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Sperm selection by swim-up in terms of deoxyribonucleic acid fragmentation as measured by the sperm chromatin dispersion test is altered in heavy smokers

      Toxic habits and their relationship with male factor infertility have been a matter of investigation in recent years, and smoking is one of the most common lifestyle toxic exposures to harmful substances. The analysis of sperm deoxyribonucleic acid fragmentation after capacitation detected a detrimental effect produced by tobacco, and this deleterious effect alters the sperm swim-up selection process in smokers, although the molecular and cellular basis of this phenomenon remain to be elucidated.
      Male factor infertility and its relationship with toxic habits has been a matter of investigation in recent years. Cigarette smoking is one of the most common toxic habits in members of the general population within reproductive age and is potentially able to induce deleterious effects on gametes.
      One clear and well-established effect of smoking habits on sperm recently has been published by our group (
      • Viloria T.
      • Rubio M.C.
      • Rodrigo L.
      • Calderon G.
      • Mercader A.
      • Mateu E.
      • et al.
      Smoking habits of parents and male:female ratio in spermatozoa and preimplantation embryos.
      ). A sensitive bias in the male-female ratio has been confirmed in embryos obtained with spermatozoa from smokers: in male heavy smokers, the XY-XX embryo ratio was decreased compared with that in nonsmokers.
      Recently, the sperm chromatin dispersion test has been developed to determine sperm DNA fragmentation extent (
      • Muriel L.
      • Garrido N.
      • Fernández J.L.
      • Remohí J.
      • Pellicer A.
      • De los Santos M.J.
      • et al.
      Value of the sperm DNA fragmentation level, measured by the sperm chromatin dispersion (SCD) test, in the IVF and ICSI outcome.
      ,
      • Muriel L.
      • Meseguer M.
      • Fernández J.L.
      • Alvarez J.
      • Remohí J.
      • Pellicer A.
      • et al.
      Value of the sperm chromatin dispersion test in predicting pregnancy outcome in intrauterine insemination (IUI): a double-blind prospective study.
      ). Previous work based on the sperm chromatin dispersion test in sperm samples before and after swim-up treatment found a significant negative correlation with fertilization rate and embryo quality, whereas no predictive value was found for pregnancy, probably because of embryo selection before transfer (
      • Muriel L.
      • Garrido N.
      • Fernández J.L.
      • Remohí J.
      • Pellicer A.
      • De los Santos M.J.
      • et al.
      Value of the sperm DNA fragmentation level, measured by the sperm chromatin dispersion (SCD) test, in the IVF and ICSI outcome.
      ). On the other hand, sperm DNA fragmentation did not predict pregnancy outcome in IUI (
      • Muriel L.
      • Meseguer M.
      • Fernández J.L.
      • Alvarez J.
      • Remohí J.
      • Pellicer A.
      • et al.
      Value of the sperm chromatin dispersion test in predicting pregnancy outcome in intrauterine insemination (IUI): a double-blind prospective study.
      ). Interestingly, these conclusions were found only after the analysis of the post-swim-up fraction, and are especially relevant given that merely the study of the capacitated sample is in fact useful for drawing conclusions about the cycle’s outcome, and that this is the sperm fraction used on the assisted reproduction. It must be stressed that many studies about DNA fragmentation presented results based only on the analysis of the ejaculated sperm (
      • Sergerie M.
      • Ouhilal S.
      • Bissonnette F.
      • Brodeur J.
      • Bleau G.
      Lack of association between smoking and DNA fragmentation in the spermatozoa of normal men.
      ,
      • Belcheva A.
      • Ivanova-Kicheva M.
      • Tzvetkova P.
      • Marinov M.
      Effects of cigarette smoking on sperm plasma membrane integrity and DNA fragmentation.
      ), thus not being representative.
      A potential cause of DNA fragmentation is the exposure to environmental toxins, and some of these toxins could be produced by cigarette smoking. The relationship between cigarette smoking and the DNA fragmentation damage in the spermatozoa was investigated by some groups. Sergerie et al. (
      • Sergerie M.
      • Ouhilal S.
      • Bissonnette F.
      • Brodeur J.
      • Bleau G.
      Lack of association between smoking and DNA fragmentation in the spermatozoa of normal men.
      ) reported no association between smoking and DNA fragmentation, and Belcheva et al. (
      • Belcheva A.
      • Ivanova-Kicheva M.
      • Tzvetkova P.
      • Marinov M.
      Effects of cigarette smoking on sperm plasma membrane integrity and DNA fragmentation.
      ) found that DNA damage in the spermatozoa of smokers were increased, although this difference was not statistically significant.
      Our goal with this study was to investigate the association between cigarette smoking and sperm DNA fragmentation in infertile couples undergoing assisted reproductive technology analyzed by the sperm chromatin dispersion test before and after the swim-up sperm selection procedure in cases in which no severe male factor infertility is found.

      Materials and methods

      Semen samples were obtained from male partners of couples who went to the Instituto Valenciano de Infertilidad, Valencia, Spain, for infertility treatment. After the men completed a questionnaire to discard compounds known to affect sperm quality, they were accepted for this study. Patients with alcohol and/or drug consumption in the previous 3 months were not eligible for this study. Also, patients were excluded if they had a history of a recent fever or exposure to gonadotoxins such as chemotherapy, radiotherapy, pesticides, or occupational exposure to heavy metals. Andrological investigations about the presence of varicocele, testicular torsion, and any other alteration in the genital urinary tract were also conducted, and all were excluded.
      A total of 99 men provided 99 semen samples to be analyzed between April 2003 and September 2004. Median age was 36 years old (range 26 to 49 years). Sperm samples were accepted only with a count of >25 million/mL and 25% progressive motility to remove severe male factor infertility.
      All of the men signed an informed consent form, and the study was approved by the Ethics Committee of the Instituto Valenciano de Infertilidad.
      Patients were asked about smoking habits, specifically, the number of cigarettes smoked per day in a constant manner within the last 3 months before they provided the sample. The average was categorized into two groups, nonsmoking men (n = 51) and smoking men (n = 48)
      Sperm parameters were evaluated according to the World Health Organization guidelines as described elsewhere (
      • Sergerie M.
      • Ouhilal S.
      • Bissonnette F.
      • Brodeur J.
      • Bleau G.
      Lack of association between smoking and DNA fragmentation in the spermatozoa of normal men.
      ), and all samples were processed by swim-up. Determination of DNA fragmentation was done by the improved sperm chromatin dispersion test (Halosperm kit, INDAS Laboratories, Madrid, Spain); two semen aliquots (from 10 to 100 μL) were taken from each semen sample before and after swim-up.
      The results from the Kolmogorov-Smirnov test performed on all data followed a normal distribution, then a Levene test was used for equality of variance, and comparisons among sperm DNA fragmentation in the two study groups were performed in the samples before and after swim-up by Student’s t-test.

      Results

      The DNA fragmentation in sperm samples before swim-up according to cigarette smoking showed no statistical differences between the two groups studied. Consequently, nonsmoking men and smoking men had similar sperm fragmentation levels (Fig. 1).
      Figure thumbnail gr1
      FIGURE 1Results of DNA fragmentation in sperm samples before and after swim-up according to male smoking status. The percentage of DNA fragmented sperm is shown in a box plot fashion.
      Viloria. Sperm DNA fragmentation in smokers. Fertil Steril 2007.
      Figure 1 also shows the results of DNA fragmentation in sperm samples after swim-up according to cigarette smoking. Comparisons between these groups showed that sperm DNA fragmentation in the men who smoked were increased with respect to those who did not smoke (P = .028), thus showing impaired DNA quality among smokers in comparison with nonsmokers.

      Discussion

      The mechanism by which tobacco could affect sperm is not fully understood, and there are controversial reports about the effects of cigarette use on human seminal quality and male factor fertility in the literature, but given that it contains more than 30 chemical agents known to be mutagens, aneugens, or carcinogens, the interaction of tobacco with the rapidly dividing spermatogenic cells could be affecting their physiological development. Nicotine is one of the major hazardous components of tobacco, responsible for most of the deleterious effects of cigarette smoking that are exerted on other human tissues (
      • Arabi M.
      Nicotinic infertility: assessing DNA and plasma membrane integrity of human spermatozoa.
      ).
      First, we performed the sperm chromatin dispersion test in the raw samples, and our results of DNA fragmentation on the two groups of men studied according to cigarette smoking were comparable. Saleh et al. (
      • Saleh R.A.
      • Agarwal A.
      • Sharma R.K.
      • Nelson D.R.
      • Thomas Jr, A.J.
      Effect of cigarette smoking on levels of seminal oxidative stress in infertile men: a prospective study.
      ) studied this association on infertile men and reported that differences among the groups of infertile smokers and infertile nonsmokers were not statistically significant. Nevertheless, they found that infertile smoking men had higher levels of seminal oxidative stress than infertile nonsmokers. This adds strength to our results, and reinforces the hypothesis that the effects exerted by several detrimental alterations affecting male gametes can be cryptic in the ejaculate, but it is noticeable in the sperm selection by swim-up. It must be noted that the sample used in any assisted reproductive technology is previously handled in the laboratory for the elimination of seminal plasma and nonspermatic cells and the selection of the best spermatozoa (better motility and morphology), mimicking the procedures within the female reproductive tract in natural conditions. In our preceding work (
      • Muriel L.
      • Garrido N.
      • Fernández J.L.
      • Remohí J.
      • Pellicer A.
      • De los Santos M.J.
      • et al.
      Value of the sperm DNA fragmentation level, measured by the sperm chromatin dispersion (SCD) test, in the IVF and ICSI outcome.
      ,
      • Muriel L.
      • Meseguer M.
      • Fernández J.L.
      • Alvarez J.
      • Remohí J.
      • Pellicer A.
      • et al.
      Value of the sperm chromatin dispersion test in predicting pregnancy outcome in intrauterine insemination (IUI): a double-blind prospective study.
      ), we found that the fertilization rate and some indicators from embryo quality are related to the extent of sperm DNA fragmentation after swim-up, we get similar results of our actual work, therefore it seems that determination of DNA fragmentation must be performed in a postisolation aliquot.
      Previous studies (
      • Sergerie M.
      • Ouhilal S.
      • Bissonnette F.
      • Brodeur J.
      • Bleau G.
      Lack of association between smoking and DNA fragmentation in the spermatozoa of normal men.
      ,
      • Belcheva A.
      • Ivanova-Kicheva M.
      • Tzvetkova P.
      • Marinov M.
      Effects of cigarette smoking on sperm plasma membrane integrity and DNA fragmentation.
      ,
      • Arabi M.
      Nicotinic infertility: assessing DNA and plasma membrane integrity of human spermatozoa.
      ,
      • Saleh R.A.
      • Agarwal A.
      • Sharma R.K.
      • Nelson D.R.
      • Thomas Jr, A.J.
      Effect of cigarette smoking on levels of seminal oxidative stress in infertile men: a prospective study.
      ) on DNA fragmentation and tobacco consumption analyzed either fresh semen or capacitated spermatozoa independently, but no study included our design, the comparison of prepreparation and postpreparation samples. Regarding the laboratory handling, the study by Belcheva et al. (
      • Belcheva A.
      • Ivanova-Kicheva M.
      • Tzvetkova P.
      • Marinov M.
      Effects of cigarette smoking on sperm plasma membrane integrity and DNA fragmentation.
      ) has analyzed the relevance of sperm preparation in the percentage of sperm cells with fragmented DNA. They studied normozoospermic donors with previous Percoll isolation and concluded that DNA fragmentation of spermatozoa of healthy smoking donors remained in the normal range. However, they observed that tobacco smoking induced deterioration in the sperm cell plasma membrane.
      The analysis of sperm DNA fragmentation in ejaculated fresh samples masks the real detrimental effect of tobacco because no differences regarding consumption are found, leading to the incorrect assumption that tobacco has no influence on sperm DNA fragmentation. After capacitation, we can detect the detrimental effect produced by tobacco. This deleterious consequence affects the sperm selection by the interference within the swim-up procedure, showing that tobacco selectively alters the sperm swim-up capacitating ability of smokers. The molecular and cellular basis of this phenomenon remain to be elucidated; it is possible that DNA fragmentation (as a continuous process) also is produced after ejaculation, and is probably increased in smoking patients in cells that are minimally fragmented (medium-size halo) and became totally fragmented after incubation for 1 hour in the capacitation process. Based on the evidence reported in this article, quitting smoking is advisable in men attempting to reach parenthood.

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