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The human corpus luteum: life cycle and function in natural cycles

      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.

      Key Words

      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 successful 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 Ca2+ and diacylglycerol (IP3) produced by phospholipase C activation. Estradiol synthesis increases progressively from the 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.
      The phenotype of preovulatory granulosa cells encompasses the expression of various stimulatory factors for cell cycle progression, including cyclin B 1–2, follicle-stimulating hormone receptor (FSHr), and the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase (3β-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 (
      • Robker R.L.
      • Richards J.S.
      Hormone-induced proliferation and differentiation of granulose cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1.
      ,
      • Jirawatnotai S.
      • Moons D.S.
      • Stocco C.O.
      • Franks R.
      • Hales D.B.
      • Gibori G.
      • et al.
      The cyclin-dependent kinase inhibitors p27Kip1 and p21Cip1 cooperate to restrict proliferative life span in differentiating ovarian cells.
      ,
      • Filicori M.
      • Cognigni G.E.
      • Gamberini E.
      • Parmegiani L.
      • Troilo E.
      • Roset B.
      Efficacy of low-dose human chorionic gonadotropin alone to complete controlled ovarian stimulation.
      ). 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.
      After the LH surge, P and 17α-hydroxyprogesterone (17α-OHP) 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 (
      • Christenson L.K.
      • Devoto L.
      Cholesterol transport and steroidogenesis by the corpus luteum.
      ). 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 β (C/EBPβ), 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 codons (
      • Kastner P.
      • Krust A.
      • Turcotte B.
      • Stropp U.
      • Tora L.
      • Gronemeyer H.
      • et al.
      Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B.
      ). 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 (
      • Lydon J.P.
      • DeMayo F.J.
      • Funk C.R.
      • Mani S.K.
      • Hughes A.R.
      • Montgomery Jr., C.A.
      • et al.
      Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities.
      ).
      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 (
      • Croxatto H.B.
      • Kovács L.
      • Massai R.
      • Resch B.A.
      • Fuentealba B.
      • Salvatierra A.M.
      • et al.
      Effects of long-term low-dose mifepristone on reproductive function in women.
      ) 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 (
      • Croxatto H.B.
      • Kovács L.
      • Massai R.
      • Resch B.A.
      • Fuentealba B.
      • Salvatierra A.M.
      • et al.
      Effects of long-term low-dose mifepristone on reproductive function in women.
      ,
      • Park O.K.
      • Mayo K.E.
      Transient expression of progesterone receptor messenger RNA in ovarian granulosa cells after the preovulatory luteinizing hormone surge.
      ,
      • Park O.K.
      • Mayo K.E.
      Regulation of progesterone receptor gene by gonadotropins and cyclic adenosine 39, 59-monophosphate in rat granulosa cells.
      ).
      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 (
      • Lydon J.P.
      • DeMayo F.J.
      • Funk C.R.
      • Mani S.K.
      • Hughes A.R.
      • Montgomery Jr., C.A.
      • et al.
      Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities.
      ).
      Thus, Conneely et al. (
      • Conneely O.M.
      • Mulack-Jericevic B.
      • DeMayo F.
      • Lydon J.P.
      • O'Malley B.W.
      Reproductive functions of progesterone receptors.
      ,
      • Conneely O.M.
      • Mulac-Jericevic B.
      • Lydon J.P.
      Progesterone-dependent regulation of female reproductive activity by two distinct progesterone receptor isoforms.
      ), 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) (
      • Liu J.H.
      • Garzo G.
      • Morris S.
      • Stuenkel C.
      • Ulmann A.
      • Yen S.S.
      Disruption of follicular maturation and delay of ovulation after administration of the antiprogesterone RU486.
      ,
      • Richards J.A.
      • Russell D.L.
      • Ochsner S.
      • Espey L.L.
      Ovulation: new dimensions and new regulators of the inflammatory-like response.
      ) 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 (
      • Peluso J.J.
      Pappalardo. Progesterone mediates its anti-mitogenic and anti-apoptotic actions in rat granulosa cells through a progesterone-binding protein with aminobutyric acid A receptor-like features.
      ). 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 suggests that P could function through a nongenomic, membrane-initiated mechanism (
      • Bramley T.A.
      • Menzies G.S.
      • Rae M.T.
      • Scobie G.
      Non-genomic steroid receptors in the bovine ovary.
      ). 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 (
      • Zhu Y.
      • Rice C.D.
      • Pang Y.
      • Pace M.
      • Thomas P.
      Cloning, expression and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes.
      ). Furthermore, steroids acting through membrane steroid receptors have been implicated in mammalian oocyte maturation (
      • Jamnongjit M.
      • Hammes S.R.
      Oocyte maturation: the coming of age of a germ cell.
      ).

      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 (
      • Retamales I.
      • Carrasco I.
      • Troncoso J.L.
      • Las Heras J.
      • Devoto L.
      • Vega M.
      Morpho-functional study of human luteal cell subpopulations.
      ). 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 (
      • Hutchison J.S.
      • Nelson P.B.
      • Zeleznik A.J.
      Effects of different gonadotropin pulse frequencies on corpus luteum function during the menstrual cycle of rhesus monkeys.
      ). 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 (
      • Zeleznik A.J.
      In vivo responses of the primate corpus luteum to luteinizing hormone and chorionic gonadotropin.
      ). 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, insulin-like growth factor 1 (IGF-1), and IGF-binding proteins (
      • Devoto L.
      • Vega M.
      • Kohen P.
      • Castro A.
      • Castro O.
      • Christenson L.K.
      • et al.
      Endocrine and paracrine-autocrine regulation of the human corpus luteum during the mid-luteal phase.
      ). Interestingly, interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α) 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 (
      • Seli E.
      • Arici A.
      Endometriosis: interaction of immune and endocrine systems.
      ). Concentrations of these cytokines are increased in the peritoneal fluid of patients with endometriosis (
      • Eisermann J.
      • Gast M.J.
      • Pineda J.
      • Odem R.R.
      • Collins J.L.
      Tumor necrosis factor in peritoneal fluid of women undergoing laparoscopic surgery.
      ). 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 (
      • Carrasco I.
      • Troncoso J.L.
      • Devoto L.
      • Vega M.
      Differential steroidogenic response of human luteal cell subpopulations.
      ). Approximately 30% of these cells are steroidogenic. Small luteal cells are presumably derived from the theca–interna, whereas large luteal 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 17α-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 17α-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 (
      • Sanders S.L.
      • Stouffer R.L.
      Localization of steroidogenic enzymes in macaque luteal tissue during the menstrual cycle and simulated early pregnancy: immunohistochemical evidence supporting the two-cell model for estrogen production in the primate corpus luteum.
      ,
      • Kohen P.
      • Castro O.
      • Palomino A.
      • Munoz A.
      • Christenson L.K.
      • Sierralta W.
      • et al.
      The steroidogenic response and corpus luteum expression of the steroidogenic acute regulatory protein after human chorionic gonadotropin administration at different times in the human luteal phase.
      ) (Fig. 1).
      Figure thumbnail gr1
      Figure 1Two-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 3β-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.

      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 granulosa-lutein cells of the CL. Inhibition of VEGF in vivo during the luteal phase in nonhuman primates prevents luteal angiogenesis and suppresses P secretion (
      • Fraser H.M.
      • Dickson S.E.
      • Lunn S.F.
      • Wulff C.
      • Morris K.D.
      • Carroll V.A.
      • et al.
      Suppression of luteal angiogenesis in the primate after neutralization of vascular endothelial growth factor.
      ).
      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 (
      • Fraser H.M.
      • Bell J.
      • Wilson H.
      • Taylor P.D.
      • Morgan K.
      • Anderson R.A.
      • et al.
      Localization and quantification of cyclic changes in the expression of endocrine gland vascular endothelial growth factor in the human corpus luteum.
      ). 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 (
      • Kupesic S.
      • Kurjak A.
      • Vujisic S.
      • Petrovic Z.
      Luteal phase defect: comparison between Doppler velocimetry, histological and hormonal markers.
      ).
      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-1β and TNF-α 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 (
      • Kohen P.
      • Castro A.
      • Caballero-Campo P.
      • Castro O.
      • Vega M.
      • Makrigiannakis A.
      • et al.
      Interleukin-1beta (IL-1beta) is a modulator of human luteal cell steroidogenesis: localization of the IL type I system in the corpus luteum.
      ). Interleukin-1 and TNF-α 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 (
      • Seli E.
      • Arici A.
      Endometriosis: interaction of immune and endocrine systems.
      ,
      • Kohen P.
      • Castro A.
      • Caballero-Campo P.
      • Castro O.
      • Vega M.
      • Makrigiannakis A.
      • et al.
      Interleukin-1beta (IL-1beta) is a modulator of human luteal cell steroidogenesis: localization of the IL type I system in the corpus luteum.
      ). 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.

      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 (
      • Gwynne J.T.
      • Strauss 3rd, J.F.
      The role of lipoproteins in steroidogenesis and cholesterol metabolism in steroidogenic glands.
      ). Human steroidogenic luteal cells take up lipoprotein-carried cholesterol (low-density lipoprotein) by endocytosis and also maintain stores of esterified cholesterol. High-density 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. The StAR is essential to this sterol translocation in response to tropic hormones, including LH and hCG (
      • Stocco D.M.
      • Clark B.J.
      Regulation of the acute production of steroids in steroidogenic cells.
      ,
      • Strauss 3rd, J.F.
      • Kallen C.B.
      • Christenson L.K.
      • Watari H.
      • Devoto L.
      • Arakane F.
      • et al.
      The steroidogenic acute regulatory protin (StAR): a window into the complexities of intracellular cholesterol trafficking.
      ).

      Luteal progesterone synthesis

      Three critical endocrine events support P secretion in primate ovarian physiology (
      • Stouffer R.L.
      Progesterone as a mediator of gonadotrophin action in the corpus luteum: beyond steroidogenesis.
      ): [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 3β-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 (
      • Chaffin C.L.
      • Dissen G.A.
      • Stouffer R.L.
      ). Conversely, StAR is found in high levels in the periovulatory human theca cells that are able to synthesize androgens from cholesterol (
      • Kiriakidou M.
      • McAllister J.M.
      • Sugawara T.
      • Strauss 3rd, J.F.
      Expression of steroidogenic acute regulatory protein (StAR) in the human ovary.
      ). 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 (
      • Devoto L.
      • Kohen P.
      • Gonzalez R.R.
      • Castro O.
      • Retamales I.
      • Vega M.
      • et al.
      Expression of steroidogenic acute regulatory protein in the human corpus luteum throughout the luteal phase.
      ).
      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 (
      • Sierralta W.D.
      • Kohen P.
      • Castro O.
      • Muñoz A.
      • Strauss III, J.F.
      • Devoto L.
      Ultrastructural and biochemical evidence for the presence of mature steroidogenic acute regulatory protein (StAR) in the cytoplasm of human luteal cells.
      ). 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 3β-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 (
      • Duncan W.C.
      • Cowen G.M.
      • Illingworth P.J.
      Steroidogenic enzyme expression in human corpora lutea in the presence and absence of exogenous human chorionic gonadotrophin (hCG).
      ). Several investigations have shown that luteal cells of different ages incubated with P5 result in a dramatic increase in P secretion, suggesting that 3β-HSD is not rate limiting in P production. Therefore, P450ssc and 3β-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 3β-HSD like epostane can interfere with P production in primates and humans and result in pregnancy termination (
      • Webster M.A.
      • Phipps S.L.
      • Gillmer M.D.
      Interruption of first trimester human pregnancy following epostane therapy. Effect of prostaglandin E2 pessaries.
      ,
      • Snyder B.W.
      • Schane H.P.
      Inhibition of luteal phase progesterone levels in the rhesus monkey by epostane.
      ).
      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 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 (
      • Andréen L.
      • Sundström-Poromaa I.
      • Bixo M.
      • Nyberg S.
      • Bäckström T.
      Allopregnanolone concentration and mood—a bimodal association in postmenopausal women treated with oral progesterone.
      ). The mRNA for 5α-reductase and 5β-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 (
      • Bäckström T.
      • Anderson A.
      • Baird D.T.
      • Selstam G.
      The human corpus luteum secretes 5 alpha-pregnane-3,20-dione.
      ,
      • Pearson-Murphy B.E.
      • Allison C.M.
      Determination of progesterone and some of its neuroactive ring A-reduced metabolites in human serum.
      ,
      • Ottander U.
      • Poromaa I.S.
      • Bjurulf E.
      • Skytt A.
      • Backstrom T.
      • Olofsson J.I.
      Allopregnanolone and pregnanolone are produced by the human corpus luteum.
      ).

      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 (
      • Sanders S.L.
      • Stouffer R.L.
      • Brannian J.D.
      Androgen production by monkey luteal cell subpopulations at different stages of the menstrual cycle.
      ), while large luteal cells are thought to be the primary site of luteal estrogen synthesis (
      • Ohara A.
      • Mori T.
      • Taii S.
      • Ban C.
      • Narimoto K.
      Functional differentiation in steroidogenesis of two types of luteal cells isolated from mature human corpora lutea of menstrual cycle.
      ), 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 (
      • Sanders S.L.
      • Stouffer R.L.
      Localization of steroidogenic enzymes in macaque luteal tissue during the menstrual cycle and simulated early pregnancy: immunohistochemical evidence supporting the two-cell model for estrogen production in the primate corpus luteum.
      ). 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 (
      • Ohara A.
      • Mori T.
      • Taii S.
      • Ban C.
      • Narimoto K.
      Functional differentiation in steroidogenesis of two types of luteal cells isolated from mature human corpora lutea of menstrual cycle.
      ,
      • Vega M.
      • Urrutia L.
      • Iniguez G.
      • Gabler F.
      • Devoto L.
      • Johnson M.C.
      Nitric oxide induces apoptosis in the human corpus luteum in vitro.
      ). It is well established that E2 synthesis by granulosa cells of the ovarian follicle is stimulated by FSH (
      • Hillier S.
      Paracrine control of follicular estrogens synthesis.
      ). However, FSH does not stimulate E2 synthesis by luteal cells in culture or sustain luteal steroidogenesis in vivo (
      • Devoto L.
      • Vega M.
      • Navarro V.
      • Sir T.
      • Alba F.
      • Castro O.
      Regulation of steroid hormone synthesis by human corpora lutea: failure of follicle-stimulating hormone to support steroidogenesis in vivo and in vitro.
      ). 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 (
      • Devoto L.
      • Kohen P.
      • Vega M.
      • Castro O.
      • González R.R.
      • Retamales I.
      • et al.
      Control of human luteal steroidogenesis.
      ). The action of FSH is evidently subserved by LH and IGF-1, which can sustain E2 by luteal cells in culture (
      • Johnson M.C.
      • Devoto L.
      • Retamales I.
      • Kohen P.
      • Troncoso J.L.
      • Aguilera G.
      Localization of insulin-like growth factor (IGF-I) and IGF-I receptor expression in human corpora lutea: role on estradiol secretion.
      ). 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 (
      • Baerwald A.R.
      • Adams G.P.
      • Pierson R.A.
      Characterization of ovarian follicular wave dynamics in women.
      ,
      • Ginther O.J.
      • Beg M.A.
      • Gastal E.L.
      • Gastal M.O.
      • Baerwald A.R.
      • Pierson R.A.
      Systemic concentration of hormones during the development of follicular waves in mares and women: a comparative study.
      ). 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 (
      • Baerwald A.R.
      • Adams G.P.
      • Pierson R.A.
      A new model for ovarian follicular development during the human menstrual cycle.
      ). 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 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 (
      • Hosokawa K.
      • Ottander U.
      • Wahlberg P.
      • Ny T.
      • Cajander S.
      • Olofsson I.J.
      Dominant expression and distribution of oestrogen receptor beta over oestrogen receptor alpha in the human corpus luteum.
      ).

      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 (
      • Stocco C.
      • Tellerías C.
      • Gibori G.
      The molecular control of corpus luteum formation, function and regresion.
      ). 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 (
      • Duncan W.C.
      • McNeilly A.S.
      • Fraser H.M.
      • Illingworth P.J.
      Luteinizing hormone receptor in the human corpus luteum: lack of down-regulation during maternal recognition of pregnancy.
      ).
      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-α, (PGF2-α), TNF-α, IL-1β, endothelin, monocyte chemoattractant (MCP-1), estrogens, and reactive oxygen species have been implicated in the luteolytic process (
      • Devoto L.
      • Vega M.
      • Kohen P.
      • Castro O.
      • Carvallo P.
      • Palomino A.
      Molecular regulation of progesterone secretion by the human corpus luteum throughout the menstrual cycle.
      ). In humans, the physiologic role of PGF2-α in luteolysis is uncertain because the ovarian production of PGF2-α is limited. In contrast, PGF2-α induces 20α-hydroxysteroid dehydrogenase (20α-HSD) expression in rodent luteal cells, which lose their capacity to secrete P (
      • Stocco C.
      • Tellerías C.
      • Gibori G.
      The molecular control of corpus luteum formation, function and regresion.
      ). It is interesting that PGF2-α suppresses StAR expression in cultured human granulosa-luteal cells (
      • Sasson R.
      • Winder N.
      • Kees S.
      • Amsterdam A.
      Induction of apoptosis in granulosa cells by TNF alpha and its attenuation by glucocorticoids involve modulation of Bcl-2.
      ). Estrogens inhibit the activity of luteal 3β-HSD by human luteal cells in vitro (
      • Vega M.
      • Devoto L.
      • Castro O.
      • Kohen P.
      Progesterone synthesis by human luteal cells: modulation by estradiol.
      ). Other investigators have postulated that the luteolytic action of exogenously administrated E2 reduces the secretion of pituitary-derived LH in women (
      • Gore B.Z.
      • Caldwell B.V.
      • Speroff L.
      Estrogen-induced human luteolysis.
      ). 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 (
      • Vega M.
      • Urrutia L.
      • Iniguez G.
      • Gabler F.
      • Devoto L.
      • Johnson M.C.
      Nitric oxide induces apoptosis in the human corpus luteum in vitro.
      ). Thus, the endothelial cell function of the CL may be affected, resulting in diminished expression of VEGF and molecules that promote endothelial cell survival (
      • Fraser H.M.
      • Bell J.
      • Wilson H.
      • Taylor P.D.
      • Morgan K.
      • Anderson R.A.
      • et al.
      Localization and quantification of cyclic changes in the expression of endocrine gland vascular endothelial growth factor in the human corpus luteum.
      ).
      Figure thumbnail gr2
      Figure 2Effects 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 = 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.
      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 (
      • Gaytan F.
      • Morales C.
      • García-Pardo L.
      • Reymundo C.
      • Bellido C.
      • Sánchez-Criado J.E.
      Macrophages, cell proliferation, and cell death in the human menstrual corpus luteum.
      ,
      • Fraser H.M.
      • Lunn S.F.
      • Harrison D.J.
      • Kerr J.B.
      Luteal regression in the primate: different forms of cell death during natural and gonadotropin-releasing hormone antagonist or prostaglandin analogue–induced luteolysis.
      ). 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 (
      • Shikone T.
      • Yamoto M.
      • Kokawa K.
      • Yamashita K.
      • Nishimori N.
      • Nakano R.
      Apoptosis of human corpora lutea during cyclic luteal regression and early pregnancy.
      ,
      • Yuan W.
      • Giudice L.C.
      Programmed cell death in human ovary is a function of follicle and corpus luteum status.
      ,
      • Vaskivuo T.
      • Ottander U.
      • Oduwole O.
      • Isomaa V.
      • Vihko P.
      • Olofsson J.
      • et al.
      Role of apoptosis, apoptosis-related factors and 17beta-hydroxysteroid dehydrogenases in human corpus luteum regression.
      ). 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 (
      • Rodger F.E.
      • Fraser H.M.
      • Duncan W.C.
      • Illingworth P.J.
      Immunolocalization of bcl-2 in the human corpus luteum.
      ), 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 (
      • Sugino N.
      • Suzuki T.
      • Kashida S.
      • Karube A.
      • Takiguchi S.
      • Kato H.
      Expression of Bcl-2 and Bax in the human corpus luteum the menstrual cycle and in early pregnancy: regulation by human chorionic gonadotropin.
      ). Moreover, the proapoptotic protein Bax has been reported to remain unchanged in the human CL throughout the luteal phase (
      • Rodger F.E.
      • Fraser H.M.
      • Krajewski S.
      • Illingworth P.J.
      Production of the proto-oncogene BAX does not vary with changing in luteal function in women.
      ), 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 N-cadherin 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 (
      • Makrigiannakis A.
      Novel trends in follicular development, atresia and corpus luteum regression: a role for apoptosis.
      ).
      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 (
      • Vega M.
      • Urrutia L.
      • Iniguez G.
      • Gabler F.
      • Devoto L.
      • Johnson M.C.
      Nitric oxide induces apoptosis in the human corpus luteum in vitro.
      ). 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 (
      • Fraser H.M.
      • Lunn S.F.
      • Harrison D.J.
      • Kerr J.B.
      Luteal regression in the primate: different forms of cell death during natural and gonadotropin-releasing hormone antagonist or prostaglandin analogue–induced luteolysis.
      ,
      • Del Canto F.
      • Sierralta W.
      • Kohen P.
      • Muñoz A.
      • Strauss III, J.
      • Devoto L.
      Features of natural and GnRH antagonist-induced corpus luteum regression and effects of in vivo human chorionic gonadotropin.
      ). 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 β-hCG vaccine to women inactivates endogenous hCG, resulting in a fall in P and menses (
      • Pal R.
      • Singh O.
      Absence of corpus luteum rescue by chorionic gonadotropin in women immunized with a contraceptive vaccine.
      ). 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 hypothalamic-pituitary-ovarian axis that begin during the periovulatory period of nonconception cycles (
      • Chen J.
      • Oiu O.
      • Lohstroh P.N.
      • Overstreet J.W.
      • Lasley B.L.
      Hormonal characteristics in the early luteal phase of conceptive an nonconceptive menstrual cycles.
      ). 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 17α-OHP, P, or E2. However, the serum level of 17α-OHP 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 (
      • Glock J.L.
      • Nakajima S.T.
      • Stewart D.R.
      • Badger G.J.
      • Brumsted J.R.
      The relationship of corpus luteum volume to relaxin, estradiol, progesterone, 17-hydroxyprogesterone and human chorionic gonadotropin levels in early normal pregnancy.
      ).
      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 (
      • Zeleznik A.J.
      In vivo responses of the primate corpus luteum to luteinizing hormone and chorionic gonadotropin.
      ). 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 (
      • Kohen P.
      • Castro O.
      • Palomino A.
      • Munoz A.
      • Christenson L.K.
      • Sierralta W.
      • et al.
      The steroidogenic response and corpus luteum expression of the steroidogenic acute regulatory protein after human chorionic gonadotropin administration at different times in the human luteal phase.
      ).
      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 (
      • Sugino N.
      • Suzuki T.
      • Kashida S.
      • Karube A.
      • Takiguchi S.
      • Kato H.
      Expression of Bcl-2 and Bax in the human corpus luteum the menstrual cycle and in early pregnancy: regulation by human chorionic gonadotropin.
      ).
      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 (
      • Makrigiannakis A.
      • Coukos G.
      • Christofidou-Solomidou M.
      • Montas S.
      • Coutifaris C.
      Progesterone is an autocrine/paracrine regulator of human granulosa cell survival in-vitro.
      ) and influence the expression of LHr (
      • Jones L.S.
      • Ottobre J.S.
      • Pate J.L.
      Progesterone regulation of luteinizing hormone receptors on cultured bovine luteal cells.
      ) and steroidogenic enzymes (
      • Chaffin C.L.
      • Dissen G.A.
      • Stouffer R.L.
      ). In addition, P has been implicated in the control of tissue inhibitors of metalloproteinase-1 expression in luteinized granulosa cells (
      • Morgan A.
      • Keble S.C.
      • London S.N.
      • Muse K.N.
      • Curry T.E.
      Antiprogesterone (RU486) effects on metalloproteinase inhibitor activity in human and rat granulosa cells.
      ). 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 (
      • Ottander U.
      • Hosokawak
      • Link N.T.
      • Olofsson J.L.
      A putative stimulatory role of progesterone acting via progesterone receptor in the steroidogenic cells of the human corpus luteum.
      ,
      • Misao R.
      • Nakanishi Y.
      • Fujimoto J.
      • Tamaya T.
      Expression of progesterone receptor isoform in corpora lutea of human subjects: correlation with serum oestrogen and progesterone concentration.
      ). Both receptor isoforms decrease in late luteal phase CL. In addition, CL does have membrane-linked P-binding 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 (
      • Vega M.
      • Devoto L.
      • Navarro V.
      • Castro O.
      • Kohen P.
      In vitro net progesterone production by human corpora lutea: effects of human chorionic gonadotropin, dibutyryl adenosine 3′,5′-monophosphate, cholera toxin, and forskolin.
      ,
      • Hild-Petito S.
      • Fazleabas A.T.
      Expression of steroid receptors and steroidogenic enzymes in the baboon (Papio anubis) corpus luteum during the menstrual cycle and early pregnancy.
      ). 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 (
      • Rothchild I.
      The corpus luteum revisited: are the paradoxical effects of RU486 a clue to how progesterone stimulates its own secretion?.
      ). 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 (
      • Duncan W.C.
      • Gay E.
      • Maybin J.A.
      The effect of human chorionic gonadotrophin on the expression of progesterone receptors in human luteal cells in vivo and in vitro.
      ). 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 (
      • Noyes R.W.
      • Hertig A.T.
      • Rock J.
      Dating the endometrial biopsy.
      ). Jones (
      • Jones G.
      The luteal phase defect.
      ) was the first to hypothesize that delayed 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 (
      • Fritz M.A.
      Inadequate luteal function and recurrent abortion: diagnosis and treatment of luteal phase deficiency.
      ,
      • Arredondo F.
      • Noble L.
      Endocrinology of recurrent pregnancy lost.
      ).
      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. (
      • Coutifaris C.
      • Myers E.R.
      • Guzick D.S.
      • Diamond M.P.
      • Carson S.A.
      • Legro R.S.
      • et al.
      NICHD National Cooperative Reproductive Medicine Network. Histological dating of timed endometrial biopsy tissue is not related to fertility status.
      ) 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. (
      • Murray M.J.
      • Meyer W.R.
      • Zaino R.J.
      • Lessey B.A.
      • Novotny D.B.
      • Ireland K.
      • et al.
      A critical analysis of the accuracy, reproducibility, and clinical utility of histologic endometrial dating in fertile women.
      ) 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 (
      • Tavaniotou A.
      • Smitz J.
      • Bourgain C.
      • Devroey P.
      Ovulation induction disrupts luteal phase function.
      ). 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. (
      • Metcalf M.G.
      • Evans J.J.
      • Mackenzie J.A.
      Indices of ovulation: comparison of plasma and salivary levels of progesterone with urinary pregnanediol.
      ) 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 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 (
      • Queenan J.T.
      • O'Brien G.D.
      • Bains L.M.
      • Simpson J.
      • Collins W.P.
      • Campbell S.
      Ultrasound scanning of ovaries to detect ovulation in women.
      ). With the advances in imaging technology, ovulation and the presence of the CL could be detected in almost 100% of women (
      • Queenan J.T.
      • O'Brien G.D.
      • Bains L.M.
      • Simpson J.
      • Collins W.P.
      • Campbell S.
      Ultrasound scanning of ovaries to detect ovulation in women.
      ). 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 (
      • Baerwald A.R.
      • Adams G.P.
      • Pierson R.A.
      Form and function of the corpus luteum during the human menstrual cycle.
      ).
      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 (
      • Baerwald A.R.
      • Adams G.P.
      • Pierson R.A.
      Form and function of the corpus luteum during the human menstrual cycle.
      ).
      Quantitative changes in luteal echo texture represent changes in the morphologic and physiologic status of the CL, as previously documented in domestic animal species (
      • Miyazaki T.
      • Tanaka M.
      • Miyakoshi K.
      • Minegishi K.
      • Kasai K.
      • Yoshimura Y.
      Power and colour Doppler ultrasonography for the evaluation of the vasculature of the human corpus luteum.
      ,
      • Singh J.
      • Pierson R.
      • Adams G.
      Ultrasound image attributes of the bovine corpus luteum: structural and functional correlates.
      ). Baerwald et al. (
      • Baerwald A.R.
      • Adams G.P.
      • Pierson R.A.
      Form and function of the corpus luteum during the human menstrual cycle.
      ) 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 (
      • Glock J.
      • Brumsted J.
      Color flow pulsed Doppler ultrasound in diagnosing luteal phase defect.
      ) 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. (
      • Kupesic S.
      • Kurjak A.
      • Vujisic S.
      • Petrovic Z.
      Luteal phase defect: comparison between Doppler velocimetry, histological and hormonal markers.
      ) 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 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 (
      • Weiner Z.
      • Thaler I.
      • Levron J.
      • Lewit N.
      • Itskovitz-Eldor J.
      Assessment of ovarian and uterine blood flow by transvaginal color Doppler in ovarian-stimulated women: correlation with the number of follicles and steroid hormone levels.
      ,
      • Tinkanen H.
      • Kujansuu E.
      • Laippala P.
      The association between hormone levels and vascular resistance in uterine and ovarian arteries in spontaneous menstrual cycles. A Doppler ultrasound study.
      ), but others were unable to find it (
      • Tan S.L.
      • Zaidi J.
      • Campbell S.
      • Doyle P.
      • Collins W.
      Blood flow changes in the ovarian and uterine arteries during the normal menstrual cycle.
      ). 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 (
      • Bourne T.H.
      • Hagström H.-G.
      • Hahlin M.
      • Josefsson B.
      • Granberg S.
      • Hellberg P.
      • et al.
      Ultrasound studies of vascular and morphological changes in the human corpus luteum during the menstrual cycle.
      ).
      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. (
      • Miyazaki T.
      • Tanaka M.
      • Miyakoshi K.
      • Minegishi K.
      • Kasai K.
      • Yoshimura Y.
      Power and colour Doppler ultrasonography for the evaluation of the vasculature of the human corpus luteum.
      ) 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. (
      • Merce L.T.
      • Bau S.
      • Bajo J.M.
      Doppler study of arterial and venous intraovarian blood flow in stimulated cycles.
      ) 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 (
      • Noyes R.W.
      • Hertig A.T.
      • Rock J.
      Dating the endometrial biopsy.
      ), endometrial protein expression (
      • Gonzalez R.R.
      • Palomino A.
      • Boric A.
      • Vega M.
      • Devoto L.
      A quantitative evaluation of alpha1, alpha4, alphaV and beta3 endometrial integrins of fertile and unexplained infertile women during the menstrual cycle. A flow cytometric appraisal.
      ), and ultrasound examination of the endometrium (
      • Ng E.H.
      • Chan C.C.
      • Tang O.S.
      • Yeung W.S.
      • Ho P.C.
      Comparison of endometrial and subendometrial blood flow measured by three-dimensional power Doppler ultrasound between stimulated and natural cycles in the same patients.
      ). Different ultrasound parameters have been suggested to assess endometrial receptivity, including endometrial thickness, endometrial echogenic pattern, and endometrial volume (
      • Ng E.H.
      • Chan C.C.
      • Tang O.S.
      • Yeung W.S.
      • Ho P.C.
      Endometrial and subendometrial blood flow measured during early luteal phase by three-dimensional power Doppler ultrasound in excessive ovarian responders.
      ,
      • Coulam C.B.
      • Bustillo M.
      • Soenksen D.M.
      • Britten S.
      Ultrasonographic predictors of implantation after assisted reproduction.
      ,
      • Ueno J.
      • Oehninger S.
      • Brzyski R.G.
      • Acosta A.A.
      • Philput C.B.
      • Muasher S.J.
      ).

       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 triple-line 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 more homogeneous, hyperechogenic endometrium is observed as endometrial glands expand under the influence of luteal P production in the secretory phase (
      • Baerwald A.R.
      • Pierson R.A.
      Endometrial development in association with ovarian follicular waves during the menstrual cycle.
      ).

       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 (
      • Goswamy R.K.
      • Steptoe P.C.
      Doppler ultrasound studies of the uterine artery in spontaneous ovarian cycles.
      ,
      • Sladkevicius P.
      • Valentin L.
      • Marsal K.
      Blood flow velocity in the uterine and ovarian arteries during the normal menstrual cycle.
      ,
      • Thompson R.S.
      • Trudinger B.J.
      • Cook C.M.
      Doppler ultrasound waveform indices: A/B ratio, pulsatility index, and Pourcelot ratio.
      ) 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 (
      • Long M.G.
      • Boultbee J.E.
      • Hanson M.E.
      • Begent R.H.J.
      Doppler time velocity waveform studies of the uterine artery and uterus.
      ,
      • Scholtes M.C.
      • Wladimiroff J.W.
      • van Rijen H.J.
      • Hop W.C.
      Uterine and ovarian flow velocity waveforms in the normal menstrual cycle: a transvaginal Doppler study.
      ).
      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 (
      • Dickey R.P.
      Doppler ultrasound investigation of uterine and ovarian blood flow in infertility and early pregnancy.
      ). Steer et al. (
      • Steer C.V.
      • Tan S.L.
      • Dillon D.
      • Mason B.A.
      • Campbell S.
      Vaginal colour Doppler assessment of uterine artery impedance correlates with immunohistochemical markers of endometrial respectively required for the implantation of an embryo.
      ) 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. (
      • Gannon B.J.
      • Carati C.J.
      • Verco C.J.
      Endometrial perfusion across the normal human menstrual cycle assessed by laser Doppler fluxometry.
      ) 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 three-dimensional (3D) ultrasound with power Doppler (
      • Schild R.L.
      • Holthanus S.
      • Alquen J.D.
      • Fimmers R.
      • Dorn C.
      • van der Ven H.
      • et al.
      Quantitative assessment of subendometrial blood flow by three-dimensional-ultrasound is an important predictive factor of implantation in an in-vitro fertilization programme.
      ,
      • Wu H.M.
      • Chiang C.H.
      • Huang H.Y.
      • Chao A.S.
      • Wang H.S.
      • Soong Y.K.
      Detection of the subendometrial vascularization flow index by three dimensional ultrasound may be useful for predicting the pregnancy rate for patients undergoing in vitro fertilization–embryo transfer.
      ). Power Doppler imaging is more sensitive than color Doppler imaging at detecting low velocity flow and hence improves the visualization of small vessels (
      • Guerriero S.
      • Ajossa S.
      • Lai M.P.
      • Risalvato A.
      • Paoletti A.M.
      • Melis G.B.
      Clinical applications of colour Doppler energy imaging in the female reproductive tract and pregnancy.
      ). 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.
      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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