{"paper_id":"57b1873c-0b5c-4109-a6cf-8eb918227224","body_text":"The gonadotropin-releasing hormone (GnRH) agonist and antagonist protocols are extensively\nutilized in the ovarian stimulation cycle to inhibit the endogenous rapid increase in the\nluteinizing hormone (LH surge) levels. The unique properties and functional mechanisms of\nGnRH analogues in the  in vitro  fertilization (IVF) cycles are well defined\n( 1 ).\nGnRH agonist have a longer half-life and higher potential than native GnRH. They initially stimulate pituitary\ngonadotrophs and production of follicle-stimulating hormone (FSH) and LH hormones, thereby cause an expected response of gonads ( 2 ). In contrast, GnRH antagonist\nimmediately suppress pituitary gonadotropin in the competition with the GnRH receptor, thereby prevent early\nexcitatory phase of agonists. Recently, there have been\nan increasing interest in using GnRH antagonist in control\novarian hyperstimulation (COH). GnRH antagonist have\nbeneficial effects compared to the GnRH agonist. Most\nnotably they cause fewer follicles and lower daily usage of\nestradiol, and thus lower incidence of ovarian hyperstimulation syndrome (OHSS), a serious complication which\neventually helps the reproductive treatment ( 3 ). However,\nit has been reported that GnRH antagonist administration\nis along with a reduced live birth rate and an increase in\nthe risk of pregnancy loss, which might be the result of\nimpaired implantation and lower estradiol concentrations\non the first day of COH ( 2 ). In addition to the pituitary, the role of GnRH in other tissues including ovary, uterus,\nand placenta have been demonstrated in previous studies.\nAlthough the mode of action of GnRH and its analogues\nare well determined on the pituitary level, its role in the\nextra pituitary tissues is still not fully understood ( 4 ).\nGnRH receptors are present on the ovarian epithelial cells, granulosa cells (GCs), and\ncumulus cells (CCs). CCs are involved in the follicular development, maturity, and quality\nof the oocyte ( 5 ). There is a bidirectional paracrine communication between the CCs and\noocytes during folliculogenesis ( 6 ). By secreting paracrine markers including growth\ndifferentiation factor 9 (GDF-9) and bone morphogenetic protein-15 (BMP-15), the oocyte\ninduces CC gene expression to ensure its development and maturation ( 7 ). For this reason,\noptimal development and the quality of the oocyte can be evaluated by the CC gene expression\nas a non-invasive method ( 6 ).  Versican  (VCAN)  and\nactivated leukocyte cell adhesion molecule ( ALCAM ) are expressed in the CCs\nand contribute to the extracellular matrix (ECM) formation ( 8 ).  VCAN , which\nis a proteoglycan, is expressed in the GCs after ovulation induction.  VCAN \nis cleaved following LH surge by a precise molecular pathway and the cleaved\n VCAN , as the functional form, is observed in the COCs ( 9 ). Since\nimportant growth factor receptors are attached to this functional form, a change in the\n VCAN  expression might also alter COC matrix properties during the oocyte\nmaturation, ovulation, and fertilization ( 8 ).  ALCAM  is known as an\nECM-related protein. Cell to cell and cell to matrix adhesion may be promoted by\n ALCAM  in the reproductive tissues.  ALCAM  has been shown\nto be expressed in the epithelium and blastocysts and is involved in the implantation\nprocess ( 10 ). A significant association is reported between the expression of these genes\nand oocyte quality ( 11 ).\nMoreover, it seems that apoptosis of CCs reduces the success rate in IVF ( 12 ) and the\nhigher the incidence of apoptotic CCs, the lower the fertilization rate ( 13 ). The vital role\nof programmed cell death in different physiological events of reproduction is well\nestablished. For instance, during folliculogenesis, the number of follicles in a follicular\ncohort primarily diminishes due to the apoptosis of GCs ( 14 ).  SURVIVIN  is a\nmember of inhibitors of apoptosis proteins (IAPs) and has an important caspase inhibitory\nfunction ( 15 ). Critical functions of survivin in folliculogenesis and follicular development\nare not limited to apoptosis inhibition, but also this protein participates in the\nregulation of the mitotic spindle checkpoint ( 16 ). Follicular development or atresia are\nregulated by different hormonal and microenvironmental factors ( 17 ). AMH, GnRH, androgens,\nand apoptotic ( BAX, P53, FOXO3 ) and anti-apoptotic ( BCL-2,\nSURVIVIN ) genes are identified as the follicular atretogenic factors ( 18 ). The\nanti-apoptotic role of  BCL-2  against a variety of cell death-inducing\nfactors has been proved in numerous studies. A correlation has been found between apoptosis\nacceleration and overexpression of  BAX , as a pro-apoptotic agent ( 19 ).\nTherefore, the present study was performed to examine the impact of GnRH agonist and antagonist on IVF cycles\nfrom clinical and molecular points of view. For this purpose, the oocyte quality, embryo development, CC gene\nexpression, and pregnancy rate were compared between\nthe two groups of patients who received GnRH agonist or\nantagonist throughout the IVF cycle.\n\nIn this study, 21 eligible infertile women\nundergoing IVF cycle were chosen. This study was conducted in\nVali-e-Asr Reproductive Health Research\nCenter, Tehran University of Medical Sciences (Tehran,\nIran) from December 2014 to February 2016. Study was\napproved by Ethics Committee of Tehran University of\nMedical Sciences (IR.TUMS.VCR.REC.1396.2309).\nPeople who agreed to take part in this study signed a\nconsent form. Participants were divided into two groups\nof subjects, who received either GnRH agonist or GnRH\nantagonist. Subjects had an equal chance of being in\nboth groups.\nThe inclusion criteria for the subjects were age<40 years and body mass index\n(BMI) <30 kg/m 2  . Furthermore, the cause of undergoing IVF was tubal\nfactor infertility, and according to WHO criteria, partners had normal sperm parameters.\nThe exclusion criteria were as follows: patients with ovarian dysfunction or other\nendocrinopathies, infertile couples with severe male factor infertility, poor responders,\npolycystic ovarian syndrome (PCOS), and endometriosis.\nThe choices of sample size and study duration were\nbased on the primary outcome obtained from the study\nof Danhua Pu 2011. A sample size of 11 with 80% power\nwas achieved by a Two-Sided One-Sample t test, which\ncan detect an effect size (i.e. mean difference) of 1.9 between the null hypothesis with no mean difference and the\nalternative hypothesis mean=-1.9 with an assessed standard deviation of 2.4 and alpha= 0.05. The sample size estimation was conducted using PASS 15 software.\nTen individuals were picked out from the GnRH agonist treated\npatients that received triptorelin 0.1 mg/day\nsubcutaneously (Decapeptyl, Ipsen, Italy) in the luteal phase of\ntheir preceding cycles based on a standardized long protocol. Following gonadotrophin\ninhibition, which was confirmed by transvaginal ultrasound, the patients received 150-225\nIU recombinant FSH (rFSH) (Gonal-F, Merck Serono Laboratories, Switzerland) on the\n3 rd  day of their periods ( 20 ).\nEleven women in the GnRH antagonist group were treated with 150-225 IU/day rFSH subcutaneously beginning\non the second day of their monthly periods followed by a\nsingle dose adjustment from day 5 of the cycle. Each patient received 0.25 mg/day of cetrorelix (Cetrotide, Serono)\non the sixth day of COH according to a fixed protocol ( 20 ).\nTo assess the ovarian response to the stimulation protocol, prior to the injection of the human chorionic gonadotropin (HCG) hormone, the follicle sizes were measured and clinical tests such as serum estradiol and FSH\nconcentration measurements and transvaginal ultrasounds\nwere performed. Serum FSH and estradiol concentrations\nwere measured using immunoassay kits (CALBIOTECH,\nUSA) with an automated multi-analysis system.\nA single dose of HCG (Gonasi HP 5000, AMSA, Italy)\n10,000 IU was injected intramuscularly following the observation of at least three follicles with an optimal diameter of 18mm and serum estradiol ≥0.40 nmol. Oocytes\nwere picked up 34-36 hours after HCG administration.\nEmbryological, clinical, and molecular variables were\nevaluated to compare the effects of GnRH antagonist and\nagonist. The pregnancy rate and the number of ovarian\nfollicles were evaluated as clinical parameters.\nFor evaluation of oocyte competence, the percentage of\nmetaphase II (MII) ( Fig .1A ), metaphase I (MI) ( Fig .1B )\nand germinal vesicle (GV) oocytes ( Fig .1C ), were calculated. Furthermore, the percentage of 2 pronuclei (2PN,\n Fig .1D ) from the total number of MII retrieved oocyteswere considered as fertilization rate.\nThe pregnancy rate was evaluated as the percentage of\nthe subjects with positive βHCG test after receiving either\nthe agonist or the antagonist protocol. The number of years\nthat a woman was infertile was considered as the infertility duration. According to the constructor’s instructions,\nserum prolactin concentrations were measured using an\nELISA kit (Calbiotech, USA). The endometrial thickness\nand ovarian follicle count (number of follicles more than\n18 mm) were measured using gynecological ultrasound.\nThe percentage of 7-cell embryos with less than 10%\nfragmentation (graded as A,  Fig .1E ) and the percentage of\nembryos with at least 7 cells having >10% fragmentation\n(graded as AB,  Fig .1F ) from the total number of fertilized\noocytes on day 3 after insemination were assessed and\ncompared between the two groups.\nDifferent stage of oocyte and embryo development that was evaluated in the study.\n A.  Metaphase II (MII),  B.  Metaphase I (MI), and\n C.  Germinal vesicle (GV) oocyte.  D.  2 pronuclei (2-PN),\n E.  7-cell embryos with less than 10% fragmentation (graded as A),\nand  F.  7 cells with more than 10% fragmentation (scale bar: 20 µm)\nPrimer sequences used in quantitative real time polymerase chai reaction\nFor the molecular investigation, expression of  VCAN, ALCAM, SURVIVIN,\nBAX  and  BCL-2  genes were evaluated in the CCs.\nThe retrieved COCs were washed several times in commercial human tubal fluid (HTF Lonza, Verviers, Belgium)\nin order to eliminate any blood cells, GCs, and debris contamination. Then, they were incubated in the fertilization\nmedium for 5 minutes (Universal IVF medium, Medicul,\nDenmark). CCs samples were mechanically dissected less\nthan 1 hour after OPU. Isolation of CCs was performed by\nwashing these cells in the culture medium and centrifugation 10 minutes at 250 × g several times ( 21 ). Then, the\ncells were pooled and preserved by rapid freezing just after\ndissection and prior to the RNA extraction.\nCC RNA extraction was performed using Arcturus\nPico Pure RNA Isolation Kit (Applied Biosystems,\nUSA) based on the manufacturer’s instructions (from ~\n4 ng pooled oocyte to 100 ng of CCs, and 3 repetitions\nfor this experiment). DNase I (Fermentas, St. Leon-Rot,\nGermany) was used three times to eliminate genomic\nDNA contaminations. The purified RNA was used for\ncDNA synthesis using oligo dT primers (Applied Biosystems, Foster City, CA) prior to real-time polymerase\nchain reaction (PCR) ( 22 ).\nThe primers were designed to the human sequence of  VCAN, ALCAM, SURVIVIN, BAX\n and  BCL-2  genes using the Gene Runner (version 3) and Primer\nExpress (version 3.05), and were blasted in http://www.ncbi.nlm. nih.gov/BLASt/. The\nprimer characteristics are presented in Table 1.\nReal-time PCR was accomplished with the SYBR Green Reagent (Applied Biosystems, USA)\nusing ABI PRISM 7300 Analyzer (Applied Biosystems, USA). The PCR cycle was repeated for\n45-55 cycles. The Q-PCR reaction was carried out at least three times using specific\nprimers. The quantification of 5 genes was evaluated using the comparison with the\nhousekeeping gene, beta-actin. Finally, 2 −∆∆CT  technique was used for\ncomparative quantification between the two groups.\nData were analysed using IBM SPSS Statistics software (version 25, IBM SPSS Statistics, Armonk, USA)\nand the graphs were drawn by GraphPad (Prism) (version 8, https://www.graphpad.com). Normality of the\nnumeric variables was checked and confirmed by Kolmogorov-Smirnov test and measures of distribution including skewness and kurtosis were within ± 1.5 and ±\n2, respectively. Data are presented as the mean (SD) and\nfrequency (percent) for numeric normal and categorical\nvariables, respectively. Comparisons of the variables between the groups were conducted by Independent Samples t test. The assumption of the homogeneity of the\nvariances were assessed by Levene’s test, and Welch\ncorrection was used when the assumption was not satisfied. For comparing the categorical variables between\nthe two groups, Pearson Chi-square test with exact P\nvalue was utilized. In all analyses, a P<0.05 indicates\nstatistically significant.\n\nClinical characteristics of the woman in different groups\nare shown in Table 2. No significant difference was observed\nin the infertility duration, age, BMI and hormonal\nlevels between the two groups. In addition, the number of\nthe dominant ovarian follicles, endometrial thickness, and\npregnancy rate were not different significantly between\nthe groups.\nIn GnRH antagonist group, the mean number of obtained COCs was higher than the GnRH agonist group,\nwhich was not statistically significant (P=0.14,  Table 3 ).\nIn order to compare the oocyte nuclear maturity, the percentages of MII, MI, and GV oocytes were calculated and\ncompared between the groups. As shown in Table 3, MII\npercentage is clinically higher in the GnRH antagonist\ngroup compared to the agonist group (84.8 ± 20 vs. 78.6\n± 27.6, P=0.57). No considerable difference was found in\nthe percentage of MI and GV oocytes between the two\ngroups (12.6 ± 17.8 vs. 9.2 ± 16.2, and 5.6 ± 9.6 vs 5.5 ±\n8.9, P=0.65 and P=0.99, respectively). Moreover, a statistically significant difference was observed in the percentage of 2 PN between the GnRH agonist and GnRH\nantagonist groups (54.5 ± 19.2 vs. 72.5 ± 9.1, respectively, P<0.05). Finally, the percentage of grade A and AB\nembryos from the total number of fertilized oocytes were\ncompared in each group. No significant difference was\nfound in the percentage of type A (52.9 ± 34.3 vs. 55.5 ±\n29.6%, P=0.24 and AB embryo (21.2 ± 20 vs. 22.9 ± 25%,\nP=0.87) between the GnRH agonist and GnRH antagonist\ngroups.\nAs shown in Figure 2, the relative  BAX  gene expression in the CCs of\npatients that received GnRH agonist was significantly higher than those with GnRH\nantagonist treatment (39.1 ± 2 vs. 27.01 ± 4.2, respectively, P<0.001).\nFurthermore, expression of  BCL-2  was higher in the CCs of the patients\nreceived GnRH antagonist against those who received GnRH agonist (61.4 ± 2.2 vs. 44.3 ±\n4.2, P<0.001). The  ALCAM  expression was significantly different\nbetween the GnRH agonist and antagonist groups (16.8 ± 0.6 vs. 22.2 ± 1.3, respectively,\nP<0.001). No significant difference was seen in the expression of\n VCAN  (40.5 ± 7.9 vs. 41.8 ± 6.7, P=0.789) and  SURVIVIN \n(64.8 ± 6 vs. 66.9 ± 7.6, P=0.131) between the GnRH agonist and antagonist groups,\nrespectively ( Fig .2 ).\nRelative gene expression of apoptotic and developmental gens by\nreal time polymerase chain reaction (PCR). **; Show significant difference.\nClinical characteristics and pregnancy outcomes of woman receiving GnRH agonist or GnRH antagonist\nGnRH; Gonadotropin-releasing hormone, BMI; Body mass index, FSH; Follicle-stimulating hormone, COH; Control ovarian hyperstimulation, CI; Confidence interval, #; P value from the\nindependent samples t test. In all variables, equal variances assumed based on the results from the Levene's Test for Equality of Variances (All P>0.05).\nComparison of embryological parameters between GnRH agonist and GnRH antagonist groups\nCOC; Cumulus oophorus complex, GV; Germinal vesicle, MI; Metaphase I, MII; Metaphase II, PN; Pro\nnucleus, CI: Confidence interval,  #  ; P value from the independent samples\nt test, and  *  ; Significant P<0.05. The Levene's Test for Equality\nof Variances showed that the assumption was not satisfied for MI, MII, PN (all\nP<0.05).\n\nThis study showed that  BAX  gene expression in the CCs of patients treated\nwith GnRH agonist was higher than those treated with GnRH antagonist. Furthermore, mRNA\nexpression of  BCL-2  and  ALCM  genes were considerably\ngreater in the CCs of the antagonist group in comparison to the agonist group. The gene\nexpression of CCs in the individuals treated with assisted reproductive technology (ART)\nhave been evaluated in numerous previous studies ( 23 ). To best of our knowledge, this is the\nfirst study to investigate the effect of GnRH analogues on CC gene expression.\nThe correlation between apoptosis of CCs and ART outcome has been demonstrated in numerous\nstudies ( 23 ). Clavero et al. ( 24 ) reported that the apoptosis rate of the GCs is not\nassociated with the oocyte maturity, quality, and pregnancy outcomes. However, Lee et al.\n( 25 ) found a strong correlation between the apoptosis of CCs and poor oocyte quality.\nMoreover, up-regulation of pro-apoptotic genes and downregulation of anti-apoptotic genes in\nthe CCs of the non-early cleavage embryos have been previously described ( 26 ). It was shown\nthat survivin plays an essential role in the function of GCs and the inhibition of apoptosis\n( 15 ). In addition, a positive relationship has been observed between the\n SURVIVIN  gene expression in the GCs and pregnancy rate ( 27 ). According to\na study by Assou et al. ( 28 ), the overexpression of  BCL-2  is associated\nwith pregnancy outcomes. The present study indicated that the relative expression of\n BCL-2  is higher in the GnRH antagonist group as compared to the agonist\ngroup. Moreover,  BAX  was overexpressed in the GnRH agonist group as\ncompared to the antagonist group. Furthermore, we found no positive correlation between the\nexpression of apoptotic genes and oocyte quality, embryo development, and pregnancy outcome.\nThe effect of different protocols of GnRH analogues on\nthe ART cycle is controversial ( 29 ). Similar results have\nbeen reported by Kara et al. ( 30 ) regarding the serum\nprogesterone and estradiol levels and the pregnancy\nrate. Prapas et al. ( 31 ) reported positive effects of GnRH\nantagonist on the live birth rate as well as embryologic\nand clinical outcomes. Furthermore, higher quality\nof blastocysts have been noticed in the recurrent\nimplantation failure (RIF) patients that received GnRH\nantagonist compared to those receiving agonist treatment\n( 32 ). Contrary to the mentioned study, de Souza Jordão et\nal. ( 33 ) revealed a higher total oocyte number and quality,\nmore embryo development, higher implantation rate,\nand better pregnancy outcomes following GnRH agonist\nadministration. A recent meta-analysis showed an equal\npregnancy rate, endometrial thickness, live birth rate,\nand cancelation rate after the use of GnRH agonist and\nantagonist in normal-responder patients ( 34 ). Although\nthe clinical and embryological results of our study are not\nconsistent with the aforementioned articles, the molecular\nfindings are compatible. This contradiction may be due to\ndifferent and incomparable sample sizes.\nTwo of the five genes that were found to be expressed during oocyte maturation were\nanalyzed in the present study ( ALCAM, VCAN ) ( 8 ). A negative correlation was\nexplained between  VCAN  expression level and the percentage of mature oocyte\nformation. Moreover, decreased  VCAN  expression was shown in the CCs of the\nsubjects with mature oocytes ( 35 ). In our study, a relatively lower expression of\n VCAN , which was not statistically significant, was observed in the GnRH\nantagonist group.  ALCAM  is known as an ECM-related protein. Cell to cell\nand cell to matrix adhesion may be promoted by  ALCAM  in the reproductive\ntissues.  ALCAM  has been shown to be expressed in the epithelium and\nblastocysts and it has an important role in the implantation process ( 36 ). A previous study\nstated expression of  ALCAM  in the CCs and GCs during the ovulatory response\n( 37 ). Moreover, a positive correlation between the  ALCAM  expression and\nproper embryo cleavage has been indicated. It was also introduced as a promising new marker\nfor non-invasive embryo selection ( 35 ). We also found a significantly higher\n ALCAM  expression in the CCs of the GnRH antagonist-treated group in\ncomparison to the agonist group.\n\nDespite no considerable difference in the oocyte quality,\nembryo development, and clinical outcomes between\nGnRH agonist and antagonist, the GnRH antagonist\nprotocol is more favorable considering our molecular\nfindings. In fact, further molecular studies should be\nperformed on this controversial subject to define the exact\neffect of GnRH analogues on the reproductive system\nand to identify any advantage or superiority between the\nGnRH agonist and antagonist protocols.","source_license":"CC-BY-4.0","license_restricted":false}