Activin A in Mammalian Physiology.

Activins are dimeric glycoproteins belonging to the transforming growth factor beta superfamily and resulting from the assembly of two beta subunits, which may also be combined with alpha subunits to form inhibins. Activins were discovered in 1986 following the isolation of inhibins from porcine follicular fluid, and were characterized as ovarian hormones that stimulate follicle stimulating hormone (FSH) release by the pituitary gland. In particular, activin A was shown to be the isoform of greater physiological importance in humans. The current understanding of activin A surpasses the reproductive system and allows its classification as a hormone, a growth factor, and a cytokine. In more than 30 yr of intense research, activin A was localized in female and male reproductive organs but also in other organs and systems as diverse as the brain, liver, lung, bone, and gut. Moreover, its roles include embryonic differentiation, trophoblast invasion of the uterine wall in early pregnancy, and fetal/neonate brain protection in hypoxic conditions. It is now recognized that activin A overexpression may be either cytostatic or mitogenic, depending on the cell type, with important implications for tumor biology. Activin A also regulates bone formation and regeneration, enhances joint inflammation in rheumatoid arthritis, and triggers pathogenic mechanisms in the respiratory system. In this 30-yr review, we analyze the evidence for physiological roles of activin A and the potential use of activin agonists and antagonists as therapeutic agents.

Translocator protein expression and localization in human endometrium and endometriosis: A potential target for a noninvasive diagnosis?

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.

Addition of synthetic polymers to a conventional cryoprotectant solution in the vitrification of bovine ovarian tissue.

Some synthetic polymers can be used at low concentrations to reduce the toxicity of conventional cryoprotectant agents. In this study we investigated whether the addition of synthetic polymers to a conventional cryoprotectant solution would improve the cryopreservation of bovine ovarian tissue. Freshly collected ovaries from ten adult crossbred cows were incised using a scalpel in the frontal section. From each cow, ovarian cortical slices of 1 mm thickness were divided into 30 fragments of 3 × 3 mm, of which 10 served as fresh controls, 10 were vitrified with conventional cryoprotectant agents (2.93 M glycerol, 27 % w/v; 4.35 M ethylene glycol, 27 % w/v), and 10 were vitrified using the same cryoprotectant agents in addition to synthetic polymers (0.2 % PVP K-12, 0.2 % SuperCool X-1000 ™ w/v and 0.4 % SuperCool Z-1000 ™ w/v). After warming, histology was used to assess follicular quantity and integrity, while in vitro culture of mechanically isolated follicles encapsulated in an alginate matrix was performed for 15 days to assess their growth and hormonal production. Vitrified ovarian tissues presented abnormal morphology, a higher percentage of atretic follicles, and their isolated follicles had lower survival rates and lower frequency of antrum formation during in vitro culture compared to those from fresh tissue. At the end of culture, the follicles that had been cryopreserved produced less estradiol and progesterone than the fresh ones. The addition of synthetic polymers during tissue vitrification did not modify any of these parameters. We conclude that, under the conditions of this study, the use of this combination of synthetic polymers for tissue vitrification did not enhance the preservation of the morphological or functional integrity of bovine ovarian follicles.

Presence of ovarian stromal aberrations after cessation of testosterone therapy in a transgender mouse model†.

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.

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.

Impaired in vitro fertilization outcomes following testosterone treatment improve with washout in a mouse model of gender-affirming hormone treatment.

BACKGROUND: The impact of gender-affirming testosterone on fertility is poorly understood, with ovarian histopathologic studies showing variable results, some with a detrimental effect on reproductive capacity and uncertain reversibility. Assisted reproductive outcome data are restricted to small case series that lack the ability to inform clinical practice guidelines and limit fertility preservation counseling for transgender and nonbinary individuals. OBJECTIVE: This study aimed to determine the impact of current testosterone and testosterone washout on in vitro fertilization outcomes in a mouse model for gender-affirming hormone treatment. We hypothesized that current or previous testosterone treatment would not affect in vitro fertilization outcomes. STUDY DESIGN: C57BL/6N female mice (n=120) were assigned to 4 treatment groups: (1) current control, (2) current testosterone, (3) control washout, and (4) testosterone washout. Testosterone implants remained in situ for 6 or 12 weeks, representing the short- and long-term treatment arms, respectively. Current treatment groups underwent ovarian stimulation with implants in place, and washout treatment groups were explanted and had ovarian stimulation after 2 weeks. Oocytes were collected, fertilized, and cultured in vitro, with one arm continuing to the blastocyst stage and the other having transfer of cleavage-stage embryos. Statistical analysis was performed using GraphPad Prism, version 9.0 and R statistical software, version 4.1.2, with statistical significance defined by P<.05. RESULTS: Current long-term testosterone treatment impaired in vitro fertilization outcomes, with fewer mature oocytes retrieved (13.7±5.1 [standard deviation] vs 28.6±7.8 [standard deviation]; P<.0001) leading to fewer cleavage-stage embryos (12.1±5.1 vs 26.5±8.2; P<.0001) and blastocysts (10.0±3.2 vs 25.0±6.5; P<.0001). There was recovery of in vitro fertilization outcomes following washout in the short-term treatment cohort, with incomplete reversibility in the long-term cohort. Testosterone did not negatively affect maturity, fertilization, or blastulation rates. CONCLUSION: In a mouse model of gender-affirming hormone treatment, testosterone negatively affected oocyte yield without affecting oocyte quality. Our findings suggest that testosterone reversibility is duration-dependent. These results demonstrate the feasibility of in vitro fertilization without testosterone discontinuation while supporting a washout period for optimization of mature oocyte yield.

Pharmacokinetic comparison of three delivery systems for subcutaneous testosterone administration in female mice.

Transmasculine individuals are often prescribed testosterone (T) for masculinizing hormone therapy. Mouse models mimicking transmasculine T therapy require reliable long-term T administration. The objectives of this study were three-fold, namely, to compare: 1) the release dynamics of three different subcutaneous delivery systems of T enanthate administration (subcutaneous injections, commercially available pellets, and silastic implants) over a 6-week period in postpubertal C57BL/6N mice, 2) to compare the timing for T levels in plasma to return to baseline and cyclicity to resume after cessation of T between injections and pellets, 3) to utilize silastic implants to achieve sustainable increase in T levels utilizing T enanthate and crystalline T. All three modes of T administration resulted in an increase in T levels in plasma. Pharmacokinetic analyses showed a similar overall exposure to T enanthate over 6 weeks (integrated area) for, subcutaneous injection (0.45 mg two times per week and 0.90 mg one time per week), pellet (5 mg 60-day release), and silastic implant (5 mg 21 week) groups. Crystalline T had lower solubility and a decreased integrated area compared to T enanthate, even when implanted at a higher dosage, indicating different pharmacokinetic profiles based on type of T formulation when utilizing the same silastic delivery method. Surgical removal of pellets and silastic tubing led to a quick drop in T levels and resumption of estrous cyclicity, while cessation of injections required a long washout period for T levels to drop and estrous cycles to resume. Sustained elevation in T levels was achieved for at least 21 weeks with silastic implants. As all three delivery methods are able to elevate T levels in female mice for at least 6 weeks, choice of T administration method should be based on outcomes of interest and study design.

Altered Endometrial Expression of α-Inhibin Subunit and Its Co-Receptor Betaglycan in Infertile Women with Endometriosis.

BACKGROUND: Inhibins and their co-receptor betaglycan are members of the transforming growth factor ß superfamily, a group of signaling molecules that control the differentiation of human endometrium in the secretory phase of the menstrual cycle. OBJECTIVE: 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. DESIGN: This was a cross-sectional study. Participants/Materials: Endometrial samples of women with (n = 17) and without (n = 22) endometriosis were subdivided according to the menstrual cycle phase into proliferative and secretory. SETTING: University hospital. METHODS: We used real-time RT-PCR to quantify mRNA levels and immunohistochemistry to localize the proteins. RESULTS: α-inhibin mRNA levels were significantly increased in the secretory phase (p < 0.01 vs. proliferative phase) only among women with endometriosis. Conversely, betaglycan mRNA levels were downregulated in the secretory endometrium of controls (p < 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 (p = 0.038 vs. proliferative phase) but not in the endometriosis group. LIMITATIONS: 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. CONCLUSION: 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.

Immunolocalization of stem/progenitor cell biomarkers Oct-4, C-kit and Musashi-1 in endometriotic lesions.

BACKGROUND: Human endometrium harbors stem/progenitor cells (SPCs) that may contribute to the establishment of endometriosis when seeded outside the uterus. Oct-4, C-kit and Musashi-1 are some of the many proteins used to characterize SPCs, but their association with endometriosis is uncertain. OBJECTIVE AND DESIGN: In this study, specimens of normal endometrium (n = 12), eutopic endometrium from women with endometriosis (n = 9), superficial peritoneal endometriosis (SUP, n = 12) and deep endometriosis (DE, n = 13) lesions were evaluated for localization and intensity of immunostaining for Oct-4, C-kit and Musashi-1. RESULTS: The three markers were abundantly expressed in normal endometrium, eutopic endometrium from endometriosis patients, SUP and DE specimens. Oct-4 and C-kit expression did not vary across groups as regards intensity or frequency. C-kit staining signal was seldom detected in vascular endothelium of normal or eutopic endometrium from endometriosis patients; however, it was positive in 67% of the SUP lesions and in 25% of the DE lesions (p = 0.042). Musashi-1 was expressed in some endometriotic glands as cell clusters, but its signal was similar between the four types of tissue (p = 0.971) CONCLUSION: The wide distribution of Oct-4, C-kit and Musashi-1 in endometria of patients with and without endometriosis and in SUP and DE endometriotic lesions suggests that these markers are not suitable for the in situ characterization of endometrial SPCs and should not be taken as surrogates for the study of SPCs in the pathogenesis of endometriosis.

ADVERSE EVENTS OF THE ARGUS II RETINAL PROSTHESIS: Incidence, Causes, and Best Practices for Managing and Preventing Conjunctival Erosion.

PURPOSE: To analyze and provide an overview of the incidence, management, and prevention of conjunctival erosion in Argus II clinical trial subjects and postapproval patients. METHODS: This retrospective analysis followed the results of 274 patients treated with the Argus II Retinal Prosthesis System between June 2007 and November 2017, including 30 subjects from the US and European clinical trials, and 244 patients in the postapproval phase. Results were gathered for incidence of a serious adverse event, incidence of conjunctival erosion, occurrence sites, rates of erosion, and erosion timing. RESULTS: Overall, 60% of subjects in the clinical trial subjects versus 83% of patients in the postapproval phase did not experience device- or surgery-related serious adverse events. In the postapproval phase, conjunctival erosion had an incidence rate of 6.2% over 5 years and 11 months. In 55% of conjunctival erosion cases, erosion occurred in the inferotemporal quadrant, 25% in the superotemporal quadrant, and 20% in both. Sixty percent of the erosion events occurred in the first 15 months after implantation, and 85% within the first 2.5 years. CONCLUSION: Reducing occurrence of conjunctival erosion in patients with the Argus II Retinal Prosthesis requires identification and minimization of risk factors before and during implantation. Implementing inverted sutures at the implant tabs, use of graft material at these locations as well as Mersilene rather than nylon sutures, and accurate Tenon's and conjunctiva closure are recommended for consideration in all patients.