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Maternal microchimerism in human fetal tissues
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BASIC SCIENCE: OBSTETRICS
Maternal microchimerism in human fetal tissues Anna Maria Jonsson, MD; Mehmet Uzunel, PhD; Cecilia Götherström, PhD; Nikos Papadogiannakis, MD, PhD; Magnus Westgren, MD, PhD OBJECTIVE: The aim of the present study was to analyze the presence of maternal cells in human fetal tissues in the second trimester. STUDY DESIGN: Tissues from 11 second-trimester fetuses terminated because of social reasons or because of malformations and/or trisomy were investigated. By cell sorting and polymerase chain reaction amplification, we studied the presence of maternal CD3⫹, CD19⫹, CD34⫹, and CD45⫹ in different fetal tissues and in placenta. RESULTS: In the group of fetuses with normal karyotype and normal
autopsy findings, 4 of 5 fetuses were positive for maternal microchi-
merism. In the group in which the fetuses were diagnosed with trisomy 21 and/or malformations, we found cells of maternal origin in 3 of 6 fetuses. CONCLUSION: The results from this study indicate that maternal microchimerism is a common phenomenon in the second-trimester fetuses. Maternal cells of lymphoid and myeloid lineages and hematopoietic progenitors are widely distributed in the second-trimester fetuses.
Key words: fetus, immunology, microchimerism, pregnancy
Cite this article as: Jonsson AM, Uzunel M, Götherström C, et al. Maternal microchimerism in human fetal tissues. Am J Obstet Gynecol 2008;198:325.e1325.e6.
T
he presence of a small amount of cells or deoxyribonucleic acid (DNA) that is derived from a genetic different individual is referred to as microchimerism. In pregnancy reciprocal cell traffic over the placenta may give rise both to fetal microchimerism in the mother and maternal microchimerism in the fetus.1,2 Fetal cell trafficking to the mother seems to be a common phenomenon, and it has been studied extensively.3-8 Conversely, microchimerism of maternal origin has gained less attention. In cord blood, presence of maternal cells is From the Departments of Obstetrics and Gynecology (Drs Jonsson and Westgren), Clinical Immunology (Drs Uzunel and Götherström), and Pathology (Dr Papadogiannakis), the Center for Fetal Medicine, Karolinska University Hospital, Huddinge, and the Karolinska Institute, Stockholm, Sweden. Received Feb. 26, 2007; revised June 28, 2007; accepted Sept. 26, 2007. Reprints: Anna Maria Jonsson Department of Gynecology and Obstetrics, Karolinska University Hospital, Huddinge, 141086 Stockholm, Sweden; [email protected]. 0002-9378/$34.00 © 2008 Mosby, Inc. All rights reserved. doi: 10.1016/j.ajog.2007.09.047
a rather common phenomenon, and various detection rates (2-100%), depending on method have been reported.9-12 It is unknown when during gestation maternal cells are passing over to the fetus, but cells of maternal origin have been detected in various concentrations in fetal blood from week 13.13 In later life some of these cells seem to persist, and maternal cells have been detected in healthy subjects up to the age of 46 years.14 Information about maternal cells in various human fetal tissues is scarce. We reported recently on presence of maternal cells in tissues of a second-trimester fetus with malformations.15 Interestingly, some of these cells expressed cell surface markers of hematopoietic stem cells. Srivatsa et al16 detected maternal cells in various organs in 4 infants who died within the first week of life. It has been speculated that maternal microchimerism may be related to the origin of several disorders during childhood such as severe combined immunodeficiency,17 juvenile dermatomyositis,18,19 pityriasis lichenoides,20 neonatal lupus syndrome,21 and biliary atresia.22 These studies are difficult to evaluate because there is a lack of information on maternal microchimerism in normal children and fetuses with regard to this aspect.
The aim of the present study was to further investigate the presence of maternal cells in different organs of secondtrimester fetuses and to characterize the subset of maternal cells in fetal tissues.
M ATERIALS AND M ETHODS Fetuses Tissue samples were collected from 5 human fetuses (cases 1-5) in which the pregnancy was terminated in the second trimester (weeks 14-17) for social reasons. Another 5 cases of termination of pregnancy from the second trimester (cases 6-10) with trisomy 21 and/or malformations were investigated (weeks 1518). A dichorionic twin pregnancy (cases 9 and 10) with trisomy 21 in both twins was included. Finally, a case of intrauterine fetal death (IUFD) diagnosed in week 27 was examined (case 11). The cases are described in Table 1. The gestational age of the fetuses was determined by ultrasound performed in the second trimester. The abortion was performed by oral administration of mifepristone 600 mg on day 1 and misoprostol 800 mg vaginally followed by 400 mg misoprostol orally every third hour until the abortion was completed on day 3. The IUFD was induced with 600 mg mifepristone followed by vaginal administration of gemeprost the following day.
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TABLE 1
Characteristics of mothers and fetuses Case #
Gestational age, wks
Reason for abortion
1
14
Social
2
14
3
Fetal weight, g
Fetal sex
HLA class mother/fetus
Fetal autopsy findings/diagnosis
42
F
HLA-A*11,32/HLA-A*11,11
Normal
Social
109
M
DRB1*04,10/DRB1*04,16
Normal
15
Social
57
M
DRB1*14,15/DRB1*01,14
Normal
4
16
Social
89
M
DRB1*0103,04/DRB1*04,08
Normal
5
17
Social
119
M
HLA-B*39,51/HLA-B*18,51
Normal
6
15
Trisomy 21
62
F
DRB1*0403,10/DRB1*10,10
Cleft palate, micrognathia, pterygium colli, artrogryposis, cystic kidneys, hypoplastic ureter
................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................
7
17
Trisomy 21
94
F
DRB1*03,1501/DRB1*03,11
Hygroma colli, no internal malformations
................................................................................................................................................................................................................................................................................................................................................................................
8
18
Malformations
236
F
HLA-A*012601 /HLA-A*01,02
Lumbosacral meningomyelocele. Normal karyotype
................................................................................................................................................................................................................................................................................................................................................................................
9
18
Dichorionic twin pregnancy, trisomy 21
195
M
DRB1*0401,13/DRB1*11,13
Hygroma colli, ventricular septum defect, abnormal lung lobation
................................................................................................................................................................................................................................................................................................................................................................................
10
18
Dichorionic twin pregnancy, trisomy 21
206
Stillbirth
680
M
DRB1*0401,13/DRB1*11,13
Hygroma colli, no internal malformations
................................................................................................................................................................................................................................................................................................................................................................................
11
27
M
DRB1*03,1103/DRB1*03,15
Facial dysmorfism, no internal malformations
Jonsson. Maternal microchimerism in human fetal tissues. Am J Obstet Gynecol 2008.
The body of the fetus was properly washed prior to autopsy according to standard procedures. The fetuses were dissected under sterile conditions, and fetal tissues were collected and placed in sterile NaCl. Peripheral blood was collected from the mothers. The study was approved by the Ethics Committee at Huddinge University Hospital (Dnr 129/01).
Cell separation and DNA extraction The fetal tissues were disintegrated by passage through a 100-m nylon mesh to form a single cell suspension and diluted in NaCl. To evaluate lineage-specific chimerism in cases 1, 3, 4, 5, 8, 9, and 10 separations of CD3⫹ (T cells), CD19⫹ (B cells), CD34⫹ (hematopoietic progenitor cells), and CD45⫹ (leukocytes) cells derived from various organs of the fetuses were made by means 325.e2
of immunomagnetic beads according to the manufacturer’s instructions (Dynal Biotech, Oslo, Norway). Each cell-separated pellet was lysed and prepared according to previously described method.23 DNA was extracted from all tissue samples using a standard saltingout procedure. Genomic DNA concentrations were measured in a Gene Quant II spectrophotometer (Pharmacia, Uppsala, Sweden)
Human leukocyte antigen (HLA)-DR typing HLA typing was performed by polymerase chain reaction (PCR) amplification with sequence-specific primers.24 We started with HLA-DR and if the sequence difference was not large enough between the fetus and the mother, we also typed for HLA-A and HLA-B. Primers specific for maternal HLA sequences were designed for each fetus-mother pair. These
American Journal of Obstetrics & Gynecology MARCH 2008
primers were tested for specificity and sensitivity. The HLA differences we used for analysis are shown in Table 1.
Detection of maternal alleles in the human fetus by PCR amplification PCR amplifications were performed with maternal-specific primers in a volume of 50 L containing 1g genomic DNA, 0.5 M of each primer, 200 M of each dNTP (Perkin-Elmer, Branchburg, CA), 1 ⫻ PCR buffer (1.5 mM MgCl, 50 mM Mg, 10 mM KCl, 0.001% gelatin), 5% glycerol, 5 g cresol red, and 1.5 U AmpliTaq polymerase (Perkin-Elmer). After an initial 3 minute hot-start/denaturation step at 94°C, 40 PCR amplification cycles were carried out in a PTC-200 thermal cycler (MJ Research, Watertown, CA). The first 10 cycles were done in a 2-segment step at 94°C for 20 seconds and at 62°C for 1 minute. The fol-
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TABLE 2
Distribution of maternal alleles in fetal tissues Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Case 7
Case 8
Case 9
Case 10
Case 11
Tissue
................................................................................................................................................................................................................................................................................................................................................................................
Lung
⫹
⫺
⫹⫹
⫺
⫹⫹
⫹⫹
⫺
⫺
⫺
⫺
⫺
Thymus
⫺
⫺
⫹⫹
⫹
⫹⫹⫹
NT
⫹⫹⫹
⫺
⫺
⫹
⫺
Kidney
⫺
NT
⫹⫹
⫺
⫹⫹⫹⫹
⫹⫹
⫺
⫺
⫺
⫹
⫺
Heart
NT
⫺
⫹⫹
⫺
⫹⫹⫹
⫹⫹
⫹⫹
⫺
⫺
⫺
⫺
Liver
⫺
⫺
⫹⫹
⫹
⫹⫹⫹⫹
⫹⫹
⫹
⫺
⫺
⫺
NT
Adrenal gland
⫺
⫺
⫹⫹
⫹
⫹⫹⫹⫹
⫹⫹
⫹⫹⫹
⫺
⫺
⫹
NT
Pancreas
NT
⫺
NT
⫹
NT
⫹⫹
⫹
⫺
⫺
⫺
NT
Spleen
⫹
⫺
⫹⫹
⫹
⫹⫹⫹
NT
⫺
⫺
⫺
⫹
NT
Brain
NT
NT
⫹⫹
NT
NT
NT
NT
NT
⫺
⫹
NT
Ovary
NT
NT
⫹⫹
NT
NT
NT
NT
⫺
NT
NT
NT
Testis
NT
⫺
⫺
⫺
⫹⫹
NT
NT
NT
⫺
⫹
NT
Placenta
NT
NT
⫹⫹
NT
NT
⫹⫹⫹⫹
NT
⫹⫹⫹⫹
⫹⫹⫹⫹
NT
NT
................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ⫺4
⫺, No maternal DNA detected; ⫹, maternal cells detected at a level of 10 NT, not tested.
⫺5
to 10
; ⫹⫹, 10
⫺3
⫺4
to 10
; ⫹⫹⫹, 10
⫺2
⫺3
to 10
⫺2
; ⫹⫹⫹⫹, greater than 10
.
Jonsson. Maternal microchimerism in human fetal tissues. Am J Obstet Gynecol 2008.
lowing 30 cycles were done in a 3-segment step at 94°C for 20 seconds, 60°C for 50 seconds, and 72°C for 20 seconds. The positive control contained maternal DNA and the negative control distilled H2O. The PCR products were separated on a ready-to-use polyacrylamide gel electrophoresis gel for 1 hour and visualized by an automated silver staining method. The band patterns were then analyzed under visible light. We considered the analyzed organs positive for maternal cells when we were able to identify a band pattern of maternal alleles on the PCR gel either in whole tissue or cell-separated fractions.
Sensitivity of PCR amplification Serial dilution experiments were performed to establish the sensitivity of the PCR amplification. Decreasing amounts of maternal DNA were mixed with DNA from a third individual, with the same HLA type as the fetus to give fixed amounts of 1 g DNA with final concentrations of maternal DNA of 1%, 0.1%, 0.01%, and 0.001%. The resulting DNA mixture was subsequently amplified using the maternal-specific primers and analyzed as described in previous text. A semiquantitative analysis was performed in which the intensity of the sample
bands on the gel was compared with the 10-fold dilution series.
R ESULTS Maternal cells were identified in 7 of 11 fetuses (Table 2). We detected maternal microchimerism in all organs investigated: lung, thymus, kidney, heart, liver, adrenal gland, pancreas, spleen, brain, ovary, and testis. Placenta was positive, as expected, in cases analyzed. In 5 cases we could not detect any maternal cells at all in fetal tissues. The earliest gestational age at which maternal alleles were found was in a 14 week old fetus. The latest was in an 18 week old fetus. The intrauterine death of a gestational age of 27 weeks was negative, but at the time of delivery, it was severely macerated, which may have affected the result. No particular fetal organs appeared more positive for maternal cells than others. There was no difference between normal and abnormal fetuses and the presence or distribution of maternal cells in the fetal tissues. Furthermore, there was no obvious correlation between positivity for maternal microchimerism and HLA type (Tables 1 and 2). The association of pregnancy history to occurrence of microchimerism is
demonstrated in Table 3. Six of the 7 positive cases were found among the multigravida (more than 1 pregnancy) women. Likewise, there was a tendency for an association between positivity for maternal microchimerism and previous fetal losses (miscarriage, termination of pregnancy) in which 6 of 7 with such history expressed maternal cells in fetal tissues. From the cell separation experiment (Table 4), it was obvious that maternal cells with both mature immunophenotype, such as T cells (CD3⫹), B (CD19⫹), cells and leucocytes (CD45⫹) as well as cells of progenitor type (CD34⫹), were present in fetal tissues. The results of the semiquantitative analysis showed no obvious differences between the 2 groups of fetuses. However, in the cases with the stronger positivity (ie, higher levels of maternal cells), there was a tendency for a uniformly strong positivity in most fetal tissues examined.
C OMMENT The present study indicates that presence of maternal cells in various fetal tissues in the second trimester is a rather common phenomenon. The maternal
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www.AJOG.org cell population was various, and we could identify cells expressing surface markers of T and B lymphocytes, leukocytes, and hematopoietic progenitor cells. Whether these cells are integrated in fetal tissues or circulating in blood of the organs is not possible to state from the present study. Maternal cells occurred in about the same frequency in all studied organs except in the placenta in which all cases were positive for maternal cells. Why fetuses are negative or positive for maternal cells is an open question. We could not identify any obvious reasons, and the material is too limited in size to explore the influences of gestational age, HLA compatibility between mother and fetus, and malformations
TABLE 3
Pregnancy history and occurrence of microchimerism Gravidity
Parity
Previous fetal loss (abortion and misscarriage)
Ocurrence of microchimerism
Case 1
1
0
0
Positive
Case 2
1
0
0
Negative
Case 3
6
1
3
Positive
Case 4
3
0
2
Positive
Case 5
4
0
3
Positive
Case 6
9
5
3
Positive
Case 7
4
2
1
Positive
Case 8
1
0
0
Negative
Case 9
5
3
1
Negative
Case 10
5
3
1
Positive
Case 11
3
2
0
Negative
.............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. ..............................................................................................................................................................................................................................................
Jonsson. Maternal microchimerism in human fetal tissues. Am J Obstet Gynecol 2008.
TABLE 4
Distribution of maternal alleles in fetal tissues after cell separation Tissue
Antigen
Case 1
Case 3
Case 4
Case 5
Case 8
Case 9
Case 10
Lung
CD3
⫹
⫹⫹
⫺
NT
⫺
NT
⫺
Lung
CD19
⫺
⫹⫹
⫺
NT
⫺
⫺
⫺
Lung
CD34
⫺
⫹⫹
⫺
⫹⫹⫹⫹
⫺
⫺
⫺
Lung
CD45
⫺
⫹⫹⫹
⫺
⫹⫹⫹⫹
⫺
⫺
⫺
Thymus
CD3
NT
⫺
⫹
⫹⫹⫹⫹
⫺
⫺
⫺
Thymus
CD19
NT
⫹
⫺
⫹⫹⫹⫹
⫺
⫺
⫺
Thymus
CD34
⫺
⫺
⫺
⫹⫹⫹⫹
⫺
NT
⫺
Thymus
CD45
NT
⫹⫹⫹
⫹
⫹⫹⫹⫹
⫺
NT
⫺
Liver
CD3
⫺
⫹⫹
⫺
⫹⫹⫹
⫺
NT
⫺
Liver
CD19
⫺
⫹⫹
⫺
⫹⫹⫹
⫺
⫺
⫺
Liver
CD34
⫺
⫹⫹⫹
⫺
⫹⫹⫹
⫺
NT
⫺
Liver
CD45
⫺
⫹⫹⫹
⫺
⫹⫹⫹⫹
⫺
⫺
⫺
Spleen
CD3
NT
⫹⫹
⫹
⫹⫹⫹⫹
⫺
NT
⫹
Spleen
CD19
NT
⫺
⫺
⫹⫹⫹⫹
⫺
⫺
⫺
Spleen
CD34
⫹
⫹
⫺
⫹⫹⫹⫹
⫺
NT
⫺
Spleen
CD45
NT
⫹⫹⫹
⫹
⫹⫹⫹⫹
⫺
⫺
⫺
Kidney
CD34
⫺
⫺
⫺
⫹⫹⫹⫹
NT
⫺
⫺
Heart
CD34
⫺
⫺
⫺
⫹⫹⫹⫹
NT
⫺
⫺
Adrenal gland
CD34
⫺
⫹
⫺
NT
NT
⫺
⫺
Pancreas
CD34
NT
NT
⫹
NT
NT
NT
⫺
Ovary
CD34
NT
NT
NT
NT
NT
NT
NT
Testis
CD34
NT
⫺
⫺
⫹⫹⫹⫹
NT
⫺
⫹
Brain
CD34
NT
⫹⫹
NT
NT
⫹
................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................
NT
NT
................................................................................................................................................................................................................................................................................................................................................................................
⫺, no maternal DNA detected; ⫹, maternal cells detected at a level of 10 NT, not tested.
⫺4
to 10
⫺5
; ⫹⫹, 10
Jonsson. Maternal microchimerism in human fetal tissues. Am J Obstet Gynecol 2008.
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American Journal of Obstetrics & Gynecology MARCH 2008
⫺3
to 10
⫺4
; ⫹⫹⫹, 10
⫺2
to 10
⫺3
; ⫹⫹⫹⫹, greater than 10
⫺2
.
www.AJOG.org with normal or abnormal karyotypes. Interestingly, in cases 9 and 10, a set of dichorionic twin pregnancy, 1 of the twins was positive for maternal cells, whereas the sibling was negative. How this could happen is unknown, but it could reflect the sensitivity of the detection method. In the present study, we used a method with a sensitivity of 1:100,000 cells. It might be that lower grades of maternal cells occurred in the negative cases of the present study. An interesting observation is the association between pregnancy history, more specifically multiple fetal loss, and occurrence of maternal microchimerism. This is in agreement with previous observations showing the influence of fetal loss on the presence of fetal cell microchimerism.25 It has been speculated that conditions associated with placental pathology facilitate the trafficking of fetal chimeric cells over the membranes during pregnancy for example preeclampsia,26-28 villitis,29,30 and aneuploidy.31,32 The present study showed that 3 of 4 fetuses with trisomy 21 were positive for maternal alleles in different tissues. Srivatsa et al16 detected the highest quantity of maternal cells in an organ of an infant with trisomy 21. This may indicate that vascular and hematological abnormalities in the trisomy 21 placenta may also facilitate the trafficking of maternal cells to the fetus. How maternal cells reach the fetal compartment is an open question. With our present knowledge, we can not state whether the trafficking of maternal cells to the fetus is a passive or active process. If it is not related to asymptomatic maternal-fetal bleedings, it could be an active migration of cells through distinctive anatomical layers. The pathophysiological consequences of maternal microchimerism in the fetus are unknown. In fetuses with severe combined immunodeficiency and a defective T cell function, it has been reported that maternal cells can engraft in fetal tissues and create life-threatening graft vs host disease (GvHD).17 How fetuses with normal immunological function are protected from this complication is unknown, but studies on fetal liver tissue have revealed that the human
Basic Science: Obstetrics fetus is capable of alloresponsiveness from the second-trimester.33 Interestingly, previous studies in human fetuses have revealed that the gestational age at which T- and natural killer– cell function is acquired varies greatly between different individuals.34 Thus, it might be that the degree of immunological maturation can influence the adaptive mechanism of the fetus to cope with maternal cells. The detailed mechanisms and the frequency of maternal GvHD against fetal tissues are not known. Villitis of unknown etiology (VUE) is probably associated with maternal GvHD reaction against placental tissue.35 The frequency of VUE is estimated to be 3-5% in term pregnancy and much lower in preterm pregnancy. One possible way to control eventual maternal GvHD against fetal/ placental tissues could be local immunosuppressive mechanisms, such as prostaglandins or alpha-fetoprotein, selectively “protecting” fetal cells.36 Because trafficking of maternal cells to the human fetus is common and occurring in normal pregnancies and although it might be a random phenomenon, one could speculate on physiological roles of this event. Exposure of the fetus to maternal cells might contribute to the development of immunological tolerance to the mother that seems to be a part of normal interaction between mother and child. Tolerance to maternal cells might also be important immediately after birth when the child is exposed to maternal antigens in breast milk. A partial tolerance is supported by the observation that at skin and organ transplantations between mother and child at HLA incompatibility do better than between father and child.37,38 The transfer of committed maternal cells to the fetus and engraftment in fetal tissues open up the possibility that maternal cells may contribute to and epigenetically affect the development of the fetal immunological and hematopoietic systems. This could be of an evolutionary advantage but also a risk for the offspring. For instances CD 34⫹ cells may give rise to endothelial precursor cells. If CD 34⫹ endothelial cells of a mother with impaired endothelial function as in hypertension pass over these precursor cells to her un-
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born child, this might result in disturbed function in the offspring. Finally, it has been reported that maternal microchimerism is associated with several disorders after birth. These relationships need to be further studied, but it is important when performing these studies to consider that maternal cells are also common in normal fetuses. The physiological role of this phenomenon is largely unknown, but it might be that it is an important part of normal physiological interf action between mother and child. REFERENCES 1. Papadogiannakis N. Traffic of leukocytes through the materno-fetal placental interface and its possible consequences. Curr Top Microbiol Immunol 1997;222:141-57. 2. Lo YM, Lau TK, Chan LY, Leung TN, Chang AM. Quantitative analysis of the bidirectional fetomaternal transfer of nucleated cells and plasma DNA. Clin Chem 2000;46:1301-9. 3. Johnson KL, Bianchi DW. Fetal cells in maternal tissue following pregnancy: what are the consequences? Hum Reprod Update 2004; 10:497-502. 4. Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S, DeMaria MA. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci USA 1996; 93:705-8. 5. O’Donoghue K, Chan J, de la Fuente J, et al. Microchimerism in female bone marrow and bone decades after fetal mesenchymal stemcell trafficking in pregnancy. Lancet 2004; 364:179-82. 6. Nelson JL, Furst DE, Maloney S, et al. Microchimerism and HLA-compatible relationships of pregnancy in scleroderma. Lancet 1998;351: 559-62. 7. Jimenez SA, Artlett CM. Microchimerism and systemic sclerosis. Curr Opin Rheumatol 2005;17:86-90. 8. Johnson KL, Bianchi DW. Fetal cells in maternal tissue following pregnancy: what are the consequences? Hum Reprod Update 2004; 10:497-502. 9. Socie G, Gluckman E, Carosella E, Brossard Y, Lafon C, Brison O. Search for maternal cells in human umbilical cord blood by polymerase chain reaction amplification of two minisatellite sequences. Blood 1994;83:340-4. 10. Hall JM, Lingenfelter P, Adams SL, Lasser D, Hansen JA, Bean MA. Detection of maternal cells in human umbilical cord blood using fluorescence in situ hybridization. Blood 1995; 86:2829-32. 11. Petit T, Gluckman E, Carosella E, Brossard Y, Brison O, Socie G. A highly sensitive polymerase chain reaction method reveals the ubiquitous presence of maternal cells in human umbilical cord blood. Exp Hematol 1995;14: 1601-5.
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12. Petit T, Dommergues M, Socie G, Dumez Y, Gluckman E, Brison O. Detection of maternal cells in human fetal blood during the third trimester of pregnancy using allele-specific PCR amplification. Br J Haematol 1997;98:767-71. 13. Lo ES, Lo YM, Hjelm NM, Thilaganathan B. Transfer of nucleated maternal cells into fetal circulation during the second trimester of pregnancy. Br J Haematol 1998;100:605-6. 14. Maloney S, Smith A, Furst DE, et al. Microchimerism of maternal origin persists into adult life. J Clin Invest 1999;104:41-7. 15. Götherström C, Jonsson AM, Matsson J, Papadogiannakis N, Westgren M. Identification of maternal CD3, 19, 34 and 45 positive cells in a second trimester fetus. Fetal Diagn Ther 2005;20:355-8. 16. Srivatsa B, Srivatsa S, Johnson KL, Bianchi DW. Maternal cell microchimerism in newborn tissues. J Pediatr 2003;142:31-5. 17. Pollack MS, Kirkpatrick D, Kapoor N, Dupont B, O’Reilly RJ. Identification by HLA typing of intrauterine-derived maternal T cells in four patients with severe combined immunodeficiency. N Engl J Med 1982;307:662-6. 18. Artlett CM, Ramos R, Jiminez SA, Patterson K, Miller FW, Rider LG. Chimeric cells of maternal origin in juvenile idiopathic inflammatory myopathies. Childhood Myositis Heterogeneity Collaborative Group. Lancet 2000;356:2155-6. 19. Reed AM, McNallan K, Wettstein P, Vehe R, Ober C. Does HLA-dependent chimerism underlie the pathogenesis of juvenile dermatomyositis? J Immunol 2004;172:5041-6. 20. Khosrotehrani K, Guegan S, Fraitag S, et al. Presence of chimeric maternally derived keratinocytes in cutaneous inflammatory diseases of children: the example of pityriasis lichenoides. J Invest Dermatol 2006;126:345-8. 21. Stevens AM, Hermes HM, Rutledge JC, Buyon JP, Nelson JL. Myocardial-tissue-spe-
325.e6
cific phenotype of maternal microchimerism in neonatal lupus congenital heart block. Lancet 2003;362:1617-23. 22. Suskind DL, Rosenthal P, Heyman MB, et al. Maternal microchimerism in the livers of patients with biliary atresia. BMC Gastroenterol 2004;14:14. 23. Mattsson J, Uzunel M, Tammik L, Aschan J, Ringden O. Leukemia lineage-specific chimerism analysis is a sensitive predictor of relapse in patients with acute myeloid leukaemia and myelodysplastic syndrome after allogeneic stem cell transplantation. Leukemia 2001;15:1976-85. 24. Olerup O, Zetterquist H. HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation. Tissue Antigens 1992;39:225-35. 25. Khosotehrani K, Johnson KL, Lau L, Dupuy A, Cha DH, Bianchi DW. The influence of fetal loss on the presence of fetal cell microchimerism. A systematic review. Arthr Rheum 2003;48:3227-41. 26. Holzgreve W, Ghezzi F, Di Naro E, Ganshirt D, Maymon E, Hahn S. Disturbed feto-maternal traffic in preeclampsia. Obstet Gynecol 1998;91:669-72. 27. Jansen MW, Korver-Hakkennes K, van Leenen D, et al. Significantly higher number of fetal cells in the maternal circulation of women with pre-eclampsia. Prenat Diagn 2001;21: 1022-6. 28. Hahn S, Huppertz B, Holzgreve W. Fetal cells and cell free fetal nucleic acids in maternal blood: new tools to study abnormal placentation? Placenta 2005;26:515-26. 29. Labarrere CA, Faulk WP. Maternal cells in chorionic villi from placenta of normal and abnormal human pregnancies. Am J Reprod Immunol 1995;33:54-9.
American Journal of Obstetrics & Gynecology MARCH 2008
www.AJOG.org 30. Myerson D, Parkin RK, Benirschke K, Tschetter CN, Hyde SR. The pathogenesis of villitis of unknown etiology: analysis with a new conjoint immunohistochemistry-in situ hybridization procedure to identify specific maternal and fetal cells. Pediatr Dev Pathol 2006;9: 257-65. 31. Bianchi DW, Williams JM, Sullivan LM, Hanson FW, Klinger KW, Shuber AP. PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies. Am J Hum Genet 1997;61:822-9. 32. Zhong XY, Burk MR, Troeger C, Jackson LR, Holzgreve W, Hahn S. Fetal DNA in maternal plasma is elevated in pregnancies with aneuploid fetuses. Prenat Diagn 2000;20:795-8. 33. Stites DP, Carr MC, Fudenberg HH. Ontogeny of cellular immunity in the human fetus: development of responses to phytohemagglutinin and to allogeneic cells. Cell Immunol 1974; 11:257-71. 34. Lindton B, Markling L, Ringden O, Kjaeldgaard A, Gustafson O, Westgren M. Mixed lymphocyte culture of human fetal liver cells. Fetal Diagn Ther 2000;15:71-8. 35. Redline RW. Placental inflammation. Semin Neonatol 2004;9:265-74. 36. Papadogiannakis N, Johnsen S-A, Olding LB. Suppressor cell activity in human cord blood. In: The immunology of the fetus. Chaouat G, ed. Boca Raton, (FL): CRC Press; 1990. p. 215. 37. Peer LA, Bernhard W, Walker JC Jr. Fullthickness skin exchanges between parents and their children. Am J Surg 1958;95:239-45. 38. van Rood JJ, Loberiza FR Jr, Zhang MJ, et al. Effect of tolerance to noninherited maternal antigens on the occurrence of graft-versus-host disease after bone marrow transplantation from a parent or an HLA-haploidentical sibling. Blood 2002;99:1572-7.
www. AJOG.org
BASIC SCIENCE: OBSTETRICS
Maternal microchimerism in human fetal tissues Anna Maria Jonsson, MD; Mehmet Uzunel, PhD; Cecilia Götherström, PhD; Nikos Papadogiannakis, MD, PhD; Magnus Westgren, MD, PhD OBJECTIVE: The aim of the present study was to analyze the presence of maternal cells in human fetal tissues in the second trimester. STUDY DESIGN: Tissues from 11 second-trimester fetuses terminated because of social reasons or because of malformations and/or trisomy were investigated. By cell sorting and polymerase chain reaction amplification, we studied the presence of maternal CD3⫹, CD19⫹, CD34⫹, and CD45⫹ in different fetal tissues and in placenta. RESULTS: In the group of fetuses with normal karyotype and normal
autopsy findings, 4 of 5 fetuses were positive for maternal microchi-
merism. In the group in which the fetuses were diagnosed with trisomy 21 and/or malformations, we found cells of maternal origin in 3 of 6 fetuses. CONCLUSION: The results from this study indicate that maternal microchimerism is a common phenomenon in the second-trimester fetuses. Maternal cells of lymphoid and myeloid lineages and hematopoietic progenitors are widely distributed in the second-trimester fetuses.
Key words: fetus, immunology, microchimerism, pregnancy
Cite this article as: Jonsson AM, Uzunel M, Götherström C, et al. Maternal microchimerism in human fetal tissues. Am J Obstet Gynecol 2008;198:325.e1325.e6.
T
he presence of a small amount of cells or deoxyribonucleic acid (DNA) that is derived from a genetic different individual is referred to as microchimerism. In pregnancy reciprocal cell traffic over the placenta may give rise both to fetal microchimerism in the mother and maternal microchimerism in the fetus.1,2 Fetal cell trafficking to the mother seems to be a common phenomenon, and it has been studied extensively.3-8 Conversely, microchimerism of maternal origin has gained less attention. In cord blood, presence of maternal cells is From the Departments of Obstetrics and Gynecology (Drs Jonsson and Westgren), Clinical Immunology (Drs Uzunel and Götherström), and Pathology (Dr Papadogiannakis), the Center for Fetal Medicine, Karolinska University Hospital, Huddinge, and the Karolinska Institute, Stockholm, Sweden. Received Feb. 26, 2007; revised June 28, 2007; accepted Sept. 26, 2007. Reprints: Anna Maria Jonsson Department of Gynecology and Obstetrics, Karolinska University Hospital, Huddinge, 141086 Stockholm, Sweden; [email protected]. 0002-9378/$34.00 © 2008 Mosby, Inc. All rights reserved. doi: 10.1016/j.ajog.2007.09.047
a rather common phenomenon, and various detection rates (2-100%), depending on method have been reported.9-12 It is unknown when during gestation maternal cells are passing over to the fetus, but cells of maternal origin have been detected in various concentrations in fetal blood from week 13.13 In later life some of these cells seem to persist, and maternal cells have been detected in healthy subjects up to the age of 46 years.14 Information about maternal cells in various human fetal tissues is scarce. We reported recently on presence of maternal cells in tissues of a second-trimester fetus with malformations.15 Interestingly, some of these cells expressed cell surface markers of hematopoietic stem cells. Srivatsa et al16 detected maternal cells in various organs in 4 infants who died within the first week of life. It has been speculated that maternal microchimerism may be related to the origin of several disorders during childhood such as severe combined immunodeficiency,17 juvenile dermatomyositis,18,19 pityriasis lichenoides,20 neonatal lupus syndrome,21 and biliary atresia.22 These studies are difficult to evaluate because there is a lack of information on maternal microchimerism in normal children and fetuses with regard to this aspect.
The aim of the present study was to further investigate the presence of maternal cells in different organs of secondtrimester fetuses and to characterize the subset of maternal cells in fetal tissues.
M ATERIALS AND M ETHODS Fetuses Tissue samples were collected from 5 human fetuses (cases 1-5) in which the pregnancy was terminated in the second trimester (weeks 14-17) for social reasons. Another 5 cases of termination of pregnancy from the second trimester (cases 6-10) with trisomy 21 and/or malformations were investigated (weeks 1518). A dichorionic twin pregnancy (cases 9 and 10) with trisomy 21 in both twins was included. Finally, a case of intrauterine fetal death (IUFD) diagnosed in week 27 was examined (case 11). The cases are described in Table 1. The gestational age of the fetuses was determined by ultrasound performed in the second trimester. The abortion was performed by oral administration of mifepristone 600 mg on day 1 and misoprostol 800 mg vaginally followed by 400 mg misoprostol orally every third hour until the abortion was completed on day 3. The IUFD was induced with 600 mg mifepristone followed by vaginal administration of gemeprost the following day.
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TABLE 1
Characteristics of mothers and fetuses Case #
Gestational age, wks
Reason for abortion
1
14
Social
2
14
3
Fetal weight, g
Fetal sex
HLA class mother/fetus
Fetal autopsy findings/diagnosis
42
F
HLA-A*11,32/HLA-A*11,11
Normal
Social
109
M
DRB1*04,10/DRB1*04,16
Normal
15
Social
57
M
DRB1*14,15/DRB1*01,14
Normal
4
16
Social
89
M
DRB1*0103,04/DRB1*04,08
Normal
5
17
Social
119
M
HLA-B*39,51/HLA-B*18,51
Normal
6
15
Trisomy 21
62
F
DRB1*0403,10/DRB1*10,10
Cleft palate, micrognathia, pterygium colli, artrogryposis, cystic kidneys, hypoplastic ureter
................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................
7
17
Trisomy 21
94
F
DRB1*03,1501/DRB1*03,11
Hygroma colli, no internal malformations
................................................................................................................................................................................................................................................................................................................................................................................
8
18
Malformations
236
F
HLA-A*012601 /HLA-A*01,02
Lumbosacral meningomyelocele. Normal karyotype
................................................................................................................................................................................................................................................................................................................................................................................
9
18
Dichorionic twin pregnancy, trisomy 21
195
M
DRB1*0401,13/DRB1*11,13
Hygroma colli, ventricular septum defect, abnormal lung lobation
................................................................................................................................................................................................................................................................................................................................................................................
10
18
Dichorionic twin pregnancy, trisomy 21
206
Stillbirth
680
M
DRB1*0401,13/DRB1*11,13
Hygroma colli, no internal malformations
................................................................................................................................................................................................................................................................................................................................................................................
11
27
M
DRB1*03,1103/DRB1*03,15
Facial dysmorfism, no internal malformations
Jonsson. Maternal microchimerism in human fetal tissues. Am J Obstet Gynecol 2008.
The body of the fetus was properly washed prior to autopsy according to standard procedures. The fetuses were dissected under sterile conditions, and fetal tissues were collected and placed in sterile NaCl. Peripheral blood was collected from the mothers. The study was approved by the Ethics Committee at Huddinge University Hospital (Dnr 129/01).
Cell separation and DNA extraction The fetal tissues were disintegrated by passage through a 100-m nylon mesh to form a single cell suspension and diluted in NaCl. To evaluate lineage-specific chimerism in cases 1, 3, 4, 5, 8, 9, and 10 separations of CD3⫹ (T cells), CD19⫹ (B cells), CD34⫹ (hematopoietic progenitor cells), and CD45⫹ (leukocytes) cells derived from various organs of the fetuses were made by means 325.e2
of immunomagnetic beads according to the manufacturer’s instructions (Dynal Biotech, Oslo, Norway). Each cell-separated pellet was lysed and prepared according to previously described method.23 DNA was extracted from all tissue samples using a standard saltingout procedure. Genomic DNA concentrations were measured in a Gene Quant II spectrophotometer (Pharmacia, Uppsala, Sweden)
Human leukocyte antigen (HLA)-DR typing HLA typing was performed by polymerase chain reaction (PCR) amplification with sequence-specific primers.24 We started with HLA-DR and if the sequence difference was not large enough between the fetus and the mother, we also typed for HLA-A and HLA-B. Primers specific for maternal HLA sequences were designed for each fetus-mother pair. These
American Journal of Obstetrics & Gynecology MARCH 2008
primers were tested for specificity and sensitivity. The HLA differences we used for analysis are shown in Table 1.
Detection of maternal alleles in the human fetus by PCR amplification PCR amplifications were performed with maternal-specific primers in a volume of 50 L containing 1g genomic DNA, 0.5 M of each primer, 200 M of each dNTP (Perkin-Elmer, Branchburg, CA), 1 ⫻ PCR buffer (1.5 mM MgCl, 50 mM Mg, 10 mM KCl, 0.001% gelatin), 5% glycerol, 5 g cresol red, and 1.5 U AmpliTaq polymerase (Perkin-Elmer). After an initial 3 minute hot-start/denaturation step at 94°C, 40 PCR amplification cycles were carried out in a PTC-200 thermal cycler (MJ Research, Watertown, CA). The first 10 cycles were done in a 2-segment step at 94°C for 20 seconds and at 62°C for 1 minute. The fol-
Research
Basic Science: Obstetrics
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TABLE 2
Distribution of maternal alleles in fetal tissues Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Case 7
Case 8
Case 9
Case 10
Case 11
Tissue
................................................................................................................................................................................................................................................................................................................................................................................
Lung
⫹
⫺
⫹⫹
⫺
⫹⫹
⫹⫹
⫺
⫺
⫺
⫺
⫺
Thymus
⫺
⫺
⫹⫹
⫹
⫹⫹⫹
NT
⫹⫹⫹
⫺
⫺
⫹
⫺
Kidney
⫺
NT
⫹⫹
⫺
⫹⫹⫹⫹
⫹⫹
⫺
⫺
⫺
⫹
⫺
Heart
NT
⫺
⫹⫹
⫺
⫹⫹⫹
⫹⫹
⫹⫹
⫺
⫺
⫺
⫺
Liver
⫺
⫺
⫹⫹
⫹
⫹⫹⫹⫹
⫹⫹
⫹
⫺
⫺
⫺
NT
Adrenal gland
⫺
⫺
⫹⫹
⫹
⫹⫹⫹⫹
⫹⫹
⫹⫹⫹
⫺
⫺
⫹
NT
Pancreas
NT
⫺
NT
⫹
NT
⫹⫹
⫹
⫺
⫺
⫺
NT
Spleen
⫹
⫺
⫹⫹
⫹
⫹⫹⫹
NT
⫺
⫺
⫺
⫹
NT
Brain
NT
NT
⫹⫹
NT
NT
NT
NT
NT
⫺
⫹
NT
Ovary
NT
NT
⫹⫹
NT
NT
NT
NT
⫺
NT
NT
NT
Testis
NT
⫺
⫺
⫺
⫹⫹
NT
NT
NT
⫺
⫹
NT
Placenta
NT
NT
⫹⫹
NT
NT
⫹⫹⫹⫹
NT
⫹⫹⫹⫹
⫹⫹⫹⫹
NT
NT
................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ⫺4
⫺, No maternal DNA detected; ⫹, maternal cells detected at a level of 10 NT, not tested.
⫺5
to 10
; ⫹⫹, 10
⫺3
⫺4
to 10
; ⫹⫹⫹, 10
⫺2
⫺3
to 10
⫺2
; ⫹⫹⫹⫹, greater than 10
.
Jonsson. Maternal microchimerism in human fetal tissues. Am J Obstet Gynecol 2008.
lowing 30 cycles were done in a 3-segment step at 94°C for 20 seconds, 60°C for 50 seconds, and 72°C for 20 seconds. The positive control contained maternal DNA and the negative control distilled H2O. The PCR products were separated on a ready-to-use polyacrylamide gel electrophoresis gel for 1 hour and visualized by an automated silver staining method. The band patterns were then analyzed under visible light. We considered the analyzed organs positive for maternal cells when we were able to identify a band pattern of maternal alleles on the PCR gel either in whole tissue or cell-separated fractions.
Sensitivity of PCR amplification Serial dilution experiments were performed to establish the sensitivity of the PCR amplification. Decreasing amounts of maternal DNA were mixed with DNA from a third individual, with the same HLA type as the fetus to give fixed amounts of 1 g DNA with final concentrations of maternal DNA of 1%, 0.1%, 0.01%, and 0.001%. The resulting DNA mixture was subsequently amplified using the maternal-specific primers and analyzed as described in previous text. A semiquantitative analysis was performed in which the intensity of the sample
bands on the gel was compared with the 10-fold dilution series.
R ESULTS Maternal cells were identified in 7 of 11 fetuses (Table 2). We detected maternal microchimerism in all organs investigated: lung, thymus, kidney, heart, liver, adrenal gland, pancreas, spleen, brain, ovary, and testis. Placenta was positive, as expected, in cases analyzed. In 5 cases we could not detect any maternal cells at all in fetal tissues. The earliest gestational age at which maternal alleles were found was in a 14 week old fetus. The latest was in an 18 week old fetus. The intrauterine death of a gestational age of 27 weeks was negative, but at the time of delivery, it was severely macerated, which may have affected the result. No particular fetal organs appeared more positive for maternal cells than others. There was no difference between normal and abnormal fetuses and the presence or distribution of maternal cells in the fetal tissues. Furthermore, there was no obvious correlation between positivity for maternal microchimerism and HLA type (Tables 1 and 2). The association of pregnancy history to occurrence of microchimerism is
demonstrated in Table 3. Six of the 7 positive cases were found among the multigravida (more than 1 pregnancy) women. Likewise, there was a tendency for an association between positivity for maternal microchimerism and previous fetal losses (miscarriage, termination of pregnancy) in which 6 of 7 with such history expressed maternal cells in fetal tissues. From the cell separation experiment (Table 4), it was obvious that maternal cells with both mature immunophenotype, such as T cells (CD3⫹), B (CD19⫹), cells and leucocytes (CD45⫹) as well as cells of progenitor type (CD34⫹), were present in fetal tissues. The results of the semiquantitative analysis showed no obvious differences between the 2 groups of fetuses. However, in the cases with the stronger positivity (ie, higher levels of maternal cells), there was a tendency for a uniformly strong positivity in most fetal tissues examined.
C OMMENT The present study indicates that presence of maternal cells in various fetal tissues in the second trimester is a rather common phenomenon. The maternal
MARCH 2008 American Journal of Obstetrics & Gynecology
325.e3
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www.AJOG.org cell population was various, and we could identify cells expressing surface markers of T and B lymphocytes, leukocytes, and hematopoietic progenitor cells. Whether these cells are integrated in fetal tissues or circulating in blood of the organs is not possible to state from the present study. Maternal cells occurred in about the same frequency in all studied organs except in the placenta in which all cases were positive for maternal cells. Why fetuses are negative or positive for maternal cells is an open question. We could not identify any obvious reasons, and the material is too limited in size to explore the influences of gestational age, HLA compatibility between mother and fetus, and malformations
TABLE 3
Pregnancy history and occurrence of microchimerism Gravidity
Parity
Previous fetal loss (abortion and misscarriage)
Ocurrence of microchimerism
Case 1
1
0
0
Positive
Case 2
1
0
0
Negative
Case 3
6
1
3
Positive
Case 4
3
0
2
Positive
Case 5
4
0
3
Positive
Case 6
9
5
3
Positive
Case 7
4
2
1
Positive
Case 8
1
0
0
Negative
Case 9
5
3
1
Negative
Case 10
5
3
1
Positive
Case 11
3
2
0
Negative
.............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. ..............................................................................................................................................................................................................................................
Jonsson. Maternal microchimerism in human fetal tissues. Am J Obstet Gynecol 2008.
TABLE 4
Distribution of maternal alleles in fetal tissues after cell separation Tissue
Antigen
Case 1
Case 3
Case 4
Case 5
Case 8
Case 9
Case 10
Lung
CD3
⫹
⫹⫹
⫺
NT
⫺
NT
⫺
Lung
CD19
⫺
⫹⫹
⫺
NT
⫺
⫺
⫺
Lung
CD34
⫺
⫹⫹
⫺
⫹⫹⫹⫹
⫺
⫺
⫺
Lung
CD45
⫺
⫹⫹⫹
⫺
⫹⫹⫹⫹
⫺
⫺
⫺
Thymus
CD3
NT
⫺
⫹
⫹⫹⫹⫹
⫺
⫺
⫺
Thymus
CD19
NT
⫹
⫺
⫹⫹⫹⫹
⫺
⫺
⫺
Thymus
CD34
⫺
⫺
⫺
⫹⫹⫹⫹
⫺
NT
⫺
Thymus
CD45
NT
⫹⫹⫹
⫹
⫹⫹⫹⫹
⫺
NT
⫺
Liver
CD3
⫺
⫹⫹
⫺
⫹⫹⫹
⫺
NT
⫺
Liver
CD19
⫺
⫹⫹
⫺
⫹⫹⫹
⫺
⫺
⫺
Liver
CD34
⫺
⫹⫹⫹
⫺
⫹⫹⫹
⫺
NT
⫺
Liver
CD45
⫺
⫹⫹⫹
⫺
⫹⫹⫹⫹
⫺
⫺
⫺
Spleen
CD3
NT
⫹⫹
⫹
⫹⫹⫹⫹
⫺
NT
⫹
Spleen
CD19
NT
⫺
⫺
⫹⫹⫹⫹
⫺
⫺
⫺
Spleen
CD34
⫹
⫹
⫺
⫹⫹⫹⫹
⫺
NT
⫺
Spleen
CD45
NT
⫹⫹⫹
⫹
⫹⫹⫹⫹
⫺
⫺
⫺
Kidney
CD34
⫺
⫺
⫺
⫹⫹⫹⫹
NT
⫺
⫺
Heart
CD34
⫺
⫺
⫺
⫹⫹⫹⫹
NT
⫺
⫺
Adrenal gland
CD34
⫺
⫹
⫺
NT
NT
⫺
⫺
Pancreas
CD34
NT
NT
⫹
NT
NT
NT
⫺
Ovary
CD34
NT
NT
NT
NT
NT
NT
NT
Testis
CD34
NT
⫺
⫺
⫹⫹⫹⫹
NT
⫺
⫹
Brain
CD34
NT
⫹⫹
NT
NT
⫹
................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................
NT
NT
................................................................................................................................................................................................................................................................................................................................................................................
⫺, no maternal DNA detected; ⫹, maternal cells detected at a level of 10 NT, not tested.
⫺4
to 10
⫺5
; ⫹⫹, 10
Jonsson. Maternal microchimerism in human fetal tissues. Am J Obstet Gynecol 2008.
325.e4
American Journal of Obstetrics & Gynecology MARCH 2008
⫺3
to 10
⫺4
; ⫹⫹⫹, 10
⫺2
to 10
⫺3
; ⫹⫹⫹⫹, greater than 10
⫺2
.
www.AJOG.org with normal or abnormal karyotypes. Interestingly, in cases 9 and 10, a set of dichorionic twin pregnancy, 1 of the twins was positive for maternal cells, whereas the sibling was negative. How this could happen is unknown, but it could reflect the sensitivity of the detection method. In the present study, we used a method with a sensitivity of 1:100,000 cells. It might be that lower grades of maternal cells occurred in the negative cases of the present study. An interesting observation is the association between pregnancy history, more specifically multiple fetal loss, and occurrence of maternal microchimerism. This is in agreement with previous observations showing the influence of fetal loss on the presence of fetal cell microchimerism.25 It has been speculated that conditions associated with placental pathology facilitate the trafficking of fetal chimeric cells over the membranes during pregnancy for example preeclampsia,26-28 villitis,29,30 and aneuploidy.31,32 The present study showed that 3 of 4 fetuses with trisomy 21 were positive for maternal alleles in different tissues. Srivatsa et al16 detected the highest quantity of maternal cells in an organ of an infant with trisomy 21. This may indicate that vascular and hematological abnormalities in the trisomy 21 placenta may also facilitate the trafficking of maternal cells to the fetus. How maternal cells reach the fetal compartment is an open question. With our present knowledge, we can not state whether the trafficking of maternal cells to the fetus is a passive or active process. If it is not related to asymptomatic maternal-fetal bleedings, it could be an active migration of cells through distinctive anatomical layers. The pathophysiological consequences of maternal microchimerism in the fetus are unknown. In fetuses with severe combined immunodeficiency and a defective T cell function, it has been reported that maternal cells can engraft in fetal tissues and create life-threatening graft vs host disease (GvHD).17 How fetuses with normal immunological function are protected from this complication is unknown, but studies on fetal liver tissue have revealed that the human
Basic Science: Obstetrics fetus is capable of alloresponsiveness from the second-trimester.33 Interestingly, previous studies in human fetuses have revealed that the gestational age at which T- and natural killer– cell function is acquired varies greatly between different individuals.34 Thus, it might be that the degree of immunological maturation can influence the adaptive mechanism of the fetus to cope with maternal cells. The detailed mechanisms and the frequency of maternal GvHD against fetal tissues are not known. Villitis of unknown etiology (VUE) is probably associated with maternal GvHD reaction against placental tissue.35 The frequency of VUE is estimated to be 3-5% in term pregnancy and much lower in preterm pregnancy. One possible way to control eventual maternal GvHD against fetal/ placental tissues could be local immunosuppressive mechanisms, such as prostaglandins or alpha-fetoprotein, selectively “protecting” fetal cells.36 Because trafficking of maternal cells to the human fetus is common and occurring in normal pregnancies and although it might be a random phenomenon, one could speculate on physiological roles of this event. Exposure of the fetus to maternal cells might contribute to the development of immunological tolerance to the mother that seems to be a part of normal interaction between mother and child. Tolerance to maternal cells might also be important immediately after birth when the child is exposed to maternal antigens in breast milk. A partial tolerance is supported by the observation that at skin and organ transplantations between mother and child at HLA incompatibility do better than between father and child.37,38 The transfer of committed maternal cells to the fetus and engraftment in fetal tissues open up the possibility that maternal cells may contribute to and epigenetically affect the development of the fetal immunological and hematopoietic systems. This could be of an evolutionary advantage but also a risk for the offspring. For instances CD 34⫹ cells may give rise to endothelial precursor cells. If CD 34⫹ endothelial cells of a mother with impaired endothelial function as in hypertension pass over these precursor cells to her un-
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born child, this might result in disturbed function in the offspring. Finally, it has been reported that maternal microchimerism is associated with several disorders after birth. These relationships need to be further studied, but it is important when performing these studies to consider that maternal cells are also common in normal fetuses. The physiological role of this phenomenon is largely unknown, but it might be that it is an important part of normal physiological interf action between mother and child. REFERENCES 1. Papadogiannakis N. Traffic of leukocytes through the materno-fetal placental interface and its possible consequences. Curr Top Microbiol Immunol 1997;222:141-57. 2. Lo YM, Lau TK, Chan LY, Leung TN, Chang AM. Quantitative analysis of the bidirectional fetomaternal transfer of nucleated cells and plasma DNA. Clin Chem 2000;46:1301-9. 3. Johnson KL, Bianchi DW. Fetal cells in maternal tissue following pregnancy: what are the consequences? Hum Reprod Update 2004; 10:497-502. 4. Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S, DeMaria MA. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci USA 1996; 93:705-8. 5. O’Donoghue K, Chan J, de la Fuente J, et al. Microchimerism in female bone marrow and bone decades after fetal mesenchymal stemcell trafficking in pregnancy. Lancet 2004; 364:179-82. 6. Nelson JL, Furst DE, Maloney S, et al. Microchimerism and HLA-compatible relationships of pregnancy in scleroderma. Lancet 1998;351: 559-62. 7. Jimenez SA, Artlett CM. Microchimerism and systemic sclerosis. Curr Opin Rheumatol 2005;17:86-90. 8. Johnson KL, Bianchi DW. Fetal cells in maternal tissue following pregnancy: what are the consequences? Hum Reprod Update 2004; 10:497-502. 9. Socie G, Gluckman E, Carosella E, Brossard Y, Lafon C, Brison O. Search for maternal cells in human umbilical cord blood by polymerase chain reaction amplification of two minisatellite sequences. Blood 1994;83:340-4. 10. Hall JM, Lingenfelter P, Adams SL, Lasser D, Hansen JA, Bean MA. Detection of maternal cells in human umbilical cord blood using fluorescence in situ hybridization. Blood 1995; 86:2829-32. 11. Petit T, Gluckman E, Carosella E, Brossard Y, Brison O, Socie G. A highly sensitive polymerase chain reaction method reveals the ubiquitous presence of maternal cells in human umbilical cord blood. Exp Hematol 1995;14: 1601-5.
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