P-595 Heterogeneity of preovulatory follicle cell types between normo- and hyporesponders
Authors: Roos K , Rooda I , Keif R.S , Liivrand M , Salumets A , Smolander O.P , Velthut-Meikas A 
Published: June 2022

Abstract

Study question
Is there a difference in the proportions of preovulatory follicle cell types between normo- and hyporesponders?

Summary answer
Human preovulatory follicles consist of 14 distinct cell types, 3 of them are significantly different between normo- and hyporesponder patients.

What is known already
Human ovarian follicles are a diverse dynamic environment for oocytes to mature and ovulate. Follicles consist of multiple cell types in layers and altogether they are responsible for a repertoire of biological processes. Dysregulations in follicular cells could result in hyporesponsiveness to ovarian stimulation. Hyporesponse might lead to the failure of IVF, due to a low number of retrieved oocytes. Its pathophysiological mechanisms have not been fully elucidated so far. This condition affects up to 25% of patients and investigating their follicular cell populations in detail is highly informative for increasing their prognosis for the live birth outcome.

Study design, size, duration
Study groups consisted of IVF patients with normoresponse (n = 10) and hyporesponse (n = 9) to rFSH stimulation according to antagonist protocol undergoing in vitro fertilization treatment at Nova Vita Clinic, Estonia. Hyporesponse to treatment was diagnosed if ≥ 200 IU of rFSH was administered to retrieve an oocyte. All study participants were ≤40 years of age with matched BMI and normal ovarian reserve. Women with polycystic ovary morphology and ovarian abnormalities observed by ultrasound examination were excluded.

Participants/materials, setting, methods
Oocyte pick-up was performed for patients after 36 hours of hCG trigger. Cumulus-oocyte complex was removed from the follicular fluid for IVF and the remaining cells were collected by centrifugation and treated with hyaluronidase and DNase. Genome-wide single-cell RNA-seq was performed for &gt;6000 individual cells of 3 normoresponder patients each. RNA-seq was performed for pooled follicular cells of each participant. CIBERTSORTx software was used to deconvolute the proportion of cell types from bulk RNA-seq data.

Main results and the role of chance
By analyzing a total number of 24 213 single cells from preovulatory follicles, we identified 4 immune cell lineages (CD45+), including neutrophils, T cells, M1 and M2 macrophages along with 10 non-immune cell lineages (CD45-): epithelial cells and theca cells, cumulus cells and 7 subtypes of granulosa cells with different functional transcriptomic profiles (p &lt; 0.05). We identified granulosa cell subclusters that were highly active in progesterone or estrogen production, extracellular matrix remodeling, ovulational process, cell migration, and apoptosis. Comparing the proportion of cell populations between normo- and hyporesponders from deconvoluted RNA-seq data (adjusted to age), we identified that 3 cell clusters varied with statistical significance (p &lt; 0.05). These detected clusters with different proportions between the studied patient groups suggest their role in contributing to hyporesponse in a preovulatory follicle regarding patients undergoing IVF treatment.

Limitations, reasons for caution
The small sample size limits the study.

Wider implications of the findings
Our work advances understanding the heterogeneity of cell types in human preovulatory follicles. The differences in the proportions of cell types of preovulatory follicles of hyporesponders could provide valuable insight for assisting IVF treatment by introducing potential therapeutic targets to improve their live birth outcome.

Trial registration number
not applicable