Placenta
Volume 28, Supplement , Pages S33-S40, April 2007

Formation of Syncytial Knots is Increased by Hyperoxia, Hypoxia and Reactive Oxygen Species

  • A.E.P. Heazell

      Affiliations

    • Maternal and Fetal Health Research Centre, St Mary's Hospital, Hathersage Road, Manchester M13 0JH, UK
    • Corresponding Author InformationCorresponding author. Tel.: +44 161 276 5460; fax: +44 161 276 6134.
    • ,
  • S.J. Moll

      Affiliations

    • Maternal and Fetal Health Research Centre, St Mary's Hospital, Hathersage Road, Manchester M13 0JH, UK
    • ,
  • C.J.P. Jones

      Affiliations

    • Division of Human Development, St Mary's Hospital, Hathersage Road, Manchester M13 0JH, UK
    • ,
  • P.N. Baker

      Affiliations

    • Maternal and Fetal Health Research Centre, St Mary's Hospital, Hathersage Road, Manchester M13 0JH, UK
    • ,
  • I.P. Crocker

      Affiliations

    • Maternal and Fetal Health Research Centre, St Mary's Hospital, Hathersage Road, Manchester M13 0JH, UK

Accepted 6 October 2006. published online 01 December 2006.

Article Outline

Abstract 

The syncytiotrophoblast contains aggregates of nuclei termed syncytial knots. Increased numbers of syncytial knots have been reported in placentae of pregnancies complicated by pre-eclampsia and fetal growth restriction (FGR). As oxidative stress has been implicated in the pathophysiology of these disorders, we hypothesised that the formation of syncytial knots may be induced by exposure to hypoxia, hyperoxia or reactive oxygen species (ROS). We assessed both the number and morphology of syncytial knots induced by culture in hypoxia, hyperoxia and with ROS. We also investigated whether the presence of syncytial knots in normal tissue was associated with a down-regulation of anti-apoptotic proteins Bcl-2, Mdm2, XIAP and survivin. Using our measurement system we describe an increased number of syncytial knots when tissue is cultured in hypoxia, hyperoxia or in the presence of ROS. The morphology of these syncytial knots was similar to those seen in vitro, although the nuclei from cultured placental explants were morphologically more homogenous, had fewer nuclear pores, and a higher heterochromatin:euchromatin ratio. Despite the apoptotic appearances of nuclei we did not detect a loss of anti-apoptotic proteins in the region of syncytial knots. We conclude that the increased number of syncytial knots in placentae from pregnancies complicated by pre-eclampsia and FGR can be replicated in vitro by ROS or hypoxia, supporting their involvement in the pathogenesis of these conditions.

Keywords: Trophoblast, Syncytial knot, Apoptosis, Hypoxia, Hyperoxia, Reactive oxygen species

 

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1. Introduction 

Pre-eclampsia and intra-uterine growth restriction (IUGR) are associated with macroscopic and microscopic changes of placental structure. Macroscopically, the placenta may show areas of infarction, fibrosis or calcification [1]. Microscopically, there is increased formation of syncytial knots—aggregations of syncytiotrophoblast nuclei. This was first described by Tenney and Parker who reported that syncytial knots are present in 10–50% of normal terminal villi at term, increasing to involve nearly all terminal villi in pre-eclampsia [2]. They also observed a link between the severity of pre-eclampsia and the frequency of syncytial knots. In addition to pre-eclampsia and IUGR, similar appearances of terminal villi have been reported in post-mature placentae, in placentae infected with malaria, in women with anti-phospholipid syndrome and in pregnancies occurring at high altitude [3], [4], [5]. It was initially, but erroneously, proposed that these changes represented hyperplasia, a response of the villi to pathological conditions promoting trophoblast outgrowth [6].

Syncytiotrophoblast nuclei aggregate in several different forms on the free surface of the villus; many different terms have been used to describe these phenomena including: syncytial sprouts, syncytial bridges, syncytial clumps, proliferation knots, nuclear clumps and trophoblastic extensions [7], [8], [9]. These were subsequently grouped into three categories: (i) syncytial sprouts, which are aggregates of immature nuclei, present in the first trimester acting as precursors of newly formed villi, (ii) syncytial bridges which are formed from fusion of syncytial sprouts from neighbouring villi, and are thought to give support to the villous tree [8], and (iii) syncytial knots, defined as accumulations of degenerating nuclei which may protrude slightly from the villous surface [10].

Ultrastructural analysis has demonstrated that nuclei contained within syncytial knots show features of advanced degeneration, specifically: pyknosis, peripheral chromatin condensation and fused nuclear membranes [8], [10]. These morphological features are similar to those described in apoptosis, a form of programmed cell death [11]. Interestingly, the amount of apoptotic nuclei is also increased in trophoblast of pregnancies complicated by IUGR and pre-eclampsia [12], [13], [14], [15]. These findings have led to the hypothesis that the increased number of syncytial knots in IUGR and pre-eclampsia may reflect exaggerated ageing or apoptotic death of the syncytiotrophoblast.

Under normal conditions syncytiotrophoblast integrity is maintained by proliferation and fusion of the underlying cytotrophoblast. Following cytotrophoblast fusion, the newly acquired syncytiotrophoblast nuclei show progressive changes of peripheral chromatin condensation, shrinkage and then aggregate into syncytial knots [16]. After the formation of syncytial knots it is hypothesised that this material is lost into the maternal circulation [17]. It has been proposed that syncytiotrophoblast nuclei are programmed to enter a “pre-apoptotic” phase, which then leads to a short apoptotic execution phase. This process represents normal epithelial cell turnover, balancing proliferation and cell death [18].

The hypothesis that the formation of syncytial knots is a degenerative process is supported by the observation that nuclei within the syncytiotrophoblast are transcriptionally inactive and unable to replicate [19]. The involvement of apoptotic pathways is demonstrated by the involvement of caspase-8, a key effector of apoptosis, in cytotrophoblast fusion [20]. In addition, lamin B, a protein associated with the nuclear pore complex, is degraded by CPP32-like caspase enzymes, and is reduced in the syncytiotrophoblast compared with the cytotrophoblast [21]. However, the syncytiotrophoblast cytoplasm is rich in anti-apoptotic proteins such as Mdm2, Bcl-2, X-linked inhibitor of apoptosis protein (XIAP) and survivin [22], [23], [24], [25]. The presence of these proteins may antagonise an apoptotic process occurring within the syncytiotrophoblast. Therefore, it seems likely that this process of nuclear degeneration does not involve a similar degeneration of cytoplasm.

At the present time the mechanism of syncytial knot formation and the reasons for their increased presence in pre-eclampsia and IUGR are not well understood. As oxidative stress has been implicated in the pathophysiology of both pre-eclampsia and IUGR we hypothesised that the formation of syncytial knots may be induced by exposure to hypoxia, hyperoxia or reactive oxygen species. We also hypothesised that formation of syncytial knots in fresh placental tissue would be associated with down-regulation of anti-apoptotic proteins in the region of the syncytial knot.

In order to address these hypotheses, we employed an in vitro model of trophoblast cell turnover in term placental explants. Previous studies using this model have demonstrated that after 24–48h the original syncytiotrophoblast begins to degenerate, and a new functional layer is reformed by 72h [26]. Within this period trophoblast cell turnover can be significantly altered by varying oxygen tension [27]; however, the effects on syncytial knot formation are not known. In addition, short periods of exposure to ischaemia–reperfusion injury have been shown to induce trophoblast apoptosis in vitro [28]. As hydrogen peroxide has a similar mode of action, we anticipated that a 6-h exposure to reactive oxygen species would be suitable to assess syncytial knot formation. To determine whether syncytial knot formation in vivo was associated with local down-regulation of anti-apoptotic factors, we examined the expression of Bcl-2, Mdm2, XIAP and survivin in fresh placental tissue from normal pregnancies.

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2. Materials and methods 

2.1. Participants 

The Local Ethics Committee gave approval for this study and all participants provided written informed consent. To compare normal and abnormal pregnancies we studied placentae from nine women with uncomplicated pregnancies, eight pregnancies complicated by pre-eclampsia and six pregnancies with IUGR. Pre-eclampsia was defined as a blood pressure of >140/90mmHg on two or more occasions after the 20th week of pregnancy, with significant proteinuria (>300mg/l per 24h) [29], cases of pre-eclampsia had no evidence of IUGR. IUGR was defined as an individualised birthweight ratio of less than the 5th centile [30]. For tissue culture experiments, placentae from 15 women with uncomplicated term pregnancies were used.

2.2. Preparation of placental explants 

Unless otherwise stated all chemicals were obtained from Sigma Chemical Co., Poole, UK. The placentae were collected from the delivery suite, St Mary's Hospital, Manchester, within 20min of delivery. Areas of each placenta were randomly sampled using a sampling frame with intersecting lines at 10cm, which was placed on the maternal surface of the placenta and placental tissue taken from underneath each intersection. This usually resulted in selection of three areas of placenta; if more than three areas of placenta were selected, those to be used were selected by random lottery sampling. The resulting villous tissue was dissected into explants weighing 5mg per fragment. Villous tissue from each sampling area was fixed prior to culture, to enable comparison between fresh and cultured placental tissue.

Three explants from each placenta were cultured, suspended on a Netwell insert (Corning Inc., NY, USA) and immersed in culture medium CMRL-1066 supplemented with antibiotics, insulin (1mg/l), hydrocortisone (0.1mg/l), retinol acetate (0.1mg/l) and 10% fetal calf serum. Explants were cultured for 4days in either 20% oxygen, 6% oxygen or 1% oxygen. These conditions were chosen as 6–8% oxygen is hypothesised to represent normoxic and 1–2% hypoxic conditions for term villous tissue [31]. The culture medium was changed after 48h. Prior to this, fresh medium was equilibrated for 24h under the appropriate oxygen conditions. Explants cultured in 1% oxygen remained within this environment while the culture medium was changed. Oxygen tension was verified in situ at 37°C using a digital meter (Strathkelvin Instruments, Glasgow, UK). Explants cultured in the presence of reactive oxygen species were cultured for 48h in 6% oxygen, followed by 6h exposure to a range of concentrations of hydrogen peroxide between 0 and 1000μM.

2.3. Immunostaining 

Placental explants were fixed in 10% neutral formalin saline (3.75% Formaldehyde, pH 7.2) and embedded in paraffin wax. Sections (5μm) were cut and transferred to 3-aminopropyltriethoxysilane coated slides. Sections were deparaffinised and antigen retrieval was carried out by exposure to microwave pre-treatment with 10mM citrate buffer, pH 6.0. Sections were further treated with 3% (v/v) hydrogen peroxide in methanol for 45min. Non-specific binding was blocked by exposure for 1h at room temperature with normal rabbit or goat serum (10% (v/v) in PBS). Sections were incubated overnight at 4°C with mouse monoclonal antibodies against Mdm2 (Clone 2A10, Merck Biosciences, Nottingham, UK, 2μg/ml), Bcl-2 (Clone 100/D5, Abcam, Cambridge, UK, 1μg/ml), XIAP (Clone 28, BD Biosciences, San Jose, CA, USA, 10μg/ml) or rabbit polyclonal antibody to survivin (R&D Systems, Minneapolis, MN, USA, 0.5μg/ml). Each tissue was incubated with either a matching concentration of non-immune mouse IgG or 10% normal goat serum to serve as negative controls. Slides were probed with biotin-conjugated goat anti-mouse or anti-rabbit antibodies (Dako, Ely, UK, 1:200) for 1h at room temperature, followed by incubation with avidin-peroxidase (5μg/ml in 0.125M TBS+0.347M NaCl [32]). Immunostaining was revealed by exposure to concentrated 3,3-diaminobenzidine for 3min. Slides were counterstained with haematoxylin and sections viewed using a Leitz microscope with ImageProPlus 4.5 (Media Cybernetics Inc., Silver Spring, MD, USA).

2.4. Assessment of syncytial knots 

Sections (5μm) were prepared from wax-embedded tissue. Following deparaffinisation, the sections were counterstained with haematoxylin and eosin. Using a 40× objective, the number of syncytial knots was counted in 10 fields of view for each experimental condition. To avoid bias the microscope was taken out of focus between frames. A syncytial knot was defined as a multi-layered aggregation of at least 10 syncytiotrophoblast nuclei protruding from the villous surface that was not in direct contact with adjacent villi [10]. The number of syncytial knots was counted manually, and trophoblast area measured by sequential colour thresholding, as previously described [33]. Data were normalised to give a measure of the number of syncytial knots per mm2 of villus, termed the estimated numerical density.

2.5. Electron microscopy 

Nine placental explants from each experimental condition were fixed and processed for electron microscopy. Tissue was fixed for 3h in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.3), then washed three times over 24h in 0.1M sodium cacodylate buffer with 3mM calcium chloride (pH 7.3). Post-fixation was carried out in 1% osmium tetroxide (Agar Scientific Ltd) in 0.05M sodium cacodylate buffer for 1h at 4°C followed by a rinse in buffer. The tissue was then dehydrated in an ascending alcohol series, treated twice with propylene oxide (15min each) then left in a 1:1 mix of propylene oxide and Taab epoxy resin (Taab Laboratories Equipment Ltd., Aldermaston, UK) for 1h at room temperature. The tissue was then left overnight at 4°C in a mixture of 1:3 propylene oxide and epoxy resin, followed by three changes of fresh resin at 45°C for 1h. The tissue was then embedded in gelatine capsules and polymerised for 72h at 60°C.

Semi-thin sections, 0.5μm thick, were cut on a Reichert Ultracut microtome, and stained with 1% toluidine blue in 1% borax. After inspection of the tissue sections to identify areas of interest, pale gold ultrathin sections (70nm) were cut with a diamond knife, mounted on copper grids and stained with uranyl acetate and lead citrate. These were examined in a Philips CM10 electron microscope, at an accelerating voltage of 80kV, and appropriate areas were photographed.

2.6. Statistical analysis 

Unless otherwise stated, statistical significance was tested using the either the Student t-test or a one-way ANOVA test with Tukey's post hoc test. Results are presented as mean±SEM. A p value of less than or equal to 0.05 was considered statistically significant.

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3. Results 

3.1. Syncytial knots are increased in placentae from pregnancies complicated by pre-eclampsia and IUGR and following exposure to hypoxia, hyperoxia and reactive oxygen species 

We initially validated our technique for quantification of syncytial knots by assessing the presence of syncytial knots in normal term pregnancies and pregnancies complicated by pre-eclampsia or IUGR. We found that the estimated numerical density of syncytial knots was significantly increased in both pre-eclampsia and IUGR compared to normal pregnancy (Fig. 1A), with an increase of approximately 2-fold in both conditions.

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  • Fig. 1 

    (A) The number of syncytial knots are increased in IUGR and pre-eclampsia compared to normal term pregnancy (*p<0.05, **p<0.01). (B) The number of syncytial knots is increased in villous explants cultured in hyperoxic and hypoxic conditions compared to fresh tissue and that cultured in normoxic conditions (**p<0.01, ***p<0.001). (C) The number of syncytial knots is increased following exposure to hydrogen peroxide (*p<0.05, ***p<0.001). (D) Micrograph of terminal villi from normal term placenta showing a single syncytial knot marked by open arrow. (E) Micrograph of terminal villi from pre-eclamptic pregnancy showing increased numbers of syncytial knots (open arrows) and areas of syncytiotrophoblast with few nuclei (shown within ellipse). (F) Micrograph of terminal villi exposed to hydrogen peroxide for 6h showing increased numbers of syncytial knots (open arrows) and areas of syncytiotrophoblast depleted of nuclei (shown within ellipse).

We then compared the density of syncytial knots in normal villous explants exposed to hypoxia, hyperoxia and reactive oxygen species. The estimated numerical density of syncytial knots was significantly increased in explants exposed to hypoxia, hyperoxia and all doses of hydrogen peroxide (Fig. 1B,C). The presence of reactive oxygen species appeared to have a greater effect on syncytial knot formation than either hypoxia or hyperoxia.

The increased presence of syncytial knots in pre-eclampsia compared to normal pregnancy is shown in Fig. 1D,E. In pre-eclampsia, the aggregations of syncytial nuclei appear to be denser than in normal pregnancy and syncytial knots were frequently seen in association with fibrin deposition. Syncytial knots can be clearly seen following exposure to ROS, leaving areas of the syncytiotrophoblast with few nuclei (Fig. 1F).

3.2. Syncytial knots resulting from exposure to hypoxia, hyperoxia and reactive species in vitro have similar morphological features to those described in vivo 

We next investigated whether the increased presence of syncytial knots in the in vitro experiments detailed above had the same morphology as those described in vivo where the nuclei are densely packed, pyknotic, with peripheral chromatin condensation (Fig. 2A,B). However, the morphology of nuclei within syncytial knots induced in vitro was more homogenous than those seen in vivo (Fig. 2A compared to B), with almost all nuclei within an individual knot appearing to be at the same stage of degeneration (Fig. 2B). The ratio of euchromatin:heterochromatin appeared to be reduced in pre-eclampsia and cultured explants compared to normal term fresh tissue (Fig. 2B and C compared to A). Thin channels of cytoplasm between the nuclei were present in syncytial knots induced in vitro, leaving very little space between nuclear membranes; in some instances nuclear membranes from adjacent nuclei appeared to be in direct contact (Fig. 2B). Explants exposed to hypoxia, hyperoxia or reactive oxygen species seemed to have a thickened nuclear membrane creating a space between the peripheral condensed heterochromatin and the nuclear envelope (Fig. 2D), whereas, in fresh tissue the heterochromatin extended to the nuclear membrane (Fig. 2A,E).

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  • Fig. 2 

    Electron micrographs. (A) Syncytial knot (SKT) in normal term placenta showing nuclei at different stages of degeneration. There is a healthy cytotrophoblast (CT) underlying the SKT. (B) Syncytial knot in placental explant exposed to ROS showing a homogenous appearance of nuclei at similar stages of degeneration. Nuclear pore (NP) complexes can be seen where euchromatin is in contact with the nuclear envelope. NP complexes are absent in nuclei with dense peripheral heterochromatin (marked with open arrows). (C) Syncytial knot in pre-eclampsia. The nuclei show signs of advanced degeneration. (D) Syncytial knot in explant exposed to hypoxia demonstrating condensation of the nuclear membrane (NM) and gap between the nuclear membrane and heterochromatin. (E) Nuclei within a syncytial knot of a post-mature placenta, showing a heterochromatin (HC):euchromatin (EC) ratio. An annulate lamella (AL) is seen in association with a degenerating nucleus. (F) High magnification of syncytial knot in placental explant exposed to hyperoxia showing a longitudinal structure (LS) lying between two degenerating nuclei. All bars: 5μm.

A further difference was the presence of annulate lamellae which could be seen in the region of syncytial knots in placentae taken from normal, pre-eclamptic and IUGR pregnancies (Fig. 2E). However, annulate lamellae were not detected in the region of syncytial knots following exposure to hypoxia, hyperoxia or reactive oxygen species, although there was a short laminar structure situated between two apoptotic nuclei (Fig. 2F), which may represent a degenerating annulate lamella or a nuclear envelope limited chromatin sheet (ELCS). Nuclear pores were present in nuclei contained within syncytial knots, even on pyknotic nuclei consisting only of condensed heterochromatin. However, the number of nuclear pores appeared to be decreased compared to fresh tissue taken from normal placentae and those from pathological pregnancies.

3.3. The expression of anti-apoptotic proteins was not reduced in the region of syncytial knots 

We then investigated whether the apoptotic appearances of nuclei contained within syncytial knots were associated with a decrease in the expression of anti-apoptotic proteins, Mdm2, Bcl-2, XIAP and survivin (Fig. 3A–D). We found that in normal pregnancy all proteins were expressed in the syncytiotrophoblast cytoplasm, while only Mdm2 and survivin were expressed within cytotrophoblast cytoplasm. There did not appear to be any reduction in the expression of Mdm2, Bcl-2, XIAP or survivin in the cytoplasm surrounding syncytial knots relative to the syncytiotrophoblast. Immuno-reactivity was undetectable in mouse and rabbit negative control samples (Fig. 3E,F).

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  • Fig. 3 

    Micrographs of terminal villi showing localisation of (A) Bcl-2, (B) Mdm-2, (C) XIAP, and (D) Survivin. All are present in the syncytiotrophoblast cytoplasm surrounding syncytial knots (SKT). (E) Negative control incubated with non-immune mouse IgG. (F) Negative control with 10% normal goat serum. All micrographs: original magnification ×1000. Scale bar: 10μm.

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4. Discussion 

In this study we validated a method for the quantification of syncytial knots. This method gives a measure of syncytial knots per villous area. In normal term pregnancy we found that there were approximately 54–75 knots per mm2. This is difficult to compare with previous observations, as these have quantified syncytial knots based on total trophoblast volume or as a percentage of terminal villi containing syncytial knots [9], [18]. However, one study did describe the number of knots in normal term tissue; there was a mean value of 0.5 knots per villus [3]. The average diameter of a terminal villus has been estimated to be 30–80μm, giving an estimated cross-sectional area of a terminal villus of 706.8–5026.5μm2. If there were an average of 0.5 knots/villus, the density of knots would be expected to be in the range of 95–707 knots/mm2. If intermediate villi were included, this estimate would fall to 37–176 knots/mm2. Since the sections that we analysed contained some mature intermediate villi in addition to the terminal villi, our measurement is within the range that is anticipated on the basis of previous studies. Furthermore, using the ratio of syncytial knots:villous area, we found that syncytial knots were increased in pre-eclampsia and IUGR demonstrating that our method is consistent with previously published data [1].

Using our measurement of syncytial knots, we assessed whether exposure to hypoxia, hyperoxia and reactive oxygen species was associated with increased numbers of syncytial knots. Culture for 54 or 96h under normoxic conditions for the placenta (6% oxygen) was not associated with a significant increase in syncytial knots, suggesting that under these conditions a normal level of trophoblast turnover was present. However, in placental explants exposed to hypoxia, hyperoxia and reactive oxygen species, there was a significant increase in the number of syncytial knots to values greater or equal to those observed in pre-eclampsia or IUGR. These culture conditions were selected as they have all been proposed as potential mechanisms of placental damage in pre-eclampsia and IUGR. Therefore, it is hypothesised that the increased number of syncytial knots in pre-eclampsia and IUGR may result from exposure to a form of oxidative stress. Our study also demonstrates the dynamic nature of placental cell turnover, as explants that were exposed to hydrogen peroxide for only 6h exhibited an increase in syncytial knot formation.

The morphology of nuclei contained within syncytial knots formed in vitro was similar to that described in vivo. The nuclei were pyknotic, with the majority of nuclei containing more heterochromatin than euchromatin and there were no nucleoli present. There were fewer nuclear pores and annulate lamellae than may have been expected. Jones and Fox [8] and Mayhew et al. [18] described occasional annulate lamellae in syncytial knots closely associated with the nuclei. Annulate lamellae are structures composed of stacks of membranes with regularly spaced pores. Annulate lamellae interact with ELCS, a fold of the nuclear envelope which projects into the cytosol. ELCS are found in malignant and some normal cells, and are associated with apoptosis [34]. Although the role of both structures is unknown, it is hypothesised that they may represent an attempt to maintain the integrity of the nuclear envelope or arise from shedding of nuclear pore complexes during apoptosis respectively [18], [34]. Constituents of the nuclear pore complexes such as Lamin B are degraded by CPP32-like caspases which are present in trophoblast [21]. Therefore, the effector enzymes of apoptosis may have a role in nuclear degeneration seen in syncytial knots. Given the relatively infrequent nature of these structures and the small area of tissue sampled by electron microscopy, the absence of annulate lamellae may be a sampling phenomenon. Further studies are required to determine the role of ELCS, annulate lamellae and nuclear pore complexes in the formation of syncytial knots and nuclear ageing within the syncytiotrophoblast.

The frequency of nuclear pores appeared to be reduced in syncytial knots induced by exposure to oxidative stress in vitro compared to those present in vivo. Nuclear pores are known to decrease in number as nuclei manifest signs of advanced apoptosis, initially relocating to areas of the nuclear envelope where euchromatin persists [35]. Very few nuclei in our in vitro culture model retained this peripheral euchromatin, instead, a thick layer of heterochromatin was seen around the outer border of the nucleus. Therefore, we suggest that the advanced nature of apoptosis seen within syncytial knots in our culture model may be associated with concomitant loss of nuclear pores. Further investigation is required to confirm this finding, and whether the loss of nuclear pores is related to nuclear shrinkage as nuclei age within the syncytiotrophoblast.

Lastly, we investigated whether the formation of syncytial knots was associated with a decrease in the expression of anti-apoptotic proteins expressed within the syncytiotrophoblast cytoplasm. There did not appear to be any decrease in the expression of Mdm2, Bcl-2, XIAP or survivin in the cytoplasm surrounding syncytial knots. The presence of these proteins provides evidence of anti-apoptotic activity at every regulatory level of the intrinsic pathway of apoptosis, which may relate to the specific degeneration of nuclei within knots, rather than the entire syncytiotrophoblast. The presence of several anti-apoptotic factors within the cytoplasm preserves syncytiotrophoblast integrity maintaining a healthy epithelium. This finding also raises the possibility that the apoptotic degeneration of nuclei within the syncytial knot commences prior to the formation of the syncytial knot, with specific nuclei then gathered together into syncytial knots. This hypothesis is supported by evidence that apoptotic nuclei, as identified by electron microscopy and TUNEL or cleaved cytokeratin-18 staining, can be found throughout the syncytiotrophoblast [21], [36]. In addition, syncytial nuclei express proteins which maintain cell cycle arrest such as p21, a cyclin-dependent kinase inhibitor [37]. Cell cycle arrest may be an important part of cytotrophoblast differentiation, as exposure of villous explants to inhibitors of the cell cycle results in an increase in hCG synthesis [38]. Further investigation of the process of nuclear ageing in syncytiotrophoblast is required, focusing particularly on the initiation of apoptosis with respect to the nature and timing of the cellular signalling mechanisms involved.

In conclusion, we have quantified the density of syncytial knots within villous tissue, and have described an increase in pre-eclampsia and IUGR. We have demonstrated that the increased number of syncytial knots may be induced by exposure of the placenta to hypoxia, hyperoxia or oxidative stress. The mechanism by which these noxious environments induces these changes within the syncytiotrophoblast is not well understood, but may involve induction of cell cycle arrest and apoptosis. Further investigation of the process of cytotrophoblast differentiation and syncytial nuclear degeneration may identify factors which are important in the accelerated formation of syncytial knots in pre-eclampsia and IUGR.

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PII: S0143-4004(06)00250-5

doi:10.1016/j.placenta.2006.10.007

Refers to erratum:

  • Erratum to “Formation of Syncytial Knots is Increased by Hyperoxia, Hypoxia and Reactive Oxygen Species” [Placenta 28, Supplement A, Trophoblast Research, Volume 21 (2007) S33–S40]

    A.E.P. Heazell, S.J. Moll, C.J.P. Jones, P.N. Baker, I.P. Crocker
    Placenta August 2007 (Vol. 28, Issue 8, Page 973)

Placenta
Volume 28, Supplement , Pages S33-S40, April 2007

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