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Study question Does variation in day 5 observation timing confound embryo-morphology-based live birth prediction, and is it possible to develop a robust comprehensive numerical prediction model. Summary answer Day 5 observation timing confounds embryo-morphology-based live birth prediction. A robust comprehensive numerical prediction model can be developed after considering a number of contributing variables. What is known already Embryo development is a dynamic process, and therefore the widely used static observations potentially lead to biased prediction of live birth outcomes. So far, little is known in regard to potential confounding impact of day 5 assessment timing on the static-morphology-based live birth prediction. In addition, the inter-observer variation in morphology-based embryo assessment requires a more robust system to improve consistency of selection. Study design, size, duration This retrospective multi-center cohort study included 8866 autologous oocyte in vitro fertilisation treatment cycles performed at 14 associated clinics within the same network during 2012–2018. Only fresh cycles with single day 5 embryo transfers were included for analysis with all pregnancies followed up until birth. Repeat cycles of same patients were excluded to avoid clustering effect in statistical analysis. Participants/materials, setting, methods Dataset was randomly split into two subsets at 60:40 ratio, with one (n = 5274) used for regression analysis and model development and the other (n = 3592) used for model testing. Multiple logistic regression was performed to evaluate live birth predicting power of several potential contributors, expressed by odds ratio (OR) and 95% confidence interval (CI). A comprehensive prediction model was subsequently developed based on calculated weights of contributing factors, then tested via receiver operating characteristics (ROC) analysis. Main results and the role of chance The timings of day 5 observation of 8866 included embryos, measured by hours post insemination (HPI), distributed in a bell shape ranging from 112.0 to 120.0 h (mean±SD 115.7±1.7 h). After taking into account female age at egg collection (grouped as Limitations, reasons for caution The retrospective design does not allow for controlling of unknown confounders. HPI was based on static observations in this study so future time-lapse study may bring more insights with more accurate observation and measurement. Wider implications of the findings: The varying HPIs at day 5 observation were alarming as this could confound live birth prediction using embryology parameters. It is important to standardise the timing of embryo observations. The inclusion of HPI into a comprehensive numerical scoring system for live birth prediction may potentially improve its robustness Trial registration number Not applicable

Study question What is the live birth rate after single, low-grade blastocyst (LGB) transfer? Summary answer The live birth rate for LGBs is 28%, ranging between 15–31% for the different inner cell mass (ICM) and trophectoderm (TE) subgroups of LGBs. What is known already Live birth rates following LGB transfer are varied and have been reported to be in the range of 5–39%. However, these estimates are inaccurate as studies investigating live birth rates following LGB transfer are inherently limited by sample size (n = 10–440 for LGB transfers) due to LGBs being ranked last for transfer. Further, these studies are heterogenous with varied LGB definitions and design. Collating LGB live birth data from multiple clinics is warranted to obtain sufficient numbers of LGB transfers to establish reliable live birth rates, and to allow for delineation of different LGB subgroups, including blastocyst age and female age. Study design, size, duration We performed a multicentre, multinational retrospective cohort study in 9 IVF centres in China and New Zealand from 2012 to 2019. We studied the outcome of 6966 single blastocyst transfer cycles on days 5–7 (fresh and frozen) according to blastocyst grade, including 875 transfers from LGBs ( Participants/materials, setting, methods The main outcome measured was live birth rate. Blastocysts were grouped according to quality grade: good-grade blastocysts (GGBs; n = 3849, aa, ab and ba), moderate-grade blastocysts (MGBs; n = 2242, bb) and LGBs (n = 875, ac, ca, bc, cb and cc) and live birth rates compared using the Pearson Chi-squared test. A logistic regression analysis explored the relationship between blastocyst grade and live birth after adjustment for the confounders: clinic, female age, expansion stage, and blastocyst age. Main results and the role of chance The live birth rates for GGBs, MGBs and LGBs were 45%, 36% and 28% respectively (p Limitations, reasons for caution Despite the large multicentre design of the study, analyses of transfers occurring within the smallest subsets of the LGB group were limited by sample size. The study was not randomised and had a retrospective character. Wider implications of the findings: LGBs maintain satisfactory live birth rates (averaging 28%) in the general IVF population. Even those in the lowest grading tier maintain modest live birth rates (15%; cc). It is recommended that LGBs not be universally discarded, and instead considered for subsequent frozen embryo transfer to maximize cumulative live birth rates. Trial registration number Not applicable

Study question Do morphometric and morphokinetic profiles of pronuclei (PN) following intracytoplasmic sperm injection (ICSI) vary between male and female human zygotes? Summary answer Male and female zygotes displayed different PN morphometrics and morphokinetics. Additionally, variations were identified between sperm-originated (SPN) and oocyte-originated (OPN) pronuclei. What is known already Previous studies have investigated the use of PN-associated parameters via static observations as indicators of zygote viability, including size equality or juxtaposition. However, recent clinical application of time-lapse videography (TLV) provides a novel opportunity to assess these pronuclear events with greater accuracy and precision of morphometric and morphokinetic measurement. A number of recent TLV studies have also investigated potential live birth prediction by such PN associated measures, however whether or not there are gender associated differences in such measures which could in turn confound live birth prediction is unknown. Study design, size, duration: This retrospective cohort study included 94 consecutive autologous single day 5 transfer cycles (either fresh or frozen) performed between January 2019 and March 2020. Only ICSI cycles (maternal age Participants/materials, setting, methods Timings included 2nd polar body extrusion (tPb2), SPN(tSPNa)/OPN(tOPNa) appearance (differentiated by proximity to Pb2) and PN fading (tPNF). Morphometrics were evaluated at 8 (stage 1), 4 (stage 2) and 0 hour before PNF (stage 3), measuring PN area (um2), PN juxtaposition, and nucleolus precursor body (NPB) arrangement. Means ± standard deviation were compared using student t test or logistic regression as odds ratio (OR) and 95% confidence interval (CI), and proportional data by chi-squared analysis. Main results and the role of chance Logistic regression indicated that male zygotes had longer time intervals of tPb2_tSPNa than female zygotes (4.8±1.5 vs 4.2±1.0 h, OR = 1.442, 95% CI 1.009–2.061, p = 0.044), but not tPb2_tOPNa (4.7±1.8 vs 4.5±1.3 h, OR = 1.224, 95% CI 0.868–1.728, p = 0.250) and tPb2_tPNF (19.9±2.8 vs 19.1±2.3 h, OR = 1.136, 95% CI 0.957–1.347, p = 0.144). SPN increased in size from stage 1 through 2 to 3 (435.3±70.2, 506.7±77.3, and 556.3±86.4 um2, p = 0.000) and OPN did similarly (399.0±59.4, 464.3±65.2, and 513.8±63.5 um2, p = 0.000), with SPN being significantly larger than OPN at each stage (p 0.05 respectively). Limitations, reasons for caution The retrospective design does not allow for control of unknown confounders. Sample size is considered relatively small. PN area measurement may not truly represent volume as PN may not be perfectly spherical. Findings were based on women Wider implications of the findings: These findings augment and extend previous studies investigating PN parameters via static observations. The reported variations between male and female embryos may confound live birth prediction when using pronuclei morphometrics and morphokinetics. Larger scaled studies are warranted to verify these findings. Trial registration number Not applicable

Study question Does slow development of fresh transferred day 5 embryos lead to decreased implantation potential and birthweight? Summary answer Slow day 5 development was associated with reduced implantation potential when transferred fresh but the subsequent birthweight of the resulting baby was not impacted. What is known already Slow development of in vitro cultured cleavage stage embryos is associated with reduced blastocyst development and implantation rates. There is no current consensus regarding whether to transfer fresh slow developing day 5 embryos or to extend culture for a subsequent day with potential for cryopreservation. It is therefore important to understand the true prognosis of fresh transferred day 5 embryos at less advanced developmental stages. This would provide evidence based guidelines for the decision making process in regard to embryo transfer. Study design, size, duration This is a retrospective multi-center cohort study, including 1213 consecutive patients undergoing autologous oocyte in vitro fertilization (IVF) treatment during 2016-2019,with fresh transfer of a single day 5 embryo (selection based on developmental stage and inner cell mass and trophectoderm morphology if blastocyst was at the ≥expanding stage). Cycle data were collected from 4 associated private clinics, with repeat cycles of same patients excluded to avoid clustering effect at statistical analysis. Participants/materials, setting, methods Live birth and birthweight were followed up in all 1213 fresh day 5 SETs. Multiple regression (logistic or linear) was performed to investigate association between slow day 5 development (defined as ≤ early blastocyst) and (a)live birth, (b) birthweight, and (c) gestation-adjusted birthweight (Z score) to account for gestational age, gender and compared to embryos at ≥ expanded stage. Results were expressed as adjusted odds ratio (aOR) with 95% confidence interval (CI)or coefficients (β). Main results and the role of chance No implantation was achieved following single fresh transfer of day 5 embryos that failed to reach early blastocyst stage (n = 76) and were transferred as ≤ morula stage. Live birth rate was significantly lower following single day 5 fresh transfer of an early blastocyst (n = 237, 16%), in comparison to expanding (n = 329, 27%, P = 0.001), expanded(n = 392, 41%, P = 0.000), and hatching/hatched blastocysts (n = 169, 44%, P = 0.000). After adjusting for potential confounding factors including; maternal age, hours post insemination at day 5 assessment, number of oocytes collected, number of 2PN embryos, and number of embryos frozen; multiple logistic regression showed significantly reduced likelihood of live birth resulting from early blastocysts in reference to those at the expanding (aOR=0.584, 0.371-0.917, P = 0.020), expanded (aOR=0.322, 0.208-0.501, P = 0.000), or hatching/hatched stages (aOR=0.255, 0.147-0.443, P = 0.000). However, multivariate linear regression indicated that early blastocysts resulting in a live birth (n = 39) did not lead to altered birthweight (β=-9.091, P = 0.904; β=-34.960, P = 0.343; β=-26.074, P = 0.414; respectively) or Z score (β = 0.045, P = 0.706; β=-0.051, P = 0.426; β=-0.028, P = 0.506; respectively) in reference to the expanding (n = 90), expanded (n = 160), or hatching/hatched stages (n = 75). Limitations, reasons for caution The retrospective nature of this study does not allow controlling of unknown confounders. The 4 participating clinics are associated within the same network with shared protocols, therefore, results may not be generalized to other clinics with different settings. Wider implications of the findings The findings suggest no clinical value of fresh day 5 transfer of embryos ≤morula stage. Although early blastocysts implant at reduced rate, assuring birthweight outcomes suggest clinical value. Future studies intend to investigate slow growing day 5 fresh transfers versus embryos that were slow growing but transferred after day 6. Trial registration number NA