Success of intracytoplasmic sperm injection in couples with male and/or female chromosome aberrations

Human Reproduction vol.12 no.12 pp.2635 2640, 1997 Success of intracytoplasmic sperm injection in couples with male and/or female chromosome aberrations M.Montag 1,5, K.van der Ven 1, S.Ved 1, A.Schmutzler 1, G.Prietl 1, D.Krebs 1, B.Peschka 2, G.Schwanitz 2, P.Albers 3, G.Haidl 4 and H.van der Ven 1 1 Department of Endocrinology and Reproductive Medicine, 2 Department of Human Genetics, 3 Department of Urology, 4 Department of Dermatology, University of Bonn, 53105 Bonn, Germany 5 To whom correspondence should be addressed This paper reports on results of intracytoplasmic sperm injection (ICSI) in patients in whom constitutional or secondary chromosome aberrations were detected in the male and/or female partner. Out of 434 couples treated by ICSI (590 cycles), 16 couples (3.7%) were affected by constitutional chromosome aberrations and 96 (22.1%) by secondary chromosome aberrations. Constitutional chromosome aberrations were found in eight male and eight female patients. Couples with the aberration in the male showed significantly lower fertilization, implantation and pregnancy rates (P < 0.05). The occurrence of female constitutional chromosome aberrations led to lower fertilization rates but implantation and pregnancy rates were similar to a control group; however, a higher abortion rate was noted. In the group with secondary chromosome aberrations, 22 males and 59 females carried an abnormality and in 15 couples, both partners. Compared to the remaining (unaffected) 322 couples, fertilization and embryo transfer rates were reduced but implantation rates and pregnancy rates were not different. In all couples where an abortion occurred, mainly parental autosomal aberrations were involved (six out of eight). Our retrospective analysis shows that an unexpectedly high number of infertile couples in an ICSI programme are affected by chromosome aberrations, which in turn may explain the reduced fertilization rates observed in this subgroup of patients. Key words: constitutional chromosome aberrations/genetic risk/ intracytoplasmic sperm injection/secondary chromosome aberrations Introduction Since its introduction in 1992 (Palermo et al., 1992), intracytoplasmic sperm injection (ICSI) has turned out to be one of the most successful assisted reproductive techniques. This has raised the question of the genetic consequences of ICSI, not only with regard to the karyotype of the children born, but also to a possible predisposition of the infertile couple (Silber et al., 1995; Chandley and Hargreave, 1996; Fishel et al., 1996; Martin, 1996; Persson et al., 1996). Several studies have shown a higher incidence of chromosome abnormalities in infertile males (Chandley et al., 1975), with an overall incidence of 7.1% constitutional aberrations (Retief et al., 1984). Most frequently, sex chromosome aneuploidies have been reported (De Braekeleer and Dao, 1991). As a consequence, in cases of male infertility it is now common practice in most in-vitro fertilization (IVF) centres to perform a cytogenetic screening of the male partner prior to ICSI treatment. It has previously been recognized that the outcome of ICSI might be influenced by the occurrence of chromosome abnormalities in the female partner (Meschede et al., 1995). Recently, we reported an increased frequency of constitutional chromosome aberrations in male and female partners of couples examined prior to ICSI (Peschka et al., 1996; van der Ven et al., 1998). In 19 out of 305 couples (6.23%), at least one partner was affected by a constitutional chromosome aberration and in one case both were affected. Whereas the rate of abnormality in the male (3.27%) was within the expected range for men with impaired semen parameters, the rate for the females (3.27%) was unexpectedly high. The data indicate that the potential contribution of maternal chromosome aberrations in cases of poor reproductive outcome cannot be neglected. Further research is needed concerning the role of maternal aneuploidy in failed human embryo implantation. Oocyte aneuploidy is found commonly among older women, and similar poor reproductive outcomes apply to women affected by a structural chromosome abnormality (Plachot et al., 1988; Angell et al., 1993; Munné et al., 1995). Routine cytogenetic analysis of both the male and the female partner is part of our infertility programme. In this paper, we have evaluated the relevance of the occurrence of constitutional as well as secondary chromosome aberrations (SCA) in the male and female for the success rate of ICSI. Materials and methods Andrological, gynaecological and cytogenetic examination Between June 1994 and October 1996, a total of 434 couples underwent 590 ICSI treatment cycles at the University of Bonn. The selection for ICSI treatment was based on the diagnosis of male factor infertility with reduced sperm quality. In a collaborative approach, all patients underwent an extensive andrological, gynaecological and cytogenetic examination prior to ICSI. For all patients a chromosome analysis was performed on peripheral lymphocytes. Up to 50 metaphases (450 550 bands per genome) European Society for Human Reproduction and Embryology 2635

M.Montag et al. were analysed per patient. The classification of secondary chromosome aberration (SCA) comprises the occurrence of one or more single cells showing either a structural or numerical chromosome abnormality of the same karyotype. However, in some cases we found several abnormal cells which exhibited a combination of different chromosome aberrations, mainly combined autosomal and sex chromosomal aberrations. The occurrence of two abnormal single cells within one preparation exhibiting a complementary aberration (e.g. 45, XO and 47, XXX) was considered to be an artefact. In complex structural rearrangements, additional molecular cytogenetic analysis by fluorescence in-situ hybridization (FISH) was performed. Patients identified as carriers for chromosomal abnormalities underwent genetic counselling to explain the importance of the findings and the implications for their offspring. Furthermore, in all individuals with constitutional aberrations, additional investigations of first and second degree relatives were recommended. Ovarian stimulation Follicular stimulation was carried out by the combination of the gonadotrophin releasing hormone agonist (GnRHa) triptorelin acetate (Decapeptyl, Ferring, Germany), human menopausal gonadotrophin (HMG; Humegon, Organon) and/or follicular stimulating hormone (FSH; Fertinorm, Serono) and human chorionic gonadotrophin (HCG). Triptorelin acetate (0.1 mg/day) was administered from day 22 of the previous cycle. Twelve to 15 days later, HMG/FSH (225 IU) was administered daily. Ovarian response was monitored by transvaginal ultrasound and HMG/FSH was adjusted according to the patient s individual response based on follicular size and oestradiol levels. HCG (10 000 IU) was administered when the leading follicles were 18mm in diameter. Intracytoplasmic sperm injection All media used for oocyte retrieval, denuding, ICSI treatment and subsequent culture were of pharmaceutical grade and free of phenol red (IVF-50; Gamete-100, ICSI-1; Scandinavian IVF Science, Göteborg, Sweden). For injection, sperm cells were prepared by a modified mini-swim-up technique. The liquefied ejaculate was washed once with Gamete-100. The sperm pellet was dissolved in 1 ml of medium, recentrifuged in a microfuge (Biofuge 13, Heraeus, Osterode, Germany) and the final pellet was resuspended in 20 50 µl of medium and stored in a CO 2 incubator. A few microlitres of the sperm suspension was placed into a central polyvinylpyrrolidone (PVP) droplet (ICSI-1) in the injection dish. The technique used for injection followed essentially the protocol published by Palermo et al. (1995). ICSI was carried out on the heated stage of an inverted microscope (DMIRB; Leica, Bensheim, Germany) equipped with microinjection devices for holding the oocyte and sperm injection (Narishige, Tokyo, Japan). Following injection, oocytes were cultured in IVF-50 up to the time of transfer. Transvaginal intrauterine embryo transfer of a maximum of three embryos took place 2 days following oocyte retrieval. Luteal phase support was performed with progesterone vaginal suppositories (2 200 mg per day) starting on the day following ovulation induction with HCG. Pregnancy was defined as the occurrence of a positive β-hcg ( 10) value at day 12 after transfer and a second, higher value 2 days later. Only pregnancies reaching HCG values 100 were considered for the evaluation. Implantation was determined after ultrasonic detection of a gestational sac and viability was demonstrated by a positive heart beat. Statistics Couples with constitutional chromosome aberrations were compared with the total number of patients without chromosome aberrations (unaffected couples) and a matched control group of patients identified in a computer database and matched by sperm analysis, women s age, number of treatment cycles (16 patients; 30 cycles) and number of oocytes injected. Data were analysed by χ 2 test and P values of 0.05 were considered significant. Results Type and frequency of chromosome aberrations A total of 434 couples undergoing 590 ICSI cycles from June 1995 to October 1996 were evaluated cytogenetically. In all, 322 couples (74.2%) showed no chromosome aberrations and were regarded as unaffected (Table I). In 112 couples (25.8%), a chromosome abnormality was detected. Out of these, 16 couples (3.7%) were affected by a constitutional aberration, present in either the male (eight couples) or the female partner (eight couples). All patients with constitutional aberrations are listed in detail in Table II. The remaining 96 couples (22.1%) showed SCA. The type and frequency of SCA are shown in Table III. The frequency of SCA ranged from 2 to 20% of all cells examined and the mean value of abnormal cells per affected patient was 3.1% for autosomal, 3.6% for sex chromosomal and 6.6% for combined autosomal and sex chromosomal aberrations. In cases of sex chromosomal SCA, we found mainly numerical aberrations (76 versus 24% structural aberrations), whereas for autosomal SCA, structural abnormalities were dominant (76%). Patients with SCA were further allocated to subgroups. Accordingly, groups were defined with couples where the male partner (22 couples), the female partner (59 couples) or both partners (15 couples) were affected (see Table I). ICSI results in patients with constitutional chromosome aberrations We first addressed the question of whether constitutional chromosome aberrations had a deleterious effect on ICSI results (Table IV). A total of 30 treatment cycles was performed in 16 couples, with 20 male and 10 female affected cycles. Fertilization rates were significantly higher in the matched control group and in the unaffected group than in the male and female affected group (P 0.05). For all groups, the transfer rates were not significantly different. The implantation rates per embryo transferred were comparable in the female affected group, the matched control group and the unaffected group, whereas the male affected group exhibited a significantly lower implantation rate (P 0.05). Pregnancy rates for the matched control group, the unaffected group and the female affected couples were comparable (28.6, 26.1 and 33.3%), Table I. Incidence and type of chromosome aberration of males and females in 434 male infertility couples Unaffected Constitutional Single cell aberrations aberrations Couples (%) 322/434 (74.2) 16/434 (3.7) 96/434 (22.1) Males (%) 322/434 8/434 (1.9) 37/434 a (8.5) Females (%) 322/434 8/434 (1.9) 74/434 a (17.1) a In 15 couples both partners were affected. 2636

ICSI in couples with chromosome aberrations Table II. Type of constitutional chromosome aberration in 16 male and female patients Patient Age Karyotype No. of Pregnancy cycles Sex chromosomal C95/479 35 47,XXX 1 0 (numerical) C96/7 41 47,XXY 2 0 Autosomal C95/701 37 46,XY,t(1;2)(p34.1;p21) 2 0 Reciprocal C95/834 30 46,XY,t(4;5)(q21;q11.2) 1 0 translocations C95/904 30 46,XY,t(1;21)(1;9;21) 3 0 C96/161 40 46,XY,t(3;12)(p24;p12) 3 0 C96/189 38 46,XY,t(1;5)(p32;q31) 3 1 C95/1244 40 46,XX,t(5;19)(p10;q10) 3 1 a C96/494 37 46,XX,t(3;18)(q24;p11.3) 1 1 a Robertsonian C95/800 38 45,XY,der(13;14)(q10;q10) 2 0 translocations C95/726 25 45,XX,der(13;14)(q10;q10) 2 0 C96/217 31 45,XX,der(14;15)(q10;q10) 1 1 Inversions C95/629 40 46,XY,inv(5)(p14.2;q22) 1 0 Other C95/1092 34 46,XY,der(9) 3 0 structural C95/1122 30 46,XX,der(9)add(9)(p12) 1 0 aberrations C95/1270 24 46,XX,der(9)add(9)(p12) 1 0 a Aborted in the first trimester. Table III. Type and frequency of abnormal single cells detected in lymphocytes in couples with SCA Chromosomes No. of patients Specification and Mean percentage of Range of abnormal affected a frequency of SCA abnormal single cells single cells (%) (%) per patient (%) Autosomes 23 females Structural: 76 3.1 2 20 13 males Numerical: 24 Sex chromosomes 24 females Structural: 24 3.6 2 10 4 males Numerical: 76 Auto- and 12 females Structural: 23 sex chromosomes 5 males Numerical: 38 6.6 4 12 (combined) Combined: 39 (structural and numerical) a In another 15 couples both partners were affected. SCA secondary chromosome aberrations. Table IV. Results of intracytoplasmic sperm injection in patients with constitutional chromosome aberration compared to patients with no chromosome aberration (matched control group and unaffected couples) Constitutional chromosome aberration Control group Total Male partner Female partner Matched couples Unaffected couples affected affected No. of couples 16 8 8 16 322 Mean age of the female 34 4.6 33.5 6.2 34.2 3.4 33 4 34.2 4.3 No. of cycles 30 20 10 30 394 Fertilization rate 93/252 a 61/156 a 32/96 a 125/251 a 2298/4016 a (36.9) (39.1) (33.3) (49.8) (57.2) No. of transfers 28 19 9 28 387 No. embryos transferred/cycle 76/28 50/19 26/9 74/28 113/387 2.7 2.6 2.9 2.77 2.9 Implantation rate per 4/76 c 1/50 b 3/26 c 11/74 b,c 118/387 embryo transferred (5.3) (2) (7.7) (14.9) (10.6) Clinical pregnancy rate 4/28 1/19 d 3/9 e 8/28 d,e 101/387 per transfer (14.3) (5.3) (33.3) (28.6) (26.1) Abortions 2 0 2 1 19 Ongoing pregnancy rate (%) 7.1 5.3 11.1 25 21 a P 0.01 for Fertilization rate. b P 0.05; c n.s. for Implantation rate per embryo transferred. d P 0.05; e n.s. for Clinical pregnancy rate per transfer. Values in parentheses are percentages. 2637

M.Montag et al. Table V. Results of intracytoplasmic sperm injection in patients with secondary chromosome aberration compared to the remaining unaffected couples Secondary chromosome aberration Total affected Female affected Male affected Male and female Control unaffected affected couples No. of couples 96 59 22 15 322 Mean age of the female 33.3 4.1 32.6 4.6 34.9 3.2 34 3.5 34.2 4.3 No. of cycles 166 96 48 22 394 Fertilization rate 627/1719 375/1037 135/379 117/303 2298/4016 (36.5) b (36.2) ab (35.6) ab (38.6) ab (57.2) b No. of transfers 152 93 39 20 387 (91.6) d (96.9) cd (81.3) cde (90.9) de (98.2) d No. embryos transferred 391/152 242/93 95/39 54/20 1113/387 per transfer cycle 2.6 2.6 2.4 2.7 2.9 Implantation rate 48/391 33/242 9/95 6/54 118/387 per embryo transferred (12.3) (13.6) (9.5) (11.1) (10.6) Clinical pregnancy rate 37/152 26/939 6/39 5/20 101/387 per transfer (24.3) (27.9) (15.4) (25) (26.1) Abortions 8/37 5/26 1/6 2/5 19/101 (21.6) (19.2) (16.7) (40) (18.8) Ongoing pregnancy rate (%) 19.1 22.6 12.8 15 21 a n.s.; b P 0.05 for fertilization rate. c,d P 0.05; e n.s. for no. of transfers. Values in parentheses are percentages. whereas the male affected group showed a significantly lower pregnancy rate (5.3%) (P 0.05). The correlation of the type of constitutional aberration and the ICSI results is also shown in Table II. We have observed one pregnancy in the male affected group and three in the female affected group. For the latter, one pregnancy occurred in a case of Robertsonian translocation and two in females with reciprocal translocations; these fetuses were both aborted. ICSI results in patients with secondary chromosome aberrations We then examined the effect of SCA on ICSI. The 96 couples with this type of aberration underwent 166 ICSI cycles (Table V). In the majority of ICSI treatment cycles, chromosomal affection originated from the female partner (96 cycles versus 48 cycles). Fertilization rates were between 35.6 and 38.6% and not significantly different between couples where the male, the female or both partners were affected. However, when compared to the remaining, unaffected patients, all subgroups showed significantly lower fertilization rates (P 0.05). The same was noted for the transfer rates, which were even significantly different between the subgroups of male and female affected couples. Implantation rates and pregnancy rates were not significantly different within affected groups or when compared to the group of unaffected couples. A high abortion rate was noted when both partners were affected (40%). Overall, most abortions (six out of eight) occurred in couples where autosomal aberrations were found (data not shown). In the unaffected couples, the abortion rate was 18.8%. Discussion This paper reports the results obtained by ICSI in couples where both male and female partners received a routine 2638 cytogenetic examination prior to ICSI treatment. Out of 590 ICSI treatment cycles which we performed, 196 (33.2%) involved patients with either constitutional or secondary chromosome abnormalities. Abnormalities were found in male and female partners of couples treated for male infertility. Reduced fertilization rates in affected patients We observed reduced fertilization rates in all couples with chromosome aberrations. It is tempting to speculate that this might be an indicator for a developmental mechanism leading to selection at the earliest stage of gamete interaction following ICSI. Several authors have reported on the occurrence of chromosomal disorders in seemingly unfertilized or failedfertilized oocytes (Pellestor, 1991; Asch et al., 1995; Wall et al., 1996). These anomalies correlate with maternal age (Angell et al., 1993; Munné et al., 1995) or ovarian stimulation therapy (Wramsby et al., 1987), and were shown to cause reduced fertilization rates (Plachot and Crozet, 1992). It is uncertain whether this applies to our female affected groups, as the mean maternal age for women with SCA was on average 32.6 4.6 years and for women with constitutional aberrations 34.2 3.4 years. The mean age of the female in the unaffected control groups (34 4.3 years) was within the same age range and the protocols for ovarian stimulation were identical for all patients. Therefore, the reduced fertilization rate may possibly be caused by the presence of chromosome aberrations. This hypothesis holds true for male constitutional aberrations, which showed a significantly lower fertilization rate when compared to the female affected groups. It is well known and widely accepted that fertilization failures can also be due to a reduced fertilization potential of the spermatozoa. This can be caused either directly by constitutional chromosome aberrations (Estop et al., 1995) or by failures during spermatogenesis, for example, defects of the sperm centrosome (Van Blerkom, 1996), sperm nuclear chromatin packaging (Bianchi et al.,

ICSI in couples with chromosome aberrations 1996) or genomic imprinting abnormalities (Tesarik and Mendoza, 1996). Both autosomal and sex chromosomal genes appear important in the evolving genetic understanding of spermatogenesis (Tiepolo and Zuffardi, 1976; Saxena et al., 1996). The role of secondary chromosome aberrations To date, the significance of SCA has not been assessed in detail, although Toncheva et al. (1994) reported that the presence of sex chromosomal aberrations at low frequency might be an underestimated cause for failure in assisted reproduction. However, it needs to be clarified whether the incidence of SCA (sex chromosomal and autosomal) is increased in ICSI couples. Our results, and those of others, indicate that the presence of SCA in lymphocyte cultures is highly unlikely to be an artificial result attributed to cellular stimulation and culture or preparation of chromosomes for karyotyping (Iskra, 1985). We cannot precisely state at present whether SCA is an indicator of low-frequency mosaicism or whether it simply reflects a greater chromosomal instability within affected individuals. To what extent SCA might cause a reduced fertilization rate is still unclear. It is possible that SCA also occur in cells in the germ line and therefore might have an impact on germ cell development and subsequent fertilization ability. Therefore, it would be interesting to analyse the chromosomal constitution of germ cells derived from male and female patients with documented SCA and to compare the results to those reported in the literature for normal individuals (Brandriff et al., 1990; Martin et al., 1992; Lee et al., 1996). New techniques for the analysis of aneuploidy in spermatozoa, for example, multicolour FISH, are promising and allow the discrimination of diploid, disomic or haploid spermatozoa (Rademaker et al., 1997). Pregnancy rates in affected couples In our study, couples with male constitutional aberrations have an especially poor prognosis to achieve an ongoing pregnancy by ICSI. At first sight, our results are in contradiction to those recently reported by Testart et al. (1996). These authors did not find a difference between pregnancy rates in couples with male and/or female structural aberrations and a control group. However, both study groups differ in their composition, especially in regard to the type of male chromosome aberration. In our study group, we report five cases with male reciprocal translocations (versus two cases in Testart s group) resulting in one pregnancy following 12 ICSI attempts. With regard to Robertsonian translocations, Testart and colleagues identified only affected males (nine ICSI attempts, three clinical pregnancies), whereas we found only one male and two females affected, and for the females one pregnancy occurred in three ICSI attempts. Experimental data are available which suggest a selection against unbalanced spermatozoa during spermatogenesis in males carrying Robertsonian translocations (Pellestor et al., 1987). This may explain the high pregnancy rates reported by Testart et al. for this type of translocation. However, this indicates the need for additional studies to establish the relationship between the type of constitutional chromosome aberration and the results after ICSI. Abortion rates in affected couples We noted high abortion rates when female patients were affected by constitutional chromosome aberrations, mainly reciprocal translocations, or when both partners were affected by SCA. Data are insufficient to draw conclusions regarding the risk of spontaneous abortion in individual subgroups. However, most abortions were found in couples with autosomal (structural) aberrations and these types of aberrations are probably less viable than sex chromosomal ones. Unfortunately, it was not possible to perform cytogenetic analysis of aborted tissue. So far, we have been unable to perform a cytogenetic analysis of the children born from SCA couples in order to evaluate whether the same type of SCA detected in the male or female could be transmitted to the offspring. Based on the results of this study, it may be useful to perform pre-implantation genetic diagnosis in patients with constitutional chromosome aberrations which showed low implantation rates and high abortion rates. Our data clearly support the argument for strict cytogenetic screening of male and female ICSI patients prior to treatment. This may allow better counselling, especially of patients with a chromosome aberration who are at high risk of miscarriage. Acknowledgements The authors would like to thank Ulla Dieckmann and Dr Ricarda Lange for their help in the data acquisition process and the staff of the IVF Unit and the Institute of Human Genetics for their assistance. 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