REPRODUCTIVE ENDOCRINOLOGY
The human corpus luteum: life cycle and function
in natural cycles
Luigi Devoto, M.D.,a Ariel Fuentes, M.D.,a Paulina Kohen, B.S.,a Pablo C
espedes, M.D.,a
a
a
a
Alberto Palomino, M.D., Ricardo Pommer, M.D., Alex Mu~
noz, B.S.,
and Jerome F. Strauss, III, M.D., Ph.D.b
a
Instituto de Investigaciones Materno Infantil, Departamento de Obstetricia y Ginecologıa, Hospital Clınico San Borja-Arriaran,
Facultad de Medicina, Universidad de Chile, Santiago, Chile, and b Department of Obstetrics and Gynecology, Virginia
Commonwealth University School of Medicine, Richmond, Virginia
Objective: To summarize recent advances in the understanding of the endocrine signaling pathways between the
hypothalamus, pituitary, and human corpus luteum (CL); to examine the major paracrine and autocrine mechanisms and the key genes and proteins involved in CL development, function, and regression in natural cycles;
to review the endocrine and molecular response of the midluteal phase CL to in vivo administration of human chorionic gonadotropin (hCG); and to describe the ultrasonographic and Doppler evaluation of the ovary and endometrium throughout the luteal phase.
Design: Published data in the literature, including the basic and clinical research studies of the authors.
Setting: University-affiliated hospital and research centers.
Patient(s): None.
Intervention(s): None.
Main Outcome Measure(s): Clinical and molecular analysis of human CL function.
Result(s): The endocrine function of the subpopulations of luteal cells is critical for the maintenance of CL function, including neovacularization and steroid hormones production. We consider the key genes and proteins that
favor development of luteal structure and function throughout the menstrual cycle and in our model of hCG treatment resembling early pregnancy.
Conclusion(s): These data indicate that the functional lifespan of the CL depends on paracrine and autocrine
mechanisms. Therefore, the significance of the key genes and proteins that we analyze in lutein cells during CL
development, function, demise, and rescue by hCG is likely to bring new therapeutic applications for the management of fertility defects and the control of fertility. (Fertil Steril 2009;92:1067–79. 2009 by American Society
for Reproductive Medicine.)
Key Words: Human corpus luteum function, natural cycles, steroidogenesis
The human corpus luteum (CL), a temporary endocrine
gland derived from the ovulated follicle, is a major source
of steroid hormones, producing up to 40 mg of progesterone (P) per day. The secretion of a significant amount of
androgens and estradiol (E2) in addition to P is unique to
the CL of many primates, including humans. The pattern
of P production throughout the luteal phase determines
menstrual cyclicity and endometrial receptivity for successReceived July 10, 2007; revised June 26, 2008; accepted July 14, 2008;
published online September 15, 2008.
L.D. has nothing to disclose. A.F. has nothing to disclose. P.K. has nothing
to disclose. P.C. has nothing to disclose. A.P. has nothing to disclose.
R.P. has nothing to disclose. A.M. has nothing to disclose. J.F.S. has
nothing to disclose.
Supported by Chilean Research Council CONICYT-FONDAP grant no.
15010006 and Fogarty International Research Collaboration Award
and NIH grant no. RFA-TW-05-002.
Reprint requests: Luigi Devoto, M.D., Faculty of Medicine, University of
Chile, P.O. Box 226-3, Santiago, Chile (FAX: 56-2-4247240; E-mail:
[email protected]).
0015-0282/09/$36.00
doi:10.1016/j.fertnstert.2008.07.1745
ful implantation, and is essential for maintenance of early
pregnancy. Thus, the underlying endocrine, autocrine/paracrine, and molecular mechanisms controlling P production
at the time of follicular cell luteinization and during the development, function, and rescue of the CL is of paramount
importance to understanding a fertile cycle. In addition,
this overview includes studies of the action of P during
follicular luteinization and the endocrine and morphologic
functional status of the ovary as evaluated by ultrasound.
FOLLICULAR LUTEINIZATION
The process of granulosa-thecal cells luteinization is triggered by pituitary-derived luteinizing hormone (LH), which
in turn activates a signal-transduction pathway dependent
on protein kinase A (PKA) and a possible pathway by which
LH receptor (LHr) is coupled to change in intracellular Ca2þ
and diacylglycerol (IP3) produced by phospholipase C activation. Estradiol synthesis increases progressively from the
Fertility and Sterility Vol. 92, No. 3, September 2009
Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
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dominant follicle and initiates the LH surge. A small increase
in P levels is seen in normal women before the LH surge,
which reflects the increasing LH pulse amplitude and frequency leading up to the surge. In humans, a LH surge of
24 to 36 hours is sufficient to initiate the resumption of oocyte
meiosis, uncoupling of gap junctions between granulosa
cells with the plasma membrane of the oocyte, luteinization
of granulosa cells, ovulation, and the initial phase of CL
development.
codons (5). Of these, PR-A is required for normal ovarian
and uterine function. In contrast, PR-B is critical for mammary development. To directly address the role of PR in reproductive function, a mouse model (PRKO) was generated
in which both isoforms of the PR were ablated by PR gene
targeting. This model provided definitive proof that PRs are
essential coordinators of all reproductive events that culminate in preparation of the reproductive tract for the establishment and maintenance of pregnancy (6).
The phenotype of preovulatory granulosa cells encompasses the expression of various stimulatory factors for
cell cycle progression, including cyclin B 1–2, folliclestimulating hormone receptor (FSHr), and the steroidogenic
enzymes 3b-hydroxysteroid dehydrogenase (3b-HSD) and
aromatase before the LH surge. Additionally, FSH induces
LHr in granulosa cells of the growing follicles. The LH
surge in turn inhibits cell proliferation, probably as a result
of changes in cyclins and other genes (1–3). The LH signaling associated with the surge increases steroid biosynthesis
and initiates the resumption of meiosis, ovulation, and subsequent luteinization of theca and granulosa cells. Thus, LH
acting through the LHr on preovulatory granulosa cells subsumes the role of FSH in the latter stages of follicular
maturation.
Evidence of a direct role of PR in human and rat ovarian
function has been supported in recent years by the demonstration that [1] the antiprogestin RU486 inhibits ovulation in the
human (7) and [2] LH is the primary signal for rupture of preovulatory ovarian follicles and induces a transient expression
of PR mRNA for both protein isoforms in granulosa cells
isolated from rat preovulatory follicles (7–9).
After the LH surge, P and 17a-hydroxyprogesterone (17aOHP) plasma concentrations increase, indicating the beginning of granulosa and theca cell luteinization. The P levels
increase rapidly after the LH surge or human chorionic
gonadotropin (hCG) administration (30 minutes). The rapidity of this response suggests that most of the enzymes and
proteins necessary for P synthesis must be present in the cells
or that they are rapidly induced. The lack of the full complement of the enzymatic machinery in the human granulosa
cells before the LH surge points to the luteinization of thecal
cells as a possible source for this immediate increase in P synthesis (4). At this time, several morphologic and molecular
changes take place in the granulosa cells. Human granulosa
cells luteinization includes an increase in expression of LHr
and progesterone receptor (PR) as well as the binding of
the promoter of the steroidogenic acute regulatory (StAR)
protein genes including steroidogenic factor 1 (SF-1), a member of the nuclear receptor superfamily, GATA-4, CCAAT/
enhancer binding protein b (C/EBPb), P450 cholesterol
side-chain cleavage (P450scc), cyclooxygenase-2 (COX-2),
and members of the matrix metalloproteinase family that
are critical determinants of P synthesis, oocyte maturation,
and follicular rupture.
CLASSIC PROGESTERONE RECEPTORS AND OVARIAN
FUNCTION
The physiologic effects of P are primarily mediated by interaction with PR. There are two classic PR isoforms, PR-A and
PR-B. Both isoforms are expressed from a single gene in humans as a result of transcription from two alternative promoters and translation initiation at two different AUG
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Devoto et al.
Features of the human corpus luteum
Definitive proof that PRs are essential mediators of ovulation has been provided by the PRKO mouse. Despite exposure to superovulatory levels of gonadotropins, PRKO
mice fail to ovulate. Analysis of the ovarian histology of
these mice revealed normal development of follicles through
the tertiary follicle stage. These follicles contain a mature
oocyte that is fully functional when isolated and fertilized
in vitro. Follicular rupture is inhibited, but the preovulatory
granulosa cells within these follicles can still differentiate
into a luteal phenotype and express the luteal marker
P450scc (6).
Thus, Conneely et al. (10, 11), established that PR is required specifically for LH-dependent follicular rupture
leading to ovulation but not for granulosa cells to undergo
luteinization. Both the PR-A and PR-B proteins are induced
in preovulatory mice follicles in response to LH stimulation. Furthermore, the selective ablation of the PR-A protein in knockout (PRAKO) mice results in infertility and
severe impairment of ovulation, indicating that expression
of the A protein is essential for mouse normal ovarian
function.
These findings indicate that the PR-A and PR-B proteins
are not functionally redundant in the mouse ovary and provide the first physiologic validation that these proteins have
different roles.
The presence of PRs in the primate ovary raises the possibility that the disruption of follicular development and function after administration of antiprogestins (mifepristone) (12,
13) is due perhaps in part to direct effects on the ovary in
addition to actions on the hypothalamic-pituitary axis.
MEMBRANE PROGESTERONE RECEPTORS AND OVARIAN
FUNCTION
Progesterone also regulates mitosis and apoptosis of rat granulosa cells isolated before the gonadotropin surge (14). These
granulosa cells do not express the classic nuclear PR; thus, it
is unlikely that PR mediates P action in these cells. The binding of P to the plasma membrane of bovine granulosa cells
Vol. 92, No. 3, September 2009
suggests that P could function through a nongenomic, membrane-initiated mechanism (15). Recently, the cloning, expression, and characterization of a membrane PR and
evidence that it is an intermediary in meiotic maturation of
fish oocytes has been reported (16). Furthermore, steroids
acting through membrane steroid receptors have been implicated in mammalian oocyte maturation (17).
ENDOCRINE AUTOCRINE/PARACRINE REGULATION OF
THE CORPUS LUTEUM
The human CL is composed of steroidogenic (theca and granulosa lutein) and nonsteroidogenic (endothelial, immune, and
fibroblast) cells, both of which are essential to the synthesis
and secretion of steroids (18). The production of these hormones largely depends on pituitary-derived LH acting
through the cyclic adenosine monophosphate (cAMP) second
messenger signaling system to regulate genes essential to
hormone synthesis and luteal development. During the cycle
of conception, trophoblastic production of human hCG prevents the regression of the CL. In monkeys with surgically induced hypothalamic lesions, the maintenance of LH levels
during the luteal phase through GnRH administration at
a pulse frequency similar to that of the early luteal phase
does not prevent luteolysis (19). Blocking LH release by
the administration of GnRH antagonist results in an abrupt
decline in serum P concentrations within 24 hours. This suggests that LH is essential to the development and maintenance of the primate CL but luteal regression is not, due to
changes in LH pulse frequency and amplitude. It has been
demonstrated that luteal regression in the primates menstrual
cycle is caused by a large reduction in the responsiveness of
the aging CL to LH, which can be overcome in the fertile
cycle by elevated concentrations of hCG (20). Indeed, the
in vitro effects of LH/hCG on human luteal cell steroidogenesis are modified by a variety of molecules encompassing
growth factors, hormones, nitric oxide, cytokines, insulinlike growth factor 1 (IGF-1), and IGF-binding proteins
(21). Interestingly, interleukin-1 (IL-1) and tumor necrosis
factor a (TNF-a) have an inhibitory effect on the stimulatory
action of hCG on steroid production by granulosa-lutein cells
as well luteal cells in culture. It is well known that these
proinflammatory cytokines are highly expressed in ectopic
endometrial cells (22). Concentrations of these cytokines
are increased in the peritoneal fluid of patients with endometriosis (23). Therefore, cytokines have been implicated in the
pathogenesis and pathophysiology of ovulatory dysfunction
and luteal phase defects of patients with endometriosis.
STEROID BIOSYNTHESIS BY THECA AND LUTEINIZED
GRANULOSA CELLS
The cells comprising the human CL have different morphologic, endocrine, and biochemical phenotypes. The number,
morphology, function, and secretory capabilities of these
cells change throughout the luteal phase (24). Approximately
30% of these cells are steroidogenic. Small luteal cells are
presumably derived from the theca–interna, whereas large luFertility and Sterility
teal cells are proposed to originate from granulosa cell lineage. Basal production of P is generally greater for
granulosa-lutein cells, which are also the site of E2 production because they express aromatase. Theca-lutein cells, however, display a greater increase in steroid production when
exposed to hCG and express 17a-hydroxylase/17/20 lyase
activity (P450c17). Theca-lutein cells produce the androgen
precursors that are aromatized by granulosa-lutein cells and
also are the site of 17a-OHP synthesis. These findings indicate that the two-cell model of estrogen biosynthesis invoked
to explain follicular estrogen production is preserved in the
human and monkey CL (25, 26) (Fig. 1).
NONSTEROIDOGENIC LUTEAL CELLS
During the transformation of the ovulatory follicle to a fully
functional CL, the vascular endothelial cells undergo an intense period of proliferation, followed by the establishment
of a rich capillary network. Approximately 30% to 40% of
the cells in a mature CL are endothelial cells. The luteal vasculature is critical to the delivery of gonadotropins and substrate like plasma lipoproteins, which provide cholesterol
for P production and removal of secretory products, mainly
steroid hormones, from luteal cells. Factors that regulate luteal vasculature play a major role in the regulation of luteal
function. Vascular endothelial growth factor (VEGF)
mRNA and protein have been localized in the granulosalutein cells of the CL. Inhibition of VEGF in vivo during
the luteal phase in nonhuman primates prevents luteal angiogenesis and suppresses P secretion (27).
Recently, a novel angiogenic factor endocrine gland–endothelial growth factor (EG-VEGF), with a degree of specificity
to the ovary, was detected in human granulosa-lutein cells. In
contrast to VEGF mRNA, EG-VEGF mRNA increases in mid
and late CL. It is thought that EG-VEGF enables the CL to
respond to hCG in early pregnancy (28). The importance of
the vasculature to CL function is reflected in the altered measures of blood flow to the CL in the setting of the luteinized
unruptured follicle (LUF) and luteal phase defects (29).
Immune cells, macrophages, and T lymphocytes are present within luteal tissue. Macrophages and endothelial cells
establish close contact with other luteal cells, which facilitates luteal cell regulation by paracrine mechanisms. The
ability of macrophages to secrete IL-1b and TNF-a is significant because both cytokines can modulate luteal steroidogenesis. In vitro studies indicate that these cytokines
diminish the LH/hCG-stimulated P production of cultured
human granulosa-lutein cells (30). Interleukin-1 and TNFa are secreted mainly by activated luteal monocytes and macrophages as well as by T and B cells. Activated macrophages
characterized the midluteal and late luteal phase CL (22, 30).
Under physiologic conditions, it is plausible that these cytokines play a role in functional and structural luteolysis, making possible the beginning of a new cycle. However, the
unscheduled activation of these mechanisms may result in
CL dysfunction.
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FIGURE 1
Two-cells theory of sustained E2 production by the human steroidogenic cells. Histologic sections of mid CL
were immunostained by the peroxidase conjugate technique. Each protein was incubated with specific antibody.
Color was developed with 3-amino-9-ethylcarbazole. Intense immunolocalization for StAR, P450scc, and
3b-HSD were detected in the cytoplasm of both granulosa and thecal lutein cells of mid human corpus luteum.
Specific cytoplasmatic staining in the periphery of the gland was detected for P450c17 in thecal lutein cells,
indicating that these cells control androgens biosynthesis. Conversely, P450arom was present only in the
cytoplasm of granulosa-luteal cells, indicating the origin of E2 secretion. The midluteal phase CL is a highly active
steroidogenic gland producing P and androgens. The divergence in enzyme localization in the mid CL is
consistent with the two-cells model of E2 production.
Devoto. Features of the human corpus luteum. Fertil Steril 2009.
CHOLESTEROL TRANSPORT TO AND WITHIN LUTEAL
STEROIDOGENIC CELLS
The first challenge for any steroid-producing cell, including
luteal cells, is to obtain the cholesterol precursor. Luteal steroidogenic cells can produce cholesterol de novo; however,
this pathway plays a minor role, as evidenced by the low
levels of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, the limiting enzyme in this cholesterol pathway (31).
Human steroidogenic luteal cells take up lipoprotein-carried
cholesterol (low-density lipoprotein) by endocytosis and also
maintain stores of esterified cholesterol. Highdensity lipoproteins may also contribute precursors for steroidogenesis through the SR-B1 receptors, which mediate
the selective uptake of high-density lipoprotein cholesterol
esters. Upon gonadotropin stimulation, cholesterol from various pools—including the intracellular cholesterol esters
present in lipid droplets, which are hydrolyzed—is conveyed
to the inner membrane of the mitochondria to serve as a substrate for pregnenolone (P5) production. It is thought that the
rate-limiting step in P synthesis is the movement of cholesterol from the outer mitochondrial membrane to the inner
membrane where the cytochrome P450scc complex is located.
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Devoto et al.
Features of the human corpus luteum
The StAR is essential to this sterol translocation in response
to tropic hormones, including LH and hCG (32, 33).
LUTEAL PROGESTERONE SYNTHESIS
Three critical endocrine events support P secretion in primate
ovarian physiology (34): [1] an LH surge is the signal for follicular rupture and for luteinization of theca and granulosa
cells; [2] LH pulses during the luteal phase are critical to
the development and function of the CL; and [3] hCG secretion by the embryo’s trophoblast sustains the CL function in
early pregnancy.
Progesterone biosynthesis requires only two enzymatic
steps: the conversion of cholesterol to P5, catalyzed by
P450ssc located on the inner mitochondrial membrane,
and its subsequent conversion to P, catalyzed by 3b-HSD
present in the smooth endoplasmic reticulum. Before the
LH surge, StAR is virtually absent from the human granulosa cells, which are unable to synthesize P from cholesterol
precursors (35). Conversely, StAR is found in high levels in
the periovulatory human theca cells that are able to
Vol. 92, No. 3, September 2009
synthesize androgens from cholesterol (36). Thus, the rapid
increase in P at the time of LH surge suggests that
luteinizing theca cells are the possible source of P. Additionally, the limited vascular network of the human periovulatory granulosa cells would limit the ability of these cells
to obtain cholesterol (low-density lipoprotein) via vasculature. The establishment of an inadequate vascular supply
to the CL is postulated to have significant ramifications on
steroid secretion later in the luteal phase.
Expression of StAR transcripts and protein are greatest in
early luteal and midluteal phase CL. In the human CL, immunodetection of StAR exhibits greater levels in theca-lutein
than granulosa-lutein cells, irrespective of the stage of the luteal phase. The level of StAR immunostaining is more variable in granulosa-lutein cells throughout the menstrual
cycle. The level of StAR immunostaining was moderate in
early luteal tissue, increased in midluteal phase CL, and declining in late luteal phase CL (37).
Very recently, our group determined by immunoelectron
microscopy the presence of significant levels of StAR in
the mitochondria and cytoplasma of human luteal steroidogenic cells throughout the luteal phase (38). We found
greater levels of StAR immunolabeling in the cytoplasm
of steroidogenic cells from early luteal and midluteal phase
human CL. The 30-kd mature StAR protein was present in
both mitochondria and cytosol. These findings support the
hypothesis that either appreciable processing of StAR 37
kd pre-protein occurs outside the mitochondria, or mature
StAR protein is selectively released into the cytoplasm after
mitochondrial processing. Examination of P450scc in human CL tissue indicates that the overall expression of this
enzyme remains elevated and relatively constant throughout
the luteal phase.
The expression of 3b-HSD within human CL appears to be
greatest during the early luteal phase and declining by the
midluteal phase; it then remains constant in the late luteal
phase CL (39). Several investigations have shown that luteal
cells of different ages incubated with P5 result in a dramatic
increase in P secretion, suggesting that 3b-HSD is not rate
limiting in P production. Therefore, P450ssc and 3b-HSD
do not appear to be rate-limiting steps in luteal P biosynthesis
during the menstrual cycle. Indeed, the consensus of a large
number of studies in human and nonhuman primates is that
the critical step in luteal P secretion is the movement of cholesterol from the outer to inner mitochondrial membrane,
which is a StAR-dependent process. Although not considered
a rate-determining enzyme in steroidogenesis, potent inhibitors of 3b-HSD like epostane can interfere with P production
in primates and humans and result in pregnancy termination
(40, 41).
In addition to classic reproductive steroid hormones synthesis, the human CL produces allopregnanolone and pregnanolone. These steroids are of interest because of their
ability to modulate the function of GABA receptors, and
Fertility and Sterility
they represent a class of compounds, also produced in neural tissue, referred to as neurosteroids. This may represent
a link between the CL and central nervous system function,
which could contribute to changes in cognitive function and
mood during the luteal phase (42). The mRNA for 5areductase and 5b-reductase is present within the CL. Luteal
tissue concentrations of these neurosteroids significantly
decrease during the late luteal phase. It is interesting that
cultured human luteal cells in the presence of hCG increased significantly the production of both neurosteroids,
suggesting that these progestins are also LH/hCG dependent (43–45).
LUTEAL ESTRADIOL BIOSYNTHESIS
Primate CL retains the ability to produce estrogens, which
distinguishes it from the CL of domestic animals and rodents
species. Small luteal cells are thought to be the primary
source of luteal androgens (46), while large luteal cells are
thought to be the primary site of luteal estrogen synthesis
(47), indicating that the two-cell model of estrogen biosynthesis invoked to explain follicular estrogens synthesis is preserved in primate CL.
The enzyme-catalyzing androgens synthesis P450c17 is located in cells near the periphery of the gland along the vascular tract (25). In contrast, P450arom staining is observed
throughout the luteal parenchyma (see Fig. 1). Luteal
P450arom mRNA levels are diminished in the late luteal
phase in association with a fall in plasma E2 levels (47, 48).
It is well established that E2 synthesis by granulosa cells of
the ovarian follicle is stimulated by FSH (49). However,
FSH does not stimulate E2 synthesis by luteal cells in culture
or sustain luteal steroidogenesis in vivo (50). Thus, although
the two-cell system for estrogen production is retained after
luteinization of the follicle, the role of FSH in stimulation androgen aromatization is not conserved (51). The action of
FSH is evidently subserved by LH and IGF-1, which can
sustain E2 by luteal cells in culture (52). A new model of
the dynamics of follicular waves in the monovular mammalian ovary has emerged, based in part on ultrasound evidence
obtained in the mares and women (53, 54). These studies challenge the conventional wisdom that a single cohort of antral
follicles grows during the follicular phase of the human menstrual cycle. High-resolution ultrasound machines were used
to acquire longitudinal images of follicular development. Two
types of follicular waves were described. Major waves
occurred when one follicle grew to >10 mm and exceeded
all others follicle by >2 mm. This wave emerged during the
mid-interovulatory interval, giving origin to the ovulatory follicle. In contrast, minor waves were defined by follicles that
developed to a diameter of <10 mm, and follicle dominance
was not manifested, resulting in anovulatory waves (55).
These studies demonstrated that subtle waves of follicular
growth (4 to 8 mm) occur during the luteal phase of the human
menstrual cycle despite FSH suppression by luteal inhibin, P,
and E2 secretion. Human granulosa cells obtained from
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follicles smaller than 5 mm expressed P450arom, raising the
possibility that serum E2 levels during the luteal phase could
be derived in part from luteal-phase follicle waves rather than
exclusively from luteal tissue. It is interesting that E2 secretion by the human ovary does not appear to be essential for
pregnancy as P replacement alone maintains pregnancy.
The exact role for luteal E2 secretion is unknown. However,
it was originally postulated to be involved in luteolysis in
the primates, where the luteolytic process is independent of
uterine prostaglandin. Conversely, the recent finding of the
presence of both types of estrogen receptors in human CL
supports a local role of E2 in luteal function (56).
FIGURE 2
Effects of hCG on StAR expression and
steroidogenic response of late luteal phase corpus
luteum. (A) Northern blot of StAR mRNA transcripts
(4.4 and 1.6 kb) and Western blot of StAR preprotein
(37 kDa) and mature protein (30 kDa) collected
during late luteal phase and 24 hours after hCG
treatment. Control ¼ midluteal phase corpus luteum.
(B) Endocrine response after hCG challenge in
women during late luteal phase and 24 hours after
hCG (10,000 IU) treatment. Values are mean SEM
*P< .05 is statistically significant.
LUTEOLYSIS
In a nonfertile cycle, the CL of primates undergoes a process
of regression, known as luteolysis, which encompasses a loss
of functional and structural integrity of the gland (57). The
functional regression is associated with decrease in P production; the structural regression occurs after decrease in P synthesis and is associated with different forms of cell death. The
molecular events involved in luteal regression and how they
are prevented by exposure to hCG remain unclear. It is
thought that changes in LH pulses and declines in the LH receptor mRNA and protein do not account for luteal regression
in primates. These findings suggest that steroidogenic function and regression of the CL are determined by factors
downstream from the LH receptor (58).
The main feature of luteal functional luteal regression is
the reduced production of P, which is associated with a decline in expression of the StAR gene and protein. The decline
in StAR expression precedes a fall in expression of other steroidogenic enzymes. This indicates the critical role of StAR
in the production of luteal P. The administration of hCG during the late human luteal phase restores the level of StAR to
those found in the midluteal phase CL as well the plasma P
and E2 levels (Fig. 2). Several molecules, including prostaglandin F2-a, (PGF2-a), TNF-a, IL-1b, endothelin, monocyte chemoattractant (MCP-1), estrogens, and reactive
oxygen species have been implicated in the luteolytic process
(59). In humans, the physiologic role of PGF2-a in luteolysis
is uncertain because the ovarian production of PGF2-a is limited. In contrast, PGF2-a induces 20a-hydroxysteroid dehydrogenase (20a-HSD) expression in rodent luteal cells,
which lose their capacity to secrete P (57). It is interesting
that PGF2-a suppresses StAR expression in cultured human
granulosa-luteal cells (60). Estrogens inhibit the activity of
luteal 3b-HSD by human luteal cells in vitro (61). Other
investigators have postulated that the luteolytic action of exogenously administrated E2 reduces the secretion of pituitary-derived LH in women (62). The reduction of luteal
perfusion and other factors produced by the macrophages
or leukocytes like reactive oxygen species may contribute
to functional and structural luteolysis (48). Thus, the endothelial cell function of the CL may be affected, resulting in
diminished expression of VEGF and molecules that promote
endothelial cell survival (28).
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Features of the human corpus luteum
Devoto. Features of the human corpus luteum. Fertil Steril 2009.
Knowledge about the molecular events occurring after
functional regression of the human CL and before cellular destruction is limited. It is thought that cell death and an increase
in matrix metalloproteinase expression (MMP-2 and MMP-9)
are important components of structural regression (63, 64).
Several laboratories have documented an increasing number
of apoptotic cells, by detection of nuclear DNA fragmentation, in late and regressing CL compared with those of early
luteal and midluteal phase human CL (65–67). In contrast,
the patterns of expression of the genes that control cell survival and apoptosis in the human CL are debated. Some investigators have found no changes in Bcl-2 (68), a cell survival
factor, in CL of different ages. However, others have
described a decline in the late luteal phase and an increase
in ectopic-pregnancy CL (69). Moreover, the proapoptotic
protein Bax has been reported to remain unchanged in the
Vol. 92, No. 3, September 2009
human CL throughout the luteal phase (70), but others have
reported increasing levels in regressing CL or undetectable
levels in the CL of pregnancy.
Additionally, it has been suggested that cell–cell interactions may regulate apoptosis. Corpus luteum regression is associated with loss of cell–cell adhesions sites. Thus, it can be
postulated that cell-adhesion molecules (CAM) are implicated in luteal cells survival and death. Cadherins are a rapidly expanding family of calcium-dependent CAMs. Luteal
cells are also strongly positive for N-cadherins in the early
luteal and midluteal phases, whereas there is only weak Ncadherin staining in late luteal CL. There is a direct correlation between the presence of N-cadherin molecules and the
absence of characteristics of cellular apoptosis (71).
Thus, the existing data indicate that apoptosis is feature of
human luteal regression. However, the mechanisms that govern luteal regression remain unclear. Indeed, luteal cells with
a positive apoptotic signal and the number of iNOS-positive
luteal cells increase within the human CL during luteal regression (48). However, the percentages of luteal cells with
apoptotic signals are low (5% to 7%), which makes it uncertain that apoptosis is the only mechanism underlying luteal
cell death. Other types of cell death, such as autophagy and
necrosis, appear to play a role in luteal regression, as been reported in the monkey and human CL, respectively (64, 72).
The unscheduled activation of these mechanisms may contribute in luteal phase defects.
CL RESCUE IN A FERTILE CYCLE
During the cycle of conception, trophoblastic production of
hCG prevents the regression of the CL. Compelling evidence
that hCG rescues the CL is that administration of b-hCG vaccine to women inactivates endogenous hCG, resulting in
a fall in P and menses (73). The hormonal characteristics of
conception and nonconception cycles are different from the
early luteal phase. Both LH and E2 levels are significantly
higher in conception cycles on day 4 and 5 after the LH
peak in urine. In contrast, serum FSH, P, and relaxin are
not significantly different in this period. These findings
may reflect alterations in signaling in the hypothalamicpituitary-ovarian axis that begin during the periovulatory period of nonconception cycles (74). Serum hCG is detectable
around the time of implantation (day 8 after ovulation) and
then rises progressively to the first 12 week of pregnancy.
The CL volume determined by vaginal ultrasound exhibits
a rapid increase in early human pregnancy without a parallel
rise in 17a-OHP, P, or E2. However, the serum level of 17aOHP during the first 6 weeks of pregnancy has been considered the best marker of luteal steroidogenesis because this
steroid is not synthesized by the trophoblast, which does
not express P450c17. In contrast a positive correlation exits
between CL volume and relaxin and hCG serum concentrations. These findings suggest that growth of the CL in early
pregnancy is largely derived from the proliferation of non–
steroid-secreting cells (75).
Fertility and Sterility
There are limited data on the molecular changes underlying the functional and structural changes in the CL of pregnancy. We and other investigators have experimental
protocols including hCG administration during midluteal
and mid-late luteal phases with the goal of determining the
molecular basis of CL rescue. Administration of exponentially increasing doses of LH or hCG prolongs the lifespan
of the CL (20). The administration of hCG during the late luteal phase restores the abundance of StAR mRNA and protein
levels to those found in midluteal phase CL as well the
plasma P level (Fig. 2). Additionally, hCG administration
shows expansion of the vascular network of theca and granulosa cells layers, with intense staining detected in the cytoplasm of steroidogenic cells (26).
Structural regression of the CL is brought about by apoptosis and autophagy, and hCG has the ability to change the apoptotic program of the late luteal phase CL. A decrease in the
proapoptotic protein, Bax, has been reported in CL of pregnancy and hCG-stimulated late CL (69).
Although the exact role of P in the CL is not clear, several
effects of P on luteal cells have been described. For example, P can directly promote luteinized granulosa cell survival
(76) and influence the expression of LHr (77) and steroidogenic enzymes (35). In addition, P has been implicated in
the control of tissue inhibitors of metalloproteinase-1 expression in luteinized granulosa cells (78). In humans, it is
well known that the CL expresses PR-A and PR-B
mRNA, and PR-A was found to be more abundant than
PR-B (79, 80). Both receptor isoforms decrease in late luteal
phase CL. In addition, CL does have membrane-linked Pbinding activity. It is therefore possible that P withdrawal
has direct effects on steroidogenic cell function. The luteal
tissue P concentration falls in the late luteal phase of humans and monkeys (81, 82). This P withdrawal is potentially
facilitated further by the reduced expression of luteal genomic PR in the late luteal phase. Thus, P could play specific
roles in luteal rescue.
Progesterone may function as a paracrine molecule in the
CL; it may also have intracrinal effects, and it has been suggested that P itself is involved in its own synthesis (83).
Therefore, if P has a major role in the function of granulosa-lutein cells at the time of luteolysis, the down-regulation
of PR in the late luteal phase would not be seen during luteal
rescue. However, this does not appear to be the case. In vivo
and in vitro studies have determined that the down-regulation
of PR is not prevented by hCG (84). This finding does not
mean that P has no role in the function of luteinized granulosa
cells as the CL ages, but it does not support a major role for P
in the luteolysis-luteal rescue transition.
CLINICAL AND LABORATORY ASSESSMENT OF THE
LUTEAL PHASE
The histologic changes of the endometrium during a natural
menstrual cycle were described more than 50 years ago (85).
Jones (86) was the first to hypothesize that delayed
1073
endometrial maturation resulting from inadequate CL P production might be a cause of early pregnancy loss and infertility. Estimates of the prevalence of luteal phase deficiency
(LPD) range between 5% and 10% in infertile women and between 10% and 25% in those with a history of recurrent early
pregnancy loss (87, 88).
Whether histologic endometrial dating has the accuracy or
the precision necessary to be a valid method for the diagnosis
of LPD or to otherwise guide the clinical management of
women with reproductive failure is a matter of debate. Coutifaris et al. (89) designed a study to assess the ability of histologic dating to discriminate between women of fertile and
infertile couples. The participants consisted of volunteers
who were 20 to 39 years of age, had regular menstrual cycles,
and had used no hormonal treatments or contraceptives for 1
month before the study. The proportion of out-of-phase biopsies in fertile and infertile women was compared, and the results showed that out-of-phase biopsy results poorly
discriminated between the women from fertile and infertile
couples in either the midluteal phase (fertile: 49.4%; infertile:
43.2%) or late luteal phase (fertile: 35.3%; infertile 23.0%).
They concluded that histologic dating of the endometrium
does not discriminate between women of fertile and infertile
couples and should not be used in the routine evaluation of
infertility. Murray et al. (90) reached similar conclusions:
the traditional endometrial histologic dating criteria are
much less temporally distinct and discriminating than originally described because of the considerable intersubject, intrasubject, and interobserver variability. Neither traditional
dating criteria nor any combination of the best performing
histologic features identified by their objective, and systematic analyses could not reliably distinguish any specific cycle
day or narrow interval of days.
In contrast, studies comparing the endometrium in in vitro
fertilization (IVF) cycles with natural cycle controls have
shown premature secretory changes in the postovulatory
and early luteal phase of the IVF cycle (91). Determination
of P in the midluteal phase has been widely used as a surrogate to confirm ovulation. The cut-point value for establishing that ovulation has occurred varies from 4 to10 ng/mL in
different settings. The large amplitude of pulsatile secretion
of P during the late luteal phase, driven by large amplitude
LH pulses, conspires against the accuracy of a single determination of this steroid. As an alternative, increased daily
excretion of pregnanediol, relative to that early in the menstrual cycle, is often taken to be evidence that a woman has
ovulated. Metcalf et al. (92) collected urine, plasma, and saliva samples during a 24-hour period from 20 women during
the follicular phase and from 20 women during the luteal
phase. They compared the 24-hour excretion of pregnanediol with [1] the concentration of P in plasma, [2] the concentration of P in saliva, [3] the concentration of
pregnanediol in small urine samples, [4] the rate of excretion of pregnanediol, and [5] the ratio of pregnanediol to
creatinine in small urine samples. It was concluded that
the most satisfactory alternative to the measurement of
1074
Devoto et al.
Features of the human corpus luteum
24-hour pregnanediol output for the biochemical assessment
of ovulation based on P production was the measurement of
the concentration of P in plasma; the least satisfactory alternative was the determination of the concentration of P in
saliva. If blood was not available, measurement of the ratio
of pregnanediol to creatinine in a small urine sample was the
preferred method.
ULTRASONOGRAPHIC AND DOPPLER EVALUATION
OF THE CL
Ultrasonographic detection of the CL after ovulation has
been reported initially to occur in only 50% to 80% of natural
menstrual cycles, determined by transabdominal ultrasonography (93). With the advances in imaging technology, ovulation and the presence of the CL could be detected in almost
100% of women (93). The CL increased in diameter for the
first week after ovulation and then began to regress in nonconception cycles. Growth of the CL is associated with an increase in luteal blood flow and E2 and P production (94).
Two morphologic types of CL can be observed after ovulation: those with and without a central fluid-filled cavity
(CFFC). Most CL contained a CFFC. The incidence of CL
containing a CFFC is greatest immediately after ovulation
and then subsequently declines. This CFFC is related to the
leakage of blood into the follicular lumen after follicular rupture. The ultrasonographic detection CFFC should be interpreted as a normal physiologic event during the menstrual
cycle (94).
Quantitative changes in luteal echo texture represent
changes in the morphologic and physiologic status of the
CL, as previously documented in domestic animal species
(95, 96). Baerwald et al. (94) reported that a decrease in luteal
ultrasound echogenicity occurred during luteal development
in association with an increase P and E2 serum concentrations. Decreased echogenicity during luteinization suggests
increased vascularization of luteal tissue and a corresponding
decreased tissue density. In contrast, the increased echogenicity described during luteolysis could be attributed to decreased vascularization and replacement of luteal tissue
with fibrous connective tissue.
Glock and Brumsted (97) used color flow pulsed Doppler
ultrasound to demonstrate a correlation between different
stages of the luteal phase and the resistance index (RI) of
the CL of the natural cycle. The lowest values of RI were detected during the midluteal phase, which corresponds to the
peak of neovascularization of the CL. In addition, an increase
in blood flow impedance has been demonstrated in the late luteal phase, associated with CL regression. Kupecic et al. (29)
found a significant difference in intraovarian impedance in
patients with luteal LPD with a significant increase in RI during the midluteal phase of those patients compared with
normal controls.
Color Doppler has also been used to study the possible
relationship between hormonal secretion and velocimetric
Vol. 92, No. 3, September 2009
parameters in both the follicular and the luteal phase; however, the results have been conflicting. Two studies showed
a significant correlation between follicular E2 and Doppler
ovarian velocimetry (98, 99), but others were unable to
find it (100). It is interesting that some investigators have
reported that luteal P secretion is closely correlated with
luteal arterial velocimetry in spontaneous cycles, but others
have failed to demonstrate any significant relationship
(101).
In normal ovulatory cycles, the intraovarian venous signal
shows a continuous or slightly undulating pattern; the venous
velocity slowly increases throughout the follicular phase.
During the midluteal phase, its values increase significantly
relative to those of the follicular phase. It is interesting that
ovarian arterial flow in women with LPD does not exhibit
any differences in arterial velocimetric parameters when
compared with those of normal ovulatory cycles. Miyazaki
et al. (95) studied luteal blood flow by power Doppler in
healthy volunteer women. With this technique, arterial and
venous vascularization are evaluated simultaneously because
the color map cannot differentiate between the two flows. The
investigators determined that the product between luteal vascularity ratio and luteal volume was statistically significantly
correlated with P concentrations. In contrast, Merce et al.
(102) found no statistically significant correlation between
luteal P levels and arterial velocimetric parameters. However, a statistically significant correlation was observed for
the maximum venous velocity. These results are difficult to
reconcile, although it could be argued that the luteal deficiency cycles (LPD and luteinized unruptured follicle) are
associated with an abnormal luteal venous network, which
may contribute to the reduced secretion of P into the general
circulation.
ULTRASONOGRAPHIC AND DOPPLER EVALUATION OF THE
LUTEAL PHASE ENDOMETRIUM
There is no reliable diagnostic method to evaluate endometrial receptivity, although several techniques have been proposed: histologic assessment of endometrial biopsy (85),
endometrial protein expression (103), and ultrasound examination of the endometrium (104). Different ultrasound parameters have been suggested to assess endometrial
receptivity, including endometrial thickness, endometrial
echogenic pattern, and endometrial volume (105–107).
Endometrial Thickness and Pattern
The endometrium appears ultrasonographically as a thin,
simple, hyperechogenic single stripe immediately after menses. The stratum functionalis and basalis layers provide different views compared with the endometrium development
during the mid–late follicular phase. A pronounced tripleline echo textural pattern that reflects the separation between
the stratum basalis and functionalis layers is clearly observed
in the periovulatory period in association with rising E2
levels. The triple-line pattern disappears after ovulation. A
Fertility and Sterility
more homogeneous, hyperechogenic endometrium is observed as endometrial glands expand under the influence of
luteal P production in the secretory phase (108).
Endometrial Blood Flow
There is controversy in the literature regarding changes in
uterine Doppler flow indexes during the luteal phase of
a spontaneous cycle. Several studies (109–111) reported
that resistance to flow declined from the early follicular phase
to the midluteal phase, but became maximal around the time
of ovulation. In contrast, other investigators noted no differences in uterine Doppler flow indexes at different phases of
the cycle (112, 113).
Doppler study of uterine arteries reflect blood flow to both
the endometrium and myometrium. Blood flow to the endometrium comes from the radial artery, which divides after
passing through the myometrial–endometrial junction to
form the basal arteries that supply the basal portion of the endometrium, and the spiral arteries that continue up towards
the endometrium.
Blood flow in the uterine vessels assessed by color Doppler
ultrasound is usually expressed as downstream impedance to
flow. Measurement of blood flow volume in the uterine vessels is difficult because vessel diameter is influenced by the
angle of the probe and vessel tortuosity (114). Steer et al.
(115) reported that the chance of pregnancy was maximal
when the uterine pulsatility index (PI) was in the range of
2.00 to 2.99. Subsequently, Gannon et al. (116) used an intrauterine laser Doppler technique to measure endometrial microvascular blood flow, which varied throughout the
menstrual cycle, with increases in blood flow during the early
follicular and early luteal phases.
More recently, blood flow in the endometrial and the subendometrial regions has been determined by the use of threedimensional (3D) ultrasound with power Doppler (117, 118).
Power Doppler imaging is more sensitive than color Doppler
imaging at detecting low velocity flow and hence improves
the visualization of small vessels (119). In summary, early
measurements of endometrial and subendometrial blood
flow suffered from methodologic weaknesses, as they were
equipment and operator dependent. The selection of endometrial and subendometrial vessels identified on color Doppler
mode was arbitrary. The new 3D Doppler technology may
provide more consistent and reliable evaluation of endometrial blood flow.
CONCLUDING REMARKS
Adequate CL function is essential for the establishment and
maintenance of pregnancy. The life cycle of the CL during
the menstrual cycle and its rescue with conception reflect
an orchestrated interaction between pituitary and embryonic
gonadotropins as well as intraluteal autocrine and paracrine
signals that modulate the endocrine function of luteal cells
and their survival or demise.
1075
After ovulation, the oocyte enters the oviduct and progresses into the ampulla, where it may be fertilized if it met
by capacitated and competent spermatozoa. Therefore, the
initial and critical steps of human reproduction, including
gametes transport, the characteristics of tubal milieu, fertilization, and embryo transport, are biological events concomitant and partially modulated by the hormones secreted by the
CL. It is interesting that, because the human oviduct does not
respond with accelerated transport to acute increases in E2, it
is likely that the embryo itself, rather than ovarian steroids,
controls its passage into the uterus.
Conversely, progestins can cause retention of the embryo
in the oviduct, increasing the probability of intratubal pregnancy. Thus, the temporal patterns and dose of P administration for luteal phase support are important determinants of
clinical outcome. Simultaneously, the rising level of luteal
P during the midsecretory phase induces morphologic predecidualization changes in the endometrium that are followed
by further transformation of the stroma that occurs in a fertile
cycle. If implantation takes place, the trophoblastic cells of
the embryo start to secrete hCG, which is necessary to maintain CL and P production. Currently, the conventional clinical
and laboratory methods to assess the luteal phase—such as
monitoring of basal body temperature, histologic dating of
the endometrium, and measurement of P plasma levels—do
not have a sufficient sensitivity to diagnosis luteal phase dysfunction. Recently, ultrasonography, and particularly 3D ultrasonography with color Doppler flow pulsed technology,
has been proposed as a new tool for evaluating the human
CL in vivo. This noninvasive approach has the advantage of
simultaneous appraisal of the endometrium and the CL
throughout the luteal phase. The lack of reliable laboratory
tools to evaluate luteal function represents an important clinical constraint in assessing the normal menstrual cycle and
luteal phase defects as well implantation defects and the endocrinology of early pregnancy loss.
Thus, the examination of molecular and cellular aspects of
CL development, function, and demise remains a major research field. In primates, in whom the mechanisms of luteolysis appear to differ significantly from nonprimates, basic
research is required in the area of CL regulation, which
may provide new leads for infertility therapies and contraceptive development.
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