The Effect of Artificial Oocyte Activation on Embryomorphokinetic Parameters and Blastulation Time

The Effect of Artificial Oocyte Activation on Embryomorphokinetic Parameters and Blastulation Time | 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 The Effect of Artificial Oocyte Activation on Embryomorphokinetic Parameters and Blastulation Time Funda Gode, İbrahim Pala, Burcu Tamer, Ahmet Zeki Işık This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4191561/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Does artificial oocyte activation (AOA) with calcium ionophore (Cai) application affect embryomorphokinetic parameters and clinical pregnancy outcomes? Methods This retrospective cohort study investigated if AOA with Cai affects the embryomorphokinetic parameters and clinical pregnancy outcomes of 413 patients. A total of 225 patients underwent intracytoplasmic sperm injection (ICSI) + AOA and 187 patients underwent only ICSI at Izmir University of Economics between 2018–2020. Results There were no significant differences between groups in terms of baseline parameters. When the duration from 2PN to tb (time to blastulation) was examined, t2, t3, t4, or t5 times were not significantly different between the groups that received and did not receive AOA treatment. Ca ionophore application was found to extend cavitation time and tb (p < 0.05). Optimal time lapse parameters (t5, cc2, S2 and t5-t2) also did not differ between two groups. There were no significant differences between groups according to embryo grades which were determined by optimal time lapse parameters. According to the fertilization and pregnancy results, no significant differences were found between the two groups. Conclusions Ca ionophore application does not seem to affect most of the embryomorphokinetic parameters except blastulation time. The clinical importance of this finding needs further research. calcium ionophore artificial oocyte activation embryomorphokinetics clinical pregnancy outcome fertilization blastulation Figures Figure 1 Figure 2 What does this study add to the clinical work There were no significant differences in embryomorphokinetic parameters between artificial oocyte activation(AOA) group and ICSI only group except extension of the cavitation and tb times. The clinical importance of prolongation of cavitation and blastulation times in AOA group needs further research. Introduction Intracytoplasmic sperm injection (ICSI) is commonly used in assisted reproductive technology (ART) and involves the microinjection of a single sperm cell directly into a mature oocyte; it is primarily used to address severe cases of male factor infertility. This technique is also useful for achieving fertilization when previous in vitro fertilization (IVF) attempts have been unsuccessful [ 1 – 3 ]. Although ICSI results in fertilization rates of 70–80%, it does sometimes fail [ 3 , 4 ], due to either oocyte abnormalities or, most commonly, activation deficiency [ 5 – 8 ]. Oocyte activation is an intricately regulated process that is triggered by intracellular calcium (Ca) oscillations from endoplasmic reticulum stores after the sperm enters the ooplasm [ 5 ]. Sperm-specific phospholipase C-zeta (PLCz), a sperm factor located in the peri-nuclear theca of spermatozoa, is the primary factor responsible for inducing intracellular Ca oscillations via an inositol-1,4,5-triphosphate (IP3)-mediated pathway [ 9 – 11 ]. When IP3 binds to receptors on the surface of the endoplasmic reticulum, it triggers the release of Ca from the internal stores of the cell [ 12 ]. Consequently, the primary reason for unsuccessful fertilization and the failure of oocyte activation is often considered a deficiency in the PLCz cascade related to sperm [ 13 , 14 ]. Ca activation is important for fertilization and is related to the activation of CAMKII and inactivation of MPF and subsequent meiotic resumption and pronucleus formation. Subsequent Ca oscillations are also important for the first mitotic division and later embryologic development processes [ 15 ]. Therefore, these oscillations, which are regulated by changes in the intracellular Ca2 + concentration in the oocyte, are responsible for controlling the processes that enable a smooth transition from oocyte to embryo development [ 16 – 19 ]. Researchers have attempted to improve the clinical outcomes of patients who experience fertilization failure or low fertilization rates using artificial oocyte activation (AOA), including the use of PLCz and artificial activators such as Ca ionomycin, Ca ionophore (A23187), strontium chloride, electrical pulse activation, and mechanical activation [ 20 ]. According to studies and meta-analyses, Ca ionophore (Cai) application significantly improves the overall fertilization rates in patients who experience fertilization failure [ 21 – 23 ]. Positive effects of Cai application have been detected at various embryonic development stages [ 19 ]. Although male factor infertility and fertilization failure history are the main indications for Cai, its use also improves fertilization success in patients with infertility for various other reasons [ 3 , 24 – 26 ]. In a recent study, an AOA group had better embryo quality than a non-AOA group in women with advanced maternal age and diminished ovarian reserve, with higher rates of cell cleavage and top-quality day 3 embryos [ 26 ]. Time-lapse monitoring systems (TLMSs) have been developed to objectively observe the progression of embryo development, enabling embryologists to continuously observe embryos while analyzing their dynamic and morphokinetic events. This allows for more comprehensive analyses of embryo development [ 27 ]. With conventional incubators, many morphological changes are frequently overlooked, including irregular cell division, blastocyst collapse and re-expansion, and the formation and absorption of fragments. However, TLMS can detect and analyze these changes [ 28 ]. In addition, time lapse parameters can be used to predict aneuploidy based on embryo grading [ 29 , 30 ]. By combining TLMS with PGT-A, the selection of euploid embryos with the greatest potential for implantation may be increased [ 28 ]. TLMS also enhances the criteria for selecting blastocysts and can accurately predict blastocyst formation [ 31 ]. Although many studies have investigated the effect of Cai after ICSI on fertilization, few have investigated the impact of Cai on embryomorphokinetic parameters and clinical pregnancy outcomes [ 19 , 22 ]. Therefore, we investigated the effects of oocyte activation with Cai on embryomorphokinetic parameters and clinical pregnancy outcomes in a large cohort including patients with various causes of infertility. Materials and Methods Patients A total of 413 patients underwent ICSI/ICSI + AOA at the Izmir University of Economics Medical Point IVF Unit between 2018–2020. ICSI + AOA was used in 225 patients while ICSI only was used in 187. The cycle results were compared retrospectively. The main criteria for Cai use were male factor infertility, previous fertilization failure, low fertilization rates (< 30%) and poor embryological development pattern in previous cycles. PGT-A cases and azoospermia patients were not included in the analysis. Oocyte stimulation Gonadotropins (follitropin alfa or menotropin 150–450IU) were administered starting on day 3 of the menstrual cycle, with 0.25mg GnRH antagonist cetrorelix acetate (Cetrotide, Merck, Germany) on cycle day 7. Human chorionic gonadotropin was used to trigger ovulation once the dominant follicle reached 18mm and oocyte pick-up was performed 35h after triggering. Oocyte collection, fertilization and time lapse embryo culture Oocyte–cumulus complexes were selected in Quinn’s Advantage™ Medium with HEPES plus HSA (Sage, USA) and surplus cumulus was dissected from around the oocytes using a stereomicroscope in a class II hood with a heated stage. The oocytes were washed and cultured in SAGE 1 Step™, with HSA (Origio, Denmark) covered with liquid paraffin oil (Origio, Denmark) for between 2 and 4 h in an atmosphere of 5.5% CO2 and 5.0% O2 in a Sanyo MCO- 19M (Panasonic) incubator. To prepare Cai, ionophore compound (GM508 CultActive) medium (Ca Ionophore) was first incubated in a standard incubator at 37°C with 5.5% CO2 for 4 h Then the microinjected oocytes were immediately placed in a solution containing Ca active substance for 15 minutes. The oocytes were incubated in the time lapse monitoring system (EmbryoScope; Unisense FertiliTech, Denmark) in an atmosphere of 5% O2, 5.5% CO2, at 37℃ until embryo transfer. Time-lapse monitoring embryo culture system EmbryoViewer software (Unisense Fertilitech Aarhus) was used to inspect the embryo images to elucidate the embryo growth after microinjection over time (hours). Pronuclear fading (Pnf), the moment when both pronuclei disappear, first cleavage, and the times at which the zygote divides into 2–8 cells (t2 to t8, respectively) were evaluated according to Basile et al.’s classification [ 29 ]. The duration of the cycle for the second cell (cc2 = t3–t2) is the time of embryo cleavage from two blastomeres into three blastomeres. The time from this cleavage to a four-blastomere embryo is the second synchrony (s2 = t4–t3). The time for the three- blastomere embryo to split into a five-blastomere embryo is the prolongation of the third cell cycle (cc3 = t5–t3). A previous study determined the optimal morphological values for the best implantation rate for t5 (t5 = 48.8–56.6 h), s2 (s2 < 0.76 h), and cc2 (cc2 < 12 h, 20.5 < t5–t2 < 29, and 11 < cc3[t5–t3] 20.5 h were defined as A or B, while those outside this optimal range were defined as C or D. Embryos with a duration of cc3 between 11 and 18 h were classified as A or C, while those beyond the optimal range were classified as B or D, depending on t5-t2 [ 29 ]. Embryo transfer Embryos were transferred between the second and fifth days of development with a Sure-Pro Ultra Embryo Replacement Catheter with an Obturator (Cooper Surgical Company, The Netherlands) and a medium Sage One Step. All patients received luteal support with the daily use of a 400 mg vaginal progesterone tablet (Progestan vaginal capsule 400 mg) and 30 mg oral dydrogesterone (Duphaston tablet 10 mg; Abbott Laboratories, Eczacibasi, Turkey). A biochemical pregnancy is characterized by a serum level of β-hCG > 20 IU/I on day 12 following embryo transfer. Clinical pregnancy is established when the activity of the heart of the fetus is detected through ultrasonography 8 weeks post-embryo transfer. Statistical Analysis All data statistical analysis were performed using Statistical Package for Social Sciences (SPSS) 26.0 studio program and p < 0.05 was considered statistically significant. Continuous data were shown as the mean and standart deviation (SD). Independent sample T-test was used for the analysis of the baseline parameters and the embryomorphokinetic parameters. Categorical variables (fertilization, embryo grade A, clinical pregnancy rates) were presented as percentages and were evaluated using the pearson’s chi-square test. Power Analysis The G*Power 3.1.9 package program was employed for the power analysis of the study. Following the collection of data, a post-hoc power analysis based on pregnancy outcomes revealed that the study had a power of 0.89 for 413 patients. Ethical Approval Institutional Review Board approval was obtained from Bahçeşehir University Faculty of Medicine before the beginning of the study. Results A total of 413 patients were included. AOA group consisted 225 patients and non-AOA group included 187 patients. When female age, BMI, duration of infertility, duration of induction(days), oocyte count, MII count and 2PN count were examined, there were no significant differences between groups (Table 1). When the durations from 2PN to the blastulation time (tb) were examined, no significant differences were found between the two groups in terms of t2, t3, t4, or t5. When the optimal time lapse parameters (t5, cc2, S2, cc3, and t5-t2) were evaluated no significant relation was found between the probability of optimal time lapse parameters and the use of Cai (Table2). The use of Cai was found to extend (p<.05) cavitation (Figure 1) and blastulation times (tb) (p<.05) (Figure 2). According to the fertilization results, there were no significant differences between the groups that received and that did not receive AOA treatment (69.87% vs. 74.4%, respectively). When it comes to embryo grades, the grade A embryo rates were not significantly different between AOA and non-AOA groups (75.3% vs. 71.7% respectively). There were also no significant differences in the clinical pregnancy outcomes (46.2% vs. 54.6%, respectively) (Table 3). Discussion In the present study, we investigated the effects of AOA application with Cai on embryomorphokinetic parameters and embryological development. To the best of our knowledge, our sample population is the largest of any study conducted on this subject to date. Some clinical studies and meta-analyses have reported that AOA may help improve fertilization, embryonic development, and clinical pregnancy outcomes in many clinical conditions [21, 23]. Therefore, we performed AOA mostly in patients with poor prognoses. Also, because changes in intracellular Ca levels influence the entire development process from oocyte to embryo transition, we examined the correlations between Cai application and morphokinetic parameters. The use of AOA is controversial, as synthetic activating agents do not fully replicate the natural Ca signaling and physiological mechanisms observed in mammalian zygotes [19, 32]. Activation of the embryo by Cai may lead to variation in behavior and division patterns that differ from normal processes. Therefore, this technique is still experimental, and gathering data from a larger number of patient embryos could yield valuable insights. Few studies have evaluated the relationship between AOA and the morphokinetic behavior of the embryo. Martinez et al. reported that AOA had no effect, with the exception of second polar body extrusion (tPB2) and time to second cell division (t3); however, they concluded that these changes did not seem affect the morphokinetic pattern of the preimplantation embryo and ascribed the findings to a rapid transient nonphysiological increase in free intracytoplasmic Ca2+ [19]. Similarly, Shebl et al. performed a sibling oocyte study in patients with previous fertilization problems. Half of the oocytes were treated with calcimycin. They found that ionophore-treated oocytes underwent significantly earlier pronuclei transformation and had a better synchronized third cell cycle [22]. In both of these studies, the patient population included couples who had experienced fertilization failure and severe male factor infertility similar with our study. However, in contrast to their findings, in our study pronuclei transformation or third cell cycle synchronization were not different and we found a prolonged tb in AOA cycles. Research on humans has highlighted the potential of tb as a determining factor for selecting embryos, surpassing other components of embryo morphological grading [35-37]. Lee et al. and Moustafa et al. noted delayed tb in embryos with chromosome abnormalities [36, 38]. Recently, Quintana-Vehí and colleagues conducted a study comparing groups in donor cycles, including calcimycin, ionomycin, and conventional ICSI cycles. ICSI was not applied in AOA groups, only parthenogenetic activation was examined. According to the results, none of the calcimycin parthenotes formed blastocysts, while an extension in blastulation was observed in the ionomycin group. Although there are some differences on designs of this study and our study we also found an extension in tb [39]. The clinical importance of these changes in embryomorphokinetic parameters is not clear. AOA process results in non-physiological calcium changes in the cells. In normal physiological processes, calcium release during oocyte activation occurs in the form of oscillations from internal stores. However, in artificial oocyte activation, it increases in a peak- shaped manner. This situation may be related to the faster expulsion of the second polar body in the initial fertilization and faster cleavage stages (t3) mentioned in previous reports, while the lack of long-lasting oscillations may be associated with the prolongation of the tb period [19,22]. Previous studies and our study results may suggest the importance of taking caution when applying Cai and the prolonged tb duration may serve as a warning for future clinical practice. It would be more appropriate to consider its utilization in cases where there is a clear indication rather than opting for elective use. Morphokinetic behavior has also been used to predict euploidy rates in different studies [29,30] because aneuploidy can potentially occur at any stage from the disjunction of chromatids to the mitotic divisions within the embryo [40-43]. We used the morphokinetic embryo grading system that predicts aneuploidy devised by Basile et al. and did not find any differences between the two groups. The literature contains little information on embryo aneuploidy and Cai activation. However, our results are in line with a recent study that found that Cai application did not increase the risk for aneuploidy and there were no developmental differences in live births as a result of transfers made with Cai administration [20]. Thus Ca2 ionophores have the capability to bind Ca2 cations, and due to their hydrophobic properties, they form a complex at the lipid bilayer of the membrane. This structure enables ionophores to transfer Ca2 across the membrane and release them into the cytosol [44]. Consequently, ionophores themselves may not necessarily enter the oocyte, potentially explaining the absence of a detectable effect of ionophores on chromosomal segregation [45]. Reports have indicated that implanted embryos after IVF, using Cai as an artificial oocyte activator, do not exhibit any chromosome abnormalities [7,47]. Moreover, no perinatal morbidity or mortality was reported among the patients involved in these studies. Comparable outcomes have also been observed in children aged 3–10 years, who were born following ICSI cycles using Cai. No differences have been noted compared to their peers [32,48]. The principal limitation of our study lies in its retrospective design. Consequently, definitive conclusions are challenging to establish. However, it is important to acknowledge that this limitation is mitigated by our substantial sample size. Our findings may help understanding the embryomorphokinetic behavior and embryo selection in AOA cycles in future applications. In conclusion, we did not find significant differences in most of the embryomorphokinetic parameters except prolongation of tb and cavitation time. However, the clinical significance of these changes is controversial because of the lack of correlations between these parameters and clinical outcomes. More studies with larger populations are needed on this subject. Declarations Author Contribution: FG designed, planned and wrote the manuscript; IP and BT collected data and applied the treatment in the laboratory; AZI drafted the manuscript and supervised the manuscript writing. All authors read and approved the final manuscript. Acknowledgements: We would like to thank Selin Madran (Nursing) and Buket Ekmekçi (Biostatistics) for their contributions. Funding: None Data Availability: The data that support the findings of this study are available from the corresponding author(F.G) upon reasonable request. Conflict of Interest: The authors have no relevant financial or non-financial interests to this close. Ethical Approval: This study was performed in line with the principles of Bahçeşehir University Faculty of Medicine and Institutional Review Board of Bahçeşehir University approved the study. Consent to participate: Informed consent was obtained from all individual participants included in the study. Consent to publish: Participants signed informed consent regarding publishing their data. References Palermo G, Joris H, Devroey P, Van Steirteghem AC (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340:17-18. https://doi.org/10.1016/0140-6736(92)92425-F Van Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, Wisanto A, Devroey P (1993) High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 8:1061-1066. https://doi.org/10.1093/oxfordjournals.humrep.a138192 Lv M, Zhang D, He X, Chen B, Li Q, Ding D, Zhang Z (2020) Artificial oocyte activation to improve reproductive outcomes in couples with various causes of infertility: a retrospective cohort study. Reprod BioMed online 40:501-509. https://doi.org/10.1016/j.rbmo.2020.01.001 Montag M, Köster M, van der Ven K, Bohlen U, & van der Ven H (2012) The benefit of artificial oocyte activation is dependent on the fertilization rate in a previous treatment cycle. Reprod BioMed online 24:521-526. https://doi.org/10.1016/j.rbmo.2012.02.002 Tesarik J, Sousa M, Testart J (1994) Human oocyte activation after intracytoplasmic sperm injection. Hum Reprod 9:511-518. https://doi.org/10.1093/oxfordjournals.humrep.a138537 Dozortsev D, De Sutter P, Dhont M (1994) Behaviour of spermatozoa in human oocytes displaying no or one pronucleus after intracytoplasmic sperm injection. Hum Reprod 9:2139-2144. https://doi.org/10.1093/oxfordjournals.humrep.a138406 Lu Q, Chen ZJ, Gao X, Ma SY, Li M, Hu JM, & Li Y (2006) Zhonghua fu chan ke za zhi, 41:182–185. https://doi.org/10.1007/s10495-012-0702-9 Nasr-Esfahani MH, Razavi S, Mardani M, Shirazi R, Javanmardi S (2007) Effects of failed oocyte activation and sperm protamine deficiency on fertilization post-ICSI. Reprod BioMed online 14:422-429. https://doi.org/10.1016/s1472-6483(10)60888-7 Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, Lai FA (2002) PLCζ: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo. Development 129:3533–3544. https://doi.org/10.1242/dev.129.15.3533 Kashir J, Heindryckx B, Jones C, De Sutter P, Parrington J, Coward K (2010) Oocyte activation, phospholipase C zeta and human infertility. Hum Reprod update 16:690-703. https://doi.org/10.1093/humupd/dmq018 Ramadan WM, Kashir J, Jones C, Coward K (2012) Oocyte activation and phospholipase C zeta (PLCζ): diagnostic and therapeutic implications for assisted reproductive technology. Cell Commun Signal 10:1-20. https://doi.org/10.1186/1478-811X-10-12 Berridge MJ (2009) Inositol trisphosphate and calcium signalling mechanisms. Biochim Biophy Acta Mol Cell Res 1793:933-940. https://doi.org/10.1016/j.bbamcr.2008.10.005 Yoon SY, Eum JH, Lee JE, Lee HC, Kim YS, Han JE, Won H, Park S, Shim S, Lee WS, Fissore R, Lee DR, Yoon TK (2012) Recombinant human phospholipase C zeta 1 induces intracellular calcium oscillations and oocyte activation in mouse and human oocytes. Hum Reprod 27:1768-1780. https://doi.org/10.1093/humrep/des092 Nomikos M, Kashir J, Swann K, Lai FA (2013) Sperm PLCζ: from structure to Ca2+ oscillations, egg activation and therapeutic potential. FEBS letters 587:3609-3616. https://doi.org/10.1016/j.febslet.2013.10.008 Neri QV, Lee B, Rosenwaks Z, Machaca K, Palermo GD (2014) Understanding fertilization through intracytoplasmic sperm injection (ICSI). Cell calcium 55:24-37. https://doi.org/10.1016/j.ceca.2013.10.006 Putney Jr JW (1986) A model for receptor-regulated calcium entry. Cell calcium 7:1-12. https://doi.org/10.1016/0143-4160(86)90026-6 Saunders CM, Swann K, & Lai FA (2007) PLCζ, a sperm-specific PLC and its potential role in fertilization. Biochem Soc Symp 74:23-36. https://doi.org/10.1042/BSS0740023 Malcuit C, Kurokawa M, Fissore RA (2006) Calcium oscillations and mammalian egg activation, J Cell Physiol 206:565-573. https://doi.org/10.1002/jcp.20471 Martinez M, Durban M, Santaló J, Rodríguez A, Vassena R (2021) Assisted oocyte activation effects on the morphokinetic pattern of derived embryos. J Assist Reprod Genet 38:531-537. https://doi.org/10.1007/s10815-020-02025-9 Zhang J, Yao G, Zhang T, Hu J, Yang G, He J, Qina H, Fan H, Bai Y, Sun Y (2023) Effect of calcium ionophore (A23187) on embryo development and its safety in PGT cycles. Front Endocrinol 13:3286. https://doi.org/10.3389/fendo.2022.979248 Murugesu S, Saso S, Jones BP, Bracewell-Milnes T, Athanasiou T, Mania A, Serhal P, Ben-Nagi J (2017) Does the use of calcium ionophore during artificial oocyte activation demonstrate an effect on pregnancy rate? A meta-analysis. Fertil Steril 108:468-482. https://doi.org/10.1016/j.fertnstert.2017.06.029 Shebl O, Trautner PS, Enengl S, Reiter E, Allerstorfer C, Rechberger T, Oppelt P, Ebner T (2021) Ionophore application for artificial oocyte activation and its potential effect on morphokinetics: a sibling oocyte study. J Assist Reprod Genet 38:3125-3133. https://doi.org/10.1007/s10815-021-02338-3 Chen X, Zhao H, Lv J, Dong Y, Zhao M, Sui X, Cui R, Liu B, Wu K (2022) Calcium ionophore improves embryonic development and pregnancy outcomes in patients with previous developmental problems in ICSI cycles. BMC Pregnancy and Childbirth 22:894. https://doi.org/10.1186/s12884-022-05228-3 Ebner T, Köster M, Shebl O, Moser M, Van der Ven H, Tews G, Montag M (2012) Application of a ready-to-use calcium ionophore increases rates of fertilization and pregnancy in severe male factor infertility. Fertil Steril 98:1432-1437. https://doi.org/10.1016/j.fertnstert.2012.07.1134 Xu Z, Yao G, Niu W, Fan H, Ma X, Shi S, Jin H, Song W, Sun Y (2021) Calcium ionophore (A23187) rescues the activation of unfertilized oocytes after intracytoplasmic sperm injection and chromosome analysis of blastocyst after activation. Front Endocrinol 12:692 https://doi.org/10.3389/fendo.2021.692082 Tsai TE, Lin PH, Lian PF, Li CJ, Vitale SG, Mikuš M, Su WP, Tsai H, Tsui K, Lin LT (2022) Artificial oocyte activation may improve embryo quality in older patients with diminished ovarian reserve undergoing IVF- ICSI cycles. J Ovarian Res 15:1-9. https://doi.org/10.1186/s13048-022-01036-7 Sciorio R (2021) Use of time-lapse monitoring in medically assisted reproduction treatments: A mini- review. Zygote 29:93-101. https://doi.org/10.1017/S0967199420000623 Zaninovic N, Irani M, & Meseguer M (2017) Assessment of embryo morphology and developmental dynamics by time-lapse microscopy: is there a relation to implantation and ploidy?. Fertility and sterility 108:722– 729. https://doi.org/10.1016/j.fertnstert.2017.10.002 Basile N, del Carmen Nogales M, Bronet F, Florensa M, Riqueiros M, Rodrigo L, García-Velasco J, Meseguer M (2014) Increasing the probability of selecting chromosomally normal embryos by time-lapse morphokinetics analysis. Fertil Steril 101:699-704. https://doi.org/10.1016/j.fertnstert.2013.12.005 Patel DV, Shah PB, Kotdawala AP, Herrero J, Rubio I, Banker MR (2016) Morphokinetic behavior of euploid and aneuploid embryos analyzed by time-lapse in embryoscope. J Hum Reprod Sci 9:112. https://doi.org/10.4103/0974-1208.183511 Motato Y, de los Santos MJ, Escriba MJ, Ruiz BA, Remohí J, & Meseguer M (2016) Morphokinetic analysis and embryonic prediction for blastocyst formation through an integrated time-lapse system. Fertil Steril 105:376-384. https://doi.org/10.1016/j.fertnstert.2015.11.001 Vanden Meerschaut F, D'Haeseleer E, Gysels H, Thienpont Y, Dewitte G, Heindryckx B, Oostra A, Roeyers H, Van Lierde K, De Sutter P (2014) Neonatal and neurodevelopmental outcome of children aged 3-10 years born following assisted oocyte activation. Reprod Biomed Online 28:54-63. https://doi.org/10.1016/j.rbmo.2013.07.013 Meseguer M, Herrero J, Tejera A, Hilligsøe KM, Ramsing NB, Remohí J (2011) The use of morphokinetics as a predictor of embryo implantation. Hum Reprod 26:2658-71. https://doi.org/10.1093/humrep/der256 Muthukumar K, Kamath MS, Mangalaraj AM, Aleyamma TK, Chandy A, George K (2013) Comparison of clinical outcomes following vitrified warmed day 5/6 blastocyst transfers using solid surface methodology with fresh blastocyst transfers. J Hum Reprod Sci 6:59. https://doi.org/10.4103/0974-1208.112384 Ho JR, Arrach N, Rhodes-Long K, Salem W, McGinnis LK, Chung K, Bendikson K, Paulson JR, Ahmady A (2018) Blastulation timing is associated with differential mitochondrial content in euploid embryos. J Assist Reprod Genet 35:711-720. https://doi.org/10.1007/s10815-018-1113-9 Moustafa SM, Rosen EM, Boylan C, Mersereau JE (2020) Time to blastulation is superior to individual components of embryo grading for live-birth prediction. F&S Reports 1:243-248. https://doi.org/10.1016/j.xfre.2020.09.016 Huayhua C, Rodríguez M, Vega J, Briones M, Rodriguez-Alvarez L, Mellisho E (2023) Blastulation time measured with time-lapse system can predict in vitro viability of bovine blastocysts. PloS one 18. https://doi.org/10.1371/journal.pone.0289751 Lee CI, Chen CH, Huang CC, Cheng EH, Chen HH, Ho ST, Lee TH (2019) Embryo morphokinetics is potentially associated with clinical outcomes of single-embryo transfers in preimplantation genetic testing for aneuploidy cycles. Reprod BioMed online 39:569-579. https://doi.org/10.1016/j.rbmo.2019.05.020 Quintana-Vehí A, Martínez M, Zamora MJ, et al (2023) 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 40:1661–1668. https://doi.org/10.1007/s10815-023-02833-9 Hassold T, Hunt P (2001) To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet 2:280-291. https://doi.org/10.1038/35066065 Wang WH, & Sun QY (2006) Meiotic spindle, spindle checkpoint and embryonic aneuploidy. Front Biosci 11:620-636. https://doi.org/10.2741/1822 Chavez SL, Loewke KE, Han J, Moussavi F, Colls P, Munne S, & Reijo Pera RA (2012) Dynamic blastomere behaviour reflects human embryo ploidy by the four-cell stage. Nature commun 3:1251. https://doi.org/10.1038/ncomms2249 Campbell A, Fishel S, Bowman N, Duffy S, Sedler M, & Hickman CFL (2013) Modelling a risk classification of aneuploidy in human embryos using non-invasive morphokinetics. Reprod BioMed online 26:477- 485. https://doi.org/10.1016/j.rbmo.2013.02.006 Brasseur R, Notredame M, Ruysschaert JM (1983) Lipid-water interface mediates reversible ionophore conformational change. Biochem Biophys Res Commun 114:632–637. https://doi.org/10.1016/0006-291x(83)90827-6 Capalbo A, Ottolini CS, Griffin DK, Ubaldi FM, Handyside AH, Rienzi L (2016) Artificial oocyte activation with calcium ionophore does not cause a widespread increase in chromosome segregation errors in the second meiotic division of the oocyte. Fertil Steril 105:807–814. https://doi.org/10.1016/j.fertnstert.2015.11.017 ESHRE Add-ons working group, Lundin K, Bentzen JG, Bozdag G, Ebner T, Harper J, Le Clef N, Moffett A, Norcross S, Polyzos NP, Rautakallio-Hokkanen S, Sfontouris I, Sermon K, Vermeulen N, Pinborg A (2023) Good practice recommendations on add-ons in reproductive medicine. Human Reproduction 38:2062–2104. https://doi.org/10.1093/humrep/dead184 Nakagawa K, Yamano S, Moride N, Yamashita M, Yoshizawa M, Aono T (2001) Effect of activation with Ca ionophore A23187 and puromycin on the development of human oocytes that failed to fertilize after intracytoplasmic sperm injection. Fertil Steril 76:148-152. https://doi.org/10.1016/S0015-0282(01)01839-8 Deemeh MR, Tavalaee M, Nasr-Esfahani MH (2015) Health of children born through artificial oocyte activation: a pilot study. Reprod Sciences 22:322-328. https://doi.org/10.1177/1933719114542017. 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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-4191561","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":303662404,"identity":"6af62992-2ec5-4bb2-9716-9fbe28ddd28a","order_by":0,"name":"Funda Gode","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYBAC9gYGBgkw6wDzASApIUNQC88BuBa2BJAWHlK08BiABQhrkcg9eOMHQ60c3/Gez69u1FjwMLAfProBv5a8ZMsehuPGkmfObrPOOQZ0GE9a2g18WuwlcsyAyo4lbriRu804hw3IluAxw6uFB6hF8g9Iy/03z4xz/hGpRZqHoQZoCw/z49w2YrTwvDG2ljE4APRLmhlzbp8EDxshv/Cw5xjefFNRBwyxw48/53yrk+NnP3wMrxYIMDgMItnAEcRGWDkY1IEI5g9Eqh4Fo2AUjIIRBgC2PkVryibbdQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-9371-6942","institution":"Dokuz Eylul University","correspondingAuthor":true,"prefix":"","firstName":"Funda","middleName":"","lastName":"Gode","suffix":""},{"id":303662405,"identity":"fdea7fd4-748a-430b-9555-99ce641d7c5b","order_by":1,"name":"İbrahim Pala","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"İbrahim","middleName":"","lastName":"Pala","suffix":""},{"id":303662406,"identity":"99d8cf75-0df5-4332-a1b8-f58bd7f0f083","order_by":2,"name":"Burcu Tamer","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Burcu","middleName":"","lastName":"Tamer","suffix":""},{"id":303662407,"identity":"ec2078b2-5629-4cd2-9ca5-78804690a402","order_by":3,"name":"Ahmet Zeki Işık","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ahmet","middleName":"Zeki","lastName":"Işık","suffix":""}],"badges":[],"createdAt":"2024-03-30 09:33:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4191561/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4191561/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57707094,"identity":"0c7da3c6-2b3f-4e62-8e82-02eb6657ac48","added_by":"auto","created_at":"2024-06-04 15:06:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":127809,"visible":true,"origin":"","legend":"\u003cp\u003eCavitation time\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4191561/v1/f63742fe611c221073126a83.png"},{"id":57707095,"identity":"a51a0578-23c5-4dad-9572-b6a592121b1d","added_by":"auto","created_at":"2024-06-04 15:06:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":130029,"visible":true,"origin":"","legend":"\u003cp\u003eBlastulation time\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4191561/v1/e2fcfd3cfb5a0a14c15b5d62.png"},{"id":71874338,"identity":"1127abc2-77f4-4005-aeed-a3969b57c534","added_by":"auto","created_at":"2024-12-19 10:47:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":664247,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4191561/v1/d243deb0-e204-4576-8db6-2c7c93c37c4e.pdf"}],"financialInterests":"","formattedTitle":"The Effect of Artificial Oocyte Activation on Embryomorphokinetic Parameters and Blastulation Time","fulltext":[{"header":"What does this study add to the clinical work","content":"\u003cp\u003eThere were no significant differences in embryomorphokinetic parameters between artificial oocyte activation(AOA) group and ICSI only group except extension of the cavitation and tb times. The clinical importance of prolongation of cavitation and blastulation times in AOA group needs further research.\u003c/p\u003e\n"},{"header":"Introduction","content":"\u003cp\u003eIntracytoplasmic sperm injection (ICSI) is commonly used in assisted reproductive technology (ART) and involves the microinjection of a single sperm cell directly into a mature oocyte; it is primarily used to address severe cases of male factor infertility. This technique is also useful for achieving fertilization when previous in vitro fertilization (IVF) attempts have been unsuccessful [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although ICSI results in fertilization rates of 70\u0026ndash;80%, it does sometimes fail [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], due to either oocyte abnormalities or, most commonly, activation deficiency [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOocyte activation is an intricately regulated process that is triggered by intracellular calcium (Ca) oscillations from endoplasmic reticulum stores after the sperm enters the ooplasm [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Sperm-specific phospholipase C-zeta (PLCz), a sperm factor located in the peri-nuclear theca of spermatozoa, is the primary factor responsible for inducing intracellular Ca oscillations via an inositol-1,4,5-triphosphate (IP3)-mediated pathway [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. When IP3 binds to receptors on the surface of the endoplasmic reticulum, it triggers the release of Ca from the internal stores of the cell [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Consequently, the primary reason for unsuccessful fertilization and the failure of oocyte activation is often considered a deficiency in the PLCz cascade related to sperm [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCa activation is important for fertilization and is related to the activation of CAMKII and inactivation of MPF and subsequent meiotic resumption and pronucleus formation. Subsequent Ca oscillations are also important for the first mitotic division and later embryologic development processes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, these oscillations, which are regulated by changes in the intracellular Ca2\u0026thinsp;+\u0026thinsp;concentration in the oocyte, are responsible for controlling the processes that enable a smooth transition from oocyte to embryo development [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResearchers have attempted to improve the clinical outcomes of patients who experience fertilization failure or low fertilization rates using artificial oocyte activation (AOA), including the use of PLCz and artificial activators such as Ca ionomycin, Ca ionophore (A23187), strontium chloride, electrical pulse activation, and mechanical activation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. According to studies and meta-analyses, Ca ionophore (Cai) application significantly improves the overall fertilization rates in patients who experience fertilization failure [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Positive effects of Cai application have been detected at various embryonic development stages [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Although male factor infertility and fertilization failure history are the main indications for Cai, its use also improves fertilization success in patients with infertility for various other reasons [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In a recent study, an AOA group had better embryo quality than a non-AOA group in women with advanced maternal age and diminished ovarian reserve, with higher rates of cell cleavage and top-quality day 3 embryos [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTime-lapse monitoring systems (TLMSs) have been developed to objectively observe the progression of embryo development, enabling embryologists to continuously observe embryos while analyzing their dynamic and morphokinetic events. This allows for more comprehensive analyses of embryo development [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. With conventional incubators, many morphological changes are frequently overlooked, including irregular cell division, blastocyst collapse and re-expansion, and the formation and absorption of fragments. However, TLMS can detect and analyze these changes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In addition, time lapse parameters can be used to predict aneuploidy based on embryo grading [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. By combining TLMS with PGT-A, the selection of euploid embryos with the greatest potential for implantation may be increased [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. TLMS also enhances the criteria for selecting blastocysts and can accurately predict blastocyst formation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough many studies have investigated the effect of Cai after ICSI on fertilization, few have investigated the impact of Cai on embryomorphokinetic parameters and clinical pregnancy outcomes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, we investigated the effects of oocyte activation with Cai on embryomorphokinetic parameters and clinical pregnancy outcomes in a large cohort including patients with various causes of infertility.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eA total of 413 patients underwent ICSI/ICSI\u0026thinsp;+\u0026thinsp;AOA at the Izmir University of Economics Medical Point IVF Unit between 2018\u0026ndash;2020. ICSI\u0026thinsp;+\u0026thinsp;AOA was used in 225 patients while ICSI only was used in 187. The cycle results were compared retrospectively.\u003c/p\u003e \u003cp\u003eThe main criteria for Cai use were male factor infertility, previous fertilization failure, low fertilization rates (\u0026lt;\u0026thinsp;30%) and poor embryological development pattern in previous cycles. PGT-A cases and azoospermia patients were not included in the analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eOocyte stimulation\u003c/h2\u003e \u003cp\u003eGonadotropins (follitropin alfa or menotropin 150\u0026ndash;450IU) were administered starting on day 3 of the menstrual cycle, with 0.25mg GnRH antagonist cetrorelix acetate (Cetrotide, Merck, Germany) on cycle day 7. Human chorionic gonadotropin was used to trigger ovulation once the dominant follicle reached 18mm and oocyte pick-up was performed 35h after triggering.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eOocyte collection, fertilization and time lapse embryo culture\u003c/h2\u003e \u003cp\u003eOocyte\u0026ndash;cumulus complexes were selected in Quinn\u0026rsquo;s Advantage\u0026trade; Medium with HEPES plus HSA (Sage, USA) and surplus cumulus was dissected from around the oocytes using a stereomicroscope in a class II hood with a heated stage. The oocytes were washed and cultured in SAGE 1 Step\u0026trade;, with HSA (Origio, Denmark) covered with liquid paraffin oil (Origio, Denmark) for between 2 and 4 h in an atmosphere of 5.5% CO2 and 5.0% O2 in a Sanyo MCO- 19M (Panasonic) incubator. To prepare Cai, ionophore compound (GM508 CultActive) medium (Ca Ionophore) was first incubated in a standard incubator at 37\u0026deg;C with 5.5% CO2 for 4 h Then the microinjected oocytes were immediately placed in a solution containing Ca active substance for 15 minutes. The oocytes were incubated in the time lapse monitoring system (EmbryoScope; Unisense FertiliTech, Denmark) in an atmosphere of 5% O2, 5.5% CO2, at 37℃ until embryo transfer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTime-lapse monitoring embryo culture system\u003c/h2\u003e \u003cp\u003eEmbryoViewer software (Unisense Fertilitech Aarhus) was used to inspect the embryo images to elucidate the embryo growth after microinjection over time (hours). Pronuclear fading (Pnf), the moment when both pronuclei disappear, first cleavage, and the times at which the zygote divides into 2\u0026ndash;8 cells (t2 to t8, respectively) were evaluated according to Basile et al.\u0026rsquo;s classification [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The duration of the cycle for the second cell (cc2\u0026thinsp;=\u0026thinsp;t3\u0026ndash;t2) is the time of embryo cleavage from two blastomeres into three blastomeres. The time from this cleavage to a four-blastomere embryo is the second synchrony (s2\u0026thinsp;=\u0026thinsp;t4\u0026ndash;t3). The time for the three- blastomere embryo to split into a five-blastomere embryo is the prolongation of the third cell cycle (cc3\u0026thinsp;=\u0026thinsp;t5\u0026ndash;t3). A previous study determined the optimal morphological values for the best implantation rate for t5 (t5\u0026thinsp;=\u0026thinsp;48.8\u0026ndash;56.6 h), s2 (s2\u0026thinsp;\u0026lt;\u0026thinsp;0.76 h), and cc2 (cc2\u0026thinsp;\u0026lt;\u0026thinsp;12 h, 20.5\u0026thinsp;\u0026lt;\u0026thinsp;t5\u0026ndash;t2\u0026thinsp;\u0026lt;\u0026thinsp;29, and 11\u0026thinsp;\u0026lt;\u0026thinsp;cc3[t5\u0026ndash;t3]\u0026thinsp;\u0026lt;\u0026thinsp;18) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We also considered these optimal values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEmbryo grading\u003c/h2\u003e \u003cp\u003eEmbryos with a duration of t5-t2\u0026thinsp;\u0026gt;\u0026thinsp;20.5 h were defined as A or B, while those outside this optimal range were defined as C or D. Embryos with a duration of cc3 between 11 and 18 h were classified as A or C, while those beyond the optimal range were classified as B or D, depending on t5-t2 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEmbryo transfer\u003c/h2\u003e \u003cp\u003eEmbryos were transferred between the second and fifth days of development with a Sure-Pro Ultra Embryo Replacement Catheter with an Obturator (Cooper Surgical Company, The Netherlands) and a medium Sage One Step.\u003c/p\u003e \u003cp\u003eAll patients received luteal support with the daily use of a 400 mg vaginal progesterone tablet (Progestan vaginal capsule 400 mg) and 30 mg oral dydrogesterone (Duphaston tablet 10 mg; Abbott Laboratories, Eczacibasi, Turkey).\u003c/p\u003e \u003cp\u003eA biochemical pregnancy is characterized by a serum level of β-hCG\u0026thinsp;\u0026gt;\u0026thinsp;20 IU/I on day 12 following embryo transfer. Clinical pregnancy is established when the activity of the heart of the fetus is detected through ultrasonography 8 weeks post-embryo transfer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data statistical analysis were performed using Statistical Package for Social Sciences (SPSS) 26.0 studio program and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Continuous data were shown as the mean and standart deviation (SD). Independent sample T-test was used for the analysis of the baseline parameters and the embryomorphokinetic parameters. Categorical variables (fertilization, embryo grade A, clinical pregnancy rates) were presented as percentages and were evaluated using the pearson\u0026rsquo;s chi-square test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePower Analysis\u003c/h2\u003e \u003cp\u003eThe G*Power 3.1.9 package program was employed for the power analysis of the study. Following the collection of data, a post-hoc power analysis based on pregnancy outcomes revealed that the study had a power of 0.89 for 413 patients.\u003c/p\u003e \u003c/div\u003e\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInstitutional Review Board approval was obtained from Bah\u0026ccedil;eşehir University Faculty of Medicine before the beginning of the study.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 413 patients were included. AOA group consisted 225 patients and non-AOA group included 187 patients. When female age, BMI, duration of infertility, duration of induction(days), oocyte count, MII count and 2PN count were examined, there were no significant differences between groups (Table 1).\u003c/p\u003e\n\u003cp\u003eWhen the durations from 2PN to the blastulation time (tb) were examined, no significant differences were found between the two groups in terms of t2, t3, t4, or t5. When the optimal time lapse parameters (t5, cc2, S2, cc3, and t5-t2) were evaluated no significant relation was found between the probability of optimal time lapse parameters and the use of Cai (Table2). The use of Cai was found to extend (p\u0026lt;.05) cavitation (Figure 1) and blastulation times (tb) (p\u0026lt;.05) (Figure 2).\u003c/p\u003e\n\u003cp\u003eAccording to the fertilization results, there were no significant differences between the groups that received and that did not receive AOA treatment (69.87% vs. 74.4%, respectively). When it comes to embryo grades, the grade A embryo rates were not significantly different between AOA and non-AOA groups (75.3% vs. 71.7% respectively). There were also no significant differences in the clinical pregnancy outcomes (46.2% vs. 54.6%, respectively) (Table 3).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we investigated the effects of AOA application with Cai on embryomorphokinetic parameters and embryological development. To the best of our knowledge, our sample population is the largest of any study conducted on this subject to date. Some clinical studies and meta-analyses have reported that AOA may help improve fertilization, embryonic development, and clinical pregnancy outcomes in many clinical conditions [21, 23]. Therefore, we performed AOA mostly in patients with poor prognoses. Also, because changes in intracellular Ca levels influence the entire development process from oocyte to embryo transition, we examined the correlations between Cai application and morphokinetic parameters.\u003c/p\u003e\n\u003cp\u003eThe use of AOA is controversial, as synthetic activating agents do not fully replicate the natural Ca signaling and physiological mechanisms observed in mammalian zygotes [19, 32]. Activation of the embryo by Cai may lead to variation in behavior and division patterns that differ from normal processes. Therefore, this technique is still experimental, and gathering data from a larger number of patient embryos could yield valuable insights.\u003c/p\u003e\n\u003cp\u003eFew studies have evaluated the relationship between AOA and the morphokinetic behavior of the embryo. Martinez et al. reported that AOA had no effect, with the exception of second polar body extrusion (tPB2) and time to second cell division (t3); however, they concluded that these changes did not seem affect the morphokinetic pattern of the preimplantation embryo and ascribed the findings to a rapid transient nonphysiological increase in free intracytoplasmic Ca2+ [19]. Similarly, Shebl et al. performed a sibling oocyte study in patients with previous fertilization problems. Half of the oocytes were treated with calcimycin. They found that ionophore-treated oocytes underwent significantly earlier pronuclei transformation and had a better synchronized third cell cycle [22]. In both of these studies, the patient population included couples who had experienced fertilization failure and severe male factor infertility similar with our study. However, in contrast to their findings, in our study pronuclei transformation or third cell cycle synchronization were not different and we found a prolonged tb in AOA cycles. Research on humans has highlighted the potential of tb as a determining factor for selecting embryos, surpassing other components of embryo morphological grading [35-37]. Lee et al. and Moustafa et al. noted delayed tb in embryos with chromosome abnormalities [36, 38]. Recently, Quintana-Veh\u0026iacute; and colleagues conducted a study comparing groups in donor cycles, including calcimycin, ionomycin, and conventional ICSI cycles. ICSI was not applied in AOA groups, only parthenogenetic activation was examined. According to the results, none of the calcimycin parthenotes formed blastocysts, while an extension in blastulation was observed in the ionomycin group. Although there are some differences on designs of this study and our study we also found an extension in tb [39].\u003c/p\u003e\n\u003cp\u003eThe clinical importance of these changes in embryomorphokinetic parameters is not clear. AOA process results in non-physiological calcium changes in the cells. In normal physiological processes, calcium release during oocyte activation occurs in the form of oscillations from internal stores. However, in artificial oocyte activation, it increases in a peak- shaped manner. This situation may be related to the faster expulsion of the second polar body in the initial fertilization and faster cleavage stages (t3) mentioned in previous reports, while the lack of long-lasting oscillations may be associated with the prolongation of the tb period [19,22]. Previous studies and our study results may suggest the importance of taking caution when applying Cai and the prolonged tb duration may serve as a warning for future clinical practice. It would be more appropriate to consider its utilization in cases where there is a clear indication rather than opting for elective use.\u003c/p\u003e\n\u003cp\u003eMorphokinetic behavior has also been used to predict euploidy rates in different studies [29,30] because aneuploidy can potentially occur at any stage from the disjunction of chromatids to the mitotic divisions within the embryo [40-43]. We used the morphokinetic embryo grading system that predicts aneuploidy devised by Basile et al. and did not find any differences between the two groups. The literature contains little information on embryo aneuploidy and Cai activation. However, our results are in line with a recent study that found that Cai application did not increase the risk for aneuploidy and there were no developmental differences in live births as a result of transfers made with Cai administration [20]. Thus Ca2 ionophores have the capability to bind Ca2 cations, and due to their hydrophobic properties, they form a complex at the lipid bilayer of the membrane. This structure enables ionophores to transfer Ca2 across the membrane and release them into the cytosol [44]. Consequently, ionophores themselves may not necessarily enter the oocyte, potentially explaining the absence of a detectable effect of ionophores on chromosomal segregation [45]. Reports have indicated that implanted embryos after IVF, using Cai as an artificial oocyte activator, do not exhibit any chromosome abnormalities [7,47]. Moreover, no perinatal morbidity or mortality was reported among the patients involved in these studies. Comparable outcomes have also been observed in children aged 3\u0026ndash;10 years, who were born following ICSI cycles using Cai. No differences have been noted compared to their peers [32,48].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;The principal limitation of our study lies in its retrospective design. Consequently, definitive conclusions are challenging to establish. However, it is important to acknowledge that this limitation is mitigated by our substantial sample size. Our findings may help understanding the embryomorphokinetic behavior and embryo selection in AOA cycles in future applications.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we did not find significant differences in most of the embryomorphokinetic parameters except prolongation of tb and cavitation time. However, the clinical significance of these changes is controversial because of the lack of correlations between these parameters and clinical outcomes. More studies with larger populations are needed on this subject.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contribution:\u0026nbsp;\u003c/strong\u003eFG designed, planned and wrote the manuscript; IP and BT collected data and applied the treatment in the laboratory; AZI drafted the manuscript and supervised the manuscript writing. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eWe would like to thank Selin Madran (Nursing) and Buket Ekmek\u0026ccedil;i (Biostatistics) for their contributions.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eNone\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available from the corresponding author(F.G) upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to this close.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval:\u003c/strong\u003e This study was performed in line with the principles of Bah\u0026ccedil;eşehir University Faculty of Medicine and Institutional Review Board of Bah\u0026ccedil;eşehir University approved the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e Informed consent was obtained from all individual participants included in the study. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish:\u003c/strong\u003e Participants signed informed consent regarding publishing their data.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePalermo G, Joris H, Devroey P, Van Steirteghem AC (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340:17-18. https://doi.org/10.1016/0140-6736(92)92425-F\u003c/li\u003e\n\u003cli\u003eVan Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, Wisanto A, Devroey P (1993) High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 8:1061-1066. https://doi.org/10.1093/oxfordjournals.humrep.a138192 \u003c/li\u003e\n\u003cli\u003eLv M, Zhang D, He X, Chen B, Li Q, Ding D, Zhang Z (2020) Artificial oocyte activation to improve reproductive outcomes in couples with various causes of infertility: a retrospective cohort study. Reprod BioMed online 40:501-509. https://doi.org/10.1016/j.rbmo.2020.01.001 \u003c/li\u003e\n\u003cli\u003eMontag M, K\u0026ouml;ster M, van der Ven K, Bohlen U, \u0026amp; van der Ven H (2012) The benefit of artificial oocyte activation is dependent on the fertilization rate in a previous treatment cycle. Reprod BioMed online 24:521-526. https://doi.org/10.1016/j.rbmo.2012.02.002 \u003c/li\u003e\n\u003cli\u003eTesarik J, Sousa M, Testart J (1994) Human oocyte activation after intracytoplasmic sperm injection. Hum Reprod 9:511-518. https://doi.org/10.1093/oxfordjournals.humrep.a138537 \u003c/li\u003e\n\u003cli\u003eDozortsev D, De Sutter P, Dhont M (1994) Behaviour of spermatozoa in human oocytes displaying no or one pronucleus after intracytoplasmic sperm injection. Hum Reprod 9:2139-2144. https://doi.org/10.1093/oxfordjournals.humrep.a138406 \u003c/li\u003e\n\u003cli\u003eLu Q, Chen ZJ, Gao X, Ma SY, Li M, Hu JM, \u0026amp; Li Y (2006) Zhonghua fu chan ke za zhi, 41:182\u0026ndash;185. https://doi.org/10.1007/s10495-012-0702-9 \u003c/li\u003e\n\u003cli\u003eNasr-Esfahani MH, Razavi S, Mardani M, Shirazi R, Javanmardi S (2007) Effects of failed oocyte activation and sperm protamine deficiency on fertilization post-ICSI. Reprod BioMed online 14:422-429. https://doi.org/10.1016/s1472-6483(10)60888-7 \u003c/li\u003e\n\u003cli\u003eSaunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, Lai FA (2002) PLC\u0026zeta;: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo. Development 129:3533\u0026ndash;3544. https://doi.org/10.1242/dev.129.15.3533 \u003c/li\u003e\n\u003cli\u003eKashir J, Heindryckx B, Jones C, De Sutter P, Parrington J, Coward K (2010) Oocyte activation, phospholipase C zeta and human infertility. Hum Reprod update 16:690-703. https://doi.org/10.1093/humupd/dmq018\u003c/li\u003e\n\u003cli\u003eRamadan WM, Kashir J, Jones C, Coward K (2012) Oocyte activation and phospholipase C zeta (PLC\u0026zeta;): diagnostic and therapeutic implications for assisted reproductive technology. Cell Commun Signal 10:1-20. https://doi.org/10.1186/1478-811X-10-12 \u003c/li\u003e\n\u003cli\u003eBerridge MJ (2009) Inositol trisphosphate and calcium signalling mechanisms. Biochim Biophy Acta Mol Cell Res 1793:933-940. https://doi.org/10.1016/j.bbamcr.2008.10.005 \u003c/li\u003e\n\u003cli\u003eYoon SY, Eum JH, Lee JE, Lee HC, Kim YS, Han JE, Won H, Park S, Shim S, Lee WS, Fissore R, Lee DR, Yoon TK (2012) Recombinant human phospholipase C zeta 1 induces intracellular calcium oscillations and oocyte activation in mouse and human oocytes. Hum Reprod 27:1768-1780. https://doi.org/10.1093/humrep/des092 \u003c/li\u003e\n\u003cli\u003eNomikos M, Kashir J, Swann K, Lai FA (2013) Sperm PLC\u0026zeta;: from structure to Ca2+ oscillations, egg activation and therapeutic potential. FEBS letters 587:3609-3616. https://doi.org/10.1016/j.febslet.2013.10.008\u003c/li\u003e\n\u003cli\u003eNeri QV, Lee B, Rosenwaks Z, Machaca K, Palermo GD (2014) Understanding fertilization through intracytoplasmic sperm injection (ICSI). Cell calcium 55:24-37. https://doi.org/10.1016/j.ceca.2013.10.006\u003c/li\u003e\n\u003cli\u003ePutney Jr JW (1986) A model for receptor-regulated calcium entry. Cell calcium 7:1-12. https://doi.org/10.1016/0143-4160(86)90026-6\u003c/li\u003e\n\u003cli\u003eSaunders CM, Swann K, \u0026amp; Lai FA (2007) PLC\u0026zeta;, a sperm-specific PLC and its potential role in fertilization. Biochem Soc Symp 74:23-36. https://doi.org/10.1042/BSS0740023 \u003c/li\u003e\n\u003cli\u003eMalcuit C, Kurokawa M, Fissore RA (2006) Calcium oscillations and mammalian egg activation, J Cell Physiol 206:565-573. https://doi.org/10.1002/jcp.20471\u003c/li\u003e\n\u003cli\u003eMartinez M, Durban M, Santal\u0026oacute; J, Rodr\u0026iacute;guez A, Vassena R (2021) Assisted oocyte activation effects on the morphokinetic pattern of derived embryos. J Assist Reprod Genet 38:531-537. https://doi.org/10.1007/s10815-020-02025-9\u003c/li\u003e\n\u003cli\u003eZhang J, Yao G, Zhang T, Hu J, Yang G, He J, Qina H, Fan H, Bai Y, Sun Y (2023) Effect of calcium ionophore (A23187) on embryo development and its safety in PGT cycles. Front Endocrinol 13:3286. https://doi.org/10.3389/fendo.2022.979248\u003c/li\u003e\n\u003cli\u003eMurugesu S, Saso S, Jones BP, Bracewell-Milnes T, Athanasiou T, Mania A, Serhal P, Ben-Nagi J (2017) Does the use of calcium ionophore during artificial oocyte activation demonstrate an effect on pregnancy rate? A meta-analysis. Fertil Steril 108:468-482. https://doi.org/10.1016/j.fertnstert.2017.06.029\u003c/li\u003e\n\u003cli\u003eShebl O, Trautner PS, Enengl S, Reiter E, Allerstorfer C, Rechberger T, Oppelt P, Ebner T (2021) Ionophore application for artificial oocyte activation and its potential effect on morphokinetics: a sibling oocyte study. J Assist Reprod Genet 38:3125-3133. https://doi.org/10.1007/s10815-021-02338-3 \u003c/li\u003e\n\u003cli\u003eChen X, Zhao H, Lv J, Dong Y, Zhao M, Sui X, Cui R, Liu B, Wu K (2022) Calcium ionophore improves embryonic development and pregnancy outcomes in patients with previous developmental problems in ICSI cycles. BMC Pregnancy and Childbirth 22:894. https://doi.org/10.1186/s12884-022-05228-3 \u003c/li\u003e\n\u003cli\u003eEbner T, K\u0026ouml;ster M, Shebl O, Moser M, Van der Ven H, Tews G, Montag M (2012) Application of a ready-to-use calcium ionophore increases rates of fertilization and pregnancy in severe male factor infertility. Fertil Steril 98:1432-1437. https://doi.org/10.1016/j.fertnstert.2012.07.1134\u003c/li\u003e\n\u003cli\u003eXu Z, Yao G, Niu W, Fan H, Ma X, Shi S, Jin H, Song W, Sun Y (2021) Calcium ionophore (A23187) rescues the activation of unfertilized oocytes after intracytoplasmic sperm injection and chromosome analysis of blastocyst after activation. Front Endocrinol 12:692 https://doi.org/10.3389/fendo.2021.692082\u003c/li\u003e\n\u003cli\u003eTsai TE, Lin PH, Lian PF, Li CJ, Vitale SG, Miku\u0026scaron; M, Su WP, Tsai H, Tsui K, Lin LT (2022) Artificial oocyte activation may improve embryo quality in older patients with diminished ovarian reserve undergoing IVF- ICSI cycles. J Ovarian Res 15:1-9. https://doi.org/10.1186/s13048-022-01036-7\u003c/li\u003e\n\u003cli\u003eSciorio R (2021) Use of time-lapse monitoring in medically assisted reproduction treatments: A mini- review. Zygote 29:93-101. https://doi.org/10.1017/S0967199420000623\u003c/li\u003e\n\u003cli\u003eZaninovic N, Irani M, \u0026amp; Meseguer M (2017) Assessment of embryo morphology and developmental dynamics by time-lapse microscopy: is there a relation to implantation and ploidy?. Fertility and sterility 108:722\u0026ndash; 729. https://doi.org/10.1016/j.fertnstert.2017.10.002\u003c/li\u003e\n\u003cli\u003eBasile N, del Carmen Nogales M, Bronet F, Florensa M, Riqueiros M, Rodrigo L, Garc\u0026iacute;a-Velasco J, Meseguer M (2014) Increasing the probability of selecting chromosomally normal embryos by time-lapse morphokinetics analysis. Fertil Steril 101:699-704. https://doi.org/10.1016/j.fertnstert.2013.12.005\u003c/li\u003e\n\u003cli\u003ePatel DV, Shah PB, Kotdawala AP, Herrero J, Rubio I, Banker MR (2016) Morphokinetic behavior of euploid and aneuploid embryos analyzed by time-lapse in embryoscope. J Hum Reprod Sci 9:112. https://doi.org/10.4103/0974-1208.183511 \u003c/li\u003e\n\u003cli\u003eMotato Y, de los Santos MJ, Escriba MJ, Ruiz BA, Remoh\u0026iacute; J, \u0026amp; Meseguer M (2016) Morphokinetic analysis and embryonic prediction for blastocyst formation through an integrated time-lapse system. Fertil Steril 105:376-384. https://doi.org/10.1016/j.fertnstert.2015.11.001\u003c/li\u003e\n\u003cli\u003eVanden Meerschaut F, D\u0026apos;Haeseleer E, Gysels H, Thienpont Y, Dewitte G, Heindryckx B, Oostra A, Roeyers H, Van Lierde K, De Sutter P (2014) Neonatal and neurodevelopmental outcome of children aged 3-10 years born following assisted oocyte activation. Reprod Biomed Online 28:54-63. https://doi.org/10.1016/j.rbmo.2013.07.013\u003c/li\u003e\n\u003cli\u003eMeseguer M, Herrero J, Tejera A, Hilligs\u0026oslash;e KM, Ramsing NB, Remoh\u0026iacute; J (2011) The use of morphokinetics as a predictor of embryo implantation. Hum Reprod 26:2658-71. https://doi.org/10.1093/humrep/der256\u003c/li\u003e\n\u003cli\u003eMuthukumar K, Kamath MS, Mangalaraj AM, Aleyamma TK, Chandy A, George K (2013) Comparison of clinical outcomes following vitrified warmed day 5/6 blastocyst transfers using solid surface methodology with fresh blastocyst transfers. J Hum Reprod Sci 6:59. https://doi.org/10.4103/0974-1208.112384 \u003c/li\u003e\n\u003cli\u003eHo JR, Arrach N, Rhodes-Long K, Salem W, McGinnis LK, Chung K, Bendikson K, Paulson JR, Ahmady A (2018) Blastulation timing is associated with differential mitochondrial content in euploid embryos. J Assist Reprod Genet 35:711-720. https://doi.org/10.1007/s10815-018-1113-9\u003c/li\u003e\n\u003cli\u003eMoustafa SM, Rosen EM, Boylan C, Mersereau JE (2020) Time to blastulation is superior to individual components of embryo grading for live-birth prediction. F\u0026amp;S Reports 1:243-248. https://doi.org/10.1016/j.xfre.2020.09.016\u003c/li\u003e\n\u003cli\u003eHuayhua C, Rodr\u0026iacute;guez M, Vega J, Briones M, Rodriguez-Alvarez L, Mellisho E (2023) Blastulation time measured with time-lapse system can predict in vitro viability of bovine blastocysts. PloS one 18. https://doi.org/10.1371/journal.pone.0289751\u003c/li\u003e\n\u003cli\u003eLee CI, Chen CH, Huang CC, Cheng EH, Chen HH, Ho ST, Lee TH (2019) Embryo morphokinetics is potentially associated with clinical outcomes of single-embryo transfers in preimplantation genetic testing for aneuploidy cycles. Reprod BioMed online 39:569-579. https://doi.org/10.1016/j.rbmo.2019.05.020\u003c/li\u003e\n\u003cli\u003eQuintana-Veh\u0026iacute; A, Mart\u0026iacute;nez M, Zamora MJ, et al (2023) 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 40:1661\u0026ndash;1668. https://doi.org/10.1007/s10815-023-02833-9 \u003c/li\u003e\n\u003cli\u003eHassold T, Hunt P (2001) To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet 2:280-291. https://doi.org/10.1038/35066065\u003c/li\u003e\n\u003cli\u003eWang WH, \u0026amp; Sun QY (2006) Meiotic spindle, spindle checkpoint and embryonic aneuploidy. Front Biosci 11:620-636. https://doi.org/10.2741/1822\u003c/li\u003e\n\u003cli\u003eChavez SL, Loewke KE, Han J, Moussavi F, Colls P, Munne S, \u0026amp; Reijo Pera RA (2012) Dynamic blastomere behaviour reflects human embryo ploidy by the four-cell stage. Nature commun 3:1251. https://doi.org/10.1038/ncomms2249\u003c/li\u003e\n\u003cli\u003eCampbell A, Fishel S, Bowman N, Duffy S, Sedler M, \u0026amp; Hickman CFL (2013) Modelling a risk classification of aneuploidy in human embryos using non-invasive morphokinetics. Reprod BioMed online 26:477- 485. https://doi.org/10.1016/j.rbmo.2013.02.006\u003c/li\u003e\n\u003cli\u003eBrasseur R, Notredame M, Ruysschaert JM (1983) Lipid-water interface mediates reversible ionophore conformational change. Biochem Biophys Res Commun 114:632\u0026ndash;637. https://doi.org/10.1016/0006-291x(83)90827-6 \u003c/li\u003e\n\u003cli\u003eCapalbo A, Ottolini CS, Griffin DK, Ubaldi FM, Handyside AH, Rienzi L (2016) Artificial oocyte activation with calcium ionophore does not cause a widespread increase in chromosome segregation errors in the second meiotic division of the oocyte. Fertil Steril 105:807\u0026ndash;814. https://doi.org/10.1016/j.fertnstert.2015.11.017 \u003c/li\u003e\n\u003cli\u003eESHRE Add-ons working group, Lundin K, Bentzen JG, Bozdag G, Ebner T, Harper J, Le Clef N, Moffett A, Norcross S, Polyzos NP, Rautakallio-Hokkanen S, Sfontouris I, Sermon K, Vermeulen N, Pinborg A (2023) Good practice recommendations on add-ons in reproductive medicine. Human Reproduction 38:2062\u0026ndash;2104. https://doi.org/10.1093/humrep/dead184 \u003c/li\u003e\n\u003cli\u003eNakagawa K, Yamano S, Moride N, Yamashita M, Yoshizawa M, Aono T (2001) Effect of activation with Ca ionophore A23187 and puromycin on the development of human oocytes that failed to fertilize after intracytoplasmic sperm injection. Fertil Steril 76:148-152. https://doi.org/10.1016/S0015-0282(01)01839-8\u003c/li\u003e\n\u003cli\u003eDeemeh MR, Tavalaee M, Nasr-Esfahani MH (2015) Health of children born through artificial oocyte activation: a pilot study. Reprod Sciences 22:322-328. https://doi.org/10.1177/1933719114542017.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1716972865.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1716972902.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1716972935.png\"\u003e\u003cbr\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"calcium ionophore, artificial oocyte activation, embryomorphokinetics, clinical pregnancy outcome, fertilization, blastulation","lastPublishedDoi":"10.21203/rs.3.rs-4191561/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4191561/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eDoes artificial oocyte activation (AOA) with calcium ionophore (Cai) application affect embryomorphokinetic parameters and clinical pregnancy outcomes?\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis retrospective cohort study investigated if AOA with Cai affects the embryomorphokinetic parameters and clinical pregnancy outcomes of 413 patients. A total of 225 patients underwent intracytoplasmic sperm injection (ICSI)\u0026thinsp;+\u0026thinsp;AOA and 187 patients underwent only ICSI at Izmir University of Economics between 2018\u0026ndash;2020.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThere were no significant differences between groups in terms of baseline parameters. When the duration from 2PN to tb (time to blastulation) was examined, t2, t3, t4, or t5 times were not significantly different between the groups that received and did not receive AOA treatment. Ca ionophore application was found to extend cavitation time and tb (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Optimal time lapse parameters (t5, cc2, S2 and t5-t2) also did not differ between two groups. There were no significant differences between groups according to embryo grades which were determined by optimal time lapse parameters. According to the fertilization and pregnancy results, no significant differences were found between the two groups.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCa ionophore application does not seem to affect most of the embryomorphokinetic parameters except blastulation time. The clinical importance of this finding needs further research.\u003c/p\u003e","manuscriptTitle":"The Effect of Artificial Oocyte Activation on Embryomorphokinetic Parameters and Blastulation Time","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-04 15:06:36","doi":"10.21203/rs.3.rs-4191561/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1ce2919b-671f-4d13-953f-4628d8a05ef7","owner":[],"postedDate":"June 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-19T10:39:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-04 15:06:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4191561","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4191561","identity":"rs-4191561","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}