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
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Abstract |
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Key words: assisted reproduction/ICSI outcome/infertility/sex chromosome mosaicism
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Introduction |
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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.1999; Tinkanen et al.1999
). The present study analyses the biological relevance of low-level sex chromosome mosaicism in the female partner for the course and outcome of ICSI.
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Materials and methods |
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Cytogenetic methods
Chromosome analysis was carried out on cultured peripheral lymphocytes using standard techniques (Benn and Perle, 1992; Gosden et al.1992
). 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 550700 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.1998
). 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.1993). The presence of two pronuclei, indicating fertilization, was checked 1618 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., 1992
). 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 MannWhitney rank sum test respectively. Differences in cycle cancellation rates, fertilization rates, implantation rates, clinical pregnancy rates and abortion rates were tested by 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.
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Results |
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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 II). 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 II
]. 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 III
). 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 III
). 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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Discussion |
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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.1996). 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., 1995
; Bonduelle et al.1998
). Beyond this aspect of chromosomal analysis, prior to ICSI to estimate genetic risks for the offspring (Johnson, 1998
), 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.1998; Peschka et al.1999
). 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.1998
).
At present, the main predictors of ovarian response to gonadotrophin stimulation are female age and basal early follicular phase FSH (Bassil et al.1999; Tinkanen et al.1999
), 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.1996; Yoshida et al.1997
). 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., 1987
; Angell, 1989
). 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.1997
).
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, 1990), 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.
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Acknowledgements |
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Notes |
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References |
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Submitted on February 22, 2001; accepted on April 26, 2001.