P-549 Differentially expressed genes in the immune cells obtained from preovulatory follicles undergoing progestin-primed ovarian stimulation and GnRH-antagonist: Insights from single-cell RNA sequencing analysis
Authors: Handa M , Takiuchi T , Miyake T , Ando Y , Shin J  W , Kimura T 
Published: July 2024

Abstract

Study question
Could controlled ovarian stimulation protocol (progestin-primed ovarian stimulation [PPOS] vs GnRH-antagonist [GnRH-ant]) affect the gene expression of human immune cells (ICs) in preovulatory follicles?

Summary answer
Proinflammatory cytokine-related gene expression was significantly higher and proinflammatory pathways were also more prominently involved in ICs in PPOS than the GnRH-ant.

What is known already
PPOS has garnered attention, with several studies suggesting reproductive outcomes comparable to GnRH-ant. However, in our previous study, pregnancy outcomes with PPOS were significantly lower than GnRH-ant (38th ESHRE Annual Meeting [O-130]) and single-cell RNA sequencing (scRNA-seq) of granulosa cells revealed increased mitochondrial DNA gene expression in PPOS than GnRH-ant (39th ESHRE Annual Meeting [P-281]). Previous studies suggested that ICs play a pivotal role in follicular and embryonic developments, via proinflammatory cytokines such as TNF-α, IL-1β, and IL-6. Notably, changes in gene expressions of ICs under different controlled ovarian stimulation (COS) protocols remain unresolved to date.

Study design, size, duration
From November 2021 to January 2022, all samples were obtained from the patients who provided written informed consent before the inclusion and underwent COS cycles at a single-institution. scRNA-seq was performed on 6,828 cells corresponding to ICs of the MII-oocyte follicles (56 from PPOS and 39 from GnRH-ant), involving 16 patients with normal ovarian reserves (aged &lt; 40 years, AMH ≥1.1 ng/mL) undergoing PPOS with chlormadinone acetate (n = 8) or GnRH-ant with cetrorelix (n = 8).

Participants/materials, setting, methods
The ICs were collected via follicular aspiration from 16 patients. The follicular fluid (FF) from each follicle was collected and all FF were mixed together in MII-oocyte for sequencing. Sample sequencing was performed using Chromium Next GEM Single Cell V(D)J Reagent Kits (10X Genomics, Pleasanton, CA, USA). Libraries were sequenced using a DNBSEQ-G400, with reads subsequently analyzed using Cellxgene. The clustering was performed using fastcluster in scDblFinder. Functional enrichment analysis was carried out using Enrichr.

Main results and the role of chance
Two population of ICs were used in this investigation: PPOS and GnRH-ant of MII-oocyte. After the quality control, 4,447 cells for PPOS and 2,381 cells for GnRH-ant, respectively, were analyzed. Following single-cell clustering based on the Seurat package, cell proportion analysis revealed eight clusters, which consist of monocyte, myeloid dendritic cell, T cell, natural killer cell and naive B cell, in the PPOS and GnRH-ant groups. No significant difference was observed in gene expression and cell distribution in each cluster. Differentially expressed genes (DEGs) were identified in the two IC population. Interestingly, a significant increase in expression of proinflammatory cytokine including IL-1β and IL-6 was observed in PPOS compared to GnRH-ant, particularly in CD14-positive monocytes. Functional significance of DEGs was validated by identifying significantly regulated pathways based on Kyoto Encyclopedia of Genes and Genomes categories, revealing involvement in TNF signaling, IL-17 signaling, and NF-κB signaling pathways.

Limitations, reasons for caution
Our conclusions are limited due to the inclusion of Japanese patients at a single-institution. The results need to be validated across different centers and other ethnicities. Due to the small sample size, these results should prompt further study to confirm our findings.

Wider implications of the findings
This is the first report assessing the DEGs of ICs under different COS protocols at the single-cell level. This study suggests that progestin for PPOS induces elevation of the proinflammatory cytokine gene expression of ICs in preovulatory follicles, which may be associated with reproductive outcomes.

Trial registration number
Not applicable