Low-level sex chromosome mosaicism in female partners of couples undergoing ICSI therapy does not significantly affect treatment outcome

B. Sonntag1, D. Meschede2, V. Ullmann1, P. Gassner3, J. Horst2, E. Nieschlag3 and H.M. Behre1,4

1 Assisted Reproduction Unit of the Department of Obstetrics and Gynaecology and the 3 Institute of Reproductive Medicine of the University, and 2 Institute of Human Genetics of the University, Münster, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: There is an increased rate of chromosomal anomalies, in particular low-level sex chromosome mosaicism, in the female partners of couples undergoing intracytoplasmic sperm injection (ICSI). METHODS: Among 811 consecutive couples presenting for pre-ICSI chromosome analysis, chromosomal abnormalities were detected in 54 individuals, of which 26 were low-level sex chromosome mosaicism in the females. Attention was focused on the treatment course and outcome of ICSI in 20 couples with low-level sex chromosome mosaicism in the females actually embarking on ICSI treatment (group I, n = 38 ICSI treatment cycles). Applying a case–control design, each of the 20 couples was matched according to female age and source of spermatozoa to couples without a chromosomal abnormality in either of the partners (group II, n = 38 ICSI treatment cycles). RESULTS: No significant differences were found between the groups in ovarian response, fertilization rate and number of embryos transferred. Pregnancy rates, as well as implantation and abortion rates did not differ significantly between the groups. CONCLUSIONS: The data suggest that low-level sex chromosome mosaicism in females has no major effect on the course and outcome of ICSI.

Key words: assisted reproduction/ICSI outcome/infertility/sex chromosome mosaicism


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In infertile couples the frequency of chromosome abnormalities is increased compared with the population baseline. The association between male factor infertility due to azoospermia or severe oligozoospermia and abnormal karyotypes is well documented (Bourrouillou et al., 1985Go; De Braekeleer and Dao, 1991Go; Chandley, 1998Go; Colombero et al.1999Go; Kleiman et al.1999Go). A high frequency of chromosomal abnormalities in the male partners of couples undergoing intracytoplasmic sperm injection (ICSI) is therefore to be expected. Additionally, recent studies have shown an unexpectedly high prevalence of chromosomal aberrations in the female partners (Meschede et al.1998Go; Scholtes et al.1998Go; Peschka et al.1999Go; Gekas et al., 2001Go). This might contribute to the fertility problem even in couples with a predominant male infertility factor, as is usually the case in ICSI patients.

Prior to ICSI treatment it is essential to have information about prognostic factors for therapy outcome, not only to individualize the stimulation protocol and thereby to achieve optimal ovarian response, but also to give adequate counselling to the patients about their chances for pregnancy. Factors such as female age, basal FSH and duration of infertility are thought to be prognostic for the outcome of assisted reproductive technologies (Bassil et al.1999Go; Tinkanen et al.1999Go). The present study analyses the biological relevance of low-level sex chromosome mosaicism in the female partner for the course and outcome of ICSI.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subjects and study design
The study groups were recruited from a cohort of 811 infertile couples consecutively enrolled for genetic counselling and chromosome analysis prior to planned ICSI therapy at our institution. A chromosomal abnormality was diagnosed in 54 patients out of 811 couples [6.66% per couple, 3.33% per individual; data partly published in (Meschede et al.1998Go)] of whom 26 were females with low-level sex chromosome mosaicism. Those couples with low-level sex chromosome mosaicism in the female partner and starting ICSI treatment constitute group I of the study (n = 20). To obtain a cohort of suitable controls (group II), each of the 20 couples was matched according to female age (<30, 30–35, >35 years) and source of spermatozoa (either ejaculate or testicular biopsy, epididymal aspiration or cryopreserved spermatozoa) with a couple without any chromosomal abnormality in either of the partners. For randomization purposes, a chronological patient list was used. Each couple from group I was assigned a control by picking the next chromosomally normal couple on this list fulfilling the matching criteria.

Cytogenetic methods
Chromosome analysis was carried out on cultured peripheral lymphocytes using standard techniques (Benn and Perle, 1992Go; Gosden et al.1992Go). At least 11 cells were karyotyped, and in cases of suspected mosaicism the number of analysed metaphases was increased to a total of 150. A resolution of 400 bands per haploid genome was achieved as a minimum, and often a more detailed structural analysis at the 550–700 band level could be performed. Routine analysis was performed on G-banded metaphases. Details of the cytogenetic findings in a major part of the cohort study have been reported previously (Meschede et al.1998Go). Aberrant single cell findings were disregarded and not considered as indicators of chromosomal mosaicism. Only if the same type of chromosomal abnormality was observed in at least two cells in the first 15 metaphases analysed, was it classified as mosaicism. As a 45,X cell line is often associated with single cell findings for other sex chromosomal abnormalities (e.g. 47,XXX), these latter are also reported. The term `low-level sex chromosome mosaicism' is only loosely defined, and we follow the most widely employed definition: a state of chromosomal mosaicism where the aberrant cell line constitutes 10% or less of all analysed metaphases.

Treatment protocol
Ovarian stimulation was performed according to standard protocols using gonadotrophin preparations in combination with gonadotrophin-releasing hormone (GnRH) analogues for the prevention of premature luteinization or ovulation. Follicular development was monitored by vaginal ultrasonography and serum oestradiol measurements. For ovulation induction, 10 000 IU human chorionic gonadotrophin (HCG) were given; oocyte retrieval was performed 36 h after HCG injection by transvaginal, ultrasound-guided aspiration. ICSI was carried out according to a published standard protocol (Van Steirteghem et al.1993Go). The presence of two pronuclei, indicating fertilization, was checked 16–18 h after ICSI. At 2 days after follicular aspiration, up to three embryos were transferred to the uterine cavity. Embryo quality was categorized according to the cumulative embryo scoring system (Steers et al., 1992Go). Pregnancies were detected 12 days after embryo transfer by serum HCG measurement (biochemical pregnancy) and confirmed as clinical pregnancy by ultrasound detection of embryonic heart beat.

Statistical analysis
Differences between study groups were analysed using either a t-test or the Mann–Whitney rank sum test respectively. Differences in cycle cancellation rates, fertilization rates, implantation rates, clinical pregnancy rates and abortion rates were tested by {chi}2-test. Statistical analysis was performed using the statistical software package Sigmastat 2.03 (Jandel Scientific, Erkrath, Germany). In general, results are presented as mean ± SEM. A P-value < 0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chromosomal abnormalities
The frequency of low-level sex chromosome mosaicism among the 811 female individuals presenting for chromosome analysis prior to planned ICSI therapy was 3.2%. The karyotypes of all female patients with low-level sex chromosome mosaicism who were starting ICSI therapy (group I) are given in Table IGo.


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Table I. Results of chromosomal analysis in group I
 
Reproductive history and disorders
The indication for ICSI therapy was andrological in all couples. Additional female factor indications (tubal factor, endometriosis, hypergonadotrophic state) were present in six patients of each group (30.0%). All patients presented with primary infertility at the time of the first treatment cycle except for four in group I (20.0%) and five in group II (25.0%, P = NS). There was no history of recurrent abortions in any patient. The duration of infertility before the first treatment cycle was 5.43 ± 0.73 (group I) versus 4.95 ± 0.60 years (group II, P = NS). Patients did not differ in terms of female age at the beginning of ICSI treatment (group I, 33.6 ± 0.95 versus group II, 33.3 ± 0.80 years, P = NS) and source of spermatozoa (epididymal aspiration/testicular biopsy or cryopreserved spermatozoa in two patients of each group). Basal FSH concentrations in the females did not differ between groups (group I, 8.84 ± 0.82 versus group II, 8.31 ± 1.08 IU/l, P = NS).

Treatment cycles
Thirty-eight ICSI treatment cycles were performed in each group (1.9 cycles per couple). Four cycles were cancelled before oocyte retrieval in group I (10.5%), compared with one cycle in group II (2.6%, P = NS). Reasons for cancellation were inadequate ovarian response in one case of group I and one case of group II, and in three other patients of group I reasons for cancellation were unrelated to stimulation therapy.

Measures of ovarian response and outcome
Follicular development did not differ between groups, as judged by the number of follicles >17 mm in diameter and by oestradiol serum concentrations on the day of ovulation induction (Table IIGo). The number of days with gonadotrophin administration and total gonadotrophin dose were not different. The number of oocytes retrieved was similar in all groups, with an equal number of mature oocytes [metaphase II (MII) oocytes; see Table IIGo]. Fertilization rates (two pronuclei after injection of an MII oocyte) did not differ between groups, and a comparable number of embryos could be transferred, showing no significant differences in the cumulative embryo score (Table IIIGo). In four cycles of group I (11.8%) and two cycles of group II (5.4%, P = NS) respectively, embryo transfer was not performed because of fertilization failure. Treatment resulted in comparable implantation, clinical pregnancy and abortion rates (see Table IIIGo). In group I, seven children were born (one twin pregnancy); while in group II, 12 children were born (four twin pregnancies).


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Table II. Measures of ovarian response
 

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Table III. Treatment outcome
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The prevalence of sex chromosome aberrations (SCA) in female partners of couples undergoing ICSI therapy is elevated compared with the rate of chromosome abnormalities found in newborn screenings (0.2% for sex chromosome abnormalities and 0.8% for all chromosome abnormalities; Nielsen and Wohlert, 1991). The major part of these SCAs represent low-level mosaics. To date, only scant information exists concerning the biological significance of these findings. The prevalence of low-level sex chromosome mosaicism in blood lymphocytes increases with age (Guttenbach et al.1995Go), and is believed to be clinically insignificant if <10% of the analysed cell lines are involved (Gardner and Sutherland, 1996Go). Karyotyping of couples of reproductive age with successful spontaneous conceptions shows a rate of female low-level sex chromosome mosaicism comparable with that in female patients undergoing ICSI therapy (Schubert et al., 1999Go), but this preliminary result remains to be confirmed. The frequency of sex chromosome mosaicism is elevated in women with recurrent spontaneous abortions (Holzgreve et al.1984Go). Data on the frequency of these aberrations in germ cells do not exist to date. The suspicion of preparational artefacts leading to the high incidence of low-level sex chromosome mosaicism would not explain the differences between the male and the female chromosomal anomaly rate.

At present, the main concern about chromosomal anomalies in ICSI patients relates to possible adverse effects on the pregnancies and children resulting from ICSI therapy (Engel et al.1996Go). An increased rate of sex chromosome anomalies was reported from two series in which women pregnant through ICSI underwent prenatal karyotyping (In't Veld et al., 1995Go; Bonduelle et al.1998Go). Beyond this aspect of chromosomal analysis, prior to ICSI to estimate genetic risks for the offspring (Johnson, 1998Go), there is the question of whether abnormal chromosomal findings such as low-level sex chromosome mosaicism might influence the course and outcome of therapy itself.

As yet, the limited data available on the biological relevance of aberrant parental karyotypes for the course of ICSI therapy are conflicting (Scholtes et al.1998Go; Peschka et al.1999Go). As ovarian response to gonadotrophin stimulation is crucial to successful therapy, knowledge of any factors predicting poor response is invaluable prior to treatment, not only for estimating success for the individual couple, but also to modify treatment, for example gonadotrophin dosage, in advance and thereby to optimize outcome. The influence of gonosomal abnormalities such as low-level sex chromosome mosaicism in the female partners on this decisive part of treatment has not yet been investigated. Some sex chromosome aberrations, as found for example in Turner syndrome, are known to compromise female reproductive function severely, leading to a state of hypergonadotrophic hypogonadism. A higher incidence of low-level sex chromosome mosaicism in patients with idiopathic premature ovarian failure compared with age-matched controls has been previously described (Devi et al.1998Go).

At present, the main predictors of ovarian response to gonadotrophin stimulation are female age and basal early follicular phase FSH (Bassil et al.1999Go; Tinkanen et al.1999Go), and both parameters were not significantly different between the current study groups. As the groups did not differ with regard to the time of stimulation and FSH dosage, and achieved the same number of follicles and serum oestradiol concentrations on the day of ovulation induction, as well as the same number of oocytes and mature oocytes at oocyte retrieval, low-level sex chromosome mosaicism does not appear to influence ovarian response. There was a higher rate of cycle cancellation in group I, not reaching statistical significance, with four cycles cancelled compared with only one cycle cancelled in group II. Considering the raw numbers, this might indicate an underlying difference in follicular response, though this was not statistically significant. However, the reason for cycle cancellation was inadequate ovarian response in only one patient of each group, while all other reasons were independent of stimulation therapy.

The overall fertilization rate was not different between the groups, and similar to that in patients with structural abnormalities in whom normal fertilization is achieved (Testart et al.1996Go; Yoshida et al.1997Go). Studies in preimplantation embryos with monosomic or trisomic karyotypes indicated that even severe aneuploidies of oocytes or spermatozoa do not prevent fertilization and zygote formation (Plachot et al., 1987Go; Angell, 1989Go). This is in contrast to the data of a previous study showing a significant difference in fertilization rate between patients with either constitutional chromosomal aberrations or single-cell aberrations and a control group (Montag et al.1997Go).

It is of major importance to estimate the degree to which low-level sex chromosome aberrations affect early embryo development. In the current study, about the same number of embryos of comparable quality were obtained at the day of embryo transfer as far as can be determined by a commonly used embryo scoring system. Whereas (independent of the use of assisted reproductive techniques) structural chromosomal abnormalities in one of the partners are known to be associated with a higher rate of spontaneous abortion (De Braekeleer and Dao, 1990Go), implantation, pregnancy and abortion rates were equal between the groups in the current study.

In conclusion, the higher prevalence of low-level sex chromosome mosaicism detected in the female partners of couples before ICSI therapy in the current study does not influence the course and outcome of ICSI treatment compared with normal controls. In contrast to structural autosomal aberrations, leading to a lower implantation rate and more abortions, parental low-level sex chromosome mosaicism in the female partner does not seem to impede embryo development. This information may be relevant for pretherapeutic genetic counselling of patients with chromosomal abnormalities.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank Susan Nieschlag for language editing of the manuscript.


    Notes
 
4 To whom correspondence should be addressed at: Andrology Unit, Martin-Luther-University, Magdeburger Str. 16, D-06097 Halle, Germany. E-mail: Hermann.Behre{at}medizin.uni-halle.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Angell, R.R. (1989) Chromosome abnormalities in human preimplantation embryos. Prog. Clin. Biol. Res., 294, 181–187.[Medline]

Bassil, S., Godin, P.A. and Gillerot, S. (1999) In vitro fertilization outcome according to age and follicle-stimulating hormone levels on cycle day 3. J. Assist. Reprod. Genet., 16, 236–241.[ISI][Medline]

Benn, P.A. and Perle, M.A. (1992) Chromosome staining and banding techniques. In Rooney, D.E. and Czepulkowski, B.H. (eds), Human Cytogenetics. A Practical Approach. Vol. I: Constitutional Analysis. IRL Press, Oxford, pp. 91–118.

Bonduelle, M., Aytoz, A., van Assche, E. et al. (1998) Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection. Hum. Reprod., 13, 781–782.[Free Full Text]

Bourrouillou, G., Mansat, A., Calvas, P. et al. (1985) Chromosome studies in 952 infertile males with a sperm count below 10 million/ml. Hum. Genet., 71, 366–367.[ISI][Medline]

Chandley, A.C. (1998) Chromosome anomalies and Y chromosome microdeletions as causal factors in male infertility. Hum. Reprod., 13 (Suppl. 1), 45–50.[Medline]

Colombero, L.T., Hariprashad, J.J., Tsai, M.C. et al. (1999) Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome. Fertil. Steril., 72, 90–96.[ISI][Medline]

De Braekeleer, M. and Dao, T.N. (1990) Cytogenetic studies in couples experiencing repeated pregnancy losses. Hum. Reprod., 5, 519–528.[Abstract]

De Braekeleer, M. and Dao, T.N. (1991) Cytogenetic studies in male infertility: a review. Hum. Reprod., 6, 245–250.[ISI][Medline]

Devi, A.S., Metzger, D.A., Luciano, A.A. et al. (1998) 45,X/46,XX mosaicism in patients with idiopathic premature ovarian failure. Fertil. Steril., 70, 89–93.[ISI][Medline]

Engel, W., Murphy, D. and Schmid, M. (1996) Are there genetic risks associated with microassisted reproduction? Hum. Reprod., 11, 2359–2370.[Abstract]

Gardner, R.J.M. and Sutherland, G.R. (1996) Chromosome Abnormalities and Genetic Counselling. Oxford University Press, New York.

Gekas, J., Thepot, F., Turleau, C. et al. (2001) Chromosomal factors of infertility in candidate couples for ICSI: an equal risk of constitutional aberrations in women and men. Hum. Reprod., 16, 82–90.[Abstract/Free Full Text]

Gosden, C.M., Davidson, C. and Robertson, M. (1992) Lymphocyte culture. In Rooney, D.E. and Czepulkowski, B.H. (eds), Human Cytogenetics. A Practical Approach. Vol. I: Constitutional Analysis. IRL Press, Oxford, pp. 31–54.

Guttenbach, M., Koschorz, B., Bernthaler, U. et al. (1995) Sex chromosome loss and aging: in situ hybridization studies on human interphase nuclei. Am. J. Hum. Genet., 57, 1143–1150.[Medline]

Holzgreve, W., Schonberg, S.A., Douglas, R.G. et al. (1984) X-chromosome hyperploidy in couples with multiple spontaneous abortions. Obstet. Gynecol., 63, 237–240.[Abstract]

In't Veld, P., Brandenburg, H., Verhoeff, A. et al. (1995) Sex chromosomal anomalies and intracytoplasmic sperm injection. Lancet, 346, 773.[ISI][Medline]

Johnson, M.D. (1998) Genetic risks of intracytoplasmic sperm injection in the treatment of male infertility: recommendations for genetic counseling and screening. Fertil. Steril., 70, 397–411.[ISI][Medline]

Kleiman, S.E., Yogev, L, Gamzu, R. et al. (1999) Genetic evaluation of infertile men. Hum. Reprod., 14, 33–38.[Abstract/Free Full Text]

Meschede, D., Lemcke, B., Exeler, J.R. et al. (1998) Chromosome abnormalities in 447 couples undergoing intracytoplasmic sperm injection – prevalence, types, sex distribution and reproductive relevance. Hum. Reprod., 13, 576–582.[Abstract]

Montag, M., van der Veen, K., Ved, S. et al. (1997) Success of intracytoplasmic sperm injection in couples with male and/or female chromosome aberrations. Hum. Reprod., 12, 2635–2640.[Abstract]

Nielsen, J. and Wohlert, M. (1991) Chromosome abnormalities found among 34,910 newborn children: results from a 13-year incidence study in Arhus, Denmark. Hum. Genet., 87, 81–83.[ISI][Medline]

Peschka, P., Leygraaf, J., van der Ven, K. et al. (1999) Type and frequency of chromosome aberrations in 781 couples undergoing intracytoplasmic sperm injection. Hum. Reprod., 14, 2257–2263.[Abstract/Free Full Text]

Plachot, M., Junca, A.M., Mandelbaum, J. et al. (1987) Chromosome investigations in early life. II. Human preimplantation embryos. Hum. Reprod., 2, 29–35.[Abstract]

Scholtes, M.C.W., Behrend, C., Dietzel-Dahmen, J. et al. (1998) Chromosomal aberrations in couples undergoing intracytoplasmic sperm injection: influence on implantation and ongoing pregnancy rates. Fertil. Steril., 70, 933–937.[ISI][Medline]

Schubert, R., Peschka, B., Schartmann, B. et al. (1999) Vergleich von Mosaiken der Geschlechtschromosomen bei Paaren, die sich einer ICSI-Therapie unterziehen, und einem Kontrollkollektiv (Abstract Volume). Jahrestagung der Deutschen Gesellschaft zum Studium der Fertilität und Sterilität, 19–21 August 1999, Bonn, Germany.

Steers, C.V., Mills, C.L., Tan, S.L. et al. (1992) The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in-vitro fertilization and embryo transfer programme. Hum. Reprod., 7, 117–119.[Abstract]

Testart, J., Gautier, E., Brami, C. et al. (1996) Intracytoplasmic sperm injection in infertile patients with structural chromosome abnormalities. Hum. Reprod., 11, 2609–2612.[Abstract]

Tinkanen, H., Blauer, M., Laippala, P. et al. (1999) Prognostic factors in controlled ovarian hyperstimulation. Fertil. Steril., 72, 932–936.[ISI][Medline]

Van Steirteghem, A.C., Nagy, Z.P., Joris, H. et al. (1993) High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 8, 1061–1066.[Abstract]

Yoshida, A., Araki, Y., Motoyama, M. et al. (1997) Structural abnormalities of autosomal chromosomes in the male partner do not influence fertilization and early development of embryos after intracytoplasmic sperm injection. Hum. Reprod., 12, 2499–2503.[Abstract]

Submitted on February 22, 2001; accepted on April 26, 2001.