The first two blastomeres contribute unequally to the human embryo.

Retrospective lineage reconstruction of humans predicts that dramatic clonal imbalances in the body can be traced to the 2-cell stage embryo. However, whether and how such clonal asymmetries arise in the embryo is unclear. Here, we performed prospective lineage tracing of human embryos using live imaging, non-invasive cell labeling, and computational predictions to determine the contribution of each 2-cell stage blastomere to the epiblast (body), hypoblast (yolk sac), and trophectoderm (placenta). We show that the majority of epiblast cells originate from only one blastomere of the 2-cell stage embryo. We observe that only one to three cells become internalized at the 8-to-16-cell stage transition. Moreover, these internalized cells are more frequently derived from the first cell to divide at the 2-cell stage. We propose that cell division dynamics and a cell internalization bottleneck in the early embryo establish asymmetry in the clonal composition of the future human body.

Current status of spent embryo media research for preimplantation genetic testing.

In recent years, a growing body of literature has emerged investigating the clinical utility of spent embryo media (SEM) for preimplantation genetic testing for aneuploidy (PGT-A) (Hammond et al. in Fertil Steril. 107(1):220-8, 2017; Xu et al. in Proc Natl Acad Sci USA. 113(42):11907-12, 2016; Shamonki et al. in Fertil Steril. 106(6):1312-8, 2016; Feichtinger et al. in Reprod BioMed Online. 34(6):583-9, 2017; Vera-Rodriguez et al. in Hum Reprod. 33(4):745-56, 2018; Kuznyetsov et al. in PLoS One. 13(5):e0197262, 2018; Ho et al. in Fertil Steril. 110(3):467-75, 2018; Capalbo et al. in Fertil Steril. 110(5):870-9, 2018). Most of these studies have reported moderate success rates, suggesting the need for improvements in sensitivity and specificity. The concordance between spent media and embryo biopsy or whole embryo was reported to be between 30.4 and 90%, with 50-70% correlation being the most representative (Xu et al. in Proc Natl Acad Sci USA. 113(42):11907-12, 2016; Shamonki et al. in Fertil Steril. 106(6):1312-8, 2016; Feichtinger et al. in Reprod BioMed Online. 34(6):583-9, 2017; Vera-Rodriguez et al. in Hum Reprod. 33(4):745-56, 2018; Kuznyetsov et al. in PLoS One. 13(5):e0197262, 2018; Ho et al. in Fertil Steril. 110(3):467-75, 2018). Here, we will analyze all spent media testing strategies including SEM collection methods, whole genome amplification (WGA) strategies, chromosome copy number detection, and bioinformatics analysis tools. We will propose improvements to further increase the accuracy and sensitivity of the assay before bringing PGT-A with SEM into the clinical sphere.

Pushing the limits of detection: investigation of cell-free DNA for aneuploidy screening in embryos.

OBJECTIVE: To determine the accuracy of cell-free DNA (cfDNA) in spent embryo medium (SEM) for ploidy and sex detection at the cleavage and blastocyst stages. To determine if assisted hatching (AH) and morphologic grade influence cfDNA concentration and accuracy. DESIGN: Prospective cohort. SETTING: Academic fertility center. PATIENT(S): Nine patients undergoing IVF; 41 donated two-pronuclei embryos and 20 embryos from patients undergoing preimplantation genetic testing for aneuploidy (PGT-A). INTERVENTIONS(S): In a donated embryo arm, SEM was collected on days 3 and 5, with one-half of the embryos undergoing AH before and one-half after. In a clinical arm, SEM was collected on day 5 before trophectoderm (TE) biopsy. Samples underwent PGT-A with the use of next-generation sequencing. cfDNA results were compared with corresponding whole embryos and TE biopsies. MAIN OUTCOME MEASURE(S): Concordance rates, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for ploidy and sex detection with the use of cfDNA. RESULT(S): Of 141 samples, cfDNA was amplified in 39% and 80.4% of days 3 and 5 SEM, respectively. Concordances for ploidy and sex, respectively, were 56.3% and 81.3% between day 3 cfDNA and whole embryos, and 65% and 70% between day 5 cfDNA and TE biopsies. Day 5 cfDNA sensitivity and specificity for aneuploidy were 0.8 and 0.61, respectively. PPV and NPV were 0.47 and 0.88, respectively. Timing of AH and morphology did not influence cfDNA concentration or accuracy. CONCLUSION(S): cfDNA is detectable on days 3 and 5, but more accurate on day 5. Although our data suggest moderate concordance rates, PGT-A with the use of cfDNA must be further optimized before clinical implementation.

Blastulation timing is associated with differential mitochondrial content in euploid embryos.

PURPOSE: Preimplantation genetic screening (PGS) and assessment of mitochondrial content (MC) are current methods for selection of the best embryos for transfer. Studies suggest that time-lapse morphokinetics (TLM) may also be helpful for selecting embryos more likely to implant. In our study, we sought to examine the relationship between TLM parameters and MC to determine if they could be used adjunctively in embryo selection. We also examined the relationship between MC with ploidy and blastulation. METHODS: Cryopreserved human embryos at the zygote stage were thawed and cultured in a time-lapse system. Blastomere and trophectoderm biopsies were performed on days 3 and 6. Biopsied cells and all whole embryos from day 6 were analyzed for MC (ratio of mitochondrial to nuclear DNA) and ploidy using next-generation sequencing. RESULTS: In embryos, MC per cell declined between day 3 and day 6. While early cleavage parameters did not predict MC, embryos with longer blastulation timing had higher MC on day 6. Day 6 MC was lower in euploid vs. aneuploid embryos and lower in blastocysts vs. arrested embryos. CONCLUSIONS: A lower MC at the blastocyst stage was associated with euploid status and blastocyst formation, indicating better embryo quality compared to those with a higher MC. Higher MC in aneuploid and arrested embryos may be explained by slower cell division or degradation of genomic DNA over time. Blastulation timing may be helpful for selection of higher quality embryos. Combining blastulation timing and MC along with morphologic grading and euploid status may offer a new direction in embryo selection.

Gestational surrogacy and the role of routine embryo screening: Current challenges and future directions for preimplantation genetic testing.

Preimplantation genetic screening (PGS) is a component of IVF entailing selection of an embryo for transfer on the basis of chromosomal normalcy. If PGS were integrated with single embryo transfer (SET) in a surrogacy setting, this approach could improve pregnancy rates, minimize miscarriage risk, and limit multiple gestations. Even without PGS, pregnancy rates for IVF surrogacy cases are generally satisfactory, especially when treatment utilizes embryos derived from young oocytes and transferred to a healthy surrogate. However, there could be a more general role for PGS in surrogacy, since background aneuploidy in embryos remains a major factor driving implantation failure and miscarriage for all infertility patients. At present, the proportion of IVF cases involving GS is limited, while the number of IVF patients requesting PGS appears to be increasing. In this report, the relevance of PGS for surrogacy in the rapidly changing field of assisted fertility medicine is discussed.

Specific responses of Salmonella enterica to tomato varieties and fruit ripeness identified by in vivo expression technology.

BACKGROUND: Recent outbreaks of vegetable-associated gastroenteritis suggest that enteric pathogens colonize, multiply and persist in plants for extended periods of time, eventually infecting people. Genetic and physiological pathways, by which enterics colonize plants, are still poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: To better understand interactions between Salmonella enterica sv. Typhimurium and tomatoes, a gfp-tagged Salmonella promoter library was screened inside red ripe fruits. Fifty-one unique constructs that were potentially differentially regulated in tomato relative to in vitro growth were identified. The expression of a subset of these promoters was tested in planta using recombinase-based in vivo expression technology (RIVET) and fitness of the corresponding mutants was tested. Gene expression in Salmonella was affected by fruit maturity and tomato cultivar. A putative fadH promoter was upregulated most strongly in immature tomatoes. Expression of the fadH construct depended on the presence of linoleic acid, which is consistent with the reduced accumulation of this compound in mature tomato fruits. The cysB construct was activated in the fruit of cv. Hawaii 7997 (resistant to a race of Ralstonia solanacearum) more strongly than in the universally susceptible tomato cv. Bonny Best. Known Salmonella motility and animal virulence genes (hilA, flhDC, fliF and those encoded on the pSLT virulence plasmid) did not contribute significantly to fitness of the bacteria inside tomatoes, even though deletions of sirA and motA modestly increased fitness of Salmonella inside tomatoes. CONCLUSIONS/SIGNIFICANCE: This study reveals the genetic basis of the interactions of Salmonella with plant hosts. Salmonella relies on a distinct set of metabolic and regulatory genes, which are differentially regulated in planta in response to host genotype and fruit maturity. This enteric pathogen colonizes tissues of tomatoes differently than plant pathogens, and relies little on its animal virulence genes for persistence within the fruit.

High-throughput screening for salmonella avirulent mutants that retain targeting of solid tumors.

Salmonella has a natural ability to target a wide range of tumors in animal models. However, strains used for cancer therapy have generally been selected only for their avirulence rather than their tumor-targeting ability. To select Salmonella strains that are avirulent and yet efficient in tumor targeting, a necessary criterion for clinical applications, we measured the relative fitness of 41,000 Salmonella transposon insertion mutants growing in mouse models of human prostate and breast cancer. Two classes of potentially safe mutants were identified. Class 1 mutants showed reduced fitness in normal tissues and unchanged fitness in tumors (e.g., mutants in htrA, SPI-2, and STM3120). Class 2 mutants showed reduced fitness in tumors and normal tissues (e.g., mutants in aroA and aroD). In a competitive fitness assay in human PC-3 tumors growing in mice, class 1 mutant STM3120 had a fitness advantage over class 2 mutants aroA and aroD, validating the findings of the initial screening of a large pool of transposon mutants and indicating a potential advantage of class 1 mutants for delivery of cancer therapeutics. In addition, an STM3120 mutant successfully targeted tumors after intragastric delivery, opening up the oral route as an option for therapy administration.

Salmonella serovar identification using PCR-based detection of gene presence and absence.

There are more than 2,500 known Salmonella serovars, and some of these can be further subclassified into groups of strains that differ profoundly in their gene content. We refer to these groups of strains as "genovars." A compilation of comparative genomic hybridization data on 291 Salmonella isolates, including 250 S. enterica subspecies I strains from 32 serovars (52 genovars), was used to select a panel of 384 genes whose presence and absence among serovars and genovars was of potential taxonomic value. A subset of 146 genes was used for real-time PCR to successfully identify 12 serovars (16 genovars) in 24 S. enterica strains. A further subset of 64 genes was used to identify 8 serovars (9 genovars) in 12 multiplex PCR mixes on 11 S. enterica strains. These gene panels distinguish all tested S. enterica subspecies I serovars and their known genovars, almost all by two or more informative markers. Thus, a typing methodology based on these predictive genes would generally alert users if there is an error, an unexpected polymorphism, or a potential new genovar.

Salmonella promoters preferentially activated inside tumors.

Salmonella enterica and avirulent derivatives prefer solid tumors over normal tissue in animal models. The identification of endogenous Salmonella promoters that are preferentially activated in tumors could further our understanding of this phenomenon. Toward this goal, a random library of S. enterica typhimurium 14028 genomic DNA was cloned upstream of a promoterless gene encoding the green fluorescent protein (GFP) TurboGFP. A population of Salmonella containing this library was injected i.v. into tumor-free nude mice and into human PC3 prostate tumors growing subcutaneously in nude mice. After 2 days, fluorescence-activated cell sorting was used to enrich for bacterial clones expressing GFP from spleens or tumors. The resulting libraries were hybridized to an oligonucleotide tiling array of the Salmonella genome. Eighty-six intergenic regions were found to be enriched in tumor samples but not in spleen. Twenty of these candidate promoters were also detected in the sequences of 100 random clones from a library enriched for expression in bacteria growing in tumors. Three candidate promoter clones were individually tested in vivo, and enhanced GFP expression in bacteria growing in tumor relative to spleen was confirmed. Two of the three clones (pflE and ansB promoter regions) are known to be induced in hypoxic conditions that pertain to many tumors. For many of the other candidate promoters preferentially induced in bacteria growing in tumors, regulatory mechanisms may not be related to hypoxia. The expression of therapeutics in Salmonella under the regulation of one or more promoters that are activated preferentially in tumors has the potential to improve the targeting of drug delivery.