Tunisian Infertile Men Research Articles

P-566 Is the seminal oxidative stress the mirror of psychological stress perceived by infertile men?

Abstract Study question To assess the association between anxiety and depression scores and the levels of oxidative stress in seminal plasma of Tunisian infertile men. Summary answer Depression in hypofertile men is associated to higher levels of catalase in seminal plasma and thus to oxidative stress. What is known already Within the last decades, the knowledge concerning the link between psychological and oxidative stress in infertile men has been surging. In a limited number of studies aiming to elucidate the psychological aspect of male infertility, data related to increased incidence of depression and anxiety have been reported. It has also been reported that anxiety and depression may trigger the production of reactive free oxygen radicals leading to disruption of the balance between free radicals and antioxidants semen properties. Study design, size, duration This was a cross-sectional study performed in the Laboratory of Cytogenetics and Reproductive Biology of Fattouma Bourguiba University Teaching Hospital (Monastir Tunisia). A total of 282 patients were assessed for levels of anxiety and depression and evaluated for semen parameters. Among these, 105 patients were assessed for levels of superoxide dismutase (SOD) and 51 patients were evaluated for the levels of catalase in seminal plasma from September 2022 to January 2023. Participants/materials, setting, methods Were included patients addressed for semen quality assessment. All patients with a previously diagnosed psychological disorder, those who presented severe depression and/or anxiety symptoms or had a stressful life event were not included. Informed consent was obtained from all the participants. The subjects were evaluated for anxiety and depression symptoms using the valid Arab version of the HAD (Hospital Anxiety and Depression) scale. Semen analysis and results interpretation were performed according to 2021 WHO guidelines. Main results and the role of chance The mean HAD-D (depression) and HAD-A (anxiety) scores were of 6.56 ± 3.07 (IIQ [4-8]) and 7.94 ± 3.73 (IIQ [5-10]) respectively. The mean levels of SOD and catalase were of 42.32 ± 24.08 and 19599.82 ± 12745.36 respectively. The results showed that patients exhibiting elevated HAD-D scores have higher levels of catalase in seminal plasma compared to those with normally ranging HAD-D scores (29856.07±15904.51 VS 17968.14±11564.79 respectively; p= 0.02). However, SOD levels were similar between the two groups and no correlation was found between seminal oxidative stress as assessed by catalase and SOD levels and both HAD-D and HAD-A scores. Limitations, reasons for caution The main limitation reason could be related to the limited number of patients evaluated for levels of catalase in seminal plasma (N = 51). Furthermore, assessing hormone’s levels (LH, FSH and testosterone) would be of great interest in elucidating the implicated pathways leading to oxidative stress and impaired semen quality. Wider implications of the findings Our results shed the light on the elevated levels of the catalase in patients with high depression scores. The absence of correlation between antioxidant enzymes and psychological well-being of infertile patients is a reassuring finding. However, awareness and recognition of depressive symptoms in infertile men is crucial when managing infertility. Trial registration number not applicable

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.

Relationship between nuclear DNA fragmentation, mitochondrial DNA damage and standard sperm parameters in spermatozoa of infertile patients with leukocytospermia

The association of leukocytospermia with male fertility is still under debate. Our objective was to evaluate the association of leukocytospermia with sperm parameters, mitochondrial DNA (mtDNA) variations, and seminal concentration of several oxidative stress and inflammatory cytokines in Tunisian infertile men. The studied patients were divided into two groups: patients without leukocytospermia (Group 1) and patients with leukocytospermia (Group 2). DNA fragmentation significantly increased in group 2 (31.41 %) compared to group 1 (14.68 %) ; (p < 0.001). A total of 115 nucleotide substitutions in mitochondrial DNA were depicted, among which 113 were previously identified. The number of substitutions was more elevated in group 2. Leukocytospermic group had significantly higher MDA (nmole/mL) levels than patients without leukocytospermia (34±24.43 vs 18.94±15.96 ; p=0.001), GSH (μg/mL) levels were also higher compared to the control group (126.53±22.87 vs 79.4±19.38 ; p < 0.001), SOD (U/mg of protein) levels were higher but without reaching the statistical significance (89.74±74.85 vs 67.56±37.11 ; p = 0.25) ; whereas seminal CAT (μmole H2O2/min/mg of protein) levels were lower in this group (10.66±14.32 vs 27.35±25.28 ; p = 0.012). No statistically significant differences between the two groups of patients were found in the levels of inflammatory cytokines. However, IL-8 level was positively correlated with DNA fragmentation and negatively correlated with vitality. These findings confirm the association between leukocytospermia and sperm DNA damage.

Y chromosome microdeletions screening in Tunisian infertile men

The aim of this study was to establish the prevalence of chromosomal abnormalities and microdeletions on the Y chromosome in Tunisian infertile men with severe oligozoospermia or non-obstructive azoospermia. In cases of azoospermia, we aimed also to correlate histological results after negative testicular sperm extraction with the type of Y chromosome microdeletion. 84 infertile patients and 52 controls were screened for karyotypic abnormalities using G-banding and Yq chromosome microdeletions using multiplex PCR. 7 infertile males (8.3%) carried chromosomal abnormalities and 8 (9.5%) presented Y chromosome microdeletions. The frequency of chromosome abnormalities in azoospermic patients was 11.1% vs 3.3% in the severe oligozoospermic group. Klinefelter syndrome was the most frequent chromosomal abnormalities in 85.7% of cases. Only one patient had a 46,X,del Y/45,X karyotype. The frequency of microdeletions was 11.1% in the azoospermic group and 6.7% in the severe oligozoospermic group. Six out of 84 (7.14%) of the infertile patients had microdeletions in the AZFc region, one azoospermic male had microdeletion in the AZFbc regions and one in the AZFb region, no deletions in the AZFa region. Among the 6 azoospermic patients with microdeletions: 4 had Sertoly cell only syndrome (SCOS) and 2 had maturation arrest (MA). Genetic abnormalities in infertile Tunisian patients are similar to those reported in other countries. The knowledge of the existence of genetic abnormalities and microdeletions is useful to provide a correct diagnosis and it allows the clinician to refer the patient to adequate assisted reproduction technique and examine the value of testicular biopsy pertinence.

Effect of Seminal Transforming Growth Factorß1 (TGFß1) and Glutathione on Apoptosis in Spermatozoa from Tunisian Infertile Men

We aimed to compare the effect of TGFB1 and glutathione on sperm necrosis in three groups of patients with increased necrospermia. The study included 120 men aged 37.6 ± 4.6 years consulting for sterility of the couple. Patients were subdivided into three groups according to the percentage of necrotic spermatozoa: necrospermia 80%) For each patient, the sperm parameters were evaluated according to WHO standards, TGFB1 and glutathione Oxidized and reduced was carried out by the ELISA technique and by spectrophotometry respectively. We found a significant increase in the levels of TGFβ1 and DFI in moderate and severe necrospermia groups and positive correlations between these two factors and sperm parameters (numeration, mobility and morphology). In addition, a significant decrease in seminal GHSt was observed in the necrozoospermic groups compared to the control group. A strong correlation was observed between the degree of necrozoospermia and the fragmentation of sperm DNA (r=0.878, p=0.001). In addition, a significant positive correlation between TGFβ1 and DFI in the two groups of patients with moderate necrospermia (r=0.43, p<0.05) and severe necrospermia (r=0.52, p<0.05). This confirms that seminal TGFβ1 is a factor in the fragmentation of spermatozoa DNA. These results suggest that decreased glutathione and increased seminal TGFβ1 are important risk factors that can lead to a succession of morphological and functional alterations of spermatozoa resulting in necrozoospermia. Their perturbation in seminal plasma can lead to mediocre results of medically assisted procreation.

‘Infertile’ studies on mitochondrial DNA variation in asthenozoospermic Tunisian men

We reviewed five studies undertaken by the same research group on the possible links between mitochondrial DNA (mtDNA) variation and asthenozoospermia, all carried out on Tunisian men. A thorough assessment of these articles reveals that all five studies were carried out on virtually the same cohort of patients, although this information was concealed by the authors. Thus, the results were ‘sliced’ in order to unjustifiably maximize the number of publications. In addition, a phylogenetic analysis of their data indicates that the reported results are notably incomplete and deficient. Overall, contrary to the original claims, the association of mtDNA variants with asthenozoospermia finds no support on this saga on Tunisian infertile men.

Identification of a novel m.9588G &gt; A missense mutation in the mitochondrial COIII gene in asthenozoospermic Tunisian infertile men

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.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".

Effects of reduced seminal enzymatic antioxidants on sperm DNA fragmentation and semen quality of Tunisian infertile men

To evaluate levels of sperm DNA fragmentation and enzymatic antioxidant status in seminal plasma of Tunisian fertile and infertile men in order to assess the effects of seminal oxidative stress on sperm DNA integrity and semen quality. Semen samples from 100 infertile patients (40 oligoasthenoteratozoospermics, 31 teratozoospermics and 29 asthenozoospermics) and 50 fertile men (controls) were analyzed for DNA fragmentation by TUNEL assay and biochemical parameters. Seminal antioxidant activities (Superoxide dismutase, Glutathione peroxidase and Catalase) and malondialdehyde concentrations were measured spectrophotometrically. Sperm DNA fragmentation and malondialdehyde levels in infertile groups were more elevated than controls. Nevertheless, the activities of the antioxidant enzymes were significantly lower in abnormal groups compared to normozoospermics. Sperm DNA fragmentation was closely and positively correlated to malondialdehyde levels (r = 0.37, P = 0.008); meanwhile, reduced seminal antioxidant profile was negatively associated to sperm DNA fragmentation. Interestingly, we noted also that sperm DNA fragmentation was negatively correlated to sperm motility (r = -0.54, P < 0.001) and positively associated to the abnormal sperm morphology (r = 0.57, P = 0.002). This report revealed that increased sperm DNA fragmentation can be due to the impaired seminal enzymatic antioxidant profile and increased Lipid peroxidation. Our results sustain that the evaluation of sperm DNA fragmentation and seminal oxidative biomarkers in infertile men is recommended as a consistent prognostic tool for male infertility assessment.

Differential expression of mRNA aromatase in ejaculated spermatozoa from infertile men in relation to either asthenozoospermia or teratozoospermia

Oestrogen biosynthesis in ejaculated spermatozoa is an autonomous process, which may influence sperm functions. The purpose of this study was to evaluate the relationship between the expression of aromatase, sperm quality and seminal neutral α-glucosidase marker in semen of Tunisian infertile men: asthenozoospermia (A; n = 16), teratozoospermia (T; n = 12) and asthenoteratozoospermia (AT; n = 11) in comparison with 18 normozoospermic ones. Aromatase mRNA levels estimated by real-time PCR were reduced in groups T (52%) and AT (67%) compared to controls and inversely correlated with the percentage of normal forms. A higher coefficient of correlation was noted in presence of microcephaly or acrosome malformations (r = -0.64). The asthenozoospermic group was divided into two subgroups according to the relative amount of aromatase. The subgroup (A2) with higher aromatase transcript level was associated with an increased seminal pH, a decreased sperm viability, low sperm percentage motility and low neutral α-glucosidase semen levels. Our data highlight the involvement of aromatase in motility and morphology of spermatozoa. Thus, this enzyme could bring new insights about quality and fertilising capacity of human spermatozoa.

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.

Impact of seminal trace element and glutathione levels on semen quality of Tunisian infertile men

BackgroundGrowing evidence indicates that oxidative stress can be a primary cause of male infertility. Non-enzymatic antioxidants play an important protective role against oxidative damages and lipid peroxidation. Human seminal plasma is a natural reservoir of antioxidants. The aim of this study was to determine glutathione (GSH) concentrations, trace element levels (zinc and selenium) and the lipid peroxidation end product, malondialdehyde (MDA), in the seminal plasma of men with different fertility potentials.MethodsSemen samples from 60 fertile men (normozoospermics) and 190 infertile patients (74 asthenozoospermics, 56 oligozoospermics, and 60 teratozoospermics) were analyzed for physical and biochemical parameters. Zinc (Zn) and selenium (Se) levels were estimated by atomic absorption spectrophotometry. Total GSH (GSHt), oxidized GSH (GSSG), reduced GSH (GSHr) and MDA concentrations were measured spectrophotometrically.ResultsZn and Se concentrations in seminal plasma of normozoospermics were more elevated than the three abnormal groups. Nevertheless, only the Zn showed significant differences. On the other hand, Zn showed positive and significant correlations with sperm motility (P = 0.03, r = 0.29) and count (P < 0.01, r = 0.49); however Se was significantly correlated only with sperm motility (P < 0.01, r = 0.36). GSHt, GSSG and GSHr were significantly higher in normozoospermics than in abnormal groups. We noted a significant association between seminal GSHt and sperm motility (P = 0.03). GSSG was highly correlated to sperm motility (P < 0.001) and negatively associated to abnormal morphology (P < 0.001). GSHr was significantly associated to total sperm motility (P < 0.001) and sperm count (P = 0.01). MDA levels were significantly higher in the three abnormal groups than in normozoospermics. Rates of seminal MDA were negatively associated to sperm motility (P < 0.01; r = -0.24) and sperm concentration (P = 0.003; r = -0.35) Meanwhile, there is a positive correlation between seminal lipid peroxidation and the percentage of abnormal morphology (P = 0.008).ConclusionsThis report revealed that decreased seminal GSH and trace element deficiencies are implicated in low sperm quality and may be an important indirect biomarker of idiopathic male infertility. Our results sustain that the evaluation of seminal antioxidant status in infertile men is necessary and can be helpful in fertility assessment from early stages.

Chromosomal defects in infertile men with poor semen quality

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.

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.