Infertility represents a major problem for ∼10–15% of reproductive-age couples 1. It occurs because of female factor, male factor or both. Male factor infertility is responsible for about 50% of infertility cases 2–5. There are many causes of male infertility; including hormonal, genetic, environmental and other causes 6. One of the most common causes is varicocele. Varicocele is defined as dilatation of the pampiniform plexus of veins. Varicocele is present in 10–15% of the normal male population. This increases to 30–40% in men with primary infertility, and the percentage increases further to reach up to 80% in men with secondary infertility 3–5.
According to the degree of dilatations, varicocele is classified into three grades: grade I, palpable only during a valsalva manoeuvre; grade II, palpable distension with the patient standing upright; and grade III, visible distension 7. Clinically undetectable varicocele cases are diagnosed only by investigations such as colour Doppler ultrasonography, scintigraphy, venography or plethysmography 8,9.
There is debate on the mechanism by which varicocele affects spermatogenesis and sperm parameters. It has been proposed that semen quality decreases uniformly in animals with induced varicocele 10. Many other theories have been hypothesized to explain this effect. One theory claimed that varicocele increases the scrotal temperature, which affects spermatogenesis inversely. It was found that the decrease in scrotal temperature following varicocele ligation supports this theory 11. Another theory suggested that varicocele causes testicular hypoxia, which may lead to impairment in spermatogenesis in patients with varicocele 12. The most recent theory, which is widely accepted, hypothesizes that varicocele increases the level of reactive oxygen species (ROS). This increase may reach critical levels that cause oxidative stress (OS). Previous studies have shown that infertile men with varicocele have markedly elevated levels of seminal ROS 13–15. There is evidence that clinical varicocele has variable effects on semen parameters including decreased sperm concentration, decreased sperm motility, decreased normal sperm morphology or all of the above, which is known as oligoasthenoteratozoospermia syndrome 9,10,16.
Human spermatozoa are especially susceptible to ROS as their plasma membrane contains polyunsaturated fatty acid in abundance. This can lead to a chain of chemical reactions, called lipid peroxidation, which may result in sperm damage. Human spermatozoa also lack cytoplasm that generates robust preventive and repair mechanisms against ROS 17. Malondialdehyde (MDA) is the end product of a lipid peroxidation reaction and is considered one of the most important markers for OS. High levels of MDA indicate a high lipid peroxidation rate, which may induce changes in sperm parameters and may lead to decreased fertility 18.
Recently, there has been growing interest in OS as one of the underlying causes for deterioration of semen parameters in infertile men with varicocele, especially sperm DNA damage 19. OS was found to have many pathological effects on spermatozoa; the most important were sperm lipid peroxidation, DNA fragmentation, mitochondrial anomalies, apoptosis and sperm ultrastructural changes 20–22.
There is controversy on the efficacy of varicocelectomy on sperm parameters and the evidences have been inconclusive. Although many researchers detected improvements of sperm parameters after varicocelectomy 23–26, others could not 27,28. There are two techniques for varicocelectomy: laparoscopic or open surgery. Surgical varicocelectomy has many approaches including abdominal, inguinal, subinguinal and scrotal approaches. A relatively recent approach (microsurgical subinguinal varicocelectomy) was introduced by Marmar et al. in 1985 29 and was modified by Goldstein et al. in 1992 30. Since then, it has become the gold standard technique in adults for the following reasons: it leads to lower rates of recurrence and complications 30–33, it is associated with better results in terms of improvements in sperm parameters 34–37 and the incidences of hydrocele artery ligation, secondary bacterial infection and other postoperative complications are significantly lower with microsurgical varicocelectomy than with other procedures 37.
In the present study, we aim to examine the effect of microsurgical varicocelectomy in infertile male patients on seminal ROS, semen parameters and sperm ultrastructure.
Patients and methods
This was a cross-sectional study of patients with varicocele attending the Department of Dermatology, Venereology and Andrology, South Valley University Hospital, Egypt, in the period from March 2010 to March 2011. Twenty-five patients have been recruited according to the following inclusion criteria: infertility for more than 1 year, patients with third-degree varicocele, sperm concentration of less than 20×106/ml, progressive motility of less than 50%, normal morphology of less than 30% and no other causes for male infertility such as hormonal causes, cryptorchidism, smoking and other causes. To exclude other causes of male infertility, complete assessment of history and full clinical examination were carried out for every patient. Laboratory investigations (routine blood testing, colorimetric estimation of MDA, sperm analysis, sperm culture, urethral swabs and sperm analysis) were carried out and any patient with a detectable pathology was excluded from the present study. Colour Doppler ultrasound was carried out for all patients.
Semen samples were collected from all patients by masturbation into a sterile wide-mouth metal-free plastic container after 4 days of abstinence. Two samples were obtained from each patient: before and 4 months after varicocelectomy. After liquefaction at 25°C for 30 min, semen analysis was carried out according to the WHO regulations to obtain volume, pH, sperm concentration, motility and morphology 38. The remaining semen sample was centrifuged at 3000 rpm for 10 min to obtain the seminal plasma at which MDA was measured. Sperm pellet was used for transmission electron microscopy (TEM) examination. Sperm concentration was determined using a Makler Counting Chamber (Sefi-Medical Instruments LTD, Irvine Scientific, Santa Ana, California, USA)
Motility was expressed as a percentage of motile spermatozoa and their mean velocity. Morphology was determined according to the WHO criteria after incubation of the sample with trypsin for 10 min at 25°C according to the methylene blue eosin staining procedure, feathering and fixation by flame. At least 100 cells were examined at a final magnification of ×1000. Semen viscoelasticity was assessed using glass pipettes as recommended by the WHO and semen samples showing a thread of more than 2 cm long were considered highly viscoelastic, whereas viscoelasticity was considered normal when the thread length was 2 cm or less.
Mini-incision (2–3 cm), subinguinal microsurgical varicocelectomy with delivery of the testicle was performed using surgical loupes with ×2.5 magnification. The testicle was then delivered and the gubernacular veins and external spermatic perforators were inspected, isolated and divided. The testicle was then returned to the scrotum and the spermatic cord was placed on a large Penrose drain. The internal and external spermatic fascias were incised and the cord structures were examined to identify its contents. The contents of the internal spermatic fascia were dissected. The internal spermatic arteries was identified and spared on the basis of their pulsations. All internal spermatic veins were ligated with 3–0 silk ties and then divided. The cord was clarified so that only the identified artery (or arteries) and lymphatic was preserved. Dissection of the contents of the external spermatic fascia was performed. The vas deferens, its associated vessels and the cremasteric artery were identified and preserved. The remaining cremasteric fibres and veins were ligated and cut. The cord was inspected to ensure that it contained only the vas deferens, its associated vessels, the testicular artery and spermatic cord lymphatics. The cord was placed back to its normal position before closure of the wound.
MDA levels were determined by the colorimetric method using the kit purchased from Bio-Diagnostic Co. (Cairo, Egypt), with the thiobarbituric acid-reactive substance 39. Using lipid peroxidase, 0.1 ml of seminal plasma was added to 0.9 ml of distilled water in glass tubes. For each tube, 0.5 ml of thiobarbituric acid reagent (0.67 g of 2-thiobarbituric acid dissolved in 100 ml of distilled water with 0.5 g NaOH and 100 ml glacial acetic acid added) was added to the solution and then heated for 1 h in a boiling water bath. After cooling, each tube was centrifuged for 10 min at 4000 rpm and the supernatant absorbance was read on a spectrophotometer at 534 nm.
Transmission electron microscopy
Sample preparation for TEM examination was carried out as follows: samples were fixed in 4% paraformaldehyde, which was resolved in 0.1 mol/l cacodylate buffer+1% glutaraldehyde. Then, agarose pellets were prepared: 1% agarose (high gelling agarose resolved in PBS 10%) was warmed to 80°C. The semen samples were washed in PBS four times; then, samples were warmed up to 60°C and 1% agarose was placed on the samples and then mixed, centrifuged and cooled in crushed ice for 3 h. Subsequently, pellets were collected in 0.1 mol/l cacodylate buffer.
All samples were embedded for 1 day. The embedding procedure included exchange of the 0.1 mol/l cacodylate buffer (15 min), followed by 3 h in 1% osmium tetroxide (2% OsO4 resolved in 3% potassium-hexacyanoferrate) and then twice washing for 15 min. Then, samples were dehydrated in ascending grades of ethanol (every step: twice for 15 min: 70, 80, 96 and 100% alcohol); samples were then placed in intermedium 1,2-propylenoxid twice for 15 min, in a mixture 1 : 1,2-propylenoxid–epoxy resin for 3 h and then in absolute epoxy resin overnight on a shaker. The following day, all samples were embedded in fresh epoxy resin in special embedding forms and placed in an oven (60°C) for 24 h.
All samples were sectioned semithin (∼1.5 µm) and then ultrathin (∼80 nm) using the Ultracut E (Reichert - Jung, Vienna, Austria). The ultrathin sections were placed on copper grids and stained with uranyl-acetate and lead citrate.
Finally, samples were washed and examined under a JeolJem 1010 (Jeol ltd, Japan). In each sample, 200 sections were examined to detect the percentage of normal spermatozoa, head abnormalities and tail abnormalities before and 4 months after varicocele repair.
Data were analysed using the SPSS version 17 software 40. To prepare the data for analysis, basic statistics were calculated (frequencies, cross-tabulation and histogram). Frequency tables were examined to explore missing data, errors in the data (outliers and incorrect data entry) and data consistency. Descriptive statistics – that is, mean, SD and median – were calculated. Test of significances: as we had a small sample (n=25), a nonparametric test (Wilcoxon signed-ranks test) was used to compare the median differences in the quality of sperm parameters, the levels of MDA (nmol/ml) and the sperm ultrastructure before and 4 months after varicocele repair. A P value was considered significant when it was less than 0.01.
This study received approval from the Medical School Ethics Committee of the Faculty of Medicine, South Valley University. A signed written consent was obtained from all participants before obtaining the sample. Confidentiality was assured for all participants.
Twenty-five patients were recruited for the current study. The mean age of the patients was 37.1±10.5 (Table 1). The MDA concentration and semen parameters before and 4 months after varicocelectomy are summarized in Table 2. The mean MDA concentration (nmol/ml) was 2.54±0.4 before varicocelectomy and decreased to 1.55±0.3 after the operation and this variation was statistically significant (P<0.001). Semen volume showed a mild increase after varicocelectomy than before (3.73±1.5 vs. 3.93±1.2, respectively; P<0.01).
The mean sperm concentration was 6.27±3.2 before varicocelectomy and increased to 8.54±2.6 after varicocele repair, with a statistically significant difference (P<0.01). The percentage of progressive sperm motility increased from 16.52±5.8 before varicocelectomy to reach 24.32±2.7 after the operation, and this was statistically significant (P<0.001). The percentage of total sperm motility increased from 28.55±6.2 before varicocelectomy to reach 42.78±3.6 after the operation, and this was also statistically significant (P<0.001). The percentage of normal sperm morphology increased from 14.40±3.7 to 19.04±4.3 with statistical significance (P<0.001). In terms of the different sperm morphological abnormalities observed under light microscope (LM), the percentage of sperm head abnormalities significantly (P<0.001) mildly reduced after varicocelectomy, with mean values of 43.8±8.1 and 40.7±6.5 before and after the operation, respectively. In contrast, there was no significant difference in the percentage of tail anomalies before (8.84±3.8) and (8.80±3.8) after varicocelectomy (P>0.05).
TEM abnormalities examined in this study included sperm head abnormalities in the form of nuclear vaculations and binucleated head, redundancy in the acrosomal membrane and acrosomal inclusions. Tail abnormalities included absence of outer dense fibres, absence of outer doublets, absence of central singlet and total absence of axoneme. The normal head and normal tail ultrastructures are shown in Figs 1 and 2, respectively, as observed in patients after varicocelectomy (Table 3).
The percentage of head anomalies including binucleated head and nuclear vaculations decreased significantly after varicocele repair, with mild mean differences, with values of 6.96±1.6 and 20.4±4.2 before and 6.04±1.1 and 18.6±2.7 after varicocelectomy, respectively (P<0.01). A statistically significant reduction was found in the percentage of acrosomal abnormalities after varicocelectomy. The mean values of redundant acrosomal membrane and acrosomal inclusions were 22.4±2.4 and 10.2±1.8 before and decreased to 20.5±1.3 and 8.6±1.6 after varicocelectomy, and this was statistically significant (P<0.001) (Table 3 and Figs 3 and 4).
In terms of the percentage of tail ultrastructural abnormalities, the percentage of completely absent axoneme decreased from 6.6±1.6 to 5.6±1.1 (P<0.01), which was considered statistically significant (Fig. 5). Other tail ultrastructural abnormalities showed a statistically significant reduction after varicocelectomy; these abnormalities included missing outer doublets before (24.3±3.9) and after (22.3±3.6) varicocelectomy, missing central singlet before (11.2±2.4) and after (9.2±1.8) varicocelectomy and deletion of outer dense fibres before (14.0±2.4) and after (11.9±1.8) varicocelectomy. The P value for all variants was less than 0.001 (Table 3).
Varicocele is considered to be the most common treatable cause of male infertility and it is well established that varicocele exerts negative effects on sperm parameters in infertile patients 41. These effects are correlated positively with the grade of varicocele, with the worst parameters in patients with grade III varicocele 42.
The effect of varicocele repair on sperm parameters and pregnancy outcomes has been studied 43. There are many modalities for the treatment of varicocele including both medical and surgical methods. Microsurgical varicocelectomy remains the best method for varicocele repair. It has been found that patients with grade III varicocele show better improvement in sperm parameters after varicocelectomy than those with lower grades 44.
The increased levels of ROS and the resulting OS are the most recently accepted explanations for the negative effects of varicocele on sperm 45,46. In this study, we used the MDA level in seminal plasma as a marker for OS before and after varicocelectomy. We found that the MDA concentration decreased significantly after varicocelectomy. These results were in agreement with the previously published data correlating varicocele with high seminal OS 47,48. In agreement with our study, many studies have correlated the beneficial effect of varicocele repair with the seminal OS 49,50.
In the present study, we detected significant improvements in all sperm parameters under LM except for the percentage of tail abnormalities. This was not in agreement with the results of Seftel et al. 51, who reported a significant improvement in sperm concentration and motility, but not in morphology. In agreement with our findings, the majority of published studies have reported improvements in all sperm parameters after varicocelectomy 52–54. However, few studies failed to detect an improvement in sperm parameters and normal or assisted pregnancy outcomes after varicocelectomy 55.
Moreover, few studies have been published to evaluate the effect of varicocele on sperm ultrastructure. It was found that higher percentages of sperm ultrastructural anomalies were detected in patients with varicocele compared with control groups 56. Many studies have identified the negative effect of varicocele on sperm DNA and chromatin condensation using different methods 24,26,57.
To our knowledge, there is no published research on the efficacy of varicocelectomy (evaluated by TEM) on sperm ultrastructure. Using TEM, we could evaluate the percentage of various ultrastructural sperm abnormalities including head and tail abnormalities. A significant decrease in the percentage of all anomalies was detected after varicocelectomy.
Furthermore, we detected significant improvements in sperm head anomalies including nuclear vaculations and binucleated head after varicocelectomy. It is well known that sperm chromatin is condensed in the nucleus and this can be explored under TEM. Sperm chromatin and DNA is correlated with the fertilizing capacity of sperm. These results indirectly support previous reports of improved DNA integrity and sperm chromatin condensation after varicocelectomy with a consequent increase in pregnancy outcomes 24,26,57. Moreover, in the current paper, a significant decrease in the percentage of acrosomal anomalies was found after varicocelectomy. The rule of acrosome in fertilization is well established and normal acrosomal function is vital for fertilization 58. Varicocele has been correlated with reduced acrosomal integrity and abnormal acrosomal function 59,60.
The tail of sperm (from the centre to the outside of the axoneme) is composed of outer dense fibres and mitochondria. Axoneme is composed of central singlet tubules surrounded by nine outer doublets (9+2) 61. It is well established that sperm tail is the part of sperm responsible for motility. Sperm motility results from anatomical and cytochemical interactions between axonemal structure, dense fibres and mitochondria 62,63. Axonemal abnormalities including the absence of central singlet, absence of outer doublets and total axonemal absence have been reduced significantly after varicocelectomy, which in turn leads to an increase in the percentage of sperm motility. Interestingly, our results indicated that tail abnormalities under LM showed no significant difference after varicocelectomy. This could be attributed to the fact that axonemal ultrastructure could not be observed except under TEM.
Strengths and limitations of the study
The strength of the present study was that it is the first study, to our knowledge, to detect the efficacy of microsurgical varicocelectomy on sperm ultrastructure using TEM. However, this study has several limitations. First, the cross-sectional nature of the study hinders the inference of causality from the associations identified between infertility and sperm characteristics. Second, the small sample (n=25) does not allow generalization of the results. We have used a nonparametric test to deal with the problem of the small sample size and to obtain significant results to aid in the inference of the research findings. Finally, the lack of a control group affects the interpretation of the results.
The current findings indicate that varicocele exerts deteriorating effects on sperm parameters and sperm ultrastructure. This could be attributed to the elevation of ROS. In addition, microsurgical varicocelectomy had a beneficial effect on seminal ROS, sperm parameters and sperm ultrastructure. Sperm ultrastructure could be a valuable method to detect the deteriorating effect of varicocele and the expected results of varicocele repair methods. To our knowledge, this is the first study to detect the efficacy of microsurgical varicocelectomy on sperm ultrastructure using TEM. The main limitations of our study are the small sample and the lack of a control group.
Conflicts of interest
There are no conflicts of interest.
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