Cryopreservation of animal oocytes and embryos: current progress and future prospects

Cryopreservation describes techniques that permit freezing and subsequent warming of biological samples without loss of viability. The application of cryopreservation in assisted reproductive technology encompasses the freezing of gametes, embryos and primordial germ cells. Whilst some protocols still rely on slow-freezing techniques, most now use vitrification, or ultra-rapid freezing, for both oocytes and embryos due to an associated decreased risk of damage caused by the lack of ice crystal formation, unlike in slow-freezing techniques. Vitrification has demonstrated its use in many applications, not only following in vitro fertilisation (IVF) procedures in human embryology clinics, but also following in vitro production (IVP) of embryos in agriculturally important, or endangered animal species, prior to embryo transfer. Here we review the various cryopreservation and vitrification technologies that are used in both humans and other animals and discuss the most recent innovations in vitrification with a particular emphasis on their applicability to animal embryology

Development of an expert system for the identification of bacteria by focal plane array Fourier transform infrared spectroscopy

This study presents new techniques for the analysis of data acquired by focal plane array Fourier transform infrared (FPA-FTIR) spectroscopy. FPA-FTIR spectrometers are capable of acquiring several orders of magnitude more data than conventional FTIR spectrometers, necessitating the use of novel data analysis techniques to exploit the information-rich nature of these infrared imaging systems. The techniques investigated in this study are demonstrated in the context of bacteria identification by FPA-FTIR spectroscopy. Initially, an examination is made of the image fidelity of three FPA-FTIR instruments and demonstrates the high degree of within-image and between-image variability that is encountered with this technology. This is followed by a description of the development of pixel filtration routines that allow for the extraction of the most representative data from the infrared images of non-uniform samples. A genetic algorithm (GA) approach is introduced for determining the relevancy of spectral features in relation to bacterial classification and is compared to other forms of classifier optimizations. A proof-of-concept study demonstrating the potential use of infrared imaging to detect bacterial samples originating from a mixed culture is then presented. Finally, an overall methodology involving the combination of these data analysis techniques and including additional approaches towards the development, maintenance, and validation of databases based on infrared imaging data is described. This methodology has been developed with an emphasis on accessibility by implementing the elements of an expert system which allows for this technology to be employed by a non-technical user.Cette étude présente une nouvelle approche d'analyse de données spectrales résultant de l'utilisation de la spectroscopie infrarouge à transformée de Fourier couplée à un détecteur type «matrice à plan focal» (FPA-FTIR) à balayage rapide. Les spectromètres FPA-FTIR ont une capacité de capture de données de plusieurs ordres de grandeur supérieurs aux spectromètres traditionnels et nécessitent donc des techniques avancées d'analyse de données pour exploiter cette mine d'information que représente l'imagerie infrarouge. La spectroscopie FPA-FTIR a été utilisée dans cette étude pour l'identification des bactéries. L'étape initiale, celle de la comparaison de trois spectromètres FPA-FTIR sur les points de vue fidélité de l'image, tant image-image qu'entre images, a révélé de grandes variabilités qui sont propres à cette technologie. Cette étape est suivie du développement de routines de filtration de pixels permettant d'extraire les données caractéristiques de l'imagerie infrarouge des échantillons non-uniformes. Un algorithme génétique (GA) est ensuite introduit pour déterminer la pertinence des caractéristiques spectrales sur le plan de la classification bactérienne et a été comparé à d'autres formes de classification optimisée. Une étude de démonstration de la capacité de la technologie d'imagerie infrarouge pour la détection des échantillons de bactéries provenant de cultures mixtes s'en est suivie. Pour terminer, une méthodologie globale combinant ces techniques d'analyse de données et incluant d'autres étapes telles le développement, la mise à niveau et la validation des bases de données d'imagerie infrarouge a été présentée. Cette méthodologie met l'emphase sur le développement et l'implantation d'un système expert accessible d'utilisation à de non-experts

Standardization of Post-Vitrification Human Blastocyst Expansion as a Tool for Implantation Prediction

The increased use of vitrified blastocysts has encouraged the development of various criteria for selecting the embryo most likely to implant. Post-thaw assessment methods and timetables vary among investigators. We investigated the predictive value of well-defined measurements of human blastocyst re-expansion, following a fixed incubation period. Post-thaw measurements were taken exactly at 0 and 120 ± 15 min. Minimum and maximum cross-sectional axes were measured. Three groups were defined: Group 1: embryos that continued to shrink by 10 µm or more; group 2: embryos that ranged from −9 to +9 µm; and group 3: re-expansion of 10 µm or more. Patient and morphokinetic data were collected and integrated into the analysis. A total of 115 cases were included. The clinical pregnancy rate for group 1 was 18.9%; group 2, 27%; and group 3, 51.2% (p = 0.007). Pre-thaw morphologic grading and morphokinetic scores of the study groups did not reveal differences. p-values were 0.17 for the pre-thaw morphologic score, 0.54 for KID3, and 0.37 for KID5. The patients’ demographic and clinical data were similar. The clinical pregnancy rate correlated with the degree of thawed blastocyst re-expansion measured 2 h after incubation. This standardized measure is suggested as a tool to predict the potential of treatment success before embryo transfer

What we learned from extended culture of ‘rejected’ day-3 cleavage stage embryos: a prospective cohort study

Abstract Background To test whether poor quality day-3 embryos can undergo successful blastulation and implantation. Methods A prospective cohort study was conducted. Whether or not a good quality embryo was transferred on day-3, poor quality (rejected) embryos were further cultured and followed. The clinical outcome of each embryo was assessed. Results A total of 694 rejected embryos (from 205 patients) were included, with a blastulation rate of 21.2% (147 embryos) compared to 64.2% general blastulation rate reported by our laboratory (P < 0.01). In a multivariate logistic regression model, only their grade on day-3 significantly affected blastulation (P = 0.01). A total of 97 embryos attained eligibility for fresh transfer or cryopreservation, only 6 of which resulted from a day-3 embryo scored < 2. Of these, 52 were transferred, resulting in 21 pregnancies (16 clinical and 5 chemical). In summary, 694 cultured embryos yielded 16 clinical pregnancies; a 2.3% clinical pregnancy rate. Conclusions Low score day-3 embryos can result in successful blastulation and clinical pregnancies. However, the normal blastulation rate is poor