Human Reproduction vol.15 no.5 pp.1200–1204, 2000
CASE REPORT
Equal distribution of congenital blood cell chimerism in
dizygotic triplets after in-vitro fertilization
Rita Kühl-Burmeister1,6, Eva Simeoni2,
Klaus Weber-Matthiesen3, Antje Milde2,
Catherine Herwartz4, Jürgen Neppert1 and
Meinolf Suttorp5
Department of 1Transfusion Medicine, 2Forensic Medicine,
3Human Genetics, 4Immunology and 5Paediatrics, University of
Kiel Medical School, Michaelisstrasse 5, D-24105 Kiel, Germany
6To
whom correspondence should be addressed
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 fertilization
Introduction
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 mono1200
chorionic 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 immunotolerance 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).
Case report
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 invitro 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
© European Society of Human Reproduction and Embryology
Blood chimerism after IVF
Table I. Serological findings
Child 1,
female
Child 2,
male
Child 3,
male
Mother
Father
ABO
Rh system
A/
AB
A/
AB
A/
AB
A
B
CcD.ee/
CcD.Ee
CcD.ee/
CcD.Ee
CcD.ee/
CcD.Ee
CCD.ee
CcD.Ee
E-positive
fluorescence
(%)
Leukocytes
XX/XY
(%)/(%)
Germ line
Tf
Pi
PLG
Gc
AB
C2
M1M3
6–1
2–1S
9
16/84
7
8/92
A
C2-1
M1M2
2–1
2
9
11/89
A
C2-1
M1M2
2–1
2
1
98
100/0
0/100
A
B
C2-1
C2
M2M3
M1
6–2
1
2–1S
2–1S
Blood group antigens and serum groups in the triplet family [antigens of the germ line cells analysed on hairs; E-positive population of erythrocytes
demonstrated by flow cytometric analysis; distribution of the peripheral leukocytes detected by in-situ hybridization with X- and Y-specific DNA probes;
polymorphisms of the serum groups transferrin (Tf), protease inhibitor (Pi), plasminogen (PLG) and glycoprotein (Gc) analysed].
Figure 1. Dot plot quadrant analysis by flow cytometry of
peripheral erythrocytes (triplet 3, male, 6 years) following
incubation with monoclonal IgM anti-E and anti-human IgM
fluorescein conjugate. Cells in quadrant 2 are considered to be
E-positive. The quantitative distribution of the two red cell
populations corresponds to that in the samples from his siblings.
phenotype of the mother’s erythrocytes was A1, CCD.ee, the
father’s B, ccD.Ee (Table I).
The two erythrocyte populations were detected by the flow
cytometry method with human Anti-E (primary antibody,
monoclonal IgM, Biotest, Dreieich, Germany) and goat antihuman 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.
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;
Figure 2. PCR of Y-chromosomal DNA in leukocytes from the
triplets and their parents: an amplification signal (arrow) is
detectable in a specimen from the female triplet. Lane 1: negative
control (female); lane 2: positive control (male); lane 3: 123 bp
ladder; lane 4: triplet 1, female; lane 5: triplet 2, male; lane 6:
triplet 3, male; lane 7: father; lane 8: mother.
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
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R.Kühl-Burmeister et al.
Table II. Leukocyte DNA analysis of the chimeric triplets and their parents
with the single locus probe technique in eight VNTR regions
Figure 3. Fluorescence-in-situ hybridization of peripheral
leukocytes (triplet 1, female) performed with human Y and X
specific DNA probes DYZ3 and DXZ1. X- (green) and Y-specific
(red, arrow) fluorescence of the chromosomes is seen in the upper
cell, the other shows the normal female XX chromosomes.
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).
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).
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).
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Probe/system
Mother
Child 1
female
Child 2
male
Child 3
male
Father
MS1
D1S7
MS31
D7S21
13.2
8.6
6.84
5.87
8.29
6.89
G3
D7S22
6.28
3.19
YNH24
D2S44
LH1
D4S110
MS205
D16S309
3.12
2.50
4.38
3.99
4.43
2.22
TBQ7
D10S28
4.29
4.07
8.48
4.84
7.57
6.85
5.87
8.85
8.31
6.89
6.84
3.19
1.63
3.37
3.14
4.00
2.95
4.43
3.17
2.23
2.10
4.73
4.55
4.29
4.08
8.52
4.85
7.61
6.84
5.87
8.79
8.27
6.89
6.85
3.19
1.63
3.37
3.12
3.97
2.93
4.43
3.17
2.21
2.10
4.69
4.55
4.29
4.05
6.9
4.8
7.55
6.43
MS43a
D12S11
8.59
4.84
7.61
6.84
5.87
8.90
8.30
6.89
6.84
3.19
1.63
3.37
3.14
3.99
2.94
4.43
3.17
2.22
2.11
4.73
4.55
4.29
4.08
8.79
5.32
6.89
1.63
4.06
3.37
4.05
2.93
3.17
2.10
4.74
4.55
Values are given in kb, following digestion with Hinf I (maternal values in
boldface type).
The analysis with HumAmelX/Y showed X and Y bands in
the female child (Figure 4).
Discussion
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 after IVF
Figure 4. PCR amplification with the HumAmelX/Y gender
identification system (I) and HumVWA (II): peripheral blood
samples (B) from the female chimeric child showed X and Y bands
in I, and the same mixed band pattern for each child in II: three
bands were demonstrated, corresponding to the alleles 15, 16 and
19 (weakly expressed). PCR amplification with DNA extracted
from buccal swabs (A) indicated a normal constellation among the
whole family. 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. M: allelic ladder, 1: mother, 2: child 1,
female, 3: child 2, male, 4: child 3, male, 5: father.
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).
Acknowledgements
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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Received on October 4, 1999; accepted on January 27, 2000
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