The Clinical Significance of a Low Percentage of Mature Oocytes Retrieved using Common Ovarian Stimulation Protocols

The objective of this study was to investigate whether percentage of mature oocytes retrieved from ovaries stimulated with long agonist or multi-dose antagonist protocols affect the implantation, clinical pregnancy and live birth of ICSI (Intracytoplasmic sperm injection) cycles. The 654 cycles of agonist (long lupron) and 610 cycles of multi-dose flexible antagonist (antagon) were analyzed after stratification according to the percentage of the mature oocytes retrieved. The clinical pregnancy of the groups with less than 30 % mature oocytes retrieved, both antagonist and agonist protocol was statistically lower (at least p< 0.05) compared to the groups with more than 30% mature oocytes retrieved. In the agonist protocol, the implantation and live births for this group were significantly (p<0.009) lower than in the group with ≥70% mature oocytes retrieved. The live births in groups with more mature oocytes retrieved (30-69% and ≥70 %) of the antagonist protocol were lower (22.2% vs. 35.9% and 23.9% vs. 41.5%, p<0.0001, respectively) compare to the agonist protocol. The results of our study showed that a very low percentage of mature oocytes retrieved impacts the clinical outcome of antagonist and long agonist protocols. DOI : 10.14302/issn.2576-2818.jfb-17-1435 Corresponding author: Teresa Wiesak PhD, Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, street: Bydgoska 5, 10-240 Olsztyn, Poland. Tel: 48 (89) 539 3164, Email : t.wiesak@pan.olsztyn.pl

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 hyperstimulation 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].
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.

Materials and Methods
The study was approved by the Institutional Review Board of the Abington Memorial Hospital, PA.Therefore, the cut-off points were developed based on our clinical experiences and visualizing the results.
Patients with ≤30% mature oocytes retrieved were assigned to the group 1 (low ovarian sensitivity to the stimulation protocol).On average, 70-80% of retrieved oocytes are mature when the patients respond well to a stimulation protocol.This rationale led to creating group 3 (high ovarian sensitivity) and the intermediate group 2 (31-69% mature oocytes retrieved).

Statistical Analysis.
The results obtained were analyzed using

Results
The distribution of diagnoses (endometriosis, idiopathic, male factor, ovulatory dysfunction, tubal, and third (≥70%) group compared to corresponding agonist groups (Table II).
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 III).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 III).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 III).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  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 III).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 IV

Discussion
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 Note: NS = not statistically significant.The column P value shows the level of statistical differences between the three studied groups.Values with different letters are different.The asterisks stand for the difference between the antagonist and agonist protocols (p<0.0009 for the groups 30-69% and p< 0.0001 for the groups with ≥70% matured oocytes).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. [39] demonstrated 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.[50], Roulier et al. [51] showed 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 cohort [27] so 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 mitogenactivated protein kinase in human granulosa luteal cells [57,58] and affect the follicles environment.Young et al.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.

Statistica 10 .Vol- 1
0 software (Statsoft, Tulsa, OK, USA and IBM SPSS Statistics 21.0, Predictive Solutions).Distribution of data was verified with the Shapiro-Wilk test and the Kolmogorov-Smirnov test with the Lilliefors correction.In view of non-Gaussian distribution of data, the non-parametric Mann-Whitney U test (a two group comparison) and the Kruskal-Wallis test (three groups comparison) were employed.The Pearson chi-square test was used to determine statistical differences in fertilization, clinical pregnancy, implantation and live birth rates among the studied groups and treatment protocols.The significance of correlations between the percentage of mature oocytes retrieved and cycle characteristics were examined by the Spearman (non-Issue 1 Pg.no.-37 parametric) test.Results of these analyzes were reported as median values with interquartile 25-75% in parentheses or percentage.P<0.05 was considered statistically significant.
Abbreviations: Clinical preg.(sac) -clinical pregnancy where the gestational sac was visible, Clinical preg.(Htb) -clinical pregnancy where the gestational sac and fetal heart beat were present.

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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=36long 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.

Table 3 .
Antagonist and agonist cycle characteristics after stratification data according to the percentage of mature oocytes retrieved.Values are medians with interquartile 25-75% in parentheses.NS = not statistically significant.The asterisks stand for the difference between the antagonist and agonist protocols.Specific p values are in the results section.

Table 3 .
Comparison of an average number of retrieved, matured, and fertilized oocytes and embryos trans- ferred between the study groups and treatment protocols (antagonist and agonist).Reported values are medians with interquartile range (25-75%) in parentheses with an exception for the fertilization rate.treatment.The numbers in column P value show statistical significance level.NS = not statistically significant.The asterisks stand for the difference between the antagonist and agonist protocols (details are in the results section).Abbreviations: M II -mature oocytes with the nuclear maturity of the metaphase II, 2PN -oocytes with two pronuclei, Fert.Rate -fertilization rate, Number of embryo transf.-number of embryo transferred.Freely Available Online www.openaccesspub.org| JFB CC-license DOI : 10.14302/issn.2576-2818.jfb-17-1435Vol-1 Issue 1 Pg.no.-39 (p< 0.0009 and p<0.0007, respectively), matured (p<0.0001,p<0.0004, respectively) and fertilized

Table 4 .
Clinical outcome of the antagonist and agonist ICSI cycles in relation to the percentage of mature oocytes retrieved.