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Effect of cigarette smoke on human placenta morphometry
International Journal of Gynecology & Obstetrics 62 Ž1998. 237]242
Article
Effect of cigarette smoke on human placenta morphometry J.E.S. Rocha, M. MatheusU , M.A. Sala Department of Gynecology and Obstetrics, Uni¨ ersity of Sao ˜ Paulo, Ribeirao ˜ Preto, Brazil Received 14 January 1998; received in revised form 29 April 1998; accepted 8 May 1998
Abstract Objecti¨ e: To evaluate the placental alterations in smoking mothers. Method: A case-control stereologic study of 10 placentas from smokers and 10 from non-smokers. Result: The authors found a significant increase in stromal volume density, villus size and trophoblast thickness, and a significant decrease in villus and vessel surface densities, total surfaces, and numberrmm2 histological field in the smokers’ placenta. Conclusions: The alterations found in the smokers’ placenta greatly contribute to impairing the exchanges between maternal and fetal blood. Q 1998 International Federation of Gynecology and Obstetrics. Keywords: Placenta; Morphometry; Cigarette smoke
1. Introduction The adverse effects of cigarette smoke on pregnancy are well documented. In 1935, Sontag and Wallace w1x demonstrated the occurrence of a defined increase of fetal heart beat when the mother smoked a cigarette. Simpson w2x reported a twofold higher frequency of low weight newborns and increased number of preterm deliveries among mothers who smoked during pregnancy. A direct relationship between the number of cigarettes smoked per day by the mother during U
Corresponding author.
pregnancy and the decrease of newborn weight was also demonstrated. The probable cause of the intrauterine growth retardation of fetuses from smoking mothers is the fetal hypoxia andror the ischemia due to the reduction of the uteroplacental blood flow. It was demonstrated that the act of smoking a single cigarette causes an acute reduction of the placental intervillous blood flow lasting approx. 15 min w3x. Underperfusion of the intervillous space decreases the oxygenation of fetal blood, which in turn acts as a vasoconstrictor on the chorial vessels, increasing the vascular resistance of the placenta w4x. The reduction of the intervillous blood flow
0020-7292r98r$19.00 Q 1998 International Federation of Gynecology and Obstetrics PII S0020-7292Ž98.00107-6
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J.E.S. Rocha et al. r International Journal of Gynecology & Obstetrics 62 (1998) 237]242
could be also responsible for morphologic and functional alterations of the smoker placenta. Nevertheless, studies on the effect of maternal smoking on the placenta show contradictory results. Placentas from smoking pregnant show no increased incidence of necrosis or other anomalies, but have microscopic lesions reflecting a periodic low perfusion, such as obliterative endarteritis, slight cytotrophoblast hyperplasia, stromal fibrosis, and small villous infarcts w5x. According to Van der Veen and Fox w6x, the most frequent lesions in smoker placenta are degeneration of the cytotrophoblast, cytotrophoblast hyperplasia, focal syncytial necrosis, reduced number of vasculo-syncytial membranes, irregular thickness of the basement membrane, and increased collagen content in the villous stroma. In contrast, placentas from women who stopped smoking only a few weeks before conception were similar to those from non-smokers w7x. Thus, it is difficult to attribute the placental alterations to any chronic damage of the uterine vessels in the smoking mother. There is a consensus that the most characteristic microscopic alterations found in smoker placentas are thickened trophoblast basement membrane, reduced lumen and lower number of fetal vessels w6,8]10x. On the other hand, morphometric studies of smokers’ placentas are still scanty. These studies can solve some of the remaining doubts on the morphologic alterations of the placenta that probably are responsible for the retarded intrauterine growth of fetuses from smoking women. Thus, the objective of the present study was to evaluate the placental alterations in smoking mothers by stereologic methods. 2. Material and methods Twenty placentas were examined in this study. Ten placentas were selected from healthy primigravid women who smoked 20 cigarettes per day, at least 2 years before and during pregnancy. The number of cigarettes smoked was stated by the patient and confirmed by her partner. Each placenta of the smoking group was assigned a control placenta from a healthy primigravid patient
who had never smoked. The control matched each case in terms of race, marital and socioeconomic status, age, preconceptional weight Ž"3 kg., weight gain during pregnancy Ž"1 kg., height Ž"5 cm., and gestational age and sex of the newborn ŽTable 1.. All mothers were under 33 years of age and had no history of alcohol or drug abuse. All pregnancies ended in spontaneous vaginal births between the 37th and the 42nd week at the University Hospital of Ribeirao ˜ Preto city Žmean altitude: 518 m above sea level.. Length of gestation was assessed from the first day of the last menstrual period and confirmed by ultrasound measurement of the fetal biparietal diameter during early pregnancy. All the infants were live-born and had no detectable congenital malformations. The project had the approval of the hospital Ethics Committee. The placentas were collected at birth, the umbilical cord being clamped 1]2 min after delivery of the baby, and immediately transferred to the laboratory. The weight and volume of each placenta were obtained after the membranes were trimmed to the edge of the disc and the cord cut to within 2 cm of its insertion. Two cotyledons, midway between the center and the periphery of the placental disc, free from any major pathology, were isolated. The volume of each cotyledon was determined before and after fixation in 10% neutral buffered formalin. After fixation, two blocks of tissue including material from the basal plate to the chorial plate, were randomly taken from each cotyledon. After paraffin embedding, 6-m mthick histological sections were cut and stained by Table 1 Patient characteristics Parameter
Non-smokers
Smokers
z
P
Age Žyears. Weight Žkg. Weight gain Žkg. Height Žcm. Gestational age Žweeks.
19 Ž3. 53 Ž4. 10.5 Ž2. 162 Ž4. 39.5 Ž1.5.
19 Ž3. 51 Ž3. 11 Ž4. 160 Ž5. 39.5 Ž1.5.
1.42 1.42 0.49 1.42 0.00
n.s. n.s. n.s. n.s. n.s.
Values represent the medians and interquartile ranges Žbetween parentheses ..
J.E.S. Rocha et al. r International Journal of Gynecology & Obstetrics 62 (1998) 237]242
the PAS-hematoxylin method. A correction factor for the conversion of processed into fresh volume was calculated for each placenta. Histopathological examination of coded sections of the two placentas from each pair was performed by one observer ŽM.A.S.. blinded about maternal smoking habits. The characteristics of the trophoblastic epithelium and its basement membrane, cytotrophoblastic cells and vasculosyncytial membranes, as well as those of the villous connective tissue and capillaries, were semiquantitatively evaluated w8x. Stereological estimates of the relative and absolute volumes of intervillous space, fibrinoid, chorionic villi, trophoblast layer, stroma and fetal vessels were performed by the point-counting method using a 100 point test-grid w11x. In a similar fashion, the surface densities Žsurface area per mm3 of placenta. and total surface areas of both chorial villi and fetal vessels were estimated by the point-intersection counting method w11x. The vessel surface]villus surface area ratio w12x and the villous vascularization index w13x were also determined. The diameters of both villi and vessels were estimated, as well as the arithmetic mean thickness of the trophoblast layer w14x. The stereological methods employed as well as the respective equations are shown in Table 2. Because the frequency distributions of values were not normal, median and interquartile ranges were used. Statistical analysis was performed by the non-parametric Wilcoxon test for related samples.
239
3. Results The macroscopic characteristics of the placentas showed no significant differences between smokers and non-smokers ŽTable 3.. Histological examination of the placentas from the smoker group showed a slightly thicker trophoblast layer, of regular thickness, with scanty vasculo-syncytial membranes. In some placentas there was a slightly increased frequency of cytotrophoblast cells. Two placentas from this group revealed a slightly thickened trophoblast basement membrane. The villous stroma showed less numerous centrally arranged capillaries, and in eight placentas they appeared to be smaller. The connective tissue was increased, with hyperplasia and fibrosis. In two placentas the stroma was edematous, showing frequent Hofbauer cells. Stereological evaluation of the relative volumes of the different placental structures only showed a significant difference for the connective tissue ŽTable 4.. The absolute volumes of the different placental structures showed no differences between smoking and non-smoking groups ŽTable 5.. The values corresponding to surface densities, diameter of villi and of villus vessels as well as the mean trophoblast thickness are presented in Table 6. The total surface of both villous and placental vessels as well as the number of villirmm2 and the number of vesselsrmm 2 were significantly reduced in the placentas from smoking mothers ŽTable 7..
Table 2 Sterological methods used to evaluate the placentas Parameter
Equation
Reference
Relative volume Surface density Vessel surface]villus surface ratio Vascularization index Diameter Mean thickness
ParPt 2 IarLt IcrI¨ PcrP¨ Pa ? zrIa Ž P¨ ? zr2 I¨ . ? w1 y Ž PerP¨ .1r2 x
w11x w11x w12x w13x w14x w14x
Pa , points falling on structure a; Pc , points falling on villous capillaries; P¨ , points falling on villi; Pe , points falling on villous axis; Pt , total number of points; Ia , intersections on the surface of structure a; Ic , intersections on the surface of capillaries; I¨ , intersections on the surface of villi; z, length between two successive points; L t , total length of the test lines.
J.E.S. Rocha et al. r International Journal of Gynecology & Obstetrics 62 (1998) 237]242
240
Table 3 Placental characteristics Parameter
Table 5 Stereological parameters of the placentas
Non-smokers
Weight Žg. 394 Ž40. Volume Žcm3 . 384 Ž22. Placental coefficient 0.121 Ž0.016. Longest diameter 19.0 Ž1.6. Žcm. Shortest diameter 17.2 Ž1.1. Žcm.
Smokers
z
P
Parameter
Non-smokers
Smokers
z
P
416 Ž55. 401 Ž50. 0.142 Ž0.024. 19.0 Ž1.5.
0.36 0.87 1.27 0.30
n.s. n.s. n.s. n.s.
Intervillous space Fibrinoid Villi Trophoblast Villous axis Connective tissue Vessels
199.3 Ž190.2. 1.1 Ž0.8. 176.4 Ž16.2. 52.9 Ž3.4. 121.0 Ž9.6. 89.3 Ž6.8. 31.9 Ž7.5.
198.7 Ž35.1. 1.4 Ž1.1. 196.6 Ž50.0. 50.6 Ž12.4. 131.6 Ž39.1. 104.5 Ž29.2. 30.9 Ž9.5.
0.05 1.07 1.12 0.15 1.02 1.38 0.76
n.s. n.s. n.s. n.s. n.s. n.s. n.s.
16.8 Ž1.8.
0.76 n.s.
Values represent the medians and interquartile ranges Žbetween parentheses ..
4. Discussion Although some authors found significant alterations of weight in the smoker placenta w6,15]19x, in the present series no significant difference was demonstrated between smoker and non-smoker groups for weight, volume or diameters of the placenta. The ratio between placental weight and newborn weight Žplacental coefficient. was higher in the smoking women w15,16,19,20x, probably revealing an adaptation of the placenta in order to increase the oxygen supply to the fetus. In agreement with Teasdale and Ghislaine w17x and Van der Velde et al. w18x, the present study demonstrated no significant difference in the placental coefficient of smoking mothers. Table 4 Stereological parameters of the placentas Parameter
Non-smokers
Smokers
z
P
Intervillous space Fibrinoid Villi Trophoblast Villous axis Connective tissue Vessels Vascular index
53.1 Ž2.5.
51.4 Ž3.2.
0.97
n.s.
0.3 Ž0.2. 46.4 Ž2.8. 14.2 Ž0.8. 33.2 Ž2.8. 24.1 Ž1.7.
0.5 Ž0.2. 46.8 Ž3.2. 13.9 Ž2.1. 33.4 Ž4.6. 26.4 Ž3.5.
1.18 1.01 0.61 0.77 1.78
n.s. n.s. n.s. n.s. - 0.05
0.97 1.38
n.s. n.s.
8.7 Ž2.0. 0.182 Ž0.042.
7.4 Ž1.8. 0.150 Ž0.026.
Medians and interquartile ranges Žbetween parentheses . of the relative volumes Ž%. of different structures of the placentas.
Medians and interquartile ranges Žbetween parentheses . of the absolute volumes Žcm3 . of the different structures of the placentas.
Gross examination of the smokers’ placentas revealed no morphologic alterations. In the present series the rare presence of infarcts in the smokers’ placentas w21x was confirmed: whereas four control placentas showed infarcts, only one placenta from the smoker group did. Histological examination of the placentas from smoking mothers revealed a slightly thickened trophoblast layer, with rare vasculo-syncytial membranes. In some placentas, there was a slightly increased number of cytotrophoblast cells. In two placentas a slightly thickened basement membrane was observed. This alteration may be the consequence of cytotrophoblast proliferation due to hypoxia w6x. The morphometric study showed that the trophoblast thickness was significantly increased in the placentas from smoking mothers, as suggested by histological examination. Both retarded thinning of the trophoblastic epithelium and a decrease of the frequency of vasculo-syncytial membranes could be due to the inhibition of apoptosis by nicotine w22x. The villous stroma showed less numerous capillaries, centrally arranged, and generally smaller. The stroma was hyperplastic and fibrotic. The significant increase in relative volume of connective tissue in the smoker placentas confirmed the histological observations. It was suggested that the increase of connective tissue was due to the higher content of collagen fibers. Although Van der Veen and Fox w6x and Teasdale and Ghislaine w17x reported a decreased volume of the capillaries, in the present study no alteration was observed
J.E.S. Rocha et al. r International Journal of Gynecology & Obstetrics 62 (1998) 237]242
241
Table 6 Stereological parameters of the placentas Parameter
Non-smokers
Smokers
z
P
Villus surface density Capillary surface density Mean villus diameter Mean capillary diameter Trophoblast thickness
321.5 Ž41.6. 280.5 Ž56.5. 59.4 Ž9.0. 13.3 Ž1.6. 4.7 Ž0.9.
253.1 Ž33.8. 207.6 Ž29.3. 69.7 Ž14.4. 14.1 Ž2.8. 5.5 Ž0.5.
2.29 2.29 2.14 0.71 1.78
- 0.05 - 0.05 - 0.05 n.s. - 0.05
Medians and interquartile ranges Žbetween parentheses . of the surface densities Žcm2 rcm3 . and diameters Ž m m. of both villi and capillaries and thickness of the trophoblast layer Ž m m..
in the relative volume of the villous vessels. According to Burton et al. w7x, the reduced volume of the capillaries reported by those authors was due to the number of cigarettes smoked Ž21]60. and to the method of fixation of the placenta, causing partial collapse of the vessels. Mean villus diameter was significantly increased in the placentas from smoking mothers, whereas the mean diameter of villous vessels showed no significant alterations. Asmussen w8x, in an ultrastructural study, claimed that the villous capillaries are reduced in size. The surface densities of both villi and vessels showed a significant reduction in the placentas from the smoking group. Teasdale and Ghislaine w17x showed a significant reduction of the villous surface in the placenta from smoking mothers, with no changes in capillary surface density. The total surface of villi as well as the total surface of vessels was significantly decreased in the placentas from smoking mothers. Furthermore, the mean number of villi and vessels per mm2 of microscopic field showed a significant reduction in the smokers’ placentas. The changes
detected in the smokers’ placenta, such as increased villus diameter and trophoblast thickness, reduction of total number of terminal villi and vessels, diminution of the surface density of villi, a decrease of the total surface of villi and vessels, diminution of the frequency of vasculo-syncytial membranes, sparse centrally arranged vessels, increased fibrosis andror raised cellularity of villi, are definitely related to a reduction of maternal]fetal exchanges, particularly those employing the simple diffusion mechanism. In this manner, the capacity of the placenta as a respiratory organ is reduced in smoking mothers, and consequently their fetuses can be exposed to a chronic hypoxic condition. Burton et al. w7x demonstrated that placentas of mothers who stopped smoking only a few weeks before conception were similar to those of nonsmokers. Thus, it is difficult to attribute the placental alterations to any chronic uterine vascular damage. It was proposed that cadmium, which rapidly concentrates in the smoker’s placenta, could be responsible for vascular and trophoblastic alterations. However, experimental studies do
Table 7 Stereological parameters of the placentas Parameter
Non-smokers
Smokers
z
P
Villus surface Vessel surface Vessel surfacervillus surface Vesselsrvillus Villirmm2 Vesselsrmm2
11.82 Ž1.42. 10.31 Ž2.04. 0.84 Ž0.13. 3.7 Ž0.7. 190.2 Ž49.8. 725.4 Ž171.4.
10.38 Ž1.95. 8.93 Ž1.74. 0.80 Ž0.12. 4.5 Ž0.8. 108.0 Ž39.0. 451.4 Ž91.0.
1.89 1.99 0.87 0.56 2.40 2.40
- 0.05 - 0.05 n.s. n.s. - 0.01 - 0.01
Medians and interquartile ranges Žbetween parentheses . of the total surface Žm2 . of both villi and vessels, ratio of vessel surface to villus surface, number of vesselsrvillus, number of villirmm2 of microscopic field and number of vesselsrmm 2 of microscopic field.
242
J.E.S. Rocha et al. r International Journal of Gynecology & Obstetrics 62 (1998) 237]242
not support this suggestion w23x. Daily injections of small doses of cadmium administered to pregnant rats did not cause alterations in morphometric features Žvolume densities of trophoblast, lacunae, fetal capillaries, connective tissue and trophoblast thickness . or electron microscopic features Žamount and localization of lipid, degenerative vesicles, thickness of the trophoblast and endothelial layer and thickening of the basement membrane. w23x. The intervillous space flow is decreased when the mother smokes, and this alteration lasts 15 min after the end of a smoking episode w4x. According to our results, it is possible to conclude that smoking, by reducing blood flow in the intervillous space, induces morphofunctional alterations in the placenta such as cytotrophoblast hyperplasia, thickening of the trophoblastic barrier and reduction of the exchange surfaces, interfering with the gas exchanges between mother and fetus and reducing the intrauterine growth of the conceptus. Acknowledgements This work was supported by a grant from the CNPq ŽConselho Nacional de Desenvolvimento Cientıfico } Proc. 420011-94.3. ´ e Tecnologico ´ References w1x Sontag LW, Wallace RF. The effect of cigarette smoking during pregnancy upon the fetal heart rate. Am J Obstet Gynecol 1935;29:77]83. w2x Simpson WJ. A preliminary report on cigarette smoking and the incidence of prematurity. Am J Obstet Gynecol 1957;73:808]815. w3x Morrow RJ, Knox Ritchie JW, Bull SB. Maternal cigarette smoking: the effects on umbilical and uterine blood flow velocity. Am J Obstet Gynecol 1988;159: 1069]1071. w4x Lehtovirta P, Forss M. The acute effect of smoking on intervillous blood flow of the placenta. Br J Obstet Gynaecol 1978;85:729]731. w5x Naeye RL. Effect of maternal cigarette smoking on the fetus and placenta. Br J Obstet Gynaecol 1978;85: 732]737. w6x Van der Veen E, Fox H. The effects of cigarette smoking on the human placenta. A light and electron microscopic study. Placenta 1982;3:243]256.
w7x Burton GF, Palmer ME, Dalton KJ. Morphometric differences between the placental vasculature of nonsmokers, smokers and ex-smokers. Br J Obstet Gynaecol 1989;96:907]915. w8x Asmussen I. Ultrastructure of the villi and fetal capillaries in placentas from smoking and non-smoking mothers. Br J Obstet Gynaecol 1980;87:239]245. w9x Jauniaux E, Burton GJ. The effect of smoking in pregnancy on early placental morphology. Obstet Gynecol 1992;79:645]648. w10x Mochizuki M, Marvo T, Masuku K, Ohtsu T. Effects of smoking on fetoplacental]maternal system during pregnancy. Am J Obstet Gynecol 1984;149:413]420. w11x Weibel ER. Stereological methods, vol. 2. Theoretical Foundations. London: Academic Press, 1980. w12x Sala MA. Topographic differences in the morphology of the cotyledon of normal human term placenta. PhD Thesis. Ribeirao ˜ Preto, 1981. w13x Bender HG. Placental insufficiency. Morphometric researches on the model of Rhesus placenta. Arch Gynakol ¨ 1974;216:289]295. w14x Sala MA, Matheus M, Valeri V. A new stereological method for estimating the thickness of a cellular layer on random section. Mikroskopie 1981;38:127]130. w15x Kullander S, Kallen ¨ ´ B. A prospective study of smoking and pregnancy. Acta Obstet Gynecol Scand 1972;50: 83]94. w16x Mulcahy K, Murphy JF, Martin F. Placental changes and maternal weight in smoking mothers. Am J Obstet Gynecol 1970;106:703]704. w17x Teasdale F, Ghislaine JJ. Morphological changes in the placentas of smoking mothers: A histomorphometric study. Biol Neonate 1989;55:251]259. w18x Van der Velde WJ, Copius Peereboom-Stegeman JHJ, Treffers PE, James J. Structural changes in the placenta of smoking mothers: a quantitative study. Placenta 1983;4:231]240. w19x Wingerd J, Christianson R, Lovitt WV, Schoen EJ. Placental ration in white and black women: relation to smoking and anemia. Am J Obstet Gynecol 1976;124: 671]675. w20x Van der Salm P, Ubachs HM, Van Wersch JW. Cord blood clotting factors in neonates of smoking and nonsmoking mothers. Int J Clin Lab Res 1994;94:177]179. w21x Christianson RE. Gross differences observed in the placentas of smokers and non-smokers. Am J Epidemiol 1979;110:178]187. w22x Wright SC, Zhong J, Zheng H, Larrick JW. Nicotine inhibition of apoptosis suggests a role in tumor promotion. FASEB J 1993;11:1045]1051. w23x Hazelhoff Roelfzema W, Zahn-Breidenbach U, Copius Peereboom-Stegeman JH. Light and electron microscopic investigation of the rat placenta after cadmium administration during pregnancy. Anat Embryol 1988;178:345]351.
Article
Effect of cigarette smoke on human placenta morphometry J.E.S. Rocha, M. MatheusU , M.A. Sala Department of Gynecology and Obstetrics, Uni¨ ersity of Sao ˜ Paulo, Ribeirao ˜ Preto, Brazil Received 14 January 1998; received in revised form 29 April 1998; accepted 8 May 1998
Abstract Objecti¨ e: To evaluate the placental alterations in smoking mothers. Method: A case-control stereologic study of 10 placentas from smokers and 10 from non-smokers. Result: The authors found a significant increase in stromal volume density, villus size and trophoblast thickness, and a significant decrease in villus and vessel surface densities, total surfaces, and numberrmm2 histological field in the smokers’ placenta. Conclusions: The alterations found in the smokers’ placenta greatly contribute to impairing the exchanges between maternal and fetal blood. Q 1998 International Federation of Gynecology and Obstetrics. Keywords: Placenta; Morphometry; Cigarette smoke
1. Introduction The adverse effects of cigarette smoke on pregnancy are well documented. In 1935, Sontag and Wallace w1x demonstrated the occurrence of a defined increase of fetal heart beat when the mother smoked a cigarette. Simpson w2x reported a twofold higher frequency of low weight newborns and increased number of preterm deliveries among mothers who smoked during pregnancy. A direct relationship between the number of cigarettes smoked per day by the mother during U
Corresponding author.
pregnancy and the decrease of newborn weight was also demonstrated. The probable cause of the intrauterine growth retardation of fetuses from smoking mothers is the fetal hypoxia andror the ischemia due to the reduction of the uteroplacental blood flow. It was demonstrated that the act of smoking a single cigarette causes an acute reduction of the placental intervillous blood flow lasting approx. 15 min w3x. Underperfusion of the intervillous space decreases the oxygenation of fetal blood, which in turn acts as a vasoconstrictor on the chorial vessels, increasing the vascular resistance of the placenta w4x. The reduction of the intervillous blood flow
0020-7292r98r$19.00 Q 1998 International Federation of Gynecology and Obstetrics PII S0020-7292Ž98.00107-6
238
J.E.S. Rocha et al. r International Journal of Gynecology & Obstetrics 62 (1998) 237]242
could be also responsible for morphologic and functional alterations of the smoker placenta. Nevertheless, studies on the effect of maternal smoking on the placenta show contradictory results. Placentas from smoking pregnant show no increased incidence of necrosis or other anomalies, but have microscopic lesions reflecting a periodic low perfusion, such as obliterative endarteritis, slight cytotrophoblast hyperplasia, stromal fibrosis, and small villous infarcts w5x. According to Van der Veen and Fox w6x, the most frequent lesions in smoker placenta are degeneration of the cytotrophoblast, cytotrophoblast hyperplasia, focal syncytial necrosis, reduced number of vasculo-syncytial membranes, irregular thickness of the basement membrane, and increased collagen content in the villous stroma. In contrast, placentas from women who stopped smoking only a few weeks before conception were similar to those from non-smokers w7x. Thus, it is difficult to attribute the placental alterations to any chronic damage of the uterine vessels in the smoking mother. There is a consensus that the most characteristic microscopic alterations found in smoker placentas are thickened trophoblast basement membrane, reduced lumen and lower number of fetal vessels w6,8]10x. On the other hand, morphometric studies of smokers’ placentas are still scanty. These studies can solve some of the remaining doubts on the morphologic alterations of the placenta that probably are responsible for the retarded intrauterine growth of fetuses from smoking women. Thus, the objective of the present study was to evaluate the placental alterations in smoking mothers by stereologic methods. 2. Material and methods Twenty placentas were examined in this study. Ten placentas were selected from healthy primigravid women who smoked 20 cigarettes per day, at least 2 years before and during pregnancy. The number of cigarettes smoked was stated by the patient and confirmed by her partner. Each placenta of the smoking group was assigned a control placenta from a healthy primigravid patient
who had never smoked. The control matched each case in terms of race, marital and socioeconomic status, age, preconceptional weight Ž"3 kg., weight gain during pregnancy Ž"1 kg., height Ž"5 cm., and gestational age and sex of the newborn ŽTable 1.. All mothers were under 33 years of age and had no history of alcohol or drug abuse. All pregnancies ended in spontaneous vaginal births between the 37th and the 42nd week at the University Hospital of Ribeirao ˜ Preto city Žmean altitude: 518 m above sea level.. Length of gestation was assessed from the first day of the last menstrual period and confirmed by ultrasound measurement of the fetal biparietal diameter during early pregnancy. All the infants were live-born and had no detectable congenital malformations. The project had the approval of the hospital Ethics Committee. The placentas were collected at birth, the umbilical cord being clamped 1]2 min after delivery of the baby, and immediately transferred to the laboratory. The weight and volume of each placenta were obtained after the membranes were trimmed to the edge of the disc and the cord cut to within 2 cm of its insertion. Two cotyledons, midway between the center and the periphery of the placental disc, free from any major pathology, were isolated. The volume of each cotyledon was determined before and after fixation in 10% neutral buffered formalin. After fixation, two blocks of tissue including material from the basal plate to the chorial plate, were randomly taken from each cotyledon. After paraffin embedding, 6-m mthick histological sections were cut and stained by Table 1 Patient characteristics Parameter
Non-smokers
Smokers
z
P
Age Žyears. Weight Žkg. Weight gain Žkg. Height Žcm. Gestational age Žweeks.
19 Ž3. 53 Ž4. 10.5 Ž2. 162 Ž4. 39.5 Ž1.5.
19 Ž3. 51 Ž3. 11 Ž4. 160 Ž5. 39.5 Ž1.5.
1.42 1.42 0.49 1.42 0.00
n.s. n.s. n.s. n.s. n.s.
Values represent the medians and interquartile ranges Žbetween parentheses ..
J.E.S. Rocha et al. r International Journal of Gynecology & Obstetrics 62 (1998) 237]242
the PAS-hematoxylin method. A correction factor for the conversion of processed into fresh volume was calculated for each placenta. Histopathological examination of coded sections of the two placentas from each pair was performed by one observer ŽM.A.S.. blinded about maternal smoking habits. The characteristics of the trophoblastic epithelium and its basement membrane, cytotrophoblastic cells and vasculosyncytial membranes, as well as those of the villous connective tissue and capillaries, were semiquantitatively evaluated w8x. Stereological estimates of the relative and absolute volumes of intervillous space, fibrinoid, chorionic villi, trophoblast layer, stroma and fetal vessels were performed by the point-counting method using a 100 point test-grid w11x. In a similar fashion, the surface densities Žsurface area per mm3 of placenta. and total surface areas of both chorial villi and fetal vessels were estimated by the point-intersection counting method w11x. The vessel surface]villus surface area ratio w12x and the villous vascularization index w13x were also determined. The diameters of both villi and vessels were estimated, as well as the arithmetic mean thickness of the trophoblast layer w14x. The stereological methods employed as well as the respective equations are shown in Table 2. Because the frequency distributions of values were not normal, median and interquartile ranges were used. Statistical analysis was performed by the non-parametric Wilcoxon test for related samples.
239
3. Results The macroscopic characteristics of the placentas showed no significant differences between smokers and non-smokers ŽTable 3.. Histological examination of the placentas from the smoker group showed a slightly thicker trophoblast layer, of regular thickness, with scanty vasculo-syncytial membranes. In some placentas there was a slightly increased frequency of cytotrophoblast cells. Two placentas from this group revealed a slightly thickened trophoblast basement membrane. The villous stroma showed less numerous centrally arranged capillaries, and in eight placentas they appeared to be smaller. The connective tissue was increased, with hyperplasia and fibrosis. In two placentas the stroma was edematous, showing frequent Hofbauer cells. Stereological evaluation of the relative volumes of the different placental structures only showed a significant difference for the connective tissue ŽTable 4.. The absolute volumes of the different placental structures showed no differences between smoking and non-smoking groups ŽTable 5.. The values corresponding to surface densities, diameter of villi and of villus vessels as well as the mean trophoblast thickness are presented in Table 6. The total surface of both villous and placental vessels as well as the number of villirmm2 and the number of vesselsrmm 2 were significantly reduced in the placentas from smoking mothers ŽTable 7..
Table 2 Sterological methods used to evaluate the placentas Parameter
Equation
Reference
Relative volume Surface density Vessel surface]villus surface ratio Vascularization index Diameter Mean thickness
ParPt 2 IarLt IcrI¨ PcrP¨ Pa ? zrIa Ž P¨ ? zr2 I¨ . ? w1 y Ž PerP¨ .1r2 x
w11x w11x w12x w13x w14x w14x
Pa , points falling on structure a; Pc , points falling on villous capillaries; P¨ , points falling on villi; Pe , points falling on villous axis; Pt , total number of points; Ia , intersections on the surface of structure a; Ic , intersections on the surface of capillaries; I¨ , intersections on the surface of villi; z, length between two successive points; L t , total length of the test lines.
J.E.S. Rocha et al. r International Journal of Gynecology & Obstetrics 62 (1998) 237]242
240
Table 3 Placental characteristics Parameter
Table 5 Stereological parameters of the placentas
Non-smokers
Weight Žg. 394 Ž40. Volume Žcm3 . 384 Ž22. Placental coefficient 0.121 Ž0.016. Longest diameter 19.0 Ž1.6. Žcm. Shortest diameter 17.2 Ž1.1. Žcm.
Smokers
z
P
Parameter
Non-smokers
Smokers
z
P
416 Ž55. 401 Ž50. 0.142 Ž0.024. 19.0 Ž1.5.
0.36 0.87 1.27 0.30
n.s. n.s. n.s. n.s.
Intervillous space Fibrinoid Villi Trophoblast Villous axis Connective tissue Vessels
199.3 Ž190.2. 1.1 Ž0.8. 176.4 Ž16.2. 52.9 Ž3.4. 121.0 Ž9.6. 89.3 Ž6.8. 31.9 Ž7.5.
198.7 Ž35.1. 1.4 Ž1.1. 196.6 Ž50.0. 50.6 Ž12.4. 131.6 Ž39.1. 104.5 Ž29.2. 30.9 Ž9.5.
0.05 1.07 1.12 0.15 1.02 1.38 0.76
n.s. n.s. n.s. n.s. n.s. n.s. n.s.
16.8 Ž1.8.
0.76 n.s.
Values represent the medians and interquartile ranges Žbetween parentheses ..
4. Discussion Although some authors found significant alterations of weight in the smoker placenta w6,15]19x, in the present series no significant difference was demonstrated between smoker and non-smoker groups for weight, volume or diameters of the placenta. The ratio between placental weight and newborn weight Žplacental coefficient. was higher in the smoking women w15,16,19,20x, probably revealing an adaptation of the placenta in order to increase the oxygen supply to the fetus. In agreement with Teasdale and Ghislaine w17x and Van der Velde et al. w18x, the present study demonstrated no significant difference in the placental coefficient of smoking mothers. Table 4 Stereological parameters of the placentas Parameter
Non-smokers
Smokers
z
P
Intervillous space Fibrinoid Villi Trophoblast Villous axis Connective tissue Vessels Vascular index
53.1 Ž2.5.
51.4 Ž3.2.
0.97
n.s.
0.3 Ž0.2. 46.4 Ž2.8. 14.2 Ž0.8. 33.2 Ž2.8. 24.1 Ž1.7.
0.5 Ž0.2. 46.8 Ž3.2. 13.9 Ž2.1. 33.4 Ž4.6. 26.4 Ž3.5.
1.18 1.01 0.61 0.77 1.78
n.s. n.s. n.s. n.s. - 0.05
0.97 1.38
n.s. n.s.
8.7 Ž2.0. 0.182 Ž0.042.
7.4 Ž1.8. 0.150 Ž0.026.
Medians and interquartile ranges Žbetween parentheses . of the relative volumes Ž%. of different structures of the placentas.
Medians and interquartile ranges Žbetween parentheses . of the absolute volumes Žcm3 . of the different structures of the placentas.
Gross examination of the smokers’ placentas revealed no morphologic alterations. In the present series the rare presence of infarcts in the smokers’ placentas w21x was confirmed: whereas four control placentas showed infarcts, only one placenta from the smoker group did. Histological examination of the placentas from smoking mothers revealed a slightly thickened trophoblast layer, with rare vasculo-syncytial membranes. In some placentas, there was a slightly increased number of cytotrophoblast cells. In two placentas a slightly thickened basement membrane was observed. This alteration may be the consequence of cytotrophoblast proliferation due to hypoxia w6x. The morphometric study showed that the trophoblast thickness was significantly increased in the placentas from smoking mothers, as suggested by histological examination. Both retarded thinning of the trophoblastic epithelium and a decrease of the frequency of vasculo-syncytial membranes could be due to the inhibition of apoptosis by nicotine w22x. The villous stroma showed less numerous capillaries, centrally arranged, and generally smaller. The stroma was hyperplastic and fibrotic. The significant increase in relative volume of connective tissue in the smoker placentas confirmed the histological observations. It was suggested that the increase of connective tissue was due to the higher content of collagen fibers. Although Van der Veen and Fox w6x and Teasdale and Ghislaine w17x reported a decreased volume of the capillaries, in the present study no alteration was observed
J.E.S. Rocha et al. r International Journal of Gynecology & Obstetrics 62 (1998) 237]242
241
Table 6 Stereological parameters of the placentas Parameter
Non-smokers
Smokers
z
P
Villus surface density Capillary surface density Mean villus diameter Mean capillary diameter Trophoblast thickness
321.5 Ž41.6. 280.5 Ž56.5. 59.4 Ž9.0. 13.3 Ž1.6. 4.7 Ž0.9.
253.1 Ž33.8. 207.6 Ž29.3. 69.7 Ž14.4. 14.1 Ž2.8. 5.5 Ž0.5.
2.29 2.29 2.14 0.71 1.78
- 0.05 - 0.05 - 0.05 n.s. - 0.05
Medians and interquartile ranges Žbetween parentheses . of the surface densities Žcm2 rcm3 . and diameters Ž m m. of both villi and capillaries and thickness of the trophoblast layer Ž m m..
in the relative volume of the villous vessels. According to Burton et al. w7x, the reduced volume of the capillaries reported by those authors was due to the number of cigarettes smoked Ž21]60. and to the method of fixation of the placenta, causing partial collapse of the vessels. Mean villus diameter was significantly increased in the placentas from smoking mothers, whereas the mean diameter of villous vessels showed no significant alterations. Asmussen w8x, in an ultrastructural study, claimed that the villous capillaries are reduced in size. The surface densities of both villi and vessels showed a significant reduction in the placentas from the smoking group. Teasdale and Ghislaine w17x showed a significant reduction of the villous surface in the placenta from smoking mothers, with no changes in capillary surface density. The total surface of villi as well as the total surface of vessels was significantly decreased in the placentas from smoking mothers. Furthermore, the mean number of villi and vessels per mm2 of microscopic field showed a significant reduction in the smokers’ placentas. The changes
detected in the smokers’ placenta, such as increased villus diameter and trophoblast thickness, reduction of total number of terminal villi and vessels, diminution of the surface density of villi, a decrease of the total surface of villi and vessels, diminution of the frequency of vasculo-syncytial membranes, sparse centrally arranged vessels, increased fibrosis andror raised cellularity of villi, are definitely related to a reduction of maternal]fetal exchanges, particularly those employing the simple diffusion mechanism. In this manner, the capacity of the placenta as a respiratory organ is reduced in smoking mothers, and consequently their fetuses can be exposed to a chronic hypoxic condition. Burton et al. w7x demonstrated that placentas of mothers who stopped smoking only a few weeks before conception were similar to those of nonsmokers. Thus, it is difficult to attribute the placental alterations to any chronic uterine vascular damage. It was proposed that cadmium, which rapidly concentrates in the smoker’s placenta, could be responsible for vascular and trophoblastic alterations. However, experimental studies do
Table 7 Stereological parameters of the placentas Parameter
Non-smokers
Smokers
z
P
Villus surface Vessel surface Vessel surfacervillus surface Vesselsrvillus Villirmm2 Vesselsrmm2
11.82 Ž1.42. 10.31 Ž2.04. 0.84 Ž0.13. 3.7 Ž0.7. 190.2 Ž49.8. 725.4 Ž171.4.
10.38 Ž1.95. 8.93 Ž1.74. 0.80 Ž0.12. 4.5 Ž0.8. 108.0 Ž39.0. 451.4 Ž91.0.
1.89 1.99 0.87 0.56 2.40 2.40
- 0.05 - 0.05 n.s. n.s. - 0.01 - 0.01
Medians and interquartile ranges Žbetween parentheses . of the total surface Žm2 . of both villi and vessels, ratio of vessel surface to villus surface, number of vesselsrvillus, number of villirmm2 of microscopic field and number of vesselsrmm 2 of microscopic field.
242
J.E.S. Rocha et al. r International Journal of Gynecology & Obstetrics 62 (1998) 237]242
not support this suggestion w23x. Daily injections of small doses of cadmium administered to pregnant rats did not cause alterations in morphometric features Žvolume densities of trophoblast, lacunae, fetal capillaries, connective tissue and trophoblast thickness . or electron microscopic features Žamount and localization of lipid, degenerative vesicles, thickness of the trophoblast and endothelial layer and thickening of the basement membrane. w23x. The intervillous space flow is decreased when the mother smokes, and this alteration lasts 15 min after the end of a smoking episode w4x. According to our results, it is possible to conclude that smoking, by reducing blood flow in the intervillous space, induces morphofunctional alterations in the placenta such as cytotrophoblast hyperplasia, thickening of the trophoblastic barrier and reduction of the exchange surfaces, interfering with the gas exchanges between mother and fetus and reducing the intrauterine growth of the conceptus. Acknowledgements This work was supported by a grant from the CNPq ŽConselho Nacional de Desenvolvimento Cientıfico } Proc. 420011-94.3. ´ e Tecnologico ´ References w1x Sontag LW, Wallace RF. The effect of cigarette smoking during pregnancy upon the fetal heart rate. Am J Obstet Gynecol 1935;29:77]83. w2x Simpson WJ. A preliminary report on cigarette smoking and the incidence of prematurity. Am J Obstet Gynecol 1957;73:808]815. w3x Morrow RJ, Knox Ritchie JW, Bull SB. Maternal cigarette smoking: the effects on umbilical and uterine blood flow velocity. Am J Obstet Gynecol 1988;159: 1069]1071. w4x Lehtovirta P, Forss M. The acute effect of smoking on intervillous blood flow of the placenta. Br J Obstet Gynaecol 1978;85:729]731. w5x Naeye RL. Effect of maternal cigarette smoking on the fetus and placenta. Br J Obstet Gynaecol 1978;85: 732]737. w6x Van der Veen E, Fox H. The effects of cigarette smoking on the human placenta. A light and electron microscopic study. Placenta 1982;3:243]256.
w7x Burton GF, Palmer ME, Dalton KJ. Morphometric differences between the placental vasculature of nonsmokers, smokers and ex-smokers. Br J Obstet Gynaecol 1989;96:907]915. w8x Asmussen I. Ultrastructure of the villi and fetal capillaries in placentas from smoking and non-smoking mothers. Br J Obstet Gynaecol 1980;87:239]245. w9x Jauniaux E, Burton GJ. The effect of smoking in pregnancy on early placental morphology. Obstet Gynecol 1992;79:645]648. w10x Mochizuki M, Marvo T, Masuku K, Ohtsu T. Effects of smoking on fetoplacental]maternal system during pregnancy. Am J Obstet Gynecol 1984;149:413]420. w11x Weibel ER. Stereological methods, vol. 2. Theoretical Foundations. London: Academic Press, 1980. w12x Sala MA. Topographic differences in the morphology of the cotyledon of normal human term placenta. PhD Thesis. Ribeirao ˜ Preto, 1981. w13x Bender HG. Placental insufficiency. Morphometric researches on the model of Rhesus placenta. Arch Gynakol ¨ 1974;216:289]295. w14x Sala MA, Matheus M, Valeri V. A new stereological method for estimating the thickness of a cellular layer on random section. Mikroskopie 1981;38:127]130. w15x Kullander S, Kallen ¨ ´ B. A prospective study of smoking and pregnancy. Acta Obstet Gynecol Scand 1972;50: 83]94. w16x Mulcahy K, Murphy JF, Martin F. Placental changes and maternal weight in smoking mothers. Am J Obstet Gynecol 1970;106:703]704. w17x Teasdale F, Ghislaine JJ. Morphological changes in the placentas of smoking mothers: A histomorphometric study. Biol Neonate 1989;55:251]259. w18x Van der Velde WJ, Copius Peereboom-Stegeman JHJ, Treffers PE, James J. Structural changes in the placenta of smoking mothers: a quantitative study. Placenta 1983;4:231]240. w19x Wingerd J, Christianson R, Lovitt WV, Schoen EJ. Placental ration in white and black women: relation to smoking and anemia. Am J Obstet Gynecol 1976;124: 671]675. w20x Van der Salm P, Ubachs HM, Van Wersch JW. Cord blood clotting factors in neonates of smoking and nonsmoking mothers. Int J Clin Lab Res 1994;94:177]179. w21x Christianson RE. Gross differences observed in the placentas of smokers and non-smokers. Am J Epidemiol 1979;110:178]187. w22x Wright SC, Zhong J, Zheng H, Larrick JW. Nicotine inhibition of apoptosis suggests a role in tumor promotion. FASEB J 1993;11:1045]1051. w23x Hazelhoff Roelfzema W, Zahn-Breidenbach U, Copius Peereboom-Stegeman JH. Light and electron microscopic investigation of the rat placenta after cadmium administration during pregnancy. Anat Embryol 1988;178:345]351.