Non-invasive molecular assessment of human embryo development and implantation potential.

In vitro fertilization (IVF) is the most common assisted reproductive technology used to treat infertility. Embryo selection for transfer in IVF cycles relies on the morphological evaluation by embryologists, either by conventional microscopic assessment or more recently by time-lapse imaging systems. Despite the introduction of time-lapse imaging improvements in IVF success rates have failed to materialize, therefore alternative approaches are needed. Recent studies have shown that embryos resulting in successful pregnancy differ in their secretome and metabolism compared to embryos that fail to implant, suggesting that molecular analysis of embryo culture medium could assist in non-invasive single embryo selection. However, this approach has yet to be adopted clinically due to the lack of appropriate highly sensitive screening technologies needed to assess volume-limited samples. Here we report the detection of hCGß, IL-8 and TNFα from conditioned culture media of single human embryos using electrochemical impedance spectroscopy. The impedimetric immunosensors revealed that morphologically non-viable embryos produce higher levels of IL-8 and TNFα, associated with abnormal cell division and cell death, respectively. More importantly, hCGß detection was able to discriminate apparently morphologically identical viable embryos. This work brings an objective dimension to embryo selection, which could overcome the major limitations of morphology-based embryo selection for implantation. Future work should include the validation of these biomarkers in a large patient cohort.

Combined noninvasive metabolic and spindle imaging as potential tools for embryo and oocyte assessment.

STUDY QUESTION: Is the combined use of fluorescence lifetime imaging microscopy (FLIM)-based metabolic imaging and second harmonic generation (SHG) spindle imaging a feasible and safe approach for noninvasive embryo assessment? SUMMARY ANSWER: Metabolic imaging can sensitively detect meaningful metabolic changes in embryos, SHG produces high-quality images of spindles and the methods do not significantly impair embryo viability. WHAT IS KNOWN ALREADY: Proper metabolism is essential for embryo viability. Metabolic imaging is a well-tested method for measuring metabolism of cells and tissues, but it is unclear if it is sensitive enough and safe enough for use in embryo assessment. STUDY DESIGN, SIZE, DURATION: This study consisted of time-course experiments and control versus treatment experiments. We monitored the metabolism of 25 mouse oocytes with a noninvasive metabolic imaging system while exposing them to oxamate (cytoplasmic lactate dehydrogenase inhibitor) and rotenone (mitochondrial oxidative phosphorylation inhibitor) in series. Mouse embryos (n = 39) were measured every 2 h from the one-cell stage to blastocyst in order to characterize metabolic changes occurring during pre-implantation development. To assess the safety of FLIM illumination, n = 144 illuminated embryos were implanted into n = 12 mice, and n = 108 nonilluminated embryos were implanted into n = 9 mice. PARTICIPANTS/MATERIALS, SETTING, METHODS: Experiments were performed in mouse embryos and oocytes. Samples were monitored with noninvasive, FLIM-based metabolic imaging of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence. Between NADH cytoplasm, NADH mitochondria and FAD mitochondria, a single metabolic measurement produces up to 12 quantitative parameters for characterizing the metabolic state of an embryo. For safety experiments, live birth rates and pup weights (mean ± SEM) were used as endpoints. For all test conditions, the level of significance was set at P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: Measured FLIM parameters were highly sensitive to metabolic changes due to both metabolic perturbations and embryo development. For oocytes, metabolic parameter values were compared before and after exposure to oxamate and rotenone. The metabolic measurements provided a basis for complete separation of the data sets. For embryos, metabolic parameter values were compared between the first division and morula stages, morula and blastocyst and first division and blastocyst. The metabolic measurements again completely separated the data sets. Exposure of embryos to excessive illumination dosages (24 measurements) had no significant effect on live birth rate (5.1 ± 0.94 pups/mouse for illuminated group; 5.7 ± 1.74 pups/mouse for control group) or pup weights (1.88 ± 0.10 g for illuminated group; 1.89 ± 0.11 g for control group). LIMITATIONS, REASONS FOR CAUTION: The study was performed using a mouse model, so conclusions concerning sensitivity and safety may not generalize to human embryos. A limitation of the live birth data is also that although cages were routinely monitored, we could not preclude that some runt pups may have been eaten. WIDER IMPLICATIONS OF THE FINDINGS: Promising proof-of-concept results demonstrate that FLIM with SHG provide detailed biological information that may be valuable for the assessment of embryo and oocyte quality. Live birth experiments support the method's safety, arguing for further studies of the clinical utility of these techniques. STUDY FUNDING/COMPETING INTEREST(S): Supported by the Blavatnik Biomedical Accelerator Grant at Harvard University and by the Harvard Catalyst/The Harvard Clinical and Translational Science Center (National Institutes of Health Award UL1 TR001102), by NSF grants DMR-0820484 and PFI-TT-1827309 and by NIH grant R01HD092550-01. T.S. was supported by a National Science Foundation Postdoctoral Research Fellowship in Biology grant (1308878). S.F. and S.A. were supported by NSF MRSEC DMR-1420382. Becker and Hickl GmbH sponsored the research with the loaning of equipment for FLIM. T.S. and D.N. are cofounders and shareholders of LuminOva, Inc., and co-hold patents (US20150346100A1 and US20170039415A1) for metabolic imaging methods. D.S. is on the scientific advisory board for Cooper Surgical and has stock options with LuminOva, Inc.

Preimplantation genetic testing for a family with usher syndrome through targeted sequencing and haplotype analysis.

BACKGROUND: Preimplantation genetic testing for monogenic defects (PGT-M) has been available in clinical practice. This study aimed to validate the applicability of targeted capture sequencing in developing personalized PGT-M assay. METHODS: One couple at risk of transmitting Usher Syndrome to their offspring was recruited to this study. Customized capture probe targeted at USH2A gene and 350 kb flanking region were designed for PGT-M. Eleven blastocysts were biopsied and amplified by using multiple displacement amplification (MDA) and capture sequencing. A hidden Markov model (HMM) assisted haplotype analysis was performed to deduce embryo's genotype by using single nucleotide polymorphisms (SNPs) identified in each sample. The embryo without paternal rare variant was implanted and validated by conventional prenatal or postnatal diagnostic means. RESULTS: Four embryos were diagnosed as free of father's rare variant, two were transferred and one achieved a successful pregnancy. The fetal genotype was confirmed by Sanger sequencing of fetal genomic DNA obtained by amniocentesis. The PGT-M and prenatal diagnosis results were further confirmed by the molecular diagnosis of the baby's genomic DNA sample. The auditory test showed that the hearing was normal. CONCLUSIONS: Targeted capture sequencing is an effective and convenient strategy to develop customized PGT-M assay.

Multi­organ assessment via a 9.4­Tesla MRS evaluation of metabolites during the embryonic development of cleft palate induced by dexamethasone.

The aim of the present study was to determine the association between maternal metabolism and development of the fetal palate, and to suggest a potential non­invasive prenatal diagnostic method for fetal cleft palate (CP). Dexamethasone (DXM) was used to create a CP mouse model. A 9.4­Tesla (T) magnetic resonance spectroscopy (MRS) imager was used to measure an array of metabolites in the maternal serum, placental tissue, amniotic fluid and fetal palates. Multivariate statistical analysis was performed using SIMCA­P 14.1 software. Following DXM treatment, variations were detected in multiple metabolites in the female mice and their fetuses based on 9.4T MRS. It was indicated that in the experimental group during CP formation, leucine, valine, creatine, acetate and citrate levels in the palatal tissue were lower, whereas lactate, alanine, proline/inositol and glutamate­containing metabolite levels were higher, compared with the levels in the control group. In placental tissue and amniotic fluid, succinate and choline levels were lower in the experimental group. The relative concentrations of cholesterol and lipids in palatal tissues from mice treated with DXM were higher compared with the concentrations in tissues from mice in the control group, with the exception of (CH2)n lipids. In the placental tissue, the alteration in cholesterol level exhibited the opposite trend. Lipid levels for the different lipid forms varied and most of them were unsaturated lipids.

Expression of miR-Let-7a, miR-15a, miR-16-1, and their target genes in fresh and vitrified embryos and its surrounding culture media for noninvasive embryo assessment.

microRNAs (miRNAs) play a critical role in implantation and development of mouse embryos. In this study, we aim to evaluate the possibility of miRNAs as potential biomarkers in the blastocyst culture to assess embryo quality. We also intend to investigate whether improved clinical outcomes of vitrified embryos agree with altered miRNA expressions. Mouse embryos from in vitro fertilization were vitrified at the two-cell stage. After thawing, the embryos were individually cultured and developed to the blastocyst stage. We used quantitative real-time polymerase chain reaction to evaluate miRNA expression levels in both vitrified and fresh groups, and culture medium (CM). The fibronectin binding assay was performed to examine for blastocyst attachment. The findings showed reduced expressions of miR-16-1 (0.2 ± 0.06) and miR-Let-7a (0.65 ± 0.1) after vitrification compared to fresh embryos. We observed significant upregulation of the target genes Vav3 (4.33 ± 0.25), integrin ß-3 (Itg ß3; 4.73 ± 0.2), and Bcl2 (2.29 ± 0.16) in the vitrified embryos compared to the fresh groups. Evaluation of blastocyst CM showed upregulation of miR-Let-7a (15.68 ± 0.89), miR-16-1 (16.18 ± 0.75), and miR-15a (13.36 ± 0.73) in the vitrified group in comparison to the fresh blastocysts (P < .05). The expression levels of miR-16-1 (3.28 ± 0.63), miR-15a (5.91 ± 0.38), and miR-Let-7a (9.07 ± 0.6) in CM of the vitrified blastocysts conducted on fibronectin were significantly higher than the fresh group (P < .05).This study showed that vitrification of embryos changes implantation and proliferation biomarkers. In addition, upregulated miRNAs in CM could be potentially used for noninvasive early assessment of embryo quality.

QUEST MRI assessment of fetal brain oxidative stress in utero.

PURPOSE: To achieve sufficient precision of R1 (=1/T1) maps of the fetal brain in utero to perform QUEnch-assiSTed (QUEST) MRI in which a significant anti-oxidant-induced reduction in R1 indicates oxidative stress. METHODS: C57BL/6 mouse fetuses in utero were gently and non-surgically isolated and secured using a homemade 3D printed clip. Using a commercial receive-only surface coil, brain maps of R1, an index sensitive to excessive and continuous free radical production, were collected using either a conventional Cartesian or a non-Cartesian (periodically rotated overlapping parallel lines with enhanced reconstruction) progressive saturation sequence. Data were normalized to the shortest TR time to remove bias. To assess oxidative stress, brain R1 maps were acquired on the lipopolysaccharide (LPS) model of preterm birth ±â€¯rosiglitazone (ROSI, which has anti-oxidant properties); phosphate buffered saline (PBS) controls ±â€¯ROSI were similarly studied. RESULTS: Sufficient quality R1 maps were generated by a combination of the 3D printed clip, surface coil detection, non-Cartesian sequence, and normalization scheme ensuring minimal fetal movement, good detection sensitivity, reduced motion artifacts, and minimal baseline variations, respectively. In the LPS group, the combined caudate-putamen and thalamus region R1 was reduced (p < 0.05) with ROSI treatment consistent with brain oxidative stress; no evidence for oxidative stress was found in the pons region. In the PBS control group, brain R1's did not change with ROSI treatment. CONCLUSION: The sensitivity and reproducibility of the combined approaches described herein enabled first-time demonstration of regional oxidative stress measurements of the fetal brain in utero using QUEST MRI.

Assessment of Histiotrophic Nutrition Using Fluorescent Probes.

Histiotrophic nutrition is a process whereby the rodent visceral yolk sac (VYS) internalizes exogenous macromolecules, degrades them, and sends the degradation products to the embryo. Quantification and visualization of histiotrophic nutrition can be accomplished using fluorescent tracer molecules such as fluorescein isothiocyanate-conjugated albumin (FITC-albumin). The methods are simple and can provide complimentary functional and structural information in studies of the effects of embryotoxicants on visceral yolk sac function.

Thyroid Hormone Economy in the Perinatal Mouse Brain: Implications for Cerebral Cortex Development.

Thyroid hormones (THs, T4 and the transcriptionally active hormone T3) play an essential role in neurodevelopment; however, the mechanisms underlying T3 brain delivery during mice fetal development are not well known. This work has explored the sources of brain T3 during mice fetal development using biochemical, anatomical, and molecular approaches. The findings revealed that during late gestation, a large amount of fetal brain T4 is of maternal origin. Also, in the developing mouse brain, fetal T3 content is regulated through the conversion of T4 into T3 by type-2 deiodinase (D2) activity, which is present from earlier prenatal stages. Additionally, D2 activity was found to be essential to mediate expression of T3-dependent genes in the cerebral cortex, and also necessary to generate the transient cerebral cortex hyperthyroidism present in mice lacking the TH transporter Monocarboxylate transporter 8. Notably, the gene encoding for D2 (Dio2) was mainly expressed at the blood-cerebrospinal fluid barrier (BCSFB). Overall, these data signify that T4 deiodinated by D2 may be the only source of T3 during neocortical development. We therefore propose that D2 activity at the BCSFB converts the T4 transported across the choroid plexus into T3, thus supplying the brain with active hormone to maintain TH homeostasis.

Morphologic and transcriptomic assessment of bovine embryos exposed to dietary long-chain fatty acids.

The main objectives of this study were to determine the influence of diets enriched in α-linolenic, linoleic or oleic acid on the development and transcriptomic profile of embryos collected from dairy cattle. Non-lactating Holstein cows received one of the three diets supplemented with 8% rolled oilseeds: flax (FLX, n = 8), sunflower (SUN, n = 7) or canola (CAN, n = 8). After a minimum 35-day diet adaptation, cows were superovulated, artificially inseminated and ova/embryos recovered non-surgically after 7.5 days. Cows fed FLX had less degenerated embryos and more viable embryos than those fed CAN or SUN. In total, 175 genes were differentially expressed in blastocysts from cows fed FLX than in cows fed CAN or SUN. These differentially expressed genes were mainly involved in cellular growth and proliferation, cellular development, and cell survival and viability. In conclusion, dietary n-3 polyunsaturated fatty acids reduced early embryonic degeneration possibly through improving embryonic cell survival and viability.

Global assessment of imprinted gene expression in the bovine conceptus by next generation sequencing.

Genomic imprinting is an epigenetic mechanism that leads to parental-allele-specific gene expression. Approximately 150 imprinted genes have been identified in humans and mice but less than 30 have been described as imprinted in cattle. For the purpose of de novo identification of imprinted genes in bovine, we determined global monoallelic gene expression in brain, skeletal muscle, liver, kidney and placenta of day ∼105 Bos taurus indicus × Bos taurus taurus F1 conceptuses using RNA sequencing. To accomplish this, we developed a bioinformatics pipeline to identify parent-specific single nucleotide polymorphism alleles after filtering adenosine to inosine (A-to-I) RNA editing sites. We identified 53 genes subject to monoallelic expression. Twenty three are genes known to be imprinted in the cow and an additional 7 have previously been characterized as imprinted in human and/or mouse that have not been reported as imprinted in cattle. Of the remaining 23 genes, we found that 10 are uncharacterized or unannotated transcripts located in known imprinted clusters, whereas the other 13 genes are distributed throughout the bovine genome and are not close to any known imprinted clusters. To exclude potential cis-eQTL effects on allele expression, we corroborated the parental specificity of monoallelic expression in day 86 Bos taurus taurus × Bos taurus taurus conceptuses and identified 8 novel bovine imprinted genes. Further, we identified 671 candidate A-to-I RNA editing sites and describe random X-inactivation in day 15 bovine extraembryonic membranes. Our results expand the imprinted gene list in bovine and demonstrate that monoallelic gene expression can be the result of cis-eQTL effects.

The ART of selecting the best embryo: A review of early embryonic mortality and bovine embryo viability assessment methods.

Animal reproductive biotechnology is continually evolving. Significant advances have been made in our understanding of early embryonic mortality and embryo development in domestic animals, which has improved the selection and success of in vitro technologies. Yet our knowledge is still relatively limited such that identifying a single embryo with the highest chance of survival and development for transfer remains challenging. While invasive methods such as embryo biopsy can provide useful information regarding the genetic status of the embryos, morphological assessment remains the most common evaluation. A recent shift, however, favors alternative, adjunct approaches for non-invasive assessment of an embryo's viability and developmental potential. Various analytical techniques have facilitated the evaluation of cellular health through the metabolome, the assessment of end products of cellular metabolism, or by analyzing spent media for small RNAs. This review discusses the application of noninvasive approaches for ascertaining the health and viability of in vitro-produced bovine embryos. A comparative analysis of noninvasive techniques for embryo assessment currently being investigated in cattle and humans is also discussed.

Evaluación de la calidad de vida de pacientes bipolares chilenos: propiedades psicométricas y utilidad diagnóstica de la versión chilena del cuestionario Quality of Life Bipolar Disorder (QoL.BD-CL) / Assessment of a version adapted and translated into Spanish of the Quality of Life Bipolar Disorder Questionnaire

Background: The Quality of life Bipolar Disorder (QoL.BD) Questionnaire specifically measures quality of life in patients with bipolar disorder. Aim: To adapt a version translated into Spanish of the questionnaire and assess its validity in Chilean patients. Material and Methods: The QoL. BD was adapted to the Chilean population through the back-translation method and then administered to 32 adult patients with a bipolar disorder and 31 subjects without the disease, both groups with similar socioeconomic status. To confirm the diagnosis, the International Neuropsychiatric Interview (MINI), Young (YMRS) and Hamilton (HAM-D) scales were applied. Quality of life was assessed using the SF-36v.2 survey. We determined internal consistency, reliability, convergent validity, the cut-off point, and the sensibility and specificity of the scale. Results: The Chilean version of the Questionnaire [QoL. BD-CL] had a high reliability (α = 0.95) and a high validity in reference to external criteria (correlation coefficients with SF-36 ranging from 0.453 and 0.819; p < 0.01). A cut-off point of 170, with sensitivity of 87.9% and specificity of 80% was determined. Conclusions: QoL.BD-CL has adequate psychometric properties, as well as an adequate sensitivity and specificity to distinguish between negative and positive perceptions of life quality in Chilean patients with bipolar disorders.

Bovine embryo sex determination by multiplex loop-mediated isothermal amplification.

In cattle, the ability to determine the sex of embryos before embryo transfer is beneficial for increasing the number of animals with the desired sex. This study therefore developed a new modification of loop-mediated isothermal amplification in a multiplex format (multiplex LAMP) for highly efficient bovine embryo sexing. Two chromosomal regions, one specific for males (Y chromosome, S4 region) and the other common to both males and females (1.715 satellite DNA), were amplified in the same reaction tube. Each target was amplified by specifically designed inner primers, outer primers, and loop primers, where one of the S4 loop primers was labeled with the fluorescent dye 6-carboxyl-X-rhodamine (emitting a red color), whereas both satellite loop primers were labeled with the fluorescent dye fluorescein isothiocyanate (emitting a green color). After amplification at 63 °C for 1 hour, the amplified products were precipitated by a small volume of cationic polymer predispensed inside the reaction tube cap. Green precipitate indicated the presence of only control DNA without the Y chromosome, whereas orange precipitate indicated the presence of both target DNAs, enabling interpretation as female and male, respectively. Accuracy of the multiplex LAMP assay was evaluated using 46 bovine embryos with known sex (25 male and 21 female) generated by somatic cell nuclear transfer and confirmed by multiplex polymerase chain reaction. The multiplex LAMP showed 100% accuracy in identifying the actual sex of the embryos and provides a fast, simple, and cost-effective tool for bovine embryo sexing.

Cost, effectiveness and environmental relevance of multidrug transporters in sea urchin embryos.

ATP-binding cassette transporters protect cells via efflux of xenobiotics and endogenous byproducts of detoxification. While the cost of this ATP-dependent extrusion is known at the molecular level, i.e. the ATP used for each efflux event, the overall cost to a cell or organism of operating this defense is unclear, especially as the cost of efflux changes depending on environmental conditions. During prolonged exposure to xenobiotics, multidrug transporter activity could be costly and ineffective because effluxed substrate molecules are not modified in the process and could thus undergo repeated cycles of efflux and re-entry. Here we use embryos of the purple sea urchin, Strongylocentrotus purpuratus, as a model to determine transport costs and benefits under environmentally relevant xenobiotic concentrations. Strikingly, our results show that efflux transporter activity costs less than 0.2% of total ATP usage, as a proportion of oxygen consumption. The benefits of transport, defined as the reduction in substrate accumulation due to transporter activity, depended largely, but not entirely, on the rate of passive flux of each substrate across the plasma membrane. One of the substrates tested exhibited rapid membrane permeation coupled with high rates of efflux, thus inducing rapid and futile cycles of efflux followed by re-entry of the substrate. This combination significantly reduced transporter effectiveness as a defense and increased costs even at relatively low substrate concentrations. Despite these effects with certain substrates, our results show that efflux transporters are a remarkably effective and low-cost first line of defense against exposure to environmentally relevant concentrations of xenobiotics.

Ammonium concentration of spent medium provides a noninvasive assessment of embryonic developmental potential in IVF.

BACKGROUND: Currently, morphological criteria are used to select embryos for transfer in in vitro fertilization (IVF)-embryo transfer. However, the implantation rate is only about 30%, indicating a need for a more efficient method of selecting high-quality embryos. This study investigated the relationship between medium ammonium concentration and IVF implantation rates and evaluated the possibility of using ammonium concentration to provide an objective, noninvasive, and simple means of assessment of embryo viability. METHODS: On day 3 after fertilization, we sampled the spent medium bathing embryos obtained from patients undergoing IVF or intracytoplasmic sperm injection (ICSI) at the Reproductive Medical Center of Sun Yat-sen Memorial Hospital, between September 2010 and January 2012. The concentration of ammonium was determined using a dry chemistry system. RESULTS: The ammonium concentration increased significantly during the period of culture of all embryos, but the increase was significantly greater in the group that, subsequently, did not achieve pregnancy (P < .001). An receiver-operating characteristic (ROC) curve indicated that ammonium concentration was highly predictive of embryo implantation failure (area under ROC curve = 0.838). Failure to achieve implantation had a predictive sensitivity of 71% and specificity of 79.6%. The accuracy of prediction of successful or failed implantation was 75.4%. In both IVF and ICSI, the embryo implantation rate decreased significantly as the medium ammonium concentration increased (P < .05). CONCLUSION: Measurement of the ammonium concentration in the spent medium may provide a new research direction for exploring a simple, rapid, and low-cost method for reliable prediction of embryo implantation with high sensitivity and specificity.

Noninvasive metabolomic profiling of human embryo culture media using a simple spectroscopy adjunct to morphology for embryo assessment in in vitro fertilization (IVF).

Embryo quality is crucial to the outcome of in vitro fertilization (IVF); however, the ability to precisely distinguish the embryos with higher reproductive potential from others is poor. Morphologic evaluation used to play an important role in assessing embryo quality, but it is somewhat subjective. The culture medium is the immediate environment of the embryos in vitro, and a change of the substances in the culture medium is possibly related to the embryo quality. Thus, the present study aims to determine whether metabolomic profiling of the culture medium using Raman spectroscopy adjunct to morphology correlates with the reproductive potential of embryos in IVF and, thus, to look for a new method of assessing embryo quality. Fifty seven spent media samples were detected by Raman spectroscopy. Combined with embryo morphology scores, we found that embryos in culture media with less than 0.012 of sodium pyruvate and more than -0.00085 phenylalanine have a high reproductive potential, with up to 85.7% accuracy compared with clinical pregnancy. So, sodium pyruvate and phenylalanine in culture medium play an important role in the development of the embryo. Raman spectroscopy is an important tool that provides a new and accurate assessment of higher quality embryos.

An economical and practical method for whole-mount in situ hybridization for mouse embryos and organs.

Whole-mount in situ hybridization (WISH) is a useful method for detecting specific gene expression patterns at their site of action during embryonic development. Traditional WISH methods are costly and suitable only for mouse embryos younger than 11.5 days. We present here an economical and practical in situ hybridization method using DIG-labeled RNA probes. We changed the conditions in several steps to make the WISH method suitable for whole mouse embryos from embryonic days 9.5 to 12.5 and for older stage mouse embryonic organs. We performed all steps in one microcentrifuge tube up to the staining steps to avoid losing or damaging the mouse embryos. We re-used the solutions and materials to make the method more economical and suitable for less sophisticated laboratories. We also performed ß-galactosidase staining on Tb × 18 Cre/Rosa26/LacZ mouse embryos; the results agreed with the in situ hybridization results. Finally, we sectioned the specimens after hybridization and ß-galactosidase staining; the results agreed with the literature.

Genetic Cre-loxP assessment of epicardial cell fate using Wt1-driven Cre alleles.

RATIONALE: Wt1-Cre-based tools are important reagents for studying epicardial cell fate and gene function. OBJECTIVE: To better describe the properties of Wt1-Cre-based tools to enhance their use in Cre-loxP-based experiments. METHODS AND RESULTS: In contrast to recently reported results, we show that constitutive Wt1(GFPCre) in combination with certain Cre-activated reporters can be used to trace (pro) epicardial cell fate. Wt1(CreERT2) can be efficiently induced by tamoxifen administration. We show substantial labeling of coronary endothelial cells when induction is performed at late but not early stages of heart development. CONCLUSIONS: Wt1-based Cre alleles are useful tools for genetic lineage tracing of epicardial cells and mesothelium of other organs. Using these tools with proper understanding of their properties and limitations enables genetic labeling of epicardial cells and their derivatives.

Cost-effectiveness of cytogenetic evaluation of products of conception in the patient with a second pregnancy loss.

OBJECTIVE: To compare the cost of two strategies for managing the patient with recurrent pregnancy loss (RPL). DESIGN: Cost analysis using a decision analytic model was used to compare obtaining an evidence-based workup (EBW) for RPL versus obtaining a karyotype of the products of conception (POC) and proceeding with an EBW only in the setting of euploid POC. SETTING: Outpatient care. PATIENT(S): A simulated cohort of patients experiencing a second pregnancy loss. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Total cost of investigating the cause of RPL after a second pregnancy loss. RESULT(S): For all age categories, obtaining a karyotype of POC was less costly than an evidenced-based RPL evaluation. Monte Caro analysis demonstrated a net economic benefit for the karyotype strategy ($4,498 [±$792] vs. $5,022 [±$1,130]). CONCLUSION(S): Our model suggests an economic advantage for obtaining a karyotype of POC in women with second miscarriage.

Assessment of xenobiotic biotransformation including reactive oxygen species generation in the embryo using benzene as an example.

Quantification of embryonic metabolic capacity is an important tool in developmental toxicology research. Bioactivation of xenobiotics into reactive intermediates often contributes to embryo toxicity; thus, identification and quantification of these toxic metabolites is essential to gain further understanding of developmental toxicity. This chapter uses the environmental chemical benzene as a model xenobiotic to describe the detection of both metabolites and reactive oxygen species (ROS) in fetal liver. Briefly, mice are bred and the presence of a vaginal plug in a female mouse indicates gestational day 1. On the desired gestational day, pregnant dams are exposed to benzene followed by sacrifice at the desired time-point after exposure. Using gas chromatography coupled to mass spectrometry, the detection of benzene metabolites can be achieved. Additionally, we describe the measurement of ROS by flow cytometry using the fluorescent probe 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein diacetate, which readily diffuses into cells and, upon oxidation by any ROS, is converted to the highly fluorescent, negatively charged carboxydichlorofluorescein, which remains trapped within the cells.