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Infertility is a problem that affects ∼13–15% of couples in the world and is defined as the failure of a couple to conceive after 1 year of unprotected regular sexual intercourse . There are various factors that affect male fertility, such as genitourinary infections, immune factors, genetic abnormalities, gonadotoxin exposure, endocrine disorders, systemic diseases, cancer and varicocele. Varicocele alone accounts for 35% of infertility cases [2, 3].
A varicocele is an abnormal dilatation, elongation and tortuosity of the pampiniform plexus veins of the spermatic cord, and is more frequent on the left side. Varicocele affects ∼15–20% of the adolescents in the world population, accounting for ∼19–41% of cases of primary infertility and >80% of secondary infertility [3, 4]. Patients with varicocele should consider this association, because fertility may decrease over time.
Different mechanisms have been proposed by which varicocele can cause infertility in men, such as scrotal hyperthermia, hypoxia, hormonal imbalances and reflow of metabolites from renal and/or adrenal glands . These conditions could cause an imbalance between the molecules that generate oxidative stress and antioxidant protection [5-7], leading to oxidative stress, which can affect cell function through mechanisms such as lipid peroxidation of the sperm membrane and fragmentation of nuclear material of the sperm [6, 8, 9].
Various methods have been used to assess DNA fragmentation , one of which is the sperm chromatin dispersion (SCD) test, which is a simple assay that does not require complex instrumentation. The test is based on subjecting the sperm to the removal of nuclear proteins and then to colouration, so that a halo around the sperm head may be observed microscopically in sperm with intact DNA, and in the case of chromatin with fragmented DNA the halo is observed to be non-existent or very small .
Most of the studies examining the association between varicocele and fertility are principally focused on the damage to the testis; however, it is clear that varicocele can affect the epididymis (which is an intrascrotal organ). The epididymis plays a crucial role in sperm maturation and acquiring of progressive motility and is the place where the spermatozoa acquire fertilizing capacity . During their transit in the epididymis, sperm undergo maturation processes of the plasma membrane, through modification of sugars, proteins and lipids . The sperm nucleus also undergoes changes in chromatin condensation , increasing the disulfide bridges of the DNA  and leading to stabilization of the genetic material. In addition, when comparing the oxidative stress levels in the seminal plasma of men with and without vasectomy it was observed that peroxidation levels in semen increased after vasectomy, indicating that the epididymis contributes to antioxidant capacity in transit and storage sperm in this organ as well as in the ejaculate .
The epididymis produces various molecules which have been used to evaluate their function, such as α-glucosidase (neutral and acid), L-carnitine and glycerophosphocholine . One marker widely used is the enzyme neutral α-glucosidase (NAG), whose origin is almost exclusively the epididymis and which can be measured in semen. This is useful for assessing dysfunction and/or obstruction in the epididymis [18, 19].
An additional aspect that may be affected by the presence of varicocele is the quality and integrity of the sperm membrane, as shown by two studies, one using flow cytometry  and the other fluorometry . A test that is often used to assess the functional integrity and the sperm-fertilizing potential is the hypoosmotic swelling test (HOST), because the ability of the sperm tail to swell in the presence of a hypoosmotic solution is a sign that transport of water across the membrane occurs normally, i.e. is a sign of membrane integrity and normal functional activity . Hyaluronan-binding assay (HBA), another test used to evaluate the sperm membrane, was created a decade ago, and assesses sperm-binding to hyaluronic acid, and hence the maturity of the membrane. Spermatozoa that are able to bind to hialuronic acid are mature and have completed the spermiogenetic processes of sperm plasma membrane remodeling, cytoplasmic extrusion, and nuclear histone–protamine replacement and show unreacted acrosomes .
The aim of the present study was to assess the relationship between a marker of epididymal function, NAG, with SCD and the integrity and maturity of the sperm membrane (tested using the HOST and an HBA) in patients with or without varicocele. Additionally, the correlation between epididymal function and the results of the above tests and semen quality measures was performed.
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The relationship between the measures of semen quality, the epididymal marker, NAG, and HBA results in the varicocele and control groups is shown in Table 1. There was no difference in age between the groups. With regard to semen analysis, patients with varicocele (grades II + III) had lower-quality sperm than those in the control group in terms of volume, sperm concentration, motility, viability, normal morphology and HOST results (all statistically significant). Men with varicocele had more sperm defects when compared with the control group. In addition, patients in the varicocele group had higher pH values and PMN. The epididymal marker NAG activity was found to be significantly lower in the varicocele than in the control group. Additionally, the results of the HBA test, which was used to test the maturity of sperm membrane, were significantly lower in the varicocele group than in the control group.
Table 1. Semen characteristics, NAG levels, HOST and sperm HBA results in the varicocele and control groups
|Variable||Control group, n = 30||Varicocele group, n = 60||P|
|Mean (sd)||Mean (sd)|
|Age, years||31.4 (4.4)||29.1 (6.4)||ns|
|Semen volume, mL||3.7 (0.9)||2.6 (1.0)||<0.001|
|pH||7.5 (0.1)||7.8 (0.2)||<0.001|
|Sperm × 106/mL||78.7 (21.7)||62.9 (37.5)||<0.001|
|Sperm × 106/ejaculate||290.5 (104.7)||167.9 (133.6)||0.03|
|Motility a + b, %||67.4 (5.8)||46.2 (13.4)||<0.001|
|Vitality, %||75.9 (2.7)||70.2 (6.8)||<0.001|
|Kruger morphology, %||12.1 (1.0)||8.6 (2.7)||<0.001|
|PMN x106/ejaculate||0.4 (0.2)||1.7 (0.7)||<0.001|
|HOST, % sperm with hypoosmotic swelling||65.5 (2.9)||47.8 (11.0)||<0.001|
|NAG, mUI/ejaculate||32.1 (7.7)||14.1 (7.1)||<0.001|
|HBA, % sperm binding to hyaluronic acid||79.8 (7.5)||65.1 (22.6)||<0.001|
Table 2 shows a comparison of sperm DNA fragmentation, assessed using the SCD test, between the control and the varicocele groups. Two main categories are shown: sperm without fragmented DNA and sperm with fragmented DNA (DNA fragmentation index). The percentage of sperm without fragmented DNA was calculated using the sum of the categories large and medium halos. The percentage of sperm with fragmented DNA was calculated using the sum of the categories small halo, without halo and core degraded (no halo and present a core weakly stained), as explained above.
Table 2. Data from sperm DNA fragmentation by SCD from infertile patients with varicocele (II+III) and from the control group without varicocele
|SCD test||Control group, n = 30||Varicocele group, n = 60||P|
|Mean (sd)||Mean (sd)|
|Sperm without fragmented DNA, %||86.7 (4.3)||63.8 (3.8)||<0.001|
|Big halo||73.6 (4.9)||54.4 (3.3)||<0.001|
|Medium halo||13.1 (3.4)||9.4 (2.9)||<0.001|
|Sperm with fragmented DNA, %||13.3 (4.2)||36.2 (3.8)||<0.001|
|Small halo||8.0 (3.1)||12.1 (2.8)||<0.001|
|No halo||3.8 (2.0)||12.1 (2.7)||<0.001|
|Degraded||1.5 (0.9)||12.0 (4.6)||<0.001|
Significantly greater DNA fragmentation was observed in sperm samples from the varicocele group than from the control group: DNA fragmentation index 13.3% in the control group and 36.2% in the varicocele group. The fragmentation observed in the control group was mainly in the ‘small halo’, followed by ‘no halo’ and ‘degraded’ categories, while in the varicocele group, a similar percentage of fragmentation was observed in each of these three categories. The ratio of total percentage of sperm with fragmented DNA to degraded (% of sperm with no halo and core irregularly or weakly stained) was 3.0 (36.2/12.0) in the varicocele group and 8.7 in the control group (13.3/1.5).
Additionally the correlation between the semen quality measures, NAG levels, the HOST findings and the DNA fragmentation index (SCD test) in all the sperm samples was calculated for both groups (n = 90). The results of the Pearson correlation analysis are shown in Table 3. An increase in NAG was found to be correlated with an increase in semen quality (volume, sperm concentration, normal morphology, motility, viability, HOST results) and maturity of sperm membrane as evaluated by the HBA. Furthermore, NAG was negatively correlated with pH, PMN and the DNA fragmentation index.
The HBA results were positively correlated with some semen variables (normal morphology, motility, viability as well as HOST results) but not with others (volume and sperm concentration). A negative correlation between HBA and pH, as well as between PMN and DNA fragmentation index was shown. The DNA fragmentation index (% SCD) was negatively correlated with all the variables evaluated, except for pH values.
Remarkably, NAG activity presented a very strong negative correlation with DNA fragmentation index (Pearson's r = −0.70). Additional important results were the positive correlation between the SCD and PMN percentages (r = 0.69) and the negative correlation with HOST results (r = −0.64). HBA showed correlation with several seminal parameters, but these correlations were always lower than found with NAG and SCD tests.
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In the present study, we evaluated the activity of the epididymal marker NAG and its relationship with the percentage of DNA fragmentation (SCD), quality and maturity of the plasma membrane (HOST and HBA) in patients with varicocele. The results show that the epididymis has a reduced function during varicocele that is associated with decreased NAG activity and lower-quality sperm membrane and nucleus.
The epididymis is an important organ involved in sperm maturation and can be divided into three regions: the head (caput), body and tail (cauda). In all segments ‘principal cells’ are metabolically active, with molecules capable of producing reactive oxygen species (ROS) and enzymatic antioxidants (superoxide dismutase, glutathione peroxidase 5 and glutathione transferase) and non-enzymatic molecules (taurine and glutathione) . The production of antioxidants by the epididymis is essential to counteract the damaging events resulting from excessive ROS production originated locally or in transit from the testis .
The epididymal marker NAG is absent in the semen of patients with obstructive azoospermia, indicating that this enzyme is derived exclusively from the epididymis, mainly from the head and body of this organ . One study showed that NAG levels are lower in the seminal plasma of patients with severe oligozoospermia or epididymal pathologies , possibly as a result of lower testicular testosterone levels. Testosterone is necessary for the function of the epididymis , as was shown by a study in rats, in which NAG activity was reduced significantly in all segments of the epididymis 7 days after castration . Previous studies show that NAG activity is positively correlated with traditional semen variables [17, 29, 31, 32], and are consistent with the results of the present study, which suggest that a normal secretion of this epididymal marker is associated with semen quality.
A study conducted using the an experimental varicocele model in rats showed that varicocele induces degeneration of the epididymal epithelium, a reduction in the tubular diameter and ultrastructural changes in principal cells. Furthermore, carnitine (an antioxidant) and the α-glucosidase activity in the caput, corpus and cauda epididymis of the rats with varicocele were lower than in the controls . Additionally, the index of the apoptosis of the epididymis epithelium was significantly higher and the content of NAG was significantly lower in rat with varicocele and a positive correlation was found between epididymal damage and the duration of varicocele [34, 35]. The expression of hypoxia-induced factor-1-α was increased during experimental varicocele in rat, and was associated with a decrease in carnitine and NAG levels .
Reducing the concentration of NAG in human semen has been shown to be associated with a significant increase in DNA fragmentation , results that are consistent with those of the present study, where we show a correlation between NAG levels and SCD with a value of r = −0.70. One of the most important antioxidant enzymes produced by the human epididymis (body and tail) is glutathione peroxidase ; it is possible that damage to the epididymis, reflected by a decrease in NAG levels, could be associated with a decrease in production of glutathione peroxidase; increased production of ROS and DNA fragmentation then occurs.
Varicocele is associated with a significant increase in DNA damage with increased oxidative stress and a reduction in mitochondrial activity and acrosome integrity [8, 38]. Sakkas and Alvarez (2010)  show that during varicocele there are several mechanisms that can cause damage to nuclear and mitochondrial DNA; however, the main source of DNA fragmentation is post-testicular, during transit through the epididymis. It has been reported that DNA fragmentation is higher in spermatozoa from the cauda of the epididymis and ejaculate, compared with the testis [40, 41]. Hypoxia and thermal stress [36, 42] during varicocele could cause an increase in ROS production by principal cells, causing damage to the epididymis and the spermatozoa stored there .
An important source of oxidative stress is polymorphonuclear leukocytes. The semen of patients with varicocele present a higher number of PMN compared with men with semen defined as normal, as observed in the present study and in previous studies [8, 31, 32]. An increase in PMN in semen can promote the generation of oxidative stress and exceed the antioxidant capacity, with a rise in DNA fragmentation and plasma membrane peroxidation . In the present study, the PMN concentration in sperm was correlated negatively with the NAG concentration (r = −0.56), which is consistent with previous reports [29, 31, 32], and positively with the DNA fragmentation index (r = 0.69). The origin of the leukocytes present in the semen has long been debated. It is believed that they come mainly from the epididymis and prostate , and it is possible that a decrease in NAG levels from the epididymis is associated with an increase in PMN, possibly as a result of epididymal inflammation or impairment.
Interestingly, in the present study, sperm samples found to be in the category degraded (those spermatozoa that show no halo and present a core irregularly or weakly stained) were present at a ratio of 1 to 3 (vs total fraction of sperm with fragmented DNA) in patients with varicocele, while the ratio was 1 to 8.7 in the control group, results that were similar to those of a previous study . This increase in the proportion of degraded DNA in the group with varicocele may indicate that the level of damage of the sperm nucleus is so strong that it affects the nuclear protein matrix. Sperm nucleus degradation may be caused by a poor compaction of sperm nucleus in patients with varicocele , making sperm more susceptible to peroxidation agents. This hypothesis is reinforced by a recent study that has shown that varicocelectomy improves integrity and sperm chromatin compaction .
The HOST evaluates the functional integrity of the plasma membrane and is a useful marker of fertility potential . In the present study, a highly significant inverse correlation (r = −0.69) between HOST results and SCD was observed, indicating that the HOST serves as an indicator of nuclear quality. These results are consistent with a recent report showing that sperm with HOST grades D, E and F (the most swollen) were those that had lower DNA fragmentation as assessed by TUNEL assay and SCD .
An additional test used to evaluate the quality of the sperm membrane is the HBA test. Hyaluronic acid binds spermatozoa that have good morphology, less DNA fragmentation, better DNA integrity, lower frequency of aneuploidies and better binding to the zonapellucida [23, 48-50]. Sperm bind to the hyaluronic acid receptor using the hyaluronidase enzymes. Hyaluronidases are a family of enzymes that catalyse the degradation of hyaluronic acid, abundant in the extracellular matrix. Evidence indicates that hyaluronidases are membrane proteins with multifunctional essential, enzymatic and non-enzymatic, roles in fertilization, as cumulus penetration, zona binding and hyaluronic acid receptor . Some hyaluronidases have been described in sperm , e.g. SPAM1 (or PH-20), which has been proposed to be captured by the sperm in the passage by the testis and epididymis  and has been associated with the ability to penetrate the matrix of cumulus oocyte .
To summarize, we observed that in patients with varicocele there was a reduction in the percentage of sperm bound to hyaluronic acid, indicating decreasing sperm hyaluronidases, which could be associated with a diminution of the ability to penetrate the cumulus and perform fertilization. Additionally, we found a positive correlation between HBA results and NAG levels and a negative correlation between HBA results and SCD, indicating an association between reduced hyaluronidase with an increase in DNA fragmentation (r = −0.30), as shown in a previous report ; however, this correlation was much lower than that observed between HOST results and SCD (r = −0.64). As in a previous report we found a positive correlation between HBA and semen quality .
We concluded that men with varicocele have lower levels of NAG activity in the semen as well as a reduction of sperm binding to hyaluronic acid, whereas DNA fragmentation levels were much higher. Additionally, we observed a strong correlation between lower levels of NAG and greater DNA fragmentation and a positive correlation between NAG and HBA results of lower magnitude. These results indicate that varicocele may compromise not only the testis but also the epididymis, causing a reduction in seminal quality measures and impaired quality of the sperm membrane and nucleus.