1 Instituto Valenciano de Infertilidad, Guardia Civil, 23, E-46020 Valencia, 2 Department of Surgery (Urology) and 3 Department of Pediatrics, Obstetrics and Gynecology, University of Valencia, Blasco Ibáñez, 17, E-46020 Valencia, Spain and 4 Cell Biology Unit, Universitat Autònoma of Barcelona, E-08193 Bellaterra, Spain
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: aneuploidy/FISH/ICSI/miscarriage/spermatozoa
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
On the other hand, chromosome abnormalities are increasingly found in the spermatozoa of many infertile men. This makes the direct analysis of sperm aneuploidy of clinical relevance, since male infertility is now treated by intracytoplasmic sperm injection (ICSI), which has the implicit risk of transmitting chromosomal aberrations from the paternal side. In fact, a higher incidence of sex chromosomal aneuploidies and structural de novo chromosomal abnormalities has been found in prenatal karyotypes following ICSI compared with the general population, which could be attributed to the characteristics of the infertile men treated (In't Veld et al., 1995; Liebaers et al., 1995
; Bonduelle et al., 1998
). The importance of analysing the cytogenetic constitution of ejaculated spermatozoa is emphasized by meiotic studies, showing that 17.626.7% of patients with severe oligozoospermia (
1x106 sperm/ml), have synaptic chromosome anomalies restricted to the germ cell line, which are not detectable by peripheral blood karyotype (Egozcue et al., 1983
, 2000a
; Vendrell et al., 1999
). In this regard, the available literature on FISH analysis of human spermatozoa confirms higher rates of sperm aneuploidy in infertile men as compared with fertile men (Moosani et al., 1995
; Lahdetie et al., 1997
; Bernardini et al., 1998
; Arán et al., 1999
; Pang et al., 1999
; Pfeffer et al., 1999
; Ushijima et al., 2000
; Vegetti et al., 2000
). An inverse correlation between sperm quality and sperm aneuploidy rates has been reported (Bernardini et al., 2000
; Nishikawa et al., 2000
; Ushijima et al., 2000
; Vegetti et al., 2000
), but contradictory results have been published concerning the relationship of sperm aneuploidy with specific sperm defects.
Another important issue is how sperm aneuploidy may influence ICSI outcome in infertile patients. There are only a few reports on small ICSI series performed on oligoasthenoteratozoospermic patients with increased sperm aneuploidy rates (Bernardini et al., 1998, 2000
; Pang et al., 1999
; Pfeffer et al., 1999
), but their results suggest that sperm aneuploidy may be associated with implantation failure and/or early fetal loss. However, larger series comparing ICSI outcome in infertile patients with and without increased sperm aneuploidy rates are necessary in order to confirm these preliminary experiences.
In the present study, we have retrospectively analysed our FISH results on spermatozoa in a series of patients with normal karyotypes presenting with a risk of sperm chromosomal abnormalities because of several factors independent of sperm quality, such as recurrent spontaneous miscarriages, repeated implantation failures after ICSI, previous pregnancies with chromosomal abnormalities or meiotic abnormalities in testicular biopsies. Objectives of the study were: (i) to investigate whether these indications were actually associated with an increased incidence of sperm aneuploidy and diploidy; (ii) to examine the correlation between sperm chromosomal abnormalities and the basic sperm parameters in this at risk population; and (iii) since some of the couples included in this series had undergone several ICSI treatments, to evaluate the effect of sperm chromosome abnormalities on ICSI outcome in terms of fertilization, implantation, pregnancy and miscarriage rates.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sperm samples were evaluated according to WHO criteria (World Health Organization, 1992). To establish whether sperm chromosomal abnormalities in this series were related to sperm quality, sperm samples were classified into five groups according to the three standard sperm parameters: Normozoospermia (n = 14), isolated asthenozoospermia (<50% progressive motile spermatozoa, n = 14), isolated teratozoospermia (<30% normal forms, n = 8), asthenoteratozoospermia (AT) (<50% progressive motile spermatozoa and <30% normal forms, n = 6) and oligoasthenoteratozoospermia (OAT, <20 x106/ml spermatozoa, <50% progressive motile spermatozoa and <30% normal forms, n = 21). To correlate the rate of chromosomal abnormalities with the severity of oligozoospermia, OAT patients were further classified into three subgroups according to sperm count: <5 x106/ml (n = 9), 510 x106/ml (n = 5), and
1020 x106/ml (n = 7).
Individual results in each patient were compared with those of a control group of nine normozoospermic donors without a history of infertility (Blanco et al., 1996, 1997
). Individual FISH results were considered as abnormal when a statistically significant increase in any of the analysed parameters (disomy rates for chromosomes 18, 21, X and Y and total diploidy rate) was observed when compared with the control group. The disomy rate for chromosome 13 was not evaluated in the controls, and therefore no statistical comparisons were performed for this parameter.
A total of 131 ICSI cycles was performed in 38 patients. ICSI outcome, including fertilization, embryo cleavage, pregnancy, implantation and miscarriage rates, of 108 cycles carried out in 31 patients with normal FISH results was compared with that of 23 cycles in seven patients with abnormal FISH results.
Informed consent regarding sperm FISH analysis and the ICSI procedure was obtained from all patients. The study was approved by our local ethical committee.
FISH protocol
For FISH analysis one semen sample from each patient was centrifuged with Sperm medium (Medicult, Copenhagen, Denmark) the pellet was fixed in methanol:acetic acid (3:1), sperm nuclei were decondensed by slide incubation in 5 mmol/l dithiothreitol (DTT) and 1% Triton X-100 and hybridization was performed following the manufacturer's protocol. Numerical abnormalities for chromosomes 13, 18, 21, X and Y were evaluated in different slides from the same sample, using triple-colour FISH for 18, X and Y chromosomes and dual-colour FISH for chromosomes 13 and 21. Centromeric DNA probes for chromosome 18 (locus D18Z1, CEP 18 Spectrum Aqua; Vysis Inc. Downers Grove, IL, USA), chromosome X (locus DXZ1, CEP X Spectrum Green; Vysis Inc.) and Chromosome Y (locus DYZ1, CEP Y Spectrum Orange; Vysis Inc.) were used for the triple-colour FISH analysis. Locus-specific DNA probes for chromosome 13 (locus RB, LSI 13 Spectrum Green; Vysis Inc.) and chromosome 21 (loci D21S259, D21S341, D21S342, LSI 21 Spectrum Orange; Vysis Inc.) were used for the dual-colour FISH analysis. FISH incubation and detection were performed according to the manufacturer's instructions.
Analysis was carried out using an Olympus AX70 epifluorescence microscope equipped with a triple-band pass filter for 4'6-diamidino-2-phenylindole (DAPI)/Texas Red/fluorescein isothiocyanate (FITC) and single-band pass filters for FITC, Texas Red and Aqua Blue. Sperm nuclei scoring was done strictly according to established criteria (Blanco et al., 1996) and only sperm samples with hybridization efficiency over 95% were evaluated. Because of the difficulty of discriminating between nullisomic spermatozoa and hybridization failures, nullisomies were not directly scored. In order to decrease the subjectivity of the observations the incidence of nullisomy for each chromosome was estimated conservatively to be similar to the incidence of disomy for the same chromosome. A total of 99 939 spermatozoa were assessed for chromosomes X, Y and 18, and 95 366 spermatozoa for chromosomes 13 and 21. When possible, 2000 sperm cells per patient were scored for each hybridization. In sperm samples from severely oligozoospermic patients, however, only a small number of cells could be evaluated. This may reduce the power of statistical analysis and therefore, final conclusions have to be drawn with caution. Hybridization efficiency was >95% in all the samples evaluated.
Statistical analysis
The incidence of disomy and diploidy for the analysed chromosomes in different groups of patients according to the indications for FISH studies and to sperm quality, was compared using 2 test (with Yates' correction when necessary) and Fisher's exact test (Graphpad Instat v. 2.05a; Graphpad Software, San Diego, CA, USA). Individual results in each patient were also compared with the results of the control group using the same tests. For comparisons of fertilization, embryo cleavage, pregnancy, implantation and miscarriage rates after ICSI, MannWhitney and Fisher's exact tests were used when indicated. A P < 0.05 was considered as statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
ICSI outcome of the cycles performed in these couples with normal or abnormal FISH results is detailed in Tables IV, V and VII. Fertilization rates were similar in both groups, however pregnancy rate per transfer (36.5 versus 26.3%) and implantation rate (14.9 versus 9.9%) were higher in the couples with normal FISH results. There was also a higher incidence of miscarriages in the group of abnormal FISH results (80 versus 54.8%). However, none of these differences reached statistical significance.
|
It is of interest to point out that although in most couples ICSI was performed with the patient's own oocytes, there were two patients with donated oocytes and abnormal FISH results (Tables IV and V). In this subgroup, four pregnancies were achieved after eleven embryo transfers, and all of them miscarried.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We considered RM of unknown aetiology as a risk group for sperm chromosome abnormalities because an increased incidence of meiotic abnormalities (Vendrell et al., 1999; Egozcue et al., 2000a
,b
) and sperm aneuploidy (Giorlandino et al., 1998
; Bernardini et al., 2000
) had been previously reported in these patients. In a preliminary report by our group, an increased incidence of sex chromosome disomies was found in RM patients, and an increase in the diploidy rate was also found in a subset of patients with RM after ovum donation (Rubio et al., 1999
). Results of the present study confirm an increased incidence in sex chromosome disomies in RM patients in comparison with controls.
We also found increased incidences of sex chromosome disomies in couples with IF after ICSI. The poor embryo quality associated with the repeated IF observed in these couples could be related to defective sperm quality (Pellestor, 1991). Impaired sperm parameters have been described to adversely affect the chromosome constitution of embryos, not only because an oocyte can be fertilized by a chromosomally abnormal spermatozoon, leading to IF (In't Veld et al., 1997
; Pang et al., 1999
) but also because paternal factors derived from the centrosome can contribute to numerical chromosome abnormalities in the embryo (Obasaju et al., 1999
).
Our third indication for sperm FISH analysis was previous pregnancies affected by Down's syndrome. None of the three patients included in this group presented an increase in the incidence of chromosomal abnormalities. These results agree in part with the report by Blanco et al. (1998), in which the overall incidence of chromosome 21 disomy in the fathers of affected children was not significantly different from the control population (Blanco et al., 1998). In the same study, however, analysis of individual data demonstrates significant increases of disomy 21 in spermatozoa of the two fathers in whom the paternal origin of the extra chromosome 21 was established. In our series, this correlation could not be checked since the origin of the extra chromosome 21 was not tested.
Analysis of spermatozoa in the patient with abnormal meiosis showed an extremely significant increase in sex chromosome disomies. These results confirm that some abnormal cell lines can progress through meiosis producing chromosomally abnormal spermatozoa despite severe meiotic arrest or synaptic anomalies (Vendrell et al., 1999; Bernardini, 2000).
Focusing on sperm quality, OAT was associated with significant increases in sex chromosome disomies, disomy for chromosomes 18 and 21, and in the percentage of diploid sperm, particularly in those samples with markedly reduced sperm concentration (<5 x106/ml spermatozoa). Our results are in accordance with previous studies that have shown increased chromosome abnormalities in OAT patients and a more frequent involvement of sex chromosomes (Arán et al., 1999; Pang et al., 1999
; Pffefer et al., 1999; Bernardini, 2000; Nishikawa et al., 2000
; Ushijima et al., 2000
; Vegetti et al., 2000
). In our series the mean incidence of sex chromosome disomies in the OAT patients was 0.68%, and in the subset of OAT patients with sperm counts <5x106/ml, it was raised to 1.13%. In general, most investigators agree with the concept of a linear relationship between the severity of meiotic process disturbance and the alteration of seminal parameters (Arán et al., 1999
; Pfeffer et al., 1999
; Vegetti et al., 2000
). Nishikawa et al. (2000) also found an increase of sex chromosome disomies in OAT patients, more remarkable in sperm samples with <10 x106/ml (Nishikawa et al., 2000
). In our study, among the autosomes, chromosome 21 showed a higher incidence of disomy (0.24%), in agreement with other studies (Arán et al., 1999
; Pfeffer et al., 1999
, Ushijima et al., 2000
; Vegetti et al., 2000
). These results reflect that chromosome 21 and sex chromosomes are more prone to meiotic errors due to non-disjunction (Spriggs et al., 1995
; Blanco et al., 1996
). Diploidy is another sperm chromosomal abnormality usually increased in OAT and infertile patients. The mean diploidy rate in our general OAT series was 0.28%, reaching 0.45% in the subgroup of patients with <5x106/ml, both being significantly higher than that observed in the group of normozoospermic patients (0.10%).
The correlation of sperm motility and morphology with chromosomal abnormalities is more controversial. In our series, patients with isolated impaired sperm motility or morphology did not show significant increases in disomy or diploidy rates in comparison with normozoospermic patients. While some authors believe that sperm aneuploidy correlates more with asthenozoospermic rates (Vegetti et al., 2000), others found a stronger association with abnormal sperm morphology (Yurov et al., 1996
; Estop et al., 1997
; In't Veld et al., 1997
). Several factors may be involved in these contradictory results in the literature. Firstly, different morphology assessment criteria have been used in these studies (WHO versus Kruger's). Secondly, there are difficulties in analysing astheno and/or teratozoospermia as isolated factors, since there are few patients with single sperm defects. In fact, in many studies, astheno and/or teratozoospermic patients were also oligozoospermic. Thirdly, perhaps sperm aneuploidy is more related to specific types of abnormal morphology (Estop et al., 1997
) such as macrocephalia or the presence of multiple tails (In't Veld et al., 1997
). This is the reason why some investigators, rather than investigating specific correlations with semen parameters, look at the overall quality of the seminal sample as expressed by the total normal motility count (TNMC) and apply in-situ hybridization (ISH) rather than FISH, in order to control the normality of sperm morphology during the analysis of signal-products (Bernardini et al., 1998
).
On the other hand, it is important to know the influence of sperm chromosome abnormalities detected by FISH on ICSI outcome. Pfeiffer et al. (1999) assessed significantly higher diploidy and total aneuploidy rates in ten OAT patients undergoing 11 ICSI cycles (Pfeiffer et al., 1999). Overall fertilization rate was 70%, but only two successful pregnancies were achieved. Pang et al. (1999) reported a total aneuploidy rate of 3374% in a series of nine severe OAT patients (Pang et al., 1999). ICSI was performed in five of them, and no ongoing pregnancies were established, similar data have been reported by another group (Bernardini et al., 2000
).
In our study, ICSI outcome was assessed retrospectively in 131 cycles carried out on patients in whom sperm chromosomal constitution was later evaluated by FISH. The poor general results in this series are related to the patient selection criteria chosen in our study. To date, this is the largest series of cycles in which ICSI results are compared in patients with normal and abnormal FISH results. Fertilization rates were similar in both groups, but apparently higher pregnancy rates (36.5 versus 26.3%) and lower miscarriage rates (54.8 versus 80%) were observed in the group of patients with normal FISH results when compared with couples with abnormal FISH results, although these differences were not significantly different, probably because the number of pregnancies in the group of patients with abnormal FISH results was low. A power calculation showed that a total of 1128 ICSI cycles (205 with abnormal and 923 with normal FISH results) should be analysed to achieve significant differences in pregnancy rates. In addition, the high miscarriage rate in the group of patients with normal FISH results is related with the selection criteria used in the study, because one of such criteria was just unexplained recurrent abortion. Nonetheless, the trend observed in our series suggests that sperm aneuploidy may be associated with IF and fetal losses. Although one patient with a moderate increase in the percentage of diploid sperm was able to father a healthy child, four ICSI cycles were required to achieve a successful pregnancy. Further studies are needed to confirm this tendency.
Moreover, some patients in our series were included in our ovum donation programme. Analysis of these cases is of interest, because the sperm contribution to ICSI outcome can be analysed as an isolated factor. In this subgroup, pregnancy rates were similar in patients with normal and abnormal FISH results. Four pregnancies were achieved in patients with abnormal sperm FISH results. All four were miscarried, further suggesting a correlation between increased incidence of sperm chromosomal abnormalities and abortion.
In conclusion, our study confirms that a normal karyotype does not exclude the presence of chromosomal abnormalities in spermatozoa of patients included in an ICSI programme. Further studies are required to understand more about the risk of higher rates of sperm aneuploidy in male partners of couples with recurrent pregnancy losses and normal seminal parameters. In fact, the low semen quality represents an independent risk factor and it is often associated. It is unlikely that the small increase found in the rates of sex chromosome aneuploidy in spermatozoa of RM and IF patients might be reflected in significant changes of clinical results (pregnancy, implantation, abortion rates) but future studies on additional chromosomes in spermatozoa and embryos from these patients may help to clarify this better. Our study provides a demonstration that OAT is associated with higher sperm aneuploidy and diploidy rates, particularly in the presence of oligozoospermia. Such conditions might, in part, explain the low implantation and high abortion rates observed during ICSI cycles.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bernardini, L., Borini, A., Preti, S. et al. (1998) Study of aneuploidy in normal and abnormal germ cells from semen of fertile and infertile men. Hum. Reprod., 13, 34063413.[Abstract]
Bernardini, L., Gianaroli, L., Fortini, D. et al. (2000). Frequency of hyper-, hypohaploidy and diploidy in ejaculate, epididymal, and testicular germ cells of infertile patients. Hum. Reprod., 15, 21652172.
Blanco, J., Egozcue, J. and Vidal, F. (1996) Incidence of chromosome 21 disomy in human spermatozoa as determined by fluorescent in-situ hybridisation. Hum. Reprod., 11, 722726.[Abstract]
Blanco, J., Rubio, C., Simón, C. et al. (1997) Increased incidence of disomic sperm nuclei in a 47,XYY male assessed by fluorescent in situ hybridisation (FISH). Hum. Genet., 99, 413416.[ISI][Medline]
Blanco, J., Gabau, E., Gómez, D. et al. (1998) Chromosome 21 disomy in the spermatozoa of the fathers of children with trisomy, 21, in a population with a high prevalence of Down Syndrome: increased incidence in cases of paternal origin. Am. J. Hum. Genet., 63, 10671072.[ISI][Medline]
Bonduelle, M., Aytoz, A., Van Assche, E. et al. (1998) Incidence of chromosomal aberrations in children born after assissted reproduction through intracytoplasmic sperm injection. Hum. Reprod., 13, 781782.
Egozcue, J., Templado, C., Vidal, F. et al. (1983) Meiotic studies in a series of 1100 infertile and sterile men. Hum. Genet., 65, 185187.[ISI][Medline]
Egozcue, J., Blanco, J. and Vidal, F. (1997) Chromosome studies in human sperm nuclei using fluorescence in-situ hybridization (FISH). Hum. Reprod. Update, 3, 441452.
Egozcue, S., Vendrell, J.M., García, F. et al. (2000a) Increased incidence of meiotic anomalies in oligoasthenozoospermic males preselected for intracytoplasmic sperm injection. J. Assist. Reprod. Genet., 17, 307309.[ISI][Medline]
Egozcue, S., Blanco, J, Vendrell, J.M. et al. (2000b) Human male infertility: Chromosome anomalies, meiotic disorders, abnormal spermatozoa and recurrent abortions. Hum. Reprod. Update, 6, 93105.
Estop, A.M., Vandermark, K.K., Munné, S. et al. (1997) Sperm morphology and chromosome aneuploidy in men with infertility as determined by fluorescence in situ hybridization (FISH). Fertil. Steril., (Suppl.), Abstract P-242, p. S208.
Giorlandino, C., Calugi, G., Laconianni, L. et al. (1998) Spermatozoa with chromosomal abnormalities may result in a higher rate of recurrent abortion. Fertil. Steril., 70, 576577.[ISI][Medline]
Hassold, T., Chen, N., Funkhouser, J. et al. (1980) A cytogenetic study of 1000 spontaneous abortions. Ann. Hum. Genet., 44, 151178.[ISI][Medline]
In't Veld, P.A., Branderburg, H., Verhoeff, A. et al. (1995) Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet, 346, 773.[ISI][Medline]
In't Veld, P.A., Broekmans, F.J.M., de France, H.F. et al. (1997) Intracytoplasmic sperm injection and abnormal spermatozoa. Hum. Reprod., 12, 752754.[Abstract]
Lahdetie, J., Saari, N., Ajonsepää-Saari, M. et al. (1997) Incidence of aneuploidy spermatozoa among infertile men studied by multicolour fluorescence in situ hybridisation. 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. Lancet, 346, 1095.
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 hybridisation. Fertil. Steril., 64, 811817.[ISI][Medline]
Nicolaidis, P. and Petersen, M.B. (1998) Origin and mechanisms of non-disjunction in human autosomal trisomies. Hum. Reprod., 13, 313319.[ISI][Medline]
Nishikawa, N., Murakami, I., Ikuta, K. et al. (2000) Sex chromosomal analysis of spermatozoa from infertile men using fluorescence in situ hybridization. J. Assist. Reprod. Genet., 17, 97102.[ISI][Medline]
Obasaju, M., Kadam, A., Sultan, K. et al. (1999) Sperm quality may adversely affect the chromosome constitution of embryos that result from intracytoplasmic sperm injection. Fertil. Steril., 72, 11131115.[ISI][Medline]
Pang, M.G., Hoegerman, 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 fluorescence in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection. Hum. Reprod., 14, 12661273.
Pellestor, F. (1991) Differential distribution of aneuploidy in human gametes according to their sex. Hum. Reprod., 6, 12521258.[Abstract]
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]
Rubio, C., Simón, C., Blanco, J. et al. (1999) Implications of sperm chromosome abnormalities in recurrent miscarriage. J. Assist. Reprod. Genet., 16, 253258.[ISI][Medline]
Spriggs, E.L., Rademaker, A.W. and Martin, R.H. (1995) Aneuploidy in human sperm: results of two- and three-color fluorescence in situ hybridization using centromeric probes for chromosomes, 1,12,15,18, X and Y. Cytogenet. Cell Genet., 71, 4753.[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 hybridisation. Hum. Reprod., 15, 1107 1111.
Vegetti, W., Van Assche, E., Frias, A. et al. (2000) Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridisation in infertile men. Hum. Reprod., 15, 351365.
Vendrell, J.M., Garcia, F., Veiga, A. et al. (1999) Meiotic abnormalities and spermatogenic parameters in severe oligoasthenozoospermia. Hum. Reprod., 14, 375378.
World Health Organization (1992) WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 3rd ed. Cambridge University Press, Cambridge, pp 4045.
Yurov, Y.B., Saias, M.J., Vorsanova, S.G. et al. (1996) Rapid chromosomal analysis of germ-line cells by FISH: an investigation of an infertile male with large-headed spermatozoa. Mol. Hum. Reprod., 2, 665668.[Abstract]
Submitted on March 16, 2001; accepted on June 26, 2001.