Cumulus cell acetyl-CoA enrichment from acetate found to decrease with maternal age using a novel approach to measure metabolism in individual cumulus cell complexes

Is the activity of sirtuin 3 (SIRT3) altered in granulosa and cumulus cells from young women with reduced ovarian reserve or women of advanced maternal age? SIRT3 mRNA and active protein in granulosa and cumulus cells were decreased in women with reduced ovarian reserve and advanced maternal age. Young women with reduced ovarian reserve or women of advanced maternal age have reduced oocyte viability, possibly due to altered granulosa and cumulus cell metabolism. The mitochondrial SIRT3 protein may be implicated in these processes as it is able to sense the metabolic state of the cell and alter mitochondrial protein function post-translationally. This is a prospective cohort study, in which women (n = 72) undergoing routine IVF/ICSI were recruited and allocated to one of three cohorts based on age and ovarian reserve (as assessed by serum anti-Mullerian hormone level). Women were classified as young (≤35 years) or of advanced maternal age (≥40 years). Granulosa and cumulus cells were collected. SIRT3 mRNA and protein levels and protein activity was analysed in granulosa and cumulus cells via quantitative PCR, immunohistochemistry and western blotting, and deacetylation activity, respectively. Activity of the glutamate dehydrogenase (GDH) enzyme, a known target of SIRT3, was assessed, and acetylated proteins in mitochondria isolated from granulosa and cumulus cells were separated by immunoprecipitation and acetylation of GDH assessed by western blotting. Data for women with good prognosis (young women with normal ovarian reserve) were compared with those from young women with reduced ovarian reserve and those of advanced maternal age. SIRT3 mRNA and active protein were present in granulosa and cumulus cells and co-localized to the mitochondria. SIRT3 mRNA in granulosa cells was decreased in young women with reduced ovarian reserve and advanced maternal age versus young women with normal ovarian reserve (P < 0.05). SIRT3 mRNA in cumulus cells was decreased in women of advanced maternal age versus young women with normal ovarian reserve only (P < 0.05). Granulosa cell GDH activity was decreased in young women with reduced ovarian reserve and in women of advanced maternal age (P < 0.05), whereas cumulus cell GDH activity was reduced in the advanced maternal age group only (P < 0.05). The acetylation profile of GDH in mitochondria revealed increased acetylation of GDH in granulosa and cumulus cells from women of advanced maternal age (P < 0.05) while young women with reduced ovarian reserve had increased GDH acetylation in granulosa cells only (P < 0.05). Although patients were allocated to groups based on maternal age and ovarian reserve and matched for BMI, other maternal factors may also alter the 'molecular health' of ovarian cells. Our data suggest that SIRT3 post-translational modification of mitochondrial enzymes in human granulosa and cumulus cells may regulate GDH activity, thus altering the metabolic milieu surrounding the developing oocyte. Owing to the association between the decline in oocyte quality and pregnancy rates in women of advanced maternal age and the possible association with reduced ovarian reserve, knowledge of perturbed SIRT3 function in granulosa and cumulus cells may lead to novel therapies to improve mitochondrial metabolism in the oocyte and follicular cells in women undergoing IVF treatment. No conflicts of interest to declare. Research was funded by an NHMRC project grant.

Cumulus cell (CC) clumps that associate with oocytes provide the oocytes with growth and signaling factors. Thus, the metabolism of the CCs may influence oocyte function, and CC metabolism may be predictive of oocyte competence for in vitro fertilization. CCs are thought to be highly glycolytic, but data on the use of other potential carbon substrates are lacking in humans. This prospective and blinded cohort study was designed to examine the substrate utilization of CCs by age and oocyte competence. Individual sets of CC clumps from participants were removed after oocyte retrieval procedure then, incubated with stable isotope labeled substrates, and analyzed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) for isotopologue enrichment of major metabolic intermediates, including acetyl-CoA. The acyl-chain of acetyl-CoA contains 2 carbons that can be derived from 13C-labeled substrates resulting in an M + 2 isotopologue that contains 2 13C atoms. Comparing the fate of three major carbon sources, mean enrichment of M + 2 acetyl-CoA (mean, standard deviation) was for glucose (3.6, 7.7), for glutamine (9.4, 6.2), and for acetate (20.7, 13.9). Due to this unexpected high and variable labeling from acetate, we then examined acetyl-CoA mean % enrichment from acetate in 278 CCs from 21 women ≤34 (49.06, 12.73) decreased with age compared to 124 CCs from 10 women >34 (43.48, 16.20) (p = 0.0004, t-test). The CCs associated with the immature prophase I oocytes had significantly lower enrichment in M + 2 acetyl CoA compared to the CCs associated with the metaphase I and metaphase II oocytes (difference: −6.02, CI: −1.74,-13.79, p = 0.013). Acetate metabolism in individual CC clumps was positively correlated with oocyte maturity and decreased with maternal age. These findings indicate that CC metabolism of non-glucose substrates should be investigated relative to oocyte function and age-related fertility. Abbreviations: CCs: cumulus cells; COC: cumulus-oocyte complex; LC-MS: liquid chromatography-mass spectrometry; acetyl-CoA: acetyl-Coenzyme A; CoA: Coenzyme A;

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific paracrine factors which regulate ovarian cumulus cell (CC) functions. This study aimed to investigate if BMP15 and GDF9 bound to CCs can be characterized, quantified, and show an association with IVF outcomes in infertile women. BMP15 and GDF9 ELISAs were validated and applied to discarded CC extracts. Pooled CCs from individual patients were collected from 120 (cohort 1; BMP15 only) and 81 infertility patients (cohort 2; BMP15 and GDF9) undergoing superovulation. BMP15 and GDF9 levels expressed per CC DNA were correlated with maternal age, clinical and embryology data. Total BMP15 and GDF9 were highly correlated with each other (r = 0.9, p < 0.001). The GDF9:BMP15 ratio was unrelated to oocyte number or age. BMP15/CC DNA and GDF9/CC DNA were unaffected by the type of superovulation and were not related to oocyte/embryo outcomes.

Complex cumulus cell-oocyte interactions govern energy utilization during oocyte development. This study investigates the relationship of cumulus cell mitochondria with oocyte development during ovarian stimulation for in vitro fertilization (IVF). This is a prospective cohort study. The setting was an academic center. Thirty women underwent ovarian stimulation for IVF. Pooled cumulus cells were collected; numbers of total and mature oocytes and two-pronuclear (day 1), six- to eight-cell cleavage (day 3), and blastocyst (day 5) embryos were recorded. A mitochondrial bioassay was developed with Jurkat cells and used with cumulus cells from IVF patients to correlate mitochondrial membrane potential resistance to carbonyl cyanide 3-chlorophenylhydrazone (CCCP) stress with oocyte development and embryogenesis. Adjusting for FSH administered and maternal age, cumulus cell mitochondrial membrane potential resistance to CCCP positively correlated with numbers of total (P < .025) and mature (P < .025) oocytes retrieved. The highest oocyte numbers that correlated with cumulus cell mitochondrial membrane potential occurred in women with the greatest ovarian response to FSH (mitochondrial membrane potential resistance to CCCP-log FSH interactions: total oocytes P < .025; mature oocytes P < .05). Multiple regression modeling of mature oocyte numbers, age, and cumulus cell mitochondrial membrane potential resistance to CCCP showed that numbers of mature oocytes best correlated with numbers of embryos at all stages (P < .0001). During ovarian stimulation for IVF, cumulus cell mitochondrial membrane potential resistance to stress correlates with numbers of total and mature oocytes retrieved, suggesting that cumulus cell-oocyte interactions involving energy facilitate oocyte development.

Gamete Interactions In Vitro

Progress in assisted reproductive technologies strongly relies on understanding the regulation of the dialogue between oocyte and cumulus cells (CCs). Little is known about the role of long non-coding RNAs (lncRNAs) in the human cumulus-oocyte complex (COC). To this aim, publicly available RNA-sequencing data were analyzed to identify lncRNAs that were abundant in metaphase II (MII) oocytes (BCAR4, C3orf56, TUNAR, OOEP-AS1, CASC18, and LINC01118) and CCs (NEAT1, MALAT1, ANXA2P2, MEG3, IL6STP1, and VIM-AS1). These data were validated by RT-qPCR analysis using independent oocytes and CC samples. The functions of the identified lncRNAs were then predicted by constructing lncRNA-mRNA co-expression networks. This analysis suggested that MII oocyte lncRNAs could be involved in chromatin remodeling, cell pluripotency and in driving early embryonic development. CC lncRNAs were co-expressed with genes involved in apoptosis and extracellular matrix-related functions. A bioinformatic analysis of RNA-sequencing data to identify CC lncRNAs that are affected by maternal age showed that lncRNAs with age-related altered expression in CCs are essential for oocyte growth. This comprehensive analysis of lncRNAs expressed in human MII oocytes and CCs could provide biomarkers of oocyte quality for the development of non-invasive tests to identify embryos with high developmental potential.

Fertility loss during female ageing is associated with increasing basal FSH and decreasing anti-Müllerian hormone (AMH) concentrations, together with compromised oocyte quality, presumably due to increased oxidative stress (OS) and DNA damage, as well as reduced metabolic and meiotic competences. Basal FSH and AMH circulatory concentrations have been broadly utilized as IVF success predictors, regardless of fluctuations in prognostic accuracy; basal FSH and AMH perform better in pre-advanced maternal age (AMA: >35 years) and AMA patients, respectively. The relationships between FSH and AMH intrafollicular levels and IVF outcomes suggest, nevertheless, that both hormones regulate oocyte competence, supporting the hypothesis that changes in FSH/AMH levels cause, at least in part, oocyte quality degradation during ageing. To understand the reasons behind the fluctuations in FSH and AMH prognostic accuracies and to clarify their participation in mechanisms determining oocyte competence and age-related subfertility, a deeper knowledge of the regulation of FSH and AMH intrafollicular signalling during the female reproductive lifespan, and of their effects on the cumulus-oocyte complex, is required. An extensive body of information on the regulation of FSH and AMH intrafollicular availability and signalling, as well as on the control of folliculogenesis and oocyte metabolism, has been accumulated. However, these datasets have been explored within the relatively narrow boundaries of their specific subjects. Given the aforementioned gaps in knowledge and their clinical relevance, herein we integrate clinical and basic data, within a wide biological perspective, aiming to shed light on (i) the reasons for the variability in the accuracy of serum FSH and AMH as fertility markers, and on (ii) the potential roles of these hormones in mechanisms regulating oocyte quality, particularly those associated with ageing. The PubMed database encompassing the period between 1960 and 2021 was searched. Principal search terms were FSH, FSH receptor, AMH, oocyte, maternal age, cumulus, transzonal projections (TZPs), actin, OS, redox, reactive oxygen species, mitochondria, DNA damage, DNA repair, aneuploidy, spindle, meiosis, gene expression, transcription, translation, oocyte secreted factors (OSFs), cAMP, cyclic guanosine monophosphate, natriuretic peptide C, growth differentiation factor 9, bone morphogenetic protein 15 and fibroblast growth factor. Our analysis suggests that variations in the accuracy of fertility prognosis reflect a modest association between circulatory AMH levels and oocyte quality as well as increasing basal FSH inter-cycle variability with age. In addition, the basic and clinical data articulated herein support the hypothesis that increased intrafollicular FSH levels, as maternal age advances, may override the physiological protective influences of AMH and OSFs against excessive FSH signalling in cumulus cells. This would result in the disruption of oocyte homeostasis via reduced TZP-mediated transfer of cumulus-derived molecules essential for meiotic competence, gene expression, redox activity and DNA repair. In-depth data analysis, encompassing a wide biological perspective has revealed potential causative mechanisms of age-related subfertility triggered by alterations in FSH/AMH signalling during the female reproductive life. Insights from new mechanistic models arising from this analysis should contribute to advancing our comprehension of oocyte biology in humans and serve as a valuable reference for novel AMA subfertility treatments aimed at improving oocyte quality through the modulation of AMH/FSH action.

A systematic review was conducted pertaining to the biologic processes of cumulus cells and measures of oocyte quality. The initial search yielded 18,549 articles, and after screening, 65 studies were included in this review. Extracted data were synthesized descriptively. Articles were categorized based on the biologic feature of cumulus cells addressed: genetic expression, biochemical activity, and morphologic characteristics. Manuscripts addressing cumulus cell function in association with gynecologic conditions and their impact on oocyte quality were also reviewed. Findings from six studies showed an association between increased expression of pathways related to extracellular matrix development (HAS2, VCAN) and improved oocyte quality. Six manuscripts reported improved oocyte quality related to genes involved in cumulus cell expansion (GREM1, PTGS2) and 6 articles noted patterns in expression of regulators of apoptotic pathways (BCL2, BIRC5, MDM2, PGAM5). Twelve studies examined biochemical features of cumulus cells, including cumulus mitochondrial membrane resistance, respirometric index, and acetyl-CoA levels, which have been associated with oocyte development. Morphologic assessment of cumulus cells was evaluated by 5 studies, demonstrating a relationship between mitochondrial dimensions, telomere length, and cumulus density/dispersion with oocyte quality. Twenty-two articles assessed cumulus cell function and its relationship with oocyte quality in diseases such as polycystic ovarian syndrome (PCOS) and endometriosis conditions, and increased maternal age (with or without decreased ovarian reserve). This comprehensive systematic review has integrated findings from studies investigating the relationship between cumulus cells and oocyte quality, identifying patterns in gene expression, biochemical features and morphologic assessments that may offer meaningful insight into this relationship.

Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte–cumulus cell complex in mice: genetic evidence for an oocyte–granulosa cell regulatory loop

The use of cumulus cell (CC) mRNA levels to predict blastocyst development and live birth outcomes in women undergoing intracytoplasmic sperm injection (ICSI) and single embryo transfer

This paper suggests an approach for the use of a single paucicellular histological sample to investigate two characteristics indicative of the cell’s functional potential: 1) the content of telomerase reverse transcriptase (TERT) and 2) the telomere length. An algorithm has been suggested for the successive detection of the catalytic telomerase subunit with immunohistochemical staining and assessment of telomere length with Q-FISH on the same set of cells. The described approach uses three incontestable advantages of the FISH technique, namely, the possibility of analyzing target sequences in individual cells and paucicellular samples, the performance of several successive hybridization rounds with different FISH probes on the same sample, and the examination of genomic regions consisting of repeats. This approach has been applied to assess the TERT content and telomere length in cumulus cells from human cumulus-oocyte complexes obtained from assisted reproduction programs. This approach provides an opportunity to examine the correlation between the TERT content and telomere length in cumulus cells surrounding the oocyte and the oocyte's capacity for fertilization, as well as the subsequent pre- and post-implantation development of the resulting embryo.

The effects of LH on tricarboxylic acid (TCA) cycle activity within denuded and cumulus cell-enclosed bovine oocytes were investigated by measuring the metabolism of L-[14C(U)]glutamine and [2-14C]pyruvate to 14CO2 after in vitro maturation (IVM) with LH in serum-free, defined culture conditions. Initially, intact cumulus cell-enclosed oocytes or oocytectomized cumulus cell complexes were incubated for 24 h for IVM in control medium alone or supplemented with LH (10 micrograms/ml) before assessment of glutamine metabolism. Glutamine oxidation was increased (p < 0.01) only after IVM of intact cumulus cell-enclosed oocytes with LH when compared to either intact untreated controls, untreated oocytectomized controls, or oocytectomized cumulus cell complexes after LH treatment (0.23 +/- 0.02 vs. 0.13 +/- 0.01, 0.12 +/- 0.01, and 0.12 +/- 0.01 nmol 14CO2/micrograms protein/3 h, respectively). Glutamine metabolism was greater (p < 0.001) in denuded oocytes from cumulus cell complexes that were exposed to LH during IVM versus nontreated controls (6.4 +/- 0.3 vs. 5.1 +/- 0.2 pmol 14CO2/oocyte/3 h, respectively). Glutamine metabolism was increased (p < 0.05) in cumulus cell-enclosed oocytes after LH exposure during IVM but was unchanged after exposure to either FSH or thyroid-stimulating hormone when compared to control treatments (0.23 +/- 0.01, 0.20 +/- 0.02, 0.19 +/- 0.01 vs. 0.16 +/- 0.02 nmol 14CO2/micrograms protein/3 h, respectively). Pyruvate metabolism was not different between LH and control treatments. Evidence demonstrated that LH acts via the cumulus cells to increase glutamine metabolism within intact cumulus cell-enclosed oocytes and in mature oocytes denuded after LH exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Can sperm selection through cumulus cells improve embryo quality compared to conventional methods, and is its effectiveness influenced by parental age? This prospective clinical trial included 99 ICSI cycles from 95 couples. Sibling oocytes were randomly allocated at the oocyte level to either the study group (cumulus cell-mediated sperm selection after conventional density gradients centrifugation (DGC), 554 oocytes) or the control group (only sperm selection by DGC, 543 oocytes), using a dish designed to facilitate sperm interaction with cumulus cells. The inclusion criteria for this study were patients using their own oocytes, with a medical indication for ICSI, who had at least 6 mature oocytes (MII) in that cycle. For semen samples, inclusion required the ability to adjust the concentration to 10 million/mL. Exclusion criteria included the use of vitrified oocytes, donated oocytes, and semen samples obtained by testicular biopsy or aspiration. Embryo quality was assessed at the blastocyst stage on day 5 according to ASEBIR. A subanalysis evaluated the influence of parental age on outcomes. The study group showed a significantly higher proportion of good-quality day-5 blastocysts compared to controls (55.2% vs. 45.3%, p = 0.028). No statistically significant differences were observed in overall blastocyst formation or pregnancy rates, although favourable trends were noted. In an age-stratified analysis, a significant improvement in day-5 blastocyst quality among evaluable blastocysts was observed in women aged 40-45 (51.4% vs. 30.4%, p = 0.017), with a non-significant trend toward improved outcomes in men aged 40-53 (44.7% vs. 32.6%, p = 0.083). No differences were seen in younger age groups. Cumulus cell-mediated sperm selection after DGC using a specialized Oosafe® ICSI Dish with Sperm Selection Channels was associated with an increased proportion of good-quality day 5 blastocysts compared with conventional sperm preparation. While clinical outcomes did not differ significantly, these findings suggest a potential benefit in specific ART subpopulations, particularly those of advanced maternal age. Further adequately powered studies are required to confirm these observations and to determine their impact on clinical outcomes.

The success of human IVF is essentially determined by the quality of the embryo (itself a function of the quality of the oocyte and sperm), the endometrium and of the interaction between them. While there are established means of establishing the quality of each individually, some of these are limited and, in particular, the embryo-endometrial dialogue is difficult to capture and observe. As a result, it is under-studied and, with this in mind, the aims of this thesis were threefold: First, to investigate the corona cell transcriptome of euploid oocytes, RNA sequencing of corona cells from individual cumulus oocyte complexes that developed into euploid blastocysts was employed. Alongside bioinformatic and statistical analysis to compare IVF outcomes, a mean number of sequence reads of 21.2 million were produced. Differentially expressed gene analysis revealed 343 statistically significant transcripts and enriched pathway analysis showed WNT signaling, MAPK signaling, focal adhesion and TCA cycle to be related to IVF outcome. Specifically, key genes within the WNT/beta-catenin signaling pathway, including AXIN, were associated with oocyte competence. Furthermore, key genes and signaling pathways were identified in corona cell profiles in association with IVF outcome following the transfer of euploid blastocysts previously vitrified in a frozen embryo transfer. A second study investigated an association between advanced maternal age (AMA) and endometriosis on the embryo-endometrial molecular dialogue before implantation. Co-culture experiments were performed with endometrial epithelial cells (EEC) and cryopreserved day 5 blastocysts from AMA or endometriosis patients. Extracellular vesicles isolated from the co-culture supernatant were analyzed for miRNA expression and revealed significant alterations correlating to AMA or endometriosis. Functional annotation analysis of miRNA-target genes revealed enriched pathways for both infertility etiologies, including disrupted cell cycle regulation and proliferation. These extracellular vesicle-bound secreted miRNAs are key transcriptional regulators in embryo-endometrial dialogue. The third study was concerned with the placental epigenome, which plays a critical role in regulating mammalian growth and development. Alterations to placental methylation, often observed at imprinted genes, can lead to adverse pregnancy complications such as intrauterine growth restriction and preterm birth. Similar associations have been observed in offspring derived from advanced paternal age fathers. As parental age at time of conception continues to rise, the impact of advanced paternal age on these reproductive outcomes is a growing concern, but limited information is available on the molecular mechanisms affected in utero. This longitudinal murine research study investigated the impact of paternal aging on genomic imprinting. Paternal age significantly impacted embryonic placental weight, fetal weight and length, significant hypermethylation was observed upon natural paternal aging and several transcript level alterations attributable to advanced paternal age were identified. This thesis also presents a series of co-authored studies on related subjects such as embryonic epigenetic dysregulation, unexplained male factor and the sperm epigenome, the dynamic transcriptome of IVF blastocysts in association with infertility, redox balance and aging-related changes in the mouse ovary/oocyte, obesity and the human sperm proteome. Collectively, these data could inform the development novel biomarkers for non-invasive assessment of oocyte competence and implantation success. They also demonstrate a paternal age effect with dysregulation at numerous imprinted loci, providing a mechanism for future adverse placental and offspring health conditions.

What is the transcriptome signature associated with poor performance of rescue IVM (rIVM) oocytes and how can we rejuvenate them? The GATA-1/CREB1/WNT signalling axis was repressed in rIVM oocytes, particularly those of poor quality; restoration of this axis may produce more usable rIVM oocytes. rIVM aims to produce mature oocytes (MII) for IVF through IVM of immature oocytes collected from stimulated ovaries. It is not popular due to limited success rate in infertility treatment. Genetic aberrations, cellular stress and the absence of cumulus cell support in oocytes could account for the failure of rIVM. We applied single-cell RNA sequencing (scRNA-seq) to capture the transcriptomes of human in vivo oocytes (IVO) (n = 10) from 7 donors and rIVM oocytes (n = 10) from 10 donors. The effects of maternal age and ovarian responses on rIVM oocyte transcriptomes were also studied. In parallel, we studied the effect of gallic acid on the maturation rate of mouse oocytes cultured in IVM medium with (n = 84) and without (n = 85) gallic acid. Human oocytes were collected from donors aged 28-41 years with a body mass index of <30. RNA extraction, cDNA generation, library construction and sequencing were performed in one preparation. scRNA-seq data were then processed and analysed. Selected genes in the rIVM versus IVO comparison were validated by quantitative real-time PCR. For the gallic acid study, we collected immature oocytes from 5-month-old mice and studied the effect of 10-μM gallic acid on their maturation rate. The transcriptome profiles of rIVM/IVO oocytes showed distinctive differences. A total of 1559 differentially expressed genes (DEGs, genes with at least 2-fold change and adjusted P < 0.05) were found to be enriched in metabolic processes, biosynthesis and oxidative phosphorylation. Among these DEGs, we identified a repression of WNT/β-catenin signalling in rIVM when compared with IVO oocytes. We found that oestradiol levels exhibited a significant age-independent correlation with the IVO mature oocyte ratio (MII ratio) for each donor. rIVM oocytes from women with a high MII ratio were found to have over-represented cellular processes such as anti-apoptosis. To further identify targets that contribute to the poor clinical outcomes of rIVM, we compared oocytes collected from young donors with a high MII ratio with oocytes from donors of advanced maternal age and lower MII ratio, and revealed that CREB1 is an important regulator. Thus, our study identified that GATA-1/CREB1/WNT signalling was repressed in both rIVM oocytes versus IVO oocytes and in rIVM oocytes of lower versus higher quality. Consequently we investigated gallic acid, as a potential antioxidant substrate in human rIVM medium, and found that it increased the mouse oocyte maturation rate by 31.1%. Raw data from this study can be accessed through GSE158539. In the rIVM oocytes of the high- and low-quality comparison, the number of samples was limited after data filtering with stringent selection criteria. For the oocyte stage identification, we were unable to predict the presence of oocyte spindle, so polar body extrusion was the only indicator. This study showed that GATA-1/CREB1/WNT signalling was repressed in rIVM oocytes compared with IVO oocytes and was further downregulated in low-quality rIVM oocytes, providing us the foundation of subsequent follow-up research on human oocytes and raising safety concerns about the clinical use of rescued oocytes. This work was supported by the Collaborative Research Fund, Research Grants Council, C4054-16G, and Research Committee Funding (Research Sustainability of Major RGC Funding Schemes), The Chinese University of Hong Kong. The authors have no conflicts of interest to declare.