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Keywords:

  • neutral α-glucosidase activity;
  • hypoosmotic swelling test;
  • sperm hyaluronan-binding assay;
  • sperm chromatin dispersion test;
  • varicocele

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Objective

  • To assess the relationship between a marker of epididymal function and both the fragmentation of the sperm nucleus and the integrity and maturity of the sperm membrane in patients with or without varicocele.

Patients and Methods

  • Semen samples were obtained from men with varicocele grades II and III (n = 60) and from a control group with zoospermia defined as normal (n = 30).
  • Samples were evaluated by a spermiogram, a hypoosmotic swelling test (HOST), neutral α-glucosidase (NAG) enzyme activity, sperm hyaluronan-binding assay (HBA) and DNA fragmentation using a sperm chromatin dispersion (SCD) test.

Results

  • Seminal plasma NAG levels, percentage of sperm bound to hyaluronic acid, HOST-positive cells and sperm quality were significantly lower in the varicocele compared with the control group.
  • Higher levels of sperm DNA fragmentation, as measured by SCD, were also observed in the varicocele group compared with the control group.
  • Seminal NAG activity levels showed a strong negative correlation with DNA fragmentation and a significant positive correlation with the HBA test and the HOST.

Conclusions

  • Varicocele causes a reduction in NAG activity by the epididymis that is associated with damage to both the membrane and sperm nucleus and a reduction in the seminal parameters.
  • NAG levels were correlated with the quality of the sperm membrane and nucleus.
  • Data suggest that a reduction of fertilization capacity in men during varicocele can result from damage to both the testis and the epididymis.

Abbreviations
SCD

sperm chromatin dispersion

NAG

neutral α-glucosidase

HOST

hypoosmotic swelling test

HBA

hyaluronan-binding assay

ROS

reactive oxygen species

PMN

polymorphonuclear neutrophils

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

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 [1]. 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 [2]. 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 [10], 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 [11].

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 [12]. During their transit in the epididymis, sperm undergo maturation processes of the plasma membrane, through modification of sugars, proteins and lipids [13]. The sperm nucleus also undergoes changes in chromatin condensation [14], increasing the disulfide bridges of the DNA [15] 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 [16].

The epididymis produces various molecules which have been used to evaluate their function, such as α-glucosidase (neutral and acid), L-carnitine and glycerophosphocholine [17]. 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 [20] and the other fluorometry [21]. 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 [22]. 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 [23].

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.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Study Population

A prospective study was carried out in two groups: a study group comprising 60 patients with grades II or III varicocele (26 grade II and 34 grade III) and a control group of 30 men with zoospermia defined as normal and without varicocele. Both groups attended a single centre (Centro Diagnóstico de Infertilidad y Enfermedades Genéticas, ULA, Mérida) for andrological evaluation, between May 2010 and July 2011. Informed written consent was obtained as well as approval for the investigation project from the research ethics committee of the Universidad de Los Andes. All subjects were evaluated by two male infertility specialists (medical records, physical examination and inclusion/exclusion criteria) and a semen analysis was performed according to the WHO fifth edition (2010) guidelines for semen analysis. Exclusion criteria were: the presence of urogenital infections; a positive assay for anti-Chlamydia trachomatis antibodies (ImmunoComb II® Monovalent IgA kit; Orgenics, Yavne, Israel); azoospermia or hypogonadism; and systemic diseases such as cancer and endocrine pathology (and their treatments), which would lead to testicular alterations. The presence of a clinical varicocele was determined by palpation and observations in the standing position before and during the Valsalva manoeuvre and confirmed by colour Doppler ultrasonography. Varicocele was classified as grade II (palpable without Valsalva manoeuvre) or grade III (visible through the scrotal skin).The study was conducted according to the guidelines established by the Declaration of Helsinki for human research outlined in the Code of Bioethics and Biosafety FONACIT (Fondo Nacional de Ciencia Tecnología e Innovación).

Semen Analysis

Semen analyses were performed according to the WHO criteria (2010). The semen sample was collected by masturbation after 2–7 days of sexual abstinence and analysed within 1 h of ejaculation. The semen volume, pH, sperm count and total motile sperm count were determined. Seminal plasma was separated by centrifugation of semen at 1200× g, and stored at −20 °C to until NAG activity determination.

Hypoosmotic Swelling Test

A HOST was performed by mixing 0.1 mL of semen with 1.0 mL of 150 mmol/kg hypoosmotic solution (obtained by mixing 7.35 g sodium citrate and 13.5 g fructose in 1000 mL distilled water). The mixture was incubated for 30 min at 37 °C. Then 0.2 mL of the mixture was examined at a ×400 magnification under a phase contrast microscope. The percentages of reacted sperm (swollen tails) and non-reacted sperm (unswollen tails) were assessed by counting a minimum of 200 spermatozoa with phase contrast optics at ×400 magnification [23, 24].

Polymorphonuclear Neutrophils

Polymorphonuclear neutrophils (PMN) were counted after peroxidase staining. Briefly, 0.0375% H2O2 was added to 4 mL benzidine stock solution (0.0125% w/v benzidine [Sigma Aldrich, St Louis. MO, USA], in 50% ethanol). Then, 10 μL of ejaculate was mixed with 20 μL of fresh benzidine-H2O2 solution. After 5 min, 160 μL of PBS was added, and the peroxidase-positive cells (round brown cells) and peroxidase-negative (unstained) cells were counted, using a haemocytometer chamber under a phase contrast microscope.

Neutral α-Glucosidase Activity

Levels of NAG were measured in all samples of seminal plasma according to the photometric method described by Guerin et al. [25]. Seminal plasma contains both a NAG isoenzyme, which originates in the epididymis, and an acid isoenzyme provided by the prostate. The latter can be selectively inhibited by SDS to allow the measurement of NAG activity, which indicates epididymal function. Accounting for non-glucosidase-related substrate breakdown, by using the inhibitor castanospermine, makes the assay more sensitive. The substrate (paranitrophenyl a-p-nitrophenol-glucopyranoside) was hydrolysed specifically to NAG into paranitrophenyl, during a 2-h incubation at 37 °C, pH 6.8. The quantity of paranitrophenyl was measured by spectrophotometer at a wavelength of 405 nm. One international unit (U) of glucosidase activity was defined as the production of 1 μmol product (p-nitrophenyl) per min at 37 °C, pH 6.8. In the latest WHO guidelines (2010) [24], a NAG >20 mU/ejaculate, was established as a reference value.

Sperm HBA

The sperm HBA test was carried out using a commercial HBA kit (Biocoat, Fort Washington, PA, USA) according to the manufacturer's instructions. Briefly, 10 μL of semen (well mixed) was added to the centre of the HBA chamber and the Cell-Vu grid cover slip was put on without trapping air bubbles. The cover slip provided a grid of 100 squares (each 0.1 mm × 0.1 mm) within a viewing circle. After incubation of the slide for 15 min, the unbound motile sperm and the bound motile sperm were counted in the same grid squares. For the HBA, 400 motile sperm were counted. The percentage of hyaluronan-binding sperm was calculated using the bound motile sperm divided by the sum of bound and unbound motile sperm counted in the same squares and then multiplied by 100 [26].

Sperm Chromatin Dispersion Test

An SCD test (Halosperm® kit; INDAS Laboratories, Madrid, Spain) [11] was used to analyse sperm DNA fragmentation. Gelled aliquots of low-melting point agarose in Eppendorf tubes were provided in the kit, each one to process a semen sample. Eppendorf tubes were placed in a water bath at 90–100 °C for 5 min to fuse the agarose, and then in a water bath at 37 °C. After 5-min incubation for temperature equilibration at 37 °C, 60 μL of the thawed semen sample was added to the Eppendorf tube and mixed with the fused agarose. A total of 20 μL of the semen-agarose mix was pipetted onto precoated slides, provided in the kit, and covered with a 22 × 22 mm coverslip. The slides were placed on a cold plate in the refrigerator (4 °C) for 5 min to allow the agarose to produce a microgel with the sperm cells embedded within. The coverslips were gently removed and the slides immediately immersed horizontally in an acid solution, previously prepared by mixing 80 μL of HCl 0.08 N (from the kit) with 10 mL of distilled water, and incubated for 7 min. The slides were horizontally immersed in 10 mL of the lysing solution for 25 min. After washing for 5 min in a tray with abundant distilled water, the slides were dehydrated in increasing concentrations of ethanol (70, 90 and 100%) for 2 min each, air-dried, and stored at room temperature in tightly closed boxes, in the dark.

For brightfield microscopy, slides were covered horizontally with a mix of Wright's staining solution (Merck, Darmstadt, Germany) and phosphate buffer solution (1:1) for 5–10 min with continuous airflow. Then the slides were briefly washed in tap water and allowed to dry. Strong staining was preferred so as to easily visualize the periphery of the dispersed DNA loop halos. The distilled water, ethanol, Wright staining solution (Merck; 1.01383.0500) and phosphate buffer solution (Merck; 1.07294.1000) were not provided in the kit. A minimum of 500 sperm per sample were scored under the ×100 objective of the microscope.

Five SCD patterns were established [11]: (i) sperm cells with large halos: those with a halo width similar to or wider than the minor diameter of the core; (ii) sperm cells with medium halos: a halo size between those with large and very small halos; (iii) sperm cells with very small halos: a halo width similar to or smaller than one-third of the minor diameter of the core; (iv) sperm cells without a halo; (v) sperm without halo and degraded (those sperm that show no halo and present a core irregularly or weakly stained). Sperm cells with very small halos, without halos and without halo and nuclear core degradation contain fragmented DNA. Finally, nucleoids that did not correspond to sperm cells were separately scored.

Statistical Analyses

Statistical analysis was performed using spss 10.0 statistical software for Windows (SPSS, Chicago, IL, USA). Basic descriptive statistics (mean values ± sd) were calculated for the varicocele group and the control group. Differences between the varicocele and control groups were determined using a t-test. Pearson's correlation coefficient analysis was performed, and a P value < 0.05 was considered to indicate statistical significance.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

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
VariableControl group, n = 30Varicocele group, n = 60P
Mean (sd)Mean (sd)
  1. ns, nonsignificant.

Age, years31.4 (4.4)29.1 (6.4)ns
Semen volume, mL3.7 (0.9)2.6 (1.0)<0.001
pH7.5 (0.1)7.8 (0.2)<0.001
Sperm × 106/mL78.7 (21.7)62.9 (37.5)<0.001
Sperm × 106/ejaculate290.5 (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/ejaculate0.4 (0.2)1.7 (0.7)<0.001
HOST, % sperm with hypoosmotic swelling65.5 (2.9)47.8 (11.0)<0.001
NAG, mUI/ejaculate32.1 (7.7)14.1 (7.1)<0.001
HBA, % sperm binding to hyaluronic acid79.8 (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 testControl group, n = 30Varicocele group, n = 60P
Mean (sd)Mean (sd)
Sperm without fragmented DNA, %86.7 (4.3)63.8 (3.8)<0.001
Big halo73.6 (4.9)54.4 (3.3)<0.001
Medium halo13.1 (3.4)9.4 (2.9)<0.001
Sperm with fragmented DNA, %13.3 (4.2)36.2 (3.8)<0.001
Small halo8.0 (3.1)12.1 (2.8)<0.001
No halo3.8 (2.0)12.1 (2.7)<0.001
Degraded1.5 (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.

Table 3. Correlation coefficients between seminal variables, NAG, SCD and sperm HBA in all the men evaluated (n = 90)
 CorrelationPearson's r
  1. [UPWARDS ARROW], increasing; [DOWNWARDS ARROW], decreasing. ns, nonsignificant (all other correlations were significant [P < 0.05]).

[UPWARDS ARROW] NAG, mUI/ejaculate[DOWNWARDS ARROW] pH−0.44
[UPWARDS ARROW] Semen volume (mL)0.78
[UPWARDS ARROW] Sperm × 106/ejaculate0.61
[UPWARDS ARROW] Kruger morphology (%)0.53
[UPWARDS ARROW] Motility a + b (%)0.49
[UPWARDS ARROW] Vitality (%)0.35
[DOWNWARDS ARROW] PMN/ejaculate−0.56
[UPWARDS ARROW] HOST (% sperm with hypoosmotic swelling)0.58
[UPWARDS ARROW] % HBA0.26
[DOWNWARDS ARROW] Sperm with fragmented DNA (%)−0.70
[UPWARDS ARROW]HBA, % sperm binding to hyaluronic acid[DOWNWARDS ARROW] pH−0.46
Semen volume (mL)ns
Sperm × 106/ejaculatens
[UPWARDS ARROW] Kruger morphology (%)0.38
[UPWARDS ARROW] Motility a + b (%)0.34
[UPWARDS ARROW] Vitality (%)0.41
[DOWNWARDS ARROW] PMN/ejaculate−0.22
[UPWARDS ARROW] HOST (% sperm with hypoosmotic swelling)0.31
[UPWARDS ARROW] NAG (mUI/ejaculate)0.26
[DOWNWARDS ARROW] Sperm with fragmented DNA (%)−0.30
[UPWARDS ARROW] Sperm with fragmented DNA (%)[UPWARDS ARROW] pH0.42
[DOWNWARDS ARROW] Semen volume (mL)−0.41
[DOWNWARDS ARROW] Sperm × 106/ejaculate−0.40
[DOWNWARDS ARROW] Kruger morphology (%)−0.54
[DOWNWARDS ARROW] Motility a + b (%)−0.64
[DOWNWARDS ARROW] Vitality(%)−0.40
[UPWARDS ARROW] PMN/ejaculate0.69
[DOWNWARDS ARROW] HOST (% sperm with hypoosmotic swelling)−0.64
[DOWNWARDS ARROW] HBA (%)−0.30
[DOWNWARDS ARROW] NAG (mUI/ejaculate)−0.70

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.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

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) [27]. 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 [2].

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 [28]. One study showed that NAG levels are lower in the seminal plasma of patients with severe oligozoospermia or epididymal pathologies [17], possibly as a result of lower testicular testosterone levels. Testosterone is necessary for the function of the epididymis [29], 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 [30]. 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 [33]. 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 [36].

Reducing the concentration of NAG in human semen has been shown to be associated with a significant increase in DNA fragmentation [37], 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 [27]; 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) [39] 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 [2].

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 [10]. 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 [8], 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 [43]. 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 [44], 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 [45].

The HOST evaluates the functional integrity of the plasma membrane and is a useful marker of fertility potential [46]. 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 [47].

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 [51]. Some hyaluronidases have been described in sperm [52], e.g. SPAM1 (or PH-20), which has been proposed to be captured by the sperm in the passage by the testis and epididymis [53] and has been associated with the ability to penetrate the matrix of cumulus oocyte [52].

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 [48]; 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 [23].

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

This investigation was partially supported by CDCHT (FA-339-05-07-B) from the Universidad de los Andes and Decanato de Investigación y Desarrollo, Universidad Simón Bolívar. We thank Dr Judith Velasco for the critical review of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References