1 Department of Human Genetics, 2 Department of Endocrinology and Reproductive Medicine, University of Bonn, 53111 Bonn, Germany
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Abstract |
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Key words: chromosome aberrations/fragile sites/intracytoplasmic sperm injection/non-disjunction of gonosomes
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Introduction |
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Nevertheless, any incidence of chromosomal abnormality in the female partner could influence the outcome of ICSI, and investigation of the female partners of ICSI patients has already been recommended (Meschede et al., 1995; Mau et al., 1997
). 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
). Whereas, in our investigation group, the rate of abnormalities in the male was within the expected range for men with impaired semen parameters, the rate for the females was unexpectedly high. These data indicate that the potential contribution of maternal chromosome aberrations in cases of poor reproductive outcome cannot be neglected.
The aim of this study was to evaluate the type and frequency of chromosomal aberrations in couples before ICSI treatment as well as cytogenetic peculiarities whose genetic importance has not yet been completely clarified.
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Materials and methods |
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Chromosome investigations were carried out from peripheral blood lymphocytes using standard techniques (Benn et al., 1992; Gosden et al., 1992
). Blood cells were cultivated for 72 h with two different media (Roswell Park Memorial Institute medium 1640, SF-3). The routine analysis was performed on Q-banded chromosome preparations. In single cases specific banding techniques as RBA, CBG, DA/DAPI and NOR were additionally applied. In the majority of cases only one cell system was analysed (lymphocytes), but in single patients with chromosome mosaicism a second cell system (skin or testis biopsy) was investigated. If possible, 20 metaphases (450550 bands per genome) were analysed. If a structural or numerical aberration was found in only one cell, it was disregarded as a single cell aberration. If the cell was trisomic for the chromosomes 8, 9, 13, 18 and 21 we analysed at least 30 metaphases respectively, and in cases with gain or loss of one sex chromosome and suspected sex chromosome mosaicisms, 50100 metaphases were examined. In complex structural rearrangements, additional molecularcytogenetic analyses by fluorescence in-situ hybridization (FISH) were performed according to a previously published method (Lichter et al., 1988
).
Chromosome aberrations observed in our study sample were subdivided into four groups: (1) constitutional aberrations (sex chromosomal aberrations with hyperploidies of X or Y chromosomes, sex chromosome mosaicisms, derivative X or Y chromosomes and autosomal aberrations with translocations, inversions, marker chromosomes, autosomal mosaicisms and other structural aberrations); (2) fragile sites of autosomes; (3) low level mosaicism of sex chromosomes; (4) secondary chromosome aberrations (gaps, breaks, exchanges).
Sex chromosomal mosaicism was defined as the occurrence of two or more different cell lines, with the additional cell lines showing a frequency of at least 6%. If occurring at lower percentages, they were classified as low level mosaicism of sex chromosomes.
Secondary chromosome aberrations were registered at 10% per sample. At a frequency of 15% per sample we asked the patient for exogenous loads (e.g. X-rays) and a second chromosome analysis was recommended after 3 months.
Patients with aberrations of groups 1, 3 and 4 underwent genetic counselling to explain the significance of the findings for themselves and for their offspring. Family investigations combined with analyses of the pedigrees were performed in cases of structural aberrations.
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Results |
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The observed aberrations comprised 35 cases with sex chromosomal and 34 cases with autosomal aberrations. The sex chromosomal aberrations included four patients with complete aneuploidies (one case of triple-X and three cases of Klinefelter syndrome). Two males had a sex chromosome mosaicism with an aberrant main cell line (47,XXY and 47,XYY). Of the other patients with mosaicism, two males and 23 females showed aberrant cell lines with frequencies between 6 and 10.7%. In an additional two cases we found a derivative Y chromosome. In one of them the proband had an isodicentric Y chromosome with two short arms and a deletion of the major part of the long arm including the testis determination factor region, which was proved by microsatellite analysis. In the second case the proband had a derivative Y chromosome with a duplication of the segment q11.2. In two mosaic cases we found in addition to the normal cell line a second one with a derivative sex chromosome [46,XY/46,der(X),Y and 46,XX/46,X,der(X)].
In 34 patients autosomal aberrations were identified. The types of aberrations were reciprocal translocations, Robertsonian translocations, inversions (pericentric and paracentric), marker chromosomes, autosomal mosaicisms and other structural aberrations.
Translocations exhibited the highest frequency of autosomal aberrations with 55.9% (19/34), 11 men and nine women. Within the group of translocations we identified five Robertsonian translocations. In four cases chromosomes 13 and 14 were involved, in one case chromosomes 14 and 15. The remaining 14 rearrangements were reciprocal translocations and the chromosomes 1, 2, 3, 4, 5, 7, 8, 9, 12, X, 15, 17, 18, 19 and 21 were involved. In 10 out of 19 cases with translocations, family investigations could be performed and showed that the majority of aberrations (8/10) was of familial origin. In five cases, the aberration was paternally, in three cases maternally, inherited. Two reciprocal translocations occurred de novo. In one of these cases we diagnosed a complex chromosome rearrangement with three chromosomes involved. In nine further cases, family investigations have not yet been completed or the relatives of the proband could not be investigated.
In six cases, we diagnosed inversions, three females and three males; three of them were pericentric and three paracentric. Of the inversions three were familial, one of them being maternally inherited, two paternally. One male patient showed an additional marker chromosome in all metaphases examined. It was delineated as an isodicentric derivative of chromosome 15. In one further case the proband showed a trisomy 18 mosaicism with a frequency of aneuploid mitoses of 4%.
In the group of other structural aberrations, we identified one case with a dicentric chromosome 21 and six cases with an additional band in chromosome 9, three of them in the short arm, three in the long arm. With molecularcytogenetic analyses (FISH) we determined the origin of the extra band as material from chromosome 9. In this group of other structural aberrations, family investigations were performed in two cases of aberrant chromosome 9: the alterations were both of paternal origin.
Fragile sites of autosomes
Fragile sites showed a frequency of 3.0%. Three autosomes, 6, 10 and 17, were involved. In one case (0.05%) the region 17p12 was affected in a woman, in another case a woman showed a fragile site in the region 10q24 (0.05%). In 46 cases (2.9%) a fragile site on the long arm of chromosome 6 (6q13) was found. The rate for men with the fragile site 6q13 was 1.5%, the rate for women was 1.4%.
Low level mosaicism of sex chromosomes
Low level mosaicism of sex chromosomes as identified in 62 cases (4.0%). Eleven men (0.7%) and 51 women (3.3%) were affected (Table I). One additional cell line was present in 51.6%, two additional cell lines in 48.4%. Table III
shows the different combinations of cell lines as well as their frequencies in males and females.
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Discussion |
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Previous studies have shown an increasing frequency of chromosomal abnormalities associated with declining sperm count (Chandley, 1979). The incidence of constitutional chromosomal aberrations among 1599 subfertile males was 2.2%, which rose to 15% among azoospermic males. In another study, more than 1444 infertile males were investigated and chromosome anomalies at a frequency of 2.9% were observed, which is increased ~10-fold compared to the average population (Bourrouillou et al., 1987
). A new study showed similar results: cytogenetic data for 447 couples referred for ICSI treatment had 2.1% (9/432) of the males with constitutional chromosomal aberrations (Meschede et al., 1998
).
Our findings of 3.8% (30/781) constitutional aberrations in the group of infertile males prior to ICSI treatment are in good agreement with these observations and underline the need for cytogenetic investigations in this population.
The high number of constitutional aberrations among the female partners of ICSI patients (5.0%, 39/781) was unexpected. This is remarkable, because several authors showed that chromosomal aberrations are far more common among infertile men than in women (Opitz et al., 1979; Healy et al., 1994
). However, a constitutional abnormality rate of 1.1% (3/261) for the female ICSI population has been reported (Testart et al., 1996
). Also, cytogenetic data in 150 couples referred for ICSI treatment has been published, where 2.6% (4/150) of the females carried constitutional chromosomal aberrations excluding low level sex chromosome mosaicism (Mau et al., 1997
). Recently a total of 24 abnormal karyotypes among 436 females studied (5.5%) has been found (Meschede et al., 1998
). This is comparable to the data obtained in our study.
The fact that 55.9% of all constitutional aberrations were contributed by the females in our ICSI cohort, which was primarily selected for male infertility, has several implications. The signs of a chromosomal imbalance may be less obvious in oocytes than in spermatozoa where aberrant morphology and reduced sperm number or motility are easy to assess. Our data underline the potential existence of an additional oocyte factor which might contribute to the fertility problems observed (van der Ven et al., 1998). As a consequence, infertility diagnosis and treatment in such cases may be determined by more obviously disturbed clinical parameters such as reduced sperm quality.
Different types of chromosomal abnormalities were present in the patients cohort.
Constitutional aberrations
Sex chromosome abnormalities are the most frequent chromosome-related cause of infertility. In our study we diagnosed sex chromosome anormalities in 50% (35/69) of the constitutional aberrations.
Only one proband showed a deletion of the major part of the long arm including the testis determination factor region. With ICSI chromosomal or gene defects which might normally be lost or eliminated by natural means could be transmitted in offspring. It is important to collect the cases with Y chromosome microdeletions find out whether ICSI may lead to a rise in the frequency of them in future generations (Chandley, 1998; Kremer et al., 1998
).
It is interesting that 25 cases of sex chromosome abnormalities showed a mosaicism for a numerical sex chromosome anomaly. There was a strong preponderance of females among the patients with this type of anomaly (23 females, two males). The overall incidence of X chromosome loss in females was two orders of magnitude higher than that determined for the Y chromosome. This difference could be due to a truly greater propensity of the X chromosome for mitotic errors (Guttenbach et al., 1995).
The significance of sex chromosome mosaicism for the individual genetic risk of offspring is not clear. Genetic counselling included the problem of possible numerical sex chromosome syndromes and clinical relevance.
Autosomal aberrations were identified in 34 cases. In our study sample, we found 14 translocation carriers (0.9%), which is 9-fold higher compared to the general population (0.1%) (Bourrouillou et al., 1985; Therman and Susman, 1993
). The individual risk for the offspring can be given more precisely in genetic counselling after extended family investigations and may thus enable the patients to decide about infertility treatment with ICSI, prenatal diagnostics by ultrasound and chromosome analysis and acceptance of remaining risk factors.
We observed Robertsonian translocations which are frequently diagnosed aberrations in all infertility investigations. We found two women and two men with 13q14q fusion, which is the most frequently found Robertsonian translocation and one woman with a 14q15q fusion. Thus, the frequency of Robertsonian translocations in our study was nearly 3-fold higher than in the general population (0.1%) (Therman and Susman, 1993). Others have shown a Robertsonian translocation rate of 0.3% (Bourrouillou et al., 1985
; Meschede et al., 1998
). The increased risk for spontaneous abortions and for viable offspring with aneuploidy is 12% or less (Gardner and Sutherland, 1996
). A small risk (<1%) exists for uniparental disomy (UPD) for chromosomes 14 and 15 in the offspring (Ledbetter and Engel, 1995
).
Inversions are less common constitutional aberrations. In our group, we diagnosed three pericentric and three paracentric inversions. According to large surveys, the risk for development of a zygote with unbalanced karyotype is usually low in those cases and might be reduced to microdeletions and duplications caused by unequal crossing over in meiosis I of the carrier (Gardner and Sutherland, 1996).
We found a marker chromosome in one male patient, which was a bisatellited marker of chromosome 15. An earlier study analysed the significance of accessory bisatellited marker chromosomes, and found that an extra structurally abnormal chromosome might lead to reduced fertility in males (Steinbach et al., 1983). A risk for carriers with supernumerary derivative 15 trisomies and UPD is described (Ledbetter and Engel, 1985).
In our study we found one phenotypically normal man with 4% trisomy 18 mosicism in lymphocytes. Some cases of trisomy 18 mosaicism with normal intelligence and non-specific dysmorphisms have been described (Beratis et al., 1982; Kohn and Shohat, 1987
; Gersdorf et al., 1990
). These cases were diagnosed either because they were parents of children with trisomy 18 or because of multiple miscarriages (Kohn and Shohat, 1987
; Gersdorf et al., 1990
).
In six cases, we identified an additional euchromatic band in chromosome 9p or 9q. This is the first description ever of this structural abnormality in cytogenetic investigations before ICSI. Only a few authors have described an additional band in 9p or 9q. Although it is suprising that the presence of a significant amount of euchromatic material should be without phenotypic effect, most of them seem to be without genetic relevance and were therefore classified as polymorphisms (Madan, 1978; Hoo et al., 1993
). Because of the high frequency of this structural abnomality in our group of infertility patients a connection with infertility cannot be excluded. However, further studies must still confirm this hypothesis.
Fragile site 6q13
This is the first description of an increased occurrence of a fragile site in 6q13 in the ICSI cohort.
The fragile site of chromosome 6 [fra(6) (q13)] was first described in 1985 (Sutherland, 1985). To date, this fragile site could not be associated with any pathological phenotype. In a prelimniary study of our own the frequency of the mentioned fragile site in our 96 ICSI patients was compared to the frequency in a control group of 94 patients at the same age and without chromosome aberrations. The fragile site on chromosome 6 [fra(6) (q13)] was nearly four times more frequent in the ICSI patients than in controls (46 out of 96:12 out of 94). This chromosomal aberration occurred with equal frequency in males and females. At present a possible correlation between the fragile site 6q13 and infertility is still unclear, but the increased frequency of this fragile site in our investigation group is notable. Further investigations are underway.
Low level mosaicism of sex chromosomes
We found low level sex chromosome mosaicism in 4.0% of patients (11 men and 51 women). The frequency of aberrant cell lines was between 4.0 and 5.7%. Recent reports demonstrate similar data in regard to mosaicism for numerical sex chromosome anomaly (Mau et al., 1997; Meschede et al., 1998
). The attempt to put these data into a proper perspective through comparison with adequate control groups has proven to be difficult. Thus, sex chromosomal aberrations have in the past been investigated in newborns (Jacobs and Hassold, 1987
; Niesen and Wohlert, 1991
), women participating in IVF programmes (Lange et al., 1993
) and/or women with recurrent abortions (Horsman et al., 1987
) as control groups. We have investigated a control group with more precisely defined criteria such as (i) spontaneous pregnancy within 2 years, (ii) age 2040 years for females and 2050 years for males and (iii) no abortions. As a result, a comparable rate of low level sex chromosome mosaicisms in the women is becoming evident. These preliminary data are confirmed by previous investigators (Guttenbach et al., 1995
), who found that in the age group between 31 and 40 years, which corresponds to average age in our ICSI cohort, the incidence of X chromosome loss in females amounts to 3.1%. Accordingly, in our ICSI cohort we found an almost identical percentage of 45,X mosaicism (3.5%). Both of these results are of the same order of magnitude and lead us to postulate that this low level mosaicism is typical for the average female population. If a sufficiently high number of cases support these data, they would influence the situation for genetic counselling in couples with sex chromosomal pecularities.
Single cell aberrations
Single cell aberrations reflect a chromosomal instability or a cultivation artefact. Other studies have found an increased amount of single cell translocations in couples with multiple spontaneous abortions, as well as other types of structural aberrations such as deletions and inversions (Higgins and Palmer, 1987). In a previous publication we presented the effect of single pathological cells on results of ICSI (Montag et al., 1997
). The fertilization rates and the transfer rates showed significantly lower outcomes, when compared to patients in whom no chromosomal abnormality had been detected. To what extent single cell aberrations might cause a reduced fertilization rate is still unclear. However, our results indicate that single cell aberrations analysed in lymphocyte cultures cannot be regarded as negligible artefacts occurring during one preparation of chromosomes for karyotyping. They might be a very mild form of a so far undetected mosaicism. As a consequence, one cannot rule out the possibility that germ cells are affected as well, and that they therefore might possess a reduced or impaired ferilization ability. Clearly, this type of aberration needs further inverstigation.
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Conclusion |
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In 69 out of 1562 patients (4.4%), constitutional chromosome aberrations could be identified. Unexpectedly, these chromosome aberrations occurred with similar frequency in males and females. As a consequence, chromosome analyses should be performed routinely in both the male and the female partner of couples planning an ICSI treatment which must be followed by appropriate genetic counselling and prenatal diagnosis in the case of abnormalities.
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Notes |
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References |
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Submitted on December 30, 1998; accepted on June 10, 1999.