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Department of Surgery, Biology Section; Interdepartmental Centre for Research and Therapy of Male Infertility, University of Siena, Siena, Italy.
| Correspondence to: Giulia Collodel, Department of Surgery, Biology Section, University of Siena, Policlinico Le Scotte, Viale Bracci, 14, 53100 Siena, Italy (e-mail: collodel{at}unisi.it). |
| Received for publication April 12, 2007; accepted for publication September 13, 2007. |
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Abstract |
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Key words: Fertile donors, sperm, TEM, FISH.
Meiotic studies and chromosome analyses by FISH should be considered for proper reproductive counseling. It is known that infertile men with poor sperm quality, due to different causes, seem to have increases in the frequency of aneuploidy (Bernardini et al, 1997; Gianaroli et al, 2005). Because individuals with abnormal semen parameters make up the majority of intracytoplasmic sperm injection (ICSI) candidates, the study of the chromosomal constitution of their spermatozoa is of great interest. The study of the incidence of disomy and diploidy in spermatozoa from fertile, apparently normal individuals is important for making comparisons to determine the real increase of aneuploidy in infertile cohorts.
Because considerable heterogeneity among normal men has been demonstrated, it is essential for each laboratory to obtain baseline data on a large number of men to evaluate variability in the normal population (Martin et al, 1996). The different groups involved in this field of research have published about internal controls used to compare the frequency of sperm aneuploidies from normal men with those from men at an increased risk of nondisjunction (Vegetti et al, 2000; Baccetti et al, 2003; Carrell et al, 2004).
In this article, we present data on aneuploidy frequencies for chromosomes 18, X, and Y in spermatozoa from 25 men of proven fertility. Spermiogram data and sperm morphology, evaluated by transmission electron microscopy (TEM), were also generated from the same samples. An attempt to compare the values of sperm apoptosis and necrosis obtained by TEM with those obtained with an annexin V/propidium iodide (PI) assay was also carried out.
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Methods |
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Semen Analysis
Light and Electron Microscopy—Semen samples from men of
proven fertility were collected by masturbation after 4 days of sexual
abstinence and were examined after liquefaction for 30 minutes at 37 °C.
Volume, pH, concentration, and motility were evaluated according to World
Health Organization (WHO) guidelines
(1999).
For electron microscopy, sperm samples were fixed in cold Karnofsky fixative and maintained for 2 hours at 4 °C. 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, and 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 Scientific, Eindhoven, The Netherlands).
Three hundred ultrathin sperm sections were analyzed for each sample. Major submicroscopic characteristics were recorded by highly trained examiners who were blinded to the experiment. TEM data were evaluated using the statistical mathematical formula by Baccetti et al (1995), based on the Bayesian method, which calculates the number of "healthy" spermatozoa (ie, free of structural defects) and the percentages of 3 main phenotypic sperm pathologies: immaturity, necrosis, and apoptosis (Baccetti et al, 2006). We observed that the lowest number of spermatozoa free of defects, assuring normal fertility, was slightly higher than 2 million sperm/mlxvolume.
FISH Analysis of Sperm—To evaluate aneuploidy frequency,
FISH was performed according to Baccetti et al
(2003) on the sperm nuclei of
the 25 men of proven fertility. A mix of 
-satellite DNA probes
(chromosome enumeration probes; Vysis Inc, Des Plaines, Ill) for chromosomes
18, X, and Y, directly labeled with different fluorochromes, was used. All
samples were analyzed by a highly trained examiner.
The overall hybridization efficiency was greater than 99%. Sperm nuclei were scored according to published criteria (Martin and Rademaker, 1995); they were only scored if they were intact and nonoverlapped and had clearly defined borders. When aneuploidy was detected, the presence of a sperm tail was confirmed. A sperm was considered disomic if the 2 fluorescent spots were of the same color; similar in size, shape, and intensity; and located inside the edge of the sperm head, at least 1 domain apart. Diploidy was identified by the presence of 2 double fluorescent spots, according to the criteria above. Observation and scoring were performed using a Leitz Aristoplan optical microscope (Leica, Wetzlar, Germany) equipped with a fluorescence apparatus, with a triple bandpass filter for aqua, orange, and green fluorochromes (Vysis Inc) and a monochrome filter for 4,6 diamidoino-2-phenylindole.
Detection of Membrane Phosphatidylserine Externalization and Membrane Integrity
The detection of phosphatidylserine (PS) externalization was performed with
the Vybrant apoptosis assay (Invitrogen Ltd, Paisley, United Kingdom), which
consists of fluorescein isothiocyanate (FITC)–annexin V and PI that are
able to differentiate viable from necrotic cells.
Seventeen of 25 samples were washed with phosphate-buffered saline, centrifuged, and suspended in annexin-binding buffer (ABB) to obtain a cell density of approximately 1 x 106. According to the manufacturer's instructions, 10 µL of FITC–annexin V and 1 µL of PI working solution (100 µg/mL) to each 100 µL of cell suspension were added. The spermatozoa were incubated for 15 minutes at room temperature. After a careful wash with ABB, a drop of sperm cell suspension was smeared on each glass slide. Slides were mounted in glycerol containing 5% n-propylgallate. Observations and photographs were made with a Leitz Aristoplan light microscope (Leica) equipped with a fluorescence apparatus. A total of 300 spermatozoa from each sample were counted. By staining cells simultaneously with FITC–annexin V (green fluorescence) and nonvital dye PI (red fluorescence), it is possible to recognize intact cells (FITC–annexin V negative, PI negative), early apoptotic cells (FITC–annexin V positive, PI negative), damaged sperm with PS externalization (FITC–annexin V positive, PI positive), and damaged necrotic sperm (FITC–annexin V negative, PI positive).
Statistical Analysis
Statistical analysis was performed using SAS software version 8 (SAS
Institute Inc, Cary, NC). Because of the small number of cases, the values
from TEM and FISH analyses were used to build normal ranges and determine the
25th and 75th percentiles.
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Results |
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Submicroscopic characteristics of sperm organelles were evaluated using a statistical mathematical formula (Baccetti et al, 1995) that calculates the number of spermatozoa free of structural defects ("healthy") and the percentages of sperm pathologies such as immaturity, necrosis, and apoptosis. These results are shown in Table 1. In 5 subjects (1, 3, 9, 10, and 25), we observed a number of "healthy" sperm in counts slightly lower than 2 x 106, a fertility index reported by us as the minimum number of sperm devoid of ultrastructural defects to result in normal fertility. In particular, the lowest number found was 1,603,886 (subject 3). The means ± SD, medians, and 25th and 75th percentiles of the values of sperm apoptosis, necrosis, and immaturity are also reported in Table 1.
The amounts of apoptosis and necrosis obtained by TEM were correlated with values obtained by the annexin V–PI assay (Table 2). This test was carried out in 17 of 25 individuals. The TEM necrosis percentages correlated with the percentages of PI-positive spermatozoa (r = .74), and the correlation for apoptosis detected by the 2 methods was positive (r = .75). However, the annexin V assay was also able to detect, in the same sperm cells, the simultaneous presence of PS externalization by FITC–annexin V and PI staining, which indicated a late stage of apoptosis. These PI-stained sperm showed broken plasma membranes, which is considered to be necrotic by TEM analysis. For this reason, we repeated correlation analysis (r = .68), adding to the values referring to necrotic cells (FITC–annexin V negative, PI positive) and those of FITC–annexin V–positive and PI-positive sperm.
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Meiotic segregation in the same samples was investigated by FISH, and 128,352 sperm nuclei were scored. The mean of frequencies related to the aneuploidy of chromosomes 18, X, and Y reported for each case are summarized in Table 3, in which the values of total disomy, total diploidy, and total aneuploidy are also shown. The mean frequency for sex chromosomal aneuploidies was 0.06% XX, 0.05% YY, and 0.14% XY; for chromosome 18, it was 0.10%, with a mean of 0.35% for total disomy. The mean frequency of diploid sperm was 0.28%, in particular 0.09% 1818XX, 0.07% 1818YY, and 0.11% 1818XY.
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In comparing the frequencies of disomy and diploidy, we found that the frequency of total diploidy among our cases ranged from 0.0996% to 0.432% and total disomy from 0.151% to 0.603%. Finally, the mean total sperm aneuploidy rate was 0.627%, ranging from 0.329% to 0.927% (Table 3). The man of proven fertility who showed the highest incidence of total aneuploidy was subject 25 with a total aneuploidy frequency of 0.927% (0.324% total diploidy and 0.603% total disomy).
Values of aneuploidy presented in articles from the literature, in which a consistent number of control cases were reported, are summarized in Table 4.
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Discussion |
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The aim of this study was to analyze sperm morphology and meiotic segregation in samples from 25 men of proven fertility, using our mathematical method applied to TEM and FISH analyses. As expected, not all of the semen samples from fertile donors reached the values that WHO considers normal, although they were very close: in particular, in our group of men of proven fertility, the lowest concentration found was 18 x 106 and the most reduced progressive motility was 29%.
The ultrastructural mathematical method (Baccetti et al, 1995) was used to evaluate morphology; this method allowed us to determine that the lowest number of spermatozoa free of defects ("healthy"), ensuring normal fertility, was slightly higher than 2 million. This mathematical approach also recently provided the thresholds for sperm pathologies such as apoptosis, necrosis, and immaturity in 20 men of proven fertility (Baccetti et al, 2006). These thresholds were confirmed in this study.
We used the annexin V/PI assay, rather than terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling assay, to confirm TEM apoptosis and necrosis observations. DNA fragmentation is a well known feature of apoptosis. This process occurs in the late stage of apoptosis and results from the activation of endogenous endonucleases. Several authors have proposed DNA stand breaks as the main evidence for apoptosis in human sperm; however, DNA fragmentation in mature sperm can have other origins beside apoptosis because it can occur during or after DNA packaging in spermiogenesis or as a result of oxidative stress (Varum et al, 2007). A critical stage of apoptosis involves the acquisition of surface changes; in particular, PS is translocated from the inner side of the plasma membrane to the outer layer. The annexin V protein preferentially binds to negatively charged phospholipids, such as PS, in the presence of Ca2+, and it shows minimal binding to phosphatidylcholine and sphingomyelin. PI penetrates cells with broken membranes, one of the first symptoms of necrosis.
The values of apoptosis and necrosis obtained by TEM were also positively correlated with those obtained by the annexin V assay. The fact that the values from TEM analysis, particularly those of necrosis, were higher compared with values observed with the annexin V assay could be due to the application of the Bayesian formula, which is based on probability calculation. For this reason, these values express percentages of probability of the presence of a given pathology, characterized by specific ultrastructural sperm defects; for example, the sperm alterations typical of necrosis are broken plasma membranes, absent or reacted acrosomes, disrupted chromatin textures, and axoneme and accessory axonemal structures (Moretti et al, 2006). Therefore, a detailed sperm analysis by TEM could show more severe results than that performed with a less sophisticated method.
In the analyzed cases, we observed that 5 subjects did not reach the previously reported fertility index, confirming that thresholds can sometimes be wrong, particularly in the field of fertilization mechanisms.
An important aspect of this study concerned the analysis of meiotic segregation in the same seminal samples examined by TEM. Chromosomal aneuploidy occurs when a sperm cell possesses more or less than a single copy of each autosomal or sex chromosome or more than 1 copy of the entire genome. Sperm aneuploidy frequencies from "fertile" or "healthy" men, "men with normal semen parameters," and "donors" are reported in the literature, but these designations were based on nonobjective criteria. This is an important issue because some ejaculates with normal concentrations, motilities, and morphologies are unable to fertilize. Only a few authors (Bernardini et al, 1998; Vegetti et al, 2000; Viville et al, 2000; Ryu et al, 2001; Carrell et al, 2004) have described the frequency of aneuploidy in men of proven fertility, and in our opinion, this is the most objective criteria for studying the incidence of meiotic segregation derangement in the normal population.
We performed FISH on sperm nuclei in the same semen samples from 25 men of recent proven fertility that were enrolled for the TEM study. The frequency of 18 disomies in our study (0.1%) is a little higher than the value reported by other groups (Martin et al, 1996; Estop et al, 2000; Hristova et al, 2002), but it appears to be slightly lower that the mean value reported in a study by Petit et al (2005).
The frequency of sex chromosomal disomies is comparable to that found by other authors (Table 3). Although Martin et al (1996) observed a mean value of 0.18% YY disomies, we obtained a mean value of 0.05% in the present study. Hristova et al (2002) showed a frequency of 0.3% XY disomies; however, in our subjects, we found 0.14%. The mean frequency of diploidies was comparable to those reported by Hristova et al (2002) and Morel et al (2004) and confirmed the value previously published by our group (Baccetti et al, 2003).
The production of diploid spermatozoa is the most common anomaly of all chromosome aberrations related to human male infertility, but a low percentage is also present in fertile men. Diploid XY sperm come from an error that occurs in the first meiotic division in primary spermatocytes, whereas XX or YY diploid sperm are derived from an error in the second meiotic division. In infertile men, any severe meiotic disorder can affect the anaphase I checkpoint and result in the production of diploid spermatozoa. This could explain why diploid sperm are so frequently found in infertile men and why they are the main and most constant anomaly observed whenever serious disturbances of the meiotic process occur (Egozcue et al, 2000).
Considering each patient individually, the result of total aneuploidy determined for 3 chromosomes varied, and the highest value observed among our cases of men of proven fertility was 0.927%. Petit et al (2005), estimating the frequency of disomy and diploidy of 7 chromosomes in spermatozoa from 10 fertile men, demonstrated total aneuploidy rates ranging from 0.4% to 1.5%. These results agree with data reported by Burrello et al (2003), who, evaluating 5 chromosomes in spermatozoa from 14 normozoospermic healthy men, found an aneuploidy rate of 0.91% (range, 0.3%–1.55%). Our values of total aneuploidy appeared to be lower, although we considered the sum of the aneuploidy values related to a number of chromosomes lower than that considered by Petit et al (2005) and Burrello et al (2003). Moreover, we studied a large group of men who showed good semen quality and recently documented proven fertility.
It is not possible to determine if sperm with abnormal morphology or motility or those affected by particular sperm pathologies are the same as those containing aneuploid nuclei according to these 2 methods. Only a few authors have used an electronic microstage locator to conclude that normally shaped sperm from men with oligoasthenoteratozoospermia also have increased aneuploidy rates (Ryu et al, 2001; Burrello et al, 2004).
In conclusion, our data are in agreement with the results reported in recent literature, considering the variations observed in the healthy male population (Martin et al, 1996). As has been reported by all authors performing sperm FISH analysis, the variation in estimated aneuploidy frequency may be due to variability in technique. Also, considering that the scoring criteria were those reported by Martin and Rademaker (1995), an inadequate number of sperm samples could be responsible for the different results from different laboratories.
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Footnotes |
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