Evaluation of an Undisturbed Embryo Culture System for Embryo Development in IVF/ICSI Cycles

During the study, participants will be excluded from further participation if any of the following conditions are met. Elimination Criteria: 1）Retrieval of ≤1 oocyte; 2）Incomplete clinical data; 3）Patients in either the control group or the intervention group whose embryos required transfer or cryopreservation on Day 3, resulting in interruption of the study protocol for extended culture to the blastocyst stage; 4）Patients who withdrew informed consent. Randomization Procedure 1. The detailed oocyte randomization procedure is as follows：all cumulus-oocyte complexes (COCs) from each patient will be randomly allocated in a 1:1 ratio to the control group or the experimental group. If the number of oocytes retrieved is odd, after 1:1 randomization, the remaining single oocyte will be allocated on the basis of the parity of the corresponding number in a pre-generated computer randomization table. An even number indicates assignment to the control group, whereas an odd number indicates assignment to the intervention group. Furthermore, in accordance with the recommendations of the 2024 Istanbul Expert Consensus, zygotes without visible two pronuclei (2PN) after fertilization will be regarded as usable embryos in both the control and experimental groups. Such embryos will not be discarded and will continue subsequent culture within their respective assigned culture systems. Oocyte retrieval, oocyte processing, and fertilization methods in ART cycles will be carried out according to the patients' clinical indications and the standard operating procedures of the reproductive medicine center. 2. The detailed randomization procedure for embryo transfer is as follows：If the best-quality embryos from the two groups are of the same grade, the group from which the embryo for transfer is selected will be determined based on a pre-generated computer randomization sequence. An even random number indicates selection of the best-quality embryo from the control group for single blastocyst transfer, whereas an odd random number indicates selection of the best-quality embryo from the intervention group. Criteria for Discontinuation or Modification of the Intervention：1) In the event of laboratory accidents, such as incubator malfunction or culture medium contamination, embryos will be immediately transferred to a backup incubator or culture dish, and the incident will be documented in the case report form (CRF). 2) If a participant withdraws consent, the intervention will be discontinued immediately. Data generated before withdrawal will be retained for analysis according to the intention-to-treat principle. Strategies to Improve Adherence and Monitoring Measures：1) Laboratory quality control: Temperature and CO2 levels will be automatically recorded daily and manually calibrated; all incubators will be equipped with 24-hour remote alarm monitoring. 2) Procedure standardization: Standard operating procedures (SOPs) will be established and strictly implemented; culture medium replacement will be verified independently by two staff members. 3\) Blinded assessment: Blastocyst grading will be performed by senior embryologists blinded to group allocation, and all assessment images will be archived. Prohibited Concomitant Interventions During the Trial：The following interventions will not be permitted during the trial: assisted hatching (AH), embryo glue, or other special embryo manipulation procedures; use of incubators or culture media not specified in the study protocol; and, in the experimental group, opening of the culture chamber or dish lid during culture for manual observation or medium replacement. Calculation of Outcome Measures 1. Primary Outcome Measure Clinical pregnancy rate：The proportion of clinical pregnancy cycles among the total number of embryo transfer cycles. Time Frame: 4-7 weeks after embryo transfer Definition: Clinical pregnancy is defined as the presence of an intrauterine gestational sac detected by ultrasonography 4-7 weeks after embryo transfer, or pathological confirmation of products of miscarriage. 2. Secondary Outcome Measures <!-- --> 1. Normal fertilization rate in IVF：The proportion of oocytes with two pronuclei (2PN) and two polar bodies (2PB) observed on Day 1 among the total number of inseminated oocytes in IVF. Time Frame: Day 1 after fertilization Normal fertilization rate in ICSI：The proportion of oocytes with 2PN and 2PB observed on Day 1 among the total number of injected metaphase II (MII) oocytes. Time Frame: Day 1 after fertilization 2. Cleavage rate：The proportion of cleavage-stage embryos on Day 2 among the total number of normally fertilized oocytes. Time Frame: Day 2 after fertilization 3. Day 3 good-quality embryo rate：The proportion of good-quality embryos on Day 3 among the total number of normally fertilized oocytes. Time Frame: Day 3 after fertilization 4. Day 3 usable embryo rate：The proportion of usable embryos on Day 3 among the total number of normally fertilized oocytes. Time Frame: Day 3 after fertilization 5. Good-quality blastocyst formation rate：The proportion of good-quality blastocysts among the total number of normally fertilized oocytes. Time Frame: Day 5/6 after fertilization 6. Day 5/6 usable blastocyst rate：The proportion of usable blastocysts on Day 5/6 among the total number of normally fertilized oocytes. Time Frame: Day 5/6 after fertilization 7. Blastocyst formation rate：The proportion of total blastocysts formed on Day 5/6 among the total number of normally fertilized oocytes. Time Frame: Day 5/6 after fertilization 8. Implantation rate：The proportion of gestational sacs detected after embryo transfer among the total number of transferred blastocysts. Time Frame: Approximately 4-7 weeks after embryo transfer （9）Biochemical pregnancy rate：The proportion of cycles with positive serum β-hCG after embryo transfer but no gestational sac detected by ultrasonography among the total number of embryo transfer cycles. Time Frame: Approximately 14 days after embryo transfer （10）Early miscarriage rate：The proportion of spontaneous miscarriage cycles occurring within 12 weeks of pregnancy among the clinical pregnancy cycles. Time Frame: Within 12 weeks of pregnancy （11）Ongoing pregnancy rate：The proportion of cycles with a viable pregnancy continuing to at least 12 weeks after clinical pregnancy among the total number of embryo transfer cycles. Time Frame: At or beyond 12 weeks of pregnancy （12）Live birth rate (first embryo transfer) The proportion of first embryo transfer cycles that result in at least one live birth. Time Frame: At delivery Recruitment: Participants will be recruited from infertile patients planning to undergo IVF or ICSI cycles at the 12 reproductive medicine centers participating in this trial. Initial screening will be conducted by trained physicians familiar with the study procedures among patients attending the reproductive medicine centers. For potential participants who meet the eligibility criteria, the investigator will introduce the study to both partners, including its purpose, procedures, potential risks, and benefits, and will provide sufficient time for them to consider whether they are willing to participate. All questions raised by the couple regarding the study will be fully addressed. After full and detailed informed consent has been obtained, both partners and the investigator will jointly sign the informed consent form. Blinding: None (Open-label) Data Collection 1. Baseline data will be collected after written informed consent is obtained, including serum sex hormone levels (E2, LH, FSH, PRL, P, and T on menstrual cycle days 2-5), antral follicle count (AFC), and routine semen analysis. Other laboratory tests required for routine clinical management during ovarian stimulation, oocyte retrieval, and frozen embryo transfer cycles will be performed according to standard practice but will not be included as study outcome measures. 2. During the intervention period, the total number of retrieved oocytes and the number allocated to the control and experimental groups will be recorded. If the total number of oocytes is odd, the method used for random allocation of the last oocyte will also be documented. From 16-18 hours after fertilization (Day 1) to the blastocyst stage (Day 5/6), fertilization outcomes, daily embryo development, and incubator environmental parameters (including temperature and gas concentration) will be recorded in the case report form (CRF) in real time by embryologists. 3. Outcome data will include all primary and secondary outcome measures. To ensure data quality, key outcome data will be double-checked, all study personnel will receive standardized training, and regular data cleaning will be performed to identify missing, inconsistent, or incorrect entries. To enhance participant retention and follow-up completion, investigators will provide detailed study explanations at enrollment, written study materials, dedicated contact information, and appropriate financial compensation. Data Management （1）Data entry will be performed concurrently with clinical data collection. Queries identified during data entry or review will be documented in writing and resolved by the principal investigator. All changes will be documented and retained together with the CRFs. All study data will be kept confidential and may not be disclosed to third parties without written authorization from the sponsor. （2）After completion of data review and confirmation of data accuracy, the database will be locked jointly by the leading center, collaborating centers, and the statistician. No changes will be permitted after database lock. The statistical analysis plan will be finalized before database lock. Statistical Analysis （1）Analysis Populations The Full Analysis Set (FAS) will include all randomized participants who receive at least one study intervention and have post-intervention assessment data, following the intention-to-treat principle. Missing data in the FAS will be handled using the last observation carried forward (LOCF) method. The Per-Protocol Set (PPS) will include participants who complete treatment and follow-up according to the protocol without major protocol deviations affecting efficacy. The PPS will be used as the primary analysis set for efficacy evaluation. Statistical Methods 1. All statistical tests will be two-sided, and P ≤ 0.05 will be considered statistically significant. Continuous variables will be summarized using mean, standard deviation, median, minimum, and maximum values, as appropriate. Categorical variables will be summarized using frequencies and percentages. Statistical analyses will be performed using SPSS 25.0 or R. 2. Baseline characteristics will be analyzed descriptively. Normality of continuous variables will be assessed using histograms and the Shapiro test. Normally distributed variables will be presented as mean ± standard deviation; non-normally distributed variables will be presented as median and interquartile range. 3. Clinical pregnancy rate, the primary outcome, will be compared between groups using the Cochran-Mantel-Haenszel (CMH) stratified chi-square test, with adjustment for baseline characteristics and center effects using multivariable logistic regression. 4. Secondary outcomes, including fertilization rate, cleavage rate, Day 3 embryo development rate, blastocyst formation rate, implantation rate, biochemical pregnancy rate, miscarriage rate, ongoing pregnancy rate, and live birth rate, will also be compared between groups using the CMH stratified chi-square test. 5. Dropout rates and adverse event-related withdrawal rates will be compared between groups using the chi-square test or Fisher's exact test. Study duration will be compared using the independent-samples t test or Wilcoxon rank-sum test, as appropriate. Data Monitoring No formal independent Data Monitoring Committee (DMC) will be established for this study, because this is an investigator-initiated clinical study involving routine ART procedures and embryo culture interventions with relatively low anticipated risk. Data monitoring will be performed by personnel qualified in Good Clinical Practice (GCP) from Suzhou Basecare Medical Corporation Ltd. or The Sixth Affiliated Hospital, Sun Yat-sen University. Interim Analysis and Stopping Guidelines An interim analysis is planned when 50% of the target sample size has been enrolled. The interim analysis will evaluate the primary outcome (clinical pregnancy rate) and selected secondary outcomes, including fertilization rate, cleavage rate, Day 2/Day 3 embryo development, and blastocyst formation rate. Safety data, including all adverse events (AEs) and serious adverse events (SAEs), will also be reviewed. The study may be suspended or terminated early under the following circumstances: Safety concerns: if a significant safety issue is identified in either group, such as an unexpectedly high rate of abnormal embryo development; Clear evidence of benefit: if the experimental group shows a significantly higher clinical pregnancy rate than the control group (p \< 0.001) and continuation is considered unnecessary; Clear evidence of inferiority/futility: if the experimental group shows a significantly lower clinical pregnancy rate than the control group (p \< 0.001). The principal investigator will review the interim results and make recommendations regarding study continuation, suspension, or termination. Harms (Adverse Events) All adverse events (AEs) and serious adverse events (SAEs) occurring during the study will be collected, assessed, recorded, reported, and followed up. An AE is defined as any unfavorable medical occurrence or worsening of a pre-existing condition during ovarian stimulation or other study-related procedures, including symptoms, signs, or abnormal laboratory findings. An SAE is defined as any event that results in death, is life-threatening, requires hospitalization or prolongation of hospitalization, results in persistent or significant disability/incapacity, leads to congenital anomaly or birth defect, or is otherwise medically important. For each AE/SAE, the investigator will record the onset time, severity, duration, actions taken, outcome, and relationship to the study intervention. Causality will be classified as definitely related, possibly related, possibly unrelated, definitely unrelated, or unassessable. Participants will be monitored from the time of informed consent until the end of follow-up through telephone or face-to-face contact. For SAEs, all relevant clinical information and follow-up data will be documented in the source records. Auditing Internal audits will be conducted on a regular basis by the hospital to ensure continued protocol compliance. Audit content will include: Data integrity: review of participant files, CRFs, laboratory records, and informed consent forms to confirm completeness, accuracy, and logical consistency; Procedural compliance: review of key study procedures, including embryo culture, randomization, and data entry, to ensure adherence to SOPs; Participant management: verification of enrollment, follow-up, and withdrawal procedures; Equipment maintenance: review of operation and maintenance records for incubators, microscopes, and other relevant equipment. Audit findings will be submitted to the study team and sponsor within 10 working days after audit completion, together with recommendations for corrective actions. Protocol Amendments Any important protocol amendments, including changes to eligibility criteria, outcome measures, or statistical methods, will be discussed by the study team and confirmed by the principal investigator (PI) and the statistical team. Written notification of major amendments will be provided to all study team members within 72 hours through internal communication channels, together with the implementation timeline. Relevant staff will receive additional training as needed. Enrolled participants will be informed of important amendments by telephone or WeChat follow-up. Ethics committees/IRBs and trial registries will also be notified in accordance with applicable requirements. Informed Consent Potential participants will first be approached by a clinician familiar with the study protocol and procedures. The study purpose, procedures, potential risks, and possible benefits will be explained, and a copy of the informed consent form will be provided for careful review. Participants will be given sufficient time to ask questions and consider participation. Written informed consent will be obtained voluntarily before any study-specific procedures are performed. Both the participant and the investigator will sign the informed consent form, which will be retained in the study files. In this study, embryos may be cultured using an undisturbed closed culture system, which is expected to improve blastocyst formation and good-quality embryo rates. Other than the assigned culture system, all procedures will follow routine hospital practice and are not expected to introduce additional risks. Confidentiality Only essential study information will be collected, and participant data will be de-identified as early as possible. Access to personal data will be restricted according to role-based permissions. Transmission of identifiable data through unsecured email will be prohibited. After study completion, the final database will be anonymized and locked. Independent data monitors will supervise data protection procedures and sign confidentiality agreements. Declaration of Interests The principal investigator and the principal investigators of the participating centers declare that they have no financial or other conflicts of interest related to this study. Access to Data All study data collected during the trial will be submitted to The Sixth Affiliated Hospital, Sun Yat-sen University. The principal investigator will be responsible for preparation of the clinical study report and manuscripts, with input from participating centers. Relevant trial documents, including source records, signed informed consent forms, CRFs, and other study records, will be retained for inspection, oversight, and analysis. Access to the final trial dataset will be limited to authorized investigators and statisticians. Ancillary and Post-Trial Care Not applicable. Dissemination Policy All study-related information will be treated as confidential until study completion and review of the final statistical analyses. Study results may be published or presented after authorization by the leading study center. Confidential or proprietary information will not be disclosed.