Using the Eeva Test™ adjunctively to traditional day 3 morphology is informative for consistent embryo assessment within a panel of embryologists with diverse experience.

PURPOSE: Since many transferred, good morphology embryos fail to implant, technologies to identify embryos with high developmental potential would be beneficial. The Eeva™ (Early Embryo Viability Assessment) Test, a prognostic test based on automated detection and analysis of time-lapse imaging information, has been shown to benefit embryo selection specificity for a panel of three highly experienced embryologists (Conaghan et al., 2013). Here we examined if adjunctive use of Eeva Test results following morphological assessment would allow embryologists with diverse clinical backgrounds to consistently improve the selection of embryos with high developmental potential. METHODS: Prospective, double-blinded multi-center study with 54 patients undergoing blastocyst transfer cycles consented to have embryos imaged using the Eeva System, which automatically measures key cell division timings and categorizes embryos into groups based on developmental potential. Five embryologists of diverse clinical practices, laboratory training, and geographical areas predicted blastocyst formation using day 3 morphology alone and day 3 morphology followed by Eeva Test results. Odds ratio (OR) and diagnostic performance measures were calculated by comparing prediction results to true blastocyst outcomes. RESULTS: When Eeva Test results were used adjunctively to traditional morphology to help predict blastocyst formation among embryos graded good or fair on day 3, the OR was 2.57 (95 % CI=1.88-3.51). The OR using morphology alone was 1.68 (95 % CI=1.29-2.19). Adjunct use of the Eeva Test reduced the variability in prediction performance across all five embryologists: the variability was reduced from a range of 1.06 (OR=1.14 to 2.20) to a range of 0.45 (OR=2.33 to 2.78). CONCLUSIONS: The Eeva Test, an automated, time-lapse enabled prognostic test, used adjunctively with morphology, is informative in helping embryologists with various levels of experience select embryos with high developmental potential.

Computer-automated time-lapse analysis results correlate with embryo implantation and clinical pregnancy: a blinded, multi-centre study.

Computer-automated time-lapse analysis has been shown to improve embryo selection by providing quantitative and objective information to supplement traditional morphology. In this multi-centre study, the relationship between such computer-derived outputs (High, Medium, Low scores), embryo implantation and clinical pregnancy were examined. Data were collected from six clinics, including 205 patients whose embryos were imaged by the Eeva(TM) System. The Eeva scores were blinded and not considered during embryo selection. Embryos with High and Medium scores had significantly higher implantation rates than those with Low scores (37% and 35% versus 15%; P < 0.0001; P = 0.0004). Similar trends in implantation rates were observed in different IVF centres each using their own protocols. Further analysis revealed that patients with at least one High embryo transferred had significantly higher clinical pregnancy rates than those with only Low embryos transferred (51% versus 34%; P = 0.02), although patients' clinical characteristics across groups were comparable. These data, together with previous research and clinical studies, confirm that computer-automated Eeva scores provide valuable information, which may improve the clinical outcome of IVF procedures and ultimately facilitate the trend of single embryo selection.

Atypical embryo phenotypes identified by time-lapse microscopy: high prevalence and association with embryo development.

OBJECTIVE: To characterize atypical dynamic embryo phenotypes identified by time-lapse microscopy, evaluate their prevalence, and determine their association with embryo development. DESIGN: Retrospective multicenter cohort study. SETTING: Five IVF clinics in the United States. PATIENT(S): Sixty-seven women undergoing IVF treatment with 651 embryos. INTERVENTION(S): Embryo videos were retrospectively analyzed for atypical phenotypes. MAIN OUTCOME MEASURE(S): Identification of four groups of atypical embryo phenotypes: abnormal syngamy (AS), abnormal first cytokinesis (A1(cyt)), abnormal cleavage (AC), and chaotic cleavage (CC). Prevalence and association with embryo morphology and development potential were evaluated. RESULT(S): A high prevalence of atypical phenotypes was observed among embryos: AS 25.1% (163/649), A1(cyt) 31.0% (195/639), AC 18% (115/639) and CC 15% (96/639). A high percentage of embryos with atypical phenotype(s) had good quality on day 3 (overall grade good or fair): AS 78.6% (70/89); A1(cyt) 79.7% (94/119), AC 86.4% (70/81), and CC 35.2% (19/54), but the blastocyst formation rates for these embryos were significantly lower compared with their respective control groups: AS 21.5% vs. 44.9%, A1(cyt) 21.7% vs. 44.6%, AC 11.7% vs. 43.1%, and CC 14.0% vs. 42.3%. CONCLUSION(S): Embryos exhibiting atypical phenotypes are highly prevalent in human embryos and show significantly lower developmental potential than control embryos. CLINICAL TRIAL REGISTRATION NUMBER: NCT01369446.

Improving embryo selection using a computer-automated time-lapse image analysis test plus day 3 morphology: results from a prospective multicenter trial.

OBJECTIVE: To assess the first computer-automated platform for time-lapse image analysis and blastocyst prediction and to determine how the screening information may assist embryologists in day 3 (D3) embryo selection. DESIGN: Prospective, multicenter, cohort study. SETTING: Five IVF clinics in the United States. PATIENT(S): One hundred sixty women ≥ 18 years of age undergoing fresh IVF treatment with basal antral follicle count ≥ 8, basal FSH <10 IU/mL, and ≥ 8 normally fertilized oocytes. INTERVENTION(S): A noninvasive test combining time-lapse image analysis with the cell-tracking software, Eeva (Early Embryo Viability Assessment), was used to measure early embryo development and generate usable blastocyst predictions by D3. MAIN OUTCOME MEASURE(S): Improvement in the ability of experienced embryologists to select which embryos are likely to develop to usable blastocysts using D3 morphology alone, compared with morphology plus Eeva. RESULT(S): Experienced embryologists using Eeva in combination with D3 morphology significantly improved their ability to identify embryos that would reach the usable blastocyst stage (specificity for each of three embryologists using morphology vs. morphology plus Eeva: 59.7% vs. 86.3%, 41.9% vs. 84.0%, 79.5% vs. 86.6%). Adjunctive use of morphology plus Eeva improved embryo selection by enabling embryologists to better discriminate which embryos would be unlikely to develop to blastocyst and was particularly beneficial for improving selection among good-morphology embryos. Adjunctive use of morphology plus Eeva also reduced interindividual variability in embryo selection. CONCLUSION(S): Previous studies have shown improved implantation rates for blastocyst transfer compared with cleavage-stage transfer. Addition of Eeva to the current embryo grading process may improve the success rates of cleavage-stage ETs.

Biomarkers identified with time-lapse imaging: discovery, validation, and practical application.

"Time-lapse markers," which are defined by time-lapse imaging and correlated with clinical outcomes, may provide embryologists with new opportunities for improving embryo selection. This article provides an overview of noninvasive biomarkers defined by time-lapse imaging studies. In addition to comprehensively reviewing the discovery of each time-lapse marker, it focuses on the criteria necessary for their successful integration into clinical practice, including [1] statistical and biological significance, [2] validation through prospective clinical studies, and [3] development of reliable technology to measure and quantify the time-lapse marker. Because manual analysis of time-lapse images is labor intensive and limits the practical use of the image data in the clinic, automated image analysis software platforms may contribute substantially to improvements in embryo selection accuracy. Ultimately, time-lapse markers that are based on a foundation of basic research, validated through prospective clinical studies, and enabled by a reliable quantification technology may improve IVF success rates, encourage broader adoption of single-embryo transfer, and reduce the risks associated with multiple gestation pregnancies.

Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues.

In the absence of perfusable vascular networks, three-dimensional (3D) engineered tissues densely populated with cells quickly develop a necrotic core. Yet the lack of a general approach to rapidly construct such networks remains a major challenge for 3D tissue culture. Here, we printed rigid 3D filament networks of carbohydrate glass, and used them as a cytocompatible sacrificial template in engineered tissues containing living cells to generate cylindrical networks that could be lined with endothelial cells and perfused with blood under high-pressure pulsatile flow. Because this simple vascular casting approach allows independent control of network geometry, endothelialization and extravascular tissue, it is compatible with a wide variety of cell types, synthetic and natural extracellular matrices, and crosslinking strategies. We also demonstrated that the perfused vascular channels sustained the metabolic function of primary rat hepatocytes in engineered tissue constructs that otherwise exhibited suppressed function in their core.

Humanized mice with ectopic artificial liver tissues.

"Humanized" mice offer a window into aspects of human physiology that are otherwise inaccessible. The best available methods for liver humanization rely on cell transplantation into immunodeficient mice with liver injury but these methods have not gained widespread use due to the duration and variability of hepatocyte repopulation. In light of the significant progress that has been achieved in clinical cell transplantation through tissue engineering, we sought to develop a humanized mouse model based on the facile and ectopic implantation of a tissue-engineered human liver. These human ectopic artificial livers (HEALs) stabilize the function of cryopreserved primary human hepatocytes through juxtacrine and paracrine signals in polymeric scaffolds. In contrast to current methods, HEALs can be efficiently established in immunocompetent mice with normal liver function. Mice transplanted with HEALs exhibit humanized liver functions persistent for weeks, including synthesis of human proteins, human drug metabolism, drug-drug interaction, and drug-induced liver injury. Here, mice with HEALs are used to predict the disproportionate metabolism and toxicity of "major" human metabolites using multiple routes of administration and monitoring. These advances may enable manufacturing of reproducible in vivo models for diverse drug development and research applications.

Multiplexed, high-throughput analysis of 3D microtissue suspensions.

Three-dimensional (3D) tissue models have significantly improved our understanding of structure/function relationships and promise to lead to new advances in regenerative medicine. However, despite the expanding diversity of 3D tissue fabrication methods, approaches for functional assessment have been relatively limited. Here, we describe the fabrication of microtissue (µ-tissue) suspensions and their quantitative evaluation with techniques capable of analyzing large sample numbers and performing multiplexed parallel analysis. We applied this platform to 3D µ-tissues representing multiple stages of liver development and disease including: embryonic stem cells, bipotential hepatic progenitors, mature hepatocytes, and hepatoma cells photoencapsulated in polyethylene glycol hydrogels. Multiparametric µ-tissue cytometry enabled quantitation of fluorescent reporter expression within populations of intact µ-tissues (n≥ 10²-10³) and sorting-based enrichment of subsets for subsequent studies. Further, 3D µ-tissues could be implanted in vivo, respond to systemic stimuli, retrieved and quantitatively assessed. In order to facilitate multiplexed 'pooled' experimentation, fluorescent labeling strategies were developed and utilized to investigate the impact of µ-tissue composition and exposure to soluble factors. In particular, examination of drug/gene interactions on collections of 3D hepatoma µ-tissues indicated synergistic influence of doxorubicin and siRNA knockdown of the anti-apoptotic gene BCL-XL. Collectively, these studies highlight the broad utility of µ-tissue suspensions as an enabling approach for high n, populational analysis of 3D tissue biology in vitro and in vivo.

Modulation of hepatocyte phenotype in vitro via chemomechanical tuning of polyelectrolyte multilayers.

It is increasingly appreciated that since cell and tissue functions are regulated by chemomechanical stimuli, precise control over such stimuli will improve the functionality of tissue models. However, due to the inherent difficulty in decoupling these cues as presented by extracellular materials, few studies have explored the independent modulation of biochemical and mechanical stimuli towards the manipulation of sustained cellular processes. Here, we demonstrate that both mechanical compliance and ligand presentation of synthetic, weak polyelectrolyte multilayers (PEMs) can be tuned independently to influence the adhesion and liver-specific functions of primary rat hepatocytes over extended in vitro culture (two weeks). These synthetic PEMs exhibited elastic moduli E ranging over 200kPa

T-cadherin modulates hepatocyte functions in vitro.

Primary hepatocytes from several different species rapidly lose viability and phenotypic functions on isolation from their native microenvironment of the liver. Stromal cells derived from both within and outside the liver can induce phenotypic functions in primary hepatocytes in vitro; however, the molecular mediators underlying this "coculture effect" have not been fully elucidated. We have previously developed a functional genomic screen utilizing cocultures of hepatocytes and 3T3 fibroblasts to identify such candidate hepatocyte-function-inducing molecules. In particular, truncated-cadherin (T-cadherin) was identified as a potential molecule of interest in induction of hepatic functions. Here we demonstrate that liver-specific functions of primary rat hepatocytes are induced on cocultivation with Chinese hamster ovary cells engineered to express T-cadherin on their surface as compared with wild-type controls. Additionally, culture of cells on substrata presenting recombinant T-cadherin protein (acellular presentation) enhanced hepatic functions in both pure hepatocyte cultures and in hepatocyte-stromal cocultures lacking endogenous T-cadherin expression. Collectively, these data indicate that both cellular and acellular presentation of T-cadherin can be used to modulate the hepatocyte phenotype in vitro for tissue engineering applications. Our work suggests potential avenues for investigating the role of T-cadherin on hepatocellular function in vivo in settings such as embryogenesis and liver pathology.

Targeted quantum dot conjugates for siRNA delivery.

Treatment of human diseases such as cancer generally involves the sequential use of diagnostic tools and therapeutic modalities. Multifunctional platforms combining therapeutic and diagnostic imaging functions in a single vehicle promise to change this paradigm. in particular, nanoparticle-based multifunctional platforms offer the potential to improve the pharmacokinetics of drug formulations, while providing attachment sites for diagnostic imaging and disease targeting features. We have applied these principles to the delivery of small interfering RNA (siRNA) therapeutics, where systemic delivery is hampered by rapid excretion and nontargeted tissue distribution. Using a PEGlyated quantum dot (QD) core as a scaffold, siRNA and tumor-homing peptides (F3) were conjugated to functional groups on the particle's surface. We found that the homing peptide was required for targeted internalization by tumor cells, and that siRNA cargo could be coattached without affecting the function of the peptide. Using an EGFP model system, the role of conjugation chemistry was investigated, with siRNA attached to the particle by disulfide cross-linkers showing greater silencing efficiency than when attached by a nonreducible thioether linkage. Since each particle contains a limited number of attachment sites, we further explored the tradeoff between number of F3 peptides and the number of siRNA per particle, leading to an optimized formulation. Delivery of these F3/siRNA-QDs to EGFP-transfected HeLa cells and release from their endosomal entrapment led to significant knockdown of EGFP signal. By designing the siRNA sequence against a therapeutic target (e.g., oncogene) instead of EGFP, this technology may be ultimately adapted to simultaneously treat and image metastatic cancer.

Fabrication of 3D hepatic tissues by additive photopatterning of cellular hydrogels.

We have fabricated a hepatic tissue construct using a multilayer photopatterning platform for embedding cells in hydrogels of complex architecture. We first explored the potential of established hepatocyte culture models to stabilize isolated hepatocytes for photoencapsulation (e.g., double gel, Matrigel, cocultivation with nonparenchymal cells). Using photopolymerizable PEG hydrogels, we then tailored both the chemistry and architecture of the hydrogels to further support hepatocyte survival and liver-specific function. Specifically, we incorporated adhesive peptides to ligate key integrins on these adhesion-dependent cells. To identify the appropriate peptides for incorporation, the integrin expression of cultured hepatocytes was monitored by flow cytometry and their functional role in cell adhesion was assessed on full-length extracellular matrix (ECM) molecules and their adhesive peptide domains. In addition, we modified the hydrogel architecture to minimize barriers to nutrient transport for these highly metabolic cells. Viability of encapsulated cells was improved in photopatterned hydrogels with structural features of 500 microm in width over unpatterned, bulk hydrogels. Based on these findings, we fabricated a multilayer photopatterned PEG hydrogel structure containing the adhesive RGD peptide sequence to ligate the alpha5beta1 integrin of cocultured hepatocytes. Three-dimensional photopatterned constructs were visualized by digital volumetric imaging and cultured in a continuous flow bioreactor for 12 d where they performed favorably in comparison to unpatterned, unperfused constructs. These studies will have impact in the field of liver biology as well as provide enabling tools for tissue engineering of other organs.

Assessment of hepatocellular function within PEG hydrogels.

Tissue-engineered therapies for liver failure offer the potential to augment or replace whole organ transplantation; however, fabrication of hepatic tissue poses unique challenges largely stemming from the complexity of liver structure and function. In this study, we illustrate the utility of highly-tunable, photopolymerizable poly(ethylene glycol) (PEG) hydrogels for 3D encapsulation of hepatic cells and highlight a range of techniques important for examining hepatocellular function in this platform. Owing to our long-term interest in incorporating proliferative progenitor cell types (e.g. hepatoblasts, oval cells, or cells derived from embryonic stem cells) and maintaining the phenotype of differentiated cells, we explored the behavior of bipotential mouse embryonic liver (BMEL) cells as a model progenitor cell and mature, fully differentiated, primary hepatocytes in this biomaterial system. We demonstrated the importance of cell-cell and cell-matrix interactions in the survival and function of these cell types, and the capacity to influence encapsulated cell phenotypes through modulation of hydrogel characteristics or gene silencing. Additionally, we demonstrated imaging techniques critical for the in situ assessment of encapsulated hepatocyte function combined with the ability to control cellular organization and overall architecture through microscale patterning technologies. Further analysis of liver progenitor as well as mature hepatocyte processes within the versatile PEG hydrogel platform will aid in the development of tissue engineered implantable liver systems.

Quantum dots to monitor RNAi delivery and improve gene silencing.

A critical issue in using RNA interference for identifying genotype/phenotype correlations is the uniformity of gene silencing within a cell population. Variations in transfection efficiency, delivery-induced cytotoxicity and 'off target' effects at high siRNA concentrations can confound the interpretation of functional studies. To address this problem, we have developed a novel method of monitoring siRNA delivery that combines unmodified siRNA with seminconductor quantum dots (QDs) as multi color biological probes. We co-transfected siRNA with QDs using standard transfection techniques, thereby leveraging the photostable fluorescent nanoparticles to track delivery of nucleic acid, sort cells by degree of transfection and purify homogenously-silenced subpopulations. Compared to alternative RNAi tracking methods (co-delivery of reporter plasmids and end-labeling the siRNA), QDs exhibit superior photostability and tunable optical properties for an extensive selection of non-overlapping colors. Thus this simple, modular system can be extended toward multiplexed gene knockdown studies, as demonstrated in a two color proof-of-principle study with two biological targets. When the method was applied to investigate the functional role of T-cadherin (T-cad) in cell-cell communication, a subpopulation of highly silenced cells obtained by QD labeling was required to observe significant downstream effects of gene knockdown.