This study investigated how follicle health and development in human ovarian tissue cryopreserved for fertility preservation varied between patients before and after six days of in vitro culture. Ovarian tissue from 12 patients (9-25 years) was used. In 3 patients, a 1hr neutral red (NR) incubation was used to identify tissues with viable follicles. Tissues were fixed, sectioned, and follicles staged and graded for health. Inter-patient differences were observed in the non-cultured tissue in the number of both healthy follicles (p=0.005) and growing follicles (p=0.005). After culture there was significant variation in the number of transitional, primary, and secondary follicles between patients (p<0.001).Asymmetric primary follicles with a single complete layer of granulosa cells plus two or more additional partial layers were 5.5 times more likely to be observed in cultured compared to non-cultured tissue (p=0.0063). Non-cultured (p=0.0125) and cultured (p<0.001) tissue selected using NR had more healthy follicles compared to tissue not selected using NR. Non-cultured and cultured tissue selected using NR had more healthy follicles compared to tissue not selected using NR (p=0.0125; p<0.001). We demonstrate that inter-patient variation exists in the health and development of follicles before and after culture. Culture systems need to be optimized to support cryopreserved ovarian tissue and these findings should prompt researchers to consider patient variation when evaluating culture systems.

STUDY QUESTION Can ovarian tissue morphology be better preserved whilst enabling histological molecular analyses following fixation with a novel fixative, neutral buffered formalin (NBF) with 5% acetic acid (referred to hereafter as Form-Acetic)? SUMMARY ANSWER Fixation with Form-Acetic improved ovarian tissue histology compared to NBF in multiple species while still enabling histological molecular analyses. WHAT IS KNOWN ALREADY NBF fixation results in tissue shrinkage in various tissue types including the ovary. Components of ovarian tissue, notably follicles, are particularly susceptible to NBF-induced morphological alterations and can lead to data misrepresentation. Bouin’s solution (which contains 5% acetic acid) better preserves tissue architecture compared to NBF but is limited for immunohistochemical analyses. STUDY DESIGN, SIZE, DURATION A comparison of routinely used fixatives, NBF and Bouin’s, and a new fixative, Form-Acetic was carried out. Ovarian tissue was used from three different species: human (n = 5 patients), sheep (n = 3; 6 ovaries; 3 animals per condition) and mouse (n = 14 mice; 3 ovaries from 3 different animals per condition). PARTICIPANTS/MATERIALS, SETTING, METHODS Ovarian tissue from humans (aged 13 weeks to 32 years), sheep (reproductively young i.e. 3–6 months) and mice (10 weeks old) were obtained and fixed in 2 ml NBF, Bouin’s or Form-Acetic for 4, 8, and 24 h at room temperature. Tissues were embedded and sectioned. Five-micron sections were stained with haemotoxylin and eosin (H&E) and the percentage of artefact (clear space as a result of shrinkage) between ovarian structures was calculated. Additional histological staining using Periodic acid-Schiff and Masson’s trichrome were performed on 8 and 24 h NBF, Bouin’s and Form-Acetic fixed samples to assess the compatibility of the new fixative with stains. On ovarian tissue fixed for both 8 and 24 h in NBF and Form-Acetic, immunohistochemistry (IHC) studies to detect FOXO3a, FoxL2, collagen IV, laminin and anti-Müllerian hormone (AMH) proteins were performed in addition to the terminal deoxynucleotidyl transferase nick end labelling (TUNEL) assay to determine the compatibility of Form-Acetic fixation with types of histological molecular analyses. MAIN RESULTS AND THE ROLE OF CHANCE Fixation in Form-Acetic improved ovarian tissue morphology compared to NBF from all three species and either slightly improved or was comparable to Bouin’s for human, mouse and sheep tissues. Form-Acetic was compatible with H&E, Periodic acid-Schiff and Masson’s trichrome staining and all proteins (FOXO3a, FoxL2, collagen IV and laminin and AMH) could be detected via IHC. Furthermore, Form-Acetic, unlike NBF, enabled antigen recognition for most of the proteins tested without the need for antigen retrieval. Form-Acetic also enabled the detection of damaged DNA via the TUNEL assay using fluorescence. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION In this study, IHC analysis was performed on a select number of protein types in ovarian tissue thus encouraging further studies to confirm the use of Form-Acetic in enabling the detection of a wider range of protein forms in addition to other tissue types. WIDER IMPLICATIONS OF THE FINDINGS The simplicity in preparation of Form-Acetic and its superior preservative properties whilst enabling forms of histological molecular analyses make it a highly valuable tool for studying ovarian tissue. We, therefore, recommend that Form-Acetic replaces currently used fixatives and encourage others to introduce it into their research workflow. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Oxford Medical Research Council Doctoral Training Programme (Oxford MRC-DTP) grant awarded to B.D.B. (Grant no. MR/N013468/1), the Fondation Hoffmann supporting R.A. and the Petroleum Technology Development Fund (PTDF) awarded to B.V.A.

In vitro follicle growth is a potential fertility preservation method for patients for whom current methods are contraindicated. Currently this method has only been successful using fresh ovarian tissue. Since many patients who may benefit from this treatment currently have cryopreserved ovarian tissue in storage, optimising in vitro follicle growth (IVG) for cryopreserved-thawed tissue is critical. This study sought to improve the first step of IVG by comparing different short-term culture systems for cryopreserved-thawed human ovarian tissue, in order to yield a higher number of healthy multilayer follicles. We compared two commonly used culture media (αMEM and McCoy’s 5A), and three plate conditions (300 µL, 1 mL on a polycarbonate membrane and 1 mL in a gas-permeable plate) on the health and development of follicles after six days of culture. A total of 5,797 follicles from three post-pubertal patients (aged 21.3 ± 2.3 years) were analysed across six different culture conditions and non-cultured control. All culture systems supported follicle development and there was no difference in developmental progression between the different conditions tested. Differences in follicle morphology were evident with follicles cultured in low volume conditions having significantly greater odds of being graded as morphologically normal compared to other conditions. Furthermore, culture in a low volume of αMEM resulted in the highest proportion of morphologically normal primary and multilayer follicles (23.8% compared to 6.3-19.9% depending on condition). We therefore recommend culture of cryopreserved human ovarian tissue in a low volume of αMEM to support follicle health and development.

Background. Ovarian tissue cryopreservation (OTC) is important for fertility preservation and conservation. Delay in OTC can occur for transport or workflow management, yet little is understood about the effect of this on the tissue. Objective. To determine if a delay of 24-48 h to OTC affects primordial follicle (PF) health. Methods. Ovaries (n = 6 sheep) were processed immediately or after storage at 4°C (24 h, 48 h). Tissue was fixed fresh, after cryopreservation or 10-day xenotransplantation. Morphological assessment of follicle health and development was performed. Findings. A total of 1,541 follicles were analysed. A 24 h processing delay did not impact PF health in fresh or cryopreserved tissue. In fresh tissue, a 48 h delay had an adverse effect on PF health (OR = 2.47, 95% CI 1.29-4.71). Interestingly, a 48 h delay resulted in cryopreserved tissue being less likely to be graded as abnormal compared to control (OR = 0.56, 95% CI 0.36-0.87). There was no difference in PF health or development across groups following xenotransplantation. Conclusion. Ovarian tissue can be stored for up to 48 h prior to cryopreservation with no net impact on PF morphology which is indicative of health.