The objective was to evaluate the relation between varicocele and sperm DNA fragmentation index (DFI) in infertile men. Materials and methods: A controlled observational study recruited 205 patients who sought medical consultation at Hanoi Medical University Hospital.
JOURNAL OF MEDICAL RESEARCH THE RELATION BETWEEN VARICOCELE AND SPERM DNA FRAGMENTATION INDEX IN INFERTILE MEN Nguyen Hoai Bac1, Nguyen Cao Thang1, Hoang Long2 Hanoi Medical University Hospital, 2Hanoi Medical University The objective was to evaluate the relation between varicocele and sperm DNA fragmentation index (DFI) in infertile men Materials and methods: A controlled observational study recruited 205 patients who sought medical consultation at Hanoi Medical University Hospital One-hundred and forty-two men were diagnosed with male infertility who had unilateral varicocele belonged to the varicocele group and 63 men with normal reproductive health belonged to the control group Results: The mean DFI was significantly higher in the varicocele group compared with the control group (31.8 ± 18.8% compared with 22.6 ± 10.6%,with p < 0.01) The statistical difference in the percentage of severe DFI ( ≥ 30%) between the two groups was recognized in this study (45.8% in the varicocele group compared with 20.6% in the control group, with p < 0.03) Some factors which were found to increase the level of DFI were age and infertility duration in patients with varicocele However, there was no difference in the level of DFI among the three groups of varicocele grade Conclusion: Varicocele has negative effects on sperm DFI Therefore, varicocele should be treated early in infertile men, disregarding the grade of varicocele Keywords: Varicocele, semen analysis, sperm DNA fragmentation, DFI I INTRODUCTION There are many causes leading to male infertility including reproductive tract infections, malignancies, endocrinal disorders, genetic disorders, hypogonadism, varicocele, genital abnormalities, etc Among them, varicocele is one of a few causes that can be treated Varicocele is found in 19 - 41% of primary infertile men and up to 45 - 84% in secondary infertile men [1] Varicocele is defined by the dilation of the pampiniform venous plexus, which is caused by the reflux of the blood flow from the left renal vein on the left side and from the inferior vena cava on the right side The venous stasis leads to many consequences that negatively affect the reproductive function of men [2] Recently, the sperm DNA fragmentation Corresponding author: Nguyen Hoai Bac, Hanoi Medical University Hospital Email: drbac.uro@gmail.com Received: 27/11/2018 Accepted: 12/03/2019 JMR 118 E4 (2) - 2019 index (DFI) has been used to evaluate the reproductive function of men in addition to conventional sperm analysis DFI provides more credibility because, unlike standard semen analysis, DFI exhibits low biological variability and has superior predictive value on reproductive outcomes [3] Varicocele has been proved to have associated with higher sperm DNA damage [4] The etiologies of sperm DNA damage are not completely understood but hypothesized to be the increased level of reactive oxygen species and a significant reduction in the level of DNA polymerase in seminal fluid The highly damaged sperm DNA negatively affect spontaneous pregnancy and the success rate of assisted reproductive technique[5] Many studies have assessed the correlation between varicocele and sperm DNA fragmentation index The majority of them were conducted on the European communities which have fundament differences in environment 15 JOURNAL OF MEDICAL RESEARCH compared with Asian communities in general and Vietnamese communities in particular In Vietnam, the researchers focused on evaluating the impact of varicocele on standard semen parameters Their studies commonly had limited sample size and no control group For the practical reasons mentioned above, the objective of this study was to analyze the effect of varicocele on sperm DNA fragmentation index II METHODS Time and location Our study was conducted from December 2013 to August 2018 at Hanoi Medical University Hospital Material and method This was a controlled observational study based on 205 men who had been examined at Hanoi Medical University hospital, in which 142 infertile men with varicocele belonged to the varicocele group and 63 men with normal reproductive health belonged to the control group Inclusion criteria of the varicocele group - Diagnosed with infertility according to the WHO manual 2010 - Diagnosed with unilateral varicocele confirmed on clinical examination and ultrasound - Had sufficient laboratory tests - Exclusion criteria of the varicocele group Had subclinical varicocele (grade 0) or had clinical varicocele with normal ultrasonography - Laboratory tests were not performed at Hanoi Medical University’s lab Inclusion criteria of the control group - Having good health determining via periodical examination - Their partner having pregnancy - Without varicocele by clinical examination and ultrasonography Variables Sperm DNA fragmentation index (DFI) is based on the evaluation of the SCD technique which was generated with Halosperm kit Data analysis Collected data was processed and analyzed by STATA 13 All results were expressed as the mean ± SD Difference between groups was estimated by the paired sample T - student test for normal distribution and Mann Whitney test for non-normal distribution All hypothesis testing was considered statistically significant if p < 0.05 III RESULTS General features of the patients Table General features of the varicocele group and the control group Variables Age (years) ≤ 30 > 30 16 Varicocele group (n = 142) n (%) (Mean ± SD) Control group (n = 63) n (%) (Mean ± SD) p 29.8 ± 5.4 28.9 ± 4.2 0.07 87 (61.2) 26.6 ± 2.7 55 (38.7) 34.9 ± 4.5 42 (66.7) 26.6 ± 2.6 21 (33.3) 33.6 ± 2.7 0.41 JMR 118 E4 (2) - 2019 JOURNAL OF MEDICAL RESEARCH Varicocele group (n = 142) n (%) (Mean ± SD) 142 21.9 ± 2.4 Variables BMI (kg/m2) Control group (n = 63) n (%) (Mean ± SD) 63 22.5 ± 2.5 p 0.07 Avg testicular 12.8 ± 3.8 13.7 ± 3.8 0.08 volume(ml) The mean age of the varicocele group and the control group were 29.8 ± 5.4 and 28.9 ± 4.2, respectively The mean BMI of the two groups were also similar The average testicular volume of the two groups was similar and within normal range Clinical findings of varicocele group Chart Clinical findings of the varicocele group Grade III varicocele occupied the highest proportion with 64.9%; grade I and grade II varicocele took over 18.2% and 16.9%, respectively The majority of patients had left side varicocele (98.6%), only two cases had right side varicocele (1.4%) Primary infertility occupied the major proportion with 73.7% Most men had duration of infertility under 24 months Measuring DFI of the varicocele group and the control group Table DFI of the varicocele group and the control group Variables DFI (%) Varicocelegroup Control group n (%) Mean ± SD n (%) Mean ± SD 142 31.8 ± 18.8 63 22.6 ± 10.6 JMR 118 E4 (2) - 2019 p 0.0002 17 JOURNAL OF MEDICAL RESEARCH Variables Varicocelegroup Control group Mean ± SD p n (%) Mean ± SD n (%) < 15 22 (15.5) 11.1 ± 2.3 16 (25.4) 11.8 ± 2.2 0.16 15 - 30 55 (38.7) 21.6 ± 4.2 34 (53.9) 21.3 ± 4.8 0.35 ≥ 30 65 (45.8) 47.4 ± 16.8 13 (20.6) 39.5 ± 6.4 0.05 The mean DFI of the varicoecele group was significantly higher than the controls (31.8% ± 18.8% and 22.6 ± 10.6%, respectively, with p = 0.0002) Chart Ratio of DFI in both groups In the varicocele group, the ratio of severely damaged DFI was the highest at 45.8% This ratio was only 20.6% in the control group Correlation of some clinical factors and DFI Table Correlation of DFI and some clinical facors Varicocele group Variables n (%) DFI (Mean ± SD) 10 (7) 27.4 ± 18.5 p BMI (kg/m2) < 18,5 18 JMR 118 E4 (2) - 2019 JOURNAL OF MEDICAL RESEARCH Varicocele group Variables n (%) DFI (Mean ± SD) p 18,5 - 22,9 81 (57) 34.7 ± 20.5 0.09 > 22,9 51 (36) 28.1 ± 15.3 ≤ 30 87 (61.3) 29.3 ± 15.9 > 30 55 (38.7) 35.7 ± 22.3 Grade I 24 (16.9) 35.2 ± 21.0 Grade II 25 (17.6) 31.7 ± 16.3 Grade III 93 (65.5) 30.9 ± 19.0 Primary infertility 105 (74) 31.2 ± 18.3 Secondary infertility 37 (26) 33.4 ± 20.5 ≤ 24 108(76) 30.1 ± 16.4 > 24 34(24) 37.1 ± 24.6 Age (year) 0.02 Varicocele grade 0.49 Type of infertility 0.27 Duration of infertility (month) 0.03 Age and duration of infertility are two risk factors that can affect DFI in varicocele men with p = 0.03 and p = 0.02, respectively IV DISCUSSION This study involved 142 infertile men with clinical varicocele with the mean age of 29.9 ± 5.5 and 63 men of the control group with the mean age of 28.8 ± 4.3 (Table 1) It was appropriate to determine the effect of varicocele on semen quality due to the fact that the majority of patients were in reproductive age Previous authors also studied a group of patients similar to this study (Abdel-Meguid et al and Ghazi et al) [6, 7] The mean BMI of the varicocele group and the control group were also identical (Table 1) This was the basis to assess the impact of varicocele on the JMR 118 E4 (2) - 2019 sperm DNA integrity on the varicocele group compared with normal fertile men In this study, 140 of 142 patients (98.6%) had left side varicocele and the two patients left had right side (Chart 1) As the spermatic veins dilated, the obstruction of the blood flow hinders the heat-exchange process of the pampiniform plexus and consequently leads to discomfort or pain in the scrotum and testicular over-heat Furthermore, an increased hydrostatic pressure in the spermatic veins obstructs the arterial blood flow of the testis and restricts 19 JOURNAL OF MEDICAL RESEARCH normal testicular growth In our study, however, the testicular volume of the affected group was 12.8 ± 3.8 ml, similar to the control group which was 13.7 ± 3.7 ml (Table 1) These results were also observed in a study by Mohammed et al which was 12.7±1.02 ml [8] The percentage of grade I, grade II and grade III varicocele were 18.3%, 17.6% and 64.1 %, respectively The majority of varicocele patients were primary infertile (73.7%) (Chart 1) Recent evidence have shown that only clinical varicocele would have a negative effect on male reproductive health [9] The deleterious effects of varicocele are manifested in the decline in quality and the quantity of sperm Conventional semen analysis is generally used to evaluate male fertility However, these parameters exhibit many limitations such as high degree of biological variability and can be affected by the patient’s duration of sexual abstinence and mental state In recent years, with the development of genetics and molecular biology, sperm DNA damage has been evaluated and considered a promising biomarker for infertility Sperm DNA fragmentation index (DFI) has been proved to be superior to the conventional semen analysis that it exhibits a lower degree of biological variability and has more predictive value in pregnancy rate [4] When DFI < 15%, the sperm has a low level of DNA damage, the couple can achieve spontaneous pregnancy The fertilization capacity of sperm significantly decreases when DFI reaches 15 - 30% DFI higher than 30% results in poor pregnancy rate even with assisted reproductive technique [10] The correlation between increased sperm DNA fragmentation and varicocele has been proved in many previous studies In a 20 prospective controlled trial on infertile men with varicocele, there was a considerable elevation in sperm mean DFI in the varicocele group compared with the control group (25% in varicocele group compared with 15% in control group), the difference was statistically significant [5] In another meta-analysis which was conducted to evaluate the alteration of sperm DNA damage in varicocele patients and to determine the efficacy of varicocelectomy, Wang et al have shown that the sperm DNA damage is significantly higher in varicocele patient and the mean difference was 9.84% (95% CI; 9.19 - 10.49; p < 0.00001) [11] In our study, the mean DFI of the varicocele patients was remarkably higher comparing with the control group (32.8 ± 19.2% and 21.4 ± 8.7% with p = 0.001) (Table 2) When sperm DNA damage is categorized into severe, moderate and mild degree, it was also apparent that the proportion of severely damaged sperm DNA in varicocele group was higher than the control group (45.8% compared with 20.6%, p = 0.003) (Chart 2) Our results once again confirmed the negative effect of varicocele on the integrity of sperm DNA Besides the direct effect of varicocele, some risk factors which contribute to the damage of sperm DNA are age, BMI, grade of varicocele, duration of infertility, etc [12] In our study, the level of DNA fragmentation had a relation with age and the duration of infertility Men aged over 30 or men with duration of infertility above 24 months had increased level of DFI (p = 0.02 with age and p = 0.03 with duration of infertility) However, there was no correlation between DFI and BMI or different varicocele grades These results indicated that varicocele could damage sperm DNA even in early grade and the damage was JMR 118 E4 (2) - 2019 JOURNAL OF MEDICAL RESEARCH more severe in elder men or men with longer duration of infertility Therefore, in clinical practices, it is important to advise patients with varicocele to have early intervention which would prevent further adverse effects V CONCLUSIONS Varicocele has a negative effect on sperm quality which is demonstrated by the increase in sperm DNA fragmentation index Infertile patients with varicocele need to have early intervention even with low grade varicocele to prevent that negative effect REFERENCES Mohammed, A and F Chinegwundoh (2009), Testicular varicocele: an overview Urol Int, 82(4), 373 - 379 Ficarra, V., et al (2012), Varicocele repair for infertility: what is the evidence? Curr Opin Urol, 22(6), 489 - 494 Agarwal, A., et al (2007), Efficacy of Varicocelectomy in Improving Semen Parameters: New Meta-analytical Approach Urology, 70(3), 532 - 538 Zini, A., et al (2011), Effect of microsurgical varicocelectomy on human sperm chromatin and DNA integrity: a prospective trial Int J Androl, 34(1), 14 - 19 Saleh, R.A., et al (2003), Evaluation of nuclear DNA damage in spermatozoa from infertile men with varicocele Fertil Steril, 80(6), 1431 - 1436 Abdel-Meguid, T.A., et al (2011), Does varicocele repair improve male infertility? JMR 118 E4 (2) - 2019 An evidence-based perspective from a randomized, controlled trial Eur Urol, 59(3), 455 - 461 Ghazi, S and H Abdelfattah (2011), Effect of microsurgical varicocelectomy on sperm DNA fragmentation in infertile men Middle East Fertility Society Journal, 16(2), 149 -153 Mohamed, E.E., M Gawish, and A Mohamed (2017), Semen parameters and pregnancy rates after microsurgical varicocelectomy in primary versus secondary infertile men Hum Fertil (Camb), 20(4), 293 296 Practice Committee of the American Society for Reproductive, M., R (2014), Society for Male, and Urology, Report on varicocele and infertility: a committee opinion Fertil Steril, 102(6), 1556 - 1560 10 Simon, L., et al (2010), Clinical significance of sperm DNA damage in assisted reproduction outcome Hum Reprod, 25(7), 1594 - 1608 11 Wang, Y.J., et al (2012), Relationship between varicocele and sperm DNA damage and the effect of varicocele repair: a metaanalysis Reprod Biomed Online, 25(3), 307 314 12 Al Omrani, B., et al (2018), Associations of sperm DNA fragmentation with lifestyle factors and semen parameters of Saudi men and its impact on ICSI outcome Reprod Biol Endocrinol, 16(1), 49 21 ... examination - Their partner having pregnancy - Without varicocele by clinical examination and ultrasonography Variables Sperm DNA fragmentation index (DFI) is based on the evaluation of the SCD... effect on sperm quality which is demonstrated by the increase in sperm DNA fragmentation index Infertile patients with varicocele need to have early intervention even with low grade varicocele. .. 1) As the spermatic veins dilated, the obstruction of the blood flow hinders the heat-exchange process of the pampiniform plexus and consequently leads to discomfort or pain in the scrotum and