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.

Impact of first chemotherapy exposure on follicle activation and survival in human cryopreserved ovarian tissue.

STUDY QUESTION: Does chemotherapy exposure prior to ovarian tissue cryopreservation (OTC) impact the signaling pathways governing follicle activation and survival for prepubertal and postpubertal patients? SUMMARY ANSWER: Chemotherapy exposure prior OTC increases follicle apoptosis rates but not follicular activation, although the PI3K/AKT/mTOR and Hippo signaling pathways were modified in the cortex. WHAT IS KNOWN ALREADY: OTC is currently the only available fertility preservation procedure for children and for patients who have already started their treatment. While previous studies have not observed harmful impacts of first chemotherapy exposure on OTC outcomes, the consequences of treatment on follicle activation and survival need to be further investigated. To address this question, we evaluated signaling pathway modifications induced by chemotherapy exposure according to pubertal status. STUDY DESIGN, SIZE, DURATION: Cryopreserved ovarian tissues from postpubertal (12-29 years old, n = 8) and prepubertal (3-10 years old, n = 8) cancer patients donated for research were thawed and cultured for 24 h. Analyses of the survival of the follicles and stroma, and of the PI3K/AKT/mTOR and Hippo signaling pathways, were conducted at thawing and after culture. Ovarian fragments exposed to chemotherapy before collection were compared to non-exposed controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological investigations were performed to assess the distribution of the follicles, stroma fibrosis, vessel integrity, and apoptosis levels. It included follicular counting, collagen staining, immunostaining on CD31 and gH2AX, as well as TUNEL staining. To explore follicle activation in the different groups, the PI3K/AKT/mTOR and Hippo signaling pathways were investigated by gene expression analyses of isolated follicles and protein analyses on whole fragments through western blots and immunostaining. MAIN RESULTS AND THE ROLE OF CHANCE: We first assessed the impact of a first exposure to chemotherapy on the collagen density and vessels in ovarian tissues at thawing and after culture. While no differences in collagen density were observed according to age or previous treatment, the vascularization area (CD31+) was significantly lower in tissue from previously exposed patients compared to non-treated ones. Apoptosis analyses (TUNEL) revealed an acute deleterious impact on follicle survival after chemotherapy exposure without affecting the follicular density. Surprisingly, leukemic patients had a significantly higher percentage of gH2AX-positive follicles, indicating a DNA damage response, compared to the other patients. The proportion of activated follicles appeared to decrease following exposure to chemotherapy, suggesting that it at least did not increase activation process. Stable KIT LIGAND gene and protein expression and cKIT protein levels were observed among the groups, confirming the absence of activation. Analysis of the PI3K pathway did not reveal a difference in the AKT phosphorylation level between the groups, but pRPS6 was significantly higher in tissue from patients previously exposed to chemotherapy compared to that from non-exposed patients. Finally, protein and gene analyses on Hippo pathway signaling showed a higher LATS1 protein level in the cortex after chemotherapy exposure. LIMITATIONS, REASONS FOR CAUTION: The heterogeneity of the human fragments, and the limited number of patients included in the cohort have to be considered as important study limitations. Moreover, this study did not explore the long-term consequences of chemotherapy on follicular development. Therefore, the results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: These results underscore the deleterious effect of previous chemotherapeutic treatment on follicle survival. Although follicular density was not reduced, these data suggested that exposure to chemotherapy impacts follicular apoptosis and the DNA damage response. Chemotherapy-induced activation was not observed despite the impact on mTOR and Hippo signaling pathways in the whole cortex. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by an Excellence of Science (EOS) Grant (ID: 30443682) and was supported by Fonds Erasme. I.D. and M.-M.D. are associate researchers at Fonds National de la Recherche Scientifique de Belgique (FNRS). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

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.

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.

Two-site evaluation of the Roche Elecsys Vitamin D total III assay.

OBJECTIVES: The high request for vitamin D testing in the last decades has led manufacturers to develop assays on automated immunoassay platforms. The objective of this study was to evaluate the performance of the new Elecsys Vitamin D total III assay for the measurement of total 25(OH)D. METHODS: A total of 844 serum samples collected in two clinical laboratories were used to evaluate the new Roche Elecsys Vitamin D total III assay. Comparisons with Roche Elecsys Vitamin D total II and liquid chromatography tandem mass spectrometry (LC-MS/MS) were carried out. Additionally, assay imprecision, linearity, matrix effects, biotin interference, cross-reactivity with 24,25(OH)2D3 and 3-epi-25(OH)D3, and outlier rate were evaluated for the Elecsys Vitamin D total III assay. RESULTS: Only the comparison between LC-MS/MS and Roche Elecsys Vitamin D total III achieved the optimal specification for bias (i.e., <3.4%). Imprecision, linearity and matrix effects showed acceptable results. The biotin interference threshold was increased up to 1,200 ng/mL and the outlier rate was low (0.26%). The cross-reactivity with 24,25(OH)2D3 and 3-epi-25(OH)D3 was weak or modest in available patient samples. However, using SRM972a with a high level of 3-epi-25(OH)D3 (enriched) revealed an important cross-reactivity with both Roche Elecsys Vitamin D total II and III assays (+74.7% and +73.7%). CONCLUSIONS: In conclusion, the Roche Elecsys Vitamin D total III assay presents several advantages compared to the previous assay generation: higher biotin interference threshold, broader measuring range, and better comparability with LC-MS/MS. However, the cross-reactivity toward 3-epi-25(OH)D3 is still problematic in high titer samples.

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.

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.

Cumulative live birth rates after IVF in patients with polycystic ovaries: phenotype matters.

RESEARCH QUESTION: Do cumulative live birth rates (CLBR) vary among women with different polycystic ovary syndrome (PCOS) phenotypes who undergo IVF/intracytoplasmic sperm injection (ICSI) treatment? DESIGN: In this retrospective cohort study, data from 567 patients undergoing an assisted reproductive technology (ART) cycle between January 2010 and December 2015 were collected. Demographical traits, cycle characteristics and clinical and laboratory data were analysed. RESULTS: After conventional ovarian stimulation using a gonadotrophin-releasing hormone antagonist protocol, the median number of oocytes retrieved ranged between 11 and 13.5 and did not differ significantly among the studied groups. Live birth rate (LBR) after fresh embryo transfer and CLBR after transfer of all fresh and vitrified embryos were significantly lower in women with hyperandrogenic PCOS phenotypes A (LBR 16.7%, CLBR 25.8%) and C (LBR 18.5%, CLBR 27.8%) compared with women with normoandrogenic PCOS phenotype D (LBR 33.7%, CLBR 48%) (P-value for LBR 0.01 and 0.03, respectively; P-value for CLBR 0.002 and 0.01, respectively) and controls with a polycystic ovarian morphology (LBR 37.1%, CLBR 53.3%) (P-value for LBR 0.002 and 0.01, respectively; P-value for CLBR <0.001 and 0.001, respectively). Multivariate regression analysis indicated that after adjustment for relevant confounders, PCOS phenotype was an independent predictor for CLBR. CONCLUSIONS: Hyperandrogenic PCOS phenotypes confer significantly lower CLBR compared with their normoandrogenic counterparts. These findings may imply the need for adapted counselling and tailored approaches when treating PCOS patients with hyperandrogenism who require ART.

The effect of serum vitamin D levels on ovarian reserve markers: a prospective cross-sectional study.

STUDY QUESTION: Is there any association between serum 25-OH vitamin D levels and ovarian reserve markers in infertile women? SUMMARY ANSWER: Vitamin D is not associated with the ovarian reserve markers, anti-mullerian hormone (AMH) and antral follicle count (AFC), in infertile women. WHAT IS KNOWN ALREADY: The mechanism underlying the relationship between vitamin D deficiency and reproduction is still unclear; however, evidence indicates a potential direct negative impact on ovarian function. This is mainly due to the fact that gonadal function may be altered by vitamin D deficiency, as observed by the expression of vitamin D receptor mRNA in human ovaries, mixed ovarian cell cultures and granulosa cell cultures. On the other hand, results from clinical studies are conflicting, with some suggesting that vitamin D status is associated with ovarian reserve, whereas other cross-sectional studies have not found any significant correlation between vitamin D and AMH levels. STUDY DESIGN, SIZE, DURATION: This study was a prospective cross-sectional study from the Centre for Reproductive Medicine at the University Hospital of Brussels. The duration of the study was one year. PARTICIPANTS/MATERIALS, SETTING, METHODS: Overall, the study included 283 consecutive infertile women younger than 42 years old and undergoing their first treatment cycle in our institution. All patients were recruited within a time interval of 12 months from the initiation of the study, before undergoing infertility treatment. Women consuming vitamin D supplements or taking medication for systematic disease or women who had undergone ovarian surgery were excluded from the study. All infertile women had serum AMH and vitamin D sampled on the same day. AFC was measured on the second or third day of the first cycle following the blood sampling for the determination of AMH and 25-OH vitamin D levels. MAIN RESULTS AND THE ROLE OF CHANCE: Among all patients, 30.7% (n = 87) were vitamin D deficient (<20 ng/mL) whereas 69.3% (n = 196) had normal vitamin D levels (≥20 ng/mL). The mean AMH and AFC levels did not differ significantly between the two groups: AMH 3.9 µ/L (±3.8) versus 4.3 µ/L (±4.8), (P value = 0.5) and AFC 13.9 (±13.3) versus 12.7 (±11.4), (P = 0.7), respectively. No correlation was observed between 25-O H vitamin D and AMH (spearman's r = 0.02, P value = 0.7) or AFC (spearman's r = -0.02, P value = 0.7). In multiple linear regression analysis, after adjusting for potential confounders (age, BMI, smoking status, infertility cause and season of blood sampling), the regression slope in all participants for total 25OH-D predicting log10 AMH was 0.006 [standard error (SE) = 0.07, P value = 0.9]. Similarly, no significant association was observed between AFC and vitamin D levels, even after controlling for relevant co-variants (regression coefficient -0.09. SE 0.08, P value = 0.2). LIMITATIONS, REASONS FOR CAUTION: Although this is the first prospective study to evaluate the relationship between vitamin D and the most important ovarian reserve markers (AMH and AFC), we need to acknowledge that the data used to generate the study findings are cross-sectional in nature. In this regard, we cannot generate or exclude any causal effect hypothesis. Nevertheless, our data support that an association between vitamin D and ovarian reserve markers is highly unlikely to exist. WIDER IMPLICATIONS OF THE FINDINGS: Although data from basic research indicate that vitamin D deficiency may have an effect on steroidogenesis and follicular development, our study, by prospectively recruiting a large number of infertile women, clearly demonstrates that vitamin D deficiency is highly unlikely to have a detrimental effect on ovarian reserve. Ongoing prospective and translational research projects are currently being conducted in order to evaluate the potential effect of vitamin D deficiency on reproductive outcome mediated through either an effect on the oocyte quality or on endometrial receptivity and embryo implantation. STUDY FUNDING/COMPETING INTERESTS: No external funding was used for this study. No conflicts of interest are declared. TRIAL REGISTRATION NUMBER: N/A.

DNA methylation reprogramming during oogenesis and interference by reproductive technologies: Studies in mouse and bovine models.

The use of assisted reproductive technology (ART) to overcome fertility problems has continued to increase since the birth of the first baby conceived by ART over 30 years ago. Similarly, embryo transfer is widely used as a mechanism to advance genetic gain in livestock. Despite repeated optimisation of ART treatments, pre- and postnatal outcomes remain compromised. Epigenetic mechanisms play a fundamental role in successful gametogenesis and development. The best studied of these is DNA methylation; the appropriate establishment of DNA methylation patterns in gametes and early embryos is essential for healthy development. Superovulation studies in the mouse indicate that specific ARTs are associated with normal imprinting establishment in oocytes, but abnormal imprinting maintenance in embryos. A similar limited impact of ART on oocytes has been reported in cattle, whereas the majority of embryo-focused studies have used cloned embryos, which do exhibit aberrant DNA methylation. The present review discusses the impact of ART on oocyte and embryo DNA methylation with regard to data available from mouse and bovine models.

Vitamin D deficiency and pregnancy rates in women undergoing single embryo, blastocyst stage, transfer (SET) for IVF/ICSI.

STUDY QUESTION: What is the influence of vitamin D deficiency on pregnancy rates among women undergoing IVF/ICSI and Day 5 (blastocyst stage) single embryo transfer (SET)? SUMMARY ANSWER: Vitamin D deficiency results in significantly lower pregnancy rates in women undergoing single blastocyst transfer. WHAT IS KNOWN ALREADY: Preliminary experiments have identified the presence of vitamin D receptors in the female reproductive system. However, results regarding the effect of vitamin D deficiency on clinical outcomes are conflicting. None of the previous studies adopted a SET strategy. STUDY DESIGN, SIZE, DURATION: Serum vitamin D concentration was measured retrospectively in patients who underwent SET on Day 5. Overall 368 consecutive infertile women treated within a period of 15 months were included in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients underwent ovarian stimulation for IVF/ICSI and Day 5 SET. Serum samples were obtained 7 days prior to embryo transfer and stored frozen at -20°C. Samples were collectively analyzed for their 25-OH vitamin D content. Vitamin D deficiency was defined as serum 25-OH vitamin D levels <20 ng/ml in accordance with the Institute of Medicine and the Endocrine Society clinical practice guidelines. MAIN RESULTS AND THE ROLE OF CHANCE: Clinical pregnancy rates were significantly lower in women with vitamin D deficiency compared with those with higher vitamin D values (41 versus 54%, P = 0.015).Logistic regression analysis was performed to identify whether vitamin D deficiency is independently associated with clinical pregnancy rates after controlling for 16 potential confounding factors. According to our results vitamin D deficiency was independently associated with lower clinical pregnancy rates, odds ratios [ORs (95% confidence interval (CI) 0.61 (0.39-0.95)] for vitamin D deficiency (deficient versus non-deficient women), P = 0.030. Finally, even when restricting our analysis to women undergoing elective SET (274 patients), vitamin D deficiency was again independently associated with pregnancy rates [OR (95% CI) 0.56 (0.33-0.93), P = 0.024]. LIMITATIONS, REASONS FOR CAUTION: Our results refer only to patients undergoing Day 5 SET. Although vitamin D deficiency appears to compromise pregnancy rates in this population, no guidance can be provided regarding a potential relationship between vitamin D deficiency and ovarian reserve or response to ovarian stimulation. WIDER IMPLICATIONS OF THE FINDINGS: Vitamin D deficiency impairs pregnancy rates in women undergoing single blastocyst transfer. Future prospective confirmatory studies are needed to validate our results and examine the exact underlying mechanism by which vitamin D levels may impair pregnancy rates in infertile women undergoing IVF/ICSI. STUDY FUNDING/COMPETING INTERESTS: None declared.

The effect of ovarian puncture on the endocrine profile of PCOS patients who undergo IVM.

BACKGROUND: To examine whether ovarian puncture for immature oocyte retrieval and in-vitro maturation (IVM) has an effect on the endocrine profile of patients with polycystic ovary syndrome (PCOS). METHODS: Twenty-two consecutive patients with PCOS undergoing IVM treatment were included. Serum anti-Müllerian hormone (AMH), sex hormone-binding globulin (SHBG), total testosterone (TT) and luteinized hormone (LH) levels were analyzed at the start of the cycle, on the day of immature oocyte retrieval (OR) and at fixed intervals thereafter, for up to three months after OR. RESULTS: Five days after OR circulating AMH, TT, calculated free testosterone (FTc), and LH levels were significantly reduced and circulating SHBG was significantly increased. Two weeks after OR, TT, FTc and LH remained reduced, whereas circulating AMH and SHBG levels recovered to pre-puncture values. Three months after OR, all circulating hormone levels had recovered to baseline values. CONCLUSION: Ovarian puncture for the retrieval of immature oocytes and IVM in patients with PCOS has a significant impact on the ovarian endocrine profile, but this impact is brief and transient.

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation.

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) is still the only option to preserve fertility in women who need to start gonadotoxic treatment urgently or in prepubertal children. During ovarian cortex preparation for cryopreservation, medullar tissue is removed. Growing antral follicles reside at the border of the cortex-medullar interface of the ovary and are broken during this process, releasing their cumulus-oocyte complex (COC). By thoroughly inspecting the medium and fragmented medullar tissue, these immature cumulus-oocyte complexes can be identified without interfering with the OTC procedure. The ovarian tissue-derived immature oocytes can be successfully matured in vitro, creating an additional source of gametes for fertility preservation. If OTC is performed within or near a medical assisted reproduction laboratory, all necessary in vitro maturation (IVM) and oocyte vitrification tools can be at hand. Furthermore, upon remission and child wish, the patient has multiple options for fertility restoration: ovarian tissue transplantation or embryo transfer after the insemination of vitrified/warmed oocytes. Hence, ovarian tissue oocyte-in vitro maturation (OTO-IVM) can be a valuable adjunct fertility preservation technique.

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).