Infertility Pregnancy After Varicocelectomy: Impact of Postoperative Motility and DFI

Infertility Pregnancy After Varicocelectomy: Impact of Postoperative Motility and DFI Karen Baker, John McGill, Rakesh Sharma, Ashok Agarwal, and Edmund Sabanegh, Jr. OBJECTIVE METHODS RESULTS CONCLUSION To examine clinical and laboratory variables to determine which, if any, predict improved semen parameters and after varicocelectomy. Surgical logs were reviewed for men who underwent varicocele ligation for a fertility concern. Clinical, demographic, and laboratory data was obtained through medical chart review and paired with postoperative reproductive outcomes obtained through chart review and survey. Eighty-three subjects met inclusion criteria of which 63 and 24 completed both preoperative and postoperative basic and advanced semen testing, respectively. There was a statistically significant improvement in semen concentration, total motile count, sperm DNA fragmentation index (DFI), and total normal sperm count (TNSC) after varicocelectomy. Motility improved significantly in patients with preoperative asthenospermia. Concentration and motility demonstrated mean increases of 5.2 M/mL and 18%, respectively. DFI decreased from a preoperative mean of 40.8% to a postoperative mean of 24.5%. Fifty-one percent of couples were able to conceive using natural conception, intrauterine insemination, or in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). Pregnancy, regardless of the method used to conceive, was associated with a higher postoperative motility. The mean postoperative DFI in couples who reported a spontaneous was 34%, which was statistically higher than the mean DFI of 17.5% in couples who reported a with IVF/ICSI (P ¼.04). Varicocele ligation improves multiple semen parameters. An increase in motility was the only variable associated with postoperative irrespective of the method by which was obtained. Mean DFI in couples able to achieve spontaneous was above 30% and statistically higher than couples who reported via IVF/ICSI. UROLOGY 81: 760e766, 2013. Published by Elsevier Inc. Controversy remains over which patients benefit from varicocele ligation and where surgery fits into the treatment paradigm of the infertile couple. Although some meta-analyses have confirmed a correlation between varicocele ligation and improvement in semen parameters and rates, it is difficult to identify which couples will benefit from surgery. 1-3 Body mass index (BMI), testis volume, varicocele grade, and hormone levels have all been suggested to predict success; however, there is no consensus on which parameters portend an improvement in fecundity after varicocele ligation. Given the high prevalence of varicoceles in subfertile men and the increasing use of in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI), predicting success is an important issue for couples. 4 The mechanisms of infertility in men with varicoceles are likely multifactorial. 4,5 Varicoceles are associated with Financial Disclosure: The authors declare that they have no relevant financial interests. From the Department of Urology, Center for Male Fertility, Glickman Urological and Kidney Institute, The Cleveland Clinic, Cleveland, OH; and Urology Institute, University Hospitals - Case Medical Center, Cleveland, OH Reprint requests: Karen Baker, M.D., Department of Urology, Center for Male Fertility, Glickman Urological and Kidney Institute, The Cleveland Clinic, Cleveland, OH 44195. E-mail: bakerk8@ccf.org Submitted: October 18, 2012, accepted (with revisions): December 4, 2012 increased oxidative stress in the male reproductive system, which is manifest by high seminal reactive oxygen species (ROS) and decreased antioxidant capacity. Oxidative stress has been linked to sperm DNA damage, which is associated with poor rates with both natural conception and assisted reproductive techniques. The sperm DNA fragmentation index (DFI) is a promising parameter for fertility investigation because the biologic variation in sperm DNA damage is less than traditional semen parameters, 6,7 the results of sperm DNA assays are highly reproducible 7,8 and emerging evidence links varicocele ligation to decreased DFI and improved rates. 9,10 The purpose of our study was to identify preoperative parameters that predict improvement in postoperative semen values and variables that predict after subinguinal microsurgical varicocelectomy. MATERIALS AND METHODS Patient Selection Our retrospective analysis included men who sought consultation for infertility and underwent subinguinal microsurgical varicocelectomy at our institution from October 2008 through October 2011. The study was approved by the Institutional 760 Published by Elsevier Inc. 0090-4295/12/$36.00 http://dx.doi.org/10.1016/j.urology.2012.12.005

Review Board. Exclusion criteria were as follows: patients younger than 18 years, pain as the sole indication for surgery, surgery for recurrent varicoceles, and patients taking concurrent medication for infertility. Varicoceles had to be apparent on physical examination and were graded in accordance with current convention of grade 1 through 3. Demographic, clinical, laboratory, radiographic, and operative data were extracted from the electronic records. Data regarding postoperative and use of assisted reproductive techniques were gathered via chart review, mailed survey, or telephone interview. Specimen Collection and Standard Semen Parameters Semen specimens were collected by masturbation and 5 ml aliquots loaded on 20 ml MicroCell chambers (Vitrolife, San Diego, CA). Manual semen analysis was performed for sperm concentration, total sperm count, percent motility, viability, and Kruger/strict morphology by 4 dedicated technicians using World Health Organization (WHO), 4th edition, methodologies. Normal values herein are based on the WHO 2010 reference limits. 11 Measurement of ROS ROS was measured by chemiluminescence assay, as previously described. 12 Ten microliters of 100-mmoL/L stock solution of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione; Sigma Chemical Co., St. Louis, MO) was added to 400 ul of neat seminal fluid. 13,14 Chemiluminescence was measured for 15 minutes using a luminometer (Autolumat LB 953; Berthold Technologies, LLC, Oak Ridge, TN) and the results were expressed as RLU/sec/10 6 sperm. Measurement of Total Antioxidant Capacity Seminal plasma total antioxidant capacity (TAC) was measured using the Cayman Chemical Antioxidant Assay Kit (cat #709001; Cayman Chemical, Ann Arbor, MI), as previously described. 15 The principle is based on the ability of aqueous and lipid-antioxidants in the seminal plasma to inhibit the oxidation of the 2,2 -Azino-di-(3-ethylbenzthiazoline sulphonate) (ABTS) to ABTS þ. Under the reaction conditions used, the antioxidants in the seminal plasma cause suppression of the absorbance at 750 nm to a degree that is proportional to their concentration. The capacity of the antioxidants in the sample to prevent ABTS oxidation was compared with that of Trolox (a water-soluble tocopherol analogue) and expressed as micromolar trolox equivalents. Measurement of Sperm DNA Damage Sperm DNA fragmentation was quantified using the terminal deoxynucleotidyl transferase-mediated fluorescein-dutp nick end labeling assay kit (Apo-Direct; Pharmingen, San Diego, CA). 8 Briefly, after fixation with 3.7% paraformaldehyde for 30 minutes on ice, spermatozoa were washed and resuspended in ice cold 70% ethanol then phosphate buffered saline. The specimens were then centrifuged at 1600 rpm for 7 minutes and the pellet resuspended for 60 minutes at 37 Cin50mL of solution containing terminal deoxynucleotidyl transferase enzyme, terminal deoxynucleotidyl transferase reaction buffer, fluorescein isothiocyanate tagged 2 0 -deoxyuridine, 5 0 -triphosphate nucleotides and distilled water. After centrifugation, the cells were washed twice in rinse buffer, resuspended in 0.5 ml of propidium iodide/rnase solution, and incubated for 30 minutes in the dark at room temperature in anticipation of flow cytometry. Flow Cytometry Analysis All fluorescence signals of labeled spermatozoa were analyzed by the flow cytometer FACScan (Becton Dickinson, San Jose, CA). A total of 10,000 spermatozoa were examined for each assay at a flow rate of <100 cells/second. The excitation wavelength was 488 nm supplied by an argon laser at 15 mw. Green fluorescence (480-530 nm) was measured in the FL-1 channel and red fluorescence (580-630 nm) in the FL-2 channel. Gating was done to exclude debris and aggregates using 90 and forward-angle light scatter. DFI was calculated from the ratio of red to total fluorescence on a 1023-channel scale using the flow cytometer software FlowJo, version 6.2.4 (FlowJo, LLC, Ashland, OR). 8 Statistics and Calculations When more than 1 preoperative semen test was available, the test closest to the surgery date was used for this study. When more than 1 postoperative semen analysis was available, the first analysis at least 80 days after surgery was used for this study. ROS-TAC scores were calculated, as previously described. 15 The total normal sperm count (TNSC) was calculated by the following formula: semen volume sperm concentration (1-DFI). The differences in preoperative and postoperative semen parameters were analyzed using a Wilcoxon signed-rank test. Categorical data were analyzed using frequency distribution in the form of chi-square or Fisher exact test. Univariate analysis was performed using analysis of variance. Variables that demonstrated significance during univariate analysis were subjected to a multivariate logistical regression analysis model. All hypothesis testing was two-tailed and the threshold for statistical significance was set at 0.05. Statistical analysis was performed using JMP 9. RESULTS Ninety-five adults underwent varicocele ligation during the study period and 83 met the inclusion criteria. Demographic and clinical information are provided in Table 1. Mean patient and partner ages were 33.4 and 31.3 years, respectively. Varicoceles were isolated to the left side in 64 patients (77%) and were bilateral in the remaining 19 patients (23%). Of the 76 patients with preoperative semen testing completed at our facility, 30% solely had a concentration defect, 7% solely had a motility defect, 14% had both a concentration and motility defect, 13% were azoospermic, and 35% demonstrated normal semen parameters by WHO 2010 reference limits. Concentration and motility defects are further stratified in Table 1. Preoperative folliclestimulating hormone was measured in 45 patients and the median value was 6.4 mu/ml (range 1.2-48.4, SD 9.5). Preoperative testosterone was measured in 40 patients and the median value was 360 ng/ml (range 141-738, SD 160). Too few patients had postoperative hormone testing to allow meaningful comparison to preoperative values. Age of the patient, age of the spouse, grade of varicocele, presence of a right varicocele, the presence or severity of preoperative semen concentration UROLOGY 81 (4), 2013 761

Table 1. Demographic and clinical information of the study cohort Patient age Preoperative concentration category (% of patients) Mean years (range) 33.4 (24-45) Normal 42 Mild oligospermia 8 BMI (SD) Moderate oligospermia 13 28.5 (5.9) Severe oligospermia 24 Azoospermia 13 Female partner age Mean years (range) 31.3 (24-42) Length infertile period at time of initial evaluation Preoperative motility category (% of patients) Mean months (range) 30 (0-120) Normal 66 Mild asthenospermia 11 Type of infertility (% of couples) Moderate asthenospermia 7 Primary 75.9 Severe asthenospermia 4 Secondary 24.1 Distribution of bilateral and unilateral varicocele (% of patients) Testicular volume by sonography (n ¼ 62) Mean volume in cc (SD) Unilateral 77.1 Left 16.9 (5.5) Bilateral 22.9 Right 18.6 (5.8) Combined 35.5 (10.6) Varicocele laterality and grade (% of patients) Left Right Grade 1 16.9 18.1 Grade 2 50.6 4.8 Grade 3 32.5 0 between left and right testis (95% CI) BMI, body mass index; CI, confidence interval; DFI, DNA fragmentation index; SD, standard deviation. * P ¼.01 (t test). 1.7 ( 3 to 0.4)* and/or motility defects, and preoperative hormone values were not significantly different between couples with primary and secondary infertility (data not shown). Sixty-three patients completed both preoperative and postoperative semen testing at our institution of which 24 also completed preoperative and postoperative testing for ROS, TAC, and DFI. Analysis of paired samples revealed that sperm concentration, total motile sperm count (TMC), DFI, and TNSC improved after varicocele ligation and these differences were statistically significant (Table 2). Subgroup analyses revealed that patients with preoperative oligospermia demonstrated a mean improvement of 5.2 M/mL (P ¼.0001) and patients with preoperative asthenospermia demonstrated a mean improvement in motility of 18.4% (P <.0001). There was a large decrease in DFI from a preoperative mean of 40.8% to a postoperative mean of 24.5% (P ¼.001). The mean TAC decreased from 2292 um preoperatively to 1885 um postoperatively (P ¼.03) and the percentage of patients with a TAC above the normal value (1420 um) decreased from 86% preoperatively to 71% postoperatively. 15 Additional mean preoperative and postoperative semen values were as follows: ejaculatory volume 3.5 ml and 3.6 ml, Kruger morphology 2.4% and 2.9%, ROS 11851 RLU/sec/10 6 and 2710.8 RLU/sec/10 6, and ROS-TAC score 49.9 and 47.3. There was no statistically significant change in these variables after surgery. Univariate analysis of paired samples demonstrated that grade 3 left varicoceles were associated with a greater mean improvement in semen concentration (P ¼.0009) and TMC (P <.0001) than grades 1 and 2. Varicocele grade was not independently associated with a change in DFI or motility. Analysis also demonstrated that a higher preoperative DFI was associated with a larger decrease in postoperative DFI (Fig. 1). Change in DFI was not associated with a change in any other semen parameter to include concentration or motility. Univariate analysis found no other variables (to include patient age, BMI, primary vs secondary infertility, presence of right varicocele, preoperative testosterone or follicle-stimulating hormone levels, or testicular volume) associated with a statistically significant change in semen parameters after surgery. No variables were associated with a statistically significant change in postoperative semen parameters during multivariate analysis. Pregnancy information was available in 70 of 83 couples (84%) of which only 2 couples (3%) reported they had not attempted since surgery. Of the 68 couples attempting, 35 (51%) were able to get pregnant by the following methods: 17 couples (25%) spontaneously, 3 couples (4.4%) by insemination, and 15 couples (22.1%) by IVF/ICSI. When tabulated based on method by which they were obtained, 49% of pregnancies were spontaneous, 9% were by intrauterine insemination, and 43% by IVF/ICSI. Female age was significantly younger in couples able to obtain a (regardless of the method used to conceive) as compared to couples unable to obtain a (mean age 30.4 and 32.8 years, respectively, P ¼.02). There was no significant difference in female age between couples who reported spontaneous and those who reported using IVF/ICSI to achieve (31.2 and 29.4 years, P ¼.2). 762 UROLOGY 81 (4), 2013

Table 2. Comparison of preoperative and postoperative semen parameters in patients with paired semen tests Parameter Subgroups (Number of Patients) Mean Preoperative Value Mean Postoperative Value Mean Change (95% CI) P Value Concentration (million/ml) All patients (63) 22.7 26.2 3.5 ( 1.3 to 8.2).01 Patients with preoperative oligospermia (38) 4.4 9.7 5.2 (2.1-8.3).0001 Motility (%) All patients (63) 43.4 48.3 4.9 ( 0.9 to 10.7).07 Patients with preoperative asthenospermia (26) 24.1 42.4 18.4 (11.4-25.3) <.0001 Total motile sperm count (million) All patients (63) 41 57.8 16.7 (1.9-31.6).03 Patients with TMC < 10 million (27) 3.5 14.1 10.6 (5.5-15.7).0002 Sperm DNA fragmentation (%) All patients (22) 40.8 24.5 16.2 ( 7.3 to 25.2).001 Patients with preoperative DFI >19% (21) 41.9 23.6 18.3 ( 10 to 26.6).0002 Patients with preoperative DFI >30% (17) 45.6 24.3 21.2 ( 11.5 to 31).0003 Total normal sperm count All patients (22) 3849.8 5354.8 1505.01 Patients with preoperative DFI >19% (21) 4098.8 5434.2 1443.4.03 Patients with preoperative DFI >30% (17) 4083.6 5739.4 1655.8.04 TMC, total motile count; TNSC, total normal sperm count; other abbreviations as in Table 1. Comparison of preoperative and postoperative semen parameters in patients with paired semen tests demonstrates a statistically significant increase in sperm concentration, sperm DNA fragmentation index, and total normal sperm count after varicocelectomy. Patients with preoperative asthenospermia demonstrated a significant improvement in sperm motility after varicocelectomy. Change in DFI Bivariate analysis of change in DFI Preoperative DFI r 2 = 0.53 p = 0.001 Figure 1. Bivariate analysis of change in sperm DNA fragmentation index (DFI) demonstrates that higher preoperative DFI is associated with a greater decrease in DFI after varicocelectomy. (Color version available online.) Examination of postoperative semen parameters and length of follow-up in couples with data is summarized in Table 3. Analysis revealed that couples who obtained a, regardless of the method, had a higher percent motility (53.3% vs 39.3%, P ¼.007) and a longer follow-up period (26.8 vs 21.9 months, P ¼.03) than couples unable to obtain a. Length of follow up was not statistically different among categories (data not shown). Of interest, couples who obtained without the use of IVF/ICSI had a higher mean postoperative DFI than couples who reported using IVF/ ICSI to achieve (34% vs 17.5%, P ¼.04). There was no significant difference in the mean sperm concentration, motility, TMC, or follow-up period between couples who conceived spontaneously and those who used IVF/ICSI. Univariate and multivariate analysis found no additional variables, including age of the patient, age of the spouse, primary vs secondary infertility, postoperative semen parameters (excepting motility), or change in semen parameters, associated with or method by which was obtained. COMMENTS Given the inherent variability of basic semen parameters, interest is growing in the use of advanced semen tests, such as DFI, to predict fecundity. Multiple studies have shown that elevated DFI negatively correlates with and this effect is more profound for spontaneous as compared with IVF/ICSI. Yet we are only aware of 1 publication examining the association of DFI and after varicocelectomy. Given the prevalence of varicoceles and the ongoing debate regarding the impact of varicocelectomy on, the association between postoperative DFI and warrants further investigation. Our study revealed a statistically significant decrease in DFI after varicocelectomy and that higher preoperative DFI was associated with a greater postoperative decrease. UROLOGY 81 (4), 2013 763

764 UROLOGY 81 (4), 2013 Table 3. Postoperative semen parameters examined by ability to obtain and the method of conception Category (number of couples) Concentration M/mL, (SD) (95% CI) P Value Motility, % (SD) Mean Postoperative Semen Parameters (95% CI) P Value TMC, Million (SD) (95% CI) All couples (83) 26 47.4 57.1 24.5 (32.7) (20.4) (87.3) (14) No follow-up 13 53.1 60.1 31 data (13) (17.3) (28.5) (90.1) (0) Follow-up data (70) Not attempting (2) Attempting (68) Failed to achieve (33) Achieved (35) Achieved without IVF/ICSI (20) Achieved with IVF/ ICSI (15) P Value DFI, % (SD) (95% CI) P Value 28.2 15.2.2 46.6 6.6.4 56.8 3.3.9 24.2 6.8.6 (34.2) ( 8.2 to 38.6) (19.1) ( 22.1 to 9) (87.8) ( 74.3 to 67.7) (14.2) ( 37.1 to 23.6) 0 0 0 0 28.7 46.6 56.8 24.2 (34.3) (19.1) (87.8) (14.2) 25.2 39.3 36.9 22.2 (36.5) (17.8) (73.2) (14.4) 32.2 7.4 53.3 13.9.007 75.2 38.3.1 25.7 3.5.6 (32.2) ( 11.7 to 25.8) (18.2) (3.9 to 24) (97.1) ( 9.9 to 86.5) (14.5) ( 9.9 to 16.9) 33.3 50 66.6 34 (32) (20.6) (69.2) (14.8) 31.2 2.0.9 56.5 6.7.4 83.1 16.5.7 17.5 16.5.04 (33.7) ( 28.1 to 24) (15.6) ( 7.7 to 21.2) (119) ( 61.8 to 94.7) (9) ( 32.2 to 0.8) ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; other abbreviations as in Tables 1 and 2. The difference in mean postoperative sperm concentration, sperm motility, total motile count, and sperm DNA fragmentation index are categorized by ability to conceive and method of conception. The values are compared between the groups and statistical significance presented. Motility is statistically higher in couples able to achieve a (regardless of the method) as compared to couples unable to conceive; however, there was no difference in motility in couples reporting IVF/ICSI as compared to spontaneous. There was no statistically significant difference in mean postoperative DFI in couples able to achieve a (regardless of the method) as compared to couples unable to conceive; however, couples who reported a spontaneous had a higher mean DFI than couples who used IVF/ICSI to conceive.

Our analysis found a higher mean postoperative DFI in couples reporting a spontaneous (34%) as compared to couples reporting conception through IVF/ICSI (17.5%) and this difference reached statistical significance. Notably, however, we found no statistically significant difference in mean postoperative DFI in couples able to achieve a (regardless of the method of conception) as compared to couples who reported no pregnancies. Increased postoperative motility was the only semen parameter associated with postoperative in our series and this association remained true regardless of the method of conception. The association of DFI and was recently explored by Smit et al 10 in 49 men with infertility, oligospermia, and varicoceles. Similar to our study, the authors confirmed an improvement in mean sperm count, concentration, progressive motility, and DFI after surgery. The authors noted that improvement in progressive motility was inversely associated with a postoperative decrease in DFI. We found no association between change in DFI and postoperative motility. Fewer couples reported spontaneous in our cohort (25% vs 37% in Smit et al 10 ), however, both studies had similar rates of IVF/ICSI pregnancies (22% vs 24%, respectively). In contrast to our results, Smit et al 10 found that couples unable to achieve a had a significantly higher mean postoperative DFI than couples able to conceive, regardless of the method of conception (37.3% vs 26.6%, P ¼.01). Similar to our study, Smit et al 10 also noted that couples who obtain a spontaneous had a higher DFI than couples who conceived via assisted reproductive technology (30.1 vs 21.3) but the statistical significance of this difference was not described. We are intrigued that couples with spontaneous pregnancies had a mean postoperative DFI >30 in both our study and that of Smit et al. 10 We propose this finding suggests that varicocelectomy makes sperm good enough that couples with no, or only minor, competing fertility conditions are able to conceive spontaneously. In our opinion, the finding in both studies that couples pregnant via IVF/ICSI had a lower mean postoperative DFI than couples with spontaneous lends strength to his viewpoint. The challenge remains to identify and quantify the other variables that influence success after varicocelectomy. That the majority of published fertility outcomes are analyzed through the prism of 1 partner highlights the challenges of fully characterizing the infertile couple. This shortcoming deserves more attention from male and female fertility specialists alike. The impact of varicocelectomy on postoperative sperm parameters in our surgical series were in keeping with numerous published studies. Several contemporary reviews demonstrate improvements in sperm concentration, total motility, progressive motility, and morphology after varicocele ligation. 1,3,16 Although the presence of increased oxidative stress and low antioxidant levels in men with varicoceles is generally well accepted; the impact of varicocelectomy on markers of oxidative stress is mixed. 17-23 Pasqualotto et al 24 found lower ROS-TAC scores in men with varicoceles; however, there was no statistically significant difference between fertile and infertile men with varicoceles. In contrast, Mancini et al 25 found no improvement in TAC after varicocele ligation. Our study demonstrated no statistically significant change in ROS or ROS-TAC scores after varicocelectomy. Mean TAC was statistically lower after surgery; however, postoperative TAC remained above the normal reference value for the majority of our subjects, making the clinical significance of this postoperative finding dubious. Improvement in markers of sperm DNA damage after varicocelectomy is corroborated in several recent publications, however, with the exception of Smit et al, 10 these studies did not assess postoperative. 9,26,27 Excepting age, the reproductive health of female partners in our cohort was inadequately characterized despite considerable effort. Additional limitations of our study include its small size and retrospective nature. Semen parameters are inherently variable; therefore, the differences between preoperative and postoperative parameters may be the result of regression to the mean e a methodical flaw shared by many studies. A longer follow-up period may have revealed a higher rate in our cohort. Our study was not designed to elicit data about failed assisted reproductive technology attempts e this information may have shed further light on the impact of DFI on postoperative. CONCLUSION Varicocele ligation resulted in a statistically significant improvement in sperm concentration and motility, TMC, DFI, and TNSC in our surgical series. Furthermore, patients with preoperative defects in concentration, motility, and DFI showed greater improvement in the corresponding parameter postoperatively. Increased postoperative motility was associated with higher chance of conception, regardless of the method used to obtain the. Mean postoperative DFI was above 30 and was statistically higher for couples able to conceive spontaneously as compared to couples who obtain a with IVF/ICSI. It is reasonable to presume that there is an equivalent degree of expertise in the preoperative selection and surgical treatment of varicoceles among fertility specialists; therefore, couples considering varicocelectomy with the intent of improving semen parameters should be encouraged by the results of this study. Acknowledgments. The authors thank Mr. Karthikeyan Venkatachalam for his expertise and assistance retrieving data from the electronic medical records. References 1. Baazeem A, Belzile E, Ciampi A, et al. Varicocele and male factor infertility treatment: a new meta-analysis and review of the role of varicocele repair. Eur Urol. 2011;60:796-808. UROLOGY 81 (4), 2013 765

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