Mounir Ajina

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.

Detection of aneuploidy rate for chromosomes X, Y and 8 by fluorescence in-situ hybridization in spermatozoa from patients with severe non-obstructive oligozoospermia

PurposeTo evaluate the frequency of sperm nuclei disomy for chromosomes 8, X, and Y in patients with severe non-obstructive oligozoospermia and to assess possible correlations between sperm nuclei aneuploidy and semen parameters or a particular clinical phenotype.Materials and methodsThe sperm aneuploidy rate for chromosomes X, Y, and 8 were assessed in 16 infertile men with severe non-obstructive oligozoospermia and 7 healthy men with normal semen parameters. The frequency of sperm aneuploidy was compared between several patients groups according to their clinical and biological factors.ResultsThe total rate of chromosomally abnormal spermatozoa was significantly higher in patients with severe oligozoospermia compared to control group (P < 0.001). A significant relationship was found between the age of patients, sperm concentration, and morphology and the mean rate of sex chromosomes disomy. In addition to the low sperm count (<5 × 106/ml), an elevated FSH level and an exposed to an elevated temperature are two major predictive factors leading to the production of higher numbers of chromosomally abnormal gametes.ConclusionPatients with severe oligozoospermia, who are potential candidates for assisted reproduction technology, presented a high level of sex numerical chromosome abnormalities, and consequently are at high risk of chromosome abnormalities in their offspring.

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

PurposeTo 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.MethodsSemen 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.ResultsSperm 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).ConclusionsThis 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.

Vascular endothelial growth factor (VEGFA) gene variation in polycystic ovary syndrome in a Tunisian women population

BackgroundPolycystic ovary syndrome (PCOS) is characterized by the growth of a number of small cysts on the ovaries which leads to sex hormonal imbalance. Women who are affected by this syndrome suffer from irregular menstrual cycles, decline in their fertility, excessive hair growth, obesity, acne and most importantly cardiac function problems. The vascular endothelial growth factor (VEGF) plays a pivotal role in tissue vascularization in general and in the pathogenesis of many diseases. The PCOS was found to be associated with high expression levels of VEGF. In women who undergo assisted reproductive procedures (ART), VEGF was found to be a key mediator of other factors to control ovary angiogenesis. Here, we set out to examine the association of VEGFA gene polymorphism with PCOS and its components in a population of Tunisia women to enhance our understanding of the genetic background leading angiogenesis and vascularization abnormalities in PCOS.MethodsThe association of VEGFA gene with PCOS and its components was examined in a cohort of 268 women from Tunisia involving 118 PCOS patients and 150 controls. VEGFA gene variations were assessed through the analysis of the following SNPs rs699947 (A/C), rs833061 (C/T), rs1570360 (G/A), rs833068 (G/A), rs3025020 (C/T), and rs3025039 (C/T). The linkage disequilibrium between SNPs was assessed using HAPLOVIEW software while combination of SNPs into haplotypes in the population and the reconstruction of the cladogram were carried-out by PHASE and ARLEQUIN programs, respectively. Genetic association and genotype-phenotype correlations were calculated by logistic regression and non-parametric tests (Kruskall-Wallis and Mann–Whitney tests), respectively, using StatView program.ResultsWe observed 10 haplotypes in our studied cohort whereH1 (ACGG), H2 (ACAG), H7 (CTGG) and H8 (CTGA) were the most frequent. We observed the association of the genotype CT of the SNP rs30225039 with PCOS phenotype (P = 0.03; OR 95 % CI = 2.05 [1.07–3.90]) and a trend for correlation of the pair of haplotypes H2/H2 with prolactin levels in plasma (P = 0.077; 193.5 ± 94.3 vs 45.7 ± 7.2). These data are consistent with literature and highlight one more time the role of vascularization in the pathogeny of PCOS.ConclusionsLD pattern in VEGF locus showed a similar LD pattern between the Tunisian population and the CEU. More haplotypes in the Tunisian population than in CEU was observed (22 haplotypes vs 16 haplotypes) suggesting higher recombination rate in Tunisians. The study showed that there was any advantage of using haplotypes compared with SNPs taken alone.

Effets à court et à long termes de la cure de varicocèle sur les caractéristiques spermatiques

RésuméNous avons étudié les caractéristiques spermatiques chez 122 patients, avant cure de varicocèle et 3 mois, 6 mois, 9 mois et 12 mois après, dans le but de dégager un profil spermatique chez ces porteurs de varicocèle et d’étudies les effets de ce traitement sur leurs spermes. II ressort de notre étude que les porteurs de varicocèle ont globalement un sperme asthénozoospermique sévère.Sur les 122 patients étudiés, −18% sont oligozoospermiques extrêmes, −26% sont oligozoospermiques sévères, −18% sont oligozoospermiques modérés, −38% sont normospermiques.La cure de varicocèle n’intervient ni sur le volume du sperme, ni sur la vitalité des spermatozoïdes, ni sur leur survie, ni sur leur taux de formes anormales, ni sur les marqueurs du tractus génital masculin, mais elle améliore d’une façon statistiquement significative la numération et la mobilité et ce, chez les oligozoospermiques extrêmes ou sévères.Ceci justifie ce type de traitement dans les cas des oligoasthénozoospermies extrêmes ou sévères dans le but d’obtenir une grossesse naturelle, si non d’augmenter les chances de réussite des techniques d’assistance médicale à la procréation.AbstractThe authors studied the sperm features of 122 patients, before and 3 months, 6 months, 9 months and 12 months after varicocele repair, in order to define the sperm profiles of these patients and to study effects of this treatment on these profiles.Patients with varicocele generally present severe asthenozoospermia. Of the 122 patients studied: −18% were extreme oligozoospermic, −26% were severe oligozoospermic, −18% were moderate oligozoospermic, −38% were normozoospermic.Varicocele repair does not influence the volume of semen, the vitality of spermatozoa, their survival, their rate of abnormal shapes, or male genital tract markers, but significantly improves sperm counts and mobility, in men with extreme or severe oligozoospermia.This type of treatment is generally indicated in patients with extreme or severe oligoasthenozoospermia in order to obtain a natural pregnancy, or at least to increase the success of medically assisted procreation techniques.

Etats épididymaire, prostatique et vésiculaire en relation avec les paramètres du spermogramme chez les hommes consultant pour infertilité

ResumeLe sperme résulte du mélange lors de l’éjaculation de différentes sécrétions du testicule, du tractus génital et des glandes annexes. Il comprend une phase cellulaire: les spermatozoïdes et une phase liquidienne: le plasma séminal. Le spermogramme constitue le premier examen à réaliser pour poser le diagnostic d’infertilité masculine. Il est complété par l’étude biochimique du sperme pour chercher une origine excrétoire à l’infertilité.Dans ce travail, nous avons étudié les fréquences des anomalies prostatique, vésiculaire et épididymaire, à travers le dosage de leurs marqueurs dans le sperme, chez 146 hommes consultant pour infertilité, ainsi que les relations éventuelles entre ces marqueurs et les caractéristiques du spermogramme.Nous avons trouvé que la prostate est la portion du tractus génital la plus touchée chez les hommes infertiles et présente une dysfonction dans 32,26% des cas, traduisant le plus souvent un état inflammatoire de cette glande.L’obstruction épididymaire est détectée dans 35% des cas d’azoospermie.Dans tous les cas, nous avons trouvé une corrélation statistiquement significative entre le volume spermatique et l’activité de l’alpha 1–4 glucosidase ainsi qu’entre le pH du sperme et l’acide citrique séminal. Chez les patients ayant une leucospermie élevée, nous n’avons pas trouvé de relation entre le taux de leucocytes dans le sperme et les différents marqueurs biochimiques. Si le taux de fructose séminal n’a pas d’effet sur la mobilité initiale des spermatozoïdes, il garde un effet statistiquement significatif sur leur survie. En effet, la diminution du fructose séminal coïncide avec une mauvaise survie des spermatozoïdes ce qui signifie l’importance de ce substrat spermatique sur le maintien de la mobilité des spermatozoïdes.AbstractThe semen results from the mixture on ejaculation of various secretions from the testicle, the genital tract and the adnex glands. It consists on a cellular phase: spermatozoa and a liquidian phase: the seminal plasma.The spermogram is the first exam to make in order to set diagnosis of male infertility. It is completed with a biochemical study of the semen to find an excretory origin to infertility.In this work, we have studied the frequences of prostatic, vesicular and epididymious anomalys, through dosage of their markers in the semen, in 146 males consulting for infertility and eventual relationships between these markers and the characteristics of the spermogram.We found out that the prostate is the most hurt portion of the genital tract in infertile males and presents a dysfunctioning in 32,26% of cases, most often resulting in an inflammatory states of this gland. The epididymious obstruction is detected in 35% of azoospermia cases.In all the cases, we found out significant statistical correlation between the semen volume and the activity of alpha 1–4 glucosidase and also the pH of the semen and the seminal citric acid. For patients having a high leucospermia we didn’t find any relationship between the leucocytes rate in the semen and the different biochemical markers. If the seminal fructose rate has no effect on the initial mobility of spermatozoa, it has a statistically significant effect on their survival. In fact, the diminution of seminal fructose coincides with a bad survival of spermatozoa, which shows the importance of this spermatic substrate on the maintaining of spermatozoa mobility.

Apoptotic sperm biomarkers and the correlation between conventional sperm parameters and clinical characteristics

The principal aim of this retrospective study was to examine the relationship between sperm apoptotic biomarkers and the patients biclinical characteristics, the conventional sperm parameters and the results of assisted reproductive technology. Sperm analysis, activated caspases, annexin V staining for phosphatidylserine (PS) externalisation and labelling assay for DNA fragmentation were assessed in 122 males of infertile couples. Fifty‐seven couples were allocated to the natural conception group, and 65 couples underwent IVF or ICSI. Semen of IVF/ICSI patients showed a higher proportion of apoptotic spermatozoa in their spermatozoa when compared with a natural conception group (p < .05). Sperm apoptotic biomarkers correlated with age, FSH, and conventional sperm parameters. DNA fragmentation correlated positively with the percentage of semen having externalised PS (r = .78, p = 0) and activated caspases (r = .71, p = 0). Patients without clinical pregnancy had higher frequency of DNA fragmentation, externalised PS and activated caspases compared to patients with clinical pregnancy (p < .001). The best specificity and greater sensitivity were obtained with the test of the DNA fragmentation compared to the other biomarkers. Among the apoptotic biomarkers, only DNA fragmentation was found to predict natural or assisted pregnancy better than conventional sperm parameters.

Analyse du statut oxydatif spermatique chez des patients infertiles

RésuméIntroductionL’infertilité masculine constitue un problème de santé publique. Plusieurs facteurs sont à l’origine de ce phénomène. Actuellement, le stress oxydatif est incriminé comme l’une des principales causes. Dans notre étude, nous avons cherché une corrélation entre les marqueurs du stress oxydatif et les caractéristiques spermatiques (numération, mobilité, morphologie).Matériel et méthodesNous avons évalué le statut oxydatif spermatique de 129 sujets infertiles. Ces sujets sont caractérisés par une infertilité de durée variable. Ils ont été subdivisés en quatre groupes: des sujets normozoospermiques considérés comme témoins (n=34); des asthénozoospermiques (Asthéno, n=43); des oligozoospermiques (Oligo, n=22) et tératozoospermiques (Térato, n=30). Parmi les marqueurs du stress oxydatif, nous avons évalué, dans le plasma séminal, le zinc, le calcium, le magnésium et le sélénium par spectrométrie d’absorption atomique à flamme et à four. Le malondialdéhyde (MDA) est dosé par spectrofluorométrie.RésultatsLes résultats de notre étude montrent que les concentrations séminales du zinc et du sélénium sont plus élevées chez les normozoospermiques que les concentrations de ces mêmes éléments chez les autres groupes. La concentration séminale en zinc est significativement corrélée avec la numération spermatique (r=0,49; p<0,001) et le MDA (r=−0,35; p<0,05). La mobilité des spermatozoïdes est corrélée avec le calcium (r=0,41; p<0,001) et le magnésium (r=0,31; p<0,05). La concentration du MDA est plus élevée chez les trois groupes de patients: oligospermiques (3,22±1,37 μg/ml), asthénospermiques (3,52±1,93 μg/ml) et tératospermiques (3,64±1,73 μg/ml) par rapport aux témoins (2,32±0,94 μg/ml). Une seule corrélation positive a été observée entre le MDA et la morphologie (r=0,19; p<0,05).ConclusionNotre étude confirme que le stress oxydatif joue un rôle important dans le processus des altérations des spermatozoïdes. Les radicaux libres peuvent, en effet, modifier la structure membranaire ainsi que celle de l’acide désoxyribonucléique. Ces altérations conduisent aussi à une augmentation du pourcentage de spermatozoïdes de formes anormales.AbstractIntroductionMale infertility constitutes public health problems. Several factors are at the origin of this phenomenon. Currently, the oxidative stress is accused to be one of the leading causes. In our study, we sought a correlation between the markers of the oxidative stress and the sperm characteristics (morphology).Material and methodsWe evaluated the antioxidant status in the seminal plasma of 129 infertile men. Patients were characterized by infertility of variable duration. They were divided into four groups: normozoospermics who were considered as controls (n=34), asthenozoospermics (Astheno; n=43), oligozoospermics (Oligo; n=22) and teratozoospermics (Terato; n=30). Among the oxidative stress markers, we evaluated, in seminal plasma, zinc, calcium, magnesium and selenium by spectrophotometry of atomic absorption to flame and furnace. The malondialdehyde (MDA) is proportioned by spectrofluorometry.ResultsOur results show that the seminal concentrations of zinc and selenium are higher in the control group than the concentrations of these same elements in the three other groups. The seminal zinc concentration was significantly correlated with the sperm count (r=0.49; p < 0.001) and MDA (r=- 0.35; p<0.05). Sperm motility was correlated with calcium (r=0.41; p<0.001) and magnesium (r=0.31; p<0.05). The MDA concentration is higher in the three groups of patients: oligozoospermics (3.22±1.37 μg/ml), asthenozoospermics (3.52±1.93 μg/ml) and teratozoospermics (3.64±1.73 μg/ml) compared with controls (2.32±0.94 μg/ml). A single positive correlation was observed between the MDA and morphology (r=0.19; p<0.05).ConclusionOur study confirms that the oxidative stress plays an important role in the process of deteriorations of the spermatozoa. The free radicals can, indeed, modify the membrane structure as well as the membrane structure of the deoxyribonucleic acid. These deteriorations also lead to an increase in the percentage of sperm of abnormal forms.

Effets in vitro des dermaseptines sur les caractéristiques du sperme

ResumeAu cours de ce travail et dans le cadre stratégique du développement des molécules multiactives et plurifonctionnelles, nous nous sommes proposés de tester pour la première fois les dermaseptines (DS), peptides de vertébrés biologiquement actifs, en tant que spermicide.Nous avons étudiéin vitro l’activité de ces nouvelles structures peptidiques, sur la mobilité et la viabilité des spermatozoïdes humains et avons pu montrer que ces DS:- sont actives à des doses micromolaires contre les spermatozoïdes humains,- possèdent un spectre d’activité spermicide comparable sinon meilleur que celui du Nonoxynol-9, spermicide classique utilisé actuellement et présentant plusieurs types de contraintes. Les DS:- ont un effet dose dépendant,- ont un mécanisme d’action différent de celui du nonoxynol-9,- entraînent l’inhibition de la mobilité des spermatozoïdes humains après un temps d’incubation relativement court, suggérant ainsi un mode d’interaction direct du peptide avec la membrane du spermatozoïde,- ont une parfaite action synergique avec d’autres agents chélateurs tel que l’EDTA qui amplifie considérablement leur activité spermicide et pourraient ainsi pallier au problème de toxicité, d’autant plus que la DS4 présente une relative activité hémolytique. Ces résultats devraient être exploités d’une manière approfondie, afin de concevoir de nouvelles séquences analogues, dénuées de cytotoxicité et ayant une bioactivité élevée; ceci devrait ouvrir la voie à la conception d’une nouvelle génération de molécules à spectre d’action large et varié, utilisable comme nouveau spermicide à action plurifonctionnelle.AbstractThe spermicidal efficacy of synthetic peptides, dermaseptin (DS1) and (DS4), was studied under in vitro conditions using human spermatozoa. The data showed that sperm motility was inhibited with various concentrations of dermaseptins at different intervals ranging from 2 to 240 min. The effective 100% inhibitory concentration (EC100) of DS4 sperm immobilization assay was equal to 50 mg/ml at 30 min, while the EC 100 of DS1 was equal to 100 mg/ml. The presence of 0.1% of chelating agent, EDTA, reduced the EC100 of DS4 to 5 mg/ml, while less than a twofold enhancement in DS 1 activity was observed in combination with EDTA.The action of dermaseptins on sperm motility was observed to be dose-dependent. Addition of pentoxifylline, which is known to enhance sperm motility, and Ca2+, which is a key element for sperm movement, did not prevent the spermicidal action of dermaseptins.