Effect of in vivo GnRH agonist and GnRH antagonist on hCG and insulin-stimulated progesterone production by human granulosa-lutein cells in vitro

Purpose : To investigate hCG and insulin-stimulated progesterone (P) production by human granulosa-lutein cells (hGLC) in vitro. Methods : hGLCs were isolated from patients undergoing IVF-ET cycles in which GnRH agonist or GnRH antagonist was used to prevent a midcycle gonadotropin surge. The cells were cultured for 3 days, and then treated with hCG 0.5, 1, and 10 IU/I, and insulin 0.01, 0.1, and 1 M in serum free conditions. In vitro P production was measured by enzyme immunoassay. Results : hCG stimulated P production by hGLCs from cycles in which GnRH antagonist was used, but a blunted response was seen in GnRH-agonist treated cycles. Insulin-stimulated P production was similar in cells from cycles in which GnRH-agonist or GnRH-antagonist treatment was used. Conclusions : Because insulin and hCG may share common pathways beyond the level of receptor activation, we hypothesize that GnRH agonist, but not GnRH antagonist, may affect the expression and/or activation of LH receptors in the hGLCs.     

Possible involvement of placental peptidases that degrade gonadotropin-releasing hormone (GnRH) in the dynamic pattern of placental hCG secretion via GnRH degradation

The presence of an extrahypothalamic gonadotropin releasing hormone (GnRH) in human placenta is well known and this decapeptide is presumed to play an important role in the regulation of the function and growth of human placenta. Immunohistochemistry showed that neutral endopeptidase 24.11 (NEP), a candidate of the responsible enzyme of GnRH degradation, is highly expressed on the cell surface of trophoblasts. Hydrolysis of GnRH by human villi was studied by measuring liberated amino acids using high performance liquid chromatography. The GnRH degrading activity was 1.53 times higher after incubation with the membrane fraction of first trimester villi than that after incubation with the membrane fraction of term villi. Phosphoramidon, a potent inhibitor of NEP, reduced the liberated amino acids to about a half, suggesting that NEP is a responsible enzyme for GnRH degradation. Ubenimex, which can inhibit several aminopeptidases, also reduced the liberated amino acids to about 50 per cent. O-phenanthroline, EDTA, and thiorphan could inhibit GnRH degradation but inhibitors of post proline endopeptidase could not. Furthermore, GnRH degrading activity of the membrane fraction was reduced remarkably after the membrane fraction was immunotitrated by anti NEP and anti placental leucine aminopeptidase (P-LAP) IgG. In conclusion, NEP and P-LAP are responsible enzymes for GnRH degradation in human villi.     

The human first-term placenta in vitro: regulation of hCG secretion by GnRH and its antagonist.

Gonadotropin releasing hormone (GnRH) has been proposed to play a role in the regulation of human chorionic gonadotropin (hCG) release from the human placenta. To test this assumption, we utilized an in vitro perifusion system, together with cultures of placental explants, to investigate short- and long-term effects of GnRH and its respective antagonist on the hCG secretion from the early human placenta. Tissue slices of human placenta (100 mg), obtained from first-trimester terminations of pregnancies, were continuously perifused and the effluent collected in fractions of 2-20 min. After initial perifusion periods of 30-40 min, either GnRH, a GnRH antagonist (SB-75; Asta Pharma, Frankfurt, Germany) or both compounds at equimolar concentrations were added to the perifusion medium at final concentration of 10(-4)-10(-8) mol/l). Administration was effected either continuously or intermittently in 10-min pulses. Further, 50-mg pieces of placental tissue explants were cultured in tissue culture plates for up to 6 days. During the perifusions, hCG (determined by enzymeimmunoassay) was found to be released spontaneously in a pulsatile fashion. Pulse amplitudes and frequencies of this episodic hCG secretion were increased in response to GnRH, but not affected by GnRH antagonist. Also, GnRH stimulated the hCG secretion during cultures of placental explants. When pharmacological doses of GnRH (10(-4) mol/l) were utilized, this stimulatory effect of GnRH was no longer evident, while perifusion with medium containing GnRH antagonist at identical concentrations stimulated the hCG secretion, indicating an intrinsic agonistic activity of the antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gestational changes in steroid hormone biosynthesis, secretion, metabolism, and action

This article examines the regulatory mechanisms and anatomic interrelationships governing pregnancy-related steroid hormones from conception through parturition.     

Dual triggering with GnRH agonist plus hCG versus triggering with hCG alone for IVF/ICSI outcome in GnRH antagonist cycles: a systematic review and meta-analysis

Purpose

To summarize available evidence from randomized-controlled trials which have evaluated triggering of final oocyte maturation with concomitant GnRH agonists and hCG in patients undergoing IVF, and to analyze whether dual triggering is as efficacious as hCG triggering in terms of oocyte and pregnancy outcomes.

Methods

A comprehensive literature search was performed to identify randomized-controlled trials comparing IVF outcomes between women receiving combined administration of hCG with GnRH agonists and those receiving hCG alone for triggering of final oocyte maturation.

Results

Four studies including 527 patients eligible for inclusion in meta-analysis were identified. No significant difference in the number of mature oocytes or fertilized oocytes retrieved was found between groups. Clinical pregnancy rate with dual triggering was significantly higher as compared with hCG-alone triggering (pooled OR?=?0.48, 95% CI 0.31–0.77, P?=?0.002), but there was no significant difference in the ongoing pregnancy rate between groups.

Conclusion

Results of meta-analysis indicate comparable or significantly improved outcomes with the use of GnRH agonists plus hCG as compared with hCG alone for triggering of final oocyte maturation.

     

Suppressive actions of a gonadotropin-releasing hormone antagonist on luteinizing hormone, follicle-stimulating hormone, and prolactin release in estrogen-deficient postmenopausal women

We investigated time- and dose-dependent actions of a gonadotropin-releasing hormone antagonist, the "Nal-Glu" peptide [Ac-D2Nal1, 4CIDPhe2, D3Pal3, Arg5, DGlu6(AA), DAla10], in nine healthy estrogen-withdrawn postmenopausal women. Gonadotropin-releasing hormone antagonist was administered subcutaneously at doses of 10, 30, 100, and 300 micrograms/kg. Suppression of immunoactive luteinizing hormone concentrations was achieved with a 30 micrograms/kg dose of antagonist. Suppression of immunoactive follicle-stimulating hormone levels was less (40%) even at the highest antagonist dose (300 micrograms/kg). Bioactive luteinizing hormone concentrations also significantly decreased (greater than 60%) at the two antagonist doses tested (30 and 300 micrograms/kg). However, the lower antagonist dose showed an "escape" of bioactive luteinizing hormone values after 18 hours. No suppressive effects of the antagonist on prolactin secretion occurred at any dose tested. We conclude that this gonadotropin-releasing hormone antagonist can achieve effective, potent, and long-lasting suppression of pituitary secretion of biologically active luteinizing hormone at higher doses, but secretion of biologically active luteinizing hormone may "escape" at lower doses.     

Liver X receptors mediate inhibition of hCG secretion in a human placental trophoblast cell line

Liver X receptors (LXR) alpha and beta are important regulators of lipid homeostasis in liver, adipose and other tissues. However, no such information is available for the human placenta. We determined expression of both LXR alpha and beta in placental trophoblast cell lines, BeWo and JAR. Exposure of BeWo cells to a synthetic LXR agonist, T0901317, resulted in an increase in the amount of mRNA of LXR target genes, sterol regulatory element-binding protein-1 and fatty acid synthase. T0901317 also increased the synthesis of lipids. Moreover, T0901317 resulted in a reduced secretion of hCG during differentiation of these cells. Our data for the first time demonstrate a new role for LXRs in the human placenta.     

Differential control of placental lactogen release and progesterone production by ovine placental tissue in vitro

The hypothesis that placental secretion of progesterone (P4) and ovine placental lactogen (oPL) are controlled through different mechanisms was tested. Placental tissue was obtained at days 133-138 of pregnancy, and explant incubations were established using 200 mg tissue per flask in 5 ml O2-saturated DMEM containing 24 mM HEPES and lacking phenol red (pH 7.4). Following a 30-min preincubation, and a 15-min control period, test substances were added and incubations continued, with periodic gassing, for 4 h at 37 degrees C in a shaking water bath. Dopamine (DA), norepinephrine (NE) and epinephrine significantly stimulated P4 production (P less than 0.05). The enhancement of placental P4 production was mimicked by the addition of 8-bromo-cyclic adenosine monophosphate and forskolin (P less than 0.05). The response to catecholamines was abolished by the addition of propranolol (P less than 0.05) but not by phentolamine (P greater than 0.05). Inclusion of a membrane-permeant substrate for P4 synthesis, 25-hydroxycholesterol, increased basal (P less than 0.05) but did not enhance agonist-induced P4 production (P greater than 0.05). High performance liquid chromatographic analysis of placental tissue demonstrated the presence of DA (80.8 +/- 7.07 pg/mg) and NE (48.8 +/- 5.77 pg/mg), as well as catecholamine metabolites. Addition of 1,2-dioctanoyl-sn-glycerol (DAG) or phorbol 12-myristate-13-acetate (PMA) enhanced oPL secretion (P less than 0.05) without affecting P4 production. The response to DAG and PMA, representing the release of considerably more oPL than can be detected by extracting the tissue, was not influenced by treatment with cycloheximide (P greater than 0.05) indicating that secretion of preformed oPL is regulated by the protein kinase C pathway. These results support the hypothesis that the secretion of oPL and the production of P4 are controlled by different mechanisms.     

Inhibition of estradiol-induced gonadotropin release in ovariectomized rhesus macaques by a gonadotropin-releasing hormone antagonist

Six adult ovariectomized rhesus macaques were given a gonadotropin-releasing hormone (GnRH) antagonist [Ac-beta-(2)DNAL,pFDPhe,DTrp,DArg]-GnRH, by intravenous infusion for 3 to 3.5 days to determine whether the positive feedback action of estradiol benzoate (E2B) on pituitary luteinizing hormone (LH) secretion could be inhibited by blockage of GnRH binding to pituitary gonadotropes. The LH release was suppressed when the antagonist was given either as a bolus injection every 6 hours or as a constant infusion, beginning 24 hours after the E2B was administered. Both LH release and follicle-stimulating hormone release were suppressed if the GnRH antagonist infusion began when the E2B was administered. The results suggest that continued hypothalamic GnRH stimulation of the pituitary is necessary for the full expression of the preovulatory-like gonadotropin surge that occurs in ovariectomized macaques in response to E2.     

Characterizing pituitary response to a gonadotropin-releasing hormone (GnRH) antagonist in monkeys: tonic follicle-stimulating hormone/luteinizing hormone secretion versus acute GnRH challenge tests before, during, and after treatment

Pituitary sensitivity to a gonadotropin-releasing hormone (GnRH) challenge test before, during, and after GnRH antagonist administration was compared in four ovariectomized female monkeys receiving GnRH antagonist intramuscularly (IM) at increasing doses of 0.3, 1.0, and 3.0 mg/kg/day over 9 days. Three days before and 3 days after treatment, monkeys received vehicle alone. On experiment days 4, 7, 10, 13, and 16, 100 micrograms of GnRH was administered intravenously (IV) and blood drawn at 0 and 30 minutes. Before treatment, tonic follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were 248 +/- 105 and 178 +/- 31 ng/ml, respectively; after 0.3 mg/kg/day of GnRH antagonist, FSH and LH decreased to 30 +/- 6 and 41 +/- 4 ng/ml, respectively. After treatment with either 1 mg/kg/day or 3 mg/kg/day of GnRH antagonist, both gonadotropins were undetectable in serum. Monkeys with lower initial levels of gonadotropins were suppressed by 48 hours after GnRH antagonist, while those with higher tonic gonadotropins were suppressed 6 days later (FSH: r = 0.992; LH: r = 0.833). The data show that initial physiologic status is predictive of the rapidity of the suppression response induced by a GnRH antagonist and that, after achieving pituitary suppression, responsivity to an IV GnRH challenge test may be restored before normal tonic FSH/LH secretion is regained.     

A role for calcium in gonadotrophin-releasing hormone (GnRH) stimulated secretion of chorionic gonadotrophin by first trimester human placental minces in vitro

In vitro studies using first-trimester human placental minces have shown that stimulation of human chorionic gonadotrophin (hCG) secretion by gonadotrophin-releasing hormone (GnRH) is dependent upon the presence of extracellular calcium. Addition of GnRH to first-trimester placental minces in vitro was found to stimulate 45Ca2+ uptake into placental minces, and the process was associated with an increase in immunoreactive hCG in the medium. Addition of GnRH to placental minces preloaded with 45Ca2+ stimulated the efflux of 45Ca2+ within one minute. The calmodulin inhibitors chlorpromazine and trifluoperazine inhibited the basal uptake and efflux of 45Ca2+, suggesting the involvement of calmodulin in the mobilization of calcium in the placenta.     

Prognostic value of serum progesterone and LH values on the day of hCG administration in IUI GnRH antagonist cycles

Objetive: to evaluate the effect of LH surge and progesterone rise in IUI cycles under gonadotropin stimulation with GnRH antagonist coadministration on pregnancy rates (PR). Study Design: The population under study consisted of 152 women prospectively studied and subjected to IUI. Results The higher the progesterone cutoff value, the lower the PR were 26.5% and 10.9% when the cutoff was 1 ng/mL, 26.0% and 8.6% when the cutoff was 1.2 ng/mL, 25.6% and 7.1% when the cutoff was 1.4 ng/mL and 25.3% and 0% when the cutoff was 1.6 ng/mL. Conclusion: In IUI cycles under GnRH antagonist coadministration, serum progesterone levels over 1.0 ng/mL are associated with lower PR, the higher the progesterone levels, the lower the PR.     

Gonadotropin levels at the start of ovarian stimulation predict normal fertilization after hCG re-trigger in GnRH antagonist cycles

PurposeTo assess the appropriateness of human chorionic gonadotropin (hCG) re‐trigger in poor responders to gonadotropin‐releasing hormone agonist (GnRHa) trigger in controlled ovarian stimulation (COS) cycles.MethodsThe 2251 cycles in 2251 patients triggered with GnRHa for oocyte stimulation, with or without requiring hCG re‐trigger between 2013 and 2018, were retrospectively analyzed to compare gonadotropin levels at the start of COS and the rate of normal fertilization between the re‐trigger and non–re‐trigger group. Furthermore, patients in the re‐trigger group were stratified by the rate of normal fertilization (good: ≥60% or poor: <60%) to compare patient demographics, hormone profiles, and clinical outcome between the subgroups.ResultsIn the re‐trigger group, FSH and LH levels at the start of COS were significantly lower in the good fertilization group than in the poor fertilization group (P < .01). Receiver operating characteristic curves identified cutoff values of the FSH and LH levels of 1.30 and 0.35 mIU/mL, respectively, for predicting ≥60% normal fertilization.ConclusionGonadotropin levels at the start of COS are predictors of response to GnRHa trigger and hCG re‐trigger necessity, and may serve as indicators to help clinicians appropriately choose hCG re‐trigger rather than abandoning the cycles or continuing the first oocyte aspiration attempt.