Endometriosis-associated infertility alters the cumulus cells miRNA signature with a differential footprint between normally fertilized oocytes and oocytes failing fertilization
The study was performed in 36 cumulus cells (CCs) samples obtained from metaphase II oocytes (MII), collected at the time of oocyte retrieval from 33 patients undergoing Assisted Reproduction (ART) between 2019-2023 at University Hospital Zurich. Among the 33 patients, 15 were diagnosed with endometriosis (E) (2 patients donated 2 samples each, 17 samples) and 18 with other infertility problems (controls, C) (11 male and 7 non-hormonal factors) (1 patient donated 2 samples each, 19 samples). Retrospective classification of fertilization status of all patients, allowed to classify the CCs samples into four experimental groups: 1) E patients with oocytes fertilized (2PN) (n=13) (referred to as E2); 2) E patients with oocytes that failed fertilization (0PN) (referred to as E0) (n=4); 3) C patients with 2PN oocytes (referred to as C2) (n=12); 4) C patients with 0PN oocytes (referred to as C0) (n=7). Endometriosis phenotype and disease grade were based on clinical history, according to the revised American Society for Reproductive Medicine (rASRM) classification. In total, 7 patients were diagnosed with E with ovarian lesions (O) and 10 without (N), while 8 with mild E (M, stage I-II) and 9 with severe E (S, stage III-IV). Small RNA-sequencing was performed for a total of 36 single CCs samples. RNA-seq data was analyzed using the Galaxy Europe server (usegalaxy.eu) and differential expression analysis was performed with the Bioconductor package EdgeR. Additionally, the differential abundance of two specific miRNAs, miR-143-3p and miR-10b-5p, was further validated in 24 remaining samples by quantitative polymerase chain reaction (qPCR). A total of 85 differential abundant (DA) miRNAs were identified between E and C patients (false discovery rate, FDR < 0.05). Regardless the disease or health status, 37 miRNAs were DA between 2PN and 0PN oocytes (FDR <0.2). Particularly in E patients, 25 DA miRNAs were found between E2 and E0 oocytes (FDR < 0.05). Besides, statistical comparison of O vs. N patients resulted in 2 DA miRNAs while for S vs M no statistical differences were found. In C patients, 13 DA miRNAs were found between C2 and C0 oocytes (FDR < 0.05). Comparisons among these DA miRNAs revealed three sets of miRNAs (FDR<0.01): 1) with 35 DA miRNAs affected only by E, 2) with 27 DA miRNAs affected by both E and the potential to be fertilized; and 3) with 6 DA miRNAs displaying a profile of a competent oocytes that despite the E disease has still the potential to be fertilized. Around 80% of the DA miRNAs identified in CCs in the three sets have been previously associated to E based on the analysis of endometrium or plasma samples. Target gene analysis of DA miRNAs unveiled genes involved in notable biological functions and pathways for the oocyte, such as: oocyte maturation, embryo development, mitochondria and spindle alterations, calcium signaling, oxidative stress and epigenetic modifications. Particularly in oocyte maturation, 10 DA miRNAs identified in Set 1 and 3 DA miRNAs in Set 3 target genes involved in oocyte meiosis. Besides, 6 DA miRNAs identified in Set 1, 8 DA miRNAs identified in Set 2 and 1 DA miRNAs in Set 3 target genes associated to progesterone-mediated oocyte maturation pathway. The current findings can inspire new diagnosis approaches and novel personalized treatments with therapeutic miRNAs underexpressed in CCs and/or with miRNA inhibitors for overexpressed miRNAs in CCs of oocytes derived from E patients. The potential applications of this study can be of significant value to E patients who wish to conceive but have a low number of oocytes retrieved and with poor quality.