Clinical data point to adverse cardiovascular events elicited by testosterone replacement therapy. Testosterone is the main hormone used in gender-affirming hormone therapy (GAHT) by transmasculine people. However, the cardiovascular impact of testosterone in experimental models of GAHT remains unknown. Sex hormones modulate T-cell activation, and immune mechanisms contribute to cardiovascular risk. The present study evaluated whether testosterone negatively impacts female cardiovascular function by enhancing Th17 cell-linked effector mechanisms. Female (8 wk old) C57BL/6J mice received testosterone (48 mg/kg/wk) for 8 wk. Male mice were used for phenotypical comparisons. The hormone treatment in female mice increased circulating testosterone to levels observed in male mice. Testosterone increased lean body mass and body mass index, and decreased perigonadal fat mass, mimicking clinical findings. After 8 wk, testosterone decreased endothelium-dependent vasodilation and increased peripheral Th17 cells. After 24 wk, testosterone increased blood pressure in female mice. Ovariectomy did not intensify phenotypical or cardiovascular effects by testosterone. Female mice lacking T and B cells [Rag1 knockout (^−/−)], as well as female mice lacking IL-17 receptor (IL-17Ra^−/−), did not exhibit vascular dysfunction induced by testosterone. Testosterone impaired endothelium-dependent vasodilation in female mice lacking γδ T cells, similarly to the observed in wild-type female mice. Adoptive transfer of CD4⁺ T cells restored testosterone-induced vascular dysfunction in Rag1^−/− female mice. Together, these data suggest that CD4⁺ T cells, most likely Th17 cells, are central to vascular dysfunction induced by testosterone in female mice, indicating that changes in immune-cell balance are important in the GAHT in transmasculine people. NEW & NOTEWORTHY Sex hormone-induced cardiovascular events are important undesirable effects in transgender people under GAHT. Studies addressing the cardiovascular impact of GAHT will certainly contribute to improve healthcare services offered to this population. Our study showing that vascular dysfunction, via Th17 cell-related mechanisms, precedes increased blood pressure induced by testosterone in a GAHT mouse model, reveals potential mechanisms involved in GAHT-related cardiovascular events and may provide new markers/targets for clinical practices in transmasculine people.

Background: Fetal stage is a critical developmental window for the skeletal muscle, but little information is available about the impact of maternal vitamin D (Vit. D) deficiency (VDD) on offspring lean mass development in the adult life of male and female animals.Methods: Female rats (Wistar Hannover) were fed either a control (1000 IU Vit. D3/kg) or a VDD diet (0 IU Vit. D3/kg) for 6 weeks and during gestation and lactation. At weaning, male and female offspring were randomly separated and received a standard diet up to 180 days old.Results: Vitamin D deficiency induced muscle atrophy in the male (M‐VDD) offspring at the end of weaning, an effect that was reverted along the time. Following 180 days, fast‐twitch skeletal muscles [extensor digitorum longus (EDL)] from the M‐VDD showed a decrease (20%; P < 0.05) in the number of total fibres but an increase in the cross‐sectional area of IIB (17%; P < 0.05), IIA (19%; P < 0.05) and IIAX (21%; P < 0.05) fibres. The fibre hypertrophy was associated with the higher protein levels of MyoD (73%; P < 0.05) and myogenin (55% %; P < 0.05) and in the number of satellite cells (128.8 ± 14 vs. 91 ± 7.6 nuclei Pax7 + in the M‐CTRL; P < 0.05). M‐VDD increased time to fatigue during ex vivo contractions of EDL muscles and showed an increase in the phosphorylation levels of IGF‐1/insulin receptor and their downstream targets related to anabolic processes and myogenic activation, including Ser ⁴⁷³Akt and Ser ^21/9GSK‐3β. In such muscles, maternal VDD induced a compensatory increase in the content of calcitriol (two‐fold; P < 0.05) and CYP27B1 (58%; P < 0.05), a metabolizing enzyme that converts calcidiol to calcitriol. Interestingly, most morphological and biochemical changes found in EDL were not observed in slow‐twitch skeletal muscles (soleus) from the M‐VDD group as well as in both EDL and soleus muscles from the female offspring.Conclusions: These data show that maternal VDD selectively affects the development of type‐II muscle fibres in male offspring rats but not in female offspring rats and suggest that the enhancement of their size and fatigue resistance in fast‐twitch skeletal muscle (EDL) is probably due to a compensatory increase in the muscle content of Vit. D in the adult age.

Recent studies have shown that maternal vitamin D deficiency (VDD) causes long-term metabolic changes in offspring. However, little is known about the impact of maternal VDD on offspring endocrine pancreas development and insulin secretion in the adult life of male and female animals. Female rats (Wistar Hannover) were fed either control (1000 IU Vitamin D3/kg), VDD (0 IU Vitamin D3/kg), or a Ca²⁺-enriched VDD diet (0 IU Vitamin D3/kg + Ca²⁺ and P/kg) for 6 weeks and during gestation and lactation. At weaning, VDD status was confirmed based on low serum calcidiol levels in dams and pups. Next, male and female offspring were randomly separated and fed a standard diet for up to 90 days. At this age, serum calcidiol levels were restored to normal levels in all groups, but serum insulin levels were decreased in VDD males without affecting glucagon levels, glycemia, or glucose tolerance. Islets isolated from VDD males showed lower insulin secretion in response to different glucose concentrations, but this effect was not observed in VDD females. Furthermore, VDD males, but not females, showed a smaller total pancreatic islet area and lower β cell mass, an effect that was accompanied by reduced gene expression of Ins1, Ins2, Pdx1, and SLC2A2. The decrease in Pdx1 expression was not related to the methylation profile of the promoter region of this gene. Most of these effects were observed in the male VDD+Ca²⁺ group, indicating that the effects were not due to alterations in Ca²⁺ metabolism. These data show that maternal VDD selectively impairs the morphology and function of β cells in adult male offspring rats and that female offspring are fully protected from these deleterious effects.

Perinatal vitamin D (Vit. D) deficiency (VDD) disrupts the development of key tissues involved in metabolic regulation, including the endocrine pancreas, white adipose tissue, and skeletal muscle. Brown adipose tissue (BAT), essential for thermoregulation and energy homeostasis, may also be affected, but the impact of perinatal VDD on BAT physiology remains unclear. In this study, forty female Wistar rats were fed either a standard AIN93G diet (1000 IU Vit. D3/kg; control group, CT) (n = 20) or a modified AIN93G diet lacking Vit. D (VDD group) (n = 20) for six weeks prior to conception and throughout gestation and lactation. Male offspring were evaluated at weaning (PN21) and adulthood (PN180) after Vit. D status was normalized through a standard diet. We found that perinatal VDD reduced total lipid droplet area, increased oxygen consumption, and upregulated thermogenic gene expression in BAT at weaning. Correspondingly, VDD offspring exhibited greater cold tolerance and enhanced BAT recruitment upon cold exposure (4 °C). Notably, normalization of Vit. D status by adulthood fully reversed these changes, indicating that while perinatal VDD transiently enhances BAT thermogenic activity during early life, it does not produce lasting effects into adulthood.