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.

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.

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.

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.

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.

Glucose metabolism characterization during mouse in vitro maturation identifies alterations in cumulus cells†.

In vitro maturation (IVM) is an assisted reproduction technique with reduced hormone-related side-effects. Several attempts to implement IVM in routine practice have failed, primarily due to its relatively low efficiency compared with conventional in vitro fertilization (IVF). Recently, capacitation (CAPA)-IVM-a novel two-step IVM method-has improved the embryology outcomes through synchronizing the oocyte nuclear and cytoplasmic maturation. However, the efficiency gap between CAPA-IVM and conventional IVF is still noticeable especially in the numerical production of good quality embryos. Considering the importance of glucose for oocyte competence, its metabolization is studied within both in vivo and CAPA-IVM matured mouse cumulus-oocyte-complexes (COCs) through direct measurements in both cellular compartments, from transcriptional and translational perspectives, to reveal metabolic shortcomings within the CAPA-IVM COCs. These results confirmed that within in vivo COC, cumulus cells (CCs) are highly glycolytic, whereas oocytes, with low glycolytic activity, are deviating their glucose towards pentose phosphate pathway. No significant differences were observed in the CAPA-IVM oocytes compared with their in vivo counterparts. However, their CCs exhibited a precocious increase of glycolytic activity during the pre-maturation culture step and activity was decreased during the IVM step. Here, specific alterations in mouse COC glucose metabolism due to CAPA-IVM culture were characterized using direct measurements for the first time. Present data show that, while CAPA-IVM CCs are able to utilize glucose, their ability to support oocytes during final maturation is impaired. Future CAPA-IVM optimization strategies could focus on adjusting culture media energy substrate concentrations and/or implementing co-culture strategies.

Correction to: Genome-wide assessment of DNA methylation in mouse oocytes reveals effects associated with in vitro growth, superovulation, and sexual maturity.

After publication of the original article [1], we were notified that.

Genome-wide assessment of DNA methylation in mouse oocytes reveals effects associated with in vitro growth, superovulation, and sexual maturity.

BACKGROUND: In vitro follicle culture (IFC), as applied in the mouse system, allows the growth and maturation of a large number of immature preantral follicles to become mature and competent oocytes. In the human oncofertility clinic, there is increasing interest in developing this technique as an alternative to ovarian cortical tissue transplantation and to preserve the fertility of prepubertal cancer patients. However, the effect of IFC and hormonal stimulation on DNA methylation in the oocyte is not fully known, and there is legitimate concern over epigenetic abnormalities that could be induced by procedures applied during assisted reproductive technology (ART). RESULTS: In this study, we present the first genome-wide analysis of DNA methylation in MII oocytes obtained after natural ovulation, after IFC and after superovulation. We also performed a comparison between prepubertal and adult hormonally stimulated oocytes. Globally, the distinctive methylation landscape of oocytes, comprising alternating hyper- and hypomethylated domains, is preserved irrespective of the procedure. The conservation of methylation extends to the germline differential methylated regions (DMRs) of imprinted genes, necessary for their monoallelic expression in the embryo. However, we do detect specific, consistent, and coherent differences in DNA methylation in IFC oocytes, and between oocytes obtained after superovulation from prepubertal compared with sexually mature females. Several methylation differences span entire transcription units. Among these, we found alterations in Tcf4, Sox5, Zfp521, and other genes related to nervous system development. CONCLUSIONS: Our observations show that IFC is associated with altered methylation at specific set of loci. DNA methylation of superovulated prepubertal oocytes differs from that of superovulated adult oocytes, whereas oocytes from superovulated adult females differ very little from naturally ovulated oocytes. Importantly, we show that regions other than imprinted gDMRs are susceptible to methylation changes associated with superovulation, IFC, and/or sexual immaturity in mouse oocytes. Our results provide an important reference for the use of in vitro growth and maturation of oocytes, particularly from prepubertal females, in assisted reproductive treatments or fertility preservation.

Dynamics of imprinted DNA methylation and gene transcription for imprinting establishment in mouse oocytes in relation to culture duration variability.

Several studies have linked assisted reproductive technologies to aberrant imprinting. We previously showed that 12-day in vitro follicle culture supports normal imprinting establishment in mouse oocytes. The aim of the present study was to assess whether shortened in vitro follicle growth (8 days of culture compared with 12 days, as a model for human in vitro maturation) or preovulatory intrafollicular oocyte "aging" in culture (14 days of culture) leads to imprinting mutations in oocytes. Limiting-dilution bisulphite sequencing showed that shortened in vitro follicle growth (8 days) does not induce oocyte epimutations at the imprinted Snrpn and Mest genes. In contrast, extension of oocyte residence in large unluteinized follicles in vitro was associated with a low level (1 of 54 alleles) of epimutations for Mest but not for Snrpn. The latter condition may occur during controlled ovarian stimulation where the oocyte growth phase may be extended for several days. Furthermore, we studied the dynamics during follicle culture of transcript levels for genes previously shown to be essential for imprinting establishment in oocytes, including Dnmt3a, Dnmt3L, and Zfp57. Oocyte total mRNA levels during in vitro follicle culture showed the timely shutdown in transcription at the antral follicle stage, and total mRNA levels were comparable to those of in vivo grown equine chorionic gonadotropin-stimulated oocytes.