Chromosomal abnormalities in couples undergoing intracytoplasmic sperm injection. A study of 370 couples and review of the literature

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1 international journal of andrology, 27: (2004) Chromosomal abnormalities in couples undergoing intracytoplasmic sperm injection. A study of 370 couples and review of the literature F. MOREL,* N. DOUET-GUILBERT,* M.-J. LE BRIS, V. AMICE, M. T. LE MARTELOT,à S. ROCHE,à A. VALÉRI, V. DERRIEN, J. AMICE* and M. DE BRAEKELEER* *Faculté de Médecine, Université de Bretagne Occidentale, Brest, Service de Cytogénétique, Cytologie et Biologie de la Reproduction, CHU Morvan, Brest, àservice de Gynécologie- Obstétrique et Médecine de la Reproduction, CHU Morvan, Brest, Service d Urologie, CHU La Cavale Blanche, Brest, Service de Gynécologie, CHG, Lorient, France Summary Intracytoplasmic sperm injection (ICSI) is now widely acknowledged as the most effective therapeutic approach to severe male infertility or unsuccessful in vitro fertilization. Cytogenetic investigations were performed in 370 females and 335 males prior to ICSI between January 1997 and April Nine men (2.7%) and 48 women (13%) had an abnormal karyotype, 44 females having some degree of numerical sex chromosome mosaicism. A review of the literature showed the prevalence of all types of chromosomal abnormalities to be much higher among male and female partners of couples examined prior to ICSI than among newborns. As most ICSIs are performed with ejaculated spermatozoa from oligospermic men, the distribution and the prevalence of the several types of chromosomal abnormalities are closer to those of oligospermic rather than azoospermic males. Our results combined with those of the literature stress the importance of karyotyping both male and female partners before ICSI is started. Adequate genetic counselling, possibly followed by prenatal diagnosis, should be offered if a chromosomal anomaly is detected. Keywords: chromosomal abnormality, cytogenetics, intracytoplasmic sperm injection, male infertility, prevalence, sex chromosome mosaicism Introduction Intracytoplasmic sperm injection (ICSI) has become a highly effective therapeutic approach to male infertility and to couples who experienced unsuccessful in vitro fertilization (IVF) (De Braekeleer & Dao, 1991). Several cytogenetic Correspondence: Pr Marc De Braekeleer, Laboratoire de Cytogènétique, Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, 22, avenue Camille Desmoulins, CS 93837, F Brest cedex 3, France. marc.debraekeleer@univ-brest.fr studies have now shown a high prevalence of chromosome abnormalities not only in the male partner of couples undergoing ICSI, but also in the female partner (Mau et al., 1997; Meschede et al., 1998; Scholtes et al., 1998; Hocquet et al., 1999; Peschka et al., 1999; Schreurs et al., 2000; Gekas et al., 2001). We conducted a study to determine the prevalence and type of chromosomal abnormalities among 370 couples undergoing ICSI. Our results are compared to those previously reported in the literature and those found in infertile males. Ó 2004 Blackwell Publishing Ltd.

2 Cytogenetics of couples prior to ICSI 179 Patients and methods All the 370 couples seen between January 1997 and April 2003 at the CHU Morvan, for whom ICSI was indicated, were included in the present study. Each couple underwent a routine diagnostic evaluation including an andrological examination and, for women, a standard endocrinological, gynaecological and ultrasound examination to rule out potential infertility factors. A karyotype was systematically performed in both members of the couple. However, because some individuals had their karyotype tested outside our institution, 370 females, but only 335 males, were included in the analysis. Chromosome analysis was carried out on phytohaemagglutinin-stimulated peripheral lymphocytes cultured for 72 h. Chromosomes were harvested according to standard procedures and the R-banded karyotypes were described according to the recommendations of ISCN (1995). Sixteen cells were karyotyped. However, if at least one of these 16 cells showed a loss or a gain of a gonosome, the number of analysed metaphases was increased to 25. If a second abnormal cell was observed, the analysis was considered to be completed; otherwise, the number of metaphases was increased to 50 (Morel et al., 2002). Chromosome polymorphisms or variants, such as inv(9)(p11q13), which increased the centromeric heterochromatin and fragile sites, were not considered in this study. Results Among the 335 men, 326 (97.3%) were found to have a normal karyotype (Table 1). Three had a robertsonian translocation and three others a balanced reciprocal translocation. Two had a 47,XXY constitution and one a supernumerary marker chromosome. Four of the 370 women had a robertsonian translocation while 44 showed some degree of numerical sex chromosome mosaicism (Table 1). The remaining 322 women (87%) had a normal karyotype. Table 1. Cytogenetic results in couples referred for ICSI at the University Hospital in Brest Males Females Type Nr Type Nr Robertsonian translocations 45,XY, der(13;14)(q10;q10) 45,XX, der(13;14)(q10;q10) 3 (0.8) 45,XY, der(14;21)(q10;q10) 2 (0.6) 45,XX, der(14;22)(q10;q10) 3 (0.9) 4 (1.1) Reciprocal translocations 46,XY, t(1;7)(p34;q21) 46,XY, t(9;16)(q34.2;p12) 46,XY, t(11;19)(q11;p13.2) 3 (0.9) Supernumerary markers 47,XY, +idic(15)(q11) Numerical sex chromosome abnormalities 47,XXY 2 (0.6) 45,X/46,XX 26 (7.0) 45,X/46,XX/47,XXX[1] 10 (2.7) 45,X[3]/46,XX[45]/47,XXX[2] 46,XX[23]/48,XXXX[2] 45,X[1]/46,XX/47,XXX[2] 3 (0.3) 45,X[2]/46,XX[45]/47, XXX[2]/49,XXXXX[1] 45,X[2]/46,XX[46]/47, XXX[1]/47,XX +i(xq)[1] 47,XXX 2 (0.6) 44 (11.9) Normal karyotype 46,XY 326 (97.3) 46,XX 322 (87.0)

3 180 F. Morel et al. Discussion Couples seeking ICSI are offered a genetic work-up during clinical investigations. Optimally, it should include not only a cytogenetic analysis, but also a molecular study looking for AZF deletions on the Y chromosome and, in the case of obstructive azoospermia, for the major cystic fibrosis transmembrane conductance regulator mutations commonly found in congenital bilateral agenesis of the vas deferens. Several studies have now reported an overall increased frequency of chromosomal aberrations in male and female partners of couples referred for ICSI (Mau et al., 1997; Meschede et al., 1998; Scholtes et al., 1998; Hocquet et al., 1999; Peschka et al., 1999; Schreurs et al., 2000; Gekas et al., 2001). Data on chromosomal abnormalities were available for 7895 males and 4662 females referred for ICSI (Table 2). Overall, 95.71% of the men and 94.12% of the females had a normal karyotype. The remaining individuals had an abnormal karyotype consisting mostly of translocations, inversions and numerical sex chromosome aberrations. More females compared to males had a cytogenetic aberration; this was the result of a high frequency of numerical sex chromosome abnormalities (4.44% in females vs. 1.82% in males), usually as low-level mosaicism. Indeed, mosaicism for a numerical X chromosome abnormality has been observed in women prior to planned ICSI treatment in several studies (Meschede et al., 1998; Scholtes et al., 1998; Peschka et al., 1999; Gekas et al., 2001; Sonntag et al., 2001). Nevertheless, it is difficult to estimate its frequency from the literature because there is no consensus on the required minimum number of cells identified with an anomaly to be considered as mosaicism, or on the number of metaphases to be analysed (Morel et al., 2002). Using the same standardized protocol, we already found an increased rate of X chromosome loss mosaicism (defined as two 45,X cells and higher) among 228 women prior to ICSI compared to control women (9.6 vs. 4.8%; p ¼ 0.01) (Morel et al., 2002). Our present results on 370 women confirm the high rate of 45,X/46,XX mosaicism (10.5%). However, the tissue distribution and the consequences of sex chromosome mosaicism are still unknown. Therefore, all women with X chromosome mosaicism were asked to consult the medical geneticist of the hospital to have proper counselling. Thus, even if we exclude mosaic X chromosome aneuploidy, 1.44% of the women have a chromosomal abnormality (Table 2). The prevalence of robertsonian translocation (0.32%) among women is 3.5 times higher than in newborns (0.092%) (De Braekeleer & Dao, 1990), 53.3 and 26.7% being der(13;14)(q10;q10) and der(14;21) (q10;q10), respectively. The probability of finding a balanced reciprocal translocation is 7.8 times higher among ICSI women (0.66%) than newborns (0.085%). All the chromosomes, except for the X chromosome, are implicated in at least one rearrangement. The distribution of the chromosome arms involved in reciprocal translocations, using Monte Carlo simulations as previously described (De Braekeleer & Dao, 1990, 1991), does not show any arm to be more rearranged than expected on the basis of their relative lengths (p > 0.05). The prevalence of pericentric and paracentric inversions is 32.5 times higher among ICSI women than newborns (0.39 vs %). Thirteen women have a pericentric inversion; six of them (46.2%) have a rearrangement of chromosome 10, including five with inv(10)(p11q21-22). Overall, 4.29% of the men have a chromosomal abnormality (Table 2). The prevalence of robertsonian translocation (0.95%) among men is 10.3 times higher than in newborns, 65.8 and 18.4% being der(13;14)(q10;q10) and Table 2. of chromosomal abnormalities reported in couples referred for ICSI Males Females Number 95% confidence interval Number 95% confidence interval Individuals Robertsonian translocations Reciprocal translocations Pericentric inversions Paracentric inversions Supernumerary markers Sex chromosome abnormalities Other Normal karyotype Data from Baschat et al. (1996), Testart et al. (1996), Pauer et al. (1997), Mau et al. (1997), Tuerlings et al. (1998), Scholtes et al. (1998), Meschede et al. (1998), Peschka et al. (1999), Hocquet et al. (1999), Schreurs et al. (2000), Bor et al. (2002), Gekas et al. (2001), De Braekeleer & Dao (1991) and this study.

4 Cytogenetics of couples prior to ICSI 181 der(14;21)(q10;q10), respectively. The probability of finding a balanced reciprocal translocation is 11.5 times higher among ICSI men (0.98%) than newborns. All the chromosomes are implicated in at least one rearrangement. Although, using Monte Carlo simulations, three chromosomes (chromosomes 12, 22 and Y) are found to be more involved in reciprocal translocations than expected on the basis of their relative lengths (p < 0.05), no particular band has a higher number of breakpoints than expected (p > 0.05). The prevalence of pericentric and paracentric inversions (0.27%) is 22.5 times higher among ICSI men Table 3. Numerical sex chromosome abnormalities among men referred for ICSI Abnormalities Total 47,XXY ,XY/47,XXY ,X/46,XY/47,XXY ,XY/47,XXY/48,XXXY ,X/46,XY ,XYY ,XY/47,XYY ,XY/47,XYY/49,XYYYY ,X/47,XYY ,XY/47,XXX ,X/46,XX/46,XY Data from Baschat et al. (1996), Testart et al. (1996), Pauer et al. (1997), Mau et al. (1997), Meschede et al. (1998), Scholtes et al. (1998), Tuerlings et al. (1998), Peschka et al. (1999), Hocquet et al. (1999), Bor et al. (2002), Gekas et al. (2001) and this study. than newborns. Five of the 13 men (38.5%) have a pericentric inversion of chromosome 10, all being inv(10)(p11q21-22). Men referred for ICSI are also at a higher risk of having a supernumerary marker (0.19% compared to 0.023% among newborns). Sex chromosome abnormalities are present in 1.82% of the 7895 men referred for ICSI, a rate that is 9.2 times higher than among male newborns (0.198%) (Table 2). The distribution of numerical sex chromosome abnormalities is available for 140 men (Table 3). About 58% of the males have a 47,XXY constitution, in a homogeneous or mosaic state. The 47,XXY karyotype was found in 0.84% of all men seeking ICSI compared to 0.105% in the male newborn population, a risk that is eight times higher. A 45,X/46,XY mosaicism is seen in 18.6% of men with a numerical sex chromosome abnormality, which gives a frequency of 0.33% of all men (<0.01% in the male newborns). Although 12.9% of the men with a numerical sex chromosome abnormality have a 47,XYY constitution, they represent only 0.23% of all males compared to 0.082% in the male newborn population, a slightly increased risk (2.8 times). Table 4 gives the frequency of several selected chromosomal abnormalities reported in males prior to ICSI and in males with oligospermia or azoospermia. The relative risk of each type of karyotypic aberration and category of males is calculated on the basis of the frequencies reported in newborns of both sexes and male newborns (De Braekeleer & Dao, 1991). Males with azoospermia are much more likely to have a sex chromosome abnormality, either numerical or structural, than those with oligospermia. On the contrary, males with oligospermia are more likely to have an autosomal rearrangement (translocations, inversions, supernumerary markers) than those with azoospermia. Table 4. of chromosomal abnormalities reported in males prior to ICSI and in males with oligo or azoospermia Males ICSI Males with oligospermia Males with azoospermia Newborns Chromosomal abnormalities Freq RR Freq RR Freq RR Freq Robertsonian translocations Reciprocal translocations Inversions Supernumerary markers Sex chromosome abnormalities ,XXY Y structural abnormalities <0.01 Other sex chromosome abnormalities a Total number of individuals b The RR (relative risk) was calculated using the newborns abnormalities as the reference. a Including 46,XY/47,XXY and 45,X/46,XY. b Based on the results obtained for newborns of both sexes and male newborns (De Braekeleer & Dao, 1990).

5 182 F. Morel et al. Therefore, as most ICSIs are performed with ejaculated spermatozoa from oligospermic men, we expect the distribution and the prevalence of the several types of chromosomal abnormalities to be closer to those of oligospermic rather than azoospermic males, as observed. In conclusion, the data available at the present time show that a significant number of chromosomal abnormalities is present in couples seeking ICSI treatment. Our results combined with those of the literature stress the importance of karyotyping both male and female partners before ICSI is started. Adequate genetic counselling, possibly followed by prenatal diagnosis, should be offered if a chromosomal anomaly is detected. Acknowledgements The authors thank Anne Dumay, Marie-France Fourel, Claire Le Roy, Edith Montenoise and Gisèle Toullec for their excellent technical work and Christine Nedelec and Nicole Le Her for secretarial expertise. Baschat, A. A., Kupker, W., Al Hasani, S., Diedrich, K. & Schwinger, E. (1996) Results of cytogenetic analysis in men with severe subfertility prior to intracytoplasmic sperm injection. Human Reproduction 11, Bor, P., Hindkjaer, J., Kolvraa, S. & Ingerslev, H. J. (2002) Y-chromosome microdeletions and cytogenetic findings in unselected ICSI candidates at a Danish fertility clinic. Journal of Assisted Reproduction and Genetics 19, De Braekeleer, M. & Dao, T. N. (1990) Cytogenetic studies in couples experiencing repeated pregnancy losses. Human Reproduction 5, De Braekeleer, M. & Dao, T. N. (1991) Cytogenetic studies in male infertility: a review. Human Reproduction 6, Gekas, J., Thépot, F., Turleau, C., Siffroi, J. P., Dadoune, J. P., Wasels, R., Benzacken, B. & Association des Cytogénéticiens de Langue Française(2001) Chromosomal factors of infertility in candidate couples for ICSI: an equal risk of constitutional aberrations in women and men. Human Reproduction 16, Hocquet, D., Roux, C., Collonge-Rame, M. A., Fellmann, F. & Bresson, J. L. (1999) Bilan des examens chromosomiques de 277 couples candidats à l injection intracytoplasmique de spermatozoïde. Andrologie 9, ISCN (1995) An International System for Human Cytogenetic Nomenclature. S. Karger, Basel. Mau, U. A., Backert, I. T., Kaiser, P. & Kiesel, L. (1997) Chromosomal findings in 150 couples referred for genetic counselling prior to intracytoplasmic sperm injection. Human Reproduction 12, Meschede, D., Lemcke, B., Exeler, J. R., De Geyter, C., Behre, H. M., Nieschlag, E. & Horst, J. (1998) Chromosome abnormalities in 447 couples undergoing intracytoplasmic sperm injection prevalence, types, sex distribution and reproductive relevance. Human Reproduction 13, Morel, F., Gallon, F., Amice, V., Le Bris, M. J., Le Martelot, M. T., Roche, S. et al. (2002) Sex chromosome mosaicism in couples undergoing intracytoplasmic sperm injection. Human Reproduction 17, References Pauer, H. U., Hinney, B., Michelmann, H. W., Krasemann, E. W., Zoll, B. & Engel, W. (1997) Relevance of genetic counselling in couples prior to intracytoplasmic sperm injection. Human Reproduction 12, Peschka, B., Leygraaf, J., van der Ven, K., Montag, M., Schartmann, B., Schubert, R., van der Ven, H. & Schwanitz, G. (1999) Type and frequency of chromosome aberrations in 781 couples undergoing intracytoplasmic sperm injection. Human Reproduction 14, Scholtes, M. C. W., Behrend, C., Dietzel-Dahmen, J., van Hoogstraten, D. G., Marx, K., Wohlers, S., Verhoeven, H. & Zeilmaker, G. H. (1998) Chromosomal aberrations in couples undergoing intracytoplasmic sperm injection: influence on implantation and ongoing pregnancy rates. Fertility and Sterility 70, Schreurs, A., Legius, E., Meuleman, C., Fryns, J. P. & D Hooghe, T. M. (2000) Increased frequency of chromosomal abnormalities in female partners of couples undergoing in vitro fertilization or intracytoplasmic sperm injection. Fertility and Sterility 74, Sonntag, B., Meschede, D., Ullmann, V., Gassner, P., Horst, J., Nieschlag, E. & Behre, H. M. (2001) Low-level sex chromosome mosaicism in female partners of couples undergoing ICSI therapy does not significantly affect treatment outcome. Human Reproduction 16, Testart, J., Gautier, E., Brami, C., Rolet, F., Sedbon, E. & Thebault, A. (1996) Intracytoplasmic sperm injection in infertile patients with structural chromosome abnormalities. Human Reproduction 11, Tuerlings, J. H. A. M., de France, H. F., Hamers, A., Hordijk, R., Van Hemel, J. O., Hansson, K. et al. (1998) Chromosome studies in 1792 males prior to intra-cytoplasmic sperm injection: the Dutch experience. European Journal of Human Genetics 6, Received 18 December 2003; revised 16 January 2004; accepted 20 January 2004