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.

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.

Angiotensin-(1-7) in human follicular fluid correlates with oocyte maturation.

STUDY QUESTION: Do angiotensin (Ang)-(1-7) levels in human ovarian follicular fluid (FF) correlate with the number and proportion of mature oocytes obtained for IVF? SUMMARY ANSWER: The present study shows for the first time that Ang-(1-7) levels in human FF correlate with the proportion of mature oocytes collected upon ovarian stimulation for IVF. WHAT IS KNOWN ALREADY: Ang-(1-7) is an active peptide of the renin-angiotensin system that stimulates oocyte maturation in isolated rabbit and rat ovaries. However, its role in human ovulation remains unexplored. STUDY DESIGN, SIZE, DURATION: This was a prospective cohort study including 64 participants from a single IVF center. Sample size was calculated to achieve a statistical power of 80% in detecting 20% differences in the proportion of mature oocytes between groups. The participants were enrolled in the study during six consecutive months. PARTICIPANTS/MATERIALS, SETTING, METHODS: Plasma samples were obtained from all subjects at Day 21 of the last menstrual cycle before starting pituitary blockade and controlled ovarian stimulation (COS). Plasma and FF samples were quickly mixed with a protease inhibitor cocktail and stored at -80°C. Ang-(1-7) was quantified in plasma and FF samples by a highly sensitive and specific radioimmunoassay, which was preceded by solid phase extraction, speed vacuum concentration and sample reconstitution in assay buffer. FF Ang-(1-7) levels were stratified into tertiles and the patients of each tertile were compared for COS/IVF outcomes using Kruskal-Wallis ANOVA. Multiple regression analysis was used to adjust correlations for potential confounders. The mRNA encoding for Mas, a receptor for Ang-(1-7), was investigated by real-time PCR in luteinized granulosa cells purified from the FF. MAIN RESULTS AND THE ROLE OF CHANCE: There was a four-fold increase in plasma Ang-(1-7) after ovulation induction (median 160.9 vs 41.4 pg/ml, P < 0.0001). FF Ang-(1-7) levels were similar to (169.9 pg/ml) but did not correlate with plasma Ang-(1-7) levels (r = -0.05, P = 0.665). Patients at the highest FF Ang-(1-7) tertile had a higher proportion of mature oocytes compared to patients at the lower FF Ang-(1-7) tertile (median 100% vs 70%, P < 0.01). There was a linear correlation between FF Ang-(1-7) and the proportion of mature oocytes (r = 0.380, P < 0.01), which remained significant after adjustment for age and duration of infertility (r = 0.447, P < 0.001). The luteinized granulosa cells expressed Mas receptor mRNA, which was positively correlated to the number of mature oocytes in women with more than three mature oocytes retrieved (r = 0.42, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: This is an observational study, therefore, no causal relationship can be established between Ang-(1-7) and human oocyte maturation. Mas protein expression was not quantified due to limited availability of granulosa cells. WIDER IMPLICATIONS OF THE FINDINGS: Since this peptide promotes oocyte maturation in other species, it deserves further investigation as a potential maturation factor to human oocytes. STUDY FUNDING AND COMPETING INTEREST(S): Research supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). The authors have nothing to disclose.

Urocortin and corticotrophin-releasing hormone receptor type 2 mRNA are highly expressed in deep infiltrating endometriotic lesions.

Ovarian endometrioma (OMA) and deep infiltrating endometriosis (DIE) are the most severe forms of endometriosis, but different pathogenetic mechanisms and clinical symptoms distinguish these two forms. Corticotrophin-releasing hormone (CRH) and urocortin (Ucn) are endometrial neuropeptides involved in tissue differentiation and inflammation. The expression of CRH, Ucn, Ucn2, CRH-receptors (type-1 and type-2) and inflammatory enzymes phospholipase-A2 group IIA (PLA2G2A) and cycloxygenase-2 (COX2) were evaluated in OMA (n = 22) and DIE (n = 26). The effect of CRH or Ucn on COX2 mRNA expression was evaluated in cultured human endometrial stromal cells. In DIE lesions, CRH, Ucn and CRH-R2 mRNA levels were significantly higher than in OMA (P < 0.01, P < 0.001 and P < 0.05, respectively); DIE lesions showed a higher expression of COX2 (P < 0.01) and PLA2G2A (P < 0.05) mRNA than OMA, which was positively correlated with CRH-R2 mRNA expression (P < 0.05). Intense immunostaining for CRH and Ucn was shown in DIE. Treatment of cultured endometrial stromal cells with Ucn significantly increased COX2 mRNA expression (P < 0.01); this effect was reversed by the CRH-R2 antagonist astressin-2B. In DIE, DIE lesions highly express neuropeptide and enzyme mRNAs, supporting a strong activation of inflammatory pathways.

Apoptosis modulation by activin A and follistatin in human endometrial stromal cells.

Activin A is a growth factor that stimulates decidualization and is abundantly expressed in endometrial proliferative disorders. Nevertheless, whether it directly affects endometrial cell survival is still unknown. This study investigated the effects of activin A on total death and apoptosis rates and on tumor necrosis factor (TNF) release by human endometrial stromal cells (HESC). We performed a controlled prospective in vitro study using primary HESC cultures obtained from healthy reproductive age women (n = 11). Cells were treated with medium alone (control) or activin A (25 ng/mL) or activin A (25 ng/mL) and its antagonist follistatin (250 ng/mL). Apoptosis and total cell death were measured by flow cytometry, while TNF concentrations in culture media were quantified by ELISA. Activin A decreased the percentage of apoptotic/dead cells from 31% to 22% (p < 0.05, paired t-test) and reduced TNF levels in culture medium by 14%, but there was no linear correlation between TNF release and apoptotic rates. Both effects of activin A were reversed by follistatin. These findings indicate that activin A promotes HESC survival, possibly by a TNF-independent pathway. This mechanism may be critical to the actions of activin A upon stromal cell growth and differentiation in physiology and disease.

Expression, localization and control of activin A release from human umbilical vein endothelial cells.

Activin-A is a member of the TGFß superfamily found in maternal and umbilical cord blood throughout gestation. We investigated whether human umbilical vein endothelial cells (HUVEC) express activin-A in vivo and tested the effects of vasoactive (endothelin-1), pro-inflammatory (interferon-γ, interleukin-8) and anti-inflammatory (dexamethasone, urocortin) factors on activin-A release by isolated HUVEC in vitro. Activin ßA subunit protein and mRNA were strongly localized in the endothelial cells of umbilical veins and were also detectable in scattered cells of the cord connective tissue. Dimeric activin-A was detected in the HUVEC culture medium at picomolar concentrations. Activin-A release by HUVEC decreased after cell incubation with urocortin (p < 0.01), whereas no effect was observed with interleukin-8, interferon-γ, endothelin-1 or dexamethasone. In summary, activin-A is present in the human umbilical vein endothelium in vivo and is produced and released by isolated HUVEC. Activin-A secretion is inhibited in vitro by urocortin, a neuropeptide with predominantly anti-inflammatory action.

The role of TGFß superfamily members in the pathophysiology of endometriosis.

The transforming growth factor-beta (TGFß) superfamily comprises over 30 dimeric proteins with conserved structures, which play important roles in the control of cellular proliferation, differentiation and apoptosis. These proteins are expressed and finely regulated in human endometrium during the menstrual cycle, which is consistent with their effects on endometrial cell proliferation and tissue remodeling. This review is focused on summarizing the role of key members of the TGFß superfamily in the pathophysiology of endometriosis. Evidence suggests that TGFß, activins, inhibins, nodal, bone morphogenetic proteins, growth differentiation factors, and anti-Müllerian hormone are produced by endometriotic lesions and could be involved in the establishment and progression of the disease. Their receptors and signaling pathways may also be altered in the presence of endometriosis and may be potential targets to the development of therapeutic agents.

Serum antimüllerian hormone measurements with second generation assay at two distinct menstrual cycle phases for prediction of cycle cancellation, pregnancy and live birth after in vitro fertilization.

PURPOSE: This study investigated the usefulness of serum antimüllerian hormone (AMH) measurements at two distinct menstrual cycle phases to predict in vitro fertilization (IVF) outcomes. METHODS: This was a prospective observational study enrolling 135 consecutive patients referred for conventional IVF or ICSI in a university hospital. Blood samples were obtained for serum AMH measurements on days 3 and 18-20, while transvaginal ultrasound was performed for antral follicle count (AFC) at day 3 of the menstrual cycle immediately before treatment. AMH was measured with the new Beckman Coulter Generation II (GenII) assay. The main outcome measures were cycle cancellation due to poor ovarian response, clinical pregnancy, and live birth. RESULTS: There was a strong correlation between AMH levels measured at day 3 and day 18-20 of the menstrual cycle (r = 0.837; P < 0.0001). Day 18-20 serum AMH was comparable to day 3 serum AMH and AFC for the prediction of cycle cancellation (areas under the ROC curve were 0.84 for day 3 AMH, 0.89 for day 18-20 AMH, and 0.80 for AFC). Day 18-20 AMH had a modest predictive value for pregnancy or live birth (area under ROC curve 0.71 for both), which was comparable to that of day 3 AMH; however, AFC had no predictive value for these outcomes. CONCLUSIONS: Day 18-20 AMH was comparable to day 3 AMH for the prediction of cycle cancellation, clinical pregnancy, and live birth after IVF. Both AMH measurements were accurate for the prediction of cancellation but were significantly less useful for the prediction of pregnancy or live birth.

A Mouse Model to Investigate the Impact of Gender Affirming Hormone Therapy with Estradiol on Reproduction.

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.

In vitro fertilization outcomes in a mouse model of gender-affirming hormone therapy in transmasculine youth.

OBJECTIVE: To investigate in vitro fertilization (IVF) outcomes in an adolescent transmasculine mouse model mimicking gender-affirming hormone therapy in prepubertal youth, both on testosterone (T) and after T washout. DESIGN: Experimental laboratory study using a validated mouse model. SETTING: University-based basic science research laboratory. ANIMAL(S): A total of 80 prepubertal 26-day-old C57BL/6N female mice were used in this study. INTERVENTION(S): Animals (n = 10/group) were implanted subcutaneously with gonadotropin-releasing hormone agonist at 3.6 mg or received sham surgery. After 21 days, they were implanted with silastic tubing containing either T 10 mg or placebo for 6 weeks. After 6 weeks, a group of animals were superovulated for immediate IVF, and another group had the implant removed and went through superovulation for IVF after 2 weeks (washout IVF). The total number of oocytes yielded, oocyte maturity rate, fertilization rate, and numbers of 2-cell embryos, 4-8-cell embryos, morula, blastocysts, and hatching blastocysts were recorded. RESULT(S): Testosterone treatment negatively impacted IVF outcomes in animals stimulated when receiving T, but not after T washout. Pretreatment with gonadotropin-releasing hormone agonist did not affect IVF outcomes. CONCLUSION(S): Although current T had a negative impact on IVF outcomes compared with controls, animals were still able to produce viable oocytes for fertilization and develop into blastocysts. Future efforts to study the impact of long-term T exposure on oocyte quality, especially aneuploidy rates, pregnancy outcomes, and live birth rates, are necessary.

Reversibility of testosterone-induced acyclicity after testosterone cessation in a transgender mouse model.

OBJECTIVE: To establish if the cessation of testosterone (T) therapy reverses T-induced acyclicity in a transgender mouse model that allows for well-defined T cessation timing. DESIGN: Experimental laboratory study using a mouse model. SETTING: University-based basic science research laboratory. ANIMALS: A total of 10 C57BL/6NHsd female mice were used in this study. INTERVENTION(S): Postpubertal C57BL/6NHsd female mice were subcutaneously implanted with T enanthate (n = 5 mice) or placebo (n = 5 mice) pellets. Pellets were surgically removed after 6 weeks to ensure T cessation, after which the mice were followed for four estrous cycles after the resumption of cyclicity. MAIN OUTCOME MEASURE(S): Primary outcomes included daily vaginal cytology and weekly T levels before, during, and after T enanthate or placebo pellet implantation and removal. Secondary outcomes included ovarian follicle distribution and corpora lutea numbers, body metrics, and terminal diestrus hormone levels. RESULT(S): T-treated mice (100%) resumed cycling within one week of T pellet removal after six weeks of T therapy. T levels were significantly elevated during T therapy and decreased to control levels after surgical pellet removal. No detectable differences were observed in the follicle count, corpora lutea formation, diestrus hormone levels, or body metrics after four estrous cycles, with the exception of persistent increased clitoral area between T-treated mice and controls. One T-treated mouse was sacrificed early due to vaginal prolapse and not included in subsequent analyses. CONCLUSION(S): Our results demonstrated a close temporal relationship between estrous cycle return and T levels dropping to control levels following T pellet removal. The return of regular cyclic ovulatory function is also supported by the formation of corpora lutea and the lack of detectable differences in key reproductive parameters as compared to controls four cycles after T cessation. These results may be relevant to understanding the reversibility of T-induced amenorrhea and possible anovulation in transgender men interested in pausing T to pursue pregnancy or oocyte donation. Results may be limited by the duration of T treatment, lack of functional testing, and physiological differences between mice and humans.

Seminal protein levels assessed with dipstick test correlate with sperm recovery after cryopreservation.

The process of freezing/thawing causes structural and functional damage to sperm samples, which can be mitigated by seminal plasma proteins. This study investigated the proposition that seminal protein measurements using a urinary dipstick prior to freezing could help predict the post-thaw recovery of live spermatozoa. This was a prospective study including 149 men undergoing semen analysis due to male and/or female infertility. The seminal samples were analysed according to World Health Organisation standards and protein concentrations were measured using a commercially available urinary dipstick following quantitative validation. The median live sperm recovery rates were 79%, 81% and 94%, respectively, in samples with protein concentrations of ≤1.0 g/L (+/++), 3.0 g/L (+++) and ≥20.0 g/L (++++) measured in fresh specimen dipstick analysis (p < 0.05) indicating that the probability of recovering at least 50% of frozen spermatozoa increased progressively with higher protein concentrations in the fresh sample (chi-square for linear association = 7.17. p = 0.007). In conclusion, fresh seminal protein concentration levels assessed with a dipstick test correlate with the proportion of live spermatozoa recovered from cryopreserved samples. This simple, low-cost test may add prognostic information to baseline semen analysis prior to sperm banking.

Conditional Fgfr1 Deletion in GnRH Neurons Leads to Minor Disruptions in the Reproductive Axis of Male and Female Mice.

In humans and mice, inactivating mutations in fibroblast growth factor receptor 1 (Fgfr1) lead to gonadotropin-releasing hormone (GnRH) deficiency and a host of downstream reproductive disorders. It was unclear if Fgfr1 signaling directly upon GnRH neurons critically drove the establishment of a functional GnRH system. To answer this question, we generated a mouse model with a conditional deletion of Fgfr1 in GnRH neurons using the Cre/loxP approach. These mice, called Fgfr1cKO mice, were examined along with control mice for their pubertal onset and a host of reproductive axis functions. Our results showed that Fgfr1cKO mice harbored no detectable defects in the GnRH system and pubertal onset, suffered only subtle changes in the pituitary function, but exhibited significantly disrupted testicular and ovarian morphology at 25 days of age, indicating impaired gametogenesis at a young age. However, these disruptions were transient and became undetectable in older mice. Our results suggest that Fgfr1 signaling directly on GnRH neurons supports, to some extent, the reproductive axis function in the period leading to the early phase of puberty, but is not critically required for pubertal onset or reproductive maintenance in sexually mature animals.

Caspase-3 gene expression in human luteinized granulosa cells is inversely correlated with the number of oocytes retrieved after controlled ovarian stimulation.

Granulosa cells control oocyte maturation through paracrine signalling and changes to the microenvironment around the oocyte. Apoptosis occurs as a physiological mechanism of granulosa cell renewal, but how it relates with the ovarian response to induced ovulation is still unclear. Therefore, this study evaluated apoptosis-related gene expression levels in granulosa cells of patients undergoing controlled ovarian stimulation. We enrolled prospectively 59 consecutive IVF patients referred to a tertiary academic hospital for couple infertility treatment. Luteinized granulosa cells were isolated from follicular fluid and the RNA was extracted, reverse-transcribed and the gene expression of apoptosis inducers (caspase-3, caspase-8 and bax) and inhibitor (Bcl-2) was quantified by real-time polymerase chain reaction. Caspase-3 gene expression correlated negatively with the number of pre-ovulatory follicles (Spearman's r = -0.308), the number of collected oocytes (r = -0.451), the number of mature oocytes (r = -0.526), the number of fertilized oocytes (r = -0.439) and the number of viable embryos (r = -0.443, all statistically significant at p < 0.02 level). No such associations were found with caspase-8, bax or bcl-2. These preliminary findings suggest that increased caspase-3 gene expression in granulosa cells is associated with a worse ovulatory response in humans.