Y-chromosome haplogroups and Azoospermia Factor (AZF) analysis in Tunisian infertile male.

The aim of the present study was to clarify the implication of Y chromosome genetic variations and haplogroups in Tunisian infertile men. A total of 27 Y-chromosomal binary markers partial microdeletions (gr/gr, b1/b3 and b2/b3) and copy number variation of DAZ and CDY genes in the AZFc region were analysed in 131 Tunisian infertile men with spermatogenic failure and severe reduced sperm concentrations and in 85 normospermic men as controls. Eleven different haplogroups in the overall population study (E3b2; J1J*, E1, E3b*, F, G, K, P/Q, R*, R1* and R1a1) were found. Interestingly, the J1J* haplogroup was significantly more frequent in azoo/oligospermic patients than in normospermic men (35.1% and 22.3%, respectively (p value = 0.04)). Results showed also that patients without DAZ/CDY1 copies loss and without partial microdeletions belonged to the R1 haplogroup. The relative high frequencies of two haplogroups, E3b2 (35.1%) and J (30%) was confirmed in Tunisia. We reported in the present study and for the first time, that J1J* haplogroup may confer a risk factor for infertility in the Tunisian population and we suggested that R1 haplogroup may ensure certain stability to Y-chromosome in Tunisian men.

Routine assessment of occupational exposure and its relation to semen quality in infertile men: a cross-sectional study.

BACKGROUND/AIM: Concerns about the detrimental effects of occupational and environmental exposure on male reproductive function have been raised by reports of declining sperm quality over the last decades. The aim of this study was to investigate the association between altered semen parameters and exposure to occupational risk factors as assessed by questionnaire. MATERIALS AND METHODS: We conducted a cross-sectional questionnaire-based study among a population of 2122 men who underwent andrological investigation for couple infertility. All participants were interviewed and their semen samples were analyzed. Information about medical history and occupational exposure was used to classify participants into exposed and unexposed groups. RESULTS: Exposure to pesticides was associated with a significantly higher risk of asthenozoospermia (adjusted odds ratio [OR] = 1.6; 95% CI, 1.0-2.4) and necrozoospermia (OR = 2.6; 95% CI, 1.4-4.7). Exposure to cement was found to be correlated with a higher risk of oligozoospermia (OR = 1.1; 95% CI, 0.9-1.4). There was no association between semen impairment and exposure to solvents, excess heat, or mechanical vibrations. CONCLUSION: We found an association between self-reported occupational exposure and altered semen parameters. These results support the usefulness of questionnaires for routine assessment and management of occupational exposures in infertile men.

Human sperm Toll-like receptor 4 (TLR4) mediates acrosome reaction, oxidative stress markers, and sperm parameters in response to bacterial lipopolysaccharide in infertile men.

PURPOSE: To study the role of Toll-like receptor 4 (TLR4) in human spermatozoa and to assess sperm parameters, oxidative stress markers, and acrosome reaction in response to the stimulation of TLR4 by its ligand, the lipopolysaccharide (LPS), as a major endotoxin of Gram-negative bacteria. METHODS: Our study was carried out in 73 sperm samples from patients undergoing semen analysis for couple infertility investigations. The studied patients were divided into three groups: normozoospermic fertile patients (n = 13), patients with abnormal and leukospermic semen (n = 13), and patients with abnormal and non-leukospermic semen (n = 47). TLR4 expression in human spermatozoa was initially analyzed by western blot. Sperm samples were incubated in the presence of LPS (200 ng/ml) for 18 h. Then, sperm motility and vitality were evaluated by microscopic observation and oxidative stress markers as malondialdehyde (MDA) and carbonyl groups (CG) were spectrophotometrically assessed in neat and selected sperm. A triple-stain technique was also performed to evaluate acrosome reaction in 15 sperm samples from infertile patients. RESULTS: TLR4 expression was confirmed in human spermatozoa with a molecular weight of 69 kDa. In the normozoospermic group, no significant differences in sperm parameters and oxidative stress markers were shown after incubation with LPS in neat and selected sperms. Regarding samples from the non-leukospermic group, LPS reduced spermatozoa motility and vitality rates in selected sperm (P = 0.003; P = 0.004, respectively). A significant increase of MDA and CG levels was also detected (P = 0.01; P = 0.02, respectively). However, only the MDA levels were significantly increased (P = 0.01) in neat LPS-stimulated sperm. The same results were shown within the leukospermic group. The comparison between the two groups, leukospermic and non-leukospermic, in selected sperms showed a more important LPS effect in the leukospermic group significantly on motility and MDA rates (P = 0.006; P = 0.009, respectively). Furthermore, a significant decrease in reacted spermatozoa rate was detected in response to LPS in selected sperm samples from infertile men (P = 0.03). CONCLUSIONS: These findings indicate that human spermatozoa express TLR4 and respond to LPS stimulation with alterations in viability, motility, and the acrosome reaction implicating reactive oxygen species (ROS) production in sperm samples from infertile patients.

gr/gr-DAZ2-DAZ4-CDY1b deletion is a high-risk factor for male infertility in Tunisian population.

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.

[Inter-and intra-operator variability in the analysis of semen parameters: results from a quality control program]. / Variabilité inter et intra-opérateur de l'analyse des paramètres spermatiques: résultat d'un programme de contrôle de qualité.

INTRODUCTION: Semen analysis is a key part of male infertility investigation. The necessity of quality management implementation in the andrology laboratory has been recognized in order to ensure the reliability of its results. The aim of this study was to evaluate intra- and inter-individual variability in the assessment of semen parameters in our laboratory through a quality control programme. METHODS: Four participants from the laboratory with different experience levels have participated in this study. Semen samples of varying quality were assessed for sperm motility, concentration and morphology and the results were used to evaluate inter-participant variability. In addition, replicates of each semen sample were analyzed to determine intra-individual variability for semen parameters analysis. RESULTS: The average values of inter-participant coefficients of variation for sperm motility, concentration and morphology were 12.8%, 19.8% and 48.9% respectively. The mean intra-participant coefficients of variation were, respectively, 6.9%, 12.3% and 42.7% for sperm motility, concentration and morphology. Despite some random errors of under- or overestimation, the overall results remained within the limits of acceptability for all participants. Sperm morphology assessment was particularly influenced by the participant's level of experience. CONCLUSION: The present data emphasize the need for appropriate training of the laboratory staff and for regular participation in internal quality control programmes in order to improve the reliability of laboratory results.

Deletion of CDY1b copy of Y chromosome CDY1 gene is a risk factor of male infertility in Tunisian men.

UNLABELLED: 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.

Molecular detection of Chlamydia trachomatis and other sexually transmitted bacteria in semen of male partners of infertile couples in Tunisia: the effect on semen parameters and spermatozoa apoptosis markers.

This study was undertaken to determine the prevalence of Chlamydia trachomatis, Mycoplasmas, and Ureaplasmas in semen samples of the male partners of infertile couples and to investigate whether Chlamydia trachomatis could initiate apoptosis in human spermatozoa. A total of 85 males partners of infertile couples undergoing routine semen analysis according to World Health Organization guidelines were included. Specimens were examined for the presence of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum and Ureaplasma parvum by Real time PCR (qPCR). Semen specimens were analysed for the appearance of apoptotic markers (sperm DNA fragmentation, activated caspase 3 levels, mitochondrial membrane potential (ΔΨm)) using flow cytometry. C. trachomatis, N. gonorrhoeae, U. urealyticum, M genitalium were detected in semen samples of 13 (15.2%), 5 (5.8%), 5 (5.8%) and 3 (3.5%) male partners of infertile couples, respectively. M. hominis and U. parvum were detected in semen sample of only one patient (1.1%). The semen of infertile men positive for C. trachomatis showed lower mean of semen count and lower rapid progressive motility (category [a]) of spermatozoa compared to uninfected men with statistically significances (p = 0.02 and p = 0.04, respectively). Flow cytometry analyses demonstrated a significant increase of the mean rate of semen with low ΔΨm and caspase 3 activation of infertile men positive for C. trachomatis compared to uninfected men (p = 0.006 and p = 0.001, respectively). DNA fragmentation was also increased in sperm of infertile men positive for C. trachomatis compared to uninfected men but without statistical significances (p = 0.62). Chlamydial infection was associated to loss of ΔΨm and caspase 3activation. Thus, C. trachomatis infection could be incriminated in apoptosis induction of spermatozoa. These effects may explain the negative direct impact of C. trachomatis infection on sperm fertilizing ability.

Combined deletion of DAZ2 and DAZ4 copies of Y chromosome DAZ gene is associated with male infertility in Tunisian men.

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.

Identification of a novel m.9588G > a missense mutation in the mitochondrial COIII gene in asthenozoospermic Tunisian infertile men.

PURPOSE: 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. METHODS: 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. RESULTS: A novel m.9588G>A mutation was found in the mtDNA sperm's in all asthenozoospermic patients and was absent in the normozoospermic and in fertile men. The m.9588G>A mutation substitutes a highly conserved Glutamate at position 128 to Lysine. In addition, PolyPhen-2 analysis predicted that this variant is "probably damaging".

Assessment of chromatin maturity in human spermatozoa: useful aniline blue assay for routine diagnosis of male infertility.

During spermatogenesis, sperm chromatin undergoes structural changes and results in a high condensation. This nuclear compaction would be useful as a predictor of sperm fertilization capacity and pregnancy outcome. We purpose to evaluate firstly the relationship among chromatin maturity assessed by aniline blue staining (AB) and the semen parameters in infertile men. Secondly, we analyzed whether the sperm gradient density centrifugation is effective to select mature spermatozoa. Fifty-one ejaculates were investigated by semen analysis and stained for chromatin condensation with AB to distinguish between unstained mature sperm and stained immature sperm. AB was applied also on 12 ejaculates which proceeded by density gradient centrifugation to compare the rates of immature sperm before and after selection. Neat semen were divided into two groups: G1 (n = 31): immature sperm <20% and G2 (n = 20): immature sperm ≥20%. No significant differences were detected in sperm concentration, motility, and normal morphology between G1 and G2. However, the rates of some morphology abnormalities were higher in G2: head abnormalities (P = 0.01) and microcephalic sperm (P = 0.02). We founded significant correlation between sperm immaturity and acrosome abnormalities (r = 0.292; P = 0.03). Sperm selection has significantly reduced the rates of immature sperm. A better understanding of chromatin structure and its impact on the sperm potential is needed to explore male infertility.

Mitochondrial DNA mutations and polymorphisms in asthenospermic infertile men.

In this study we performed a systematic sequence analysis of 6 mitochondrial genes (cytochrome oxidase I, cytochrome oxidase II, cytochrome oxidase III, adenosine triphosphate synthase6, ATP synthase8, and cytochrome b] in 66 infertile men suffering from asthenospermia (n=34) in comparison to normospermic infertile men (n=32) and fertile men (n=100) from Tunisian population. A total of 72 nucleotide substitutions in blood cells mitochondrial DNA were found; 63 of them were previously identified and reported in the human mitochondrial DNA database ( www.mitomap.org ) and 9 were novel. We also detected in 3 asthenospermic patients a novel heteroplasmic missense mitochondrial mutation (m.9387 G>A) in COXIII gene (8.8%) that was not found in any of normospermic infertile and fertile men. This mutation substituting the valine at position 61 to methionine in a conserved amino acid in the transmembrane functional domain of the polypeptide, induces a reduction of the hydropathy index (from +1.225 to +1.100) and a decrease of the protein 3D structures number (from 39 to 32) as shown by PolyPhen bioinformatic program.

A novel m.6307A>G mutation in the mitochondrial COXI gene in asthenozoospermic infertile men.

Infertility affects 10-15% of the population, of which approximately 40% is due to male etiology and consists primarily of low sperm count (oligozoospermia) and/or abnormal sperm motility (asthenozoospermia). Recently, it has been demonstrated that mtDNA substitutions can influence semen quality. In this study, we performed a sequence analysis of the mitochondrial cytochrome oxidase I (COXI) gene in 31 infertile men suffering from asthenozoospermia in comparison to 33 normozoospermic infertile men and 100 fertile men from the Tunisian population. A novel m.6307A>G mutation was found in sperm mitochondrial DNA (mtDNA). This mutation was found in six asthenozoospermic patients, and was absent in normozoospermic and fertile men. We also detected 21 known substitutions previously reported in the Human Mitochondrial Database. The m.6307A>G mutation substitutes a highly conserved asparagine at position 135 to serine. In addition, PolyPhen-2 analysis predicted that this variant is "probably damaging.

Possible association of a novel missense mutation A6375G in the mitochondrial cytochrome C oxidase I gene with asthenospermia in the Tunisian population.

Cytochrome c oxidase encoded by multiple mitochondrial genes (COXI, COXII, and COXIII) and nuclear genes is an essential component of the mitochondrial respiratory chain that catalyzes the reduction of molecular oxygen by reduced cytochrome c. Subunits COXI and COXII of cytochrome c oxidase are known to play the most essential role in proton pumping and electron transfer. In this study we screened the somatic mitochondrial COXI gene of infertile men suffering from asthenospermia (n=34) in comparison to normozoospermic infertile men (n=32) and fertile men (n=100) from the Tunisian population. A novel homoplasmic missense mitochondrial mutation (m.6375A>G) was found in 5 asthenospermic patients (14%) but not in any of normozoospermic infertile men and fertile men. This mutation substituting the isoleucine at position 158 to valine in a highly conserved amino acid induces a reduction of the hydropathy index (from +1.920 to +0.239) and a decrease of the protein 3D structure number (from 50 to 26) as shown by PolyPhen bioinformatic program.

Effect of freezing-thawing process and quercetin on human sperm survival and DNA integrity.

We aimed in the first part of our work to study the effect of cryopreservation on the human sperm DNA integrity and the activation of caspase 3, the main apoptosis indicator. In the second part, we were interested in testing the effect of quercetin, as an antioxidant, in preventing sperm damage during the freeze-thawing process. Seventeen semen samples were obtained from 17 men recruited for infertility investigations. Liquefied sperm was cryopreserved using spermfreeze®. Nine of the used samples were divided into two aliquots; the first one was cryopreserved with spermfreeze only (control) and the second one was cryopreserved with spermfreeze supplemented with quercetin to a final concentration of 50 µM. Sperm motility and viability were assessed according to WHO criteria. We used TUNEL assay and the Oxy DNA assay to assess sperm DNA integrity. Activated caspase 3 levels were measured in spermatozoa using fluorescein-labeled inhibitor of caspase (FLICA). Cryopreservation led to a significant increase in sperm DNA fragmentation, DNA oxidation and caspase 3 activation (p<0.01). Supplementation of the cryopreservation medium with quercetrin induced a significant improvement in post thaw sperm parameters, compared to those of control, regarding sperm motility (p=0.007), viability (p=0.008) and DNA integrity (p=0.02); however, it had no effect on caspase 3 activation (p=0.3). We conclude that oxidative stress plays a major role in inducing sperm cryodamage but implication of apoptosis in this impairment requires further investigations. Quercetin could have protective effect during cryopreservation but further research is needed to confirm this effect.

Partial microdeletions in the Y-chromosome AZFc region are not a significant risk factor for spermatogenic impairment in Tunisian infertile men.

Azoospermia factor (AZF) subdeletions were reported to be significant risk factors for spermatogenesis. In this study, we screened classical and partial microdeletions of the Y-chromosome AZF region in a group of 261 infertile men. Partial deletions were also screened in a control group of fertile men (n=124). In addition, Y haplogroups were analyzed in 24 gr/gr deleted patients. Among the 261 studied infertile men, seven subjects were found to have classical microdeletions. The most common partial deletion of AZFc (gr/gr) was observed in 13.02% of infertile men and in 12.90% of fertile men. The b1/b3 deletion was identified in 4.98% of infertile men and in 2.41% of fertile men. In addition, the b2/b3 deletion was identified in 1.53% of infertile patients but not in the control group. Our results suggest that partial AZFc deletions are not associated with spermatogenic failure in the Tunisian population.

Chromosomal defects in infertile men with poor semen quality.

PURPOSE: 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). METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Day 3 compared with day 2 cryopreservation does not affect embryo survival but improves the outcome of frozen-thawed embryo transfers.

A retrospective study was performed to determine the differences in embryo survival and frozen-thawed embryo transfers outcome between cryopreservation performed on day 3 versus day 2. We conclude that freezing supernumerary embryos on day 3 provides similar thawing survival parameters, better implantation, pregnancy, and live-birth rates compared with day 2 cryopreservation.