Analysis of reproductive outcomes in cases with repeated high proportion of multiple pronuclei (MPN) after previous IVF and subsequent ICSI

Analysis of reproductive outcomes in cases with repeated high proportion of multiple pronuclei (MPN) after previous IVF and subsequent ICSI | 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 Article Analysis of reproductive outcomes in cases with repeated high proportion of multiple pronuclei (MPN) after previous IVF and subsequent ICSI Mingzhao Li, Shengjia Shi, Sen Qiao, Xia Xue, Juanzi Shi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6216425/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 Intracytoplasmic sperm injection (ICSI) is beneficial for most women with a high multiple pronuclei (MPN) incidence from previous conventional in vitro fertilization (IVF). Nevertheless, a small group of patients still undergo the same problem in the subsequent ICSI treatment. In this research, we aimed to explore whether ICSI is still effective in improving embryonic development and reproductive outcomes in such patients. A prospective cohort study was performed between January 2017 and December 2023, including 12 couples with a high proportion of MPN zygotes in previous C-IVF and subsequent ICSI cycles. The MPN rate was not less than 50% and the number of MPN zygotes was not less than 3 in each initiated cycle for the couples included in the study. We observed no significant differences in the 2PN (22.29 versus 25.14%; p = 0.526), MPN (45.14 versus 40.98%; p = 0.427) and 1PN (3.43 versus 2.73; p = 0.703) rates between previous IVF and subsequent ICSI cycles. After conventional IVF, a total D3 good quality embryo rate of 0% (0/39) was achieved, which was significantly lower than that of 23.91% (11/46) after ICSI (0 versus 23.91%; p = 0.012). There was no significant difference in the D3 available embryo rate between the two groups (38.46 versus 60.87%; p = 0.066). ICSI cycles demonstrated significantly lower blastomere multinucleation rate compared with previous IVF cycles (65.21 versus 87.18%; p = 0.037). We also observed that ICSI showed a significant improvement in the ongoing pregnancy (62.50 versus 0%; p = 0.031) and live birth (62.50 versus 0%; p = 0.031) rates compared with conventional IVF. We further identified a novel heterozygous missense mutation of TUBB8 (c.33G > T [p.Q11H]) that cause a repeated high proportion of MPN zygotes. For patients with high proportion of MPN zygotes in previous C-IVF and subsequent ICSI cycles, ICSI still can improve embryo development and reproductive outcomes compared with C-IVF. Biological sciences/Developmental biology Health sciences/Endocrinology Multiple pronuclei IVF ICSI Embryo quality Reproductive outcomes Figures Figure 1 Introduction Two pronuclei typically form following fertilization in humans, with one pronucleus originating from the penetrating spermatozoon and the other from the oocyte. Nevertheless, in vitro fertilization (IVF) and embryo culture routinely lead to variations of this outcome 1 . And the incidence of multiple pronuclei (MPN) is a common phenomenon and it ranges from 2–10% after conventional in vitro fertilization (C-IVF) in human 2 . The majority of MPN during C-IVF is due to polyspermy. For patients who experience a high incidence of MPN zygotes after C-IVF, intracytoplasmic sperm injection (ICSI) may serve as an effective tool to overcome this abnormal condition in subsequent treatment cycles 3 . Although ICSI can effectively eliminates dispermic triploidy, it does not prevent oocyte-induced MPN formation 4 . It has been confirmed that ICSI attempts that result in MPN zygotes are the product of retention of the oocyte’s second polar body 5 . Therefore, there are still some patients who will face similar outcomes following subsequent ICSI. Some studies showed that ICSI could enhance the embryo quality in patients with non-male factor infertility 6 , 7 , 8 . Another study also demonstrated that ICSI could significantly improve the good quality embryo rate of the patients with poor quality embryos in the previous C-IVF cycles 8 . In this study, we focused on the population who obtained high proportion of MPN zygotes in previous C-IVF and subsequent ICSI cycles. In the previous C-IVF cycles, none of the couples were able to obtain a good-quality embryo on day 3. Furthermore, the infertility factor identified in the enrolled couples was non-male factor infertility. Therefore, ICSI may be beneficial to the patients with such issues. Nevertheless, the current literature lacks sufficient evidence regarding the effectiveness of ICSI in this specific subpopulation of patients. Therefore, we aimed to explore whether ICSI was still effective in improving embryonic development and reproductive outcomes in such patients. Materials and methods A retrospective cohort study was performed between January 2017 and December 2023, including 12 couples with a high proportion of MPN zygotes in previous C-IVF and subsequent ICSI cycles. Patients were only included when they underwent one C-IVF and one ICSI cycle at the Center for Reproductive Medicine at Northwest Women’s and Children’s Hospital. Further, the rate of MPN zygotes was not less than 50% and the number of MPN zygotes was not less than 3 in each initiated cycle. Embryo development and reproductive outcomes after ICSI were compared to previous C-IVF cycles. For couples with no available embryos in previous C-IVF and subsequent ICSI cycles, the genetic screening was recommended for them. To identify any genetic abnormalities in these cases, we performed whole-exome sequencing for TUBB8 gene and validated the results by Sanger sequencing. We focused on TUBB8 (MIM: 347688; NM_177987.3) missense mutations after filtering out allele frequencies higher than 1% in the gnomAD, ExAC, and 1000 Genomes databases and intronic or synonymous mutations. The peripheral blood samples were taken from female patients and their family members, after obtaining informed consent, following the guidelines approved by the Ethics Review Board of the Northwest Women’s and Children’s Hospital. This study was approved by the Ethics Committee of Northwest Women’s and Children’s Hospital (No. 2023003). All participants in our study underwent controlled ovarian hyperstimulation. The ovarian stimulation protocols in our reproductive medicine center include the GnRH agonist long protocol, GnRH agonist short protocol, and GnRH antagonist protocol, as detailed in previous literature 9 . Notably, recombinant follicle-stimulating hormone (FSH) or urinary FSH and/or human menopausal gonadotropins were used with daily doses between 100 and 450 IU based on patients’ characteristics as calculated previously 9 . C-IVF fertilization was performed 2-2.5 h after oocyte retrieval. Each oocyte was incubated with approximately 40 000 sperm and fertilization was allowed to occur naturally. Shortterm fertilization was adopted and the cumulus granule cells were peeled off 4-4.5 h after fertilization. Our skilled ICSI operators injected the metaphase II oocytes by the direct penetration technique. Oocytes were placed individually into 5 µl droplets of G-MOPS (Vitrolife, Goteborg, Sweden) solution covered under warm mineral oil. Sperm were placed in a central 5 µl droplet of polyvinylpyrrolidone solution, and the details of ICSI protocol were as previously described 10 . Oocytes were examined for the presence of two pronuclei (PN) to confirm fertilization 19–20 h after insemination. Normal fertilization was defined by the presence of 2PN and the extrusion of the second polar body. The nucleus status of the blastomeres was evaluated as non-multinuclear at the two-cell stage when each blastomere contained at most one nucleus. After 64–68 h of culture, the morphologic score was given for day-3 embryo according to the number of blastomeres, homogeneous degree of blastomeres and degree of cytoplasmic fragmentation: grade I (8–10 blastomeres, even homogeneous blastomeres 10 blastomeres with even homogeneous blastomeres of no cytoplasmic fragmentation, 8–10 blastomeres, even homogeneous blastomeres with 10%-20% cytoplasmic fragmentation), grade III (uneven and nonhomogeneous blastomeres with 20–50% cytoplasmic fragmentation), and grade IV (uneven and non-homogeneous blastomeres with > 50% cytoplasmic fragmentation). The D3 good quality embryos were graded I and II. The D3 available embryos were graded I, II, and III. Blastocysts were observed on the fifth morning after oocyte retrieval, and the scoring system for blastocyst evaluation was a combination of the stage of development from 1 to 6 (early, blastocyst, full blastocyst, expanded, hatching/hatched) and of the grade of the inner cell mass (ICM; A, tightly packed, many cells; B, loosely grouped, several cells; or C, very few cells.) and of the trophectoderm (TE; A, many cells forming a cohesive epithelium; B, few cells forming a loose epithelium; or C, very few large cells.). Three methods of luteal support are implemented in our center. I. Vaginal progesterone gel (90 mg q.d; Crinone, Serono, Hertfordshire, UK); II. Vaginal progesterone soft capsules (0.2 g t.i.d; Utrogestan, Besins, France); III. Intramuscular progesterone (60 mg q.d; Xianju, Zhejiang, China). Patients from both groups could select one of these three luteal support methods and receive oral progesterone (10 mg t.i.d; Dydrogesterone, Abbott Biologicals B.V., Amsterdam, Netherlands) simultaneously. The luteal support was maintained until week 10 of gestation. Clinical pregnancy was characterized as the presence of an intrauterine gestational sac on ultrasonography during the first trimester. Ongoing pregnancy was defined as a clinical pregnancy that continued for at least 12 weeks. Live birth was defined as a pregnancy that ended with the birth of a live infant. Statistical analysis between groups in the case of continuous variables was performed with Student’s t test for data with normal distribution. Non-parametric Mann-Whitney U-test was performed for data with skewed distribution. Statistical analysis between groups in the case of categorical variables was expressed as number and percentage and Chi-square test or Fisher exact test was performed. The statistical analysis was performed with SPSS version 23 (IBM Corp.; NY, USA). A p -value of less than 0.05 was considered to indicate statistical significance. Results The baseline characteristics of the patients were shown in Table 1 , including female age, female body mass index (BMI), basal FSH, basal E 2 , total Gn dosage, stimulation duration, number of oocytes retrieved, male age, sperm concentration, progressive motility and the mean number of embryos transferred ( p > 0.05). Table 1 General characteristics of the enrolled patients. Parameter C-IVF ICSI P -Value Patients (n) 12 12 / Cycles (oocyte retrievals) 175 218 / Female age (y) 30.92 ± 3.37 31.25 ± 3.17 0.805 BMI for women (kg/m²) 23.53 ± 3.55 23.58 ± 3.26 0.971 Basal FSH (IU/L) 6.28 ± 2.20 6.18 ± 2.13 0.909 Basal E 2 (pg/mL) 51.26 ± 32.34 51.10 ± 29.82 0.990 Total Gn dosage (IU) 2201.67 ± 1100.66 2210.41 ± 697.42 0.982 Stimulation duration (days) 11.17 ± 2.69 10.50 ± 1.68 0.474 Number of oocytes retrieved (n) 14.58 ± 7.42 18.16 ± 7.02 0.237 Male age (y) 33.17 ± 2.69 33.50 ± 2.78 0.768 Sperm concentration (10 6 /mL) 59.42 ± 34.91 63.67 ± 33.06 0.762 Progressive motility (a + b) (%) 57.58 ± 9.74 56.92 ± 9.31 0.865 Mean number of embryos transferred (n) 1.63 ± 0.52 1.50 ± 0.53 0.359 From the ICSI cycles, a total of 218 cumulus-oocyte complexes (COCs) (mean 18.16) were collected, from which 183 MII oocytes (mean 15.25) were used for treatment. From previous IVF cycles, a total of 175 COCs (mean 14.58) were obtained. Following conventional IVF, an overall normal fertilization rate of 22.29% (39/175) was achieved, which was similar compared that of 25.14% (46/183) observed after ICSI (22.29 versus 25.14%; p = 0.526). We observed no significant differences in the MPN (45.14 versus 40.98%; p = 0.427) and 1PN (3.43 versus 2.73; p = 0.703) rates between the two groups. The developmental capacity of fertilized oocytes was evaluated by assessing embryo formation on Day 3 and Day 5 post-fertilization. After conventional IVF, a total D3 good quality embryo rate of 0% (0/39) was achieved, which was significantly lower than that of 23.91% (11/46) after ICSI (0 versus 23.91%; p = 0.012). There was no significant difference in the D3 available embryo rate between the two groups (38.46 versus 60.87%; p = 0.066). We further observed no significant differences in the mean number of D3 embryo blastomere (6.73 versus 7.40; p = 0.269) and D3 embryo fragmentation rate (9 versus 8%; p = 0.628) between the two groups. Importantly, ICSI cycles demonstrated significantly lower blastomere multinucleation rate compared with previous IVF cycles (65.21 versus 87.18%; p = 0.037). In conventional IVF cycles, a total of 13 embryos were transferred in fresh cycles. Additionally, no embryos were cryopreserved, and 2 embryos underwent extended culture to the blastocyst stage. In ICSI cycles, a total of 12 embryos were transferred in fresh cycles. Additionally, 3 embryos were cryopreserved, and 13 embryos underwent extended culture to the blastocyst stage (Table 2 ). The details of embryo development after previous C-IVF and subsequent ICSI cycles in couples with repeated high proportion of MPN were shown in a supplementary table. Table 2 Comparison of embryo development after previous C-IVF and subsequent ICS cycles in couples with repeated high proportion of ≥ 3PN (P1-P12). Parameter C-IVF ICSI P -Value Patients (n) 12 12 / Cycles (oocyte retrievals) 12 12 / Cumulus-oocyte complexes (n) 175 218 / 2PN rate (%, n) 22.29 (39/175) 25.14 (46/183) 0.526 MPN rate (%, n) 45.14 (79/175) 40.98 (75/183) 0.427 1PN rate (%, n) 3.43 (6/175) 2.73 (5/183) 0.703 D3 good quality embryo rate (D3 good quality embryos/2PN) 0 (0/39) 23.91 (11/46) 0.012 D3 available embryo rate (D3 embryos/2PN) 38.46 (15/39) 60.87 (28/46) 0.066 Mean number of D3 embryo blastomere (n) 6.73 ± 1.87 7.40 ± 1.89 0.269 Mean fragmentation of D3 embryo (%) 9% 8% 0.628 Blastomere multinucleation rate (Multinucleation/2PN) 87.18 (34/39) 65.21 (30/46) 0.037 No. of embryos transferred in fresh (n) 13 12 / No. of cryopreserved embryos (n) 0 3 / Embryos of extended culture to blastocyst-stage (n) 2 13 / No. of Blastocyst formation (n) 1 1 / Overall, following ICSI, a total pregnancy rate of 62.50% (5/8) was achieved, which was significantly higher than the pregnancy rate of 0% (0/8) observed after previous IVF cycles (62.50 versus 0%; p = 0.031). We also observed that ICSI showed a significant improvement in the ongoing pregnancy (62.50 versus 0%; p = 0.031) and live birth (62.50 versus 0%; p = 0.031) rates compared with previous IVF (Table 3 ). Table 3 Comparison of clinical outcomes after previous C-IVF and subsequent ICSI cycles in couples with repeated high proportion of ≥ 3PN (P1-P12). Parameter C-IVF ICSI-AOA P -Value Cycles (oocyte retrievals) 12 12 / Cycles (Embryo transfers) 8 8 / No available embryos (cycles, n) 4 4 / Cleavage-stage embryo transfer (n) 8 8 / Transfers with D3 good quality embryo (%, n) 0 (0/8) 87.50 (7/8) 0.003 Pregnancy rate (+ hCG/initiated cycle) 0 (0/8) 62.50 (5/8) 0.031 Clinical pregnancy rate (%, n) 0 (0/8) 62.50 (5/8) 0.031 Ongoing pregnancy rate (> 12 weeks) (%, n) 0 (0/8) 62.50 (5/8) 0.031 Live birth rate (%, n) 0 (0/8) 62.50 (5/8) 0.031 For three couples with no available embryos in previous IVF and subsequent ICSI cycles, one patient refused to perform the genetic screening. we performed whole-exome sequencing for TUBB8 gene and validated the results by Sanger sequencing in two female patients and their family members. In one family, no genetic mutations were found in the TUBB8 gene. In the other family, we identified a novel heterozygous missense mutation of TUBB8 (c.33G > T [p.Q11H]) that cause a repeated high proportion of MPN zygotes after previous IVF and subsequent ICSI. This heterozygous mutation of c.33G > T (p.Q11H) was inherited from her father. The variant c.33G > T was located in a conserved GTPase domain of TUBB8 families and was highly conserved among different species (Fig. 1 ). Discussion In this paper, we firstly showed that ICSI could improve embryo development and reproductive outcomes compared with conventional IVF in non-male infertile patients with high proportion of MPN zygotes. Secondly, we indicated that ICSI could significantly decrease blastomere multinucleation rate compared with previous IVF cycles in such patients. Lastly, we identified a novel heterozygous missense mutation of TUBB8 (c.33G > T [p.Q11H]) in such patients with no available embryos. Previous studies have demonstrated that ICSI could enhance the embryo quality in patients with non-male factor infertility 6 , 7 , 8 . Yang et al. showed that embryos developed via ICSI exhibited superior quality compared to those derived from C-IVF when comparing sibling oocytes from non-male-factor couples 6 . Kim et al. reported that ICSI could improve the fertilization and D3 good quality embryo rates in patients with non-male factor infertility compared with C-IVF 7 . Wang et al. investigated patients with poor-quality embryos from previous C-IVF cycles and observed that ICSI significantly improved the D3 good quality embryo rate relative to C-IVF 8 . In this study, we focused on couples with characteristics similar to those in the aforementioned reports. The enrolled population also had non-male factor infertility and poor-quality embryos in their initial treatment cycle. The key difference, however, was that poor embryo quality was associated with a higher incidence of MPN, and the MPN rate did not decrease after subsequent ICSI treatment. Overall, our findings are consistent with previous observations in the literature It was well established that both the number of D3 embryo blastomeres and the degree of embryo fragmentation were critical factors influencing embryo quality. Some studies have demonstrated that early cleavage, defined as occurring 25–27 hours post C-IVF/ICSI, serves as a strong indicator of embryo viability 11 , 12 , 13 . In theory, the oocyte is fertilized earlier by the sperm in ICSI compared with C-IVF, as the spermatozoon was directly injected into the oocyte. Consequently, the early cleavage rate would be higher in ICSI cycle than in IVF cycle. Previous studies demonstrated that the D3 good quality embryo rate was significantly higher in the early cleavage group, which was associated with higher number of D3 embryo blastomeres. Nevertheless, we observed no significant difference in the mean number of D3 embryo blastomeres between previous IVF and subsequent ICSI cycles which might be attributed to the limited sample size. Some studies also indicated that ICSI did not have a significant impact on the D3 embryo fragmentation rate which was consistent with our observation 14 , 15 . Interestingly, we found a significant difference in the blastomere multinucleation rate between previous C-IVF and subsequent ICSI. After ICSI, the blastomere multinucleation rate was significantly decreased than that of previous C-IVF. Blastomere multinucleation is a common nuclear abnormality observed in early human embryos. It was reported that a multinucleation in day 2 and day 3 cleavage embryos of 11%-34% 16,17,18 . The blastomere multinucleation rate was 75.29% across all cycles in this study, which was significantly higher than the rate reported in previous studies. Previous studies showed that the occurrence of blastomere multinucleation events was associated with a high incidence of MPN zygotes, which might explain the elevated blastomere multinucleation rate observed in this study 19 , 20 . The way a single embryo was affected might reflect how the whole cohort was influenced, even if the other embryos did not exhibit the same characteristics. The mononucleated embryos always had a higher incidence of normally aligned nucleolar precursor bodies, which was correlated with both continued embryo development and better reproductive outcomes in various studies 21 , 22 . Salumets et al. indicated that a higher number of embryos with a normal halo (a translocation of cellular organelles) were also observed in mononucleated embryos compared with multinucleated embryos 23 . And it was confirmed that the halo was associated with embryo quality 23 . Thus, the primary reasons for the increase in the rate of high-quality embryos might be related to the reduction in the proportion of multinucleated embryos on day 3. In this research, a total of 13 embryos were transferred and none of the embryos succeeded in the implantation in previous IVF cycles. Of them, 11 were multinucleated embryos and 2 were mononucleated embryos. In subsequent ICSI cycles, 12 embryos were transferred and 5 embryos succeeded in the implantation. The above data indicated that it was transferred fewer multinucleated embryos in C-IVF cycles compared with ICSI cycles. And it was also demonstrated that embryo multinucleation was associated with reduced developmental rates and lower implantation rates in Days 2–3 embryo transfers in assisted reproduction treatment 24 , 25 . The requirement for extended culture to the blastocyst-stage typically involved at least two good-quality embryos on day 3, and all embryo transfers were performed on day 3 in this research. Thus, the primary reason for the increased implantation rate might be associated with the reduction in the proportion of multinucleated embryo transfers on day 3. The incidence of a high proportion of MPN zygotes results in a sharp decrease in the number of available embryos and it increases the risk of cancelled transfers. In this research, 3 couples obtained no available embryos in previous IVF and subsequent ICSI cycles. For two female patients and their family members, we performed whole-exome sequencing for TUBB8 gene. Tubulin β eight class VIII (TUBB8) is the predominant β-tubulin isotype present in early embryos, playing a crucial role in the human oocyte spindle assembling 26 . The Mutations in TUBB8 have been confirmed to be associated with various aspects of reproductive biology, including the maturation of human oocytes, fertilization, pre-implantation embryonic development, and even failures in embryo implantation failure 27 . In one family, we identified a novel heterozygous missense mutation of TUBB8 (c.33G > T [p.Q11H]) and this heterozygous mutation of c.33G > T (p.Q11H) was inherited from her father. Previous study also identified additional novel heterozygous missense mutations in TUBB8 that were associated with a MPN phenotype in zygotes following C-IVF or ICSI, in three sterile patients who experienced repeated early embryo developmental arrest 28 . These findings suggested that TUBB8 played a significant role in the incidence of MPN phenotype. A recent study has provided a comprehensive mechanism elucidating how TUBB8 missense variants lead to oocyte abnormalities and has proposed new therapeutic avenues for treating female infertility in clinical settings 29 . It is believed that more effective treatment approaches will be offered for the patients with TUBB8 variants in the near future. Thus, this information will be valuable for future genetic counseling of infertile patients diagnosed with MPN especially for patients undergoing multiple cycles with high proportion of MPN zygotes and no available embryos. There are certain weaknesses in the current study that should be underlined. First, the primary drawback is the retrospective design and small sample size. Second, there may be some potential bias and confounders that cannot be excluded. Lastly, the data about blastocyst culture is limited and the blastocyst formation rate is relatively low in subsequent ICSI cycles. It may be associated with the limited number of D3 good quality embryos, and most of these embryos need to be used for transfer on day 3. Given the limited data and methodological constraints, further data accumulation is needed to obtain more reliable conclusions. Declarations Ethics approval and consent to participate All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. This study was approved by the Ethics Review Board of the Northwest Women’ s and Children’ s Hospital, Xi’ an, China (2023003). Competing interests The authors declare that there is no conflict of interest. Funding This project was supported by Shaanxi Technology Committee Industrial Public Relation Project (Project Number: 2023-YBSF-034) and Young Physicians Program of Chinese Medical Association (No. 17020470716). Author Contribution M.L. and J.S. designed the research; S.S. and S.Q. performed the research. 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Novel mutations and structural deletions in TUBB8: expanding mutational and phenotypic spectrum of patients with arrest in oocyte maturation, fertilization or early embryonic development. Hum. Reprod. 32 , 457–464 (2017). Sha, Q. et al. Novel mutations in TUBB8 expand the mutational and phenotypic spectrum of patients with zygotes containing multiple pronuclei. Gene 769 , 145227 (2021). Luo, H. et al. Pathogenic variants of TUBB8 cause oocyte spindle defects by disrupting with EB1/CAKP5 interactions and potential treatment targeting microtubule acetylation through HDAC6 inhibition. Clin. Transl Med. 15 , e70193 (2025). Additional Declarations No competing interests reported. Supplementary Files Supplementary.docx 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. 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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-6216425","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":481846944,"identity":"9fcf3020-2574-4548-885e-43bb30048e94","order_by":0,"name":"Mingzhao Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Mingzhao","middleName":"","lastName":"Li","suffix":""},{"id":481846946,"identity":"e89d47fd-9a4a-48d7-a101-4703f34f43ca","order_by":1,"name":"Shengjia Shi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shengjia","middleName":"","lastName":"Shi","suffix":""},{"id":481846947,"identity":"8f7a6c2c-eae2-4ecc-975f-40e27daf4c09","order_by":2,"name":"Sen Qiao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sen","middleName":"","lastName":"Qiao","suffix":""},{"id":481846948,"identity":"1d53b4de-9042-4126-a4aa-995d04ecf2f0","order_by":3,"name":"Xia Xue","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"Xue","suffix":""},{"id":481846949,"identity":"815e0f37-5f5d-4b3f-b1de-d91ff95a68d4","order_by":4,"name":"Juanzi Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYNACHgY5Nvb2A6RpMebjOZNAmj2J8yQcDIhTKt9//JnkD5m69DYJhgSGHxXbCGsxOHAg2ZiHhy23TbrxAGPPmdtEaGFsOPiYgYcnt03mQAIzYxsRWuSbgXp+8Eiks0kkGBCnheEYM+MDHh6DBOK1GJxhYwb6JcGwDRjIB4nyCzjEfvbUycu3tx988KOCGIeBAGMPhD5ApHoQ+EGC2lEwCkbBKBh5AABl/jVqASBJ6gAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Juanzi","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2025-03-13 04:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6216425/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6216425/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86388089,"identity":"b9cb80d9-1008-4e05-929e-e11db1a1000b","added_by":"auto","created_at":"2025-07-10 06:18:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":562936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic screening and analysis for patients with no available embryos A. Genetic analysis and sanger sequencing chromatograms of the family. B. Localization of mutations in the gene. C. Localization of mutations in the protein. D. Locations and conservation of mutation in TUBB8.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6216425/v1/e1de0661ab88205ff7ace725.png"},{"id":89265413,"identity":"71d4a437-04df-4515-aafc-b910c56a7716","added_by":"auto","created_at":"2025-08-18 08:02:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1193699,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6216425/v1/7d9fe063-66f2-4ace-9d01-9b53b69613a0.pdf"},{"id":86388088,"identity":"51d14bfa-847b-4e2b-a54e-d4e13dc90923","added_by":"auto","created_at":"2025-07-10 06:18:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17558,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-6216425/v1/c579013b17c6e7c33d20942b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Analysis of reproductive outcomes in cases with repeated high proportion of multiple pronuclei (MPN) after previous IVF and subsequent ICSI","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTwo pronuclei typically form following fertilization in humans, with one pronucleus originating from the penetrating spermatozoon and the other from the oocyte. Nevertheless, in vitro fertilization (IVF) and embryo culture routinely lead to variations of this outcome\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. And the incidence of multiple pronuclei (MPN) is a common phenomenon and it ranges from 2\u0026ndash;10% after conventional in vitro fertilization (C-IVF) in human\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe majority of MPN during C-IVF is due to polyspermy. For patients who experience a high incidence of MPN zygotes after C-IVF, intracytoplasmic sperm injection (ICSI) may serve as an effective tool to overcome this abnormal condition in subsequent treatment cycles\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Although ICSI can effectively eliminates dispermic triploidy, it does not prevent oocyte-induced MPN formation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. It has been confirmed that ICSI attempts that result in MPN zygotes are the product of retention of the oocyte\u0026rsquo;s second polar body\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Therefore, there are still some patients who will face similar outcomes following subsequent ICSI. Some studies showed that ICSI could enhance the embryo quality in patients with non-male factor infertility\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Another study also demonstrated that ICSI could significantly improve the good quality embryo rate of the patients with poor quality embryos in the previous C-IVF cycles\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we focused on the population who obtained high proportion of MPN zygotes in previous C-IVF and subsequent ICSI cycles. In the previous C-IVF cycles, none of the couples were able to obtain a good-quality embryo on day 3. Furthermore, the infertility factor identified in the enrolled couples was non-male factor infertility. Therefore, ICSI may be beneficial to the patients with such issues. Nevertheless, the current literature lacks sufficient evidence regarding the effectiveness of ICSI in this specific subpopulation of patients. Therefore, we aimed to explore whether ICSI was still effective in improving embryonic development and reproductive outcomes in such patients.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eA retrospective cohort study was performed between January 2017 and December 2023, including 12 couples with a high proportion of MPN zygotes in previous C-IVF and subsequent ICSI cycles. Patients were only included when they underwent one C-IVF and one ICSI cycle at the Center for Reproductive Medicine at Northwest Women\u0026rsquo;s and Children\u0026rsquo;s Hospital. Further, the rate of MPN zygotes was not less than 50% and the number of MPN zygotes was not less than 3 in each initiated cycle. Embryo development and reproductive outcomes after ICSI were compared to previous C-IVF cycles.\u003c/p\u003e\u003cp\u003eFor couples with no available embryos in previous C-IVF and subsequent ICSI cycles, the genetic screening was recommended for them. To identify any genetic abnormalities in these cases, we performed whole-exome sequencing for \u003cem\u003eTUBB8\u003c/em\u003e gene and validated the results by Sanger sequencing. We focused on \u003cem\u003eTUBB8\u003c/em\u003e (MIM: 347688; NM_177987.3) missense mutations after filtering out allele frequencies higher than 1% in the gnomAD, ExAC, and 1000 Genomes databases and intronic or synonymous mutations. The peripheral blood samples were taken from female patients and their family members, after obtaining informed consent, following the guidelines approved by the Ethics Review Board of the Northwest Women\u0026rsquo;s and Children\u0026rsquo;s Hospital. This study was approved by the Ethics Committee of Northwest Women\u0026rsquo;s and Children\u0026rsquo;s Hospital (No. 2023003).\u003c/p\u003e\u003cp\u003eAll participants in our study underwent controlled ovarian hyperstimulation. The ovarian stimulation protocols in our reproductive medicine center include the GnRH agonist long protocol, GnRH agonist short protocol, and GnRH antagonist protocol, as detailed in previous literature\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Notably, recombinant follicle-stimulating hormone (FSH) or urinary FSH and/or human menopausal gonadotropins were used with daily doses between 100 and 450 IU based on patients\u0026rsquo; characteristics as calculated previously\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eC-IVF fertilization was performed 2-2.5 h after oocyte retrieval. Each oocyte was incubated with approximately 40 000 sperm and fertilization was allowed to occur naturally. Shortterm fertilization was adopted and the cumulus granule cells were peeled off 4-4.5 h after fertilization. Our skilled ICSI operators injected the metaphase II oocytes by the direct penetration technique. Oocytes were placed individually into 5 \u0026micro;l droplets of G-MOPS (Vitrolife, Goteborg, Sweden) solution covered under warm mineral oil. Sperm were placed in a central 5 \u0026micro;l droplet of polyvinylpyrrolidone solution, and the details of ICSI protocol were as previously described\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOocytes were examined for the presence of two pronuclei (PN) to confirm fertilization 19\u0026ndash;20 h after insemination. Normal fertilization was defined by the presence of 2PN and the extrusion of the second polar body. The nucleus status of the blastomeres was evaluated as non-multinuclear at the two-cell stage when each blastomere contained at most one nucleus.\u003c/p\u003e\u003cp\u003eAfter 64\u0026ndash;68 h of culture, the morphologic score was given for day-3 embryo according to the number of blastomeres, homogeneous degree of blastomeres and degree of cytoplasmic fragmentation: grade I (8\u0026ndash;10 blastomeres, even homogeneous blastomeres\u0026thinsp;\u0026lt;\u0026thinsp;10% cytoplasmic fragmentation), grade II (6\u0026ndash;7 or \u0026gt;\u0026thinsp;10 blastomeres with even homogeneous blastomeres of no cytoplasmic fragmentation, 8\u0026ndash;10 blastomeres, even homogeneous blastomeres with 10%-20% cytoplasmic fragmentation), grade III (uneven and nonhomogeneous blastomeres with 20\u0026ndash;50% cytoplasmic fragmentation), and grade IV (uneven and non-homogeneous blastomeres with \u0026gt;\u0026thinsp;50% cytoplasmic fragmentation). The D3 good quality embryos were graded I and II. The D3 available embryos were graded I, II, and III.\u003c/p\u003e\u003cp\u003eBlastocysts were observed on the fifth morning after oocyte retrieval, and the scoring system for blastocyst evaluation was a combination of the stage of development from 1 to 6 (early, blastocyst, full blastocyst, expanded, hatching/hatched) and of the grade of the inner cell mass (ICM; A, tightly packed, many cells; B, loosely grouped, several cells; or C, very few cells.) and of the trophectoderm (TE; A, many cells forming a cohesive epithelium; B, few cells forming a loose epithelium; or C, very few large cells.).\u003c/p\u003e\u003cp\u003eThree methods of luteal support are implemented in our center. I. Vaginal progesterone gel (90 mg q.d; Crinone, Serono, Hertfordshire, UK); II. Vaginal progesterone soft capsules (0.2 g t.i.d; Utrogestan, Besins, France); III. Intramuscular progesterone (60 mg q.d; Xianju, Zhejiang, China). Patients from both groups could select one of these three luteal support methods and receive oral progesterone (10 mg t.i.d; Dydrogesterone, Abbott Biologicals B.V., Amsterdam, Netherlands) simultaneously. The luteal support was maintained until week 10 of gestation.\u003c/p\u003e\u003cp\u003eClinical pregnancy was characterized as the presence of an intrauterine gestational sac on ultrasonography during the first trimester. Ongoing pregnancy was defined as a clinical pregnancy that continued for at least 12 weeks. Live birth was defined as a pregnancy that ended with the birth of a live infant.\u003c/p\u003e\u003cp\u003eStatistical analysis between groups in the case of continuous variables was performed with Student\u0026rsquo;s t test for data with normal distribution. Non-parametric Mann-Whitney U-test was performed for data with skewed distribution. Statistical analysis between groups in the case of categorical variables was expressed as number and percentage and Chi-square test or Fisher exact test was performed. The statistical analysis was performed with SPSS version 23 (IBM Corp.; NY, USA). A \u003cem\u003ep\u003c/em\u003e-value of less than 0.05 was considered to indicate statistical significance.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe baseline characteristics of the patients were shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, including female age, female body mass index (BMI), basal FSH, basal E\u003csub\u003e2\u003c/sub\u003e, total Gn dosage, stimulation duration, number of oocytes retrieved, male age, sperm concentration, progressive motility and the mean number of embryos transferred ( \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eGeneral characteristics of the enrolled patients.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC-IVF\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eICSI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Value\u003c/p\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 \u003cp\u003ePatients (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCycles (oocyte retrievals)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e175\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e218\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale age (y)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.92\u0026thinsp;\u0026plusmn;\u0026thinsp;3.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.25\u0026thinsp;\u0026plusmn;\u0026thinsp;3.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.805\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBMI for women (kg/m\u0026sup2;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.53\u0026thinsp;\u0026plusmn;\u0026thinsp;3.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.58\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.971\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBasal FSH (IU/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.28\u0026thinsp;\u0026plusmn;\u0026thinsp;2.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.909\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBasal E\u003csub\u003e2\u003c/sub\u003e (pg/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.26\u0026thinsp;\u0026plusmn;\u0026thinsp;32.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.10\u0026thinsp;\u0026plusmn;\u0026thinsp;29.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.990\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal Gn dosage (IU)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2201.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1100.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2210.41\u0026thinsp;\u0026plusmn;\u0026thinsp;697.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.982\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStimulation duration (days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.474\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of oocytes retrieved (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.58\u0026thinsp;\u0026plusmn;\u0026thinsp;7.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.16\u0026thinsp;\u0026plusmn;\u0026thinsp;7.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.237\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale age (y)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.768\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSperm concentration (10\u003csup\u003e6\u003c/sup\u003e/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59.42\u0026thinsp;\u0026plusmn;\u0026thinsp;34.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63.67\u0026thinsp;\u0026plusmn;\u0026thinsp;33.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.762\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eProgressive motility (a\u0026thinsp;+\u0026thinsp;b) (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57.58\u0026thinsp;\u0026plusmn;\u0026thinsp;9.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56.92\u0026thinsp;\u0026plusmn;\u0026thinsp;9.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.865\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean number of embryos transferred (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.359\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eFrom the ICSI cycles, a total of 218 cumulus-oocyte complexes (COCs) (mean 18.16) were collected, from which 183 MII oocytes (mean 15.25) were used for treatment. From previous IVF cycles, a total of 175 COCs (mean 14.58) were obtained. Following conventional IVF, an overall normal fertilization rate of 22.29% (39/175) was achieved, which was similar compared that of 25.14% (46/183) observed after ICSI (22.29 versus 25.14%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.526). We observed no significant differences in the MPN (45.14 versus 40.98%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.427) and 1PN (3.43 versus 2.73; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.703) rates between the two groups. The developmental capacity of fertilized oocytes was evaluated by assessing embryo formation on Day 3 and Day 5 post-fertilization. After conventional IVF, a total D3 good quality embryo rate of 0% (0/39) was achieved, which was significantly lower than that of 23.91% (11/46) after ICSI (0 versus 23.91%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012). There was no significant difference in the D3 available embryo rate between the two groups (38.46 versus 60.87%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.066). We further observed no significant differences in the mean number of D3 embryo blastomere (6.73 versus 7.40; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.269) and D3 embryo fragmentation rate (9 versus 8%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.628) between the two groups. Importantly, ICSI cycles demonstrated significantly lower blastomere multinucleation rate compared with previous IVF cycles (65.21 versus 87.18%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037). In conventional IVF cycles, a total of 13 embryos were transferred in fresh cycles. Additionally, no embryos were cryopreserved, and 2 embryos underwent extended culture to the blastocyst stage. In ICSI cycles, a total of 12 embryos were transferred in fresh cycles. Additionally, 3 embryos were cryopreserved, and 13 embryos underwent extended culture to the blastocyst stage (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The details of embryo development after previous C-IVF and subsequent ICSI cycles in couples with repeated high proportion of MPN were shown in a supplementary table.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison of embryo development after previous C-IVF and subsequent ICS cycles in couples with repeated high proportion of \u0026ge;\u0026thinsp;3PN (P1-P12).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC-IVF\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eICSI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Value\u003c/p\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 \u003cp\u003ePatients (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCycles (oocyte retrievals)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCumulus-oocyte complexes (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e175\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e218\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2PN rate (%, n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.29 (39/175)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.14 (46/183)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.526\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMPN rate (%, n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.14 (79/175)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.98 (75/183)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.427\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1PN rate (%, n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.43 (6/175)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.73 (5/183)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.703\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD3 good quality embryo rate (D3 good quality embryos/2PN)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0/39)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.91 (11/46)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD3 available embryo rate (D3 embryos/2PN)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.46 (15/39)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60.87 (28/46)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.066\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean number of D3 embryo blastomere (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.269\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean fragmentation of D3 embryo (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.628\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBlastomere multinucleation rate (Multinucleation/2PN)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.18 (34/39)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65.21 (30/46)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.037\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo. of embryos transferred in fresh (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo. of cryopreserved embryos (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEmbryos of extended culture to blastocyst-stage (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo. of Blastocyst formation (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eOverall, following ICSI, a total pregnancy rate of 62.50% (5/8) was achieved, which was significantly higher than the pregnancy rate of 0% (0/8) observed after previous IVF cycles (62.50 versus 0%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.031). We also observed that ICSI showed a significant improvement in the ongoing pregnancy (62.50 versus 0%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.031) and live birth (62.50 versus 0%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.031) rates compared with previous IVF (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison of clinical outcomes after previous C-IVF and subsequent ICSI cycles in couples with repeated high proportion of \u0026ge;\u0026thinsp;3PN (P1-P12).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC-IVF\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eICSI-AOA\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Value\u003c/p\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 \u003cp\u003eCycles (oocyte retrievals)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCycles (Embryo transfers)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo available embryos (cycles, n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCleavage-stage embryo transfer (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTransfers with D3 good quality embryo (%, n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0/8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.50 (7/8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePregnancy rate (+\u0026thinsp;hCG/initiated cycle)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0/8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62.50 (5/8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClinical pregnancy rate (%, n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0/8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62.50 (5/8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOngoing pregnancy rate (\u0026gt;\u0026thinsp;12 weeks) (%, n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0/8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62.50 (5/8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLive birth rate (%, n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0/8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62.50 (5/8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003eFor three couples with no available embryos in previous IVF and subsequent ICSI cycles, one patient refused to perform the genetic screening. we performed whole-exome sequencing for \u003cem\u003eTUBB8\u003c/em\u003e gene and validated the results by Sanger sequencing in two female patients and their family members. In one family, no genetic mutations were found in the \u003cem\u003eTUBB8\u003c/em\u003e gene. In the other family, we identified a novel heterozygous missense mutation of \u003cem\u003eTUBB8\u003c/em\u003e (c.33G\u0026thinsp;\u0026gt;\u0026thinsp;T [p.Q11H]) that cause a repeated high proportion of MPN zygotes after previous IVF and subsequent ICSI. This heterozygous mutation of c.33G\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Q11H) was inherited from her father. The variant c.33G\u0026thinsp;\u0026gt;\u0026thinsp;T was located in a conserved GTPase domain of \u003cem\u003eTUBB8\u003c/em\u003e families and was highly conserved among different species (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this paper, we firstly showed that ICSI could improve embryo development and reproductive outcomes compared with conventional IVF in non-male infertile patients with high proportion of MPN zygotes. Secondly, we indicated that ICSI could significantly decrease blastomere multinucleation rate compared with previous IVF cycles in such patients. Lastly, we identified a novel heterozygous missense mutation of \u003cem\u003eTUBB8\u003c/em\u003e (c.33G\u0026thinsp;\u0026gt;\u0026thinsp;T [p.Q11H]) in such patients with no available embryos.\u003c/p\u003e\u003cp\u003ePrevious studies have demonstrated that ICSI could enhance the embryo quality in patients with non-male factor infertility\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Yang et al. showed that embryos developed via ICSI exhibited superior quality compared to those derived from C-IVF when comparing sibling oocytes from non-male-factor couples\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Kim et al. reported that ICSI could improve the fertilization and D3 good quality embryo rates in patients with non-male factor infertility compared with C-IVF\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Wang et al. investigated patients with poor-quality embryos from previous C-IVF cycles and observed that ICSI significantly improved the D3 good quality embryo rate relative to C-IVF\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we focused on couples with characteristics similar to those in the aforementioned reports. The enrolled population also had non-male factor infertility and poor-quality embryos in their initial treatment cycle. The key difference, however, was that poor embryo quality was associated with a higher incidence of MPN, and the MPN rate did not decrease after subsequent ICSI treatment. Overall, our findings are consistent with previous observations in the literature\u003c/p\u003e\u003cp\u003eIt was well established that both the number of D3 embryo blastomeres and the degree of embryo fragmentation were critical factors influencing embryo quality. Some studies have demonstrated that early cleavage, defined as occurring 25\u0026ndash;27 hours post C-IVF/ICSI, serves as a strong indicator of embryo viability\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In theory, the oocyte is fertilized earlier by the sperm in ICSI compared with C-IVF, as the spermatozoon was directly injected into the oocyte. Consequently, the early cleavage rate would be higher in ICSI cycle than in IVF cycle. Previous studies demonstrated that the D3 good quality embryo rate was significantly higher in the early cleavage group, which was associated with higher number of D3 embryo blastomeres. Nevertheless, we observed no significant difference in the mean number of D3 embryo blastomeres between previous IVF and subsequent ICSI cycles which might be attributed to the limited sample size. Some studies also indicated that ICSI did not have a significant impact on the D3 embryo fragmentation rate which was consistent with our observation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eInterestingly, we found a significant difference in the blastomere multinucleation rate between previous C-IVF and subsequent ICSI. After ICSI, the blastomere multinucleation rate was significantly decreased than that of previous C-IVF. Blastomere multinucleation is a common nuclear abnormality observed in early human embryos. It was reported that a multinucleation in day 2 and day 3 cleavage embryos of 11%-34%\u003csup\u003e16,17,18\u003c/sup\u003e. The blastomere multinucleation rate was 75.29% across all cycles in this study, which was significantly higher than the rate reported in previous studies. Previous studies showed that the occurrence of blastomere multinucleation events was associated with a high incidence of MPN zygotes, which might explain the elevated blastomere multinucleation rate observed in this study\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The way a single embryo was affected might reflect how the whole cohort was influenced, even if the other embryos did not exhibit the same characteristics.\u003c/p\u003e\u003cp\u003eThe mononucleated embryos always had a higher incidence of normally aligned nucleolar precursor bodies, which was correlated with both continued embryo development and better reproductive outcomes in various studies\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Salumets et al. indicated that a higher number of embryos with a normal halo (a translocation of cellular organelles) were also observed in mononucleated embryos compared with multinucleated embryos\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. And it was confirmed that the halo was associated with embryo quality\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Thus, the primary reasons for the increase in the rate of high-quality embryos might be related to the reduction in the proportion of multinucleated embryos on day 3.\u003c/p\u003e\u003cp\u003eIn this research, a total of 13 embryos were transferred and none of the embryos succeeded in the implantation in previous IVF cycles. Of them, 11 were multinucleated embryos and 2 were mononucleated embryos. In subsequent ICSI cycles, 12 embryos were transferred and 5 embryos succeeded in the implantation. The above data indicated that it was transferred fewer multinucleated embryos in C-IVF cycles compared with ICSI cycles. And it was also demonstrated that embryo multinucleation was associated with reduced developmental rates and lower implantation rates in Days 2\u0026ndash;3 embryo transfers in assisted reproduction treatment\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The requirement for extended culture to the blastocyst-stage typically involved at least two good-quality embryos on day 3, and all embryo transfers were performed on day 3 in this research. Thus, the primary reason for the increased implantation rate might be associated with the reduction in the proportion of multinucleated embryo transfers on day 3.\u003c/p\u003e\u003cp\u003eThe incidence of a high proportion of MPN zygotes results in a sharp decrease in the number of available embryos and it increases the risk of cancelled transfers. In this research, 3 couples obtained no available embryos in previous IVF and subsequent ICSI cycles. For two female patients and their family members, we performed whole-exome sequencing for \u003cem\u003eTUBB8\u003c/em\u003e gene. Tubulin β eight class VIII (TUBB8) is the predominant β-tubulin isotype present in early embryos, playing a crucial role in the human oocyte spindle assembling\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The Mutations in \u003cem\u003eTUBB8\u003c/em\u003e have been confirmed to be associated with various aspects of reproductive biology, including the maturation of human oocytes, fertilization, pre-implantation embryonic development, and even failures in embryo implantation failure\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In one family, we identified a novel heterozygous missense mutation of \u003cem\u003eTUBB8\u003c/em\u003e (c.33G\u0026thinsp;\u0026gt;\u0026thinsp;T [p.Q11H]) and this heterozygous mutation of c.33G\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Q11H) was inherited from her father. Previous study also identified additional novel heterozygous missense mutations in \u003cem\u003eTUBB8\u003c/em\u003e that were associated with a MPN phenotype in zygotes following C-IVF or ICSI, in three sterile patients who experienced repeated early embryo developmental arrest\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. These findings suggested that \u003cem\u003eTUBB8\u003c/em\u003e played a significant role in the incidence of MPN phenotype. A recent study has provided a comprehensive mechanism elucidating how \u003cem\u003eTUBB8\u003c/em\u003e missense variants lead to oocyte abnormalities and has proposed new therapeutic avenues for treating female infertility in clinical settings\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. It is believed that more effective treatment approaches will be offered for the patients with \u003cem\u003eTUBB8\u003c/em\u003e variants in the near future. Thus, this information will be valuable for future genetic counseling of infertile patients diagnosed with MPN especially for patients undergoing multiple cycles with high proportion of MPN zygotes and no available embryos.\u003c/p\u003e\u003cp\u003eThere are certain weaknesses in the current study that should be underlined. First, the primary drawback is the retrospective design and small sample size. Second, there may be some potential bias and confounders that cannot be excluded. Lastly, the data about blastocyst culture is limited and the blastocyst formation rate is relatively low in subsequent ICSI cycles. It may be associated with the limited number of D3 good quality embryos, and most of these embryos need to be used for transfer on day 3. Given the limited data and methodological constraints, further data accumulation is needed to obtain more reliable conclusions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\u003cp\u003e All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. This study was approved by the Ethics Review Board of the Northwest Women\u0026rsquo; s and Children\u0026rsquo; s Hospital, Xi\u0026rsquo; an, China (2023003).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003e This project was supported by Shaanxi Technology Committee Industrial Public Relation Project (Project Number: 2023-YBSF-034) and Young Physicians Program of Chinese Medical Association (No. 17020470716).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.L. and J.S. designed the research; S.S. and S.Q. performed the research. X.X. analyzed the data; All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData available on request corresponding author due to privacy and ethical restrictions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen, S. et al. Tripronuclear Zygotes in IVF Laboratory Quality Control: Experimental Evaluation and Potential Applications. \u003cem\u003eInt. J. Gen. Med.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 949\u0026ndash;954 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, H. T. et al. Could the presence and proportion of three/multiple pronuclei (3PN/MPN) zygotes indicate the cytoplasmic maturation state of oocyte cohort in conventional IVF patients? \u003cem\u003eJ. Gynecol. Obstet. Hum. Reprod.\u003c/em\u003e \u003cb\u003e53\u003c/b\u003e, 102738 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJun, S. H. et al. Benefit of intracytoplasmic sperm injection in patients with a high incidence of triploidy in a prior in vitro fertilization cycle. \u003cem\u003eFertil. Steril.\u003c/em\u003e \u003cb\u003e86\u003c/b\u003e, 825\u0026ndash;829 (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRosen, M. P. et al. Triploidy formation after intracytoplasmic sperm injection may be a surrogate marker for implantation. \u003cem\u003eFertil. Steril.\u003c/em\u003e \u003cb\u003e85\u003c/b\u003e, 384\u0026ndash;390 (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSachs, A. R. et al. Factors associated with the formation of triploid zygotes after intracytoplasmic sperm injection. \u003cem\u003eFertil. Steril.\u003c/em\u003e \u003cb\u003e73\u003c/b\u003e, 1109\u0026ndash;1114 (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang, D. et al. Intracytoplasmic sperm injection improving embryo quality: comparison of the sibling oocytes of non-male-factor couples. \u003cem\u003eJ. Assist. Reprod. Genet.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 351\u0026ndash;355 (1996).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim, J. Y. et al. Can intracytoplasmic sperm injection prevent total fertilization failure and enhance embryo quality in patients with non-male factor infertility? \u003cem\u003eEur. J. Obstet. Gynecol. Reprod. Biol.\u003c/em\u003e \u003cb\u003e178\u003c/b\u003e, 188\u0026ndash;191 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, J. et al. 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Novel mutations and structural deletions in TUBB8: expanding mutational and phenotypic spectrum of patients with arrest in oocyte maturation, fertilization or early embryonic development. \u003cem\u003eHum. Reprod.\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e, 457\u0026ndash;464 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSha, Q. et al. Novel mutations in TUBB8 expand the mutational and phenotypic spectrum of patients with zygotes containing multiple pronuclei. \u003cem\u003eGene\u003c/em\u003e \u003cb\u003e769\u003c/b\u003e, 145227 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLuo, H. et al. Pathogenic variants of TUBB8 cause oocyte spindle defects by disrupting with EB1/CAKP5 interactions and potential treatment targeting microtubule acetylation through HDAC6 inhibition. \u003cem\u003eClin. Transl Med.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, e70193 (2025).\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"Multiple pronuclei, IVF, ICSI, Embryo quality, Reproductive outcomes","lastPublishedDoi":"10.21203/rs.3.rs-6216425/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6216425/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntracytoplasmic sperm injection (ICSI) is beneficial for most women with a high multiple pronuclei (MPN) incidence from previous conventional in vitro fertilization (IVF). Nevertheless, a small group of patients still undergo the same problem in the subsequent ICSI treatment. In this research, we aimed to explore whether ICSI is still effective in improving embryonic development and reproductive outcomes in such patients. A prospective cohort study was performed between January 2017 and December 2023, including 12 couples with a high proportion of MPN zygotes in previous C-IVF and subsequent ICSI cycles. The MPN rate was not less than 50% and the number of MPN zygotes was not less than 3 in each initiated cycle for the couples included in the study. We observed no significant differences in the 2PN (22.29 versus 25.14%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.526), MPN (45.14 versus 40.98%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.427) and 1PN (3.43 versus 2.73; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.703) rates between previous IVF and subsequent ICSI cycles. After conventional IVF, a total D3 good quality embryo rate of 0% (0/39) was achieved, which was significantly lower than that of 23.91% (11/46) after ICSI (0 versus 23.91%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012). There was no significant difference in the D3 available embryo rate between the two groups (38.46 versus 60.87%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.066). ICSI cycles demonstrated significantly lower blastomere multinucleation rate compared with previous IVF cycles (65.21 versus 87.18%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037). We also observed that ICSI showed a significant improvement in the ongoing pregnancy (62.50 versus 0%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.031) and live birth (62.50 versus 0%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.031) rates compared with conventional IVF. We further identified a novel heterozygous missense mutation of \u003cem\u003eTUBB8\u003c/em\u003e (c.33G\u0026thinsp;\u0026gt;\u0026thinsp;T [p.Q11H]) that cause a repeated high proportion of MPN zygotes. For patients with high proportion of MPN zygotes in previous C-IVF and subsequent ICSI cycles, ICSI still can improve embryo development and reproductive outcomes compared with C-IVF.\u003c/p\u003e","manuscriptTitle":"Analysis of reproductive outcomes in cases with repeated high proportion of multiple pronuclei (MPN) after previous IVF and subsequent ICSI","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 06:18:34","doi":"10.21203/rs.3.rs-6216425/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":"e2594025-9649-4215-b928-4678481192c3","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51159990,"name":"Biological sciences/Developmental biology"},{"id":51159991,"name":"Health sciences/Endocrinology"}],"tags":[],"updatedAt":"2025-08-18T07:54:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-10 06:18:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6216425","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6216425","identity":"rs-6216425","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}