Tohoku

J. Exp.

Med., 1992,

167, 247-257

Quantitative Investigations Terminal Villi in Maternal by Scanning Microscopy

and

of Placental Diabetes Mellitus

Transmission

Electron

MASASHIHONDA,CHISATOTOYODA*,MASAO NAKABAYASHIt and YASUE OMORI Diabetes Center, *Department of Pathology and 'Maternal and Perinatal Center, Tokyo Women's Medical College, Tokyo 162

HONDA,M., TOYODA,C., NAKABAYASHII, M. and OMORI,Y. Quantitative Investigations of Placental Terminal Villi in Maternal Diabetes Mellitus by Scanning and Transmission Electron Microscopy. Tohoku J. Exp. Med., 1992, 167 (4), 247-257 The structure of the terminal villi was observed in placentae from non-diabetic mothers and mothers with diabetes mellitus using scanning and transmission electron microscopy. The metabolic condition of maternal diabetes was tightly controlled. In the diabetic group, the diameter of the terminal villi was significantly smaller than in the control group. The ramification pattern of villi, classified into hypo-, moderate- and hyper-ramifications, was shown to be mostly moderate in the non-diabetics whereas most of the diabetic placentae had either hypo- or hyper-ramifications. Mothers with a longer duration of diabetes and complicated with retinopathy tended to have hypo-ramification ; in these mothers, the weight of the neonates was significantly less than normal. Moreover, in the diabetic placentae, syncytial knots were found more frequently, the percentage of vasculo-syncytial membranes tended to be lower, and the trophoblastic basement membrane was significantly thicker than in the control. These abnormalities in the diabetic placentae were independent of the methods of delivery ; they seem to be related with fetal growth retardation and poor neonatal outcome, which are commonly seen in diabetic pregnancy. diabetic placenta ; electron microscopy ; quantitative investigation ; terminal villi

Neonatal complications and intrauterine death have been associated with placental insufficiencies. These are more common in diabetic than in nondiabetic pregnancies. In fact, the diabetic placenta has been the subject matter of a large number of light microscopicstudies (Burstein et al. 1957; Martin 1967; Fox 1969; Jacomo et al. 1976; Haust 1981; Bjork and Persson 1984). However, only a small number of electron microscopic studies (Okudaira et al. 1966; Liebhart 1974; Jones and Fox 1976) have been reported, and all of them lack quantitative analysis of microstructure, which may explain why they have not Received

April

22, 1992;

revision

accepted 247

for publication

August

7, 1992.

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M. Honda et al.

been successful in defining diabetic placenta.

the

morphological

abnormalities

specific

for the

In the present study, we quantitatively examined the changes of the terminal villi in diabetic placentae by scanning and also, partially, by transmission electron microscopy, and the findings were correlated retardation of the fetus and with the complications diabetic pregnancies.

with the intrauterine of neonates commonly

growth seen in

MATERIALS AND METHODS Twenty-one diabetic placentae were investigated. Of these, 15 were from subjects with non-insulin dependent diabetes mellitus (NIDDM) and 6 from those with insulin dependent diabetes mellitus (IDDM). In both groups, some had simple diabetic retinopathy. In four patients, in whom there had been no history of diabetes, the disease appears to have developed during pregnancy. Besides diabetes mellitus, no complications were observed during pregnancy. Maternal age ranged from 23 to 42 years (30.6±4.7: Mean±S.D.). Gestational age at delivery, calculated from the last menstrual period, was 38.7+0.8 weeks (Table 1). In patients of both groups, the postprandial blood glucose levels were less than 120 mg/100 ml throughout the pregnancy, HbAI was 8.0+0.9% and in the 3rd trimester, it was 7.8+0.8% with the normal range in our laboratory being 6.5-8.6%. All mothers gave birth to live newborns but with some complications : respiratory disturbance in 3 neonates, hyperbilirubinemia in 12, polycythemia in 3 and hypoglycemia

TABLE1. Clinical data of the diabetic subjects

Electron

Microscopy

of Diabetic

Placenta

249

in 12. Five infants were delivered by cesarean section. Other parameters defining the study population are presented in Table 1. In addition, normal placentae from 14 non-diabetic mothers were obtained to form a control group whose ages ranged from 24 to 35 years (28.8± 2.9), and the mean gestational period was 40.3+1.1 weeks. The mean neonatal weight was 3,285±591 g and the mean placental weight was 586.4± 94 g. All these mothers delivered live neonates vaginally and there were no perinatal complications. Immediately after a placenta was delivered, tissue slices about 3 X 3 mm and 3 mm thick were taken midway through the central cotyledon. These were fixed first in 2.0% glutaraldehyde in phosphate buffer (pH 7.2 ; 0.1 M), then in 2.0% osmic acid. After being dehydrated through an alcohol gradient and critical point-dried, the tissues were mounted with silver colloid and coated in vacuo with gold palladium. The coated samples were observed with a scanning electron microscope (HHS-2R ; Hitachi Co., Ltd., Tokyo). Tissues for transmission microscopy were first fixed in phosphate buffer with 2.0% glutaraldehyde, then in 0.1% osmic acid, dehydrated by passing through an alcohol gradient, embedded in epoxylysin and cut with an ultramicrotone (DIATOME, DIATOME Co., Ltd., Bienne, Switzerland). Ultrathin sections were double-stained with uranyl and lead salts then were observed with an electron microscope (H-12; Hitachi Co., Ltd., Tokyo). Terminal villi, i.e., the final branching of the villous tree, were photographed at a magnification of x 100 with a scanning electron microscope. In each case, one hundred terminal villi, which were crosssectioned, were selected, and their diameters were measured on photographs with vernier callipers. Photographs of trophoblastic basement membranes (TBM) were taken with a transmission electron microscope at x 10,000, and the thickness of TBM was measured at 30 places in each placenta using a magnifying glass with a scale. The syncytial nuclei are irregularly dispersed and often appear aggregated forming multinucleated protrusions on the villous surface ; these structures are known as "syncytial knots" (Fig. 1), and are thought to play no role in the functional activity of the syncytium (Fox 1978). The percentage of the terminal villi having syncytial knots from the total was calculated. In the terminal villi, the syncytium bulging over the dilated capillaries is anuclear and attenuated. Normally, on light microscopy, it appears fused with the vessel wall (Fig. 2)

Fig. 1.

Light

knotting

micrograph is indicated

of the terminal by an arrow.

villi X 100

of a normal

placenta.

Syncytial

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M. Honda

et al.

Fig. 2. Light micrograph of the terminal vasculo-syncytial membrane is indicated

villi of a normal placenta. by an arrow. x 400

The

forming the well-known "vasculo-syncytial membrane" (Getzowa and Sadowsky 1950). The percentage of terminal villi having vasculo-syncytial membranes (VSM) from the total was also calculated. Statistical analyses of the results were performed using Student's t-test and the x 2 test. RESULTS Scanning that

electron

the diameters

control

placentae.

Fig . 3. Scanning The terminal

micrographs

of the terminal The

patterns

of the normal villi

were

of terminal

and diabetic

smaller

in the

branching

electron micrograph of the terminal villi have moderate-ramifications.

villi x 100

placentae diabetic

of the

villous

of a normal

revealed

than tree

placenta.

in the were

Electron

Microscopy

of Diabetic

Placenta

251

classified according to Kaufman et al. (1987) into hypo- (Class I), moderate- (Class II) and hyper-ramification (Class III), where the hypo-, moderate- and, hyperramifications, were defined as terminal villi arising from the stem, intermediate and other terminal villi, respectively. In the control placentae, the ramifications were almost moderate (Fig. 3), but in most diabetic placentae, either hypo- (Fig. 4) or hyper- ramifications were dominant (Fig. 6 and Table 2). The villous ramifications of diabetic placentae from patients with IDDM were

Fig. 4.

Scanning

showing

TABLE 2.

electron

micrograph

hypo-ramifications.

of the terminal

vill i of a diabetic

placenta

X 100

Quantitation of the ultrastructural findings and diabetic placentae

in the terminal

villi of

normal

252

M. Honda

Fig. 5. Scanning electron micrograph sh owing moderate-ramifications.

TABLE 3.

Quantitative distribution of diabetes mellitus according to placentae

et al.

of the terminal x 100

selected the

class

villi of a d iabetic

placenta

variables found in patients of villous ramifications in

with their

Electron

Fig. 6.

Scanning

sh owing

electron

Microscopy

of Diabetic

microgr aph of the terminal

hyper-ramifications.

Placenta

villi

253

of a d iabetic

placenta

x 100

mostly Class III. Some of them were Class I but none were Class II. Class II ramifications were observed in villi from patients with NIDDM (Fig. 5). Placentae from subjects with diabetes of long duration and retinopathy showed mostly Class I ramifications. In addition, in the diabetic group, the weights of neonates and placentae from mothers with Class I villous ramifications were significantly smaller than those with Class II or III ramifications (Table 3). Both in the control and the diabetic group, the villous ramifications of placenta did not correlate with the gestational age at delivery or maternal age.

Fig

7. Transparent electron micrograph showing the membrane (arrow) of the terminal vill i in a normal

trophoblastric placenta.

basement X 10,000

M. Honda

254

Fig.

et al.

8. Transparent electron micrograph showing the thickening of the trophob lastic basement membrane (arrow) of the terminal abets villi in a c diabeti placenta. X 10,000

The percentage of terminal villi having syncytial knots was significantly increased in the diabetic placentae. There was no significant difference in the percentage of villi with VSM, though VSM tended to be fewer in diabetic placentae (Table 2). The thickness of trophoblastic basement membranes (TBM) was compared between the control and diabetic placentae (Figs. 7 and 8). The diabetic placental TBM were thicker than in the control (Table 2). Both in the control and diabetic groups, there was no correlation between the thickness of TBM and the gestational age at delivery or maternal age. No significant difference was found between placentae delivered by cesarean section and those by vaginal delivery, in the diameter of the terminal villi, villous ramifications, percentage of syncytial knots, VSM or TBM thickness.

DISCUSSION Normal fetal growth and development depend largely on placental function. Some forms of intrauterine fetal growth retardation have been correlated with poor placental blood flow and transfer of nutrients from mother to fetus (Lunell et al. 1979; Honda et al. 1990). Generally, placental villi are grouped into stem and terminal villi (Kaufmann et al. 1979). The terminal villi are the final branches of the villous tree through which the fetus establishes contact with the maternal circulation. The regional structural variations of the placenta could probably be evaluated in more accurate terms if they were subjected the morphometric evaluations (Wigglesworth 1969; Bjork and Persson 1984). Therefore, the same portion of the placenta is used for

Electron

Microscopy

of Diabetic

Placenta

255

each study. The diameter of normal terminal villi is 30-60 um (Aherne and Dunnill 1966; Kaufmann et al. 1979). We have found that in diabetic placentae the diameter of terminal villi is significantly smaller, despite tight metabolic control of the mothers' diabetes mellitus. A linear correlation was reported between the surface area of the placental villi and the fetal body weight (Aherne and Dunnill 1966).. The increased surface area of villi in diabetes was attributed to villous hyper-ramifications (Bjork and Persson 1984 ; Boyd et al. 1986 ) and was associated with variations in blood glucose levels (Madsen 1986). It was demonstrated in the present investigation that the villi are moderately branched in non-diabetic placentae, whereas in most diabetic, and especially in IDDM placentae, they are overly branched. Some hypo- and moderateramifications were also observed in diabetic placentae. However, there was no significant difference in the placental and neonatal weights between such placentae and those from diabetic mothers whose placentae had moderate- or hyperramifications. Similarly, there was no significant difference in placental and neonatal weights between the controls and diabetic subjects. These results appear likely to reflect the strict control of blood glucose levels made in our clinic. We suppose that between the diabetic placentae with moderateand those with hyper-ramifications, there may be no significant difference in the total surface area, but only the ramification patterns differ. Intrauterine fetal growth retardation and poor neonatal outcome are often found in diabetic pregnancies complicated by vascular disease (White 1974). We also observed that in patients with a longer history of diabetes and retinopathy, the terminal villi of placentae were less branched than in those with a shorter history, without retinopathy and with moderate or hyper-ramifications. Moreover, the weight of the placenta and that of the neonates in the former group were significantly smaller than in the latter. The reason why decreased fetal weight is related with villous hypo-ramifications is not known, but it could be because of a decreased placental transfer of oxygen and nutrients in diabetics as a consequence of placental hypoplasia and underdeveloped villous branching. The exchange of oxygen and carbon dioxide in the placenta depends on the volume of blood flow (Madsen 1986). We found that the percentage of syncytial knots is significantly elevated in the diabetic placenta. This may reduce the blood flow through villi to a certain degree, as assumed by Cantle et al. (1987). Fox (1967) reported an inverse relationship between the frequency of VSM and the incidence of neonatal asphyxia. Our results, though not significant, indicate a decreased VSM in diabetic pregnancy, providing a possible cause of fetal hypoxia. It has been reported that in the diabetic placentae, the terminal villi have more thickened TBM (Okudaira et al. 1966; Fox 1969; Jones and Fox 1976). Our measurements on transmission electron micrographs also showed a

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M. Honda et al.

significant thickening of TBM in diabetic placentae, thus confirming diffusion distance between the maternal and fetal blood.

an increased

In the absence of significant differences in any of the measured parameters between the diabetic placentae delivered by cesarean section and those by vaginal delivery,

we assume that the morphological

abnormalities

found

placenta are a direct result of diabetes itself. The quantitative data presented above provide information

in the diabetic on the structural

abnormalities of the diabetic placenta in much more accurate terms than previously. On this basis, we are looking forward to gaining more insight into the functional problems in materno-fetal transport. Acknowledgments We would like to thank Prof. W. Page Faulk of the Department of Pathology, Center for Reproduction and Transplantation Immunology, Methodist Hospital, Indianapolis, for his valuable suggestions ; Ms. L. Pancho for her assistance in writing the manuscript ; and Ms. S. Minei for experimental assistance and Ms. B. Levene for English correction. References 1) Aherne, W. & Dunnill, MS. (1966) Quantitative aspects of placental structure. J. Pathol. Bacteriol., 91, 123-139. 2) Bjork, 0. & Persson, B. (1984) Villous structure in different parts of the cotyledon in placentas of insulin-dependent diabetic women : A morphometric study. Acta Obstet. Gynecol.&and., 63, 37-43. 3) Boyd, D.A., Scott, A. & Keeling, G.W. (1986) Quantitative structural studies on placentas from pregnancies complicated by diabetes mellitus. Br. J. Obstet. Gynecol., 93, 31-35. 4) Burstein, R., Soule, S.D. & Blumenthal, H.T. (1957) Histogenesis of pathological processes in placentas of metabolic disease in pregnancy. II. The diabetic state. Am. J. Obstet. Gynecol.,74, 96-104. 5) Cantle, S.G., Kaufmann, P., Luckhardt, M. & Schweikhart, G. (1987) Interpretation of syncytial sprouts and syncytial bridges in the human placenta. Placenta, 8, 221234. 6) Fox, H. (1967) The incidence and significance of vasculosyncytial membranes in the human placenta. J. Obstet. Gynaecol.Br. Commonw.,74, 28-33. 7) Fox, H. (1969) Pathology of the placenta in maternal diabetes mellitus. Obstet. Gynecol.,34, 792-798. 8) Fox, H. (1978) Histology of the placental villi. In : Pathology of the Placenta, edited by H. Fox, W.B. Saunders Co., London-Philadelphia-Toronto pp. 13-21. 9) Getzowa, S., & Sadowsky, A. (1950) On the structure of the human placenta with full-term and immature foetus, living or dead. J. Obstet.Gynaecol.Br. Emp., 57, 388396. 10) Haust, M.O. (1981) Meternal diabetes mellitus-Effects on the fetus and placenta. Pathology, 22, 201-285. 11) Honda, M., Lowy, C. & Thomas, C.R. (1990) The effects of maternal diabetes on placental transfer of essential and non-essential fatty acids in the rat. Diabetes Res., 15, 47-51. 12) Jacomo, K.H., Benedetti, W.L., Sala, M.A. & Alvarez, H. (1976) Pathology of the trophoblast and fetal vessels of the placenta in maternal diabetes mellitus. Acta Diabetol. Lat. 13, 216-235.

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13) Jones, C.J.P & Fox, H. (1976) Placental changes in gestational diabetes. An ultrastructural study. Obstet. Gynecol.,48, 274-280. 14) Kaufmann, P., Sen, D.K. & Schweikhart, G. (1979) Classification of human placental villi. Cell Tissue Res., 200, 409-423. 15) Kaufmann, P., Luckhardt, M., Schweikhart, G. & Cantle, S.G. (1987) Cross-sectional features and three:dimensional structure of human placental villi. Placenta, 8, 235247. 16) Liebhart, M. (1974) Ultrastructure of the stromal connective tissue of normal placenta and of placenta in diabetes mellitus of mothers. Pathol. Eur., 9, 177-184. 17) Lunell, NO., Sarby, B., Lewander, R. & Nylund, L.E. (1979) Comparison of uteroplacental blood flow in normal and in intrauterine growth retarded pregnancy. Gynecol.Obstet. Invest., 10, 106-108. 18) Madsen, H. (1986) Fetal oxygenation in diabetic pregnancy. Dan. Med. Bull.,33, 64-74. 19) Martin, V. (1967) Plakopathia diabetica. Abnormalities of the placenta by maternal diabetes mellitus. VirchowsArch. Pathol. Anat., 343, 51-63. 20) Okudaira, Y., Hirota, K. & Cohen, S. (1966) Ultrastructure of the human placenta in maternal diabetes mellitus. Lab. Invest., 15, 910-926. 21) White, P. (1974) Diabetes mellitus in pregnancy. Clin. Perinatol., 1, 331-347. 22) Wigglesworth, J.S. (1969) Vascular anatomy of the human placenta and its significance for placental pathology. J. Obstet. Gynaecol.Br. Commonw.,76, 979-989.

Quantitative investigations of placental terminal villi in maternal diabetes mellitus by scanning and transmission electron microscopy.

The structure of the terminal villi was observed in placentae from non-diabetic mothers and mothers with diabetes mellitus using scanning and transmis...
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