Computer-assisted semen analysis parameters in men with varicocele: is surgery helpful?

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1 FERTILITY AND STERILITY Copyright 1996 American Society for Reproductive Medicine Vol. 66, No, 3, September 1996 Printed on acid-free paper in U. S. A. Computer-assisted semen analysis parameters in men with varicocele: is surgery helpful? Firuza R. Parikh, M,D, *t Sudesh A Kamat, M,Sc, * Geeta G, Kodwaney, RSc, * Donta Balaiah, M'sc,t Jaslok Hospital and Research Centre, and Institute for Research in Reproduction, Bombay, India Objective: To assess sperm characteristics and fertility before and after varicocelectomy using computer-assisted semen analysis (CASA). Design: Preoperative and postoperative sperm parameters of infertile men with varicocele were analyzed statistically and the outcome of pregnancy was determined. Setting: Department of Infertility Management and Assisted Reproduction, J aslok Hospital and Research Centre, Bombay. Patients: Semen samples of 49 men with varicocele-related infertility were analyzed using CASA Interventions: Sperm parameters of 26 men who underwent varicocelectomy were evaluated 3, 6, and 9 months postoperatively. Conception, occurring either naturally or therapeutically, was recorded. Main Outcome Measures: Evaluation of improvement of sperm parameters after surgery. Results: Sperm count, motility parameters, curvilinear velocity, straightline velocity, lateral head displacement, and normal morphology were significantly lower in men with varicocele. Postoperatively, there was significant improvement in count, motility, and normal morphology, with a decrease in proportion of acrosome-deficient heads and tapering forms. Mter varicocelectomy, 46.2 of the men had normal semen parameters, with the overall pregnancy rate being 50. Conclusions: Computer-assisted semen analysis provides the potential for accurate quantitative evaluation of semen in men with varicocele. Varicocelectomy results in improvement in semen quality with pregnancy rates of 50. Fertil Steril 1996;66:440-5 Key Words: Varicocele, CASA, infertility Received June 19, 1995; revised and accepted April 18, * Department of Infertility Management and Assisted Reproduction, Jaslok Hospital and Research Centre. t Reprint requests: Firuza R. Parikh, M.D., Department of Infertility Management and Assisted Reproduction, J aslok Hospital and Research Centre, 15, Dr. G. Deshmukh Marg, Bombay , India (FAX: ). t Institute for Research in Reproduction. 440 Parikh et al. CASA studies in varicocele The role of varicocele in male infertility has been the subject of much debate. In the normal male population, the incidence of varicocele is 15 to 20; however, its prevalence among infertile males is 40 (1, 2). The pathophysiological mechanism by which it exerts its effect on spermatogenesis is not yet completely understood. There is substantial evidence that varicocele impairs spermatogenesis and, consequently, semen quality (3), although, for reasons not completely understood, this detrimental effect is observed only in approximately 50 of patients with varicocele (4). Many infertile men with varicocele manifest low values for sperm density, motility, and! or normal morphology in their semen analysis (2). Some have abnormal results with the sperm penetration assay (5). Varicocelectomy has been reported to improve semen quality in 60 to 80 of infertile men with varicocele, with pregnancy rates of 30 to 55 (6). However, there are some patients who remain infertile despite varicocele surgery. Also, there are reports that varicocele treatment brings neither improvement of sperm quality nor pregnancy (7, 8). In view of the contradictory results found in the Fertility and Sterility

2 literature, the present study was carried out to evaluate the relationship of varicocele to semen quality and fertility and to ascertain the usefulness ofvaricocelectomy in infertile men with varicocele. Computer-assisted semen analysis (CASA) of preoperative and postoperative semen parameters (3, 6, and 9 months) was carried out, using the HTM-C Motility Analyzer (Hamilton Thorn Research, Danvers, MA), to judge the effectiveness of CASA in the characterization of semen abnormalities associated with varicocele and also to judge objectively improvement or otherwise in semen parameters. To the best of our knowledge the analysis of various sperm motion parameters by CASA in men before and after varicocele surgery so far has not been reported in the literature. Patients MATERIALS AND METHODS Forty-nine patients with varicocele-associated male infertility (27 bilateral, 22 unilateral) were compared with 50 normal control subjects. Normal controls were men who demonstrated >50 fertilization in vitro and who did not have a male factor where IVF was performed for female factor. Twentysix men with varicocele underwent varicocelectomy and were advised semen evaluation at 3, 6, and 9 months postoperatively. However, in 6 of these patients, 3- and 6-month follow-up data of sperm parameters was not available. Men who had additional pathologies that might have interfered with fertility were not included in this study. All men had normal FSH values. The fertility of the female partners was evaluated carefully and they were found to be potentially fertile. The standard protocol used to assess the fertility potential of the female partners included endocrine assessment of day 3 FSH, LH, thyroidstimulating hormone, and PRL and luteal phase P, tubal patency either by hysterosalpingogram or laparoscopy as indicated, sonographic evaluation of pelvic structures, and ovulatory profile and general physical examination. Men whose partners were found to have any reproductive pathology were not included in this study. Varicocele Diagnosis Varicocele was diagnosed by physical palpation in standing position and during valsalva maneuver, and also by color Doppler ultrasonography. The size of the varicocele was classified as large (grade III), moderate (grade II), or small (grade I) (9). Semen Analysis with HTM-C Motility Analyzer Semen quality was assessed preoperatively and at 3, 6, and 9 months postoperatively. Semen quality Vol. 66, No.3, September 1996 was checked every 3 months after varicocelectomy for ~9 months and there was 12 months follow up of fecundity. Semen samples were obtained by masturbation after 3 days of sexual abstinence. A 5-J.lL aliquot of semen sample was placed in a 10-J.lm deep Makler chamber (Sefi Medical Instruments, Haifa, Israel) and the sperm concentration and motility parameters were analyzed with the HTM-C Motility Analyzer. Sperm Morphology Smears of semen samples were stained by the Papanicolaou technique. At least 100 sperm were examined under the phase-contrast microscope. The World Health Organization (WHO) guidelines (10) were used for classification, but additional emphasis was given to acrosome adequacy. Sperm having regular oval-shaped heads and intact acrosomes, covering ~40 to 70 of the head, without midpiece or tail defects were considered normal whereas those fulfilling the above criteria, but having <40 acrosome covering were considered acrosome deficient. Sperm with abnormal shape or size of head or midpiece or tail defects were considered abnormal. Varicocelectomy Varicocelectomy was performed by the urologist. The approach was inguinal and veins were ligated bilaterally in all men, as it is our clinical experience that unilateral ligation very often is followed by the detection of varicocele in the contralateral side within a short span of time. Statistical Analysis The statistical analysis was carried out using the Student's t-test. P values of :50.05 were considered significant. RESULTS Of26 men who underwent varicocelectomy, 17 had bilateral and 9 had unilateral varicocele. The mean age of these patients was 33 years (range, 26 to 36 years). The duration of infertility ranged from 3 to 5 years. The preoperative values of different sperm parameters in men with unilateral (n = 22) and bilateral (n = 27) varicocele compared with controls (n = 50) is depicted in Tables 1 and 2. The mean sperm count, motility, path velocity, progressive motility, type A motility, curvilinear velocity (VCL), straightline velocity (VSL), and amplitude oflateral head displacement (ALH) were significantly lower (P < 0.05) in patients with varicocele (both unilateral as well as bilateral) compared with normal controls. The beat Parikh et a1. CASA studies in varicocele 441

3 Table 1 Sperm Count and Motility Parameters in Semen Samples of Patients With Unilateral and Bilateral Varicocele Compared With Normal Controls* Normal controls Unilateral Bilateral Variables (n = 50) (n = 22) (n = 27) Count ± ± 3.3t 20.9 ± 3.8t Motility 76.6 ± ± 4.2t 27.0 ± 3.9t Path velocity 41.5 ± ± 1.2t 26.6 ± l.4t Progressive motility 26.3 ± ± 2.0t 7.6 ± 1.9t Type A motility 58.7 ± ± 3.H 15.3 ± 2.9t Curvilinear velocity 54.9 ± ± 2.H 35.5 ± 1.9t Straight-line velocity 29.4 ± ± 1.H 19.3 ± LIt Linearity 55.5 ± ± ± 2.6 Straightness 69.9 ± ± ± 3.0 Amplitude of lateral head displacement 4.3 ± ± 0.2t 2.5 ± 0.2t Beat cross frequency 6.0 ± ± 0.5t 5.3 ± 0.5 t p < 0.05 compared with normal controls. cross frequency was significantly lower in patients with unilateral varicocele (P < 0.05), whereas there was no statistically significant difference in the linearity and straightness values between patient and control group. In patients with varicocele, the normal sperm morphology was significantly lower (P < 0.05) compared with normal controls, with a corresponding increase in head abnormalities such as acrosome deficiency, round head, tapering forms, and amorphous head (P < 0.05). Conjoined and small head abnormalities occurred in significant proportions (P < 0.05) of patients with unilateral varicocele only. The comparison of preoperative and postoperative (3, 6, and 9 month) sperm parameters in those patients who underwent varicocelectomy is shown in Tables 3 and 4. There was a significant increase in sperm count after 3 months (P < 0.05) and 6 and 9 months (P < 0.01) postoperatively. Motility also was found to improve significantly after 6 months (P < 0.01), reaching a peak after 9 months (P < 0.001). The path velocity and type A motility increased significantly after 6 and 9 months of surgery (P < 0.01). Progressive motility also improved significantly after 9 months (P < 0.01). Straightline velocity (P < 0.01) and VCL values (P < 0.05) increased significantly after 9 months. There was a significant difference in the linearity values after 6 months (P < 0.05). Straightness, ALH, and beat cross frequency did not differ significantly preoperatively and postoperatively. Normal sperm morphology was found to improve significantly (P < 0.001) after varicocelectomy, reaching normal levels (>20). The proportion of 442 Parikh et al. CASA studies in varicocele acrosome-deficient heads and tapering forms decreased significantly after 9 months (P < 0.05). Preoperatively, oligospermia «20 X 1Q 6 /ml) was encountered in 65.4 (17/26) of men with varicocele, asthenospermia «50 motility, <25 type A) was found in 76.9 (20/26), and teratospermia «20 normal) was found in 65.4 (17/26). Postoperatively, after 9 months of follow up, oligospermia was present in only 42.3 (11/26) of the men, asthenospermia was present in 34.6 (9/26), and teratospermia was present in 26.9 (7/26) only. Preoperatively, seven out of twenty-six patients (26.9) had only a single sperm defect (either oligospermia or asthenospermia or teratospermia), 26.9 (7/26) had double sperm defect, and 42.3 (11/26) had all three sperm defects. Preoperatively, one patient had normal sperm parameters despite varicocele and had infertility. Postoperatively, single, double, and triple sperm defects were seen in only 15.4 (4/26), 26.9 (7/26), and 11.5 (3/26), respectively. Nine months after varicocelectomy, 46.2 (12/26) of patients had normal sperm parameters. During the follow-up period of 1 year, 13 pregnancies were achieved, of which 10 delivered and 3 aborted. Ofthe 13 pregnancies, 7 patients conceived naturally, 5 conceived with the sperm processing and lui procedures, and 1 conceived with IVF. The pregnancy rate was 53 (9/17) for patients with bilateral varicocele and 44.5 (4/9) for unilateral varicocele. The overall pregnancy rate was 50. We note that the seven couples who conceived naturally had attempted luis before varicocele surgery and none of the women had conceived. Table 2 Sperm Morphological Characteristics in Semen Samples of Patients With Unilateral and Bilateral Varicocele as Compared With Normal Controls* Variables Normal forms Acrosome-deficient forms Round head Tapering head Amorphous forms Small head Large head Conjoined head Pin head Pyriform Postacrosomal elongation Acrosome-absent head Total head defects Normal controls Unilateral Bilateral (n = 50) (n = 22) (n = 27) 44.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.7t 20.0 ± 1.5t 10.9 ± 1.5t 8.2 ± 1.5t 4.5 ± 0.9t 5.3 ± 1.2t 3.9 ± ± O.4t 1.4 ± ± ± ± ± 2.6t t p < 0.05 compared with normal controls ± 1.8t 18.0 ± 1.6t 10.3 ± 1.6t 6.6 ± 1.3t 3.5 ± 0.6t 3.9 ± ± ± ± ± ± ± ± 2.7t Fertility and Sterility

4 Table 3 Sperm Count and Motility Parameters of Semem Samples of Infertile Men With Varicocele, Preoperatively and Postoperatively (3, 6, and 9 Months)* Postoperative Variables Preoperative (n = 26) 3 Months (n = 20) 6 Months (n = 20) 9 Months (n = 26) Count Motility Path velocity Progressive motility Type A motility Curvilinear velocity Straightline velocity Linearity Straightness Amplitude of lateral displacement Beat cross frequency 17.4 ± ± ± ± ± ± ± ± ± ± ± ± 5.4t 33.6 ± ± ± ± ± ± 1.6t 56.8 ± ± ± ± ± 6.0:1: 47.2 ± 6.6:1: 32.0 ± 1.8:1: 14.8 ± ± 5.0:1: 43.3 ± ± 1.2t 57.5 ± 2.2t 75.0 ± ± ± ± 6.4:1: 51.8 ± ± 1.3:1: 17.4 ± 2.7:1: 32.8 ± 4.3:1: 42.9 ± 1.8t 24.1 ± 1.0:1: 56.9 ± 1.3t 75.3 ± ± ± 0.4 t p < 0.05 compared with preoperative sperm values. :I: P < 0.01 compared with preoperative sperm values. P < compared with preoperative sperm values. DISCUSSION Male factor disorders account for almost 50 of infertile couples. The presence of a varicocele is one of the most readily identifiable causes of male subfertility. Varicocele generally is associated with reduced sperm count, motility, and normal morphology, often in combination. Numerous studies have been carried out to establish the relationship between varicocele and male infertility and to understand the mechanism by which it exerts its detrimental effect on sperm quality. Although varicocele is observed in approximately 40 of infertile men, it also is observed in 15 of normal males who father children. The WHO study (3) clearly identifies varicocele as an important identifiable cause of male infertility. Patients with varicocele may have other factors, such as antisperm antibodies or hormonal imbalance, which also may contribute to infertility. In our study, strict exclusion criteria were followed so that only men with varicocele-associated infertility and no other contributing factors were included, thus accounting for their small number. The preoperative and postoperative semen samples were analyzed, without prior knowledge, to avoid any observation bias. The use of the Hamilton Thorn motility analyzer allowed determination of the various sperm motility parameters such as path velocity, VeL, and VSL not studied previously. Infertile men with varicocele were found to have significantly lower sperm count Table 4 Sperm Morphological Characteristics of Semen Samples of Infertile Men With Varicocele, Preoperatively and Postoperatively (3, 6, and 9 Months)* Postoperative Variables Normal forms Acrosome-deficient head Round head Tapering head Amorphous forms Small head Large head Conjoined head Pin head Pyriform Postacrosomal elongation Acrosome-absent head Total head defects Preoperative (n = 26) 17.3 ± ± ± ± ± ± ± ± ± ± ± ± ± Months 6 Months 9 Months (n = 20) (n = 20) (n = 26) 19.8 ± ± ± l.4t 18.4 ± ± ± 1.7:1: 8.0 ± ± ± ± ± ± 0.8:1: 2.9 ± ± ± ± ± ± l.lt 4.6 ± ± ± ± ± ± ± 0.2:1: 1.0 ± ± ± ± ± ± ± ± ± 1.8t 52.5 ± ± ± ± ± 2.5 t P < 0.01 compared with preoperative sperm values. Vol. 66, No.3, September 1996 :I: P < 0.05 compared with preoperative sperm values. Parikh et al. CASA studies in varicocele 443

5 (P < 0.05) and motility (P < 0.05) compared with normal controls. Also, other sperm motion parameters such as path velocity, progressive motility, type A motility, VCL, VSL, and ALH were significantly lower in the patient group (P < 0.05). Various hypotheses have been put forward to explain the cause of infertility in men with varicocele. Most plausible among these, are elevation of scrotal temperature (11,12) and reflux of toxic metabolites from the renal and/or adrenal vein (13, 14). Higher incidence of varicocele has been reported in male factor secondary infertility compared with primary infertility (15, 16). Mcleod (17) described a stress pattern in semen samples of men with varicocele wherein there was an increased incidence of tapering heads and amorphous and immature sperm cells in semen. Although some investigators (18) could confirm Mcleod's (17) findings, others (19) could not and found no increase in number of tapering forms compared with men without varicocele. In our study, both men with unilateral as well as bilateral varicocele had significantly lower normal forms (P < 0.05) compared with controls. There was a significant increase in sperm head abnormalities such as acrosome-deficient forms, round head, tapering head, and amorphous forms (P < 0.05). Of 26 men who underwent varicocelectomy, 65.4 had oligospermia, 76.9 had asthenospermia, and 65.4 had teratospermia. Single, double, and triple sperm defects (oligospermia, asthenospermia, teratospermia) were present in 26.9, 26.9, and 42.3 ofthe patients, respectively. Only one patient had all three sperm parameters normal. Our results support the concept that varicocele is responsible for decrease in sperm quality, thereby affecting fertility in men with varicocele. There are varying reports as to whether surgical correction of the varicocele is helpful therapeutically. Although a study have shown improvement in both the semen quality as well as pregnancy rates after varicocelectomy (6), others have found no difference between prevaricocelectomy and postvaricocelectomy sperm parameters nor an improvement in pregnancy rates after surgery (7, 8). Varicocelectomy has been reported to improve semen quality in 60 to 80 ofthe patients with pregnancy rates of30 to 55 (6). Improvement in sperm parameters is seen within 6 to 18 months after surgery. In our study, the duration of infertility of these patients ranged from 3 to 5 years, during which time no conception occurred either naturally or with treatment. Also no significant change was seen in semen parameters during that period. Postoperatively, there was a significant improvement in sperm count after 6 months (P < 0.01). Sperm counts were found to increase in most pa- 444 Parikh et al. CASA studies in varicocele tients after surgical treatment (20, 21). The effect seemed more pronounced when initial sperm densities were> 10 X 106/mL. Motility also was found to improve significantly after 9 months (P < 0.001). Improvement in sperm count and motility has been reported previously by others (21). Significant improvement in the path velocity (P < 0.01), progressive motility (P < 0.01), type A motility (P < 0.01), VCL (P < 0.05), VSL (P < 0.01), and linearity (P < 0.05) of the sperm was observed 9 months postoperatively. Curvilinear velocity has been reported to be an useful predictor of male fertility (22). In IVF, VCL has been observed to be significantly higher in the fertilization group than in the group having failed fertilization (23). Hyperactivated motility is marked by increased curvature in swimming trajectories. The high VCL values observed postoperatively are indicative of efficient sinuous, pliant motion, which can render more efficient interaction of sperm with the oocytes. The high VSL and VCL values observed are consistent with the concept that sperm moves in a forward direction but in an oscillating path. Morphology, including acrosome status, of normal sperm is an important predictor of fertilizing potential. When the normal forms were <20, there was complete failure offertilization under in vitro conditions (23). Hence, 20 normal forms has been taken as the cutofflevel for normal sperm morphology. Improvement in sperm morphology, after varicocelectomy, has been reported by some authors (20). Others have found no difference in preoperative and postoperative sperm morphology (24). In our study, normal forms of the spermatozoa were found to improve significantly (P < 0.01) postoperatively, after 9 months, reaching normal level. There was a significant reduction in the number of acrosome-deficient heads and tapering forms. Only small heads were found to have increased in number. Postoperatively, the proportion of men with oligospermia (42.3), asthenospermia (34.6), and teratospermia (26.9) also was reduced. Single, double, and triple sperm defects were present in only 15.4, 26.9, and 11.5, respectively. Nine months after surgery, 46.2 of the patients had normal sperm parameters. In our study, the overall pregnancy rate was 50. A high pregnancy rate was seen in patients with either unilateral (44.5) or bilateral (53) varicocele. Our results suggest that varicocelectomy is useful for men with only varicocele-associated infertility. In conclusion, varicocele affects sperm quality, thereby affecting male fertility, in the majority of men with varicocele-associated infertility. Varicocelectomy is the treatment of choice for these men, re- Fertility and Sterility

6 suiting in significant improvement in sperm quality and fertilizing potential and corresponding improvement in pregnancy rates in approximately 50 ofthe patients. Computer-assisted semen analysis allows accurate quantitative evaluation of semen samples before and after varicocelectomy, thus providing a better insight of the various sperm motion characteristics in men with varicocele-associated infertility. REFERENCES 1. Greenberg SH. Varicocele and male fertility. Fertil Steril 1977;28: Dubin L, Amelar RD. Etiologic factors in 1,294 consecutive cases of male infertility. Fertil Steril 1971;22: World Health Organization. The influence of varicocele on parameters of fertility in a large group of men presenting to infertility clinics. Fertil Steril 1992;57: Verstoppen GR, Steeno OP. Varicocele and the pathogenesis of the associated sub-fertility. A review of the various theories. II. Results of surgery. Andrologia 1977;9: Rogers BJ, Mygatt GG, Soderdahl DW, Hale RW. Monitoring of suspected infertile men with varicocele by the sperm penetration assay. Fertil Steril 1985;44: Hanley HG, Harrison RG. The nature and surgical treatment of varicocele. Br J Surg 1962;50: Baker H, Burger H, De Kretser D, Hudson B, Rennie G, Straffon W. Testicular vein ligation and fertility in men with varicocele. Br Med J 1985;291: Vermeulen A, Vandeweghe M. Improved fertility after varicocele correction: fact or fiction. Fertil Steril 1984;42: Dhabuwala CB, Parulkar BJ. Doppler flow analysis and conventional ultrasonography for evaluation of the infertile male. In: Jaffe R, Pierson RA, Abramowicz JS, editors. Imaging in infertility and reproductive endocrinology. Philadelphia: Lippincott, 1994: World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 2nd ed. Cambridge: The Press Syndicate of the University of Cambridge, Zorgniotti AW, Macleod J. Studies in the temperature, hu- man semen quality and varicocele. Fertil Steril1974;24: Yamaguchi M, Sakatoku J, Takihara H. The application of intrascrotal deep body temperature measurement for the non invasive diagnosis of varicocele. Fertil Steril 1989;52: Comhaire F, Vermeulen A. Varicocele sterility: cortisol and catecholamines. Fertil Steril 1974;25: Ito H, Fuse H, Minagawa H, Kawamura K, Murakami M, Shimazaki J. Internal spermatic vein prostaglandins in varicocele patients. Fertil Steril1982;37: Gorelick JI, Goldstein M. Loss offertility in men with varicocele. Fertil Steril 1993;59: Witt MA, Lipshultz LI. Varicocele: a progressive or static lesion? Urology 1993;42: Macleod J. Seminal cytology in the presence of varicocele. Fertil Steril 1965; 16: Naftulin BN, Samuels SJ, Hellstrom WJG, Lewis EL, Overstreet JW. Semen quality in varicocele patients is characterized by tapered sperm cells. Fertil Steril 1991;56: Jecht EW, Muller R, Zieglwalner E. Varicocele and seminal cytology. In: Jecht EW, Zeilter E, editors. Varicocele and male infertility. Recent advances in diagnosis and therapy. New York: Springer-Verlag, 1982: Dhabuwala CB, Hamid S, Moghissi KS. Clinical versus subclinical varicocele: improvement in fertility after varicocelectomy. Fertil Steril 1992;57: Nieschlag E, Behre HM, Schlingheider A, Nashan D, Pohl J, Fischedick AR. Surgical ligation versus angiographic embolization of the vena spermatica: a prospective randomized study for the treatment of varicocele related infertility. Andrologia 1993;25: Holt WV, Moore HD, Hillier SG. Computer-assisted semen measurement of sperm swimming speed in human semen: correlation of results with in vitro fertilization assays. Fertil Steril 1985;44: Kamat SA, Kodwaney G, Balaiah D, Joshi N, Parikh F. Importance of computer assisted semen analysis and sperm function in IVF. Mol Androl 1995;7: Nilsson S, Edvinsson A, Nilsson B. Improvement of semen and pregnancy rate after ligation and division of the internal spermatic vein: fact or fiction? Br J Urol 1979;51: Vol. 66, No.3, September 1996 Parikh et al. CASA studies in varicocele 445