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Keywords:

  • assisted reproductive technologies;
  • oxidative stress;
  • varicocele

Abstract

  1. Top of page
  2. Abstract
  3. Role of sperm factors in ICSI
  4. Effects of varicocele on the outcomes of ART
  5. Evidence from human and animal studies
  6. Proliferation and apoptosis of germ cells
  7. Testicular hypoxia
  8. Other mechanisms
  9. Interstitial lesions
  10. Oxidative stress
  11. NO
  12. Anti-oxidants
  13. Heat stress
  14. Conclusions
  15. Conflict of interest
  16. References

Varicocele is the most common and treatable cause of male infertility. Studies of a rat experimental left varicocele model and human testicular biopsy samples have shown the involvement of various factors in its pathophysiology. Among them, oxidative stress plays a major role in impairing spermatogenesis and sperm function. Therefore, in addition to palpation, scrotal ultrasonography and color Doppler ultrasound, evaluation of testicular oxidative stress (e.g. scrotal temperature is a surrogate parameter) is recommended to enable diagnosis and suitable treatment of varicocele. Varicocelectomy increases the fertilization, pregnancy and live birth rates, indicating improved sperm function; it is therefore important even in couples undergoing intracytoplasmic sperm injection. Routine sperm-function tests are warranted to monitor the sperm quality after varicocelectomy and consequent improvement in the outcomes of assisted reproductive technology. Furthermore, the indications of varicocelectomy in assisted reproductive technology should be widened.


Abbreviations & Acronyms
4-HNE =

4-hydroxy-2-nonenal

8-OHdG =

8-hydroxy-2′-deoxyguanosine

ART =

assisted reproductive technology

CCM =

cerebral cavernous malformation

CREM =

cyclic adenosine 3′,5′-monophosphate response element modulator

ELV =

experimental left varicocele

eNOS =

endothelial nitric oxide synthase

GDNFα1 =

glial cell line-derived neurotropic factor receptor α1

GSR =

glutathione reductase

GPX =

glutathione peroxidase

GST =

glutathione S-transferase

HIF1 =

hypoxia-inducible factor 1

HO-1 =

heme oxygenase 1

ICSI =

intracytoplasmic sperm injection

IL-1 =

interleukin-1

iNOS =

inducible nitric oxide synthase

IUI =

intrauterine insemination

IVF =

in vitro fertilization

MDA =

malondialdehyde

nNOS =

neuronal nitric oxide synthase

NO =

nitric oxide

PDRN =

polydeoxyribonucleotide

PCNA =

proliferating cell nuclear antigen

PN =

pronuclear

O2- =

superoxide anion

ROS =

reactive oxygen species

RNS =

reactive nitrogen species

SOD =

superoxide dismutase

TUNEL =

terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling

VEGF =

vascular endothelial growth factor

Role of sperm factors in ICSI

  1. Top of page
  2. Abstract
  3. Role of sperm factors in ICSI
  4. Effects of varicocele on the outcomes of ART
  5. Evidence from human and animal studies
  6. Proliferation and apoptosis of germ cells
  7. Testicular hypoxia
  8. Other mechanisms
  9. Interstitial lesions
  10. Oxidative stress
  11. NO
  12. Anti-oxidants
  13. Heat stress
  14. Conclusions
  15. Conflict of interest
  16. References

Varicocele is the most common cause of male infertility. Its prevalence is notably high (∼40%) in couples with male factor infertility compared with the general population (≤20%).1 While debating the pathophysiology of varicocele, its influence on infertility treatment should be considered. Twenty years ago, we reviewed that varicocele causes spermatogenic disorder, and attempted to detect and treat as many cases as possible.2 In the 1990s, the widespread application of ICSI lowered the importance of varicocele treatment. Furthermore, the usefulness of varicocele treatment itself was regarded as doubtful.3,4 In contrast, two recent meta-analyses have shown that varicocelectomy significantly improves sperm concentration and motility in infertile men with palpable varicocele and abnormal preoperative semen parameters,5,6 and increases the odds ratio of spontaneous pregnancy.6,7 Furthermore, we attempted to identify treatable varicocele, because the indications of varicocelectomy are limited.8,9 The effectiveness of this surgical procedure has been confirmed in palpable or large varicocele. However, even subclinical varicocele shows remarkable postoperative improvement, and several studies have shown that the improvements in testicular histology10 and spermiogram11 are not related to varicocele size. In the era of ART, many urologists and andrologists consider that the purposes of varicocele treatment are to achieve patient satisfaction and cost-effectiveness by natural pregnancy or IUI instead of IVF or ICSI.

Daitch et al. observed that although varicocelectomy did not improve semen characteristics in all men studied, it appeared to improve pregnancy and live birth rates in couples with male factor infertility who underwent IUI.12 They concluded that men with varicocele should be screened for functional factors not measured by routine semen analysis. Actually, ICSI does not need motile sperm, sperm capacitation, acrosome reaction, cumulus penetration and zona binding, but no remarkable improvements in pregnancy and live birth rates have been reported since the 1990s. In addition to the role of female factors (e.g. advanced marriage age and obesity), the involvement of sperm factors has been recently revisited.13 Men with sperm DNA damage show reduced in vitro14,15 and in vivo16–18 fertility success. Furthermore, sperm DNA damage results in defective embryonic development,19 increased probability of implantation failure and risk of recurrent miscarriages.20 In fact, several studies, including our experience, support the importance of varicocelectomy even in couples undergoing ICSI (Table 1).21–23 These results suggest that varicocelectomy improves sperm function and DNA content, which cannot be evaluated by routine semen analysis. After the validation of these phenomena by randomized controlled studies, varicocelectomy will be highly recommended even in couples undergoing ICSI.

Table 1.  ICSI outcome in infertile couples in whom the male partner had treated or untreated clinical varicocele
 With varicocelectomyWithout varicocelectomyP-value
  1. †Series of oligoasthenozoospermia, ‡series of non-obstructive azoospermia, §χ2-test.

Esteves et al.21(n = 80)(n = 162) 
 % Two PN fertilization78.066.00.04
 % Clinical pregnancy60.045.00.04§
 % Live birth46.231.40.03§
Haydardedeoglu et al.22(n = 31)(n = 65) 
 % Two PN fertilization62.161.80.94
 % Clinical pregnancy74.252.30.03§
 % Live birth64.541.50.03§
Pasqualotto et al.23(n = 169)(n = 79) 
 % Two PN fertilization64.973.20.04§
 No. clinical pregnancy30.931.10.98§
Our experience (2007–2011)(n = 21)(n = 53) 
 % Two PN fertilization70.368.80.93
 % Clinical pregnancy61.928.30.02§
 % Live birth52.324.50.04§

Sperm or differentiated germ cells are susceptible to the harmful effects of varicocele, which might be directly involved in the outcomes of ICSI. Information on how spermatogenesis is disrupted is important to understand the pathophysiology of varicocele in the context of ART. Figure 1 lists the possible mechanisms of impaired spermatogenesis and sperm function. Various combinations of these factors, and not a single pathway, are involved in such impairment. Here, we review the pertinent literature, based on studies of a rat ELV model and human testicular biopsy samples mainly during the past decade.

image

Figure 1. Summary of the pathophysiology of varicocele to cause the spermatogenic disturbance, categorized by the mechanistic disturbance, causable factors and cell fate. A number of associated molecules, which are not listed here, have been reported to be involved in the process, but only the represented mechanisms are shown. Necrosis of the germ cell has not been reported in the testis with varicocele.

Download figure to PowerPoint

Effects of varicocele on the outcomes of ART

  1. Top of page
  2. Abstract
  3. Role of sperm factors in ICSI
  4. Effects of varicocele on the outcomes of ART
  5. Evidence from human and animal studies
  6. Proliferation and apoptosis of germ cells
  7. Testicular hypoxia
  8. Other mechanisms
  9. Interstitial lesions
  10. Oxidative stress
  11. NO
  12. Anti-oxidants
  13. Heat stress
  14. Conclusions
  15. Conflict of interest
  16. References

As shown in Table 1, three of four studies, including our experience, support the beneficial effects of varicocelectomy on the outcomes of ICSI.21,22 The fertilization rate is inconsistent among these studies, perhaps because it is dependent on the egg quality and stimulation methods of fertilized eggs. We and Haydardedeoglu et al. found no significant difference in the fertilization rate, but varicocelectomy ensures significantly higher pregnancy and live birth rates.22 These results show that sperm factors affect the pregnancy and live birth rates, but not the fertilization rate; in other words, they affect the development after fertilization.

Men with varicocele have apparent sperm DNA damage, for which oxidative stress is considered one of the most important factors.24 Information regarding sperm DNA damage in ejaculates is accumulating, because the samples can be obtained readily. In contrast, the mechanisms of such damage during spermatogenesis in humans are still hypothetical, because of the difficulty in obtaining testicular biopsy samples. The etiology of sperm DNA damage is multifactorial. Zini et al. have reported that varicocelectomy is associated with improved sperm DNA integrity and chromatin compaction, and could improve spermatogenesis, particularly spermiogenesis (i.e. the stage in spermatogenesis where compaction and stability of sperm DNA and chromatin occur).25 Researchers at Aitken's laboratory have found that disrupted spermatogenesis might cause the production of spermatozoa with impaired protamination, poor chromatin compaction (first step) and increased susceptibility to DNA damage (second step), and proposed a two-step hypothesis to explain the phenomenon of sperm DNA damage during spermiogenesis.26,27 In brief, impairment of protamination and chromatin compaction increases the susceptibility of affected cells (i.e. those with incomplete replacement of histones by protamines) to oxidative stress and causes defective spermiogenesis. Morphologically, abnormal spermatozoa show disruption of sperm-head actin by cadmium, which is present in high concentrations in men with varicocele.28 Defects in sperm–cervical mucus interaction, sperm survival in the female genital tract, sperm zona-binding ability and acrosin activity, as well as decreased response to the capacitating challenge, lower incidence and intensity of tyrosine phosphorylation, acrosome exocytosis, and microdeletion of the alpha-1 subunit of the sperm calcium channels have also been reported.29,30 Recently, Sadek et al. reported a significant increase in abnormal sperm chromatin condensation by acidic aniline blue staining before varicocelectomy; this increase was markedly improved after varicocelectomy.31 Sperm DNA fragmentation could result from aberrant chromatin packing during spermatogenesis.

Evidence from human and animal studies

  1. Top of page
  2. Abstract
  3. Role of sperm factors in ICSI
  4. Effects of varicocele on the outcomes of ART
  5. Evidence from human and animal studies
  6. Proliferation and apoptosis of germ cells
  7. Testicular hypoxia
  8. Other mechanisms
  9. Interstitial lesions
  10. Oxidative stress
  11. NO
  12. Anti-oxidants
  13. Heat stress
  14. Conclusions
  15. Conflict of interest
  16. References

According to the comprehensive reviews published in the past 10 years regarding the pathophysiology of varicocele,24,29,30,32–35 several hypotheses on the impairment of spermatogenesis have been proposed, including endocrine and testicular paracrine disturbances, heat stress, hypoxia, oxidative stress, accumulation of toxic substances, genetic disturbances, and autoimmunity, leading to decreased proliferation of germ cells and apoptosis (Fig. 1). By preliminary studies of heat-stressed rat testes, Shiraishi showed that autophagic cell death plays a role, at least in part, in the impairment of spermatogenesis.36 Retrograde flow of adrenal metabolites, such as adrenomedullin, catecholamines, cortisol, prostaglandins E and F, and serotonin was considered to be an underlying mechanism, but these metabolites do not appear to be associated with the effects of varicocele.1 The pathophysiology of varicocele is multifactorial, and none can independently explain the mechanisms of impaired spermatogenesis. Therefore, a combination of several factors affects spermatogenesis and sperm function, and the relative involvement of these factors is different in each patient. Although only a small percentage of men with varicocele are infertile, varicocele accelerates spermatogenic injury along with other underlying cofactors37 and/or intrinsic testicular underdevelopment.38 Several studies of human seminal plasma and sperm have shown the involvement of oxidative stress, the most plausible factor impairing sperm function, but the effects of oxidative stress in the testes and ejaculates are unknown. In the present review, we summarize the intratesticular events on the basis of studies of men with varicocele and rats with ELV.

Testicular biopsy in men with varicocele has shown depressed spermatogenesis with maturation arrest, sloughing of spermatogenic epithelium, profusion of Leydig cells, thickening of the tubular basement membrane and interstitial blood vessel wall with luminal narrowing, and increased deposition of interstitial fibrous tissue.10,39,40 Given the inherent limitations of clinical studies, longitudinal evaluation of testicular biopsy samples is difficult, but several reports have shown the temporal changes in testicular histology before and after varicocelectomy; maturation of germ cells,10,39 including absence of meiotic abnormalities40 and normalization of the number of Leydig cells.39 The changes in the tubular basement membrane and interstitial blood vessel wall were unaffected.39 These histological changes are not specific to varicocele, but mechanistic information is difficult to obtain from human subjects, because the study design must be non-invasive and the subjects should differ in terms of the varicocele status, patient age and/or other health-related issues.

Because of these limitations, animal models of varicocele have been developed in several species, the most common being rats. With regard to the rat ELV model, Turner observed the following: (i) spermatic vein reflux does not occur; (ii) pathophysiological changes occur without the involvement of the ipsilateral adrenal gland; and (iii) collateral circulation to the contralateral testis is absent.41 These findings show that spermatic vein reflux is not an important factor in the pathophysiology of varicocele. However, ELV does not always reflect the pathophysiology of varicocele in infertile men; it presumably indicates the early testicular events after varicocele formation and represents the pathophysiology caused by disturbed venous drainage.

Proliferation and apoptosis of germ cells

  1. Top of page
  2. Abstract
  3. Role of sperm factors in ICSI
  4. Effects of varicocele on the outcomes of ART
  5. Evidence from human and animal studies
  6. Proliferation and apoptosis of germ cells
  7. Testicular hypoxia
  8. Other mechanisms
  9. Interstitial lesions
  10. Oxidative stress
  11. NO
  12. Anti-oxidants
  13. Heat stress
  14. Conclusions
  15. Conflict of interest
  16. References

Several studies have shown decreased testicular volume in men with varicocele.1 Transitions in mitotic and miotic divisions during spermatogenesis are accompanied by dynamic changes in the expressions of different cell-cycle genes. PCNA is directly involved in DNA synthesis, and its expression is relatively restricted to the S-phase of the cell cycle. Decreased expression of PCNA, especially in spermatogonia, has been reported in men with varicocele.42–44 Decreased expression of CREM suggests decreased cellular kinetics in testes with varicocele.45 Heat stress, a major deleterious factor of varicocele, is reported to induce double-strand breaks in DNA because of denaturation and dysfunction of heat-labile repair proteins, such as DNA polymerases. The expressions and activities of testicular DNA polymerases α, β and γ are decreased in heat-stressed tissue cultures.46

In addition to impaired germ-cell proliferation, varicocele-associated apoptosis causes male infertility,47 and apoptosis of germ cells in the ELV model and human testicular samples is the most investigated cellular response caused by varicocele. ELV causes apoptosis of germ cells at 7–28 days in adult rats,48 and the number of apoptotic cells reduces after varicocele repair in adolescent49 and adult50 rats. Oxidative stress is supposedly involved in varicocele-induced apoptosis of germ cells. Cam et al. reported that ELV induces ROS production and apoptosis in the testes, and vitamin E has a protective role in spermatogenesis.51 The increased expression of proapoptotic protein Bax by ELV is downregulated by melatonin administration.52 In humans, p53-mediated apoptosis has been reported mainly in the primary spermatocyte,44 and a study of ELV confirmed the occurrence of p53-dependent cell death in spermatocytes.53 In contrast, Kilinc et al. found no association among varicocele, apoptosis and p53 expression in adolescent rats with ELV,54 and Fujisawa et al. reported no significant increase in the number of apoptotic germ cells in human testicular biopsy samples.55 Differences in patient characteristics, rat age and species might be responsible for the discrepancies among the studies. Basically, apoptotic germ cells are quickly phagocytosed by Sertoli cells,56 and the results of TUNEL assay, the most common histochemical method for detection of apoptotic cells, depend on the number of apoptotic cells and the phagocytotic function of Sertoli cells. Use of early markers of apoptosis (e.g. externalization of annexin V and detection of caspase-3 fragment)57 and evaluation of Sertoli-cell function might reduce these discrepancies. By using poly(adenosine diphosphate-ribose) polymerase to detect the p85 fragment, an early marker of apoptosis, a significant positive correlation has been found between germ-cell apoptosis and increased testicular temperature.57

Evaluation of germ-cell apoptosis is clinically important. Men who respond to varicocelectomy have significantly lower levels of apoptosis in their testicular biopsy samples,58 showing that germ-cell apoptosis predicts improvement after varicocelectomy.

Testicular hypoxia

  1. Top of page
  2. Abstract
  3. Role of sperm factors in ICSI
  4. Effects of varicocele on the outcomes of ART
  5. Evidence from human and animal studies
  6. Proliferation and apoptosis of germ cells
  7. Testicular hypoxia
  8. Other mechanisms
  9. Interstitial lesions
  10. Oxidative stress
  11. NO
  12. Anti-oxidants
  13. Heat stress
  14. Conclusions
  15. Conflict of interest
  16. References

Increased venous hydrostatic pressure by varicocele disturbs arterial inflow for normal intratesticular circulation. Recent studies have shown the presence of testicular hypoxia, which is the most investigated mechanism causing impairment of spermatogenesis in men with varicocele and rats with ELV. However, controversies regarding arterial blood flow still exist; it was found to be increased59 or decreased60 in rats with ELV, but unchanged in men with varicocele.30

Varicocele can cause tissue hypoxia and related events, such as angiogenesis.61 HIF1 binds to the VEGF gene, and VEGF regulates the following pathways to enable cellular adaptation to hypoxia. HIF1α expression has been detected in the germ-cell cytoplasm and vascular endothelium.61 Furthermore, increased VEGF expression has been observed in testicular endothelial cells in men with varicocele and in the germ-cell cytoplasm in rats with ELV;61 VEGF expression possibly rises as a compensatory mechanism to counteract the stasis and consequent hypoxic state that might impair testicular function.62 VEGF might have either a paracrine effect on the testicular microvasculature, providing an adequate microenvironment in the seminiferous, tubular and interstitial compartments of the testes, or an autocrine effect on the activity of the testicular cell types.63 In fact, the mean microvessel density is significantly higher in men with varicocele than in controls.61 Furthermore, exogenous administration of VEGF decreases the profusion of apoptotic cells induced by ELV.49 PDRN stimulates the A2A receptor to induce VEGF production during pathological conditions of low tissue perfusion, and treatment with PDRN is as effective as varicocelectomy in abrogating the testicular damage caused by ELV.64

All the previous studies focused on unilateral testicular hypoxia. However, by venography, Gat et al. found that varicocele is a bilateral vascular disease and a right-sided venous return problem affects 86% of infertile men with clinically evident left-side varicocele.65 In fact, increased expression of HIF1 has been observed in the right testis in the ELV model, suggesting the occurrence of contralateral testicular hypoxia. Given that HIF1 expression is inducible by various stressors, the pathophysiology of varicocele should not be considered to always involve hypoxia.

Other mechanisms

  1. Top of page
  2. Abstract
  3. Role of sperm factors in ICSI
  4. Effects of varicocele on the outcomes of ART
  5. Evidence from human and animal studies
  6. Proliferation and apoptosis of germ cells
  7. Testicular hypoxia
  8. Other mechanisms
  9. Interstitial lesions
  10. Oxidative stress
  11. NO
  12. Anti-oxidants
  13. Heat stress
  14. Conclusions
  15. Conflict of interest
  16. References

IL-1 plays a role in the autocrine and paracrine regulation of normal testicular function. Sahin et al. reported increased IL-1α expression in Leydig cells, Sertoli cells and spermatogonia of adolescent rats with ELV, and proposed that IL-1α might affect both germ-cell and immature Leydig-cell proliferation, and modulate adult Leydig- and Settle-cell function.66 IL-1 produces free radicals in many tissues, showing that increased IL-1α expression in testes with varicocele indirectly causes sperm dysfunction through oxidative stress.

Notch signaling is an evolutionarily conserved mechanism, and Notch is involved in the regulation of proliferation, differentiation and apoptosis. Sahin et al. reported that Notch1 is associated with the maturation of spermatids. Notch2 is related to both proliferation and maturation of spermatogenic cells, and Notch3 seems to be related to Leydig cell functions.67 Decreased expressions of both Notch1 and Notch2 depend on the degree of varicocele development, showing the potential role in varicocele-associated testicular dysfunction. However, the exact function of Notch and the implication of the decreased expression after ELV are unexplored.

Two groups of proteins mainly acting in the central nervous system have been reported. CCM proteins, CCM2 and CCM3, are related to cavernomas in the brain. In the normal testes, CCM2 and CCM3 are found in the heads of postmiotic germ cells and the cytoplasm of round spermatids, respectively. Adolescent rats with ELV show increased expression of CCM2 in round and elongated spermatid acrosomes, Leydig cells and vessels.68 Furthermore, increased expression of CCM3 has been observed in the cytoplasm of round spermatids, microvessels and interstitium.68 GDNFα1, a brain growth factor, is involved in normal spermatogenesis, and its expression in spermatids and Leydig cells decreases in ELV.69 The effects of the changes in CCM2, CCM3 and GDNFα1 expressions on spermatogenesis are still unclear, but an understanding of the involvement of these neuronal factors in spermatogenesis might help clarify normal, as well as abnormal, spermatogenesis.

Interstitial lesions

  1. Top of page
  2. Abstract
  3. Role of sperm factors in ICSI
  4. Effects of varicocele on the outcomes of ART
  5. Evidence from human and animal studies
  6. Proliferation and apoptosis of germ cells
  7. Testicular hypoxia
  8. Other mechanisms
  9. Interstitial lesions
  10. Oxidative stress
  11. NO
  12. Anti-oxidants
  13. Heat stress
  14. Conclusions
  15. Conflict of interest
  16. References

Interstitial deterioration does not directly influence sperm function, but plays a crucial role in the impairment of spermatogenesis. The relationship between interstitial changes and sperm function is unclear, but an increased number of interstitial lesions usually represents a poor response to varicocelectomy. These changes include proliferation of Leydig cells, thickening of the tubular basement membrane and interstitial blood vessel wall with luminal narrowing, and increased deposition of interstitial fibrous tissue.10,39,40 As the basement membrane thickens, the sperm concentration and spermatogenesis usually decrease significantly. The basement membrane contains several proteins including laminin, type IV collagen, heparin sulfate proteoglycans and ectatin/nidogen. In men with varicocele, the basement membrane thickens, apparently because of increased deposition of collagen fibers.70,71 Furthermore, men with varicocele have a thicker basement membrane with more intense staining of α1(IV) and α2(IV) chains than men with normal testes.72

Men with varicocele have decreased expressions of E-cadherin and α-catenin at the junctions between adjacent Sertoli cells, indicating disruption of the blood–testis barrier in varicocele.73 These changes have also been observed in the ELV model, showing decreased E-cadherin and α-catenin expressions at 4–8 weeks after ELV.74

Mast cells contribute to fibrogenesis by producing and secreting bioactive mediators. The average number of mast cells containing both tryptase and chymase, which are detectable in the interstitium and lamina propria, per seminiferous tubular section is significantly high in men with varicocele compared with controls.75 The selective expansion of the mast-cell population containing both tryptase and chymase might be related to spermatogenic disorders and testicular fibrosis.

Oxidative stress

  1. Top of page
  2. Abstract
  3. Role of sperm factors in ICSI
  4. Effects of varicocele on the outcomes of ART
  5. Evidence from human and animal studies
  6. Proliferation and apoptosis of germ cells
  7. Testicular hypoxia
  8. Other mechanisms
  9. Interstitial lesions
  10. Oxidative stress
  11. NO
  12. Anti-oxidants
  13. Heat stress
  14. Conclusions
  15. Conflict of interest
  16. References

In the past 10 years, oxidative stress has been the most investigated factor involved in the pathophysiology of varicocele. It has been implicated in various pathophysiological states causing male infertility.33 Despite the aforementioned mechanisms, oxidative stress is the only factor explaining sperm dysfunction in men with varicocele.

Because of the high cellular turnover in normal spermatogenesis, ROS and RNS, such as superoxide, hydroxyl, peroxyl, hydroperoxyl, NO and nitrogen dioxide, which are produced by the peroxidation and oxidation of many cellular lipids, proteins, carbohydrates and nucleic acids, are generated. Oxidative stress is essential to maintain cellular homeostasis, but excess oxidative stress leads to cellular dysfunction. Oxidative modification of DNA and proteins is considered one of the earliest events caused by oxidative stress. Agarwal et al. reported that the level of seminal ROS correlates with the varicocele grade in men with varicocele.24 Specifically, men with grade 2 and 3 varicocele have greater levels of ROS in the seminal plasma than men with grade 1 varicocele.76 Although most studies show that seminal ROS levels are higher in men with varicocele than controls, others question the association between varicocele and the seminal ROS level.77 Evidence shows that the effectiveness of varicocelectomy greatly depends on the preoperative testicular oxidative stress level.34,78 If oxidative stress has only a minor role in the pathophysiology of varicocele, no association should exist between the outcomes of varicocelectomy and the level of testicular oxidative stress.

The plasma membrane of testicular cells is rich in polyunsaturated fatty acids, and is therefore vulnerable to oxidative stress. Koksal et al. reported that the levels of lipid peroxidation and MDA increase depending on the varicocele grade in men with varicocele.79,80 A specific and stable end product of lipid peroxidation, the aldehyde 4-HNE, can diffuse within, or even escape from, the cells and attack targets far from the site of the original free radical event. It is a potent alkylating agent that reacts with DNA and proteins, generating various forms of adducts (cysteine, lysine and histidine residues) capable of inducing specific cellular stress responses, such as signaling and apoptosis.81 Analysis of testicular biopsy samples has shown that the expressions of 4-HNE-modified proteins increase with the varicocele grade and correlate with increasing patient age, evidencing the progressive effects of varicocele.82 Immunoreactivities of 4-HNE-modified proteins have been observed in almost all the testicular components, especially in Sertoli cells and spermatocytes.82 Testicular 4-HNE-modified p53 expression is also greater in men with varicocele than in fertile men.44 Furthermore, 8-OHdG, a product of oxidative DNA damage following specific enzyme cleavage after 8-hydroxylation to the guanine base, is a marker of oxidative stress. Increased expression of 8-OHdG has been reported in the testicular tissue of men with varicocele;83 it localizes mainly in spermatocytes and the incidence of 8-OHdG-positive cells is associated with the varicocele grade.83 The expression of 8-OHdG also depends on the clinical grade of varicocele.

Cadmium deposition in the testes induces oxidative stress and apoptosis. The negative effects of cadmium can be mediated by the induction of the Fas ligand to trigger apoptosis; by indirect generation of the hydroxyl radical, O2-, hydrogen peroxide and/or NO; and/or by decreased concentration, which acts as an anti-oxidant. Benoff et al. reported elevated apoptosis levels and cadmium accumulation in the bilateral testes of infertile men with left varicocele.58 In terms of direct effects, increased cadmium concentration could disrupt Sertoli-cell actin, block spermiation, disrupt membrane integrity by causing loss of sperm-head actin and damage the actin cytoskeleton. Cadmium could also affect sperm quality directly by affecting spermatogenesis or indirectly by increasing oxidative stress.

To evaluate testicular oxidative stress, stable products generated by oxidative stress (e.g. MDA, 4-HNE-modified proteins, 8-OHdG and nitrite/nitrate) have been used as surrogate markers. However, direct measurement of the unstable elements causing oxidative stress has been carried out in few studies. In an ELV-based study, testicular O2- was found to be threefold higher in the ELV group than in the control group.60 Furthermore, Cam et al. showed increased O2-, by direct measurement with lucigenin-enhanced chemiluminescence assay, and ROS levels in testes with ELV.51

In many testicular disorders causing male infertility, extracellular ROS are produced by leukocytes, mainly neutrophils. Leukocytes are normally present in the prostate and seminal vesicles, and are, at least in part, involved in oxidative stress in the ejaculates. However, given that few inflammatory cells are observable in testes with varicocele, heat stress and tissue hypoxia, toxic substances can be considered the major sources of oxidative stress in men with varicocele. Infiltration of mast cells might also be partly involved in the generation of oxidative stress in such patients.75

NO

  1. Top of page
  2. Abstract
  3. Role of sperm factors in ICSI
  4. Effects of varicocele on the outcomes of ART
  5. Evidence from human and animal studies
  6. Proliferation and apoptosis of germ cells
  7. Testicular hypoxia
  8. Other mechanisms
  9. Interstitial lesions
  10. Oxidative stress
  11. NO
  12. Anti-oxidants
  13. Heat stress
  14. Conclusions
  15. Conflict of interest
  16. References

NO is a short-lived free radical and well-known cause of oxidative stress in many diseases (cytotoxic). It reacts with O2- to yield the active metabolites peroxynitrite and peroxynitrous acid, both of which are strong oxidants. In contrast, NO has also been implicated in various reproductive functions (cytoprotective). For example, low levels of NO promote human sperm capacitation in vitro.84 Basically, NO acts as a vasodilator, and locally produced NO is involved in the regulation of the testicular vasculature. Men with varicocele have excessive release of NO into the spermatic vein,85–87 showing the testicular production of NO. Recent studies of the rat ELV model have shown increased production of NO in testes with varicocele;88 however, information regarding whether this increase is associated with oxidative stress and has harmful effects on spermatogenesis is lacking. The testicular concentration of NO increases before the histological changes in ELV.88 The effects mediated by NO are dose dependent: it acts as a mediator of the aforementioned functions at physiological concentrations, but it is harmful to the reproductive system at supraphysiological concentrations.85–87 Santoro et al. reported that increased levels of iNOS in Leydig cells might cause deterioration of spermatogenesis and sperm dysfunction in varicocele.89 In contrast, iNOS expression is upregulated, perhaps, to maintain testicular arterial blood flow as a compensatory mechanism against testicular hypoxia caused by venous stasis or reflux. In the ELV model, iNOS activity is markedly increased in Leydig cells of rats with varicocele.90 Furthermore, some adolescents with varicocele show increased MDA levels together with elevated NO levels, indicating excessive lipid peroxidation.91 In the ELV model, treatment with aminoguanidine, a specific inhibitor of iNOS and an anti-oxidant, improved sperm DNA fragmentation,92 showing that NO has detrimental effects on sperm as well as spermatogenesis.

Studies have shown an increase in the NO concentration in the left spermatic vein compared with serum.85–87 This concentration is not cytotoxic and is usually derived from eNOS and nNOS, but not iNOS, which causes a 100- to 1000-fold increase in the NO concentration. Findings of intense nicotinamide adenine dinucleotide phosphate diaphorase activity in endothelial cells suggest that eNOS in testicular endothelial cells is a source of NO.93 These observations imply that locally synthesized NO might be involved in the control of vascular tone, and it passes into the internal spermatic veins, leading to their dilatation. In eNOS transgenic mice, with a 1000-fold level of intratesticular NO, eNOS-derived NO exceptionally impairs spermatogenesis.94 Furthermore, adolescents with grade II and III varicocele have upregulated expression of iNOS in Leydig cells.87,89 Taken together, iNOS expression is absent in the adult testes,93 but present in the adolescent testes, and excessive NO produced by iNOS might impair spermatogenesis and dilate the internal spermatic veins, generating varicocele.

Anti-oxidants

  1. Top of page
  2. Abstract
  3. Role of sperm factors in ICSI
  4. Effects of varicocele on the outcomes of ART
  5. Evidence from human and animal studies
  6. Proliferation and apoptosis of germ cells
  7. Testicular hypoxia
  8. Other mechanisms
  9. Interstitial lesions
  10. Oxidative stress
  11. NO
  12. Anti-oxidants
  13. Heat stress
  14. Conclusions
  15. Conflict of interest
  16. References

In healthy men, ROS are strictly controlled by a complex anti-oxidant defense system. Fertile men with and without varicocele have comparable testicular volume and oxidative stress, indicating increased ROS production or impaired ROS-scavenging activity in infertile men with varicocele.38 The testes contain several anti-oxidants that protect germ cells from oxidative damage. Two types of mechanisms are responsible for such protection: enzymatic and non-enzymatic. The testicular antioxidant enzymes in mammals are SOD, GSR, GPX, GST, catalase and HO-1.95,96 Germ cells, in general, do not appear to use the enzymatic anti-oxidant system as the major defense against ROS damage, and are consequently more susceptible to oxidative stress than somatic cells.95

The results of four proteomic studies show gene alterations in the testes after heat stress.97–100 The upregulated genes include HO-1 (accession no. AA213167), oxidative stress-induced protein (accession no. U40930) and GSTP1 (accession no. P19157). In humans, a varicocele grade-dependent increase in HO-1 expression, mainly in Leydig cells, has been observed in men with varicocele.82 This increase plays an important role in protecting the cells from the deleterious effects of oxidative stress. The downregulated genes include extracellular SOD Cu-Zn (SOD3; accession no. U38261), GSR1 (accession no. X76341), GSTα2 (accession no. J03958), GSTPi1 (accession no. D30687), microsomal GST12 (accession no. J03752) and thioredoxin-like protein 2 (accession no. Q9CQM9). In general, these proteins reduce oxidative stress. There is a distinct possibility that SOD plays a role in the male reproductive system.101,102 Germ cells appear to be highly susceptible to ROS and die without expressing SOD.103 Taken together with the results of decreased expression of SOD104 and GSH104,105 in the ELV model, rats with ELV and men with varicocele show similar molecular changes after heat stress. Therefore, heat stress presumably plays a major role in the impairment of spermatogenesis in men with varicocele.

Non-enzymatic anti-oxidant molecules include α-tocopherol (vitamin E), β-carotene (vitamin A), ascorbate (vitamin C), glutathione, estrogens, creatine (related to carotene), flavonoids (aromatic oxygen heterocyclic compounds widely distributed in higher plants), resveratrol (a botanical anti-oxidant), metallothionein (cadmium-binding protein involved in heavy metal detoxification), taurine (aminosulfonic acid) and its precursors; and other thiols, such as non-structural polyunsaturated lipids and melatonin. Oral administration of vitamin E reduces the levels of testicular free radicals in rats with ELV,51 and the effectiveness of anti-oxidants in infertile men has been shown in randomized trials.106 Therefore, anti-oxidants can be effectively applied for non-surgical treatment of varicocele. However, the mechanisms of impaired spermatogenesis induced by oxidative stress in men with varicocele should be clarified (Fig. 2).

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Figure 2. Relationships between the etiologies of varicocele and the involvement of oxidative stress. Note that various etiologies are overlapped in each patient. Among them, oxidative stress plays a major role in causing the impairment of spermatogenesis by heat and hypoxic stress. Current established management for varicocele is varicocelectomy, but etiology-based management is carried out if the pathophysiology of each patient can be evaluated.

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Leptin might have local effects on spermatogenesis, and its anti-oxidant role has been well reported. The impairment of spermatogenesis is associated with an increase in leptin (spermatocytes) and leptin receptor (Leydig cells) expressions in the testes, and the expressions in germ cells are significantly increased in men with varicocele.107 Increased expression of leptin (seminiferous tubules and interstitium) and its receptor (interstitium) have also been reported in infertile men with varicocele and adult rats with ELV.108 The exact reason for the increased expression of leptin in testes with varicocele remains to be explored.

Genetic factors might predispose men to severe damage from varicocele. Men with deletions in GSTM1 have elevated levels of 8-OHdG in sperm DNA, deletions in mitochondrial DNA and impaired sperm motility.109 Furthermore, genotypes of GST could predict clinical benefit from varicocelectomy.110 In addition, Lenzi et al. reported that alterations of sperm membrane fatty acid composition associated with varicocele might increase susceptibility to ROS-induced damage.111

Varicocelectomy induces a significant decrease in oxidative damage in sperm DNA, and an increase in anti-oxidant capacity in seminal plasma, showing that varicocele repair is an effective treatment in men with varicocele.112 Furthermore, it normalizes the incidence of 4977-bp mitochondrial DNA deletion, 8-OHdG content in sperm DNA, oxidative DNA damage, and thiobarbituric acid reactive substance and nitrate/nitrite levels.112–115 Hurtado de Catalfo et al. reported that varicocele repair also normalizes the levels of non-enzymatic anti-oxidants (e.g. α-tocopherol, ascorbate, retinol, selenium and zinc).115 These results suggest that varicocelectomy not only reduces ROS production, but also increases anti-oxidant expressions, thus reducing oxidative stress.

Heat stress

  1. Top of page
  2. Abstract
  3. Role of sperm factors in ICSI
  4. Effects of varicocele on the outcomes of ART
  5. Evidence from human and animal studies
  6. Proliferation and apoptosis of germ cells
  7. Testicular hypoxia
  8. Other mechanisms
  9. Interstitial lesions
  10. Oxidative stress
  11. NO
  12. Anti-oxidants
  13. Heat stress
  14. Conclusions
  15. Conflict of interest
  16. References

Heat stress is the most plausible cause of the impairment of spermatogenesis in men with varicocele, and has been investigated since 1941.116 In general, cells with high mitotic activity (e.g. cancer cells and germ cells) are more susceptible to heat stress than are somatic cells. ELV bilaterally increases testicular temperature, which reasonably explains how the unilateral lesion causes deterioration of the total sperm output. Spermatogenesis is temperature sensitive,117–119 and proceeds optimally at approximately 36°C in men. The internal spermatic artery (surrounded by the pampiniform plexus) maintains the testes at 35–36°C, 1–2°C lower than the core temperature, by the countercurrent heat-exchange system. In animal studies, testicular temperature is measured directly in the testes, but clinical studies are limited to surface determination of scrotal temperature. In the rat ELV model, testicular blood flow and temperature increase bilaterally and spermatogenesis decreases.56 Varicocele causes an average scrotal temperature increase of 2.6°C by the dilatation of the venous plexus, and varicocele repair in humans lowers testicular temperature by 0.5°C.30,118

Although measurement of scrotal temperature is the only method to evaluate the functional aspect of varicocele, the effects of changes in scrotal temperature on spermatogenesis in men with varicocele are controversial. Considering that continuous monitoring of scrotal temperature in the same men showed a huge variation in the temperature by body position and activity over a 24-h period,120,121 the absolute scrotal temperature is difficult to interpret and unreliable in showing heat stress. Several researchers found no difference in scrotal temperature between infertile men with and without varicocele,122,123 because of the different modalities of its measurement and conflicting results. Furthermore, several reports have described scrotal temperature measured from the skin surface, which does not always represent testicular temperature. Direct intratesticular measurement by using needle thermistors has shown increased testicular temperature in men with varicocele.117 Contact thermometers measure temperature at a depth of 5–10 mm from the skin. Therefore, measurement of the core temperature (CoreTemp CTM204; Terumo, Tokyo, Japan) could reflect testicular temperature (Fig. 3).118 Yamaguchi et al. have reported the usefulness of the core temperature to evaluate men with varicocele; in the standing position, the core temperature increases in men with varicocele, but decreases in men without varicocele.118

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Figure 3. Measurement of scrotal deep body temperature. The temperature is measured at prone position for 15 min, then at standing position for 15 min. The device is developed to measure the testicular, but not scrotal surface, temperature.

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The detailed mechanism of how heat stress affects germ cells is described elsewhere.36 In testes with varicocele, heat stress could be a major cause of oxidative stress, because increased scrotal temperature is closely associated with testicular oxidative stress.57 This result suggests that scrotal temperature, rather than the varicocele grade, might indicate suitable candidates for varicocelectomy. Furthermore, several human studies have shown higher scrotal temperature in infertile men than in fertile controls, regardless of the cause of infertility.119,122 Varicocele per se is not pathological, and fertile men with varicocele have lower oxidative stress than infertile men.38 Even subclinical varicocele can be detected and treated in a reasonable way by assessing testicular oxidative stress (e.g. scrotal temperature measurement) in addition to the conventional diagnostic methods (e.g. palpation, scrotal ultrasonography, color Doppler ultrasound and venography; Fig. 4).

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Figure 4. Concepts to evaluate the varicocele. Combinations of different standpoints will give us a better understanding to manage men with varicocele.

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Conclusions

  1. Top of page
  2. Abstract
  3. Role of sperm factors in ICSI
  4. Effects of varicocele on the outcomes of ART
  5. Evidence from human and animal studies
  6. Proliferation and apoptosis of germ cells
  7. Testicular hypoxia
  8. Other mechanisms
  9. Interstitial lesions
  10. Oxidative stress
  11. NO
  12. Anti-oxidants
  13. Heat stress
  14. Conclusions
  15. Conflict of interest
  16. References

Several guidelines limit the applicability of varicocelectomy, but its indications in ART should be widened. Multicenter prospective studies of the effects of varicocelectomy on the outcomes of IVF/ICSI are required. Furthermore, as sperm factors can influence the outcomes of IVF/ICSI, evaluation of sperm function is warranted to monitor the effectiveness of varicocelectomy and consequent improvement in the outcomes of IVF/ICSI. The pathophysiology of varicocele is still enigmatic, and we cannot clearly show the relationships between the pathophysiology of varicocele and the outcomes of IVF/ICSI. Both basic and clinical studies are required to investigate this condition to improve not only spermatogenesis, but also the live birth rate after IVF/ICSI.

References

  1. Top of page
  2. Abstract
  3. Role of sperm factors in ICSI
  4. Effects of varicocele on the outcomes of ART
  5. Evidence from human and animal studies
  6. Proliferation and apoptosis of germ cells
  7. Testicular hypoxia
  8. Other mechanisms
  9. Interstitial lesions
  10. Oxidative stress
  11. NO
  12. Anti-oxidants
  13. Heat stress
  14. Conclusions
  15. Conflict of interest
  16. References