Luteal Rescue Research Articles

HCG Administration in Luteal Rescue: Intracavitary or Subcutaneously in Agonist Induced Ovulation in IVF Cycles

Background: Ovarian hyperstimulation syndrome (OHSS) is characterized by collection of fluid in third spaces in in vitro fertilization (IVF) cycles and can result in the cancellation of the cycle and be fatal in 3 women out of 100,000. The aim of this study is to compare the admission of human chorionic gonadotropin (hCG) subcutaneously versus intracavitary during ovum pick-up (OPU) in agonist induced ovulation in IVF cycles in terms of pregnancy outcomes. Methods: This study was carried out in Kocaeli University Faculty of Medicine, Department of Obstetrics and Gynecology Assisted Reproductive Techniques Clinic as a retrospective study. 157 patients who underwent IVF treatment between January 2018 and February 2020, with ≥25 follicles detected in ultrasound and 1 mg of triptorelin acetate was administered for ovulation trigger, and 36 hours later of whom ≤20 oocytes were obtained in OPU were enrolled in this study. 109 patients who were administered 1500 IU of hCG subcutaneously belonged to Group 1, and 1500 IU hCG was administered intracavitary to 48 patients as Group 2. Results: Infertility causes, and characteristics of both groups were similar. Number of retrieved oocytes, Metaphase 2 (MII) oocytes and fertilization rates were similar in both groups. Implantation rate per embryo transferred was higher in the intracavitary group (p = 0.01). There was no significant difference for pregnancy rate, clinical pregnancy rate, ongoing pregnancy rate, livebirth rate and OHSS frequency between both groups. Twin pregnancy rate was significantly higher in the intracavitary group. Conclusions: Administration of 1500 IU hCG intracavitary at the time of OPU is associated with improved implantation rates when compared to subcutaneous hCG administration without a significant raise in OHSS occurrence. Prospective, randomized studies with bigger patient cohort are needed.

O-241 Elevated serum progesterone levels before frozen embryo transfer do not negatively impact reproductive outcomes: a large retrospective cohort study

Abstract Study question Do patients with high serum progesterone levels before frozen embryo transfer (FET) under hormonal replacement therapy (HRT) present worse reproductive outcomes? Summary answer Elevated serum progesterone levels before FET in artificially prepared cycles with vaginal or vaginal plus subcutaneous progesterone do not impair reproductive outcomes. What is known already Low serum progesterone levels before FET do negatively affect reproductive outcomes in terms of live birth rate. However, there is not robust data regarding the impact of high serum progesterone levels in the luteal phase of patients who undergo HRT for FET. Study design, size, duration Retrospective cohort study of 3183 blastocyst FET cycles under HRT performed in a university-affiliated fertility centre between March 2009 and December 2020. All the cycles presented adequate serum progesterone levels before FET (≥10.6 ng/ml). A total of 1360 cycles corresponded to frozen homologous embryo transfer (ET) (hom-FET), 1024 were euploid ET (eu-FET) after preimplantational genetic testing for aneuploidies (PGT-A), and 799 cycles were frozen heterologous ET (het-FET). The primary objective was live birth rate (LBR). Participants/materials, setting, methods Standard HRT was used. Luteal phase was covered with vaginal progesterone 200 mg/8h, or vaginal plus a daily subcutaneous injection of progesterone (25 mg). Serum progesterone levels were measured the day before FET. Elevated progesterone levels were considered in the 90th and 95th centiles. A generalized additive model (GAM) was performed to study the functional relationships between progesterone and LBR. A multivariable logistic regression was used to evaluate the effect of high progesterone over LBR. Main results and the role of chance Mean serum progesterone level before FET was 16.77±8.43 ng/ml. Progesterone levels were significantly higher in the group under vaginal plus subcutaneous progesterone (21.87±14.17 vs. 15.56±5.72, p < 0.001). No differences in clinical pregnancy, miscarriage and LBR were found according to the use of vaginal or vaginal plus subcutaneous progesterone for each of the groups (hom-FET, eu-FET and het-FET). Live birth rates were comparable among patients in the highest centile of serum progesterone levels (≥p90, ≥22.33 ng/ml) and the rest of patients (p < 90) (43.9 vs 41.3%; p = 0.381). Patients with progesterone levels ≥p90 presented lower BMI compared to those in the lower centiles (<p90) (22.62±3.82 vs. 23.32±4.06; P = 0.009). After dividing patients in deciles according to serum progesterone levels before, no differences in LBR were observed among groups (P = 0.938). No association was observed with GAM model between progesterone levels and LBR. A multivariable logistic regression adjusted by oocyte age, type of treatment and number of embryos transferred was applied for centile 90 and centile 95 of progesterone, and showed that serum progesterone in their highest levels did not negatively impact LBR. Limitations, reasons for caution The main limitation of this study is its retrospective design. The results only apply for patients under HRT with vaginal micronized progesterone alone or plus subcutaneous progesterone. Progesterone determination was measured before blastocyst FET. Extrapolation to other HRT protocols or timings of progesterone measurement needs to be validated. Wider implications of the findings The results of this study suggest that once a threshold of serum progesterone before FET is achieved, progesterone levels are not predictive of the clinical outcome. Actually, LBR are not negatively affected when progesterone levels are found in their highest centiles after luteal phase rescue with vaginal plus subcutaneous progesterone. Trial registration number Not applicable

Roadmap to pregnancy during the period of maternal recognition in the cow: Changes within the corpus luteum associated with luteal rescue

A viable corpus luteum (CL) producing an adequate amount of progesterone is absolutely essential for establishment and maintenance of pregnancy. One function of embryonic signaling to the mother is to ensure that the CL is maintained. In ruminants, the secretion of uterine prostaglandin F2alpha (PGF2A) is the signal that initiates luteolysis. Despite many studies to determine if PGF2A secretion from the uterus is altered in early pregnancy, conflicting interpretations have led to controversy regarding the exact mechanisms by which maternal recognition of pregnancy is achieved. In addition to alteration of uterine prostaglandin metabolism, changes within the CL itself may facilitate the establishment of a successful pregnancy. These changes include alteration of luteal blood flow, prostaglandin metabolism, sensitivity to prostaglandin actions, increased steroidogenic capacity, significant intracellular molecular changes and modification of the immune cells that are within the CL. Whether these intraluteal changes are necessary to establish pregnancy is undetermined. The focus of this review will be to provide a brief historical perspective on the utero-ovarian relationships that regulate luteal lifespan and review current knowledge of the CL of pregnancy in sheep and cattle.

Molecular profiling demonstrates modulation of immune cell function and matrix remodeling during luteal rescue†.

The corpus luteum (CL) is essential for maintenance of pregnancy in all mammals and luteal rescue, which occurs around day 16-19 in the cow, is necessary to maintain luteal progesterone production. Transcriptomic and proteomic profiling were performed to compare the day 17 bovine CL of the estrous cycle and pregnancy. Among mRNA and proteins measured, 140 differentially abundant mRNA and 24 differentially abundant proteins were identified. Pathway analysis was performed using four programs. Modulated pathways included Tcell receptor signaling, vascular stability, cytokine signaling, and extracellular matrix remodeling. Two mRNA that were less in pregnancy were regulated by prostaglandin F2A in culture, while two mRNA that were greater in pregnancy were regulated by interferon tau. To identify mRNA that could be critical regulators of luteal fate, the mRNA that were differentially abundant during early pregnancy were compared to mRNA that were differentially abundant during luteal regression. Eight mRNA were common to both datasets, including mRNA related to regulation of steroidogenesis and gene transcription. A subset of differentially abundant mRNA and proteins, including those associated with extracellular matrix functions, were predicted targets of differentially abundant microRNA (miRNA). Integration of miRNA and protein data, using miRPath, revealed pathways such as extracellular matrix-receptor interactions, abundance of glutathione, and cellular metabolism and energy balance. Overall, this study has provided a comprehensive profile of molecular changes in the corpus luteum during maternal recognition of pregnancy and has indicated that some of these functions may be miRNA-regulated.

Prolonged luteal lifespan and pseudopregnancy in Asian elephants (Elephas maximus)

Pseudopregnancy is a physiological occurrence in mammals which have copulation induced ovulation, but is rarely described in spontaneous ovulating species. In this study, three cases of prolonged luteal lifespan are reported in non-pregnant Asian elephants (Elephas maximus). Case 1 was a 25-year-old female that had produced three calves previously; Case 2 was a nulliparous and 32-year-old at the start of the pseudopregnancy episode; and Case 3 occurred in a 49-year-old nulliparous elephant. Serum progesterone metabolite concentrations remained elevated for 10 months in Case 1. Urinary progestagens were high for >16 months in Case 2 and for five months in Case 3. In Case 1, multiple persistent corpora lutea were visualized monthly by ultrasonography. In all three cases, uterine leiomyoma were present and progestagen concentrations decreased spontaneously. In Case 1, the elephant became pregnant 3 years later, whilst with Case 2, the female resumed estrous cycling normally, and for the Case 3 female, there was continuation with another prolonged luteal phase before ovarian function was purposely suppressed. These examples indicate that persistently elevated progestagen concentrations may not always be indicative of pregnancy in elephants. The reasons for prolonged luteal lifespan are not understood, although serum prolactin concentrations quantified in the Case 1 female were elevated compared to values from previous reports and two other herd mates. Furthermore, all three elephants had varying degrees of uterine pathologies. It is believed that the resulting damage to the endometrium may have led to a reaction similar to implantation, which includes prolactin secretion. Prolactin may exert luteotropic properties and is thought to initiate luteal rescue during pregnancy in elephants.

Ovarian hyperstimulation syndrome after GnRH agonist trigger and rhCG luteal rescue protocol

Ovarian hyperstimulation syndrome (OHSS) is a rare, but serious iatrogenic complication of In Vitro Fertilization treatments. Since 1943, when the first cases of OHSS were reported, strategies to prevent it have not been very successful. At present, the most used strategy to prevent OHSS is the “freeze-all strategy”, which consists in the utilization of a gonadotropin releasing hormone (GnRH) agonist trigger for oocytes maturation in a GnRH antagonist protocol, followed by cryopreservation of all embryos. In this paper, we present a case of a young woman who developed OHSS after GnRH agonist trigger followed by rhCG luteal rescue protocol. By reporting this case, we want to highlight the importance of OHSS prevention and patients’ selection for hCG luteal rescue protocol.

The loss of luteal progesterone production in women is associated with a galectin switch via α2,6-sialylation of glycoconjugates.

Luteal progesterone is fundamental for reproduction, but the molecular regulation of the corpus luteum (CL) in women remains unclear. Galectin-1 and galectin-3 bind to the sugar chains on cells to control key biological processes including cell function and fate. The expression and localization of LGALS1 and LGALS3 were analyzed by quantitative PCR and histochemical analysis, with special reference to α2,6-sialylation of glycoconjugates in carefully dated human CL collected across the menstrual cycle and after exposure to human chorionic gonadotrophin (hCG) in vivo. The effects of hCG and prostaglandin E2 on the expression of galectins and an α2,6-sialyltransferase 1 (ST6GAL1) in granulosa lutein cells were analyzed in vitro. Galectin-1 was predominantly localized to healthy granulosa lutein cells and galectin-3 was localized to macrophages and regressing granulosa lutein cells. Acute exposure to luteotrophic hormones (hCG and prostaglandin E2) up-regulated LGALS1 expression (P < .001). ST6GAL1, which catalyzes α2,6-sialylation to block galectin-1 binding, increased during luteolysis (P < .05) as did LGALS3 (P < .05). Luteotrophic hormones reduced ST6GAL1 and LGALS3 in vivo (P < .05) and in vitro (P < .001). There was an inverse correlation between the expression of ST6GAL1 and HSD3B1 (P < .01) and a distinct cellular relationship among α2,6-sialylation, 3β-hydroxysteroid dehydrogenase, and galectin expression. Galectin-1 is a luteotrophic factor whose binding is inhibited by α2,6-sialylation in the human CL during luteolysis. ST6GAL1 and galectin-3 expression is increased during luteolysis and associated with a loss of progesterone synthesis. Luteotrophic hormones differentially regulate galectin-1 and galectin-3/α2,6-sialylation in granulosa lutein cells, suggesting a novel galectin switch regulated by luteotrophic stimuli during luteolysis and luteal rescue.

HCG (1500IU) administration on day 3 after oocytes retrieval, following GnRH-agonist trigger for final follicular maturation, results in high sufficient mid luteal progesterone levels - a proof of concept

BackgroundControlled ovarian hyperstimulation (COH) which combining GnRH antagonist co-treatment and GnRH agonist trigger with an additional 1500 IU hCG luteal rescue on day of oocytes retrieval, has become a common tool aiming to reduce severe ovarian hyperstimulation syndrome (OHSS). In the present, proof of concept study, we evaluate whether by deferring the hCG rescue bolus for 3 days, we are still able to rescue the luteal phase.MethodsPatients undergoing the GnRH-antagonist protocol, who were considered at high risk for developing severe OHSS and received GnRH-agonist for final oocyte maturation, were included. For luteal phase support, all patients received an “intense” luteal support. Those who had no signs of early moderate OHSS on day 3 after oocytes retrieval were instructed to inject 1500 IU of HCG bolus (hCG group). Ovarian stimulation characteristics and mid luteal progesterone levels were compared between those who received the HCG bolus and those who did not.ResultsEleven IVF cycles were evaluated, 5 in the hCG group and 6 in the intense luteal support only group. While no in-between group differences were observed in ovarian stimulation characteristics, significantly higher mid luteal progesterone levels (>127 nmol/L vs 42.1 ± 14.5 nmol/L, respectively) and a non-significant increase in pregnancy rate (40% vs 16.6%, respectively), were observed in those who receive the hCG bolus compared to those who did not.ConclusionshCG luteal rescue should be offered 3 days after oocytes retrieval only to those patients with no signs of early moderate OHSS. Further studies are required to elucidate the appropriate regimen of luteal HCG administration in patients undergoing final follicular maturation with GnRH-agonist.

Severe early ovarian hyperstimulation syndrome following GnRH agonist trigger with the addition of 1500 IU hCG

Is severe early ovarian hyperstimulation syndrome (OHSS) completely prevented with the GnRH agonist trigger and 1500 IU hCG luteal rescue protocol? Severe early OHSS can occur even after the GnRH agonist trigger and 1500 IU hCG luteal rescue protocol. Prior studies including over 200 women who received the GnRH agonist trigger and 1500 hCG luteal rescue protocol have reported complete prevention of severe early OHSS. Only a few late OHSS cases have been reported and it has been suggested that this protocol can be safely applied to any women under risk. This retrospective cohort study included all women who were at high risk of OHSS and were given the GnRH agonist trigger plus hCG luteal rescue protocol between December 2008 and August 2012 in the two participating centers. There were 23 women with a mean estradiol level of 4891 ± 2214 pg/ml and a mean number of >12 mm follicles of 20 ± 6 on the day of ovulation triggering. OHSS was categorized according to the Golan criteria. Overall 6 of the 23 (26%) women developed severe OHSS. Five women had severe early OHSS requiring ascites drainage and hospitalization and three of these women did not undergo embryo transfer. The number of follicles measuring 10-14 mm on the day of triggering was significantly different between women who developed severe early OHSS and those who did not. The small number of women with severe early OHSS may have prevented identification of other significant risk factors. Although the GnRH agonist plus 1500 IU hCG luteal rescue protocol significantly decreases the risk of severe OHSS, this life threatening complication can still occur in high-risk patients. It would be prudent to avoid hCG luteal rescue and freeze all embryos for future transfer in such women particularly when there are ≥18 follicles with 10-14 mm diameters even with few larger follicles.

Ovarian hyperstimulation syndrome- an optimal solution for an unresolved enigma

Ovarian hyperstimulation syndrome (OHSS) is a serious complication of controlled ovarian hyperstimulation (COH). The syndrome almost always presents either after hCG administration in susceptible patients or during early pregnancy. Despite many years of clinical experience, there are no precise methods to completely prevent severe OHSS, except by withholding the ovulation-inducing trigger of hCG. Recently, COH which combining GnRH antagonist co-treatment and GnRH agonist trigger has become a common tool aiming to eliminate severe early OHSS. However, the observed decrease in implantation and pregnancy rates following this approach has encouraged different modifications of luteal support aiming to improve outcome. One of the suggest approach is the 1500 IU hCG luteal rescue, which appears to be a promising protocol, aiming to reduce (rather than eliminating) severe early OHSS, without compromising outcome. In the present paper we discuss the different suggested strategies and offer a strict triage, aimed at eliminating the occurrence of severe OHSS based on several clinical observations, including the role of GnRH-antagonist in COH protocols, the use of different luteal rescue protocols and the ability to transfer embryos in the blastocyst stage.

Gestating for 22 months: luteal development and pregnancy maintenance in elephants

The corpus luteum, a temporally established endocrine gland, formed on the ovary from remaining cells of the ovulated follicle, plays a key role in maintaining the early mammalian pregnancy by secreting progesterone. Despite being a monovular species, 2-12 corpora lutea (CLs) were found on the elephant ovaries during their long pregnancy lasting on average 640 days. However, the function and the formation of the additional CLs and their meaning remain unexplained. Here, we show from the example of the elephant, the close relationship between the maternally determined luteal phase length, the formation of multiple luteal structures and their progestagen secretion, the timespan of early embryonic development until implantation and maternal recognition. Through three-dimensional and Colour Flow ultrasonography of the ovaries and the uterus, we conclude that pregnant elephants maintain active CL throughout gestation that appear as main source of progestagens. Two LH peaks during the follicular phase ensure the development of a set of 5.4 ± 2.7 CLs. Accessory CLs (acCLs) form prior to ovulation after the first luteinizing hormone (LH) peak, while the ovulatory CL (ovCL) forms after the second LH peak. After five to six weeks (the normal luteal phase lifespan), all existing CLs begin to regress. However, they resume growing as soon as an embryo becomes ultrasonographically apparent on day 49 ± 2. After this time, all pregnancy CLs grow significantly larger than in a non-conceptive luteal phase and are maintained until after parturition. The long luteal phase is congruent with a slow early embryonic development and luteal rescue only starts 'last minute', with presumed implantation of the embryo. Our findings demonstrate a highly successful reproductive solution, different from currently described mammalian models.

Oocyte maturation employing a GnRH agonist in combination with low-dose hCG luteal rescue minimizes the severity of ovarian hyperstimulation syndrome while maintaining excellent pregnancy rates

The traditional hCG 'trigger' for initiating final oocyte maturation exacerbates ovarian hyperstimulation syndrome (OHSS) in patients with an excessive follicular response because of its sustained stimulatory effect on the corpora lutea. However, a GnRH agonist trigger can produce a short duration endogenous LH surge which is adequate to initiate oocyte maturation, but allows the corpora lutea to regress, reducing the severity of OHSS. This approach produces an excellent embryology outcome, but generally results in low pregnancy rates even with the early initiation of estrogen and progesterone luteal support. The purpose of this study was to determine if a low dose of hCG (1500 IU) support on the day of oocyte retrieval can maintain good pregnancy rates, while not abolishing the protective effect of an agonist trigger on the development of severe OHSS. This retrospective study included 71 women who were at high risk of severe OHSS (≥ 14 follicles ≥ 12 mm) and who received an agonist trigger for final oocyte maturation. The transfer of a solitary embryo produced a biochemical pregnancy rate of 60.6% and a clinical ongoing pregnancy rate of 52.1%. Only one patient was hospitalized with severe OHSS (1.4%), despite the average patient producing nearly 17 oocytes per cycle. Oocyte maturation employing a GnRH agonist (protocol) in combination with low-dose hCG luteal support produces excellent clinical pregnancy rates, while not compromising the ability of GnRH agonist to prevent severe OHSS.

Human Chorionic Gonadotropin Up-Regulates Expression of Myeloid Cell Leukemia-1 Protein in Human Granulosa-Lutein Cells: Implication of Corpus Luteum Rescue and Ovarian Hyperstimulation Syndrome

The corpus luteum is a dynamic endocrine structure with periodic development and regression during menstrual cycles. Its lifespan can be prolonged by human chorionic gonadotropin (hCG). However, the signal mechanisms of this phenomenon remain unclear. Our objective was to investigate the molecular mechanisms of hCG in the maintenance of the viability of granulosa-lutein cells. Granulosa-lutein cells were obtained from women undergoing in vitro fertilization. We examined the effects of hCG on the survival of cultured granulosa-lutein cells. The signal pathway inducing antiapoptotic protein was investigated. hCG enhanced viability of granulosa-lutein cells through antiapoptosis but not proliferation, because the apoptotic marker of annexin V was decreased, but the proliferative markers of Ki67 and proliferating cell nuclear antigen were not increased. Myeloid cell leukemia-1 (Mcl-1) protein, but not B-cell lymphoma protein-2 or B-cell lymphoma protein-xL, was significantly induced by hCG and LH. The granulosa-lutein cells secreted vascular endothelial growth factor that induced endothelial permeability. Mcl-1 small interfering RNA increased DNA fragmentation and diminished the antiapoptotic effect of hCG. hCG induced Mcl-1 expression through the LH/hCG receptor, adenylate cyclase, protein kinase A, and cAMP response element-binding protein signal pathway. Flavopiridol inhibited Mcl-1 production, released cytochrome c, and induced apoptosis of granulosa-lutein cells. We first demonstrate that hCG prevents apoptosis of granulosa-lutein cells through the induction of Mcl-1 protein via the LH/hCG receptor and a cAMP response element-binding protein-dependent pathway. We may have found the molecular mechanism for luteal rescue during early pregnancy. Mcl-1 prevents apoptosis and increases cell viability but not proliferation as mechanisms for luteal rescue. Mcl-1 is a key molecule of hCG signaling.

Vascular Changes in the Corpus Luteum During Early Pregnancy in the Cow

The present study determined vascular changes in the bovine corpus luteum (CL) at Day 16 (early maternal recognition period) and Day 40 in early pregnancy and compared them to the CL from Day 12 and Day 16 of the estrous cycle. The CLs were analyzed in the central and peripheral regions, where site-depending features of vessels and angiogenic factors are evident. The same protein level of the endothelial cell marker von Willebrand factor was retained in the CL from Day 16 of the estrous cycle to Day 40 of early pregnancy. The protein level of pericytes and smooth muscle cells was determined using smooth muscle alpha-actin; the level decreased at Day 40 of early pregnancy in both regions of the CL. No significant change in the expressions of vascular endothelial growth factors VEGF(164) and VEGF(120) mRNA occurred from Day 16 of the estrous cycle until Day 40 of early pregnancy. Angiopoietin (ANGPT)-2 / ANGPT-1 mRNA ratio (an index of instability of vasculature) increased in the periphery at Day 16 of the estrous cycle and then decreased until Day 40 of early pregnancy. The results suggest that there is no difference in vascular structure between non-pregnant and pregnant luteal tissue during the early maternal recognition period (Day 16). Also, luteal rescue by early pregnancy may be not associated with further blood vessel formation but rather may be related to the decrease of blood vessels per unit of area and blood vessel stabilization in the bovine CL.

Genome-Wide Gene Expression Analysis Reveals a Dynamic Interplay between Luteotropic and Luteolytic Factors in the Regulation of Corpus Luteum Function in the Bonnet Monkey (Macaca radiata)

Although LH is essential for survival and function of the corpus luteum (CL) in higher primates, luteolysis occurs during nonfertile cycles without a discernible decrease in circulating LH levels. Using genome-wide expression analysis, several experiments were performed to examine the processes of luteolysis and rescue of luteal function in monkeys. Induced luteolysis with GnRH receptor antagonist (Cetrorelix) resulted in differential regulation of 3949 genes, whereas replacement with exogenous LH (Cetrorelix plus LH) led to regulation of 4434 genes (1563 down-regulation and 2871 up-regulation). A model system for prostaglandin (PG) F(2alpha)-induced luteolysis in the monkey was standardized and demonstrated that PGF(2alpha) regulated expression of 2290 genes in the CL. Analysis of the LH-regulated luteal transcriptome revealed that 120 genes were regulated in an antagonistic fashion by PGF(2alpha). Based on the microarray data, 25 genes were selected for validation by real-time RT-PCR analysis, and expression of these genes was also examined in the CL throughout the luteal phase and from monkeys treated with human chorionic gonadotropin (hCG) to mimic early pregnancy. The results indicated changes in expression of genes favorable to PGF(2alpha) action during the late to very late luteal phase, and expressions of many of these genes were regulated in an opposite manner by exogenous hCG treatment. Collectively, the findings suggest that curtailment of expression of downstream LH-target genes possibly through PGF(2alpha) action on the CL is among the mechanisms underlying cross talk between the luteotropic and luteolytic signaling pathways that result in the cessation of luteal function, but hCG is likely to abrogate the PGF(2alpha)-responsive gene expression changes resulting in luteal rescue crucial for the maintenance of early pregnancy.

Novel Regulated Expression of the SLIT/ROBO Pathway in the Ovary: Possible Role during Luteolysis in Women

The human corpus luteum (CL) undergoes luteolysis, associated with marked tissue and vascular remodeling, unless conception occurs and the gland is rescued by human chorionic gonadotropin (hCG). In Drosophila the Slit gene product, a secreted glycoprotein, acts as a ligand for the roundabout (robo) transmembrane receptor. Together they influence the guidance and migration of neuronal and nonneuronal cells. In vertebrates three Slit (Slit1, Slit2, Slit3) and four Robo (Robo1, Robo2, Robo3/Rig-1, Robo4/Magic Robo) genes have been identified. ROBO1, SLIT2, and SLIT3 are also inactivated in human cancers and may regulate apoptosis and metastasis. Because processes such as apoptosis and tissue remodeling occur during the regression of the CL, the aim of this study was to investigate the expression, regulation, and effects of the SLIT and ROBO genes in human luteal cells. Immunohistochemistry and RT-PCR revealed that SLIT2, SLIT3, ROBO1, and ROBO2 are expressed in luteal steroidogenic cells and fibroblast-like cells of the human CL. Furthermore, using real-time quantitative PCR, expression of SLIT2, SLIT3, and ROBO2 was maximal in the late-luteal phase and significantly reduced after luteal rescue in vivo with exogenous hCG (P<0.05). Additionally, hCG significantly inhibited SLIT2, SLIT3, and ROBO2 expression in cultured luteinized granulosa cells (P<0.05). Blocking SLIT-ROBO activity increased migration and significantly decreased levels of apoptosis in primary cultures of luteal cells (P<0.05). Overall, these results suggest the SLIT/ROBO pathway could play an important role in luteolysis in women.

Angiogenesis in the human corpus luteum.

Angiogenesis is important for the formation and development of the corpus luteum and for maintenance of luteal function. Blood vessel regression is an important physiological phenomenon in the corpus luteum, which is associated with tissue involution during structural luteolysis. Angiogenesis actively occurs during the early luteal phase and is completed by the mid-luteal phase. Perivascular cells (pericytes) increase in number from the early luteal phase to the mid-luteal phase, suggesting that blood vessels are gradually stabilized until the mid-luteal phase. In the corpus luteum undergoing luteolysis, blood vessels and pericytes decrease in number, which is related to structural involution. In the corpus luteum of early pregnancy, the number of blood vessels with pericytes increases, suggesting that angiogenesis occurs again, accompanied by blood vessel stabilization. These changes in vasculature of the corpus luteum are regulated by the collaboration with vascular endothelial growth factor, which is involved in proliferation of vascular endothelial cells, and angiopoietins, which are involved in stabilization of blood vessels. This review focuses on angiogenesis, blood vessel stabilization and blood vessel regression during the divergent phases of luteal formation, luteal regression and luteal rescue by pregnancy. (Reprod Med Biol 2008; 7: 91-103).

Expression and regulation of oestrogen receptors in the human corpus luteum

The molecular mechanisms underlying the control of corpus luteum lifespan in women are not fully understood. Oestradiol has various luteolytic, or luteotrophic, functions in some species, and as it is synthesised within the human corpus luteum, it is an excellent candidate molecule to be a paracrine regulator of luteal function. This study aimed to comprehensively investigate the expression, regulation and effects of oestrogen receptors (ER) in human luteal cells. Genomic oestrogen receptors ERalpha, ERbeta1 and ERbeta2 were immunolocalised in human corpora lutea from throughout the luteal phase. mRNA expression was investigated throughout the luteal phase and after luteal rescue with exogenous human chorionic gonadotrophin (hCG). The regulation of ER expression and oestradiol action was investigated in cultures of luteinised granulosa cells. ER subtypes ERbeta1 and ERbeta2 were localised throughout the luteal phase to steroidogenic cells in the human corpus luteum and cells of the surrounding stroma. Unlike follicular granulosa cells, steroidogenic cells in the corpus luteum showed minimal ERalpha immunostaining. The presence of endothelial cells in the granulosa cell layer with ERbeta1 and ERbeta2 positive nuclei was noted. ERbeta1 and ERbeta2 were differentially regulated across the luteal phase with ERbeta1 maximally expressed in the mid-luteal phase, while ERbeta2 expression was maximal in the early luteal phase. In vivo and in vitro, hCG had no long-term effect on ER expression, although in vitro hCG and oestradiol acutely down-regulated ERs. Treatment with oestradiol in vitro down-regulated 11beta-hydroxysteroid dehydrogenase type 1 and inhibin betaA subunit confirming a functional oestradiol response. These data highlight functional and differentially regulated oestradiol reception in human luteal cells.

Role of Luteal Glucocorticoid Metabolism during Maternal Recognition of Pregnancy in Women

The human corpus luteum (hCL) is an active, transient, and dynamic endocrine gland. It will experience extensive tissue and vascular remodeling followed by 1) demise of the whole gland without any apparent scarring or 2) maintenance of structural and functional integrity dependent on conceptus-derived human chorionic gonadotropin (hCG). Because cortisol has well-characterized roles in tissue remodeling and repair, we hypothesized that it may have a role in controlling luteal dissolution during luteolysis and would be locally produced toward the end of the luteal cycle. Glucocorticoid-metabolizing enzymes [11beta-hydroxysteroid dehydrogenase (11betaHSD) types 1 and 2] and the glucocorticoid receptor (GR) were assessed in hCL and cultures of luteinized granulosa cells (LGC) using immunofluorescence and quantitative RT-PCR. Furthermore, the effect of cortisol on steroidogenic cell survival and fibroblast-like cell activity was explored in vitro. The hCL expressed 11betaHSD isoenzymes in LGC and nuclear GR in several cell types. hCG up-regulated the expression and activity of 11betaHSD type 1 (P < 0.05) and down-regulated type 2 enzyme (P < 0.05) in vitro and tended to do the same in vivo. Cortisol increased the survival of LGC treated with RU486 (P < 0.05) and suppressed the activity of a proteolytic enzyme associated with luteolysis in fibroblast-like cells (P < 0.05). Our results suggest that, rather than during luteolysis, it is luteal rescue with hCG that is associated with increased local cortisol generation by 11betaHSD type 1. Locally generated cortisol may therefore act on the hCL through GR to have a luteotropic role in the regulation of luteal tissue remodeling during maternal recognition of pregnancy.

IS THE UPREGULATION OF VEGF BY hCG IN HUMAN LUTEAL CELLS DEPENDENT ON HIF-1ALPHA EXPRESSION?

Angiogenesis is essential for normal growth and function of the corpus luteum. Several lines of research have demonstrated the importance of Vascular Endothelial Growth Factor (VEGF) in corpus luteum angiogenesis. Generally it is believed that hypoxia is the primary inducer of VEGF production in most tissues. Indeed, experiments using non-human primate cells suggested that hypoxia was a major regulator of luteal VEGF expression. However we have previously shown that VEGF is upregulated by human chorionic gonadotropin (hCG) in the human corpus luteum. As Hypoxia Inducible Factor-1alpha (HIF-1alpha) has been shown to regulate the expression of VEGF under non-hypoxic (i.e. ligand stimulated) conditions, as well as under hypoxic conditions, in cell lines, we hypothesised that the effect of hCG on luteal VEGF expression was mediated through HIF-1alpha. We therefore studied the effect of hCG on VEGF and HIF-1á expression in cultured luteinized granulosa cells (LGCs) in vitro and in human corpora lutea in vivo. LGCs were obtained from women undergoing oocyte recovering as part of an assisted conception program. Isolated LGCs were cultured under serum-free conditions, on Matrigel-coated plastic tissue culture plates, for one week and the culture medium was refreshed every two days. Cells were then treated with hCG alone (100 ng/ml) or hCG in the presence of synthetic inhibitory molecules before mRNA was collected. The expression of VEGF and HIF-1alpha mRNA was analyzed by Taqman quantitative real-time PCR. In vitro, hCG up-regulated both VEGF (P<0.0001) and HIF-1alpha (P<0.0001) mRNA transcripts in LGCs. In vivo, expression was investigated in corpora lutea from the late-luteal phase in the presence (n=5) or absence (n=5) of exogenous hCG concentrations similar to those seen in early pregnancy. Both VEGF (P=0.02) and HIF-1alpha (P=0.034) mRNA expression was upregulated by hCG during luteal rescue when compared to that in the late-luteal phase. To investigate whether the effect of hCG on HIF-1alpha/VEGF expression was under influence of progesterone, LGCs were stimulated with hCG in the presence or absence of the progesterone synthesis inhibitor aminoglutethamide (100 μM). The up-regulation of both HIF-1alpha and VEGF by hCG was independent of progesterone in vitro. As both the Raf/MEK/MAPK and the PI3 kinase/Akt signalling pathways have been shown to stimulate VEGF mRNA expression through the regulation of HIF-1alpha activity in a range of cell lines, LGCs were stimulated with hCG in the presence or absence of specific synthetic inhibitors of the Raf/MEK/MAPK (PD98059 (50 μM)) and PI3 kinase/Akt (LY294002 (50 μM)) pathways. PD98059 and LY294002 inhibited both VEGF and HIF-1alpha mRNA expression, whereas the mRNA expression of steroidogenic acute regulatory protein (StAR), which is regulated through a cAMP-dependent PKA pathway, was not affected. In summary, our results suggest that the expression of HIF- 1alpha is hormonally regulated in human luteal cells under normoxic conditions in vitro and in vivo and this may in turn regulate VEGF expression. (poster)