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OBJECTIVE: To report a live birth from a patient with complete zona-free oocytes due to abnormal zona production and to reveal full time-lapse blastocyst development footage of its originating embryo. METHODS: A 34-year-old woman presented with a history of failed fertilization via standard in vitro fertilization insemination and a potential absence of zona pellucida. A total of 3 intracytoplasmic sperm injection cycles were undertaken with all oocytes collected being zona-free. Embryos created in the initial 2 cycles were cultured in the G1+/G2+ sequential media in a benchtop incubator. During the final successful cycle, the culture strategy was shifted to single step media (G-TL) in an Embryoscope+ incubator. RESULTS: The first 2 attempts led to a biochemical pregnancy or no blastocyst available for transfer. In the third cycle, 13 out of 24 collected oocytes were subjected to injection, with 4 being normally fertilized. Two blastocysts were subsequently formed, in which one was cryopreserved and the other transferred. A live baby girl (1570g) was subsequently delivered at 34 weeks of gestation by cesarean section. CONCLUSION: Live birth can be achieved for patients with zona production deficiency. Adjustment in ovarian stimulation and subsequent embryo culture strategies may have potentially contributed to the success of the 3rd cycle. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02114-3.

Refereed/Peer-reviewed Research question: Does variation in day 5 assessment timing confound live birth prediction using snapshot blastocyst morphology and is it possible to develop a numerical prediction algorithm? Design: Retrospective multicentre cohort study including 4851 autologous oocyte single day 5 fresh embryo transfers performed at 11 Monash IVF clinics between 2016 and 2020. Repeat cycles of the same patients were excluded to avoid clustering effects in regression analysis. Results: Hours post insemination (HPI) at day 5 assessment (115.9 ± 2.6 h) significantly correlated with blastocyst developmental stage (r = 0.118, P < 0.001). Independent association (expressed as adjusted odds ratio [aOR] and 95% confidence interval [CI]) was identified between live birth and HPI (aOR 0.950, 95% CI 0.925–0.976, P < 0.001) after accounting for blastocyst morphology and a range of patient/cycle characteristics. Algorithms were constructed using four significant live birth predictors: HPI at day 5 assessment, blastocyst developmental stage (aOR 1.347, 95% CI 1.217–1.491, P < 0.001), morphological grade (aOR 1.314, 95% 1.197–1.443, P < 0.001) and maternal age (aOR 0.922, 95% CI 0.907–0.936, P < 0.001). Receiver operating characteristic (ROC) analysis showed consistent predicting performance of algorithms via five-fold cross-validation, with similar area under the ROC curve (AUC 0.718, 0.715, 0.720, 0.712, 0.726, P < 0.001, respectively, in development subsets; and AUC 0.718, 0.731, 0.709, 0.741, 0.684, P < 0.001, respectively, in validation subsets). A score (ranging from 0.1 to 4.7) calculator based on the final algorithm was subsequently created. Conclusions: Day 5 assessment timing is a confounding factor for live birth prediction using snapshot blastocyst morphology. A numerical algorithm incorporating day 5 assessment HPI, blastocyst morphology and maternal age can be developed for live birth prediction.

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

The recent clinical introduction of time-lapse videography into in vitro fertilization laboratories has offered a novel opportunity for embryologists to explore improved methods for embryo selectio...

IntroductionThe perinatal–postnatal family environment is associated with childhood outcomes including impacts on physical and mental health and educational attainment. Family longitudinal cohort studies collect in-depth data that can capture the influence of an era on family lifestyle, mental health, chronic disease, education and financial stability to enable identification of gaps in society and provide the evidence for changes in government in policy and practice.Methods and analysisThe Queensland Family Cohort (QFC) is a prospective, observational, longitudinal study that will recruit 12 500 pregnant families across the state of Queensland (QLD), Australia and intends to follow-up families and children for three decades. To identify the immediate and future health requirements of the QLD population; pregnant participants and their partners will be enrolled by 24 weeks of gestation and followed up at 24, 28 and 36 weeks of gestation, during delivery, on-ward, 6 weeks postpartum and then every 12 months where questionnaires, biological samples and physical measures will be collected from parents and children. To examine the impact of environmental exposures on families, data related to environmental pollution, household pollution and employment exposures will be linked to pregnancy and health outcomes. Where feasible, data linkage of state and federal government databases will be used to follow the participants long term. Biological samples will be stored long term for future discoveries of biomarkers of health and disease.Ethics and disseminationEthical approval has been obtained from the Mater Research Ethics (HREC/16/MHS/113). Findings will be reported to (1) QFC participating families; (2) funding bodies, institutes and hospitals supporting the QFC; (3) federal, state and local governments to inform policy; (4) presented at local, national and international conferences and (5) disseminated by peer-review publications.

Embryo quality is a key determinant of the success of IVF. Although the focus has been on selecting the best embryo for transfer, the classification of low-grade blastocysts (LGB) in existing scoring systems has received less attention. This is worrisome; embryo freezing allows optimal use of all created embryos, thus maximizing the cumulative live birth rate, which is arguably the most important outcome for infertile couples. A PubMed search was conducted in August 2020, using '((('poor-quality' OR 'poor quality') OR ('low-grade' OR 'low grade')) AND ('embryo' OR 'blastocyst')) AND ('pregnancy' OR 'live birth')'. This scoping review shows that LGB have similar euploidy and pregnancy success rates after implantation and have no adverse effects on pregnancy or perinatal outcomes. Evidence for pregnancy outcomes is lacking for different grades of LGB, with most studies clustering all LQB as one to compare with optimal blastocysts.

This study investigated the efficacy of four published day 3 embryo time-lapse algorithms based on different types of datasets (known implantation data [KID] and single embryo transfer [SET]), and the confounding effect of female age and conventional embryo morphology. Four algorithms were retrospectively applied to three types of datasets generated at Fertility North between February 2013 and December 2014: (a) KID dataset (

PURPOSE: To investigate the between-laboratory reproducibility of embryo selection/deselection effectiveness using qualitative and quantitative time-lapse parameters. METHODS: A systematic search was performed on MEDLINE, EMBASE, and the Cochrane Library (up to February 2020) without restriction on date, language, document type, and publication status. Measuring outcomes included implantation, blastulation, good-quality blastocyst formation, and euploid blastocyst. RESULTS: We detected 6 retrospective cohort studies externally validating the first clinical time-lapse model (Meseguer) emphasizing quantitative parameters, of which 3 (including one involving 2 independent centers) were included for the pooled analysis. Receiver operating characteristics analysis showed reduced predictive power of the model when either including or not including sister clinic validation. Fifteen cohort studies evaluating qualitative parameters were included for meta-analysis, and the mean Newcastle-Ottawa Scale was 5.3. Overall, meta-analysis showed significantly adverse association between the presence of ≥ 1 cleavage abnormalities and embryo implantation rates (11 studies, n = 7266; RR = 0.39[0.28, 0.55](95% CI); I(2) = 57%). Further analysis showed adverse impacts of direct cleavage (7 studies, n = 7065; RR = 0.28 [0.15, 0.54] (95% CI); I(2) = 46%), reverse cleavage (2 studies, n = 3622; RR = 0.16 [0.03, 0.75] (95% CI); I(2) = 0%), chaotic cleavage (2 studies, n = 3643; RR = 0.11 [0.02, 0.69] (95% CI); I(2) = 24%), and multinucleation (5 studies, n = 2576; RR = 0.59 [0.50, 0.69] (95% CI); I(2) = 0%), but not the

Objective To study the performance of thawed zygotes and cleavage stage embryos transferred either on the day of thaw or after overnight culture. Methods A retrospective study of 864 frozen embryo transfer cycles. Cryosurvival rates per thawed embryo and implantation rates were analysed for embryos frozen on Day 1, Day 2 or Day 3 relative to oocyte collection (Day 0) and transferred on the day of thaw or after overnight culture, together with clinical pregnancy rates and prevalence of multiple gestations. Results Survival of Day 3 embryos was significantly lower than those frozen on Day 1 (P=0.017) or Day 2 (P=0.015). Following overnight culture, resumption of mitosis of zygotes was more frequent than Day 2 (P=0.000) which are in turn higher than Day 3 (P=0.000) embryos. The implantation rate for Day 2 embryos dividing overnight was significantly higher than those that did not divide for women Conclusions Later cleavage stage post-thaw embryos survive and resume mitosis less frequently compared to earlier stages. Embryos not resuming mitosis after culture overnight can implant, particularly Day 3 embryos, suggesting that they can further increase the cumulative pregnancy rate per oocyte collection and that discarding them is wasteful. Overnight culture is best used for logistical reasons rather than a strategy to improve pregnancy rates.

A total of 488 Day 3 human embryos with known implantation data from two independent in vitro fertilization laboratories were included for analysis, with 270 from Fertility North (FN) and 218 from Canberra Fertility Centre (CFC). Implanting embryos grew at different rates between FN and CFC as indicated in hours of the time intervals between pronuclear fading and the 4- (13.9 ± 1.1 vs. 14.9 ± 1.8), 5- (25.7 ± 1.9 vs. 28.4 ± 3.7) and 8-cell stages (29.0 ± 3.2 vs. 32.2 ± 4.6), as well as the durations of 2- (10.8 ± 0.8 vs. 11.6 ± 1.1), 3- (0.4 ± 0.5 vs. 0.9 ± 1.2), and 4-cell stages (11.8 ± 1.4 vs. 13.6 ± 2.9), all p0.05. The application of a previously published time-lapse algorithm on ICSI embryos from the two participating laboratories failed to reproduce a predictive pattern of implantation outcomes (FN: AUC=0.565, p=0.250; CFC: AUC=0.614, p=0.224). However, for the qualitative measures including poor conventional morphology, direct cleavage, reverse cleavage and6 intercellular contact points at the end of the 4-cell stage, there were similar proportions of embryos showing at least one of these biological events in either implanting (3.1% vs. 3.3%, p0.05) or non-implanting embryos (30.4% vs. 38.3%, p0.05) between FN and CFC. Furthermore, implanting embryos favored lower proportions of the above biological events compared to the non-implanting ones in both laboratories (both p0.01). To conclude, human embryo morphokinetics may vary between laboratories, therefore time-lapse algorithms emphasizing quantitative timing parameters may have reduced inter-laboratory transferability; qualitative measures are independent of cell division timings, with potentially improved inter-laboratory reproducibility.

To present a time-lapse deselection model involving both qualitative and quantitative parameters for assessing embryos on day 3.Retrospective cohort study and prospective validation.Private IVF center.A total of 270 embryos with known implantation data (KID) after day 3 transfer from 212 IVF/intracytoplasmic sperm injection (ICSI) cycles were retrospectively analyzed for building the proposed deselection model, followed by prospective validation using an additional 66 KID embryos.None.Morphological score on day 3, embryo morphokinetic parameters, abnormal cleavage patterns, and known implantation results.All included embryos were categorized either retrospectively or prospectively into 7 grades (A+, A, B, C, D, E, F). Qualitative deselection parameters included poor conventional day 3 morphology, abnormal cleavage patterns identified via time-lapse monitoring, and8 cells at 68 hours postinsemination. Quantitative parameters included time from pronuclear fading (PNF) to 5-cell stage and duration of 3-cell stage. KID implantation rates of embryos graded from A+ to F were 52.9%, 36.1%, 25.0%, 13.8%, 15.6%, 3.1%, and 0 respectively (area under the curve [AUC] = 0.762; 95% confidence interval [CI], 0.701-0.824), and a similar pattern was seen in either IVF (AUC = 0.721; 95% CI, 0.622-0.821) or ICSI embryos (AUC = 0.790; 95% CI, 0.711-0.868). Preliminary prospective validation using 66 KID embryos also showed statistically significant prediction in Medicult (AUC = 0.750; 95% CI, 0.588-0.912) and Vitrolife G-Series (AUC = 0.820; 95% CI, 0.671-0.969) suites of culture media.The proposed model involving both qualitative and quantitative deselection effectively predicts day 3 embryo implantation potential and is applicable to all IVF embryos regardless of insemination method by using PNF as the reference starting time point.

Time-lapse videography showed that human early cleavage embryos were quicker following intracytoplasmic sperm injection to reach developmental milestones compared to in vitro fertilization when using insemination as the timing start point (t0), due to differences in the time taken for embryos to reach pronuclear fading (PNF). These differences disappeared when PNF was used as t0. Using a biological rather than procedural t0 will allow a unified assessment strategy to be applied to all cycles irrespective of the insemination method.

A total of 341 fertilized and 37 unfertilized oocytes from 63 intracytoplasmic sperm injection (ICSI) treatment cycles were included for retrospective assessment using the Embryoscope time-lapse video system. The second polar body (pb2) extrusion occurred at 2.9±0.1 h (range 0.70-10.15 h) relative to sperm injection. All oocytes reduced in size following sperm injection (p0.05) with shrinkage ceasing after 2h in the unfertilized and at pb2 extrusion in the fertilized oocytes. Pb2 extrusion was significantly delayed for women aged38 years compared to those35 years (3.4±0.2 vs. 2.8±0.1, p0.01) or 35-38 years (3.4±0.2 vs. 2.8±0.1, p0.01), but timing was not related to the Day 3 morphological grades (1-4) of subsequent embryos (2.9±0.1, 2.9±0.1, 2.8±0.2 and 3.0±0.1; p0.05 respectively). A shorter time of first cleavage division relative to either sperm injection or pb2 extrusion is associated with both top grade (AUC=0.596 or 0.601, p=0.006 or 0.004) and usable embryos (AUC=0.638 or 0.632, p=0.000 respectively) on Day 3. In summary, (i) pb2 of human oocytes extrudes at various times following sperm injection, (ii) the timing of pb2 extrusion is significantly delayed when female age38 years, but not related to subsequent embryo development, (iii) all human oocytes reduce in size following sperm injection, (iv) completion of pb2 extrusion in the fertilized oocytes is a pivotal event in terminating shrinkage of the vitellus, and (v) time to first cleavage division either from sperm injection or pb2 extrusion is a significant predictive marker for embryo quality on Day 3.

Objective To investigate the prevalence and potential causes of reverse cleavage (RC) by human early-cleavage embryos and its associations with embryonic development and implantation after transfer. Design Clinical retrospective cohort study. Setting Private fertility treatment center. Patient(s) A total of 126 consecutive in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles, with 353 IVF and 436 ICSI embryos cultured in the Embryoscope until day 3. Intervention(s) None. Main Outcome Measure(s) Embryo assessment on day 3, incidence of abnormal division, embryo morphokinetic parameters, and fetal heart beat. Result(s) RC, referring to either blastomere fusion or failed cytokinesis, occurred up to three times per individual embryo in 27.4% of embryos during the first three cleavage cycles. A higher incidence was associated with GnRH antagonist cycles compared with agonist cycles (odds ratio [OR] 1.683), or with ICSI compared with IVF (OR 1.600). After ICSI, sperm progressive motility was associated with RC (area under the receiver operating characteristic curve: 0.573). Compared with RC-negative embryos, a lower proportion of RC-positive embryos reached 6-cell stage or beyond by day 3 (47.7% vs. 71.7%), and were more likely to have multinucleation at the 4-cell stage (10.1% vs. 5.0%). Embryos showing RC had significantly poorer performance in both conventional grading and morphokinetic parameters, and they implanted less (0/22 vs. 29/131) than those not showing RC. Conclusion(s) RC significantly compromised embryo development, culminating in poor implantation potential. For each embryo, it can occur on more than one occasion at any stage during the first 3 days of culture. It is associated with factors affecting both oocyte and sperm.