ORIGINAL ARTICLE
Effect of repeated sequential ejaculation on sperm DNA integrity in subfertile males with asthenozoospermia T. M. Hussein1, A. F. Elariny1, M. M. Elabd2, Y. F. Elgarem1 & M. M. Elsawy3 1 Department of Dermatology, STD’s & Andrology, Faculty of Medicine, Alexandria University, Alexandria, Egypt; 2 Department of Gynecology & Obstetrics, Faculty of Medicine, Alexandria University, Alexandria, Egypt; 3 Department of Clinical Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
Keywords Asthenozoospermia—comet assay—DNA integrity—repeated ejaculation. Correspondence Dr Tarek M. Hussein, Department of Dermatology, STD’s & Andrology, Faculty of Medicine, Alexandria University, Alexandria, Egypt. Tel.: +20 12 392 8375; E-mail:
[email protected] Accepted: June 9, 2008
Summary The aim of this work was to study the possible beneficial effect of repeated sequential ejaculation on sperm DNA integrity in subfertile males and its possible implementation in assisted reproduction. The study included 20 infertile males with idiopathic asthenozoospermia or oligoasthenozoospermia. They underwent detailed history taking, complete clinical assessment and hormonal assessment. Patients were asked to bring two semen samples (taken within 1–3 h). Two consecutive samples were assessed with regard to semen volume, sperm count, motility grading, and morphology and sperm DNA integrity using the comet assay. There was a significant improvement in the sperm motility pattern and DNA integrity in the second sample in comparison with the first sample. Therefore, it is concluded that due to its positive impact on sperm motility and DNA integrity, repeated sequential ejaculation is recommended in subfertile males with idiopathic asthenozoospermia who pursue assisted reproduction.
Introduction Unlike the chromatin structure of somatic cells, sperm chromatin is very tightly compacted by virtue of the unique associations among the DNA, the nuclear matrix, and sperm nuclear proteins (Balhorn, 1982). During the later stages of spermatogenesis, histones are replaced by protamines (Oliva & Dixon, 1991). Protamines are small, positively charged, testis-specific nuclear proteins. In human beings, two protamines, protamine 1 (P1) and protamine 2 (P2), replace approximately 85% of sperm histones (Barone et al., 1994). The introduction of protamines into the sperm nucleus allows the DNA strands to form toroidal (donut-shaped) structures, facilitating the tight compaction of the sperm nuclear head (Balhorn, 1982). Interand intramolecular disulphide cross-links between the cysteine-rich protamines allow further compaction and stabilisation of the sperm nucleus, protecting sperm DNA from external stress and subsequent DNA break-
age (Balhorn, 1982; The Practice Committee of the American Society for Reproductive Medicine, 2006). Sperm DNA integrity is essential for the accurate transmission of genetic information (Amann, 1989). Any form of sperm chromatin abnormalities or DNA damage may result in male infertility. It has been reported that in vivo fecundity decreases progressively when more than 30% of the spermatozoa are identified as having DNA damage (Evenson et al., 2002). Furthermore, Zini et al. (2001) reported that sperm chromatin/DNA is an independent measure of sperm quality that may have better diagnostic and prognostic capabilities than standard sperm parameters (concentration, motility and morphology). The clinical significance of this assessment lies in its association not only with natural conception rates, but also with assisted reproduction success rates (Evenson et al., 2002). Therefore, screening for sperm DNA damage may provide useful information in cases of male idiopathic infertility and in those men pursuing assisted reproduction (Agarwal & Said, 2003). ª 2008 The Authors
312
Journal Compilation ª 2008 Blackwell Publishing Ltd
Æ Andrologia 40, 312–317
T. M. Hussein et al.
Male infertility
The exact mechanism by which chromatin abnormalities arise in human spermatozoa is not precisely understood, but three main theories have been proposed, namely defective sperm chromatin packaging, apoptosis and oxidative stress (Agarwal & Said, 2003). Both direct and indirect assay methods have been used to assess DNA damage. Direct methods for detecting DNA breaks include the single-cell gel electrophoresis assay (SCGE or comet assay) and the terminal deoxynucleotidyl transferase-mediated 2¢-deoxyuridine 5¢-triphosphate (dUTP)-nick end-labelling (TUNEL) assay. Indirect methods for assessing DNA damage include sperm chromatin integrity assay (e.g. sperm chromatin structure assay, SCSA), which use chromatin and/or DNA intercalating dyes, such as acridine orange to differentiate singleand double-stranded DNA (Evenson et al., 1999; Carrell & Liu, 2001; Spano et al., 2002). The comet assay measures the DNA damage by quantifying the single- and double-stranded breaks associated with DNA damage (McKelvey-Martin et al., 1997). The relationship between frequency of intercourse and fertility potential has been studied for a long time. In the early 1940s, Hotchkiss stated that closely spaced sequential ejaculates in normospermic men could result in a significant decrease in the total sperm counts available for insemination. So, the usual recommendation to couples trying to conceive was, and still is, to have intercourse every other day around the time of expected ovulation (Glass, 1986). On the other hand, in cases of asthenozoospermia or oligoasthenozoospermia, many studies showed that closely spaced ejaculation could result in an increase in total motile spermatozoa in the second ejaculate. It was stated that pooling of two sequential ejaculates from subfertile men suffering from asthenozoospermia or oligoasthenozoospermia is a simple method to increase the quality of sperm for assisted reproduction techniques (Tur-Kaspa et al., 1994; Maj et al., 1998). The aim of this study was to assess the possible beneficial effect of repeated sequential ejaculation on sperm motility and DNA integrity in subfertile males with idiopathic asthenozoospermia. Patients and methods 1. The study included 20 infertile males with idiopathic asthenozoospermia or oligoasthenozoospermia from the Andrology Outpatient Clinic (Alexandria main university hospital) who had given informed consent. 2. All the patients underwent detailed history taking about their fertility problem, complete clinical assessment and hormonal assessment. Cases with evident varicocele, hypogonadism or infection were excluded. ª 2008 The Authors Journal Compilation ª 2008 Blackwell Publishing Ltd
Æ Andrologia 40, 312–317
3. All the patients were asked to bring two semen samples (taken within 1–3 h) after an abstinence period of 3 days. Two samples were collected in the laboratory. 4. Assessment of the two consecutive semen samples with regard to semen volume, sperm count, sperm motility grading and sperm morphology was performed according to the criteria of the World Health Organization (1999) as a reference. The analysis was performed by means of a computer-aided sperm analyser. 5. Assessment of the two consecutive semen samples with regard to sperm DNA integrity was performed using comet assay, which includes quantitative detection of single- and double-stranded DNA breaks associated with DNA damage (McKelvey-Martin et al., 1993). The SCGE or comet assay basically measures DNA nicks in individual cells. The comet assay was performed using slides and protocol provided by Trevigen (Gaithersburg, MD, USA) (Sun et al., 1997). Briefly, a suspension of the spermatozoa was embedded in 1% low-melt agarose on glass slides and exposed to alkaline conditions for 60 min to denature the DNA molecules. The slides were subjected to electrophoresis to allow separation of the intact DNA from the fragmented molecules. The cells were stained with a DNA-intercalating fluorescent dye (SYBR green) and evaluated individually under a fluorescent microscope. At least 50 spermatozoas/dose were evaluated by the scion imaging software (NIH) for tail length and tail moment. The more extensive the DNA damage, the larger and more intense the ‘tail’ of the comet. For this purpose, the ‘tail moment’ was measured, which combines the tail length and the distribution of the DNA in the tail (Sun et al., 1997). Software to calculate the tail moment is commercially available. This assay was used to evaluate the extent of the DNA damage: 1. According to tail moments, spermatozoa were classified into: (i) Spermatozoa with mild DNA damage (tail moments 0–19) (ii) Spermatozoa with moderate DNA damage (tail moments 20–40). (iii) Spermatozoa with severe DNA damage (tail moments 41–60). This subdivision was arbitrarily guided by previous studies on comet assay (Morris et al., 2002). 2. Ten healthy fertile males of similar age served as a control. 3. The research plan was approved by the medical and ethical committee in the Faculty of Medicine (Alexandria University, Egypt). 4. Statistical analysis was performed using t-test. Differences were considered statistically significant when P < 0.05. 313
Male infertility
T. M. Hussein et al.
Results 1. The age of the patients ranged from 23 to 43 years (mean 31.90 ± 5.62), the age of the control group ranged from 24 to 41 years (mean 31.60 ± 6.43). 2. The duration of infertility in the patients group ranged from 15 to 156 months (mean 61.92 ± 40.24). 3. Assessment of the first and second semen samples in the patients group revealed the following (Table 1): (i) There was a significant decrease in the semen volume in the second ejaculate. (ii) There was a nonsignificant decrease in the sperm count in the second ejaculate. (iii) There was a significant increase in spermatozoa with rapid progressive (M1) and slow progressive (M2) motility in the second semen sample in comparison with the first sample. There was a significant decrease in spermatozoa with nonprogressive motility (M3) in the second sample. There was a nonsignificant decrease in nonmotile spermatozoa (M4) in the second sample.
(iv) There was a significant increase in spermatozoa with mild DNA damage (D1) in the second ejaculate in comparison with the first ejaculate. There was a significant decrease in spermatozoa with severe DNA damage in the second ejaculate. 4. Assessment of the first and second semen samples in the control group revealed the following (Table 2): (i) There was a significant decrease in the semen volume in the second ejaculate. (ii) There was a significant decrease in the sperm count in the second ejaculate in comparison with the first ejaculate. (iii) There was a nonsignificant increase in spermatozoa with rapid progressive (M1) and slow progressive (M2) motility in the second semen sample in comparison with the first sample. There was a significant decrease in spermatozoa with nonprogressive motility (M3) in the second sample. There was a nonsignificant decrease in nonmotile spermatozoa (M4) in the second sample.
Table 1 Comparison between the first and second semen samples in the patient group Semen volume (ml) First semen sample Range 1.5–6.0 Mean ± SD 4.15 ± 1.18 Second semen sample Range 1.5–5 Mean ± SD 3.32 ± 0.905 t-test 3.98 P 0.0016*
Sperm count (mil/ml)
Sperm motility (%)
Sperm DNA integrity (%)
M1
M2
M3
M4
D1
D2
D3
2–57 18.7 ± 14.9
0–25 7.97 ± 5.91
0–65 29.73 ± 16.7
10–75 47.43 ± 14.5
5–74 15.3 ± 15.2
3–45 21.5 ± 12.8
15–80 52.4 ± 16.4
2–69 26 ± 16.34
2.5–55 17.3 ± 12.5 0.69 0.3495
0–25 12.9 ± 7.59 3.07 0.0031*
5–70 37 ± 15.9 1.803 0.0447*
10–60 37.7 ± 14.30 2.98 0.0056*
0–79 13.3 ± 14.3 0.87 0.2981
5–56 31.2 ± 14 3.68 0.0037*
30–90 51.4 ± 15.4 0.88 0.4010
5–51 17.7 ± 11.3 2.08 0.0131*
M1, rapid progressive; M2, slow progressive; M3, nonprogressive motility; M4, nonmotile. D1, mild DNA damage; D2, moderate DNA damage; D3, severe DNA Damage. *Statistically significant.
Table 2 Comparison between the first and second semen samples in the control group Semen volume (ml) First semen sample Range 3–5 Mean ± SD 3.6 ± 0.70 Second semen sample Range 2–4 Mean ± SD 2.95 ± 0.50 t-test 4.85 P 0.0001*
Sperm count (mil/ml)
Sperm motility%
Sperm DNA integrity (%)
M1
M2
M3
M4
D1
D2
D3
21–65 39.9 ± 13.34
23–35 27.4 ± 4.38
25–60 37 ± 10.06
10–40 27.9 ± 8.81
0–20 8.7 ± 5.66
60–75 68.6 ± 4.67
15–30 20.4 ± 5.42
5–20 11 ± 5.16
18–55 33.3 ± 11.83 5.21 0.0001*
20–37 29.7 ± 5.25 1.68 0.06
35–50 41.3 ± 5.70 0.98 0.14
15–25 20 ± 4.71 2.01 0.01*
0–20 8 ± 5.87 0.85 0.28
65–80 72 ± 4.22 1.96 0.03*
15–30 22.3 ± 4.16 1.27 0.17
0–10 4.8 ± 3.39 3.52 0.001*
See details of abbreviations in footnote of Table 1. *Statistically significant. ª 2008 The Authors
314
Journal Compilation ª 2008 Blackwell Publishing Ltd
Æ Andrologia 40, 312–317
T. M. Hussein et al.
Male infertility
(iv) There was a significant increase in spermatozoa with mild DNA damage (D1) in the second ejaculate in comparison with the first ejaculate. There was a significant decrease in spermatozoa with severe DNA damage in the second ejaculate. Discussion Accumulating evidence suggests that disturbances in the organisation of the genomic material into sperm nuclei are negatively correlated with the fertility potential of the spermatozoa (Agarwal & Said, 2003). Evaluation of sperm DNA integrity could be considered an important objective test in cases with unexplained infertility with normal and abnormal semen parameters. Saleh et al. (2002) compared the standard semen parameters and SCSA parameters in fertile donors and in infertile men with normal and abnormal semen parameters and concluded that sperm DNA damage analysis could reveal hidden abnormalities in men with idiopathic infertility. It is documented that there is a negative correlation between defective sperm chromatin structure and fertility, in vivo (Evenson et al., 1999; Spano et al., 2002) and in vitro (Check et al., 2005; Evenson & Wixon, 2006). So, any technique that can lead to improvement in DNA integrity or separation of spermatozoa with a relatively higher percentage of normal DNA could lead to positive impact on the results of assisted reproduction. This study revealed a significant improvement in the quality of sperm motility (rapid progressive and slow progressive spermatozoa) in the second ejaculate in comparison with the first one in cases with idiopathic asthenozoospermia. Many studies had evaluated the effect of closely spaced sequential ejaculation (after variable periods) on sperm motility. Tur-Kaspa et al. (1994) tested the value of repeated sequential ejaculation (after periods 1–4 and 24 h) on the number of total motile spermatozoa in oligozoospermia and oligoasthenozoospermic infertile males versus normozoospermic men and found a significant improvement in the number of total motile spermatozoa in the second ejaculate in the group of oligoasthenozoospermia. They indicated that sequential ejaculation may overcome the impaired sperm transport causing low total motile sperm counts observed in some oligozoospermic and/or asthenozoospermic men. On the other hand, there was a significant decrease in the total motile sperm counts in the second ejaculate in normozoospermic men. In this study, we used quality of sperm motility instead of number of total motile spermatozoa because we believe that the type of sperm motility is more important than the sperm count in the era of microassisted reproduction that requires only few spermatozoa ª 2008 The Authors Journal Compilation ª 2008 Blackwell Publishing Ltd
Æ Andrologia 40, 312–317
with high quality. In contrast to a study by Tur-Kaspa et al. (1994), our study revealed improvement in the motility quality in the second ejaculate in the control group, but this was statistically nonsignificant. Also, Maj et al. (1998) demonstrated a significant improvement in motility and total motility scores in the second ejaculates in cases with asthenozoospermia and oligoasthenozoospermia, but not in cases of isolated oligozoospermia. The results of the study by Barash et al. (1995) revealed a statistically significant improvement in sperm cell motility and in motile count after swim-up in the second ejaculate. They also demonstrated a significant improvement in the fertilisation rate and cleavage rate when oocytes had been exposed to sperm from the second ejaculate. CorreaPe´rez et al. (2004) demonstrated a significant improvement in sperm count, motility and viability in the second ejaculate (after 60 min) in cases with epididymal necrozoospermia. We believe that factors leading to epididymal dysfunction and occurrence of epididymal necrozoospermia may be working to a lesser extent in cases with idiopathic asthenozoospermia or oligoasthenozoospermia. The hostile environment in the epididymis may be caused by a dysfunction or partial obstruction of the epididymis itself, stasis of seminal fluids, accumulation of senescent-degenerating spermatozoa, packing of cells involved in the removal of ageing sperm (Wilton et al., 1988; de Kretser et al., 1998; Mallidis et al., 2000; Fang & Baker, 2003). This study could be a preliminary report about the beneficial effect of repeated sequential ejaculation on sperm DNA integrity. There was a significant improvement in DNA integrity in cases with idiopathic asthenozoospermia or oligoasthenozoospermia. It is possible that spermatozoa in the second ejaculate had a shorter exposure period to the hostile epididymal environment in these cases. So, using the second ejaculate in the previous conditions could provide us with spermatozoa with a relatively better DNA integrity for intrauterine insemination (IUI) and micro-assisted reproduction. There is an evident relationship between sperm DNA integrity and success rates of assisted reproduction. Many studies confirmed the negative correlation between defective sperm chromatin structure (DNA breaks) and success rates of assisted reproductive techniques (Barash et al., 1995; Bungum et al., 2004; Check et al., 2005). Bungum et al. (2007) had indicated that DNA integrity testing can be used as an independent predictor of fertility in couples undergoing IUI. They proposed that all infertile men should be tested with SCSA as a supplement to the standard semen analysis. When the DNA fragmentation index exceeds 30%, intracytoplasmic sperm injection should be the method of choice. 315
Male infertility
Also, the results of this study revealed that the beneficial effect of repeated sequential ejaculation was present in the fertile control group, but this result needs to be assessed on a larger sample for verification. It is concluded that repeated sequential ejaculation is recommended to subfertile males with idiopathic asthenozoospermia who pursue assisted reproduction due to its positive impact on sperm motility and DNA integrity. References Agarwal A, Said TM (2003) Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 9:331–345. Amann RP (1989) Can fertility potential of a seminal sample be predicted accurately?. J Androl 16:89–98. Balhorn R (1982) A model for the structure of chromatin in human sperm. J Cell Biol 93:298–305. Barash A, Lurie S, Weissman A, Insler V (1995) Comparison of sperm parameters, in vitro fertilization results, and subsequent pregnancy rates using sequential ejaculates, collected two hours apart, from oligoasthenozoospermic men. Fertil Steril 64:1008–1011. Barone GJ, De Lara J, Cummings KB, Ward WS (1994) DNA organization in human spermatozoa. J Androl 15:139–144. Bungum M, Humaidan P, Spano M, Jepson K, Bungum L, Giwercman A (2004) The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI. Hum Reprod 19:1401–1408. Bungum M, Humaidan P, Axmon A, Spano M, Bungum L, Erenpreiss J, Giwercman A (2007) Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome. Hum Reprod 22:174–179. Carrell DT, Liu L (2001) Altered protamine 2 expression is uncommon in donors of known fertility, but common among men with poor fertilizing capacity, and may reflect other abnormalities of spermiogenesis. J Androl 22:604–610. Check JH, Graziano V, Cohen R, Krotec J, Check ML (2005) Effect of an abnormal sperm chromatin structural assay (SCSA) on pregnancy outcome following (IVF) with ICSI in previous IVF failures. Arch Androl 51:121–124. Correa-Pe´rez JR, Ferna´ndez-Pelegrina R, Aslanis P, Zavos PM (2004) Clinical management of men producing ejaculates characterized by high levels of dead sperm and altered seminal plasma factors consistent with epididymal necrospermia. Fertil Steril 81:1148–1150. Evenson D, Wixon R (2006) Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay. Reprod Biomed Online 12:466–472. Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, deAngelis P, Claussen OP (1999) Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod 14:1039–1049.
T. M. Hussein et al.
Evenson DP, Larson KL, Jost LK (2002) Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with the other techniques. J Androl 23:25–43. Fang S, Baker HW (2003) Male infertility and adult polycystic kidney disease are associated with necrospermia. Fertil Steril 79:643–644. Glass RH (1986) Infertility. In: Reproductive Endocrinology. Yen SSC, Jaffe RB (eds). Saunders, Philadelphia, pp 571–613. de Kretser DM, Huidobro C, Southwick GJ, Temple-Smith PD (1998) The role of the epididymis in human infertility. J Reprod Fertil 53:271–275. Maj K, Milewczyk P, Slusarski M, Smolarczyk R, Pekala M (1998) The effect of frequent ejaculation on semen’s parameters in men with inadequate fertility. Ginekol Pol 69:466–469. Mallidis C, Lim TC, Hill ST, Skinner DJ, Brown DJ, Johnston WI, Baker HW (2000) Necrospermia and chronic spinal cord injury. Fertil Steril 74:221–227. McKelvey-Martin VJ, Green MH, Schmezer P, Pool-Zobel BL, De Me´o MP, Collins A (1993) The single cell gel electrophoresis assay (comet assay): a European review. Mutat Res 288:47–63. McKelvey-Martin VJ, Melia N, Walsh IK, Johnston SR, Hughes CM, Lewis SEM, Thompson W (1997) Two potential clinical applications of the alkaline single-cell gel electrophoresis assay: (1) human bladder washings and transitional cell carcinoma of the bladder: and (2) human sperm and male infertility. Mutat Res 375:93–104. Morris ID, Ilott s, Dixon L, Brison DR (2002) The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development. Hum Reprod 17:990–998. Oliva R, Dixon GH (1991) Vertebrate protamine genes and the histone-to-protamine replacement reaction. Prog Nucleic Acid Res Mol Biol 40:25–94. Saleh R, Agarwal A, Nelson D, Nada E, El-Tonsy M, Alvarez JG, Thomas AJ, Sharma R (2002) Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study. Fertil Steril 78:313–318. Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E, Leter G (2002) Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team. Fertil Steril 73:43–50. Sun JG, Jurisicova A, Casper RF (1997) Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 56:602–607. The Practice Committee of the American Society for Reproductive Medicine (2006) The clinical utility of sperm DNA integrity testing. Fertil Steril 86(Suppl. 4):S–35. Tur-Kaspa I, Maor Y, Levran D, Yonish M, Mashiach S, Dor J (1994) How often should infertile men have intercourse to achieve conception? Fertil Steril 62:370–375. Wilton LJ, Temple-Smith PD, Baker HW, De Kretser DM (1988) Human male infertility caused by degeneration and death of sperm in the epididymis. Fertil Steril 49:1052–1058. ª 2008 The Authors
316
Journal Compilation ª 2008 Blackwell Publishing Ltd
Æ Andrologia 40, 312–317
T. M. Hussein et al.
Male infertility
World Health Organization (1999) WHO Manual for the Examination of Human Semen and Sperm–cervical Mucus Interaction. Cambridge University Press, Cambridge.
ª 2008 The Authors Journal Compilation ª 2008 Blackwell Publishing Ltd
Æ Andrologia 40, 312–317
Zini A, Kamal K, Phang D, Willis J, Jarvi K (2001) Biologic variability of sperm DNA denaturation in infertile men. Urology 58:258–261.
317
