Since the early 1980’s a variety of controlled ovarian hyperstimulation (COH) protocols with the use of gonadotrophin-releasing hormone (GnRH) analogues (agonists or antagonists) in conjunction with gonadotropins/menotropins have been developed ^{1, 2, 3, 4}. The addition of GnRH analogues significantly reduced the incidence of premature LH surges and cycle cancellations, leading to a higher number of oocytes retrieved, with an improved outcome of infertility treatments ^{5, 6, 7, 8}. Despite a significant number of studies ³,¹²,¹³,³¹,³⁴,³⁸,⁴⁰,⁴²,⁴⁸,⁵² comparing both long GnRH agonist and GnRH antagonist protocols, the superiority of one over another is still widely debated in the literature.

The mechanism of action of these two analogues (agonist and antagonist) is different. In the long agonist protocol, antral follicles are recruited by exogenous gonadotropin after early depletion of endogenous hormones. In the antagonist protocol the endogenous hormones recruit follicles. GnRH antagonist controls gonadotropin secretion by its immediate suppression in the pituitary. The use of the antagonist is associated with a shorter time of stimulation, reduced gonadotropin consumption and a reduced ovarian hyper-stimulation incidence (OHSS) as well as a different pattern of steroid hormones concentration in the blood and follicular fluid ⁹ when compared to the agonist protocol ^{10, 11, 12, 13, 14}. The differences in the endocrine environment for maturing oocytes may alter the ovarian folliculogenesis and influence the oocyte quality ^{15, 16, 17}and subsequently embryo development ¹⁸. It has been demonstrated that embryos derived from the women stimulated with the flexible GnRH antagonist protocol underwent faster with the earliest cleavage than embryos derived from women stimulated with a long GnRH agonist protocol ¹⁹.

Previously, a positive correlation between the follicle size, oocyte maturity and pregnancy rate of women undergoing IVF, has been reported ^{20, 21, 22, 23, 24, 25}. Others ^{26, 27, 28} demonstrated that larger leading follicles yield better pregnancy rates, but not necessarily a higher percentage of mature oocytes or availability of embryos for transfer ²⁵. Teissier et al. ¹⁷ showed a discrepancy between the size and maturity status of the oocytes collected from the patients stimulated with the agonist protocol. They found mature oocytes were present in smaller follicles, and immature in larger follicles. Nogueira et al showed a significantly higher percentage of immature oocytes were retrieved from large follicles in antagonist cycles when compared to the agonist protocol ²⁷. They found also a greater heterogeneity in maturity of oocytes retrieved from patients stimulated with antagonist. Therefore, taking into consideration the information available in the literature, we undertook the present study to determine whether a low, medium or high percentage of mature oocytes retrieved may influence the clinical outcome of the two commonly used ovarian stimulation protocols (flexible multi-dose antagonist and long agonist) in IVF patients.

The study was approved by the Institutional Review Board of the Abington Memorial Hospital, PA. The data of 1264 ICSI cycles (2000 – 2013 years) with information on the nuclear maturity of retrieved oocytes, were extracted from the electronic database. Any types of donors (oocytes, sperm) or gestational carrier cycles were excluded. The follicular growth was stimulated using common IVF protocols either agonist (long lupron, 654 cycles) or multidose flexible antagonist (antagon, 610 cycles) and pure gonadotropins and menotropins (Gonal-F, Serono Laboratiories INC. Norwell, MA, Follistim, Organon Inc, West Orange, NJ, Repronex and Brevale, Ferring Pharmaceuticals Inc., Tarrytown, NY). The ovarian stimulation started on day 3. The initial dose of hormones was determined based on the follicles size, estradiol level in the blood, age of patient and type of diagnosis. Dosages were adjusted every 2-3 days in association with the follicles size and estradiol level in the blood. HCG 10,000 IU (Profasi, Serono, USA) was administered when at least one follicle was ≥18 mm in diameter, and endometrial thickness of >8 mm and appropriate estradiol levels achieved. The oocytes were retrieved 34-36 h later. To precisely assess the maturity of retrieved oocytes, denudation of oocytes using hyaluronidase (80 IU/ml; Conception Technology, San Diego, CA, USA) was performed 1-2 h after oocytes collection. Spermatozoa for the ICSI procedure was prepared using density gradient (90 & 45%; PureCeption, Conception Technologies, CA, USA) protocol. After injection, oocytes were cultured in a fertilization medium following cleavage media (both from Cooper Surgical Inc., Trumbull, CT, USA). Fertilization was assessed 17-20 h after injection. The embryo development (number and regularity of blastomeres, degree of embryo fragmentation and cytoplasm quality) was assessed daily. Ultrasound-guided embryo transfer (ET) was carried out on the day 3 after oocytes retrieval. The number of embryos transferred was in accordance to the guidelines of the Practice Committee of American Society for Reproductive Medicine (ASRM) and Practice Committee of the Society for Assisted Reproductive Technology.

The distribution of diagnoses (endometriosis, idiopathic, male factor, ovulatory dysfunction, tubal, polycystic ovary syndrome, diminished ovarian reserve or advanced maternal age) in our antagonist and agonist protocol study groups is presented in Table 1. The majority of patients in both treatment protocols were diagnosed as male factor (approx. 45%) and diminished ovarian reserve (approx. 25%).

The use of the antagonist or agonist in the ovarian stimulation protocols did not have any effect (p<0.35) on the overall percentage of mature oocytes retrieved. Percentage of mature oocytes retrieved correlated positively (p<0.003, data not shown) with the number of days of stimulation in both protocols. There were no differences between the three studied groups (≤30%, 31-69% and ≥70 %) of the antagonist and agonist protocols in relation to the age, day 3 FSH level, endometrium thickness, estradiol level on day of hCG, or estradiol concentration per retrieved oocyte (Table 2). However, in the antagonist protocol treated group of ≥70 % matured oocytes retrieved, there was a tendency (p<0.053) in enrolling more older patients. Consequently, the difference in the age (p<0.001) between the two treatment protocols of the groups with ≥70 % mature oocytes was established. The endometrium was significantly thinner in the antagonist protocol groups (p<0.004, p<0.001and p<0.002, respectively) compared to the agonist. The estradiol level at time of HCG injection was significantly (p<0.001) lower for the antagonist of second (31-69%) and third (≥70%) group compared to corresponding agonist groups (Table 2).

There were no differences in the number of eggs retrieved between the antagonist groups. However, the first agonist protocol group (≤30% mature oocytes) had the lowest number of retrieved oocytes (p<0.04) when compared to other groups using this protocol (Table 3). The number of mature and fertilized (2PNs) oocytes gradually increased (p<0.001) as more mature oocytes were retrieved in each of both protocol groups (Table 3). There were no differences in the number of transferred embryos between each protocol group , as well as between the treatment protocols. There were no differences in the fertilization rate between the antagonist and agonist protocol groups (Table 3). The differences between the treatment protocols (antagonist vs. agonist) were determined among the groups 31-69% and ≥70 % mature oocytes in the number of retrieved (p< 0.0009 and p<0.0007, respectively), matured (p<0.0001, p<0.0004, respectively) and fertilized oocytes (p< 0.0004, p<0.0007, respectively). Additionally, the fertilization rate of the agonist group with ≥70% of mature oocytes was significantly higher (p<0.03) compared to the corresponding antagonist protocol group (Table 3). The overall number of retrieved, mature and, fertilized oocytes was significantly higher (p<0.05) in the agonist protocol when compared to the antagonist protocol (data not shown).

There were no differences in implantation, and live births between the three studied antagonist protocol groups (Table 4). The clinical pregnancy rate (presence of sac and heart beat) in the first antagonist protocol group (≤30% mature oocytes) was lower (p<0.03 and p<0.05, respectively) when compared to the other two studied groups. In the agonist protocol, implantation and live births of the first group were significantly (p<0.009) lower than compared to third group but not the second group. The clinical pregnancy rate in first agonist protocol group (≤30% mature oocytes) was lower (p<0.008 and p<0.023, respectively) compare to the other two groups. Live birth rates were significantly different between the antagonist and agonist protocols: 22.2 vs. 35.95% (p<0.0009) for groups 31-69% and 23.9 vs. 41.5% (p<0.0001) for groups with ≥70% mature oocytes.

In our study, the groups with low ovarian response (less than 30 % of mature oocytes retrieved) of the antagonist (antagon) and the agonist (long lupron) were comparable in terms of characteristics assessed (endocrinological and embryological) and the clinical outcome (implantation, clinical pregnancy and live births). Similarly, Al-Inany et al. ²⁹ showed no differences in clinical pregnancy rates between the GnRH antagonist and the GnRH agonist treatment in patients with low ovarian responses or PCO patients ³⁰. However, in our study, the groups with low percentages of mature oocytes retrieved from antagonist and agonist groups were significantly different in terms of the number of the matured (M II) and fertilized oocytes (2PNs) and clinical pregnancy from other studied groups. Additionally, in the agonist protocol, the implantation rate and live births of the <30% group were significantly lower from the group with the highest percentage of the mature oocytes retrieved. A low clinical outcome of the groups with lower number of mature oocytes retrieved might be a result of a lower sensitivity of follicles to ovulation induction protocols. It appears that this effect was more evident in the agonist than in the antagonist protocol, as the variables examined showed statistical differences. Thus, the same ovulation induction protocol might produce different treatment outcome in patients with different ovarian sensitivity to the stimulation protocol ²⁹.

There were no statistical differences in clinical outcomes (implantation, clinical pregnancy and live births) between the second and third group (groups with more mature oocytes retrieved) of antagonist and agonist protocols. However, when the second and third group of antagonist was compared with corresponding group of agonist protocol, there were significantly lower live birth rate in the antagonist protocol (second groups 22.2 vs. 35.9% and third groups 23.9 vs. 41.5 %). Similarly, Orvieto & Patrizio ³¹ reported that live birth rates and ongoing pregnancy were significantly lower in the group treated with the GnRH antagonist when compared to the agonist long protocol. After analyzing nine trials and twenty eight RCTs for GnRH antagonist application in IVF, Youssef & Elashmawi ³² determined that, the live birth rate was 1.5% and 2% lower in the GnRH antagonist when compared to the agonist treatment. However, a recent review of Al-Inany et al. ²⁹ that is contrary to their previous reports ¹,³³,has demonstrated no evidence of statistically significant differences in the rates of live births or ongoing pregnancies when comparing GnRH agonist long protocols with antagonist protocols. A meta-analysis by Xiao et al. ³⁴ showed that ongoing pregnancy and live births were similar in the GnRH antagonist when compared with the standard long GnRH agonist protocols. Nonetheless, Conrad et al. ³⁵ demonstrated significantly higher live birth rates in women provided with LH supplementation in antagonist cycles, where their intra-cycle LH levels were very low. The lower live birth rates in the antagonist protocol group in our study might be a result of a thinner endometrium and lower estradiol levels on HCG injection day when compared to the agonist protocol. It is well documented that inadequate estradiol levels from ovarian stimulation may impair endometrial receptivity ^{36, 37}. Similarly, Orvieto et al. ³⁸ showed a significantly lower endometrial thickness for the antagonist treatment when compared to the agonist. However, Simon et al.³⁹demonstrated that endometrial development after GnRH antagonist mimics the natural endometrium to a greater extent than after GnRH agonist.

In our study, there was no difference in the clinical pregnancy between the second and third group of antagonist and agonist protocols as well between these two treatment protocols. Engel et al. ⁴⁰ performed a sub-analysis of patients with equal demographic and clinical features, which resulted in similar pregnancy rates independent of GnRH used. Shanbhag et al.⁴¹ and Orvieto et al.³⁸ observed a lower pregnancy rate in the antagonist protocol compared to the GnRH agonist long protocol. Ludwig et al.⁴² in their meta-analysis study showed a reduction in the pregnancy rate using antagonist - ganirelix/antagon vs. long agonist protocol. However, the antagonist cetrorelix resulted in the same pregnancy rate as with the long agonist protocol. The studies of Al-Inany & Aboulghar ⁴³ and Depalo et al. ¹³ showed a trend towards a lower pregnancy rate in the antagonist protocol. A similar trend was also observed in patients with LH deficiency as documented by low E₂ to oocyte ratio and was explained by the endometrial impact of a lower LH level ⁴⁴. Higher pregnancy rates in the agonist cycles may be a result of larger number of oocytes and embryos for selection for transfer ⁴⁵. Demonstrated differences in the number of M II and 2PNs between the studied groups of both protocols in our study were more likely to be a result of enforced stratification of the data. Therefore, in each group of both protocols, the number of mature and fertilized oocytes were progressively increasing with parallel increase in estradiol level in the blood produced by growing follicles. However, the overall number of oocytes retrieved, MII oocytes and 2PN oocytes in the antagonist protocol was significantly lower compared to the agonist in our study. Similarly, European and Middle East Study ⁴⁶, Mochtar et al. ⁴⁷, Barmat et al. ⁴⁸ and Orvieto & Patrizio ³¹ reported a lower mean number of cumulus-oocyte-complexes (COC) and 2 pronuclear (PN) oocytes in the GnRH-antagonist group compared to the GnRH – agonist group. The reports of Albano et al. 10, Olivennes et al. ⁴⁹, Fluker et al. ⁵⁰, Roulier et al. ⁵¹showed similar results to those obtained in our study, suggesting that they stem from a lower serum estradiol level on the day of hCG administration. A possible reason for a lower number of oocytes retrieved from the patients on the GnRH antagonist protocol compared to the long agonist protocol was suggested by Hurine et al.⁵². The authors claim that this is a result of a relatively higher level of FSH during early follicular phase that coincides with a range of initially developing follicles of the antagonist regiment, causing decreased synchronization of the follicular cohort²⁷so that lower number oocytes were retrieved. Moreover, the differences between these two protocols might be due to a different mechanism of GnRH actions. In the agonist (long protocol) after a variable period of endogenous gonadotrophin depletion, small antral follicles are recruited by the exogenous gonadotropins. In contrast, in the antagonist cycle, the recruited follicles have already been exposed for a few days to endogenous inter-cycle FSH rise ^{1, 53, 54}. Administration of the GnRH antagonist at the end of the stimulation period could have had an effect on the cell cycle of granulosa cells ⁵⁵. In vitro studies showed that GnRH-antagonist restrains cell growth by decreasing the synthesis and the stimulatory effects of IGFs on follicle growth ⁵⁶. GnRH may act as an autocrine factor by regulating mitogen-activated protein kinase in human granulosa luteal cells ^{57, 58} and affect the follicles environment. Young et al. ⁵⁹ demonstrated a difference in the follicular microenvironment between GnRH agonist long protocol and GnRH antagonist protocol. However, the authors were not able to show an effect of the follicular microenvironment on the clinical outcome (pregnancy and implantation rates), most likely due to a small number of analyzed cycles (n=32-antagonist and n=36- long GnRH agonist). The recent study on the morphokinetics of the embryos ¹⁷ demonstrated that abnormality in cleavage embryo (reverse cleavage) was associated with the regiment used for ovarian stimulation. Reverse cleavage was more frequently seen in the embryos where GnRH antagonists were used compared to GnRH agonists. The authors suggested that mechanisms controlling reverse cleavage may be sensitive to the environment of the oocyte during folliculogenesis. Therefore, it supports the earlier statement, that population of follicles and oocytes of long agonist and antagonist protocols differ from each other.

In conclusion, clinical outcomes appear to be influenced by the percentage of mature oocytes retrieved especially when the percentage of retrieved mature oocytes is low. It is essential to establish ovarian sensitivity to gonadotropins before any type of individualized approach of controlled ovarian stimulation protocol will be applied.

Authors report no financial or commercial conflicts of interest.

TW data collection and elaboration of the manuscript; RM statistical analysis; SGS data collection, editing of the manuscript. All authors approved the final version of the manuscript.

The authors wish to thank the staff of the embryology laboratory for retrieving data from the electronic data base and the Toll Center for Reproductive Science team for their enthusiasm and support in collecting data.