Hum. Reprod. Advance Access originally published online on August 19, 2004
Human Reproduction 2004 19(10):2298-2302; doi:10.1093/humrep/deh438
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Morphologically normal spermatozoa of patients with secretory oligo-astheno-teratozoospermia have an increased aneuploidy rate
1 Section of Endocrinology, Andrology and Internal Medicine, Department of Biomedical Sciences, and Master in Andrological and Human Reproduction Sciences,, 3 Department of Human Anatomy, University of Catania and 2 Unit of Cardiology, Garibaldi Hospital, Catania, Italy
4 To whom correspondence should be addressed at: Sezione di Endocrinologia, Andrologia e Medicina Interna, Dipartimento di Scienze Biomediche, Università di Catania, Ospedale Garibaldi, Piazza S. Maria di Gesù, 95123 Catania, Italy. Email: acaloger{at}unict.it
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Abstract |
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BACKGROUND: Normal morphology is a major criterion for selecting spermatozoa to be injected. Given that teratozoospermia is one of the most critical parameters associated with sperm aneuploidy, the purpose of this study was to evaluate the aneuploidy rate of morphologically normal spermatozoa of patients with oligo-astheno-teratozoospermia (OAT). METHODS: Ten patients with secretory OAT and six age-matched normozoospermic men with a normal karyotype were enrolled. After assignment to normal or abnormal category, the location of each spermatozoon was recorded using an electronic microstage locator. Slides were then subjected to triple-colour fluorescence in situ hybridization for chromosomes X, Y and 12. RESULTS: OAT patients had a lower number of morphologically normal and abnormal spermatozoa carrying the X chromosome, compared with normozoospermic men. They also exhibited increased XY and XX disomy rates. Morphologically abnormal spermatozoa from normozoospermic men also had an increased XX disomy rate compared with normally shaped spermatozoa obtained from the same men. The total sperm aneuploidy rate of morphologically abnormal spermatozoa of normozoospermic men was 4.4-fold higher than that of spermatozoa with normal morphology. The total aneuploidy rates of spermatozoa with normal or abnormal head shape from OAT patients were similar to each other and to that of abnormally shaped spermatozoa from normozoospermic men, but they were higher than the rate found in normally shaped spermatozoa of normal men. CONCLUSIONS: Normally shaped spermatozoa of OAT patients have an increased aneuploidy rate.
Key words: chromosomes 12/multicolour fluorescence in situ hybridization/oligo-astheno-teratozoospermia/sex chromosomes/aneuploidy/morphology
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Introduction |
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ICSI has revolutionized the treatment of severe male infertility (Palermo et al., 1992
). Soon after its introduction, this technique raised concerns about the risk of transmitting genetic diseases to the offspring, because a spermatozoon with an abnormally genetic constitution may be selected and used for oocyte injection (Meschede and Horst, 1997). The risk seems particularly elevated since an increasing body of literature has shown that patients with oligo-astheno-teratozoospermia (OAT), who more frequently require ICSI to overcome their infertile condition, have an increased sperm aneuploidy rate, despite a normal blood karyotype (for a review see Calogero et al., 2003). In particular, an inverse relationship has been reported between the sperm aneuploidy rate and sperm concentration (Pang et al., 1999; Vegetti et al., 2000; Calogero et al., 2001) or, to a lower extent, sperm motility (Vegetti et al., 2000). Spermatozoa with an abnormal morphology have also been shown to correlate negatively with sperm aneuploidy (Bernardini et al., 1998; Ushijima et al., 2000; Calogero et al., 2001). Indeed, patients with teratozoospermia have an aneuploidy rate significantly higher than that of normal controls (Gole et al., 2001). We have shown that teratozoospermia is the sperm parameter best associated with sperm aneuploidy. Indeed, a group of patients with isolated teratozoospermia had a sperm aneuploidy rate similar to that of a group of OAT patients (Calogero et al., 2001). Furthermore, Harkonen et al. (2001) showed that patients with severe teratozoospermia (normal forms <10%) have a sperm aneuploidy rate significantly higher than that found in patients with a less marked degree of teratozoospermia. ICSI bypasses all natural sperm selection processes, since the embryologist chooses the spermatozoon to be injected into the oocyte. Only motile and morphologically normal spermatozoa will be selected. However, these criteria will not exclude the presence of an abnormal karyotype, since the altered intratesticular environment may disrupt the fine-tuned mechanisms of chromosome segregation during spermatogenesis. In this case, spermatozoa with a normal morphology may also be aneuploid, cancelling out the careful selection of a normally shaped spermatozoon aimed at reducing the risk of transmitting aneuploidy to the ICSI offspring. The present study was undertaken to assess the sperm aneuploidy rate of spermatozoa with normal and abnormal morphology. To accomplish this, we evaluated the sperm aneuploidy rate for chromosomes 12, X and Y in single spermatozoa identified according to their morphology in patients with secretory OAT. Six normozoospermic men were selected as a matched control group.
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Patient selection
Ten OAT patients, aged 29 (24–40) years (median and range), with a normal blood karyotype were recruited for this study. Semen samples were collected by masturbation after 4–5 days of abstinence, and the main sperm parameters are shown in Table I. As previously reported (Calogero et al., 2001
), all patients fulfilled the following criteria as an expression of semen abnormality mainly of testicular origin: normal or reduced (
12 ml) testicular volume, normal or increased FSH levels and negative andrological history for pathology of the excretory genital tract. The patients with normal testicular volume and serum FSH had to have no clinical or ultrasound signs of epididymal abnormalities. Patients with abnormal spermatozoon morphology of possible epididymal origin (cytoplasmic droplets, coiled tails or short tails) were excluded from the study. Six healthy men, aged 26 (22–37) years (median and range), with normal sperm density, motility and morphology (Table I) served as controls. The protocol was approved by the Institutional Review Board and an informed written consent was obtained from each man enrolled in this study. Sperm parameter assessment was performed on fresh seminal semen according to the World Health Organization criteria (World Health Organization, 1999). The initial sperm morphology assessment was performed using the Papanicolaou staining method at 40x magnification. A spermatozoon was considered morphologically normal if it had: a smooth oval head (length, 4.0–5.0 µm; width 2.5–3.5 µm), a well-defined acrosomal region comprising 40–70% of the head area, no defects of the neck and the midpiece; cytoplasmic droplets less than half the size of the normal head; and a straight, thin tail with a length of 45 µm.
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FISH analysis
Following liquefaction, semen samples were washed three times in phosphate-buffered saline (PBS), pH 7.2, centrifuged at 650 g for 10 min at 20°C and the sediment was then fixed in methanol/acetic acid (3:1). The specimens were spread on slides and single sperm morphology carefully evaluated using the same criteria recommended by the World Health Organization (1999)
at 40x magnification, but without Papanicolaou staining. The results of the morphological assessment in methanol/acetic acid-fixed spermatozoa were not significantly different from the initial evaluation (fixed-stained spermatozoa). The location of each spermatozoon was recorded using the electronic microstage locator with which the DMRXA2 fluorescent microscope used for this study is equipped (Leica Microsystems SpA, Milan, Italy). The images were recorded using the software Mic-Control (Leica, Milan) and the coordinates written down and retrieved after fluorescence in situ hybridization (FISH) using the same software. The slides were then washed in 2x standard saline citrate (SSC) solution and incubated for 5 min in 1 mol/l Tris buffer, pH 9.5, containing 25 mmol/l dithiothreitol (DTT), to achieve a suitable sperm head decondensation, as previously reported (Calogero et al., 2001).
A triple-colour FISH was carried out on each patient and control, using 
-centromeric probes for chromosomes 12, X and Y. Alpha centromeric probes were obtained from plasmids containing a specific 
6 kb DNA insert: pBR12 for chromosome 12, pDMX1 for chromosome X and pLAY5,5 for chromosome Y. Escherichia coli with the specific plasmid were grown in a Petri dish with LB medium containing 50 µg/ml ampicillin, and incubated overnight at 37°C. Subsequently, a colony was selected and amplified in 10 ml of LB medium containing 50 µg/ml ampicillin overnight at 37°C. The extraction of the plasmid was carried out by miniprep, using the alkaline lysis method, as previously reported (Sambrook et al., 1989). Plasmids containing the 12 and X chromosome probes (DNA 1 µg) were labelled with Fluor X-dCTP, whereas plasmids containing the 12 and Y chromosome probes were labelled with Cy3-dUTP, using a nick-translation kit purchased from Pharmacia Biotech (Milan, Italy). The size of the fragments obtained was checked in a 1% agarose gel.
Each slide was denatured with a solution of 70% formamide/2x SSC (pH 7.5) at 80°C for 150 s. The slides were immersed in a 70, 90 and 100% ethanol series for 3 min each and dried by air. The probes, precipitated and denatured at 80°C for 8 min, 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 in a post-hybridization wash of 50% formamide/2x SSC three times at 37°C for 5 min, 2x SSC three times at 42°C for 5 min and 2x SSC/0.1% Tween-20 once at room temperature for 5 min. The slides were then mounted in 4',6-diamidino-2-phenylindole (DAPI) counterstain and antifade and stored in the dark at 4°C until microscope observation was carried out.
The slides were observed on a Leica DMRXA2 fluorescent microscope with the appropriate set of filters: single band DAPI, fluorescein isothiocyanate (FITC) and Cy3. Previously identified intact spermatozoa bearing a similar degree of decondensation and clear hybridization signals were scored; disrupted or overlapping spermatozoa were excluded from analysis. Spermatozoa were regarded as abnormal if they presented two (or more) distinct hybridization signals for the same chromosome, each equal in intensity and size to the single signal found in normal monosomic nuclei. We considered only clear hybridization signals, similar in size, separated from each other by at least one signal domain and clearly positioned within the sperm head. Divided (split) signals were not scored as disomies. Spermatozoa were scored as nullisomic if they showed no signal for a given chromosome, whereas the signal of the other chromosome tested was present. Finally, a spermatozoon was considered diploid in cases where it manifested two signals for each tested chromosome and in cases where the tail as well as the normal oval shape of a sperm head were evident. No FISH signals in a spermatozoon head showing DAPI stain were considered a case of no hybridization. The hybridization efficiency was >99%.
Statistical analysis
Results are shown as median and range throughout the study, unless otherwise indicated. The data were analysed with the Mann–Whitney test. SPSS 9.0 for Windows was used for statistical calculation. A significant statistical difference was accepted when the P-value was <0.05.
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The main sperm parameters of normozoospermic men and secretory OAT patients are shown in Figure 1. Among OAT patients, six had oligozoospermia and all had astheno- and teratozoospermia.
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A total of 2094 spermatozoa were classified as being morphologically normal in the six normozoospermic men enrolled (median = 324 spermatozoa; range = 289–489), whereas 1931 had an abnormal head shape (median = 328 spermatozoa; range = 186–397), including amorphous, round, micro- and enlarged heads. In the group of patients with OAT, a total of 2346 spermatozoa were found to be normally shaped (median = 230 spermatozoa; range 78–335), whereas 3993 had an abnormal head morphology (median = 406 spermatozoa; range 301–481). Because of the abnormal sperm parameters, we could not score a greater number of spermatozoa with a normal morphology among OAT patients. The coordinates of each spermatozoon categorized were recorded and it was retrieved after FISH to establish its chromosome complement.
Sperm aneuploidy rates for chromosomes 12, X and Y are reported in Table I. OAT patients had a slight, but significantly (P<0.05), lower number of morphologically normal and abnormal spermatozoa carrying the X chromosome, compared with normozoospermic men. They also exhibited increased XY and XX disomy rates (P<0.01), whereas YY disomy, sex chromosome nullisomy and chromosome 12 disomy and nullisomy rates were similar. Spermatozoa with an abnormal sperm head from normozoospermic men also had a significantly (P<0.05) higher XX disomy rate compared with morphologically normal spermatozoa obtained from the same men.
The total aneuploidy rates for chromosomes X, Y and 12 in spermatozoa with a normal or abnormal head from normozoospermic men and OAT patients is shown in Figure 2. Morphologically abnormal spermatozoa of normozoospermic men had an aneuploidy rate 4.4-fold higher than that found in spermatozoa with a normal morphology (P<0.001). The total aneuploidy rates of spermatozoa with normal or abnormal head shape from OAT patients were similar to each other and to that of abnormally shaped spermatozoa from normozoospermic men, but they were significantly higher than the rate found in normally shaped spermatozoa of normal men (P<0.001). In particular, normally and abnormally shaped spermatozoa from OAT patients had an aneuploidy rate 3.6- and 4.7-fold higher, respectively, than that found in normally shaped spermatozoa of normozoospermic men.
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Discussion |
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The increased sperm aneuploidy rate in patients with secretory OAT has raised concern about the possible transmission of chromosomal abnormalities to the offspring generated through assisted reproductive techniques. The risk is particularly elevated since patients with severe spermatogenesis impairment, who more often undergo ICSI for the treatment of their infertility, have the greatest sperm aneuploidy rate and particularly those with the highest degree of teratozoospermia (Bernardini et al., 1998
; Ushijima et al., 2000; Calogero et al., 2001). Morphologically abnormal spermatozoa are not, however, selected for oocyte injection, apparently lowering the risk of generating an aneuploid embryo. Nevertheless, the aneuploidy rate of morphologically normal spermatozoa of patients with secretory OAT is not well known. For this reason, we evaluated the sperm aneuploidy rate of morphologically normal and abnormal spermatozoa of patients with secretory OAT or normozoospermia. Surprisingly, we found that morphologically normal spermatozoa of patients with secretory OAT have a sperm aneuploidy rate similar to that found in spermatozoa with head shape abnormalities. On the other hand, the aneuploidy rate of morphologically normal spermatozoa of normozoospermic men was significantly lower than that found in their spermatozoa with an abnormally shaped head. It can be postulated that the factors which cause OAT in these patients affect, at the same time, the fine-tuned mechanisms of chromosome segregation in all germ cells. Therefore, our data show that normal morphology does not indicate the selection of a chromosomally normal spermatozoon in patients with secretory OAT, suggesting a real increased risk for generating aneuploid offspring. Accordingly, the limited available data on ICSI fetal karyotypes reveal that, in comparison with a general neonatal population, there is a slight but significant increase in de novo sex chromosomal aneuploidy (0.6% instead of 0.2%) and structural autosomal abnormalities (0.4% instead of 0.07%) (Bonduelle et al., 2002; Van Steirteghem et al., 2002).
The results of the present study are in close agreement with those of Ryu et al. (2001). The authors studied the frequency of aneuploidy for chromosomes 18, X and Y in morphologically normal spermatozoa of eight infertile patients having <4% normal forms according to Kruger's strict criteria. The aneuploidy rate of these spermatozoa was compared with that of normally shaped spermatozoa of six normal donors of proven fertility. The FISH analysis showed that the sperm aneuploidy rate of the infertile teratozoospermic patients was 3.3% (range 1.8–5.5%). This rate was nearly 3-fold greater than that found in morphologically normal spermatozoa of fertile men who had an aneuploidy rate of 1.3% (range 0–2.6%). The sex chromosome aneuploidy rate was 2-fold higher in the infertile group as compared with the control group. The authors did not show the aneuploidy rate of morphologically abnormal spermatozoa (Ryu et al., 2001).
The increased aneuploidy rate found in normally shaped spermatozoa of OAT patients may also explain the lower success rate of ICSI in patients with OAT (for a review see Calogero et al., 2003). However, the abnormal sperm parameters found in these patients may as well have a negative effect on the ICSI outcome. For instance, a recent study showed that ICSI clinical results correlate negatively with the degree of abnormal sperm morphology. Implantation and pregnancy rates were 9.6 and 20.2%, respectively, in patients with teratozoospermia, whereas they were 18.7 and 36.7% in patients with normal sperm morphology (De Vos et al., 2003). However, we have shown recently that an increased sperm aneuploidy rate has a negative impact on ICSI outcome. Unselected patients undergoing ICSI were sorted into two groups according to their sperm aneuploidy rate: one with a normal and the other with an elevated sperm aneuploidy rate. Although fertilization and cleavage rates were similar between the two groups, patients with a normal sperm aneuploidy rate had higher pregnancy and implantation rates compared with patients who had an elevated aneuploidy rate, whereas the abortion rate was significantly higher in the latter. Other factors that may have a negative outcome on the clinical ICSI outcome, such as female partner age, abnormalities of conventional semen parameters or female factor of infertility, were similar in both groups (Burrello et al., 2003).
In conclusion, the present study showed that morphologically normal spermatozoa of OAT patients carry an abnormal chromosomal constitution with the same frequency as that found in spermatozoa with an abnormal head shape. Therefore, the risk of selecting aneuploid spermatozoa to be used for oocyte injection is high in OAT patients. These findings further strengthen the needs for genetic counselling before carrying out ICSI, to decrease the risk of transmitting genetic disease to the offspring. This may be achieved, for example, by establishing methods to identity spermatozoa with a normal chromosomal complement (Cayli et al., 2003).
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Presented at the 19th annual meeting of the European Society of Human Reproduction and Embryology, Madrid, Spain, June 29 to July 2, 2003. Supported in part by a grant from MURST (Cofinanziamento 1999), Rome, and from the University of Catania, Faculty of Medicine, Catania, Italy.
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Submitted on January 2, 2004; resubmitted on February 12, 2004; accepted on April 2, 2004.
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