Double vitrification and warming of blastocysts does not affect pregnancy, miscarriage or live birth rates.

RESEARCH QUESTION: Does double blastocyst vitrification and warming affect pregnancy, miscarriage or live birth rates, or birth outcomes, from embryos that have undergone preimplantation genetic testing for aneuploidies (PGT-A) testing? DESIGN: This retrospective observational analysis of embryo transfers was performed at a single centre between January 2017 and August 2022. The double-vitrification group included frozen blastocysts that were vitrified after 5-7 days of culture, warmed, biopsied (either once or twice) and re-vitrified. The single vitrification (SV) group included fresh blastocysts that were biopsied at 5-7 days and then vitrified. RESULTS: A comparison of the 84 double-vitrification blastocysts and 729 control single-vitrification blastocysts indicated that the double-vitrification embryos were frozen later in development and had expanded more than the single-vitrification embryos. Of the 813 embryo transfer procedures reported, 452 resulted in the successful delivery of healthy infants (56%). There were no significant differences between double-vitrification and single-vitrification embryos in the pregnancy, miscarriage or live birth rates achieved after single-embryo transfer (55% versus 56%). Logistic regression indicated that while reduced live birth rates were associated with increasing maternal age at oocyte collection, longer culture prior to freezing and lower embryo quality, double vitrification was not a significant predictor of live birth rate. CONCLUSIONS: Blastocyst double vitrification was not shown to impact pregnancy, miscarriage or live birth rates. Although caution is necessary due to the study size, no effects of double vitrification on miscarriage rates, birthweight or gestation period were noted. These data offer reassurance given the absence of the influence of double vitrification on all outcomes after PGT-A.

Morphokinetic Profiling Suggests That Rapid First Cleavage Division Accurately Predicts the Chances of Blastulation in Pig In Vitro Produced Embryos.

The study of pig preimplantation embryo development has several potential uses: from agriculture to the production of medically relevant genetically modified organisms and from rare breed conservation to acting as a physiologically relevant model for progressing human and other (e.g., endangered) species' in vitro fertilisation technology. Despite this, barriers to the widespread adoption of pig embryo in vitro production include lipid-laden cells that are hard to visualise, slow adoption of contemporary technologies such as the use of time-lapse incubators or artificial intelligence, poor blastulation and high polyspermy rates. Here, we employ a commercially available time-lapse incubator to provide a comprehensive overview of the morphokinetics of pig preimplantation development for the first time. We tested the hypotheses that (a) there are differences in developmental timings between blastulating and non-blastulating embryos and (b) embryo developmental morphokinetic features can be used to predict the likelihood of blastulation. The abattoir-derived oocytes fertilised by commercial extended semen produced presumptive zygotes were split into two groups: cavitating/blastulating 144 h post gamete co-incubation and those that were not. The blastulating group reached the 2-cell and morula stages significantly earlier, and the time taken to reach the 2-cell stage was identified to be a predictive marker for blastocyst formation. Reverse cleavage was also associated with poor blastulation. These data demonstrate the potential of morphokinetic analysis in automating and upscaling pig in vitro production through effective embryo selection.

Supplementation of porcine in vitro maturation medium with FGF2, LIF, and IGF1 enhances cytoplasmic maturation in prepubertal gilts oocytes and improves embryo quality.

In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed 'FLI'. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.

Eukaryotic response to hypothermia in relation to integrated stress responses.

Eukaryotic cells respond to hypothermic stress through a series of regulatory mechanisms that preserve energy resources and prolong cell survival. These mechanisms include alterations in gene expression, attenuated global protein synthesis and changes in the lipid composition of the phospholipid bilayer. Cellular responses to hyperthermia, hypoxia, nutrient deprivation and oxidative stress have been comprehensively investigated, but studies of the cellular response to cold stress are more limited. Responses to cold stress are however of great importance both in the wild, where exposure to low and fluctuating environmental temperatures is common, and in medical and biotechnology settings where cells and tissues are frequently exposed to hypothermic stress and cryopreservation. This means that it is vitally important to understand how cells are impacted by low temperatures and by the decreases and subsequent increases in temperature associated with cold stress. Here, we review the ways in which eukaryotic cells respond to hypothermic stress and how these compare to the well-described and highly integrated stress response systems that govern the cellular response to other types of stress.

Shiraz wines made from grape berries (Vitis vinifera) delayed in ripening by plant growth regulator treatment have elevated rotundone concentrations and "pepper" flavor and aroma.

Preveraison treatment of Shiraz berries with either 1-naphthaleneacetic acid (NAA) or Ethrel delayed the onset of ripening and harvest. NAA was more effective than Ethrel, delaying harvest by 23 days, compared to 6 days for Ethrel. Sensory analysis of wines from NAA-treated fruit showed significant differences in 10 attributes, including higher "pepper" flavor and aroma compared to those of the control wines. A nontargeted analysis of headspace volatiles revealed modest differences between wines made from control and NAA- or Ethrel-treated berries. However, the concentration of rotundone, the metabolite responsible for the pepper character, was below the level of detection by solid phase microextraction-gas chromatography-mass spectrometry in control wines, low in Ethrel wines (2 ng/L), and much higher in NAA wines (29 ng/L). Thus, NAA, and to a lesser extent Ethrel, treatment of grapes during the preveraison period can delay ripening and enhance rotundone concentrations in Shiraz fruit, thereby enhancing wine "peppery" attributes.

Ripening of grape berries can be advanced or delayed by reagents that either reduce or increase ethylene levels.

Grape (Vitis vinifera L.) berries are considered to be nonclimacteric fruit as they do not exhibit a large rise in ethylene production or respiration rate at the onset of ripening (veraison). However, ethylene may still play a role in berry development and in ripening in particular. (2-Chloroethyl)phosphonic acid (CEPA), an ethylene-releasing reagent, delayed ripening when applied early in berry development. In agreement with a role for ethylene in controlling the timing of ripening, the application of an inhibitor of ethylene biosynthesis, aminoethoxyvinylglycine (AVG), advanced ripening, as did abscisic acid, when applied during the preveraison period. Applications of CEPA nearer to the time of veraison enhanced berry colouration. Changes in the expression of ethylene biosynthesis and receptor genes were observed throughout berry development. Transcript levels of some of these genes were increased by CEPA and decreased by AVG, suggesting changes in ethylene synthesis and perception during the preveraison period that might contribute to the biphasic response to CEPA (ethylene). The significant delay of ripening in field-grown grapes through the application of CEPA also indicates that this may be useful in controlling the timing of veraison, and therefore harvest date, in warmer climates.