Human Reproduction Update, Vol.7, No.5 pp.

461±472, 2001

Varicocele and male infertility: Part II

The pathophysiology of varicoceles in the light of current

molecular and genetic information

Joel L.Marmar
Division of Urology, Robert Wood Johnson Medical School at Camden, Camden, NJ, USA

Address for correspondence: Division of Urology, Robert Wood Johnson Medical School, 3 Cooper Plaza, Suite 411, Camden,
NJ 08103. E-mail: marmar-joel@cooperhealth.edu

Varicoceles are a common cause of male infertility, but despite data being obtained from animal models and human
studies the pathophysiology remains unclear. Recently, molecular and genetic information has been reported on men
with varicoceles which may shed new light onto the causes of decreased semen parameters and poor sperm function.
Here, a number of studies are reviewed in an attempt to develop a working hypothesis for the relationship of
varicoceles and infertility. New studies on testicular tissue of men with varicoceles have demonstrated increased
apoptosis among developing germ cells, which may be the cause of oligospermia. Other studies with semen have
shown increased levels of reactive oxygen species (ROS) in association with poor sperm motility. Recent studies of
morphologically abnormal spermatozoa have demonstrated disruption of the sperm head actin by cadmium, a cation
reported to be present in high concentrations among some men with varicoceles. Finally, microdeletions of the alpha-
1 subunit of the sperm calcium channels in a proportion of men with varicoceles suggests a genetic defect leading to
abnormal acrosomal function. The intent of this review was to explain the pathophysiology of varicoceles, and the
®ndings seem to support a `co-factor' hypothesis. In order for varicoceles to be associated with infertility, they exist
as `co-factors' along with other molecular/genetic problems.

Key words: acrosome/apoptosis/cadmium/reactive oxygen species/varicocele

TABLE OF CONTENTS suggested favourable semen and pregnancy data following

Introduction
Current understanding of the pathophysiology of varicoceles
The consistent effects of varicoceles in animal models
The clinical diversity of humans with varicoceles
The effect of varicoceles on sperm concentration
Apoptosis and spermatogenesis
The effect of varicoceles on sperm motility
The effect of varicoceles on sperm morphology
Acrosome reaction induction test in men with varicoceles
Conclusions
References
Introduction
Varicoceles have been recognized as a cause of male infertility
for many years. Although varicocelectomies have been used to
correct these lesions, the management of varicoceles remains
controversial for several reasons. A number of studies have
varicocelectomy (Pryor and Howards, 1987; Schlesinger et al.,
1994), whilst other studies have reported negative results (Nilsson
et al., 1979; Baker et al., 1985; Nieschlag et al., 1998).
Furthermore, it is unclear why some men with varicoceles father
children (Uehling, 1968; Kursh, 1987) and have normal semen
studies, whereas others fail to improve despite surgery. The
controversy persists because the current concepts that are
available to explain the pathophysiology of varicoceles seem
incomplete for all of these clinical situations. In addition, the
criteria for surgery are not standardized between clinics. Although
out-patient microsurgical varicocelectomies have gained wide
acceptance (Marmar et al., 1985; Goldstein et al., 1992), surgery
is usually recommended empirically based upon the infertility
history and/or semen ®ndings that demonstrate at least one
persistently low parameter (Dubin and Amelar, 1977; Newton et
al., 1980; Marks et al., 1986; Goldstein et al., 1992; Ross and
Ruppman, 1993; Marmar and Kim, 1994). Recently, several new
molecular and genetic laboratory studies have been reported in

Ó European Society of Human Reproduction and Embryology 461

J.L.Marmar
men with varicoceles. Some of these studies utilized testicular
¯uid and seminiferous tissue acquired by minimally invasive
percutaneous methods (Craft et al., 1995; Marmar, 1998). It is
hoped that information from these studies will provide new data
to explain the pathophysiology of varicoceles and lead to
recommendations for standardized surgical criteria. This review
will outline some of the new research, examine critically the data,
and propose new ideas for future investigations. However, before
examining this new information, it seems appropriate to review
some earlier concepts.
Current understanding of the pathophysiology of
varicoceles
Varicoceles develop from retrograde blood ¯ow down the internal
spermatic veins and creamasteric veins into the pampiniform
plexus. The reversal of venous ¯ow occurs because of absent or
incomplete valves. Anatomical dissections on men with varico-
celes demonstrated absence of the valve at the junction of the left
renal vein and the internal spermatic vein (Kohler, 1967).
Retrograde venography studies on men with varicoceles
(Ahlberg et al., 1966; Coolsaet, 1980; Comhaire et al., 1981)
have documented either absent or incompetent valves along the
entire internal spermatic vein. The retrograde blood ¯ow leads to
varying degrees of increased hydrostatic pressure, prolonged
stasis and increased temperature within the testis. In response to
heat, adverse effects were reported on the seminiferous epithe-
lium, leading to the loss of spermatocytes and spermatids
(Mieusset and Bujan, 1995). In separate reports (Fujisawa et al.,
1988; Steinberger, 1991), biochemical changes in response to heat
were described, including diminished DNA synthesis within germ
cells, and reduced inhibin and androgen binding protein output by
Sertoli cells. Although scrotal hyperthermia has been recognized
as an important factor in the pathophysiology of varicoceles,
several other effects have been studied in animal models and
humans, and some of these data are presented in the next section.
The consistent effects of varicoceles in animal models
Varicocele models have been created in at least four species of
laboratory animals: rat (Turner and Lopez, 1990); dog (Saypol et
al., 1981); rabbit (Snydle and Cameron, 1983; So®kitis and
Miyagwa, 1994); and monkeys (Kay et al., 1979; Fussell et al.,
1981; Harrison et al., 1986). The `varicocele effect' was created
by partial constriction of the left renal vein and/or left testicular
vein, leading to sustained partial obstruction with congestion and
dilatation of the pampiniform plexus and testis vasculature. In the
models that achieved visible evidence of venous distension, there
was a reproducible increase in intratesticular temperature and
interstitial pressures in all species. Although these animals were
fertile prior to creation of the `varicocele effect', over time, 25±
50% of the seminiferous tubules demonstrated a predictable
pattern of hypospermatogenesis with premature sloughing of
spermatocytes and spermatids. The sperm density in a monkey
model fell from a mean of 4403106/ml to 2273106/ml after 90
days, and in the rabbits from 300±4503106/ml to 17±1003106/
ml. These effects were reversible in the rat model following
removal of the obstructing suture, as long as it was removed
462
within 30 days after creation of the varicocele (Green et al.,
1984).
The clinical diversity of humans with varicoceles
Unlike the animal models, humans with varicoceles have greater
clinical diversity. The varicoceles range in size from large visible
lesions to palpable lesions and to non-palpable subclinical
varicoceles (Dubin and Amelar, 1970), but the effect of size on
semen parameters has been debated. Maximum bene®t from
varicocelectomies was reported among men with large lesions and
lower sperm densities (Steckel et al., 1993), whilst others (Tinga
et al., 1984) demonstrated improvement mostly among men with
large varicoceles as long as the sperm densities were <403106/
ml. These authors suggested a `ceiling effect' or no improvement
when the initial sperm densities were >403106/ml. In contrast,
others have reported improvement with varicocelectomies among
selected men with small or subclinical varicoceles (Yarborough et
al., 1989; McClure et al., 1991; Dhabuwala et al., 1992).
Other manifestations of variability have been noted in the
internal spermatic vein hydrostatic pressures, degrees of stasis and
the intratesticular temperatures. The mean venous tension among
38 infertile varicocele patients was reported to be 81.88 mmHg at
rest (range 70±92 mmHg) (Sha®k, 1983). During the Valsalva
manoeuvre, the mean venous tension rose to 85.8 mmHg, the
mean values for controls being 60.5 mmHg (range 52±74 mmHg).
These data indicated an overlap between patients and controls,
and furthermore, the increased pressures were not necessarily
sustained in humans because they may be dependent on changes
in position. Another group (Sayfan and Adam, 1978) studied 20
subfertile men with varicoceles, ®ve fertile men with varicoceles
and ®ve controls. There were no differences in spermatic vein
pressures among these groups while the men were supine or at 45°
in the Trendelenberg position.
In another study, radioisotopes were used to demonstrate stasis
in 18% of patients with low-grade varicoceles compared with
88% of men with large varicoceles (Comhaire et al., 1983). This
stasis was quanti®ed with radionucleotides (Mali et al., 1984): the
mean blood volume of the pampiniform plexus for controls was
0.3 (range 0.2±0.4) units, whereas the mean for patients with
varicoceles was 0.9 (range 0.3±2.0) units. In our own institution,
varying degrees of stasis were observed within the pampiniform
plexus during retrograde venography, because the venous
washout of contrast from the pampiniform plexus ranged between
2 and 45 min while the patient was in the supine position. The
clinical signi®cance of the stasis is unclear, but it is believed that
it may affect scrotal hyperthermia and blood ¯ow.
Increased scrotal temperatures have been considered as
important factors in the pathophysiology of varicoceles, but these
temperatures may also be quite variable. In a study of scrotal
temperature in 300 consecutive infertile men, 76 men had left
varicoceles (Zorgniotti and MacLeod, 1973). These data suggest
that the mean temperature for the left hemiscrotum was 33.5
(range 32.2±34.8) °C for controls versus a mean of 35.0°C for
men with varicoceles and a mean sperm count of <63106/ml.
Although the difference between the means was signi®cant, there
was considerable overlap in temperatures between patients and
controls. Moreover, other physical factors seem to in¯uence this
testicular temperature, such as clothing and shaving of the scrotal

hair. Wearing boxer shorts did not affect scrotal temperatures
(Mounkelwitz and Gilbert, 1998), but wearing an athletic
supporter raised the scrotal temperatures by 1°C (Wang et al.,
1997). In contrast, shaving of the scrotal hair lowered the
temperature (Kurz and Goldstein, 1986). One group (Mieusset
and Bujan, 1995) reviewed scrotal temperatures of 550 con-
secutive infertile men, and noted that 43% of the infertile
population had scrotal hyperthermia; however, 25% of these cases
had normal semen parameters. Others (Wright et al., 1997)
reported that testicular temperatures were elevated in men with
either unilateral or bilateral varicoceles, and that the temperatures
declined following microsurgical varicocelectomies, though it
seems unlikely that scrotal hyperthermia alone can completely
explain the pathophysiology of varicoceles.
The testis biopsy specimens from humans with varicoceles may
be quite variable, because they demonstrate histological patterns
including Sertoli cell-only (Kim et al., 1999), spermatogenic
arrest (Etriby et al., 1967), hypospermatogenesis and normal
spermatogenesis (Dubin and Hotchkiss, 1969). When bilateral
testicular biopsies were performed among men with left-sided
varicoceles, both testes demonstrated similar histology and had
similar quantitative Johnsen scores, suggesting that either a left
varicocele causes bilateral affects, or that varicoceles coexist with
other underlying pathological factors affecting all of the
seminiferous tissue (Aggar and Johnsen, 1978).
The semen data from men with varicoceles re¯ects the
variability of the biopsy ®ndings. In a review of 190 cases of
varicocelectomy (Newton et al., 1980), only 76 men (40%) had
sperm densities <203106/ml, and 56 men (29.5%) had motilities
<30%. Others (Schlesinger et al., 1994) reviewed data from 16
studies, including 1077 treated patients; the mean sperm densities
ranged from 18 to 563106/ml, the mean percentage motilities
from 21 to 73%, and the mean normal morphology from 18 to
48%. Follow-up data on 466 men treated by varicocelectomy
were also provided (Marmar and Kim, 1994). The preoperative
semen value from among this group included 123 men (26.5%)
with sperm densities <203106/ml as their only abnormal ®ndings,
97 (20.8%) with reduced motilities <50% as their only problem,
and 19 (4.1%) with normal morphology <40%. The remaining
227 patients had mixed seminal pattern de®cits.
These data suggest that the underlying pathology may be
multifactorial, and that there may be speci®c causes responsible
for individual sperm defects which would require individualized
treatment plans. Thus, the working hypothesis in this review will
be that varicoceles are co-factors with existing molecular/genetic
problems which, in combination, determine the degree of
infertility in these cases as well as the reversibility. In the
following sections, some of the molecular/genetic ®ndings will be
discussed in relation to speci®c de®cits in semen parameters.
The effect of varicoceles on sperm concentration
The range of sperm concentrations among patients who appear to
bene®t from varicocelectomy is extreme, from azoospermia to up
to 403106 spermatozoa per ml. For example, in a classic paper
(Tulloch, 1955) when a varicocelectomy was performed on an
azoospermic male, after surgery the semen parameters improved,
leading to pregnancy. This report has been the stimulus for
varicocele surgery over the years, but until recently most
Pathophysiology of varicoceles
azoospermic men were excluded as candidates for varicocelect-
omy. With the development of IVF/intracytoplasmic sperm
injection (ICSI), there has been renewed interest in varicocelect-
omy for azoospermic men, and the data from these studies seem
to illustrate the co-factor effect of varicoceles (Czaplicki et al.,
1979). Although these azoospermic men represent an extreme
group, these data suggest that the outcome of varicocelectomy
may depend upon the pre-existing genetic status. For example,
one study reported improved semen data following varicocelect-
omy in 55% of azoospermic men with an accompanying
pregnancy rate of 14% (3/22) (Mathews et al., 1998). Others
(Kim et al., 1999) operated on 28 men with varicoceles who had
non-obstructive azoospermia, and 12 (43%) of these demonstrated
spermatozoa in the ejaculate after 24 months of follow-up. The
improvement occurred in men who had testis biopsies with
hypospermatogenesis or spermatogenic arrest at the spermatid
level, but there was no improvement among men with Sertoli cell-
only syndrome (SCOS). Although some azoospermic men
improve after varicocelectomy, it may be reasonable for those
men with varicoceles to undergo testicular biopsies to determine
the underlying histology. At present, percutaneous testis biopsies
may be performed in an of®ce setting with local anaesthesia
(Marmar, 1998).
In addition, these azoospermic men may require Y-chromo-
some mapping. In an examination of microdeletions of the Y
chromosome in 200 consecutive infertile men (Pryor et al., 1997),
70 of the men had varicoceles and two tested positive for Y-
chromosome microdeletions. This ®nding illustrates that identi®-
able genetic problems may coexist in patients with varicoceles. In
a recent study, a group of azoospermic and oligospermic men
with varicoceles and Y-chromosome microdeletions or aneuploi-
dy failed to improve following varicocelectomy (Turek et al.,
2000). These data suggest that Y-chromosome mapping and
karyotype studies may be important in the work-up of men with
varicoceles and azoospermia or severe oligospermia, and that the
genetic ®ndings may predict varicocelectomy outcome.
Most men with varicoceles are not azoospermic but manifest
varying degrees of oligospermia. Nevertheless, other molecular/
genetic causes may be present to explain the infertility and
reduced sperm density. For example, apoptosis has been identi®ed
as a signi®cant cause of oligospermia in infertile men (Lin et al.,
1997a,b), while other studies have linked apoptosis with
varicoceles in animal models and humans (Baccetti et al., 1996;
Simsek et al., 1998; Caruso et al., 1999). Although a
comprehensive review of apoptosis is beyond the scope of this
review, a brief summary seems appropriate because this process
may form the basis for oligospermia in men with varicoceles.
Apoptosis and spermatogenesis
Spermatogenesis is an ongoing proliferative process that leads to
the development of millions of spermatozoa each day. Since
apoptosis has been recognized as a fundamental process in both
normal and pathological conditions, apoptosis may determine the
ultimate sperm output in infertile men, including those men with
varicoceles. Apoptosis can affect all three classes of germ cells
including spermatogonia, spermatocytes and spermatids (Sinha-
Hikim et al., 1998). During normal spermatogenesis in the rat, it
has been estimated that up to 75% of all preleptotene
463
J.L.Marmar
spermatocytes would be lost by apoptosis (Huckins, 1998).
Apoptosis has been examined as a cause of germ cell loss in
elderly men (Brinkworth et al., 1997), whilst in other human
studies (Lin et al., 1997a,b) increased apoptotic germ cells were
demonstrated among men with hypospermatogenesis and sper-
matogenic arrest at the spermatid stage, but not in men with
SCOS. The presence of endothelial nitric oxide synthase (eNOS)
has been reported in apoptotic germ cells, but not in normal cells
(Zini et al., 1996); these data suggest a role for nitric oxide (NO)
and eNOS in germ cell apoptosis, but more studies are needed in
con®rmation. The microscopic appearance of apoptotic cells
include cell shrinkage, membrane blebbing, nuclear condensation,
and fragmentation in the form of vesicles called `apoptotic
bodies' (Allan et al., 1987; Bodey et al., 1998). The common
biological event associated with apoptosis is fragmentation of
nucleosomal units of DNA into segments of about 200 base pairs
which, when separated on agarose gel electrophoresis, form a
distinctive DNA ladder (Batistantou and Greene, 1993). When
studying ®xed tissue, apoptotic cells may be identi®ed and
quanti®ed by in-situ terminal deoxy nucleotidyl dUTP nick-end
labelling (TUNEL assay), which is available in a kit from several
commercial sources.
There are many pathways leading to apoptosis, and these
processes seem to be regulated at three levels (Brill et al., 1999).
At the cell membrane, there are speci®c membrane receptors
mediating death signals of the tumour necrosis factor receptor
(TNFR) family known as Fas and Fas ligand (Lee et al., 1997).
With immunohistochemistry, Fas has been located on germ cells,
and Fas ligand on Sertoli cells (Lee et al., 1999). At the
cytoplasmic level, there are signal transduction pathways
involving cysteine proteases called caspases (Thornberry and
Lazebnik, 1998). When Fas binds to its Fas ligand, the union
forms a molecular complex on the inner surface of the cell
membrane known as the death-inducing signalling complex,
which involves procaspase-8 (Fuchs et al., 1997). The activated
caspase-8 stimulates other capsases, including caspase-3 which
mediates apoptosis. At the nuclear level, there are speci®c
apoptotic regulatory genes including p53 and Bcl-2 which include
pro-apoptotic Bax genes and anti-apoptotic Bcl-2 genes. The p53
tumour suppressor gene responds to DNA damage and tempora-
rily arrests the cell cycle at the G1 phase, thereby giving suf®cient
time for DNA repair (King and Cidlowski, 1998). A p53-
dependent pathway has been described for the initial phase of
apoptosis (Yin et al., 1998); however, if the DNA damage is
irreparable, p53 initiates cell death by stimulating the Fas receptor
complex and the Fas gene (Fuchs et al., 1997). Bcl-2 may
potentiate or inhibit apoptosis (Adams and Cory, 1998) by either
stimulating or inhibiting the release of cytochrome-c from
mitochondria. The presence of this enzyme will stimulate
caspase-9, which in turn will stimulate caspase-3 as part of the
pathway to apoptosis (Woo et al., 1998). From these studies,
future investigations on men with varicoceles may include the
molecular events of apoptosis.
For example, recent studies have suggested that apoptosis may
play an important role in the development of oligospermia among
varicocele patients. Increased germ cell apoptosis was demon-
strated in a rat model using the TUNEL assay (Caruso et al.,
1999). After 28 days, the model demonstrated 0.27 apoptotic cells
per seminiferous tubule compared with 0.14 cells for controls
464
(P < 0.002). In humans, increased apoptosis was demonstrated
among varicocele patients (Simsek et al., 1998), the mean
percentage apoptotic cells per total germ cells counted per high-
power ®eld being 14.7% for men with varicoceles compared with
2% for controls. In a report on ejaculated spermatozoa, it was
shown that up to 10% of sperm cells in the ejaculate of men with
varicoceles were apoptotic compared with 0.1% among fertile
controls (Baccetti et al., 1996). Most recently, the association of
apoptotic cells and cadmium deposition in testicular specimens
from men with varicoceles was documented (Hurley et al., 1999).
Thus, there appears to be a link between varicoceles and
apoptosis, and speci®c pathways will be examined for these
men because there are three pathways leading to apoptosis that
may be associated with varicocele effects, including heat stress,
androgen deprivation, and accumulation of toxic stimuli. Each of
these pathways will be examined in detail.
Heat stress and apoptosis
Although an average scrotal temperature increase of 2.5°C has
been demonstrated among men with varicoceles (Goldstein and
Eid, 1989), it is unclear whether this increase is suf®cient to
initiate apoptosis. Therefore, other conditions and other molecular
markers have been examined to document the relationship of heat
and apoptosis. In one study (Lue et al., 1999), the scrota of rats
were exposed to 43°C for 15 min. The results of the heat stress led
to apoptosis which was cell-speci®c and stage-speci®c. Pachytene
spermatocytes and spermatids at stages I±IV, XII±XIV of the
spermatogenic cycles were susceptible to apoptosis. Similar
apoptotic effects were noted following heat stress in mice with the
cryptorchid model (Yin et al., 1997). Other markers of heat effect
have been studied, and it has been well documented that speci®c
heat shock proteins (HSP70-2) are distributed over sperm cells
and appear to be up-regulated in heat stress (Miller et al., 1992).
In the absence of heat shock proteins in a `knock-out' model there
was a dramatic increase in apoptosis (Dix et al., 1996). Although
there are no studies on heat shock proteins in patients with
varicoceles, future investigations may provide useful information
about individual heat shock protein responses. Thus, identi®cation
of these secondary markers may de®ne conditions for heat-
induced apoptosis, especially when testis tissue is examined.
In a separate report (Sinha-Hikim and Swerdloff, 1995) it was
noted that there was an early and increased expression of the pro-
apoptotic Bax gene in susceptible germ cells after 30 min of
scrotal heating. Over time, there was a delayed expression of Bcl-
2, which may have been an attempt to escape apoptosis.
Currently, there have been no studies on this type of gene
expression among men with varicoceles, but the technology is
available for these investigations.
Androgen deprivation and apoptosis
Another apoptotic pathway that may occur in men with
varicoceles results from androgen deprivation, and a group of
studies have examined both peripheral and intratesticular
testosterone changes. For example, some investigators (Hudson
et al., 1985; Fujisawa et al., 1994) demonstrated an abnormal LH
response to gonadotrophin-releasing hormone (GnRH) stimula-
tion among men with varicoceles, and used these tests to predict
the outcome of varicocelectomy; however, the changes on serum
testosterone in these men have been variable. Other investigators

measured serum testosterone in the internal spermatic vein
(Swerdloff and Walsh, 1975; Hudson, 1988) but found no
differences between varicocele patients and controls. Yet other
studies have demonstrated low serum testosterone values in
speci®c situations. For example, low serum testosterone was
reported in men with varicoceles and sexual inadequacy
(Comhaire and Vermeulen, 1975), while others reported low
testosterone in men over 30 years of age with varicoceles (World
Health Organization, 1992), and reduced serum testosterone in
men with varicoceles but with at least one ®rm testis (Su et al.,
1995).
Several other studies examined the intratesticular effects. One
group (Zirkin et al., 1989) showed that an intratesticular
testosterone concentration of at least 20 ng/ml was required to
maintain spermatogenesis in the rat. Others (Rajfer et al., 1987)
reported a sharp decline in intratesticular testosterone in the
experimental rat model with the varicoceles, without an effect on
serum testosterone concentration. The ability of testicular tissue
homogenates to convert [3H]pregnenolone to 3H-T was also
evaluated (Weiss et al., (1978), and these data suggest a decreased
intratesticular testosterone in animal models and humans with
varicoceles. Leydig cells were quanti®ed in men with varicoceles,
and the ®ndings used to predict varicocelectomy outcomes
(Rodriguez-Rigau et al., 1978). In other studies, semi-thin
sections of testis biopsy material were used to evaluate the
function of Leydig cells based upon their histological appearance
(Hadziselimovic et al., 1986). In some cases, the Leydig cells
appeared `burned out', which implied that this group would not
bene®t from varicocelectomy.
Speci®c studies of apoptosis and androgen deprivation have
been carried out on rat models. Induction of apoptosis in
immature rats 4 days after a hypophysectecomy was demonstrated
(Tapanainen et al., 1993), while in mature rats, apoptosis was
induced with a GnRH antagonist (Sinha-Hikim and Swerdloff,
1995). As soon as 5 days after initiating the antagonist treatment,
there was selective apoptosis of prelepytene and pachytene
spermatocytes and spermatids in stages VII±VIII. This pattern of
apoptosis is never seen in normal rats. When recombinant (r)LH
and recombinant (r)FSH were introduced, there was attenuation of
apoptosis or a reduction of 67% with rLH and 79% with rFSH.
The intratesticular testosterone concentrations returned to normal.
In an in-vitro study, it was demonstrated that isolated segments of
seminiferous tubules incubated in serum-free conditions induced
apoptosis of germ cells within 48 h (Erkkila et al., 1997). This
reaction was suppressed with testosterone supplement, but the
value of intratesticular testosterone necessary to initiate apoptosis
is still unclear.
Recently, results were reported of intratesticular androgen
measurements from percutaneous testicular aspirates in healthy
men (Jarow et al., 1999). The mean testosterone concentrations
were 485 ng/ml, or substantially greater than serum concentra-
tions. Presently, this technique is being applied to men with
varicoceles and infertility, and these data may determine the true
concentration of intratesticular testosterone necessary to induce
apoptosis. In the past, supplemental human chorionic gonado-
trophin (HCG) was recommended for men following varicoce-
lectomy (Dubin and Amelar, 1977). Thus, in future, selective
stimulation may be used on speci®c patients who are diagnosed
Pathophysiology of varicoceles
with intratesticular androgen deprivation, and the percutaneous
aspirates may be useful in these cases to develop threshold levels.
Although selective testosterone stimulation may have potential
for some infertile men in the future, recent studies have sequenced
the molecular structure of the androgen receptor (Tut et al., 1997;
Yong et al., 1997; Yoshida et al., 1999) and have suggested
receptor dysfunction in some cases based on excessive glutamine
repeats. If there were more than 27 polyglutamine repeats in the
receptor, then there was a higher risk for male infertility and
androgen receptor dysfunction. One group (Yong et al., 1997)
suggested that these mutations might affect up to 30% of infertile
men, while others (Yoshida et al., 1999) studied the androgen
receptor repeats in 41 azoospermic males including seven with
varicoceles. It was also indicated that one of these varicocele
patients had excessive repeats (K.-I.Yoshida, personal commu-
nication). Thus, defective androgen receptors may occur coin-
cidentally in men with varicoceles, and if testosterone stimulation
is planned in the future then molecular studies of the androgen
receptor may be indicated.
Toxic agents and apoptosis
Toxic agents such as 2-methoxyethanol, an ethylene glycol ether
and its by-product 2-methoxyacetic acid, have been proven to
cause apoptosis in rats and mice, leading to spermatocyte death
(Brinkworth et al., 1997; Ku et al., 1995; Li et al., 1996). In men
with varicoceles, it was suggested (Comhaire and Vermeulen,
1974; Cohen et al., 1975) that toxic adrenal by-products might
re¯ux down the internal spermatic vein to the testicle. However,
the toxic effects of cortisol and catecholamines were never
proven. Other groups (Ito et al., 1982; Cockett et al., 1984)
demonstrated higher concentrations of prostaglandin and seroto-
nin in the spermatic veins of varicocele patients, but these
compounds did not produce an anti-spermatogenic effect.
Perhaps, the most widely studied toxic agent related to
infertility has been cigarette smoke. In men, the semen quality
may shift into an infertile range by smoking (Vine et al., 1996),
and among men with varicoceles oligospermia is ten-fold more
common in smokers than in non-smokers (Kleiber et al., 1987). In
animal models with varicoceles, impairment of spermatogenesis
was greatest in those animals exposed to nicotine when compared
with operated controls (Peng et al., 1990).
In a separate study, cadmium has been suggested as a toxic
agent to spermatogenesis because serum cadmium concentrations
of cadmium-exposed workers and smokers were more than double
those of non-smokers (Chia et al., 1994). Recently, increases in
seminal plasma and testicular cadmium have been documented
among men with varicoceles that were comparable with those in
heavy smokers (Benoff et al., 1997; Hurley et al., 2000). Several
investigators have reported toxic effects of this cation, but these
data will be discussed in more detail in later sections. Other recent
data have linked cadmium with apoptosis. The administration of
cadmium chloride to a rat model induced the characteristic
apoptosis ladder-like patterns of DNA on the agarose gel (Jones et
al., 1997). In humans, testicular biopsy specimens from men with
varicoceles correlated cadmium deposition with the percentage of
apoptotic cells per round tubule (Hurley et al., 1999). The mean
for control specimens was 0.3 mg Cd2+/mg dry weight with a
mean of 10% apoptotic cells. These ®gures were comparable with
those in a group of varicocele patients with normal spermatogen-
465
J.L.Marmar
esis on testis biopsy. In contrast, a group of varicocele patients
with hypospermatogenesis on biopsy had a mean of 1.3 mg Cd2+/
mg dry weight and 20±50% apoptotic cells per round tubule. In
addition, 60 patients with varicoceles were classi®ed on the basis
of seminal zinc and cadmium concentrations. Group 1 had
seminal zinc >1.6 mmol/l and seminal cadmium <0.4 mg/l, group
2 had seminal zinc <0.4 mmol/l and seminal plasma cadmium
0.4±0.7 mg/l, and group 3 had seminal zinc <0.4 mmol/l, but
seminal plasma cadmium >0.7 mg/l. Although pregnancies
followed varicocelectomies in patients within groups 1 and 2,
none of the men in group 3 achieved pregnancy after surgery.
Thus, these data suggest a potential for cadmium as a speci®c
toxic agent in men with varicoceles, and a brief view of cadmium
toxicity follows.
Review of cadmium toxicity
In laboratory animals, the accumulation of cadmium has produced
testicular damage and alterations in sperm function (Laskey et al.,
1984; Hew et al., 1993), and occupational exposure to cadmium
has been associated with impaired reproductive function
(Saaranen et al., 1989). These facts may be of some concern
because the general public is exposed to excess cadmium on a
daily basis in the form of contaminated drinking water, cigarette
smoke and aerosols (Elinder et al., 1985; Ragan and Mast, 1990).
Airborne cadmium particles are absorbed by the lung in a two-
fold greater concentration than particles absorbed through the
gastrointestinal tract. Cadmium may reach the testicle because of
increased testicular blood ¯ow which has been demonstrated in
experimental animal models with varicoceles (Saypol et al., 1981;
Nagler et al., 1987); however, increases in testicular arterial ¯ow
have not been de®nitively characterized and indeed, some studies
suggest decreased testicular ¯ow in both laboratory animals (Hsu
et al., 1995) and humans (Comhaire et al., 1983; Ross et al., 1994;
Li et al., 1999). Nevertheless, within the testicle, cadmium
produces damage to the endothelium of the testicular vasculature,
causing intratesticular oedema and an increase in intratesticular
¯uid pressure (Mason et al., 1964; Setchell and Waites, 1970).
Once this damage occurs, cadmium has easy access to all
compartments within the testicle, but these heavy metals are
usually bound to amino acids, peptides or phospholipids
(Ballatori, 1991; Vallee, 1995).
Metallothioneins are speci®c low-molecular weight, soluble,
metal-binding proteins that are produced by speci®c genes (Searle
et al., 1987; Senguin and Hammer, 1987; Waalkes et al., 1988). In
rat knock-out models for metallothionein genes, there is an
increased sensitivity to cadmium (Masters et al., 1994).
Furthermore, different strains of mice demonstrate variable
resistance to cadmium toxicity (Gunn et al., 1965; Taylor et al.,
1973). In humans, ethnic differences have been reported with
regard to susceptibility of germ cells to apoptosis (Sinha-Hikim et
al., 1998). Hypothetically, men with varicoceles may have genetic
variability leading to different metallothionein production rates.
There are no references on this subject because presently, there
are no data since this subject has not been studied in men with
varicoceles, and future studies of this type may provide new
information about individual molecular responses.
The role of nutritional supplements has been studied in the past,
but these studies may be revisited in the light of current
information on men with varicoceles. For example, in laboratory
466
animals, the administration of zinc (Saksena et al., 1983; Al-
Bader et al., 1999), ascorbic acid (Shiraishi et al., 1993) and
selenium (Jones et al., 1997) either before or after exposure to
cadmium may produce protection against the toxicity of
cadmium. In the past, some clinicians have used zinc supplements
as empirical therapy for men with varicoceles (Marmar et al.,
1975; Takihara et al., 1987). In the future, zinc, ascorbic acid and
selenium supplements may be used on a selective basis for men
with either documented elevations of seminal and/or testicular
cadmium and low concentrations of semen zinc, but carefully
designed clinical trials are needed to study the ef®cacy of these
supplements.
Although apoptosis may impact the sperm density of infertile
men with varicoceles, many of these patients present with other
seminal abnormalities such as decreased sperm motility and
reduced numbers of morphologically normal spermatozoa.
Speci®c causes for motility de®cits and abnormal sperm
morphology will be discussed in the next sections.
The effect of varicoceles on sperm motility
Many infertile men with varicoceles present with low sperm
motility, either alone or in combination with other low semen
parameters (Schlesinger et al., 1994), and several studies have
pointed to speci®c causes of low motility, including antisperm
antibodies, reactive oxygen species (ROS) and poor mitochon-
drial function. These factors will be discussed in the following
paragraphs.
One report of sperm-bound immunoglobulins in 27 of 84 (32%)
infertile men with palpable varicoceles was made using an
enzyme-linked immunosorbent assay (ELISA) (Gilbert et al.,
1989), while others (Oshinsky et al., 1993) reported IgG and IgA
sperm- bound antibodies in ®ve of 29 (17%) patients with palpable
varicoceles using immunobeads. These data suggest that sperm
antibodies may occur in a limited number of men with varicoceles,
but it is still unclear whether varicocelectomy can alter these
antibodies. In a later study (Knudson et al., 1994), nine of 32
infertile men with varicoceles had positive immunobead tests.
Following varicocelectomy, the motile sperm count improved for
71% of the men, but there was no change in the antibody status. A
report was also made (Agarwal, 1992) of 15 pregnancies among 45
antibody-positive couples (30%) treated with sperm washing and
intrauterine insemination (IUI). Others (Nagy et al., 1995) reported
on 55 ICSI cycles from 32 couples with high antibody titres (>80%
binding), the pregnancy rate being 30%.
Among patients attending an infertility clinic, 40% had
detectable levels of ROS in their semen (Aitken and Clarkson,
1987), whereas ROS were not detected in the semen of normal
volunteers (Iwasaki and Gagnon, 1992). Furthermore, ROS
production in semen seems inversely related to sperm motility
(de Lamirande and Gagnon, 1992). ROS may be a by-product of
seminal leukocytes or defective spermatozoa (Aitken et al., 1992),
and it has been demonstrated (Huszar and Vigue, 1994) that
cytoplasmic mid-pieces retained by spermatozoa may lead to the
production of ROS. Antioxidants are usually present in the semen
to scavenge oxygen free radicals in the form of urates, ascorbates,
sulphydryl groups, tocopherol and carotenoids (Lewis et al.,
1997). In men with varicoceles, ROS levels may be elevated
(Weese et al., 1993; Sharma and Agarwal, 1996) while the

seminal antioxidants may be low (Barbieri et al., 1999; Hendin et
al., 1999). In addition, defective mitochondrial oxidative
phosphorylation was demonstrated in a rat model with varicocele
(Hsu et al., 1995), and low levels of mitochondrial coenzyme Q10
(an antioxidant) were documented in the semen of men with
varicoceles (Mancini et al., 1994). Other natural enzymatic
scavengers such as superoxide dismutase (SOD) and catalase may
be also be de®cient (Alvarez and Storey, 1982; Gagnon et al.,
1991).
The effects of varicocelectomy on sperm motility in 12 studies
totalling 1010 patients has been reviewed (Schlesinger et al.,
1994). These data included changes of percentage motility after
surgery, but the outcomes were inconclusive. Five studies showed
a statistically signi®cant improvement in percentage motility after
surgery, but seven showed no improvement. In one study (Ismail
et al., 1999), computer-assisted semen analysis (CASA) was used
to evaluate qualitative sperm motility before and after varicoce-
lectomy. The results suggested a statistically signi®cant increase
in progressive motility and track speed following surgery. In
contrast, others (Fuse et al., 1995) noted improved sperm velocity
with CASA following varicocelectomy only in patients whose
partners became pregnant. Progressive improvement in CASA
values was also noted over 9 months of postoperative follow-up
(Parikh et al., 1996).
In some cases with elevated seminal ROS and de®ciencies of
antioxidants, investigators have used antioxidant therapy (Lenzi et
al., 1992) and glutathione (Kessopoulou et al., 1995). These data
suggest that varicocele patients with poor sperm motility and
evidence of oxidative stress may bene®t from a combination of
surgery and antioxidant therapy, but additional controlled clinical
trials are needed to evaluate these therapies.
The effects of varicoceles on sperm morphology
The classic morphological ®nding associated with varicoceles has
been the `stress pattern', which includes increased numbers of
elongated tapered sperm heads and amorphous cells (MacCloud,
1965). For example, 50 men with varicoceles had 36% tapered
forms in their ejaculate compared with 15% in controls with other
forms of idiopathic infertility (Naftulin et al., 1991); however,
these studies were performed with Papanicolaou stains and with
protocols that recorded abnormal morphological forms. In recent
years, morphology has been evaluated by `strict' criteria which
identify normal forms (Kruger et al., 1986). With these protocols,
sperm heads have oval shapes with length measurements of 3±
5 mm and width measurements of 2±3 mm. The acrosome covered
at least 40% of the sperm head, and there was minimal retention
of cytoplasmic droplets. The sperm tail was straight and
elongated. Recent studies in men with varicoceles indicated that
they had reduced numbers of morphologically normal forms, as
assessed by `strict' criteria (Vasquez-Levin et al., 1997; Schatte et
al., 1998).
In other studies, several investigators have examined speci®c
factors to explain some of the morphological defects found among
men with varicoceles. In one such study (Benoff et al., 1995)
donor spermatozoa were exposed to increasing concentrations of
cadmium, whereupon the spermatozoa took on the morphological
appearance of tapered spermatozoa as a result of depletion of
actin stores from the sperm cytoskeleton. These data suggest that
Pathophysiology of varicoceles
the presence of excess cadmium may induce the `stress pattern'.
The heat shock proteins may appear in response to heat, and it has
been suggested (Haus et al., 1993) that these proteins contain
sequences that are similar to actin. It is unclear whether these heat
shock proteins are protective or whether they inhibit polymeriza-
tion of actin by competitive binding (Kantengwa et al., 1991).
Nevertheless, future studies of heat shock proteins and sperm
morphology in men with varicoceles may reveal important
information regarding the abnormal morphology seen in these
patients. An increase was reported in the number of morpholo-
gical forms with retained cytoplasmic mid-pieces in men with
varicoceles (Zini et al., 1999). This morphological ®nding was
associated with reduced sperm motility but, following varicoce-
lectomy, the percentage motility increased from 22.6 to 32.9%,
while the percentage of spermatozoa with retained cytoplasm was
reduced from 25.8 to 18.1%.
In utilizing `strict' criteria to evaluate sperm morphology,
abnormalities in the structure of the acrosome may be identi®ed
(Menkveld et al., 1996), but in most instances specialized sperm
function tests are required to detect normal acrosome function.
Although sperm function tests have been used less in clinical
practice in recent years, the acrosome reaction induction test
seems important in the study of men with varicoceles.
Acrosome reaction induction test in men with varicoceles
An ionophore was used to induce the acrosome reaction among
oligospermic men, including some with varicoceles (Aitken et al.,
1984). However, this test may be less physiological than others
because it bypasses the need for calcium transfer. Others (Virgil
et al., 1994) used an immuno¯uorescence technique and human
follicular ¯uid stimulation to detect the acrosome reaction among
38% of infertile men with varicoceles. This test may be more
physiological, but it requires specialized equipment. As a simpler
alternative, hypo-osmotic swelling and double staining were
combined to document the viable acrosome-reacted spermatozoa
after exposure to the capacitation protocol including human
follicular ¯uid (Glazier et al., 2000). These authors reported that
48% of men with varicoceles had an abnormal acrosome reaction
induction test as part of their work-up. Following surgery, 35% of
this group converted to a normal acrosome reaction induction test
result, suggesting that varicocelectomy might help some men to
improve their acrosome function, especially in response to
progesterone stimulation. The acrosome reaction is complex
however, and more detailed studies have investigated the various
steps involved in the process.
A more physiological test was developed to study speci®c
events of the acrosome reaction (Benoff et al., 1993). These
authors noted that the reaction is a calcium-dependent process
which requires functional calcium channels in the sperm
membrane. Only a limited subpopulation of motile spermatozoa
seemed capable of completing the necessary exocytosis of the
acrosome reaction (Benoff et al., 1996). It was found that,
following the loss of surface cholesterol and in response to a
mannose stimulation, there was an in¯ux of calcium which
stimulated movement of subplasma membrane stores of D-
mannose-speci®c lectins, driven by myosin motors. With this assay,
the investigators identi®ed three distinct patterns, and infertile men
with varicoceles seemed incapable of achieving the second (type 2)
467
J.L.Marmar
pattern whereby the mannose ligands ordinarily congregated on the
sperm head surface distal to the equatorial region. These data
suggested that men with varicoceles may have an abnormal acrosome
reaction because of limited calcium in¯ux; this situation is similar to
that in men receiving calcium channel blockers, who were also
unable to manifest a type 2 pattern (Benoff et al., 1995).
These ®ndings have stimulated further investigations into the
structure and sequence of calcium channels among men with
varicoceles (Benoff, 1998). The acrosome reaction appears to be
associated with an L-type voltage-dependent calcium ion channel
(L-VDCC). This channel is composed of four subunits, and the a-
1 subunit has been sequenced. Speci®c changes in the molecular
structure of this unit have been identi®ed in other human disease
states (Ophoff et al., 1996; Zhuchenko et al., 1997). Most
recently, amino acid deletions have been noted in the lining of the
ion-conducting pore of the calcium channel among men with
varicoceles (Benoff et al., 2000), suggesting that there is a
potential genetic cause of infertility in these men.
Calcium channels may also be susceptible to environmental
toxins, because L-VDCC channels transport zinc, cadmium,
nickel, cobalt, aluminium and lead (Florman et al., 1992;
Florman, 1994; Platt and Busselberg, 1994). Recently, increased
concentrations of cadmium and lower concentrations of zinc have
been reported in the semen of infertile men with varicoceles
(Benoff et al., 1997). Thus, it has been postulated that cadmium
may enter the sperm head and exert a deleterious effect on the
calcium channel. A limited number of autometallography studies
have been carried out on testicular specimens from men with
varicoceles which identi®ed increased deposits of cadmium, and
the site of entry of cadmium and zinc have been co-localized on
the L-VDCC channel. It remains unclear whether varicocelect-
omy can reverse the effects of cadmium, or whether zinc
supplements may protect the L-VDCC channels from cadmium
toxicity. The laboratory technology is available to study these
interactions however, and molecular studies are needed to
evaluate these individuals.
Conclusions
Varicoceles are commonly diagnosed among men with infertility,
but the causes of infertility in these cases may be multifactorial.
Data from recent molecular and genetic studies may shed new
light onto the understanding of the pathophysiology of varico-
celes, with new diagnostic approaches and treatment plans being
developed from these ®ndings. For example, reduced sperm
concentration in men with varicoceles may be caused by
apoptosis which may be documented by TUNEL assays in either
ejaculated spermatozoa or testicular tissue. Several factors
associated with varicoceles may induce pathways which lead to
apoptosis, including heat stress, androgen deprivation and
exposure to toxic elements. The percutaneous aspiration of
testicular tissue and ¯uid may be utilized in the future to identify
apoptosis in men, in association with heat shock protein response
and low intratesticular testosterone. Since increased cadmium
deposition has been recognized as a toxic agent in men with
varicoceles, seminal and tissue assays for cadmium may be used
in future work-ups on these men.
De®cits in sperm motility among men with varicoceles may be
related to sperm antibodies, excess ROS, and dysfunction of
468
sperm mitochondria. Laboratory tests to identify these speci®c
problems are currently available. Some men with varicoceles may
present with abnormal sperm morphology based upon `strict'
criteria. These men usually show abnormalities in sperm function
and presently, acrosome reaction induction tests may be utilized
to document abnormal sperm function.
Several treatment options are available for these patients,
including outpatient microsurgical varicocelectomy, sperm wash-
ing, IUI and ICSI. It has been shown (Schlegel, 1997) that the cost
of IVF/ICSI was $89 091 per delivered child, whereas the cost of
varicocelectomy was $26 268 per delivered child. Nevertheless,
ICSI is an important option for the therapeutic armamentarium
and may be required in selective cases.
Several new non-surgical therapeutic options may also be
available. For example, supplemental antioxidant therapy may be
appropriate for men with poor motility and documented
elevations of ROS. Testosterone stimulation may be utilized with
HCG injection therapy for men with speci®c deprivation of
intratesticular testosterone. Supplementary zinc may be utilized in
men with reduced seminal zinc concentrations and increased
seminal cadmium. However, controlled studies will be needed to
evaluate the ef®cacy of these supplements.
There are several new opportunities for future research in men
with varicoceles. Since apoptosis may occur among men with
varicoceles, additional studies should be carried out using
ejaculated spermatozoa and testicular tissue in order to evaluate
Fas±Fas-ligand activity, cytoplasmic caspase activity and gene
expressions by the Bax/Bcl2 family. In addition, studies may be
carried out to evaluate heat shock protein production in these
men. Since cadmium toxicity has been suggested as a factor in the
pathophysiology of varicoceles, the measurement of metal-
binding proteins (metallothioneins) may provide new information
in this group of men. Since calcium channels may be defective in
men with varicoceles, initial studies of the a-1 subunit
microdeletions may determine the pattern of these abnormal
sequences. These ®ndings imply a genetic defect which may not
be correctable in some men.
The ®eld of andrology is entering a new phase which will
include clinical applications of molecular and genetic informa-
tion. Although some of these concepts related to varicoceles
appear rather futuristic, it is hoped that patients with varicoceles
will bene®t from new protocols and treatment options developed
from these molecular/genetic studies. Finally, the current
literature supports the hypothesis of varicoceles as co-factors
that may occur in men with varying degrees of molecular/genetic
problems. In combination, they may lead to infertility and
determine the potential for reversibility.
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