Direct visualization of pre-protamine 2 detects protamine assembly failures and predicts ICSI success.

Histone-to-protamine transition is an essential step in the generation of fully functional spermatozoa in various mammalian species. In human and mouse, one of the two protamine-encoding genes produces a precursor pre-protamine 2 (pre-PRM2) protein, which is then processed and assembled. Here, we design an original approach based on the generation of pre-PRM2-specific antibodies to visualize the unprocessed pre-PRM2 by microscopy, flow cytometry and immunoblotting. Using mouse models with characterized failures in histone-to-protamine replacement, we show that pre-PRM2 retention is tightly linked to impaired nucleosome disassembly. Additionally, in elongating/condensing spermatids, we observe that pre-PRM2 and transition protein are co-expressed spatiotemporally, and their physical interaction suggests that these proteins act simultaneously rather than successively during histone replacement. By using our anti-human pre-PRM2 antibody, we also measured pre-PRM2 retention rates in the spermatozoa from 49 men of a series of infertile couples undergoing ICSI, which shed new light on the debated relation between pre-PRM2 retention and sperm parameters. Finally, by monitoring 2-pronuclei embryo formation following ICSI, we evaluated the fertilization ability of the sperm in these 49 patients. Our results suggest that the extent of pre-PRM2 retention in sperm, rather than pre-PRM2 accumulation per se, is associated with fertilization failure. Hence, anti-pre-PRM2 antibodies are valuable tools that could be used in routine monitoring of sperm parameters in fertility clinics, as well as in experimental research programmes to better understand the obscure process of histone-to-protamine transition.

Epigenetic modifications at DMRs of imprinting genes in sperm of type 2 diabetic men.

High rates of infertility in type 2 diabetic (T2DM) men have led to attempts to understand the mechanisms involved in this process. This condition can be investigated from at least two aspects, namely sperm quality indices and epigenetic alterations. Epigenetics science encompasses the phenomena that can lead to inherited changes independently of the genetics. This study has been performed to test the hypothesis of the relationship between T2DM and the epigenetic profile of the sperm, as well as sperm quality indices. This research included 42 individuals referred to the infertility clinic of Royan Institute, Iran in 2019-2021. The study subjects were assigned to three groups: normozoospermic non-diabetic (control), normozoospermic diabetic (DN) and non-normozoospermic diabetic (D.Non-N). Sperm DNA fragmentation was evaluated using the sperm chromatin structure assay technique. The global methylation level was examined using 5-methyl cytosine antibody and the methylation status in differentially methylated regions of H19, MEST, and SNRPN was assessed using the methylation-sensitive high-resolution melting technique. The results showed that the sperm global methylation in spermatozoa of D.Non-N group was significantly reduced compared with the other two groups (P < 0.05). The MEST and H19 genes were hypomethylated in the spermatozoa of D.Non-N individuals, but the difference level was not significant for MEST. The SNRPN gene was significantly hypermethylated in these individuals (P < 0.05). The results of this study suggest that T2DM alters the methylation profile and epigenetic programming in spermatozoa of humans and that these methylation changes may ultimately influence the fertility status of men with diabetes.

Rare missense variant in MSH4 associated with primary gonadal failure in both 46, XX and 46, XY individuals.

STUDY QUESTION: Can whole-exome sequencing (WES) reveal a shared pathogenic variant responsible for primary gonadal failure in both male and female patients from a consanguineous family? SUMMARY ANSWER: Patients with primary ovarian insufficiency (POI) and non-obstructive azoospermia (NOA) were homozygous for the rare missense variant p. S754L located in the highly conserved MSH4 MutS signature motif of the ATPase domain. An oligozoospermic patient was heterozygous for the variant. WHAT IS KNOWN ALREADY: MSH4 is a meiosis-specific protein expressed at a certain level in the testes and ovaries. Along with its heterodimer partner MSH5, it is responsible for double-strand Holliday junction recognition and stabilization, to ensure accurate chromosome segregation during meiosis. Knockout male and female mice for Msh4 and Msh5 are reportedly infertile due to meiotic arrest. In humans, MSH4 is associated with male and female gonadal failure, with distinct variations in the MutS domain V. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of a consanguineous family with multiple cases of gonadal failure in both genders. The subject family was recruited in Iran, in 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: The proband who is affected by POI, an NOA brother, a fertile sister and their parents were subjected to WES. The discovered variant was validated in these individuals, and the rest of the family was also genotyped by Sanger sequencing. The variant was not detected in 800 healthy Iranian individuals from the Iranome database nor in 30 sporadic NOA and 30 sporadic POI patients. Suggested effect in aberrant splicing was studied by RT-PCR. Moreover, protein homology modeling was used to further investigate the amino acid substitution in silico. MAIN RESULTS AND THE ROLE OF CHANCE: The discovered variant is very rare and has never been reported in the homozygous state. It occurs in the ATPase domain at Serine 754, the first residue within the highly conserved MutS signature motif, substituting it with a Leucine. All variant effect prediction tools indicated this variant as deleterious. Since the substitution occurs immediately before the Walker B motif at position 755, further investigations based on protein homology were conducted. Considering the modeling results, the nature of the substituted amino acid residue and the distances between p. S754L variation and the residues of the Walker B motif suggested the possibility of conformational changes affecting the ATPase activity of the protein. LARGE SCALE DATA: We have submitted dbSNP entry rs377712900 to ClinVar under SCV001169709, SCV001169708 and SCV001142647 for oligozoospermia, NOA and POI, respectively. LIMITATIONS, REASONS FOR CAUTION: Studies in model organisms can shed more light on the role of this variant as our results were obtained by variant effect prediction tools and protein homology modeling. WIDER IMPLICATIONS OF THE FINDINGS: Identification of variants in meiotic genes should improve genetic counseling for both male and female infertility. Also, as two of our NOA patients underwent testicular sperm extraction (TESE) with no success, ruling out the existence of pathogenic variants in meiotic genes in such patients prior to TESE could prove useful. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by Royan Institute in Tehran, Iran, and Institut Pasteur in Paris, France. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Vasectomy reversal: Unilateral versus bilateral vasovasostomy.

The aim of this study was to evaluate and compare the efficiencies of unilateral and bilateral vasovasostomies as the vasectomy reversal procedures. A total of 95 patients with a history of bilateral vasectomy were evaluated. 42 of them had undergone unilateral surgery, and bilateral surgery had been done for the other 53 patients. Their information including the age, the time interval between the initial vasectomy to the reversal surgery and other underlying illnesses or medications was gathered. Patency rates in the unilateral and bilateral groups were 88.1% (38 patients) and 88.7% (48 patients), respectively, the difference of which was not statistically significant (p = .907). Successful pregnancies occurred in 22 (52.4%) and 29 (54.7%) patients, respectively, which did not show any statistically significant difference too (p = .713). Based on the multivariate logistic regression model, only the time interval between vasectomy and the reversal (duration of obstruction) was predictive of patency (OR = 1.112, p = .037). The outcomes of the unilateral and bilateral vasovasostomies in terms of patency and pregnancy rates were not significantly different. We suggest that performing unilateral, instead of bilateral, vasovasostomy can reduce the time of anaesthesia and surgery and save costs and consumables without having a significant negative impact on the surgical outcomes.

Observational retrospective study of Intracytoplasmic sperm injection outcomes of 117 infertile men with severe short tail sperm defect.

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.

Sertoli cell-only syndrome: etiology and clinical management.

Almost 50% of infertility cases are due to male factors, and spermatogenesis failure is one of the most severe forms of male infertility. Sertoli cell-only syndrome (SCOS) also known as germ cell aplasia is characterized by azoospermia in which the seminiferous tubules of testicular biopsy are lined only with Sertoli cells. The definitive diagnosis of SCOS is by diagnostic testicular biopsy. Although SCOS may be a result of Klinefelter syndrome, most of the SCOS men have a normal karyotype. Along with genetic aberrations, signaling pathways and endocrine processes might be major factors in the development of SCOS. Sperm retrieval and intracytoplasmic sperm injection (ICSI) are available treatments for SCOS. However, some SCOS patients do not have therapeutic options to help them having a biological child. This review aims to summarize our present knowledge about SCOS and to highlight the importance of future researches in the diagnosis and treatment of this disorder.

Corrigendum to: Deletion of dpy-19 like 2 (DPY19L2) gene is associated with total but not partial globozoospermia.

The dpy-19 like 2 (DPY19L2) gene is the most common genetic cause of globozoospermia characterised by the production of round-headed spermatozoa without an acrosome. The present study was performed on 63 men with globozoospermia and 41 normozoospermic individuals to evaluate the frequency of the DPY19L2 gene and exons; deletion and genetic changes in exons 1, 5, 7-11, 19, 21 and interval introns; and some epidemiological factors (e.g. varicocele, smoking, drug use, alcohol consumption and a family history of infertility). Homozygous deletion of DPY19L2 was identified in 35% of men with globozoospermia. Exon 7 was deleted in 4.8% of men with globozoospermia in which DPY19L2 was not deleted. No genetic variations were observed within the DPY19L2 exons examined, but five intronic polymorphisms were detected: 1054-77T>C in intron 9, 1131+65T>C and 1131+53A>G in intron 10 and 1218+22T>C and 1218+73T>C in intron 11. There were significant differences in the frequency of 1054-77T>C and 1218+22T>C polymorphisms between the globozoospermic and normozoospermic groups. In addition, there were significant differences between the two groups in sperm count, sperm motility, a history of infertility in the family and varicocele. Based on these findings, DPY19L2 deletion is the major cause of total globozoospermia and there is no association between exons 1, 5, 8-11, 19 and 21 polymorphisms of the DPY19L2 gene in the occurrence of this defect.

Deletion of dpy-19 like 2 (DPY19L2) gene is associated with total but not partial globozoospermia.

The dpy-19 like 2 (DPY19L2) gene is the most common genetic cause of globozoospermia characterised by the production of round-headed spermatozoa without an acrosome. The present study was performed on 63 men with globozoospermia and 41 normozoospermic individuals to evaluate the frequency of the DPY19L2 gene and exons; deletion and genetic changes in exons 1, 5, 7-11, 19, 21 and interval introns; and some epidemiological factors (e.g. varicocele, smoking, drug use, alcohol consumption and a family history of infertility). Homozygous deletion of DPY19L2 was identified in 35% of men with globozoospermia. Exon 7 was deleted in 4.8% of men with globozoospermia in which DPY19L2 was not deleted. No genetic variations were observed within the DPY19L2 exons examined, but five intronic polymorphisms were detected: 1054-77T>C in intron 9, 1131+65T>C and 1131+53A>G in intron 10 and 1218+22T>C and 1218+73T>C in intron 11. There were significant differences in the frequency of 1054-77T>C and 1218+22T>C polymorphisms between the globozoospermic and normozoospermic groups. In addition, there were significant differences between the two groups in sperm count, sperm motility, a history of infertility in the family and varicocele. Based on these findings, DPY19L2 deletion is the major cause of total globozoospermia and there is no association between exons 1, 5, 8-11, 19 and 21 polymorphisms of the DPY19L2 gene in the occurrence of this defect.

Evaluation of sperm DNA fragmentation and chromatin structure in infertile men with immotile short-tail sperm defect.

Teratozoospermia is characterised by the presence of spermatozoa with abnormal morphology. One of the morphological disorders that lead to male infertility is immotile short-tail sperm (ISTS) defect. In this study, we evaluated the levels of chromatin packing and DNA fragmentation in patients with immotile short-tail sperm defect. Semen samples were obtained from 31 infertile men with ISTS as case group and 31 normozoospermic men as a control group. Protamine status was evaluated using chromomycin A3 (CMA3) staining and sperm DNA fragmentation assessed by sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling (TUNEL). The percentage of positive CMA3 spermatozoa was significantly higher in patients' samples (22.6 ± 6.9) compared with controls (16.3 ± 4.2) (p < .05) and also mean (±SD) of sperm DNA fragmentation was significantly higher in patients compared with controls, as measured by TUNEL assay (10.45 ± 4.60 vs. 7.03 ± 2.86, p < .05) and SCSA (24.80 ± 13.1 vs. 15.2 ± 7.2, p < .05). According to our study, sperm DNA fragmentation and chromatin packing abnormality are significantly higher in the ISTS samples compared with normal samples. A possible explanation for this relationship is that sperm chromatin condensation and sperm flagellum formation occur during the same phase of spermatogenesis.

Oxidative stress-related miRNAs in spermatozoa may reveal the severity of damage in grade III varicocele.

Varicocele is associated with excessive production of reactive oxygen species (ROS). Although the harmful effects of ROS on sperm DNA, proteins and lipids are well documented, its impact on the expression of miRNAs in spermatozoa has not been fully understood. In this study, the expression patterns of microRNAs (miRNAs), miR-21, miR-34a and miR-122a as well as the level of ROS in the fertile control (FC; proven fertility without varicocele, n = 15) and grade III varicocele patients with normal (VN; n = 15) and abnormal (VA; n = 15) spermogram were investigated. The real-time PCR was performed to analyse the expression of the miRNAs, while oxidative stress was evaluated by measuring the concentrations of MDA. Our results showed that the expression levels of miR-21 (p = .001), miR-34a, (p = .007) and miR-122a (p < .001) were significantly decreased in spermatozoa of VN and VA patients in comparison with the fertile group. Also, increased levels of oxidative stress were detected in semen samples of varicocele patients compared with the fertile control (p < .0001). Overall, these findings demonstrate oxidative stress changes the expression pattern of some miRNAs, and these alterations could be a valuable diagnostic marker for the diagnosis and prognosis of varicocele-induced oxidative stress to retain the male fertility during the spermatogenesis process.

Identification of a missense variant in CLDN2 in obstructive azoospermia.

Obstructive azoospermia (OA), defined as an obstruction in any region of the male genital tract, accounts for 40% of all azoospermia cases. Of all OA cases, ~30% are thought to have a genetic origin, however, hitherto, the underlying genetic etiology of the majority of these cases remain unknown. To address this, we took a family-based whole-exome sequencing approach to identify causal variants of OA in a multiplex family with epidydimal obstruction. A novel gain-of-function missense variant in CLDN2 (c.481G>C; p.Gly161Arg) was found to co-segregate with the phenotype, consistent with the X-linked inheritance pattern observed in the pedigree. To assess the pathogenicity of this variant, the wild and mutant protein structures were modeled and their potential for strand formation in multimeric form was assessed and compared. The results showed that dimeric and tetrameric arrangements of Claudin-2 were not only reduced, but were also significantly altered by this single residue change. We, therefore, envisage that this amino acid change likely forms a polymeric discontinuous strand, which may lead to the disruption of tight junctions among epithelial cells. This missense variant is thus likely to be responsible for the disruption of the blood-epididymis barrier, causing dislodged epithelial cells to clog the genital tract, hence causing OA. This study not only sheds light on the underlying pathobiology of OA, but also provides a basis for more efficient diagnosis in the clinical setting.

The role of DEFB126 variation in male infertility and medically assisted reproduction technique outcome.

RESEARCH QUESTION: Human DEFB126 is an important component of the glycocalyx of human spermatozoa. Beta-defensins play a primary role in male infertility due to their involvement in maturation and capacitation of spermatozoa. A 2-nt deletion of DEFB126 affects sperm function and so this study investigated the possible association between DEFB126 variants and its protein expression on medically assisted reproduction (MAR) technique outcome in Iranian infertile males. DESIGN: The presence of a 2-nt deletion of DEFB126, and its protein expression in spermatozoa, were investigated by standard polymerase chain reaction (PCR) sequencing and immunocytochemistry, respectively. MAR technique outcome according to clinical pregnancy rates was assessed in 277 Iranian males with unexplained infertility, including 139 patients who underwent intrauterine insemination (IUI) and 103 patients who underwent IVF/intracytoplasmic sperm injection (ICSI), as well as 35 infertile males who declined to use any MAR treatment. As the control group, 100 fertile males with a normal spermiogram were enrolled. RESULTS: The 2-nt deletion of DEFB126 was significantly higher in infertile patients than controls (P ≤ 0.05). The presence of this deletion resulted in significantly lower clinical pregnancy rates following IUI (P ≤ 0.05); however, there were no differences in IVF/ICSI outcomes according to genotype. The protein expression in del/del males was also remarkably lower than that of the other genotypes. CONCLUSIONS: This sequence variation of DEFB126 may impair male reproductive function and can be related to male infertility. Interestingly, males with the del/del genotype have a normal spermiogram; however, their spermatozoa are evidently functionally impaired, which can affect IUI treatment outcome, but not treatment by IVF/ICSI.

Micro-quantity straw as a carrier for cryopreservation of oligozoospermic semen samples: Effects of storage times and cryoprotectant.

Application of an appropriate freezing carrier is crucial for improving post-thaw recovery of oligozoospermic samples. In this study, our purpose is developing a user-friendly, easy handling and close micro-quantity (MQ) straw along with different freezing media, for cryopreservation of oligozoospermic samples. Twenty oligozoospermic semen samples were collected and mixed with glycerol egg yolk citrate (GEYC) or Spermfreeze® (SPF) medium. The mixture was loaded into MQ straws, sealed and stored in liquid nitrogen (LN) vapor. After freezing, the straws were transferred into cryotube and plunged into LN. Post-thawed sperm parameters including motion characteristics, viability, membrane and DNA integrity were evaluated one and three months after cryopreservation. The post-thawed sperm parameters were significantly reduced in GEYC and SPF medium compared to fresh samples. No statistically significant differences were seen in sperm characteristics between the two storage times (i.e. month 1 vs. month 3). Furthermore, GEYC medium yielded higher motility, viability and membrane integrity compared to SPF at both storage time-points. Sperm DNA integrity was also improved in GEYC group compared to SPF after 1 month of storage. The findings of our study showed that application of MQ straw along with GEYC, as the cryoprotectant, was beneficial for cryopreservation of low count semen samples.

Spermatogenesis disorder is associated with mutations in the ligand-binding domain of an androgen receptor.

Androgens play a key role in spermatogenesis, and their functions are mediated by the androgen receptor (AR). Some mutations in the AR gene have the potential to alter the primary structure and function of the protein. The aim of this study was to investigate the AR gene mutations in a cohort of males with idiopathic azoospermia referred to Royan Institute. Fifty-one biopsy samples were obtained for routine clinical purposes from 15 men with hypospermatogenesis (HS), 17 patients with maturation arrest (MA) and 19 patients with Sertoli cell-only syndrome (SCOS). The AR cDNAs were prepared from tissue mRNAs and were sequenced. One synonymous variant and three nonsynonymous protein coding single nucleotide polymorphisms (nsSNPs) were detected. Protein structure prediction demonstrated that the S815I and M746T nonsynonymous variants would affect protein structure and its normal function. Our study suggests that mutations in the AR gene would change or disturb the receptor's normal activity. Although these variations may influence spermatogenesis, it is difficult to say that they lead to a lack of spermatogenesis.

Study of trinucleotide expansions and expression of androgen receptor in infertile men with abnormal spermogram referred to Royan institute.

Androgen receptor (AR) mediates androgen activities such as the growth of accessory sex organs, and initiation and promotion of spermatogenesis. There are two trinucleotide polymorphisms (CAG and GGN repeats) in the first exon of AR gene that their association with infertility is still controversial. The variants of both polymorphic repeats were investigated by PCR-Sequencing in 220 infertile men (80 azoospermic, 60 oligospermic and 80 asthenospermic) and 80 healthy fertile controls. AR Expression level was quantified by RT-qPCR on 30 patients (20 patients with nonobstructive azoospermia (NOA) and 10 obstructive azoospermia patients as controls). Our results demonstrated that the medians of CAG and GGN repeats length in infertile group were significantly higher than fertile men (p < 0.05). AR expression results showed a significant increase in SCOS group compared to control (p < 0.05). Long stretches of tandem repeats of AR gene may negatively affect the function of the gene and consequently lead to male infertility. In patients with SCOS, AR expression increases because of the lack of germ cells. Therefore, with increasing AR expression, the probability of SCOS occurrence is also increased. It can be concluded that increasing AR expression in testes tissue decreases the probability of sperm presence.

Sperm retrieval rate and reproductive outcome of infertile men with azoospermia factor c deletion.

To evaluate the success rate in sperm retrieval (SR) through microdissection testicular sperm extraction (micro-TESE) in infertile azoospermia factor c (AZFc)-deleted men and determining their reproductive outcomes following ICSI, medical records of couples with AZFc-deleted male partners were reviewed on patient's age, serum hormone levels, karyotype, testicular pathology and pregnancy outcomes. A comparison on age and serum hormone level was conducted between groups with positive and negative sperm retrieval in both azoospermic and oligozoospermic AZFc-deleted men. Of 225 who had AZFc deletion, 195 cases followed clinical treatments. From 195 cases, 116 were azoospermic, 79 were oligozoospermic. Pathology profile was available in 103 of 195 subjects which the predominant trait was SCOS and was seen in 66.9% of cases (69 of 103). Success rate of sperm retrieval in azoospermic patients who underwent micro-TESE was 36.3% (28/77). Forty-three oligozoospermic and 17 azoospermic patients started ART cycle. Pregnancy rate in oligozoospermic group was 35.4% (17 cases), whilst there was no clinical pregnancy in azoospermic group. In conclusion, the pregnancy and delivery in oligozoospermic patients with AZFc deletion are comparable with other studies, but despite of sperm retrieval in azoospermic patients with AZFc deletion, the chance of pregnancy or delivery in these patients was very low.

Deficient expression of JMJD1A histone demethylase in patients with round spermatid maturation arrest.

JMJD1A (jumonji domain-containing 1A), a known histone H3K9 demethylase, has been identified as a critical epigenetic regulator in male germ cells, activating the sperm chromatin-packaging genes encoding protamines (PRM) and transition proteins (TNP) required for spermatid elongation and condensation. This research investigated the expression pattern of JMJD1A protein in testicular biopsies of 79 infertile men who had undergone testicular sperm extraction. Samples were classified into obstructive azoospermia (OA, n = 26), round spermatid maturation arrest (SMA, n = 29) and Sertoli cell only syndrome (SCOS, n = 24). Experiments including the detection of mRNA and protein expressions of JMJD1A revealed a severe decrease of JMJD1A/JMJD1A in samples with SMA and SCOS compared with samples with OA (P < 0.005, Kruskal-Wallis test). Additional experiments, including incorporation of JMJD1A on the promoter regions of TNP and PRM genes, and the expression of these genes, showed a significant decrease in the SMA and SCOS versus the OA testes (P < 0.005, Kruskal-Wallis test). These findings show the low expression of JMJD1A/JMJD1A, as well as its low incorporation into chromatin in testes with round spermatid maturation arrest, suggesting that a deficient expression of JMJD1A/JMJD1A might be reflecting and/or contributing to round spermatid maturation arrest.

The impact of RABL2B gene (rs144944885) on human male infertility in patients with oligoasthenoteratozoospermia and immotile short tail sperm defects.

PURPOSE: Male infertility is a multifactorial disorder with impressively genetic basis; besides, sperm abnormalities are the cause of numerous cases of male infertility. In this study, we evaluated the genetic variants in exons 4 and 5 and their intron-exon boundaries in RABL2B gene in infertile men with oligoasthenoteratozoospermia (OAT) and immotile short tail sperm (ISTS) defects to define if there is any association between these variants and human male infertility. METHODS: To this purpose, DNA was extracted from peripheral blood and after PCR reaction and sequencing, the results of sequenced segments were analyzed. In the present study, 30 infertile men with ISTS defect and 30 oligoasthenoteratozoospermic infertile men were recruited. All men were of Iranian origin and it took 3 years to collect patient's samples with ISTS defect. RESULTS: As a result, the 50776482 delC intronic variant (rs144944885) was identified in five patients with oligoasthenoteratozoospermia defect and one patient with ISTS defect in heterozygote form. This variant was not identified in controls. The allelic frequency of the 50776482 delC variant was significantly statistically higher in oligoasthenoteratozoospermic infertile men (p < 0.05). Bioinformatics studies suggested that the 50776482 delC allele would modify the splicing of RABL2B pre-mRNA. In addition, we identified a new genetic variant in RABL2B gene. CONCLUSIONS: According to the present study, 50776482 delC allele in the RABL2B gene could be a risk factor in Iranian infertile men with oligoasthenoteratozoospermia defect, but more genetic studies are required to understand the accurate role of this variant in pathogenesis of human male infertility.

Whole-exome sequencing of familial cases of multiple morphological abnormalities of the sperm flagella (MMAF) reveals new DNAH1 mutations.

STUDY QUESTION: Can whole-exome sequencing (WES) of patients with multiple morphological abnormalities of the sperm flagella (MMAF) identify causal mutations in new genes or mutations in the previously identified dynein axonemal heavy chain 1 (DNAH1) gene? SUMMARY ANSWER: WES for six families with men affected by MMAF syndrome allowed the identification of DNAH1 mutations in four affected men distributed in two out of the six families but no new candidate genes were identified. WHAT IS KNOWN ALREADY: Mutations in DNAH1, an axonemal inner dynein arm heavy chain gene, have been shown to be responsible for male infertility due to a characteristic form of asthenozoospermia called MMAF, defined by the presence in the ejaculate of spermatozoa with a mosaic of flagellar abnormalities including absent, coiled, bent, angulated, irregular and short flagella. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of patients presenting a MMAF phenotype. Patients were recruited in Iran and Italy between 2008 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: WES was performed for a total of 10 subjects. All identified variants were confirmed by Sanger sequencing. Two additional affected family members were analyzed by direct Sanger sequencing. To establish the prevalence of the DNAH1 mutation identified in an Iranian family, we carried out targeted sequencing on 38 additional MMAF patients of the same geographical origin. RT-PCR and immunochemistry were performed on sperm samples to assess the effect of the identified mutation on RNA and protein. MAIN RESULTS AND THE ROLE OF CHANCE: WES in six families identified a causal mutations in two families. Two additional affected family members were confirmed to hold the same homozygous mutation as their sibling. In total, DNAH1 mutations were identified in 5 out of 12 analyzed subjects (41.7%). If we only include index cases, we detected two mutated subjects out of six (33%) tested MMAF individuals. Furthermore we sequenced one DNAH1 exon found to be mutated (c.8626-1G > A) in an Iranian family in an additional 38 MMAF patients from Iran. One of these patients carried the variant confirming that this variant is relatively frequent in the Iranian population. The effect of the c.8626-1G > A variant was confirmed by RT-PCR and immunochemistry as no RNA or protein could be observed in sperm from the affected men. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: WES allows the amplification of 80-90% of all coding exons. It is possible that some DNAH1 exons may not have been sequenced and that we may have missed some additional mutations. Also, WES cannot identify deep intronic mutations and it is not efficient for detection of large genomic events (deletions, insertions, inversions). We did not identify any causal mutations in DNAH1 or in other candidate genes in four out of the six tested families. This indicates that the technique and/or the analysis of our data can be improved to increase the diagnosis efficiency. WIDER IMPLICATIONS OF THE FINDINGS: Our findings confirm that DNAH1 is one of the main genes involved in MMAF syndrome. It is a large gene with 78 exons making it challenging and expensive to sequence using the traditional Sanger sequencing methods. We show that WES sequencing is good alternative to Sanger sequencing to reach a genetic diagnosis in patients with severe male infertility phenotypes. STUDY FUNDING/COMPETING INTERESTS: This work was supported by following grants: the 'MAS-Flagella' project financed by the French ANR and the DGOS for the program PRTS 2014 and the 'Whole genome sequencing of patients with Flagellar Growth Defects (FGD)' project financed by the Fondation Maladies Rares for the program Séquençage à haut débit 2012. The authors have no conflict of interest.

Leukocyte Gene Expression and Plasma Concentration in Multiple Sclerosis: Alteration of Transforming Growth Factor-ßs, Claudin-11, and Matrix Metalloproteinase-2.

Multiple sclerosis is a neurodegenerative disease characterized by the present of leukocytes in the brain tissue and subsequently the formation of sclerotic plaques. Leukocytes penetration into the blood-brain barrier is related to several factors, such as, the conversion of leukocyte gene expression or plasma characteristics. In this frame, we explore alteration of matrix metalloproteinase-2 (MMP-2), transforming growth factor beta (TGF-ß) family, and Claudin-11 (as a main myelin structural protein) in leukocytes and blood plasma of multiple sclerosis patients compared to the normal group. Blood samples were collected from thirteen men affected by MS and fifteen healthy men. Leukocyte gene expression was measured using real-time PCR and plasma parameters were examined by ELISA. The results of this study showed that the gene expression of Claudin-11 was significantly higher in MS group compared with normal. Interestingly, the MMP-2 pattern was similar to Claudin-11 and correlated positively with it. It was observed that, although the expressions of TGF-ß1 and TGF-ß2 are down-regulated in the leukocytes of subjects with MS, they showed higher levels of these cytokines in blood plasma. The plasma level of TGF-ß3 in MS patients was higher than normal and correlated with Claudin-11 concentration. In conclusion, the aberrant pattern of Claudin-11, TGF-ßs family, and MMP-2 expression in leukocytes of the MS patients was observed in this study. Moreover, the plasma levels of TGF-ßs family increased in the MS group. The findings of this study provide clues for further investigations to assay MS pathogenesis.