Comparisons of GnRH antagonist protocol versus GnRH agonist long protocol in patients with normal ovarian reserve: A systematic review and meta-analysis

Objective To evaluate the effectiveness and safety of gonadotropin-releasing hormone antagonist (GnRH-ant) protocol and gonadotropin-releasing hormone agonist (GnRH-a) long protocol in patients with normal ovarian reserve. Methods We searched the PubMed (1992–2016), Cochrane Library (1999–2016), Web of Science (1950–2016), Chinese Biomedical Database (CBM, 1979–2016), and China National Knowledge Infrastructure (CNKI, 1994–2016). Any randomized controlled trials (RCTs) that compared GnRH-ant protocol and GnRH-a long protocol in patients with normal ovarian reserve were included, and data were extracted independently by two reviewers. The meta-analysis was performed by Revman 5.3 software. Results Twenty-nine RCTs (6399 patients) were included in this meta-analysis. Stimulation days (mean difference (MD) [95% confidence interval (CI)] = -0.8 [-1.36, -0.23], P = 0.006), gonadotrophin (Gn) dosage (MD [95% CI] = -3.52 [-5.56, -1.48], P = 0.0007), estradiol (E2) level on the day of human chorionic gonadotrophin (HCG) administration (MD [95% CI] = -365.49 [-532.93, -198.05], P<0.0001), the number of oocytes retrieved (MD [95% CI] = -1.41 [-1.84, -0.99], P<0.00001), the embryos obtained (MD [95% CI] = -0.99 [-1.38, -0.59], P<0.00001), incidence of ovarian hyperstimulation syndrome (OHSS) (OR [95% CI] = 0.69 [0.57, 0.83], P<0.0001) were statistically significantly lower in GnRH-ant protocol than GnRH-a long protocol. However, the clinical pregnancy rate (OR [95% CI] = 0.90 [0.80, 1.01], P = 0.08), ongoing pregnancy rate (OR [95% CI] = 0.88 [0.77, 1.00], P = 0.05), live birth rate (OR [95% CI] = 0.95 [0.74, 1.09], P = 0.27), miscarriage rate (OR [95% CI] = 0.98 [0.69, 1.40], P = 0.93), and cycle cancellation rate (OR [95% CI] = 0.86 [0.52, 1.44], P = 0.57) showed no significant differences between the two groups. Conclusion GnRH-ant protocol substantially decreased the incidence of OHSS without influencing the pregnancy rate and live birth rate compared to GnRH-a long protocol among patients with normal ovarian reserve.


Introduction
Since gonadotropin-releasing hormone agonist (GnRH-a) was developed in the 1980s [1], it has played an important role in controlled ovarian hyperstimulation (COH) among patients who are undergoing assisted reproductive technology (ART). The advantage of GnRH agonist is to prevent premature luteinizing hormone (LH) surge, thereby increasing the number of retrieved oocytes and pregnancy rates and decreasing the number of cycle cancellations [2,3]. These advantages, however, may lead to ovarian hyperstimulation syndrome (OHSS) or other side effects [4]. GnRH antagonist (GnRH-ant), which was discovered in the 1990s, can competitively block GnRH receptors and cause rapid suppression of Gn release [5]. This protocol has fewer complications and is more convenient for patients because of the shorter treatment time and fewer injections [6]. However, its effectiveness is still debated.
Multiple studies, including meta-analyses and randomized controlled trials (RCTs), of the GnRH-a protocol and GnRH-ant protocol on pregnancy rate and live birth rate have yielded controversial findings [6][7][8]. A 2006 Cochrane systematic review of 27 RCTs showed that GnRH-ant protocol has a significantly lower clinical pregnancy rate and live birth rates than those in GnRH-a long protocol, while the incidence of OHSS is significantly lower in GnRHant protocol [9]. However, a 2011 Cochrane systematic review of 45 RCTs found that there was no significant difference in the live birth rates between the GnRH-a and GnRH-ant groups [10]. A recent Cochrane systematic review of 73 RCTs in 2016 also concluded that these two protocols have equivalent live birth rates, and GnRH-ant protocol has a lower incidence of OHSS [11].
The finding that GnRH-ant protocol reduces the pregnancy rate may result from the fact that some centers only choose GnRH-ant protocol as their second treatment option in COH, or use it to treat the patients with an unfavorable prognosis, such as repeated implantation failures, older patients, and low responders [12]. This study's purpose is to determine the effectiveness and safety of GnRH-a long protocol and GnRH-ant protocol among patients with normal ovarian reserve to unify the influencing factors.

Materials and methods
Search strategy "GnRH agonist", "GnRHa", "GnRH antagonist", "GnRH-ant", "GnRHA", "randomized controlled trial", "RCT", and "Normal ovarian reserve" were used as the keywords for the literature searches in the PubMed (1992-2016), Cochrane Library (1999-2016), Web of Science , Chinese Biomedical Database (CBM,1979(CBM, -2016, and China National Knowledge Infrastructure (CNKI,1994-2016) databases. The retrieval time was from the first publication of the journal to the end of December 2016. References included in the studies were also searched.

Inclusion and exclusion criteria
Inclusion criteria were RCTs that compared the effectiveness and safety of GnRH-a long protocol and GnRH-ant protocol in patients with normal ovarian reserve. Exclusion criteria included failure to report appropriate randomized procedures, classification of participants as low or high ovarian response or endometriosis, and unclear or inappropriate outcomes. Editorials, letters to the editor, review articles, case reports and animal experimental studies were also excluded.

Data extraction and quality assessment
Studies were screened by two reviewers (R.W. and Y.W.) independently, and any disagreement was settled by consensus. First, the title and abstract of each study was read carefully to exclude the studies that clearly did not meet the inclusion criteria. Then, the full text of the remaining studies was read to determine which studies would be included in this study.
The quality assessment of RCTs was compiled using Cochrane's risk of bias tool [13], which included sequence generation, allocation concealment blinding of participants, personnel and outcome assessors, incomplete outcome data, selective outcome reporting, and other sources of bias.

Outcome measures
The main efficacy outcome measures included the clinical pregnancy rate (defined as the presence of a gestational sac on ultrasound or gestational sac with fetal heart tones), the ongoing pregnancy rate (determined as pregnancies with over 12 weeks of gestation), and the live birth rate. The secondary efficacy outcome measures were ovarian stimulation outcomes, which included stimulation days, gonadotropin dosage, endometrial thickness and estradiol (E2) level on the day of human chorionic gonadotrophin (HCG) administration, the number of oocytes retrieved, and the embryos obtained. The safety outcome measures included the incidence of OHSS, the miscarriage rate, and the cycle cancellation rate.

Statistical analysis
All statistical analyses were performed using Revman 5.3 software. Dichotomous outcomes were expressed as odds ratios (ORs) with 95% confidence intervals (CIs). Continuous variables were expressed as weighted mean differences (MDs) with 95% CIs. Heterogeneity was evaluated using the Q-test and I 2 -index values, and reported for each outcome as a P-value and percentage, respectively. If heterogeneity was adopted (I 2 <25% or >50% with P>0.1), metaanalysis used a fixed-effects model. Otherwise (I 2 !50% or I 2 >25% with P 0.1), meta-analysis used a random-effects model. Sensitivity analysis was used to determine the stability of the results. Begg's funnel plot and Begg's test were used to assess publication bias by STATA software (version 12.0, Stata Corp).

Eligible studies
The initial literature search yielded 1,304 studies. Screening of the titles and abstracts resulted in 68 published articles that could possibly compare the GnRH-a long protocol and GnRH-ant protocol in patients with normal ovarian reserve. There is no official definition of normal ovarian reserve. Therefore, the studies included in our article mainly depended on a consensus, which met any two of the following criteria: Age < 40 years, normal menstrual cycle, basal FSH 10 IU/L, basal E2 < 60 pg/ml, AFC > 5, previous IVF attempts < 3, no previous poor response/OHSS history, no polycystic ovary syndrome (PCOS) / severe endometriosis. After reading the full papers, only 29 studies (6399 patients) finally met our inclusion criteria [8,. Although the cited studies used different drugs and doses for COH, they all belong to either GnRH-ant protocol or GnRH-a protocol, so we included these studies in a general way. The literature screening process and the results are shown in Fig 1, and the basic characteristics of the included papers are shown in Table 1. The quality assessment results are shown in S1 Table. Main outcome measures of effectiveness The clinical pregnancy rate. A total of 25 studies [8, 14-23, 25-29, 32-39, 41](5,814 cases) were included in the clinical pregnancy rate meta-analysis. There was no heterogeneity (P = 0.91, I 2 = 0%) among the trials; therefore, the fixed-effects model was used for the metaanalysis. The results indicated that there was no statistically significant difference in the clinical pregnancy rate between the GnRH-ant group and the GnRH-a long-protocol group (OR [95% CI] = 0.90 [0.80, 1.01], P = 0.08, Fig 2).
The live birth rate. A total of 6 studies [8,14,31,35,37,41](2,237 cases) were included in the live birth rate meta-analysis. There was no heterogeneity (P = 0.88, I 2 = 0%) among the trials; therefore, the fixed-effects model was used for the meta-analysis. The results indicated that there was no statistically significant difference in the live birth rate between the GnRH-ant group and the GnRH-a long-protocol group (OR [95% CI] = 0.95 [0.74, 1.09], P = 0.27, Fig 4).
The endometrial thickness on the day of HCG administration. A total of 6 studies [23,24,27,35,36,40](698 cases) were included in the meta-analysis of endometrial thickness on the day of HCG administration. There was no heterogeneity (P = 0.68, I 2 = 0%) among the trials; therefore, the fixed-effects model was used for the meta-analysis. The results indicated that there was no statistically significant difference in the endometrial thickness on the day of HCG administration between the GnRH-ant group and the GnRH-a long-protocol group (MD [95% CI] = -0.06 [-0.22, 0.11], P = 0.5, Fig 7).
The E2 level on the day of HCG administration. A total of 15 studies [8, 14, 16, 22-27, 32-34, 36, 39, 40](1,770 cases) were included in the E2 level on the day of HCG administration meta-analysis. There was heterogeneity (P<0.00001, I 2 = 91%) among the trials; therefore, the random-effects model was used for the meta-analysis. The results indicated that the E2 level    Fig 9).
The miscarriage rate. A total of 14 studies [8, 14-20, 23, 32, 34-37](3,198 cases) were included in the miscarriage rate meta-analysis. There was no heterogeneity (P = 0.94, I 2 = 0%) among the trials; therefore, the fixed-effects model was used for the meta-analysis. The results indicated that there was no statistically significant difference in the miscarriage rate between the GnRH-ant group and the GnRH-a long-protocol group (OR [95% CI] = 0.98 [0.69, 1.40], P = 0.93, Fig 12).

Sensitivity analysis and publication bias
The sensitivity analysis was performed by excluding the maximum weight studies [8,17,31,36,37,41] in each outcome. The results showed that there was no influence on the pooled OR value; therefore, the outcomes are stable.

Summary of main results
Ovarian reserve can be tested by biochemical measures, such as basal follicle stimulating hormone (bFSH), anti-Mullerian hormone (AMH), and inhibin B level, as well as imaging measures, such as antral follicle count (AFC). The role of testing ovarian reserve is to help clinicians determine the appropriate protocol for individual patients, because ovarian reserve is the foundation of ovarian response to COH which may be vital for assisted reproductive technology (ART) outcome. Ovarian response can be divided into three types, low response, normal response, and high response. In 2011, the European Society of Human Reproduction and Embryology (ESHRE) standardized the definition of poor ovarian response [42], which usually occurs due to diminished ovarian reserve (DOR), and high response, which mainly refers to PCOS. However, there is no standard definition of normal ovarian reserve or normal response. Therefore, patients with normal ovarian reserve are included in our study to a certain extent based on a consensus.
Results of our meta-analysis mainly focused on the effectiveness and safety of GnRH antagonist protocol and GnRH agonist long protocol in COH. Regarding effectiveness, stimulation days, Gn dosage, E2 level on the day of HCG administration, and the number of oocytes retrieved, the embryos obtained were statistically significantly lower in GnRH-ant protocol than GnRH-a long protocol. The shorter stimulation period and lower Gn dosage in the GnRH-ant group may lead to the lower E2 level on the day of HCG administration, fewer retrieved oocytes, and fewer obtained embryos. However, regarding the main outcome measures of effectiveness, clinical pregnancy rate, ongoing pregnancy rate, and live birth rate had no significant differences between the two groups. The endometrial thickness on the day of HCG administration also showed no significant difference. This indicated that GnRH-ant protocol did not lead to a thicker endometrium on the day of HCG administration, and there was no difference in the reproductive outcome between the two groups. This means that even if the GnRH-ant protocol leads to fewer retrieved oocytes and fewer obtained embryos, this protocol does not reduce the chance of achieving a live birth. Moreover, GnRH-ant reduced the stimulation period and the Gn dosage, which is more convenient and cost-effective for patients.
The incidence of OHSS was statistically significantly lower in GnRH-ant protocol than in GnRH-a long protocol. This may because GnRH-ant protocol resulted in fewer retrieved oocytes, and a lower E2 level on the day of HCG administration, which had less possibility to induce OHSS than GnRH-a long protocol. No significant difference was found between the miscarriage rate, and the cycle cancellation rate between the two groups. Therefore, GnRH-ant protocol is safer because of the lower occurrence of OHSS without leading to miscarriage or cycle cancellation. According to our results, GnRH-ant protocol can reduce the incidence of OHSS, faster and more cost-effectively, without affecting the reproductive outcome in normal ovarian reserve patients. Therefore, GnRH-ant protocol should be used as widely as GnRH-a protocol in these patients.

Comparison with other studies
The recent Cochrane systematic review of 73 RCTs in 2016 has concluded that GnRH-ant protocol and GnRH-a protocol have equivalent live birth rates, while GnRH-ant protocol has a lower incidence of OHSS [11]. Their results are similar to ours. However, the patients with all types of ovarian reserve were included in their study, which covered DOR and PCOS.
A meta-analysis focusing on the supposed normal ovarian responders, including 23 RCTs (n = 3961), was conducted in 2014 [43]. The results indicated that the incidence of OHSS was significantly lower, whereas the ongoing pregnancy and live birth rates were similar in the GnRH antagonist and the standard long GnRH agonist protocols, which is similar to the results of our meta-analysis.
Another systematic review and meta-analysis of 22 RCTs (n = 3176) comparing GnRH-ant and GnRH-a reported the live birth as their main outcome measure [7]. No significant difference was present in the probability of live birth between the two GnRH analogs [OR = 0.86; 95% CI = 0.72 to 1.02]. This result remains stable in subgroup analysis that ordered the studies by type of population studied, Gn type used for stimulation, type of agonist protocol used, type of agonist used, type of antagonist protocol used, type of antagonist used, presence of allocation concealment, presence of co-intervention, and the way the information on live birth was retrieved. However, their study also included all types of ovarian reserve.
Additionally, a meta-analysis of four RCTs (n = 874) in 2008 mainly measured ongoing pregnancy rate [44]. The ongoing pregnancy rate per randomized woman was not found to be significantly different between patients with and without oral contraceptive pill (OCP) pretreatment (OR: 0.74, 95% CI: 0.53 to 1.03). Duration of Gn stimulation (WMD: 1.41 days, 95% CI: 1.13 to 1.68) and Gn consumption (WMD: 542 IU, 95% CI: 127 to 956) were significantly increased after OCP pretreatment. No significant differences were observed regarding the number of retrieved oocytes. However, the patients included in their study were also not classified by ovarian reserve.

Limitations of this study
First of all, this meta-analysis lacked a uniform definition of normal ovarian reserve, which made it difficult to define inclusion and exclusion criteria, and the studies included may be not strict enough. Second, although the random-effects model was used to minimize the heterogeneity, it cannot be abolished. The heterogeneity may relate to the size of studies that ranged from 40 to 1,050 patients, and to the time span between 2000 and 2016. Additionally, the quality of reported studies was uneven, which may also lead to heterogeneity. The methods of randomization, concealment, and blinding were unclear in some included studies, some of which also had incomplete and selective outcome data. Third, although comprehensive searches were undertaken to ensure that all eligible studies were included, there is still the possibility that some potentially eligible studies were left out. All of these factors may lead to bias.

Implications
The GnRH-ant protocol is a short, convenient, and cost-effective protocol in COH among patients with normal ovarian reserve, which can also reduce the incidence of OHSS without affecting the live birth rate when compared to GnRH-a long protocol. When referring to patients with normal ovarian reserve, in future clinical practice, GnRH-ant protocol may also be a good choice for COH. Moreover, a uniform definition for ovarian reserve, especially for normal ovarian reserve, should be standardized as soon as possible. RCTs of GnRH-ant protocol and GnRH-a long protocol in patients with different ovarian reserve are still lacking. And the study design of future RCTs should be more rigorous according to the CONSORT statement [45].

Conclusion
In conclusion, our meta-analysis demonstrated that GnRH antagonist protocol substantially decreased the incidence of OHSS without influencing the pregnancy rate and live birth rate compared to GnRH agonist long protocol among patients with normal ovarian reserve. Therefore, for patients with normal ovarian reserve, GnRH-ant protocol should play a more important role in COH when clinicians are making individualizing and optimizing treatment decisions.
Supporting information S1 Table. Quality assessment of studies.