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We report on the results of array-CGH and Whole exome sequencing (WES) studies carried out in a Tunisian family with 46,XX premature ovarian insufficiency (POI). This study has led to the identification of a familial Xp22.12 tandem duplication with a size of 559.4 kb, encompassing only three OMIM genes (RPS6KA3, SH3KBP1and EIF1AX), and a new heterozygous variant in SPIDR gene: NM_001080394.3:c.1845_1853delTATAATTGA (p.Ile616_Asp618del) segregating with POI. Increased mRNA expression levels were detected for SH3KBP1 and EIF1AX, while a normal transcript level for RPS6KA3 was detected in the three affected family members, explaining the absence of intellectual disability (ID). To the best of our knowledge, this is the first duplication involving the Xp22.12 region, reported in a family without ID, but rather with secondary amenorrhea (SA) and female infertility. As EIF1AX is a regulatory gene escaping X-inactivation, which has an extreme dosage sensitivity and highly expressed in the ovary, we suggest that this gene might be a candidate gene for ovarian function. Homozygous nonsense pathogenic variants of SPIDR gene have been reported in familial cases in POI. It has been suggested that chromosomal instability associated with SPIDR molecular defects supports the role of SPIDR protein in double-stranded DNA damage repair in vivo in humans and its causal role in POI. In this family, the variant (p.Ile616_Asp618del), present in a heterozygous state, is located in the domain that interacts with BLM and might disrupt the BLM binding ability of SPIDR protein. These findings strengthen the hypothesis that the additional effect of this variant could lead to POI in this family. Although the work represents the first evidence that EIF1AX duplication might be responsible for POI through its over-expression, further functional studies are needed to clarify and prove EIF1AX involvement in POI phenotype.

A high prevalence of genetic kidney disease in Tunisia has been detected, and their study provides very important clinical and genetic information. Autosomal dominant polycystic kidney disease (ADPKD) is one of the main causes of morbidity and mortality associated with the kidneys in Tunisia. We present here clinical and genetic characteristics of a cohort of Tunisian patients with ADPKD. Nineteen Tunisian patients with ADPKD, among 4 familial cases and 11 sporadic cases, and 50 Healthy individuals were included in this cohort. Genetic studies of PKD1/2 were carried on using Sanger sequencing and MLPA. In our study, the mean age at diagnosis was 47 ± 18 years. In addition, 84.21% of cases present a family history of ADPKD. Overall, 57.89% of the affected individuals had HTA and 26.31% patients had hematuria. 15.78 % of the patient has extra-renal cysts i.e. one patient with splenic cysts and two patients had liver cysts. 57.89 % of patients were diagnosed with various extra-renal clinical presentations i.e. myopia, hernia, deafness, intracranial aneurysm, respiratory distress, hyperthyroidism, urinary tract infection and lower back pains. The PKD1 genotype showed earlier onset of ESRD compared to PKD2 genotype (43 vs. 55 years old). Six mutations have been detected in PKD1 gene. Among them, three were novels e.g. c.688 TG, p.C230G and c.690CG, p.C230W among exon 5 and c.8522AG, p.N2841S among exon 23. In addition, thirteen single nucleotides polymorphisms have been reported in PKD1 gene. Among them, eleven previously reported in heterozygous state and two novel single nucleotides polymorphisms in heterozygous and homozygous state and predicted to be probable polymorphisms by computational tools: c.496CT, p.L166= among the exon 4, and c.10165GC and p.E3389Gln among the exon 31. Only three single nucleotides polymorphisms previously reported in ADPKD database have been identified in PKD2 gene. The description and analysis of our cohort can help in rapid and reliable diagnosis for early management of patients in Tunisia. Indeed, predictive genetic testing can facilitate donor evaluation and increase living related kidney transplantation.

The azoospermia factor c (AZFc) region harbors multi-copy genes that are expressed in the testis. Deletions of this region lead to reduced copy numbers of these genes. In this present study we aimed to determine the frequency of AZFc subdeletion in infertile and fertile men from Tunisia and to identify whether deletions of DAZ and CDY1 gene copies are deleterious on spermatogenesis and on semen quality. We studied a group of 241 infertile men and 115 fertile healthy males using a sequence tagged site (STS)±method. To gain insight into the molecular basis of the heterogeneous phenotype observed in men with the deletion we defined the type of DAZ and CDY1 genes deleted. We reported in the present study and for the first time a new type of AZFc deletion (gr/gr-DAZ2-DAZ4-CDY1b) and hypothesis that this new deletion is the result of two successive events. We also demonstrated that this deletion constitutes a relative high-risk factor for male infertility in Tunisian population.

Infertility affects 10-15 % of the population, of which, approximately 40 % is due to male etiology consisting primarily of low sperm count (oligozoospermia) and/or abnormal sperm motility (asthenozoospermia). It has been demonstrated that mtDNA base substitutions can greatly influence semen quality.In the present study we performed a systematic sequence analysis of the mitochondrial cytochrome oxidase III (COIII) gene in 31 asthenozoospermic infertile men in comparaison to normozoospermic infertile men (n=33) and fertile men (n=150) from Tunisian population.A novel m.9588GA mutation was found in the mtDNA sperm's in all asthenozoospermic patients and was absent in the normozoospermic and in fertile men. The m.9588GA mutation substitutes a highly conserved Glutamate at position 128 to Lysine. In addition, PolyPhen-2 analysis predicted that this variant is "probably damaging".

The aim of this study was to investigate the oxidative stress status and antioxidant enzyme activities in infertile men's semen and to determine their relationship with spermatozoa characteristics. Four groups of infertile men, normozoospermic (n=9), azoospermic (n=13), oligoasthenozoospermic (n=38), and asthenozoospermic (n=42), were tested for malonaldialdehyde (MDA) concentration and catalase (CAT) and superoxide dismutase (SOD) activities in semen using spectrophotometric methods. We found that MDA levels in semen and SOD activity in seminal plasma (SP) were significantly higher in oligoasthenozoospermic and asthenozoospermic groups compared with normozoospermic group. In contrast, the mean values of CAT activity were not significantly different in all groups. We found positive correlations between semen MDA concentration and SOD activity (P

L’exploration de l’homme azoospermique est aujourd’hui d’un interet capital compte tenu des progres recents des techniques d’Assistance Medicale a la Procreation, essentiellement l’injection intra-cytoplasmique de spermatozoide, qui permet desormais a des hommes consideres definitivement infertiles, de devenir peres avec leurs propres gametes. Nous rapportons ici les resultats d’une etude retrospective, effectuee sur 54 patients infertiles tunisiens, suivis pour exploration d’une azoospermie et nous discutons les aspects clinico-biologiques et etiologiques de ces perturbations majeures du sperme. L’etude a comporte l’analyse descriptive, retrospective des donnees de l’examen clinique et des explorations complementaires (biologie seminale, bilan hormonal, examens genetiques, etc). Nous avons trouve que 43 cas d’azoospermie etaient d’origine secretoire et 7 cas d’origine excretoire. Un cas etait d’origine mixte et 3 autres d’origine indeterminee. L’azoospermie etait associee a une atrophie/hypotrophie testiculaire clinique ou echographique dans 43,9% des cas, a une FSH elevee dans 58,5% des cas et a des anomalies chromosomiques dans 31,4% des cas. Les aberrations etaient a type de syndrome de Klinefelter (9 cas), syndrome de mâle 46,XX (1 cas) et inversion pericentrique d’un chromosome 12 (1 cas). Les microdeletions du chromosome Y, la varicocele, la cryptorchidie, l’infection urogenitale et les causes obstructives, etaient observees chez, respectivement 25%; 48,8%;13,9%; 9,5% et 13% des patients azoospermiques. Aucune anomalie genetique et hormonale n’a ete trouvee dans les cas de deficiences spermatiques d’origine excretoire; cependant un tableau d’infection uro-genitale a ete trouve dans environ la moitie des cas. Conformement a la litterature, dans la population tunisienne, l’azoospermie est essentiellement d’origine secretoire et est causee principalement par les anomalies genetiques.

Nine Y-STR loci from the "minimal haplotype" (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393) included in Y-STR Haplotype Reference Databases (YHRD) with 4 additional Y-STRs (DYS436, DYS437, DYS438, DYS439) were analyzed by PCR using duplex and Y-PLEX 12 kit, followed by automatic genotyping in a sample of 105 Tunisian males originating from Sfax region (south Tunisia). Allelic frequencies and gene diversities for each Y-STR locus were determined. The high haplotype diversity (0.9932) and discrimination capacity (0.7714) show the usefulness of these loci for human identification in forensic studies and paternity tests in Tunisia. The most common haplotype was shared by 4.7% (5 individuals) of the sample was only found in samples from the Tunisian population reported in YHRD. One private allele for DYS392 (allele 17) was discovered and duplications were observed for five loci (DYS19, DYS389I, DYS393, DYS437 and DYS439).

The relationship between male infertility and AZFc micro-deletions that remove multiple genes of the Y chromosome varies among countries and populations. The purpose of this study was to analyze the prevalence and the characteristics of different Deleted in azoospermia ( DAZ ) gene copy deletions and their association with spermatogenic failure and male infertility in Tunisian men. 241 infertile men (30.7% azoospermic (n = 74), 31.5% oligozoospermic (n = 76) and 37.7% normozoospermic (n = 91)) and 115 fertile healthy males who fathered at least one child were included in the study. Three DAZ -specific single nucleotide variant loci and six bi-allelic DAZ -SNVs (I–VI) were analyzed using polymerase chain reaction (PCR)–restriction fragment length polymorphism and PCR. Our findings showed high frequencies of infertile men (73.85%) and controls (78.26%) having only three DAZ gene copies ( DAZ1 / DAZ2 / DAZ3 or DAZ1 / DAZ3 / DAZ4 variants); so deletion of DAZ2 or DAZ4 were frequent both in infertile (36.5% and 37.3%, respectively) and fertile groups (33.9% and 44.3%, respectively) and removing DAZ4 copy was significantly more frequent in oligospermic than in normospermic men (p = 0.04) in infertile group. We also report for the first time that simultaneous deletion of both DAZ2 and DAZ4 copies was significantly more common in infertile men (12.4%) than in fertile men (4.3%) (p = 0.01). However, deletions of DAZ1 / DAZ2 and DAZ3 / DAZ4 clusters were very rare. Analysis of DAZ gene copies in Tunisian population, suggested that the simultaneous deletion of DAZ2 and DAZ4 gene copies is associated with male infertility, and that oligospermia seems to be promoted by removing DAZ4 copy.

The relationship between male infertility and microdeletions in the Y chromosome that remove multiple genes varies among countries and populations. The aim of this study was to investigate the different types of Chromodomain protein , Y-linked 1 ( CDY1 ) gene deletions and their effect on male infertility and spermatogenesis in Tunisian men. A total of 241 infertile men with different spermatogenic impairments and 115 fertile men were included in this study. We determined the prevalence of CDY1a and CDY1b copy deletions by PCR-RFLP using PvuII as restriction endonuclease. Results : Among the 356 Tunisian individuals, 93.25% had the two copies ( CDY1a and CDY1b ) of CDY gene (91.2% in infertile patients and 97.3% in fertile men). We also found that deletion of CDY1b was significantly more frequent in infertile patients (azoo/oligospermic and normospermic) than in fertile men (7% vs 1.7% respectively; p value = 0.02). However, deletion of CDY1a copy was very rare, and was detected in only one fertile man and four normospermic infertile patients. Our findings showed that deletion of CDY1b copy gene is a significant risk factor for male infertility independent of sperm concentration, whereas deletion of CDY1a gene seems to have no effect on fertility in the Tunisian population.

Il existe de nombreuses controverses concernant la relation entre la presence de leucocytes dans le sperme et la fertilite masculine. Dans le but de determiner le retentissement de la pyospermie sur la qualite du sperme, nous avons entrepris une etude retrospective dans laquelle nous avons compare les parametres du sperme ainsi que la frequence des anomalies spermatiques entre des patients infertiles presentant au spermogramme une pyospermie (leucospermie ≥1 million/ml de sperme) et des patients non pyospermiques (leucospermie

l’infection genitale a Chlamydia trachomatis (CT) est une cause frequente d’infertilite du couple. Son retentissement sur les parametres du sperme et sur la fertilite masculine reste controverse. Le but de cette etude prospective est d’evaluer l’impact de l’infection genitale a CT sur le sperme chez des hommes consultant pour infertilite du couple. l’etude a porte sur 97 couples infertiles. La recherche de CT etait realisee sur les prelevements genitaux par immuno-fluorescence directe (IFD), culture cellulaire, PCR et ELISA, et sur les prelevements sanguins par micro-immunofluorescence (MTF). Chez tous les hommes, un spermogramme complet etait realise selon la methode standardisee de l’OMS (1). 34% des hommes etaient positifs pour un ou plusieurs marqueurs de l’infection chlamydienne (CT+). Dans environ 76% des cas l’infection etait asymptomatique et dans 60,6% des cas les epouses etaient CT+. Les valeurs moyennes des parametres du spermogramme dans les deux groupes de patients CT-t et CT-ne montraient pas de differences significatives; elles etaient respectivement de 39,6%±17,5% et de 40,4%±14,9% pour la mobilite des spermatozoides, de 61,9%±18,1% et 62,4%±18,5% pour la vitalite, de 80,7×106±67,5×106 et 67,1×106±65,2×106 pour la numeration et de 34,7%±16,7% et 33%±0,1% pour le taux des formes normales. L’oligospermie etait significativement plus frequente dans le groupe CT+(54,5%) contre (26,9%) dans le groupe CT-. Les taux eleves de flagelles enroules etaient observes chez 18,5% des patients CT+et chez 7,4% des patients CT-, mais la difference n’est pas significative. La prevalence de l’infection genitale a CT est frequente dans notre etude. L’absence de retentissement de l’infection chlamydienne sur les valeurs moyennes des parametres du spermogramme permet de suggerer qu’il n’existe pas d’effet deletere de CT sur les spermatozoides, mais nous ne pouvons pas exclure un retentissement sur leurs capacites fecondantes ou sur leur ultrastructure. La frequence relativement elevee de l’oligospermie dans le groupe CT+ suggere l’existence d’un effet nefaste sur la fonction gametogene du testicule ou sur la permeabilite des voies spermatiques. Une autre hypothese serait que l’oligospermie, reflet d’une spermatogenese perturbee pourrait etre associee a une diminution de l’immunite locale favorisant ainsi les infections. Des etudes plus rigoureuses avec une exploration complete de toutes les fonctions spermatiques et des differents compartiments du tractus genital sont necessaires pour determiner avec plus de precision le retentissement reel de l’infection chlamydienne sur la fonction de reproduction de l’homme.

To assess the incidence and the type of chromosomal aberrations in males with infertility we reviewed cytogenetic results in 76 Tunisian infertile men (54 nonobstructive azoospermia and 22 oligo-asthenospermia).Karyotyping was performed on peripheral blood lymphocytes according to the standard methods. Molecular diagnosis of classical and partial Y-chromosomal microdeletions was performed by amplifying Y-specific STSs markers.Various numerical and structural chromosome abnormalities were identified in 15 patients (19.48%). The occurrence of chromosomal abnormality in the azoospermics and severe oligo-asthnospermic was 21.7% and 13.5%, respectively. The most common was Klinefelter syndrome, accounting for 10 of the 15 cytogenetic defects. The total frequency of Y chromosomal microdeletions was 17.1%, with respective frequencies in azoospermic and severe oligospermic groups, 11.1% and 31.8%. The most frequent of Y chromosomal deletions were the partial ones (11.1% in azoospermic and 27.2% in oligospermic).The occurrence of chromosomal abnormalities among infertile males strongly suggests the need for routine genetic testing and counseling prior to the employment of assisted reproduction techniques.

The purpose of this study was threefold: to compare semen and first void urine (FVU) specimens from asymptomatic infertile men for the detection of Chlamydia trachomatis, genital ureaplasma, and genital mycoplasma infections using in-house inhibitor-controlled polymerase chain reaction (PCR)-microtiter plate hybridization assay; to determine the prevalence of those organisms in infertile men in Tunisia; and to study the relationship between these bacteria and male infertility. Paired urine and semen specimens from 104 patients were examined by in-house PCR for the presence of DNA of Chlamydia trachomatis, genital ureaplasmas (Ureaplasma urealyticum and Ureaplasma parvum) and genital mycoplasmas (Mycoplasma hominis and Mycoplasma genitalium). Semen analysis was assessed according to the guidelines of the World Health Organization. Nominal scale variables, the Mann-Whitney test, and the Kruskal-Wallis nonparametric analysis of variance test were used for statistical analysis. There was a very high concordance (>95%) and a very good agreement (kappa > 0.9) between the detection of Chlamydia trachomatis, genital ureaplasmas, and Mycoplasma hominis in semen and corresponding FVU specimens. Our findings also show a high concordance (81.1%) and a good agreement (kappa = 0.79) between the detection of Mycoplasma genitalium in both specimens. C trachomatis, genital mycoplasmas, and genital ureaplasmas were found to be widespread among infertile male patients in Tunisia, as shown by their respective prevalences of 43.3%, 18.3%, and 14.4%. The mean values of seminal volume, sperm concentration, sperm viability, sperm motility, sperm morphology, and leukocyte count were not significantly related either to the detection of C trachomatis DNA or to that of genital ureaplasma or mycoplasma DNA in semen specimens. Using our in-house PCR, both semen and FVU were found to be sensitive diagnostic specimens for the detection of C trachomatis, ureaplasmas, and mycoplasmas. The FVU, a less invasive and self-collected specimen, can serve as a marker for the presence of these organisms in the genital tract and can be used as a reliable way of detecting asymptomatic carriers of infection.

The aim of this study was to determine the appropriate tag sequence tagged site (STS) associated with azoospermia. We evaluated the incidence of Y chromosome microdeletions in Tunisian infertile male patients by polymerase chain reaction using 14 STSs in the azoospermia factor (AZF) region of Yq11. A logistic regression analysis was performed to test the association of STSs with semen quality. Haploview version 3.11 was used to identify the possible blocks of deletion involving a minimum number of STSs and that can be used to tag the deletion block in future analysis. Using the 14 STSs, 48% infertile patients (102 of 210) had microdeletions of Y chromosome but, following the European Academy of Andrology guidelines, only 16% of patients had microdeletions. A statistically significant difference was found with some STSs for azoospermia and oligozoospermia. A candidate combinaison composed of 4 STSs (RBMY-sy157-sy84-sy130) was associated with azoospermia in a Tunisian population. According to this study, this tag can be used in the screening of Y chromosome microdeletions before assisted reproduction treatment in a Tunisian population.