Efficacy of assisted oocyte activation in improving the reproductive outcome in conventional IVF cases with recurrent developmental problems

Efficacy of assisted oocyte activation in improving the reproductive outcome in conventional IVF cases with recurrent developmental problems | 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 Efficacy of assisted oocyte activation in improving the reproductive outcome in conventional IVF cases with recurrent developmental problems Mingzhao Li, Sen Qiao, Xia Xue, Juanzi Shi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6458796/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background Assisted oocyte activation (AOA) is employed to enhance fertilization rates following fertilization failure after intracytoplasmic sperm injection (ICSI). Several studies have also demonstrated that AOA may play a role in embryo development. Poor embryo quality remains one of the major challenges for patients with recurrent developmental problems. We aimed to investigate whether ICSI followed by assisted oocyte activation (ICSI-AOA) can improve embryo quality in conventional in vitro fertilization (C-IVF) cases with recurrent developmental problems. Methods This retrospective cohort single-center study compared ICSI-AOA cycles with previous C-IVF cycles in couples who had nearly normal fertilization rates (≥ 40%) but impaired embryonic development (at most one D3 good quality embryo for all previous cycles) in at least two prior IVF cycles. A total of 24 couples experiencing embryo developmental issues were included in this study from January 2019 to December 2022. Results After ICSI-AOA, the D3 good quality embryo rate was significantly improved compared with previous C-IVF cycles (19.30 versus 4.04%; p < 0.001). We observed that the mean number of D3 embryo blastomere was significantly increased after ICSI-AOA compared with previous C-IVF cycles (7.85 versus 6.94; p = 0.002). No significant difference was observed in the D3 embryo fragmentation rate between previous C-IVF and subsequent ICSI-AOA cycles (14% versus 13%, p = 0.088). The ICSI-AOA cycles showed significantly higher pregnancy (41.67 versus 19.23%, p = 0.039) and live birth (29.17 versus 3.85%, p = 0.002) rates compared with previous C-IVF treatment. Conclusion Our data suggest that ICSI-AOA may be beneficial to some patients with recurrent developmental problems in C-IVF treatment. Assisted oocyte activation IVF ICSI Embryo quality Clinical outcomes Background Oocyte activation is a vital step in fertilization, involving a series of molecular events essential for successful egg fertilization [ 1 ]. A key player in this process is calcium signaling, which plays a crucial role in initiating and regulating the complex mechanisms required for oocyte activation. In the past two decades, the combination of intracytoplasmic sperm injection (ICSI) with assisted oocyte activation (ICSI-AOA) has become increasingly popular as a therapeutic approach for patients with total fertilization failure (TFF), low fertilization (LF), and sperm-specific oocyte activation failure [ 2 , 3 ]. Calcium signaling is closely linked to embryonic development, playing a critical role in regulating early cell divisions and the cell cycle during embryogenesis [ 4 ]. Disruptions in calcium signaling can lead to abnormalities in cell cleavage and subsequent developmental issues [ 5 , 6 ]. Currently, the AOA techniques, such as those utilizing calcium rises, are primarily applied in cases of LF or TFF. Given the strong correlation between calcium availability and cell division, we believe exploring ICSI-AOA as a potential solution for recurrent embryo development issues is highly valuable. By improving the activation process, ICSI-AOA could enhance embryo quality and potentially improve the outcomes of some embryos. For patients with poor embryo quality problems at least two initiated in vitro fertilization (IVF) cycles, they probably will not obtain good quality embryos in the incoming conventional IVF (C-IVF) cycle. So far, there is no effective way to improve embryo quality for such patients. There is controversy over whether using ICSI is beneficial to improve embryo quality for patients with poor quality embryos in previous C-IVF cycles [ 7 – 10 ]. As the effectiveness of ICSI-AOA in improving embryo development for previous ICSI treatments with poor embryo quality has been reported, we attempt to investigate whether ICSI-AOA can improve embryo quality in C-IVF cases with recurrent developmental problems. Methods Study design A retrospective cohort study was performed between January 2019 and December 2022, including 24 couples (P1-P24) with a history of embryo developmental problems. Patients were only included when they underwent at least two fresh C-IVF cycles at the Center for Reproductive Medicine at Northwest Women’s and Children’s Hospital. Further, close normal fertilization (≥ 40% two pronuclei (2PN)) with at most one day 3 good quality embryo in the whole previous C-IVF cycles, which were required before inclusion in the study. Normal fertilization, embryo development, pregnancy, ongoing pregnancy and live birth rates after ICSI-AOA were compared to previous C-IVF cycles. This study was approved by the Ethics Committee of Northwest Women’s and Children’s Hospital (No. 2022007). The requirement for consent was waived by the institutional review board because of its retrospective design. All patient data were collected from electronic medical records and IVF treatment sheets. Ovarian stimulation protocol Ovarian stimulation protocol All the participants received standardized controlled ovarian hyperstimulation (COH) treatment following one of three established regimens: the GnRH agonist long protocol, GnRH agonist short protocol, and GnRH antagonist protocol, as detailed in previous literature [ 11 ]. Notably, recombinant follicle-stimulating hormone (FSH) or urinary FSH and/or human menopausal gonadotropins were used with individualized dosages (100–450 IU/day) determined by ovarian reserve markers and patient profile, consistent with prior methodology [ 11 ]. IVF and ICSI procedure C-IVF was initiated 2-2.5 hours post-retrieval, with oocytes exposed to approximately 40,000 motile sperm for natural fertilization. Following a short-term incubation period (4-4.5 hours), cumulus cells were mechanically removed. For ICSI, mature (MII) oocytes were injected by the direct penetration technique. Gametes were prepared in separate microdroplets: oocytes in 5 µL G-MOPS medium (Vitrolife, Goteborg, Sweden) and sperm in polyvinylpyrrolidone solution, both maintained under warm mineral oil (Vitrolife, Goteborg, Sweden). The injection procedure followed established methodology [ 12 ]. Assisted oocyte activation process Following ICSI, MII oocytes were immediately treated with a commercial ionophore (GM508 Cult-Active; Gynemed, Lensahn, Germany) for 15 min under mineral oil. After activation, oocytes were washed three times in G-1™ Plus medium (Vitrolife, Goteborg, Sweden). Embryos were individually cultured in G-1™ Plus medium until day 3, then transferred to group culture in G-2™ Plus (Vitrolife, Goteborg, Sweden) for extended blastocyst development. Fertilization check and embryo grading Normal fertilization was confirmed 18–19 hours post-insemination through identification of two pronuclei (2PN) accompanied by second polar body extrusion. After 64–68 h of culture, the morphologic score was given for cleavage-stage embryos. The morphologic score of blastocysts were given on the fifth morning after oocyte retrieval. The detailed scoring criteria were based on our published literature [ 12 ]. The D3 good quality embryos were graded I and II. The D3 available embryos were graded I, II, and III. The blastocyst scored ≥ 3BB was defined as high-quality blastocyst. Embryo cryopreservation Vitrification and warming of embryos were performed using ultra-rapid technologies (Cryotop® and vitrification and warming media, Kitazato BioPharma Co. Ltd., Shizouka, Japan), as described by the manufacturer. D3 embryos were warmed on the prior afternoon, while the blastocysts underwent same-day warming and transfer. Embryo transfer The mucus in the cervical os was cleaned in advance with a cotton swab soaked in warm and humid saline. Embryos were transferred under ultrasound guidance with a transfer catheter (Limerick, Ireland). Ongoing pregnancy was defined as a clinical pregnancy that continued for a minimum of 12 weeks. Miscarriage was defined as spontaneous loss of a clinical pregnancy before 22 completed weeks of gestation. Live birth was defined as the delivery of a live-born infant (> 24 weeks of gestation). Statistical analysis Reproductive outcomes (fertilization, embryo quality, and clinical results) were compared between C-IVF and ICSI-AOA cycles using Fisher's exact test (SPSS Statistics 25; IBM). P -values < 0.05 were considered as statistically significant. Results Patients’ baseline clinical characteristics In total, 24 ICSI-AOA cycles were compared with 52 previous IVF cycles from the same patient cohort (P1-P24). The baseline characteristics of the patients were shown in Table 1 , including female age, female body mass index (BMI), basal FSH, total Gn dosage, stimulation duration, number of oocytes retrieved, male age, sperm concentration and progressive motility. The number of oocytes retrieved was significantly lower in the previous C-IVF cycles than that in the subsequent ICSI-AOA cycles (8.08 versus 12.33%, p = 0.004). Other parameters were similar between the two groups ( p > 0.05). Table 1 General characteristics of the enrolled patients. Parameter C-IVF ICSI-AOA P-Value Patients (n) 24 24 / Cycles (oocyte retrievals) 52 24 / Female age (y) 30.67 ± 2.97 31.96 ± 3.31 0.096 BMI for women (kg/m²) 23.34 ± 3.38 23.17 ± 3.17 0.646 Basal FSH(IU/L) 6.07 ± 2.04 6.25 ± 2.50 0.750 Total Gn dosage (IU) 2357.54 ± 761.42 2274.83 ± 866.88 0.674 Stimulation duration (days) 10.19 ± 2.03 9.25 ± 2.09 0.066 Number of oocytes retrieved (n) 8.08 ± 4.73 12.33 ± 7.68 0.004 Male age (y) 31.98 ± 3.32 33.04 ± 3.79 0.220 Sperm concentration (10 6 /mL) 57.73 ± 32.71 53.93 ± 27.68 0.624 Progressive motility (a + b) (%) 50.25 ± 11.24 54.13 ± 12.07 0.175 Mean number of embryos transferred (n) 1.62 ± 0.49 1.76 ± 0.66 0.359 Embryo development after previous C-IVF and subsequent ICSI-AOA cycles From the ICSI-AOA cycles, a total of 296 cumulus-oocyte complexes (COCs) (mean 12.33) were collected, from which 216 MII oocytes (mean 9.00) were used for treatment. From previous IVF cycles, a total of 420 COCs (mean 8.26) were obtained. Following ICSI-AOA, an overall normal fertilization rate of 79.17% (171/216) was achieved, which was significantly higher compared that of 53.10% (223/420) observed after conventional IVF cycles (79.17 versus 53.10%; p < 0.001) (Table 1 ). The developmental capacity of fertilized oocytes was evaluated by assessing embryo formation on Day 3 and Day 5 post-fertilization. After ICSI-AOA, a total D3 available embryo rate of 47.37% (81/171) and blastocyst formation of 22.22% (8/36) were achieved, which were similar to D3 available embryo of 45.29% (101/223) and blastocyst formation rates of 20.59% (7/34) obtained after conventional IVF. In conventional IVF cycles, a total of 48 embryos were transferred in fresh cycles. Additionally, 19 embryos were cryopreserved, and 34 embryos underwent extended culture to the blastocyst stage. In ICSI-AOA cycles, a total of 18 embryos were transferred in fresh cycles. Additionally, 27 embryos were cryopreserved, and 36 embryos underwent extended culture to the blastocyst stage. Importantly, ICSI-AOA cycles demonstrated significantly higher D3 good quality embryo rate compared with previous IVF cycles (19.30 versus 4.04%; p < 0.001). We further observed that the mean number of D3 embryo blastomere was significantly increased after ICSI-AOA compared with previous IVF cycles (7.85 versus 6.94; p = 0.002). No significant difference was observed in the D3 embryo fragmentation rate between previous C-IVF and subsequent ICSI-AOA cycles (14% versus 13%, p = 0.088) (Table 2 ). Table 2 Comparison of embryo development after previous C-IVF and subsequent ICSI-AOA cycles in couples with embryo developmental problems (P1-P24). Parameter C-IVF ICSI-AOA P-Value Patients (n) 24 24 Cycles (oocyte retrievals) 52 24 / Cumulus-oocyte complexes (n) 420 296 / Normal fertilization rate (%) 53.10 (223/420) 79.17 (171/216) < 0.001 D3 available embryo rate (D3 embryos/2PN) 45.29 (101/223) 47.37 (81/171) 0.682 D3 good quality embryo rate (D3 good quality embryos/2PN) 4.04 (9/223) 19.30 (33/171) < 0.001 Mean number of D3 embryo blastomere (n) 6.94 ± 1.85 7.85 ± 2.03 0.002 Mean fragmentation of D3 embryo (%) 14% 13% 0.088 No. of D3 embryos transferred in fresh (n) 48 18 / No. of D3 embryos cryopreserved (n) 19 27 / Embryos of extended culture to blastocyst-stage (n) 34 36 / Blastocyst formation rate (D5 or 6 embryos/cultured embryo to day 5 or 6) 20.59 (7/34) 22.22 (8/36) 0.868 Good quality blastocyst formation rate (D5 or 6 good quality embryos/cultured embryo to day 5 or 6) 2.94 (1/34) 11.11 (4/36) 0.185 Clinical outcomes after previous C-IVF and subsequent ICSI-AOA cycles Overall, following ICSI-AOA, a total pregnancy rate of 41.67% (10/24) was achieved, which was significantly higher than the pregnancy rate of 19.23% (10/52) observed after previous IVF cycles ( p = 0.039). Previous IVF cycles resulted in three biochemical pregnancies, five miscarriages, and two live births (3.85%, 2/52), while the ICSI-AOA cycles yielded one biochemical pregnancy, two miscarriages, and seven live births (29.17%, 7/24). After ICSI-AOA, the live birth rate showed a significant improvement compared to previous IVF treatment (29.17 versus 3.85%, p = 0.002) (Table 3 ). Table 3 Comparison of clinical outcomes after previous C-IVF and subsequentICSI-AOA cycles in couples with embryo developmental problems (P1-P24). Parameter C-IVF ICSI-AOA P-Value Cycles (Embryo transfers) 45 17 / Transfers in fresh (n) 31 9 / Transfers after thawing (n) 14 8 / Cancelled transfers with no embryos available (n) 10 6 / Oocyte retrievals with fresh or frozen embryo transfer (ET/initiated cycle) 86.54 (45/52) 70.83 (17/24) 0.101 Cleavage-stage embryo transfer (n) 42 15 / Blastocyst-stage embryo transfer (n) 3 2 / Transferred blastocyst rate (D5 or 6 embryo transfers/ total embryo transfers) 6.67 (3/45) 11.76 (2/17) 0.511 Pregnancy rate (+ hCG/initiated cycle) 19.23 (10/52) 41.67 (10/24) 0.039 Biochemical pregnancy (n) 3 1 / Ectopic pregnancy (n) 0 0 / Miscarriage (n) 5 2 / Ongoing pregnancy (> 12 weeks) (n) 2 7 / Live birth rate (live births/initiated cycle) 3.85 (2/52) 29.17 (7/24) 0.002 Discussion In this study, we found that ICSI-AOA improved the D3 good quality embryo rate compared with previous IVF treatment in some patients included in the study. The number of D3 embryo blastomeres is a significant factor affecting embryo quality. We observed that the mean number of D3 embryo blastomere was significantly increased after ICSI-AOA compared with previous IVF cycles. Nevertheless, we observed no significant difference in the proportion of D3 embryos with different blastomeres which might be associated with limited cases (Supplementary Fig. 1). Embryo fragmentation is also an important factor affecting embryo quality. The origin of fragmentation is still not fully understood and is likely to be multifactorial. Some factors including embryo culture condition, female age, gamete quality, ovulation induction, aneuploidy, and fertilization method appear to play a significant role in the etiology of cytoplasmic fragmentation. In this research, the main differences between the two groups were fertilization methods and the number of oocytes retrieved. Some researches demonstrated that ICSI did not have a significant impact on the D3 embryo fragmentation rate which suggested that ICSI was not a major contributor to adverse outcomes related to embryo fragmentation [ 13 , 14 ]. Another retrospective study indicated that the lower number of oocytes was associated with increased embryo fragmentation [ 15 ]. There was a significant difference in the number of oocyte retrieved, so we further analyzed the D3 embryo fragmentation rate after ICSI-AOA compared with that of previous C-IVF cycles. No significant difference was observed in the D3 embryo fragmentation rate between previous C-IVF and subsequent ICSI-AOA cycles. And we also observed that there was no significant difference in the proportion of embryos with different degree of fragmentation (Supplementary Fig. 2). A recent systematic review concluded that the origin and the significance of iatrogenic or external factors on fragmentation of cleavage-stage embryos varies in the published papers [ 16 ]. Importantly, our data also indicated that ICSI-AOA significantly improved the pregnancy and live birth rates compared with previous IVF cycles, highlighting its potential to not only improve embryo quality but also increase clinical success rates. These findings suggest that ICSI-AOA could offer a valuable approach for patients struggling with poor embryo development, potentially improving both embryo quality and the overall success of IVF treatments. Additionally, Multiple recent studies have demonstrated that ICSI-AOA can improve the developmental ability of embryos and pregnancy outcomes [ 17 – 20 ]. The abovementioned studies support our findings that AOA using calcium ionophores is positively associated with improved embryo quality. Although some researches showed promising results, two sibling-oocyte studies failed to confirm the benefits of AOA in addressing embryo developmental issues. One study enrolled 140 patients with normal fertilization rate > 70%, but D3 good quality embryo rate ≤ 30% in previous ICSI cycles. After ICSI-AOA, no significant difference was observed in the rate of D3 good quality embryos between the AOA and non-AOA groups. Additionally, AOA did not improve the clinical pregnancy and live birth rates [ 21 ]. The other study involved 19 patients with normal fertilization rate > 30% and D5 poor embryo quality in at least one previous ICSI cycle. After ICSI-AOA, no significant differences were observed in the rates of D3 good quality embryos and D5 good quality blastocysts between the AOA and non-AOA groups [ 22 ]. A recent cohort study compared ICSI-AOA cycles and previous ICSI cycles in couples who had normal fertilization rates (60%) but impaired embryonic development (15% blastocyst formation) in at least two previous ICSI cycles. It also demonstrated that AOA did not improve the rates of D3 good quality embryos and D5 good quality blastocysts in the populations with recurrent embryo developmental problems after ICSI [ 23 ]. Nevertheless, no studies have reported whether ICSI-AOA can improve the embryo quality in standard IVF cases with recurrent developmental problems. In this research, most of the enrolled male partners exhibited normal semen parameters and only three of them presented a slightly higher rate of abnormal sperm morphology. Multiple researches have shown that isolated teratozoospermia may not adversely affect the overall success rates of IVF procedures [ 24 – 26 ]. The current findings indicate that patients with moderate and severe teratozoospermia can be effectively treated with IVF and rescue-ICSI can be performed when necessary. 26 Meanwhiles, it has been confirmed that oocyte competency is the key to embryo potential [ 27 , 28 ]. Thus, we hypothesize that the poor embryo quality is primarily attributable to factors related to the oocytes for standard IVF cases with recurrent developmental problems. It is important to acknowledge that oocyte activation deficiencies are not the only reasons contributing to poor embryo development. Factors such as sperm DNA damage, oocyte abnormalities in structural proteins and mitochondria, and issues with transcription factors may also contribute to poor embryo prognosis [ 29 ]. Importantly, none of these can be rescued by assisted oocyte activation. Although nearly half (11/24) of ICSI-AOA cycles showed an improved number of D3 good quality embryos compared to previous IVF cycle, this benefit was not observed in all patients participating in this study. Problems with oocyte-derived and deficiencies in downstream signaling pathways may be an important cause of poor embryo development [ 30 ]. Although some cases of oocyte-related could be overcome by AOA, it was also confirmed that AOA was not beneficial for all patients with a suspected oocyte-related activation deficiency [ 31 ]. As mentioned before, AOA will in theory only benefit patients suffering from a Ca + deficiency. We further displayed the details of embryo development after previous C-IVF and subsequent ICSI-AOA cycles (Supplementary table). After ICSI-AOA, several patients (P1, P4, and P7) exhibited an increase in the number of D3 available embryo, D3 good quality embryo, and good quality blastocyst. All three patients (P1, P4, and P7) achieved successful live births. AOA treatment also proved beneficial for certain patients (P3, P6, P8, P9, P10, P14, P18, and P20), as they experienced an increase in the D3 good quality embryo. Among them, two patients (P14 and P20) achieved successful live births. Conversely, some patients (P12, P21, P22, and P24) did not benefit from ICSI-AOA, due to a decrease in the number of D3 good quality embryos compared with previous IVF cycles. Notably, two (P12 and P24) of these patients achieved successful live births. ICSI-AOA treatment was ineffective for the patients (P16, P17, and P19), all of whom produced fewer available embryos than during prior IVF cycles without achieving successful live births. Additionally, six other patients (P2, P5, P11, P13, P15, and P23) similarly did not benefit from ICSI-AOA since they had available embryos in previous IVF cycles but yielded no available embryos on day 3 after ICSI-AOA. It should be noted that the cancellation rate of no available embryos was not decreased after ICSI-AOA. We suspected that it might be associated with the low number of oocytes retrieved and 2PN zygotes. In 6 cycles with no available embryos after ICSI-AOA, there were 4 cycles in which no more than three oocytes were retrieved. To the best of the authors’ knowledge, this is a new design to assess the effect of AOA on improving embryo development in standard IVF cases with recurrent developmental problems. Although various modifications to the culture strategy and ovarian stimulation protocol have been explored in attempts to improve embryo quality, it's difficult to get consistent and effective conclusions. It is challenging to identify the sole appropriate reason that can effectively improve embryo development for these patients. Despite this, it provides a new therapeutic idea which suggest that ICSI-AOA combined with the optimization of ovarian stimulation protocol may be worthy to attempt to improve embryo quality for the patients with recurrent developmental problems in standard IVF cases. A recent meta-analysis has demonstrated that AOA could not only significantly improve live birth and overall pregnancy, but also did not affect the incidence of miscarriage, congenital birth defects, and neonatal sex ratio [ 32 ]. Our data showed that there were 2 twin births in the 7 live deliveries after ICSI-AOA. The mean gestational age was 272 days in singleton and the mean gestational age was 254 days in twins. The mean birthweight was 3169 g in singleton and the mean birthweight was 2533 g in twins. And no congenital malformation occurred after ICSI-AOA. Thus, ICSI-AOA can be considered as an effective and safe method which may benefit to couples with recurrent developmental problems in standard IVF. Strengths and limitations There are certain weaknesses in the current study that should be underlined. First, the primary drawback is the retrospective design and small sample size. Second, there may be some potential bias and confounders that cannot be excluded. For previous IVF cycles, the number of oocytes retrieved is 8.08. For subsequent ICSI-AOA cycles, the number of oocytes retrieved is 12.33. We further displayed the details of each patient and observed that it mainly exhibited a significant increase in the number of eggs retrieved for 7 patients (P1, P3, P8, P9, P12, P13 and P20). Of these patients, only two patients (P1 and P8) showed an increase in the D3 available embryos after ICSI-AOA. Additionally, we choose the commercial A23187 for AOA in this research because the application of calcimycin is performed for AOA due to its established capacity to enhance embryo development [ 32 ]. Nevertheless, recent researches have demonstrated that AOA treatment using ionomycin yields higher activation compared to calcimycin [ 33 , 34 ]. Furthermore, failed ICSI-AOA cycles employing calcimycin can potentially be rescued through the use of ionomycin [ 35 ]. These data suggested that the embryo development might be further improved in these patients if ionomycin was utilized for AOA. Although the optimal AOA method was not employed, our data indicated that certain improvements could be achieved by AOA with calcimycin. Lastly, the data about blastocyst culture is limited so we are not certain whether AOA can improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after conventional IVF. Conclusion In conclusion, our study demonstrated that ICSI-AOA may be beneficial to some patients with recurrent developmental problems in C-IVF treatment. Decision-makers need to pay more attention to these patients with repeated failures in C-IVF treatment. Given the limited data and methodological constraints, further data accumulation is needed to obtain more reliable conclusions. Abbreviations AOA Assisted oocyte activation IVF In vitro fertilization ICSI Intracytoplasmic sperm injection TFF Total fertilization failure LF Low fertilization OAF Oocyte activation failure FSH Follicle-stimulating hormone GnRH Gonadotropin-releasing hormone BMI Body mass index ET Embryo transfer 2PN Two pronuclei Declarations Acknowledgements Not applicable. Author contributions M.L. and J.S. designed the research; M.L. and S.Q. performed the research. X.X. analyzed and prepared the data; All authors have reviewed the manuscript and agreed to be accountable for all aspects of the work. Funding This project was supported by Shaanxi Technology Committee Industrial Public Relation Project (Project Number: 2023-YBSF-034) and Young Physicians Program of Chinese Medical Association (No. 17020470716). Data availability Data available on request corresponding author due to privacy and ethical restrictions. Ethical approval and consent to participate The study involving human participants was reviewed and approved by the Ethics Committee of Northwest Women’s and Children’s Hospital (No. 2022007). The study was complied with the Helsinki Declaration and the patient records were innominate ahead of the data analysis. Patient consent was waived due to the retrospective nature of the research. Consent for publication Not applicable since there are no details, images, or videos relating to an individual person. Competing interests The authors declare no competing interests. References Stricker SA. 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Villani MT, Morini D, Spaggiari G, Falbo AI, Melli B, La Sala GB, et al. Are sperm parameters able to predict the success of assisted reproductive technology? A retrospective analysis of over 22,000 assisted reproductive technology cycles. Andrology. 2022;10:310–21. Li D, Wang Y, Han L, Gao D, Yang C, Wang W, et al. Role of sperm morphological parameters in the selection of fertilization methods. Rev Int Androl. 2024;22:68–73. Keefe D, Kumar M, Kalmbach K. Oocyte competency is the key to embryo potential. Fertil Steril. 2015;103:317–22. 10.1016/j.fertnstert.2014.12.115 . Nikiforov D, Grøndahl ML, Hreinsson J, Andersen CY. Human Oocyte Morphology and Outcomes of Infertility Treatment: a Systematic Review. Reprod Sci. 2022;29:2768–85. Bashiri Z, Amidi F, Amiri I, Zandieh Z, Maki CB, Mohammadi F, et al. Male Factors: the Role of Sperm in Preimplantation Embryo Quality. Reprod Sci. 2021;28:1788–811. Vanden Meerschaut F, Nikiforaki D, De Gheselle S, Dullaerts V, Van den Abbeel E, Gerris J, et al. Assisted oocyte activation is not beneficial for all patients with a suspected oocyte-related activation deficiency. Hum Reprod. 2012;27:1977–84. Jones KT, Lane SI. Molecular causes of aneuploidy in mammalian eggs. Development. 2013;140:3719–30. Shan Y, Zhao H, Zhao D, Wang J, Cui Y, Bao H. Assisted Oocyte Activation With Calcium Ionophore Improves Pregnancy Outcomes and Offspring Safety in Infertile Patients: A Systematic Review and Meta-Analysis. Front Physiol. 2022;12:751905. Lv M, Zhang D, He X, Chen B, Li Q, Ding D, et al. Artificial oocyte activation to improve reproductive outcomes in couples with various causes of infertility: a retrospective cohort study. Reprod Biomed Online. 2020;40:501–9. Jia L, Chen P, Su W, He S, Guo Y, Zheng L, et al. Artificial oocyte activation with ionomycin compared with A23187 among patients at risk of failed or impaired fertilization. Reprod Biomed Online. 2023;46:35–45. Quintana-Vehí A, Martínez M, Zamora MJ, Rodríguez A, Vassena R, Miguel-Escalada I, et al. Significant differences in efficiency between two commonly used ionophore solutions for assisted oocyte activation (AOA): a prospective comparison of ionomycin and A23187. J Assist Reprod Genet. 2023;40:1661–8. Wang M, Zhu L, Liu C, He H, Wang C, Xing C, et al. A Novel Assisted Oocyte Activation Method Improves Fertilization in Patients With Recurrent Fertilization Failure. Front Cell Dev Biol. 2021;9:672081. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfigure.docx SuppTab.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 15 May, 2025 Editor assigned by journal 13 May, 2025 Editor invited by journal 24 Apr, 2025 Submission checks completed at journal 23 Apr, 2025 First submitted to journal 23 Apr, 2025 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-6458796","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":457365645,"identity":"36762652-28ac-43b1-bcb6-7fa85a2170ac","order_by":0,"name":"Mingzhao Li","email":"","orcid":"","institution":"Northwest Women’s and Children’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mingzhao","middleName":"","lastName":"Li","suffix":""},{"id":457365646,"identity":"456b67b3-3385-4ec3-9a21-8ff41acf062b","order_by":1,"name":"Sen Qiao","email":"","orcid":"","institution":"Northwest Women’s and Children’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sen","middleName":"","lastName":"Qiao","suffix":""},{"id":457365647,"identity":"a1fa4437-212e-4709-9f69-e465cae95033","order_by":2,"name":"Xia Xue","email":"","orcid":"","institution":"Northwest Women’s and Children’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"Xue","suffix":""},{"id":457365648,"identity":"377c20bc-f296-4684-9095-e14f9b4649aa","order_by":3,"name":"Juanzi Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYNACHgY5Nvb2A6RpMebjOZNAmj2J8yQcDIhTKt9//JnkD5m69DYJhgSGHxXbCGsxOHAg2ZiHhy23TbrxAGPPmdtEaGFsOPiYgYcnt03mQAIzYxsRWuSbgXp+8Eiks0kkGBCnheEYM+MDHh6DBOK1GJxhYwb6JcGwDRjIB4nyCzjEfvbUycu3tx988KOCGIeBAGMPhD5ApHoQ+EGC2lEwCkbBKBh5AABl/jVqASBJ6gAAAABJRU5ErkJggg==","orcid":"","institution":"Northwest Women’s and Children’s Hospital","correspondingAuthor":true,"prefix":"","firstName":"Juanzi","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2025-04-16 02:38:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6458796/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6458796/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83014683,"identity":"bc8fc33b-3509-4d82-95d5-ea5b902f9c75","added_by":"auto","created_at":"2025-05-19 06:03:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":749934,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6458796/v1/6fb87920-a5cd-4a01-b85a-f6c2fde05784.pdf"},{"id":83012416,"identity":"382cc3a3-56fd-47b6-8851-785db81ac897","added_by":"auto","created_at":"2025-05-19 05:37:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":148421,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-6458796/v1/200005bd65fa6f4091d01e0c.docx"},{"id":83012410,"identity":"33360a23-741e-4ca3-8dc9-c7153c88ba3f","added_by":"auto","created_at":"2025-05-19 05:37:26","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19817,"visible":true,"origin":"","legend":"","description":"","filename":"SuppTab.docx","url":"https://assets-eu.researchsquare.com/files/rs-6458796/v1/433a5937173361ffdc0affb0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Efficacy of assisted oocyte activation in improving the reproductive outcome in conventional IVF cases with recurrent developmental problems","fulltext":[{"header":"Background","content":"\u003cp\u003eOocyte activation is a vital step in fertilization, involving a series of molecular events essential for successful egg fertilization [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. A key player in this process is calcium signaling, which plays a crucial role in initiating and regulating the complex mechanisms required for oocyte activation. In the past two decades, the combination of intracytoplasmic sperm injection (ICSI) with assisted oocyte activation (ICSI-AOA) has become increasingly popular as a therapeutic approach for patients with total fertilization failure (TFF), low fertilization (LF), and sperm-specific oocyte activation failure [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCalcium signaling is closely linked to embryonic development, playing a critical role in regulating early cell divisions and the cell cycle during embryogenesis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Disruptions in calcium signaling can lead to abnormalities in cell cleavage and subsequent developmental issues [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Currently, the AOA techniques, such as those utilizing calcium rises, are primarily applied in cases of LF or TFF. Given the strong correlation between calcium availability and cell division, we believe exploring ICSI-AOA as a potential solution for recurrent embryo development issues is highly valuable. By improving the activation process, ICSI-AOA could enhance embryo quality and potentially improve the outcomes of some embryos.\u003c/p\u003e \u003cp\u003eFor patients with poor embryo quality problems at least two initiated in vitro fertilization (IVF) cycles, they probably will not obtain good quality embryos in the incoming conventional IVF (C-IVF) cycle. So far, there is no effective way to improve embryo quality for such patients. There is controversy over whether using ICSI is beneficial to improve embryo quality for patients with poor quality embryos in previous C-IVF cycles [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. As the effectiveness of ICSI-AOA in improving embryo development for previous ICSI treatments with poor embryo quality has been reported, we attempt to investigate whether ICSI-AOA can improve embryo quality in C-IVF cases with recurrent developmental problems.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eA retrospective cohort study was performed between January 2019 and December 2022, including 24 couples (P1-P24) with a history of embryo developmental problems. Patients were only included when they underwent at least two fresh C-IVF cycles at the Center for Reproductive Medicine at Northwest Women\u0026rsquo;s and Children\u0026rsquo;s Hospital. Further, close normal fertilization (\u0026ge;\u0026thinsp;40% two pronuclei (2PN)) with at most one day 3 good quality embryo in the whole previous C-IVF cycles, which were required before inclusion in the study. Normal fertilization, embryo development, pregnancy, ongoing pregnancy and live birth rates after ICSI-AOA were compared to previous C-IVF cycles. This study was approved by the Ethics Committee of Northwest Women\u0026rsquo;s and Children\u0026rsquo;s Hospital (No. 2022007). The requirement for consent was waived by the institutional review board because of its retrospective design. All patient data were collected from electronic medical records and IVF treatment sheets.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOvarian stimulation protocol\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eOvarian stimulation protocol\u003c/div\u003e \u003cp\u003eAll the participants received standardized controlled ovarian hyperstimulation (COH) treatment following one of three established regimens: the GnRH agonist long protocol, GnRH agonist short protocol, and GnRH antagonist protocol, as detailed in previous literature [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Notably, recombinant follicle-stimulating hormone (FSH) or urinary FSH and/or human menopausal gonadotropins were used with individualized dosages (100\u0026ndash;450 IU/day) determined by ovarian reserve markers and patient profile, consistent with prior methodology [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eIVF and ICSI procedure\u003c/h3\u003e\n\u003cp\u003eC-IVF was initiated 2-2.5 hours post-retrieval, with oocytes exposed to approximately 40,000 motile sperm for natural fertilization. Following a short-term incubation period (4-4.5 hours), cumulus cells were mechanically removed. For ICSI, mature (MII) oocytes were injected by the direct penetration technique. Gametes were prepared in separate microdroplets: oocytes in 5 \u0026micro;L G-MOPS medium (Vitrolife, Goteborg, Sweden) and sperm in polyvinylpyrrolidone solution, both maintained under warm mineral oil (Vitrolife, Goteborg, Sweden). The injection procedure followed established methodology [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAssisted oocyte activation process\u003c/h3\u003e\n\u003cp\u003eFollowing ICSI, MII oocytes were immediately treated with a commercial ionophore (GM508 Cult-Active; Gynemed, Lensahn, Germany) for 15 min under mineral oil. After activation, oocytes were washed three times in G-1\u0026trade; Plus medium (Vitrolife, Goteborg, Sweden). Embryos were individually cultured in G-1\u0026trade; Plus medium until day 3, then transferred to group culture in G-2\u0026trade; Plus (Vitrolife, Goteborg, Sweden) for extended blastocyst development.\u003c/p\u003e\n\u003ch3\u003eFertilization check and embryo grading\u003c/h3\u003e\n\u003cp\u003eNormal fertilization was confirmed 18\u0026ndash;19 hours post-insemination through identification of two pronuclei (2PN) accompanied by second polar body extrusion. After 64\u0026ndash;68 h of culture, the morphologic score was given for cleavage-stage embryos. The morphologic score of blastocysts were given on the fifth morning after oocyte retrieval. The detailed scoring criteria were based on our published literature [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The D3 good quality embryos were graded I and II. The D3 available embryos were graded I, II, and III. The blastocyst scored\u0026thinsp;\u0026ge;\u0026thinsp;3BB was defined as high-quality blastocyst.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEmbryo cryopreservation\u003c/h2\u003e \u003cp\u003eVitrification and warming of embryos were performed using ultra-rapid technologies (Cryotop\u0026reg; and vitrification and warming media, Kitazato BioPharma Co. Ltd., Shizouka, Japan), as described by the manufacturer. D3 embryos were warmed on the prior afternoon, while the blastocysts underwent same-day warming and transfer.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEmbryo transfer\u003c/h3\u003e\n\u003cp\u003eThe mucus in the cervical os was cleaned in advance with a cotton swab soaked in warm and humid saline. Embryos were transferred under ultrasound guidance with a transfer catheter (Limerick, Ireland). Ongoing pregnancy was defined as a clinical pregnancy that continued for a minimum of 12 weeks. Miscarriage was defined as spontaneous loss of a clinical pregnancy before 22 completed weeks of gestation. Live birth was defined as the delivery of a live-born infant (\u0026gt;\u0026thinsp;24 weeks of gestation).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eReproductive outcomes (fertilization, embryo quality, and clinical results) were compared between C-IVF and ICSI-AOA cycles using Fisher's exact test (SPSS Statistics 25; IBM). \u003cem\u003eP\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u0026rsquo; baseline clinical characteristics\u003c/h2\u003e \u003cp\u003eIn total, 24 ICSI-AOA cycles were compared with 52 previous IVF cycles from the same patient cohort (P1-P24). The baseline characteristics of the patients were shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, including female age, female body mass index (BMI), basal FSH, total Gn dosage, stimulation duration, number of oocytes retrieved, male age, sperm concentration and progressive motility. The number of oocytes retrieved was significantly lower in the previous C-IVF cycles than that in the subsequent ICSI-AOA cycles (8.08 versus 12.33%, \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.004). Other parameters were similar between the two groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGeneral characteristics of the enrolled patients.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParameter\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eC-IVF\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eICSI-AOA\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP-Value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatients (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCycles (oocyte retrievals)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale age (y)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.96\u0026thinsp;\u0026plusmn;\u0026thinsp;3.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.096\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI for women (kg/m\u0026sup2;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.34\u0026thinsp;\u0026plusmn;\u0026thinsp;3.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.17\u0026thinsp;\u0026plusmn;\u0026thinsp;3.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.646\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasal FSH(IU/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.07\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.750\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Gn dosage (IU)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2357.54\u0026thinsp;\u0026plusmn;\u0026thinsp;761.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2274.83\u0026thinsp;\u0026plusmn;\u0026thinsp;866.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.674\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStimulation duration (days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.19\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.066\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of oocytes retrieved (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.08\u0026thinsp;\u0026plusmn;\u0026thinsp;4.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.33\u0026thinsp;\u0026plusmn;\u0026thinsp;7.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale age (y)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.04\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.220\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSperm concentration (10\u003csup\u003e6\u003c/sup\u003e/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57.73\u0026thinsp;\u0026plusmn;\u0026thinsp;32.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53.93\u0026thinsp;\u0026plusmn;\u0026thinsp;27.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.624\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProgressive motility (a\u0026thinsp;+\u0026thinsp;b) (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50.25\u0026thinsp;\u0026plusmn;\u0026thinsp;11.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54.13\u0026thinsp;\u0026plusmn;\u0026thinsp;12.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.175\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean number of embryos transferred (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.359\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEmbryo development after previous C-IVF and subsequent ICSI-AOA cycles\u003c/h2\u003e \u003cp\u003eFrom the ICSI-AOA cycles, a total of 296 cumulus-oocyte complexes (COCs) (mean 12.33) were collected, from which 216 MII oocytes (mean 9.00) were used for treatment. From previous IVF cycles, a total of 420 COCs (mean 8.26) were obtained. Following ICSI-AOA, an overall normal fertilization rate of 79.17% (171/216) was achieved, which was significantly higher compared that of 53.10% (223/420) observed after conventional IVF cycles (79.17 versus 53.10%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The developmental capacity of fertilized oocytes was evaluated by assessing embryo formation on Day 3 and Day 5 post-fertilization. After ICSI-AOA, a total D3 available embryo rate of 47.37% (81/171) and blastocyst formation of 22.22% (8/36) were achieved, which were similar to D3 available embryo of 45.29% (101/223) and blastocyst formation rates of 20.59% (7/34) obtained after conventional IVF. In conventional IVF cycles, a total of 48 embryos were transferred in fresh cycles. Additionally, 19 embryos were cryopreserved, and 34 embryos underwent extended culture to the blastocyst stage. In ICSI-AOA cycles, a total of 18 embryos were transferred in fresh cycles. Additionally, 27 embryos were cryopreserved, and 36 embryos underwent extended culture to the blastocyst stage. Importantly, ICSI-AOA cycles demonstrated significantly higher D3 good quality embryo rate compared with previous IVF cycles (19.30 versus 4.04%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). We further observed that the mean number of D3 embryo blastomere was significantly increased after ICSI-AOA compared with previous IVF cycles (7.85 versus 6.94; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). No significant difference was observed in the D3 embryo fragmentation rate between previous C-IVF and subsequent ICSI-AOA cycles (14% versus 13%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.088) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of embryo development after previous C-IVF and subsequent ICSI-AOA cycles in couples with embryo developmental problems (P1-P24).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParameter\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eC-IVF\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eICSI-AOA\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP-Value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatients (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCycles (oocyte retrievals)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCumulus-oocyte complexes (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e420\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e296\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal fertilization rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53.10 (223/420)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e79.17 (171/216)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD3 available embryo rate (D3 embryos/2PN)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.29 (101/223)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.37 (81/171)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.682\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD3 good quality embryo rate (D3 good quality embryos/2PN)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.04 (9/223)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.30 (33/171)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean number of D3 embryo blastomere (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean fragmentation of D3 embryo (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.088\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of D3 embryos transferred in fresh (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of D3 embryos cryopreserved (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEmbryos of extended culture to blastocyst-stage (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlastocyst formation rate (D5 or 6 embryos/cultured embryo to day 5 or 6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.59 (7/34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.22 (8/36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.868\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGood quality blastocyst formation rate (D5 or 6 good quality embryos/cultured embryo to day 5 or 6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.94 (1/34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.11 (4/36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.185\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eClinical outcomes after previous C-IVF and subsequent ICSI-AOA cycles\u003c/h2\u003e \u003cp\u003eOverall, following ICSI-AOA, a total pregnancy rate of 41.67% (10/24) was achieved, which was significantly higher than the pregnancy rate of 19.23% (10/52) observed after previous IVF cycles (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.039). Previous IVF cycles resulted in three biochemical pregnancies, five miscarriages, and two live births (3.85%, 2/52), while the ICSI-AOA cycles yielded one biochemical pregnancy, two miscarriages, and seven live births (29.17%, 7/24). After ICSI-AOA, the live birth rate showed a significant improvement compared to previous IVF treatment (29.17 versus 3.85%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of clinical outcomes after previous C-IVF and subsequentICSI-AOA cycles in couples with embryo developmental problems (P1-P24).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParameter\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eC-IVF\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eICSI-AOA\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP-Value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCycles (Embryo transfers)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTransfers in fresh (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTransfers after thawing (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCancelled transfers with no embryos available (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOocyte retrievals with fresh or frozen embryo transfer (ET/initiated cycle)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e86.54 (45/52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70.83 (17/24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.101\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCleavage-stage embryo transfer (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlastocyst-stage embryo transfer (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTransferred blastocyst rate (D5 or 6 embryo transfers/ total embryo transfers)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.67 (3/45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.76 (2/17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.511\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePregnancy rate (+\u0026thinsp;hCG/initiated cycle)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.23 (10/52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.67 (10/24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.039\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiochemical pregnancy (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEctopic pregnancy (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMiscarriage (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOngoing pregnancy (\u0026gt;\u0026thinsp;12 weeks) (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLive birth rate (live births/initiated cycle)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.85 (2/52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.17 (7/24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e "},{"header":"Discussion","content":"\u003cp\u003eIn this study, we found that ICSI-AOA improved the D3 good quality embryo rate compared with previous IVF treatment in some patients included in the study. The number of D3 embryo blastomeres is a significant factor affecting embryo quality. We observed that the mean number of D3 embryo blastomere was significantly increased after ICSI-AOA compared with previous IVF cycles. Nevertheless, we observed no significant difference in the proportion of D3 embryos with different blastomeres which might be associated with limited cases (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eEmbryo fragmentation is also an important factor affecting embryo quality. The origin of fragmentation is still not fully understood and is likely to be multifactorial. Some factors including embryo culture condition, female age, gamete quality, ovulation induction, aneuploidy, and fertilization method appear to play a significant role in the etiology of cytoplasmic fragmentation. In this research, the main differences between the two groups were fertilization methods and the number of oocytes retrieved. Some researches demonstrated that ICSI did not have a significant impact on the D3 embryo fragmentation rate which suggested that ICSI was not a major contributor to adverse outcomes related to embryo fragmentation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Another retrospective study indicated that the lower number of oocytes was associated with increased embryo fragmentation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. There was a significant difference in the number of oocyte retrieved, so we further analyzed the D3 embryo fragmentation rate after ICSI-AOA compared with that of previous C-IVF cycles. No significant difference was observed in the D3 embryo fragmentation rate between previous C-IVF and subsequent ICSI-AOA cycles. And we also observed that there was no significant difference in the proportion of embryos with different degree of fragmentation (Supplementary Fig.\u0026nbsp;2). A recent systematic review concluded that the origin and the significance of iatrogenic or external factors on fragmentation of cleavage-stage embryos varies in the published papers [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImportantly, our data also indicated that ICSI-AOA significantly improved the pregnancy and live birth rates compared with previous IVF cycles, highlighting its potential to not only improve embryo quality but also increase clinical success rates. These findings suggest that ICSI-AOA could offer a valuable approach for patients struggling with poor embryo development, potentially improving both embryo quality and the overall success of IVF treatments. Additionally, Multiple recent studies have demonstrated that ICSI-AOA can improve the developmental ability of embryos and pregnancy outcomes [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The abovementioned studies support our findings that AOA using calcium ionophores is positively associated with improved embryo quality. Although some researches showed promising results, two sibling-oocyte studies failed to confirm the benefits of AOA in addressing embryo developmental issues. One study enrolled 140 patients with normal fertilization rate\u0026thinsp;\u0026gt;\u0026thinsp;70%, but D3 good quality embryo rate\u0026thinsp;\u0026le;\u0026thinsp;30% in previous ICSI cycles. After ICSI-AOA, no significant difference was observed in the rate of D3 good quality embryos between the AOA and non-AOA groups. Additionally, AOA did not improve the clinical pregnancy and live birth rates [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The other study involved 19 patients with normal fertilization rate\u0026thinsp;\u0026gt;\u0026thinsp;30% and D5 poor embryo quality in at least one previous ICSI cycle. After ICSI-AOA, no significant differences were observed in the rates of D3 good quality embryos and D5 good quality blastocysts between the AOA and non-AOA groups [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A recent cohort study compared ICSI-AOA cycles and previous ICSI cycles in couples who had normal fertilization rates (60%) but impaired embryonic development (15% blastocyst formation) in at least two previous ICSI cycles. It also demonstrated that AOA did not improve the rates of D3 good quality embryos and D5 good quality blastocysts in the populations with recurrent embryo developmental problems after ICSI [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Nevertheless, no studies have reported whether ICSI-AOA can improve the embryo quality in standard IVF cases with recurrent developmental problems.\u003c/p\u003e \u003cp\u003eIn this research, most of the enrolled male partners exhibited normal semen parameters and only three of them presented a slightly higher rate of abnormal sperm morphology. Multiple researches have shown that isolated teratozoospermia may not adversely affect the overall success rates of IVF procedures [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The current findings indicate that patients with moderate and severe teratozoospermia can be effectively treated with IVF and rescue-ICSI can be performed when necessary.\u003csup\u003e26\u003c/sup\u003e Meanwhiles, it has been confirmed that oocyte competency is the key to embryo potential [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Thus, we hypothesize that the poor embryo quality is primarily attributable to factors related to the oocytes for standard IVF cases with recurrent developmental problems. It is important to acknowledge that oocyte activation deficiencies are not the only reasons contributing to poor embryo development. Factors such as sperm DNA damage, oocyte abnormalities in structural proteins and mitochondria, and issues with transcription factors may also contribute to poor embryo prognosis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Importantly, none of these can be rescued by assisted oocyte activation.\u003c/p\u003e \u003cp\u003e Although nearly half (11/24) of ICSI-AOA cycles showed an improved number of D3 good quality embryos compared to previous IVF cycle, this benefit was not observed in all patients participating in this study. Problems with oocyte-derived and deficiencies in downstream signaling pathways may be an important cause of poor embryo development [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Although some cases of oocyte-related could be overcome by AOA, it was also confirmed that AOA was not beneficial for all patients with a suspected oocyte-related activation deficiency [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. As mentioned before, AOA will in theory only benefit patients suffering from a Ca\u003csup\u003e+\u003c/sup\u003e deficiency.\u003c/p\u003e \u003cp\u003eWe further displayed the details of embryo development after previous C-IVF and subsequent ICSI-AOA cycles (Supplementary table). After ICSI-AOA, several patients (P1, P4, and P7) exhibited an increase in the number of D3 available embryo, D3 good quality embryo, and good quality blastocyst. All three patients (P1, P4, and P7) achieved successful live births. AOA treatment also proved beneficial for certain patients (P3, P6, P8, P9, P10, P14, P18, and P20), as they experienced an increase in the D3 good quality embryo. Among them, two patients (P14 and P20) achieved successful live births. Conversely, some patients (P12, P21, P22, and P24) did not benefit from ICSI-AOA, due to a decrease in the number of D3 good quality embryos compared with previous IVF cycles. Notably, two (P12 and P24) of these patients achieved successful live births. ICSI-AOA treatment was ineffective for the patients (P16, P17, and P19), all of whom produced fewer available embryos than during prior IVF cycles without achieving successful live births. Additionally, six other patients (P2, P5, P11, P13, P15, and P23) similarly did not benefit from ICSI-AOA since they had available embryos in previous IVF cycles but yielded no available embryos on day 3 after ICSI-AOA. It should be noted that the cancellation rate of no available embryos was not decreased after ICSI-AOA. We suspected that it might be associated with the low number of oocytes retrieved and 2PN zygotes. In 6 cycles with no available embryos after ICSI-AOA, there were 4 cycles in which no more than three oocytes were retrieved.\u003c/p\u003e \u003cp\u003eTo the best of the authors\u0026rsquo; knowledge, this is a new design to assess the effect of AOA on improving embryo development in standard IVF cases with recurrent developmental problems. Although various modifications to the culture strategy and ovarian stimulation protocol have been explored in attempts to improve embryo quality, it's difficult to get consistent and effective conclusions. It is challenging to identify the sole appropriate reason that can effectively improve embryo development for these patients. Despite this, it provides a new therapeutic idea which suggest that ICSI-AOA combined with the optimization of ovarian stimulation protocol may be worthy to attempt to improve embryo quality for the patients with recurrent developmental problems in standard IVF cases.\u003c/p\u003e \u003cp\u003eA recent meta-analysis has demonstrated that AOA could not only significantly improve live birth and overall pregnancy, but also did not affect the incidence of miscarriage, congenital birth defects, and neonatal sex ratio [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our data showed that there were 2 twin births in the 7 live deliveries after ICSI-AOA. The mean gestational age was 272 days in singleton and the mean gestational age was 254 days in twins. The mean birthweight was 3169 g in singleton and the mean birthweight was 2533 g in twins. And no congenital malformation occurred after ICSI-AOA. Thus, ICSI-AOA can be considered as an effective and safe method which may benefit to couples with recurrent developmental problems in standard IVF.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStrengths and limitations\u003c/h2\u003e \u003cp\u003eThere are certain weaknesses in the current study that should be underlined. First, the primary drawback is the retrospective design and small sample size. Second, there may be some potential bias and confounders that cannot be excluded. For previous IVF cycles, the number of oocytes retrieved is 8.08. For subsequent ICSI-AOA cycles, the number of oocytes retrieved is 12.33. We further displayed the details of each patient and observed that it mainly exhibited a significant increase in the number of eggs retrieved for 7 patients (P1, P3, P8, P9, P12, P13 and P20). Of these patients, only two patients (P1 and P8) showed an increase in the D3 available embryos after ICSI-AOA. Additionally, we choose the commercial A23187 for AOA in this research because the application of calcimycin is performed for AOA due to its established capacity to enhance embryo development [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Nevertheless, recent researches have demonstrated that AOA treatment using ionomycin yields higher activation compared to calcimycin [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Furthermore, failed ICSI-AOA cycles employing calcimycin can potentially be rescued through the use of ionomycin [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These data suggested that the embryo development might be further improved in these patients if ionomycin was utilized for AOA. Although the optimal AOA method was not employed, our data indicated that certain improvements could be achieved by AOA with calcimycin. Lastly, the data about blastocyst culture is limited so we are not certain whether AOA can improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after conventional IVF.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study demonstrated that ICSI-AOA may be beneficial to some patients with recurrent developmental problems in C-IVF treatment. Decision-makers need to pay more attention to these patients with repeated failures in C-IVF treatment. Given the limited data and methodological constraints, further data accumulation is needed to obtain more reliable conclusions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAOA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAssisted oocyte activation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIVF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIn vitro fertilization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eICSI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIntracytoplasmic sperm injection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTotal fertilization failure\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLow fertilization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOAF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOocyte activation failure\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFSH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFollicle-stimulating hormone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGnRH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGonadotropin-releasing hormone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBMI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBody mass index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eET\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEmbryo transfer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e2PN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTwo pronuclei\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.L. and J.S. designed the research; M.L. and S.Q. performed the research. X.X. analyzed and prepared the data; All authors have reviewed the manuscript and agreed to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported by Shaanxi Technology Committee Industrial Public Relation Project (Project Number: 2023-YBSF-034) and Young Physicians Program of Chinese Medical Association (No. 17020470716).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData available on request corresponding author due to privacy and ethical restrictions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study involving human participants was reviewed and approved by the Ethics Committee of Northwest Women\u0026rsquo;s and Children\u0026rsquo;s Hospital (No. 2022007). The study was complied with the Helsinki Declaration and the patient records were innominate ahead of the data analysis. Patient consent was waived due to the retrospective nature of the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable since there are no details, images, or videos relating to an individual person.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStricker SA. Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev Biol. 1999;211:157\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Sui Y, Xiao M, Sun X, Fu J. Assessing the impact of calcium ionophore on pregnancy outcomes in artificial oocyte activation cycles: a 10-year update of systematic review and meta-analysis. J Assist Reprod Genet. 2024;42:165\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMehdinejadiani S, Goudarzi N, Masjedi F, Govahi A, Mirani M, Mehdinejadiani K, et al. Clinical Applications of Assisted Oocyte Activation in Couples with Various Reproductive Problems: A Systematic Review. Reprod Sci. 2024;31:2916\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalakier H, Dziak E, Sojecki A, Librach C, Michalak M, Opas M. Calcium-binding proteins and calcium-release channels in human maturing oocytes, pronuclear zygotes and early preimplantation embryos. Hum Reprod. 2002;17:2938\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei Y, Wang J, Qu R, Zhang W, Tan Y, Sha Y, et al. 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Obstet Gynecol Sci. 2024;67:588.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNicholson CL, Dean M, Attia A, Milne PA, Martins da Silva S. Artificial oocyte activation improves ICSI outcomes following unexplained fertilization abnormalities. Reprod Biomed Online. 2024;49:104327.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAntonova I, Yunakova M, Bochev I, Magunska N, Yaneva G, Ivanov D. Assisted oocyte activation significantly improves zygote formation, cleavage, and implantation rates in patients with a history of fertilization failures. Physiol Int. 2024;111:207\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYin M, Li M, Li W, Wu L, Yan Z, Zhao J, et al. Efficacy of artificial oocyte activation in patients with embryo developmental problems: a sibling oocyte control study. Arch Gynecol Obstet. 2022;305:1225\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMateizel I, Santos-Ribeiro S, Segers I, Wouters K, Mackens S, Verheyen G. Effect of A23187 ionophore treatment on human blastocyst development-a sibling oocyte study. J Assist Reprod Genet. 2022;39:1225\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCardona Barber\u0026aacute;n A, Bonte D, Boel A, Thys V, Paredis R, Machtelinckx F, et al. Assisted oocyte activation does not overcome recurrent embryo developmental problems. Hum Reprod. 2023;38:872\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKasman AM, Li S, Zhao Q, Behr B, Eisenberg ML. Relationship between male age, semen parameters and assisted reproductive technology outcomes. Andrology. 2021;9:245\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillani MT, Morini D, Spaggiari G, Falbo AI, Melli B, La Sala GB, et al. Are sperm parameters able to predict the success of assisted reproductive technology? A retrospective analysis of over 22,000 assisted reproductive technology cycles. Andrology. 2022;10:310\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi D, Wang Y, Han L, Gao D, Yang C, Wang W, et al. Role of sperm morphological parameters in the selection of fertilization methods. Rev Int Androl. 2024;22:68\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeefe D, Kumar M, Kalmbach K. Oocyte competency is the key to embryo potential. 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Reprod Biomed Online. 2020;40:501\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia L, Chen P, Su W, He S, Guo Y, Zheng L, et al. Artificial oocyte activation with ionomycin compared with A23187 among patients at risk of failed or impaired fertilization. Reprod Biomed Online. 2023;46:35\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuintana-Veh\u0026iacute; A, Mart\u0026iacute;nez M, Zamora MJ, Rodr\u0026iacute;guez A, Vassena R, Miguel-Escalada I, et al. Significant differences in efficiency between two commonly used ionophore solutions for assisted oocyte activation (AOA): a prospective comparison of ionomycin and A23187. J Assist Reprod Genet. 2023;40:1661\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang M, Zhu L, Liu C, He H, Wang C, Xing C, et al. A Novel Assisted Oocyte Activation Method Improves Fertilization in Patients With Recurrent Fertilization Failure. Front Cell Dev Biol. 2021;9:672081.\u003c/span\u003e\u003c/li\u003e\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-pregnancy-and-childbirth","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prch","sideBox":"Learn more about [BMC Pregnancy and Childbirth](http://bmcpregnancychildbirth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/prch/default.aspx","title":"BMC Pregnancy and Childbirth","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Assisted oocyte activation, IVF, ICSI, Embryo quality, Clinical outcomes","lastPublishedDoi":"10.21203/rs.3.rs-6458796/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6458796/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAssisted oocyte activation (AOA) is employed to enhance fertilization rates following fertilization failure after intracytoplasmic sperm injection (ICSI). Several studies have also demonstrated that AOA may play a role in embryo development. Poor embryo quality remains one of the major challenges for patients with recurrent developmental problems. We aimed to investigate whether ICSI followed by assisted oocyte activation (ICSI-AOA) can improve embryo quality in conventional in vitro fertilization (C-IVF) cases with recurrent developmental problems.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis retrospective cohort single-center study compared ICSI-AOA cycles with previous C-IVF cycles in couples who had nearly normal fertilization rates (\u0026ge;\u0026thinsp;40%) but impaired embryonic development (at most one D3 good quality embryo for all previous cycles) in at least two prior IVF cycles. A total of 24 couples experiencing embryo developmental issues were included in this study from January 2019 to December 2022.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAfter ICSI-AOA, the D3 good quality embryo rate was significantly improved compared with previous C-IVF cycles (19.30 versus 4.04%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). We observed that the mean number of D3 embryo blastomere was significantly increased after ICSI-AOA compared with previous C-IVF cycles (7.85 versus 6.94; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). No significant difference was observed in the D3 embryo fragmentation rate between previous C-IVF and subsequent ICSI-AOA cycles (14% versus 13%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.088). The ICSI-AOA cycles showed significantly higher pregnancy (41.67 versus 19.23%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.039) and live birth (29.17 versus 3.85%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) rates compared with previous C-IVF treatment.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur data suggest that ICSI-AOA may be beneficial to some patients with recurrent developmental problems in C-IVF treatment.\u003c/p\u003e","manuscriptTitle":"Efficacy of assisted oocyte activation in improving the reproductive outcome in conventional IVF cases with recurrent developmental problems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-19 05:37:21","doi":"10.21203/rs.3.rs-6458796/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2025-05-15T07:20:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-13T09:58:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-24T10:31:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-23T08:09:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pregnancy and Childbirth","date":"2025-04-23T08:08:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pregnancy-and-childbirth","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prch","sideBox":"Learn more about [BMC Pregnancy and Childbirth](http://bmcpregnancychildbirth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/prch/default.aspx","title":"BMC Pregnancy and Childbirth","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e2594025-9649-4215-b928-4678481192c3","owner":[],"postedDate":"May 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-05-19T05:37:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-19 05:37:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6458796","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6458796","identity":"rs-6458796","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}