The Effect of Dual Trigger on In Vitro Fertilization/Intracytoplasmic Sperm Injection Outcomes in Patients with Suboptimal Ovarian Response (POSEIDON Classification Group I): A Randomized Clinical Trial.

In the recent classification of the Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number (POSEIDON) group, patients with suboptimal ovarian response are introduced as women less than 35 years old in whom the number of retrieved oocytes after a standard ovarian stimulation is less than 9; despite adequate ovarian reserve ( 1 ). Researchers are exploring new methods to enhance both the quantity and quality of oocytes retrieved from infertile women, with the aim of increasing the success rate of fertility treatment cycles ( 2 ). A key component of the controlled ovarian stimulation (COS) cycle is the triggering of the final stage of follicular maturation, an area of research that remains contentious and merits further investigation ( 3 ). Replacement of human chorionic gonadotropin (hCG) with gonadotropin-releasing hormone agonist (GnRH-a) for the final stimulation of oocyte development has led to the creation of novel protocols, enhancing the array of options available in assisted reproductive technology (ART) for the advantage of both current and future patients ( 2 ). In clinical practice, the use of a GnRH agonist for final stimulation has been recognized as the most effective method for preventing ovarian hyperstimulation syndrome (OHSS) ( 3 ). One additional benefit noted for the final stimulation of the oocyte using a GnRH agonist is its ability to promote the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in a manner that closely resembles the physiological processes of a normal menstrual cycle ( 4 ). The concept of "dual trigger" which combines a bolus dose of GnRH agonist with a reduced dose of hCG hormone at the time of final oocyte stimulation has been presented and investigated in patients with excessive ovarian response in in vitro fertilization (IVF) cycles ( 5 ). Utilizing this oocyte triggering technique not only diminishes the likelihood of OHSS but has also significantly enhanced the rates of pregnancy and live births ( 6 ). However, the current evidence does not sufficiently demonstrate that the use of a dual trigger leads to enhancements in clinical pregnancy rates, fertilization rates, live birth rates, or early pregnancy loss rates when compared to the GnRH-a trigger. To adequately assess the effectiveness of this protocol for high responders, more randomized clinical trials (RCTs) are necessary ( 7 ) .On the other hand, this trigger method has been proposed as a suitable method for improving COS outcomes in patients with poor ovarian response (POR); however, large-scale, high-quality RCTs are required to confirm efficacy and fully address the magnitude of this effect ( 8 ). One of the challenges is that patients with normal ovarian reserve do not respond appropriately to ovarian stimulation with a standard protocol. Griffin et al. ( 9 ) reported that in patients with a 25% maturation in previous IVF cycle, the use of dual trigger can increase the number of mature oocytes. In 2019, ESHRE indicated that dual triggering is not advisable for individuals with normal ovarian response. Nevertheless, there was a lack of explicit guidance concerning PORs, highlighting the necessity for a rigorously designed randomized controlled trial to assess the efficacy of dual triggering in this specific population ( 10 ). Considering that there are limited studies regarding patients with unexpected POR (POSEIDON classification group I), the researchers opted to develop a RCT to examine the impact of dual trigger on the final maturation of oocytes and its subsequent effects on IVF/ intracytoplasmic sperm injection (ICSI) cycle outcomes in patients who have previously experienced suboptimal ovarian response.

As indicated in the sampling flowchart, out of 52 patients randomly assigned to two groups, a total of 46 women completed the study procedure in the dual trigger group (n =24) or control group (n=22, Fig .1 ). Baseline characteristics included female age, type and duration of infertility, BMI, as well as TSH, AMH, FSH, LH, and a history of IVF/ICSI cycles were similar between the two groups. Also, the two groups were comparable regarding the mean time period between the prior cycle and the present cycle (months) ( Table 1 ). Flowchart of the study sampling. The comparison of the baseline characteristics of the patients in two groups *; Descriptive data were compared using an independent Student’s t test and presented as mean ± SD. Qualitative variables were compared by Chi-square test and are presented number (%). P<0.05 was considered statistically significant. BMI; Body mass index, COH; Controlled ovarian hyperstimulation, FSH; Follicle-stimulating hormone, LH; Luteinizing hormone, TSH; Thyroid-stimulating hormone, AMH; Anti-müllerian hormone, IVF/ICSI; In vitro fertilization/intracytoplasmic sperm injection, MII; Metaphase II, HCG; Human chorionic gonadotropin, and GnRH; Gonadotropin-releasing hormone. According to Table 2, the comparison of COS cycle outcomes in the two groups showed that there was no statistically significant difference between the two groups in terms of the total dose of gonadotropin, serum estradiol level on the day of oocyte triggering, the number of human menopausal gonadotropin (HMG) ampoules, and stimulation duration. In this table, statistically significant differences were found between the two groups by comparison of the mean ± SD of the number of follicles > 13 mm (on oocyte trigger day, P=0.013), the number of retrieved oocyte (P=0.001), and the number of MII oocytes (P=0.022) compared to the controls, there was also a slight increase in oocyte recovery ratio in women undergoing dual trigger (P=0.057). In addition, the specifications of the embryos obtained from the participants were compared between two groups. The total number of obtained embryos was significantly higher in women undergoing dual trigger than in the control group (P=0.010). There was no significant difference between the two groups in terms of the quality of frozen embryos (P=0.321). The comparison of the ovarian stimulation and IVF/ICSI cycle’s outcomes between two groups Values are presented as the mean ± standard deviation or number (%). *; It was obtained by independent t test and Chi-square tests. The statistically significant level was 0.05. MII; Metaphase II, ET; Embryo transfer, and IVF/ICSI; In vitro fertilization/intracytoplasmic sperm injection. In the follow-up, the comparison of embryo transfer cycle outcomes in women between the two groups revealed that there were no statistically significant differences in the number of transferred embryos, and chemical, clinical pregnancy, and live birth rates. However, the rates of clinical pregnancy and live birth in the dual group were higher than the control group (40.9 vs. 25 and 40.9% vs. 20%, P=0.275 and P=0.143, respectively, Table 3 ). The comparison of embryo transfer cycle outcomes between two groups Values are presented as the mean ± standard deviation/error and number (%). *; It was obtained by independent t test and Chi-square. The statistically significant level was 0.05. ET; Embryo transfer. To determine the changes in ovarian stimulation results between the previous cycle and the current cycle within the group, the total number of retrieved and MII oocytes, and the number of obtained embryos in the previous cycle and the current cycle were compared in each group, separately. The analysis showed that the total number of retrieved and MII oocytes as well as the number of obtained embryos in patients in the dual group were significantly improved compared to their previous cycle (P<0.001 for all), while these changes were not remarkable in the control group (P=0.241, P=0.184, and P=0.219, respectively, Table 4 ). Comparing the results of ovarian stimulation in the previous cycle with the current cycle separately in each group *; Data are presented as the mean ± standard deviation by using paired sample t test. MII; Metaphase II.

The findings of the current study indicated that the dual trigger notably enhanced the quantity of retrieved oocytes, the maturation rate, and the number of embryos obtained when compared to both the control group and the prior cycle of patients who had experienced a suboptimal ovarian response. In the follow-up, while the rates of clinical pregnancy and live birth among women receiving dual trigger was greater than that observed in the control group, the difference did not achieve statistical significance. To the best of our knowledge, research is still ongoing in the field of investigating the effectiveness of the dualtrigger method on the outcomes of ovarian stimulation cycles in patients exhibiting both high ( 6 , 7 , 12 ) and normal ovarian responses ( 13 - 20 ), as well as, more recently, those with PORs ( 10 , 21 - 24 ). Recently, dual trigger with GnRH agonist and standard dose of hCG has become an option in antagonist cycles, especially in normal responders ( 25 ). In the field of patients with POR, initially, three retrospective studies indicated the beneficial impact of dual trigger in patients exhibiting a high ratio of immature oocytes ( 9 , 21 , 26 ). However, Wu et al. ( 22 ) did not observe a positive impact of dual trigger on mature oocyte yield in women exhibiting diminished ovarian reserve in minimal ovarian stimulation during IVF cycles. In the most recent retrospective study, Mutlu et al. ( 27 ), revealed the potential benefits of dual trigger in POR patients according to the Bologna criteria on live birth rates. They concluded that the difference in pregnancy outcomes and live birth rate can be attributed to the higher rates of oocyte maturation and high quality in the dual trigger group. The beneficial outcomes associated with the dual trigger may be attributed to either the enhancement of oocyte maturity or its direct influence on the implantation process ( 27 ). Additionally, GnRH-a offers the advantage of facilitating a physiological ovulation trigger by stimulating the surge of FSH alongside the LH surge during the mid-cycle. The presence of FSH promotes the development of LH receptors on granulosa cells, thereby increasing the follicles' responsiveness to the LH surge. The LH activity and sensitivity can be strengthened by adding GnRHa to the hCG trigger, which helps overcome endocrine milieu disturbances, granulosa cell dysfunction, oocyte maturation, and cumulus expansion insufficiency. This simultaneous FSH surge achieved through this approach is beneficial in addressing these issues. The clinical manifestations of these situations are an increased number of mature oocytes, improved embryo quality, and pregnancy rates ( 27 ). POR patients, a challenging subgroup in IVF cycles, have different clinical characteristics compared to normal responders, including older age, reduced ovarian reserve, and poor oocyte quality. In the review of the literature, the effect of the dual trigger was investigated in patients with a history of a low ratio of retrieved oocytes per the number of observed follicles on oocyte trigger day, and also in another study by the same authors, in patients with a low ratio of mature oocytes ( 21 , 28 ). The beneficial effects of dual triggers were reported in these studies, which are consistent with our findings. Elsewhere, Zhang et al. ( 24 ) retrospectively investigated the effect of dual trigger with a combination of GnRH-a and standard dose hCG on IVF outcomes in poor responders according to the Bologna criteria and found that the number of retrieved oocytes and mature oocytes in 5000 dual groups trigger (5,000 or 10,000 IU hCG plus 0.1 mg GnRH-a) was significantly higher compared to the hCG alone trigger group. The number of transferred embryos, implantation, and pregnancy rates were similar between the groups. Differently, Mutlu et al. ( 27 ), administrated 0.2 mg of GnRH-a together with 250 mg of recombinant hCG (Ovitrel) in the dual trigger group and found that pregnancy outcomes were similar to other studies that used the same dose of GnRH-a for final oocyte stimulation ( 10 , 13 ). As a result of these data, it has been hypothesized that the dose of GnRH-a needed to enhance pregnancy rates in the dual trigger group should not be less than 0.2 mg. On the other hand, Zhou et al. ( 29 ) conducted a RCT that demonstrated a dual trigger enhances the quantity of high-quality embryos in women of advanced age (aged ≥35 years) undergoing IVF/ICSI treatment; however, this approach does not lead to an increased number of oocytes retrieved when compared to the use of a single hCG or GnRHa trigger. In Mutlu et al.’s study ( 27 ), contrary to Zhang et al. ( 24 ), patients with a history of two POR after maximum stimulation in the absence of advanced age or abnormal ovarian reserve tests were also included in the study, and dual trigger significantly improved the live birth rate in this group of patients. In our study, this group of patients was examined and satisfactory results were observed. Recently, in a RCT with the same inclusion criteria as our study, a significant increase in the number of high-quality embryos in dual trigger with GnRH-a and hCG compared to stimulation with GnRH-a alone or hCG alone was reported ( 30 ). In alignment with the findings of our research, this pilot study indicates a trend towards higher rates of clinical pregnancies and live births. Nevertheless, the observed differences were not statistically significant due to the relatively small size of the study population; nonetheless, these results hold clinical significance. Another study including patients with reduced ovarian reserve showed that the rates of pregnancy and live birth in the dual trigger group were significantly higher than the hCG alone group ( 31 ). In a retrospective analysis of 384 cycles of the POSEIDON group 4 patients, there was a statistically significant improvement in the number of retrieved oocytes, mature oocytes, number of high-quality embryos, clinical pregnancy rate, and live birth in the dual trigger group. Dual trigger may be beneficial in this subgroup of patients undergoing IVF treatment by increasing the number of high-quality embryos ( 32 ). In present study, due to the small sample size, no statistically significant difference was observed in terms of clinical pregnancy and live birth rates; however, according to the higher number of frozen embryos in the dual trigger group, it seems that this method is also effective in improving pregnancy outcomes. The current study evaluated patients in the POSEIDON classification group I who had at least one POR and appropriate ovarian reserve through an RCT; it can be considered as its strength. The prevalence of challenging patients with unexpected POR was limited, so the weakness of the present study was the low sample size; however, the post-hoc power analysis showed that the present clinical trial has high power to draw conclusions regarding to the main outcomes.

By virtue of present findings, dual trigger significantly improved the number and quality of the retrieved oocytes as well as the number of obtained embryos in the patients with a history of unexpected poor response (POSEIDON group I). Although dual triggerr has been associated with an increase in clinical pregnancy and live birth rates, this association lacks statistical significance; therefore, to validate the current findings, further clinical trials with larger sample sizes are required in this area.

The current investigation was carried out as a RCT following the endorsement of the research protocol by the scientific review board and the ethics committee of Royan Institute, spanning from November 2019 to November 2021 (IR.ACECR.ROYAN.REC.1397.004 and NCT04549649 ). The study population consisted of all clients with infertility problems who had indications for second IVF/ICSI cycles, for whom the retrieved oocytes were less than optimal in the previous COS cycle. The principal investigator outlined the objectives of the project to the eligible patient, and upon obtaining written consent, the patient was incorporated into the study. Individuals diagnosed with suboptimal or POR as per the POSEIDON stratification system (designated as POSEIDON group I) were incorporated into the study based on the following criteria: i. Female participants aged below 35 years, ii. A history of unanticipated poor or suboptimal ovarian response following the application of standard protocols at the Royan Institute, iii. Sufficient ovarian reserve, defined as an antral follicle count (AFC) of five or more on menstrual cycle days 2-3, and a basal serum anti-müllerian hormone (AMH) level of at least 1.2 ng/ml ( 1 ). Patients with an established diagnosis of premature ovarian failure, moderate to severe endometriosis, uterine factor infertility, and severe male factor diagnosis as well as indications for pre-implantation genetic screening, blastocyst and/or embryo donation treatment cycles, cigarette and drug addiction, and body mass index (BMI) >30 kg/m 2 were excluded. Referral infertile women were screened in the clinic and eligible women were selected. The selected patients were educated about, the aims and methods of the study. They were only included in the study after giving the written consent. The researchers gathered data by filling out the case report forms. All patients underwent COS utilizing the standard GnRH antagonist protocol. Blood samples for FSH, LH, and estradiol levels were collected on the second day of the menstrual cycle, immediately prior to the initiation of gonadotropin stimulation. Ovarian stimulation commenced on either the second or third day of menstruation, with a maximum dosage of 225 IU of recombinant FSH (rFSH, Cinnal-f, Cinagen, Iran). If a follicle measuring 13 mm was detected, GnRH antagonist injections (Cetrotide®, 0.25 mg cetrorelix acetate, Serono, Inc) were initiated and continued until the day of triggering. From the seventh day of the cycle onward, the rFSH dosage was adjusted based on the ovarian response observed through vaginal ultrasonography. Once at least two follicles of 18 mm or greater were identified, the final oocyte maturation trigger was administered. Subsequently, a blocked randomization method with blocks of size 4 was employed to assign patients into two groups. The final ovarian stimulation (oocyte triggering) was conducted in groups A and B as follows: Group A (experimental): A subcutaneous administration of 0.2 mg of GnRH-a (Decapeptyl®; Ferring GmbH) and two ampoules of recombinant hCG (Ovitrelle®, 250 μg/0.5 ml, Merck, Serono, Inc) was performed simultaneously. Group B (control): Only two ampoules of recombinant hCG (Ovitrelle®, 250 μg/0.5 ml, Merck, Serono, Inc) were injected subcutaneously. The ovarian stimulation cycles that involved fewer than two follicles were canceled. Ovum retrieval was conducted 32 to 34 hours following oocyte triggering, after which IVF or ICSI was carried out for all patients. Morphological characteristics of MII oocytes such as zona placida thickness, size of perivitelline space, polar body shape, and internal and external cytoplasmic factors were investigated. Especially abnormalities such as central organelle cluster, smooth endoplasmic reticulum (SER), and incorporation were more important. The embryologists classified the morphology of oocytes into three groups based on the numbers and proportions of these abnormalities in the retrieved MII oocytes: Eumorphic, slightly dysmorphic, dysmorphic, and highly dysmorphic. The assessment of embryo quality was based on following criteria, including the total cell count, the degree of fragmentation, the variability in cell size, and the overall symmetry. This evaluation categorized embryos as follows: excellent (day 3: 6-8 blastomeres of uniform size with fragmentation at or below 10%), good (day 3: 6-8 blastomeres that may be either uniform or varied in size with fragmentation between 10 and 20%), and poor (characterized by uneven and limited blastomeres with fragmentation exceeding 20%) ( 11 ). The clinician determined the method of embryo transfer according to the condition of the endometrium and the progesterone level on the day of ovum retrieval. If the endometrial state was deemed unsuitable, a freeze-all approach was implemented. For fresh embryo transfers, vaginal progesterone suppositories (Cyclogest ®; Actavis, Barnstaple, UK) were administered twice daily to support the luteal phase. In cycles involving frozen embryo transfer, the endometrium was prepared using a hormonal protocol that included pretreatment with a GnRH agonist. The primary outcome included the count of dominant follicles (≥13 mm) on the day of oocyte trigger and the number of mature (MII) oocytes. The secondary endpoints encompassed the total number of oocytes retrieved, the oocyte maturity rate (calculated as the number of MII oocytes divided by the total number of oocytes), oocyte yield (the total number of oocytes retrieved divided by the antral follicle count [AFC]), and mature oocyte yield [the number of mature oocytes retrieved divided by (AFC)], Additional parameters assessed were the total dosage of gonadotropins administered, the duration of ovarian stimulation, the quality of obtained embryos, the fertilization rate [the ratio of the total number of two-pronuclear (2PN) stage zygotes to the total number of injected MII oocytes], the chemical pregnancy rate [indicated by a positive β-hCG test 14 days post-embryo transfer (ET)], the clinical pregnancy rate (determined by the presence of a fetal heartbeat via transvaginal ultrasound 6 to 7 weeks after ET), and the live birth rate (defined as the delivery of a fetus that breathes or exhibits other signs of life, irrespective of gestational age). This RCT was performed as a pilot study to detect the main differences between the two groups by primary outcome. At the end of the study, post hoc power analysis showed that group sample sizes of 24 and 22 achieved 70% power to detect a difference of 2.7 between the two groups by primary outcome with a significance level (alpha) of 0.05 using a two-sample t test. Statistical analysis was performed using SPSS software (version 22; Inc. Chicago, IL, USA). All quantitative variables were assessed for normality through the Kolmogorov-Smirnov test. The findings indicated that all quantitative variables in this study exhibited a normal distribution, which were subsequently reported as the mean ± Standard deviation/error (SD/E). Qualitative variables were represented using frequency and percentage. Data were analyzed using the 2-tailed Student t test, and Chisquare test between two groups, where appropriate. A P<0.05 was considered statistically significant.