Antifreeze protein from Anatolia polita (ApAFP914) improved outcome of vitrified in vitro sheep embryos

Embryo cryopreservation is an important tool to preserve endangered species. As a cryoprotectant for mouse oocytes, antifreeze protein from Anatolica polita (ApAFP914) has demonstrated utility. In the present study, the effects of controlled slow freezing and vitrification methods on the survival rate of sheep oocytes fertilized in vitro after freezing-thawing were compared. Different ApAFP914 concentrations were added to the vitrification liquid for exploring the effect of antifreeze protein on the warmed embryos. The results showed that the survival and hatching rates of in vitro derived embryos were significantly higher than that of the slow freezing method. Furthermore, among the cryopreserved embryos at different developmental stages, the survival and hatching rates of the expanded blastocyst were significantly higher than those of the blastocysts, early blastocysts and morula. The survival and the hatching rates of the fast-growing embryos were both significantly higher than that of the slow-growing embryos. Additionally, treatment of ApAFP914 (5–30 μg/mL) did not increase the freezing efficiency of the 6–6.5 d embryos. However, addition of 10 μg/mL of ApAFP914 significantly increased the hatching rate of slow-growing embryos. In conclusion, our study suggests that the vitrification is better than the slow freezing method for the conservation of in vitro sheep embryos, and supplementation of ApAFP914 (10 μg/mL) significantly increased the hatching rate of slow-growing embryos after cryopreservation.     

Rabbit blastocyst: Allocation of cells to the inner cell mass and trophectoderm

The proportion of total cells in the blastocyst allocated to the inner cell mass (ICM) and trophectoderm (TE) is important for future development and may be a sensitive indicator to evaluate culture conditions. The number of cells and their distribution within the two primary cell lineages were determined for the rabbit embryo developing in vivo after superovulation or nonsuperovulation or embryo transfer and compared with embryos developing in vitro. Comparisons were made with cultured embryos or embryos grown in vivo until 3.5, 4.0, and 4.5 days of age. Embryos from superovulated rabbits developed in vivo for 3.5, 4.0, and 4.5 days, respectively, had 361, 758, and 902 total cells (P<0.05), and in nonsuperovulated rabbits 130, 414, and 905 total cells (P<0.05), with increasing proportions of ICM cells over time (P<0.05). One-cell embryos recovered from superovulated females and transferred to nonsuperovulated recipients developed more slowly with 70, 299, and 550 total cells after 3.5, 4.0, and 4.5 days of culture (P<0.05), respectively. The proportion of ICM cells increased with age of the embryo. Corresponding values for one-cell embryos cultured in vitro resulted in 70, 299, and 550 total cells (P<0.05). However, in vitro culture of morula-stage embryos in the presence of fetal bovine serum for 24 hr did not delay growth. In addition, the proportions of ICM/total cells were 0.17, 0.25, and 0.29 for embryos developing in vitro at 3.5, 4.0, and 4.5 days, respectively, similar to those for embryos developing in vivo at each of the three recovery times. These data establish for the first time the number and proportion of cells allocated to the ICM of the rabbit embryo developing in vivo or under defined conditions in vitro. © 1995 Wiley-Liss, Inc.     

Growth and DNA replication in rabbit blastocysts.

DNA content and DNA polymerase activity were measured on rabbit blastocysts removed from the uterus at 24-hr intervals over the period of days 4-7 postcoitum (pc). Median DNA content increased 53 times over the 72-hr period, from 25.3 ng on day 4 to 1,360 ng on day 7. Median DNA polymerase activity (fmole of radiolabeled nucleotide incorporated in 30 min at 37 degrees C) increased 393-fold from day 4 to day 7: 32.8 to 12,900. These embryos also increased in surface area and volume by 334-fold and 6,078-fold, respectively. Litters containing individuals with high DNA content also tended to have similar individuals with high DNA polymerase activity. Therefore, DNA polymerase activity may be a useful measure of the potential for the next cell division. A large amount of variation existed between blastocysts in all parameters measured. An analysis of variance, conducted to partition variation between litters and within litters, determined that within-litter variation was actually greater than that between litters, resulting in intraclass correlation coefficients less than 0.5. There was also a positive regression of DNA content and DNA polymerase activity on surface area in 6- and 7-day-old blastocysts after eliminating variation attributable to litters. The developmental pattern of DNA polymerase activity in the rabbit may be quantitatively different from that described in the mouse. The pattern in mammals is very different from that described in several nonmammalian species.     

Endometrial ultrastructure in the early uterine response to blastocysts and artificial deciduogenic stimuli in rats

Summary The early uterine response to transplanted, delayed and estrogenactivated blastocysts was studied ultrastructurally and compared with that induced by intrauterine instillations of deciduogenic agents (arachis oil, air). The uterine responses to delayed and activated blastocysts showed no ultrastructural or temporal differences. Already within 4 h after transfer to a sensitized uterus, the delayed blastocysts exhibited signs of activation, and both types of blastocysts had started to attach onto an undamaged epithelial lining. Signs of stromal cell differentiation into decidual cells were also seen as early as 4 h after transfer, while the Pontamine-blue reaction did not appear until after 8 h. The results therefore indicate that the transplanted blastocysts induced decidualization atraumatically and that the delayed blastocysts were either deciduogenic already before transfer or rapidly acquired deciduogenic properties after transfer.Artificial decidual induction with oil and air led to damage or death of a large number of cells in the uterine luminal epithelium. Within only 15 min after instillation pronounced signs of cell damage were seen, and later numerous cells were extruded from the epithelial lining. In the stroma ultrastructural signs of decidual cell differentiation and a Pontamine-blue reaction were observed as early as 4 h after induction. It is therefore suggested that oil and air induce decidualization via the epithelium by means of trauma.Supported by grants from the Swedish Medical Research Council (Project 12X-70)     

Predicting live birth by combining cleavage and blastocyst-stage time-lapse variables using a hierarchical and a data mining-based statistical model

Prolonged embryo culture is increasingly used as a way of improving pregnancy rates, especially in the context of single embryo transfer. So far, only a handful of studies examined the relation between implantation potential and time-lapse parameters extracted from later stages (morula and blastocyst) of embryo development. For this retrospective study all 285 single vitrified-thawed blastocyst transfers (SVBT) from all consecutive unselected patients whose fertilized oocytes were submitted to time-lapse monitoring (TLM) from a two-year cohort were analysed. Two different statistical models were created; a hierarchical one including the two strongest live birth (LB) predictors (t2 and texpB₂) and a more complex model based on principal component analysis (PCA) and logistic regression methods. The first, four-category, hierarchical model effectively distinguished between blastocysts of increasing LB rates (8, 30, 40, 53%). For the second data-mining model quartiles of the created Sc parameter had increasing LB rates (12, 19, 40, 49%). AUC values were comparable for both models (0.723, 95CI%:0.66–0.79 versus 0.717, 95CI%:0.65–0.78). The combination of cleavage- and blastocyst-stage variables through hierarchical or data mining-based algorithms was used successfully to predict live birth. However, due to the lack of internal / external validation the predictive capacities of this model could differ largely in different datasets.