Hydatidiform mole: An ultrastructural analysis of syncytiotrophoblast surface organization

Phi-mm (t989), IO, 19.5

212

Hydatidiform Mole: An Ultrastructural Analysis of Syncytiotrophoblast Surface Organization

COLIN OCKLEFORD”ld, CAROLINE BARKER”, JANET GRIFFITHS”, GEORGE McTURKb, ROSEMARY FISHER” & SYLVIA LAWLER’ *’Depurtment II[ Anatomy, The Medicul School and %The Scanning E. M. Unit, Unioersr~y qf‘Letcester, P.O. Bos 1.38, I%zit>ersi
INTRODUCTION Hydatidiform mole is an aberrant conceptus with a tumourogenic potential, occurring in about one in every I 500 pregnancies in Caucasians. Approximately IO per cent of patients with hydatidiform mole develop sequelae that require chemotherapeutic intervention. The tumours are either the malignant choriocarcinoma or the benign invasive mole. Variation of incidence of hydatidiform mole has been correlated with birth rate (Hsu, 1964), overpopulation (Poen and Djojopranoto, 1965), undernourishment (Acosta-Sison, 1959), geographical location (McCorriston, 1968), maternal age and prior abortion (Bandy, Clarke-Pearson and Hammond, 1984) and parental blood group combinations (Bagshawe et al, 1971). Hydatidiform moles have been divided pathologically into two entities (Szulman and Surti, t978a). Complete moles were defined as those having no fetus, total hydatidiform change from oedema to central cistern formation and conspicuous hyperplasia, while partial moles had a range of villi from normal to cystic, focal hyperplasia and evidence of the presence of a fetus (Vassilakos, Riotton and Kajii, 1977). Genetically the complete hydatidiform mole is androgenetic in origin (Kajii and Ohama, 1977; Jacobs et al, 1980). If complete hydatidiform moles are derived by duplication of a haploid sperm they have a homozygous genome (Lawler et al, 1979, r982b). They are heterozygous if they arise by dispermy (Ohama et al, 1981). Partial moles are triploid (Szulman and Surti; r978b), have a maternal contribution to the genome and usually arise by dispermy (Jacobs et al, 1982; Lawler et al, r982a). Macroscopic identification of swollen villi is not a sufficient criterion to identify complete or partial hydatidiform mole because hydropically degenerate villi have long been recognized as a common feature of abortuses. The crucial diagnostic distinction is an histological one (Ockleford et al, 1983). It hinges on whether there is trophoblastic hyperplasia in the tissue or not. It 0143~4004/89/020195 + 18$os.oo/o

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has been the aim of several investigators histologically to type hydatidiform moles in the hope of being able to select a group at risk of requiring further treatment for choriocarcinoma or persistent trophoblastic disease (Hertig and Sheldon, 1947; Ringertz, 1970) but Elston (1978) has reviewed the conclusions and found none of the systems fully predictive of eventual clinical outcome. The possibility remains however that some ultrastructural features of the molar trophoblast may be of importance (Ferenczy and Richart, 1972; Ockleford, 1980) either in indicating more clearly the basis of the pathogenesis of trophoblastic tumours or in correlating more effectively with clinical diagnosis. This paper therefore is a record of an ultrastructural study. We have employed secondary electron detecting scanning electron microscopy performed in the usual manner and made use of a special specimen stage situated closer to the cathode than is conventional in an attempt to gain higher resolution than in many previous studies. Novel structures are described and discussed in relation to the organization of the tissue and mechanisms of transformation and cell physiology.

MATERIALS

AND METHODS

Tissue collection and fixation Specimens of hydatidiform mole being used in a genetic study were obtained from the Royal Marsden Hospital, London as part of a study of the genetic origin of molar pregnancies (Lawler et al, r98za,b). They included 8 partial moles ranging from 10.5 to 27 weeks menstrual age and 23 complete hydatidiform moles ranging from 8-21 weeks menstrual age. Genetic studies showed that 3 of the complete hydatidiform moles were dispermic in origin and 20 were most likely to be homozygous, arising by fertilization of an anucleate egg by a single sperm which subsequently duplicated. One of the patients with dispermic complete hydatidiform moles and 5 of the patients with homozygous complete moles required chemotherapy after evaluation of the mole. Specimens (12) of healthy 1st trimester trophoblast were obtained from the Royal Infirmary and the General Hospital at Leicester for purposes of comparison. Sixteen of the hydatidiform mole specimens were stored frozen in 0.C.T (Miles Laboratories) under liquid nitrogen at - 196°C. The 0.C.T was completely removed after thawing and prior to fixation. All tissue was fixed in 3 per cent formalin or 3 per cent glutaraldehyde in Sterilin vials and was stored at 4°C. Examination of placental tissue using light microscopy Portions of tissue approximately 1.5 x 1.5 cm were examined under fixative using the Zeiss Tessovar light microscope. The volume of fixative in the Petri dish was adjusted to minimize stray reflected light in the image. Images were recorded using Kodak Panatomic-X black and white film. Macrographs of the general appearance of mole and healthy placental samples of equivalent gestational age were taken at magnifications ranging from (I x to 5 X ). The film was developed using Acutol developer and the images were printed on Ilford multigrade photographic paper using a Durst 35 mm enlarger. Tissue preparation for scanning electron microscopy (a) Post Fixation. Hydatidiform mole tissue and normal tissue specimens were dissected into

Orkleford et al: HydattdtformMole

pieces no larger than 5 mm across. The tissue was then post fixed in ide for I hour at 4C.

197 2

per cent osmium tetrox-

(6) LMzydration. Post fixed placental tissue was washed 3 times with distilled water, then dehydrated with ethanol in covered containers as follows. (i) 50 per cent ethanol for 15 minutes; (ii) 70 per cent ethanol for 15 minutes; (iii) 90 per cent ethanol for 15 minutes; (iv) I oo per cent ethanol for I 5 minutes; (v) two changes of IOO per cent ethanol dried over sodium sulphate, each for 15 minutes; (vi) 100 per cent acetone for 30 minutes; (vii) a change into fresh IOO per cent acetone. In this state the tissue was stored for critical point drying. (c) Critical

Point Drying. Dehydrated placental tissue was transferred to an open ‘boat’ containing IOO per cent acetone, within a Polaron critical point dryer. The acetone was replaced with liquid CO, and samples were dried out at a pressure of 1200 p.s.i. and a temperature of 3t.s°C (Anderson, 1950, 1951).

(d) Surface Coating. Dried placental tissue specimens were mounted on aluminium stubs with the aid of double-sided Scotch tape. Specimens were coated with an electrically conducting layer of gold using a Polaron Es150 sputter coater. Coating thickness was estimated at between IO and 30 nm on the basis of periodic measurements with a quartz crystal film thickness monitor (Polaron). The sputter coating process was repeated if specimen charging was noted. The mounted specimens were stored in an air-tight, sealed dessicator containing silica gel.

Examination of tissue using scanning electron microscopy Both normal and molar specimens of placental tissue were examined in an IS1 60 scanning electron microscope at an accelerating voltage of 30 kV. Specimens were further examined in an IS1 DS130 scanning electron microscope at accelerating voltages of 15 and 9 kV when higher resolution was required. The tissue was examined at various magnifications, the greatest being approximately 120000 x . A few exceptionally well preserved and thinly coated specimens held the promise of potential analysis at higher resolution. These were examined using the top stage of the IS1 DS130 scanning electron microscope.

Photography Kodak Technical Pan or Panatomic-X film were used for image recording and developed using Acutol developer. Prints were made on Ilford multigrade photographic paper using a Durst 35 mm enlarger.

Procedures for transmission electron microscopy The techniques used for transmission electron microscopy have been fully described in a previous publication (Ockleford, Wakely and Badley, 1981).

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RESULTS Examination at low power using the Zeiss Tessovar microscope and the standard resolution stage of the scanning electron microscope has revealed the relatively large-scale differences in size between the chorionic villi in the non-pathological sample set and the hydatidiform mole samples. Only 3 of the 18 mole samples contained some chorionic villi less than 0.5 mm in diameter, whilst none of the control group contained any chorionic villi with a diameter greater than 0.5 mm (Figures I and 2). The ultrastructural analysis of the surface has revealed several interesting features. These will be considered individually. Ridging of the surface One of the most consistent differences between the control and pathological tissue was that the depth and extent of ridging of the surface was much greater in the hydatidiform moles (Figures 3 and 4). These frequently formed circumferential or more complicated patterns (Figure 5). The ridges are approximately 5-10 ,um in height and vary considerably in breadth with a minimum width of the order of IO pm. Outgrowths A striking feature of the outgrowths of the hydatidiform molar surfaces was the frequency of small structures the overall size of sprouts or small primary chorionic villi which had a flattened or paddle shape (Figures 6 and 7). This shape is reminiscent of the flattened shape of intestinal villi and raises the question whether there is metaplasia occuring. These paddle shaped outgrowths also occur to a lesser extent in the normal tissue samples, but in the latter circumstances villus outgrowths are far more commonly disc shaped in transverse section. Microvillous surface Both normal tissue and molar tissue were covered with areas of microvilli. Fields of unbranched microvilli of simple shape, uniform length and upright posture were relatively uncommon in both groups (Figure 8). Patches or areas of smooth (non-microvillous) surface on the other hand were common in both groups (Figure 9). These areas were of particular interest because they permitted the better examination of a number of invaginated regions of the surface (vide infra). Microgibbosities, described earlier by Ockleford and Clode (1983) were identified in 1/3 of the pathological samples but in none of the healthy villi (Figure IO). Many areas of healthy and molar tissue revealed a complex surface where fine elements of the approximate thickness of microvilli were fused into a meshwork. Microvilli with swollen tips were encountered amongst some hydatidiform mole samples (Figure I I). Repair processes Repair processes including the laying down of fibrous (fibrin?) material over the surface of the syncytiotrophoblast (Figure 12) and apparent epithelial repair (Figure 13) were noted in a small number of healthy and pathological samples. Structures observed at higher resolution Using the top-stage, rather more detail than has been previously reported was observed in healthy and molar tissue sur&ces. For example the surfiiGes of the microvillar and intermicrovillar membrane are clearly not smooth. Structures visual&d as small mounds cover a

Ock.&ford et al: Hydatidijbrm Mole

Figiure I. Tessovar macrograph

of a sample of healthy tst trimester human placental tissue showing the fine chow villi and branching of the chorionic villus tree. x m.

Fig, we 2. Tessovar macrograph of a complete homozygous ally swollen, resembling a bunch of grapes. x 5.

hydatidiform

mole specimen. The chorionic villi are I:ypic-

Placenta (1989), Vol. 10

Figure 3. Scanning electron micrograph

of a portion of the chorionic villus tree of healthy 1st trimester placenta.

Figure t 4. Scanning electron micrograph of the ridged trophobiastic e&helium hetero zygous molar trophoblast specimen. x 75.

of an expanded chorionic villus

x 97.

an

Oikle/ord et al: ffydatidiform Mole

ZOI

F~gurry. Scanning electron micrograph of the surface morphology of a swollen chorionic villus of a complete (homozygous) hydatidiform mole specimen shown at relatively low magnification. Note the deep ridges in the trophoblastic cpitheiium.

x 91.

Figure 6(A). Scanning electron micrograph of paddle-shaped trophoblastic sprouts extending outwards from a healthy 1st trimester chorionic villus. x 298. (B) Scanning electron micrograph of a syncytial sprout from a healthy 1st trimester trophoblast sample. x I zzq.

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7. A portion of the surface of a partial (heterozygous) *the trophoblastic surface. x 131.

Figurer 8. A scanning electron micrograph mole. x II 880.

mole showing numerous sprouts developing from a ridge :d

of a relatively undysplastic

area of microvilli From a complete hydatidifor m

Or.kit$ord et al: ffydaridrform Mole

9. Area of syncytial trophoblast from a portion of hydatidiform lich contained openings of a variety of sizes. x 576.

203

mole which was smooth in overall appe .an,ce

Figure IO. A number of microgibbosities (Ockleford and Clode, 1983) are visible on the surface of this complec te which was dispermic in origin and which eventually required treatment for persistent trophoblastic disease. 7 featw 2s and a small sprout (s) are raised above the surrounding surface level. x a 331.

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Figure II. A high magnification scanning electron micrograph of the microvillar surface of a complete (homozyg ous) hydatb diform mole. The structure of these cytoplasmic projections is that of typical microvilli but with bulbous (inset) x 5 319 (inset x 17 793).

Figure ~.a. A scanning electron micrograph showing the individual fibres of the mesh-like network applied to the microv ,illar surface of a complete hydatidiform mole at higher magnification. Note the highly folded surface of the cell (a) atta iched to the interwoven tibres. x 5 464.

Orklefvrd et al: Hydatzdiform Mole

FtgU’e 13. Scanning electron micrograph (dispe rmic) hydatidiform mole. x 690.

205

of a cluster of cells filling a break in the syncytiotrophoblast

of this partial

Figure 14. Scanning electron micrograph of the syncytial cell surface of a complete hydatidiform mole at high mlagnification, showing the structure of microvilli and openings in the intermicrovillous spaces. At this resolution the ‘G tveolar collars ' (arrowheads) can be clearly identified surrounding the apertures of the size of micropinocytic invagin ations. x 118890.

Placenta (1989), Vol. 10

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substantial proportion of the microvillar surface. These are between 30 and IOO nm in diameter (Figures 14 and 15). A number of openings in the surface have been detected. Some of the larger ones ( > 750 nm diameter) appear to be the entrances to quite deep channels. The smallest are of the correct order of size to represent the openings of micropinocytic caveolae (Figure 16). The most striking feature of these smaller apertures is that they subtend a distinct collar composed of moundlike structures which are packed together surrounding the aperture and are raised above the general level of the surrounding surface. The small particles or mounds in these ‘caveolar collars’ have diameters in the range I* 80 nm. Ultracircumvallate features Within the healthy placental samples, on areas of surface that were otherwise smooth or nodular, relatively large structures (up to about IO pm diameter) were observed. These consisted of circular raised ridges in the intermicrovillar membrane surrounding a central depression (Figure 17). Because of their superficial similarity in appearance to the aberrant macroscopic form-the circumvallate placenta-we have found it convenient to describe these structures as ultracircumvallate. Reticulate surface organization Certain regions of rather nodular surface showed a most interesting surface organization. A clearly defined reticular pattern of strands of material 2-4 pm in breadth is raised above the surrounding cell surface (Figure 18). The polygonal enclosures bounded by these strands vary in size. The smallest being 2-3 pm and the largest being about 20 pm across their longest dimension. The structures are distinct from the ultracircumvallate areas of surface in being surrounded by much thinner raised boundary ridges which are polygonal and not annular in outline. Transmission electron microscopy Uitrathin transverse sections of the molar syncytial cell surface reveal the range of cytoskeletal structures already described in the healthy placenta as part of the syncytioskeled layer (Ockleford, Wakely and Badley, 1981). These include microfilaments and microtubules (Figure 19). The orientations of the microtubules lie mainly with their long axes within the plane underlying the plane of the intermicrovillar membrane.

DISCUSSION Higher resolution structure One of the most positive features of this study is the description of new structures in the large molecular to supramolecular size range. It is well known that the surf& of a transporting epithelium such as the syncytiotrophoblast bears a heterogeneous collection of sites. These include receptors, bound ligand, surface antigens, the exposed parts of transmembrane proteins comprising amongst other things ion transporting channels and transmembrane iinkage molecules. The richly heterogeneous population of mounds revealed on the surface cot&I r&t the presence of such structures. The possibility that these features were coating artefacts was considered and rejected, for similar detail was not seen on the surface of the specimen mount which was coated at the same time and because the distribution of particles could be correlated in an apparently consistent way with the presence of other structures.

rq~e

‘7. Detail of Figure 14. The microvilli shown exhibit a cobbled cell surface organization.

x 45 04.5

F&e 16. The portion of maternal oriented syncytiotrophoblast surface of a complete hydatidiform mole contained rounded subunits which were raised up to form caveolar collars (#). These surround the rim of structures the size of coated pits. x IOI 796.

Placenta (19X9), Vol. ro

Figure 17. A region of syncytiotrophoblast circum wallate’ appearance. x z 796.

Figure 18. An area of the syncytiotrophoblast

reticul ate formation.

x * j*o.

from a healthy first trimester sample bearing structures

(*) with an ‘uIltra-

of a mole specimen that was covered in a series of ridges that made

w a

F’I~UW ‘9. Transmission

superficial cytoplasm.

electron micrograph showing a coated pit (cp) microtubules x 66 oao (inset X 83 300).

the most obvious

Citing

caveolac,

cess of internalization Ockleford,

from

forming

If this interpretation

hinderance transmission

(Willingham

of particles electron

and Pastan,

tron microscopy (Willingham

surrounding

was observed.

of receptor-l&and

1984) to enter

endocytosis. mutual

example,

a collar of particles

a bolus

and Pastan,

the apertures

The ‘caveolar

complexes

coated

during

is apt the geometry

micrographs

the process

of these

Lateral uptake

of label is frequently

as the openings

of receptor

images

implies

views of fairly mature

show a flask shaped

1980) and in labelled

interpreted

collar’ may reflect the dynamic

of pro-

at least some of which are known (Fine and

vesicles

at the entrance.

(s) and an endosome (e) in the

studies

seen resting

1980). This type of distribution

structure

mediated

that there

caveolae

with a narrow

monitored

neck

by transmission

on the outer

is not surprising

surface

is

derived elec-

of the lip

when the crowded

nature of the collar zone is appreciated. The reasons previous Burton,

why this additional

scanning

electron

1987) are probably

the top stage are significant

detail has been discovered

microscope technical.

studies The control

improvements

(e.g.

in the present

Clint,

Wakely

of coating

thickness

and

study and not in Ockleford,

1979;

and the application

of

to the method.

Hyperplasia Finding scanning electron microscopic evidence of extreme ridging in 17, and microgibbosities or similar structures in 12 of the 31 molar specimens where no similar structures were found in the 12 healthy

chorionic

crucial in diagnosis

villus samples,

of hydatidiform

may reflect

the hyperplasia

which

is histologically

mole.

The overgrowth of surface structures in these and other fashions recorded here is probably at least in part dependent on overgrowth of cortical polymeric and other structural proteins (Edwards and Booth, 1987) that form support for the tissue and the microvilli (Ockleford,

l’lircmtu (I&),

210

1 ‘01.10

Wakely and Badley, 1981). This is because, although the surface area of the chorionic villus is vastly increased, the syncytioskeletal (cortical) layer of structural proteins in not obviously thinner and the surface itself is not smoothed as it would be were the growth achieved by stretching the surface. This is an important conclusion because in the process of transformation to tumour status, errors in the organizational control of the cytoskeleton are frequently observed, and there is thought to be loss of the usual genetic repression of growth controlling genes (Burridge, 1986; Ben-Ze’ve, 1985). There are a variety of ways in which the imbalance in the inheritance pattern of parental genes known to occur in this condition could lead to the loss of normal control and the reduction or loss of the suppression of the activity of structural protein genes. These observations confirm the current view that the development of swollen villi is not a result simply of interruption of fluid loss by the tissue owing to fetal death, but that it is a trophoblast growth control abnormality (Park, 1967). We can extend this view by suggesting that it probably affects at least some of the polymeric structural proteins of the cortical layer of the syncytiotrophoblast. Ultrastructural

pathology

In analysing these specimens it was clearly possible to distinguish between molar trophoblast and healthy tissue using the scanning ultrastructural images generated. The more subtle distinctions between complete and partial moles and the groups of samples from patients, divided on the basis of the need for further treatment, were not apparent ultrastructurally (using as indicators the prevalence of any of the surface features described above, or a combination thereof). The relatively small samples (in macroscopic terms) of the molar tissue (usually < 6 vesicles) may have contributed to this negative conclusion because focal morphological changes are a feature of partial moles. Reticulate surface architecture The remarkable appearance of the surface where the reticulate architectures were observed (Figure 18) may be of interest with regard to the cytoskeletal organization of vlli. In a previous study (Ockleford, Wakely and Badley, 1981) it has been shown that the cortical layer of the syncytiotrophoblast is richly endowed with the polymeric structural proteins actin and tubulin. Transmission electron micrographs of this layer demonstrated microtubules and microfilaments. In this study the ultrathin sections of molar tissue demonstrate the presence of similar structures (Figure 19). In both healthy and molar trophoblast the microtubules appear to be concentrated near the syncytiotrophoblast maternal oriented surface. The immunofluorescence data from healthy chorionic villi (Ockleford, Wakely and Badley, 1981) indicate that microtubules take up a more open pattern than the microfilaments and that in places the microtubules are organized in a reticular fashion. The dimensions of the immunofluorescence patterns and the raised polygonal ridges seen here in scanning electron micrographs are similar and the two sets of images may therefore reflect the same underlying structures. The fact that the reticulate structure is seen in relief on scanning electron micrographs can be explained by differential shrinkage of the syncytial cytoplasm during dehydration (Boyde et al, 1977) drawing down the surface to reveal the pattern of the underlying cortical cytoskeleton.

SUMMARY The scanning ultrastructural

examination

of a series of 31 hydatidiform

mole and 12 healthy

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MO/~

?I,

placental specimens of similar gestational age has revealed a variety of surface architectures more common in molar tissue. Characteristic paddle-shaped sprouts, ridging of the syncytial maternal oriented surface and microgibbosities are described. These structures are explicable in terms of organellar hyperplasia of cortical cytoskeletal elements found in healthy tissue. Specific morphological evidence of involvement of these elements in a condition where aberrant growth control leads to the characteristic trophoblastic hyperplasia is a further indication that cytoskeletal elements may mediate transformation. An increase in resolution obtained over previous scanning electron microscope studies has allowed the description of detailed features such as ‘caveolar collars’ on the maternal oriented healthy and molar trophoblast surfaces. These observations are of relevance to understanding the mechanisms of several cell physiological processes, including transepithelial transport. Yea observations of a reticular organization in the surface layer of molar trophoblast indicatc that a syncytioskeletal layer, with organization resembling that previously described in healthy chorionic villi, is also present in molar villi.

ACKNOWLEDGEMENTS We thank Professor J. MacVicar and the obstetricians and gynaccologists of the Royal Infirmary and the General Hospital I.&ester for clinical coordination; Ian Indans and Chris d’lacey for skilled technical assistance and for help with manuscript preparation.

REFERENCES

Acosta-Sison, H.

(1959) Observations which may indicate the etiology of hydatidiform mole and explain its high incidence in the Phillipines and asiatic countries. Phillipine~ournal ofsurgery, 14,209~297. Anderson, T. F. (1950) The use of the critical point phenomena in preparing specimens for the electron microscope.

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Anderson, T. F. (1951) Techniques for the preservation of 3-dimensional structure in preparing specimens for the electron microscope. Transactrons of the New York Academy of Sciences II, 13, 13*134. Bagshawe, K. D., Rawlins, G., Pike, M. C. & Lawler, S. D. (1971) ABO blood-groups in trophoblastic neoplasia. Lancer, I, 553-557.

Bandy, L. C., Clarke-Pearson, D. L. & Hammond, C. B. (1984) Malignant potential of gestational trophoblastic disease at the extreme ages of reproductive life. Obstetrics and Gynecology, 64(3X 395-399. Ben-Ze’ev, A. (1985) The cytoskeleton in cancer cells. Biochemira Bzophysica Arta, 780,305-341. Boyd, A., Bailey, E., Jones, S. J. & Tamarin, A. (1977) Dimensional changes during specimen preparation for scanning electron microscopy. In Scanning electron microscopy Vol. I. (Ed.) Johari, 0. M. pp. 507-518. Chicago, Illinois: Proceedings of the Workshop on Biological Specimen Preparation Techniques, IIT Research Institutes. Burridge, K. (1986) Substrate adhesions in normal and transformed fibroblasts: Organization and regulation of cytoskeletal, membrane and extracellular matrix components at focal contacts. Cancer Review, 4, 18-78. Burton, G. J. (1987) The fine structure of the human placental villus as revealed by scanning electron microscopy. Scannrflg Microscopy, l(4), 1811~r828. Clint, J. M., Wakely, J & Ockleford, C. D. (1979) Differentiated regions of human placental cell surface associated with attachment of chorionic villi, phagocytosis of maternal erythrocytes and syncytiotrophoblast repair. Proceedtngz of the Royal Soctety, London, Sews B, zo4,345-353.

Echlin, P. (1975) Sputter-coating techniques for scanning electron microscopy. In Scanning electron mrrroscop,y. (Eds) Johari, O.M. & Corvin, I. pp. 217~224. Chicago, Illinois: Proceedings of the 8th. annual SEM Symposium, IIT Research Institute. Edwards, H. C. & Booth, A. G. (1987) Calcium sensitive, lipid binding cytoskeletal probes of the human placental microvillar region.3ournal of Cell Biology, 1og,303~312. Elston, C. W. (1978) Trophoblastic tumours of the placenta. In Pathology of the Placenta. (Ed.) Fox, H. Vol. VII. of major problems in Pathology. pp. 368-425. London: W. B. Saunders Co. Ltd. Elston, C. W. SCBagshawe, K. D. (1972) The value of histological grading in the management of hydatidiform mole. 3ournal of Obstetrics and Gynaecology of the British Commonwealth, 71,717-724,

Ferenczy, A & Richart, R. M. (1972) Scanning electron microscopic study of normal and molar trophoblast. cologr1.Oncolog)l, 1,95--I IO.

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