Assessment of active translation in cumulus-enclosed and denuded oocytes during standard in vitro maturation and early embryo development.

Which actively translated maternal transcripts are differentially regulated between clinically relevant in vitro and in vivo maturation (IVM) conditions in mouse oocytes and zygotes? Our findings uncovered significant differences in the global transcriptome as well as alterations in the translation of specific transcripts encoding components of energy production, cell cycle regulation, and protein synthesis in oocytes and RNA metabolism in zygotes. Properly regulated translation of stored maternal transcripts is a crucial factor for successful development of oocytes and early embryos, particularly due to the transcriptionally silent phase of meiosis. This is a basic science study utilizing an ICR mouse model, best suited for studying in vivo maturation. In the treatment group, fully grown germinal vesicle oocytes from stimulated ovaries were in vitro matured to the metaphase II (MII) stage either as denuded without gonadotropins (IVM DO), or as cumulus-oocyte complexes (IVM COC) in the presence of 0.075 IU/ml recombinant FSH (rFSH) and 0.075 IU/ml recombinant hCG (rhCG). To account for changes in developmental competence, IVM COC from non-stimulated ovaries (IVM COC-) were included. In vivo matured MII oocytes (IVO) from stimulated ovaries were used as a control after ovulation triggering with rhCG. To simulate standard IVM conditions, we supplemented media with amino acids, vitamins, and bovine serum albumin. Accordingly, in vitro pronuclear zygotes (IMZ) were generated by IVF from IVM DO, and were compared to in vivo pronuclear zygotes (IVZ). All experiments were performed in quadruplicates with samples collected for both polyribosome fractionation and total transcriptome analysis. Samples were collected over three consecutive months. All ICR mice were bred under legal permission for animal experimentation (no. MZE-24154/2021-18134) obtained from the Ministry of Agriculture of the Czech Republic. Actively translated (polyribosome occupied) maternal transcripts were detected in in vitro and in vivo matured mouse oocytes and zygotes by density gradient ultracentrifugation, followed by RNA isolation and high-throughput RNA sequencing. Bioinformatic analysis was performed and subsequent data validation was done by western blotting, radioactive isotope, and mitotracker dye labelling. Gene expression analysis of acquired polysome-derived high-throughput RNA sequencing data revealed significant changes (RPKM ≥ 0.2; P ≤ 0.005) in translation between in vitro and in vivo matured oocytes and respectively produced pronuclear zygotes. Surprisingly, the comparison between IVM DO and IVM COC RNA-seq data of both fractionated and total transcriptome showed very few transcripts with more than a 2-fold difference. Data validation by radioactive isotope labelling revealed a decrease in global translation bof20% in IVM DO and COC samples in comparison to IVO samples. Moreover, IVM conditions compromised oocyte energy metabolism, which was demonstrated by both changes in polysome recruitment of each of 13 mt-protein-coding transcripts as well as by validation using mitotracker red staining. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE241633 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241633). It is extremely complicated to achieve in vivo consistency in animal model systems such as porcine or bovine. To achieve a high reproducibility of in vivo stimulations, the ICR mouse model was selected. However, careful interpretation of our findings with regard to assisted reproductive techniques has to be made by taking into consideration intra-species differences between the mouse model and humans. Also, the sole effect of the cumulus cells' contribution could not be adequately addressed by comparing IVM COC and IVM DO, because the IVM DO were matured without gonadotropin supplementation. Our findings confirmed the inferiority of standard IVM technology compared with the in vivo approach. It also pointed at compromised biological processes employed in the critical translational regulation of in vitro matured MII oocytes and pronuclear zygotes. By highlighting the importance of proper translational regulation during in vitro oocyte maturation, this study should prompt further clinical investigations in the context of translation. This work was supported by the Czech Grant Agency (22-27301S), Charles University Grant Agency (372621), Ministry of Education, Youth and Sports (EXCELLENCE CZ.02.1.01/0.0/0.0/15_003/0000460 OP RDE), and Institutional Research Concept RVO67985904. No competing interest is declared.

Dvoran M ，Iyyappan R ，Masek T ，Pospisek M ，Kubelka M ，Susor A ... -  《-》

A pre-in vitro maturation medium containing cumulus oocyte complex ligand-receptor signaling molecules maintains meiotic arrest, supports the cumulus oocyte complex and improves oocyte developmental competence.

Can a pre-in vitro maturation (pre-IVM) medium containing signaling molecules rather than chemical/pharmaceutical agents, sustain meiotic arrest and improve developmental competence of in vitro matured oocytes in CF1 outbred mice? A short 2 h period of pre-IVM prevents spontaneous meiotic resumption, improves mitochondria activity in subsequently matured oocytes, and increases developmental competence, pregnancy rate and implantation of resulting embryos. Spontaneous resumption of meiosis in vitro is detrimental for oocyte developmental competence. Pre-IVM systems that prevent spontaneous meiotic resumption with chemical/pharmaceutical agents are a promising approach to improving IVM oocyte competence; however, the success of these methods has proven to be inconsistent. This study consisted of a series of experiments using cumulus oocyte complexes (COC) derived from outbred mice following ovarian stimulation. The study was designed to examine if a novel, ligand/receptor-based pre-IVM treatment could sustain meiotic arrest in vitro and improve oocyte developmental competence, compared to control IVM. Two pre-IVM durations (2 h and 24 h) were evaluated, and the effect of the mitochondrial stimulator PQQ during 24 h pre-IVM was studied. Murine (outbred CF1) immature COC were cultured in vitro in the presence of C-type natriuretic peptide (CNP) (30 nM), estradiol (100 nM), FSH (1 × 10-4 IU/ml) and bone morphogenic protein 15 (BMP15) (100 ng/ml) for 2 h or 24 h prior to IVM. Meiotic status during pre-IVM and IVM was analyzed using orcein staining, and functionality of gap junction communication was confirmed using the functional gap junction inhibitor carbenoxolone (CBX). Oocytes exposed to pre-IVM treatment were compared to control oocytes collected on the same day from the same females and undergoing standard IVM. Developmental competence and embryo viability was assessed by oocyte mitochondrial activity and ATP concentration, in vitro embryo development following IVF and in vitro culture, blastocyst cell number and allocation, embryo morphokinetics, and embryo transfer. Differences were determined to be significant when P < 0.05. Both a short (2 h) and long (24 h) pre-IVM period successfully prevented spontaneous resumption of meiosis. Moreover, gap junctions remained open during the pre-IVM period, as shown by the resumption of meiosis (95.9 ± 2.1%) in the presence of CBX during pre-IVM. A 2 h pre-IVM treatment improved blastocyst development after 96 h of culture per cleaved embryo compared to control (71.9 ± 7.4% versus 53.3 ± 6.2%, respectively), whereas a longer 24 h pre-IVM had no effect on development. A short 2 h period of pre-IVM increased mitochondrial activity in mature oocytes. On the contrary, mitochondrial activity was reduced in mature oocytes following 24 h of arrest and IVM. Treatment of arrested COC with pyrroloquinoline quinone (PQQ) during the 24 h pre-IVM period successfully maintained mitochondrial activity equal to control. However, PQQ was not able to improve blastocyst development compared to pre-IVM 24 h without PQQ. Moreover, ATP concentration in mature oocytes following pre-IVM and/or IVM, did not differ between treatments. A 2 h pre-IVM period prior to IVM improved pregnancy rate following transfer to recipient females. Implantation was also improved after transfer of embryos derived from oocytes arrested for either 2 h or 24 h prior to IVM, compared to control IVM derived embryos (41.9 ± 9%, 37.2 ± 9.5% and 17.2 ± 8.3%, respectively), although fetal development did not differ. Slower meiotic resumption and enhanced mitochondrial activity likely contribute to improved developmental competence of oocytes exposed to pre-IVM for 2 h, but further experiments are required to identify specific mechanisms. Maintaining oocytes in meiotic arrest for 24 h with this approach could be a potential window to improve oocyte quality. However, an initial attempt to utilize this period of arrest to manipulate quality with PQQ, a mitochondrial stimulator, did not improve oocyte competence. IVM could be an attractive clinical alternative to conventional IVF, with reduced time, cost and reliance on high doses of exogenous hormones to stimulate follicle growth, thus eliminating ovarian hyperstimulation syndrome (OHSS). Currently IVM is not widely used as it results in reduced embryo development and lower pregnancy outcomes compared to embryos produced from in vivo matured oocytes. Our approach to IVM, incorporating a ligand/receptor pre-IVM period, could improve human oocyte quality following IVM leading to routine adoption of this patient friendly technology. In addition, our methodology of pre-IVM containing signaling molecules rather than chemical/pharmaceutical agents may prove to be more consistent at improving oocyte quality than those focusing only on cAMP modulation with pharmacological agents. Finally, a reliable method of maintaining oocytes in meiotic arrest in vitro provides a novel window of opportunity in which the oocyte may be manipulated to address specific physiological deficiencies prior to meiotic resumption. N/A. This study was supported by the Colorado Center for Reproductive Medicine (CCRM, Lone Tree, Colorado USA). We declare no conflict of interest.

Santiquet NW ，Greene AF ，Becker J ，Barfield JP ，Schoolcraft WB ，Krisher RL ... -  《-》

Differences in cumulus cell gene expression indicate the benefit of a pre-maturation step to improve in-vitro bovine embryo production.

Does the gene expression profile of cumulus cells (CC) accompanying oocytes with different degrees of chromatin compaction within the germinal vesicle (GV) reflect the oocyte's quality and response in culture during in-vitro embryo production (IVP). The transcriptomic profile of the CC is related to oocyte competence, setting the stage for the development of customized pre-maturation strategies to improve IVP. Oocytes complete the acquisition of their competence during antral follicle development. During this period, the chromatin configuration within the GV changes dynamically and is indicative of oocyte's developmental potential. The interactions between somatic and germ cells modulate chromatin morphology and function and are critical for acquisition of oocyte competence. Bovine cumulus-oocyte complexes (COC) were isolated from 0.5 to 6 mm antral follicles. Surrounding CC were separated from the oocyte and classified as GV0, GV1, GV2 and GV3 according to the degree of the oocyte's chromatin compaction. RNA extracted from CC of each group was amplified and hybridized on a bovine embryo-specific 44 K Agilent slide. The CC_GV1, CC_GV2 and CC_GV3 classes were each hybridized against the CC_GV0 class, representing an early oocyte differentiation stage with poor development competence. The data were normalized and fold changes of the differentially expressed genes were determined. Microarray data were validated using quantitative RT-PCR on selected targets. Microarray data were further analyzed through: (i) between-group analysis (BGA), which classifies the samples according to their transcriptomic profiles; (ii) cluster analysis according to the expression profile of each gene; and (iii) Ingenuity Pathway Analysis (IPA) to study gene regulation patterns and predicted functions. Furthermore, CC of each GV group were cultured and apoptotic cells were assessed after 3 h by caspase analysis. Finally, based on the analysis of CC transcriptomic profiles and the relationship between morphological features of the COC and the oocyte chromatin configuration, a customized, stage-dependent oocyte pre-maturation (pre-IVM) system was used to improve oocyte developmental potential before IVM. For this, the blastocyst rate and quality were assessed after in-vitro maturation and fertilization of pre-matured oocytes. Overall, quantitative RT-PCR results of a subset of five selected genes were consistent with the microarray data. Clustering analysis generated 16 clusters representing the main profiles of transcription modulation. Of the 5571 significantly differentially expressed probes, the majority (25.49%) best fitted with cluster #6 (downregulation between CC_GV0 and CC_GV1 and stable low levels in successive groups). IPA identified the most relevant functions associated with each cluster. Genes included in cluster #1 were mostly related to biological processes such as 'cell cycle' and 'cell death and survival', whereas genes included in cluster #5 were mostly related to 'gene expression'. Interestingly, 'lipid metabolism' was the most significant function identified in clusters #6, #9 and #12. IPA of gene lists obtained from each contrast (i.e., CC_GV0 vs. CC_GV1; CC_GV0 vs. CC_GV2; CC_GV0 vs. CC_GV3) revealed that the main affected function in each contrast was 'cell death and survival'. Importantly, apoptosis was predicted to be inhibited in CC_GV1 and CC_GV2, but activated in CC_GV3. Caspase analysis indicated that a low percentage of CC_GV0 was prone to undergo apoptosis but apoptosis increased significantly in CC from oocytes with condensed chromatin, reaching a peak in CC_GV3 (P < 0.05). Finally, the tailored oocyte pre-maturation strategy, based on morphological features of the COC and the oocyte chromatin configuration, demonstrated that pre-IVM improved the developmental capability of oocytes at early stages of differentiation (GV1-enriched COC) but was detrimental for oocytes at more advanced stages of development (GV2 and GV3-enriched COC). The data are available through the GEO series accession number GSE79886. This study was conducted with bovine samples. Whether or not the results are applicable to human oocytes requests further elucidation. Embryo transfer experiments are required to determine whether the improvement in blastocyst rates in the tailored system leads to increased live birth rates. The identification of multiple non-invasive biomarkers predictive of oocyte quality can greatly strengthen the pre-IVM approach aimed to improve IVM outcomes. These results have potentially important implications in treating human infertility and in developing breeding schemes for domestic mammals. This work was supported in part by NSERC Strategic Network EmbryoGENE, Canada and in part by CIG-Marie Curie Actions-Reintegration Grants within the EU 7FP (n. 303640, 'Pro-Ovum'). The authors declare no potential conflict of interest.

Dieci C ，Lodde V ，Labreque R ，Dufort I ，Tessaro I ，Sirard MA ，Luciano AM ... -  《-》

Improved cryotolerance and developmental potential of in vitro and in vivo matured mouse oocytes by supplementing with a glutathione donor prior to vitrification.

Can supplementation of media with a glutathione (GSH) donor, glutathione ethyl ester (GEE), prior to vitrification protect the mouse oocyte from oxidative damage and critical changes in redox homeostasis, and thereby improve cryotolerance? GEE supplementation supported redox regulation, rapid recovery of spindle and chromosome alignment after vitrification/warming and improved preimplantation development of mouse metaphase II (MII) oocytes. Cryopreservation may affect mitochondrial functionality, induce oxidative stress, and thereby affect spindle integrity, chromosome segregation and the quality of mammalian oocytes. GEE is a membrane permeable GSH donor that promoted fertilization and early embryonic development of macaque and bovine oocytes after IVM. Two experimental groups consisted of (i) denuded mouse germinal vesicle (GV) oocytes that were matured in vitro in the presence or absence of 1 mM GEE (IVM group 1) and (ii) in vivo ovulated (IVO) MII oocytes that were isolated from the ampullae and exposed to 1 mM GEE for 1 h prior to vitrification (IVO group 2). Recovery of oocytes from both groups was followed after CryoTop vitrification/warming for up to 2 h and parthenogenetic activation. Reactive oxygen species (ROS), spindle morphology and chromosome alignment were analyzed by confocal laser scanning microscopy (CLSM) and polarization microscopy in control and GEE-supplemented MII oocytes. The relative overall intra-oocyte GSH content was assessed by analysis of monochlorobimane (MBC)-GSH adduct fluorescence in IVM MII oocytes. The GSH-dependent intra-mitochondrial redox potential (EmGSH) of IVM MII oocytes was determined after microinjection with specific mRNA at the GV stage to express a redox-sensitive probe within mitochondria (mito-Grx1-roGFP2). The absolute negative redox capacity (in millivolts) was determined by analysis of fluorescence of the oxidized versus the reduced form of sensor by CLSM and quantification according to Nernst equation. Proteome analysis was performed by quantitative 2D saturation gel electrophoresis (2D DIGE). Since microinjection and expression of redox sensor mRNA required removal of cumulus cells, and IVM of denuded mouse oocytes in group 1 induces zona hardening, the development to blastocysts was not assessed after IVF but instead after parthenogenetic activation of vitrified/warmed MII oocytes from both experimental groups. IVM of denuded mouse oocytes in the presence of 1 mM GEE significantly increased intra-oocyte GSH content. ROS was not increased by CryoTop vitrification but was significantly lower in the IVM GEE group compared to IVM without GEE before vitrification and after recovery from vitrification/warming (P < 0.001). Vitrification alone significantly increased the GSH-dependent intra-mitochondrial redox capacity after warming (EmGSH, P < 0.001) in IVM oocytes, presumably by diffusion/uptake of cytoplasmic GSH into mitochondria. The presence of 1 mM GEE during IVM increased the redox capacity before vitrification and there was no further increase after vitrification/warming. None of the reproducibly detected 1492 spots of 2D DIGE separated proteins were significantly altered by vitrification or GEE supplementation. However, IVM of denuded oocytes significantly affected spindle integrity and chromosome alignment right after warming from vitrification (0 h) in group 1 and spindle integrity in group 2 (P < 0.05). GEE improved recovery in IVM group as numbers of oocytes with unaligned chromosomes and aberrant spindles was not significantly increased compared to unvitrified controls. The supplementation with GEE for 1 h before vitrification also supported more rapid recovery of spindle birefringence. GEE improved significantly development to the 2-cell stage for MII oocytes that were activated directly after vitrification/warming in both experimental groups, and also the blastocyst rate in the IVO GEE-supplemented group compared to the controls (P < 0.05). None LIMITATIONS, REASONS FOR CAUTION: The studies were carried out in a mouse model, in IVM denuded rather than cumulus-enclosed oocytes, and in activated rather than IVF MII oocytes. Whether the increased GSH-dependent intra-mitochondrial redox capacity also improves male pronuclear formation needs to be studied further experimentally. The influence of GEE supplementation requires also further examination and optimization in human oocytes before it can be considered for clinical ART. Although GEE supplementation did not alter the proteome at MII, the GSH donor may support cellular homeostasis and redox regulation and, thus, increase developmental competence. While human MII oocyte vitrification is an established procedure, GEE might be particularly beneficial for oocytes that suffer from oxidative stress and reduced redox capacity (e.g. aged oocytes) or possess low GSH due to a reduced supply of GSH from cumulus. It might also be of relevance for immature human oocytes that develop without cumulus to MII in vitro (e.g. in ICSI cycles) for ART. The study has been supported by the German Research Foundation (DFG FOR 1041; EI 199/3-2). There are no conflict of interests.

Trapphoff T ，Heiligentag M ，Simon J ，Staubach N ，Seidel T ，Otte K ，Fröhlich T ，Arnold GJ ，Eichenlaub-Ritter U ... -  《-》

An improved IVM method for cumulus-oocyte complexes from small follicles in polycystic ovary syndrome patients enhances oocyte competence and embryo yield.

Are meiotic and developmental competence of human oocytes from small (2-8 mm) antral follicles improved by applying an optimized IVM method involving a prematuration step in presence of C-Type Natriuretic Peptide (CNP) followed by a maturation step in presence of FSH and Amphiregulin (AREG)? A strategy involving prematuration culture (PMC) in the presence of CNP followed by IVM using FSH + AREG increases oocyte maturation potential leading to a higher availability of Day 3 embryos and good-quality blastocysts for single embryo transfer. IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for the patients, but the approach is not widely used because of an efficiency gap compared to conventional ART. In vitro systems that enhance synchronization of nuclear and cytoplasmic maturation before the meiotic trigger are crucial to optimize human IVM systems. However, previous PMC attempts have failed in sustaining cumulus-oocyte connections throughout the culture period, which prohibited a normal cumulus-oocyte communication and precluded an adequate response by the cumulus-oocyte complex (COC) to the meiotic trigger. A first prospective study involved sibling oocytes from a group of 15 patients with polycystic ovary syndrome (PCOS) to evaluate effects of a new IVM culture method on oocyte nuclear maturation and their downstream developmental competence. A second prospective study in an additional series of 15 women with polycystic ovaries characterized and fine-tuned the culture conditions. Fifteen women with PCOS (according to Rotterdam criteria) underwent IVM treatment after 3-5 days of highly purified human menopausal gonadotropin (HP-hMG) stimulation and no human chorionic gonadotropin (hCG) trigger before oocyte retrieval. A first study was designed with sibling oocytes to prospectively evaluate the impact of an IVM culture method: 24 h PMC with CNP + 30 h IVM with FSH and AREG, on embryo yield, in comparison to the standard (30 h) IVM clinical protocol (Group I, n = 15). A second prospective study was performed in 15 women with polycystic ovaries, to characterize and optimize the PMC conditions (Group II, n = 15). The latter study involved the evaluation of oocyte meiotic arrest, the preservation of cumulus-oocyte transzonal projections (TZPs), the patterns of oocyte chromatin configuration and cumulus cells apoptosis following the 24 and 46 h PMC. Furthermore, oocyte developmental potential following PMC (24 and 46 h) + IVM was also evaluated. The first 20 good-quality blastocysts from PMC followed by IVM were analysed by next generation sequencing to evaluate their aneuploidy rate. PMC in presence of CNP followed by IVM using FSH and AREG increased the meiotic maturation rate per COC to 70%, which is significantly higher than routine standard IVM (49%; P ≤ 0.001). Hence, with the new system the proportion of COCs yielding transferable Day 3 embryos and good-quality blastocysts increased compared to routine standard IVM (from 23 to 43%; P ≤ 0.001 and from 8 to 18%; P ≤ 0.01, respectively). CNP was able to prevent meiosis resumption for up to 46 h. After PMC, COCs had preserved cumulus-oocyte TZPs. The blastocysts obtained after PMC + IVM did not show increased aneuploidy rates as compared to blastocysts from conventional ART. The novel IVM approach in PCOS patients was tested in oocytes derived from small antral follicles which have an intrinsically low developmental potential. Validation of the system would be required for COCs from different (larger) follicular sizes, which may involve further adjustment of PMC conditions. Furthermore, considering that this is a novel strategy in human IVM treatment, its global efficiency needs to be confirmed in large prospective randomized controlled trials. The further application in infertile patients without PCOS, e.g. cancer patients, remains to be evaluated. The findings of this pilot study suggest that the efficiency gap between IVM and conventional IVF can be reduced by fine-tuning of the culture methods. This novel strategy opens new perspectives for safe and patient-friendly ART in patients with PCOS. IVM research at the Vrije Universiteit Brussel has been supported by grants from: the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680); the Fund for Research Flanders (Fonds Wetenschappelijk Onderzoek-Vlaanderen-FWO, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69). The authors have no conflicts of interest.

Sánchez F ，Lolicato F ，Romero S ，De Vos M ，Van Ranst H ，Verheyen G ，Anckaert E ，Smitz JEJ ... -  《-》