2PN Zygotes Research Articles

O-287 Prognostic value of perivitelline-thread-associated kinetic activity of the plasma membrane of the pronuclear human zygote: a retrospective, observational study

Abstract Study question Is perivitelline-thread-associated kinetic activity of the zygote membrane prior to pronuclear fading (tPNf) indicative of the developmental potential of human embryos in vitro? Summary answer Embryos displaying kinetic activity of the zygote membrane had a significantly decreased clinical utilization rate, compared to those without. What is known already Morphology (pronuclei, cytoplasm etc.) of the pronuclear human zygote has been shown to correlate with ongoing embryo development and implantation potential. Further morphokinetic observations have been noted during this dynamic period of preimplantation development with the use of time-lapse imaging. The hypothesized mechanical actions of perivitelline threads (PVT) have been associated with blastomere fragmentation during early cleavage divisions and subsequent poor embryo development. Their presence and activity prior to tPNf and prognostic value with regard to treatment outcome, have not been studied in detail. Study design, size, duration This retrospective study assessed the development of 1047 two pronuclear (2PN) zygotes post IVF/ICSI until culture day 5, from a total of 172 patients which underwent time-lapse imaging monitoring with the EmbryoScope + (Vitrolife) between 01/06/2019 and 20/03/2020. Participants/materials, setting, methods Time-lapse videos of embryo development were analyzed manually for the detection and characterization of the ‘event’ of interest: an evident wave of membrane displacement coordinated by PVT prior to tPNf and first cleavage division. Assessed embryos were then categorized into two groups: an event group and a non-event group. The primary outcome of embryo utilization rate was compared between the two groups. Live birth and miscarriage rates per blastocyst transferred were assessed as secondary outcomes. Main results and the role of chance Morphokinetic analysis of the event established a mean commencement time at 22.5 ± 9.6 hours, cessation at 27.3 ± 4.7 hours, followed by tPNf at 27.9 ± 5.1 hours post fertilization. A total of 157 embryos in the event group and 890 in the non-event group were assessed (event occurrence rate of 15.0%). The average age of oocyte utilized was not significantly different between the event and non-event group at 34.6 years versus 34.5 years, respectively. The embryo utilization rate in the non-event group was significantly higher than that observed in the event group; 50.6% versus 29.9%, respectively (p < 0.001). The odds of an embryo not being utilized were higher in embryos with an observed event, compared to those without (OR = 2.3; 95% CI = 1.66, 3.45). Live birth rates for fresh blastocyst transfers were 42.9% (6/14) and 29.8% (42/141) for the event and non-event groups, respectively (p > 0.05). Miscarriage rates for fresh blastocyst transfers were 33.3% (3/9) and 35.4% (23/65) for the event and non-event groups, respectively (p > 0.05). Overall, these results indicate that event observation is associated with a significantly decreased embryo utilization rate due to subsequent poor embryo development in extended culture conditions. Limitations, reasons for caution A number of events occurring outside the plane of focus prevented accurate assessment, resulting in data exclusion. High levels of variability between the degree of kinetic activity, and transient nature of the PVT were observed. Differences in treatment outcomes were not powered for statistical analysis. Wider implications of the findings Notably, embryos displaying kinetic activity of the zygote membrane prior to tPNf are less likely to develop into embryos which can be utilized for transfer or cryopreservation. Observation of the described event may aid with clinical decision-making and managing patient expectations during the period of in vitro monitoring and development. Trial registration number Not applicable

Does the cell number of 0PN embryos on day 3 affect pregnancy and neonatal outcomes following single blastocyst transfer?

Background0PN zygotes have a low cleavage rate, and the clinical outcomes of cleavage-stage embryo transfers are unsatisfactory. Blastocyst culturing is used to screen 0PN embryos, but whether the cell number of 0PN embryos on day 3 affects the clinical outcomes following single blastocyst transfer is unknown and would be helpful in evaluating the clinical value of these embryos.MethodsThis retrospective study compared 46,804 0PN zygotes, 242 0PN frozen-thawed single blastocyst transfers, and 92 corresponding 0PN singletons with 232,441 2PN zygotes, 3563 2PN frozen-thawed single blastocyst transfers, and 1250 2PN singletons from January 2015 to October 2019 at a tertiary-care academic medical centre. The 0PN and 2PN embryos were divided into two groups: the group with < 6 cells on day 3 and that with ≥ 6 cells. Embryo development, subsequent pregnancy and neonatal outcomes were compared between the two groups.ResultsThe cleavage and available blastocyst rates of the 0PN zygotes were much lower than those of the 2PN zygotes (25.9% vs. 97.4%, P < 0.001; 13.9% vs. 23.4%, P < 0.001). In the < 6 cells group, the available blastocyst rate of the cleaved 0PN embryos was significantly lower than that of the 2PN embryos (2.5% vs. 12.7%, P < 0.001). However, in the ≥ 6 cells group, the available blastocyst rate of the 0PN cleaved embryos significantly improved, although it was slightly lower than that of the 2PN embryos (33.9% vs. 35.7%, P = 0.014). Importantly, compared to those of the 2PN single blastocyst transfers, the clinical pregnancy rate, live birth rate, Z-score and malformation rate of the 0PN single blastocyst transfers were not significantly different in either the < 6 cells group (30.4% vs. 39.8%, P = 0.362; 30.4% vs. 31.3%, P = 0.932; 0.89 ± 0.90 vs. 0.42 ± 1.02, P = 0.161; 0% vs. 2.6%, P = 1.000) or the ≥ 6 cells group (50.7% vs. 46.6%, P = 0.246; 39.7% vs. 38.3%, P = 0.677; 0.50 ± 1.23 vs. 0.47 ± 1.11, P = 0.861; 2.4% vs. 1.8%, P = 1.000).ConclusionsThe cell number on day 3 of 0PN embryos affected the subsequent formation of blastocysts but did not influence the subsequent pregnancy and neonatal outcomes of 0PN single blastocyst transfers, which may be beneficial to clinicians counselling patients on the clinical value of 0PN embryos.

Calcium gluconate infusion is not as effective as dopamine agonists in preventing ovarian hyperstimulation syndrome.

The aim of the study was to compare the effectiveness of calcium gluconate and cabergoline therapy in the prevention of ovarian hyperstimulation syndrome (OHSS). Eight hundred and forty-five women who underwent GnRH antagonist protocol and at high risk for developing OHSS were divided into two groups, those given cabergoline (n=435) or calcium gluconate (n=410). In cabergoline group, 0.5 mg of cabergoline was administered once daily p.o. starting on the day of ovulation trigger and continued until the following 8 days. In calcium gluconate group, intravenous calcium gluconate was administered daily for four days starting on the day of oocyte pickup (OPU). 10 ml of 10% calcium gluconate solution was dissolved in 200 ml of physiological saline and administered by intravenous route within 40 minutes. Infusion was started within the first 30 minutes following the OPU and continued on the 1st, 2nd and 3rd days after OPU. Mild OHSS was developed in 367 (89%) patients receiving calcium gluconate infusion, while 251 patients (57%) in the cabergoline group developed mild OHSS. The frequency of mild OHSS in the calcium group was significantly higher than the cabergoline group (p<.001). Moderate OHSS was observed in 32 people (7.8%) in the calcium gluconate group, while it was observed in 184 people in the cabergoline group (42.3%). Calcium gluconate infusion significantly reduced the development of moderate OHSS compared to cabergoline therapy (p<.001). Severe OHSS developed in 11 patients (2.7%) in the calcium gluconate group, while severe OHSS did not develop in those given cabergoline (0%, p<.001). Clinical pregnancy, live birth and abortion rates were similar in the two groups. When logistic regression analysis was performed, a significant correlation was found between age, BMI, AMH, the number of antral follicle count, OHSS history, paracentesis, progesterone on the day of hCG, 2 PN zygotes, and HbA1c levels and the development of OHSS. No correlation was found between the use of metformin or cetrotide and the development of OHSS. Calcium gluconate treatment is not effective in the prevention of OHSS.

Efficacy of using recombinant granulocyte colony-stimulating factor in IVF/ICSI protocols in patients with predicted suboptimal response to controlled ovarian stimulation

BACKGROUND: According to most studies, the efficacy of IVF/ICSI protocols in patients with suboptimal response was significantly lower than in women with normal response. However, methodological approaches optimizing the efficacy of IVF/ICSI protocols in patients with suboptimal response have not been developed.&#x0D; AIM: The aim of this study was to evaluate the efficacy of using recombinant granulocyte colony-stimulating factor in IVF/ICSI protocols in women with predicted suboptimal response to controlled ovarian stimulation.&#x0D; MATERIALS AND METHODS: This prospective study included 102 patients with predicted suboptimal response: treatment group (n = 34) and comparison group (n = 68). Controlled ovarian stimulation was performed according to the standard protocol with gonadotropin-releasing hormone antagonists. Women in treatment group daily received 60 mcg of recombinant granulocyte colony-stimulating factor subcutaneously from the onset of stimulation until the day of human chorionic gonadotropin blood test. The intergroup comparative analysis included clinical-anamnestic data, indicators of hormonal status, ovarian reserve, main characteristics of protocols stimulation, embryological data and the efficacy of IVF/ICSI programs.&#x0D; RESULTS: In the treatment group, the number of retrieval oocytes was higher (8.1 5.3 vs. 4.7 2.6, respectively; p 0.001), and the effective dose of follicle-stimulating hormone preparations was lower than in the comparison group (403.6 351.1 IU vs. 694.3 950.4 IU, respectively; p = 0.013). Besides, the number of mature oocytes (6.8 5.2 vs. 3.7 1.9, respectively; p 0.01), 2PN zygotes (5.1 3.9 vs. 2.8 1.7, respectively; p 0.01), and good quality embryos (3.4 3.3 vs. 1.8 1.5, respectively; p 0.01) were also higher in the treatment group. Сlinical pregnancy rate in women receiving recombinant granulocyte colony-stimulating factor was significantly higher (41.2% vs. 22.1%, respectively; OR 2.47; 95% CI 1.016.03; p 0.05).&#x0D; CONCLUSIONS: Using recombinant granulocyte colony-stimulating factor improves the efficacy of IVF/ICSI protocols in women with predicted suboptimal response.

Are tri‐pronuclear embryos that show two normal‐sized pronuclei and additional smaller pronuclei useful for embryo transfer?

PurposeThis study aimed to analyze whether tripronuclear (3PN) zygotes, with two normal‐sized PNs and an additional smaller PN (2.1PN), can be used for embryo transfer.Method(s)A retrospective embryo cohort study was conducted on 695 patients who underwent intracytoplasmic sperm injection treatment. Blastocyst formation rates were compared between 2.1PN and 2PN zygotes and PGT‐A analysis was performed on 15 blastocysts derived from 2.1PN zygotes.Result(s)Blastocyst formation rates were comparable between 2.1PN (43.8%) and 2PN zygotes (54.8%; p = 0.212). The rates of blastocysts with good morphology derived from 2.1 PN and 2PN zygotes were 18.8% and 25.5%, respectively. No significant differences were detected (p = 0.383). All of the analyzed blastocysts were diploid; however, 13 of these were found to be aneuploid, with a further two being mosaic.ConclusionOur results suggest that 2.1PN embryos can reach blastocyst stage. These blastocysts were diploid, however, predominantly aneuploid, and therefore could not be used for embryo transfer.

Tripronuclear Zygotes in IVF Laboratory Quality Control: Experimental Evaluation and Potential Applications.

This study set out to evaluate quality control within a new in vitro fertilization (IVF) laboratory environment and of new incubators based on the culture results of tripronuclear zygotes. The representative environmental indicators within new and old IVF laboratories were monitored, and tripronuclear zygotes were cultured in the two laboratories; the results were analyzed and compared. Subsequently, tripronuclear (3PN) zygotes were cultured in both new and old incubators and the culture results were compared. No differences were found in embryo development between 3PN zygotes in the old and new laboratories. However, in the quality control test, the degeneration rate and developmental arrest rate in the new incubator early phase group were significantly increased when compared with the old incubators. Moreover, the grade I embryo rate also decreased significantly. Nevertheless, all the above comparisons in the new incubator later phase group showed no statistical significance as compared to those observed in old incubators. Tripronuclear zygotes are sensitive to the environment in IVF laboratories and can be considered useful during quality control trials of new IVF laboratories and new equipment including incubators.

Embryo attrition in planned PGT-A: predicting the number of available blastocysts for transfer.

During a typical IVF cycle, there is unavoidable attrition from oocytes retrieved to blastocysts formed. Some patients will not have blastocysts available to biopsy or embryos for transfer. The purpose of this study was to predict the number of transferable blastocysts available for patients based on their age and number of 2pn zygotes. This was a retrospective cohort study of all fresh autologous IVF and ICSI cycles in which PGT-A was planned from 1/2012 to 3/2020. In total, 746 cycles from 571 patients were analyzed. Patient cycles were stratified into two groups: less than four 2pn zygotes (n = 85) and at least four 2pn zygotes (n = 661). Cycles were then stratified by patient age. Cycle outcomes, including number of cleavage-stage embryos, blastocysts, euploid blastocysts, and low level mosaic blastocysts, were determined. Cleavage-rate was independent of age and number of 2pn zygotes and ranged between 96 and 100%. Blastocyst conversion and euploid blastocyst conversion rates were directly correlated to age, ranging from 52 to 83% for blastocyst conversion and 0-28% for euploid blastocyst conversion. For patients above the age of 40years with less than four 2pn zygotes, the risk of having no transferable embryos was 99.7%. While the literature demonstrates higher live birth rates with the use of PGT-A in women of advancing age, this is inconsequential if there is no embryo available to transfer. Women over 40years with less than four 2pn zygotes should consider transfer of one or more untested embryos either on day 3 or on day 5.

A short length of gonadotropin hyperstimulation is sufficient to achieve an optimal IVF outcome in advance-aged women

ObjectiveTo analyze the duration of gonadotropin hyperstimulation's impacts on oocyte quality and clinical outcomes in aged in vitro fertilization (IVF) patients. Materials and methodsThis retrospective study was carried out using IVF records of the Chang Gung Memorial Hospital IVF center from January 2017 to December 2019. A total of 308 IVF cycles with patients aged 40–44 years were included. Clinical characteristics of patients who received a short controlled ovarian hyperstimulation (COH) (i.e., 6–7 days; s-COH group) or a long COH treatment (i.e., 9–10 days; l-COH group) were compared. In addition, analysis was conducted using data within two age subgroups: 40–42 years and 42–44 years subgroups. ResultsThe s-COH group received significantly lower total doses of gonadotropin and had smaller leading follicles at the time of ovulation trigger when compared to the l-COH group. The s-COH group also produced a significantly lower number of oocytes, mature metaphase II (MII) oocytes, and 2 PN zygotes compared to the l-COH group. However, there was no significant difference in the number of transferable and good-quality embryos between the two treatment groups. Likewise, the pregnancy rate and live birth rate were comparable in the s-COH and l-COH groups. Similar results were obtained when the analysis was limited to select age subgroups (i.e., 40–42 and 42–44 years subgroups). ConclusionWhile a long COH generates more oocytes per cycle, a 6–7 days COH treatment, which is at the lower end of the recommended window of stimulation, could achieve a pregnancy outcome comparable to that applied 9–10 days of COH in aged patients.

Blastocyst Prediction of Day-3 Cleavage-Stage Embryos Using Machine Learning

Background: Embryo selection plays an important role in the success of in vitro fertilization (IVF). However, morphological embryo assessment has a number of limitations, including the time required, lack of accuracy, and inconsistency. This study determined whether a machine learning-based model could predict blastocyst formation using day-3 embryo images. Methods: Day-3 embryo images from IVF/intracytoplasmic sperm injection (ICSI) cycles performed at My Duc Phu Nhuan Hospital between August 2018 and June 2019 were retrospectively analyzed to inform model development. Day-3 embryo images derived from two-pronuclear (2PN) zygotes with known blastocyst formation data were extracted from the CCM-iBIS time-lapse incubator (Astec, Japan) at 67 hours post ICSI, and labeled as blastocyst/non-blastocyst based on results at 116 hours post ICSI. Images were used as the input dataset to train (85%) and validate (15%) the convolutional neural network (CNN) model, then model accuracy was determined using the training and validation dataset. The performance of 13 experienced embryologists for predicting blastocyst formation based on 100 day-3 embryo images was also evaluated. Results: A total of 1,135 images were allocated into training ([Formula: see text] 967) and validation ([Formula: see text] 168) sets, with an even distribution for blastocyst formation outcome. The accuracy of the final model for blastocyst formation was 97.72% in the training dataset and 76.19% in the validation dataset. The final model predicted blastocyst formation from day-3 embryo images in the validation dataset with an area under the curve of 0.75 (95% confidence interval [CI] 0.69–0.81). Embryologists predicted blastocyst formation with the accuracy of 70.07% (95% CI 68.12%–72.03%), sensitivity of 87.04% (95% CI 82.56%–91.52%), and specificity of 30.93% (95% CI 29.35%–32.51%). Conclusions: The CNN-based machine learning model using day-3 embryo images predicted blastocyst formation more accurately than experienced embryologists. The CNN-based model is a potential tool to predict additional IVF outcomes.

Dynamic Oxygen Conditions Promote the Translocation of HIF-1α to the Nucleus in Mouse Blastocysts.

Oxygen tension is one of the most critical factors for mammalian embryo development and its survival. The HIF protein is an essential transcription factor that activated under hypoxic conditions. In this study, we evaluated the effect of dynamic oxygen conditions on the expression of embryonic genes and translocation of hypoxia-inducible factor-1α (HIF-1α) in cultured mouse blastocysts. Two-pronuclear (2PN) zygotes harvested from ICR mice were subjected to either high oxygen (HO; 20%), low oxygen (LO; 5%), or dynamic oxygen (DO; 5% to 2%) conditions. In the DO group, PN zygotes were cultured in 5% O2 from days 1 to 3 and then in 2% O2 till day 5 after hCG injection. On day 5, the percentage of blastocysts in the cultured embryos from each group was estimated, and the embryos were also subjected to immunocytochemical and gene expression analysis. We found that the percentage of blastocysts was similar among the experimental groups; however, the percentage of hatching blastocysts in the DO and LO groups was significantly higher than that in the HO group. The total cell number of blastocysts in the DO group was significantly higher than that of both the HO and LO groups. Further, gene expression analysis revealed that the expression of genes related to the embryonic development was significantly higher in the DO group than that in the HO and LO groups. Interestingly, HIF-1α mRNA expression did not significantly differ; however, HIF-1α protein translocation from the cytoplasm to the nucleus was significantly higher in the DO group than in the HO and LO groups. Our study suggests that dynamic oxygen concentrations increase the developmental capacity in mouse preimplantation embryos through activation of the potent transcription factor HIF-1α.

CLINICAL BENEFITS OF CULTURING INTRACYTOPLASMIC SPERM INJECTION-DERIVED NONPRONUCLEATED (0PN) AND MONOPRONUCLEATED (1PN) ZYGOTES, AND THE IMPORTANCE OF VERIFYING BIPARENTAL CHROMOSOME INHERITANCE IN 1PN-DERIVED BLASTOCYSTS

Evaluate the developmental competence and aneuploidy status of blastocysts derived from 0PN and 1PN zygotes following intracytoplasmic sperm injection (ICSI) and preimplantation genetic testing for aneuploidy (PGT-A). Elucidate biparental chromosome inheritance in female 1PN-derived blastocysts.

A FALSE START: CAN SINGLE PRONUCLEAR ZYGOTES GET BACK ON TRACK?

Single pronuclear (1PN) zygotes have the potential to develop into blastocysts and lead to live birth.1 We investigated whether the blastocyst formation rate was different in embryos with 1 versus 2 pronuclei observed at standard fertilization time point, approximately 18-20 hours post insemination.

P–521 Association between maternal age and euploid blastocyst availability in cycles with less than four two-pronucleate zygotes

Abstract Study question For patients with less than four two-pronucleate (2pn) zygotes, is there an age-cutoff above which preimplantation genetic diagnosis for aneuploidy (PGT-A) is futile? Summary answer Women over 40y with less than four 2pn zygotes should consider transfer of a day 3 embryo over culture to blastocyst with PGT-A. What is known already During a typical IVF cycle, there is unavoidable attrition from oocytes retrieved, to embryos obtained, to blastocysts formed such that some patients, particularly those with advanced age or poor ovarian response, may not have blastocysts available to biopsy. While randomized trials have shown improved pregnancy rates with the use of PGT-A in patients of advancing age, these trials primarily included patients with good ovarian reserve and multiple blastocysts available. The optimal age group within poor responders who would benefit most from PGT-A has yet to be determined. Study design, size, duration This was a retrospective cohort study of all fresh autologous IVF or IVF/ICSI cycles in which PGT-A was planned from 1/2012 to 3/2020. Only patients with less than four 2pn zygotes were included. A total of 85 cycles from 75 patients were analyzed. Participants/materials, setting, methods Number of cleavage-stage embryos, blastocysts, biopsy-quality blastocysts and euploid embryos were assessed, after stratification by age. Adjusted relative risks (aRR) and 95% confidence intervals (CI) were calculated adjusting for BMI, AMH, FSH, stimulation protocol, and ICSI. Poisson regression was used for counts. Generalized estimating equations were used to account for patients contributing multiple cycles. Main results and the role of chance There were no differences in number of 2pn zygotes (p = 0.98) or cleavage stage embryos (p = 0.94) across age groups. Patients aged 41–42y had a significantly lower number of blastocysts (1.18 vs. 2.00; aRR 0.59 95%CI: 0.37–0.95) and biopsy-quality blastocysts (0.73 vs. 1.53; aRR 0.50 95% CI: 0.26–0.98) compared to patients &amp;lt;35y.These patients also had fewer euploid embryos available (0.09 vs 0.67), although the difference was not significant in the adjusted model (aRR 0.14 95% CI: 0.01–1.57). None of the patients &amp;gt;42y had euploid blastocysts. When considering the mean and three standard deviations (0.09 [SD 0.3]), 99.7% of patients over 40y have no euploid embryo available for transfer. Limitations, reasons for caution This study was retrospective in nature and limited by small sample sizes when patients were stratified by age. A prospective randomized trial of patients with less than four 2pn zygotes to day 3 fresh embryo transfer vs PGT-A frozen embryo transfer is needed to confirm these findings. Wider implications of the findings: Patients over 40y with less than four 2pn zygotes are at high risk of having no euploid blastocysts. While the literature demonstrates higher live birth rates with the use of PGT-A in women of advancing age, this is inconsequential if there is no embryo available to transfer. Trial registration number Not applicable

P–784 Neonatal follow-up of babies born derived from mono-pronuclear zygotes

Abstract Study question Are the neonatal outcomes normal of babies derived from the transfer of blastocysts derived from mono-pronuclear(1PN) zygotes? Summary answer There was no effect on growth or increase in congenital anomalies up to 18-months in babies of 1PN-derived births. What is known already 1PN zygotes are observed in ART, albeit at a low rate. We have previously reported that 80.7% of 1PN zygotes derived from IVF or ICSI had a biparental chromosome using Live Cell imaging techniques, and some of these developed to the blastocyst stage (Tokoro et al. ASRM 2013). Furthermore, we have reported that these blastocysts can result in a viable pregnancy and healthy live birth (Tsuji et al. ASRM2020). However, there is some uncertainty about the developmental mechanism of 1PN zygotes, and there is no clear consensus on their clinical utility. Study design, size, duration This was a retrospective study which included 55 cases where there was a live birth after single embryo transfer of a blastocyst derived from 1PN zygote. The incidence of birth defects, birth weight was recorded as well as a physical development survey of 25 children who responded to the 18-months follow-up survey. The time period was 72 months (January 2013 to December 2018). Participants/materials, setting, methods Patients seeking fertility treatment at an established private IVF clinic. We compared the birth weight, birth after18-months height and weight of children born to 1PN zygotes with data from a control, 2PN group. Statistical significance was determined using the t-test (level of P &amp;lt; 0.05). Main results and the role of chance The incidence of birth defects in 1PN embryo-derived infants was 1.8% (1/55). The average birth weight of boys in the 1PN group was 3105.6+/–360.3g, which was not significantly different from 3041.0+/–443.3 g in the 2PN group. In girls, the average birth weight was 3085.7+/–454.9 g in the 1PN group, which was not significantly different from the 2PN group (2938.9+/–311.5 g). The average height at 18-months, was 81.6+/–2.5 cm vs 80.5+/–3.4 cm for boys; 79.0+/–1.8 cm vs 79.0+/–3.4 cm for girls in the 1PN and 2PN groups, respectively. The average body weights of the 1PN and 2PN groups were 11.1+/–1.1 kg vs 10.7+/–1.1 kg for boys; 9.7+/–0.9 kg vs 10.1+/–1.0 kg for girls, respectively. There was no significant difference in average height and weight up-to the 18-months follow-up survey. Limitations, reasons for caution The incidence of 1PN derived births is low and the study was limited to cases of single blastocyst embryo transfer. Wider implications of the findings: The incidence of congenital anomalies in Japan was around 1.7 to 2%, and the incidence was similar in the 1PN. There was no difference in the birth weight and 18-months follow-up survey of the 1PN compared with the 2PN. We have demonstrated that there is clinical utility of 1PN embryo. Trial registration number Not applicable

P–133 Monopronuclear (1PN) embryos can derive in healthy pregnancies

Abstract Study question Should 1PN embryos be considered suitable for transfer when normal development is observed at day 3 or day 5? Summary answer In IVF/ICSI cycles, 1PN zygotes are encountered in 2.7% of inseminated oocytes. Transfer of 1PN-embryos should be considered in the absence of suitable 2PN embryos. What is known already During in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) zygotes containing only a single pronucleus (monopronuclear, 1PN) are encountered in 1–7.7% of cases, while the display of two pronuclei is expected in a normally fertilised oocyte. A 1PN zygote can be of gynogenetic or androgenetic origin, but it can also be biparental. Gynogenetic and androgenetic 1PN embryos can be haploid or diploid, so a diploid 1PN embryo is not guaranteed to be normally fertilised. Generally, 1PN are discarded, as they have an increased risk for aneuploidy. However, sporadically they can develop into healthyl babies. Study design, size, duration 1PN-zygotes (n = 1287, 2.7% from all inseminated oocytes) from 1–1–2016 up to 15–12–2020 were retrospectively evaluated. The development and fate (discarded/transferred/cryopreserved) of all embryos were recorded. Embryos were evaluated at day 2, 3 or 5 of development. The policy of our unit is that, in absence of 2PN embryos, normal developed 1PN-embryos can be transferred on day 3. Supernumerary 1PN embryos can be cryopreserved at blastocyst stage. Ongoing pregnancies from fresh embryo transfers (ET) were analysed. Participants/materials, setting, methods In 946 IVF/ICSI cycles, at least one 1PN zygote was observed (total 1287 embryos). ICSI with ejaculated, PESA or TESE sperm counted for a total of 795 embryos, IVF cycles for 494 embryos. Embryo evaluation was performed using a home-made numerical algorithm: A (top embryo; 150–200 points), B (regular embryo; 100–149 points) or C (poor embryo; 0–99 points). Monopronuclear embryos always scored lower than equal developed 2PN embryos. Blastocyst evaluation was according to Gardner score. Main results and the role of chance From the 795 ICSI embryos, 49 (6.1%) were used for fresh ET (26 scored quality A or B), and a total of 60 embryos developed to blastocyst and were cryopreserved. From these 49 ICSI transfers, 4 (8.1%) ongoing pregnancies were obtained, all 4 from DET (1PN+2PN embryo), from which one twin pregnancy was confirmed. From the 494 IVF embryos, 41 (8.3%) were used for fresh ET (24 scored A or B), and 62 blastocysts were cryopreserved. A total of 9/41 (22%) ongoing pregnancies were obtained: 5 from SET (1PN) and 4 from DET (1PN+ 2PN embryo). Therefore, in only five IVF cycles a confirmed pregnancy was observed from a 1PN embryo (all A-quality embryos). Considering six ongoing pregnancies with complete certainty of monopronuclear origin from fresh tranfers could be confirmed from our retrospective data, we can conclude that although the live birth rate of these embryos is very low (around 0,5- 1.0%), they should not be discarded when development is normal and no dipronuclear embryos are present. Limitations, reasons for caution Cryo-thawing data is missing as these embryos were not differentially marked at freezing. Therefore, the cumulative pregnancies from monopronuclear embryos could be higher. Embryos were not evaluated in a time lapse system, so asynchronicity of PN formation could explain missing the right moment for evaluation, while normal fertilized. Wider implications of the findings: Notably, IVF monopronuclear embryos display a higher developmental potential than those derived from ICSI. We suggest that, in absence of dipronuclear embryos, culture to blastocyst stage before considering fresh ET or cryopreservation will help differentiate viable 1PN embryos, reducing the higher chance of genetic anomalies and miscarriages. Trial registration number N.A.

P–257 An unknown cause lead to polyspermy in IVF cycles and 0PN zygotes in ICSI cycles in male patient

Abstract Study question The patient sperm has normal morphology and motility, which paternal factors cause the abnormal fertilization in IVF/ICSI and what is the underlying mechanism? Summary answer A genetic mutation of BEX1 and decreased PLC-zeta has been found in patient, which may provide novel insights of polyspermy and pronucleus formation during fertilization. What is known already In mammals, pronucleus formation, a landmark event for fertilization, is critical for embryonic development. Abnormal fertilization refers to the abnormal number of pronucleus and polar bodies in zygotes during in vitro fertilization, with an incidence of 5–15%, among which the incidence of polyspermy and 0PN is about 2–10% and 30%. However, the mechanisms underlying pronucleus formation still unclear. More research has focused on oocyte activation, while paternal relevant abnormal fertilizations have been rarely established. The mechanism of how sperm and/or substances carried by sperm influence the physiological process of fertilization is also unclear. Study design, size, duration In our study, we first work on the preliminary observation and analysis of sperm morphology, structure and sperm chromosome number, and then made further analysis at the genetic level to find out the cause of this particular phenotype in this patient. We use of zone-free golden hamster ova test the fertilizing capacity and rescue the pronucleus formation with SrCl2. Participants/materials, setting, methods The patient, golden hamster, Papanicolaou stain, scanning electron microscope (SEM), Transmission Electron Microscope (TEM), Fluorescence in situ hybridization (FISH), Whole Exome Sequencing (WES), IVF, ICSI, Assisted Oocyte Activation (AOA). Main results and the role of chance During 2016–2018, they did 4 cycle assistant reproduction technology. Cycle1, conventional IVF(C-IVF), 9 MII oocytes, 9 3PN zygotes; Cycle2, ICSI, 10 MII oocytes, 10 0PN zygotes; Cycle3, donor-oocytes C-IVF, 6 MII oocytes, 6 3PN zygotes, and the donor did C-IVF get normal zygotes and embryos; Cycle4, donor-sperm C-IVF, 7 MII oocytes, 4 2PN zygotes, 3 useable embryos. Remarkably, clinical examination about male shows normal sperm semen parameters. Papanicolaou stain and SEM shows that the sperm of the patient has normal morphology. The TEM data shows that the spermatozoa with normal head morphology and intact 9 + 2 sperm flagella structure. In the sperm FISH analysis, Chromosome ploidy is haploid. We performed WES on the male, after exclusion of frequent variants and application of technical and biological filters, two homozygous missense mutations were identified in BEX1 (c.191G&amp;gt;A [p. W64X]), which has been few reports of male infertility. The western blot result show that the PLC-zeta was decreased in patient. After 10mM SrCl2 assisted oocyte activation, the zygote has the pronucleus formation in ICSI. Limitations, reasons for caution At present, we only observe sperm related factors (morphology, structure, chromosome number, genetic mutation). Next step is to detect the substances sperm carried (e.g. RNA-seq, proteomics). In this case, what is of great concern to us is the inconsistencies of the abnormal fertilization during the conventional IVF and ICSI cycles. Wider implications of the findings: Many studies of fertilization mechanism, the main focus is on the maternal cytoplasmic factors, such as the Ca 2+ release initiate the fast block of oocytes. There are few reports about abnormal fertilization due to sperm factors. Our case may offer new insights for the study of fertilization. Trial registration number Not applicable

O-154 First mitotic spindle formation led by sperm centrosome-dependent microtubule organising centres may cause high incidence of zygotic division errors in humans

Abstract Study question Why do multinucleated blastomeres appear at high frequency in two-cell-stage embryos in humans? Summary answer Failure in microtubule assembly during the first mitotic spindle body formation by sperm centrosome-dependent microtubule organising centres (MTOCs) may lead to chromosomal instability. What is known already Unlike that in mice, multinucleated blastomeres appear at high frequency in two-cell-stage embryos in humans. However, the underlying mechanism remains elusive. In mice, multiple acentriolar MTOCs appear around the male and female pronuclei after pronuclear disappearance and contribute to dual-spindle formation, engulfing each parental chromosome. This spindle formation may ensure an error-free division, keeping the chromosomes stable during the first cleavage, as observed in mice, but it is unclear whether a similar mechanism exists in humans. Study design, size, duration To examine how sperm centrosomes contribute to MTOC formation in humans, two types of 3PN zygotes derived fromeither conventional in vitro fertilization (c-IVF, n = 30) or intracytoplasmic sperm injection (ICSI, n = 10) were used. The zygotes were collected from October 2018 to January 2020. MTOC and mitotic spindle formation at consecutive stages of development during the first cleavage were analysed under static and dynamic conditions using immunofluorescence assay and fluorescent live-cell imaging. Participants/materials, setting, methods Under ethics approval, 3PN zygotes were donated by infertile couples undergoing c-IVF or ICSI cycles at the Yamashita Shonan Yume Clinic in Japan. All participants provided informed consent. Immunofluorescence assay was performed using antibodies against α-tubulin, pericentrin, and H3K9me3 after fixation with MTSB-XF solution. Fluorescent live-cell imaging was performed using TagGFP2-H2B mRNA (chromosome marker) and FusionRed-MAP4 mRNA (microtubule marker). Main results and the role of chance Immunofluorescence revealed that while 3PN zygotes derived from c-IVF showed four pericentrin dots, those derived from ICSI exhibited two pericentrin dots. In pro-metaphase, an independent group of chromosomes derived from each pronucleus and MTOCs were formed by the sperm centrosome at the core. Microtubules from each MTOC extended toward the chromosomes in the early metaphase; a quadrupolar spindle was formed in the c-IVF-derived zygotes, and a bipolar spindle was formed in the ICSI-derived zygotes by the MTOCs at the zygote apex after chromosome alignment. In pro-metaphase, the microtubules extended from the MTOCs to the nearest chromosome. Since microtubule assembly was found on oocyte-derived chromosomes, we hypothesised that whether a chromosome is surrounded by microtubules depends on the location of the MTOCs, irrespective of its origin. Live-cell imaging of histone H2B and MAP4 revealed that four MTOCs appeared around the three pronuclei just before the disappearance of the pronuclear membrane; microtubules then extended from the MTOCs toward the chromosomes, beginning to form a mitotic spindle as the chromosomes moved to the centre of the oocyte. Interestingly, one of the three assembled chromosome groups showed no microtubule assembly in the pro-metaphase. Similar results were obtained in all six 3PN zygotes subjected. Limitations, reasons for caution We demonstrated the high risk of developing bare chromosomes not surrounded by microtubules during the formation of the first mitotic spindle, using human tripronuclear zygotes. However, owing to unavailability of normal fertilized oocytes for this study because of the clinical use, we were unable to confirm this in normal zygotes. Wider implications of the findings Although two sperm centrosome-dependent MTOCs are expected to be formed in normal fertilized oocytes, these MTOCs are not sufficient to completely enclose physically separated female and male chromosomes with the microtubules. This explains the high frequency of zygotic division errors that lead to unstable human chromosomes. Trial registration number not applicable

P–201 The beneficial effects of ZP-free culture on cytoplasmic fragmentation in human embryos. : An innovative trial using 3PN zygotes

Abstract Study question Is it possible to culture ZP-free embryos to eliminate perivitelline threads, which are known to be involved in generating cytoplasmic fragments at the first cleavage? Summary answer ZP-free culturing, an innovative system that decreases the amount of cytoplasmic fragments without disrupting the blastomeres, using incubators with time-lapse imaging. What is known already A study in 2017 observed perivitelline threads in more than 50% of cleavage-stage human embryos using time-lapse imaging, and the rate of cytoplasmic fragmentation (at the first cleavage) was significantly decreased in embryos without perivitelline threads (P &amp;lt; 0.001). While it has been proposed that perivitelline threads play an important role in crosslinking the cumulus cells and oocyte during maturation, the mechanism underlying such a role remains unclear. It is also unknown whether the threads still function in mature MII oocytes. Study design, size, duration A prospective study was conducted using 2,852 normal (2PN/2PB) embryos from c-IVF/ICSI and 113 abnormal (3PN) embryos obtained from c-IVF between 2017 and 2019. The zona pellucida (ZP) of 71 abnormal embryos was removed at the pronuclear stage (“ZP-free”), and the rest (n = 42) were cultured as “ZP-intact”. Normal and abnormal embryos were cultured for five days in bench-top incubators (MINC, COOK) and an incubator equipped with a time-lapse imaging system. Participants/materials, setting, methods Embryos used in this study were donated by 412 couples who underwent c-IVF cycles in our clinic between 2017 and 2019. For ZP removal, 3PN embryos were placed in 0.125M sucrose-containing HEPES media drops to reduce the ooplasm size. Then, ooplasms were completely separated from ZPs by a laser and pipetting. Embryo development and morphology of the three groups (normal, ZP-intact and ZP-free abnormal) were compared based on the degree of cytoplasmic fragmentation. Main results and the role of chance The first cleavage occurred in 97.8% (n = 2,790/2,852) of 2PN/2PB, 83.3% (n = 35/42) of ZP-intact 3PN and 97.2% (n = 69/71) of ZP-free 3PN. Normal (2PN/2PB), ZP-intact and ZP-free 3PN embryos were classified into three groups based on the modified Veeck’s criteria thus: &amp;lt;20% fragmented compared to the total volume of cytoplasm at the first cleavage (Grade 1 and 2, Good); 20–39% fragmented (Grade 3, Fair) and ≧40% fragmented (Grade 4, Poor). Of 69 cleaved ZP-free 3PN embryos, 68.1% (n = 47) showed less than 20% fragments which was significantly higher than 2PN/2PB (43.7%, n = 1,218/2,790) and ZP-intact 3PN (45.7%, n = 16/35; P &amp;lt; 0.05). Furthermore, 24.6% (n = 17/69) of ZP-free 3PN embryos showed 20–39% fragments which was significantly lower than 2PN/2PB (45.9%, n = 1,281/2,790; P &amp;lt; 0.05). In addition, 50.7% of ZP-free 3PN embryos (n = 36) developed to the morula stage after the third cleavage, and 29.6% (n = 21) formed blastocoel and became blastocysts. Thus, removing the ZP before the first cleavage did not adversely affect embryo development and decreased the cytoplasmic fragmentation. Limitations, reasons for caution Due to ethical and clinical limitations, we only examined abnormally fertilized embryos in this study. Moreover, since the relationship between the perivitelline threads and cytoplasmic fragments is unclear, we plan to conduct molecular biological analysis of the perivitelline threads in further studies. Wider implications of the findings: This study revealed that ZP is not always necessary after the pronuclear stage because ZP-free embryos studied herein developed normally and maintained cell adhesion well. This innovative culture method might provide the breakthrough needed for patients to improve embryo quality who obtain embryos with severe fragmentation caused by perivitelline threads. Trial registration number Not applicable

P–185 Overcoming a sudden COVID–19 lockdown and other similar unforeseen adverse events: Oocyte-sperm cryopreservation 4 hours after conventional in vitro insemination

Study questionCan human oocytes be cyropreserved when they were previously inseminated while obtaining normal fertilization, embryo development and pregnancies?Summary answerCryopreservation of recently inseminated human oocytes produces normal 2PN zygotes, viable embryos and ongoing pregnancies.What is known alreadyOocyte vitrification is a well-established technique in the infertility field supported by its effectiveness to freeze and thaw embryos at various stages of development. However, it is yet unknown whether cryopreservation performed in a period between conventional in vitro insemination and the appearance of pronuclei, affects any of the steps leading to fertilization, thereby arresting this process.Study design, size, durationCase report that includes 5 IVF cycles where cryopreservation and thawing were performed in a pre-zygote stage, 4 hours after the conventional insemination of oocytes. The rationale for this procedure was to comply with a government restriction to circulate and work in order to prevent the spread of the SARS-CoV–2 virus, unexpectedly 3 days ahead (effective on 03/19/2020). Participants/materials, setting, methods: Off-site embryologists cancelled all pending embryo transfers and returned home (2,000 km away) before the stay-home quarantine. Five couples were informed and consent was obtained on the urgent need to cryopreserve their already inseminated oocytes despite of the limited scientific information. We vitrified 28 oocyte-sperm pairs in vitrification devices containing 2 to 3 inseminated oocytes each.Main results and the role of chanceEight months later all non-essential activities, including non-urgent procedures in hospital facilities, were permitted again, allowing embryologists to travel and resume their postponed work. We thawed 19 of the so-called “pre-zygotes” corresponding to 4 of the 5 initial couples. Female ages were 29, 39, 38 and 37 years old. Our primary outcomes were detection of pronuclei and the occurrence of fertilization. The secondary outcome was pregnancy success. On day 1, we observed the presence of 2PN in 16 oocytes (84%). Only one patient with 2 oocytes failed to fertilize. We transferred 2 good-to-fair-grade cleaved embryos to each of the 3 patients, obtaining one pregnancy with a positive fetal heartbeat from the 39 years old patient. Two of the couples were also able to cryopreserve 2 and 1 surplus embryos on day 5, respectively.Limitations, reasons for cautionOur strategy was used exclusively to overcome an unexpected adverse event while complying with State and County regulations on COVID–19. As a case report, a large sample size is needed to confirm and extend these results including the putative detrimental effects of arresting the fertilization process through vitrification.Wider implications of the findings: Although cryopreservation implies to arrest of any stage of a development continuum, to the best of our knowledge, this is the first time that both fertilization and pregnancy were achieved after halting the fertilization process long before the appearance of pronuclei (at ∼4 hours after insemination).Trial registration numberNot applicable

Nuclear-to-cytoplasmic ratios of 1PN and 2PN zygotes after in vitro fertilization of mouse oocytes.

Numerous studies have reported comparisons of the nuclear-to-cytoplasmic (NC) ratio during mitosis. However, little information is known about how the pronuclear size is regulated and determined at the end of meiosis II in mammalian zygotes. The present study aims to analyze the NC ratio of female and male pronuclei, and also to compare the size of single pronuclei using photographs that were obtained during experiments to create chimeric hermaphrodites from 2-cell oocytes. The volume of both the female and the male pronucleus was found to correlate with the volume of the oocyte cytoplasm. The NC ratio of the male pronucleus was greater than that of the female pronucleus. The NC ratio of the average volume of the female and male pronuclei was greater than the NC ratio of the mononucleate oocytes. The occurrence of 1PN oocytes was significantly higher when the volume of cytoplasm was lower than the cut-off value. These results indicated that the NC ratio is retained during pronuclear formation. A higher NC ratio in male compared with the female pronucleus indicated structural and/or molecular difference between the two pronuclei. 1PN formation may occur when sperm enters close to the MII spindle.