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Influence of ejaculatory abstinence on seminal total antioxidant capacity and sperm membrane lipid peroxidation
ORIGINAL ARTICLE: ANDROLOGY
Influence of ejaculatory abstinence on seminal total antioxidant capacity and sperm membrane lipid peroxidation Paul B. Marshburn, M.D.,a,b Allie Giddings, M.D.,b Stephanie Causby, M.S.,a,b Michelle L. Matthews, M.D.,a,b Rebecca S. Usadi, M.D.,a,b Nury Steuerwald, Ph.D.,c and Bradley S. Hurst, M.D.a,b a Division of Reproductive Endocrinology and Infertility, b Department of Obstetrics and Gynecology, and Research Center, Carolinas Healthcare System, Charlotte, North Carolina
c
Cannon
Objective: To determine whether the period of ejaculatory abstinence (EA) influences the total antioxidant capacity (TAC) of semen or lipid peroxidation (LPO) of sperm membranes. Design: A prospective experimental trial. Setting: Academic medical center for reproductive endocrinology and infertility. Patient(s): Forty men from infertile couples planning intrauterine insemination. Intervention(s): Men provided semen specimens after EA periods of 1 and 4 days. Main Outcome Measure(s): Semen analysis, peroxidase staining, and assays for seminal TAC and sperm membrane LPO, with measures compared between days 1 and 4 within individuals (internal control) using paired t tests. Result(s): The shorter period of EA (1 day vs. 4 days) resulted in statistically significant decreases in semen volume (24%), sperm density (28%), and total sperm count (3.2%). There was a statistically significant increase in TAC with the shorter period of EA (1 day) compared with 4 days of EA. No difference was detected in sperm membrane LPO comparing 1 day of EA and 4 days of EA. Conclusion(s): Higher seminal TAC obtained after a shorter period of EA could diminish oxidative stress–induced sperm damage by a mechanism independent of LPO. Shorter periods of EA may thus improve sperm quality by protecting from reactive oxygen species damage, even though lower numbers of motile sperm are produced after a shorter period of EA. This would be consistent with prior research indicating improved results after intrauterine insemination under these circumstances. Use your smartphone (Fertil SterilÒ 2014;-:-–-. Ó2014 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Abstinence, male infertility, seminal plasma oxidative stress, spermatozoa, total and connect to the antioxidant capacity Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/marshburnp-ejaculatory-abstinence/
F
or selected couples with male factor infertility, ovulation induction with intrauterine insemination (IUI) produces significantly higher cycle fecundity compared to timed intercourse (1). We previously reported that shortening the period of
ejaculatory abstinence (EA) resulted in a higher pregnancy rate after IUI despite the fact that fewer motile sperm cells were inseminated (2). These findings are consistent with the premise that improved sperm quality following a shortened EA period, rather than
Received December 6, 2013; revised May 13, 2014; accepted May 27, 2014. P.B.M. has nothing to disclose. A.G. has nothing to disclose. S.C. has nothing to disclose. M.L.M. has nothing to disclose. R.S.U. has nothing to disclose. N.S. has nothing to disclose. B.S.H. has nothing to disclose. Supported by Cannon Research Grant Committee, Charlotte, NC (Number 08–033). Reprint requests: Paul B. Marshburn, M.D., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Carolinas Healthcare System, P.O. Box 32861, Charlotte, North Carolina 28232–2861 (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.05.039 VOL. - NO. - / - 2014
discussion forum for this article now.*
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motile sperm number, is the principal factor that produced a higher IUI cycle fecundity. Sperm cells in the cauda of the epididymis and vas deferens may be subject to a harmful seminal microenvironment before or after ejaculation that could impair sperm function in proportion to increasing time exposure. Maturing, epididymal spermatozoa require a level of reactive oxygen species (ROS) for the formation of disulfide-bridging for DNA compaction, but excessive ROS would induce oxidative damage to spermatozoa that could negatively affect fertilization potential. This balanced ROS 1
ORIGINAL ARTICLE: ANDROLOGY microenvironment in the epididymis is regulated by the glutathione peroxidase system (3). Mature spermatozoa are particularly susceptible to ROS damage because they cannot respond to oxidative challenge with transcriptional activity secondary to loss of cytosolic organelles and because the external sperm membrane has a particular composition that is highly reactive to oxidative injury. Excessive ROS may be generated from leukocytes and dying spermatozoa in this seminal microenvironment. The ROS and/or deficient antioxidant defense systems in semen may cause sperm damage and have been implicated as an important cause of male factor infertility (4–6). Potential harmful effects of oxidative stress on human spermatozoa include damage to sperm membranes by lipid peroxidation (LPO), sperm DNA susceptibility to free radical attack, or sperm protein damage (7, 8). Theoretically, an increased antioxidant capacity in seminal fluid could neutralize superoxide free radicals and mitigate sperm damage. Increasing the frequency of ejaculation may reduce spermatozoal exposure to harmful reactive oxygen species, thereby improving sperm viability and function. To test this hypothesis, we have determined whether the period of ejaculatory abstinence (EA) influences the total antioxidant capacity (TAC) of semen or impacts lipid peroxidation (LPO) of sperm membranes. A reduction of reactive oxygen stress by shortening the period of EA could be a mechanism to explain why a higher pregnancy rate after IUI results from semen specimens obtained after a short period of EA despite fewer motile sperm cells being inseminated (2).
MATERIALS AND METHODS Study Participants Forty men from couples with infertility being treated at the Reproductive Medicine and Infertility clinic at Carolinas Healthcare System and planning to undergo IUI were recruited to participate in this prospective experimental study after institutional review board approval. The average frequency of intercourse for the men before producing their first semen specimen for study was between once and twice weekly. Men were excluded from study entry for the following reasons: failure to follow the ejaculatory abstinence (EA) protocol, specimen less than 2 cc in volume, current smoking, presence of varicocele, history of vasectomy reversal, consumption of certain vitamins or supplements (vitamin E or C, coenzyme Q, taurine or glutathione), or genital infection within 6 months. The decision to exclude men with ejaculates of less than 2 cc in volume was secondary to technical problems with having sufficient volume and/or sperm for the TAC and LPO assays.
Study Protocol All men provided two semen samples. The initial semen specimen was collected after 4 days of EA and the second specimen 1 day later. Semen analysis was performed after liquefaction at 37 C in accordance with World Health Organization guidelines (9). Quantification of seminal leukocytes was assessed by peroxidase staining. Raw semen (seminal 2
plasma plus spermatozoa) was subjected to nondensity centrifugation at 300 g for 7 minutes, and the supernatant (seminal plasma) was removed from the sperm pellet. Two aliquots of neat seminal plasma of 200 mL were transferred to cryovials and were cryopreserved as indicated herein for subsequent TAC assay. The sperm pellet was resuspended in the residual seminal plasma and was then diluted by careful addition of the TEST-yolk buffer freezing medium (Irvine Scientific) at a 1:1 (volume/volume) ratio. Gentle, uniform resuspension was performed for 5 minutes. The cryopreservation vials with the seminal plasma (for later TAC assay) and the resuspension of sperm with TEST-yolk buffer freezing medium (for later LPO assay) were placed in a 20 C freezer for 8 minutes and thereafter in liquid nitrogen vapor at 79 C for 2 hours. The vials were then transferred to liquid nitrogen at 196 C. For TAC and LPO testing, each reconstituted semen plus TEST-yolk buffer vial or seminal plasma vial was removed from liquid nitrogen and thawed at room temperature for 5 minutes, followed by incubation for 20 minutes at 37 C. Assay for seminal total antioxidant capacity (TAC). Seminal plasma was assayed in triplicate for total antioxidant capacity (TAC) using a Randox Total Antioxidant Status kit (Crumlin) per the manufacturer's instructions. Briefly, samples were incubated with ABTS (2,20 -Azino-di-[3-ethylbenzthiazoline] sulphonate) radical cations, which have a relatively stable blue-green color in the absence of antioxidants. The degree of color suppression by antioxidants in the sample, which is proportional to the concentration, was measured at 600 nm using a DU 800 UV/Visible Spectrophotometer (Beckman Coulter). Unknown sample antioxidant concentrations were interpolated from a standard curve created from samples of known concentrations. Assay for sperm membrane lipid peroxidation (LPO). Sperm membrane lipid peroxidation was measured using a Calbiochem Lipid Hydroperoxide (LPO) Assay Kit (Darmstadt) per the manufacturer's instructions. Thawed sperm resuspensions were diluted in Ham's F-10 (pH 7.4) with a 1:1 (volume/volume) ratio of resuspension to buffer medium, followed by centrifugation at 300 g for 7 minutes. After discarding the supernatant, we resuspended the sperm pellet in fresh Ham's F-10 (pH 7.4) buffer to an adjusted final concentration of 20 106 spermatozoa/mL (10). Sample lipid hydroperoxides were first extracted in chloroform to eliminate interference by hydrogen peroxide or endogenous ferric ions. Lipid hydroperoxides present in this extract were measured directly using redox reactions with ferrous ions. The ferric ions produced were detected using thiocyanate ion as a chromogen. The absorbance of the reactions was measured at 500 nM using a Synergy HT Multi-Detection Microplate reader (BioTek). Unknown lipid hydroperoxide sample concentrations were calculated using a standard curve generated with samples of known concentrations.
Statistical Analysis Measures for semen parameters, total antioxidant capacity (TAC), and lipid peroxidation (LPO) were compared statistically on semen specimens obtained after 4 days of ejaculatory VOL. - NO. - / - 2014
Fertility and Sterility® abstinence (EA 4) and again in the same individual 1 day later (EA 1). A paired two-tailed t test was employed to statistically compare results for EA4 and EA1 specimens for a given individual to allow each man to serve as his own internal control. The values for semen parameters under columns EA1 and EA4 in Table 1 were derived by averaging determinations from within each group. The values for the mean difference and percentage change columns were the average of the determinations for each individual trial. A sample size of 32 was calculated to have 80% power to detect a difference of 20% in TAC levels between the paired EA groups as determined by a pilot study.
RESULTS The average age of the men in our study was 34 years. Twenty-four out of 40 men had semen parameters associated with male factor infertility defined as having at least one of the following: sperm density less than 20 million sperm/mL, total motility of less than 50%, or recurrent poor sperm morphology. A statistically significant reduction in semen volume (24%; P< .001), sperm density (3%; P< .02), and total sperm count (28%; P< .001) was noted with a shorter period of ejaculatory abstinence when semen parameters from EA1 with EA4 specimens were compared (Table 1). No differences in the percentage motility, percentage normal forms, or leukocyte density were detected comparing semen specimens for an EA period of 1 day vs. 4 days. In our cohort, no man exhibited significant leukocytospermia (>1 106 white blood cells/mL). A shorter period of ejaculatory abstinence (1 day) increased seminal total antioxidant capacity (TAC) compared with the longer 4 day period of abstinence (P< .02) (Fig. 1). There was no measurable difference in sperm membrane lipid peroxidation (LPO) when comparing 1 day vs. 4 days of ejaculatory abstinence (Fig. 2).
DISCUSSION We showed in a previous study that a period of EA of 2 days or less before obtaining a specimen for IUI produced higher pregnancy rates than IUI with specimens after longer periods of EA (2), a report that supports the findings of Jurema et al. (11). These higher conception rates after IUI with a shorter period of EA occurred with specimens that possessed a lower
total, motile sperm (TMS) fraction in the IUI specimen. We hypothesized that other factors besides TMS must be involved with the observed enhancement in pregnancy rates provided by shortening the EA period. Shortening the residence time of sperm in the cauda of the epididymis or vas deferens may remove those sperm cells from an environment harmful to sperm function such as superoxide stress. There are limited data on the independent contribution of antioxidant/ROS content from the individual male accessory sex gland secretions. Most of the data in the human indicate that the primary sources of ROS in semen originate from senescent sperm or polymorphonuclear leukocytes. Our study provides no data to elucidate whether the increased TAC that we measure in semen after a shorter period of EA have an impact on the ROS environment of the epididymis or other male accessory sex glands. Purging sperm from the cauda of the epididymis or vas deferens by shortening the period of EA could diminish the population of senescent sperm, thus reducing ROS and perhaps lessening the protective effect of TAC. After ejaculation but before the isolation of motile sperm from the seminal plasma, increased TAC may protect sperm from ROS damage. Because semen is an admixture of secretions from the epididymis, vas deferens, seminal vesicles, and the prostate gland, the contribution from each glandular secretion is a complicated issue to resolve. It is an important point to acknowledge that if the cauda/vas deferens microenvironment has a high level of harmful ROS, then the protection of sperm may not occur until after ejaculation when they are exposed to accessory glandular secretions containing antioxidants and/or ROS scavengers. In that case, any improvement in sperm quality would potentially result from either reversing or slowing the detrimental effects of oxidative stress after ejaculation and until sperm are separated from the seminal plasma. We show in this experimental study that reducing the period of ejaculatory abstinence from 4 days to 1 day will increase seminal total antioxidant capacity (TAC) while producing no detectable change in sperm membrane lipid peroxidation (LPO). Higher seminal TAC would reduce exposure of sperm to harmful ROS and may be a protective mechanism for improving sperm quality. Such a protective mechanism appears to be independent of oxidative sperm membrane damage by lipid peroxidation.
TABLE 1 The impact of ejaculatory abstinence of 1 day (EA1) vs. 4 days (EA4) on semen analysis parameters (n [ 40). Parameter Volume (mL) Sperm density (millions/mL) Motility (%) Normal morphology, Kruger strict (%) Total sperm count (millions) WBCs (no. per 100 sperm)
EA1 (±SD)
EA4 (±SD)
Mean differencea
% Changeb
P valuec
3.1 1.0 36.6 27.1 61.6 14.1 2.1 1.6 112.5 77.4 0.6 0.3
4.0 1.9 50.5 24.4 61.7 12.4 1.2 0.9 201.8 83.2 0.5 0.2
0.89 14.16 0.018 0.9 87.5 0.15
24.0 3.2 4.2 12.1 28.4 3.7
< .001 < .02 .99 .06 < .001 .31
Note: EA1 ¼ 1 day of ejaculatory abstinence; EA4 ¼ 4 days of ejaculatory abstinence; SD ¼ standard deviation. a EA1 EA4. b [(EA1 EA4)/EA4] 100. c Semen analysis parameters compared within a given male subject by a two-tailed t test with a man serving as his own control. Marshburn. Ejaculate period and semen antioxidants. Fertil Steril 2014.
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ORIGINAL ARTICLE: ANDROLOGY
FIGURE 1 *
Comparison of the total antioxidant capacity (TAC) in seminal fluid after 1 day (EA1) vs. 4 days (EA4) of ejaculatory abstinence. The counted photons per minute measures the total antioxidant capacity [TAC]; (see Materials and Methods). TAC determinations were compared within a given male subject by a two-tailed t test, with a man serving as his own control from EA1 to EA4. *P<.02. Marshburn. Ejaculate period and semen antioxidants. Fertil Steril 2014.
Agarwal et al. (6) have provided strong evidence that male factor infertility is associated with higher levels of ROS. A low level of ROS in semen is associated with enhanced human sperm capacitation, acrosome reaction, and hyperactivated sperm motility (12, 13), which are all important functions involved with fertilization. Oxidative stress caused by excess generation of free radicals from leukocytes, dying spermatozoa, and/or deficient antioxidant defense systems in semen is significantly correlated with male factor infertility (4–6).
FIGURE 2
Comparison of the sperm membrane lipid peroxidation (LPO) after 1 day (EA1) vs. 4 days (EA4) of ejaculatory abstinence. The level of malondialdehyde (nM/h) measures the degree of sperm membrane lipid peroxidation (see Materials and Methods). LPO determinations were compared within a given male subject by a two-tailed t test, with a man serving as his own control from EA1 to EA4. P¼.98. Marshburn. Ejaculate period and semen antioxidants. Fertil Steril 2014.
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The harmful effect of oxidative stress on human spermatozoa may include lipid peroxidation (LPO) of the sperm plasma membrane by reducing the sperm membrane fluidity required for sperm-oocyte fusion (7). Sperm DNA also is susceptible to free radical attack because of the loss of antioxidants and scavenging enzymes after the removal of most of the sperm cytoplasm during sperm maturation events in the epididymis (8). Men with increased levels of seminal ROS have increased DNA fragmentation and apoptosis in spermatozoa (14–16) and a compromise in the mitochondrial membrane potential of spermatozoa (17). A depleted antioxidant capacity would lessen the capability of semen to neutralize harmful ROS, and therefore sperm would be more vulnerable to oxidative damage with low TAC levels. A logical consideration from our findings would include the possibility that free radical attack of sperm DNA or mitochondrial membranes may occur at a lower level of ROS than would be required to impact the LPO of sperm plasma membranes. Further study would be required to evaluate this possibility. Investigations on the impact of the period of EA on sperm quality or the seminal microenvironment have revealed differing results. These differing results are, at least in part, secondary to variations in the protocols for the time period of EA, the intensity of ejaculatory frequency before analysis, the measures of sperm quality studied, and the evaluation of men in both the fertile and infertile populations. Frequent ejaculations (twice daily for 4 days) in healthy men and those with spinal cord injuries improved sperm viability and quality, and these results were comparable with a single, second ejaculation provided 60 minutes after an initial one (18, 19). Several studies indicated that sperm DNA fragmentation was increased with longer periods of EA, consistent with the notion that the longer the mature sperm cell resides within the cauda of the epididymis or the vas deferens the greater the risk of DNA deterioration (20–24). In a different study, however, the degree of DNA fragmentation measured by sperm chromatin structure assay remained unchanged following 1, 3, 5, and 8 day periods of EA (25). Within a single, healthy sperm donor, seminal ROS was not found to depend on sperm concentration, motility, or the period of EA (26). Our data in men from infertile couples would indicate that a shorter period of EA is associated with a higher seminal TAC, which would enhance the protection of sperm from ROS damage. The augmentation of such a seminal defense mechanism could improve sperm quality and, at least in part, explain the higher pregnancy rates after IUI observed after shorter periods of EA. Further study is warranted to: [1] define the measure(s) of sperm viability and quality that may be improved after 1 day of EA, [2] evaluate the TAC levels in the secretions from individual accessory sex glands, [3] determine whether higher TAC levels of semen used for IUI are directly correlated with increased pregnancy rates, and [4] identify the precise interval of EA and the degree of ejaculatory frequency to produce optimum sperm quality. Because sperm membrane LPO was not different for the intervals of EA that we studied, other indices such as a reduction in DNA fragmentation should be investigated in men from infertile couples under the conditions of our EA protocol. VOL. - NO. - / - 2014
Fertility and Sterility® The strengths of our study include the prospective, experimental design, the fact that the trial was in men of infertile couples who served as their own control, and the testing population was selected to exclude exogenous influences that may affect the seminal superoxide environment. Men were excluded from study if they were current smokers, had a varicocele, or took antioxidant vitamin supplements, all factors that could have an impact on seminal ROS (27, 28). A weakness of our study is that we did not directly measure seminal ROS levels and could not determine the origin(s) nor the relative amount(s) of ROS contributed from the individual accessory glands of the excurrent ductal system. The ROS/TAC ratio has been proposed to be a more reliable indicator for the potential free radical damage to spermatozoa. Furthermore, our results should be interpreted in light of the specific interval of EA and the frequency of ejaculation before testing. As seen in other trials, a more intensive frequency of ejaculations or varying periods of EA could produce different results with regards to the seminal microenvironment and indices of sperm quality. We conclude that higher seminal TAC obtained after a shorter period of EA may protect sperm from oxidative stress-induced sperm damage by a mechanism independent of lipid peroxidation of sperm membranes. It is, however, not clear whether the mechanism is driven by a production of higher seminal TAC levels in the excurrent ductal system related to shorter EA period or by shorter epididymal/vas deferens residence time for spermatozoa before ejaculation. Shorter periods of EA could improve sperm quality by protecting from ROS damage, even though lower numbers of motile sperm are produced after a shorter period of EA. This proposed mechanism would be consistent with prior research findings from our group showing a higher pregnancy rate after IUI under these circumstances (2). Measuring LPO and TAC levels in the semen specimen used for IUI could provide direct correlation of these measures with IUI success. The sperm requirements for the LPO assay precluded using that specimen for insemination in this experimental study. For this reason, we cannot provide a direct correlation of higher IUI success with semen specimens having higher seminal TAC levels from a shorter EA period. Therefore, such correlations should be interpreted with caution even though we did re-create the periods of EA for the current study that were used in our prior IUI report that had indicated a higher per cycle pregnancy rates with a shorter EA interval. Our findings help to define an optimum period of EA before IUI and provide justification for recommending an EA period of 1 or 2 days before oocyte insemination for IVF. This EA protocol should improve the chances for conception in assisted reproductive cycles by means of a method that is simple and free of cost.
REFERENCES 1.
2.
Verhulst SM, Cohlen BJ, Hughes E, Heineman MJ, Velde ET. Intra-uterine insemination for unexplained subfertility. Cochrane Database Syst Rev 2006:CD001838. Marshburn PB, Alanis M, Matthews ML, Usadi RS, Papadakis MH, Kullstam S, et al. A short period of ejaculatory abstinence before intrauterine
VOL. - NO. - / - 2014
3.
4.
5. 6. 7. 8. 9.
10. 11.
12. 13.
14.
15.
16.
17.
18.
19. 20.
21.
22.
23.
24.
insemination is associated with higher pregnancy rates. Fertil Steril 2010;93: 286–8. Noblanc A, Kocer A, Chabory E, Vernet P, Saez F, Cadet R, et al. Glutathione peroxidases at work on epididymal spermatozoa: an example of the dual effect of reactive oxygen species on mammalian male fertilizing ability. J Androl 2011;32:641–50. Aitken RJ, Clarkson JS, Hargreave TB, Irvine DS, Wu FC. Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia. J Androl 1989;10:214–20. Griveau JF, Le Lannou D. Reactive oxygen species and human spermatozoa: physiology and pathology. Int J Androl 1997;20:61–9. Agarwal A, Saleh RA, Bedaiway MA. Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril 2003;79:829–43. Aitken J, Fisher H. Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioessays 1994;16:259–67. Barone JG, De Lara J, Cummings KB, Ward WS. DNA organization in human spermatozoa. J Androl 1994;15:139–44. World Health Organization. WHO laboratory manual for the examination of human sperm and semen-cervical mucus interaction. 4th ed. New York: Cambridge University Press; 1999. Wang Y, Sharma RK, Agarwal A. Effect of cryopreservation and sperm concentration on lipid peroxidation in human semen. Urology 1997;50:409–13. Jurema MW, Vieira AD, Bankowski B, Petrella C, Zhao Y, Wallach E, et al. Effect of ejaculatory abstinence period on the pregnancy rate after intrauterine insemination. Fertil Steril 2005;84:678–81. Zini A, De Lamirande E, Gagnon C. Low levels of nitric oxide promote human sperm capacitation in vitro. J Androl 1995;16:424–31. de Lamirande E, Gagnon C. Human sperm hyperactivation in whole semen and its association with low superoxide scavenging capacity in seminal plasma. Fertil Steril 1993;59:1291–5. Wang X, Sharma RK, Sikka SC, Thomas AJ Jr, Falcone T, Agarwal A. Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertil Steril 2003;80: 531–5. Mahfouz R, Sharma R, Thiyagarajan A, Kale V, Gupta S, Sabanegh E, et al. Semen characteristics and sperm DNA fragmentation in infertile men with low and high levels of seminal reactive oxygen species. Fertil Steril 2010; 94:2141–6. Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, et al. Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril 2003;79(Suppl 3):1597–605. Wang X, Sharma RK, Gupta A, George V, Thomas AJ Jr, Falcone T, et al. Alterations in mitochondria membrane potential and oxidative stress in infertile men: a prospective observational study. Fertil Steril 2003;80(Suppl 2):844–50. Wilton LJ, Temple-Smith PD, Baker HW, de Kretser DM. Human male infertility caused by degeneration and death of sperm in the epididymis. Fertil Steril 1988;49:1052–8. Mallidis C, Lim TC, Hill ST, Skinner DJ, Brown DJ, Johnston WI, et al. Necrospermia and chronic spinal cord injury. Fertil Steril 2000;74:221–7. Richthoff J, Spano M, Giwercman YL, Frohm B, Jepson K, Malm J, et al. The impact of testicular and accessory sex gland function on sperm chromatin integrity as assessed by the sperm chromatin structure assay (SCSA). Hum Reprod 2002;17:3162–9. Aktan G, Dogru-Abbasoglu S, Kucukgergin C, Kadioglu A, OzdemirlerErata G, Kocak-Toker N. Mystery of idiopathic male infertility: is oxidative stress an actual risk? Fertil Steril 2013;99:1211–5. Spano M, Kolstad AH, Larsen SB, Cordelli E, Leter G, Giwercman A, et al. The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Hum Reprod 1998;13:2495–505. Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E, Leter G. Sperm chromatin damage impairs human fertility: The Danish First Pregnancy Planner Study Team. Fertil Steril 2000;73:43–50. Bonde JP, Joffe M, Apostoli P, Dale A, Kiss P, Spano M, et al. Sperm count and chromatin in men exposed to inorganic lead: lowest adverse effect levels. Occup Environ Med 2002;59:234–42.
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ORIGINAL ARTICLE: ANDROLOGY 25. 26.
6
De Jonge C, LaFromboise M, Bosmans E, Ombelet W, Cox A, Nijs M. Influence of the abstinence period on human sperm quality. Fertil Steril 2004;82:57–65. Desai NR, Mahfouz R, Sharma R, Gupta S, Agarwal A. Reactive oxygen species levels are independent of sperm concentration, motility, and abstinence in a normal, healthy, proven fertile man: a longitudinal study. Fertil Steril 2010;94:1541–3.
27.
28.
Saleh RA, Agarwal A, Sharma RK, Nelson DR, Thomas AJ Jr. Effect of cigarette smoking on levels of seminal oxidative stress in infertile men: a prospective study. Fertil Steril 2002;78:491–9. Agarwal A, Sharma RK, Desai NR, Prabakaran S, Tavares A, Sabanegh E. Role of oxidative stress in pathogenesis of varicocele and infertility. Urology 2009; 73:461–9.
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Influence of ejaculatory abstinence on seminal total antioxidant capacity and sperm membrane lipid peroxidation Paul B. Marshburn, M.D.,a,b Allie Giddings, M.D.,b Stephanie Causby, M.S.,a,b Michelle L. Matthews, M.D.,a,b Rebecca S. Usadi, M.D.,a,b Nury Steuerwald, Ph.D.,c and Bradley S. Hurst, M.D.a,b a Division of Reproductive Endocrinology and Infertility, b Department of Obstetrics and Gynecology, and Research Center, Carolinas Healthcare System, Charlotte, North Carolina
c
Cannon
Objective: To determine whether the period of ejaculatory abstinence (EA) influences the total antioxidant capacity (TAC) of semen or lipid peroxidation (LPO) of sperm membranes. Design: A prospective experimental trial. Setting: Academic medical center for reproductive endocrinology and infertility. Patient(s): Forty men from infertile couples planning intrauterine insemination. Intervention(s): Men provided semen specimens after EA periods of 1 and 4 days. Main Outcome Measure(s): Semen analysis, peroxidase staining, and assays for seminal TAC and sperm membrane LPO, with measures compared between days 1 and 4 within individuals (internal control) using paired t tests. Result(s): The shorter period of EA (1 day vs. 4 days) resulted in statistically significant decreases in semen volume (24%), sperm density (28%), and total sperm count (3.2%). There was a statistically significant increase in TAC with the shorter period of EA (1 day) compared with 4 days of EA. No difference was detected in sperm membrane LPO comparing 1 day of EA and 4 days of EA. Conclusion(s): Higher seminal TAC obtained after a shorter period of EA could diminish oxidative stress–induced sperm damage by a mechanism independent of LPO. Shorter periods of EA may thus improve sperm quality by protecting from reactive oxygen species damage, even though lower numbers of motile sperm are produced after a shorter period of EA. This would be consistent with prior research indicating improved results after intrauterine insemination under these circumstances. Use your smartphone (Fertil SterilÒ 2014;-:-–-. Ó2014 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Abstinence, male infertility, seminal plasma oxidative stress, spermatozoa, total and connect to the antioxidant capacity Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/marshburnp-ejaculatory-abstinence/
F
or selected couples with male factor infertility, ovulation induction with intrauterine insemination (IUI) produces significantly higher cycle fecundity compared to timed intercourse (1). We previously reported that shortening the period of
ejaculatory abstinence (EA) resulted in a higher pregnancy rate after IUI despite the fact that fewer motile sperm cells were inseminated (2). These findings are consistent with the premise that improved sperm quality following a shortened EA period, rather than
Received December 6, 2013; revised May 13, 2014; accepted May 27, 2014. P.B.M. has nothing to disclose. A.G. has nothing to disclose. S.C. has nothing to disclose. M.L.M. has nothing to disclose. R.S.U. has nothing to disclose. N.S. has nothing to disclose. B.S.H. has nothing to disclose. Supported by Cannon Research Grant Committee, Charlotte, NC (Number 08–033). Reprint requests: Paul B. Marshburn, M.D., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Carolinas Healthcare System, P.O. Box 32861, Charlotte, North Carolina 28232–2861 (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.05.039 VOL. - NO. - / - 2014
discussion forum for this article now.*
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motile sperm number, is the principal factor that produced a higher IUI cycle fecundity. Sperm cells in the cauda of the epididymis and vas deferens may be subject to a harmful seminal microenvironment before or after ejaculation that could impair sperm function in proportion to increasing time exposure. Maturing, epididymal spermatozoa require a level of reactive oxygen species (ROS) for the formation of disulfide-bridging for DNA compaction, but excessive ROS would induce oxidative damage to spermatozoa that could negatively affect fertilization potential. This balanced ROS 1
ORIGINAL ARTICLE: ANDROLOGY microenvironment in the epididymis is regulated by the glutathione peroxidase system (3). Mature spermatozoa are particularly susceptible to ROS damage because they cannot respond to oxidative challenge with transcriptional activity secondary to loss of cytosolic organelles and because the external sperm membrane has a particular composition that is highly reactive to oxidative injury. Excessive ROS may be generated from leukocytes and dying spermatozoa in this seminal microenvironment. The ROS and/or deficient antioxidant defense systems in semen may cause sperm damage and have been implicated as an important cause of male factor infertility (4–6). Potential harmful effects of oxidative stress on human spermatozoa include damage to sperm membranes by lipid peroxidation (LPO), sperm DNA susceptibility to free radical attack, or sperm protein damage (7, 8). Theoretically, an increased antioxidant capacity in seminal fluid could neutralize superoxide free radicals and mitigate sperm damage. Increasing the frequency of ejaculation may reduce spermatozoal exposure to harmful reactive oxygen species, thereby improving sperm viability and function. To test this hypothesis, we have determined whether the period of ejaculatory abstinence (EA) influences the total antioxidant capacity (TAC) of semen or impacts lipid peroxidation (LPO) of sperm membranes. A reduction of reactive oxygen stress by shortening the period of EA could be a mechanism to explain why a higher pregnancy rate after IUI results from semen specimens obtained after a short period of EA despite fewer motile sperm cells being inseminated (2).
MATERIALS AND METHODS Study Participants Forty men from couples with infertility being treated at the Reproductive Medicine and Infertility clinic at Carolinas Healthcare System and planning to undergo IUI were recruited to participate in this prospective experimental study after institutional review board approval. The average frequency of intercourse for the men before producing their first semen specimen for study was between once and twice weekly. Men were excluded from study entry for the following reasons: failure to follow the ejaculatory abstinence (EA) protocol, specimen less than 2 cc in volume, current smoking, presence of varicocele, history of vasectomy reversal, consumption of certain vitamins or supplements (vitamin E or C, coenzyme Q, taurine or glutathione), or genital infection within 6 months. The decision to exclude men with ejaculates of less than 2 cc in volume was secondary to technical problems with having sufficient volume and/or sperm for the TAC and LPO assays.
Study Protocol All men provided two semen samples. The initial semen specimen was collected after 4 days of EA and the second specimen 1 day later. Semen analysis was performed after liquefaction at 37 C in accordance with World Health Organization guidelines (9). Quantification of seminal leukocytes was assessed by peroxidase staining. Raw semen (seminal 2
plasma plus spermatozoa) was subjected to nondensity centrifugation at 300 g for 7 minutes, and the supernatant (seminal plasma) was removed from the sperm pellet. Two aliquots of neat seminal plasma of 200 mL were transferred to cryovials and were cryopreserved as indicated herein for subsequent TAC assay. The sperm pellet was resuspended in the residual seminal plasma and was then diluted by careful addition of the TEST-yolk buffer freezing medium (Irvine Scientific) at a 1:1 (volume/volume) ratio. Gentle, uniform resuspension was performed for 5 minutes. The cryopreservation vials with the seminal plasma (for later TAC assay) and the resuspension of sperm with TEST-yolk buffer freezing medium (for later LPO assay) were placed in a 20 C freezer for 8 minutes and thereafter in liquid nitrogen vapor at 79 C for 2 hours. The vials were then transferred to liquid nitrogen at 196 C. For TAC and LPO testing, each reconstituted semen plus TEST-yolk buffer vial or seminal plasma vial was removed from liquid nitrogen and thawed at room temperature for 5 minutes, followed by incubation for 20 minutes at 37 C. Assay for seminal total antioxidant capacity (TAC). Seminal plasma was assayed in triplicate for total antioxidant capacity (TAC) using a Randox Total Antioxidant Status kit (Crumlin) per the manufacturer's instructions. Briefly, samples were incubated with ABTS (2,20 -Azino-di-[3-ethylbenzthiazoline] sulphonate) radical cations, which have a relatively stable blue-green color in the absence of antioxidants. The degree of color suppression by antioxidants in the sample, which is proportional to the concentration, was measured at 600 nm using a DU 800 UV/Visible Spectrophotometer (Beckman Coulter). Unknown sample antioxidant concentrations were interpolated from a standard curve created from samples of known concentrations. Assay for sperm membrane lipid peroxidation (LPO). Sperm membrane lipid peroxidation was measured using a Calbiochem Lipid Hydroperoxide (LPO) Assay Kit (Darmstadt) per the manufacturer's instructions. Thawed sperm resuspensions were diluted in Ham's F-10 (pH 7.4) with a 1:1 (volume/volume) ratio of resuspension to buffer medium, followed by centrifugation at 300 g for 7 minutes. After discarding the supernatant, we resuspended the sperm pellet in fresh Ham's F-10 (pH 7.4) buffer to an adjusted final concentration of 20 106 spermatozoa/mL (10). Sample lipid hydroperoxides were first extracted in chloroform to eliminate interference by hydrogen peroxide or endogenous ferric ions. Lipid hydroperoxides present in this extract were measured directly using redox reactions with ferrous ions. The ferric ions produced were detected using thiocyanate ion as a chromogen. The absorbance of the reactions was measured at 500 nM using a Synergy HT Multi-Detection Microplate reader (BioTek). Unknown lipid hydroperoxide sample concentrations were calculated using a standard curve generated with samples of known concentrations.
Statistical Analysis Measures for semen parameters, total antioxidant capacity (TAC), and lipid peroxidation (LPO) were compared statistically on semen specimens obtained after 4 days of ejaculatory VOL. - NO. - / - 2014
Fertility and Sterility® abstinence (EA 4) and again in the same individual 1 day later (EA 1). A paired two-tailed t test was employed to statistically compare results for EA4 and EA1 specimens for a given individual to allow each man to serve as his own internal control. The values for semen parameters under columns EA1 and EA4 in Table 1 were derived by averaging determinations from within each group. The values for the mean difference and percentage change columns were the average of the determinations for each individual trial. A sample size of 32 was calculated to have 80% power to detect a difference of 20% in TAC levels between the paired EA groups as determined by a pilot study.
RESULTS The average age of the men in our study was 34 years. Twenty-four out of 40 men had semen parameters associated with male factor infertility defined as having at least one of the following: sperm density less than 20 million sperm/mL, total motility of less than 50%, or recurrent poor sperm morphology. A statistically significant reduction in semen volume (24%; P< .001), sperm density (3%; P< .02), and total sperm count (28%; P< .001) was noted with a shorter period of ejaculatory abstinence when semen parameters from EA1 with EA4 specimens were compared (Table 1). No differences in the percentage motility, percentage normal forms, or leukocyte density were detected comparing semen specimens for an EA period of 1 day vs. 4 days. In our cohort, no man exhibited significant leukocytospermia (>1 106 white blood cells/mL). A shorter period of ejaculatory abstinence (1 day) increased seminal total antioxidant capacity (TAC) compared with the longer 4 day period of abstinence (P< .02) (Fig. 1). There was no measurable difference in sperm membrane lipid peroxidation (LPO) when comparing 1 day vs. 4 days of ejaculatory abstinence (Fig. 2).
DISCUSSION We showed in a previous study that a period of EA of 2 days or less before obtaining a specimen for IUI produced higher pregnancy rates than IUI with specimens after longer periods of EA (2), a report that supports the findings of Jurema et al. (11). These higher conception rates after IUI with a shorter period of EA occurred with specimens that possessed a lower
total, motile sperm (TMS) fraction in the IUI specimen. We hypothesized that other factors besides TMS must be involved with the observed enhancement in pregnancy rates provided by shortening the EA period. Shortening the residence time of sperm in the cauda of the epididymis or vas deferens may remove those sperm cells from an environment harmful to sperm function such as superoxide stress. There are limited data on the independent contribution of antioxidant/ROS content from the individual male accessory sex gland secretions. Most of the data in the human indicate that the primary sources of ROS in semen originate from senescent sperm or polymorphonuclear leukocytes. Our study provides no data to elucidate whether the increased TAC that we measure in semen after a shorter period of EA have an impact on the ROS environment of the epididymis or other male accessory sex glands. Purging sperm from the cauda of the epididymis or vas deferens by shortening the period of EA could diminish the population of senescent sperm, thus reducing ROS and perhaps lessening the protective effect of TAC. After ejaculation but before the isolation of motile sperm from the seminal plasma, increased TAC may protect sperm from ROS damage. Because semen is an admixture of secretions from the epididymis, vas deferens, seminal vesicles, and the prostate gland, the contribution from each glandular secretion is a complicated issue to resolve. It is an important point to acknowledge that if the cauda/vas deferens microenvironment has a high level of harmful ROS, then the protection of sperm may not occur until after ejaculation when they are exposed to accessory glandular secretions containing antioxidants and/or ROS scavengers. In that case, any improvement in sperm quality would potentially result from either reversing or slowing the detrimental effects of oxidative stress after ejaculation and until sperm are separated from the seminal plasma. We show in this experimental study that reducing the period of ejaculatory abstinence from 4 days to 1 day will increase seminal total antioxidant capacity (TAC) while producing no detectable change in sperm membrane lipid peroxidation (LPO). Higher seminal TAC would reduce exposure of sperm to harmful ROS and may be a protective mechanism for improving sperm quality. Such a protective mechanism appears to be independent of oxidative sperm membrane damage by lipid peroxidation.
TABLE 1 The impact of ejaculatory abstinence of 1 day (EA1) vs. 4 days (EA4) on semen analysis parameters (n [ 40). Parameter Volume (mL) Sperm density (millions/mL) Motility (%) Normal morphology, Kruger strict (%) Total sperm count (millions) WBCs (no. per 100 sperm)
EA1 (±SD)
EA4 (±SD)
Mean differencea
% Changeb
P valuec
3.1 1.0 36.6 27.1 61.6 14.1 2.1 1.6 112.5 77.4 0.6 0.3
4.0 1.9 50.5 24.4 61.7 12.4 1.2 0.9 201.8 83.2 0.5 0.2
0.89 14.16 0.018 0.9 87.5 0.15
24.0 3.2 4.2 12.1 28.4 3.7
< .001 < .02 .99 .06 < .001 .31
Note: EA1 ¼ 1 day of ejaculatory abstinence; EA4 ¼ 4 days of ejaculatory abstinence; SD ¼ standard deviation. a EA1 EA4. b [(EA1 EA4)/EA4] 100. c Semen analysis parameters compared within a given male subject by a two-tailed t test with a man serving as his own control. Marshburn. Ejaculate period and semen antioxidants. Fertil Steril 2014.
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FIGURE 1 *
Comparison of the total antioxidant capacity (TAC) in seminal fluid after 1 day (EA1) vs. 4 days (EA4) of ejaculatory abstinence. The counted photons per minute measures the total antioxidant capacity [TAC]; (see Materials and Methods). TAC determinations were compared within a given male subject by a two-tailed t test, with a man serving as his own control from EA1 to EA4. *P<.02. Marshburn. Ejaculate period and semen antioxidants. Fertil Steril 2014.
Agarwal et al. (6) have provided strong evidence that male factor infertility is associated with higher levels of ROS. A low level of ROS in semen is associated with enhanced human sperm capacitation, acrosome reaction, and hyperactivated sperm motility (12, 13), which are all important functions involved with fertilization. Oxidative stress caused by excess generation of free radicals from leukocytes, dying spermatozoa, and/or deficient antioxidant defense systems in semen is significantly correlated with male factor infertility (4–6).
FIGURE 2
Comparison of the sperm membrane lipid peroxidation (LPO) after 1 day (EA1) vs. 4 days (EA4) of ejaculatory abstinence. The level of malondialdehyde (nM/h) measures the degree of sperm membrane lipid peroxidation (see Materials and Methods). LPO determinations were compared within a given male subject by a two-tailed t test, with a man serving as his own control from EA1 to EA4. P¼.98. Marshburn. Ejaculate period and semen antioxidants. Fertil Steril 2014.
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The harmful effect of oxidative stress on human spermatozoa may include lipid peroxidation (LPO) of the sperm plasma membrane by reducing the sperm membrane fluidity required for sperm-oocyte fusion (7). Sperm DNA also is susceptible to free radical attack because of the loss of antioxidants and scavenging enzymes after the removal of most of the sperm cytoplasm during sperm maturation events in the epididymis (8). Men with increased levels of seminal ROS have increased DNA fragmentation and apoptosis in spermatozoa (14–16) and a compromise in the mitochondrial membrane potential of spermatozoa (17). A depleted antioxidant capacity would lessen the capability of semen to neutralize harmful ROS, and therefore sperm would be more vulnerable to oxidative damage with low TAC levels. A logical consideration from our findings would include the possibility that free radical attack of sperm DNA or mitochondrial membranes may occur at a lower level of ROS than would be required to impact the LPO of sperm plasma membranes. Further study would be required to evaluate this possibility. Investigations on the impact of the period of EA on sperm quality or the seminal microenvironment have revealed differing results. These differing results are, at least in part, secondary to variations in the protocols for the time period of EA, the intensity of ejaculatory frequency before analysis, the measures of sperm quality studied, and the evaluation of men in both the fertile and infertile populations. Frequent ejaculations (twice daily for 4 days) in healthy men and those with spinal cord injuries improved sperm viability and quality, and these results were comparable with a single, second ejaculation provided 60 minutes after an initial one (18, 19). Several studies indicated that sperm DNA fragmentation was increased with longer periods of EA, consistent with the notion that the longer the mature sperm cell resides within the cauda of the epididymis or the vas deferens the greater the risk of DNA deterioration (20–24). In a different study, however, the degree of DNA fragmentation measured by sperm chromatin structure assay remained unchanged following 1, 3, 5, and 8 day periods of EA (25). Within a single, healthy sperm donor, seminal ROS was not found to depend on sperm concentration, motility, or the period of EA (26). Our data in men from infertile couples would indicate that a shorter period of EA is associated with a higher seminal TAC, which would enhance the protection of sperm from ROS damage. The augmentation of such a seminal defense mechanism could improve sperm quality and, at least in part, explain the higher pregnancy rates after IUI observed after shorter periods of EA. Further study is warranted to: [1] define the measure(s) of sperm viability and quality that may be improved after 1 day of EA, [2] evaluate the TAC levels in the secretions from individual accessory sex glands, [3] determine whether higher TAC levels of semen used for IUI are directly correlated with increased pregnancy rates, and [4] identify the precise interval of EA and the degree of ejaculatory frequency to produce optimum sperm quality. Because sperm membrane LPO was not different for the intervals of EA that we studied, other indices such as a reduction in DNA fragmentation should be investigated in men from infertile couples under the conditions of our EA protocol. VOL. - NO. - / - 2014
Fertility and Sterility® The strengths of our study include the prospective, experimental design, the fact that the trial was in men of infertile couples who served as their own control, and the testing population was selected to exclude exogenous influences that may affect the seminal superoxide environment. Men were excluded from study if they were current smokers, had a varicocele, or took antioxidant vitamin supplements, all factors that could have an impact on seminal ROS (27, 28). A weakness of our study is that we did not directly measure seminal ROS levels and could not determine the origin(s) nor the relative amount(s) of ROS contributed from the individual accessory glands of the excurrent ductal system. The ROS/TAC ratio has been proposed to be a more reliable indicator for the potential free radical damage to spermatozoa. Furthermore, our results should be interpreted in light of the specific interval of EA and the frequency of ejaculation before testing. As seen in other trials, a more intensive frequency of ejaculations or varying periods of EA could produce different results with regards to the seminal microenvironment and indices of sperm quality. We conclude that higher seminal TAC obtained after a shorter period of EA may protect sperm from oxidative stress-induced sperm damage by a mechanism independent of lipid peroxidation of sperm membranes. It is, however, not clear whether the mechanism is driven by a production of higher seminal TAC levels in the excurrent ductal system related to shorter EA period or by shorter epididymal/vas deferens residence time for spermatozoa before ejaculation. Shorter periods of EA could improve sperm quality by protecting from ROS damage, even though lower numbers of motile sperm are produced after a shorter period of EA. This proposed mechanism would be consistent with prior research findings from our group showing a higher pregnancy rate after IUI under these circumstances (2). Measuring LPO and TAC levels in the semen specimen used for IUI could provide direct correlation of these measures with IUI success. The sperm requirements for the LPO assay precluded using that specimen for insemination in this experimental study. For this reason, we cannot provide a direct correlation of higher IUI success with semen specimens having higher seminal TAC levels from a shorter EA period. Therefore, such correlations should be interpreted with caution even though we did re-create the periods of EA for the current study that were used in our prior IUI report that had indicated a higher per cycle pregnancy rates with a shorter EA interval. Our findings help to define an optimum period of EA before IUI and provide justification for recommending an EA period of 1 or 2 days before oocyte insemination for IVF. This EA protocol should improve the chances for conception in assisted reproductive cycles by means of a method that is simple and free of cost.
REFERENCES 1.
2.
Verhulst SM, Cohlen BJ, Hughes E, Heineman MJ, Velde ET. Intra-uterine insemination for unexplained subfertility. Cochrane Database Syst Rev 2006:CD001838. Marshburn PB, Alanis M, Matthews ML, Usadi RS, Papadakis MH, Kullstam S, et al. A short period of ejaculatory abstinence before intrauterine
VOL. - NO. - / - 2014
3.
4.
5. 6. 7. 8. 9.
10. 11.
12. 13.
14.
15.
16.
17.
18.
19. 20.
21.
22.
23.
24.
insemination is associated with higher pregnancy rates. Fertil Steril 2010;93: 286–8. Noblanc A, Kocer A, Chabory E, Vernet P, Saez F, Cadet R, et al. Glutathione peroxidases at work on epididymal spermatozoa: an example of the dual effect of reactive oxygen species on mammalian male fertilizing ability. J Androl 2011;32:641–50. Aitken RJ, Clarkson JS, Hargreave TB, Irvine DS, Wu FC. Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia. J Androl 1989;10:214–20. Griveau JF, Le Lannou D. Reactive oxygen species and human spermatozoa: physiology and pathology. Int J Androl 1997;20:61–9. Agarwal A, Saleh RA, Bedaiway MA. Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril 2003;79:829–43. Aitken J, Fisher H. Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioessays 1994;16:259–67. Barone JG, De Lara J, Cummings KB, Ward WS. DNA organization in human spermatozoa. J Androl 1994;15:139–44. World Health Organization. WHO laboratory manual for the examination of human sperm and semen-cervical mucus interaction. 4th ed. New York: Cambridge University Press; 1999. Wang Y, Sharma RK, Agarwal A. Effect of cryopreservation and sperm concentration on lipid peroxidation in human semen. Urology 1997;50:409–13. Jurema MW, Vieira AD, Bankowski B, Petrella C, Zhao Y, Wallach E, et al. Effect of ejaculatory abstinence period on the pregnancy rate after intrauterine insemination. Fertil Steril 2005;84:678–81. Zini A, De Lamirande E, Gagnon C. Low levels of nitric oxide promote human sperm capacitation in vitro. J Androl 1995;16:424–31. de Lamirande E, Gagnon C. Human sperm hyperactivation in whole semen and its association with low superoxide scavenging capacity in seminal plasma. Fertil Steril 1993;59:1291–5. Wang X, Sharma RK, Sikka SC, Thomas AJ Jr, Falcone T, Agarwal A. Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertil Steril 2003;80: 531–5. Mahfouz R, Sharma R, Thiyagarajan A, Kale V, Gupta S, Sabanegh E, et al. Semen characteristics and sperm DNA fragmentation in infertile men with low and high levels of seminal reactive oxygen species. Fertil Steril 2010; 94:2141–6. Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, et al. Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril 2003;79(Suppl 3):1597–605. Wang X, Sharma RK, Gupta A, George V, Thomas AJ Jr, Falcone T, et al. Alterations in mitochondria membrane potential and oxidative stress in infertile men: a prospective observational study. Fertil Steril 2003;80(Suppl 2):844–50. Wilton LJ, Temple-Smith PD, Baker HW, de Kretser DM. Human male infertility caused by degeneration and death of sperm in the epididymis. Fertil Steril 1988;49:1052–8. Mallidis C, Lim TC, Hill ST, Skinner DJ, Brown DJ, Johnston WI, et al. Necrospermia and chronic spinal cord injury. Fertil Steril 2000;74:221–7. Richthoff J, Spano M, Giwercman YL, Frohm B, Jepson K, Malm J, et al. The impact of testicular and accessory sex gland function on sperm chromatin integrity as assessed by the sperm chromatin structure assay (SCSA). Hum Reprod 2002;17:3162–9. Aktan G, Dogru-Abbasoglu S, Kucukgergin C, Kadioglu A, OzdemirlerErata G, Kocak-Toker N. Mystery of idiopathic male infertility: is oxidative stress an actual risk? Fertil Steril 2013;99:1211–5. Spano M, Kolstad AH, Larsen SB, Cordelli E, Leter G, Giwercman A, et al. The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Hum Reprod 1998;13:2495–505. Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E, Leter G. Sperm chromatin damage impairs human fertility: The Danish First Pregnancy Planner Study Team. Fertil Steril 2000;73:43–50. Bonde JP, Joffe M, Apostoli P, Dale A, Kiss P, Spano M, et al. Sperm count and chromatin in men exposed to inorganic lead: lowest adverse effect levels. Occup Environ Med 2002;59:234–42.
5
ORIGINAL ARTICLE: ANDROLOGY 25. 26.
6
De Jonge C, LaFromboise M, Bosmans E, Ombelet W, Cox A, Nijs M. Influence of the abstinence period on human sperm quality. Fertil Steril 2004;82:57–65. Desai NR, Mahfouz R, Sharma R, Gupta S, Agarwal A. Reactive oxygen species levels are independent of sperm concentration, motility, and abstinence in a normal, healthy, proven fertile man: a longitudinal study. Fertil Steril 2010;94:1541–3.
27.
28.
Saleh RA, Agarwal A, Sharma RK, Nelson DR, Thomas AJ Jr. Effect of cigarette smoking on levels of seminal oxidative stress in infertile men: a prospective study. Fertil Steril 2002;78:491–9. Agarwal A, Sharma RK, Desai NR, Prabakaran S, Tavares A, Sabanegh E. Role of oxidative stress in pathogenesis of varicocele and infertility. Urology 2009; 73:461–9.
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