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.

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.

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.

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".

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.

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.

Pericentric inversion of chromosom 12 [Inv (12) (p12q12)] associated with idiopathic azoospermia in one infertile Tunisian man.

Chromosome aberrations are found in 2-7% of couples with fertility problems and pericentric inversions are structural chromosomal abnormalities, potentially associated with infertility or multiple miscarriages. In this study, we report the first case of pericentric inversion of chromosome 12 associated with non-obstructive azoospermia. A karyogram revealed pericentric inversion of chromosome 12 with breakpoints at 12p12 and 12q12. Testicular histopathology confirmed the Sertoli cell-only syndrome.

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.

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.

Genetic association between AZF region polymorphism and Klinefelter syndrome.

Because of conflicting results about the association between azoospermic patients with Klinefelter syndrome (KFS) and azoospermia factor (AZF) polymorphism, and because nothing is known about the association of KFS with partial AZFc deletions, an association study was performed in Tunisian KFS patients. A total of 29 azoospermic patients and 13 fertile men were enrolled in this study. The classical microdeletions were found in six out of nine KFS patients (67%). Gr/Gr deletions and b2/b3 deletions are partial AZFc deletions. One KFS patient without classical microdeletions had a gr/gr deletion. This deletion (gr/gr) was observed in four out of 18 azoospermic patients without chromosomal abnormalities. In addition, two b2/b3 and one AZFc deletion were identified in this group. All KFS patients had elevated plasma FSH and LH concentrations, but normal plasma testosterone concentration. The testis biopsy of three samples with Y microdeletions revealed Sertoli cell-only syndrome. No Y microdeletions or partial AZFc deletions were found in the fertile group. It is concluded that in the patient population KFS patients may harbour Y microdeletions, and screening for these should be part of the diagnostic work-up, particularly in those considering assisted reproductive techniques. However, partial AZFc deletions might not play a role in predisposing genetic background for the phenotype of azoospermic KFS subjects.