Chromatin Accessibility Landscape in Human Early Embryos and Its Association with Evolution.

The dynamics of the chromatin regulatory landscape during human early embryogenesis remains unknown. Using DNase I hypersensitive site (DHS) sequencing, we report that the chromatin accessibility landscape is gradually established during human early embryogenesis. Interestingly, the DHSs with OCT4 binding motifs are enriched at the timing of zygotic genome activation (ZGA) in humans, but not in mice. Consistently, OCT4 contributes to ZGA in humans, but not in mice. We further find that lower CpG promoters usually establish DHSs at later stages. Similarly, younger genes tend to establish promoter DHSs and are expressed at later embryonic stages, while older genes exhibit these features at earlier stages. Moreover, our data show that human active transposons SVA and HERV-K harbor DHSs and are highly expressed in early embryos, but not in differentiated tissues. In summary, our data provide an evolutionary developmental view for understanding the regulation of gene and transposon expression.

The development of early human lymphatic vessels as characterized by lymphatic endothelial markers.

Lymphatic vessel development studies in mice and zebrafish models have demonstrated that lymphatic endothelial cells (LECs) predominantly differentiate from venous endothelial cells via the expression of the transcription factor Prox1. However, LECs can also be generated from undifferentiated mesoderm, suggesting potential diversity in their precursor cell origins depending on the organ or anatomical location. Despite these advances, recapitulating human lymphatic malformations in animal models has been difficult, and considering lymphatic vasculature function varies widely between species, analysis of development directly in humans is needed. Here, we examined early lymphatic development in humans by analyzing the histology of 31 embryos and three 9-week-old fetuses. We found that human embryonic cardinal veins, which converged to form initial lymph sacs, produce Prox1-expressing LECs. Furthermore, we describe the lymphatic vessel development in various organs and observe organ-specific differences. These characterizations of the early development of human lymphatic vessels should help to better understand the evolution and phylogenetic relationships of lymphatic systems, and their roles in human disease.

Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human.

Understanding new modulators of axon regeneration is central to neural repair. Our previous work demonstrated critical roles of atypical cadherin Celsr2 during neural development, including cilia organization, neuron migration and axon navigation. Here, we address its role in axon regeneration. We show that Celsr2 is highly expressed in both mouse and human spinal motor neurons. Celsr2 knockout promotes axon regeneration and fasciculation in mouse cultured spinal explants. Similarly, cultured Celsr2 mutant motor neurons extend longer neurites and larger growth cones, with increased expression of end-binding protein 3 and higher potassium-induced calcium influx. Mice with Celsr2 conditional knockout in spinal motor neurons do not exhibit any behavioural deficits; however, after branchial plexus injury, axon regeneration and functional forelimb locomotor recovery are significantly improved. Similarly, knockdown of CELSR2 using shRNA interference in cultured human spinal motor explants and motor neurons increases axonal fasciculation and growth. In mouse adult spinal cord after root avulsion, in mouse embryonic spinal cords, and in cultured human motor neurons, Celsr2 downregulation is accompanied by increased levels of GTP-bound Rac1 and Cdc42, and of JNK and c-Jun. In conclusion, Celsr2 negatively regulates motor axon regeneration and is a potential target to improve neural repair.

The so-called absorption process of the pulmonary vein into the left atrium of the heart: a histological study using human embryos and fetuses.

BACKGROUND: Embryonic pulmonary veins (PVs) are believed to be absorbed into the left atrium (LA) to provide an adult morphology in which "four" veins drain separately into the atrium. MATERIALS AND METHODS: Serial histological sections were obtained from 27 human embryos and fetuses. RESULTS: Between 5 and 6 weeks, the four PVs joined together to form a trunk-like structure (initial spatium pulmonalis) that was larger than the initial LA (two-ostia pattern). The cardiac nerves ran inferiorly along the posterior aspect of the four veins, as well as the spatium. At and until 7 weeks, the cardiac nerves were concentrated to elongate the nerve fold, and the latter separated the left PV trunk from the expanding LA (left spatium). Similarly, the right PV opened to a thick and deep LA recess (right spatium). At 8-12 weeks, depending on the growth of the LA, the opening of the left and right PVs became distant, and the spatium was elongated transversely. The left spatium was enlarged to open widely to the proper left atrium in contrast to the right spatium pushed anteriorly by the right atrium. The three-ostia pattern was transiently observed because of the lost delimitation between the left spatium and proper atrium. The myocardium was thin in the left spatium behind the left atrial nerve fold, whereas the right spatium was tube-like with a thick myocardium. CONCLUSIONS: The four-ostia pattern seemed to be established at birth due to a drastically increased venous return from the lung, resulting in a flat smooth left atrial posterior wall.

On the origins and fate of chromosomal abnormalities in human preimplantation embryos: an unsolved riddle.

About 8 out of 10 human embryos obtained in vitro harbour chromosomal abnormalities of either meiotic or mitotic origin. Abnormalities of mitotic origin lead to chromosomal mosaicism, a phenomenon that has sparked much debate lately as it confounds results obtained through preimplantation genetic testing for aneuploidy (PGT-A). PGT-A in itself is still highly debated, not only on the modalities of its execution but also on whether it should be offered to patients at all. We will focus on post-zygotic chromosomal abnormalities leading to mosaicism. First, we will summarize what is known about the rates of chromosomal abnormalities at different developmental stages. Next, based on the current understanding of the origin and cellular consequences of chromosomal abnormalities, which is largely based on studies on cancer cells and model organisms, we will offer a number of hypotheses on which mechanisms may be at work in early human development. Finally, and very briefly, we will touch upon the impact our current knowledge has on the practice of PGT-A. What is the level of abnormal cells that an embryo can tolerate before it loses its potential for full development? And is blastocyst biopsy as harmless as it seems?

We have reached a dead end for preimplantation genetic testing for aneuploidy.

The hypothesis of preimplantation genetic testing for aneuploidy (PGT-A) was first proposed 20 years ago, suggesting that during IVF elimination of aneuploid embryos prior to transfer will improve implantation rates of remaining embryos and, therefore, increase pregnancy and live birth rates, while also reducing miscarriages. Subsequently, unvalidated and increasingly unrestricted clinical utilization of PGT-A called for at least one properly randomized controlled trial (RCT) to assess cumulative live birth rates following a single oocyte retrieval, utilizing all fresh and frozen embryos of an IVF cycle. Only recently two such RCTs were published, however both, when properly analysed, not only failed to demonstrate significant advantages from utilization of PGT-A, but actually demonstrated outcome deficits in comparison to non-use of PGT-A, when patient selection biases in favour of PGT-A were reversed. Moreover, because of high embryo mosaicism at the blastocyst stage and, therefore, high false-positive rates from trophectoderm biopsies, large numbers of chromosomal-normal embryos with normal pregnancy potential are unnecessarily left unused or discarded, indisputably causing harm to affected couples. We, therefore, strongly call for restricting PGT-A to only research protocols and, as of this point in time, encourage professional societies in the field to follow suit with appropriate practice guidelines.

Cleavage-stage human embryo arrest, is it embryo genetic composition or others?

Embryo transfer is a crucial step in IVF cycle, with increasing trend during the last decade of transferring a single embryo, preferably at the blastocyst stage. Despite increasing evidence supporting Day 5 blastocyst-stage transfer, the optimal day of embryo transfer remains controversial. The crucial questions are therefore, whether the mechanisms responsible to embryos arrest are embryo aneuploidy or others, and whether those embryos arrested in-vitro between the cleavage to the blastocyst stage would survive in-vivo if transferred on the cleavage-stage. We therefore aim to explore whether aneuploidy can directly contribute to embryo development to the blastocyst stage. Thirty Day-5 embryos, that their Day-3 blastomere biopsy revealed a single-gene defect, were donated by 10 couples undergoing preimplantation genetic testing treatment at our center. Affected high quality Day-3 embryos were cultured to Day-5, and were classified to those that developed to the blastocyst-stage and those that were arrested. Each embryo underwent whole genome amplification. Eighteen (60%) embryos were arrested, did not develop to the blastocyst stage and 12 (40%) have developed to the blastocyst stage. Nineteen embryos (63.3%) were found to be euploid. Of them, 12 (66.6%) were arrested embryos and 7 (58.3%) were those that developed to the blastocyst-stage. These figures were not statistically different (p = 0.644). Our observation demonstrated that the mechanism responsible to embryos arrest in vitro is not embryo aneuploidy, but rather other, such as culture conditions. If further studies will confirm that Day-5 blastocyst transfer might cause losses of embryos that would have been survived in vivo, cleavage-stage embryo transfer would be the preferred timing. This might reduce the cycle cancellations due to failure of embryo to develop to the blastocyst stage and will provide the best cumulative live birth-rate per started cycle.

GDF9 concentration in embryo culture medium is linked to human embryo quality and viability.

PURPOSE: We aimed to evaluate the link between the GDF9 concentration in day 3 human embryo culture medium and embryo quality and viability. METHODS: Two independent, prospective, observational studies were conducted. In study 1, a total of 280 embryos from 70 patients who obtained at least 4 embryos with 6-10 blastomeres (2 transferable and 2 non-transferable embryos) at day 3 were enrolled. In study 2, a total of 119 embryos from 61 patients (29 fully implanted and 32 non-implanted patients) were enrolled. The corresponding GDF9 concentrations in spent culture medium of embryos were quantified by ELISA assay. The expression pattern of GDF9 in human embryos was investigated using Q-PCR and immunofluorescence. RESULTS: GDF9 mRNA and protein were detected from human oocytes to eight-cell embryos and displayed a slow decreasing trend. In study 1, GDF9 concentration in culture medium is lower for transferable embryos compared with non-transferable embryos (331 pg/mL (quartiles: 442, 664 pg/mL) vs. 518 pg/mL (quartiles: 328, 1086 pg/mL), P < 0.001), and increased commensurate with the diminution of the embryo quality (P < 0.001). In study 2, significantly lower GDF9 concentration was detected for implanted embryos than non-implanted embryos (331 pg/mL (quartiles: 156, 665 pg/mL) vs. 518 pg/mL (quartiles: 328, 1086 pg/mL), P < 0.001). The same trend was found between the embryos that led to live birth and those that failed. CONCLUSION: The GDF9 concentration in culture medium is linked to embryo quality and viability, and exhibited the potential to be a non-invasive biomarker for embryo selection.

Ethical Issues in Commercialisation of Stem Cell Therapies: Mapping the Terrain.

Bioethical debates on stem cell research have focused primarily on the moral status of human embryos. This article highlights seven distinct policy and ethical issues associated with the commercialisation of stem cell therapies, describes some of the underlying moral questions on which they turn, and argues that there is an urgent need to refocus the debate on stem cell research beyond the controversy over embryo destruction.

Dampened X-chromosomes in human pluripotent stem cells: dampening or erasure of X-upregulation?

The recent report of X-chromosome dampening in human preimplantation embryos remains controversial. Subsequently, Sahakyan et al. found evidence of X-chromosome dampening in human naïve pluripotent stem cells (hPSCs) as well. Here, we discuss whether X-dampening reported in hPSCs truly reflects the dampening of X-chromosomes or it is a consequence of the erasure of X-chromosome upregulation.

Is there still a rationale for non-invasive PGT-A by analysis of cell-free DNA released by human embryos into culture medium?

Human embryos utilise an array of processes to eliminate the very high prevalence of aneuploid cells in early embryo stages. Human embryo self-correction was recently demonstrated by their ability to eliminate/expel abnormal blastomeres as cell debris/fragments. A whole genome amplification study has demonstrated that 63.6% of blastocysts expelled cell debris with abnormal chromosomal rearrangements. Moreover, 55.5% of euploid blastocysts expel aneuploid debris, strongly suggesting that the primary source of cell free DNA in culture media is expelled aneuploid blastomeres and/or their fragments. Such a substantial ability to self-correct downstream from the blastocyststage, therefore, renders any chromosomal diagnosis at the blastocyststage potentially useless, and this, unfortunately, also must particularly include non-invasive PGT-A based on cell-free DNA in spent medium. High rates of false-positive diagnoses of human embryos often lead to non-use and/or disposal of embryos with entirely normal pregnancy potential. Before adopting yet another round of unvalidated PGT-A as a routine adjunct to IVF, we here present facts that deserve to be considered.

Cumulus cells of euploid versus whole chromosome 21 aneuploid embryos reveal differentially expressed genes.

RESEARCH QUESTION: Can cumulus cells be used as a non-invasive target for the study of determinants of preimplantation embryo quality? DESIGN: Cumulus cells were collected from monosomy 21, trisomy 21 and euploid embryos and subjected to RNA sequencing analysis and real-time polymerase chain reaction assays. The differential gene expression was analysed for different comparisons. RESULTS: A total of 3122 genes in monosomy 21 cumulus cells and 19 genes in trisomy 21 cumulus cells were differentially expressed compared with euploid cumulus cells. Thirteen of these genes were differentially expressed in both monosomy and trisomy 21, compared with euploid, including disheveled segment polarity protein 2 (DVL2), cellular communication network factor 1 (CCN1/CYR61) and serum response factor (SRF), which have been previously implicated in embryo developmental competence. In addition, ingenuity pathway analysis revealed cell-cell contact function to be affected in both monosomy and trisomy 21 cumulus cells. CONCLUSIONS: These findings support the use of cumulus cell gene expression analysis for the development of biomarkers evaluating oocyte quality for patients undergoing fertility preservation of oocytes.

Repeated cryopreservation process impairs embryo implantation potential but does not affect neonatal outcomes.

RESEARCH QUESTION: Does repeated cryopreservation process affect embryo implantation potential and neonatal outcomes of human embryos? DESIGN: This retrospective cohort study was conducted in the Reproductive Medicine Centre, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All assisted reproductive technology (ART) cycles were carried out between January 2014 and December 2018. Preferentially matched participants were divided into three groups according to the times of embryo cryopreservation: the fresh group (n = 249), the cryopreservation group (n = 244) and the re-cryopreservation group (n = 216). Embryo implantation rate, live birth rate, miscarriage rate and neonatal complication rate were compared among these three groups. RESULTS: The embryo implantation rate, clinical pregnancy rate and live birth rate in the re-cryopreservation group were significantly lower, and the miscarriage rate also slightly increased. Logistic regression analysis indicated that embryos with repeated cryopreservation and lower trophectoderm scores were at higher risk of embryo implantation failure in single embryo transfer cycles (OR 1.79 and 1.56, respectively). No significant differences were observed in gender, gestational age, birthweight, neonatal abnormality and neonatal complications among the groups. CONCLUSIONS: Our findings demonstrate the adverse effect of repeated cryopreservation on embryo implantation potential. The study offers embryologists and reproductive clinicians a warning of detrimental role of repeated cryopreservation. If unnecessary, it is strongly recommended to avoid repeated practice of vitrification and warming on embryos.

Early Forebrain Neurons and Scaffold Fibers in Human Embryos.

Neural progenitor proliferation, neuronal migration, areal organization, and pioneer axon wiring are critical events during early forebrain development, yet remain incompletely understood, especially in human. Here, we studied forebrain development in human embryos aged 5 to 8 postconceptional weeks (WPC5-8), stages that correspond to the neuroepithelium/early marginal zone (WPC5), telencephalic preplate (WPC6 & 7), and incipient cortical plate (WPC8). We show that early telencephalic neurons are formed at the neuroepithelial stage; the most precocious ones originate from local telencephalic neuroepithelium and possibly from the olfactory placode. At the preplate stage, forebrain organization is quite similar in human and mouse in terms of areal organization and of differentiation of Cajal-Retzius cells, pioneer neurons, and axons. Like in mice, axons from pioneer neurons in prethalamus, ventral telencephalon, and cortical preplate cross the diencephalon-telencephalon junction and the pallial-subpallial boundary, forming scaffolds that could guide thalamic and cortical axons at later stages. In accord with this model, at the early cortical plate stage, corticofugal axons run in ventral telencephalon in close contact with scaffold neurons, which express CELSR3 and FZD3, two molecules that regulates formation of similar scaffolds in mice.

Gene editing of human embryos is not contrary to human rights law: A reply to Drabiak.

In an article in this journal, Katherine Drabiak argues that green lighting genome editing of human embryos is contrary to "fundamental human rights law." According to the author, genome editing of human embryos violates what we should recognize as a fundamental human right to inherit a genome without deliberate manipulation. In this reply article, we assess Drabiak's legal analysis and show methodological and substantive flaws. Methodologically, her analysis omits the key international legal instruments that form the so-called International Bill of Rights and thus fails to provide a full and accurate account of the fundamental international human rights standards. Substantively, Drabiak invokes, as a basis for prohibiting gene editing of human embryos, a legal standard (the rights and integrity of the future child) that is unknown to international law. Contrary to Drabiak's account, genome editing of human embryos is not prohibited under international law. Indeed, the right to health and the right to benefit from scientific progress may be interpreted as the basis of a legal duty to provide equality of access to germline gene editing, once determined it is beneficial and safe to use.

Human germline editing in the era of CRISPR-Cas: risk and uncertainty, inter-generational responsibility, therapeutic legitimacy.

BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats-associated (CRISPR-Cas) technology may allow for efficient and highly targeted gene editing in single-cell embryos. This possibility brings human germline editing into the focus of ethical and legal debates again. MAIN BODY: Against this background, we explore essential ethical and legal questions of interventions into the human germline by means of CRISPR-Cas: How should issues of risk and uncertainty be handled? What responsibilities arise regarding future generations? Under which conditions can germline editing measures be therapeutically legitimized? For this purpose, we refer to a scenario anticipating potential further development in CRISPR-Cas technology implying improved accuracy and exclusion of germline transmission to future generations. We show that, if certain concepts regarding germline editing are clarified, under such conditions a categorical prohibition of one-generation germline editing of single-cell embryos appears not to be ethically or legally justifiable. CONCLUSION: These findings are important prerequisites for the international debate on the ethical and legal justification of germline interventions in the human embryo as well as for the harmonization of international legal standards.

Systems based analysis of human embryos and gene networks involved in cell lineage allocation.

BACKGROUND: Little is understood of the molecular mechanisms involved in the earliest cell fate decision in human development, leading to the establishment of the trophectoderm (TE) and inner cell mass (ICM) stem cell population. Notably, there is a lack of understanding of how transcriptional networks arise during reorganisation of the embryonic genome post-fertilisation. RESULTS: We identified a hierarchical structure of preimplantation gene network modules around the time of embryonic genome activation (EGA). Using network models along with eukaryotic initiation factor (EIF) and epigenetic-associated gene expression we defined two sets of blastomeres that exhibited diverging tendencies towards ICM or TE. Analysis of the developmental networks demonstrated stage specific EIF expression and revealed that histone modifications may be an important epigenetic regulatory mechanism in preimplantation human embryos. Comparison to published RNAseq data confirmed that during EGA the individual 8-cell blastomeres are transcriptionally primed for the first lineage decision in development towards ICM or TE. CONCLUSIONS: Using multiple systems biology approaches to compare developmental stages in the early human embryo with single cell transcript data from blastomeres, we have shown that blastomeres considered to be totipotent are not transcriptionally equivalent. Furthermore we have linked the developmental interactome to individual blastomeres and to later cell lineage. This has clinical implications for understanding the impact of fertility treatments and developmental programming of long term health.

Non-invasive mitochondrial DNA quantification on Day 3 predicts blastocyst development: a prospective, blinded, multi-centric study.

In ART, embryo quality evaluation is routinely based on morphological criteria. We previously demonstrated that the mitochondrial DNA (mtDNA)/genomic DNA (gDNA) ratio in culture medium was significantly associated with embryo quality and viability potential. The purpose of this prospective, blinded, multi-centric study was to validate the use of mtDNA/gDNA ratio in Day 3 spent medium as a predictor of human embryo developmental competence. The mtDNA/gDNA ratio was assessed in Day 3 culture media (n=484) of embryos from 143 patients by quantitative PCR. A mixed effect logistic regression model was applied. We found that mtDNA/gDNA ratio in Day 3 culture medium combined with embryo morphology improves the prediction upon blastulation compared to morphology alone (P < 0.0001), independent of patient and cycle characteristics. With regard to routine use in clinics, we evaluated the ability of the novel, combined grading score to improve selection of developmentally competent embryos of a single cohort. Including embryos from 44 patients, the sensibility and specificity of the scoring system based on Day 3 morphological stage were 92% and 13%, respectively. Integration with the culture medium mtDNA/gDNA ratio increased the performance of the method (sensibility: 95%; specificity: 65%). The results of this study suggest the possibility of carrying out a non-invasive evaluation of embryonic mtDNA content through the culture medium. When combined with embryo morphology, it has the potential to help embryologists rank embryos and choose which embryo(s) has the greater development potential, and thus should be transferred on Day 3, among sibling embryos with the same morphological grade.

Correction of the Marfan Syndrome Pathogenic FBN1 Mutation by Base Editing in Human Cells and Heterozygous Embryos.

There are urgent demands for efficient treatment of heritable genetic diseases. The base editing technology has displayed its efficiency and precision in base substitution in human embryos, providing a potential early-stage treatment for genetic diseases. Taking advantage of this technology, we corrected a Marfan syndrome pathogenic mutation, FBN1T7498C. We first tested the feasibility in mutant cells, then successfully achieved genetic correction in heterozygous human embryos. The results showed that the BE3 mediated perfect correction at the efficiency of about 89%. Importantly, no off-target and indels were detected in any tested sites in samples by high-throughput deep sequencing combined with whole-genome sequencing analysis. Our study therefore suggests the efficiency and genetic safety of correcting a Marfan syndrome (MFS) pathogenic mutation in embryos by base editing.

Defining the limits of detection for chromosome rearrangements in the preimplantation embryo using next generation sequencing.

STUDY QUESTION: Is next generation sequencing (NGS) capable of detecting smaller sub-chromosomal rearrangements in human embryos than the manufacturer's quoted resolution suggests? SUMMARY ANSWER: NGS was able to detect unbalanced chromosome segments smaller than the manufacturer's resolution. WHAT IS KNOWN ALREADY: Array Comparative Genomic Hybridization (array-CGH) has been the gold standard platform used for PGD of chromosome rearrangements. NGS is a viable alternative to array-CGH for PGD of chromosome arrangements given that the manufacturer's guidelines quote a resolution of ≥20 Mb. However, as many patients carry a chromosome rearrangement <20 Mb, the detection limits of NGS warrant further investigation. STUDY DESIGN, SIZE, DURATION: This study involved a retrospective assessment of stored DNA samples from embryos that had previously been diagnosed as unbalanced by array-CGH as part of routine PGD in two separate IVF clinics between November 2013 and April 2017. SurePlex whole genome amplification (WGA) products derived from DNA extracted from an embryo biopsy sample known to carry an unbalanced form of a chromosome rearrangement were subjected to a specific NGS workflow (VeriSeq PGS). The results from the two technologies were compared for each sample. PARTICIPANTS/MATERIALS, SETTING, METHODS: WGA products from 200 embryos known to carry unbalanced rearrangements were sequenced and analysed. These embryos had been created by 75 patients known to carry a chromosome rearrangement (68 reciprocal translocations, 3 pericentric inversions, 1 paracentric inversion, 2 insertions and 1 dual reciprocal and inversion). Each sample was assessed for the size of the segmental gain/loss (Mb), copy number for each segment and chromosome, segregation pattern, the number of bins in the analysis software used and concordance with array-CGH results. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 294 unbalanced chromosome segments were assessed. NGS was capable of detecting 285/294 (97%) unbalanced segments previously identified using array-CGH. The final PGD diagnosis was concordant for 200/200 (100%) embryos. In total, 44/75 (59%) patients contained an unbalanced chromosome segment below the quoted 20 Mb manufacturer's stated resolution. Of these, 35/44 (80%) patients had segments that were able to be detected using NGS, whilst maintaining clinical outcome concordance. LIMITATIONS, REASONS FOR CAUTION: Our study subset did not include any rearrangements involving the Y chromosome. NGS has less available bins per chromosome compared to the array-CGH platform used, thus it remains possible that chromosome rearrangements predicted to be small but still detectable by array-CGH may not be feasible for testing using NGS. This should be considered when undertaking a theoretical feasibility assessment for detecting the chromosome rearrangement in question. Only one specific workflow for WGA and NGS was investigated in this study. WIDER IMPLICATIONS OF THE FINDINGS: This study has shown that NGS is available for the detection of unbalanced chromosome rearrangements ≥10 Mb. STUDY FUNDING/COMPETING INTEREST(S): Part sponsorship of the VeriSeq PGS kits used was provided by Illumina. The remainder of the kits were provided by two commercial IVF clinics. None of the authors has any conflicting interests to declare. TRIAL REGISTRATION NUMBER: N/A.