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Purpose: This article aims to assess how differences in maternal age distributions between IVF clinics affect the performance of an artificial intelligence model for embryo viability prediction and proposes a method to account for such differences. Methods: Using retrospectively collected data from 4805 fresh and frozen single blastocyst transfers of embryos incubated for 5 to 6 days, the discriminative performance was assessed based on fetal heartbeat outcomes. The data was collected from 4 clinics, and the discrimination was measured in terms of the area under ROC curves (AUC) for each clinic. To account for the different age distributions between clinics, a method for age-standardizing the AUCs was developed in which the clinic-specific AUCs were standardized using weights for each embryo according to the relative frequency of the maternal age in the relevant clinic compared to the age distribution in a common reference population. Results: There was substantial variation in the clinic-specific AUCs with estimates ranging from 0.58 to 0.69 before standardization. The age-standardization of the AUCs reduced the between-clinic variance by 16%. Most notably, three of the clinics had quite similar AUCs after standardization, while the last clinic had a markedly lower AUC both with and without standardization. Conclusion: The method of using age-standardization of the AUCs that is proposed in this article mitigates some of the variability between clinics. This enables a comparison of clinic-specific AUCs where the difference in age distributions is accounted for. [ABSTRACT FROM AUTHOR]

Purpose: Despite the success of ICSI in treating severe male factor infertile patients, total fertilization failure (FF) still occurs in around 1–3% of ICSI cycles. To overcome FF, the use of calcium ionophores has been proposed to induce oocyte activation and restore fertilization rates. However, assisted oocyte activation (AOA) protocols and ionophores vary between laboratories, and the morphokinetic development underlying AOA remains understudied. Methods: A prospective single-center cohort study involving 81 in vitro matured metaphase-II oocytes from 66 oocyte donation cycles artificially activated by A23187 (GM508 CultActive, Gynemed) (n=42) or ionomycin (n=39). Parthenogenesis was induced, and morphokinetic parameters (tPNa, tPNf, t2-t8, tSB, and tB) were compared between the 2 study groups and a control group comprising 39 2PN-zygotes from standard ICSI cycles. Results: Ionomycin treatment resulted in higher activation rates compared to A23187 (38.5% vs 23.8%, p=0.15). Importantly, none of the A23187-activated parthenotes formed blastocysts. When evaluating the morphokinetic dynamics between the two ionophores, we found that tPNa and tPNf were significantly delayed in the group treated by A23187 (11.84 vs 5.31, p=0.002 and 50.15 vs 29.69, p=0.005, respectively). t2 was significantly delayed in A23187-activated parthenotes when compared to the double heterologous control embryo group. In contrast, the morphokinetic development of ionomycin-activated parthenotes was comparable to control embryos (p>0.05). Conclusion: Our results suggest that A23187 leads to lower oocyte activation rates and profoundly affects morphokinetic timings and preimplantation development in parthenotes. Despite our limited sample size and low parthenote competence, standardization and further optimization of AOA protocols may allow wider use and improved outcomes for FF cycles. [ABSTRACT FROM AUTHOR]

Introduction: Morphokinetic analysis using a closed time-lapse monitoring system (EmbryoScope + ™) provides quantitative metrics of meiotic progression and cumulus expansion. The goal of this study was to use a physiologic aging mouse model, in which egg aneuploidy levels increase, to determine whether there are age-dependent differences in morphokinetic parameters of oocyte maturation. Methods: Denuded oocytes and intact cumulus-oocyte complexes (COCs) were isolated from reproductively young and old mice and in vitro matured in the EmbryoScope + ™. Morphokinetic parameters of meiotic progression and cumulus expansion were evaluated, compared between reproductively young and old mice, and correlated with egg ploidy status. Results: Oocytes from reproductively old mice were smaller than young counterparts in terms of GV area (446.42 ± 4.15 vs. 416.79 ± 5.24 µm2, p < 0.0001) and oocyte area (4195.71 ± 33.10 vs. 4081.62 ± 41.04 µm2, p < 0.05). In addition, the aneuploidy incidence was higher in eggs with advanced reproductive age (24–27% vs. 8–9%, p < 0.05). There were no differences in the morphokinetic parameters of oocyte maturation between oocytes from reproductively young and old mice with respect to time to germinal vesicle breakdown (GVBD) (1.03 ± 0.03 vs. 1.01 ± 0.04 h), polar body extrusion (PBE) (8.56 ± 0.11 vs. 8.52 ± 0.15 h), duration of meiosis I (7.58 ± 0.10 vs. 7.48 ± 0.11 h), and kinetics of cumulus expansion (0.093 ± 0.002 vs. 0.089 ± 0.003 µm/min). All morphokinetic parameters of oocyte maturation were similar between euploid and aneuploid eggs irrespective of age. Conclusion: There is no association between age or ploidy and the morphokinetics of mouse oocyte in vitro maturation (IVM). Future studies are needed to evaluate whether there is an association between morphokinetic dynamics of mouse IVM and embryo developmental competence. [ABSTRACT FROM AUTHOR]

Purpose: We aimed to compare the DNA methylation profiles of human embryos cultured in uninterrupted or interrupted incubators. Methods: This study included 9 women, ≤ 30 years old (range: 20–30 years), without a history of genetic diseases or smoking, undergoing ICSI treatment, and each woman donated one oocyte. Embryos were randomly assigned to culture in either time-lapse imaging or standard incubators after ICSI. We compared the DNA methylation profiles of human eight-cell embryos cultured in uninterrupted condition using time-lapse imaging (TLI) incubator (EmbryoScope) to those cultured in interrupted culture model using standard incubators (SI, G185 K-System). Nine single-cell whole-genome bisulfite sequencing (WGBS) datasets were analyzed, including four SI-cultured embryos and five TLI-cultured embryos at the eight-cell stage. Results: A total of 581,140,020 and 732,348,182 clean reads were generated from the TLI and SI groups, respectively. TLI-cultured embryos had similar genome-wide methylation patterns to SI-cultured embryos. There were no significant differences in the methylation and transcription levels of transposable elements and imprinted control regions. Although a total of 198 differentially methylated genes (DMGs) were identified, only five DMGs had significantly different transcription levels between the two groups. Conclusions: This is the first study to compare the DNA methylation profiles of embryos cultured in TLI and SI and will provide a foundation for evaluating the safety of TLI application in assisted reproductive technologies. However, further study with a larger cohort of samples was needed for the data validation. [ABSTRACT FROM AUTHOR]

Purpose: We aimed to identify the correlation between morphological pronuclear (PN) status and the genetically determined ploidy configuration in preimplantation embryos.A retrospective observational study was conducted on 1982 embryos displaying normal fertilization and 380 embryos showing an atypical PN pattern, tested for aneuploidies and ploidy status via preimplantation genetic testing (PGT) between May 2019 and May 2024. Ploidy prediction was performed using a validated targeted-NGS approach and a proprietary bioinformatic pipeline analyzing SNPs B-allele frequency information. Ploidy results were obtained in relation to the morphological PN pattern and further stratified by mode of PN observation, maternal age, and embryo quality parameters.Abnormal ploidy results in 2PN-derived embryos were 1% (n = 20/1982): 0.8% showed triploidy and 0.2% haploidy. Ploidy results in relation to PN number in atypical fertilization were as follows: 0PN (n = 150/380) associated with 87.3% of diploidy, 8.7% of haploidy, and 4.0% of triploidy; 1PN-derived blastocysts (n = 73/153) were haploid in 47.7% of cases, 6.5% were triploid, and 45.7% diploid; 2.1PN (n = 23/280) and 3PN patterns (n = 54/280) predicted a triploid result in 34.8% and 74.1% of cases, respectively. PN observation with time-lapse increased ploidy status predictivity from 28.3% to 80.4% (p < 0.01) and reduced expected diploid rates to 19.6% (p < 0.01). Diploidy rate was higher for maternal age ≤ 35 years and for morphologically high-grade embryos.Morphological PN check can be improved by incorporating ploidy analysis within the conventional PGT workflow. Euploid 2PN-derived embryos can be further selected removing haploids and triploids, and some atypical PN pattern can be better classified.Methods: We aimed to identify the correlation between morphological pronuclear (PN) status and the genetically determined ploidy configuration in preimplantation embryos.A retrospective observational study was conducted on 1982 embryos displaying normal fertilization and 380 embryos showing an atypical PN pattern, tested for aneuploidies and ploidy status via preimplantation genetic testing (PGT) between May 2019 and May 2024. Ploidy prediction was performed using a validated targeted-NGS approach and a proprietary bioinformatic pipeline analyzing SNPs B-allele frequency information. Ploidy results were obtained in relation to the morphological PN pattern and further stratified by mode of PN observation, maternal age, and embryo quality parameters.Abnormal ploidy results in 2PN-derived embryos were 1% (n = 20/1982): 0.8% showed triploidy and 0.2% haploidy. Ploidy results in relation to PN number in atypical fertilization were as follows: 0PN (n = 150/380) associated with 87.3% of diploidy, 8.7% of haploidy, and 4.0% of triploidy; 1PN-derived blastocysts (n = 73/153) were haploid in 47.7% of cases, 6.5% were triploid, and 45.7% diploid; 2.1PN (n = 23/280) and 3PN patterns (n = 54/280) predicted a triploid result in 34.8% and 74.1% of cases, respectively. PN observation with time-lapse increased ploidy status predictivity from 28.3% to 80.4% (p < 0.01) and reduced expected diploid rates to 19.6% (p < 0.01). Diploidy rate was higher for maternal age ≤ 35 years and for morphologically high-grade embryos.Morphological PN check can be improved by incorporating ploidy analysis within the conventional PGT workflow. Euploid 2PN-derived embryos can be further selected removing haploids and triploids, and some atypical PN pattern can be better classified.Results: We aimed to identify the correlation between morphological pronuclear (PN) status and the genetically determined ploidy configuration in preimplantation embryos.A retrospective observational study was conducted on 1982 embryos displaying normal fertilization and 380 embryos showing an atypical PN pattern, tested for aneuploidies and ploidy status via preimplantation genetic testing (PGT) between May 2019 and May 2024. Ploidy prediction was performed using a validated targeted-NGS approach and a proprietary bioinformatic pipeline analyzing SNPs B-allele frequency information. Ploidy results were obtained in relation to the morphological PN pattern and further stratified by mode of PN observation, maternal age, and embryo quality parameters.Abnormal ploidy results in 2PN-derived embryos were 1% (n = 20/1982): 0.8% showed triploidy and 0.2% haploidy. Ploidy results in relation to PN number in atypical fertilization were as follows: 0PN (n = 150/380) associated with 87.3% of diploidy, 8.7% of haploidy, and 4.0% of triploidy; 1PN-derived blastocysts (n = 73/153) were haploid in 47.7% of cases, 6.5% were triploid, and 45.7% diploid; 2.1PN (n = 23/280) and 3PN patterns (n = 54/280) predicted a triploid result in 34.8% and 74.1% of cases, respectively. PN observation with time-lapse increased ploidy status predictivity from 28.3% to 80.4% (p < 0.01) and reduced expected diploid rates to 19.6% (p < 0.01). Diploidy rate was higher for maternal age ≤ 35 years and for morphologically high-grade embryos.Morphological PN check can be improved by incorporating ploidy analysis within the conventional PGT workflow. Euploid 2PN-derived embryos can be further selected removing haploids and triploids, and some atypical PN pattern can be better classified.Conclusion: We aimed to identify the correlation between morphological pronuclear (PN) status and the genetically determined ploidy configuration in preimplantation embryos.A retrospective observational study was conducted on 1982 embryos displaying normal fertilization and 380 embryos showing an atypical PN pattern, tested for aneuploidies and ploidy status via preimplantation genetic testing (PGT) between May 2019 and May 2024. Ploidy prediction was performed using a validated targeted-NGS approach and a proprietary bioinformatic pipeline analyzing SNPs B-allele frequency information. Ploidy results were obtained in relation to the morphological PN pattern and further stratified by mode of PN observation, maternal age, and embryo quality parameters.Abnormal ploidy results in 2PN-derived embryos were 1% (n = 20/1982): 0.8% showed triploidy and 0.2% haploidy. Ploidy results in relation to PN number in atypical fertilization were as follows: 0PN (n = 150/380) associated with 87.3% of diploidy, 8.7% of haploidy, and 4.0% of triploidy; 1PN-derived blastocysts (n = 73/153) were haploid in 47.7% of cases, 6.5% were triploid, and 45.7% diploid; 2.1PN (n = 23/280) and 3PN patterns (n = 54/280) predicted a triploid result in 34.8% and 74.1% of cases, respectively. PN observation with time-lapse increased ploidy status predictivity from 28.3% to 80.4% (p < 0.01) and reduced expected diploid rates to 19.6% (p < 0.01). Diploidy rate was higher for maternal age ≤ 35 years and for morphologically high-grade embryos.Morphological PN check can be improved by incorporating ploidy analysis within the conventional PGT workflow. Euploid 2PN-derived embryos can be further selected removing haploids and triploids, and some atypical PN pattern can be better classified. [ABSTRACT FROM AUTHOR]

Purpose: To compare morphokinetic parameters in embryos obtained from women with and without endometriosis. Methods: We evaluated a total of 3471 embryos resulting from 434 oocyte retrievals performed at a single academic center. One thousand seventy-eight embryos were obtained from women affected by endometriosis and 2393 came from unaffected controls. All embryos were cultured in a time-lapse incubator chamber for up to 6 days. IVF cycle outcomes and morphokinetic parameters collected prospectively were retrospectively reviewed. Results: Morphokinetic data suggest that embryo development is impaired in embryos obtained from women with endometriosis (EE). EE were slower to achieve the 2–8 cell stages compared to control embryos (CE) (p < 0.001); additionally, time to compaction was delayed compared to CE (p = 0.015). The timing of late developmental events, including morulation and blastulation was also delayed in the endometriosis cohort (p < 0.001). In addition to demonstrating delayed cell cycle milestones, EE were less likely than controls to progress to morula, blastocyst, and expanded blastocyst stages (p < 0.001). Furthermore, a smaller proportion of embryos in the endometriosis group fell into optimal kinetic ranges for cc2 (p = 0.003), t5 (p = 0.019), tSB (p < 0.001), and tEB (p = 0.007). There were no significant differences in clinical pregnancy or live birth rates between groups. Conclusion: Embryos from endometriosis patients demonstrate impairments in both early and late developmental events, and progress to the morula, blastocyst, and expanded blastocyst stages at lower rates than control embryos. Despite these differences, IVF outcomes are similar for patients with and without endometriosis. [ABSTRACT FROM AUTHOR]

Purpose: To study the morphometric and morphokinetic profiles of pronuclei (PN) between male and female human zygotes. Method(s): This retrospective cohort study included 94 consecutive autologous single day 5 transfer cycles leading to a singleton live birth. All oocytes were placed in the EmbryoScope + incubator post-sperm injection with all annotations performed retrospectively by one embryologist (L-SO). Timing parameters included 2nd polar body extrusion (tPB2), sperm-originated PN (tSPNa) or oocyte-originated PN (tOPNa) appearance, and PN fading (tPNF). Morphometrics were evaluated at 8 (stage 1), 4 (stage 2), and 0 h before PNF (stage 3), measuring PN area (um2), PN juxtaposition, and nucleolar precursor bodies (NPB) arrangement. Results: Male zygotes had longer time intervals of tPB2_tSPNa than female zygotes (4.8 ± 0.2 vs 4.2 ± 0.1 h, OR = 1.442, 95% CI 1.009–2.061, p = 0.044). SPN increased in size from stage 1 through 2 to 3 (435.3 ± 7.2, 506.7 ± 8.0, and 556.3 ± 8.9 um2, p = 0.000) and OPN did similarly (399.0 ± 6.1, 464.3 ± 6.7, and 513.8 ± 6.5 um2, p = 0.000), with SPN being significantly larger than OPN at each stage (p < 0.05 respectively). More male than female zygotes reached central PN juxtaposition at stage 1 (76.7% vs 51.0%, p = 0.010), stage 2 (97.7% vs 86.3%, p = 0.048), and stage 3 (97.7% vs 86.3%, p = 0.048). More OPN showed aligned NPBs than in SPN at stage 1 only (44.7% vs 28.7%, p = 0.023). Conclusion(s): Embryos with different sexes display different morphokinetic and morphometric features at the zygotic stage. Embryo selection using such parameters may lead to unbalanced sex ratio in resulting offspring. [ABSTRACT FROM AUTHOR]

Objectives: Using time-lapse microscopy, previous research has shown that IVF mouse embryos that cleave earlier at the first division ('fast') develop into blastocysts with increased glucose consumption and lower likelihood of post-implantation loss as compared to slower cleaving embryos ('slow'). Further, metabolomics analysis employing LC–MS conducted on groups of 'fast' blastocysts revealed that more aspartate was consumed. With the worldwide adoption of single blastocyst transfer as the standard of care, the need for quantifiable biomarkers of viability, such as metabolism of specific nutrients, would greatly assist in embryo selection for transfer. Methods: Here we describe the development of a targeted enzymatic assay to quantitate aspartate uptake of single blastocysts. Results: Results demonstrate that the rates of aspartate and glucose consumption were significantly higher in individual 'fast' blastocysts. Blastocysts, together with placental and fetal liver tissue collected following transfer, were analysed for the expression of genes involved in aspartate and carbohydrate metabolism. In 'fast' blastocysts, expressions of B3gnt5, Slc2a1, Slc2a3, Got1 and Pkm2 were found to be significantly higher. In placental tissue derived from 'fast' blastocysts, expression of Slc2a1, Got1 and Pkm2 were significantly higher, while levels of Got1 and Pkm2 were lower in fetal liver tissue compared to tissue from 'slow' blastocysts. Conclusions: Importantly, this study shows that genes regulating aspartate and glucose metabolism were increased in blastocysts that have higher viability, with differences maintained in resultant placentae and fetuses. Consequently, the analysis of aspartate uptake in combination with glucose represents biomarkers of development and may improve embryo selection efficacy and pregnancy rates. [ABSTRACT FROM AUTHOR]

Purpose: Is the presence of cytoplasmic strings (CS) in human blastocysts associated with the probability of clinical pregnancy with fetal heart (CPFH) after transfer. Methods: This case-control study involved 300 single blastocyst transfers. 150 of these resulted in a CPFH (cases) while 150 did not (controls). All embryos were cultured in Embryoscope+ and AI software (IVY) was used to select the blastocyst with the highest score from the cohort for transfer. An embryologist, blind to the transfer outcome, recorded the CS number, location, and duration of their activity. Results: There was a significant difference in the number of blastocysts that contained CS, with 97.3% of women's blastocysts resulting in +CPFH containing the CS compared to 88.7% of blastocysts in women who did not have a pregnancy (p = 0.007, OR; 4.67, CI 95% 1.5–14.2). CS appeared 2.4 h earlier in embryo development in the +CPFH group compared to their negative counterparts (p = 0.007). There was a significant difference in the average number of CS/blastocyst with a higher number being present in those that achieved a clinical pregnancy (mean: 6.2, SD 2.9) compared to those that did not (mean: 4.6, SD 3.0) (p ≤ 0.0001). There was a significant increase in the number of vesicles seen traveling along the CS with more seen in the blastocysts resulting in a +CPFH (mean: 4.3 SD 2.1) compared to those in the −CPFH group (mean: 3.1, SD 2.1). Conclusion: This study has shown that the presence of cytoplasmic strings in human blastocysts is associated with the probability of clinical pregnancy with fetal heart. [ABSTRACT FROM AUTHOR]

Purpose: Our retrospective study is to investigate an end-to-end deep learning model in identifying ploidy status through raw time-lapse video. Methods: By randomly dividing the dataset of time-lapse videos with known outcome of preimplantation genetic testing for aneuploidy (PGT-A), a deep learning model on raw videos was trained by the 80% dataset, and used to test the remaining 20%, by feeding time-lapse videos as input and the PGT-A prediction as output. The performance was measured by an average area under the curve (AUC) of the receiver operating characteristic curve. Result(s): With 690 sets of time-lapse video image, combined with PGT-A results, our deep learning model has achieved an AUC of 0.74 from the test dataset (138 videos), in discriminating between aneuploid embryos (group 1) and others (group 2, including euploid and mosaic embryos). Conclusion: Our model demonstrated a proof of concept and potential in recognizing the ploidy status of tested embryos. A larger scale and further optimization on the exclusion criteria would be included in our future investigation, as well as prospective approach. [ABSTRACT FROM AUTHOR]

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

Purpose: The improvement of clinical outcome provided by time-lapse technology (TLT) in IVF over conventional incubation (CI) still remains controversial. This study aimed at evaluating whether the exclusive use of time-lapse technology (TLT) during whole IVF care improves total cumulative live birth rate (TCLBR) and shortens time to live birth (TTLB) as compared to the use of CI in couples undergoing ICSI. Methods: This retrospective cohort study was conducted in couples with male infertility undergoing their first ICSI cycle in 2014–2015 and for whom embryo culture system remained the same during their whole IVF care, i.e., TLT or CI. Couples were followed up up to 2020, including all following frozen-embryo transfers and ICSI cycles (if any). Survival analysis was used to compare clinical outcome and time-related endpoints between both groups. Results: A total of 151 and 250 couples underwent their whole IVF care with the exclusive use of TLT and CI, respectively. Survival analysis showed that TCLBR after whole IVF care was significantly higher in TLT than in CI group (66.9 vs 56.4%, p=0.02, log-rank test). Median live birth time was significantly shorter in TLT than CI group (464 vs 596 days, p=0.01). Conclusions: We found that TCLBR and TTLB were significantly improved with TLT over CI in couples undergoing ICSI for male factor. This study fuels the debate on the clinical benefit of using TLT. The use of time-related endpoints adds important information for both patients and practitioners. [ABSTRACT FROM AUTHOR]

Objective: Assisted oocyte activation (AOA) can restore fertilization rates after IVF/ICSI cycles with fertilization failure. AOA is an experimental technique, and its downstream effects remain poorly characterized. Clarifying the relationship between AOA and embryo, morphokinetics could offer complementary insights into the quality and viability of the embryos obtained with this technique. The aim of this study is to compare the preimplantation morphokinetic development of embryos derived from ICSI-AOA (experimental group) vs. ICSI cycles (control group). Methods: A retrospective cohort study was carried out with 141 embryos from fresh oocyte donation cycles performed between 2013 and 2017; 41 embryos were derived from 7 ICSI-AOA cycles and 100 embryos from 18 ICSI cycles. Morphokinetic development of all embryos was followed using a time-lapse system. Results: We show that embryos from both groups develop similarly for most milestones, with the exception of the time of second polar body extrusion (tPB2) and the time to second cell division (t3). Conclusions: We conclude that ionomycin mediated AOA does not seem to affect the morphokinetic pattern of preimplantation embryo development, despite the alterations found in tPB2 and t3, which could directly reflect the use of a Ca2+ ionophore as a transient and quick non-physiologic increase of free intracytoplasmic Ca2+. [ABSTRACT FROM AUTHOR]

Purpose: To study whether members of the miR-290-295 cluster in spent culture medium (SCM) of embryos are correlated with morphokinetics and apoptosis. Methods: Cryopreserved 1-cell stage mouse embryos were cultured to the blastocyst stage, development was monitored by time-lapse, 59 SCM were collected, and miR-291a and miR-294 were detected with polymerase chain reaction (PCR). Blastocysts were immuno-stained for sexing (H2AK119ub) and for apoptosis (TUNEL). Each embryo and SCM were individually processed. Correlations were run between the miRNAs and developmental events (t2, t3, t4, t5, t8, tSB, tB, ECC2, ECC3, s2, s3, dB) and apoptosis (apoptotic cells/total cell number %). MiR-294 SCM and cell levels were compared in 40 blastocysts. Apoptosis was induced in 15 blastocysts with UV radiation and SCM samples were analyzed for miR-294. Results: MiR-291a and miR-294 are released in variable levels by mouse blastocysts. Their release is similar between male and female embryos. No significant correlations were found between these miRNAs and development. MiR-294 was significantly positively correlated with apoptosis (r = 0.560, p < 0.001). Cellular expression was lower in blastocysts that released miR-294 in high levels compared with null, low, and medium release embryos (p < 0.01). UV radiation caused apoptosis which triggered higher secretion of miR-294 in 15 blastocysts versus 13 control embryos (p < 0.01). Conclusion(s): MicroRNAs are important regulators of preimplantation development. Apoptosis triggers the release of miR-294 by blastocysts which possibly serves a secretory role for embryo-maternal communication. SCM miRNA analysis is possible for individually cultured embryos and future studies can investigate miRNAs as noninvasive markers of embryo quality. [ABSTRACT FROM AUTHOR]

To assess whether morphokinetic features at the cleavage stage together with specific gene expression in cumulus cells (CCs) may be used to predict whether human embryos are able to achieve the expanded blastocyst stage on day 5. Eighty-one embryos were cultured using the Geri plus® time-lapse system. Twenty-seven embryos progressing to the expanded blastocyst stage (BL group) were compared with thirty-five embryos showing developmental arrest (AR group) and nineteen reaching the stage of early or not fully expanded blastocyst (nBL group). The analyzed morphokinetic variables were pronuclear appearance (tPNa), pronuclear fading (tPNf), and completion of cleavage to two, three, four, and eight cells (t2, t3, t4, and t8). CCs were analyzed by RT-qPCR for bone morphogenetic protein 15 (BMP15), cytochrome c oxidase subunit II (COXII), ATP synthase subunit 6 (MT-ATP6), connexin 43 (Cx43), and heme oxygenase-1 (HO-1). Embryos of BL group showed a significantly faster kinetic. BMP15, COXII, and MT-ATP6 mRNA expression was significantly higher in CCs of BL group embryos, whereas Cx43 and HO-1 mRNA levels were higher in AR group. Kinetic parameters and gene expression were not significantly different between either the BL and nBL groups or the AR and nBL groups. ROC curves showed that the most predictive cut-offs were t2 < 26.25 for morphokinetics and COXII > 0.3 for gene expression. Multivariable logistic regression analysis showed that morphokinetic variables and gene expression were both valuable, independent predictors of embryo development to expanded blastocyst. Our results suggest the possibility of developing integrated prediction models for early embryo selection at the cleavage stage. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the between-laboratory reproducibility of embryo selection/deselection effectiveness using qualitative and quantitative time-lapse parameters. Methods: A systematic search was performed on MEDLINE, EMBASE, and the Cochrane Library (up to February 2020) without restriction on date, language, document type, and publication status. Measuring outcomes included implantation, blastulation, good-quality blastocyst formation, and euploid blastocyst. Results: We detected 6 retrospective cohort studies externally validating the first clinical time-lapse model (Meseguer) emphasizing quantitative parameters, of which 3 (including one involving 2 independent centers) were included for the pooled analysis. Receiver operating characteristics analysis showed reduced predictive power of the model when either including or not including sister clinic validation. Fifteen cohort studies evaluating qualitative parameters were included for meta-analysis, and the mean Newcastle-Ottawa Scale was 5.3. Overall, meta-analysis showed significantly adverse association between the presence of ≥ 1 cleavage abnormalities and embryo implantation rates (11 studies, n = 7266; RR = 0.39[0.28, 0.55]95% CI; I2 = 57%). Further analysis showed adverse impacts of direct cleavage (7 studies, n = 7065; RR = 0.28 [0.15, 0.54] 95% CI; I2 = 46%), reverse cleavage (2 studies, n = 3622; RR = 0.16 [0.03, 0.75] 95% CI; I2 = 0%), chaotic cleavage (2 studies, n = 3643; RR = 0.11 [0.02, 0.69] 95% CI; I2 = 24%), and multinucleation (5 studies, n = 2576; RR = 0.59 [0.50, 0.69] 95% CI; I2 = 0%), but not the < 6 intercellular contact points at the 4-cell stage (1 study, n = 185; RR = 0.17 [0.02, 1.15] 95% CI). Conclusions: Qualitative time-lapse parameters are reliably associated with embryo developmental potential among laboratories, whereas the reproducibility of time-lapse embryo selection model that emphasizes quantitative parameters may be compromised when externally applied. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to establish a new method of decreasing cytoplasmic fragmentation in early-stage human embryos. Methods: The zona pellucida (ZP) of abnormally-fertilized oocytes (zygotes with three pronuclei (3PN)), which were donated by patients, was removed at the pronuclear stage. ZP-free embryos were observed in a time-lapse imaging and culturing system in order to examine developmental morphology and embryonic quality. Results: Based on a modification of Veeck's criteria, 47 of 69 ZP-free 3PN embryos (68.1%) showed fragmentation of less than 20% of the total volume of cytoplasm at the first cleavage (grades 1 and 2), 17 (24.6%) showed 20–40% cytoplasmic fragments (grade 3), and only 5 (7.2%) showed more than 40% fragments (grade 4). These results suggest that the rate of fragmentation is decreased by ZP removal before the first cleavage, compared with normal (ZP-intact) 3PN and 2-pronuclear/2-polar body embryos. Conclusions: 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. This innovative culture system might provide the major breakthrough needed for patients who have difficulty obtaining good-quality embryos. [ABSTRACT FROM AUTHOR]

Purpose: While several studies reported the association between morphokinetic parameters and implantation, few predictive models were developed to predict implantation after day 5 embryo transfer, generally without external validation. The objective of this study was to evaluate the respective performance of 2 commercially available morphokinetic-based models (KIDScore™ Day 5 versions 1 and 2) for the prediction of implantation and live birth after day 5 single blastocyst transfer. Methods: This monocentric retrospective study was conducted on 210 ICSI cycles with single day 5 embryo transfer performed with a time-lapse imaging (TLI) system between 2013 and 2016. The association between both KIDScore™ and the observed implantation and live birth rates was calculated, as well as the agreement between embryologist's choice for transfer and embryo ranking by the models. Results: Implantation and live birth rate were both 35.7%. A significant positive correlation was found between both models and implantation rate (r = 0.96 and r = 0.90, p = 0.01) respectively. Both models had statistically significant but limited predictive power for implantation (AUC 0.60). There was a fair agreement between the embryologists' choice and both models (78% and 61% respectively), with minor differences in case of discrepancies. Conclusions: KIDScore™ Day 5 predictive models are significantly associated with implantation rates after day 5 single blastocyst transfer. However, their predictive performance remains perfectible. The use of these predictive models holds promises as decision-making tools to help the embryologist select the best embryo, ultimately facilitating the implementation of SET policy. However, embryologists' expertise remains absolutely necessary to make the final decision. [ABSTRACT FROM AUTHOR]

Purpose: To assess the effect of body mass index (BMI) on morphokinetic parameters of human embryos evaluated with time-lapse technology during in vitro culture. Methods: A retrospective analysis of ART cycles utilizing time-lapse technology was undertaken to assess the potential impact of maternal BMI on morphokinetic and static morphological parameters of embryo development. The cohort of patients was divided into four groups: 593 embryos from 128 underweight women in group A; 5248 embryos from 1107 normal weight women in group B; 1053 embryos from 226 overweight women in group C; and 286 embryos from 67 obese women in group D. Results: After adjusting for maternal age, paternal age, and cause of infertility, time to reach five blastomeres (t5) and time to reach eight blastomeres (t8) were longer in obese women compared with normoweight women [50.84 h (46.31–55.29) vs. 49.24 h (45.69–53.22) and 57.89 h (51.60–65.94) vs. 55.66 h (50.89–62.89), adjusted p < 0.05 and adjusted p < 0.01, respectively]. In addition, t8 was also delayed in overweight compared with normoweight women [56.72 h (51.83–63.92) vs. 55.66 h (50.89–62.89), adjusted p < 0.01]. No significant differences were observed among groups with regard to embryo morphology and pregnancy rate. Miscarriage rate was higher in underweight compared with normoweight women (OR = 2.1; 95% CI 1.12–3.95, adjusted p < 0.05). Conclusion: Assessment with time-lapse technology but not by classical static morphology evidences that maternal BMI affects embryo development. Maternal obesity and overweight are associated with slower embryo development. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was to investigate the cause of repeated multipronucleus (MPN) formation in zygotes in a patient after both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Method: This is a case study. A patient had unexplained primary infertility with recurring total MPN zygotes after IVF and ICSI cycles. Time-lapse monitoring of pronucleus formation was carried out. Embryos developed from MPN zygotes were analyzed by fluorescence in situ hybridization (FISH). Single-cell RNA-seq analysis was used to identify gene expression profiles of the patient's oocyte and zygote, and these were compared to the data from oocytes and zygotes from donors with normal fertilization (patient, n = 1; donors, n = 4). Oocyte-specific genes with differential expression were selected by the Amazonia! database. Results: From time-lapse analysis, we observed the formation of multiple micronuclei near the site of the second polar body extrusion. These micronuclei migrated, expanded, and juxtaposed with the male pronucleus leading to a multipronucleus. None of these MPN zygotes could develop to the blastocyst stage, and FISH analysis revealed a chaotic chromosomal complement in the arrested embryos. RNA-seq analysis showed 113 differentially expressed genes (DEGs) between the patient and the donor oocytes and zygotes. Moreover, 25 of the 113 DEGs were unique or highly expressed in oocytes and early embryos. From 25 DEGs, three genes, DYNC2LI1, NEK2, and CCNH, which are involved in meiosis and the chromosome separation process, were further validated by real-time PCR. Conclusion: We identified several candidate genes affecting pronucleus formation as a new cause of infertility. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was to investigate the chromosomal constitution and the developmental potential of intracytoplasmic sperm injection (ICSI) deriving embryos displaying a single pronucleus at the zygote stage. Methods: Eighty-eight embryos from single pronucleus (1PN) two polar bodies (2PB) ICSI zygotes from 64 preimplantational genetic screening (PGS) cycles (October 2012-December 2014), were retrospectively analyzed. Zygotes were cultured in a time-lapse incubator. Embryo biopsy was performed on day 3 and genetic analysis approached by array comparative genomic hybridization. Results: Chromosomal analysis revealed that 17% (15/88) of embryos derived from 1PN 2PB zygotes were diagnosed as euploid. After blastomere biopsy at day 3, the blastocyst rate at day 5 was 3.4% (3/88). Only 2.3% (2/88) euploid blastocysts were obtained. In two couples and after counseling and patient agreement, the transfer of a euploid blastocyst from a 1PN 2PB ICSI zygote was performed resulting in the birth of a healthy child. Conclusions: These results open the possibility to consider embryos coming from 1PN 2PB ICSI zygotes for transfer when no other embryos from 2PN 2PB ICSI zygotes are available and if a PGS diagnosis of euploidy is obtained. Confirmation of biparental inheritance is strongly recommended. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to compare timings of key events of embryo development from those originating from either fresh or cryopreserved ejaculate sperm using time-lapse technology. Methods: In this retrospective observational cohort study, time-lapse technology was used to monitor 1927 embryos from 234 women undergoing intracytoplasmic sperm injection (ICSI) and utilizing either fresh ( n = 172 cycles) or cryopreserved ejaculate sperm ( n = 62 cycles) for insemination were included in the study . Key developmental events as described in time-lapse were compared with the use of generalized estimating equations (GEE) to adjust for any auto-correlation between the observations. In addition, multivariable logit regression models were used to account for any known baseline differences between the two groups. Results: There were no differences in conventional embryo development such as number of 8-cell embryos by 72 h ( p = 0.359), the number of blastocysts by 120 h ( p = 0.417), and the number of top quality blastocysts ( p = 0.956) between the two groups compared. There were no statistical differences in the timings of any of the key embryo developmental events (PN_t1, NEBD, cytokinesis, t2, t3, t4, t5, t6, t7, t8, tM, tSB, tEB, tHB, s1, s2, s3, cc2, and cc3) when either fresh or cryopreserved ejaculate sperm was used for ICSI. This was also confirmed with conventional morphological assessment. Conclusions: This observational cohort study has shown that there are no differences in the morphokinetic parameters of early embryo development when either fresh or frozen ejaculate sperm are used for ICSI insemination. [ABSTRACT FROM AUTHOR]

Purpose: This is a retrospective analysis of the morphokinetics, prevalence, and implantation potential of embryos with irregular first and second cleavages as identified by time-lapse microscopy. Methods: The study included 253 women who underwent 387 assisted reproduction treatments with intracytoplasmic sperm injection (ICSI). Each patient was assigned to one of three groups based on embryo cleavage results. In group I, one to two embryos per cycle showed irregular cleavage; group II, at least three embryos with abnormal cleavage; and in group III (the control group), all embryos cleaved normally. The number of embryos that cleaved from 1 to ≥3 cells or from 2 to ≥5 cells for each patient was recorded. Their prevalence and association with women's characteristics and pregnancy outcome were evaluated. Results: The prevalence of irregular cleavage was 15.6 % among 1772 ICSI embryos. In 101 cycles, 1-2 embryos per cycle showed irregular cleavage (group I). In 32 cycles, at least 3 embryos showed abnormal cleavage (group II). In 254 cycles, all embryos cleaved normally (group III). The average age of the women in group II was significantly lower in comparison with groups I and III (32.5 ± 4.2 vs. 35.1 ± 4.9 and 35.5 ± 5.1, respectively, p < 0.02). In comparison of groups I and II, the odds ratio for ≥3 embryos with irregular cleavage in women younger than 35 was 3.48 (95 % CI, 1.28 to 9.46). Embryos with irregular cleavage were transferred in 16 women. Three live births were achieved following the transfer of single blastocysts derived from embryos with irregular cleavage from two to five cells. Conclusions: Early embryos with irregular cleavage are significantly more prevalent in younger women. When these embryos develop to the blastocyst stage, they may have normal implantation potential, leading to the birth of healthy babies. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of our study was to use time-lapse in order to evaluate the impact of sperm origin (fresh ejaculate or surgically retrieved) on embryo morphokinetic parameters and clinical outcome in intracytoplasmic sperm injection (ICSI) cycles. Methods: This retrospective monocentric study was conducted in 485 unselected couples undergoing 604 ICSI cycles with embryo culture in the Embryoscope®. Among them, 445 couples underwent ICSI cycle with fresh ejaculated sperm and 40 with surgically retrieved sperm (26 with testicular sperm and 14 with epididymal sperm). Embryo morphokinetic parameters and clinical cycle outcome were compared between fresh ejaculated sperm and surgically retrieved sperm. A subgroup analysis was also conducted between testicular and epididymal sperm ICSI cycles. Results: Clinical outcome was comparable between groups according to sperm origin. Although most early morphokinetic parameters were comparable between ejaculated and surgical sperm groups, a few parameters were significantly different between both groups, but with a considerable overlap in their distribution. Late cellular events occurred significantly later in the surgical sperm group than in the ejaculated sperm group. Conclusions: Morphokinetic analysis did not allow us to identify clinically relevant differences between fresh ejaculate and surgically retrieved sperm groups. Further studies are needed, especially concerning the relationship between sperm origin and late morphokinetic parameters, such as blastocyst development. [ABSTRACT FROM AUTHOR]

Purpose: The aims of this prospective study were to evaluate whether time-lapse parameters can aid in the prediction of day 5 embryo quality and also to assess their discriminatory capacity. Methods: In this prospective cohort study, we used time-lapse technology to record specific timings of key events for 380 day 5 blastocysts (originating from 108 patients). Generalized estimating equation regression models were used to evaluate the capacity of these markers to identify a top-quality blastocyst. Multivariable regression models were also constructed, aiming to identify the model with the highest capacity to predict a top-quality blastocyst. The discriminatory capacity of single predictors or composite models was assessed with the use of receiver operating characteristic (ROC) analyses. Results: Eight significant predictive parameters of a top-quality blastocyst were identified: s3, t6, t7, t8, tM, tSB, tB and tEB. A ROC analysis of the identified parameters found s3 (area under the curve-AUC 0.585, 95 % CI 0.534-0.635) to have the best individual discriminatory capacity to predict a top-quality blastocyst prior to embryo compaction. The parameter tEB (AUC 0.727, 95 % CI 0.675-0.775) was the best predictor regardless of embryo stage. A model containing s3, t8 and tEB showed a slightly increased discriminatory capacity for top-quality blastocyst prediction (AUC 0.748, 95 % CI 0.697-0.794). Conclusions: The identified morphokinetic parameters and their cutoffs, albeit of limited clinical value, add to the increasing knowledge concerning the potential predictive markers of a top-quality blastocyst. Additional evidence is necessary before validated time-lapse parameters can be used for embryo selection in IVF laboratories. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to undertake a critical appraisal of the available evidence for the use of time-lapse imaging for embryo selection in clinical IVF. Methods: A literature search in PubMed, Scopus, Cochrane Central, ClinicalTrials.gov, Current Controlled Trials, and WHO International Clinical Trials Registry Platform was performed to identify randomized controlled trials that investigated the effect of time-lapse embryo selection and/or the time-lapse incubation system on ongoing pregnancy rate. We then performed a systematic review and assessed the relative risks (RRs) and 95 % confidence intervals (CIs) for ongoing pregnancy rates and the risk of bias of the eligible studies. Results: We identified four eligible randomized studies, three of which investigated the effect of both time-lapse incubation system and selection on ongoing pregnancy rate; the pooled result revealed a benefit of this intervention (relative risk (RR) 1.20; 95 % CI 1.05-1.37). However, the evidence was judged to be of low quality due to study limitations; a beneficial effect was observed in only one study deemed to be at high risk of bias. The single study assessing the effect of only the time-lapse incubation system revealed a non-significant negative effect (RR 0.71; 95 % CI 0.49-1.03). Conclusions: The findings from this systematic review of the current evidence do not support routine use of time-lapse technology in clinical IVF. We therefore believe that the use of time-lapse imaging for embryo selection should remain experimental and that couples should not be subject to a surcharge for having their embryos cultured in a time-lapse imaging system. Future studies evaluating this technology in well-designed trials should be performed. [ABSTRACT FROM AUTHOR]

Purpose: Oocyte vitrification does not affect embryo quality after oocyte warming, making this method effective in the preservation of female fertility. Morphokinetic parameters can be used to predict the competence of an embryo produced from fresh oocytes. Our aim was to study the effect of oocyte vitrification on zygote-embryo kinetics (pl). Methods: The embryo-kinetics of fresh and sibling vitrified/warmed oocytes were compared to determine the consequences of oocyte preservation on the timing of embryo development. A 44-hours time-lapse analysis, from the time of ICSI (t0), of 179 fertilized fresh oocytes was compared to 168 fertilized sibling vitrified/warmed oocytes. Results: Oocyte vitrification accelerated pronuclear disappearance, one-cell stage timing and modified nucleoli activity by increasing their number and decreasing their diameter at the zygote stage. In contrast, embryo kinetics during cleavage were similar to those observed for fresh sibling oocytes based on the parameters examined in this study. Conclusions: At the zygote stage, oocyte vitrification induces changes in pronuclei stability, probably due to pronuclei envelop instability as well as modifications in nucleoli functionality. Therefore, the predictive morphokinetic parameters on embryo competence found from fresh oocytes must be revised when applied on embryos from vitrified/warmed oocytes. [ABSTRACT FROM AUTHOR]

Purpose: To determine if Aneuploidy Risk Classification Models are predictive of euploidy/aneuploidy amongst IVF facilities. Methods: We retrospectively applied key time lapse imaging events of embryos (Campbell et al.[, ]) to stratify embryos into 3 groups: low, medium and high risk of aneuploidy. The actual ploidy results (from array comparative genomic hybridization) were compared with expectations [, ]. Sources of variability in morphokinetic parameters were determined using Analysis of Variance (ANOVA). Results: The model failed to segregate euploid embryos from aneuploid embryos cultured at our facility. Further analysis indicated that the variability of embryos among patients was too great to allow selection of euploid embryos based on simple morphokinetic thresholds. Clinical selection of embryos based on morphokinetics alone is unlikely to identify euploid embryos accurately for transfer or yield higher rates of live delivery. Conclusions: The use of non-invasive morphokinetics is unlikely to discriminate aneuploid from euploid embryos. Further, it does not approach the accuracy of preimplantation genetic screening with array comparative genomic hybridization. [ABSTRACT FROM AUTHOR]

Purpose: To analyze the cleavage patterns in dipronuclear (2PN) and tripronuclear (3PN) embryos in relation to fertilization method. Method: Time-lapse analysis. Results: Compared to 2PN, more 3PN IVF embryos displayed early cleavage into 3 cells ( p < 0.001), displayed longer duration of the 3-cell stage ( p < 0.001), and arrested development from the compaction stage and onwards ( p < 0.001). For the IVF embryos, the 2nd and 3rd cleavage cycles were completed within the expected time frame. However, timing of the cell divisions within the cleavage cycles differed between the two groups. In contrast, the completion of the 1st, 2nd, and 3rd cleavage cycle was delayed, but with a similar division pattern for 3PN ICSI compared with the 2PN ICSI embryos. 3PN, more often than 2PN ICSI embryos, displayed early cleavage into 3 cells ( p = 0.03) and arrested development from the compaction stage and onwards ( p = 0.001). More 3PN IVF than ICSI embryos displayed early cleavage into 3 cells ( p < 0.001). Conclusions: This study reports differences in cleavage patterns between 2PN and 3PN embryos and for the first time demonstrates differences in the cleavage pattern between 3PN IVF and ICSI embryos. [ABSTRACT FROM AUTHOR]

Purpose: To review relevant studies examining the relationship between embryo morpho-kinetics and aneuploidy. Methods: Search of Pubmed and Medline using relevant keywords pertaining to morphology, morphokinetics and embryonic aneuploidy, as well as examination of various reference lists and conference proceedings. Results: An abundance of publications, both preliminary and peer-reviewed, have emerged regarding the usefulness of time-lapse imaging in tracking embryo development and improving embryo selection. Recently, these publications have explored ability to not only predict blastocyst formation and implantation, but also the ability to detect embryonic chromosomal aneuploidy. Of the two peer-reviewed retrospective studies on morpho-kinetics and embryonic aneuploidy, one demonstrates that early cleavage timings can indicate chromosomal complement, while the other demonstrates that key events following the maternal-zygotic transition can be markers of aneuploidy. A recent paper also demonstrates improved outcomes following IVF using a selection algorithm to identify embryos at 'low risk' of chromosomal abnormalities. However, the predictive nature of these events and timings is far from ideal. Additionally, results may be dependent upon the day of biopsy and method utilized for chromosomal assessment. Conclusion: With continued effort, the combination of multiple morphologic endpoint assessments and developmental timings and refinement of modeling systems may improve the predictive ability to determine embryonic aneuploidy. This may help select a subset of embryos that are less likely to carry chromosomal abnormalities and improve assisted reproductive outcomes. However, embryo biopsy, followed by preimplantation genetic screening/comprehensive chromosomal screening still remains the most reliable method to assess chromosomal complement of preimplantation embryos. [ABSTRACT FROM AUTHOR]