Addition Of GnRH Agonist Research Articles

O-292 Comparison of effect of two different trigger regimens; single (hCG) versus dual (hCG + Leuprolide) on outcome of fresh IVF cycles: A randomized controlled trial

Study questionDoes adding gonadotropin-releasing hormone agonist (GnRHa) to hCG trigger increases the number of high-grade embryos in GnRH antagonist protocol in fresh non-donor IVF?Summary answerFinal oocyte maturation triggered by dual trigger increases the number of MII oocytes thus transferring good-quality embryos and cryopreserving surplus embryos compared to hCG trigger.What is known alreadyhCG has been conventionally used as a ‘faux’ LH surge to bring about final oocyte maturation due to structural similarity between the two. GnRH agonist, on the other hand, induces a more physiological gonadotropin surge for follicular maturation, but is associated with luteal phase deficiency. Recent studies have shown that combining GnRHa with hCG trigger improves oocyte maturation and embryo quality with the added benefit of a luteal phase support, thereby improving IVF outcomes in terms of both embryological and reproductive outcomes.Study design, size, durationA single-center, open labelled, randomized controlled trial including 100 normal responder patients between 21-38 years undergoing IVF using GnRH antagonist protocol between January 2020 to August 2021. The study excluded patients with the presence of other variables of adverse outcomes like diminished ovarian reserve (AFC < 5 or AMH < 1.2 ng/ml), endocrine disorders, thin endometrium (<6mm), previous history of uterine surgeries, and high responders.Participants/materials, setting, methods100 patients undergoing fresh IVF cycle using GnRH antagonist protocol were randomized after informed consent to receive either dual trigger (Leuprolide acetate 1 mg + rhCG 250 mcg, n = 50) or single hCG trigger (rhCG 250 mcg, n = 50). Oocyte retrieval was done 35-37 hours after trigger followed by IVF/ICSI, as indicated. Oocyte and embryo grading was done using Istanbul consensus. Analysis was done by ITT. Outcomes were analyzed using Independent t-test and Chi-square test.Main results and the role of chanceThe baseline characteristics were comparable in both arms. the number of MII oocytes retrieved (7.82 versus 5.92, p = 0.003) and the number of day-3 grade-1 embryos (4.24 versus 1.8, p < 0.001) were higher in the dual trigger group, whereas fertilization rates between the two groups (91.82% versus 88.51%, p=NS) were comparable. Consequently, the number of embryos cryopreserved (2.68 versus 0.94, p < 0.001) were significantly higher in the dual trigger group. However, the implantation rate between the two groups (21% versus 19.6%, p = 0.770) was comparable. The serum LH levels 12 hours post trigger were measured in both the arms and as expected, high serum LH values were documented in the dual trigger group (46.23 mIU/ml vs 0.93 mIU/ml, p < 0.0001).Limitations, reasons for cautionDue to the impact of the Covid-19 pandemic causing an intermittent pause in IVF services at our center, a smaller sample size of 100 patients could be enrolled in the study, and reproductive outcomes in terms of live births and cumulative live births could not be assessedWider implications of the findingsThis study, though small, has contributed to some evidence of redesigning the dual trigger in all antagonist cycles, with the exception of high responders and PCOS patients. The addition of GnRHa to hCG trigger has led to the possibility of cryopreserving surplus embryos thereby increasing the cumulative live births.Trial registration numberCTRI/2020/08/027030

Is addition of GNRH agonist to HRT significant in improving the outcome in FTE transfer cycles? Randomized study.

Background: Despite the high number of frozen embryo transfer cycles being conducted, there is much debate about which protocol for endometrial preparation is ideal.Aim: The aim of this study is detect the value of gonadotropin-releasing hormone agonist (GNRH-a) in the preparation of the endometrium regarding the cycle outcome.Materials and Methods: One hundred forty women participated in the study. They were randomly divided into two groups; group 1 and group 2. The patients of group 1 received injection of GnRH-a triptoreline (3.75 mg) on the day-21 of the menstrual cycle proceeding the replacement cycle. Both groups received exogenous estradiol starting from the second day of the cycle then the dose was increased incrementally till the endometrial thickness was ≥ 8mm or more. Progesterone was given to the patients before embryo transfer and continued thereafter. Twelve days later, serum pregnancy test was done and if positive, transvaginal ultrasonography was done two weeks later for detection of embryo cardiac activity.Results: Both groups showed no statistically significant differences regarding demographic, clinical and laboratory data and their relations to pregnancy. Serum pregnancy tests detected chemical pregnancy that was confirmed by transvaginal ultrasonography at 6 weeks gestation in only 49.3% of women (clinical pregnancy rate =49.3%). Only six women aborted at 8-12 weeks gestation (ongoing pregnancy rate =45%). There were no significant differences between both groups regarding the clinical outcome.Conclusion: Addition of GNRH-a to HRT to prepare the endometrium in the FTE transfer cycle had no significant effect on the clinical outcome.

661 Vitamin a and Iron as an Adjuvant Therapy in Addition of Gnrh Agonist in Precocious Puberty

BackgroundTo examine the effect of vitamin A and Iron supplementation on growth outcome of central precocious puberty (CPP) patients who receive GnRH agonist.Method36 female CPP patients were randomized in control...

Extracellular signal-regulated kinase and c-Src, but not Jun N-terminal kinase, are involved in basal and gonadotropin-releasing hormone-stimulated activity of the glycoprotein hormone alpha-subunit promoter.

Addition of a GnRH agonist (GnRH-A) to alphaT3-1 cells stimulates different MAPK cascades: ERK, Jun N-terminal kinase (JNK), and p38. Activation of JNK, ERK, and p38 shows a unique fold activation ratio of 25:12:2, which might encode signal specificity. ERK is translocated to the nucleus within 20 min with a peak at 120 min of GnRH-A stimulation. We used the human alpha-subunit promoter linked to chloramphenicol acetyl transferase (alphaCAT) to examine the role of ERK, JNK, and c-Src, which is implicated in MAPK activation, in basal and GnRH-stimulated alphaCAT. Addition of GnRH-A resulted in a 3-fold increase in alphaCAT, whereas the Ca(2+) ionophore ionomycin and the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA) had no effect. Addition of GnRH-A and TPA, but not GnRH-A and ionomycin, produced a synergistic response, whereas removal of Ca(2+), but not down-regulation of TPA-sensitive PKCs, abolished GnRH-A-stimulated alphaCAT. Thus, regulation of alpha-promoter activity by GnRH is Ca(2+) dependent and is further augmented by PKC. Cotransfection of alphaCAT and constitutively active or dominant negative plasmids of ERK and JNK cascade members, or the use of the ERK inhibitor PD98059, revealed that ERK, but not JNK, is involved in basal and GnRH-A-stimulated alphaCAT. Because c-Src participates in MAPK activation by GnRH, we also studied its role. Cotransfection of alphaCAT and the dominant negative form of c-Src or incubation with the c-Src inhibitor PP1 reduced GnRH-A-stimulated alphaCAT. The 5'-deletion analysis revealed that the -846/-420 region participated in basal alpha-transcription. In addition, the -346/-156 region containing the pituitary glycoprotein hormone basal element, alpha-basal elements, glycoprotein-specific element, and upstream response element is involved in basal and GnRH-A-stimulated alphaCAT. ERK contribution to GnRH maps to -346/-280 containing the pituitary glycoprotein hormone basal element and alpha-basal elements 1/2. Surprisingly, although c-Src is involved in GnRH-A-stimulated ERK, its involvement is mapped to another region (-280/-180) containing the glycoprotein-specific element. Thus, ERK and c-Src but not JNK are involved in basal and GnRH-A-stimulated-alphaCAT, whereas c-Src contribution is independent of ERK activation.

Activation of MAPK Cascades by GnRH: ERK and Jun N-Terminal Kinase Are Involved in Basal and GnRH-Stimulated Activity of the Glycoprotein Hormone LHβ-Subunit Promoter

The role of ERK and Jun N-terminal kinase (JNK) in basal- and GnRH-stimulated LHbeta-promoter activity was examined in the gonadotroph cell line LbetaT-2. GnRH agonist (GnRH-A) stimulates the MAPK cascades ERK, JNK, and p38MAPK, with a peak at 7 min for ERK and at 60 min for JNK and p38MAPK. The rat glycoprotein hormone LHbeta-subunit promoter, linked to the chloramphenicol acetyl transferase (CAT) reporter gene, was used to follow its activation. Addition of GnRH-A (10 nM) to LbetaT-2 cells resulted in a 6-fold increase in LHbeta-CAT activity at 8 h, which was markedly reduced by a GnRH antagonist. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA), but not the Ca(2+) ionophore ionomycin, stimulated LHbeta-CAT activity. Addition of GnRH-A and TPA together did not produce an additive response. Down-regulation of PKC, but not removal of Ca(2+), abolished the GnRH-A and the TPA response. Cotransfection of the LHbeta-promoter and the constitutively active form of Raf-1 stimulated basal and GnRH-A-induced LHbeta-CAT activity. The dominant negative forms of the ERK cascade members Ras, Raf-1, and MAPK/ERK kinase (MEK) markedly reduced basal and GnRH-A-induced LHbeta-CAT activity, Similar results were obtained with the MEK inhibitor PD 098059. Cotransfection of the LHbeta-promoter and the constitutively active CDC42 stimulated basal and GnRH-A-induced LHbeta-CAT activity. The dominant negative forms of the JNK cascade members Rac, CDC42, and SEK markedly diminished basal and GnRH-A-induced LHbeta-CAT activity. Interestingly, the constitutively active form of c-Src stimulated the basal and the GnRH-A response, whereas the dominant negative form of c-Src, or the c-Src inhibitor PP1 diminished basal and the GnRH-A response. We conclude that ERK and JNK are involved in basal and GnRH-A stimulation of LHbeta-CAT activity. c-Src participates also in LHbeta-promoter activation by a mechanism which might be linked to ERK and JNK activation.

Addition of gonadotrophin‐releasing hormone agonist and/or two inseminations with husband's sperm do not improve the pregnancy rate in superovulated cycles

A prospective randomized study was performed to evaluate the addition of a gonadotropin releasing hormone agonist (GnRH-a) during treatment with human menopausal gonadotropins (hMG) in cycles with artificial inseminations with husband's washed sperm (AIH). We also compared the pregnancy rate per cycle after one versus two AIHs. We designed a 22 factorial trial. A total of 172 couples with unexplained infertility (n=88), endometriosis (n=39), or cervical (n=24) or male (n=21) factors were included, of whom 161 fulfilled the inclusion criteria and treatment. Eighty-one women were treated with GnRH-a/hMG and another 80 with hMG only, respectively. The pregnancy rates did not differ between the two stimulation protocols (12% for GnRH-a/hMG and 9% for hMG). With GnRH-a/hMG more follicles >15 mm (3.4 and 2.4, respectively; p<0.01) and a higher multiple pregnancy rate after 20 weeks of gestation were observed (55% vs. 0%; p<0.05). Eighty-seven women were treated with one AIH, whereas 65 women received two AIHs on two consecutive days. The pregnancy rates were similar in these two groups (11% and 9% respectively; n.s.) It is concluded that neither addition of GnRH-a before and during controlled ovarian hyperstimulation nor two AIHs compared with one single AIH per cycle has a beneficial effect on the pregnancy rate. However, GnRH-a increases the risk for multiple pregnancies.

Induction of apoptosis in ovarian carcinoma cell line by gonadotropin-releasing hormone agonist.

Gonadotropin releasing hormone (GnRH) agonist suppresses the growth of the cancer cells in vitro. To evaluate the effect of a GnRH agonist (GnRHA) in ovarian carcinomas, we investigated the interactions of GnRHA with the KOC-2s human ovarian cancer cells. The addition of GnRHA (10(-8)-10(-6)M) produced an increase 20-30% in the number of cells (p < 0.05). GnRHA (10(-5) M) produced a slight, statistically insignificant decrease of < 10% in the cell count. No DNA fragmentation was produced by GnRHA (10(-8)-10(-6)M). However, GnRHA (10(-5)M) produced internucleosomal cleavage of DNA into fragments with multiples of 180 to 200 bp. This DNA "ladder" pattern is characteristic of apoptosis. The amount of Fas antigen was reduced by each concentration of GnRHa. The addition of GnRHA (10(-6)M and 10(-5)M) significantly increased the secretion of TNF-alpha (p < 0.001). The time- and dose-dependent effects of GnRHA might be confined to the KOC-2s cells as demonstrated by growth inhibitions and characteristic internucleosomal DNA fragmentation. However, the effects of GnRHA on secretion of TNF-alpha and the expression of Fas antigen differed. The present results provide a basis for future studies on the mechanism of apoptotic effect of GnRHA.

The inhibition of growth and down-regulation of gonadotropin releasing hormone (GnRH) receptor in alphaT3-1 cells by GnRH agonist.

Gonadotropin releasing hormone (GnRH) and its analogs inhibit the growth of hormone-dependent tumors in vivo and in vitro. The inhibition of growth and proliferation of tumor cells in vitro by GnRH and its analogs indicates that the tumor suppressing effect of the hormone is only partially due to suppression of pituitary gonadotropin release which reduces circulating steroid levels that are required for proliferation. Demonstration of GnRH-binding sites on some tumors suggests a direct inhibitory effect of GnRH and its analogs. However, the mechanism by which GnRH and its analogs inhibit tumor cell growth is not known. Our hypothesis is that the inhibition of growth and proliferation of tumor cells by GnRH and its analogs are mediated through down-regulation of its receptor expression. To test this hypothesis, mouse pituitary gonadotrope cell line (alphaT3-1) was selected as a model since this is the only cell line which expresses a sufficiently high level of GnRH receptors for precise measurements of the mRNA for the receptor. Addition of GnRH agonist (D-Lys6)GnRH to the cell cultures caused a time-dependent decrease in both cell growth, as measured by cell number, and cell proliferation, as measured by [3H]thymidine incorporation into DNA. After 1 h of treatment of alphaT3-1 cells with 1 microM of (D-Lys6)GnRH, the cell number was reduced to 83.0 +/- 13.4 compared to control, decreased to 75.1 +/- 3.2 at 2 h, 63.2 +/- 0.66 at 4 h and 52.2 +/- 0.87 at 24 h. This decrease in cell number was accompanied by a parallel decrease in [3H]thymidine incorporation into DNA. The inhibition of cell growth and [3H]thymidine incorporation by treatment with 1 microM of (D-Lys6)GnRH was sustained for at least 72 h. Inhibition of alphaT3-1 cell growth and [3H]thymidine incorporation was dose-dependent; thus 10(-9) M (D-Lys6)GnRH resulted in about 30% inhibition within 4h which was comparable to 10(-6) M (D-Lys6)GnRH, whereas 10(-12) M (D-Lys6)GnRH was ineffective. Measurement of mRNA for the GnRH receptor by Northern blot analysis showed a decrease in levels of mRNA by 5% within 2 h of treatment of alphaT3-1 cells with 1 microM (D-Lys6)GnRH, by 30% at 4 h and by 50% at 24 h. In conclusion these data demonstrate that treatment of alphaT3-1 cells with (D-Lys6)GnRH causes an inhibition of cell growth and proliferation, and down-regulates the GnRH receptor mRNA levels.

Testicular GnRH-Receptors and Direct Effects of a GnRH-Agonist on Human Testicular Steroidogenesis

GnRH-like substances have been isolated from interstitial fluid from the rat testis and have been found to exert direct, mainly inhibitory effects on steroidogenesis. In rat testis, specific GnRH receptors have been shown, but so far no GnRH-receptors have been isolated from the human testis. In this study testicular tissue from nine elderly men was incubated with either 3H-pregnenolone or 3H-progesterone and the steroid metabolic patterns were analyzed. In parallel incubations a GnRH-agonist was added to the incubate in concentrations of 10(-6) M or 10(-7) M. Furthermore, receptor studies were performed in tissue specimens from four of these untreated patients as well as in tissue specimens from five GnRH-agonist treated patients. No consistent effects were elicited by the addition of GnRH-agonist on the steroid metabolic patterns in vitro after three hours incubation. In the receptor studies the GnRH-agonist was bound to the testicular tissue, although the binding sites were of low affinity and high capacity, indicating a less specific kind of binding than classical receptor binding.

Epidermal growth factor and gonadotropin‐releasing hormone inhibit cyclic AMP–dependent luteinizing hormone receptor formation in ovarian granulosa cells

The induction of luteinizing hormone (LH) receptors was studied in granulosa cells prepared from the ovaries of hypophysectomized diethylstilbestrol-treated immature rats. Incubation of granulosa cells for 48 h with increasing concentrations of follicle-stimulating hormone (FSH) or choleragen caused parallel rises in cAMP levels and LH receptors. These observations, with the finding that 8-Bromo-cAMP also induced LH receptor formation, indicate that hormonal stimulation of LH binding sites is mediated by cAMP. Peptide hormones that inhibited FSH-stimulated cAMP production, such as epidermal growth factor (EGF) and a gonadotropin-releasing hormone agonist (GnRHa), also prevented LH receptor formation. GnRHa and EGF had negligible effects on FSH-stimulated cAMP production from 0 to 24 h of culture, but reduced cAMP accumulation by 80% and 90%, respectively, from 24 to 48 h when the majority of LH receptors appeared. FSH-sensitive adenylate cyclase activity, as measured by the conversion of (3H)-ATP to (3H)-cAMP, was inhibited by GnRHa and EGF at 48 h of culture. EGF and GnRHa also reversed the inhibiton of ectophosphodiesterase activity caused by FSH in granulosa cells between 48 and 72 h of culture. Both EGF and GnRHa inhibited induction of LH receptors by 8-Bromo-cAMP, suggesting that their effects are also on cAMP action. Addition of GnRHa, but not EGF, between 36 and 48 h of culture completely prevented further increases in LH receptors induced by 8-Bromo-cAMP, indicating that the inhibitory action of GnRHa can be initiated at later times during granulosa cell differentiation, whereas full expression of EGF action requires a longer period. These results demonstrate that EGF and GnRH inhibit FSH-induced LH receptor formation in the granulosa cell by reducing hormone-dependent cAMP production and also by impairing the ability of cAMP to stimulate LH receptor formation.