1 Departments of Cytogenetics and Reproductive Medicine, University Hospital of Amiens, 2 Department of Cytogenetics, University Hospital of Necker-Enfants-Malades, Paris, 3 Departments of Cytogenetics and Reproductive Medicine, University Hospital of Tenon, Paris, 4Department of Human Genetics, University Hospital of Tours, France, 5Department of Cytogenetics, Hospital of Caen, 6Department of Human Genetics, University Hospital of Toulouse, 7 Department of Cytogenetics, University Hospital of Reims, 8 Cytogenetic Laboratory of Metz, 9 Department of Cytogenetics, University Hospital of Jean Verdier, Bondy, France
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Abstract |
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Key words: chromosomal aberration/female infertility/genetic counselling/intracytoplasmic sperm injection/male infertility
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Introduction |
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In order to assess the frequency of chromosomal aberrations in French ICSI candidates (including the female partners), and to explore the existence of a hidden female chromosomal factor in some cases of couple infertility, a collaborative, retrospective, clinical and cytogenetic study was performed, initiated by the Association des Cytogénéticiens de Langue Franciaise (ACLF), with 20 reproduction centres and cytogenetic laboratories in France. The results are presented here, and their implications discussed.
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Materials and methods |
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Karyotyping was conducted by analysis of G and/or R banding using the peripheral blood lymphocyte culture technique. All chromosomal abnormalities were reported in accordance with the current international standard nomenclature (Mitelman, 1995). At least 20 metaphases were analysed for each patient. In cases of numerical mosaics (especially for sex chromosomes), >30 cells were examined. Only numerical abnormalities present in more than two cells were considered as mosaics (Mitelman, 1995
). Sex chromosome mosaics occurring at a level of <10% were considered to be low-level mosaicism or minor mosaicism. The smallest sex chromosome mosaic reported contained a clone of 3% of cells (see Table III
). Pericentric inversions of chromosome 9 or other structural chromosomal variants and polymorphisms (Dutch Society of Obstetrics and Gynecology, 1996
), low-level mosaicism for fragile sites, and structural rearrangements confined to a single cell were not included in chromosomal abnormalities, but considered as normal cytogenetic events.
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Inclusion criteria
All patients were referred for sterility (no spontaneous pregnancy despite >2 years unprotected intercourse) and ICSI treatment because of male factor infertility: semen alteration which does not permit conventional IVF and/or previous failure of fertilization in at least one conventional IVF attempt. Management of women patients excluded gynaecological causes of female infertility. Full medical and laboratory records were available in all cases.
Study population
A total of 2196 (88.5%) correctly and fully completed questionnaires from 2196 men and 1012 women was collected between April 1995 and August 1998. More male than female partners were collected because some centres did not consider the women. Incomplete questionnaires (n = 284; 11.5%) were not used for analysis.
Male partners of couples (n = 1012) included in the survey
Andrological examination, full medical history, serum FSH, vasography findings, and abnormal testicular volume or biopsy results were used to classify as explained (30%, 274/917) or unexplained (70%, 643/917) the origin of infertility of non-azoospermic male partners of couples included in the survey (sperm concentration >0.106/ml; 90.6%, 917/1012). Explained infertility referred to patients with infertility that was obstructive (retrograde ejaculation, testis, epididymis or prostate infection, agenesis of vas deferens and/or abnormal vasography results) or non-obstructive (testicular atrophy, high serum FSH concentration, abnormal testicular biopsy and/or clinical hypogonadism). Unexplained infertility referred to patients for whom there was no detectable reason for any obstructive or non-obstructive form of infertility.
Statistical analysis
The 2-test was used for statistical evaluation. The level of significance was P < 0.05.
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Results |
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Non-mosaic numerical sex chromosome abnormalities and Y chromosome abnormalities
The incidence of 47,XXY in azoospermic men was 141 times higher than in male newborns, while the incidence of Y chromosome abnormalities in azoospermic men was 46.6 times higher than in male newborns.
Frequency of sex chromosome mosaicism in men and women
Forty-five cases had mosaicism for number of sex chromosomes, with a strong preponderance of women (2.77%, 28/1012) compared with men (0.77%, 17/2196). These cases in women represented 100% (28/28) of abnormal cytogenetic results with a sex chromosome anomaly, compared with 23.3% (17/73) in the men. The increase noted for mosaicism for the sex chromosome was significantly higher in azoospermic men and among the female partners than in the men as a whole (2-test, respectively P < 0.05 and P < 0.001). Even if these cases were disregarded in the analysis, 2.08% (21/1012) of abnormal karyotypes remained in women (see Table II
). In the group of women with sex chromosome mosaicism, 79% (22/28) had a low level of mosaicism.
The mean age (32.6 years) of the 28 women with sex chromosome mosaicism in our cohort was above the average of 32.0 years calculated for the remainder of our female study population (n = 984), but the number of women with a mosaicism for sex chromosome anomaly in the >32-year-olds (16/648, 2.47%) or <32-year-olds (12/364, 3.29%; 2-test) was not significantly different.
Autosomal balanced structural abnormalities
The incidence of autosomal balanced structural abnormalities in women and men was respectively 7.3 and 7.7 times higher than in newborns: the increase was not significantly different between these two groups (2-test).
The incidence of reciprocal translocations in women and men was respectively 4.5 and 8 times higher than in newborns: the increase was not significantly different between these two groups (2-test). The incidence of Robertsonian translocations in women and men was respectively 7.7 and 9.1 times higher than in newborns: the increase was not significantly different between these two groups (
2-test).
The incidence of inversions in women was 16.4 times higher than in newborns. This increase compared to the infertile male group was significant: 16.4 times and 3.3 times higher than in newborns for women and men respectively (2, P < 0.01).
Sperm concentration, motility and morphology
The incidence of chromosome abnormalities in terms of sperm concentration for the study population is reported in Table IV. The incidence observed in azoospermic patients (18.71%) was significantly higher than that in the normal sperm concentration group (3.02%) (
2, P < 0.001). However, the incidence noted for patients with a sperm concentration >0 and <5x106/ml (4.55%,
2, not significant) or
5x106/ml but <20x106/ml (2.37%,
2, not significant) was not significantly different from that of the normal sperm concentration group (3.02%).
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Female chromosomal factor
If there was no female chromosomal factor in some cases of couple infertility, a stochastic distribution of female partners with a chromosome aberration would have been expected in the present population of explored infertile couples (n = 1012). After having subdivided the men in terms of sperm concentration, a significantly higher frequency of chromosomal aberration in women was noted in couples when the sperm concentration was normal (7.55%, 21/278), compared with couples with an abnormal sperm concentration (3.81%, 28/734, 2, P < 0.02) or with azoospermic (1.05%, 1/95,
2, P < 0.02) or oligozoospermic (4.22%, 27/639,
2, P < 0.05) male partners (Table V
). Also, a significantly higher frequency of chromosomal aberration in women was noted in couples referred for fertilization failure, compared with those referred for semen alteration [respectively 9.37% (21/224) and 3.55% (28/788);
2, P < 0.01] (see Table I
).
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Discussion |
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This excess of chromosome aberrations in women may have contributed to the infertility observed, even if selection against aneuploid oocytes at fertilization has been considered to be less likely than during the pre-implantation period or early stages of pregnancy (Plachot, 1997). An increased incidence of abnormality has been reported in oocytes obtained after IVF failure from couples with unexplained infertility compared with those with male factor infertility (Speed, 1986a
,b
; Mau et al., 1997
; Plachot, 1997
) and some authors report the possible implication of maternal chromosome abnormalities in reduced fertilization rates (Mittwoch et al., 1990
; Meschede et al., 1995
; Van der Ven et al., 1998
).
The incidence of mosaicism for sex chromosomes is not related to women's age, is significantly higher in women than in men (where the incidence is significantly higher in azoospermic men than in the whole male population), and represents a large proportion of all chromosome abnormalities in women (Table II), as noted previously (2.4%, Meschede et al., 1998; 7.2%, Scholtes et al., 1998). This may argue for the involvement of these mosaics in infertility. Low-level mosaicism is seen in 79% (22/28, Table III
) of women with sex chromosome mosaicism. Low-frequency sex chromosomal aberrations have been described as an underestimated cause of failure in assisted reproduction (Simpson, 1992
) and have been associated with a low implantation rate (Scholtes et al., 1998
). In spite of this, and as reported previously (Healy et al., 1994
; Van der Ven et al., 1998
), it would be highly speculative to postulate a connection between this type of chromosomal aberration and the infertility of our couples. First, the incidence of such aberrations is still poorly known in the general population: the largest published study was based on cytogenetic analysis of a few cells (Hook and Hamerton, 1977
). Second, many cytogeneticists do not even mention the aberrations in karyotyping results because they are considered as normal cytogenetic variants in asymptomatic patients. Third, there is no clearly established minimum percentage of aberrant cells required for the karyotype to be classified as a real mosaicism and not a low-level mosaicism (Mitelman, 1995
). Also, one study reports comparable rates of low-level sex chromosomal mosaicism between women and men included in ICSI programmes and control groups of couples with a spontaneous pregnancy within 2 years (Peschka et al., 1999
). More data are needed, particularly on the general population, to define the role of low-level sex chromosomal mosaicism in the infertility of some couples.
It has been known for some 25 years (Chandley et al., 1975; Chandley, 1979
) that there is a causal relation between chromosomal aberrations and male infertility, that the incidence of chromosomal aberrations is inversely correlated with sperm count, and that the major indication for karyotyping an infertile man is still usually an abnormal sperm analysis. The frequency and type of chromosome abnormalities in our population of ICSI infertile men do not differ from published data on non-ICSI infertile men (Chandley et al., 1975
; Chandley, 1979
). Infertile male ICSI candidates therefore do not seem to have a particular chromosomal profile compared with non-ICSI infertile men. Although the frequency of chromosome abnormality in normozoospermic men was elevated (3.02%) (Table IV
), as reported previously in non-ICSI candidates (1.68%, Yoshida et al., 1997), the incidence noted for patients with a sperm concentration >0 and <5x106/ml or
5x106/ml but <20x106/ml was not significantly different from that of the normal sperm concentration group. No significant correlation was noted between sperm motility and morphology anomalies and the incidence of chromosome abnormalities, in accordance with some data (Bourrouillou et al., 1992
; Testart et al., 1996
), but in contradiction with others for sperm motility (Chandley et al., 1975
; Bourrouillou et al., 1992
) and morphology (Bourrouillou et al., 1992
). This confirms the low weighting accorded to semen disturbance in predicting a risk of chromosomal abnormality (Pandiyan and Jequier, 1996
), and perhaps suggests that fertilization failure is the only sign of chromosomal abnormality in men and may in itself justify a cytogenetic analysis.
A 47,XYY karyotype was found in seven men, a mosaic 47,XYY/46,XY in one, and a Y-chromosome aberration in nine (Table III): we confirm (Tuerlings et al., 1998
) the relatively high frequency of men with a 47,XYY karyotype or Y-chromosome aberration in ICSI candidates (Table II
), although most men with a 47,XYY karyotype are reported to be normally fertile (Van Wijek et al., 1962
; Jones, 1997
). Failure of spermatogenesis in patients with a Yq chromosome aberration could be explained by alteration of the azoospermia factors a, b and c located on the euchromatic segment of Yq (Viguié et al., 1982
; Tuerlings et al., 1998
). As noted previously (Scholtes et al., 1998
), we report more reciprocal than Robertsonian translocations in male ICSI candidates, in contrast to that reported by others (Tuerlings et al., 1998
). Supernumerary marker chromosomes were found in three men (Table III
). An excess of such markers was previously noted for infertile men (Pandiyan and Jequier, 1996
; Mau et al., 1997
; Tuerlings et al., 1998
). A surprisingly high frequency of inversions was noted in women compared with men and newborns (Table III
). An association between autosomal inversion and infertility in men has been reported (Faed et al., 1979
), but inversions in women are not reported to be implicated in infertility. The inversion concerned chromosome 10 in three cases and chromosome 4 in two (Table III
), each time at different breakpoints: more data may be useful in defining their status concerning female infertility, and particularly their influence on oocyte production and quality.
In the present study, an overall increased frequency of chromosomal aberrations was found in French ICSI candidates, and it was confirmed that in some cases of poor reproductive outcome there was a potential contribution of maternal chromosome aberrations that cannot be identified by standard clinical evaluation. Our results, like those of other authors, first emphasize the need for thorough genetic work-up in men (Meschede et al., 1998; Tuerlings et al., 1998
; Van der Ven et al., 1998
) and women (Meschede et al., 1998
; Van der Ven et al., 1998
) undergoing ICSI. In both cases, this work-up should include karyotyping. Second, the results show that aneuploid oocytes may be selected at fertilization, and third, that fertilization failure could be the only sign of chromosomal abnormality in men or women. By selecting a single spermatozoon for injection, the ICSI technique by-passes the usual process of natural selection which is thought to occur both during natural conception and in conventional IVF, resulting in a greater chance of fertilization involving an abnormal spermatozoon or oocyte. Fertilization and pregnancy rates are independent of both sperm motility and morphology (Baschat et al., 1996
), and morphological parameters of embryo quality prior to implantation show no relation to karyotype. Also, preliminary follow-up data on pregnancies conceived through ICSI suggest that sex chromosome anomalies may be more common than in naturally occurring pregnancies (Jacobs et al., 1992
; In't Veld et al., 1995; Liebaers et al., 1995a
; Van Opstal et al., 1997
). Karyotypes done on children conceived by ICSI have found an increased incidence of abnormalities (Zenzes et al., 1992
; Liebaers et al., 1995a
,b
), and in a study reporting a significant difference in development one year after birth between newborns conceived by ICSI and IVF and naturally conceived newborns (Bowen et al., 1998
) it was supposed that the slight development delays may be due to chromosomal abnormalities. In chromosomally abnormal couples, as there is a high likelihood of producing an embryo with chromosome abnormalities, the difficulty of prenatal diagnosis in multiple fetus pregnancy must be considered when making any decisions concerning the number of embryos that should be transferred.
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Notes |
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2 Association de Cytogeneticiens de Langue Francaise:
M.Montagnon, Department of Cytogenetics, University Hospital of Ambroise Paré, Boulogne-Billancourt, France
N.Rives, Department of Cytogenetics, University Hospital of Rouen, France
L. Clotteau, Department of Cytogenetics, University Hospital of Rouen, France
M.C.Malingue, Department of Human Genetics, Centre University Hospital of Angers, France
P.Darabi, Department of Reproductive Medicine, Centre University Hospital of Cochin, Paris, France
C.Poitot, Department of Reproductive Medicine, Centre University Hospital of Cochin, Paris, France
F.Baverel, Department of Cytogenetics, University Hospital of Cochin, Paris, France
S.Lesourd, Departments of Cytogenetics and Reproductive Medicine, University Hospital of Pitié-Salpêtrière, Paris, France
M.A.Collonge-Rame, Departments of Reproductive Medicine, Hospital of Besancion, France
J.Lespinasse, Department of Cytogenetics, University Hospital of Chambéry, France
C.Carel, Department of Human Genetics, University Hospital of Nice, France
A.Devaux, Department of Reproductive Medicine, University Hospital of Bichat, Paris, France
M.H.Couturier-Turpin, Department of Cytogenetics, University Hospital of Bichat, Paris, France
F.Mugneret, Department of Cytogenetics, Hospital of Dijon, France
S.Delcleve-Paulhac, Department of Reproductive Medicine, University Hospital of Limoges, France
C.Yardin, Department of Cytogenetics, University Hospital of Limoges, France
F.Cartault, Department of Human Genetics, Hospital of St Denis la Réunion, France
10 To whom correspondence should be addressed E-mail: gekas.jean{at}chu-amiens.fr
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References |
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Submitted on May 2, 2000; accepted on September 25, 2000.