P-467 Optimizing in vitro growth of immature follicles from cryopreserved vs fresh human cortical ovarian tissue
Authors: Del Valle J.S , Asseler J.D , Fan X , Louwe L.A , Pilgram G.S.K , Metzemaekers J , Van der Westerlaken L.A.J , Chuva de Sousa Lopes S.M 
Published: June 2022

Abstract

Study question
Can cryopreserved-thawed human ovarian cortex be cultured in vitro to produce growing follicles as efficiently as from fresh samples?

Summary answer
In contrast to fresh samples, cryopreserved-thawed ovarian cortex did not develop morphologically normal growing follicles, even when adding follicular growth stimulators.

What is known already
A multistep culture system has been shown to produce mature oocytes from immature follicles present in the human cortex, without the addition of follicular growth stimulators. However, these promising results were achieved using fresh human ovarian cortex tissue donated from cisgender women, which is not available for the purpose of fertility preservation. It remains unclear whether this multistep culture system can be used to mature follicles from cryopreserved-thawed human ovarian cortex. In addition, the applicability of the culture system to mature follicles in vitro has not been investigated using ovarian tissue from transmasculine people.

Study design, size, duration
Fresh and cryopreserved ovarian cortex tissue isolated from the same transmasculine people were cultured during 8 days (first step of the culture system) with or without the addition of a PI3K/Akt promotor, Sphingosine-1-Phosphate (S1P). The ovarian cortex fragments were collected a day 0 and day 8 for downstream morphological and quantitative analysis of the follicular population. This study also included data of in vitro culture of cryopreserved samples from oncological cisgender women.

Participants/materials, setting, methods
Ovarian cortex tissue was collected from six testosterone user transmasculine people (25,8 ± 4,9 years) and three oncological cisgender women (24,0 ± 6,0 years). Fresh cortex was either directly cultured or previously cryopreserved. All ovarian fragments were cultured in the same conditions for 8 days. After the culture period, follicular population was quantified on histological sections (haematoxylin-eosin) and compared to samples of day 0. Follicular cell types, cell proliferation and apoptosis were assessed by immunofluorescence.

Main results and the role of chance
Fresh ovarian cortex tissue isolated from transmasculine people showed morphologically normal primary follicles (PFs) and secondary follicles (SFs) after 8 days of culture and no antral follicles were observed. Moreover, the addition of the follicular growth stimulator, S1P, to the culture medium significantly increased the number of growing follicles (PFs, P &lt; 0.05; SFs, P &lt; 0.01). The addition of S1P stimulated the emergence of SFs that showed expression of anti-mullerian hormone (AMH), contained proliferative (PCNA+) granulosa cells and showed low levels of apoptotic cells (TUNEL assay). Structurally, these in vitro grown SFs showed an intact basement membrane assessed by the expression of collagen IV. By contrast, using the same culture protocol but starting from cryopreserved-thawed cortex tissue from the same patients resulted in a very low number of viable growing follicles (PFs and SFs) even in the presence of S1P. Lastly, when using the same culture protocol on cryopreserved-thawed samples from oncological cisgender women, we observed a low efficiency of follicular growth, comparable to that observed in cryopreserved ovarian tissue from (age-matched) transmasculine people.

Limitations, reasons for caution
Limitations of this study are: small sample size and lack of fresh cortex tissue from oncological cisgender women. Due to the high variability of follicular density among patients, the use of a larger number of samples is recommended.

Wider implications of the findings
Optimization of culture systems to grow and mature follicles in the ovarian cortex of cryopreserved samples (both from cis and trans persons) is urgently needed. Current culture protocols can be applied to fresh samples, but further characterization is needed to consider this as an alternative in fertility preservation treatment.

Trial registration number
Not applicable