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The health risks associated with transferring embryos classified as mosaic by preimplantation genetic testing for aneuploidies (PGT-A) are currently unknown. Such embryos produce PGT-A results indicating the presence of both euploid and aneuploid cells and have historically been deselected from transfer and grouped with uniformly aneuploid embryos as ‘abnormal’. In recent years, numerous groups have reported the intentional transfer of mosaic embryos in the absence of uniformly euploid embryos, largely observing births of seemingly healthy babies. However, it remains to be understood whether the embryonic mosaicism invariably becomes resolved during the ensuing pregnancy, or whether the placenta and/or fetal tissues retain aneuploid cells, and if so to what potential clinical effect. Here, we report two cases of mosaicism persisting from the embryonic stage to the established pregnancy. Case 1 involved an embryonic low-level segmental mosaic loss in Chromosome (Chr) 1, which was confirmed in amniocentesis as well as in brain tissue of the products of conception. This pregnancy was terminated due to the chromosomal pathologies associated with 1p36 deletion syndrome, such as severe intellectual disability. Case 2 involved a low-level mosaic Chr 21 trisomy, which was confirmed with chorionic villus sampling and amniocentesis. The ensuing pregnancy was terminated after ultrasound identification of severe abnormalities in the placenta and fetus. Together, these two cases should be taken into account for risk-benefit assessments of prospective mosaic embryo transfers.

Chromosomal mosaicism, or the co-existence of cells with different chromosomal content, is a phenomenon that has been documented in human embryos for three decades. Early versions of preimplantation genetic testing (PGT-A) did not measure mosaicism, either because typically only a single cell was assessed, or because the technique could not accurately identify it. While this led to a straightforward diagnosis (an embryo was considered either normal or abnormal), it simply avoided the matter and in hindsight may have led to numerous misdiagnoses with negative clinical consequences. Modern PGT-A evaluates a multicellular biopsy with technologies capable of recognizing intermediate copy number signals for chromosomes or sub-chromosomal regions. We are therefore inevitably confronted with the issue of mosaicism and the challenge of managing embryos producing such results in the clinic. Here we discuss recent data showing that not only mosaicism in general, but specific features of mosaicism detected with PGT-A are associated with variable clinical outcomes. The conclusion is evident: mosaicism should be considered for more informed and improved embryo selection in the clinic.

The spread of SARS-CoV-2 has led to a devastating pandemic, with infections resulting in a range of symptoms collectively known as COVID-19. The full repertoire of human tissues and organs susceptible to infection is an area of active investigation, and some studies have implicated the reproductive system. The effects of COVID-19 on human reproduction remain poorly understood, and particularly the impact on early embryogenesis and establishment of a pregnancy are not known. In this work, we explore the susceptibility of early human embryos to SARS-CoV-2 infection. By using RNA-seq and immunofluorescence, we note that ACE2 and TMPRSS2, two canonical cell entry factors for SARS-CoV-2, are co-expressed in cells of the trophectoderm in blastocyst-stage preimplantation embryos. For the purpose of viral entry studies, we used fluorescent reporter virions pseudotyped with Spike (S) glycoprotein from SARS-CoV-2, and we observe robust infection of trophectoderm cells. This permissiveness could be attenuated with blocking antibodies targeting S or ACE2. When exposing human blastocysts to the live, fully infectious SARS-CoV-2, we detected cases of infection that compromised embryo health. Therefore, we identify a new human target tissue for SARS-CoV-2 with potential medical implications for reproductive health during the COVID-19 pandemic and its aftermath.

Extra or missing chromosomes—a phenomenon termed aneuploidy—frequently arises during human meiosis and embryonic mitosis and is the leading cause of pregnancy loss, including in the context of in vitro fertilization (IVF). While meiotic aneuploidies affect all cells and are deleterious, mitotic errors generate mosaicism, which may be compatible with healthy live birth. Large-scale abnormalities such as triploidy and haploidy also contribute to adverse pregnancy outcomes, but remain hidden from standard sequencing-based approaches to preimplantation genetic testing (PGT-A). The ability to reliably distinguish meiotic and mitotic aneuploidies, as well as abnormalities in genome-wide ploidy may thus prove valuable for enhancing IVF outcomes. Here, we describe a statistical method for distinguishing these forms of aneuploidy based on analysis of low-coverage whole-genome sequencing data, which is the current standard in the field. Our approach overcomes the sparse nature of the data by leveraging allele frequencies and linkage disequilibrium (LD) measured in a population reference panel. The method, which we term LD-informed PGT-A (LD-PGTA), retains high accuracy down to coverage as low as 0.05× and at higher coverage can also distinguish between meiosis I and meiosis II errors based on signatures spanning the centromeres. LD-PGTA provides fundamental insight into the origins of human chromosome abnormalities, as well as a practical tool with the potential to improve genetic testing during IVF.Significance StatementWhole chromosome gains and losses—termed aneuploidies—are the leading cause of human pregnancy loss and congenital disorders. Recent work has demonstrated that in addition to harmful meiotic aneuploidies, mitotic aneuploidies (which lead to mosaic embryos harboring cells with different numbers of chromosomes) may also be common in preimplantation embryos but potentially compatible with healthy birth. Here we developed and tested a method for distinguishing these forms of aneuploidy using genetic testing data from 8154 IVF embryos. We re-classified embryos based on signatures of meiotic and mitotic error, while also revealing lethal forms of chromosome abnormality that were hidden to existing approaches. Our method complements standard protocols for preimplantation and prenatal genetic testing, while offering insight into the biology of early development.

Study question To explore the effect of chromosomal mosaicism detected in preimplantation genetic testing (PGT-A) on prenatal and postnatal outcome of mosaic embryo pregnancies Summary answer No significant difference between euploid and mosaic embryos was observed in terms of weeks of gestation, average weight, and developmental defect of the babies born What is known already Mosaic embryos have the potential to implant and develop into healthy babies. Transfer of these embryos is now offered as an option for women who undergo IVF resulting in no euploid embryos. While, prenatal diagnosis has shown the depletion of chromosomal mosaicism in mosaic embryos, several concerns remain. For instance, the direct effects of different kind of mosaicism on prenatal/postnatal outcome and the possibility that intra-biopsy mosaicism in the TE is a poor predictor of the ploidy status of the ICM. Thus, there is certainly a need for comprehensive analyses of obstetrical and neonatal outcome data of transferred mosaic embryos. Study design, size, duration Compiled analysis from multicenter data on transfers of mosaic embryos (n = 1,000) and their outcome, with comparison to a euploid control group (n = 5,561). To explore the effect of embryonic mosaicism on newborns, we matched mosaic embryos resulting in a birth with a euploid embryo by a series of parameters (maternal age, embryo morphology, and indication for PGT-A). Prenatal tests and birth characteristics of > 200 neonates from mosaic embryo transfers were compared to > 200 euploid embryos. Participants/materials, setting, methods PGT-A was performed on blastocyst-stage embryos with 24-Chromosome whole genome amplification (WGA)-based Next Generation Sequencing (NGS). In accordance with established guidelines, embryos were categorized as mosaic when PGT-A results indicated 20-80% aneuploid content. Prenatal testing where performed in 30% of pregnancies with amniocentesis, 4% did an extra analysis for potential UPD for the suspected mosaic chromosome, and an additional 16% performed chorionic villus sampling (CVS) and 9.5% performed noninvasive prenatal testing (NIPT). Main results and the role of chance Of the 465 mosaic embryos that implanted, about 20% miscarried, and out of those, 75% were early spontaneous abortions. Of the pregnancies, 3 out of 368 were stillborn (2 out of them were twins that were extremely premature at 23 weeks, and the other died during pregnancy from a heart defect). The remaining 99% of those have been born or are late ongoing pregnancies at the time of analysis. Prenatal tests were performed in > 200 pregnancies and the vast majority tested normal. All 5 abnormal cases were amniocentesis tests showing microdeletions or insertions of sizes smaller than the resolution used during PGT-A, so they were unrelated to the mosaicism detected with PGT-A. In fact, in none of the cases did the prenatal test reflect the mosaicism detected at the embryonic stage. Matching each of the 162 mosaic embryos resulting in a birth with a euploid embryo, we found that the length of gestation was similar on average, and so was the average weight of the babies at birth. We also gathered information on the routine physical examination performed on babies at birth, and of those 162 babies from mosaic embryo transfers, none had obvious developmental defects or gross abnormalities. Limitations, reasons for caution Even though newborns resulting from mosaic embryo transfers in this study invariably appeared healthy by routine examination, concerns for long-term health cannot yet be entirely dispelled. The question must therefore be carefully considered by each clinic and patient situation. Wider implications of the findings Prenatal testing of > 200 pregnancies from mosaic embryo transfers showed no incidence of mosaicism that matched the PGT-A findings, indicating the involvement of self-corrective mechanisms. Pregnancy and obstetric data indicates that mosaic embryos prevailing through gestation and birth have similar chromosomal and physiological health compared to euploid embryos. Trial registration number none

ProblemHow do we manage treatment and stabilization in clinical settings and static population communities like assisted living facility settings of their patient or resident populations during and post the SARS-CoV-2 pandemic?ScopeThis proposal explores the possible and predicted changes to standard operating procedures to the facility management and associated landscape and focuses on series of deployments, during and post peak SARS-CoV-2 activity, and will outline possible models for the current medical facility model that we operate with. This article primarily focuses on non-emergency facility management.Assumptions and understanding of the fieldWith a reduction in the numbers nationally, patients are highly motivated and likely to seek non-emergency and planned medical procedural treatment as early as possible as social distancing measures are eased and restrictions on non-urgent procedures are lifted.Conclusions and next stepsAn initial pan-national shutdown and suspension of services was necessary in an effort to ensure that essential medical services and resources were not strained. The authors feel that a strategic resumption of regular non-emergency treatments around the United States and continued provision of services at care facilities is possible with innovative testing strategies like pooled screening of large populations at a manageable price point. Moreover, pooling as a strategy when used widely, would be extremely effective at predicting outbreaks of the virus and as an effect help in mitigating the spread of the virus in its “second-wave”. We have developed one such innovative pooling strategy that can be easily deployed across laboratories and reduce the cost of population wide COVID-19 testing significantly

To study how the attributes of mosaicism identified during preimplantation genetic testing for aneuploidy relate to clinical outcomes, in order to formulate a ranking system of mosaic embryos for intrauterine transfer.Compiled analysis.Multi-center.A total of 5,561 euploid blastocysts and 1,000 mosaic blastocysts used in clinical transfers in patients undergoing fertility treatment.None.Implantation (gestational sac), ongoing pregnancy, birth, and spontaneous abortion (miscarriage before 20 weeks of gestation).The euploid group had significantly more favorable rates of implantation and ongoing pregnancy/birth (OP/B) compared with the combined mosaic group or the mosaic group affecting only whole chromosomes (implantation: 57.2% vs. 46.5% vs. 41.8%; OP/B: 52.3% vs. 37.0% vs. 31.3%), as well as lower likelihood of spontaneous abortion (8.6% vs. 20.4% vs. 25%). Whole-chromosome mosaic embryos with level (percent aneuploid cells)50% had significantly more favorable outcomes than the ≥50% group (implantation: 44.5% vs. 30.4%; OP/B: 36.1% vs. 19.3%). Mosaic type (nature of the aneuploidy implicated in mosaicism) affected outcomes, with a significant correlation between number of affected chromosomes and unfavorable outcomes. This ranged from mosaicism involving segmental abnormalities to complex aneuploidies affecting three or more chromosomes (implantation: 51.6% vs. 30.4%; OP/B: 43.1% vs. 20.8%). Combining mosaic level, type, and embryo morphology revealed the order of subcategories regarding likelihood of positive outcome.This compiled analysis revealed traits of mosaicism identified with preimplantation genetic testing for aneuploidy that affected outcomes in a statistically significant manner, enabling the formulation of an evidence-based prioritization scheme for mosaic embryos in the clinic.

There is a high incidence of chromosomal abnormalities in early human embryos, whether they are generated by natural conception or by assisted reproductive technologies (ART). Cells with chromosomal copy number deviations or chromosome structural rearrangements can compromise the viability of embryos; much of the naturally low human fecundity as well as low success rates of ART can be ascribed to these cytogenetic defects. Chromosomal anomalies are also responsible for a large proportion of miscarriages and congenital disorders. There is therefore tremendous value in methods that identify embryos containing chromosomal abnormalities before intrauterine transfer to a patient being treated for infertility—the goal being the exclusion of affected embryos in order to improve clinical outcomes. This is the rationale behind preimplantation genetic testing for aneuploidy (PGT-A) and structural rearrangements (-SR). Contemporary methods are capable of much more than detecting whole chromosome abnormalities (e.g., monosomy/trisomy). Technical enhancements and increased resolution and sensitivity permit the identification of chromosomal mosaicism (embryos containing a mix of normal and abnormal cells), as well as the detection of sub-chromosomal abnormalities such as segmental deletions and duplications. Earlier approaches to screening for chromosomal abnormalities yielded a binary result of normal versus abnormal, but the new refinements in the system call for new categories, each with specific clinical outcomes and nuances for clinical management. This review intends to give an overview of PGT-A and -SR, emphasizing recent advances and areas of active development.

Behind every successful IVF embryo transfer, there is a great game of chance. Methods seeking to tilt the balance and increase the likelihood of implantation have been proposed and implemented with varying results, including embryo morphology, FISH-PGS, comprehensive chromosomal screening (CCS), morphokinetics, endometrial receptivity testing. It has been suggested that mitochondrial DNA (mtDNA) copy number could serve as a biomarker for embryo viability, but this concept was recently challenged. The world of IVF is left with unanswered questions: Why are there discrepancies in the reports? Should mtDNA copy number be considered to rank embryos for transfer? And in a broader sense, how well must a technique be validated before its implementation in the IVF clinic? Here, we explore these questions attempting to piece together the published data and suggest future directions to help unravel the subject matter.

Germline genome editing (GGE) holds the potential to mitigate or even eliminate human heritable genetic disease, but also carries genuine risks if not appropriately regulated and performed. It also raises fears in some quarters of apocalyptic scenarios of designer babies that could radically change human reproduction. Clinical need and the availability of alternatives are key considerations in the ensuing ethical debate. Writing from the perspective of a fertility clinic, we offer a realistic projection of the demand for GGE. We lay out a framework proposing that GGE, hereditary genetic disorders, and

Germline genome editing (GGE) holds the potential to mitigate or even eliminate human heritable genetic disease, but also carries genuine risks if not appropriately regulated and performed. It also raises fears in some quarters of apocalyptic scenarios of designer babies that could radically change human reproduction. Clinical need and the availability of alternatives are key considerations in the ensuing ethical debate. Writing from the perspective of a fertility clinic, we offer a realistic projection of the demand for GGE. We lay out a framework proposing that GGE, hereditary genetic disorders, and in vitro fertilization are fundamentally entwined concepts. We note that the need for GGE to cure heritable genetic disease is typically grossly overestimated, mainly due to the underappreciated role of preimplantation genetic testing. However, we might still find applications for GGE in the correction of chromosomal abnormalities in early embryos, but techniques for that purpose do not yet exist.

Research question Conflicting data exist on the utility of quantification of mitochondrial DNA (mtDNA) levels as a predictor of blastocyst implantation in the IVF clinic. The current study determined whether blastocysts with highly elevated mtDNA levels could result in healthy pregnancies and births, and whether mitochondrial functional output might be a readout of cell stress in the embryo. Design mtDNA levels were determined in 109 blastocysts used in clinical transfers into 100 women, noting their clinical outcomes. In a separate set of embryos, mitochondrial function was quantified in a model of embryo stress, aneuploidy. Measurement of mtDNA levels made use of surplus material from the process of preimplantation genetic testing for aneuploidies, and followed recently proposed unifying guidelines for mtDNA quantification. Results Unusually high mtDNA levels did not preclude blastocyst implantation and healthy births. An analysis of 109 blastocysts showed no significant difference between mtDNA levels in implanted (n = 55) versus non-implanted (n = 54) blastocysts. No obvious differences in the degree of mitochondrial functional output were detected in a model of embryo stress. Conclusions Measurement of mtDNA copy number might not provide any advantage in embryo prioritization and could lead to a deselection of blastocysts that would result in healthy pregnancies and births. Furthermore, the quantification of mitochondrial functional output in a model of cellular stress might suggest that mitochondria are not clear targets for biomarker identification as it relates to blastocyst viability. Any suggested link between mtDNA levels, mitochondria or their output with blastocyst transfer outcome requires further validation.

STUDY QUESTION\ud Is a clinical trophectoderm (TE) biopsy a suitable predictor of chromosomal aneuploidy in blastocysts?\ud \ud SUMMARY ANSWER\ud In the analyzed group of blastocysts, a clinical TE biopsy was an excellent representative of blastocyst karyotype in cases of whole chromosome aneuploidy, but in cases of only segmental (sub-chromosomal) aneuploidy, a TE biopsy was a poor representative of blastocyst karyotype.\ud \ud WHAT IS KNOWN ALREADY\ud Due to the phenomenon of chromosomal mosaicism, concern has been expressed about the possibility of discarding blastocysts classified as aneuploid by preimplantation genetic testing for aneuploidy (PGT-A) that in fact contain a euploid inner cell mass (ICM). Previously published studies investigating karyotype concordance between TE and ICM have examined small sample sizes and/or have utilized chromosomal analysis technologies superseded by Next Generation Sequencing (NGS). It is also known that blastocysts classified as mosaic by PGT-A can result in healthy births. TE re-biopsy of embryos classified as aneuploid can potentially uncover new instances of mosaicism, but the frequency of such blastocysts is currently unknown.\ud \ud STUDY DESIGN, SIZE, DURATION\ud For this study, 45 patients donated 100 blastocysts classified as uniform aneuploids (non-mosaic) using PGT-A by NGS (n = 93 whole chromosome aneuploids, n = 7 segmental aneuploids). In addition to the original clinical TE biopsy used for PGT-A, each blastocyst was subjected to an ICM biopsy as well as a second TE biopsy. All biopsies were processed for chromosomal analysis by NGS, and karyotypes were compared to the original TE biopsy.\ud \ud PARTICIPANTS/MATERIALS, SETTING, METHODS\ud The setting for this study was a single IVF center with an in-house PGT-A program and associated research laboratory.\ud \ud MAIN RESULTS AND THE ROLE OF CHANCE\ud When one or more whole chromosomes were aneuploid in the clinical TE biopsy, the corresponding ICM was aneuploid in 90 out of 93 blastocysts (96.8%). When the clinical TE biopsy contained only segmental (sub-chromosomal) aneuploidies, the ICM was aneuploid in three out of seven cases (42.9%). Blastocysts showing aneuploidy concordance between clinical TE biopsy and ICM were also aneuploid in a second TE biopsy in 86 out of 88 cases (97.7%). In blastocysts displaying clinical TE–ICM discordance, a second TE biopsy was aneuploid in only two out of six cases (33.3%).\ud \ud LIMITATIONS, REASONS FOR CAUTION\ud All embryos in this study had an initial classification of ‘aneuploid’ and not ‘euploid’ or ‘mosaic’. Therefore, the findings of this study refer specifically to a TE biopsy predicting aneuploidy in the remaining blastocyst, and cannot be extrapolated to deduce the ability of a TE biopsy to predict euploidy in the blastocyst. No conclusions should be drawn from this study about the ability of a mosaic TE biopsy to predict the karyotype of the corresponding blastocyst. Caution should be exercised in generalizing the findings of the sample group of this study to the general IVF blastocyst population. The segmental aneuploidy group only contained seven samples.\ud \ud WIDER IMPLICATIONS OF THE FINDINGS\ud The high rate of intra-blastocyst concordance observed in this study concerning whole chromosome aneuploidy contributes experimental evidence to the validation of PGT-A at the blastocyst stage. Concomitantly, the results suggest potential clinical value in reassessing blastocysts deemed aneuploid by TE re-biopsy in select cases, particularly in instances of segmental aneuploidies. This could impact infertility treatment for patients who only have blastocysts classified as aneuploid by PGT-A available.\ud \ud STUDY FUNDING/COMPETING INTEREST(S)\ud This study was supported by the Zouves Foundation for Reproductive Medicine and Zouves Fertility Center. The authors have no competing interest to disclose.

Objective: To determine whether human pre-implantation embryos have the potential to be infected by SARS-CoV-2, the virus responsible for COVID-19 Design: Assessment of expression levels of SARS-CoV-2 entry mediators in human embryo biopsies by RNAseq analysis, and infection of cultured embryos with SARS-CoV-2 Spike glycoprotein pseudotyped reporter virions expressing green fluorescent protein (GFP) Materials and Methods: Trophectoderm biopsies from blastocyst-stage embryos (n=24) were processed for RNAseq using a commercial kit and sequenced;results were analyzed for expression of factors implicated in SARS-CoV-2 cellular entry For viral infection experiments, blastocyst-stage embryos (n=94) were hatched from zonas mechanically, and infected by spinoculation with GFP-reporter virions pseudotyped with the SARS-CoV-2 Spike glycoprotein (required for SARS-CoV-2 entry) Embryos were subsequently monitored for fluorescence at 24-48 hours post-infection Various control conditions were used as specified in the ‘results’ section A mixed population of euploid, aneuploid, and untested embryos used in the study were from IVF treatment, donated to research by signed informed consent The project was approved by an Institutional Review Board Results: Cells collected from blastocyst-stage embryos robustly expressed the canonical SARS-CoV-2 entry receptor ACE2 and the putative activator protease TMPRSS2, in addition to other reported entry factors Embryos exposed to reporter virions pseudotyped with SARS-CoV-2 Spike glycoprotein displayed robust GFP signal, often in numerous cells with cytoplasmic localization Specificity was confirmed by the absences of fluorescence in embryos treated with virions lacking the Spike glycoprotein (“bald” virus), or when embryos were spinoculated with media alone in the absence of virus Embryos exposed to Spike glycoprotein-positive reporter virus in the presence of neutralizing anti Spike- or anti-ACE2-blocking antibodies exhibited negligible GFP signal, while control monoclonal IgG antibody-treated embryos maintained GFP expression These results implicated the canonical Spike-ACE2 axis in the viral entry Lastly, embryos exposed to reporter virions pseudotyped with Spike glycoprotein of SARS-CoV-1 (which also enters cells via ACE2) displayed GFP fluorescence, while embryos exposed to reporter viruses pseudotyped with Spike glycoprotein of MERS (which utilizes Dipeptidyl Peptidase IV (DPP4) instead of ACE2) resulted in no fluorescence Conclusions: Our results indicate that cells present in pre-implantation embryos are permissive to the canonical Spike-ACE2 viral entry mechanism utilized by SARS-CoV-2 These results encourage further investigation into the potential of SARS-CoV-2 infection in human embryos and may have wider implications in natural conception and ART practice

Objective\ud To investigate the parameters of mosaicism and the biological mechanisms leading to healthy pregnancies from mosaic embryo transfers.\ud \ud Design\ud Prospective study.\ud \ud Setting\ud IVF center and associated research laboratory.\ud \ud Patient(s)\ud Fifty-nine patients.\ud \ud Intervention(s)\ud Embryos underwent blastocyst-stage preimplantation genetic testing for aneuploidy by next-generation sequencing. Trophectoderm biopsies containing 20%–80% abnormal cells were deemed mosaic, and corresponding blastocysts were transferred. Mosaic embryos donated to research were examined for karyotype concordance in multiple biopsies and assessed for cell proliferation and death by immunofluorescence and computational quantitation.\ud \ud Main Outcome Measure(s)\ud Chemical start of pregnancy, implantation, fetal heartbeat, and birth.\ud \ud Result(s)\ud Globally, mosaic embryos showed inferior clinical outcomes compared with euploid embryos. Aneuploid cell percentage in trophectoderm biopsies did not correlate with outcomes, but type of mosaicism did, as embryos with single mosaic segmental aneuploidies fared better than all other types. Mosaic blastocysts generated from oocytes retrieved at young maternal ages (?34 years) showed better outcomes than those retrieved at older maternal ages. Mosaic embryos displayed low rates of karyotype concordance between multiple biopsies and showed significant elevation of cell proliferation and death compared with euploid embryos.\ud \ud Conclusion(s)\ud After euploid embryos, mosaic embryos can be considered for transfer, prioritizing those of the single segmental mosaic type. If a patient has mosaic embryos available that were generated at different ages, preference should be given to those made at younger ages. Intrablastocyst karyotype discordance and differential cell proliferation and death might be reasons that embryos classified as mosaic can result in healthy pregnancies and babies.

PURPOSE: To evaluate whether the mitoscore of cleavage stage embryos might correlate with developmental kinetics and the ploidy status. MATERIALS: This retrospective single-center study involved all cycles between April 2016 and April 2018 in which preimplantation genetic testing for aneuploidy (PGT-A) on day 3 was performed. The mitochondrial DNA (mtDNA) content and embryo ploidy were determined on 375 single blastomere biopsies by next generation sequencing (NGS). After intracytoplasmic sperm injection, a time-lapse imaging system (embryoscope) was used to follow the development. The median mtDNA content of cleavage stage embryos (49.4) was used to stratify the embryos into two groups to compare embryo development and ploidy status: low mitoscore group (≤ 49.4) and high mitoscore group (> 49.4). RESULTS: The total number of euploid embryos was equal between both mitoscore groups (32.1% versus 33.5%; p = 0.854). However, embryos in the low mitoscore group had a significantly higher cell number on day 3 (8.13 ± 1.59 versus 7.62 ± 1.5; p = 0.0013) and showed a significantly faster development up until the 8-cell stage. Mitoscore was not different between euploid and aneuploid embryos, with the same blastomere number at the time of biopsy. Furthermore, absence of cavitation within 118 h after insemination was correlated with higher mitoscore values (60.22 ± 42.23 versus 50.97 ± 13.37; p = 0.006) and a lower chance of being euploid (17.1% versus 47.4%; p = 0.001). CONCLUSION: mtDNA content of cleavage stage embryos correlates with time-lapse parameters. Early blastulation is correlated with a lower mtDNA content and a higher chance of euploidy.

Introduction Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, CFTR. An individual must inherit two defective cystic fibrosis alleles, one from each parent, to have the disease. Worldwide, over 70,000 individuals are affected by this monogenic disease. Preimplantation genetic testing (PGT) aims at selecting healthy embryos by testing for chromosome abnormalities and monogenic diseases. Often a limited number of cells or genetic material from the embryo is available to perform the detection of both aneuploidies and monogenic diseases with accuracy. To overcome this challenge, we developed a novel single tube assay enabling the simultaneous detection of both copy number variations (CNV) across the whole genome and single nucleotide variants (SNV) or small insertion/deletion (indels) located in the CFTR gene target region. Material and Methods The assay was performed using a modified SMARTer® PicoPLEX® Gold Single Cell DNA-Seq kit (Takara Bio USA, R300669). More specifically, the assay included simultaneous whole genome amplification (WGA) and targeted enrichment of selected regions of the CFTR gene. All 23 mutations of CFTR recommended to be tested by the American College of Medical Genetics (ACMG) were covered. As proof of principle, we prepared libraries from either single cells or five cells using model cell lines purchased from the Coriell Institute that contain different known CFTR variants, such as delF508, or aneuploidies of different sizes (2.5MB to 25 MB). In addition, libraries were made from trophectoderm biopsies isolated in the clinic from human blastocysts containing known CFTR mutations. The resulting libraries were sequenced on a MiSEQ to achieve 1M reads per sample (2 × 75 cycles). Results The percentage of sequencing reads allocated to both applications (CNV and SNV detections) was optimized to maximize the performance of the assay. To detect copy number variations, a shallow and even coverage of the genome is sufficient (∼0.025x). However, to detect variants, SNPs or small indels, the targeted regions of interest required a robust and deeper coverage (500x). At a sequencing depth of 1M reads, the coverage of the genome was ensured by 95% of the reads and the targeted regions of CFTR were covered by ∼5% of the reads. When using the five-cell model system, the 2.5 and 25MB chromosomal losses and the characterized heterozygous variants of CFTR were detected virtually 100% of the time (n of 18). When using single cells, we consistently detected the 25MB deletion (n of 18). Also, the expected heterozygous CFTR variants were detected over 80% of the time. Conclusion The sequencing reads allocated to both applications was optimized to maximize the performance of both CNV and SNV detection. Over 20 samples were successfully processed on a single MiSeq run. From 5 sorted cells our new approach provided representative and uniform coverage of the genome, suitable for detection of copy number variations (CNV). From the same assay, mutations contained in the CFTR gene were also accurately reported. Studies with TE biopsies with known CFTR mutations are now in progress and will be reported at the meeting.

Introduction Mitochondrial DNA (mtDNA) copy number has been proposed as a biomarker of implantation in IVF embryos. Some studies suggest that blastocysts containing disproportionately elevated levels of mtDNA per cell always fail to implant, and blastocysts implanting successfully have lower mtDNA levels on average ( Fragouli et al., 2015 , Diez-Juan et al., 2015 ). Other studies do not report such a trend, including one from our group ( Treff et al., 2017 , Victor et al., 2017 ). An explanation for the conflicting reports couldbe that the method for quantitation of mtDNA copy number varies across studies. As a result, technicalguidelines have been proposed to increase uniformity of mtDNA quantitation ( Wells, 2017 ). Here, weadhere to those guidelines in analyzing mtDNA copy number in a large number of blastocysts used fortransfer, to determine whether it is a valid predictor of implantation. Materials & Methods We quantified mtDNA levels in surplus product of the PGT-A process from blastocysts used in IVF withknown outcomes. In order to satisfy the suggested guidelines for mtDNA quantitation, we used qPCRmeasuring a locus in the mtDNA sequence and a multicopy locus in the nuclear DNA sequence, andcomputed the ratio between the two values. All molecular material was fresh and devoid of degradation. Results In our hands, unlike in previous reports, blastocysts with extremely high mtDNA levels successfully implanted and led to healthy births. If using mtDNA copy number to deselect embryos, these samples would not have been chosen for transfer. In addition, analysis of >100 blastocysts showed a statistically insignificant difference between mtDNA levels in implanted versus non-implanted blastocysts. Conclusions These findings suggest that in a single-clinic setting, measurement of mtDNA copy number might not provide any advantage to embryo ranking, and might even lead to de-selection of blastocysts that result to healthy pregnancies. The validity of measurement of mtDNA copy number requires further investigation, given different findings in individual centers.

Introduction IVF clinics are routinely transferring embryos diagnosed through PGT-A as mosaic anueploid with greaterfrequency to patients who lack available euploid embryos. Nevertheless, there is a lack of evidencebasedguidelines that establish which characteristics and genetic abnormalities affect a mosaic embryo'sclinical predictive outcome. Materials & methods 80 mosaic embryos were transferred at a single IVF program from August of 2015 through February of 2018. All embryos were tested in-house by high resolution NGS on trophectoderm biopsies obtained from blastocysts at 5-7 days of development. Karyotype profiles were analyzed for characteristics predictive of improved clinical outcomes. This study followed PGDIS guidelines defining mosaic embryosas containing whole or segmental chromosomal gains or losses between 20% and 80%. Results Of the 80 transferred mosaic embryos, 41 (51.3%) established a chemical pregnancy, 25 (31.3%)developed an embryonic sac (implantation), and 20 (25.0%) developed a fetal heartbeat (FHB). Of these20, 11 resulted in a live birth and at the time of analysis 9 were ongoing pregnancies, meaning that nopost-FHB miscarriages had occurred. Embryos derived from oocytes younger than 35 years old had asignificantly higher implantation rate than embryos from oocytes 35 years or older (50.0% to 21.2%,n=28 and 52 respectively) as well as comparable implantation rates to euploid embryos from across allage groups (younger than 35 years, n=141: 51.1%; greater than 35 years, n=341: 48.4%). There was anoticeable difference in implantation rate between embryos with either a single chromosomal mosaicloss or gain (33.3%, n=45) and mosaic embryos with multiple chromosomal losses or gains (23.1%,n=26). Mosaic embryos displaying only a single segmental chromosomal abnormality had a higherchance of implanting and developing a FHB (n=26, 38.5%, 34.6%) than mosaic embryos with multiplesegmental abnormalities or whole chromosome gains or losses (n=52, 26.9%, 21.2%) and also had a farlower chance of losing a pregnancy after an initial positive beta (23.1% to 46.2%, n=13 and 26respectively). When the embryos were broken down by severity of mosaicism, we found that 20% to50% (n=36) severity mosaic embryos were significantly more likely to implant and develop a FHB thanthe more severe mosaic 50%-80% embryos (n=12). Additionally, mosaic embryos were divided up bynumber of estimated abnormal cell lines present in the trophectoderm biopsy. The implantation rate forembryos estimated with only one aneuploid cell population was significantly higher than those with two(n=12, 33.3% vs n=12, 8.3%). Lastly, embryos diagnosed with mosaic trisomies did not have a significantdifference in implantation or FHB development (n=30, 33.3%, 30.0%) from those with monosomies (n=28, 32.1%, 25.9%). Conclusions Embryos classified as mosaic are capable of producing healthy babies and should be considered for transfer when a euploid embryo is not available. When there are multiple mosaic embryos to select from for any given patient, our data suggests embryos diagnosed with a single segmental abnormality should be preferred, followed by those with less severe mosaicism as measured by number of affected chromosomes and severity of aneuploidy in the mosaic mix.

Objectives: Sepsis is associated with cardiac contractile dysfunction attributed to alterations in Ca2+ handling. We examined the subcellular mechanisms involved in sarcoplasmic reticulum Ca2+ loss that mediate altered Ca2+ handling and contractile dysfunction associated with sepsis. Design: Randomized controlled trial. Setting: Research laboratory Subjects: Male wild type and transgenic mice Interventions: We induced sepsis in mice using the colon ascendens stent peritonitis model. Measurements and Main Results: Twenty-four hours after colon ascendens stent peritonitis surgery, we observed that wild type mice had significantly elevated proinflammatory cytokine levels, reduced ejection fraction, and fractional shortening (ejection fraction %, 54.76 ± 0.67; fractional shortening %, 27.53 ± 0.50) compared with sham controls (ejection fraction %, 73.57 ± 0.20; fractional shortening %, 46.75 ± 0.38). At the cardiac myocyte level, colon ascendens stent peritonitis cells showed reduced cell shortening, Ca2+ transient amplitude and sarcoplasmic reticulum Ca2+ content compared with sham cardiomyocytes. Colon ascendens stent peritonitis hearts showed a significant increase in oxidation-dependent calcium and calmodulin-dependent protein kinase II activity, which could be prevented by pretreating animals with the antioxidant tempol. Pharmacologic inhibition of calcium and calmodulin-dependent protein kinase II with 2.5 μM of KN93 prevented the decrease in cell shortening, Ca2+ transient amplitude, and sarcoplasmic reticulum Ca2+ content in colon ascendens stent peritonitis myocytes. Contractile function was also preserved in colon ascendens stent peritonitis myocytes isolated from transgenic mice expressing a calcium and calmodulin-dependent protein kinase II inhibitory peptide (AC3-I) and in colon ascendens stent peritonitis myocytes isolated from mutant mice that have the ryanodine receptor 2 calcium and calmodulin-dependent protein kinase II-dependent phosphorylation site (serine 2814) mutated to alanine (S2814A). Furthermore, colon ascendens stent peritonitis S2814A mice showed preserved ejection fraction and fractional shortening (ejection fraction %, 73.06 ± 6.31; fractional shortening %, 42.33 ± 5.70) compared with sham S2814A mice (ejection fraction %, 71.60 ± 4.02; fractional shortening %, 39.63 ± 3.23). Conclusions: Results indicate that oxidation and subsequent activation of calcium and calmodulin-dependent protein kinase II has a causal role in the contractile dysfunction associated with sepsis. Calcium and calmodulin-dependent protein kinase II, through phosphorylation of the ryanodine receptor would lead to Ca2+ leak from the sarcoplasmic reticulum, reducing sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude and contractility. Development of organ-specific calcium and calmodulin-dependent protein kinase II inhibitors may result in a beneficial therapeutic strategy to ameliorate contractile dysfunction associated with sepsis. Fil: Sepúlveda, Marisa Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentina Fil: Gonano, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentina Fil: Viotti, Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentina Fil: Morell, Malena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentina Fil: Blanco, Paula Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentina Fil: López Alarcón, Maria Micaela. Universidade Federal do Rio de Janeiro; Brasil Fil: Peroba Ramos, Isalira. Universidade Federal do Rio de Janeiro; Brasil Fil: Bastos Carvalho, Adriana. Universidade Federal do Rio de Janeiro; Brasil Fil: Medei, Emiliano. Universidade Federal do Rio de Janeiro; Brasil Fil: Vila Petroff, Martin Gerarde. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentina

A central unresolved question in the molecular cascade that drives establishment of left–right (LR) asymmetry in vertebrates are the mechanisms deployed to relay information between the midline site of symmetry-breaking and the tissues which will execute a program of asymmetric morphogenesis. The cells located between these two distant locations must provide the medium for signal relay. Of these, the gut endoderm is an attractive candidate tissue for signal transmission since it comprises the epithelium that lies between the node, where asymmetry originates, and the lateral plate, where asymmetry can first be detected. Here, focusing on the mouse as a model, we review our current understanding and entertain open questions concerning the relay of LR information from its origin. genesis 52:503–514, 2014.

Guanine-rich nucleic acids are known to form highly stable G-quadruplex structures, also known as G-quartets. Recently, there has been a tremendous amount of interest in studying G-quadruplexes owing to the realization of their biological importance. G-rich sequences (GRSs) capable of forming G-quadruplexes are found in the vicinity of polyadenylation regions and are involved in regulating 3' end processing of mammalian pre-mRNAs. G-rich motifs are also known to play an important role in alternative, tissue-specific splicing by interacting with hnRNP H protein subfamily. Whether quadruplex structure directly plays a role in regulating RNA processing events requires further investigation. To date there has not been a comprehensive effort to study G-quadruplexes near RNA processing sites. We have applied a computational approach to map putative Quadruplex forming GRSs within the transcribed regions of a large number of alternatively processed human and mouse gene sequences that were obtained as fully annotated entries from GenBank and RefSeq. We have used the computed data to build the GRSDB database that provides a unique avenue for studying G-quadruplexes in the context of RNA processing sites. GRSDB website offers visual comparison of G-quadruplex distribution patterns among all the alternative RNA products of a gene with the help of dynamic graphics. At present, GRSDB contains data from 1310 human and mouse genes, of which 1188 are alternatively processed. It has a total of 379 223 predicted G-quadruplexes, of which 54 252 are near RNA processing sites. GRSDB is a good resource for researchers interested in investigating the functional relevance of G-quadruplexes, especially in the context of alternative RNA processing. It can be accessed at http://bioinformatics.ramapo.edu/grsdb/.

Gastrulation leads to three germ layers--ectoderm, mesoderm and endoderm--that are separated by two basement membranes. In the mouse embryo, the emergent gut endoderm results from the widespread intercalation of cells of two distinct origins: pluripotent epiblast-derived definitive endoderm (DE) and extra-embryonic visceral endoderm (VE). Here we image the trajectory of prospective DE cells before intercalating into the VE epithelium. We show that the transcription factor SOX17, which is activated in prospective DE cells before intercalation, is necessary for gut endoderm morphogenesis and the assembly of the basement membrane that separates gut endoderm from mesoderm. Our results mechanistically link gut endoderm morphogenesis and germ layer segregation, two central and conserved features of gastrulation.

A central unresolved question in the molecular cascade that drives establishment of left–right (LR) asymmetry in vertebrates are the mechanisms deployed to relay information between the midline site of symmetry-breaking and the tissues which will execute a program of asymmetric morphogenesis. The cells located between these two distant locations must provide the medium for signal relay. Of these, the gut endoderm is an attractive candidate tissue for signal transmission since it comprises the epithelium that lies between the node, where asymmetry originates, and the lateral plate, where asymmetry can first be detected. Here, focusing on the mouse as a model, we review our current understanding and entertain open questions concerning the relay of LR information from its origin. genesis 52:503–514, 2014.

Analysis of Sox17 mutant mice reveals that gap junction coupling across the gut endoderm of the embryo transmits the left-right asymmetric signal from the node to the site of asymmetric organogenesis in mice., Establishment of left-right (LR) asymmetry occurs after gastrulation commences and utilizes a conserved cascade of events. In the mouse, LR symmetry is broken at a midline structure, the node, and involves signal relay to the lateral plate, where it results in asymmetric organ morphogenesis. How information transmits from the node to the distantly situated lateral plate remains unclear. Noting that embryos lacking Sox17 exhibit defects in both gut endoderm formation and LR patterning, we investigated a potential connection between these two processes. We observed an endoderm-specific absence of the critical gap junction component, Connexin43 (Cx43), in Sox17 mutants. Iontophoretic dye injection experiments revealed planar gap junction coupling across the gut endoderm in wild-type but not Sox17 mutant embryos. They also revealed uncoupling of left and right sides of the gut endoderm in an isolated domain of gap junction intercellular communication at the midline, which in principle could function as a barrier to communication between the left and right sides of the embryo. The role for gap junction communication in LR patterning was confirmed by pharmacological inhibition, which molecularly recapitulated the mutant phenotype. Collectively, our data demonstrate that Cx43-mediated communication across gap junctions within the gut endoderm serves as a mechanism for information relay between node and lateral plate in a process that is critical for the establishment of LR asymmetry in mice., Author Summary Superficially, humans, like other vertebrates, are bilaterally symmetrical. Nonetheless, the internal configuration of visceral organs reveals a stereotypical asymmetry. For example, human hearts are generally located on the left and the liver on the right side within the body cavity. How this left-right asymmetry is established is an area of interest, for both intrinsic biological significance and its medical application. In the mouse, the initial event that breaks left-right symmetry occurs at the node, a specialized organ located in the midline of the developing embryo. Somehow this initial asymmetry leads to a cascade of events that results in the activation of a genetic circuit on the left side of the embryo, which then leads to asymmetric organ formation. Here we show that the laterality information that is generated at the node is transferred to the lateral extremity of the embryo across the gut endoderm, which is the precursor tissue of the respiratory and digestive tracts and associated organs such as lungs, liver, and pancreas. Sox17 mutant mouse embryos exhibit defects in gut endoderm formation and fail to establish left-right asymmetry. Analysis of the mutants reveals that gap junction coupling across the gut endoderm is the mechanism of left-right information relay from the midline site of symmetry breaking to the site of asymmetric organogenesis in mice.

SummaryThe cell movements underlying the morphogenesis of the embryonic endoderm, the tissue that will give rise to the respiratory and digestive tracts, are complex and not well understood. Using live imaging combined with genetic labeling, we investigated the cell behaviors and fate of the visceral endoderm during gut endoderm formation in the mouse gastrula. Contrary to the prevailing view, our data reveal no mass displacement of visceral endoderm to extraembryonic regions concomitant with the emergence of epiblast-derived definitive endoderm. Instead, we observed dispersal of the visceral endoderm epithelium and extensive mixing between cells of visceral endoderm and epiblast origin. Visceral endoderm cells remained associated with the epiblast and were incorporated into the early gut tube. Our findings suggest that the segregation of extraembryonic and embryonic tissues within the mammalian embryo is not as strict as believed and that a lineage previously defined as exclusively extraembryonic contributes cells to the embryo.

Despite the importance of the gut and its accessory organs, our understanding of early endoderm development is still incomplete. Traditionally, endoderm has been difficult to study because of its small size and relative fragility. However, recent advances in live cell imaging technologies have dramatically expanded our understanding of this tissue, adding a new appreciation for the complex molecular and morphogenetic processes that mediate gut formation. Several spatially and molecularly distinct subpopulations have been shown to exist within the endoderm before the onset of gastrulation. Here, we review findings that have uncovered complex cell movements within the endodermal layer, before and during gastrulation, leading to the conclusion that cells from primitive endoderm contribute descendants directly to gut.

The tumor suppressor Lkb1 is a regulator of cellular energy, proliferation, and polarity, yet the mechanism by which it controls tissue morphogenesis or cancer remains poorly defined. By culturing embryonic tissues from mice harboring a mutant Lkb1 that can be rapidly inhibited, we have previously shown that acute loss of Lkb1 results in destabilized tissue architecture and the formation of highly dynamic pancreatic cysts (Lo et al, JCB 2012). Remarkably, the pancreatic cysts evolve within a few days into precancerous-like lesions that we now demonstrate to have a transcriptional profile that is strikingly similar to those reported for mouse and human PanINs (Pancreatic Intraepithelial Neoplasias). Utilizing fluorescence activated cell sorting, we have begun to track gene expression signatures associated with canonical signaling pathways in both single cells and subsets of cells from the embryonic explants. Since our experimental system offers precise temporal control and accessibility to manipulation within a tissue context, we are able to gain unprecedented insight into the earliest events in pancreatic oncogenesis. Citation Format: Bryan Lo, Manuel Viotti, Geraldine Strasser, Ira Mellman. Inhibition of Lkb1 in cultured embryonic pancreas destabilizes tissue architecture to generate precancerous-like lesions. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A41.

Live imaging of genetically encoded fluorescent protein reporters is increasingly being used to investigate details of the cellular behaviors that underlie the large-scale tissue rearrangements that shape the embryo. However, the majority of mouse fluorescent reporter strains are based on the green fluorescent protein (GFP). Mouse reporter strains expressing fluorescent colors other than GFP are therefore valuable for co-visualization studies with GFP, where relative positioning and relationship between two different tissues or compartments within cells are being investigated. Here, we report the generation and characterization of a transgenic Afp::mCherry mouse strain in which cis-regulatory elements from the Alpha-fetoprotein (Afp) locus were used to drive expression of the monomeric Cherry red fluorescent protein. The Afp::mCherry transgene is based on and recapitulates reporter expression of a previously described Afp::GFP strain. However, we note that perdurance of mCherry protein is not as prolonged as GFP, making the Afp::mCherry line a more faithful reporter of endogenous Afp expression. Afp::mCherry transgenic mice expressed mCherry specifically in the visceral endoderm and its derivatives, including the visceral yolk sac, gut endoderm, fetal liver, and pancreas of the embryo. The Afp::mCherry reporter was also noted to be expressed in other documented sites of Afp expression including hepatocytes as well as in pancreas, digestive tract, and brain of postnatal mice.