(O-06) Virilizing Testosterone Decreases Pre-antral Follicle Size and Oocyte Mitochondrial Volume in a Transgender Male Swine Model

Abstract Authors: Annie E. Newell-Fugate, Nirvana Mahabir, Maddie Nguyen, Angela Pinon, Irshad Ahmad
Affiliation for all authors: College Station, Texas, USA

Abstract Text: There are roughly 1.6 million transgender Americans, 80% of whom are on gender-affirming hormone therapy. Although transgender men (femalemale) are counseled to undergo fertility preservation prior to initiation of testosterone hormone therapy, a large percentage of transgender youth do not undergo fertility preservation prior to beginning treatment. There are conflicting data in the human literature on the impact of virilizing testosterone on ovarian antral follicle size and number, and no information exists on the effect of virilizing testosterone on pre-antral follicle classes or oocyte mitochondria. The objective of this pilot study was to assess the impact of virilizing testosterone on ovarian follicle size and number and on oocyte mitochondrial volume and number in a transgender male swine model. Three sexually mature female pigs were housed individually, were fed 1.5 kg of feed daily, and were heat checked daily. Testosterone enanthate (TE) was administered via silastic implants as follows: placebo (0 g TE, n=1), transgender male (2.24 g TE, n=2). Fasting blood samples for measurement of androgens and estrogens by LC-MS/MS were collected on days 1-3 and then every three days post-implantation for 42 days. Ovaries were collected and fixed in 4% paraformaldehyde, and H&E histology was performed for follicle counts, whereas transmission electron microscopy was performed for oocyte mitochondrial assessment. An individual blinded to treatment assessed follicle and oocyte mitochondrial counts and size in Qupath software. Follicles were classified as primordial, primary, secondary, tertiary, or tertiary-atretic. Each pig had three separate images analyzed for follicles and oocyte mitochondria, respectively. Serum hormone measurements were analyzed with repeated measures in time ANOVA. The non-parametric Mann-Whitney test was used to analyze ovarian and oocyte morphology and count measurements. The level of significance was set at 0.05 for all statistical tests. Serum testosterone levels consistent with those of transgender men were achieved with the 2.24 g TE dose (transgender male pigs). Transgender male pigs remained in anestrus during the study. Serum estrogens, androstenedione, and dihydrotestosterone were not different between placebo female and transgender male pigs. Serum dehydroepiandrosterone (DHEA; a weak androgen) was lower in transgender male pigs compared to the placebo female pig. Transgender male pigs had greater numbers of primordial, antral, and atretic antral follicles than the placebo female pig. However, with respect to follicle size, transgender male pigs had smaller primary and secondary follicles than the placebo female pig. Ongoing histology image analysis is focused on granulosa and theca cell size in primary and secondary follicles and the number of granulosa cell layers in secondary follicles. Although the number of oocyte mitochondria did not differ between treatment groups, the oocyte mitochondrial volume was decreased for transgender male pigs as compared to the placebo female pig. These results suggest that virilizing testosterone adversely affects aspects of ovarian folliculogenesis and markers of oocyte health that may negatively impact the fertility of transgender men wishing to conceive. Future research will focus on replicating these findings in a larger cohort of pigs and exploring whether testosterone withdrawal reverses these effects.