{"paper_id":"bd6b8b3d-c7e3-4b40-8fb9-444ce0754258","body_text":"Production, maturation, and transport of sperm\noccur in the male reproductive tract ( 1 ). Molecular\nand structural anomalies in this system may\nresult in male infertility ( 2 ). The most common\nstructural anomaly associated with the reproductive\ntract is abnormal enlargement of the pampiniform\nplexus of veins within the scrotum,\ncommonly referred to as varicocele ( 3 ). Although\nthe association between male infertility\nand varicocele has been known since the past\ncentury, a limited number of studies exist that report the molecular and genetic bases of varicocele.\nTherefore, further research in this field may\nopen new strategies for treatment of male infertility\ndue to varicocele ( 4 ).\nOne of the key events in the pathology of varicocele\nis excessive production of reactive oxygen\nspecies (ROS) ( 5 ). Oxidative stress results\nfrom an imbalance between ROS production and\nantioxidant capacity ( 6 ). However, it is important\nto bear in mind that ROS acts as a doubleedged\nsword. Although it serves as a key signal\nmolecule in physiological processes, ROS\nalso has a role in pathological processes ( 7 ). In\npathological conditions, two roles have been\nenvisaged for overproduction of ROS: i. ROS\ninduced damage to sperm membrane reduces\nsperm motility and ability of the sperm to fuse\nwith the oocyte, and ii. ROS directly damages\nsperm DNA and subsequently effects genomic\nintegrity of the embryo ( 8 ,  9 ). Therefore, antioxidant\ntherapy may overcome the deleterious\neffects of ROS in individuals with varicocele\n( 10 ). Varicocelectomy, especially through microsurgery,\nhas been shown to restore testicular\nvolumes and semen parameters, as well as reduce\nthe degree of DNA fragmentation ( 11 ,  12 ).\nDespite these beneficial effects of varicocelectomy,\nfewer studies have focused on the role of\nantioxidants as adjunct therapy along with varicocelectomy\n( 13 ).\nN-acetyl-L-cysteine (NAC) is a derivative of the\nnaturally occurring amino acid L-cysteine that has\nfree radical scavenging activity. Therefore, it is\nsupplemented to alleviate glutathione (GSH) depletion\nduring oxidative stress ( 14 ,  15 ).\nPrevious studies have shown both  in vivo  ( 6 ) and\n in vitro  ( 16 ,  17 ) addition of NAC may improve semen\nparameters and thereby improve male fertility.\nDespite these beneficial effects of NAC, there\nhas been no report on the effect of NAC in individuals\nwith varicocele. Therefore, the aim of this\nstudy is to evaluate the effects of NAC on semen\nquality, protamine content, DNA damage, oxidative\nstress and fertility following microsurgical\nvaricocelectomy.\n\nThis prospective clinical trial carried out between\n2011 and 2013, was approved by the Ethics\nCommittee for Research Involving Human\nSubjects at Royan Institute and the Isfahan Fertility\nand Infertility Center. All individuals gave\ninformed consent prior to participation in the\nstudy.\nInclusion criteria included male gender, age\nyounger than 45 years, primary infertility, and\nleft-sided varicocele (grades II and III) diagnosed\nby palpation and Doppler duplex ultrasound. Exclusion\ncriteria included grade I varicocele, azoospermia,\nrecurrent varicocele, leukocytospermia,\nurogenital infections, testicular size discrepancy,\nabnormal hormonal profile, anatomical disorders,\nKlinefelter’s syndrome, cancer, fever in the 90\ndays prior to surgery, seminal sperm antibodies,\nexcessive alcohol and drug consumption, previous\nhistory of scrotal trauma or surgery, and occupational\nexposure.\nWe included female partners who were less than\n35 years of age that had normal ovulatory cycles\nand patent tubes (hysterosalpingography or laparoscopy)\nin this study. Individuals with endometriosis,\ncycle irregularity, or gross anatomical abnormalities\nwere excluded.\nThis study was designed similar to a blinded\nclinical trial. A total of 40 individuals with grades\nII and III varicocele enrolled in this study. Following\nmicrosurgery, the patients were randomly\nallocated to the control or treatment groups. In the\ncontrol group, individuals received no drug after\nvaricocelectomy (n=20). In the treatment or NAC\ngroup (n=15), the individuals received three tablets\nof NAC (200 mg daily) post-varicocelectomy\nfor three months based on a previous study ( 6 ). In\nthis study, five individuals were excluded from\nthe treatment group due to lack of compliance\nwith NAC use, according to the study protocol.\nAll parameters assessed in this study were carried\nout by a single trained individual unaware of\ntreatment assignment. Duration of infertility was\n2.1 ± 0.2 years and duration of marriage was 3.8\n± 0.3 years in individuals with varicocele.\nWe initially aimed to include a group in which\nthe individuals did not want to undergo surgery,\nas either a control (without surgery) or treatment (without surgery+NAC) group. However, there\nwere few individuals that refused to undergo surgery\nsince the majority of these individuals had\nreferred for infertility treatment.\nPrior to surgery and at three months post-surgery,\nall participants provided semen samples by\nmasturbation after 3-4 days of abstinence. Semen\nsamples were analyzed according to World\nHealth Organization (WHO) criteria ( 18 ). After\nimmobilizing the sperm with a fixing solution,\nwe evaluated the sperm concentration by\na Makler counting chamber. Sperm were expressed\nas million/ml. Sperm motility and morphology\nwere assessed by the Computer Aided\nSperm Analysis (CASA) system (LABOMED,\nSDC313B). Sperm morphology was evaluated\nby Diff-Quik staining. DNA fragmentation\nand protamine deficiency were assessed with\nthe TUNEL assay ( 19 ) and chromomycin A3\n(CMA3) staining ( 20 ).\nA sperm suspension (20-30 μl) was smeared on\nthe slide, allowed to air dry and stained with the\nprepared kit ( 18 ). Briefly, slides were immersed for\n30 seconds into methanol (fixative), eosin (stain basic\nproteins) and a thiazin-like stain (stain DNA),\nrespectively. Subsequently, slides were dipped into\nwater to remove excess dye and allowed to air dry.\nWe evaluated 200 sperm per slide.\nDNA fragmentation was evaluated with the\naid of a terminal deoxynucleotidyltransferasemediated\ndUTP nick-end labeling (TUNEL)\nkit (Apoptosis Detection System Fluorescein,\nG3250, Promega, Mannheim, Germany) according\nto Kheirollahi-Kouhestani et al. ( 19 ).\nOn each slide, 500 sperm were assessed under\nan epifluorescent microscope (BX51, Olympus,\nJapan) at ×100 magnification. Sperm with\nred heads were considered to have intact DNA.\nThose with green heads were considered to have\nfragmented DNA.\nThe percentage of sperm with protamine deficiency\nwas assessed with CMA3 staining, according\nto Nasr-Esfahani et al. ( 20 ). On each\nslide, we assessed 500 sperm under an epifluorescent\nmicroscope (BX51, Olympus, Japan) at\n×100 magnifications. Those sperm that stained\nlight yellow were considered as CMA3 positive\nor protamine deficient; however, sperm that\nstained dark yellow were considered to have normal\nprotamine content.\nROS status was assessed using a 2', 7'-dichlorodihydrofluorescein-\ndiacetate (DCFH-DA, D6883,\nSigma Co., USA) probe according to Kiani-Esfahani\net al. ( 21 ). Briefly, a 2.5 mM stock solution\nof H2DCF-DA was prepared in dimethyl sulfoxide\nand stored at -70°C. A total of one million sperm\nwere treated with 5 μM H2DCFDA for 30 minutes,\nwhile percentages of ROS positive sperm\nwere defined by flow cytometery.\nThe Kolmogorov-Smirnov Z test was used to assess\nnormal data distribution. The student’s t test\nwas carried out using the Statistical Package for the\nSocial Studies (SPSS11.5, Chicago, IL, USA). For\ncomparison between control and NAC groups, we\nused the independent t test. For comparison between\nthe preand post-surgery in control and NAC groups,\nthe paired t test was used. The differences with values\nof P<0.05 were considered statistically significant.\n\nThe study population consisted of 35 individuals\nwith grades II and III varicocele. The\nmean ages of male participants was 30.1 ± 4.4\n(range: 22-45) years; for females, it was 26.6 ±\n4.9 (range: 17-35) years. During this study, individuals\nwith varicocele were randomly assigned\nto control (non-NAC) and NAC groups.\nIn order to show that there were no significant\ndifferences between the two groups before treatments,\nthe sperm parameters between control and\nNAC groups were compared. The results showed\nno significant difference between the two groups\n( Table 1 ). Age range of females (27.4 ± 5.7 vs.\n26.05 ± 4.2 years) and males (30.7 ± 1.4 vs. 29.6\n± 0.7 years) were also similar between the two\ncontrol and NAC groups.\nComparison of pre-surgery semen parameters and ages in men\nwith varicocele in the control and N-acetyl-L-cysteine (NAC) groups\nIn the NAC group, sperm concentration prior\nto surgery (23.9 ± 5.9) compared to after surgery\n(45.4 ± 7.1, P<0.01), percentage of sperm motility\nprior to surgery (36.9 ± 5.5) versus after surgery\n(58.2 ± 5.4, P<0.01) and normal morphology\nprior to surgery (0.71 ± 0.1) versus after surgery\n(2.71 ± 0.3, P<0.01), showed significant improvement\n( Fig .1A ). Similarly, in the control group,\nsperm concentration (29.7 ± 6.9 vs. 42.4 ± 7.02,\nP<0.05) and normal morphology (0.7 ± 0.1 vs. 1.9\n± 0.2, P<0.01) also significantly improved following\nsurgery. Unlike the NAC group, however, the\npercentage of sperm motility (34.9 ± 4.6 vs. 43.6\n± 4.9, P=0.1) did not increase following surgery in\nthe control group ( Fig .1B ).\nComparison of sperm parameters before and after surgery\nin A. N-acetyl-L-Cysteine (NAC) and B. Control group.\n*; Indicate significant difference before and after surgery.\nIn the NAC group, percentages of normal protamine\ncontent (48.7 ± 4.1 vs. 63.5 ± 1.6, P<0.01),\npercentages of DNA integrity (80.6 ± 1.8 vs.\n89.8 ± 1.4, P<0.01), percentage of ROS-negative\nsperm (77.2 ± 7.5 vs. 92.3 ± 2.6, P<0.05), and\nintensity of sperm ROS (40.02 ± 7.1 vs.78.1 ±\n14.6, P<0.01) significantly increased following\nsurgery ( Fig .2A ). Similarly, in the control group,\npercentages of normal protamine content (48.7 ±\n2.9 vs. 53.2 ± 3.1, P<0.01), percentages of DNA\nintegrity (82.2 ± 1.7 vs. 85.9 ± 1.7, P<0.01) and\nintensity of sperm ROS (37.2 ± 3.6 vs. 61.3 ± 5.3,\nP<0.01) also increased significantly following\nsurgery. Unlike the NAC group, the percentage\nof ROS-negative sperm (57.6 ± 6.6 vs. 60.9 ± 6.4,\nP=0.3) did not increase following surgery in the\ncontrol group ( Fig .2B ).\nComparison of different parameters before and after surgery\nin A. N-acetyl-L-cysteine (NAC) and B. Control groups.\n*; Indicate significant difference before and after surgery and\nROS; Reactive oxygen species.\nIn order to evaluate improvement in these parameters\nbetween the NAC and control groups,\nwe calculated the difference between the mean\nvalues of these parameters before and after surgery,\ndivided by the mean values of these parameters\nbefore surgery, times 100. There was no\nsignificant difference in improvement of sperm concentration (220.5 ± 75.9 vs. 149.7 ± 81.3,\nP=0.5), percentage of sperm motility (100.4 ±\n29.5 vs. 44.6 ± 21.8, P=0.1), abnormal morphology\n(100 ± 16.3 vs. 81.8 ± 27.1, P=0.5), percentage\nof ROS-negative sperm (36.6 ± 23.1 vs. 16.5\n± 9.5, P=0.3) and intensity of sperm ROS (76.6\n± 29.5 vs. 64.9 ± 19.5, P=0.7) observed between\nthe NAC and control groups, respectively\n(Figes .3 ,  4 ). However, we found significant differences\nin percentages of improvement of normal\nprotamine content (45.6 ± 13.5 vs. 11.9 ±\n4.7, P<0.05) and DNA integrity (11.8 ± 2.01 vs.\n4.7 ± 1.3, P<0.01) between the NAC and control\ngroups, respectively ( Fig .4 ).\nComparison of percentage of improvement in semen parameters\nin N-acetyl-L-cysteine (NAC) and control groups.\nComparison of percentage improvement in different parameters\nin N-acetyl-L-cysteine (NAC) and control groups.\n*; Indicate significant difference between NAC and control\ngroups and ROS; Reactive oxygen species.\nThe percentage of clinical pregnancy in the NAC\ngroup was 33.4% (5/15), in the control group, this\nresult was 10% (2/20).\n\nOxidative stress induced by heat stress is considered\nthe central element that contributes to the\netiology of infertility in individuals with varicocele\n( 22 ). Therefore, surgical varicocele repair is\nexpected to be beneficial to these individuals by\nalleviating heat, and thereby oxidative stress ( 10 ).\nA second approach to alleviate the varicocele associated\nROS is antioxidant therapy ( 10 ,  13 ). In\norder to improve the efficiency of surgical treatment,\nconcomitant therapy with antioxidants has\nbe e n suggested ( 13 ).\nWe aimed to evaluate the effect of NAC on semen\nquality, protamine content, DNA damage,\noxidative stress and fertility following varicocelectomy,\nas well as to compare these parameters in\nindividuals with varicocele who did not use NAC\nafter surgery. NAC is one of the oldest and most\npowerful antioxidants that treat various diseases,\nincluding respiratory disorders, heart disease,\nheavy metal poisoning, overdose with acetaminophen\nand epilepsy ( 6 ,  23 ).\nThe results of this study showed that sperm\nparameters (concentration, motility and normal\nmorphology) significantly improved after surgery\ncompared to before surgery in both the NAC\nand control groups, with the exception of the\npercentage sperm motility which insignificantly\nimproved in the control group. Despite controversies\non the degree of improvement of each semen\nparameters post-varicocelectomy ( 24 ), this\ninsignificant improvement of motility in the control\ngroup was consistent with previous reports\n( 25 ). However, the results of this study suggested\nthat NAC might have an additional value by improving\nsperm motility post-varicocelectomy. In\ncontrast to these results, Comhaire et al. ( 26 ) reported\nthat although NAC improved sperm concentration\nand acrosome reaction, it had no effect\non motility and morphology.\nDespite the importance of semen parameters in\nfertility, many researchers have suggested that other\nsperm function characteristics should be considered\nalong with these parameters when assessing\nfertility ( 27 ,  28 ). Therefore, in this study, we have\nassessed genomic integrity and ROS production.\nSperm DNA becomes susceptible to damage by\nthree postulated routes: i. Improper packaging of\nDNA during spermiogenesis, ii. Oxidative stress\nand iii. Apoptosis ( 29 ). We have assessed DNA\ndamage, ROS production and sperm nuclear maturity\nbefore and after surgery in the NAC and control\ngroups. Of note, all parameters improved after\nsurgery in both groups, except for the percentage\nof ROS negative sperm in the control group. The percentage of sperm motility did not significantly\nimprove before and after surgery in the control\ngroup. Therefore, this lack of significant improvement\nin motility post-surgery in the control group\nmight be related to the lack of significant improvement\nin percentage of ROS negative sperm. In addition,\nthe improved sperm motility in the NAC\ngroup was associated with a reduced percentage\nof sperm producing ROS or increased percentage\nof ROS negative sperm, which might be related to\nNAC treatment. The existence of this correlation\nmight be explained by the cascade of events that\nbegin with ROS associated with lipid peroxidation\n( 30 ), which in turn, reduces membrane fluidity.\nThis prevents axonemal protein phosphorylation\nand leads to sperm immobilization ( 30 ,  31 ). NAC\nmay break these chains of events.\nIn this study, there was higher mean ROS intensity\nin semen samples after surgery compared to\nbefore surgery. This contrasted expectations since\nROS production should decrease post-surgery.\nThis was likely due to leakage of ROS or reduced\nproduction of ROS after loss of enzymes in the\nsperm of these individuals, which were in their final\nstage of apoptosis. This supposition has been\npreviously presented by Aitken et al. ( 32 ) who reported\nthat initially ROS positive sperm progressively\nbecome TUNEL positive. This indicated\nthat in individuals with varicocele, despite higher\nproduction of ROS, the ROS might leak from\nthese cells or sperm in the final stage of apoptosis,\nhence the enzymes that produced ROS were not as\nefficient. This might account for the reduced intensity\nof ROS.\nIn order to further differentiate between the role\nof surgery and antioxidant therapy, we calculated\nthe percentage of improvement relative to before\nsurgery for sperm parameters, sperm protamine\ncontent, DNA integrity, and ROS in each group\nand compared them between the NAC and control\ngroups. The percentage of improvement was calculated\nby the difference between the mean values\nof a parameter before and after surgery divided\nby its initial value before surgery. The results revealed\nno significant difference for percentage of\nimprovement for the semen parameters between\nthe NAC and control groups. However, among the\nsperm functional parameters assessed, the percentage\nof improvement for the normal protamine content\nand DNA fragmentation significantly differed\nbetween the NAC and control groups, despite no\ninitial difference between the two groups before\nsurgery. These results suggested that despite the\nsimilar process of surgery in the two groups, the\ndifference between percentages of improvement\nin the two groups was due to antioxidant activity\nof NAC. Possibly other etiological factors might\naccount for this difference, which were improved\nby NAC, or NAC might overcome the secondary\nside effects of surgery. However, the role of NAC\non its own in treatment of varicocele has yet to be\nelucidated.\nDespite the higher rate of pregnancy in the NAC\ngroup compared to the control group, we did not\ncompare clinical pregnancy rates between the\ngroups. Due to the limited number of cases, further\nstudy would be warranted.\n\nNAC can scavenge free radicals, increase GSH\nproduction and reduce disulfide bonds, as well as\nviscosity and elasticity of semen, which are important\nfor fertility. This, in conjunction with the results\nof the current study (improved sperm parameters,\nDNA integrity and chromatin packaging),\nmay account for the higher pregnancy rate in NAC\ngroup. In order to reach this conclusion, additional\nstudies are recommended.","source_license":"CC-BY-4.0","license_restricted":false}