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From the * Section of Endocrinology, Andrology and
Internal Medicine, Department of Biomedical Sciences, and Master in
Andrological and Human Reproduction Sciences, University of Catania, Catania,
Italy, and Private IVF Center of Rabat, Rabat,
Morocco.
| Correspondence to: Prof Aldo E. Calogero, Cattedra di Endocrinologia, Ospedale Garibaldi, Piazza S. Maria di Gesù, 95123 Catania, Italy (fax: 39-95-310899; e-mail: acaloger{at}unict.it). |
| Received for publication October 10, 2002; accepted for publication January 28, 2003. |
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Abstract |
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Key words: Enlarged sperm head, fluorescence in situ hybridization, male infertility
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Materials and Methods |
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Semen Preparation for FISH Analysis
An aliquot of spermatozoa was washed 3 times in phosphate-buffered saline
(pH 7.2) and centrifuged at 650 x g for 10 minutes. The
sediment was then fixed in methanol/acetic acid (3: 1). The fixed specimens
were stored at –20°C until further processing. The sperm heads were
decondensed by incubating the spermatozoa with 25 mmol/L dithiothreitol, as
reported elsewhere (Calogero et al,
2001b). The treatment did not disrupt the sperm structure,
including the tail, which allowed an unequivocal differentiation between
spermatozoa and the other cells present in the ejaculate.
DNA Probes
Double- and a triple-color FISH were done on each patient and control,
using alpha-centromeric probes for chromosomes 8, 12, 18, X, and Y. The probe
mixture for triple FISH consisted of a repetitive DNA sequence of centromeric
probes: for chromosome X (pDMX1), labeled fluorescein isothiocyanate (FITC);
for chromosome Y (pLAY5.5), labeled Cy3; and for chromosome 12 (pBR12),
labeled FITC and Cy3. The probe mixture for the double-color FISH consisted
also of a repetitive DNA sequence of centromeric probes for chromosome 8
(pZ8.4) and for chromosome 18 (2Xba), labeled FITC or Cy3, respectively. The
probes were provided by Prof M. Rocchi, University of Bari (Bari, Italy).
Hybridization Procedure
Hybridization was performed as reported elsewhere
(Calogero et al, 2001b). In
brief, each slide was denatured by means of a solution of 70%
formamide/2x standard saline citrate (SSC; pH 7.5) at 80°C for 150
seconds. The slides were immersed in a 70%, 90%, and 100% ethanol series for 3
minutes each and dried by air. The probes, precipitated and denatured at
80°C for 8 minutes, were applied directly to the slides, which were then
covered with a coverslip and sealed with rubber cement. Hybridization occurred
overnight in a dark humidified container at 37°C, after which the
coverslip was removed and the slides were immersed 3 times in a
posthybridization wash of 50% formamide/2x SSC at 37°C for 5
minutes, 2x SSC 3 times at 42°C for 5 minutes, and 2x SSC/0.1%
Tween 20 at room temperature for 5 minutes. The slides were then mounted in
4',6-diamidino-2-pheneylindole (DAPI) counterstain and antifade and
stored in the dark at 4°C for the microscope observation. The slides were
observed using an Axiophot fluorescence microscope (C. Zeiss, Oberkochen,
Germany) with an appropriate set of single-band filters for DAPI, FITC, and
Cy3. The hybridization efficiency was calculated dividing the total number of
cells observed by the total number of cells with at least 1 hybridization
signal and multiplying this ratio by 100. It was always higher than 99%.
Spermatozoa were scored as reported elsewhere
(Calogero et al, 2001b). Only
intact spermatozoa with clear hybridization signals were scored. We excluded
disrupted or overlapping spermatozoa. Spermatozoa were considered to be
polysomic if they presented 2 or more distinct hybridization signals of equal
intensity separated by at least 1 signal domain. Diploid spermatozoa displayed
2 signals for each tested chromosome with normal head and tail morphology.
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Results |
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The results of triple- and double-color FISH for each patient are reported in Tables 2 and 3. Patient K53 had an extremely elevated sperm aneuploidy rate. Indeed, 25.13% of the spermatozoa had heterochromosome aneuploidy (normal range, 0%–0.62%), 33.68% had chromosome 12 aneuploidy, and 3.83% had the simultaneous presence of chromosome 12 and heterochromosome aneuploidy. The double-color FISH showed an aneuploidy rate of 6.09% for chromosomes 8 and 18 (normal range, 0.15–0.70). A large percentage of spermatozoa (62.87%) had aneuploidies regarding both autosomes simultaneously. The total aneuploidy rate was 69.55% (normal range, 0.3%–1.55%), whereas the mean diploidy rate was 19.06% (normal range, 0%–0.20%). Patients K56 and K61 had sperm aneuploidy rates substantially lower than that observed in patient K53 but higher than those found in normal control subjects. K56 had an aneuploidy rate of 2.64% for the sex chromosomes and 2.52% for chromosomes 8 and 18. The total aneuploidy rate was 5.16%, whereas the mean diploidy rate was 0.47%. K61 had a decreased number of spermatozoa bearing the chromosome X. The aneuploidy rate was 7.08% for the sex chromosomes and 6.04% for chromosomes 8 and 18. The total aneuploidy rate was 13.12%, whereas the mean diploidy rate was 1.23%.
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Discussion |
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These results are in close agreement with other observations that have shown an increased sperm aneuploidy rate in patients with teratozoospermia. Aribarg et al (2000) examined the sperm heterochromosome aneuploidy (by double FISH) in 20 patients with an almost absolute teratozoospermia (abnormal forms ranging from 98% to 100%, WHO criteria) and found increased XY, XX, and YY disomy rates compared with 20 normal men. We reported that the sperm aneuploidy rate for chromosomes 8, 12, 18, X, and Y in 9 patients with isolated teratozoospermia (normal forms ranging from 1% to 9%, Kruger et al's [1988] strict criteria) was similar to that of 19 patients with oligo-astheno-teratozoospermia, which suggests that teratozoospermia is the hallmark for the abnormal chromosomal constitution (Calogero et al, 2001b). However, Gole et al (2001) found that 8 patients with isolated teratozoospermia (normal forms <10%, WHO criteria) had a sperm aneuploidy rate lower than that of patients with oligo-astheno-teratozoospermia but significantly higher than that found in normal controls. Härkönen et al (2001) reported that patients with less than 10% abnormally shaped spermatozoa (WHO criteria) had a significantly higher chromosome 7, chromosome 18, XY, and YY disomy rates than patients with a less severe degree of teratozoospermia or normal controls. An increased sperm chromosome 13, XX, and YY disomy rate has also been reported in 9 patients with teratozoospermia (normal forms <13%, WHO criteria) (Templado et al, 2002). However, those patients had also asthenozoospermia, and it has been shown that patients with asthenozoospermia (total motility ranging 3%–45%, WHO criteria) have increased chromosome 12 and XX disomy rates (Hristova et al, 2002).
An increased sperm aneuploidy rate in patients with teratozoospermia has further been supported by studies that have evaluated the chromosome constitution of spermatozoa with normal and abnormal morphology in the same patients. Bernardini et al (1998) showed an almost 30-fold increase of chromosomes 1 and 17 aneuploidy rates in spermatozoa with an abnormal morphology, compared with germ cells with normal morphology, in controls and infertile patients. That study was conducted using a colorimetric in situ hybridization method that preserves sperm head morphology and cytoplasmic structures (sperm tail). A greater sex chromosome aneuploidy rate has been reported in spermatozoa with abnormal morphology in a patient with Klinefelter syndrome. Spermatozoon images, captured before FISH analysis, showed an abnormal morphology in 18 of 24 identified spermatozoa. In particular, 7 spermatozoa had head vacuoles, 4 had a slightly amorphous head, 3 had a severely amorphous head, 2 were microcephalic, 1 was bicephalic, and 1 had neck abnormality. Eleven (61%) of these spermatozoa showed to carry an abnormal karyotype, whereas only 1 (16.6%) of 6 spermatozoa with a normal morphology was aneuploid (sex chromosome nullisomy) (Estop et al, 1998).
The greater sperm aneuploidy rate found in patient K53, whose teratozoospermia was mainly characterized by an increased rate of spermatozoa with enlarged heads, agrees with other observations. Viville et al (2000) reported an extremely elevated sperm aneuploidy rate (66.9%) only in a patient whose absolute teratozoospermia was characterized by an elevated number of spermatozoa with enlarged heads (64%). In contrast, the other 3 patients reported in that study, who presented an absolute teratozoospermia mainly related to sperm tail or acrosome (absence or irregularly shaped) defects, had a normal sperm aneuploidy rate. In't Veld et al (1977) also reported an elevated sperm aneuploidy rate in a patient with 100% macrocephalic spermatozoa. The presence of multiple tails in a high proportion of these spermatozoa suggested, however, that they originated from a lack of cell division during meiosis. Accordingly, 40% of these mature germ cells were diploid. Another case of a patient with absolute teratozoospermia, characterized by 30% headless spermatozoa and 70% abnormal head structure (large, round, and with or without acrosome), showed aneuploidy in virtually all spermatozoa (99.2%) and diploidy in about 20% of them (Weissenberg et al, 1998). Therefore, altogether, these data suggest that patients with teratozoospermia characterized by enlarged heads have a particularly elevated aneuploidy rate.
Previous studies have shown that poor sperm morphology results in poor fertilization rates (Oehninger et al, 1988), pregnancy rates (Kahraman et al, 1999), and embryo quality (Cohen et al, 1991; Parinaud et al, 1993) in in vitro fertilization programs. In addition, a higher spontaneous abortion rate has been reported in conventional in vitro fertilization performed using semen with an abnormal morphology (Oehninger et al, 1988). Given that about 50% of spontaneous abortions are chromosomally abnormal (see Hassold, 1986 for review), it can be hypothesized that one reason for this is the elevated sperm aneuploidy rate found in spermatozoa with an abnormal morphology. Indeed, Yakin and Kahraman (2001) reported a 60% chromosomal abnormality rate in embryos developed from semen samples with enlarged heads. For this reason, they suggested that ``couples should be counseled about low rates of fertilization and pregnancy with ICSI when only macrocephalic spermatozoa are present in the ejaculate.'' The results of the present study add further evidence that these patients definitely need thorough genetic evaluation and counseling.
In conclusion, the present study adds further evidence that teratozoospermia is associated with an increased sperm aneuploidy rate. Abnormal chromosome constitution reaches a very elevated degree in spermatozoa with enlarged heads. The presence of these sperm abnormalities at an elevated rate may be viewed as a bad omen for pregnancy outcome (Calogero et al, 2001a).
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Acknowledgments |
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Footnotes |
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