High sperm aneuploidy rate in unselected infertile patients and its relationship with intracytoplasmic sperm injection outcome

Aldo E. Calogero1,5, Adele De Palma1, Caterina Grazioso1,2, Nunziata Barone1, Nunziatina Burrello1, Irina Palermo1, Antonio Gulisano3, Carlo Pafumi4 and Rosario D'Agata1

1 Division of Endocrinology and Master in Andrological Sciences, New Methodologies in Human Reproductive Medicine, University of Catania, Catania, 2 Master in Endocrinological and Metabolic Sciences, University of Naples, Naples, 3 Department of Obstetrics and Gynaecology and 4 Department of Microbiology and Obstetrical Science, University of Catania, Catania, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Men with oligoasthenoteratozoospermia (OAT) frequently undergo intracytoplasmic sperm injection (ICSI) as a treatment for their infertility. However, there is an increased risk of transmitting chromosomal abnormalities to the offspring given that natural selection is bypassed by the use of this technique and patients have an increased rate of sperm aneuploidy which, in addition, may negatively affect ICSI outcome. For this reason, the rate of sperm aneuploidy in unselected patients undergoing ICSI and its impact on ICSI performance have been evaluated. METHODS: Aneuploidy and diploidy were evaluated in spermatozoa separated by swim-up for oocyte injection, using DNA probes for chromosomes 8, 12, 18, X and Y. RESULTS: ICSI patients had sperm aneuploidy and diploidy rates significantly higher than those of 13 normozoospermic men who served as controls. Although the total aneuploidy rate varied considerably between the 18 patients, 15 of them (83%) had values above the upper range of the control group. Eighteen ICSI cycles were performed with an overall fertilization rate of 95% and a pregnancy rate of 39%. The aneuploidy rate of the 11 patients whose wives did not achieve pregnancy was slightly higher than that of pregnant couples, but the difference did not reach statistical significance. However, 10 patients in this group (91%) had a sperm aneuploidy rate well above the upper limit of the controls as compared with two patients in the `pregnant' group (29%). CONCLUSIONS: This study has shown that unselected patients undergoing ICSI had an elevated sperm aneuploidy rate. Lack of pregnancy was associated with a tendency towards an increased aneuploidy rate; however, pregnancy occurred even in the presence of an elevated sperm aneuploidy rate.

Key words: aneuploidy/chromosome 8, 12, 18, X and Y/fluorescent in-situ-hybridization/intracytoplasmic sperm injection/oligoasthenoteratozoospermia


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The advent of intracytoplasmic sperm injection (ICSI) has made it possible to achieve pregnancy in severe cases of male infertility. The use of spermatozoa from these men has raised concerns about the possible high risk of chromosomal aneuploidies for the concepti, especially because natural selection is bypassed with the use of this technique (Chandley and Hargreave, 1996Go). This potential hazard seems to be confirmed by reports of a higher incidence of sex chromosomal aneuploidy and structural de novo chromosomal abnormalities, which have been shown to be of paternal origin (Van Opstal et al., 1997Go), in children conceived after ICSI, compared with the general population (In't Veld et al., 1995Go; Liebaers et al., 1995Go; Bonduelle et al., 1998Go). The study of the chromosomal complement in the spermatozoa of men with abnormal sperm parameters, who make up the majority of ICSI candidates, is, therefore, becoming a fundamental step.

The development of fluorescence in-situ hybridization (FISH) and its application to the study of the sperm aneuploidy rate enabled us to look into a comparatively large number of spermatozoa, using a chromosome-specific DNA probe that can be detected by fluorescence microscopy. Despite the fact that some studies have not shown any significant difference in the sperm aneuploidy rate between fertile and infertile patients (Miharu et al., 1994Go; Guttenbach et al., 1997Go), many authors have reported a higher frequency of sperm chromosome aneuploidy rate in patients with abnormal sperm parameters compared to normal controls (Moosani et al., 1995Go; Bernardini et al., 1997Go; Lahdetie et al, 1997Go; McInnes et al, 1998Go; Storeng et al., 1998Go; Pang et al., 1999Go; Ushijima et al., 2000Go; Vegetti et al., 2000Go; Calogero et al., 2001Go). Since aneuploidy might have a negative impact on oocyte fertilization and/or on embryonic development, we thought it was worth evaluating the rate of sperm aneuploidy in unselected patients undergoing ICSI and its impact on ICSI performance.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patient selection
Eighteen patients whose median age was 35 years (range: 23–42) were included in this study. From January to June 2000, they had attended our Andrology and Reproductive Endocrinology Unit (AREU) with a view to participating in an ICSI programme as a treatment for their infertile status. Semen samples were collected by masturbation on the day of oocyte retrieval, usually after 4–5 days of abstinence. Morphology assessment was performed on raw semen and on the same swim-up preparation used for the oocyte injection according to Kruger's strict criteria (Kruger et al., 1986Go). The other parameters of the ejaculated semen were evaluated according to the World Health Organization criteria (WHO, 1992). The median age of the female partners was 31 years (range: 20–40). To define normal semen aneuploidy rate in ejaculated semen, 13 healthy men of the median age of 25 years (range: 19–35), with normal sperm density, motility and morphology and normal karyotype were recruited. Semen samples with volume >=2 ml, sperm concentration >=20x106/ml, percentage of spermatozoa with total motility >=50% and with normal morphology >=14% were regarded as normal.

Ovulation was induced in all the female partners using a combination of a gonadotrophin-releasing hormone analogue (buserelin, Suprefact® nasal spray; Hoechst, Milan, Italy), initiated in the luteal phase (suppression protocol) and follicle-stimulating hormone (FSH) (Metrodin®; Serono, Rome, Italy). A total of 10 000 IU of human chorionic gonadotrophin (HCG; Profasi®; Serono) were administered i.m. when three or more follicles were >16 mm in diameter. Transvaginal follicular aspiration was performed 35–36 h later. Progesterone supplementation (100 mg/day i.m., Prontogest®; Amsa s.r.l., Rome, Italy) was initiated on the day of the transfer and continued until the 12th week of pregnancy, in the cases in which it had occurred. Pregnancy was confirmed by the measurement of increasing concentrations of serum ßHCG on at least two occasions from day 14 after the embryo transfer.

Karyotype analyses
Blood (3 ml) was withdrawn into tubes containing heparin to prevent clotting. Metaphase spreads were made from phytohaemagglutinin-stimulated peripheral lymphocytes using standard cytogenetic techniques (Rooney, 1992).

Semen preparation for FISH analysis
An aliquot of the swim-up preparation used for the oocyte intracytoplasmic injection was washed three times in phosphate buffered saline (PBS), pH 7.2, and centrifuged at 650 g for 10 min; the sediment was then fixed in methanol/acetic acid (3:1). The fixed specimens were stored at –20°C until further processing.

Sperm head decondensation
Methanol/acetic acid-fixed spermatozoa were spread on slides and the slides were washed in 2x standard saline citrate solution (SSC) and incubated for 5 min in 1 mol/l Tris buffer, pH 9.5, containing 25 mmol/l dithiothreitol (DTT) (Martin et al., 1995Go). As this treatment did not disrupt the sperm structure, including the tail, the differentiation between the spermatozoa and the other cells present in the ejaculate was unequivocal and easy to detect.

DNA probes
A double- and a triple-colour FISH were carried out 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), labelled FITC, for chromosome Y (pLAY5.5) labelled Cy3 and for chromosome 12 (pBR12) labelled FITC and Cy3. The probe mixture for double-colour FISH consisted also of a repetitive DNA sequence of centromeric probes for chromosome 8 (pZ8.4) and for chromosome 18 (2Xba), labelled FITC and Cy3, respectively. The probes were provided by Professor M.Rocchi, University of Bari (Bari, Italy).

Hybridization procedure
Each slide was denatured in a PCR machine 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 one coverslip was removed and the slides were immersed three times in a post-hybridization wash of 50% formamide/2x SSC at 37°C for 5 min, 2x SSC three times at 42°C for 5 min and 2x SSC/0.1% Tween 20 at room temperature for 5 min. The slides were then mounted in 4',6-diamidino-2-pheneylindole (DAPI) counterstain and antifade and stored in the dark at 4°C prior to carrying out microscopic observation.

Scoring criteria
The slides were observed using an Axiophot® fluorescence microscope (C. Zeiss, Oberkochen, Germany) with the appropriate set of filters: single band DAPI, FITC, and Cy3. Only slides with a hybridization rate >98% were analysed and, with few exceptions, about 2000 sperm nuclei per slide (about 4000 per patient) were scored. Sperm slides were scored according to previous recommendations (Williams et al., 1993Go). Only 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 took into consideration only clear hybridization signals, similar in size, separated from each other by at least one signal domain and clearly positioned within the sperm head. Split signals were not scored as disomies. Spermatozoa were scored as nullisomic if they showed no signal for a given chromosome, provided that the signal of the other chromosome tested was present. Finally, a spermatozoon was considered diploid when it manifested two signals for each tested chromosome and when the tail as well as the normal oval shape of a sperm head was evident. No FISH signals in a spermatozoon head showing DAPI stain was considered a case of no-hybridization.

Statistical analysis
Results are shown as median and range throughout the study. The data were analysed using the Mann–Whitney test. The correlation between sperm aneuploidy rate and sperm concentration, and the percentage of normal forms or total motility were calculated by means of the Spearman correlation coefficient. SPSS 9.0 was used for statistical calculation. A significant statistical difference was accepted when the P value was <0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The median age of the patients was significantly higher compared with the controls (P < 0.005; Table IGo). As for their sperm parameters, only two patients were normal, while the remaining patients had at least one abnormal semen parameter. Teratozoospermia was the commonest alteration, it being present, alone or in combination, in 13 out of the 18 patients (72.2%) (Table IGo). Blood karyotype was normal in all the patients.


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Table I. Clinical and sperm parameters of the patients enrolled in the study
 
A total of 60475 spermatozoa from the 18 patients included in the study was scored. A mean of 3360 sperm nuclei (range: 830–4113) was counted per sample. The results are shown in Table IIGo. The median hybridization efficiency was 99.95% (range: 99.4–100) and 99.81% (range: 98.99–100), for the triple- and double-colour FISH respectively and it was not different from the one obtained in the controls. The readability of the signals, the quality of decondensation, and the rate of FISH failure were comparable with those previously obtained in this laboratory (Calogero et al., 2001Go).


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Table II. Sperm aneuploidy and diploidy rates for chromosome X, Y, 12, 8 and 18 in 18 unselected patients undergoing intracytoplasmic sperm injection
 
A median 49.2% (range: 47.6–50.4) of the spermatozoa carried an X chromosome whereas a median 49.4% (range: 48.1–50.8) carried a Y chromosome respectively. These percentages were not different from those of the control group (Table IIGo). The patients had significantly higher disomy rates for their sex chromosomes and for autosomes 8 and 18 (P < 0.001) compared with the controls (Table IIGo). Their total aneuploidy (disomy + nullisomy) rate was significantly higher (P < 0.001 versus control; Figure 1Go). Although the total aneuploidy rate varied largely between patients, 15 of them (83.3%) had values above the upper range of the control group (Figure 1Go). A highly significant negative correlation was observed between the total aneuploidy rate and normal forms in the swim-up preparation (r = -0.68, P < 0.001). The total diploidy rate was also significantly higher in patients with respect to the controls (P < 0.05, Mann–Whitney test; Figure 1Go).



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Figure 1. Total sperm aneuploidy (upper panel) and diploidy (lower panel) rates for chromosomes 8, 12, 18, X and Y in 13 normozoospermic healthy men (control) and in 18 unselected patients undergoing intracytoplasmic sperm injection (ICSI). Dashed lines represent median values.

 
Eighteen ICSI cycles were performed in the 18 couples enrolled in this study. The median fertilization rate was 95% (range: 28–100) and the pregnancy rate was 38.9%. All patients had embryos transferred. Patients were divided into two groups according to the achievement of their partners' clinical pregnancy. Seven couples became pregnant, whereas the other 11 did not. Female age was not statistically different, 34 years (range: 25–40) and 31 years (range: 20–39) respectively. The male partners of the seven women who became pregnant showed a slight trend towards a lower total aneuploidy rate with respect to those in the non-pregnant couples, but this proved to be non-significant. However, the evaluation of the individual aneuploidy rate showed that five patients (71.4%) in the pregnant group had an aneuploidy rate close to the upper limit of the normal range, whereas 10 patients (90.9%) in the non-pregnant group had an aneuploidy rate well above that of the controls. The sperm diploidy rate did not show any statistically significant difference between the two groups (Figure 2Go). Two additional patients (A24 and A30), whose wives' biochemical pregnancy ended in abortion, had high sperm aneuploidy rates: 2.46 and 2.65% respectively.



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Figure 2. Total sperm aneuploidy (upper panel) and diploidy (lower panel) rates for chromosomes 8, 12, 18, X and Y in 18 male patients divided on the basis of their partners' clinical pregnancy outcome following intracytoplasmic sperm injection. Dashed lines represent median values whereas continuous lines indicate the lower and upper range of 13 normozoospermic men.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
FISH analysis showed a significantly higher sperm aneuploidy rate among infertile men requesting ICSI as a treatment for their infertile condition. A high percentage of these men (83%) had an aneuploidy rate above the one detected in normozoospermic men. Sex chromosome aneuploidy was about twice that of autosomes 8 and 18, whereas the aneuploidy rate of chromosome 12 was very low and similar to the one found in the control group. The majority of our patients had one or more abnormal sperm parameters and teratozoospermia was the commonest feature, confirming previous reports which have shown a high incidence of aneuploidy in the spermatozoa of infertile men with abnormal sperm parameters (Moosani et al., 1995Go; Bernardini et al., 1997Go; Lahdetie et al., 1997Go; McInnes et al., 1998Go; Storeng et al., 1998Go; Pang et al., 1999Go; Ushijima et al., 2000Go; Vegetti et al., 2000Go; Calogero et al., 2001Go). The frequency of aneuploid spermatozoa after sperm selection was directly correlated with the number of abnormal forms found after separation and inversely correlated with the conventional parameters in the ejaculate. The significantly greater mean age of the ICSI patients may be a potential bias in light of reports showing a positive correlation between age and sperm aneuploidy rate (Robbins et al., 1995Go; Griffin et al., 1995Go). However such a relationship does not seem to involve chromosomes 8 (Robbins et al., 1995Go) and 18 (Griffin et al., 1995Go). As far as sexual chromosomes are concerned, the only documented disomy found increased in aged donors was YY, whereas XY or XX disomies do not seem to be affected by age (Martin et al., 1995Go).

The elevated aneuploidy rate detected confirmed that swim-up separation did not reduce the likelihood of having spermatozoa with chromosomal abnormalities and suggested that this technique did not select against aneuploid gametes. In fact, the aneuploidy rate of swim-up selected spermatozoa has been reported to be similar to that found in raw semen for chromosomes 1, 13, 18, 21, X and Y (Pfeffer et al., 1999Go; Van Dyk et al., 2000Go). Density gradient centrifugation does not seem to be superior to swim-up in selecting haploid spermatozoa since an elevated incidence of chromosomal abnormalities have been found after its use (Colombero et al., 1999Go). In contrast, a recent study has demonstrated that the zona pellucida seems to be able to select against spermatozoa with chromosome aberration, in particular against aneuploidy. Although the mechanism of this effect is still unknown, this method might be used to reduce the risk of handling aneuploid spermatozoa during ICSI, at least if this finding is confirmed (Van Dyk et al., 2000Go).

In an attempt to gain more insight into the clinical consequence of sperm aneuploidy, we have evaluated the relationship between aneuploid sperm and ICSI performance. Aneuploidy did not seem to affect the overall fertilization (95%) and pregnancy (39%) rates. However, when patients were divided into two groups on the basis of pregnancy outcome, the partners of non-pregnant women showed a tendency towards a higher sperm aneuploidy rate compared with patients whose partners were pregnant. It is noteworthy that ten out of the eleven patients whose partners did not achieve pregnancy had an aneuploidy rate well above that of the control group, whereas patients whose wives had became pregnant had, in the majority of the cases (70%), values within or slightly above the upper limit of the controls. Since the women's age was not statistically different between the two groups, these data suggest that among our infertile patients, pregnancy was more often associated with a normal or nearly-normal sperm aneuploidy rate. However, a high sperm aneuploidy rate did not preclude the possibility of one of our patients impregnating his wife.

The extent to which aneuploid spermatozoa are responsible for the frequency of implantation failure and the incidence of `early' spontaneous abortion is still not clear. However, a reduced fertilizing ability of aneuploid spermatozoa has been suggested by Storeng and collaborators who detected an increased XY, XX and YY sperm disomy rates in patients who needed ICSI because of a previous conventional IVF failure compared with semen able to fertilize oocytes by conventional IVF (Storeng et al., 1998Go). Five patients with elevated sperm aneuploidy rates were unable to achieve live births because of fertilization failure (one patient), lack of pregnancy (two patients), preclinical abortion (one patient) and first trimester spontaneous abortion (one patient) (Pang et al., 1999Go). On the other hand, another series of ten aneuploid patients undergoing 11 ICSI cycles had an overall fertilization rate of 70% (range: 33–94) and all couples produced embryos to be transferred. However, no pregnancy resulted in eight of them, whereas the other two couples had ongoing pregnancy at the time of publication (Pfeffer et al., 1999Go). More recently, Van Dyk et al. reported an increased sperm diploidy rate (46,XY, 46,XX and 46,YY) in patients whose partners had not achieved pregnancy through ICSI, compared with those who had done so (Van Dyk et al., 2000Go). These data, therefore, suggest that oocyte fertilization can be achieved in the presence of aneuploid spermatozoa and results in a low pregnancy rate. In contrast, Colombero et al. have reported that an increased frequency of sperm aneuploidy did not appear to affect assisted reproductive technique outcome (Colombero et al., 1999Go).

In conclusion, this study has shown that infertile patients entering an ICSI programme had a high rate of sperm aneuploidy that appeared to be inversely related to raw semen parameters and to the rate of normal forms present after sperm selection. Lack of pregnancy was associated with a tendency towards an increased aneuploidy rate; however pregnancy occurred even in the presence of an elevated sperm aneuploidy rate, thus increasing the risk of generating offspring with chromosomal abnormalities.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank David Farrugia, Lecturer, Faculty of Economics, University of Catania, for the English text editing. This study was supported in part by a grant from MURST (Cofinanziamento 1999), Rome, and from the University of Catania, Faculty of Medicine, Catania, Italy.


    Notes
 
5 To whom correspondence should be addressed at: Cattedra di Endocrinologia, Ospedale Garibaldi, Piazza S. Maria di Gesù, 951213 Catania, Italy. E-mail: acaloger{at}unict.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on October 23, 2000; accepted on March 20, 2001.