SUMOylation of linker histone H1 drives chromatin condensation and restriction of embryonic cell fate identity.

The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultra-condensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity.

Ex utero mouse embryogenesis from pre-gastrulation to late organogenesis.

The mammalian body plan is established shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. Although pre- and peri-implantation mouse embryos are routinely cultured in vitro1,2, approaches for the robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. Here we present highly effective platforms for the ex utero culture of post-implantation mouse embryos, which enable the appropriate development of embryos from before gastrulation (embryonic day (E) 5.5) until the hindlimb formation stage (E11). Late gastrulating embryos (E7.5) are grown in three-dimensional rotating bottles, whereas extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture platforms. Histological, molecular and single-cell RNA sequencing analyses confirm that the ex utero cultured embryos recapitulate in utero development precisely. This culture system is amenable to the introduction of a variety of embryonic perturbations and micro-manipulations, the results of which can be followed ex utero for up to six days. The establishment of a system for robustly growing normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool for investigating embryogenesis, as it eliminates the uterine barrier and allows researchers to mechanistically interrogate post-implantation morphogenesis and artificial embryogenesis in mammals.

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.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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). INTERPRETATION: 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. FUNDING: Barts Charity, Pharmasure Pharmaceuticals, Hong Kong OG Trust Fund, Hong Kong Health and Medical Research Fund, Hong Kong Matching Fund.

Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts.

SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation.

Quantitative Proteomics and Metabolomics of Culture Medium from Single Human Embryo Reveal Embryo Quality-Related Multiomics Biomarkers.

An effective tool to assess embryo quality in the assisted reproduction clinical practice will enhance successful implantation rates and mitigate high risks of multiple pregnancies. Potential biomarkers secreted into culture medium (CM) during embryo development enable rapid and noninvasive methods of assessing embryo quality. However, small volumes, low biomolecule concentrations, and impurity interference collectively preclude the identification of quality-related biomarkers in single blastocyst CM. Here, we developed a noninvasive trace multiomics approach to screen for potential markers in individual human blastocyst CM. We collected 84 CM samples and divided them into high-quality (HQ) and low-quality (LQ) groups. We evaluated the differentially expressed proteins (DEPs) and metabolites (DEMs) in HQ and LQ CM. A total of 504 proteins and 189 metabolites were detected in individual blastocyst CM. Moreover, 9 DEPs and 32 DEMs were identified in different quality embryo CM. We also categorized HQ embryos into positive implantation (PI) and negative implantation (NI) groups based on ultrasound findings on day 28. We identified 41 DEPs and 4 DEMs associated with clinical implantation outcomes in morphologically HQ embryos using a multiomics analysis approach. This study provides a noninvasive multiomics analysis technique and identifies potential biomarkers for clinical embryo developmental quality assessment.

In vitro culture alters cell lineage composition and cellular metabolism of bovine blastocyst†.

Profiling bovine blastocyst transcriptome at the single-cell level has enabled us to reveal the first cell lineage segregation, during which the inner cell mass (ICM), trophectoderm (TE), and an undefined population of transitional cells were identified. By comparing the transcriptome of blastocysts derived in vivo (IVV), in vitro from a conventional culture medium (IVC), and in vitro from an optimized reduced nutrient culture medium (IVR), we found a delay of the cell fate commitment to ICM in the IVC and IVR embryos. Developmental potential differences between IVV, IVC, and IVR embryos were mainly contributed by ICM and transitional cells. Pathway analysis of these non-TE cells between groups revealed highly active metabolic and biosynthetic processes, reduced cellular signaling, and reduced transmembrane transport activities in IVC embryos that may lead to reduced developmental potential. IVR embryos had lower activities in metabolic and biosynthetic processes but increased cellular signaling and transmembrane transport, suggesting these cellular mechanisms may contribute to improved blastocyst development compared to IVC embryos. However, the IVR embryos had compromised development compared to IVV embryos with notably over-active transmembrane transport activities that impaired ion homeostasis.

Interleukin-6 supplementation improves bovine conceptus elongation and transcriptomic indicators of developmental competence†.

A high incidence of pregnancy failures occurs in cattle during the second week of pregnancy as blastocysts transition into an elongated conceptus. This work explored whether interleukin-6 supplementation during in vitro embryo production would improve subsequent conceptus development. Bovine embryos were treated with 0 or 100 ng/mL recombinant bovine interleukin-6 beginning on day 5 post-fertilization. At day 7.5 post-fertilization, blastocysts were transferred into estrus synchronized beef cows (n = 5 recipients/treatment, 10 embryos/recipient). Seven days after transfer (day 14.5), cows were euthanized to harvest reproductive tracts and collect conceptuses. Individual conceptus lengths and stages were recorded before processing for RNA sequencing. Increases in conceptus recovery, length, and the proportion of tubular and filamentous conceptuses were detected in conceptuses derived from interleukin-6-treated embryos. The interleukin-6 treatment generated 591 differentially expressed genes in conceptuses (n = 9-10/treatment). Gene ontology enrichment analyses revealed changes in transcriptional regulation, DNA-binding, and antiviral actions. Only a few differentially expressed genes were associated with extraembryonic development, but several differentially expressed genes were associated with embryonic regulation of transcription, mesoderm and ectoderm development, organogenesis, limb formation, and somatogenesis. To conclude, this work provides evidence that interleukin-6 treatment before embryo transfer promotes pre-implantation conceptus development and gene expression in ways that resemble the generation of a robust conceptus containing favorable abilities to survive this critical period of pregnancy.

The number of nuclei in compacted embryos, assessed by optical coherence microscopy, is a non-invasive and robust marker of mouse embryo quality.

Optical coherence microscopy (OCM) visualizes nuclei in live, unlabeled cells. As most cells are uninucleated, the number of nuclei in embryos may serve as a proxy of the cell number, providing important information on developmental status of the embryo. Importantly, no other non-invasive method currently allows for the cell number count in compacted embryos. We addressed the question of whether OCM, by providing the number of nuclei in compacted mouse embryos, may help evaluate embryo quality. We subjected compacted embryonic Day 3 (E3.0: 72 h after onset of insemination) mouse embryos to OCM scanning and correlated nuclei number and developmental potential. Implantation was assessed using an outgrowth assay (in vitro model meant to reflect embryonic ability to implant in vivo). Embryos with more cells at E3.0 (>18 cells) were more likely to reach the blastocyst stage by E4.0 and E5.0 (P ≪ 0.001) and initiate hatching by E5.0 (P < 0.05) than those with fewer cells (<12 cells). Moreover, the number of cells at E3.0 strongly correlated with the total number of cells in E4.0 and E5.0 embryos (ρ = 0.71, P ≪ 0.001 and ρ = 0.61, P ≪ 0.001, respectively), also when only E4.0 and E5.0 blastocysts were considered (ρ = 0.58, P ≪ 0.001 and ρ = 0.56, P ≪ 0.001, respectively). Additionally, we observed a strong correlation between the number of cells at E3.0 and the number of trophectoderm cells in E4.0 and E5.0 blastocysts (ρ = 0.59, P ≪ 0.001 and ρ = 0.57, P ≪ 0.001, respectively). Importantly, embryos that had more cells at E3.0 (>18 cells) were also more likely to implant in vitro than their counterparts with fewer cells (<12 cells; P ≪ 0.001). Finally, we tested the safety of OCM imaging, demonstrating that OCM scanning affected neither the amount of reactive oxygen species nor mitochondrial activity in the embryos. OCM also did not hinder their preimplantation development, ability to implant in vitro, or to develop to term after transfer to recipient females. Our data indicate that OCM imaging provides important information on embryo quality. As the method seems to be safe for embryos, it could be a valuable addition to the current repertoire of embryo evaluation methods. However, our study was conducted only on mouse embryos, so the proposed protocol would require optimization in order to be applied in other species.

Morphological quality on Day 3 affects the pregnancy outcomes of low-quality euploid blastocysts: a retrospective cohort study.

STUDY QUESTION: Does the morphological quality on Day 3 influence the pregnancy outcomes of euploid blastocysts? SUMMARY ANSWER: The morphological quality on Day 3 affects the clinical pregnancy rate (CPR) and live birth rate (LBR) of low-quality euploid blastocysts. WHAT IS KNOWN ALREADY: The morphological grading of Day 3 embryos affects the pregnancy outcome of cleavage-stage embryos and is an excellent indicator to predict embryo development potential. However, it is still unclear whether morphological quality on Day 3 is associated with pregnancy outcomes of the euploid blastocyst. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study comprised 1275 patients who received single euploid blastocyst transfer between January 2016 and August 2021 at a tertiary teaching hospital. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were grouped into two groups according to the morphological grading on Day 3 of transferred blastocysts: high-quality (HQ, including Grades I and II) Day 3 embryos and low-quality (LQ, Grade III) Day 3 embryos. The primary outcomes were CPR and LBR. Interactions of development days (Day 5 and Day 6) and morphological quality (high- and low-quality) of blastocysts with morphological quality of Day 3 embryos on pregnancy outcomes were tested in the stratified analysis and logistic regression models. The multivariate logistic regression analysis was conducted to investigate the independent effect of the morphological quality of Day 3 embryos on pregnancy outcomes after adjusting for potentially confounding factors. MAIN RESULTS AND THE ROLE OF CHANCE: The CPR and LBR of the HQ Day 3 embryos group were statistically higher than those of the LQ Day 3 embryos group (CPR: 59.73% versus 49.70%, respectively, P = 0.015; LBR: 49.73% versus 41.21%, respectively, P = 0.041). The development days of blastocysts did not exhibit a multiplicative interaction with the morphological quality of Day 3 embryos on the CPR (P for interaction = 0.648) and LBR (P for interaction = 0.925). The morphological quality of blastocysts exhibits a multiplicative interaction with the morphological quality of Day 3 embryos on the CPR (P for interaction = 0.020) and LBR (P for interaction = 0.012). After adjusting for potential confounders, the HQ Day 3 embryo group was positively associated with the CPR (adjusted odds ratio (aOR): 2.10, 95% CI: 1.31-3.36, P = 0.002) and LBR (aOR: 1.97, 95% CI: 1.20-3.25, P = 0.008) of LQ blastocysts. However, the morphological quality on Day 3 was not significantly associated with the CPR (aOR: 0.95, 95% CI: 0.58-1.55, P = 0.835) and LBR (aOR: 0.86, 95% CI: 0.53-1.40, P = 0.550) of HQ blastocysts. LIMITATIONS, REASONS FOR CAUTION: Selection and confounding bias introduced by the retrospective design cannot be completely eliminated in this study, although multivariable logistic analysis was conducted to adjust for potential confounders. Also, some subgroups had small sample sizes, which may reduce statistical power. Moreover, participants in our study only received single euploid blastocyst transfer, and whether the results could apply to blastocysts with unknown ploidy status is unclear. WIDER IMPLICATIONS OF THE FINDINGS: This study found that the morphological quality on Day 3 was significantly associated with the CPR and LBR of LQ blastocysts; Therefore, when only LQ euploid blastocysts are available for transfer, blastocysts derived from HQ Day 3 embryos are recommended. STUDY FUNDING/COMPETING INTEREST(S): No external funding was obtained. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Abnormal cleavage up to Day 3 does not compromise live birth and neonatal outcomes of embryos that have achieved full blastulation: a retrospective cohort study.

STUDY QUESTION: Do embryos displaying abnormal cleavage (ABNCL) up to Day 3 have compromised live birth rates and neonatal outcomes if full blastulation has been achieved prior to transfer? SUMMARY ANSWER: ABNCL is associated with reduced full blastulation rates but does not impact live birth rates and neonatal outcomes once full blastulation has been achieved. WHAT IS KNOWN ALREADY?: It is widely accepted that ABNCL is associated with reduced implantation rates of embryos when transferred at the cleavage stage. However, evidence is scarce in the literature reporting birth outcomes from blastocysts arising from ABNCL embryos, likely because they are ranked low priority for transfer. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study included 1562 consecutive autologous in vitro fertilization cycles (maternal age 35.1 ± 4.7 years) performed at Fertility North, Australia between January 2017 and June 2022. Fresh transfers were performed on Day 3 or 5, with remaining embryos cultured up to Day 6 before vitrification. A total of 6019 embryos were subject to blastocyst culture, and a subset of 664 resulting frozen blastocysts was included for live birth and neonatal outcome analyses following single transfers. PARTICIPANTS/MATERIALS, SETTING, METHODS: ABNCL events were annotated from the first mitotic division up to Day 3, including direct cleavage (DC), reverse cleavage (RC) and <6 intercellular contact points at the 4-cell stage (<6ICCP). For DC and RC in combination, the ratios of affected blastomeres over the total number of all blastomeres up to Day 3 were also recorded. All pregnancies were followed up until birth with gestational age, birthweight, and sex of the baby being recorded. MAIN RESULTS AND THE ROLE OF CHANCE: Full blastulation rates for embryos showing DC (19.5%), RC (41.7%), <6ICCP (58.8%), and mixed (≥2) ABNCL types (26.4%) were lower than the rates for those without ABNCL (67.2%, P < 0.01 respectively). Subgroup analysis showed declining full blastulation rates with increasing ratios of combined DC/RC affected blastomeres over all blastomeres up to the 8-cell stage (66.2% when 0 affected, 47.0% when 0.25 affected, 27.4% when 0.5 affected, 14.5% when 0.75 affected, and 7.7% when all affected, P < 0.01). However, once full blastulation had been achieved, no difference was detected between DC, RC, <6ICCP, and no ABNCL blastocysts following single frozen transfers in subsequent live birth rates (25.9%, 33.0%, 36.0% versus 30.8%, P > 0.05, respectively), gestational age (38.7 ± 1.6, 38.5 ± 1.2, 38.3 ± 3.5 versus 38.5 ± 1.8 weeks, P > 0.05, respectively) and birthweight (3343.0 ± 649.1, 3378.2 ± 538.4, 3352.6 ± 841.3 versus 3313.9 ± 509.6 g, P > 0.05, respectively). Multiple regression (logistic or linear as appropriate) confirmed no differences in all of the above measures after accounting for potential confounders. LIMITATIONS, REASONS FOR CAUTION: Our study is limited by its retrospective nature, making it impossible to control every known or unknown confounder. Embryos in our dataset, being surplus after selection for fresh transfer, may not represent the general embryo population. WIDER IMPLICATIONS OF THE FINDINGS: Our findings highlight the incremental impact of ABNCL, depending on the ratio of affected blastomeres up to Day 3, on subsequent full blastulation. The reassuring live birth and neonatal outcomes of ABNCL blastocysts imply a potential self-correction mechanism among those embryos reaching the blastocyst stage, which provides valuable guidance for clinical practice and patient counseling. STUDY FUNDING/COMPETTING INTEREST(S): This research is supported by an Australian Government Research Training Program (RTP) Scholarship. All authors report no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

In silico-designed vitrification protocols: an approach to improve survival of in vitro produced-bovine embryos.

In brief: In silico predictions validated in this study demonstrate the potential for designing shorter equilibration protocols that improve post-warming re-expansion and hatching rates of D7 and D8 in vitro-produced bovine embryos. Our results benefit the livestock industry by providing a refined and reproducible approach to cryopreserving bovine embryos, which, in addition, could be useful for other mammalian species. Abstract: The cryopreservation of in vitro-produced (IVP) embryos is vital in the cattle industry for genetic selection and crossbreeding programs. Despite its importance, there is no standardized protocol yielding pregnancy rates comparable to fresh embryos. Current approaches often neglect the osmotic tolerance responses to cryoprotectants based on temperature and time. Hereby, we propose improved vitrification methods using shorter dehydration-based protocols. Blastocysts cultured for 7 (D7) or 8 days (D8) were exposed to standard equilibration solution (ES) at 25ºC and 38.5ºC. Optimized exposure times for each temperature and their impact on post-warming re-expansion, hatching rates, cell counts, and apoptosis rate were determined. In silico predictions aligned with in vitro observations, showing original volume recovery within 8 min 30 s at 25ºC or 3 min 40 s at 38.5ºC (D7 blastocysts) and 4 min 25 s at 25ºC and 3 min 15 s at 38.5ºC (D8 blastocysts) after exposure to ES. Vitrification at 38.5ºC resulted in D7 blastocysts re-expansion and hatching rates (93.1% and 38.1%, respectively) comparable to fresh embryos (100.0% and 32.4%, respectively), outperforming the 25ºC protocol (86.2% and 24.4%, respectively; P < 0.05). No differences were observed between D7 and D8 blastocysts using the 38.5ºC protocol. Total cell number was maintained for D7 and D8 blastocysts vitrified at 38.5ºC but decreased at 25ºC (P < 0.05). Apoptosis rates increased post-warming (P < 0.05), except for D8 blastocysts vitrified at 38.5ºC, resembling fresh controls. In conclusion, based on biophysical permeability data, new ES incubation times of 3 min 40 s for D7 blastocysts and 3 min 15 s for D8 blastocysts at 38.5ºC were validated for optimizing vitrification/warming methods for bovine IVP blastocysts.

L-Carnitine sustainably affects bioenergetic profile of bovine blastocysts and transcriptome profile of elongation-stage embryos.

In brief: In the present study the sustainable effect of L-carnitine during the culture period on the post-transfer development was investigated. Taken together, we uncovered direct effects of L-carnitine on the bioenergetic profile of day 7 blastocysts along with sustainable effects on mtDNA copy numbers and transcriptome profile of bovine day 14 embryos. Abstract: L-Carnitine (LC) is known to play key roles in lipid metabolism and antioxidative activity, implicating enhanced cryotolerance of bovine blastocysts. However, sustainability of LC supplementation during culture period on preimplantation development beyond the blastocyst stage has not been investigated so far. Therefore, all embryos were cultured under fatty acid-free conditions, one group with LC (LC embryos) and the control group without LC (control) supplementation. Transfer to recipients was conducted on day 6. Elongation-stage embryos were recovered on day 14; metrics of embryo recollection, developmental rates as regards early elongation-stage as well as mean embryo length did not differ between the groups. Gene expression analyses via NGS revealed 341 genes to be differentially regulated between elongation-stage embryos derived from LC supplementation compared to controls. These played mainly a role in molecular functions and biological processes like oxidoreductase activity, ATP-dependent activity, cellular stress, and respiration. Pathways like oxidative phosphorylation and thermogenesis, extracellular matrix receptor signaling, PI3K-Akt, and focal adhesion were affected by differentially regulated genes. Moreover, all DEGs located on the mitochondria were significantly downregulated in LC embryos, being in line with lower mitochondrial copy number and mtDNA integrity compared to the control group. Finally, we uncovered alterations of the bioenergetic profile on day 7 as a consequence of LC supplementation for the first time, revealing significantly higher oxygen consumption rates, ATP linked respiration and spare capacity for LC embryos. In summary, we uncovered direct effects of LC supplementation during the culture period on the bioenergetic profile along with sustainable effects on mtDNA copy numbers and transcriptome profile of bovine day 14 embryos.

Long-term culture induces Bax-dependent apoptosis in rat preimplantation embryos.

Although rat preimplantation embryos are necessary for producing genetically modified rats, their in vitro culture remains a challenge. Rat zygotes can develop from the one-cell stage to the blastocyst stage in vitro; however, long-term culture reduces their developmental competence via an unknown mechanism. In this study, we examined how in vitro conditions affect rat preimplantation embryos, which may explain this reduced competence. Comprehensive gene expression analysis showed that genes related to apoptosis and energy metabolism were differentially expressed in rat embryos cultured long-term in vitro compared with those developed in vivo. Furthermore, we found that the expression of Bak1 and Bax, which are responsible for mitochondrial outer membrane permeabilization, were more upregulated in embryos cultured in vitro than those developed in vivo. Similarly, apoptosis-dependent DNA fragmentation was also exacerbated in in vitro culture conditions. Finally, gene disruption using CRISPR/Cas9 showed that Bax, but not Bak1, was responsible for these effects. These findings suggest that long-term in vitro culture induces Bax-dependent apoptosis through the mitochondrial pathway and may provide clues to improve the long-term culture of rat preimplantation embryos for genetic engineering research.

Multiple collapses of blastocysts after full blastocyst formation is an independent risk factor for aneuploidy - a study based on AI and manual validation.

BACKGROUND: The occurrence of blastocyst collapse may become an indicator of preimplantation embryo quality assessment. It has been reported that collapsing blastocysts can lead to higher rates of aneuploidy and poorer clinical outcomes, but more large-scale studies are needed to explore this relationship. This study explored the characteristics of blastocyst collapse identified and quantified by artificial intelligence and explored the associations between blastocyst collapse and embryo ploidy, morphological quality, and clinical outcomes. METHODS: This observational study included data from 3288 biopsied blastocysts in 1071 time-lapse preimplantation genetic testing cycles performed between January 2019 and February 2023 at a single academic fertility center. All transferred blastocysts are euploid blastocysts. The artificial intelligence recognized blastocyst collapse in time-lapse microscopy videos and then registered the collapsing times, and the start time, the recovery duration, the shrinkage percentage of each collapse. The effects of blastocyst collapse and embryo ploidy, pregnancy, live birth, miscarriage, and embryo quality were studied using available data from 1196 euploid embryos and 1300 aneuploid embryos. RESULTS: 5.6% of blastocysts collapsed at least once only before the full blastocyst formation (tB), 19.4% collapsed at least once only after tB, and 3.1% collapsed both before and after tB. Multiple collapses of blastocysts after tB (times ≥ 2) are associated with higher aneuploid rates (54.6%, P > 0.05; 70.5%, P < 0.001; 72.5%, P = 0.004; and 71.4%, P = 0.049 in blastocysts collapsed 1, 2, 3 or ≥ 4 times), which remained significant after adjustment for confounders (OR = 2.597, 95% CI 1.464-4.607, P = 0.001). Analysis of the aneuploid embryos showed a higher ratio of collapses and multiple collapses after tB in monosomies and embryos with subchromosomal deletion of segmental nature (P < 0.001). Blastocyst collapse was associated with delayed embryonic development and declined blastocyst quality. There is no significant difference in pregnancy and live birth rates between collapsing and non-collapsing blastocysts. CONCLUSIONS: Blastocyst collapse is common during blastocyst development. This study underlined that multiple blastocyst collapses after tB may be an independent risk factor for aneuploidy which should be taken into account by clinicians and embryologists when selecting blastocysts for transfer.

Investigating developmental characteristics of biopsied blastocysts stratified by mitochondrial copy numbers using time-lapse monitoring.

BACKGROUND: For in vitro fertilization (IVF), mitochondrial DNA (mtDNA) levels in the trophectodermal (TE) cells of biopsied blastocysts have been suggested to be associated with the cells' developmental potential. However, scholars have reached differing opinions regarding the use of mtDNA levels as a reliable biomarker for predicting IVF outcomes. Therefore, this study aims to assess the association of mitochondrial copy number measured by mitoscore associated with embryonic developmental characteristics and ploidy. METHODS: This retrospective study analyzed the developmental characteristics of embryos and mtDNA levels in biopsied trophectodermal cells. The analysis was carried out using time-lapse monitoring and next-generation sequencing from September 2021 to September 2022. Five hundred and fifteen blastocysts were biopsied from 88 patients undergoing IVF who met the inclusion criteria. Embryonic morphokinetics and morphology were evaluated at 118 h after insemination using all recorded images. Blastocysts with appropriate morphology on day 5 or 6 underwent TE biopsy and preimplantation genetic testing for aneuploidy (PGT-A). Statistical analysis involved generalized estimating equations, Pearson's chi-squared test, Fisher's exact test, and Kruskal-Wallis test, with a significance level set at P < 0.05. RESULTS: To examine differences in embryonic characteristics between blastocysts with low versus high mitoscores, the blastocysts were divided into quartiles based on their mitoscore. Regarding morphokinetic characteristics, no significant differences in most developmental kinetics and observed cleavage dysmorphisms were discovered. However, blastocysts in mitoscore group 1 had a longer time for reaching 3-cell stage after tPNf (t3; median: 14.4 h) than did those in mitoscore group 2 (median: 13.8 h) and a longer second cell cycle (CC2; median: 11.7 h) than did blastocysts in mitoscore groups 2 (median: 11.3 h) and 4 (median: 11.4 h; P < 0.05). Moreover, blastocysts in mitoscore group 4 had a lower euploid rate (22.6%) and a higher aneuploid rate (59.1%) than did those in the other mitoscore groups (39.6-49.3% and 30.3-43.2%; P < 0.05). The rate of whole-chromosomal alterations in mitoscore group 4 (63.4%) was higher than that in mitoscore groups 1 (47.3%) and 2 (40.1%; P < 0.05). A multivariate logistic regression model was used to analyze associations between the mitoscore and euploidy of elective blastocysts. After accounting for factors that could potentially affect the outcome, the mitoscore still exhibited a negative association with the likelihood of euploidy (adjusted OR = 0.581, 95% CI: 0.396-0.854; P = 0.006). CONCLUSIONS: Blastocysts with varying levels of mitochondrial DNA, identified through biopsies, displayed similar characteristics in their early preimplantation development as observed through time-lapse imaging. However, the mitochondrial DNA level determined by the mitoscore can be used as a standalone predictor of euploidy.

The unbearable ignorance of the composition of IVF culture media.

Culture media play an essential role in the success of IVF. Their composition has undergone major modifications over the 45 years since the birth of Louise Brown. Most IVF programmes now rely on commercially produced media, which they buy in small vials, guaranteed to be sterile and non-embryotoxic. Unfortunately, information about the components of the culture media and their concentrations is no longer available. Arguing that culture media recipes are proprietary, relevant commercial interests have stopped labelling their products with this vital information. Given the critical role that is played by culture media in the success of IVF, as well as the subsequent health of the children who are born after IVF, this information should not remain a 'company secret'. Clinicians and scientists working in IVF must insist that the labelling of culture media includes all of the constituents and their concentrations. Only in this way can we monitor the influence of culture media on IVF outcomes, innovate and continue to advance the field of IVF.

A compact, high-throughput semi-automated embryo vitrification system based on hydrogel.

RESEARCH QUESTION: What is the efficiency and efficacy of the novel Biorocks semi-automated vitrification system, which is based on a hydrogel? DESIGN: This comparative experimental laboratory study used mouse model and human day 6 blastocysts. Mouse oocytes and embryos were quality assessed post-vitrification. RESULTS: The Biorocks system successfully automated the solution exchanges during the vitrification process, achieving a significantly improved throughput of up to 36 embryos/oocytes per hour. Using hydrogel for cryoprotective agent delivery, 12 vessels could be processed simultaneously, fitting comfortably within an assisted reproductive technology (ART) workstation. In tests involving the cryopreservation of oocytes and embryos, the system yielded outcomes equivalent to the manual Cryotop method. For example, the survival rate for mouse oocytes was 98% with the Biorocks vitrification system (n = 46) and 95% for the manual Cryotop method (n = 39), of which 46% and 41%, respectively, progressed to blastocysts on day 5 after IVF. CC-grade day 6 human blastocysts processed with the Biorocks system (n = 39) were associated with a 92% 2 h re-expansion rate, equivalent to the 90% with Cryotop (n = 30). The cooling/warming rates achieved by the Biorocks system were 31,900°C/minute and 24,700°C/minute, respectively. Oocyte quality was comparable or better post-vitrification for Biorocks than Cryotop. CONCLUSIONS: The Biorocks semi-automated vitrification system offers enhanced throughput without compromising the survival and developmental potential of oocytes and embryos. This innovative system may help to increase the efficiency and standardization of vitrification in ART clinics. Further investigations are needed to confirm its efficacy in a broader clinical context.

Temperature fluctuations during embryo transfer can be mitigated by optimizing transfer protocol.

RESEARCH QUESTION: What impact do variations in embryo transfer catheter loading and movement procedures have on temperature and pH fluctuations during embryo transfer? DESIGN: Mock embryo transfers were conducted to test the impact of air flow/movement, use of catheter coverings, and the type of workstation used for catheter loading on catheter temperature. A thermocouple probe inserted into the tip of the outer catheter or taped to the exterior of the inner catheter recorded temperature within the catheter every 5 s from time of mock embryo loading (TL) to 60 s (TL + 60 s) or from the start of transit (TT). Fluctuations in culture medium pH in embryo transfer dishes were monitored. RESULTS: The rate of cooling during transit was faster (all P < 0.05) when catheters were uncovered compared with all covering methods tested. This resulted in a lower catheter temperature at TL + 20 s (28.43 ± 0.30 °C) compared with catheters covered by plastic tubing (31.4 ± 0.30 °C), paper (31.0 ± 0.26 °C) or paper + thumb (31.1 ± 0.78 °C; all P ≤ 0.05). Temperature was maintained more effectively when catheters were loaded in a crib compared with a heated stage, until initiation of transit, when the rate of temperature decrease was similar. Culture medium pH increased more rapidly when embryo transfer dishes remained on a heated stage during the procedure compared with in an open crib. CONCLUSIONS: Temperature loss during the embryo transfer procedure can be mitigated by reducing the transit time and using catheter coverings. Use of a crib for catheter loading only improved temperature stability while the catheter remained in the crib, not during transit, and reduced pH fluctuations during the procedure.

Considerations for future modification of The Association for the Study of Reproductive Biology embryo grading system incorporating time-lapse observations.

The Association for the Study of Reproductive Biology (ASEBIR) Interest Group in Embryology (in Spanish 'Grupo de Interés de Embriología') reviewed key morphokinetic parameters to assess the contribution of time-lapse technology (TLT) to the ASEBIR grading system. Embryo grading based on morphological characteristics is the most widely used method in human assisted reproduction laboratories. The introduction and implementation of TLT has provided a large amount of information that can be used as a complementary tool for morphological embryo evaluation and selection. As part of IVF treatments, embryologists grade embryos to decide which embryos to transfer or freeze. At the present, the embryo grading system developed by ASEBIR does not consider dynamic events observed through TLT. Laboratories that are using TLT consider those parameters as complementary data for embryo selection. The aim of this review was to evaluate review time-specific morphological changes during embryo development that are not included in the ASEBIR scoring system, and to consider them as candidates to add to the scoring system.

Total duration of spontaneous blastocyst collapse during the expansion stage is an independent predictor of euploidy and live birth rates.

RESEARCH QUESTION: Is the total duration of spontaneous blastocyst collapse to re-expansion before biopsy related to ploidy and live birth rates after single euploid blastocyst transfer? DESIGN: This was a retrospective cohort study of 600 preimplantation genetic testing cycles for aneuploidy (PGT-A) cycles, involving 2203 biopsied blastocysts, at a large reproductive medicine centre. Features of spontaneous blastocyst collapse from full to expanded stage, before biopsy, were observed using an embryoscope viewer for embryos cultured in a time-lapse incubator. In total, 568 cycles of frozen blastocyst transfers, either single euploid or mosaic, were performed. Correlations between collapse features and PGT-A outcomes were evaluated, as well as live birth rate, following euploid embryo transfer. RESULTS: Blastocysts with lower morphological quality or delayed development had significantly higher rates of collapse, multiple collapses, and a longer duration of collapse to re-expansion. After controlling for confounders, such as oocyte age, morphological quality of blastocyst, and day of biopsy, multivariate logistic regression revealed that the total duration of collapse to re-expansion was an independent predictor of lower euploidy rate; the multivariate OR was 0.85 (95% CI 0.77-0.95; P = 0.00). Furthermore, even with euploid embryo transfer, the probability of a live birth decreased as the total duration of collapse to re-expansion increased; the multivariate OR was 0.79 (95% CI 0.64-0.98; P = 0.033). CONCLUSION: The total duration of blastocyst collapse to re-expansion could be used as a predictor of lower euploidy and live birth rate. When developing blastocyst algorithms for pregnancy prediction, the duration of spontaneous blastocyst collapse should be included as a significant variable.