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

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

Depletion of aneuploid cells in human embryos and gastruloids

Chromosomal instability leading to aneuploidy is pervasive in early human embryos and is considered as a major cause of infertility and pregnancy wastage. Here we provide several lines of evidence that blastocysts containing aneuploid cells are worthy of in vitro fertilization transfer. First, we show clinically that aneuploid embryos can lead to healthy births, suggesting the presence of an in vivo mechanism to eliminate aneuploidy. Second, early development and cell specification modelled in micropatterned human 'gastruloids' grown in confined geometry show that aneuploid cells are depleted from embryonic germ layers, but not from extraembryonic tissue, by apoptosis in a bone morphogenetic protein 4 (BMP4)-dependent manner. Third, a small percentage of euploid cells rescues embryonic tissue in mosaic gastruloids when mixed with aneuploid cells. Finally, single-cell RNA-sequencing analysis of early human embryos revealed a decline of aneuploidy beginning on day 3. Our findings challenge two current dogmas: that a single trophectoderm biopsy at blastocyst stage to perform prenatal genetic testing can accurately determine the chromosomal make-up of a human embryo, and that aneuploid embryos should be withheld from embryo transfer in association with in vitro fertilization

The Effect of Cilostamide on Gap Junction Communication Dynamics, Chromatin Remodeling, and Competence Acquisition in Pig Oocytes Following Parthenogenetic Activation and Nuclear Transfer

In the pig, the efficiency of in vitro embryo production and somatic cell nuclear transfer (SCNT) procedures remains limited. It has been suggested that prematuration treatments (pre-IVM) based on the prolongation of a patent bidirectional crosstalk between the oocyte and the cumulus cells through gap junction mediate communication (GJC), together with the maintenance of a proper level of cAMP, could improve the developmental capability of oocytes. The aim of this study was to assess: (i) dose dependent effects of cilostamide on nuclear maturation kinetics; (ii) the relationship between treatments on GJC functionality and large-scale chromatin configuration changes; (iii) and the impact of treatments on developmental competence acquisition after parthenogenic activation (PA) and SCNT. Accordingly, COC were collected from 3-6 mm antral follicles and cultured for 24 h in defined culture medium with or without 1 \u3bcM cilostamide. GJC functionality was assessed by Lucifer Yellow microinjection, while chromatin configuration was evaluated by fluorescence microscopy after nuclear staining. Cilostamide administration sustained functional coupling up to 24 h of culture and delayed meiotic resumption as only 25.6% of cilostamide-treated oocytes reached ProMI stage compared to the control (69.7%; P<0.05). Moreover, progressive chromatin condensation was delayed before meiotic resumption based upon G2/M biomarker phosphoprotein epitope acquisition using immunolocalization. Importantly, cilostamide treatment under these conditions, improved oocyte developmental competence as reflected in higher blastocyst quality after both parthenogenetic activation and SCN

Natriuretic peptide precursor C delays meiotic resumption and sustains gap junction-mediated communication in bovine cumulus-enclosed oocytes

Oocyte in vitro maturation (IVM) has become a valuable technological tool for animal breeding and cloning and the treatment of human infertility because it does not require the administration of exogenous gonadotropin to obtain fertilizable oocytes. However, embryo development after IVM is lower compared to in vivo maturation, most likely because oocytes collected for IVM are heterogeneous with respect to their developmental competencies. Attempts to improve IVM outcome have relied upon either prematuration culture (PMC) or two-step maturation strategies in the hope of normalizing variations in developmental competence. Such culture systems invoke the pharmacological arrest of meiosis, in theory providing oocytes sufficient time to complete the acquisition of developmental competence after cumulus-enclosed oocytes isolation from the follicle. The present study was designed to test the efficiency of natriuretic peptide precursor C (NPPC) as a nonpharmacologic meiosis-arresting agent during IVM in a monoovulatory species. NPPC has been shown to maintain meiotic arrest in vivo and in vitro in mice and pigs; however, the use of this molecule for PMC has yet to have been explored. Toward this end, meiotic cell cycle reentry, gap-junction functionality, and chromatin configuration changes were investigated in bovine cumulus-enclosed oocytes cultured in the presence of NPPC. Moreover, oocyte developmental competence was investigated after IVM, in vitro fertilization, and embryo culture and compared to standard IVM-in vitro fertilization protocol without PMC. Our results suggest that NPPC can be used to delay meiotic resumption and increase the developmental competence of bovine oocytes when used in PMC protocols

Role of Natriuretic Peptide Type C (CNP) in Maintaining Meiotic Arrest and Sustaining Gap Junction Mediated Communications and Chromatin Configuration Changes in Bovine Cumulus Enclosed Oocytes

Natriuretic peptide type C (CNP) and its receptor natriuretic peptide receptor 2 (NPR2) contribute to maintaining oocyte meiotic arrest. In mice CNP is produced by granulosa cells (GC) and binds to its receptor NPR2, a guanylyl-cyclase that mediates cGMP delivery to the oocyte through gap junction mediated coupling (GJC). CNP/NPR2 signaling inhibits oocyte Phosphodiesterase 3A (PDE3A), and thus maintains oocyte meiotic arrest. In mice, exogenous CNP maintains oocyte meiotic arrest only in the presence of estradiol (E2) that induces the expression of NPR2. We investigated the role of CNP in bovine cumulus enclosed oocytes (CEOs) in 1) preventing spontaneous oocyte meiotic resumption upon removal from follicle, 2) sustaining GJC and 3) promoting chromatin configuration changes (transition from GV1 to GV3) required to achieve developmental potential before germinal vesicle (GV) breakdown. Experiments were repeated 3 times. Data were analyzed by Fisher\u2019s exact test or ANOVA. In preliminary dose-response experiments, 325 CEOs were cultured with 0-500 nM CNP \ub1 100 nM E2 for 14 h. Compared to control group, 100 nM CNP +100 nM E2 was the most effective treatment in maintaining meiotic arrest but only transiently even in presence of E2. Successively, 900 CEOs were cultured for 6-8 h either in the absence or presence of CNP or 10 \u3bcM Cilostamide, a specific PDE3A inhibitor used as a control of efficient meiotic arrest. Both treatments sustained GJC during 6-8 h of culture, when compared to control group (P<0.01) as assessed by Lucifer Yellow-dye spreading in surrounding cells after ooplasmic injection. However, while CNP arrested significantly more oocytes at the GV stage compared to control (85% vs 59% after 6 h and 75% vs 49% after 8 h, respectively, P<0.005), Cilostamide more efficiently maintained meiotic arrest for 6-8 h (96-98%, P<0.01). Progressive chromatin configuration transition from GV1 to GV3 stages was observed, by DAPI staining, during meiotic arrest in both treatments. This was confirmed by immunolocalization of G2/M mitosis-associated ser/thr phospho-proteins (MPM2) used as a marker of meiotic resumption. MPM2 signal increased in the GV along with changes in chromatin configurations and in the ooplasm during meiotic resumption but ooplasmic MPM2 augmentation was delayed during meiotic arrest with either CNP or Cilostamide. Finally, reversibility of meiotic arrest was assessed on a total of 458 CEOs. After 6h of culture with either CNP or Cilostamide, the percentage of oocytes reaching MII after 22h of IVM was similar to control for 24 h (76%, 82% and 84%, respectively). This is the first report on the putative role of CNP on bovine meiotic progression and its involvement in GJC control and large-scale chromatin remodeling. Importantly, our study indicates striking differences compared to the mouse since CNP only transiently arrests meiotic progression even in presence of E2. Moreover, RT-PCR showed the presence of Npr2 in both oocyte and GCs, while mice only expressed Npr2 in GC. Interestingly CNP represent a \u2018physiological\u2019 option instead of pharmacological inhibition of meiotic resumption for the development of culture systems that can sustain GJC and promote large-scale chromatin remodeling and oocyte differentiation. Funded by: L\u2019Oreal Italia per le Donne e la Scienza 2012; Marie Curie Actions FP7-CIG (Contract: 303640,"Pro-Ovum"); Dote Ricerca-FSE, Regione Lombardia

IVF, from the past to the future: the inheritance of the Capri Workshop Group

Today IVF use is booming all over the world and has even started to play a role in demographic analyses. Prognosis-adjusted estimates suggest that up to two-thirds of couples could achieve a live birth. However, the scenario is less exciting in reality. Discontinuation during the cycles is common, and age and ovarian response continue to be crucial in modulating this rate of success. A growing interest is now given to the risk of abuses and in particular to overtreatment and to prescriptions of useless, if not harmful, expensive additional treatments (\u2018add-ons\u2019). A more rational, evidence-based and wise approach is needed. From a scientific perspective, several obscure aspects remain and warrant future investigations. Of particular interest are the neglected role of sperm selection, the potential adult implications of early embryo life in vitro and the issue of sustainabilit

A prognosis-based approach to infertility: understanding the role of time

The current definition of infertility acknowledges the importance of duration of pregnancy seeking but fails to recognize the prevalent negative impact of female age. In fact, the diagnosis of unexplained infertility increases with women's age because of our incapacity to discern between age-related infertility and real unexplained infertility. Physicians' response to the pressures of increased female age has been to take prompt refuge in assisted reproduction despite the lack of robust evidence and the inherent risks and costs of these procedures. Moreover, the prioritization of immediate health gains over those in the future, preference for accessing active treatment rapidly and reluctance to wait for spontaneous pregnancy expose patients to additional risks of overtreatment. Solutions are not simple to find but an alternative and innovative vision of infertility based on prognosis may be a valid solution. The availability of validated dynamic models based on real-life data that could predict both natural and ART-mediated conceptions may be of benefit. They could facilitate patients' counselling and could optimize the chances of success without exposing patients to unnecessary, expensive and demanding treatment