Follicle outcomes in human ovarian tissue: effect of freezing, culture, and grafting

Abstract

Objective: To study the effect of freezing, in vitro culture (IVC) and grafting to chorioallantoic membrane (CAM) on follicle outcomes in human ovarian tissue.

Design: An experimental study.

Setting: University-based research laboratory.

Patients: Fresh and cryopreserved ovarian tissue from 10 patients was donated to research with their consent and institutional review board approval.

Interventions: Fresh and frozen-thawed ovarian cortical pieces were in vitro-cultured and compared (fresh-IVC vs FT-IVC). The FT-IVC fragments were then examined against fragments grafted to CAM (FT-CAM). After both IVC and CAM grafting, ovarian cortical pieces (4×2×1 mm³) were analyzed on days 0, 1, and 6.

Main outcome measures: Follicle analyses included histology (count and classification) and immunohistochemistry (Ki67 [proliferation], caspase-3 [apoptosis], 1A and 1B light chain 3B [autophagy], p-Akt, FOXO1, and p-rpS6 [PI3K activation]). Droplet digital polymerase chain reaction further explored expression of PI3K pathway- and oocyte-related genes in tissue sections.

Results: No major differences were detected between fresh-IVC and FT-IVC tissues in any conducted analyses. Although a significant drop was observed in primordial follicle (PF) proportions in the fresh-IVC and FT-IVC groups (d0 vs. d6, P<.002), they held steady in the FT-CAM group (d0 vs. d6, P>.05). The PF rates were also significantly higher in the FT-CAM group than the FT-IVC group on d6 (P=.02). Importantly, avian erythrocytes were already present in 30% of implants from d1. Apoptotic and autophagic follicle rates increased during IVC (P<.008), but remained significantly lower in the FT-CAM group (P<.01), confirming superior follicle preservation in CAM-grafted tissue. Upregulation of the PI3K/FOXO pathway was established in the IVC groups, demonstrating PF activation, whereas significant pathway downregulation was detected in the FT-CAM group (P<.03). The droplet digital polymerase chain reaction tests confirmed oocyte growth during IVC and follicle autophagy in all groups; however, the PI3K pathway appeared to be differentially modulated in tissues and follicles.

Conclusions: In vitro culture induces PF depletion with no additional impact of freezing. Grafting to CAM preserves the PF pool by curbing follicle activation, apoptosis, and autophagy, probably thanks to rapid graft revascularization and/or the circulating embryonic antimüllerian hormone. These findings highlight the importance of enhancing neoangiogenesis in ovarian grafts and investigating the potential benefits of administering antimüllerian hormone to prevent PF burnout.

Keywords: Follicle activation; chorioallantoic membrane (CAM); in vitro culture (IVC); ovarian tissue cryopreservation.