Placental apoptosis in normal and abnormal pregnancy

Current Obstetrics & Gynaecology (1998) 8, 13~0 © 1998 Harcourt Brace & Co. Ltd

Mini-symposium: The placenta

Placental apoptosis in normal and abnormal pregnancy

S. C. Smith and E N. Baker

INTRODUCTION

Cell death (like clinical death) is difficult to define, and there is no easy way to find out exactly when a cell dies. When a cell has been dead for a given period of time (12-24 h) it undergoes a series of secondary morphological changes, such as the breakdown of the nucleus; this is the process of necrosis. Necrosis is easily recognized by light microscopy, but the identification of cells that have just died is difficult. Oncosis should not be confused with necrosis. Necrosis is the series of events that occur after cell death has taken place. To describe a cell as dying by necrosis is the same as describing a patient's death as being due to postmortem autolysis.

Until recently, the subject of cell death was considered to be dnll and morbid. Indeed, disease processes tended to be thought of in terms of cell overgrowth rather than in terms of death aberration. 1Times have changed, and the subject of cell death has become fashionable for a variety of reasons, including the realization that many of us die as a direct result of cell death in the form of infarcts. Another major incentive for the study of cell death has been the increase in organ grafting, and the need to understand organ viability. Cell death occurs in a variety of settings, somewhat reminiscent of the human condition: • by accident, such as failure of the blood supply • by suicide: every cell type is capable of this • by specialized killer cells.

HISTORICAL BACKGROUND

Apoptosis ('programmed' cell death/cell suicide) was first described in a landmark paper in 1972 by Kerr, Wylie and Currie, 2 but the concept of a form of cell death distinct from oncosis was appreciated by Kerr as early as 1965. 3 He described a form of cell death distinct from the swollen cells of oncosis, and gave this process the preliminary name 'shrinkage necrosis'. Apoptosis is a fundamental biological phenomenon that occurs under a wide variety of physiological and pathological situations. Despite the fact that it was first described a quarter of a century ago, it was not widely accepted as a concept within medicine and science until comparatively recently. The first international meeting to highlight apoptosis (Modulating factors in multistage chemical carcinogenesis) was held in Sardinia in September 1989. 4 The concept of apoptosis, that the death of selected individual cells within a tissue can be an active process mediated by the cell's own biochemical mechanisms,

There are two major forms of cell death. The most common form is cell death resulting from an irreversible reaction to injury, lack of nutrients or lack of oxygen. This form of cell death can be regarded as either accidental cell death or cell murder. In practice, only one form of accidental cell death is well understood, and that is ischaemic cell death. Ischaemic cell death is accompanied by cellular swelling, and this form of death is called oncosis (from the Greek 6nkos, swelling), a name coined almost a century ago by von Reckfinghausen. The other major form of cell death is cell death by 'suicide', which occurs by the process of apoptosis. Assassination' by killer cells also occurs by apoptosis~

Dr S. Smith, Research Fellow and Mr P. N. Baker, Senior Lecturer, School of Human Development, Academic Department of Obstetrics & Gynaecology, New Maternity Unit, City Hospital, Hucknall Road, Nottingham NG5 1PB, UK

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has been reported by some 4 to be one of the most important milestones in cell and tissue research this century. The term apoptosis derives from the Greek description of the 'dropping off' or 'falling away' of petals from flowers, or leaves from trees. Prior to the description of apoptosis, all cell death was assumed to be the outcome of some form of external insult, and to be oncotic in nature. Reports of the investigation of apoptosis within placental tissue have only started to appear in the literature over the last 2 years? ,6

•

APOPTOSIS AND ONCOSIS

Fig. 2

Whilst the identification of" dead cells can be difficult, cells which have undergone apoptosis can be recognized by the experienced observer using light microscopy, because of their characteristic morphology (see Fig. 1). The most characteristic light microscopy feature of apoptosis is the condensation of nuclear heterochromatin, first as a crescent apposed to the nuclear membrane, and later into single or multiple dense bodies. With practice this can be detected by conventional oil-immersion light microscopy of haematoxylin and eosin stained cells, and in the absence of a definitive molecular marker, morphological examination is the best way to identify apoptosis. The distinctive morphology is best revealed by electron microscopy which remains as the 'gold standard' in the identification of a p o p t o s i s . 7 Figure 2 is a comparison of apoptosis and oncosis. Cell death by apoptosis has been described in a wide range of circumstances? It plays an important role in embryology and developmental biology, good examples of this being the involution of the Mfillerian and Wolffian systems. Apoptosis also contributes to the atrophy of fully developed mammalian tissues following the removal of endocrine stimulation, such as in the post-lactational breast. The process also balances mitosis in cyclically stimulated epithelia such as the human endometrium and must occur, to some extent, in all tissues. Apoptosis also occurs in tissues treated with a wide variety of toxic stimuli, particularly when these are applied at low dosage e.g. cytotoxic drugs, ionizing radiation, hyperthermia and hypoxia.

Comparison of the morpholo~v of apoptosis and oncosis Apoptosis

• • • • •

Oncosis

Single cells affectedwithin living tissues Pyknotic nuclei Dyes initiallyexcluded Cytoplasm compacted - urganelles intact No inflammation Phagocytosis by specialist macrophages or neighbouring cells

• •

• •

Sheets of cells - tissue structure disruption Nuclei intact and faintly staining Dyes enter Cellular oedema - ruptured plasma and internal membranes Acute inflammation No phagoeytosis

The molecular mechanisms of apoptosis have recently been reviewed by Tanuma. 9 Figure 3 provides an overview. Induction occurs when cells receive various signals at cell-surface receptors. Two important cell surface receptors are the Fas/APO-1 cell-surface protein and the tumour-necrosis-factor receptor (TNFR). During signal transduction in plasma membranes, second messengers (cAMP, cGMP etc.) are produced through signal transduction systems: adenylate cyclase, guanylate cyclase etc. The events that occur downstream of these receptors are still unclear, but the 'cross-talk' of the second messengers results in the expression of apoptosis regulator genes, the activation of pre-existing apoptotic gene products and/or suppression of the expression of survival genes. A cell with the competence to undergo apoptosis is able to operate processes for survival until determination occurs. In the determination process, cells pass through a 'point of no return' for apoptosis. Several oncogenes (c-myc, p53, bcl-2, c-fos etc.) have been shown to be important in commitment events. Both the induction and the determination processes of apoptosis are diverse in different cell types and states. However, once the apoptotic program is initiated, execution appears to occur irreversibly via a common pathway.

Molecular mechanism of apoptosis Apoptosis Signals ( h o r m o n e , c y t o k i n e , antigen, virus, radiation, drug)

! !

Morphological features of apoptosis

Induction

1 Loss of specialised surface structures eg microviUi and

Determination (point o f n o return)

contact regions 2

Cell volume reduction

3

Conservation

! !

of cytoplasmic organelle integrity

Execution

4 Condensation of nuclear chromatin 5 Phagocytosis by parenchymal cells or specialised macrophages. Fig. 1

APOPTOSIS Fig. 3

Placental apoptosis in pregnancy

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In many types of cell a sustained increase in the intracellular calcium (Ca 2+) occurs early in apoptosis. This may be a trigger for the activation of a nuclear 'apoptotic-specific' endonuclease that cleaves genomic DNA in the linker regions between nucleosomes to produce nucleosomal oligomers. Each nucleosome has 180 base pairs associated with it, and these oligomers consist of multiples of 180 base pairs. ICElike proteases (ICE - interleukin converting enzyme), other kinds of proteases such as carpain, and a tissue transglutaminase that cross-links cytoplasmic proteins are also activated. The series of execution events converts a cell to an irreversible apoptotic state. Apoptotic cells or their fragments (apoptotic bodies) are rapidly recognized and phagocytosed by macrophages or their neighbours. It therefore follows that whilst apoptosis is a mechanism of cell death, it is also a mechanism of cell clearance. The process is rapid, taking only 1-2 h for completion? Apoptosis requires energy, and can depend on new gene e•pression following induction. In normal lymphocytes it can be suppressed by the inhibition of RNA or protein synthesis, suggesting that the transcription and translation of some genes, including those of activator proteins, are required for progression of apoptosis. By contrast, in neutrophils, gastrointestinal crypt cells and certain other cell types, apoptosis is rapidly induced by inhibitors of RNA or protein synthesis. In these situations, the apoptotic machinery is believed to be already constructed, but suppressed by protein inhibitors with short half-lives.

for gestation at delivery, fetal sex, parity, ethnic origin, maternal height and booking weight. The IBR enables a more accurate prediction of pregnancies that end in a poor outcome than birthweight for gestational age alone. 1°IUGR is the end point for a number of clinical scenarios within pregnancy, and an attempt to propose a single pathological process to explain reduced fetal growth in such diverse clinical situations as uterine abnormality, multiple pregnancy and pre-eclampsia would be unrealistic. Placentae from pregnancies complicated by IUGR tend to be smaller than the placentae from normal uncomplicated pregnancies, 1113 and presumably contain fewer cells. Does apoptosis play a role in the pathophysiology of IUGR?

APOPTOSIS IN THE HUMAN PLACENTA

Light microscopy

Placental apoptosis has been investigated in Nottingham over the last 18 months. The initial hypothesis driving the research was that apoptosis might occur to a greater extent within the placentae of pregnancies complicated by intrauterine growth restriction (IUGR), than it does within the placentae of normal uncomplicated pregnancies, resulting in smaller placentae and, hence, smaller babies. The project has had three broad aims:

Those specimens that were examined using light microscopy were collected into either methyl carnoys fixative or 10% formalin. Light microscopy was carried out on all samples. Following fixation, each sample was paraffin embedded and 4 g m sections were mounted on microscope slides. Five sections were cut for each sample. These sections were cut from different blocks of tissue from the same placenta to ensure that the samples examined were randomly selected. The sections were stained using haematoxylin and eosin and then mounted under cover slips. Each section was examined at a magnification of xl000 - oil immersion. For each placenta, 50 fields of view were examined (10 fields from each section), and the number of apoptotic nuclei identified was expressed as a percentage of the total number of nuclei counted. Approximately 5000-7000 nuclei were counted for each patient. In pilot experiments, the inter-observer error was 17%, although the ranking of the observers (from highest to lowest percentage of apoptotic nuclei) was very similar. All counts and incidence percentages quoted here are the results obtained by a single observer to remove any inter-observer error. The observer was blinded and intra-observer error was 2%. To see if there was any

1. To conclusively demonstrate apoptosis within human placental tissue. 2. To look for possible differences in the incidence of apoptosis throughout pregnancy. 3. To look for possible differences in the incidence of apoptosis within the placentae of pregnancies complicated by IUGR. The definition used to identify cases of IUGR has depended on three criteria: clinical evidence of suboptimal growth; ultrasound scan evidence of deviation from an appropriate growth centile; and individualized birthweight ratios (IBRs) below the 10th centile. The IBR is a ratio relative to a predicted birthweight, calculated using independent coefficients

PATIENTS AND METHODS Placental tissue samples were obtained from 31 firsttrimester pregnancies, 11 second-trimester pregnancies, 43 uncomplicated third-trimester pregnancies and 26 third-trimester pregnancies complicated by IUGR. The first and second trimester samples were obtained following termination of pregnancy, where there was considered to be no pathological features. The thirdtrimester samples were collected from the delivery suite of the City Hospital, Nottingham. Placental samples were taken from both vaginal deliveries (assisted and normal) and Caesarean sections. Infected specimens were avoided as areas of acute inflammation (such as occur in chorio-amnionitis) contain many apoptotic neutrophils. All samples were rapidly fixed.

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consistent variation in the incidence of apoptosis in different parts of the same placenta, five of the thirdtrimester placentae were divided into outer, middle and inner thirds, and random samples were taken from each of these three areas.

Electron microscopy Samples were taken into primary electron microscopy fixative. Electron microscopic examination of seven separate placental samples was performed. Multiple examinations were .carried out on each specimen. Following fixation, sections of tissue were cut, mounted and stained using toluidine blue. Apoptotic cells were first identified in these sections using light microscopy; validation of the light microscopy detection of apoptotic cells was then performed. Serial sections were cut from the same block and prepared with lead salts, prior to examination using transmission electron microscopy.

TUNEL staining This was performed on 21 placental samples, which were collected into 10% formalin. Terminal deoxynucleotidyl transferase mediated d UTP-marker nick-end labelling (TUNEL) is a technique first described by Gavrieli et al in 199274 The 'nick end' refers to the DNA 3'-OH groups generated as a result of endonuclease activity at linker DNA regions between nucleosomes. D N A fragmentation being a characteristic of apoptosis. 15 In the detection system used the 'marker' attached to the dUTP was digoxigenin. The enzyme terminal deoxynucleotidyl transferase (TdT) formed a polymeric tail at the 3'-OH DNA 'nick' ends with dUTP-digoxigenin. After labelling, an 'anti-digoxigenin antibody'/peroxidase conjugate was bound to the digoxigenin molecules in the marker tail. In the final step diaminobenzidine (DAB) was added. The reporter peroxidase bound to the digoxigenin and then generated an intense brown signal from this chromogenic substrate. The tissue was then counter-stained using either methyl green or haematoxylin and the slides were examined using light microscopy at xl000 (as described previously). Apoptotic cells/nuclei were easily seen, as they labelled brown, as compared With other cells/nuclei which wel'e green or blue (ApopTag® Plus In-Situ Apoptosis Detection Kit - Peroxidase, Oncor Inc.). T U N E L staining was mainly used for the identification/confirmation of apoptosis within the cells of the placenta, but in four cases the T U N E L technique was used to quantify apoptosis.

RESULTS

Demonstration of apoptosis Using light microscopy, the occurrence of apoptosis within the cells of placental samples throughout

Fig. 4 Lightmicroscopypicture of apoptoticnucleiwithina syncytialknot (syncytiotrophoblast).This is term placenta. The apoptotic nucleiare easilyrecognized.Someof the nucleiare uniformlydense,whilstothers demonstratemarginationof nuclear chromatin. The stain is toluidineblue, and the magnificationis xl000 - oil immersion.

pregnancy has been demonstrated (Fig. 4). Apoptosis was observed in all cell types within placental tissue. The demonstration of apoptotic cells was validated by transmission electron microscopy (Fig. 5). The occurrence of apoptosis was also confirmed using T U N E L staining (Fig. 6).

Quantification of apoptosis The incidence of apoptotic cells/nuclei in placental samples from the three trimesters of pregnancy, obtained using light microscopy, are illustrated in Figure 7. The incidences of apoptosis in the first and second trimesters were similar. The incidence of apoptosis was significantly higher in the third trimester than in the first and second (P < 0.01, Mann-Whitney U test), and significantly higher in the placentae of third trimester pregnancies complicated by I U G R when compared with normal. The proportions of the total numbers of apoptotic cells/nuclei in each cell compartment (for all groups)were as follows: endothelial cells 6%, stromal cells 31% and trophoblast cells/nuclei 63%. The majority of the apoptotic trophoblast nuclei seen were within the syncytiotrophoblast.

Placental apoptosis in pregnancy

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INCIDENCES OF APOPTOSlS 0.30.25

Statistics:

~ ~

Medi. . . . d Interquartile

0.2 ~

0.0 First n=31

[

Second n=11

I

Third n=43

I

IUGR n=26

'r

Fig. 7 Bar chart demonstrating the incidences of apoptosis in the different groups that have been quantified. The quantification was done by means of light microscopy (see methods). For each placenta examined, the incidence of apoptotic cells is the total number of apoptotic cells identified (of all kinds), expressed as a percentage of the total number of cells counted for that sample. Statistics: values are medians and interquartile ranges.

Fig. 5 Transmission electron microscopy picture of apoptotic nuclei within the syncytiotrophoblast. This is term placenta. The distinctive nuclear changes are easily recognized. Magnification bar is 2 gm x8500.

apoptosis obtained by counting TUNEL stained sections was consistently almost double the incidence obtained by counting using conventional light microscopy techniques (Table 2).

DISCUSSION

The occurrence of apoptosis within the cells of the placenta has been conclusively demonstrated. The incidence of apoptosis has been shown to be signifi: cantly higher in the third trimester of pregnancy when compared with the first and second, and the overall incidence of apoptosis has been shown to be significantly higher amongst the cells in the placentae of pregnancies complicated by IUGR than it is amongst the cells in the placentae of normal uncomplicated pregnancies. The three initial aims of the Nottingham project have been successfully completed, and this Fig. 6 Light microscopy picture of an apoptotic nucleus labelled by the TUNEL technique. This is first trimester placental tissue. The apoptotic nucleus is stained brown. The counter stain here is methyl green, and the magnification is xl000 - oil immersion.

There were no significant differences in the incidence of apoptosis within different areas of the same placenta (Table 1). A significantly higher proportion of the IUGR pregnancies were delivered by means of Caesarean section when compared with the normal pregnancies. Analysis of the results of the normal third-trimester cohort revealed no significant differences in the incidence of apoptosis between vaginally delivered pregnancies and Caesarean sections. Analysis of the results comparing the four cases, which were quantified using both light microscopy and TUNEL staining, revealed that the incidence of

Table 1 Incidences of apoptosis within different areas of the placenta Placental area

Incidence of apoptotic celis

Outer third Middle third Inner third

0.14% (0.11%-0.52%) 0.16% (0.09%-0.20%) 0.24% (0.07%o-0.25%)

n=5 (medians and ranges)

Table 2 Comparison of the observed incidences of apoptotic cells using light microscopy and TUNEL staining Patient group Normal Normal Normal IUGR

IBR

Light microscopy

28th centile 56th centile 58th centile 9th centile

0.13% 0.19% 0.15% 0.15%

TUNEL staining 0.22% 0.29% 0.29% 0.32%

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work has been accepted for publication. ~6,17A number of issues should be considered in the interpretation of these findings. Whilst apoptosis in the placenta has only recently been reported, its occurrence has been demonstrated by several independent groups, 5,6'18'~9,2°and apoptosis is now considered to play an important role in the physiology and pathophysiology of the placenta. In this study, apoptosis was seen to occur throughout pregnancy in trophoblast, endothelial cells and stromal cells. This is consistent with the fact that it is a 'basic biological phenomenon'. 3Apoptosis must occur in all tissues and all cell types at a basic background level. The information available in the literature concerning the background incidence of apoptosis within other tissues is limited, but the incidence in normal rat glomeruli has been quoted as 0.01%? 1 The incidences of apoptosis that have been demonstrated here are in keeping with this low background rate. The finding that the incidence of placental apoptosis is significantly higher in the third trimester of pregnancy when compared to the first and second trimesters, is in keeping with the report by Kim et aF° of a decrease in the expression of bcl-2 within placental tissue in the third trimester. The primary role of bcl-2 is the inhibition of apoptosis, and a decrease in its expression within the third trimester would be consistent with our histological findings of increased apoptosis. The fact that the incidence of apoptosis rises as pregnancy continues is not surprising, there being some suggestion that apoptosis may play a role in senescence. Tissues can become more susceptible to apoptotic stimuli with increasing age? One of the obvious questions raised by this study is whether the observed increase in the incidence of apoptosis seen with IUGR is as a result of the pathological processes leading to IUGR, or an aetiological component in the development of IUGR? One of the known triggers of apoptosis is hypoxiaS; if placentation were to occur in a location of relatively poor blood supply, this in itself could account for IUGR. Increased placental cell apoptosis seen in such a situation could be interpreted as an effect of poor placentation and IUGR, rather than as an aetiological phenomenon. An increase in fetoplacental vascular impedance at the capillary level resulting in impaired gas and nutrient transfer could be a cause of apoptor sis within the cells of the placenta. Again, if this were the case, apoptosis under such conditions would be occurring as a result of the pathological processes leading to IUGR. Nelsons 6 has suggested that the areas of apoptosis within the syncytiotrophoblast could be part of a physiological process resulting in easier transport across the placenta. If placentation were to occur in an area of relatively poor blood supply, resulting in ischaemia at the placental bed, it may well be that increased apoptosis under such situations could be interpreted as an attempt to compensate for this relative ischaemia, and so increase placental

exchange. Alternatively, it is possible that our starting hypothesis is correct, and that an increase in placental cell apoptosis is a primary pathological process that results in smaller placentae and, hence, smaller babies. If this is the case, it is important that the factors leading to the apoptosis in such situations are carefully investigated. Whilst apoptotic cells have a distinctive morphology, they remain difficult to identify. Counting apoptotic cells was a subjective and repetitive process that was open to a great deal of individual interpretation. All of the quantification results presented were the work of a single individual to remove any interobserver error. However, validation of the light microscopy findings using electron microscopy (and other microscopy techniques) was vital. Electron microscopy remains the 'gold standard' in the identification of apoptotic cells.7 Unfortunately, owing to the extremely low incidence of apoptosis within placental tissue (in common with other ex vivo material), it would prove to be prohibitively expensive to use electron microscopy to quantify apoptosis in the placenta by this method. As was demonstrated in the results section, the observed incidence of apoptosis, as determined by TUNEL staining, was twice the value determined using light microscopy alone. A number of recent papers have reported the labelling of necrotic cells by the TUNEL method, Gold et a122and Yasuda et al, 23 and this could, in part, account for this discrepancy. Also, TUNEL staining kits may well identify apoptotic cells by nick end labelling prior to their morphological features becoming apparent. These two points may explain this discrepancy. The syncytiotrophoblast is a unique and fascinating tissue, consisting of a collection of nuclei within a 'sea' of cytoplasm. Very little is known about apoptosis within multi-nuclear tissues. Schwartz and his co-workers have produced a number of publications looking at cell death within the intersegmental muscles (ISMs) of the tobacco hawkmoth, Manduca sexta, which is a multi-nuclear tissue. The cells within this tissue undergo a form of programmed cell death, which Schwartz distinguishes from apoptosis 24,25and which he refers to as 'non-apoptotic' programmed cell death. When examined ultrastructurally, these dying cells do not display membrane blebbing or chromatin margination. The DNA from the ISM cells of Manduca sexta that have undergone this nonapoptotic-programmed cell death does not demonstrate cleavage into nucleosomal fragments, suggesting that endonuclease activity is not involved. During their programmed cell death, these nuclei lose their multilobed form, and become small and round in shape. Their chromatin becomes pyknotic, but does not disperse along the inner margin of the nuclear envelope; instead it forms focal condensations throughout the nucleus. Such nuclear changes would not be classified as apoptotic by the criteria of Kerr et al. 2 The trans-

Placental apoptosis in pregnancy mission electron microscopy pictures that we have produced of 'apoptotic' nuclei within the syncytiotrophoblast of the placenta have demonstrated some nuclei that have a classical apoptotic appearance, and some dying nuclei that have features similar to those described by Schwatrz et al within the ISM of Manduca sexta. This leads us to consider that we may be demonstrating a variant of programmed cell death similar to the one demonstrated by Schwartz. Whilst the electron microscopy findings would, in part support this conclusion, the fact that we have had positive TUNEL staining within placental tissue is more appropriate to classical apoptosis. In apoptosis the cells are cleared from the tissue by phagocytosis either by 'professional' phagocytes, such as macrophages, or by parenchymal cells, which become opportunistic phagocytes. Indeed, apoptotic cells or bodies when viewed using the electron microscope are usually seen within phagocytic cells. The syncytiotrophoblast is not made up of distinct individual cells, and the opportunity for phagocytosis, therefore, does not exist. Despite this fact, the distinctive electron microscopy features of apoptotic nuclei have been demonstrated within the syncytiotrophoblast. It is a possibility that these apoptotic nuclei are lost into the maternal circulation and then dealt with by the maternal reticuloendothelial system. As mentioned in the results section, most of the apoptosis that has been identified in placental tissue has been within either the trophoblast compartment or the villous stroma. This is not particularly surprising as the placenta is made up of the following proportions of total nuclei: 39% trophoblast, 50% stroma and 11% endothelial at 12-16 weeks; the corresponding values near term are 40%, 47% and 13%.26 Syncytiotrophoblast nuclei are, therefore, more prevalent than cytotrophoblast nuclei throughout gestation at a ratio of 9:1,27apoptosis within the syncytial layer could help to maintain this ratio. Apoptosis is a rare phenomenon, and would, therefore, be expected to be seen most frequently in the most common cell/nuclear types within a given tissue. The low levels of apoptosis identified within endothelial cells could also be due to the rapid expulsion of such cells into the fetal circulation once they have undergone apoptosis. The increase in the incidence of apoptosis demonstrated within IUGR placentae appears to be due to an increased incidence of apoptosis within the syncytiotrophoblast, but until a formal quantification of the process within the trophoblast ~s carried out, this can only be an Impression. Apoptosis has been seen in all cell types within placental tissue, from both normal placentae and placentae from pregnancies complicated by IUGR, and it may well be that apoptosis is increased to a similar level in all cell types making up the placenta. Work is currently being carried out in Nottingham looking at several in-vitro models of placental apoptosis, with the aim of elucidating some of the features of the mechanisms of placental apoptosis. Guilbert et •

)

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.

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al have already established that apoptosis can be induced in primary human trophoblast cells in culture by a combination of TNF-~ and human interferon7.28They have also demonstrated that this effect can be reversed by the use of epidermal growth factor? 9 The role of IGF-1 needs to be investigated, as this is known to reduce death in cell culture? ° The report of reduced levels of IGF-1 in women whose pregnancies are complicated by IUGR is consistent with the hypothesis that placental apoptosis is increased in I U G R ? l The effects of platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) will also be investigated. These have been proposed as being fundamental to the pathogenesis of IUGR. 32 ACKNOWLEDGEMENTS

The authors would like to thank Emlyn Price and Trevor Gray for their technical assistance with this project. REFERENCES

1. Majno G, Joris I. Cells, tissues, and disease. Principles of general pathology. Cambridge: Blackwell Science 1996 2. Kerr JFR, Wylie AH, Currie AR. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26:239-257 3. Kerr JFR. A histochemical study of hypertrophy and ischaemic injury of rat liver with special reference to changes in lysosomes. J Pathol Bact 1965; 90: 419-435. 4. Harmon BV, Allan DJ. Apoptosis: a 20th Century Scientific Revolution. In: Sluyser M (ed.) Apoptosis in normal development and cancer. London: Taylor & Francis, 1996: 1-19 5. Price E, Smith SC, Symonds EM, Baker PN. Placental apoptosis in pre-eclampsia and intrauterine growth retardation. Hyper in Preg, 1997; 16(2): 290 6. Savill J. Apoptosis in disease. Eur J Clin Invest 1994; 24: 715 723 7. Wylie AH, Duvall E. Cell injury and death. In: McGee JO'D, Isaacson PG, Wright NA (eds.) Oxford textbook of pathology, principles of pathology. Oxford: Oxford University Press, 1992; 1:141-147 8. Tanuma S-I. Molecular mechanisms of apoptosis. In: Sluyser M (ed.) Apoptosis in normal development and cancer. London: Taylor & Francis. 1996:39-59 9. Nelson DM Apoptotic changes occur in syncytiotrophoblast of human placental villi where fibrinoid is deposited at discontinuities in the villous trophoblast. Placenta 1996; 17: 38%391 10.Wilcox MA, Johnson IR, Maynard PV, Smith SJ, Chilvers CED. The individualised birthweight ratio: a more logical outcome measure of pregnancy than birthweight alone. Br J Obstet Gynaecol 1993; 100:342-347 11. Baker PN, Johnson IR, Gowland PA, Hykin J, Adams V, Mansfield P, Worthington BS. Measurement of fetal liver, brain and placental volumes with echo-planar magnetic resonance imaging. Br J Obstet Gynaecol 1995; 102: 35-39 12. Teasdale E Idiopathic intrauterine growth retardation: histomorphometry of the human placenta. Placenta 1984; 5: 83-92 13. Shen Y. Stereological study of the placenta in intrauterine growth retardation with different ponderal index. Chinese J Obstet Gynecol 1992, 27:351-354 14. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. J Cell Biol 1992; 119(3): 493-501 15. Arends MJ et al. Apoptosis: the role of the endonuclease. Amer J Pathol 1990; 136:593~08

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25. Schwartz LM, Smith SW, Jones MEE, Osborne BA. Do all programmed cell deaths occur by apoptosis? Proc Natl Acad Sci USA 1993; 90:980-984 26. Mayhew TM, Wadrop E, Simpson RA. Proliferative versus hypertrophic growth in tissue subcompartments of human placental villi during gestation. J Anat 1994; 184:535-543 27. Mayhew TM, Simpson RA. Quantitative evidence for the spatial dispersal of trophoblast nuclei in human placental villi during gestation. Placenta 1994; 15:837 844 28. Yui J, Garcia-LLoret M, Wegmann TG, Guilbert LJ. Cytotoxicity of tumour necrosis factor-alpha and human gamma-interferon against primary human placental trophoblasts. Placenta 1994; 15:819-835 29. Garcia-LLoret M, Yui J, Winkler-Lowen B, Guilbert LJ. Epidermal growth factor inhibits cytokine-induced apoptosis of primary human trophoblasts. Journal of Cellular Physiology 1996; 167:324-332 30. Harrington EA, Bennett MR, Fanidi A, Evan GI. C-Mycinduced apoptosis in fibroblasts is inhibited by specific cytokines. Embo J 1994; 13(14): 3286-3295 31. Delmis J, Drazancic A, Ivanisevic M, Suchanek E. Glucose, insulin, HGH and IGF-1 levels in maternal serum, amniotic fluid and umbilical venous serum: a comparison between late normal pregnancy and pregnancies complicated with diabetes and fetal growth retardation. J Perinatat Med 1992; 20:47-56 32. Charnock-Jones DS, Sharkey AM, Boocock CA, Ahmed A, Plevin R, Ferrara N, Smith S Vascular endothelial growth factor receptor localisation and activation in human trophoblast and choriocarcinoma cells. Biol Reprod 1994; 100: 342-347