Recovery Of Spermatogenesis Research Articles

Longitudinal trends in testicular volume z scores from puberty to adulthood, sperm quality, and paternity outcomes after childhood cancer.

Childhood cancer therapy may cause subfertility. This study correlated cancer therapy exposures with testicular volumes from puberty to adulthood, spermatogenesis, and paternity outcomes in adulthood. The study population comprised 255 male childhood cancer survivors (CCS) (survival ≥5 years, diagnosed in 1964-2000 at the Helsinki Children's Hospital) whose testicular volume was measured at ages 12 years (n=38), 14 years (n=57), 16 years (n=63), 18 years (n=105), and in adulthood (n=43; median age, 27 years). Testicular volumes were converted to age-specific z scores. In addition, 92 CCS provided semen sample in adulthood (median age, 25.2 years); and paternity was evaluated through national register data (mean age at assessment, 37.6 years; n=252). Compared with age-specific reference values, CCS generally exhibited low testicular volume z scores at ages 12-18 years. Testicular volume z scores in CCS treated exclusively with chemotherapy returned to the reference range in adulthood. In contrast, patients exposed to testicular radiation ≥1 gray (Gy) (median dose, 12 Gy) showed no late recovery in testicular size. Testicular radiation ≥1 Gy and a cyclophosphamide equivalent dose ≥12 g/m2 were identified as risk factors for azoospermia in adulthood. Patients exposed to testicular radiation ≥1 Gy and a cyclophosphamide equivalent dose ≥4 g/m2 had lower paternity rates. Testicular volume growth after prolonged follow-up suggests a potential late recovery of spermatogenesis in CCS treated exclusively with chemotherapy. However, alkylating agents increased the risk of having prolonged azoospermia and nonpaternity. High-dose testicular radiation causes long-term depletion of spermatogonia and was the strongest risk factor for azoospermia and nonpaternity.

Phosphodiesterase inhibitors and testicular torsion: A systematic review and meta-analysis of animal studies.

Testicular torsion is a common urological emergency in children and teenagers that, in most cases, requires surgical exploration and detorsion. However, even after a successful intervention, a high number of patients still develop irreversible damage. To investigate whether the administration of phosphodiesterase inhibitors (PDE) in animal models of testicular torsion can reduce damages caused by the ischemia-reperfusion syndrome. A systematic search of the Medline, Web of Science, Embase, and Ovid databases for studies up to December 2023 was performed using PRISMA guidelines. A detailed search was conducted using the following key terms: "testicles," "ischemia," "reperfusion," and "phosphodiesterase inhibitors." There was no restriction regarding language or year of publication. We investigated spermatogenesis by the Johnsen's biopsy score (JTBS) and histological damage by the Cosentino's biopsy score (CBS). Malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), and glutathione peroxidase (GPx) testicular levels were also analyzed. Initially, 296 articles were identified. However, only 20 studies met the inclusion criteria. PDE inhibitors improved JTBS (MD=2.53, CI=1.94 to 3.13), CBS (MD=-1.11, CI=-1.25 to -0.97) and SOD (SMD=3.27, CI=1.59 to 4.95) outcomes. PDE did not improve MDA (SMD=-0.93, CI=-2.19 to 0.34), NO (SMD=-0.54, CI=-1.56 to 0.48), and GPx (SMD=0.77, CI=-0.38 to 1.92) outcomes. A higher dose of PDE inhibitors only improved CBS outcome. This review indicates that the administration of PDE inhibitors in rats ameliorated the outcomes, representing a promising complementary strategy for spermatogenesis recovery following testicular torsion. Our review suggests that JTBS and CBS could be translated into future human studies, validating and extending these findings within the context of human physiopathology, while providing applicability of interventions in real clinical practice.

Optimal Restoration of Spermatogenesis following Testosterone Therapy using hCG and FSH

To study improvements in spermatogenesis in men with a history of testosterone therapy using a novel fertility treatment regimen. A single-center retrospective cohort analysis. Seventy-seven men with previous testosterone use seeking fertility treatment from January 2020 to March 2024. A treatment regimen of 3000IU human Chorionic Gonadotropin (hCG) and 75IU Follicle Stimulating Hormone (FSH) three times a week was utilized. The primary outcome measured was change in sperm concentration during hCG/FSH therapy. The secondary outcome measured was whether concurrent testosterone therapy during hCG/FSH therapy affected recovery of spermatogenesis. Within the entire cohort (n=77), 74% of men demonstrated improvements in their sperm concentrations. There was not a significant difference in recovery of sperm concentration in men who stayed on testosterone therapy during hCG/FSH reboot (No testosterone therapy [n=50], 74% improved vs. Concurrent testosterone therapy [n=27], 74% improved). We report optimal recovery of spermatogenesis with hCG/FSH therapy in infertile men with a history of testosterone use. Concurrent testosterone therapy does not impede hCG/FSH-mediated spermatogenic recovery.

Identification of Candidate Genes Related to Hybrid Sterility by Genomic Structural Variation and Transcriptome Analyses in Cattle-yak

Hybrids between closely related but genetically incompatible species are often inviable or sterile. Cattle-yak, an interspecific hybrid of yak and cattle, exhibits male-specific sterility, which limits the fixation of its desired traits and prevents genetic improvement in yak through crossbreeding. Transcriptome profiles of testicular tissues have been generated in cattle, yak, and cattle-yak; however, the genetic variations underlying differential gene expression associated with hybrid sterility have yet to be elucidated. We detected differences in the cellular composition and gene expression of testes from yak and cattle-yak at 3 mo of age, 10 mo of age and adulthood. Histological analysis revealed that the most advanced germ cells were gonocytes (prospermatogonia) at 3 mo and spermatocytes at 10 mo. Complete spermatogenesis occurred in the seminiferous tubules of adult yak, whereas only spermatogonia and a limited number of spermatocytes were detected in the testis of adult cattle-yak. Transcriptome analysis revealed 180, 6310, and 6112 differentially expressed genes (DEGs) in yak and cattle-yak at each stage, respectively. Next, we examined the spermatogenic cell types in the backcross generation (BC1) and detected the appearance of round spermatids, indicating the partial recovery of spermatogenesis in these animals. Compared with those in cattle-yak, 272 DEGs were identified in the testes of BC1 animals. Notably, we discovered that the expression of X chromosome-linked (X-linked) genes was upregulated in the testis of cattle-yak compared with yak, suggesting a possible abnormality in the process of meiotic sex chromosome inactivation (MSCI) in hybrid animals. We next screened DEGs harboring structural variations (SVs) and identified a list of SV genes associated with spermatogonial development, meiotic recombination, and double-strand break (DSB) repair. Furthermore, we found that the SV genes ESCO2 (establishment of sister chromatid cohesion N-acetyltransferase 2) and BRDT (bromodomain testis associated) may be involved in meiotic arrest of cattle-yak spermatocytes. Overall, our research provides a valuable database for identifying structural variant loci that contribute to hybrid sterility.

Application of testicular organ culture system for the evaluation of spermatogenesis impairment

Recently, it was reported that a testicular organ culture system (TOCS) using polydimethylsiloxane (PDMS) chips with excellent oxygen permeability and biocompatibility, called the PDMS-chip ceiling (PC) method, enables improved spermatogenesis efficiency. We investigated whether this PC method is useful for detecting impaired spermatogenesis caused by busulfan (Bu), a typical testicular toxicant. In this study, testicular tissue fragments from Acro3-EGFP mice, which express the green fluorescent protein (GFP) and reflect the progression of spermatogenesis, were subjected to the PC method. When treated with Bu, cultured tissues shrank in volume, and their GFP-expressing area decreased or disappeared. Histological examination confirmed the regression of spermatogenesis. In addition, immunohistochemical examination revealed that spermatogonia, including spermatogonial stem cells (SSCs), were the primary targets of Bu toxicity. Time-course analysis demonstrated that the recovery of spermatogenesis, dependent on Bu concentration, correlated closely with the severity of damage to these target cells. These results suggest that the PC method is a useful approach for detecting spermatogenesis impairment accurately through faithful recapitulation of spermatogenesis in vivo.

The dose-response relationship between cumulative alkylating agent exposure and sperm concentration in adult survivors of childhood cancer: A report from the St. Jude Lifetime (SJLIFE) cohort study.

10066 Background: Male survivors of childhood cancer treated with alkylating agents (AA) are at risk for azoospermia; however, the long-term dose-response relationship is unknown. Methods: Of the 685 male SJLIFE participants treated with AA but no radiation therapy, 387 (56.5%) provided an evaluable semen sample (mean age at diagnosis: 8.3 years, mean age at evaluation: 28.6 years, and mean years from diagnosis to evaluation: 20.3 years). 73 had one or more subsequent semen analyses (median interval between semen analyses 5.4 years; interquartile range 4.2 – 6.6 years). Survivors were categorized as azoospermia, oligospermia (sperm concentration > 0 and < 15 million/ml), or normospermia (sperm concentration ≥ 15 million/ml). AA exposure was estimated using the cyclophosphamide equivalent dose (CED). Risks were estimated using the odds ratio (OR) and 95% confidence intervals (CI) from multinomial logistic regression analyses. Results: Among survivors 22.5% had azoospermia, 26.6% oligospermia, and 50.9% normospermia (Table). Motility and progressive motility were not associated with increasing CED categories among normospermic or oligospermic participants. Multinomial logistic regression including CED, age at diagnosis and at follow-up demonstrated that, for each 1,000 mg/m2 increase in CED, the odds of azoospermia increased by 1.14 (95% CI 1.09, 1.20), and the odds of azoospermia and oligospermia increased by 1.22 (95% CI, 1.14, 1.30). Azoospermia and oligospermia were best distinguished from normospermia using a CED cutoff of 7,000 mg/m2 based on the Youden Index. Nearly all participants initially identified with azoospermia (24 out of 25) maintained this diagnosis upon later evaluation. Conclusions: Nearly half of adult childhood cancer survivors who received alkylating agents without radiation therapy experience impaired sperm production, either low sperm count (oligospermia) or no sperm at all (azoospermia). The association increases with increasing CED. Recovery of spermatogenesis is unlikely among those who present azoospermic. CED = 7,000 mg/m2 is the optimal cutoff for differentiating risk for oligo- or azoospermia from normospermia. [Table: see text]

Impact of varicocelectomy on sperm parameters and hormone levels in infertile patients with sickle cell disease

Purpose: This study analyzes the semen parameters and gonadal hormone levels in infertile patients with sickle cell disease (SCD) and palpable varicoceles who underwent varicocelectomy for impaired semen parameters. Patients and methods: Improvements in the semen parameters—serum levels of total testosterone, follicle-stimulating hormone (FSH), and inhibin B—and testicular volume (TV) in 18 infertile patients with SCD were evaluated before and at 6 months after varicocelectomy. Results: Before varicocelectomy, all sperm parameters were affected, with high rates of low sperm concentration (83.3%), poor motility (88.9%), and abnormal morphology (77.8%). All sperm parameters were altered in 61.1%. On follow-up at 55.5 months after varicocelectomy, improvement in all the semen parameters was successful and complete in 22.2% but was not significant in 77.8%. The median follow-up period for sperm recovery was relatively long at 60.3 months. Total testosterone, FSH, and inhibin B were not significantly different before and after treatment (P > 0.05). However, testosterone, FSH, and TV significantly correlated with the semen parameters (P < 0.05). Moreover, a significant positive correlation was found between TV and inhibin B (P < 0.05), and the improvement in the semen parameters was significantly correlated with primary hypogonadism (P < 0.05). Conclusion: In adult men with SCD, varicocelectomy had a positive impact on the impaired seminal parameters and may be an option before sperm cryopreservation to improve future male fertility. Furthermore, primary testicular failure may be a feature of reversible spermatogenesis recovery in these patients.

5- methylcytidine effectively improves spermatogenesis recovery in busulfan-induced oligoasthenospermia mice

The function and regulatory mechanisms of 5-methylcytidine (m5C) in oligoasthenospermia remain unclear. In this study, we made a mouse model of oligoasthenospermia through the administration of busulfan (BUS). For the first time, we demonstrated that m5C levels decreased in oligoasthenospermia. The m5C levels were upregulated through the treatments of 5-methylcytidine. The testicular morphology and sperm concentrations were improved via upregulating m5C. The cytoskeletal regenerations of testis and sperm were accompanying with m5C treatments. m5C treatments improved T levels and reduced FSH and LH levels. The levels of ROS and MDA were significantly reduced through m5C treatments. RNA sequencing analysis showed m5C treatments increased the expression of genes involved in spermatid differentiation/development and cilium movement. Immunofluorescent staining demonstrated the regeneration of cilium and quantitative PCR (qPCR) confirmed the high expression of genes involved in spermatogenesis. Collectively, our findings suggest that the upregulation of m5C in oligoasthenospermia facilitates testicular morphology recovery and male infertility via multiple pathways, including cytoskeletal regeneration, hormonal levels, attenuating oxidative stress, spermatid differentiation/development and cilium movement. m5C may be a potential therapeutic agent for oligoasthenospermia.

(081) Fertility Trends in Men with Prior History of Anabolic Steroid Use: Results from a Standardized Treatment Regimen

Abstract Introduction The management of infertility in men who have previously used anabolic steroids poses a complicated clinical challenge. Addressing the infertility issues in this specific population requires a comprehensive approach that considers the underlying physiological alterations caused by anabolic steroid use and tailors treatment interventions accordingly to restore and optimize reproductive function. In this study, we report an analysis of sperm parameter recovery and fertility outcomes in men who had azoospermia or severe oligospermia caused by anabolic steroid use and underwent a standardized treatment regimen for recovery of spermatogenesis. Objective The study aims to assess the longitudinal trends in sperm concentrations, determine the proportion of men achieving normozoospermia, and evaluate subsequent pregnancy rates in this specific population. Additionally, the study seeks to identify potential predictors for improved semen parameters and successful pregnancy outcomes. Methods A retrospective analysis of the period spanning from 2018 to 2022 was conducted on a cohort of men with a prior history of anabolic steroid use who complained of infertility. The standardized treatment approach involved the cessation of testosterone replacement therapy (TRT) and the administration of a combination regimen comprising clomiphene citrate and human chorionic gonadotropin (hCG) for a minimum of 3 to 6 months. Following adequate treatment, men were contacted in March 2023 to determine the most up-to-date pregnancy outcomes. Statistical analysis was performed utilizing SPSS, employing univariate logistic regression analysis to establish a predictive mathematical model. The level of statistical significance was defined as p < 0.05. Results A total of 45 men (median age 37 years, IQR 32-45) met inclusion criteria for this analysis. Median duration of prior T use was 4 years (IQR 1.3-10), with the two most common modalities consisting of injection therapy (43.5%) and oral therapy (34.8%). Median initial sperm concentration was 0 million/cc (IQR 0-1.15), and 23 (51.1%) men initially presented with azoospermia. The median duration of combination hCG/clomid therapy was 5 months (IQR 3-12). In initially azoospermic men (N:23), 5 were lost to follow-up, 6 (33.3%) progressed to severe oligospermia (<5 million/cc), 6 (33.3%) to oligospermia (<15 million/cc), 1 (5.6%) to normozoospermia (>15 million/cc), and 5 (27.8%) remained azoospermic following medical treatment for 6 months. Among the 24 couples who responded to the follow-up call, a total of 9 (37.5%) achieved a successful subsequent pregnancy. Of these, 33.3% (3 couples) used assisted reproductive technology (ART), while 66.7% (6 couples) conceived naturally. On logistic regression analysis, no significant predictors for improved sperm parameters or successful pregnancy were identified. Conclusions Despite the implementation of appropriate treatment regimens, a significant proportion of men with a prior history of anabolic steroid use continue to exhibit severe oligospermia, with more than half failing to demonstrate substantial improvement in their semen parameters even after undergoing a six-month medical treatment protocol. Moreover, only a fraction of men, estimated to be 1 in 6, achieve normozoospermia following medical treatment after anabolic steroid use. Future work should continue to explore predictors for improved sperm parameters on follow-up as well as subsequent successful pregnancy in men with prior history of anabolic steroid use. Disclosure No.

Ursonic acid attenuates spermatogenesis in oligozoospermia mice through inhibiting ferroptosis

Ursonic acid (UNA) is a natural pentacyclic triterpene found in some medicinal plants and foods. The reproductive protective effect of UNA was evaluated in a mouse model of oligozoospermia induced by busulfan (BUS) at 30 mg/kg b.w.. The mice were initially divided into groups with UNA concentrations of 10, 30, 50, 100 mg/kg. Subsequently, based on sperm parameters, the optimal concentration of 50 mg/kg was identified. As a control, an additional group was supplemented with ursolic acid at a concentration of 50 mg/kg. The results indicated that BUS caused the loss of spermatogenic cells in testis, the decrease of sperm in epididymis, the disorder of testicular cytoskeleton, the decrease of serum sex hormones such as testosterone which induced an increase in feedback of androgen receptor and other testosterone-related proteins, the increase of malondialdehyde and reactive oxygen species levels and the increase of ferroptosis in testis while UNA successfully reversed these injuries. High-throughput sequencing revealed that UNA administration significantly upregulated the expression of genes associated with spermatogenesis, such as Tnp1, Tnp2, Prm1, among others. These proteins are crucial in the histone to protamine transition during sperm chromatin remodeling. Network pharmacology analysis reveals a close association between UNA and proteins related to the transformation of histones to protamine. Molecular docking studies reveal that UNA can interact with the ferroptosis-inhibiting gene SLC7A11, thereby modulating ferroptosis. Taken together, UNA alleviated BUS-induced oligozoospermia by regulating hormone secretion, mitigating oxidative stress and promoting recovery of spermatogenesis by inhibiting the ferroptosis.

Evaluating Sperm Recovery Time and Efficacy of Monotherapy vs. Combination Therapies in Men with Congenital Hypogonadotropic Hypogonadism: A Systematic Review and Meta-Analysis.

There is a lack of pooled data exploring the time and rates for human chorionic gonadotropin (hCG) monotherapy vs. combination therapies (hCG+human menopausal gonadotropin or recombinant human follicle-stimulating hormone) to restore spermatogenesis in azoospermic men with congenital hypogonadotropic hypogonadism (CHH). We aimed to investigate the time and rates to recover spermatogenesis among azoospermic CHH men receiving monotherapy vs. combination therapy. We conducted a systematic review and meta-analysis following the PRISMA guidelines. The search was performed on PubMed, EMBASE, Web of Science, and Scopus databases up to November 2023. Forrest plots were generated to visually present the pooled effect sizes for time to recover spermatogenesis, specifically employing the standardized mean difference (SMD). Publication bias was assessed utilizing funnel plots. PROSPERO ID: CRD42023473615. The search identified 720 studies meeting inclusion criteria. Our meta-analysis of 1,240 men with CHH revealed significant differences in the time to recover spermatogenesis between combination therapies and monotherapy. The weighted mean recovery time was significantly shorter for combination therapies (10 months) compared to monotherapy (33 months). The SMD under the common effect model was 8.8 for combination therapies and 24.98 for monotherapy, indicating a more rapid recovery with combination therapies, p<0.01. The rates of sperm recovery were 66.76% for combination therapies and 51.9% for monotherapy, p=0.03. Significant heterogeneity was observed in both groups (I²=86% for combination therapies and I²=68% for monotherapy), suggesting considerable variation in individual responses. The present meta-analysis reveals that in men with CHH, combination therapies expedite spermatogenesis recovery more than monotherapy. Additionally, combination therapies yield a higher rate of sperm appearing in the ejaculate as compared to hCG monotherapy. The significant heterogeneity observed in both groups underscores the variability in individual responses, warranting further investigation and caution in interpreting these results.

Protective Role of Gallic Acid with Rutin or Quercetin on Testicular Glutathione and Histopathology in Rats Exposed to Busulfan

Busulfan (BUS) is an alkylating agent used in chemotherapy that induces oxidative stress and testicular damage. This study investigates the protective effects of co-administering Gallic Acid (GAL) with Rutin (RUT) or Quercetin (QUE) against BUS-induced reductions in testicular glutathione (GSH) in Wistar rats. Thirty-two (32) male Wistar rats were randomly divided into four groups: control, BUS only, BUS+GAL+RUT, and BUS+GAL+QUE. Over a 56-day treatment period, the rats were assessed for changes in GSH concentration and testicular histopathology. GSH levels were measured to evaluate oxidative stress, while histological evaluations were performed to assess spermatogenic recovery. BUS administration significantly reduced GSH concentration in the testis, indicating oxidative stress. Co-administration of GAL+RUT or GAL+ QUE reversed this decrease, restoring GSH levels to near-control values. Although there were no significant changes in testicular and body weight across groups, the histopathological analysis showed that GAL+QUE improved spermatogenesis recovery more effectively than GAL+RUT. Additionally, GAL+RUT exhibited more mature germ cells and potential for testicular repopulation. The co-administration of GAL+RUT or GAL+QUE effectively mitigates BUS-induced oxidative stress in rat testes, with GAL+QUE demonstrating superior recovery in spermatogenic function. These findings suggest that incorporating these flavonoid combinations could help reduce chemotherapy-induced testicular damage.

Pre- and post-chemotherapy spermatogenesis in male patients with malignant bone and soft tissue tumors.

Introduction: Malignant bone and soft tissue tumors, commonly called sarcomas, predominantly originate in bone and soft tissues and typically affect individuals at a younger age. Following the resection of the primary tumor, treatment often necessitates radiation therapy and gonadotoxic chemotherapy, the specifics of which depend on the disease's stage Conversely, there is a notable concern regarding the potential loss of fertility due to these treatments. Consequently, it is recommended that men consider sperm cryopreservation before initiating treatment. This study aims to assess spermatogenesis in male patients diagnosed with malignant bone and soft tissue tumors before and after chemotherapy. Methods: This study involved 34 male patients diagnosed with malignant bone and soft tissue tumors and subsequently underwent sperm cryopreservation before initiating treatment. Medical records included details about the primary disease, age, marital status at presentation, semen analysis results, treatment regimen and number of courses, post-treatment semen analysis, renewal status and outcomes. Results: The mean age at the time of sperm cryopreservation was 22.8years. The median semen volume was 2.5mL, sperm concentration was 32.6 million/ml, and sperm motility was 38.5%. Following chemotherapy, semen analysis was conducted on 12 patients, with ifosfamide being the predominant drug used in all cases. Among these 12 patients, eight retained viable spermatozoa, and two successfully achieved spontaneous pregnancies resulting in live births. In one of the remaining four cases where no sperm were detected in ejaculate, a live birth was achieved through intracytoplasmic sperm injection using cryopreserved sperm. Discussion: While ifosfamide, the primary chemotherapy drug for patients with malignant bone and soft tissue tumors, was associated with severe impairments in spermatogenesis, recovery of spermatogenesis was observed in many cases. However, there were instances of prolonged azoospermia. Even in such cases, assisted reproduction using cryopreserved sperm remained viable for achieving parenthood. In light of these findings, offering patients the opportunity for fertility preservation is advisable.

Fertility outcomes in men with prior history of anabolic steroid use

To study sperm parameters recovery and fertility outcomes in men with azoospermia or severe oligospermia caused by anabolic steroid use who underwent a standardized treatment regimen for spermatogenesis recovery. A retrospective analysis of a cohort of men with a prior history of anabolic steroid use and infertility complaints (between 2018 and 2022) was conducted. The standardized treatment approach involved discontinuing testosterone replacement therapy and administering a combination regimen of clomiphene citrate and human chorionic gonadotropin for a minimum of 3 to 6 months. The main outcome measures included changes in sperm parameters, predominantly sperm concentration, and subsequent pregnancy outcomes. A total of 45 men (median age 37 years, IQR 32-45) met the inclusion criteria for this analysis. Median duration of prior T use was 4 years (IQR 1.3-10), with the 2 most common modalities consisting of injection therapy (43.5%) and oral therapy (34.8%). The median initial sperm concentration was 0 million/cc (IQR 0-1.15), and 23 (51.1%) men initially presented with azoospermia. The median duration of combination human chorionic gonadotropin/clomid therapy was 5 months (IQR 3-12). In initially azoospermic men (N: 23), 5 were lost to follow-up, 6 (33.3%) progressed to severe oligospermia (<5 million/cc), 6 (33.3%) to oligospermia (<15 million/cc), 1 (5.6%) to normozoospermia (>15 million/cc), and 5 (27.8%) remained azoospermic after medical treatment for 6 months. Among the 24 couples who responded to the follow-up call, a total of 9 (37.5%) achieved a successful subsequent pregnancy. Of these, 33.3% (3 couples) used assisted reproductive technology, whereas 66.7% (6 couples) conceived naturally. On logistic regression analysis, no significant predictors for improved sperm parameters or successful pregnancy were identified. Despite appropriate treatment regimens, a significant proportion of men with a prior history of anabolic steroid use continue to exhibit severe oligospermia, with more than half showing limited improvement in semen parameters after 6 months of treatment. Only a fraction of men achieves normozoospermia after treatment. Further research is needed to explore predictors for improved sperm parameters and successful pregnancy outcomes in men with a history of anabolic steroid use.

Gypenosides Production and Spermatogenesis Recovery Potentials of Extracts from Cell Suspension Cultures of Gynostemma pentaphyllum

Background: Gynostemma pentaphyllum (GP), also called Giao-co-lam, is a traditional Vietnamese herb, also known as the "Herb of Immortality", that grows throughout Asian countries and is used for the treatment of hematuria, edema in the pharynx and neck, tumors, and trauma.&#x0D; Methods: In this study, we investigated the effects of culture conditions on cell growth and total gypenoside accumulation in the GP suspension cells. Cells were cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg/L KIN and 0.5 mg/L IBA, and different inoculum sizes (2-4 g) for 10-24 days.&#x0D; Results: The results showed that the optimum inoculum size and shaking speed were 3 g of callus and 120 rpm, respectively. The highest cell biomass reached was 5.833 g of fresh weight, corresponding to 0.136 g of dry weight after 20 days of culture. The total gypenoside and Rb1 accumulation was the highest after 18 days of culture, with 46.498 mg/g and 0.038 mg/g dry weight, respectively. In addition, the crude extract from GP cell suspension cultures remarkably improved pathological changes in mouse testicular tissue after scrotal heat exposure. Blood testosterone levels were significantly increased in heat-exposed mice treated with the GP cell suspension culture extract.&#x0D; Conclusion: Taken together, these results suggest that GP bio-mass production by cell suspension cultures leads to the accumulation of gypenosides in large amounts, and provides the potential for the recovery of spermatogenesis following heat stress.

(073) The Effects of Intranasal Testosterone (Natesto™) for Treatment of Hypogonadism on Maintenance of Spermatogenesis: Demographic Data from a Single-Institution, Prospective, Non-blinded Study

Abstract Introduction Testosterone therapy (TTh) is becoming increasingly common among men of reproductive age in the United States. An estimated 3 million men are on TTh; however, exogenous testosterone use can disrupt the hypothalamic-pituitary-gonadal axis, leading to reduced spermatogenesis and infertility. The spontaneous recovery of spermatogenesis after cessation of TTh is possible, but may take months to years and can lead the patient to experience hypogonadal symptoms. Recent preliminary results have suggested that a 4.5% intranasal testosterone gel (Natesto™) may offer the potential to offset these symptoms and re-establish eugonadal levels of circulating testosterone. It has also been shown that men on Natesto™ maintain follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels and total motile sperm count within the normal range. This prospective, non-blinded study seeks to confirm the role of Natesto™ to alleviate hypogonadal symptoms in men on gonadotropin therapy for restoration of spermatogenesis. Objective To determine if Natesto™ can alleviate hypogonadal symptoms while preserving the recovery of spermatogenesis. The primary objective of this study is to examine changes in total motile sperm count at baseline, 14 weeks, and 26 weeks. Secondary outcomes include changes in LH, FSH, testosterone, estrogen, and quality of life measurements at these time intervals. Methods IRB approval was obtained for this study. Male patients between 18 and 64 years of age with a diagnosis of testosterone-induced hypo-spermatogenesis were recruited from a tertiary referral male infertility clinic between December 2021 and June 2022. Patients ceased intramuscular testosterone therapy and initiated gonadotropin therapy with FSH 75IU and hCG 3000IU every other day as well as 4.5% testosterone gel (Natesto™) three times daily. Patients were evaluated at baseline, 14 weeks, and 26 weeks. Clinic visits consisted of a hormone profile with total testosterone, estradiol, FSH, LH, questionnaires (IIEF, SF36, and ADAM) as well as a semen analysis. Results A total of 9 patients who met inclusion criteria were identified and elected to take part in the study. Four of the patients obtained baseline intake questionnaires, bloodwork, and a semen analysis with at least 1 additional follow-up visit. Five patients are pending initial follow-up. Patient intake demographic data included a median age of 35, LH of 3.15, FSH of 3.2, testosterone 260ng/dl, IIEF 20, SF-36 energy/fatigue score of 42.5%, and ADAM of 5.5. Four patients had complete gonadotropin suppression, and 6 were azoospermic. Interval semen analyses revealed total motile sperm count increases from 17.9 to 25 million and 9.7 to 10.9 million, with 2 patients demonstrating persistent azoospermia. Mean testosterone increased by 297.5ng/dl. No patients reported adverse events. Conclusions Preliminary results are limited and suggest that men undergoing cessation of testosterone therapy for restoration of spermatogenesis have decreased sexual and overall function scores. Continuation of this study, with recruitment of a larger sample size, will provide important, novel data regarding the effect of Natesto™ therapy on the hypogonadal symptoms seen in men during TTh cessation for spermatogenesis restoration. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Endo Pharmaceuticals

Notch pathway is required for protection against heat stress in spermatogonial stem cells in medaka.

Gamete production is a fundamental process for reproduction; however, exposure to stress, such as increased environmental temperature, can decrease or even interrupt this process, affecting fertility. Thus, the survival of spermatogonial stem cells (SSCs) is crucial for the recovery of spermatogenesis upon stressful situations. Here, we show that the Notch pathway is implicated in such survival, by protecting the SSCs against thermal stress. First, we corroborated the impairment of spermatogenesis under heat stress in medaka, observing an arrest in metaphase I at 10days of heat treatment, an increase in the number of spermatocytes, and downregulation of ndrg1b and sycp3. In addition, at 30days of treatment, an interruption of spermatogenesis was observed with a strong loss of spermatocytes and spermatids. Then, the exposure of adult males to thermal stress condition induced apoptosis mainly in spermatogenic and supporting somatic cells, with the exception of the germinal region, where SSCs are located. Concomitantly, the Notch pathway-related genes were upregulated, including the ligands (dll4, jag1-2) and receptors (notch1a-3). Moreover, during thermal stress presenilin enhancer-2 (pen-2), the catalytic subunit of γ-secretase complex of the Notch pathway was restricted to the germinal region of the medaka testis, observed in somatic cells surrounding type A spermatogonia (SGa). The importance of Notch pathway was further supported by an ex vivo approach, in which the inhibition of this pathway activity induced a loss of SSCs. Overall, this study supports the importance of Notch pathways for the protection of SSCs under chronic thermal stress.

Desire for children and fertility preservation in transgender and gender-diverse people: A systematic review.

The decision to pursue one's desire for children is a basic human right. For transgender and gender-diverse (TGD) people, gender-affirming care may alter the possibilities to fulfill one's desire for children due to the impact of this treatment on their reproductive organs. We systematically included 76 studies of varying quality describing the desire for children and parenthood; fertility counseling and utilization; and fertility preservation options and outcomes in TGD people. The majority of TGD people expressed a desire for children. Fertility preservation utilization rates were low as there are many barriers to pursue fertility preservation. The most utilized fertility preservation strategies include oocyte vitrification and sperm banking through masturbation. Oocyte vitrification showed successful outcomes, even after testosterone cessation. Sperm analyses when banking sperm showed a lower quality compared to cis male samples even prior to gender-affirming hormone treatment and an uncertain recovery of spermatogenesis after discontinuing treatment.

Heavy Metal Pollution and Male Fertility: An Overview on Adverse Biological Effects and Socio-Economic Implications.

Trace metals can be divided into two subgroups considering their pathophysiological effects: the first consists of microelements essential for life (arsenic, cobalt, chromium, copper, fluorine, iron, iodine, manganese, molybdenum, nickel, selenium, silicon, tin, vanadium and zinc), implicated in important metabolic processes; the second includes toxic microelements, such as cadmium (Cd), mercury (Hg), chromium (Cr), and lead (Pb) for living organisms, even at low concentrations. These metals contribute to serious consequences for human health, including male infertility. Studies performed in several in vitro and in vivo models revealed that environmental exposure to toxic pollutants, as heavy metals, negatively affects human male fertility. Stem cells, due to their ability to self-renew and differentiate in several cell types, have been proposed as a useful tool in assisted reproductive technology, permitting the spermatogenesis recovery in patients with irreversible infertility. Considering the effects of heavy metals on male fertility and, from a demographic point of view, the decreased fertility ratio, further strategies are required to maintain a sustainable turn-over of 2 children for woman. We discuss here the findings on the biological effects of heavy metal pollution in the male fertility and underline the related socioeconomic impact on population demography.

Models and Methods for Evaluating Regeneration of Spermatogenesis After Cytotoxic Treatments.

Cytotoxic exposure, predominantly during radiation and/or chemotherapy treatment for cancer, interferes with fertility in men. While moderate doses cause temporary azoospermia allowing eventual recovery of spermatogenesis, higher doses of sterilizing agents can cause permanent sterility by killing the spermatogonial stem cells (SSCs). In this chapter, the methods involved in the following aspects of cytotoxic regeneration are described: (i) designing rodent and non-human primate models for regeneration of spermatogenesis after cytotoxic treatment by radiation and chemotherapy; (ii) analysis of SSCs with respect to the impact of the cytotoxic treatment, including analysis of spermatogonial clones, scoring the testicular section to analyze the extent of spermatogenic recovery, preparation of testicular and epididymal sperm, and collection of semen in non-human primates for sperm analysis; and (iii) preparation and delivery of a GnRH antagonist and steroids for enhancement or induction of spermatogonial differentiation, leading to the regeneration of spermatogenesis, largely applicable in the rat model.