Structure-activity relationship and mechanistic study of organotins as inhibitors of human, pig, and rat gonadal 3ß-hydroxysteroid dehydrogenases.

Organotins have been widely used in various industrial applications. This study investigated the structure-activity relationship as inhibitors of human, pig, and rat gonadal 3ß-hydroxysteroid dehydrogenases (3ß-HSD). Human KGN cell, pig, and rat testis microsomes were utilized to assess the inhibitory effects of 18 organotins on the conversion of pregnenolone to progesterone. Among them, diphenyltin, triethyltin, and triphenyltin exhibited significant inhibitory activity against human 3ß-HSD2 with IC50 values of 114.79, 106.98, and 5.40 µM, respectively. For pig 3ß-HSD, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin demonstrated inhibitory effects with IC50 values of 172.00, 100.19, 87.00, 5.75, and 1.65 µM, respectively. Similarly, for rat 3ß-HSD1, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin displayed inhibitory activity with IC50 values of 81.35, 43.56, 55.55, 4.09, and 0.035 µM, respectively. They were mixed inhibitors of pig and rat 3ß-HSD, while triphenyltin was identified as a competitive inhibitor of human 3ß-HSD2. The mechanism underlying the inhibition of organotins on 3ß-HSD was explored, revealing that they may disrupt the enzyme activity by binding to cysteine residues in the catalytic sites. This proposition was supported by the observation that the addition of dithiothreitol reversed the inhibition caused by all organotins except for triethyltin, which was partially reversed. In conclusion, this study provides valuable insights into the structure-activity relationship of organotins as inhibitors of human, pig, and rat gonadal 3ß-HSD. The mechanistic investigation suggests that these compounds likely exert their inhibitory effects through binding to cysteine residues in the catalytic sites.

Reproductive outcomes in patients with high levels of sperm DNA fragmentation using testicular sperm for intracytoplasmic injection: a retrospective analysis.

This study aims to compare the embryological and clinical parameters of intracytoplasmic sperm injection (ICSI) cycles using testicular versus ejaculated sperm in male patients with elevated sperm DNA fragmentation (SDF). A total of 73 ICSI cycles were examined in couples where the male partner exhibited high levels of SDF. ICSI was performed using either ejaculated or testicular sperm. The primary outcomes were rates of blastocyst formation, high-quality embryo development, and clinical pregnancy. The DNA fragmentation index (DFI) for testicular sperm (16.81 ± 17.51) was significantly lower than that of ejaculated sperm (56.96 ± 17.56). While the blastocyst formation rate was significantly higher in the testicular sperm group compared to the ejaculated sperm group, no statistically significant differences were noted in fertilization rate (72.15% vs. 77.23%), rate of high-quality embryo formation (47.17% vs. 46.53%), clinical pregnancy (50% vs. 56.52%), Cumulative pregnancy (70.2% vs. 55.6%), or live birth rate (43.75% vs.43.48%). Testicular spermatozoa have no additional advantage over ejaculated spermatozoa except for blastocyst quality in patients with high SDF, the use of testicular spermatozoa for the first ICSI cycle in male infertility patients with high SDF should be undertaken after much consideration at present.

The metabolic activation of pentachlorophenol to chloranil as a potent inhibitor of human and rat placental 3ß-hydroxysteroid dehydrogenases.

Pentachlorophenol (PCP) is a widely used pesticide. However, whether PCP and its metabolite chloranil have endocrine-disrupting effects by inhibiting placental 3ß-hydroxysteroid dehydrogenase 1 (3ß-HSD1) remains unclear. The study used in vitro assays with human and rat placental microsomes to measure 3ß-HSD activity as well as human JAr cells to evaluate progesterone production. The results showed that PCP exhibited moderate inhibition of human 3ß-HSD1, with an IC50 value of 29.83 µM and displayed mixed inhibition in terms of mode of action. Conversely, chloranil proved to be a potent inhibitor, demonstrating an IC50 value of 147 nM, and displaying a mixed mode of action. PCP significantly decreased progesterone production by JAr cells at 50 µM, while chloranil markedly reduced progesterone production at ≥1 µM. Interestingly, PCP and chloranil moderately inhibited rat placental homolog 3ß-HSD4, with IC50 values of 27.94 and 23.42 µM, respectively. Dithiothreitol (DTT) alone significantly increased human 3ß-HSD1 activity. Chloranil not PCP mediated inhibition of human 3ß-HSD1 activity was completely reversed by DTT and that of rat 3ß-HSD4 was partially reversed by DTT. Docking analysis revealed that both PCP and chloranil can bind to the catalytic domain of 3ß-HSDs. The difference in the amino acid residue Cys83 in human 3ß-HSD1 may explain why chloranil is a potent inhibitor through its interaction with the cysteine residue of human 3ß-HSD1. In conclusion, PCP is metabolically activated to chloranil as a potent inhibitor of human 3ß-HSD1.

Comparison of structure-activity relationship for bisphenol analogs in the inhibition of gonadal 3ß-hydroxysteroid dehydrogenases among human, rat, and mouse.

Bisphenol A (BPA) is a widely used plastic material and its potential endocrine disrupting effect has restricted its use and increasing use of BPA alternatives has raised health concerns. However, the effect of bisphenol alternatives on steroidogenesis remains unclear. The objective of this study was to compare inhibitory potencies of 10 BPA alternatives in the inhibition of gonadal 3ß-hydroxysteroid dehydrogenase (3ß-HSD) in three species (human, rat and mouse). The inhibitory potency for human 3ß-HSD2, rat 3ß-HSD1, and mouse 3ß-HSD6 ranged from bisphenol FL (IC50, 3.32 µM for human, 5.19 µM for rat, and 3.26 µM for mouse) to bisphenol E, F, and thiodiphenol (ineffective at 100 µM). Most BPA alternatives were mixed inhibitors of gonadal 3ß-HSD and they dose-dependently inhibited progesterone formation in KGN cells. Molecular docking analysis showed that all BPA analogs bind to steroid and NAD+ active sites. Lipophilicity of BPA alternatives was inversely correlated with IC50 values. In conclusion, BPA alternatives mostly can inhibit gonadal 3ß-HSDs and lipophilicity determines their inhibitory strength.

The role of hepatitis B virus surface protein in inducing Sertoli cell ferroptosis.

BACKGROUNDS: Hepatitis B virus infection could result in male infertility with sperm defects and dysfunction. Sertoli cells are essential for testis function and play a crucial role in spermatogenesis. Sertoli cell death contributes to spermatogenesis impairment, leading to poor sperm quality. Ferroptosis has been implicated as a mechanism of Sertoli cell death. The issue in studying the relationship between hepatitis B virus and Sertoli cell ferroptosis has not yet been addressed. OBJECTIVES: To explore the mechanisms underlying ferroptosis in hepatitis B virus-exposed Sertoli cells. MATERIALS AND METHODS: Human Sertoli cells were treated in vitro with levels of 25, 50, and 100 µg/mL of hepatitis B virus surface protein (HBs). Cell viability and levels of glutathione, malondialdehyde, cellular ferrous ion (Fe2+ ), lipid peroxidation, and N6-methyladenosine in Sertoli cells were detected. The level of glutathione peroxidase 4, transferrin receptor 1, ferritin heavy chain, tripartite motif (TRIM) 37, methyltransferase like 3, and insulin-like growth factor 2 mRNA binding protein 2 was examined. Cell transfection was carried out to alter expression of ferroptosis-related proteins. qPCR and immunoblotting were performed to measure protein expression level. Immunoprecipitation was applied to determine the protein and protein-RNA interaction. Luminescence analysis was performed to identify the target of methyltransferase like 3. RESULTS: HBs exposure triggered ferroptosis featured with increased intracellular Fe2+ ion, reduced cell viability and expression of glutathione peroxidase 4 in Sertoli cells. HBs treatment significantly increased TRIM37 expression, which suppressed glutathione peroxidase 4 expression through ubiquitination. TRIM37 silencing attenuated the effect of HBs exposure-regulated cell viability and ferroptosis. HBs upregulated N6-methyladenosine modification in TRIM37 3'-UTR by increasing methyltransferase like 3 expression. The binding of N6-methyladenosine reader insulin-like growth factor 2 mRNA binding protein 2 and TRIM37 3'-UTR enhanced the stability of TRIM37 mRNA. CONCLUSION: HBs can decrease human Sertoli cell viability by promoting ferroptosis induced by the loss of glutathione peroxidase 4 activity through TRIM37-mediated ubiquitination of glutathione peroxidase 4. The findings highlight the role of TRIM37/glutathione peroxidase 4 signaling responsible for ferroptosis regulation in hepatitis B virus-infected Sertoli cells.

Halogenated bisphenol A derivatives potently inhibit human, rat, and mouse gonadal 3ß-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico molecular docking analysis.

Bisphenol A (BPA) is a widely used plastic material, and halogenated BPA derivatives are formed either by synthesis or environmental processes. However, the effect of halogenated bisphenols on steroidogenesis remains unclear. The aim of this study was to compare inhibition of 6 BPA derivatives on gonadal 3ß-hydroxysteroid dehydrogenases (3ß-HSDs) in three species (human, rat, and mouse). The inhibition on human 3ß-HSD2 was tetrabromo BPA (TBBPA, IC50, 1.01 µM)>trichloro BPA (TrCBPA, 3.95 µM)>tetrachloro BPA (TCBPA, 4.14 µM)>monochloro BPA (MCBPA, 4.74 µM)>others with TrCBPA of competitive, TBBPA of noncompetitive and MCBPA/TCBPA of mixed inhibition. The inhibition on rat 3ß-HSD1 was TCBPA (1.68 µM)>TrCBPA (1.72 µM)>MCBPA (2.80 µM)>BPA>others with mixed inhibition. The inhibition on mouse 3ß-HSD6 was TrCBPA (1.59 µM) >MCBPA (3.36 µM)>TCBPA (3.72 µM)>others with mixed inhibition. Molecular docking analysis showed that TBBPA, TrCBPA, and TCBPA bind to steroid active sites, contacting with catalytic residue Tyr154 of human 3ß-HSD2. MCBPA, TrCBPA, and TCBPA bind to steroid active site of rat 3ß-HSD1. MCBPA and TrCBPA bind to active site of mouse 3ß-HSD6. Regression of lowest binding energy values with Ki values revealed a significant negative linear regression (P < 0.05). In conclusion, halogenated BPA derivatives are more potent inhibitors of three 3ß-HSDs than BPA and there is structure-dependent inhibition. SYNOPSIS: Chlorinated bisphenol derivatives after water chlorination process and other halogenated bisphenols effectively inhibit human and rat 3ß-HSD activity, thereby leading to steroid hormone deficiency.

Bisphenol S stimulates Leydig cell proliferation but inhibits differentiation in pubertal male rats through multiple mechanisms.

Bisphenol S (BPS) is a novel bisphenol A (BPA) analogue, a ubiquitous environmental pollutant that disrupts male reproductive system. Whether BPS affects Leydig cell maturation in male puberty remains unclear. Male Sprague-Dawley rats (age of 35 days) were daily gavaged to 0, 1, 10, 100, and 200 mg/kg/day from postnatal days 35-56. BPS at 1-10 mg/kg/day and higher doses markedly reduced serum testosterone and progesterone levels but it at 200 mg/kg/day significantly increased estradiol level. BPS at 100 and 200 mg/kg/day significantly elevated serum luteinizing hormone (LH) levels. BPS at 1-10 mg/kg/day and higher doses significantly reduced inhibin A and inhibin B levels. BPS at 100 and 200 mg/kg/day markedly increased CYP11A1+ Leydig cell number, but did not affect HSD11B1+ (a mature Leydig cell marker) cell number. BPS at 10 mg/kg/day and higher doses significantly downregulated the expression of Cyp11a1 and at 100 and 200 mg/kg/d significantly lowered Cyp17a1, Hsd11b1, and Nr5a1 in the testes. BPS at 100 and/or 200 mg/kg/day significantly elevated Lhb in the pituitary. BPS at 100 and 200 mg/kg/day significantly increased the phosphorylation of AKT1, AKT2, and CREB without affecting total AKT1, AKT2, and CREB levels. BPS at 1-100 µM significantly suppressed testosterone production and induced proliferation of primary immature Leydig cells after 24 h of treatment and these actions were reversed by estrogen receptor α antagonist, ICI 182780, and partially reversed by vitamin E. BPS at 0.1-10 µM significantly increased oxidative stress of Leydig cells in vitro. BPS also directly inhibited 17ß-hydroxysteroid dehydrogenase 3 activity at 10-100 µM. In conclusion, BPS causes hypergonadotropic androgen deficiency in male rats during pubertal exposure via activating ESR1 and inducing ROS in immature Leydig cells and directly inhibiting 17ß-hydroxysteroid dehydrogenase 3 activity.

Curcumin analogues exert potent inhibition on human and rat gonadal 3ß-hydroxysteroid dehydrogenases as potential therapeutic agents: structure-activity relationship and in silico docking.

Curcuminoids are functional food additives, and the effect on gonadal hormone biosynthesis remains unclear. Gonads contain 3ß-hydroxysteroid dehydrogenase isoforms, h3ß-HSD2 (humans) and r3ß-HSD1 (rats), which catalyse pregnenolone into progesterone. The potency and mechanisms of curcuminoids to inhibit 3ß-HSD activity were explored. The inhibitory potency was bisdemethoxycurcumin (IC50, 1.68 µM) >demethoxycurcumin (3.27 µM) > curcumin (13.87 µM) > tetrahydrocurcumin (109.0 µM) > dihydrocurcumin and octahydrocurcumin on KGN cell h3ß-HSD2, while that was bisdemethoxycurcumin (1.22 µM) >demethoxycurcumin (2.18 µM) > curcumin (4.12 µM) > tetrahydrocurcumin (102.61 µM) > dihydrocurcumin and octahydrocurcumin on testicular r3ß-HSD1. All curcuminoids inhibited progesterone secretion by KGN cells under basal and forskolin-stimulated conditions at >10 µM. Docking analysis showed that curcuminoids bind steroid-active site with mixed or competitive mode. In conclusion, curcuminoids inhibit gonadal 3ß-HSD activity and de-methoxylation of curcumin increases inhibitory potency and metabolism of curcumin by saturation of carbon chain losses inhibitory potency.

Benzophenone-1 and -2 UV-filters potently inhibit human, rat, and mouse gonadal 3ß-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico docking analysis.

Benzophenone (BP) ultraviolet (UV) -filters have been widely used to prevent adverse effects of UV. Whether they can disrupt gonadal steroidogenesis remains unclear. Gonadal 3ß-hydroxysteroid dehydrogenases (3ß-HSD) catalyse the conversion of pregnenolone to progesterone. This study explored the effect of 12 BPs on human, rat, and mouse 3ß-HSD isoforms, and analysed the structure-activity relationship (SAR) and underlying mechanisms. The inhibitory potency was BP-1 (IC50, 5.66 ± 0.95 µM) > BP-2 (5.84 ± 2.22 µM) > BP-6 (185.8 ± 115.2 µM) > BP3-BP12 on human KGN 3ß-HSD2, BP-2 (5.90 ± 1.02 µM) > BP-1 (7.55 ± 1.26 µM) > BP3-B12 on rat testicular 3ß-HSD1, and BP-1 (15.04 ± 5.20 µM) > BP-2 (22.64 ± 11.81 µM) > BP-6（125.1 ± 34.65 µM）> BP-7 (161.1 ± 102.4 µM) > other BPs on mouse testicular 3ß-HSD6. BP-1 is a mixed inhibitor of human, rat, and mouse 3ß-HSDs, and BP-2 is a mixed inhibitor of human and rat 3ß-HSDs and a noncompetitive inhibitor of mouse 3ß-HSD6. 4-Hydroxyl substitution in the benzene ring plays a key role in enhancing potency of inhibiting human, rat, and mouse gonadal 3ß-HSDs. BP-1 and BP-2 can penetrate human KGN cells to inhibit progesterone secretion at ≥ 10 µM. Docking analysis revealed that the 4-hydroxyl group of BP-1 and BP-2 forms hydrogen bonds with residue Ser123 of human 3ß-HSD2 and residue Asp127 of rat 3ß-HSD1. In conclusion, this study demonstrates that BP-1 and BP-2 are the most potent inhibitors of human, rat, and mouse gonadal 3ß-HSDs and that there is a significant SAR difference.

MicroRNA profiling reveals the role of miR-133b-3p in promoting apoptosis and inhibiting cell proliferation and testosterone synthesis in mouse TM3 cells.

Late-onset hypogonadism (LOH) is an age-related clinical and biological syndrome in which serum testosterone deficiency is an important characteristic and diagnostic indicator. In this study, we firstly analyzed the difference in the expression level of three miR-133 s (including miR-133a-3p, miR-133a-5p, and miR-133b-3p) in rat testis samples, blood samples from mice before and 1 wk after testis removal, and mouse TM3 cells. Secondly, the mimics and inhibitors corresponding to the three miR-133 s of mouse were transfected into TM3 cells separately to determine the correlation between the three miRNAs. Finally, using mouse TM3 cells to analyze the effect of miR-133b overexpression or inhibition on the proliferation and apoptosis of mouse testicular Leydig cells, the effect on genes related to testosterone synthesis, and the effect on the level of testosterone in the culture medium. We found that, compared with the testis tissue of newborn rats, miR-133a-5p was increased in adult rats, and miR-133a-3p and miR-133b-3p were decreased. In addition, 1 wk after the testis was removed, the expression levels of these three miRNAs in the blood of adult mice decreased. The correlation of the three miRNAs was summarized, and it was found that miR-133b-3p played an important role in it. In TM3 cells, overexpression of miR-133b-3p suppressed the proliferation and promotes apoptosis of cells, suppressed the expression level of most genes related to cell proliferation and testosterone synthesis, and the concentration of testosterone in the culture medium decreased while these phenomena can be reversed by the inhibition of miR-133b-3p expression. It was found that miR-133b-3p can regulate testosterone production in TM3 cells at least by targeting FSCN1. The above results suggest that miR-133b-3p plays an important role in regulating testosterone synthesis. These findings also provide new candidate diagnostic indicators for late-onset hypogonadism in men and provide new clues for the further study of pathogenesis.

LncRNA HOTAIR promotes proliferation and suppresses apoptosis of mouse spermatogonium GC-1 cells by sponging miR-761 to modulate NANOS2 expression.

LncRNA HOX antisense intergenic RNA (HOTAIR) can regulate cancer-related gene expression and promote stem cell and tumor cell proliferation via mechanisms including the competing endogenous RNA (ceRNA) mechanism. HOTAIR is abundantly expressed in the genital tubercle of E11.5, E12.5, and E13.5 embryos, whereas it became barely detectable at E13.5 and expressed again in adult mouse testis. However, the underlying function and mechanism of HOTAIR in spermatogenesis have not been elucidated. Interestingly, other researchers reported that the function of gene Nanos C2HC-Type Zinc Finger 2 (nanos2) includes the maintenance of both the primordial germ cells (PGCs) and germline stem cells, and Nanos2 protein and transcripts (NANOS2) were detected only in PGCs from day E11.5 and undifferentiated spermatogonia in spermatogenesis. We therefore investigated the relationship between HOTAIR and NANOS2 in maintaining spermatogonial stem cell population. We found that, compared to the adult mouse, the expression levels of HOTAIR and NANOS2 in embryo mouse were significantly higher and miR-761expression level was lower. In mouse GC-1 spermatogonia cells, overexpression of miRNA-761 significantly inhibited the expression of NANOS2 and HOTAIR, suppressed the proliferation, and promotes apoptosis of cells. Knock down and overexpression of HOTAIR indicated that HOTAIR expression was positively correlated with NANOS2 expression; overexpressed HOTAIR could promote proliferation and suppresses apoptosis of GC-1 cells. By a rescue experiment and dual luciferase reporter assay, miR-761 was identified as a direct target of HOTAIR, and NANOS2 was identified as the direct target of miR-761. The above results indicate that HOTAIR promotes proliferation and suppresses apoptosis of mouse spermatogonium GC-1 cells by sponging miR-761 to modulate NANOS2 expression. Our findings elucidate one of possible mechanisms and importance of HOTAIR in maintaining spermatogonial stem cell population, and provide new candidate genes and possible pathogenesis for male infertility.

Rutin inhibits androgen synthesis and metabolism in rat immature Leydig cells in vitro.

In the early stage of androgen-sensitive prostate cancer, cancer cells require androgens to grow. Hormone therapy that lowers androgen output or blocks androgen receptor can suppress the growth of this type of prostate cancer. Rutin, a flavonoid derivative of many plants, has numerous pharmacological effects. The objective of this study was to investigate the effect of rutin on androgen biosynthesis in Leydig cells isolated from the testes of pubertal rats. Immature Leydig cells isolated from 35 days-old male Sprague-Dawley rats were cultured in vitro with 0.5-50 µM rutin for 3 hr. Rutin significantly inhibited androgen secretion at 0.5, 5 and 50 µM under basal condition (medium only). At 50 µM, rutin also markedly compromised androgen secretion stimulated by 10 ng/ml luteinising hormone and 10 mM 8-bromoadenosine 3', 5'-cyclic monophosphate. Further analysis demonstrated that rutin compromised the transcript levels of Scarb1, Cyp11a1 and Hsd3b1 and their proteins expression. Rutin directly inhibited rat testicular CYP17A1, HSD17B3 and AKR1C14 activities at 50 µM. Rutin did not alter mitochondrial membrane potential at up to 50 µM. In conclusion, rutin suppresses androgen biosynthesis in Leydig cells through multiple mechanisms, thereby having benefits for the treatment of androgen-sensitive prostate cancer.

Analysis of semen quality of 38 905 infertile male patients during 2008-2016 in Wenzhou, China.

This study analyzed the trend in semen quality of infertile male patients in Wenzhou, China, based on the data obtained from 38 905 patients during 2008-2016 in The First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China). The results showed that only 24.9% of the patients had normal semen quality. For the semen quality of infertile male patients, that of the workers and 40-year-olds was significantly worse than the other occupational and age groups. For all the infertile patients, low semen volume, asthenozoospermia, and teratozoospermia accounted for 8.4%, 50.5%, and 54.1%, respectively. During 2008-2016, the annual mean percentage of fast forward motile spermatozoa, percentage of total forward motile spermatozoa, and percentage of spermatozoa with normal morphology decreased linearly with slopes of -2.11, -2.59, and -0.70, respectively. The proportion of patients with asthenozoospermia and multi-abnormal spermatozoa increased during 2008-2016 with slopes of 4.70 and 4.87, respectively, while for low semen volume, it decreased with a slope of -0.47 in the same time period. The proportion of patients with teratozoospermia increased from 2008 to 2011 and from 2011 to 2016 with slopes of 17.10 and 2.09, respectively. In general, the deteriorating trend of semen quality of infertile male patients in Wenzhou was obvious. Future efforts should be made to reveal the adverse influences on semen quality, such as occupational exposure, environmental quality, and living habits. Furthermore, more pervasive reproduction health education is necessary.

Neurotrophin-3 stimulates stem Leydig cell proliferation during regeneration in rats.

Neurotrophin-3 (NT-3) acts as an important growth factor to stimulate and control tissue development. The NT-3 receptor, TRKC, is expressed in rat testis. Its function in regulation of stem Leydig cell development and its underlying mechanism remain unknown. Here, we reported the role of NT-3 to regulate stem Leydig cell development in vivo and in vitro. Ethane dimethane sulphonate was used to kill all Leydig cells in adult testis, and NT-3 (10 and 100 ng/testis) was injected intratesticularly from the 14th day after ethane dimethane sulphonate injection for 14 days. NT-3 significantly reduced serum testosterone levels at doses of 10 and 100 ng/testis without affecting serum luteinizing hormone and follicle-stimulating hormone levels. NT-3 increased CYP11A1-positive Leydig cell number at 100 ng/testis and lowered Leydig cell size and cytoplasmic size at doses of 10 and 100 ng/testis. After adjustment by the Leydig cell number, NT-3 significantly down-regulated the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, Insl3, Trkc and Nr5a1) and the proteins. NT-3 increased the phosphorylation of AKT1 and mTOR, decreased the phosphorylation of 4EBP, thereby increasing ATP5O. In vitro study showed that NT-3 dose-dependently stimulated EdU incorporation into stem Leydig cells and inhibited stem Leydig cell differentiation into Leydig cells, thus leading to lower medium testosterone levels and lower expression of Lhcgr, Scarb1, Trkc and Nr5a1 and their protein levels. NT-3 antagonist Celitinib can antagonize NT-3 action in vitro. In conclusion, the present study demonstrates that NT-3 stimulates stem Leydig cell proliferation but blocks the differentiation via TRKC receptor.

Methylation levels of IGF2 and KCNQ1 in spermatozoa from infertile men are associated with sperm DNA damage.

Abnormal imprinted genes methylation in spermatozoa has been shown to be associated with subfertility. However, the relationship between sperm DNA damage and specific imprinted genes methylation remains unclear. In this study, DNA methylation levels were determined at seven imprinted genes loci (H19, INS-IGF2, KCNQ1, MEG3, MEST, PEG3 and SNRPN) in 66 semen samples using the MSRE-qPCR method. The semen samples were divided into two groups according to the threshold value (25%) of DNA fragmentation index (DFI). We found that the mean methylation level at IGF2 (cg17037101) in the group with DFI ≥ 25% was lower than that in the group with DFI < 25% (13.7 ± 3% vs. 31.5 ± 5.3%, p = 0.0053). However, the methylation levels of other CpGs did not differ from the imprinted genes. Correlation analysis of DFI with the methylation levels of imprinted genes demonstrated that the IGF2 (cg17037101) methylation level was negatively correlated with sperm DFI (r = -0.448, p = 0.0038), and the KCNQ1 (cg24932449) methylation level was positively correlated with sperm DFI (r = 0.354, p = 0.0273). Our results suggest that the aberrant methylation of IGF2 and KCNQ1 genes may be associated with sperm DNA damage.

[A260G and A386G single nucleotide polymorphisms of the DAZL gene are not correlated with male infertility in the Chinese population of Zhejiang Province].

OBJECTIVE: To assess the association of the A260G and A386G single nucleotide polymorphisms (SNP) of the DAZL gene with male infertility in the Chinese population of Zhejiang Province. METHODS: We collected the peripheral blood samples from 317 idiopathic infertile males with azoospermia or oligozoospermia and 246 normal fertile men, and genotyped the polymorphic loci of the A260G and A386G polymorphisms of the DAZL gene using the SNaPshot technique. RESULTS: The DAZL gene A260G was found genetically polymorphic in the Chinese population of Zhejiang Province, with the gene frequencies and their distribution consistent to the Hardy-Weinberg equilibrium. The frequencies of the AA, AG and GG genotypes of the A260G polymorphism were 92.3%, 7.3%, and 0.4% respectively in the normal controls and 94.3%, 5.7%, and 0% in the infertile patients, with no statistically significant differences between the two groups (P = 0.43, OR = 0.78, 95% CI 0.413-1.46). Heterozygosis (AG) of A386G was found in 1 of the control males but not in the infertile patients, while homozygosis (GG) of A386G was not observed in either group (P = 0.259, OR = 0.698, 59% CI: 0.374-1.306). CONCLUSION: A260G and A386G SNPs of the DAZL gene are not associated with spermatogenic failure and neither represents a molecular marker for the genetic diagnosis of male infertility in the Chinese population of Zhejiang Province.

Effect of sperm DNA fragmentation on the clinical outcomes for in vitro fertilization and intracytoplasmic sperm injection in women with different ovarian reserves.

OBJECTIVE: To investigate effect of sperm DNA fragmentation (SDF) on clinical outcomes of assisted reproductive technology in women with normal ovarian reserve (NOR) versus reduced ovarian reserve (ROR). DESIGN: Retrospective clinical study. SETTING: University-affiliated tertiary teaching hospital. PATIENT(S): A total of 2,865 consecutive couples undergoing their first in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycle. INTERVENTION(S): SDF assessed using sperm chromatin dispersion in sperm samples 1-2 months before treatment. MAIN OUTCOME MEASURE(S): SDF, IVF, and ICSI outcomes. RESULT(S): The grouping criteria were [1] basal follicle stimulating hormone >10 IU/L, [2] antral follicle count <6, and [3] female age ≥38 years. Women fulfilling two of the three criteria were considered to have ROR, and those not meeting any criteria were considered to have NOR. The area under the receiver operating characteristic curve was 0.594 (0.539-0.648) for the ROR group and 0.510 (0.491-0.530) for the NOR group. A cutoff value for SDF to predict the clinical pregnancy rate (CPR) in the ROR group was 27.3%. When the SDF exceeded 27.3%, the live-birth and implantation rates in the ROR group were statistically significantly decreased, but the clinical pregnancy, live-birth, and implantation rates were not affected in the NOR group. The risk of early abortion increased significantly in the NOR group when the SDF exceeded 27.3%. CONCLUSION(S): Sperm DNA fragmentation has a greater impact on IVF and ICSI outcomes among women with ROR, so SDF testing may be of particular clinical significance for these couples.

Effect of sperm DNA fragmentation on clinical outcome of frozen-thawed embryo transfer and on blastocyst formation.

During the last decades, many studies have shown the possible influence of sperm DNA fragmentation on assisted reproductive technique outcomes. However, little is known about the impact of sperm DNA fragmentation on the clinical outcome of frozen-thawed embryo transfer (FET) from cycles of conventional in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI). In the present study, the relationship between sperm DNA fragmentation (SDF) and FET clinical outcomes in IVF and ICSI cycles was analyzed. A total of 1082 FET cycles with cleavage stage embryos (C-FET) (855 from IVF and 227 from ICSI) and 653 frozen-thawed blastocyst transfer cycles (B-FET) (525 from IVF and 128 from ICSI) were included. There was no significant change in clinical pregnancy, biochemical pregnancy and miscarriage rates in the group with a SDF >30% compared with the group with a SDF ≤30% in IVF and ICSI cycles with C-FET or B-FET. Also, there was no significant impact on the FET clinic outcome in IVF and ICSI when different values of SDF (such as 10%, 20%, 25%, 35%, and 40%) were taken as proposed threshold levels. However, the blastulation rates were significantly higher in the SDF ≤30% group in ICSI cycle. Taken together, our data show that sperm DNA fragmentation measured by Sperm Chromatin Dispersion (SCD) test is not associated with clinical outcome of FET in IVF and ICSI. Nonetheless, SDF is related to the blastocyst formation in ICSI cycles.

[Diagnostic value of sperm DNA fragmentation for male infertility].

OBJECTIVE: To assess the diagnostic value of sperm DNA fragmentation (SDF) for male infertility. METHODS: Two hundred and ninety-nine males attending infertility clinic were classified into 157 primary infertile cases and 142 fertile controls. Semen analysis was performed as recommended by the World Health Organization (WHO). SDF was assessed by sperm chromatin dispersion (SCD) assay, and the results were expressed as DNA fragmentation index (DFI). RESULTS: The DFI was significantly higher in infertile males than that in fertile controls [(17.1± 9.3)% vs. (14.2± 9.0)%](P< 0.01). No significant difference was detected in the age of male and female partners, seminal volume, sperm count, motility and morphology between infertile males and fertile controls (P> 0.05). The area under the receiver operating characteristic curve (AUC) was 0.861 [95% confidence interval (CI)= 0.814-0.907] for 15.1% of SDF. The threshold level of 15.1% was derived as cut-off value to discriminate infertile men from fertile controls. By this threshold, specificity was 88.2% and sensitivity was 81.8%. The 299 men were divided into group A (n= 120) with DFI≥ 15.1% and group B (n= 179) with DFI< 15.1% based on the cut-off value. The percentage of infertile men in group A was significantly higher than that in group B (79.2% vs. 34.6%) (P< 0.01). The odds ratio (OR) for infertility in the two groups was 7.2 (95%CI= 4.2-12.3). CONCLUSION: Sperms with high-level of DNA fragmentation can impair male fertility. DFI can be used as a good diagnostic marker for male infertility.

[PDE5 inhibitors for the management of temporary penile erectile dysfunction during treatment with assisted reproductive technology].

OBJECTIVE: To evaluate phosphodiesterase type 5 (PDE5) inhibitors in the management of temporary penile erectile dysfunction (ED) in patients undergoing assisted reproductive technology (ART). METHODS: This study included 75 male patients that experienced ejaculation failure due to temporary ED during ART treatment. We treated the patients with PDE5 inhibitors sildenafil, tadanafil and vardenafil, and then evaluated the hardness of penile erection using Erection Hardness Score (EHS) and analyzed the end-point efficacy. RESULTS: Sildenafil was administered to 28 of the patients, tadanafil to 25, and vardenafil to 22. Of the total number of patients, 61 (81.3%) achieved effective erection, but no significant differences were observed in the rate of effectiveness among the sildenafil (24 cases, 85.7%), tadanafil (20 cases, 80.0%) and vardenafil (17 cases, 77.3%) groups (P > 0.05). After medication, 53 (70.7%) of the patients successfully ejaculated, but there were no remarkable differences in the success rate among the sildenafil (21 cases, 75.0%), tadanafil (17 cases, 68.0%) and vardenafil (15 cases, 68.2%) groups (P > 0.05). Of the 75 patients, 37 received the recommended initial dose and 38 the maximum recommended dose of PDE5 inhibitors, but no significant differences were found in the rate of successful sperm retrieval between the former (28 cases, 75.7%) and the latter group (25 cases, 65.8%) (P > 0.05). Mild adverse events, including transient flush and dizziness, occurred in 5 cases (6.7%). CONCLUSION: PDE5 inhibitors can help temporary ED patients to achieve penile erection and ejaculation during ART treatment.