Placental alkaline phosphatase (PLAP) staining and human chorionic gonadotropin (hCG) production in cultures of fresh and cryopreserved cytotrophoblasts isolated by CD9/MHC class I/MHC class II immunoelimination

We have further characterized villous trophoblasts isolated by trypsinization and purified by elimination of CD9/MHC class I/MHC class II expressing cells. The cells isolated were >99.99% cytotrophoblasts by criteria of cytokeratin (positive) and vimentin (negative) expression. Purified cells directly after isolation (fresh) were compared with cryopreserved and thawed cells (frozen) for production of human chorionic gonadotropin (hCG) and expression of placental alkaline phosphate (PLAP) after 4 h of culture. We found that fresh cells may adhere slightly more strongly than frozen cells, contained approximately 8-fold more PLAP-positive cells (indicating syncytial fragments) after adherence but neither preparation would secrete hCG until day 4 of culture. We conclude that the cells isolated by cell elimination were cytotrophoblasts with only a small number of PLAP (<0.2% of the fresh population plated) positive cells and that both populations shared the property of very low hCG production until cultured past day 4. We speculate that cells isolated by other methods (accompanying paper) may be contaminated by even more syncytial fragments, detectable by PLAP staining and by production of hCG in the first 48 h of culture.     

The role of Bcl-2 expression in EGF inhibition of TNF-alpha/IFN-gamma-induced villous trophoblast apoptosis.

The inflammatory cytokines tumour necrosis factor alpha (TNF-alpha) and immune interferon gamma (IFN-gamma) stimulate villous cytotrophoblast apoptosis while epidermal growth factor (EGF) protects. We hypothesize that TNF-alpha, IFN-gamma and EGF regulate apoptosis in part by modulating cellular expression levels of the anti-death gene bcl-2. While Bcl-2 is reported to be strongly expressed in villous syncytiotrophoblasts, it is not known whether the protein is expressed in cultured villous cytotrophoblasts (CT) and, if so, whether it is functional. We show by Northern blot analysis that bcl-2 mRNA is expressed in cultured CT and by immunoblot analysis that the protein is strongly expressed in highly purified first trimester and term villous cytotrophoblasts. The expression levels of Bcl-2 protein were the same in first trimester and term cytotrophoblasts. Culture with TNF-alpha/IFN-gamma and EGF did not alter expression of either Bcl-2 protein or of the pro-apoptotic Bcl-2 family member Bak. Double label flow cytometric analysis that measured apoptosis and Bcl-2 content simultaneously showed that cells expressing low levels of Bcl-2 underwent TNF-alpha/IFN-gamma-induced apoptosis at a higher frequency than cells expressing lower levels. We conclude that Bcl-2 is expressed in cytotrophoblasts, that its expression is constitutive and that modulation of its expression levels does not mediate cytokine and growth factor regulation of apoptosis in these cells.     

Villous trophoblasts cultured on semi-permeable membranes form an effective barrier to the passage of high and low molecular weight particles

An effective in vitro model of the placental villous syncytium cultured on semi-permeable substrata is essential for studies of infectious pathogen transmission from mother to fetus. Current models using amniotic membranes or thinner artificial membranes show significant leakage, suggesting disruption of tight junctions or the presence of gaps between syncytial units. Such disruption and discontinuity of trophoblast cultures are probably the result of high stromal cell contamination, poor viability and lack of proliferation in culture. We have successfully cultured confluent layers of tight-junctioned syncytium on semi-permeable insert membranes using highly viable purified cytotrophoblasts and an alternating multiple seeding and differentiation technique. Using criteria including transepithelial diffusion of high and low molecular weight substances, electrical resistance and directional secretion of the matrix metalloproteinase, MMP-9, we demonstrate that these cultures form effective and functional physical barriers that can be maintained for up to 1 month.     

Caveolae and caveolin-1 in human term villous trophoblast

Caveolae are flask-shaped invaginations of the plasma membrane found in many cell types, particularly endothelium. A major structural component is the membrane protein caveolin-1 which associates with numerous signalling molecules, including endothelial nitric oxide (eNOS). Caveolin-1, which co-immunoprecipitates with eNOS in preparations from endothelial cells, regulates eNOS activity, holding it inactive. Controversy now exists regarding the presence of caveolae and caveolin-1 in trophoblasts, hence this study was carried out to examine whether the high levels of eNOS expressed in human syncytiotrophoblast are associated with caveolin-1, and to find out if caveolae are present in villous cytotrophoblasts and syncytiotrophoblast.Immunohistochemistry of term placentae revealed only weak labelling for caveolin-1 in the syncytiotrophoblast although the endothelium of the terminal villus vessels stained strongly. By electron microscopy, numerous caveolae were identified in the villus capillary endothelium but were extremely rare in the syncytium. Caveolin-1 staining was extensive in purified, isolated term villous cytotrophoblasts, with the purity of these cytokeratin positive cells confirmed by cytospin analysis and flow cytometry. Caveolae were clearly demonstrated in ultrastructural sections of the purified cytotrophoblasts. The time course of expression of caveolin-1 and eNOS during differentiation of villous cytotrophoblast into syncytiotrophoblast in culture was studied. Western analysis showed that caveolin-1 expression evident in day 1 whole cell lysates decreased at day 3 when the cells had syncytialized and declined further by day 6, while the levels of actin (control) remained high. eNOS expression in the same samples followed a different pattern, with the low levels in day 1 cells increasing substantially by 3 days in culture, subsiding again by day 6. eNOS association with caveolin-1 in day 1 and day 3 trophoblast cultures was evidenced by the demonstration that eNOS co-immunoprecipitates with caveolin-1 and vice versa. We conclude that human villous cytotrophoblasts express caveolin-1, which assembles into caveolae. Differentiation into syncytium results in a decrease, but not disappearance, of expression of caveolin-1 and a marked reduction of the caveolae.     

ED(27) trophoblast-like cells isolated from first-trimester chorionic villi are genetically identical to HeLa cells yet exhibit a distinct phenotype

ED(27) trophoblast-like cells were prepared from human chorionic villus samples obtained at 9 weeks gestation and have been grown continuously in vitro without phenotypic drift for nearly a decade. These cells express many trophoblast markers, including cytokeratin, placental alkaline phosphatase (PLAP), secretion of 17beta-estradiol, and a microvillous apical surface. The ED(27) cell line is a useful model system for studies of placental cell biology and has been distributed to laboratories world-wide. However, experiments to investigate their relationship to primary villous cytotrophoblast have shown that these cells do not secrete detectable amounts of human chorionic gonadotropin in culture and, when digested with trypsin, disperse into individual cells. Furthermore, immunocytochemical studies demonstrated that, unlike villous cytotrophoblasts, ED(27) cells were immunoreactive with monoclonal antibodies recognizing some HLA Class I antigens. This was not HLA-G, however, as would be expected if these cells originated from extravillous cytotrophoblasts, but rather classical HLA-A, B which is thought not to be expressed by any trophoblast subpopulations. These inconsistencies prompted us to question the authenticity of the continuous cell line as it now exists. Genetic haplotype analysis using the polymerase chain reaction (PCR) revealed that ED(27) was genetically identically to the HeLa cell line. Inasmuch as HeLa cells have never been grown in the laboratory (DAK), the only possible origin of HeLa cell contamination of ED(27) cells was the WISH cell line, and further PCR analysis revealed that this cell line was also genetically identical to HeLa. Like ED(27) cells, HeLa cells and WISH cells synthesized small amounts of estrogen and were found to express PLAP and antigens recognized by the monoclonal antibodies ED822, directed against the syncytiotrophoblast, and J1B5 directed against villous cytotrophoblast. These results point out the need for adherence to rigorous and consistent quality control measures to assure the authenticity of cell lines used as in vitro model systems.     

A positive immunoselection method to isolate villous cytotrophoblast cells from first trimester and term placenta to high purity

We developed a method for isolating highly pure villous cytotrophoblast cells from first trimester and term placenta that excludes extravillous trophoblast and syncytiotrophoblast fragments. The method is based on positive immunoselection using an antibody (mAb C76/18) reacting with hepatocyte growth factor activator inhibitor 1, HAI-1, a membrane antigen on villous cytotrophoblast. As a comparison, we also immunopurified cells using an antibody against CD105, present on syncytiotrophoblast and some extravillous trophoblast cells. The isolates were characterized by flow cytometry. HAI-1-positive cells from first trimester and term placentae were highly pure (>98 per cent cytokeratin 7-positive) mononuclear trophoblast cells. These isolations were contaminated with only very small percentages of vimentin and CD45-positive cells. HAI-1-positive trophoblast cells lacked CD105 and also HLA class I, a marker for extravillous trophoblast. In culture HAI-1-positive cells adhered, displayed an epithelial morphology, and survived for more than three days. In contrast, CD105-positive cell fractions from first trimester placenta were a heterogeneous mixture of mononuclear and multinuclear elements consisting of syncytiotrophoblast fragments, extravillous trophoblast cells, as well as around 5 per cent non-trophoblastic contaminants. In conclusion, the positive immunoselection method using antibody C76/18 yielded highly pure villous cytotrophoblast cells devoid of elements derived from syncytiotrophoblast or extravillous trophoblast.     

Vitamins C and E inhibit apoptosis of cultured human term placenta trophoblast

Preeclampsia can be lethal to both mother and baby. The prominent symptoms of this syndrome are hypertension, proteinuria and oedema, resulting from an exaggerated aseptic systemic inflammatory response, triggered by placental factors shed into the maternal circulation. Syncytiotrophoblast microparticles (STBM) are one possible factor, shed when the placenta is exposed to stressors such as hypoxia/reperfusion. These can disrupt mitochondria, triggering apoptosis and necrosis, placental pathologies which are increased in preeclampsia. We tested the effects of antioxidant vitamins C (50muM) and E (50muM) on trophoblast in culture, using term villous cytotrophoblast preparations. Following Percoll gradient centrifugation and MHC class I expressing cell depletion of placenta digests, syncytial fragments were removed using anti-placental alkaline phosphatase antibody. This yielded cytotrophoblasts of consistently high purity. EGF (10ng/ml) stimulated syncytialisation and hCG and progesterone production. However, mitochondrial induced apoptosis (MIA) was evident 96h post-isolation, as mitochondrial membrane potential loss and caspase 9 and caspase 3 activation. ROCK-1 cleavage and syncytiotrophoblast particle shedding increased concurrently with apoptosis induction. Vitamins blocked MIA and syncytiotrophoblast particle shedding and significantly increased hCG (p<0.005) and progesterone (p<0.02) concentrations in culture supernatants, reflecting the increased survival rates. Although more cells survived in culture, syncytialisation rate (%) was significantly reduced (p<0.005). We conclude that vitamins C and E can significantly reduce mitochondrial damage generated following syncytialisation in vitro. However, further work is required to determine whether antioxidant vitamins interfere with normal fusion processes.     

Effect of oxygen levels in villous trophoblast apoptosis

In pregnancies complicated by intrauterine growth restriction (IUGR), the villous trophoblast shows increased apoptosis and immature cytotrophoblasts (CT) may be exposed to both higher or lower oxygen levels than normal placentae. We propose that villous CT undergo higher frequencies of apoptosis at extreme oxygen tensions. The apoptosis of CT isolated from normal term placentae was examined before culture and after 24 h of culture at different oxygen tensions with or without TNFalpha. The apoptosis frequencies of cells cultured for 24 h at O2 levels of approximately 15 mm and approximately 38 mm Hg were similar to the frequency before culture. Both constitutive and TNFalpha-induced apoptosis and cell loss were highest at low (<10 mm Hg) and high ( approximately 140 mm Hg) oxygen tensions. Further, the ratios of induced to constitutive apoptosis, constant from approximately 15 mm to approximately 140 mm Hg, indicate induced apoptosis to be rather insensitive to changes in oxygen levels. These results show that primary villous trophoblasts from normal placentae undergo minimal apoptosis unless subjected to extreme oxygen tensions <15 mm or 140 mm Hg. The results indicate that normal villous trophoblasts are remarkably resistant to hypoxia-induced apoptosis.     

Stimulation of hCG and inhibition of hPL in isolated human trophoblast cells in vitro by glycodelin A

The immunosuppressive protein glycodelin A (formerly named PP14) is produced by human decidua and secreted in the maternal circulation. Glycodelin A concentrations in serum have been used as indicators of endometrial function. The purpose of this study was to investigate the effect of glycodelin A on human chorionic gonadotropin (hCG) and human placental lactogen (hPL) release by freshly isolated cytotrophoblasts (in vitro). Cytotrophoblasts have been prepared from human term placenta by the three-step trypsin-DNase dispersion method of villous tissue followed by a percoll gradient centrifugation step. When placed in culture, the isolated mononuclear trophoblasts differentiated into syncytial counterparts within 12-72 h after plating. Trophoblasts were incubated with varying concentrations (60-300 microg/ml) of glycodelin A. Glycodelin A was isolated and purified by chromatographic methods from amnion fluid. Supernatants of the trophoblast cell cultures were assayed for hCG and hPL by immunological methods. The release of hCG is increased in glycodelin A-treated trophoblast cell cultures compared to untreated trophoblast cells. Glycodelin A inhibits the production of hPL in vitro. Differences in Glycodelin A stimulated cells and untreated controls are statistical significant. hCG and hPL are markers for the differentiation process of trophoblast cells to syncytial trophoblasts. The results imply that glycodelin A secreted by decidualised endometrium modulates endocrine function, as well as the differentiation of trophoblasts in culture.     

Human trophoblast-endometrial interactions in an in vitro suspension culture system

We developed an in vitro suspension co-culture system to examine the interaction of 1st, 2nd and 3rd trimester purified cytotrophoblasts with human endometrium. Endometrium explants were added to cytotrophoblast cell suspensions and placed on an angled gyrating platform in a 37 degrees C incubator. When endometrium was cultured alone it was able to remain viable for up to 3 days. When trophoblasts were cultured alone, they formed small and large aggregates, and occasionally spherical shells with hollow centers. When trophoblasts and endometrium were cultured together, the trophoblasts adhered to the exposed stromal surfaces of the tissue fragments. The surface epithelium was not receptive to trophoblast attachment except in one experiment when day 19 endometrium was used for the co-incubation, suggesting that surface attachment is usually restricted. A common finding was the presence of an acellular zone in the endometrium only adjacent to the attached trophoblasts. We speculate that this zone may be caused by proteolysis and resynthesis of ECM proteins by the trophoblasts. Based on our results, this in vitro suspension should prove useful for examining those factors which: (1) induce endometrial permissiveness, (2) promote paracrine effects on the endometrium, and (3) facilitate human trophoblast invasion.     

Changes in the expression of apoptosis-related proteins in the life cycle of human villous trophoblast

The outer layer of the human placenta is the multinucleated syncytiotrophoblast. The syncytiotrophoblast is formed by the fusion of mononuclear cytotrophoblasts, and aged syncytiotrophoblast nuclei are extruded into the maternal blood as membrane-enclosed "syncytial nuclear aggregates" that are then eliminated from the maternal circulation. Apoptosis proteins are hypothesized to be involved in both of these processes, but the mechanism of death in the syncytiotrophoblast is unclear and death processes in this multinucleated layer are likely to differ from related processes in mononuclear cells. We have used a combination of villous explant culture and immunohistochemical staining of semi-serial sections from the explants to study the changing expression of 4 proteins that are markers of apoptotic processes in first-trimester human placentae. These studies show that Bcl-2 expression is limited to the syncytiotrophoblast and syncytial nuclear aggregates, while conversely Bax is expressed in some cytotrophoblasts. Activated caspase 3 and the M30 cytokeratin neoepitope were localized to isolated regions of the syncytiotrophoblast and some syncytial nuclear aggregates but were never present in the same area. Combining our results with those of others, we suggest a refined scheme whereby proteins of the apoptosis cascade participate in both the processes of syncytial formation and death.     

Vascular endothelial growth factor but not placental growth factor promotes trophoblast syncytialization in vitro.

OBJECTIVE: The object of this study was to determine the effect of epithelial growth factor (EGF), vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) on the differentiation of first-trimester and term cytotrophoblasts. METHODS: The first-trimester trophoblasts were isolated from villous tissue obtained at suction termination (n = 5), and the term trophoblasts were isolated from placentas (n = 6) at elective cesarean. Cultured cells were stimulated with EGF, VEGF, or PlGF at 0.5, 5, and 50 ng/mL, in the presence or absence of N(G)-nitro-L-arginine methyl ester hydrochloride (10(-4) M). Syncytialized trophoblasts were identified by immunostaining with antidesmosomal protein and anti-cytokeratin-7, whereas nuclei were counted in each syncytia using hematoxylin. RESULTS: Without treatment, background levels of syncytialization were significantly higher in term preparations than first-trimester cells. With VEGF and EGF, the number and size of syncytia increased significantly for the first-trimester cytotrophoblasts (P <.05). Neither VEGF nor EGF had any effect on the syncytialization of cultured cells at term. Nitric oxide showed no involvement in syncytial induction, and PlGF had no effect on syncytialization of cytotrophoblasts, from either the first or third trimester. CONCLUSION: Both EGF and VEGF appeared to enhance the in vitro syncytialization of first trimester cytotrophoblasts.     

Expression of the p53 gene and apoptosis in gestational trophoblastic disease

In order to understand the involvement of the p53 tumour suppressor gene in the pathogenesis of gestational trophoblastic disease (GTD), we investigated its genetic status, protein expression and its role in apoptosis in samples of complete and partial hydatidiform mole as compared with those of normal placenta. Direct sequencing of polymerase chain reaction (PCR) products of the coding and non-coding regions of the p53 gene demonstrated no mutations in any of the studied samples. Immunohistochemical studies revealed increased expression of the p53 protein predominantly in the nuclei of villous cytotrophoblasts. This over-expression of p53 was found in all samples of complete mole, in 50 per cent of partial mole samples and in about 30 per cent of normal placenta cases, although no significant difference in the staining intensity and pattern was observed. An in situ detection of DNA nicking (TUNEL) staining, demonstrating apoptosis, was also detected predominantly in villous cytotrophoblasts and in stromal areas. The per centage of apoptotic cells in all studied samples, determined by flow cytometry, demonstrated a significant increase in apoptotic cells in samples of complete and partial hydatidiform mole compared with those of normal placenta (P< 0.0003 and P< 0.004, respectively).In conclusion, the current study may provide a possible explanation to the pathogenesis of GTD, probably associated with extensive p53-dependent apoptosis to modulate excessive trophoblastic proliferation.     

早孕期人细胞滋养层细胞的分化及分离、培养、鉴定

目的:分离正常早孕期人细胞滋养层细胞并分析其分化功能。方法:用不同的消化方法分离正常早孕期人细胞滋养层细胞,分别在体外培养24h,用相差显微镜、Giemsa染色、免疫细胞化学法、扫描电镜、Transwell分析其形态及分化功能。结果:①采用低浓度胰酶一次性消化法获得绒毛外滋养层细胞,特异表达细胞角蛋白7,不表达波形蛋白,细胞互相聚集但不聚合,具有高度增殖活性和侵袭能力;②采用高浓度胰酶、长时间连续消化法分离得到绒毛滋养层细胞,具有高度融合、分泌hCG-β的合体滋养层细胞特性;③采用低浓度胰酶连续消化法获得的细胞包含绒毛外滋养层细胞和绒毛滋养层细胞,其共性是表达细胞总角蛋白,不表达波形蛋白。结论:利用不同的分离方法可有效、经济地获得具有不同分化功能的早孕期人细胞滋养层细胞。     

The contribution of apoptosis-inducing factor (AIF) to villous trophoblast differentiation

Apoptosis is postulated to be a delayed but important part of the differentiation of placental villous cytotrophoblasts (CT) into functional syncytiotrophoblast (ST). This hypothesis is based on the observation that the externalization of phosphatidylserine and the activation of caspase 8 are required for trophoblast differentiation. In contradiction to this hypothesis we have previously found that differentiation occurs in the presence of both broad spectrum and caspase 8 specific inhibitors. Apoptosis-inducing factor (AIF) is a mitochondria-associated protein which is known to translocate to the nucleus and induce caspase-independent nuclear condensation, phosphatidylserine externalization and cell death. Thus AIF nuclear translocation may result in the apoptotic-like features associated with trophoblast differentiation and may be an obligatory event for differentiation to proceed. AIF translocation was assessed in isolated primary trophoblasts by optical section microscopy of antibody stained cells. We found AIF to be strongly expressed in the villous trophoblast and that small amounts of AIF were localized to the nucleus of the cells. Significantly, inhibitors of AIF translocation (calpeptin and zFA-fmk) blocked translocation but not differentiation of the cells. We conclude that AIF translocation is not involved in CT differentiation in isolated cell culture.     

Direct preparations from chorionic villi--relationship between villous morphology and mitotic index

It has been postulated that chorionic villi with abundant sprouts have a higher mitotic index and are therefore preferable for obtaining direct chromosome preparations from chorionic villus samples. This theory was tested by correlating villous morphology with mitotic index. Surprisingly, no statistically significant relationship was found. Choice of culture medium, however, was found to be important, with serum-free RPMI yielding a higher mitotic index than 40 per cent FCS in MEM. We conclude that villous morphology, as assessed in this study, is not a major factor in determining the success of direct chromosome preparations.     

Double Immuno-labelling of Proliferating Villous Cytotrophoblasts in Thick Paraffin Sections: Integrating Immuno-histochemistry and Stereology in the Human Placenta

In order to understand the pathological basis of abnormal villous trophoblast development in diseased placentas, the organ must be sampled by non-biased methods and subject to analysis by stereological tools. This approach permits quantification of cytotrophoblast density and syncytiotrophoblast structure including evidence of apoptotic shedding via syncytial knots. The stereological quantification of cells (or their) nuclei requires that each should be unambiguously identified and counted within a defined volume of tissue. A major limitation of such studies at present is the inability to accurately identify and phenotype subsets of villous cytotrophoblasts that either proliferate or are destined to fuse into the overlying syncytiotrophoblast.We describe the development of a novel double immuno-labelling protocol to selectively identify proliferating villous cytotrophoblast cells in human placental villi using thick (25 μm) paraffin sections suitable for stereological quantification. Cytotrophoblast cells were selectively stained using a monoclonal anti-cytokeratin 7 (CK 7) antibody without antigen retrieval, followed by nuclear Ki-67 co-localisation. Both antibodies displayed full depth penetration with sharp, clearly defined staining precipitates and no cross-reactivity. This double immuno-labelling protocol is reproducible, cost effective and time efficient (8 h). Use of a variety of antibodies following antigen retrieval will be a significant advancement in the ability to accurately quantify sub-populations of villous cytotrophoblast in normal and pathological placentas.     

Hypoxia attenuates PGE(2)but increases prostacyclin and thromboxane production in human term villous trophoblast.

Prostanoids have been proposed to play a major role in the regulation of uteroplacental blood flow. We examined the effect of hypoxia on the production of prostaglandin E(2)(PGE(2)) thromboxane B(2)(TXB(2)), and prostacyclin (measured as 6-keto-PGF(1alpha)) by human term trophoblast cells and villous placental explants. Explants (n=8) and purified trophoblast cells (n=5) were incubated for 24-72 h under either normoxic (21 per cent O(2)) or hypoxic (2 per cent O(2)) conditions. In trophoblast monolayer cultures, hypoxia attentuated PGE(2)production rates to 52+/-9.4 per cent (mean+/-sem, P< 0.05) but recovered to control rates within 48 h. In villous explants, PGE(2)production was significantly decreased after 48 and 72 h of hypoxia versus the normoxic control, accompanied by increased production of 6-keto-PGF(1alpha)to 173.9+/-26.7 per cent after 48 h. TXB(2)production was increased to 172.3+/-25.9 per cent and 653.2+/-135.7 per cent (P< 0.05) control after 48 and 72 h of hypoxia, respectively. These results were confirmed in villous explants (n=3) cultured in the presence of exogenous 10 microm arachidonic acid. Hypoxia had no significant effect on TXB(2)and 6-keto-PGF(1alpha)in trophoblast cells. In summary, our findings suggest that hypoxia could be responsible for abnormal profiles of prostanoid production commonly observed in women with pre-eclampsia. These results indicate a putative link between hypoxia and compromised placental perfusion.     

Unique epitopes of lactoferrin expressed in human cytotrophoblasts involved in immunologic reactions.

OBJECTIVE: Lactoferrin is an iron-binding protein that has been implicated in protection against infections and allogeneic recognition reactions and in the control of cell growth. We studied the biochemical characteristics and expression of the unique lactoferrin epitopes (LF(1)) in human placentas. STUDY DESIGN: Immunohistologic studies of normal human term placentas were done by using monoclonal antibodies to LF(1). Double-antibody experiments were done by using monoclonal antibodies to markers of inflammation (macrophages, human leukocyte antigen [HLA-DR]). LF(1) was studied immunochemically by using lactoferrin fragments produced by the reaction of lactoferrin with trypsin or N-glycanase. RESULTS: Anti-LF(1) monoclonal antibodies reacted with most interstitial cytotrophoblasts in the basal plate and with villous cytotrophoblasts of some but not all chorionic villi. Cytotrophoblasts expressing LF(1) were associated with large numbers of HLA-DR-reactive macrophages. Anti-LF(1) monoclonal antibodies reacted with 2 distinct tryptic fragments of lactoferrin, and these reactivities were not affected by treatment with N-glycanase. CONCLUSION: Placental cytotrophoblasts express unique epitopes of lactoferrin (LF(1)). Such expression is increased in the presence of activated macrophages. This expression could be an extraembryonic response to inflammation and maternal allogeneic recognition as an effort to protect trophoblastic cells. The LF(1) epitopes might represent conserved polypeptide epitopes on 2 homologous lobes of lactoferrin.