CAPA-IVM and conventional IVF groups on the basis of the CpG probe after linear regression analysis using a significant cut-off of false-discovery rate <0.05 and |Δß|≥0.05. In cord blood, only one CpG site showed a significant gain of methylation in the CAPA-IVM group. In the placenta, CAPA-IVM was significantly associated with changes in methylation at five CpG sites. Significantly more variable DNA methylation was found in five probes in the placenta, 54 in cord blood, and two in buccal smears after IVM of oocytes. In cord blood samples, 20 CpG sites had more variable methylation in the conventional IVF vs. IVM group. Isolated CpG sites showing differences in methylation in cord blood were not associated with changes in gene expression of the overlapping genes. CONCLUSION(S): Capacitation-IVM appeared to be associated with only a small amount of epigenetic variation in cord blood, placental tissue, and neonate buccal smears. CLINICAL TRIAL REGISTRATION NUMBER: NCT03405701 (www. CLINICALTRIALS: gov).

Mouse oocytes restore antral stage partial mechanical denudation in vitro.

In brief: Partially denuded mouse cumulus-oocyte complexes restore likely functional transzonal projections in culture, under meiotic inhibition, with no detectable impact on oocyte competence. This proof-of-concept study constitutes positive premises for improving the developmental competence of human capacitation (CAPA)-in vitro maturation (IVM) oocytes with inadequate somatic cell connections. Abstract: In vitro oocyte culture might be the sole option for fertility preservation in some patients. This relies on constant oocyte-somatic bidirectional communication, and its precocious disruption alters oocyte competence. In non-human chorionic gonadotropin-triggered human in vitro maturation (IVM), retrieval of cumulus-oocyte complexes (COCs) by needle aspiration from the targeted small follicles (2-8 mm) leads to the collection of some partially denuded (PD) COCs with poor developmental competence. Hypothetically, re-establishing connectivity in these COCs could rescue oocyte quality. To test this, we used a well-characterized mouse preantral follicle culture system. On day 8, at antral stage, in part of the follicles, the oocytes were mechanically denuded while in other follicles in vitro grown oocytes were replaced with age matched fully stripped in vivo grown ones. The denuded oocytes were cultured on top of the somatic compartment until day 12, when oocyte-somatic reconnection was assessed. Furthermore, to better mimic the current biphasic IVM setup, fully surrounded (FS) COCs were collected from 19- to 21- day-old unprimed mice. Following partial mechanical denudation, COCs were cultured under meiotic inhibition for 2-4 days, to test oocyte-cumulus cell (CC) reconnection. Meiotic and developmental competence endpoints were compared between reconnected and FS-cultured COCs. We concluded that (i) in vivo- and in vitro- grown antral oocytes reconnect with in vitro-grown somatic companions; (ii) PD-COCs restore the FS morphology in culture, under meiotic inhibition; and (iii) oocyte quality from reconnected and intact cultured COCs is comparable. These observations encourage translational work to rescue partially denuded oocytes in human IVM.

Glucose and redox metabolism in meiotically blocked in vitro grown mouse antral follicles.

PURPOSE: Glucose and redox metabolism characterization in mouse antral follicles with meiotically blocked oocytes, after in vitro follicle culture (IFC) from the early secondary stage. METHODS: Following IFC (10 days), oocytes, corresponding cumulus (CC), and granulosa cells (GC) were collected from antral follicles: (i) on day 9-immature, germinal vesicle (GV) stage; (ii) on day 10, after hCG/EGF stimulation-mature, metaphase II (MII) stage and meiotically blocked (MB) immature GV stage. The metabolic profiles of all samples (GV, MII, and MB) were compared by measuring changes in metabolites involved in glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP), and redox activity via enzymatic spectrophotometric assays in each cell type. RESULTS: Within MB follicles, GCs drive higher levels of glycolysis and lactic acid fermentation (LAF) while oocytes exert more PPP activity. MB-oocytes had significantly larger diameters compared to day 9 GVs. MB follicles revealed limited metabolic changes in the somatic compartment compared to their GV counterparts (before stimulation). MB-CCs showed increased aconitase and glucose-6-phosphate dehydrogenase activities with lower malate levels comparted to GV-CCs. MB and MII in vitro grown follicles displayed comparable metabolic profiles, suggesting culture induces metabolic exhaustion regardless of the maturation stage. CONCLUSIONS: Current results suggest that in addition to impaired nuclear maturation, metabolic disruption is present in MB follicles. MB follicles either compensate with high levels of TCA cycle and PPP activities in CCs, or are unable to drive proper levels of aerobic metabolism, which might be due to the current culture conditions.

Effects of lactate, super-GDF9, and low oxygen tension during bi-phasic in vitro maturation on the bioenergetic profiles of mouse cumulus-oocyte complex†.

In vitro maturation (IVM) is an alternative assisted reproductive technology with reduced hormone-related side effects and treatment burden compared to conventional IVF. Capacitation (CAPA)-IVM is a bi-phasic IVM system with improved clinical outcomes compared to standard monophasic IVM. Yet, CAPA-IVM efficiency compared to conventional IVF is still suboptimal in terms of producing utilizable blastocysts. Previously, we have shown that CAPA-IVM leads to a precocious increase in cumulus cell (CC) glycolytic activity during cytoplasmic maturation. In the current study, considering the fundamental importance of CCs for oocyte maturation and cumulus-oocyte complex (COC) microenvironment, we further analyzed the bioenergetic profiles of maturing CAPA-IVM COCs. Through a multi-step approach, we (i) explored mitochondrial function of the in vivo and CAPA-IVM matured COCs through real-time metabolic analysis with Seahorse analyzer, and to improve COC metabolism (ii) supplemented the culture media with lactate and/or super-GDF9 (an engineered form of growth differentiation factor 9) and (iii) reduced culture oxygen tension. Our results indicated that the pre-IVM step is delicate and prone to culture-related disruptions. Lactate and/or super-GDF9 supplementations failed to eliminate pre-IVM-induced stress on COC glucose metabolism and mitochondrial respiration. However, when performing pre-IVM culture under 5% oxygen tension, CAPA-IVM COCs showed similar bioenergetic profiles compared to in vivo matured counterparts. This is the first study providing real-time metabolic analysis of the COCs from a bi-phasic IVM system. The currently used analytical approach provides the quantitative measures and the rational basis to further improve IVM culture requirements.

Oocyte in vitro maturation: physiological basis and application to clinical practice.

Oocyte in vitro maturation (IVM) is an assisted reproductive technology with a long and sometimes checked history. It is a minimally invasive technique involving the deliberate collection of immature oocytes from patients that have received no or minimal ovarian stimulation and the culture of oocytes to maturity in vitro, before standard procedures thereafter. Now, IVM is classified as nonexperimental and is primarily indicated for patients with a high antral follicle count, especially patients with polycystic ovaries or polycystic ovary syndrome, as well as for fertility preservation in cancer patients. In the recent past, IVM practice has had a confusing array of clinical protocols and has been slow to adapt to new scientific insights; however, recently, significant advances have been made in IVM culture methods based on new knowledge from animal studies, combined with defining a simple patient treatment protocol. These improvements have led to significant recent progress in IVM practice to the extent that IVM is now routinely practiced in a growing number of centers with specialized expertise around the world.

Genome-wide assessment of DNA methylation alterations induced by superovulation, sexual immaturity and in vitro follicle growth in mouse blastocysts.

BACKGROUND: In their attempt to fulfill the wish of having children, women who suffer from fertility issues often undergo assisted reproductive technologies such as ovarian stimulation, which has been associated with adverse health outcomes and imprinting disorders in children. However, given the crucial role of exogenous hormone stimulation in improving human infertility treatments, a more comprehensive analysis of the potential impacts on DNA methylation in embryos following ovarian stimulation is needed. Here, we provide genome-wide DNA methylation profiles of blastocysts generated after superovulation of prepubertal or adult mice, compared with blastocysts derived from non-stimulated adult mice. Additionally, we assessed the impact of the in vitro growth and maturation of oocytes on methylation in blastocysts. RESULTS: Neither hormone stimulation nor sexual maturity had an impact on the low global methylation levels characteristic of the blastocyst stage or was associated with extensive DNA methylation alterations. However, we found hormone- and age-associated changes at specific positions but dispersed throughout the genome. In particular, we detected anomalous methylation at a limited number of CpG islands. Additionally, superovulation in adult mice was associated with alterations at the Sgce and Zfp777 imprinted genes. On the other hand, in vitro culture of follicles from the early pre-antral stage was associated with globally reduced methylation and increased variability at imprinted loci in blastocysts. CONCLUSIONS: Our results indicate a minimal effect of ovarian stimulation of adult and prepubertal mice on the DNA methylation landscape attained at the blastocyst stage, but potentially greater impacts of in vitro growth and maturation of oocytes. These findings have potential significance for the improvement of assisted reproductive techniques, in particular for those related to treatments in prepubertal females, which could be crucial for improving human fertility preservation strategies.

Bioinformatic Analysis of Human Cumulus Cells to Unravel Cellular's Processes that Could Be Used to Establish Oocyte Quality Biomarkers with Clinical Application.

Metadata analysis of public microarray datasets using bioinformatics tools has been successfully used in several biomedical fields in the search for biomarkers. In reproductive science, there is an urgent need for the establishment of oocyte quality biomarkers that could be used in the clinical environment to increase the chances of successful outcomes in treatment cycles. Adaptive cellular processes observed in cumulus oophorus cells reflect the conditions of the follicular microenvironment and may thus bring relevant information of oocyte's conditions. Here we analyzed human cumulus cells gene expression datasets in search of predictors of oocyte quality, a strategy which uncovered several cellular processes positively and negatively associated with embryo development and pregnancy potential. Secondly, the expression levels of genes that were present in the majority of processes observed were validated in house with clinical samples. Our data confirmed the association of the selected biomarkers with blastocyst formation and pregnancy potential rates, independently of patients' clinical characteristics such as diagnosis, age, BMI, and stimulation protocol applied. This study shows that bioinformatic analysis of cellular processes can be successfully used to elucidate possible oocyte quality biomarkers. Our data reinforces the need to consider clinical characteristics of patients when selecting relevant biomarkers to be used in the clinical environment and suggests a combination of positive (PTGS2) and negative (CYPB1) quality biomarkers as a robust strategy for a complementary oocyte selection tool, potentially increasing assisted reproduction success rates. Also, GPX4 expression as pregnancy potential biomarker is indicated here as a possibility for further investigations.

Protective Effects of Antioxidants on Cyclophosphamide-Induced Ovarian Toxicity.

The most common limitation of anticancer chemotherapy is the injury to normal cells. Cyclophosphamide, which is one of the most widely used alkylating agents, can cause premature ovarian insufficiency and infertility since the ovarian follicles are extremely sensitive to their effects. Although little information is available about the pathogenic mechanism of cyclophosphamide-induced ovarian damage, its toxicity is attributed to oxidative stress, inflammation, and apoptosis. The use of compounds with antioxidant and cytoprotective properties to protect ovarian function from deleterious effects during chemotherapy would be a significant advantage. Thus, this article reviews the mechanism by which cyclophosphamide exerts its toxic effects on the different cellular components of the ovary, and describes 24 cytoprotective compounds used to ameliorate cyclophosphamide-induced ovarian injury and their possible mechanisms of action. Understanding these mechanisms is essential for the development of efficient and targeted pharmacological complementary therapies that could protect and prolong female fertility.

Immunolocalization of melatonin receptor type 1 in the sheep ovary and involvement of the PI3K/Akt/FOXO3a signaling pathway in the effects of melatonin on survival and in vitro activation of primordial follicles.

This study characterized the expression of melatonin receptor type 1 (MT1 ) protein in sheep ovaries, evaluated melatonin effects on primordial follicle survival and development after in vitro culture of ovarian tissue and verified the possible involvement of the phosphatidylinositol-3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FOXO3a) pathway in the melatonin actions. Ovine ovarian fragments were cultured in α-modified minimum essential medium alone (α-MEM+ ) or supplemented with 100, 500, or 1000 pg/ml melatonin for 7 days. PI3K inhibition was performed through pretreatment of ovarian fragments with LY294002. Thereafter, immunohistochemistry was performed to evaluate the expression of cleaved caspase-3, Akt, phosphorylated-Akt, and phosphorylated-FOXO3a (p-FOXO3a). The immunohistochemical localization of the MT1 receptor protein was documented in sheep preantral and antral follicles. After in vitro culture, 100 pg/ml melatonin showed higher follicular survival and activation than α-MEM+ and other melatonin concentrations. After PI3K inhibition, there was an increase in cleaved caspase-3-positive follicles, and a decrease in the primordial follicle activation, Akt phosphorylation, and nuclear exclusion of p-FOXO3a. In conclusion, MT1 receptor protein is present in the sheep ovary. Furthermore, 100 pg/ml melatonin maintains survival and stimulates activation of primordial follicles through the PI3K/Akt/FOXO3a signaling pathway after in vitro culture of sheep ovarian tissue.

Epigallocatechin-3-gallate attenuates cyclophosphamide-induced damage in mouse ovarian tissue via suppressing inflammation, apoptosis, and expression of phosphorylated Akt, FOXO3a and rpS6.

This study was conducted to evaluate the protective effects of epigallocatechin-3-gallate (EGCG) against ovarian toxicity in cyclophosphamide-treated mice and to verify the possible involvement of phosphorylated Akt, FOXO3a and rpS6 in the EGCG actions. Mice received saline solution (i.p.; control) or a single dose of cyclophosphamide (200 mg/kg body weight, i.p.) or mice were pretreated with N-acetylcysteine (150 mg/kg body weight, i.p.; positive control) or with EGCG (5, 25 or 50 mg/kg body weight, i.p.) once daily for three days followed by injection with single dose of cyclophosphamide (200 mg/kg body weight, i.p.). Thereafter, the mice were euthanized, and the ovaries were harvested and destined to histological (follicular morphology and activation), immunohistochemistry (cleaved caspase-3 and TNF-α) and fluorescence (mitochondrial activity and GSH concentrations) analyses. Furthermore, we examined the participation of p-Akt, p-FOXO3a and p-rpS6 in the protective effects of EGCG in cyclophosphamide-induced ovarian damage by immunohistochemical staining. The results showed that pretreatment with N-acetylcysteine or EGCG at 25 and 50 mg/kg before cyclophosphamide administration preserved the normal follicular morphology, prevented primordial follicle loss, reduced atresia, inflammation, and mitochondrial damage, and increased GSH concentrations compared to the only cyclophosphamide treatment. Additionally, pretreatment with 25 mg/kg EGCG regulated phosphorylated Akt, FOXO3a and rpS6 after cyclophosphamide treatment. In conclusion, short-time pretreatment with 25 mg/kg EGCG can prevent follicle loss in cyclophosphamide-treated mice by reducing oxidative damage, inflammation, and apoptosis, and regulating of p-Akt, p-FOXO3a and p-rpS6.

Characterization of carbohydrate metabolism in in vivo- and in vitro-grown and matured mouse antral follicles†.

Establishing an ideal human follicle culture system for oncofertility patients relies mainly on animal models since donor tissue is scarce and often of suboptimal quality. The in vitro system developed in our laboratory supports the growth of prepubertal mouse secondary follicles up to mature oocytes. Given the importance of glucose in preparing the oocyte for proper maturation, a baseline characterization of follicle metabolism both in the culture system and in vivo was carried out. Markers of glucose-related pathways (glycolysis, tricarboxylic acid [TCA] cycle, pentose phosphate pathway [PPP], polyol pathway, and hexosamine biosynthetic pathway), as well as the antioxidant capacity, were measured in the different follicle cell types by both enzymatic activities (spectrophotometric detection) and gene expression (qPCR). This study confirmed that in vivo the somatic cells, mainly granulosa, exhibit intense glycolytic activity, while oocytes perform PPP. Throughout the final maturation step, oocytes in vivo and in vitro showed steady levels for all the key enzymes and metabolites. On the other hand, ovulation triggers a boost of pyruvate and lactate uptake in cumulus cells in vivo, consumes reduced nicotinamide adenine dinucleotide phosphate, and increases TCA cycle and small molecules antioxidant capacity activities, while in vitro, the metabolic upregulation in all the studied pathways is limited. This altered metabolic pattern might be a consequence of cell exhaustion because of culture conditions, impeding cumulus cells to fulfill their role in providing proper support for acquiring oocyte competence.

Development of children born from IVM versus IVF: 2-year follow-up of a randomized controlled trial.

STUDY QUESTION: Is there any difference in developmental outcomes in children born after capacitation IVM (CAPA IVM) compared with conventional IVF? SUMMARY ANSWER: Overall development up to 24 months of age was comparable in children born after CAPA IVM compared with IVF. WHAT IS KNOWN ALREADY: IVM has been shown to be a feasible alternative to conventional IVF in women with a high antral follicle count (AFC). In addition to live birth rate, childhood development is also a relevant metric to compare between the two approaches to ART and there are currently no data on this. STUDY DESIGN, SIZE, DURATION: This study was a follow-up of babies born to women who participated in a randomized controlled trial comparing IVM with a pre-maturation step (CAPA IVM) and IVF. Developmental assessments were performed on 231 children over 24 months of follow-up. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants in the randomized controlled trial had an indication for ART and a high AFC (≥24 follicles in both ovaries). They were randomized to undergo one cycle of either IVM (n = 273) or IVF (n = 273). Of these, 96 women and 118 women, respectively, had live births. Seventy-six women (94 children, 79.2%) and 104 women (137 children, 88.1%), respectively, completed Ages & Stages Third Edition Questionnaire assessment (ASQ-3), and underwent evaluation of Developmental Red Flags at 6, 12 and 24 months of age. MAIN RESULTS AND THE ROLE OF CHANCE: Baseline characteristics of participants in the follow-up study between the IVM and IVF groups were comparable. Overall, there were no significant differences in ASQ-3 scores at 6, 12 and 24 months between children born after IVM or IVF. The proportion of children with developmental red flags was low and did not differ between the two groups. Slightly, but significantly, lower ASQ-3 problem solving and personal-social scores in twins from the IVM versus IVF group at 6 months were still within the normal range and had caught up to the IVF group in the 12- and 24-month assessments. The number of children confirmed to have abnormal mental and/or motor development after specialist assessment was four in the IVM group and two in the IVF group (relative risk 2.91, 95% CI 0.54-15.6; P = 0.23). LIMITATIONS, REASONS FOR CAUTION: This study is an open-label follow-up of participants in a randomized controlled trial, and not all original trial subjects took part in the follow-up. The self-selected nature of the follow-up population could have introduced bias, and the sample size may have been insufficient to detect significant between-group differences in developmental outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Based on the current findings at 2 years of follow-up, there does not appear to be any significant concern about the effects of IVM on childhood development. These data add to the evidence available to physicians when considering different approaches to fertility treatment, but require validation in larger studies. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number FWO.106-YS.2017.02. L.N.V. 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; T.M.H. has received speaker fees from Merck, Merck Sharp and Dohme, and Ferring; R.J.N. has receives grant funding from the National Health and Medical Research Council (NHMRC) of Australia; B.W.M. has acted as a paid consultant to Merck, ObsEva and Guerbet and is the recipient of grant money from an NHMRC Investigator Grant; J.E.J.S. reports lecture fees from Ferring Pharmaceuticals, Biomérieux and Besins Female Healthcare, grants from Fund for Research Flanders (FWO) and is co-inventor on granted patents on CAPA-IVM methodology in the USA (US10392601B2) and Europe (EP3234112B1); T.D.P., M.H.N.N., N.A.N., T.T.L., V.T.T.T., N.T.N., H.L.T.H. and X.T.H.L. have no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years, and no other relationships or activities that could appear to have influenced the submitted work. TRIAL REGISTRATION NUMBER: NCT04296357 (www.clinicaltrials.gov). TRIAL REGISTRATION DATE: 5 March 2020. DATE OF FIRST PATIENT'S ENROLMENT: 7 March 2020.

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.

Melatonin Attenuates Cyclophosphamide-Induced Primordial Follicle Loss by Interaction with MT1 Receptor and Modulation of PTEN/Akt/FOXO3a Proteins in the Mouse Ovary.

This study evaluated the protective effect of melatonin before cyclophosphamide administration on ovarian function and its potential mechanism in a mouse model. Two studies were performed. In the first, mice were pretreated with melatonin (10, 20, or 30 mg/kg body weight, i.p.) once daily for 3 days, followed by injection with a single dose of cyclophosphamide (200 mg/kg body weight, i.p.) 30 min after the last melatonin injection. The second study analyzed whether melatonin type 1 and/or 2 receptors mediate the effects of melatonin on the ovary through administration of non-selective MT1/MT2 antagonist (luzindole) or selective MT2 antagonist (4-PPDOT) before the treatment with melatonin plus cyclophosphamide. After treatment groups, the ovaries were harvested and destined to histology, immunohistochemistry, and fluorescence analyses. Lastly, we examined the p-PTEN, p-Akt, and p-FOXO3a participation in the protective effect of melatonin in cyclophosphamide-induced ovarian damage. Results demonstrated that pretreatment with 20 mg/kg melatonin before cyclophosphamide administration showed more morphologically normal follicles, attenuated primordial follicle loss, decreased growing follicle atresia and mitochondrial damage, and increased GSH concentrations. Furthermore, treatment with luzindole blocked the protective effects of melatonin against the damage caused by cyclophosphamide. Additionally, pretreatment with 20 mg/kg melatonin regulated the PTEN/Akt/FOXO3a signaling pathway components after cyclophosphamide treatment. In conclusion, pretreatment with 20 mg/kg melatonin prevented primordial follicle loss and reduced apoptosis and oxidative damage in the mouse ovary during experimental chemotherapy with cyclophosphamide. Furthermore, the MT1 receptor and PTEN/Akt/FOXO3a proteins mediated these cytoprotective effects.

Positive effects of amphiregulin on human oocyte maturation and its molecular drivers in patients with polycystic ovary syndrome.

STUDY QUESTION: Does use of medium containing amphiregulin improve meiotic maturation efficiency in oocytes of women with polycystic ovary syndrome (PCOS) undergoing in vitro maturation (IVM) preceded by a capacitation culture step capacitation IVM (CAPA-IVM)? SUMMARY ANSWER: Use of medium containing amphiregulin significantly increased the maturation rate from oocytes retrieved from follicles with diameters <6 or ≥6 mm pre-cultured in capacitation medium. WHAT IS KNOWN ALREADY: Amphiregulin concentration in follicular fluid is correlated with human oocyte developmental competence. Amphiregulin added to the meiotic trigger has been shown to improve outcomes of IVM in a range of mammalian species. STUDY DESIGN, SIZE, DURATION: This prospective, randomized cohort study included 30 patients and was conducted at an academic infertility centre in Vietnam from April to December 2019. Patients with PCOS were included. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the first stage, sibling oocytes from each patient (671 in total) were allocated in equal numbers to maturation in medium with (CAPA-AREG) or without (CAPA-Control) amphiregulin 100 ng/ml. After a maturation check and fertilization using intracytoplasmic sperm injection (ICSI), all good quality Day 3 embryos were vitrified. Cumulus cells (CCs) from both groups were collected at the moment of ICSI denudation and underwent a molecular analysis to quantify key transcripts of oocyte maturation and to relate these to early embryo development. On return for frozen embryo transfer (second stage), patients were randomized to have either CAPA-AREG or CAPA-Control embryo(s) implanted. Where no embryo(s) from the randomized group were available, embryo(s) from the other group were transferred. The primary endpoint of the study was meiotic maturation efficiency (proportion of metaphase II [MII] oocytes; maturation rate). MAIN RESULTS AND THE ROLE OF CHANCE: In the per-patient analysis, the number of MII oocytes was significantly higher in the CAPA-AREG group versus the CAPA-Control group (median [interquartile range] 7.0 [5.3, 8.0] versus 6.0 [4.0, 7.0]; P = 0.01). When each oocyte was evaluated, the maturation rate was also significantly higher in the CAPA-AREG group versus the CAPA-Control group (67.6% versus 55.2%; relative risk [RR] 1.22 [95% confidence interval (CI) 1.08-1.38]; P = 0.001). No other IVM or embryology outcomes differed significantly between the two groups. Rates of clinical pregnancy (66.7% versus 42.9%; RR 1.56 [95% CI 0.77-3.14]), ongoing pregnancy (53.3% versus 28.6%; RR 1.87 [95% CI 0.72-4.85]) and live birth (46.7% versus 28.6%; RR 1.63 [95% CI 0.61-4.39]) were numerically higher in the patients who had CAPA-AREG versus CAPA-Control embryos implanted, but each fertility and obstetric outcome did not differ significantly between the groups. In the CAPA-AREG group, there were significant shifts in CC expression of genes involved in steroidogenesis (STAR, 3BHSD), the ovulatory cascade (DUSP16, EGFR, HAS2, PTGR2, PTGS2, RPS6KA2), redox and glucose metabolism (CAT, GPX1, SOD2, SLC2A1, LDHA) and transcription (NRF2). The expression of three genes (TRPM7, VCAN and JUN) in CCs showed a significant correlation with embryo quality. LIMITATIONS, REASONS FOR CAUTION: This study included only Vietnamese women with PCOS, limiting the generalizability. Although 100 ng/ml amphiregulin addition to the maturation culture step significantly improved the MII rate, the sample size in this study was small, meaning that these findings should be considered as exploratory. Therefore, a larger patient cohort is needed to confirm whether the positive effects of amphiregulin translate into improved fertility outcomes in patients undergoing IVM. WIDER IMPLICATIONS OF THE FINDINGS: Data from this study confirm the beneficial effects of amphiregulin during IVM with respect to the trigger of oocyte maturation. The gene expression findings in cumulus indicate that multiple pathways might contribute to these beneficial effects and confirm the key role of the epidermal growth factor system in the stepwise acquisition of human oocyte competence. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED; grant number FWO.106-YS.2017.02) and by the Fund for Research Flanders (FWO; grant number G.OD97.18N). L.N.V. has received speaker and conference fees from Merck, grants, speaker and conference fees from Merck Sharpe and Dohme, and speaker, conference and scientific board fees from Ferring. T.M.H. has received speaker fees from Merck, Merck Sharp and Dohme and Ferring. J.S. reports speaker fees from Ferring Pharmaceuticals and Biomérieux Diagnostics and grants from FWO Flanders, is co-inventor on granted patents on CAPA-IVM methodologies in USA (US10392601B2), Europe (EP3234112B1) and Japan (JP 6806683 registered 08-12-2020) and is a co-shareholder of Lavima Fertility Inc., a spin-off company of the Vrije Universiteit Brussel (VUB, Brussels, Belgium). NA, TDP, AHL, MNHN, SR, FS, EA and UDTH report 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: NCT03915054.

The Role of Mitochondria in Oocyte Maturation.

With the nucleus as an exception, mitochondria are the only animal cell organelles containing their own genetic information, called mitochondrial DNA (mtDNA). During oocyte maturation, the mtDNA copy number dramatically increases and the distribution of mitochondria changes significantly. As oocyte maturation requires a large amount of ATP for continuous transcription and translation, the availability of the right number of functional mitochondria is crucial. There is a correlation between the quality of oocytes and both the amount of mtDNA and the amount of ATP. Suboptimal conditions of in vitro maturation (IVM) might lead to changes in the mitochondrial morphology as well as alternations in the expression of genes encoding proteins associated with mitochondrial function. Dysfunctional mitochondria have a lower ability to counteract reactive oxygen species (ROS) production which leads to oxidative stress. The mitochondrial function might be improved with the application of antioxidants and significant expectations are laid on the development of new IVM systems supplemented with mitochondria-targeted reagents. Different types of antioxidants have been tested already on animal models and human rescue IVM oocytes, showing promising results. This review focuses on the recent observations on oocytes' intracellular mitochondrial distribution and on mitochondrial genomes during their maturation, both in vivo and in vitro. Recent mitochondrial supplementation studies, aiming to improve oocyte developmental potential, are summarized.

Fresh embryo transfer versus freeze-only after in vitro maturation with a pre-maturation step in women with high antral follicle count: a randomized controlled pilot study.

PURPOSE: In vitro maturation (IVM) is an alternative to in vitro fertilization (IVF) for women at high risk of developing ovarian hyperstimulation syndrome (OHSS). This study determined the effectiveness and safety of a freeze-only strategy versus fresh embryo transfer (ET) after IVM with a pre-maturation step (CAPA-IVM) in women with a high antral follicle count (AFC). METHODS: This randomized, controlled pilot study (NCT04297553) was conducted between March and November 2020. Forty women aged 18-37 years with a high AFC (≥24 follicles in both ovaries) undergoing one cycle of CAPA-IVM were randomized to a freeze-only strategy with subsequent frozen ET (n = 20) or to fresh ET (n = 20). The primary endpoint was ongoing pregnancy resulting in live birth after the first ET of the started treatment cycle. RESULTS: The ongoing pregnancy rate in the freeze-only group (65%) was significantly higher than that in the fresh ET group (25%; p = 0.03), as was the live birth rate (60% versus 20%; p = 0.02). Clinical pregnancy rate was numerically, but not significantly, higher after frozen versus fresh ET (70% versus 35%; p = 0.06), while the number of day 3 or good quality embryos, endometrial thickness on the day of oocyte pick-up, implantation rate, and positive pregnancy test rate did not differ significantly between groups. No cases of OHSS were observed, and miscarriage and multiple pregnancy rates were similar in the two groups. CONCLUSIONS: These findings suggest that the effectiveness of CAPA-IVM could be improved considerably by using a freeze-only strategy followed by frozen ET in subsequent cycles. TRIAL REGISTRATION NUMBER: NCT04297553 ( www.clinicaltrials.gov ).

Reversing complete mechanical transzonal projections disruption during mouse in vitro follicle culture with unaltered oocyte competence†.

In vitro oocyte growth is widely studied as an alternative fertility preservation approach. Several animal models are used to generate extensive information on this complex process regulated by the constant and dynamic interaction between the oocyte and its somatic compartment throughout follicle growth and maturation. A two-dimensional attachment mouse secondary follicle culture system was used to assess the oocyte's capacity to overcome disconnection from its somatic companions at different developmental stages for final competence acquisition. To test this, complete mechanical denudation of oocytes from preantral (PA) and early antral (EA) follicles was performed. Established endpoints were the oocyte's potential to reconnect with somatic cells and the impact of connectivity disruption on mature oocyte quality. This study proves that oocytes from PA and EA cultured mouse follicles can overcome complete denudation, restoring likely functional transzonal projections with no significant differences in meiotic and developmental competence compared with those from intact cultured follicles. These novel findings constitute good premises for developing successful strategies to rescue human oocyte competence in the context of in vitro culture approaches such as nonhuman chorionic gonadotropin triggered in vitro maturation.