GnRH Agonist Stimulation Research Articles

GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LβT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of β-actin, with elevated levels occurring at 10 min. The level of β-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of β-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of β-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LβT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.

Comparing ART outcomes in women with endometriosis after GnRH agonist versus GnRH antagonist ovarian stimulation: a systematic review.

Endometriosis is an oestrogen-dependent disease that can cause subfertility in women who may require assisted reproductive technology (ART) to achieve their pregnancy goals. The aim of this study was to compare ART outcomes in women with endometriosis following the long GnRH-agonist controlled ovarian stimulation (COS) protocol with those taking the GnRH-antagonist COS protocol. MEDLINE, Embase and Web of Science were systematically searched in June 2022. Randomized controlled trials (RCTs) and observational studies comparing the long GnRH-agonist COS protocol and the GnRH-antagonist COS protocol in women with all stages/subtypes of endometriosis were included. Data were synthesized into comprehensive tables for systematic review. The Scottish Intercollegiate Guidelines Network (SIGN) checklists were used for the risk of bias assessment of non-randomized studies and randomized studies, and all the included studies were deemed to have acceptable quality. Eight studies (one RCT and seven observational) with 2695 patients (2761 cycles) were included. Most studies generally reported non-significant differences in clinical pregnancy or live birth rates regardless of the COS protocol used. However, the GnRH-agonist protocol may yield a higher total number of oocytes retrieved, especially mature oocytes. Conversely, the GnRH-antagonist protocol required a shorter COS duration and lower gonadotrophin dose. Adverse outcomes, such as rates of cycle cancellation and miscarriage, were similar between both COS protocols. Both the long GnRH-agonist and GnRH-antagonist COS protocols generally yield similar pregnancy outcomes. However, the long GnRH-agonist protocol may be associated with a higher cumulative pregnancy rate due to the higher number of retrieved oocytes available for cryopreservation. The underlying mechanisms of the two COS protocols on the female reproductive tract remain unclear. Clinicians should consider treatment costs, stage/subtype of endometriosis and pregnancy goals of their patients when selecting a GnRH analogue for COS. A well-powered RCT is needed to minimize the risk of bias and compare the risk for ovarian hyperstimulation syndrome. This review was prospectively registered at PROSPERO under Registration No. CRD42022327604.

Insulin-like peptide 3 (INSL3) in congenital hypogonadotrophic hypogonadism (CHH) in boys with delayed puberty and adult men.

Delayed puberty in males is almost invariably associated with constitutional delay of growth and puberty (CDGP) or congenital hypogonadotrophic hypogonadism (CHH). Establishing the cause at presentation is challenging, with "red flag" features of CHH commonly overlooked. Thus, several markers have been evaluated in both the basal state or after stimulation e.g. with gonadotrophin releasing hormone agonist (GnRHa).Insulin-like peptide 3 (INSL3) is a constitutive secretory product of Leydig cells and thus a possible candidate marker, but there have been limited data examining its role in distinguishing CDGP from CHH. In this manuscript, we assess INSL3 and inhibin B (INB) in two cohorts: 1. Adolescent boys with delayed puberty due to CDGP or CHH and 2. Adult men, both eugonadal and having CHH. Retrospective cohort studies of 60 boys with CDGP or CHH, as well as 44 adult men who were either eugonadal or had CHH, in whom INSL3, INB, testosterone and gonadotrophins were measured. Cohort 1: Boys with delayed puberty aged 13-17 years (51 with CDGP and 9 with CHH) who had GnRHa stimulation (subcutaneous triptorelin 100mcg), previously reported with respect to INB. Cohort 2: Adult cohort of 44 men (22 eugonadal men and 22 men with CHH), previously reported with respect to gonadotrophin responses to kisspeptin-54. Median INSL3 was higher in boys with CDGP than CHH (0.35 vs 0.15 ng/ml; p=0.0002). Similarly, in adult men, median INSL3 was higher in eugonadal men than CHH (1.08 vs 0.05 ng/ml; p<0.0001). However, INSL3 more accurately differentiated CHH in adult men than in boys with delayed puberty (auROC with 95% CI in adult men: 100%, 100-100%; boys with delayed puberty: 86.7%, 77.7-95.7%).Median INB was higher in boys with CDGP than CHH (182 vs 59 pg/ml; p<0.0001). Likewise, in adult men, median INB was higher in eugonadal men than CHH (170 vs 36.5 pg/ml; p<0.0001). INB performed better than INSL3 in differentiating CHH in boys with delayed puberty (auROC 98.5%, 95.9-100%), than in adult men (auROC 93.9%, 87.2-100%). INSL3 better identifies CHH in adult men, whereas INB better identifies CHH in boys with delayed puberty.

Ovarian functions and polycystic ovary syndrome in adult women with type 1 diabetes mellitus in a Turkish population.

The effect of gonadotropin-releasing hormone agonist (GnRHa) stimulation has not been studied in adult women with type 1 diabetes mellitus (DM). We investigated the baseline and stimulated hormone levels after GnRHa and the frequency and relationship between polycystic ovary syndrome (PCOS) and type 1 DM in adult women with type 1 DM. We included 55 adult women (age, 17-35years) with type 1 DM and 15 healthy women (age, 20-29years). Hormones including total testosterone, androstenedione, dehydroepiandrosterone sulphate (DHEAS), follicle-stimulating hormone (FSH), luteinising hormone (LH), estradiol, prolactin, and thyroid-stimulating hormone were measured in the early follicular phase of the menstrual cycle. All participants underwent GnRHa stimulation test, and FSH, LH, estradiol and 17-OHP response levels were measured every 6h for 24h. PCOS was diagnosed according to ESHRE/ASRM (Rotterdam) criteria. Between patients with type 1 DM and healthy controls, no significant differences were noted in mean age and body mass index (BMI) as well as baseline and stimulated hormone levels after buserelin stimulation, except for baseline serum 17-OHP levels, which was higher in patients with type 1 DM. Polycystic ovary morphology (PCOM) was detected in 14 (25%) patients, clinical hyperandrogenism in 16 (29%), hyperandrogenemia in 25 (45%), anovulatory cycle in 72%, and PCOS in 20 (36%). All parameters representing androgen excess disorders, except 17-OHP level, of both groups were similar, and frequencies of PCOS and anovulatory cycle in adult women with type 1 DM were 36% and 72%, respectively.

ANTAGONIST OR PROTAGONIST: ASSESSING STIMULATION PROTOCOLS THROUGH THE LENS OF ARTIFICIAL INTELLIGENCE

To determine if there are differences in oocyte/embryo quality and clinical pregnancy outcomes among patients who undergo either GnRH antagonist or low-dose luteal GnRH agonist (Lupron) stimulation (LDLL) protocols by using deep machine learning algorithms.

Activin A specifically suppresses the expression of annexin A5 mRNA and augments gonadotropin-releasing hormone stimulation of A1 expression in LβT2 gonadotrope cells.

Recently, we reported that gonadotropin-releasing hormone (GnRH) stimulates annexin A1 (Anxa1) and A5 (Anxa5) mRNA expression through the GnRH-receptor-mitogen-activated protein kinase cascade in LβT2 cells. As LβT2 cells respond to activin, we examined the effect of activin A on Anxa1 and a5 expression in LβT2 cells. Activin A (0.4 and 4 ng/mL) treatment decreased Anxa5 mRNA levels in a dose-dependent manner, but did not affect Anxa1 mRNA levels at concentrations up to 40 ng/mL. After activin A treatment (4 ng/mL), Anxa5 mRNA levels significantly decreased at 6 h, gradually declined until 24 h, and remained low until 48 h, whereas Anxa1 mRNA levels did not significantly change following treatment. Pretreatment with activin A for 24 h increased GnRH agonist (GnRHa)-induced Anxa1 increase by approximately 7-fold compared with GnRHa stimulation alone, but Anxa5 was not affected. As previously reported, these activin A treatments increased gonadotropin β subunit and GnRH receptor mRNA levels and slightly decreased common α-glycoprotein subunit mRNA levels. Furthermore, we examined the effect of activin A on Nr4a3, which is repressed by ANXA5 and which reduces Fshb expression, and found that activin A augmented Nr4a3 expression and slightly decreased the GnRHa-induced increase in Nr4a3. These results suggest that in gonadotrope cells, the mechanism regulating Anxa1 and a5 expression is differentially coupled with activin A signal transduction. Activin A suppresses Anxa5 expression under increased Nr4a3 expression, whereas activin A and GnRH synergistically stimulate Anxa1 expression. These GnRH-inducible annexins may have different specific functions in gonadotropes.

The Diagnostic Utility of the Basal Luteinizing Hormone Level and Single 60-Minute Post GnRH Agonist Stimulation Test for Idiopathic Central Precocious Puberty in Girls.

ObjectiveThe present study aimed to assess the diagnostic utility of the Luteinizing hormone (LH) levels and single 60-minute post gonadotropin-releasing hormone (GnRH) agonist stimulation test for idiopathic central precocious puberty (CPP) in girls.MethodsData from 1,492 girls diagnosed with precocious puberty who underwent GnRH agonist stimulation testing between January 1, 2016, and October 8, 2020, were retrospectively reviewed. LH levels and LH/follicle-stimulating hormone (FSH) ratios were measured by immuno-chemiluminescence assay before and at several timepoints after GnRH analogue stimulation testing. Mann–Whitney U test, Spearman’s correlation, χ2 test, and receiver operating characteristic (ROC) analyses were performed to determine the diagnostic utility of these hormone levels.ResultsThe 1,492 subjects were split into two groups: an idiopathic CPP group (n = 518) and a non-CPP group (n = 974). Basal LH levels and LH/FSH ratios were significantly different between the two groups at 30, 60, 90, and 120 minutes after GnRH analogue stimulation testing. Spearman’s correlation analysis showed the strongest correlation between peak LH and LH levels at 60 minutes after GnRH agonist stimulation (r = 0.986, P < 0.001). ROC curve analysis revealed that the 60-minute LH/FSH ratio yielded the highest consistency, with an area under the ROC curve (AUC) of 0.988 (95% confidence interval [CI], 0.982–0.993) and a cut-off point of 0.603 mIU/L (sensitivity 97.3%, specificity 93.0%). The cut-off points of basal LH and LH/FSH were 0.255 mIU/L (sensitivity 68.9%, specificity 86.0%) and 0.07 (sensitivity 73.2%, specificity 89.5%), respectively, with AUCs of 0.823 (95% CI, 0.799–0.847) and 0.843 (95% CI, 0.819–0.867), respectively.ConclusionsA basal LH value greater than 0.535 mIU/L can be used to diagnose CPP without a GnRH agonist stimulation test. A single 60-minute post-stimulus gonadotropin result of LH and LH/FSH can be used instead of a GnRH agonist stimulation test, or samples can be taken only at 0, 30, and 60 minutes after a GnRH agonist stimulation test. This reduces the number of blood draws required compared with the traditional stimulation test, while still achieving a high level of diagnostic accuracy.

A Randomized Controlled Trial on the Efficacy and Safety of Low-Dose hCG in a Short Protocol with GnRH Agonist and Ovarian Stimulation with Recombinant FSH (rFSH) During the Follicular Phase in Infertile Women Undergoing ART.

Τhis study aims to investigate whether the addition of low-dose hCG throughout stimulation in infertile women undergoing IVF improves IVF outcome parameters. This is a prospective, multicenter, randomized, double-blind, placebo-controlled, Phase IIIb clinical study, conducted in three university IVF units. We studied whether the addition of 100 IU hCG/day to a short GnRH agonist IVF protocol from the onset of the follicular phase (group 1, n=40) or placebo (group 2, n=41) had any impact on the number of high-quality transferred embryos at day 2 and clinical pregnancy rates. The comparison encompassed descriptive statistics, and univariate and multivariate analyses. Concerning the primary outcomes, we found no differences in both the number of high-quality embryos (≥2) at day 3 [21/40 (52.5%) vs. 14/41 (34.2%), p=0.095] and clinical pregnancy rates [10/40 (25%) vs. 10/41 (24.4%), p=0.949], respectively. Similarly, there were no differences concerning the secondary outcomes preset for this trial. According to the results of the multivariate logistic regression analysis, no significant associations were noted for primary outcomes (clinical pregnancy: adjusted OR=0.89, 95% CI: 0.29–2.75; (≥2 excellent quality embryos at day 3: adjusted OR=0.54, 95% CI: 0.21–1.42, with group 1 set as reference category); similarly, no differences were noted with respect to secondary outcomes, except from the increased odds of ≥2 poor-quality embryos at day 3 occurring in group 2 (adjusted OR= 11.69, 95%CI: 1.29–106.19). The addition of low-dose hCG to a short GnRH agonist protocol for IVF does not improve the number of top-quality embryos and clinical pregnancy rates.Supplementary InformationThe online version contains supplementary material available at 10.1007/s43032-021-00683-3.

Metformin treatment before and during IVF or ICSI in women with polycystic ovary syndrome.

The use of insulin-sensitising agents, such as metformin, in women with polycystic ovary syndrome (PCOS) who are undergoing ovulation induction or in vitro fertilisation (IVF) cycles has been widely studied. Metformin reduces hyperinsulinaemia and suppresses the excessive ovarian production of androgens. It is suggested that as a consequence metformin could improve assisted reproductive techniques (ART) outcomes, such as ovarian hyperstimulation syndrome (OHSS), pregnancy, and live birth rates. To determine the effectiveness and safety of metformin as a co-treatment during IVF or intracytoplasmic sperm injection (ICSI) in achieving pregnancy or live birth in women with PCOS. We searched the Cochrane Gynaecology and Fertility Group Specialised Register, CENTRALvia the Cochrane Register of Studies Online (CRSO), MEDLINE, Embase, PsycINFO, LILACS, the trial registries for ongoing trials, and reference lists of articles (from inception to 13 February 2020). Types of studies: randomised controlled trials (RCTs) comparing metformin treatment with placebo or no treatment in women with PCOS who underwent IVF or ICSI treatment. women of reproductive age with anovulation due to PCOS with or without co-existing infertility factors. Types of interventions: metformin administered before and during IVF or ICSI treatment. live birth rate, incidence of ovarian hyperstimulation syndrome. Two review authors independently selected the studies, extracted the data according to the protocol, and assessed study quality. We assessed the overall quality of the evidence using the GRADE approach. This updated review includes 13 RCTs involving a total of 1132 women with PCOS undergoing IVF/ICSI treatments. We stratified the analysis by type of ovarian stimulation protocol used (long gonadotrophin-releasing hormone agonist (GnRH-agonist) or short gonadotrophin-releasing hormone antagonist (GnRH-antagonist)) to determine whether the type of stimulation used influenced the outcomes. We did not perform meta-analysis on the overall (both ovarian stimulation protocols combined) data for the outcomes of live birth and clinical pregnancy rates per woman because of substantial heterogeneity. In the long protocol GnRH-agonist subgroup, the pooled evidence showed that we are uncertain of the effect of metformin on live birth rate per woman when compared with placebo/no treatment (risk ratio (RR) 1.30, 95% confidence interval (CI) 0.94 to 1.79; 6 RCTs; 651 women; I2 = 47%; low-quality evidence). This suggests that if the chance for live birth following placebo/no treatment is 28%, the chance following metformin would be between 27% and 51%. Only one study used short protocol GnRH-antagonist and reported live birth rate. Metformin may reduce live birth rate compared with placebo/no treatment (RR 0.48, 95% CI 0.29 to 0.79; 1 RCT; 153 women; low-quality evidence). This suggests that if the chance for live birth following placebo/no treatment is 43%, the chance following metformin would be between 13% and 34% (short GnRH-antagonist protocol). We found that metformin may reduce the incidence of OHSS (RR 0.46, 95% CI 0.29 to 0.72; 11 RCTs; 1091 women; I2 = 38%; low-quality evidence). This suggests that for a woman with a 20% risk of OHSS without metformin, the corresponding risk using metformin would be between 6% and 14%. Using long protocol GnRH-agonist stimulation, metformin may increase clinical pregnancy rate per woman compared with placebo/no treatment (RR 1.32, 95% CI 1.08 to 1.63; 10 RCTs; 915 women; I2 = 13%; low-quality evidence). Using short protocol GnRH-antagonist, we are uncertain of the effect of metformin on clinical pregnancy rate per woman compared with placebo/no treatment (RR 1.38, 95% CI 0.21 to 9.14; 2 RCTs; 177 women; I2 = 87%; very low-quality evidence). We are uncertain of the effect of metformin on miscarriage rate per woman when compared with placebo/no treatment (RR 0.86, 95% CI 0.56 to 1.32; 8 RCTs; 821 women; I2 = 0%; low-quality evidence). Metformin may result in an increase in side effects compared with placebo/no treatment (RR 3.35, 95% CI 2.34 to 4.79; 8 RCTs; 748 women; I2 = 0%; low-quality evidence). The overall quality of evidence ranged from very low to low. The main limitations were inconsistency, risk of bias, and imprecision. This updated review on metformin versus placebo/no treatment before or during IVF/ICSI treatment in women with PCOS found no conclusive evidence that metformin improves live birth rates. In a long GnRH-agonist protocol, we are uncertain whether metformin improves live birth rates, but metformin may increase the clinical pregnancy rate. In a short GnRH-antagonist protocol, metformin may reduce live birth rates, although we are uncertain about the effect of metformin on clinical pregnancy rate. Metformin may reduce the incidence of OHSS but may result in a higher incidence of side effects. We are uncertain of the effect of metformin on miscarriage rate per woman.

Live-Birth Outcomes and Congenital Malformations After Progestin-Primed Ovarian Stimulation in Maternal Endometriosis.

PurposeIn patients who had advanced endometriosis, we use different protocols including GnRH agonist, GnRH antagonist and progestin-primed ovarian stimulation (PPOS) protocols to assess live-birth congenital malformations delivered after in vitro fertilization (IVF) and vitrified embryo transfer cycles.MethodsA retrospective cohort study is conducted by us. It includes 1495 live-born infants in maternal endometriosis. From January 2010 to January 2017, we brought into infants who underwent either gonadotropin-releasing hormone agonist long protocol, gonadotropin-releasing hormone antagonist protocol or PPOS. We chose neonatal outcomes and congenital malformations as our major measures.ResultsNeonatal outcomes, as well as congenital malformations, were considered as the main measures, and gestational age, birth weight, birth length, multiple births and early neonatal death are included. All groups were comparable. The GnRH antagonist group (1.41%) and the GnRH antagonist protocol group (1.8%) had the same incidence of live-birth defects as the PPOS groups (1.33%) were similar. There were no apparent differences when it came to congenital malformations among the three groups. Multivariate logistic regression showed that infertility-time factors as well as multiple births combined to add the risk of congenital malformations; the adjusted odds were 1.143 (95% confidence interval [CI]: 0.988–1.323) and 3.253 (95% CI: 1.359–7.788). Besides, no association was found among various ovarian stimulations as well as congenital birth defect programs, maternal age, body mass index, parity or infant sex.ConclusionThis study suggests that, in contrast to conventional ovarian stimulation, PPOS neither has any effect on neonatal outcomes in IVF adverse effects nor does it elevate the rate of congenital malformations in late endometriosis. However, randomized controlled trials of the long-term outcomes of children born after PPOS protocols for maternal endometriosis are needed and the follow-up studies were conducted to confirm this result.

SAT-195 Oocyte-Secreted Factors GDF9 and BMP15 Measured in Serum Correlate with Reproductive Potential in Women

Bone morphogenetic protein-15 (BMP15) and growth differentiation factor-9 (GDF9) are oocyte-secreted factors critical for folliculogenesis, oocyte developmental competence and fertility. BMP15 and GDF9 are produced only by gametes and despite known associations with reproductive pathologies (1), concentrations in serum have not previously been reported. We have developed novel immunoassays that allow quantitative measurement of BMP15 and GDF9 in serum and have applied these to samples collected from women undergoing IVF to investigate the possibility that these proteins may be useful biomarkers of female reproductive function. The BMP15 and GDF9 immunoassays were developed in-house and validated for sensitivity (24 and 26 pg/ml, respectively), specificity (<0.01% and <0.03%, respectively) and reproducibility (inter-assay CV <10%). BMP15 and GDF9 were not detectable in a significant number of samples (33% for BMP15; 71% for GDF9) and these were assigned the sensitivity value for subsequent analyses. The effect of superovulation of patients undergoing IVF treatment on serum BMP15 and GDF9 was assessed using samples collected immediately before and on multiple days during FSH stimulation in antagonist treatment cycles (56 bloods from 14 women). BMP15 and GDF9 varied 64-and 15-fold respectively between women, but, within an individual were unchanged throughout superovulation, and were independent of FSH dose (P>0.05). Further analyses including an additional 141 women treated for infertility demonstrated no difference in BMP15 or GDF9 between GnRH antagonist (n=69) and agonist (n=17) stimulation cycles, or between these stimulated cycles and unstimulated cycles (n=41). Serum GDF9 positively correlated with the number of oocytes retrieved from non-PCO/PCOS women after superovulation (r=0.439, P=0.05, n=27), but not in PCO/PCOS patients. Serum GDF9, but not BMP15, was significantly lower in poor responders (women with <4 oocytes retrieved or cycle cancellation due to insufficient follicles on ultrasound (2)), compared with normal responders (27.2±0.8 vs 50.6±7.0 pg/ml; P<0.05). Serum BMP15 and GDF9 did not correlate with age, however BMP15 positively correlated with day 2 baseline FSH (r=0.305, P<0.05, n=52). This is the first report of quantitative measurement of BMP15 and GDF9 levels in human serum, correlating with reproductive potential. Although assay sensitivities require improvement, this study demonstrates the diagnostic potential of oocyte-secreted BMP15 and GDF9 as serum biomarkers in reproductive medicine.

Vitamin D level affects IVF outcome partially mediated via Th/Tc cell ratio

The role of vitamin D (VD) in IVF outcome and immune parameters has not been elucidated well. Women undergoing IVF treatment with GnRH agonist (Agonist) and progestin-primed ovarian stimulation (PPOS) protocols were divided into VD lower (VDL, 25(OH)VD ≤20ng/mL) and VD higher (VDH, 25(OH)VD >20ng/mL) groups. Follicular fluid (FF) VD level, IVF outcomes, and peripheral blood immunophenotypes by flow cytometry were analyzed. FF VD levels of the whole subjects were positively correlated with peripheral blood VD level (r=0.86, P<0.001). The number of mature oocytes and the blastocyst formation rate were significantly higher in women with VDH group as compared with those of VDL group in both Agonist and PPOS groups (P<0.05, respectively). In women with PPOS protocol, peripheral blood NK and B-cell proportions and T helper/T cytotoxic (Th/Tc) cell ratios of VDL group were significantly higher than those of VDH group (P<0.05, respectively). In women with Agonist protocol, peripheral blood B-cell proportion and Th/Tc ratios of VDL group were significantly higher than those of VDH group (P<0.05, respectively). VD level is associated with IVF outcomes possibly derived by T-cell immunity, particularly Th/Tc ratios.

Baseline ovarian cyst formation following gonadotropin-releasing hormone analogues: their prevalence and impact on in vitro fertilization outcome

One known complication of gonadotropin-releasing hormone (GnRH) agonist treatment for in vitro fertilization (IVF) cycles is the formation of functional ovarian cysts. The effect of baseline ovarian cysts on the outcome of IVF treatment is controversial. No prior studies have compared the prevalence of cyst formation in GnRH agonist versus GnRH antagonist stimulation protocols. Our objective was to determine the prevalence and significance of baseline follicular cysts on the response to controlled ovarian hyperstimulation and the outcome of IVF in women treated with a GnRH agonist versus a GnRH antagonist. Retrospective case-controlled study. Three-hundred and eighteen consecutive IVF cycles performed between January 2010 and December 2014 were analyzed. The subjects were grouped according to stimulation protocol (GnRH agonist vs. GnRH antagonist) and by the presence or absence of baseline ovarian cysts. We compared baseline estradiol levels, total gonadotropin dose used, total number of days of ovulation induction, number of cycle cancellations, number of oocytes retrieved, and the clinical and live birth rates. There was no significant difference in the prevalence of baseline ovarian cysts using a GnRH agonist versus antagonist protocol (14% vs. 9.5%, p = 0.269). The average time delay to initiate an IVF cycle was 8.9 days for the agonist cycles and 10.2 days for the antagonist cycles (range 0-28 days). The number of oocytes retrieved was lower in the group of patients with a baseline cyst treated with the antagonist protocol (4.7 with a cyst vs. 10.2 without a cyst, p = 0.039) as well as a significantly higher cycle cancellation rate (36% vs. 8.4%, p = 0.028, OR 6.16, 95% CI 1.51-25.12). This effect was not seen with the GnRH agonist protocol. There was no difference in the number of ampules of gonadotropins used for stimulation or total days of stimulation for either protocol. Finally, there was no significant difference between the groups according to the clinical pregnancy rate or live birth rate. The presence of a baseline ovarian cyst delays the time to initiate an IVF cycle; however, there is no significant difference in the prevalence of cyst formation between GnRH agonist and GnRH antagonist protocols. Although presence of a baseline ovarian cyst was associated with a lower number of oocytes retrieved and higher cycle cancellation rate, there is no negative impact on pregnancy rates when compared to cycles without cyst formation. Larger prospective studies are needed to confirm these findings.

The baseline antiMullerian hormone (AMH) level as an independent predictor of live birth rate of frozen-thawed embryo transfers: a multivariate analysis of 828 cycles

The aim of this study was to assess the prognostic effect of AMH in frozen-thawed embryo transfers. A retrospective cohort study. All non-polycystic ovary patients who underwent their first frozen-thawed embryo transfers in our unit and had basal serum AMH evaluated between 2010 and 2015 were evaluated in this retrospective study. AMH was measured using the enzyme amplified two-site immunoassay (ELISA) provided by Beckman Coulter (AMH Gen II ELISA, Beckman Coulter). Individualized controlled ovarian hyper-stimulation (COH) protocols included long GnRH agonist, GnRH antagonist and minimal stimulation protocols, based on individual patient characteristics. When at least two follicles reached 18 mm in diameter, 5000-10,000 IU hCG (Ovidrel, Merck Serono) was adopted to achieve final follicular maturation and oocyte retrieval performed 32-36 hours later. Intracytoplasmic sperm injection was performed if the concentration of motile sperm was <1×106/mL, otherwise a conventional in vitro fertilization was used. Vitrification was performed for embryo freezing with the use of a Cryotop (Kitazato Corp.) device. Embryo transfer was operated under trans-vaginal sonography guidance. The primary outcome is Live birth rate, which was defined as rate of deliveries that resulted in at least one live born baby per transfer cycle. Chi-square and binary regression were used for data analysis. A P<0.05 indicated significant result. In total, 828 patients were included. We grouped them into three groups based on their baseline AMH concentration: low AMH group (<25th), middle AMH (25th- 75th) and high AMH level (>75th). The results showed lower AMH level was associated with significantly lower implantation rate (21.9% vs. 43.2% vs. 58.8%, P<0.001), clinical pregnancy rate (32.0% vs. 55.2% vs. 65.7%, P<0.001), live birth rate (21.8% vs. 43.6% vs. 52.7%, P<0.001) but higher miscarriage rate (31.8% vs. 17.5% vs. 15.4% P=0.014). After stratifying the patients into two age groups, being younger or older than 35 years, we still observed significant influence of AMH on outcomes in all patients in both age groups. Furthermore, serum AMH kept significant in multivariate analysis when adjusting covariates (i.e. age, FSH, AFC, endometrium thickness, endometrium preparation protocols, number of embryos transferred, etiology of infertility). The area under the curve (AUC) for serum AMH, age, AFC and FSH were 0.635, 0.634, 0.615 and 0.543 respectively, for predicting live birth. Our results demonstrated that baseline AMH was an independent predictive factor of live birth rate of frozen embryo transfers, irrespective of maternal age. It had only moderate predictive value on predicting live birth, superior to that of AFC and FSH.

Does thalassemia influence ovarian response? An analysis of 127 cycles involving pre-implantation genetic diagnosis of thalassemia in southern China

To evaluate the impact of thalassemia carrier status on the response to controlled ovarian stimulation (COS) and outcome of intracytoplasmic sperm injection (ICSI). Seventy couples that both carried a mutation attributed to thalassemia (PGD group) and 57 couples in which only the father was a thalassemia carrier (CON group) were enrolled. All female subjects received long protocol GnRH agonist stimulation and received equivalent doses of recombinant follicle-stimulating hormone and the number of retrieved oocytes and utilisable embryos did not differ significantly. The endometrial thickness at human chorionic gonadotropin (hCG) administration day and the number of transferable embryos was lower in the PGD groups. However, pregnancy outcomes, including the clinical pregnancy rate and ongoing pregnancy rate, did not differ significantly between the two groups per cycle. Ovarian response to COS is not impaired by maternal thalassemia carrier status and embryo biopsy did not impair preimplantation embryo development or pregnancy outcomes.

Strategies for Controlled Ovarian Stimulation in the Setting of Ovarian Aging

In the context of assisted reproduction, the term ovarian aging is often used to refer to declining potential of ovaries to produce oocytes in adequate number or quality in response to controlled ovarian stimulation (COS). Different aspects of COS have been modified with the intention to increase the number and quality of oocytes obtained for in vitro fertilization. In the setting of ovarian aging, suppression of the luteinizing hormone (LH) surge with gonadotropin-releasing hormone (GnRH) antagonist or short GnRH agonist protocol and stimulation with a daily gonadotropin dosage of ≤ 300 IU/day seem to be appropriate first choices, and there is a strong need for well-designed randomized controlled trials investigating effects of addition of LH activity, estradiol priming, transdermal testosterone administration, and growth hormone supplementation. Given the lack of high-quality evidence showing effectiveness of one approach over another, other factors such as duration of stimulation, total gonadotropin consumption and cost of medication, patient friendliness, and possible side effect profiles must be considered in tailoring the COS protocol according to each individual's needs and desires.

The association between anti-Mullerian hormone and IVF-ICSI outcome in poor responder patients performing long protocol

Purpose of investigation: Ovarian reserve reflects the capacity of the ovaries for a successful pregnancy. Anti-Mullerian hormone (AMH) could be a useful marker to predict ovarian reserve and to adjust controlled ovarian stimulation. The aim of this study was to assess the relationship between AMH and intracytoplasmic sperm injection-in vitro fertilization (IVF-ICSI) outcome in poor responder women. Materials and Methods: This study was conducted prospectively for a period of 12 months. Inclusion criteria were FSH value > 15 iu/l or antral follicle number nd day of cycle. All patients underwent GnRH agonist stimulation with long protocol. Serum AMH levels were measured in the treatment cycle just before the stimulation. After the treatment, patients who were pregnant formed the study group and patients who were not pregnant formed the control group. Serum AMH level was the main outcome measure. Results: The study and control group consisted of 34 and 70 patients, respectively. No significant difference was found in duration of infertility, antral follicular count, basal E2 and FSH levels. The mean serum AMH level was significantly higher in study group (p = 0.005). The retrieved oocyte number, metaphase 2 oocyte number, and fertilization rate were also significantly higher in the study group. Discussion: Evaluation of serum AMH seems to be a useful marker to predict IVF-ICSI outcome in poor responder patients.

Addition of gonadotropin releasing hormone agonist for luteal phase support in in-vitro fertilization: an analysis of 2739 cycles.

Luteal phase is defective in in vitro fertilization (IVF) cycles, and many regimens were tried for the very best luteal phase support (LPS). Gonadotropin releasing hormone (GnRH) agonist use, which was administered as an adjunct to the luteal phase support in IVF cycles, was suggested to improve pregnancy outcome measures in certain randomized studies. We analyzed the effects of addition of GnRH agonist to standard progesterone luteal support on pregnancy outcome measures, particularly the live birth rates. This is a retrospective cohort study, including 2739 IVF cycles. Long GnRH agonist and antagonist stimulation IVF cycles with cleavage-stage embryo transfer were included. Cycles were divided into two groups: Group A included cycles with single-dose GnRH agonist plus progesterone LPS and Group B included progesterone only LPS. Live birth rates were the primary outcome measures of the analysis. Miscarriage rates and multiple pregnancy rates were the secondary outcome measures. Live birth rates were not statistically different in GnRH agonist plus progesterone (Group A) and progesterone only (Group B) groups in both the long agonist and antagonist stimulation arms (40.8%/41.2% and 32.8%/34.4%, p<0.05 respectively). Moreover, pregnancy rates, implantation rates, and miscarriage rates were found to be similar between groups. Multiple pregnancy rates in antagonist cycles were significantly higher in Group A than those in Group B (12.0% and 6.9%, respectively). A beneficial effect of a single dose of GnRH agonist administration as a luteal phase supporting agent is yet to be determined because of the wide heterogeneity of data present in literature. Well-designed randomized clinical studies are required to clarify any effect of luteal GnRH agonist addition on pregnancy outcome measures with different doses, timing, and administration routes of GnRH agonists.

Inhibin B plus LH vs GnRH agonist test for distinguishing constitutional delay of growth and puberty from isolated hypogonadotropic hypogonadism in boys.

The distinction between constitutional delay of growth and puberty (CDGP) and isolated hypogonadotropic hypogonadism (IHH) in males with delayed puberty is difficult but important for timely treatment. We assessed the accuracy of the GnRH agonist test (triptorelin 0·1 mg) in comparison with inhibin B alone or in combination with basal LH for the diagnosis of IHH. Ninety-seven prepubertal males aged 13·7-17·5 year, with testicular volumes ≤4 ml, were examined every 6 months. CDGP was defined by a testicular volume ≥8 ml after 18 months, and IHH was defined by a testicular volume <5 ml after 24 months follow-up. Inhibin B concentrations were measured by ELISA, and LH concentrations were measured by CLIA. At follow-up, the cohort comprised 52 boys with CDGP and nine with IHH. The other patients were lost for follow-up (n = 10), had not reached follow-up yet (n = 20) or did not reach a definite testicular volume (n = 6). Basal LH <0·3 IU/l, stimulated LH (4 h) <5·3 IU/l or inhibin B <111 pg/ml had 100% sensitivity for IHH. Only LH (4 h) <5·3 IU/l had a specificity of 100%, and the specificities of basal LH <0·3 IU/l (88%) or inhibin B <110 pg/ml (92%) were lower. The combination of LH <0·3 IU/l with inhibin B <111 pg/ml increased the specificity to 98·1%. The LH response 4 h after GnRH agonist stimulation has an excellent accuracy for the diagnosis of IHH in prepubertal boys with delayed puberty. However, the measurement of inhibin B and basal LH in combination is a valid, reliable and less-invasive alternative test.

Evidence for gonadotrophin secretory and steroidogenic abnormalities in brothers of women with polycystic ovary syndrome

Are there abnormalities in gonadotrophin secretion, adrenal steroidogenesis and/or testicular steroidogenesis in brothers of women with polycystic ovary syndrome (PCOS)? Brothers of women with PCOS have increased gonadotrophin responses to gonadotrophin releasing hormone (GnRH) agonist stimulation and alterations in adrenal and gonadal steroidogenesis. PCOS is a complex genetic disease. Male as well as female first-degree relatives have reproductive features of the syndrome. We previously reported that brothers of affected women have elevated circulating dehydroepiandrosterone sulfate levels. This was a case-control study performed in 29 non-Hispanic white brothers of 22 women with PCOS and 18 control men. PCOS brothers and control men were of comparable age, weight and ethnicity. Adrenocorticotrophic hormone (ACTH) and GnRH agonist stimulation tests were performed. Gonadotrophin responses to GnRH agonist as well as changes in precursor-product steroid pairs (delta, Δ) across steroidogenic pathways in response to ACTH and GnRH agonist were examined. Basal total (T) levels did not differ, but dehydroepiandrosterone (DHEA) levels (0.13 ± 0.08 brothers versus 0.22 ± 0.09 controls, nmol/l, P = 0.03) were lower in brothers compared with control men. ACTH-stimulated Δ17-hydroxypregnenolone (17Preg)/Δ17-hydroxyprogesterone (17Prog) (7.8 ± 24.2 brothers versus 18.9 ± 21.3 controls, P = 0.04) and ΔDHEA/Δandrostenedione (AD) (0.10 ± 0.05 brothers versus 0.14 ± 0.08 controls, P = 0.04) were lower in brothers than in the controls. GnRH agonist-stimulated Δ17Prog/ΔAD (0.28 ± 8.47 brothers versus 4.79 ± 10.28 controls, P = 0.003) was decreased and luteinizing hormone (38.6 ± 20.6 brothers versus 26.0 ± 9.8 controls, IU/l, P = 0.02), follicle-stimulating hormone (10.2 ± 7.5 brothers versus 4.8 ± 4.1 controls, IU/l P = 0.002), AD (1.7 ± 1.4 brothers versus 0.9 ± 1.5 controls, nmol/l, P = 0.02) and ΔAD/ΔT (0.16 ± 0.14 brothers versus 0.08 ± 0.12 controls, P = 0.005) responses were increased in brothers compared with controls. The modest sample size may have limited our ability to observe other possible differences in steroidogenesis between PCOS brothers and control men. Decreased ACTH-stimulated Δ17Preg/Δ17Prog and ΔDHEA/ΔAD responses suggested increased adrenal 3β-hydroxysteroid dehydrogenase activity in the brothers. Decreased Δ17Prog/ΔAD and increased ΔAD/ΔT responses to GnRH agonist stimulation suggested increased gonadal 17,20-lyase and decreased gonadal 17β-hydroxysteroid dehydrogenase activity in the brothers. Increased LH and FSH responses to GnRH agonist stimulation suggested neuroendocrine alterations in the regulation of gonadotrophin secretion similar to those in their proband sisters. These changes in PCOS brothers may reflect the impact of PCOS susceptibility genes and/or programming effects of the intrauterine environment. This research was supported by P50 HD044405 (A.D.), K12 HD055884 (L.C.T.), U54 HD034449 (A.D., R.S.L.) from the National Institute of Child Health and Development. Some hormone assays were performed at the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core that is supported by U54 HD28934 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Partial support for some of the clinical studies was provided by UL1 RR025741 and UL1 TR000150 (Northwestern University Clinical and Translational Sciences Institute) from the National Center for Research Resources, National Institutes of Health, which is now the National Center for Advancing Translational Sciences. The authors have no conflict of interest to declare.