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The effect of post-adolescence bisphenol A (BPA) exposure on the reproductive system is not well-defined. We therefore performed this meta-analysis to elucidate the associations between post-adolescence BPA exposure and reproductive-related outcomes. A search was performed on the PubMed, EMBASE, and Web of science databases to identify relevant literature. The standardized mean differences (SMDs) and the 95 % confidence intervals (CIs) were measured by fixed-effects or random-effects models. Publication bias was assessed using funnel plots and Egger's regression test. A total of 40 studies were included in the final analysis. The results showed that post-adolescence BPA exposure was negatively associated with reproductive-related organ weighty (Testis weight: SMD: -0.61; 95 % Cl: -0.85, -0.36; epididymis weight; SMD: -0.43; 95 % Cl: -0.69, -0.17; seminal vesicles weight; SMD: -0.77; 95 % Cl: -1.05, -0.49) and sperm parameters (Sperm motility: SMD: -1.44; 95 % Cl: -1.95, -0.93; epididymal sperm concentration: SMD: -2.26; 95 % Cl: -2.79, -1.72; and abnormal sperm morphology: SMD: 2.41; 95 % Cl: 1.41, 2.86). Moreover, BPA exposure decreased the level of testosterone (T) and superoxide dismutase (SOD), but increased the ratio of serum estradiol (E2) to T. This systematic review demonstrates that post-adolescence exposure to BPA may adversely affect reproductive functions in male rodents.

Oligoasthenoteratozoospermia (OAT) refers to the combination of various sperm abnormalities, including a decreased sperm count, reduced motility, and abnormal sperm morphology. Only a few genetic causes have been shown to be associated with OAT. Herein, we identified a novel homozygous frameshift mutation in meiosis-specific nuclear structural 1 (MNS1; NM_018365: c.603_604insG: p.Lys202Glufs*6) by whole-exome sequencing in an OAT proband from a consanguineous Chinese family. Subsequent variant screening identified four additional heterozygous MNS1 variants in 6/219 infertile individuals with oligoasthenospermia, but no MNS1 variants were observed among 223 fertile controls. Immunostaining analysis showed MNS1 to be normally located in the whole-sperm flagella, but was absent in the proband's sperm. Expression analysis by Western blot also confirmed that MNS1 was absent in the proband's sperm. Abnormal flagellum morphology and ultrastructural disturbances in outer doublet microtubules were observed in the proband's sperm. A total of three intracytoplasmic sperm injection cycles were carried out for the proband's wife, but they all failed to lead to a successful pregnancy. Overall, this is the first study to report a loss-of-function mutation in MNS1 causing OAT in a Han Chinese patient.

BACKGROUNDThe precise movement of proteins and vesicles is an essential ability for all eukaryotic cells. Nowhere is this more evident than during the remarkable transformation that occurs in spermiogenesis—the transformation of haploid round spermatids into sperm. These transformations are critically dependent upon both the microtubule and the actin cytoskeleton, and defects in these processes are thought to underpin a significant percentage of human male infertility.OBJECTIVE AND RATIONALEThis review is aimed at summarising and synthesising the current state of knowledge around protein/vesicle transport during haploid male germ cell development and identifying knowledge gaps and challenges for future research. To achieve this, we summarise the key discoveries related to protein transport using the mouse as a model system. Where relevant, we anchored these insights to knowledge in the field of human spermiogenesis and the causality of human male infertility.SEARCH METHODSRelevant studies published in English were identified using PubMed using a range of search terms related to the core focus of the review—protein/vesicle transport, intra-flagellar transport, intra-manchette transport, Golgi, acrosome, manchette, axoneme, outer dense fibres and fibrous sheath. Searches were not restricted to a particular time frame or species although the emphasis within the review is on mammalian spermiogenesis.OUTCOMESSpermiogenesis is the final phase of sperm development. It results in the transformation of a round cell into a highly polarised sperm with the capacity for fertility. It is critically dependent on the cytoskeleton and its ability to transport protein complexes and vesicles over long distances and often between distinct cytoplasmic compartments. The development of the acrosome covering the sperm head, the sperm tail within the ciliary lobe, the manchette and its role in sperm head shaping and protein transport into the tail, and the assembly of mitochondria into the mid-piece of sperm, may all be viewed as a series of overlapping and interconnected train tracks. Defects in this redistribution network lead to male infertility characterised by abnormal sperm morphology (teratozoospermia) and/or abnormal sperm motility (asthenozoospermia) and are likely to be causal of, or contribute to, a significant percentage of human male infertility.WIDER IMPLICATIONSA greater understanding of the mechanisms of protein transport in spermiogenesis offers the potential to precisely diagnose cases of male infertility and to forecast implications for children conceived using gametes containing these mutations. The manipulation of these processes will offer opportunities for male-based contraceptive development. Further, as increasingly evidenced in the literature, we believe that the continuous and spatiotemporally restrained nature of spermiogenesis provides an outstanding model system to identify, and de-code, cytoskeletal elements and transport mechanisms of relevance to multiple tissues.

Exposure to lead (Pb) is a major risk factor in reproductive toxicity, somatic, and germ cell genotoxicity. Exposure via deteriorating Pb paints and contaminated air, soil, and water had been the primary routes. However, with increasing reports of Pb accumulation in mushrooms and other food items may increase the etiology of Pb poisoning. The study herein investigated somatic genotoxicity and reproductive abnormalities in mice fed extracts of Pb-contaminated Pleurotus ostreatus. Male mice were fed aqueous extracts of P. ostreatus cultivated in 0, 10, 20, 100, 200, 500, and 1000 mg/L of Pb-contaminated rice straw for 35 days. Testes were analyzed for Pb accumulation, histopathology and relative weight gain, caudal epididymis for abnormal sperm morphology, and bone marrow for micronucleus test. Concentration-related significant increase in Pb accumulation was observed in P. ostreatus and testes of exposed mice. Decrease testicular weight, congestion of blood vessels, necrosis, and disorganization of the seminiferous tubules were observed in treated mice. In addition, fold increase of 2.78, 3.39, 6.67, 7.21, 9.63, and 9.70 in abnormal sperm morphology in accordance with the Pb concentrations respectively, confirmed reproductive toxicity. Significant increase in micronucleated polychromatic (PCE) and normochromatic (NCE) erythrocytes and concentration-related decrease PCE-NCE ratio in the bone marrow of treated mice suggest genome instability. Pb-contaminated P. ostreatus increased somatic and germ cell genotoxicity in mice. This may predispose the mice to genetic related syndromes and reproductive syndromes. It further suggests caution in the consumption of metal laden wild mushrooms and crop plants.

Varicocele is one of the most important causes of infertility in men which gradually leads to testicular dysfunction. Testicular heat stress-induced oxidative stress is considered the main cause of pathology in these individuals. In this study, the effects of curcumin and nano-curcumin, as natural antioxidants, were investigated on spermatogenesis and sperm function in varicocele-induced rats. Seventy Wistar rats were randomly divided into seven groups; sham, control, varicocele, varicocele + curcumin 50 mg, varicocele + curcumin 100 mg, varicocele + nano-curcumin 4 mg and varicocele + nano-curcumin 8 mg. After 2 months of antioxidant therapy, all the rats were sacrificed. The results demonstrated that the mean sperm concentration and motility were significantly lower while the mean of abnormal morphology, lipid peroxidation, intracytoplasmic ROS and DNA damage was significantly higher in varicocelised rats compared to control and sham groups (p < .05). Both doses of curcumin and also nano-curcumin were significantly effective in improving the aforementioned parameters except for abnormal sperm morphology, and motility where nano-curcumin (4 mg) was significantly more effective than other groups (p < .05). The results of the current study suggest the application of nano-curcumin is more preferable to curcumin in infertile individuals with varicocele.

Cadmium (Cd) as a heavy metal damages testis and decreases fertility, however, antioxidants can improve sperm parameters and decrease male infertility. In this study we investigated the effect of astaxanthin (AST) on sperm parameters, expression levels of CatSper1 and CatSper2 genes in presence of Cd in mice. Thirty adults’ mice were divided into 4 groups, sham group received olive oil and saline (olive oil is the solvent of AST and saline is the solvent of Cd), Cd group received 1 mg/kg Cdcl2, a group received 10 mg/kg AST and 1 mg/kg Cdcl2 and a group received 10 mg/kg AST. The treatments were done intraperitoneally for 14 days. After 14 days sperm parameters were analyzed. Malondialdehyde level, catalase enzyme activity, the alteration of CatSper1 and CatSper2 genes expression were measured in testis. Results showed that Sperm count, viability, CatSper1 gene expression and catalase activity significantly decreased by Cd compared to sham group. Cd significantly increased sperm DNA fragmentation (SDF), abnormal sperm morphology and malondialdehyd level compared to sham group. AST significantly increased sperm count, viability and CatSper1 gene expression and decreased SDF and abnormal sperm in comparison with Cd group. AST protected testis and decreased oxidative stress induced by Cd. Our findings indicated that AST could protect sperm DNA, enhanced CatSper1 gene expression and sperm quality in presence of Cd. No significant differences were found in CatSper2 expression among treatments. Therefore, AST as a strong antioxidant can help to protect the potential of fertility against Cd toxicity.

MUSASHI (MSI) family plays the main role in the spermatogenesis process. The purpose of this study was the assessment of sperm MSI1 and MSI2, and sperm functional tests in infertile men (n = 30) with varicocele and fertile men (n = 30). Furthermore, MSI1 and MSI2 proteins were assessed in testicular tissue of azoospermic men (n = 9) as well as epididymal spermatozoa and testis of mice. Expression of MSI1 and MSI2 was assessed at RNA and protein levels in human spermatozoa. Sperm concentration and motility were significantly lower, while abnormal sperm morphology, lipid peroxidation, DNA fragmentation and protamine deficiency were significantly higher in men with varicocele compared to fertile individuals. Any significant difference was not observed in the expression of MSI1 and MSI2 mRNA between the two groups. Unlike MSI1 protein that was not detectable in humans, the relative expression of MSI2 protein was similar in varicocele and fertile individuals. The expression level of both Msi1 and Msi2 proteins was also observable in mouse spermatozoa. No significant relationship was observed between sperm functional parameters with expression of these genes. The data of this study demonstrated that although MSI1 and MSI2 play important roles during spermatogenesis, their relative expression in spermatozoa was not affected by varicocele.

Quetiapine is one of the most commonly prescribed antipsychotics to treat schizophrenia in adults, in particular. In this study, quetiapine's effects were assessed on healthy sperm production in rats at repeated-pharmacological doses. Additionally, the effects of quetiapine on oxidative status and hormonal balance were also evaluated in rats. Quetiapine was administered to rats orally at 10, 20, and 40 mg/kg body weight doses for 28 days. At the end of this period, body and organ weights were measured, sperm concentration, motility, and morphology were determined, sperm damage was assessed, and histopathological analysis of testicular tissue was performed. Additionally, serum FSH, LH, and testosterone levels as male reproductive hormones were measured. Catalase, superoxide dismutase, glutathione, and malondialdehyde levels were determined for evaluating the oxidative status of testicular tissue. The findings obtained in this study showed that relative epididymis weights and sperm concentration decreased and abnormal sperm morphology increased in quetiapine-administered rats. Irregularity of typical architecture of the seminiferous tubules and germinal cell disorganization was observed in testicular sections of 20 and 40 mg/kg quetiapine-administered rats. Further, serum LH and testosterone levels decreased in 20 and 40 mg/kg quetiapine-administered rats. Additionally, decreased catalase and superoxide dismutase activities in testicular tissue of quetiapine-administered rats and increased malondialdehyde levels in testicular tissue of 40 mg/kg quetiapine-administered rats were measured. In conclusion, quetiapine treatment decreased sperm quality, altered hormone levels, and induced oxidative stress may be considered potential contributors to this adverse effect.

This study aimed to investigate the effects of heavy metals on measures of male fertility. One hundred and two infertile men with occupational exposure and thirty fertile men were included in this study. Blood and urinary levels of lead, cadmium, zinc and copper were measured by atomic absorption spectrophotometry. Semen parameters and a motile sperm organelle morphology examination were also performed. Measures of hormonal levels, oxidation-reduction potential, DNA fragmentation index and chromatin condensation were assessed for all participants. Heavy metals levels, oxidative stress and DNA quality were significantly higher in the infertile group compared to controls. FSH and testosterone levels were lower in the infertile group. A urinary cadmium level was positively associated with abnormal sperm morphology (r = .225, p < .05). Normal morphology was inversely correlated with the duration of the exposure (r = -.227, p = .022). The blood lead level was positively related to the level of testosterone (r = .223, p = .031). Cadmium and lead blood levels were positively correlated with the level of chromatin decondensation (r = .528, p < .001; r = .280, p = .017). Our study showed that occupational exposure to heavy metals is very harmful to reproductive health. DNA quality and oxidative stress investigations must be recommended for reprotoxic exposed patients prior to in vitro fertilisation treatment.

Sperm fibrous sheath, a unique cytoskeletal structure, is implicated in various sperm physiological functions, such as sperm maturation, motility and capacitation. AKAP4 has been described to be required for structural and functional integrity of the fibrous sheath. We generated Akap4-knockout mice line using CRISPR-Cas9 system. Cytomorphology and motility of sperm and testes were studied, confirming loss of Akap4 led to abnormal sperm morphology, motility and infertility. The proteomic components of testes were studied and Akap4 was found to be significantly decreased in the Akap4-knockout mice. Testis single-cell RNA sequencing and analysis revealed three genes with significant change in the general cell population, i.e., Akap4, Haspin, and Ccdc38. The single-cell RNA expression profiles also showed that the major difference between Akap4-knockout and wild-type testes existed in the elongating cell cluster, where in the Akap4-knockout testes, a subgroup of elongating cells with marker genes involved in cell adhesion and migration were increased, while a subgroup of elongating cells marked by mitochondrial sheath genes were decreased. Our results revealed the complex and well-coordinated procedures of spermatogenesis, and substantiated Akap4's indispensable roles in the integrity of sperm flagellum and the step-wise maturation of spermatozoa.

Background Evidences from human and animal studies suggest that reproductive function may be affected by mercury. The aim of this review was to explore the mercury influence on human fertility. Methods A systematic search was made in PubMED for papers published between 1975–2017, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Results Increased mercury levels were associated with infertility or subfertility status. Further, infertile subjects with unexplained infertility showed higher levels of mercury in hair, blood and urine than fertile ones. Mercury exposure induced sperm DNA damage and abnormal sperm morphology and motility. Additionally, mercury levels were related with higher incidence of menstrual and hormonal disorders and increased rates of adverse reproductive outcomes. Conclusions Our review showed that mercury negatively impacts human reproduction, affecting the reproductive and endocrine systems in both male and female. However, the molecular mechanisms underlying the mercury-associated decline on fertility remains unknown.

Despite the association between tobacco use and the harmful effects on general health as well as male fertility parameters, smoking remains globally prevalent. The main content of tobacco smoke is nicotine and its metabolite cotinine. These compounds can pass the blood-testis barrier, which subsequently causes harm of diverse degree to the germ cells. Although controversial, smoking has been shown to cause not only a decrease in sperm motility, sperm concentration, and an increase in abnormal sperm morphology, but also genetic and epigenetic aberrations in spermatozoa. Both animal and human studies have highlighted the occurrence of sperm DNA-strand breaks (fragmentation), genome instability, genetic mutations, and the presence of aneuploids in the germline of animals and men exposed to tobacco smoke. The question to be asked at this point is, if smoking has the potential to cause all these genetic aberrations, what is the extent of damage? Hence, this review aimed to provide evidence that smoking has a mutagenic effect on sperm and how this subsequently affects male fertility. Additionally, the role of tobacco smoke as an aneugen will be explored. We furthermore aim to incorporate the epidemiological aspects of the aforementioned and provide a holistic approach to the topic.

International audience; Varicocele is characterized by testicular dysfunction that originates from hyperthermia and hypoxia, leading to defects in testicular tissue and altered spermatozoa structure and function. The varicocele testis is characterized by the presence of intracellular iron deposits that contribute to the associated oxidative stress. Therefore, we tested the hypothesis that administration of an iron-chelating agent, such as deferasirox (DFX), could potentially mitigate the consequences of varicocele on testicular tissue and spermatozoa. Using a well-established rat model of varicocele (VCL), we show that treatment with DFX partially improved the structure and function of the testis and spermatozoa. In particular, sperm motility was markedly restored whereas abnormal sperm morphology was only partially improved. No significant improvement in sperm count was observed that could be associated with the proapoptotic response observed following iron chelation treatment. No reduction in oxidative damage to spermatozoa was observed since lipid peroxidation and DNA integrity were not modified. This was suggested to be a result of increased oxidative stress. Finally, we also saw no indication of attenuation of the endoplasmic reticulum/unfolded protein (ER/UPR) stress response that we recently found associated with the VCL testis in rats.

Protein phosphatase 4 (PPP4) is a protein phosphatase that, although highly expressed in the testis, currently has an unclear physiological role in this tissue. Here, we show that deletion of PPP4 catalytic subunit gene Ppp4c in the mouse causes male-specific infertility. Loss of PPP4C, when assessed by light microscopy, did not obviously affect many aspects of the morphology of spermatogenesis, including acrosome formation, nuclear condensation and elongation, mitochondrial sheaths arrangement and ‘9 + 2’ flagellar structure assembly. However, the PPP4C mutant had sperm tail bending defects (head-bent-back), low sperm count, poor sperm motility and had cytoplasmic remnants attached to the middle piece of the tail. The cytoplasmic remnants were further investigated by transmission electron microscopy to reveal that a defect in cytoplasm removal appeared to play a significant role in the observed spermiogenesis failure and resulting male infertility. A lack of PPP4 during spermatogenesis causes defects that are reminiscent of oligoasthenoteratospermia (OAT), which is a common cause of male infertility in humans. Like the lack of functional PPP4 in the mouse model, OAT is characterized by abnormal sperm morphology, low sperm count and poor sperm motility. Although the causes of OAT are probably heterogeneous, including mutation of various genes and environmentally induced defects, the detailed molecular mechanism(s) has remained unclear. Our discovery that the PPP4C-deficient mouse model shares features with human OAT might offer a useful model for further studies of this currently poorly understood disorder.

Male infertility is a global health problem, and the underlying molecular mechanisms are not clearly known. Ion channels and microRNAs (miRNAs), known to function in many vital functions in cells, have been shown to play a significant role in male infertility through changes in their expressions. The study aimed to evaluate the alterations of testicular and/or spermatozoal potassium voltage-gated channel subfamily J member 11 (KCNJ11), Cystic fibrosis transmembrane conductance regulator (CFTR), miR-let-7a and miR-27a expressions in carbamazepine-related male infertility. Here, we showed that carbamazepine reduced sperm motility, increased abnormal sperm morphology, and impaired hormonal balance as well as increased relative testis weight and decreased relative seminal vesicle weight. On the other hand, downregulated KCNJ11 and upregulated miR-let-7a expressions were determined in testis (p < .05). Also, downregulated KCNJ11 and upregulated CFTR and miR-27a expressions were found in spermatozoa (p < .05). Interestingly, altered testicular KCNJ11 and miR-let-7a expressions were correlated with decreased sperm motility and elevated sperm tail defect. Besides, spermatozoal CFTR and miR-27a expressions positively correlated with sperm tail defects. The results indicated a significant relationship between ion channel and/or miRNA expression alterations and impaired sperm parameters due to carbamazepine usage.

Intracytoplasmic sperm injection (ICSI) is increasingly used to treat male-factor infertility when sperm parameters are not proper for intrauterine insemination (IUI) or in vitro fertilization (IVF). Among sperm abnormalities, short tail sperm defect is a rare kind of teratozoospermia, which is a severe cause of male infertility. In this study, we evaluated the ICSI outcomes of infertile men with severely short tail sperm defect. 117 infertile men with primary infertility were included in this study. We evaluated the impact of short tail sperm defect on large ICSI series (228 cycles) outcomes. The fertilisation rate (FR) was 49.0%, the clinical pregnancy rate (PR) was 21.7%, and the delivery rate (DR) was 17.5%. The results of statistical analysis show that there is no relationship between short tail sperm defect and clinical pregnancy. According to the present study, there were patients with successful ICSI outcomes despite the severe defect in their spermatozoa flagella. Our results can be considered in two main aspects: (a) it seems that ICSI could be a proper therapy for infertile men with short-tailed sperm defect and (b) the abnormal sperm morphology (especially in sperm flagellum) is not a reliable predictor for the ICSI outcomes. In conclusion, our study suggests that ICSI should be considered as a proper treatment way for infertile men with severe short tail sperm defect and probably other sperm flagella abnormalities.

The main purpose of this project is to verify whether there is a difference in the expression of aryl hydrocarbon receptor (AhR) between varicocele (VC) and normal male semen, and determine whether there is a connection with the parameters of semen analysis. The risk factors of infertility in patients with VC were also studied. Semen samples were collected for semen analysis and Western blot. Logistic regression was used to investigate the risk factors associated with infertility in patients with VC. Men with VC had lower AhR expression compared to healthy men; correlation analysis results showed that: AhR expression in patients with VC group was significantly positively correlated with sperm concentration and sperm motility; significantly negatively correlated with the diameter of spermatic veins during Valsalva and the percentage of abnormal sperm morphology; the research results of related risk factors show that the risk of infertility of patients with grade III is 1.67 times that of patients with grades I and II. For each unit of abnormal sperm morphology, the risk of infertility increases 1.04 times. Sperm concentration, total sperm viability and each unit the expression of AhR protein decreases the risk of infertility by 3%, 9% and 11% respectively.

Ethanol consumption is a major cause of male infertility, but the exact mechanism is still largely unknown. This study attempted to investigate the effect of ethanol on sperm morphology, acrosome reaction status and the alteration of the testicular protein expressions. Fourteen male rats were divided into control and ethanol groups (n = 7/each group). Ethanol-treated rats received ethanol (5 g/kg, 40% v/v) via oral gavage for consecutive 14 days. Testosterone hormone, sperm parameters, and testicular and epididymal histopathologies were evaluated. In addition, the expressions of testicular proteins including androgen receptor (AR), heat shock protein 70 (HSP70), steroidogenic acute regulatory protein (StAR) and tyrosine-phosphorylated (TyrPho) proteins were investigated. The results showed that ethanol significantly increased percentage of abnormal sperm morphology and acrosome-reacted spermatozoa. Some seminiferous and ductus epididymal histopathologies were observed in ethanol-treated rats. Significantly, ethanol reduced serum testosterone and expressions of testicular AR and TyrPho proteins. However, the overexpression of StAR and HSP70 proteins in ethanol testis was found. It was concluded that the changes in testicular protein expressions may be involved in mechanism of male infertility caused from ethanol consumption.

Importin α proteins play a central role in the transport of cargo from the cytoplasm to the nucleus. In this study, we observed that male knock-out mice for importin α4, which is encoded by the Kpna4 gene (Kpna4-/- ), were subfertile and yielded smaller litter sizes than those of wild-type (WT) males. In contrast, mice lacking the closely related importin α3 (Kpna3-/- ) were fertile. In vitro fertilization and sperm motility assays demonstrated that sperm from Kpna4-/- mice had significantly reduced quality and motility. In addition, acrosome reaction was also impaired in Kpna4-/- mice. Transmission electron microscopy revealed striking defects, including abnormal head morphology and multiple axoneme structures in the flagella of Kpna4-/- mice. A five-fold increase in the frequency of abnormalities in Kpna4-/- mice compared to WT mice indicates the functional importance of importin α4 in normal sperm development. Moreover, Nesprin-2, which is a component of the linker of nucleus and cytoskeleton complex, was expressed at lower levels in sperm from Kpna4-/- mice and was localized with abnormal axonemes, suggesting incorrect formation of the nuclear membrane-cytoskeleton structure during spermiogenesis. Proteomics analysis of Kpna4-/- testis showed significantly altered expression of proteins related to sperm formation, which provided evidence that genetic loss of importin α4 perturbed chromatin status. Collectively, these findings indicate that importin α4 is critical for establishing normal sperm morphology in mice, providing new insights into male germ cell development by highlighting the requirement of importin α4 for normal fertility.

This study was carried out to evaluate the level of nuclear sperm DNA damage in men with isolated polymorphic teratozoospermia and examining its relationship with apoptosis and oxidative stress. A total of 89 subjects were divided into two groups: men with isolated teratozoospermia (n = 69) and men with normal semen parameters (n = 20) as controls. Sperm DNA breaks were determined by using acridine orange staining. The proportion of viable spermatozoa with mitochondrial transmembrane depolarization was detected by fluorescence microscopy through the use of MitoPTJC-1 staining method. Bivariate Annexin V/6-CFDA analysis was then set out in order to measure the percentage of both viable and dead spermatozoa with phosphatidylserine (PS) externalization. Seminal antioxidant profile (reduced glutathione (GSHr); oxidized glutathione (GSSG); glutathione S-transferase (GST)) and total protein sulfhydryl (P-SH) concentrations were measured spectrophotometrically. Men with isolated teratozoospermia, when compared to controls, showed significantly increased level of single sperm DNA breaks and higher proportions of spermatozoa with phosphatidylserine externalization and mitochondrial depolarization. Among the differently studied oxidative stress seminal parameters, the rates of seminal GSHr, GST, and P-SH were significantly decreased in the teratozoospermic group. However, the seminal rates of GSSG and GST have decreased, but only GST did not show a significant difference. Interestingly, significant correlations were found between the studied apoptotic markers and the rate of atypical sperm forms with the incidences of head abnormalities. Furthermore, positive inter-correlations were found between sperm DNA defects, impaired seminal antioxidant status, and the apoptotic sperm markers. Such data provide clear evidence that the apoptotic alterations are closely correlated to abnormal sperm morphology and DNA damage. Moreover, decreased seminal antioxidant profile may be a crucial factor involved in the mechanism of sperm cell death-mediated DNA breaks in teratozoospermic semen.

DNA fragmentation can be deleterious on spermatozoon morphology but the pathogenesis of teratozoospermia associated with DNA breaks is not fully understood, even if oxidative attacks and defects in chromatin maturation are hypothesized. Therefore, this study is one of the first to clarify on the underlying hypothesizes behind such observations. The objectives of our study were to assess the role of oxidative attacks in DNA damage pathogenesis in ejaculated spermatozoa from patients with isolated teratozoospermia. We aimed to assess the correlation of DNA breaks with morphologically abnormal spermatozoa, as well as ROS level and impairment chromatin condensation. A total of 90 patients were divided into two groups, men with isolated teratozoospermia (n = 60) and men with normal semen parameters (n = 30) as controls. DNA fragmentation was evaluated by TUNEL assay; chromatin immaturity was studied using acridine orange and toluidine blue staining. We evaluated the ability of spermatozoa to produce reactive oxygen species with nitro blue tetrazolium staining. Patient with teratozoospermia when compared to fertile men showed significantly higher rates of semen ROS production, sperm hypocondensated chromatin, denaturated DNA, and fragmented DNA. All these parameters were positively correlated with abnormal sperm morphology. The studied DNA integrity markers were also correlated with ROS production. Fragmented DNA is the main pathway leading to morphology defects in the sperm. In fact, impaired chromatin compaction may induce DNA breaks and free radicals, which can break the DNA backbone indirectly, by reducing protamination and disulphide bond formation, as oxidative attack appears to be the major cause of poor semen morphology.

The human sperm nucleus contains cytoplasm. However, the origin and incidence of human sperm intranuclear cytoplasmic retention (INCR) remain unknown. The objectives of this study were to observe the morphological origin of INCR within the seminiferous epithelium and investigate the incidence of INCR in fertile and teratozoospermia men using transmission electron microscopy (TEM). By TEM, INCR initially appeared in elongating round spermatid nuclei and varied in size, number, shape, content, location and distribution within sperm nuclei. The teratozoospermia group (n = 16) demonstrated a higher incidence of INCR than did the fertile group (n = 16) (17.6 ± 5.2% vs. 9.7 ± 3.4%; p = 0.000). In the fertile group, no correlations were found between the incidence of INCR and abnormal sperm morphology, nuclear vacuole, acrosome integrity, motility or concentration (p > 0.05). However, the incidence of INCR exhibited a positive relationship with sperm abnormal morphology in the teratozoospermia group (r = 0.616, p = 0.011). These results demonstrate that INCR occurs in the early process of spermatogenesis and is an alteration found in the nucleus. Spermatozoa from teratozoospermia men contained more INCRs than those from fertile males. More attention should be paid to the possibility of spermatozoa containing INCR when using spermatozoa with abnormal head morphology for clinical or diagnostic purposes.

Teratozoospermia is a disorder associated with high abnormal sperm morphology which affects fertility in males. In recent years, it has been described that biomarker-based sperm quality evaluation can alleviate male infertility treatment. Phospholipase C zeta (PLCζ) is a sperm-specific factor which appears to be a predicting biomarker for fertilization potential of males. Following fertilization, PLCζ enters into oocyte cytoplasm and induces oocyte activation, a fundamental stage in initiation of embryo development. Currently, PLCζ parameters, including localization patterns, the proportion of PLCζ-expressing sperm and the expression level, are not defined in polymorphic teratozoospermic men. This study aimed to evaluate PLCζ parameters in polymorphic teratozoospermic men, and compare these parameters with fertile normozoospermic men. Semen samples from thirteen normozoospermic fertile men and twenty-three polymorphic teratozoospermic men were included in this study and evaluated using western blotting and immunofluorescence analyses. Our data indicated significantly lower expression of PLCζ in polymorphic teratozoospermic men, as compared with control men; however, there was no significant difference in localization patterns and the proportion of PLCζ-expressing sperm between polymorphic teratozoospermic patients and control men. Collectively, findings from the present study demonstrated that polymorphic teratozoospermic men did not show abnormal localization patterns or the absence of PLCζ, as compared to the control men; nonetheless, lower expression of PLCζ, considering its role in oocyte activation, might be one of the possible causes of infertility in these patients.

Our previous work has demonstrated that microcystin-leucine arginine (MC-LR) is a potent toxin for the reproductive system of male mice and it exerts cytotoxicity in spermatogonial cells, resulting in the constitutional and functional changes of the mouse testis. The present study was designed to investigate the functions of P-element-induced wimpy (piwi)-interacting RNAs (piRNAs) in MC-LR-induced reproductive toxicity in male mice. We observed an increase in the mmu_piR_003399 level in spermatogonial cells and mouse testicular tissues following treatment with MC-LR. Moreover, our data confirmed that cyclin-dependent kinase 6 (CDK6) was the target gene of mmu_piR_003399. Increases in the concentration of mmu_piR_003399 were correlated with the reduced expression of CDK6 both in vitro and in vivo. mmu_piR_003399 induced cell cycle arrest at the G1-phase, down-regulated sperm counts and sperm motility, and compromised sperm morphology. On the contrary, suppressing the expression of mmu_piR_003399 could substantially attenuate MC-LR-induced pathology in mice including cell cycle arrest, reduced mature sperm counts, sperm viability loss and abnormal sperm morphology. Furthermore, our data supported that mmu_piR_003399 existed in mouse serum and plasma, and its level was increased in MC-LR-treated mice. In conclusion, we demonstrate that mmu_piR_003399 plays a crucial role in regulating MC-LR-induced reproductive toxicity.

The endocrine-disrupting properties of paracetamol have been previously demonstrated in rodent studies of abnormal sperm morphology and diminished testosterone production, in addition to epidemiologic studies of diminished couple fecundity. In this study, we examined the relationship between paracetamol and its metabolite p-aminophenol quantified in a single spot urine and semen quality among 501 male partners of couples planning for pregnancy. Men provided a urine specimen and two fresh semen samples collected approximately one month apart and underwent 24-h analysis for 35 semen quality parameters. Paracetamol and p-aminophenol were quantified in urine by ultra-high-performance liquid chromatography coupled with an electrospray triple quadrupole mass spectrometry. The relationship between natural-log-transformed urinary paracetamol and p-aminophenol rescaled by their standard deviation and 21 Box-Cox-transformed, 14 non-transformed semen parameters was assessed using linear mixed-effects models. The median concentrations (IQR) of urinary paracetamol and p-aminophenol were 15.5 ng/mL (5.44, 73.5) and 978 ng/mL (500, 1596), respectively. Following adjustment for creatinine and age, a 1-standard deviation increase in log-transformed urinary paracetamol was associated with a reduction in beat cross-frequency and an increase in DNA fragmentation [β (95% CI): -0.59 Hz (-1.16, -0.03) and 0.05% (0.01, 0.09), respectively]. These findings were corroborated in models of categorical chemical concentrations; higher concentrations of paracetamol remained associated with reduced beat cross-frequency and increased DNA fragmentation. A 1-standard deviation increase in log-transformed urinary p-aminophenol was associated with a reduction in sperm head area [β (95% CI): -0.1 μm2 (-0.18, -0.02) and width -0.02 μm (-0.04, -0.01)]. However, only the association with sperm head area remained statistically significant in models of p-aminophenol quartiles. Our findings suggest that adult male urinary paracetamol is associated with sperm motility and DNA fragmentation, while the metabolite, p-aminophenol, is predominantly associated with sperm head morphometry.

This study was aimed at determining whether an in vivo subcutaneous exposure to n-butylparaben (n-ButP) during one complete spermatogenic cycle could be harmful to the reproductive system of young male rats. Animals were subcutaneously given 0, 150, 300 and 600 mg/kg/day of n-ButP with vehicle (peanut oil). Body and organ weights, n-ButP excretion, biochemical parameters, sperm and spermatid count, sperm motility, viability, maturity and morphology were examined. Results showed that after a completed spermatogenic cycle, although n-ButP did not induce dose-related changes in the different biochemical parameters, a significant decrease of triacylglicerides (TAG) –due to the vehicle-was found. Furthermore, no effects of n-ButP on body weight gain and relative organ weight changes were noted. Regarding sexual organs, prostate relative weight was significantly increased at the high dose of n-ButP. On the other hand, a significant increase of abnormal sperm morphology due to n-ButP exposure, accompanied by different alterations in sexual organs histopathology, was found. The current results indicate that subcutaneous exposure of n-ButP in young male rats induced toxic effects on the reproductive system, which could affect the capacity of fertilization of animals.

In 1979, more than 2000 persons ingested rice oil contaminated with polychlorinated biphenyls and polychlorinated dibenzofurans; this event was called the “Yucheng accident.” An increased percentage of oligospermia, reduced ability of sperm to penetrate oocytes, and reduced percentage of male offspring were reported in Yucheng men. This study examined whether the sperm sex ratio and chromosome aneuploidy are responsible for our observed findings in Yucheng men. In 1999–2000, Yucheng men and their neighborhood referents aged 37–50 years were recruited for physical examination, followed by semen analysis. The semen samples were analyzed for chromosomal aneuploidy through fluorescent in situ hybridization according to an established procedure in our laboratory. A total of 50 Yucheng men and 34 neighborhood referents volunteered to participate in the study. Although abnormal morphology was mildly increased, no differences were observed in sperm percentages, with normal numbers of chromosomes X, Y, and 8 in the two groups. The percentage of sperm with aneuploidy of the sex chromosomes or chromosome 8 and of that with diploidy did not vary between both groups. The normal X/Y sperm ratio was not different between the groups. However, among Yucheng men, 8% had a normal X/Y sperm ratio of >1.4, and no neighborhood referent showed such an elevated X/Y ratio. Chromosomal aneuploidy was not elevated in Yucheng men. The mechanisms underlying the reduced sperm capability of oocyte penetration and changed offspring sex ratio in Yucheng men remain undetermined.

Vitamin K is present in the testes though its actual function in male reproduction is poorly understood. This study investigated the harmful effect of extrahepatic vitamin K insufficiency on the testicular structure. Sprague-Dawley rats were fed with a diet containing warfarin for 2, 4 and 8 weeks; control animals received a standard diet without warfarin. It was found that extrahepatic vitamin K deficiency that is induced by warfarin results in histopathological features that range from delayed spermiation, presence of multinucleated giant cells in the seminiferous tubules, germ cells degeneration, asthenozoospermia, oligozoospermia and increase in the percentage of abnormal sperm morphology when compared to the controls. Data obtained from the two groups were analysed using the Student t test. It is concluded that warfarin-induced vitamin K deficiency has a negative impact on spermatogenesis.

Objective To investigate the applicability of phospholipase C zeta (PLCζ) analysis in assisting the clinical decision-making process when considering artificial oocyte activation (AOA) for infertile males in assisted reproductive technology. Design Fifty-six males (43 infertile/13 fertile) were screened using our PLCζ assay. Setting Fertility unit/university laboratory. Patient(s) Infertile males with abnormal sperm morphology or total fertilization failure, low fertilization rate ( Intervention(s) We analyzed PLCζ levels in sperm from fertile and infertile males. Eligible patients subsequently underwent intracytoplasmic sperm injection (ICSI)/artificial oocyte activation (AOA) with calcimycin (GM508). Main Outcome Measure(s) PLCζ localization and level and the proportion of sperm expressing PLCζ. Thresholds of PLCζ deficiency, fertilization rates, pregnancy rates, and live birth rates of AOA and non-AOA cycles. Result(s) Compared with 13 fertile controls, 34 of the 43 infertile males had significantly lower levels of PLCζ and/or a significantly lower proportion of sperm exhibiting PLCζ. Of these 34 patients, 15 showed a significant PLCζ reduction in both parameters, which we termed “PLCζ deficiency.” Five PLCζ-deficient patients opted for AOA; all five achieved fertilization, and four achieved clinical pregnancies and live births. The fertilization rate improved significantly from 18.6% (ICSI) to 56.8% (ICSI/AOA). The clinical pregnancy rate and live birth rate with AOA were both 40% per initiated cycle. Youden index analysis revealed that the cutoffs below which infertile males were likely to benefit from AOA were 71% for the proportion of sperm expressing PLCζ and 15.57 arbitrary units for mean PLCζ level. Conclusion(s) PLCζ analysis is a useful diagnostic tool to determine patient eligibility for subsequent AOA treatment.

We evaluated the live birth rate and the prevalence of congenital anomalies in couples undergoing intrauterine insemination with abnormal sperm morphology (less than 4% normal forms).We retrospectively reviewed intrauterine insemination outcomes from January 2012 to March 2015. Patients who were found to have an ultrasound confirmed clinical pregnancy were contacted to determine the live birth rate and the prevalence of congenital abnormalities. We used chi-square analysis to assess categorical variables and the Student t-test to assess continuous variables. Logistic regression was done to assess the odds of achieving pregnancy and the risk of spontaneous abortion while assessing female age, the total motile count and sperm morphology.In 984 intrauterine insemination procedures performed in a total of 501 couples we found no difference in the ultrasound clinical pregnancy rate in couples with sperm morphology less than 4% vs 4% or greater (12.3% vs 13.6%, p=0.59). We collected live birth and birth abnormality data on 95 of the 130 couples with ultrasound confirmed clinical pregnancy for a 73% response rate. We found no difference in the live birth rate or the spontaneous abortion rate after an ultrasound confirmed clinical pregnancy in couples with abnormal sperm morphology (less than 4% normal forms). There was also no increased risk of birth abnormalities for patients with abnormal sperm morphology.Abnormal sperm morphology impacted neither the pregnancy rate nor the live birth rate in couples undergoing intrauterine insemination. These results can be used to reassure couples who undergo intrauterine insemination that there is a minimal impact of abnormal sperm morphology on the live birth rate and the prevalence of birth abnormalities.

Objective: The current study investigated the effects of clomiphene citrate on the hypothalamic-pituitary-testicular axis, steroidogenesis, sperm parameters, and testicular antioxidant enzyme activity of male Wistar rats submitted to lead acetate (Pb)-induced reproductive toxicity. Methods: Twenty adult male Wistar rats were divided into four groups of equal size as follows: Control; Clomid (0.35 mg/kg); Pb (10 mg/kg); and Clomid + Pb. Serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone, testicular 17-β hydroxysteroid dehydrogenase (17-β HSD) activity, androgen receptors, catalase activity, superoxide dismutase (SOD), malondialdehyde (MDA), sperm motility, viability, counts and morphology were estimated after oral administration of Clomid and/or lead acetate for 35 consecutive days. Data were analyzed using ANOVA at p

The testis is a temperature-sensitive organ that needs to be maintained 2–7 °C below core body temperature to ensure the production of normal sperm. Failure to maintain testicular temperature in mammals impairs spermatogenesis and leads to low sperm counts, poor sperm motility and abnormal sperm morphology in the ejaculate. This review discusses the recent knowledge on the response of testicular somatic cells to heat stress and, specifically, regarding the relevant contributions of heat, germ cell depletion and inflammatory reactions on the functions of Sertoli and Leydig cells. It also outlines mechanisms of testicular thermoregulation, as well as the thermogenic factors that impact testicular function.

Male reproductive damage in the general population comprises different disorders in various biomarkers, which could be respectively caused by a number of exposure factors. However, researchers considering the environmental/behavioral/psychological exposures together to evaluate their contribution to male reproductive damage are still lacking. The present study investigated the comprehensive association between 138 environmental/behavioral/psychological exposures and 32 male reproductive biomarkers in 796 young Chinese men using graph-guided fused lasso (GFLASSO) and hierarchical clustering methods. All biomarkers were found to be associated with various exposures. A combination of these exposures not only predicted the levels of single biomarkers in another test dataset, but also identified the comprehensive reproductive features by clustering the men into five subgroups with distinct damages representing disrupted spermatogenesis with abnormal sperm morphology, low sperm motility with DNA fragmentation, chromatin immaturity, aberrant endocrine, or DNA strand breakage. The findings can be used to suggest a novel way to identify the males with a high risk of reproductive damage and develop personalized preventive strategies.

The fertility study design recommended in the ICH S5(R2) Harmonised Guideline for Detection of Toxicity to Reproduction for Medicinal Products emphasizes the importance of histopathological endpoints next to a pairing assessment in evaluating male fertility. However, in a male rat fertility study with JNJ-26489112, a CNS-active agent, while there were no effects on histological endpoints, mating performance or pregnancy outcomes, sperm assessment was included. The high dose males presented with reversible decreases in epididymal, but not testicular, sperm concentration and motility and an increase in abnormal sperm morphology. In view of the differences in fertility between rats and humans, these types of sperm effects in rats suggest the potential for an impact on human male fertility that would be undetected if not for the sperm assessment. Therefore, the current example suggests that including semenology as a standard endpoint in nonclinical fertility studies may be warranted.

A Chilean 35-year-old male patient with a history of primary infertility made an appointment at the Unit of Reproductive Medicine at Clinica Las Condes, Santiago, Chile. Multiple semen analyses revealed abnormal sperm morphology as the most prevalent finding. Multiflagellated and macrocephalic spermatozoa were observed and indicated a possible macrozoospermic phenotype. The constant presence of abnormal sperm morphology led the scope of the study to include Aurora Kinase C (AURKC) gene sequencing. The patient was diagnosed with a homozygous mutation of this gene. The mutation was detected in exon 6, type c.744C>G+/+ (P.Y248*) variant. As previously described in the Human Gene Mutation Database (HGMD), this pathogenic variant is associated with macrozoospermia. Although this mutation is not the most frequently observed, it is the first of its kind reported in Latin America.

The tumor suppressor p53, and the cyclin-dependent kinase inhibitor Ink4c, have been both implicated in spermatogenesis control. Both p53-/- and Ink4c-/- single knockout male mice are fertile, despite testicular hypertrophy, Leydig cell differentiation defect, and increased sperm count in Ink4c-/- males. To investigate their collaborative roles, we studied p53-/- Ink4c-/- dual knockout animals, and found that male p53-/- Ink4c-/- mice have profoundly reduced fertility. Dual knockout male mice show a marked decrease in sperm count, abnormal sperm morphology and motility, prolongation of spermatozoa proliferation and delay of meiosis entry, and accumulation of DNA damage. Genetic studies showed that the effects of p53 loss on fertility are independent of its downstream effector Cdkn1a. Absence of p53 also partially reverses the hyperplasia seen upon Ink4c loss, and normalizes the Leydig cell differentiation defect. These results implicate p53 in mitigating both the delayed entry into meiosis and the secondary apoptotic response that occur in the absence of Ink4c. We conclude that the cell cycle genes p53 and Ink4c collaborate in sperm cell development and differentiation, and may be important candidates to investigate in human male infertility conditions.

Study question Can a standardized assessment of abnormal human sperm morphology provide additional useful information by identifying men with more severe disturbances in different types of abnormalities? Summary answer Definition-based categorization of sperm head, midpiece and tail defects has shown how differently these abnormalities are distributed in fertile men and other groups of men, thus providing high and low thresholds, a starting point for diagnosis or research purposes. What is known already Several recent studies have reported indisputable genetic origins for various sperm defects. A few studies have also identified associations between environmental factors and low percentages of morphologically normal spermatozoa. Nevertheless, with the exception of rare situations in which the vast majority of spermatozoa have specific, easily characterized defects, such as 'globozoospermia', little attention has been paid to the description and precise quantification of human sperm abnormalities. The lack of standardization in the phenotyping of sperm morphological defects by conventional microscopy is a limiting factor for diagnosis and for intra- or inter-observer or centre consistency in studies investigating the causal factors and possible functional consequences of the abnormalities detected. There are currently no baseline data for abnormalities of sperm morphology based on a standardized classification, in the general population, among fertile or other groups of men. Study design, size, duration This study is based on detailed sperm abnormality datasets acquired by a standardized classification method, from several groups of men, over the same 5-year period. Participants/materials, setting, methods We studied cross-sectional data from fertile men (n = 926), male partners from infertile couples (n = 1747) and testicular cancer patients (n = 239). We used a standardized classification to analyse Shorr-stained slides, taking into account all the abnormalities encountered. Main results and the role of chance Most sperm defects were significantly more frequent in infertile than in fertile men, with 20-30% of infertile men having frequencies of abnormalities above the 95th percentile in fertile men for 9 out of the 15 categories of abnormalities. Interestingly, several head abnormalities were significantly more frequent in patients with testicular cancer than in infertile men, highlighting the particular impact of this condition on sperm morphogenesis. We used the 95th percentile in fertile men as the lower threshold and the 99th percentile in infertile men as an extreme upper threshold, for the classification of morphological abnormality frequencies into three levels: low, intermediate and high. The assessment of several semen samples, with or without a genetic background, for abnormal sperm morphology, based on the percentage of normal spermatozoa, a teratozoospermia index, and the detailed profile of abnormalities categorized according to the three levels proposed, has highlighted the value of detailed phenotyping for diagnosis and research purposes. Limitations, reasons for caution The thresholds proposed for the various categories of sperm abnormality should be considered relative rather than absolute, owing to the known sampling error related to the limited number of spermatozoa assessed per sample, or when studying the general population or populations from regions other than Western Europe. The standardized assessment of abnormal sperm morphology requires time and experience. We therefore suggest that this assessment is carried out during a first andrological check-up or for epidemiological or research studies, rather than in the routine management of infertile couples for assisted reproductive technologies. Wider implications of the findings The study design used for the fertile group of men was similar to that previously used for the WHO reference values, providing a rationale for considering the 95th percentile in fertile men as the level below which abnormalities may be considered to occur at a frequency representing random background variations of a normal spermiogenesis process. The crude frequencies obtained, and the three levels of abnormality frequency proposed for each standardized category of sperm defect, provide baseline data useful for diagnosis and a starting point for future studies aiming to identify associations with genetic or environmental factors. Study funding/competing interests Part of this study was supported by contract BMH4-CT96-0314 from the European Union. The authors have no competing interests to declare.

Background Infertility affects 15% of human couples, with men being responsible in approximately 50% of cases. Moreover, the aetiology of male factor infertility is poorly understood. The majority of male factor infertility remains idiopathic and potentially genetic in origin. The association of the Y chromosome and mitochondrial haplogroups with male infertility has been previously reported. This association differs between studied populations and their geographical distributions. These effects have been only rarely analysed in mixed populations, such as South Americans. Methods In this study, we analysed the contributions of the Y chromosome and mitochondrial haplogroups to male infertility in a mixed population. A case control study was conducted. Regular PCR and high-resolution-melting-real-time PCR were performed to type haplogroups from fertile and infertile men. The sperm parameters from infertile men were compared in each haplogroup by logistic regression analysis and ANOVA. Results The genotyping confirmed the known admixture characteristic of the Uruguayan population. The European paternal contribution was higher than the maternal contribution in both fertile and infertile men. Neither maternal nor paternal ancestry presented differences between the cases and controls. Men belonging to the Y chromosome haplogroup F(xK) more frequently presented with an abnormal sperm morphology than men from other haplogroups. The sperm parameters were not associated with the mitochondrial haplogroups. Conclusions The data presented in this study showed an association between male infertility and ancestry in the Uruguayan population. Specifically, abnormal sperm morphology was associated with the Y chromosome haplogroup F(xK). Since the Y chromosome lacks recombination, these data suggest that some genes that determine sperm morphology might be inherited in blocks with the region that determines specific haplogroups. However, the possible association between the Y chromosome haplogroup F(xK) and sperm morphology requires further confirmatory testing. Data linking infertility with ancestry are needed to establish the possible causes of infertility and define male populations susceptible to infertility. Whether the admixed characteristics of the Uruguayan population exert any pressure on male fertility potential must be further investigated.

Obesity, defined as excessive accumulation of fat in adipose tissue, is a metabolic disorder resulting from behavioral, environmental and heritable causes. Obesity increases the risks of hypertension, diabetes, cardiovascular disease, sleep apnea, respiratory problems, osteoarthritis and cancer. Meanwhile, the negative impact of obesity on male reproduction is gradually recognized. According to the clinical investigations and animal experiments, obesity is correlated with reductions in sperm concentration and motility, increase in sperm DNA damage and changes in reproductive hormones. Several mechanisms can elucidate the effects of obesity on sperm functions and male subfertility, i.e., the excessive conversion of androgens into estrogens in redundant adipose tissue causes sexual hormone imbalance, subsequently resulting in hypogonadism. Secondly, adipokines produced by adipose tissue induce severe inflammation and oxidative stress in male reproductive tract, directly impairing testicular and epididymal tissues. Moreover, increased scrotal adiposity leads to increase gonadal heat, continuously hurting spermatogenesis. Therefore, obesity alters the systematic and regional environment crucial for spermatogenesis in testis and sperm maturation in epididymis, and finally results in poor sperm quality including decreased sperm motility, abnormal sperm morphology and acrosome reaction, changed membrane lipids and increased DNA damage. Furthermore, recent studies indicate that epigenetic changes may be a consequence of increased adiposity. A major effort to identify epigenetic determinants of obesity revealed that sperm DNA methylation and non-coding RNA modification are associated with BMI changes and proposed to inherit metabolic comorbidities across generations. This review will explain how obesity-related changes in males to influence sperm function and male fertility as well.

Hypercholesterolemia is a marker for several adult chronic diseases. Recently we demonstrated that sub/infertility is also associated to Hypercholesterolemia in rabbits. Seminal alterations included: abnormal sperm morphology, decreased sperm number and declined percentage of motile sperm, among others. In this work, our objective was to evaluate the effects of hypercholesterolemia on testicular efficiency and spermiogenesis, as the latter are directly related to sperm number and morphology respectively. Tubular efficiency was determined by comparing total number of spermatogenic cells with each cell type within the proliferation/differentiation compartments. We found lower testicular efficiency related to both a decrease in spermatogonial cells and an increase in germ cell apoptosis in hypercholesterolemic rabbits. On the other hand, spermiogenesis-the last step of spermatogenesis involved in sperm shaping-was detaily analyzed, particularly the acrosome-nucleusmanchette complex. The manchette is a microtubular-based temporary structure responsible in sperm cell elongation. We analyzed the contribution of actin filaments and raft microdomains in the arrangement of the manchette. Under fluorescence microscopy, spermatocyte to sperm cell development was followed in cells isolated from V to VIII tubular stages. In cells from hypercholesterolemic rabbits, abnormal development of acrosome, nucleus and inaccurate tail implantation were associated with actin-alpha-tubulin-GM1 sphingolipid altered distribution. Morphological alterations were also observed at electron microscopy. We demonstrated for the first time that GM1-enriched microdomains together with actin filaments and microtubules are involved in allowing the correct anchoring of the manchette complex. In conclusion, cholesterol enriched diets promote male fertility alterations by affecting critical steps in sperm development: spermatogenesis and spermiogenesis. It was also demonstrated that hypercholesterolemic rabbit model is a useful tool to study serum cholesterol increment linked to sub/infertility. Fil: Simón, Layla Yamila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad del Aconcagua; Argentina Fil: Funes, Abi Karenina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Yapur, Martín A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Cabrillana, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad Nacional de Cuyo; Argentina Fil: Monclus, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Boarelli, Paola Vanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad del Aconcagua; Argentina Fil: Vincenti, Amanda Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Saez Lancellotti, Tania Emilce Estefania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad del Aconcagua; Argentina Fil: Fornes, Miguel Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina

Exposure to pesticides affects many body organs including reproductive system. Disorder of the reproductive system leads to infertility and therefore has been in the center of attention within the recent decades. Pesticides are one of the compounds that might reduce the semen quality in the exposed workers according to current knowledge. Although many underlying mechanisms have been proposed, the mechanisms of action are not clarified yet. The object of the present review was to criticize all the results of studies which evaluated the pesticide effects on male reproductive system. Results indicate that semen changes are multifactorial in the workers exposed to pesticides as there are numerous factors affecting sperm quality in occupational exposures. Majority of pesticides including organophosphoruses affect the male reproductive system by mechanisms such as reduction of sperm density and motility, inhibition of spermatogenesis, reduction of testis weights, reduction of sperm counts, motility, viability and density, and inducing sperm DNA damage, and increasing abnormal sperm morphology. Reduced weight of testes, epididymis, seminal vesicle, and ventral prostate, seminiferous tubule degeneration, change in plasma levels of testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), decreased level and activity of the antioxidant enzymes in testes, and inhibited testicular steroidogenesis are other possible mechanisms. Moreover, DDT and its metabolites have estrogenic effects on males. Although effect of pesticides on sperm quality is undeniable, well-designed long-term studies are needed to elucidate all the possible affecting variables such as socioeconomic, cultural, nutritional, occupational, physical, and clinical characteristics alongside pesticides.

Summary The study was conducted to evaluate the vanadium-induced testicular toxicity and its effect on sperm parameters, sperm nuclear DNA damage and histological alterations in Sprague Dawley rats and to assess the protective effect of G-hesperidin against this damage. Treatment of rats with vanadium at a dose of 1 mg kg bw−1 for 90 days resulted in significant reduction in serum testosterone levels, sperm count and motility. Further, a parallel increase in abnormal sperm morphology and adverse histopathological changes in testis was also associated with vanadium administration when compared to normal control. Moreover, sperm chromatin dispersion assay revealed that vanadium induces sperm nuclear DNA fragmentation. A marked increase in testicular malondialdehyde levels and decreased activity of antioxidant enzymes such as superoxide dismutase and catalase indicates vanadium-induced oxidative stress. Co-administration of G-hesperidin at a dose of 25 and 50 mg kg bw−1 significantly attenuated the sperm parameters and histological changes by restoring the antioxidant levels in rat testis. These results suggested that vanadium exposure caused reduced bioavailability of androgens to the tissue and increased free radical formation, thereby causing structural and functional changes in spermatozoa. G-hesperidin exhibited antioxidant effect by protecting the rat testis against vanadium-induced oxidative damage, further ensures antioxidant potential of bioflavonoids.

Summary Bromodomain testis-specific (BRDT) protein is essential for the normal process of spermatogenesis. Mutant mice that expressed truncated BRDT had impaired testicular histology with severely reduced sperm concentration and abnormal sperm morphology, while a model of knockout Brdt mice with no BRDT protein had complete meiotic arrest. A BRDT single nucleotide polymorphism (SNP) (rs3088232) was reported as being associated with infertility in men. We assessed testicular specimens of 276 azoospermic men who underwent testicular sperm extraction to search for specimens that showed spermatogenic impairments similar to those of mutant BRDT mice. Ten similar specimens were selected for BRDT gene sequencing and they revealed three NCBI-reported SNPs (rs10783071, rs3088232 and rs10747493) variously distributed among them. Bioinformatics analysis predicted that they would not affect protein activity. Further assessment of rs3088232 frequency in a large group of non-obstructive azoospermia men and fertile controls demonstrated no significant difference between them (27.2 and 21.7% respectively; p = 0.122, Fisher's exact test). We conclude that the testicular impairments observed in the 10 specimens were not a consequence of BRDT gene mutation. The association between BRDT rs3088232 and infertility that had been reported in other studies was not supported.

In recent years, wide utilization of herbal drugs has encouraged scientists to determine their impressive effects on health. Since Nigella sativa L. seed (N. sativa) has many uses including infertility in traditional medicine, the effects of Nigella sativa L. seed oil on abnormal semen quality in infertile men with abnormal semen quality are of interest. This study was conducted on Iranian infertile men with inclusion criteria of abnormal sperm morphology less than 30% or sperm counts below 20×10(6)/ml or type A and B motility less than 25% and 50% respectively. The patients in N. sativa oil group (n=34) received 2.5mlN. sativa oil and placebo group (n=34) received 2.5ml liquid paraffin two times a day orally for 2 months. At baseline and after 2 months, the sperm count, motility and morphology and semen volume, pH and round cells as primary outcomes were determined in both groups. Results showed that sperm count, motility and morphology and semen volume, pH and round cells were improved significantly in N. sativa oil treated group compared with placebo group after 2 months. It is concluded that daily intake of 5ml N. sativa oil for two months improves abnormal semen quality in infertile men without any adverse effects.

Lead (Pb) exposure at high concentrations is associated with poor sperm quality, acrosome alterations, and low fertilization rate. Sperm capacitation and the acrosome reaction (AR) are required for successful fertilization. Actin polymerization is crucial for correct capacitation, and small GTPases, such as RhoA, Rac1, and Cdc42, are involved. This study aimed to evaluate the effects of Pb on sperm fertilization ability, capacitation, AR, and the mechanisms involved in mice exposed to low Pb concentrations. CD1 mice were exposed to 0.01% Pb2+ for 45 days through their drinking water and their spermatozoa were collected from the cauda epididymis-vas deferens to evaluate the following: AR (oAR: initial, sAR: spontaneous, and iAR: induced) using the PNA-FITC assay, sperm capacitation (P-Tyr levels), actin polymerization (phalloidin-TRITC), MDA production (stress oxidative marker), the RhoA, Rac1, and Cdc42 protein levels, and the in vitro fertilization (IVF). After the treatment, the blood Pb (PbB) concentration was 9.4 ± 1.6 μg/dL. Abnormal sperm morphology and the oAR increased (8 and 19%, respectively), whereas the iAR decreased (15%) after a calcium ionophore challenge, and the actin polymerization decreased in the sperm heads (59%) and tails (42%). Rac1 was the only Rho protein to significantly decrease (33%). Spermatozoa from the Pb-treated mice showed a significant reduction in the fertilization rate (19%). Our data suggest that Pb exposure at environmental concentrations (PbB

Oligo-astheno-teratozoospermia (OAT), a condition that includes low sperm number, low sperm motility and abnormal sperm morphology, is the commonest cause of male infertility. Because genetic analysis is frequently impeded by the infertility phenotype, the genetic basis of many of OAT conditions has been hard to verify. Here, we show that deficiency of ORP4, a sterol-binding protein in the oxysterol-binding protein (OSBP)-related protein family, causes male infertility due to severe OAT in mice. In ORP4-deficient mice, spermatogonia proliferation and subsequent meiosis occurred normally, but the morphology of elongating and elongated spermatids was severely distorted, with round-shaped head, curled back head or symplast. Spermatozoa derived from ORP4-deficient mice had little or no motility and no fertilizing ability in vitro. In ORP4-deficient testis, postmeiotic spermatids underwent extensive apoptosis, leading to a severely reduced number of spermatozoa. At the ultrastructural level, nascent acrosomes appeared to normally develop in round spermatids, but acrosomes were detached from the nucleus in elongating spermatids. These results suggest that ORP4 is essential for the postmeiotic differentiation of germ cells.

The septin gene belongs to a highly conserved family of polymerizing GTP-binding cytoskeletal proteins. SEPTs perform cytoskeletal remodeling, cell polarity, mitosis, and vesicle trafficking by interacting with various cytoskeletons. Our previous studies have indicated that SEPTIN12+/+/+/− chimeras with a SEPTIN12 mutant allele were infertile. Spermatozoa from the vas deferens of chimeric mice indicated an abnormal sperm morphology, decreased sperm count, and immotile sperm. Mutations and genetic variants of SEPTIN12 in infertility cases also caused oligozoospermia and teratozoospermia. We suggest that a loss of SEPT12 affects the biological function of microtublin functions and causes spermiogenesis defects. In the cell model, SEPT12 interacts with α- and β-tubulins by co-immunoprecipitation (co-IP). To determine the precise localization and interactions between SEPT12 and α- and β-tubulins in vivo, we created SEPTIN12-transgene mice. We demonstrate how SEPT12 interacts and co-localizes with α- and β-tubulins during spermiogenesis in these mice. By using shRNA, the loss of SEPT12 transcripts disrupts α- and β-tubulin organization. In addition, losing or decreasing SEPT12 disturbs the morphogenesis of sperm heads and the elongation of sperm tails, the steps of which are coordinated and constructed by α- and β-tubulins, in SEPTIN12+/+/+/− chimeras. In this study, we discovered that the SEPTIN12-microtubule complexes are critical for sperm formation during spermiogenesis.

Since the discovery of spermatozoon by Anton van Leeuwenhoek in 1677, there has been an ever increasing understanding of its role in reproduction. Many factors adversely affect sperm quality, including varicocele, accessory gland infection, immunological factors, congenital abnormalities, and iatrogenic systemic and endocrine causes, such as diabetes mellitus, obesity, metabolic syndrome, and smoking. The mechanisms responsible for the association between poor sperm parameters and ill health may include oxidative stress, low-grade inflammation, low testosterone, and low sex-hormone-binding globulin. Oxidative stress in the testicular microenvironment may result in decreased spermatogenesis and sperm DNA damage, loss of sperm motility, and abnormal sperm morphology. Low testosterone caused by advanced age, visceral obesity, and inflammation is associated with the development of cardiovascular disease. Hence, semen analysis has an important role in the routine evaluation of idiopathic male infertility, usually manifested as low sperm counts, impaired sperm motility, or absence of sperm, and remains the most common single diagnostic tool. Several studies have shown an inverse relationship between semen quality and medical disorders. This review elucidates the effect of medical disorders and social habits on sperm quality, the mechanisms that are involved in the impairment of sperm quality, and whether or not sperm quality can be used as an index of good health and longevity in a man.

Objective To determine differences and frequency of excessive (≥50%) sperm DNA damage between ejaculated and motile sperm. Design Sperm DNA damage was assessed by acridine orange fluorescence and the results of ejaculated and motile sperm were compared. Setting Public and private clinical assisted reproduction centers. Patient(s) A total of 272 subfertile men were studied. Intervention(s) None. Main Outcome Measure(s) Semen analysis and sperm DNA damage. Result(s) Sperm DNA damage was negatively correlated with sperm motility and normal morphology. Overall, 39.7% (108 of 272) of semen samples had excessive sperm DNA damage. In contrast, only 15% (41 of 272) of motile sperm fractions had excessive DNA damage. Based on DNA results of motile sperm and semen characteristics, the proportion of men with excessive sperm DNA damage was 26% in severe teratozoospermia, 17.5% in oligozoospermia, 12.5% in moderate teratozoospermia, and 4.6% in normozoospermia. Severe teratozoospermia had five times more frequent excessive DNA damage than normozoospermia. Conclusion(s) Abnormal sperm morphology is highly associated with sperm DNA damage. Results of DNA damage of ejaculated sperm do not accurately reflect DNA status of motile sperm. Therefore, sperm DNA damage should be assessed in motile sperm fraction rather than whole ejaculated sperm.

The evaluation of strict morphology for predicting successful pregnancy has been controversial, nevertheless remains an essential component of semen analysis. Patients with teratozoospermia (abnormal strict morphology) have traditionally been counseled to undergo assisted reproduction. However, recent studies suggest that patients with abnormal sperm morphology alone should not be precluded from attempting natural conception before undergoing assisted reproduction. The goal of this review is to provide an update on the evaluation of sperm morphology for prognosis in assisted reproductive techniques such as intrauterine insemination and in vitro fertilization with or without intracytoplasmic sperm injection. Additionally, we propose a logical approach to the evaluation of a patient with teratozoospermia seeking fertility treatment.