Study design and collection of donor ovaries
Donor ovaries were collected from female-to-male transgender donors during their combined gender reassignment operation, which has been published previously and included total laparoscopic hysterectomy, bilateral salpingo-oophorectomy and bilateral mastectomy (29).
Patients were recruited between February 2017 and December 2018. Inclusion criteria were: (i) gender-completing operation; (ii) age 18–40 years; (iii) willingness to donate ovarian tissue for research purposes.
Ethical approval
Oral and written informed consent was obtained from all participants. The study was approved by the ethics committee of the Medical University of Vienna (EK 2240/2016), was conducted in accordance with the Declaration of Helsinki and was retrospectively registered in the Current Controlled Trials Register (NCT03649087).
Surgical technique and tissue preparation
Total laparoscopic hysterectomy and bilateral salpingo-oophorectomy was performed in all patients. To ensure short ischemia times, the ovarian perfusion through the infundibulo-pelvic (IP) ligament was obtained until dissection of the uterus was completed. After dissection of the IP ligament and opening of the vaginal cuff, uterus, fallopian tubes and ovaries were removed en block through the vagina. Dissection of ovarian tissue was performed on a side table. The remaining part of the ovary was sent for histo-pathologic examination and morphological evaluation of the follicles. After an incubation in PBS for a maximum of 10 minutes the ovarian tissue was split into pieces of 10mm x 5-10mm x 2mm for subsequent vitrification.
Vitrification and Warming
Two different vitrification protocols were used and both were performed in an open and in a closed system. For the open system, the tubes with the tissue pieces (see below) were closed with a lid after immersion in liquid nitrogen, whereas for the closed system, this was done directly before the nitrogen step. The study flow chart is provided in Fig. 1.
The first protocol contained DMSO as cryoprotective agent and was previously described by Silber et al. (protocol 1) (1). The second contained ethylene glycol and propylene glycol (propane-1,2-diol) as cryoprotective agents and was purchased by ORIGIO (Origio, Måløv, Denmark (protocol 2).
Except otherwise stated, all chemicals were obtained from Sigma (Sigma Chemical Co., St Louis, USA).
Protocol 1
For vitrification according to the protocol previously published by Silber and colleagues (1) the washed pieces were equilibrated for 10 min. in equilibration solution (ES) containing 7,5% Dimethylsulfoxid (DMSO) and 10% fetal calf serum (FCS) in DMEM. Afterwards the pieces were transferred with a tweezer to the vitrification solution (VS) containing 20% ethylene glycol (EG), 20% DMSO, 0.5M Sucrose and 10%FCS in DMEM. After 10 to 15 minutes of incubation, the tissue pieces were removed from the VS and transferred to a 1.8 ml tube (Nunc, ThermoFisher Waltham, Massachusetts, US) with tweezers. The tubes were then quickly immersed in liquid nitrogen using a tube holder.
For warming, the samples were removed from liquid nitrogen and 1 ml of a 37°C pre-warmed warming solution containing 1 M sucrose and 20% FCS were added. After incubation for 1–3 min. in a water bath (37°C) the pieces were transferred to 1 ml of a solution containing 0.5M sucrose and 20% FCS for 3 min. After a washing step with PBS the ovarian pieces were either used for formalin fixation and histological examination or for FACS analyses.
Protocol 2
For vitrification according to the DMSO-free protocol by Origio, we used the MediCult Vitrification Cooling kit for human oocytes, cleavage stage embryos and blastocyst and adapted the protocol for our purposes as follows: The equilibration and vitrification solutions were warmed to room temperature. The ovarian tissue pieces were equilibrated for 15 min. in equilibration solution and transferred afterwards with a tweezer to the vitrification solution (VS) for a maximum of 1 min. The tissue pieces were then removed from the VS and transferred to a 1.8 ml tube (Nunc, ThermoFisher Waltham, Massachusetts, US) with tweezers. The tubes were immersed in liquid nitrogen using a tube holder.
For warming we used the MediCult Vitrification Warming kit by Origio and adapted the protocol as follows: After removing the samples from liquid nitrogen 1 ml of a 37°C pre-warmed warming solution was added directly into the tube, the tube was incubated in a 37°C water bath for a maximum of 3 min. Afterwards the ovarian tissue pieces were transferred into dilution media 1 for 3 min. Then the tissue was transferred for another 3 min in dilution media 2. After washing steps with washing media and PBS the ovarian pieces were either used for formalin fixation and histological examination or for enzymatic digestion and subsequent FACS analyses.
Enzymatic digestion with Liberase
For the enzymatic digestion with Liberase, the tissue was sliced into small pieces with a scalpel and was transferred in 10 ml PBS. After addition of 215µl of Liberase DH (2,8 Wünsch Units) the suspension was incubated for 1 h at 37°C. The suspension was shaken every 15 min. with a pipette for additionally mechanically disruption. After the digestion step, the cell suspension was filtered through a 100µm filter and rinsed with PBS. The cells were collected by centrifugation (300rcf) and washed twice in PBS.
Fluorescence Activated Cell Sorting (FACS) analysis
The cells were suspended in 1 ml PBS and analyzed on a BD FACSVerse Flow cytometer. DAPI (4,6 Diamino-2-Phenylindole, Dihydrochloride) was added to the samples ten minutes before start of the analysis to distinguish vital and non-vital cells. A minimum of 10.000 events was collected. Data were analyzed using a BD FACSuite V1.06 and FLOWJO software (www.flowjo.com).
Histological analysis
Fresh and thawed ovarian pieces were fixed in 4% buffered formaldehyde and embedded in paraffin blocks. The paraffin blocks were serially cut into 4 µm sections and stained with hematoxylin and eosin. Within these sections, the number of follicles present were recorded and classified as primordial (oocyte surrounded by a single flat layer of follicle epithelial cells/pre-granulosa cells), primary (single layer of cuboidal granulosa cells), secondary (two or more layers of granulose cells, no antrum), or antral (presence of an antrum), similar to previously described methods (19, 30, 31). Morphologic evaluation of the follicles was based on examination of the integrity of the basement membrane, cellular density, presence or absence of pyknotic bodies, and integrity of the oocyte. Based on these criteria, follicles were classified as morphologically normal or abnormal.
Statistical analysis
The statistical analyzes were performed using the SPSS 27.0 software. Data are provided as median and interquartile ranges (IQR). The data of the FACS analyzes were analyzed using ANOVA for independent samples followed by Tukey’s HSD Test. Differences were considered statistically significant at p < 0.05.