Acrosome reaction (AR) is an exocytotic event that involves the fusion and fenestration of the spermatozoon plasma membrane with the outer acrosomal membrane. AR is of utmost importance for sperm ovum interaction and fertilization. Triggering of the AR could be regulated by the action of diverse compounds, namely, metabolites, neurotransmitters, and hormones. γ-Amino butyric acid (GABA) and progesterone are among the compounds that induce AR 1.
Although there is direct evidence of GABA initiation of mouse sperm AR, there are conflicting results on GABA-induced exocytosis in human spermatozoa 2. In-vitro studies have suggested that this neurotransmitter receptor plays a role in the AR initiated by progesterone. Sperm GABAA receptor may also be involved in capacitation and hyperactivated motility. It has been shown that GABA increases capacitation in epididymal guinea-pig and ejaculated human sperm and ram sperm 3,4. Moreover, GABA increases hyperactivated motility in ejaculated human sperm 5.
Progesterone induces AR through CatSper calcium channels located on the sperm membrane. Besides AR, progesterone exerts other effects on sperm, including the induction of hyperactivated motility 6 and chemotaxis 7. All these effects may be mediated by the CatSper calcium channel 8,9.
Varicocele is the most frequent finding in male infertility, with a prevalence of 19–41% in men with primary infertility and about 45–81% in men with secondary infertility 10,11. The supposed mechanisms by which varicocele affects testicular function include increased scrotal temperature, reflux of metabolites from the kidney and adrenal gland, decreased volume of blood flow, and anoxia 12. Patients with varicocele may show abnormalities in the sperm count, motility, and morphology 13 and sperm functions 14.
The aim of this study was to evaluate the in-vitro effect of GABA and progesterone on sperm motility and AR in infertile patients with varicocele. The aim of the study was also to evaluate the in-vitro effect of GABA and progesterone on sperm motility and AR after varicocelectomy and to compare it with the prevaricocelectomy effect.
Patients and methods
Forty-two infertile patients with varicocele and oligozoospermia and/or asthenozoospermia were recruited randomly from the Andrology Unit of the Dermatology and Andrology Department, Assiut University Hospital. Exclusion criteria included specific genital diseases that may impair reproductive capacity, such as genital infection, undescended testis, and testicular atrophy. Patients with other systemic diseases that may impair reproductive capacity, such as hepatic, renal, endocrine, and autoimmune diseases, were also excluded. Patients with particular habits (e.g. smoking) were excluded. The Scientific Research Ethics Committee of Assiut Faculty of Medicine approved the study and all participants signed an informed consent.
Assessment of history and clinical examination
Full, detailed assessments of personal and medical histories were performed from all participants with a special focus on reproductive history. They were also subjected to a thorough general medical and genital examination. Varicocele was detected clinically and confirmed by scrotal ultrasound using a Fukuda Denshi Tellus UF-850XTD (Fukuda Denshi, Tokyo, Japan), equipped with color flow imaging.
Conventional semen analysis
All semen samples were collected by masturbation in nontoxic polypropylene containers after 3–5 days of sexual abstinence. After liquefaction at 37°C, conventional semen analysis was carried out following the WHO’s 1999 guidelines 15.
Each semen sample was divided into three equal parts. GABA (2.5 μmol/l; Sigma, St Louis, Missouri, USA) and progesterone (2.5 μmol/l; Sigma) were added separately to Ham’s F-10 medium (Sigma). Semen samples were diluted 1 : 1 and mixed gently with the media containing the additives or Ham’s F-10 medium alone as a control. Samples were centrifuged at 300g for 10 min. After washing the sperm with Ham’s F-10 medium or the media containing the additives twice, the pellet generated was resuspended in Ham’s F-10 medium or the media containing the additives. Sperm suspensions were incubated for 1 h at 37°C and then the total motility of the sperm was evaluated according to the WHO’s 1999 guidelines 15.
Acrosome reaction assessment
AR detection was carried out using fluorescein isothiocyanate-conjugated Pisum sativum lectin (ICN Biomedicals Inc., Irvine, California, USA) staining procedure was carried out as designated by Mendoza et al. 16. Smears of the treated sperm suspension were permeabilized in methanol and incubated with a lectin (50 µg/ml) to bind the fluorescein isothiocyanate-PSA probe. Spermatozoa were examined under fluorescence epi-illumination at 1000× magnification for the detection of AR.
Four to 6 months after varicocelectomy, conventional semen analysis and semen preparation procedure were repeated. Sperm motility and AR were evaluated as before.
Forty-two patients were included in the study. The age of the participants ranged between 24 and 43 years, mean 34.4±2.7. Sperm count was significantly higher after varicocelectomy (33.29±19.29 million/ml) compared with that before varicocelectomy (25.71±16.10 million/ml; P=0.002). Total motility also showed a significant improvement after varicocelectomy (43.57±9.64%) compared with that before varicocelectomy (39.52±11.39%; P=0.041).
In patients included in the study, total sperm motility showed a significant increase after double wash with HAM’s F-10 with GABA or progesterone compared with samples treated with HAM’s F-10 (control) only as shown in Table 1. Similarly, AR was higher with GABA and progesterone compared with HAM’s F-10 only (Table 1).
After varicocelectomy, samples treated with HAM’s F-10 and GABA or progesterone showed a significant improvement in total motility and AR compared with samples treated with HAM’s F-10 only (control) (Table 2).
Table 3 shows a significant improvement in total motility and AR in samples treated with GABA after varicocelectomy compared with those treated with GABA before varicocelectomy. Similarly, total motility and AR were significantly higher in samples treated with progesterone after varicocelectomy compared with those treated with progesterone before varicocelectomy.
The human sperm is the target of many neurotransmitters and hormones that act through different receptors. They affect many sperm parameters such as motility, chemotaxis, capacitation, and AR. Among these mediators are GABA and progesterone 5.
GABA have been found in the seminal plasma of normozoospermic patients 17 and GABA receptors have been described in the plasma membrane of non-human spermatozoa 18 and human spermatozoa 19. The effects of GABA are mediated by two types of receptors named GABAA and GABAB 20. Activation of the GABAA receptor results in the opening of the chloride channel, leading to an influx of chloride ions (Cl−) and cell hyperpolarization 21.
Preoperatively, GABA increased total sperm motility and AR compared with the control. This is consistent with a study carried out by Calogero et al. 21 in which GABA significantly increased the percentage of active spermatozoa and spermatozoa showing hyperactivated motility. Our results are also consistent with previous studies carried out on rat 22 and human 2 spermatozoa, where GABA induced AR in both rat and human sperm.
Our data are at variance with those reported by Aanesen et al. 19, who showed no effects of GABA on sperm motility. The reason for this discrepancy may be the differences in the experimental protocol between the two studies. First, Aanesen et al. 19 used a swim-up sperm preparation, whereas we tested the effects of GABA on a crude sperm preparation after double wash. Second, and perhaps more importantly, different GABA concentrations were used in the two studies.
Similar to GABA, progesterone increased sperm motility and induced AR compared with the control. This is consistent with a study carried out by Calogero et al. 21, Uhler et al. 23, and Vigil et al. 1 in which progesterone increased the percentage of active spermatozoa and of spermatozoa showing hyperactivated motility. Our results are also consistent with the results of Vigil et al. 24, where the percentage of AR was 58.2±0.84 in progesterone treatment versus 29.0±0.71 in the control (P<0.05).
After varicocelectomy, semen parameters (count and motility) improved statistically, which is inconsistent with previous studies 13,14,25. Motility and AR after double wash with HAM’s F-10 were significantly higher compared with before varicocelectomy. GABA and progesterone improved sperm motility and induced AR as before surgery. This shows the role of varicocelectomy in improving semen quality of infertile patients. Daitch et al. 26 reported that varicocele treatment may not improve semen characteristics in all men, but it appears to improve pregnancy and live birth rates among couples undergoing intrauterine insemination for male factor infertility probably because functional factors not measured on routine semen analysis may affect pregnancy rates. Our results are also consistent with a study by Glazier et al. 27, where 48% of men with varicocele had abnormal AR; 35% of these men became normal after varicocelectomy.
This improvement in AR after varicocelectomy may be multifactorial. A study by De Amicis et al. 28 showed that varicocele may lead to male factor infertility by a mechanism involving a decreased progesterone expression in human spermatozoa. Moreover, varicocele may lead to quantitative and qualitative molecular differences in the expression of important sperm plasma proteins affecting signal transduction and calcium ion influx, ultimately affecting the AR 14. In addition, excessive production of reactive oxygen species in patients with varicocele leads to lipid peroxidation of the cell membrane, affecting the fusiogenic ability of the sperm cell membrane and resulting in inhibition of the AR 29–31.
GABA and progesterone improve sperm motility and induce AR. Sperm motility and AR showed significant improvement after varicocelectomy in response to GABA and progesterone compared with before varicocelectomy. Clinically, GABA and progesterone might be used during semen processing to improve the results of intrauterine insemination. Further studies are needed in the future to evaluate the effect of GABA and progesterone on the pregnancy rate in patients undergoing intrauterine insemination.
Conflicts of interest
There are no conflicts of interest.
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