Embryo Survival Research Articles

Analysis of short videos of bovine embryos can detect presence of cryodamage

Cryopreservation of bovine embryos has transformed animal breeding strategies by allowing storage of genetic material which enables efficient recipient utilization, sale and market­ing of embryos, and shipping and transport of embryos around the world. Advancements have been made in cryopreserva­tion techniques including the introduction of direct transfer ethylene glycol media, controlled rate freezers allowing pre­cise temperature control of the freezing curve and vitrifica­tion techniques. However, little advancement has been made to evaluate embryo survival of cryopreservation which results in the transfer of embryos which have suffered cryodamage and have no chance of establishing a pregnancy. The objective of this study was to use graphic image processing (GIP) and machine-learning (ML) techniques to evaluate embryo health post-cryopreservation.

Combinatorial microRNA activity is essential for the transition of pluripotent cells from proliferation into dormancy

Combinatorial microRNA activity is essential for the transition of pluripotent cells from proliferation into dormancy

Effect of temperature and spawning agents on wild female asp (Leuciscus aspius) reproductive efficiency under controlled conditions

Artificial reproduction is a bottleneck to produce stocking material for many species of freshwater fish. One of these species is the asp, Leuciscus aspius. Research in the field of artificial reproduction of this species is very scarce and often incomplete. There are no breeding protocols specifying optimal environmental conditions and hormonal stimulation for many species of rheophilic cyprinids, including asp. Since the number of natural asp populations is constantly decreasing, it is important to support natural stocks by restocking with high quality stocking material. For this reason, optimized protocols are needed to breed this species under controlled conditions to produce stocking material with high biodiversity and good health. Such an approach will make it possible to maintain the population of natural asp at a constant level. The aim of this study was to develop the protocol of asp artificial reproduction using optimized thermal conditions and appropriate hormonal stimulation. In experiment I, the influence of constant temperature (10.0, 12.0 and 14.0 °C) on the effectiveness of artificial reproduction of asp. In experiment II, the effectiveness of asp reproduction was checked after the application of spawning agents: Ovopel, Ovaprim or a combination of these two agents The obtained results indicate that for the final maturation of oocytes (FOM) and artificial reproduction of asp in controlled conditions, water temperatures of 10–12 °C are the most useful. Under these thermal conditions, the highest percentages of female’s ovulation and embryo survival, as well as the percentage of hatching, were obtained. Hormone injections are necessary to perform final oocyte maturation (FOM) in female asp in captivity. All spawning agents used were especially useful for artificial reproduction of asp, however, the best values of the studied indices, such as ovulation rate and embryo survival, were obtained after the application of Ovaprim or the combination of Ovopel and Ovaprim in water temperature at a range of 10–12 °C. It was found that the pH of ovarian fluid may be a preliminary indicator of the biological quality of eggs in asps. The optimal pH value is 8.0–8.4. At pH below 7.4, no viable embryos were observed.

A nox2/cybb zebrafish mutant with defective myeloid cell reactive oxygen species production displays normal initial neutrophil recruitment to sterile tail injuries.

Reactive oxygen species are important effectors and modifiers of the acute inflammatory response, recruiting phagocytes including neutrophils to sites of tissue injury. In turn, phagocytes such as neutrophils are both consumers and producers of reactive oxygen species. Phagocytes including neutrophils generate reactive oxygen species in an oxidative burst through the activity of a multimeric phagocytic nicotinamide adenine dinucleotide phosphate oxidase complex. Mutations in the NOX2/CYBB (previously gp91phox) nicotinamide adenine dinucleotide phosphate oxidase subunit are the commonest cause of chronic granulomatous disease, a disease characterized by infection susceptibility and an inflammatory phenotype. To model chronic granulomatous disease, we made a nox2/cybb zebrafish (Danio rerio) mutant and demonstrated it to have severely impaired myeloid cell reactive oxygen species production. Reduced early survival of nox2 mutant embryos indicated an essential requirement for nox2 during early development. In nox2/cybb zebrafish mutants, the dynamics of initial neutrophil recruitment to both mild and severe surgical tailfin wounds was normal, suggesting that excessive neutrophil recruitment at the initiation of inflammation is not the primary cause of the "sterile" inflammatory phenotype of chronic granulomatous disease patients. This nox2 zebrafish mutant adds to existing in vivo models for studying reactive oxygen species function in myeloid cells including neutrophils in development and disease.

Beyond Immune Balance: The Pivotal Role of Decidual Regulatory T Cells in Unexplained Recurrent Spontaneous Abortion.

Recurrent spontaneous abortion (RSA) is defined as two or more consecutive pregnancy failures, which brings tremendous stress to women of childbearing age and seriously affects family well-being. However, the reason in about 50% of cases remains unknown and is defined as unexplained recurrent spontaneous abortion (URSA). The immunological perspective in URSA has attracted widespread attention in recent years. The embryo is regarded as a semi-allogeneic graft to the mother. A successful pregnancy requires transition to an immune environment conducive to embryo survival at the maternal-fetal interface. As an important member of regulatory immunity, regulatory T (Treg) cells play a key role in regulating immune tolerance at the maternal-fetal interface. This review will focus on the phenotypic plasticity and lineage stability of Treg cells to illustrate its relationship with URSA.

The importance of developmental programming in the beef industry

The beef industry relies on multiple focused segments (e.g., cow-calf, stocker/feeder, and meat packing) to supply the world with beef. Thus, the potential impact of developmental programming on the beef industry needs to be evaluated with regards to the different production traits that drive profitability within each segment. For example, when nutrient restriction of dams occurred early in gestation embryo survival was decreased and the ovarian reserve of heifer progeny was negatively affected. Restriction during mid- to late gestation negatively impacted first service conception rates and pregnancy success of daughters. Even non-nutrient stress has been reported to impact transgenerational embryo development through the male progeny. Primary and secondary muscle fibers form during months two to eight (Days 60–240) of gestation. Therefore, external stimuli (nutrition or environmental) during this window have the potential to decrease the postnatal number of muscle fibers; which has an irreversible impact on animal growth and performance. Nutrient restriction during the last third of gestation resulted in decreased weaning weights, and in some instances decreased dry mater intake, hot carcass weight, and marbling scores. Protein supplementation during late gestation; however, increased weaning weight and ADG to weaning, but progeny of dams restricted in protein in late gestation had greater ribeye area. The importance of developmental programming is recognized; however, its precise application depends on comprehension of its integrated effects across the multiple-focused segments of the beef industry.

Low toxicity of magnetite-based modified bionanocomposites with potential application for wastewater treatment: Evaluation in a zebrafish animal model

In recent years, the escalating concerns surrounding environmental pollution and the need for sustainable wastewater treatment solutions have underscored the significance of developing technologies that can efficiently treat wastewater while also reducing negative ecological effects. In this context, our study aims to contribute to the advancement of sustainable technologies for wastewater treatment, by investigating the effects that bare magnetite nanoparticles and those functionalized with the enzyme laccase could have in an aquatic animal, zebrafish, at various life cycle stages. Exposure to magnetite nanoparticles shows some effects on embryo hatching, survival rates, or larval behavior at higher concentrations. For both treatments, the hatching percentages were close to 80% compared to 93% for the control group. At the end of the observations in larvae, survival in all the evaluated groups was higher than 90%. Additionally, we evaluated the accumulation of nanoparticles in various stages of zebrafish. We found that, although there was accumulation during embryonic stages, it did not affect normal development or subsequent hatching. Iron levels in different organs such as gills, muscles, gastrointestinal tract, and brain were also evaluated in adults. Animals treated with a mix of food and nanoparticles at 10 μg/mL (Food group) presented a higher concentration of iron accumulation in muscle, gastrointestinal tract, and gills compared to the untreated control group. Although iron levels increased depending on the dose and exposure method applied, they were not statistically significant from the control groups. Our findings suggest that bionanocomposites evaluated here can be considered safe for removal of contaminants in wastewater without toxic effects or detrimental accumulation fish's health.

Local and global stressors as major drivers of the drastic regression of brown macroalgae forests in an oceanic island

Similar to other coastal regions worldwide, forests created by brown macroalgae have severely declined in recent decades across the Macaronesian oceanic archipelagos (northeastern Atlantic), eroding the provision of ecosystem services. However, the putative effects of natural and anthropogenic stressors (both local and global) on such declines across spatial and temporal scales remain unresolved. Our research endeavored to investigate the connection between local and global stressors and the distribution and extent of the brown macroalgae Gongolaria abies-marina in the rocky intertidal and adjacent subtidal zones of Gran Canaria over the past four decades. We also quantified the presence of populations at small scales, according to local micro-habitat topography (“open rock” versus “refuge”). Through herbarium records, we additionally analyzed the historical variation in the thallus size of the species. Finally, we experimentally assessed the thermotolerance of embryonic stages to warming. The main environmental drivers explaining the regression of G.abies-marina were the increasing number of marine heatwaves, while the number of local human impacts (quantified through the HAPI index) also accounted for further regression in the extent of marine forests. Warming experimentally reduced the survival and size of macroalgal embryos. A progressive miniaturization of the species, currently restricted to micro-habitat refuges as a survival strategy, seems likely to be the final stage in the progressive disappearance of this macroalgae from the island’s rocky shores.

Slow freezing cryopreservation of Korean bovine blastocysts with an additional sucrose pre-equilibration step.

Embryo cryopreservation is a valuable technique used for preserving genetic resources for long periods. However, the survival rate of embryos is dependent on the method used. Therefore, in this study, we evaluated the efficiency of slow freezing method but with an additional dehydration step prior to freezing to overcome the formation of ice crystals. Oocytes collected from the ovaries of native Korean cattle subjected to in vitro fertilization were cultured for 7 days until the formation of expanded blastocysts. Before freezing, the blastocysts were placed in four pre-equilibration media: a control medium with no addition of sucrose, and three experimental media with the addition of 0.1, 0.25, and 0.5 M sucrose, respectively. Then, the pre-equilibrated embryos were frozen. Embryo survival and hatching rates were evaluated morphologically at 24, 48, and 72 h after thawing. Immunofluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and gene expression analysis of the re-expanded blastocytes were examined 24 h after freeze-thawing. The survival rate was significantly higher in the 0.1 M group than in the control group (p < 0.05), and the hatching rate at 72 h was significantly higher in the 0.25 and 0.5 M groups than in the control group (p < 0.05). TUNEL-positive cells were significantly lower in the 0.25 M group than in the control group (12.5 ± 0.9 vs. 8.3 ± 0.8; p < 0.05). The gene expression of BCL2 associated X, heat shock protein 70 kDa, and aquaporin 3 in the 0.25 M group was significantly lower than that in the control group (p < 0.05). Our study revealed that treatment with 0.25 M sucrose before slow freezing improved the viability of bovine embryos after freeze-thawing.

Impact of reducing lipid content during in vitro embryo production: A systematic review and meta-analysis

There is still no consensus regarding the role of lipid modulators during in vitro embryo production. Thus, we investigated how lipid reducers during the in vitro maturation of oocytes (IVM) or in vitro culture (IVC) of embryos impact their cryotolerance. A literature search was performed using three databases, recovering 43 articles for the systematic review, comprising 75 experiments (13 performed in IVM, 62 in IVC) and testing 13 substances. In 39 % of the experiments, an increase in oocyte and/or embryo survival after cryopreservation was reported, in contrast to 48 % exhibiting no effect, 5 % causing negative effects, and 8 % influencing in a dose-dependent manner. Of the 75 experiments extracted during IVM and IVC, 41 quantified the lipid content. Of those that reduced lipid content (n = 26), 50 % increased cryotolerance, 34 % had no effect, 8 % harmed oocyte/embryo survival, and 8 % had different results depending on the concentration used. Moreover, 28 out of the 43 studies were analyzed under a meta-analytical approach at the IVC stage in cattle. There was an improvement in the cryotolerance of bovine embryos when the lipid content was reduced. Forskolin, l-carnitine, and phenazine ethosulfate positively affected cryotolerance, while conjugated linoleic acid had no effect and impaired embryonic development. Moreover, fetal bovine serum has a positive impact on cryotolerance. SOF and CR1aa IVC media improved cryotolerance, while mSOF showed no effect. In conclusion, lipid modulators did not unanimously improve cryotolerance, especially when used in IVM, but presented positive effects on cryotolerance during IVC when reaching lipid reduction.

Metal nanoparticle-induced effects on green toads (Amphibia, Anura) under climate change: conservation implications.

The toxicity of aluminum oxide (Al2O3), copper oxide (CuO), iron oxide (Fe3O4), nickel oxide (NiO), zinc oxide (ZnO), and titanium dioxide (TiO2) nanoparticles (NPs) on amphibians and their interaction with high temperatures, remain unknown. In this study, we investigated the survival, developmental, behavioral, and histological reactions of Bufotes viridis embryos and larvae exposed to different NPs for a duration of 10 days, using lethal concentrations (LC25%, LC50%, and LC75% mg/L) under both ambient (AT: 18 °C) and high (HT: 21 °C) temperatures. Based on LC, NiONPs > ZnONPs > CuONPs > Al2O3NPs > TiO2NPs > Fe3O4NPs showed the highest mortality at AT. A similar pattern was observed at HT, although mortality occurred at lower concentrations and Fe3O4NPs were more toxic than TiO2NPs. The results indicated that increasing concentrations of NPs significantly reduced hatching rates, except for TiO2NPs. Survival rates decreased, abnormality rates increased, and developmental processes slowed down, particularly for NiONPs and ZnONPs, under HT conditions. However, exposure to low concentrations of Fe3O4NPs for up to 7 days, CuONPs for up to 72 h, and NiO, ZnONPs, and TiO2NPs for up to 96 h did not have a negative impact on survival compared with the control group under AT. In behavioral tests with larvae, NPs generally induced hypoactivity at AT and hyperactivity at HT. Histological findings revealed liver and internal gill tissue lesions, and an increase in the number of melanomacrophage centers at HT. These results suggest that global warming may exacerbate the toxicity of metal oxide NPs to amphibians, emphasizing the need for further research and conservation efforts in this context.

Collective effects of rising average temperatures and heat events on oviparous embryos.

Survival of the immobile embryo in response to rising temperature is important to determine a species' vulnerability to climate change. However, the collective effects of 2 key thermal characteristics associated with climate change (i.e., rising average temperature and acute heat events) on embryonic survival remain largely unexplored. We used empirical measurements and niche modeling to investigate how chronic and acute heat stress independently and collectively influence the embryonic survival of lizards across latitudes. We collected and bred lizards from 5 latitudes and incubated their eggs across a range of temperatures to quantify population-specific responses to chronic and acute heat stress. Using an embryonic development model parameterized with measured embryonic heat tolerances, we further identified a collective impact of embryonic chronic and acute heat tolerances on embryonic survival. We also incorporated embryonic chronic and acute heat tolerance in hybrid species distribution models to determine species' range shifts under climate change. Embryos' tolerance of chronic heat (T-chronic) remained consistent across latitudes, whereas their tolerance of acute heat (T-acute) was higher at high latitudes than at low latitudes. Tolerance of acute heat exerted a more pronounced influence than tolerance of chronic heat. In species distribution models, climate change led to the most significant habitat loss for each population and species in its low-latitude distribution. Consequently, habitat for populations across all latitudes will shift toward high latitudes. Our study also highlights the importance of considering embryonic survival under chronic and acute heat stresses to predict species' vulnerability to climate change.

A sex-linked supergene with large effects on sperm traits has little impact on reproductive traits in female zebra finches.

Despite constituting an essential component of fitness, reproductive success can vary remarkably between individuals and the causes of such variation are not well understood across taxa. In the zebra finch-a model songbird, almost all the variation in sperm morphology and swimming speed is maintained by a large polymorphic inversion (commonly known as a supergene) on the Z chromosome. The relationship between this polymorphism and reproductive success is not fully understood, particularly for females. Here, we explore the effects of female haplotype, and the combination of male and female genotype, on several primary reproductive traits in a captive population of zebra finches. Despite the inversion polymorphism's known effects on sperm traits, we find no evidence that inversion haplotype influences egg production by females or survival of embryos through to hatching. However, our findings do reinforce existing evidence that the inversion polymorphism is maintained by a heterozygote advantage for male fitness. This work provides an important step in understanding the causes of variation in reproductive success in this model species.

The embryo-oil drop assembly: the timing and morphology of a critical event for fish early-life history survival

Egg specific gravity is of relevance for fish recruitment since the ability to float influences egg and larvae development, dispersal and connectivity between fishing grounds. Using zootechnics, histological approaches, optical and electronic transmission microscopy, this study describes the morphogenetic mechanism of adhesion of the oil-drop covering layer (OCL) to the oil droplet (OD) in embryos of Merluccius merluccius under physical conditions reflecting the marine environment. The herein described primordial (p)OCL is a substructure of the inner yolk syncytial layer which contains egg organella aimed to mobilize lipidic reserves from the oil drop (OD) towards the embryo blood. It is shown that the timely OD-OCL assembly is a critical morphogenetic process for embryo and larvae survival. Such assembly depends on egg buoyance because of its influence on the embryo capacity to rotate within the perivitelline space. Therefore, oil droplet adhesion (ODA) eggs are capable to complete their development while oil droplet non-adhesion eggs (ODNA) dye soon after hatching. We show that gravity-dependent egg buoyance categories exhibit different ODA/ODNA ratios (0–77%) and that relationship diminishes under incubation systems such as sprayers, that do not assure a dynamic seawater surface mixing to avoid egg desiccation. As an adaptive trait, egg gravity strongly depends on oceanic properties such as current dynamics, turbulence, oxygen, rainfall, and salinity, whose rapid changes would likely challenge the sustainability of fisheries recruitment.

Cuban Policosanol (Raydel®) Exerts Higher Antioxidant and Anti-Glycation Activities than Chinese Policosanol (BOC Sciences) in Reconstituted High-Density Lipoproteins: In Vivo Anti-Inflammatory Activities in Zebrafish and Its Embryos.

The present study compares sugarcane-wax purified policosanols sourced from Cuba (Raydel®) and China (BOC Sciences) and utilized following the synthesis of reconstituted high-density lipoproteins (rHDL). The two policosanols exhibited distinctly different ingredient ratios of long-chain aliphatic alcohols, particularly 1-octacosanol (C28) and 1-tetratriacotanol (C34). After synthesizing rHDL with apolipoprotein A-I (apoA-I), the two policosanols bound well with phospholipid and apoA-I to form the discoidal rHDL. Notably, rHDL-1, containing Cuban policosanol, displayed the largest particle diameter at approximately 78 ± 3 nm. In contrast, both control rHDL (rHDL-0) and rHDL containing Chinese policosanol (rHDL-2) exhibited smaller particles, with diameters of approximately 58 ± 3 nm and 61 ± 2 nm, respectively. Furthermore, rHDL-1 demonstrated enhanced anti-glycation activity, safeguarding apoA-I from degradation within HDL, and displayed the antioxidant ability to inhibit LDL oxidation. A microinjection of each rHDL into zebrafish embryos in the presence of carboxymethyllysine (CML) revealed rHDL-1 to have the strongest antioxidant activity with the highest embryo survivability and normal developmental morphology. Dermal application to recover the wound revealed rHDL-1 to have the highest wound-healing activity (75%) and survivability (92%) in the cutaneous wound area in the presence of CML. In adult zebrafish, injecting CML (250 μg) caused acute death and hyperinflammation, marked by heightened neutrophil infiltration and interleukin (IL)-6 production in liver. However, co-administering rHDL-1 notably increased survival (85%) and exhibited strong anti-inflammatory properties, reducing IL-6 production while improving the blood lipid profile. However, a co-injection of rHDL-2 resulted in the lowest survivability (47%) with more hepatic inflammation. In conclusion, Cuban policosanol (Raydel®) has more desirable properties for the in vitro synthesis of rHDL with stronger anti-glycation and antioxidant activities than those of Chinese policosanol (BOC Sciences). Moreover, Raydel-policosanol-integrated rHDL demonstrates a noteworthy effect on accelerated wound healing and robust anti-inflammatory properties, leading to increased survivability in zebrafish embryos and adults by effectively suppressing CML-induced hyperinflammation.

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation.

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.

Assessment of air pollution emitted during cooking using biomass and cleaner fuels in the Shiselweni region of Eswatini (Swaziland)

AbstractIn Eswatini, 62.3% of households still rely on solid fuel for cooking, especially wood (61.8%). Smoke emissions during biomass burning remain the primary source of indoor air pollution, comprising pollutants detrimental to health. This study provides a quantitative exposure assessment of fuels used in the Shiselweni region with the objectives of monitoring the carbon monoxide (CO) and carbon dioxide (CO2) concentrations during cooking events and evaluating cancer and noncancer risks due to exposure to particulate-bound polycyclic aromatic hydrocarbons (PAHs) during cooking events among cooking personnel in households that cook indoors by burning biomass. Real-time CO, CO2, and particulate matter (PM) monitoring was performed in seventeen kitchens during cooking events across the Shiselweni region using different cooking methods: biomass in open fires and stoves, liquefied petroleum gas (LPG), and electric stoves. PM2.5, PM10, and CO exceeded indoor exposure guidelines for biomass fuel-related homesteads. Particulate PAH intake concentrations were evaluated, and biomass fuel users exhibited high cancer risks and low embryo survival chances due to particulate PAH exposure. The average total cost of reducing the incremental lifetime cancer risk (ILCR) and hazard quotient (HQ) to acceptable levels was lower when shifting from biomass to LPG stoves than when shifting to electric stoves. Graphical abstract

Preimplantation Genetic Testing: Personal Views Regarding the Invasiveness of Trophectoderm Biopsy and Risks on Embryos Development “An Operators Survey”

This study aims to assess the Trophectoderm (TE) biopsy practice in Jordan in terms of the following effectiveness parameters: timing of zona breaching, risk of inner cell mass herniation if zona breaching was done on day 3, timing of TE sampling, method of biopsy (pulling or flicking), number of laser pulses, assessment of embryo survival after biopsy, and degeneration rate. An online cross-sectional survey was conducted in November 2022. The collected data presented the perception of embryologists (&gt;10 years experience) about the difficulty of the technique and the awareness of the risks it imposes on embryonic development. Potential predictors of embryologists’ awareness of the risks of trophectoderm biopsy in preimplantation genetic testing (PGT) and procedure difficulty were investigated. 125 embryologists were eligible, and 72 (57.6%) adequately filled the questionnaire, of which 51 (70.8%) perceived the procedure as moderately difficult. However, 8 (11.1%) embryologists perceive it as very difficult. Regarding the preferred time of zona breaching, 39 (54.2%) of embryologists perform zona breaching on day 5 of embryonic life. 68% claim they primarily use flicking when performing TE biopsy. Moreover, 33 (45.8%) of the 72 surveyed embryologists claimed they use 2-3 laser pulses, and 56 (77.8%) claimed it takes 2 to 3 minutes to finish the procedure. Regarding the embryologists’ awareness of the risk of Inner Cell Mass (ICM) herniation, most embryologists 46 (64%) believed there is a moderate risk if zona breaching is done on day 3. 23 (32%) acknowledge the procedure as having a low risk for embryonal development. 29 (40.3%) of embryologists assess survival by checking the re-expansion of the biopsied blastocyst after 2 hours, while 18% check blastocyst re-expansion after 15 minutes. 39 (54.2%) claimed that the incidence of degeneration rate post-TE biopsy is rare. TE biopsy strategy is one of the most promising biopsy techniques in PGT. Most embryologists in Jordan perceive the procedure as moderately difficult due to the technical considerations involved in performing the optimum TE biopsy.

Developmental progression continues during embryonic diapause in the roe deer

Embryonic diapause in mammals is a temporary developmental delay occurring at the blastocyst stage. In contrast to other diapausing species displaying a full arrest, the blastocyst of the European roe deer (Capreolus capreolus) proliferates continuously and displays considerable morphological changes in the inner cell mass. We hypothesised that developmental progression also continues during this period. Here we evaluate the mRNA abundance of developmental marker genes in embryos during diapause and elongation. Our results show that morphological rearrangements of the epiblast during diapause correlate with gene expression patterns and changes in cell polarity. Immunohistochemical staining further supports these findings. Primitive endoderm formation occurs during diapause in embryos composed of around 3,000 cells. Gastrulation coincides with elongation and thus takes place after embryo reactivation. The slow developmental progression makes the roe deer an interesting model for unravelling the link between proliferation and differentiation and requirements for embryo survival.

Embryo long-term storage does not affect assisted reproductive technologies outcome: analysis of 58,001 vitrified blastocysts over 11 years

BackgroundRecently the potential detrimental effect that the duration of storage time may have on vitrified samples has raised some concern, especially when some studies found an association between cryo-storage length and decreased clinical results. ObjectiveThis study aimed to evaluate the effects of the storage time length of day-5 vitrified blastocysts in two study groups: freeze-all cycles and non elective frozen embryo transfers (FET). Study designThis was a retrospective study including 58001 vitrified/warmed day-5 blastocysts from two different populations according to the reason for FET. Elective FET comprised freeze-all cycles (N=16615 blastocysts and 16615 patients) where only single embryo transfers (SET) and only the first FET were included. The non elective FET group included 41386 embryos from 25571 patients where FET took place using supernumerary embryos after fresh embryo transfer. All the possible FETs were included. Both SET and double embryo transfers (DET) were included. Donor and autologous oocytes were used. The period covered by this study was 11 years. The blastocysts sample was clustered into deciles which provided specific storage duration categories. Main outcome was live birth rate (LBR) and secondary outcomes were embryo survival, miscarriage, clinical and ongoing pregnancy rates according to storage duration. The impact of storage time was assessed by univariable analyses in both groups. The comparison was made between each decile to the last one. A multivariable logistic regression analysis was conducted including the variables with significant association found in the univariate analysis. Student's t-test and Chi squared tests, or an analysis of variance were used wherever appropriate. P < 0.05 was considered statistically significant. ResultsThere were statistical differences in baseline characteristics of patients included in the study groups. Storage durations ranged from ≤ 0.67 to ≥ 4.34 and from ≤ 1.8 to ≥ 34.81 months in freeze-all and non elective FET respectively. Embryo survival did not show statistical differences across the categories of storage time in freeze-all and non elective FET groups. Statistical differences were found for the LBR across some, but not all the subgroups of storage duration. The multivariable analysis showed no association between storage time and the LBR in both groups (NS). Blastocyst quality, body mass index (BMI), number of retrieved oocytes, endometrial preparation, male factor and uterine factor were related to the drop in the LBR in the freeze-all group (p<0.05). In the non elective FET group the variables that showed significant association with the LBR were, age at retrieval and at FET, type of FET (SET or DET), number of retrieved oocytes, BMI, endometrial preparation, origin of sperm sample and female factor. ConclusiónThis large study demonstrated no association between storage time and clinical outcome. Other variables such as patient's age, embryo quality, BMI and etiology, are somewhat responsible for impacting outcome. This provides evidence for the safety of embryo vitrification, even after long storage periods. This is reassuring for both IVF practitioners and the patients undergoing FET of either elective or non elective embryos.