Morphologic studies of the ruptured amnion

Morphologic studies of the ruptured amnion RALPH M. WYNN, M.D. PETERS. SEVER, B.A. LOUIS M. HELLMAN, M.D. Brooklyn, New York

An operative technique of experimental rupture of the fetal membranes in the guinea pig was devised to permit continuation of the pregnancy and subsequent histologic examination for evidence of regeneration. The definitive amnion after premature rupture may regenerate morphologically normal tissue. Although the origin and disposition of the fetal membranes differ among mammalian species, comparative histologic and electron microscopic studies indicate basic structural characteristics common to all mammalian amnions. Earlier concepts of the fate of the ruptured human amnion are reviewed. Direct application to man of these experimental findings may not be valid, but comparative anatomical evidence suggests that one explanation for cessation of leakage and continuation of pregnancy following premature rupture of the human membranes may be healing of the defect by regenerated amnion.

R E A s s E s s M E N T of the role of premature rupture of the human fetal membranes in increasing perinatal mortality has kindled interest in the fate of the affected amnion. Histopathologic studies of the human fetal membranes have been concerned primarily with the etiologic roles of infection and anatomic defects in their premature rupture. 4 Although Blanc4 a has suggested that regrowth of epithelium may occur at the edges of necrotic amnion, regeneration of the completely ruptured membrane has not been unequivocally demonstrated. Clinical circumstances surrounding premature mpture of the membranes in human pregnancy preclude prolonged observations in women. An initial approach to the problem was therefore attempted in laboratory animals. Success of the experiment depended upon development of an operative technique

that permits rupture or excision of fragments of amnion without loss of ammotlc fluid and that avoids infection and abortion. The pregnancy could thus continue for a period of time sufficient to allow histologic observations of the fate of the membranes. Although the data provided by this study are derived from experiments in the guinea pig, in which the origin and disposition of the fetal membranes differ from those in man, 10 the success of the technique has led us to organize similar experiments in the macaque that are likely to provide information more directly applicable to human pregnancy. Premature rupture of the membranes, though not necessarily preventable, is an important obstetric factor in prematurity. Its careful management may reduce perinatal loss. 7 The facts upon which such therapy is based, however, are presently meager. Materials and methods

From the Department of Obstetrics and Gynecology, State University of New York, Downstate Medical Center. This research was supported in part by Grant HD 00018 from the United States Public Health Service, National Institute of Child Health and Human Development, and Grant R-198-66 from the United Cerebral Palsy Research and Educational Foundation.

Preliminary experiments with the rat were discontinued because the small size of the conceptus led to difficulty in orientation during histologic examination. The pregnant guinea pig, although highly susceptible to infection, anesthesia, and abortion, proved

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more suitable because of its larger size and its longer gestational period. Hysterotomy in the guinea pig before the fiftieth day of gestation was often followed by abortion. Laparotomy after the sixtieth day, however, was frequently followed by premature labor before histologic observations could be made. The successful experiments were therefore performed between the fiftieth and fifty-fifth days of gestation. The surgical procedures were successfully

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performed on 15 rats and 12 guinea pigs. In these 27 animals loss of amniotic fluid was minimal and the me~nbranes at autopsy appeared grossly to have regenerated. In only 6 guinea pigs, however, was histologic examination sufficiently extensive to provide unequivocal evidence of regenerating amn!on. The animals were anesthetized with opendrop ether after premedication with sodium pentobarbital (28 mg. per kilogram) and atropine sulfate ( 0.04 mg. per kilogram ) .

Fig. 1. Section of uterus and conceptus of guinea pig at about 50 days' gestation showing relations of fetal membranes. Placenta (P), yolk sac (Y), amnion (A) , fetal skin (F) , and uterine wall ( U) are shown . Arrows indicate the structures incorporated in initial stitches in the procedure described in text. (x5.)

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Under surgical conditions, the abdomen was opened and with minimal manipulation one uterine cornu was grasped. Avoiding the placenta and large uterine vessels, two nonabsorbable sutures were placed through uterus and membranes, including the fetus, which was elevated into the surgical field to produce tamponade and prevent loss of amniotic fluid (Fig. 1). A purse-string suture was placed in the uterus surrounding these two stitches but not tied. The paper-thin uterine wall was then incised down to the membranes. A 2 to 3 mm. fragment of amnion overlying the fetus was then excised and preserved for histologic examination. The l:imits of rupture of the amnion were identified by two sutures of No. 5-0 silk or nylon placed through the membrane. The purse-string suture in the uterine wali was tied, the sutures elevating the fetus were removed, and the abdomen was closed in layers. No antibiotics were administered. Approximately 96 hours after hysterotomy, laparotomy was repeated. The entire operative site, including uterine wall and membranes, was excised and fixed in 10 per cent phosphate-buffered formalin. Several days later the tissues were embedded in paraffin and serial sections 5 p, in thickness were made throug;h the entire block, perpendicular to the site of incision. Sections were stained with hematoxylin and eosin or with toluidine blue. In several of the experiments specimens of amnion were excised for ultrastructural study. These tissues were fixed immediately in 5 per cent glutaraldehyde at 0° C. and postfixed in phosphate-buffered 1 per cent osmic acid. After dehydration and embedding in Araldite, thin sections were prepared on a PorterBlum MT-1 ultramicrotome, stained with uranyl acetate, and examined with the Zeiss EM-9 electron microscope.

Obs;ervations Normal amnion at the fiftieth day of gestation consists of a single layer of epithelium, ranging from low cuboidal to columnar, with pale-staining cytoplasm and dense nuclei, which occupy varying positions in the cell. The connective tissue is areolar and avascu-

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lar with unremarkable mesenchymal components (Fig. 2). In sections of tissue removed on the fourth or fifth day after operation, amnion and highly vascular yolk sac were recognized at the sites of the previous excision, which were identified by sutures (Fig. 3, A). An acute inflammatory response was elicited by the hysterotomy. Neutrophils were found between uterine wall and yolk sac, between yolk sac and amnion, and within the amniotic cavity (Fig. 3, B). The earliest evidence of regeneration was the appearance of avascular mesenchyme at the operative site. Shortly thereafter, a single layer of epithelium with pale eosinophilic cystoplasm and few nuclei with sparse mitotic figures could be detected (Fig. 4). By the fifth day after rupture, the regenerated amnion was histologically indistinguishable from that of the amnionic control at the same stage of gestation. Most obvious of the ultrastructural characteristics of the amnionic epithelium are the numerous large cytoplasmic projections from the free surface. Short microvilli arise from portions of the epithelial surface that are not involved in these promontories. ?\.1ost

of the cytoplasm is occupied by ribosomes, but well-developed endoplasmic reticulum is scant. Golgi bodies are only moderately complex and mitochondria are sparse. Prominent desmosomes connect epithelial cells. Large intercellular spaces are found in addition to a system of intracellular channels that extend from pinocytotic vesicles at the bases of epithelial microvilli to epithelial basement lamina. The nuclei are ovoid and moderately electron dense (Fig. 5). Cells of great electron density, which are generally lower, abut less dense elements. The distribution of the two cellular types is apparently random. There are fewer cytoplasmic projections from the more electrondense elements into the amniotic cavity, but the number and distribution of organelles in the two types of cells are similar. The basal plasma membranes are moderately convoluted. The collagenous stroma contains typical fibrocytes and occasional macrophages

362 Wynn, Sever, and Hellman

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Fig. 3. A, Experimental hom showing suture (arrow) and folds of regenerated amnion (A ) at the site of excision. ( x l65.) B, Relation of regenerated amnion (A) to fetal skin (F ). Arrow indicates pus in uterine lumen. (x250.)

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Fig. 4. Histologic details of regenerated amnion. A, The scalloped epithelial border is shown. Fibroblasts (F) are seen in the edematous avascular mesenchyme. (x730.) Compare with Fig. 2, B. B, Single layer of epithelium in regenerated amnion surrounded by numerous neutrophi!s. (x730.) Compare with Fig. 3, B.

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that resemble Hofbauer cells (Fig. 6 ) Y It is sep;Hated from the epithelium by a typical basement lamina. Con1ment

No satisfactory explanation has been provided for the well-known phenomenon of premature rupture of the human fetal membranes followed by cessation of leakage and normal labor many weeks later. Schuman/ 3 casting doubt on the significance of "high rupture" of the amnion, suggested th<1:t the long :latent period in such cases could be

Morphologic studies of ruptured amnion 365

explained by rupture of the chorion alone with leakage of fluid that had accumulated between amnion and chorion. According to this concept of a double sac, the "intact membranes" at the time of delivery represent only the amnion. Bourne, 5 however, postulated that a small leak could lead to closer apposition of the presenting part to the lower uterine segment. In that case, when dilatation of the cervix occurs, the membranes strip away from the opposite side of the lower segment to occupy the space formed by the dilating os, thus creating a new sac

Fig. 5. Electron micrograph of amnion of guinea pig at about 50 days' gestation. The epithelial cytoplasm (E) is studded with numerous ribosomes but contains few well-developed

?rganelle~. ~hort .microvilli (light arrow) and large cytoplasmic projections (heavy arrow )

mto ammotlc cav1ty are seen. A convoluted basement lamina (B) separates epithelium from collagen-rich stroma (S) . (x4,500.)

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Fig. 6. Ultrastructure of amnion adjacent to that shown in Fig. 5. More electron-dense (D ) and less dense ( L ) epithelial cells are randomly distributed. Typical fibroblasts (F) are seen in th e stroma. (x4,500.)

of "forewaters," with the presenting part acting as a valve at the original site of leakage. A third explanation is based on healing or sealing of the membranes. For none of these views, however, has adequate histologic documentation been provided. The studies of Danforth, McElin, and States, 6 furthermore, have indicated that premature rupture of the membranes is not related to inherent weakness of the amnion, which was shown to withstand pressures exceeding those produced by normal uterine contractions.

Despite differences in formation and disposition of the definitive membranes of guinea pig and man, the amnions in the two species are histologically similar. According to Mossman, 10 amniogenesis in the guinea pig is highly specialized because the amniaembryonic portion of the inner cell mass separates from the trophoblast long before cavitation occurs in either. As a result, two cavities are formed before mesoderm and amnionic folds appear. The human amnion presumably arises by cavitation. Although

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implantation in both man and guinea pig is interstitial and antimesometrial, the embryonic disc in the guinea pig is opposite the site of implantation, and the definitive placenta forms mesometrially. Placentation in the guinea pig, furthermore, is complicated by extremely early complete inversion of the yolk sac, a structure that is rudimentary in man. In Hertig's classical vie\0 the human amnion is derived from cytotrophoblast immediately adjacent to the dorsal aspect of the germ disc. Another concept, however, which is b.ased on comparative anatomy and still supported by Mossman/ 1 holds that the amnion, at least in rodents and perhaps also in man, is basically extraembryonic somatopleure reflected from the embryonic body wall or the umbilical cord. Ample histologic and ultrastructural evidence indicates basic morphologic similarities among all mammalian amnions. The human amnion, as described histologically by Schmidt12 and iater by Bourne," closely resembles that of the guinea pig. 1 • 3 Comparative electron microscopic studies 2 · 9 ' 14 and unpublished data from this laboratory likewise indicate fundamental morphologic features common to all mammalian amnions. Ultrastructural simplicity characterizes the amnion in general. The morphologic adaptations are primarily to transport of large volumes of fluid. Interspecific differences center m:Jinly about the extent of development

Morphologic studies of ruptured amnion

36;

of intercellular spaces and of podocytic specializations, which are more extensive in man, for example, than in the guinea pig. It appears, furthermore, that the histologic epithelial variations, which range from low cuboidal to high columnar, may be related more to the state of fixation or to stretching or folding of the membranes than to physiologic functions. The histogenetic relations of the light and dark cells observed with the electron microscope and their roles in secretion and absorption remain to be ascertained. The presence of necrotic cells in the normal amnion creates difficulty in attempts to interpret them as etiologic factors in premature rupture. The amnion of the guinea pig, presumably nourished by amniotic fluid or adjacent vascular structures, appears capable of regenerating histologically normal membranes after rupture. Histologic and ultrastructural similarities suggest that possibility in human pregnancy aiso. The cessation of leakage of fluid and the continuation of pregnancy for many additional \veeks may thus be explained in certain instances by regrowth of histologically normal fetal membranes. We are grateful to David H. Stern for preparation of many of the histologic sections and to Jennifer A. Harris for expert assistance with the photomicrography.

REI'I:RI:NCI:S

1. Bautzmann, H., and Hertenstein, C.: Ztschr. 2. 3.

4. 4a. 5. 6. 7. 8.

Zellforsch. 45: 676, 1957. Bautzmann, H., and Schmidt, W.: Ztschr. Zel.\forsch. 51: 571, 1960. Bautzmann, H., and Schroder, R.: Acta anat. 33: 38, 1958. Ben!rschke, K.: .A.M. J. 0BST. & GYNEC. 84: 1595, 1962. Blanc, W. A.: New York J. Med. 61: 1492, 1961. Bourne, G.: The Human Amnion and Chorion, London, 1962, Lloyd-Luke, Chap. 16. Danforth, D. N., McElin, T. W., and States, M. N.: AM. J. OI!sT. & GYNEC. 65: 480, 1953. Eastman, N. J., and Hellman, L. M.: Williams Obstetrics, ed. 13, New York, 1966, AppletonCentllry-Crofts, Inc., Chap. 21. Hertig, A. T.: Yale J. Bioi. & Med. 18: 107, 1945.

9. Larsen, ]. F., and Davies, J.: Anat. Rec. 143: 27, 1962. 10. Mossman, H. W.: Contrib. Embryo!. 26: 129, 1937. 11. Mossman, H. W.: In Wynn, R. M., editor: Second Conference on Fetal Homeostasis, New York, 1967, The Ne\"/ York .A.cademy of Sci~ ences, p. 13. 12. Schmidt, W.: Ztschr. Anat. 119: 203, 1956, 13. Schuman, W.: AM. J 0BsT. & GYNEC. 62: 633, 1951. 14. Thomas, C. E.: J. Ultrastructure Res. 13: 65, 1965. 15. Wynn, R. M.: AM. ]. DBST. & GYNEC. 97: 235, 1967. 450 Clarkson Avenue Brooklyn, New York 11203