STUDY QUESTION Is it possible to identify by mass spectrometry a wider range of proteins and key proteins involved in folliculogenesis and oocyte growth and development by studying follicular fluid (FF) from human small antral follicles (hSAF)? SUMMARY ANSWER The largest number of proteins currently reported in human FF was identified in this study analysing hSAF where several proteins showed a strong relationship with follicular developmental processes. WHAT IS KNOWN ALREADY Protein composition of human ovarian FF constitutes the microenvironment for oocyte development. Previous proteomics studies have analysed fluids from pre-ovulatory follicles, where large numbers of plasma constituents are transferred through the follicular basal membrane. This attenuates the detection of low abundant proteins, however, the basal membrane of small antral follicles is less permeable, making it possible to detect a large number of proteins, and thereby offering further insights in folliculogenesis. STUDY DESIGN, SIZE, DURATION Proteins in FF from unstimulated hSAF (size 6.1 ± 0.4 mm) were characterised by mass spectrometry, supported by high-throughput and targeted proteomics and bioinformatics. The FF protein profiles from hSAF containing oocytes, capable or not of maturing to metaphase II of the second meiotic division during an IVM (n = 13, from 6 women), were also analysed. PARTICIPANTS/MATERIALS, SETTING, METHODS We collected FF from hSAF of ovaries that had been surgically removed from 31 women (∼28.5 years old) undergoing unilateral ovariectomy for fertility preservation. MAIN RESULTS AND THE ROLE OF CHANCE In total, 2461 proteins were identified, of which 1108 identified for the first time in FF. Of the identified proteins, 24 were related to follicular regulatory processes. A total of 35 and 65 proteins were down- and up-regulated, respectively, in fluid from hSAF surrounding oocytes capable of maturing (to MII). We found that changes at the protein level occur already in FF from small antral follicles related to subsequent oocyte maturation. LIMITATIONS, REASONS FOR CAUTION A possible limitation of our study is the uncertainty of the proportion of the sampled follicles that are undergoing atresia. Although the FF samples were carefully aspirated and processed to remove possible contaminants, we cannot ensure the absence of some proteins derived from cellular lysis provoked by technical reasons. WIDER IMPLICATIONS OF THE FINDINGS This study is, to our knowledge, the first proteomics characterisation of FF from hSAF obtained from women in their natural menstrual cycle. We demonstrated that the analysis by mass spectrometry of FF from hSAF allows the identification of a greater number of proteins compared to the results obtained from previous analyses of larger follicles. Significant differences found at the protein level in hSAF fluid could predict the ability of the enclosed oocyte to sustain meiotic resumption. If this can be confirmed in further studies, it demonstrates that the viability of the oocyte is determined early on in follicular development and this may open up new pathways for augmenting or attenuating subsequent oocyte viability in the pre-ovulatory follicle ready to undergo ovulation. STUDY FUNDING/COMPETING INTEREST(S) The authors thank the financial support from ReproUnion, which is funded by the Interreg V EU programme. No conflict of interest was reported by the authors. TRIAL REGISTRATION NUMBER N/A
STUDY QUESTION How does the human granulosa cell (GC) transcriptome change during ovulation? SUMMARY ANSWER Two transcriptional peaks were observed at 12 h and at 36 h after induction of ovulation, both dominated by genes and pathways known from the inflammatory system. WHAT IS KNOWN ALREADY The crosstalk between GCs and the oocyte, which is essential for ovulation and oocyte maturation, can be assessed through transcriptomic profiling of GCs. Detailed transcriptional changes during ovulation have not previously been assessed in humans. STUDY DESIGN, SIZE, DURATION This prospective cohort study comprised 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital-affiliated fertility clinic in 2016–2018. PARTICIPANTS/MATERIALS, SETTING, METHODS From each woman, one sample of GCs was collected by transvaginal ultrasound-guided follicle aspiration either before or 12 h, 17 h or 32 h after ovulation induction (OI). A second sample was collected at oocyte retrieval, 36 h after OI. Total RNA was isolated from GCs and analyzed by microarray. Gene expression differences between the five time points were assessed by ANOVA with a random factor accounting for the pairing of samples, and seven clusters of protein-coding genes representing distinct expression profiles were identified. These were used as input for subsequent bioinformatic analyses to identify enriched pathways and suggest upstream regulators. Subsets of genes were assessed to explore specific ovulatory functions. MAIN RESULTS AND THE ROLE OF CHANCE We identified 13 345 differentially expressed transcripts across the five time points (false discovery rate, <0.01) of which 58% were protein-coding genes. Two clusters of mainly downregulated genes represented cell cycle pathways and DNA repair. Upregulated genes showed one peak at 12 h that resembled the initiation of an inflammatory response, and one peak at 36 h that resembled the effector functions of inflammation such as vasodilation, angiogenesis, coagulation, chemotaxis and tissue remodelling. Genes involved in cell–matrix interactions as a part of cytoskeletal rearrangement and cell motility were also upregulated at 36 h. Predicted activated upstream regulators of ovulation included FSH, LH, transforming growth factor B1, tumour necrosis factor, nuclear factor kappa-light-chain-enhancer of activated B cells, coagulation factor 2, fibroblast growth factor 2, interleukin 1 and cortisol, among others. The results confirmed early regulation of several previously described factors in a cascade inducing meiotic resumption and suggested new factors involved in cumulus expansion and follicle rupture through co-regulation with previously described factors. LARGE SCALE DATA The microarray data were deposited to the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/gds/, accession number: GSE133868). LIMITATIONS, REASONS FOR CAUTION The study included women undergoing ovarian stimulation and the findings may therefore differ from a natural cycle. However, the results confirm significant regulation of many well-established ovulatory genes from a series of previous studies such as amphiregulin, epiregulin, tumour necrosis factor alfa induced protein 6, tissue inhibitor of metallopeptidases 1 and plasminogen activator inhibitor 1, which support the relevance of the results. WIDER IMPLICATIONS OF THE FINDINGS The study increases our understanding of human ovarian function during ovulation, and the publicly available dataset is a valuable resource for future investigations. Suggested upstream regulators and highly differentially expressed genes may be potential pharmaceutical targets in fertility treatment and gynaecology. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by EU Interreg ÔKS V through ReproUnion (www.reprounion.eu) and by a grant from the Region Zealand Research Foundation. None of the authors have any conflicts of interest to declare.
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