Hum. Reprod. Advance Access originally published online on February 1, 2007
Human Reproduction 2007 22(4):1037-1041; doi:10.1093/humrep/del480
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Prenatal diagnosis and normal outcome of a 46,XX/46,XY chimera: A Case Report
1 Service de Cytogénétique 2 Service de Génétique Moléculaire, University René Descartes-Paris 5, Paris, France 3 Service de Gynécologie Obstétrique, University René Descartes-Paris 5, Paris, France 4 Service de Chirurgie Viscérale, Hôpital Necker Enfants Malades, APHP University René Descartes-Paris 5, Paris, France 5 Université René Descartes-Paris 5, Paris, France
6 To whom correspondence should be addressed at: Service de Cytogénétique et d'Embryologie, Hôpital Necker-Enfants Malades, 149, rue de Sèvres, 75743 Paris Cedex15, France. Tel.: +33 1 44 49 58 54; Fax: +33 1 44 49 04 17; E-mail: valerie.malan{at}nck.aphp.fr
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Abstract |
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The phenotypic spectrum of 46,XX/46,XY chimeric patients is variable. It ranges from normal male or female genitalia to different degrees of ambiguous genitalia. Chimerism results from the amalgamation of two different zygotes in a single embryo, whereas mosaicism results from a mitotic error in a single zygote. Several other mechanisms resulting in a chimera have been discussed in the literature. Here, we report on a new case of chimerism (46,XX/46,XY) diagnosed at 17 weeks' gestation on amniocentesis performed because of advanced maternal age. Ultrasound examination revealed normal female external genitalia, and a healthy baby girl was delivered at term.
We used polymorphic markers spanning the X chromosome and several autosomes in order to identify the genetic mechanism involved. Mosaicism was excluded because of the presence of 3 alleles at 11 autosomal and 4 X chromosome loci. On autosomes, the origin of this third allele was maternal for two pericentromeric markers (located on 2p11.2 band and 8p11.2 band), paternal for six markers and paternal or maternal for the other three markers. On the X chromosome, the origin of the third allele was maternal for all four markers. Thus, two different paternal and maternal haploid sets were observed. These results are compatible with a tetragametic chimera.
Key words: chimera/mosaic/prenatal diagnosis
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Introduction |
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During prenatal diagnosis, the simultaneous presence of 46,XX and 46,XY cell lines is observed in 0.24% of amniotic fluid cell cultures (Lillian, 1998
). Most often, the presence of female XX cells results from maternal cell contamination of a 46,XY sample, but rare cases of chimerism have been reported (Hunter et al., 1982; Lawce, 1985; Freiberg et al., 1988; Amor et al., 1999; Yaron et al., 1999 Pinhas-Hamiel et al., 2002; Simon-Bouy et al., 2003; Chen et al., 2005). Chimerism results from fusion of two different zygotes in a single embryo, whereas mosaicism results from a mitotic error in a single zygote (de Grouchy, 1980). In 1962, Gartler et al. described the first case of chimerism. Since then, about 30 cases have been reported in the literature. Very few cases have been studied using microsatellite markers, in order to identify the genetic mechanism involved. Here we describe a 46,XX/46,XY chimera observed during prenatal diagnosis performed because of advanced maternal age. The pregnancy resulted in a healthy baby girl at term with normal female external genitalia. |
Case report |
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After undergoing amniocentesis at 17 weeks' gestation because of advanced maternal age, a 41-year old patient was referred to our centre because two different cells lines (46,XX [90%] and 46,XY [10%]) were observed in amniotic fluid cell culture. Ultrasound examination at 22 weeks' gestation revealed normal female external genitalia. The family history was unremarkable. This was the second pregnancy. The first pregnancy resulted in dizygotic twins (girl and boy), now 12 years old. The twin pregnancy occurred after infertility treatments with clomiphene citrate (Clomid®).
At our centre, a second amniocentesis as well as a cordocentesis was performed at 25 weeks' gestation. The results of fetal karyotyping carried out on blood and amniotic fluid cells confirmed previous findings. HCG levels observed in fetal serum and maternal serum allowed to exclude a maternal cell contamination.
Another ultrasound examination was performed at 25 weeks' gestation in order to visualize the external genitalia. It revealed that the fetus had external female genitalia. Furthermore, a complete uterus and the gonads were visualized. Given the normal second ultrasound findings, the couple decided to keep the pregnancy. The parents were informed of the risk of sterility and gonadoblastoma in the case of ovotestis. After a full-term pregnancy, a healthy baby girl was delivered. Pelvic ultrasound examination showed normal internal genitalia. Gonadal biopsy was not performed.
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Materials and methods |
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Karyotyping after GTG and RHG banding was performed according to standard procedures on cultured amniotic fluid cells, fetal cord blood sampling and parental peripheral blood lymphocytes. In order to estimate the percentage of each cell line, Fluorescence in situ hybridization (FISH) study using X and Y chromosome centromeric probes was performed. Also SRY and heterochromatin Y (Yqh) probes were used to rule out any possible Y structural abnormality.
DNA was extracted from amniotic fluid cells and parental peripheral blood cells. Genotype analysis was performed using 35 autosomal markers and 15 X-linked markers (Tables I–III). PCR conditions were as following: 95°C for 5 min, 95°C for 30 s, 55°C for 30 s and 72°C for 30 s for 30 cycles. PCR products were loaded onto an ABI prism 3130 (Applied Biosystem). Results were analysed using Genescan and Genotyper softwares.
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Results |
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Karyotypes obtained from the second amniocentesis confirmed the presence of 2 cell lines from 20 cells examined (46,XX [19] and 46,XY [1]). FISH analysis performed on blood sample showed that 93% of the cells were XX and 7% XY (300 nuclei and metaphases analysed). FISH analysis using Yqh and SRY probes demonstrated a single hybridization signal for each probe (300 cells analysed). No other cell line, in particular 45,X, was observed. The parental karyotypes were normal. These findings confirmed the results obtained on the first amniotic fluid cells sample.
Out of 35 autosomal loci analysed, 10 were fully informative (D1S2696, D4S2971, D5S822, D4S3038, D6S257, D6S402, ZNF220, D10S1711, D14S1007 and D14S260), whereas 15 (D1S442, D1S2845, D2S2393, D5S2086, D6S344, D7S2563, D11S4125, D13S285, D14S1023, D16S3081, D18S453, D20S871, D21S1903, D22S1169 and D22S1161) were partially informative. We considered a marker as fully informative when it allowed us to identify or to exclude a third paternal or maternal allele, whereas a partially informative marker did allow us to identify or to exclude a third allele, but did not allow to define the maternal or paternal origin. A third allele was present for 11 markers. The origin of this third allele was maternal for two markers (D2S2216 and ZNF220), paternal for six markers (D2S2175, D4S3038, D6S402, D6S344, D10S1711 and D22S1161) and paternal or maternal for the other three markers (D2S2393, D21S1903 and D22S1169). Data are shown in Tables I and II and in Figure 1. Out of 15 chromosome X markers, 2 maternal alleles were found for 4 markers (DXS1060, DXS8051, DXS987 and DXS1001) (Table III). The interpretation of the results was difficult because of the low number of 46,XY cells (
10%).
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Discussion |
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Only a small number of chimeric patients with a 46,XX/46,XY karyotype have been reported to date. In most cases, this condition has been diagnosed at birth, due to the presence of ambiguous external genitalia. These cases account for about 13% of true hermaphrodites (Danon, 1996). The phenotypic spectrum from these patients varies from normal male or female external genitalia (Froesch et al.,1983; Freiberg et al., 1988
; Bromilow and Duguid, 1989) to different degrees of ambiguous genitalia (Fitzgerald et al., 1979; Farag et al., 1987; Poissonnier et al., 1987; Green et al., 1994; Hadjiathanasiou et al., 1994; Sawai et al., 1994). Only a few cases of 46,XX/46,XY individuals have been reported with normal external genitalia. Very often patients are infertile, even though offspring have been infrequently produced by men (Watkins et al., 1981; Schoenle et al., 1983) and women (Bromilow and Duguid, 1989; Verp et al., 1992).
Prenatally, the finding of a mixture of both 46,XX and 46,XY cells in amniotic fluid culture is observed at a frequency of 0.24% (Lillian, 1998). Maternal cell contamination of a male fetus amniotic fluid explains the great majority of cases. Other cases of 46,XX/46,XY karyotype may result from a laboratory error due to a cross contamination of two samples. The death of a male twin (a ‘vanishing twin’) could not result in the presence of XX and XY cells in the amniotic fluid (Lillian, 1998). After excluding these hypotheses, the diagnosis of chimerism or mosaicism can be proposed (Malan et al., 2006).
Usually, individuals with a 46,XX/46,XY karyotype are considered as chimera rather than mosaic, even though a molecular analysis has not been provided. A mosaic contains genetically different cells originating from a single zygote. It results from a mitotic error during the first blastomeric division or at a later stage. To our knowledge, Niu et al. (2002) reported the first case of mosaicism proven at the molecular level in a hermaphrodite individual (46,XX [39]/46,XY [9]). Molecular studies showed a single maternal contribution (i.e. one haploid set of maternal chromosomes) and a single paternal allelic contribution (i.e. one haploid set of paternal chromosomes). Chimerism is a rare condition where cell lines originate from two distinct zygotes. Chimerism is thought to result from the fertilization of two oocytes by two sperms and subsequent fusion of two zygotes into one single embryo. This condition is called tetragametic chimera. Different molecular studies have confirmed this mechanism (Green et al., 1994; Uehara et al., 1995; Bonthron, 1997; Strain et al., 1998; Yu et al., 2002). In these cases, a double paternal contribution (i.e. two different haploid sets of paternal chromosomes) and a double maternal contribution (i.e. two different haploid sets of maternal chromosomes) are observed. However, four alleles are found only at some loci, since the two oocytes and the two sperms share some chromosomes. It is worth noting that medically assisted reproduction techniques were performed in the cases of Bonthron et al. (1997) and Strain et al. (1998). It is likely that these new technologies increase the risk of chimerism since several embryos are usually transferred to improve the chance of pregnancy. Furthermore, elevated maternal age is a factor favouring dizygotic twinning (Hankins and Saade, 2005) and consequently the risk of chimerism by amalgamation of the two embryos.
Fertilization of the second polar body is another mechanism. It has been discussed by several authors, but never confirmed at the molecular level (Chen et al., 2005). Molecular studies using microsatellite markers should reveal in this situation a double paternal contribution and a unique maternal contribution, except for markers localized at the telomeric ends of the chromosomes due to crossing over during the meiosis I prophase. A large number of polymorphic markers are necessary to confirm this hypothesis.
The last mechanism reported by Giltay et al. (1998) consists in the fertilization of a parthenogenetic ovum by two sperms: a parthenogenetic division of a female pronucleus produces two identical nuclei which are fertilized by two spermatozoa. Therefore, the genotype analysis shows a single maternal and a double paternal contribution. A slightly different mechanism was proposed by Strain et al. (1995). Here, division of the haploid nucleus results in two identical cells. One nucleus is fertilized by a sperm while there is a diploidization (or endoreplication) of the other cell.
Prenatal genetic counseling is quite difficult in these cases. The possibility of sexual ambiguity, infertility and gonadoblastoma should be discussed. It is worth noting that intelligence is normal. With respect to the external genitalia of the fetus, detailed ultrasound examination is very useful (Jouannic et al., 2005). Unfortunately, the respective proportions of 46,XX and 46,XY cell lines observed in fetal blood or in amniotic fluid does not allow to predict the nature of the fetal internal and external genitalia.
In our patient, two populations of cells, namely 46,XX and 46,XY cells, were found in the amniotic fluid as well as in the fetal blood. Molecular studies were performed in order to distinguish chimerism from mosaicism. A third allele was observed at 11 autosomal loci as well as 4 X chromosome loci. Therefore, we excluded the diagnosis of mosaicism. The origin of the third autosomal allele is maternal for two markers, paternal for six markers and maternal or paternal for three markers. The origin of the third X chromosome allele is maternal for all markers. Thus, two paternal and two maternal genetic contributions are observed. The two markers ZNF220 and D2S2216, for which two maternal alleles are found, are pericentromeric and located respectively on 8p11.2 and 2p11.2. These results are compatible with a tetragametic chimera. Also, elevated maternal age could have favoured dizygotic twinning. Molecular results showed two maternal and paternal centromeric alleles for some markers. Indeed, in the case of embryo amalgamation, chromosomes segregate randomly and independently in each ovum. Therefore, centromeric alleles are different for some markers but similar for others since two oocytes (or the two sperms) may share the same chromosome. However, the hypothesis of fertilization of the second polar body cannot be formally excluded because of the small number of two maternal centromeric alleles observed. These two different maternal alleles may be the result of crossing over occurring very near the centromere.
In conclusion, we report on prenatal diagnosis of a chimera with normal female phenotype. Polymorphic DNA marker analysis is helpful to distinguish chimera from mosaic and to determine the mechanism leading to 46,XX/46,XY chimera.
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We thank Michel Alcaraz and Laurent Leroy for their data processing assistance.
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Submitted on September 23, 2006; resubmitted on November 5, 2006; accepted on November 16, 2006.
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