P–713 The effect of aging and polycystic ovarian syndrome on the maturation and apoptosis of granulosa cells from women undergoing controlled ovarian hyperstimulation with recombinant hFSH
Authors: Papageorgiou K , Mastora E , Kesikiadou E , Zisiadi A , Genopoulou A , Zikopoulos A , Grigoriou M , Zikopoulos K , Georgiou I , Michaelidis T M 
Published: August 2021

Abstract

Study question
What is the effect of biological aging and polycystic ovarian syndrome (PCOS) on steroidogenic and apoptotic pathways of granulosa cells isolated from women undergoing IVF?

Summary answer
This analysis revealed that aging as well as PCOS influence the response of women to ovarian stimulation, affecting steroidogenesis and apoptosis on granulosa cells.

What is known already
Infertility and subfertility affect a significant proportion of humans worldwide. Age-related factors in women lead to a decrease in the ovarian oocyte reserve, and PCOS, a pathological condition characterized by anovulation and hyperandrogenism, are the two most common reasons of subfertility. Despite the progress in recent years, the causes are still ill-defined, and there is a strong need for interventions to ensure good quality oocytes in healthy aging, as well as in PCOS women. Gene expression analyses of granulosa cells isolated during oocyte retrieval are highly informative for understanding the communication between oocytes and follicular cells and optimizing IVF outcomes.

Study design, size, duration
This report is part of an ongoing study starting from June 2020. 42 women undergoing IVF/ICSI at the University Hospital of Ioannina, Greece, volunteered to participate with informed consent. Participants were divided into three groups: 30- to 35-year-old patients with PCOS, 30- to 35-year-old non-PCOS women, and older women aged 38- to 42-year-old. All participants received 150 – 300IU/d recombinant hFSH. Participants/materials, setting, methods: Granulosa cells (GC) were collected from the follicular fluid at the day of oocyte retrieval using the protocol described by Ferrero et al., 2012 with modifications that allow us to isolate nearly pure populations, also from small or very few follicles. GC purity was determined by FACS and immunocytochemistry. Reverse Transcription-quantitative-PCR was employed to correlate the expression of steroidogenesis- and apoptosis-related genes with the clinical characteristics of each participant, including FSH and progesterone/estradiol serum levels.

Main results and the role of chance
FACS and immucytochemistry analysis using specific markers of granulosa cells, like FSHR, as well as leucocyte and erythrocyte cell markers (CD45 and Glycophorin) confirmed the purity of the isolated granulosa cell populations. No differences were found in hormone serum levels between 30- to 35-year-old PCOS (Group A) and non-PCOS women of the same age (Group B) whereas progesterone and estradiol serum levels were lower in 38- to 42-year-old women (Group C) compared to group B. RT-qPCR analysis of maturation-related genes (ZP3 and AHR) revealed the presence of more immature follicles in Group A compared to the other groups. The granulosa cells from women of group A were characterized by increased expression of genes involved in steroidogenic pathways (such as FSHR, CYP17A1) compared to group B, as well as of the genes for the estradiol and progesterone receptors (ESR1 and PGRMC1, respectively), and by decreased levels of the apoptosis-related genes BAX and BCL-xL. Importantly, the granulosa cells from the women of Group C were characterized by lower mRNA levels of steroidogenesis-related genes, as well as of ESR1, and by increased apoptosis as revealed by the increased ratio of BAX/BCL2 transcripts, compared to Group A.

Limitations, reasons for caution
The presented results of gene expression analysis are based on preliminary data as the number of participants is small (6 women per group), but they are part of an ongoing study where the number of participants is increasing in order to improve quantification and reproducibility of our results.
Wider implications of the findings: Potential differences in signaling pathways that are crucial for the maturation of granulosa cells and follicles during biological aging or in pathological conditions such as PCOS are expected to provide valuable information concerning the response of individual women to gonadotropin stimulation and might help to design more personalized therapeutic strategies.

Trial registration number
Not applicable