Excessive Hormones Concentration in Serum May Increase the Risk of Polyspermy for Single Oocyte

Excessive Hormones Concentration in Serum May Increase the Risk of Polyspermy for Single Oocyte | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Excessive Hormones Concentration in Serum May Increase the Risk of Polyspermy for Single Oocyte Haijing Zhao, Qi Qiu, Ping Yuan, Nengyong Ouyang, Yuqin Zhu, Wenjun Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-136349/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 Background: Fertilization of an oocyte by multiple sperms is known as polyspermy. Here, the purpose of this work was to investigate the possible correlation between hormones concentration in serum and the fertilization outcome of single oocyte in IVF cycles. Methods: In this retrospective analysis, the baseline characteristics and serum hormones levels of female patients retrieving single oocyte in IVF cycles were analyzed. Female age, duration of infertility, body mass index (BMI), basal follicle stimulating hormone (FSH), basal luteinizing hormone (LH), basal estradiol (E2), the level of FSH, LH, E2 and progesterone (P) on the day of Gonadotropin (Gn), the level of FSH, LH, E2 and P on the day of human chorionic gonadotropin (HCG) were measured. Results: According to the number of pronucleus in single oocyte on Day1, patients were assigned to IVF-2PN group (two pronucleus) and IVF-3PN group (three or more pronucleus). There were extremely higher levels of basal E2, LH and E2 on the day of Gn in IVF-3PN group compared with IVF-2PN group ( P <0.01). The significantly higher level of LH on the day of HCG was present in IVF-3PN compared to IVF-2PN group ( P <0.05). Forthermore, the optimal cutoff value of basal E2 (70.5 IU/L), LH (6.315 IU/L) and E2 (128.5 IU/L) on the day of Gn and LH (3.28 IU/L) on the day of HCG were determined by ROC curve analysis. The results showed that the percentage of IVF-2PN significantly decreased and the percentage of IVF-3PN considerably increased when the basal E2 concentration was higher than the cutoff value ( P <0.05), the same tendency were present in the analysis of LH concentration, E2 concentration on the day of Gn and LH concentration on the day of HCG ( P <0.01). Conclusions: Our data indicated for the first time that the excessive concentration of basal E2, excessive concentrations of LH and E2 on the day of Gn and excessive concentration of LH on the day of HCG may significantly increase the risk of polyspermy for single oocyte in IVF cycles. Sexual & Reproductive Medicine IVF Single oocyte Hormone concentration Fertilization outcome Figures Figure 1 Plain English Summary Oocyte that fuses with more than a single sperm contain an excessive amount of genetic material contributed by the male and are said to be polyspermic, which is catastrophic to human embryonic development in vitro. In this study, total of 433 patients from in vitro fertilization (IVF) cycles were analyzed. We have made comparisons on basic information and serum hormones levels between IVF-2PN group (two pronucleus) and IVF-3PN group (three or more pronucleus). We also determined the optimal cutoff value of basal E2, LH and E2 on the day of Gn and LH on the day of HCG by Receiver Operator Characteristic (ROC) curve analysis. The data indicated that the percentage of IVF-2PN significantly decreased and the percentage of IVF-3PN considerably increased when these hormones concentration were higher than the cutoff value. In conclusion, for the patients retrieving single oocyte in IVF cycles, excessive hormones concentration may significantly increase the risk of polyspermy. Background Fertilization is an essential biological process in sexual reproduction and contains a series of molecular interactions between the sperm and oocyte [ 1 , 2 ]. Mammalian fertilization has been studied as a sequence of steps: sperm bind and penetrate through the zona pellucida of the oocyte, adhere to the oocyte plasma membrane and finally fuse with the oocyte. Sperm-oocyte fusion is indispensable for completing mammalian fertilization. Among the various steps of fertilization, gamete membrane fusion must be an extremely robust and precise mechanism, as it is the climax of fertilization [ 3 ]. Fertilization of an oocyte by multiple sperms, a condition is known as polyspermy, is catastrophic to embryonic development in most sexually reproducing species [ 4 ]. The rate of polyspermy from in vitro fertilized human oocytes is around 10% and therefore remarkably high [ 5 ]. Two primary safeguards protect oocytes against polyspermy: the “oocyte membrane block” and the “zona reaction”. The former involves a depolarization of the oocyte membrane, polyspermy is prevented at the plasma membrane level, where fusion of the first sperm causes depolarization of the membrane and shedding of the oocyte’s sperm receptor Juno, thereby preventing fusion of further sperm [ 6 , 7 ], transiently contributing to polyspermy block. The “oocyte membrane block” or so-called “fast block” to polyspermy occurs in seconds [ 8 , 9 ]; The second proposed mechanism involves a Ca 2+ oscillation event activated by attachment of the sperm to the oocyte membrane. The subsequent increase in intracellular Ca 2+ concentration triggers the exocytosis of cortical granules from just below the oocyte membrane into the perivitelline space. Cortical granules contain enzymes such as hydrolase, proteinase, and peroxidase that modify and thereby harden the zona pellucida [ 10 ]. This “slow block” occurs within approximately 5 to 8 min of oocyte activation and is considered the main mechanism of polyspermy block in humans [ 11 ]. A variety of oocyte-related factors contribute to the pathogenesis of polyspermy, including the degree of oocyte maturation, changes in the molecular biology of the ZP, and alterations in the cortical reaction [ 6 , 12 – 14 ]. It also has been shown that the phosphatidylinositol signaling pathway plays an important role in the cortical reaction, and alterations of this pathway might disrupt the membrane block leading to polyspermy [ 15 ]. Understanding the mechanisms that protect mammalian oocytes from polyspermy is not only of interest for fundamental research but also of direct medical relevance. In human assisted reproductive technology, polyspermy is lethal to the embryo development in vitro, particularly to the patients who retrieved single oocyte which is so precious. Therefore, our study was the first time to focus on the possible correlation between hormones concentration in serum and the fertilization outcome of single oocyte in IVF cycles. Methods Study Population In this retrospective study, total of 433 patients from conventional IVF between 2015–2019 were analyzed: The inclusion criteria: a) the female patients retrieved single oocyte during the operation; b) the fertilization outcome of single oocyte was 2PN or 3PN. Controlled ovarian hyperstimulation protocol In all of IVF cycles, the types of controlled ovarian hyperstimulation protocol were mini-stimulation cycles, natural / semi-natural ovulation cycles, superovulation cycles and antagonist cycles. Criteria for grouping In selected IVF cycles, according to the number of pronucleus in single oocyte on Day1, patients were assigned to two groups: a) IVF-2PN group: one female pronucleus and one male pronucleus were present in the oocyte; b) IVF-3PN group: three or more pronucleus were present in the oocyte. Statistical analyses Differences in age, duration of infertility, BMI, basal hormone levels and the levels of FSH、LH、E2 and P during medication were analyzed by T-test using a SPSS version 22 (IBM Corp., USA). The values were expressed as the mean ± standard error. The values with P < 0.05 were considered statistically significant. Results Baseline characteristics of patients Comparison of demographic information from the patients, such as age, duration of infertility, BMI and basal hormone levels between IVF-2PN group and IVF-3PN group were listed in Table 1. There was a significantly higher level of basal E2 in IVF-3PN group compared with IVF-2PN group ( P < 0.01). Information of patients during medication In the period of controlled ovulation, the main information of female patients, such as total usage of Gn、the levels of FSH、LH、E2 and P in serum on the day of Gn; total usage of HCG、the levels of FSH、LH、E2 and P in serum on the day of HCG were comparative in the two groups (Table 2). There were considerably higher levels of LH and E2 in serum on the day of Gn in IVF-3PN group compared with IVF-2PN group ( P < 0.01). In addition, a significantly higher level of LH in serum on the day of HCG was showed in IVF-3PN group compared to IVF-2PN group ( P < 0.05). The thresholds of several hormone concentrations in serum The optimal cutoff value of basal E2 (70.5 IU/L)、LH (6.315 IU/L) and E2 (128.5 IU/L) in serum on the day of Gn and LH (3.28 IU/L) in serum on the day of HCG were determined by ROC curve analysis. The percentage of IVF-2PN significantly decreased and the percentage of IVF-3PN considerably increased when the basal E2 concentration was higher than the cutoff value ( P < 0.05) (Table 3), the same tendency were present in the analysis of LH concentration、E2 concentration on the day of Gn (Table 4, Table 5) and LH concentration on the day of HCG ( P < 0.01) (Table 6). Discussion Sexual reproduction culminates in fertilization and occurs when two haploid gametes recognize each other and fuse to form a single diploid zygote [ 16 ]. Eggs that fuse with more than a single sperm contain an excessive amount of genetic material contributed by the male and are said to be polyspermic, resulting in the formation of a nonviable embryo. Aviable human embryo can only develop from an oocyte that is fertilized by a single sperm [ 17 ]. However, the incidence of polyspermy is 3–10% in IVF cycles [ 18 , 19 ]. The phospholipase C (PLC)-induced cascade of events was one of the earliest known signaling pathways initiated by fertilization. Fast polyspermy block after fertilization in X. laevis eggs is mediated by activation of PLC, which increases inositol 1,4,5-trisphosphate (IP3) and evokes Ca 2+ release from the endoplasmic reticulum (ER). The calcium rise is caused by the opening of IP3-gated calcium channels in the ER [ 20 ]. This ER-derived Ca 2+ then activates a Cl − channel to induce the fast polyspermy block [ 21 ]. So, a major question remain to be elucidated. How does the sperm-egg interaction activated PLC to increase IP3 and release of Ca 2+ from the ER in the oocyte? Our results indicated that the significantly differences in the level of LH and E2 on the day of Gn, the level of LH on the day of HCG between IVF-2PN group and IVF-3PN group may be relative to the fertilization outcome of single oocyte. During the development of primary follicles, gap junctions construct channels for material and information exchange between granulosa cells and oocytes. Some hormones, ions and signal molecules (such as cAMP, Ca 2+ , IP3, etc.) can communicate between cells and cause important biological effects. For example, the signal molecules produced by oocytes can be transported to granulosa cells to maintain the continuous proliferation and development of granulosa cells and prevent the premature differentiation of granulosa cells. In the secondary follicular stage, the follicular membrane began to differentiate into two layers: the inner and outer membranes. At this time, LH receptor has been expressed on endometrial cells. LH can act on endometrial cells and promote the synthesis and secretion of androgen. Androgen diffuses into granulosa cells, and then they are transformed into E2 by the aromatase in granulosa cells. There are receptors for E2 in the cytoplasm and nucleus of granulosa cells. The synthesized E2 has a positive feedback effect on granulosa cells and can stimulate the proliferation of granulosa cells. Combined with the above professional knowledge and our previous research results, we put forward such a scientific hypothesis: the more LH content, the more androgens synthesized by the endometrial cells, and the more E2 synthesized by the granulosa cells, then E2 promotes the proliferation of granulosa cells. At this time, in order to maintain the continuous proliferation and development of granulosa cells, the signal molecule IP3 produced by oocytes may be transported to granulosa cells through gap junctions, so the number of IP3 binding to endoplasmic reticulum ligands is reduced, which leads to the insufficient release of Ca 2+ in endoplasmic reticulum, the blocking mechanism of polyspermy can not be started, and which eventually results in the occurrence of polyspermy (Fig. 1 ). The limitation of this paper is to analyze the differences of hormone levels in a retrospective way. In the next prospective cohort study, we will detect the hormones concentration secreted by granulosa cells from the cell level, and discover the gene variation in the peripheral blood from the genetic point of view. Conclusion The study was the first time to focus on possible correlation between hormones concentration in serum and the fertilization outcome of single oocyte in IVF cycles. Our data indicated that excessive concentrations of LH ( ≧ 6.315 IU/L) and E2 ( ≧ 128.5 IU/L) on the day of Gn and excessive concentration of LH ( ≧ 3.28 IU/L) on the day of HCG may increase the risk of polyspermy for single oocyte. Our findings also provide a strong evidence and a clear clinical guidance for clinicians: when the patient retrieving single oocyte suffered a 3PN fertilization fate in IVF, ICSI is recommended for the next cycle to avoid polyspermy if the relevant hormone concentrations were above the threshold. Abbreviations IVF: in vitro fertilization ICSI: intracytoplasmic sperm injection E2: Estradiol LH: Luteinizing Hormone FSH: Follicle Stimulating Hormone P: Progesterone Gn: gonadotropin HCG: human chorionic gonadotropin ROC: Receiver Operator Characteristic BMI: body mass index ZP: zona pellucida PLC: phospholipase C IP3: inositol 1,4,5-trisphosphate ER: endoplasmic reticulum Declarations Ethics approval and consent to participate This study was approved by the Institutional Medical Ethics Committee of Sun Yat-Sen Memorial Hospital affiliated with SunYat-sen University. Participants gave written informed consent. Consent for publication The article was approved for publication by Institutional Medical Ethics Committee of Sun Yat-Sen Memorial Hospital. Availability of data and material Data will not be shared, because the study contains human data, it is private. Funding The study was funded by Mechanism of inflammatory factors mediated by SP1 regulating telomerase activity in granulosa cells and affecting follicular development in endometriosis through NF-kB (2019A1515012005). Authors' contributions Haijing Zhao conceived of the manuscript and wrote the first draft, Qi Qiu and Ping Yuan performed the statistical analyses, Nengyong Ouyang and Yuqin Zhu collected and proofread data, Wenjun Wang participated in its design and helped to draft the manuscript. All authors reviewed the final manuscript. All authors read and approved the final manuscript. Acknowledgements Not applicable. Competing Interest The authors declare no conflict of interest. References Wassarman PM, Jovine L, Litscher ES: A profile of fertilization in mammals. Nature Cell Biology 2001, 3: E59-64. Evans, P. J: Sperm-Egg Interaction. Annual Review of Physiology 2012, 74: 477-502. Inoue N, Hagihara Y, Wright D, Suzuki T, Wada I: Oocyte-triggered dimerization of sperm IZUMO1 promotes sperm–egg fusion in mice. Nature Communications 2015, 6: 8858. Wozniak KL, Carlson AE: Ion channels and signaling pathways used in the fast polyspermy block. Molecular Reproduction and Development 2020, 87 . Cheeseman LP, Boulanger JM, Bond LM, Schuh M: Two pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes. Nature Communications 2016, 7: 13726. Gardner AJ, Evans JP: Mammalian membrane block to polyspermy: new insights into how mammalian eggs prevent fertilisation by multiple sperm. Reproduction Fertility & Development 2006, 18: 53. Bianchi, Enrica, Doe, Brendan, Goulding, David, Wright, Gavin, J.: Juno is the egg Izumo receptor and is essential for mammalian fertilization. Nature 2014. Jaffe LA: Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature 1976, 261: 68. Jaffe LA, Gould-Somero M, Holland LZ: Studies of the Mechanism of the Electrical Polyspermy Block Using Voltage Clamp during Cross-Species Fertilization. Journal of Cell Biology 1982, 92: 616-621. Wassarman P, Chen J, Cohen N, Litscher E, Liu C, Qi H, Williams Z: Structure and function of the mammalian egg zona pellucida. J Exp Zool 1999, 285: 251-258. Mio Y, Iwata K, Yumoto K, Kai Y, Sargant HC, Mizoguchi C, Ueda M, Tsuchie Y, Imajo A, Iba Y, Nishikori K: Possible mechanism of polyspermy block in human oocytes observed by time-lapse cinematography. Journal of Assisted Reproduction and Genetics 2012, 29: 951-956. Ducibella: The cortical reaction and development of activation competence in mammalian oocytes. Human Reproduction Update 1996, 2: 29-42. Niwa K, Park CK, Okuda K: Penetration in vitro of bovine oocytes during maturation by frozen-thawed spermatozoa. Journal of Reproduction & Fertility 1991, 91: 329-336. Bauskin AR, Franken DR, Uwe E, Peter D: Characterization of human zona pellucida glycoproteins. Molecular Human Reproduction 1999 : 6. Tatone C, Eekelen CGV, Colonna R: Plasma membrane block to sperm entry occurs in mouse eggs upon parthenogenetic activation. Molecular Reproduction & Development 1994, 38: 200-208. Wright GJ, Bianchi E: The challenges involved in elucidating the molecular basis of sperm–egg recognition in mammals and approaches to overcome them. Cell and Tissue Research 2015, 363: 227-235. Ven HHVD, Al-Hasani S, Diedrich K, Hamerich U, Krebs D: Polyspermy in in Vitro Fertilization of Human Oocytes: Frequency and Possible Causes. Ann N Y Acad Sci 1985, 442: 88-95. Trounson A, Bongso A: 3 Fertilization and Development in Humans. Current Topics in Developmental Biology 1996. Ho P, Yeung W, Chan Y, So W, Chan S: Factors affecting the incidence of polyploidy in a human in vitro fertilization program. International Journal of Fertility & Menopausal Studies 1994, 39: 14. Jaffe LA: The fast block to polyspermy: New insight into a century-old problem. Journal of General Physiology 2018 : jgp.201812145-. Carlson AE, Lee MT, Phelps WA, Tembo M, Wozniak KL: PLC and IP3-evoked Ca2+ release initiate the fast block to polyspermy in Xenopus laevis eggs. Journal of General Physiology 2018, 150: 1239-1248. Tables Table 1 Baseline characteristics of patients from IVF-2PN group and IVF-3PN group. IVF-2PN group IVF-3PN group P Female Mean age (y) 38.72 ± 0.28 39.06 ± 0.41 0.546 Duration of infertility 5.08 ± 0.24 5.06 ± 0.37 0.964 The total cycles 3.17 ± 0.15 3.80 ± 0.31 0.054 Infertility type primary/secondary primary/secondary — 95/236 33/74 BMI (Kg/m 2 ) 22.42 ± 0.17 22.23 ± 0.36 0.608 Basal FSH (IU/L) 12.81 ± 0.49 12.30 ± 0.68 0.596 Basal LH (IU/L) 5.26 ± 0.26 6.05 ± 0.58 0.159 Basal E2 (IU/L) 69.29 ± 3.73 102.47 ± 14.46 0.002 Basal PRL (IU/L) 14.24 ± 1.11 17.06 ± 3.30 0.299 Male Mean age (y) 40.28 ± 0.34 40.91 ± 0.56 0.354 BMI (Kg/m 2 ) 24.52 ± 0.21 24.01 ± 0.29 0.196 Acrosomal enzyme 65.40 ± 2.27 74.12 ± 3.99 0.060 P < 0.05: significant difference Table 2 Information of patients from IVF-2PN group and IVF-3PN group during medication. IVF-2PN group IVF-3PN group P Gn Total usage 1311.56 ± 63.03 1060.98 ± 82.52 0.038 Total day 7.08 ± 0.21 6.25 ± 0.39 0.058 Start usage 163.78 ± 3.70 161.33 ± 5.55 0.735 FSH (IU/L) 11.96 ± 0.38 12.34 ± 0.66 0.628 LH (IU/L) 5.64 ± 0.24 7.15 ± 0.55 0.004 E2 (IU/L) 123.16 ± 7.35 170.65 ± 19.58 0.006 P (IU/L) 1.12 ± 0.13 1.04 ± 0.17 0.738 HCG Total usage 9048.19 ± 123.27 9163.55 ± 217.87 0.644 FSH (IU/L) 25.95 ± 4.85 20.45 ± 1.17 0.521 LH (IU/L) 7.46 ± 0.35 9.22 ± 0.66 0.016 E2 (IU/L) 565.10 ± 22.16 508.48 ± 28.71 0.181 P (IU/L) 1.09 ± 0.13 1.32 ± 0.37 0.463 P < 0.05: significant difference Table 3 Comparsion of the percentage of IVF-2PN and IVF-3PN between Low concentration group and High concentration group according to basal E2 level. Basal E2 (IU/L) Low concentration High concentration P Fertilization outcome (< 70.5 IU/L) ( ≧ 70.5 IU/L) IVF-2PN number (%) 232 (78.4) 94 (68.6) 0.028 IVF-3PN number (%) 64 (21.6) 43 (31.4) 0.028 P < 0.05: significant difference Table 4 Comparsion of the percentage of IVF-2PN and IVF-3PN between Low concentration group and High concentration group according to LH level on the day of Gn. LH (IU/L) (Gn ) Low concentration High concentration P Fertilization outcome (< 6.315 IU/L) ( ≧ 6.315 IU/L) IVF-2PN number (%) 230 (79.9) 96 (66.2) 0.002 IVF-3PN number (%) 58 (20.1) 49 (33.8) 0.002 P < 0.05: significant difference Table 5 Comparsion of the percentage of IVF-2PN and IVF-3PN between Low concentration group and High concentration group according to E2 level on the day of Gn. E2 (IU/L) (Gn ) Low concentration High concentration P Fertilization outcome (< 128.5 IU/L) ( ≧ 128.5 IU/L) IVF-2PN number (%) 225 (79.5) 101 (67.3) 0.005 IVF-3PN number (%) 58 (20.5) 49 (32.7) 0.005 P < 0.05: significant difference Table 6 Comparsion of the percentage of IVF-2PN and IVF-3PN between Low concentration group and High concentration group according to LH level on the day of HCG. LH (IU/L) (HCG ) Low concentration High concentration P Fertilization outcome (< 3.28 IU/L) ( ≧ 3.28 IU/L) IVF-2PN number (%) 98 (86.7) 228 (71.3) 0.001 IVF-3PN number (%) 15 (13.3) 92 (28.7) 0.001 P < 0.05: significant difference Cite Share Download PDF Status: Posted Version 1 posted 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 In Review Editorial Policies 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-136349","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":7112115,"identity":"e8718f88-ce3d-4f47-ae77-309dee1f4d6d","order_by":0,"name":"Haijing Zhao","email":"","orcid":"","institution":"Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haijing","middleName":"","lastName":"Zhao","suffix":""},{"id":7112116,"identity":"419517bc-1673-457a-af03-8a76a1cf82c0","order_by":1,"name":"Qi Qiu","email":"","orcid":"","institution":"Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Qiu","suffix":""},{"id":7112117,"identity":"b0b4f9ff-36f0-4a65-bbfb-7514d296738a","order_by":2,"name":"Ping Yuan","email":"","orcid":"","institution":"Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Yuan","suffix":""},{"id":7112118,"identity":"6cc9a6ca-264f-43c2-8f9d-6ede33ec134b","order_by":3,"name":"Nengyong Ouyang","email":"","orcid":"","institution":"Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nengyong","middleName":"","lastName":"Ouyang","suffix":""},{"id":7112119,"identity":"838cf5ce-84aa-4caa-a4bf-a8664a2d98fc","order_by":4,"name":"Yuqin Zhu","email":"","orcid":"","institution":"Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuqin","middleName":"","lastName":"Zhu","suffix":""},{"id":7112120,"identity":"5f82813e-3d68-41a7-bef6-aa232686cb8f","order_by":5,"name":"Wenjun Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYJACAwjFfODAhx+kaWFLPDizhzTLeIwPc7ARY8Px3gPFvDtq7fln93w4zMDDIM8vdoCAljPnEox5zxxnlrhzdsPhAgsGw5mzE/BrMbuRY2DM23aMzUAid8PhGTwMCQa3CWm5/washcdAIufBYR42YrTc4AFpqZEAamEgTov9mRwDw7ltBwwkbqQZAANZgrBfJNvPmBm8bauz55+R/PjDhx828vzSBLQAARswKg/DOBIElYMA8wMGhjqiVI6CUTAKRsEIBQB3xEQV25dWOgAAAABJRU5ErkJggg==","orcid":"","institution":"Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Wenjun","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2020-12-26 11:47:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-136349/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-136349/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":4615952,"identity":"70277298-d025-4c20-82b8-c5fe172e8cb3","added_by":"auto","created_at":"2020-12-30 23:57:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":61861,"visible":true,"origin":"","legend":"Excessive concentration of E2 negatively regulates the phosphatidylinositol signaling pathway and affects the blocking mechanism of polyspermy.","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-136349/v1/6f4843ec6eb91b4e91ca80a1.jpg"},{"id":13641366,"identity":"af989f9c-1fe9-4473-aa22-7a63c07de106","added_by":"auto","created_at":"2021-09-17 09:03:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":728429,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-136349/v1/8a1ee2d1-bdbd-485d-a6a3-56640f5d67f9.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eExcessive Hormones Concentration in Serum May Increase the Risk of Polyspermy for Single Oocyte\u003c/p\u003e","fulltext":[{"header":"Plain English Summary","content":"\u003cp\u003eOocyte that fuses with more than a single sperm contain an excessive amount of genetic material contributed by the male and are said to be polyspermic, which is catastrophic to human embryonic development in vitro.\u003c/p\u003e\n\u003cp\u003eIn this study, total of 433 patients from in vitro fertilization (IVF) cycles were analyzed. We have made comparisons on basic information and serum hormones levels between IVF-2PN group (two pronucleus) and IVF-3PN group (three or more pronucleus). We also determined the optimal cutoff value of basal E2, LH and E2 on the day of Gn and LH on the day of HCG by Receiver Operator Characteristic (ROC) curve analysis. The data indicated that the percentage of IVF-2PN significantly decreased and the percentage of IVF-3PN considerably increased when these hormones concentration were higher than the cutoff value.\u003c/p\u003e\n\u003cp\u003eIn conclusion, for the patients retrieving single oocyte in IVF cycles, excessive hormones concentration may significantly increase the risk of polyspermy.\u003c/p\u003e"},{"header":"Background","content":"\u003cp\u003eFertilization is an essential biological process in sexual reproduction and contains a series of molecular interactions between the sperm and oocyte [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]. Mammalian fertilization has been studied as a sequence of steps: sperm bind and penetrate through the zona pellucida of the oocyte, adhere to the oocyte plasma membrane and finally fuse with the oocyte. Sperm-oocyte fusion is indispensable for completing mammalian fertilization. Among the various steps of fertilization, gamete membrane fusion must be an extremely robust and precise mechanism, as it is the climax of fertilization [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eFertilization of an oocyte by multiple sperms, a condition is known as polyspermy, is catastrophic to embryonic development in most sexually reproducing species [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. The rate of polyspermy from in vitro fertilized human oocytes is around 10% and therefore remarkably high [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]. Two primary safeguards protect oocytes against polyspermy: the \u0026ldquo;oocyte membrane block\u0026rdquo; and the \u0026ldquo;zona reaction\u0026rdquo;. The former involves a depolarization of the oocyte membrane, polyspermy is prevented at the plasma membrane level, where fusion of the first sperm causes depolarization of the membrane and shedding of the oocyte\u0026rsquo;s sperm receptor Juno, thereby preventing fusion of further sperm [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e], transiently contributing to polyspermy block. The \u0026ldquo;oocyte membrane block\u0026rdquo; or so-called \u0026ldquo;fast block\u0026rdquo; to polyspermy occurs in seconds [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]; The second proposed mechanism involves a Ca\u003csup\u003e2+\u003c/sup\u003e oscillation event activated by attachment of the sperm to the oocyte membrane. The subsequent increase in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration triggers the exocytosis of cortical granules from just below the oocyte membrane into the perivitelline space. Cortical granules contain enzymes such as hydrolase, proteinase, and peroxidase that modify and thereby harden the zona pellucida [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. This \u0026ldquo;slow block\u0026rdquo; occurs within approximately 5 to 8\u0026nbsp;min of oocyte activation and is considered the main mechanism of polyspermy block in humans [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eA variety of oocyte-related factors contribute to the pathogenesis of polyspermy, including the degree of oocyte maturation, changes in the molecular biology of the ZP, and alterations in the cortical reaction [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. It also has been shown that the phosphatidylinositol signaling pathway plays an important role in the cortical reaction, and alterations of this pathway might disrupt the membrane block leading to polyspermy [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. Understanding the mechanisms that protect mammalian oocytes from polyspermy is not only of interest for fundamental research but also of direct medical relevance.\u003c/p\u003e\n\u003cp\u003eIn human assisted reproductive technology, polyspermy is lethal to the embryo development in vitro, particularly to the patients who retrieved single oocyte which is so precious. Therefore, our study was the first time to focus on the possible correlation between hormones concentration in serum and the fertilization outcome of single oocyte in IVF cycles.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eStudy Population\u003c/h2\u003e\n\u003cp\u003eIn this retrospective study, total of 433 patients from conventional IVF between 2015\u0026ndash;2019 were analyzed: The inclusion criteria: a) the female patients retrieved single oocyte during the operation; b) the fertilization outcome of single oocyte was 2PN or 3PN.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eControlled ovarian hyperstimulation protocol\u003c/h2\u003e\n\u003cp\u003eIn all of IVF cycles, the types of controlled ovarian hyperstimulation protocol were mini-stimulation cycles, natural\u003cstrong\u003e/\u003c/strong\u003esemi-natural ovulation cycles, superovulation cycles and antagonist cycles.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eCriteria for grouping\u003c/h2\u003e\n\u003cp\u003eIn selected IVF cycles, according to the number of pronucleus in single oocyte on Day1, patients were assigned to two groups: a) IVF-2PN group: one female pronucleus and one male pronucleus were present in the oocyte; b) IVF-3PN group: three or more pronucleus were present in the oocyte.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analyses\u003c/h2\u003e\n\u003cp\u003eDifferences in age, duration of infertility, BMI, basal hormone levels and the levels of FSH、LH、E2 and P during medication were analyzed by \u003cem\u003eT-test\u003c/em\u003e using a SPSS version 22 (IBM Corp., USA). The values were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. The values with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eBaseline characteristics of patients\u003c/h2\u003e\n\u003cp\u003eComparison of demographic information from the patients, such as age, duration of infertility, BMI and basal hormone levels between IVF-2PN group and IVF-3PN group were listed in Table\u0026nbsp;1. There was a significantly higher level of basal E2 in IVF-3PN group compared with IVF-2PN group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eInformation of patients during medication\u003c/h2\u003e\n\u003cp\u003eIn the period of controlled ovulation, the main information of female patients, such as total usage of Gn、the levels of FSH、LH、E2 and P in serum on the day of Gn; total usage of HCG、the levels of FSH、LH、E2 and P in serum on the day of HCG were comparative in the two groups (Table\u0026nbsp;2). There were considerably higher levels of LH and E2 in serum on the day of Gn in IVF-3PN group compared with IVF-2PN group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In addition, a significantly higher level of LH in serum on the day of HCG was showed in IVF-3PN group compared to IVF-2PN group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eThe thresholds of several hormone concentrations in serum\u003c/h2\u003e\n\u003cp\u003eThe optimal cutoff value of basal E2 (70.5\u0026nbsp;IU/L)、LH (6.315\u0026nbsp;IU/L) and E2 (128.5\u0026nbsp;IU/L) in serum on the day of Gn and LH (3.28\u0026nbsp;IU/L) in serum on the day of HCG were determined by ROC curve analysis. The percentage of IVF-2PN significantly decreased and the percentage of IVF-3PN considerably increased when the basal E2 concentration was higher than the cutoff value (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;3), the same tendency were present in the analysis of LH concentration、E2 concentration on the day of Gn (Table\u0026nbsp;4, Table\u0026nbsp;5) and LH concentration on the day of HCG (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Table\u0026nbsp;6).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSexual reproduction culminates in fertilization and occurs when two haploid gametes recognize each other and fuse to form a single diploid zygote [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. Eggs that fuse with more than a single sperm contain an excessive amount of genetic material contributed by the male and are said to be polyspermic, resulting in the formation of a nonviable embryo. Aviable human embryo can only develop from an oocyte that is fertilized by a single sperm [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, the incidence of polyspermy is 3\u0026ndash;10% in IVF cycles [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe phospholipase C (PLC)-induced cascade of events was one of the earliest known signaling pathways initiated by fertilization. Fast polyspermy block after fertilization in X. laevis eggs is mediated by activation of PLC, which increases inositol 1,4,5-trisphosphate (IP3) and evokes Ca\u003csup\u003e2+\u003c/sup\u003e release from the endoplasmic reticulum (ER). The calcium rise is caused by the opening of IP3-gated calcium channels in the ER [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. This ER-derived Ca\u003csup\u003e2+\u003c/sup\u003e then activates a Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e channel to induce the fast polyspermy block [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. So, a major question remain to be elucidated. How does the sperm-egg interaction activated PLC to increase IP3 and release of Ca\u003csup\u003e2+\u003c/sup\u003e from the ER in the oocyte? Our results indicated that the significantly differences in the level of LH and E2 on the day of Gn, the level of LH on the day of HCG between IVF-2PN group and IVF-3PN group may be relative to the fertilization outcome of single oocyte.\u003c/p\u003e\n\u003cp\u003eDuring the development of primary follicles, gap junctions construct channels for material and information exchange between granulosa cells and oocytes. Some hormones, ions and signal molecules (such as cAMP, Ca\u003csup\u003e2+\u003c/sup\u003e, IP3, etc.) can communicate between cells and cause important biological effects. For example, the signal molecules produced by oocytes can be transported to granulosa cells to maintain the continuous proliferation and development of granulosa cells and prevent the premature differentiation of granulosa cells. In the secondary follicular stage, the follicular membrane began to differentiate into two layers: the inner and outer membranes. At this time, LH receptor has been expressed on endometrial cells. LH can act on endometrial cells and promote the synthesis and secretion of androgen. Androgen diffuses into granulosa cells, and then they are transformed into E2 by the aromatase in granulosa cells. There are receptors for E2 in the cytoplasm and nucleus of granulosa cells. The synthesized E2 has a positive feedback effect on granulosa cells and can stimulate the proliferation of granulosa cells. Combined with the above professional knowledge and our previous research results, we put forward such a scientific hypothesis: the more LH content, the more androgens synthesized by the endometrial cells, and the more E2 synthesized by the granulosa cells, then E2 promotes the proliferation of granulosa cells. At this time, in order to maintain the continuous proliferation and development of granulosa cells, the signal molecule IP3 produced by oocytes may be transported to granulosa cells through gap junctions, so the number of IP3 binding to endoplasmic reticulum ligands is reduced, which leads to the insufficient release of Ca\u003csup\u003e2+\u003c/sup\u003e in endoplasmic reticulum, the blocking mechanism of polyspermy can not be started, and which eventually results in the occurrence of polyspermy (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe limitation of this paper is to analyze the differences of hormone levels in a retrospective way. In the next prospective cohort study, we will detect the hormones concentration secreted by granulosa cells from the cell level, and discover the gene variation in the peripheral blood from the genetic point of view.\u003c/p\u003e"},{"header":"Conclusion","content":" \u003cp\u003eThe study was the first time to focus on possible correlation between hormones concentration in serum and the fertilization outcome of single oocyte in IVF cycles. Our data indicated that excessive concentrations of LH (\u0026thinsp;≧\u0026thinsp;6.315\u0026nbsp;IU/L) and E2 (\u0026thinsp;≧\u0026thinsp;128.5\u0026nbsp;IU/L) on the day of Gn and excessive concentration of LH (\u0026thinsp;≧\u0026thinsp;3.28\u0026nbsp;IU/L) on the day of HCG may increase the risk of polyspermy for single oocyte. Our findings also provide a strong evidence and a clear clinical guidance for clinicians: when the patient retrieving single oocyte suffered a 3PN fertilization fate in IVF, ICSI is recommended for the next cycle to avoid polyspermy if the relevant hormone concentrations were above the threshold.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eIVF: \u003c/strong\u003ein vitro fertilization\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eICSI: \u003c/strong\u003eintracytoplasmic sperm injection\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE2:\u003c/strong\u003e Estradiol\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLH: \u003c/strong\u003eLuteinizing Hormone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFSH: \u003c/strong\u003eFollicle Stimulating Hormone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eP:\u003c/strong\u003e Progesterone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGn: \u003c/strong\u003egonadotropin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHCG: \u003c/strong\u003ehuman\u0026nbsp;chorionic\u0026nbsp;gonadotropin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROC: \u003c/strong\u003eReceiver Operator Characteristic\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBMI: \u003c/strong\u003ebody mass index\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZP:\u003c/strong\u003e zona pellucida\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePLC:\u003c/strong\u003e phospholipase C\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIP3:\u003c/strong\u003e inositol 1,4,5-trisphosphate\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eER: \u003c/strong\u003eendoplasmic reticulum\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Medical Ethics Committee of Sun Yat-Sen Memorial Hospital affiliated with SunYat-sen University. Participants gave written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe article was approved for publication by Institutional Medical Ethics Committee of Sun Yat-Sen Memorial Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will not be shared, because the study contains human data, it is private.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was funded by Mechanism of inflammatory factors mediated by SP1 regulating telomerase activity in granulosa cells and affecting follicular development in endometriosis through NF-kB (2019A1515012005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHaijing Zhao\u003c/strong\u003e conceived of the manuscript and wrote the first draft, \u003cstrong\u003eQi Qiu\u003c/strong\u003e and \u003cstrong\u003ePing Yuan\u003c/strong\u003e performed the statistical analyses, \u003cstrong\u003eNengyong Ouyang\u003c/strong\u003e and \u003cstrong\u003eYuqin Zhu \u003c/strong\u003ecollected and proofread data, \u003cstrong\u003eWenjun Wang\u003c/strong\u003e participated in its design and helped to draft the manuscript. All authors reviewed the final manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWassarman PM, Jovine L, Litscher ES: \u003cstrong\u003eA profile of fertilization in mammals.\u003c/strong\u003e \u003cem\u003eNature Cell Biology \u003c/em\u003e2001, \u003cstrong\u003e3:\u003c/strong\u003eE59-64.\u003c/li\u003e\n\u003cli\u003eEvans, P. J: \u003cstrong\u003eSperm-Egg Interaction.\u003c/strong\u003e \u003cem\u003eAnnual Review of Physiology \u003c/em\u003e2012, \u003cstrong\u003e74:\u003c/strong\u003e477-502.\u003c/li\u003e\n\u003cli\u003eInoue N, Hagihara Y, Wright D, Suzuki T, Wada I: \u003cstrong\u003eOocyte-triggered dimerization of sperm IZUMO1 promotes sperm\u0026ndash;egg fusion in mice.\u003c/strong\u003e \u003cem\u003eNature Communications \u003c/em\u003e2015, \u003cstrong\u003e6:\u003c/strong\u003e8858.\u003c/li\u003e\n\u003cli\u003eWozniak KL, Carlson AE: \u003cstrong\u003eIon channels and signaling pathways used in the fast polyspermy block.\u003c/strong\u003e \u003cem\u003eMolecular Reproduction and Development \u003c/em\u003e2020, \u003cstrong\u003e87\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eCheeseman LP, Boulanger JM, Bond LM, Schuh M: \u003cstrong\u003eTwo pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes.\u003c/strong\u003e \u003cem\u003eNature Communications \u003c/em\u003e2016, \u003cstrong\u003e7:\u003c/strong\u003e13726.\u003c/li\u003e\n\u003cli\u003eGardner AJ, Evans JP: \u003cstrong\u003eMammalian membrane block to polyspermy: new insights into how mammalian eggs prevent fertilisation by multiple sperm.\u003c/strong\u003e \u003cem\u003eReproduction Fertility \u0026amp; Development \u003c/em\u003e2006, \u003cstrong\u003e18:\u003c/strong\u003e53.\u003c/li\u003e\n\u003cli\u003eBianchi, Enrica, Doe, Brendan, Goulding, David, Wright, Gavin, J.: \u003cstrong\u003eJuno is the egg Izumo receptor and is essential for mammalian fertilization.\u003c/strong\u003e \u003cem\u003eNature \u003c/em\u003e2014.\u003c/li\u003e\n\u003cli\u003eJaffe LA: \u003cstrong\u003eFast block to polyspermy in sea urchin eggs is electrically mediated.\u003c/strong\u003e \u003cem\u003eNature \u003c/em\u003e1976, \u003cstrong\u003e261:\u003c/strong\u003e68.\u003c/li\u003e\n\u003cli\u003eJaffe LA, Gould-Somero M, Holland LZ: \u003cstrong\u003eStudies of the Mechanism of the Electrical Polyspermy Block Using Voltage Clamp during Cross-Species Fertilization.\u003c/strong\u003e \u003cem\u003eJournal of Cell Biology \u003c/em\u003e1982, \u003cstrong\u003e92:\u003c/strong\u003e616-621.\u003c/li\u003e\n\u003cli\u003eWassarman P, Chen J, Cohen N, Litscher E, Liu C, Qi H, Williams Z: \u003cstrong\u003eStructure and function of the mammalian egg zona pellucida.\u003c/strong\u003e \u003cem\u003eJ Exp Zool \u003c/em\u003e1999, \u003cstrong\u003e285:\u003c/strong\u003e251-258.\u003c/li\u003e\n\u003cli\u003eMio Y, Iwata K, Yumoto K, Kai Y, Sargant HC, Mizoguchi C, Ueda M, Tsuchie Y, Imajo A, Iba Y, Nishikori K: \u003cstrong\u003ePossible mechanism of polyspermy block in human oocytes observed by time-lapse cinematography.\u003c/strong\u003e \u003cem\u003eJournal of Assisted Reproduction and Genetics \u003c/em\u003e2012, \u003cstrong\u003e29:\u003c/strong\u003e951-956.\u003c/li\u003e\n\u003cli\u003eDucibella: \u003cstrong\u003eThe cortical reaction and development of activation competence in mammalian oocytes.\u003c/strong\u003e \u003cem\u003eHuman Reproduction Update \u003c/em\u003e1996, \u003cstrong\u003e2:\u003c/strong\u003e29-42.\u003c/li\u003e\n\u003cli\u003eNiwa K, Park CK, Okuda K: \u003cstrong\u003ePenetration in vitro of bovine oocytes during maturation by frozen-thawed spermatozoa.\u003c/strong\u003e \u003cem\u003eJournal of Reproduction \u0026amp; Fertility \u003c/em\u003e1991, \u003cstrong\u003e91:\u003c/strong\u003e329-336.\u003c/li\u003e\n\u003cli\u003eBauskin AR, Franken DR, Uwe E, Peter D: \u003cstrong\u003eCharacterization of human zona pellucida glycoproteins.\u003c/strong\u003e \u003cem\u003eMolecular Human Reproduction \u003c/em\u003e1999\u003cstrong\u003e:\u003c/strong\u003e6.\u003c/li\u003e\n\u003cli\u003eTatone C, Eekelen CGV, Colonna R: \u003cstrong\u003ePlasma membrane block to sperm entry occurs in mouse eggs upon parthenogenetic activation.\u003c/strong\u003e \u003cem\u003eMolecular Reproduction \u0026amp; Development \u003c/em\u003e1994, \u003cstrong\u003e38:\u003c/strong\u003e200-208.\u003c/li\u003e\n\u003cli\u003eWright GJ, Bianchi E: \u003cstrong\u003eThe challenges involved in elucidating the molecular basis of sperm\u0026ndash;egg recognition in mammals and approaches to overcome them.\u003c/strong\u003e \u003cem\u003eCell and Tissue Research \u003c/em\u003e2015, \u003cstrong\u003e363:\u003c/strong\u003e227-235.\u003c/li\u003e\n\u003cli\u003eVen HHVD, Al-Hasani S, Diedrich K, Hamerich U, Krebs D: \u003cstrong\u003ePolyspermy in in Vitro Fertilization of Human Oocytes: Frequency and Possible Causes.\u003c/strong\u003e \u003cem\u003eAnn N Y Acad Sci \u003c/em\u003e1985, \u003cstrong\u003e442:\u003c/strong\u003e88-95.\u003c/li\u003e\n\u003cli\u003eTrounson A, Bongso A: \u003cstrong\u003e3 Fertilization and Development in Humans.\u003c/strong\u003e \u003cem\u003eCurrent Topics in Developmental Biology \u003c/em\u003e1996.\u003c/li\u003e\n\u003cli\u003eHo P, Yeung W, Chan Y, So W, Chan S: \u003cstrong\u003eFactors affecting the incidence of polyploidy in a human in vitro fertilization program.\u003c/strong\u003e \u003cem\u003eInternational Journal of Fertility \u0026amp; Menopausal Studies \u003c/em\u003e1994, \u003cstrong\u003e39:\u003c/strong\u003e14.\u003c/li\u003e\n\u003cli\u003eJaffe LA: \u003cstrong\u003eThe fast block to polyspermy: New insight into a century-old problem.\u003c/strong\u003e \u003cem\u003eJournal of General Physiology \u003c/em\u003e2018\u003cstrong\u003e:\u003c/strong\u003ejgp.201812145-.\u003c/li\u003e\n\u003cli\u003eCarlson AE, Lee MT, Phelps WA, Tembo M, Wozniak KL: \u003cstrong\u003ePLC and IP3-evoked Ca2+ release initiate the fast block to polyspermy in Xenopus laevis eggs.\u003c/strong\u003e \u003cem\u003eJournal of General Physiology \u003c/em\u003e2018, \u003cstrong\u003e150:\u003c/strong\u003e1239-1248.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cp\u003eTable 1\u003c/p\u003e\n\u003cp\u003eBaseline characteristics of patients from IVF-2PN group and IVF-3PN group.\u003c/p\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eIVF-2PN group\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eIVF-3PN group\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003eP\u003c/span\u003e\u003c/div\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003eFemale\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eMean age (y)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e38.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e39.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.546\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eDuration of infertility\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e5.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e5.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.964\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eThe total cycles\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e3.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e3.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.054\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eInfertility type\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eprimary/secondary\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eprimary/secondary\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u0026mdash;\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e95/236\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e33/74\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eBMI (Kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e22.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e22.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.608\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eBasal FSH (IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e12.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e12.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.596\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eBasal LH (IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e5.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e6.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.159\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eBasal E2 (IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e69.29\u0026thinsp;\u0026plusmn;\u0026thinsp;3.73\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e102.47\u0026thinsp;\u0026plusmn;\u0026thinsp;14.46\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.002\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eBasal PRL (IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e14.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e17.06\u0026thinsp;\u0026plusmn;\u0026thinsp;3.30\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.299\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003eMale\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eMean age (y)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e40.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e40.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.354\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eBMI (Kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e24.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e24.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.196\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eAcrosomal enzyme\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e65.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.27\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e74.12\u0026thinsp;\u0026plusmn;\u0026thinsp;3.99\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.060\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003e\u003cspan class=\"Italic\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05: significant difference\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tabb\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cp\u003eTable 2\u003c/p\u003e\n\u003cp\u003eInformation of patients from IVF-2PN group and IVF-3PN group during medication.\u003c/p\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eIVF-2PN group\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eIVF-3PN group\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003eP\u003c/span\u003e\u003c/div\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003eGn\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eTotal usage\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e1311.56\u0026thinsp;\u0026plusmn;\u0026thinsp;63.03\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e1060.98\u0026thinsp;\u0026plusmn;\u0026thinsp;82.52\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.038\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eTotal day\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e7.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e6.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.058\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eStart usage\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e163.78\u0026thinsp;\u0026plusmn;\u0026thinsp;3.70\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e161.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5.55\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.735\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eFSH (IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e11.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e12.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.628\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eLH (IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e5.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e7.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.004\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eE2 (IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e123.16\u0026thinsp;\u0026plusmn;\u0026thinsp;7.35\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e170.65\u0026thinsp;\u0026plusmn;\u0026thinsp;19.58\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.006\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eP (IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.738\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003eHCG\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eTotal usage\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e9048.19\u0026thinsp;\u0026plusmn;\u0026thinsp;123.27\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e9163.55\u0026thinsp;\u0026plusmn;\u0026thinsp;217.87\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.644\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eFSH (IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e25.95\u0026thinsp;\u0026plusmn;\u0026thinsp;4.85\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e20.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.521\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eLH (IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e7.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e9.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.016\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eE2 (IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e565.10\u0026thinsp;\u0026plusmn;\u0026thinsp;22.16\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e508.48\u0026thinsp;\u0026plusmn;\u0026thinsp;28.71\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.181\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eP (IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e1.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.463\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003e\u003cspan class=\"Italic\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05: significant difference\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tabc\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cp\u003eTable 3\u003c/p\u003e\nComparsion of the percentage of IVF-2PN and IVF-3PN between Low concentration group and High concentration group according to basal E2 level.\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eBasal E2 (IU/L)\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eLow concentration\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eHigh concentration\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003eP\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eFertilization outcome\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e(\u0026lt; 70.5\u0026nbsp;IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e(\u0026thinsp;≧\u0026thinsp;70.5\u0026nbsp;IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eIVF-2PN number (%)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e232 (78.4)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e94 (68.6)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.028\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eIVF-3PN number (%)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e64 (21.6)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e43 (31.4)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.028\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003e\u003cspan class=\"Italic\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05: significant difference\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tabd\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cp\u003eTable 4\u003c/p\u003e\n\u003cp\u003eComparsion of the percentage of IVF-2PN and IVF-3PN between Low concentration group and High concentration group according to LH level on the day of Gn.\u003c/p\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eLH (IU/L) (Gn )\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eLow concentration\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eHigh concentration\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003eP\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eFertilization outcome\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e(\u0026lt; 6.315\u0026nbsp;IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e(\u0026thinsp;≧\u0026thinsp;6.315\u0026nbsp;IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eIVF-2PN number (%)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e230 (79.9)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e96 (66.2)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.002\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eIVF-3PN number (%)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e58 (20.1)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e49 (33.8)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.002\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003e\u003cspan class=\"Italic\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05: significant difference\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tabe\" style=\"width: 673px;\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cp\u003eTable 5\u003c/p\u003e\n\u003cp\u003eComparsion of the percentage of IVF-2PN and IVF-3PN between Low concentration group and High concentration group according to E2 level on the day of Gn.\u003c/p\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth style=\"width: 230px;\" align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth style=\"width: 312px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eE2 (IU/L) (Gn )\u003c/div\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 103px;\" align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 230px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 147px;\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eLow concentration\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 165px;\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eHigh concentration\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 103px;\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003eP\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 230px;\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eFertilization outcome\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 147px;\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e(\u0026lt; 128.5\u0026nbsp;IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 165px;\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e(\u0026thinsp;≧\u0026thinsp;128.5\u0026nbsp;IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 103px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 230px;\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eIVF-2PN number (%)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 147px;\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e225 (79.5)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 165px;\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e101 (67.3)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 103px;\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.005\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 230px;\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eIVF-3PN number (%)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 147px;\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e58 (20.5)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 165px;\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e49 (32.7)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 103px;\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.005\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 645px;\" colspan=\"4\"\u003e\u003cspan class=\"Italic\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05: significant difference\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tabf\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cp\u003eTable 6\u003c/p\u003e\n\u003cp\u003eComparsion of the percentage of IVF-2PN and IVF-3PN between Low concentration group and High concentration group according to LH level on the day of HCG.\u003c/p\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eLH (IU/L) (HCG )\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eLow concentration\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eHigh concentration\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003eP\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eFertilization outcome\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e(\u0026lt; 3.28\u0026nbsp;IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e(\u0026thinsp;≧\u0026thinsp;3.28\u0026nbsp;IU/L)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eIVF-2PN number (%)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e98 (86.7)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e228 (71.3)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.001\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eIVF-3PN number (%)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e15 (13.3)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e92 (28.7)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.001\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003e\u003cspan class=\"Italic\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05: significant difference\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\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":"IVF, Single oocyte, Hormone concentration, Fertilization outcome","lastPublishedDoi":"10.21203/rs.3.rs-136349/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-136349/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Fertilization of an oocyte by multiple sperms is known as polyspermy. Here, the purpose of this work was to investigate the possible correlation between hormones concentration in serum and the fertilization outcome of single oocyte in IVF cycles.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eIn this retrospective\u0026nbsp;analysis, the baseline characteristics and serum hormones levels of female patients retrieving single oocyte in IVF cycles were analyzed. Female age, duration of infertility, body mass index (BMI), basal follicle stimulating hormone (FSH), basal luteinizing hormone (LH), basal estradiol (E2), the level of FSH, LH, E2 and progesterone (P) on the day of Gonadotropin (Gn), the level of FSH, LH, E2 and P on the day of\u0026nbsp;human\u0026nbsp;chorionic\u0026nbsp;gonadotropin (HCG) were measured. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAccording to the number of pronucleus in single oocyte on Day1, patients were assigned to IVF-2PN group (two pronucleus) and IVF-3PN group (three or more pronucleus). There were extremely higher levels of basal E2, LH and E2 on the day of Gn in IVF-3PN group compared with IVF-2PN group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). The significantly higher level of LH on the day of HCG was present in IVF-3PN compared to IVF-2PN group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). Forthermore, the optimal cutoff value of basal E2 (70.5 IU/L), LH (6.315 IU/L) and E2 (128.5 IU/L) on the day of Gn and LH (3.28 IU/L) on the day of HCG were determined by ROC curve analysis. The results showed that the percentage of IVF-2PN significantly decreased and the percentage of IVF-3PN considerably increased when the basal E2 concentration was higher than the cutoff value (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05), the same tendency were present in the analysis of LH concentration, E2 concentration on the day of Gn and LH concentration on the day of HCG (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Our data indicated for the first time that the excessive concentration of basal E2, excessive concentrations of LH and E2 on the day of Gn and excessive concentration of LH on the day of HCG may significantly increase the risk of polyspermy for single oocyte in IVF cycles.\u003c/p\u003e","manuscriptTitle":"Excessive Hormones Concentration in Serum May Increase the Risk of Polyspermy for Single Oocyte","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-30 23:57:05","doi":"10.21203/rs.3.rs-136349/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":"290b4d02-d5ab-48c6-b169-fb7173ef44f2","owner":[],"postedDate":"December 30th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":1666503,"name":"Sexual \u0026 Reproductive Medicine"}],"tags":[],"updatedAt":"2021-01-09T22:27:12+00:00","versionOfRecord":[],"versionCreatedAt":"2020-12-30 23:57:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-136349","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-136349","identity":"rs-136349","version":["v1"]},"buildId":"i_I_jhV-QviBjPLXJga9V","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}