Chimerism in a fertile woman with 46,XY karyotype and female phenotype: Case report
R. Sudik1,
S. Jakubiczka2,
F. Nawroth1,
E. Gilberg3 and
P.F. Wieacker2,4
1 Department of Obstetrics and Gynecology, Klinikum Neubrandenburg,
2 Institute of Human Genetics, Otto-von-Guericke University Magdeburg, and
3 Department of Pediatrics, Klinikum Neubrandenburg, Germany
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Abstract
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We report on the unexpected finding of a 46,XY karyotype in a 30 year-old woman with normal ovarian function and a former pregnancy at 17 years of age. Chromosome analysis was performed prior to intracyoplasmic sperm injection (ICSI), due to infertility of her husband. Repeated chromosome analysis in lymphocytes of the female resulted in a normal male karyotype. Fluorescence in-situ hybridization (FISH) analysis of cultured lymphocyte interphase nuclei detected in 99% of the cells one X and one Y chromosome-specific signal respectively, whereas two X chromosome-specific signals were observed in only 1% of the nuclei. Chromosome analysis of fibroblasts of ovarian and muscular tissues as well as of skin revealed a normal female karyotype (46,XX). Chimerism could be proven by variable number of tandem repeats (VNTR) analysis. Since the case history of the patient revealed that her twin brother died shortly after birth, it can be assumed that chimerism is caused by fetofetal transfusion during pregnancy and delivery of the proposita.
Key words:
chimerism/fertility/FISH/VNTR
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Introduction
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Intracytoplasmic sperm injection (ICSI) has proven to be the most successful treatment of male infertility. Defects in sperm production can be caused by genetic factors (e.g. Wieacker and Jakubiczka, 1997) and there is an inverse proportion between the incidence of chromosomal abnormalities and sperm count. Also an elevated frequency of chromosome aberrations has been detected in female partners of infertile couples, even if the ovarian function appears to be normal (Van der Ven et al., 1998
). Here, we report on the unexpected finding of a 46,XY karyotype in a 30 year-old woman exhibiting normal ovarian function and requiring ICSI because of her husband's infertility.
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Materials and methods
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Chromosome preparations from phytohaemagglutinin (PHA)-stimulated lymphocytes or cultured fibroblasts and GTG banding were performed following standard procedures. For fluorescence in-situ hybridization (FISH) on interphase nuclei of lymphocytes commercially available chromosome-specific DNA-probes X and Y (AneuVysion Assay Kit; Vysis, Stuttgart, Germany) were used.
DNA was extracted from peripheral blood lymphocytes by a salting-out procedure and from cultured fibroblasts by phenol/chloroform, both according to standard protocols. Polymerase chain reaction (PCR) conditions for amplification of variable number of tandem repeats (VNTR) loci D2S367, D14S269, D15S156, and DXS1238 (STR44) were: ~200 ng of DNA, 0.4 µmol/l of each primer, 0.2mmol/l dATP, 0.2mmol/l dGTP, 0.2mmol/l dTTP, 0.01mmol/l dCTP, 0.1 µCi
[32P]-dCTP (3000 Ci/mmol), 1.5 mmol/l MgCl2, and 1 IU Taq polymerase (Gibco-BRL, Karlsruhe, Germany) in 25 µl of the manufacturer's buffer. Amplification was carried out by an initial denaturation for 5 min at 94°C, followed by 30 cycles of 60 s at 94°C, 60 s at annealing temperature (53°C for D14S269 and D2S367, 56°C for D15S156 and 55°C for DXS1238) and 60 s at 72°C, and a final elongation step for 10 min at 72°C. Products were separated on 6% denaturing polyacrylamide gels and visualized by autoradiography.
The polymorphic CAG repeat of the androgen receptor (AR) gene was analysed according to Yamamoto et al. (Yamamoto et al., 1992
) with the following minor modifications. Amplification products from PCR amplification were separated on 2% agarose gels, bands were cut out and DNA was re-extracted by centrifugation through spun columns. Instead of end-labelling primer ARS1 0.125 µCi
[32P]-dCTP (3000 Ci/mmol) was added per 25 µl reaction mix. Finally, four rounds of primer extension reactions were applied rather than one.
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Case report and results
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A 30 year old woman was referred to our centre of reproductive medicine because of unfulfilled wish for children for 5 years. The spermiogram of the husband showed oligoasthenoteratozoospermia. In contrast, gynaecological history and examination of the proposita were inconspicuous. Menarche occurred at 14 years. She reported on a pregnancy at 17 years of age in a former partnership. Normal ovarian function was confirmed by regular menstruation, normal concentrations of gonadotrophins, prolactin, oestradiol, testosterone and progesterone on day 22 of the cycle. Because of her husband's infertility, ICSI treatment was recommended and chromosome analysis was performed in lymphocytes of both partners. The unexpected finding of a male karyotype (46,XY) in the woman was confirmed in a repeated analysis. Furthermore, interphase FISH analysis with X and Y chromosome-specific probes was performed in cultured blood cells. One X and one Y signal were detected in 99 cells, whereas one cell showed two X-specific signals. For examination of internal genitalia laparoscopy and hysteroscopy were performed, revealing normal female internal genitalia. In the course of these procedures biopsies were taken from skin, rectus muscle and ovaries. Chromosome analysis of fibroblasts showed a 46,XX karyotype in all cultures. After having been informed about these results, the proposita reported on a twin brother who died four days after birth. Because of the suspicion of twin chimerism VNTR analysis was performed in DNA of the lymphocytes and the cultured fibroblasts using the markers D2S367, D14S269, D15S156, DXS1238 (STR44), and the polymorphic CAG repeat of the AR gene (Table I
). VNTR analysis revealed different allelic patterns in blood and fibroblasts, demonstrating the presence of chimerism in this patient.
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Discussion
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The term chimera is used when an organism contains cells from two or more zygotes. It can be classified as artificial chimerism, tetragametic chimerism and twin chimerism. Artificial chimerism can arise from transfused blood stem cells either by intrauterine transfusion or by allogenic bone marrow transplantation as well as other organ transplantations. Tetragametic chimerism is induced by the fertilization of two oocytes by two spermatozoa and the fusion of these products into one body. Tetragametic chimerism seems to be very rare in humans (Tippett, 1983
). Such a chimerism can be the cause of true hermaphroditism. Twin chimerism confined to blood cells has been thought to be an exception in humans, while it is reported frequently in cattle. However, using a sensitive fluorescence technique, blood group chimerism was detected in 8% of twin pairs and 21% of triplets (van Dijk et al., 1996
).
The unexpected finding of a 46,XY karyotype in a woman with normal ovarian function gave rise to the suspicion of chimerism. Furthermore, the case history of the patient revealed that a twin brother died four days after birth, suggesting a twin chimerism confined to blood cells and due to fetofetal transfusion.The 46,XX karyotype in fibroblasts derived from muscle, ovaries and skin strongly supported this suspicion. Finally, chimerism could be proven unequivocally by analysis of VNTR markers demonstrating allele patterns which can be only explained by different genomes. To our knowledge, only few similar cases have been reported so far. One group (Gencik et al., 1980
), detected a 46,XX/46,XY chimerism in a female affected by mammary hypoplasia and sterility. However, whole body chimerism could be demonstrated by the investigation of different tissues. Another group (Farber et al., 1989
), reported on a phenotypically normal woman with a 46,XY karyotype. The proposita and her twin brother exhibited identical DNA fingerprints.
It is surprising that in our case the acceptor's lymphocytes have been almost completely eradicated by the donor's lymphocyte population. Even though the exact ratio of 46,XY and 46,XX cells in blood cannot be derived from the results of FISH and chromosome analysis in cultured lymphocytes, VNTR analysis in blood showing only alleles of one genome suggests very strongly that the pool of lymphocytes consists mainly of 46,XY cells. Cytogenetic analysis of oocytes was not possible. However, we expect that oocytes are derived from 46,XX cells. At first, chimerism should be restricted to blood cells in the case of fetofetal transfusion. Furthermore, follicles with premeiotic germ cells containing only one X chromosome are prone to atresia, as it is the case for Turner syndrome or XY gonadal dysgenesis. These cases of twin chimerism are instructive for our understanding of the fetal immune system. Obviously, the immunological tolerance during intrauterine life (Ober, 1998
) can be maintained in these cases, preventing a graft versus host reaction for the rest of the individual's life.

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Figure 1. Allelic patterns of variable number of tandem repeat (VNTR) markers in blood (1) and fibroblast (2) DNA.
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Notes
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4 To whom correspondence should be addressed at: Universitätsklinikum Magdeburg, Institut für Humangenetik, Leipziger Strasse 44, 39120 Magdeburg, Germany. E-mail: Peter.Wieacker{at}medizin.uni-magdeburg.de 
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References
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Submitted on June 1, 2000;
accepted on September 29, 2000.