Functional changes to Achilles tendon and enthesis in an adolescent mouse model of testosterone hormone therapy LeeAnn A. Hold, Tessa Phillips, Paige Cordts, Stephanie S. Steltzer, Seung-Ho Bae, et al. Connective Tissue Research, 2025 PURPOSE/AIM Some youth seek puberty suppression to prolong decision-making prior to starting hormone therapy to help align their physical sex characteristics with their gender identity. During peripubertal growth, connective tissues such as tendon rapidly adapt to applied mechanical loads (e.g. exercise) yet if and how tendon adaptation is influenced by sex and gender-affirming hormone therapy during growth remains unknown. The goal of this study was to understand how pubertal suppression followed by testosterone influences the structural and functional properties of the Achilles tendon using an established adolescent mouse model of testosterone hormone therapy. MATERIALS AND METHODS C57BL/6N female mice were assigned at postnatal day 26 to the following experimental groups: control (vehicle treated), gonadotropin release hormone analogue (GnRHa) treatment alone to delay puberty, testosterone (T) alone after puberty, or delayed puberty with T treatment (i.e. GnRHa followed by T). RESULTS We found that pubertal suppression using GnRHa with and without T, as well as treatment with T alone post-puberty, increased the ultimate load of tendon in female mice. Additionally, we found that GnRHa, but not T treatment resulted in a significant increase in cell density at the Achilles enthesis. CONCLUSIONS These findings demonstrate that delayed puberty and T have no negative influence on structural or functional properties of mouse tendon.
A Mouse Model to Investigate the Impact of Gender Affirming Hormone Therapy with Estradiol on Reproduction Daniel R. Pfau, Amanda R. Schwartz, Cynthia Dela Cruz, Vasantha Padmanabhan, Molly B. Moravek, et al. Advanced Biology, 2024 Gender‐affirming hormone therapy (GAHT) can help transgender and/or gender diverse (TGD) individuals achieve emobidment goals that align with their transition needs. Clinical evidence from estradiol (E)‐GAHT patients indicate widespread changes in tissues sensitive to E and testosterone (T), particularly in the reproductive system. Notably, E‐GAHTs effects on hormones and reproduction vary greatly between patients. With the goal of informing clinical research and practice for TGD individuals taking E, this study examines intact male mice implanted with capsules containing one of three different E doses (low 1.25 mg; mid 2.5 mg; high 5 mg), or a blank control capsule. All E‐GAHT doses suppress T and follicle stimulating hormone levels while elevating E levels. Only the high E‐GAHT dose significantly supresses luteinizing hormone levels. All E‐GAHT doses affect epididymis tubule size similarly while seminiferous tubule morphology and bladder weight changes are dose‐dependent. E‐GAHT does not alter the presence of mature sperm, though E‐exposed sperm have altered motility. These data represent the first evidence that mouse models offer an effective tool to understand E‐GAHTs impact on reproductive health and the dose‐dependent effects of this model permit examinations of diverse patient outcomes.
Translocator protein expression and localization in human endometrium and endometriosis: A potential target for a noninvasive diagnosis? Maíra Casalechi, Cynthia Dela Cruz, Wiviane A. Assis, Millene Vieira‐Lopes, Felipe Eduardo F. Lopes, et al. Cell Biology International, 2024 The limitations of current imaging methods to detect small or superficial endometriotic lesions prompt the search for new molecular targets. TSPO is an 18 KDa protein located in the outer mitochondrial membrane, which can be traced by positron emission tomography (PET) using specific ligands. TSPO is located mostly in neurons and inflammatory sites outside the brain. We hypothesized that it might also be expressed in the human endometrium and endometrial‐like tissue, being a target for molecular imaging of endometriosis. This prospective cross‐sectional study included 28 women with endometriosis and 11 endometriosis‐free controls. Endometriotic lesions (n = 49) and normal peritoneum (n = 13) from endometriosis patients were obtained during laparoscopy, while samples of eutopic endometrium from patients with endometriosis (n = 28) and from control women (n = 11) were collected in the operating room using a flexible device. TSPO mRNA expression was evaluated by quantitative reverse‐transcription real‐time PCR while protein expression was evaluated by immunohistochemistry with a monoclonal antibody antihuman TSPO. TSPO mRNA expression was detected in an invariable fashion in all tissue types evaluated; however, TSPO protein was found to be more abundant in the glandular epithelium than in the stroma, both in the endometrium and in the endometriotic lesions. Interestingly, hormone therapies did not alter the expression of TSPO, and its presence was mostly negative in tissues adjacent to endometriotic implants. As a proof of concept, the protein expression pattern of TSPO in endometriotic tissue and along the adjacent areas suggests that TSPO‐based molecular imaging might be used for noninvasive endometriosis detection.
Presence of ovarian stromal aberrations after cessation of testosterone therapy in a transgender mouse model Hadrian M Kinnear, Prianka H Hashim, Cynthia Dela Cruz, Faith L Chang, Gillian Rubenstein, et al. Biology of Reproduction, 2023 Some transmasculine individuals may be interested in pausing gender-affirming testosterone therapy and carrying a pregnancy. The ovarian impact of taking and pausing testosterone is not completely understood. The objective of this study was to utilize a mouse model mimicking transmasculine testosterone therapy to characterize the ovarian dynamics following testosterone cessation. We injected postpubertal 9–10-week-old female C57BL/6N mice once weekly with 0.9 mg of testosterone enanthate or a vehicle control for 6 weeks. All testosterone-treated mice stopped cycling and demonstrated persistent diestrus within 1 week of starting testosterone, while control mice cycled regularly. After 6 weeks of testosterone therapy, one group of testosterone-treated mice and age-matched vehicle-treated diestrus controls were sacrificed. Another group of testosterone-treated mice were maintained after stopping testosterone therapy and were sacrificed in diestrus four cycles after the resumption of cyclicity along with age-matched vehicle-treated controls. Ovarian histological analysis revealed stromal changes with clusters of large round cells in the post testosterone group as compared to both age-matched controls and mice at 6 weeks on testosterone. These clusters exhibited periodic acid–Schiff staining, which has been previously reported in multinucleated macrophages in aging mouse ovaries. Notably, many of these cells also demonstrated positive staining for macrophage markers CD68 and CD11b. Ovarian ribonucleic acid-sequencing found upregulation of immune pathways post testosterone as compared to age-matched controls and ovaries at 6 weeks on testosterone. Although functional significance remains unknown, further attention to the ovarian stroma may be relevant for transmasculine people interested in pausing testosterone to carry a pregnancy.
A mouse model mimicking gender-Affirming treatment with pubertal suppression followed by testosterone in transmasculine youth Cynthia Dela Cruz, Hadrian M Kinnear, Prianka H Hashim, Abigail Wandoff, Likitha Nimmagadda, et al. Human Reproduction, 2023 STUDY QUESTION Can mice serve as a translational model to examine the reproductive consequences of pubertal suppression with GnRH agonist (GnRHa) followed by testosterone (T) administration, a typical therapy in peripubertal transmasculine youth? SUMMARY ANSWER An implanted depot with 3.6 mg of GnRHa followed by T enanthate at 0.45 mg weekly can be used in peripubertal female mice for investigating the impact of gender-affirming hormone therapy in transmasculine youth. WHAT IS KNOWN ALREADY There is limited knowledge available in transgender medicine to provide evidence-based fertility care, with the current guidelines being based on the assumption of fertility loss. We recently successfully developed a mouse model to investigate the reproductive consequences of T therapy given to transgender men. On the other hand, to our knowledge, there is no mouse model to assess the reproductive outcomes in peripubertal transmasculine youth. STUDY DESIGN, SIZE, DURATION A total of 80 C57BL/6N female mice were used in this study, with n = 7 mice in each experimental group. PARTICIPANTS/MATERIALS, SETTING, METHODS We first assessed the effectiveness of GnRHa in arresting pubertal development in the female mice. In this experiment, 26-day-old female mice were subcutaneously implanted with a GnRHa (3.6 mg) depot. Controls underwent a sham surgery. Animals were euthanized at 3, 9, 21 and 28 days after the day of surgery. In the second experiment, we induced a transmasculine youth mouse model. C57BL/6N female mice were subcutaneously implanted with a 3.6 mg GnRHa depot on postnatal day 26 for 21 days and this was followed by weekly injections of 0.45 mg T enanthate for 6 weeks. The control for the GnRH treatment was sham surgery and the control for T treatment was sesame oil vehicle injections. Animals were sacrificed 0.5 weeks after the last injection. The data collected included the day of the vaginal opening and first estrus, daily vaginal cytology, weekly and terminal reproductive hormones levels, body/organ weights, ovarian follicular distribution and corpora lutea (CL) counts. MAIN RESULTS AND THE ROLE OF CHANCE GnRHa implanted animals remained in persistent diestrus and had reduced levels of FSH (P = 0.0013), LH (P = 0.0082) and estradiol (P = 0.0155), decreased uterine (P < 0.0001) and ovarian weights (P = 0.0002), and a lack of CL at 21 days after GnRHa implantation. T-only and GnRHa+T-treated animals were acyclic throughout the treatment period, had sustained elevated levels of T, suppressed LH levels (P < 0.0001), and an absence of CL compared to controls (P < 0.0001). Paired ovarian weights were reduced in the T-only and GnRHa+T groups compared with the control and GnRHa-only groups. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Although it is an appropriate tool to provide relevant findings, precaution is needed to extrapolate mouse model results to mirror human reproductive physiology. WIDER IMPLICATIONS OF THE FINDINGS To our knowledge, this study describes the first mouse model mimicking gender-affirming hormone therapy in peripubertal transmasculine youth. This model provides a tool for researchers studying the effects of GnRHa-T therapy on other aspects of reproduction, other organ systems and transgenerational effects. The model is supported by GnRHa suppressing puberty and maintaining acyclicity during T treatment, lower LH levels and absence of CL. The results also suggest GnRHa+T therapy in peripubertal female mice does not affect ovarian reserve, since the number of primordial follicles was not affected by treatment. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Michigan Institute for Clinical and Health Research grants KL2 TR 002241 and UL1 TR 002240 (C.D.C.); National Institutes of Health grants F30-HD100163 and T32-HD079342 (H.M.K.); University of Michigan Office of Research funding U058227 (A.S.); American Society for Reproductive Medicine/Society for Reproductive Endocrinology and Infertility grant (M.B.M.); and National Institutes of Health R01-HD098233 (M.B.M.). The University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core Facility was supported by the Eunice Kennedy Shriver NICHD/NIH grants P50-HD028934 and R24-HD102061. The authors declare that they have no competing interests.
Altered Endometrial Expression of a-Inhibin Subunit and Its Co-Receptor Betaglycan in Infertile Women with Endometriosis Cynthia Dela Cruz, Quesia T. Vilamil, Maíra Casalechi, Carolina P. Rezende, Wiviane A. Assis, et al. Gynecologic and Obstetric Investigation, 2022 <b><i>Background:</i></b> Inhibins and their co-receptor betaglycan are members of the transforming growth factor β superfamily, a group of signaling molecules that control the differentiation of human endometrium in the secretory phase of the menstrual cycle. <b><i>Objective:</i></b> Since endometriosis is associated with endometrial dysfunction and infertility, this study aimed at evaluating the expression of α-inhibin and betaglycan mRNA and proteins in endometrial samples of infertile women with and without endometriosis. <b><i>Design:</i></b> This was a cross-sectional study. <b><i>Participants/Materials:</i></b> Endometrial samples of women with (<i>n</i> = 17) and without (<i>n</i> = 22) endometriosis were subdivided according to the menstrual cycle phase into proliferative and secretory. <b><i>Setting:</i></b> University hospital. <b><i>Methods:</i></b> We used real-time RT-PCR to quantify mRNA levels and immunohistochemistry to localize the proteins. <b><i>Results:</i></b> α-inhibin mRNA levels were significantly increased in the secretory phase (<i>p</i> &#x3c; 0.01 vs. proliferative phase) only among women with endometriosis. Conversely, betaglycan mRNA levels were downregulated in the secretory endometrium of controls (<i>p</i> &#x3c; 0.01 vs. proliferative) but failed to change between cycle phases of patients with endometriosis. Both proteins were present in the glandular epithelium and stroma in the endometrium of women with and without endometriosis. Immunostaining analysis showed that while α-inhibin protein expression did not vary significantly, the intensity of betaglycan immunostaining decreased in the secretory phase in the control group (<i>p</i> = 0.038 vs. proliferative phase) but not in the endometriosis group. <b><i>Limitations:</i></b> We cannot determine whether endometriosis causes the abnormal expression of α-inhibin and betaglycan in the eutopic endometrium or if this alteration already existed before the establishment of endometriotic lesions. <b><i>Conclusion:</i></b> Our findings suggest an abnormally increased expression of α-inhibin mRNA (not protein) and betaglycan (mRNA and protein) in the secretory-phase endometrium of women with endometriosis.
