Characterization of EVs (Extracellular Vesicles)/miRNAs (microRNAs) From Human Follicular Fluid

• Background Extracellular vesicles (EVs) are membrane-bound vesicles found in all biological fluids. They contain various regulatory molecules, including microRNAs (miRNAs). It is hypothesized that EVs in human follicular fluid (the fluid surrounding the oocyte within the ovary) play a crucial role in oocyte development through these miRNAs.
• Research Question Is there a difference in miRNA expression in EVs in human follicular fluid based on the patient's age, the maturation stage of the corresponding oocyte, and/or the ploidy status of the resulting embryo (euploid in the case of a normal chromosome count vs. aneuploid in the case of an abnormal chromosome count)?
• Methodology This prospective study will include patients in two age groups (≥ 38 years vs. ≤ 32 years; 7 patients per group) undergoing an ICSI treatment (intracytoplasmic sperm injection) in combination with a coPGT-M treatment at Ghent University Hospital. PGT-M (pre-implantation genetic testing for monogenic disorders) is an IVF/ICSI procedure in which an embryo biopsy is performed on day 5 or 6 of embryo development to test for known genetic disorders that could be inherited from one or both parents. With the coPGT-M technology or comprehensive PGT-M, in addition to detecting known genetic defects, any additional chromosomal abnormalities in the embryo are also identified. An embryo is considered aneuploid if its chromosome number is abnormal.

For the participating patients, follicular fluid and plasma will be collected, and consent will be requested to store non-developing oocytes and embryos from the ICSI treatment.

Inclusion will take place on day 10 of ovarian stimulation if the patient meets the inclusion and exclusion criteria and at least 5 follicles are confirmed to be growing.

• Follicular Fluid (residual material): After oocyte retrieval, follicular fluid will be collected from 2-10 follicles per patient. EV isolation from this follicular fluid will be performed using the optimal technique determined in a preceding experiment (ID 18220, see 'Relations', comparison of 3 existing techniques to determine the optimal EV isolation technique from human follicular fluid). RNA extraction followed by miRNA sequencing will be used to assess differences in miRNA expression based on age (≤ 32 years vs. ≥ 38 years), oocyte maturation stage (mature vs. immature), and the ploidy status of the resulting embryo (euploid vs. aneuploid). In addition, the following hormone concentrations will be determined in the follicular fluid: estradiol, progesterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH).
• Plasma: For participating patients, an additional blood tube will be taken during each blood sample/infusion placement within the standard-of-care IVF/ICSI treatment from day 10 of ovarian stimulation until the day of oocyte retrieval. EV isolation and RNA extraction will be performed on plasma samples of the ovulation trigger day and oocyte retrieval day. MiRNA sequencing of these samples will be correlated with the miRNA expression of EVs in follicular fluid.