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

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CONTRIBUTIONS TO EMBRYOLOGY, NO. 224
 
CONTRIBUTIONS TO EMBRYOLOGY, NO. 224
  
I —-»
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I —-»
  
histological and histochemical observations on the corpus
+
histological and histochemical observations on the corpus luteum of human pregnancy with special reference to corpora lutea associated with early normal and abnormal ova
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
 
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,
+
Free’ I-Io.s'pz'ral for I-Vomcw, Brool(1inc,' Dcpmwnxcvzts of Pathology, O[25tctric5, and Gynecology, Hm'z2ard Medical School
O[25tctric5, and Gynecology, Hm'z2ard Medical School
 
  
With scvcn plates
+
With scvcn plates CONTENTS PAGE REVIEW 01-‘ THE LITIERATURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 57
CONTENTS
 
PAGE
 
REVIEW 01-‘ THE LITIERATURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 57
 
  
 
NI.-XTIZRIAL AND M15'r1101)s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
 
NI.-XTIZRIAL AND M15'r1101)s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Line 105: Line 99:
 
St:.\11~.1A1ur AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 73
 
St:.\11~.1A1ur AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 73
  
LITER.-‘.TL'RIi CITED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
+
LITER.-‘.TL'RIi CITED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 HISTOLOGICAL AND HISTOCHEMICAL OBSERVATIONS ON THE CORPUS LUTEUM OF HUMAN PREGNANCY
HISTOLOGICAL AND HISTOCHEMICAL OBSERVATIONS ON THE CORPUS
 
LUTEUM OF HUMAN PREGNANCY
 
  
WITH SPECIAL REFERENCE TO CORPORA LUTEA ASSOCIATED
+
WITH SPECIAL REFERENCE TO CORPORA LUTEA ASSOCIATED WITH EARLY NORMAL AND ABNORMAL OVA
WITH EARLY NORMAL AND ABNORMAL OVA
 
  
 
REVIE\V OF THE LITERATURE
 
REVIE\V OF THE LITERATURE
  
Speculation and investigation concerning the role
+
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).
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
+
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.
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
+
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.
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
 
57
  
As the corpus luteum matures and attains the socalled blossom stage, the cells of the theca interna are
+
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.
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
+
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 58 CORPUS LUTEUM OF HUMAN PREGNANCY
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
+
the corpus luteum commences to regress about 8 to 10 days after ovulation.
10 days after ovulation.
 
  
Gillman and Stein (1941) tabulate the number of
+
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.
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
+
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.
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
+
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.
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
+
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.
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
+
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
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
+
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.
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
+
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).
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
+
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 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
+
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.
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
+
McKay and Robinson (1947) studied a series of human corpora lutea of the normal menstrual cycle, employing some of the newer techniques for detecting
human corpora lutea of the normal menstrual cycle,
 
employing some of the newer techniques for detecting
 
  
presumptive ketosteroid compounds. The presence
+
presumptive ketosteroid compounds. The presence CORPUS LUTEUM OF HUMAN PREGNANCY 59
CORPUS LUTEUM OF HUMAN PREGNANCY 59
 
  
of birefringent crystals, autofluorescent materials,
+
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.
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
+
E.\'amination 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
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
+
in the granulosa lutein layer from the 22d day through late menstruation.
late menstruation.
 
  
Studies of autofluorescent substances and substances
+
Studies of autofluorescent substances and substances that reacted with phenylhydrazine and sulfuric acid essentially paralleled the observations made on birefringence.
that reacted with phenylhydrazine and sulfuric acid
 
essentially paralleled the observations made on birefringence.
 
  
McKay and Robinson (1947) also noted in the
+
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.
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
+
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.
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
+
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/;
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
+
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. 60 CORPUS LUTEUM OF HUMAN PREGNANCY
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
 
MATERIAL AND Ml:ZTI-IODS
  
Since 1939 two of the authors (I-Iertig and Rock)
+
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.
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
 
Nine corpora lutea associated with older pregnancies
  
25 days to 41/; months) were also obtained from
+
25 days to 41/; months) were also obtained from surgical material from the Free Hospital for \-‘Vomen (Brookline, Massachusetts) or the Boston Lying-In Hospital.
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
+
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.
histochemical changes in the gland during the normal
 
menstrual cycle with those observed in pregnancy.
 
  
All corpora lutea of nonpregnant cycles and those
+
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 I-Iertig, 1944).
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
+
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
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
+
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.
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
+
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).
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
+
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.
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
+
Two specimens were treated for the histochemieal demonstration of phospholipid according to the
demonstration of phospholipid according to the
 
  
method of Baker (1946).
+
method of Baker (1946). CORPUS LUTEUM OF HUMAN PREGNANCY 61
CORPUS LUTEUM OF HUMAN PREGNANCY 61
 
  
OBSERVATIONS ON CORPORA LUTEA OF THE NONPREGNANT
+
OBSERVATIONS ON CORPORA LUTEA OF THE NONPREGNANT MENSTRUAL CYCLE
MENSTRUAL CYCLE
 
  
 
T/ze Mature Grcmfirm Follicle, S48-731
 
T/ze Mature Grcmfirm Follicle, S48-731
  
Figure 1, plate I, is a photomicrograph of a typical
+
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.”
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
+
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
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.
 
in the membrana granulosa at this stage of development.
  
Corpus Luteum of 16x}: Day of Cycle, Estimatca’ Age
+
Corpus Luteum of 16x}: Day of Cycle, Estimatca’ Age 2 Days, S48-2636
2 Days, S48-2636
 
  
Macroscopically, this corpus luteum appeared as a
+
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.
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)
+
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.
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
+
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.
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
+
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, 62 CORPUS LUTEUM OF HUMAN PREGNANCY
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
+
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.
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
+
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).
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
+
Corpus Lzztezmz of zot/2 Day of Cycle, Estinzaterl Age 6 Days, S48-2262
6 Days, S48-2262
 
  
On cut section, the corpus luteum measured 1.7 by
+
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.
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.
+
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
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.
 
red blood cell can be noted in them.
  
thus is undergoing early organization. No capillaries
+
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.
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
+
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
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
+
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.
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
+
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.
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
+
Corpus Lmfczmz of 23a? Day of Cycle, Estinzazerl Age 9 Days, S48-3028
9 Days, S48-3028
 
  
The corpus luteum measured 1.5 cm. in its greatest
+
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.
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
+
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.
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
+
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.
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
+
Only an occasional theca lutein cell contains alkaline phosphatase (fig. 9, pl. 2). The true granulosa CORPUS LUTEUM OF HUMAN PREGNANCY 63
CORPUS LUTEUM OF HUMAN PREGNANCY 63
 
  
lutein cells are devoid of this enzyme. A number of
+
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.
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
 
Franz 24:/2 Day of Cycle to M emtmation
  
Between the 23d day of the cycle and the onset of
+
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.
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
+
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.
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
 
Corpus Lzztermz during Early Memtrrzrztion, S48-535
  
At this stage the theca interna is very indistinct.
+
At this stage the theca interna is very indistinct. True theca lutein cells are found only in scattered
True theca lutein cells are found only in scattered
 
  
clumps, chiefly in the connective-tissue septa that
+
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.
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
+
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.
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
+
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
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
 
OBSERVATIONS ON CORPORA LUTEA OF NORMAL PREGNANCY
Line 736: Line 253:
 
A 2-03}! Egg, 17-Day Corpus Luream, 349-2439
 
A 2-03}! Egg, 17-Day Corpus Luream, 349-2439
  
This ovum was recovered from the middle third
+
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.
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
+
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.
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
+
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.
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 413-Day Blm't0c_v5t, 19-Day Corpus Lzrteunz, 548-5000
548-5000
 
  
This specimen is the first normal human blastocyst
+
This specimen is the first normal human blastocyst yet recovered from the uterine cavity. The associated endometrium is typical 19-day secretory endometrium.
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.
+
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.
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
+
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.
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
 
7‘/3- and 913-Day Pregrzazzcies
  
Figures 13 and 14, plate 3, are photomicrographs
+
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
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
+
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 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).
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,
+
A 12- to I 3-Day Pregnancy, Carnegie N 0. 8558, S46-2767
S46-2767
 
  
The corpus luteum was moderately cystic and
+
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.
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
+
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.
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
+
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. CORPUS LUTEUM OF HUMAN PREGNANCY 65
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
+
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).
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
+
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).
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
 
A 16-Day Pregnancy, Carnegie No. 8602, S48-2088
  
The cystic corpus luteum measured 3 cm. in its
+
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.
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
+
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.
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
+
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.
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
+
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
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
+
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.
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:
 
A 26-Day Pregnancy, S48-263:
  
This specimen was obtained with the uterus at time
+
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.
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
+
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.
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
+
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.
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
+
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. 66 CORPUS LUTEUM OF HUMAN PREGNANCY
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
+
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.
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
+
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.
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
+
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.
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
 
A 28- to 35-Day Preg.rzm1c_v, S48-4854
  
On gross examination the corpus luteum measured
+
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 organizecl.
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,
+
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.
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
+
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,
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
+
showing a clearly defined colloid globule. Other K cells in various stages of degeneration are present in this section.
K cells in various stages of degeneration are present
 
in this section.
 
  
The Sudan black preparations show large numbers
+
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.
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
+
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.
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
 
/I 4- to 4‘/_-;_.-Mont/z Pregrzrmcy, 548-2624
  
This specimen was obtained incidentally to a total
+
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.
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
+
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 CORPUS LUTEUM OF HUMAN PREGNANCY 67
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
+
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.
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
+
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
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
+
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.
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
 
OBSERVATIONS ON CORPORA LUTEA ASSOCIATED VVITH ABNORMAL OVA
  
In view of the fact that our material included 13
+
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).
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
+
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.
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.
+
CORPORA I_.U’l'I-IA ASSOCIATED wrri-1 .A13:~.'-orm.-xi. FREE-LYING Ova
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),
+
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.
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
 
A 5-Cell Egg, I9-Day Corpus Lute:-:m, S48-3948
  
The corpus luteum measured 2.0 by 1.8 cm. in its
+
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.
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,
+
The ovum consisted of five abnormal blastomeres, several of which were multinucleated and showed
several of which were multinucleated and showed
 
  
other evidences of degeneration and retardation of
+
other evidences of degeneration and retardation of development.
development.
 
  
The corpus luteum differs in no respect from a
+
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.
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,
+
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.
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
+
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.
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
+
CORPORA LUTEA Assocuman WITH ABNoR:\r;tL Ova w1TH ADEQUATE TROPHOBLAST
w1TH ADEQUATE TROPHOBLAST
 
  
 
An 8-Day Ozmm, Carnegie No. 8370, S46-676
 
An 8-Day Ozmm, Carnegie No. 8370, S46-676
  
The ovum is the youngest implanted embryo of
+
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 ade68 CORPUS LUTEUM OF HUMAN PREGNANCY
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
+
quate but poorly organized, that is, laminated instead of being concentrically arranged with the syncytiotrophoblast surrounding the cytotrophoblast.
of being concentrically arranged with the syncytiotrophoblast surrounding the cytotrophoblast.
 
  
The corpus luteum appears to be normal in all
+
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.
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
 
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
+
This ovum is not markedly defective. The trophoblast is moderately hypoplastic and the germ disk is maloriented.
is maloriented.
 
  
The theca interna appears to be normal in all
+
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.
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
 
An Ir-Day Ozmm, Carnegie N 0. 8299, 545-1220
  
The trophoblast appears to be normal although
+
The trophoblast appears to be normal although somewhat poorly organized. The germ disk is markedly maloriented.
somewhat poorly organized. The germ disk is
 
markedly maloriented.
 
  
The corpus luteum is very large and consists of a
+
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
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).
 
very active corpus luteum (fig. 30, pl. 6).
Line 1,144: Line 389:
 
Carnegie N0. 7850, 540-2699
 
Carnegie N0. 7850, 540-2699
  
This ovum is associated with an endometrium that
+
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.
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
+
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
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.
 
evidence of colloid degeneration is to be noted anywhere in the section.
  
CORPORA Luri-:.-i ASSOCIATED wrrn ABNORMAL,
+
CORPORA Luri-:.-i ASSOCIATED wrrn ABNORMAL, SHaLLowi.v I.\tPL.~xN*rEn Ova
SHaLLowi.v I.\tPL.~xN*rEn Ova
 
  
 
Ar: 11- to I2-Day Ourmz, Carnegie N0. 8000, S42-217
 
Ar: 11- to I2-Day Ourmz, Carnegie N0. 8000, S42-217
  
The ovum is very shallowly implanted, although all
+
The ovum is very shallowly implanted, although all the elements appear to be normal.
the elements appear to be normal.
 
  
The theca lutein layer appears to be normal and
+
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.
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
 
A I21/3-Day Oz/um, Carnegie No. 290, 844-2785
  
Although this ovum is superficially implanted, it
+
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.
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
+
The corpus luteum is in no way markedly different from that associated with Carnegie no. 8000.
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
 
A Conpus LU'n~:U.\t Assoenvrsn w1TH .»'tN OVUM CoNSISTING or Si'Nev'r1oTRopi-ioBL.-xsT ONLY
Line 1,186: Line 413:
 
A 12-Day Oz/um, Cczrnegic No. 8329, S45-1809
 
A 12-Day Oz/um, Cczrnegic No. 8329, S45-1809
  
This embryo is markedly abnormal in that there
+
This embryo is markedly abnormal in that there is no cytotrophoblast, no segmentation cavity, and no germ disk, but as a mass of syncytiotrophohlast it is fairly large.
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
+
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
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,
+
of colloid degeneration of any of the K cells (fig. 31, pl. 6).
pl. 6).
 
  
Coupons. Lure.-x Assoel.-cren wrrt-I OVA SHOWING
+
Coupons. Lure.-x Assoel.-cren wrrt-I OVA SHOWING HvPoi>1..»xs'r1e TROI’liUBL.-\S'l‘
HvPoi>1..»xs'r1e TROI’liUBL.-\S'l‘
 
  
 
Carncgz'e No. 7771, S4079!
 
Carncgz'e No. 7771, S4079!
  
This markedly abnormal ovum is associated with a
+
This markedly abnormal ovum is associated with a 27-day late-secretory endometrium. The ovum is a CORPUS LUTEUM OF HUMAN PREGNANCY 69
27-day late-secretory endometrium. The ovum is a
 
CORPUS LUTEUM OF HUMAN PREGNANCY 69
 
  
polypoid structure; both elements of the trophoblast
+
polypoid structure; both elements of the trophoblast are present, but poor in quality and organization. The germ disk is totally lacking.
are present, but poor in quality and organization.
 
The germ disk is totally lacking.
 
  
The corpus luteum is poorly vascularized. Both
+
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).
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
 
Carnegie N0. 7800, 540-1327
  
This ovum is associated with an endometrium that
+
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.
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
+
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.
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
 
About 3 Mont/is Pregnancy, Blig/ztcd Ouum, S48-824
  
This specimen was obtained with the corresponding
+
This specimen was obtained with the corresponding left tube, left ovary, and uterus. Pregnancy was interrupted because of impending cardiac failure. The
left tube, left ovary, and uterus. Pregnancy was
 
interrupted because of impending cardiac failure. The
 
  
placenta was found to be immature and associated
+
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.
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)
+
I-Iaematoxylin 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. Connectivetissue invasion of both layers is marked. The capillaries of the granulosa lutein layer are collapsed.
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
+
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.
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,
+
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.
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,
+
Unfortunately, no formalin-fixed material was available at the time this specimen came to our attention, and lipid studies could not be made.
and lipid studies could not be made.
 
  
 
NOTES ON THE K CELLS
 
NOTES ON THE K CELLS
  
Although this study was undertaken to outline the
+
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.
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
+
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
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.
+
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 70 CORPUS LUTEUM OF HUMAN PREGNANCY
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
 
70 CORPUS LUTEUM OF HUMAN PREGNANCY
 
  
 
substance in these K cells is characteristic of phospholipid.
 
substance in these K cells is characteristic of phospholipid.
  
Because of these indications of high 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.
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)
+
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
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
+
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.
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
+
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.
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
+
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
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.
+
ketonic lipid, that is, ketosteroid compounds. CORPUS LUTEUM OF HUMAN PREGNANCY 71
CORPUS LUTEUM OF HUMAN PREGNANCY 71
 
  
 
DISCUSSION
 
DISCUSSION
  
Our observations of the corpus luteum of the nonpregnant cycle parallel substantially the classic description of Meyer (1911). Observations of vascular
+
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.
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
+
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).
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).
 
  
VVe have no evidence that these cells are specifically
+
VVe 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.
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
+
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.
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
+
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 72 CORPUS LUTEUM OF HUMAN PREGNANCY
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
 
72 CORPUS LUTEUM OF HUMAN PREGNANCY
 
  
elements from the theca interna into the granulosa
+
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.
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
+
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.
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
+
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.
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,
+
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
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
+
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.
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
+
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.
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
+
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.
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
+
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.
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
+
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.
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 trophoCORPUS LUTEUM OF HUMAN PREGNANCY 73
 
These observations suggest that a normal trophoCORPUS LUTEUM OF HUMAN PREGNANCY 73
  
blast is essential to the maintenance of the corpus
+
blast is essential to the maintenance of the corpus luteum, the functional integrity of which, in turn,
luteum, the functional integrity of which, in turn,
 
  
is responsible for maintaining normal decidua during
+
is responsible for maintaining normal decidua during the early weeks of pregnancy.
the early weeks of pregnancy.
 
  
 
SUMMARY AND CONCLUSIONS
 
SUMMARY AND CONCLUSIONS
  
1. The morphological and histochemical changes
+
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.
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,
+
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.
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
+
3. Evidence is presented that these cells constitute a distinct cell type and represent the site of intense
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.
 
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
+
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.
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
+
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.
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
+
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.
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.
 
  
 
LITERATURE CITED
 
LITERATURE CITED
  
Asor.1.1., S. A. 1928. The growth and function of the corpus
+
Asor.1.1., S. A. 1928. The growth and function of the corpus luteum. Physiol. Rev., vol. 8, pp. 313-341.
luteum. Physiol. Rev., vol. 8, pp. 313-341.
+
 
 +
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.
  
As:-1m«;1., R., and A. M. St-:1.1o.\1.-m. 1949. A new reagent for
+
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.
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.
+
BAKER, I. R. 1946. The histochemical recognition of lipine. Quart. Iour. Micr. Sci., vol. 87, pp. 441-470.
Physiol., vol. 126, pp. 162-170.
 
  
BAKER, I. R. 1946. The histochemical recognition of lipine.
+
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 .
Quart. Iour. Micr. Sci., vol. 87, pp. 441-470.
 
  
BENNETT, H. S. 1940. The life history and secretion of the
+
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.
cells of the adrenal cortex of the cat. Amer. Iour. Anat.,
 
vol. 67, pp. 151-22 .
 
  
BREWER, J. I. 1942. Studies of the human corpus luteum.
+
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.)
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
+
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.
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
+
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.
it is in swine. Carnegie Inst. VVash. Pub. 222, Contrib.
 
to Embryol., vol. 2, pp. 69-94.
 
  
1948. Alkaline phosphatase in the ovarian follicle
+
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 in the corpus luteum. Carnegie Inst. Wash. Pub.
 
575, Contrib. to Embr}/'01., vol. 32, pp. 1-8.
 
  
DALTON, A. L, E. R. M1Tct1ELL, B. F. Joints, and V. B. Perms.
+
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.
1944. Changes in adrenal glands of rats following exposure to lowered oxygen tension. Iour. Natl. Cancer
 
Inst., vol. 4, pp. 527-536.
 
  
D1—:1.es1'ar_, M. 1910. Recherches sur le follicule de Graaf et
+
D1-:1~.11>s1-:v, E. ‘W. 1948. The chemical cytology of endocrine glands. Recent Prog. Hormone Res., vol. 3, pp. 127-157.
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
+
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 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!
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,
 
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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CONTRIBUTIONS TO EMBRYOLOGY, NO. 224

I —-»

histological and histochemical observations on the corpus 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, 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

O1ss1»:1tv.-\.'r1oNs 01¢ CORPORA LUTEA 01= THE NONPREGN:\NT i\"lEI\'STRU.~\L C1'c1.1=. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 61

The .\«'Iature Graafian Follicle, S48-731 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ . . . . . . . . . . . . . . . . . 61

Corpus Luteum of 16th Day of Cycle, Estimated Age 2 Days, S48-2636 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Corpus Lutcum of 20th Day of Cycle, Estimated Age 6 Days, S48-2262 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

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

From 24th Day of Cycle to Menstruation . . . . . . . . . . . . _ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

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

01ss1=.1w;\'r1o.\*s ON CORPORA LUTI-EA or NORM.-11. PREGNANCY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

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

A .va,%_-Day Blastocyst, 19-Day Corpus Lutcum, S48-5000 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.;

71/;;- and 9‘/2-Day Pregnancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 6.}

A 12- to 13-Day Pregnancy, Carnegie No. 8558, 846-2767 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 6.}

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

A 26-Day Pregnancy, S48-2631 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

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

A 4- to 4‘/2-Montli Pregnancy, S48-2624 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . 66

OBSliRVATI()NS ON CORPORA LU'r13.~\ Assocmrizn wm-1 ABNORMAL Om . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Corpora Lutea Associated with Abnormal Free-Lying Ova _ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 7

A 5-Cell Egg, 19-Day Corpus Lutcum, S48-39.-1,8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Corpora Lutea Associated with Abnormal Ova with Adequate Trophoblast . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

An 8-Day Ovum, Carnegie N0. 8370, 846-676 . . . . . _ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

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

An 11-Day Ovum, Carnegie N0. 8299, S45-1220 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Carnegie N0. 7850, S40-2699 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 68

Corpora Lutea Associated with Abnormal, Shallowly Implanted Ova . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 68

An 11- to 12-Day Ovum, Carnegie N0. 8000, S42-217 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

A 12‘/3-Day Ovurn, Carnegie No. 8290, S44-2785 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

A Corpus Lutcum Associated with an Ovum Consisting of Syncytiotrophoblast Only . . . . . . . . . . . . . . . . . . . . 68

A 12-Day Ovum, Carnegie N0. 8329, 845-1809 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Corpora Lutea Associated with Ova Showing Hypoplastic Trophoblast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 68

Carnegie N0. 7771, 840-791 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ., 68

Carnegie No. 7800, 840-1327 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 69

About 3 Months Pregnancy, Blighted Ovum, S48-82.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

NOTES 031 THE K CELLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

St:.\11~.1A1ur AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 73

LITER.-‘.TL'RIi CITED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 HISTOLOGICAL AND HISTOCHEMICAL OBSERVATIONS ON THE CORPUS LUTEUM OF HUMAN PREGNANCY

WITH SPECIAL REFERENCE TO CORPORA LUTEA ASSOCIATED WITH EARLY NORMAL AND ABNORMAL OVA

REVIE\V 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.

57

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 58 CORPUS LUTEUM OF HUMAN PREGNANCY

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 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 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 (I-Iertig and Rock, 1949b), to that associated with a 41/;

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. 60 CORPUS LUTEUM OF HUMAN PREGNANCY

MATERIAL AND Ml:ZTI-IODS

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 41/; months) were also obtained from 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 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 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). CORPUS LUTEUM OF HUMAN PREGNANCY 61

OBSERVATIONS ON CORPORA LUTEA OF THE NONPREGNANT MENSTRUAL CYCLE

T/ze 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 16x}: Day of Cycle, Estimatca’ 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, 62 CORPUS LUTEUM OF HUMAN PREGNANCY

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 Lzztezmz of zot/2 Day of Cycle, Estinzaterl 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 Lmfczmz of 23a? Day of Cycle, Estinzazerl 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 CORPUS LUTEUM OF HUMAN PREGNANCY 63

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.

Franz 24:/2 Day of Cycle to M emtmation

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 Lzztermz during Early Memtrrzrztion, 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). 64 CORPUS LUTEUM OF HUMAN PREGNANCY

OBSERVATIONS ON CORPORA LUTEA OF NORMAL PREGNANCY

A 2-03}! Egg, 17-Day Corpus Luream, 349-2439

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 413-Day Blm't0c_v5t, 19-Day Corpus Lzrteunz, 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‘/3- and 913-Day Pregrzazzcies

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 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, 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. CORPUS LUTEUM OF HUMAN PREGNANCY 65

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. 66 CORPUS LUTEUM OF HUMAN PREGNANCY

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 Preg.rzm1c_v, 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 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 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.

/I 4- to 4‘/_-;_.-Mont/z Pregrzrmcy, 548-2624

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 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 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

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 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), 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 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 Assocuman WITH ABNoR:\r;tL Ova w1TH ADEQUATE TROPHOBLAST

An 8-Day Ozmm, 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 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 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 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

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 N0. 7850, 540-2699

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.

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

Ar: 11- to I2-Day Ourmz, Carnegie N0. 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 I21/3-Day Oz/um, Carnegie No. 290, 844-2785

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.

A Conpus LU'n~:U.\t Assoenvrsn w1TH .»'tN OVUM CoNSISTING or Si'Nev'r1oTRopi-ioBL.-xsT ONLY

A 12-Day Oz/um, Cczrnegic 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 syncytiotrophohlast 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).

Coupons. Lure.-x Assoel.-cren wrrt-I OVA SHOWING HvPoi>1..»xs'r1e TROI’liUBL.-\S'l‘

Carncgz'e No. 7771, S4079!

This markedly abnormal ovum is associated with a 27-day late-secretory endometrium. The ovum is a CORPUS LUTEUM OF HUMAN PREGNANCY 69

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 N0. 7800, 540-1327

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 Mont/is Pregnancy, Blig/ztcd Ouum, 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.

I-Iaematoxylin 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. 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 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 70 CORPUS LUTEUM OF HUMAN PREGNANCY

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. CORPUS LUTEUM OF HUMAN PREGNANCY 71

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).

VVe 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 72 CORPUS LUTEUM OF HUMAN PREGNANCY

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 trophoCORPUS LUTEUM OF HUMAN PREGNANCY 73

blast 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.

LITERATURE CITED

Asor.1.1., S. A. 1928. The growth and function of the corpus luteum. Physiol. Rev., vol. 8, pp. 313-341.

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.

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.

BAKER, I. R. 1946. The histochemical recognition of lipine. Quart. Iour. Micr. Sci., vol. 87, pp. 441-470.

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 .

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.

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.)

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.

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.

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.

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 during the reproductive cycle. Endocrinology, vol. 33, 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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