Molecular Human Reproduction, Vol. 6, No. 6, 535-540,
June 2000
© 2000 European Society of Human Reproduction and Embryology
Pregnancy |
HLA-G expression in extravillous trophoblasts is an intrinsic property of cell differentiation: a lesson learned from ectopic pregnancies
1 Department of Obstetrics and Gynecology and 2 Department of Pathology Hadassah University Hospital, Mt. Scopus, Jerusalem, 91240, Israel. E-mail: syagel{at}yam-suff.huji.ac.il
Abstract
Human leukocyte antigen (HLA)-G is a major histocompatibility gene expressed almost exclusively in extravillous trophoblasts at the fetal–maternal interface. HLA-G may play a role in protecting the fetus from attack by the maternal natural killer cells. The extravillous trophoblasts invade the decidua and maternal spiral arteries. The factors which regulate the cell-specific expression of HLA-G are unknown. In this study we asked if HLA-G is expressed in extravillous trophoblasts that develop outside of their normal cellular environment, as in the case of ectopic pregnancies. Since all ectopic pregnancies implant in the absence of underlying decidua we also used a placenta accreta as an experimental control. We found that HLA-G mRNA and protein were expressed in the extravillous trophoblasts in the 13 ectopic specimens studied. In a case of placenta accreta (which develops without decidua basalis and is therefore adherent to the underlying myometrium), HLA-G mRNA and protein were also expressed. These results suggest that HLA-G expression is induced in a cell autonomous manner rather than determined by appropriate environmental cues.
ectopic pregnancy/HLA-G/placenta/placenta accreta/trophoblast
Introduction
Human leukocyte antigen (HLA)-G is a nonclassical major histocompatibility antigen with a restricted pattern of expression (Kovats et al., 1990
). In the placenta, throughout the three trimesters of pregnancy, HLA-G is found in the invasive extravillous trophoblast cell lineage (Yelavarthi et al., 1991; Chumbley et al., 1993; McMaster et al., 1995). Extravillous trophoblasts are epithelial cells derived from the trophoectoderm of the developing blastocyst. They form cell columns which anchor the placenta to the uterus and invade the decidua (interstitial invasion) and the maternal spiral arteries (endovascular invasion). Expression of HLA-G is related to the degree of invasiveness of the trophoblasts, as was observed both in vivo and in vitro. In vivo, both protein and mRNA expression of HLA-G peak in the first trimester when the cells are most invasive. In addition, when primary trophoblasts are isolated and cultured, HLA-G expression increases as the cells differentiate along the invasive pathway (McMaster et al., 1995). When invasion is defective and shallow as in the toxaemia of pregnancy, pre-eclampsia, protein and mRNA expression of HLA-G is reduced both in vivo and in isolated trophoblasts in vitro (Hara et al., 1996; Lim et al., 1997; Goldman-Wohl, 2000). These studies suggest a strong positive correlation between the invasive phenotype of trophoblasts and HLA-G expression. Furthermore, HLA-G expression is not found in the non-invasive syncytiotrophoblast of the chorionic villi (McMaster et al., 1995).
Although in direct contact with maternal blood and tissue, invasive trophoblasts are not rejected by the mother. Trophoblasts, of fetal origin, should theoretically be rejected by the mother as hemiallografts. But all trophoblasts, villous as well as extravillous, lack expression of the highly polymorphic HLA class I antigens A and B (Redman, 1983; Bulmer and Johnson, 1985; reviewed in Christiansen, 1999; Fernadez et al., 1999). Lack of HLA expression by trophoblasts could explain why the maternal T cells do not recognize trophoblasts as foreign. However, NK (natural killer) cells are the major lymphocyte population at the fetal–maternal interface. NK cells lyse cells without HLA molecules (reviewed in King et al., 1997; Loke and King, 1997). The invasive trophoblasts encounter the maternal NK cells when they invade the uterine tissue. Expression of HLA-G by invasive trophoblasts may afford protection from attack by the maternal NK cells in the uterus. A pivotal role for HLA-G in protecting invasive trophoblasts from maternal immune rejection is supported by recent observations that HLA-G prevents lysis by NK cells in vitro (Pazmany et al., 1996; Rouas-Freiss et al., 1997; reviewed in Yokoyama, 1997). Recent work using cell transfectants co-expressing HLA-G and HLA-E and assayed with NK clones suggests that protection afforded by HLA-G may be due to its regulation of HLA-E through its leader sequence (Navarro et al., 1999). The significance of these results with respect to the situation in the placenta is unknown. Furthermore, HLA-G, unlike other HLA molecules, is not highly polymorphic and thus the paternal HLA-G may not be recognized as foreign (Shukla et al., 1990; Pook et al., 1991; Ishitani and Geraghty, 1992; Morales et al., 1993; Tamaki et al., 1993; Ober et al., 1996; Yamashita et al., 1996; Hviid et al., 1997). These studies lend support to the notion that HLA-G expression in invasive trophoblasts may protect these fetal cells from maternal immune surveillance. Distribution of the HLA-G protein and its role in the placenta has been reviewed (Bouteiller et al., 1999).
Attempts have been made to reveal elements in the promoter of HLA-G which govern its restricted expression pattern but no specific DNA sequences which confer the unique transcriptional regulation have yet been found (Schmidt et al., 1993, 1995; and our unpublished results). However, the elements which govern the transcriptional regulation of classical major histocompatibility complex (MHC) class I promoters are well characterized (Israel et al., 1986, 1989; Kimura et al., 1986; Baldwin and Sharp, 1987; Blaner et al., 1989). Although specific factors which control the tight transcriptional regulation of HLA-G are not yet known, they may be induced by appropriate environmental cues which are unique to the maternal–fetal interface. Alternatively, expression of HLA-G may be an intrinsic property of the state of differentiation of the extravillous trophoblast cell lineage. This fundamental question in developmental biology, of whether environment or cell autonomy induces gene expression, is rarely amenable to study in the human system. We were able to address this question for HLA-G by studying specimens from 13 ectopic pregnancies as well as in a placenta accreta used as a control.
Materials and methods
Specimen collection
A paraffin-embedded block from a placenta accreta was selected from the archives of the Pathology Department at Hadassah Hospital, Mount Scopus. A placenta accreta develops without decidua basalis and is therefore adherent to the underlying myometrium. Normal first trimester placentae were taken from elective pregnancy terminations all in accordance with the protocol of the Human Subjects Committee approved by our institution.
Reports of 430 cases of ectopic pregnancies were detected in a medical records search at Mount Scopus Hospital, covering the past 6 years. We selected ectopic pregnancies in which a heartbeat had been detected by ultrasonography to obtain samples of presumed healthy tissue for in-situ hybridization analysis (which requires intact mRNA). To detect extravillous trophoblasts (the cells which normally express HLA-G), we performed immunohistochemistry for cytokeratin on the paraffin-embedded sections. The slides were examined by a perinatal pathologist and those specimens with extravillous trophoblasts were chosen for in-situ analysis. To obtain further samples we expanded our search parameters and examined 40 ectopic specimens by cytokeratin immunohistochemistry and selected those with extravillous trophoblasts. In total we characterized through mRNA in-situ hybridization analysis 13 ectopic specimens for HLA-G expression, seven with heartbeats and six without.
In-situ hybridization
Radioactive RNA in-situ hybridization with a [35S]UTP-labelled HLA-G antisense riboprobe was performed as described by us (Goldman-Wohl et al., 2000).
Immunohistochemistry
Anti-cytokeratin immunohistochemistry, with a broad spectrum keratin monoclonal mouse primary antibody, was performed on serial sections according to the Histostain Plus kit, Zymed Lab-SA System (South San Francisco, CA, USA), and developed with their AEC chromogen mixture. Anti-HLA-G immunohistochemistry was performed with an HLA-G monoclonal antibody (McMaster et al., 1995) and the Histostain Plus kit. We added an antigen retrieval (microwave) protocol (Copeman et al., 2000), 5 min in 0.01 mol/l citrate buffer pH 6.0 after dewaxing.
Results
HLA-G expression in trophoblasts in normal first trimester pregnancies
We observed HLA-G expression in extravillous trophoblasts (Figure 1A and B) through mRNA in-situ hybridization analysis of first trimester placental sections with an antisense HLA-G riboprobe. No expression was detected in the syncytiotrophoblast nor in the villous cytotrophoblasts. Hybridization with an HLA-G sense riboprobe did not reveal hybridization above background levels, thus confirming our probe specificity (Figure 2A and B).
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Trophoblasts in ectopic pregnancies
Table I
summarizes the clinical profile of the patient cohort. The ultrasound findings record seven cases with an embryonic heartbeat and six without. Laparoscopy and pathology confirmed the diagnosis of an ectopic pregnancy. Trophoblasts were identified in all 13 specimens by cytokeratin immunohistochemistry. Positive staining for cytokeratin, which stains all trophoblasts, is observed in Figure 3A and B.
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mRNA expression of HLA-G in ectopic pregnancies and placenta accreta
Using mRNA in-situ hybridization analysis, we found that intermediate trophoblasts were positive for HLA-G expression present either in the lumen of the Fallopian tube or invading its wall (Figure 4A–C
). All 13 ectopic specimens were positive for HLA-G mRNA expression. As a further example of implantation in an inappropriate environment, we studied a placenta accreta. We observed extravillous trophoblasts positive for HLA-G expression (Figure 3D and E
) at the implantation site directly overlying the myometrium without intervening decidua.
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HLA-G protein expression in ectopic pregnancies and placenta accreta
Using antigen retrieval and an antibody against HLA-G on the paraffin-embedded sections, we detected positive HLA-G expression in all 13 ectopic specimens (Figure 5A and B
) and in the placenta accreta (Figure 5C). Thus HLA-G protein is also expressed in extravillous trophoblasts at an ectopic site.
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Discussion
In all 13 ectopic pregnancies examined, HLA-G mRNA and protein were expressed in extravillous trophoblasts. We conclude that HLA-G expression is most likely an intrinsic property of the differentiation of extravillous trophoblasts and is not dependent on appropriate environmental cues determined by interaction with the decidua. Since ectopic placentae are either accreta (where the villous tissue has grown into the tubal musculature without intervening decidua) or percreta (where the villi penetrate and rupture the Fallopian tube) we used a placenta accreta as a control and found that extravillous trophoblasts in the placenta accreta expressed HLA-G. The extravillous trophoblasts in ectopic pregnancies, unlike in a normal pregnancy, invade the Fallopian tubes and not the decidua. The extravillous trophoblasts in a placenta accreta also encounter an abnormal environment because the trophoblasts enter the myometrium without underlying decidua. Thus HLA-G expression correlates with the invasive trophoblast phenotype whether or not the normal tissue is invaded.
The cellular environment encountered by extravillous trophoblasts in an ectopic pregnancy differs from that of the normal placenta in that there is no decidua. Benirschke and Kaufman (1995) reviewed the literature in which the placenta of ectopic pregnancies was discussed. They concluded that any reports of decidual cells in ectopic pregnancies are most likely mistaken and that the reported decidual cells are almost certainly X cells (extravillous or intermediate trophoblasts). The extravillous trophoblasts in an ectopic pregnancy invade extrauterine tissue. The precise cell–cell interaction at the fetal–maternal interface is therefore lacking in ectopic pregnancies. However, it is possible that extravillous trophoblasts encountering any maternal tissue may induce expression of HLA-G which would also be the case in an ectopic pregnancy. Our conclusion that HLA-G is expressed without invasion into the decidua agrees with the observation that HLA-G is found in reverse transcription–polymerase chain reaction (RT–PCR) analysis of preimplantation embryos developed in vitro(Jurisicova et al., 1996). However RT–PCR, unlike in-situ analysis, does not allow one to define the actual cells expressing HLA-G. This determination would be necessary to form a supporting conclusion from the preimplantation embryo studies. Furthermore, the preimplantation study was done on embryos developed in vitro which may not accurately depict HLA-G expression in vivo.
One of the methods that developmental biologists use to study the effects of cell environment on cell differentiation is transplantation of cells to an ectopic site in a living embryo (reviewed in Gurdon et al., 1993). By studying extravillous trophoblasts in ectopic pregnancies we had the unusual opportunity of asking this basic question of developmental biology in the human. We conclude that induction of HLA-G expression in extravillous trophoblasts is most likely determined by autonomous regulation as opposed to its cellular environment.
Acknowledgments
We thank our colleague Mrs Ruth Har-Nir for advice and assistance and Mrs Mally Sappir of the Hadassah pathology department for technical assistance. We thank Mike McMaster for the HLA-G antibody. We acknowledge the support of the United States Israel Binational Science Foundation, BSF 93-00192/3 (S.Y.), Ministry of the Chief Scientist, Israel (D.G.W.), and the Ministry of Immigrant Adsorption, Israel (D.G.W).
Notes
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Hebrew University Hadassah Hospital, Mt Scopus, POB 24035, Jerusalem, Israel 91240. E-mail: syagel{at}yam-suff.huji.ac.il 
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Submitted on November 18, 1999; accepted on March 8, 2000.
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