The effects of cigarette smoking on the human placenta: a light and electron microscopic study

Placenta 0982), 3, 243-256

The Effects of Cigarette Smoking on the Human Placenta: a Light and Electron Microscopic Study

F. VAN DER VEEN & H. F O X Department of Pathology, University of Manchester, Stopford Building, Oxford Road, Manchester MI3 9PT, UK

INTRODUCTION The clinical effects of cigarette smoking during pregnancy, now fully documented and recently reviewed by Pirani (1978), include an increased perinatal mortality, an unduly high incidence of 'small for dates' babies and a general tendency for fetal weight to be below average. It is clearly tempting to consider that placental damage may be a contributory factor to this excess of fetal complications but most studies of placentae from cigarette-smoking women have been concerned principally with the weight and gross characteristics of the organ and there have been relatively few reports of either the histological appearances(L6hr, Ardelt and Dehnhard, 1972; L6hr et al, i975; Spira et al, I977; Naeye, 1978 , 1979) or the fine structure (Asmussen, i978 , 198o) Of these placentae. The reported findings have been contradictory and have not as yet yielded a consistent pattern of placental pathology in cigarette smokers, and we therefore report here a study of the morphological changes seen in the placentae of women who smoke during their pregnancy.

MATERIALS AND METHODS For the purposes of light microscopic examination i00 placentae were collected, 72 from cigarette smokers and 28 from non-smokers. In addition, 31 placentae were collected for electronoptical examination, 23 of which were from smokers and eight of which were from nonsmokers. Data on smoking habits were obtained by direct questioning of the mother on two occasions during pregnancy, once during the first trimester and again at term; women who stopped smoking during the course of their pregnancy were excluded. Non-smokers were defined as being women who had never smoked whilst smokers were divided into three groups: those smoking fewer than i i cigarettes a day during pregnancy were classed as 'light' smokers, those smoking i i t o 20 a day were considered to be 'medium' smokers and those smoking in excess of 2o cigarettes a day were regarded as 'heavy' smokers. O f the 72 placentae from smokers examined by light microscopy, 22 were from light smokers, 32 from medium smokers and 18 were from heavy smokers. Of the further 23 placentae from smokers examined electronoptically, 4 were from light smokers, 9 from medium smokers and io from heavy smokers. Quite apart from maternal smoking habits, placentae in both the smoking and non-smoking groups were collected only from cases fulfilling the following criteria: oi43-4oo4/82/o3o3-243 $o2.oo

(~) 1982 W. B. Saunders Company Ltd

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F. van der Veen, H. Fox

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mother was fully healthy. pregnancy was uncomplicated. child was delivered between the 39th and 41st week of gestation. child was born alive and free from congenital defect.

There were no differences in ethnic or socioeconomic mix between the women in the smoking and the non-smoking groups: approximately four-fifths of the women in each group admitted to occasional intake of alcohol and none gave a history of alcohol abuse. No occasional, or addicted, user of drugs was included in either series. Placentae collected for light microscopy were fixed whole in io per cent formalin and later weighed after trimming off the cord and membranes. The placentae were then cut into vertical slices and four blocks were taken: in an attempt to limit sampling error these blocks were selected in a highly standardized manner, using a method previously described (Fox, i978). Sections were stained with haematoxylin and eosin and with periodic acid Schift, the stained sections were code-numbered in the laboratory and when examined the observer was unaware if they came from the smoking or the non-smoking group. Each section was scanned for evidence of lesions in the basal or chorionic plates, villitis or abnormalities of the fetal vessels; if an 'endarteritis obliterans' was present in the fetal stem arteries this was classed, separately for severity and extent, as being of' + ' , ' + + ' o r ' + + + ' grade. The villous maturity was assessed only in terminal villi in the maternal zone of each section. One hundred terminal villi in the maternal zone of each placenta were counted and the number of villi containing cytotrophoblastic cells, vasculosyncytial membranes and syncytial knots were noted and later expressed as a percentage. The data regarding placental weight, birth weight and villous histology were then analysed in a Joint System ( J C L I 9o6A/CDC 76oo) computer of the University of Manchester Regional Computer Centre, using SPSS (Statistical Package for Social Sciences) version 7. Placentae for electron microscopy were obtained immediately after delivery and a small piece of villous tissue was taken from an area just deep to the basal plate in the central part of the maternal surface; great care was taken to ensure that the site of sampling was standard. The tissue was fixed in 2.5 per cent glutaraldehyde and prepared for electron microscopy as previously described (Jones and Fox, I976). Sections were examined in a Philips EM 3oi electron microscope, by an observer who was, at that time, unaware if they were from the smoking or non-smoking group.

RESULTS Placental weight and fetoplacental weight ratio The maternal, fetal and placental variables are detailed in Table i (this referring only to the i oo placentae selected for light microscopy). Although the mean birth weight was lower in the smoking than in the non-smoking group the difference was, probably because of the small size of the series, not statistically significant. Similarly, although the incidence of low birth weight infants was considerably greater in the smoking than the non-smoking group this difference also proved to be not statistically significant. The mean placental weight in the smoking group was higher than that in the non-smoking group and multivariant regression analysis showed that the effect of smoking on placental weight, when adjusted for birth weight and sex of the infant, was an increase in mean placental weight of 13 g, 38 g and 29 g for light, medium and heavy smokers respectively, differences which were not, however, statistically significant. The only statistically

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Table 2. Incidenceof villousvasculosyncytialmembranes,cytotrophoblasticcellsand syncytialknots in smokingand non-smokinggroups

Number of cases Non-smokers Light smokers Medium smokers Heavy smokers Total smokers

Mean proportionof villi Mean proportion of villi Mean proportionof villi with vasculosyncytial with cytotrophoblastic with syncytialknots membranes cells

29 22 32 i8 72

71.9 68.0 63.7 56.5a 63.3~

14.8 20.5 I9.8 23.8~ 2i.o

37-5 37.8 40-4 39-9 39.5

Significant(P < 0.05). b Significant(P < o.0. significant difference between the two groups was that of the lower fetoplacental weight ratio in the smoking group. Light microscopy The villi were of normal maturity in all cases examined, in both the smoking and the nonsmoking groups. A focal villitis was present in two placentae (7- i per cent) of the non-smoking group and in eight placentae (I i per cent) of the smoking group. The mean counts of villous vasculosyncytial membranes, cytotrophoblastic cells and syncytial knots are detailed in Table 2; taking the smoking group as a whole there was a significant decrease in the incidence of vasculosyncytial membranes and a significant increase in the incidence of villous cytotrophoblastic cells in these placentae compared with those from the non-smokers, but the number of syncytial knots in the smoking group did not differ from that found in the non-smoking group. Breaking up the smoking group in terms of the number of cigarettes smoked, it was apparent that significant differences in morphological parameters were confined to the heavy smoking group, and that it was solely the presence of this group that led to significant differences between the smoking group as a whole and the non-smoking group. Within the fetal vasculature an obliterative endarteritis was found in 64 per cent of the placentae from non-smokers and in 76 per cent of those from smokers (Table 3); there was no significant difference in either the extent or the severity of this vascular abnormality between the two groups of placentae. No abnormality of the basal plate or decidua was seen in any placenta and there was only one instance of chorio-amnionitis (in the smoking group). Table 3. Incidence,severityand extentofobliterativeendarteritisof the fetalstemvesselsin smokingand non-smoking

groups Obliterativeendarteritis present absent (%) (%) Smokers Non-smokers

76.4 64.3

23.6 35.7

Obliterative endarteritis of + + and + + + severity (%)

Obliterative endarteritis of + + and + + + extent (%)

22.2 17.9

52.7 5~

Electron microscopy N o n - s m o k i n g group

Villous ultrastructure in the placentae from non-smokers corresponded to that noted in previous descriptions (Boyd and Hamilton, x97o; Jones, i976 ).

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Figure I. Villous trophoblast in a placenta from a smoker with a prominent cytotrophoblastic cell and syncytiotrophoblast of regular outline with a full complement of microvilli of normal appearance on the free surface. • 16 800.

Smoking group Villous cytotrophoblast. Electron microscopy confirmed the presence of an excess of prominent cytotrophoblastic cells (Figure I) in nine of the 23 placentae in this group. Most of the cytotrophoblastic cells, in all the placentae, were of the intermediate type (Figure 2) with many mitochondria, a prominent Golgi body, a few narrow cisternae of endoplasmic reticulum, a number of electron--dense membrane-limited droplets and an abundance of free ribosomes; a mitotic figure was seen in one placenta. The cytotrophoblastic organelles had, for the most part, a fully normal appearance but in three placentae the mitochondria showed some degree of swelling of the cristae and in a further four placentae there was some dilatation of the cisternae of rough endoplasmic reticulum. The limiting plasma membranes of the cytotrophoblastic cells were generally smooth but in seven placentae showed marked basal digitation and striking interdigitation with the adjacent syncytiotrophoblast. In three placentae, degenerative changes, such as generalized organellar swelling and disruption, cytoplasmic rarefaction and margination of nuclear chromatin or karyolysis, were seen in isolated cytotrophoblastic cells (Figure 3).

Villous syncytiotrophoblast. The syncytiotrophoblast had, in general, a regular outline with a full complement of microvilli of normal appearance on the free surface (Figure I). In I5 placentae, however, there were focal areas, of very variable extent, in which there was a markedly complicated infolding of the surface plasma membrane with local loss of syncytial cytoplasm (Figures 2 and 4)- In these foci the microvilli were either lost or markedly reduced in number

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Figure 2. Villous syncytiotrophoblast in a placenta from a smoker: on the left there is a focal area of infolding of the free plasma membrane (indicated by arrows) and on the right there is dilatation of the rough endoptasmic reticulum (RER). The underlying cytotrophoblastic cell is of the 'intermediate' type. x I6 8oo.

Figure 3. A degenerating villous cytotrophoblastic cell in a placenta from a smoker. The nucleus shows karyolysis and the cell organelles are disrupted, x 87oo.

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Figure 4. A focal area of infolding of the plasma membrane of the villous syncytiotrophoblast (arrowed) with marked loss of cytoplasm in a placenta from a smoker, x 166oo.

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Figure 5. Villous syncytiotrophoblast in a placenta from a smoker. There is a transition between an area of marked infolding of the free plasma membrane (on the left and arrowed) and syncytiotrophoblast with a more normal outline (on the right), x 173oo.

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and those remaining were bizarrely distorted or elongated. Areas of the syncytiotrophoblast showing this striking appearance were usually immediately adjacent to syncytiotrophoblast with a normal outline and with non-deformed microvilli (Figure 5). In three placentae the microvilli, outside of the scalloped foci, did show some degree of abnormality of form with irregular budding, stunting, elongation or forking. In occasional placentae there were tongue-like protrusions ofsyncytial cytoplasm into the intervillous space. Focal syncytial necrosis was seen in two placentae (Figure 6) but even in these the extent of necrosis was small and the vast bulk of the syncytiotrophoblast appeared fully viable. In io placentae there was dilatation of the syncytial rough endoplasmic reticulum, the distended cisternae containing moderately electronlucent flocculent material (Figure 7). The syncytial mitochondria were normal in both number and configuration but in most placentae there was a decrease in the number of syncytial pinocytotic vesicles and a reduced number of syncytial electron-dense secretory droplets. The syncytial nuclei were normal; in I I placentae there was a striking degree of digitation of the basal plasma membrane. No clear-cut correlation could be established between either the degree or extent of the various syncytial abnormalities noted and the number of cigarettes smoked by the mother, though it was noted that the most marked changes in the villous syncytiotrophoblast were seen in three placentae from women who smoked heavily and gave birth to infants weighing less than 2500 g. Trophoblastic basement membrane. In 20 of the placentae there was irregular thickening of the trophoblastic basement membrane (Figure 8). Collagen fibres were sometimes seen within the thickened membrane as were electron--dense inclusions thought to be remnants of the ironcalcium complexes normally found in early pregnancy. Villous stroma. The stromal content of collagen fibres tended to be increased. Stromalfetal capillaries. The majority of these vessels were small. The endothelial cells were

generally of normal contour but were, in five placentae, bulbous with reduced pseudopodial extensions. Intraluminal cytoplasmic blebbing from the endothelial cells was seen in t2 placentae (Figure 9). Myofilaments were markedly well developed in the endothelial cells of eight placentae but otherwise these cells had a normal content of cytoplasmic organelles. In six placentae degenerative changes were seen in the endothelial cells (Figure xo) which were thin and ragged with cytoplasmic rarefaction and marked organellar damage and loss. In five placentae the capillary basement membrane showed a moderate degree of laminar thickening.

DISCUSSION Our findings with regard to birth weight, placental weight and the fetoplacental weight ratio are in accord with those established in much larger series (Kullander and Kallen, I97I; Wilson, I97i; Spira et al, 1975; Wingerd et al, I976; Christianson, x979) , in so far as smoking is associated with a decrease in fetal weight but has no significant effect on placental weight, this resulting in a decreased fetoplacental weight ratio. Histological and electronoptical examination of placentae from women who smoke revealed a paucity of vasculosyncytial membranes, cytotrophoblastic cell proliferation, decreased syncytial pinocytotic and secretory activity, dilatation of rough endoplasmic reticulum in the

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Figure 6. A focal area o f necrosis of the villous syncytiotrophoblast in a placenta from a smoker, x 26 8oo.

Figure 7. Dilated cisternae of the r o u g h endoplasmic reticulum in the villous syncytiotrophoblast o f a placenta from a smoker, x i7ooo.

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Ftgure 8. Villus in a placenta from a smoker. T h e trophoblastic basement membrane (BM) is markedly thickened. There is some degree of digitation (arrowed) of the syncytial basal plasma membrane, x x4 ioo.

Figure 9. A villous fetal capillary in a placenta from a smoker. The endothelial cells show intraluminal blebbing. x 29 5 oo.

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A villous fetal capillaryin a placenta from a smoker.The endothelial cells are thin and raggedwith marked loss of cell organelles. • 168oo. F~gure lO.

syncytiotrophoblast, abnormalities of syncytial microvilli, focal syncytial necrosis, focal intblding of the free plasma membrane of the syncytiotrophoblast, degeneration of isolated cytotrophoblastic cells, thickening of the trophoblastic basement membrane and degeneration of villous capillary endothelial cells. Not all these abnormalities were found in every placenta from a woman who had smoked and indeed most of these morphological changes were confined to a minority of such placentae which did not usually show the full constellation of possible changes. Our findings are at some variance with those described in other studies; we were unable to confirm that an obliterative endarteritis of the fetal stem vessels (L6hr, Ardelt and Dehnhard, I972; L6hr et al, i975; Naeye, I978), an excess of villous syncytial knots (Spira et al, I977) , a deficiency of villous cytotrophoblastic cells (Asmussen, i977) or decidual necrosis (Naeye, i978, I979) are characteristic features of the smoker's placenta but we could agree that thickening of the trophoblastic basement membrane (Asmussen, i977, x98o) and villous cytotrophoblastic proliferation (Naeye, I978 , i979) are frequently found in such placentae. Many of the pathological features seen in the placentae of women who smoke, such as cytotrophoblastic cell hyperplasia, trophoblastic basement membrane thickening, focal syncytial necrosis, decreased syncytial pinocytotic and secretory activity and microvillous abnormalities, are those which are also typically encountered in placentae subjected to ischaemia, for example in the hypertensive complications of pregnancy (Jones and Fox, 198o, 198I); furthermore, they resemble closely those seen in placental villi maintained in culture under conditions of low oxygen tension (MacLennan, Sharp and Shaw-Dunn, i972 ). That there is some degree of uteroplacental ischaemia in many cigarette smokers appears highly probable for it has been shown experimentally that administration of nicotine results in a sharp decrease in uterine blood flow both in the pregnant sheep (Resnik, Brink and Wilkes, 1979) and in the gravid rhesus monkey (Suzuki, Minei and Johnson, i98o ) whilst in the pregnant human the smoking of a single cigarette causes an acute decrease in blood flow through the intervillous

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space (Lehtovirta and Forss, 1978). These effects of nicotine are presumably mediated by its vasoconstrictive properties and are probably accentuated by the raised carboxyhaemoglobin level in the maternal blood (Longo, 1977; Meyer, 1978). Not all the placental abnormalities found in the placentae of cigarette smokers, however, are explicable solely on the basis of ischaemia. A reduction in placental blood flow could not account for degenerative changes in a number of markedly hypoxia-resistant cytotrophoblastic cells, for degenerative changes in the endothelial cells or for the curious focal infoldings of the syncytial plasma membrane. Damage of this type could be due to any of the several thousand compounds found in tobacco smoke (Longo, I98o) but it is possible that cadmium and the polycyclic aromatic hydrocarbons may be implicated. It may appear invidious to select these particular substances but cadmium is a known constituent of tobacco smoke and has been shown experimentally to have a particular propensity for causing placental necrosis (Parizek, 1964); furthermore, it has been shown that the placental content of cadmium is very much higher in smokers than in non-smokers (Copius Peereboom et al, 1979). It is therefore possible that cadmium in tobacco smoke could cause injury to the human placenta and that the degenerative changes seen in cytotrophoblastic and endothelial cells are due to cadmium toxicity. Polycyclic aromatic hydrocarbons are also present in relatively high concentration in tobacco smoke and the placental activity of aryl-hydrocarbon hydroxylase correlates clearly with the number of cigarettes which the mother smokes (Nebert, Winker and Gelboin, 1969; Pelkonen, Jouppila and K~irki, I972). Exposure to polycyclic aromatic hydrocarbons induces the enzyme aryl-hydrocarbon hydroxylase and it has been suggested that the hydroxyhtion of hydrocarbons by this enzyme, and by others, may lead to inhibition of placental oxidative enzyme systems (Longo, x98o). This is an attractive hypothesis and it may be that the abnormal infolding of the syncytial surface plasma membrane is due to a loss of membrane stability as a consequence of a deficiency of oxidative enzymes. A possible further indication of membrane damage is the unduly high levels of heat-stable alkaline phosphatase in women who smoke (Pirani and MacGillivray, i978), this enzyme being normally bound to the free plasma membrane. That the placenta suffers a degree of injury in cigarette smokers appears clear but the contribution of this damage to the poor fetal growth that is a frequent feature of pregnancies in smoking women is uncertain. Any ischaemic damage suffered by the villous syncytiotrophoblast appears to be readily repaired as a result of cytotrophoblastic cell proliferation and transformation into syncytiotrophoblast, and there is no evidence that in cigarette smokers this repair process is impaired or inefficient. Nevertheless, the damage, in some cases, to microvilli and the reduced pinocytotic activity in the syncytium suggest that there may be some impairment of transfer activity; if, indeed, alkaline phosphatase is also being lost from the syncytial plasma membrane as a result of membrane damage this would further decrease the syncytial transfer capacity in so far as this enzyme appears to play a vital, but as yet ill-defined, role in membrane transfer systems. It must be stressed, however, that syncytial damage in these placentae is very limited in extent and that most of the trophoblast appears to be fully viable. Hence any presumed loss of transfer capacity could well be compensated for by the increased placental growth which occurs in cigarette smokers. The fact that placental weights in smoking women are not significantly different from those in non-smoking women cannot be taken as indicating that smoking has no effect on placental growth. There is now considerable evidence that although the placenta has some capacity for autonomous growth the size of the placenta is largely controlled by fetal factors (Cefalo et ai, 1977) and that therefore a small baby has a small placenta, though nevertheless one that is fully capable of meeting the nutritional demands of the small fetus. The fact that in smokers fetal weight tends to be low whilst placental weight is normal indicates that there has in fact been a compensatory hyperplasia or hypertrophy of the

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placenta, a p h e n o m e n o n known to occur in other circumstances in which there is a chronic i m p a i r m e n t of oxygen supply to the placenta from the onset of gestation, for example pregnancy at high altitude, severe maternal anaemia (McCiung, 1969; Beischer et al, I97O). Placental function may be somewhat impaired in cigarette smokers but it is nevertheless probable that the ability of the placenta to repair ischaemic damage and to undergo a compensatory hyperplasia may well offset the functional effects of trophoblastic damage, the reduced fetal growth probably being partly due to an inadequate maternal supply of oxygen and partly to the direct effects of nicotine and carbon monoxide, or any of the m a n y other substances in cigarette smoke, on the fetus.

SUMMARY A histological and electronoptical study of placentae from women who smoke cigarettes d u r i n g pregnancy shows a tendency towards a paucity of vasculosyncytial membranes, villous cytotrophoblastic cell proliferation, focal syncytial necrosis, decreased syncytial pinocytotic and secretory activity, dilatation of syncytial rough endoplasmic reticulum, abnormalities of syncytial microvilli, focal infolding of the free plasma m e m b r a n e of the syncytiotrophoblast, degeneration of isolated cytotrophoblastic cells, irregular thickening of the trophoblastic basement m e m b r a n e and degeneration of villous capillary endothelial cells. It is thought that many of these changes are due to placental ischaemia consequent u p o n nicotine-induced constriction of the uterine vessels; some of the changes cannot be explained on this basis and it is suggested that these may possibly be due either to c a d m i u m toxicity or to accumulation of polycyclic aromatic hydrocarbons. T h e r e is apparently some i m p a i r m e n t of placental function in cigarette smokers b u t this is probably offset by the ability of the placenta to repair ischaemic damage and to undergo a compensatory hyperplasia.

ACKNOWLEDGEMENTS The authors wish to thank Miss S. Murphy and the nursing staff of the Central Delivery Unit, St Mary's Hospital, Manchester, for their cooperation in obtaining the specimens at the moment of delivery, Mrs. S. Walker for her technical expertise and Mrs L. Chawner for printing the electron micrographs. F.v.d.V was supported by the Stichting 'De Drie Lichten' and the Jan Dekker Stichting en Dr Ludgardine Bouwman Stichting.

REFERENCES

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