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INTRODUCTION: The purpose of the study was to compare the 5-hydroxytryptamine (5-HT) plasma levels of the internal spermatic vein and peripheral vein for patients with varicocele and infertility with a comparison group of fertile patients needing scrotal surgery for other reasons, and to examine the correlation between the 5-HT levels and semen parameters.
METHODS: Participants were 30 patients with varicocele and infertility and a comparison group of 30 fertile patients with inguinal hernia, hydrocele, or epididymal cyst. Their ages ranged from 24-35 years. Patients with varicocele received at least 2 semen analyses before surgery. Spermatic vein blood samples were taken intraoperatively and peripheral vein blood samples were taken preoperatively from all patients. Computer-assisted sperm analysis (CASA) was used for semen analysis; the enzyme linked immunosorbent assay (ELISA) method was used to measure blood plasma 5-HT levels. Analysis of variance and Spearman's rank correlation tests were used to analyze the data.
RESULTS: The plasma free serotonin concentration of the internal spermatic vein was significantly higher than the concentration in the peripheral vein for patients with varicocele (P < .01), and significantly higher than the spermatic vein concentration of the comparison group (P < .01). In the comparison group, no statistically significant difference was found between the plasma free serotonin concentration of the spermatic and peripheral veins (P > .05). There were significant negative correlations between the plasma 5-HT levels of the internal spermatic vein and sperm density (Spearman r = -0.465, P = .0097) and sperm motility (Spearman r = -0.539, P = .0024) in the patients with varicocele.
CONCLUSIONS: Patients with varicocele have elevated free 5-HT plasma concentrations in the spermatic vein that may be a cause of fertility decline.
KEYWORDS: Varicocele; 5-hydroxytryptamine; Internal spermatic vein; Peripheral vein; Semen parameters .
CORRESPONDENCE: Salifou Issiaka TRAORE, MD, PhD. Wuhan University Zhongnan Hospital, Department of Urology, Wuchang East Lake Road No. 169, Wuhan City, P.O. Box 430071, China () or Professor Zheng Xin-min ().
CITATION: Urotoday Int J. 2010 Aug;3(4). doi:10.3834/uij.1944-5784.2010.08.11
ABBREVIATIONS AND ACRONYMS: 5-HT, 5-hydroxytryptamine; 5-HT2R, 5-hydroxytryptamine receptor type 2; CRF, corticotropin releasing factor; ISVD, internal spermatic vein diameter; OD, optical density; WHO, World Health Organization.
Varicocele is the enlargement and dilatation of the pampiniform plexus and internal spermatic vein within the spermatic cord. It occurs most often on the left side and is associated with testicular dysfunction [1-3]. Varicoceles have been ranked on the first line of the World Health Organization (WHO) list of infertility diseases, with a 15% incidence in the general population and nearly a 40% incidence among infertile patients .
Pathophysiological findings [5,6] have proven that defective valves in the internal spermatic vein allow backflow and stagnation of renal blood containing high levels of waste products into the testis. This renal blood is mainly responsible for testicular damage. The mechanisms underlying varicocele involvement in male infertility are not yet fully understood. Different hypotheses have been used to explain how the varicocele affects testicular function, but no single hypothesis has been fully accepted [7-9].
Although fertility improvement after varicocelectomy has been reported frequently, there is still a debate about the effectiveness of surgical treatment [10,11]. Therefore, further research is needed to clarify the contribution of varicocele to infertility mechanisms and to improve treatment outcomes. The purposes of the present study were: (1) to compare the 5-hydroxytryptamine (5-HT) plasma levels of the spermatic vein and peripheral vein for patients with varicocele and infertility with a comparison group of fertile patients needing scrotal surgery for other reasons, and (2) to examine the correlation between the 5-HT levels and semen parameters
The investigation was a prospective study with a comparison group. It was approved by the author's institutional ethics committee. All participants provided written informed consent.
The participants were 60 patients from the urology and general surgery departments of Wuhan University Zhongnan Hospital. They were evaluated between February, 2007 and January, 2008. Exclusion criteria were: (1) patients with abnormal platelet count, arteriosclerosis, or chronic renal failure; (2) patients taking drugs (eg, sertraline, paroxetine ) that could influence blood 5-HT levels. The patients were assigned to one of 2 groups, based on their diagnosis.
Group 1: experimental group (n = 30). Patients in group 1 had varicocele and infertility. Varicocele was confirmed through color Doppler ultrasound . Patients had internal spermatic vein diameter (ISVD) ≥ 2.0 mm with time of reflux ≥ 1 s under normal respiration, or blood backflow or ISVD evident enhancement during the valsalva test. Infertility was defined as the inability to achieve conception after at least 1 year of unprotected sexual intercourse with a gynecologically normal partner. The primary symptoms of these patients were 3-6 months of scrotal and/or lower abdominal pain or discomfort. Twenty patients had grade II varicocele, defined as a moderate varicocele with enlarged veins that are palpable even when the patient does not perform a valsalva test; 10 patients had grade III varicocele, defined as a large varicocele with dilated veins that are directly visible through scrotal skin. Varicoceles were bilateral in 11 patients and unilateral in 19 patients. According to the WHO 1999 standard , all patients presented various degrees of semen impairment and were candidates for inguinal or palomo varicocelectomy. The choice of open varicocelectomy was made by the patients after counseling.
Group 2: comparison group (n = 30). Patients in group 2 did not have a varicocele and had a recent (ie, approximately 1 year) history of fertility. These patients were candidates for scrotal surgery because of inguinal hernia (n = 18), hydrocele (n = 8), and epididymal cyst (n = 4).Table 1 contains the means and standard deviations (SD) for age, weight, height, and body mass index of both patient groups. Patients in the experimental group had a mean age of 29.5 years (range, 24-35 years; patients in the comparison group had a mean age of 29.4 years (range, 23-35 years). There were no significant group differences for any of the characteristics.
Specimen collection and measurement. The semen of the patients with varicocele was analyzed at least 2 times before surgery. The semen samples were taken following masturbation, after 2-3 days of sexual abstinence. The samples were put into a clean tube and kept at 37ºC. A computer-assisted semen analysis (CASA) was conducted within 60 minutes of collection.
Peripheral vein blood samples were taken preoperatively and spermatic vein blood samples were taken intraoperatively for all patients. Each blood sample was 4 mL. The blood was put into a clean test tube containing heparin and carried immediately to the laboratory for centrifugation (3000 rotations/minute for 5 minutes). Aliquot supernatant plasma was then retrieved. A total of 0.5 mL of this plasma was put into a test tube containing 0.5 mL of 4% ethylenediaminetetraacetic acid (EDTA). It was then centrifuged at 3000 rotations/minute for 5 minutes. The supernatant poor platelet plasma was retrieved and frozen at -20ºC. Afterwards, 5-HT levels were measured using enzyme linked immunosorbent assay (ELISA) kit number QRCT-548751ELA/UTL (Shanghai Tian-cheng Ltd; Shanghai, China).
Experimental procedures. The steps of the experimental procedures were: (1) place 100 μL of diluted sample (1:100) and 100 μL of standard solution into different wells of a coated microtitration strip, incubated at 37ºC for 30 minutes; (2) empty the strip, wash it 3 times, and dry it with absorbent paper; (3) add 100 μL of enzyme conjugate solution to each well and incubate at 37ºC for 30 minutes; (4) empty the strip, wash it with rinsing buffer 5 times, and dry it with absorbent paper; (5) add 5 μL of substrate from the patient to each well and incubate at 37ºC for 15 minutes; (6) at the end, add 50 μL of stop solution to each well and use the WeScan-MK2 (Denley Dragon; Finland) to read the optical density (OD) value at 450 nanometers wavelength; (7) draw a standard curve on linear graph paper by plotting each reference standard mean absorbance against its concentration, with optical density on the vertical Y axis and concentration on the horizontal X axis. Knowing the OD value for each sample, the corresponding 5-HT concentration was determined.
The semen outcome measures were sperm density, sperm motility, and percentage of abnormal sperm. The blood outcome measures were 5-HT plasma concentration from the peripheral vein and internal spermatic vein.
SPSS 13.0 software (SPSS Inc, Chicago, IL, USA) was used for statistical analyses. An analysis of variance (ANOVA) was performed to analyze group differences in plasma 5-HT levels. Spearmanâ€™s rank correlation coefficient was used to determine the correlation between the plasma-free serotonin level and semen parameters. Probability < .05 was used to indicate statistical significance.
Plasma 5-HT Concentration in the Internal Spermatic Vein and Peripheral Vein
Figure 1 contains the results of the intragroup comparisons between the plasma 5-HT concentrations of the internal spermatic vein and peripheral vein for each patient group. The mean (standard deviation ) plasma free serotonin concentration in the internal spermatic vein of the patients with varicocele was 548 (194.35) nmol/L. This mean value highly exceeded the mean serotonin concentration of 240.53 (76.40) nmol/L in the peripheral vein; the difference was statistically significant (P < .01). The mean plasma free 5-HT concentration in the internal spermatic vein of the patients in the comparison group was 259.86 (78.44) nmol/L. This mean value was not significantly different from the mean serotonin concentration of 224.76 (83.41) nmol/L in the peripheral vein.
Comparison of the mean plasma free serotonin concentration of the internal spermatic vein between groups revealed that the concentration was significantly higher in the patients with varicocele (P < .01). There were no significant group differences in mean plasma free 5-HT concentration in the peripheral vein.
Correlation Between Internal Spermatic Vein Plasma 5-HT and Semen Parameters
The scatter plots showing the correlation between internal spermatic vein plasma free 5-HT concentration and sperm motility and semen density for the patients with varicocele are shown in Figure 2 and Figure 3, respectively. A statistically significant negative correlation was found between the internal spermatic vein plasma free 5-HT levels and sperm motility (Spearman r = -0.539; P = .0024). Similarly, a statistically significant negative correlation was found between the internal spermatic vein plasma free serotonin concentration and semen density (Spearman r = -0.465; P = .0097). No statistically significant correlation was found between the spermatic vein plasma free 5-HT levels and abnormal morphology sperm rate (Spearman r = -0.138; P = .466).
The findings of significantly higher free serotonin concentrations in the internal spermatic vein when compared with the peripheral vein for the patients with varicocele, and significantly higher spermatic vein free serotonin concentrations in patients with varicocele when compared with patients in the group without varicocele are in agreement with Cai Xiao-qing et al  and Gonzalez and Garcia-Hjarles . Other intragroup and intergroup comparisons were not significantly different.
Testicular histopathologic examination was not conducted in this investigation. However, given well-established values of semen parameters in objective evaluation of male gonads , their variation patterns could reflect serotonin toxicity. The significant negative correlations between plasma serotonin levels and both sperm density and sperm motility in the present study are similar to results demonstrated by Caldamone et al . These findings indicate that elevated plasma-free serotonin concentration in the internal spermatic vein of patients with varicocele has a negative effect on the spermatozoa production and maturation processes, and may be at least partly responsible for sperm quality and fertility impairment in these patients.
5-HT has both vasoactive neurotransmitter and growth factor functions. The peripheral vein component comes mainly from enterochromaffin cell secretion. Two forms exist in blood circulation, taken up and stored by a platelet inactive form (90%), and a free plasma serotonin active form (10%). If a patient has a varicocele, blood from the renal vein is refluxed toward the spermatic vein, carrying waste products directly to the liver. Therefore, blood pools into the testis venous system, leading to an increase in intraluminal oncotic pressure and free serotonin levels. A slowdown in the return of testicular venous blood to systemic circulation may cause hypoxia and endothelium adherence of platelets, which are thought to be potential providers of free serotonin. Moreover, once testicular microcirculation 5-HT levels overcome the endothelium monoamine oxidase (MAO) enzyme scavenging ability of the 5-HT, free serotonin concentration starts to increase gradually.
The exact mechanism underlying 5-HT involvement in the decreased fertility of patients with varicocele is not yet certain. However, there is evidence in the literature supporting this action. 5-HT might decrease fertility potential through some paracrine and autocrine pathways. This hypothesis is based on the findings of previous studies of 5-HT receptor distribution and function [18-20]. Seminiferous tubule, testis accessory organ, and epithelial cell stimulation by serotonin via 5-HT receptor type 2 (5-HT 2R) may induce smooth muscle contraction and imbalance of the fluid component. The sperm production and maturation processes are then affected. The contractile and mitogenic roles of 5-HT in the smooth muscle of the peripheral blood vessel have been proven [21-24]. Excessive free serotonin continuously stimulates the smooth muscle of blood vessels either directly via 5-HT 2R or indirectly via thromboxane A2. This action might induce vasoconstriction and smooth muscle cell proliferation, contributing to microcirculation disturbances and waste metabolic acid collection in the testis. Furthermore, high-affinity 5-HT 2R located on platelets allows serotonin to induce platelet aggregation, which later releases a huge amount of 5-HT. This might result in vasoconstriction and thrombogenesis, thus worsening the decrease in testicular blood flow and microcirculation disorder.
5-HT is also thought to be a powerful inflammatory mediator . Its high levels in the hypoxia condition may induce testis interstitial tissue inflammation and fibrosis, leading to a decrease in blood supply and atrophy of leydig cells. According previous evidence [26,27], leydig cell 5-HT2R stimulation by serotonin may induce the corticotropin releasing factor (CRF). In addition to its negative effect on the hypothalamic-pituitary-gonadal axis, this CRF is known also to exert a local inhibitory role on androgen secretion of luteinizing hormone (LH) related interstitial endocrine cells. Moreover, CRF could stimulate leydig cell secretion of beta-endorphin, which might inhibit follicle-stimulating hormone (FSH) regulatory action on Sertoli cells in spermatogenesis.
Several well-designed investigations and meta-analysis studies [28,29] have proven that surgical treatment of varicocele is not as ideal as it was once thought. Moreover, despite being the main treatment option for male infertility in clinical practice, a significant number of patients fail to achieve natural pregnancy following varicocelectomy. According to Coolsaet , some patients may fail to achieve fertility because of irreversible damage caused by prolonged disease or misdiagnosed subclinical varicocele. However, another potential reason for infertility may be vascular reflux of the vas deferens [30-32]. The healing process of testis tissue might be compromised by continuous exposure to potentially toxic substances such as 5-HT. The results of the present study indicate that adding 5-HT 2R blocker drugs (eg, sarpogrelate, ketanserin) to surgical treatment of infertile patients with varicocele might be helpful in improving the treatment outcome.
Patients with varicocele had high 5-HT plasma concentration in the internal spermatic vein. This high serotonin concentration might have deleterious effects on semen quality, particularly semen density and motility. Thus, decline in fertility for patients with varicocele might be attributed to high concentrations of free serotonin in the internal spermatic vein.
The authors express gratitude to the Chinese Scholarship Council (CSC) for providing research facilities. They thank Professor Hu Han-lin at the laboratory division for technical assistance during the experiment, and Professor Chen-dong E, Director of the Wuhan University Medical Statistics Department.
Conflict of Interest: none declared.
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