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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: aneuploidy/chromosome 8, 12, 18, X and Y/fluorescent in-situ-hybridization/intracytoplasmic sperm injection/oligoasthenoteratozoospermia
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1994; Guttenbach et al., 1997
), 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., 1995
; Bernardini et al., 1997
; Lahdetie et al, 1997
; McInnes et al, 1998
; Storeng et al., 1998
; Pang et al., 1999
; Ushijima et al., 2000
; Vegetti et al., 2000
; Calogero et al., 2001
). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 3536 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., 1995). 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., 1993). 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 MannWhitney 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1999; Van Dyk et al., 2000
). 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., 1999
). 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., 2000
).
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., 1998). 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., 1999
). On the other hand, another series of ten aneuploid patients undergoing 11 ICSI cycles had an overall fertilization rate of 70% (range: 3394) 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., 1999
). 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., 2000
). 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., 1999
).
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bonduelle, M., Aytoz, A., Van Assche, E. et al. (1998) Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection (editorial). Hum. Reprod., 13, 781782.
Calogero, A.E., De Palma, A., Grazioso, C. et al. (2001) Aneuploidy rate in spermatozoa of selected men with abnormal semen parameters. Hum. Reprod., 16, 11721179.
Chandley, A.C. and Hargreave, T.B. (1996) Genetic anomaly and ICSI. Hum. Reprod., 11, 930932.[ISI][Medline]
Colombero, L.T., Hariprashd, J.J., Tsai, M.C. et al. (1999) Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome. Fertil. Steril., 72, 9096.[ISI][Medline]
Griffin, D.K., Abruzzo, M.A., Millie, E.A. et al. (1995) Non-disjunction in human sperm: evidence for an effect of increasing age. Hum. Mol. Genet., 4, 22272232.[Abstract]
Guttenbach, M., Martinez-Exposito, M-J., Michelmann, H.W. et al. (1997) Incidence of diploid and disomic sperm nuclei in 45 infertile men. Hum. Reprod., 12, 468473.[ISI][Medline]
Kruger, T.F., Menkveld, R., Stander, F.S. et al. (1986) Sperm morphology feature as a prognostic factor in in-vitro fertilization. Fertil. Steril., 46, 11181123.[ISI][Medline]
In't Veld, P.A., Brandenburg, H., Verhoeff, A. et al. (1995) Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet, 346, 773.[ISI][Medline]
Lahdetie, J., Saai, N., Ajosenpaa-Saara, M. et al. (1997) Incidence of aneuploid spermatozoa among infertile men studied by multicolor fluorescence in situ hybridization. Am. J. Med. Genet., 71, 115121.[ISI][Medline]
Liebaers, I., Bonduelle, M., Van Assche, E. et al. (1995) Sex chromosome abnormalities after intracytoplasmic sperm injection (letter). Lancet, 346, 1095.
Martin, R.H., Spriggs, E., Ko, E. et al. (1995) The relationship between paternal age, sex ratios, and aneuploidy frequencies in human sperm, as assessed by multicolor FISH. Am. J. Hum. Genet., 57, 13951399.[Medline]
McInnes, B., Rademaker, A., Greene, C.A. et al. (1998) Abnormalities for chromosomes 13 and 21 detected in spermatozoa from infertile men. Hum. Reprod., 13, 27872790.
Miharu, N., Best, R.G., Young, S.R. (1994) Numerical chromosome abnormalities in spermatozoa of fertile and infertile men detected by fluorescence in situ hybridization. Hum. Genet., 93, 502506.[ISI][Medline]
Moosani, N., Pattinson, H.A., Carter, M.D. et al. (1995) Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in situ hybridization. Fertil. Steril., 64, 811817.[ISI][Medline]
Pang, M.G., Hegerman, S.F., Cuticchia, A.J. et al. (1999) Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y by fluorescent in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection. Hum. Reprod., 14, 12661273.
Pfeffer, J., Pang, M.G., Hoegerman, S.F. et al. (1999) Aneuploidy frequencies in semen fractions from ten oligoasthenoteratozoospermic patients donating sperm for intracytoplasmic sperm injection. Fertil. Steril., 72, 472478.[ISI][Medline]
Robbins, W.A., Baulch, J.E., Moore, D. 2nd et al. (1995) Three-probe FISH to assess chromosome X, Y, and 8 aneuploidy in sperm of 14 men from two healthy groups: evidence for a paternal age effect on sperm aneuploidy. Reprod. Fertil. Dev., 7, 799809.[ISI][Medline]
Rooney, D. and Czepulkowski, B. (1992) Human cytogenetics. A practical approach, vol. 1. Oxford University Press, Oxford.
Storeng, R.T., Plachot, M., Theophile, D. et al. (1998) Incidence of sex chromosome abnormalities in spermatozoa from patients entering an IVF or ICSI protocol. Acta Obstet. Gynecol. Scand., 77, 191197.[ISI][Medline]
Ushijima, C., Kumasako, Y., Kihaile, P.E. et al. (2000) Analysis of chromosomal abnormalities in human spermatozoa using multi-colour fluorescence in-situ hybridization. Hum. Reprod., 15, 11071111.
Van Dyk, Q., Lanzendorf, S., Kolm, P. et al. (2000) Incidence of aneuploid spermatozoa from subfertile men: selected with motility versus hemizona-bound. Hum. Reprod., 15, 15291536.
Van Opstal, D., Los, F.J., Ramlakhan, S. et al. (1997) Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection. Hum. Reprod., 12, 682686.[Abstract]
Vegetti, W., Van Assche, E., Frias, A. et al. (2000) Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men. Hum. Reprod., 15, 351365.
Williams, B.J., Ballenger, C.A., Malter, H.E. et al. (1993) Non disjunction in human sperm: results of fluorescence in situ hybridization studies using two and three probes. Hum. Mol. Genet., 2, 19291936.[Abstract]
World Health Organization (1992) WHO laboratory manual for the examination of human semen and spermcervical mucus interaction, 3rd edn. Cambridge University Press, Cambridge.
Submitted on October 23, 2000; accepted on March 20, 2001.