Effects of different fertilization methods on oocyte maturity

Effects of different fertilization methods on oocyte maturity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of different fertilization methods on oocyte maturity Xuhui Zhang, Jingchuan Yuan, Xiaoling Zhang, Yinjing Dong, Yaonan Cao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8677406/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Objective: To investigate the effects of different fertilization methods on oocyte maturity. Methods: A retrospective analysis was conducted on patients who underwent in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) at the Reproductive Medicine Center of The Fourth Hospital of Shijiazhuang between January 2022 and June 2024. A total of 2553 cycles were included and categorized into four groups based on medical history and fertilization strategy: Group I (1111 cycles): Short-term fertilization for patients with primary infertility (no prior pregnancy between the couple); Group II (619 cycles): Overnight fertilization for patients with secondary infertility (prior pregnancy history); Group III (584 cycles): ICSI for patients with male factor infertility; Group IV (239 cycles): Short-term fertilization combined with early rescue ICSI (Re-ICSI). The short-term fertilization group was further subdivided by observation timing: the D0 subgroup (assessed on the day of oocyte retrieval) and the D1 subgroup (assessed on the day of fertilization observation). Comparisons were performed between the D0 and D1 subgroups, D1 subgroup and overnight fertilization group, ICSI and Re-ICSI groups, and D0 subgroup and Re-ICSI group, focusing on general clinical data and oocyte maturation parameters. Results: The D1 subgroup exhibited significantly higher metaphase II (MII) and metaphase I (MI) oocyte rates, and a significantly lower germinal vesicle (GV) oocyte rate compared to the D0 subgroup ( P <0.05). The overnight fertilization group had a significantly higher female age and lower number of retrieved oocytes than the short-term fertilization group ( P <0.05), alongside a significantly higher MII oocyte rate and lower GV oocyte rate than the D1 subgroup ( P <0.05). The Re-ICSI group showed significantly fewer retrieved oocytes and lower oocyte maturity than the D0 subgroup ( P 0.05). Conclusion: Different fertilization methods exert distinct impacts on oocyte maturity, with overnight fertilization yielding the highest maturity, followed by short-term fertilization, and Re-ICSI yielding the lowest. This variation may be associated with asynchronous oocyte development. Fertilization method Oocytes maturity Short-term fertilization Overnight fertilization ICSI Re-ICSI Figures Figure 1 Introductions Fertilization is a crucial step in assisted reproductive technology (ART) treatment. Conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are two commonly used fertilization methods in ART [ 1 , 2 ]. IVF is further classified into short-term fertilization and overnight fertilization based on the time of cumulus cell removal. Currently, most reproductive centers adopt short-term IVF for primary infertility patients without male factor infertility to avoid IVF fertilization failure and resort to rescue-intracytoplasmic sperm injection (Re-ICSI) [ 3 ]. Couples with secondary infertility (those with a history of pregnancy) undergo overnight fertilization. For patients with male factor infertility (poor sperm quantity or quality), ICSI is directly chosen. Oocytes are classified based on their maturity into mature oocytes (metaphase II oocytes, MII oocytes), metaphase I oocytes (MI oocytes), germinal vesicle stage oocytes (GV oocytes), empty zona pellucida oocytes, and degenerated oocytes. The combination of mature oocytes with sperm to form zygotes marks the beginning of embryo development [ 4 ]. After cumulus cell removal in ICSI fertilization, the MII oocyte rate can be directly used as an indicator to assess oocyte maturity [ 5 ]. However, there is no fixed standard for assessing oocyte maturity in IVF. On the day of oocyte retrieval (D0) in short-term IVF fertilization, cumulus cell removal can determine oocyte maturity, but after overnight culture on the day of fertilization observation (D1), MI oocytes and GV oocytes further mature, and oocyte maturity may change [ 6 ]. Modifying the methods and strategies in the laboratory fertilization process can improve fertilization outcomes and increase oocyte utilization [ 7 ]. However, the impact of fertilization methods on oocyte maturity remains unclear. This study aims to explore the influence of different fertilization methods on oocyte maturity to better guide clinical practice. Material and methods I. Research Subjects and Grouping A retrospective analysis was conducted on patients who underwent IVF/ICSI at the Reproductive Medicine Center of the Fourth Hospital of Shijiazhuang from January 2022 to June 2024. The inclusion criteria were as follows: (1) female age 5; (3) normal karyotype in both partners; (4) first cycle of ovulation induction. The exclusion criteria were: patients with endometriosis, oocyte donors, oocyte freezing cycles, oocytes with abnormal morphology (waxy zona pellucida), in vitro matured (IVM) oocytes, and ICSI fertilization failure. Grouping was based on fertilization methods: patients with male partner's semen parameters meeting conventional IVF criteria underwent short-term fertilization or overnight fertilization; for male factor (severe oligoasthenoteratozoospermia), direct ICSI was performed. For IVF patients, short-term fertilization + Re-ICSI was used for primary infertility (patients without pregnancy history between the couple), and overnight fertilization for secondary infertility (patients with pregnancy history). According to the time of oocyte maturity assessment, it was further divided into the D0 subgroup and D1 subgroup: among them, the short-term fertilization group, ICSI group, and Re-ICSI group were observed after stripping the cumulus cells on D0, while the D1 subgroup and overnight fertilization group were observed on D1. The D0 subgroup and the D1 subgroup were the same group of the patients observed on D0 and D1, respectively. (Figure 1) The D0 subgroup and D1 subgroup, the D1 subgroup and overnight fertilization group, ICSI group and Re-ICSI group, the D0 subgroup and Re-ICSI group were compared to analyze the mature oocyte rate, immature oocyte rate, empty zona pellucida/regression rate, etc. of each group. II. Research Methods 1. Controlled Ovarian Hyperstimulation: The center's routine ovulation induction protocols were used, mainly including the long protocol and the antagonist protocol. After administration of gonadotropins, B-ultrasound was used to monitor the number, size of follicles, and thickness of the endometrium, as well as serum hormone levels, and the dose of gonadotropins was adjusted. When the dominant follicle developed to 18 mm - 20 mm, 5,000 - 10,000 U of human chorionic gonadotropin (HCG, Zhuhai Lize) was administered, and 36 - 38 hours later, oocyte retrieval was performed under vaginal B-ultrasound guidance. 2. Semen Optimization Treatment: According to the male partner's semen condition, optimization treatment was performed before IVF/ICSI fertilization. 3. ICSI Fertilization: 39 - 40 hours after HCG injection, the oocytes were treated with 80 U/mL hyaluronidase (H3757, Sigma, USA) for 30 seconds, and followed by cumulus cell removal via aspiration. The oocyte maturity was evaluated, and MII oocytes were directly subjected to ICSI fertilization. The presence of two pronuclei 18 - 20 hours after fertilization was considered as the ICSI fertilization group. 4. Short-term Fertilization: 38 - 40 hours after HCG injection, the processed sperm was added to the double-well dish (Falcon, USA) containing oocytes, at a concentration of 150,000–300,000 sperm/mL of culture medium. Each dish contained about 5 oocytes. After co-culture of sperm and oocytes for 4 - 6 hours, the cumulus cells were removed and the second polar body was observed. If the proportion of oocytes with two polar bodies among mature oocytes was ≥ 30%, it was considered as the short-term fertilization group. The presence of two pronuclei 18 - 20 hours after fertilization was considered as the short-term fertilization (D1) group. If the proportion was < 30%, Re-ICSI was performed, and it was considered as the Re-ICSI group [2, 3]. 5. Overnight Fertilization: 18 - 20 hours after IVF fertilization, the cumulus cells were removed and fertilization was observed. The presence of two pronuclei was considered as the IVF overnight fertilization group. 6. Observation Indicators: Oocyte maturity assessment on D0 was conducted after cumulus cell removal, and on D1 during fertilization observation. On day 0, the rates of MII oocytes, MI oocytes, GV oocytes, and the rate of empty zona pellucida/ovum degeneration in the IVF short-term fertilization group, ICSI group, and Re-ICSI group were calculated as follows: MII oocyte rate = (number of MII oocytes / number of retrieved oocytes) × 100%; MI oocyte rate = (number of MI oocytes / number of retrieved oocytes) × 100%; GV oocyte rate = (number of GV oocytes / number of retrieved oocytes) × 100%; rate of empty zona pellucida/ovum degeneration = [(number of empty zona pellucida + number of degenerated oocytes) / number of retrieved oocytes] × 100%. On day 1, the MII oocyte rate in the IVF short-term fertilization (D1) group and overnight fertilization group was calculated as: MII oocyte rate = (number of fertilized oocytes + number of MII oocytes) / number of retrieved oocytes × 100%; the rest were the same as above. III. Statistical Analysis The analysis was conducted using SPSS 27.0 statistical software. Continuous data with a normal distribution were expressed as mean ± standard deviation (x̄ ± s), and comparisons among multiple groups were performed using one-way ANOVA and a Bonferroni correction was applied. Categorical data were expressed as n (%), and the comparison of rates between groups was conducted using the χ² test. P value < 0.05 was considered statistically significant. Figure 1. Research Subjects and Grouping Flowchart Results A total of 2,553 IVF/ICSI cycles were included in this study, including 1,111 short-term fertilization cycles, 619 overnight fertilization cycles, 584 ICSI cycles, and 239 Re-ICSI cycles. the short-term fertilization group was further divided into the D0 subgroup and D1 subgroup based on different observation times. 1. Comparison of general conditions among groups The age of women in the overnight fertilization group was significantly higher than that in the short-term fertilization group (30.32±2.92 vs. 29.28±2.97), and the number of retrieved oocytes was significantly lower (12.08 ± 6.26 vs. 13.46 ± 6.78). The number of retrieved oocytes in the Re-ICSI group was significantly lower than that in the short-term fertilization group (12.36 ± 6.14 vs. 13.46 ± 6.78), with statistically significant differences ( P 0.05) (Table 1). 2. Comparison between the D0 subgroup and the D1 subgroup Compared with the D0 subgroup, the D1 subgroup had a significantly higher MII oocyte rate (88.12% vs. 83.89%, χ2 value = 30.480, P = 0.000), a significantly higher MI oocyte rate (7.37% vs. 6.69%, χ2 value = 5.217, P = 0.022), and a significantly lower GV oocyte rate (4.22% vs. 9.14%, χ2 value = 290.522, P = 0.000), with statistically significant differences. There was no statistically significant difference in the rate of degenerated/empty zona pellucida (0.29% vs. 0.27%, χ2 value = 0.106, P = 0.745) (Table 2). 3. Comparison between the D1 subgroup and overnight fertilization group The MII oocyte rate in the overnight fertilization group was significantly higher than that in the D1 subgroup (90.44% vs. 88.12%, χ2 value = 27.178, P = 0.000), and the GV oocyte rate was significantly lower (2.70% vs. 4.22%, χ2 value = 32.122, P = 0.000), with statistically significant differences ( P < 0.05). There were no statistically significant differences in the MI oocyte rate and the rate of degenerated/empty zona pellucida between the two groups (χ2 value = 2.849, P = 0.091) (Table 3). 4. Comparison of oocytes between ICSI group and Re-ICSI group There were no statistically significant differences in the number of retrieved oocytes, MII oocyte rate (83.26% vs. 82.30%, χ2 value = 1.364, P = 0.243), MI oocyte rate (6.93% vs. 7.78%, χ 2 value = 2.296, P = 0.130), GV oocyte rate (9.53% vs. 9.61%, χ2 value = 0.015, P = 0.901), and the rate of degenerated/empty zona pellucida (0.28% vs. 0.30%, χ 2 value = 0.044, P = 0.833) between the ICSI group and the Re-ICSI group (Table 4). 5. Comparison between short-term fertilization (D0) group and Re-ICSI group Compared with the D0 subgroup, the Re-ICSI group had a significantly lower MII oocyte rate (82.30% vs. 83.89%, χ2 value = 4.578, P = 0.032) and a significantly higher MI oocyte rate (7.78% vs. 6.69%, χ2 value = 4.592, P = 0.032), with statistically significant differences. The GV oocyte rate (9.53% vs. 9.61%, χ2 value = 0.657, P = 0.418) and the degenerated/empty zona pellucida rate (0.28% vs. 0.30%, χ2 value = 0.082, P = 0.774) showed no statistically significant differences (Table 5). Table 1 Comparison of General Conditions among Different Groups ( ±s) Group Short-term fertilization group Overnight fertilization group ICSI group Re-ICSI group F P Number of patients 1111 619 584 239 Female age （ ± s ） 29.28±2.97 30.32±2.92 * 29.37±3.31 29.87±2.64 3.554 0.014 Duration of infertility（ ± s ） 3.27±2.17 3.28±2.42 3.24±2.29 3.56±2.77 2.17 0.096 BMI （ ± s ） 23.76±3.89 23.54±3.77 23.32±3.66 23.95±4.12 1.149 0.328 Number of retrieved oocytes（ ± s ） 13.46±6.78 12.08±6.26 * 12.23±5.93 12.36±6.14 ※ 8.37 ＜0.001 Note: Compared with the overnight fertilization group, the short-term fertilization group showed a significant difference ( * P <0.05); compared with the Re-ICSI group, the short-term fertilization group also demonstrated a significant difference ( ※ P <0.05). Table 2 Comparison of Oocyte Maturation Status in D0 subgroup and D1 subgroup [% (n)] Group D0 subgroup D1 subgroup χ2 P MII oocyte rate %（ n ） 83.89 (12549/14958) 88.12 (13181/14958) 30.480 0.000 MI oocyte rate %（ n ） 6.69 (1001/14958) 7.37 (1102/14958) 5.217 0.022 GV oocyte rate %（ n ） 9.14 (1367/14958) 4.22 (631/14958) 290.522 0.000 Degeneration/empty zona pellucida rate %（ n ） 0.27 (41/14958) 0.29 (44/14958) 0.106 0.745 Table 3 Comparison of Oocyte Maturation Status in D1 subgroup and Overnight fertilization group [% (n)] Group D1 subgroup Overnight fertilization group χ2 P MII oocyte rate %（ n ） 88.12 (13181/14958) 90.44 (6764/7479) 27.178 0.000 MI oocyte rate %（ n ） 7.37 (1102/14958) 6.69 (500/7479) 3.497 0.061 GV oocyte rate %（ n ） 4.22 (631/14958) 2.70 (202/7479) 32.122 0.000 Degeneration/empty zona pellucida rate %（ n ） 0.29 (44/14958) 0.27 (13/7479) 2.829 0.093 Table 4 Comparison of Oocyte Maturation Status in ICSI group and Re-ICSI group [% (n)] Group ICSI group Re-ICSI group χ2 P MII oocyte rate %（ n ） 83.26 (5949/7145) 82.30 (2432/2955) 1.364 0.243 MI oocyte rate %（ n ） 6.93 (495/7145) 7.78 (230/2955) 2.296 0.130 GV oocyte rate %（ n ） 9.53 (681/7145) 9.61 (284/2955) 0.015 0.901 Degeneration/empty zona pellucida rate %（ n ） 0.28 (20/7145) 0.30 (9/2955) 0.044 0.833 Table 5 Comparison of Oocyte Maturation Status in D0 subgroup and Re-ICSI group [% (n)] Group D0 subgroup Re-ICSI group χ2 P MII oocyte rate %（ n ） 83.89 (12549/14958) 82.30 (2432/2955) 4.578 0.032 MI oocyte rate %（ n ） 6.69 (1001/14958) 7.78 (230/2955) 4.592 0.032 GV oocyte rate %（ n ） 9.14 (1367/14958) 9.61 (284/2955) 0.657 0.418 Degeneration/empty zona pellucida rate %（ n ） 0.27 (41/14958) 0.30 (9/2955) 0.082 0.774 Discussions Gamete quality and maturity assessment are critical for ART success, as oocyte maturation directly influences fertilization and subsequent embryo development [ 9 ]. MII oocytes—compared to immature counterparts—exhibit significantly higher fertilization potential, making maturity assessment a key determinant of fertilization timing [ 9 , 10 ]. Expert consensus recommends short-term or overnight fertilization for patients with normal sperm parameters [ 11 ]. Primary infertility patients face a higher risk of fertilization failure and thus typically undergo short-term fertilization, while secondary infertility patients (with reduced fertilization failure risk) often receive overnight fertilization [ 12 ]. This study focused on comparing the effects of multiple fertilization strategies—including short-term fertilization (D0 vs. D1), overnight fertilization, ICSI, and Re-ICSI—on oocyte maturity. Our findings indicate that short-term and overnight fertilization differentially impact oocyte maturity. Despite differences in female age and retrieved oocyte number between the two groups (attributed to inclusion criteria), the overnight fertilization group exhibited a significantly higher MII oocyte rate than the short-term fertilization group (P < 0.05). This discrepancy may stem from differences in cumulus cell removal timing: short-term fertilization involves cumulus cell removal 4–6 hours after sperm-oocyte co-culture (D0), while overnight fertilization delays removal until 16–18 hours post-co-culture (D1). Earlier cumulus cell removal in short-term fertilization may impair ooplasmic maturation, reducing the MII oocyte rate and oocyte utilization efficiency [ 13 ]. Cumulus cell co-culture is known to promote in vitro oocyte development and maturation [ 14 ], supporting the use of overnight fertilization for secondary infertility patients (non-male factor) to enhance oocyte utilization [ 11 ]. Consistent with previous studies [ 6 ], our data show that immature oocytes identified in short-term fertilization cycles on D0 may undergo further maturation by D1. For example, MI oocytes observed on D0 can achieve improved maturation, fertilization, and embryo quality when cultured overnight in the original fertilization dish [ 6 ]. Clinical practice may therefore prioritize cumulus cell removal from mature cumulus-oocyte complexes (COCs) during short-term fertilization, while retaining immature COCs in the fertilization dish for overnight culture [ 11 ]. Re-ICSI involves cumulus cell removal and injection 4–5 hours later than conventional ICSI. Theoretically, this delay should enhance oocyte maturity; however, our results showed a slightly lower MII oocyte rate in the Re-ICSI group (82.30% vs. 83.26% in the ICSI group), albeit without statistical significance (P > 0.05). This contrasts with findings by Balli et al. [ 11 ], who reported higher maturity with delayed Re-ICSI injection. The discrepancy may reflect patient selection bias: our Re-ICSI group included only short-term fertilization failure cases, which may have inherently lower oocyte maturity potential, whereas Balli et al.’s study did not restrict patient populations. Notably, the Re-ICSI group had significantly fewer retrieved oocytes and lower oocyte maturity than the short-term fertilization group. Low oocyte maturity—characterized by a high proportion of immature sibling oocytes—can impair fertilization, embryo development, clinical pregnancy, and live birth rates [ 16 ]. This highlights that MII oocyte developmental competence depends on both nuclear and ooplasmic/membrane maturation. The reduced oocyte maturity and retrieval number in the Re-ICSI group may be associated with asynchronous ovulation induction and oocyte development. Patients with low oocyte maturity are less likely to achieve high fertilization rates with short-term fertilization, increasing the need for Re-ICSI [ 2 , 12 ]. Zeng et al. [ 17 ] reported that complete fertilization failure in first-cycle non-male factor short-term fertilization cycles warrants full Re-ICSI, while partial fertilization failure may require partial Re-ICSI. Complete Re-ICSI cycles are often linked to sperm-oocyte binding barriers, prompting recommendations for direct ICSI in subsequent cycles. In contrast, partial Re-ICSI cycles may reflect asynchronous oocyte maturation, with some oocytes failing short-term fertilization but succeeding with overnight fertilization. For such cases, adjusted ovulation induction protocols and overnight fertilization are recommended for the second cycle. Additionally, GnRH-a combined with HCG trigger (double trigger) can improve oocyte maturity and utilization in patients with prior IVF failure or no high-quality embryos [ 2 ], providing valuable guidance for optimizing ovarian stimulation strategies to maximize MII oocyte yield and improve pregnancy outcomes. In summary, different fertilization methods—driven by variations in cumulus cell removal timing, culture duration, and observation time—exert distinct impacts on oocyte maturity. The order of maturity (from highest to lowest) is: overnight fertilization group > short-term fertilization (D1) subgroup > short-term fertilization (D0) subgroup > Re-ICSI group ≤ ICSI group. The study’s inclusion criteria (female age 35 years [ 19 ], and other factors—including obesity, lifestyle, genetics, systemic disease, ovulation induction protocols, laboratory procedures, and culture conditions—may also influence oocyte quality. For example, high salt intake adversely affects oocyte maturation despite not impacting clinical pregnancy outcomes in first ART cycles [ 20 ]. While oocyte number does not directly affect maturity rate, fewer than 5 retrieved oocytes complicates second polar body assessment, justifying the inclusion criterion of > 5 retrieved oocytes. Oocyte morphology and maturity remain key indicators of oocyte quality [ 18 ]. Abnormal oocytes and reduced maturity are common in ICSI cycles [ 21 ], prompting the exclusion of patients with abnormal oocyte morphology (waxy zona pellucida) [ 8 ], IVM, and ICSI fertilization failure [ 4 ] from this study. Patients with endometriosis (EMS) were also excluded, as EMS is associated with reduced ovarian reserve, impaired oocyte/embryo quality, and higher cycle cancellation rates—even in the absence of significant differences in oocyte maturity rates compared to non-EMS patients. This study has several limitations. First, the retrospective design introduces selection bias (e.g., physician preference for fertilization methods may be influenced by unrecorded patient characteristics). Second, confounding factors (e.g., age, retrieved oocyte number) were not adjusted via multivariate logistic regression. Third, inter-observer agreement for oocyte maturity assessment was not evaluated. Fourth, long-term outcomes (e.g., implantation rate, live birth rate) were not analyzed. Finally, no prospective randomized controlled trial (RCT) was conducted, and baseline differences existed among enrolled patients. Future prospective RCTs with larger sample sizes are needed to validate these findings. Conclusion This study compared oocyte maturity across multiple fertilization strategies (short-term fertilization D0 vs. D1, D1 vs. overnight fertilization, ICSI vs. Re-ICSI, D0 vs. Re-ICSI) and analyzed general clinical characteristics, mature/immature oocyte rates, and empty zona pellucida/degeneration rates. The findings clearly demonstrate the impact of fertilization methods on oocyte maturity, providing evidence-based guidance for clinical medication and ovulation induction protocols: Patients with secondary infertility (not due to male factors): It is recommended to use overnight fertilization to increase the MII (mature meiotic division) rate of oocytes; Patients with failed Re-ICSI: In the next cycle, the ovulation induction protocol can be adjusted (such as double triggering [ 2 ]), rather than directly switching to ICSI. Declarations Availability of Data and Materials All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author. Author Contributions XHZ and JCY designed the present study. XLZ and YNC collected raw data. XHZ, GS and YJD checked and analyzed all data. XHZ was a major contributor to manuscript writing.YJ revised the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work. Ethics Approval and Consent to Participate The study was carried out in accordance with the guidelines of the Declaration of Helsinki. A waiver of informed consent was granted for this study because it did not disclose any personal or identifying patient information and posed no risk to the participants. Approval was granted by the Research Ethics Committee of the Fourth Hospital of Shijiazhuang (approval number: 20240022). Acknowledgment We thank all staff of the Center for Reproductive Medicine, the Fourth Hospital of Shijiazhuang of the Fourth Hospital of Shijiazhuang. Funding This work was supported by grants from Medical Science Research Project of Hebei (Grant No.20251111). Conflict of Interest The authors have no conflicts of interest to declare. 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Ueno S, Berntsen J, Ito M, et al. Pregnancy prediction performance of an annotation-free embryo scoring system on the basis of deep learning after single vitrified-warmed blastocyst transfer: a single-center large cohort retrospective study[J]. Fertil Steril. 2021 Oct;116(4):1172-1180. Wang G, Yeung CK, Zhang JL, Hu XW, Ye YX, Yang YX, Li JC, Lee KK, Yang X, Wang LJ. High salt intake negatively impacts ovarian follicle development. Ann Anat. 2015 Jul;200:79-87. Kim TE, Lee HK, Jee BC. Clinical and laboratory factors associated with the presence of dysmorphic oocytes in intracytoplasmic sperm injection cycles[J]. Clin Exp Reprod Med. 2023 Dec;50(4):270-276. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 22 Feb, 2026 Reviewers agreed at journal 19 Feb, 2026 Reviewers invited by journal 17 Feb, 2026 Editor invited by journal 27 Jan, 2026 Editor assigned by journal 26 Jan, 2026 Submission checks completed at journal 26 Jan, 2026 First submitted to journal 23 Jan, 2026 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-8677406","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":595150480,"identity":"dd51ea9e-8d15-45eb-94a5-aa1515219038","order_by":0,"name":"Xuhui Zhang","email":"","orcid":"","institution":"Fourth Hospital of Shijiazhuang","correspondingAuthor":false,"prefix":"","firstName":"Xuhui","middleName":"","lastName":"Zhang","suffix":""},{"id":595150482,"identity":"148669d1-44df-40d0-92f0-8ce951269e48","order_by":1,"name":"Jingchuan Yuan","email":"","orcid":"","institution":"Fourth Hospital of Shijiazhuang","correspondingAuthor":false,"prefix":"","firstName":"Jingchuan","middleName":"","lastName":"Yuan","suffix":""},{"id":595150483,"identity":"1f8a314a-68e4-417c-90cc-fa91aa6a1123","order_by":2,"name":"Xiaoling Zhang","email":"","orcid":"","institution":"Fourth Hospital of Shijiazhuang","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Zhang","suffix":""},{"id":595150485,"identity":"786ef488-37d5-4fdf-8aae-81061dc2a195","order_by":3,"name":"Yinjing Dong","email":"","orcid":"","institution":"Fourth Hospital of Shijiazhuang","correspondingAuthor":false,"prefix":"","firstName":"Yinjing","middleName":"","lastName":"Dong","suffix":""},{"id":595150490,"identity":"95de0778-af7e-4c3b-b0c1-72f6452034fb","order_by":4,"name":"Yaonan Cao","email":"","orcid":"","institution":"Fourth Hospital of Shijiazhuang","correspondingAuthor":false,"prefix":"","firstName":"Yaonan","middleName":"","lastName":"Cao","suffix":""},{"id":595150496,"identity":"3450dbac-063d-4d34-bef4-74fad5c00b5e","order_by":5,"name":"Ge Song","email":"","orcid":"","institution":"Fourth Hospital of Shijiazhuang","correspondingAuthor":false,"prefix":"","firstName":"Ge","middleName":"","lastName":"Song","suffix":""},{"id":595150502,"identity":"6d42f379-7d96-4dc5-b22c-95bb97f5f215","order_by":6,"name":"Yan Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIie3QIQvCQBTA8TsOLp2uvqHoV3hDEJtf5QZiUlhcEN2YbEHFuuZXMBoHg6WzGydWgzaDQc2KN5vhfly8P+/uEWIYf4hbUV5eEGZhsr6W0p/okzoUQyf1ejRaZh0sVaFPWmSEDXHxaZLKrn2cswoPIwpRILBaIIe+G3BiJQv5PWGxVwICt4OsOLi7JgG132qm5FtEBOGEYXxwFScIY10iEZ4H+jnjnhuzKskAIUNAGnNOqiWvJQfPQXQpGEhVCO1f2usoP93vU0k3Z3q9+ZOWlay+J2/Eb9cNwzCMjx5VBElJLGxBcQAAAABJRU5ErkJggg==","orcid":"","institution":"Fourth Hospital of Shijiazhuang","correspondingAuthor":true,"prefix":"","firstName":"Yan","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2026-01-23 09:24:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8677406/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8677406/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103301966,"identity":"f9ce2fe2-cdac-42bc-859a-44fc2ded88eb","added_by":"auto","created_at":"2026-02-24 08:21:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":299412,"visible":true,"origin":"","legend":"\u003cp\u003eResearch Subjects and Grouping Flowchart\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8677406/v1/0585318dfaa17c52c1a3607b.png"},{"id":103506350,"identity":"db0f295e-323c-4d53-a928-44240e789180","added_by":"auto","created_at":"2026-02-26 13:35:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":707627,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8677406/v1/277d2f63-3475-4f5a-bab2-05af56a17eb2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of different fertilization methods on oocyte maturity","fulltext":[{"header":"Introductions","content":"\u003cp\u003eFertilization is a crucial step in assisted reproductive technology (ART) treatment. Conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are two commonly used fertilization methods in ART [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. IVF is further classified into short-term fertilization and overnight fertilization based on the time of cumulus cell removal. Currently, most reproductive centers adopt short-term IVF for primary infertility patients without male factor infertility to avoid IVF fertilization failure and resort to rescue-intracytoplasmic sperm injection (Re-ICSI) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Couples with secondary infertility (those with a history of pregnancy) undergo overnight fertilization. For patients with male factor infertility (poor sperm quantity or quality), ICSI is directly chosen.\u003c/p\u003e \u003cp\u003eOocytes are classified based on their maturity into mature oocytes (metaphase II oocytes, MII oocytes), metaphase I oocytes (MI oocytes), germinal vesicle stage oocytes (GV oocytes), empty zona pellucida oocytes, and degenerated oocytes. The combination of mature oocytes with sperm to form zygotes marks the beginning of embryo development [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. After cumulus cell removal in ICSI fertilization, the MII oocyte rate can be directly used as an indicator to assess oocyte maturity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, there is no fixed standard for assessing oocyte maturity in IVF. On the day of oocyte retrieval (D0) in short-term IVF fertilization, cumulus cell removal can determine oocyte maturity, but after overnight culture on the day of fertilization observation (D1), MI oocytes and GV oocytes further mature, and oocyte maturity may change [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eModifying the methods and strategies in the laboratory fertilization process can improve fertilization outcomes and increase oocyte utilization [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, the impact of fertilization methods on oocyte maturity remains unclear. This study aims to explore the influence of different fertilization methods on oocyte maturity to better guide clinical practice.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eI. Research Subjects and Grouping\u003c/p\u003e\n\u003cp\u003eA retrospective analysis was conducted on patients who underwent IVF/ICSI at the Reproductive Medicine Center of the Fourth Hospital of Shijiazhuang from January 2022 to June 2024. The inclusion criteria were as follows: (1) female age \u0026lt; 35 years; (2) number of retrieved oocytes \u0026gt; 5; (3) normal karyotype in both partners; (4) first cycle of ovulation induction. The exclusion criteria were: patients with endometriosis, oocyte donors, oocyte freezing cycles, oocytes with abnormal morphology (waxy zona pellucida), in vitro matured (IVM) oocytes, and ICSI fertilization failure. Grouping was based on fertilization methods: patients with male partner\u0026apos;s semen parameters meeting conventional IVF criteria underwent short-term fertilization or overnight fertilization; for male factor (severe oligoasthenoteratozoospermia), direct ICSI was performed. For IVF patients, short-term fertilization + Re-ICSI was used for primary infertility (patients without pregnancy history between the couple), and overnight fertilization for secondary infertility (patients with pregnancy history). According to the time of oocyte maturity assessment, it was further divided into the D0 subgroup \u0026nbsp;and D1 subgroup: among them, the short-term fertilization group, ICSI group, and Re-ICSI group were observed after stripping the cumulus cells on D0, while the D1 subgroup and overnight fertilization group were observed on D1. The D0 subgroup and the D1 subgroup were the same group of the patients observed on D0 and D1, respectively. (Figure 1) The D0 subgroup and D1 subgroup, the D1 subgroup and overnight fertilization group, ICSI group and Re-ICSI group, the D0 subgroup and Re-ICSI group were compared to analyze the mature oocyte rate, immature oocyte rate, empty zona pellucida/regression rate, etc. of each group.\u003c/p\u003e\n\u003cp\u003eII. Research Methods\u003c/p\u003e\n\u003cp\u003e1. Controlled Ovarian Hyperstimulation: The center\u0026apos;s routine ovulation induction protocols were used, mainly including the long protocol and the antagonist protocol. After administration of gonadotropins, B-ultrasound was used to monitor the number, size of follicles, and thickness of the\u0026nbsp;endometrium, as well as serum hormone levels, and the dose of gonadotropins was adjusted. When the dominant follicle developed to 18 mm - 20 mm, 5,000 - 10,000 U of human chorionic gonadotropin (HCG, Zhuhai Lize) was administered, and 36 - 38 hours later, oocyte retrieval was performed under\u0026nbsp;vaginal B-ultrasound guidance.\u003c/p\u003e\n\u003cp\u003e2. Semen Optimization Treatment: According to the male partner\u0026apos;s semen condition, optimization treatment was performed before IVF/ICSI fertilization.\u003c/p\u003e\n\u003cp\u003e3. ICSI Fertilization: 39 - 40 hours after HCG injection, the oocytes were treated with 80 U/mL hyaluronidase (H3757, Sigma, USA) for 30 seconds, and followed by cumulus cell removal via aspiration. The oocyte maturity was evaluated, and MII oocytes were directly subjected to ICSI fertilization. The presence of two pronuclei 18 - 20 hours after fertilization was considered as the ICSI fertilization group.\u003c/p\u003e\n\u003cp\u003e4. Short-term Fertilization: 38 - 40 hours after HCG injection, the processed sperm was added to the double-well dish (Falcon, USA) containing oocytes, at a concentration of 150,000\u0026ndash;300,000 sperm/mL of culture medium. Each dish contained about 5 oocytes. After co-culture of sperm and oocytes for 4 - 6 hours, the cumulus cells were removed and the second polar body was observed. If the proportion of oocytes with two polar bodies among mature oocytes was \u0026ge; 30%, it was considered as the short-term fertilization group. The presence of two pronuclei 18 - 20 hours after fertilization was considered as the short-term fertilization (D1) group. If the proportion was \u0026lt; 30%, Re-ICSI was performed, and it was considered as the Re-ICSI group [2, 3].\u003c/p\u003e\n\u003cp\u003e5. Overnight Fertilization: 18 - 20 hours after IVF fertilization, the cumulus cells were removed and fertilization was observed. The presence of two pronuclei was considered as the IVF overnight fertilization group.\u003c/p\u003e\n\u003cp\u003e6. Observation Indicators: Oocyte maturity assessment on D0 was conducted after cumulus cell removal, and on D1 during fertilization observation. On day 0, the rates of MII oocytes, MI oocytes, GV oocytes, and the rate of empty zona pellucida/ovum degeneration in the IVF short-term fertilization group, ICSI group, and Re-ICSI group were calculated as follows: MII oocyte rate = (number of MII oocytes / number of retrieved oocytes) \u0026times; 100%; MI oocyte rate = (number of MI oocytes / number of retrieved oocytes) \u0026times; 100%; GV oocyte rate = (number of GV oocytes / number of retrieved oocytes) \u0026times; 100%; rate of empty zona pellucida/ovum degeneration = [(number of empty zona pellucida + number of degenerated oocytes) / number of retrieved oocytes] \u0026times; 100%. On day 1, the MII oocyte rate in the IVF short-term fertilization (D1) group and overnight fertilization group was calculated as: MII oocyte rate = (number of fertilized oocytes + number of MII oocytes) / number of retrieved oocytes \u0026times; 100%; the rest were the same as above.\u003c/p\u003e\n\u003cp\u003eIII. Statistical Analysis\u003c/p\u003e\n\u003cp\u003eThe analysis was conducted using SPSS 27.0 statistical software. Continuous data with a normal distribution were expressed as mean \u0026plusmn; standard deviation (x̄ \u0026plusmn; s), and comparisons among multiple groups were performed using one-way ANOVA and a Bonferroni correction was applied. Categorical data were expressed as n (%), and the comparison of rates between groups was conducted using the \u0026chi;\u0026sup2; test. \u003cem\u003eP\u003c/em\u003e value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e\n\u003cp\u003eFigure 1. Research Subjects and Grouping Flowchart\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 2,553 IVF/ICSI cycles were included in this study, including 1,111 short-term fertilization cycles, 619 overnight fertilization cycles, 584 ICSI cycles, and 239 Re-ICSI cycles. the short-term fertilization group was further divided into the D0 subgroup and D1 subgroup based on different observation times.\u003c/p\u003e\n\u003cp\u003e1. Comparison of general conditions among groups\u003c/p\u003e\n\u003cp\u003eThe age of women in the overnight fertilization group was significantly higher than that in the short-term fertilization group (30.32\u0026plusmn;2.92\u0026nbsp;vs.\u0026nbsp;29.28\u0026plusmn;2.97), and the number of retrieved oocytes was significantly lower (12.08 \u0026plusmn; 6.26 vs. 13.46 \u0026plusmn; 6.78). The number of retrieved oocytes in the Re-ICSI group was significantly lower than that in the short-term fertilization group (12.36 \u0026plusmn; 6.14 vs. 13.46 \u0026plusmn; 6.78), with statistically significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). There were no statistically significant differences in the age of men, duration of infertility, and BMI (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) (Table 1).\u003c/p\u003e\n\u003cp\u003e2. Comparison between the D0 subgroup and the D1 subgroup\u003c/p\u003e\n\u003cp\u003eCompared with the D0 subgroup, the D1 subgroup had a significantly higher MII oocyte rate (88.12% vs. 83.89%, \u0026chi;2 value = 30.480, \u003cem\u003eP\u003c/em\u003e = 0.000), a significantly higher MI oocyte rate (7.37% vs. 6.69%, \u0026chi;2 value = 5.217, \u003cem\u003eP\u003c/em\u003e = 0.022), and a significantly lower GV oocyte rate (4.22% vs. 9.14%, \u0026chi;2 value = 290.522,\u003cem\u003e\u0026nbsp;P\u003c/em\u003e = 0.000), with statistically significant differences. There was no statistically significant difference in the rate of degenerated/empty zona pellucida (0.29% vs. 0.27%, \u0026chi;2 value = 0.106, \u003cem\u003eP\u003c/em\u003e = 0.745) (Table 2).\u003c/p\u003e\n\u003cp\u003e3. Comparison between the D1 subgroup and overnight fertilization group\u003c/p\u003e\n\u003cp\u003eThe MII oocyte rate in the overnight fertilization group was significantly higher than that in the D1 subgroup (90.44% vs. 88.12%, \u0026chi;2 value = 27.178, \u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.000), and the GV oocyte rate was significantly lower (2.70% vs. 4.22%, \u0026chi;2 value = 32.122, \u003cem\u003eP\u003c/em\u003e = 0.000), with statistically significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). There were no statistically significant differences in the MI oocyte rate and the rate of degenerated/empty zona pellucida between the two groups (\u0026chi;2 value = 2.849, \u003cem\u003eP\u003c/em\u003e = 0.091) (Table 3).\u003c/p\u003e\n\u003cp\u003e4. Comparison of oocytes between ICSI group and Re-ICSI group\u003c/p\u003e\n\u003cp\u003eThere were no statistically significant differences in the number of retrieved oocytes, MII oocyte rate (83.26% vs. 82.30%, \u0026chi;2 value = 1.364, \u003cem\u003eP\u003c/em\u003e = 0.243), MI oocyte rate (6.93% vs. 7.78%, \u0026chi;\u003csup\u003e2\u003c/sup\u003e value = 2.296, \u003cem\u003eP\u003c/em\u003e = 0.130), GV oocyte rate (9.53% vs. 9.61%, \u0026chi;2 value = 0.015, \u003cem\u003eP\u003c/em\u003e = 0.901), and the rate of degenerated/empty zona pellucida (0.28% vs. 0.30%, \u0026chi;\u003csup\u003e2\u003c/sup\u003e value = 0.044,\u003cem\u003e\u0026nbsp;P\u003c/em\u003e = 0.833) between the ICSI group and the Re-ICSI group (Table 4).\u003c/p\u003e\n\u003cp\u003e5. Comparison between short-term fertilization (D0) group and Re-ICSI group\u003c/p\u003e\n\u003cp\u003eCompared with the D0 subgroup, the Re-ICSI group had a significantly lower MII oocyte rate (82.30% vs. 83.89%, \u0026chi;2 value = 4.578, \u003cem\u003eP\u003c/em\u003e = 0.032) and a significantly higher MI oocyte rate (7.78% vs. 6.69%, \u0026chi;2 value = 4.592, \u003cem\u003eP\u003c/em\u003e = 0.032), with statistically significant differences. The GV oocyte rate (9.53% vs. 9.61%, \u0026chi;2 value = 0.657, \u003cem\u003eP\u003c/em\u003e = 0.418) and the degenerated/empty zona pellucida rate (0.28% vs. 0.30%, \u0026chi;2 value = 0.082, \u003cem\u003eP\u003c/em\u003e = 0.774) showed no statistically significant differences (Table 5).\u003c/p\u003e\n\u003cp\u003eTable 1 Comparison of General Conditions among Different Groups \u0026nbsp;(\u003cimg width=\"13\" height=\"19\" src=\"data:image/wmf;base64,R0lGODlhFAAdAHcAMSH+GlNvZnR3YXJlOiBNaWNyb3NvZnQgT2ZmaWNlACH5BAEAAAAALAMABgAPABAAhAAAAAAAAB0AAAAdMgAAMwAcSAAzWh0zWh1GbDQdHTMAADIdADMzSDNdXTVIWzNGRjNbgEgcAEgdHVozHVozAEg1W11dRl1/f0huf1lubmxGHX9/XX9uSGaIiIBbM4iIZgVBICAGZGmKaKqubOu2yaAGqlIaKFmwAZEuLRug8gJQArIigKZc+pSRAOQlGeFUE0AGgABwBLsV6QoQMlKawGGmCgEAOw==\" alt=\"image\"\u003e\u0026plusmn;s)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"586\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003eShort-term fertilization group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003eOvernight fertilization group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003eICSI group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003eRe-ICSI group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eNumber of patients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e1111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e584\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e239\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eFemale age \u0026nbsp;\u0026nbsp;（\u003cimg width=\"13\" height=\"19\" src=\"data:image/wmf;base64,R0lGODlhFAAdAHcAMSH+GlNvZnR3YXJlOiBNaWNyb3NvZnQgT2ZmaWNlACH5BAEAAAAALAMABgAPABAAhAAAAAAAAB0AAAAdMgAAMwAcSAAzWh0zWh1GbDQdHTMAADIdADMzSDNdXTVIWzNGRjNbgEgcAEgdHVozHVozAEg1W11dRl1/f0huf1lubmxGHX9/XX9uSGaIiIBbM4iIZgVBICAGZGmKaKqubOu2yaAGqlIaKFmwAZEuLRug8gJQArIigKZc+pSRAOQlGeFUE0AGgABwBLsV6QoQMlKawGGmCgEAOw==\" alt=\"image\"\u003e\u0026plusmn;\u003cem\u003es\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e29.28\u0026plusmn;2.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e30.32\u0026plusmn;2.92\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e29.37\u0026plusmn;3.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e29.87\u0026plusmn;2.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e3.554\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eDuration of infertility（\u003cimg width=\"13\" height=\"19\" src=\"data:image/wmf;base64,R0lGODlhFAAdAHcAMSH+GlNvZnR3YXJlOiBNaWNyb3NvZnQgT2ZmaWNlACH5BAEAAAAALAMABgAPABAAhAAAAAAAAB0AAAAdMgAAMwAcSAAzWh0zWh1GbDQdHTMAADIdADMzSDNdXTVIWzNGRjNbgEgcAEgdHVozHVozAEg1W11dRl1/f0huf1lubmxGHX9/XX9uSGaIiIBbM4iIZgVBICAGZGmKaKqubOu2yaAGqlIaKFmwAZEuLRug8gJQArIigKZc+pSRAOQlGeFUE0AGgABwBLsV6QoQMlKawGGmCgEAOw==\" alt=\"image\"\u003e\u0026plusmn;\u003cem\u003es\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e3.27\u0026plusmn;2.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e3.28\u0026plusmn;2.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e3.24\u0026plusmn;2.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e3.56\u0026plusmn;2.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e2.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e0.096\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eBMI\u003c/p\u003e\n \u003cp\u003e（\u003cimg width=\"13\" height=\"19\" src=\"data:image/wmf;base64,R0lGODlhFAAdAHcAMSH+GlNvZnR3YXJlOiBNaWNyb3NvZnQgT2ZmaWNlACH5BAEAAAAALAMABgAPABAAhAAAAAAAAB0AAAAdMgAAMwAcSAAzWh0zWh1GbDQdHTMAADIdADMzSDNdXTVIWzNGRjNbgEgcAEgdHVozHVozAEg1W11dRl1/f0huf1lubmxGHX9/XX9uSGaIiIBbM4iIZgVBICAGZGmKaKqubOu2yaAGqlIaKFmwAZEuLRug8gJQArIigKZc+pSRAOQlGeFUE0AGgABwBLsV6QoQMlKawGGmCgEAOw==\" alt=\"image\"\u003e\u0026plusmn;\u003cem\u003es\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e23.76\u0026plusmn;3.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e23.54\u0026plusmn;3.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e23.32\u0026plusmn;3.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e23.95\u0026plusmn;4.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e1.149\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e0.328\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eNumber of retrieved oocytes（\u003cimg width=\"13\" height=\"19\" src=\"data:image/wmf;base64,R0lGODlhFAAdAHcAMSH+GlNvZnR3YXJlOiBNaWNyb3NvZnQgT2ZmaWNlACH5BAEAAAAALAMABgAPABAAhAAAAAAAAB0AAAAdMgAAMwAcSAAzWh0zWh1GbDQdHTMAADIdADMzSDNdXTVIWzNGRjNbgEgcAEgdHVozHVozAEg1W11dRl1/f0huf1lubmxGHX9/XX9uSGaIiIBbM4iIZgVBICAGZGmKaKqubOu2yaAGqlIaKFmwAZEuLRug8gJQArIigKZc+pSRAOQlGeFUE0AGgABwBLsV6QoQMlKawGGmCgEAOw==\" alt=\"image\"\u003e\u0026plusmn;\u003cem\u003es\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e13.46\u0026plusmn;6.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e12.08\u0026plusmn;6.26\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e12.23\u0026plusmn;5.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e12.36\u0026plusmn;6.14\u003csup\u003e※\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e8.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e＜0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: Compared with the overnight fertilization group, the short-term fertilization group showed a significant difference (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05); compared with the Re-ICSI group, the short-term fertilization group also demonstrated a significant difference (\u003csup\u003e※\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003eTable 2 Comparison of Oocyte Maturation Status in\u0026nbsp;D0 subgroup and D1 subgroup [% (n)]\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"595\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eD0 subgroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eD1 subgroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026chi;2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eMII oocyte rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e83.89\u003c/p\u003e\n \u003cp\u003e(12549/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e88.12\u003c/p\u003e\n \u003cp\u003e(13181/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e30.480\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eMI oocyte rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e6.69\u003c/p\u003e\n \u003cp\u003e(1001/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e7.37\u003c/p\u003e\n \u003cp\u003e(1102/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e5.217\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eGV oocyte rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e9.14\u003c/p\u003e\n \u003cp\u003e(1367/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e4.22\u003c/p\u003e\n \u003cp\u003e(631/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e290.522\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eDegeneration/empty zona pellucida rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003cp\u003e(41/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e0.29\u003c/p\u003e\n \u003cp\u003e(44/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.106\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.745\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eTable 3 Comparison of Oocyte Maturation Status in D1 subgroup and\u0026nbsp;Overnight fertilization group\u0026nbsp;[% (n)]\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"595\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eD1 subgroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eOvernight fertilization group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026chi;2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eMII oocyte rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e88.12\u003c/p\u003e\n \u003cp\u003e(13181/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e90.44\u003c/p\u003e\n \u003cp\u003e(6764/7479)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e27.178\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eMI oocyte rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e7.37\u003c/p\u003e\n \u003cp\u003e(1102/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e6.69\u003c/p\u003e\n \u003cp\u003e(500/7479)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e3.497\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.061\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eGV oocyte rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e4.22\u003c/p\u003e\n \u003cp\u003e(631/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e2.70\u003c/p\u003e\n \u003cp\u003e(202/7479)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e32.122\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eDegeneration/empty zona pellucida rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e0.29\u003c/p\u003e\n \u003cp\u003e(44/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003cp\u003e(13/7479)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e2.829\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.093\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eTable 4 Comparison of Oocyte Maturation Status in\u0026nbsp;ICSI group and Re-ICSI group\u0026nbsp;[% (n)]\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"595\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eICSI group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eRe-ICSI group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026chi;2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eMII oocyte rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e83.26\u003c/p\u003e\n \u003cp\u003e(5949/7145)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e82.30\u003c/p\u003e\n \u003cp\u003e(2432/2955)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e1.364\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.243\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eMI oocyte rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e6.93\u003c/p\u003e\n \u003cp\u003e(495/7145)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e7.78\u003c/p\u003e\n \u003cp\u003e(230/2955)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e2.296\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.130\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eGV oocyte rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e9.53\u003c/p\u003e\n \u003cp\u003e(681/7145)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e9.61\u003c/p\u003e\n \u003cp\u003e(284/2955)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.901\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eDegeneration/empty zona pellucida rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003cp\u003e(20/7145)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003cp\u003e(9/2955)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.833\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 5 Comparison of Oocyte Maturation Status in D0 subgroup and\u0026nbsp;Re-ICSI group\u0026nbsp;[% (n)]\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"595\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eD0 subgroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eRe-ICSI group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026chi;2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eMII oocyte rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e83.89\u003c/p\u003e\n \u003cp\u003e(12549/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e82.30\u003c/p\u003e\n \u003cp\u003e(2432/2955)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e4.578\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.032\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eMI oocyte rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e6.69\u003c/p\u003e\n \u003cp\u003e(1001/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e7.78\u003c/p\u003e\n \u003cp\u003e(230/2955)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e4.592\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.032\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eGV oocyte rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e9.14\u003c/p\u003e\n \u003cp\u003e(1367/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e9.61\u003c/p\u003e\n \u003cp\u003e(284/2955)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.657\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.418\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eDegeneration/empty zona pellucida rate\u003c/p\u003e\n \u003cp\u003e%（\u003cem\u003en\u003c/em\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003cp\u003e(41/14958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003cp\u003e(9/2955)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.082\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.774\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussions","content":"\u003cp\u003eGamete quality and maturity assessment are critical for ART success, as oocyte maturation directly influences fertilization and subsequent embryo development [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. MII oocytes\u0026mdash;compared to immature counterparts\u0026mdash;exhibit significantly higher fertilization potential, making maturity assessment a key determinant of fertilization timing [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExpert consensus recommends short-term or overnight fertilization for patients with normal sperm parameters [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Primary infertility patients face a higher risk of fertilization failure and thus typically undergo short-term fertilization, while secondary infertility patients (with reduced fertilization failure risk) often receive overnight fertilization [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This study focused on comparing the effects of multiple fertilization strategies\u0026mdash;including short-term fertilization (D0 vs. D1), overnight fertilization, ICSI, and Re-ICSI\u0026mdash;on oocyte maturity.\u003c/p\u003e \u003cp\u003eOur findings indicate that short-term and overnight fertilization differentially impact oocyte maturity. Despite differences in female age and retrieved oocyte number between the two groups (attributed to inclusion criteria), the overnight fertilization group exhibited a significantly higher MII oocyte rate than the short-term fertilization group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This discrepancy may stem from differences in cumulus cell removal timing: short-term fertilization involves cumulus cell removal 4\u0026ndash;6 hours after sperm-oocyte co-culture (D0), while overnight fertilization delays removal until 16\u0026ndash;18 hours post-co-culture (D1). Earlier cumulus cell removal in short-term fertilization may impair ooplasmic maturation, reducing the MII oocyte rate and oocyte utilization efficiency [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Cumulus cell co-culture is known to promote in vitro oocyte development and maturation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], supporting the use of overnight fertilization for secondary infertility patients (non-male factor) to enhance oocyte utilization [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsistent with previous studies [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], our data show that immature oocytes identified in short-term fertilization cycles on D0 may undergo further maturation by D1. For example, MI oocytes observed on D0 can achieve improved maturation, fertilization, and embryo quality when cultured overnight in the original fertilization dish [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Clinical practice may therefore prioritize cumulus cell removal from mature cumulus-oocyte complexes (COCs) during short-term fertilization, while retaining immature COCs in the fertilization dish for overnight culture [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRe-ICSI involves cumulus cell removal and injection 4\u0026ndash;5 hours later than conventional ICSI. Theoretically, this delay should enhance oocyte maturity; however, our results showed a slightly lower MII oocyte rate in the Re-ICSI group (82.30% vs. 83.26% in the ICSI group), albeit without statistical significance (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). This contrasts with findings by Balli et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], who reported higher maturity with delayed Re-ICSI injection. The discrepancy may reflect patient selection bias: our Re-ICSI group included only short-term fertilization failure cases, which may have inherently lower oocyte maturity potential, whereas Balli et al.\u0026rsquo;s study did not restrict patient populations.\u003c/p\u003e \u003cp\u003eNotably, the Re-ICSI group had significantly fewer retrieved oocytes and lower oocyte maturity than the short-term fertilization group. Low oocyte maturity\u0026mdash;characterized by a high proportion of immature sibling oocytes\u0026mdash;can impair fertilization, embryo development, clinical pregnancy, and live birth rates [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This highlights that MII oocyte developmental competence depends on both nuclear and ooplasmic/membrane maturation. The reduced oocyte maturity and retrieval number in the Re-ICSI group may be associated with asynchronous ovulation induction and oocyte development. Patients with low oocyte maturity are less likely to achieve high fertilization rates with short-term fertilization, increasing the need for Re-ICSI [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eZeng et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] reported that complete fertilization failure in first-cycle non-male factor short-term fertilization cycles warrants full Re-ICSI, while partial fertilization failure may require partial Re-ICSI. Complete Re-ICSI cycles are often linked to sperm-oocyte binding barriers, prompting recommendations for direct ICSI in subsequent cycles. In contrast, partial Re-ICSI cycles may reflect asynchronous oocyte maturation, with some oocytes failing short-term fertilization but succeeding with overnight fertilization. For such cases, adjusted ovulation induction protocols and overnight fertilization are recommended for the second cycle. Additionally, GnRH-a combined with HCG trigger (double trigger) can improve oocyte maturity and utilization in patients with prior IVF failure or no high-quality embryos [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], providing valuable guidance for optimizing ovarian stimulation strategies to maximize MII oocyte yield and improve pregnancy outcomes.\u003c/p\u003e \u003cp\u003eIn summary, different fertilization methods\u0026mdash;driven by variations in cumulus cell removal timing, culture duration, and observation time\u0026mdash;exert distinct impacts on oocyte maturity. The order of maturity (from highest to lowest) is: overnight fertilization group\u0026thinsp;\u0026gt;\u0026thinsp;short-term fertilization (D1) subgroup\u0026thinsp;\u0026gt;\u0026thinsp;short-term fertilization (D0) subgroup\u0026thinsp;\u0026gt;\u0026thinsp;Re-ICSI group\u0026thinsp;\u0026le;\u0026thinsp;ICSI group.\u003c/p\u003e \u003cp\u003eThe study\u0026rsquo;s inclusion criteria (female age\u0026thinsp;\u0026lt;\u0026thinsp;35 years) were chosen to minimize confounding by advanced maternal age, a well-established risk factor for poor oocyte quality [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Ovarian function declines with age\u0026thinsp;\u0026gt;\u0026thinsp;35 years [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and other factors\u0026mdash;including obesity, lifestyle, genetics, systemic disease, ovulation induction protocols, laboratory procedures, and culture conditions\u0026mdash;may also influence oocyte quality. For example, high salt intake adversely affects oocyte maturation despite not impacting clinical pregnancy outcomes in first ART cycles [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. While oocyte number does not directly affect maturity rate, fewer than 5 retrieved oocytes complicates second polar body assessment, justifying the inclusion criterion of \u0026gt;\u0026thinsp;5 retrieved oocytes.\u003c/p\u003e \u003cp\u003eOocyte morphology and maturity remain key indicators of oocyte quality [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Abnormal oocytes and reduced maturity are common in ICSI cycles [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], prompting the exclusion of patients with abnormal oocyte morphology (waxy zona pellucida) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], IVM, and ICSI fertilization failure [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] from this study. Patients with endometriosis (EMS) were also excluded, as EMS is associated with reduced ovarian reserve, impaired oocyte/embryo quality, and higher cycle cancellation rates\u0026mdash;even in the absence of significant differences in oocyte maturity rates compared to non-EMS patients.\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, the retrospective design introduces selection bias (e.g., physician preference for fertilization methods may be influenced by unrecorded patient characteristics). Second, confounding factors (e.g., age, retrieved oocyte number) were not adjusted via multivariate logistic regression. Third, inter-observer agreement for oocyte maturity assessment was not evaluated. Fourth, long-term outcomes (e.g., implantation rate, live birth rate) were not analyzed. Finally, no prospective randomized controlled trial (RCT) was conducted, and baseline differences existed among enrolled patients. Future prospective RCTs with larger sample sizes are needed to validate these findings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study compared oocyte maturity across multiple fertilization strategies (short-term fertilization D0 vs. D1, D1 vs. overnight fertilization, ICSI vs. Re-ICSI, D0 vs. Re-ICSI) and analyzed general clinical characteristics, mature/immature oocyte rates, and empty zona pellucida/degeneration rates. The findings clearly demonstrate the impact of fertilization methods on oocyte maturity, providing evidence-based guidance for clinical medication and ovulation induction protocols: Patients with secondary infertility (not due to male factors): It is recommended to use overnight fertilization to increase the MII (mature meiotic division) rate of oocytes; Patients with failed Re-ICSI: In the next cycle, the ovulation induction protocol can be adjusted (such as double triggering [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]), rather than directly switching to ICSI.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAvailability of Data and Materials\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eXHZ and JCY\u0026nbsp;designed the present study.\u0026nbsp;XLZ and YNC\u0026nbsp;collected raw data.\u0026nbsp;XHZ, GS\u0026nbsp;and\u0026nbsp;YJD checked and analyzed all data.\u0026nbsp;XHZ was a major contributor to manuscript writing.YJ\u0026nbsp;revised the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthics Approval and Consent to Participate\u003c/p\u003e\n\u003cp\u003eThe study was carried out in accordance with the guidelines of the Declaration of Helsinki. A waiver of informed consent was granted for this study because it did not disclose any personal or identifying patient information and posed no risk to the participants. Approval was granted by the Research Ethics Committee of the Fourth Hospital of Shijiazhuang (approval number: 20240022).\u003c/p\u003e\n\u003cp\u003eAcknowledgment\u003c/p\u003e\n\u003cp\u003eWe thank all staff of the Center for Reproductive Medicine, the Fourth Hospital of Shijiazhuang\u0026nbsp;of the Fourth\u0026nbsp;Hospital\u0026nbsp;of Shijiazhuang.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from Medical Science Research Project of Hebei (Grant No.20251111).\u003c/p\u003e\n\u003cp\u003eConflict of Interest\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSauerbrun-Cutler MT, Huber WJ 3rd, Has P, Shen C, Hackett R, Alvero R, Wang S. Is intracytoplasmic sperm (ICSI) better than traditional in vitro fertilization (IVF): confirmation of higher blastocyst rates per oocyte using a split insemination design. J Assist Reprod Genet. 2020 Jul;37(7):1661-1667. \u003c/li\u003e\n\u003cli\u003eZhang XD, Liu JX, Liu WW, Gao Y, Han W, Xiong S, Wu LH, Huang GN. Time of insemination culture and outcomes of in vitro fertilization: a systematic review and meta-analysis. Hum Reprod Update. 2013 Nov-Dec;19(6):685-95. \u003c/li\u003e\n\u003cli\u003eJiang Y, Yuan JC, Song G, et al. Comparing the pregnancy outcomes of Re‑ICSI and ICSI embryos in fresh ET and FET cycles[J]. Biomed Rep. 2023 Aug 9;19(4):66. \u003c/li\u003e\n\u003cli\u003eBalli M, Cecchele A, Pisaturo V, Makieva S, Carullo G, Somigliana E, Paffoni A, Vigano\u0026apos; P. Opportunities and Limits of Conventional IVF versus ICSI: It Is Time to Come off the Fence. J Clin Med. 2022 Sep 27;11(19):5722.\u003c/li\u003e\n\u003cli\u003eGlenn TL, Kotlyar AM, Seifer DB. The Impact of Intracytoplasmic Sperm Injection in Non-Male Factor Infertility-A Critical Review. J Clin Med. 2021 Jun 14;10(12):2616. \u003c/li\u003e\n\u003cli\u003eCarles M, Lefranc E, Bosquet D, Capelle S, Scheffler F, Copin H, Cabry R, Benkhalifa M. In vitro maturation of oocytes from stimulated IVF-ICSI cycles using autologous cumulus cell co-culture: A preliminary study. Morphologie. 2023 Mar;107(356):28-37. \u003c/li\u003e\n\u003cli\u003eAvci B, Kasapoglu I, Cakir C, Ozbay A, Ata B, Uncu G. Fertilisation and early embryonic development of immature and rescue in vitro-matured sibling oocytes. Hum Fertil (Camb). 2022 Feb;25(1):107-116.\u003c/li\u003e\n\u003cli\u003eJiang Y, Yuan JC, Song G, Zhang XH, Miao SB, Wu XH. Comparing the pregnancy outcomes of Re‑ICSI and ICSI embryos in fresh ET and FET cycles. Biomed Rep. 2023 Aug 9;19(4):66. \u003c/li\u003e\n\u003cli\u003eSciorio R, Tramontano L, Greco PF, et al. Morphological assessment of oocyte quality during assisted reproductive technology cycle[J]. JBRA Assist Reprod. 2024 Aug 26;28(3):511-520. \u003c/li\u003e\n\u003cli\u003eAnagnostopoulou C, Maldonado Rosas I, Singh N, et al. Oocyte quality and embryo selection strategies: a review for the embryologists, by the embryologists[J]. PANMINERVA MED. 2022 Jun;64(2):171-184. \u003c/li\u003e\n\u003cli\u003eBalli M, Cecchele A, Pisaturo V, Makieva S, Carullo G, Somigliana E, Paffoni A, Vigano\u0026apos; P. Opportunities and Limits of Conventional IVF versus ICSI: It Is Time to Come off the Fence. J Clin Med. 2022 Sep 27;11(19):5722. \u003c/li\u003e\n\u003cli\u003ePractice Committee of the American Society for Reproductive Medicine. Practice Committee of the American Society for Reproductive Medicine. Evidence-based treatments for couples with unexplained infertility: a guideline. Fertil Steril. 2020 Feb;113(2):305-322.\u003c/li\u003e\n\u003cli\u003eKusuma AC, Oktari N, Mihardja H, Srilestari A, Simadibrata CL, Hestiantoro A, Wiweko B, Muna N. Electroacupuncture Enhances Number of Mature Oocytes and Fertility Rates for In Vitro Fertilization. Med Acupunct. 2019 Oct 1;31(5):289-297.\u003c/li\u003e\n\u003cli\u003eYılmaz N, \u0026Ouml;zyer Ş, Taş D, et al. Fertilization and early embryonic development of in vitro matured metaphase I oocytes in patients with unexpected low oocyte maturity rate. ZYGOTE. 2022 Jun;30(3):319-323. \u003c/li\u003e\n\u003cli\u003eBartolacci A, Intra G, Coticchio G, dell\u0026apos;Aquila M, Patria G, Borini A. Does morphological assessment predict oocyte developmental competence? A systematic review and proposed score. J Assist Reprod Genet. 2022 Jan;39(1):3-17. \u003c/li\u003e\n\u003cli\u003eCapper E, Krohn M, Summers K, et al. Low oocyte maturity ratio is associated with a reduced in vitro fertilization and intracytoplasmic sperm injection live birth rate. FERTIL STERIL. 2022 Oct;118(4):680-687.\u003c/li\u003e\n\u003cli\u003eZeng J, Yao Z, Zhang Y, et al. Fertilization and neonatal outcomes after early rescue intracytoplasmic sperm injection: a retrospective analysis of 16,769 patients[J]. Arch Gynecol Obstet,2022,306:249-258. \u003c/li\u003e\n\u003cli\u003eMinasi MG, Anagnostopoulou C, Boitrelle F, et al. Oocytes evaluation and in-vitro fertilization/intra cytoplasmic sperm injection outcomes[J]. Panminerva Med. 2023 Jun;65(2):179-187. \u003c/li\u003e\n\u003cli\u003eUeno S, Berntsen J, Ito M, et al. Pregnancy prediction performance of an annotation-free embryo scoring system on the basis of deep learning after single vitrified-warmed blastocyst transfer: a single-center large cohort retrospective study[J]. Fertil Steril. 2021 Oct;116(4):1172-1180.\u003c/li\u003e\n\u003cli\u003eWang G, Yeung CK, Zhang JL, Hu XW, Ye YX, Yang YX, Li JC, Lee KK, Yang X, Wang LJ. High salt intake negatively impacts ovarian follicle development. Ann Anat. 2015 Jul;200:79-87.\u003c/li\u003e\n\u003cli\u003eKim TE, Lee HK, Jee BC. Clinical and laboratory factors associated with the presence of dysmorphic oocytes in intracytoplasmic sperm injection cycles[J]. Clin Exp Reprod Med. 2023 Dec;50(4):270-276.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-womens-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmwh","sideBox":"Learn more about [BMC Women's Health](http://bmcwomenshealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmwh/default.aspx","title":"BMC Women's Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Fertilization method, Oocytes maturity, Short-term fertilization, Overnight fertilization, ICSI, Re-ICSI","lastPublishedDoi":"10.21203/rs.3.rs-8677406/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8677406/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eTo investigate the effects of different fertilization methods on oocyte maturity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eA retrospective analysis was conducted on patients who underwent in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) at the Reproductive Medicine Center of The Fourth Hospital of Shijiazhuang between January 2022 and June 2024. A total of 2553 cycles were included and categorized into four groups based on medical history and fertilization strategy: Group I (1111 cycles): Short-term fertilization for patients with primary infertility (no prior pregnancy between the couple); Group II (619 cycles): Overnight fertilization for patients with secondary infertility (prior pregnancy history); Group III (584 cycles): ICSI for patients with male factor infertility; Group IV (239 cycles): Short-term fertilization combined with early rescue ICSI (Re-ICSI). The short-term fertilization group was further subdivided by observation timing: the D0 subgroup (assessed on the day of oocyte retrieval) and the D1 subgroup (assessed on the day of fertilization observation). Comparisons were performed between the D0 and D1 subgroups, D1 subgroup and overnight fertilization group, ICSI and Re-ICSI groups, and D0 subgroup and Re-ICSI group, focusing on general clinical data and oocyte maturation parameters.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The D1 subgroup exhibited significantly higher metaphase II (MII) and metaphase I (MI) oocyte rates, and a significantly lower germinal vesicle (GV) oocyte rate compared to the D0 subgroup (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). The overnight fertilization group had a significantly higher female age and lower number of retrieved oocytes than the short-term fertilization group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05), alongside a significantly higher MII oocyte rate and lower GV oocyte rate than the D1 subgroup (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). The Re-ICSI group showed significantly fewer retrieved oocytes and lower oocyte maturity than the D0 subgroup (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). No significant differences in oocyte maturity were observed between the ICSI and Re-ICSI groups (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Different fertilization methods exert distinct impacts on oocyte maturity, with overnight fertilization yielding the highest maturity, followed by short-term fertilization, and Re-ICSI yielding the lowest. This variation may be associated with asynchronous oocyte development.\u003c/p\u003e","manuscriptTitle":"Effects of different fertilization methods on oocyte maturity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 08:21:18","doi":"10.21203/rs.3.rs-8677406/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-02-22T14:33:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"215561624250176280951628181281582392559","date":"2026-02-19T14:18:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-17T13:13:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-27T10:57:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-26T06:46:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-26T06:45:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Women's Health","date":"2026-01-23T09:09:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-womens-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmwh","sideBox":"Learn more about [BMC Women's Health](http://bmcwomenshealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmwh/default.aspx","title":"BMC Women's Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4a8a67b5-b158-4023-8061-6b76b8a748dc","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-24T08:21:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 08:21:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8677406","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8677406","identity":"rs-8677406","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}