Leila Ammar-Keskes

Chromosomal defects in infertile men with poor semen quality

PurposeTo 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).MethodsKaryotyping 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.ResultsVarious 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).ConclusionThe 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.

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.

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. Mol. Reprod. Dev. 80: 581–587, 2013.

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”.

Screening of ΔF508 mutation and IVS8-poly T polymorphism in CFTR gene in Tunisian infertile men without CBAVD.

It is well established that cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations are involved in congenital bilateral absence of the vas deferens (CBAVD), causing obstructive azoospermia and male infertility. Also, several studies reported a relatively high prevalence of CFTR gene mutations in healthy men presenting reduced sperm quality. In this study, we investigate ΔF508 mutation and IVS8‐polyT polymorphism in CFTR gene in Tunisian infertile men without CBAVD. Genetic analyses were performed in 148 infertile patients and 126 fertile individuals. The polymorphic IVS8‐polyT tract in CFTR gene was analysed in only 129 infertile patients and 54 individuals of control group. As well, we screened for Y chromosome microdeletions in all infertile patients. No ΔF508 mutation was diagnosed either in infertile patients or in control group. 5T allele of IVS8‐polyT tract was found in both infertile men (4.26%) and fertile individuals (8.33%). 5T/5T genotype was observed only in two azoospermic patients without Y microdeletions. The most frequent genotype of IVS8‐polyT tract in infertile men and controls was 7T/7T (69.75% and 59.25% respectively). There was no association between IVS8‐polyT polymorphism and reduced semen quality. Neither ΔF508 mutation nor 5T allele is involved in pathogenesis of male infertility in Tunisian infertile patients without CBAVD.

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.

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.

The CAG repeat polymorphism of mitochondrial polymerase gamma (POLG) is associated with male infertility in Tunisia

Male fertility largely depends on sperm quality, which may be affected by environmental and genetic factors. Recent data emphasised the implication of the polymorphism of mitochondrial DNA polymerase gamma (POLG) CAG repeats in male infertility. In this report, we explored a possible role of the (POLG) gene polymorphism in male infertility in Tunisian men. The polymorphic CAG repeat in the nuclear POLG gene was studied in 339 male subjects (216 patients with infertility (69 azoospermic, 115 oligoasthenoteratospermic and 32 normospermic) and 123 fertile) after DNA amplification by PCR, followed by genotyping using an automatic sequencer. The heterozygous and the homozygous mutant genotypes (10/≠10 and ≠10/≠10) were significantly more frequent among infertile patients than among fertile controls (11.2% versus 1.6%, P = 1.3 × 10−3 and 4.6% versus 0.8%, P = 4.2 × 10−7 respectively). We also found a significant difference between the frequencies of 10/≠10 genotype in azoospermic (4.4%) and in oligoasthenoteratospermic (15.6%) infertile patients (P = 2.6 × 10−2). However, the homozygous mutant genotype (≠10/≠10) was seen at similar frequencies in azoospermic, normospermic and oligoasthenospermic men (4.4%, 3.1% and 5.2% respectively). Under our conditions, the findings showed an association between POLG CAG repeat polymorphism and male infertility in 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.

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.