Placental Fas and Fas Ligand Expression in Normal Early, Term and Molar Pregnancy

Placenta (2004), 25, 321–330 doi:10.1016/j.placenta.2003.08.020

Placental Fas and Fas Ligand Expression in Normal Early, Term and Molar Pregnancy S. Pongcharoen a, R. F. Searle b and J. N. Bulmer c,* a

School of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne, UK; b School of Medical Education Development, University of Newcastle upon Tyne, Newcastle upon Tyne, UK; c School of Clinical and Laboratory Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne, UK Paper accepted 6 August 2003

To clarify the Fas and Fas-ligand status of normal and molar trophoblast, the expression of Fas and FasL by placental trophoblast populations in partial and complete hydatidiform moles was compared with that in normal first trimester and term pregnancies using an avidin-biotin peroxidase technique on frozen and formalin-fixed paraffin-embedded placental tissues with both monoclonal and polyclonal antibodies. The TUNEL technique was used to detect apoptotic cells in the same tissues. The immunoreactivity for Fas and Fas-ligand was comparable with both monoclonal and polyclonal antibodies on frozen as well as paraffin-embedded sections. In normal early and molar pregnancy there was strong FasL expression by villous cytotrophoblast and syncytiotrophoblast. However, there were significant differences in FasL expression by trophoblast subpopulations in both early and term normal pregnancy and between the same trophoblast subpopulation at different gestations, with FasL staining generally being weaker at term. Strong FasL staining by cytotrophoblast cells in the distal parts of cell columns contrasted with unstained cytotrophoblast in the proximal part of columns. Distinct trophoblast subpopulations in partial hydatidiform mole also differentially expressed FasL with reduced FasL expression in proliferating syncytiotrophoblast. In contrast there was no differential FasL expression in complete hydatidiform mole, all trophoblast subpopulations strongly expressing FasL. Unlike the differential expression of FasL there were no differences in Fas expression by trophoblast populations in normal early or term placental tissues. Fas expression was reduced in villous cytotrophoblast at term. Differential expression of Fas by different trophoblast subpopulations was noted in partial and complete hydatidiform mole. In complete mole villous cytotrophoblast and syncytiotrophoblast stained strongly compared with proliferating trophoblast. Using TUNEL labelling apoptosis was rarely detected in placental trophoblast. Differential Fas and FasL expression by trophoblast subpopulations in normal and pathological pregnancy does not appear to be related to apoptosis of trophoblast. Placenta (2004), 25, 321–330  2003 Elsevier Ltd. All rights reserved.

INTRODUCTION Hydatidiform mole is a gestational trophoblastic disease in which trophoblast cells proliferate abnormally [1] unlike normal human pregnancy where trophoblast cells proliferate and differentiate to form the placenta in a tightly regulated manner [2]. Complete hydatidiform moles (CHM) are usually diploid with a 46,XX karyotype, derive entirely from the paternal genome and are characterized by generalized trophoblastic hyperplasia with no fetus [1,3,4]. In contrast, partial hydatidiform moles (PHM) are triploid, with an extra paternal chromosome component. PHM usually result from fertilization of a normal ovum by two spermatozoa [5], affect only * To whom correspondence should be addressed at: Department of Pathology, Royal Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne, NE1 4LP, UK. Tel.: +44-(0)191-282-4749; Fax: +44-(0)191-222-8100; E-mail: [email protected] 0143-4004/$–see front matter

part of the placenta, and a fetus, often with congenital malformation, is usually found [1]. Because its chromosomes are entirely paternal in origin, unlike the normal semiallogeneic conceptus, CHM is a complete intrauterine allograft and hence may be expected to elicit a maternal immune response. Our previous studies have shown an increase in activated CD4+ T cells in decidua associated with partial and complete hydatidiform moles compared with normal early pregnancy suggesting an altered maternal immune response against molar trophoblast [6]. Apoptosis is recognized as a key mechanism in development, tissue homeostasis and immune responses [7,8], and has been proposed to play an integral role in successful placental development [9–11]. Fas ligand (CD95-ligand/FasL/CD178), a type II transmembrane trimeric protein belonging to the tumour necrosis factor (TNF) family [12,13], induces apoptosis of Fas (CD95/Apo-1) expressing cells [8]. Fas, a monomeric molecule, is trimerized when bound to its ligand and  2003 Elsevier Ltd. All rights reserved.

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sends a signal through its death domain leading to a cellular suicide cascade [14]. Although a number of studies have demonstrated constitutive expression of FasL by different placental trophoblast populations in normal pregnancy [15–21], others have reported that villous syncytiotrophoblast showed only spot-like membrane located staining in distinct areas or was mostly unstained [22–24]. The conflicting FasL status of villous syncytiotrophoblast requires clarification especially as it has been suggested that expression of FasL may allow trophoblast in placental villi to evade maternal immune surveillance and protect the fetal semi-allograft by maternal T cell apoptosis. In contrast the FasL status of trophoblast in molar pregnancy has received little attention [18]. We have reported increased Fas expression in decidual CD4+ T cells in hydatidiform mole compared with normal pregnancy [25]. Differential FasL expression in molar trophoblast could regulate local maternal immune responses against trophoblast and FasL mediated apoptosis could be an important mechanism for immune tolerance of the fetoplacental unit in molar pregnancy. Conversely increased FasL expression by decidual CD4+ T cells in molar pregnancy [25] may have implications for placental development since previous studies have suggested that activated decidual T cells expressing FasL may induce apoptosis of Fas+ villous syncytiotrophoblast [10,22]. Huppertz et al. [22], however, questioned the significance of Fas/FasL pathway in initiating trophoblast apoptosis in view of the discordant localization of FasL on villous cytotrophoblast and Fas on syncytiotrophoblast. Recently Balkundi et al. [26] demonstrated that Fas expression by villous trophoblast in normal pregnancy can be upregulated by proinflammatory cytokines during chorioamnionitis suggesting that the role of Fas and FasL in trophoblast survival may be more complex than originally anticipated. The Fas status of trophoblast in molar pregnancy remains unknown. In view of the conflicting reports on Fas and FasL expression, the aim of this study was to compare FasL and Fas expression and to assess apoptosis by placental trophoblast subpopulations in partial and complete hydatidiform moles with that in normal early and term pregnancy.

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Table 1. Primary antibodies used on paraffin-embedded and frozen tissues Antibody

FasL (sc-834) FasL (NCL-FAS-L) Fas (sc-715) Fas (NCL-FAS-310)

Dilution Paraffinembedded

Frozen

Pretreatment for paraffin-embedded tissues

1/200 1/25 1/500 1/5

1/400 1/40 1/750 1/10

None Pressure cookera Trypsinb 10 minutes Pressure cookera

a

citrate buffer (citric acid monohydrate: Sigma Chemical Co., Poole, UK) in distilled water, pH 6.0. b 2.5% trypsin (DIFCO Laboratories, Detroit, USA) in distilled water, pH 7.8 containing 2.5% calcium chloride.

in paraffin-embedded tissues. All paraffin tissue blocks were sectioned at 3 µm and mounted on 3-aminopropyltriethoxysilane (APES; Sigma Chemical Co., Poole, UK) coated slides. Frozen tissues were sectioned at 5–7 µm, air dried and fixed in acetone at room temperature for 10 min. At least one section from each case was stained with haematoxylin and eosin (H&E) to allow morphological assessment.

Antibodies FasL and Fas were localized using rabbit polyclonal antibodies raised against FasL (sc-834) and Fas (sc-715) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). In addition, FasL (NCL-FAS-L) and Fas (NCL-FAS-310) monoclonal antibodies (Novocastra Laboratories, Newcastle upon Tyne, UK) were used in selected normal and molar pregnancy samples to compare the reaction patterns with those of FasL and Fas polyclonal antibodies. The optimal dilution and incubation time for each antibody was carefully established by titration using similarly fixed and processed positive control tonsil tissues. Optimal antibody dilutions and pretreatments to avoid non-specific background staining are summarized in Table 1.

Single immunohistochemical labelling MATERIALS AND METHODS Tissues Formalin-fixed paraffin-embedded placental tissues from six normal first trimester pregnancy terminations, six normal term pregnancies, 11 partial and 10 complete hydatidiform moles were retrieved from archive files of the Department of Pathology, Royal Victoria Infirmary, Newcastle upon Tyne and the Department of Obstetrics and Gynaecology, Siriraj Hospital, Mahidol University, Bangkok. Frozen placental tissues from two elective first trimester pregnancy terminations were retrieved from archive files of the Department of Pathology, Royal Victoria Infirmary to compare staining results with those

FasL and Fas expression was examined using an avidin-biotin peroxidase technique (Vectastain Elite, Vector Laboratories, Peterborough, UK). Paraffin-embedded sections were deparaffinized, rehydrated and incubated for 10 min with 0.5 per cent hydrogen peroxide in methanol to block endogenous peroxidase activity. For pressure cooking pretreatment, sections were heated for 1 min in citrate buffer, pH 6.0 (see Table 1). For trypsin pretreatment, sections were incubated with trypsin for 10 min (see Table 1). After a thorough wash in running water, sections were incubated for 30 min with 1.5 per cent normal goat (for polyclonal antibodies/pAb) or horse (for monoclonal antibodies/mAb) blocking serum in 0.1  Tris 0.05M saline, pH 7.6 (TBS) followed by incubation with

Pongcharoen et al.: Placental Fas and Fas Ligand Expression in Pregnancy

anti-FasL or anti-Fas antibody for 60 min. After washing twice in TBS, sections were incubated for 30 min with biotinylated anti-rabbit (for pAb) or anti-mouse (for mAb) immunoglobulins, and washed again in TBS before avidin-biotin peroxidase was applied for 30 min. After further washing in TBS the reaction was developed with 3,3#-diaminobenzidine (DAB; Sigma Chemical Co.) containing 0.01 per cent H2O2 to give a brown reaction product. Sections were lightly counterstained with Mayer’s haematoxylin, dehydrated, cleared and mounted with DPX synthetic resin (Raymond A. Lamb Ltd, London, UK). Positive controls were performed in each staining run using normal tonsil tissues. Negative controls were performed for each sample by replacing the primary antibody with non-immune rabbit (for pAb) or mouse (for mAb) IgG as appropriate. Dilutions of primary antibodies for frozen sections are summarized in Table 1. The staining steps were identical to those described for paraffin-embedded sections with the omission of deparaffinization, rehydration, quenching in H2O2 and trypsin or heat pretreatment.

Detection of apoptotic cells using TUNEL assay Apoptosis was detected in formalin-fixed paraffin-embedded placental tissues using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labelling (TUNEL) assay (Apoptosis Detection System, Fluorescein; Promega, Southampton, UK), according to the manufacturer’s instructions. The sections were mounted using Vectashield mounting medium with propidium iodide (Vector Laboratories) to lightly counterstain the nuclei, covered with glass coverslips and then stored at 40(C in the dark until analysis and photography within 24 h. Two positive controls for the detection of DNA fragmentation were included: a section of high grade endometrial carcinoma showing histological evidence of extensive apoptosis and a section of normal early pregnancy decidua treated with DNase type I to produce fragmentation of chromosomal DNA. The DNase positive control was washed separately from the other sections to prevent any residual DNase activity affecting other sections in the assay. Negative controls were performed for each test section by replacing TdT enzyme in the TdT incubation solution with distilled water in order to assess both non-specific binding and the extent of autofluorescence present within the tissue section.

Quantitation of placental FasL and Fas expression The staining reactivity was assessed at 400 magnification using a semi-quantitative H score as previously described [27,28]. Villous syncytiotrophoblast and cytotrophoblast were assessed in all sample groups. In normal early gestation placentae two other cytotrophoblast populations were distinguished: (1) cytotrophoblast columns, comprising vacu-

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olated cytotrophoblast cells at the tips of the anchoring villi; and (2) cytotrophoblast islands in the intervillous space, comprising pleomorphic cells, often lacking a syncytiotrophoblast covering [2]. Syncytiotrophoblast sprouts were assessed separately in normal early pregnancy samples. In both complete and partial molar pregnancy, proliferating cytotrophoblast refers to cytotrophoblast proliferating circumferentially from molar chorionic villi; excessive non-villous syncytiotrophoblast, often seen as syncytial sprouts, was designated proliferating syncytiotrophoblast [29]. At least 200 cells of each trophoblast subpopulation were examined. The reactivity of the trophoblast cells was compared with the positive control tissue and graded as negative, weakly positive, moderately positive and strongly positive (categories 0, 1, 2 and 3, respectively). The percentage of trophoblast cells in each staining category was then calculated by counting positive cells in each category and expressing these as a percentage of the total number of cells counted. The H score was then calculated as H=(P00)+(P11)+(P22)+(P33), where P0–3 is the percentage of cells in category 0, 1, 2, 3, respectively, thus giving H scores that ranged from zero (no staining) to 300 (intense staining of all cells). Statistical analysis was performed using the nonparametric Mann–Whitney test with the conventional significance level of P<0.05.

RESULTS The immunoreactivity for FasL and Fas was similar with both monoclonal and polyclonal antibodies (Figures 1–3). The staining patterns of FasL and Fas on paraffin-embedded and frozen sections were also comparable. All quantification was performed on paraffin-embedded tissues using the polyclonal antibodies. Negative controls showed no immunoreactivity (Figure 1B).

FasL expression by trophoblast subpopulations The analysis of FasL expression by placental trophoblast subpopulations in normal and molar pregnancy is summarized in Table 2. Normal pregnancy. In all normal first trimester placental tissues FasL was localized to villous cytotrophoblast, villous syncytiotrophoblast, cytotrophoblast islands and cytotrophoblast columns with varying staining intensities. Villous cytotrophoblast cells were intensely positive for FasL (median, range; 245, 187–286) (Figure 2A), whereas FasL staining of cytotrophoblast columns (108, 29–136) (Figure 2A) and cytotrophoblastic islands (192, 154–200) (Figure 2B) was significantly lower (columns, P=0.0051; islands, P=0.0306). Although not separately quantified, it was notable that cytotrophoblast cells in the proximal part of cell columns adjacent to the villous core were mostly negative for FasL, whereas trophoblast cells in the distal columns consistently expressed FasL (Figure 2A).

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Figure 1. Immunoperoxidase staining for FasL (A) and Fas (C, D) monoclonal antibody (A, C) and polyclonal antibody (D) on formalin-fixed paraffin-embedded (A, C) and frozen (D) sections of placenta from normal early (C, D), and partial hydatidiform mole (A) pregnancy. Negative control (B; paraffin-embedded placenta from partial hydatidiform mole) stained with anti-rabbit IgG showed no staining. Original magnification 200 (A, B) and 400 (C, D).

Villous syncytiotrophoblast was also significantly more positive for FasL (268, 246–293) than cytotrophoblast columns (P=0.0051) and islands (P=0.0051) (Figure 2A, B). FasL positivity of villous syncytiotrophoblast did not differ significantly from villous cytotrophoblast or syncytial sprouts (268, 248–289) (Figure 2A). At term trophoblast reactivity for FasL was generally weaker than in the first trimester (Figure 2C). In common with normal early pregnancy, term villous cytotrophoblast cells, although less abundant, expressed FasL moderately (200, 199–202). FasL expression was lower in term villous syncytiotrophoblast (179, 162–200) than both term villous cytotrophoblast (P=0.0367) and first trimester villous syncytiotrophoblast (P=0.0081). Syncytial sprouts in term placenta also expressed FasL moderately to strongly but were scarce and hence were not quantified. Hydatidiform mole. FasL expression by villous cytotrophoblast (265, 192–277) in PHM was comparable with that in normal early pregnancy. Similarly FasL expression by proliferating cytotrophoblast (197, 174–267) (Figure 2D) did not differ in PHM compared with normal early pregnancy. In contrast, FasL expression was reduced in proliferating syncytiotrophoblast (241, 110–276) in PHM compared with syncytiotropho-

blast sprouts in normal early pregnancy (P=0.0394). In common with normal early pregnancy, FasL expression of villous cytotrophoblast in PHM was stronger than that of proliferating cytotrophoblast (P=0.0031). Expression of FasL by villous cytotrophoblast, villous syncytiotrophoblast and proliferating syncytiotrophoblast did not differ significantly within PHM. FasL expression in CHM was generally intense, with villous cytotrophoblast, villous syncytiotrophoblast and proliferating syncytiotrophoblast all strongly expressing FasL (238, 158–289; 242, 171–283; and 253, 165–292, respectively) (Figure 2E, F). FasL expression was significantly increased in proliferating cytotrophoblast (252, 182–284; P=0.0197) in CHM compared with normal early pregnancy. Unlike normal early pregnancy and PHM, within CHM there were no differences in FasL expression between trophoblast subpopulations which all strongly expressed FasL. Fas expression by trophoblast subpopulations Expression of Fas by placental trophoblast subpopulations in normal and molar pregnancy is summarized in Table 3. Negative controls showed no labelling (data not shown) and positive controls showed the expected immunoreactivity.

Pongcharoen et al.: Placental Fas and Fas Ligand Expression in Pregnancy

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Figure 2. Immunoperoxidase staining for FasL on formalin-fixed paraffin-embedded sections of placenta from normal early (A, B), term (C) and molar (D, E, F) pregnancy. (A) Note intense FasL expression in villous cytotrophoblast, villous syncytiotrophoblast and syncytial sprouts. Trophoblast cells in the proximal part of the cytotrophoblast columns are negative or weakly positive for FasL unlike cytotrophoblast cells in the distal columns. (B) Cytotrophoblast islands express FasL moderately compared with syncytial sprouts. (C) Term placenta shows moderate FasL expression by villous cytotrophoblast and weak FasL expression by villous syncytiotrophoblast. (D) Partial hydatidiform mole shows strong FasL expression by proliferating cyto- and syncytiotrophoblast. (E) Complete hydatidiform mole shows strong FasL expression by villous cyto- and syncytiotrophoblast, proliferating cytotrophoblast and syncytiotrophoblast. (F) Strong FasL expression by proliferating cytotrophoblast in complete hydatidiform mole. Original magnification 100 (A, B, C, E) and 200 (D, F). Thick arrow, villous cytotrophoblast; thin arrow, villous syncytiotrophoblast; CTC, cytotrophoblast column; Ds, distal part; Px, proximal part; CTI, cytotrophoblast island; PCT, proliferating cytotrophoblast; PST, proliferating syncytiotrophoblast; SS, syncytial sprout.

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There was a significant decrease (P=0.0081) in Fas expression by villous cytotrophoblast at term (200, 200–256) compared with normal early pregnancy (246, 209–288) (Table 3). In contrast, Fas expression by villous syncytiotrophoblast was comparable in normal term (252, 199–255) and first trimester placenta (285, 204–300). Syncytial sprouts of term placenta also expressed Fas moderately but due to their scant number they were not quantified. Hydatidiform mole. In contrast with normal early pregnancy, differential Fas expression by trophoblast subpopulations was observed in molar pregnancy. In PHM, villous cytotrophoblast (181, 161–288; P=0.0488) and proliferating cytotrophoblast (201, 113–214; P=0.0475) expressed Fas significantly less than proliferating syncytiotrophoblast (220, 200–280; Table 3; Figure 3B). Fas expression by other trophoblast subpopulations was comparable (Table 3). Differential expression of Fas by trophoblast subpopulations was also noted in CHM. Villous cytotrophoblast (277, 170–286; P=0.0273) and villous syncytiotrophoblast (283, 262– 290; P=0.0009) expressed Fas significantly more intensely than proliferating cytotrophoblast (222, 178–245) (Table 3; Figure 3C). Fas expression by other trophoblast subpopulations was comparable (Table 3). Interestingly, Fas expression by villous cytotrophoblast in PHM was significantly weaker than that in normal early pregnancy (P=0.0394; Table 3) and CHM (P=0.0441; Table 3). There were no significant differences in Fas expression by proliferating cytotrophoblast, villous syncytiotrophoblast and proliferating syncytiotrophoblast between partial and complete hydatidiform mole and normal early pregnancy (Table 3).

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36 Figure 3. Avidin-biotin immunoperoxidase staining for Fas on formalinfixed paraffin-embedded sections of placenta from normal early pregnancy (A), partial (B) and complete (C) hydatidiform mole. (A) Fas is localized to villous cyto- (thick arrows) and syncytiotrophoblast (thin arrows) and syncytial sprout (SS). (B) Fas expression is reduced in villous cytotrophoblast (thick arrow) in partial hydatidiform mole. (C) Complete hydatidiform mole differentially expressed Fas. Villous cyto (thick arrow)- and syncytiotrophoblast (thin arrow) express Fas significantly more strongly than proliferating cytotrophoblast (PCT). Original magnification 200.

Apoptosis in the placenta Normal decidua treated with DNase and the high grade endometrial carcinoma showed numerous TUNEL positive cells, whereas the negative control showed no labelling (data not shown). In normal early placenta apoptosis was rarely seen. Apoptotic cells were villous syncytiotrophoblast and syncytial sprouts rather than villous cytotrophoblast. Few apoptotic cells were seen in cytotrophoblast islands. Similarly, in hydatidiform mole apoptosis was only occasionally seen in villous syncytiotrophoblast and proliferating syncytiotrophoblast and was not observed in villous cytotrophoblast. A few apoptotic cells were seen in proliferating cytotrophoblast islands. Since apoptotic trophoblast cells were rare, they were not quantified in either normal or molar pregnancy. DISCUSSION

Normal pregnancy. Unlike the differential expression of FasL, there were no differences in Fas expression by trophoblast subpopulations in normal early pregnancy or term placental tissues (Table 3; Figure 3A).

The present semi-quantitative study has demonstrated differential expression of Fas and FasL by trophoblast subpopulations in normal and molar pregnancy and extends previous

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Table 2. FasL expression by placental trophoblast subpopulations from normal early and term pregnancy, partial and complete hydatidiform mole (median H score and range) Trophoblast subpopulations

Normal early pregnancya

Normal term pregnancyb

Partial hydatidiform molec

Complete hydatidiform moled

Statistical significancee

Villous CT CT columns Proliferating CT/cell islands Villous ST Proliferating ST/ ST sprouts

245 108 192 268 268

200 (199-202) NA NA 179 (162-200) NA

265 (192-277) NA 197 (174-267) 246 (157-279) 241 (110-276)

238 (158-289) NA 252 (182-284) 242 (171-283) 253 (165-292)

NA a vs d, P=0.0197 a vs b, P=0.0081 a vs c, P=0.0394

(187-286) (29-136) (154-200) (246-293) (248-289)

CT, cytotrophoblast; ST, syncytiotrophoblast; vs, versus; NA, not applicable. P values as stated, unless otherwise not significant (comparison was performed between normal early and term pregnancy and between normal early and molar pregnancy). e

Table 3. Fas expression by placental trophoblast subpopulations from normal early and term pregnancy, partial and complete hydatidiform mole (median H score and range) Trophoblast subpopulations

Normal early pregnancya

Normal term pregnancyb

Partial hydatidiform molec

Complete hydatidiform moled

Statistical significancee

Villous CT

246 (209-288)

200 (200-256)

181 (161-228)

277 (170-286)

CT columns Proliferating CT/cell islands Villous ST Proliferating ST/ST sprouts

266 (206-294) 259 (116-294)

NA NA

NA 201 (113-214)

NA 222 (178-245)

a vs b, P=0.0081, a vs c, P=0.0394, c vs d, P=0.0441 NA NS

285 (204-300) 278 (202-300)

252 (199-255) NA

277 (161-300) 220 (200-280)

283 (262-290) 232 (212-295)

NS NS

CT, cytotrophoblast; ST, syncytiotrophoblast; vs, versus; NA, not applicable. P values as stated, unless otherwise not significant (comparison was performed between normal early and term pregnancy and between normal early and molar pregnancy). e

reports [15,17–20]. The finding of differential immunoreactivity using polyclonal and monoclonal anti-FasL antibodies on frozen and paraffin-embedded tissues concurs with previous reports that FasL is expressed by villous cytotrophoblast and syncytiotrophoblast in normal early and term pregnancy [15–21]. However, our findings conflict with reports that FasL expression is questionable on villous syncytiotrophoblast [22–24]. The explanation for the discrepancy is not obvious; in the latter study the authors stated that all their anti-FasL antisera needed careful titration to avoid false positive staining. They reported that the polyclonal antisera sc-834, sc-956 and sc-957 gave the same staining outcomes although only anti sc-956 did not give ‘a somewhat muddy reaction’ with high background. In the present study a similar pattern of immunoreactivity was obtained using both the polyclonal sc-834 antibody and the monoclonal NCL-FasL antibody. Using systematically titrated anti-FasL antibodies at a dilution of 1 : 200 or greater, positive immunoreactivity in endothelial cells of fetal vessels and Hofbauer cells within the villous core was demonstrable in paraffin-embedded and frozen tissue sections in the present study, as reported by Hammer and Dohr [24]. Stromal cells within the villous core also stained positively, a finding which the latter authors assumed indicated non-specific immunoreactivity. Since trophoblast in the proxi-

mal cytotrophoblast columns showed no immunoreactivity in the same tissue section as showed positive villous stromal cells and, as expected, only nucleated cells within maternal villous blood stained positively we doubt this interpretation. Moreover reports by others indicate that FasL is more widely expressed by human tissues, including stromal cells, than originally anticipated [30,31]. Our findings suggest that FasL is constitutively expressed by villous syncytiotrophoblast during early normal pregnancy. The strong FasL expression by trophoblast cells distal to the villous core in cytotrophoblast columns in early pregnancy contrasted with the negative or weakly stained cytotrophoblast cells in the proximal part of the columns. These findings concur with another report of differential FasL expression by extravillous trophoblast in the distal part of the cell columns [32] and suggest that the cytotrophoblast cells in the columns that give rise to invasive trophoblast cells upregulate FasL as they approach the maternal tissues. Others, however, have reported strong FasL expression in cytotrophoblast cells proximal to the villous core [23]. Although FasL expression is maintained throughout gestation, the reduction in FasL expression at term, particularly by villous syncytiotrophoblast, was striking and may be associated with reduced invasive behaviour of trophoblast with increasing gestational age [33].

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The increased incidence of apoptosis at term in invasive extravillous trophoblast probably reflects FasL and Fas expression [34]. Others have noted abundant FasL staining by term syncytiotrophoblast [16,35]. This discrepancy may relate to the reported reduction in FasL expression in term placental tissues after labour compared with non-labour term samples [36]. Moreover a recent study has reported absent FasL mRNA in term cytotrophoblast [37]. Comparison of the pattern of FasL expression between normal early and molar pregnancy indicates differential FasL expression between trophoblast subpopulations in normal early pregnancy and strong FasL expression in all trophoblast subpopulations in CHM. The increased FasL expression on proliferating cytotrophoblast in CHM suggests that loss of the tight control of cytotrophoblast proliferation is associated with a concomitant increase of FasL expression. Interestingly FasL expression in PHM was similar to normal early pregnancy as FasL expression by proliferating cytotrophoblast was significantly weaker than that of villous cytotrophoblast. Overall the pattern of FasL positivity indicates that FasL expression in a CHM differs from normal early pregnancy while that associated with PHM more closely resembles normal early pregnancy. In this context it is noteworthy that biologically active FasL is expressed in choriocarcinoma [38] Although the immune privileged status of placental trophoblast in normal and molar pregnancy may be explained by its ability to regulate maternal immune responses through FasL mediated apoptosis, accumulating evidence indicates that Fas/ FasL interaction at the fetoplacental interface is not essential for successful murine allopregnancy [39]. Moreover Fas+ T cells, endometrial granulated lymphocytes and macrophages in maternal decidua in close proximity to FasL+ trophoblast in molar pregnancy fail to undergo apoptosis (Wongweragiat, Searle and Bulmer, unpublished data). It is possible that the increased population of Fas+ maternal immune leucocytes [25] secrete cytokines that influence the abnormal proliferation of molar trophoblast. In contrast to the pattern of FasL labelling in normal and molar pregnancy there was no differential Fas expression by trophoblast subpopulations in normal early and late pregnancy, although Fas expression was reduced in villous cytotrophoblast of term compared with early pregnancy. As in the case of FasL expression, the Fas status of trophoblast has resulted in discrepant reports. Fas expression in both early and term villous syncytiotrophoblast but no staining in villous cytotrophoblast has been reported [22,40]. Others have reported no Fas reactivity on extravillous cytotrophoblast in early pregnancy [23]. In contrast Roh et al. [35] reported intense Fas staining in fetal vessels with a lack of staining on villous

Placenta (2004), Vol. 25

syncytiotrophoblast at term. The present study provides no support for the notion of discordant localization in the expression of Fas and FasL between trophoblast subpopulations either in normal or molar pregnancy. The significance of reduced Fas expression for normal placental function remains to be established. Previous studies have suggested that FasL+ activated decidual T cells may induce apoptosis of Fas+ trophoblast in normal and molar pregnancy [10,22,25]. Although apoptosis of villous trophoblast in normal and molar pregnancy has been reported [11,41–43], in this study using the TUNEL method apoptosis was detected only focally in villous syncytiotrophoblast, and was rare in villous cytotrophoblast and extravillous trophoblast, the findings being similar in both normal early pregnancy and hydatidiform mole. The present observations that apoptosis is uncommon, however, is in accord with other studies which showed that Fas expression by human placental cytotrophoblast does not necessarily mediate apoptosis [44], the Fas response being specifically blocked by unknown mechanisms to avoid killing by FasL. It is also possible that trophoblast apoptosis is inhibited by EGF [45] or that Fas expression is down-regulated by soluble FasL [46,47]. The present study also supports a previous study that apoptosis in normal term placenta may be inhibited by trophoblast expression of bcl-2, which is reduced in placentae associated with pre-eclampsia and intrauterine growth retardation [40]. Recent study of human trophoblast cells has shown that they are resistant to Fas-mediated apoptosis and that this resistance can be increased by treatment with interleukin (IL-6) and IL-10 [48]. This apoptosis inhibition by IL-10 may be due to induction of FLICE-like inhibitory protein (FLIP) which inhibits caspase 8 activity [48]. Despite the presence of an increased population of FasL+ CD4+ T cells in molar pregnancy decidua [25] there was no substantial increase in apoptotic Fas+molar trophoblast. The significance of differential Fas expression in molar pregnancy remains unclear. Additional mechanisms may be involved in controlling trophoblast proliferation and invasion. These include the balance of cytokines produced by placental trophoblast and maternal derived cells at the uteroplacental interface [49], the expression of non-classical HLA molecules by trophoblast [50], the interaction of cell surface adhesion molecules and their ligands expressed by maternal and fetal cells [51], the local production of matrix metalloproteinases and their inhibitors [33] and trophoblast hormone production [52]. Further studies on the control mechanisms operating during placental development are needed in order to understand both normal and pathological pregnancy.

ACKNOWLEDGEMENTS The authors would like to thank Dr Somchaya Neungton, Department of Obstetrics and Gynaecology, Mahidol University, Bangkok, Thailand for his kind cooperation in supplying some hydatidiform mole tissues. We also thank Mrs Barbara Innes for her excellent technical assistance. Dr Sutatip Pongcharoen was generously supported by the Royal Thai Government and the Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand to which we extend our grateful thanks.

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