Compact Morula Research Articles

P-229 Effect of morula pattern on clinical outcomes in single vitrified-warmed blastocyst transfer cycles

Abstract Study question Does morula pattern affect clinical outcomes in single vitrified-warmed blastocyst transfer cycles? Summary answer Ongoing pregnancy rate is statistically increased when transferring the blastocyst derived from fully compacted morula in single vitrified-warmed blastocyst transfer cycles. What is known already Time-lapse microscopy has enabled the detailed identification and measurement of dynamic events of embryo development. Recent studies have shown that the exclusion of one or more cells from the compacted morula is associated with reduced developmental competence and a decrease in pregnancy rate. Additionally, it is also associated with decreased rates of PGT-A euploid and implantation. These findings demonstrate the correlation between morula pattern and the outcomes of assisted reproductive technologies. Study design, size, duration A retrospective cohort study was performed between January 2020 and November 2023. A total of 99 single vitrified-warmed blastocyst transfer cycles (women ≥ 35 years) were analyzed. The rates of biochemical pregnancy, clinical pregnancy, implantation, miscarriage, and ongoing pregnancy were evaluated. Participants/materials, setting, methods Images of morula stage embryos were collected at 7-focal planes and every 5-minute interval using a time-lapse microscope. The morula stage embryos were divided into two groups according to their compaction patterns. Morula stage embryos without excluded or extruded cells were defined as fully compacted morula (FCM, n = 47). In contrast, Morula stage embryos with excluded or extruded cells were defined as partially compacted morula (PCM, n = 52). Main results and the role of chance There were no differences of patient characteristics between FCM and PCM regarding female age (37.8 ± 2.1 vs. 37.8 ± 1.8, p = 0.96), number of previous IVF failure (0.5 ± 1.1 vs. 0.9 ± 1.6, p = 0.23), number of retrieved oocytes (16.1 ± 4.0 vs. 14.3 ± 5.1, p = 0.14), maturation rate (86.5% vs. 89.1%, p = 0.18), fertilization rate (73.8% vs. 69.9%, p = 0.39), survival rate (100% vs. 100%, p = Not applicable), and number of transferred embryos (1.0 ± 0.0 vs. 1.0 ± 0.0, p = Not applicable), with the exception of rate of good-quality embryos on Day 3 (44.7% vs. 37.6%, p = 0.04). No statistical differences were observed in rates of biochemical pregnancy (44.7% vs. 34.6%, p = 0.31), clinical pregnancy (40.4% vs. 26.9%, p = 0.15), miscarriage (11.4% vs. 15.4, p = 0.57), and implantation (40.4% vs. 26.9%, p = 0.15) between FCM and PCM, respectively. However, FCM demonstrated a significantly increased ongoing pregnancy rate compared to PCM (29.8% vs. 11.5%, p = 0.02). Limitations, reasons for caution This study was conducted with a small sample size, which limited the generalizability of the findings. Therefore, further studies are needed with a larger sample size and more diverse populations to confirm our findings. Wider implications of the findings The compaction pattern of morula stage embryos can be an indicator for embryo selection. The blastocyst derived from fully compacted morula has the potential to improve ongoing pregnancy rate in single vitrified-warmed blastocyst transfer cycles. Trial registration number not applicable

Survival and Development of in Vivo Produced Boran and Boran* Holstein Cross Embryos

One of biotechnology technique that is frequently used to enhance the number of animals with superior genetic ability and high productivity is embryo transfer. Embryos can be obtained in vivo or in vitro, and they can be frozen and then thawed before being delivered to the recipient animals. Conception rates are influenced by a number of variables, including the quality and developmental stage of the embryo, the location of the embryo's deposit in the uterus, the degree of difficulty of the transfer, whether to use a fresh or frozen embryo, the operator's experience, the corpus luteum's quality, whether to use a heifer or a cow, and the time of year the transfer occurs. The fertility of domestic animals is severely impacted by early embryonic death. For this reason, this study was carried out to gather data on early embryonic development that is normal, the amount and timing of embryonic mortality, potential endogenous and exogenous causes of embryonic loss, and to develop strategies to lower embryonic mortality. A total of 40 embryos (20 fresh and 20 frozen, 26 quality grade 1 and 14 quality grade 2, 29 compact Morula stage and 11 early Blastocyst stage) were transferred to 40 recipient cows (22 Boran and 18 H-B cross) with different body condition score. Return to heat was used as method of pregnancy diagnosis and all recipients were followed around day 14 post embryo transfer and 14 animals were shown heat sign, the rest 26 animals were suspected for pregnancy (65%). Using ultrasound, a pregnancy diagnosis was made on day 45, and 20 recipient animals were found to be 50% positive for PD. On day 60, PD positive animals were re-examined with ultrasound and only 10 were confirmed as PD positive (25%). The PD negative on day 45 and day 60 were suspected to be early embryonic mortality. Other pregnancy loss occurred in this study was abortion nearly after five month of pregnancy. It is not doubtful that, the technique of embryo transfer is utilized to increase the reproductive rates of important female animals. However, it needs proper management for both donor and recipient animals. Therefore, for the successful application of the technology optimum level of feeding both quantity and quality, health management and conducive environment should be fulfilled for all animals.

Association of early cleavage, morula compaction and blastocysts ploidy of IVF embryos cultured in a time-lapse system and biopsied for genetic test for aneuploidy

IVF embryos have historically been evaluated by morphological characteristics. The time-lapse system (TLS) has become a promising tool, providing an uninterrupted evaluation of morphological and dynamic parameters of embryo development. Furthermore, TLS sheds light on unknown phenomena such as direct cleavage and incomplete morula compaction. We retrospectively analyzed the morphology (Gardner Score) and morphokinetics (KIDScore) of 835 blastocysts grown in a TLS incubator (Embryoscope+), which were biopsied for preimplantation genetic testing for aneuploidy (PGT-A). Only the embryos that reached the blastocyst stage were included in this study and time-lapse videos were retrospectively reanalysed. According to the pattern of initial cleavages and morula compaction, the embryos were classified as: normal (NC) or abnormal (AC) cleavage, and fully (FCM) or partially compacted (PCM) morulae. No difference was found in early cleavage types or morula compaction patterns between female age groups (< 38, 38–40 and > 40 yo). Most of NC embryos resulted in FCM (≅ 60%), while no embryos with AC resulted in FCM. Aneuploidy rate of AC-PCM group did not differ from that of NC-FCM group in women < 38 yo, but aneuploidy was significantly higher in AC-PCM compared to NC-FCM of women > 40 yo. However, the quality of embryos was lower in AC-PCM blastocysts in women of all age ranges. Morphological and morphokinetic scores declined with increasing age, in the NC-PCM and AC-PCM groups, compared to the NC-FCM. Similar aneuploidy rates among NC-FCM and AC-PCM groups support the hypothesis that PCM in anomalous-cleaved embryos can represent a potential correction mechanism, even though lower morphological/morphokinetic scores are seen on AC-PCM. Therefore, both morphological and morphokinetic assessment should consider these embryonic development phenomena.

Blastocysts from partial compaction morulae are not defined by their early mistakes

Research questionIs partial compaction during morula formation associated with an embryo's developmental ability and implantation potential? DesignRetrospective analysis of data from 196 preimplantation genetic testing for aneuploidy (PGT-A) cycles. Embryos starting compaction were grouped according to the inclusion or not of all the blastomeres in the forming morula (full compaction or partial compaction). The possible effect of maternal age and ovarian response on compaction was analysed. Morphokinetic characteristics, blastocyst formation rate, morphology and cytogenetic constitution of the obtained blastocysts were compared. Comparisons of reproductive outcomes after the transfer of euploid blastocysts from both groups were established. Finally, in a subset of embryos, the chromosomal constitution concordance of the abandoned cells and the corresponding blastocyst through trophectoderm biopsies was assessed. ResultsA total of 430 embryos failed to include at least one cell during compaction (partial compaction group [49.3%]), whereas the 442 remaining embryos formed a fully compacted morula (full compaction group [50.7%]). Neither female age nor the number of oocytes collected affected the prevalence of partial compaction morulae. Morphokinetic parameters were altered in embryos from partial compaction morulae compared with full compaction. Although an impairment in blastocyst formation rate was observed in partial compaction morulae (57.2% versus 70.8%, P < 0.001), both chromosomal constitution (euploidy rate: partial compaction [38.4%] versus full compaction [34.2%]) and reproductive outcomes (live birth rate: partial compaction [51.9%] versus full compaction [46.2%]) of the obtained blastocysts were equivalent between groups. A high ploidy correlation of excluded cells-trophectoderm duos was observed. ConclusionsPartial compaction morulae show a reduced developmental ability compared with full compaction morulae. Resulting blastocysts from both groups, however, have similar euploidy rates and reproductive outcomes. Cell exclusion might be a consequence of a compromised embryo development regardless of the chromosomal constitution of the excluded cells.

Interleukin-6 stimulates in vitro development of late-stage ovine embryos

The effect of interleukin-6 (IL-6) supplementation during the different phases of in vitro embryo culturing (IVC) on embryo development and embryonic gene expression was studied in ovine. IL-6 was added to IVC medium during the late phases (72–192 h; 5, 10, and 25 ng/ml IL-6) or entire period (0–192 h; 10 ng/ml IL-6) of IVC to determine its effect on embryo development. Further, the effect of IL-6 (10 ng/ml) supplementation at the 72 h of IVC on gene expressions associated with JAK/STAT signalling and pluripotency in 8–16 cell embryos (1 h post-supplementation) and compact morulae (48 h post-supplementation), and apoptosis and primitive endoderm (PrE) development in compact morulae was investigated. The supplementation of 10 ng/ml IL-6 during the late phases of IVC significantly (P < 0.05) increased blastocyst formation (35.2 ± 1.52%) compared to the control (21.1 ± 1.11%), and 5 ng/ml (25.9 ± 2.98%) or 25 ng/ml (16.5 ± 0.73%) IL-6 groups. Conversely, IL-6 (10 ng/ml) treatment throughout the IVC period significantly (P < 0.05) decreased the rate of cleavage (55.4 ± 1.57%) and blastocyst formation (14.5 ± 1.28%) compared to the control group (65.8 ± 1.35% and 21.5 ± 0.97%, respectively). In 8–16 cell embryos and compact morulae, the IL-6 treatment significantly (P < 0.05) affected the expression of genes associated with JAK/STAT signalling and pluripotency. Further, the treatment significantly (P < 0.05) downregulated BAX and CASP3, and upregulated GATA6 expression in compact morulae. In conclusion, IL-6 supplementation affected the in vitro development of ovine embryos in a dose- and time-dependent manner. The beneficial effect of IL-6 on the development of late-stage embryos was mediated through the changes in gene expressions associated with JAK/STAT signalling, pluripotency, apoptosis and PrE development.

Developmental competence of IVF and SCNT goat embryos is improved by inhibition of canonical WNT signaling.

The specific role of the canonical WNT/β-catenin signaling pathway during the preimplantation development of goat remains unclear. Our objective was to investigate the expression of β-CATENIN, one of the critical components of Wnt signaling pathway, in IVF embryos and compare it with SCNT embryos in goat. In addition, we evaluated the consequence of inhibition of β-catenin using IWR1. Initially, we observed cytoplasmic expression of β-CATENIN in 2 and 8-16 cell stage embryos and membranous expression of β-CATENIN in compact morula and blastocyst stages. Furthermore, while we observed exclusively membranous localization of β-catenin in IVF blastocysts, we observed both membranous and cytoplasmic localization in SCNT blastocysts. We observed that Inhibition of WNT signaling by IWR1 during compact morula to blastocyst transition (from day 4 till day 7 of in vitro culture) increased blastocyst formation rate in both IVF and SCNT embryos. In conclusion, it seems that WNT signaling system has functional role in the preimplantation goat embryos, and inhibition of this pathway during the period of compact morula to blastocyst transition (D4-D7) can improve preimplantation embryonic development.

Developmental potential of artificially produced chimeric embryos following transfer to surrogate Goats

An experiment was conducted to study the developmental potential of goat chimeric embryos. A total of 8393 mature oocytes were randomly divided into two groups. Gr 1: oocytes (n=5222) were activated with 5 μM calcium ionophore for 5 to 7 min followed by treatment with 2.0 mM DMAP (dimethylaminopurine) for 4 h in mCR2 aa (modified Charles Rosenkrans 2 amino acid) medium. Gr 2: oocytes (n=3171) were used for in vitro fertilization (IVF). Inner cell mass from hatched blastocysts of parthenogenetic activated embryos were used to produce embryonic stem cell-like cells while 2 cell embryos obtained from IVF were utilized to produce tetraploid embryos. Aggregation efficiency, compact morula and blastocyst formations were 84.93±8.13, 80.53±10.55 and 7.05±3.08%, respectively. A total of 47 chimeric goat embryos were transferred into 17 Sirohi female recipients, resulted in in vivo development of foetus in two recipients. In first recipient, foetus looked like a very thick and hyperechoic amniotic ring until 49 days; thereafter, it was completely resorbed. In second recipient, foetus and amniotic ring were visible as hypoechoic structure from 50 days onwards; thereafter it was completely resorbed at 58 days of embryo transfer. In conclusion, the study indicated that foetal resorption was observed at various stages of pregnancy after transfer of chimeric embryos into recipients. It appears that placental atrophy may be the principal cause of the loss of foetuses.

The proteomic analysis of bovine embryos developed in vivo or in vitro reveals the contribution of the maternal environment to early embryo

BackgroundDespite many improvements with in vitro culture systems, the quality and developmental ability of mammalian embryos produced in vitro are still lower than their in vivo counterparts. Though previous studies have evidenced differences in gene expression between in vivo- and in vitro-derived bovine embryos, there is no comparison at the protein expression level.ResultsA total of 38 pools of grade-1 quality bovine embryos at the 4–6 cell, 8–12 cell, morula, compact morula, and blastocyst stages developed either in vivo or in vitro were analyzed by nano-liquid chromatography coupled with label-free quantitative mass spectrometry, allowing for the identification of 3,028 proteins. Multivariate analysis of quantified proteins showed a clear separation of embryo pools according to their in vivo or in vitro origin at all stages. Three clusters of differentially abundant proteins (DAPs) were evidenced according to embryo origin, including 463 proteins more abundant in vivo than in vitro across development and 314 and 222 proteins more abundant in vitro than in vivo before and after the morula stage, respectively. The functional analysis of proteins found more abundant in vivo showed an enrichment in carbohydrate metabolism and cytoplasmic cellular components. Proteins found more abundant in vitro before the morula stage were mostly localized in mitochondrial matrix and involved in ATP-dependent activity, while those overabundant after the morula stage were mostly localized in the ribonucleoprotein complex and involved in protein synthesis. Oviductin and other oviductal proteins, previously shown to interact with early embryos, were among the most overabundant proteins after in vivo development.ConclusionsThe maternal environment led to higher degradation of mitochondrial proteins at early developmental stages, lower abundance of proteins involved in protein synthesis at the time of embryonic genome activation, and a global upregulation of carbohydrate metabolic pathways compared to in vitro production. Furthermore, embryos developed in vivo internalized large amounts of oviductin and other proteins probably originated in the oviduct as soon as the 4–6 cell stage. These data provide new insight into the molecular contribution of the mother to the developmental ability of early embryos and will help design better in vitro culture systems.

Factors Affecting the Efficiency of In Vitro Embryo Production in Prepubertal Mediterranean Water Buffalo.

Embryos from prepubertal water buffalo can be produced using laparoscopic ovum pickup (LOPU) and in vitro embryo production (IVEP). However, to date, it is unclear what factors and environmental conditions can affect LOPU-IVEP efficiency in prepubertal animals, especially buffalo. In this study, we explored the impact of season, age and individual variation among female donor animals, as well as the effect of the sire used for in vitro fertilization. Donor animals between 2 and 6 months of age were stimulated using gonadotropins prior to LOPU, which was performed at two-week intervals. Following in vitro maturation and fertilization, the resulting embryos were then cultured to the blastocyst stage until they were either vitrified or transferred into recipient animals. The number of follicles available for aspiration and embryo development rates was stable throughout the year. As animals became older, there was a slight trend for fewer COCs recovered from LOPU and better embryo development. There was a large individual variation in both ovarian response and the developmental competence of oocytes among donors. The bull used for fertilization also had a significant impact on embryo development. Upon embryo transfer, pregnancy rates were not affected by the number of embryos transferred per recipient. The best pregnancy rates were achieved when transferring blastocysts, compared to compact morula or hatched blastocysts. Finally, vitrification had no effect on pregnancy rate compared to fresh embryos.

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 &amp;lt;35 (n = 140), 35-37 (n = 175), 38-40 (n = 314) and &amp;gt;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 &amp;lt; 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 &amp;lt; 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 &amp;lt; 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 (&amp;lt;35:54.7%, 35-37:56.0%, 38-40:33.3%, &amp;gt;40:24.1%); NC-PCM (&amp;lt;35:37.5%, 35-37:19.0%, 38-40:25.0%, &amp;gt;40:19.0%); AC-PCM (&amp;lt;35:66.7%, 35-37:48.5%, 38-40:39.0%, &amp;gt;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-243 Automating tracking of cell division for human embryo development in time lapse videos

Abstract Study question Can tracking cell division and predicting human embryo cleavage stages be automated in time-lapse videos (TLV) using AI object detection methods? Summary answer We developed software predicting blastomere count and tracking cell cleavages up until 4-5 stage. The software employs object detection technique called YOLOv5 to detect cells. What is known already Embryo morphology plays an important part in determining viability. Parameters such as number of cells present following fertilization, abnormal cell division (reverse/direct) and evaluating cleavage stages have correlation with pregnancy rates. However, continuous manual evaluation can be time-consuming, and automation will assist in embryo viability assessment. YOLOv5 has proven to accurately detect objects in videos. YOLOv5 uses mean average precision (mAP) as a metric to quantify the portions of frames in videos having the correct count of the objects. Study design, size, duration We have developed a software that uses YOLOv5 to detect cells present in frames of TLV, then marks each cell boundary with different colored circular overlays using OpenCV. We trained YOLOv5 to detect objects: cell, morula and blastocyst using 150 images of different cell-stages, morula, blastocyst. For object cell mAP was 0.65. Annotated location of objects in images and YOLOv5 predictions were reviewed by embryologists. We evaluated the software on TLV from 11 patients. Participants/materials, setting, methods After YOLOv5 detects cells in frames of TLV, our software computes cell count and assigns each cell a different color which is maintained until cell division into daughter cells. Later, daughter cells were also assigned different colors. If the frame has a preceding frame, software calculates detected cells' proximity with each cell in the preceding frame and copies color scheme provided proximity is within some threshold. The software provides TLV with colored overlays as output. Main results and the role of chance In starting frames of TLV with single cell, software accurately detected 1-cell (high precision=0.99, high recall=0.83, high F1-score=0.90). We observed some misclassification between 1-cell and morula. The reason could be that compacted morula looks like 1-cell. Best performance is observed for 2-cells (high precision=0.91, high recall=0.98, high F1-score=0.95). 4-cells were sometimes misclassified with 3 or 5-cells (high precision=0.88, low recall=0.59, high F1-score=0.71). One reason for the misclassification can be that overlapping between cells increases with number of cells. 3-cell and 5-cell are confused with other stages, still cleavage stage detection is better than random: 3-cell (average precision=0.43, high recall=0.83, average F1-score=0.49), 5-cell (average precision=0.44, average recall=0.40, average F1-score=0.40). For cell-stages&amp;gt;5, YOLOv5 detects less cells than actual count and software predicts cleavage later than actual by 9-10 frames on average. The proximity threshold used was 0.10 for cell-count&amp;lt;4 and 0.05 for count&amp;gt;4. In 5 TLV, overlay color for cells changes abruptly between frames, possibly because once YOLOv5 detected a stage, in consecutive frames less cell-number was recorded, and then again reported correct count. Sometimes, software selected the wrong parent for daughter cells (incorrect colored overlay). 2 TLV had direct and reverse cleavages and software could detect these two patterns. Limitations, reasons for caution Overall, our software can precisely detect cells, cell divisions and cleavage stages up to 4-cell stages. We hypothesize that training YOLOv5 on a bigger dataset and including several focal plane information will enable our software to detect overlapping cells and cleavage stages &amp;gt; =5. Wider implications of the findings Object detection proved to be pragmatic for ART and tracking cell division using our software will reduce time consumed in manual annotations, easier prediction of abnormal cleavages and more objective assessments. Qualitative evaluation by embryologists resulted in the overall verdict that this is useful and promising for further development. Trial registration number not applicable

O-234 The guinea pig embryo: a potential new model for human development

Abstract Study question Is the guinea pig a good model for human preimplantation embryo development? Summary answer Preimplantation guinea pig embryos better recapitulate the human embryo compared to the mouse, offering a promising new model to study preimplantation, naive pluripotency and infertility. What is known already Mouse embryos have historically been used to model reproduction, but caution is warranted as several discrepancies with the human exist during preimplantation. Guinea pigs have been a long standing model of human development and are known to better recapitulate placentation and brain development compared to the mouse. We now speculate that guinea pigs may also represent a superior animal model to study preimplantation development. However, to date, guinea pig preimplantation embryos remain to be investigated in great detail. We now aim to assess the developmental dynamics of the guinea pig preimplantation embryo with cross-species comparisons to both human and mouse. Study design, size, duration In vivo guinea pig embryos were flushed 3-6 days post-fertilization (N = 10-12/ time point). Embryos (embryonic day (E)3-6 were collected and processed for downstream experiments. Participants/materials, setting, methods Immunofluorescence of Sox2 (epiblast (EPI) marker), Sox17 and Gata6 (primitive endoderm (PE) marker) and Cdx2 and Gata3 (trophectoderm (TE)) was performed. Cells were counted and the dynamics of lineage marker expression and blastocyst formation were determined. In parallel, scRNA-seq (Smartseq2) of guinea pig embryos was performed. Main results and the role of chance At the compacted morula stage (late E4.25-E4.75), similar to the human and in contrast to the mouse, we observe co-expression Sox2, Gata6 and Gata3, with no detection of Sox17 and Cdx2. Similar to the human, blastocyst formation begins between E5.0-E5.25. By mid-blastocysts (E5.25-E5.5), cells are poised for lineage specification with a high number of cells co-expressing Sox2/Sox17, and no cells co-expressing markers of all three lineages. By late blastocyst, E5.5-E5.75, specification of all three lineages is achieved and no co-expression Sox2/Sox17/Cdx2 or Sox2/Gata6/Gata3 is observed. Further, similar to the human and in contrast to the mouse, there is a disconnect between morphology and lineage specification in the early blastocyst. These data suggest that signaling pathways governing lineage specification in the human and guinea pig differ compared to the mouse. Finally, cross-species analysis of scRNA-seq revealed conserved pluripotency gene signatures between the human and guinea pig embryos, suggesting that the guinea pig may also contain naive stem cells and that plasticity of these lineages may be more similar to the human compared to the mouse. Limitations, reasons for caution Our model only explores similarities/differences between the human and guinea pig preimplantation embryo with regards to timing of development and lineage segregation. Wider implications of the findings In addition to helping us understand human preimplantation development, this new model may serve to examine the longer-term consequences of preimplantation exposures. Further, insights from this study have broad application in the fields of Reproduction, Development, ART and Stem cell biology. Trial registration number NA

Effects of single or serial embryo splitting on the development and morphokinetics of in vitro produced bovine embryos

Embryo splitting can be used in cattle in vitro production (IVP) to improve embryo availability and to increase selection intensity. Despite this widespread utility, a comparative investigation of the viability of IVP embryos split at Day 2 (2-cell stage), Day 3 (8-cell stage), and blastocyst stage has not been undertaken. Similarly, the suitability of splitting Day 3 embryos with atypical numbers of blastomeres, and the feasibility of serial-splitting cleavage stage embryos, have not been investigated in cattle. Here, we demonstrate that the strategy most likely to produce the greatest output of viable embryos is the splitting of Day 3 embryos into four parts, regardless of whether embryos with exactly eight cells or an atypical number of blastomeres are used. This approach was found to produce 1.8 blastocysts per zygote on average compared to just 0.4 blastocysts per zygote for non-split controls. Single-splitting was also found to be superior to serial-splitting which, whilst feasible, impaired embryo viability as judged by cell number at day 7 post-insemination. Interestingly, zygotes (≥2 cells) split once on either Day 2 or Day 3 post-insemination, whilst resulting in smaller blastocysts than control embryos, displayed higher cell counts than expected at the blastocyst stage, suggesting a compensatory mechanism might be at play. Indeed, time-lapse imagery revealed that zygotes split at 2-cells reached the compact morula and expanded blastocyst stages earlier than either those split at Day 3 or non-split controls. Developmental events between splits originating from the same progenitor appeared well synchronized only up to the third cleavage division.

Asynchrony between in vivo and in vitro rabbit embryos

Background: Comparative features of embryos developed under in vitro and in vivo conditions are particularly important in designing embryo transfer procedures that fulfil embryo-recipient synchronization requirements. Objective: To determine the degree of asynchrony in rabbit embryo development between cultured and in vivo embryos. Methods: A total of 55 non-lactating multiparous female rabbits were used. Embryos were classified as 16-cells or early morulae at 48 hours post-coitum (hpc). Embryos were cultured during 30 or 32 h and embryo development was compared with in vivo embryos of 72 hpc. In vitro and in vivo embryos at 72 hpc were classified as early or compacted morulae. Bayesian statistics was used. Difference between in vivo and in vitro embryos and the actual probability of the difference between the in vivo and in vitro embryo higher than zero (P) was estimated. Results: The percentage of compacted morulae was higher in in vivo embryos than in in vitro embryos with +6 h of asynchrony (73.5 and 32.8%, P=1.00). But the percentage of compacted morulae was similar with +8 h asynchrony. Conclusions: In vitro embryos delay their development by + 8 hours compared to in vivo embryos.

Dynamic Changes in the Proteome of Early Bovine Embryos Developed In Vivo.

Early embryo development is a dynamic process involving important molecular and structural changes leading to the embryonic genome activation (EGA) and early cell lineage differentiation. Our aim was to elucidate proteomic changes in bovine embryos developed in vivo. Eleven females were used as embryo donors and pools of embryos at the 4–6 cell, 8–12 cell, morula, compact morula and blastocyst stages were analyzed by nanoliquid chromatography coupled with label free quantitative mass spectrometry. A total of 2,757 proteins were identified, of which 1,950 were quantitatively analyzed. Principal component analysis of data showed a clear separation of embryo pools according to their developmental stage. The hierarchical clustering of differentially abundant proteins evidenced a first cluster of 626 proteins that increased in abundance during development and a second cluster of 400 proteins that decreased in abundance during development, with most significant changes at the time of EGA and blastocyst formation. The main pathways and processes overrepresented among upregulated proteins were RNA metabolism, protein translation and ribosome biogenesis, whereas Golgi vesicle transport and protein processing in endoplasmic reticulum were overrepresented among downregulated proteins. The pairwise comparison between stages allowed us to identify specific protein interaction networks and metabolic pathways at the time of EGA, morula compaction and blastocyst formation. This is the first comprehensive study of proteome dynamics in non-rodent mammalian embryos developed in vivo. These data provide a number of protein candidates that will be useful for further mechanistic and functional studies.

Fatty Acid Profile of Blood Plasma at Mating and Early Gestation in Rabbit.

The aim of this study was to analyse the fatty acid (FA) profile of blood plasma at mating and 72 hpm by gas chromatography. Moreover, the correlation between FA and ovulation rate, normal embryos and compacted morulae was estimated. Palmitic, linoleic, oleic and stearic were the highest FA concentrations at mating and 72 hpm. Most long chain saturated and PUFA were higher at 72 hpm than at mating, while MUFA were higher at mating. SFA, MUFA and PUFA were high and positively correlated. Correlation was 0.643 between MUFA at mating and ovulation rate, and 0.781 between MUFA and normal embryos, respectively. Compacted morulae were slightly correlated with SFA at mating (0.465). In conclusion, the FA profile of plasma varies depending on the reproductive cycle of the rabbit female, adapting to energetic requirements at mating and early gestation. Moreover, positive correlations are found between fatty acids and ovulation rate and embryo development and quality.

Expression map of entry receptors and infectivity factors for pan-coronaviruses in preimplantation and implantation stage human embryos.

PurposeTo predict if developing human embryos are permissive to multiple coronaviruses.MethodWe analyzed publicly available single-cell RNA-seq datasets of human embryos for the known canonical and non-canonical receptors and spike protein cleavage enzymes for multiple coronaviruses like SARS-CoV, SARS-CoV-2, MERS-CoV, hCoV-229E, and hCoV-NL63. We also analyzed the expression of host genes involved in viral replication, host proteins involved in viral endosomal sorting complexes required for transport (ESCRT), genes of host proteins that physically interact with proteins of SARS-CoV-2, and the host genes essential for coronavirus infectivity.ResultsOf the known receptors of SARS viruses, ACE2, BSG, GOLGA7, and ZDHHC5 were expressed in different proportions in the zygote, 4-cell, 8-cell, morula, and blastocysts including the trophectoderm. The MERS-CoV receptor, DPP4, and hCoV-229E receptor, ANPEP, were expressed mainly from the compact morula to the blastocyst stages. Transcripts of the MERS-CoV alternate receptor LGALS1 were detected in most cells at all stages of development. TMPRSS2 transcripts were detected in the epiblast, primitive endoderm, and trophectoderm, while transcripts of the endosomal proteases CTSL, CTSB, and FURIN were expressed in most cells at all stages of development. ACE2 and TMPRSS2 were co-expressed in a proportion of epiblast and trophectoderm cells. The embryonic cells expressed genes involved in ESCRT, viral replication, SARS-CoV-2 interactions, and coronavirus infectivity. The ACE2 and TMPRSS2 co-expressing cells were enriched in genes associated with lipid metabolism, lysosome, peroxisome, and oxidative phosphorylation pathways.ConclusionPreimplantation and implantation stage human embryos could be permissive to multiple hCoVs.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10815-021-02192-3.

Production of viable bovine embryos by intracytoplasmic sperm injection of oocytes harvested from slaughtered old cows

Abstract: (1) Background: Intracytoplasmic sperm injection (ICSI) is currently used to increase fertilization success by avoiding several oocyte or sperm deficiencies that would normally prevent conception after in vivo fertilization or classical in vitro fertilization. This paper aimed at improving the in vitro fertilization protocol of bovine oocytes, harvested from old cows after slaughtering, using intracytoplasmic sperm injection; (2) Methods: Oocytes were harvested by puncture of follicles from ovaries obtained from slaughtered old cows, followed by aspiration. Out of the 127 cumulus-oocyte complexes that were harvested, 84 (66.14%) were declared suitable for cultivation, after morphological evaluation. Following oocyte maturation for 22 hours, 77 cumulus-oocyte complexes were morphologically intact and could undergo the steps required for intracytoplasmic injection of spermatozoa. Frozen-thawed bull semen was used for ICSI and the 77 fertilized oocytes were kept for 24 hours in an atmosphere enriched with 5% CO2.; (3) Results: Fertilized oocytes transformed into 46 zygotes (fertilization rate of 59.74%), while after 168 h of cultivation 38 transferable compact morulae or early blastocysts were obtained; (4) Conclusions: Intracytoplasmic sperm injection can represent a viable alternative to classical IVF, when oocytes or sperm with lower fertility are used.

Promoter-specific expression of the imprinted IGF2 gene in bovine oocytes and pre-implantation embryos.

The bovine IGF2 locus is a genomic region with alternative transcripts controlled by five promoters (P0, P1, P2, P3 and P4). As transcriptional regulation can affect messenger RNA (mRNA) stability and translation, and thus, subsequent biological effects, this study evaluated the bovine IGF2 promoter-specific expression patterns in oocytes and pre-implantation embryos produced in vitro by our standard IVP procedures. Immature and matured oocytes, and pre-implantation embryos at the 1-, 2-, 4-, 8- and 16-cell, and at early morula, compact morula, blastocyst and expanded blastocyst stages were collected in three pools of five structures per stage, in four replicates. Total RNA was extracted and subjected to RT-qPCR, using four sets of IGF2 promoter-specific primers covering transcripts driven by promoters P0/P1, P2, P3 and P4, with fragments sequenced for confirmation. Expression of P2- and P4-derived transcripts showed an initial peak between immature (P4) or matured (P2/P4) oocytes and 2-cell embryos, gradually falling until embryo genome activation (EGA), rising again at compaction and cavitation. P0/P1-derived transcripts were identified after EGA, during compaction, whereas P3 activity was not detected at any stage. Our findings suggest that P0/P1 and P2 likely have secondary roles during early stages, whereas P3 may be more relevant later in development. P4 seems to be the main pathway for bovine IGF2 expression during oocyte maturation and embryo development and, therefore, the main target to influence IVP in modulation of embryo growth and in studies in developmental biology.

ANTI-MÜLLERIAN HORMONE (AMH) AS ENDOCRINE MARKER FOR EMBRYO PRODUCTION IN SUPEROVULATED FRIESIAN COWS

The present study aimed to evaluate follicular dynamics, yield, quality and stage of embryos in superovulated Friesian cows with high and low AMH levels. A total of 10 Friesian cows synchronized with PGF2α to bring them in heat before start superovulation protocol. On Day 10 post oestrus, cows were injected with 2500 IU PMSG, then after 48h cow were injected with 3 ml PGF2α and with 5 ml GnRH on day 14. Cows were artificially inseminated twice at 12 and 24 h after GnRH. Embryos were collected after 7 days from AI. To measure AMH concentration 1 blood sample was collected at the beginning of second follicular wave according to ultrasonography. Results showed that number of antral follicles and total follicles was greater in high AMH level than in low AMH level donors. Differences in CL number of cows in high AMH level was greater than in low AMH level on flushing day. AMH concentration had a significant positive correlation with antral follicles number pre-treatment, total CL number on flushing day, and ovulation rate . The average number and recovery rate of recovered embryos from donor cows with high AMH level was greater than those recovered from cows in low AMH level. Also results indicated highly, significant, and positive correlation of AMH level with total number, recovery rate, excellent, good grade of embryos, and blastocysts, compact morulae, and morulae. In conclusion, circulating AMH concentration, as endocrine marker, is highly associated individually with superovulatory response and embryo production potential in cows.