GnRH-Agonist Ovulation Trigger in Patients Undergoing Controlled Ovarian Hyperstimulation for IVF with Stop GnRH-Agonist Combined with Multidose GnRH-Antagonist Protocol

Objective: This study aimed to characterize those patients undergoing the stop gonadotropin-releasing hormone (GnRH)-agonist combined with multidose GnRH-antagonist protocol, with suboptimal response to GnRH-agonist trigger in in vitro fertilization (IVF) cycles. Design: This is a cohort study. Setting: The study was conducted in a university hospital. Patients: All consecutive women admitted to our IVF unit from February 2020 through November 2020 who reached the ovum pick-up stage were reviewed. Interventions: Triggering final oocyte maturation by GnRH-ag alone (GnRH-ag trigger group), or combined with hCG (dual trigger group), in patients undergoing the stop GnRH-agonist combined with multidose GnRH-antagonist protocol was performed. Main Outcome Measure: The main outcome measure was LH level 12 h after the trigger. Results: Five out of the 32 patients (15.6%) demonstrated suboptimal response as reflected by LH levels <15 IU/L 12 h after GnRH-agonist trigger. Moreover, while no differences were observed in oocyte recovery rate, maturity, or embryo quality between the different study groups (GnRH-ag trigger and dual trigger groups), those achieving a suboptimal response to the GnRH-agonist trigger (post-trigger LH <15 mIU/mL) demonstrated significantly higher number of follicles and peak estradiol levels at the day of trigger, compared to those with optimal response (post-trigger LH >15 mIU/mL). Conclusions: The stop GnRH-agonist combined with GnRH-antagonist protocol enables the substitution of hCG with GnRH-ag for final oocyte maturation. However, caution should be taken in high responders, where the dual trigger with small doses of hCG (1,000–1,500 IU) should be considered, aiming to avoid suboptimal response (post-trigger LH levels <15 IU/L).

Background Controlled ovarian hyperstimulation (COH) enables the recruitment of multiple healthy fertilizable oocytes in patients undergoing in vitro fertilization (IVF)-embryo transfer [1]. However, despite the great improvements in ovarian stimulation protocols and fertilization procedures, live birth rates per embryo transferred remain at approximately 15-50%, and severe ovarian hyperstimulation syndrome (OHSS) remains one of their major threats [2,3].
Moreover, while there are no precise methods to completely prevent severe OHSS, except by withholding human chorionic gonadotropin (hCG), during the last decade, observations have suggested that gonadotropin-releasing hormone agonist (GnRH-ag) by its consequent LH surge may effectively trigger oocyte maturation and ovulation, with the consequent elimination of severe OHSS [4,5]. It is noteworthy that previous studies have shown that patients receiving a GnRH-agonist trigger alone, who have a post-trigger LH <15 mIU/mL, were more likely to have suboptimal response or a canceled retrieval [6][7][8][9].
Recently, we introduced the stop GnRH-agonist combined with multiple-dose GnRH-antagonist protocol to our COH armamentarium. The rationale behind the sequential treatment stems from the advantages of its components. The mid-luteal GnRH-ag pretreatment causes downregulation of the GnRH receptors with the consequent suppression of pituitary LH secretion for as long as 10 days after the last dose of the agonist. This effect, together with the immediate LH suppression provided by the GnRH-ant, will eliminate premature LH surge and might improve the quality of the embryos generated [10].
This protocol was successfully used in poor-responder patients [10], those with poor embryo quality [11], and those with elevated peak serum progesterone levels [12]. Moreover, following the observations of comparable or improved oocyte/embryo quality following hCG trigger as compared to GnRH-ag trigger, and the different effects of LH and hCG on the downstream signaling of the LH receptor, GnRH-ag is now offered concomitantly with the standard hCG trigger dose (dual trigger), to improve oocyte/embryo yield and quality [13], or solely instead of hCG, aiming to eliminate the risk to develop severe OHSS [3]. The aim of the present study was to evaluate whether GnRH-agonist trigger in patients undergoing the stop GnRH-agonist combined with multiple-dose GnRH-antagonist protocol will result in an optimal response/trigger, as reflected by post-trigger LH <15 mIU/mL [6][7][8][9].

Patients and Methods
We reviewed the computerized files of all consecutive women admitted to our IVF unit from February 2020 through November 2020 who reached the ovum pick-up stage. Exclusion criteria were use of donor oocytes or transfer of frozen-thawed embryos and use of other protocol than the stop GnRH-agonist combined with multiple-dose GnRH-antagonist protocol (as previously described [10]). Only those patients who underwent triggering of final follicular maturation with GnRH-agonist trigger, either solely (GnRH-ag trigger group) or combined with hCG (dual trigger group), were included. LH levels were measured 12 h after GnRHagonist trigger injection. Those with post-trigger LH <15 mIU/mL were considered as achieving a suboptimal response. The study was approved by the Institutional Research Ethics Board of the Sheba Medical Center.
Data on patient age and infertility treatment-related variables were collected from the files. The decision regarding final follicular maturation triggering was based on physician judgment. Generally, patients exhibiting estradiol level >10,000 pmol/L were prescribed the GnRH-agonist-only trigger, as were patients developing >15 follicles of >10 mm in diameter. LH level 12 h after trigger, embryological parameters, and number of oocytes retrieved and oocyte recovery rate (defined as the number of oocytes retrieved per number of follicles >10 mm in diameter at the day of trigger) were also retrieved and analyzed. Classification of embryo quality was based on previously published scoring parameters [14]; a topquality embryo was defined as 4-5 blastomeres on day 2, 7 or more blastomeres on day 3, equally sized blastomeres and ≤10% fragmentation on day 3, and no multinucleation.
Statistical analysis was performed with Student's t test and χ 2 test, as appropriate. Results are presented as mean ± standard deviation; p value <0.05 was considered significant.

Results
Of the 1,070 cycles performed in our IVF unit from February 2020 through November 2020, 32 IVF cycles were eligible for analysis. Patients' age, BMI, and basal day-3 FSH levels were 39.3 ± 3.9 years, 22.7 ± 4.2 kg/m 2 , and 11.0 ± 4.7 IU/L, respectively. Five patients underwent the GnRH-agonist trigger solely, and 27 the dual trigger, of whom 1 and 4 presented a suboptimal LH rise following the GnRH-agonist trigger, respectively. The clinical characteristics of the IVF cycles of these 2 study groups are shown in Table 1. As expected, those undergoing the GnRH-agonist solely trigger demonstrated significantly higher peak estradiol level (10,303 ± 9,354 vs. 4,507 ± 3,924, respectively, p < 0.02) and more follicles <14 mm in diameter at the trigger day (8.4 ± 6.4 vs. 4.6 ± 3.3, respectively, p < 0.03). These differences are biased owing to the fact that we offered the GnRH-agonist solely trigger approach mainly to patients at risk to develop OHSS or those destined to freeze-all (such as those undergoing Gynecol Obstet Invest 2021;86:427-431 DOI: 10.1159/000517177 preimplantation genetic testing). No in-between group differences were observed in the number of oocytes retrieved, oocyte recovery rates, or the percentage of MII oocytes per oocyte retrieved, nor the percentages of 2PN per MII oocyte or TQE per 2PN.
While comparing those with post-trigger LH <15 mIU/mL, who were considered as achieving a suboptimal response to those with optimal response (LH >15 IU/L), no in-between group differences were observed in the number of oocytes retrieved, oocyte recovery rates, or the percentage of MII oocytes per oocyte retrieved, nor the percentages of 2PN per MII oocyte or TQE per 2PN (Table 2). While considering ovarian stimulation characteristics, those achieving a suboptimal response to the GnRH-agonist trigger demonstrated significantly higher number of follicles and peak estradiol levels (11,413 + 8,106 vs. 4,301 + 3,601 pmol/L, respectively, p < 0.003) at the day of trigger (Table 2).

Discussion
In the present study of patients undergoing the stop GnRH-agonist combined with multiple-dose GnRH-antagonist protocol, who underwent triggering of final follicular maturation with GnRH-agonist trigger, either solely or combined with hCG (dual trigger), 5 out of the 32 patients (15.6%) demonstrated suboptimal response as reflected by LH levels <15 IU/L 12 h after GnRH-agonist trigger. This figure is higher than the reported 5.2% in patients undergoing the multiple-dose GnRH-antagonist protocol [9].
GnRH-agonist causes suppression of pituitary LH secretion for as long as 10 days after the last dose of the agonist [15]. It might be therefore assumed that a residual prolonged pituitary suppression following the stop GnRH-agonist protocol might be the culprit of the observed higher suboptimal response.
We could not observe any differences in oocyte recovery rate, maturity, or embryo quality between the different study groups. The reason is that the aforementioned variables are affected by the trigger mode, and most of our patients were triggered with both GnRH-agonist and hCG (dual trigger). On the other hand, we could indeed demonstrate significantly higher number of follicles and peak estradiol levels at the day of trigger in those achieving a suboptimal response to the GnRH-agonist trigger (post-trigger LH <15 mIU/mL).
Meyer et al. [9] assessed the risk factors for suboptimal response to GnRH-agonist trigger and demonstrated that prolonged use of hormonal contraception and very low LH levels on the day of trigger were independent factors for suboptimal response to GnRH-agonist trigger. Cozzolini et al. [16] have evaluated the role of urinary LH surge testing and found it easy, safe, and reliable in detecting suboptimal response to GnRH-agonist trigger. More- over, they demonstrated that it could identify patients who could benefit from hCG rescue after an unsuccessful GnRH-agonist trigger. Therefore, it might be suggested that high responders undergoing the stop GnRH-agonist combined with GnRH-antagonist are at risk to a suboptimal response to GnRH-agonist solely trigger. To avoid this suboptimal response, we recommend considering the dual trigger with low-dose hCG (1,000-1,500 IU) in this subgroup of patients, aiming to prevent the suboptimal response.
The stop GnRH-agonist combined with GnRH-antagonist was successfully used in several groups of patients [10][11][12]. The rationale behind the sequential treatment stems from the advantages of its components: (a) the long GnRH-ag protocol pretreatment results in better synchronized response and a scheduled cycle [17,18]; (b) since continuing the GnRH-ag during COH is often associated with a significant increase in the number of gonadotropin ampoules required for achieving adequate follicular development, its cessation might improve ovarian response and avoids the need of increasing gonadotropin daily dose. GnRH-ag causes suppression of pituitary LH secretion for as long as 10 days after the last dose of the agonist [15]; and (c) the stop GnRH-ag together with the multiple-dose GnRH-ant provides immediate LH suppression, eliminating premature LH surge, and might improve the quality of the embryos generated. In the present study, we could demonstrate another advan-tage by the feasibility of triggering final oocyte maturation by GnRH-agonist together with hCG (dual trigger), with improved IVF outcome [13].
A limitation of our analysis is its retrospective design and the small sample size. However, based on our patient selection process, only consecutive patients fulfilling the inclusion criteria were enrolled, which considerably decreases the likelihood of selection bias. We could not compare the pre-to post-trigger delta, since pre-trigger LH was measured only in a minority of patients. Despite its nonrandomized and uncontrolled nature, we still suggest that our results should be used to advise patients and physicians, since a randomized controlled study does not seem feasible.
In conclusion, the stop GnRH-agonist combined with GnRH-antagonist protocol was successfully used in poorresponder patients [10], those with poor embryo quality [11], and those with elevated peak serum progesterone levels [12]. In the present study, we could demonstrate that it also enables the substitution of hCG with GnRH-ag for final oocyte maturation. However, caution should be taken in high responders, especially in those patients after prolonged hormonal contraception use or those with low LH levels on the day of trigger. In the latter, the addition of small doses of hCG (1,000-1,500 IU) should be considered (dual trigger), aiming to avoid suboptimal response (post-trigger LH levels <15 IU/L). Further studies are required to identify other risk factors for suboptimal response to GnRH-agonist trigger.