GnRH agonist versus GnRH antagonist in IVF/ICSI cycles with recombinant LH supplementation: DNA fragmentation and apoptosis in granulosa cells

Objective: To compare the level of apoptosis and DNA fragmentation in the human granulosa cell (GC) layer exposed to an agonist or antagonist of GnRH in intracytoplasmic sperm injection (ICSI) cycles supplemented with recombinant LH (rLH).Study design: Patients without ovulatory dysfunction, aged <= 37 years and in their first ICSI cycle were prospectively randomised to receive either a long GnRH agonist protocol or a multi-dose antagonist protocol. In both groups, recombinant FSH supplemented with rLH was used for ovarian stimulation, and the GCs were collected during oocyte denudation. The GCs were then analysed for DNA fragmentation by TUNEL assay and for apoptosis using the annexin-V assay. The outcomes were given as the percentage of GCs with DNA fragmentation and apoptosis out of the total number of GCs analysed. Comparison of the agonist versus the antagonist group was performed using the Mann-Whitney test.Results: DNA fragmentation: 32 patients were included in either the GnRH agonist group (n = 16) or the antagonist group (n = 16). The percentage of GCs with positive DNA fragmentation did not differ significantly (P = 0.76) between the agonist group (15.5 +/- 9.4%) and the antagonist group (18.8 +/- 13.3%). Apoptosis: 28 patients were included in either the GnRH agonist group (n = 14) or the antagonist group (n = 14). The percentage of GCs positive for apoptosis did not differ significantly (P = 0.78) between the agonist group (34.6 +/- 14.7%) and the antagonist group (36.5 +/- 22%).Conclusions: The results suggest that therapy with either an agonist or antagonist of GnRH is associated with comparable levels of DNA fragmentation and apoptosis in granulosa cells in ICSI cycles supplemented with rLH. (C) 2012 Elsevier B.V. All rights reserved.

GnRH agonist versus GnRH antagonist in assisted reproduction cycles: oocyte morphology

Background: The selection of developmentally competent human gametes may increase the efficiency of assisted reproduction. Spermatozoa and oocytes are usually assessed according to morphological criteria. Oocyte morphology can be affected by the age, genetic characteristics, and factors related to controlled ovarian stimulation. However, there is a lack of evidence in the literature concerning the effect of gonadotropin-releasing hormone (GnRH) analogues, either agonists or antagonists, on oocyte morphology. The aim of this randomized study was to investigate whether the prevalence of oocyte dysmorphism is influenced by the type of pituitary suppression used in ovarian stimulation.Methods: A total of 64 patients in the first intracytoplasmic sperm injection (ICSI) cycle were prospectively randomized to receive treatment with either a GnRH agonist with a long-term protocol (n: 32) or a GnRH antagonist with a multi-dose protocol (n: 32). Before being subjected to ICSI, the oocytes at metaphase II from both groups were morphologically analyzed under an inverted light microscope at 400x magnification. The oocytes were classified as follows: normal or with cytoplasmic dysmorphism, extracytoplasmic dysmorphism, or both. The number of dysmorphic oocytes per total number of oocytes was analyzed.Results: Out of a total of 681 oocytes, 189 (27.8 %) were morphologically normal, 220 (32.3 %) showed cytoplasmic dysmorphism, 124 (18.2%) showed extracytoplasmic alterations, and 148 (21.7%) exhibited both types of dysmorphism. No significant difference in oocyte dysmorphism was observed between the agonist- and antagonist- treated groups (P > 0.05). Analysis for each dysmorphism revealed that the most common conditions were alterations in polar body shape (31.3%) and the presence of diffuse cytoplasmic granulations (22.8%), refractile bodies (18.5%) and central cytoplasmic granulations (13.6%). There was no significant difference among individual oocyte dysmorphisms in the agonist- and antagonist-treated groups (P > 0.05).Conclusions: Our randomized data indicate that in terms of the quality of oocyte morphology, there is no difference between the antagonist multi-dose protocol and the long-term agonist protocol. If a GnRH analogue used for pituitary suppression in IVF cycles influences the prevalence of oocyte dysmorphisms, there does not appear to be a difference between the use of an agonist as opposed to an antagonist.

GnRH agonist versus GnRH antagonist in poor ovarian responders: A meta-analysis

The aim of this meta-analysis was to compare the efficacy of gonadotrophin antagonist (GnRH-ant) versus GnRH agonist (GnRHa) as coadjuvant therapy for ovarian stimulation in poor ovarian responders in IVF/intracytoplasmic sperm injection cycles. Search strategies included on-line surveys of databases such as MEDLINE, EMBASE and others. A fixed effects model was used for odds ratio (OR) and effect size (weighted mean difference, WMD). Six trials fulfilled the inclusion criteria (randomized controlled trials). There was no difference between GnRH-ant and GnRHa (long and flare-up protocols) with respect to cycle cancellation rate, number of mature oocytes and clinical pregnancy rate per cycle initiated, per oocyte retrieval and per embryo transfer. When the mete-analysis was applied to the two trials that had used GnRH-ant versus long protocols of GnRHa, a significantly higher number of retrieved oocytes was observed in the GnRH-ant protocols [P = 0.018; WMD: 1.12 (0.18, 2.05)]. However, when the meta-analysis was applied to the four trials that had used GnRH-ant versus flare-up protocols, a significantly higher number of retrieved oocytes (P = 0.032; WMD: -0.51, 95% CI -0.99, -0.04) was observed in the GnRHa protocols. Nevertheless, additional randomized controlled trials with better planning are needed to confirm these results.

Recombinant LH supplementation to recombinant FSH during induced ovarian stimulation in the GnRH-antagonist protocol: A meta-analysis

This study aims to compare the efficacy of recombinant LH (rLH) supplementation for ovarian stimulation in gonadotrophin-releasing hormone-antagonist protocol for IVF/intracytoplasmic sperm injection cycles. Search strategies included online surveys of databases. The fixed effects model was used for odds ratio (OR) and effect size (weighted mean difference, WMD). Five trials fulfilled the inclusion criteria. When the meta-analysis was carried out, advantages were observed for the LH supplementation protocol with respect to higher serum oestradiol concentrations on the day of human chorionic gonadotrophin administration (P < 0.0001; WMD: 514, 95% CI 368, 660) and higher number of mature oocytes (P = 0.0098; WMD: 0.88, 95% CI 0.21, 1.54). However, these differences were not observed in the total amount of recombinant FSH (rFSH) administered, days of stimulation, number of oocytes retrieved, the clinical pregnancy rate per oocyte retrieval, the implantation rate and miscarriage rate. This result demonstrates that the association of rLH with rFSH may prevent any decrease in oestradiol after antagonist administration and that a significantly higher number of mature oocytes was available for laboratory work. Nevertheless, it failed to show any statistically significant difference in clinically significant end-points in IVF (implantation and pregnancy rates). Additional randomized controlled trials are needed to confirm these results further. © 2007 Published by Reproductive Healthcare Ltd.

Administration of single-dose GnRH agonist in the luteal phase in ICSI cycles: a meta-analysis

Background: The effects of gonadotrophin-releasing hormone agonist (GnRH-a) administered in the luteal phase remains controversial. This meta-analysis aimed to evaluate the effect of the administration of a single-dose of GnRH-a in the luteal phase on ICSI clinical outcomes.Methods: The research strategy included the online search of databases. Only randomized studies were included. The outcomes analyzed were implantation rate, clinical pregnancy rate (CPR) per transfer and ongoing pregnancy rate. The fixed effects model was used for odds ratio. In all trials, a single dose of GnRH-a was administered at day 5/6 after ICSI procedures.Results: All cycles presented statistically significantly higher rates of implantation (P < 0.0001), CPR per transfer (P = 0.006) and ongoing pregnancy (P = 0.02) in the group that received luteal-phase GnRH-a administration than in the control group (without luteal-phase-GnRH-a administration). When meta-analysis was carried out only in trials that had used long GnRH-a ovarian stimulation protocol, CPR per transfer (P = 0.06) and ongoing pregnancy (P = 0.23) rates were not significantly different between the groups, but implantation rate was significant higher (P = 0.02) in the group that received luteal-phase-GnRH-a administration. on the other hand, the results from trials that had used GnRH antagonist multi-dose ovarian stimulation protocol showed statistically significantly higher implantation (P = 0.0002), CPR per transfer (P = 0.04) and ongoing pregnancy rate (P = 0.04) in the luteal-phaseGnRH- a administration group. The majority of the results presented heterogeneity.Conclusions: These findings demonstrate that the luteal-phase single-dose GnRH-a administration can increase implantation rate in all cycles and CPR per transfer and ongoing pregnancy rate in cycles with GnRH antagonist ovarian stimulation protocol. Nevertheless, by considering the heterogeneity between the trials, it seems premature to recommend the use of GnRH-a in the luteal phase. Additional randomized controlled trials are necessary before evidence-based recommendations can be provided.

Uma revisão sistemática sobre o uso de agonistas e antagonistas do GnRH em pacientes más respondedoras à estimulação ovariana

The purpose of this investigation was to make a systematic review of the medical literature in order to compare the efficacy of GnRH antagonists and agonists for poor responders to ovarian stimulation. According to the data collected, the use of GnRH antagonist protocols showed better results in comparison to long protocols with a GnRH agonist regarding the following aspects: lower cycle cancellation rate due to poor ovarian response; higher number of oocytes retrieved; higher clinical pregnancy rate per initiated cycle. Nevertheless, these results were not observed when the flare-up protocols of GnRH agonists were used. Moreover the number of oocytes retrieved with GnRH agonist was significantly higher in relation to the GnRH antagonist.

O emprego de hormônio luteinizante recombinante associado ao hormônio folículo-estimulante recombinante nos protocolos de estimulação ovariana com antagonista do GnRH

The purpose of this investigation was to verify the efficacy of recombinant LH supplementation for controlled ovarian stimulation in GnRH-antagonist protocol for assisted reproductive technologies cycles. Search strategies included on-line surveys of databases from 1990 to 2006. In this review and meta-analysis, the observed advantages for the LH supplementation protocol were a higher serum estradiol levels on the day of hCG administration and a higher number of mature oocytes. However, there were no differences observed in the total amount of r-FSH administered, days of stimulation, number of oocyte retrieved, the clinical pregnancy rate per oocyte retrieval, the implantation rate and miscarriage rate. This result demonstrates that the association of r-LH with r-FSH may prevent any decrease in estradiol after antagonist administration and a significant higher number of mature oocytes was obtained. Nevertheless, additional randomized controlled trials are needed confirm these observations.

Cost-effectiveness comparison between pituitary down-regulation with a gonadotropin-releasing hormone agonist short regimen on alternate days and an antagonist protocol for assisted fertilization treatments

Objective: To compare cost-effectiveness between pituitary down-regulation with a GnRH agonist (GnRHa) short regimen on alternate days and GnRH antagonist (GnRHant) multidose protocol on in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) outcome. Design: Prospective, randomized. Setting: A private center. Patient(s): Patients were randomized into GnRHa (n = 48) and GnRHant (n = 48) groups. Intervention(s): GnRHa stimulation protocol: administration of triptorelin on alternate days starting on the first day of the cycle, recombinant FSH (rFSH), and recombinant hCG (rhCG) microdose. GnRHant protocol: administration of a daily dose of rFSH, cetrorelix, and rhCG microdose. Main Outcome Measure(s): ICSI outcomes and treatment costs. Result(s): A significantly lower number of patients underwent embryo transfer in the GnRHa group. Clinical pregnancy rate was significantly lower and miscarriage rate was significantly higher in the GnRHa group. It was observed a significant lower cost per cycle in the GnRHa group compared with the GnRHant group ($5,327.80 ± 387.30 vs. $5,900.40 ± 472.50). However, mean cost per pregnancy in the GnRHa was higher than in the GnRHant group ($19,671.80 ± 1,430.00 vs. $11,328.70 ± 907.20). Conclusion(s): Although the short controlled ovarian stimulation protocol with GnRHa on alternate days, rFSH, and rhCG microdose may lower the cost of an individual IVF cycle, it requires more cycles to achieve pregnancy. Clinical Trial Registration Number: NCT01468441. © 2013 by American Society for Reproductive Medicine.

Agonista versus antagonista do GnRH em ciclos de reprodução assistida: morfologia oocitária

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

AÇÃO REGULATÓRIA DO GnRH NO DESENVOLVIMENTO EMBRIONÁRIO PRECOCE E VITRIFICAÇÃO DE EMBRIÕES SUÍNOS PELO MÉTODO DE MICROGOTA (ROLE OF GnRH DURING EARLY EMBRYONIC DEVELOPMENT AND DEVELOPMENT OF SWINE EMBRYOS FOLLOWING VITRIFICATION BY MICRODROPLET METHOD)

The objective of this study was to investigate the role of GnRH on the preimplantation development of mouse embryos in vitro. GnRH-I, GnRH-II, and GnRH agonists: Des-Gly, Des-Trp and histrelin did not improve embryo development. However, treatment with the specific GnRH antagonist SB-75 blocked embryo development at morula stage. The inhibition of embryo development by SB-75 could be rescued by the addition of histrelin. To determine which intracellular signaling cascade is involved following binding of GnRH to the GnRHR, embryos were cultured in the presence of specific PKC (GFX) or PKA (SQ22536) inhibitors. The PKC inhibitor blocked embryo development at a similar stage as SB-75, whereas SQ22536 had an inhibitory effect, diminishing blastocyst formation and hatched rates. There are evidences that GnRH has an essential autocrine effect on mouse embryonic development via GnRHR, probably by activating PKC signaling cascade while the inhibition of the GnRH signaling does not activate apoptotic mechanisms involving caspase-3. In another experiment, development in vitro of embryos from Chinese Meishan (M) and occidental white crossbred (WC) females were investigated after improving the vitrification protocol for pig embryos. Efficient cryopreservation of zona pellucida-intact porcine embryos and studies of the difference among breeds could greatly impact the swine industry. The percentage of embryos surviving 24 h after cryopreservation without lysis or degeneration was higher for M (72%) than WC (44%). However, in vitro development of embryos that survived cryopreservation was not different between M and WC at the expanded (64%) or hatched (22%) blastocyst stages. Developmental rates were significantly higher for control embryos than frozen embryos from both breeds at expanded blastocyst stage, but not at hatched blastocyst stage. Rates of expanded blastocyst formation did not differ between M and WC control embryos (98 and 95%, respectively). With a new procedure to warm vitrified pig embryos, the survival rates may be improved. The optimal stages to vitrify pig embryos using the microdroplet method ranges from late compact morula to early expanded blastocyst. The results suggest that M embryos have a higher capacity to survive the vitrification process than WC embryos. O objetivo do presente estudo foi investigar a importância do GnRH no desenvolvimento embrionário precoce em camundongos. GnRH-I, GnRH-II e os GnRH agonistas: Des-Gly, Des-Trp e histrelina não incrementaram o desenvolvimento embrionário. Entretanto, o tratamento com SB-75, um antagonista específico do GnRH, bloqueou o desenvolvimento embrionário no estádio de mórula. A inibição do desenvolvimento embrionário pelo SB-75 pôde ser revertida com a adição de histrelina. Para determinar a cascata do sinal intracelular desencadeada pela ligação do GnRH com o seu receptor, embriões foram cultivados na presença de inibidores específicos da PKC (GFX) e da PKA (SQ22536). O inibidor da PKC bloqueou o desenvolvimento embrionário em estádio similar ao bloqueio mediado pelo SB- 75, enquanto o SQ22536 teve efeito inibitório diminuindo a formação de blastocisto e taxas de eclosão. Os resultados sugerem que o GnRH tem um efeito autócrino essencial no desenvolvimento embrionário através do GnRHR, provavelmente, ativando a cascata da PKC. Por outro lado, a inibição do sinal do GnRH não ativa mecanismos apoptóticos que involvam caspase-3. Em outro experimento, foi investigado o desenvolvimento in vitro de embriões da raça Meishan (M) e branco cruzado (WC) após vitrificação pelo método microgota. O desenvolvimento de protocolos eficientes para criopreservação de embriões suínos com a zona pelúcida intacta e a avaliação das diferenças entre raças pode ter um significativo impacto na suinocultura. A percentagem de embriões que sobreviveram à criopreservação depois de 24 h foi maior na M (72%) do que na WC (44%). No entanto, o desenvolvimento in vitro dos embriões que sobreviveram à criopreservação não foi diferente entre M e WC nos estádios de blastocisto expandido (64%) ou eclodido (22%). Os índices de desenvolvimento foram significativamente mais altos para os embriões controle do que para os embriões vitrificados nas duas raças no estádio de blastocisto expandido, porém não foram diferentes para o estádio de blastocisto eclodido. A formação de blastocisto expandido não diferiu entre os embriões controle M e WC (98 e 95%, respectivamente). Com o novo procedimento (“hot warm”) para descongelar embriões vitrificados pelo método de microgota, pode-se aumentar dos índices de sobrevivência. Os melhores estádios embrionários para a vitrificação de embriões suínos variam de mórula compacta tardia até blastocisto expandido inicial. Os resultados sugerem que embriões M têm mais capacidade de sobreviver ao processo de vitrificação do que embriões WC.

Dynamic changes in mitogen-activated protein kinase (MAPK) activities in the corpus luteum of the bonnet monkey (Macaca radiata) during development, induced luteolysis, and simulated early pregnancy: A role for p38 MAPK in the regulation of luteal function

Changes in MAPK activities were examined in the corpus luteum (CL) during luteolysis and pregnancy, employing GnRH antagonist (Cetrorelix)-induced luteolysis, stages of CL, and hCG treatment to mimic early pregnancy as model systems in the bonnet monkey. We hypothesized that MAPKs could serve to phosphorylate critical phosphoproteins to regulate luteal function. Analysis of several indices for structural (caspase-3 activity and DNA fragmentation) and functional (progesterone and steroidogenic acute regulatory protein expression) changes in the CL revealed that the decreased luteal function observed during Cetrorelix treatment and late luteal phase was associated with increased caspase-3 activity and DNA fragmentation. As expected, human chorionic gonadotropin treatment dramatically increased luteal function, but the indices for structural changes were only partially attenuated. All three MAPKs appeared to be constitutively active in the mid-luteal-phase CL, and activities of ERK-1/2 and p38-MAPK (p38), but not Jun N-terminal kinase (JNK)-1/2, decreased significantly (P < 0.05) within 12 - 24 h after Cetrorelix treatment. During the late luteal phase, in contrast to decreased ERK-1/2 and p38 activities, JNK-1/2 activities increased significantly (P < 0.05). Although human chorionic gonadotropin treatment increased ERK-1/2 and p38 activities, it decreased JNK-1/2 activities. The activation status of p38 was correlated with the phosphorylation status of an upstream activator, MAPK kinase-3/6 and the expression of MAPK activated protein kinase-3, a downstream target. Intraluteal administration of p38 kinase inhibitor (SB203580), but not MAPK kinase-1/2 inhibitor (PD98059), decreased the luteal function. Together, these data suggest an important role for p38 in the regulation of CL function in primates.

A role for LH in the regulation of expression of mRNAs encoding components of the insulin-like growth factor (IGF) system in the ovine corpus luteum

P.M. Hastie and W. Haresign (2006). A role for LH in the regulation of expression of mRNAs encoding components of the insulin-like growth factor (IGF) system in the ovine corpus luteum. Animal Reproduction Science, 96(1-2), 196-209. Sponsorship: DEFRA RAE2008

Efeito dos andrógenos na estabilização da preferência manual em machos adultos de sagui comum (Callithrix jacchus)

The role of steroids hormones on the behavior of vertebrates have been described as organizational and activational effects. These actions occur in different periods of the ontogenetic development as fetal, early post natal and during puberty (organizational effect) or modifying the expression of behavioral patterns during time life (activational effects). Studies on brain lateralization in hand use in human and non-human primates have shown that sexual hormones seems to participate in the process of handedness strength that begins in the puberal period and is stabilized at the adult age. The aim of this study was to investigate in adult male Callithrix jacchus if the strength of use of the hand in common marmoset adult male is stable (organizational effect) or androgens variations could affect its stability (activational effect). The preferential use of one hand in 14 common marmoset (Callithrix jacchus was studied in two contexts: (1) spontaneous holding food and directing the food to mouth (feeding episodes), and (2) forced reaching food tests where the animal have to reach the food through a hole within a cover plate with a central hole that allow the use of one hand only to reach the food. The records were made during 5 sessions/20 bouts each during baseline totalizing 100 episodes before two treatments. Firstly it was used GnRH antagonist: a single subcutaneous injection of 100µg de Cetrotide – acetate of cetrorrelix (Baxter Oncology GmbH, Germany) (n=10). Secondly, a single GnRH injection of 0.2mg of GnRH (Sigma – Aldrich) (n= 8) was used. After injections 20 successful attempts of hand use episodes was recorded in the 1st , 2 nd, 7th, 15th and 30 th days, totalizing in the whole period 100 episodes for each context, after both treatments. Fecal sampling to measure extracted fecal androgens was performed in all days of data collection across the length of the basal and during the experimental periods. Statistical analysis by mixed model, Tukey test to compare mean values after the two treatments, and Levene test to compare mean variance were used, all for p-value < 0.05. In basal phase 6 animals used preferentially the right hand, 5 the left and 3 were ambidextrous. Mean handedness index in basal phase were different from that after both treatment starting at 7th day. Mean variance of handedness index for spontaneous and forced activities does not differs before and after both treatments but the mean values for GnRH index were higher than that observed for its antagonist. These findings suggested that androgens have an activational effect on handedness in adult male C. jacchus

Effect of constant infusion of gonadotropin releasing hormone (GnRH) agonist Buserelin and antagonist CDB 2085 A using osmotic minipumps on testicular function in adult male bonnet monkey (Macaca radiata)

The effect of chronic infusion of gonadotropic hormone agonist Buserelin or antagonist CDB 2085 A for 15 weeks via alzet minipumps in adult male bonnet monkeys was studied. Infusion of Buserelin resulted in a decrease in the difference between serum testosterone values at 22.00 hours and 10.00 hours, decrease in responsiveness to injected Buserelin as judged by change in serum testosterone values from pre-injection values and decrease in sperm counts. Infusion of antagonist resulted in a decrease in the difference between serum testosterone values at 22.00 hours and 10.00 hours.