Alignment among the zygotic cleavage plane, pronuclear axis, and polar axis predicts live birth outcome of blastocyst

Alignment among the zygotic cleavage plane, pronuclear axis, and polar axis predicts live birth outcome of blastocyst | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Alignment among the zygotic cleavage plane, pronuclear axis, and polar axis predicts live birth outcome of blastocyst Yanhe Liu, Kelli Peirce, Jay Natalwala, Vincent Chapple This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7936004/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Purpose To investigate whether alignment among zygotic cleavage plane (CP), pronuclear (PN) axis and polar axis (PB) is associated with higher live birth rate (LBR) in single transferred blastocysts? Methods A total of 103 fresh and 138 frozen blastocysts created via either conventional IVF insemination or intracytoplasmic sperm injection (ICSI) using autologous oocytes at between September 2022 and December 2023 were retrospectively analyzed. Live birth rates were compared according to alignment amongst three zygotic axes. The PN axis was defined by the longitudinal axis before fading and the PB axis was determined by the position of second polar body. Alignment relative to the zygotic CP was classified as CPPN + or CPPN- for the PN axis, and CPPB + or CPPB- for the PB axis. Results In the fresh transfer dataset, CPPN + blastocysts had a significantly higher LBR than CPPN- blastocysts (61.8% vs 12.5%, P < 0.001), while CPPB + outperformed CPPB- (49.3% vs 13.3%, P < 0.001). The highest LBR was observed in CPPN+/CPPB + blastocysts (77.5%), compared with CPPN+/CPPB- (20.0%, P < 0.001), CPPN-/CPPB+ (15.2%, P < 0.001) and CPPN-/CPPB- (6.7%, P < 0.001). Similar patterns were observed in the frozen transfer dataset, where CPPN+/CPPB + blastocysts again had the highest LBR (48.3%), compared with CPPN+/CPPB- (16.1%, P = 0.003), CPPN-/CPPB+ (19.4%, P = 0.007) and CPPN-/CPPB2- (11.1%, P = 0.005). Logistic regression confirmed CPPN and CPPB alignment as independent predictors of live birth in both datasets, after adjusting for several potential confounders. Conclusion Our findings support zygotic alignment among CP, PN and PB alxes as a potential viability marker for blastocyst selection. Zygote cleavage plane pronuclear polar body live birth Figures Figure 1 Figure 2 Introduction Blastocyst culture is widely used as a tool for embryo selection prior to intrauterine transfer [ 1 ]. Recent clinical introduction of time-lapse videography has offered a novel opportunity for further improved embryo selection, with an increasing number of selection algorithms proposed [ 2 – 4 ]. However, a recent well designed randomized controlled trial, did not find significant improvement by either time-lapse culture alone, or in combination with the added selection using embryo morphokinetics profiles [ 5 ]. Meanwhile, novel viability markers continue to emerge [ 6 , 7 ], highlighting the growing potential of time-lapse embryo selection via additional newly identified non-invasive time-lapse markers. Recently, there has been a focus on the first mitotic division of the human zygote following its finer morphokinetic profiles being revealed via time-lapse imaging [ 8 , 9 ]. Amongst these early morphokinetic features, the alignment amongst zygotic cleavage plane (CP), pronuclear (PN) axis and polar (PB) axis were highlighted as potential biomarkers for embryo viability. However, early studies were based on static observations [ 10 ], making it a challenge to detect temporal changes in the PN axis orientation, and even impossible to define PB axis being unable to accurately locate the second polar body extrusion. In a 2021 study using a dataset including predominately Day 2/3 transfers, higher pregnancy rates were seen in embryos with aligned zygotic CP and PN axis [ 11 ]. A more recent study demonstrated a reduced euploidy rate in blastocysts having misaligned zygotic CP and PN axes [ 12 ]. Another study also reported better alignment between zygotic CP and PN in embryos leading to a live birth [ 13 ]. However, all the embryos involved in these studies were fertilized via intracytoplasmic sperm injection (ICSI), leaving a knowledge gap in those fertilized via the more natural conventional IVF insemination. Also, live birth data on this topic following blastocyst transfer is also currently scarce in the literature. Furthermore, it remains unclear whether the alignment between zygotic CP and PB axis is indicative of subsequent viability of an embryo. Therefore, using live birth as an endpoint, this study aims to test the hypothesis that alignment amongst zygotic CP, PN axis and PB axis is associated with higher live birth rate following single blastocyst transfers, regardless of IVF or ICSI insemination. Materials and methods This retrospective study included two separate datasets, namely a fresh transfer dataset including 103 single transferred fresh blastocysts and a frozen transfer dataset comprising 138 single transferred frozen blastocysts. All blastocysts were created at between September 2022 and December 2023, using autologous oocytes via either IVF with 4-hour co-incubation of oocytes and sperm or ICSI. Blastocysts undergoing preimplantation geneic testing were excluded from analysis. Time-lapse imaging frequency was set at every 10 minutes and started from either the completion of the 4-hour IVF insemination or immediately after ICSI until the end of culture on Day 5 or 6. In either the fresh (Day 5 only) or frozen (Day 5 or 6) dataset, only one blastocyst (first transferred during the forementioned timeframe) was included for analysis to avoid clustering effect at statistical analysis. All clinical and laboratory procedures were conducted according to the standard protocols of the clinic. Table 1 describes baseline characteristics of cycles associated with the included blastocysts in each dataset. Retrospective data analysis was approved by the Ramsay Health Care Human Research Ethics Committee (2022/ETH/0073). Gamete preparation, insemination, and embryo culture Ovarian stimulation, transvaginal oocyte aspiration and sperm preparation were performed according to previous publication [14]. For IVF insemination, cumulus-oocyte-complexes (COCs) were co-incubated with the prepared sperm sample for four hours in G-IVF TM Plus media (Vitrolife, Sweden) overlaid with Ovoil TM (Vitrolife) at 6% CO 2 5% O 2 and 89% N 2 at 37℃. Oocytes were then separated from cumulus cells and sperm, and placed into G-TL TM Plus media (Vitrolife), for uninterrupted culture at 6% CO 2 5% O 2 and 89% N 2 at 37℃ in the Embryoscope+ incubator (Vitrolife) up to Day 6. COCs for ICSI insemination were denuded by brief exposure to Synvitro Hyadase (Origio, Denmark) for a maximum of 10 seconds, followed by mechanical removal of cumulus cells from the oocytes. Metaphase II oocytes were injected with a single spermatozoon, with the first polar body oriented at either 12- or 6-o’clock, before uninterrupted culture in the Embryoscope+ incubator up to Day 6. Fresh blastocyst transfer, vitrification/warming of blastocysts, and frozen transfer Blastocysts were assessed according to Gardner system [1] on Day 5 or Day 6, and those with at least a stage-3 expansion and A/B-grade inner cell mass and trophectoderm were given priority for transfer or cryopreservation. The embryo transfer procedure was conducted as previously described [14]. Suitable blastocysts were vitrified using the Rapid-i TM device and RapidVit TM Blast media (Vitrolife) and subsequently warmed using the RapidWarm TM Blast media (Vitrolife), as per manufacturer’s protocol. All frozen blastocysts were transferred two to four hours post warm as previous described [15]. All pregnancies were followed up until birth with a definitive outcome recorded for each blastocyst. Time-lapse annotation for zygotic alignment All annotations for zygotic alignment were retrospectively carried out by the same embryologist (YL), who was blinded to birth outcomes and blastocyst grades at data collection. The PN axis was defined by their longitudinal axis before fading, while the PB axis was represented by either (a) the position of the second polar body extrusion or (b) where the female PN emerged if the extrusion footage was unavailable due to IVF insemination ( Figure 1, Video 1 ). Alignment relative to the zygotic CP was classified as CPPN+ or CPPN- for the PN axis, and CPPB+ or CPPB- for the PB axis. A total of four categories were classified by combining alignment outcomes of CPPN and CPPB; comprising the CPPN+/CPPB+, CPPN+/CPPB-, CPPN-/CPPB+, and CPPN-/CPPB- subgroups.Examples under each category areillustrated in Figure 2 . An example of time-lapse footage demonstrating CPPN+/CPPB+ is also presented in Video 2, capturedfrom an embryo leading to a live birth. Statistical analysis Proportional measures were compared between groups by Fisher’s Exact Test. Continuous parameters were assessed via Student T-test. Multivariate logistic regression was employed to evaluate independent associations between studied factors and the subsequent live birth outcome; adjusting for maternal age at oocyte retrieval, insemination methods, day 3 cell count, blastocyst expansion, morphology, and blastulation timing. Statistics were expressed via adjusted odds ratios (aOR) and 95% confidence interval (CI). Statistical analysis was conducted using Statistical Package for the Social Sciences (version 25, IBM, Washington, USA) with P <0.05 considered as statistically significant. Results A total of 81 (33.6%) live births were recorded from the 241 single blastocyst transfer cycles, including 40 (38.8%) from the fresh transfer subset (n=103, aged 34.2±3.9 years) and 41 (29.7%) from the frozen transfer subset (n=138, aged 35.3±4.5 years). Fresh transfer dataset In the fresh transfer dataset ( Table 2 ), CPPN+ blastocysts had a higher LBR than CPPN- blastocysts (61.8% vs 12.5%, P <0.001), while CPPB+ outperformed CPPB- (49.3% vs 13.3%, P <0.001). The highest LBR was observed in CPPN+/CPPB+ blastocysts (77.5%), compared to CPPN+/CPPB- (20.0%, P <0.001), CPPN-/CPPB+ (15.2%, P <0.001) and CPPN-/CPPB- (6.7%, P <0.001). Frozen transfer dataset In the frozen transfer dataset ( Table 3 ), CPPN+ blastocysts had a higher LBR (37.1%) than CPPN- (16.3%, P =0.011), and CPPB+ produced a higher LBR (38.2%) over CPPB- blastocysts (14.3%, P =0.003). The highest LBR was again observed in CPPN+/CPPB+ blastocysts (48.3%), compared to CPPN+/CPPB- (16.1%, P =0.003), CPPN-/CPPB+ (19.4%, P =0.007) and CPPN-/CPPB2- (11.1%, P =0.005). Insemination methods Table 4 shows a detailed breakdown in live birth rates of each subgroup according to zygotic alignment, comparing the IVF- and ICSI-originated blastocysts in either the fresh or frozen transfer dataset. No significant difference ( P >0.05) was detected in live birth rates between the IVF and ICSI groups. Logistic regression In the fresh transfer dataset, both CPPN alignments (aOR=17.843, 95% CI 4.649-68.480, P <0.001) and CPPB alignments (aOR=14.884, 95% CI 3.264-67.870, P <0.001) were independently associated with higher live birth rates after adjusting for potential confounding factors. Similarly, in the frozen transfer dataset, independent associations were identified for CPPN (aOR=2.782, 95% CI 1.054-7.344, P =0.039) and CPPB (aOR=3.619, 95% CI 1.381-9.485, P =0.009). Discussion Our data support the hypothesis that alignment amongst zygotic CP, PN axis and PB axis is associated with higher live birth rates following single blastocyst transfers. This is in agreement with previous studies [ 11 , 12 ], which reported that alignment between the CP and PN axis correlates with increased euploidy or pregnancy rates. While a mouse study suggested a potential role of the sperm entry site in determining the zygotic CP [ 16 ], human studies to date have been limited to embryos derived exclusively from ICSI [ 11 , 12 , 17 ]. To address the artificial nature of sperm entry in ICSI, we extended the investigation to include blastocysts derived from conventional IVF for comparison. Notably, our findings demonstrate that the correlation between zygotic alignment and live birth is independent of the insemination method, thereby bridging an important gap in the current literature. Moreover, our study included an unselected patient cohort, enhancing the generalizability of our findings compared to studies involving selected patient populations, such as those limited to PGT-eligible patients [ 12 ]. A further strength of our study is the use of live birth as the primary outcome, which is widely regarded as the most clinically relevant endpoint in IVF research, surpassing surrogate markers such as euploidy or pregnancy rates [ 11 , 12 ]. Our analysis also accounted for potential confounding variables, including insemination methods, maternal age at oocyte retrieval, and blastocyst morphology. These adjustments reinforce the robustness of zygotic alignment as a potential complementary selection marker to conventional morphology assessment [ 18 ]. The biological significance of zygotic alignment between the CP and PN axis remains poorly understood. Using immunofluorescence it was identified that two pericentric signals were located at the interface between the paternal and maternal pronuclei in the majority of analyzed zygotes [ 11 ]. This finding underscores the potential importance of spatial alignment between the PN axis and the first mitotic spindle, which is established by the corresponding centrosome at syngamy. Accurate chromosomal segregation during the first zygotic cleavage is paramount [ 19 ] and segregation errors may lead to aneuploid daught cells [ 20 ]. Therefore, it is speculated that alignment between the zygotic CP and PN axis couild promote correct zygotic cleavage. In our study, no association was found between maternal age and CPPN or CPPB alignment, consistent with findings of a previous study [ 12 ]. This further implies that mitotic-origin aneuploidy may be independent of maternal age, in contrasts to the well-established age-related aneuploidy risk associated with meiotic errors. Zygotic polarity is well established in animal models, with the second PB marking the embryonic pole, opposite the abembryonic pole [ 21 ], although it remains unclear in human. It is hypothesized that misalignment between the zygotic CP and PB axis may lead to unequal distribution of polarized cytoplasmic determinants between daughter cells, potentially impairing subsequent embryonic development [ 22 ]. However, clinical studies testing this hypothesis remain limited. A recent conference abstract suggested that the zygotic CP is independent of the PB axis, although this statement was not supported by a direct assessment of CP-PB alignment [ 13 ]. Instead, their analysis was limited to evaluating the angle between the PN and PB axes. In contrast, our study employed the zygotic CP as a direct reference for its alignment with the PB axis, providing a more robust evaluation of this relationship. Our study outlined detailed protocols for defining zygotic CP, PN axis and PB axis, offering a potential framework for standardization and improved comparability in future research. Recognizing the three-dimensional architecture of human zygotes, we presented all possible alignment patterns from various viewing angles in the Fig. 2 , categorized according to different alignment outcomes. We also established a practical protocol to define the PB axis in zygotes fertilized via conventional IVF, using the emergence location of maternal PN as a marker to distinguish the second PB from the first ( Video 1 ). Furthermore, given the highly dynamic nature of PN axis orientation prior to PN breakdown, it is critical to define the PN axis based solely on its final orientation immediately before fading ( Video 1 ). Although both our fresh and frozen transfer datasets demonstrated similar trends, we observed a relatively lower LBR for the CPPN+/CPPB + category within the frozen transfer dataset (48.4%) compared to the fresh transfer dataset (77.5%). This discrepancy may be partially attributed to several factors: (a) blastocysts in the frozen transfer dataset were generally graded with lower preference than those selected for fresh transfer (as shown in Table 1 ); (b) the frozen transfer dataset included a higher proportion of slower-developing blastocysts (ie. Day 6); (c) potential effects associated with vitrification and warming procedures; and (d) the frozen transfer dataset had an older maternal age at oocyte retrieval. Nevertheless, the associations observed in our study remained statistically relevant across both fresh and frozen transfers, reinforcing the potential value of zygotic alignment, specifically among the CP, PN axis, and PB axis, as a novel marker in future interpretable blastocyst selection models [ 23 ]. Several limitations of our study should be acknowledged. First, although the sample size provided sufficient statistical power to detect differences in the proposed comparisons, it is still considered relatively small. Second, the retrospective design introduces the possibility of unmeasured or residual confounding variables beyond those adjusted for in our regression analysis. Additionally, the annotation of zygotic CP, PN axis and PB axis involve a degree of subjectivity. To minimize variability, all annotations in this study were performed by a single operator, thereby eliminating inter-operator variation. However, broader clinical implementation of this selection marker will require formal assessment of inter-operator reproducibility, supported by structured training program to ensure consistent annotation. In the long term, automated annotation assisted by artificial intelligence technology offers a promising solution, enhancing both objectivity and efficiency. In conclusion, our results suggest that alignment of the CP, PN, and PB axes in zygote is associated with a higher live birth potential of blastocysts. This finding indicates that axis alignment may serve as a valuable complementary biomarker, to improve blastocyst selection strategies. Furthermore, our data help bridge a gap in the literature by demonstrating that IVF-derived zygotes exhibit behavior similar to their ICSI-derived counterparts. Declarations Author Contribution YL, JN and VC conceived this study. YL contributed to the implementation of this study and data collection. KP, JN, VC and YL all contributed to data analysis, interpretation and manuscript writing. Acknowledgement The authors thank the doctor and embryology teams at Fertility North for their kind support of this study. Data Availability Data would be made available upon reasonable responses to the corresponding author. References Gardner DK, Schoolcraft WB. Culture and transfer of human blastocysts. Curr Opin Obstet Gynecol. 1999;11(3):307–11. Liu Y, et al. Time-lapse deselection model for human day 3 in vitro fertilization embryos: the combination of qualitative and quantitative measures of embryo growth. Fertil Steril. 2016;105(3):656–e6621. Meseguer M, et al. The use of morphokinetics as a predictor of embryo implantation. Hum Reprod. 2011;26(10):2658–71. Tran D, et al. Deep learning as a predictive tool for fetal heart pregnancy following time-lapse incubation and blastocyst transfer. Hum Reprod (Oxford England). 2019;34(6):1011–8. Bhide P, et al. Clinical effectiveness and safety of time-lapse imaging systems for embryo incubation and selection in in-vitro fertilisation treatment (TILT): a multicentre, three-parallel-group, double-blind, randomised controlled trial. Lancet. 2024;404(10449):256–65. Bickendorf K, et al. Spontaneous collapse as a prognostic marker for human blastocysts: a systematic review and meta-analysis. Hum Reprod. 2023;38(10):1891–900. Marconetto A, et al. Cytoplasmic strings in human blastocysts: hypotheses of their role and implications for embryo selection. Hum Reprod. 2024;39(11):2453–65. Coticchio G, et al. The first mitotic division: a perilous bridge connecting the zygote and the early embryo. Hum Reprod. 2023;38(6):1019–27. Coticchio G, et al. Fertilization signatures as biomarkers of embryo quality. Hum Reprod. 2022;37(8):1704–11. Gianaroli L, et al. Pronuclear morphology and chromosomal abnormalities as scoring criteria for embryo selection. Fertil Steril. 2003;80(2):341–9. Nakaoka M et al. P–193 First cleavage division perpendicular to the pronuclear axis adversely affects the clinical outcome in human embryos. Hum Reprod, 2021. 36(Supplement_1). Mizobe Y, et al. Formation of the first plane of division relative to the pronuclear axis predicts embryonic ploidy. Reprod Biomed Online. 2024;49(3):104110. Porokh V et al. P-190 The first cleavage plane in human embryos is dictated by the topology of pronuclei, not by the polar body position. Hum Reprod, 2022. 37(Supplement_1). Liu Y et al. Prevalence, consequence, and significance of reverse cleavage by human embryos viewed with the use of the Embryoscope time-lapse video system. Fertil Steril, 2014. 102(5): pp. 1295–1300 e2. Lee T, et al. Abnormal cleavage up to Day 3 does not compromise live birth and neonatal outcomes of embryos that have achieved full blastulation: a retrospective cohort study. Hum Reprod. 2024;39(5):955–62. Plusa B, et al. Site of the previous meiotic division defines cleavage orientation in the mouse embryo. Nat Cell Biol. 2002;4(10):811–5. Porokh V, et al. Zygotic spindle orientation defines cleavage pattern and nuclear status of human embryos. Nat Commun. 2024;15(1):6369. Consensus T et al. W.G.o.t.u.o.t.E.A.I.,., The Istanbul consensus update: a revised ESHRE/ALPHA consensus on oocyte and embryo static and dynamic morphological assessment†,‡. Human Reproduction, 2025. 40(6): pp. 989–1035. Currie CE, et al. The first mitotic division of human embryos is highly error prone. Nat Commun. 2022;13(1):6755. Ono Y, et al. Shape of the first mitotic spindles impacts multinucleation in human embryos. Nat Commun. 2024;15(1):5381. Gardner R. The early blastocyst is bilaterally symmetrical and its axis of symmetry is aligned with the animal-vegetal axis of the zygote in the mouse. Development. 1997;124(2):289–301. Garello C, et al. Pronuclear orientation, polar body placement, and embryo quality after intracytoplasmic sperm injection and in-vitro fertilization: further evidence for polarity in human oocytes? Hum Reprod. 1999;14(10):2588–95. Lee T, et al. A brief history of artificial intelligence embryo selection: from black-box to glass-box. Hum Reprod. 2024;39(2):285–92. Tables Table 1. Patient characteristics Parameters Fresh transfer dataset Frozen transfer dataset Number of patients Number of live births (%) Maternal age at oocyte retrieval (years, mean±SD, min-max) Insemination methods IVF (%) ICSI (%) Number of cells on Day 3 (at 66 hpi) 5 or less (%) 6 (%) 7 (%) 8 (%) 9 or more (%) Expansion stage at transfer Full blastocyst (%) Expanded (%) Hatching (%) Hatched (%) Blastocyst morphology (ICM/TE) AA (%) AB/BA/BB (%) tB (hours, mean±SD, min-max) 103 40 (38.8%) 34.2±3.9 (26-41) 45 (43.7%) 58 (56.3%) 1 (1.0%) 5 (4.9%) 11 (10.7%) 57 (55.3%) 29 (28.1%) 22 (21.4%) 44 (42.7%) 37 (35.9%) 0 65 (63.1%) 38 (36.9%) 101.9±6.6 (84.0-118.6) 138 41 (29.7%) 35.3±4.5 (22-46) 55 (40.0%) 83 (60.0%) 6 (4.3%) 4 (2.9%) 21 (15.2%) 80 (58.0%) 27 (19.6%) 21 (15.2%) 51 (37.0%) 65 (47.1%) 1 (0.7%) 71 (51.4%) 67 (48.6%) 106.3±10.5 (86.4-145.6) Note: hpi=hours post insemination, ICM=inner cell mass, TE=trophectoderm, tPNF=timing of pronuclear fading, tB=timing of blasulation Table 2. Live birth rates and maternal age (years) according to alignment amongst zygotic cleavage plane, pronuclear axis and polar axis in freshly transferred blastocysts (n=103) Alignment between zygotic cleavage plane and pronuclear axis (CPPN) Yes (CPPN+) No (CPPN-) Total Alignment between zygotic cleavage plane and polar axis (CPPB) Yes (CPPB+) 77.5% (31/40) 33.8±4.3 15.2% (5/33) 34.8±4.0 49.3% (36/73) 34.3±4.2 No (CPPB-) 20.0% (3/15) 33.8±3.4 6.7% (1/15) 34.6±2.9 13.3% (4/30) 34.2±3.1 Total 61.8% (34/55) 33.8±4.1 12.5% (6/48) 34.7±3.7 38.8% (40/103) 34.2±3.9 Note: Chi squared analysis was used to test statistical significance between different groups. CPPN+ vs CPPN-, p<0.001; CPPB+ vs CPPB-, p<0.001; CPPN-CPPB- vs CPPN+CPPB+, p<0.001; CPPN+CPPB- vs CPPN+CPPB+, p<0.001; CPPN-CPPB+ vs CPPN+CPPB+, p<0.001. Student T -test was used to test statistical significance in maternal age at oocyte retrieval between different groups. CPPN+ vs CPPN-, p=0.248; CPPB+ vs CPPB-, p=0.907; CPPN-CPPB- vs CPPN+CPPB+, p=0.510; CPPN+CPPB- vs CPPN+CPPB+, p=1.000; CPPN-CPPB+ vs CPPN+CPPB+, p=0.311. Table 3. Live birth rates and maternal age (years) according to alignment amongst zygotic cleavage plane, pronuclear axis and polar axis in frozen transferred blastocysts (n=138) Alignment between zygotic cleavage plane and pronuclear axis (CPPN) Yes (CPPN+) No (CPPN-) Total Alignment between zygotic cleavage plane and polar axis (CPPB) Yes (CPPB+) 48.3% (28/58) 34.5±4.1 19.4% (6/31) 36.5±4.6 38.2% (34/89) 35.2±4.4 No (CPPB-) 16.1% (5/31) 35.0±4.8 11.1% (2/18) 36.5±4.4 14.3% (7/49) 35.6±4.7 Total 37.1% (33/89) 34.7±4.3 16.3% (8/49) 36.5±4.5 28.7% (41/138) 35.3±4.5 Note: Chi squared analysis was used to test statistical significance in live birth rates between different groups. CPPN+ vs CPPN-, p=0.011; CPPB+ vs CPPB-, p=0.003; CPPN-CPPB- vs CPPN+CPPB+, p=0.005; CPPN+CPPB- vs CPPN+CPPB+, p=0.003; CPPN-CPPB+ vs CPPN+CPPB+, p=0.007. Student T -test was used to test statistical significance in maternal age at oocyte retrieval between different groups. CPPN+ vs CPPN-, p=0.022; CPPB+ vs CPPB-, p=0.619; CPPN-CPPB- vs CPPN+CPPB+, p=0.080; CPPN+CPPB- vs CPPN+CPPB+, p=0.607; CPPN-CPPB+ vs CPPN+CPPB+, p=0.039. Table 4. Comparison of live birth rates between insemination methods (IVF vs ICSI) according to different alignment outcomes amongst zygotic cleavage plane, pronuclear axis and polar axis. IVF ICSI Total Fresh blastocysts (n=103) CPPN+ CPPN- 64.3% (18/28) 5.9% (1/17) 59.3% (16/27) 16.1% (5/31) 61.8% (34/55) 12.5% (6/48) CPPB+ CPPB- 50.0% (17/34) 18.2% (2/11) 48.7% (19/39) 10.5% (2/19) 49.3% (36/73) 13.3% (4/30) Frozen blastocysts (n=138) CPPN+ CPPN- 42.1% (16/38) 17.6% (3/17) 33.3% (17/51) 15.6% (5/32) 37.1% (33/89) 16.3% (8/49) CPPB+ CPPB- 47.1% (16/34) 14.3% (3/21) 32.7% (18/55) 14.3% (4/28) 38.2% (34/89) 14.3% (7/49) Note: Chi squared analysis showed no significant difference between IVF and ICSI groups in any of the above subgroups. Additional Declarations No competing interests reported. Supplementary Files LiuZygoticCPVideo1.mp4 LiuZygoticCPvideo2.mp4 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Nov, 2025 Reviews received at journal 24 Nov, 2025 Reviews received at journal 23 Nov, 2025 Reviewers agreed at journal 12 Nov, 2025 Reviewers agreed at journal 05 Nov, 2025 Reviewers invited by journal 30 Oct, 2025 Editor assigned by journal 24 Oct, 2025 Submission checks completed at journal 24 Oct, 2025 First submitted to journal 23 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-7936004","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":542265770,"identity":"e9a4fc31-1f00-4e6d-85bb-1c7d64072dae","order_by":0,"name":"Yanhe Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYDACZsYHDAk8EnL8DAxsxGphNmz4IGNjLNlAtBYGZsPGGTZpiRsOEKtF3p2Z/TFPzuHEzTeSnz34UMEgzy92gPEzDx4thoeZGZt5zhw23nYjzdxwxhkGw5mzE5il8Wpp5j/YzNtzWHbbjQQzad42hgSD2wkMBLQAbeH9d5hx84z0bzAtzL/xaZFnZmZsnMGTprhBIgduCxteWwyAWmZ84LExljjzpkxyxhkJoF8S2yzn4LOl/zDDB3BUtqdvk/hQYSPPL518+MYbfLYcgLEEEkCkBBAzNuDRALQFLs1/ALeqUTAKRsEoGNkAAKKqSNeTOApWAAAAAElFTkSuQmCC","orcid":"","institution":"Fertility North","correspondingAuthor":true,"prefix":"","firstName":"Yanhe","middleName":"","lastName":"Liu","suffix":""},{"id":542265771,"identity":"9edba42e-f999-4a1e-8eb3-7566926b12c5","order_by":1,"name":"Kelli Peirce","email":"","orcid":"","institution":"Fertility North","correspondingAuthor":false,"prefix":"","firstName":"Kelli","middleName":"","lastName":"Peirce","suffix":""},{"id":542265772,"identity":"67983573-50a9-4237-bde0-be88be41b05d","order_by":2,"name":"Jay Natalwala","email":"","orcid":"","institution":"Fertility North","correspondingAuthor":false,"prefix":"","firstName":"Jay","middleName":"","lastName":"Natalwala","suffix":""},{"id":542265773,"identity":"48201173-83a9-4768-b55c-6f58c9e4d3c8","order_by":3,"name":"Vincent Chapple","email":"","orcid":"","institution":"Fertility North","correspondingAuthor":false,"prefix":"","firstName":"Vincent","middleName":"","lastName":"Chapple","suffix":""}],"badges":[],"createdAt":"2025-10-24 02:23:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7936004/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7936004/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":95789782,"identity":"83e4fba2-0e15-4025-b4c2-9d23355ed463","added_by":"auto","created_at":"2025-11-13 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06:29:59","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":70362,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7936004/v1/13df1c76145d9ffb9e09e965.html"},{"id":95789779,"identity":"4b45b2f8-f1ea-4f6f-99de-a97d7f312fe2","added_by":"auto","created_at":"2025-11-13 06:29:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":370655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIllustration of cleavage plane, pronuclear axis and polar axis.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7936004/v1/33d3f4a2b5d04eb0b1b6d946.jpg"},{"id":95789780,"identity":"2329394a-0517-4622-b830-b5ba42b252d2","added_by":"auto","created_at":"2025-11-13 06:29:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":571010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClassification of blastocysts according to zygotic alignment outcomes with possible phenotypes illustrated for each category\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7936004/v1/ca5006cca5157d972986e728.jpg"},{"id":95805471,"identity":"4ade7b49-d680-44fd-a317-614a67daf8c5","added_by":"auto","created_at":"2025-11-13 08:41:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1894259,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7936004/v1/808295e4-b18d-4b0b-9071-920c88ce8419.pdf"},{"id":95789781,"identity":"007a324e-055e-474e-b479-01842eb58ff3","added_by":"auto","created_at":"2025-11-13 06:29:59","extension":"mp4","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":554994,"visible":true,"origin":"","legend":"","description":"","filename":"LiuZygoticCPVideo1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7936004/v1/c2482eed5344e230553d936c.mp4"},{"id":95789784,"identity":"947728d1-7fd4-426f-ad3a-03c5355ba9ec","added_by":"auto","created_at":"2025-11-13 06:29:59","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3284968,"visible":true,"origin":"","legend":"","description":"","filename":"LiuZygoticCPvideo2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7936004/v1/4ac20314c9232d7de2bb26b4.mp4"}],"financialInterests":"No competing interests reported.","formattedTitle":"Alignment among the zygotic cleavage plane, pronuclear axis, and polar axis predicts live birth outcome of blastocyst","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBlastocyst culture is widely used as a tool for embryo selection prior to intrauterine transfer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Recent clinical introduction of time-lapse videography has offered a novel opportunity for further improved embryo selection, with an increasing number of selection algorithms proposed [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, a recent well designed randomized controlled trial, did not find significant improvement by either time-lapse culture alone, or in combination with the added selection using embryo morphokinetics profiles [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Meanwhile, novel viability markers continue to emerge [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], highlighting the growing potential of time-lapse embryo selection via additional newly identified non-invasive time-lapse markers.\u003c/p\u003e\u003cp\u003eRecently, there has been a focus on the first mitotic division of the human zygote following its finer morphokinetic profiles being revealed via time-lapse imaging [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Amongst these early morphokinetic features, the alignment amongst zygotic cleavage plane (CP), pronuclear (PN) axis and polar (PB) axis were highlighted as potential biomarkers for embryo viability. However, early studies were based on static observations [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], making it a challenge to detect temporal changes in the PN axis orientation, and even impossible to define PB axis being unable to accurately locate the second polar body extrusion. In a 2021 study using a dataset including predominately Day 2/3 transfers, higher pregnancy rates were seen in embryos with aligned zygotic CP and PN axis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. A more recent study demonstrated a reduced euploidy rate in blastocysts having misaligned zygotic CP and PN axes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Another study also reported better alignment between zygotic CP and PN in embryos leading to a live birth [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, all the embryos involved in these studies were fertilized via intracytoplasmic sperm injection (ICSI), leaving a knowledge gap in those fertilized via the more natural conventional IVF insemination. Also, live birth data on this topic following blastocyst transfer is also currently scarce in the literature. Furthermore, it remains unclear whether the alignment between zygotic CP and PB axis is indicative of subsequent viability of an embryo. Therefore, using live birth as an endpoint, this study aims to test the hypothesis that alignment amongst zygotic CP, PN axis and PB axis is associated with higher live birth rate following single blastocyst transfers, regardless of IVF or ICSI insemination.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eThis retrospective study included two separate datasets, namely a fresh transfer dataset including 103 single transferred fresh blastocysts and a frozen transfer dataset comprising 138 single transferred frozen blastocysts. All blastocysts were created at between September 2022 and December 2023, using autologous oocytes via either IVF with 4-hour co-incubation of oocytes and sperm or ICSI. Blastocysts undergoing preimplantation geneic testing were excluded from analysis. Time-lapse imaging frequency was set at every 10 minutes and started from either the completion of the 4-hour IVF insemination or immediately after ICSI until the end of culture on Day 5 or 6. In either the fresh (Day 5 only) or frozen (Day 5 or 6) dataset, only one blastocyst (first transferred during the forementioned timeframe) was included for analysis to avoid clustering effect at statistical analysis. All clinical and laboratory procedures were conducted according to the standard protocols of the clinic.\u0026nbsp;\u003cstrong\u003eTable 1\u003c/strong\u003e describes baseline characteristics of cycles associated with the included blastocysts in each dataset. Retrospective data analysis was approved by the Ramsay Health Care Human Research Ethics Committee (2022/ETH/0073).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGamete preparation, insemination, and embryo culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvarian stimulation, transvaginal oocyte aspiration and sperm preparation were performed according to previous publication [14]. For IVF insemination, cumulus-oocyte-complexes (COCs) were co-incubated with the prepared sperm sample for four hours in G-IVF\u003csup\u003eTM\u003c/sup\u003e Plus media (Vitrolife, Sweden) overlaid with Ovoil\u003csup\u003eTM\u003c/sup\u003e (Vitrolife) at 6% CO\u003csub\u003e2\u003c/sub\u003e 5% O\u003csub\u003e2\u003c/sub\u003e and 89% N\u003csub\u003e2\u003c/sub\u003e at 37℃. Oocytes were then separated from cumulus cells and sperm, and placed into G-TL\u003csup\u003eTM\u003c/sup\u003e Plus media (Vitrolife), for uninterrupted culture at 6% CO\u003csub\u003e2\u003c/sub\u003e 5% O\u003csub\u003e2\u003c/sub\u003e and 89% N\u003csub\u003e2\u003c/sub\u003e at 37℃ in the Embryoscope+ incubator (Vitrolife) up to Day 6. COCs for ICSI insemination were denuded by brief exposure to Synvitro Hyadase (Origio, Denmark) for a maximum of 10 seconds, followed by mechanical removal of cumulus cells from the oocytes. Metaphase II oocytes were injected with a single spermatozoon, with the first polar body oriented at either 12- or 6-o’clock, before uninterrupted culture in the Embryoscope+ incubator up to Day 6.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFresh blastocyst transfer, vitrification/warming of blastocysts, and frozen transfer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlastocysts were assessed according to Gardner system [1] on Day 5 or Day 6, and those with at least a stage-3 expansion and A/B-grade inner cell mass and trophectoderm were given priority for transfer or cryopreservation. The embryo transfer procedure was conducted as previously described [14]. Suitable blastocysts were vitrified using the Rapid-i\u003csup\u003eTM\u003c/sup\u003e device and RapidVit\u003csup\u003eTM\u003c/sup\u003e Blast media (Vitrolife) and subsequently warmed using the RapidWarm\u003csup\u003eTM\u003c/sup\u003e Blast media (Vitrolife), as per manufacturer’s protocol. All frozen blastocysts were transferred two to four hours post warm as previous described [15]. All pregnancies were followed up until birth with a definitive outcome recorded for each blastocyst.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTime-lapse annotation for zygotic alignment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll annotations for zygotic alignment were retrospectively carried out by the same embryologist (YL), who was blinded to birth outcomes and blastocyst grades at data collection. The PN axis was defined by their longitudinal axis before fading, while the PB axis was represented by either (a) the position of the second polar body extrusion or (b) where the female PN emerged if the extrusion footage was unavailable due to IVF insemination (\u003cstrong\u003eFigure 1,\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Video 1\u003c/strong\u003e). Alignment relative to the zygotic CP was classified as CPPN+ or CPPN- for the PN axis, and CPPB+ or CPPB- for the PB axis. A total of four categories were classified by combining alignment outcomes of CPPN and CPPB; comprising the CPPN+/CPPB+, CPPN+/CPPB-, CPPN-/CPPB+, and CPPN-/CPPB- subgroups.Examples under each category areillustrated in \u003cstrong\u003eFigure 2\u003c/strong\u003e. An example of time-lapse footage demonstrating CPPN+/CPPB+ is also presented in \u003cstrong\u003eVideo 2,\u0026nbsp;\u003c/strong\u003ecapturedfrom an embryo leading to a live birth.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProportional measures were compared between groups by Fisher’s Exact Test. Continuous parameters were assessed via Student T-test. Multivariate logistic regression was employed to evaluate independent associations between studied factors and the subsequent live birth outcome; adjusting for maternal age at oocyte retrieval, insemination methods, day 3 cell count, blastocyst expansion, morphology, and blastulation timing. Statistics were expressed via adjusted odds ratios (aOR) and 95% confidence interval (CI). Statistical analysis was conducted using Statistical Package for the Social Sciences (version 25, IBM, Washington, USA) with \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 considered as statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 81 (33.6%) live births were recorded from the 241 single blastocyst transfer cycles, including 40 (38.8%) from the fresh transfer subset (n=103, aged 34.2±3.9 years) and 41 (29.7%) from the frozen transfer subset (n=138, aged 35.3±4.5 years).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFresh transfer dataset\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the fresh transfer dataset (\u003cstrong\u003eTable 2\u003c/strong\u003e), CPPN+ blastocysts had a higher LBR than CPPN- blastocysts (61.8% vs 12.5%, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), while CPPB+ outperformed CPPB- (49.3% vs 13.3%, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). The highest LBR was observed in CPPN+/CPPB+ blastocysts (77.5%), compared to CPPN+/CPPB- (20.0%, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), CPPN-/CPPB+ (15.2%, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) and CPPN-/CPPB- (6.7%, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFrozen transfer dataset\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the frozen transfer dataset (\u003cstrong\u003eTable 3\u003c/strong\u003e), CPPN+ blastocysts had a higher LBR (37.1%) than CPPN- (16.3%, \u003cem\u003eP\u003c/em\u003e=0.011), and CPPB+ produced a higher LBR (38.2%) over CPPB- blastocysts (14.3%, \u003cem\u003eP\u003c/em\u003e=0.003). The highest LBR was again observed in CPPN+/CPPB+ blastocysts (48.3%), compared to CPPN+/CPPB- (16.1%, \u003cem\u003eP\u003c/em\u003e=0.003), CPPN-/CPPB+ (19.4%, \u003cem\u003eP\u003c/em\u003e=0.007) and CPPN-/CPPB2- (11.1%, \u003cem\u003eP\u003c/em\u003e=0.005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInsemination methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e shows a detailed breakdown in live birth rates of each subgroup according to zygotic alignment, comparing the IVF- and ICSI-originated blastocysts in either the fresh or frozen transfer dataset. No significant difference (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05) was detected in live birth rates between the IVF and ICSI groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLogistic regression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the fresh transfer dataset, both CPPN alignments (aOR=17.843, 95% CI 4.649-68.480, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) and CPPB alignments (aOR=14.884, 95% CI 3.264-67.870, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) were independently associated with higher live birth rates after adjusting for potential confounding factors. Similarly, in the frozen transfer dataset, independent associations were identified for CPPN (aOR=2.782, 95% CI 1.054-7.344, \u003cem\u003eP\u003c/em\u003e=0.039) and CPPB (aOR=3.619, 95% CI 1.381-9.485, \u003cem\u003eP\u003c/em\u003e=0.009).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur data support the hypothesis that alignment amongst zygotic CP, PN axis and PB axis is associated with higher live birth rates following single blastocyst transfers. This is in agreement with previous studies [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], which reported that alignment between the CP and PN axis correlates with increased euploidy or pregnancy rates. While a mouse study suggested a potential role of the sperm entry site in determining the zygotic CP [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], human studies to date have been limited to embryos derived exclusively from ICSI [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. To address the artificial nature of sperm entry in ICSI, we extended the investigation to include blastocysts derived from conventional IVF for comparison. Notably, our findings demonstrate that the correlation between zygotic alignment and live birth is independent of the insemination method, thereby bridging an important gap in the current literature.\u003c/p\u003e\u003cp\u003eMoreover, our study included an unselected patient cohort, enhancing the generalizability of our findings compared to studies involving selected patient populations, such as those limited to PGT-eligible patients [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. A further strength of our study is the use of live birth as the primary outcome, which is widely regarded as the most clinically relevant endpoint in IVF research, surpassing surrogate markers such as euploidy or pregnancy rates [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Our analysis also accounted for potential confounding variables, including insemination methods, maternal age at oocyte retrieval, and blastocyst morphology. These adjustments reinforce the robustness of zygotic alignment as a potential complementary selection marker to conventional morphology assessment [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe biological significance of zygotic alignment between the CP and PN axis remains poorly understood. Using immunofluorescence it was identified that two pericentric signals were located at the interface between the paternal and maternal pronuclei in the majority of analyzed zygotes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This finding underscores the potential importance of spatial alignment between the PN axis and the first mitotic spindle, which is established by the corresponding centrosome at syngamy. Accurate chromosomal segregation during the first zygotic cleavage is paramount [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and segregation errors may lead to aneuploid daught cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, it is speculated that alignment between the zygotic CP and PN axis couild promote correct zygotic cleavage. In our study, no association was found between maternal age and CPPN or CPPB alignment, consistent with findings of a previous study [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This further implies that mitotic-origin aneuploidy may be independent of maternal age, in contrasts to the well-established age-related aneuploidy risk associated with meiotic errors.\u003c/p\u003e\u003cp\u003eZygotic polarity is well established in animal models, with the second PB marking the embryonic pole, opposite the abembryonic pole [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], although it remains unclear in human. It is hypothesized that misalignment between the zygotic CP and PB axis may lead to unequal distribution of polarized cytoplasmic determinants between daughter cells, potentially impairing subsequent embryonic development [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, clinical studies testing this hypothesis remain limited. A recent conference abstract suggested that the zygotic CP is independent of the PB axis, although this statement was not supported by a direct assessment of CP-PB alignment [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Instead, their analysis was limited to evaluating the angle between the PN and PB axes. In contrast, our study employed the zygotic CP as a direct reference for its alignment with the PB axis, providing a more robust evaluation of this relationship.\u003c/p\u003e\u003cp\u003eOur study outlined detailed protocols for defining zygotic CP, PN axis and PB axis, offering a potential framework for standardization and improved comparability in future research. Recognizing the three-dimensional architecture of human zygotes, we presented all possible alignment patterns from various viewing angles in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, categorized according to different alignment outcomes. We also established a practical protocol to define the PB axis in zygotes fertilized via conventional IVF, using the emergence location of maternal PN as a marker to distinguish the second PB from the first (\u003cb\u003eVideo 1\u003c/b\u003e). Furthermore, given the highly dynamic nature of PN axis orientation prior to PN breakdown, it is critical to define the PN axis based solely on its final orientation immediately before fading (\u003cb\u003eVideo 1\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eAlthough both our fresh and frozen transfer datasets demonstrated similar trends, we observed a relatively lower LBR for the CPPN+/CPPB\u0026thinsp;+\u0026thinsp;category within the frozen transfer dataset (48.4%) compared to the fresh transfer dataset (77.5%). This discrepancy may be partially attributed to several factors: (a) blastocysts in the frozen transfer dataset were generally graded with lower preference than those selected for fresh transfer (as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e); (b) the frozen transfer dataset included a higher proportion of slower-developing blastocysts (ie. Day 6); (c) potential effects associated with vitrification and warming procedures; and (d) the frozen transfer dataset had an older maternal age at oocyte retrieval. Nevertheless, the associations observed in our study remained statistically relevant across both fresh and frozen transfers, reinforcing the potential value of zygotic alignment, specifically among the CP, PN axis, and PB axis, as a novel marker in future interpretable blastocyst selection models [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSeveral limitations of our study should be acknowledged. First, although the sample size provided sufficient statistical power to detect differences in the proposed comparisons, it is still considered relatively small. Second, the retrospective design introduces the possibility of unmeasured or residual confounding variables beyond those adjusted for in our regression analysis. Additionally, the annotation of zygotic CP, PN axis and PB axis involve a degree of subjectivity. To minimize variability, all annotations in this study were performed by a single operator, thereby eliminating inter-operator variation. However, broader clinical implementation of this selection marker will require formal assessment of inter-operator reproducibility, supported by structured training program to ensure consistent annotation. In the long term, automated annotation assisted by artificial intelligence technology offers a promising solution, enhancing both objectivity and efficiency.\u003c/p\u003e\u003cp\u003eIn conclusion, our results suggest that alignment of the CP, PN, and PB axes in zygote is associated with a higher live birth potential of blastocysts. This finding indicates that axis alignment may serve as a valuable complementary biomarker, to improve blastocyst selection strategies. Furthermore, our data help bridge a gap in the literature by demonstrating that IVF-derived zygotes exhibit behavior similar to their ICSI-derived counterparts.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYL, JN and VC conceived this study. YL contributed to the implementation of this study and data collection. KP, JN, VC and YL all contributed to data analysis, interpretation and manuscript writing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank the doctor and embryology teams at Fertility North for their kind support of this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData would be made available upon reasonable responses to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGardner DK, Schoolcraft WB. Culture and transfer of human blastocysts. Curr Opin Obstet Gynecol. 1999;11(3):307\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu Y, et al. Time-lapse deselection model for human day 3 in vitro fertilization embryos: the combination of qualitative and quantitative measures of embryo growth. Fertil Steril. 2016;105(3):656\u0026ndash;e6621.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeseguer M, et al. The use of morphokinetics as a predictor of embryo implantation. Hum Reprod. 2011;26(10):2658\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTran D, et al. Deep learning as a predictive tool for fetal heart pregnancy following time-lapse incubation and blastocyst transfer. Hum Reprod (Oxford England). 2019;34(6):1011\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBhide P, et al. Clinical effectiveness and safety of time-lapse imaging systems for embryo incubation and selection in in-vitro fertilisation treatment (TILT): a multicentre, three-parallel-group, double-blind, randomised controlled trial. Lancet. 2024;404(10449):256\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBickendorf K, et al. Spontaneous collapse as a prognostic marker for human blastocysts: a systematic review and meta-analysis. Hum Reprod. 2023;38(10):1891\u0026ndash;900.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarconetto A, et al. Cytoplasmic strings in human blastocysts: hypotheses of their role and implications for embryo selection. Hum Reprod. 2024;39(11):2453\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCoticchio G, et al. The first mitotic division: a perilous bridge connecting the zygote and the early embryo. Hum Reprod. 2023;38(6):1019\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCoticchio G, et al. Fertilization signatures as biomarkers of embryo quality. Hum Reprod. 2022;37(8):1704\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGianaroli L, et al. Pronuclear morphology and chromosomal abnormalities as scoring criteria for embryo selection. Fertil Steril. 2003;80(2):341\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNakaoka M et al. P\u0026ndash;193 First cleavage division perpendicular to the pronuclear axis adversely affects the clinical outcome in human embryos. Hum Reprod, 2021. 36(Supplement_1).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMizobe Y, et al. Formation of the first plane of division relative to the pronuclear axis predicts embryonic ploidy. Reprod Biomed Online. 2024;49(3):104110.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePorokh V et al. P-190\u0026emsp;The first cleavage plane in human embryos is dictated by the topology of pronuclei, not by the polar body position. Hum Reprod, 2022. 37(Supplement_1).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu Y et al. \u003cem\u003ePrevalence, consequence, and significance of reverse cleavage by human embryos viewed with the use of the Embryoscope time-lapse video system.\u003c/em\u003e Fertil Steril, 2014. 102(5): pp. 1295\u0026ndash;1300 e2.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee T, et al. Abnormal cleavage up to Day 3 does not compromise live birth and neonatal outcomes of embryos that have achieved full blastulation: a retrospective cohort study. Hum Reprod. 2024;39(5):955\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePlusa B, et al. Site of the previous meiotic division defines cleavage orientation in the mouse embryo. Nat Cell Biol. 2002;4(10):811\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePorokh V, et al. Zygotic spindle orientation defines cleavage pattern and nuclear status of human embryos. Nat Commun. 2024;15(1):6369.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eConsensus T et al. W.G.o.t.u.o.t.E.A.I.,., \u003cem\u003eThe Istanbul consensus update: a revised ESHRE/ALPHA consensus on oocyte and embryo static and dynamic morphological assessment\u0026dagger;,\u0026Dagger;.\u003c/em\u003e Human Reproduction, 2025. 40(6): pp. 989\u0026ndash;1035.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCurrie CE, et al. The first mitotic division of human embryos is highly error prone. Nat Commun. 2022;13(1):6755.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOno Y, et al. Shape of the first mitotic spindles impacts multinucleation in human embryos. Nat Commun. 2024;15(1):5381.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGardner R. The early blastocyst is bilaterally symmetrical and its axis of symmetry is aligned with the animal-vegetal axis of the zygote in the mouse. Development. 1997;124(2):289\u0026ndash;301.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarello C, et al. Pronuclear orientation, polar body placement, and embryo quality after intracytoplasmic sperm injection and in-vitro fertilization: further evidence for polarity in human oocytes? Hum Reprod. 1999;14(10):2588\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee T, et al. A brief history of artificial intelligence embryo selection: from black-box to glass-box. Hum Reprod. 2024;39(2):285\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Patient characteristics\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 274px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFresh transfer dataset\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFrozen transfer dataset\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 274px;\"\u003e\n \u003cp\u003eNumber of patients\u003c/p\u003e\n \u003cp\u003eNumber of live births (%)\u003c/p\u003e\n \u003cp\u003eMaternal age at oocyte retrieval\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; (years, mean\u0026plusmn;SD, min-max)\u003c/p\u003e\n \u003cp\u003eInsemination methods\u003c/p\u003e\n \u003cp\u003eIVF (%)\u003c/p\u003e\n \u003cp\u003eICSI (%)\u003c/p\u003e\n \u003cp\u003eNumber of cells on Day 3 (at 66 hpi)\u003c/p\u003e\n \u003cp\u003e5 or less (%)\u003c/p\u003e\n \u003cp\u003e6 (%)\u003c/p\u003e\n \u003cp\u003e7 (%)\u003c/p\u003e\n \u003cp\u003e8 (%)\u003c/p\u003e\n \u003cp\u003e9 or more (%)\u003c/p\u003e\n \u003cp\u003eExpansion stage at transfer\u003c/p\u003e\n \u003cp\u003eFull blastocyst (%)\u003c/p\u003e\n \u003cp\u003eExpanded (%)\u003c/p\u003e\n \u003cp\u003eHatching (%)\u003c/p\u003e\n \u003cp\u003eHatched (%)\u003c/p\u003e\n \u003cp\u003eBlastocyst morphology (ICM/TE)\u003c/p\u003e\n \u003cp\u003eAA (%)\u003c/p\u003e\n \u003cp\u003eAB/BA/BB (%)\u003c/p\u003e\n \u003cp\u003etB (hours, mean\u0026plusmn;SD, min-max)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e103\u003c/p\u003e\n \u003cp\u003e40 (38.8%)\u003c/p\u003e\n \u003cp\u003e34.2\u0026plusmn;3.9 (26-41)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e45 (43.7%)\u003c/p\u003e\n \u003cp\u003e58 (56.3%)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1 (1.0%)\u003c/p\u003e\n \u003cp\u003e5 (4.9%)\u003c/p\u003e\n \u003cp\u003e11 (10.7%)\u003c/p\u003e\n \u003cp\u003e57 (55.3%)\u003c/p\u003e\n \u003cp\u003e29 (28.1%)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e22 (21.4%)\u003c/p\u003e\n \u003cp\u003e44 (42.7%)\u003c/p\u003e\n \u003cp\u003e37 (35.9%)\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e65 (63.1%)\u003c/p\u003e\n \u003cp\u003e38 (36.9%)\u003c/p\u003e\n \u003cp\u003e101.9\u0026plusmn;6.6\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(84.0-118.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e138\u003c/p\u003e\n \u003cp\u003e41 (29.7%)\u003c/p\u003e\n \u003cp\u003e35.3\u0026plusmn;4.5 (22-46)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e55 (40.0%)\u003c/p\u003e\n \u003cp\u003e83 (60.0%)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e6 (4.3%)\u003c/p\u003e\n \u003cp\u003e4 (2.9%)\u003c/p\u003e\n \u003cp\u003e21 (15.2%)\u003c/p\u003e\n \u003cp\u003e80 (58.0%)\u003c/p\u003e\n \u003cp\u003e27 (19.6%)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e21 (15.2%)\u003c/p\u003e\n \u003cp\u003e51 (37.0%)\u003c/p\u003e\n \u003cp\u003e65 (47.1%)\u003c/p\u003e\n \u003cp\u003e1 (0.7%)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e71 (51.4%)\u003c/p\u003e\n \u003cp\u003e67 (48.6%)\u003c/p\u003e\n \u003cp\u003e106.3\u0026plusmn;10.5\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(86.4-145.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: hpi=hours post insemination, ICM=inner cell mass, TE=trophectoderm, tPNF=timing of pronuclear fading, tB=timing of blasulation\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Live birth rates and maternal age (years) according to alignment amongst zygotic cleavage plane, pronuclear axis and polar axis in freshly transferred blastocysts (n=103)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003eAlignment between zygotic cleavage plane and pronuclear axis (CPPN)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eYes (CPPN+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eNo (CPPN-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003eAlignment between zygotic cleavage plane and polar axis\u0026nbsp;(CPPB)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eYes (CPPB+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e77.5% (31/40)\u003c/p\u003e\n \u003cp\u003e33.8\u0026plusmn;4.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e15.2% (5/33)\u003c/p\u003e\n \u003cp\u003e34.8\u0026plusmn;4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e49.3% (36/73)\u003c/p\u003e\n \u003cp\u003e34.3\u0026plusmn;4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eNo (CPPB-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e20.0% (3/15)\u003c/p\u003e\n \u003cp\u003e33.8\u0026plusmn;3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e6.7% (1/15)\u003c/p\u003e\n \u003cp\u003e34.6\u0026plusmn;2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e13.3% (4/30)\u003c/p\u003e\n \u003cp\u003e34.2\u0026plusmn;3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e61.8% (34/55)\u003c/p\u003e\n \u003cp\u003e33.8\u0026plusmn;4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e12.5% (6/48)\u003c/p\u003e\n \u003cp\u003e34.7\u0026plusmn;3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e38.8% (40/103)\u003c/p\u003e\n \u003cp\u003e34.2\u0026plusmn;3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: Chi squared analysis was used to test statistical significance between different groups. CPPN+ vs CPPN-, p\u0026lt;0.001; CPPB+ vs CPPB-, p\u0026lt;0.001; CPPN-CPPB- vs CPPN+CPPB+, p\u0026lt;0.001; CPPN+CPPB- vs CPPN+CPPB+, p\u0026lt;0.001; CPPN-CPPB+ vs CPPN+CPPB+, p\u0026lt;0.001. Student T -test was used to test statistical significance in maternal age at oocyte retrieval between different groups. CPPN+ vs CPPN-, p=0.248; CPPB+ vs CPPB-, p=0.907; CPPN-CPPB- vs CPPN+CPPB+, p=0.510; CPPN+CPPB- vs CPPN+CPPB+, p=1.000; CPPN-CPPB+ vs CPPN+CPPB+, p=0.311. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Live birth rates and maternal age (years) according to alignment amongst zygotic cleavage plane, pronuclear axis and polar axis in frozen transferred blastocysts (n=138)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 346px;\"\u003e\n \u003cp\u003eAlignment between zygotic cleavage plane and pronuclear axis (CPPN)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eYes (CPPN+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eNo (CPPN-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003eAlignment between zygotic cleavage plane and polar axis\u0026nbsp;(CPPB)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eYes (CPPB+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e48.3% (28/58)\u003c/p\u003e\n \u003cp\u003e34.5\u0026plusmn;4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e19.4% (6/31)\u003c/p\u003e\n \u003cp\u003e36.5\u0026plusmn;4.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e38.2% (34/89)\u003c/p\u003e\n \u003cp\u003e35.2\u0026plusmn;4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eNo (CPPB-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e16.1% (5/31)\u003c/p\u003e\n \u003cp\u003e35.0\u0026plusmn;4.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e11.1% (2/18)\u003c/p\u003e\n \u003cp\u003e36.5\u0026plusmn;4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e14.3% (7/49)\u003c/p\u003e\n \u003cp\u003e35.6\u0026plusmn;4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e37.1% (33/89)\u003c/p\u003e\n \u003cp\u003e34.7\u0026plusmn;4.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e16.3% (8/49)\u003c/p\u003e\n \u003cp\u003e36.5\u0026plusmn;4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e28.7% (41/138)\u003c/p\u003e\n \u003cp\u003e35.3\u0026plusmn;4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: Chi squared analysis was used to test statistical significance in live birth rates between different groups. CPPN+ vs CPPN-, p=0.011; CPPB+ vs CPPB-, p=0.003; CPPN-CPPB- vs CPPN+CPPB+, p=0.005; CPPN+CPPB- vs CPPN+CPPB+, p=0.003; CPPN-CPPB+ vs CPPN+CPPB+, p=0.007. Student T -test was used to test statistical significance in maternal age at oocyte retrieval between different groups. CPPN+ vs CPPN-, p=0.022; CPPB+ vs CPPB-, p=0.619; CPPN-CPPB- vs CPPN+CPPB+, p=0.080; CPPN+CPPB- vs CPPN+CPPB+, p=0.607; CPPN-CPPB+ vs CPPN+CPPB+, p=0.039. \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4. Comparison of live birth rates between insemination methods (IVF vs ICSI) according to different alignment outcomes amongst zygotic cleavage plane, pronuclear axis and polar axis.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eIVF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eICSI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003eFresh blastocysts (n=103)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003eCPPN+\u003c/p\u003e\n \u003cp\u003eCPPN-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e64.3% (18/28)\u003c/p\u003e\n \u003cp\u003e5.9% (1/17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e59.3% (16/27)\u003c/p\u003e\n \u003cp\u003e16.1% (5/31)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e61.8% (34/55)\u003c/p\u003e\n \u003cp\u003e12.5% (6/48)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003eCPPB+\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; CPPB-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e50.0% (17/34)\u003c/p\u003e\n \u003cp\u003e18.2% (2/11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e48.7% (19/39)\u003c/p\u003e\n \u003cp\u003e10.5% (2/19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e49.3% (36/73)\u003c/p\u003e\n \u003cp\u003e13.3% (4/30)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003eFrozen blastocysts (n=138)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003eCPPN+\u003c/p\u003e\n \u003cp\u003eCPPN-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e42.1% (16/38)\u003c/p\u003e\n \u003cp\u003e17.6% (3/17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e33.3% (17/51)\u003c/p\u003e\n \u003cp\u003e15.6% (5/32)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e37.1% (33/89)\u003c/p\u003e\n \u003cp\u003e16.3% (8/49)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003eCPPB+\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; CPPB-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e47.1% (16/34)\u003c/p\u003e\n \u003cp\u003e14.3% (3/21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e32.7% (18/55)\u003c/p\u003e\n \u003cp\u003e14.3% (4/28)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e38.2% (34/89)\u003c/p\u003e\n \u003cp\u003e14.3% (7/49)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: Chi squared analysis showed no significant difference between IVF and ICSI groups in any of the above subgroups.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"journal-of-assisted-reproduction-and-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Journal of Assisted Reproduction and Genetics","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"Zygote, cleavage plane, pronuclear, polar body, live birth","lastPublishedDoi":"10.21203/rs.3.rs-7936004/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7936004/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eTo investigate whether alignment among zygotic cleavage plane (CP), pronuclear (PN) axis and polar axis (PB) is associated with higher live birth rate (LBR) in single transferred blastocysts?\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eA total of 103 fresh and 138 frozen blastocysts created via either conventional IVF insemination or intracytoplasmic sperm injection (ICSI) using autologous oocytes at between September 2022 and December 2023 were retrospectively analyzed. Live birth rates were compared according to alignment amongst three zygotic axes. The PN axis was defined by the longitudinal axis before fading and the PB axis was determined by the position of second polar body. Alignment relative to the zygotic CP was classified as CPPN\u0026thinsp;+\u0026thinsp;or CPPN- for the PN axis, and CPPB\u0026thinsp;+\u0026thinsp;or CPPB- for the PB axis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIn the fresh transfer dataset, CPPN\u0026thinsp;+\u0026thinsp;blastocysts had a significantly higher LBR than CPPN- blastocysts (61.8% vs 12.5%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while CPPB\u0026thinsp;+\u0026thinsp;outperformed CPPB- (49.3% vs 13.3%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The highest LBR was observed in CPPN+/CPPB\u0026thinsp;+\u0026thinsp;blastocysts (77.5%), compared with CPPN+/CPPB- (20.0%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), CPPN-/CPPB+ (15.2%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and CPPN-/CPPB- (6.7%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similar patterns were observed in the frozen transfer dataset, where CPPN+/CPPB\u0026thinsp;+\u0026thinsp;blastocysts again had the highest LBR (48.3%), compared with CPPN+/CPPB- (16.1%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003), CPPN-/CPPB+ (19.4%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007) and CPPN-/CPPB2- (11.1%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005). Logistic regression confirmed CPPN and CPPB alignment as independent predictors of live birth in both datasets, after adjusting for several potential confounders.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eOur findings support zygotic alignment among CP, PN and PB alxes as a potential viability marker for blastocyst selection.\u003c/p\u003e","manuscriptTitle":"Alignment among the zygotic cleavage plane, pronuclear axis, and polar axis predicts live birth outcome of blastocyst","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 06:29:54","doi":"10.21203/rs.3.rs-7936004/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-27T15:56:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-24T05:12:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-23T20:08:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"214079434067860185704661942712841188227","date":"2025-11-12T15:33:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"95817580990743988201591087685544356516","date":"2025-11-05T22:15:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-30T19:52:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-24T04:57:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-24T04:57:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Assisted Reproduction and Genetics","date":"2025-10-24T02:14:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"journal-of-assisted-reproduction-and-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Journal of Assisted Reproduction and Genetics","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9a7f0c0d-ee5b-4d0d-bf01-391ad1cf14e6","owner":[],"postedDate":"November 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-03T12:55:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-13 06:29:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7936004","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7936004","identity":"rs-7936004","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}