BACKGROUND AND AIM This study aimed to compare two embryo decision support tools from Vitrolife, iDAScore® and KIDScore™ D5, using EmbryoScope for embryo culture and assessment. The deep learning tool iDAScore® (Intelligent Data Analysis for Embryo Evaluation) has learned to rank embryos according to their likelihood of implantation. KIDScore™ D5 uses information on selected morphokinetic events annotated by the user in the software to calculate a score that reflects the implantation potential of embryos. In this prospective multi-centre randomized controlled study, the embryo decision tools are compared regarding clinical pregnancy rate as the primary endpoint. METHODS Patients undergoing IVF/ICSI treatments at two Swedish IVF clinics were asked to participate by signing informed consent. After IVF/ICSI, embryos were cultivated in EmbryoScope following each clinic´s practise. On day 5, an embryologist scored all normally fertilised embryos by visual assessment according to the Gardner and Schoolcraft criteria using the EmbryoScope software. If ≥ 2 good quality blastocysts (≥3BB) were available, the cycle was randomized to the control group and assessed with KIDScore™ D5 (n = 62) or the study group and assessed with iDAScore® (n = 64). The highest scored blastocyst was transferred and surplus good quality blastocysts were frozen. If a freeze-all strategy was decided, the first frozen embryo transfer was performed with the highest ranked embryo according to group allocation. RESULTS Elective single blastocyst transfer resulted in similar rates of positive hCG-results (KIDScore D5 64.5% vs iDAScore® 59.4%, p = 0.59), and the same clinical pregnancy rates, 50.0 % in both groups, p = 1. Clinical pregnancy was defined as ultrasound evidence of an intrauterine pregnancy with a fetal heartbeat at > 6 weeks of gestation. See Table 1 for details. CONCLUSIONS Embryos selected using the deep learning tool iDAScore® resulted in the same pregnancy rate and clinical pregnancy rate as KIDScore™ D5. Since iDAScore® is independent of user annotation and hence independent of the embryologist´s knowledge, expertise and workload, the embryo selection is less subjective. Using iDAScore® is timesaving and hence could benefit clinics with a high workload or with less experienced embryologists.

PURPOSE:The aim of this study was to investigate whether cells originating from human embryonic stem cells (hESCs) could be successfully transplanted onto a partially wounded human cornea. A second aim was to study the ability of the transplanted cells to differentiate into corneal epithelial-like cells.METHODS:Spontaneously, differentiated hESCs were transplanted onto a human corneal button (without limbus) with the epithelial layer partially removed. The cells were cultured on Bowman's membrane for up to 9 days, and the culture dynamics documented in a time-lapse system. As the transplanted cells originated from a genetically engineered hESC line, they all expressed green fluorescent protein, which facilitated their identification during the culture experiments, tissue preparation and analysis. To detect any differentiation into human corneal epithelial-like cells, we analysed the transplanted cells by immunohistochemistry using antibodies specific for CK3, CK15 and PAX6.RESULTS:The transplanted cells established and expanded on Bowman's membrane, forming a 1-4 cell layer surrounded by host corneal epithelial cells. Expression of the corneal marker PAX6 appeared 3 days after transplantation, and after 6 days, the cells were expressing both PAX6 and CK3.CONCLUSION:This shows that it is possible to transplant cells originating from hESCs onto Bowman's membrane with the epithelial layer partially removed and to get these cells to establish, grow and differentiate into corneal epithelial-like cells in vitro.

This report summarises our efforts in deriving, characterising and banking of 20 different human embryonic stem cell lines. We have derived a large number of human embryonic stem cell lines between 2001 and 2005. One of these cell lines was established under totally xeno-free culture conditions. In addition, several subclones have been established, including a karyoptypical normal clone from a trisomic mother line. A master cell banking system has been utilised in concert with an extensive characterisation programme, ensuring a supply of high quality pluripotent stem cells for further research and development. In this report we also present the first data on a proprietary novel antibody, hES-Cellect, that exhibits high specificity for undifferentiated hES cells. In addition to the traditional manual dissection approach of propagating hES cells, we here also report on the successful approaches of feeder-free cultures as well as single cell cultures based on enzymatic digestion. All culture systems used as reported here have maintained the hES cells in a karyotypical normal and pluripotent state. These systems also have the advantage of being the principal springboards for further scale up of cultures for industrial or clinical applications that would require vastly more cells that can be produced by mechanical means.

There have been recent reports of human embryonic stem cell (hESC) lines developing chromosomal aberrations after long-term culture, indicating an unstable genomic status due to the in vitro milieu. This raises concern, since it would limit their use in therapeutics. In this study the chromosomal status of five well-characterized hESC lines, SA002, SA002.5, AS034.1.1, SA121 and SA461, was monitored during long-term in vitro culture. The criteria of defined hESCs were met by all of the five hESC lines (four diploid and one trisomic for chromosome 13). The genomes were screened for chromosomal aberrations and rearrangements using comparative genomic hybridization (CGH), interphase fluorescence in situ hybridization (FISH) and traditional karyotyping on several occasions while in culture. The genomic integrity was shown to be maintained after repeated freeze-thaw procedures and continuous culture in vitro for up to 22 months (148 passages). We discuss the most common de novo chromosomal aberrations reported in hESCs, as well as their possible origin.

The use of human embryonic stem cells (hESCs) in most applications is dependent on their undifferentiated proliferation in vitro. Recent studies have illustrated the possibility that chromosomal changes may occur in hESCs during in vitro propagation of these cells. However, no studies so far have screened for chromosomal abnormalities in hESCs using high‐resolution techniques that can detect alterations on a few base‐pair levels. We have used the recently developed multiplex ligation‐dependent probe amplification procedure to analyze the possible occurrence of deletions or duplications in the subtelomeric regions of hESCs in early and late passages. In this study we show that no subtelomeric anomalies were detected in any of the nine hESC lines investigated, supporting the conclusion that hESCs, under appropriate conditions, maintain genomic stability during in vitro propagation.

Human embryonic stem cells (hESC) are isolated as clusters of cells from the inner cell mass of blastocysts and thus should formally be considered as heterogeneous cell populations. Homogenous hESC cultures can be obtained through subcloning. Here, we report the clonal derivation and characterization of two new hESC lines from the parental cell line SA002 and the previously clonally derived cell line AS034.1, respectively. The hESC line SA002 was recently reported to have an abnormal karyotype (trisomy 13), but within this population of cells we observed rare individual cells with an apparent normal karyotype. At a cloning efficiency of 5%, we established 33 subclones from SA002, out of which one had a diploid karyotype and this subline was designated SA002.5. From AS034.1 we established one reclone designated AS034.1.1 at a cloning efficiency of 0.1%. These two novel sublines express cell surface markers indicative of undifferentiated hESC (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), Oct-4, alkaline phosphatase, and they display high telomerase activity. In addition, the cells are pluripotent and form derivatives of all three embryonic germ layers in vitro as well as in vivo. These results, together with the clonal character of SA002.5 and AS034.1.1 make these homogenous cell populations very useful for hESC based applications in drug development and toxicity testing. In addition, the combination of the parental trisomic hESC line SA002 and the diploid subclone SA002.5 provides a unique experimental system to study the molecular mechanisms underlying the pathologies associated with trisomy 13.

The use of human embryonic stem cells (HESC) in research is increasing exponentially and HESC will certainly be of importance in biological, clinical and toxicological research for many years to come. Once established, HESC lines are expected to be chromosomally stable. However, our own experience of culturing HESC and some published reports indicate that HESC may show chromosomal instability while being cultured continuously in vitro. We conclude that the effects of different culture techniques and long‐term culture on the chromosome stability of HESC still remain to be elucidated and we recommend regular analysis of the chromosome constitution in cell lines using traditional karyotyping, CGH, FISH and PCR. We also recommend freezing of HESC at low passage number and in larger batches after thawing and expansion in order to secure material in case mutations occur in the cell line at a later stage of culture.

Establishment of human embryonic stem cells (hES) from surplus human IVF embryos has been successful when both fresh and frozen-thawed cleavage stage embryos have been cultured to the blastocyst stage. This study reports the characteristics of the starting material, the blastocysts, for hES cell lines that were first derived at the University of Gothenburg, Sahlgrenska University Hospital in 1999. Twenty-two hES cell lines were derived by Cellartis AB from 114 blastocysts, giving an overall success rate of 19.3%. The blastocysts from which the hES cell lines were established were of varying morphological quality, both fresh and frozen-thawed. Two techniques of hES establishment were applied, i.e. direct application of the blastocysts on feeder cells or the standard immunosurgery method. It was further found that the efficiency by which frozen-thawed embryos gave rise to new hES cell lines was 3.7 times better than with fresh surplus embryos. These findings suggest that frozen-thawed embryos are superior to fresh surplus human embryos in hES cell establishment, which also avoids specific ethical problems associated with embryo donation in a fresh IVF cycle.

BACKGROUND IVF laboratories performing embryo transfer at day 2 or 3 after fertilization are currently discarding pre-embryos considered suboptimal using morphological criteria. The objective of this study was to investigate whether blastocysts, cultured from such pre-embryos (surplus), were chromosomally and morphologically normal. As a control group we used morphologically good quality embryos (GQE), cultured to the blastocyst stage. METHODS Human pre-embryos considered suboptimal were cultured to the blastocyst stage. As a control group, frozen-thawed pre-embryos of good quality were cultured under identical conditions. The chromosomal status of the blastocysts obtained was studied by multi-colour fluorescence in-situ hybridization for chromosomes 13, 16, 18, 21, 22, X and Y. RESULTS There is, on average, a significantly higher degree of chromosomal aberrations in blastocysts derived from surplus pre-embryos compared to blastocysts derived from GQE, and the chromosomal aberrations are generally found in a higher number of blastomeres per blastocyst. In addition, blastocysts from surplus pre-embryos had significantly poorer morphology compared to GQE. Improvement in morphology and/or developmental rate in surplus pre-embryos between day 2 and day 3 did not predict a morphologically/chromosomally normal blastocyst. However, this study shows that close to half of the surplus pre-embryos that reach the blastocyst stage can be considered chromosomally normal when assessed for these seven chromosomes. Furthermore, we found that chromosomal aberrations were more concentrated in a particular cell population within blastocysts derived from GQE, compared with surplus blastocysts. CONCLUSIONS The study suggests that even if the IVF laboratory is on average making the correct decision about the potential of a pre-embryo, surplus pre-embryos that might become chromosomally normal blastocysts are still being discarded.