Electro-Metabolic Coupling of Cumulus-Oocyte Complex.

Oocyte-cumulus cell interaction is essential for oocyte maturation and competence. The bidirectional crosstalk network mediated by gap junctions is fundamental for the metabolic cooperation between these cells. As cumulus cells exhibit a more glycolytic phenotype, they can provide metabolic substrates that the oocyte can use to produce ATP via oxidative phosphorylation. The impairment of mitochondrial activity plays a crucial role in ovarian aging and, thus, in fertility, determining the success or failure of assisted reproductive techniques. This review aims to deepen the knowledge about the electro-metabolic coupling of the cumulus-oocyte complex and to hypothesize a putative role of potassium channel modulators in order to improve fertility, promote intracellular Ca2+ influx, and increase the mitochondrial biogenesis and resulting ATP levels in cumulus cells.

Testosterone Supplementation Promotes Estrogen Synthesis of Buffalo Cumulus Cells Surrounding In Vitro-Matured Oocytes.

Cumulus cells (CCs) synthesize estrogens that are essential for follicular development. However, the effects of androgen on estrogen production in buffalo CCs remain unknown. In the present study, the impacts of testosterone on estrogen synthesis of buffalo CCs surrounding in vitro-matured oocytes were investigated. The results showed that testosterone supplementation improved both the expression levels of estrogen synthesis-related genes (CYP11A1, CYP19A1, and 17ß-HSD) and the secretion levels of estradiol in buffalo CCs surrounding in vitro-matured oocytes. Furthermore, testosterone treatment enhanced the sensitivity of buffalo CCs surrounding in vitro-matured oocytes to follicle-stimulating hormone (FSH). This study indicated that testosterone supplementation promoted the estrogen synthesis of buffalo CCs surrounding in vitro-matured oocytes mainly through strengthening the responsiveness of CCs to FSH. The present study serves as a foundation of acquiring high-quality recipient oocytes for buffalo somatic cell nuclear transfer.

Oocyte Competence of Prepubertal Sheep and Goat Oocytes: An Assessment of Large-Scale Chromatin Configuration and Epidermal Growth Factor Receptor Expression in Oocytes and Cumulus Cells.

The oocyte competence of prepubertal females is lower compared to that of adults, mainly because they originate from small follicles. In adult females, the germinal vesicle (GV) and epidermal growth factor receptor (EGFR) have been associated with oocyte competence. This study aimed to analyze GV chromatin configuration and EGFR expression in prepubertal goat and sheep oocytes obtained from small (<3 mm) and large (≥3 mm) follicles and compare them with those from adults. GV chromatin was classified from diffuse to condensed as GV1, GVn, and GVc for goats and NSN, SN, and SNE for sheep. EGFR was quantified in cumulus cells (CCs) by Western blotting and in oocytes by immunofluorescence. Oocytes from prepubertal large follicles and adults exhibited highly condensed chromatin in goats (71% and 69% in GVc, respectively) and sheep (59% and 75% in SNE, respectively). In both species, EGFR expression in CCs and oocytes was higher in prepubertal large follicles than in small ones. In adult females, EGFR expression in oocytes was higher than in prepubertal large follicles. In conclusion, GV configuration and EGFR expression in CCs and oocytes were higher in the large than small follicles of prepubertal females.

Cyanidin-3-O-glucoside protects Zearalenone-induced in vitro maturation disorders of porcine oocytes by alleviating NOX4-dependent oxidative stress and endoplasmic reticulum stress in cumulus cells.

Zearalenone (ZEN) is widely found in foodstuffs and has serious harmful effects on female fertility, especially in pigs. Cyanidin-3-O-glucoside (C3G), a type of anthocyanin, exists in most dark fruits and vegetables; it has many positive dietary effects including as an antioxidant, anti-inflammatory, or anti-apoptotic agent. However, the beneficial effects of C3G alongside ZEN-induced damage in porcine oocytes and the underlying molecular mechanism have not been investigated. In this work, porcine cumulus-oocyte complexes (COCs) were divided into Control (Ctrl), ZEN, ZEN + C3G (Z + C), and C3G, and treated for 44-46 h in vitro. The results showed that C3G could alleviate ZEN-induced disorders of first polar body (PBI) extrusion, abnormalities of spindle assembly, cortical granule distribution, and mitochondrial distribution; these results were produced via restoring transzonal projections (TZPs), and inhibiting nicotinamide adenine dinucleotide phosphate oxidase (NOX4)-dependent oxidative stress and 'glucose regulatory protein 78/protein kinase-like endoplasmic reticulum kinase/α subunit of eukaryotic initiation factor 2α/activating transcription factor 4/C/EBP-homologous protein' (GRP78/PERK/eIF2α/ATF4/CHOP)-mediated endoplasmic reticulum stress (ERS) during oocyte maturation. Moreover, the over-expression of NOX4 in cumulus cells could result in a significant increase in ROS levels and ER fluorescence intensity in oocytes. In conclusion, C3G promoted in vitro maturation of porcine oocytes exposed to ZEN via mitigating NOX4-dependent oxidative stress and ERS in cumulus cells. These results contribute to our comprehension of the molecular mechanisms underlying the protective effects of C3G against ZEN toxicity in porcine oocytes, and they provide a novel theoretical foundation and strategy for future applications of C3G in the improvement of female reproduction.

Autophagy activity is increased in the cumulus cells of women with poor ovarian response.

OBJECTIVE: To evaluate the autophagy status of cumulus cells (CCs) in women with poor ovarian response (POR). MATERIALS AND METHODS: CCs were divided into normal ovarian response (NOR) group and POR group. The ultrastructure of autophagy was analyzed by transmission electron microscopy (NOR: n = 18, POR: n = 26). The mRNA and protein of autophagy markers were detected by Quantitative real-time polymerase chain reaction (NOR: n = 15, POR: n = 19) and Western blotting (NOR: n = 41, POR: n = 38), respectively. RESULTS: Transmission electron microscopy demonstrated abundant autophagosomes and even autophagic death in the POR group. There were no differences in LC3 and P62 mRNA expression between the two groups (p > 0.05). The BCL2 mRNA expression was lower in the POR group (p < 0.05). Moreover, the LC3 II/I ratio and the P62 protein expression were significantly higher in the POR group (p < 0.05). CONCLUSIONS: Autophagy in CCs of POR women is activated and the autophagic flux is blocked. The up-regulation of autophagy in CCs may be related to the pathogenesis of POR.

Characterization of metabolic patterns in porcine cumulus cells during meiotic maturation.

Metabolic coupling between oocytes and the surrounding somatic cells allows for normal two-way communication, and their interactions is necessary for generating developmentally competent eggs. However, the metabolic framework that support oocyte maturation in surrounding cumulus cells is still lacking. Herin, we established a temporal metabolome profile of porcine cumulus cells at three key stages during oocyte maturation, illustrating the picture of global metabolic network in cumulus cells. Importantly, we discovered the novel metabolic signature in cumulus cells during meiotic maturation, in specific, significant consumption of fatty acids, elevated activity of hexosamine biosynthetic pathway (HBP), and enhanced polyamine biosynthesis. Meanwhile, we observed the different utilization of tryptophan, active biosynthesis of progesterone, and progressive decrease in purine and pyrimidine metabolism as the oocytes progress through meiosis. Collectively, our metabolomic data serves an entree to elaborate on the dynamic changes in these metabolic pathways, which not only reveals the metabolic networks controlling oocyte development, but also lays a foundation for the discovery of biomarkers in the improvement in porcine oocyte culture system.

Mitochondrial Dysfunction in Advanced Maternal Aged Cumulus Cells: A Possible Link to ATP Synthase Impairment?

Age-related changes in the mitochondrial status of human cumulus cells (hCCs) impact oocyte quality; however, the relationship between hCC mitochondrial (dys)function and reproductive aging remains poorly understood. This study aimed to establish the interplay between hCC mitochondrial dysfunction and women's reproductive potential. In this investigation, 266 women were enrolled and categorized into two groups based on their age: a young group (<35 years old) and an advanced maternal age (AMA) group (≥35 years old). Comprehensive analysis of reproductive outcomes was conducted in our population. Various mitochondrial-related parameters were analyzed across distinct subsets. Specifically, mitochondrial membrane potential (∆Ψm) and mitochondrial mass were examined in 53 samples, mtDNA content in 25 samples, protein levels in 23 samples, bioenergetic profiles using an XF24 Extracellular Flux Analyzer in 6 samples, and levels of reactive oxygen species (ROS) and adenosine triphosphate (ATP) in 39 and 43 samples, respectively. In our study, the reproductive potential of AMA women sharply decreased, as expected. Additionally, an impairment in the mitochondrial function of hCCs in older women was observed; however, no differences were found in terms of mitochondrial content. Regarding oxidative phosphorylation, metabolic profiling of hCCs from AMA women indicated a decrease in respiratory capacity, which was correlated with an age-dependent decrease in the ATP synthase (ATP5A1) protein level. However, intracellular ROS and ATP levels did not differ between groups. In conclusion, our study indicates that age-related dysfunction in hCCs is associated with impaired mitochondrial function, and, although further studies are required, ATP synthase could be relevant in this impairment.

Follicular Fluid-Derived Extracellular Vesicles Influence on In Vitro Maturation of Equine Oocyte: Impact on Cumulus Cell Viability, Expansion and Transcriptome.

Cumulus cell (CC) expansion is pivotal for oocyte maturation, during which CCs release factors that initiate paracrine signaling within the follicular fluid (FF). The FF is abundant in extracellular vesicles (EVs) that facilitate intercellular communication. Although bovine and murine EVs can control cumulus expansion, these effects have not been observed in equines. This study aimed to assess the impact of FF-derived EVs (ffEVs) on equine CC expansion, viability, and transcriptome. Cumulus-oocyte complexes (COCs) that underwent in vitro maturation (IVM) in the presence (200 µg protein/mL) or absence (control) of ffEVs were assessed for cumulus expansion and viability. CCs were isolated after 12 h of IVM, followed by RNA extraction, cDNA library generation, and subsequent transcriptome analysis using next-generation sequencing. Confocal microscopy images illustrated the internalization of labeled ffEVs by CCs. Supplementation with ffEVs significantly enhanced cumulus expansion in both compacted (Cp, p < 0.0001) and expanded (Ex, p < 0.05) COCs, while viability increased in Cp groups (p < 0.01), but decreased in Ex groups (p < 0.05), compared to the controls. Although transcriptome analysis revealed a subtle effect on CC RNA profiles, differentially expressed genes encompassed processes (e.g., MAPK and Wnt signaling) potentially crucial for cumulus properties and, consequently, oocyte maturation.

Unravelling the role of HAS2, GREM1, and PTGS2 gene expression in cumulus cells: implications for human oocyte development competency - a systematic review and integrated bioinformatic analysis.

The leading indicator for successful outcomes in in-vitro fertilization (IVF) is the quality of gametes in oocytes and sperm. Thus, advanced research aims to highlight the parameter in assessing these qualities - DNA fragmentation in sperm and oocyte development capacity (ODC) via evaluation of microenvironments involving its maturation process. Regarding oocytes, most evidence reveals the role of cumulus cells as non-invasive methods in assessing their development competency, mainly via gene expression evaluation. Our review aims to consolidate the evidence of GDF-9 derivatives, the HAS2, GREM1, and PTGS2 gene expression in cumulus cells used as ODC markers in relevant publications and tailored to current IVF outcomes. In addition to that, we also added the bioinformatic analysis in our review to strengthen the evidence aiming for a better understanding of the pathways and cluster of the genes of interest - HAS2, GREM1, and PTGS2 in cumulus cell level. Otherwise, the current non-invasive method can be used in exploring various causes of infertility that may affect these gene expressions at the cumulus cell level. Nevertheless, this method can also be used in assessing the ODC in various cohorts of women or as an improvement of markers following targeted tools or procedures by evaluating the advancement of these gene expressions following the targeted intervention.

Role and action mechanisms of miR-149 and miR-31 in regulating function of pig cumulus cells and oocytes.

Understanding the mechanisms for oocyte maturation and optimizing the protocols for in vitro maturation (IVM) are greatly important for improving developmental potential of IVM oocytes. The miRNAs expressed in cumulus cells (CCs) play important roles in oocyte maturation and may be used as markers for selection of competent oocytes/embryos. Although a recent study from our group identified several new CCs-expressed miRNAs that regulate cumulus expansion (CE) and CC apoptosis (CCA) in mouse oocytes, validation of these findings and further investigation of mechanisms of action in other model species was essential before wider applications. By using both in vitro and in vivo pig oocyte models with significant differences in CE, CCA and developmental potential, the present study validated that miR-149 and miR-31 improved CE and developmental potential while suppressing CCA of pig oocytes. We demonstrated that miR-149 and miR-31 targeted SMAD family member 6 (SMAD6) and transforming growth factor ß2 (TGFB2), respectively, in the transforming growth factor-ß (TGF-ß) signaling. Furthermore, both miR-149 and miR-31 increased CE and decreased CCA via activating SMAD family member 2 (SMAD2) and increasing the expression of SMAD2 and SMAD family member 4. In conclusion, the present results show that miR-149 and miR-31 improved CE and developmental potential while suppressing CCA of pig oocytes by activating the TGF-ß signaling, suggesting that they might be used as markers for pig oocyte quality.

Biochemical profiling of the follicular environment to predict oocyte competence in cattle.

To identify markers of oocyte competence, we compared the biochemical characteristics of fluid and cells from follicles containing oocytes with different capacities to form an embryo. Follicles (5-6 mm) were dissected, and follicular fluid (FF), granulosa cells (GC), cumulus cells (CC) from immature and mature cumulus-oocyte-complexes (COC) were individually collected. The oocytes were matured, fertilized, and cultured individually until day 8 (D8) of development. On D8, the samples were grouped according to embryo production into those that gave rise to blastocysts (EMB) and those that did not reach the blastocyst stage (NEMB). In CCs from immature and mature COCs and GCs, expression of CASP3, SERPINE2, VCAN, LUM, FSHR, EGFR, PGR, and GHR genes was quantified. Cell-free DNA (cfDNA), progesterone, and estradiol concentrations in the FF were determined. Data were analyzed by Mann-Whitney U test (GraphPad Prism 9). GHR was highly expressed in immature CCs from the EMB group, whereas CASP3 was highly expressed in mature CCs from the NEMB group (P<0.05). During maturation, the expression of CASP3 and GHR genes increased only in the NEMB group. ART2 cfDNA was highly detected in FF of the NEMB compared to the EMB group. Progesterone concentration was similar between the groups, whereas estradiol concentration was higher (P<0.05) in the EMB than in the NEMB group. It was concluded that a higher level of GHR transcripts in immature CCs, lower CASP3 expression in CCs from matured COCs, lower levels of ART2, and higher estradiol concentrations in FF may indicate oocytes with greater potential for development.

Growth differentiation factor 9 regulates the expression of estrogen receptors via Smad2/3 signaling in goat cumulus cells.

Both oocyte secretory factors (OSFs) and estrogen are essential for the development and function of mammalian ovarian follicles, playing synergistic role in regulating oocyte growth. OSFs can significantly affect the biological processes regulated by estrogen in cumulus cells (CCs). It is a scientific question worth investigating whether oocyte secretory factors can influence the expression of estrogen receptors in CCs. In our study, we observed a significant increase in the mRNA and protein expressions of estrogen receptor ß (Esr2/ERß) and G-protein-coupled estrogen receptor (GPER) in cumulus cells of goat cumulus-oocyte complexes (COCs) cultured in vitro for 6 h. Furthermore, the addition of 10 ng/mL growth-differentiation factor 9 (GDF9) and 5 ng/mL bone morphogenetic protein 15 (BMP15) to the culture medium of goat COCs resulted in a significant increase in the expressions of ERß and GPER in cumulus cells. To explore the mechanism further, we performed micromanipulation to remove oocyte contents and co-cultured the oocytectomized complexes (OOXs) with denuded oocytes (DOs) or GDF9/BMP15. The expressions of ERß and GPER in the co-culture groups were significantly higher than those in the OOXs group, but there was no difference compared to the COCs group. Mechanistically, we found that SB431542 (inhibitor of GDF9 bioactivity), but not LDN193189 (inhibitor of BMP15 bioactivity), abolished the upregulation of ERß and GPER in cumulus cells and the activation of Smad2/3 signaling. In conclusion, our results demonstrate that the oocyte secretory factor GDF9 promotes the activation of Smad2/3 signaling in cumulus cells during goat COCs culture in vitro, and the phosphorylation of Smad2/3 induces the expression of estrogen receptors ERß and GPER in cumulus cells.

SIRT2 regulates apoptosis by inducing mitophagy in sheep cumulus cells.

Cumulus cells surrounding oocytes furnish nutritional support crucial for oocyte maturation in vitro, and thereby enhance oocyte quality significantly. Our previous studies affirmed the role of SIRT2 in regulation of mitochondrial function in sheep granulosa cells. However, the effect of SIRT2 action on mitophagy in these cells remain unclear. Here, RNA-seq was used to scrutinize pathways where differentially expressed genes (DEGs) are enriched following SIRT2 knockdown in cumulus cells. Prior to SIRT2 knock down, cumulus cells were treated with the mitophagy inhibitor Mdivi-1. Potential mechanisms by which SIRT2 affects apoptosis via mitophagy were explored. Results indicated that DEGs after SIRT2 knockdown were enriched in various pathways including mitochondria, mitophagy, and apoptosis. The expression levels of CASP3/CASP9 were significantly increased after mitophagy activation (P < 0.01), whereas inhibition of mitophagy had no effect on apoptosis (P > 0.05). Pretreatment of cumulus cells with Mdivi-1 prior to SIRT2 knockdown significantly reduced the expression of mitophagy-related genes, the number of autolysosomes, the expression of CASP3/CASP9, and the levels of Ca2+ and cytochrome C (P < 0.05). In addition, an improvement in mitochondrial morphology and increases in ATP levels and mitochondrial DNA (mtDNA) copy numbers were observed. Interestingly, double knockdown of SIRT2 and MAPK15 was found to reverse increased mitophagy and apoptosis activity caused by SIRT2 knockdown. Our findings indicate that SIRT2 modulate apoptosis in cumulus cells by regulating mitophagy, with MAPK15 likely playing a pivotal role in this process.

Energy metabolism disorders during in vitro maturation of bovine cumulus-oocyte complexes interfere with blastocyst quality and metabolism.

Glucose and fatty acids (FA) metabolism disturbances during oocyte in vitro maturation (IVM) affect their metabolism and surrounding cumulus cells, but only inhibition of glucose metabolism decreases embryo culture efficiency. Therefore, the present experiment aimed to reveal if glucose or FA metabolism inhibition leads to the disruption of embryo developmental potential, and to characterize the metabolic landscape of embryos reaching the blastocyst stage. Inhibitors of glucose (IO + DHEA) or FA (ETOMOXIR) metabolism were applied during IVM, and the control group was matured under standard conditions. Blastocysts obtained from experimental and control groups were analyzed with regard to lipidome and metabolome (mass spectrometry), transcriptome (RNA-Seq) and fluorescence lipid droplets staining (BODIPY). We showed that inhibition of glucose and fatty acid metabolism leads to cellular stress response compromising the quality of preimplantation embryos. The inhibition of energy metabolism affects membrane fluidity as well as downregulates fatty acids biosynthesis and gene expression of trophectoderm cell line markers. Therefore, we conclude that oocyte maturation environment exerts a substantial effect on preimplantation development programming at cellular and molecular levels.

Retinoic Acid Action in Cumulus Cells: Implications for Oocyte Development and In Vitro Fertilization.

In the field of human in vitro fertilization (IVF), selecting the best oocyte for freezing or embryo for transfer remains an important focus of clinical practice. Although several techniques are and have been used for this goal, results have generally not been favorable and/or are invasive such that damage to some embryos occurs, resulting in a reduced number of healthy births. Therefore, the search continues for non-invasive oocyte and embryo quality markers that signal the development of high-quality embryos. Multiple studies indicate the important positive effects of retinoic acid (RA) on oocyte maturation and function. We previously showed that a high follicular fluid (FF) RA concentration at the time of oocyte retrieval in IVF protocols was associated with oocytes, giving rise to the highest quality embryos, and that cumulus granulosa cells (CGCs) are the primary source of follicle RA synthesis. Data also demonstrated that connexin-43 (Cx43), the main connexin that forms gap junctions in CGCs, is regulated by RA and that RA induces a rapid increase in gap junction communication. Here, we hypothesize that CGC RA plays a causal role in oocyte competency through its action on Cx43 and, as such, may serve as a biomarker of oocyte competence. Multiple studies have demonstrated the requirement for Cx43 in CGCs for the normal progression of folliculogenesis, and that the increased expression of this connexin is linked to the improved developmental competence of the oocyte. The data have shown that RA can up-regulate gap junction intercellular communication (GJIC) in the cumulus-oocyte complex via a non-genomic mechanism that results in the dephosphorylation of Cx43 and enhanced GJIC. Recognizing the positive role played by gap junctions in CGCs in oocyte development and the regulation of Cx43 by RA, the findings have highlighted the possibility that CGC RA levels may serve as a non-invasive indicator for selecting high-quality oocytes for IVF procedures. In addition, the data suggest that the manipulation of Cx43 with retinoid compounds could provide new pharmacological approaches to improve IVF outcomes in cases of failed implantation, recurrent miscarriage, or in certain diseases that are characterized by reduced fecundity, such as endometriosis.

Transcriptomic responses of cumulus granulosa cells to SARS-CoV-2 infection during controlled ovarian stimulation.

Cumulus granulosa cells (CGCs) play a crucial role in follicular development, but so far, no research has explored the impact of SARS-CoV-2 infection on ovarian function from the perspective of CGCs. In the present study, we compared the cycle outcomes between infected and uninfected female patients undergoing controlled ovarian stimulation, performed bulk RNA-sequencing of collected CGCs, and used bioinformatic methods to explore transcriptomic changes. The results showed that women with SARS-CoV-2 infection during stimulation had significantly lower number of oocytes retrieved and follicle-oocyte index, while subsequent fertilization and embryo development were similar. CGCs were not directly infected by SARS-CoV-2, but exhibited dramatic differences in gene expression (156 up-regulated and 65 down-regulated). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses demonstrated a high enrichment in antiviral, immune and inflammatory responses with necroptosis. In addition, the pathways related to telomere organization and double strand break repair were significantly affected by infection in gene set enrichment analysis. Further weighted gene co-expression network analysis identified a key module associated with ovarian response traits, which was mainly enriched as a decrease of leukocyte chemotaxis and migration in CGCs. For the first time, our study describes how SARS-CoV-2 infection indirectly affects CGCs at the transcriptional level, which may impair oocyte-CGC crosstalk and consequently lead to poor ovarian response during fertility treatment.

A Circular RNA Derived from the Pumilio 1 Gene Could Regulate PTEN in Human Cumulus Cells.

CircRNAs are a class of non-coding RNAs able to regulate gene expression at multiple levels. Their involvement in physiological processes, as well as their altered regulation in different human diseases, both tumoral and non-tumoral, is well documented. However, little is known about their involvement in female reproduction. This study aims to identify circRNAs potentially involved in reproductive women's health. Candidate circRNAs expressed in ovary and sponging miRNAs, already known to be expressed in the ovary, were selected by a computational approach. Using real time PCR, we verified their expression and identified circPUM1 as the most interesting candidate circRNA for further analyses. We assessed the expression of circPUM1 and its linear counterpart in all the follicle compartments and, using a computational and experimental approach, identified circPUM1 direct and indirect targets, miRNAs and mRNAs, respectively, in cumulus cells. We found that both circPUM1 and its mRNA host gene are co-expressed in all the follicle compartments and proposed circPUM1 as a potential regulator of PTEN, finding a strong positive correlation between circPUM1 and PTEN mRNA. These results suggest a possible regulation of PTEN by circPUM1 in cumulus cells and point out the important role of circRNA inside the pathways related to follicle growth and oocyte maturation.

Addition of rapamycin or co-culture with cumulus cells from younger reproductive age women does not improve rescue in vitro oocyte maturation or euploidy rates in older reproductive age women.

Both spontaneously conceived pregnancies and those achieved using assisted reproduction decline with advancing maternal age. In this study, we tested if rapamycin and/or cumulus cells (CCs) from young donors could improve oocyte maturation and euploidy rates of germinal vesicle (GV) stage oocytes obtained from older women of reproductive age. A total of 498 GVs from 201 women >38 years (40.6 ± 1.8, mean ± SD) were included. GVs were randomly assigned into five groups for rescue IVM: control (with no CCs and no rapamycin); with autologous CCs; with autologous CCs and rapamycin; with CCs from young women (<35 years); and with CCs from young women and rapamycin. After 24 h of culture, the first polar body (PB) was biopsied in metaphase II oocytes, and the cytogenetic constitution was assessed using next-generation sequencing for both oocytes and PBs. Comparable maturation rates were found (56.2%, 60.0%, 46.5%, 51.7%, and 48.5% for groups 1-5, respectively; P = 0.30). Similarly, comparable euploidy rates were observed in the five groups (41.5%, 37.8%, 47.2%, 43.6%, and 47.8% for Groups 1-5, respectively; P = 0.87). Our findings indicate that rescue IVM is effective for obtaining mature euploid oocytes in older women of reproductive age, and that incubation with rapamycin or CCs obtained from young donors does not improve the maturation or euploidy rate.

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

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

Long pentraxin 3 and vitamin D receptor mRNA expression pattern of cumulus granulosa cells isolated from PCOS oocytes at different stages of nuclear maturation.

BACKGROUND: A fine-tuned pro-inflammatory and anti-inflammatory balance in the follicular unit is essential for cumulus expansion and successful ovulation. While the long pentraxin 3 (PTX3) gene is required for the expansion of cumulus cells (CCs), ovulation, resumption of meiosis and fertilization, the vitamin D receptor gene (VDR-X2) is required for intra-follicle redox balance. This study was planned to determine the expression pattern of VDR-X2 and PTX3 mRNA in CCs isolated from germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) oocytes of PCOS patients with ovulatory dysfunction. METHODS: The relative expression of CC-PTX3 and CC-VDR-X2 mRNA were evaluated using qRT-PCR in a total of 79 CC samples collected from individual cumulus-oocyte complex of 40 infertile patients (20 PCOS and 20 non-PCOS normal responders) who underwent ovarian stimulation with the GnRH antagonist protocol. RESULTS: Relative PTX3 mRNA expressions of CCMI-control and CCMII-control showed 3- and 9-fold significant upregulation compared to CCGV-control, respectively. The relative PTX3 mRNA expression of CCMII-control increased approximately three fold compared to CCMI-control. Compared to CCGV-pcos, a 3-fold increase was noted in the relative PTX3 mRNA expression of CCMI-pcos and an approximately 4-fold increase in the PTX3 mRNA expression of CCMII-pcos. Relative PTX3 mRNA expression values of CCMII-pcos and CCMI-pcos were similar. A 6-fold upregulation of relative PTX3 mRNA and a 4-fold upregulation of VDR-X2 mRNA were detected in CCMII-control compared to CCMII-pcos. CC-VDR-X2 expression patterns of the PCOS and control groups overlapped with the CC-PTX3 pattern. Fertilization rates of the PCOS group exhibiting failed transcript expression were similar to normal responders. CONCLUSION: The fact that relative CC-PTX3 and CC-VDR mRNA expression does not increase during the transition from MI to MII stage in PCOS as in normal responders suggests that PTX3 and VDR expression may be defective in cumulus cells of PCOS patients with ovulatory dysfunction.