Paper - The histology and cytology of the human and monkey placenta

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Wislocki GE. and Bennett SB. The histology and cytology of the human and monkey placenta, with special reference to the trophoblast. (1943) Amer. J Anat. 337-448.

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This historic 1943 paper by Wislocki and Bennett describes the human and monkey placenta.

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The Histology And Cytology Of The Human And Monkey Placenta, with Special Reference To The Trophoblast

George E. Wislocki And H. Stanley Bennett

Department of Anatomy, Harvard Medical School, Boston, Massacimsetts

One Text Figure and Thirteen Plates (Forty-Four. Figures) 1943

  • The principal observations and results embodied in this paper formed part of the second Robert J. Terry Lecture, entitled "The Primate Placenta, with Special Reference to the Trophoblast", delivered by the senior author at Washington University, School of Medicine, St. Louis, on April 22, 1942. Three abstracts, referring (1) to the nature of the lipoid droplets in the syncytial trophoblast (Bennett and Wisloeki), (2) to the relationship of the blood vessels to the trophohlnst in the chorionic villi of the humrm term placenta (Wislocki and Bennett), and (3) to the nature of the foamy, vacnolated and vesicular appearances of the trophoblast ( and Bennett), have appeared in the Proceedings of the American Association of Anatomists in 1942 anal 1943. References to these are listed in the bibliography.


The present study concerns the histology and cytology of the normal human and monkey placenta, with special reference to the finer structure and functions of the trophoblast. The cytology of the placenta has been investigated less, perhaps, than the finer structure of any other major organ or tissue. This is attributable probably to the complex organization of the placenta, both in its comparative anatomical aspects and in its ontogenetic development. In View of these complexities, investigators up to the present time have concerned themselves mainly with the topographical and histological relationships of placental structures, employing for the most part standard histological techniques involving preponderantly hematoxylin and eosin preparations. This natural preoccupation With the topography of the placenta and embryo has culminated in recent years, as far as the Primates are concerned, in a series of important studies of the development of macaque and human ova, a goodly number of which have appeared from the Department of Embryology of the Carnegie Institution of Washington.

The recently completed history of the development of primate eggs should serve as a substantial groundwork for resuming more careful investigations of the finer cellular structure of the placenta. The need for the extension of cytological studies into the field of placentation is illustrated by the fact that the masterly and extensive compilation by Grosser (’27) which deals with human and comparative placentation offers relatively little in regard to placental cytology. Moreover, if one examines practically any of the recent papers on the early development of the primate placenta (e. g., Hill, ’32; VVislocki and Streeter, ’38; Hertig and Rock, ’41), it is clear that interest centers mainly around the topography and general histology of ‘the specimens, f'ew of the remarks and even fewer of the illustrations being devoted to the elucidation of cytological features at a magnification greater than 300 or 400 times or by the application of special fixatives and differential stains.

The present study concerns the investigation of a series of human and monkey placentae,'utilizing a variety of fixatives and stains, and centering upon the cytological features of the trophoblast. This study makes no pretence of being complete; indeed, the very wealth of existing cytological methods, as well as the difficulties of obtaining adequate material, makes that impossible. Yet we hope, in hand our material, to illustrate and to discuss certain features and properties of the trophoblast which have been generally neglected and which certainly deserve to be investigated more extensively. lVe have also attempted to separate our actual observations from the various interpretations and speculations which we have chosen to attach to the data.

Material and Methods

The human material at our disposal consists of fourteen well-preserved placentae ranging in age from 30 days up to term. The specimens were obtained largely through the generosity of Dr. Franklin F. Snyder from the Chicago Lying-In Hospital, and from Dr. Arthur Hertig from the Boston LyingIn Hospital. Acknowledgment for one specimen each should also be made to Dr. Kenneth Farnsworth and to the Surgical Service of the Peter Bent Brigham Hospital.

The monkey material comprises four pregnant uteri, three of which were obtained through the generosity of Dr. Carl G. Hartman from the Department of Embryology of the Carnegie Institution. These three macaque monkeys (Macaca mulatta) were killed on the twenty-eighth, fifty-eighth, and sixty-fourth day of pregnancy respectively. The fourth pregnant animal, received from another source, was in the last third of gestation.

The principal fixatives used were formaldehyde, formoldichromate, Zenker-formol, Champy’s fluid, Bodian fixative no. 2, Mann-Kopsch osmic-sublimate, I-Ieidenhain’s, Susa, Carnoy’s mixtures, Bouin’s fluid and absolute alcohol.

The fixed material was stained by a variety of methods. Heidenhain’s iron hematoxylin, hematoxylin and eosin, Masson’s trichrome mixture, Mallory’s connective tissue stain, the Azan technique, Mayer’s paracarmine and Orange G, Best’s carmine stain and Paps’ silver reticulum impregnation were used on appropriate fixed material. Mitochondria were demon— strated by the Altman-Kull method. The Golgi apparatus was impregnated with osmic acid.

Fresh whole villi were stained with Sudan III. Other fresh villi and sections of gelatin-imbedded, formaldehyde-fixed villi were treated with phenylhydrazine hydrochloride to locate water insoluble ketones, according to the method of Bennett (’40) for demonstrating ketones and aldehydes in tissue sections.

The drawings of the sections were all prepared by Miss E. R. Piotti using the camera lucida. The figures, with the exceptions which will be mentioned, were dra.wn t.o scale at a magnification of X 1600.

Introductory Consideriations

The youngest specimens in which we have attempted to explore the cytology of the trophoblast consist of two blastocysts, one from a monkey, the other human, from the end of the first month of pregnancy. These two very fresh placentae were cut up and fixed in various ways and stained by a variety of techniques. Into our description and discussion of the cytology of the trophoblast derived from study of these specimens we have drawn the cytological information which is available in the literature in regard to earlier stages of development in both the macaque and the human. For the macaque, Wlislocki and Streeter (’38) have given a day—by—day account of the early history of the development of the placenta including the growth of the trophoblast and its interactions with the endometrium. Thus one of the present investigators had a firsthand acquaintance with the placenta of the macaque, and, as our cytological study of the trophoblast required it, we rea examined the sections of the early stages of the macaque which are on deposit in the Carnegie Laboratory of Embryology. In reference to the human, we have reviewed the passages and remarks pertaining to the cytology oi‘ the trophololast in young human embryos in the papers by Streeter ( ’26), Florian (’28), Stieve (’36), Jones and Brewer (’35), Brewer (’37), Johnston (’41), Hertig and Rock (’41), and Hamilton and Gladstone (’42). Thus we have attempted to present certain problems regarding the trophoblast by utilizing‘ additional sources of available information beyond our own immediate material.

1. The tropholzlast: (t6fi’£l.'tt‘tO3'l.S cmd tevr-m/mology

Wle shall divide the trophoblast, following current custom, into the cytotrophoblast and the syncytial trophoblast. The latter we shall call, for short, syncytium. Other writers have attempted to divide both of these types into further categories or subdivisions. \Ve shall avoid the classification created by Grosser (’27), who distinguishes first and second generations of syncytium and cytotrophoblast and who attempts to discriminate also between an “implantation syncytiurn” and a “resorptive syncytium”. Similarly we shall not adopt the terms “prolifcrative plasmodinin” and “resorptive plasmodium” as defined by Florian (’28). In regard to the early stages of development of the macaque, VVislocki and Streeter (’38), in discussing the classification of Grosser, reached the conclusion that from examination of their monkey material it was clear that “both cytotrophoblast and syncytial trophoblast are always recognizable from the period of the free blastocyst on, any clistinction between a stage of implantation cytotrophoblast and a stage of implantation syncytium being more fanciful than real.” The histology of the earliest human embryos (Hertig and Rock, ’41) also indicates that such a distinction is not usefully applicable to early human material.

In regard to the use of “implantation syncytium” as contrasted with “resorptive syncytium” as used by Grosser, Wislocki and Streeter have pointed out also that “resorptive ” would apply equally well to the entire development of the trophoblast and should not be restricted to any particular stage. In reference to Florian’s use of “proliferative plasn1odiun1” and “absorptive plasmodium”, we do not regard the properties of difierent fractions of the trophoblast as suffieiently well established or documented to warrant the use of implicit adjectives indicating differing functions. A good case can be made out that much of the trophoblast is in varying degrees both undergoing proliferation and serving to nourish the fetal tissues at the same time.

In any event, we regard it as sounder to proceed with the investigation of the syncytium and the cytotrophoblast unburdened by preconceived terms indicating possible functions. Jonsequently, we shall define and discuss the trophoblast on a topographical basis, recognizing the following categories depending upon the sites or localities in which it occurs.

(a) The cytotrophoblast and syncytium associated with the primary villi before the appearance of mesoderm in the villi.

(b) The cytotrophoblast and syncytium associated with the secondary, or definitive, villi and the chorionic plate. This eytotrophoblast will be referred to as Langhans’.cells, epithelium or layer.

(c) The cytotrophoblastic cell columns and their associated covering syncytium, which connect the distal ends of the developing secondary chorionic villi with the trophoblastic shell.

((1) The trophoblastic shell composed mainly of cytotrophoblast and formed by the fusion of the expanded distal ends of the cytotrophoblastic cell columns. A variable amount of syncytium is intermingled in the cytotrophoblastic shell and extends variable distances, more so in man than in the macaque (Wlislocki and Streeter), into the decidua. We see no objection on topographical grounds to calling this outlying syncytial trophoblast the peripheral syncytium.

(e) The trophoblastic cell islands or nodes, composed mainly of cytotrophoblast with a variable amount of associated syncytium.

2, Prefctory re/rniarks on the fzmctiorns of the trophoblast

The trophoblast is the parenchyma of the placenta and constitutes the essential tissue which sustains the growth and nutrition of the growing embryo by securing nutriment for it from the maternal organism. The trophoblast in primates obtains this nutriment in tlie earliest stages, before the definitive ehorionic villi and the fetal circulation are established, directly from disintegrating maternal tissues, as well as from more or less stagnant maternal blood at the periphery of the implantation site. Somewhat later when the circulation of blood in the intervillous space has begun and the fetal circulation has commenced, the essential barrier between mother and fetus becomes progressively established in the trophoblast clothing the chorionic villi. Through the trophoblast covering the villi pass the various substances derived from the maternal blood stream and necessary for the growth and maintenance of the fetus. In the opposite direction certain end-products of fetal metabolism are excreted into the maternal blood.

It has been demonstrated in recent years that in addition. to these functions the placental tissues are a source of several hormones, including estrogens, progesterone, and a pituitarylike hormone commonly called chorionic gonadotropin. It is not known precisely where in the placenta these hormones are stored or discharged. The existing experimental and physiological evidence does indicate, however, that it is the fetal portion of the placenta, rather than the maternal, in which these substances are present. A cytological study of this organ should bear in mind the presence of placental hormones and should seek to demonstrate them by histochemical means.

3. The early diflere-ntiation owed growth of the trophoblast

Streeter (’38, ’41) has recently emphasized the importance of the early segregation of the auxiliary from the formative elements of the ovum. He points out, using the macaque as his illustration, that during the passage of the ovum through the oviduct into the uterus in the first week of development the precocious trophoblast cells become specialized and distinct from the more primitive, less specialized and still dormant embryo-forming cells. The trophoblastic cells or auxiliary elements of the ovum are destined to form the structures for the attachment and nourishment of the embryo. Thus in the macaque a blastocyst is formed which on the ninth day begins to undergo implantation in the uterine wall.

In the macaque it is clear that in the stage of the free blastecyst and at the very beginning of implantation the trophoblast is composed of discrete cells (VVislocki and Streeter, ’38). Soon thereafter (by the end of the tenth day) syncytial tropho— blast makes its appearance. In the human, both cytotropho~ blast and syncytial trophoblast are present in the youngest known eggs of the eleventh and twelfth days (Streeter, ’26; Sticve, ’36; Hertig and Rock, ’41). In a 10-§—day chimpanzee egg, cellular and syncytial trophoblast is also already present (Elder, Hartman and Heuser, ’38).

4. The relationship of the cytotrophoblast to the syncytial trophohlast

It is generally agreed that the syncytial trophoblast is derived from the cytotrophoblast. This belief is based upon several forms of observation and reasoning. Histological transitions have been observed between individual cytotrophoblastie cells and the syncytium (Delporte, ’12; Florian, ’28; Brewer, ’37; Hertig and Rock, ’41). The acquisition of a brush border by cells in transition has been illustrated by Brewer (’37).

Shadows of former cell boundaries have been noticed in the syncytium (Hamilton and Gladstone, ’42). Occasionally neighboring Langhans cells have been observed in process of fusion preliminary to becoming part of the syncytial layer (Florian, ’28; Brewer, ’37).

The distribution of mitoses in the trophoblast points also toward the derivation of the syncytinm from the cytotrophoblast, for mitoses have repeatedly been seen in the latter (Hofbauer, ’05; Grosser, ’27, p. 347; Jones and Brewer, ’35; Brewer, ’37; Hertig and Rock, ’41; Hamilton and Gladstone, ’42), whereas none has been observed in the syncytium. In our own experience mitoses are Very frequent in the Langhans cells covering the villi (fig. 4), and even more frequent in the portions of the cytotrophoblastic cell columns in the Vicinity of their attachment to the inesodermal chorionic villi (fig. 23). Vtiislocki and Streeter (’38) pointed out in the monkey that the proximal parts of the eytotrophoblastic cell columns are composed of relatively compactly arranged and densely staining cells, whereas as one proceeds distally toward the trophoblastie shell the cells of the columns become clearer and are more loosely arranged. The same is true of the cell columns in the human, as indicated by Jones and Brewer ( ’35), Hamilton and Gladstone (’42), as well as in our own material. In the proximal, compact portion of the cell columns at the growing tips of the secondary villi mitoses are abundant, whereas in the distal portions of the columns and in the cytotrophcblastic shell mitoses are encountered rarely, if ever.

Our own observations agree with those of others that mitotic figures are absent in the syncytial trophoblast. The possibility of amitotic nuclear division in the syncytium has been accepted by some (Grosser, ’27, p. 350; Florian, ’28; Stieve, ’36; Hertig and Rock, ’41; Johnston, ’41) in order to account for its growth. Florian postulates the occurrence of amitosis on the basis of having observed occasional dumbbell—shaped nuclei, while Johnston says that the presence, now and then, of two elongated nuclei lying side by side or of V— and Y-shaped nuclei gives support to the View that amitotic division is the rule in the syncytium. It should be added that the nuclei of the syncytiuni compared to those of the eytotrophoblast are as a rule much smaller, more deeply staining, and more irregular in form.

The gradual disappearance of the Langhans layer without signs that the cells actually degenerate in any great number has been offered as additional supporting evidence by some writers that the cytotrophoblast covering the secondary villi is mainly transformed into syncytium.

The evidence for the derivation of the syncytium from the cytotrophoblast, although not absolute, appears to be sound. Thus the cytotrophoblast possesses the important function of being a germinal bed Which, besides perpetuating itself for a certain period, gives rise to the syncytium. As has been pointed out, this cytomorphosis of the cellular trophoblast occurs more abundantly in certain localities than in others.

5. The relationship of the cytotrophoblast to the primary mesoderm and cmgiogenesis

Streeter (’26), in describing the “Miller” ovum, suggests that the primary mesoderm derives probably from the trophoblast. Hertig (’35), in an extensive study of a series of early macaque and human embryos, examined this suggestion. He concluded that the origin of the primary mesoderm as well as of the earliest angioblasts is by a process of delarnination and differentiation from the chorionic trophoblast. Wlislocki and Streeter (’38) adopted Hert_ig’s View in their study of early stages of the placentation of the macaque. On the other hand, Bloom and Bartelmez (’40), in a study of heinatopoiesis in slightly older human embryos, remark that they have only occasionally seen cells that might possibly be interpreted as transition stages between cytotrophoblast and mesenchyme, while they have no convincing evidence at all of the developmcnt of vessel-forming cells from the cytotrophoblast as described by Hertig.

The Syncytial Trophoblast

Certain histological characteristics of-the syneytial trophoblast have been known for a long time. It is also recognized that the syneytium clothing the secondary chorionie villi cliffers in certain respects from that associated with the tropho— blastic cell columns and the trophoblastie shell. These differe11ces have been made the basis for the introduction of the terms “resorptive” and “proliferative” syneytium by certain authors whose ideas were mentioned in a previous passage. The syneytium also changes its histological character during the course of gestation, that clothing the villi in the early period differing in many respects from that covering the villi late in pregnancy.

As would be anticipated, many previous descriptions and remarks concerning the histology of the syneytium are to be found in the literature. In the commonly studied hematoxylin and eosin preparations, the nuclei of the syneytium are described as being in general deeply staining, small, and irregular,'while the cytoplasm is found to be darker than that of the cytotrophoblast and more purplish in color. Attention has been called in the past to a variety of special characteristics and properties of the syneytium. Thus it has been noted that its cytoplasm often has a foamy appearance and in places is markedly vaeuolated, especially in its outlying peripheral parts associated with tl1e cell columns and the trophoblastic shell. its surface has been described as possessing a brushborder. Golgi material, mitochondria and lipoids have been demonstrated in the syneytium, and evidence of phagocytosis by its cytoplasm has been noticed. Degenerative changes in the cytoplasm and in the nuclei have been observed, involving alterations of the nuclei and either vaeuolization or hyaliniza— tion of the cytoplasm.

It is our purpose in hand with the present material to reexamine a number of these topics and to introduce some new ones. We shall begin with a consideration of the outer surface of the syncytium, and then proceed to a series of further topics in an order which seems reasonable to us.

1. The outer surface of the syncytémn, including the occurrence of a so—called brush—border

Since there is ample reason to regard the syncytium, especially that portion covering the secondary villi, as exercising resorptive and excretory functions as well as even possibly secretory ones, the detailed structure of the surface of the syncytium deserves to be carefully investigated.

Of the surface of the syncytium in the “Miller” ovum, Streeter (’26) gives no particular description, 11or is any mention made of a brush—border. Nor does Stieve (’36), describing the “Werner” egg, refer to the finer structure of the trophoblast or mention a “brush”, or otherwise differentiated, border.

Hertig and Rock (’41) observe, on the other hand, in their 11- and 12-day human ova that the lining of the syncytial spaces shows a delicate vertical striation, a so—called brushhorder. This border, they state, is somewhat more accentuated in the older specimen. Otherwise they present no details of the structure of the syncytial surface. Jones and Brewer (’35) and Brewer (’37), describing embryos at the primitive streak stage, state that the syncytium covering the short villi and the chorionic wall has a brush—border whereas the deeply penetrating peripheral syneytial masses do not. The brushborder consists of closely grouped filaments continuous with the protoplasm a11d, according to Brewer (’37), each filament seems to be composed of granular or short rods arranged end to end. He also calls attention to the beginning acquisition of a brush—border by certain cytotrophoblastic cells which are becoming transformed into syncytium. Johnston (’41), referring to an embryo in the primitive streak period, also observes that the syncytium covering the villi bears a well—de— fined brush—b0rder whereas the peripheral syncytium does not. Hamilton and Gladstone ( ’42) remark of their specimen that a typical brush-border is observed in many parts, whereas in other places it is indistinct or absent. Grosser (’27, pp. 270—271) cites references to the effect that the “implantation” and “peripheral” syncytium in younger ova is more amply provided with brush-border than the “resorptive” syncytium.

In the macaque Wislocki and Streeter (’38) stated that in their series of early stages they had not observed brush-borders. In view of our present interest in this topic we have reexamined the macaque series of the Carnegie Laboratory of Embryology with the following results:

In the early stages (11 to 14 days) the syncytiurn is foamy or delicately vacuolated and finely granular. The free surface bounding the lacunar spaces and later the beginning intervil— lous space is irregular. The appears in the main to extend or fade out into lacy cytoplasmic streamers, which are delicately stippled by scattered granules. No organized border definitely differentiated or set off from the underlying cytoplasm is observed. In a 15-day specimen (C-571), however, it is remarked that in rare places there is a suggestion of a delicately stippled or granular border which is redder (hematoxylin and eosin) than the rest of the cytoplasm. Similarly in a 17-day macaque (C-457) in one or two places there is a slight suggestion_of a similar border. In a 20-day specimen (C—424), in some areas clothing the villi, a pinkish or reddish outer border is definitely visible, but without obvious striations or hairs. Alternating with these areas are the usual, markedly irregular surfaces which exhibit pale, ill-defined streamers or delicate fronds of stippled cytoplasm. In many places in these specimens it appears as though the surface cytoplasm of‘ the syncytium might be releasing its finely granular substance to a certain extent into the intervillous space. Similarly in specimens of the twenty—second and twenty-ninth days areas possessing a pinkish border are visible, whereas in a 35-day specimen in some places a brush—like border is encountered. Here the border supervenes on a foamy, granular zone of cytoplasm which in turn overlies a much broader, denser zone in which the nuclei are irregularly scattered. It should be added that in the 30—day macaque specimen of the present material a brush—like border is clearly visible on some parts of the syncytium.

These observations siiggest that the “brush-border” ap pears later in the maea que than in the human. Since the mode of fixation is alleged to play such a variable and uncertain role in preserving “brush—borders”, it should be pointed out that the macaque series and the two human ova obtained by Hertig and Rock were fixed in Bouin’s fluid under almost identical conditions of handling. Consequently, these two sets of tissue should be comparable; yet in the human a “brushborder,” according to Hertig and Rock, is present in 11- and 12-day specimens, whereas we have not observed it in the macaque with any definiteness until the end of the first month.

All of the writers cited above give only very brief accounts of the syncytial border. A detailed description of the placental brush-border based on study of somewhat older stages is presented by Hofbauer (’O5). He describes the great variability of the brush-border and gives its detailed morphology. By some it has been referred to as ciliated, although no ciliary movements have ever been observed by those who have searched for motion in fresh material. Others have applied the term “steroeilia” to the hair—like appearances, although clearly discernible basal granules have rarely been described. The majority have been content to refer to the striated parts of the syncytium as a brush-border.

Hofbauer points out that the structural varia.bi1_ity of the brush-border cannot be attributed to fixation or to postmortem changes alone, although some fixatives, especially these containing acetic acid, are said to preserve it better than others. Grosser (’27) also arrives at the conclusion that the inconstancy of the border cannot be ascribed to postmortem changes. The brush-border is said to disappear by the fifth or sixth month of gestation.

Our own experience, too, has been that the so-called brushborder is very variable in structure and inconstant in its oecurrence and distribution within any given placenta. Over considerable portions of the chorionic villi no definite brush can be seen at all. Yet in view of the clearness with which a brush—border is often seen and the uncertainty of the role of fixation and other factors in its preservation, few of the previous observers have paid much attention to the character and properties of the surface of the syncytiuin in the areas where a brush-like border is lacking. \Ve have studied these areas with particular care since we believe that they may be quite as significant functionally as those bearing a bruslrborder, albeit the functions may not be entirely similar.

In view of these considerations we shall present our own observations regarding the appearance of the surface of the syncytium. The reader is referred to a number of the accompanying illustrations. Typical instances of brush—like borders are seen in figures 2, 5, 6, 25, and 37. Often the hair—like processes or striations appear to be variably cemented together by a homogenous, refractile substance. Rarely, as can be barely perceived in figure 6, there is an accentuation immediately beneath the hairs of the granular stippling which characterizes the general eytoplasm of the syncytium. These bodies are in our estimation too indefinite and inconstant to be regarded as true basal granules. Some have described beneath the brush a narrow, clear submarginal zone, the ectoplasm of certain writers, which is set off from the darker, granular cytoplasm of the main portion of the syncytium. In our preparations a submarginal zone is seldom so clearly defined as this statement implies. However, in certain areas, especially where the brush—border is ill-defined or absent, there may be considerable vacuolization of the marginal and submarginal cytoplasm.

From the well—defined brush seen in figures 5 and 25, and other less regular ones in figures 6 and 37, one can pass to areas like those of figures 7 and 26, where the hair—like or filamentous projections are less regular in regard to both length and spacing and are arranged in bundles or clusters. Between the “hairs”, or bundles of them, there is usually a delicate precipitate. Occasionally the clusters fan out distally into the intervillous space (fig. 7) and the individual hairs appear to be composed of separate particles set end to end. The presence of delicate precipitate and particles associated with the variable brush-like border suggests tl1e possibility that they may be substances which are being released or secreted by the syncytium into the intervillous space, although the faint precipitate may be mainly blood plasma which has been trapped and precipitated between the hair—like processes. A third possible interpretation, namely, that the precipitate belongs to thin surface films of the syncytial cytoplasm, cannot be categorically denied.

Finally one comes to areas in great abundance to which scant attention has been paid in the past in which there is little or no resemblance to a brush-border. Here, instead, the cytoplasm exhibits streamers or fronds of protoplasm extending out irregularly from the surface (figs. 2, 8 and 16). In these regions the marginal or submarginal cytoplasm is sometimes markedly vaeuolated, as can be observed in figure 16, as Well as at the lower margin of figure 2. In figure 16 the lacelike cytoplasmic prolongations give the impression of being in process of enclosing or engulfing droplets of fluid. Yet the impression is also gained here that particles of the lacy streamers may be in process of being detached and liberated into the intervillous space.

In preparations fixed and stained for mitochondria, the surface protoplasm frequently exhibits a markedly vacuolated appearance (figs. 13 and 14). In these preparations it is characteristic for the mitochondria to extend out actually into the cytoplasmic streamers.

The tinctorial reactions of the syncytial surface deserve to be specifically mentioned. It was remarked that in the early placentae of the rhesus monkey stained with hematoxylin and eosin, we observed an accentuation of the eosin stain in the granules imbedded in the irregular surface cytoplasm.

In the present material stained with Mallory’s connective tissue or with Masson’s trichrome mixture the border of the syneytium stains differently than the deeper part of the syncytial cytoplasm. In figure 37 it may be observed that after Mall0ry’s stain the brush-border is blue Whereas the underlying cytoplasm is purple. With Masson’s stain the border stains green while the underlying cytoplasm is reddish. Ofttimes when the border is rather well-defined the blue or green is limited rather sharply to the hair-like border (fig. 37). However, in all localities where the brush is less sharply delineated, including the areas where streamers and vacuoles predominate, the outer surface of the syncytium is also stained blue or green, but this coloring extends more deeply and irregularly into the submarginal zone to mingle transitionally with the respective color, either purple or red, of the cytoplasm beneath. These tinctorial differences suggest that the surface cytoplasm has different properties than that lying beneath it.

The various manifestations of the syncytial border to which We have called attention are found in our experience covering all parts of the syncytium which present a. free surface. The most regular brush-borders are found perhaps upon the secondary villi, whereas longer and more irregular filiform or hair-like appendages and streamers are encountered frequently on the surface of the peripheral syncytium. The longest projections of all have been seen in the monkey material of 58 and 64 days. Jones and Brewer (’35) and Johnston (’41) indicate that in their young human embryos brush-border is less frequent on the peripheral syncytium than on that covering tl1e-secondary villi. Grosser (’27, pp. 270-271), on the contrary, cites references to the effect that the “implantation” and “peripheral” syncytium in younger ova is more amply provided with brush-border than the “resorptive” syncytium. These conflicting statements may emanate from the adoption of different criteria as to what constitutes a “brush-border”. Our own figures and descriptions of the various appearances illustrate the difficulties involved.

Regarding the genesis of the varying structure of the border of the trophoblastic syncytium, We wish to present a possible explanation. This concerns the probability that the syncytium is a plastic mass subject to a certain degree of flowing and to changes in its form and contour, as suggested by the descriptions of it in the Writings of many observers. In this event its surface would be to a variable degree unstable, and the varying appearances met with would represent various phases of this activity. All investigators observing sections of younger stages of human and monkey placentae mention the invasive qualities of the trophoblast and remark at length upon the protoplasmic streamers, strands, and knobs of syncytium which penetrate the decidua, or form on the surface of the primary villi. Hofbauer (’O5) and Grosser (’27, p. 272) accept such appearances as possibly indicating amoeboid activity on the part of the syncytium, While the phrasing of the descriptions in the Works of many writers implies a belief or tacit assumption that the syncytium possesses considerable plasticity and the capacity to flow or stream. Indeed, Friedheim (’29) describes very convincingly flowing movements of the syncytium in explanted chorionic villi in tissue cultures. For 1 to 2 days after explantation he actually observed the formation of pseudopodia and filiform processes on the surface of the syncytium, resembling the syncytial sprouts and buds seen on the surface of normal villi in histological sections. These pseudopodia were relatively large and contained nuclei which had been carr;ied along in the streaming cytoplasm. Occasionally small bits of protoplasm were seen to detach themselves completely from the main cytoplasmic masses. He does not mention the detailed appearance of the protoplasmic surface of these pseudopodia or sprouts, but from his pictures, which are unfortunately at magnifications no greater than 460 X, it does not seem as though anything approaching the appearance of a brush-border Was present. These movements of the cytoplasm occur for several days, after which the syncytium regresses while the growth of the cytotrophoblast (Langhans cells) comes into the ascendancy. Thus We have some justification from direct observation to assume that the syncytium is unstable and that its contours must be subject to fairly constant modification. In View of this it does not seem remarkable that the surface appearance of such a tissue should be subject to great variability. “Te suggest that where the brush is most apparent and complete the syncytium, temporarily at least, may be less motile, whereas in the regions where the fronds and streamers occur the cytoplasm may be in a greater state of flux.

The frond—like and lacy arrangements seen in the fixed, stained material are reminiscent of the surfaces of various kinds of cells as observed in recent years in tissue cultures and other living preparations. They recall to some extent the undulating borders and surface streamers of fibroblasts, macrophages, and cells from both sarcomas and carcinomas as observed in tissue cultures by Lewis (’31), in which he called attention to the actual entry of fluid into the cytoplasm in the form of droplets or vacuoles. To this phenomenon he gave the name pinocytosis, meaning drinking by cells. Similar observations on fibroblasts proliferating in the ear of the living rabbit have been recorded by Stearns ( ’40). Vfislocki and Streeter (’38) in a study of the trophoblast in the macaque suggested that the prominent vacuoles enclosed by a thin covering of syncytium, which are present for a brief period in the syncytium clothing the cytotrophoblastie cell columns, represent a form of resorption or imbibition of fluids from the interVillous space possibly by a process of pinocytosis. In keeping with this thought We have observed in the present study that those portions of the syncytial trophoblast which possess a border composed of streamers or lacy projections often evhibit foamy or vacuolated appearances of the subjacent cytoplasm, suggesting active fluid transfer in these areas.

The foregoing considerations suggest to us that the various surface irregularties of the syncytium are implicated in the taking up of fluid and nutrient material from the intervillous space. Yet this interpretation does not preclude the possibility that other substances, for example excretory products of the fetus and possibly secretory products of the trophoblast as Well, may be transferred in the opposite direction. In the case of fibroblasts Stearns (’-10) observed that bits of highly Vacuolated protoplasm detached themselves from the actively moving cytoplasm to disintegrate in the interstitial fluid. This vacuolated protein material which is given off by the fibroblasts appears to initiate fiber formation and hence may contain enzymes or collagen. Many of the cytological pictures presented by the variable hairs, filaments and cytoplasmic streamers constituting the surface of the syncytium suggest that substances may be similarly liberated from the surface of the trophoblast. Thus we are inclined to the belief that the surface of the syncytium serves as a barrier through which certain substances are being absorbed at the same time that other substances may be in process of being secreted or discharged in the reverse direction. This interpretation of structural appearances is in accord with the conclusion arrived at of necessity on biochemical grounds that the trophoblast not only transmits nourishment to the fetus but excretes fetal Waste products besides liberating certain hormonal substances into the maternal blood stream.

To the objection that all or part of the appearances seen on the surface of the syncytium are artifacts and hence of slight importance, we have the following brief remarks to make. Wlhether the variable structures ranging from hairs, threads, lacy streamers, etc. seen after fixation, be regarded as artifacts or not, they indicate in our estimation that the surface of the syncytium has a special and peculiar structure related to its functional activities and dependent upon the as yet unanalyzed constitution of the syncytial surface in the living state. Moreover, it differs in appearance in many respects after fixation from other characteristic cellular barriers in the body similarly treated, and hence possesses certain structural pecularities of its own. The character of the surface protoplasm, whether it appears in the form of a brushborder or in the shape of irregular protoplasmic streamers, suggests that the margin of the syncytium presents variable molecular arrangements which govern the processes of absorption and excretion. In the border of the syncytium we have observed birefringent material in fresh, unfixed villi, a finding which indicates the presence there of an oriented molecular pattern.

2. The foamy, lacanar, nacnolatecl ancl pesto-n-tar appearances of the syncyttmn.

lnder this title we Wish to draw together a number of phenomena, which by others have hitherto been treated separately, but which we believe to be related one to another and hence deserve to be discussed under one heading.

The cytoplasmic appearances here considered pertain to various parts of the trophoblast at different periods of gestation. VVC shall enumerate and define the principal places and at what periods structures of the character of lacunae, foamy cytoplasm, vacuoles and vesicles occur in the tropho— blast and what we deem their underlying significance to be.

We shall present these structures in reference to the place and time of their occurrence under the following headings:

a. Vacnolteation and the for-motion of lacunae tn the syncytial trophohlast during the period of t'mplan.tat~ion.

b. F17ne—grav27ned 'uavonolieation (delicate foamy appearances) of the syncyttal trophoblast.

c. The occurrence of larger vacuoles in the sy-neyttmn of the macaque, at the height of (lerelopmrent of the trophoblasttc cell columns.

cl. The ooc:nrre»nce of irregularly scattered, large vacuoles in various parts of the syncytinnt.

a. Vacnolieatton and the formation of lacunae in the synoytial trophohlast (luring the perlocl of implantation. One of the principal features of the early trophoblast is the presence of vacuoles in the syneytium. Regarding the formation of these, Streeter (’26) says, in describing the Miller ovum,

“if one examines the primitive syncytium at its transitional base, Where it is elaborated from the cytotrophoblast, one finds everywhere a great many small, fluid—containing spaces, which tend to separate the cell—units off from one another. In addition to the small spaces, there are larger ones, and in fact one can pick out transitional ones all the Way up to large lacunae. The smaller spaces might be considered as vacuoles of intracellular origin. Those of intermediate and larger size, however, lack the necessary characteristic of degeneration vacuoles and fluid inclusions, in that one can not be sure that they are entirely surrounded by cytoplasm.”

All or a certain fraction of these vacuoles coalesce in a manner to produce the trophoblastic lacunae which are the forerunners of the intervillous space. He observes also that by the formation of vacuoles and lacunae, the syncytium becomes spongelike and is spun out into relatively thin, irregular strands. l/Vislocki and Streeter ( ’38) note that in the macaque the solid trophoblastie plate on the eleventh day develops lacunae and becomes eonverted into an open, reticulated mesh consisting of syncytial trophoblast. These lacunae, the forerunners of the intervillous space, tap maternal capillaries and promptly fill with plasma and red cells. Hertig and Rock ( ’4:1) express themselves to the effect that the syncytium in their young embryos is vacuolated and that by enlargement and coalescence these vacuoles form large, intracellular spaces which are in turn converted into intercommunicating trophoblastic lacunae which will ultimately contain maternal blood.

Thus it is suggested in the earliest period that by the formation of vacuoles in the syncytium and their coalescence, primitive lacunae are formed which are destined subsequently to become the intervillous space.

I). Fiarz/e-grained vacuolization (delicate foamy appearance) of the syawytial trophoblast. An extremely fine-grained vacuolization or delicate foamy appearance of the syncytium begins at the time of the formation of the syneytial trophoblast and persists until the definitive villi and the chorionic circulation have been established. This type of vacuolization can be documented by material from the series of macaque ova which We have reexamined. In an 11- to 12-day embryo (C661), the syncytial cytoplasm is delicately foamy and finely granular. In a 13-day embryo (C-562) it can be described as to finely vaeuolated and variably granular, while in a 15-day embryo (C-571) the syncytium is similarly vacuolated and contains eosinophilic dust—like granules. In a 20-day specimen (C-424) and in a 35—day specimen (C-479) there is a barely perceptible foamy quality to the synoytium clothing the villi in addition to numerous dust-like cytoplasmic granules. It is our impression that this type of vaeuolization diminishes rapidly about the time that the circulation in the definitive chorionic villi becomes well established. It persists, however, to a variable extent in the syncytium accompanying the cell columns and the trophoblastic shell, where it is associated often with the formation or appearance of much larger vacuoles of varyincr size.

In the human, foamy vacuolization in the early stages is less well documented, although it is mentioned in several of the youngest human embryos (Streeter, ’26; Hertig and Rock, ’41). In somewhat older human embryos, Florian (’28) mentions and pictures (fig. 7) a fine—grained vacuolization of the syncytium, more especially associated with trophoblastic cell columns and the shell. Johnston ( ’41) observes that the syncytial (plasmodial) trophoblast has the appearance of a. very fine foam. Hamilton and Gladstone (’42) also remark that in some areas the outer border of the syncytium of the villi and chorionic Wall shows a foamy or finely vaeuolated appearance, While in other areas larger vacuoles are formed. In our own 30»day human specimen the syncytium clothing the villi exhibits variably distributed areas where a certain degree of fine vacuolization is visible (fig. 5), besides locations Where clusters or single vacuoles ranging in size from several H up to 5 or 10 u can bee seen (figs. 6 and 16). Traces of this are encountered also in the syncytium covering the chorionic villi of an 8-weeks’ old placenta. Hofbauer (’05) remarks that foamy, vaeuolated appearances are more frequently encountered in younger than in older plaeentae. F‘ujimura’s text and figures (’2]) give the impression that vacuolization of the syneytium clothing the human villi in the early months is more prevalent than we have observed it to be. Places resembling his schematized drawings are present, but they are by no means the rule, as his manner of selection and portrayal of fields tends to suggest. In our hands, the majority of techniques show the syncytium as being on the Whole finely stippled rather than foamy, only the technique for mitochondria resulting frequently in an appearance as though those bodies Were arranged around small or minute clear spaces resembling vacuoles. The fact that Fujimura stained almost exclusively for mitochondria may account for his Widespread portrayal of vacuolization of the cytoplasm.

c. The occurrence of larger vacuoles in the syncytium of the macaque at the height of clevelopmenlt of the trophoblastic cell columns. Vllislocki and Streeter (’38), describing the macaque, point out the characteristic occurrence of large vacuoles enclosed by syncytium on the surface of the cytotrophoblastic cell columns, mainly in the neighborhood of the junction of the developing mesodermal villi with the cell columns. These vacuoles are especially abundant between the eighteenth and tWenty—second days of development and thereafter diminish in number. VVislocki and Streeter interpret these vacuoles as a manifestation of absorptive activity of a physiological nature by the trophoblast, involving the absorption of fluid from the intervillous space by a process of pinocytosis. They also suggest that their disappearance between the twenty-second and twenty—ninth days may be related to the establishment of the fetal circulation and the drainage which the circulation promotes iii the villi. More rarely, in the macaque, similar vacuoles are encountered in the peripheral syncytium which is penetrating the decidua at the junctional zone.

cF. The occurrence of irregularly scattered, large vacuoles in vairiious parts of the smzicytimn. Sizable vacuoles have been described in human material by many authors, more especially in the peripheral syncytium associated with the trophoblastic shell and the junctional or penetration zone, as Well as in the distal parts of the cell columns. In the syncytium covering the chorionic villi vacuoles when they develop (see above) seldom become sizable under normal conditions. In the peripheral syncytium, on the contrary, they are present in various sizes rangiiig from foamy vacuolization up to quite large vacuoles. An excellent account of these vacuoles is given by Florian ( ’28). They appear frequently in the redundant knobs, sprouts or tabs of syncytium which occur with especial frequency in the peripheral trophoblast (fig. 2). The presence of the vacuoles is frequently associated, according to Florian a11d others, with signs of degeneration of the syncytium, documented by nuclear changes, especially the formation of swollen giant nuclei. The vacuoles theinselves a1'e regarded by Florian as a sign of degeneration. Grosser (’27) and Florian have occasionally noticed large vacuoles which filled with maternal blood sugjgestiiig that the Wall at one place has broken down so that the vacuolar cavity communicates with the intervillous space. This We have observed also in a number of places. This particular observation stiggests thatlnot unlike the vacuoles described in the earliest stage Which become converted into lacunae containing maternal blood, some, at least, of these vacuoles break down and become incorporated into the intervillous space. Yet, even if all of these vacuoles at the period under discussion were to in this manner, it would be impossible for them to add iiiaterially to the growth or volume of the intervillous space, as the similar vacuoles do importantly in the initial stage of the formation of the lacunar spaces.

Similar vacuoles are encountered in the placental. labyrinth in thickened tabs of svncytiuin associated with the tropho— blastic cell islands.

3. C70-malclercitio/n of the g¢av'z«c5'i-3 aml significance of the 'ua.riou~.s types of eiacuolieat/ion cn.-comzlerccl in the .91/-nag/tixzt-11;.

During the earliest stages of implantation in both man and macaque, vacuolization of the syneytium is associated with the formation of blood lacunae Which are the forerunners of the intervillous space (VVis1ocki and Streeter, ’38; I-Iertig and Rock, ’41). Thus at this period vacuolization plays a definite and significant part in the development of the placenta. Hertig and Edmonds (’40) refer the formation of vacuoles in the trophoblast at implantation to the property of the syncytium to imbibe fluid.

Regarding the foamy appearances and the vacuolization of the trophoblast in various later stages, sundry ideas have been expressed. Grosser (’27, p. 349) mentions Langhans as having interpreted them as being due to postmortem changes, while Kossmann regarded them as being either resorptive or degenerative in nature. Fujimura (’21) concludes that the vacuoles encountered in the syncytium clothing the secondary villi are secretory in nature and arise by the successive transformation of mitochondria into lipoid droplets and then into secretory vacuoles which are discharged gradually into the intervillous space. Florian (’28) and others (cit. Hofbauer, ’05), in speaking of the vacuoles, are of the opinion that they are associated with and represent degenerative changes.

In regard to the vacuoles in the syncytium of the trophoblastic cell columns in the macaque, Vtlislocki and Streeter (’38) have interpreted them as being resorptive and in regard to the time of their disappearance as being related to the establishment of the fetal circulation.

In connection with the question of the genesis of placental vacuoles, the observations of Hertig and Edmonds (’40) on hydatidiform moles are of interest. They conclude from a study of this condition that the edema and vesicular transformation of the stroma of the villi, as Well as the excessive va.cuolization of the syncytium, attributable to the failure of the fetal circulation to become established or to its cessation following developmental abnormalities or death of the embryo. They arrive at the conclusion that one of the normal physiological properties of the trophoblast is to imbibe fluid from the intervillous space and that this property is demonstrated by the accumulation of fluid in the event that the cherionic vessels fail to develop as in hydatidiform moles. They also stress the parallelism between accumulation of imbibed fluid in this pathological condition and the vacuolization attending the formation of lacunae in the trophoblast at the time of implantation.

Certain of the observations recorded here appear to be in harmony with and to support the thesis of Hertig and Edmonds. Foamy and vacuolated appearances of the syncytium are much more in evidence before tl1e circulation is established than afterwards. 1n the early stages the syncytium is almost invariably finely vacuolated, whereas in the syncytium covering the secondary villi after the circulation has formed, vacuolization is very much less evident. Wiislocki and Streeter ("38) had already suggested a time relationship between the disappearance of the sizable vacuoles seen in the cell columns of the macaque and the formation of the fetal circulation. Contrariwise, vacuoles persist longest in those portions of the syncytium which are least favored by the fetal circulation. For example sizable vacuoles tend to persist in the peripheral syncytium in localities remote from the vascularized chorionic stroma. In the latter case the walls of these vacuoles appear ultimately to break down in association with the movements of the syncytium or by its degeneration, releasing the contained fluid into the intervillous space from which it originally derived. In the formation of the primary lacunae also, in the absence of any fetal circulation, vacuoles are formed which subsequently coalesce and break down giving rise to the primary intervillous spaces.

An observation by Hofbauer (’05) is of interest in this connection. He reported that vacuolization was augmented when bits of human placenta were immersed for a short period in physiological salt solution before fixation. He interpreted this as evidence of imbibition of fluid by the syncytium under the conditions of his experiment. ' ’

Thus there is some support for the belief that vacuolization is an indication of fluid transfer from the intervillous space into the syncytium. Nevertheless, as pointed out in the previous -section dealing with the nature of the surface of the syncytiurn, transfer of various substances must take place through the trophoblast from fetus to mother, as well as from mother to fetus. The probability of the taking in of fluid by the brush-border and the protoplasmic streamers was advanced, but evidence was also adduced to illustrate the possibility that other substances may be transferred in the opposite direction.

VVe have suggested that the minute vacuoles which occur in variable number in the marginal and subrnarginal cytoplasm of the syncytium probably represent fluid which is being taken in. Moreover, We have observed that this foamy Vacuolization is much more pronounced in the earlier stages than in the later ones after the fetal circulation has formed. Consequently, it is a likely assumption that the presence‘ of the fetal circulation facilitates the transfer of fluid and hence reduces the degree of vacuolization. It was also pointed out that some of the vacuoles present in the syneytium are undoubtedly returned to the maternal side when conditions do not favor their absorption on the fetal side. This is illustrated, for example, by the elimination of vacuoles in the peripheral syncytium as Well as by the conversion of vacuoles into the primary blood lacunae in the earliest stages of placental growth.

Fujirnura, in our estimation, adopts a one—sided interpretation of the small vacuoles which occur in the syncytium of the secondary villi. He considers them all as representing secretion derived from the conversion of mitochondria into lipoid droplets and these in turn into secretory vacuoles which are discharged into the intervillous space. He ignores completely the importance of the syncytiuin as a resorptive surface. In our experience foamy vacuoles and vesicles occur in the syncytium of the secondary villi with less regula.rity than Fujimura’s diagrammatic illustrations show. He employed techniques for mitochondria Which, as we have observed, accentuate the vacuolated appearance of the syncytium (figs. 13 and 14). As discussed in previous passages, we regard the marginal and submarginal vacuoles of the syncytium as being manifestations of fluid transfer rather than as representing specific. secretory structures in the sense of Fujimura. In so far as the mitochondria of the syncytium may play a role in the production of enzymes (Bensley ’42), some of these enzymes might be discharged from the syncytium into the maternal blood stream. Yet, F‘11jimura’s conclusion that the mitochondria are converted into lipoid droplets which in turn become secretion vacuoles goes far beyond the bounds of actual evidence.

4. The phagocytvic properties of the s,z,m.c;z/timn

The syneytium in the early stages of placental development exhibits phagoeytic properties. VVe wish to limit the term phagocytosis in the discussion at this poi11t to the absorption of microscopic particles, and shall 11ot include such forms of resorption as the taking in of nltramicroseopic substances (e.g., vital dyes) or the absorption of fluid (e.g., plasma) by pinocytosis, ultrafiltration or dialysis, unless such absorption is accompanied by the display of’ visible microscopic. particles. and Streeter (’38) state that in the ea.rly stages of the macaque they observe syncytial trophoblast which can be seen surrounding clumps of degene1‘atin§>; maternal epithelium. They remark also on the presence within the trophoblast of small darkly stained masses surrounded by clear spaces, which they regard as being‘ degenerating ingested clumps of uterine epithelium. Brewer (’37) cites evidence of phagocytosis in his young human specimens by both syncytium and cytotrophoblast at the decidua.l border, remarking that the maternal tissues most connnonly phagocytized are red blood cells, leucocytes and reticulum. Hertig and Rock (’41), in their two embryos, observe within the syncytial trophoblast irregular, eosinophilic, g'ranula.r or amorphous niasses which they regard as the remains of engulfed maternal tissue. They remark also that degenerating leueoe-ytes are seen, suggesting that the ingested material is maternal blood. The other papers with which we are familiar do not mention this topic. Yet the observations cited above indicate that in the earliest stages the syncytial trophoblast, especially those peripheral parts which penetrate the decidua, not infrequently engulfs and digests coarse particles of the maternal tissues. Yet most of the decidua. which is destroyed by the growing ovum doubtlessly undergoes extensive cytolysis before being absorbed, as postulated by Hill (’32) and others, instead of being directly phag'oc.ytized in the form of gross particles.

Jones and Brewer (’37) state that placental brusl1—borders are associated with pliagocytic activity, as suggested to them by Bartelmez. In our estimation the brush—borders do in all likelihood exercise resorptive functions as explained in a preVious section of this paper, but yet rarely, if ever, engulf actual cells or clumps of tissue in the sense in which pliagocytosis is usually defined. In so far as the absorption of lluid by the surface cytoplasm of the syneyticuni niay occur by pinoeytosis, opportunity is naturally offered for the transfer of somewhat larger molecules than ordinarily are tlioiiglit of as passing‘ through the boundaries of cells. Yet such possible taking in of fluid droplets by the trophoblast does not appear to involve the transfer of sizable particles by phagocytosis, because, although it seems established that many categories of proteins are transferred from mother to fetus in the hemochorial type of placenta, actual particulate matter and formed bodies do not ordinarily penetrate the trophoblast excepting the peripheral syncytium to a slip;ht degree in early human stages as described above. India ink particles, for example, injected into the blood stream of pregnant rabbits are not phagfocytizerl by the trophoblast of the placental labyrinth (lllislocki, ’21). In the present investigation, we administered 12 cc. of undiluted fliggriiis’ Waterproof India ink intravenously into a pregnant rhesus monkey of 58 days of gestation. \Ve killed the animal 4 hours later, but upon microscopic exainination of the chorionic villi found no evidence of phagocytosis of carbon particles by the trophoblast. Thus pliag'oc_yt(>sis by the human and monkey placenta appears to be restricted to the peripheral trophoblast in the relatively early stages of ggfestation.

5. The f)'T‘(%.S‘("77(7G of lipo/icl (lroplczfis in the .91/m:_1/tmm Figures 3, 9, 10, 11, 12 and 21 illustrate the distribution of lipoid in the trophoblast as demonstrated in our material in unstained sections after fixation in (lhampy’s fluid. Figures 9, 10, 11 and 21 are from hunian placeutae, sliowing the distribution of lipoid droplets at 30 days, 13 Weeks, 6 months and at term, While figure 12 shows the lipoids in the syneytium of a macaque at 4 weeks of gestation.

It will be observed that fat droplets are present in the syncytium throughout gestation, although they are larger and more numerous in the younger than in the older stages. No Fat droplets are Visible in the brush—border, and but relatively few i11 the subrna1'g'inal zone. In the main they lie in the middle of the syneytium at the level of the nuclear layer. l\lany of the droplets appear to have a clea r halo of cytoplasiii around them. There is a certain degree of inequality in their distribution, for in some regions of the syncytiuin they are more numerous than in other localities. ln the peripliieral syncytium, especially Where it occurs in knobs or sprouts, the lipoid droplets are exceedingly abundant. Two such solid sprouts of syncytium packed with fat droplets are shown in figure 3. Wle have observed no fat whatsoever in the“hans cells, altliough lipoid droplets occasionally occur in outlying cells of the cytotropho— blast in the cell columns and trophoblastic shell. Droplets of fat occur infrequently in the stroma of the villi, although in a specimen from the sixth month We have observed scattered, small droplets in the mesenchymal cells as well as in the intereellular matrix (fig. 11). The fat droplets in the syncytiurn diminish in size and number during the course of pregnancy, but fine droplets are still present at term (fig. 21).

Before proceeding to the discussion of the nature of the fat visible in the syncytium, We wish to present some observations on the occurrence of keto-compounds in the trophoblast as demonstrated by the plienylhydrazine reaction.

6. The p/rr2..s'cm"<,a of ket0nc.s- in the .9yw/cg/timn We were led to an examination of the chorionic villi for ketones because of evidence in recent years which indicates that certain steroid hormones, more especially progesterone, are produced in the placenta. lWe followed in this investigation the techniques developed by one of us for the demonstration of ketone bodies and aldehydes in the adrenal cortex (Bennett, ’40) and in the interstitial cells of the testes (Pollock, ’42). Wlhole villi from several plaeentae, both in a fresh condition and after fixation in 10% formalin, were immersed in bufI"ered phenylhydrazine hydrochloride after removal of ascorbic acid from the Inaterial with iodine. After this treatment, yellow phenylhydrazones formed on the surface of the villi but not in the stroina (fig. 1). Examination of the cell columns and cell islands revealed that it is the syneytium and not the eytotrophoblast which. shows this characteristic yellow color. Moreover, villi extracted with acetone and then irninersed in phenylhydrazine developed no yellow color. Bennett had shown previously in the adrenal that the ketones disappear upon acetone extraction, resulting in a negative color reaction. From our experience with phenylhydrazine we conclude that aeetone—soluble, water—insoluble ketones and aldehydes are present in the placenta but are confined to the syneytial trophoblast.

Fig. 1 A frozen section of a. ('horionic villus tr:-z1t.e(l with phe11}'ll1y(l1'aziue to show the production of yellow plienylliyrlrazones, which are lin1it.ed to the sync_V— tium, and are iiidieatlve of the presence and localization of ketones and aldehydes. N0 yellow color appears in control sections extracted with acetone and treated with phenylliydraziiio. Hunian, 9 weeks. X l8().

7. The nature of the lipoid droplets present in the syncytiam

In reference to lipoidal and fatty substances in the placenta, we have considered the papers by Ilofbauer (’05), van Cauwenberghe (’08), Bondi (’1l.), Acconci (’14), Ballerini (’12), Fujimura (’2] ), and Froboese (’24). A dissertation by Lewandowski (Halle, ’20) on this topic was not available to us.

All of these authors agree essentially that fat is constantly and characteristically present in the syncytial trophoblast. Experience shows that the droplets of fat show a tendency to be arra.nged in a layer in the neighborhood of the nuclei of the syncytium, as is well illustrated in our own figures. The results are essentially identical in all hands, whether osmic fixation, Sudan III, or Scarlet R be employed. Fujimura pictures more widespread and more irregularly distributed lipoidal bodies than any of the other investigators. The writers quoted agree that this layer of fat droplets diminishes during the course of gestation. We have observed that the droplets diminish in size and relative number, but it does not necessarily follow from this that the total amount of lipoid present iii the placenta diminishes, since as Needham (’31) points out chemical analyses have shown that the absolute amount of lipoidal material in the placenta is greater at term than at any preceding time. Hofbauer (’O5) and van Oauwenberghe (’08), as well as ourselves, find extremely delicate droplets of osmicated fat constantly present at term. Fujimura, on the contrary, finds no fat in the syncytium beyond the seventh month. Acconci, Bondi and Ballerini do not make is specifically clear whether any stainable fat actually exists in their estimation in the placenta at term. Froboese observes fat variably present at term in localized form, but considers it as indicative of degeneration of parts oi’ the syncytium. However, he utilized Sudan III, which in our experience as employed on frozen sections, does not reveal as delicate lipoidal particles as can be brought out by osmic acid in thin paraffin sections studied with the oil immersion lens.

Froboese states that the lipoid droplets of the syncytium are not doubly refractile. Grosser ( ’28), on the contrary, remarks briefly that the syncytium of the human placenta is birefringent, basing this opinion, we are led to infer, upon a statement by Wolff ( ’13) to the efiect that oxydase granules are present in the syncytium and that such substances are known to be birefringent. VVe have observed a considerable quantity of doubly refractile material in the syncytium of fresh villi and to a lesser extent in formalin—fiXed material. In unfixed frozen sections of chorionic villi particularly, birefringence manifests itself as a cloud of sparkling particles" most conspicuous in that portion of the syneytium in which fat droplets are most abundantly seen after osniic acid fixation or Sudan III staining. Sometimes a lesser and more delicate birefringence appears to be associated with the line of attachment of the “brush-border” on the surface of the syncytium, where little or no stainable fat is demonstrable. The birefringence disappears after treatment of the sections with acetone. Certain lipoids as well as many steroid hormones, it may be pointed out, may form birefringent crystals and are soluble in acetone. It may be recalled that the phenylhydrazine reaction for ketones and aldehydes disappears also after treatment of sections of the chorionic villi with acetone. The stroma of the villi contains occasional brilliant birefringent particles which are always much larger in size than the fine particles abundantly present in the syncytium.

In reference to the Langhans cells, most authors agree that they contain extremely little if any fat. Yet Fujimura’s diagrams indicate the presence of not infrequent droplets, as is also true of the account of IIofbauer. Van Cauvvenberghe, Acconci, Froboese and Ballerini, on the contrary, find at most only very occasional traces of fat in the Langhans cells clothing the villi. VVe have not observed any.

In the stroma of the chorionic villi fat is apparently variable in amount and distribution. In Sudan III preparations Acconci describes and illustrates in somewhat artificial drawings great quantities of fat in What one must assume to be Hofbauer cells, whereas none whatsoever is shown in the mesenchymal type of stromal cells. Hofbauer, in osmic preparations, describes fat droplets as present in quite variable but never large amounts in round vacuolated cells (Hotbauer cells) and in the ordinary mesenchymal cells, as well as also frequently as free droplets in the intercellular matrix. Bondi remarks that the stroma of the villi contains little fat, but that it may be increased in certain pathological states including death of the fetus. Ballerini states that fat is scant in the stroma of the villi, and that insofar as it is present, it reaches its maximum at the time of completion of placenta.l growth. Froboese declares that the norma.l chorionic connective tissue contains no fat (Sudan III) in the first months of pregnancy, but that some fat may appear later on, particularly in the stroma of degeneration villi. Our own experience, primarily with Champy preparations, is that fat is not present in the stroma of the villi in the first months. However, in a preparation of the sixth month We have observed scattered, small fat droplets in the mesenchyrnal cells, besides free in the stroma of a considerable number of villi (fig. 11).

One may summarize the above observations by concluding that the only constant and therefore presumably functionally significant depot of lipoid in the chorionic villus is situated in the syneytium, while none, for practical purposes, occurs in the Langhans layer. The relative amount of lipoid in the syncytium decreases up to term. The stroma contains inconstant amounts of fat, which are generally more numerous in advanced stages of gestation.

Regarding the nature and genesis of the lipoid in the syncytium of the villi, several hypotheses have been advanced. These fall under four general headings :

(1) It is a sign of degeneration of the trophoblast.

(2) It represents fat that is being transmitted from mother to fetus, for the nutrition and growth of the latter.

(3) It is an antecedent of vacuolar secretion which is discharged into the maternal circulation.

(4) It is intrinsic, metabolic or stabile fat, formed or stored in the syncytium and subserving here some as yet unknown function.

1. That the lipoid is a sign of degeneration of tlie trophoblast is a View expressed mainly by investigators in the last century and generally discarded by the more recent writers. Hofbauer, Bondi and Ballerini, amongst others, have pointed out with justification that the fat of the syncytium is associated with normal, healthy, physiologically active villi, and that in the terminal period of gestation, if it were degenerative, it should increase in amount instead of diminishing. Furthermore, Bondi and Ballerini call attention to the fact that visible fat actually diminishes in the syncytium of villi in which the trophoblast is undergoing regressive metamorphosis.

2. The majority of investigators have regarded the lipoicl in the syncytium as fat that is being transmitted from mother to fetus to serve in the growth and maintenance of tlie fetal organism. Hofbauer championed this View, and it has had followers in Bondi and Froboese. This concept rests mainly on the following arguments:

(a) The fetus derives its nourishment from the maternal organism, hence fats must be transmitted across the placenta, and the fat visible in the chorionic villi must represent that fat in transit.

(b) Since the presence of stainable fat in the syncytium resembles the stainable fat in the epithelial cells of the small intestine, which is also an organ subserving the transmission of food substances, the two must be alike.

(e) According to I-Iofbauer, Sudan III, which is soluble in fatty acids, appears, when administered to pregnant animals, in the fat depots of the fetuses.

((1) Also according to IIofbauer, cocoa-butter, when fed to pregnant animals, passes the placenta to be assimilated and stored in the fetuses.

Subsequent work on the possible passage of cocoa-butter, as well as other unsaturated fats, has not borne out Hofbauer’s observation. Apparently such fats are not transmitted through the placenta. For a critical discussion of the chemical aspects of this question, the reader is referred to Needham (’31, vol. 2, pp. 1190-1193; vol. 3, pp. 1472-1478). It emerges from NeedhaIn’s review that fats and lipoids are unequally distributed in the blood streams of mother and fetus on the two sides of the barrier. Furthermore, feeding fat experimentally to pregnant animals, or contrariwise Withholding it, has no influence on the amount of fat in the fetal circulation. Thus the maintenance of fat in the fetal blood stream appears to be independent of the condition prevailing in the maternal blood stream. Moreover, the thesis of Hofbauer regarding the passage of Sudan III is nullified by the fact that more recent observers (Gage and Gage, ’O9; Mendel and Daniels. ’12), after repeated, have not substantiated his claim. Sudan III and similar dyes administered in combination with an oil or fat in the food of the mother stain the mother’s fat depots, but are not transmitted through the placenta. Sudan III does, however, appear in the milk, besides reaching the yolk of the eggs in birds (Gage and Fish, ’24). Thus on chemical grounds the passage of fats and lipoids into the fetus appears to be more complex than the passag'e of fatty acids through the intestinal epithelium and this has led some authors to postulate that the fetus may build its own fats from proteins or carbohydrates (N eedham, loc. cit.; “lesson, ’26). More recent Work by Boyd ( ’35) and Boyd and Wlilson ( ’35) indicates that lipoid is transmitted to the fetus in the form of phospholipids, as well as in lesser amount as free cholesterol and cholesterol esters.

On histological grounds, also, the assumption that the lipoid in the syncytium represents fat in process of transmission through the placenta is not too firmly grounded. The reasoning from histological similarity with the intestine appears to us_to be Wholly gratuitous. Moreover, as Bondi and Ballerini have observed, the fat Visible in the human chorionic syncytium appears to be quite independent of the state of nutrition of the mother, and its amount does not change in the event of the death of the fetus. Also, fat has been observed in large quantities in the proliferating syncytial masses encountered in hydatidiform moles (Bondi, ’11 ; Ballerini, ’l2), illustrating that its presence does not depend upon a normal placenta nor upon a viable, growing fetus.

3. The thesis that the lipoid of the chorionic syncytium is an antecedent of a vacuolar secretion which is discharged into the maternal circulation emanates from Fujimnra. On what to us appear to be wholly fanciful grounds, he postulates that the mitochondria in the syiicytial trophoblast becomes converted into fat droplets. Many of these droplets appear to be surrounded by a clear halo in osinicated preparations, and this appearance Fujimura has interpreted as proof that the fat droplets become transformed into vacuoles which approach the border of the syncytium and discharge into the intervillous space. This whole course of events, which has little proof of its actually occuring, Fujimura regards as demonstrating “internal secretion” on the part of the placenta.

4, The fourth thesis assumes that the lipoid present in the syncytial border is a form of metabolic fat, representing‘ an intrinsic, relatively stabile deposit of lipoidal material. Ballerini (’12) advances this concept on the grounds mentioned previously that the syncytial fat is independent of and does not fluctuate with nutritional changes in the mother nor with the death of the fetus. It also occurs typically and regularly in the viable, proliferating masses of syncytium encountered in hydatidiform moles, Where it is presumably neither degenerative in nature nor being transmitted as a nutritive ele— ment. In view of these considerations Ballerini concludes that the fat of the chorionic syncytium is related to some vital metabolic activity, rather‘ than to the immediate ends of fetal nutrition.

Several writers, including Ballerini, in discussing this topic, have pointed out that if the fetus derives its supply of fat from the visible lipoidal layer in the syncytium, this bed should increase during the course of gestation instead of diminishing as it is generally accepted as doing. It should be pointed out, however, that although there is an apparent diminution there may be no absolute reduction in the amount of fat, since the placenta very much larger at the end than at the beginning of pregnancy. As Needham points out, al— though the relative percentage of various lipoids, including‘ cholesterol, determined chemically reaches its peak in the placenta in the first months of pregnancy, the absolute amount of fat in the placenta is greater at term than at any preceding period.

Although Ballerini considers the fat of the chorionic villi to be metabolic rather than nutritive, he offers no satisfactory explanation a.s to What purpose such a reservoir of lipoidal material might subserve. “Te, ourselves, not only reg_>;ard the arguments in favor of the metabolic nature of the lipoids in the syncytium as being’ more plausible than those adduced in support of the alternate hypotheses, but we believe that our observations on the presence of ketones in the syncytium lend additional support to this hypothesis.

We have found that in the syncytium of the chorionic villi, upon treatment with buffered phenylhydrazine hydrochloride solution after removal of ascorbic acid with iodine, yellow phenylhydrazoncs are formed. Villi extracted with acetone and then immersed in phenylhydrazine give rise to no yellow color. Thus it appears that. acetone-soluble, Vvateninsoluble ketones and aldehydes are confined to the syncytial c0ve1'in9; of the villi. It does not seem a mere coincidence that this reaction occurs in a tissue which is oliaracterized by the constant presence of a layer of cytoplasmic lipoid droplets which are birefringent. In the adrenal (Bennett, ’40) and the testes (Pollock, ’42) the cells which appear to contain steroid l1ormones arc cliaracterizecl (1) by the presence of lipoid substances which reduce osmic acid and stain with. Sudan III; (2) by the presence of ketones and aldeliydes which form phenylhydrazones; and (3) by the presence of bi1'efringent crystals. In the placenta, the syncytial trophoblast also displays all three of these characteristics, Whereas no other elements of the chorionic villi do so. In the placenta, the association of lipoids, some of which are birefringent, and of compounds with the solubility properties and reaction with phenylhydrazine typical of steroid hormones, indicates that the syncytial trophoblast is probably the site of synthesis and secretion of chorionic progesterone and estrogen. Thus we postulate that the lipoids in the chorionic syncytium may be related to an important endocrine function of the placenta. It is established in regard to human placental estrogen and progesterone that these substances increase in amount until shortly before birth, a course not inconsistent with the known presence of lipoids in the syncytium. Insofar as lipoids are associated with the formation of these hormones, the fetal placental tissue offers no other constant depot of lipoid from which they might emanate.

In conclusion, we have a suggestion to offer concerning the identification of the cellular elements in tissue cultures of human placenta. Gey and his associates (’38) had some difficulty in their preparations in distinguishing between cytotrophoblast (Langhans cells) and syncytium. Syncytium, if it arises typically in tissue cultures, should contain fat droplets, whereas the antecedent cytotrophoblast should not; this characteristic difference should be explored as a. possible means of identifying the two forms of trophoblast. Moreover, if we are correct in our thesis that the production of steroid hormones is attached to the presence of lipoid droplets in the syncytium, these hormones should be absent from the cultures in the event that syncytial trophoblast does not grow or differentiate. Interestingly enough Jones, Grey and Gey (’43) report in a recent paper that they were able to identify chorionic gonadotropin in their tissue cultures, but did not find estrogen or progesterone in significant amounts. This fits substantially with the conclusion of Sengupta ( ’35) that in his tissue cultures the syncytial layer degenerated, as well as with the observations of Friedheim ( ’29). The latter found that the syncytium disappeared after several days, Whereas the cytotrophoblast proliferated abundantly. In later passages, in some cultures in which growth was feeble, there was a tendency for some return to the syncytial state. If the observations of Sengupta (135) and Friedheim (’29) are essentially correct that cytotrophoblast is exclusively or preponderantly present in tissue cultures, identification of chorionic gonadotropic hormone by Jones, Grey and Grey would indicate that this hormone is a product of the cytotrophoblast.

In the identification of the type of trophoblast growing in tissue cultures, the application of stains for glycogen might also prove to be useful. As explained in detail in a subsequent section, eytotrophoblast is often rich in glycogen, whereas the syncytium contains practically none.

8. The presence of -mitochomliria in the syncytium

Previous mention or accounts of mitochondria in the human placenta exist in papers by Van Cauwenberghe (’08), DeKervily (’16), Fujimura (’2]) and VVislocki and Key (’21). Van C-auwenberghe remarks that in his experience they are difficult to stain, but that he has observed mitochondria in both the syncytium and the Langhans cells. DeKervily offers a brief statement confined solely to documenting the occurrence of mitochondria in the Langhans cells of the human placenta. Fujimura gives a detailed description of the mitochondria in all of the elements, both fetal and maternal, which comprise the human placenta. Wlislocki and Key were interested mainly in the mitochondria occurring in the trophoblast of different mammals including man, and in the human placenta at term they observed abundant mitochondria in the syneytium.

In our present material we find mitochondria in the syncy— tium from the earliest stage which we have examined (1 month) until term. The mitochondria are extremely abundant in the syneytium at 1 month, consisting of a multitude of intensively staining short rods and some granules (fig. 13). The rod—shaped ones are straight or somewhat curved; they vary in length and thickness, and individual ones may be thicker a.t one end or in the middle. They occur in great abundance throughout the entire thickness of the syncytium. The mitochondria are arranged to a considerable degree circularly around clear spaces, or vacuoles, in the cytoplasm. A certain number of these vacuoles, especially in the middle zone of the syncytium, are probabiy to be accounted for by the presence of the fatty droplets, demonstrable after osmic acid treatment shown in figure 11, Yet all of the clear spaces, especially those situated close to the surface of the syneytiuin, cannot be accounted for by the presence of lipoid droplets. These clear spaces resembling vacuoles are cliaraeteristic of preparations fixed and stained for rnitochondria; in preparations otherwise fixed and stained, vacuolization occurs only patchily and as a rule the vacuoles, whether they be the r1ega— ‘rive images of fat droplets or true vacuoles, are smaller. The syncytiuni contains many more mitochondria than the Lang‘hans cells or the cells of the stroma of the chorionic villi. in a specimen of 13 Weeks the diameter of the syncytium has diminished somevvhat, but the mitochondria are still very abundant (fig. 14). They are in general distinctly smaller in size than at the previous stage and the majority could novv be described as being very shortrods or even granules. They are still arranged to a considerable extent circularly around clear fields of cytoplasm. They preponderate in number over those encountered in the Langhans cells. As pointed out in a previous passage, the surface of the syneytium in preparations showing mitochondria presents a vacuolated, reticular appearance associated with streamers and fronds of cytoplasm projecting from the surface of the villi. Mitochondria are invariably encountered in the denser strands a.nd threads of eyto~ plasm of these reticulated borders.

At term Initochondria are still present in the syncytial trophoblast altliough the syneytiuni has been reduced for the most part to an exceedingly thin, delicate layer (fig. 19). It will be observed that at term the mitochondria are smaller than they were in the previous stages; the majority have become reduced from rod—shaped to granular forms. Nevertheless they are distinctly present in the syncytium at term.

Our observations differ in one important respect from those of Fujimura. who states that at no time after the fourth month of pregnancy could plastosomes be detected. The observations of Wislocki and Key (’21), as well as the present ones, do not bear out Fujimura. ’s statement, for we have encountered niitocliondria in the syncytium at a.ll stages of gestation.

We are also in sharp disagreement with Fujimura in regard to a matter of interpretation. He postulates that the plastesomes of the trophoblastic syncytium are the forerunners of the lipoid droplets and that these in turn, as mentioned previ— ously, become transformed into vacuoles. ‘We find no acceptable evidence, either in his account or in our material, upon which to base such a conclusion.

Recent investigations of mitochondria indicate that they contain appreciable amounts of such substances as riboflavin, succinoxidase and cytochrome oxidase (Bensley, ’42), and hence may be the chief site of localization of enzymes essential to basic cellular oxidation. In the case of the placenta, mitochondria are present in greater abundance in the syncytium than iii any other elements of the chorionic villi. If the num— ber of the mitochondria could be taken as an index of the amounts of these enzymes present iii the cell, this would indi~ cate that respiratory enzymes may be far more abundant in the syncytium than elsewhere in the fetal portion of the pri— mate placenta. This would further silggest that the oxygen utilization or cellular metabolism of the syncytium might be greater than elsewhere in the villi. This does not seem to be an unreasonable speculation when one considers that the synoytiurn may move about and flow, synthesize certain hor~ mones, and form a selectively permeable barrier, maintaining diffe1*ing' concentrations of substances on its two sides. This last function would involve the expenditure of considerable osmotic work, the energy for which would have to be supplied by oxidation reactions catalyzed by the type of enzymes found by Bcnsley to be associated with mitochondria.

In this connection observations by Wblfi’ (’13) upon certain granules in the trophoblast which give the oxydase reaction, are of interest. lndophenol blue appears in granules in the cytoplasm of both the syncytium and the l.ang‘hans cells. These oxydase granules are more abundant in the former than the latter, but within the former they are most numerous in the marginal cytoplasm at the inner and outer borders of the syncytium. In the interior of the syneytium where fat droplets are most. plentiful the oxydase granules are fewest in number. Wlolff cites Grierke to the effect that oxydase granules in general are doubly refractile, and may be lipoidal in nature.

9. The Golgi apparratus of the syncytium

“'0 have made no searching investigation of the Golgi apparatus, but our preparations bear out the more extensive investigation of Acconci (’12) in regard to this subject. He observed in human material that the Langhans cells have a Well—de1nonstrable Golgi net at one side of the nucleus. In re gard to the syneytium, the Golgi material is of a dispersed type, comprising various forms of granular rods, short threads and occasional loose networks enclosing groups of nuclei. Aeconci remarks that their shapes bear a considerable resemblance to mitochondria. One of our preparations from a monkey of 2 months’ gestation age illustrates the thread—like and widely dispersed form which the Golgi material assumes in the syneytium (fig. 15). In contrast to the general dispersion of the Golgi net in the cytoplasm of the syneytium, in the Langhans cells it forms discrete masses near the nucleus, suggesting to us that it becomes dispersed when the Langhans cells fuse to form the syncytium and the nuclei are no longer capable of dividing by mitosis.

10. The timer surface of the syncytium

As Wislocki and Streeter have shown, the auxiliary tissues of the implanting macaque egg are composed of a thin—walled, collapsed chorion one side of which develops into a solid plate of trophoblast which penetrates the endometrial epithelial cushion at the site of implantation. The trophoblastie plate soon becomes transformed into a lacunar, reticulated mesh consisting of syncytial trophoblast. The auxiliary tissue expands extremely rapidly during the first 2 weeks of development, whereas the inner cell mass, which becomes the embryo, increases Very slowly in volume. Thus most of the nourish~ ment derived from the maternal tissues at this period seems to be utilized to meet the needs of the rapidly growing trophoblast. After the third and fourth weeks of development, when the fetal heart begins to beat and the circulation is gradually established, nutriment is presumably carried in greater and increasing amounts to the embryo. At this time the growth rate of the embryo begins to overtake that of the auxiliary tissue.

Thus the initial activity of the trophoblast appears to be designed for its own immediate maintenance and growth, while the nutritive requirements of the embryo are at first relatively slight. On the twelfth day, in the macaque, the blastocyst cavity (exoeoelome) which was colla.psed at the time of implantation becomes distended again With fluid. This indicates that fluid passes through the trophoblast. into the exocoelome where it bathes the embryonic mass and probably subserves the slight nutritive needs of the slowly differentiating embryo.

By the thirteenth and fourteenth days the primary villi are composed of cytotrophoblast associated with an incomplete covering of syneytium. The primary villi project into a pool of stagnant maternal blood (intervillous space). Soon thereafter on the fifteenth and sixteenth days as the mesenchyina delaminates from the cytotrophoblast, secondary chorionic villi begin to form. The covering of the secondary villi consists of both cytotrophoblast and syncytium, yet the cytotrophoblast forms by no means an unbroken layer of epithelium segregating the syncytial trophoblast from the developing mesenchyma. Between the actively dividing cells of the eytotrophoblast (Langhans cells) there are many gaps where the syncytial layer abuts directly the mesenchymal stroma. Thus from the outset, in the macaque, the Langhans cell layer never completely segregates the syncytium from contact with the chorionic stroma.

This we regard as important in reference to the mode of transportation of substances through the epithelial covering of the chorionie villus into the stroma and the developing chorionic capillaries. If the Lang-hans layer were complete, the nutritive fluid which is transmitted to the fetal blood stream would be obliged to traverse the syneytium and the Langhans epithelium in succession. Through the observed gaps fluid being transmitted may easily by—pass the Langhans cells and with the gradual reduction of the Langhans cells as gestation advances the direct contact of syncytium and stroma constantly increases.

In our 30-day human specimen it is apparent that the syncytium, which there is reason to suppose is very plastic and flexible, appears to mold itself to the contours of the Langhans cells, dipping down between neighboring Langhans cells or clusters of them as tongues of cytoplasm which establish contact with the stroma (figs. 4, 5 and 13).

In approximation with the stroma, the deep or inner surface of the syncytium appears to present a simple border at all periods of gestation. In Paps’ preparations designed to impregnate the reticular fibers of the mesenchymal stronia, the peripheral portion of the delicate reticulum can be seen to be condensed in the form of a reticular network in contact with the deep surface of the syncytium and the Langhans cells (fig. 22). No hyaline membrane or special secretion seems to intervene between the simple surface of the syncytium and the reticular membrane. Naturally, the Langhans cells diminish in number in the course of pregnancy, contact between the syncytium and the stroma of the villi increases greatly.

The surface of contact between the Langhans cells and the syneytium also merits examination. The cytoplasm of the Langhans cells, whether or not it be signally Vacuolated, is, after staining, much clearer and lighter in color than the cytoplasm of the syncytium. Consequently the round, oval, or somewhat polyhedral contours of the Langhans cells usually stand out sharply against the darker staining, abutting cytoplasm of the syncytium (plates 3 and 4). As a rule the surface of the Langlians cells is delimited by a distinct sharp line, as though the cytoplasm were enclosed in a sliglitly denser, more deeply staining surface film or membrane (fig). 37). This has led some authors to speak of the cells as being encapsulated. Grosser, indeed, suggests that this “capsule”, since it becomes tinged a bluish color after application of l\‘Iallory’s connective tissue stain, may be a surface change related to the release or formation of a material which he designates as “fibrinoid”. (}i*osse1”s interpretation will be dismissed in a later section of our paper when we consider the possible functional activities of the cytotropliobla st in general. Insofar as certain Langhans cells possess a sharply delimited cell border, it occurs over the entire circumference of the cell. Thus the presence of a sharply defined surface membrane bears no distinct relationship to whether the border of the cell in contact with syncytiuni, a rieig*l1b()1‘i11g Langhans cell, or with the basally situated reticular Inembrane (fig. 37).

We conclude from our observations that the syncytium clothing the secondary villi contacts the underlying stronia at some points from the very outset and that as gestation advances this contact steadily increases pari passu with the {gradual diminution of the Langhans cells. We also conclude that the deep or inner surface of the syneytiurn, regardless of whether it happens to Contact lianghans cells or the stroma, is of a simple character, rjytologically speaking, and that no special hyaline or cuticular structures mediate between the s_Vncyti111n and the subjacent stroma. The outer surface of the stroma of the villi is condensed into a. delicate network composed of a1'g~yrophile reticular fibers against which the sync_Vtium and Langhans cells impinge. “Te find no reason to subscribe to the concept of van ('7a,11wenberglie ( ’07) which creates a “deep syneytial membrane” between the syneytiurii and the l.1a1’1g'llaIlS epitlieliuin, and a “basal Vitreous membrane” between the syneytiuni and the stroma. These membranes appear to us to be mainly constructs on this author’s part with little genuine evidence to substantiate their existence, as one can appreciate by examining" his Very diagrranimatie drawings.

11. The syncytium of normal villi at term

The syncytium, which is a relatively thick layer covering the villi i11 the first months of pregnancy, gradually becomes much thinner as gestation advances. Also tl1e Langhans cells dwindle in number so that in the last months of gestation they are only exceptionally observed. The capillaries of the stroma become in the course of time very much enlarged assuming the appearance of so—called sinusoids or venous sinuses. The outer portions of the walls of these sinusoids in many places become very intimately pressed against the syncytium covering the villi. At these points the syncytium appears to be thinned-out forming a delicate protoplasmic membrane, stretched over the outer contour of the blood vessels (plate 5). The nuclei of the trophoblast tend to leave these areas and to collect in groups in the somewhat thicker syncytium in the intervals between the thinned-out places. These tenuous membranous areas in close contact with the Walls of the fetal blood vessels have, with probable justification, been regarded as regions in which the most active interchange of metabolites occurs between the intervillous space and the fetal circulation. Bremer (’16) has referred to them as the epithelial plates of the chorionic villi, and has likened them to similar, thinnedout areas of protoplasm resembling plates, Which have been described on the surface of the glomeruli of the kidneys. He regards them as excretory areas, and has attempted to correlate their appearance with the disappearance of the \Volftian bodies, which he believes subserve the excretory needs of the fetus prior to the appearance of the plates. Grersh (’37) takes exception to this explanation, because he found by experiments that both the mesonephros and metanephros may function simultaneously and continuously. Of his observations he states that they do not imply that the thin membranous plates do not serve as a site of placental transfer of substances; but they do question the causal relationship established by Bremer between the appearance and frequency of the placental plates and the functional failure of the rnesonephros.

We have undertaken to reexamine carefully the cytological structure of the syneytium at term. In the first part of pregnancy We described the outer surface of the syncytium as possessing either a brush—border or of being provided with lacy or wavy protoplasmic streamers. As gestation advances the brush is more ditlieult to demonstrate and the cytoplasmic projections of the character of streamers also become less conspicuous. Nevertheless, a distinct irregularity of the syncytial surface persists throughout the last months of pregnancy. Nearly everywhere, even at term, the outer border of the syncytium is composed of fine, closely but unevenly set, hair—like irregularities (plate 5). These filaments are seldom more than one—third or one—quarter as long as the antecedent hair-like or Wavy processes observed in the first months of pregnancy. Moreover, they are extremely irregular in length giving an appearance which might be aptly described by the term “stubble.” This stubble—like protoplasmic border occurs alike over the thinned-out as Well as over the thicker portions of the syneytium. The technique for clisplaying mitochondria emphasizes the border more than the other methods employed, a. response which was observed also in the earlier period.

Wle find no reference, in the various papers which we have consulted, to the stubble—like border which we have observed on the syncytium in the last months of pregnancy. The majority of authors, if they mention the point at all, describe the syncytium as having essentially a smooth surface. An exception is van Cauwenberghe (’07) who describes and depicts the presence of a regiular brush—border on the surface of the syncytium throughout gestation. At term he shows the syncytial surface as possessing a border composed of reg;ularl_v— spaced, short, straight hairs, all of equal length, differing‘ little from the brush—border of the earlier period except that the hairs are perhaps only one—third as long. He probably observed What we have seen, but interpreted it as being arranged in a much more orderly fashion than it actually is. Vllliat We have observed is to us not a typical brush-border at all, but a succession of minute protoplasmic irregularities on the surface of the syncytium, very well depicted in figure 44. This formation indicates, we believe, that even at term the surface protoplasm is distinctly modified and possibly more plastic and unstable than is at present recognized. Interestingly enough, Hill (’32, figs. 116 and 117) presents two figures Without special comment, of chorionie villi of gorilla and man at term, in which perceptible “hairs” are visible on the surface of the syncytium.

At term, the syncytium still contains scattered, exceedingly delicate droplets of fat, demonstrable by osmie acid, which are frequently surrounded as in the earlier period by pale cytoplasmic halos (fig. 21). The trophoblast also still manifests a considerable number of granular mitochondria (fig. 19). On the other hand, it rarely contains vacuoles, the cytoplasm having an excessively delicate stippled appearance resemblin_9; ground—glass.

The latter finding is at variance with a brief report by Hedenberg and Strindberg (’16) who describe vacuoles pr0~ truding from the surface of the syncytium in a human placenta at term. In a drawing which accompanies their text, a very even and regular brush—border is depicted in the intervals between the vacuoles. They regard the vacuoles as secretion, anticoagulant in nature, which is being discl1aI'g2;ed from the syncytium. “Te have not encountered similar vacuoles in our own material. The vacuoles which they show barely eomiect with the syncytium and to us convey the impression of beingartefacts produced in the blood plasma iii the intervillons space, rather than as being a product of the syncytium itself. Also, for reasons cited in a previous paragraph, we do not regard the even brush-border, which they depict, as illustrating‘ accurately the true condition of the surface of the syncytium at term.

The thinned-out, membranous areas overlying the bulging capillaries differ in nowise from the rest of the syncytium excepting that at these points the trophoblast is very thin and is usually devoid of nuclei. Like the bulk of the syncytium, these areas possess a stubble-like border and in the cytoplasm occasional fat droplets are visible, besides a goodly quantity of mitochondria (figs. 17, 18, 19 and 21). The subjacent blood Vessels which contain fetal erythrocytes are lined by conspicuous endothelial cells which are rich in cytoplasm and possess nuclei which are thicker and have more rounded ends than one customarily encounters in ordinary blood vessels. in sections stained with hematoxylin and eosin or iron hematoxylin it looks as though the endothelial walls of these capillaries were pressed directly against the nei,g"hboring syncytium (fig. 18). However, in preparations stained by Mallory’s connective tissue, by the Azan method, or with Masson’s triehrome mixture, it is observed that the capillaries are separated from the trophoblast by an intervening material which is blue by the first two methods and green after l\’lasson’s stain (fig. 17). The nature of this interstitial material does not become entirely clear unless one adopts a silver impregnation method. After application of Paps’ method for reticulum, it at once becomes apparent that the intervening substance, which is diffusely colored by the connective tissue stains, contains sharply delineated argyrophil reticular fibers. Examination reveals that all of the sinusoidal capillaries which press against the syneytium are separated from it by a delicate intervening‘ network of reticulum which is connected around the sides of the bulging vessels with the more plentiful meshwork of reticulum occupying the core of the villus (fig. 20). No one to our knowledge has heretofore demonstrated the presence of this intervening reticular net.

These thinned—out areas Bremer ( ’16) has called “epithelial plates”, and has likened them to similar structures occurring on the glomeruli of the kidney. In the renal glomeruli, Well as in the pulmonary alveoli, however, discussion has centered inconolusively around the presence or absence of so—called “epithelial plates”, “epicytes”, and “naked endothelial or mesenchymal ground membranes”. In. our estimation, the normal huinan chorionie villus at term can be removed from these discussions, because three definite layers, namely, endothelium, a reticular network, and syncytium always intervene between the lumen of a fetal capillary and the intervillous space.

The Cytotrophoblast

The question of the cytotrophoblast as a germinal layer giving rise, on the one hand, to the syncytial trophoblast and, on the other, to Inesenchyma and angioblasts at the time of the initial formation of the secondary villi, has been briefly outlined in our introductory considerations. That the s_vncytium delaminates from the cytotrophoblast has been accepted by the majority of investigators. Vllhether the possesses any other important functions has not been clearly established. It is our purpose to present the results of our investigation of the cytological properties of the cytotrophoblast and to discuss their possible functional significance.

1. The clmmcteristics of the Langhans cells

The Langhans cells possess fairly uniform characteristics. They may exhibit rounded, ovoid or polyhedral shapes. Some authors refer to them as cuboidal (Jones and Brewer, ’35), but that suggests a regularity and angularity of shape which the majority of them do not possess. They vary considerably in size, the smallest appearing to be no more than one-third as voluminous as the largest ones. This variability is probably related to the fact that they are actively dividing. Their cyto~ plasm is usually quite clear, with relatively little affinity for stains so that they stand out in contrast to the syncytium by their pallor. The cytoplasm varies from a very delicate, uniformly stippled appearance to others in which the fine granules are dusted in a delicate reticulated pattern through the cytoplasm. In many, fairly sizable, faint vacuoles appear to lie in the meshes of the finely granular cytoplasm (fig. 37). They possess numerous rod—shaped and granular mitochondria, although not as abundantly as the syncytium. They possess no intracytoplasmic lipoidal granules, but do contain some stainable glycogen, as observed by Driessen (’07, ’O8) and Flesch (’11) whom we can confirm. A Golgi apparatus can be demonstrated in the cytoplasm on on.e side of the nucleus, although it does not appear to polarize tl1e cells in any constant or specific direction. Nor does the Golgi apparatus in our preparations stand out as clearly and distinct as pertrayed in the drawings of Aceonci (’l2). The cytoplasm is set off from its surroundings by a delicate membrane or capsule, which can be seen after applying Mallor_v’s connective tissue stain (fiw. 37), Masson ’s stain, or the Azan method. With Mallory’s stain it is colored blue, while with Masson’s trichrome mixture it is green. This delicate membrane surrounds the Langhans cells and separates them from the cytoplasm of the syncytium. It is dillicult to decide whether this structure represents (1) a definite cell membrane, and hence

an integral part of the cytoplasm of the Langhans cells; (2) a secretion derived from the Langhaiis cells; or (3) an intercellular matrix continuous with the gjr<>u11d substance of the stroma of the villus.

Vile have had difficulty in staining‘ a similar substance around the Langahan's cells in our monkey In-aterial and are somewhat perplexed by not finding it there. It will be noted that the cytoplasm of the T.a11g,'l1ans cells in figure 37 stains a faint lavender or lilac color, whereas the membrane under consideration is a definite blue. In Mallory and Masson preparations the blue or gi'ee11 respectively appears to be confluent with the ground substance of the stroma. of the villi. Impregnation methods for reticulum yield one bit of valuable information.. lt can be readily demonstrated, as figure 22 illustrates, that the matrix of the stroma of the ehorionic villi contains abundant arg'y1'opl1il fibers, an.d that these fibers and fibrils are conden sed peripherally to constitute a close-meshed network upon which the La.ngl1a.ns cells and syncytium rest, without extending up between the Lang'hans cells and syneytium into the blue capsular material under discussion. Thus this substance is devoid of fibrils demonstrable by appropriate silver techniques.

It should be noted that the question as to the nature of the blue staini_n.g substance surrounding,‘ the Tlanahans cells in the human placenta has been raised by Grosser (’25 a, b, ’27). In plate 3 of his chapter in the Volumes of Ilalban and Seitz (Grosser, ’25) he shows a field stained by Mallory’s connective tissue stain which is similar to our figure 37, although at a much lower magnification. Regarding his preparation he concludes that the cytotrophoblastic cells are separated by partitions which stain like connective tissue (with Mallory’s stain) and which consist of “fibrinoid”. This substance, which he designates as “fibrinoid”, he regards as a product or secretion of the cytotrophoblast.

We shall have occasion in a. subsequent passage to return to this subject and to a further discussion of Professor Grosser’s concept.

One other topic concerning the Langhans cells we shall briefly touch upon. It is well known that the Tlanghans cells gradually diminish in number during the course of gestation. There is little unanimity, however, as to the time of their complete disappearance. Froboese (’24) places it as early as the third or fourth month, while the majority speak of the fifth or sixth month. Merttens (1894) and Van Cauwenberghe (’07), on the contrary, believe that some Langhans cells persist until term. Grosser (’27) remarks, however, that he has encountered no convincing proof of their presence at term, and that confusion with stromal cells can hardly be avoided. VVitli these opinions in mind, we carefully examined a term placenta and succeeded in finding in iron heniatoxylin sections an occasional clear cell beneath the syncytium of the character of the one seen near the upper margin of figure 18. This cell has undoubtedly a resemblance to a Langlians cell, but bearing Grosser’s remark in mind that there might be a confusion with stromal cells, we adopted the procedure of searching for possible Langhans cells in sections impregnated for reticulum. From our experience with silver preparations we felt reasonably confident that the stromal cells would be surrounded by reticulum, whereas any Langhans cells, if present, would not be so enclosed. Consequently, we examined sections from the same placenta, which had been impregnated by Paps’ method for reticulum. On searching diligently, we came across an occasional cell resembling those of Langhans, which was partially imbedded in the syncytial layer and was not surrounded by a network of reticular fibers. The reticulum in these preparations appeared to be so completely and well impregnated down to the finest fibrils that we are unwilling to entertain the idea that the reticular net was incompletely stained. These occasional clear cells stood out from the darker, adjacent in much the same manner as in the earlier periods, although in the Paps’ preparations the nuclei appeared to be as irregularly shaped as those of the syncytium. The few cells of this character Varied considerably in size, some being quite large with big nuclei, whereas others were smaller. One of the latter is illustrated i.n .figu1'e 44..

2. The cytotrophoblast of the cell columns and the trophoblastlc shell

The character of the cytotrophoblast differs materially in the cell columns and shell from that of the typical Langlians cells. The cell columns are essentially the remains of the primary villi forming the continuations of the secondary villi. The columns extend from the tips of the secondary villi to the floor of the intervillous space, where their distal ends spread out and unite to constitute a lamina designated as the trophoblastic shell which surrounds the entire ovum. The outer surface of th.e shell constitutes the boundary between the fetal placenta and the uterine decidua. The cell columns and shell are composed preponderantly of cytotrophoblast, althoiigli the cell columns are clothed outwardly by a layer of syncytium and the shell has a variable amount of syncytium interspersed in it.

The cytotrophoblast of the proximal portions of the cell columns is continuous with the layer of Langhans cells associated with the secondary Villi (fig. 34). Consequently the cells of the proximal portions of the columns are very similar in their cytology to the Langhans cytotrophoblast; like the latter, they are frequently observed to be undergoing mitosis, and are quite compactly arranged, with few interstices between them (fig. 53.‘ ). As one passes from tl1e proximal end of a cell column toward the distal end which merges with the tropho— blastie shell, the character of the cells changes appreciably. Tl1e cells become variably separated and dispersed by the accumulation between them of a considerable amount of matrix or ground substance (fig. 34). The cytoplasm becomes increasingly vacuolated u11til many of the cells appear to be markedly distended by large, clear, unstained, or at best, faintly staining vacuoles (figs. 38 a.nd 39). The vacuoles predominate in the periphery of the cells, the faintly granular cytoplasm tending to persist and to collect in the perinuelear zone. In this stippled perinuclear area, the Golgi apparatus and the majority of the mitochondria can be demonstrated. Glycogen is plentiful i11 the cytotrophoblast, n1ost especially in the heavily vacuolated cells of the cell columns and the trophoblastic shell. The glycogen appears in Best ’s carmine preparations i11 the form of intracytoplasmic granules or droplets scattered variously in the cytoplasmic threads between the vacuoles (fig. 25). The bulk of the vacuolated cells forming the cell colu1n11s give the appearance of being viable, active cells which are not undergoing degeneration; their nuclei do not difier materially from those of the Langhans cells and show no appreciable evidence of pyenosis. 111 the trophoblastic shell, on the contrary, a certain number of the cells appear to be in process of degeneration.

The shapes of the cells constituting the cytotrophoblast are worthy of special mention. The eytotrophoblastic cells where they are close to one another and compactly arranged have fairly regular polyhedral surfaces. On the other hand, where the cells have been. dispersed, as i.n parts of the cell columns a11d trophoblastic shell, their contours become more irregular and variable. The extremely vacuolated cells have a tendency to be somewhat rounded, whereas the less vacuolated ones may take on various shapes. ‘Some of the latter become quite stellate, while many others become variably elongated and irregular (fig. 24).

In this connection recent observations of placental tissue cultures are of considerable interest. Friedheim (’29), Sengupta (’35), and Jones, Grey and (ley (’42) have shown that it is the eytotrophoblast principally which survives and proliferates when cultured. Friedheim ha.s observed that the cytotrophohlastie cells in the deeper parts of the cultures are compactly arranged resembling a cellular mosaic, but that on the periphery they tend to be loosely arranged and isolated from one another. Wherever this occurs the cytotropliohlastic cells become actively motile (Friedheim) and send out a Variety of cytoplasmic streamers and threads (Friedheiin; Jones, Gey and Grey). This motility and streaming of the cytoplasm account perhaps for the Variety of shapes, enumerated above, which one encounters in fixed preparations amongst the cytotrophoblastie cells in various parts of the cell columns and trophoblastie shell.

The trophoI)la..stic shell (1,/ml» the I/if-I’(V.’tL/i/()?’l(/Ill zone

The junction of the trophohlastie shell and the decidua, variously designated as the “junctional”, “transitional” or “penetration” zone, is Variable in character. In some areas this zone contains a goodly quantity of cellular debris, While in other areas the trophoblast and decidua meet without the presence of any great amount of intervening detritus. The discriinination as to which cell elements in this zone are fetal and which are maternal otters considerable difficulty, as many observers in the past have indicated. In man the transition is somewhat less clearly defined than in the rhesus monkey (\*Vislocki and Streeter, ’38), because in the former the tabs and strands of Vacuolated syncytiuin associated with the trophoblastie shell are more numerous and penetrate more deeply and irregularly into the deeidua than in the monkey. By liistologieal and cytological means typical cytotrophoblastic cells can often be distinguished from decidual cells (plate 10). Yet, so many of the decidual cells and cytotrophoblastic cells in the junctional zone are or atypical that it is easy to confuse them. The decidual cells in or near this ‘cone often become highly vacuolated (fig. 31) and they may contain lipoid droplets (fig. 32). The latter circumstance might be helpful in telling them apart were it not for the fact that degenerating cytotrophoblastic cells of the shell also acquire a variable number of fat droplets stainable by Sudan III, or osmic acid, as observed by Froboese (’24), Jones and Brewer (’35), and Brewer (37). Jones and Brewer (’35) have suggested utilizing Best’s earmine, for glycogen, as a means of distinguishing decidual cells from cytotrophoblast, but it is obvious that staining for glycogen after formol—Zenker fixation, as adopted by them, otters no valid means of discrimination since glycogen is highly soluble in aqueous solutions. Driessen (’O7) and Flesch (’11) have amply shown that glycogen is abundantly present in the decidual cells as well as in the cytotrophoblast of the human placenta. In our hands, in both human and monkey material, we have found Best’s glycogen stain inadequate to discriminate clearly between the maternal and fetal elements of the junetional zone. (Compare figs. 25 and 43)?

The only completely satisfactory means of distinguishing the boundary between the fetal and maternal tissues in the junctional zone is by the use of silver impregnation methods for reticular fibers. Keibel (’11), using Bielschowsky’s silver impregnation technique, first observed that by this method the connective tissue fibers of the decidua are impregnated, but that the reticular fibers cease at the border of the trophobla stic

“Windle (’40) makes the categorical statement that only the maternal portion of the placenta contains glycogen. This is apparently true of mouse (Driessen, ‘O8) and rabbit (Chlpman, ’02; Driesscn, ’O8). However, in the rat, in a colored figure (plate 5, fig. 2) Goldrnann (’.‘12) shows glycogen as abundantly present in the fetal syncytinm, stained with Best’s earznlne. In man ‘it is well known that glycogen is alnizndmitly present in the cytoti-opliohlast (Driessen, ’07 ; Flesch, ’1l).

Thus no generalization about its presence or absence in the fetal placenta can be drawn.

A statement by Tenney ( ’36), to the eflcct that glycogen disappears from the human syncytinm during its involution, suggests that the syncytial trophoblast normally contains glycogen. Yet, others have not observed it in the human sync);tium with the exception of Flesch ( ’ll), who reports having encountered traces of it with Best’s carmine following n niodificd fixation.

shell where the fetal tissue begins. Jones and Brewer U35), repeating this observation, conclude that the connective tissue ends abruptly at the decidual edge, While in the cytotrophoblast no argyrophil fibers can be impregnated. Using Paps’ impregnation for reticulum we have obtained similar results in both the human and the macaque (figs. 28 and 29). In the macaque the reticulum ends sharply, only a few, short, disjointed fraginents occurring amongst the outermost cells of the trophoblastie shell (fig. 28). In the eytotrophoblast the only elements blackened by the silver are small punctate bodies which occur variably in the cytoplasm of the tropho— blastic cells. In the human the picture is essentially the same, the frayed and amputated ends of the argyrophilic decidual reticular net terminating at the border of the trophoblastic shell (fig. 29). As Brewer (’37) has pointed out, stray bits of frayed or detached reticular fibers on the extreme periphery of the trophoblastic shell give a semblance here and there of having been phagocytized by both syncytial and cellular trophoblast.

It is reasonable to assume that the dissolution of the decidua, including the fibrous reticular network as well as its cells, occurs by the agency of cytolytic substances given off by the trcphoblast. ‘Whether these hypothetical substances eman— ate solely or mainly from the syncytium or from the cvtotro— phoblast has been a matter for difference of opinion. Gr'zifen— berg (’10) and Kiss ( ’21) have advanced the view that it is the cytotrophoblast rather than the syncytium which destroys the decidua. In the days immediately following implantation in the rhesus monkey (Vtlislocki and Streeter, ’38) and in man (Hertig and Rock, ’41), syneytial trophoblast is almost eX— clusively present on the periphery of the blastocyst, and hence presumably plays the major cytolytic role. During the third and fourth weeks of development, however, the trophoblastie shell differentiates into a variably thick lamina surrounding the ovum. Although it contains some syncytium and in the human, especially, exhibits occasional syncytial strands which penetrate deeply into the decidua, the border of the trophoblastic shell consists mainly of cytotrophoblastic cells which come in contact with the decidua. This is especially true of the rhesus monkey, and is well illustrated in plate 13 of the paper by Vllislocki and Streeter ( ’38), showing placental specimens of the twenty—ninth and thirty—fifth days. Thus, at this period, the thesis of Grrafenberg and Kiss that the cytotrophoblast rather than the syncytium may be principally responsible for the cytolysis and destruction of the adjacent deeidua receives some support. Friedheim (’29) has observed that the medium of tissue cultures is liquefied by the cytotro— phoblast but not by the syncytium; this he attributes to the proteolytie activity of the former.

4. The dceidua

Our study deals essentially with the trophoblast and does not include a comprehensive survey of the decidua. Yet, because we have included the trophoblastic shell and the junctional zone within the scope of our examination, we have had to familiarize ourselves to some extent with the appearance of the decidua. Since the literature contains few satisfactory drawings or photographs illustrating the cytology of this tissue, we have included six figures showing the details of decidual cells, as we have encountered them in human and monkey material (plate 10 and figs. 43 and 45).

Figure 30 shows a conspicuous and common type of decidual cell in human material, which possesses smooth, delicately stippled cytoplasm, some of the cells being darker than others. Figure 31 illustrates the presence of mitochondria in decidual cells, all of which are more vacuolated than the preceding type. Figure 32 represents decidual cells most of which contain fat droplets. Figure 33 illustrates a field of decidual cells in the rhesus monkey. Figure 43 shows decidual cells of the rhesus monkey, stained for glycogen with Best’s earmine, while figure 45 depicts another group of decidual cells with the Golgi apparatus impregnated by the l\=Iann—Kopsch method. It will be observed from the six figures, all drawn to scale, that the decidual cells of the monkey are distinctly smaller than those in the human. Some of the difficulties of telling decidual cells and cytotrophoblastic cells apart under certain conditions will be appreciated by comparing figure 33 with 26, and figure 31 with figures 27 and 29.

5. On the HLZ-t'ZH”C’. mzd genesis of the mtc'r.9titial m,artm'a; associated with the cytotrophoblalst. The question of “fibrm0'icZ”

Vlle come now to the presenta.tion and discussion of one of the most obscure problems of placental histology, namely, the nature and genesis of those substances designated as fibrin, canalized fibrin and fibrinoid.

These substances are familiar to most observers of placental tissue from the widely used hematoxylin and eosin preparations in which they stain in a variety of shades of pink or red (fig. 41). They manifest themselves in Various ways as ground substances associated variously with the trophoblast in the cell columns, trophoblastic shell, cell islands, and subchorial tissue of the closing plate, besides occurring in the decidua capsularis and white infarcts.

Professor Grosser (’25 a, b, ’27) has been the main investigator of these substances in the last 20 years, and his opinions in regard to them have been generally accepted by other writers. He distinguishes between “fibrin” and “fibrinoid” substances. “Fibrin” he defines as precipitated fibrous material derived from blood, lymph or tissue fluid. “Fibrinoid” he classifies a.s a substance occurring in rather homogeneous masses or deposits which stains less red with eosin than fibrin and, on the other hand, stains intensely blue with Mallory’s connective tissue stain. Fibrinoid occurs, according to him, in many localities in the placenta and arises in a variety of ways from two main sources, namely, (1) from the trophoblast, and (2) from the maternal tissues.

The major essentials of his classification of “fibrinoid” may be summarized in the following manner:

I. Fibrinoid derived from the zfrophoblast, its major source.

1. In early stages of development some of the syneytium produces fibrinoid or is converted into it.

2. The cytotrophoblast of the cell columns and trophoblastic shell secretes fibrinoid, and is also transformed into it by degeneration.

3. Similarly the cytotrophoblast of the cell islands produces fibrinoid, and is also converted into it by degeneration.

4. In later stages, the ehorionic epithelium in general, including the syncytiuni in later stages, may become converted into fibrinoid, mainly by degeneration. This includes the appearance of subchorial iibrinoid as well as the transformation of the trophoblast covering some of the villi into fibrinoid accompanying the formation of white infarcts.

II. l?‘ibri1-zioicl derived from fetal mesoderm. Since the stroma of certain villi participating in the formation of white infarcts ultimately vanishes in the ground substance, the fetal mesoderm can also be regarded as a source of fibrinoid, albeit a fairly negligible one.

III. Fibriinoid derixvcd from degeneraitimg matter?/zva~l tz'3sues, its secomlary source. Laminated, homogeneous masses of fibrinoid, partly fetal and partly maternal in origin, are observed on the periphery of human ova of the second and third Weeks. Similar Inasses appear also in later stages, giving rise to Nitabuch’s and Rohr’s layers. Degenerating maternal tissue contributes also to the formation of fibrinoid in the decidua eapsularis.

According to Grosser, then, “fibrinoid” is a noncellular, nonfibrous, homogeneous substance, staining pink with eosin and blue with Mallory’s connective tissue stain, derived from heterogeneous sources, partly as a secretion, and partly as a product of degeneration of trophoblast (both syncytial and cellular), but secondarily also from degenerating maternal tissues. It can be differentiated from fibrin which becomes variably deposited in or near it; fibrin is fibrillar or granular, stains red with eosin, and is derived from blood plasma, lymph or tissue fluid, and is presumably entirely maternal in origin.

“Fibrinoid” contains neither reticular (argyrophil) fibers nor bundles of collagenous fibers, hence is not ordinary connective tissue. In spite of its heterogeneous origins, (}rosser’s state ments indicate that he believes it to be a rather definite entity into which a. number of diverse cellular products and cells are transformed. The designation “fibrinoid”, which literally signifies “fibrin—like”, Grosser introduced in the first edition of his well-known book, adopting the term for no better reason than that the “substance” under consideration differed from fibrin and that the term “fibrinoid” had already been applied to the placenta by Neumann, although in a somewhat different sense (Grosser, ’25 a, ’27).

Our own observations on this topic agree with those of Grosser in two major respects. First of all, it is possible, in our estimation, to distinguish fibrin histologieally by the application of Mallory’s connective tissue stain. Wle may add that we have found the Azan method and Masson’s trichrome stain to be equally satisfactory. Wlith Mallory’s stain and Azan, fibrin is stained a brilliant red (figs. 40 and 42), while with Masson’s stain it is a brownish or yellowish red. The tinctorial reactions of these stains stand in marked contrast to eosin as used in hematoxylin and eosin preparations, which does not differentiate clearly between fibrin and other interstitial deposits ( 41). Indeed, the great uncertainty in the past as between fibrin, canalized fibrin, and fibrinoid substances must be attributed to the long continued adherence to the use of hematoxylin and eosin.

Our second point of agreemnt concerns t.he absence of argyrophil reticular fibers or of collagenous bundles in the ground substance associated with the trophoblast. The combined ob— servations of Keibel, Grosser, Jones and Brewer and ourselves should dispel all doubts on this score. Although there are no argyrophil fibers in the matrix associated with the tropho— blast, silver impregnation methods, as one might anticipate, may occasionally stain “cement lines” between the eytotrophoblastic cells when they are compactly arranged (Grosser, ’25 a, 4). A paper by Tenney (’35), which does not refer to the pertinent literature on this subject, concludes that the ground substance associated with the cytotroplioblast must be collagenous, because it stains blue wit.h a modification of Mallory’s connective tissues stain, —— this in spite of the fact that the author realized that this stain is not specific.

In regard to the genesis of the ground substance (“fibrin— oid”) present in the placental structures under discussion, we also agree with Grosser that this matrix is derived from both maternal and fetal sources. We disagree with him, however, as to the relative importance of the respective sources, as well as in regard to the nature and functional significance of the various components contributed by the fetal and maternal tissues. Grrosser places the emphasis on the homogeneity of the ground substance, stressing its uniform optical appearance and tinctorial reactions. Yet, because of its diverse origins, it sems to us that the ground substance may well be different in composition and properties in various localities, in spite of the fact that histochemical means are not yet available to demonstrate such differences. We are of the opinion also that fibrinogen, from which fibrin arises, may be present in the ground substance in Varying amounts, but not clemenstrable there by any known staining reaction.

Since the interstitial ground substance appears to be the sole medium of chemical exchange between the cytotrophoblast and the decidua or the intervillous space in the case of the cell islands, we look upon it containing substances diffusing from the maternal blood or tissues on the one hand as well as substances diffusing in the reverse direction from the cytotrophoblastic cells to the maternal blood. Included in the latter are not only excretory products _of these cells, but also any hormones, enzymes or other specific metabolites which these cells might synthesize and secrete.

We introduce these speculative considerations concerning the ground substance advisedly in order t.o weigh and appraise the equally speculative and tentative interpretations advanced by Grosser. VVe believe that such discussion will usefully stimulate further investigation of this topic. As a preface to our further reflections on this subject We present below an outline of our own concept of the nature and genesis of the ground substance and fibrin associated with the trophoblast.

I. The ground substance of the trophoblastic cell columns and the trophoblastic shell represents: (1) mainly, a translidate from the cndometrium consisting‘ of tissue fluid (edema fluid) which contains nutritive 1nateria.l derived mainly from cytolized decidua, glandular secretions and hemolized maternal blood; (2) in other part a product of the cytotrophoblast including cytolizing agents and possibly other physiological substances; and (3) a certain amount of necrotic trophoblast, small at first, and increasing with age until the cell columns and shell disappear.

11. The ground substance of the cell island represents: (1) in part a transudate of maternal blood plasma: (2) in other part a product. of the eytotrophobla st, possibly including physiologically active substances; and (3) degenerating‘ cellular and syncytial trophoblast, minimal at first and increasing’ with age.

TH. The ground substance of white infarcts represents an elaboration and extension of H, in which Widespread degeneration of the trophoblast and of entire villi occurs.

IV. The ground substance of the capsularis consists of ( 1) a transudate from the maternal tissue, as in I, containing much necrotic decidua and extravasated blood; and (2) of products of degenerating trophoblast. The capsularis has a limited physiological existence and represents in the main a. degenerating structure.

V. Fibrin, which is derived from fibrinogen, appears in variable amounts a.nd distribution in all of these structures. Fibrinogen probably occurs in proportion to the amount of blood plasma or extravasated blood that has entered into the formation of the matrix. It is probably more concentrated in some localities than others, although it can not be distinguished by histochemical means. Fibrin appears to be progressively but variably precipitated in the ground substance, clearly indicative that fibrinogen is one of its components. The fibrin so formed is recognizable by its staining reaction (Mallory, Masson, Azan) as illustrated in figures 40 and 42. Fibrin may also form directly on the surface of degenerating placental tissue by the clotting of stagnant blood, thus contributing, for example, to the progressive increase in size of White infarcts.

Our experience with the trophoblas-tic cell columns and trophoblastic shell goes back to the day—by-day study of these structures in a well-fixed and stained series of macaque embryos (Wlislocki and Streeter, ’38). It was observed that from the sixteenth or seventeenth day on, when the trophoblastic cell columns and shell begin to assume their characteristic form, the cells begin to become loosened up by the accumulation of fluid between them. This separation or loosening up of the cells commences in the trophoblastic shell and the distal parts of the cell columns, diminishing toward the proximal parts of the cell columns which attach to the secondary villi. This edematous condition of the cell columns and shell is well discernible by the twentieth day, and is extremely pronounced by the end of the first month. The presence of interstitial fluid in the cell columns and shell of the macaque characterizes these structures as long as they persist. Wislocki and Streeter, referring to the lakes of interstitial fluid in the proximal parts of the cell columns and trophoblastic shell, speak of them as representing fluid, containing nutriment, that presumably nourishes the trophobl.ast. They remark, further: “Indeed, the junctional zone, which contains degenerating maternal tissue and edematous fluid, must be regarded as "a continuing source of nutriment for the proliferating cells of the trophoblastic shell.”

Wle see no reason for changing this interpretation of the nature of the protein-containing ground substance lodged between the cytotrophoblastic cells. If one believes, as many have postulated, that the decidual endometrium by its glandular secretions, necrosis of its cells, and the extravasation of maternal blood Within it, nourishes the trophoblast during its active phase of growth, how else than in the manner postulated can one envision the transfer of this nutrient material to the growing trophoblast?

This fluid can be seen in figures 34 and 39 from sections of a monkey placenta. It stains a vivid green with Masson’s stain and a bright blue after Mallory’s stain and with Azan. In keeping with this concept also is the observation that the green staining protein—containing fluid, confined in the interstices of the cell columns and shell, is continuous with a similar green or blue staining ground substance present in the decidua, the major difference being that in the decidua the matrix is associated with demonstrable reticular or collagenous fibers, whereas these are absent in the matrix of the cytotroplioblast. Grosser, on the contrary, regards this ground substance primarily as a product of the trophoblast, basing this supposition upon the reasoning that the masses of “fibrinoid” may be completely surrounded by trophoblast making it impossible for them “to be derived from any other source than from transformed fetal cells” (Grosser, ’25 a, p. 311). Yet, we doubt the validity of Grosser’s conclusion because in the main the ground substance can be traced with ease and Without interruption from the cell columns through the trophoblastic shell into the decidua. In Grosser’s example, Where in one section the ground substance is seemingly enclosed in an unbroken Wall of cellular troplioblast, We are not impressed by the proof, Which Would demand a statement derived from study of a complete series of serial sections. Thus we postulate that the ground substance of the cell columns and trophoblastic s.hell represents in major part a transudate from the decidua.

We are prepared, nevertheless, to believe that the cytotrophoblast may contribute to the formation of the ground substance, although we do not regard it, as Grosser does, as necessarily the principal source of the matrix. He bases his conclusion on the presence of a network of blue staining matrix (after Mallory’s stain) which surrounds the cellular troplioblast (fig. 38), as well as upon the presence of what he calls blue partitions between the Lang-hans cells of the secondary villi (fig. 37). He cites the excellent description of Graf Spee (’15), who first observed the cytotrophoblastic cell columns after application of Ma.llory’s stain. Grrosser’s main support for this conclusion appears to be that since the eytotrophoblastic cells are surrounded by a matrix, this associated substance must be mainly a product of these cells.

In seeking for possible evidence for the participation of the cytotrophoblast in the formation of the ground substance, perhaps the first thing that comes to mind is the repeated postulate of many recent Writers that it gives off cytolytic substances which are responsible for the necrosis and other changes in the deeidua in the immediate neighborhood of the Primate ovum. On the basis of the observed changes it seems reasonable to assume that the syncytium and the cytot.ropho— blast, especially of the cytotrophoblastic shell, may give rise to and liberate proteolytic enzymes.

It should be remarked also that the cytotrophoblast may secrete the chorionie gonadotropic hormone. In tissue cultures, Friedheim (’29), Sengupta (’35), and Seegar, Grey a.nd Gey (’42) have observed the proliferation of cellular trophoblast, Whereas little, if any, syncytium is formed. Moreover, Gey, Seegar and Hellman (’37) and Jones, Grey and Grey (’42) have demonstrated the production of ehorionic gonadotropic hormone in such cultures over a period of several months. A more detailed discussion of the possible role of the cytotro— phoblast as the source of chorionic gonadotropie hormone will be presented in a. subsequent section.

In searchin for other possible evidence of secretion by the cytotrophoblast one’s attention is attracted to the vacuolated appearance of its cytoplasm. The cytotrophoblast of the cell columns and of the trophoblastic shell is conspieuosly vacuolated (figs. 38 and 39), whereas the Lang-hans cells exhibit a, lesser degree of vacuolization (fig. 37). The cells at the junction of the primary and secondary villi, where mitoses are plentiful, show the fewest vacuoles (fig. 38). Vaeuolization is, however, somewhat variable, for in the monkey, even in the trophoblastic shell wehave observed fields in which vacuolization is relatively slight, whereas at the proximal ends of the cell columns in occasional areas, ‘where the cells have become loosened up, vacuolization may be prominent (fig. 24).

A characteristic feature of these vacuoles is that they are uncolored by the stains employed, whereas the protein—c0n— taining. ground substance between the cells stains brilliantly (figs. 38 and 39). Thus the colorless intracytoplasmic vacuoles appear to differ in composition from the matrix. They are distinguishable also ‘-from the vacuoles encountered in the peripheral syncytium becauselnany of the latter contain a certain amount of-flocculent precipitate which is tinged ‘by the stains employed.

Certain writers have interpreted the vacuoles in the cytotrophoblasticcells as a sign of degeneration. Yet in our estimation, the majority of the cells possessing vacuoles do not exhibit nuclei which are abnormallyswollen or noticeably pycnotic (figs. 24, .26, 38vand 39). Nevertheless, in the tropho— blastic shell, especially in-' the vicinity of the junctional zone, many of the vacuolated cells are undoubtedly disintegrating and their nuclei are perceptibly altered (figs. 27 and .29).

We regard the majority of the cytotrophoblastic cells. as being actively metabolizing and secretingcells Which are taking in certain substances at the same time that they are engaged in discharging others. Friedheim ( ’29) and Jones, Grey and Grey (’43) have observed living cytotrophoblast proliferating in tissue cultures. In such preparations numerous cytoplasmic streamers, threads and pseudopodia are described on the surface of the cells, associated with the presence of abundant intracytoplasmic vacuoles. Cytoplasmic streamers, pseudopodia and vacuoles are notably present", according to Friedheim, in the cytotrophoblastl. of tissue cultures when the proliferating cells -are loosely arranged, whereas they are absent in areas where the cells have grown in compact fashionand are crowded. This behavior has a certainlcounterpart in-the appearances of fixed preparations of the placenta. In our sections, in those areas where the cytotrophoblast is compactly arrranged, there is little or no interstitial substance and the cell boundaries are snugly fitted together giving a mosaie—like appearance (fig. 23). In areas of the sections Where the cells are loosely arranged with abundant interstitial matrix, they are, on the contrary, as a rule variably stellate or irregular in form and bear a certain resemblance to the loosely arranged cytotrophoblastic cells proliferating in tissue culture. In figure 24, for example, the cytoplasm of .the loosely arranged cells, especially on the right and top of the figure, is drawn out into delicate vacuolated sheets and streamers resembling the -condition described in the living cells in tissue cultures.

It seems noteworthy to us that cytotrophoblast cells seen in tissue cultures are almost interchangeable in their appearance with fibroblasts as described in the living rabbit’s car by Stearns (’40) and in macrophages, fibroblasts and cancer cells in tissue cultures by Lewis ( ’3l).

From the foregoing considerations we are inclined to regard the majority of the vacuolated trophoblastic cells as being physiologically active and normal and to assume that the vacuoles are related to their functional activities. These cells are necessarily taking in fluid and certain metabolites at the same time that they are excreting or secreting other substances. IVhich of these functions the vacuoles subserve is difficult to decide from the histological pictures. The rea.der’s attention is called to figure 24, in which at some places on the cell surfaceslthe vacuoles and the cytoplasm are seemingly in process of being liberated into the matrix. In the event that We are correct in our postulate that enzymes and ehorionic gonadotropic hormone are liberated by the cytotrophoblast, it is from these surfaces that such substances must be dis-charged. Yet we have no real proof that the vacuoles necessarily represent specific means by which these secretions are being released.

In the cell islands We lean. similarly towards the View that the ground substance is a mixture of plasma derived from the maternal blood in the intervillous space and secretory and excretory products of the cytotrophoblastic cells (figs. 36 and 40). Here, too, the cells be variably vacuolated and the majority of them, especially in the early stages of gestation, do not show noticeable signs of degeneration. The syncytium covering the cell islands is at best incomplete, so that it does not form a continuous sheet which might influence the diffusion of plasma i11to the interstices of these structures.

Ultimately much of the cytotrephoblast degenerates and vanishes, contributing its substance to the matrix. Moreover as the matrix ages, the Iibrinogen which one must assume accumulates is gradually converted into fibrin. Figure 40 illustrates fibrin just beginning to be deposited at a number of points in the ground substance of a cell island. The fibrin increases and involves more and more of the ground substance (fig. 42). In the junctional zone and in the deeidua capsularis, where actual blood has been extravasatecl or where blood stagnates in the intervillous space in contact with cell islands or degenerating villi, extensive deposition of fibrin may occur.

A subsidiary observati.on regarding the ground substance deserves to he mentioned in concluding our presentation of this topic. In the two monkey placentae of the fifty—eighth and sixty—second days, many of the secondary villi exhibit a sharp line of vividly staining matrix between the stroma of the villus and the cytotroplloblast of the proximal ends of the cytotrophoblastic cell columns (figs. 24 and 34). This delicate lamina of brightly staining hyaline-like material lies definitely between the reticular basement network a.nd the cytotroph0blast, as demonstrated to our satisfaction with silver impregnations for reticulum. It does not apparently communicate with the ground substance of the stroma by fading into it; instead, its border is sharply delimited from the latter (fig. 24). In the same monkey placentae some of the cytotrophoblastic cell columns have loosened up so that their interstitial substance extends all of the way to the proximal ends where the columns join the secondary villi (fig. 24). There, the interstitial substance of the cell columns appears to break through the last barrier of vacuolated cytotroplioblastic cells at some points to meet and mix with the peculiar lamina of matrix to which we have called attention. This hyaline lamina stains more vividly and deeply green or blue with M-asson’s or Mallory’s stains respectively than the regular ground substance of the cell columns, indicating that it may contain a higher concentration of protein than the latter. A slight distance from the tips of the secondary villi this structure fades from View (fig. 334). Indications of a similar, although much less Well—developed, concentration of matrix has been observed at the tips of human secondary villi iii the second and third months of gestation. \Ve have no explanation to offer regarding the nature or significance of this structure.


We have presented evidence in this paper suggesting that the lipoid droplets in the syncytium are associated with the presence of ketone bodies or aldehydes as revealed by a yellow reaction of the syncytium with phenylhydrazine. This observation suggests that the syneytium may be the site of production of those steroid hormones believed to arise from the placenta.

Certain clinical pathological observations fit in Well with such an assumption. Bondi (’11) and Ballerini (’12) both emphasize that in areas of degenerating syneytium in both normal and abnormal human placentae, the fat droplets diminish or disappear entirely. Tenney (’36) also remarks briefly that no fat can be demonstrated in degenerating syncytium. It is an accepted fact that normal placentae, as they approach term and undergo physiological aging, show a certain amount of degeneration of the syncytium involving, according to Tenney (’36), 10 to 50% of the villi. Tenney (’36) and Tenney and Parker ( ’40) believe that they have demonstrated that in toxemia, severe preeelampsia and eclampsia there is a very definite and significant increase in the amount of degenerated syncytium. These lesions involve the ultimate disappearance of the nuclei from the syncytial layer and the conversion of its cytoCYTOLOGY on PLACENTAL TROPHOBLAST 407

plasm into a thin, irregular layer of hyaline material. Smith and Smith (’-11), studying the endocrine imbalance of toxemia, preeclampsia and eclampsia, report that these conditions of late pregnancy are closely associated with a deficiency of progestin, a concomitant decrease of estrogen, and a consequent changed metabolism of estrogen involving greater and more rapid destruction. l

The decrease in lipoid demonstrable histologically in degenerating trophoblast, considered together with the appreciable increase in degenerating syncytium iii the toxemias, combined with the observations that progesterone and estrogens diminish in toxemias, supports the concept that the syncytium and particularly its lipoidal droplets have a definite relationship to the normal production of steroid hormones.

Interesting in this connection are certain observations on the presence of lipoid droplets in the syncytium in hydatidiform moles. Bondi ( ’11) reports that the fat in the syncytium in that condition is histologically normal in amount and distribution, While Ballerini ( ’12) states that visible lipoids increase in the invasive syncytium, although they are absent in the walls of the vesicles. Hcrtig and Edmonds (’40) point o11t that in this disease the invasive syncytium appears to be essentially normal, whereas that covering the vesicularly-trans formed stroma tends to undergo degeneration. The citations presented above regarding the disappearance of lipoids in degenerating syncytium would seem to explain the absence of fat in the degenerated portions of the syncjvtial trophoblast. On the whole, the lipoid of the syncytium in hydatidiform moles, one might anticipate, should be variably decreased depending upon the amount of syncytium present and the extent to which it had undergone degeneration. Hence, in View of the concept developed above, we should expect to find steroid hormone production by the placenta in this disease variably diminished. Smith and Smith (’35) found comparatively low levels of “oestrin” in cases of ehorion epitliclionia and l1ydatidiform mole.

Of further interest in regard to the site of formation of steriod hormones is the recent observation of Jones, Grey and Grey (’43) that estrogen and progesterone could not be recovered from placental tissue cultures in amounts indicating their production there, although cliorionic gonadotropic hormone was evidently being produced. Friedheim (’29) and Sengupta (’35) had previously emphasized that the syncytium tends to degenerate soon after explantation and is not reformed as long as the cultures are actively proliferating. Insofar as syncytium reappears eventually, it seems to be derived from the cytotrophoblast, is slight in amount, and, from the descriptions which these authors give, appears to us to be rather a.typical. It would be of interest to find out whether these occasional atypical syncytial masses ever contain lipoid droplets. The negative finding for steroid hormones (Jones, Grey and Grey) in placental tissue cultures, combined with the observation that the syncytium is scanty in amount or absent, supports our concept of the syncytium as the site of formation of placental steroid hormones.

"We pass now to a consideration of the evidence regarding the possible site of formation of the chorionic gonadotropic hormone. It is generally firmly accepted that this hormone originates in the troplioblast, although it is uncertain whether it emanates from the cellular or syncytial trophoblast, or both.

Observations by Friedheim (’29), Sengupta (’35), and Jones, G-ey and Grey, (’43) who have studied chorionic villi in tissue cultures, indicate that it is the cytotrophoblast, rather than the syncytium, which undergoes proliferation. Syncytial trophoblast, insofar as it arises, appears to be derived from the cellular form, is small in amount and atypical in appearance. Indeed, in his most actively growing cultures Friedheim observed no conversion whatsoever of cytotrophoblast into syncytium. Grey, Jones and Hellman (’38) and Jones, Gey and Grey (V13) have demonstrated that cultures containing aetively growing cytotrophoblast produce appreciable quantities of chorionic gonadotropic hormone, even after several months have elapsed. These observations indicate that the eytotrophoblast is the source of the hormone.

Kido (’37) reports that gonadotropic hormone was produced by human placental tissue transplanted in one experiment to the anterior chamber of the eye of a rabbit. There, it is stated, marked proliferation of Tianghans cells occurred, but it seems very doubtful to us that such a heterologous graft could have actually maintained itself.

Chorionic gonadotropic hormone is, as a rule, abundantly present in the urine of women suffering from hydatidiform mole or chorion epitlielioma (Tenney and Parker, ’39, ’40; Rubin, 310). Upon the successful surgical removal of these growths the hormone promptly disappears. Tenney and l’arl«:er (’39) remark that the amount of hornione found corresponds roughly to the amount of trophoblastic “cells” in the placenta or mole and that a mole with cystic villi and slight trophoblastie proliferation gives a low titer. These findings suggest that actively proliferating cytotrophoblast may be more important in the production of the ehorionic grenadetropie hormone than the derivative syneytium. It should be emphasized also that the curve of excretion of gonadotropic hormone in the pregnant woman parallels approximately the rise and decline of the cytotrophohlast, whereas it bears no such relationship to the syncytium which continues to exist about evenly throughout the entire course of pregnancy. The observations of Elder and Brnhn (’39) on the excretion of’ chorionic gonadotropie hormone in the chimpanzee agree with this, for they obtained positive Friedman tests from the thirtieth to the one hundred and twentieth day of conception. During the last 2 or 3 months of pregnancy their tests were negative. The span during which the tests are positive certainly coincides with the maximum activity and expansion of the cytotrophoblast, and with no other cellular components of the placenta that we are aware of.

In the rhesus monkey positive tests are reported for only a brief period (Hainlett, ’37) from days 19 to 25 following ovulation. Obviously the cytotrophoblast in the rhesus monkey flourishes for a much longer period than the course of 1 week, so that it would be necessary to postulate that in this a.nimal the chorionic hormone is masked in some manner, or is not excreted in the same amounts or in the same way as in the anthropoid apes and man. In reference to the excretion of cherionic gonadotropie. hormone the chimpanzee is evidently much closer to man than to the rhesus monkey?

Another interesting consideration is _an observation by Philipp and Huber (’36) to the effect that the decidua gives positive tests for gonadotropic hormone in the immediate neighborhood of the placenta, but that tests are negative in decidua obtained some distance away. Similarly Parker and Tenney (’38) report that of all of the maternal human tissues in pregnancy which they extracted for prolan ( chorionic gonadotropic hormone), they never obtained a positive result in liver, spleen, adrenal or kidney, Whereas the uterus and decidua were the only maternal tissues from which it could be recovered in any amount. Both of these observations indicate that there is a local concentration of gonadotropic hormone in the uterine wall. Its presence there might well be expected if the hormone were discharged into the matrix or ground substance of the cytotrophoblastic cell columns and trophobla.stic shell as it would have to be if it Were secreted by the cytotro— phoblast. It is apparent that this ground substance stands in open continuation with the matrix of the decidua. Thus through these avenues secretions, either cytolytic or hormonal, could he transferred readily into the tissue spaces of the uterine wall.

3 Newton ( ’38) states that in ma.n the sudden appearance and rise in concentration of gonadotropic hormone in the urine occu'r between the fortieth and fiftieth days of pregnancy. This, he says, corresponds approximately to the time at which the circulation is established in the chorionie villi. In our estimation, this places the establishment of the circulation rather late. In the rhesus monkey the circulation heeomes established soon after the twenty—second day (Wislocki and Streeter, ’38), towards the close of, the brief period during which Hamlett reports the presence of gonadotropic hormone in the urine. Thus, in this animal a relationship such as Newton suggests in man does not prevail.

Considering all aspects of the possible clues presented in the previous paragraphs We are Willing to suggest in a speculative and purely tenta.tive manner that the chorionic gonadotropie hormone derives from the eytotrophoblast rather than from the syncytium.


A large body of literature has accumulated regarding the passage of substances through the placenta from mother to fetus and vice versa. Debate has centered mainly upon the question as to whether the placenta is essentially a semipermeable membrane, governed solely by osmosis and ultraf1ltration acting in accordance with Donnan’s phenomenon of overall equilibrium, or whether, in addition, the cellular membrane exercises a selective regulation of some substances and secretion of others by virtue of special physical or chemical properties. Most of the conclusions regarding the nature of the barrier have been arrived at indirectly, utilizing chemical means, by comparison of the concentrations of various substances in the maternal and fetal blood streams. Certain data have led some investigators to postulate that the barrier behaves like a semipermeable membrane, While other data have been interpreted as pointing to the opposite conclusion.

Cunningham (’20) rendered a distinct service to this problem by his careful investigation of two substances, the passage of which could be studied both chemically and histologically. He emphasized the important point that in considering a given living membrane, We have to deal with three phases of activity, namely, the entrance of a substance into the membrane, its passage through the substance of the membrane, and its exit from the opposite surface of the membrane. “The factors involved in each of these may be entirely different, and those relating to one membrane may differ entirely from those relating to another.” He proceeded to demonstrate that iron ammonium citrate and sodium ferrocyanide which pass quickly and with equal rapidity through the endothelium of living blood vessels, as well as through serous membranes, are taken up by the placental trophoblast of the eat only very slowly. Vflien injected into the maternal blood stream. these. salts do not enter the trophoblast for several hours. Moreover, having been absorbed, the iron ammonium citrate remains in the trophoblast, never entering‘ the fetal blood stream, whereas the ferrous salt eventually penetrates the basal surface of the syncytiuni thus gaining access to the fetal circulation. Thus of these two salts, both of which readily pass man_y semipermeable membranes (endotheliuni, mesothelium), both eventually penetrate the cytoplasm of the syncytial trophoblast, but only one is subsequently transferred to the fetus. These results siiggest, as Cunningliam points out, that the iron ammonium citrate meets a specific regulating mechanism in the trophoblast and hence the latter can not be regarded as an inert, physiologically inactive membrane.

Groldmann (’O9) showed that when pyrrhol blue or trypan blue is injected repeatedly into pregnant mice and rats, these dyes are taken up and stored in the cytoplasm of the trophoblast. Vl-’islocki (’20, ’21) demonstrated, similarly, the storage of trypan blue in the trophoblast of the placenta of cats, rabbits and guinea pigs. ln the cat none of this stored dye is transmitted to the fetus, whereas in the rodents after long administration traces of it appear in the body of the fetus. These e.\'periment.<, as pointed out by Cunningham, support his contention that the entry of a substance into the cytoplasm of the trophoblast, its passage through the cytoplasm, and its possible exit represent different phases of activity. 01 further interest about trypan blue, si.milar to Cunninghanfs experience with his iron salts, is that it is stored in the trophoblast only after repeated injections, whereas it passes through endothelial and mesothelial barriers elsewhere in the body with the greatest of ease. Thus the presents a relatively high threshold of absorption for certain substances.

A more recent observation by Yamaguchi and Koyama (’26), if it can be verified, appears to represent a similar behavior. Thoy report that upon feeding‘ egg yolk to pregnant rabbits for a long time, the chorionic epithelium becomes difCY'L‘()L(.)(i-Y or PLACENTAL TROPHOBLAST 413

fusely filled with almost pure cholesterin esters, whereas no evident cholesterol steatosis is noticeable in the fetal tissues. This observation suggests a storage of cholesterol very similar to that of trypan blue.

Subsequently Cunningham (’23) reported in a brief note upon the passage of ferrous and ferric iron salts through the placenta of the rabbit. in this animal both salts traversed the trophoblast, albeit the ferric salt more slowly than the ferrous lI‘OI1.

The present investigation gives a certain amount of morphological support to the theory of Cunningliam that the trophoblast represents a membrane of unusual complexity. Cytological study of the syncytial trophoblast suggests that it has a Very intricate structure. It contains nuclei, Golgi material, mitochondria, lipoid droplets, cytoplasmic granulation and vacuoles in complex relationships to one another. Furthermore, its outer surface is peculiarly modified, possessing cytoplasmic streamers and hairlike processes differentiated in a, variety of ways. it clear, moreover, that the outer surface is very different in structure from the inner surface facing the Lanefhans cells and the stroma of the chorionic villi.

Even the thinned—out syncytial plates of the chorionic villi at term, which Bremer (’l6) regards as being physiologically inert, present evidence, by virtue of possessiiig mitochondria, a few lipoidal droplets and a stubbl,e—like surface, of being: more liigfhly organized than a semipermeable membrane in its simplest configuration. Bremer postulates that these plates, resembling' the epithelium of glomeruli, are areas through which the fetal Waste products are excreted. Cunningham points out, however, that, insofar as transmission of substances through these plates takes place by diffusion and is regulated by osmosis, they must be equally capable of transmitting substances from the mother in the opposite direction. If they subserve excretion in the sense implied, they must also be resorptive for the readily diffusible components of the maternal blood.

In this connection a peculiarity of the placenta of rodents is of some interest. Like certain other groups of mammals, rodents possess a yolk sac placenta in addition to an allantoic placenta. Goldmann (’07, ’09) and \lVislocki (’21) observed in a variety of rodents that the epithelium of the inverted yolk sac absorbs and stores trypan blue administered to the mother. W'islocki (’21) stated that these cells stain very rapidly and deeply with trypan blue, a single injection suflicing after a few hours to cause the cytoplasm of the cells to become heavily laden with fine blue granules, in contrast to the tropho— blastie syneytium which stores the dye only after repeated injections. Subsequently Everett (’3:')) studied the absorption of a variety of dyes by the epithelium of the yolk sac and arrived at a number of interesting conclusions. Toluidin blue, perfusing the uterine circulation, entered the embryo through the columnar epithelium of the yolk sac before it appeared in the allantoic vessels. Everett points out that his experiments indicate that the membranous trephoblastic epithelial plates of the allantoic placenta are not as permeable to toluidin blue as the relatively thick yolk sac epithelium. Thus the thinness of a membrane may not signify that substances will enter it (trypan blue) or be transmitted through it (toluidin blue) with greater speed than through a thicker cellular membrane. The quality of the protoplasm would appear to be more important than the quantity. Everett concludes also from his studies that in the yolk sac placenta, similarly to the allantoic placenta, certain substances encounter regulatory mechanisms.

In addition to the complex functions of regulating the transfer of inorganic and organic salts, proteins, fats and carbohydrates from mother to fetus, and facilitating the excretion of fetal waste products, the trophoblast either stores, or actually synthesizes, hormones which are subsequently released into both fetal and maternal blood streams. Vile have suggested, for reasons given in previous sections, thatthe placental steroid hormones are formed in the syneytium. The formation of hormones by the syncytial trophoblast is a further reason to regard the placental barrier as being more than a simple semipermeable membrane operating solely by diffusion and osmosis.


A variety of observations and discussions are presented in this paper regarding the cytology of the trophoblast in the human and monkey (Macaca mulatta) placenta. The various topics which we have investigated concerning the syncytium and the cytotrophoblast are enumerated in an introductory outline.

An attempt has been made to explore as far as possible the nature and functions of the trophoblast, as revealed by study of its cytology. A discussion is presented of the possible sites of formation of the several placental hormones and the nature of the placental barrier has been touched upon at numerous points.

Certain of our findings may be summarized as follows:

1. The outer surface of the syncytium exhibits a variable structure ranging from a brush—like border to one consisting of irregular streamers of cytoplasm. This variability We regard as normal and as attributable to instability and plasticity of the surface by virtue of which its cytoplasm can flow and stream and hence assumes various appearances. These variable manifestations bespeak an active transfer of fluid and metabolites across the surface of the trophoblast.

2. A variety of vacuoles are enumerated and described in various parts of the syncytium at different periods and their possible genesis and functions are discussed. They appear to arise in the main by the taking in of fluid from the intervillous space or the deeidua. Tn the trophoblast in the earliest stages, small vacuoles coalesce to form larger ones Which then break down to form the trophoblastie lacunae which are the forerunners of the intervillous space (Streeter; Hertig and Rock). Vacuoles appear to be most abundant in the period before the fetal circulation becomes established and they diminish thereafter, although normally some persist in localities Where opportunity for passage of fluid into the fetal circulation is unfavorable and minimal. Such isolated vacuoles are especially prevalent in outlying parts of the syncytium; they may eventually discharge their contents into the intervillous space and disappear.

3. The failure of particles of India ink to be taken in by the trophoblast in the rhesus monkey and other animals illustrates that phagocytosis of particles of this size is 11ot a general property of the trophoblast and that it does not play an important role in placental transfer. Nevertheless, in young human and monkey embryos at the period when the trophoblast is actively invading the endometrium at the implantation site and While the trophoblastic shell is forming, a certain degree of phagocytosis of degenerating maternal tissues by the syncytium has been observed. On the other hand, the storage of vital dyes (trypan blue) by the trophoblast of rodents and carnivores suggests that the taking in of particles of these dyes is a general property of the trophoblast.

4. Lipoid droplets are always demonstrable in the normal syncytium from early stages until term. No lipoid droplets occur normally i.n the Langhans epithelium, whereas they are encountered in inconstant number in the stroma of the villi.

Under the polarizing microscope it can be demonstrated that the syncytium contains birefringent substances occurring Widely throughout the syncytium, but evidently most abundant in the field of distribution of the stainable fatty substances. This birefringence disappears upon treatment of the sections with acetone.

6. If frozen sections of chorionic villi are treated with phenylhydrazine, yellow phenylhydrazones are formed in the syncytial layer of the trophoblast, but after treatment of the sections with acetone no phenylhydrazones are formed. This reaction indicates the presence of acetone-soluble ketones or aldehydes in the syncytium.

7. The association in the syncytium of fat droplets and birefringent substances soluble in acetone, and of acetone-soluble substances capable of reacting with phenylliydrazine to form yellow phenylhydrazones suggests that the syiicytilim is the site of formation of placenta] steroid hormones.

8. As a corollary to the preceding conclusion, as well as on other grounds, we consider the stainable lipoid in the placenta as probably in good part “intrinsic” or “metabolic” lipoid rather than as “extrinsic” fat which is in process of transmission from mother to fetus for purposes of nutrition.

9. The inner border of the syncytiurn, in contrast to the outer border, is relatively smooth a.nd cytologically little differentiated.

10. The syncytium at term still contains demonstrable particles of lipoid as well as mitochondria and its surface exhibits minute stubble-like irregularities but no typical brush-border. The thinned—out membranes (“epithelial plates”) over the bulging fetal capillaries consist of three layers, (1) outermost, a thin layer of syncytium in which some mitochondria and a few fat droplets are demonstrable; (2) innermost, the endothelium of the capillaries; and (3) between them, a network of argyrophile reticular fibers continuous with the stroma of the villi. In our estimation occasional Langhans cells persist until term beneath the layer of syncytium.

11. The cellular trophoblast, on what to us appears to be sound grounds, is widely regarded a germinal bed from which the syncytial layer is derived, as well as possibly the earliest mesenchynie and angioblastic tissue (Hertig).

12. The cytology‘ of the cellular trophoblast is described and discussed. The cytotrophoplast is associated in the trophoblastic cell columns and shell with an interstitial matrix or ground substance (so—ca.l1ed “fibrinoid”) the nature and genesis of which is discussed.

13. In our estimation this ground substance is a proteincontaining fluid of mixed origins, derived mainly from the decidua and maternal blood stream, but containing also substances released by the cellular This ground substance contains no argyrophile or collagenous fibers. Fibrin becomes variously deposited in it.

14. In our estimation the cellular trophoblast is composed of functionally active cells which perforce take in certain substances, While elaborating and discharging other substances. The vacuoles which characterize these cells are, we believe, an exprcsion of metabolic exchange rather than a sign of degeneration. Although some of the cellular trophoblast, especially on the periphery of the trophoblastic shell, undergoes degeneration relatively early, and the remainder later, We regard the mitochondria, glycogen and vacuoles in the extensive masses of cellular trophoblast during the early stages of placental development as reflecting the functions of normal cells. For reasons presented in the text we suggest very tentatively that the cytotrophoblast, rather than the syncytium, may be the site of formation of chorionic gonadotropin.

15. It is pointed out that the cytology of the syncytium bespeaks its great complexity and supports the view advanced by Cunningham that the placental barrier (syncytiurn) exercises selective and regulatory functions of a complex nature. These involve the selective entry of substances into the syncytium, their regulation in the cytoplasm and the control of their egress from its inner surface in the ease of substances coming from the mother, and in the opposite sense for excretory products of the fetus. In addition to these functions, it is becoming apparent that certain substances including a variety of enzymes, as Well as hormones, are actually formed in the syncytial cytoplasm. Some of these may be retained in the cytoplasm to exercise certain functions there, While others may be excreted into the maternal and fetal blood streams. To the latter belong the steroid hormones, for the production of which by the syncytium we have presented certain indications.


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HER'l'1G, A. T. 1935 Angiogcnesis in the early human chorion and in the primary placenta of the macaque monkey. Carnegie Cont:-ib. to Embryo1., vol. 25, pp. 37-81.

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H;.;R-mg, ARTHUR T,’ AND JOHN Ro<,:K 1941 Two human ova. of the prcvillous stage, having an ovulation age of about eleven and twelve. days respectively. Carnegie Contrib. to Embryology, vol. 29, pp. 127-156.

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1931 Locomotion of lyxiipliocytes. Bull. .1. H. Hospital, vol. 49, pp. 29-36.

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NEEDHAM, JOSEPH 1,931 Cliemical E111b'ryology. New York and Cambridge, Einrlaixd. The Macmillan Company and the University Press. 3 vols.

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- A bit of the peripheral syncytium stained by the Altmann-Kull method for initochondria. Observe the abundant mitochondria, most of which are rod-shaped. Note also the marked vacuolization of the cytoplasm, as well as the variable morphology of the sylieytial s11rfa,ce. Ilardly a true bruslrborder, tl1c surface is covered by cytoplasmic lmirs and streamers of unequal length which become a lacelike veil of cytoplasm on the lower border of the syncytium. Human, 30 days. X 1440.

3 Two characteristic tabs or tags of syncytium, projecting into the intcrvillous space from the surface of secondary chorionic. villi. The preparation, which is from an unfixed and unstained spread, shows the typical presence of dark droplets in the syncytium. The bulk of these is ievealed to be fat droplets which stain with Sudan III as well as with osmic acid. As a rule, these lipoidal droplets avoid the submarginal zone of the syncytium, which may be foamy or possess a groundglass—like quality. Human, 9 weeks. X 450.


4 A section of :1. villus from :1 3O~d21,y l1un1an placenta. The syncytlxxm is deli(1n,tel_\' g1':.1llul1l.I' and faitltly vzicuolatcd. The Langhans cells are frequently 0h— served in p1‘u(=,ess of undergm'ng mitosis. Iron lmnnatoxylln. X M300.

5, G, 7 and 8 These illustmte, in addition to rnany other figures, the V2)J'l€Lbl0 cl1:,nmL:te'1* of the su1'f:1ce of the syncytiun1, ranging from {L fz1irl,_v stl-021;; reselnblancc to :1‘ true br11sl1-bmder to that of 21 lacy border, conlposed of cytoplasmic. StI'e2:'llIl(-‘,I‘S. The oytoplzasm of the syncytitun is finely g'runul:).r 01' stippled, but in addition exhibits variable degrees of Vacuolization. The de,epe.1' lying c.lea1‘ spaces or vaelloles "m the sync}'tin111 can be accounted for to at c.0nside.I'able extent as negnliive images 01" fat d'1*0p1ets, whereas the delicate vz1(:u0les 0(:(i.ur1“ing in the suh1n:1rgin:1l zone are Ilslmlly lipoid free. These figures are all from a‘ 30-day human spevinlen fixed in formznlin~dichromate»sublin1:1tc, and stained with iron llematuxylin. X lh()0.



9, 10, 11 and 12 The tropllobmst at various stages of pregnancy, stained for lipoids.

9 The trophoblast of a human ehorionir=, villus of 30 days. Fixed in Champy’s fluid, unstailled. The syncytium contains numerous fat. droplets, Whereas the Langl1a,11s cells, II0fh.‘/n1e‘r cells and stronm contain 110119,. The fat droplets Freqllently appear to have :1 Cytopklsnfic halo around them. X 1600.

10 A human specimen of‘ 13 weeks fixed in Ch:1.1npy’s fluid, unstained. The lipoid droplets are s1n:a,Her ’r,h:m at the previous stage. X ]60CL

11 A human speeimen of 6 months fixed in (7hamp_V’s fluid, unstained. The i11~ d'1Vidun.1 fat droplets in ‘(.119 syneytiuln lmve diIniuishe(1 ])ereeptlb1y in size. Small fat droplets 0<=,(%.111' V':).1'i::lb1}' and ine0nsta11t1y in the stroum of the villus during the last months of p1*eg11:mey. X ]6UU.

12 The t1*op11obla‘st of a. rhesus lnonkey at 4 weeks fixed 1'11 Ch:.1n1py’s fluid, unst:/1i11ed. The II1Ullkl-‘,_V plat-,m1t,:L c011tai11s fat dist'191bute(1 in the same pattern as in the human. Lipoid droplets are (=,(111st:111t1y and ub1,111du11t1y preselnt in the syneytial 1u_ye1*, while none are present in the ].:mghnns layer. >< 1b’l)().


13 The tropllolalust, liun'm.u, 3!] days, st."1.ine-rl by the Altmann-K1111 method for mitocllondrin. (')bser\'(->, the ,«_=,'rez1it wealth of rod-slumped mitochondrial in the syncitinm. ’l‘he II1lt()('.1l()Yl(1I'll:L in the Langhans cells are fewer and shorter. Many of the clear spaces, around which the niitoeliondrial are a1*1'uJ1ged in the deeper portion of the syncytium, are assot-,iate(l with the Ilegative images of fat droplets (fig. 9), but m:uiy of the c.lezu- spaces in the subnw,rg'iun1 zone, must be true vacuoles. X 1600.

14: A simila1' mitoellondrial preparatiori from it 13-week human placenta. The syncytium has (liminishecl somewhat in Width, and the mitochondria, although ab1111(.’l:a,11t, are smz‘-Lller in size. The mitochondrial extend out into the cytoplasmic streamers on the surface of the s_y11e_ytium. X 1600.

15 The trophohlust of a.‘rl1es11s rnonlcey, 58 days of age, stained for the Golgi apparatus by the Nlnnn-Kopsch metliod. The Golgi 111a,te1'i:il in the syneytium occurs i11 broken threads dispersed throughout the eytoplasiii. X 1600.

16 The trophohlnst of a 3(.)-day lumi.-in placenta stainefl with iron liematoxylin, giving another view of the surface of the syneytiuin. Beneath and between the delicate st1'‘r's or lmir-like processes, the fornmtioxl of vacuoles is particularly accentuated. In the stroms in the lower part of the figure are two Hofbauer cells. These cells contain :1, V‘:-ll"l1Ll)le rnumber of va,c.uoles of different sizes. As a. rule, the contents of the vacuoles do not stain with iron liemntoxylin, but occasionally, as in the present instrmee, they do. A typical Hofbn.11e'r cell is visible in the tinstaineél pm-pzL1':Ltio11in figure 9. X ‘I600.


17, 18 19, 20 and 21 show chorionie villi of the human placenta at term.

17 A villus, stained with M:1llory"s connective tissue stain, to illustrate the presence of blue staining connective tissue (black hi the figure) intervening between the syncytial covering and the underlying, dilated blood vessels. X 1600.

18 A villus at term stained with iron hematoxylin. Note in this and the other villi observed at term that the syneytium possesses a stubble—like surface o'r border. It is exceptional to find the surface really smooth. Langhans cells are said to disappear entirely around the sixth month, although some have doubted this. 011r own preparations suggest that a few Langhans cells persist until term. Such a possible occasional Langhans cell at term is present in this figure, represented by the large clear cell indenting the syncytium on the upper border of the villus. X 1600.

19 A villus at term stained for mitochondria by the Altman»Kull method. Ob~ serve that mitochondria are still abundantly present in the syncytium. They occur even in the thinned out, menihranous areas overlying the dilated chorionic blood vessels, suggesting that these “chorionic plates” are composed of viable, active

cytoplasm. X 1600.


20 A villus at term, fixed by Bodian 2 and stained by Paps’ method for reticulum. The figure illustrates that the ill—defined blue niztterial stained by Mallory’s connective tissue stain (fig. 17) contains in reality a1-gyropliil reticular fibers. These enclose Without exception the dilzited ehorionie eapillaries and hence inter vane in the “ehorionic plates” between the thinned~out syiieytillin and the sub— jaeent endothelium lining the ehorionie blood vessels. In the present figure the reticulum enclosing the capillary forms :1 black line suggesting an intervening meinbrane; but in reality it can be denionstratecl that this argyrophil substance consists of a network of fibers. X 1600.

21 A villus at term from an unstained Champy preparation. This specimen illustrates that lipoid droplets are still typically present in the syneytium at term, although they are smaller than in the first part of gestation (plate 3). Occasional droplets occur in the thinne-d—out Il10]Yll’)I‘d11011S areas of the syneytium. X 1600.

22 Portion of a villus of a human plaeent.a of 9 weeks, fixed by Bodian 2 and stained by Paps’ method for reticulum. It will be observed that the trophoblast rests upon a reticular basement network but that argyrophil reticular fibers do not penetrate between adjaeeiit Langhans cells, nor between the Langlians cells and the syneytium. X 1600.


23 The cytotrophoblast of 21 cell column at the site of union with a secondary chorionic villus, from a. 30-day human placenta, stained by Masso11’s trichromc mixture. To the right are the mesenchymal cells of the stroma of the villus; to the left is part of the cell column. Like the Langhans cells, the cells of the proximal portions of the cell columns undergo frequent mitosis and they show great varia< tions in size. The cytoplasm of some cells is clearer than that of others, and in general it exhibits a finc<grm'ned opacity with relatively little vacuolization. X 1600.

24 The cytotrophohlast of the cell columns becomes loosened up by the accumulation of an abundant intercellular matrix or ground substance (placenta of rhesus monkey, 64 days). Matrix has also accumulated between the trophoblast and the border of the reticular stroma. of the tips of the secondary villi, where it forms a sharply defined narrow lamina or sheath, consisting of a clear, smooth, in tcnscly staining substance, for a discussion of which see text. Accompanying the accumulation of ground substance, the cytoplasm of the cells comes extremely vacu» elated. For a. discussion of the genesis and properties of these vacuoles, see text. Compare this condition at 64 days in the monkey with the compact arrangement of and absence of vacuolization in the cells near the tips of the secondary villi in the human at 30 days, as shown in the preceding figure.


25 A eytotroplmhlastlc coll column of the placenta. of 21 rhesus monkey of 58 (lays, stained for g'lyc<>g'en by Rest-‘s <':nrrni.11e. The cytot1'o1;>l1ol>last, especially when it is vacuolatod, C01'lt-élills large quzu1tities of g;1ycog'en, The less Va.c11ulated V:-1I'ieties of the eytotrophoblast, '111cluding' the llangllans cells, contain less glycogen. Only tmccs of glycogen, at best, have ever been seen in the s,\,'ncytl111x1. The cells of the decidua also contain l.-a,r,«_-:0 amounts of glycogen. X l(1‘00.

26 A portion of a cytotrophoblast.'lc cell column of 21. rhesus monkey of 58 days, stained with iron hematoxylin. Outwnrflly, the cove-rillg syllcytium can be seen with delicate cytoplastnlc threads on its surface which are grouped. into clusters. Unlike the eytotropholxlast shown in figure 24 from :1 monkey of 62 days, the cytotrophoblastic cells in this locality are less vacuoluted and are not yet w1'dr-,ly sup.’-1rated by :1coun1'ulated ground substance. X 1600.

27 The cyt0t1' of :1. cell column of a. human pl:1eontn of 30 days st;-1.incc1 with iron hemntoxylin. The cells are l)econ1'1ng v2Lcuol.'1.ted, some of them being more so than others. Between the cells a certain zunount of matrix or intu'rsti.ti:a.l SUI.)stance is beginning to appear. The vacuoles a,ce1unulu,te in the p<-,ripl1ery of the cell cytoplasm, whereas the rclnnnnts of the finely gra.nuln.r cytoplnsnl tend to remain in the neighborlmod of the nucleus. X 1600.


28 The junction of the trophoblastie shell (left) and the decidua (right) in the placenta of at rhesus monkey of 28 days, fixed in Bodizln 2 and in1pregn:a,te<l for reticulum by Paps’ method. Observe that the argyrophil reticular fibers of the decidually trzmsformetl endometrium cease abruptly at the border of the tro1uho— blastic shell. 1n o11r experience, this is the most acciimte, method for displaying sharply the actual bOu21d£L1‘y betweeii the cytotroplioljlastic shell and the deeidim. The niatrix or interstitial substance in the eytotrophoblastic shell and cell columns does not contain fibrils, demoiistrable by silver impregnzttions, or collagenous bundles. Small, round bodies of blaekeiied substance, va.ria,bly scattered, occur in the cytoplntsiii of the cytoti-oplioblastic cells of the shell and cell eolmnns, as well as iiiterstitially. The significance of such blackened particles is not apparent, but tliay cannot be construed in zuiy sense as representing filorillnr elenicnts. X 1600.

29 The jinn‘-,tion of the trophoblastic shell (left) and the de(‘.i(lu2t (riglit) of a human placenta of 9 weeks, fixed by Eodian 2 and stained by Paps’ method for reticulum. Observe, similarly to the monkey placenta. shown in the p‘1'ec.edlng figure, that the argyrupliil reticular fibers of the dceidllally trnnsformerl endoinetriiiin cease abruptly at the boundary of the trophoblastie shell. X 1600.


30, 31, 32 and 33 show some of the clxziracteristics of deaidual cells.

30 ,l)ec.iduul cells commonly encountered in the human placenta, with relatively small nuclei and u la'rge amount of uniform, finely stipplc-d cytoplasm. The cytoplasm of these uolls is variably dark. Human placenta of 28 days, stained with iron llematoxylin. X 1600.

31 Dccidual cells from anotller portion of the same placzenta, stained by the Altn'1a.nn—Kull method fdi" mitocl10nd1'i21. Small, granular, or slightly elongatetl mitoclnoudria are widely disporsetl in the cytoplasm. SO1116 of the decidual cells are markedly vacuolatod. X 1600.

32 Deciclual cells from the same placenta, fixed in Cha1npy’s fluid to illustrate that some of the cells contain fat droplets. X 1600.

33 The dccidua, of the plat-,enta of a'1'hesus monkey of 58 days, stained with iron hematoxylin. Observe that the dccic:lu:1.l cells are similar to those of the liunizm, although they are snmllcr and the nuclei relatively larger. X 1600.


34 A troplioblastic cell column of the placenta of a rhesus monkey of 58 days, stained with Mass011’s trichrome mixture. Observe that in the proximal part of the column the cytoti-opl10bl:1stic cells are quite compactly arranged, whereas as one passes toward the distal end of the column (right) the cells become dispersed by the :accuu1ulation of :1 green staining‘ grmmd sulJsta.n(*e, Observe that there is at very vividly stained accumulation of protein-co11ta,ining material between the rotieular stroma of the secondary villus and the cytotrophohlustic cell column at the junctioii of the two (left). Cmiipare with figures 24 and 39. X 225.

35 A pdrtion of :1 secondary villus and mljaceiit cytotrophoblastie cell column from :4 human placenta of 15 weeks, stained by Paps’ method for reticulum. Ohserve that the stroma of the villus contains .'-xrgyrophil retic.11lzLr fibers, but that the ground substance of the cytotroplioblast (-(mtains none. X 225.

36 A cytotrophoblastie cell island of a liuman placenta of 13 weeks, stained with Azan, Observe the blue staining interstitial fluid separating the cytotropho— blastie cells. The relations of cells and matrix are similar in the eel] columns (fig. 34) and cell islands. X 225.


37 The t1'ophohlust 01" a setaoiidmy vhorionio villus of a 30-day human plaeelita, stained with Mallory’s connective tissue stain. Observe that the “brush—borde'r” of the s,yneytiLu.u stains differently than the deeper portion of the eytoplzisni. Observe the faintly vaeuolated Ilmiglums cells which are bounded by a, delicate blue uiembrane. This nnemlirmie is not argyrophil, an zt'r;_v;;y1-o[ response lielxig given solely by the 1‘etio.ul:1.r basement. Luemlira.r1e (fig. 22) upon which the Laughans eells rest, and which does not enclose the cells. Sul)jnee11t to the basement reticulum a fetal eapillary containing nucleated red blood cells is visible. X 1600.

38 A portion of :1 c<_ytotrophoblastie eell column of a i-Bl)-(lay human plaeeiita, fixed in Susa fluid and stained with Mallory’s (-onneeti'\'e tissue stain. Observe the well preserved nuclei and the vaeuolated e_vtoplasm of the large eytotrophhblastie cells. Deep blue rnatrix is just beginning to appear in the interstices b€t\V'ee11,tl1(l cells. X1600.

239 A portion of a eytotropliohlnstie eell column of the plnneiita of n 58-‘flay rhesus monkey, fixed in Susn fluid and stained with I\/lnsson ls t1'i.el1rome mixture. The figure illustrates the vaeuol:itec'l rtonrlition oi" the cytotroplioblustle cells, and shows the aeeumulation. of vividly staining, protein-containing, interstitial fluid between the cells. No fibers, argyropllil or otherwise, are deiiioiistrable in the ground substance. X l(i(JO.

40 A portion of :1 eytotroplloblastio cell island of’ £1 lllllllzlll placenta of 13 weeks, fixed in Oerno_v’s fixative and stained with Aza-n". Observe the beginning formation of red staining fibrin in the blue ground subst‘.mee. As the oytotrophoblastie cell eolumns, cell islands and shell age, variable amounts of fibrin appear in them. Ohserve that the eytotroplioblastic cells of the cell. islands are not as vat-,uolater.l as those in the cell columns of an earlier period (fig. 38). X 1600.


41 A portion of 3 truphobiastic coll island fixed in Susa and stained with heIn:L— toxyliu and eosiuy illustmting the difficinlty hy this method of dis<-,'1'imi11ating fihriu 1-‘rmn the n0n—fib1-inous ground substzinc-0, as well as in some iiistzmces from the cell cytoplasni itself’. Human placelita of‘ 13 weeks. X 225.

42 A portion of a similar 0.911 is1;111(1 stained with Azau to illustrate the (11'fi"er— ential co101‘i11g' of cells, ground suhstaiicc (blue) and fibrin (red). Compare with figure 40. Human plucuiita of 13 weeks. X 225.

43 Decidllal coils of u rhesus monkey St:liI1L'd for glycogen 1n_y Bcst’s ca,1‘n1ine. X 1440.

44 A smail hunlan villus at terni p'1'<-‘,purcr1 by Paps‘ nlotliod for rotioulllm, showing" Zl clear cell in the syncytiilm, whirh n'u1y represent the 1'eI1u1ins of’ :1 Lungliaus cell. The tar,-,t tl'1:»1t it is not surr0und9d by reticular fibers i11(1icatcs that it is not at cell belonging to the str01uzL. Observe the irregular hairlike process on the s11rfa,ce of the syncytiuin. X 1440.

45 Decidual cells of a rhesus monkey, slimving the Golgi apprfmtiis stained by the M31111-Kopscli method. X “I440.

Cite this page: Hill, M.A. 2017 Embryology Paper - The histology and cytology of the human and monkey placenta. Retrieved November 24, 2017, from

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