P-429 Transcriptomic heterogeneity within cortical ovarian follicle pool in child and adult
Authors: Hassan J , Rooda I , Velthut-Meikas A , Tuuri T , Otala M , Knuus K , Jahnukainen K , Salumets A , Damdimopoulou P 
Published: June 2023

Abstract

Study question
What are the transcriptomic differences between child and adult ovarian follicles in the human cortex?

Summary answer
Our results suggest that human ovarian cortical follicles show marked differences before and after puberty, with the greatest differences being present in the secondary stage.

What is known already
The ovaries and their main functional structures, follicles, are important for determining the reproductive health. Follicle growth and maturation are controlled by multiple mechanisms, including the hypothalamus pituitary ovarian axis and local paracrine signalling. Although dormant primordial follicles are activated to grow even in infancy, all growing follicles before the first menarche are bound to undergo atresia. Follicle growth activation in infancy and adulthood remains poorly understood. Additionally, child and adult follicles behave differently during in vitro culture suggesting differing biology, raising many questions e.g., about their suitability for fertility preservation.

Study design, size, duration
Ovarian cortical tissues from children were collected through fertility preservation program at the Helsinki Children’s hospital (Finland). Families of underage patients signed an informed written consent. Adult ovarian tissue was collected from gender reassignment patients at the Helsinki University hospital and Karolinska university hospital after informed written consent. Follicles at different maturation stages were isolated from ovarian cortex for single follicle transcriptomic analysis. Freshly-fixed cortical biopsies (3x3x1 mm3) were used for validation of transcriptomic analysis.

Participants/materials, setting, methods
Single viable follicles (n = 120, 60/age group) were mechanically and enzymatically isolated from frozen-thawed ovarian tissue from four adults (age &gt;18 yrs) and five children (age 1-11 yrs) and processed for RNA-sequencing. Individual follicles were lysed, prepared for RNA libraries following Smart-Seq2 protocol and sequenced using NextSeq 500 platform. After quality assessment, 109 follicles were analysed (Adult n = 54, Child n = 55). DDX4, AMH, FOXL2 and DAZL transcripts and proteins localization were evaluated via immunodetection and RNA-FISH assay.

Main results and the role of chance
Principal component analysis (PCA) of all sequenced follicles demonstrated an expected timely progression of their stages based on maturation, and an overall similarity between child and adult. Interestingly, PCA displayed two distinct clusters of follicles in both adult and child samples. The first group contained only growing follicles and, after differentially expressed gene (DEG) analysis, demonstrated lower expression of traditional oocyte markers (group 1). The second group consisted of primordial and growing follicles and had lower expression of traditional granulosa marker (group 2). Group-related differential expression of main oocyte (DDX4, DAZL) and granulosa (FOXL2, AMH) marker genes were validated in tissue sections with RNA-FISH.
Downstream analyses focused on group 2 follicles. When child and adult follicles were compared to each other, the highest number of DEGs were found in the secondary stage (primordial n = 32, primary n = 14, secondary n = 387). Pathway analysis of DEG displays enrichment of oestrogen-related related pathways in adult secondary follicles. Furthermore, we analysed DEGs between the follicular stages per age group, finding high number of DEGs between primordial and primary follicles (Adult n = 1663, Child n = 1914). The majority of those genes are unique to the age group (common=512).

Limitations, reasons for caution
Adult ovarian tissue is obtained from gender reassignment patients, who receive androgen treatments prior to surgery. Child ovarian tissue was collected from fertility preservation programme where some patients were exposed to chemotherapy prior to tissue collection.

Wider implications of the findings
The differences between child and adult follicles encourage further investigation of the suitability of child ovarian tissue in current fertility preservation protocols. Understanding transcriptomic changes during early folliculogenesis and reasons for follicle degeneration can help tailor protocols for in vitro growth of follicles.

Trial registration number
not applicable