Hum. Reprod. Advance Access originally published online on September 14, 2007
Human Reproduction 2007 22(11):2814-2823; doi:10.1093/humrep/dem284
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Histochemical and morphological examination of proliferation and apoptosis in human first trimester villous trophoblast
Department of Physiology, All India Institute of Medical Sciences, New Delhi 110029, India
1 Correspondence address. Tel: +91-11-2659-4625; Fax: +91-11-2658-8641/2658-8663; E-mail: jsen47{at}gmail.com
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Abstract |
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BACKGROUND: Our present knowledge about trophoblast turnover in human first trimester placental villi based on multiparametric examination of proliferation and apoptosis is limited.
METHODS: Human villous placentae collected during 6, 7 and 8 weeks (n = 10/each group) of gestation were examined for trophoblast proliferation and apoptosis based on quantitative analyses of immunopositive Fas, tumor necrosis factor receptor 1 (TNFR1), cytokeratin 18 fragment (18f), number of proliferating cell nuclear antigen (PCNA), Ki67 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positive nuclei, scores of mitotic and apoptotic indices and ultrastructural characteristics.
RESULTS: Mitotic index in cytotrophoblast higher (P < 0.05) at 6 week compared with 7 and 8 weeks of gestation showed significant (P < 0.05) negative correlation between its prevalence and gestational age. Syncytiotrophoblast exhibited higher number of TUNEL positive nuclei (P < 0.01), TUNEL positive apoptotic nuclei (P < 0.05) and apoptotic index (P < 0.05) compared with cytotrophoblast at same gestational age. Positive correlations found between cytokeratin 18f and apoptotic index (P < 0.01), Fas and apoptotic index (P < 0.01), TUNEL positive nuclei and apoptotic index (P < 0.05), cytokeratin 18f and Fas (P < 0.01), whereas cytokeratin 18f (P < 0.05) and Fas (P < 0.05) showed positive correlation only with TUNEL positive apoptotic nuclear data. Phalangeal intrusions of syncytiotrophoblast between transitional cytotrophoblasts showed apposed plasma membranes bearing thickened membrane leaflets, inter-membranous gaps enclosing membranous invaginations, liposome-like particles; patches of membrane seen to be dissolved resulting in cytoplasmic continuity typical of syncytial formation.
CONCLUSION: Cellular remodeling of first trimester villous placenta requires a complex homeodynamics involving proliferation in cytotrophoblast, development-associated syncytialization and apoptosis in syncytiotrophoblast.
Key words: apoptosis/cytotrophoblast/syncytial formation/proliferation/syncytiotrophoblast
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Introduction |
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Human villous placenta is characterized by floating chorionic villi of syncytiotrophoblast overlying a single layer of cytotrophoblast. Proliferative stem cells (Langhans’ cells) make up the villous cytotrophoblast, and a basal lamina separates it from the fetal stroma. Daughter cells of the stem cells differentiate depending upon their position into either villous syncytiotrophoblast or invasive extravillous cytotrophoblast (Enders et al., 2001
). Maintenance of structural and functional integrity of placental tissue involves a highly regulated cell turnover that is dependent upon a delicate balance involving proliferation, differentiation and loss of trophoblast cells (Benirschke and Kaufmann, 2000). Maternal anemia leading to preplacental hypoxic condition has been shown to lead to increased proliferative activity in villous and extravillous cytotrophoblast with increased incidence of pre-eclampsia, placental abruption and preterm labor (Kosanke et al., 1998). Indirect evidence suggests that hypoxic stress affects placental growth that in turn may precipitate pre-eclampsia and fetal growth retardation (Myatt, 2006). Thus, it is generally believed that placental physiology during the first trimester of pregnancy relates to the details of functional establishment of placenta (Jauniaux et al., 2006). However, the present knowledge about the physiology of human placental cell turnover during the first trimester is rather limited. Although it is generally known that a pool of cells in cytotrophoblast compartment provides stem cells for placental growth, there is no clear knowledge about the characteristics of natural cellular turnover in this compartment in the first trimester. There is evidence of apoptosis and necrosis in human first trimester villous placental trophoblast (Burton et al., 2003). On the contrary, there are reports indicating that first trimester villous placental cytotrophoblast does not show apoptosis as a discernible feature (Smith et al., 1997; Chan et al., 1999). In fact, pregnancies complicated by pre-eclampsia or intrauterine growth retardation show placental dysfunction and are often associated with increased apoptosis in villous trophoblast cells (Crocker et al., 2003). Multinucleate syncytiotrophoblast originate from cytotrophoblast fusion, but the developmental process of fusion of trophoblast cell membranes is as yet poorly understood. Huppertz et al. (1999) proposed that cytotrophoblast cells utilize the initial stages of apoptotic cellular machinery for differentiation, and later fusion with syncytiotrophoblast. Indirect evidence supports this possibility (Black et al., 2004). Clearly there is a need to understand the regulation of trophoblast proliferation and apoptosis in normal pregnancy before we can address the pathophysiological basis of the placental disorders as described above. In the present study, the turnover of human first trimester villous placental trophoblast has been examined based on histochemical and morphometric analyses of proliferation [mitotic index, Ki67, and proliferating cell nuclear antigen (PCNA)] and apoptosis [apoptotic index, tumor necrosis factor receptor p55 (TNFR1), Fas, cytokeratin 18 fragment (18f), and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) positivity] in human villous placental trophoblast cells at gestational ages of 6, 7 and 8 weeks. |
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Tissue samples
First trimester human placental samples were obtained from women undergoing elective surgical termination of pregnancy without undergoing any prior medication at 6 weeks (n = 10), 7 weeks (n = 10), 8 weeks (n = 10) of gestation (timed from last menstrual period) at the All India Institute of Medical Sciences, New Delhi. All women had provided their written consent to participate in the study. The study design had been approved by the Ethics Committee of the All India Institute of Medical Sciences. Placental samples were immediately placed in ice-cold phosphate-buffered saline (PBS, pH 7.4) and transported on ice to the laboratory within 15 min for further processing as described below.
Tissue processing
Placental samples were carefully washed in ice-cold PBS (pH 7.4) to remove adhering blood, and villi were quickly dissected from adjoining uterine tissue and decidua as described earlier (Lamba et al., 2005), and samples were fixed in (i) cold, phosphate-buffered neutral paraformaldehyde (4%, w/v) for paraffin embedding and sectioning (5 µm) for routine histology and histochemistry (Ghosh et al., 1996) and (ii) ice-cold Karnovsky’s fixative [2% (w/v) glutaraldehyde, 2% (w/v) paraformaldehyde, calcium chloride (1 mM), glucose (100 mM) in phosphate buffer (0.1 M, pH 7.4)] and post-fixed in 1% (w/v) osmium tetroxide, embedded in epoxy resin (Embed812, Electron Microscopic Sciences, PA, USA) for transmission electron microscopy (Sengupta et al., 1990). Semi-thin (1 µm) sections were stained with 1% toluidine blue, and thin (
70 nm) sections were stained with uranyl acetate and lead citrate and viewed using a Philips CM10 Transmission Electron Microscope (Sengupta et al., 1990).
Mitotic and apoptotic indices
The methodological details for assessing indices of cell mitosis and apoptosis have been described earlier (Ghosh et al., 1992; Sengupta et al., 2003). Briefly, for each placenta at least 30 fields (10 fields from each section), 500 nuclei per section were randomly examined (x1000) using a Leica DMRD Microscope attached with digital camera for all placental samples collected at 6, 7 and 8 weeks of gestation. Sections were counted for presence of mitosis by absence of nuclear membrane, and for apoptosis by presence of adnuclear basophilic granules and chromatin condensation, and the data were expressed separately as a percentage of numbers of positive nuclei relative to the total number of nuclei studied in the cytotrophoblast and syncytiotrophoblast compartment, respectively.
Immunohistochemistry
Immunohistochemical staining for detection of PCNA, Ki67, cytokeratin 18f, TNFR1 and Fas were performed using specific antibodies. Parallel sections were subjected to immunohistochemical detection of cytokeratin 7 positive cytotrophoblast cells to define cellular localization of above factors. Table 1 provides the details of target antigens and antibodies. Immunohistochemical stainings for PCNA and Ki67 were performed as described earlier (Cattoretti et al., 1992; Sengupta et al., 2003). Immunohistochemistry for the detection of Fas, cytokeratin 18f and TNFR1 was carried out using methodologies described earlier (Yonehara et al., 1989; Kadyrov et al., 2001; Lalitkumar et al., 2005). Briefly, deparaffinized and hydrated tissue sections were subjected to microwave heating in 0.1 M sodium citrate buffer (pH 6.0) for retrieval of antigens. The endogenous peroxidase activity was quenched and non-specific binding was blocked with non-immune sera as described earlier (Sengupta et al., 2003). Visualization was achieved using Vectastain ABC Peroxidase Elite Kit (Vector Laboratories, Burlingame, CA, USA) and freshly prepared 3,3'-diaminobenzidine (DAB) tetrahydrochloride (Sigma Chemical Company, St Louis, MO, USA) as substrate. All immunostaining procedures were performed in a single run. Specificity of antibody ligand binding and visualization were assessed by omitting primary antibody, by replacing primary antibody with unrelated immunoglobulin (Ig) from same species and other species, by omitting secondary antibody, by replacing labeled secondary antibody with unrelated labeled Ig from the same and other species and by antibody neutralization with specific ligands (Fas and TNFR1) on adjacent tissue sections.
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Detection of apoptotic nuclei in situ using TUNEL method
Detection of nuclear DNA fragmentation as a morphological marker of the apoptosis process in histological sections was achieved by employing in situ TUNEL method (Gavrieli et al., 1992
) using an in situ cell death detection kit (Roche Applied Sciences, Sandhofer Strasse, Mannheim, Germany) as described previously (Sengupta et al., 2003). Briefly, 5 µm tissue sections were deparaffinized, hydrated and rinsed with 0.1 M phosphate buffer (pH 7.4) followed by quenching of endogenous peroxidase activity using hydrogen peroxide. The TUNEL reaction mixture containing TdT enzyme [(EC 2.7.7.3
[EC]
1) from calf thymus] and fluorescein-labeled nucleotide mixture were added and the sections were incubated for 60 min at 37°C followed by incubation with sheep anti-fluorescein antibody (Fab fragment) conjugated with horseradish peroxidase for 30 min at 37°C. Visualization was achieved using freshly prepared DAB tetrahydrochloride for 10 min at room temperature. All solutions were prepared using sterile water to avoid artificial DNA fragmentation. For negative control, instead of using TUNEL mixture, sections were incubated only with TdT label solution without the TdT enzyme mixture, and for positive control, DNase I treated sections were used.
Microscopic quantification
Numbers of nuclei showing staining for PCNA, Ki67 and TUNEL were obtained from at least 3 sections for each sample, and at least 10 fields per section were viewed at x 200 magnification. The data were expressed as percentage of numbers of positive nuclei relative to the total number of nuclei studied of cytotrophoblast and syncytiotrophoblast compartments (Spyratos et al., 2002). For analysis of cytokeratin 18f, Fas and TNFR1 immunostaining, at least 3 sections for each sample (at least 15 fields per section) were morphometrically analysed to estimate areas of immunopositivity in different compartments using a Leica microscope and a precalibrated computer-assisted video image analysis system (Leica QWin DC 200, Cambridge, UK) as described elsewhere (Ghosh et al., 1998; Sengupta et al., 2003). The data were expressed as percentage of immunopositive area relative to the total area studied of cytotrophoblast and syncytiotrophoblast compartments. The different cell types were detected using an interactive planimeter analyzer and immunopositive areas were measured by detecting positive profiles in digitized images based on an optimized grey level threshold after shading correction and pixel calibration against the standard provided by the manufacturer. Three investigators independently performed the morphometric analyses in a blinded manner, and sections that yielded coefficient of variance 
10% in the pooled data analysis were excluded. It was assumed that these measurements reflected concentrations of the investigated proteins in cells of human first trimester placental tissues.
Statistical analysis
Statistical analyses of quantitative measurements were performed using Kruskal–Wallis test. Inter-dependence among different parameters and comparison between different cell types were examined by computing Kendall rank-order correlation coefficient and Wilcoxon signed rank test, respectively (Hollander and Wolfe, 1999). The probability level of P = 0.05 was taken as the limit of significance.
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Morphology
Morphological examination of placental villi at 6, 7 and 8 weeks of gestation showed conspicuous syncytiotrophoblast growth with a clear continuous layer of cytotrophoblast adjoining stromal matrix bearing fetal blood vessels (Fig. 1A–C), conglomeration of large, round to oval shaped cytotrophoblast with highly basophilic cytoplasm and dense crowding of syncytiotrophoblast nuclei (Fig. 1C), and features of cell mitosis in cytotrophoblast (Fig. 1D) and apoptosis in cytotrophoblast (Fig. 1E) and in syncytiotrophoblast (Fig. 1F). Undifferentiated cytotrophoblast showed electron-lucent cytoplasm enclosing Golgi bodies, mitochondria, strands of rough endoplamic reticulum, a large round nucleus bearing thin rim of heterochromatin and evenly dispersed euchromatin, and apical microvilli and membranous blebbing extended into adjoining syncytiotrophoblast (Fig. 2A). Cytotrophoblast in proliferating pool showed features of metabolically active cells characterized by large number of mitochondria, rough endoplasmic reticulum, confronting cisternae, and nuclei showing marked indentations, nucleolar margination (Fig. 2B) and typical features of karyokinesis (Fig. 2C). Occasionally, frank necrosis was detected in cytotrophoblast having calcareous deposits surrounding highly electron-dense nuclei and large intracytoplasmic vacuoles enclosing necrotic debris (Fig. 2C). Protoplasmic phalanges of syncytiotrophoblast were found extended between cytotrophoblast cells and often reached the basement membrane. The cell borders between cyto- and syncytiotrophoblast often exhibited membrane plications, dense plaques adjoined dilated inter-membraneous spaces with enclosed vesiculating membranes and electron-dense, liposome-like bodies (Fig. 2D).
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Morphologically, syncytialization in villous samples was characterized by breakdown of adjoining plasma membranes leading to cytoplasmic continuity between cyto- and syncytiotrophoblast (Fig. 3). Figure 3A shows a cytotrophoblast with markedly indented nucleus having nucleolar margination, and smooth apical cell membrane between syncytiotrophoblast and basement membrane. The ultrastructural features of ‘transition stage’ cytotrophoblast [as designated by Enders (1965)
] resembled that of overlying syncytiotrophoblast; short stretches of cell membranes closely apposed with syncytiotrophoblast, inter-membranous gaps enclosed complex, vesiculating membranous structures and electron-dense, liposome-like bodies bounded at both ends by dense plaques (Figs. 3B, C). Cytotrophoblast bearing typical flask shaped caveolae either adjoining or within complex invaginations of plasma membranes were observed at such putative sites of syncytialization (Fig. 3D). The complex membranous invaginations adjoining dense plaques between cytotrophoblast and syncytiotrophoblast often enclosed electron-dense particles; numerous lysosomes and large dilated endoplasmic reticulum were found in adjoining cell matrix (Fig. 3E). These cellular features characteristic of ‘pre-fusion’ state were also found at sites of syncytial phalangeal intrusion between ‘transition stage’ cytotrophoblast cells (Fig. 2D). In a pool of electron-dense cells in cytotrophoblast compartment, discrete areas of apparent continuity with syncytiotrophoblast bearing dilated rough endoplasmic reticulum, scattered electron-dense particles, heterolysosomes and incomplete plasma membranes on the side toward syncytium were seen, and these were often closely associated with membranous invaginations and membrane bound, plaque-like structures (Fig. 3F). This stage is considered to represent the ‘fusion state’. Presence of cellular organelles interposed between isolated stretches of such membranes observed at this stage suggests that the discontinuity was not an artifact. As described previously by Carter (1964) and Enders (1965), isolated stretches of plasma membrane bearing desmosomes were observed frequently within syncytium in the present study.
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Proliferation
As shown in Table 2, the mitotic index for cytotrophoblast cell was significantly higher (P < 0.05) at 6 weeks of gestation compared with that at 7 weeks and 8 weeks of gestation. Significant negative (P < 0.05) correlation was evident for the prevalence of proliferating cytotrophoblast cells and gestational age. However, there was no significant difference in numbers of nuclei showing immunoreactive PCNA and Ki67 in cytotrophoblast cells in early gestation (Table 2). Mitotic bodies as well as immunoreactive Ki67 nuclei were found in cytotrophoblast (Figs. 2C and 4A) but not in syncytiotrophoblast. Although PCNA antigen was detected in both cyto- and syncytiotrophoblast (Fig. 4B), the number of PCNA positive nuclei in cytotrophoblast was higher (P < 0.01) (Table 2). Among three parameters of proliferation (mitotic index, Ki67 and PCNA) examined, no correlation was observed between any two parameters in any compartment, as well as in compartment-free and stage-free pooled data analysis.
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Apoptosis
As shown in Table 3, apoptotic bodies (
1%), total TUNEL positive nuclei (3%) and TUNEL positive apoptotic nuclei (0.4%) were detected in a small population of cells in cytotrophoblast with no significant difference among 6, 7 and 8 weeks of gestation and without any relation between their prevalence and gestational age. Occurrence of apoptotic bodies (3%), total TUNEL positive nuclei (14%) and TUNEL positive apoptotic nuclei (4%) were significantly more numerous in syncytiotrophoblast (Fig. 4G) when compared with cytotrophoblast at same gestational stage (P < 0.05, apoptotic bodies; P < 0.01, total TUNEL positive nuclei; P < 0.05, TUNEL positive apoptotic nuclei), as well as, in pooled data analysis (P < 0.01, apoptotic bodies and total TUNEL positive nuclei; P < 0.05, TUNEL positive apoptotic nuclei). There was no significant difference in numbers of apoptotic cells, total TUNEL positive nuclei and TUNEL positive apoptotic nuclei in syncytiotrophoblast compartment at 6, 7 and 8 weeks of gestation (Table 3).
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As shown in Table 4, the percentage immunoreactive area showed an increasing trend with the age of gestation in both compartments for cytokeratin 18f (P < 0.01; Fig. 4E, F) and Fas (cytotrophoblast: P < 0.05; syncytiotrophoblast: P < 0.01; Fig. 4D). A similar increasing trend for TNFR1 was found in cytotrophoblast (P < 0.01; Fig. 4C), but in syncytiotrophoblast significantly (P < 0.01) higher TNFR1 was detected in 8-week samples compared with 7-week, but not 6-week samples. Generally, the values for cytokeratin 18f, Fas and TNFR1 were higher in syncytiotrophoblast compared with cytotrophoblast at the same gestational stage (P < 0.05), as well as, in pooled data analysis (P < 0.01) (Table 4). Generally, immunoreactive cytokeratin 18f, Fas and TNFR1 in both cytotrophoblast and syncytiotrophoblast compartment were seen to be discrete (Fig. 4C–F). Significant concordance was seen between cytokeratin 18f and apoptotic indices (P < 0.01), Fas and apoptotic indices (P < 0.01) and TUNEL positive nuclei and apoptotic indices (P < 0.05), as well as between the expression of Fas and cytokeratin 18f (P < 0.01), whereas cytokeratin 18f (P < 0.05) and Fas (P < 0.05) showed positive correlation only with TUNEL positive apoptotic nuclear data, not with total TUNEL positive data.
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Discussion |
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There are reports based on studies of proliferation, syncytialization and apoptosis in human first trimester placental villi; however, there is no reported study dealing with the cellular characteristics of growth, differentiation and death in human first trimester placental villous trophoblast using multiparametric approach on the same platform. In the present study, we report the spatio-temporal characteristics of proliferation, syncytialization and apoptosis in human first trimester villous trophoblast cells collected at 6, 7 and 8 weeks of normal pregnancy.
The results of the present study corroborate earlier reports that human placental cytotrophoblasts show marked cell proliferation as evidenced by ultrastructural characteristics, mitotic indices and immunohistochemical labeling for proliferation markers like Ki67 and PCNA (Enders, 1965; Boyd and Hamilton, 1970; Chan et al., 1999; Ishihara et al., 2000; Burton et al., 2003). Although there was no difference in the percentage of cytotrophoblast cells showing Ki67 positive and PCNA positive nuclei over time during this period of exponential growth of the placental mass (Braunstein et al., 1980), the mitotic index was significantly higher in 6-week samples compared with 7- and 8-week samples; number of cytotrophoblast cells showing mitotic index (2%) was less than Ki67 positive (12%) and PCNA positive (27%) cells. The values of mitotic indices were lowest among the three parameters of cell proliferation studied seemingly due to the short time span occupied by mitotic phase (M-phase) in the duration of entire cell cycle (Levine, 2004). The observation that mitotic index in 6-week placental villous cytotrophoblast was higher compared with that in 7- or 8-week samples, but with no difference in the values of Ki67 positive and PCNA positive data on the time scale, appears intriguing. The observed stochastic characteristics of cytotrophoblast cells in the mitotic pool is likely to be an epiphenomenon in time function or space function evolved from longer gap periods in cell cycle and it may be integral to the developmental process of early placenta (Ishihara et al., 2000; Anglana and Debatisse, 2001).
The apparent dissociation between our observed data for Ki67 and PCNA in both the compartments may be explained in terms of the fact that Ki67 is a more reliable marker of cell proliferation and is expressed differentially during the cell cycle, generally displaying a very short half-life of 1–2 h (Gerdes et al., 1984; Scholzen and Gerdes, 2000), whereas PCNA shows a longer half-life of 20 h (Bravo and Macdonald-Bravo, 1987). Thus, PCNA immunopositivity was seen to persist even after syncytialization in the present study. PCNA is also involved in DNA repair, including nucleotide excision repair, base excision repair and mismatch repair (Jonsson and Hubscher, 1997; Kelman, 1997), and this might be a reason for its generally higher level of expression than that of Ki67 (Kosanke et al., 1998).
Extensive cellular growth of cytotrophoblast layer and its remodeling to enrich the syncytiotrophoblast are essential for maintaining placental physiology. Specific differentiation pathways are involved in syncytium formation in myotubes, osteoclasts and syncytiotrophoblast (Mayer, 2002). Multinucleate syncytiotrophoblast originate from cytotrophoblast fusion, but the nature of the fusion process is poorly understood. It is not known whether syncytium formation in trophoblast involves, similar to the fusogenic reovirus, fusion-associated small transmembrane proteins that trigger apoptosis-induced membrane instability (Salsman et al., 2005). In human villous cytotrophoblast cells endogenous retroviral envelope proteins have been reported to decrease cell proliferation, initiate intercellular fusion with an efflux of phosphatidylserine that was found to be independent of apoptosis (Das et al., 2004), and resulted in HCG production characteristic of syncytial differentiation (Rote et al., 2004). On the basis of immunohistochemical and biochemical data, Huppertz et al. (1999) have proposed that triggering of the early stages of the apoptotic cascade results in transient externalization of phosphatidylserine to facilitate cell–cell fusion and syncytialization in human villous placenta. Furthermore, they have proposed that subsequent to syncytial fusion, entropy in the apoptosis cascade is increased by the activation of anti-apoptotic factor (Huppertz et al., 1998).
On the basis of ultrastructural characteristics of first trimester human villous trophoblast, we propose a two-stage model of cell fusion. In the first stage, ‘pre-fusion complex’ is established between ‘transition’ stage cytotrophoblast and its adjoining synctiotrophoblast, and it is characterized by closely approximating dense membrane plaques between apposed plasma membranes enclosing gaps. Such gaps often had vesiculating membranous structures and electron-dense, liposome-like structures. In the second stage, a ‘fusion complex’ with punctate areas of plasma membrane breakdown resulting in focal cytoplasmic continuity is established. It is substantiated by the additional evidence of presence of intracytoplasmic desmosomes and membrane fragments inside adjoining syncytiotrophoblast. The ultrastructural evidence of presence of membrane plaques between closely apposed membranes of cytotrophoblast and syncytiotrophoblast, and that of membrane dissolution at sites have earlier been presented as morphologic evidence for cell fusion in a co-culture of human first trimester chorionic villi with decidua explant in vitro (Babawale et al., 2002).
Placental trophoblast apoptosis has often been studied using a single parameter of this complex process: FasL expression (Runic et al., 1996), presence of TNF
messenger RNA and proteins (Lea et al., 1997), TNFR1 (Yui et al., 1996), Bcl-2 expression (Lea et al., 1997; Sakuragi et al., 1994) and identification of oligonucleosomal-length DNA fragments produced during apoptosis in histological sections by in situ enzymatic labeling using TUNEL staining (Yasuda et al., 1995; Kokawa et al., 1998) in placental samples. Ultrastructural characteristics typifying apoptosis have been reported in cells of cytotrophoblast of normal human first trimester placental samples (Ishihara et al., 2000; Burton et al., 2003). We report here, based on a multiparametric analyses of proteins linked with apoptosis, e.g. cytokeratin 18f, Fas and TNFR1, that pro-apoptotic proteins are more abundantly present in syncytial compartment (9–20%) compared with cytotrophoblast (5–8%) at 6–8 weeks of normal pregnancy. The focal immunostaining of cytokeratin 18f in syncytiotrophoblast compartment as observed in the present study corroborates the previous observations that apoptosis in syncytiotrophoblast is a localized phenomenon (Huppertz et al., 1998, 1999; Kadyrov et al., 2001). An overall significantly lower incidence of apoptosis in cytotrophoblast in comparison with syncytiotrophoblast in the first trimester could also reflect a specific resistance to Fas-mediated apoptosis due to timed expression of X-linked inhibitor of apoptosis by first trimester human trophoblast cells (Straszewski-Chavez et al., 2004). Significant correlation between the incidence of apoptotic bodies and expression of Fas, cytokeratin 18f and TUNEL score in trophoblast observed during early gestation is suggestive of their efficacy as markers of apoptosis in this cell compartment. Our observation that total TUNEL score did not associate with immunopositive areas for cytokeratin 18f and Fas is not surprising; it is known that TUNEL positivity can be bypassed during apoptosis and that cells may show TUNEL positive staining even in the absence of apoptosis (Yasuda et al., 1995; Robertson et al., 2000). However, simultaneous consideration of nuclear morphology and TUNEL positivity yielded positive correlation between data for TUNEL positive apoptotic nuclei and immunopositive areas for cytokeratin 18f, as well as, between TUNEL positive apoptotic nuclei and immunopositive areas for Fas. Thus, simultaneous analysis of nuclear morphology and TUNEL staining appears mandatory for examining apoptosis based on DNA fragmentation in paraffin embedded placental villi samples (Walker and Quirke, 2001). Although TUNEL staining was detected in trophoblast and occasionally in stromal cells, immunopositive cytokeratin 18f was seen only in the epithelial compartment. Indeed cytokeratin is not generally expressed in mesenchymal and endothelial cells (Caulin et al., 1997).
Collectively, it appears from the present study that a complex homeodynamic balance involving proliferation, differentiation and apoptosis in trophoblast cells with relatively higher degree of apoptosis in syncytiotrophoblast and proliferation in cytotrophoblast is required for development-associated syncytialization and cellular remodeling of villous placenta during the first trimester.
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This research was supported with funds received from the Indian Council of Medical Research (D.G.) and the Department of Science and Technology (DST), under SERC FAST Track Scheme (M.K.). The authors wish to thank the DST funded Electron Microscopy Facility, All India Institute of Medical Sciences, New Delhi.
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The authors express their profound thanks to Professor S. Mittal and Professor V. L. Bhargava as well as to all members of the nursing staff of the Department of Gynecology and Obstetrics, All India Institute of Medical Sciences, and the Sita Ram Bhartiya Institute of Science and Research Hospital, New Delhi for their kind assistance and cooperation in sample collection.
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References |
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Anglana M, Debatisse M. Dual control of replication timing: stochastic onset but programmed completion of mammalian chromosome duplication. J Biol Chem (2001) 276:36639–36646.
Babawale MO, Mobberly MA, Ryder TA, Elder MG, Sullivan MHF. Ultrastructure of the early human feto-maternal interface co-cultured in vitro. Hum Reprod (2002) 17:1351–1357.
Benirschke K, Kaufmann P. Pathology of the Human Placenta. (2000) 4th edn. New York, USA: Springer-Verlag.
Black S, Kadyrov M, Kaufman P, Ugele B, Emans N, Huppertz B. Syncytial fusion of human trophoblast depends on caspase 8. Cell Death Differ (2004) 11:90–98.[CrossRef][Web of Science][Medline]
Boyd JD, Hamilton WJ. The Human Placenta. (1970) Cambridge, UK: Heffer.
Braunstein GD, Rasor JL, Engvall E, Wade ME. Interrelationships of human chorionic human placental lactogen and pregnancy-specific b1 glycoprotein throughout normal human gestation. Am J Obstet Gynecol (1980) 138:1205–1213.[Web of Science][Medline]
Bravo R, Macdonald-Bravo H. Existence of two populations of cyclin proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J Cell Biol (1987) 105:1549–1554.
Burton GJ, Skepper JN, Hemstock J, Cindrova T, Jones CJP, Jauniaux E. Reappraisal of the contrasting morphological appearances of villous cytotrophoblast cells during early human pregnancy: evidence for both apoptosis and primary necrosis. Placenta (2003) 24:297–305.[CrossRef][Web of Science][Medline]
Carter JE. Morphologic evidence of syncytial formation from thecytotrophoblastic cells. J Obstet Gynecol (1964) 23:647–656.
Cattoretti G, Becker MHG, Key G, Duchrow M, Schluter C, Galle J, Gerdes J. Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB1 and MIB3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol (1992) 168:357–363.[CrossRef][Web of Science][Medline]
Caulin C, Salvesen GS, Oshima RG. Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis. J Cell Biol (1997) 138:1379–1394.
Chan CCW, Lao TT, Cheung ANY. Apoptotic and proliferative activities in first trimester placentae. Placenta (1999) 20:223–227.[CrossRef][Web of Science][Medline]
Crocker IP, Cooper S, Ong SC, Baker PN. Differences in apoptotic susceptibility of cytotrophoblasts and syncytiotrophoblasts in normal pregnancy to those in complicated with preeclampsia and intrauterine growth restriction. Am J Pathol (2003) 162:637–643.
Das M, Xu B, Lin L, Chakrabarti S, Shiaswamy V, Rote NS. Phosphatidylserine efflux and intercellular fusion in a BeWo model of human villous cytotrophoblast. Placenta (2004) 25:396–407.[CrossRef][Web of Science][Medline]
Enders AC. Formation of syncytium from cytotrophoblast in the human placenta. Obstet Gynecol (1965) 25:378–386.[Web of Science][Medline]
Enders AC, Blankenship TN, Fazleabas AT, Jones CJP. Structure of anchoring villi and the trophoblastic shell in the human, baboon and macaque placenta. Placenta (2001) 22:284–303.[CrossRef][Web of Science][Medline]
Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol (1992) 119:493–501.
Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol (1984) 133:1710–1715.[Abstract]
Ghosh D, De P, Sengupta J. Effect of RU 486 on the endometrial response to deciduogenic stimulus in ovariectomized rhesus monkeys treated with oestrogen and progesterone. Hum Reprod (1992) 7:1048–1060.
Ghosh D, Sengupta J, Hendrickx AG. Effect of single-dose early luteal phase administration of mifepristone (RU 486) on implantation stage endometrium in the rhesus monkey. Hum Reprod (1996) 11:2026–2035.
Ghosh D, Kumar PGLL, Sengupta J. Effects of early luteal phase administration of mifepristone (RU486) on leukemia inhibitory factor, transforming growth factor β and vascular endothelial growth factor in the implantation stage endometrium of the rhesus monkey. J Endocrinol (1998) 157:115–125.[Abstract]
Hollander M, Wolfe D. Nonparametric Statistical Methods. (1999) 3rd edn. New York, USA: John Wiley and Sons Inc.
Huppertz B, Frank HG, Kingdom JCP, Reister F, Kaufmann P. Villous cytotrophoblast regulation of the syncytial apoptotic cascade in the human placenta. Histochem Cell Biol (1998) 110:495–508.[CrossRef][Web of Science][Medline]
Huppertz B, Frank HG, Reister F, Kingdom J, Korr H, Kaufmann P. Apoptosis cascade progresses during turnover of human trophoblast: Analysis of villous cytotrophoblast and syncytial fragments in vitro. Lab Invest (1999) 12:1687–1702.
Ishihara N, Matsuo H, Murakashi H, Laoag-fernandez J, Samoto T, Maruo T. Changes in proliferative potential, apoptosis and Bcl-2 protein expression in cytotrophoblasts and syncytiotrophoblast in human placenta over the course of pregnancy. Endocrine J (2000) 47:317–327.[CrossRef]
Jauniaux E, Poston L, Burton GJ. Placental-related diseases of pregnancy: involvement of oxidative stress and implications in human evolution. Hum Reprod Update (2006) 12:747–755.
Jonsson ZO, Hubscher U. Proliferating cell nuclear antigen: more than a clamp for DNA polymerases. Bioessays (1997) 19:967–975.[CrossRef][Web of Science][Medline]
Kadyrov M, Kaufmann P, Huppertz B. Expression of a cytokeratin 18 neo-epitope is a specific marker for trophoblast apoptosis in human placenta. Placenta (2001) 22:44–48.[CrossRef][Web of Science][Medline]
Kelman Z. PCNA: structure, function and interaction. Oncogene (1997) 14:629–640.[CrossRef][Web of Science][Medline]
Kokawa K, Shikone T, Nakano R. Apoptosis in human chrionic villi and decidua during normal embryonic development and spontaneous abortion in the first trimester. Placenta (1998) 19:21–26.[Web of Science][Medline]
Kosanke G, Kadyrov M, Korr H, Kaufmann P. Maternal anemia results in increased proliferation in human placental villi. Trophoblast Res (1998) 11:339–357.
Lalitkumar PGL, Sengupta J, Ghosh D. Endometrial tumor necrosis factor alpha (TNF alpha) is a likely mediator of early luteal phase mifepristone-mediated negative effector action on the preimplantation embryo. Reproduction (2005) 129:323–335.
Lamba P, Kar M, Sengupta J, Ghosh D. Effect of (Ala8, 13, 18)-magainin II amide on human trophoblast cells in vitro. Indian J Physiol Pharmacol (2005) 49:27–38.[Medline]
Levine EM. Cell cycling through development. Development (2004) 131:2241–2248.
Lea RG, Tulppala M, Critchley HO. Deficient syncytiotrophoblast tumour necrosis factor-alpha characterizes failing first trimester pregnancies in a subgroup of recurrent miscarriage patients. Hum Reprod (1997) 12:1313–1320.
Mayer A. Membrane fusion in eukaryotic cells. Ann Rev Cell Dev Biol (2002) 18:289–314.[CrossRef][Web of Science][Medline]
Myatt L. Placental adaptive responses and fetal reprogramming. J Physiol (2006) 572:25–30.
Rote NS, Chakrabarti S, Stetzer BP. The role of human endogenous retroviruses in trophoblast differentiation and placental development. Placenta (2004) 25:673–683.[CrossRef][Web of Science][Medline]
Runic R, Lockwood CJ, Ma Y, Dipasquale B, Guller S. Expression of Fas ligand by human cytotrophoblasts: implications in placentation and fetal survival. J Clin Endocrinol Metab (1996) 81:3119–3122.
Robertson JD, Orrenius S, Zhivotovsky B. Review: nuclear events in apoptosis. J Struct Biol (2000) 129:346–358.[CrossRef][Web of Science][Medline]
Salsman J, Top D, Boutilier J, Duncan R. Extensive syncytium formation mediated by the reovirus FAST proteins triggers apoptosis-induced membrane instability. J Virol (2005) 79:8090–8100.
Sakuragi N, Matsuo H, Coukos G, Fierth EE, Bronner MP, VanArsdale CM, Krajewosky S, Reed JC, Strauss JF. Differentiation-dependent expression of the BCl-2-proto-oncogene in the human trophoblast lineage. J Soc Gynaecol Invest (1994) 1:164–172.
Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol (2000) 182:311–322.[CrossRef][Web of Science][Medline]
Sengupta J, Given RL, Talwar D, Ghosh D. Endometrial response to deciduogenic stimulus in ovariectomized rhesus monkeys treated with oestrogen and progesterone: an ultrastructural study. J Endocrinol (1990) 124:53–57.
Sengupta J, Dhawan L, Lalitkumar PGL, Ghosh D. A multiparametric study of the action of mifepristone used in emergency contraception using the rhesus monkey as a primate model. Contraception (2003) 68:453–469.[CrossRef][Web of Science][Medline]
Smith SC, Baker PN, Symonds EM. Placental apoptosis in normal human pregnancy. Am J Obstet Gynecol (1997) 177:57–65.[CrossRef][Web of Science][Medline]
Spyratos F, Ferrero-Pous M, Trassard M, Hacene K, Phillips E, Tubiana-Hulin M, Doussal VL. Correlation between MIB-1 and other proliferation markers. Cancer (2002) 94:2151–2159.[CrossRef][Web of Science][Medline]
Straszewski-Chavez SL, Abrahams VM, Funai EF, Mor G. X-linked inhibitor of apoptosis (XIAP) confers human trophoblast cell resistance to Fas-mediated apoptosis. Mol Hum Reprod (2004) 10:33–41.
Walker JA, Quirke P. Viewing apoptosis through a ‘TUNEL. J Pathol (2001) 195:275–276.[CrossRef][Web of Science][Medline]
Yasuda M, Umemura S, Osamura RY, Kenjo T, Tsutsumi Y. Apoptotic cells in the human endometrium and placental villi; pitfalls in applying the TUNEL method. Arch Histol Cytol (1995) 58:185–190.[Web of Science][Medline]
Yonehara S, Ishii A, Yonehara M. A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumour necrosis factor. J Exp Med (1989) 169:1747–1756.
Yui J, Hemmings D, Garcia-Lloret M, Guilbert LJ. Expression of the human p55 and p75 tomour necrosis factor receptors in primary villous trophoblasts and their role in cytotoxic signal transduction. Biol Reprod (1996) 55:400–409.[Abstract]
Submitted on May 15, 2007; resubmitted on July 31, 2007; accepted on August 17, 2007.
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