Periodic Reporting for period 1 - POMOLIM (Polycystic ovarian syndrome: novel molecular characterization and personalized in vitro maturation protocol.)
Période du rapport: 2020-04-01 au 2022-03-31
This research project aimed to perform a novel epigenomics and transcriptomics characterization of oocytes and cumulus cells from women with Polycystic ovarian syndrome (PCOS) and develop a safe in vitro system that maintains their epigenetic and transcriptional integrity and improve the outcome of in vitro maturation (IVM) cycles in these patients. Human oocytes were donated by patients with polycystic ovary syndrome (PCOS) undergoing fertility treatments and by healthy volunteers enrolled in a recruitment program. The molecular study of human PCOS oocytes is a very innovative topic and the originality of the granted project relies on the fact that very few research groups, if any, are able to study the human female gamete, given the limited access to this precious material. The results derived from this study can be implemented in the clinic and, therefore, can improve medical management strategies for patients. In addition, they could help to reduce the psychological burden and the high social security budget for infertility treatments in PCOS women.
Recruitment of patients/volunteers and healthy volunteers was achieved thanks to the ongoing collaboration between the host lab and the fertility center CRG at the UZBrussel (the university hospital of the VUB). Participants were enrolled through correspondent recruitment programs running in the CRG. Specifically, research material was collected from (i) PCOS patients undergoing IVM who donated part of their oocytes for research and (ii) both PCOS-like and healthy volunteers who donated the total amount of the retrieved oocytes for research. Eligible patients/volunteers between the ages of 18 and 37 were evaluated by a gynecologist. Blood tests were performed to determine free testosterone levels and lipid profile including fasting plasma glucose and insulin. Rotterdam criteria was followed for the diagnosis of PCOS. Women with PCOS phenotype A (biochemical hyperandrogenism + ovulatory dysfunction + polycystic ovaries) according to these criteria were enrolled in the study. The PCOS group included patients and volunteers with phenotype A, metabolic syndrome and insulin resistance. The age-matched control group included non-PCOS volunteers that did not have either metabolic syndrome or insulin resistance. Participants were minimally stimulated with gonadotrophins and cumulus-oocytes complexes (COCs) were aspirated from small antral follicles (2-8mm) without any ovulation triggering. Mechanical denudation of oocytes was performed and paired oocytes and cumulus cells (CC) were individually stored. DNA methylome and transcriptome analysis of individual PCOS and non-PCOS GV oocytes was accomplished using innovative parallel single-cell bisulfite sequencing (scBS-seq) and single-cell RNA-sequencing (scRNA-seq) techniques. The pool of retrieved oocytes from the same woman were randomized in three experimental treatment arms: (1) standard pre-IVM (pre-IVM culture medium supplemented with CNP), (2) standard pre-IVM in combination with Cumulin, and (3) both standard pre-IVM and IVM complemented with Cumulin. After the 24 hours of pre-IVM step, COCs were transferred to IVM culture medium in the presence of FSH and Amphiregulin (AREG) and incubated for 30 h. Fifteen replicate experiments were performed with a total of 75-100 COCs per protocol. Replicates were randomly assigned to an indirect or direct assay to evaluate oocyte competence and maturation.
The improved CAPA-IVM approach has the potential to reduce the burden and the subsequent cycle abandonment and the large budget of social security for infertility treatments in PCOS women. This shorter and less invasive technique could increase access to treatment to more couples from lower socio-economic regions in Europe. It could also be applied as a fertility preservation strategy in girls and women receiving gonadotoxic cancer treatment. Conventional IVF demands a strict and intense regime of medication use, constant monitoring of hormone levels and ovarian ultrasounds. This process can very quickly become mentally and physically taxing for women, as well as for their partners and social environment. In comparison, the IVM technique does not require anymore the injection of high doses of hormones to patients; hence the patient-friendliness of IVM approach would reduce the burden and the subsequent cycle abandonment. In addition, IVF financing is expensive for governments and patients. The average cost of an IVF cycle in Europe is 4000-7000€ and, in general, patients may need to undergo several cycles of treatment which multiplies the overall cost. There is some (partial) state funding/reimbursement for IVF in part of the EU member states. However, the extent of this support varies from around 90% in Belgium or France, to 20-30% in Bulgaria and Spain. Thanks to the proposed improved CAPA-IVM approach for PCOS patients, the large budget of social security for infertility treatments could be reduced and the access to treatment could be increased. The results of this project will have beneficial implications for public health policies in Europe with respect to quality, safety, regulation and financial control of treatments with IVF. Policy making agencies and health care system will use these results towards the implementation of new strategies on reproductive medicine.