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This study has shown that fresh amniotic epithelial cells which are negative by indirect immunofluorescence for HLA and Trf receptors become positive for both of these markers following prolonged culture. In addition, the amniotic antigens which are characteristic of fresh amniotic epithelial cells are lost and replaced by trophoblast antigens subsequent to maintenance of the cells in vitro. These observations suggest that, although the expression of HLA and Trf receptors are apparently negative in vivo, such gene products become activated and manifest in plasma membranes following exposure of the cells to culture conditions. In addition, the normal manifestation of the amnion antigens is lost following prolonged periods of culture. These findings indicate that the coding and expression of normally accepted transplantation antigens undergo a significant perturbation in vitro, and that markers of normal extra-embryonic epithelium in vivo are lost after the cells have been cultured in vitro.

The specificities of antisera produced in nine rabbits to seven different preparations of human amniotic epithelium and controls were studied by immunofluorescence on cryostat sections of human amniochorions, placentae and a large collection of other tissues. Three different groups of reaction patterns were identified with the use of these antibodies, indicating the presence of at least three antigenic groupings which have been tentatively designated as amnion antigens 1 (AA1), amnion antigens 2 (AA2) and amnion antigens 3 (AA3). The AA1 group was found on the ectodermal tissues of breast ductal and corneal epithelium as well as on Hassall's corpuscles, while the AA2 group was also found on breast ductal and corneal epithelium and basal keratinocytes, but not on Hassall's corpuscles. The AA3 group was found on basement membranes of ectodermally derived epithelium, but was negative on kidney. The identification of the distribution of antigens in common with ectodermal tissues and extra-embryonic membranes suggests that human amnion and chorion may have a close embryological relationship with embryonic ectoderm and its subsequent products of organogenesis.

Antibodies produced in rabbits to detergent-solubilized human amnion were found to react with Hassall's corpuscles in human thymus. Following nomenclature for placental antigens, the immunogenic group responsible for these antibodies has been tentatively designated as amnion antigens 1 (AA1). The anti-AA1 antisera did not react with other thymic components, nor did they react with any other extra-embryonic tissues than amniotic epithelium. Some adult ectodermally derived tissues, such as breast ductal and corneal epithelium, reacted with anti-AA1, but others such as skin and vagina did not. These findings link an antigenic relationship between amniotic epithelium and certain ectodermal derivatives. Amnion exists long before these tissues are formed, raising the possibility that amniotic epithelium may play an inductive role in their development.

The spiral arteries of the placental bed lose clearly recognizable arterial features early in pregnancy because of their invasion by extravillous trophoblast. Extensive structural alterations take place in the walls of invaded arteries, their walls containing an amorphous acidophilic "fibrinoid" matrix in which endovascular trophoblast is embedded. Such spiral arteries examined by immunofluorescence techniques in placental bed biopsy specimens obtained at cesarean section show a characteristic pattern of reaction with antisera to epithelial basement membrane amniotic antigens. These antisera have been previously demonstrated to react with the basement membrane of human amnion and of ectodermally derived epithelium such as breast ducts and skin. Interstitial trophoblast usually does not contain these antigens, and it is postulated that, following vascular invasion, endovascular trophoblast produces amniotic antigens to establish its presence. This may be of importance in maintaining the structural integrity of spiral arteries and thereby adequate maternal blood flow to the fetal-placental unit.

Three monoclonal antibodies to human amnion (GB4, GB9 and GB11) which recognized well-defined structures of the adult rabbit eye were used to study the embryonic eye development. The reactivity of GB4 could be identified on the migrating neural crest cells under the corneal epithelium at 2 weeks of gestation. At 3 weeks of gestation, the conjunctival epithelium, corneal epithelium and corneal endothelium reacted with GB4. In the newborn rabbit, the corneal epithelium was reactive with GB9 instead of GB4; corneal endothelium was positive with GB4 at this stage, but gradually became negative in the adult rabbit; the subcapsular epithelium of the lens was recognized by GB4 when it changed from pseudostratified to simple cuboidal epithelium. During early embryonic life, GB11 was positive on the external retinal layer of the optic cup and lens vesicle. In the adult, GB11 reacted only with the pigmented epithelium of the ciliary processes. The results of this study demonstrated that the expression of antigens recognized by GB4, GB9 and GB11 could be mapped according to the epithelial differentiation of the rabbit eye during embryogenesis.

A newborn male infant with epidermolysis bullosa letalis died at age 6 weeks of septicemia. He had many congenital ectodermal abnormalities and a rudimentary thymus. The spleen had no germinal centers and the lymph nodes lacked follicles. Cytotrophoblast of the amniochorion was sparse and the amniotic epithelium was distorted. Immunohistologic studies of amniotic epithelium revealed the unprecedented presence of trophoblast antigens. Before birth of the patient, the parents had a 5-year history of infertility; after death of the patient, the mother sustained a ruptured tubal pregnancy that necessitated blood transfusions and surgical removal of the tube. She subsequently became pregnant a third time and was delivered of a normal child. The wife shared four major histocompatibility (HLA) antigens with her husband. The parents' immunogenetic characteristics and the immunopathologic features and extraembryonic membranes of the child are considered in terms of maternal responses to trophoblast antigens.

Monoclonal antibody GB36, which was raised against human term syncytiotrophoblastic microvilli, was found to recognize a novel epithelial antigen. The antibody immunoprecipitated several membrane proteins from BeWo (choriocarcinoma) and HT-29 (colon adenocarcinoma) cells. Under non-reducing conditions, four peptides of 180, 155, 135 and 130 kDa were revealed by SDS-PAGE analysis. Three peptides of slightly higher molecular weights were revealed under reducing conditions; these three peptides had identical pI (6.2) as shown by two-dimensional gel electrophoresis. By immunofluorescence, GB36 reacted with villous cytotrophoblasts and cytotrophoblasts of chorion laeve, as well as with the basal surface of syncytiotrophoblast and amniotic epithelium. Extravillous trophoblasts of cytotrophoblastic shell in the basal plate and the cytotrophoblastic cell island were non-reactive. It is suggested that the antigen of GB36 may play a role in the polarity of epithelial cells and the adhesion of epithelial cells to extracellular matrix.