Fas-FasL System in Molar Pregnancy

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Fas–FasL System in Molar Pregnancy Simmi Soni1, Gayatri Rath1, Chandra Prakash Prasad1, Sudha Salhan2, Arun Kumar Jain3, Sunita Saxena3 1

Department of Anatomy, Vardhman Mahavir Medical College & Safdarjung Hospital, New Delhi, India; Department of Obstetrics and Gynaecology, Vardhman Mahavir Medical College & Safdarjung Hospital, New Delhi, India; 3 Institute of Pathology – ICMR, New Delhi, India 2

Keywords Apoptosis, complete hydatidiform moles, Fas, Fas L, placenta Correspondence Gayatri Rath, Professor and Head, Department of Anatomy, Vardhman Mahavir Medical College & Safdarjung Hospital, New Delhi110029, India. E-mail: [email protected] Submitted July 26, 2010; accepted October 5, 2010. Citation Soni S, Rath G, Prasad CP, Salhan S, Jain AK, Saxena S. Fas–FasL system in molar pregnancy. Am J Reprod Immunol 2011; 65: 512–520 doi:10.1111/j.1600-0897.2010.00926.x

Problem Hydatidiform mole (molar pregnancy) is the commonest form of Gestational Trophoblastic Disease, with the risk of undergoing malignant transformation. The molecular pathway leading to pathogenesis and progression of molar pregnancy is barely understood. The study focuses on Fas ⁄ FasL system which represents one of the main apoptotic pathways controlling placental morphogenesis. Method of study Placental tissues from 52 patients with complete hydatidiform moles (CHMs) and 55 age-matched controls were examined for Fas and FasL expression using immunohistochemistry, immunofluorescence and Western blotting. The protein expression was also correlated with trophoblast apoptosis (assessed by M30 Cyto DEATH), clinico-pathological parameters and disease progression. Results Immunohistochemistry and immunofluorescence revealed both cytoplasmic and membranous expression of Fas in villous syncytiotrophoblast as well as cytotrophoblast but FasL was confined merely to the cytoplasm of syncytiotrophoblast. Both Fas (cytoplasm and membrane) and FasL were significantly upregulated in syncytiotrophoblast of CHMs (P = 0.004, P < 0.0001 and P < 0.0002 respectively) and showed a positive association between them (P = 0.019). However, none of the proteins reveal any correlation with M30 index. The results were revalidated using Western blotting. Conclusion This study demonstrated differential expression of Fas and FasL in CHMs and its implications in the pathogenesis of molar pregnancy has been discussed.

Introduction The Fas ⁄ Fas ligand (Fas ⁄ FasL) system, also known as the death factor system, is one of the major pathways for the induction of apoptosis in cells and tissues.1 Fas (CD95) is a type I membrane protein of 45 kDa that belongs to the TNF receptor family of proteins. FasL, a type II membrane protein of

37 kDa, belongs to the TNF and CD40 ligand family.1 The binding of the Fas receptor by FasL results in activation of a cascade of intracellular proteolytic enzymes resulting in apoptosis.2 The Fas ⁄ FasL system in normal tissues is limited to activated T-lymphocytes, natural killers cells, and to a few immune privileged tissues such as the brain, eye, testis and placenta.3 Its expression and function responds to American Journal of Reproductive Immunology 65 (2011) 512–520


ª 2010 John Wiley & Sons A/S


changes in the microenvironment, and plays a pivotal role in controlling cell proliferation and tissue remodeling.2 Elevated FasL expressions have been found in tumor cells of colon, esophageal, stomach, lung, and ovarian cancer, which may trigger apoptosis of activated lymphocytes, offering a survival advantage to tumors.4,5 Fas and FasL are expressed by the trophoblast throughout pregnancy.6,7 The role of Fas ⁄ FasL system in trophoblast physiology is presumably more complex than originally expected. Fas expression has also been demonstrated in the chorion and amnion of fetal membranes at term as well as in the decidua.8,9 Studies in reproductive tissues have shown that the expression of Fas and FasL are mainly related to tissue remodeling and involution.10 Any discrepancy in Fas ⁄ FasL system may have relevance in placental pathologic conditions such as pre-eclampsia,11,12 recurrent abortion13 and hydatidiform moles. Hydatidiform mole (HMs) is an abnormal placental mass characterized by enlarged, edematous chorionic villi and proliferation of trophoblasts to a varying degree. It is classified into partial hydatidiform moles (PHMs) and complete hydatidiform moles (CHMs).14 Approximately 8–30% of HMs possesses the risk of developing persistent trophoblastic disease (PTD), requiring therapeutic interventions.15 However, its etiology remains intangible; thus the molecular mechanisms underneath this disorder needs to be explored. The primary aim of the study was to investigate the expression patterns of integral components of apoptosis; Fas and FasL in placental tissue of CHMs, to elucidate the biological and clinical relevance of Fas ⁄ FasL system in molar pregnancy. Materials and methods Placental Tissue Specimens A total of 107 (55 + 52) cases were included in the study. Evacuated placental samples from patients with CHMs (n = 52) were collected from Department of Obstetrics & Gynaecology (VMMC & Safdarjung Hospital, New Delhi, India). They presented with ultrasonographic abnormalities suggestive of molar pregnancy and the diagnosis was confirmed with histological examination of samples after suction evacuation. All the patients included in the study group were CHMs, because of limited number of PHMs available. Placental tissue from medically ter-

minated normal pregnancies (n = 55), ranging from 8 to 20 weeks were taken as age-matched controls. The study design was approved by the Institutional Human Ethics Committee and informed written consent was obtained from each subject (both case and control) enrolled in the study. The Patient data included maternal age, gestational age, menstrual history, obstetrics history (gravidity, parity and abortions), medical history and family history (Table I). The gestational age of the CHMs, based on the last menstrual period, ranged from 8 to 20 weeks (median age 14 weeks). The median age of the patients was 24 years (range, 19– 35 years) in CHMs group. Follow up was available in 30 of 52 patients with the diagnosis of CHMs in this study. Among 30 cases, 17 underwent spontaneous regression and 13 developed PTD. Immunohistochemistry Immediately after suction evacuation, the desired portion of the placental specimen was fixed in 10% buffered formalin for 24 hr. Following fixation, the samples were routinely processed for paraffin embedding. Later, 5-lm thick sections were mounted on poly-l-lysine coated slides, air-dried and processed for immunohistochemical analysis. The sections were deparaffinized, rehydrated and immunostained with Fas, FasL and M30 (to evaluate apoptosis) antibodies, respectively. The antibodies were tested at a series of dilutions and antigen retrieval conditions (buffer, heating intensity and time) were standardized to ensure optimal antigen detection with minimal background. The retrieval was carried out in citrate buffer (pH 6.0) and endogenous peroxidase activity was quenched with 3% H2O2 in methanol. The sections were then incubated overnight at 4C with (a) mouse anti-human Fas monoclonal antibody (B-10: sc-8009; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) at 1:100 dilution, (b) mouse anti-human FasL monoclonal antibody (WW10: sc-73974; Santa Cruz Biotechnologies) at 1:40 dilution or with (c) mouse anti-human M30 Cyto DEATH antibody (12140349001; Roche Applied Science, Mannheim, Germany) at a dilution of 1:50. Later, sections were rinsed in phosphate-buffered saline (PBS) and incubated with polymer based REAL EnvisionTM (Dextran coupled with peroxidase molecules and goat secondary antibody molecules against rabbit and mouse immunoglobulins in buffered solution) (Dako Cytomation, Glostrup,

American Journal of Reproductive Immunology 65 (2011) 512–520 ª 2010 John Wiley & Sons A/S



Denmark) at room temp. Finally, the Sections were developed with DAB (as the chromogen substrate), counterstained with Mayer’s hematoxylin and examined under light microscope (Olympus BX-51, Tokyo, Japan). For negative control, primary antibody was replaced by isotype-specific IgG. Assessment of Expression Evaluation of protein expression was carried out in two randomly chosen slides of each case and every

slide was studied by two observers (S.S. & S.S.) blinded to the experimental group. An average of twenty microscopic fields was examined (each slide) at 100·, 200· & 400· magnification under a light microscope (Olympus BX-51). The Fas and FasL expressions were scored with respect to the subjectively evaluated intensity of staining and the proportion of villous cells showing positive reaction. The cytoplasmic or membranous staining was considered as positive. Each observer used a simple intensity scoring system whereby; 0 was no staining, 1 was

Table I Correlation of Clinicopathological Parameters with Fas and FasL Expression Fas expression [Median (Interquartile Range)] Clinicopathological parameters Maternal age >30 £30 P value Gestational age (weeks) 8–12 13–20 P value Habits (smoking ⁄ tobacco) Nil Present P value Cycle Reg Irreg P value Parity Nulli Multi P value Prior abortions Nil ‡1 P value Bad obstetrics history No Yes P value Disease progression Progd Regrd P value

No. of cases

Fas L [M (IQR)]

CT (cyto)

CT (memb)

ST (cyto)

ST (memb)

ST (cyto)

46 6

1.0 (2.00) 1.0 (1.76) 0.29

0.00 (1.25) 0.00 (0.30) 0.26

2.0 (2.00) 2.0 (2.28) 0.74

1.0 (2.25) 2.0 (1.88) 0.73

0.00 (1.0) 0.00 (1.74) 0.40

11 41

1.01 (3.0) 1.0 (1.88) 0.27

0.00 (1.20) 0.00 (0.90) 0.42

1.97 (2.00) 2.00 (2.00) 0.56

1.00 (1.00) 1.37 (2.38) 0.86

0.00 (0.60) 0.00 (1.00) 0.57

47 8

1.0 (2.00) 0.50 (1.50) 0.69

0.00 (1.50) 0.00 (0.00) 0.47

2.00 (2.00) 1.00 (2.00) 0.69

1.50 (2.50) 0.50 (1.00) 0.09

0.00 (1.00) 0.00 (1.00) 0.75

35 17

1.00 (0.75) 0.50 (3.00) 0.03* fl

0.00 (0.55) 0.00 (1.50) 0.17

2.00 (1.53) 2.00 (2.25) 0.55

2.00 (2.00) 1.00 (2.25) 0.32

0.00 (1.00) 0.00 (1.00) 0.79

22 30

1.01 (1.63) 1.00 (1.38) 0.52

0.25 (2.00) 0.00 (0.38) 0.16

1.97 (1.63) 2.00 (2.00) 0.51

1.37 (2.63) 1.12 (2.00) 0.33

0.00 (1.00) 0.00 (1.00) 0.93

38 14

0.00 (1.25) 1.02 (2.00) 0.68

0.00 (1.25) 0.00 (0.55) 0.60

2.00 (2.00) 1.00 (2.00) 0.32

1.24 (2.50) 1.24 (1.75) 0.42

0.00 (1.00) 0.00 (2.50) 0.14

43 9

1.00 (2.00) 0.75 (1.00) 0.16

0.00 (1.13) 0.00 (0.38) 0.36

2.00 (2.00) 1.05 (1.05) 0.06

1.37 (2.50) 0.50 (1.38) 0.07

0.00 (1.00) 0.00 (1.01) 0.12

13 17

1.00 (1.62) 1.0 (1.375) 0.97

0.00 (0.00) 0.00 (0.90) 0.43

2.25 (1.29) 1.97 (2.25) 0.52

1.37 (3.00) 1.62 (1.87) 0.88

0.00 (1.00) 0.00 (1.00) 0.47

Bad obstetric history includes history of tubal pregnancy ⁄ prenatal death ⁄ still birth ⁄ ovarian cyst ⁄ multiple fibroids. CT, cytotrophoblast; ST, syncytiotrophoblast; cyto, cytoplasm; memb, membrane; Progd, progressed cases; Regrd, regressed cases. *Significant. **Mann–Whitney U-test and Wilcoxon signed-rank test [Asymp. Sig. (two-tailed)]. Figure in bold is statistically significant.

American Journal of Reproductive Immunology 65 (2011) 512–520


ª 2010 John Wiley & Sons A/S


weak staining, 2 moderate staining and 3 intense staining. And the proportion of stained villous cells was assessed on a scale with grades: ) (0), 50% positive cells. The final evaluation was performed using [(intensity · percentage of positivity) ⁄ 2]. For M30, the apoptotic index was calculated by counting the number of positive nuclei per field (at 200·), in 20 random fields.16 The percentage of agreement between the observers was 80%; and the samples scored inside this range were considered for the final appraisal. Immunofluorescence For immunofluorescence, the tissue sections were deparaffinized, rehydrated and immunostained with Fas and FasL antibodies at a dilution of 1:400 and 1:160, respectively. Later, sections were rinsed in PBS and incubated with secondary antibody (Biotinylated anti-rabbit, anti-mouse and anti-goat immunoglobulins in PBS) (LSAB Kit – Dako Cytomation) for 1 hr. This was followed by rinsing in PBS and incubation in streptavidin–conjugated-FITC labeled tertiary antibody (Dako Cytomation) at 4C for 45 min. Finally, the sections were counterstained with propidium iodide (PI), mounted with fluorescent mounting media (Dako Cytomation) and examined under fluorescent microscope (Axio Imager M1; Carl Zeiss Pvt Ltd, Bangalore, India). Immunoblot Analysis of Fas and FasL in Normal and Molar Placental Tissue Frozen placental tissue samples (case and controls) were homogenized and lysed in RIPA buffer. Concentration of protein was determined using the Bradford assay (Sigma Chemicals, Bangalore, India) and equal quantity of protein (80 lg ⁄ lane) from tissue lysates was electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred onto polyvinylidenedifluoride (PVDF) membranes. After blocking in 5% non-fat milk, blots were incubated with anti-Fas antibody (1:500 dilution) ⁄ anti-FasL antibody (1:200 dilution) at 4C overnight. Membranes were then incubated with HRP-conjugated secondary antibody (rabbit ⁄ mouse anti-IgG) (Dako Cytomation), diluted at an appropriate dilution in 1% BSA, for 2 hr at room temperature. Finally, the protein bands were visualized on X-ray film using an enhanced chemiluminescence

system (ECL; Santa Cruz Biotechnology) and Intensity-based quantification of Western blot bands was analyzed using ChemiImager4400 software (Alpha Innotech, San Leandro, CA, USA). Statistical Analysis Statistical analysis was performed on SPSS (18.0) statistics software (SPSS Inc., Chicago, IL, USA). The results were expressed in Median with Interquartile Range (continuous variables), or percentages of total (categorical variables). The non-parametric Mann– Whitney U-test and Wilcoxon signed-rank test [Asymp. Sig. (two-tailed)] were used to determine differences in mean values for comparison between study (CHM patients) and control group. The Pearson correlation (two-tailed) and chi-square test were used to assess inter-protein significance (Correlations between Fas, FasL and M30). Differences of P < 0.05 were considered to be statistically significant. Results Immunohistochemistry and Immunofluorescence (on Representative Samples) Evaluation of Fas expression The results revealed specific expression of Fas protein in the chorionic villi of both normal and molar placentae examined (Figs 1 and 2). The reactivity of Fas protein was almost uniform among different villi regardless of maturity level or location. However, the intensity of the staining varied among different cell types and experimental group. The Fas protein was found immunolocalized to villous syncytiotrophoblast (ST), cytotrophoblast (CT), as well as Hofbauer cells of both normal and molar placentae (Figs 1a–c and 2a,b). In the normal cases, the cytotrophoblast cells underlying the syncytium, gave distinct cytoplasmic and membrane-located staining, whereas the syncytiotrophoblast, showed only faint staining distinctively (Fig. 1a). On the other hand, in majority of CHMs, villous CT showed mild immunostaining, but ST cells displayed intense immunoreactivity for Fas, both in cytoplasm (Figs 1b and 2a) as well as syncytial membrane (Figs 1c and 2b). Its expression in CT decreased (P < 0.0001 & P < 0.0005; cytoplasm and membrane respectively) and ST increased (P < 0.004 & P < 0.0001; cytoplasm and membrane respectively) significantly in CHMs when compared with gestational-matched normal placentae (Table II). The

American Journal of Reproductive Immunology 65 (2011) 512–520 ª 2010 John Wiley & Sons A/S









current study also demonstrated for the first time nuclear expression of Fas in CHMs (Fig. 1d). Furthermore, Fas expression in CT was found significantly reduced in patients with irregular menstrual cycles in comparison with women with regular cycles (P = 0.03) (Table I). However, no correlation could be found between clinical progress of the patients with CHMs and the Fas expression (Table I). Analysis of Fas L expression Both immunohistochemical and immunofluorescence analysis revealed specific and constitutive expression of FasL protein in the placental villi examined. In contrast to Fas expression, the FasL was confined only to the syncytiotrophoblast layer. The cytotrophoblasts cells as well as Hofbauer cells

Fig. 1 Immunohistochemical localization of Fas and FasL in normal and molar placental tissue (a–f). (a) Normal placenta (12 weeks) showing intense cytoplasmic and moderate membranous immunostaining of Fas protein in CT (arrow heads) but hardly any expression in ST (arrows). (b) CHMs displaying moderate Fas expression in both CT (arrow heads) and ST (arrows). (c) CHMs (12 weeks) showing distinct membranous and mild cytoplasmic staining for Fas in ST (arrows). (d) Nuclear expression of Fas in ST of CHMs (arrows). (e) Control placentae (14 weeks) showing faint immunostaining for FasL in ST (arrows). (f) CHMs (14 weeks) demonstrating specific cytoplasmic expression of FasL in ST layer (arrows) (Original magnification ·400).

showed negative immunostaining for FasL. A cytoplasmic pattern of immunoreactivity was detected with no membranous ⁄ nuclear staining or background (Figs 1e,f and 2c). Its expression was found to be upregulated significantly in CHMs (P < 0.0002) in comparison with age-matched normal placentae (Fig. 1e,f) (Table II). Furthermore, the FasL expression did not reveal any correlation with the clinico-pathological parameters analyzed or the disease progression (Table I). Correlation among Fas, FasL and apoptosis The apoptotic cells were detected using M30 Cyto DEATH antibody and the apoptotic index was calculated. Both Fas ⁄ FasL and M30 were expressed by the villous cells of CHMs but the immunoreactivity of Fas (CT cytoplasm, CT membrane, ST cytoplasm and American Journal of Reproductive Immunology 65 (2011) 512–520


ª 2010 John Wiley & Sons A/S




Fig. 2 Immunofluorescence analysis of Fas and FasL in CHMs demonstrating FITC, PI and merged (FITC + PI) images (a–c). (a) CHMs showing moderate Fas expression in ST cytoplasm (arrows) and negative expression in CT (arrow heads). (b) Molar placental tissue displaying prominent membranous immunostaining for Fas in ST (arrows). (c) Molar tissue demonstrating significant cytoplasmic with negative membranous FasL expression in ST (arrows) (Original magnification ·400).


Table II Immunohistochemical Analysis of Fas and FasL in CHMs in Comparison with Age-Matched Control Placentae


Cell types





Cyto Memb Cyto Memb Cyto

CHMs [Median (I.Q. Range)]

Control [Median (I.Q. Range)]

P value

1.00 0.00 2.00 1.12 0.00

3.00 1.00 1.00 0.00 0.00

0.0001* 0.0005* 0.004* 0.0001* 0.0002*

(1.88) (0.60) (2.00) (2.00) (1.00)

(1.00) (2.75) (1.00) (0.75) (0.00)

CT, cytotrophoblast; ST, syncytiotrophoblast; cyto, cytoplasm; memb, membrane. *Significant. **Mann–Whitney U-test ⁄ Wilcoxon signed-rank test [Asymp. Sig. (two-tailed)].

ST membrane) and FasL in CHMs did not correlate with the extent of apoptosis (assessed by M30) (P = 0.85, P = 0.42, P = 0.084, P = 0.42 and P = 0.15, respectively). However, a significant positive association was found between Fas and FasL expression in ST cytoplasm (P = 0.019 ⁄ P = 0.04) (Tables III and IV). Western blotting The Western blotting analysis for Fas protein revealed a major band of approximately 45 kDa,

whereas, FasL showed a prominent band of around 42 kDa (Fig. 3). The significant upregulation of Fas and FasL in immunohistochemicaly confirmed cases of CHMs (M1, M2, M3, M4) was revalidated by Western blotting (Fig. 3). Intensity-based quantification of Western blot bands showed marked increase in the expression of both Fas (2 folds) and FasL (threefolds) in CHMs (Fig. 3). The values were expressed in mean ± standard deviation of four independent experiments and the mean values were used for graphic representation (Fig. 3). Discussion A number of studies have accounted the expression of Fas and FasL in trophoblast of normal human pregnancy;7,17–20 however, there is scarcity of literature on HMs.16,21 Moreover, the earlier reports on the expression of Fas and FasL in CHMs were controvertial.21,22 There have been different views regarding the precise localization of FasL in the villous trophoblast. Some investigators have reported the FasL as cytoplasmic,19,20 whereas others have described membranous expression.7,17 Besides, there were studies supporting that FasL is expressed by both cytotrophoblast and syncytiotrophoblast,16,23 whereas others have noticed FasL in cytotrophoblast cells alone.7,17 On contrary to these reports, this study demonstrated FasL expression solely in

American Journal of Reproductive Immunology 65 (2011) 512–520 ª 2010 John Wiley & Sons A/S



Table III Correlation Among Fas, FasL and M30 Index (Apoptosis) Fas expression Proteins

Fas L M30 index Fas L

CT (cyt)

M30 index CT (memb)

[Pearson correlation value (P value)] 0.152 (0.281) )0.178 (0.206) )0.028 (0.851) 0.119 (0.421) [Chi-square value (P value)] 0.265 (1.00) 0.415 (1.00)

ST (cyt)

ST (memb)

ST (cyt)

0.325 (0.019*) )0.252 (0.084)

0.179 (0.205) )0.119 (0.420)

)0.210 (0.152) 1

5.53 (0.04*)

0.283 (0.518)

CT, cytotrophoblast; ST, syncytiotrophoblast; cyto, cytoplasm; memb, membrane. *Significant. **Pearson correlation (two-tailed) ⁄ chi-square analysis (two-sided). Figures in bold are statistically significant.

Table IV Positive Association Between Fas and FasL in Syncytiotrophoblast Cytoplasm Fas (ST cyto) No. of CHMs (n = 52)

Negative (£2.0) Positive (>2.0) P value

Fas L Negative (£2.0) 33 (63.46%) Positive (>2.0) 0 (00%)

16 (30.77%) 3 (5.77%)

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