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Characterization and localization of HLA antigens on hydatidiform mole
Characterization and localization of HLA antigens on hydatidiform mole C. A. Sunderland, Ph.D., C. W. G. Redman, M.B., Ch.B., and G. M. Stirrat, M.D. Bristol and Headington, Oxford, England Frozen sections of three specimens of hydatidiform mole were stained with monoclonal antibodies to HLA Class I and Class II antigens by means of an indirect immunoperoxidase technique. Class I (HLA A, B, C) antigens were detected on proliferating extravillous trophoblast and on villous stromal cells but not on quiescent villous trophoblast. Trophoblast Class I antigen was detected with four different antibodies to monomorphic determinants but not with antibodies to the appropriate polymorphic HLA A or B type. Stromal cells were reactive with all Class I antibodies. Class II (HLA DR) antigens were not detected on any molar tissue. The expression of HLA antigens by molar tissue is similar to that of the normal firsttrimester human placenta. (AM J OBSTET GYNECOL 1985;151:130-5.)
Key words: HLA antigens, hydatidiform mole, trophoblast, monoclonal antibodies
The normal placenta, hydatidiform mole, and gestational choriocarcinoma share the unusual property of being natural allografts in the sense that they are genetically disparate tissues in intimate contact with a fully immunocompetent host. In most circumstances the natural allograft is successful; pregnancy continues to term and choriocarcinoma, if left untreated, leads to the death of the sufferer. It is now clear that the major histocompatibility antigens (HLA) of man function not only as the major inducers of, and targets for, artificial tissue allograft rejection but also as essential mediators in normal cellular immunologic reactions. Precise determination of the nature and localization of HLA antigens on placenta and trophoblast tumors is therefore necessary if we are to understand immunologic reactions toward them. Host recognition of the H LA system of the conceptus clearly occurs during both normal and molar pregnancy, as shown by the presence of paternal-specific HLA antibodies in maternal blood prior to delivery.'" Several studies have demonstrated the absence of both Class I (HLA A, B, C) and Class II (HLA DR) antigens on the villous trophoblast of the normal placenta."·' In contrast, cells of the villous stroma, which form the central part of the villi, do express class I antigens
From the Department of Obstetrics and Gynaecology, Bristol Maternity Hospital, Bristol, and the Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Maternity Hospital, Headington. Supported by Medical Research Council Grant No. 8120833SB. Received for publication January 30, 1984; revised June 15, 1984; accepted August 28, 1984. Reprint requests: Dr. C. A. Sunderland, Department of Obstetrics and Gynaecology, Bristol Maternity Hospital, Southwell St., Bristol, Engl.and BS2 8EG.
130
throughout gestation. Class II antigens are found on specific dendrite-like cells in the villous stroma in thirdtrimester placentas.:' 6 Such stromal antigens are separated from maternal tissue by the HLA-deficient villous trophoblast, which is thought to make up a continuous layer around the villi. Recent studies have shown that several populations of nonvillous trophoblast express Class I antigens. 7 8 Such trophoblast is thought to arise from primitive villous trophoblast during early gestation and comes to lie in direct contact with maternal uterine tissue for the remainder of the pregnancy. Such antigens, however, can only be detected with antibodies to monomorphic or constant determinants of HLA A, B, C, and not with antibodies directed to the polymorphic, or variable, regions of the molecule. In this paper we report a similar study of hydatidiform mole. This benign placental tumor is characterized by aberrant trophoblast proliferation and hydropic degeneration of the villi. It occurs in up to 1 in 250 pregnancies in some parts of the Far East but only 1 in 2000 in Europe and the continental United States." Two distinct types have been identified within the hydatidiform mole classification. 10· 12 Complete mole refers to a tumor of androgenetic origin whose XX chromosome constitution is exclusively paternal and which presents with the classical vesicular or grapelike appearance due to generalized hydropic degeneration and trophoblast proliferation. A fetus is never present. In contrast partial moles are triploid, bearing maternal and paternal genes; hydropic degeneration is partial and trophoblastic proliferation is focal. There is normally evidence of a fetus. Approximately 9% of complete moles develop into some form of persistent trophoblastic disease, usually choriocarcinoma.''
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Fig. I. Serial sections of Mole I were stained with monoclonal antibodies with the indirect immunoperoxidase technique. A, NDOG I monoclonal antibody stains the syncytiotrophoblast plasma membrane and some components within the column of proliferating extravillous trophoblast (PT). B, W6/32 monoclonal antibody, which detects a monomorphic determinant of Class I (HLA A, B, C) antigens, stains proliferating extravillous trophoblast (PT), villous stromal cells (VS), and some maternal leukocytes trapped in a fibrinous layer external to the villous trophoblast (L). C, NFK-1 monoclonal antibody, which detects a monomorphic determinant of Class II (HLA-DR) antigens, stains no molar tissue. Labeling is limited to maternal lymphocytes (L) as in B. (Nuclei were counterstained with hematoxylin. Original magnification x 120.)
We have collected and studied samples from three hydatidiform moles with the use of a panel of monoclonal antibodies in an indirect immunoperoxidase technique on frozen sections. Patients and methods
Specimens with the histopathologic diagnosis of hydatidiform mole were taken from three patients. They were classified into complete or partial moles on the basis of the morphologic criteria described by Szulman and Surti 11. 12 after examination of several blocks of tissue from each patient. It has been previously shown that there is an excellent correlation between morphologic and cytogenetic classification of moles. 19 Mole I. Molar tissue was evacuated at 16 weeks' menstrual age. The diagnosis was initially suggested by a routine ultrasound scan at 13 weeks and then confirmed ultrasonically and biochemically. Microscopically, all villi showed marked hydatidiform change with gross swelling and central cistern formation. There was
moderate trophoblastic proliferation around most villi. There was neither macroscopic nor microscopic evidence of a fetus. Mole I was designated as a complete mole. Mole 2. Molar tissue was evacuated from a patient with uncertain dates after detection by ultrasound. Histologic analysis showed enlarged villi containing an immature mesenchymal stroma. There was moderate trophoblastic proliferation and moderate anaplasia throughout the specimen. There was neither macroscopic nor microscopic evidence of a fetus. Mole 2 was designated a complete mole. Villi were generally smaller and the stroma more dense than those of Mole 1, suggesting the specimen was of an earlier gestation. Mole 3. Molar products were observed after the suction termination in a teenager of a pregnancy previously considered normal. There was marked variability in the size of the villi, including some with normal mesenchymal stroma. Nucleated erythrocytes were detected in fetal capillaries. Trophoblastic cysts or "fjords"
132 Sunderland, Redman, and Stirrat
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Fig. 2. Serial sections of Mole 2 were stained with monoclonal antibodies with the indirect immunoperoxidase technique. A, The antibody MEI, which specifically detects HLA B7, B22, B27, stains only cells of the villous stroma (VS), while (B) W6/32 stains, in addition, areas of proliferating trophoblast (Pn. (Nuclei were counterstained with hematoxylin. Original magnification x 120.)
Table I. Monoclonal antibody preparations Monoclonal antibody NDOGI
NFK-1 W6/32 BB 7.7 BB 7.8 BBMI MA 2.1 MEI
Reference
Specificity Villous syncytiotrophoblast plasma membrane, some nonvillous trophoblast, unreactive with most adult tissues including liver, kidney, heart, brain, colon, pancreas, and pregnant uterus Class II (HLA DR) antigens,* monomorphict Class 1 (HLA A, B, C) antigens, monomorphic Class I (HLA A, B, C,) antigens, monomorphic Class I (HLA A, B, C,) antigens, monomorphic f3rMicroglobulin Class I HLA A2 and B 17 Class I HLA B7, B22 and B27
7
15 16 16 16 16 17 18
Preparation Tissue culture supernatant
Ascites Ascites lmmunoglobulin Immunoglobulin Immunoglobulin Ascites Ascites
*The reactivity of this antibody with the DC and SB Class II locus products has not yet been determined. tThe term monomorphic means that the determinant recognized is common to all HLA antigens of this class. In contrast, antibodies MA 2. l and ME I react only with certain HLA A and B specificities and are therefore deemed to react with a "polymorphic" antigenic determinant.
as described by Szulman and Surti were observed. There was only slight trophoblastic proliferation. Mole 3 was designated as a partial mole. Small pieces of tissue were taken and frozen in liquid nitrogen. Sections (5 to 8 µ.m) were then cut in a cryostat. The slides were air dried for 2 hours, fixed in acetone at room temperature for 10 minutes, then washed in 0.15 mol/L of sodium chloride and 0.05 moll L of Tris hydrochloride, pH 7.6. Sections were incubated with monoclonal antibody (50 µ.l) at a predetermined saturating concentration for 40 minutes at room temperature. They were then washed twice in Trissaline solution and incubated similarly with 50 µJ of peroxidase-conjugated rabbit antimouse immunoglobulin (Dako) at a 1: 50 dilution in Tris-saline solution and 10% normal human serum. Slides were again washed and incubated with 0.6 mg/ml of diaminobenzidine-tetrahydrochloride and 0.007% hydrogen
peroxide in Tris-saline solution. The reaction was stopped after 8 minutes by immersion in excess water; the nuclei were counterstained with hematoxylin, and sections were then dehydrated and mounted in DPX. Photomicrographs were taken on a Zeiss photomicroscope III. Figs. 1and2 were taken through a blue filter. The monoclonal antibody preparations used in this study are described in Table I. An irrelevant monoclonal antibody was used as a negative control in all experiments.
Results Large areas of trophoblastic proliferation were evident in all three hydatidiform moles examined. One such area is illustrated in Fig. 1. In the normal firsttrimester placenta, the NDOG 1 monoclonal antibody is known to react specifically with the syncytiotrophoblast plasma membrane and with some extracellular
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HLA antigens on hydatidiform mole
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Fig. 3. Serial sections of Mole 3 were stained with three different monoclonal antibodies specific for monomorphic determinants of Class I antigens. These were (A) BBM I, (B) BB7.7, and (C) BB7.8. All three antibodies labeled an island of nonvillous trophoblast (NVn and an area of maternal decidua (D). The ME I antibody stains maternal decidual tissue only (D). (Nuclei were counterstained with hematoxylin. Original magnification x 120.)
material associated with proliferating cytotrophoblast cell columns. 7 It does not react with most other normal human tissues, including maternal decidual cells. Similarly, in the mole (Fig. I, A), NDOGI defines an area in which the continuity of the densely labeled syncytiotrophoblast plasma membrane has been broken by the outward proliferation of cytotrophoblast presenting as a column of cells with associated NDOG I staining proceeding from the site of proliferation. Staining for Class I (HLA A, B, C) antigens with the W6/32 monoclonal antibody (Fig. I, B) shows that this proliferating nonvillous trophoblast population and villous stromal cells both express the Class I antigen while the quiescent areas of villous trophoblast are negative. A similar study of several areas from all three hydatidiform moles showed the same localization of Class I HLA antigens. It was noted, however, that staining of the nonvillous trophoblast was not uniform. Antigen expression was more common on the cellular cytotrophoblast than on the syncytial giant cells and in each mole some areas of proliferating cytotrophoblast were found that did not
express the Class I antigen. No staining of any molar tissue was detected with the NFK-I antibody to Class II (HLA DR) antigens (Fig. 1, C), positive reactions being limited to some leukocytes present in the intervillous space. Molar tissue was next examined with two antibodies specific for polymorphic determinants of HLA A, B, C. These were MA 2.I, specifically reactive with HLA A2 and BI 7, and ME I, specific for HLA B7, B22, and B27. In each case the reactivity of the conceptus was assessed by immunohistologic examination of the molar villous stroma with each antibody. Similar examination of maternal leukocytes in the intervillous space or decidua gave the maternal reactivity. The results are given in Table II. The alleles A2, BI7, B7, B22, and B27 occur in the Caucasian population with gene frequencies of25%, 6%, I I%, I3%, and 4%, respectively. 21 This means that MA 2. I and ME I should each theoretically be reactive with approximately 50% of individuals. The results of Table II are consistent with this prediction. In Moles 2 and 3, it was found that despite clear
134 Sunderland, Redman, and Stirrat
January !, 1985 Am J Obstet Gynecol
Table II. Typing of mother and conceptus, in terms of reactivity with polymorphic HLA antibodies Mole Mole I Mother Conceptus Mole 2 Mother Conceptus Mole 3 Mother Concept us
MA 2.1
ME 1
+ +
+
+ +
+
Typing was performed by the indirect immunoperoxidase technique on frozen sections. Maternal reactivity was assessed by staining of decidual tissue or lymphocytes in the intervillous space, while staining of villous stromal cells gave the reactivity of the conceptus.
staining of villous stromal tissue with one of these antibodies, the same antibody on the same sections never labeled proliferating trophoblast. This is illustrated in Fig. 2, A. By contrast a serial section with W6/32, detecting monomorphic determinants of Class I (HLA A, B, C) antigens, shows clear staining of this same region of proliferating trophoblast (Fig. 2, B). It has been suggested that this discrepancy between staining with W6/ 32 antibody and antibodies to polymorphic determinants is due to some unique property of the W6/32 monoclonal antibody. We have therefore examined molar tissue with other antibodies directed to monomorphic determinants of Class I (HLA A, B, C) antigens. Fig. 3 shows an area of Mole 3 made up of maternal decidual tissue (D), an island of nonvillous trophoblast (NVT), and a portion of hydropic villus. The antibody BBM l, which is specific for [3"-microglobulin, was seen to react with both maternal decidua and nonvillous trophoblast (Fig. 3, A). Similar results were obtained with antibodies BB 7. 7 (Fig. 3, B) and BB 7 .8 (Fig. 3, C) which react with a monomorphic determinant of Class I antigens distinct from that defined by W6/32. 16 The pattern of staining with the polymorphic ME l antibody on this tissue was markedly different. This antibody reacted only with maternal tissue in these sections and served to confirm the histologic distinction between decidua and trophoblast (Fig. 3,D).
Comment One previous study reported that the villous trophoblast of hydatidiform mole lacks Class I (HLA A, B, C) antigens, but such antigens are detectable on villous stromal cells. 13 Our data are compatible with these studies bm extend them co an analysis of HLA antigens on the proliferating extravillous trophoblast of hydatidiform mole.
It is apparent that the expression of HLA antigens on molar tissue is similar to that of normal first-trimester placenta, in terms of both its nature and its localization. Thus shortly after implantation in normal pregnancy the villous cytotrophoblast proliferates, bursting through the syncytiotrophoblast membrane and forming cytotrophoblast cell columns which invade the maternal decidua and produce the cytotrophoblast shell around the conceptus. This extravillous trophoblast bears an obvious histologic relationship to the proliferating trophoblast of hydatidiform mole. Furthermore both populations express Class I but not Class II HLA antigens 7 and again, in both cases, the Class I antigens are not detectable with antibodies to polymorphic determinants of HLA even when such antibodies are clearly observed to be bound to the fetal villous stromal cells. 8 The detection of Class I antigens on nonvillous trophoblast with antibodies to monomorphic but not polymorphic determinants may be due to a number of causes which have been discussed in detail elsewhere. 8 Briefly, the discrepancy may arise because trophoblast Class I antigens are biochemically distinct from Class I antigen that have been defined in other tissues. Alternatively it may arise from an antigen dosage effect. Thus a single polymorphic determinant accounts for only 12% to 15% of the total HLA A, B, C antigens as detected with W6/32 antibody on lymphocytes of a heterozygous individual. 9 It may therefore be that antibodies to monomorphic determinants, which react with the whole Class I population, can detect the antigen but the polymorphic antibodies react with so small a subset of the total antigen population as to move below the level of detection with this technique. It is of note that complete molar tissue has a double paternal genotype and so might be expected to express polymorphic determinants at 25% to 30% of the total, that is, the equivalent of a homozygous individual. While it is clearly desirable to extend the study to additional moles and to include cytogenetic data, definite answers to these questions regarding trophoblast HLA will only come through molecular analysis at the biochemical level. This requires the preparation of purified nonvillous trophoblast. It is known that a portion of primigravid women develop antibodies to maternal Class I antigens during the course of either normal or molar pregnancy. In normal pregnancy such antibodies may arise as a result of "leakage" of fetal lymphocytes into the maternal bloodstream. In complete moles there is no fetus or fetal circulation and so immunization must arise though the molar tissue itself. Yamashita et al. 14 have shown molar tissue to specifically absorb antipaternal HLA antibody. If further study demonstrates that trophoblast Class I antigens are unusual and do not express
HLA antigens on hydatidiform mole
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polymorphic determinants, then the most likely route of immunization might be through the villous stromal cells, brought into contact with the maternal immune system through discontinuity in the villous trophoblast layer. Such discontinuity may occur in the region of the anchoring villi which are responsible for the attachment of the villous placenta to the basal plate. 20 We are grateful to Mr. N. Kelly for expert technical assistance, to Dr. J. N. Bulmer for helpful discussion, and to the following for the gift of monoclonal antibodies: Prof. J. Bodmer, Dr. A. J. Mc Michael, Dr. Sue Fuggle, and Dr. A. F. Williams. REFERENCES
9. I 0. 1I. 12. 13.
14.
I. Terasaki PI, Mickey MR, Yamazaki JN, and Vredevoe D.
2. 3. 4.
5.
6. 7. 8.
Maternal-fetal incompatibility. I. Incidence of HL-A antibodies and possible association with congenital abnormalities. Transplantation 1970;9:538. Lawler SD, Klouda PT, Bagshawe KD. Immunogenicity of molar pregnancies in the HL-A system. A!\1 J 0BSTET GYNECOL 1974;120:857. Faulk WP, Temple A. Distribution of f32 microglobulin and HLA in chorionic villi of human placentae. Nature I 976;262:799. Galbraith RM, Kantor RRS, Ferrara GB, Ades EW, Galbraith GMP. Differential anatomical expression of transplantation antigens within the normal human placental chorionic villus. Am J Rep rod Immunol 1981; I :33 I. Sunderland CA, Naiem M, Mason DY, Redman CWG, Stirrat GM. The expression of major histocompatibility antigens by human chorionic villi. J Reprod lmmunol 1981;3:323. Sutton L, Mason DY, Redman CWG. HLA-DR positive cells in the human placenta. Immunology I 98 I ;49: 103. Sunderland CA, Redman CWG, Stirrat GM. HLA-A, B, C antigens are expressed on non-villous trophoblast of the early human placenta. J Immunol 1981;127:2614. Redman CWG, McMichael AJ, Stirrat GM, Sunderland CA, Ting A. Class I Major Histocompatibility Complex
I 5.
16.
17. 18. I 9.
20. 21.
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antigens on human extra-villous trophoblast. Immunology I 984;52:457. Bagshawe KD. Choriocarcinoma. Baltimore: Williams & Wilkins, 1969. Kajii T, Ohama K. Androgenetic origin of hydatidiform mole. Nature 1977;268:633. Szulman AE, Surti U. The syndromes of hydatidiform mole. I. Cytogenetic and morphologic correlations. AM J 0BSTET GYr-iECOL 1978; I 31 :665. Szulman AE, Surti U. The syndromes of hydatidiform mole. II. Morphologic evolution of the complete and partial mole. AMJ 0BSTET GY:\ECOL 1983;146:221. Berkowitz RS, Anderson DJ, Hunter NJ, Goldstein DP. Distribution of major histocompatibility (HLA) antigens in chorionic villi of molar pregnancy. AM J OBSTET GYr-iECOL 1983;146:221. Yamashita K, Wake N, Araki T, Ichinoe K, Makoto K. Human lymphocyte antigen expression in hypatidiform mole: androgenesis following fertilization by a haploid sperm. AMJ OBSTET GY:\ECOL 1979;135:597. Fuggle SV, Errasti P, Daar AS, Fabre JW, Ting A, Morris PJ. Localization of major histocompatibility complex (HLA-A, B, C and -DR) antigens in 46 kidneys. Transplantation I 983;35:385. Parham P, Androlewicz MJ, Brodsky FM, Holmes NJ, Ways JP. Monoclonal antibodies: purification, fragmentation and application to structural and functional studies of Class I MHC antigens. J Immunol Methods 1982; 53:133. McMichael AJ, Parham PR, Rust N, Brodsky FM. A monoclonal antibody that recognizes an antigenic determinant shared by HLA-A2 and -BI 7. Hum Immunol 1980; 1: 121. Ellis SA, Taylor C, McMichael AJ. Recognition of HLAB27 and related antigens by a monoclonal antibody. Hum Immunol 1982;5:49. Jacobs PA, Hunt PA, Matsuura JS, Wilson CC. Complete and partial hydatidiform mole in Hawaii: cytogenetics, morphology and epidemiology. Br J Obstet Gynaecol I 982;89:258. Boyd JD, Hamilton WJ. The human placenta. Cambridge: W. Heffer, 1970: 190-206. Bodmer WF, Cavalli-Sforza LL. Genetics, evolution and man. San Francisco: WH Freeman, 1976:347-50.
Key words: HLA antigens, hydatidiform mole, trophoblast, monoclonal antibodies
The normal placenta, hydatidiform mole, and gestational choriocarcinoma share the unusual property of being natural allografts in the sense that they are genetically disparate tissues in intimate contact with a fully immunocompetent host. In most circumstances the natural allograft is successful; pregnancy continues to term and choriocarcinoma, if left untreated, leads to the death of the sufferer. It is now clear that the major histocompatibility antigens (HLA) of man function not only as the major inducers of, and targets for, artificial tissue allograft rejection but also as essential mediators in normal cellular immunologic reactions. Precise determination of the nature and localization of HLA antigens on placenta and trophoblast tumors is therefore necessary if we are to understand immunologic reactions toward them. Host recognition of the H LA system of the conceptus clearly occurs during both normal and molar pregnancy, as shown by the presence of paternal-specific HLA antibodies in maternal blood prior to delivery.'" Several studies have demonstrated the absence of both Class I (HLA A, B, C) and Class II (HLA DR) antigens on the villous trophoblast of the normal placenta."·' In contrast, cells of the villous stroma, which form the central part of the villi, do express class I antigens
From the Department of Obstetrics and Gynaecology, Bristol Maternity Hospital, Bristol, and the Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Maternity Hospital, Headington. Supported by Medical Research Council Grant No. 8120833SB. Received for publication January 30, 1984; revised June 15, 1984; accepted August 28, 1984. Reprint requests: Dr. C. A. Sunderland, Department of Obstetrics and Gynaecology, Bristol Maternity Hospital, Southwell St., Bristol, Engl.and BS2 8EG.
130
throughout gestation. Class II antigens are found on specific dendrite-like cells in the villous stroma in thirdtrimester placentas.:' 6 Such stromal antigens are separated from maternal tissue by the HLA-deficient villous trophoblast, which is thought to make up a continuous layer around the villi. Recent studies have shown that several populations of nonvillous trophoblast express Class I antigens. 7 8 Such trophoblast is thought to arise from primitive villous trophoblast during early gestation and comes to lie in direct contact with maternal uterine tissue for the remainder of the pregnancy. Such antigens, however, can only be detected with antibodies to monomorphic or constant determinants of HLA A, B, C, and not with antibodies directed to the polymorphic, or variable, regions of the molecule. In this paper we report a similar study of hydatidiform mole. This benign placental tumor is characterized by aberrant trophoblast proliferation and hydropic degeneration of the villi. It occurs in up to 1 in 250 pregnancies in some parts of the Far East but only 1 in 2000 in Europe and the continental United States." Two distinct types have been identified within the hydatidiform mole classification. 10· 12 Complete mole refers to a tumor of androgenetic origin whose XX chromosome constitution is exclusively paternal and which presents with the classical vesicular or grapelike appearance due to generalized hydropic degeneration and trophoblast proliferation. A fetus is never present. In contrast partial moles are triploid, bearing maternal and paternal genes; hydropic degeneration is partial and trophoblastic proliferation is focal. There is normally evidence of a fetus. Approximately 9% of complete moles develop into some form of persistent trophoblastic disease, usually choriocarcinoma.''
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Fig. I. Serial sections of Mole I were stained with monoclonal antibodies with the indirect immunoperoxidase technique. A, NDOG I monoclonal antibody stains the syncytiotrophoblast plasma membrane and some components within the column of proliferating extravillous trophoblast (PT). B, W6/32 monoclonal antibody, which detects a monomorphic determinant of Class I (HLA A, B, C) antigens, stains proliferating extravillous trophoblast (PT), villous stromal cells (VS), and some maternal leukocytes trapped in a fibrinous layer external to the villous trophoblast (L). C, NFK-1 monoclonal antibody, which detects a monomorphic determinant of Class II (HLA-DR) antigens, stains no molar tissue. Labeling is limited to maternal lymphocytes (L) as in B. (Nuclei were counterstained with hematoxylin. Original magnification x 120.)
We have collected and studied samples from three hydatidiform moles with the use of a panel of monoclonal antibodies in an indirect immunoperoxidase technique on frozen sections. Patients and methods
Specimens with the histopathologic diagnosis of hydatidiform mole were taken from three patients. They were classified into complete or partial moles on the basis of the morphologic criteria described by Szulman and Surti 11. 12 after examination of several blocks of tissue from each patient. It has been previously shown that there is an excellent correlation between morphologic and cytogenetic classification of moles. 19 Mole I. Molar tissue was evacuated at 16 weeks' menstrual age. The diagnosis was initially suggested by a routine ultrasound scan at 13 weeks and then confirmed ultrasonically and biochemically. Microscopically, all villi showed marked hydatidiform change with gross swelling and central cistern formation. There was
moderate trophoblastic proliferation around most villi. There was neither macroscopic nor microscopic evidence of a fetus. Mole I was designated as a complete mole. Mole 2. Molar tissue was evacuated from a patient with uncertain dates after detection by ultrasound. Histologic analysis showed enlarged villi containing an immature mesenchymal stroma. There was moderate trophoblastic proliferation and moderate anaplasia throughout the specimen. There was neither macroscopic nor microscopic evidence of a fetus. Mole 2 was designated a complete mole. Villi were generally smaller and the stroma more dense than those of Mole 1, suggesting the specimen was of an earlier gestation. Mole 3. Molar products were observed after the suction termination in a teenager of a pregnancy previously considered normal. There was marked variability in the size of the villi, including some with normal mesenchymal stroma. Nucleated erythrocytes were detected in fetal capillaries. Trophoblastic cysts or "fjords"
132 Sunderland, Redman, and Stirrat
January !, 1985 Am J Obstet Gynecol
Fig. 2. Serial sections of Mole 2 were stained with monoclonal antibodies with the indirect immunoperoxidase technique. A, The antibody MEI, which specifically detects HLA B7, B22, B27, stains only cells of the villous stroma (VS), while (B) W6/32 stains, in addition, areas of proliferating trophoblast (Pn. (Nuclei were counterstained with hematoxylin. Original magnification x 120.)
Table I. Monoclonal antibody preparations Monoclonal antibody NDOGI
NFK-1 W6/32 BB 7.7 BB 7.8 BBMI MA 2.1 MEI
Reference
Specificity Villous syncytiotrophoblast plasma membrane, some nonvillous trophoblast, unreactive with most adult tissues including liver, kidney, heart, brain, colon, pancreas, and pregnant uterus Class II (HLA DR) antigens,* monomorphict Class 1 (HLA A, B, C) antigens, monomorphic Class I (HLA A, B, C,) antigens, monomorphic Class I (HLA A, B, C,) antigens, monomorphic f3rMicroglobulin Class I HLA A2 and B 17 Class I HLA B7, B22 and B27
7
15 16 16 16 16 17 18
Preparation Tissue culture supernatant
Ascites Ascites lmmunoglobulin Immunoglobulin Immunoglobulin Ascites Ascites
*The reactivity of this antibody with the DC and SB Class II locus products has not yet been determined. tThe term monomorphic means that the determinant recognized is common to all HLA antigens of this class. In contrast, antibodies MA 2. l and ME I react only with certain HLA A and B specificities and are therefore deemed to react with a "polymorphic" antigenic determinant.
as described by Szulman and Surti were observed. There was only slight trophoblastic proliferation. Mole 3 was designated as a partial mole. Small pieces of tissue were taken and frozen in liquid nitrogen. Sections (5 to 8 µ.m) were then cut in a cryostat. The slides were air dried for 2 hours, fixed in acetone at room temperature for 10 minutes, then washed in 0.15 mol/L of sodium chloride and 0.05 moll L of Tris hydrochloride, pH 7.6. Sections were incubated with monoclonal antibody (50 µ.l) at a predetermined saturating concentration for 40 minutes at room temperature. They were then washed twice in Trissaline solution and incubated similarly with 50 µJ of peroxidase-conjugated rabbit antimouse immunoglobulin (Dako) at a 1: 50 dilution in Tris-saline solution and 10% normal human serum. Slides were again washed and incubated with 0.6 mg/ml of diaminobenzidine-tetrahydrochloride and 0.007% hydrogen
peroxide in Tris-saline solution. The reaction was stopped after 8 minutes by immersion in excess water; the nuclei were counterstained with hematoxylin, and sections were then dehydrated and mounted in DPX. Photomicrographs were taken on a Zeiss photomicroscope III. Figs. 1and2 were taken through a blue filter. The monoclonal antibody preparations used in this study are described in Table I. An irrelevant monoclonal antibody was used as a negative control in all experiments.
Results Large areas of trophoblastic proliferation were evident in all three hydatidiform moles examined. One such area is illustrated in Fig. 1. In the normal firsttrimester placenta, the NDOG 1 monoclonal antibody is known to react specifically with the syncytiotrophoblast plasma membrane and with some extracellular
Volume 151 Number I
HLA antigens on hydatidiform mole
..•.
\
'·
.... " '•fl
.
133
-., .,.
...,•"-/'
f';
Fig. 3. Serial sections of Mole 3 were stained with three different monoclonal antibodies specific for monomorphic determinants of Class I antigens. These were (A) BBM I, (B) BB7.7, and (C) BB7.8. All three antibodies labeled an island of nonvillous trophoblast (NVn and an area of maternal decidua (D). The ME I antibody stains maternal decidual tissue only (D). (Nuclei were counterstained with hematoxylin. Original magnification x 120.)
material associated with proliferating cytotrophoblast cell columns. 7 It does not react with most other normal human tissues, including maternal decidual cells. Similarly, in the mole (Fig. I, A), NDOGI defines an area in which the continuity of the densely labeled syncytiotrophoblast plasma membrane has been broken by the outward proliferation of cytotrophoblast presenting as a column of cells with associated NDOG I staining proceeding from the site of proliferation. Staining for Class I (HLA A, B, C) antigens with the W6/32 monoclonal antibody (Fig. I, B) shows that this proliferating nonvillous trophoblast population and villous stromal cells both express the Class I antigen while the quiescent areas of villous trophoblast are negative. A similar study of several areas from all three hydatidiform moles showed the same localization of Class I HLA antigens. It was noted, however, that staining of the nonvillous trophoblast was not uniform. Antigen expression was more common on the cellular cytotrophoblast than on the syncytial giant cells and in each mole some areas of proliferating cytotrophoblast were found that did not
express the Class I antigen. No staining of any molar tissue was detected with the NFK-I antibody to Class II (HLA DR) antigens (Fig. 1, C), positive reactions being limited to some leukocytes present in the intervillous space. Molar tissue was next examined with two antibodies specific for polymorphic determinants of HLA A, B, C. These were MA 2.I, specifically reactive with HLA A2 and BI 7, and ME I, specific for HLA B7, B22, and B27. In each case the reactivity of the conceptus was assessed by immunohistologic examination of the molar villous stroma with each antibody. Similar examination of maternal leukocytes in the intervillous space or decidua gave the maternal reactivity. The results are given in Table II. The alleles A2, BI7, B7, B22, and B27 occur in the Caucasian population with gene frequencies of25%, 6%, I I%, I3%, and 4%, respectively. 21 This means that MA 2. I and ME I should each theoretically be reactive with approximately 50% of individuals. The results of Table II are consistent with this prediction. In Moles 2 and 3, it was found that despite clear
134 Sunderland, Redman, and Stirrat
January !, 1985 Am J Obstet Gynecol
Table II. Typing of mother and conceptus, in terms of reactivity with polymorphic HLA antibodies Mole Mole I Mother Conceptus Mole 2 Mother Conceptus Mole 3 Mother Concept us
MA 2.1
ME 1
+ +
+
+ +
+
Typing was performed by the indirect immunoperoxidase technique on frozen sections. Maternal reactivity was assessed by staining of decidual tissue or lymphocytes in the intervillous space, while staining of villous stromal cells gave the reactivity of the conceptus.
staining of villous stromal tissue with one of these antibodies, the same antibody on the same sections never labeled proliferating trophoblast. This is illustrated in Fig. 2, A. By contrast a serial section with W6/32, detecting monomorphic determinants of Class I (HLA A, B, C) antigens, shows clear staining of this same region of proliferating trophoblast (Fig. 2, B). It has been suggested that this discrepancy between staining with W6/ 32 antibody and antibodies to polymorphic determinants is due to some unique property of the W6/32 monoclonal antibody. We have therefore examined molar tissue with other antibodies directed to monomorphic determinants of Class I (HLA A, B, C) antigens. Fig. 3 shows an area of Mole 3 made up of maternal decidual tissue (D), an island of nonvillous trophoblast (NVT), and a portion of hydropic villus. The antibody BBM l, which is specific for [3"-microglobulin, was seen to react with both maternal decidua and nonvillous trophoblast (Fig. 3, A). Similar results were obtained with antibodies BB 7. 7 (Fig. 3, B) and BB 7 .8 (Fig. 3, C) which react with a monomorphic determinant of Class I antigens distinct from that defined by W6/32. 16 The pattern of staining with the polymorphic ME l antibody on this tissue was markedly different. This antibody reacted only with maternal tissue in these sections and served to confirm the histologic distinction between decidua and trophoblast (Fig. 3,D).
Comment One previous study reported that the villous trophoblast of hydatidiform mole lacks Class I (HLA A, B, C) antigens, but such antigens are detectable on villous stromal cells. 13 Our data are compatible with these studies bm extend them co an analysis of HLA antigens on the proliferating extravillous trophoblast of hydatidiform mole.
It is apparent that the expression of HLA antigens on molar tissue is similar to that of normal first-trimester placenta, in terms of both its nature and its localization. Thus shortly after implantation in normal pregnancy the villous cytotrophoblast proliferates, bursting through the syncytiotrophoblast membrane and forming cytotrophoblast cell columns which invade the maternal decidua and produce the cytotrophoblast shell around the conceptus. This extravillous trophoblast bears an obvious histologic relationship to the proliferating trophoblast of hydatidiform mole. Furthermore both populations express Class I but not Class II HLA antigens 7 and again, in both cases, the Class I antigens are not detectable with antibodies to polymorphic determinants of HLA even when such antibodies are clearly observed to be bound to the fetal villous stromal cells. 8 The detection of Class I antigens on nonvillous trophoblast with antibodies to monomorphic but not polymorphic determinants may be due to a number of causes which have been discussed in detail elsewhere. 8 Briefly, the discrepancy may arise because trophoblast Class I antigens are biochemically distinct from Class I antigen that have been defined in other tissues. Alternatively it may arise from an antigen dosage effect. Thus a single polymorphic determinant accounts for only 12% to 15% of the total HLA A, B, C antigens as detected with W6/32 antibody on lymphocytes of a heterozygous individual. 9 It may therefore be that antibodies to monomorphic determinants, which react with the whole Class I population, can detect the antigen but the polymorphic antibodies react with so small a subset of the total antigen population as to move below the level of detection with this technique. It is of note that complete molar tissue has a double paternal genotype and so might be expected to express polymorphic determinants at 25% to 30% of the total, that is, the equivalent of a homozygous individual. While it is clearly desirable to extend the study to additional moles and to include cytogenetic data, definite answers to these questions regarding trophoblast HLA will only come through molecular analysis at the biochemical level. This requires the preparation of purified nonvillous trophoblast. It is known that a portion of primigravid women develop antibodies to maternal Class I antigens during the course of either normal or molar pregnancy. In normal pregnancy such antibodies may arise as a result of "leakage" of fetal lymphocytes into the maternal bloodstream. In complete moles there is no fetus or fetal circulation and so immunization must arise though the molar tissue itself. Yamashita et al. 14 have shown molar tissue to specifically absorb antipaternal HLA antibody. If further study demonstrates that trophoblast Class I antigens are unusual and do not express
HLA antigens on hydatidiform mole
Volume 151 Number I
polymorphic determinants, then the most likely route of immunization might be through the villous stromal cells, brought into contact with the maternal immune system through discontinuity in the villous trophoblast layer. Such discontinuity may occur in the region of the anchoring villi which are responsible for the attachment of the villous placenta to the basal plate. 20 We are grateful to Mr. N. Kelly for expert technical assistance, to Dr. J. N. Bulmer for helpful discussion, and to the following for the gift of monoclonal antibodies: Prof. J. Bodmer, Dr. A. J. Mc Michael, Dr. Sue Fuggle, and Dr. A. F. Williams. REFERENCES
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