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Apoptosis: Molecular Control of Placental Function—A Workshop Report
Placenta (2001), 22, Supplement A, Trophoblast Research, 15, S101–S103 doi:10.1053/plac.2001.0645, available online at http://www.idealibrary.com on
APOPTOSIS, MOLECULAR AND GENETIC CONTRIBUTIONS Apoptosis: Molecular Control of Placental Function—A Workshop Report B. Huppertza,g, N. S. Roteb, D. M. Nelsonc, F. Reisterd, S. Blacke and J. S. Huntf a
Department of Anatomy, University of Technology, Aachen, Germany Departments of Biological Sciences and Obstetrics & Gynecology, Wright State University School of Medicine, Dayton, Ohio 45435, USA c Washington University School of Medicine, St Louis, MO, USA d Department of Obstetrics and Gynecology, University of Technology, Aachen, Germany e Monash University, Box Hill Hospital, Box Hill, Australia f Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, USA Paper accepted 5 January 2001 b
Following successful completion of the workshop on apoptosis in Schladming, 1999 (Huppertz and Hunt, 2000), the organizers were requested to develop a second workshop in Rochester, 2000. This workshop was designed to link trophoblast apoptosis to functional aspects of the placenta. The basic principles and mechanisms involved in trophoblast apoptosis (for review see Huppertz and Kaufmann, 1999) are becoming clearer and there is more and more information on the functional roles apoptosis plays in placental development and integrity. The presentations in this workshop represented this development. Joan Hunt dealt with the expression of tumour necrosis factor (TNF) superfamily genes in human placenta and potential relationships with apoptosis. The TNF superfamily contains at least eight genes encoding proteins that mediate apoptosis. These include TNF, LT, LT, LIGHT, FasL, TRAIL, TWEAK and 4-1BBL. Fourteen genes encode receptors for these ligands. Of these, all eight genes encoding apoptosis-inducing ligands and 13 of the 14 genes encoding receptors for these ligands are expressed in human placentae (Hunt et al., 1999). Messages have been identified at early and/or late stages of gestation by Northern blot analysis and reverse transcriptase polymerase chain reaction. In an effort to dissect the sites of synthesis of the TNF superfamily ligands and receptors, trophoblasts (Jar and JEG-3 cell lines, term villous cytotrophoblasts) and macrophages (U937 and THP-1 cell lines and term placental macrophages) were examined for transcripts. Of the ligands, only FasL and g
To whom correspondence should be addressed at: Department of Anatomy, University of Technology, Wendlingweg 2, D-52057 Aachen, Germany. Fax: +49-241-8888-472. E-mail: bhuppertz@ post.klinikum.rwth-aachen.de
0143–4004/01/0A0S101+03 $35.00/0
LT mRNAs were absent in all trophoblasts; none of the ligand messages were absent in all macrophages. Of the receptors, 4-1BB and OPG messages were absent in all trophoblasts; only OPG was absent in all macrophages. However, term villous cytotrophoblasts lacked TRAIL and LIGHT as well as FasL and LT; placental macrophages lacked 4-1BBL and also lacked DcR1 as well as OPG transcripts, thus demonstrating some differences between normal cells and cell lines in the expression of both ligands and receptors. TNF appears to be multifunctional, regulating hormone synthesis and placental architecture through effects on integrins and matrix metalloproteinases. FasL seems to have the ability to influence placental development and may also provide a measure of immune privilege and serve as a barrier to maternal–fetal cell trafficking. TRAIL is positioned to prevent maternal immune cell attack on the placenta but may also exert profound effects on other trophoblast functions. Neal S. Rote discussed the regulation of phosphatidylserine distribution during differentiation and apoptosis of villous trophoblast. All mammalian cells actively maintain a heterogenous distribution of phospholipids in their plasma membranes. Aminophospholipids, such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), are preferentially maintained on the inner face of the plasma membrane by a flippase. Exposure of PS on the outer leaflet of the cell membrane is an early active event in cells undergoing apoptosis. Normal human villous cytotrophoblast differentiation is also characterized by PS externalization, related to intercellular fusion. Movement of PS and PE from the outer to inner plasma membrane surface by means of the aminophospholipid translocase is relatively fast (t1/2 of 5–10 min). 2001 IFPA and Harcourt Publishers Ltd
S102
An undifferentiated model of trophoblast, BeWo, has an active aminophospholipid translocase. During forskolin or cAMPinduced BeWo differentiation, PS is externalized with rather slow kinetics; antigenic or annexin V-binding PS appearing progressively on the surface of the cell over 48 h. This process is concurrent with hormonal differentiation and intercellular fusion into syncytiotrophoblast and can be completely blocked by monoclonal antibody against PS. The relatively slow rate of externalization is compatible with the activity of a floppase, which redistributes PS and PE to the outer surface with a t1/2 of 1–2 h. During apoptosis, PS externalization appears to be relatively rapid; the binding of annexin V and monoclonal anti-PS antibodies increased significantly by 5 min and reached maximum expression by 40 min. The rapid expression of apoptosis-related PS is compatible with the action of the recently described scramblase (Vogt, Ng and Rote, 1997), which has a t1/2 of less than 1 min. Apoptosis-related PS externalization is not accompanied by increased intercellular fusion. Rote and co-workers hypothesized that the floppase is responsible for PS externalization during differentiation and the scramblase during apoptosis, and investigated whether the mechanisms of PS externalization during trophoblast differentiation and apoptosis processes were mediated by different enzymes. BeWo is an in vitro model of both trophoblast differentiation and apoptosis. After 24 h treatment of forskolin-induced differentiation, PS was detected on the cell surface by binding of FITC-annexin V, which increased during 24–72 h of treatment, but was not accompanied by apoptosis monitored by TUNEL assay. Apoptosis induction by staurosporine for 1 h triggered rapid PS externalization. Inhibition of floppase activity with vanadate blocked differentiation-related but not the apoptosis-related PS externalization. The effector caspase inhibitor ZVAD-fmk completely abolished staurosporine-induced apoptosis, but had no effect on differentiation-mediated PS externalization. Thus, PS externalization appears to be a marker for both trophoblastic differentiation and apoptosis. Following these talks on mechanistic issues of trophoblast apoptosis, subsequent presentations dealt with changes in the rate of apoptosis and their effects on the maintenance of the placenta and the outcome of pregnancy. D. Michael Nelson discussed a role for prostanoids and peroxisome proliferator activator receptor gamma (PPARc) activation in the differentiation and apoptosis of human trophoblast. Underperfusion and hypoxia of the placenta are associated with pre-eclampsia and fetal growth restriction. Hypoxia in vivo and in vitro limits differentiation and enhances apoptosis in human villous trophoblast. The mechanisms regulating these biological responses in trophoblast are unclear. However, pre-eclampsia is associated with enhanced apoptosis, altered prostanoid production and increased expression of cyclooxygenase-2 in the trophoblast of placental villi. PPAR is clearly essential for murine placental development and differentiation, and PPAR is also expressed in trophoblast of human placental villi.
Placenta (2001), Vol. 22, Supplement A, Trophoblast Research, Vol. 15
Dr Nelson discussed the role of prostanoids and PPAR activation in the regulation of differentiation and apoptosis in cultured human trophoblast. The prostanoids thromboxane and 15-deoxy-12,14-prostaglandin J2 (15PGJ2), a ligand for PPAR, hinder biochemical and morphological differentiation and enhance apoptosis in cultured trophoblast. In contrast, troglitazone, another ligand for PPAR, enhances differentiation and does not affect apoptosis in trophoblast. Both thromboxane and 15PGJ2, but not troglitazone, upregulate p53 expression, suggesting that the apoptotic response of trophoblast to these ligands is p53 dependent. The data indicate that thromboxane and PPAR activation play important roles in trophoblast biology. Frank Reister discussed macrophage-mediated tryptophan depletion that may contribute to apoptosis of extravillous trophoblast and may have an impact on pre-eclampsia. Recent data (Reister et al., 1999) indicate a role of trophoblast apoptosis in reduced trophoblast invasiveness in pre-eclampsia (PE) and an impact of macrophages in this process. Macrophages are known to secrete indoleamine 2,3-dioxygenase (IDO), a tryptophan-degrading enzyme. Tryptophan starvation leads to apoptosis of trophoblast hybrid cells. Therefore these investigators speculated that macrophages might be involved in tryptophan-associated apoptosis of trophoblast hybridoma, and investigated if changes in the cytokine environment might provide activating mechanisms. The effect of tryptophan supplementation on apoptosis was studied in a co-culture model (extravillous trophoblast hybrid cells/ macrophages). The supplementation of tryptophan significantly reduced the incidence of trophoblast apoptosis in a dose-dependent manner. Immunohistochemical evaluation of the distribution of uterine natural killer (NK)-cells in the placental bed of both PE patients revealed a significant increase of uterine NK-cells in PE patients compared with healthy pregnant women. Thus, the secretion of IDO is one of the mechanisms used by macrophages in order to induce trophoblast apoptosis. This may contribute to insufficient endovascular trophoblast invasion in PE. NK-cell-derived interferon- may stimulate IDO-activation in placental bed macrophages in PE. Simon Black showed evidence for a redox regulation of magnesium-induced placental apoptosis. Placental apoptosis has been implicated in fetal growth restriction, placental abruption and pre-eclampsia. The cause of increased placental apoptosis is not known; however, oxidative stress has been implicated in placental dysfunction. Recently, nitric oxide, a component of peroxynitrite and a major source of placental oxidative stress, was shown to be increased by magnesium. Furthermore, maternal magnesium levels are elevated in preeclampsia and fetal growth restriction, and physiological levels of magnesium cause loss of trophoblast viability. These researchers report magnesium-stimulated placental apoptosis in vitro as evidenced by oligosomal DNA laddering and tissue shrinkage associated with clumping of pycnotic trophoblast nuclei. Placental apoptosis measured by oligosomal DNA laddering increased over the physiological range of
Huppertz et al.: Apoptosis and Placental Function
extracellular magnesium. Plasminogen activator inhibitor type 2 cleavage and cytokeratin 18 neoepitope formation indicated caspases were involved in the mechanism of magnesiuminduced placental apoptosis. They inhibited magnesiuminduced apoptosis with vitamin C, vitamin E, N-acetylcysteine, neomycin or Methylene Blue, and interpreted their inhibitor results as evidence that magnesiuminduced placental apoptosis was associated with an oxidative state. Black and co-workers propose that redox-regulating agents may be a fruitful source of safe, inexpensive, effective and rational agents that can be used to improve obstetric outcomes. Furthermore, we advocate further study of the regulation of maternal magnesium. In a final presentation, Berthold Huppertz reported that hypoxia favours necrotic versus apoptotic shedding of placental syncytiotrophoblast into the maternal circulation and suggested implications for the pathogenesis of pre-eclampsia. In the course of normal pregnancy, several grams of placental trophoblast are shed daily into the maternal circulation and are tolerated by the mother. The balance between apoptotic and necrotic shedding of this material is presently unknown. Since pre-eclampsia is characterized by a pronounced degree of inflammatory damage, this group investigated the possibility that excessive shedding of necrotic placental trophoblast is involved in the pathogenesis of this disorder. They studied an explant model of human trophoblast turnover in 3 per cent and 20 per cent oxygen over 2 days. Trophoblast turnover including apoptosis and necrosis were monitored by: conventional histology, immunolocalization of Ki67 (a proliferation marker), Bcl-2 (an apoptosis inhibitor), caspases 8 and 3 (apoptosis promoters), and DNA degradation. Furthermore, electron microscopy and [3H]cytidine and [3H]uridine incorporations
S103
were employed. Culture in 3 per cent oxygen resulted in increased villous cytotrophoblast proliferation and necrotic shedding of the overlying syncytiotrophoblast. The proteins necessary for execution of apoptosis were retained in the cytotrophoblast due to lack of syncytial fusion. By contrast the syncytium remained intact in 20 per cent oxygen. Reduced cytotrophoblast proliferation was noted, but syncytial fusion occurred leading to activation of caspase 3. As such, physiological trophoblast turnover, leading to apoptotic shedding was observed in the overlying syncytiotrophoblast. These investigators concluded that reduced oxygen supply to the placenta favours necrotic rather than apoptotic shedding of syncytial fragments into the maternal circulation. Since uteroplacental ischaemia is a significant risk factor for pre-eclampsia, these findings may explain the link between reduced uteroplacental blood flow and the systemic clinical manifestations of this disease. REFERENCES Hunt JS, Phillips TA, Rasmussen CA, Bowen JA & Bluethmann H (1999) Apoptosis-inducing members of the tumor necrosis factor supergene family: potential functions in placentae. Trophoblast Research, 13, 243–257. Huppertz B & Hunt JS (2000) Trophoblast apoptosis and placental development—a workshop report. Placenta, 21 (Suppl A), S74–S76. Huppertz B & Kaufmann P (1999) The apoptosis cascade in human villous trophoblast: a review of methods and findings. Trophoblast Research, 13, 215–242. Reister F, Frank HG, Heyl W, Kosanke G, Huppertz B, Schroder W, Kaufmann P & Rath W (1999) The distribution of macrophages in spiral arteries of the placental bed in pre-eclampsia differs from that in healthy patients. Placenta, 20, 229–233. Vogt E, Ng AK & Rote NS (1997) Antiphosphatidylserine antibody removes annexin-V and facilitates the binding of prothrombin at the surface of a choriocarcinoma model of trophoblast differentiation. Am J Obstet Gynecol, 177, 964–972.
APOPTOSIS, MOLECULAR AND GENETIC CONTRIBUTIONS Apoptosis: Molecular Control of Placental Function—A Workshop Report B. Huppertza,g, N. S. Roteb, D. M. Nelsonc, F. Reisterd, S. Blacke and J. S. Huntf a
Department of Anatomy, University of Technology, Aachen, Germany Departments of Biological Sciences and Obstetrics & Gynecology, Wright State University School of Medicine, Dayton, Ohio 45435, USA c Washington University School of Medicine, St Louis, MO, USA d Department of Obstetrics and Gynecology, University of Technology, Aachen, Germany e Monash University, Box Hill Hospital, Box Hill, Australia f Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, USA Paper accepted 5 January 2001 b
Following successful completion of the workshop on apoptosis in Schladming, 1999 (Huppertz and Hunt, 2000), the organizers were requested to develop a second workshop in Rochester, 2000. This workshop was designed to link trophoblast apoptosis to functional aspects of the placenta. The basic principles and mechanisms involved in trophoblast apoptosis (for review see Huppertz and Kaufmann, 1999) are becoming clearer and there is more and more information on the functional roles apoptosis plays in placental development and integrity. The presentations in this workshop represented this development. Joan Hunt dealt with the expression of tumour necrosis factor (TNF) superfamily genes in human placenta and potential relationships with apoptosis. The TNF superfamily contains at least eight genes encoding proteins that mediate apoptosis. These include TNF, LT, LT, LIGHT, FasL, TRAIL, TWEAK and 4-1BBL. Fourteen genes encode receptors for these ligands. Of these, all eight genes encoding apoptosis-inducing ligands and 13 of the 14 genes encoding receptors for these ligands are expressed in human placentae (Hunt et al., 1999). Messages have been identified at early and/or late stages of gestation by Northern blot analysis and reverse transcriptase polymerase chain reaction. In an effort to dissect the sites of synthesis of the TNF superfamily ligands and receptors, trophoblasts (Jar and JEG-3 cell lines, term villous cytotrophoblasts) and macrophages (U937 and THP-1 cell lines and term placental macrophages) were examined for transcripts. Of the ligands, only FasL and g
To whom correspondence should be addressed at: Department of Anatomy, University of Technology, Wendlingweg 2, D-52057 Aachen, Germany. Fax: +49-241-8888-472. E-mail: bhuppertz@ post.klinikum.rwth-aachen.de
0143–4004/01/0A0S101+03 $35.00/0
LT mRNAs were absent in all trophoblasts; none of the ligand messages were absent in all macrophages. Of the receptors, 4-1BB and OPG messages were absent in all trophoblasts; only OPG was absent in all macrophages. However, term villous cytotrophoblasts lacked TRAIL and LIGHT as well as FasL and LT; placental macrophages lacked 4-1BBL and also lacked DcR1 as well as OPG transcripts, thus demonstrating some differences between normal cells and cell lines in the expression of both ligands and receptors. TNF appears to be multifunctional, regulating hormone synthesis and placental architecture through effects on integrins and matrix metalloproteinases. FasL seems to have the ability to influence placental development and may also provide a measure of immune privilege and serve as a barrier to maternal–fetal cell trafficking. TRAIL is positioned to prevent maternal immune cell attack on the placenta but may also exert profound effects on other trophoblast functions. Neal S. Rote discussed the regulation of phosphatidylserine distribution during differentiation and apoptosis of villous trophoblast. All mammalian cells actively maintain a heterogenous distribution of phospholipids in their plasma membranes. Aminophospholipids, such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), are preferentially maintained on the inner face of the plasma membrane by a flippase. Exposure of PS on the outer leaflet of the cell membrane is an early active event in cells undergoing apoptosis. Normal human villous cytotrophoblast differentiation is also characterized by PS externalization, related to intercellular fusion. Movement of PS and PE from the outer to inner plasma membrane surface by means of the aminophospholipid translocase is relatively fast (t1/2 of 5–10 min). 2001 IFPA and Harcourt Publishers Ltd
S102
An undifferentiated model of trophoblast, BeWo, has an active aminophospholipid translocase. During forskolin or cAMPinduced BeWo differentiation, PS is externalized with rather slow kinetics; antigenic or annexin V-binding PS appearing progressively on the surface of the cell over 48 h. This process is concurrent with hormonal differentiation and intercellular fusion into syncytiotrophoblast and can be completely blocked by monoclonal antibody against PS. The relatively slow rate of externalization is compatible with the activity of a floppase, which redistributes PS and PE to the outer surface with a t1/2 of 1–2 h. During apoptosis, PS externalization appears to be relatively rapid; the binding of annexin V and monoclonal anti-PS antibodies increased significantly by 5 min and reached maximum expression by 40 min. The rapid expression of apoptosis-related PS is compatible with the action of the recently described scramblase (Vogt, Ng and Rote, 1997), which has a t1/2 of less than 1 min. Apoptosis-related PS externalization is not accompanied by increased intercellular fusion. Rote and co-workers hypothesized that the floppase is responsible for PS externalization during differentiation and the scramblase during apoptosis, and investigated whether the mechanisms of PS externalization during trophoblast differentiation and apoptosis processes were mediated by different enzymes. BeWo is an in vitro model of both trophoblast differentiation and apoptosis. After 24 h treatment of forskolin-induced differentiation, PS was detected on the cell surface by binding of FITC-annexin V, which increased during 24–72 h of treatment, but was not accompanied by apoptosis monitored by TUNEL assay. Apoptosis induction by staurosporine for 1 h triggered rapid PS externalization. Inhibition of floppase activity with vanadate blocked differentiation-related but not the apoptosis-related PS externalization. The effector caspase inhibitor ZVAD-fmk completely abolished staurosporine-induced apoptosis, but had no effect on differentiation-mediated PS externalization. Thus, PS externalization appears to be a marker for both trophoblastic differentiation and apoptosis. Following these talks on mechanistic issues of trophoblast apoptosis, subsequent presentations dealt with changes in the rate of apoptosis and their effects on the maintenance of the placenta and the outcome of pregnancy. D. Michael Nelson discussed a role for prostanoids and peroxisome proliferator activator receptor gamma (PPARc) activation in the differentiation and apoptosis of human trophoblast. Underperfusion and hypoxia of the placenta are associated with pre-eclampsia and fetal growth restriction. Hypoxia in vivo and in vitro limits differentiation and enhances apoptosis in human villous trophoblast. The mechanisms regulating these biological responses in trophoblast are unclear. However, pre-eclampsia is associated with enhanced apoptosis, altered prostanoid production and increased expression of cyclooxygenase-2 in the trophoblast of placental villi. PPAR is clearly essential for murine placental development and differentiation, and PPAR is also expressed in trophoblast of human placental villi.
Placenta (2001), Vol. 22, Supplement A, Trophoblast Research, Vol. 15
Dr Nelson discussed the role of prostanoids and PPAR activation in the regulation of differentiation and apoptosis in cultured human trophoblast. The prostanoids thromboxane and 15-deoxy-12,14-prostaglandin J2 (15PGJ2), a ligand for PPAR, hinder biochemical and morphological differentiation and enhance apoptosis in cultured trophoblast. In contrast, troglitazone, another ligand for PPAR, enhances differentiation and does not affect apoptosis in trophoblast. Both thromboxane and 15PGJ2, but not troglitazone, upregulate p53 expression, suggesting that the apoptotic response of trophoblast to these ligands is p53 dependent. The data indicate that thromboxane and PPAR activation play important roles in trophoblast biology. Frank Reister discussed macrophage-mediated tryptophan depletion that may contribute to apoptosis of extravillous trophoblast and may have an impact on pre-eclampsia. Recent data (Reister et al., 1999) indicate a role of trophoblast apoptosis in reduced trophoblast invasiveness in pre-eclampsia (PE) and an impact of macrophages in this process. Macrophages are known to secrete indoleamine 2,3-dioxygenase (IDO), a tryptophan-degrading enzyme. Tryptophan starvation leads to apoptosis of trophoblast hybrid cells. Therefore these investigators speculated that macrophages might be involved in tryptophan-associated apoptosis of trophoblast hybridoma, and investigated if changes in the cytokine environment might provide activating mechanisms. The effect of tryptophan supplementation on apoptosis was studied in a co-culture model (extravillous trophoblast hybrid cells/ macrophages). The supplementation of tryptophan significantly reduced the incidence of trophoblast apoptosis in a dose-dependent manner. Immunohistochemical evaluation of the distribution of uterine natural killer (NK)-cells in the placental bed of both PE patients revealed a significant increase of uterine NK-cells in PE patients compared with healthy pregnant women. Thus, the secretion of IDO is one of the mechanisms used by macrophages in order to induce trophoblast apoptosis. This may contribute to insufficient endovascular trophoblast invasion in PE. NK-cell-derived interferon- may stimulate IDO-activation in placental bed macrophages in PE. Simon Black showed evidence for a redox regulation of magnesium-induced placental apoptosis. Placental apoptosis has been implicated in fetal growth restriction, placental abruption and pre-eclampsia. The cause of increased placental apoptosis is not known; however, oxidative stress has been implicated in placental dysfunction. Recently, nitric oxide, a component of peroxynitrite and a major source of placental oxidative stress, was shown to be increased by magnesium. Furthermore, maternal magnesium levels are elevated in preeclampsia and fetal growth restriction, and physiological levels of magnesium cause loss of trophoblast viability. These researchers report magnesium-stimulated placental apoptosis in vitro as evidenced by oligosomal DNA laddering and tissue shrinkage associated with clumping of pycnotic trophoblast nuclei. Placental apoptosis measured by oligosomal DNA laddering increased over the physiological range of
Huppertz et al.: Apoptosis and Placental Function
extracellular magnesium. Plasminogen activator inhibitor type 2 cleavage and cytokeratin 18 neoepitope formation indicated caspases were involved in the mechanism of magnesiuminduced placental apoptosis. They inhibited magnesiuminduced apoptosis with vitamin C, vitamin E, N-acetylcysteine, neomycin or Methylene Blue, and interpreted their inhibitor results as evidence that magnesiuminduced placental apoptosis was associated with an oxidative state. Black and co-workers propose that redox-regulating agents may be a fruitful source of safe, inexpensive, effective and rational agents that can be used to improve obstetric outcomes. Furthermore, we advocate further study of the regulation of maternal magnesium. In a final presentation, Berthold Huppertz reported that hypoxia favours necrotic versus apoptotic shedding of placental syncytiotrophoblast into the maternal circulation and suggested implications for the pathogenesis of pre-eclampsia. In the course of normal pregnancy, several grams of placental trophoblast are shed daily into the maternal circulation and are tolerated by the mother. The balance between apoptotic and necrotic shedding of this material is presently unknown. Since pre-eclampsia is characterized by a pronounced degree of inflammatory damage, this group investigated the possibility that excessive shedding of necrotic placental trophoblast is involved in the pathogenesis of this disorder. They studied an explant model of human trophoblast turnover in 3 per cent and 20 per cent oxygen over 2 days. Trophoblast turnover including apoptosis and necrosis were monitored by: conventional histology, immunolocalization of Ki67 (a proliferation marker), Bcl-2 (an apoptosis inhibitor), caspases 8 and 3 (apoptosis promoters), and DNA degradation. Furthermore, electron microscopy and [3H]cytidine and [3H]uridine incorporations
S103
were employed. Culture in 3 per cent oxygen resulted in increased villous cytotrophoblast proliferation and necrotic shedding of the overlying syncytiotrophoblast. The proteins necessary for execution of apoptosis were retained in the cytotrophoblast due to lack of syncytial fusion. By contrast the syncytium remained intact in 20 per cent oxygen. Reduced cytotrophoblast proliferation was noted, but syncytial fusion occurred leading to activation of caspase 3. As such, physiological trophoblast turnover, leading to apoptotic shedding was observed in the overlying syncytiotrophoblast. These investigators concluded that reduced oxygen supply to the placenta favours necrotic rather than apoptotic shedding of syncytial fragments into the maternal circulation. Since uteroplacental ischaemia is a significant risk factor for pre-eclampsia, these findings may explain the link between reduced uteroplacental blood flow and the systemic clinical manifestations of this disease. REFERENCES Hunt JS, Phillips TA, Rasmussen CA, Bowen JA & Bluethmann H (1999) Apoptosis-inducing members of the tumor necrosis factor supergene family: potential functions in placentae. Trophoblast Research, 13, 243–257. Huppertz B & Hunt JS (2000) Trophoblast apoptosis and placental development—a workshop report. Placenta, 21 (Suppl A), S74–S76. Huppertz B & Kaufmann P (1999) The apoptosis cascade in human villous trophoblast: a review of methods and findings. Trophoblast Research, 13, 215–242. Reister F, Frank HG, Heyl W, Kosanke G, Huppertz B, Schroder W, Kaufmann P & Rath W (1999) The distribution of macrophages in spiral arteries of the placental bed in pre-eclampsia differs from that in healthy patients. Placenta, 20, 229–233. Vogt E, Ng AK & Rote NS (1997) Antiphosphatidylserine antibody removes annexin-V and facilitates the binding of prothrombin at the surface of a choriocarcinoma model of trophoblast differentiation. Am J Obstet Gynecol, 177, 964–972.