Morula Stage Research Articles

Classification of embryos at the morula stage is highly correlated with clinical pregnancy

Background and Aim: Evidence from conventional microscopy suggests that if cellular material is excluded from the embryo at compaction (incomplete compaction), blastocyst formation and outcome may be compromised (Ivec et. al., 2011). The introduction of time-lapse has made it possible to evaluate compaction by observing the entire process rather than relying on a single static observation. This has enabled any correlation between compaction completeness, morula grade and outcome to be studied.

Effect of Culture Medium on in vitro Fertilization in Local Iraqi Ewes.

in vitro fertilization (IVF) is considered to be the most important reproductive biotechnological method having great potential to accelerate genetic improvement in ruminants as well as for research on embryonic development. The present study aimed to investigate the effect of culture medium and the addition of natural and synthetic antioxidants on in vitro maturation (IVM), fertilization (IVF), and culture (IVC) in local Iraqi ewes. A total of 304 reproductive systems of local ewes were collected from a slaughterhouse in Fallujah, Anbar Province, Iraq from 3, January to 1, July 2021. The study was conducted in the Reproductive Biotechnology Laboratory, Department of Surgery and Theriogenology, College of Veterinary Medicine, University of Fallujah, Iraq. A total of 1368 oocytes were recovered from 608 ovaries surrounded by cumulus cells. The method of collection was aspiration and oocytes were divided into eight treatments. The first (T1), the second (T2), the third (T3), the fourth (T4), the fifth (T5), the sixth (T6), the seventh (T7), and the eighth (T8) treatments were MEM + Capparis spinosa extract 50µmol, MEM + Silymarin extract 100µmol, MEM + Coenzyme Q10 5 µmol, MEM only serves as a control, DMEM + Capparis spinosa extract 50 µmol, DMEM + Silymarin extract 100µmol, DMEM + Coenzyme Q10 5µmol and DMEM only serves as a control, respectively. The results indicated a significant difference (P≤0.05) between T5 (DMEM + Capparis spinosa extract) and other controls or treatments. Cultural medium DMEM with Capparis spinosa extract (as an antioxidant) presents the best results in the morula and blastocyst stage.

MEIOTIC AND MITOTIC ANEUPLOIDIES DRIVE ARREST OF IN VITRO FERTILIZED HUMAN PREIMPLANTATION EMBRYOS

The high incidence of chromosome aneuploidy in early human development is the primary cause of pregnancy loss, miscarriage, and still birth following natural conception as well as in vitro fertilization (IVF). Preimplantation genetic testing for aneuploidy (PGT-A) of IVF embryos at the blastocyst stage has confirmed the prevalence of aneuploidies of putative meiotic and postzygotic mitotic origins. However, only about half of normally fertilized embryos develop to the blastocyst stage in vitro, while the others arrest at cleavage to late morula or early blastocyst stages.

Cryopreservation increases accumulation of exogenous stearic acid in mouse embryos

Cryopreservation of preimplantation embryos is a widely used technique, but this procedure might impact the subsequent embryo development. The effect of slow freezing and vitrification on the lipid metabolism in preimplantation mammalian embryos is not well studied. In this work, we applied Raman spectroscopy of isotopically labeled molecules to address the effects of cryopreservation on fatty acid accumulation in mouse embryos. Embryos after slow freezing or vitrification were cultured for 20 h in a medium supplemented with bovine serum albumin saturated with deuterated stearic acid (dSA). After this period the concentration of dSA estimated from Raman spectra of frozen-thawed and vitrified-warmed embryos at the morula stage was almost twice higher compared to non-cryopreserved morulas. At the same time, frozen-thawed and vitrified-warmed 4-cell embryos did not demonstrate any difference in the level of stearic acid uptake from non-cryopreserved embryos of the same stage. After an additional 24 h culture, cryopreserved and non-cryopreserved embryos demonstrated similar dSA uptake.

Expression of microRNA let-7 in cleavage embryos modulates cell fate determination and formation of mouse blastocysts†.

After fertilization, the zygote undergoes cell division. Up to the 8-cell stage, the blastomeres of mouse preimplantation embryos are morphologically identical. The first cell differentiation starts in the morula leading to the formation of trophectoderm cells and inner cell mass cells of the blastocyst. The regulation of the differentiation event and the formation of blastocysts are not fully known. Lethal-7 (let-7) is a family of evolutionarily conserved microRNAs. Here, we showed that the expression of let-7a and let-7g decreased drastically from the 1-cell stage to the 2-cell stage, remained low up to the 8-cell stage and slightly increased after the morula stage of mouse embryos. The expression of let-7 in the inner cell mass was higher than that in the trophectoderm. Forced expression of let-7a in embryos at the 1-cell and 4-cell stage inhibited blastocyst formation and downregulated the expression of CDX2 but maintained that of OCT4 in the trophectoderm. Forced expression of other let-7 isoforms exhibited similar inhibitory action on blastulation. On the other hand, inhibition of let-7a at the 4-cell stage and the 8-cell stage enhanced blastocyst formation. Co-injection of green fluorescent protein (GFP) mRNA (lineage tracer) with either precursor of let-7a (pre-let-7a) or scramble control into one blastomere of 2-cell embryos showed that ~75% of the resulting blastocysts possessed GFP+ cells in their inner cell mass only. The biased development towards the inner cell mass with forced expression of let-7 was reproduced in 2-cell chimeric embryos consisting of one wildtype blastomere and one GFP mRNA-injected blastomere from another 2-cell embryo carrying a doxycycline-inducible let-7g gene. Bioinformatics analysis indicated that Tead4 was a potential target of let-7. Let-7 bound to the 3'UTR of Tead4 and let-7 forced expression downregulated the expression of Tead4 in mouse blastocysts. Co-injection of Tead4 mRNA partially nullified the modulatory roles of let-7a in the inner cell mass cell fate. In conclusion, a high level of let-7 at the 2-cell stage favored the formation of the inner cell mass, whereas a low level of let-7 at the 4-cell to 8-cell stage enhanced blastocyst formation. Tead4 mediated the action of let-7 on the inner cell mass cell-fate determination.

PSX-B-21 Equine Embryo Viability Based on Morphokinetics

Abstract The objective of this study was to analyze equine embryo morphokinetics and whether or not they were indicative of survivability upon successful implantation into the uterus of a recipient female. In equine reproduction, fertility is a lowly heritable trait that has an exceedingly large financial impact on the ability to grow genetic lines. In order to increase pregnancy rates, producers must have access to accurate decision-making tools. Ten (n = 10) fertilized equine embryos collected via flushing by licensed veterinarian, Dr. Gregg Veneklasen, DVM in Canyon, TX. Flushed embryos were filmed for a thirty-five (35) second observation period with an Iolight Microscope and then processed through video motion magnification (VMM) software Lambda Vue, that amplified embryo activity 300x their normal movement. These VMM embryos were then assessed for time specific morphological changes using Image J software. Embryo morphological dimension shifts previously unseen under a simple microscope became humanly perceptible and measurable. For morula stage embryos, measurements of significance were taken on the inner cell mass (ICM), total embryo area, trophoblast to ICM distance, and finally the vertical, diagonal, and horizontal axis of the sub-zonal distance. Protocols were developed for embryos in the blastocyst stages, where measurements were taken on the vertical, diagonal, and horizontal axis of the total embryo, area of the embryo, notation of general ICM location, and trophoblast thickness around the embryo face. All measurements were taken in microns every five seconds for the thirty-five second recording period. Findings presented that embryos with a greater ICM, total embryo area, and sub-zonal vertical, diagonal, and horizontal distance established pregnancies as frequently as embryos with less momentous differences in distance (P < 0.1). This research has shown the relationship between morphokinetics and pregnancy had no significance of determining pregnancy viability. This resulted in enhanced prediction of embryo transfer techniques based on traditional grading, making pregnancy rates more predictable and consistent.

Embryonic and larval development of the commercially important freshwater fish gobi, Glossogobius giuris (Hamilton, 1822)

The present study was carried out to investigate the embryonic and larval development of the commercially important freshwater fish gobi (Glossogobius giuris) in laboratory conditions at the Mini Hatchery cum Breeding Complex of the Department of Fisheries Biology and Genetics under the Faculty of Fisheries, Bangladesh Agricultural University (BAU), Mymensingh. Healthy broodstocks of both female and male gobi were collected from the grow-out ponds. Matured eggs and sperms were obtained through the induction of spawning by the injection of PG (pituitary gland) hormones. Fertilization was done by mixing of a few drops of sperm suspensions into the egg mashes in a small plastic bowl and washing several times to remove the excess sperm and unfertilized eggs. Fertilized eggs were then collected and incubated in a mini circular cemented tank with the provision of a continuous water supply. At least 10 fertilized eggs undergoing embryonic development were continuously monitored for studying the daily changes in their development. Fertilized eggs were collected randomly up to the morula stage every 4-5 h, and then after every 1 h interval until hatching and every 4 h after hatching. Soon after fertilization, the eggs were swelled, extended longitudinally, and became adhesive and light brownish to transparent in color. The unfertilized egg did not change in size and shape, rather turned whitish in color and became demersal. At fertilization, the length of eggs was increased from 0.6 to 3.0 mm and width from 0.3 to 0.5 mm, having a tendency to attach to the substrates or the shelters provided. The first cleavage of eggs has occurred within 1.3-2.5 h at 27.0 ± 1.0 °C. The development of the embryos of Gobi occurred about 15 h post-fertilization at 27.0-29.0 °C. The stages of embryonic development were observed with cleavage, followed by morula, blastula, gastrula, yolk plug stage, organogenesis and until hatching of non-pigmented larvae. Hatching started from 35 to 48 h post-fertilization and was completed within 5-6 h at the same temperature range, as all embryos did not hatch out at a time. Newly hatched larvae were measured to be 0.50-0.60 mm in length. The yolk sac was completely absorbed in 48-50 h during larval development and when the hatchlings reached 1.60 ± 0.02 mm in total length; they then started feeding within 48-60 h post-hatching. The findings of the present study have not only provided valuable information on the embryonic and larval development of gobi (G. giuris) but would immensely be helpful towards the establishment of large-scale seed production techniques for aquaculture and conservation of this important fishery.

Comparative maternal protein profiling of mouse biparental and uniparental embryos.

BackgroundMaternal proteins have important roles during early embryonic development. However, our understanding of maternal proteins is still very limited. The integrated analysis of mouse uniparental (parthenogenetic) and biparental (fertilized) embryos at the protein level creates a protein expression landscape that can be used to explore preimplantation mouse development.ResultsUsing label-free quantitative mass spectrometry (MS) analysis, we report on the maternal proteome of mouse parthenogenetic embryos at pronucleus, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages and highlight dynamic changes in protein expression. In addition, comparison of proteomic profiles of parthenogenotes and fertilized embryos highlights the different fates of maternal proteins. Enrichment analysis uncovered a set of maternal proteins that are strongly correlated with the subcortical maternal complex, and we report that in parthenogenotes, some of these maternal proteins escape the fate of protein degradation. Moreover, we identified a new maternal factor-Fbxw24, and highlight its importance in early embryonic development. We report that Fbxw24 interacts with Ddb1-Cul4b and may regulate maternal protein degradation in mouse.ConclusionsOur study provides an invaluable resource for mechanistic analysis of maternal proteins and highlights the role of the novel maternal factor Fbw24 in regulating maternal protein degradation during preimplantation embryo development.

Slow Development of Day 5 Embryo Leads to Compromised Live Birth But Not Birthweight Outcomes

Background: There is currently no consensus regarding developmental cutoff for fresh transfer of slow developing day 5 embryos on. Literature is sparse regarding true prognosis of such embryos. Aim: To investigate live birth and birthweight outcomes of slow developing day 5 transferred embryos. Method: This retrospective multi-center study included 1213 consecutive autologous-oocyte single fresh day 5 transfers performed at 4 Monash IVF clinics during 2016-2019. Repeat cycles by same patients were excluded to avoid clustering effects. Live birth and birthweight were followed up in all pregnancies. Multiple regression was performed to investigate associations between slow day 5 development (defined as ≤ early blastocyst) and (a) live birth, (b) birthweight, and (c) gestation-adjusted birthweight (Z score) to account for gestational age and gender. Results were expressed as adjusted odds ratio (aOR) with 95% confidence interval (CI). Results: No implantation was achieved when day 5 embryo failed to reach early blastocyst stage (i.e. ≤morula, n=76). Live birth rate was significantly lower following transfer of early blastocysts (n=237, 16%), in comparison to expanding (n=329, 27%, P=0.001), expanded (n=392, 41%, P=0.000), and ≥hatching blastocysts (n=169, 44%, P=0.000). After adjusting for maternal age, hours post insemination at day 5 assessment, number of oocytes collected, number of 2PNs, and number of embryos frozen; multiple logistic regression showed a significantly reduced likelihood of live birth resulting from early blastocysts in reference to expanding (aOR=0.584, 0.371-0.917, P=0.020), expanded (aOR=0.322, 0.208-0.501, P=0.000), or ≥hatching stages (aOR=0.255, 0.147-0.443, P=0.000). However, early blastocysts resulting in live birth (n=39) did not alter birthweight or Z score in reference to expanding (n=90, P>0.05 respectively), expanded (n=160, P>0.05 respectively), or ≥hatching (n=75, P>0.05 respectively). Conclusion: There is no clinical value of fresh transferring day 5 embryos at ≤morula stage. Fresh early blastocysts lead to a reduced live birth rate but not birthweight outcomes.

Major transcriptomic, epigenetic and metabolic changes underlie the pluripotency continuum in rabbit preimplantation embryos.

Despite the growing interest in the rabbit model for developmental and stem cell biology, the characterization of embryos at the molecular level is still poorly documented. We conducted a transcriptome analysis of rabbit preimplantation embryos from E2.7 (morula stage) to E6.6 (early primitive streak stage) using bulk and single-cell RNA-sequencing. In parallel, we studied oxidative phosphorylation and glycolysis, and analysed active and repressive epigenetic modifications during blastocyst formation and expansion. We generated a transcriptomic, epigenetic and metabolic map of the pluripotency continuum in rabbit preimplantation embryos, and identified novel markers of naive pluripotency that might be instrumental for deriving naive pluripotent stem cell lines. Although the rabbit is evolutionarily closer to mice than to primates, we found that the transcriptome of rabbit epiblast cells shares common features with those of humans and non-human primates.

Pregnancy of Cryopreserved Ovine Embryos at Different Developmental Stages

BACKGROUND: Developmental stage and cryopreservation method have significant impact on the pregnancy rate after transfer of embryos produced in vivo. OBJECTIVE: To determine the pregnancy outcomes from ovine embryos cryopreserved at different developmental stages. MATERIALS AND METHODS: Embryos at different developmental stages were obtained from donor ewes through simultaneous estrus treatment and laparoscopic artificial insemination. Embryos, either cryopreserved via vitrification or slow freezing method, were implanted into recipient ewes. The pregnancy rate was determined 35 days after transfer. RESULTS: The pregnancy rate of developing embryos increases after transfer from the morula stage, early blastocyst to expanded blastocyst stages (64.9%, 73.9% and 81.3%, respectively). However, cryopreservation significantly decreases the pregnancy rate of embryos at all three developmental stages, and there is no significant difference among developmental stages (43.9%, 43.7%, 52.9%, respectively). There is also no significant difference in the pregnancy rate between slowly-frozen embryos and vitrified embryos. CONCLUSION: The pregnancy outcomes of embryo transfer is better at the expanded blastocyst stage than at earlier stages. However, no difference is observed in the pregnancy rate of embryos at different developmental stage after cryopreservation, either by slow freezing and vitrification. Cryopreservation methods for ovine embryos, both slow freezing and vitrification, need further improvement.

Linker histone H1FOO is required for bovine preimplantation development by regulating lineage specification and chromatin structure†.

Linker histone H1 binds to the nucleosome and is implicated in the regulation of the chromatin structure and function. The H1 variant H1FOO is heavily expressed in oocytes and early embryos. However, given the poor homology of H1FOO among mammals, the functional role of H1FOO during preimplantation embryonic development remains largely unknown, especially in domestic animals. Here, we find that H1FOO is not only expressed in oocytes and preimplantation embryos but granulosa cells and spermatids in cattle. We then demonstrate that the interference of H1FOO results in preimplantation embryonic developmental arrest in cattle using either RNA editing or Trim-Away approach. H1FOO depletion leads to a compromised expression of critical lineage-specific genes at the morula stage and affects the establishment of cell polarity. Interestingly, H1FOO depletion causes a significant increase in the expression of genes encoding other linker H1 and core histones. Concurrently, there is an increase of H3K9me3 and H3K27me3, two markers of repressive chromatin and a decrease of H4K16ac, a marker of open chromatin. Importantly, overexpression of bovine H1FOO results in severe embryonic developmental defects. In sum, we propose that H1FOO controls the proper chromatin structure that is crucial for the fidelity of cell polarization and lineage specification during bovine preimplantation development.

Antioxidant procyanidin B2 protects oocytes against cryoinjuries via mitochondria regulated cortical tension

BackgroundIrreversible cryodamage caused by oocyte vitrification limited its wild application in female fertility preservation. Antioxidants were always used to antagonist the oxidative stress caused by vitrification. However, the comprehensive mechanism underlying the protective role of antioxidants has not been studied. Procyanidin B2 (PCB2) is a potent natural antioxidant and its functions in response to vitrification are still unknown. In this study, the effects of PCB2 on vitrified-thawed oocytes and subsequent embryo development were explored, and the mechanisms underlying the protective role of PCB2 were systematically elucidated.ResultsVitrification induced a marked decline in oocyte quality, while PCB2 could improve oocyte viability and further development after parthenogenetic activation. A subsequent study indicated that PCB2 effectively attenuated vitrification-induced oxidative stress, rescued mitochondrial dysfunction, and improved cell viability. Moreover, PCB2 also acts as a cortical tension regulator apart from strong antioxidant properties. Increased cortical tension caused by PCB2 would maintain normal spindle morphology and promote migration, ensure correct meiosis progression and finally reduce the aneuploidy rate in vitrified oocytes. Further study reveals that ATP biosynthesis plays a crucial role in cortical tension regulation, and PCB2 effectively increased the cortical tension through the electron transfer chain pathway. Additionally, PCB2 would elevate the cortical tension in embryo cells at morula and blastocyst stages and further improve blastocyst quality. What’s more, targeted metabolomics shows that PCB2 has a beneficial effect on blastocyst formation by mediating saccharides and amino acids metabolism.ConclusionsAntioxidant PCB2 exhibits multi-protective roles in response to vitrification stimuli through mitochondria-mediated cortical tension regulation.

Reprogramming barriers in bovine cells nuclear transfer revealed by single-cell RNA-seq analysis.

Many progresses have recently been achieved in animal somatic cell nuclear transfer (SCNT). However, embryos derived from SCNT rarely result in live births. Single‐cell RNA sequencing (scRNA‐seq) can be used to investigate the development details of SCNT embryos. Here, bovine fibroblasts and three factors bovine iPSCs (3F biPSCs) were used as donors for bovine nuclear transfer, and the single blastomere transcriptome was analysed by scRNA‐seq. Compared to in vitro fertilization (IVF) embryos, SCNT embryos exhibited many defects. Abnormally expressed genes were found at each stage of embryos, which enriched in metabolism, and epigenetic modification. The DEGs of the adjacent stage in SCNT embryos did not follow the temporal expression pattern similar to that of IVF embryos. Particularly, SCNT 8‐cell stage embryos showed failures in some gene activation, including ZSCAN4, and defects in protein association networks which cored as POLR2K, GRO1, and ANKRD1. Some important signalling pathways also showed incomplete activation at SCNT zygote to morula stage. Interestingly, 3F biPSCNT embryos exhibited more dysregulated genes than SCNT embryos at zygote and 2‐cell stage, including genes in KDM family. Pseudotime analysis of 3F biPSCNT embryos showed the different developmental fate from SCNT and IVF embryos. These findings suggested partial reprogrammed 3F biPS cells as donors for bovine nuclear transfer hindered the reprogramming of nuclear transfer embryos. Our studies revealed the abnormal gene expression and pathway activation of SCNT embryos, which could increase our understanding of the development of SCNT embryos and give hints to improve the efficiency of nuclear transfer.

Specifics of vitrification of in vitro-produced cattle embyos at various development stages

Producing embryos in vitro is an important technology used to improve the genetic potential of cattle and perfect the programs of their breeding. Regardless of the way they are produced, all embryos that had not been used for transplantation to recipients must be conserved. Because of significantly increased interest in the problem of cryoconservation of embryos, both coming from scientists and businesses, there are emerging new commercial environments that allow the facilitation of cryoconservation and the increase in the embryo survival. Oocyte-cumulus complexes obtained from the ovaries of slaughtered clinically healthy cows matured in 22–24 h in in vitro conditions. The oocytes were co-cultured with spermatozoids in Fertilization medium, and the obtained zygotes were cultured in Culture medium with Sodium-Pyruvate for 4 or 7 days up to the stage of morula or blastocyste, respectively. For the vitrification of cow embryos, we used a commercial kit for the vitrification of human embryos, having compared the duration of equilibration. According to the results of the studies, we observed high efficiency of cryoconservation of cow embryos using the commercial kit for vitrification of human embryos. The results revealed the significant effect of equilibration on survival and further development of embryos. In addition, we described the dependence of development stage of cattle embryo on the duration of the contact of embryo with equilibration solution. Therefore, optimal time of contact of cattle embryos at the morula stage with equilibration solution was 12 minutes. On the 24th h after thawing, 46.7 ± 3.3% of the embryos were observed to undergo blastulation, and on 48th h, this parameter increased to 96.7 ± 3.3%, which corresponded to the parameters in the group of embryos that had not been subjected to cryoconservation. In the conditions of further cultivation, the percentage of blastocystes that hatched in the experimental group was no different from that of the control. At the same time, the highest efficiency of vitrification of blastocystes of cows was seen after the contact with the equilibration solution for 15 min, since the percentage of hatched blastocystes was the same as in the control group. Therefore, using the commercial kit for vitrification of human embryos is beneficial, for it promotes the parameters of cow embryos after vitrification/thawing that are similar to such of intact embryos (without freezing). The data we analyzed and presented in the paper could help to increase the efficiency of cryoconservation of cattle embryos for both scientific and commercial purposes.

Evaluation of the In Vitro Embryo Production of Farm Animals Under the Circumstances of Embryo Transfer Technology

“Embryo transfer” technology is one of the deterministic techniques related to the production of in vitro or in vivo produced embryos. This technique controls to a large extent the integrity of the developmental component of early embryos and their ability to develop later until birth. Pregnancy rates resulting from the transferred embryos were widely dispersed and associated with many variables and factors. The developmental stage of early embryos had a significant impact on pregnancy rates, as most studies agreed to transfer embryos in the blastocyst and morula stages due to the high rates of pregnancy (40%-100%) compared to the blastomeres. Embryo cryopreservation technology (rapid and slow) competed to a large extent with fresh embryos in pregnancy rates, even surpassing the latter in some cases, as the percentage approached 100%. The resulting pregnancy rates varied greatly (up to 95%) according to the method of transferring and hormonal induction. The results were mainly based on the size and type of the animal on the one hand and on the nature of the biological activity and the specific function of the hormones (progesterone, prostaglandin F2α, gonadotropin - releasing hormone and follicle - stimulating hormone, etc.) involved in regulating estrus in donors and recipients on the other hand. This review concluded that the embryo transfer technology has given many scenarios that cannot lead to an inevitable result in judging the efficacy of the technology of in vitro embryo production, but the results are encouraging and require further efforts.

O-288 Association of initial zygote cleavage, compaction pattern and euploidy rates: possible implications for embryo correction mechanisms

Abstract Study question Is the morula compaction pattern associated with early zygote cleavage anomalies and euploidy rates? Can it represent a possible mechanism of embryonic correction? Summary answer Abnormalities in the first divisions give rise to dysfunctional blastomeres, which were excluded from compaction process. This process represent a possible embryonic correction mechanism. What is known already IVF embryos have historically been analyzed based on morphological characteristics focusing on days 2/3 (cleavage stage) and 5/6 (blastocyst). The morula stage, usually reached on day 4, represent a transitional phase resulting from the embryonic genome activation and has received little attention in clinical embryology. However, during this crucial stage, cell differentiation begins, metabolism reaches higher levels, changes in cell shape and cell-destination mechanisms are enhanced, providing opportunities for “quality control”. At morula stage, embryos with severe genetic abnormalities are sorted out by natural selection or possibly self-correction events may occurs, reducing the load of aneuploid cells in mosaic embryos. Study design, size, duration We evaluated laboratory and clinical outcomes of 836 blastocysts (195 ICSI cycles) cultured in a time-lapse system and biopsied for preimplantation genetic testing for aneuploidy (PGT-A), from April-2018 to June-2020. Time-lapse videos were retrospectively analyzed to assess morphokinetic parameters of initial zygote cleavages and morula compaction. These parameters were evaluated and associated to genetic and embryonic outcomes. Blastocyst outcomes were also assessed according to maternal age <35 (n = 140), 35-37 (n = 175), 38-40 (n = 314) and >40 (n = 207). Participants/materials, setting, methods The zygote initial cleavages were classified as normal (1-2-4 cleavage) or anomalous: 1-3 cleavage (zygote splits directly into three blastomeres); 1-2-3 cleavage (2-3 in < 3h); and 1-2-5 cleavage (after the first normal cleavage, one blastomere splits directly into three blastomeres). Morula were classified as: Fully Compacted Morulae (FCM); Partially Compacted Morulae (PCM). The PCM could have cells excluded from the compaction process (Exc-PCM), extruded from an already compacted morula (Ext-PCM), or both situations (Exc/Ext-PCM). Main results and the role of chance Most blastocysts showed normal zygotic cleavage (83.3%). Anomalous cleavage such as 1-3 (0.8%), 1-2-3 (7.2%) and 2-5 (8.7%) were less prevalent and no differences were observed according to female age (p = 0.268). Overall, FCM (61.0%) was a more frequent pattern than PCM (39.0%). According to compaction pattern of PCM, Exc-PCM (25.1%) was more prevalent than Ext-PCM (11.5%) and Exc/Ext-PCM (2.4%). There was also no difference in the prevalence of the morulae compaction pattern according to female age (p = 0.124). FCM occurred in 511 embryos (73.4%) from zygotes with normal cleavage (n = 696), but no FCM was observed among anomalous cleaved zygotes (p < 0.001). Genetic and embryonic outcomes were displayed as follows: normal cleavage and FCM (NC-FCM, n = 511), normal cleavage and PCM (NC-PCM, n = 185), and anomalous cleavage and PCM (AC-PCM, n = 140). NC-FCM and AC-PCM groups showed statistically similar euploidy rates (39.3% and 38.6%, p = 0.870), that were both significantly higher than the NC-PCM group euploidy rate (23.8%, p < 0.001). However, morphokinetic scores (KidScore) and percentage of top-quality blastocysts at morphological evaluation were discordant: NC-FCM (6.4±1.7; 81.4%), NC-PCM (5.0±1.8; 47.6%) and AC-PCM (4.3±2.0; 37.9%). Euploidy rate decreased with female age for all groups: NC-FCM (<35:54.7%, 35-37:56.0%, 38-40:33.3%, >40:24.1%); NC-PCM (<35:37.5%, 35-37:19.0%, 38-40:25.0%, >40:19.0%); AC-PCM (<35:66.7%, 35-37:48.5%, 38-40:39.0%, >40:3.7%). Limitations, reasons for caution The retrospective design of the study represents a general limitation. In addition, this study evaluated only embryos that reached the blastocyst stage and showed enough quality to be biopsied. Therefore, the association between anomalies in the first and second mitotic cleavages and the embryonic development could not be evaluated. Wider implications of the findings Similar euploidy rates among NC-FCM and AC-PCM support the hypothesis that cell exclusion/extrusion in compaction stage of anomalous-cleaved zygotes can represent a potential correction mechanism, even though lower morphological/morphokinetic scores are seen on AC-PCM. That process is supposed to rescue embryos from the mosaic aneuploidy which occurs after abnormal cleavage. Trial registration number not applicable

P-231 Embryo morphokinetics in male factor infertility

Abstract Study question Are embryo morphokinetics and division pattern susceptible to sperm origin? Summary answer Surgical retrieved sperm and Oligo-astheno-teratozoospermia have significant effect on morphokinetics and pattern of embryo development in IVF. What is known already Among couples seeking primary infertility treatment, approximately one third of them have male factor indication. Intracytoplasmic sperm injection (ICSI) has changed their prognosis to achieve successful pregnancy. Recently, there are many conflicting studies on the relation of sperm origin, embryo development and clinical outcome after fertilization burrier is passed by ICSI. Implementation of time-lapse monitoring system (TLM) allows us to analyze embryo development under stable conditions, and identify morphological, temporal, spatial parameters and cell cycle endpoints at which embryos with sperm of different origin exhibit distinct kinetics. The magnitude of variable morphokinetics in anticipating pregnancy outcome is not yet determined. Study design, size, duration This is a prospective cohort study that included 195 ICSI cycles stratified into three groups of different sperm origin (abnormal sperm (OAT), surgical sperm(SRS) and normosperm (CS)). Each group included around 400 zygotes for patients seeking IVF treatment in Fakih IVF/Dubai between December 2019 and December 2021 in Dubai / UAE. The mean age of patients is (22-40 years female/ 22-45 years male). Normospermia subset was limited to couples seeking gender selection and tubal factor. Participants/materials, setting, methods Embryo morphokinetics (time to polar body (tPB) until time to blastocyst (tB)) and cell cycle durations were analyzed. Embryos were examined for irregular cleavage phenotypes. The prevalence of each category of out of-range morphokinetics and abnormal cleavage among the different groups was compared. The portion of embryos displaying optimal ranges for the indicated morphokinetics was explored focusing on cell cycle duration. Finally, multi-variables influencing morphokinetics parameters were controlled. Chi2 test was performed in SPSS, P-value<0.05. Main results and the role of chance Embryos derived from SRS were later to reach tPNf and t2(26.66±9.16 and 29.03±8.52) than embryos derived from CS (25.15±4.20 and 27.84±4.98) and OAT (25.71±6.20 and 28.67±7.22). P < 0.05. Embryos derived from SRS reached t5(47.94±11.02) faster than both embryos derived from CS (49.61±8.89) and OAT (49.26±9.47). Embryos derived from SRS reached t6(51.64±10.44) faster than embryos from CS (53.10±8.96). P value=0.0. Embryos derived from SRS and OAT reached tSB (95.34±9.01 and 94.92 ±10.04) and tB(103.68±8.53 and 102.08±10.32) significantly later than embryos derived from CS (91.16±5.65 and 96.34±7.70). No correlation was found between sperm origin and irregular embryo phenotypes except for blastomere asymmetry and incomplete morulation. P = 0.0004. SRS and OAT had higher incidence of blastomere asymmetry at 2-, 3- and 4cell stage than CS. P = <0.05. For incomplete morulation, CS group had higher incidence of 1-3 fragments at morula stage(71.3%) than SRS and OAT (57.4% and 49%). However, OAT had higher incidence of 3-5 fragments at morula stage(35.9%) than SRS (224.1%) and CS (18.8%). Significantly, SRS displayed higher proportion of embryos out of range for most of the measured morphokinetics. P < 0.05. Accordingly, less proportion of embryos in SRS and OAT fall within optimal range for cell cycle duration P < 0.0. Antral follicle count, number of collected oocytes and male’s age had significantly affected morphokinetics and cleavage abnormality. Limitations, reasons for caution Selection of sperm by ICSI due to male- and severe male factor infertility may result in selecting live sperm with the best available normal morphology which may not reflect DNA fragmentation status of the overall sample where damage of DNA is more significant and lead to bias the results. Wider implications of the findings Embryo development potential with different sperm origin was compared based normal optimum morphokinetics or not. Further follow up study to investigate clinical outcome of implantation and clinical pregnancy rate, miscarriage rate and live birth rate is needed. Trial registration number DSREC-11/2021_07

P-133 The best of the worst: analyze of outcome in artificial reproductive technologies (ART) fresh cycles with transfers of slow-growing embryos and expanded poor-quality blastocysts

Abstract Study question Fresh transfer of slow-growing embryos versus expanded poor-quality blastocysts, in case of absence of good-quality blastocysts on day 5: which is the optimal approach? Summary answer The clinical pregnancy rate (CPR), live birth rate (LBR) are significantly higher during expanded poor-quality blastocysts transfers. There’s no statistical difference in miscarriage rate (MR). What is known already Several studies show contradictory results about which blastocyst morphological parameter best predicts pregnancy outcomes in ART cycle. However, in clinical practice embryologists encounter in ART protocols with scheduled fresh embryo transfer the absence of expanded good-quality blastocysts on day 5. In this case, slow-growing embryos or poor-quality blastocysts could be chosen for transfer. Day 5 slow-growing embryos have low implantation potential in fresh ART cycles, it could be due to embryo-endometrial asynchrony. Also, the quality of further blastocyst remains unclear. Poor-quality blastocysts have a high aneuploidy rate and lower viability compared to good-quality blastocysts. Study design, size, duration This study included ART cycles with fresh embryo transfer on day 5, with selected group of patients under 40 years of age, between 2014 and 2019. Group A consisted of 619 cycles with slow-growing embryos transfer; group B included 88 cycles with expanded poor-quality blastocysts transfer; the control group counted 1501 cycles with a transfer of good-quality blastocysts. Group A had an average of 1,5 embryos transferred per patient, control group and group B 1,3. Participants/materials, setting, methods Embryos were cultured until day 5 and scored according to Gardner and Schoolcraft’s grading system. In group A, slow-growing embryos were transferred at the stage of morula and early blastocyst with a degree of expansion 1. In group B, expanded poor-quality blastocysts were scored as 3-6CB, 3-6BC, 3-6CC. In control group expanded good-quality blastocysts were scored as 3-6AA, 3-6AB, 3-6BA, 3-6BB.The cycles with donor gametes, surrogacy, and preimplantation genetic testing were excluded. Main results and the role of chance The main women’s age of group A, B and control group was 32,2 ± 4,2, 31,5 ± 4,7 and 32,6 ± 4,6 years respectively. The CPR, the ongoing pregnancy rate (OPR), the LBR and the implantation rate (IR) were significantly increased in the group B compared to the group A [34,0% (30/88) vs 17,7% (110/619); P < 0,01], [21,6% (19/88) vs 11,9% (74/619); P = 0,012], [19,3% (17/88) vs 10,5% (65/619); P = 0,015] and [26,2% (31/118) vs 12,6% (119/946); P < 0,01] respectively. There’s no statistical difference in the MR between group A and B [35,2% (42/119) vs 35,5% (11/31); P = 0,946]. The CPR, the OPR, the LBR and the IR were significantly higher in the control group compared to the group A and B [50,5% (758/1501); P < 0,01], [41,6% (625/1501); P < 0,001], [40,3% (605/1501); P < 0,001] and [40,45% (849/2099); P < 0,01]. The MR in the control group was almost two times lower compared to the group A and B [16,2% (138/849); P < 0,01). Relationships between variables were assessed by Pearson's chi-squared test. Limitations, reasons for caution The study is limited due to the uneven distribution of patients in three groups and by a low number of participants in group B. The grading of blastocysts’ quality is also subjected to a human factor. Wider implications of the findings If in ART protocol with scheduled fresh embryo transfer have no expanded good-quality blastocysts, expanded poor-quality blastocysts could be chosen for transfer and slow-growing embryos should be cultured until day 6 with subsequent cryopreservation of blastocysts. Patients should be informed about elevated MR after the transfer of poor-quality blastocysts. Trial registration number Not applicable

P-248 Fallopian tube extracellular matrix hydrogels for in vitro embryo culture in rabbit models: A post-natal study

Abstract Study question Can tissue-specific extracellular matrix (ECM) hydrogels derived from decellularized (DC) fallopian tubes have phenotypic consequences in the offspring when used for in vitro embryo culture? Summary answer Tissue-specific ECM hydrogels from DC rabbit fallopian tubes influence assisted reproductive technologies (ART) outcomes, resulting in offspring with a higher body weight. What is known already The in vitro culture of embryos fails to mimic the physiological dynamism occurring in the reproductive tract. In response to this sub-optimal environment, mammalian embryo exhibits a high degree of developmental plasticity, leading to specific phenotypic deviations at birth and beyond (e.g. low weight). Recently, it has been demonstrated that using in vivo derived culture complements, these changes can be ameliorated. The ECM is one of the elements shown to be important in retaining structural and biological signals from the native source tissue. The current bioengineering techniques could provide hydrogels mimicking the native environment and offering an optimal embryo development. Study design, size, duration This study was approved by an ethics committee (2018/VSC/PEA/0116). Rabbit oviducts (n = 24) were collected, fragmented, DC and solubilized to create hydrogels. After physicochemical characterization, the tissue-specific ECM hydrogels were used for in vitro culture of pronuclear embryos during 48 hours (n = 146), which were subsequently transferred to recipient females, along with embryos cultured under control conditions (n = 138). After delivery, the rabbits were weighed during 20 weeks and blood tests were performed at weeks 8 and 20. Participants/materials, setting, methods To create hydrogels, we employed a decellularization protocol based on SDS detergent. After characterizing and validating decellularization (Francés-Herrero et al. 2021), ECM hydrogels were used to generate coatings (overnight at 37ºC) on culture plates. Pronuclear embryos were cultured until morula stage and then transferred by laparoscopy to obtain the offspring. The animals were weighed and their blood was analyzed by hemogram. T-test was used for the statistical analysis after normality verification (p < 0.05 statistically significant). Main results and the role of chance After 48 hours of embryo culture, no differences in development and morphology were detected between the coating and control conditions (Francés-Herrero et al. 2021). With a total of 284 embryos transferred, the implantation and live birth rates were slightly higher in the coating group than in the control group (31.26 ± 21.44% vs 26.53 ± 24.8% and 19.96 ± 12.5% vs 18.68 ± 24.68%, respectively), but the differences were not statistically significant. During the first 20 weeks of life, the animals from the coating group consistently showed higher weights than the control group. Although this difference was not significant at birth (75.15 ± 15.48 g vs 61.88 ± 17.79 g, respectively), it was significant at subsequent measurements (1117.73 ± 172.54 g vs 942 ± 105.51 g at week 5, 2513 ± 352.83 g vs 2256.25 ± 168.52 g at week 10, 3475.56 ± 297.91 g vs 3257.5 ± 189.94 g; at week 15 and 4151.11 ± 269.28 g vs 3808.75 ± 293.08 g at week 20; p < 0.05). The animals of both experimental groups showed normal blood parameters (white blood cells, % lymphocytes, % monocytes, % granulocytes, red blood cells and hematocrit) at 8 and 20 weeks of life. Limitations, reasons for caution This study has been carried out in an animal model and, although the offspring generated is healthy, the scope of this technique is still unknown. So, the next step would be to study the embryonic reprogramming or media metabolomic profile under ECM in vitro culture. Wider implications of the findings These findings show how culture media supplementation with fallopian tube ECM allows improving in vitro conditions and decreases postnatal phenotypic deviations after ART. This study could initiate a new embryo culture techniques era to guarantee that ART are utilized in the most efficient and safest possible practice. Trial registration number Not applicable