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* From the Department of Pediatrics, Obstetrics
and Reproductive Medicine, Biology Section, University of Siena, Regional
Referral Centre for Male Infertility, Azienda Ospedaliera Universitaria
Senese, Siena, Italy; and the
Phlebolymphological Centre, Department of
Surgery, University of Siena, Siena, Italy.
| Correspondence to: Prof Baccio Baccetti, Department of Pediatrics, Obstetrics and Reproductive Medicine, Biology Section, University of Siena, Policlinico S. Maria alle Scotte, 53100 Siena, Italy. |
| Received for publication April 26, 2005; accepted for publication July 25, 2005. |
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Abstract |
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Key words: FISH, pathological spermatogenesis, TEM, varicocelectomy
The evaluation of recovered sperm quality after varicocelectomy is certainly a hot topic in the field of varicocele research. The range of sperm concentration among patients who appear to benefit from varicocelectomy is extremely broad, from azoospermia to up to 40 x 106 spermatozoa/mL (Marmar et al, 2001). A recovery in sperm concentration and qualitative sperm parameters was demonstrated (Demas et al, 1991; Marsman et al, 1995; Parikh et al, 1996) after varicocele surgical correction; however, Schlesinger et al (1994) observed an enhancement in sperm density only, and Soffer et al (1984) observed improvement in the motility index.
In recent years a correlation between varicocele and the molecular analysis of Y microdeletions or chromosome aneuploidies (or both) has been detected. Y microdeletion screening should be performed for infertile men with varicocele for a proper diagnosis and to avoid unnecessary treatment that probably would not improve the semen parameters (Moro et al, 2000; Cayan et al, 2001). A genetic screening for chromosome aneuploidies, investigated by fluorescence in situ hybridization (FISH), of the sperm of infertile men affected by varicocele with poor semen quality was performed by Finkelstein et al (1998), in which a general increase of meiotic nondisjunction was found. For this reason, in the present paper, a large-scale investigation was designed to define a sperm ultramorphology, a genetic pattern specific for varicocele, and to elucidate the improvement of sperm quality after varicocelectomy.
During the years 1999–2005, the ejaculates from selected men affected by varicocele, recruited at the Regional Referral Centre for Male Infertility (RRCMI; Siena), were examined. Semen samples were analyzed by light microscopy to evaluate sperm concentration and motility. The morphological characteristics of these sperm were studied by TEM. The effect of varicocele on meiotic chromosome segregation was investigated by FISH in 39 sperm samples. The potential direct benefits of varicocelectomy on sperm quality were evaluated, analyzing sperm characteristics before and after varicocele surgical correction.
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Materials and Methods |
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We selected 306 of 352 men by following some exclusion criteria: patients with varicocele also showing other anatomical pathologies, hormonal imbalance, carriers of genetic sperm defects, and patients presenting different degrees of consanguinity in their history. Of 306 patients, 13 resulted azoospermic, and 293 seminal analyses were performed for the others. FISH analysis was performed for 39 of 293 patients with varicocele of various degrees and with different semen parameters.
In the selected group, sexual development and medical histories were normal and serum hormone concentrations (follicle-stimulating hormone, luteinizing hormone, prolactin, androstenedione, dehydro-3-epiandrosterone-sulphate, estradiol, testosterone, free testosterone, B-inhibin) were within the standard range. Microbiological investigations revealed no urogenital infections. None of the patients had ever received hormone therapy. Only patients with an apparently normal 46, XY karyotype were included in this study.
Among the selected group, 26 patients underwent varicocelectomy. We analyzed semen characteristics before and after this varicocele repair. Y microdeletion screening was performed on this group.
Semen Analysis
Light and Electron Microscopy—
Semen samples of patients with varicocele were collected by masturbation
after 4 days of sexual abstinence and examined after liquefaction for 30
minutes at 37°C. Volume, pH, concentration, and motility were evaluated
according to WHO guidelines (World Health
Organization, 1999).
In the group of 26 patients undergoing varicocele repair, semen analysis was performed before surgery and repeated 3–4 months after varicocelectomy.
For electron microscopy, sperm samples were fixed in cold Karnovsky fixative and maintained at 4°C for 2 hours Fixed semen was washed in 0.1 mol/L cacodylate buffer (pH 7.2) for 12 hours, postfixed in 1% buffered osmium tetroxide for 1 hour at 4°C, then dehydrated and embedded in Epon Araldite. Ultrathin sections were cut with a Supernova ultramicrotome (Reickert Jung, Vienna, Austria), mounted on copper grids, stained with uranyl acetate and lead citrate, and then observed and photographed with a Philips CM10 TEM (Philips Scientifics, Eindhoven, The Netherlands).
For each patient, 300 ultrathin sperm sections were analyzed. Major submicroscopic characteristics were recorded by highly trained examiners (E.M., P.P.) who were blind to the experiment, applying the same evaluation criteria. TEM data were elaborated using the mathematical formula by Baccetti et al (1995), based on the Bayesan technique. This formula considers 16 selected submicroscopic characteristics of sperm organelles able to define the sperm function (see Appendix) and calculates the number of spermatozoa free of structural defects ("healthy") and the percentages of 3 main phenotypic sperm pathologies: immaturity, necrosis, and apoptosis.
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Semen samples from 20 men of proven fertility (ages 22 to 35 years) with normal karyotype and without varicocele were used as controls.
FISH Analysis of Sperm—
To evaluate aneuploidy frequency, FISH was performed according to Baccetti
et al (2003) on the sperm
nuclei of 39 patients with varicocele. A mix of 
-satellite DNA probes
(Chromosome Enumeration Probes, Vysis, Ill) for chromosomes 18, X, and Y,
directly labeled with different fluorochromes, was used. Sperm nuclei were
scored according to published criteria
(Martin and Rademaker, 1995; Baccetti et al, 2003). All
samples were analyzed by a highly trained examiner (G.C.).
Observation and scoring were performed using a Leitz Aristoplan Optical Microscope (Rockleigh, NJ) equipped with a fluorescence apparatus, with a triple bandpass filter for aqua, orange, and green fluorochromes (Vysis) and a monochrome filter for diamino-phenylindole (DAPI).
Controls
Semen samples from seven fertile men (ages 26 to 39 years) were analyzed
and used as controls (Baccetti et al,
2003).
Surgery
Twenty-six patients underwent surgery because of testicular pain. The
surgery time and echography exam were performed in the Phlebolymphological
Centre, Department of Surgery, University of Siena. A preoperating color
duplex scan exam was performed to demonstrate the number of varicose trunks
presented inside the spermatic cord.
The surgery was carried out in the Day Hospital regime, using an anesthetic block of both the ileohypogastric and ileoinguinal nerves, performed at the anterior superior iliac spine, and local anesthesia at the basis of the scrotum where we made the skin incision. We prefer the subinguinal approach to preserve the inguinal channel, if in the future the patient could present an inguinal hernia. We made a curved incision along the base of the scrotum and opened all the superficial planes up to the spermatic cord, which was isolated distally to the external inguinal ring to explore the pavement of this area and to discover whether the external spermatic vein pierced the muscle wall. The spermatic cord was then suspended on a Farabeuf retractor and opened to search for the varicose vein, demonstrated with duplex scan exam. A wide resection was performed both proximally and distally. Moreover, it was possible to move the testis for a more extensive resection, often associated with a vaginal eversion. After careful hemostasis control and local antibiotic washing, we performed an intradermic suture.
All varicocelectomies were performed by 1 surgeon (S.M.). All patients were discharged 3 hours after surgery, dressed with a testicle jockstrap, given an antibiotic therapy for 3 days, and an anti-inflammatory drug if necessary.
Until this time, patients had not presented a vein reflux in the spermatic cord at the color duplex scan exam.
Polymerase Chain Reaction Analysis
Polymerase chain reaction (PCR) analysis was performed for the group of 26
patients undergoing surgery. DNA was extracted from peripheral blood
lymphocytes using the QIAamp DNA Blood kit (QIAGEN, Hilden, Germany). PCR was
performed according to European Academy of Andrology (EAA) guidelines for
molecular diagnosis of Y chromosomal microdeletions
(Simoni et al, 2004).
Control DNA was extracted from the blood of 10 male donors, ages 30–40 years, with a documented history of fertility. DNA extracted from the blood of 2 fertile women was used as a negative control.
Statistical Analysis
Semen parameters and sperm pathologies in the varicocele group are
expressed as percentages and means ± SD. Standardized skewedness and
standardized kurtosis values were used to determine whether the data of the
patients, before and after varicocelectomy, came from normal distribution. Not
all the data were normally distributed; therefore, the statistical analysis
was performed using the Wilcoxon rank test to verify a significant difference
in the same group between the semen values before and after varicocelectomy.
Significance was defined as P < .05.
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Results |
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Submicroscopic characteristics of sperm organelles were evaluated using our statistical mathematical formula, which calculates the number of spermatozoa free of structural defects ("healthy"). In 55 (19%) of 293 individuals, the number of healthy spermatozoa was higher than 2 x 106, assuring normal fertility. On the contrary, 238 patients (81%) showed a number of healthy sperm lower than 2 x 106, and these were considered infertile according to the formula by Baccetti et al (1995).
Semen analysis showed that 84 of 238 patients (35%) were oligozoospermic and progressive motility was reduced in 230/238 (97%). The mean values of semen parameters are reported in Table 1. TEM analysis confirmed the presence of different defects affecting sperm organelles related to immaturity, apoptosis, and necrosis. The characteristic features of immaturity (Figure 1a) were reduced acrosomes located far from the nucleus; and misshaped, round, or elliptical nuclei with uncondensed chromatin. Cytoplasmic droplets were detected around the head and at the middle piece level of the tail, which appeared rolled up with a badly assembled mitochondrial helix. The axonemes sometimes showed normal structure with visible dynein arms (Figure 1b). Double spermatozoa were frequently present. Marginated chromatin, cytoplasmatic translucent vacuoles, and swollen and badly assembled mitochondria were the typical ultrastructural markers of apoptosis (Figures 1a and 2). Spermatozoa with broken plasma membrane, reacted or absent acrosome, misshaped nuclei with disrupted chromatin, and poor axonemal and periaxonemal cytoskeletal structures were affected by necrosis (Figure 2).
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The mathematical formula by Baccetti et al (1995) also calculates the percentage of these phenotypic sperm pathologies. Immaturity was predominant in the group of 293 patients affected by varicocele (72.44% vs 55.10% fertile controls); the evaluated standard deviation was moderately low, showing that immaturity is widely present in spermatozoa from varicocele patients. Necrosis was high (42.32% vs 21% fertile controls), and the high value of standard deviation did not allow us to affirm that necrosis was widely diffused in the spermatozoa from these men. Finally, apoptosis was more than twice (12.15%) that of fertile controls (4.8%); in this case, the value of standard deviation indicated that sperm apoptosis was associated with varicocele only in some cases. These results are shown Table 2.
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Meiotic segregation was investigated by FISH carried out on the sperm nuclei of 39 patients with varicocele. A total number of 193 062 sperm nuclei was scored. The mean frequencies of the aneuploidy of chromosomes 18, X, and Y are summarized in Table 3. Of 39 patients, 8 showed FISH values within the normal range, 15 presented diploidy and disomy frequencies higher than the normal range, and only 6 had diploidy frequencies outside normal values. Generally, the mean frequency percentage of sex chromosome disomy and diploidy was higher in the spermatozoa from varicocele patients than in the control group.
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To quantify the effects of a varicocelectomy on sperm quality, we analyzed, before and after the surgical treatment, the semen of 26 men by light and electron microscopy (Table 4). Before varicocelectomy, mathematically elaborated TEM analysis showed that 10 patients were within the parameters of submicroscopical fertility (number of "healthy" sperm > 2 x 106), whereas 16 patients did not reach normal values, despite the general normal sperm concentration.
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In the "fertile" group, the number of "healthy" sperm after varicocelectomy was strongly reduced in only 1 patient (10%), whereas 6 patients (37.5%) reached values of a number of "healthy" sperm able to ensure natural fertility in the "infertile" group.
In both groups, statistical analysis comparing semen characteristics before and after varicocele repair (Table 4) was performed to assess significant differences. Among the semen characteristics evaluated (sperm concentration/mL, motility, total number and percentage of healthy sperm, pathologies) in the "fertile" group, only immaturity improved significantly (P < .05) after surgery (Table 4). In the "infertile" group, the total number and the percentage of "healthy" sperm resulted significantly higher, P < .002 and P < .003 respectively, after treatment (Table 4).
PCR analysis performed on peripheral blood lymphocytes did not reveal any microdeletions of the Y chromosome.
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Discussion |
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Different researchers have focused upon the effect of varicocele in the function of human sperm cells and sperm characteristics (Rodriguez-Rigau et al 1981; Naftulin et al, 1991; Zini et al, 2000; Hauser et al, 2001; Ziemba et al, 2002). Sperm morphology is generally evaluated by light microscopy, although a deep analysis of subcellular sperm anomalies can only be performed by electron microscopy. Using this method, Holstein and Roosen-Runge (1981) demonstrated that varicocele affects sperm morphology by the impairment of differentiation in the early spermatid stage. Reichart et al (2000) suggested that varicocele may cause deleterious alterations in early spermatid head differentiation, causing sperm acrosome and nucleus malformations.
To detect specific ultrastructural sperm anomalies related to varicocele, we analyzed the sperm characteristics of 293 patients by TEM analysis elaborated by the formula by Baccetti et al (1995). We examined only 1 semen sample from each patient, but in such a large population, we supposed that it was sufficient to evaluate the relation between varicocele and sperm quality. By this method, only 19% of the analyzed patients were within the ultrastructural range of fertility.
All pathologies were higher in the varicocele group compared to controls. Whereas immaturity had already been reported in sperm from varicocele patients (Baccetti et al, 1991, 1993), it has also recently been reported that apoptosis seems to have an important role in spermatogenetic dysfunction associated with varicocele (Simsek et al, 1998). To our knowledge, the results showed for the first time that sperm necrosis could be related to the presence of varicocele.
The data concerning meiotic segregation of analyzed chromosomes are of particular interest. It is known that infertile patients with poor sperm quality, due to different causes, seem to have an increase in the frequency of aneuploidy (Bernardini et al, 1997). However, the presence of compromised testicular environments could also favor meiotic errors (Mroz et al, 1999). We analyzed, by FISH, spermatozoa from selected patients with varicocele and showing different semen profiles. The mean frequencies of disomies and diploidies were generally out of normal range, indicating a severe disturbance of meiotic segregation. These data support the hypothesis that the negative effect of varicocele on sperm morphogenesis can affect the karyoskeleton and the cytoskeleton, involving chromatin texture during molding and chromatid separation.
Several studies have reported an improvement in sperm parameters after surgical treatment of varicocele (Nieschlag et al, 1993; Yamamoto et al, 1994; Parikh et al, 1996; Pierik et al, 1998; Ismail et al, 1999; Kim et al, 1999; Onozawa et al, 2002) and that 30% to 60% of couples achieve pregnancy as a result of varicocelectomy (Schoor et al, 2001). The treatment of varicocele in infertile patients can improve sperm count and morphological parameters, particularly abnormalities of the head, according to Kruger's strict criteria (Vasquez-Levin et al, 1997; Bouchot et al, 1999).
Other studies have shown no benefit in sperm parameters after varicocele repair (Nilsson et al, 1979; Gerhard et al, 1992).
The variability of these results could be due to a lack of uniformity in patient selection, follow-up, treatment techniques (Yavetz et al, 1992), and statistical methods (Comhaire, 1983). It is also necessary to consider that the evaluation of semen quality itself is not always performed according to the same criteria.
In our study, a mathematical ultrastructural evaluation of sperm characteristics carried out before and after varicocele repair showed a general improvement in sperm quality. However, only immaturity in the group of fertile patients and the percentage and the number of healthy sperm in the group of infertile patients reached statistical significance. These data support the hypothesis of a general improvement in sperm quality after varicocele repair in our selected group of patients, which, generally showed a normal sperm concentration and reduced progressive motility. It is hoped that a varicocelectomy will allow the infertile patient with clinical varicocele to not only improve the likelihood of pregnancy through natural conception, but also increase the chances of conception with a less invasive and less costly technique. Further studies of a larger number of heterogeneous cases should be planned.
In the group of 26 patients, screening for chromosome Y microdeletion was also performed, as recommended by Moro et al (2000), to avoid unnecessary treatment that would probably not improve semen parameters. Indeed, men with varicocele and genetic alterations appear to have a poorer response to varicocele repair than men without coexisting genetic lesions (Cayan et al, 2001). In our group, there was no increase in the incidence of Y chromosome microdeletions; however, because the population was small, it was impossible to draw a definitive conclusion.
In our study, we pointed out that varicocele has a negative effect on sperm structure and function, and on meiotic segregation. Moreover, we observed a recovery of sperm quality, particularly in infertile patients after varicocelectomy. Considering these data, we suggest that TEM and FISH analyses should be performed for all varicocele patients, to identify infertile individuals who should be proposed for surgery. The fertile patients without ultrastructural and genetic sperm alterations should be monitored every 6–12 months to promptly detect a possible deterioration in seminal parameters.
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Footnotes |
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