Paper - Histological and histochemical observations on the corpus luteum: Difference between revisions

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| [[File:Mark_Hill.jpg|50px|left]] This 1951 paper is a historic histological study of the development of the corpus luteum.
| [[File:Mark_Hill.jpg|50px|left]] This 1951 paper is a historic histological study of the development of the corpus luteum.
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'''Modern Notes:''' [[Menstrual Cycle|Menstrual Cycle]] | [[Ovary Development]]
'''Modern Notes:''' {{corpus luteum}} | {{menstrual cycle}} | {{ovary}}
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{{Historic Disclaimer}}
{{Historic Disclaimer}}
CONTRIBUTIONS TO EMBRYOLOGY, NO. 224
=Histological and Histochemical Observations on the Corpus Luteum of Human Pregnancy with special reference to Corpora Lutea associated with early Normal and Abnormal Ova=
[[File:Arthur Hertig.jpg|thumb|alt=Arthur T Hertig|link=Embryology History - Arthur Hertig|Arthur Tremain Hertig (1904-1990)]]
 
White RF. [[Embryology History - Arthur Hertig|Hertig AT.]] Rock J. and Adams E.
 
Free Hospital for Women, Brookline, Departments of Pathology, Obstetrics, and Gynecology, Harvard Medical School
 
 
With seven plates (1951)
 
==Review of the Literature==
 
Speculation and investigation concerning the role of the human corpus luteum in the normal menstrual cycle and in pregnancy have occupied the energies of many investigators since early times. For excellent reviews of the work that has led to modern concepts of the corpus luteum, the reader is referred to Asdell (1928) and Pratt (1935).
 
 
Meyer (1911) was the first to describe in detail the macroscopic and microscopic appearance of the stages from the ruptured follicle to complete involution of the human corpus luteum. He was the first to point out that there are four recognizable stages in the development of the corpus luteum, namely proliferation, vascularization, mature or so-called blossom stage, and regression. For the sake of completeness, Meyer’s concept of the development of the corpus luteum, which has been generally accepted by workers in the field, will be outlined briefly.
 
 
During the proliferative stage, which follows immediately upon rupture of the mature Graafian follicle, there is increased vascularity of the theca interna and theca externa, evidenced by dilatation of the capillaries. Storage of fat occurs in both theca interna and granulosa, but more noticeably in the former; mitoses in the granulosa layer are less evident or entirely absent, but mitotic figures are still present in the theca interna; hemorrhage into the follicle is not the rule, because capillaries do not extend into the granulosa layer at or before rupture. The stage of vascularization is marked by a well defined membrana granulosa which has increased conspicuously by hypertrophy of the former granulosa cells. The theca externa is still quite evident, but the cells of the theca interna are already shrunken and are no longer coherent. The folding of the wall of the corpus luteum is much more marked, and there is a notable increase of storage of lipoid in the granulosa cells, which may be termed granulosa lutein cells at this stage of development. Fibroblasts are found in the central coagulum as early as 48 hours after rupture of the follicle.
 
 
As the corpus luteum matures and attains the socalled blossom stage, the cells of the theca interna are irregular and decreased in number. The uniform granulosa lutein cells are large and irregularly cuboidal, with increased lipoid content. Capillaries, accompanied by a few spindle cells, pass between the luteal cells, and individual luteal cells are surrounded by connective-tissue fibrils. The inner contour of the granulosa lutein layer is lined smoothly by a conspicuous capillary network. The central coagulum has undergone considerable connective-tissue organization, or it may become cystic, or a hematoma may form in it. Unless pregnancy is superimposed, the corpus luteum goes on to regression, which is characterized by fatty degeneration and simple atrophy of lutein cells, associated with increased invasion of the luteal tissue by connective-tissue elements. Ultimately, the lipoid substance of the lutein tissue disappears and the corpus luteum is transformed into the corpus albicans by a process of hyaline degeneration which may extend over several months.
 
 
Meyer (1911, 1932) observed that the mature stage and the regressive stage were imperfectly limited and beginning regression could not be definitely recognized. Novak (1932, 1941) stated that regression begins shortly before menstruation, about the 26th day of a normal 28-day cycle. Brewer (1942), however, presents evidence that regression begins at the termination of the vascular stage, about 4 to 6 days before the onset of menstruation. Chemical assays of lutein tissue reveal a steady increase in phospholipid from immediately after ovulation until the 10th day, after which time the phospholipid content of the gland falls. During the first to days of existence of the corpus luteum there is a slight fall in the cholesterol ester content of lutein tissue, but after this time there is an abrupt increase in cholesterol ester content (Brewer, 1942). These demonstrable chemical changes in the corpus luteum, together with the microscopic appearance of the gland, led Brewer to conclude that the corpus luteum commences to regress about 8 to 10 days after ovulation.
 
 
Gillman and Stein (1941) tabulate the number of corpora lutea of pregnancy examined by various investigators prior to 1941, including their own series of 19 specimens. Brewer (1942) reports examining 8 corpora lutea of early pregnancy, but does not furnish any details concerning the histology of these specimens. I-Iertig and Rock (1941, 1945, 1949a) and Heuser, Rock, and Hertig (1945) describe briefly 8 corpora lutea associated with early pregnancies ranging from 7 days ovulation age to the definitive yolksac stage.
 
 
Meyer (1911) characterized the corpus luteum of pregnancy from the 2d month of pregnancy on as having a coarser, cruder appearance due to hypertrophy and increased lipid deposition in the lutein cells, a high degree of connective-tissue proliferation around and between the luteal cells, and thick-walled capillaries. He noted that hyaline degeneration in the corpus luteum of pregnancy was long delayed. He remarked that the theca lutein cells are sometimes retained up to the 4th month of pregnancy, but are insignificant and decrease in number until the final month of pregnancy, when the theca cells flourish again.
 
 
Gillman and Stein (1941), in a study of 13 corpora lutea associated with intra-uterine pregnancies ranging froi 35 to 282 days, state that there is a “critical period” of sudden growth in the volume of the corpus luteum between the 50th and 60th days of pregnancy, due to an excess production of hormonal fluid in the fibrotts-tissue-lined cavity, which is subsequently obliterated; that the theca lutein cells attain their maximum development synchronously with the development of this cavity, and that after its collapse they also disappear; that the granulosa cells, on the other hand, persist throughout pregnancy, and that their vacuolar secretion may degenerate into colloid or even calcium-containing deposit.
 
 
Brewer (1942) noted, in addition to sustained high phospholipid levels and low cholesterol ester levels in the corpus luteum of pregnancy, an increase in vascularity, a lack of fatty degeneration, and absence of simple atroppy of the luteal cells.
 
 
Asdell (1928) and Pratt (I935) have reviewed the literature concerning the origin of the true lutein cell of the human corpus luteum. Meyer decided that the theca lutein cells disappear early in the life of the corpus luteum, leaving only luteal cells of granulosa origin. Chydenius (1926) decided upon a dual origin of the lutein cell. Shaw (1925) described theca lutein cells, or paralutein cells as he termed them, but considered that they remain at the periphery of the gland and do not take part in the formation of the stroma of the gland. A real dilierence between species does seem to exist in the extent to which theca lutein cells invade the stroma of the corpus luteum. According to Hammond and Marshall (1925), this invasion appears to be small or entirely wanting in the monotremes, marsupials, certain rodents, the sheep, the horse, and man, whereas in bats, the guinea pig, the cow, and most of all the sow, the invasion appears to be considerable.
 
 
McNutt (1924) asserted the dual origin of the lutein cell in the cow, stating that small clumps of theca lutein cells invade the space filled by the granulosa lutein cells and become detached from the connectivetissue framework. Despite some divergence of opinion regarding the origin of the human lutein cell, it is established that early in the development of the corpus luteum, the lutein cell derived from the membrana granulosa becomes dominant and is the cell usually described as the lutein cell (Pratt, 1935).
 
 
Corner (1915) described, in addition to the granulosa lutein and theca lutein cells, cells of a third type in the corpus luteum of the sow. These are smaller than the lutein cell, of varied shape, and strongly eosinophilic. They contain small vacuoles even when fixed with osmic acid. He believed that Delestre (1910) also saw them. Gillman and Stein (1941) pointed out the existence of dark and light cells in the granulosa and regarded them as representing different phases of activity of secretion in granulosa lutein cells.
 
 
In recent years a number of new methods of chemical cytology have been developed, and these are being applied to a great variety of organs and tissues. An excellent review of advances in this field is given by Dempsey (1948). To date, reports of the application of these techniques to the human corpus luteum are few.
 
 
McKay and Robinson (1947) studied a series of human corpora lutea of the normal menstrual cycle, employing some of the newer techniques for detecting presumptive ketosteroid compounds. The presence of birefringent crystals, autofluorescent materials, acetone-soluble keto compounds, and sterol substances which reacted with sulfuric acid was noted. ‘Nhen employing Sudan IV to detect sudanophilie lipids, they noticed that lipid was confined to the theca interna in the developing follicle. In the 15-day corpus luteum, small sudanophilie drops were found in all the granulosa and theca lutein cells. During the next 6 or 7 days of the life of the corpus luteum, fine peripherally distributed lipid droplets were seen in almost all the granulosa cells. During this period there was a steady increase in the number and size of sudanophilie droplets in the cells of the theca interna, these droplets being much larger than those in the granulosa cells. About the 23cl day of the cycle a marked decrease was observed in the number of sudanophilie granules in the granulosa lutein cells, many cells being completely devoid of lipid. There were, however, patchy areas containing large drops of lipid in the granulosa layer around the blood vessels of the invading connective-tissue septa. Fewer theca interna cells contained lipid than in earlier stages, but those that did, contained large droplets. This decrease in total sudanophilie substance in both layers was maintained to the 27th day of the cycle. After the 27th day, a greatly increased amount of sudanophilie substance distributed in large, coarse droplets was observed in both layers.
 
 
Examination for birefringent materials revealed a few tiny anisotropic crystals in the theca interna of the 15-day corpus luteum. There was a steady increase in the size and number of these crystals up to the 24th day of the cycle. Very fine birefringent crystals were seen in the granulosa cells on the 17th day, increasing in number and size until the 22d day of the cycle. Throughout the period embracing the 17th to the 23d day of the menstrual cycle, more anisotropic substance was present in the theca interna than in the granulosa at any stage of development. There was a notable decrease in the amount of hirefringent crystals in the theca interna from the 24th to the 27th day, after which there was a considerable increase that persisted through menstruation. A comparative absence of birefringent crystals was observed in the granulosa lutein layer from the 22d day through late menstruation.
 
 
Studies of autofluorescent substances and substances that reacted with phenylhydrazine and sulfuric acid essentially paralleled the observations made on birefringence.
 
McKay and Robinson (1947) also noted in the granulosa layer a few extracellular large oily drops that were sudanophilie and reacted with sulfuric acid and phenylhydrazine but were negative for birefringence and autofluorescence. These were seen only after the 22d day of the cycle, when reactive materials had disappeared from the granulosa cells. The authors suggest that these large drops may be indicative of a change from active secretion to storage or inactivity, basing this interpretation on findings in the adrenal gland (Selye, 1937; Sarason, 1943; Dalton et al., 1944) that line lipid droplets are associated with active secretion and large droplets with inactivity.
 
 
Corner (1948) in a study of 3 human corpora lutea observed that the cells of the theca interna contain alkaline phosphatase up to a day or two after ovulation, but subsequently lose it. The granulosa lutein cells seemed to be almost devoid of alkaline phosphatase in the stages studied.
 
It is the purpose of this paper to describe in some detail the histological and histochemical variations of the human corpus luteum from the earliest stage of pregnancy yet obtained, a 2-cell tubal ovum (Hertig and Rock, 1949b), to that associated with a 4.5 month fetus. For purposes of comparison, a study of corpora lutea of the normal menstrual cycle is included. Evidence will be presented that the lutein cell is derived from the membrana granulosa of the follicle. A third cell type, derived from the theca interna of the follicle, has been demonstrated. Cells of this type make their way into the granulosa layer at or shortly after the time of rupture of the follicle, and become quite prominent as the gland approaches its peak of physiological activity. Evidence will be presented that these cells are the site of intense localization of ltetosteroids, which presumably are the secretory products of the corpus luteum.
 
==Materials and Methods==
 
Since 1939 two of the authors (I-Iertig and Rock) have been searching for early human ova, and to date they have succeeded in obtaining 32 ova ranging from 2 to 17 days ovulation age. Of these specimens, 19 are regarded as normal. The remaining 13 were adjudged to be abnormal because of one or more of the following conditions: multinucleated blastomeres, shallow implantation, malorientation or lack of germ disk, defective trophoblast, or abnormal segmentation cavity. These specimens were obtained as described by Hertig and Rock (1944). In all instances, the ovary containing the corpus luteum was removed with the uterus at operation. The corpora lutea thus obtained were examined macroscopically and then appropriately prepared for microscopic examination as described below.
 
 
Nine corpora lutea associated with older pregnancies 25 days to 4.5 months) were also obtained from surgical material from the Free Hospital for Women (Brookline, Massachusetts) or the Boston Lying-In Hospital.
 
 
Forty-eight corpora lutea not associated with pregnancy were studied in order to compare histologic and histochemical changes in the gland during the normal menstrual cycle with those observed in pregnancy.
 
 
All corpora lutea of nonpregnant cycles and those associated with ova still free in the tubes or uterine cavity were dated by referring to the endometrial age (Hertig, 1945). It is assumed that the normal menstrual cycle is 28 (lays in length, with ovulation on the 14th day of the cycle. It is generally agreed that, regardless of the length of the menstrual cycle, ovulation takes place about 14 days (:2 (lays) before the first day of the next expected period (Rock and Hertig, 1944).
 
 
Material obtained prior to June 1947 had been fixed and stained in accordance with routine laboratory procedures, the following stains being employed: haematoxylin and eosin, eosin-methylene blue, ironalum haematoxylin, phosphotungstic acid-haematoxylin, and Scarlet red for fats. Recently acquired specimens have been stained with eosin-methylene
 
 
blue and with haematoxylin and eosin, and have been subjected to the following special procedures: The alkaline phosphatase procedure of Gomori (1941) was applied to all recently acquired corpora lutea. Frozen sections, 10 to 20 microns in thickness, of formalin-fixed material were prepared, and alternate sections were treated with acetone, alcohol, and acetone—alcohol mixtures for comparison with untreated sections. Sudan black was used as a general lat stain. The phenylhydrazine reaction of Bennett (1940) and the plasmal reaction as described by Lison (1936) were applied to representative cases. Plain sections, both untreated and subjected to fat solvents, were mounted in glycerine jelly and examined under crossed Nicol prisms for evidence of birefringence; and under a fluorescence microscope similar to that described by Grafllin (1939), using as illumination a beam of ultraviolet rays obtained by filtering the light of a carbon-arc lamp through a copper sulfate solution and a Corex filter no. 586.
 
 
Formalin-fixed frozen sections were floated onto slides and blotted dry, and a drop of a solution consisting of equal parts of concentrated sulfuric acid and acetic anhydride was put on the tissue. The slides were examined immediately for the appearance of brown droplets in the cells of the corpus luteum and ovarian stroma. This reaction, formerly used as a test for cholesterol (Romeis, 1928), is now ltnown to depend upon the presence of unsaturated bonds in steroid molecules (S0l)0tl{:1, 193,8).
 
 
Very recently new techniques for the histochemieal detection of active carbonyl groups in lipoid and nonlipoitl materials have been developed (Seligman and Ashbel, I949; Ashbel and Seligman, 1949), and through the courtesy of Drs. A. M. Seligman and R. Ashbel, of the Beth Israel Hospital, Boston, Massachusetts, selected corpora lutea were studied employing these new techniques.
 
 
Two specimens were treated for the histochemieal demonstration of phospholipid according to the method of Baker (1946).
 
==Observations on Corpra Lutea of the Nonpregnant Menstrual Cycle==
 
===The Mature Grcmfirm Follicle, S48-731===
 
Figure 1, plate I, is a photomicrograph of a typical mature Graafian follicle. The theca interim is several cell layers thick. Many of the theca cells are highly vacuolated. Widelyr dilated, blood—filled capillaries are prominent in the theca immediately beneath the basal layer of the granulosa. i\/Iitotic figures are rare in the theca interna. Conspicuous in the theca are a number of cells that differ markedly from the neighboring theca cells. These cells have small, dense, hyperchromatic nuclei which are irregular in outline, and stand out in bold relief against the nuclei of the theca interna cells, which by contrast are plump, ovoid, and vesicular, with a single prominent, eccentric nucleolus. The cytoplasm of these stellate cells is homogeneous and more strikingly eosinophilic than that of the theca cells. Careful study has shown that these cells do not represent intravascular or perivascular elements. For lack of a more specific and descriptive name, and in the interest of definiteness without repetition, these cells will be referred to in the subsequent descriptions and discussions as “K cells.”
 
 
The membrana granulosa is sharply demarcated from the theca interna by a closely packed layer of cells contiguous with, and similar to, the cells of the membrana itself, which is many cell layers thick and thrown into convolutions by the hypertrophy and multiplication of the granulosa cells. Consequently, the inner layer of the follicle presents a scalloped, undulating edge, with tongues of theca interna projecting into the convolutions from the stroma of the ovary. Mitotic figures are numerous in the membrana granulosa. The nuclei of the granulosa cells are perhaps a little larger than those of the theca interna cells, and because of their rapid rate of division and growth are more basophilic. Their cytoplasm presents a frothy appearance, and cell outlines are indistinct, although there is no evidence of vacuolization. There is no evidence of capillary penetration into or proliferation within the membrana granulosa. None of the K cells present in the theca interna are seen in the membrana granulosa at this stage of development.
 
 
===Corpus Luteum of 16th Day of Cycle, Estimated Age 2 Days, S48-2636===
 
Macroscopically, this corpus luteum appeared as a hemorrhagic, unhealed crater I cm. in diameter and 2 mm. in depth, on the posterior surface of the ovary.


I  —-»
Haematoxylin and eosin sections (fig. 2, pl. 1) show the theca interna to be considerably thinned out and the membrana granulosa thrown into a large number of deep convolutions, owing in part to the collapse of the follicle after rupture. The vascular channels of the theca interna are widely dilated, and in several places endothelial sprouts are seen penetrating the membrana granulosa from the theca interna. The cells of the theca interna are essentially the same as those observed in the mature follicle, and rarely show mitoses. The granulosa cells are plump and polyhedral, with round, vesicular nuclei. Their cytoplasm is frothy, but no distinct vacuolization is seen. The granulosa cells are arranged in bundles or fascicles separated in many areas by large lacunae of extravasated blood. These lacunae are not lined with endothelium.


histological and histochemical observations on the corpus
The K cells noted in the theca interna of the mature follicle are very prominent at this stage of development of the corpus luteum. Although few of them are found in the theca interna, ribbons of them can be seen spreading out into the membrana granulosa, penetrating as far as the central coagulum. The attenuated cytoplasmic processes of these cells suggest amoeboid activity. It is to be noted that these cells are more numerous at this and subsequent stages of development than in the mature follicle. Only once have we observed a mitotic figure in a cell that we could definitely say was a K cell. It is possible that the small, irregular, hyperchromatic nuclei characteristic of these cells at this stage represent rapid mitotic activity. As will be noted later, the nuclei of these cells become larger and less hyperchromatic as the gland approaches the period of maximum functional activity.
luteum of human pregnancy
with special reference to corpora lutea associated
with early normal and abnormal ova


R. F. \-Vurrx-:, A. T. I-IILRTIG, I. ROCK, [mu E. AD.-nus


Free’ I-Io.s'pz'ral for I-Vomcw, Brool(1inc,' Dcpmwnxcvzts of Pathology,
Sudan black preparations (fig. 3, pl. 1) reveal that the cells of the theca interna contain much more lipid than do those of the membrana granulosa. This lipid is distributed in fine droplets in most cells, although medium-sized and coarse droplets are found in some of the theca interna cells. As has been mentioned, the granulosa cells contain much less lipid, and this is evenly distributed as a fine peripheral dusting of sudanophilic lipid. In an occasional granulosa cell a few coarse globules of lipid are seen. The K cells are distinguishable only with some diliiculty. They present a uniform, nongranular sudanophilia.
O[25tctric5, and Gynecology, Hm'z2ard Medical School


With scvcn plates
CONTENTS
PAGE
REVIEW 01-‘ THE LITIERATURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 57


NI.-XTIZRIAL AND M15'r1101)s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Alkaline phosphatase is localized exclusively in the cells of the theca interim and in the endothelium of blood vessels. The cytoplasm of the granulosa cells contains no alkaline phosphatase at this stage of development (fig. 4, pl. 1).


O1ss1»:1tv.-\.'r1oNs 01¢ CORPORA LUTEA 01= THE NONPREGN:\NT i\"lEI\'STRU.~\L C1'c1.1=. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 61
===Corpus Luteum of 20th Day of Cycle, Estimated Age 6 Days, S48-2262===


The .\«'Iature Graafian Follicle, S48-731 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ . . . . . . . . . . . . . . . . . 61
On cut section, the corpus luteum measured 1.7 by 1.5 cm. in its greater diameters. The convoluted borders were bright yellow and from 1 to 3 mm. thick. The central coagulum was pale gray with several small hemorrhagic areas.


Corpus Luteum of 16th Day of Cycle, Estimated Age 2 Days, S48-2636 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
The corpus luteum at this stage is quite compact. There is very little extravasated blood in either layer. The blood vessels of the theca interna are widely dilated. Definite capillaries are seen in the granulosa layer, but these are still small and only an occasional The central coagulum contains many proliferating fibroblasts and red blood cell can be noted in them.


Corpus Lutcum of 20th Day of Cycle, Estimated Age 6 Days, S48-2262 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
thus is undergoing early organization. No capillaries are seen in it. The cells of the theca interna appear to be widely separated by fibroblasts and other connective—tissue elements; their cytoplasm is markedly vacuolated, but the nuclei show no evidence of cellular degeneration. The granulosa cells are large and polyhedral, with distinct cell membranes and marked peripheral vacuolization of the cytoplasm (fig. 5, pl. 1). Interspersed among the granulosa cells are a number of K cells, whose nuclei appear dark, irregular, and almost pycnotic.


Corpus Luteum of 23d Day of Cycle, Estimated Age 9 Days, S48-3028 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 62


From 24th Day of Cycle to Menstruation . . . . . . . . . . . . _ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Sudanophilic substance is distributed irregularly in the cells of the theca interna. Some of the theca cells contain few lipid droplets; others contain numerous fine, peripherally distributed lipid droplets, and many contain large, coarse globules of sudanophilic substances. Nearly all the granulosa lutein cells contain fine, peripherally distributed lipid droplets. Only an occasional granulosa cell contains the coarse lipid droplets noted in the theca. The large, stellate K cells are particularly conspicuous at this stage. All of them appear uniformly sudanophilic, although close examination reveals some granular deposits of sttdanophilic substances which are almost masked by the uniformly sudanophilic background of the cytoplasm of these cells (fin. 6, pl. 1). The nuclei of the K cells are clear and devoid of lipid.


Corpus Lutcum during Early Menstruation, S48-535 . . . . _ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63


01ss1=.1w;\'r1o.\*s ON CORPORA LUTI-EA or NORM.-11. PREGNANCY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
The theca lutein cells are uniformly devoid of alkaline phosphatase. This statement applies equally well to the majority of the granulosa cells. A small number of cells scattered among the granulosa cells, however, contain alkaline phosphatase in moderate amounts. It is difiicult to state at this stage of development that these cells containing alkaline phosphatase deposits are the K cells so prominent in the Sudan black preparation.


A 2-Cell Egg, 17-Day Corpus Luteum, S49-2439 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1


A .va,%_-Day Blastocyst, 19-Day Corpus Lutcum, S48-5000 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.;
===Corpus Luteum of 23rd Day of Cycle, Estimated Age 9 Days, S48-3028===


71/;;- and 9‘/2-Day Pregnancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 6.}
The corpus luteum measured 1.5 cm. in its greatest diameters. The convoluted border was bright yellow, and averaged 3 mm. in thickness. The central coagt1him was well organized and was the site of a recent small hemorrhage.


A 12- to 13-Day Pregnancy, Carnegie No. 8558, 846-2767 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 6.}
It is to be noted in this specimen that the theca lutein layer is much less prominent than in the 20-day corpus luteum, but that those theca lutein cells that are observed present essentially the same characteristics as those noted in the earlier stage. The granulosa lutein cells are plump and show all the evidences of marked physiological activity. The K cells are quite numerous and prominent in this specimen and are readily recognized by their more angular shapes, dark, homogeneous cytoplasm, and small, dark nuclei (Fig. 7, pl. 2). Capillaries in the granulosa lutein layer are numerous and widely dilated.


A 16-Day Pregnancy, Carnegie N0. 8602, S48-2088 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65


A 26-Day Pregnancy, S48-2631 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
The theca lutein cells contain large, coarse drops of lipid, whereas the granulosa lutein cells contain fine, peripherally distributed lipid granules in large numbers (fig. 8, pl. 2). The K cells are especially prominent in this preparation, and there can be little question that these elongated, uniformly sudanophilic cells with clear, ovoid nuclei are the same cells that are so conspicuous in haemato:~:ylin and eosin preparations.


A 28- to 35-Day Pregnancy, S48-4854 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66


A 4- to 4‘/2-Montli Pregnancy, S48-2624 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . 66
Only an occasional theca lutein cell contains alkaline phosphatase (fig. 9, pl. 2). The true granulosa lutein cells are devoid of this enzyme. A number of cells, however, having the configuration and nuclear characteristics of the K cells as they appear in routine and Sudan-treated preparations contain high concentrations of alkaline phosphatase. Capillary endothelium in all layers of the corpus luteum contains the enzyme in high concentration.


OBSliRVATI()NS ON CORPORA LU'r13.~\ Assocmrizn wm-1 ABNORMAL Om . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
===From 24th Day of Cycle to Menstuation===


Corpora Lutea Associated with Abnormal Free-Lying Ova _ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 7
Between the 23d day of the cycle and the onset of menstruation, the evidence of regression in the corpus luteum becomes increasingly marked. There is no evidence of further capillary proliferation. The theca cells become less and less distinct, until they can be found only in widely separated clumps in the connective-tissue septa invaginating the granulosa from the ovarian stroma. The granulosa lutein cells show evidence of degeneration, manifested by loss of chromaticity of the nuclei and loss of most of the peripheral vacuolization noted at earlier stages. Many granulosa cells show increasing accumulation of medium—sized and coarse lipid droplets.


A 5-Cell Egg, 19-Day Corpus Lutcum, S48-39.-1,8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
The fate of the K cells becomes evident during this period. With haematoxylin and eosin stains these cells become more eosinophilie, the cytoplasm becomes more dense and homogeneous, and the nuclei become quite contracted and hyperchromatic. The cytoplasm seems to condense, until ultimately all that remains of many of these cells late in the life of the corpus luteum is a dense, eosinophilie mass similar to the colloid described by Gillman and Stein (1941). This process of colloid degeneration evidently extends over a long period, because, as will be noted in our discussion of the corpus luteum of early menstruation, large numbers of apparently normal K cells can be found in older corpora lutea. The K cells are still prominent in Sudan black preparations, but show a progressive loss of sudanophilia and a retraction of their cytoplasmic processes. The pattern of alkaline phosphatase distribution remains essentially the same as that observed in the 23-day specimen, with perhaps some diminution in the concentration of the enzyme in the K cells as the corpus luteum becomes older.


Corpora Lutea Associated with Abnormal Ova with Adequate Trophoblast . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
===Corpus Luteum during Early Menstruation, S48-535===


An 8-Day Ovum, Carnegie N0. 8370, 846-676 . . . . . _ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
At this stage the theca interna is very indistinct. True theca lutein cells are found only in scattered clumps, chiefly in the connective-tissue septa that penetrate between the folds of the granulosa from the ovarian side of the gland (fig. 10, pl. 2). Those theca cells that persist are markedly vacuolated and show wide variation in the staining characteristics of their nuclei. Most of the granulosa lutein cells have lost the peripheral vacuolization that typifies the actively secreting gland. Many cell boundaries in the granulosa lutein layer are quite indistinct. The capillaries in both cell layers are more or less uniformly collapsed, and contain few erythrocytes. Although the K cells are still prominent in routine sections, they do not present the full-blown appearance seen in the 23-day corpus luteum. Moreover, their cytoplasm is denser than has previously been noted and in most instances appears to be contracting, leaving large vacuoles between the granulosa lutein cells. Some of the K cells have degenerated to what we consider the end stage of this line of cells, a dense, strongly eosinophilie colloid droplet. All gradations between a stellate, active cell and the final degenerative end product, colloid, can be found in this specimen, a fact which indicates that this process of colloid degeneration must extend well beyond the menstrual period.


A 10- to 11-Day Ovum, Carnegie N0. 7770, S40-749 _ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68


An 11-Day Ovum, Carnegie N0. 8299, S45-1220 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
The theca lutein cells contain only medium-sized and coarsely irregular lipid droplets (fig. 11, pl. 2). The true granulosa cells contain much more lipid than at any previous stage. Although the lipid droplets are larger than in earlier stages, they are peripherally distributed in most granulosa cells, and in none are the droplets as large and coarse as those in the theca lutein cells. A few granulosa cells are heavily laden with medium-sized lipid droplets. The K cells are still prominent in the Sudan black preparations, but their cytoplasmic processes are markedly retracted, the cytoplasm has lost some of its uniform homogeneous sudanophilia, and a few fine sudanophilic granules are seen against the slate-gray cytoplasmic background of the cells. A few large, oily drops showing varying degrees of sudanophilia are present. It is believed that these represent the colloid drops noted in the haematoxylin and eosin preparations.


Carnegie N0. 7850, S40-2699 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 68
The alkaline phosphatase preparations show essentially the same pattern of distribution of the enzyme as in the 23-day specimen, with perhaps some diminution in the concentration of the enzyme in the K cells (fig. 12, pl. 2).  


Corpora Lutea Associated with Abnormal, Shallowly Implanted Ova . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 68
==Observations on Corpra Lutea in Normal Pregnancy==


An 11- to 12-Day Ovum, Carnegie N0. 8000, S42-217 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
===A 2-Cell Egg, 17-Day Corpus Luteum, S49-2439===


A 12‘/3-Day Ovurn, Carnegie No. 8290, S44-2785 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
This ovum was recovered from the middle third of the Fallopian tube and consisted of two normal blastomeres. This specimen, of about 60 hours coital age, is the earliest human ovum yet recovered. The endometrium had the characteristics of that of 2 to 21/; days after ovulation.


A Corpus Lutcum Associated with an Ovum Consisting of Syncytiotrophoblast Only . . . . . . . . . . . . . . . . . . . . 68
On cut surface, the corpus luteum measured 2.5 by 1.5 cm. in its greatest diameters. The convoluted border was red-gray, and varied in thickness from 3 mm. at the base to I mm. at the unhealed stigma. The central coagulum was moderately well organized and showed some peripheral congestion.


A 12-Day Ovum, Carnegie N0. 8329, 845-1809 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Microscopic examination demonstrates that this corpus luteum is in no respect materially different from S48-2636 (figs. 2-4). K cells streaming into the granulosa lutein layer from the theca are prominent and numerous. Sudanophilic substances are much more prominent in the theca interna than in the granulosa lutein layer. Alkaline phosphatase is restricted to the theca interna and the endothelium of the blood vessels.


Corpora Lutea Associated with Ova Showing Hypoplastic Trophoblast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 68
===A 4.5-Day Blastocyst, 19-Day Corpus Luteum, 548-5000===


Carnegie N0. 7771, 840-791 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ., 68
This specimen is the first normal human blastocyst yet recovered from the uterine cavity. The associated endometrium is typical 19-day secretory endometrium.


Carnegie No. 7800, 840-1327 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 69
On cut surface the corpus luteum measured 2.2 cm. in its greatest diameters. The convoluted borders were reddish gray, measuring 3 to 4 mm. in thickness. The stigma was completely healed, and the central coagulum was pale and gelatinous.


About 3 Months Pregnancy, Blighted Ovum, S48-82.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Microscopic examination reveals that this corpus luteum is almost identical in all respects with the 20-day corpus luteum of the normal menstrual cycle and with S48-3948, the corpus luteum associated with the abnormal 5-cell, 4‘/§_- to 5-day ovum.


NOTES 031 THE K CELLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
===7.5- and 9.5-Day Pregnancies===


DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Figures 13 and 14, plate 3, are photomicrographs of sections of corpora ltttea associated with 7‘/3- and 9‘/3-day normal pregnancies respectively (Carnegie nos. {{CE8020}}, {{CE8215}}). Unfortunately, these sections, which were stained with haematoxylin and eosin, have faded


St:.\11~.1A1ur AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 73
to a great extent, but the following features are noteworthy. There is a progressive increase in vascularity and dilatation of the capillaries in the granulosa lutein layer. The theca lutein layer remains quite distinct and prominent. Peripheral vacuolization of the granulosa lutein cells, which is normally disappearing in the corpus luteum of the nonpregnant cycle by 26 to 27 days, becomes increasingly noticeable after 9.5; days of pregnancy and is particularly striking at 11 and 12 days of pregnancy. The cell boundaries of the granulosa cells become progressively less distinct because of the line peripheral vacuolization. At this stage the granulosa cells resemble the “prickle cells” described by Hertig and Rock (1941). Although the K cells are not strikingly conspicuous in any of these sections, they are seen in considerable numbers. It appears that these cells have actually been stimulated and are assuming the appearance of full-blown activity noted in the 21-day corpus luteum (Hg. 7).


LITER.-‘.TL'RIi CITED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
===A 12- to 13-Day Pregnancy, Carnegie No. {{CE8558}}, S46-2767===
HISTOLOGICAL AND HISTOCHEMICAL OBSERVATIONS ON THE CORPUS
LUTEUM OF HUMAN PREGNANCY


WITH SPECIAL REFERENCE TO CORPORA LUTEA ASSOCIATED
The corpus luteum was moderately cystic and measured 2.0 cm. in its greatest diameters. The convoluted border was 1 to 3 mm. thick and was pale yellow with an orange tinge, but did not appear to be senescent.
WITH EARLY NORMAL AND ABNORMAL OVA


REVIE\V OF THE LITERATURE
The theca lutein layer is quite prominent and many cell layers in thickness. The cytoplasm of these cells is vacuolated, and the nuclei resemble those of actively functioning cells. The granulosa lutein cells show marked peripheral vacuolization, and the cell boundaries are thus almost completely obliterated (fig. 15, pl. 3). There has been a significant increase in the number of widely patent vascular channels in the granulosa layer. K cells are not at all conspicuous in haematoxylin and eosin sections. However, the dense, pycnotic nuclei and contracted cytoplasm characteristic of many of the K cells in figure 10 are not frequently seen here, a fact which indicates a recrudescence of activity of these cells.


Speculation and investigation concerning the role
of the human corpus luteum in the normal menstrual
cycle and in pregnancy have occupied the energies of
many investigators since early times. For excellent
reviews of the work that has led to modern concepts
of the corpus luteum, the reader is referred to Asdell
(1928) and Pratt (1935).


Meyer (1911) was the first to describe in detail the
A very striking change is noted in the Sudan black preparations. Only a few theca interna cells contain coarse lipid granules. The majority of the theca lutein and granulosa cells contain large numbers of very fine, peripherally distributed sudanophilic droplets.  
macroscopic and microscopic appearance of the stages
from the ruptured follicle to complete involution of
the human corpus luteum. He was the first to point
out that there are four recognizable stages in the development of the corpus luteum, namely proliferation,
vascularization, mature or so-called blossom stage,
and regression. For the sake of completeness, Meyer’s
concept of the development of the corpus luteum,
which has been generally accepted by workers in the
field, will be outlined briefly.


During the proliferative stage, which follows immediately upon rupture of the mature Graafian follicle, there is increased vascularity of the theca interna
and theca externa, evidenced by dilatation of the
capillaries. Storage of fat occurs in both theca interna
and granulosa, but more noticeably in the former;
mitoses in the granulosa layer are less evident or
entirely absent, but mitotic figures are still present in
the theca interna; hemorrhage into the follicle is not
the rule, because capillaries do not extend into the
granulosa layer at or before rupture. The stage of
vascularization is marked by a well defined membrana
granulosa which has increased conspicuously by hypertrophy of the former granulosa cells. The theca externa is still quite evident, but the cells of the theca
interna are already shrunken and are no longer
coherent. The folding of the wall of the corpus luteum
is much more marked, and there is a notable increase
of storage of lipoid in the granulosa cells, which may
be termed granulosa lutein cells at this stage of development. Fibroblasts are found in the central coagulum as early as 48 hours after rupture of the follicle.


57
K cells are very prominent (fig. 16, pl. 3) and show marked uniform sudanophilia and attenuated cytoplasmic processes like those seen in the physiologically active corpus luteum of the 23d clay of the nonpregnant cycle (fig. 8).


As the corpus luteum matures and attains the socalled blossom stage, the cells of the theca interna are
irregular and decreased in number. The uniform
granulosa lutein cells are large and irregularly
cuboidal, with increased lipoid content. Capillaries,
accompanied by a few spindle cells, pass between the
luteal cells, and individual luteal cells are surrounded
by connective-tissue fibrils. The inner contour of
the granulosa lutein layer is lined smoothly by a conspicuous capillary network. The central coagulum
has undergone considerable connective-tissue organization, or it may become cystic, or a hematoma may
form in it. Unless pregnancy is superimposed, the
corpus luteum goes on to regression, which is characterized by fatty degeneration and simple atrophy
of lutein cells, associated with increased invasion of
the luteal tissue by connective-tissue elements. Ultimately, the lipoid substance of the lutein tissue disappears and the corpus luteum is transformed into the
corpus albicans by a process of hyaline degeneration
which may extend over several months.


Meyer (1911, 1932) observed that the mature stage
A few of the theca lutein cells contain alkaline phosphatase, but most are devoid of the enzyme. A few K cells at the junction of the granulosa and theca lutein layers contain a high concentration of alkaline phosphatase, but of particular interest is the demonstration of the enzyme in significant amounts in some of the true granulosa lutein cells (fig. 17, pl. 3).
and the regressive stage were imperfectly limited and
beginning regression could not be definitely recognized. Novak (1932, 1941) stated that regression begins shortly before menstruation, about the 26th day
of a normal 28-day cycle. Brewer (1942), however,
presents evidence that regression begins at the termination of the vascular stage, about 4 to 6 days before
the onset of menstruation. Chemical assays of lutein
tissue reveal a steady increase in phospholipid from
immediately after ovulation until the 10th day, after
which time the phospholipid content of the gland
falls. During the first to days of existence of the
corpus luteum there is a slight fall in the cholesterol
ester content of lutein tissue, but after this time there
is an abrupt increase in cholesterol ester content
(Brewer, 1942). These demonstrable chemical changes
in the corpus luteum, together with the microscopic
appearance of the gland, led Brewer to conclude that
58 CORPUS LUTEUM OF HUMAN PREGNANCY


the corpus luteum commences to regress about 8 to
===A 16-Day Pregnancy, Carnegie No. {{CE8602}}, S48-2088===
10 days after ovulation.


Gillman and Stein (1941) tabulate the number of
The cystic corpus luteum measured 3 cm. in its greatest diameters. The convoluted borders were a brilliant yellow and varied in thickness from 4 mm. at the base to I mm. at the healed stigma.
corpora lutea of pregnancy examined by various investigators prior to 1941, including their own series
of 19 specimens. Brewer (1942) reports examining
8 corpora lutea of early pregnancy, but does not
furnish any details concerning the histology of these
specimens. I-Iertig and Rock (1941, 1945, 1949a) and
Heuser, Rock, and Hertig (1945) describe briefly 8
corpora lutea associated with early pregnancies ranging from 7 days ovulation age to the definitive yolksac stage.


Meyer (1911) characterized the corpus luteum of
pregnancy from the 2d month of pregnancy on as
having a coarser, cruder appearance due to hypertrophy
and increased lipid deposition in the lutein cells, a
high degree of connective-tissue proliferation around
and between the luteal cells, and thick-walled capillaries. He noted that hyaline degeneration in the
corpus luteum of pregnancy was long delayed. He
remarked that the theca lutein cells are sometimes
retained up to the 4th month of pregnancy, but are
insignificant and decrease in number until the final
month of pregnancy, when the theca cells flourish
again.


Gillman and Stein (1941), in a study of 13 corpora
Haematoxylin and eosin sections of this specimen are very similar to those of the preceding stage. The theca interna is quite prominent (fig. 18, pl. 4). The granulosa lutein cells show considerable activity, manifested by noticeable peripheral vacuolization which causes cell boundaries to appear very indistinct. A large number of fine capillaries form a delicate vascular network in the granulosa lutein layer. K cells are even more numerous and distinct in this specimen than in the 12- to 13-day pregnancy (fig. 15), but a number of these cells are undergoing colloid degeneration as described in the corpus luteum of early menstruation. In this and in subsequent specimens to be described, a number of regular, spherical vacuoles are seen in the granulosa lutein layer. These vacuoles apparently mark the site of colloid deposits, which have dropped out of the section in preparation.
lutea associated with intra-uterine pregnancies ranging
froi 35 to 282 days, state that there is a “critical
period” of sudden growth in the volume of the corpus
luteum between the 50th and 60th days of pregnancy,
due to an excess production of hormonal fluid in the
fibrotts-tissue-lined cavity, which is subsequently
obliterated; that the theca lutein cells attain their
maximum development synchronously with the development of this cavity, and that after its collapse
they also disappear; that the granulosa cells, on the
other hand, persist throughout pregnancy, and that
their vacuolar secretion may degenerate into colloid
or even calcium-containing deposit.


Brewer (1942) noted, in addition to sustained high
phospholipid levels and low cholesterol ester levels
in the corpus luteum of pregnancy, an increase in
vascularity, a lack of fatty degeneration, and absence
of simple atroppy of the luteal cells.


Asdell (1928) and Pratt (I935) have reviewed the
The great majority of both theca lutein cells and granulosa lutein cells contain large numbers of fine peripheral sudanophilic droplets. An occasional theca lutein cell contains a number of medium-sized lipid droplets (fig. 19, pl. 4). The K cells are quite conspicuous and appear essentially the same as in the corpus luteum of the 12- to 13-day pregnancy.
literature concerning the origin of the true lutein cell
of the human corpus luteum. Meyer decided that the


theca lutein cells disappear early in the life of the
corpus luteum, leaving only luteal cells of granulosa
origin. Chydenius (1926) decided upon a dual origin
of the lutein cell. Shaw (1925) described theca lutein
cells, or paralutein cells as he termed them, but considered that they remain at the periphery of the gland
and do not take part in the formation of the stroma
of the gland. A real dilierence between species does
seem to exist in the extent to which theca lutein cells
invade the stroma of the corpus luteum. According
to Hammond and Marshall (1925), this invasion
appears to be small or entirely wanting in the monotremes, marsupials, certain rodents, the sheep, the
horse, and man, whereas in bats, the guinea pig, the
cow, and most of all the sow, the invasion appears
to be considerable.


McNutt (1924) asserted the dual origin of the lutein
In figure 20, plate 4, the theca lutein cells are seen to be almost completely devoid of alkaline phosphatase. Nearly all of the granulosa lutein cells, however, contain alkaline phosphatase in varying amounts, thereby making it difficult to identify the K cells. Several cells at the junction of theca lutein and granulosa lutein layers contain a high concentration of the enzyme, and on the basis of their position and configuration these are very likely the K cells so conspicuously revealed by other techniques.
cell in the cow, stating that small clumps of theca
lutein cells invade the space filled by the granulosa
lutein cells and become detached from the connectivetissue framework. Despite some divergence of opinion
regarding the origin of the human lutein cell, it is
established that early in the development of the corpus
luteum, the lutein cell derived from the membrana
granulosa becomes dominant and is the cell usually
described as the lutein cell (Pratt, 1935).


Corner (1915) described, in addition to the granulosa lutein and theca lutein cells, cells of a third type
===A 26-Day Pregnancy, S48-263===
in the corpus luteum of the sow. These are smaller
than the lutein cell, of varied shape, and strongly
eosinophilic. They contain small vacuoles even when
fixed with osmic acid. He believed that Delestre
(1910) also saw them. Gillman and Stein (1941)
pointed out the existence of dark and light cells in
the granulosa and regarded them as representing
different phases of activity of secretion in granulosa
lutein cells.


In recent years a number of new methods of chemical cytology have been developed, and these are being
This specimen was obtained with the uterus at time of operation for carcinoma of the cervix. The corpus luteum was cystic, 2 to 4 cm. in diameter, and was filled with a clear, yellowish fluid. The convoluted border was yellowish gray, and 2 to 3 mm. thick.
applied to a great variety of organs and tissues. An
excellent review of advances in this field is given by
Dempsey (1948). To date, reports of the application
of these techniques to the human corpus luteum are
few.


McKay and Robinson (1947) studied a series of
human corpora lutea of the normal menstrual cycle,
employing some of the newer techniques for detecting


presumptive ketosteroid compounds. The presence
The theca interna is very conspicuous in this specimen, being several cell layers thick around the entire granulosa lutein layer (fig. 21, pl. 4). The nuclei stain uniformly and present the appearance of actively functioning cells. For the first time since the stages of the mature follicle and the 16-day corpus luteum of the nonpregnant menstrual cycle, a number of small, irregular stellate cells with wrinkled hyperchromatic nuclei and homogeneous eosinophilic cytoplasm are seen in the theca lutein layer. Except for somewhat smaller size, these cells are identical in all respects with the K cells noted in routine haematoxylin and eosin sections of the younger corpora lutea. The sudden reappearance of these cells in the theca lutein layer is unexplained. There is no evidence of transformation or degeneration of theca lutein cells to these forms. The capillaries and sinusoids of the theca lutein layer are widely dilated.
CORPUS LUTEUM OF HUMAN PREGNANCY 59


of birefringent crystals, autofluorescent materials,
acetone-soluble keto compounds, and sterol substances
which reacted with sulfuric acid was noted. ‘Nhen
employing Sudan IV to detect sudanophilie lipids,
they noticed that lipid was confined to the theca
interna in the developing follicle. In the 15-day corpus
luteum, small sudanophilie drops were found in all
the granulosa and theca lutein cells. During the next
6 or 7 days of the life of the corpus luteum, fine
peripherally distributed lipid droplets were seen in
almost all the granulosa cells. During this period
there was a steady increase in the number and size
of sudanophilie droplets in the cells of the theca
interna, these droplets being much larger than those
in the granulosa cells. About the 23cl day of the
cycle a marked decrease was observed in the number
of sudanophilie granules in the granulosa lutein cells,
many cells being completely devoid of lipid. There
were, however, patchy areas containing large drops
of lipid in the granulosa layer around the blood
vessels of the invading connective-tissue septa. Fewer
theca interna cells contained lipid than in earlier
stages, but those that did, contained large droplets.
This decrease in total sudanophilie substance in both
layers was maintained to the 27th day of the cycle.
After the 27th day, a greatly increased amount of
sudanophilie substance distributed in large, coarse
droplets was observed in both layers.


E.\'amination for birefringent materials revealed a
The granulosa lutein layer at this stage appears highly disorganized. Cell boundaries are quite indistinct. The individual granulosa lutein cells show marked variation in the stainability of their nuclei and cytoplasm. There are a large number of patent capillaries in the granulosa lutein layer. Around each of these capillaries, proliferation of perivascular connective-tissue elements is seen. There is a definite increase in the reticular network that at this stage encircles nearly every granulosa lutein cell. The central coagulum is well organized, and large, dilated vascular channels are present in this new connective tissue.
few tiny anisotropic crystals in the theca interna of
the 15-day corpus luteum. There was a steady increase in the size and number of these crystals up to
the 24th day of the cycle. Very fine birefringent
crystals were seen in the granulosa cells on the 17th
day, increasing in number and size until the 22d day
of the cycle. Throughout the period embracing the
17th to the 23d day of the menstrual cycle, more
anisotropic substance was present in the theca interna
than in the granulosa at any stage of development.
There was a notable decrease in the amount of hirefringent crystals in the theca interna from the 24th
to the 27th day, after which there was a considerable
increase that persisted through menstruation. A comparative absence of birefringent crystals was observed


in the granulosa lutein layer from the 22d day through
late menstruation.


Studies of autofluorescent substances and substances
Very few K cells that may be regarded as active are seen, but large numbers of these cells in all stages of degeneration are present. Colloid is present in larger amounts, and as a corollary the number of large, empty vacuoles is also increased over the preceding stage.  
that reacted with phenylhydrazine and sulfuric acid
essentially paralleled the observations made on birefringence.


McKay and Robinson (1947) also noted in the
granulosa layer a few extracellular large oily drops
that were sudanophilie and reacted with sulfuric acid
and phenylhydrazine but were negative for birefringence and autofluorescence. These were seen only
after the 22d day of the cycle, when reactive materials
had disappeared from the granulosa cells. The authors
suggest that these large drops may be indicative of a
change from active secretion to storage or inactivity,
basing this interpretation on findings in the adrenal
gland (Selye, 1937; Sarason, 1943; Dalton et al., 1944)
that line lipid droplets are associated with active secretion and large droplets with inactivity.


Corner (1948) in a study of 3 human corpora lutea
Most of the theca lutein and granulosa lutein cells contain very fine lipid droplets in large quantities (fig. 22, pl. An occasional theca lutein cell contains a few medium-sized lipid droplets. A significant number of the granulosa lutein cells contain large amounts of lipid in large, coarse droplets, indicating fatty degeneration. The K cells are still quite prominent and markedly sudanophilic, but they appear frayed and fibrillar. They are definitely not so numerous or attenuated as in figure 19. Many large, smooth sudanophilie globules are present. These match in number, size, shape, and location the colloid droplets seen in haematoxylin and eosin sections.
observed that the cells of the theca interna contain
alkaline phosphatase up to a day or two after ovulation, but subsequently lose it. The granulosa lutein
cells seemed to be almost devoid of alkaline phosphatase in the stages studied.


It is the purpose of this paper to describe in some
detail the histological and histochemical variations
of the human corpus luteum from the earliest stage
of pregnancy yet obtained, a 2-cell tubal ovum (I-Iertig
and Rock, 1949b), to that associated with a 41/;


month fetus. For purposes of comparison, a study
Of particular interest is the appearance of large numbers of these characteristic sudanophilic K cells scattered among the cells of the theca lutein layer. Both examples of 26-day pregnancy show them, and although carefully searched for, they were not seen in any other specimen. These cells appear to be very similar to the K cells observed in the early stages of development of the corpus luteum, and do not present the frayed, fibrillar appearance noted in the K cells in the granulosa lutein layer of this same specimen, facts which suggest that these cells represent younger forms.
of corpora lutea of the normal menstrual cycle is included. Evidence will be presented that the lutein
cell is derived from the membrana granulosa of the
follicle. A third cell type, derived from the theca
interna of the follicle, has been demonstrated. Cells
of this type make their way into the granulosa layer
at or shortly after the time of rupture of the follicle,
and become quite prominent as the gland approaches
its peak of physiological activity. Evidence will be
presented that these cells are the site of intense localization of ltetosteroids, which presumably are the secretory products of the corpus luteum.
60 CORPUS LUTEUM OF HUMAN PREGNANCY


MATERIAL AND Ml:ZTI-IODS


Since 1939 two of the authors (I-Iertig and Rock)
The theca interna is uniformly devoid of alkaline phosphatase (Hg. 23, pl. 4), although most of the granulosa lutein cells contain moderate amounts of the enzyme. It is diflicult to recognize K cells in this preparation. The endothelium of blood vessels in both layers contains alkaline phosphatase.
have been searching for early human ova, and to date
they have succeeded in obtaining 32 ova ranging from
2 to 17 days ovulation age. Of these specimens, 19
are regarded as normal. The remaining 13 were
adjudged to be abnormal because of one or more of
the following conditions: multinucleated blastomeres,
shallow implantation, malorientation or lack of germ
disk, defective trophoblast, or abnormal segmentation
cavity. These specimens were obtained as described
by Hertig and Rock (1944). In all instances, the ovary
containing the corpus luteum was removed with the
uterus at operation. The corpora lutea thus obtained
were examined macroscopically and then appropriately
prepared for microscopic examination as described
below.


Nine corpora lutea associated with older pregnancies
===A 28- to 35-Day Pregnancy, S48-4854===


25 days to 41/; months) were also obtained from
On gross examination the corpus luteum measured 2.3 by 2.0 cm in its greatest diameters. The convoluted border was grayish yellow, measuring 2 to 3 mm. in thickness. The central coagulum was well organized.
surgical material from the Free Hospital for \-‘Vomen
(Brookline, Massachusetts) or the Boston Lying-In
Hospital.


Forty—eight corpora lutea not associated with pregnancy were studied in order to compare histologic and
histochemical changes in the gland during the normal
menstrual cycle with those observed in pregnancy.


All corpora lutea of nonpregnant cycles and those
The theca lutein layer is still very prominent, although not so conspicuous as in the preceding specimen. Many of the nuclei are dense and irregularly pycnotic. The cytoplasm of most of the cells is highly vacuolated (fig. 24, pl. 5). The vascular sinusoids of the theca interna are almost completely collapsed.
associated with ova still free in the tubes or uterine
cavity were dated by referring to the endometrial age
(Hertig, 1945). It is assumed that the normal menstrual cycle is 28 (lays in length, with ovulation on
the 14th day of the cycle. It is generally agreed that,
regardless of the length of the menstrual cycle, ovulation takes place about 14 days (:2 (lays) before the
first day of the next expected period (Rock and
I-Iertig, 1944).


Material obtained prior to June 1947 had been fixed
and stained in accordance with routine laboratory
procedures, the following stains being employed:
haematoxylin and eosin, cosin—methylene blue, ironalum haematoxylin, phosphotungstic acicl—haematoxylin, and Scarlet red for fats. Recently acquired
specimens have been stained with eosin-methylene


blue and with haematoxylin and eosin, and have been
The granulosa lutein layer presents essentially the same picture as that of the preceding specimen in haematoxylin and eosin preparations. Figure 25, plate 5, is a photomicrograph of this same section, showing a clearly defined colloid globule. Other K cells in various stages of degeneration are present in this section.
subjected to the following special procedures: The
alkaline phosphatase procedure of Gomori (1941)
was applied to all recently acquired corpora lutea.
Frozen sections, 10 to 20 microns in thickness, of
formalin-fixed material were prepared, and alternate
sections were treated with acetone, alcohol, and
acetone—alcohol mixtures for comparison with untreated sections. Sudan black was used as a general
lat stain. The phenylhydrazine reaction of Bennett
(1940) and the plasmal reaction as described by Lison
(1936) were applied to representative cases. Plain
sections, both untreated and subjected to fat solvents,
were mounted in glycerine jelly and examined under
crossed Nicol prisms for evidence of birefringence;
and under a fluorescence microscope similar to that
described by Grafllin (1939), using as illumination a
beam of ultraviolet rays obtained by filtering the light
of a carbon-arc lamp through a copper sulfate solution and a Corex filter no. 586.


Formalin-fixed frozen sections were floated onto
slides and blotted dry, and a drop of a solution consisting of equal parts of concentrated sulfuric acid
and acetic anhydride was put on the tissue. The
slides were examined immediately for the appearance
of brown droplets in the cells of the corpus luteum
and ovarian stroma. This reaction, formerly used as
a test for cholesterol (Romeis, 1928), is now ltnown
to depend upon the presence of unsaturated bonds in
steroid molecules (S0l)0tl{:1, 193,8).


Very recently new techniques for the histochemieal
The Sudan black preparations show large numbers of very fine peripherally distributed lipid granules in both theca lutein and granulosa lutein cells (fig. 26, pl. 5). Occasional cells undergong fatty degeneration are seen in both layers. The K cells are still quite conspicuous in the granulosa lutein layer, and all show a line dusting of sudanophilic droplets in their cytoplasm. A few small, uniformly sudanophilic cells similar to those described in the previous specimen are seen in the theca lutein layer, but these are not sufficiently conspicuous to photograph. A number of large, oily sudanophilic droplets resting in vacuoles may be noted among the granulosa cells. It is believed that this is the colloid mentioned earlier.
detection of active carbonyl groups in lipoid and nonlipoitl materials have been developed (Seligman and
Ashbel, I949; Ashbel and Seligman, 1949), and
through the courtesy of Drs. A. M. Seligman and
R. Ashbel, of the Beth Israel Hospital, Boston, Massachusetts, selected corpora lutea were studied employing these new techniques.


Two specimens were treated for the histochemieal
demonstration of phospholipid according to the


method of Baker (1946).
The alkaline phosphatase preparations are particularly striking. A number of the cells of the theca lutein layer contain large amounts of the enzyme, but most of the theca cells are totally devoid of it. On the other hand, the granulosa lutein layer contains a very high concentration of the enzyme, which is so diffusely distributed that it is impossible to separate the granulosa lutein cells from the K cells. Numerous large vacuoles, representing the site of colloid deposits that have been lost or dissolved in the process of preparation, are evident in this section as in figures 20 and 23.
CORPUS LUTEUM OF HUMAN PREGNANCY 61


OBSERVATIONS ON CORPORA LUTEA OF THE NONPREGNANT
=== A 4- to 4.5-Month Pregnancy, S48-2624===
MENSTRUAL CYCLE


T/ze Mature Grcmfirm Follicle, S48-731
This specimen was obtained incidentally to a total hysterectomy performed during the 5th month of pregnancy because of carcinoma of the cervix. The corpus luteum measured 2 by 1.5 cm. The stigma was depressed and well healed. The periphery of the corpus luteum presented a yellowish, fatlike appearance upon the cut surface. Several small gelatinous areas were seen between the convolutions. There was a small hemorrhage, 2 by 5 cm., in the exact center of the gland.


Figure 1, plate I, is a photomicrograph of a typical
mature Graafian follicle. The theca interim is several
cell layers thick. Many of the theca cells are highly
vacuolated. Widelyr dilated, blood—filled capillaries are
prominent in the theca immediately beneath the basal
layer of the granulosa. i\/Iitotic figures are rare in the
theca interna. Conspicuous in the theca are a number
of cells that differ markedly from the neighboring
theca cells. These cells have small, dense, hyperchromatic nuclei which are irregular in outline, and
stand out in bold relief against the nuclei of the theca
interna cells, which by contrast are plump, ovoid, and
vesicular, with a single prominent, eccentric nucleolus.
The cytoplasm of these stellate cells is homogeneous
and more strikingly eosinophilic than that of the theca
cells. Careful study has shown that these cells do not
represent intravascular or perivascular elements. For
lack of a more specific and descriptive name, and in
the interest of definiteness without repetition, these
cells will be referred to in the subsequent descriptions
and discussions as “K cells.”


The membrana granulosa is sharply demarcated
This corpus luteum is fairly well preserved, as is indicated by the retention of many of the normal cellular relationships, particularly in the granulosa lutein layer. A striking feature, however, is that the theca lutein layer is represented by only a few scattered pycnotic nuclei (fig. 27, pl. 5). Nearly all the granulosa lutein cells are undergoing atrophy, and large, gaping vacuoles are scattered throughout the granulosa lutein layer. A number of colloid deposits are visible. The lutein layer is almost completely avascular. Connective-tissue organization is marked.
from the theca interna by a closely packed layer of
cells contiguous with, and similar to, the cells of the
membrana itself, which is many cell layers thick and
thrown into convolutions by the hypertrophy and
multiplication of the granulosa cells. Consequently,
the inner layer of the follicle presents a scalloped,
undulating edge, with tongues of theca interna projecting into the convolutions from the stroma of the
ovary. Mitotic figures are numerous in the membrana
granulosa. The nuclei of the granulosa cells are perhaps a little larger than those of the theca interna
cells, and because of their rapid rate of division and
growth are more basophilic. Their cytoplasm presents a frothy appearance, and cell outlines are indistinct, although there is no evidence of vacuolization.
There is no evidence of capillary penetration into or
proliferation within the membrana granulosa. None
of the K cells present in the theca interna are seen


in the membrana granulosa at this stage of development.


Corpus Luteum of 16x}: Day of Cycle, Estimatca’ Age
As in the haematoxylin and eosin preparations, the theca lutein layer is not evident in Sudan black treated sections. The granulosa lutein cells are uniformly devoid of lipid (fig. 28, pl. 5). An occasional K cell stands out conspicuously by virtue of its intense and uniform sudanophilia. Most of the K cells, however, appear only as “shadow forms,” having lost most of their sudanophilia. Though it is not evident in the photomicrograph, the fibrillar substructure of these cells is very conspicuous at higher magnification. Alkaline phosphatase is no longer demonstrable in the majority of granulosa lutein cells. A few cells along the outer margin of the lutein layer contain high concentrations of the enzyme (fig. 29, pl. 5). These cells occupy the same locus as the sudanophilic K cells mentioned above. It is of particular interest that the endothelium of blood vessels of both layers no longer contains demonstrable alkaline phosphatase.
2 Days, S48-2636


Macroscopically, this corpus luteum appeared as a
==Observations on Corpora Lutea associated with Abnormal Ova==
hemorrhagic, unhealed crater I cm. in diameter and
2 mm. in depth, on the posterior surface of the ovary.


Haematoxylin and eosin sections (fig. 2, pl. 1)
In view of the fact that our material included 13 corpora lutea associated with ova that were adjudged to be abnormal on the basis of criteria stated earlier, it was decided to study these specimens closely to determine whether any relation between anatomical integrity of the corpus luteum and the condition of the ovum could be detected. These abnormal ova have been the subject of separate communications by Hertig and Rock (1944, 1949!), 1950).
show the theca interna to be considerably thinned
out and the membrana granulosa thrown into a large
number of deep convolutions, owing in part to the
collapse of the follicle after rupture. The vascular
channels of the theca interna are widely dilated, and
in several places endothelial sprouts are seen penetrating the membrana granulosa from the theca interna. The cells of the theca interna are essentially
the same as those observed in the mature follicle, and
rarely show mitoses. The granulosa cells are plump
and polyhedral, with round, vesicular nuclei. Their
cytoplasm is frothy, but no distinct vacuolization is
seen. The granulosa cells are arranged in bundles
or fascicles separated in many areas by large lacunae
of extravasated blood. These lacunae are not lined
with endothelium.


The K cells noted in the theca interna of the mature
follicle are very prominent at this stage of development of the corpus luteum. Although few of them
are found in the theca interna, ribbons of them can
be seen spreading out into the membrana granulosa,
penetrating as far as the central coagulum. The attenuated cytoplasmic processes of these cells suggest
amoeboid activity. It is to be noted that these cells
are more numerous at this and subsequent stages of
development than in the mature follicle. Only once
have we observed a mitotic figure in a cell that we
could definitely say was a K cell. It is possible that
the small, irregular, hyperchromatic nuclei characteristic of these cells at this stage represent rapid mitotic
activity. As will be noted later, the nuclei of these
cells become larger and less hyperchromatic as the
gland approaches the period of maximum functional
activity.


Sudan black preparations (fig. 3, pl. 1) reveal that
Despite the fact that it is difficult to judge accurately the age of these ova, because of abnormalities of blastomeres, trophoblast, chorionic cavity, or germ disk, a reasonable estimate of age can be made from the appearance of the endometrium and from the history.
the cells of the theca interna contain much more
lipid than do those of the membrana granulosa. This
lipid is distributed in fine droplets in most cells,
62 CORPUS LUTEUM OF HUMAN PREGNANCY


although medium-sized and coarse droplets are found
===Corpora Lutea associated with Abnormal Free-lying Ova===
in some of the theca interna cells. As has been mentioned, the granulosa cells contain much less lipid,
and this is evenly distributed as a fine peripheral
dusting of sudanophilic lipid. In an occasional granulosa cell a few coarse globules of lipid are seen. The
K cells are distinguishable only with some diliiculty.
They present a uniform, nongranular sudanophilia.


Alkaline phosphatase is localized exclusively in the
The corpora lutea associated with S43-I372 (Carnegie no. 8190), a 9—cell, 31/2" to 4-day free-lying segmenting ovum, and S46-3332 (Carnegie no. 8450), an 8-cell egg, are in no way different from the corpora lutea of the 17th and 18th days of the normal nonpregnant menstrual cycle.
cells of the theca interim and in the endothelium of
blood vessels. The cytoplasm of the granulosa cells
contains no alkaline phosphatase at this stage of development (fig. 4, pl. 1).


Corpus Lzztezmz of zot/2 Day of Cycle, Estinzaterl Age
====A 5-Cell Egg, 19-Day Corpus Luteum, S48-3948====
6 Days, S48-2262


On cut section, the corpus luteum measured 1.7 by
The corpus luteum measured 2.0 by 1.8 cm. in its greatest diameters. The convoluted borders were pinkish gray, with a maximum thickness of 2 mm. The coagulum was pearly gray, and gelatinous in consistency.
1.5 cm. in its greater diameters. The convoluted
borders were bright yellow and from 1 to 3 mm. thick.
The central coagulum was pale gray with several
small hemorrhagic areas.


The corpus luteum at this stage is quite compact.
There is very little extravasated blood in either layer.
The blood vessels of the theca interna are widely
dilated. Definite capillaries are seen in the granulosa
layer, but these are still small and only an occasional
The central
coagulum contains many proliferating fibroblasts and


red blood cell can be noted in them.
The ovum consisted of five abnormal blastomeres, several of which were multinucleated and showed other evidences of degeneration and retardation of development.


thus is undergoing early organization. No capillaries
The corpus luteum differs in no respect from a 19-day corpus luteum of the normal nonpregnant menstrual cycle. The theca interna is quite prominent, several cell layers in thickness over the crests of the granulosa. The cytoplasm of the theca interna cells is markedly vacuolated. The granulosa lutein cells are plump and polyhedral, and only a few of them show peripheral vacuolization. A number of small capillaries and endothelial sprouts are seen in the granulosa lutein layer, but these capillaries are not dilated. K cells are not conspicuous in the routine haematoxylin and eosin sections.
are seen in it. The cells of the theca interna appear
to be widely separated by fibroblasts and other connective—tissue elements; their cytoplasm is markedly
vacuolated, but the nuclei show no evidence of cellular degeneration. The granulosa cells are large and
polyhedral, with distinct cell membranes and marked
peripheral vacuolization of the cytoplasm (fig. 5, pl.
1). Interspersed among the granulosa cells are a number of K cells, whose nuclei appear dark, irregular,
and almost pycnotic.


Sudanophilic substance is distributed irregularly in
the cells of the theca interna. Some of the theca cells
contain few lipid droplets; others contain numerous
fine, peripherally distributed lipid droplets, and many
contain large, coarse globules of sudanophilic substances. Nearly all the granulosa lutein cells contain
fine, peripherally distributed lipid droplets. Only an
occasional granulosa cell contains the coarse lipid


droplets noted in the theca. The large, stellate K cells
Most theca lutein cells contain fine lipid droplets, but many cells contain only rnedium-sized to coarse lipid granules. Nearly all the granulosa lutein cells contain large amounts of fine peripherally distributed lipid. An occasional granulosa cell contains mediumsized lipid droplets. The K cells are not particularly prominent, showing only a moderate degree of uniform slate-gray sudanophilia.
are particularly conspicuous at this stage. All of them
appear uniformly sudanophilic, although close examination reveals some granular deposits of sttdanophilic
substances which are almost masked by the uniformly
sudanophilic background of the cytoplasm of these
cells (fin. 6, pl. 1). The nuclei of the K cells are clear
and devoid of lipid.


The theca lutein cells are uniformly devoid of
Alkaline phosphatase is restricted to a few scattered clumps of cells of the theca lutein layer and the endothelium of blood vessels in both layers.
alkaline phosphatase. This statement applies equally
well to the majority of the granulosa cells. A small
number of cells scattered among the granulosa cells,
however, contain alkaline phosphatase in moderate
amounts. It is difiicult to state at this stage of development that these cells containing alkaline phosphatase deposits are the K cells so prominent in the
Sudan black preparation.


Corpus Lmfczmz of 23a? Day of Cycle, Estinzazerl Age
===Corpora Lutea Associated with Abnormal Ova with Adequate Trophoblast===
9 Days, S48-3028


The corpus luteum measured 1.5 cm. in its greatest
====An 8-Day Ovum, Carnegie No. {{CE8370}}, S46-676====
diameters. The convoluted border was bright yellow,
and averaged 3 mm. in thickness. The central coagt1him was well organized and was the site of a recent
small hemorrhage.


It is to be noted in this specimen that the theca
The ovum is the youngest implanted embryo of this series of abnormal ova. The chorionic cavity is absent. Although ectoderm is present in the germ disk, there is no endoderm. The trophoblast is adequate but poorly organized, that is, laminated instead of being concentrically arranged with the syncytiotrophoblast surrounding the cytotrophoblast.
lutein layer is much less prominent than in the 20-day
corpus luteum, but that those theca lutein cells that
are observed present essentially the same characteristics as those noted in the earlier stage. The granulosa lutein cells are plump and show all the evidences
of marked physiological activity. The K cells are
quite numerous and prominent in this specimen and
are readily recognized by their more angular shapes,
dark, homogeneous cytoplasm, and small, dark nuclei
(Fig. 7, pl. 2). Capillaries in the granulosa lutein
layer are numerous and widely dilated.


The theca lutein cells contain large, coarse drops
of lipid, whereas the granulosa lutein cells contain
fine, peripherally distributed lipid granules in large
numbers (fig. 8, pl. 2). The K cells are especially
prominent in this preparation, and there can be little
question that these elongated, uniformly sudanophilic
cells with clear, ovoid nuclei are the same cells that
are so conspicuous in haemato:~:ylin and eosin preparations.


Only an occasional theca lutein cell contains alkaline phosphatase (fig. 9, pl. 2). The true granulosa
The corpus luteum appears to be normal in all respects. The theca interna is prominent. Both layers are well vascularized. The granulosa lutein cells show a moderate degree of peripheral vacuolization. K cells are numerous and appear to be normally active. No evidence of colloid degeneration is seen.
CORPUS LUTEUM OF HUMAN PREGNANCY 63


lutein cells are devoid of this enzyme. A number of
====A 10- to 11-Day Ovum, Carnegie No. {{CE7770}}, S40-749====
cells, however, having the configuration and nuclear
characteristics of the K cells as they appear in routine
and Sudan-treated preparations contain high concentrations of alkaline phosphatase. Capillary endothelium
in all layers of the corpus luteum contains the enzyme
in high concentration.


Franz 24:/2 Day of Cycle to M emtmation
This ovum is not markedly defective. The trophoblast is moderately hypoplastic and the germ disk is maloriented.


Between the 23d day of the cycle and the onset of
The theca interna appears to be normal in all respects and consistent with that of a normal 10- or 11-day pregnancy. The granulosa lutein layer is moderately well vascularized. K cells are relatively few, but appear to be normal in all respects. There is, however, great variation in size and stainability of the nuclei of the granulosa lutein cells.
menstruation, the evidence of regression in the corpus
luteum becomes increasingly marked. There is no
evidence of further capillary proliferation. The theca
cells become less and less distinct, until they can be
found only in widely separated clumps in the connective-tissue septa invaginating the granulosa from
the ovarian stroma. The granulosa lutein cells show
evidence of degeneration, manifested by loss of
chromaticity of the nuclei and loss of most of the
peripheral vacuolization noted at earlier stages. Many
granulosa cells show increasing accumulation of
medium—sized and coarse lipid droplets.


The fate of the K cells becomes evident during this
====An 11-Day Ovum, Carnegie No. {{CE8299}}, 545-1220====
period. With haematoxylin and eosin stains these
cells become more eosinophilie, the cytoplasm becomes
more dense and homogeneous, and the nuclei become
quite contracted and hyperchromatic. The cytoplasm
seems to condense, until ultimately all that remains
of many of these cells late in the life of the corpus
luteum is a dense, eosinophilie mass similar to the
colloid described by Gillman and Stein (1941). This
process of colloid degeneration evidently extends over
a long period, because, as will be noted in our discussion of the corpus luteum of early menstruation,
large numbers of apparently normal K cells can be
found in older corpora lutea. The K cells are still
prominent in Sudan black preparations, but show a
progressive loss of sudanophilia and a retraction of
their cytoplasmic processes. The pattern of alkaline
phosphatase distribution remains essentially the same
as that observed in the 23-day specimen, with perhaps
some diminution in the concentration of the enzyme
in the K cells as the corpus luteum becomes older.


Corpus Lzztermz during Early Memtrrzrztion, S48-535
The trophoblast appears to be normal although somewhat poorly organized. The germ disk is markedly maloriented.


At this stage the theca interna is very indistinct.
The corpus luteum is very large and consists of a large cystic cavity enclosed by a thin rim of lutein tissue. Both granulosa and theca lutein layers are moderately well vascularized and appear to be functionally active. Most of the K cells show little evidence of colloid degeneration. This appears to be a very active corpus luteum (fig. 30, pl. 6).
True theca lutein cells are found only in scattered


clumps, chiefly in the connective-tissue septa that
====Carnegie No. {{CE7850}}, S40-2699====
penetrate between the folds of the granulosa from
the ovarian side of the gland (fig. 10, pl. 2). Those
theca cells that persist are markedly vacuolated and
show wide variation in the staining characteristics of
their nuclei. Most of the granulosa lutein cells have
lost the peripheral vacuolization that typifies the
actively secreting gland. Many cell boundaries in the
granulosa lutein layer are quite indistinct. The capillaries in both cell layers are more or less uniformly
collapsed, and contain few erythrocytes. Although
the K cells are still prominent in routine sections,
they do not present the full-blown appearance seen in
the 23-day corpus luteum. Moreover, their cytoplasm
is denser than has previously been noted and in most
instances appears to be contracting, leaving large
vacuoles between the granulosa lutein cells. Some
of the K cells have degenerated to what we consider
the end stage of this line of cells, a dense, strongly
eosinophilie colloid droplet. All gradations between
a stellate, active cell and the final degenerative end
product, colloid, can be found in this specimen, a fact
which indicates that this process of colloid degeneration must extend well beyond the menstrual period.


The theca lutein cells contain only medium-sized
This ovum is associated with an endometrium that evidences early decidual reaction. Except for moderate hypoplasia of the trophoblast, the ovum appears to be fairly good.
and coarsely irregular lipid droplets (fig. 11, pl. 2).
The true granulosa cells contain much more lipid
than at any previous stage. Although the lipid droplets are larger than in earlier stages, they are peripherally distributed in most granulosa cells, and in none
are the droplets as large and coarse as those in the
theca lutein cells. A few granulosa cells are heavily
laden with medium-sized lipid droplets. The K cells
are still prominent in the Sudan black preparations,
but their cytoplasmic processes are markedly retracted,
the cytoplasm has lost some of its uniform homogeneous sudanophilia, and a few fine sudanophilic
granules are seen against the slate-gray cytoplasmic
background of the cells. A few large, oily drops
showing varying degrees of sudanophilia are present.
It is believed that these represent the colloid drops
noted in the haematoxylin and eosin preparations.


The alkaline phosphatase preparations show essentially the same pattern of distribution of the enzyme
as in the 23-day specimen, with perhaps some diminution in the concentration of the enzyme in the K cells
(fig. 12, pl. 2).
64 CORPUS LUTEUM OF HUMAN PREGNANCY


OBSERVATIONS ON CORPORA LUTEA OF NORMAL PREGNANCY
The theca interna is prominent, many cell layers thick, and appears to be functionally active. Peripheral vacuolization of the granulosa lutein cells is not so marked as in chronologically similar corpora lutea associated with normal pregnancy. Mitoses are to be noted in both layers of the corpus luteum. K cells are quite numerous, but are irregularly distributed. No evidence of colloid degeneration is to be noted anywhere in the section.


A 2-03}! Egg, 17-Day Corpus Luream, 349-2439
===Corpra Lutea associated with Abnormal Shallow Implantation===


This ovum was recovered from the middle third
====An 11- to 12-Day Ovum, Carnegie No. {{CE8000}}, S42-217====
of the Fallopian tube and consisted of two normal
blastomeres. This specimen, of about 60 hours coital
age, is the earliest human ovum yet recovered. The
endometrium had the characteristics of that of 2 to
21/; days after ovulation.


On cut surface, the corpus luteum measured 2.5 by
The ovum is very shallowly implanted, although all the elements appear to be normal.
1.5 cm. in its greatest diameters. The convoluted
border was red-gray, and varied in thickness from
3 mm. at the base to I mm. at the unhealed stigma.
The central coagulum was moderately well organized
and showed some peripheral congestion.


Microscopic examination demonstrates that this
The theca lutein layer appears to be normal and contains several immature K cells. The granulosa lutein layer is moderately well vascularized, and although some lutein cells show early signs of atrophy, the majority appear to be functionally active. Distinctive K cells are rare, but little colloid degeneration is seen.
corpus luteum is in no respect materially different
from S48-2636 (figs. 2-4). K cells streaming into the
granulosa lutein layer from the theca are prominent
and numerous. Sudanophilic substances are much
more prominent in the theca interna than in the
granulosa lutein layer. Alkaline phosphatase is restricted to the theca interna and the endothelium of
the blood vessels.


A 413-Day Blm't0c_v5t, 19-Day Corpus Lzrteunz,
====A 12.5-Day Ovum, Carnegie No. {{CE290}}, S44-2785====
548-5000


This specimen is the first normal human blastocyst
Although this ovum is superficially implanted, it shows evidence of early villus formation. The germ disk has undergone a curious buckling and is still attached to the trophoblast. The latter is irregularly developed, poorest at the implantation pole and, paradoxically, good elsewhere.
yet recovered from the uterine cavity. The associated
endometrium is typical 19-day secretory endometrium.


On cut surface the corpus luteum measured 2.2 cm.
The corpus luteum is in no way markedly different from that associated with Carnegie no. {{CE8000}}.
in its greatest diameters. The convoluted borders were
reddish gray, measuring 3 to 4 mm. in thickness. The
stigma was completely healed, and the central coagulum was pale and gelatinous.


Microscopic examination reveals that this corpus
===Corpus Lutea associated with an Ovum consisting of Syncytiotrophoblast Only===
luteum is almost identical in all respects with the
20-day corpus luteum of the normal menstrual cycle
and with S48-3948, the corpus luteum associated with
the abnormal 5-cell, 4‘/§_- to 5-day ovum.


7‘/3- and 913-Day Pregrzazzcies
====A 12-Day Ovum, Carnegie No. {{CE8329}}, S45-1809====


Figures 13 and 14, plate 3, are photomicrographs
This embryo is markedly abnormal in that there is no cytotrophoblast, no segmentation cavity, and no germ disk, but as a mass of syncytiotrophoblast it is fairly large.
of sections of corpora ltttea associated with 7‘/3- and
9‘/3-day normal pregnancies respectively (Carnegie
nos. 8020, 8215). Unfortunately, these sections, which
were stained with haematoxylin and eosin, have faded


to a great extent, but the following features are noteworthy. There is a progressive increase in vascularity
Both lutein layers appear to be well vascularized and functionally active. K cells are numerous in certain parts of the section, but almost completely lacking in other areas. There is, however, no evidence of colloid degeneration of any of the K cells (fig. 31, pl. 6).
and dilatation of the capillaries in the granulosa lutein
layer. The theca lutein layer remains quite distinct
and prominent. Peripheral vacuolization of the granulosa lutein cells, which is normally disappearing in
the corpus luteum of the nonpregnant cycle by 26 to
27 days, becomes increasingly noticeable after 91/; days
of pregnancy and is particularly striking at II and
12 days of pregnancy. The cell boundaries of the
granulosa cells become progressively less distinct because of the line peripheral vacuolization. At this
stage the granulosa cells resemble the “prickle cells”
described by Hertig and Rock (1941). Although the
K cells are not strikingly conspicuous in any of these
sections, they are seen in considerable numbers. It
appears that these cells have actually been stimulated
and are assuming the appearance of full-blown activity
noted in the 21-day corpus luteum (Hg. 7).


A 12- to I 3-Day Pregnancy, Carnegie N 0. 8558,
===Corpus Lutea associated with an Ovum showing Hypoplastic rophoblast===
S46-2767


The corpus luteum was moderately cystic and
====Carnegie No. {{CE7771}}, S40-791====
measured 2.0 cm. in its greatest diameters. The convoluted border was 1 to 3 mm. thick and was pale
yellow with an orange tinge, but did not appear to
be senescent.


The theca lutein layer is quite prominent and many
This markedly abnormal ovum is associated with a 27-day late-secretory endometrium. The ovum is a polypoid structure; both elements of the trophoblast are present, but poor in quality and organization. The germ disk is totally lacking.
cell layers in thickness. The cytoplasm of these cells
is vacuolated, and the nuclei resemble those of actively
functioning cells. The granulosa lutein cells show
marked peripheral vacuolization, and the cell boundaries are thus almost completely obliterated (fig. 15,
pl. 3). There has been a significant increase in the
number of widely patent vascular channels in the
granulosa layer. K cells are not at all conspicuous in
haematoxylin and eosin sections. However, the dense,
pycnotic nuclei and contracted cytoplasm characteristic of many of the K cells in figure 10 are not frequently seen here, a fact which indicates a recrudescence of activity of these cells.


A very striking change is noted in the Sudan black
The corpus luteum is poorly vascularized. Both theca lutein and granulosa lutein cells appear to be moderately active, but not so much so as one would find in a normal pregnancy of this stage of development. A very striking feature is the complete colloid degeneration of all the K cells. Large amounts of colloid are present in all parts of the section, and no functional K cells are seen (fig. 32, pl. 6).
preparations. Only a few theca interna cells contain
coarse lipid granules. The majority of the theca lutein
and granulosa cells contain large numbers of very
fine, peripherally distributed sudanophilic droplets.
CORPUS LUTEUM OF HUMAN PREGNANCY 65


K cells are very prominent (fig. 16, pl. 3) and show
====Carnegie No. {{CE7800}}, S40-1327====
marked uniform sudanophilia and attenuated cytoplasmic processes like those seen in the physiologically
active corpus luteum of the 23d clay of the nonpregnant cycle (fig. 8).


A few of the theca lutein cells contain alkaline
This ovum is associated with an endometrium that shows early decidual reaction. The chorion is irregularly deficient, with marked hypoplasia of the trophoblast. To judge from the development of the embryo, the chorion should have early villi.
phosphatase, but most are devoid of the enzyme. A
few K cells at the junction of the granulosa and theca
lutein layers contain a high concentration of alkaline
phosphatase, but of particular interest is the demonstration of the enzyme in significant amounts in some
of the true granulosa lutein cells (fig. 17, pl. 3).


A 16-Day Pregnancy, Carnegie No. 8602, S48-2088
The theca lutein layer appears to be normal and functioning. The granulosa lutein layer is well vascularized. There is marked variation in size, regularity, and stainability of the nuclei of the granulosa lutein cells. The majority of the K cells have undergone more or less complete colloid degeneration, although this feature is not so conspicuous as in Carnegie no. {{CE7771}}.


The cystic corpus luteum measured 3 cm. in its
====About 3 Months Pregnancy, Blighted Ovum, S48-824====
greatest diameters. The convoluted borders were a
brilliant yellow and varied in thickness from 4 mm.
at the base to I mm. at the healed stigma.


Haematoxylin and eosin sections of this specimen
This specimen was obtained with the corresponding left tube, left ovary, and uterus. Pregnancy was interrupted because of impending cardiac failure. The placenta was found to be immature and associated with a blighted ovum of about 3 months menstrual age. The cystic corpus luteum measured 1.5‘ cm. in diameter and contained straw-colored fluid. The lutein border was grayish yellow, and about 2 mm. thick.
are very similar to those of the preceding stage. The
theca interna is quite prominent (fig. 18, pl. 4). The
granulosa lutein cells show considerable activity, manifested by noticeable peripheral vacuolization which
causes cell boundaries to appear very indistinct. A
large number of fine capillaries form a delicate vascular network in the granulosa lutein layer. K cells
are even more numerous and distinct in this specimen
than in the 12- to 13-day pregnancy (fig. 15), but a
number of these cells are undergoing colloid degeneration as described in the corpus luteum of early
menstruation. In this and in subsequent specimens
to be described, a number of regular, spherical vacuoles
are seen in the granulosa lutein layer. These vacuoles
apparently mark the site of colloid deposits, which
have dropped out of the section in preparation.


The great majority of both theca lutein cells and
granulosa lutein cells contain large numbers of fine
peripheral sudanophilic droplets. An occasional theca
lutein cell contains a number of medium-sized lipid
droplets (fig. 19, pl. 4). The K cells are quite conspicuous and appear essentially the same as in the
corpus luteum of the 12- to 13-day pregnancy.


In figure 20, plate 4, the theca lutein cells are seen to
Haematoxylin and eosin sections (fig. 33, pl. 6) show a corpus luteum that has lost all its distinctive morphologic features. The theca lutein layer is markedly reduced in bulk. Most of the nuclei are densely pycnotic. The granulosa lutein layer has lost all evidence of functional activity. Not a single active lutein cell can be found. In many, the nuclei are dense and pycnotic, and in others the nuclei have completely lost their capacity for staining. A large number of colloid-containing vacuoles are present. Cell boundaries are totally obliterated. Connective tissue invasion of both layers is marked. The capillaries of the granulosa lutein layer are collapsed.
be almost completely devoid of alkaline phosphatase.
Nearly all of the granulosa lutein cells, however, contain alkaline phosphatase in varying amounts, thereby
making it difficult to identify the K cells. Several
cells at the junction of theca lutein and granulosa


lutein layers contain a high concentration of the
enzyme, and on the basis of their position and configuration these are very likely the K cells so conspicuously revealed by other techniques.


A 26-Day Pregnancy, S48-263:
Only an occasional cell of the theca lutein layer contains alkaline phosphatase. Nearly every cell in the granulosa lutein layer contains some of the enzyme, but not in significant amounts.


This specimen was obtained with the uterus at time
of operation for carcinoma of the cervix. The corpus
luteum was cystic, 2 to 4 cm. in diameter, and was
filled with a clear, yellowish fluid. The convoluted
border was yellowish gray, and 2 to 3 mm. thick.


The theca interna is very conspicuous in this specimen, being several cell layers thick around the entire
Inasmuch as the ovum was found to be blighted, and the placenta was immature, it is highly probable that this specimen does not represent a normal 3 months pregnancy. It is included to demonstrate that the degenerative changes in the Corpus luteum may parallel trophoblastic degeneration.
granulosa lutein layer (fig. 21, pl. 4). The nuclei
stain uniformly and present the appearance of actively
functioning cells. For the first time since the stages
of the mature follicle and the 16-day corpus luteum
of the nonpregnant menstrual cycle, a number of
small, irregular stellate cells with wrinkled hyperchromatic nuclei and homogeneous eosinophilic cytoplasm are seen in the theca lutein layer. Except for
somewhat smaller size, these cells are identical in all
respects with the K cells noted in routine haematoxylin and eosin sections of the younger corpora
lutea. The sudden reappearance of these cells in the
theca lutein layer is unexplained. There is no evidence of transformation or degeneration of theca
lutein cells to these forms. The capillaries and sinusoids of the theca lutein layer are widely dilated.


The granulosa lutein layer at this stage appears
Unfortunately, no formalin-fixed material was available at the time this specimen came to our attention, and lipid studies could not be made.
highly disorganized. Cell boundaries are quite indistinct. The individual granulosa lutein cells show
marked variation in the stainability of their nuclei
and cytoplasm. There are a large number of patent
capillaries in the granulosa lutein layer. Around each
of these capillaries, proliferation of perivascular connective-tissue elements is seen. There is a definite
increase in the reticular network that at this stage
encircles nearly every granulosa lutein cell. The central coagulum is well organized, and large, dilated
vascular channels are present in this new connective
tissue.


Very few K cells that may be regarded as active
==Notes on the K Cells==
are seen, but large numbers of these cells in all stages
of degeneration are present. Colloid is present in
larger amounts, and as a corollary the number of
large, empty vacuoles is also increased over the preceding stage.
66 CORPUS LUTEUM OF HUMAN PREGNANCY


Most of the theca lutein and granulosa lutein cells
Although this study was undertaken to outline the morphological changes that take place in the corpus luteum during early pregnancy, it became apparent quite early in the course of the investigation that the K cells described in all specimens discussed bear more than a casual relation to the cyclical activity of the gland, in the nonpregnant cycle as well as in early pregnancy.
contain very fine lipid droplets in large quantities
(fig. 22, pl.  An occasional theca lutein cell contains a few medium-sized lipid droplets. A significant number of the granulosa lutein cells contain
large amounts of lipid in large, coarse droplets, indicating fatty degeneration. The K cells are still quite
prominent and markedly sudanophilic, but they
appear frayed and fibrillar. They are definitely not
so numerous or attenuated as in figure 19. Many
large, smooth sudanophilie globules are present. These
match in number, size, shape, and location the colloid
droplets seen in haematoxylin and eosin sections.


Of particular interest is the appearance of large
numbers of these characteristic sudanophilic K cells
scattered among the cells of the theca lutein layer.
Both examples of 26-day pregnancy show them, and
although carefully searched for, they were not seen in
any other specimen. These cells appear to be very
similar to the K cells observed in the early stages of
development of the corpus luteum, and do not present
the frayed, fibrillar appearance noted in the K cells
in the granulosa lutein layer of this same specimen,
facts which suggest that these cells represent younger
forms.


The theca interna is uniformly devoid of alkaline
These cells are conspicuous in routine sections because of their homogeneous, highly cosinophilic cytoplasm. It is also to be noted, however, that in sections treated with basic dyes, their cytoplasm is more markedly basophilic than that of neighboring lutein cells. This suggests strong acid properties of the protoplasm, a reaction characteristic of phospholipid. As previously stated, these cells are very striking in sections treated with Sudan black. They are uniformly sudanophilic, in contrast with the granular sudanophilia of the theca lutein and granulosa lutein cells. The sudanophilic material is soluble with difficulty in cold alcohol or alcohol-acetone mixtures. Traces of sudanophilia can be detected in these cells after 24 hours’ treatment of the section in lipid solvents before staining. By contrast, the granular deposits of lipid in theca lutein and granulosa lutein cells are completely dissolved by similarly pretreating the sections for 1/3 to 1 hour before staining with Sudan black. The low solubility of the sudanophilic substance in these K cells is characteristic of phospholipid.
phosphatase (Hg. 23, pl. 4), although most of the
granulosa lutein cells contain moderate amounts of
the enzyme. It is diflicult to recognize K cells in this
preparation. The endothelium of blood vessels in
both layers contains alkaline phosphatase.


A 28- to 35-Day Preg.rzm1c_v, S48-4854


On gross examination the corpus luteum measured
Because of these indications of high phospholipid content in the K cells, the technique for the demonstration of phospholipid in tissues (Baker, 1946) was employed in two selected corpora lutea. Figure 34, plate 7, is a photomicrograph of a K cell, at a magnification of 750, in a :20-day corpus luteum of the normal menstrual cycle, showing a high concentration of phospholipid. The phospholipid is uniformly distributed in these cells, as contrasted with the lutein cells of both layers, where phospholipid appears as peripherally distributed granules. It was observed incidentally that in the developing follicle phospholipicl is restricted to the theca interna layer. Pretreatment of parallel sections with pyridine completely removed all traces of phospholipid from the gland.
2.3 by 2.0 cm. in its greatest diameters. The convoluted border was grayish yellow, measuring 2 to
3 mm. in thickness. The central coagulum was well
organizecl.


The theca lutein layer is still very prominent,
although not so conspicuous as in the preceding specimen. Many of the nuclei are dense and irregularly
pycnotic. The cytoplasm of most of the cells is highly
vacuolated (fig. 24, pl. 5). The vascular sinusoids
of the theca interna are almost completely collapsed.


The granulosa lutein layer presents essentially the
All the specimens discussed in this paper were subjected to the tests for presumptive ketosteroids employed so effectively by Dempsey and Bassett (1943) and by McKay and Robinson (1947). Our findings in the developing follicle and corpus luteum of the nonpregnant cycle agree substantially with those of McKay and Robinson. It was noted, however, in the course of these studies that the K cells were reactive with the Schiff, phenylhydrazine, and LiebermanBurchardt reactions. This reactivity is manifested as a uniform, homogeneous color reaction, in contrast with the granular reactivity of theca lutein cells described by McKay and Robinson. As with Sudan black, this reactive material was more dillicult to dissolve in lipid solvents than that of the theca lutein cells. Observations of autofluorescence under ultraviolet light were equivocal. It is noteworthy, however, that birefringent crystals cannot be observed in these K cells under the polarizing microscope. In only one specimen, the 16-day normal pregnancy (Carnegie no. {{CE8602}}), was a suggestion of crystalline birefringence seen. Up to this point, all the reactions of the K cells pointed to ketosteroid substances, but failure to demonstrate birefringent crystals in them seemed to rule out that possibility. It has subsequently been pointed out to us by Seligman (personal communication) that all the evidence presented above points to the fact that the high phospholipid content of these cells may prevent the crystallization of ketosteroid necessary for development of birefringence in plane-polarized light.
same picture as that of the preceding specimen in
haematoxylin and eosin preparations. Figure 25,
plate 5, is a photomicrograph of this same section,


showing a clearly defined colloid globule. Other
K cells in various stages of degeneration are present
in this section.


The Sudan black preparations show large numbers
Very recently Seligman and Ashbel (I949) developed a technique for the demonstration of ketonic lipids. Figures 35 and 36, plate 7, are photomicrographs of sections treated by this technique. It will be noted that reactive material is concentrated in these cells. Seligman and Ashbel have observed that the corpora lutea of animals do not react to the tests for ketosteroid unless they are fixed in formalin, which unmasks the active carbonyl group. To rule out the possibility that the reactive material in these cells might be an aldehydic group, produced by hydrolysis of plasmogens, parallel sections were treated by a new technique for the demonstration of free aldehyde groups (Seligman and Ashbel, unpublished data). No free aldehydic groups could be demonstrated. Thus, all the evidence seems to indicate that these K cells represent the locus of high concentration of ketosteroid. Their reactivity as measured by these special histochemical techniques parallels closely the changes observed in sudanophilia during various stages of development of the corpus luteum.
of very fine peripherally distributed lipid granules in
both theca lutein and granulosa lutein cells (fig. 26,
pl. 5). Occasional cells undergong fatty degeneration are seen in both layers. The K cells are still quite
conspicuous in the granulosa lutein layer, and all show
a line dusting of sudanophilic droplets in their cytoplasm. A few small, uniformly sudanophilic cells
similar to those described in the previous specimen
are seen in the theca lutein layer, but these are not
sufficiently conspicuous to photograph. A number of
large, oily sudanophilic droplets resting in vacuoles
may be noted among the granulosa cells. It is believed that this is the colloid mentioned earlier.


The alkaline phosphatase preparations are particularly striking. A number of the cells of the theca
lutein layer contain large amounts of the enzyme,
but most of the theca cells are totally devoid of it.
On the other hand, the granulosa lutein layer contains a very high concentration of the enzyme, which
is so diffusely distributed that it is impossible to
separate the granulosa lutein cells from the K cells.
Numerous large vacuoles, representing the site of
colloid deposits that have been lost or dissolved in the
process of preparation, are evident in this section as
in figures 20 and 23.


/I 4- to 4‘/_-;_.-Mont/z Pregrzrmcy, 548-2624
As regards the question of origin and function of the K cells, it must be borne in mind that they could represent undifferentiated elements of the reticuloendothelial system, for example wandering macrophages, angioblasts, or young fibroblasts. It is possible that these cells arc histiocytic, and that the intense localization of ketonic lipid noted in them represents merely the phagocytosis of excess ketonic lipid. It is planned to perfuse a human ovary containing an active corpus luteum with a vital dye to determine whether these cells have the capacity to phagocytose foreign matter, utilizing the technique of ‘Nerthessen (1949). In the absence of further and more definitive studies, we believe that all the evidence presented regarding the peculiar characteristics of these cells points to their being a distinct cell line, intimately related in some manner to the function of the corpus luteum in the production or utilization of ketonic lipid, that is, ketosteroid compounds.


This specimen was obtained incidentally to a total
==Discussion==
hysterectomy performed during the 5th month of
pregnancy because of carcinoma of the cervix. The
corpus luteum measured 2 by 1.5 cm. The stigma
was depressed and well healed. The periphery of the
corpus luteum presented a yellowish, fatlike appearance upon the cut surface. Several small gelatinous
areas were seen between the convolutions. There
was a small hemorrhage, 2 by 5 cm., in the exact
center of the gland.


This corpus luteum is fairly well preserved, as is
Our observations of the corpus luteum of the nonpregnant cycle parallel substantially the classic description of Meyer (1911). Observations of vascular changes, coupled with information that can be deduced from histochemical procedures, lead us to agree with Brewer (1942) that the corpus luteum reaches its maximum activity on or about the 9th day after ovulation. ‘Ne have assumed the following criteria as evidence of functional activity of the corpus luteum: widely dilated capillaries in both theca and granulosa lutein layers, fine peripherally distributed sudanophilic substances in the lutein cells, demonstrable alkaline phosphatase in the cytoplasm, and ketonic lipid demonstrable by the techniques described above.
indicated by the retention of many of the normal
cellular relationships, particularly in the granulosa
lutein layer. A striking feature, however, is that the
theca lutein layer is represented by only a few scattered pycnotic nuclei (fig. 27, pl. 5). Nearly all the
granulosa lutein cells are undergoing atrophy, and
CORPUS LUTEUM OF HUMAN PREGNANCY 67


large, gaping vacuoles are scattered throughout the
granulosa lutein layer. A number of colloid deposits
are visible. The lutein layer is almost completely
avascular. Connective-tissue organization is marked.


As in the haematoxylin and eosin preparations, the
It has been generally held that the ovary is responsible for at least two hormones, both of which have experimentally proved specific actions in the menstrual cycle. Dempsey and Bassett (1943) working with rats, and McKay and Robinson (1947) in a study of human material, found that reactive material, presumptively ketosteroid, was localized exclusively in the theca interna of the developing follicle and appeared in insignificant amounts in the granulosa lutein cells during the active stages of the corpus luteum. Inasmuch as they could not demonstrate ketosteroid in significant amounts in the granulosa lutein layer, they suggested that the theca interna is probably responsible for the production of both progesterone and estrogenic substances. Our observations suggest that a ketosteroid substance is intensely localized in a specific line of cells having their origin in the theca interim and making their way into the granulosa lutein layer. It is possible that the hormone production of these cells in the unruptured follicle is responsible for the suggestive progestational changes that take place in the uterus of experimental animals at or shortly before ovulation (Reynolds and Friedman, I93o; Astwood, 1939).
theca lutein layer is not evident in Sudan black
treated sections. The granulosa lutein cells are uniformly devoid of lipid (fig. 28, pl. 5). An occasional
K cell stands out conspicuously by virtue of its intense
and uniform sudanophilia. Most of the K cells, however, appear only as “shadow forms,” having lost most


of their sudanophilia. Though it is not evident in the
photomicrograph, the fibrillar substructure of these
cells is very conspicuous at higher magnification.
Alkaline phosphatase is no longer demonstrable in
the majority of granulosa lutein cells. A few cells
along the outer margin of the lutein layer contain
high concentrations of the enzyme (fig. 29, pl. 5).
These cells occupy the same locus as the sudanophilic
K cells mentioned above. It is of particular interest
that the endothelium of blood vessels of both layers
no longer contains demonstrable alkaline phosphatase.


OBSERVATIONS ON CORPORA LUTEA ASSOCIATED VVITH ABNORMAL OVA
We have no evidence that these cells are specifically concerned with the production of progesterone rather than estrogen. Nevertheless, the temptation to infer that such a relationship exists is strong. There is no valid reason why a given cell cannot produce more than one hormone, as those of the pituitary gland apparently do. As has been observed, these K cells show the greatest evidence of functional activity dur ing the stages when maintenance of the progestational state of the uterine endometrium is essential. In the normal menstrual cycle, if a fertilized ovum has not become implanted in the endometrium by 6 or 7 days after ovulation, that cycle ends in menstruation (Rock and Hertig, 1948). In such an event, the need for maintenance of the progestational type of endometrium no longer exists and the corpus luteum soon begins to regress. On the other hand, if pregnancy is superimposed, the earliest evidence of sustained and accelerated functional activity is seen in the recrudescence of activity in the K cells. It has been generally agreed that pregnancy will continue uninterrupted even though the corpus luteum is removed, when the placenta is producing suflicient progesterone to maintain the decidua. The exact time at which this transfer of function from corpus luteum to placenta takes place is not so well agreed upon. Dr. G. van S. Smith (unpublished data) states that the corpus luteum is essential through the 7th to the 8th, and possibly the 9th, week of pregnancy. No doubt there is considerable overlapping of hormone production by corpus luteum and placenta. Our observations suggest that the corpus luteum ceases to produce hormone completely by the 4th month of pregnancy, and there is evidence that functional activity decreases rather sharply between the 7th and 12th weeks of gestation.


In view of the fact that our material included 13
corpora lutea associated with ova that were adjudged
to be abnormal on the basis of criteria stated earlier,
it was decided to study these specimens closely to
determine whether any relation between anatomical
integrity of the corpus luteum and the condition of
the ovum could be detected. These abnormal ova
have been the subject of separate communications by
Hertig and Rock (1944, 1949!), 1950).


Despite the fact that it is difficult to judge accurately the age of these ova, because of abnormalities
Our studies indicate that the theca interna contributes substantially to the granulosa lutein layer in the human corpus luteum. However, the theca lutein and granulosa lutein cells described by all workers remain separate and distinct entities. The elements contributed to the granulosa lutein layer by the theca lutein layer are the K cells to which so much space has been given in this discussion.
of blastomeres, trophoblast, chorionic cavity, or germ
disk, a reasonable estimate of age can be made from
the appearance of the endometrium and from the
history.


CORPORA I_.U’l'I-IA ASSOCIATED wrri-1 .A13:~.'-orm.-xi.
FREE-LYING Ova


The corpora lutea associated with S43-I372 (Carnegie no. 8190), a 9—cell, 31/2" to 4-day free-lying segmenting ovum, and S46-3332 (Carnegie no. 8450),
These K cells in the granulosa lutein layer of the human corpus luteum have been observed by many earlier workers, as cited by Gillman and Stein (1941). These latter authors have interpreted them as representing different phases of functional activity of the granulosa lutein cells. It is highly probable that earlier work failed to elicit the true nature of these cells for three reasons: First, they had not been observed in the theca interna of the developing follicle; secondly, there had been no observation of migration of cellular elements from the theca interna into the granulosa lutein layer; and, thirdly, lipid stains such as Sudan black, which is capable of staining the phospholipid so characteristic of these cells, were not available.
an 8-cell egg, are in no way different from the corpora
lutea of the 17th and 18th days of the normal nonpregnant menstrual cycle.


A 5-Cell Egg, I9-Day Corpus Lute:-:m, S48-3948


The corpus luteum measured 2.0 by 1.8 cm. in its
The specific colloid degeneration which is peculiar to these K cells is interpreted as further evidence that they represent a distinct cell type. The fact that this colloid has the same histochemical properties as the K cells suggests that the colloid represents stored secretory products. Although colloid deposits are visible in old corpora lutea of the nonpregnant cycle, they are much more conspicuous in the degenerating corpus luteum of pregnancy, a fact which suggests both an increase in the number of cells producing the precursor of this product and an increased concentration of precolloid ketosteroid-phospholipid complexes within the cell.
greatest diameters. The convoluted borders were
pinkish gray, with a maximum thickness of 2 mm.
The coagulum was pearly gray, and gelatinous in
consistency.


The ovum consisted of five abnormal blastomeres,
several of which were multinucleated and showed


other evidences of degeneration and retardation of
As observed by Corner (19.18), in the human ovary alkaline phosphatase is restricted to the theca interna of the developing follicle and disappears from the theca lutein cells by the 4th or 5th post-ovulatory day. In contrast with Corner’s findings, we observed alkaline phosphatase in a few granulosa lutein cells, but this becomes evident only as the gland approaches the peak of functional activity, that is, at or about 8 days after ovulation. As stated earlier, we have observed alkaline phosphatase in the K cells as early as 6 days after rupture of the follicle. V-Vhen pregnancy occurs, alkaline phosphatase appears in increasingly higher concentrations in the K cells, and in gradually increasing amounts in the true granulosa lutein cells as the progress of pregnancy makes additional demands on the corpus luteum. There is some recrudescence of alkaline phosphatase in the theca lutein cells, most marked at 6 or 7 weeks menstrual age. The activity of the theca lutein layer during pregnancy, as measured by the concentration of this particular enzyme, is insignificant when compared with that of the granulosa lutein layer and the K cells.
development.


The corpus luteum differs in no respect from a
19-day corpus luteum of the normal nonpregnant
menstrual cycle. The theca interna is quite prominent, several cell layers in thickness over the crests
of the granulosa. The cytoplasm of the theca interna
cells is markedly vacuolated. The granulosa lutein
cells are plump and polyhedral, and only a few of
them show peripheral vacuolization. A number of
small capillaries and endothelial sprouts are seen in
the granulosa lutein layer, but these capillaries are
not dilated. K cells are not conspicuous in the routine haematoxylin and eosin sections.


Most theca lutein cells contain fine lipid droplets,
The theca lutein layer undergoes marked hypertrophy during early pregnancy, attaining its maximum development about the 26th day of pregnancy, dating from ovulation. Early in pregnancy, the coarse lipid deposits characteristic of the menstrual corpus luteum are completely replaced by fine, granular lipid deposits which are indicative of functional activity. After the 26th day of pregnancy the theca lutein layer becomes less and less prominent, until at 4 months no trace can be found except a few small pycnotic nuclei. That the theca interna may continue to supply K cells is suggested by the fact that recrudescence of K cells in the theca lutein layer is observed in both the corpora lutea associated with 26-day pregnancies.
but many cells contain only rnedium-sized to coarse
lipid granules. Nearly all the granulosa lutein cells
contain large amounts of fine peripherally distributed
lipid. An occasional granulosa cell contains mediumsized lipid droplets. The K cells are not particularly
prominent, showing only a moderate degree of uniform slate-gray sudanophilia.


Alkaline phosphatase is restricted to a few scattered
clumps of cells of the theca lutein layer and the
endothelium of blood vessels in both layers.


CORPORA LUTEA Assocuman WITH ABNoR:\r;tL Ova
Another very important question that remains to be elucidated is the role of the granulosa lutein cell. The fact that alkaline phosphatase concentration in the granulosa lutein cell increases during that period of pregnancy when greatest demands are being made upon the gland suggests that the granulosa lutein Cell plays more than a passive role. It is possible that the short 3-hour period of incubation in the glycerophosphate medium employed in this study was insufficient to reveal low concentrations of alkaline phosphatase in the granulosa lutein cells of the nonpregnant corpus luteum. Here, again, it is a tempting suggestion that there may be a direct relation between alkaline phosphatase in the granulosa lutein cell and the demonstrable phospholipid-ketosteroid matrix of the K cells.
w1TH ADEQUATE TROPHOBLAST


An 8-Day Ozmm, Carnegie No. 8370, S46-676


The ovum is the youngest implanted embryo of
As regards the corpora lutea associated with abnormal ova, it is interesting to note that the corpora lutea associated with preimplantation ova are normal in all respects and comparable with those of the normal menstrual cycle or normal pregnancy of identical chronological age.
this series of abnormal ova. The chorionic cavity is
absent. Although ectoderm is present in the germ
disk, there is no endoderm. The trophoblast is ade68 CORPUS LUTEUM OF HUMAN PREGNANCY


quate but poorly organized, that is, laminated instead
of being concentrically arranged with the syncytiotrophoblast surrounding the cytotrophoblast.


The corpus luteum appears to be normal in all
Careful examination of the corpora lutea associated with implanted but abnormal ova discloses that there is an almost direct relation between the amount of trophoblast present and the integrity of the corpus luteum. In those ova which are almost completely devoid of trophoblast (Carnegie nos. {{CE7771}}, {{CE7800}}), the corpus luteum is poorly vascularizcd and does not manifest the heightened functional activity expected at this stage of development. The most conspicuous feature of these corpora lutea, however, is the total and uniform colloid degeneration of all K cells.
respects. The theca interna is prominent. Both layers
are well vascularized. The granulosa lutein cells
show a moderate degree of peripheral vacuolization.
K cells are numerous and appear to be normally
active. No evidence of colloid degeneration is seen.


A 10- to I1-Day Ouzrm, Carrzegie N0. 7770, S40-749


This ovum is not markedly defective. The trophoblast is moderately hypoplastic and the germ disk
On the other hand, Carnegie no. {{CE8329}} is a markedly abnormal ovum consisting only of syncytiotrophoblast. K cells are numerous in this corpus luteum, and no evidence of colloid degeneration is to be noted. The specimens associated with abnormal ova showing only moderate hypoplasia of the trophoblast appear to be good but not perfect corpora lutea; that is, peripheral vacuolization and vascularity are moderately deficient.
is maloriented.


The theca interna appears to be normal in all
respects and consistent with that of a normal 10- or
11-day pregnancy. The granulosa lutein layer is
moderately well vascularized. K cells are relatively
few, but appear to be normal in all respects. There
is, however, great variation in size and stainability
of the nuclei of the granulosa lutein cells.


An Ir-Day Ozmm, Carnegie N 0. 8299, 545-1220
These observations suggest that a normal trophoblast is essential to the maintenance of the corpus luteum, the functional integrity of which, in turn, is responsible for maintaining normal decidua during the early weeks of pregnancy.


The trophoblast appears to be normal although
==Summary and Conclusions==
somewhat poorly organized. The germ disk is
markedly maloriented.


The corpus luteum is very large and consists of a
1. The morphological and histochemical changes have been studied in a total of 89 human corpora lutea. Forty-eight of these represent corpora lutea of the normal menstrual cycle, every day from ovulation to menstruation being represented. Twenty-eight corpora lutea of normal pregnancies ranging from the 2-cell egg to 4%, months, and 13 corpora lutea associated with abnormal ova were studied. An attempt has been made to correlate the changes in histochemical reactivity in the various components of the corpus luteum with the anatomical evidences of function.
large cystic cavity enclosed by a thin rim of lutein
tissue. Both granulosa and theca lutein layers are
moderately well vascularized and appear to be functionally active. Most of the K cells show little evidence of colloid degeneration. This appears to be a


very active corpus luteum (fig. 30, pl. 6).
2. As the mature Graafian follicle nears ovulation, a number of distinctive cells, not previously described, become conspicuous. At or shortly after the time of ovulation, these cells appear in large numbers in the granulosa lutein layer, attaining that position by their own motility or being carried in as the theca interna and its accompanying blood vessels invaginate into the collapsed membrana granulosa.


Carnegie N0. 7850, 540-2699
3. Evidence is presented that these cells constitute a distinct cell type and represent the site of intense localization of ketonic lipid, if not the site of production or utilization of ketonic lipids or steroids.


This ovum is associated with an endometrium that
4. Until the time of implantation of the ovum in the endometrium, no difference can be noted between chronologically similar corpora lutea of the normal menstrual cycle and those associated with normal or abnormal ova.
evidences early decidual reaction. Except for moderate hypoplasia of the trophoblast, the ovum appears
to be fairly good.


The theca interna is prominent, many cell layers
5. After implantation, at or about 6 or 7 days after ovulation, the corpus luteum does not undergo regression but is stimulated to increasingly higher levels of functional activity until 6 weeks of menstrual age or later, after which the function of the corpus luteum is gradually taken over by the placenta.
thick, and appears to be functionally active. Peripheral vacuolization of the granulosa lutein cells is not
so marked as in chronologically similar corpora lutea
associated with normal pregnancy. Mitoses are to be
noted in both layers of the corpus luteum. K cells are
quite numerous, but are irregularly distributed. No


evidence of colloid degeneration is to be noted anywhere in the section.
6. It is quite apparent that when the implanted ovum is deficient in trophoblastic development, the corpus luteum undergoes early regression. The most striking feature of this failure of the corpus luteum is the uniform colloid degeneration of the K cells. The worse the ovum as regards the development of the trophoblast, the more complete is the colloid degeneration of the K cells in the associated corpus luteum.


CORPORA Luri-:.-i ASSOCIATED wrrn ABNORMAL,
==Literature Cited==
SHaLLowi.v I.\tPL.~xN*rEn Ova


Ar: 11- to I2-Day Ourmz, Carnegie N0. 8000, S42-217
Asor.1.1., S. A. 1928. The growth and function of the corpus luteum. Physiol. Rev., vol. 8, pp. 313-341.


The ovum is very shallowly implanted, although all
As:-1m«;1., R., and A. M. St-:1.1o.\1.-m. 1949. A new reagent for the histochemical demonstration of active carbonyl groups. A new method for staining ketonic steroids. Endocrinology, vol. 44, pp. 565-583.
the elements appear to be normal.


The theca lutein layer appears to be normal and
Asrwoon, E. B. 1939. Changes in the weight and water content of the uterus of the normal adult rat. Amer. ]0ur. Physiol., vol. 126, pp. 162-170.
contains several immature K cells. The granulosa
lutein layer is moderately well vascularized, and although some lutein cells show early signs of atrophy,
the majority appear to be functionally active. Distinctive K cells are rare, but little colloid degeneration
is seen.


A I21/3-Day Oz/um, Carnegie No. 290, 844-2785
BAKER, I. R. 1946. The histochemical recognition of lipine. Quart. Iour. Micr. Sci., vol. 87, pp. 441-470.


Although this ovum is superficially implanted, it
BENNETT, H. S. 1940. The life history and secretion of the cells of the adrenal cortex of the cat. Amer. Iour. Anat., vol. 67, pp. 151-22 .
shows evidence of early villus formation. The germ
disk has undergone a curious buckling and is still
attached to the trophoblast. The latter is irregularly
developed, poorest at the implantation pole and, paradoxically, good elsewhere.


The corpus luteum is in no way markedly different
BREWER, J. I. 1942. Studies of the human corpus luteum. Evidence for the early onset of regression of the corpus luteum of menstruation. Amer. Iour. Obstet. and Gynecol., vol. 44, pp. 1048-1059.
from that associated with Carnegie no. 8000.


A Conpus LU'n~:U.\t Assoenvrsn w1TH .»'tN OVUM CoNSISTING or Si'Nev'r1oTRopi-ioBL.-xsT ONLY
Cnvmamus, I. I. 1926. Uber die Struktur in den Corpus luteum-Zellen des Menschen und ihre Veriinderungen wfihrend des Menstruationszyklus und bei Graviclitiit. Arbeit. aus cl. pathol. Inst. u. (l. Univ. Helsingfors, vol. 4, pp. 319-414. (Cited by Astlell, 1928.)


A 12-Day Oz/um, Cczrnegic No. 8329, S45-1809
Conxra, G. \V. 1915. The corpus luteum of pregnancy, as it is in swine. Carnegie Inst. VVash. Pub. 222, Contrib. to Embryol., vol. 2, pp. 69-94.


This embryo is markedly abnormal in that there
1948. Alkaline phosphatase in the ovarian follicle and in the corpus luteum. Carnegie Inst. Wash. Pub. 575, Contrib. to Embr}/'01., vol. 32, pp. 1-8.
is no cytotrophoblast, no segmentation cavity, and no
germ disk, but as a mass of syncytiotrophohlast it is
fairly large.


Both lutein layers appear to be well vascularized
DALTON, A. L, E. R. M1Tct1ELL, B. F. Joints, and V. B. Perms. 1944. Changes in adrenal glands of rats following exposure to lowered oxygen tension. Iour. Natl. Cancer Inst., vol. 4, pp. 527-536.
and functionally active. K cells are numerous in
certain parts of the section, but almost completely
lacking in other areas. There is, however, no evidence


of colloid degeneration of any of the K cells (fig. 31,
D1—:1.es1'ar_, M. 1910. Recherches sur le follicule de Graaf et le corps jaune de la vache. Iour. de l‘anat. et de la physiol. (Paris), vol. 46, pp. 286-309.
pl. 6).


Coupons. Lure.-x Assoel.-cren wrrt-I OVA SHOWING
D1-:1~.11>s1-:v, E. ‘W. 1948. The chemical cytology of endocrine glands. Recent Prog. Hormone Res., vol. 3, pp. 127-157.
HvPoi>1..»xs'r1e TROI’liUBL.-\S'l‘


Carncgz'e No. 7771, S4079!
and D. L. B.1ss1»:'r1'. 1943. Observations on the fluorescence, birefringence and histochemistry of the rat ovary during the reproductive cycle. Endocrinology, vol. 33, pp. 334-401


This markedly abnormal ovum is associated with a
GILLMAN, 1., and H. B. STEIN. 1941. The human corpus luteum of pregnancy. Surg., Gynecol., and Obstet., vol. 73: PP- I39‘149 Gomoar, G. 1941. The distribution of phosphatase in normal organs and tissues. Iour. Cell. and Comp. Physiol., vol. 17, pp- 7I-83
27-day late-secretory endometrium. The ovum is a
CORPUS LUTEUM OF HUMAN PREGNANCY 69


polypoid structure; both elements of the trophoblast
GRAFFLIN, A. L. 1939. The thyroid and parathyroid glands of the Barasingha deer, with particular reference to autofluorescence, fat and pigment. Iour. Morphol., vol. 65, pp- 297-32!
are present, but poor in quality and organization.
The germ disk is totally lacking.


The corpus luteum is poorly vascularized. Both
theca lutein and granulosa lutein cells appear to be
moderately active, but not so much so as one would
find in a normal pregnancy of this stage of development. A very striking feature is the complete colloid
degeneration of all the K cells. Large amounts of
colloid are present in all parts of the section, and no
functional K cells are seen (fig. 32, pl. 6).


Carnegie N0. 7800, 540-1327
HAMMOND, I., and F. H. A. MansH.»\LL. 1925. Reproduction in the rabbit. 210 pp. Edinburgh.


This ovum is associated with an endometrium that
HERTIG, A.T. 1945. Diagnosing the endometrial biopsy. Proc. Soc. Diagnosis in Sterility, pp. 93-128.
shows early decidual reaction. The chorion is irregularly deficient, with marked hypoplasia of the trophoblast. To judge from the development of the embryo,
the chorion should have early villi.


The theca lutein layer appears to be normal and
and I. RocK.. 1941. Two human ova of the pre-villous stage, having an ovulation age of about eleven and twelve days respectively. Carnegie Inst. Wash. Pub. 525, Contrib. to Embryol., vol. 29, pp. 127-156.
functioning. The granulosa lutein layer is well vascularized. There is marked variation in size, regularity,
and stainability of the nuclei of the granulosa lutein
cells. The majority of the K cells have undergone
more or less complete colloid degeneration, although
this feature is not so conspicuous as in Carnegie
no. 7771.


About 3 Mont/is Pregnancy, Blig/ztcd Ouum, S48-824
1944. On the development of the early human ovum, with special reference to the trophoblast of the previllous stage: a description of 7 normal and 5 pathologic ova. Amer. Iour. Obstet. and Gynecol., vol. 47, 913- 149-184-


This specimen was obtained with the corresponding
1945. Two human ova of the pre-villous stage, having a developmental age of about seven and nine days respectively. Carnegie Inst. Wash. Pub. 557, Contrib. to Embryol., vol. 31, pp. 65-84.
left tube, left ovary, and uterus. Pregnancy was
interrupted because of impending cardiac failure. The


placenta was found to be immature and associated
1949a. Two human ova of the pre-villous stage, having a developmental age of about eight and nine days respectively. Carnegie Inst. W’ash. Pub. 583, Contrib. to Embryol., vol. 33, pp. 169-186.
with a blighted ovum of about 3 months menstrual
age. The cystic corpus luteum measured 1.5‘ cm. in
diameter and contained straw-colored fluid. The
lutein border was grayish yellow, and about 2 mm.
thick.


I-Iaematoxylin and eosin sections (fig. 33, pl. 6)
1949b. A series of potentially abortive ova recovered from fertile women prior to the first missed menstrual period. Read before Amer. Gynecol. Soc. at Hot Springs, Va., May 18, 1949. Amer. Jour. Obstet. and Gynecol., vol. 58, pp. 968-993.
show a corpus luteum that has lost all its distinctive
morphologic features. The theca lutein layer is
markedly reduced in bulk. Most of the nuclei are
densely pycnotic. The granulosa lutein layer has lost
all evidence of functional activity. Not a single active
lutein cell can be found. In many, the nuclei are
dense and pycnotic, and in others the nuclei have
completely lost their capacity for staining. A large
number of colloid-containing vacuoles are present.
Cell boundaries are totally obliterated. Connectivetissue invasion of both layers is marked. The capillaries of the granulosa lutein layer are collapsed.


Only an occasional cell of the theca lutein layer
1950. Abortive ova and associated endometrium. Proc. Conference on Menstruation and Its Disorders, 1947. Natl. Committee on Maternal Health. Pub. Charles C. Thomas.
contains alkaline phosphatase. Nearly every cell in
the granulosa lutein layer contains some of the enzyme, but not in significant amounts.


Inasmuch as the ovum was found to be blighted,
Hsussa, C. H., I. Rocx, and A. T. HERTIG. 1945. Two human embryos showing early stages of the definitive yolk sac. Carnegie Inst. Wash. Pub. 557, Contrib. to Embryol., vol. 31, pp. 85-99.
and the placenta was immature, it is highly probable
that this specimen does not represent a normal 3
months pregnancy. It is included to demonstrate that
the degenerative changes in the Corpus luteum may
parallel trophoblastic degeneration.


Unfortunately, no formalin-fixed material was available at the time this specimen came to our attention,
LISON, L. 1936. Histochimie animale; méthodes et problemes. 320 pp. Paris.
and lipid studies could not be made.


NOTES ON THE K CELLS
MCKAY, D. C., and D. ROBINSON. 1947. Observations on the fluorescence, birefringence and histochemistry of the
human ovary during the menstrual cycle. Endocrinology. "01- 41. pt» 378-394.


Although this study was undertaken to outline the
McNL'-'r'r, G. W. 1924. The corpus luteum of the ox ovary in relation to the estrous cycle. Preliminary report. Iour. Amer. Vet. Med. Assoc., vol. 65, pp. 556-597.
morphological changes that take place in the corpus
luteum during early pregnancy, it became apparent
quite early in the course of the investigation that the
K cells described in all specimens discussed bear
more than a casual relation to the cyclical activity of
the gland, in the nonpregnant cycle as well as in early
pregnancy.


These cells are conspicuous in routine sections because of their homogeneous, highly cosinophilic cytoplasm. It is also to be noted, however, that in sections
treated with basic dyes, their cytoplasm is more
markedly basophilic than that of neighboring lutein
cells. This suggests strong acid properties of the


protoplasm, a reaction characteristic of phospholipid.
I\-Ir.v1;R, R. 1911. Ueber Corpus luteum-Bildung beim
As previously stated, these cells are very striking in
Menschen. Arch. f. Gynaekol., vol. 93, pp. 354-404.
sections treated with Sudan black. They are uniformly sudanophilic, in contrast with the granular
1932. Uber das Stadium proliferationis s. hyper-
sudanophilia of the theca lutein and granulosa lutein
aemicum sowie iiber den Begrifi und die Abgrenzung
cells. The sudanophilic material is soluble with difficulty in cold alcohol or alcohol-acetone mixtures.
des Bliitestadiums des Corpus luteum beim Menschen.
Traces of sudanophilia can be detected in these cells
after 24 hours’ treatment of the section in lipid solvents before staining. By contrast, the granular deposits of lipid in theca lutein and granulosa lutein
cells are completely dissolved by similarly pretreating
the sections for 1/3 to 1 hour before staining with
Sudan black. The low solubility of the sudanophilic
70 CORPUS LUTEUM OF HUMAN PREGNANCY


substance in these K cells is characteristic of phospholipid.
Arch. f. Gynaekol., vol. 149, pp. 315-346.


Because of these indications of high phospholipid
Novax, E. 1932. Cyclical changes in the ovary. In Obstetrics
content in the K cells, the technique for the demonstration of phospholipid in tissues (Baker, 1946) was
and gynecology, A. H. Curtis (ed.), vol. 1, sec. 2, chap. 8,
employed in two selected corpora lutea. Figure 34,
PP- 337-343- 19.-11. Gynecology and female endocrinology. 605 pp.
plate 7, is a photomicrograph of a K cell, at a magnification of 750, in a :20-day corpus luteum of the normal
Boston.
menstrual cycle, showing a high concentration of
phospholipid. The phospholipid is uniformly distributed in these cells, as contrasted with the lutein
cells of both layers, where phospholipid appears as
peripherally distributed granules. It was observed
incidentally that in the developing follicle phospholipicl is restricted to the theca interna layer. Pretreatment of parallel sections with pyridine completely
removed all traces of phospholipid from the gland.


All the specimens discussed in this paper were subjected to the tests for presumptive ketosteroids employed so effectively by Dempsey and Bassett (1943)
P11.vr'r, I. P. 1935. The human corpus luteum. Arch. Pathol.,
and by McKay and Robinson (1947). Our findings
vol. 19, pp. 380-425, 545-562.
in the developing follicle and corpus luteum of the
nonpregnant cycle agree substantially with those of
McKay and Robinson. It was noted, however, in the
course of these studies that the K cells were reactive
with the Schiff, phenylhydrazine, and LiebermanBurchardt reactions. This reactivity is manifested as
a uniform, homogeneous color reaction, in contrast
with the granular reactivity of theca lutein cells described by McKay and Robinson. As with Sudan
black, this reactive material was more dillicult to dissolve in lipid solvents than that of the theca lutein
cells. Observations of autofluorescence under ultraviolet light were equivocal. It is noteworthy, however, that birefringent crystals cannot be observed in
these K cells under the polarizing microscope. In
only one specimen, the 16-day normal pregnancy
(Carnegie no. 8602), was a suggestion of crystalline
birefringence seen. Up to this point, all the reactions
of the K cells pointed to ketosteroid substances, but
failure to demonstrate birefringent crystals in them
seemed to rule out that possibility. It has subsequently
been pointed out to us by Seligman (personal communication) that all the evidence presented above


points to the fact that the high phospholipid content
REYNOLDS, S. R. M., and M. H. FRIED.\-IAN. 1930. Studies on
of these cells may prevent the crystallization of ketosteroid necessary for development of birefringence in
the uterus. III. The activity of the uterine fistula in
plane-polarized light.
unanesthetized rabbits following coitus and during
pseudopregnancy. Amer. Iour. Physiol., vol. 94, pp. 696-
707.


Very recently Seligman and Ashbel (I949) developed a technique for the demonstration of ketonic
Roex, 1., and A. T. HERTIG. 1944. Information regarding
lipids. Figures 35 and 36, plate 7, are photomicrographs of sections treated by this technique. It will
the time of human ovulation derived from a study of
be noted that reactive material is concentrated in these
3 unfertilized and II fertilized ova. Amer. Iour. Obstet.
cells. Seligman and Ashbel have observed that the
and Gynecol., vol. 47, pp. 343-356.
corpora lutea of animals do not react to the tests for
ketosteroid unless they are fixed in formalin, which
unmasks the active carbonyl group. To rule out the
possibility that the reactive material in these cells
might be an aldehydic group, produced by hydrolysis
of plasmogens, parallel sections were treated by a new
technique for the demonstration of free aldehyde
groups (Seligman and Ashbel, unpublished data). No
free aldehydic groups could be demonstrated. Thus,
all the evidence seems to indicate that these K cells
represent the locus of high concentration of ketosteroid. Their reactivity as measured by these special
histochemical techniques parallels closely the changes
observed in sudanophilia during various stages of
development of the corpus luteum.


As regards the question of origin and function of
1948. The human conceptus during the first
the K cells, it must be borne in mind that they could
two weeks of gestation. Amer. ]our. Obstet. and Gynecol.,
represent undifferentiated elements of the reticuloendothelial system, for example wandering macrophages, angioblasts, or young fibroblasts. It is possible that these cells arc histiocytic, and that the intense localization of ketonic lipid noted in them
vol. 55, pp. 6-14.
represents merely the phagocytosis of excess ketonic
lipid. It is planned to perfuse a human ovary containing an active corpus luteum with a vital dye to
determine whether these cells have the capacity to
phagocytose foreign matter, utilizing the technique of
‘Nerthessen (1949). In the absence of further and
more definitive studies, we believe that all the evidence presented regarding the peculiar characteristics
of these cells points to their being a distinct cell line,
intimately related in some manner to the function of
the corpus luteum in the production or utilization of


ketonic lipid, that is, ketosteroid compounds.
Ro.\n=.1s, B. 19:18. Taschenbuch der mikroskopischen Technik.
CORPUS LUTEUM OF HUMAN PREGNANCY 71
12th ed. Munich and Berlin.


DISCUSSION
SARASON, E. L. 1943. Morphologic changes in the rat's adrenal
cortex under various experimental conditions. Arch.
Pathol., vol. 35, pp. 373-390.


Our observations of the corpus luteum of the nonpregnant cycle parallel substantially the classic description of Meyer (1911). Observations of vascular
SELIGM.-‘LN, A. M., and R. A.sHn1~:1.. 1949. A new reagent for
changes, coupled with information that can be deduced from histochemical procedures, lead us to agree
the histochemical demonstration of active carbonyl groups
with Brewer (1942) that the corpus luteum reaches
of lipoitl and nonlipoid materials. Bull. New England
its maximum activity on or about the 9th day after
Med. Center, vol. 11, pp. 85-86.
ovulation. ‘Ne have assumed the following criteria
as evidence of functional activity of the corpus luteum:
widely dilated capillaries in both theca and granulosa
lutein layers, fine peripherally distributed sudanophilic
substances in the lutein cells, demonstrable alkaline
phosphatase in the cytoplasm, and ketonic lipid demonstrable by the techniques described above.


It has been generally held that the ovary is responsible for at least two hormones, both of which have
Samara, H. 1937. Studies on adaptation. Endocrinology, vol.
experimentally proved specific actions in the menstrual
21, pp. 169-188.
cycle. Dempsey and Bassett (1943) working with
rats, and McKay and Robinson (1947) in a study of
human material, found that reactive material, presumptively ketosteroid, was localized exclusively in
the theca interna of the developing follicle and appeared in insignificant amounts in the granulosa lutein
cells during the active stages of the corpus luteum.
Inasmuch as they could not demonstrate ketosteroid
in significant amounts in the granulosa lutein layer,
they suggested that the theca interna is probably
responsible for the production of both progesterone
and estrogenic substances. Our observations suggest
that a ketosteroid substance is intensely localized in
a specific line of cells having their origin in the theca
interim and making their way into the granulosa
lutein layer. It is possible that the hormone production of these cells in the unruptured follicle is responsible for the suggestive progestational changes
that take place in the uterus of experimental animals
at or shortly before ovulation (Reynolds and Friedman, I93o; Astwood, 1939).


VVe have no evidence that these cells are specifically
SHAW, W. 1925. The fate of the Graafian follicle in the
concerned with the production of progesterone rather
human ovary. Iour. Obstet. and Gynecol. Brit. Empire,
than estrogen. Nevertheless, the temptation to infer
vol. 32:, pp. 679-689.
that such a relationship exists is strong. There is no
valid reason why a given cell cannot produce more
than one hormone, as those of the pituitary gland
apparently do. As has been observed, these K cells
show the greatest evidence of functional activity dur
ing the stages when maintenance of the progestational
state of the uterine endometrium is essential. In the
normal menstrual cycle, if a fertilized ovum has not
become implanted in the endometrium by 6 or 7 days
after ovulation, that cycle ends in menstruation (Rock
and Hertig, 1948). In such an event, the need for
maintenance of the progestational type of endometrium no longer exists and the corpus luteum soon
begins to regress. On the other hand, if pregnancy is
superimposed, the earliest evidence of sustained and
accelerated functional activity is seen in the recrudescence of activity in the K cells. It has been generally agreed that pregnancy will continue uninterrupted even though the corpus luteum is removed,
when the placenta is producing suflicient progesterone
to maintain the decidua. The exact time at which
this transfer of function from corpus luteum to placenta takes place is not so well agreed upon. Dr. G.
van S. Smith (unpublished data) states that the corpus
luteum is essential through the 7th to the 8th, and
possibly the 9th, week of pregnancy. No doubt there
is considerable overlapping of hormone production
by corpus luteum and placenta. Our observations
suggest that the corpus luteum ceases to produce
hormone completely by the 4th month of pregnancy,
and there is evidence that functional activity decreases
rather sharply between the 7th and 12th weeks of
gestation.


Our studies indicate that the theca interna contributes substantially to the granulosa lutein layer in
So11or1<.x, H. 1938. The chemistry of the sterids. 634 pp.
the human corpus luteum. However, the theca lutein
Baltimore.
and granulosa lutein cells described by all workers
remain separate and distinct entities. The elements
contributed to the granulosa lutein layer by the theca
lutein layer are the K cells to which so much space
has been given in this discussion.


These K cells in the granulosa lutein layer of the
W1-:1mnsss1=.N, N. T. 1949. A technique of organ culture for
human corpus luteum have been observed by many
protracted metabolism studies. Endocrinology, vol. 44, pp. 109-126.
earlier workers, as cited by Gillman and Stein (1941).
These latter authors have interpreted them as representing different phases of functional activity of the
granulosa lutein cells. It is highly probable that earlier
work failed to elicit the true nature of these cells for
three reasons: First, they had not been observed in
the theca interna of the developing follicle; secondly,
there had been no observation of migration of cellular
72 CORPUS LUTEUM OF HUMAN PREGNANCY


elements from the theca interna into the granulosa
==Plates==
lutein layer; and, thirdly, lipid stains such as Sudan
===Plate 1===
black, which is capable of staining the phospholipid
so characteristic of these cells, were not available.


The specific colloid degeneration which is peculiar
gr-., granulosa; t/2. z'nt., theca interna; K, K cells
to these K cells is interpreted as further evidence that
they represent a distinct cell type. The fact that this
colloid has the same histochemical properties as the
K cells suggests that the colloid represents stored
secretory products. Although colloid deposits are visible in old corpora lutea of the nonpregnant cycle, they
are much more conspicuous in the degenerating corpus
luteum of pregnancy, a fact which suggests both an
increase in the number of cells producing the precursor of this product and an increased concentration of precolloid ketosteroid-phospholipid complexes
within the cell.


As observed by Corner (19.18), in the human ovary
Fig. 1. Section of the wall of a mature human Graafian
alkaline phosphatase is restricted to the theca interna
follicle, X300, Haematoxylin and eosin. Prominent,
of the developing follicle and disappears from the
small dark nuclei of K cells stand out conspicuously in
theca lutein cells by the 4th or 5th post-ovulatory day.
the theca interna.
In contrast with Corner’s findings, we observed alkaline phosphatase in a few granulosa lutein cells, but
this becomes evident only as the gland approaches
the peak of functional activity, that is, at or about
8 days after ovulation. As stated earlier, we have
observed alkaline phosphatase in the K cells as early
as 6 days after rupture of the follicle. V-Vhen pregnancy occurs, alkaline phosphatase appears in increasingly higher concentrations in the K cells, and in
gradually increasing amounts in the true granulosa
lutein cells as the progress of pregnancy makes additional demands on the corpus luteum. There is some
recrudescence of alkaline phosphatase in the theca
lutein cells, most marked at 6 or 7 weeks menstrual
age. The activity of the theca lutein layer during
pregnancy, as measured by the concentration of this
particular enzyme, is insignificant when compared
with that of the granulosa lutein layer and the K cells.


The theca lutein layer undergoes marked hypertrophy during early pregnancy, attaining its maximum development about the 26th day of pregnancy,
Fig. 2. Corpus luteum of 16th day of cycle, estimated
dating from ovulation. Early in pregnancy, the coarse
age 2 days, ><30o, haematoxylin and eosin. The thcca
lipid deposits characteristic of the menstrual corpus
interna is considerably thinned. The granulosa is poorly
luteum are completely replaced by fine, granular lipid
organized. Prominent K cells can be seen penetrating into
deposits which are indicative of functional activity.
the granulosa.
After the 26th day of pregnancy the theca lutein layer
becomes less and less prominent, until at 4 months no


trace can be found except a few small pycnotic
Fig. 3. Same specimen as figure 2, X 300, Sudan black.
nuclei. That the theca interna may continue to supply
Sudanophilic substances present in much higher concen-
K cells is suggested by the fact that recrudescence of
K cells in the theca lutein layer is observed in both
the corpora lutea associated with 26-day pregnancies.


Another very important question that remains to be
tration in theca interna than in granulosa.
elucidated is the role of the granulosa lutein cell. The
fact that alkaline phosphatase concentration in the
granulosa lutein cell increases during that period of
pregnancy when greatest demands are being made
upon the gland suggests that the granulosa lutein Cell
plays more than a passive role. It is possible that the
short 3-hour period of incubation in the glycerophosphate medium employed in this study was insufficient to reveal low concentrations of alkaline
phosphatase in the granulosa lutein cells of the nonpregnant corpus luteum. Here, again, it is a tempting
suggestion that there may be a direct relation between
alkaline phosphatase in the granulosa lutein cell and
the demonstrable phospholipid-ketosteroid matrix of
the K cells.


As regards the corpora lutea associated with abnormal ova, it is interesting to note that the corpora
Fig. 4. Same specimen as figure 2, X400, alkaline
lutea associated with preimplantation ova are normal
phosphatase. Alkaline phosphatase is restricted to tbeea
in all respects and comparable with those of the normal menstrual cycle or normal pregnancy of identical
intcrna and enclotbelium of blood vessels.
chronological age.


Careful examination of the corpora lutea associated
Fig. 5. Corpus luteum of zoth day of cycle, estimated
with implanted but abnormal ova discloses that there
age 6 days, X300, haematoxylin and eosin. The theca
is an almost direct relation between the amount of
interna is not prominent. The granulosa is compact and
trophoblast present and the integrity of the corpus
well vascularized. K cells are conspicuous because of
luteum. In those ova which are almost completely
their small, hyperchromatic nuclei and dense, homo-
devoid of trophoblast (Carnegie nos. 7771, 7800), the
geneous cytoplasm.
corpus luteum is poorly vascularizcd and does not
manifest the heightened functional activity expected
at this stage of development. The most conspicuous
feature of these corpora lutea, however, is the total
and uniform colloid degeneration of all K cells.


On the other hand, Carnegie no. 8329 is a markedly
Fig. 6. Same specimen as figure 5, X 3oo, Sudan black.
abnormal ovum consisting only of syncytiotrophoblast. K cells are numerous in this corpus luteum, and
The section does not include theca interna. Note periph-
no evidence of colloid degeneration is to be noted.
eral distribution of sudanophilic substances in granulosa
The specimens associated with abnormal ova showing
cells and uniform sudanophilia of attenuated K cells.
only moderate hypoplasia of the trophoblast appear
===Plate 2===
to be good but not perfect corpora lutea; that is,
peripheral vacuolization and vascularity are moderately deficient.


These observations suggest that a normal trophoCORPUS LUTEUM OF HUMAN PREGNANCY 73
Fig. 7. Corpus luteum of 23d day of cycle. estimated
age 9 days, X300, haematoxylin and eosin. The K. cells
are numerous and markedly hypertrophied. Peripheral
vacuolization of granulosa lutein cells is marked.


blast is essential to the maintenance of the corpus
Fig. 8. Same specimen as figure 7, X goo. Sudan hlack.
luteum, the functional integrity of which, in turn,
A small segment of theca interna is present in upper
part of figure. Note coarse droplets of lipid in cells of
theca interna as contrasted with the line droplets in the
granulosa lutein cells. K cells are numerous.


is responsible for maintaining normal decidua during
Fig. 9. Same specimen as figure 7. X300. alkaline
the early weeks of pregnancy.
phosphatase. Only an occasional cell of the theca interna
contains the enzyme. ()ne K cell, with extensive cyto-
plasmic processes. contains the eiizyine in high concen-
tration. An occasional granulosa cell contains alltaline
phosphatase in moderate amounts.


SUMMARY AND CONCLUSIONS
Fig. 10. Corpus luteum of early menstruation. esti-
mated age 15-16 days, ><3oo_. haematoxylin and eosin.
The theca interna is almost completely atrophic. K cells
in various stages of degeneration are to he noted in granu-
losa layer. I’eripheral vacuoli:/.ation of granulosa cells is
not marked.


1. The morphological and histochemical changes
Fig. 11. Same specimen as ligure Io. ><3oo, Sudan
have been studied in a total of 89 human corpora
black. Nearly all of the theea interna cells contain only
lutea. Forty-eight of these represent corpora lutea of
coarse lipid droplets. Iiarly fatty degeneration of some
the normal menstrual cycle, every day from ovulation to menstruation being represented. Twenty—eight
of the granulosa cells is evident. The K cells are some-
corpora lutea of normal pregnancies ranging from the
what contracted and show some granular sud:mophilic
2-cell egg to 4%, months, and 13 corpora lutea associated with abnormal ova were studied. An attempt
deposits.
has been made to correlate the changes in histochemical reactivity in the various components of the corpus luteum with the anatomical evidences of function.


2. As the mature Graafian follicle nears ovulation,
Fig. 12. Same specimen as ligure Io, ><_ioo, alkaline
a number of distinctive cells, not previously described,
phosphatase. ()nly a few scattered cells in the granulosa
become conspicuous. At or shortly after the time of
lutein layer contain demonstrable amounts of the en-
ovulation, these cells appear in large numbers in the
zyme.
granulosa lutein layer, attaining that position by their
own motility or being carried in as the theca interna
and its accompanying blood vessels invaginate into
the collapsed membrana granulosa.


3. Evidence is presented that these cells constitute
===Plate 3===
a distinct cell type and represent the site of intense


localization of ketonic lipid, if not the site of production or utilization of ketonic lipids or steroids.
gl'., granulosa; t/1. int., theca interna: K, K cells


4. Until the time of implantation of the ovum in
Fig. 13. Corpus luteum of 16- to 17-day normal preg-
the endometrium, no difference can be noted between
nancy. Carnegie no. 8602, X 300. l1:lL'l11:1I()X}'lll‘l and eosin.
chronologically similar corpora lutea of the normal
Note indistinct cell boundaries of granulosa lutein cells
menstrual cycle and those associated with normal or
and numerous K cells.
abnormal ova.


5. After implantation, at or about 6 or 7 days after
liim. it). Same specimen as ligure 18, X300. Sudan
ovulation, the corpus luteum does not undergo regression but is stimulated to increasingly higher levels
lilaek. Large amounts of finely distributed lipid are
of functional activity until 6 weeks of menstrual age
present in lioth granulosa and theca lutein cells. K. cells
or later, after which the function of the corpus luteum
are numerous. but appear frayed and more irregularly
is gradually taken over by the placenta.
stellate than in ligure 16. plate 3,.


6. It is quite apparent that when the implanted
Fig. 20. Same specimen as l‘igure 18. ‘.><.ioo. alkaline
ovum is deficient in trophoblastic development, the
phosphatase. The theta interna is uniformly devoid of
corpus luteum undergoes early regression. The most
the enzyme, whereas nearly every cell in the granulosa
striking feature of this failure of the corpus luteum
lutein layer contains alkaline phosphatase.
is the uniform colloid degeneration of the K cells.
The worse the ovum as regards the development of
the trophoblast, the more complete is the colloid
degeneration of the K cells in the associated corpus
luteum.


LITERATURE CITED
Fig. 21. Corpus luteum of 2(i-clay normal pre_J,it;nic)~‘.
><.3uo. ltaeinato.\yli11 and eosin. The theca interna is
markedly hypertrophied as compared with figure ill.
K cells are numerous and apparently very active in the
granulosa la}-‘er.


Asor.1.1., S. A. 1928. The growth and function of the corpus
Fig. 22. Same specimen as ligtire 2|, )-(goo, Sudan
luteum. Physiol. Rev., vol. 8, pp. 313-341.
hlack.


As:-1m«;1., R., and A. M. St-:1.1o.\1.-m. 1949. A new reagent for
\'lt'lt.'(.l lipitl. Of special interest is the reappearance of a
the histochemical demonstration of active carbonyl groups.
A new method for staining ketonic steroids. Endocrinology, vol. 44, pp. 565-583.


Asrwoon, E. B. 1939. Changes in the weight and water content of the uterus of the normal adult rat. Amer. ]0ur.
Roth layers contain large amounts of rinel_\} di-
Physiol., vol. 126, pp. 162-170.


BAKER, I. R. 1946. The histochemical recognition of lipine.
numlier of small. uniformly sudanophilie Ii cells in the
Quart. Iour. Micr. Sci., vol. 87, pp. 441-470.
theea interna.


BENNETT, H. S. 1940. The life history and secretion of the
Fig. 23. Same specimen as ligtire 2|. ><_;oo. alkaline
cells of the adrenal cortex of the cat. Amer. Iour. Anat.,
phosphatase. The C11’/.}'l1‘IL’ is not demonstrable in the
vol. 67, pp. 151-22 .
theca interna. hut is tlilltisely present in the entire granu-
losa layer.


BREWER, J. I. 1942. Studies of the human corpus luteum.
===Plate 7===
Evidence for the early onset of regression of the corpus
luteum of menstruation. Amer. Iour. Obstet. and Gynecol., vol. 44, pp. 1048-1059.


Cnvmamus, I. I. 1926. Uber die Struktur in den Corpus
gr., granulosa; :11. :'m., theca interna; K, K cells
luteum-Zellen des Menschen und ihre Veriinderungen
wfihrend des Menstruationszyklus und bei Graviclitiit.
Arbeit. aus cl. pathol. Inst. u. (l. Univ. Helsingfors, vol. 4,
pp. 319-414. (Cited by Astlell, 1928.)


Conxra, G. \V. 1915. The corpus luteum of pregnancy, as
Fig. 24. Corpus luteum of zoth day of cycle, estimated
it is in swine. Carnegie Inst. VVash. Pub. 222, Contrib.
age 6 days, X 750, Bakcr’s phospholipid technique. Note
to Embryol., vol. 2, pp. 69-94.
the high, uniform concentration of phospholipid in the
spindle-shaped K cell. Red blood cells in a near-by
capillary also contain reactive material.


1948. Alkaline phosphatase in the ovarian follicle
Fig. 25. Corpus luteum of the 23d day of the non-
and in the corpus luteum. Carnegie Inst. Wash. Pub.
pregnant menstrual cycle, estimated age 9 days, X300,
575, Contrib. to Embr}/'01., vol. 32, pp. 1-8.
Seligman-Ashbel technique for demonstration of active carbonyl groups. Note reactive material in K cells in
high concentration, and in lutein cells of both layers in
lesser amounts.


DALTON, A. L, E. R. M1Tct1ELL, B. F. Joints, and V. B. Perms.
FIG. 26. Corpus luteum of early menstruation, same
1944. Changes in adrenal glands of rats following exposure to lowered oxygen tension. Iour. Natl. Cancer
specimen as figure 11, plate 2, X300, Seligman-Ashbel
Inst., vol. 4, pp. 527-536.
technique for demonstration of active carbonyl groups.
Note diminution in reactive material in both layers.
K cells are less numerous, their processes retracted as
in figure 11, :1 Sudan black preparation of this same
specimen.


D1—:1.es1'ar_, M. 1910. Recherches sur le follicule de Graaf et
le corps jaune de la vache. Iour. de l‘anat. et de la
physiol. (Paris), vol. 46, pp. 286-309.


D1-:1~.11>s1-:v, E. ‘W. 1948. The chemical cytology of endocrine
glands. Recent Prog. Hormone Res., vol. 3, pp. 127-157.


and D. L. B.1ss1»:'r1'. 1943. Observations on the fluorescence, birefringence and histochemistry of the rat ovary
{{Footer}}
during the reproductive cycle. Endocrinology, vol. 33,
[[Category:Draft]][[Category:1950's]]
pp. 334-401
GILLMAN, 1., and H. B. STEIN. 1941. The human corpus
luteum of pregnancy. Surg., Gynecol., and Obstet., vol.
73: PP- I39‘149
Gomoar, G. 1941. The distribution of phosphatase in normal
organs and tissues. Iour. Cell. and Comp. Physiol., vol.
17, pp- 7I-83
GRAFFLIN, A. L. 1939. The thyroid and parathyroid glands
of the Barasingha deer, with particular reference to
autolluorescence, fat and pigment. Iour. Morphol., vol. 65, pp- 297-32!

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White RF. Hertig AT. Rock J. and Adams E. Histological and histochemical observations on the corpus luteum of human pregnancy with special reference to corpora lutea associated with early normal and abnormal ova. (1951) Contributions To Embryology, No. 224 57-71.

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Histological and Histochemical Observations on the Corpus Luteum of Human Pregnancy with special reference to Corpora Lutea associated with early Normal and Abnormal Ova

Arthur T Hertig
Arthur Tremain Hertig (1904-1990)

White RF. Hertig AT. Rock J. and Adams E.

Free Hospital for Women, Brookline, Departments of Pathology, Obstetrics, and Gynecology, Harvard Medical School


With seven plates (1951)

Review of the Literature

Speculation and investigation concerning the role of the human corpus luteum in the normal menstrual cycle and in pregnancy have occupied the energies of many investigators since early times. For excellent reviews of the work that has led to modern concepts of the corpus luteum, the reader is referred to Asdell (1928) and Pratt (1935).


Meyer (1911) was the first to describe in detail the macroscopic and microscopic appearance of the stages from the ruptured follicle to complete involution of the human corpus luteum. He was the first to point out that there are four recognizable stages in the development of the corpus luteum, namely proliferation, vascularization, mature or so-called blossom stage, and regression. For the sake of completeness, Meyer’s concept of the development of the corpus luteum, which has been generally accepted by workers in the field, will be outlined briefly.


During the proliferative stage, which follows immediately upon rupture of the mature Graafian follicle, there is increased vascularity of the theca interna and theca externa, evidenced by dilatation of the capillaries. Storage of fat occurs in both theca interna and granulosa, but more noticeably in the former; mitoses in the granulosa layer are less evident or entirely absent, but mitotic figures are still present in the theca interna; hemorrhage into the follicle is not the rule, because capillaries do not extend into the granulosa layer at or before rupture. The stage of vascularization is marked by a well defined membrana granulosa which has increased conspicuously by hypertrophy of the former granulosa cells. The theca externa is still quite evident, but the cells of the theca interna are already shrunken and are no longer coherent. The folding of the wall of the corpus luteum is much more marked, and there is a notable increase of storage of lipoid in the granulosa cells, which may be termed granulosa lutein cells at this stage of development. Fibroblasts are found in the central coagulum as early as 48 hours after rupture of the follicle.


As the corpus luteum matures and attains the socalled blossom stage, the cells of the theca interna are irregular and decreased in number. The uniform granulosa lutein cells are large and irregularly cuboidal, with increased lipoid content. Capillaries, accompanied by a few spindle cells, pass between the luteal cells, and individual luteal cells are surrounded by connective-tissue fibrils. The inner contour of the granulosa lutein layer is lined smoothly by a conspicuous capillary network. The central coagulum has undergone considerable connective-tissue organization, or it may become cystic, or a hematoma may form in it. Unless pregnancy is superimposed, the corpus luteum goes on to regression, which is characterized by fatty degeneration and simple atrophy of lutein cells, associated with increased invasion of the luteal tissue by connective-tissue elements. Ultimately, the lipoid substance of the lutein tissue disappears and the corpus luteum is transformed into the corpus albicans by a process of hyaline degeneration which may extend over several months.


Meyer (1911, 1932) observed that the mature stage and the regressive stage were imperfectly limited and beginning regression could not be definitely recognized. Novak (1932, 1941) stated that regression begins shortly before menstruation, about the 26th day of a normal 28-day cycle. Brewer (1942), however, presents evidence that regression begins at the termination of the vascular stage, about 4 to 6 days before the onset of menstruation. Chemical assays of lutein tissue reveal a steady increase in phospholipid from immediately after ovulation until the 10th day, after which time the phospholipid content of the gland falls. During the first to days of existence of the corpus luteum there is a slight fall in the cholesterol ester content of lutein tissue, but after this time there is an abrupt increase in cholesterol ester content (Brewer, 1942). These demonstrable chemical changes in the corpus luteum, together with the microscopic appearance of the gland, led Brewer to conclude that the corpus luteum commences to regress about 8 to 10 days after ovulation.


Gillman and Stein (1941) tabulate the number of corpora lutea of pregnancy examined by various investigators prior to 1941, including their own series of 19 specimens. Brewer (1942) reports examining 8 corpora lutea of early pregnancy, but does not furnish any details concerning the histology of these specimens. I-Iertig and Rock (1941, 1945, 1949a) and Heuser, Rock, and Hertig (1945) describe briefly 8 corpora lutea associated with early pregnancies ranging from 7 days ovulation age to the definitive yolksac stage.


Meyer (1911) characterized the corpus luteum of pregnancy from the 2d month of pregnancy on as having a coarser, cruder appearance due to hypertrophy and increased lipid deposition in the lutein cells, a high degree of connective-tissue proliferation around and between the luteal cells, and thick-walled capillaries. He noted that hyaline degeneration in the corpus luteum of pregnancy was long delayed. He remarked that the theca lutein cells are sometimes retained up to the 4th month of pregnancy, but are insignificant and decrease in number until the final month of pregnancy, when the theca cells flourish again.


Gillman and Stein (1941), in a study of 13 corpora lutea associated with intra-uterine pregnancies ranging froi 35 to 282 days, state that there is a “critical period” of sudden growth in the volume of the corpus luteum between the 50th and 60th days of pregnancy, due to an excess production of hormonal fluid in the fibrotts-tissue-lined cavity, which is subsequently obliterated; that the theca lutein cells attain their maximum development synchronously with the development of this cavity, and that after its collapse they also disappear; that the granulosa cells, on the other hand, persist throughout pregnancy, and that their vacuolar secretion may degenerate into colloid or even calcium-containing deposit.


Brewer (1942) noted, in addition to sustained high phospholipid levels and low cholesterol ester levels in the corpus luteum of pregnancy, an increase in vascularity, a lack of fatty degeneration, and absence of simple atroppy of the luteal cells.


Asdell (1928) and Pratt (I935) have reviewed the literature concerning the origin of the true lutein cell of the human corpus luteum. Meyer decided that the theca lutein cells disappear early in the life of the corpus luteum, leaving only luteal cells of granulosa origin. Chydenius (1926) decided upon a dual origin of the lutein cell. Shaw (1925) described theca lutein cells, or paralutein cells as he termed them, but considered that they remain at the periphery of the gland and do not take part in the formation of the stroma of the gland. A real dilierence between species does seem to exist in the extent to which theca lutein cells invade the stroma of the corpus luteum. According to Hammond and Marshall (1925), this invasion appears to be small or entirely wanting in the monotremes, marsupials, certain rodents, the sheep, the horse, and man, whereas in bats, the guinea pig, the cow, and most of all the sow, the invasion appears to be considerable.


McNutt (1924) asserted the dual origin of the lutein cell in the cow, stating that small clumps of theca lutein cells invade the space filled by the granulosa lutein cells and become detached from the connectivetissue framework. Despite some divergence of opinion regarding the origin of the human lutein cell, it is established that early in the development of the corpus luteum, the lutein cell derived from the membrana granulosa becomes dominant and is the cell usually described as the lutein cell (Pratt, 1935).


Corner (1915) described, in addition to the granulosa lutein and theca lutein cells, cells of a third type in the corpus luteum of the sow. These are smaller than the lutein cell, of varied shape, and strongly eosinophilic. They contain small vacuoles even when fixed with osmic acid. He believed that Delestre (1910) also saw them. Gillman and Stein (1941) pointed out the existence of dark and light cells in the granulosa and regarded them as representing different phases of activity of secretion in granulosa lutein cells.


In recent years a number of new methods of chemical cytology have been developed, and these are being applied to a great variety of organs and tissues. An excellent review of advances in this field is given by Dempsey (1948). To date, reports of the application of these techniques to the human corpus luteum are few.


McKay and Robinson (1947) studied a series of human corpora lutea of the normal menstrual cycle, employing some of the newer techniques for detecting presumptive ketosteroid compounds. The presence of birefringent crystals, autofluorescent materials, acetone-soluble keto compounds, and sterol substances which reacted with sulfuric acid was noted. ‘Nhen employing Sudan IV to detect sudanophilie lipids, they noticed that lipid was confined to the theca interna in the developing follicle. In the 15-day corpus luteum, small sudanophilie drops were found in all the granulosa and theca lutein cells. During the next 6 or 7 days of the life of the corpus luteum, fine peripherally distributed lipid droplets were seen in almost all the granulosa cells. During this period there was a steady increase in the number and size of sudanophilie droplets in the cells of the theca interna, these droplets being much larger than those in the granulosa cells. About the 23cl day of the cycle a marked decrease was observed in the number of sudanophilie granules in the granulosa lutein cells, many cells being completely devoid of lipid. There were, however, patchy areas containing large drops of lipid in the granulosa layer around the blood vessels of the invading connective-tissue septa. Fewer theca interna cells contained lipid than in earlier stages, but those that did, contained large droplets. This decrease in total sudanophilie substance in both layers was maintained to the 27th day of the cycle. After the 27th day, a greatly increased amount of sudanophilie substance distributed in large, coarse droplets was observed in both layers.


Examination for birefringent materials revealed a few tiny anisotropic crystals in the theca interna of the 15-day corpus luteum. There was a steady increase in the size and number of these crystals up to the 24th day of the cycle. Very fine birefringent crystals were seen in the granulosa cells on the 17th day, increasing in number and size until the 22d day of the cycle. Throughout the period embracing the 17th to the 23d day of the menstrual cycle, more anisotropic substance was present in the theca interna than in the granulosa at any stage of development. There was a notable decrease in the amount of hirefringent crystals in the theca interna from the 24th to the 27th day, after which there was a considerable increase that persisted through menstruation. A comparative absence of birefringent crystals was observed in the granulosa lutein layer from the 22d day through late menstruation.


Studies of autofluorescent substances and substances that reacted with phenylhydrazine and sulfuric acid essentially paralleled the observations made on birefringence.

McKay and Robinson (1947) also noted in the granulosa layer a few extracellular large oily drops that were sudanophilie and reacted with sulfuric acid and phenylhydrazine but were negative for birefringence and autofluorescence. These were seen only after the 22d day of the cycle, when reactive materials had disappeared from the granulosa cells. The authors suggest that these large drops may be indicative of a change from active secretion to storage or inactivity, basing this interpretation on findings in the adrenal gland (Selye, 1937; Sarason, 1943; Dalton et al., 1944) that line lipid droplets are associated with active secretion and large droplets with inactivity.


Corner (1948) in a study of 3 human corpora lutea observed that the cells of the theca interna contain alkaline phosphatase up to a day or two after ovulation, but subsequently lose it. The granulosa lutein cells seemed to be almost devoid of alkaline phosphatase in the stages studied.

It is the purpose of this paper to describe in some detail the histological and histochemical variations of the human corpus luteum from the earliest stage of pregnancy yet obtained, a 2-cell tubal ovum (Hertig and Rock, 1949b), to that associated with a 4.5 month fetus. For purposes of comparison, a study of corpora lutea of the normal menstrual cycle is included. Evidence will be presented that the lutein cell is derived from the membrana granulosa of the follicle. A third cell type, derived from the theca interna of the follicle, has been demonstrated. Cells of this type make their way into the granulosa layer at or shortly after the time of rupture of the follicle, and become quite prominent as the gland approaches its peak of physiological activity. Evidence will be presented that these cells are the site of intense localization of ltetosteroids, which presumably are the secretory products of the corpus luteum.

Materials and Methods

Since 1939 two of the authors (I-Iertig and Rock) have been searching for early human ova, and to date they have succeeded in obtaining 32 ova ranging from 2 to 17 days ovulation age. Of these specimens, 19 are regarded as normal. The remaining 13 were adjudged to be abnormal because of one or more of the following conditions: multinucleated blastomeres, shallow implantation, malorientation or lack of germ disk, defective trophoblast, or abnormal segmentation cavity. These specimens were obtained as described by Hertig and Rock (1944). In all instances, the ovary containing the corpus luteum was removed with the uterus at operation. The corpora lutea thus obtained were examined macroscopically and then appropriately prepared for microscopic examination as described below.


Nine corpora lutea associated with older pregnancies 25 days to 4.5 months) were also obtained from surgical material from the Free Hospital for Women (Brookline, Massachusetts) or the Boston Lying-In Hospital.


Forty-eight corpora lutea not associated with pregnancy were studied in order to compare histologic and histochemical changes in the gland during the normal menstrual cycle with those observed in pregnancy.


All corpora lutea of nonpregnant cycles and those associated with ova still free in the tubes or uterine cavity were dated by referring to the endometrial age (Hertig, 1945). It is assumed that the normal menstrual cycle is 28 (lays in length, with ovulation on the 14th day of the cycle. It is generally agreed that, regardless of the length of the menstrual cycle, ovulation takes place about 14 days (:2 (lays) before the first day of the next expected period (Rock and Hertig, 1944).


Material obtained prior to June 1947 had been fixed and stained in accordance with routine laboratory procedures, the following stains being employed: haematoxylin and eosin, eosin-methylene blue, ironalum haematoxylin, phosphotungstic acid-haematoxylin, and Scarlet red for fats. Recently acquired specimens have been stained with eosin-methylene


blue and with haematoxylin and eosin, and have been subjected to the following special procedures: The alkaline phosphatase procedure of Gomori (1941) was applied to all recently acquired corpora lutea. Frozen sections, 10 to 20 microns in thickness, of formalin-fixed material were prepared, and alternate sections were treated with acetone, alcohol, and acetone—alcohol mixtures for comparison with untreated sections. Sudan black was used as a general lat stain. The phenylhydrazine reaction of Bennett (1940) and the plasmal reaction as described by Lison (1936) were applied to representative cases. Plain sections, both untreated and subjected to fat solvents, were mounted in glycerine jelly and examined under crossed Nicol prisms for evidence of birefringence; and under a fluorescence microscope similar to that described by Grafllin (1939), using as illumination a beam of ultraviolet rays obtained by filtering the light of a carbon-arc lamp through a copper sulfate solution and a Corex filter no. 586.


Formalin-fixed frozen sections were floated onto slides and blotted dry, and a drop of a solution consisting of equal parts of concentrated sulfuric acid and acetic anhydride was put on the tissue. The slides were examined immediately for the appearance of brown droplets in the cells of the corpus luteum and ovarian stroma. This reaction, formerly used as a test for cholesterol (Romeis, 1928), is now ltnown to depend upon the presence of unsaturated bonds in steroid molecules (S0l)0tl{:1, 193,8).


Very recently new techniques for the histochemieal detection of active carbonyl groups in lipoid and nonlipoitl materials have been developed (Seligman and Ashbel, I949; Ashbel and Seligman, 1949), and through the courtesy of Drs. A. M. Seligman and R. Ashbel, of the Beth Israel Hospital, Boston, Massachusetts, selected corpora lutea were studied employing these new techniques.


Two specimens were treated for the histochemieal demonstration of phospholipid according to the method of Baker (1946).

Observations on Corpra Lutea of the Nonpregnant Menstrual Cycle

The Mature Grcmfirm Follicle, S48-731

Figure 1, plate I, is a photomicrograph of a typical mature Graafian follicle. The theca interim is several cell layers thick. Many of the theca cells are highly vacuolated. Widelyr dilated, blood—filled capillaries are prominent in the theca immediately beneath the basal layer of the granulosa. i\/Iitotic figures are rare in the theca interna. Conspicuous in the theca are a number of cells that differ markedly from the neighboring theca cells. These cells have small, dense, hyperchromatic nuclei which are irregular in outline, and stand out in bold relief against the nuclei of the theca interna cells, which by contrast are plump, ovoid, and vesicular, with a single prominent, eccentric nucleolus. The cytoplasm of these stellate cells is homogeneous and more strikingly eosinophilic than that of the theca cells. Careful study has shown that these cells do not represent intravascular or perivascular elements. For lack of a more specific and descriptive name, and in the interest of definiteness without repetition, these cells will be referred to in the subsequent descriptions and discussions as “K cells.”


The membrana granulosa is sharply demarcated from the theca interna by a closely packed layer of cells contiguous with, and similar to, the cells of the membrana itself, which is many cell layers thick and thrown into convolutions by the hypertrophy and multiplication of the granulosa cells. Consequently, the inner layer of the follicle presents a scalloped, undulating edge, with tongues of theca interna projecting into the convolutions from the stroma of the ovary. Mitotic figures are numerous in the membrana granulosa. The nuclei of the granulosa cells are perhaps a little larger than those of the theca interna cells, and because of their rapid rate of division and growth are more basophilic. Their cytoplasm presents a frothy appearance, and cell outlines are indistinct, although there is no evidence of vacuolization. There is no evidence of capillary penetration into or proliferation within the membrana granulosa. None of the K cells present in the theca interna are seen in the membrana granulosa at this stage of development.


Corpus Luteum of 16th Day of Cycle, Estimated Age 2 Days, S48-2636

Macroscopically, this corpus luteum appeared as a hemorrhagic, unhealed crater I cm. in diameter and 2 mm. in depth, on the posterior surface of the ovary.

Haematoxylin and eosin sections (fig. 2, pl. 1) show the theca interna to be considerably thinned out and the membrana granulosa thrown into a large number of deep convolutions, owing in part to the collapse of the follicle after rupture. The vascular channels of the theca interna are widely dilated, and in several places endothelial sprouts are seen penetrating the membrana granulosa from the theca interna. The cells of the theca interna are essentially the same as those observed in the mature follicle, and rarely show mitoses. The granulosa cells are plump and polyhedral, with round, vesicular nuclei. Their cytoplasm is frothy, but no distinct vacuolization is seen. The granulosa cells are arranged in bundles or fascicles separated in many areas by large lacunae of extravasated blood. These lacunae are not lined with endothelium.

The K cells noted in the theca interna of the mature follicle are very prominent at this stage of development of the corpus luteum. Although few of them are found in the theca interna, ribbons of them can be seen spreading out into the membrana granulosa, penetrating as far as the central coagulum. The attenuated cytoplasmic processes of these cells suggest amoeboid activity. It is to be noted that these cells are more numerous at this and subsequent stages of development than in the mature follicle. Only once have we observed a mitotic figure in a cell that we could definitely say was a K cell. It is possible that the small, irregular, hyperchromatic nuclei characteristic of these cells at this stage represent rapid mitotic activity. As will be noted later, the nuclei of these cells become larger and less hyperchromatic as the gland approaches the period of maximum functional activity.


Sudan black preparations (fig. 3, pl. 1) reveal that the cells of the theca interna contain much more lipid than do those of the membrana granulosa. This lipid is distributed in fine droplets in most cells, although medium-sized and coarse droplets are found in some of the theca interna cells. As has been mentioned, the granulosa cells contain much less lipid, and this is evenly distributed as a fine peripheral dusting of sudanophilic lipid. In an occasional granulosa cell a few coarse globules of lipid are seen. The K cells are distinguishable only with some diliiculty. They present a uniform, nongranular sudanophilia.


Alkaline phosphatase is localized exclusively in the cells of the theca interim and in the endothelium of blood vessels. The cytoplasm of the granulosa cells contains no alkaline phosphatase at this stage of development (fig. 4, pl. 1).

Corpus Luteum of 20th Day of Cycle, Estimated Age 6 Days, S48-2262

On cut section, the corpus luteum measured 1.7 by 1.5 cm. in its greater diameters. The convoluted borders were bright yellow and from 1 to 3 mm. thick. The central coagulum was pale gray with several small hemorrhagic areas.

The corpus luteum at this stage is quite compact. There is very little extravasated blood in either layer. The blood vessels of the theca interna are widely dilated. Definite capillaries are seen in the granulosa layer, but these are still small and only an occasional The central coagulum contains many proliferating fibroblasts and red blood cell can be noted in them.

thus is undergoing early organization. No capillaries are seen in it. The cells of the theca interna appear to be widely separated by fibroblasts and other connective—tissue elements; their cytoplasm is markedly vacuolated, but the nuclei show no evidence of cellular degeneration. The granulosa cells are large and polyhedral, with distinct cell membranes and marked peripheral vacuolization of the cytoplasm (fig. 5, pl. 1). Interspersed among the granulosa cells are a number of K cells, whose nuclei appear dark, irregular, and almost pycnotic.


Sudanophilic substance is distributed irregularly in the cells of the theca interna. Some of the theca cells contain few lipid droplets; others contain numerous fine, peripherally distributed lipid droplets, and many contain large, coarse globules of sudanophilic substances. Nearly all the granulosa lutein cells contain fine, peripherally distributed lipid droplets. Only an occasional granulosa cell contains the coarse lipid droplets noted in the theca. The large, stellate K cells are particularly conspicuous at this stage. All of them appear uniformly sudanophilic, although close examination reveals some granular deposits of sttdanophilic substances which are almost masked by the uniformly sudanophilic background of the cytoplasm of these cells (fin. 6, pl. 1). The nuclei of the K cells are clear and devoid of lipid.


The theca lutein cells are uniformly devoid of alkaline phosphatase. This statement applies equally well to the majority of the granulosa cells. A small number of cells scattered among the granulosa cells, however, contain alkaline phosphatase in moderate amounts. It is difiicult to state at this stage of development that these cells containing alkaline phosphatase deposits are the K cells so prominent in the Sudan black preparation.


Corpus Luteum of 23rd Day of Cycle, Estimated Age 9 Days, S48-3028

The corpus luteum measured 1.5 cm. in its greatest diameters. The convoluted border was bright yellow, and averaged 3 mm. in thickness. The central coagt1him was well organized and was the site of a recent small hemorrhage.

It is to be noted in this specimen that the theca lutein layer is much less prominent than in the 20-day corpus luteum, but that those theca lutein cells that are observed present essentially the same characteristics as those noted in the earlier stage. The granulosa lutein cells are plump and show all the evidences of marked physiological activity. The K cells are quite numerous and prominent in this specimen and are readily recognized by their more angular shapes, dark, homogeneous cytoplasm, and small, dark nuclei (Fig. 7, pl. 2). Capillaries in the granulosa lutein layer are numerous and widely dilated.


The theca lutein cells contain large, coarse drops of lipid, whereas the granulosa lutein cells contain fine, peripherally distributed lipid granules in large numbers (fig. 8, pl. 2). The K cells are especially prominent in this preparation, and there can be little question that these elongated, uniformly sudanophilic cells with clear, ovoid nuclei are the same cells that are so conspicuous in haemato:~:ylin and eosin preparations.


Only an occasional theca lutein cell contains alkaline phosphatase (fig. 9, pl. 2). The true granulosa lutein cells are devoid of this enzyme. A number of cells, however, having the configuration and nuclear characteristics of the K cells as they appear in routine and Sudan-treated preparations contain high concentrations of alkaline phosphatase. Capillary endothelium in all layers of the corpus luteum contains the enzyme in high concentration.

From 24th Day of Cycle to Menstuation

Between the 23d day of the cycle and the onset of menstruation, the evidence of regression in the corpus luteum becomes increasingly marked. There is no evidence of further capillary proliferation. The theca cells become less and less distinct, until they can be found only in widely separated clumps in the connective-tissue septa invaginating the granulosa from the ovarian stroma. The granulosa lutein cells show evidence of degeneration, manifested by loss of chromaticity of the nuclei and loss of most of the peripheral vacuolization noted at earlier stages. Many granulosa cells show increasing accumulation of medium—sized and coarse lipid droplets.

The fate of the K cells becomes evident during this period. With haematoxylin and eosin stains these cells become more eosinophilie, the cytoplasm becomes more dense and homogeneous, and the nuclei become quite contracted and hyperchromatic. The cytoplasm seems to condense, until ultimately all that remains of many of these cells late in the life of the corpus luteum is a dense, eosinophilie mass similar to the colloid described by Gillman and Stein (1941). This process of colloid degeneration evidently extends over a long period, because, as will be noted in our discussion of the corpus luteum of early menstruation, large numbers of apparently normal K cells can be found in older corpora lutea. The K cells are still prominent in Sudan black preparations, but show a progressive loss of sudanophilia and a retraction of their cytoplasmic processes. The pattern of alkaline phosphatase distribution remains essentially the same as that observed in the 23-day specimen, with perhaps some diminution in the concentration of the enzyme in the K cells as the corpus luteum becomes older.

Corpus Luteum during Early Menstruation, S48-535

At this stage the theca interna is very indistinct. True theca lutein cells are found only in scattered clumps, chiefly in the connective-tissue septa that penetrate between the folds of the granulosa from the ovarian side of the gland (fig. 10, pl. 2). Those theca cells that persist are markedly vacuolated and show wide variation in the staining characteristics of their nuclei. Most of the granulosa lutein cells have lost the peripheral vacuolization that typifies the actively secreting gland. Many cell boundaries in the granulosa lutein layer are quite indistinct. The capillaries in both cell layers are more or less uniformly collapsed, and contain few erythrocytes. Although the K cells are still prominent in routine sections, they do not present the full-blown appearance seen in the 23-day corpus luteum. Moreover, their cytoplasm is denser than has previously been noted and in most instances appears to be contracting, leaving large vacuoles between the granulosa lutein cells. Some of the K cells have degenerated to what we consider the end stage of this line of cells, a dense, strongly eosinophilie colloid droplet. All gradations between a stellate, active cell and the final degenerative end product, colloid, can be found in this specimen, a fact which indicates that this process of colloid degeneration must extend well beyond the menstrual period.


The theca lutein cells contain only medium-sized and coarsely irregular lipid droplets (fig. 11, pl. 2). The true granulosa cells contain much more lipid than at any previous stage. Although the lipid droplets are larger than in earlier stages, they are peripherally distributed in most granulosa cells, and in none are the droplets as large and coarse as those in the theca lutein cells. A few granulosa cells are heavily laden with medium-sized lipid droplets. The K cells are still prominent in the Sudan black preparations, but their cytoplasmic processes are markedly retracted, the cytoplasm has lost some of its uniform homogeneous sudanophilia, and a few fine sudanophilic granules are seen against the slate-gray cytoplasmic background of the cells. A few large, oily drops showing varying degrees of sudanophilia are present. It is believed that these represent the colloid drops noted in the haematoxylin and eosin preparations.

The alkaline phosphatase preparations show essentially the same pattern of distribution of the enzyme as in the 23-day specimen, with perhaps some diminution in the concentration of the enzyme in the K cells (fig. 12, pl. 2).

Observations on Corpra Lutea in Normal Pregnancy

A 2-Cell Egg, 17-Day Corpus Luteum, S49-2439

This ovum was recovered from the middle third of the Fallopian tube and consisted of two normal blastomeres. This specimen, of about 60 hours coital age, is the earliest human ovum yet recovered. The endometrium had the characteristics of that of 2 to 21/; days after ovulation.

On cut surface, the corpus luteum measured 2.5 by 1.5 cm. in its greatest diameters. The convoluted border was red-gray, and varied in thickness from 3 mm. at the base to I mm. at the unhealed stigma. The central coagulum was moderately well organized and showed some peripheral congestion.

Microscopic examination demonstrates that this corpus luteum is in no respect materially different from S48-2636 (figs. 2-4). K cells streaming into the granulosa lutein layer from the theca are prominent and numerous. Sudanophilic substances are much more prominent in the theca interna than in the granulosa lutein layer. Alkaline phosphatase is restricted to the theca interna and the endothelium of the blood vessels.

A 4.5-Day Blastocyst, 19-Day Corpus Luteum, 548-5000

This specimen is the first normal human blastocyst yet recovered from the uterine cavity. The associated endometrium is typical 19-day secretory endometrium.

On cut surface the corpus luteum measured 2.2 cm. in its greatest diameters. The convoluted borders were reddish gray, measuring 3 to 4 mm. in thickness. The stigma was completely healed, and the central coagulum was pale and gelatinous.

Microscopic examination reveals that this corpus luteum is almost identical in all respects with the 20-day corpus luteum of the normal menstrual cycle and with S48-3948, the corpus luteum associated with the abnormal 5-cell, 4‘/§_- to 5-day ovum.

7.5- and 9.5-Day Pregnancies

Figures 13 and 14, plate 3, are photomicrographs of sections of corpora ltttea associated with 7‘/3- and 9‘/3-day normal pregnancies respectively (Carnegie nos. 8020, 8215). Unfortunately, these sections, which were stained with haematoxylin and eosin, have faded

to a great extent, but the following features are noteworthy. There is a progressive increase in vascularity and dilatation of the capillaries in the granulosa lutein layer. The theca lutein layer remains quite distinct and prominent. Peripheral vacuolization of the granulosa lutein cells, which is normally disappearing in the corpus luteum of the nonpregnant cycle by 26 to 27 days, becomes increasingly noticeable after 9.5; days of pregnancy and is particularly striking at 11 and 12 days of pregnancy. The cell boundaries of the granulosa cells become progressively less distinct because of the line peripheral vacuolization. At this stage the granulosa cells resemble the “prickle cells” described by Hertig and Rock (1941). Although the K cells are not strikingly conspicuous in any of these sections, they are seen in considerable numbers. It appears that these cells have actually been stimulated and are assuming the appearance of full-blown activity noted in the 21-day corpus luteum (Hg. 7).

A 12- to 13-Day Pregnancy, Carnegie No. 8558, S46-2767

The corpus luteum was moderately cystic and measured 2.0 cm. in its greatest diameters. The convoluted border was 1 to 3 mm. thick and was pale yellow with an orange tinge, but did not appear to be senescent.

The theca lutein layer is quite prominent and many cell layers in thickness. The cytoplasm of these cells is vacuolated, and the nuclei resemble those of actively functioning cells. The granulosa lutein cells show marked peripheral vacuolization, and the cell boundaries are thus almost completely obliterated (fig. 15, pl. 3). There has been a significant increase in the number of widely patent vascular channels in the granulosa layer. K cells are not at all conspicuous in haematoxylin and eosin sections. However, the dense, pycnotic nuclei and contracted cytoplasm characteristic of many of the K cells in figure 10 are not frequently seen here, a fact which indicates a recrudescence of activity of these cells.


A very striking change is noted in the Sudan black preparations. Only a few theca interna cells contain coarse lipid granules. The majority of the theca lutein and granulosa cells contain large numbers of very fine, peripherally distributed sudanophilic droplets.


K cells are very prominent (fig. 16, pl. 3) and show marked uniform sudanophilia and attenuated cytoplasmic processes like those seen in the physiologically active corpus luteum of the 23d clay of the nonpregnant cycle (fig. 8).


A few of the theca lutein cells contain alkaline phosphatase, but most are devoid of the enzyme. A few K cells at the junction of the granulosa and theca lutein layers contain a high concentration of alkaline phosphatase, but of particular interest is the demonstration of the enzyme in significant amounts in some of the true granulosa lutein cells (fig. 17, pl. 3).

A 16-Day Pregnancy, Carnegie No. 8602, S48-2088

The cystic corpus luteum measured 3 cm. in its greatest diameters. The convoluted borders were a brilliant yellow and varied in thickness from 4 mm. at the base to I mm. at the healed stigma.


Haematoxylin and eosin sections of this specimen are very similar to those of the preceding stage. The theca interna is quite prominent (fig. 18, pl. 4). The granulosa lutein cells show considerable activity, manifested by noticeable peripheral vacuolization which causes cell boundaries to appear very indistinct. A large number of fine capillaries form a delicate vascular network in the granulosa lutein layer. K cells are even more numerous and distinct in this specimen than in the 12- to 13-day pregnancy (fig. 15), but a number of these cells are undergoing colloid degeneration as described in the corpus luteum of early menstruation. In this and in subsequent specimens to be described, a number of regular, spherical vacuoles are seen in the granulosa lutein layer. These vacuoles apparently mark the site of colloid deposits, which have dropped out of the section in preparation.


The great majority of both theca lutein cells and granulosa lutein cells contain large numbers of fine peripheral sudanophilic droplets. An occasional theca lutein cell contains a number of medium-sized lipid droplets (fig. 19, pl. 4). The K cells are quite conspicuous and appear essentially the same as in the corpus luteum of the 12- to 13-day pregnancy.


In figure 20, plate 4, the theca lutein cells are seen to be almost completely devoid of alkaline phosphatase. Nearly all of the granulosa lutein cells, however, contain alkaline phosphatase in varying amounts, thereby making it difficult to identify the K cells. Several cells at the junction of theca lutein and granulosa lutein layers contain a high concentration of the enzyme, and on the basis of their position and configuration these are very likely the K cells so conspicuously revealed by other techniques.

A 26-Day Pregnancy, S48-263

This specimen was obtained with the uterus at time of operation for carcinoma of the cervix. The corpus luteum was cystic, 2 to 4 cm. in diameter, and was filled with a clear, yellowish fluid. The convoluted border was yellowish gray, and 2 to 3 mm. thick.


The theca interna is very conspicuous in this specimen, being several cell layers thick around the entire granulosa lutein layer (fig. 21, pl. 4). The nuclei stain uniformly and present the appearance of actively functioning cells. For the first time since the stages of the mature follicle and the 16-day corpus luteum of the nonpregnant menstrual cycle, a number of small, irregular stellate cells with wrinkled hyperchromatic nuclei and homogeneous eosinophilic cytoplasm are seen in the theca lutein layer. Except for somewhat smaller size, these cells are identical in all respects with the K cells noted in routine haematoxylin and eosin sections of the younger corpora lutea. The sudden reappearance of these cells in the theca lutein layer is unexplained. There is no evidence of transformation or degeneration of theca lutein cells to these forms. The capillaries and sinusoids of the theca lutein layer are widely dilated.


The granulosa lutein layer at this stage appears highly disorganized. Cell boundaries are quite indistinct. The individual granulosa lutein cells show marked variation in the stainability of their nuclei and cytoplasm. There are a large number of patent capillaries in the granulosa lutein layer. Around each of these capillaries, proliferation of perivascular connective-tissue elements is seen. There is a definite increase in the reticular network that at this stage encircles nearly every granulosa lutein cell. The central coagulum is well organized, and large, dilated vascular channels are present in this new connective tissue.


Very few K cells that may be regarded as active are seen, but large numbers of these cells in all stages of degeneration are present. Colloid is present in larger amounts, and as a corollary the number of large, empty vacuoles is also increased over the preceding stage.


Most of the theca lutein and granulosa lutein cells contain very fine lipid droplets in large quantities (fig. 22, pl. An occasional theca lutein cell contains a few medium-sized lipid droplets. A significant number of the granulosa lutein cells contain large amounts of lipid in large, coarse droplets, indicating fatty degeneration. The K cells are still quite prominent and markedly sudanophilic, but they appear frayed and fibrillar. They are definitely not so numerous or attenuated as in figure 19. Many large, smooth sudanophilie globules are present. These match in number, size, shape, and location the colloid droplets seen in haematoxylin and eosin sections.


Of particular interest is the appearance of large numbers of these characteristic sudanophilic K cells scattered among the cells of the theca lutein layer. Both examples of 26-day pregnancy show them, and although carefully searched for, they were not seen in any other specimen. These cells appear to be very similar to the K cells observed in the early stages of development of the corpus luteum, and do not present the frayed, fibrillar appearance noted in the K cells in the granulosa lutein layer of this same specimen, facts which suggest that these cells represent younger forms.


The theca interna is uniformly devoid of alkaline phosphatase (Hg. 23, pl. 4), although most of the granulosa lutein cells contain moderate amounts of the enzyme. It is diflicult to recognize K cells in this preparation. The endothelium of blood vessels in both layers contains alkaline phosphatase.

A 28- to 35-Day Pregnancy, S48-4854

On gross examination the corpus luteum measured 2.3 by 2.0 cm in its greatest diameters. The convoluted border was grayish yellow, measuring 2 to 3 mm. in thickness. The central coagulum was well organized.


The theca lutein layer is still very prominent, although not so conspicuous as in the preceding specimen. Many of the nuclei are dense and irregularly pycnotic. The cytoplasm of most of the cells is highly vacuolated (fig. 24, pl. 5). The vascular sinusoids of the theca interna are almost completely collapsed.


The granulosa lutein layer presents essentially the same picture as that of the preceding specimen in haematoxylin and eosin preparations. Figure 25, plate 5, is a photomicrograph of this same section, showing a clearly defined colloid globule. Other K cells in various stages of degeneration are present in this section.


The Sudan black preparations show large numbers of very fine peripherally distributed lipid granules in both theca lutein and granulosa lutein cells (fig. 26, pl. 5). Occasional cells undergong fatty degeneration are seen in both layers. The K cells are still quite conspicuous in the granulosa lutein layer, and all show a line dusting of sudanophilic droplets in their cytoplasm. A few small, uniformly sudanophilic cells similar to those described in the previous specimen are seen in the theca lutein layer, but these are not sufficiently conspicuous to photograph. A number of large, oily sudanophilic droplets resting in vacuoles may be noted among the granulosa cells. It is believed that this is the colloid mentioned earlier.


The alkaline phosphatase preparations are particularly striking. A number of the cells of the theca lutein layer contain large amounts of the enzyme, but most of the theca cells are totally devoid of it. On the other hand, the granulosa lutein layer contains a very high concentration of the enzyme, which is so diffusely distributed that it is impossible to separate the granulosa lutein cells from the K cells. Numerous large vacuoles, representing the site of colloid deposits that have been lost or dissolved in the process of preparation, are evident in this section as in figures 20 and 23.

A 4- to 4.5-Month Pregnancy, S48-2624

This specimen was obtained incidentally to a total hysterectomy performed during the 5th month of pregnancy because of carcinoma of the cervix. The corpus luteum measured 2 by 1.5 cm. The stigma was depressed and well healed. The periphery of the corpus luteum presented a yellowish, fatlike appearance upon the cut surface. Several small gelatinous areas were seen between the convolutions. There was a small hemorrhage, 2 by 5 cm., in the exact center of the gland.


This corpus luteum is fairly well preserved, as is indicated by the retention of many of the normal cellular relationships, particularly in the granulosa lutein layer. A striking feature, however, is that the theca lutein layer is represented by only a few scattered pycnotic nuclei (fig. 27, pl. 5). Nearly all the granulosa lutein cells are undergoing atrophy, and large, gaping vacuoles are scattered throughout the granulosa lutein layer. A number of colloid deposits are visible. The lutein layer is almost completely avascular. Connective-tissue organization is marked.


As in the haematoxylin and eosin preparations, the theca lutein layer is not evident in Sudan black treated sections. The granulosa lutein cells are uniformly devoid of lipid (fig. 28, pl. 5). An occasional K cell stands out conspicuously by virtue of its intense and uniform sudanophilia. Most of the K cells, however, appear only as “shadow forms,” having lost most of their sudanophilia. Though it is not evident in the photomicrograph, the fibrillar substructure of these cells is very conspicuous at higher magnification. Alkaline phosphatase is no longer demonstrable in the majority of granulosa lutein cells. A few cells along the outer margin of the lutein layer contain high concentrations of the enzyme (fig. 29, pl. 5). These cells occupy the same locus as the sudanophilic K cells mentioned above. It is of particular interest that the endothelium of blood vessels of both layers no longer contains demonstrable alkaline phosphatase.

Observations on Corpora Lutea associated with Abnormal Ova

In view of the fact that our material included 13 corpora lutea associated with ova that were adjudged to be abnormal on the basis of criteria stated earlier, it was decided to study these specimens closely to determine whether any relation between anatomical integrity of the corpus luteum and the condition of the ovum could be detected. These abnormal ova have been the subject of separate communications by Hertig and Rock (1944, 1949!), 1950).


Despite the fact that it is difficult to judge accurately the age of these ova, because of abnormalities of blastomeres, trophoblast, chorionic cavity, or germ disk, a reasonable estimate of age can be made from the appearance of the endometrium and from the history.

Corpora Lutea associated with Abnormal Free-lying Ova

The corpora lutea associated with S43-I372 (Carnegie no. 8190), a 9—cell, 31/2" to 4-day free-lying segmenting ovum, and S46-3332 (Carnegie no. 8450), an 8-cell egg, are in no way different from the corpora lutea of the 17th and 18th days of the normal nonpregnant menstrual cycle.

A 5-Cell Egg, 19-Day Corpus Luteum, S48-3948

The corpus luteum measured 2.0 by 1.8 cm. in its greatest diameters. The convoluted borders were pinkish gray, with a maximum thickness of 2 mm. The coagulum was pearly gray, and gelatinous in consistency.


The ovum consisted of five abnormal blastomeres, several of which were multinucleated and showed other evidences of degeneration and retardation of development.

The corpus luteum differs in no respect from a 19-day corpus luteum of the normal nonpregnant menstrual cycle. The theca interna is quite prominent, several cell layers in thickness over the crests of the granulosa. The cytoplasm of the theca interna cells is markedly vacuolated. The granulosa lutein cells are plump and polyhedral, and only a few of them show peripheral vacuolization. A number of small capillaries and endothelial sprouts are seen in the granulosa lutein layer, but these capillaries are not dilated. K cells are not conspicuous in the routine haematoxylin and eosin sections.


Most theca lutein cells contain fine lipid droplets, but many cells contain only rnedium-sized to coarse lipid granules. Nearly all the granulosa lutein cells contain large amounts of fine peripherally distributed lipid. An occasional granulosa cell contains mediumsized lipid droplets. The K cells are not particularly prominent, showing only a moderate degree of uniform slate-gray sudanophilia.

Alkaline phosphatase is restricted to a few scattered clumps of cells of the theca lutein layer and the endothelium of blood vessels in both layers.

Corpora Lutea Associated with Abnormal Ova with Adequate Trophoblast

An 8-Day Ovum, Carnegie No. 8370, S46-676

The ovum is the youngest implanted embryo of this series of abnormal ova. The chorionic cavity is absent. Although ectoderm is present in the germ disk, there is no endoderm. The trophoblast is adequate but poorly organized, that is, laminated instead of being concentrically arranged with the syncytiotrophoblast surrounding the cytotrophoblast.


The corpus luteum appears to be normal in all respects. The theca interna is prominent. Both layers are well vascularized. The granulosa lutein cells show a moderate degree of peripheral vacuolization. K cells are numerous and appear to be normally active. No evidence of colloid degeneration is seen.

A 10- to 11-Day Ovum, Carnegie No. 7770, S40-749

This ovum is not markedly defective. The trophoblast is moderately hypoplastic and the germ disk is maloriented.

The theca interna appears to be normal in all respects and consistent with that of a normal 10- or 11-day pregnancy. The granulosa lutein layer is moderately well vascularized. K cells are relatively few, but appear to be normal in all respects. There is, however, great variation in size and stainability of the nuclei of the granulosa lutein cells.

An 11-Day Ovum, Carnegie No. 8299, 545-1220

The trophoblast appears to be normal although somewhat poorly organized. The germ disk is markedly maloriented.

The corpus luteum is very large and consists of a large cystic cavity enclosed by a thin rim of lutein tissue. Both granulosa and theca lutein layers are moderately well vascularized and appear to be functionally active. Most of the K cells show little evidence of colloid degeneration. This appears to be a very active corpus luteum (fig. 30, pl. 6).

Carnegie No. 7850, S40-2699

This ovum is associated with an endometrium that evidences early decidual reaction. Except for moderate hypoplasia of the trophoblast, the ovum appears to be fairly good.


The theca interna is prominent, many cell layers thick, and appears to be functionally active. Peripheral vacuolization of the granulosa lutein cells is not so marked as in chronologically similar corpora lutea associated with normal pregnancy. Mitoses are to be noted in both layers of the corpus luteum. K cells are quite numerous, but are irregularly distributed. No evidence of colloid degeneration is to be noted anywhere in the section.

Corpra Lutea associated with Abnormal Shallow Implantation

An 11- to 12-Day Ovum, Carnegie No. 8000, S42-217

The ovum is very shallowly implanted, although all the elements appear to be normal.

The theca lutein layer appears to be normal and contains several immature K cells. The granulosa lutein layer is moderately well vascularized, and although some lutein cells show early signs of atrophy, the majority appear to be functionally active. Distinctive K cells are rare, but little colloid degeneration is seen.

A 12.5-Day Ovum, Carnegie No. 290, S44-2785

Although this ovum is superficially implanted, it shows evidence of early villus formation. The germ disk has undergone a curious buckling and is still attached to the trophoblast. The latter is irregularly developed, poorest at the implantation pole and, paradoxically, good elsewhere.

The corpus luteum is in no way markedly different from that associated with Carnegie no. 8000.

Corpus Lutea associated with an Ovum consisting of Syncytiotrophoblast Only

A 12-Day Ovum, Carnegie No. 8329, S45-1809

This embryo is markedly abnormal in that there is no cytotrophoblast, no segmentation cavity, and no germ disk, but as a mass of syncytiotrophoblast it is fairly large.

Both lutein layers appear to be well vascularized and functionally active. K cells are numerous in certain parts of the section, but almost completely lacking in other areas. There is, however, no evidence of colloid degeneration of any of the K cells (fig. 31, pl. 6).

Corpus Lutea associated with an Ovum showing Hypoplastic rophoblast

Carnegie No. 7771, S40-791

This markedly abnormal ovum is associated with a 27-day late-secretory endometrium. The ovum is a polypoid structure; both elements of the trophoblast are present, but poor in quality and organization. The germ disk is totally lacking.

The corpus luteum is poorly vascularized. Both theca lutein and granulosa lutein cells appear to be moderately active, but not so much so as one would find in a normal pregnancy of this stage of development. A very striking feature is the complete colloid degeneration of all the K cells. Large amounts of colloid are present in all parts of the section, and no functional K cells are seen (fig. 32, pl. 6).

Carnegie No. 7800, S40-1327

This ovum is associated with an endometrium that shows early decidual reaction. The chorion is irregularly deficient, with marked hypoplasia of the trophoblast. To judge from the development of the embryo, the chorion should have early villi.

The theca lutein layer appears to be normal and functioning. The granulosa lutein layer is well vascularized. There is marked variation in size, regularity, and stainability of the nuclei of the granulosa lutein cells. The majority of the K cells have undergone more or less complete colloid degeneration, although this feature is not so conspicuous as in Carnegie no. 7771.

About 3 Months Pregnancy, Blighted Ovum, S48-824

This specimen was obtained with the corresponding left tube, left ovary, and uterus. Pregnancy was interrupted because of impending cardiac failure. The placenta was found to be immature and associated with a blighted ovum of about 3 months menstrual age. The cystic corpus luteum measured 1.5‘ cm. in diameter and contained straw-colored fluid. The lutein border was grayish yellow, and about 2 mm. thick.


Haematoxylin and eosin sections (fig. 33, pl. 6) show a corpus luteum that has lost all its distinctive morphologic features. The theca lutein layer is markedly reduced in bulk. Most of the nuclei are densely pycnotic. The granulosa lutein layer has lost all evidence of functional activity. Not a single active lutein cell can be found. In many, the nuclei are dense and pycnotic, and in others the nuclei have completely lost their capacity for staining. A large number of colloid-containing vacuoles are present. Cell boundaries are totally obliterated. Connective tissue invasion of both layers is marked. The capillaries of the granulosa lutein layer are collapsed.


Only an occasional cell of the theca lutein layer contains alkaline phosphatase. Nearly every cell in the granulosa lutein layer contains some of the enzyme, but not in significant amounts.


Inasmuch as the ovum was found to be blighted, and the placenta was immature, it is highly probable that this specimen does not represent a normal 3 months pregnancy. It is included to demonstrate that the degenerative changes in the Corpus luteum may parallel trophoblastic degeneration.

Unfortunately, no formalin-fixed material was available at the time this specimen came to our attention, and lipid studies could not be made.

Notes on the K Cells

Although this study was undertaken to outline the morphological changes that take place in the corpus luteum during early pregnancy, it became apparent quite early in the course of the investigation that the K cells described in all specimens discussed bear more than a casual relation to the cyclical activity of the gland, in the nonpregnant cycle as well as in early pregnancy.


These cells are conspicuous in routine sections because of their homogeneous, highly cosinophilic cytoplasm. It is also to be noted, however, that in sections treated with basic dyes, their cytoplasm is more markedly basophilic than that of neighboring lutein cells. This suggests strong acid properties of the protoplasm, a reaction characteristic of phospholipid. As previously stated, these cells are very striking in sections treated with Sudan black. They are uniformly sudanophilic, in contrast with the granular sudanophilia of the theca lutein and granulosa lutein cells. The sudanophilic material is soluble with difficulty in cold alcohol or alcohol-acetone mixtures. Traces of sudanophilia can be detected in these cells after 24 hours’ treatment of the section in lipid solvents before staining. By contrast, the granular deposits of lipid in theca lutein and granulosa lutein cells are completely dissolved by similarly pretreating the sections for 1/3 to 1 hour before staining with Sudan black. The low solubility of the sudanophilic substance in these K cells is characteristic of phospholipid.


Because of these indications of high phospholipid content in the K cells, the technique for the demonstration of phospholipid in tissues (Baker, 1946) was employed in two selected corpora lutea. Figure 34, plate 7, is a photomicrograph of a K cell, at a magnification of 750, in a :20-day corpus luteum of the normal menstrual cycle, showing a high concentration of phospholipid. The phospholipid is uniformly distributed in these cells, as contrasted with the lutein cells of both layers, where phospholipid appears as peripherally distributed granules. It was observed incidentally that in the developing follicle phospholipicl is restricted to the theca interna layer. Pretreatment of parallel sections with pyridine completely removed all traces of phospholipid from the gland.


All the specimens discussed in this paper were subjected to the tests for presumptive ketosteroids employed so effectively by Dempsey and Bassett (1943) and by McKay and Robinson (1947). Our findings in the developing follicle and corpus luteum of the nonpregnant cycle agree substantially with those of McKay and Robinson. It was noted, however, in the course of these studies that the K cells were reactive with the Schiff, phenylhydrazine, and LiebermanBurchardt reactions. This reactivity is manifested as a uniform, homogeneous color reaction, in contrast with the granular reactivity of theca lutein cells described by McKay and Robinson. As with Sudan black, this reactive material was more dillicult to dissolve in lipid solvents than that of the theca lutein cells. Observations of autofluorescence under ultraviolet light were equivocal. It is noteworthy, however, that birefringent crystals cannot be observed in these K cells under the polarizing microscope. In only one specimen, the 16-day normal pregnancy (Carnegie no. 8602), was a suggestion of crystalline birefringence seen. Up to this point, all the reactions of the K cells pointed to ketosteroid substances, but failure to demonstrate birefringent crystals in them seemed to rule out that possibility. It has subsequently been pointed out to us by Seligman (personal communication) that all the evidence presented above points to the fact that the high phospholipid content of these cells may prevent the crystallization of ketosteroid necessary for development of birefringence in plane-polarized light.


Very recently Seligman and Ashbel (I949) developed a technique for the demonstration of ketonic lipids. Figures 35 and 36, plate 7, are photomicrographs of sections treated by this technique. It will be noted that reactive material is concentrated in these cells. Seligman and Ashbel have observed that the corpora lutea of animals do not react to the tests for ketosteroid unless they are fixed in formalin, which unmasks the active carbonyl group. To rule out the possibility that the reactive material in these cells might be an aldehydic group, produced by hydrolysis of plasmogens, parallel sections were treated by a new technique for the demonstration of free aldehyde groups (Seligman and Ashbel, unpublished data). No free aldehydic groups could be demonstrated. Thus, all the evidence seems to indicate that these K cells represent the locus of high concentration of ketosteroid. Their reactivity as measured by these special histochemical techniques parallels closely the changes observed in sudanophilia during various stages of development of the corpus luteum.


As regards the question of origin and function of the K cells, it must be borne in mind that they could represent undifferentiated elements of the reticuloendothelial system, for example wandering macrophages, angioblasts, or young fibroblasts. It is possible that these cells arc histiocytic, and that the intense localization of ketonic lipid noted in them represents merely the phagocytosis of excess ketonic lipid. It is planned to perfuse a human ovary containing an active corpus luteum with a vital dye to determine whether these cells have the capacity to phagocytose foreign matter, utilizing the technique of ‘Nerthessen (1949). In the absence of further and more definitive studies, we believe that all the evidence presented regarding the peculiar characteristics of these cells points to their being a distinct cell line, intimately related in some manner to the function of the corpus luteum in the production or utilization of ketonic lipid, that is, ketosteroid compounds.

Discussion

Our observations of the corpus luteum of the nonpregnant cycle parallel substantially the classic description of Meyer (1911). Observations of vascular changes, coupled with information that can be deduced from histochemical procedures, lead us to agree with Brewer (1942) that the corpus luteum reaches its maximum activity on or about the 9th day after ovulation. ‘Ne have assumed the following criteria as evidence of functional activity of the corpus luteum: widely dilated capillaries in both theca and granulosa lutein layers, fine peripherally distributed sudanophilic substances in the lutein cells, demonstrable alkaline phosphatase in the cytoplasm, and ketonic lipid demonstrable by the techniques described above.


It has been generally held that the ovary is responsible for at least two hormones, both of which have experimentally proved specific actions in the menstrual cycle. Dempsey and Bassett (1943) working with rats, and McKay and Robinson (1947) in a study of human material, found that reactive material, presumptively ketosteroid, was localized exclusively in the theca interna of the developing follicle and appeared in insignificant amounts in the granulosa lutein cells during the active stages of the corpus luteum. Inasmuch as they could not demonstrate ketosteroid in significant amounts in the granulosa lutein layer, they suggested that the theca interna is probably responsible for the production of both progesterone and estrogenic substances. Our observations suggest that a ketosteroid substance is intensely localized in a specific line of cells having their origin in the theca interim and making their way into the granulosa lutein layer. It is possible that the hormone production of these cells in the unruptured follicle is responsible for the suggestive progestational changes that take place in the uterus of experimental animals at or shortly before ovulation (Reynolds and Friedman, I93o; Astwood, 1939).


We have no evidence that these cells are specifically concerned with the production of progesterone rather than estrogen. Nevertheless, the temptation to infer that such a relationship exists is strong. There is no valid reason why a given cell cannot produce more than one hormone, as those of the pituitary gland apparently do. As has been observed, these K cells show the greatest evidence of functional activity dur ing the stages when maintenance of the progestational state of the uterine endometrium is essential. In the normal menstrual cycle, if a fertilized ovum has not become implanted in the endometrium by 6 or 7 days after ovulation, that cycle ends in menstruation (Rock and Hertig, 1948). In such an event, the need for maintenance of the progestational type of endometrium no longer exists and the corpus luteum soon begins to regress. On the other hand, if pregnancy is superimposed, the earliest evidence of sustained and accelerated functional activity is seen in the recrudescence of activity in the K cells. It has been generally agreed that pregnancy will continue uninterrupted even though the corpus luteum is removed, when the placenta is producing suflicient progesterone to maintain the decidua. The exact time at which this transfer of function from corpus luteum to placenta takes place is not so well agreed upon. Dr. G. van S. Smith (unpublished data) states that the corpus luteum is essential through the 7th to the 8th, and possibly the 9th, week of pregnancy. No doubt there is considerable overlapping of hormone production by corpus luteum and placenta. Our observations suggest that the corpus luteum ceases to produce hormone completely by the 4th month of pregnancy, and there is evidence that functional activity decreases rather sharply between the 7th and 12th weeks of gestation.


Our studies indicate that the theca interna contributes substantially to the granulosa lutein layer in the human corpus luteum. However, the theca lutein and granulosa lutein cells described by all workers remain separate and distinct entities. The elements contributed to the granulosa lutein layer by the theca lutein layer are the K cells to which so much space has been given in this discussion.


These K cells in the granulosa lutein layer of the human corpus luteum have been observed by many earlier workers, as cited by Gillman and Stein (1941). These latter authors have interpreted them as representing different phases of functional activity of the granulosa lutein cells. It is highly probable that earlier work failed to elicit the true nature of these cells for three reasons: First, they had not been observed in the theca interna of the developing follicle; secondly, there had been no observation of migration of cellular elements from the theca interna into the granulosa lutein layer; and, thirdly, lipid stains such as Sudan black, which is capable of staining the phospholipid so characteristic of these cells, were not available.


The specific colloid degeneration which is peculiar to these K cells is interpreted as further evidence that they represent a distinct cell type. The fact that this colloid has the same histochemical properties as the K cells suggests that the colloid represents stored secretory products. Although colloid deposits are visible in old corpora lutea of the nonpregnant cycle, they are much more conspicuous in the degenerating corpus luteum of pregnancy, a fact which suggests both an increase in the number of cells producing the precursor of this product and an increased concentration of precolloid ketosteroid-phospholipid complexes within the cell.


As observed by Corner (19.18), in the human ovary alkaline phosphatase is restricted to the theca interna of the developing follicle and disappears from the theca lutein cells by the 4th or 5th post-ovulatory day. In contrast with Corner’s findings, we observed alkaline phosphatase in a few granulosa lutein cells, but this becomes evident only as the gland approaches the peak of functional activity, that is, at or about 8 days after ovulation. As stated earlier, we have observed alkaline phosphatase in the K cells as early as 6 days after rupture of the follicle. V-Vhen pregnancy occurs, alkaline phosphatase appears in increasingly higher concentrations in the K cells, and in gradually increasing amounts in the true granulosa lutein cells as the progress of pregnancy makes additional demands on the corpus luteum. There is some recrudescence of alkaline phosphatase in the theca lutein cells, most marked at 6 or 7 weeks menstrual age. The activity of the theca lutein layer during pregnancy, as measured by the concentration of this particular enzyme, is insignificant when compared with that of the granulosa lutein layer and the K cells.


The theca lutein layer undergoes marked hypertrophy during early pregnancy, attaining its maximum development about the 26th day of pregnancy, dating from ovulation. Early in pregnancy, the coarse lipid deposits characteristic of the menstrual corpus luteum are completely replaced by fine, granular lipid deposits which are indicative of functional activity. After the 26th day of pregnancy the theca lutein layer becomes less and less prominent, until at 4 months no trace can be found except a few small pycnotic nuclei. That the theca interna may continue to supply K cells is suggested by the fact that recrudescence of K cells in the theca lutein layer is observed in both the corpora lutea associated with 26-day pregnancies.


Another very important question that remains to be elucidated is the role of the granulosa lutein cell. The fact that alkaline phosphatase concentration in the granulosa lutein cell increases during that period of pregnancy when greatest demands are being made upon the gland suggests that the granulosa lutein Cell plays more than a passive role. It is possible that the short 3-hour period of incubation in the glycerophosphate medium employed in this study was insufficient to reveal low concentrations of alkaline phosphatase in the granulosa lutein cells of the nonpregnant corpus luteum. Here, again, it is a tempting suggestion that there may be a direct relation between alkaline phosphatase in the granulosa lutein cell and the demonstrable phospholipid-ketosteroid matrix of the K cells.


As regards the corpora lutea associated with abnormal ova, it is interesting to note that the corpora lutea associated with preimplantation ova are normal in all respects and comparable with those of the normal menstrual cycle or normal pregnancy of identical chronological age.


Careful examination of the corpora lutea associated with implanted but abnormal ova discloses that there is an almost direct relation between the amount of trophoblast present and the integrity of the corpus luteum. In those ova which are almost completely devoid of trophoblast (Carnegie nos. 7771, 7800), the corpus luteum is poorly vascularizcd and does not manifest the heightened functional activity expected at this stage of development. The most conspicuous feature of these corpora lutea, however, is the total and uniform colloid degeneration of all K cells.


On the other hand, Carnegie no. 8329 is a markedly abnormal ovum consisting only of syncytiotrophoblast. K cells are numerous in this corpus luteum, and no evidence of colloid degeneration is to be noted. The specimens associated with abnormal ova showing only moderate hypoplasia of the trophoblast appear to be good but not perfect corpora lutea; that is, peripheral vacuolization and vascularity are moderately deficient.


These observations suggest that a normal trophoblast is essential to the maintenance of the corpus luteum, the functional integrity of which, in turn, is responsible for maintaining normal decidua during the early weeks of pregnancy.

Summary and Conclusions

1. The morphological and histochemical changes have been studied in a total of 89 human corpora lutea. Forty-eight of these represent corpora lutea of the normal menstrual cycle, every day from ovulation to menstruation being represented. Twenty-eight corpora lutea of normal pregnancies ranging from the 2-cell egg to 4%, months, and 13 corpora lutea associated with abnormal ova were studied. An attempt has been made to correlate the changes in histochemical reactivity in the various components of the corpus luteum with the anatomical evidences of function.

2. As the mature Graafian follicle nears ovulation, a number of distinctive cells, not previously described, become conspicuous. At or shortly after the time of ovulation, these cells appear in large numbers in the granulosa lutein layer, attaining that position by their own motility or being carried in as the theca interna and its accompanying blood vessels invaginate into the collapsed membrana granulosa.

3. Evidence is presented that these cells constitute a distinct cell type and represent the site of intense localization of ketonic lipid, if not the site of production or utilization of ketonic lipids or steroids.

4. Until the time of implantation of the ovum in the endometrium, no difference can be noted between chronologically similar corpora lutea of the normal menstrual cycle and those associated with normal or abnormal ova.

5. After implantation, at or about 6 or 7 days after ovulation, the corpus luteum does not undergo regression but is stimulated to increasingly higher levels of functional activity until 6 weeks of menstrual age or later, after which the function of the corpus luteum is gradually taken over by the placenta.

6. It is quite apparent that when the implanted ovum is deficient in trophoblastic development, the corpus luteum undergoes early regression. The most striking feature of this failure of the corpus luteum is the uniform colloid degeneration of the K cells. The worse the ovum as regards the development of the trophoblast, the more complete is the colloid degeneration of the K cells in the associated corpus luteum.

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Plates

Plate 1

gr-., granulosa; t/2. z'nt., theca interna; K, K cells

Fig. 1. Section of the wall of a mature human Graafian follicle, X300, Haematoxylin and eosin. Prominent, small dark nuclei of K cells stand out conspicuously in the theca interna.

Fig. 2. Corpus luteum of 16th day of cycle, estimated age 2 days, ><30o, haematoxylin and eosin. The thcca interna is considerably thinned. The granulosa is poorly organized. Prominent K cells can be seen penetrating into the granulosa.

Fig. 3. Same specimen as figure 2, X 300, Sudan black. Sudanophilic substances present in much higher concen-

tration in theca interna than in granulosa.

Fig. 4. Same specimen as figure 2, X400, alkaline phosphatase. Alkaline phosphatase is restricted to tbeea intcrna and enclotbelium of blood vessels.

Fig. 5. Corpus luteum of zoth day of cycle, estimated age 6 days, X300, haematoxylin and eosin. The theca interna is not prominent. The granulosa is compact and well vascularized. K cells are conspicuous because of their small, hyperchromatic nuclei and dense, homo- geneous cytoplasm.

Fig. 6. Same specimen as figure 5, X 3oo, Sudan black. The section does not include theca interna. Note periph- eral distribution of sudanophilic substances in granulosa cells and uniform sudanophilia of attenuated K cells.

Plate 2

Fig. 7. Corpus luteum of 23d day of cycle. estimated age 9 days, X300, haematoxylin and eosin. The K. cells are numerous and markedly hypertrophied. Peripheral vacuolization of granulosa lutein cells is marked.

Fig. 8. Same specimen as figure 7, X goo. Sudan hlack. A small segment of theca interna is present in upper part of figure. Note coarse droplets of lipid in cells of theca interna as contrasted with the line droplets in the granulosa lutein cells. K cells are numerous.

Fig. 9. Same specimen as figure 7. X300. alkaline phosphatase. Only an occasional cell of the theca interna contains the enzyme. ()ne K cell, with extensive cyto- plasmic processes. contains the eiizyine in high concen- tration. An occasional granulosa cell contains alltaline phosphatase in moderate amounts.

Fig. 10. Corpus luteum of early menstruation. esti- mated age 15-16 days, ><3oo_. haematoxylin and eosin. The theca interna is almost completely atrophic. K cells in various stages of degeneration are to he noted in granu- losa layer. I’eripheral vacuoli:/.ation of granulosa cells is not marked.

Fig. 11. Same specimen as ligure Io. ><3oo, Sudan black. Nearly all of the theea interna cells contain only coarse lipid droplets. Iiarly fatty degeneration of some of the granulosa cells is evident. The K cells are some- what contracted and show some granular sud:mophilic deposits.

Fig. 12. Same specimen as ligure Io, ><_ioo, alkaline phosphatase. ()nly a few scattered cells in the granulosa lutein layer contain demonstrable amounts of the en- zyme.

Plate 3

gl'., granulosa; t/1. int., theca interna: K, K cells

Fig. 13. Corpus luteum of 16- to 17-day normal preg- nancy. Carnegie no. 8602, X 300. l1:lL'l11:1I()X}'lll‘l and eosin. Note indistinct cell boundaries of granulosa lutein cells and numerous K cells.

liim. it). Same specimen as ligure 18, X300. Sudan lilaek. Large amounts of finely distributed lipid are present in lioth granulosa and theca lutein cells. K. cells are numerous. but appear frayed and more irregularly stellate than in ligure 16. plate 3,.

Fig. 20. Same specimen as l‘igure 18. ‘.><.ioo. alkaline phosphatase. The theta interna is uniformly devoid of the enzyme, whereas nearly every cell in the granulosa lutein layer contains alkaline phosphatase.

Fig. 21. Corpus luteum of 2(i-clay normal pre_J,it;nic)~‘. ><.3uo. ltaeinato.\yli11 and eosin. The theca interna is markedly hypertrophied as compared with figure ill. K cells are numerous and apparently very active in the granulosa la}-‘er.

Fig. 22. Same specimen as ligtire 2|, )-(goo, Sudan hlack.

\'lt'lt.'(.l lipitl. Of special interest is the reappearance of a

Roth layers contain large amounts of rinel_\} di-

numlier of small. uniformly sudanophilie Ii cells in the theea interna.

Fig. 23. Same specimen as ligtire 2|. ><_;oo. alkaline phosphatase. The C11’/.}'l1‘IL’ is not demonstrable in the theca interna. hut is tlilltisely present in the entire granu- losa layer.

Plate 7

gr., granulosa; :11. :'m., theca interna; K, K cells

Fig. 24. Corpus luteum of zoth day of cycle, estimated age 6 days, X 750, Bakcr’s phospholipid technique. Note the high, uniform concentration of phospholipid in the spindle-shaped K cell. Red blood cells in a near-by capillary also contain reactive material.

Fig. 25. Corpus luteum of the 23d day of the non- pregnant menstrual cycle, estimated age 9 days, X300, Seligman-Ashbel technique for demonstration of active carbonyl groups. Note reactive material in K cells in high concentration, and in lutein cells of both layers in lesser amounts.

FIG. 26. Corpus luteum of early menstruation, same specimen as figure 11, plate 2, X300, Seligman-Ashbel technique for demonstration of active carbonyl groups. Note diminution in reactive material in both layers. K cells are less numerous, their processes retracted as in figure 11, :1 Sudan black preparation of this same specimen.



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