An experimental in vitro study was performed. The experiments were carried out in the Endocrine and Tumoral Molecular Biology Laboratories, installed in the Department of Physiology at UFRGS and in Experimental Pathology, at the Experimental Research Center of Hospital de Clínicas de Porto Alegre (HCPA). This project was supported by Fundo de Incentivo à Pesquisa e Eventos - HCPA (FIPE-HCPA #2018-0462).
Sample of ovarian tissue
The samples were collected from the ovarian cortex of patients from the Gender Identity Program (Programa de Identidade de Gênero - PROTIG), Hospital de Clínicas de Porto Alegre (HCPA), who underwent sexual reassignment surgery, with indication independent of this study. The criteria for inclusion in the study were patients who underwent pan-hysterectomy, aged between 20 and 45 years old and who did not present ovarian neoplasia. The sample size was determined by convenience.
After obtaining fragments of the ovarian cortex, the specimens were sent for anatomopathological analysis. The samples were identified and transported refrigerated to the laboratory in a medium composed of Hank's salt solution (Gibco BRL Grand Island, N.Y, USA) and 1% kanamycin. In a laminar flow hood, excess blood was removed, the samples were cut into fragments of 2, 3 and 4 mm of diameter with a disposable biopsy punch (Kolplast, Brazil). After cutting, the samples were plated.
In vitro ovarian fragments culture
The ovarian cortex fragments were placed in a 96-well plate. Each well was filled with 200μL of DMEM medium (Gibco BRL Grand Island, NY, USA) supplemented with 1% antibiotic (streptomycin), 25 mIU/mL recombinant FSH (GONAL-f®) and 5% fetal bovine serum (SBF - Gibco BRL Grand Island, NY, USA). The tissue fragments were grown at 37°C in a humidified incubator, with constant injection of 5% CO2 for 2, 4, 6 and 8 days. The culture medium was changed every 48 hours and the fragments were observed everyday under an inverted microscope. After 2, 4, 6 or 8 days of culture, the fragments were fixed in 10% buffered formaldehyde.
Slide preparation and histological analysis
The formaldehyde fixed samples were sent to HCPA Experimental Pathology Unit where they were embedded in paraffin and cut into 5 µm series sections for histological analysis. The slides were stained with hematoxylin/eosin.
To limit the effect of heterogeneous follicular distribution within the samples of the ovarian cortex submitted to culture, random sections were performed per piece of ovary, with the purpose of covering the entire fragment. The slides were evaluated under an optical microscope by an experienced pathologist.
A primordial follicle was defined as the presence of an oocyte surrounded by a layer of spindle-shaped granulosa cells and a primary follicle is an oocyte surrounded by cuboidal granulosa cells. Secondary follicles are characterized by an oocyte that is completely surrounded by a pellucid zone and the presence of at least two layers of granulosa cells. Antral follicles are defined by the presence of an antral cavity .
The data were entered twice, revised and analyzed using the SPSS program, version 18.0 [SPSS Inc. Launched in 2009. PASW Statistics for Windows, version 18.0. Chicago: SPSS Inc.]. Qualitative variables were described as absolute (n) and relative (n%) frequencies. Fisher's exact test was applied and Yates’s correction for continuity was used when indicated. Quantitative variables were expressed as medians, as distributed by the Shapiro Wilk normality test; so, the Kruskal-Wallis test and Dunn-Bonferroni post hoc test were applied. For all analyzes, the significance level was set at 5%.
Ethical aspects and biosafety
An informed consent form (ICF) was applied to authorize the use of ovarian fragments in the present study. Patients were informed about the research and invited to participate by signing the ICF, knowing that they could withdraw at any time.
After the experiments, the residues were packed in white bags, closed, sealed and identified with a biological residue label with all the required information and delivered to the competent collection service of the institution. Phenol residues were treated as chemical waste and collected by the collection service based at the Chemistry Institute of UFRGS.