Dual trigger for final oocyte maturation in expected normal responders with a high immature oocyte rate: A randomized controlled trial

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Methods This study included expected normal ovarian responders younger than 40 years old whose immature oocyte rate in the previous cycle was more than 50% at the reproductive center from July 2021 to January 2023. A total of 73 patients were enrolled at trigger, including 34 in the hCG trigger group and 39 in the dual trigger group (co-administration of gonadotrophin releasing hormone (GnRH) agonist and hCG, 40 and 34 hours prior to oocyte retrieval, respectively). The primary outcome was oocyte maturation rate. Results There was no significant difference in the number of oocytes retrieved between the two study groups, but the oocyte maturation rate was higher in dual trigger group (84.0% [14.0%] vs. 55.5% [19.8%], P < 0.001). Moreover, there were also higher cumulative pregnancy rate (69.4% vs. 40.0%, P = 0.035) and cumulative live birth rate (66.7% vs. 36.0%, P = 0.022) in dual trigger group. Conclusion For normal responders with low oocyte maturation rates, the dual trigger may be more effective than the conventional hCG trigger. Trial Registration Chictr.gov.cn, identifier: ChiCTR2100049292 Normal ovarian responder Dual trigger Gonadotropin releasing hormone agonist Human chorionic gonadotropin MII oocytes Figures Figure 1 Introduction During the period preceding spontaneous ovulation, the oestradiol (E 2 ) level secreted by the dominant follicle reaches its peak, triggering the surge of follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels, both of which cooperate to promote oocyte maturation and excretion during the final stages of the process. It has been shown that hCG alone can be used in ovarian stimulation cycles using a GnRH antagonist (GnRH-ant) protocol for triggering maturation of the oocyte and induced meiosis/follicular maturation as a substitute for LH surge (1). As a result of the prolonged luteinization, hCG-only trigger is associated with a higher risk of ovarian hyperstimulation syndrome (OHSS) (2). Induction of oocyte maturation using gonadotrophin releasing hormone agonist (GnRH-a) has been shown to decrease OHSS incidence in comparison to hCG triggers. However, due to the lower total amount and shorter duration of endogenous gonadotropins (Gn) produced following GnRH-a stimulation, which led to the subsequent luteal insufficiency, there is a reduction in pregnancy rates and a higher rate of miscarriage (3, 4). Addressing this issue with co-administration of GnRH-a and hCG, the dual trigger approach, both reduced the risk of OHSS in patients with a high ovarian response (5) and contributed to the comparable or even higher pregnancy rates compared with hCG trigger (6). Orvieto R et al. elucidated how to tailor each trigger mode to its appropriate subgroup of patients (7). In addition, it is of interest that a new trigger regimen, co-administration of GnRH-a and hCG, 40 and 34 hours prior to ovum pick-up (OPU), respectively, was used to prolong the time between trigger and OPU, with the success of obtaining mature oocytes, pregnancy, delivery in a patient with recurrent empty follicle syndrome (8). Furthermore, the studies relevant to normal responders who had a lower rate of mature oocytes (< 50%) indicated that this dual trigger regimen resulted in a significant increase in the number of mature oocytes and transferable embryos obtained, as well as the proportion of oocytes obtained to the number of preovulatory follicles, but the difference in pregnancy rates was not conclusive (9, 10). Nevertheless, poor oocyte maturation rates are associated with lower clinical pregnancy and live birth rates according to recent studies (11). Based on this, the present study was designed to investigate whether dual trigger could improve the MII oocytes rate and pregnancy outcomes in normal responders with poor oocyte maturation rates. Materials And Methods Ethical Approval of the Study Protocol The reproductive ethics committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (TCM) certified this study as ethical (Identifier: SDUTCM/2021.7.26). All patients provided written informed consent. All treatments were undertaken in strict accordance with the Declaration of Helsinki 1964 and its later amendments. Study Design This study was a registered randomized controlled trial (RCT, http://www.chictr.org.cn/ , identifier: ChiCTR2100049292) carried out in the reproductive center, affiliated hospital of Shandong University of Traditional Chinese Medicine, between July 2021 to January 2023 (12). Inclusion Criteria (1) Patients with an expected normal ovarian response (NOR) who had no previous history of cancellation of an in vitro fertilization / intracytoplasmic sperm injection (IVF/ICSI) cycle and a poor ovarian response; 6 ≤ AFC ≤ 15; 1.2ng/ml ≤ AMH ≤ 3.5ng/ml; Basal FSH < 10 mIU / ml; (2) Patients with < 50% mature oocytes in the only one previous fresh IVF/ICSI cycle triggered with hCG; (3) In the previous IVF/ICSI cycle, standard ovarian stimulation protocol was performed using a GnRH-ant protocol. Exclusion Criteria (1) Age ≥ 40 years old ; (2) Patients with a body mass index (BMI) ≥ 30 kg/m2; (3) Individuals with high risk of OHSS during controlled ovarian stimulation; (4) Patients with endocrine or metabolic disorders; (5) Patients with untreated severe endometriosis, submucosal myoma, multiple endometrial polyps, pelvic inflammation, uterine malformation, Asherman syndrome and hydrosalpinx prior to embryo transfer (ET); (6) Patients with abnormal immune function and chromosome karyotype of either spouse. Randomization and Blinding Randomization was commenced on the trigger day. A random sequence of codes was used to assign individuals to one of two groups, A or B, in a 1:1 ratio by computer. A central randomization database was established to store the randomization scheme (www.medresman.org). The online randomization procedure was operated by a data specialist who was not involved in patient recruitment and clinical management. Physicians were informed by e-mail of the allocation results after randomization. As a result, the operation ensured that allocation concealment was maintained since the service did not reveal the allocation until after the randomization process, i.e., after the baseline visit. Considering the nature of the intervention, we did not blind physicians and participants to the intervention. However, the trial outcome assessors were blinded to the assigned groups. Controlled Ovarian Stimulation Protocol Standard ovarian stimulation protocol with gonadotrophins was performed using a GnRH-ant protocol. Ovarian stimulation with 150–225 IU/day of recombinant FSH (Puregon, Merck Sharp & Dohme B.V., Haarlem, Netherlands) was started on the 3rd day of the menstrual cycle. The patients' attending physicians determined the starting dose of Gn based on their age and BMI before participation in the study and dynamically monitor the ovarian response based on the results of serial transvaginal ultrasound follicle measurements and assays of serum E 2 , progesterone (P 4 ), LH. The dose of Gn was adjusted according to the subject’s response. When ovarian stimulation reached the fifth day, GnRH-ant (0.25mg, Cetrorelix; Merck Serono, Darmstadt, Germany) was added and continued until trigger day. When 2 follicles were ≥ 18mm in diameter or when 3 follicles were ≥ 17mm in diameter, triggering was performed. According to the results of randomization, patients were divided into the following two groups: (1) hCG-only trigger: Patients were triggered with recombinant hCG (r-hCG, 6500 IU, Ovidrel, European Serono, France) 36 hours before OPU; (2) Dual trigger: Patients were triggered with co-administration of 0.2 mg GnRH-a (0.1mg, Diphereline, France, Epson) and hCG, 40 and 34 hours prior to OPU, respectively. Oocyte retrieval was performed by transvaginal puncture under transvaginal ultrasound guidance. Embryo Culture Cumulus cells were enzymatically removed from oocytes and mature oocytes were subjected to intracytoplasmic sperm injection. In an environment of 5.0% O2 and 5.6% CO2, the pre-equilibrated embryo Petri dishes were used to culture zoosperm. According to Gardner's criteria, embryo morphology and quality were evaluated. When selecting embryos for vitrification or transfer, it should focus on no fewer than six blastomeres with ≤ 20% fragmentation, which indicates top quality. Embryos with a fragmentation rate between 20% and 50% were not transferred or vitrified unless they reach the 8-cell stage on day 3. In our center, we have consistently adhered to the principle of transferring one top-quality day 3 embryo or two suboptimal embryos at the cleavage stage. Fresh embryo transfer would be cancelled if met: Endometrial thickness less than 7 mm on trigger day; Serum P 4 levels greater than 1.5ng/ml on trigger day; High OHSS risk, i.e., retrieval of more than 15 oocytes and serum E 2 concentrations higher than 4000 pg/ml on trigger day, etc. Endometrial Preparation Protocol for FET Transvaginal ultrasound and serum hormone measurements (LH, E 2 , and P 4 ) were performed on days 8 through 10 of the menstrual cycle, depending on the duration of the menstrual cycle, to track endometrial thickness and follicle size until ovulation triggering conditions were reached. A single intramuscular injection of 4000 IU hCG (Lizhu Pharmaceutical Trading Co., China) was used to trigger ovulation when the dominant follicle diameter was more than 17 mm, the endometrial thickness was more than 7 mm, P 4 was less than 1.5 ng/ml, and E 2 was more than 150 pg/mL. At approximately 9:00 am, the hCG injection was administered. Patients who had unanticipated spontaneous ovulation while being monitored, as well as those who had no prominent follicles by day 25 of the menstrual cycle, were eliminated. Luteal Phase Support Protocols Routine luteal phase support was given after embryo transfer (ET), using intramuscular P 4 (Zhejiang Xianju Pharmaceutical Co., Ltd.) injection 40mg/day, P 4 vaginal sustained release gel (8% Crinone, Moxerano) 90mg/day, or oral P 4 10mg three times a day (dydrogesterone, Abbott Laboratories biologicals), or in combination, continued from the day of ET until 10 weeks of gestation. The manner of P 4 administration is determined by the clinical preferences of both physicians and patients. Study Endpoints and Definitions The primary outcome was oocytes maturation rate (i.e., the percentage of the number of MII oocytes over the total number of oocytes retrieved). The secondary outcomes are number of oocytes retrieved, normal fertilization rate (the proportion of oocytes that become fertilized), number of two-pronuclear (2PN) embryos, number of D3 top quality embryos (TQE), number of D3 transferable embryos, number of remaining frozen embryos, cumulative clinical pregnancy rate and cumulative live birth rate (LBR). Top quality embryo was defined as seven or more blastomeres of uniform size and fragmentation rate less than 20% on day three. Clinical pregnancy was defined as the appearance of a gestational sac and fetal heartbeat detected by transvaginal ultrasonography. Cumulative clinical pregnancy rate and LBR were defined as the proportion of participants with clinical pregnancy or live birth after one year of follow-up. Data Statistical Analysis This clinical study was a superiority, randomized parallel controlled trial. The primary outcome measurement was MII oocytes rate. According to previous data of our center, the mean estimated rate of MII oocytes in dual trigger group was approximately 70%, using GnRH-a (40h before OPU) + hCG (34h before OPU), and 30% in hCG trigger group, with standard deviations of 40% for each group. The sample size of 40 individuals for each group was calculated by PASS 15.0 (NCSS, LLC. Kaysville, Utah, USA.) assuming α = 0.05 (two-sided) and β = 0.10 (90% power). Suppose that a dropout rate of 10%, we calculated that the sample size for each group was 45 individuals. Ultimately, the RCT need to recruit a total of 90 individuals. Because the randomization was implemented on the trigger day, recruited participants who dropped out of trial during the ovarian stimulation process were not randomized. Therefore, only outcome variables were analyzed for subjects randomized to the study protocol (per protocol analysis). All the data were analyzed with SPSS version 26.0. Data are presented as mean ± standard deviation (Mean ± SD) for continuous variables and as frequency (percentage) [n (%)] for categorical data. According to the normality and variance of the data, the continuous data were analyzed by use of Student's t-test or Mann-Whitney U-test. When data did not conform to normal distribution, a nonparametric test was used, which were expressed as the median (interquartile range) [M (IQR)]. Categorical data were analyzed using chi square and Fisher's exact tests with an expected frequency of less than 5. P < 0.05 indicated that the difference was statistically significant. Results Baseline Characteristics A total of 90 women met the inclusion criteria. Among them, 5 patients who had high risk of OHSS (i.e., more than 20 follicles over 10 mm in diameter) in the ovarian stimulation process were excluded. Moreover, GnRH-a trigger and freeze-all strategy were used. One patient who had early follicular ovulation, and 8 patients who had broken off ovarian stimulation due to uncontrollable factors such as COVID-19 were also not included. The remaining 3 patients decline to participate. No cases of early onset ovulation were observed after trigger. The actual dropout rate was 18.8%. Finally, a total of 73 patients were included in the analysis. Thirty-four patients were assigned to the hCG trigger group and 39 patients were assigned to the dual trigger group (see Fig. 1 ). The baseline characteristics and demographics did not differ significantly between the dual trigger and hCG trigger groups in terms of age, BMI, AFC, basal sex hormone level, or duration and type of infertility (Table 1 ). Although there were no significant differences in the total dose of Gn between the two groups, the total duration of ovarian stimulation was higher in dual trigger group (Table 2 ). Table 1 Participants demographics characteristics of study participants hCG Trigger (n = 34) Dual Trigger (n = 39) P value Age (yrs.) 30.97 ± 3.65 31.26 ± 4.05 0.754a Duration of infertility (yrs.) 3.00 (2.75) 3.00 (2.50) 0.537b BMI (kg/m2) 22.1 (4.00) 22.5 (4.65) 0.847b AMH (ng/ml) 3.58 (1.41) 2.96 (1.46) 0.174b AFC (n) 12.5 (7.0) 13.0 (6.0) 0.233b Basal FSH (IU/L) 6.96 (1.83) 6.46 (2.16) 0.071b Basal LH (IU/L) 4.33 (1.98) 4.95 (3.65) 0.398b Basal E 2 (pg/ml) 38.2 (16.1) 40.0 (23.3) 0.283b Basal P (ng/ml) 0.54 (0.29) 0.54 (0.42) 0.956b Type of infertility (%) 0.094c Primary 24/34 (70.6) 19/39 (48.7) Secondary 10/34 (29.4) 20/39 (51.3) Total duration of ovarian stimulation (d) 9 (1.75) 10 (2.00) < 0.001 Total dose of gonadotropin (IU) 1913 (543) 2025 (544) 0.110 Abbreviations: BMI, body mass index; AMH, anti-Müllerian hormone; AFC, antral follicle count; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E 2 , estradiol; P, progesterone Data are presented as mean ± SD, the Median (IQR) or n (%). a student’s t test; b Independent-Samples Mann-Whitney U-Test; c Fisher’s exact test Table 2 Comparison of cycles characteristics between two study groups hCG Trigger (n = 34) Dual Trigger (n = 39) P value* Number of oocytes retrieved 15 (11.8) 17 (12.5) 0.088 Number of MII oocytes retrieved 7.50 (4.00) 15 (9.00) < 0.001 MII oocytes rate (%) 55.5 (19.8) 84.0 (14.0) < 0.001 Normal fertilization rate (%) 65.0 (39.5) 75.0 (33.5) 0.032 Number of 2PN embryos 3.0 (4.0) 9.0 (7.5) < 0.001 Number of D3 embryos available for transfer 1.0 (3.75) 4.0 (4.50) < 0.001 Number of D3 TQE 0 (1.00) 1.0 (3.00) 0.003 Number of remaining frozen embryos 0 (2.00) 4.0 (4.50) < 0.001 Cycles of no embryos available for transfer (%) 9/34 (26.5) 3/39 (7.7) 0.055 Abbreviations: MII, metaphase II; 2PN, two-pronuclear; TQE, top quality embryos Data are presented as the Median (interquartile range, IQR). *Independent-Samples Mann-Whitney U-Test Ovarian Stimulation Outcomes The number of oocytes retrieved (15 [11.8] vs 17 [12.5], P = 0.088) did not differ significantly between the two study groups, but there was a significant difference in number of MII oocytes retrieved (7.5 [4.0] vs 15 [9.0], P < 0.001), MII oocytes rate (55.5% [19.8%] vs 84.0% [14.0%], P < 0.001), normal fertilization rate (65.0% [39.5%] vs 75.0% [33.5%], P = 0.032), number of 2PN embryos (3.0 [4.0] vs 9.0 [7.5], P < 0.001), number of D3 TQE (0 [1.0] vs 1 [3.0], P = 0.003), number of D3 transferable embryos (1.00 [3.75] vs 4.0 [4.50], P < 0.001), and number of remaining frozen embryos (0 [2.0] vs 4 [4.5], P < 0.001) between the hCG trigger and dual trigger groups (Table 2 ). Further, there was no difference in the incidence of no embryos available for transfer between patients with dual trigger and those with hCG trigger (26.5% vs 7.7%, P = 0.055, see Table 2 ). Pregnancy Outcomes Following fresh ET, there was no significant difference in the LBR (Table 3 ). Nevertheless, the cumulative pregnancy rate (10/25 [40.0%] versus 25/36 [69.4%], P = 0.035) and cumulative LBR (9/25 [36.0%] versus 24/36 [66.7%], P = 0.022) of dual trigger group was significantly higher than that of hCG trigger group. Table 3 Comparison of pregnancy outcomes between two study groups hCG Trigger (n = 34) Dual Trigger (n = 39) P value* Live birth rate per FreET (%) 4/11 (36.4) 2/4 (50.0) 1.000 Cumulative clinical pregnancy rate (%) 10/25 (40.0) 25/36 (69.4) 0.035 Cumulative live birth rate (%) 9/25 (36.0) 24/36 (66.7) 0.022 Abbreviations: FreET, fresh embryo transfer Data are n (%). *Fisher's exact test Discussion This study shows that co-administration of GnRH-a and hCG for final oocyte maturation, 40 and 34 hours prior to OPU, respectively (dual trigger), increased the number of MII oocytes retrieved and the rate of mature oocytes in patients with normal ovarian response who had a poor oocyte maturation rate (< 50%) in the previous cycle. Oocytes complete meiosis I and arrest at metaphase II until fertilization, at which point meiosis II is completed (13). Twenty-eight to thirty-eight hours after the onset of the LH surge preovulatory oocytes in metaphase II were obtained (14). After controlled ovarian stimulation, some of the oocytes retrieved are arrested at the germinal vesicle or metaphase I (MI) stage despite correct administration of hCG (15).The etiology of oocyte maturation arrest is complex. Recently, pathogenic variants in genes associated with oocyte maturation arrest have been identified. Pathogenic variants in PATL2 mutations mainly result in oocyte GV stage arrest by decreased amounts of protein (16). A heterozygous or homozygous mutation in the TUBB8 causes MI arrest through disruption of microtubule formation, meiotic spindle assembly, and microtubule dynamics (17). In addition, pathogenic variants in TRIP13 also responsible for oocyte meiotic arrest (18). Unfortunately, there is no effective treatment at present. When the percentage of meiotic competence failure oocytes was 25% or more, no pregnancy was achieved (19). A study shows that when the oocyte maturation rate is low (< 57.5%), it indicates a bad IVF cycle outcome (11). It is common knowledge that hCG has no FSH receptor activity. Compared with hCG alone, GnRH-a trigger induces an increase in endogenous LH and FSH, which is similar to the surge of gonadotropin in the middle of the natural cycle. Fabris et al. demonstrated that in patients with high immature oocyte rate in the previous IVF cycle, the number of retrieved mature oocytes increases when the proportion of immature oocytes declines due to dual triggering (20). Our study reached the same conclusion, but differed in that the administration of GnRH-a and hCG did not occur simultaneously and prolonged the time between trigger and OPU. Dual trigger that can increase the number of mature oocytes and increase the rate of mature oocyte may have benefited from the surge in FSH. The surge in FSH stimulates the cumulus cells of oocyte-cumulus complexes to secrete a meiotic activating substance that allows the oocyte meiotic process to resume and activate cumulus expansion during the final stages of oocyte maturation (21, 22). Furthermore, FSH has been shown to promote the formation of LH receptor sites in granulosa cells and the development of the corpus luteum, which in turn promotes estrogen and P 4 production (23). Additionally, GnRH receptors have been found in a variety of human tissues, including the granulosa cells of the pre-ovulatory phase. In mammals, oocytes remain in the prophase of the first meiosis until the gonadotropin surge at puberty. The intra-oocyte concentration of cAMP and cGMP in this prolonged period prevents the resumption of meiosis in the oocyte. As a result of LH, cGMP levels decrease and meiosis resumes (24). It has been demonstrated that peripheral GnRH receptor activation decreases intracellular cAMP levels. Several genes are induced by GnRH that are involved in follicular rupture and oocyte maturation (25). The favorable results in our study could be attributed to the FSH surge and direct action of the agonist on the ovarian GnRH receptor. Perhaps, for unknown reasons, the LH pathway in the patient is blocked, and GnRH agonists activate a different pathway. Another possibility is that the interval between the ovulation trigger and oocyte retrieval is prolonged. In a natural cycle, the onset of LH surge occurs 34–36 hours before follicular rupture. For optimal oocyte maturation, LH concentration must be maintained above a threshold for 14–27 hours (26). Oocyte maturation and follicular rupture are time-dependent processes, which require different times in different patients. It is hypothesized that certain patients require a longer period of time for cumulus expansion, which allows the oocyte to detach from the follicular wall. In these cases, oocyte immaturity may result when aspiration occurs 36 h after hCG administration. Co-administration of GnRH-a and hCG as trigger was associated with increased embryo implantation (27). Endometrial receptivity may be improved by GnRH-a acting as an autocrine and (or) paracrine regulator (25). Cheon et al. suggested that an increased expression of endometrial GnRH-II peptide, noted during the early and mid-secretory phase, may play an important role in human embryo implantation (28). Co-administration of GnRH-a and hCG for final oocyte maturation not only compensated for luteal insufficiency after GnRH-a trigger but also improved patients cycle outcomes by improving endometrial receptivity. Consistent with the study findings that Decleer et al. (29) suggested that women who received dual triggering were more likely to have a surplus of frozen embryos. The studies by Griffin D et al. and Fumei Gao et al. showed that the dual trigger did not improve patients cycle outcomes, which is in contrast to the findings of our study (15, 30). Consisting of the co-administration of GnRH agonist and hCG for final oocyte maturation, 40 and 34 h prior to OPU, respectively, differs from the simultaneous administration of GnRH agonist and hCG for final oocyte maturation, 36h prior to OPU, by the additional prolongation of the time between ovulation triggering and OPU. This later prolongation, may explain the beneficial effect in terms of both oocytes maturation and pregnancy rate. In this RCT, it is undeniable that limitations remain, including the relatively small sample size and unexpected higher dropout rate. Although our included population was normal responders, the fact is that partial normal responders also face a high risk of OHSS in the course of ovarian stimulation. When this happened, they were excluded. Therefore, this study cannot discuss the relationship between dual trigger and OHSS incidence. Because too few patients underwent fresh ET, it was not powered to show a difference in the pregnancy outcomes with fresh ET. Moreover, we did not detect and analyze relevant gene mutations in this population due to cost-benefit considerations. Although the duration of Gn administration was increased by one day in the dual trigger group, the number of oocytes retrieved was not increased compared with that in the hCG trigger group. Moreover, no statistical difference was found in the dosage of Gn between the two groups. Therefore, this may not be the reason for the increase in the number of mature oocytes. Conclusion In conclusion, we demonstrated that co-administration of GnRH-a and hCG for final oocyte maturation, 40 and 34 hours prior to OPU, respectively, can increase MII oocyte rate and enable patients to obtain more embryos with higher quantity and quality. Moreover, the dual trigger was equally beneficial for pregnancy outcomes. Declarations Funding Statement This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Data Sharing Statement The data generated and analyzed from the current study will be availed by the corresponding author upon request. Consent for Publication Written informed consent for publication was obtained from all participants. Disclosure None of the authors have a conflict of interest to declare with regard to this study. Author Contributions Jing-Yan Song and Zhen-Gao Sun conceived and designed the study. Meng-Han Yan was responsible for data analysis and writing of the manuscript. Qian-Qian Zhang and Wen-Xiu Yang were responsible for the data collection and interpretation. Jing-Yan Song critically revised the important intellectual content of this manuscript. 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GnRH Agonist and hCG (Dual Trigger) Versus hCG Trigger for Final Oocyte Maturation in Expected Normal Responders With a High Immature Oocyte Rate: Study Protocol for a Randomized, Superiority, Parallel Group, Controlled Trial. Front Endocrinol (Lausanne). 2022,13(1664-2392 (Print)):831859. Marteil G, Richard-Parpaillon L, Kubiak JZ. Role of oocyte quality in meiotic maturation and embryonic development. Reproductive Biology. 2009,9(3):203-24. Seibel MM, Smith DM, Levesque L, Borten M, Taymor ML. The temporal relationship between the luteinizing hormone surge and human oocyte maturation. American Journal of Obstetrics and Gynecology. 1982,142(5):568-72. Griffin D, Feinn R, Engmann L, Nulsen J, Budinetz T, Benadiva C. Dual trigger with gonadotropin-releasing hormone agonist and standard dose human chorionic gonadotropin to improve oocyte maturity rates. Fertil Steril. 2014,102(2):405-9. Chen B, Zhang Z, Sun X, Kuang Y, Mao X, Wang X, et al. 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Cumulus cells of oocyte-cumulus complexes secrete a meiosis-activating substance when stimulated with FSH. Molecular Reproduction and Development. 1997,46(3):296-305. Kol S, Humaidan P. LH (as HCG) and FSH surges for final oocyte maturation: sometimes it takes two to tango? Reprod Biomed Online. 2010,21(5):590-2. Zhou X, Guo P, Chen X, Ye D, Liu Y, Chen S. Comparison of dual trigger with combination GnRH agonist and hCG versus hCG alone trigger of oocyte maturation for normal ovarian responders. Int J Gynaecol Obstet. 2018,141(3):327-31. Sun Q-Y, Miao Y-L, Schatten H. Towards a new understanding on the regulation of mammalian oocyte meiosis resumption. Cell Cycle. 2009,8(17):2741-7. Yu B, Ruman J, Christman G. The role of peripheral gonadotropin-releasing hormone receptors in female reproduction. Fertil Steril. 2011,95(2):465-73. Zelinski-Wooten MB, Hutchison JS, Chandrasekher YA, Wolf DP, Stouffer RL. Administration of human luteinizing hormone (hLH) to macaques after follicular development: further titration of LH surge requirements for ovulatory changes in primate follicles. The Journal of Clinical Endocrinology & Metabolism. 1992,75(2):502-7. Lin MH, Wu FS, Hwu YM, Lee RK, Li RS, Li SH. Dual trigger with gonadotropin releasing hormone agonist and human chorionic gonadotropin significantly improves live birth rate for women with diminished ovarian reserve. Reprod Biol Endocrinol. 2019,17(1):7. Cheon KW, Lee HS, Parhar IS, Kang IS. Expression of the second isoform of gonadotrophin-releasing hormone (GnRH-II) in human endometrium throughout the menstrual cycle. Mol Hum Reprod. 2001,7(5):447-52. Decleer W, Osmanagaoglu K, Seynhave B, Kolibianakis S, Tarlatzis B, Devroey P. Comparison of hCG triggering versus hCG in combination with a GnRH agonist: a prospective randomized controlled trial. Facts Views Vis Obgyn. 2014,6(4):203-9. Gao F, Wang Y, Fu M, Zhang Q, Ren Y, Shen H, et al. Effect of a "Dual Trigger" Using a GnRH Agonist and hCG on the Cumulative Live-Birth Rate for Normal Responders in GnRH-Antagonist Cycles. Front Med (Lausanne). 2021, 8:683210. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2581457","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":186679502,"identity":"532ac5ec-f9b5-4442-995d-9226fbb2e1c0","order_by":0,"name":"Meng-Han Yan","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Meng-Han","middleName":"","lastName":"Yan","suffix":""},{"id":186679503,"identity":"d73acc2e-6487-4730-ad75-1d62c6093987","order_by":1,"name":"Qian-Qian Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qian-Qian","middleName":"","lastName":"Zhang","suffix":""},{"id":186679504,"identity":"a0bd5b6a-6a32-4b0b-a015-9fb8029584f0","order_by":2,"name":"Wen-Xiu Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wen-Xiu","middleName":"","lastName":"Yang","suffix":""},{"id":186679505,"identity":"3a95c633-470e-4e3c-b9ad-2abe5927fcda","order_by":3,"name":"Zhen-Gao Sun","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhen-Gao","middleName":"","lastName":"Sun","suffix":""},{"id":186679506,"identity":"f913d118-4ea8-4a20-95b1-d56a04d93e44","order_by":4,"name":"Jing-Yan Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIie3PsWoCQRCA4TkG7ppJbD3wIcYqBETzKCcHqY6QB1A5EKzuAQz6HCHlLAtnY7LtQSpJa7GWQoqoGEnjnmXA/buB+ZhdAJ/vH8YAgSQAuB/EcocaUV5L4ETU9PmxFRdyAfkdkKzucPXgJnfRu8jqbRix0UoTG4IKArvJzpP74imR/nKBLGWi5vxJwSzH+OXV8TDJWPqTElkVLOsdwZaEeOMiZn0kumGF+IPCZlJDqsOVAXJJoIiFqJ4crgjGy5DVlFNqkhq7/2Ky9mo7GaW3Rn9Z+93t9RZjZTcOckynf4Ygr93fNepesuXz+XxX2g+TalzZs5RdZQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2700-017X","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jing-Yan","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2023-02-13 10:06:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2581457/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2581457/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":34944890,"identity":"36a58b4c-6eff-43bb-81cc-ab2fe6ae0b4c","added_by":"auto","created_at":"2023-03-28 22:28:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1328234,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart of study population recruitment.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2581457/v1/432f830e33495113b63dff4d.jpg"},{"id":39669741,"identity":"3a612d49-46eb-4314-bc2a-bc790cfaa0c6","added_by":"auto","created_at":"2023-07-07 04:30:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":434854,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2581457/v1/b9809d97-6820-450b-98e4-d4c569ee916f.pdf"}],"financialInterests":"","formattedTitle":"Dual trigger for final oocyte maturation in expected normal responders with a high immature oocyte rate: A randomized controlled trial","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDuring the period preceding spontaneous ovulation, the oestradiol (E\u003csub\u003e2\u003c/sub\u003e) level secreted by the dominant follicle reaches its peak, triggering the surge of follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels, both of which cooperate to promote oocyte maturation and excretion during the final stages of the process. It has been shown that hCG alone can be used in ovarian stimulation cycles using a GnRH antagonist (GnRH-ant) protocol for triggering maturation of the oocyte and induced meiosis/follicular maturation as a substitute for LH surge (1). As a result of the prolonged luteinization, hCG-only trigger is associated with a higher risk of ovarian hyperstimulation syndrome (OHSS) (2). Induction of oocyte maturation using gonadotrophin releasing hormone agonist (GnRH-a) has been shown to decrease OHSS incidence in comparison to hCG triggers. However, due to the lower total amount and shorter duration of endogenous gonadotropins (Gn) produced following GnRH-a stimulation, which led to the subsequent luteal insufficiency, there is a reduction in pregnancy rates and a higher rate of miscarriage (3, 4). Addressing this issue with co-administration of GnRH-a and hCG, the dual trigger approach, both reduced the risk of OHSS in patients with a high ovarian response (5) and contributed to the comparable or even higher pregnancy rates compared with hCG trigger (6). Orvieto R et al. elucidated how to tailor each trigger mode to its appropriate subgroup of patients (7).\u003c/p\u003e \u003cp\u003eIn addition, it is of interest that a new trigger regimen, co-administration of GnRH-a and hCG, 40 and 34 hours prior to ovum pick-up (OPU), respectively, was used to prolong the time between trigger and OPU, with the success of obtaining mature oocytes, pregnancy, delivery in a patient with recurrent empty follicle syndrome (8). Furthermore, the studies relevant to normal responders who had a lower rate of mature oocytes (\u0026lt;\u0026thinsp;50%) indicated that this dual trigger regimen resulted in a significant increase in the number of mature oocytes and transferable embryos obtained, as well as the proportion of oocytes obtained to the number of preovulatory follicles, but the difference in pregnancy rates was not conclusive (9, 10). Nevertheless, poor oocyte maturation rates are associated with lower clinical pregnancy and live birth rates according to recent studies (11).\u003c/p\u003e \u003cp\u003eBased on this, the present study was designed to investigate whether dual trigger could improve the MII oocytes rate and pregnancy outcomes in normal responders with poor oocyte maturation rates.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e \u003cstrong\u003eEthical Approval\u003c/strong\u003e \u003cp\u003e \u003cb\u003eof the Study Protocol\u003c/b\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003e The reproductive ethics committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (TCM) certified this study as ethical (Identifier: SDUTCM/2021.7.26). All patients provided written informed consent. All treatments were undertaken in strict accordance with the Declaration of Helsinki 1964 and its later amendments.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design\u003c/h2\u003e \u003cp\u003eThis study was a registered randomized controlled trial (RCT, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.chictr.org.cn/\u003c/span\u003e\u003cspan address=\"http://www.chictr.org.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, identifier: ChiCTR2100049292) carried out in the reproductive center, affiliated hospital of Shandong University of Traditional Chinese Medicine, between July 2021 to January 2023 (12).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eInclusion Criteria\u003c/h2\u003e \u003cp\u003e(1) Patients with an expected normal ovarian response (NOR) who had no previous history of cancellation of an in vitro fertilization / intracytoplasmic sperm injection (IVF/ICSI) cycle and a poor ovarian response; 6\u0026thinsp;\u0026le;\u0026thinsp;AFC\u0026thinsp;\u0026le;\u0026thinsp;15; 1.2ng/ml\u0026thinsp;\u0026le;\u0026thinsp;AMH\u0026thinsp;\u0026le;\u0026thinsp;3.5ng/ml; Basal FSH\u0026thinsp;\u0026lt;\u0026thinsp;10 mIU / ml; (2) Patients with \u0026lt;\u0026thinsp;50% mature oocytes in the only one previous fresh IVF/ICSI cycle triggered with hCG; (3) In the previous IVF/ICSI cycle, standard ovarian stimulation protocol was performed using a GnRH-ant protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eExclusion Criteria\u003c/h2\u003e \u003cp\u003e(1) Age\u0026thinsp;\u0026ge;\u0026thinsp;40 years old ; (2) Patients with a body mass index (BMI)\u0026thinsp;\u0026ge;\u0026thinsp;30 kg/m2; (3) Individuals with high risk of OHSS during controlled ovarian stimulation; (4) Patients with endocrine or metabolic disorders; (5) Patients with untreated severe endometriosis, submucosal myoma, multiple endometrial polyps, pelvic inflammation, uterine malformation, Asherman syndrome and hydrosalpinx prior to embryo transfer (ET); (6) Patients with abnormal immune function and chromosome karyotype of either spouse.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRandomization and Blinding\u003c/h2\u003e \u003cp\u003eRandomization was commenced on the trigger day. A random sequence of codes was used to assign individuals to one of two groups, A or B, in a 1:1 ratio by computer. A central randomization database was established to store the randomization scheme (www.medresman.org). The online randomization procedure was operated by a data specialist who was not involved in patient recruitment and clinical management. Physicians were informed by e-mail of the allocation results after randomization. As a result, the operation ensured that allocation concealment was maintained since the service did not reveal the allocation until after the randomization process, i.e., after the baseline visit. Considering the nature of the intervention, we did not blind physicians and participants to the intervention. However, the trial outcome assessors were blinded to the assigned groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eControlled Ovarian Stimulation Protocol\u003c/h2\u003e \u003cp\u003eStandard ovarian stimulation protocol with gonadotrophins was performed using a GnRH-ant protocol. Ovarian stimulation with 150\u0026ndash;225 IU/day of recombinant FSH (Puregon, Merck Sharp \u0026amp; Dohme B.V., Haarlem, Netherlands) was started on the 3rd day of the menstrual cycle. The patients' attending physicians determined the starting dose of Gn based on their age and BMI before participation in the study and dynamically monitor the ovarian response based on the results of serial transvaginal ultrasound follicle measurements and assays of serum E\u003csub\u003e2\u003c/sub\u003e, progesterone (P\u003csub\u003e4\u003c/sub\u003e), LH. The dose of Gn was adjusted according to the subject\u0026rsquo;s response. When ovarian stimulation reached the fifth day, GnRH-ant (0.25mg, Cetrorelix; Merck Serono, Darmstadt, Germany) was added and continued until trigger day. When 2 follicles were \u0026ge;\u0026thinsp;18mm in diameter or when 3 follicles were \u0026ge;\u0026thinsp;17mm in diameter, triggering was performed. According to the results of randomization, patients were divided into the following two groups: (1) hCG-only trigger: Patients were triggered with recombinant hCG (r-hCG, 6500 IU, Ovidrel, European Serono, France) 36 hours before OPU; (2) Dual trigger: Patients were triggered with co-administration of 0.2 mg GnRH-a (0.1mg, Diphereline, France, Epson) and hCG, 40 and 34 hours prior to OPU, respectively. Oocyte retrieval was performed by transvaginal puncture under transvaginal ultrasound guidance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEmbryo Culture\u003c/h2\u003e \u003cp\u003eCumulus cells were enzymatically removed from oocytes and mature oocytes were subjected to intracytoplasmic sperm injection. In an environment of 5.0% O2 and 5.6% CO2, the pre-equilibrated embryo Petri dishes were used to culture zoosperm. According to Gardner's criteria, embryo morphology and quality were evaluated. When selecting embryos for vitrification or transfer, it should focus on no fewer than six blastomeres with \u0026le;\u0026thinsp;20% fragmentation, which indicates top quality. Embryos with a fragmentation rate between 20% and 50% were not transferred or vitrified unless they reach the 8-cell stage on day 3. In our center, we have consistently adhered to the principle of transferring one top-quality day 3 embryo or two suboptimal embryos at the cleavage stage. Fresh embryo transfer would be cancelled if met: Endometrial thickness less than 7 mm on trigger day; Serum P\u003csub\u003e4\u003c/sub\u003e levels greater than 1.5ng/ml on trigger day; High OHSS risk, i.e., retrieval of more than 15 oocytes and serum E\u003csub\u003e2\u003c/sub\u003e concentrations higher than 4000 pg/ml on trigger day, etc.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEndometrial Preparation Protocol for FET\u003c/h2\u003e \u003cp\u003eTransvaginal ultrasound and serum hormone measurements (LH, E\u003csub\u003e2\u003c/sub\u003e, and P\u003csub\u003e4\u003c/sub\u003e) were performed on days 8 through 10 of the menstrual cycle, depending on the duration of the menstrual cycle, to track endometrial thickness and follicle size until ovulation triggering conditions were reached. A single intramuscular injection of 4000 IU hCG (Lizhu Pharmaceutical Trading Co., China) was used to trigger ovulation when the dominant follicle diameter was more than 17 mm, the endometrial thickness was more than 7 mm, P\u003csub\u003e4\u003c/sub\u003e was less than 1.5 ng/ml, and E\u003csub\u003e2\u003c/sub\u003e was more than 150 pg/mL. At approximately 9:00 am, the hCG injection was administered. Patients who had unanticipated spontaneous ovulation while being monitored, as well as those who had no prominent follicles by day 25 of the menstrual cycle, were eliminated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eLuteal Phase Support Protocols\u003c/h2\u003e \u003cp\u003eRoutine luteal phase support was given after embryo transfer (ET), using intramuscular P\u003csub\u003e4\u003c/sub\u003e (Zhejiang Xianju Pharmaceutical Co., Ltd.) injection 40mg/day, P\u003csub\u003e4\u003c/sub\u003e vaginal sustained release gel (8% Crinone, Moxerano) 90mg/day, or oral P\u003csub\u003e4\u003c/sub\u003e 10mg three times a day (dydrogesterone, Abbott Laboratories biologicals), or in combination, continued from the day of ET until 10 weeks of gestation. The manner of P\u003csub\u003e4\u003c/sub\u003e administration is determined by the clinical preferences of both physicians and patients.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStudy Endpoints and Definitions\u003c/h2\u003e \u003cp\u003eThe primary outcome was oocytes maturation rate (i.e., the percentage of the number of MII oocytes over the total number of oocytes retrieved).\u003c/p\u003e \u003cp\u003eThe secondary outcomes are number of oocytes retrieved, normal fertilization rate (the proportion of oocytes that become fertilized), number of two-pronuclear (2PN) embryos, number of D3 top quality embryos (TQE), number of D3 transferable embryos, number of remaining frozen embryos, cumulative clinical pregnancy rate and cumulative live birth rate (LBR). Top quality embryo was defined as seven or more blastomeres of uniform size and fragmentation rate less than 20% on day three. Clinical pregnancy was defined as the appearance of a gestational sac and fetal heartbeat detected by transvaginal ultrasonography. Cumulative clinical pregnancy rate and LBR were defined as the proportion of participants with clinical pregnancy or live birth after one year of follow-up.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eData Statistical Analysis\u003c/h2\u003e \u003cp\u003eThis clinical study was a superiority, randomized parallel controlled trial. The primary outcome measurement was MII oocytes rate. According to previous data of our center, the mean estimated rate of MII oocytes in dual trigger group was approximately 70%, using GnRH-a (40h before OPU)\u0026thinsp;+\u0026thinsp;hCG (34h before OPU), and 30% in hCG trigger group, with standard deviations of 40% for each group. The sample size of 40 individuals for each group was calculated by PASS 15.0 (NCSS, LLC. Kaysville, Utah, USA.) assuming α\u0026thinsp;=\u0026thinsp;0.05 (two-sided) and β\u0026thinsp;=\u0026thinsp;0.10 (90% power). Suppose that a dropout rate of 10%, we calculated that the sample size for each group was 45 individuals. Ultimately, the RCT need to recruit a total of 90 individuals.\u003c/p\u003e \u003cp\u003eBecause the randomization was implemented on the trigger day, recruited participants who dropped out of trial during the ovarian stimulation process were not randomized. Therefore, only outcome variables were analyzed for subjects randomized to the study protocol (per protocol analysis). All the data were analyzed with SPSS version 26.0. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) for continuous variables and as frequency (percentage) [n (%)] for categorical data. According to the normality and variance of the data, the continuous data were analyzed by use of Student's t-test or Mann-Whitney U-test. When data did not conform to normal distribution, a nonparametric test was used, which were expressed as the median (interquartile range) [M (IQR)]. Categorical data were analyzed using chi square and Fisher's exact tests with an expected frequency of less than 5. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated that the difference was statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBaseline Characteristics\u003c/h2\u003e \u003cp\u003eA total of 90 women met the inclusion criteria. Among them, 5 patients who had high risk of OHSS (i.e., more than 20 follicles over 10 mm in diameter) in the ovarian stimulation process were excluded. Moreover, GnRH-a trigger and freeze-all strategy were used. One patient who had early follicular ovulation, and 8 patients who had broken off ovarian stimulation due to uncontrollable factors such as COVID-19 were also not included. The remaining 3 patients decline to participate. No cases of early onset ovulation were observed after trigger. The actual dropout rate was 18.8%. Finally, a total of 73 patients were included in the analysis. Thirty-four patients were assigned to the hCG trigger group and 39 patients were assigned to the dual trigger group (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The baseline characteristics and demographics did not differ significantly between the dual trigger and hCG trigger groups in terms of age, BMI, AFC, basal sex hormone level, or duration and type of infertility (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although there were no significant differences in the total dose of Gn between the two groups, the total duration of ovarian stimulation was higher in dual trigger group (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParticipants demographics characteristics of study participants\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehCG Trigger\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;34)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDual Trigger\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;39)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (yrs.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.97\u0026thinsp;\u0026plusmn;\u0026thinsp;3.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.26\u0026thinsp;\u0026plusmn;\u0026thinsp;4.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.754a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration of infertility (yrs.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.00 (2.75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.00 (2.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.537b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI (kg/m2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.1 (4.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.5 (4.65)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.847b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMH (ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.58 (1.41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.96 (1.46)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.174b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAFC (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.5 (7.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.0 (6.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.233b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasal FSH (IU/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.96 (1.83)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.46 (2.16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.071b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasal LH (IU/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.33 (1.98)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.95 (3.65)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.398b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasal E\u003csub\u003e2\u003c/sub\u003e (pg/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.2 (16.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.0 (23.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.283b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasal P (ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.54 (0.29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.54 (0.42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.956b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType of infertility (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.094c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24/34 (70.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19/39 (48.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSecondary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10/34 (29.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20/39 (51.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal duration of ovarian stimulation (d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9 (1.75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (2.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal dose of gonadotropin (IU)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1913 (543)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2025 (544)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.110\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eAbbreviations: BMI, body mass index; AMH, anti-M\u0026uuml;llerian hormone; AFC, antral follicle count; FSH, follicle-stimulating\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003ehormone; LH, luteinizing hormone; E\u003csub\u003e2\u003c/sub\u003e, estradiol; P, progesterone\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, the Median (IQR) or n (%).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003ea student\u0026rsquo;s t test; b Independent-Samples Mann-Whitney U-Test; c Fisher\u0026rsquo;s exact test\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of cycles characteristics between two study groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehCG Trigger\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;34)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDual Trigger\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;39)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP value*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of oocytes retrieved\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15 (11.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17 (12.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.088\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of MII oocytes retrieved\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.50 (4.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15 (9.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMII oocytes rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55.5 (19.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e84.0 (14.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal fertilization rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e65.0 (39.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e75.0 (33.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.032\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of 2PN embryos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.0 (4.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.0 (7.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of D3 embryos available for transfer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.0 (3.75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.0 (4.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of D3 TQE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0 (1.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0 (3.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of remaining frozen embryos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0 (2.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.0 (4.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCycles of no embryos available for transfer (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9/34 (26.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3/39 (7.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.055\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eAbbreviations: MII, metaphase II; 2PN, two-pronuclear; TQE, top quality embryos\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eData are presented as the Median (interquartile range, IQR).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e*Independent-Samples Mann-Whitney U-Test\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eOvarian Stimulation Outcomes\u003c/h2\u003e \u003cp\u003eThe number of oocytes retrieved (15 [11.8] vs 17 [12.5], P\u0026thinsp;=\u0026thinsp;0.088) did not differ significantly between the two study groups, but there was a significant difference in number of MII oocytes retrieved (7.5 [4.0] vs 15 [9.0], P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), MII oocytes rate (55.5% [19.8%] vs 84.0% [14.0%], P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), normal fertilization rate (65.0% [39.5%] vs 75.0% [33.5%], P\u0026thinsp;=\u0026thinsp;0.032), number of 2PN embryos (3.0 [4.0] vs 9.0 [7.5], P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), number of D3 TQE (0 [1.0] vs 1 [3.0], P\u0026thinsp;=\u0026thinsp;0.003), number of D3 transferable embryos (1.00 [3.75] vs 4.0 [4.50], P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and number of remaining frozen embryos (0 [2.0] vs 4 [4.5], P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) between the hCG trigger and dual trigger groups (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Further, there was no difference in the incidence of no embryos available for transfer between patients with dual trigger and those with hCG trigger (26.5% vs 7.7%, P\u0026thinsp;=\u0026thinsp;0.055, see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePregnancy Outcomes\u003c/h2\u003e \u003cp\u003eFollowing fresh ET, there was no significant difference in the LBR (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Nevertheless, the cumulative pregnancy rate (10/25 [40.0%] versus 25/36 [69.4%], P\u0026thinsp;=\u0026thinsp;0.035) and cumulative LBR (9/25 [36.0%] versus 24/36 [66.7%], P\u0026thinsp;=\u0026thinsp;0.022) of dual trigger group was significantly higher than that of hCG trigger group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of pregnancy outcomes between two study groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehCG Trigger\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;34)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDual Trigger\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;39)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP value*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLive birth rate per FreET (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4/11 (36.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2/4 (50.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCumulative clinical pregnancy rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10/25 (40.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25/36 (69.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.035\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCumulative live birth rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9/25 (36.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24/36 (66.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eAbbreviations: FreET, fresh embryo transfer\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eData are n (%).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e*Fisher's exact test\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study shows that co-administration of GnRH-a and hCG for final oocyte maturation, 40 and 34 hours prior to OPU, respectively (dual trigger), increased the number of MII oocytes retrieved and the rate of mature oocytes in patients with normal ovarian response who had a poor oocyte maturation rate (\u0026lt;\u0026thinsp;50%) in the previous cycle.\u003c/p\u003e \u003cp\u003eOocytes complete meiosis I and arrest at metaphase II until fertilization, at which point meiosis II is completed (13). Twenty-eight to thirty-eight hours after the onset of the LH surge preovulatory oocytes in metaphase II were obtained (14). After controlled ovarian stimulation, some of the oocytes retrieved are arrested at the germinal vesicle or metaphase I (MI) stage despite correct administration of hCG (15).The etiology of oocyte maturation arrest is complex. Recently, pathogenic variants in genes associated with oocyte maturation arrest have been identified. Pathogenic variants in PATL2 mutations mainly result in oocyte GV stage arrest by decreased amounts of protein (16). A heterozygous or homozygous mutation in the TUBB8 causes MI arrest through disruption of microtubule formation, meiotic spindle assembly, and microtubule dynamics (17). In addition, pathogenic variants in TRIP13 also responsible for oocyte meiotic arrest (18). Unfortunately, there is no effective treatment at present. When the percentage of meiotic competence failure oocytes was 25% or more, no pregnancy was achieved (19). A study shows that when the oocyte maturation rate is low (\u0026lt;\u0026thinsp;57.5%), it indicates a bad IVF cycle outcome (11). It is common knowledge that hCG has no FSH receptor activity. Compared with hCG alone, GnRH-a trigger induces an increase in endogenous LH and FSH, which is similar to the surge of gonadotropin in the middle of the natural cycle. Fabris et al. demonstrated that in patients with high immature oocyte rate in the previous IVF cycle, the number of retrieved mature oocytes increases when the proportion of immature oocytes declines due to dual triggering (20). Our study reached the same conclusion, but differed in that the administration of GnRH-a and hCG did not occur simultaneously and prolonged the time between trigger and OPU. Dual trigger that can increase the number of mature oocytes and increase the rate of mature oocyte may have benefited from the surge in FSH. The surge in FSH stimulates the cumulus cells of oocyte-cumulus complexes to secrete a meiotic activating substance that allows the oocyte meiotic process to resume and activate cumulus expansion during the final stages of oocyte maturation (21, 22). Furthermore, FSH has been shown to promote the formation of LH receptor sites in granulosa cells and the development of the corpus luteum, which in turn promotes estrogen and P\u003csub\u003e4\u003c/sub\u003e production (23).\u003c/p\u003e \u003cp\u003eAdditionally, GnRH receptors have been found in a variety of human tissues, including the granulosa cells of the pre-ovulatory phase. In mammals, oocytes remain in the prophase of the first meiosis until the gonadotropin surge at puberty. The intra-oocyte concentration of cAMP and cGMP in this prolonged period prevents the resumption of meiosis in the oocyte. As a result of LH, cGMP levels decrease and meiosis resumes (24). It has been demonstrated that peripheral GnRH receptor activation decreases intracellular cAMP levels. Several genes are induced by GnRH that are involved in follicular rupture and oocyte maturation (25). The favorable results in our study could be attributed to the FSH surge and direct action of the agonist on the ovarian GnRH receptor. Perhaps, for unknown reasons, the LH pathway in the patient is blocked, and GnRH agonists activate a different pathway. Another possibility is that the interval between the ovulation trigger and oocyte retrieval is prolonged. In a natural cycle, the onset of LH surge occurs 34\u0026ndash;36 hours before follicular rupture. For optimal oocyte maturation, LH concentration must be maintained above a threshold for 14\u0026ndash;27 hours (26). Oocyte maturation and follicular rupture are time-dependent processes, which require different times in different patients. It is hypothesized that certain patients require a longer period of time for cumulus expansion, which allows the oocyte to detach from the follicular wall. In these cases, oocyte immaturity may result when aspiration occurs 36 h after hCG administration.\u003c/p\u003e \u003cp\u003eCo-administration of GnRH-a and hCG as trigger was associated with increased embryo implantation (27). Endometrial receptivity may be improved by GnRH-a acting as an autocrine and (or) paracrine regulator (25). Cheon et al. suggested that an increased expression of endometrial GnRH-II peptide, noted during the early and mid-secretory phase, may play an important role in human embryo implantation (28). Co-administration of GnRH-a and hCG for final oocyte maturation not only compensated for luteal insufficiency after GnRH-a trigger but also improved patients cycle outcomes by improving endometrial receptivity. Consistent with the study findings that Decleer et al. (29) suggested that women who received dual triggering were more likely to have a surplus of frozen embryos. The studies by Griffin D et al. and Fumei Gao et al. showed that the dual trigger did not improve patients cycle outcomes, which is in contrast to the findings of our study (15, 30). Consisting of the co-administration of GnRH agonist and hCG for final oocyte maturation, 40 and 34 h prior to OPU, respectively, differs from the simultaneous administration of GnRH agonist and hCG for final oocyte maturation, 36h prior to OPU, by the additional prolongation of the time between ovulation triggering and OPU. This later prolongation, may explain the beneficial effect in terms of both oocytes maturation and pregnancy rate.\u003c/p\u003e \u003cp\u003eIn this RCT, it is undeniable that limitations remain, including the relatively small sample size and unexpected higher dropout rate. Although our included population was normal responders, the fact is that partial normal responders also face a high risk of OHSS in the course of ovarian stimulation. When this happened, they were excluded. Therefore, this study cannot discuss the relationship between dual trigger and OHSS incidence. Because too few patients underwent fresh ET, it was not powered to show a difference in the pregnancy outcomes with fresh ET. Moreover, we did not detect and analyze relevant gene mutations in this population due to cost-benefit considerations. Although the duration of Gn administration was increased by one day in the dual trigger group, the number of oocytes retrieved was not increased compared with that in the hCG trigger group. Moreover, no statistical difference was found in the dosage of Gn between the two groups. Therefore, this may not be the reason for the increase in the number of mature oocytes.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, we demonstrated that co-administration of GnRH-a and hCG for final oocyte maturation, 40 and 34 hours prior to OPU, respectively, can increase MII oocyte rate and enable patients to obtain more embryos with higher quantity and quality. Moreover, the dual trigger was equally beneficial for pregnancy outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Sharing Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated and analyzed from the current study will be availed by the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent for publication was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone of the authors have a conflict of interest to declare with regard to this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJing-Yan Song and Zhen-Gao Sun conceived and designed the study. Meng-Han Yan was responsible for data analysis and writing of the manuscript. Qian-Qian Zhang and Wen-Xiu Yang were responsible for the data collection and interpretation. Jing-Yan Song critically revised the important intellectual content of this manuscript. All authors gave final approval of the version to be published, have agreed on the journal to which the article has been submitted, and agree to be accountable for all aspects of the work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLudwig Mi, Doody KJ, Doody KM. Use of recombinant human chorionic gonadotropin in ovulation induction. Fertility and Sterility. 2003,79(5):1051-9.\u003c/li\u003e\n\u003cli\u003eCastillo JC, Humaidan P, Bernabeu R. Pharmaceutical options for triggering of final oocyte maturation in ART. Biomed Res Int. 2014,2014:580171.\u003c/li\u003e\n\u003cli\u003eHaahr T, Roque M, Esteves SC, Humaidan P. GnRH Agonist Trigger and LH Activity Luteal Phase Support versus hCG Trigger and Conventional Luteal Phase Support in Fresh Embryo Transfer IVF/ICSI Cycles-A Systematic PRISMA Review and Meta-analysis. Front Endocrinol (Lausanne). 2017,8:116.\u003c/li\u003e\n\u003cli\u003eYoussef MA, Van der Veen F, Al-Inany HG, Mochtar MH, Griesinger G, Nagi Mohesen M, et al. Gonadotropin-releasing hormone agonist versus HCG for oocyte triggering in antagonist-assisted reproductive technology. Cochrane Database Syst Rev. 2014(10):CD008046.\u003c/li\u003e\n\u003cli\u003eHumaidan P, Polyzos NP, Alsbjerg B, Erb K, Mikkelsen AL, Elbaek HO, et al. GnRHa trigger and individualized luteal phase hCG support according to ovarian response to stimulation: two prospective randomized controlled multi-centre studies in IVF patients. Hum Reprod. 2013,28(9):2511-21.\u003c/li\u003e\n\u003cli\u003eGriffin D, Benadiva C, Kummer N, Budinetz T, Nulsen J, Engmann L. Dual trigger of oocyte maturation with gonadotropin-releasing hormone agonist and low-dose human chorionic gonadotropin to optimize live birth rates in high responders. Fertil Steril. 2012,97(6):1316-20.\u003c/li\u003e\n\u003cli\u003eOrvieto R. Triggering final follicular maturation--hCG, GnRH-agonist or both, when and to whom? J Ovarian Res. 2015,8(1757-2215 (Electronic)):60.\u003c/li\u003e\n\u003cli\u003eBeck-Fruchter R, Weiss A, Lavee M, Geslevich Y, Shalev E. Empty follicle syndrome: successful treatment in a recurrent case and review of the literature. Hum Reprod. 2012,27(5):1357-67.\u003c/li\u003e\n\u003cli\u003eHaas J, Zilberberg E, Dar S, Kedem A, Machtinger R, Orvieto R. Co-administration of GnRH-agonist and hCG for final oocyte maturation (double trigger) in patients with low number of oocytes retrieved per number of preovulatory follicles--a preliminary report. J Ovarian Res. 2014,7(1757-2215 (Print)):77.\u003c/li\u003e\n\u003cli\u003eZilberberg E, Haas J, Dar S, Kedem A, Machtinger R, Orvieto R. Co-administration of GnRH-agonist and hCG, for final oocyte maturation (double trigger), in patients with low proportion of mature oocytes. Gynecol Endocrinol. 2015,31(2):145-7.\u003c/li\u003e\n\u003cli\u003eCapper E, Krohn M, Summers K, Mejia R, Sparks A, Van Voorhis BJ. Low oocyte maturity ratio is associated with a reduced in vitro fertilization and intracytoplasmic sperm injection live birth rate. Fertility and Sterility. 2022,118(4):680-7.\u003c/li\u003e\n\u003cli\u003eYan MH, Cao JX, Hou JW, Jiang WJ, Wang DD, Sun ZG, et al. GnRH Agonist and hCG (Dual Trigger) Versus hCG Trigger for Final Oocyte Maturation in Expected Normal Responders With a High Immature Oocyte Rate: Study Protocol for a Randomized, Superiority, Parallel Group, Controlled Trial. Front Endocrinol (Lausanne). 2022,13(1664-2392 (Print)):831859.\u003c/li\u003e\n\u003cli\u003eMarteil G, Richard-Parpaillon L, Kubiak JZ. Role of oocyte quality in meiotic maturation and embryonic development. Reproductive Biology. 2009,9(3):203-24.\u003c/li\u003e\n\u003cli\u003eSeibel MM, Smith DM, Levesque L, Borten M, Taymor ML. The temporal relationship between the luteinizing hormone surge and human oocyte maturation. American Journal of Obstetrics and Gynecology. 1982,142(5):568-72.\u003c/li\u003e\n\u003cli\u003eGriffin D, Feinn R, Engmann L, Nulsen J, Budinetz T, Benadiva C. Dual trigger with gonadotropin-releasing hormone agonist and standard dose human chorionic gonadotropin to improve oocyte maturity rates. Fertil Steril. 2014,102(2):405-9.\u003c/li\u003e\n\u003cli\u003eChen B, Zhang Z, Sun X, Kuang Y, Mao X, Wang X, et al. Biallelic Mutations in PATL2 Cause Female Infertility Characterized by Oocyte Maturation Arrest. Am J Hum Genet. 2017,101(4):609-15.\u003c/li\u003e\n\u003cli\u003eYao Z, Zeng J, Zhu H, Zhao J, Wang X, Xia Q, et al. Mutation analysis of the TUBB8 gene in primary infertile women with oocyte maturation arrest. J Ovarian Res. 2022,15(1):38.\u003c/li\u003e\n\u003cli\u003eZhang Z, Li B, Fu J, Li R, Diao F, Li C, et al. Bi-allelic Missense Pathogenic Variants in TRIP13 Cause Female Infertility Characterized by Oocyte Maturation Arrest. Am J Hum Genet. 2020,107(1):15-23.\u003c/li\u003e\n\u003cli\u003eBar-Ami S, Zlotkin E, Brandes JM, Itskovitz-Eldor J. Failure of meiotic competence in human oocytes. Biol Reprod. 1994,50(5):1100-7.\u003c/li\u003e\n\u003cli\u003eFabris AM, Cruz M, Legidos V, Iglesias C, Munoz M, Garcia-Velasco JA. Dual Triggering With Gonadotropin-Releasing Hormone Agonist and Standard Dose Human Chorionic Gonadotropin in Patients With a High Immature Oocyte Rate. Reprod Sci. 2017,24(8):1221-5.\u003c/li\u003e\n\u003cli\u003eByskov AG, Andersen CY, Hossaini A, Guoliang X. Cumulus cells of oocyte-cumulus complexes secrete a meiosis-activating substance when stimulated with FSH. Molecular Reproduction and Development. 1997,46(3):296-305.\u003c/li\u003e\n\u003cli\u003eKol S, Humaidan P. LH (as HCG) and FSH surges for final oocyte maturation: sometimes it takes two to tango? Reprod Biomed Online. 2010,21(5):590-2.\u003c/li\u003e\n\u003cli\u003eZhou X, Guo P, Chen X, Ye D, Liu Y, Chen S. Comparison of dual trigger with combination GnRH agonist and hCG versus hCG alone trigger of oocyte maturation for normal ovarian responders. Int J Gynaecol Obstet. 2018,141(3):327-31.\u003c/li\u003e\n\u003cli\u003eSun Q-Y, Miao Y-L, Schatten H. Towards a new understanding on the regulation of mammalian oocyte meiosis resumption. Cell Cycle. 2009,8(17):2741-7.\u003c/li\u003e\n\u003cli\u003eYu B, Ruman J, Christman G. The role of peripheral gonadotropin-releasing hormone receptors in female reproduction. Fertil Steril. 2011,95(2):465-73.\u003c/li\u003e\n\u003cli\u003eZelinski-Wooten MB, Hutchison JS, Chandrasekher YA, Wolf DP, Stouffer RL. Administration of human luteinizing hormone (hLH) to macaques after follicular development: further titration of LH surge requirements for ovulatory changes in primate follicles. The Journal of Clinical Endocrinology \u0026amp; Metabolism. 1992,75(2):502-7.\u003c/li\u003e\n\u003cli\u003eLin MH, Wu FS, Hwu YM, Lee RK, Li RS, Li SH. Dual trigger with gonadotropin releasing hormone agonist and human chorionic gonadotropin significantly improves live birth rate for women with diminished ovarian reserve. Reprod Biol Endocrinol. 2019,17(1):7.\u003c/li\u003e\n\u003cli\u003eCheon KW, Lee HS, Parhar IS, Kang IS. Expression of the second isoform of gonadotrophin-releasing hormone (GnRH-II) in human endometrium throughout the menstrual cycle. Mol Hum Reprod. 2001,7(5):447-52.\u003c/li\u003e\n\u003cli\u003eDecleer W, Osmanagaoglu K, Seynhave B, Kolibianakis S, Tarlatzis B, Devroey P. Comparison of hCG triggering versus hCG in combination with a GnRH agonist: a prospective randomized controlled trial. Facts Views Vis Obgyn. 2014,6(4):203-9.\u003c/li\u003e\n\u003cli\u003eGao F, Wang Y, Fu M, Zhang Q, Ren Y, Shen H, et al. Effect of a \u0026quot;Dual Trigger\u0026quot; Using a GnRH Agonist and hCG on the Cumulative Live-Birth Rate for Normal Responders in GnRH-Antagonist Cycles. Front Med (Lausanne). 2021, 8:683210.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Normal ovarian responder, Dual trigger, Gonadotropin releasing hormone agonist, Human chorionic gonadotropin, MII oocytes","lastPublishedDoi":"10.21203/rs.3.rs-2581457/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2581457/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eTo evaluate whether dual trigger could improve reproductive outcomes in women with low oocyte maturation rates compare to human chorionic gonadotropin (hCG) trigger.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study included expected normal ovarian responders younger than 40 years old whose immature oocyte rate in the previous cycle was more than 50% at the reproductive center from July 2021 to January 2023. A total of 73 patients were enrolled at trigger, including 34 in the hCG trigger group and 39 in the dual trigger group (co-administration of gonadotrophin releasing hormone (GnRH) agonist and hCG, 40 and 34 hours prior to oocyte retrieval, respectively). The primary outcome was oocyte maturation rate.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThere was no significant difference in the number of oocytes retrieved between the two study groups, but the oocyte maturation rate was higher in dual trigger group (84.0% [14.0%] vs. 55.5% [19.8%], P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Moreover, there were also higher cumulative pregnancy rate (69.4% vs. 40.0%, P\u0026thinsp;=\u0026thinsp;0.035) and cumulative live birth rate (66.7% vs. 36.0%, P\u0026thinsp;=\u0026thinsp;0.022) in dual trigger group.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eFor normal responders with low oocyte maturation rates, the dual trigger may be more effective than the conventional hCG trigger.\u003c/p\u003e\u003ch2\u003eTrial Registration\u003c/h2\u003e \u003cp\u003eChictr.gov.cn, identifier: ChiCTR2100049292\u003c/p\u003e","manuscriptTitle":"Dual trigger for final oocyte maturation in expected normal responders with a high immature oocyte rate: A randomized controlled trial","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-28 22:28:37","doi":"10.21203/rs.3.rs-2581457/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d4f47ec0-68eb-45a2-b766-e5eac36b4e4f","owner":[],"postedDate":"March 28th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-07-07T04:29:51+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-28 22:28:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2581457","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2581457","identity":"rs-2581457","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}