{"paper_id":"552d737b-312c-41cd-b29d-61bc96182c8b","body_text":"In assisted reproductive technology (ART), the goal of controlled ovarian stimulation\n(COS) is to promote the development of multiple dominant follicles and support\noocyte maturation, ultimately increasing the chances of conception. Throughout this\nprocess, oocytes undergo both nuclear and cytoplasmic maturation, which are\nessential for maximizing fertilization potential and developmental competence [ 1 ][ 2 ]. COS\ninvolves two key time points: (i) ovulation triggering and (ii) oocyte retrieval.\nThe period between these stages is crucial for in vivo oocyte maturation, which is\nregulated by a complex cascade of biochemical processes [ 3 ][ 4 ]. The interval\nbetween ovulation trigger and oocyte pickup (OPU) is crucial, as it encompasses the\ninitiation of luteinization, the expansion of cumulus cells, and the resumption of\noocyte meiosis through reduction division [ 5 ].\nSeveral studies have indicated that the interval between hCG administration and OPU\nsignificantly influences follicular maturation, the proportion of oocytes with fully\nexpanded cumulus, the number of metaphase II (MII) oocytes, embryo developmental\npotential, and overall IVF outcomes [ 5 ][ 6 ][ \n7 ][ 8 ]. The timing of oocyte\nretrieval following the oocyte maturation trigger plays a crucial role in\ndetermining the clinical outcomes of ART. However, findings on the optimal duration\nof this interval remain inconsistent. While some studies have reported that\nextending the time to OPU does not necessarily lead to a higher yield of mature\noocytes or improved clinical outcomes [ \n9 ][ 10 ][ 11 ][ 12 ], others have\nsuggested that a prolonged interval may enhance oocyte maturation[ 13 ][ 14 ][ 5 ], increase fertilization rates [ 15 ], improve blastocyst development [ 16 ], result in a greater number of high-quality\nembryos, and contribute to higher ongoing pregnancy rates [ 17 ]. Additionally, some studies have indicated that extending\nthe interval between human chorionic gonadotropin (hCG) administration and oocyte\nretrieval predominantly increases the proportion of metaphase II (MII) oocytes\nwithout significantly impacting pregnancy rates[ \n18 ][ 19 ]. To date, there is no\nconsensus regarding the ideal hCG-OPU interval, with reported durations in clinical\npractice ranging from 32 to 38 hours [ \n13 ][ 12 ]. Limited research exists\non the influence of trigger-to-OPU intervals in patients with poor ovarian responses\npatients, despite studies focusing on the general in vitro fertilization (IVF)\npopulation. Due to their low oocyte yield and suboptimal quality, these patients may\nbe highly affected by even slight variations in timing. A recent study demonstrated\nthat in patients with diminished ovarian reserve (DOR), prolonging the interval\nbetween hCG administration and oocyte retrieval to 36 hours led to enhanced\nfertilization rates and improved embryo development compared to intervals of 34 and\n35 hours. Nevertheless, this extension did not influence pregnancy outcomes [ 20 ]. Determining the optimal timing for oocyte\nretrieval to improve ART outcomes continues to be a complex issue, particularly\namong poor ovarian responders—a population that is often underrepresented in current\nresearch. This study seeks to investigate the effect of the interval between the\ndual trigger for final oocyte maturation and oocyte retrieval on ART outcomes in\nthis specific group. Given the limited data addressing this relationship in poor\nresponders, the findings of this research may offer valuable insights. By exploring\nassociations between treatment variables and timing intervals, the study aims to\ninform more effective scheduling protocols within ART practices.\n\nThis study is designed as a randomized, single-blind clinical trial in which patients\nare blinded to the type of intervention. The trial will be conducted 217 infertile\npatients from April 2024 to March 2025 at the infertility clinic of Shariati\nHospital, affiliated with Tehran University of Medical Sciences (TUMS), Tehran,\nIran. This registered prospective randomized controlled trial (IRCTID:\nIRCT20120215009014N512) was approved by the Institutional Ethical Review Board\n(IR.TUMS.SHARIATI.REC.1403.012). Prior to enrollment, all participants were provided\nwith a detailed explanation of the study's objectives and procedures, and written\ninformed consent was obtained. Those who met the inclusion criteria and provided\nconsent were subsequently enrolled in the study. The inclusion criteria are based on\nthe Bologna or POSEIDON III and IV definitions for poor ovarian responders. Poor\novarian response is diagnosed based on advanced maternal age (≥40 years), a history\nof poor response in prior cycles (≤3 oocytes or cycle cancellation), or abnormal\novarian reserve tests (AFC ≤5-7 or AMH ≤1.1 ng/ml). Meeting at least two of these\ncriteria confirms the diagnosis. Participants will be were excluded if they meet met\nany of the following conditions: Stage III or IV endometriosis, History of ovarian\nsurgery, Requirement for preimplantation genetic diagnosis (PGD), Sever Male factor\n(TESE). After enrollment, all participants will undergo a standard treatment cycle.\nAll cycles that were canceled in the study occurred after patient enrollment, either\ndue to the cancellation of the embryo transfer cycle or the OPU cycle (such as\novulation occurring before puncture or empty follicle syndrome). During the initial\nevaluation of participants, a transvaginal ultrasound was conducted to assess\nuterine and ovarian normalcy, while a semen analysis was performed to evaluate sperm\nparameters, including count, morphology, and motility. Furthermore, hormonal\nprofiling was undertaken on days 1-3 of the menstrual cycle, measuring levels of\nthyroid-Stimulating Hormone (TSH) TSH, FSH, AMH, vitamin D, LH, estradiol, and\nprolactin to ensure baseline physiological normality.\nIf no abnormalities are identified during the initial evaluation, patients proceed to\nthe treatment cycle, which begins on the second or third day of menstruation, as\ndetermined by the attending physician. The treatment involves the administration of\nthe maximum dose of gonadotropins within an antagonist protocol. On the seventh day\nof stimulation, a transvaginal ultrasound is conducted to monitor follicular\ndevelopment. When the follicles reach a size of 13-14 mm, a daily subcutaneous\ninjection of GnRH antagonist (cetrorelix 0.25 mg) is initiated. A follow-up\nultrasound is performed 2-3 days later, and if at least three dominant follicles\nwith a diameter of ≥18 mm is observed, oocyte maturation is triggered using a dual\ninjection of highly purified urinary hCG (10,000 IU) and GnRH agonist (Decapeptyl\n0.2 mg). Participants were randomly assigned to one of two groups using a simple\nrandomization method implemented in R (version 4.4.1) software. The control group\nundergoes oocyte retrieval 36 hours after the trigger injection, while the\nintervention group undergoes the procedure 34 hours post-trigger. Patients were\nblinded to the type of intervention and they were assigned to groups based on a\nrandom sequence available to the researcher. In both groups, follicle aspiration and\noocyte retrieval are performed under general anesthesia with transvaginal ultrasound\nguidance. All retrieved metaphase II (MII) oocytes are fertilized through\nintracytoplasmic sperm injection (ICSI). Subsequently, based on progesterone levels\non the trigger day, embryo quality, and endometrial thickness, the resulting embryos\nare either cryopreserved or prepared for fresh transfer.\nAfter denudation, the retrieved oocytes were evaluated for quality and maturity,\ncategorized as germinal vesicle (GV), metaphase I (MI), or metaphase II (MII). ICSI\nwas performed on the MII oocytes, and fertilization was assessed 16-18 hours later\nby the presence of two pronuclei (2PN). Reproductive outcomes were measured,\nincluding the number of oocytes retrieved, the number of MII oocytes, and the oocyte\nmaturity rate (percentage of normal MII oocytes out of total retrieved normal\noocytes). The fertilization rate was calculated by dividing the number of oocytes\nwith 2PN (16-18 hours post-insemination) by the number of MII oocytes injected.\nHigh-quality embryos were defined as grade A and B cleavage embryos according to\nASEBIR criteria. Additionally, the chemical pregnancy rate (positive β-hCG test 14\ndays after embryo transfer, expressed as a percentage of ET cycles), and the\nclinical pregnancy rate (evidence of pregnancy via ultrasound 5-6 weeks after ET,\nexpressed as a percentage of ET cycles) were recorded.\nIn the frozen embryo transfer (FET) protocol, estradiol (E2) was administered on days\n2-3 of the cycle, either orally or vaginally, with monitoring of endometrial\nthickness by ultrasound after 10 days. If the endometrium reached ≥7 mm with a\ntrilaminar pattern, progesterone therapy was initiated. If not, estradiol was\ncontinued until the endometrium met the required thickness. Once optimal,\nprogesterone (50 mg intramuscularly and 800 mg vaginally) was started, and embryos\nwere transferred on day 4 for cleavage-stage embryos. In stimulated cycles for\nembryo freezing, letrozole (5 mg) was administered from day 2, with ultrasound\nassessments on day 12. If a dominant follicle reached ≥14 mm and endometrial\nthickness was ≥7 mm, a follow-up ultrasound occurred 3 days later. If the follicle\nreached 18-20 mm, HCG was administered, and embryo transfer occurred 7 days later.\nIn fresh embryo transfer cycles, progesterone (50 mg intramuscularly and 800 mg\nvaginally) was started post-ovulation, with embryo transfer occurring on day 4 for\ncleavage-stage embryos. IUI was performed in the operating room for patients with no\navailable oocytes.\nThe primary outcome of this study is the oocyte maturity rate. Secondary outcomes\ninclude the total number of oocytes retrieved, the number of mature oocytes (MII),\nthe fertilization rate, the number and quality of embryos, as well as the\nbiochemical pregnancy rate and clinical pregnancy rate.\nThe oocyte maturity rate was identified as the primary outcome. Based on the findings\nof Raziel et al. (2006) [ 7 ] and accounting for\na 6% loss to follow-up, the required sample size was determined to be 202\nparticipants (101 per group) to achieve 90% statistical power with a significance\nlevel of 0.05 for detecting differences in oocyte maturity rate. Data analysis was\nconducted using IBM SPSS Statistics version 24 . The Kolmogorov-Smirnov test was\nused to assess the normality of data distribution. Qualitative variables were\npresented as frequency and percentage, while quantitative data with a normal\ndistribution were expressed as mean ± standard deviation. Differences between the\nplacebo and treatment groups were evaluated using the Mann-Whitney U test, Fisher’s\nexact test, or the Chi-square test, with statistical significance set at P<0.05.\nAdditionally, linear regression and multiple logistic regression models were applied\nto adjust for potential confounders (AFC day 3). In this study, a pre-protocol\napproach was adopted.\n\n+Calculated based on Mann–Whitney or Fisher exact or\nchi-square tests. ++Calculated based on t-test.  *  Because the\nmissing\n(not reported) percentage is not equal to 100%\n+Calculated based on Mann–Whitney. ++ Adjusted in linear or logistic\nregression models by AFC day3. *  Among 169 patients. ET embryo\ntransfer\nFigure  1 . Summary of patient flow through the study\nAs illustrated in Figure- 1 , the study included a\ntotal of 217 patients, with 109 in the intervention group and 108 in the placebo group.\nThroughout the trial, no significant statistical differences were observed between the\nintervention and control groups regarding infertility duration, average age, BMI,\nhormonal profile, and other baseline clinical and demographic factors—except for AFC at\nthe study's outset (Table- 1 ). The analysis\naccounted for the effect of AFC. Table- 2  demonstrates\nthat the intervention group had significantly higher oocyte numbers (4.78 vs. 2.50, P<0.001)\nand oocyte retrieval rate (90.93% vs. 48.94%, P<0.001) compared to the control group.\nSimilarly, the oocyte maturity rate was greater in the intervention group (72.46% vs.\n64.07%, P=0.005), though the number of MII oocytes showed no significant difference\nbetween groups (3.46 vs. 1.80, P=0.098). Additionally, the intervention group had a\nsignificantly higher fertilization rate (91.73% vs. 70.54%, P<0.001). In addition,\nthe cancellation rate (OPU + ET) was notably higher in the control group (P<0.001).\nRegarding embryos, both the number of embryos per transfer and the proportion of\nhigh-quality embryos (A and B) were significantly greater in the intervention group (P<0.001).\nClinical pregnancy rates (P=0.01) and biochemical pregnancy rates (P=0.007) were also\nsignificantly higher in the intervention group. After adjusting for AFC, changes in all\nvariables remained significant, except for the fertilization rate.\nAdditionally, we compared the variables between the two groups, categorized by the\ntransfer cycle (fresh or frozen embryo transfer) (Table- \n3 ) and the endometrial preparation protocol in frozen embryo transfer (Table- 4 ). In frozen embryo transfer cycles, both the\nnumber of embryos per transfer and the proportion of high-quality embryos (A and B) were\nsignificantly higher in the intervention group (P<0.001). In fresh embryo transfer\ncycles, this was only true for the number of embryos per transfer (P=0.02). Furthermore,\nin fresh embryo transfer cycles, biochemical pregnancy rates (P=0.035) and clinical\npregnancy rates (P=0.058) were also significantly higher in the intervention group. As\nshown in Table-5, in HRT cycles, the number of\nembryos per transfer was significantly higher in the intervention group (P=0.047).\nHowever, in stimulated cycles, the number of high-quality embryos (A and B) was\nsignificantly higher in the intervention group (P=0.046).\n\n+Calculated based on Mann–Whitney or Fisher exact tests.  *  Only among\ntransfer patients. NM:  not meaning\n+Calculated based on Mann–Whitney or Fisher exact tests. NM:  not meaning\nIn current randomized clinical trial, we examined the impact of the interval between dual\ntrigger (hCG and decapeptyl) administration and oocyte retrieval on oocyte maturation\nand ART outcomes in POR. Our findings indicate that a shortened interval (34 hours)\nbetween dual trigger (hCG and decapeptyl) administration and oocyte retrieval results in\nsignificantly higher oocyte yield, oocyte retrieval rates, MII oocyte numbers,\nfertilization rates, number of high-quality embryos, and overall improved ART outcomes\ncompared to the conventional 36-hour interval.\nThe timing between trigger and OPU is critical for the success of ART, as processes such\nas luteinization, cumulus cell expansion, and resumption of meiosis must occur before\naspiration [ 21 ]. To achieve optimal outcomes,\nprecise management of this interval is essential to ensure a higher yield of mature\noocytes while preventing spontaneous ovulation [ \n22 ]. Physiological studies suggest that ovulation generally takes place\nbetween 24 and 56 hours following the LH surge, with an average occurrence at 32 hours [ 23 ]. Nader and Berkowitz [ 21 ] suggested that ovulation may occur earlier than 36 hours in\nsome women, advising that intervals under 35 hours should be targeted to prevent\novulation.\nThere is limited research on the impact of trigger-to-OPU intervals in patients with poor\novarian response, despite studies conducted on the general IVF population, which have\nyielded conflicting results. A recent study showed that in patients with diminished\novarian reserve (DOR), extending the interval between hCG administration and oocyte\nretrieval to 36 hours resulted in improved fertilization rates and better embryo\ndevelopment compared to 34- and 35-hour intervals. However, this extension did not\naffect pregnancy outcomes [ 20 ]. Their findings\nrevealed no statistically significant differences between the groups regarding\nbiochemical pregnancy rate (P=0.252), clinical pregnancy rate (P=0.867), total pregnancy\nloss rate (P=0.859), or live birth rate (P=0.338). Although there was a trend toward\nhigher biochemical and clinical pregnancy rates in the 36-hour OPU group, the lack of\nstatistical significance suggests that variations in OPU timing may not substantially\naffect pregnancy outcomes. Consistent with their results, Wang et al.[ 19 ] found no significant impact of OPU timing on\npregnancy rates. However, other studies have reported improved pregnancy outcomes with\nlater OPU timings [ 10 ][ 24 ][ 7 ].\nWei Wang et al. conducted a meta-analysis including five RCTs with 895 participants,\nwhich showed that the oocyte maturation rate was higher in the long interval group (>36\nhours) compared to the short interval group (<36 hours) [ 19 ]. However, the findings differed from our study, likely due to\nthe inclusion of patients with low ovarian reserve in our cohort. Similarly, Runxin Gan\net al.'s meta-analysis reported comparable maturation rates in both the short and long\ninterval groups (85.6% and 87.4%, respectively) [ \n23 ]. These results also contrasted with ours, as their study focused on\npatients with polycystic ovary syndrome (PCOS), while our study specifically targeted\npatients with poor ovarian reserve.\nInconsistent with our study's findings, Garor et al. [ \n10 ] found that delayed OPU was associated with a greater number of embryos and\nhigher fertilization rates compared to early OPU. A key finding from their study was the\nsignificantly higher OPU cancellation rate due to early ovulation in the 34-hour OPU\ngroup (15.7%) compared to the 35-hour (3.5%) and 36-hour (2.2%) groups (P<0.001). To\nminimize the risk of premature ovulation, the 34-hour OPU group had its OPU procedure\nscheduled earlier due to the elevated LH levels on the trigger day. However, early OPU\ndid not prevent premature ovulation in this group, resulting in fewer mature oocytes and\nlower fertilization rates. Similarly, Choi et al. [ \n25 ] also reported that early oocyte retrieval during an early LH surge did\nnot effectively reduce cycle cancellation rates and may contribute to lower\nfertilization rates.\nIn line with previous studies, Skvirsky et al. [ 26 ]\ndemonstrated that extending the interval between hCG administration and OPU could\nenhance oocyte maturation and embryo quality in women over 36 years of age. The\nblastocyst formation rate differed significantly among the groups (P=0.025), with the\nhighest rate observed in the 36-hour OPU group. This suggests that delaying OPU to 36\nhours may benefit blastocyst development, potentially due to improved oocyte maturity\nand cytoplasmic competence. These results imply that a longer interval between trigger\nand OPU may improve embryo quality at later stages.\nThe discrepancies between our study and prior research may be attributed to key factors\nsuch as differences in study populations (POR vs. DOR or PCOS patients or general IVF\npatients), triggering protocols (dual trigger vs. hCG-only trigger), and Ovarian\nPhysiology (POR patients may need earlier OPU to prevent over-maturation and loss of\nviable oocytes). Our findings highlight that for POR patients, a shorter trigger-to-OPU\ninterval enhances ART success rates, which may serve as an important consideration in\nrefining individualized stimulation protocols for this specific subgroup of patients.\nThis study offers a novel approach to improving ART outcomes in POR patients by\noptimizing OPU timing with dual triggering, which enhances fertilization rates, embryo\nquality, and pregnancy outcomes. The inclusion of POR patients strengthens its clinical\nrelevance, as this population faces significant challenges in ART. Additionally, the RCT\nand prospective design enhance the study’s validity by reducing bias and providing a\nclear assessment of causality. However, limitations include a small sample size, lack of\nlong-term follow-up on live birth rates, and a single-center design, which may limit the\ngeneralizability of the findings. Further studies with a larger, more diverse cohort are\nneeded to validate these results and refine ART protocols.\n\nOur findings suggest that in patients with POR, a 34-hour interval between dual\ntriggering and OPU, significantly improves fertilization rates, embryo quality, and\npregnancy outcomes when compared to the standard 36-hour interval. This timing\nadjustment appears to optimize the maturation of oocytes, leading to higher-quality\nembryos, which, in turn, may enhance the likelihood of successful fertilization and\npregnancy. By fine-tuning the timing of OPU, especially in women with POR, we may\nimprove the overall success rates of ART, offering a more effective approach to\nfertility treatment for this specific patient population.\n\nThe authors declare no conflicts of interest.","source_license":"CC-BY-4.0","license_restricted":false}