Time-lapse Imaging System Research Articles

The TLI system can help day-3 single cleavage embryo transfer to obtain comparative clinical outcomes to day-4 or day-5.

The aim was to explore whether the time-lapse imaging system can help day-3 single cleavage embryo transfer to obtain comparative clinical outcomes to day-4 or 5. The data of 1237 patients who underwent single embryo transfer from January 1, 2018, to September 30, 2020, in our reproductive medicine centre were retrospectively analysed. They were divided into the day-3 single cleavage-stage embryo transfer (SCT) group (n = 357), day-4 single morula transfer (SMT) group (n = 129) and day-5 single blastocyst transfer (SBT) group (n = 751) according to the different embryo transfer stage. The clinical and perinatal outcomes of the three groups were analysed and compared. The clinical pregnancy rates of the patients in the day-3 SCT group, day-4 SMT group and day-5 SBT group were 68.07, 70.54 and 72.04%, respectively. The live birth rates were 56.86, 61.24 and 60.99%, respectively. The monozygotic twin (MZT) rate in the day-3 SCT group was significantly lower than that in the day-5 SBT group (P = 0.049). Regarding perinatal outcomes, only the secondary sex ratio had a significant difference (P < 0.05). After age stratification, no improvement was found in the pregnancy outcomes of patients >35 years of age receiving blastocyst transfer. Our findings suggest that for patients with multiple high-quality embryos on day-3, prolonging the culture time can improve the pregnancy outcome to some extent, but it will bring risks. For centres that have established morphodynamic models, day-3 SCT can also achieve an ideal pregnancy outcome and reduce the rate of monozygotic twins and sex ratio.

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

Time-lapse imaging systems for embryo incubation and selection might improve outcomes of in-vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) treatment due to undisturbed embryo culture conditions, improved embryo selection, or both. However, the benefit remains uncertain. We aimed to evaluate the effectiveness of time-lapse imaging systems providing undisturbed culture and embryo selection, and time-lapse imaging systems providing only undisturbed culture, and compared each with standard care without time-lapse imaging. We conducted a multicentre, three-parallel-group, double-blind, randomised controlled trial in participants undergoing IVF or ICSI at seven IVF centres in the UK and Hong Kong. Embryologists randomly assigned participants using a web-based system, stratified by clinic in a 1:1:1 ratio to the time-lapse imaging system for undisturbed culture and embryo selection (time-lapse imaging group), time-lapse imaging system for undisturbed culture alone (undisturbed culture group), and standard care without time-lapse imaging (control group). Women were required to be aged 18-42 years and men (ie, their partners) 18 years or older. Couples had to be receiving their first, second, or third IVF or ICSI treatment and could not participate if using donor gametes. Participants and trial staff were masked to group assignment, embryologists were not. The primary outcome was live birth. We performed analyses using the intention-to-treat principle and reported the main analysis in participants with primary outcome data available (full analysis set). The trial is registered on the International Trials Registry (ISRCTN17792989) and is now closed. 1575 participants were randomly assigned to treatment groups (525 participants per group) between June 21, 2018, and Sept 30, 2022. The live birth rates were 33·7% (175/520) in the time-lapse imaging group, 36·6% (189/516) in the undisturbed culture group, and 33·0% (172/522) in the standard care group. The adjusted odds ratio was 1·04 (97·5% CI 0·73 to 1·47) for time-lapse imaging arm versus control and 1·20 (0·85 to 1·70) for undisturbed culture versus control. The risk reduction for the absolute difference was 0·7 percentage points (97·5% CI -5·85 to 7·25) between the time-lapse imaging and standard care groups and 3·6 percentage points (-3·02 to 10·22) between the undisturbed culture and standard care groups. 79 serious adverse events unrelated to the trial were reported (n=28 in time-lapse imaging, n=27 in undisturbed culture, and n=24 in standard care). In women undergoing IVF or ICSI treatment, the use of time-lapse imaging systems for embryo culture and selection does not significantly increase the odds of live birth compared with standard care without time-lapse imaging. Barts Charity, Pharmasure Pharmaceuticals, Hong Kong OG Trust Fund, Hong Kong Health and Medical Research Fund, Hong Kong Matching Fund.

O-319 A randomised trial of time-lapse imaging of embryos in in-vitro-fertilisation treatment

Abstract Study question Do time-lapse imaging (TLI) systems used in IVF/ICSI treatment improve live birth either due to undisturbed culture conditions or improved embryo selection or both? Summary answer In women undergoing IVF/ICSI treatment the use of TLI systems does not significantly increase the odds of live birth compared to standard care without TLI. What is known already TLI systems used in IVF treatment have the potential to improve fertility outcomes either due to undisturbed culture conditions or improved embryo selection or both. However, there remains uncertainty about its benefits. Existing reviews and primary studies predominantly report no differences between the use of TLI and conventional methods. However none of the previous trials were adequaltely powered to detect meaningful differences in live birth and the quality of most trials was deemed to be low to very low. Study design, size, duration TILT (Time-lapse Imaging Trial) is a multi-centre, three-arm, parallel-group, double-blind, randomised controlled trial. Participants were randomly assigned in a 1:1:1 ratio to the TLI system for both undisturbed culture and embryo selection (TLI arm), TLI system for undisturbed culture conditions alone (undisturbed culture arm), and standard care without TLI (control arm). From June 2018 to September 2022, we randomised 1575 participants based on a pre-specified sample size to detect an 8.75% difference in live births. Participants/materials, setting, methods We randomised 1575 couples undergoing IVF/ICSI with broad inclusion criteria across seven IVF centres. The primary outcome was live birth. Secondary outcomes included clinical pregnancy, pregnancy loss, and multiple pregnancy. Primary analysis was performed based on the intention-to-treat principle for all participants with primary outcome data available and the effect estimate reported as an adjusted odds ratio (aOR). Pre-specified sensitivity analyses and subgroup analyses for age and method of embryo transfer were performed. Main results and the role of chance We recruited 2191 and randomised 1575 participants. Live birth data were available for 1558/1575 (98.92%) participants. The livebirth rates were 33.7% (175/520) in the TLI arm, 36.6% (189/516) in the undisturbed culture arm and 33.0% (172/522) in the control arm. The aOR was 1.04 (97.5% confidence interval (CI) 0.73 to 1.47) for TLI arm vs control and 1.20 (97.5% CI 0.85 to 1.70) for undisturbed culture vs control. The clinical pregnancy rates were 42.2% (219/519) in the TLI arm, 43.4% (225/518) in the undisturbed culture arm, and 40.9% (212/519) in the control arm. There were no significant differences for TLI vs control arm (aOR 1.06, 95% CI 0.82-1.38) and undisturbed culture vs control arm (aOR 1.11, 95% CI 0.86-1.46). The rates of pregnancy loss between clinical pregnancy and 24 weeks’ gestation were not statistically different when TLI (8.7%, 45/515) and undisturbed culture arms (6.4%, 33/513) were compared with control arm (8.1%, 42/517) with aOR 1.09 (95% CI 0.66-1.77) and 0.76 (95% CI 0.45-1.29) respectively. None of the other secondary outcomes demonstrated significant differences between the arms The findings were similar irrespective of the woman’s age and use of fresh or frozen embryo replacement, and for all sensitivity analyses, for primary and secondary outcomes. Limitations, reasons for caution Although the trial recruitment was delayed by the SARS-CoV-2 pandemic, this did not impact the results as patient populations, clinical care pathways and time-lapse technology remained similar. The remit of this trial did not allow assessment of the influence of TLI systems on embryology laboratory workflow or perform a health-economic analysis. Wider implications of the findings Our trial provides high-quality definitive evidence of no benefit of the use of TLI systems for embryo incubation and selection to improve success rates in women having IVF and ICSI treatment. Trial registration number ISRCTN17792989

#23 : Correlation of Multinucleated Embryo and Pregnancy Outcome

Background and Aims: Single embryo transfer is increasingly done in practice to avoid multiple pregnancy. Therefore, it is important to ensure the embryo with the best pregnancy outcome is selected. Although new criteria involving genomic, transcriptomic, and proteomic have been developed to predict embryo outcome, morphology of the embryo remains as the main indicator because it is easy to implement and less controversial. To study the correlation of multinucleated embryos and pregnancy outcomes Method: This is a retrospective review of a total of 411 embryos from 225 women undergoing their first IVF or ICSI cycle in University Malaya Medical Centre, Kuala Lumpur between January 2019 and October 2021. Multinucleation was defined as the presence of more than one nucleus within at least one of the blastomeres of a 2-cell embryo and a time-lapse imaging system was used to observe embryo development (Embryoscope; Unisense Fertilitech). Group allocation was based on the presence or absence of multinucleation on day 2 after egg retrieval. Embryos with evidence of one or more blastomere having multinucleation were included in the studied group (n = 67 embryos). Embryos without any blastomere exhibiting multinucleation were included in the control group (n = 344 embryos). Results: The data were based on 225 transfers of 411 embryos consisting of 16.3% (n = 67) from studied group and 83.7% (n = 344) from the control group. The biochemical pregnancy rate and implantation rate is lower in the studied group compared to the control group although not statistically significant (Table 1). Conclusion: Multinucleated embryos reduce the implantation rate and clinical pregnancy rate although not statistically significant.

P-254 Short-time or overnight co-incubation with sperm in conventional IVF delivers similar pre-implantation development outcomes: a sibling cumulus-oocyte complex study

Abstract Study question Does a short-time co-incubation in conventional IVF improve rates of fertilization, embryo development and euploidy, when compared to overnight co-incubation? Summary answer There is no significant difference in fertilization, embryo development and euploidy rates between sibling oocytes subjected to short co-incubation versus overnight co-incubation. What is known already Conventional IVF usually implies coincubation of cumulus-oocyte complex(es) with sperm for an overnight period. This overnight incubation might create an over exposure of COCs to reactive oxygen species (ROS), which might disturb oocyte homeostasis, and impact on embryonic developmental rate. To this extend, many early studies have compared a short co-incubation time (2-6 hours) with overnight co-incubation, with reported conflicting outcomes. None of the studies combined time-lapse monitoring with short co-incubation, which allows the identification of early morphokinetic events of the inseminated oocytes after conventional IVF, as already known for ICSI oocytes in time-lapse incubators. Study design, size, duration This single center prospective study included 604 COCs in 38 IVF/ICSI cycles with preimplantation genetic testing between December 2020 and November 2022. After oocyte retrieval a min of 6 COCs were assigned for insemination by IVF and equally divided between short(2hrs) and overnight (16-20hrs) co-incubation. Supernumerary oocytes were denuded and inseminated by ICSI. Primary endpoint was fertilization rate; secondary endpoints were maturation rate, ploidy rate after trophectoderm biopsy, embryo development and morphokinetics. Participants/materials, setting, methods Couples with a female age between 18-43 (average:34.4) years, BMI ≤30kg/m2 and normal semen parameters (WHO) in the fresh ejaculate on the day of OR, were eligible for the study. After IVF, all embryos were cultured in a time-lapse imaging system in Global Total LP media. Blastocysts (≥Bl3BCC) were subjected to trophectoderm biopsy on day 5-7 and next generation sequencing (NGS) to determine blastocyst ploidy status. Main results and the role of chance In total, 288 oocytes (47.7%) were denuded for ICSI, whereas 316 (52.3%) oocytes were randomized 1:1 to short and overnight co-incubation groups. Maturation rates (M2/COC) and normal fertilization rates (2PN/COC) in short and overnight co-incubation groups were similar (88.0% vs 83.5%, P = 0.074 and 60.8% vs 62.0%, P = 0.193, respectively). Day-3 embryo cell numbers (P = 0.383), fragmentation rates (P = 0.529), blastulation rates on day 5 (39.9 vs 38.0% P = 0.696), time to blastulation (median: 112.6 vs 111.1 hours after insemination by time lapse, P = 0.544) were similar between short and overnight co-incubation groups. In total, 48 embryos in short and 45 embryos in overnight co-incubation groups were of sufficient quality for biopsy resulting in similar euploidy rates between the groups (39.6 vs 44.4%, P = 0.791). When compared to their siblings in ICSI, short (mean difference: -6.48%, 95% CI: -18.5 to 5.59%, P = 0.290) and overnight co-incubation (mean difference: -0.87%, 95% CI: -12.9 to 11.2%, P = 0.886) attained similar fertilization rates per cycle. No significant differences regarding blastulation and euploidy rates were observed in short or overnight co-incubation compared to ICSI in unadjusted and adjusted analyses. Limitations, reasons for caution This is a prospective study with a limited patient population with a normal ovarian response. Results might not be transferable to patients with high or low ovarian reserve. Wider implications of the findings The present study demonstrated that there is no difference between short and overnight co-incubation in terms of fertilization and euploidy rates. Therefore short co-incubation might allow us to benefit from early denudation, and to investigate early morphokinetics in time lapse monitoring, which are otherwise missed by overnight co-incubation. Trial registration number NCT04627545

P-734 Blastocysts derived from tripronuclear zygote including small pronuclear zygote have high diploid and euploid rate with NGS and SNP analysis.

Abstract Study question What is diploid and euploid frequency of blastocysts derived from tripronuclear zygote (3PN) including a small pronucleus (2.1PN) with NGS and SNP analysis? Summary answer Diploid rate of the blastocysts derived from 3PN including 2.1PN was 91.3%, in which euploid, aneuploid and mosaic embryos were 18.7%, 61.3% and 11.3%, respectively. What is known already A 3PN zygote is thought to be produced when two sperm enter into the same oocyte during in vitro fertilization (IVF) or whenthe second polar body is not extruded on both IVF and intracytoplasmic sperm injection (ICSI). It has been reported that 3PN is observed around 3-10%, and the embryos are usually discarded by the reason for genetic risks for triploid. However, some studies reported that blastocyst derived from 3PN and/or 2.1PN (defined as small PN &amp;lt; 20μm) contained normal diploid and euploid embryos by the preimplantation genetic testing, and that normal healthy babies were born from these embryos. Study design, size, duration We conducted a cross-sectional study between July 2019 and June 2022 in single reproduction center. We included 80 blastocysts in this study which generated from 3PN zygotes including 2.1PN zygotes. Of these, 71 blastocysts were from ICSI and 9 blastocysts were from IVF. These blastocysts were assessed for chromosomal ploidy by next generation sequencing (NGS) and single nucleotide polymorphism (SNP) analysis and embryonic development of these blastocysts were monitored by time-lapse imaging system. Participants/materials, setting, methods Average age of women was 39.2 years (22-47 years). The smallest pronuclear diameter who provided blastocysts were 12.7±2.8μm (8.5-19.7μm) in average. The blastocysts were divided between inner cell mass (ICM) and trophectoderm (TE) using laser system under inverted microscope, followed by NGS and SNP. The t-test was performed to analyze differences between triploid and diploid. Statistical significance was defined as P &amp;lt; 0.05. All participants provided written informed consent, and Institutional Review Board approval was obtained. Main results and the role of chance Diploid rate of the blastocysts derived from 3PN including 2.1PN was 91.3%, in which euploid, aneuploid and mosaic embryos were 18.7%, 61.3% and 11.3%, respectively. Of 80 blastocysts, 64 blastocysts (80%) were able to be divided ICM from TE. In terms of triploid and diploid, the results of ICM and TE were completely concordant, although 15.6% discordancy was observed between ICM and TE. Average ages of the patients with triploid, euploid, aneuploid, and mosaic embryos were 41.1±5.1 years, 36.0±6.3 years, 40.6±3.5 years, and 35.2±3.6 years, respectively. The patients’ age with aneuploid embryos was significantly higher than those with euploid and mosaic embryos (P = 0.015 and P &amp;lt; 0.001, respectively). Mean diameter of the smallest pronucleus of triploids and diploids were 15.2±4.1μm (8.5-19.7μm) and 12.7±2.8μm (8.5-18.9μm), respectively. Of diploid embryos, euploid, aneuploid, and mosaic were comparable in mean diameter of the smallest pronucleus. There were no differences in insemination methods (IVF and ICSI), frequency of abnormal blastomere division, embryonic developmental speed, and blastocyst grade, between triploid and diploid embryos. Limitations, reasons for caution Since this was a cross-sectional study by cytogenetics analysis of blastocysts, causal association between incidence of 3PN (including 2.1PN) and postnatal findings and significance of extra micro pronucleus are unknown. We need to assess longitudinal-study with large sample size of embryo transfer with PGT-A. Wider implications of the findings We revealed that blastocyst derived from 3PN including 2.1PN had high diploid and euploid rate by NGS and SNP analysis. Therefore, 3PN including 2.1PN embryos could be used for embryo transfer after chromosome ploidy analyses. Trial registration number Not applicable

Successful use of sexed stallion semen for in vitro embryo production; a preliminary study

The use of sex-sorted semen to obtain offspring of a desired sex in commercial livestock breeding programs has been in a state of rapid growth as sorting technology has evolved (Neculai-Valeanu and Ariton, Animals 2021, 11, 1182-1194, 2021). In horses, sex-sorted semen has resulted in successful pregnancies using low-dose or hysteroscopic artificial insemination, albeit with decreased pregnancy rates (Clulow et al, Animal Reproduction Science 108: 287–297, 2008; Gibb et al, Eq Vet J 49:160–166, 2017). Even though sex-sorted semen has been used to produce viable in vitro produced (IVP) embryos, the cleavage and blastocyst rates remain low (Colleoni et al, Reproduction, Fertility and Development 21(1): 228-229, 2008). Using current sperm sorting technology (ST Inc, Navasota, TX), we investigated fertilization, cleavage, and embryo development rates after ICSI using a time-lapse imaging system. To sex sort the semen, the ejaculate was diluted in 1:1 in a casein/cholesterol-based semen extender (BotuSemen Gold) supplemented with 60ug/ml of Hoechst 33342. The diluted semen was centrifuged through a colloid suspension (EquiPure) for 25 min at 300g using 3mL of EquiPure per billion sperm. After centrifugation, the sperm pellet was split into two aliquots: NON-SEXED, resuspended in BotuCrio, cooled and frozen in 0.5 ml straws at ∼ 100 million/ml, and SEXED, resuspended in a proprietarychemically-defined medium (EquiShip, ST Genetics), and transported to the sorting facility at ∼20°C. At the laboratory, the sperm were passed through the SexedULTRA™ Genesis III flow cytometer. After approximately 40 million total events X- and Y-bearing sperm were recovered, diluted in BotuCrio at a concentration of 2 million/ml, loaded into 0.5 ml straws and frozen. Oocytes were obtained from mares using transvaginal ultrasound-guided aspiration, shipped/held for 24h, followed by in vitro maturation. The matured MII oocytes from each aspiration were randomly split and injected with SEXED and NONSEXED sperm. A total of 56 oocytes were fertilized. We observed 24/29 and 23/27 cleaved oocytes, resulting in 11 and 12 blastocysts in SEXED and NONSEXED groups, respectively. No significant difference was detected in the cleavage and blastocyst rates among the groups. Embryo kinetic analysis revealed a slightly shorter duration from injection to blastocyst formation in the SEXED versus NONSEXED groups (183h and 188h, respectively). Higher number of embryos are required to compare the morpho-kinetics of male and female embryos at each developmental stage. This is a preliminary report, showing the successful use of frozen sexed semen in a commercial in vitro embryo production setting and further studies are ongoing.

Time-lapse monitoring: An adjunct tool to select embryos for preimplantation genetic testing.

The goal for the present study was to investigate the effect of aneuploidy on embryo morphokinetics events in a time-lapse imaging (TLI) system incubator. This retrospective cohort study was performed in a private university-affiliated in vitro fertilization center, between 2019 March and December 2020. Kinetic data were analyzed in 935 embryos, derived from 316 patients undergoing intracytoplasmic sperm injection cycle with preimplantation genetic testing (PGT) for aneuploidy, individually cultured in a TLI incubator until Day 5 of development. Timing of morphokinetic variables, the incidences of multinucleation, and Known Implantation Data Score (KIDScore)-Day 5 were compared between euploid (n = 352) and aneuploid embryos (n = 583). Aneuploid embryos showed significantly longer timing to complete specific morphokinetic parameters compared to euploidy embryos. Euploidy embryos also showed a significantly higher KIDScore when compared with the aneuploidy ones. Our evidence suggests that TLI monitoring may be an adjunct approach to select embryos for PGT; however, cautious investigation is still needed.

Previous infection with SARS-CoV-2 impacts embryo morphokinetics but not clinical outcomesin a time-lapse imaging system.

The goal for the present study was to investigate whether previous infection with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) may compromise embryo morphokinetics and implantation. For that, a historical cohort study was performed in a private university-affiliated in vitro fertilizationcenter. The study included 1628 embryos from 88 patients undergoing intracytoplasmic sperm injection(ICSI) cycles. Patients were age-matched in a 1:3 ratio to either a coronavirusdisease (COVID)group, including patients with a positive SARS-CoV-2 immunoglobulin test (n = 22 patients, 386 embryos), or a control group, including patients with a negative SARS-CoV-2 immunoglobulin test (n = 66, 1242 embryos). The effect of previous infection with SARS-CoV-2 on morphokinetic events and ICSI outcomes was evaluated. Embryos derived from patients in the COVIDgroup presented longer time to pronuclei appearance and fading, time to form two, three, four and five cells, and time to blastulation. The durations of the third cell cycle and to time to complete synchronous divisions were also significantly increased in the COVIDgroup compared with the controlgroup, whereasknown implantation diagnosisscore Day 5 ranked significantly lower in the COVIDgroup. No differences were observed between the COVID and control groups on clinical outcomes. In conclusion, patients planning parenthood, who have recovered from COVID-19 infection, must be aware of a possible effect of the infection on embryo development potential.

ARE THE MORPHOKINETIC PARAMETERS OF MOSAIC EMBRYOS DIFFERENT FROM EUPLOID EMBRYOS? RESULTS OF 1167 EMBRYOS DIAGNOSED BY NEXT GENERATION SEQUENCING (NGS) FROM A SINGLE CENTER

To compare the morphokinetic parameters of mosaic and euploid embryos in time lapse imaging system (TLI) and to analyze the relation between these parameters and clinical outcomes of mosaic embryo transfers.

Electrospun Membranes Designed for Burst Release of New Gold-Complexes Inducing Apoptosis of Melanoma Cells.

Two non-commercial metallic Au-based complexes were tested against one of the most aggressive malignant melanomas of the skin (MeWo cells), through cell viability and time-lapse live-cell imaging system assays. The tests with the complexes were carried out both in the form of free metallic complexes, directly in contact with the MeWo cell line culture, and embedded in fibers of Polycaprolactone (PCL) membranes produced by the electrospinning technique. Membranes functionalized with complexes were prepared to evaluate the efficiency of the membranes against the melanoma cells and therefore their feasibility in the application as an antitumoral patch for topical use. Both series of tests highlighted a very effective antitumoral activity, manifesting a very relevant cell viability inhibition after both 24 h and 48 h. In the case of the AuM1 complex at the concentration of 20 mM, melanoma cells completely died in this short period of time. A mortality of around 70% was detected from the tests performed using the membranes functionalized with AuM1 complex at a very low concentration (3 wt.%), even after 24 h of the contact period. The synthesized complexes also manifest high selectivity with respect to the MeWo cells. The peculiar structural and morphological organization of the nanofibers constituting the membranes allows for a very effective antitumoral activity in the first 3 h of treatment. Experimental points of the release profiles were perfectly fitted with theoretical curves, which easily allow interpretation of the kinetic phenomena occurring in the release of the synthesized complexes in the chosen medium.

High oocyte immaturity rates affect embryo morphokinetics: lessons of time-lapse imaging system

Research questionDoes oocyte immaturity rate affect morphokinetic events in a time-lapse imaging (TLI) system? DesignHistorical cohort study carried out in a private university-affiliated IVF centre. Injected oocytes (n = 3368) cultured in a TLI incubator, from intracytoplasmic sperm injection (ICSI) cycles (n = 474) carried out between March 2019 and December 2020, were analysed. The effects of immature oocyte rates (the number of germinal-vesicle and metaphase I oocytes by the number of retrieved oocytes in each cycle, on morphokinetic events) were investigated considering clustering of data using mixed models. Evaluated kinetic markers were pronuclei appearance (tPNa), timing to pronuclei fading (tPNf), timing to two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), timing to morulae (tM) and timing to start of blastulation (tSB) and to blastulation (tB). Durations of the second (t3-t2) and third (t5-t3) cell cycles (cc2 and cc3, respectively) and timing to complete synchronous divisions s1 (t2-tPNf), s2 (t4-t3) and s3 (t8-t5) were also evaluated. ResultsPositive relationships were observed between oocyte immaturity rates and slower tPNa, tPNf, t2, t3, t4, t5, t6, t7, t8, tSB, tB and cc3. Multinucleation at two- and four-cell stages were positively correlated with oocyte immaturity rate. The KIDScore ranking was negatively correlated with oocyte immaturity rate. No associations were found between oocyte immaturity rate and clinical outcomes. ConclusionsIncreasing oocyte immaturity rate correlates with delayed cell cleavage and blastulation. These findings highlight the importance of TLI for the identification and de-selection of slow-growing embryos for transfer, in cycles with high oocyte immaturity rate.

Improved embryonic development and utilization rates with EmbryoScope: a within-subject comparison versus a benchtop incubator.

The objective of this study was to investigate whether, in consecutive intracytoplasmic sperm injection (ICSI) cycles, embryonic development in an incubator with a time-lapse imaging (TLI) system is better than the previous one obtained in a benchtop incubator (G-185) with similar cultivation characteristics. The study was of a retrospective within-subject design, in which each cycle served as its own control. Data were obtained via the chart review of patients undergoing ICSI in a private university-affiliated in vitro fertilization (IVF) centre who fulfilled the following criteria: second ICSI attempt in which embryos were cultured in a TLI incubator system (TLI group, n = 71), preceded by a first ICSI attempt in which embryos were cultured in a benchtop incubator (Control group, n = 71). Embryonic development up to the fifth day of development, oocyte utilization rate (OUR; transferred embryos plus frozen embryos per total number of retrieved oocytes) and embryo utilization rate (EUR; transferred embryos plus frozen embryos per normally fertilized oocyte) were compared between the groups. There were significant differences in the day 2 non-cleavage rate, day 5 embryo rate, blastocyst development rate, frozen blastocyst rate, OUR, and EUR, in favour of the TLI group. Embryonic development, frozen blastocyst rate, OUR and EUR in the second ICSI cycle were significantly improved when the culture was performed in the EmbryoScope, compared with those rates obtained with culture in a G-185 in the first ICSI cycle of the same patients. The results may also lead to higher cumulative pregnancy outcomes following embryo thawing and transfer.

A NOVEL ZONA PELLUCIDA (ZP)-FREE CULTURE SYSTEM FOR SEVERE CYTOPLASMIC FRAGMENTATION CASES: A PILOT STUDY USING 3PN EMBRYOS AND TIME-LAPSE CINEMATOGRAPHY

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 < 0.001). While it is 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. Therefore, in this study, zona pellucida of abnormally-fertilized oocytes which were donated by patients was removed at pronuclear stage. Those ZP-free oocytes were observed in time-lapse culturing system in order to examine developmental morphology. This study used 57 abnormally fertilized (3PN) embryos (cIVF: n = 51, ICSI: n = 6) donated by assisted reproduction technology patients in our clinic with informed consent since 2017. After confirming the three pronuclei, we removed the ZP from each 3PN embryo using a laser, and the resultant ZP-free embryos were cultured and observed in an incubator equipped with a time-lapse imaging system. For ZP removal, 3PN embryos were placed in drops of 0.125M sucrose-containing HEPES media that had been covered with mineral oil and warmed to 37°C. Despite a small reduction in ooplasm size, half of the ZP was removed by laser (Saturn 5; Origio、Lykos; Hamilton Thorne). Subsequently, the ooplasms were completely separated from their ZPs by pipetting, and these ZP-free 3PN embryos were cultured continuously for 5 days with time-lapse imaging. Of 58 ZP-free embryos in total, 54 (94.7%) were cleaved, and there was no significant decrease in cleavage rate compared to 2PN embryos (98%) used routinely in our clinic. Furthermore, 28 of the 54 embryos (51.9%) developed to the morula stage after third cleavage, and 18 embryos (33.3%) formed a blastocoel and became blastocysts. Thus, removing the ZP before cleavage did not adversely affect the embryo development. In terms of the amount of fragmentation, based on the modified Veeck’s criteria, 36 of 54 ZP-free 3PN embryos (66.7%) showed less than 20% of the volume in fragments compared to the total volume of cytoplasm at the first cleavage (Grade 1 and 2), 14 (25.9%) showed 20-40% fragments (Grade 3), and only 4 (7.4%) showed > 40% fragments (Grade 4). These results suggested that the rate of fragmentation was decreased by ZP removal before the first cleavage. This study revealed that the ZP is not always necessary for normal development after the pronuclear stage because the ZP-Free embryos studied herein developed normally, maintained their cell adhesion well, and showed a decreased rate of fragmentation.

HIGH OOCYTE IMMATURITY RATES IMPACT EMBRYO MORPHOKINETICS: LESSONS OF TIME-LAPSE IMAGING SYSTEM

Up to 30% of the retrieved oocytes are still immature after controlled ovarian stimulation (COS). The ovarian asynchrony can be indicative of less responsiveness to COS, and oocyte immaturity rate may reflect the developmental competence of the entire cohort. Time-lapse imaging (TLI) systems allow for the mapping of morphological changes or events with the exact time-point of occurrence. The aim of this study was to investigate the impact of oocyte immaturity rate on morphokinetic events in a TLI system. This retrospective cohort study was performed in a private university-affiliated IVF center from March 2019 to December 2020 and included 3368 injected oocytes, cultured in a TLI incubator, from 474 ICSI cycles. Immature oocytes rates were defined as the number of germinal-vesicle (GV) plus Metaphase-I oocytes (MI) divided by the number of retrieved oocytes in each cycle. The effects of immature oocytes rates on morphokinetic events were investigated taking into account clustering of data (multiple embryos per cycle), using generalized mixed models. Evaluated kinetic markers were: pronuclei appearance (tPNa), timing to pronuclei fading (tPNf), timing to two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), and timing to start of blastulation (tSB) and to blastulation (tB). Durations of the second and third cell cycles (cc2 and cc3, respectively) were also evaluated. The post hoc achieved power was > 95.0%. From 4120 retrieved oocytes, 3130 were mature (MII), and 990 were immature (660 GV and 330 MI). Positive relationships were observed between the rates of immature oocytes and slower tPNa (B: 0.053, CI: 0.045 – 0.061, p<0.001), tPNf (B: 0.081, CI: 0.070 – 0.093, p<0.001), t2 (B: 0.076, CI: 0.064 – 0.087, p<0.001), t3 (B: 0.070, CI: 0.056 – 0.084, p<0.001), t4 (B: 0.070, CI: 0.055 – 0.084, p<0.001), t5 (B: 0.083, CI: 0.063 – 0.102, p<0.001), t6 (B: 0.066, CI: 0.047 – 0.086, p<0.001), t7 (B: 0.076, CI: 0.055 – 0.098, p<0.001), t8 (B: 0.064, CI: 0.040 – 0.087, p<0.001), tSB (B: 0.090, CI: 0.014 – 0.165, p=0.020), tB (B: 0.043, CI: 0.011 – 0.075, p=0.009), and cc3 (B: 0.018, CI: 0.005 – 0.031, p=0.007). No significant associations were found between oocyte immaturity rate and clinical outcomes. Increasing oocyte immaturity rate correlates with delayed cell cleavage and blastulation. These findings highlight the importance of TLI for the identification and de-selection of slow-growing embryos for transfer, in cycles with high oocyte immaturity rate.

SERUM ANTI-MÜLLERIAN HORMONE CONCENTRATIONS IMPACTS NOT ONLY THE QUANTITY, BUT ALSO THE QUALITY OF THE OVARIAN RESERVE: THE IMPORTANCE OF TIME-LAPSE IMAGING SYSTEM

Serum anti-müllerian hormone (AMH) concentration has been implicated as the most valuable marker of the ovarian reserve, as it correlates highly with the baseline antral follicle count. The AMH has been assigned as a marker of ovarian function mainly in the assessment of the quantitative aspect of the ovarian reserve. On the other side, the predictive value of AMH on oocyte quality and embryo developmental competence is controversial. Time-lapse imaging (TLI) systems allow for the assessment of morphological changes with the exact time-point of occurrence, and may be a valuable tool to evaluate the correlation between serum AMH concentration and the competences of both oocyte and developing embryo. The goal for the present study was to evaluate whether the serum AMH concentration impacts morphokinetic events in a TLI system. This retrospective cohort study was performed in a private university-affiliated IVF center from March 2019 to December 2020 and included 902 injected oocytes from 114 ICSI cycles, cultured in a TLI incubator until the fifth day of development. The effects of serum AMH concentrations on morphokinetic events and ICSI outcomes were evaluated by taking into account clustering of data (multiple embryos per cycle), using generalized mixed models. Evaluated kinetic markers were: pronuclei appearance (tPNa), timing to pronuclei fading (tPNf), timing to two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), and timing to blastulation (tB). Durations of the second (t3-t2) and third (t5-t3) cell cycles (cc2 and cc3, respectively) and timing to complete synchronous divisions s1 (t2-tPNf), s2 (t4-t3), and s3 (t8-t5) were also evaluated. The post hoc achieved power was > 98.0%. Inverse relationships were observed between serum AMH concentrations and faster tPNf (B: -0.047, CI: -0.071 – -0.024, p<0.001), t3 (B: -0.070, CI: -0.102 – -0.039, p<0.001), t4 (B: -0.080, CI: -0.114 – -0.046, p<0.001), t5 (B: -0.075, CI: -0.123 – -0.028, p=0.002), t6 (B: -0.105, CI: -0.147 – -0.064, p<0.001), t7 (B: -0.120, CI: -0.165 – -0.075, p<0.001), t8 (B: -0.170, CI: -0.220 – -0.119, p<0.001), and tB (B: -0.153, CI: -0.224 – -0.082, p<0.001), cc2 (B: -0.041, CI: -0.062 – -0.019, p<0.001), and s3 (B: -0.086, CI: -0.129 - -0.044, p<0.001). There was a positive association between serum AMH concentration and implantation rate (B: -19.11, CI: -29.12 - -9.11, p<0.001). No significant associations were found between serum AMH concentrations and other morphokinetic parameters or clinical outcomes. Increased serum AMH concentrations correlate with faster embryo development in a TLI incubator and implantation rates.

EMBRYOS MORPHOKINETICS: DOES THE SOURCE OF GONADOTROPHIN PREPARATIONS MATTER?

Follicle growth can be obtained with different preparations or combinations of gonadotrophins. Approaches combining FSH with LH-like-action gonadotrophins have been associated with improved IVF outcomes. Whether a controlled ovarian stimulation (COS) regimen based on r-FSH and r-LH leads to better outcomes compared with a protocol using human menopausal gonadotropin (hMG) is still under debate. The goal for the present study was to compare the effect of two FSH plus LH-like protocols on morphokinetic events in a time-lapse imaging (TLI) system. This retrospective cohort study was performed in a private university-affiliated IVF center from March 2019 to December 2020 and included 1268 embryos, cultured in a TLI incubator, from 198 ICSI cycles. Cycles were split into two groups depending on the protocol applied for COS: 142 patients (904 embryos) were stimulated with rFSH + rLH, whereas 56 patients (364 embryos) were treated with hMG. The effects of the COS protocol on morphokinetic events and ICSI outcomes were compared among the groups taking into account clustering of data (multiple embryos per cycle), using generalized mixed models. Evaluated kinetic markers were: pronuclei appearance (tPNa), timing to pronuclei fading (tPNf), timing to two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), and timing to start of blastulation (tSB) and to blastulation (tB). Durations of the second (t3-t2) and third (t5-t3) cell cycles (cc2 and cc3, respectively) and timing to complete synchronous divisions s1 (t2-tPNf), s2 (t4-t3), and s3 (t8-t5) were also evaluated. The post hoc achieved power was > 99.0%. When compared with the protocol with hMG, COS using rFSH + rLH leaded to faster tPNa (7.4h ± 0.30 vs 6.5h ± 0.18, p<0.007), tPNf (25.6h ± 0.40 vs 24.4h ± 0.25, p<0.009), t5 (50.0h ± 0.60 vs 48.5h ± 0.37, p<0.035), and t6 (52.9h ± 0.58 vs 51.5h ± 0.37, p<0.043). The s3 were also decreased in rFSH + rLH protocols (10.4h ± 0.36 vs 8.9h ± 0.58, p<0.033). As for the clinical outcomes, no significant differences were noted among the groups for pregnancy (40.0% ± 11.0 vs 33.0% ± 10.9, p=0.398) and implantation rates (28.0% ± 6.2 vs 22.2% ± 8.4, p=0.76), for rFSH + rLH and hMG groups respectively. Stimulation protocols using associations of rFSH and rLH lead to faster initial embryo development when compared with protocols using hMG.

IDEAL CULTURE CONDITIONS FOR ENHANCING FULL PREIMPLANTATION DEVELOPMENT OF EMBRYOS GENERATED THROUGH SOMATIC CELL HAPLOIDIZATION

To identify the ideal culture conditions for promoting full preimplantation development of artificial embryos generated through somatic cell hapoloidization (SCH). Metaphase II (MII) oocytes from B6D2F1 mice were enucleated under Oosight™ visualization and exposure to cytochalasin B. Nuclear transfer of an individual FVB mouse cumulus cell was conducted by transfer into the perivitelline space of the resulting ooplast and fused using Sendai virus. Development of a pseudo-meiotic spindle was assessed in the reconstructed oocytes 2 hours after transfer, and the oocytes were then piezo-ICSI inseminated. Zygotes were cultured either in KSOM medium (KSOM), KSOM supplemented with Trichostatin A (TSA), or KSOM supplemented with fasudil, scriptaid, and RS-1 (cocktail). Trichostatin A and scriptaid are both histone-deacetylase inhibitors that may improve cellular reprogramming and embryo development after nuclear transfer. Fasudil, a Rho-associated kinase inhibitor, reduces apoptosis, while RS-1 aids in DNA damage repair. Zygotes were cultured in TSA and cocktail for 24 hrs only, before being changed to KSOM. Intact oocytes were ICSI inseminated as controls and cultured in KSOM (control). All four groups were monitored in a time-lapse imaging system for 96hr. A total of 527 MII oocytes were enucleated, resulting in 522 ooplasts (99.0%). Nuclear transfer of these ooplasts resulted in a survival rate of 99.6%, generating 520 reconstructed oocytes. A positive spindle was assessed in 360 artificial oocytes (69.2%), and piezo-ICSI of these spindle-positive oocytes resulted in a survival rate of 75.0%. ICSI insemination of 60 control oocytes had a comparable survival rate (83.3%). Fertilization rates were similar among all culture conditions, as well as to the control, with rates of 91.2% for the cocktail group, 93.8% for the KSOM group, 94.4% for the TSA group, and 94.0% for the control. The cocktail group exhibited significant delays at all stages (P<0.05) except the 2-cell stage when compared to the control. KSOM-cultured embryos had comparable morphokinetic development to the control up to the blastocyst stage, except for a delay in pronuclei appearance (P<0.002). Morphokinetic parameters of TSA embryos were comparable to the controls at all stages, beginning with the pseudo-second polar body extrusion (haploidization) up until the blastocyst stage. The intact oocyte control had an 82.3% blastulation rate compared to all experimental groups (P<0.00001). The cocktail cohort had a blastulation rate of only 9.6%, and the KSOM group had a blastulation rate of 14.5%; the highest blastulation rate was reached by the TSA group at 23.5%. Although the blastocyst development was still suboptimal, among the culture media tested, the addition of a histone-deacetylase inhibitor (TSA) yielded artificial embryos with morphokinetic parameters similar to the control.

P–804 Morphokinetic development by time-lapse imaging of conceptuses generated from artificial oocytes

Abstract Study question What are the ideal culture conditions to enhance full preimplantation development of embryos generated by FVB somatic cell haploidization (SCH) in the mouse model? Summary answer The presence of a histone deacetylase inhibitor yielded the best morphokinetic development of expanded blastocysts generated by FVB SCH, comparable to control blastocysts. What is known already Various culture conditions and medium supplements have been proposed to promote preimplantation development of embryos generated by SCH, including supplementation with trichostatin A (TSA), fasudil, scriptaid, and RAD–51 stimulatory compound–1 (RS–1). TSA and scriptaid, both histone-deacetylase inhibitors, have been found to improve embryo development following nuclear transfer by enhancing histone acetylation and cellular reprogramming. Additionally, fasudil is a Rho-associated kinase inhibitor that has been shown to reduce apoptosis and promote cell proliferation. Finally, RS–1 stimulates RAD51 activity, which promotes the repair of DNA damage and increases the efficacy of somatic cell reprogramming. Study design, size, duration B6D2F1 mouse metaphase II (MII) oocytes underwent enucleation and nuclear transfer, or were ICSI inseminated serving as controls. Reconstituted oocytes showing development of a meiotic-like spindle demonstrated successful SCH, and were ICSI inseminated. SCH conceptuses were cultured in one of three groups: KSOM, KSOM supplemented with TSA (TSA), or KSOM supplemented with fasudil, scriptaid, and RS–1 (Cocktail). ICSI controls (ICSIC) were cultured in KSOM medium. Fertilization and full preimplantation development were compared among all groups. Participants/materials, setting, methods Ooplasts were generated from MII oocytes by removing spindle complexes under OosightÔ visualization and cytochalasin B exposure. A single FVB mouse cumulus cell was transferred into the perivitelline space and fused with the ooplast, facilitated by Sendai virus. Reconstructed oocytes with novel pseudo-meiotic spindles underwent piezo-ICSI and were cultured in different media conditions in a time-lapse imaging system up to 96h. TSA and Cocktail embryos had media changed to regular KSOM 10 hours after insemination. Main results and the role of chance A total of 274 B6D2F1 MII oocytes were enucleated, resulting in a 95.9% survival rate. All ooplasts survived nuclear transfer and 62.1% successfully haploidized after 2 hours. ICSIC and reconstituted SCH oocytes survived piezo-ICSI at rates of 81.5% and 57.0%, respectively (P &amp;lt; 0.01). SCH embryos were then allocated into KSOM, TSA supplied, and Cocktail media. Fertilization rates for ICSIC, KSOM, and TSA embryos were 92.4%, 90.7%, and 94.4%, respectively, while the rate for embryos cultured in Cocktail was only 71.9% (P &amp;lt; 0.03). While embryos cultured in Cocktail had a comparable 2-cell timing to ICSIC, embryos in TSA reached developmental milestones with a closer timing to the ICSIC, having minor delays at the 3-, 4-, and 6-cell stages (P &amp;lt; 0.05). KSOM- and Cocktail-cultured embryos were delayed at most of the stages (P &amp;lt; 0.01), except for the two-pronuclei appearance. Although the TSA group displayed the best embryo developmental pattern, the final rate of blastocyst development was somewhat homogeneous with rates of 15.4%, 23.5%, and 13.0% for the KSOM, TSA, and Cocktail groups, respectively (P &amp;lt; 0.001), and remarkably lower than the ICSIC (81.6%). Limitations, reasons for caution Although live pups have been obtained using BDF cumulus cells, embryos generated by FVB cumulus cells show a remarkably lower blastocyst development, but maintain morphokinetic characteristics similar to ICSIC in the presence of TSA. Wider implications of the findings: While using different strains to enhance genetic variance, the morphokinetic analysis of preimplantation embryos in ideal culture conditions is paramount to the progress of neogametogenesis. The implementation of this technique may soon help create genotyped oocytes for women with compromised ovarian reserve. Trial registration number N/A

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