Effect of microsurgical varicocelectomy on semen parameters, serum, and intratesticular testosterone levels

Abstract Objective The goal of this work was to evaluate if men who underwent microsurgical varicocelectomy would have improvement in serum testosterone (T) as well as serum 17‐hydroxyprogesterone (17‐OHP—An intratesticular T biomarker) in addition to semen parameters after operation. Materials and Methods We conducted a prospective analysis of 30 men who underwent microsurgical varicocelectomy from December 2018 to September 2019. We assessed varicocele grade and laterality, serum T, serum 17‐OHP, serum follicle‐stimulating hormone (FSH), serum LH, and semen parameters in baseline and follow‐up. According to the data distribution, we reported the median and interquartile ranges and utilized the Mann‐Whitney U, Student's t test and Wilcoxon rank test. Correlation analysis was performed with the Spearman test. Results In the baseline, 9 (30%) men had 17‐OHP < 55 ng/dL and 21 (70%) men presented with 17‐OHP ≥ 55 ng/dL. Also, 19 men had TMSC < 9 million, including 6 men with azoospermia, 1 man with cryptozoospermia, and 11 men with TMSC ≥ 9 million. We found an improvement in most SA parameters of most men, which include concentration (63.3%, 19/30), motility (46.6%, 14/30), and TMSC (60%, 18/30). About seven (36.8%) men had TMSC upgraded to > 9. There was a significant change in volume (2.1 [1.5‐2.8] to 2.4 [1.7‐3.6] cc, P = .018), concentration (6.8 [0.8‐22.5] to 12.5 [1‐31] million/cc, P = .047) and TMSC (4.4 [0.3‐15.1] to 10.5 [0‐41.8] million, P = .012) after surgery. We neither found a change in serum T nor a change in intratesticular T (serum 17‐OHP) after varicocelectomy (P > .05). FSH, LH and T were similar both before and after varicocelectomy (P > .05). Conclusion Despite improvement in semen parameters following varicocelectomy, we did not see changes in either serum or intratesticular T. This suggests that improvement of semen parameters following varicocele repair could be from factors other than changes in androgen levels within the testis.


| INTRODUC TI ON
Varicocele, defined as abnormally enlarged and tortuous veins of the pampiniform plexus, is the most common identified cause of male factor infertility. It is found in 15% of the general population, but is present in up to 35% of men presenting with primary infertility and up to 80% of men presenting with secondary infertility. 1 Varicocele can negatively impact appropriate sperm production leading to decreased fertility and complete azoospermia in 4%-14% of patients. 2 The proposed detrimental effects of varicocele on testicular function include oxidative stress, high scrotal temperature with loss of the scrotal counter-cooling mechanism, reflux of adrenal metabolites, and changes in androgen production. 3,4 Leydig cells drive spermatogenesis via the secretion of testosterone (T), which acts on Sertoli and/or peritubular cells to create an environment which enables normal progression of germ cells through the spermatogenic cycle. 5 Also, testicular biopsy in patients with idiopathic varicocele showed decreased tubular diameter, increased Leydig cell atrophy with vacuolization and decreased Leydig cell number in testicular tissue when staining for T. 6 These studies suggest that varicoceles affect Leydig cell histology, and thus, likely affecting sperm production and T levels. Although little data has been published, some authors showed an association between varicocele and low T, and subsequent improvement after surgical repair. 7,8,9,10 However, a large population study among adolescents showed no association between varicocele and serum T levels. 11 We based the correlation of 17-OHP with ITT in Amory et al 12 study, that compared ITT levels through testicular aspiration with serum 17-OHP levels in men previously treated with exogenous T who were distributed in different hCG dosages groups (r = .78, P < .0001). We published a recent study in J Urol 13 evaluating the positive association between 17-OHP and ITT in a study comparing men receiving exogenous T to men receiving clomiphene citrate or human chorionic gonadotropin, where men receiving exogenous T had undetectable 17-OHP levels in follow-up, while men receiving medications to increase ITT had a significant increase in 17-OHP levels. Since one of the mechanisms by which varicocelectomy improves semen parameters is with improvement in ITT production, we hypothesized that semen analysis parameters improvement after varicocele repair could be associated to augmentation of ITT levels. The objective of this study was to evaluate whether changes in semen parameters following varicocele repair will be associated with changes in either ITT or serum T.

| MATERIAL S AND ME THODS
After institutional review board approval, we prospectively followed men who underwent microsurgical varicocelectomy from December 2018 to September 2019. Indications for varicocelectomy were according to the AUA guidelines. 14 Varicocele was diagnosed by physical exam, and in those cases when clinical diagnosis was difficult (previous scrotal surgery or tight scrotum with difficult body habitus), scrotal ultrasound was performed. All subclinical varicoceles were excluded from this study. All surgeries were performed by a single high-volume surgeon, using microsurgical subinguinal technique. For all men, we evaluated serum 17-OHP, total T, FSH, and semen parameters before and after varicocele repair. All men had at least two semen analyses before undergoing varicocele repair according to the WHO guidelines.
Men with azoospermia and severe oligozoospermia (concentration < 5 million/cc) in baseline had genetic evaluation prior to treatment. We excluded patients with chromosome abnormalities and microdeletions.
Post-varicocelectomy measurements were obtained approximately 3-6 months after surgery. Three semen parameters were considered during the analysis: Concentration (million/cc), Motility (%), and Total Motile Sperm Count (TMSC). All semen analyses were performed by the same technician.
In order to develop a "standard" level for serum 17-OHP, we performed a cross-sectional analysis of a representative sample of 181 fertile controls (men who were evaluated for vasectomy reversal, erectile dysfunction, Peyronie's disease, orchialgia, and vasectomy evaluation) aged 39 + 11.2 years. The 25th, 50th, and 75th percentile for serum 17-OHP was 55, 72, and 102 ng/dL. We used 55 ng/dL as the threshold to determine the lower limit of normal. The lower limit of normal established by commercial laboratories ranges from 27-199 ng/dL (LabCorp-www.labco rp.com) and from 33-195 ng/dL (QUEST-www.quest diagn otics.com). We preferred to use the cut point of 55 ng/dL, because we wanted to use a representative sample of fertile controls and was similar to the cutoffs used by the large commercial laboratories. Therefore, we evaluated men based on serum 17-OHP ≤ 55 ng/dL (low 17-OHP) or 17-OHP > 55 ng/dL (normal 17-OHP). Also, patients were divided according to baseline total motile sperm count (TMSC) in two groups: TMSC < 9 and TMSC > 9. We excluded men without baseline and post-varicocelectomy measurements, those receiving exogenous T.

| RE SULTS
Between December 2018 and September 2020, a total of 30 men fit the inclusion criteria and underwent varicocelectomy. Clinical and demographic characteristics of the analyzed men are evident in Table 1. In the baseline analysis, 6 (20%) of men had T < 300 ng/ dL while 24 (80%) men had T > 300 ng/dL. Also, 9 (30%) men had 17-OHP < 55 ng/dL and 21 (70%) men presented with 17-OHP ≥ 55 ng/dL. Of the nine men with low 17-OHP, only two (22.2%) presented with orchialgia. This is a very similar proportion to men with normal 17-OHP, where five (23.8%) presented with orchialgia. When divided according to TMSC, 19 men had TMSC < 9 million, including 6 azoospermic and 1 cryptozoospermic, and 11 men with TMSC ≥ 9 million (Table 2). Of the 19 patients with baseline TMSC < 9, 11 men had increase in TMSC and 7 men improved SA to TMSC ≥ 9 in the follow-up. Conversely, only one man with baseline TMSC ≥ 9 had a considerable decrease in TMSC to < 9. Men with TMSC < 9 after surgery had higher levels of FSH in baseline and follow-up, higher prevalence of bilateral varicocele, smaller testicles, and lower TMSC count in the baseline ( After performing a correlational analysis, we found a positive correlation between baseline T with baseline 17-OHP (r = .691, P < .001), follow-up T and follow-up 17-OHP (r = .400, P = .029) and follow-up 17-OHP and postoperative semen volume (r = .429, P = .018). But, we did not find significant correlation between preoperative 17-OHP and SA concentration, TMSC, varicocele grade, and laterality (P > .05) ( Table 5).
We also evaluated the effected of higher grades of varicocele in hormonal levels and semen parameters by excluding grade I varicocele from the analysis. Only TMSC variation was significant in this particular group (4.85 [0.55-22.25] to 11 [1.05-53] million, P = .028) ( Table 6).

| D ISCUSS I ON
Varicocele has been proposed to be the cause of infertility in a considerable amount of men seeking to conceive. Varicocelectomy is a cost-effective treatment for infertility, with microsurgical subinguinal or inguinal as the best techniques. 15 Since many studies showed correlation between varicocele repair and serum T levels improvement, 7 Abbreviations: 17-OHP: 17-hydroxyprogesterone (ng/dL); FSH: Follicle-stimulating hormone (mIU/mL); LH: Luteinizing hormone (mIU/mL); SA: Semen analysis; T: Testosterone (ng/dL); TMSC: Total motile sperm count (million).
conception parameters, proving that varicocele repair is an important tool in the andrologist arsenal. Although many studies showed that varicocele repair could improve T levels, we could not reproduce these finding in our study. Also, there was no significant correlation between serum 17-OHP and semen analysis improvement after the surgery. Therefore, we could not verify our initial hypothesis.
Previous studies showed that men with normal T are less likely to improve total T postoperatively. 18  Our study not only has strengths, but it also has its limitations. The strengths include the novelty of the study, associating varicocelectomy with ITT and semen parameters. All procedures were performed using microsurgical technique by a single high-volume surgeon and all semen analyses were performed by a single specialist, reducing bias in surgical technique as well as sperm analyses. We also tried to control variability in this study by performing all serum measurements between 6:00 and 10:00 am, in a single laboratory. Although we could not define a precise association between ITT and SA parameters In conclusion, microsurgical varicocele repair resulted in improvements in all evaluated semen parameters, but not in ITT/17-OHP or serum T levels. The mechanism by which varicocele repair improves semen parameters appears to be driven by factors other than changes in androgen synthesis within the testicular microenvironment.