Human Reproduction, Vol. 15, No. 5, 1200-1204,
May 2000
© 2000 European Society of Human Reproduction and Embryology
Equal distribution of congenital blood cell chimerism in dizygotic triplets after in-vitro fertilization: Case report
1 Department of Transfusion Medicine, 2 Forensic Medicine, 3 Human Genetics, 4 Immunology and 5 Paediatrics, University of Kiel Medical School, Michaelisstrasse 5, D-24105 Kiel, Germany
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Abstract |
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The special situation of multiple pregnancies following IVF has led to a growing interest in the assessment of embryonal development by means of molecular genetics. We report a case of congenital blood chimerism in dizygotic triplets (two boys, one girl) present in erythrocytes and leukocytes in both sexes. Routine pre-operative blood serology of the 6 year old female triplet revealed chimerism of the red cells. Flow cytometry of the erythrocytes and DNA analysis of the leukocytes demonstrated that all three children had the same proportions of male and female cells. Fluorescent in-situ hybridization (FISH) analyses revealed Y chromosomes in 84% of the girl's leukocytes and in 89/92% of the two boys' leukocytes. The true genetic lines were determined by analysing polymorphism of serum groups (glycoprotein, transferrin, protease inhibitor and plasminogen) secreted by non-haematopoetic tissue, by blood group typing of hair roots and by DNA analysis of endothelial cells. Evidently placental anastomoses allowed a reciprocal intra-uterine transfusion of blood stem cells in the triplets.
Key words: blood chimerism/dizygotic triplets/in-vitro fertiliz-ation
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Introduction |
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A chimera is defined as an organism whose cells originate from two or more zygotes (Anderson et al., 1951
). The definition of blood chimerism requires the presence of cells derived from two (or more) genetically different individuals populating the lympho-haematopoietic compartments. Blood chimerism may occur artificially or spontaneously. Artificial chimerism occurs transiently after blood transfusions, while allogeneic bone marrow or stem cell transplantation results in stable blood chimerism (Schattenberg et al., 1989). The spontaneous type of blood chimerism has been observed in twins after transfusion of haematopoietic stem cells between fetuses during the first trimester. Placental injection studies revealed frequent anastomoses of blood vessels between monochorionic and monozygotic twins (Robertson and Karel, 1983). However, in human dizygotic twins, cell exchange with proliferation in the genetically different fetus is rare, and only a few cases have been described (Race and Sanger, 1975). Recent findings point to a more frequent incidence of blood chimerism in multiple births (van Dijk et al., 1996).
The growth of the genetically different cell line of the other fetus is associated with special immunological phenomena in blood chimeras caused by the induction of specific immuno-tolerance during fetal development (Tippett, 1983). This tolerance results in the failure to express the regular antibodies against A or B blood group antigens of the `donor' twin cells and the mutual acceptance of organ grafts (Billingham et al., 1953).
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Case report |
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A 6 year old girl was to undergo surgical correction of the duct of Botallo. The pre-operative blood serology revealed mixed field agglutination of ABO and Rhesus markers. The girl was a triplet born at 32 weeks gestation following in-vitro fertilization (IVF). The mother had undergone several microsurgical interventions after a diagnosis of primary sterility due to inflammation of the Fallopian tubes. She received an IVF treatment cycle using a combined regimen of gonadotrophin releasing hormone (GnRH), human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG). A transfer of three quadricellular embryos was performed. At birth the girl showed intra-uterine dystrophy. Her birth weight was 1520 g. At the age of 8 days she was given a transfusion of one unit of erythrocytes for anaemia. The other two triplets were males (2050 g and 1940 g) with no apparent somatic anomaly. No information was available on the quality of the placentae.
Samples of blood, hair roots and buccal swabs from the triplets and their parents were taken for further serological, flow cytometric and molecular genetic analysis. Red cells were typed with conventional, commercially available test sera for the following blood group antigens: ABO1, Rhesus1, Kell1, P12, MNSs2, Lewis2, Duffy2, Kidd2, and Lutheran2 (1Micro Typing System, Fa DiaMed, Cressier, CH; 2 tube test). The gel test revealed two erythrocyte populations of blood groups A and AB in similar proportions in each of the three infants. Likewise, the separated A-positive/B-negative erythrocyte population of the children showed a negative response for the Rhesus E-antigen using a gel test (Lapierre et al., 1990). No haemagglutinins were detected in the triplets' sera. The phenotype of the mother's erythrocytes was A1, CCD.ee, the father's B, ccD.Ee (Table I).
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The two erythrocyte populations were detected by the flow cytometry method with human Anti-E (primary antibody, monoclonal IgM, Biotest, Dreieich, Germany) and goat anti-human IgM fluorescein-conjugated F(ab')2 as secondary antibody (Austin et al., 1995
). A total of 7–9% fluorescence signals from the erythrocytes of each of the three children were revealed collecting events at a rate of 10 000 cells in a cell sorter (FACScan®; Becton Dickinson, Montain View, CA, USA; Figure 1). The signals of the mother's cells corresponded to an E-negative cell control, the father's to an E-positive population.
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There was no difference between the infants in other blood group antigens, polymorphism of erythrocyte enzymes or HLA antigens (class I/II). The blood group of the germ line was determined by examining the roots of their hairs. The girl's cells were typed as AB, both of the brothers as A (absorption–elution test was used in forensic investigations; Pötsch-Schneider et al., 1986). Polymorphism of serum groups, such as glycoprotein (Gc), transferrin (Tf), protease inhibitor (Pi) and plasminogen (PLG), secreted by non-haematopoetical tissues revealed the genetic differences between the girl and her brothers (Prokop and Göhler, 1986
). The results of the investigation done on the two boys were exactly identical (see Table I
).
Amplification of specific DNA sequences by means of PCR demonstrated the presence of Y chromosomes (Figure 2) in the peripheral leukocytes of the girl (Suttorp et al., 1993). The conventional analysis of the chromosomes revealed the karyotype 46,XY in four of seven evaluated metaphases from the girl's cells. The karyotype 46,XX was found in one of six metaphases in cells from one of the boys, while the other showed a regular male karyotype in all six metaphases analysed. Neither the parents' nor the children's cells showed any structural chromosome anomalies. The distribution of the female and male cells was further analysed by chromosomal interphase in-situ hybridization. Using X and Y specific probes detecting the DYZ3 and the DXZ1 locus, XX (green/green) and XY (green/red) fluorescence of the chromosomes was seen in all leukocyte preparations (200 cells were analysed) from each of the children (Siebert et al., 1998). Similar to the red cell population, the leukocytes were also equally distributed. The girl showed 84% XY cells, 16% were of her own XX. The boys had 92/89% XY cells; 8/11% were XX (Figure 3).
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The origin of the cells was determined by means of DNA analysis with the single locus probe technique for restriction fragment length polymorphism (RFLP) in eight variable number of tandem repeat (VNTR) regions. All probes are used routinely in paternity testing (Wyman and White, 1980
). Five probes (MS31, MS43a, G3, MS205, TBQ7) revealed additional DNA bands inherited from the mother or father or both (Table II).
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PCR amplification with the HumAmelX/Y gender identification system and the STR system HumVWA with DNA extracted from buccal swabs indicated a normal constellation among the whole family (Mannucci et al., 1994
). The alleles 16/19 (HumVWA) and the expected single band with the gender identification system were observed in the girl and the alleles 15/16 (HumVWA) and two bands with HumAmelX/Y in the boys. DNA extracted from the blood revealed the same mixed band pattern for each child: three bands (HumVWA) corresponding to the alleles 15, 16 and 19 (weakly expressed). The analysis with HumAmelX/Y showed X and Y bands in the female child (Figure 4).
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Discussion |
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TopAbstractIntroductionCase reportDiscussionReferences |
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Dizygotic triplets after IVF with transfer of three embryos are an apparently rare occurrence. It is supposed that gonadotrophin stimulation by ovulation induction regimens leads to increased pre-embryo splitting and zygote division (Avrech et al., 1993
). In addition to this rare finding we were able to demonstrate obvious blood chimerism in all three children (one girl, two monozygotic boys). In all three the male and female erythrocyte and leukocyte populations showed approximately the same distribution (~90% XY leukocytes, E-negative erythrocytes, 10% XX leukocytes, E-positive erythrocytes). In the girl the `donor' cell line predominated. The two boys, in contrast, had only a small proportion of female donor cells. These findings are in agreement with earlier reports showing that the major proportion of blood cells in the mixture does not necessarily indicate the true genetic (somatic) cell type (Chown et al., 1963).
DNA analysis of blood cells and non-haematological tissue (e.g. buccal cells, hairs and nails) was necessary to detect blood chimerism and distinguish it from mosaicism (Dauber et al., 1999). In cases of unlike sex, PCR analysis and fluorescent in-situ hybridization (FISH) techniques using Y and X specific probes can reveal chimerism and proportions of nucleated male and female cells (Suttorp et al., 1993). The triplets showed additional erythrocyte antigens of paternal origin. HLA typing was not helpful in our cases, as all three children were HLA identical.
Blood chimerism may be a pitfall in forensic investigations like paternity testing and crime cases. On the one hand, the application of only two or three DNA probes can fail to detect chimerism (Hansen and Sondervang, 1993); on the other hand, if additional bands are found, they can be falsely interpreted as an artificial cell mixture. These details have been discussed elsewhere from the viewpoints of forensic medicine (Milde et al., 1999).
DNA fingerprinting using several single locus probes demonstrated the simultaneous presence of both paternal and maternal alleles, a finding that can only be interpreted as a DNA mixture derived from two zygotes (Hansen and Sondervang, 1993). Typing of serum groups and the DNA analysis of hairs and buccal swabs demonstrated that the two boys, who had previously been thought to be dizygotic because three quadricellular embryos had been transferred, were actually monozygotic. Evidently in cases of multiple birth following IVF it is worthwhile to clarify zygocity, even if the number of transferred embryos is identical to the number of fetuses that had developed.
We assume that this blood cell chimerism was the result of an intensive reciprocal transfer of haematopoetic stem cells between the triplets. Some transfused cells settled down in the bone marrow of the host and produced blood cells of the donor line, a phenomena that is well known from animal work (Owen, 1945) and first described and supposed in the human (Dunsford et al., 1953). Other types of spontaneous blood chimerism, such as cell mosaicism, whole body chimerism or maternal–fetal transfusion could be excluded. Further subtle forms of chimerism were described (Van Dijk et al., 1996). Their cases showed a low percentage of donor red blood cells, some of these forms were transient and were possibly caused by maternal–fetal transfusions. We assume that our results indicate that the triplets are immunologically tolerant to their sibling(s) of the opposite sex and that the chimerism will probably remain stable for their whole life.
An explanation for dystrophic embryonal growth following IVF could be the development of placental anastomoses between fetal blood vessels. Apart from chimerism vascular communications can produce a fetal–fetal transfusion syndrome also in dichorionic placentae (Robertson et al., 1983; French et al., 1998). A dichorionic diamniotic placentation in dizygotic twins with vascular anastomoses followed by amelia, cutis aplasia and blood chimerism has been demonstrated (Phelan et al., 1998). In our report the anaemia and dystrophy observed in the female triplet could have been caused by blood losses because of an unidirectional strong placental blood flow to the two monozygotic siblings (Bajoria, 1998).
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Acknowledgments |
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We would like to thank Dr E. Westphal and Dr S. Jenisch (Department of Immunology) for typing the HLA class I and II antigens.
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Notes |
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6 To whom correspondence should be addressed
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Submitted on October 4, 1999; accepted on January 27, 2000.
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