Conditioned place avoidance is associated with a distinct hippocampal phenotype, partly preserved pattern separation, and reduced reactive oxygen species production after stress.

Stress is associated with contextual memory deficits, which may mediate avoidance of trauma-associated contexts in posttraumatic stress disorder. These deficits may emerge from impaired pattern separation, the independent representation of similar experiences by the dentate gyrus-Cornu Ammonis 3 (DG-CA3) circuit of the dorsal hippocampus, which allows for appropriate behavioral responses to specific environmental stimuli. Neurogenesis in the DG is controlled by mitochondrial reactive oxygen species (ROS) production, and may contribute to pattern separation. In Experiment 1, we performed RNA sequencing of the dorsal hippocampus 16 days after stress in rats that either develop conditioned place avoidance to a predator urine-associated context (Avoiders), or do not (Non-Avoiders). Weighted genome correlational network analysis showed that increased expression of oxidative phosphorylation-associated gene transcripts and decreased expression of gene transcripts for axon guidance and insulin signaling were associated with avoidance behavior. Based on these data, in Experiment 2, we hypothesized that Avoiders would exhibit elevated hippocampal (HPC) ROS production and degraded object pattern separation (OPS) compared with Nonavoiders. Stress impaired pattern separation performance in Non-Avoider and Avoider rats compared with nonstressed Controls, but surprisingly, Avoiders exhibited partly preserved pattern separation performance and significantly lower ROS production compared with Non-Avoiders. Lower ROS production was associated with better OPS performance in Stressed rats, but ROS production was not associated with OPS performance in Controls. These results suggest a strong negative association between HPC ROS production and pattern separation after stress, and that stress effects on these outcome variables may be associated with avoidance of a stress-paired context.

Novel insights into reproductive ageing and menopause from genomics.

The post-reproductive phase or menopause in females is triggered by a physiological timer that depends on a threshold of follicle number in the ovary. Curiously, reproductive senescence appears to be decoupled from chronological age and is instead thought to be a function of physiological ageing. Ovarian ageing is associated with a decrease in oocyte developmental competence, attributed to a concomitant increase in meiotic errors. Although many biological hallmarks of general ageing are well characterized, the precise mechanisms underlying the programmed ageing of the female reproductive system remain elusive. In particular, the molecular pathways linking the external menopause trigger to the internal oocyte chromosome segregation machinery that controls fertility outcomes is unclear. However, recent large scale genomics studies have begun to provide insights into this process. Next-generation sequencing integrated with systems biology offers the advantage of sampling large datasets to uncover molecular pathways associated with a phenotype such as ageing. In this mini-review, we discuss findings from these studies that are crucial for advancing female reproductive senescence research. Targets identified in these studies can inform future animal models for menopause. We present three potential hypotheses for how external pathways governing ovarian ageing can influence meiotic chromosome segregation, with evidence from both animal models and molecular targets revealed from genomics studies. Although still in incipient stages, we discuss the potential of genomics studies combined with epigenetic age acceleration models for providing a predictive toolkit of biomarkers controlling menopause onset in women. We also speculate on future research directions to investigate extending female reproductive lifespan, such as comparative genomics in model systems that lack menopause. Novel genomics insights from such organisms are predicted to provide clues to preserving female fertility.

Epigenetic, genetic and maternal effects enable stable centromere inheritance.

Centromeres are defined epigenetically by the histone H3 variant CENP-A. The propagation cycle by which pre-existing CENP-A nucleosomes serve as templates for nascent assembly predicts the epigenetic memory of weakened centromeres. Using a mouse model with reduced levels of CENP-A nucleosomes, we find that an embryonic plastic phase precedes epigenetic memory through development. During this phase, nascent CENP-A nucleosome assembly depends on the maternal Cenpa genotype rather than the pre-existing template. Weakened centromeres are thus limited to a single generation, and parental epigenetic differences are eliminated by equal assembly on maternal and paternal centromeres. These differences persist, however, when the underlying DNA of parental centromeres differs in repeat abundance, as assembly during the plastic phase also depends on sufficient repetitive centromere DNA. With contributions of centromere DNA and the Cenpa maternal effect, we propose that centromere inheritance naturally minimizes fitness costs associated with weakened centromeres or epigenetic differences between parents.

Pharmahuasca and DMT Rescue ROS Production and Differentially Expressed Genes Observed after Predator and Psychosocial Stress: Relevance to Human PTSD.

Post-traumatic stress disorder (PTSD) is associated with cognitive deficits, oxidative stress, and inflammation. Animal models have recapitulated features of PTSD, but no comparative RNA sequencing analysis of differentially expressed genes (DEGs) in the brain between PTSD and animal models of traumatic stress has been carried out. We compared DEGs from the prefrontal cortex (PFC) of an established stress model to DEGs from the dorsolateral PFC (dlPFC) of humans. We observed a significant enrichment of rat DEGs in human PTSD and identified 20 overlapping DEGs, of which 17 (85%) are directionally concordant. N,N-dimethyltryptamine (DMT) is a known indirect antioxidant, anti-inflammatory, and neuroprotective compound with antidepressant and plasticity-facilitating effects. We tested the capacity of DMT, the monoamine oxidase inhibitor (MAOI) harmaline, and "pharmahuasca" (DMT + harmaline) to reduce reactive oxygen species (ROS) production and inflammatory gene expression and to modulate neuroplasticity-related gene expression in the model. We administered DMT (2 mg/kg IP), harmaline (1.5 mg/kg IP), pharmahuasca, or vehicle every other day for 5 days, following a 30 day stress regiment. We measured ROS production in the PFC and hippocampus (HC) by electron paramagnetic resonance spectroscopy and sequenced total mRNA in the PFC. We also performed in vitro assays to measure the affinity and efficacy of DMT and harmaline at 5HT2AR compared to 5-HT. DMT and pharmahuasca reduced ROS production in the PFC and HC, while harmaline had mixed effects. Treatments normalized 9, 12, and 14 overlapping DEGs, and pathway analysis implicated that genes were involved in ROS production, inflammation, growth factor signaling, neurotransmission, and neuroplasticity.

A Study on the Clustering of Extra Virgin Olive Oils Extracted from Cultivars Growing in Four Ionian Islands (Greece) by Multivariate Analysis of Their Phenolic Profile, Antioxidant Activity and Genetic Markers.

BACKGROUND: The phenolic fraction of extra virgin olive oil (EVOO) has disease preventive and health-promoting properties which are supported by numerous studies. As such, EVOO is defined as a functional food. The aim of the present study was to characterize the phenolic profile of olive oil from cultivars farmed in the Ionian Islands (Zakynthos, Kefalonia, Lefkada, and Kerkyra) and to investigate the association of phenols to antioxidant activity, which is central to its functionality. Furthermore, the study investigates whether multivariate analyses on the concentration of individual biophenolic compounds and genetic population diversity could classify the olive oil samples based on their geographic origin. METHODS: Phenols were determined in 103 samples from different Ionian Island tree populations by 1H nuclear magnetic resonance (NMR), and sample antioxidant activity was measured by their capacity to reduce the free radical 2,2-diphenyl-1-picrylhydrazyl) (DPPH). Genetic diversity was measured by estimating Nei's population genetic distance using 15 reproducible bands from random amplified polymorphic DNA (RAPD) genotyping. RESULTS: Principal component analysis (PCA) of the secoiridoid concentrations clustered samples according to cultivar. Clustering based on genetic distances is not concordant with phenolic clustering. A cultivar effect was also demonstrated in the association between the concentration of individual phenols with DPPH reducing activity. CONCLUSIONS: Taken together, the study shows that the olive oil phenolic content defines "cultivar-specific phenolic profiles" and that environmental factors other than agronomic conditions contribute more to phenotype variance than genetics.

Chondrocyte protein co-synthesis network analysis links ECM mechanosensing to metabolic adaptation in osteoarthritis.

BACKGROUND: Knee osteoarthritis (OA) is one of the most common structural OA disorders globally. Incomplete understanding of the fundamental biological aspects of osteoarthritis underlies the current lack of effective treatment or disease modifying drugs. RESEARCH DESIGN AND METHODS: We implemented a systems approach by making use of the statistical network concepts in Weighted Gene Co-expression Analysis to reconstruct the organization of the core proteome network in chondrocytes obtained from OA patients and healthy individuals. Protein modules reflect groups of tightly co-ordinated changes in protein abundance across healthy and OA chondrocytes. RESULTS: The unbiased systems analysis identified extracellular matrix (ECM) mechanosensing and glycolysis as two modules that are most highly correlated with ΟΑ. The ECM module was enriched in the OA genetic risk factors tenascin-C (TNC) and collagen 11A1 (COL11A1), as well as in cartilage oligomeric matrix protein (COMP), a biomarker associated with cartilage integrity. Mapping proteins that are unique to OA or healthy chondrocytes onto the core interactome, which connects microenvironment sensing and regulation of glycolysis, identified differences in metabolic and anti-inflammatory adaptation. CONCLUSION: The interconnection between cartilage ECM remodeling and metabolism is indicative of the dynamic chondrocyte states and their significance in osteoarthritis.

Identity-by-state-based haplotyping expands the application of comprehensive preimplantation genetic testing.

STUDY QUESTION: Is it possible to haplotype parents using parental siblings to leverage preimplantation genetic testing (PGT) for monogenic diseases and aneuploidy (comprehensive PGT) by genome-wide haplotyping? SUMMARY ANSWER: We imputed identity-by-state (IBS) sharing of parental siblings to phase parental genotypes. WHAT IS KNOWN ALREADY: Genome-wide haplotyping of preimplantation embryos is being implemented as a generic approach for genetic diagnosis of inherited single-gene disorders. To enable the phasing of genotypes into haplotypes, genotyping the direct family members of the prospective parent carrying the mutation is required. Current approaches require genotypes of either (i) both or one of the parents of the affected prospective parent or (ii) an affected or an unaffected child of the couple. However, this approach cannot be used when parents or children are not attainable, prompting an investigation into alternative phasing options. STUDY DESIGN, SIZE, DURATION: This is a retrospective validation study, which applied IBS-based phasing of parental haplotypes in 56 embryos derived from 12 PGT families. Genome-wide haplotypes and copy number profiles generated for each embryo using the new phasing approach were compared with the reference PGT method to evaluate the diagnostic concordance. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included 12 couples with a known hereditary genetic disorder, participating in the comprehensive PGT program and with at least one parental sibling available (e.g. brother and/or sister). Genotyping data from both prospective parents and the parental sibling(s) were used to perform IBS-based phasing and to trace the disease-associated alleles. The outcome of the IBS-based PGT was compared with the results of the clinically implemented reference haplotyping-based PGT method. MAIN RESULTS AND THE ROLE OF CHANCE: IBS-based haplotyping was performed for 12 PGT families. In accordance with the theoretical prediction of allele sharing between sibling pairs, 6 out of 12 (50%) couples or 23 out of 56 embryos could be phased using parental siblings. In families where phasing was possible, haplotype calling in the locus of interest was 100% concordant between the reference PGT method and IBS-based approach using parental siblings. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Phasing of parental haplotypes will only be possible when the disease locus lies in an informative region (categorized as IBS1). Phasing prospective parents using relatives with reduced genetic relatedness as a reference (e.g. siblings) decreases the size and the occurrence of informative IBS1 regions, necessary for haplotype calling. By including more than one extended family member, the chance of obtaining IBS1 coverage in the interrogated locus can be increased. A pre-PGT work-up can define whether the carrier couple could benefit from this approach. WIDER IMPLICATIONS OF THE FINDINGS: Phasing by relatives extends the potential of comprehensive PGT, since it allows the inclusion of couples who do not have access to the standard phasing references, such as parents or offspring. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the KU Leuven grant (C14/18/092), Research Foundation Flanders (FWO; GA09311N), Horizon 2020 innovation programme (WIDENLIFE, 692065) and Agilent Technologies. J.R.V., T.V. and M.Z.E. are co-inventors of a patent ZL910050-PCT/EP2011/060211-WO/2011/157846 'Methods for haplotyping single-cells' and ZL913096-PCT/EP2014/068315-WO/2015/028576 'Haplotyping and copy number typing using polymorphic variant allelic frequencies' licensed to Agilent Technologies. The other authors have no conflict of interest to declare.

Multi-centre evaluation of a comprehensive preimplantation genetic test through haplotyping-by-sequencing.

STUDY QUESTION: Can reduced representation genome sequencing offer an alternative to single nucleotide polymorphism (SNP) arrays as a generic and genome-wide approach for comprehensive preimplantation genetic testing for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR) in human embryo biopsy samples? SUMMARY ANSWER: Reduced representation genome sequencing, with OnePGT, offers a generic, next-generation sequencing-based approach for automated haplotyping and copy-number assessment, both combined or independently, in human single blastomere and trophectoderm samples. WHAT IS KNOWN ALREADY: Genome-wide haplotyping strategies, such as karyomapping and haplarithmisis, have paved the way for comprehensive PGT, i.e. leveraging PGT-M, PGT-A and PGT-SR in a single workflow. These methods are based upon SNP array technology. STUDY DESIGN, SIZE, DURATION: This multi-centre verification study evaluated the concordance of PGT results for a total of 225 embryos, including 189 originally tested for a monogenic disorder and 36 tested for a translocation. Concordance for whole chromosome aneuploidies was also evaluated where whole genome copy-number reference data were available. Data analysts were kept blind to the results from the reference PGT method. PARTICIPANTS/MATERIALS, SETTING, METHODS: Leftover blastomere/trophectoderm whole genome amplified (WGA) material was used, or secondary trophectoderm biopsies were WGA. A reduced representation library from WGA DNA together with bulk DNA from phasing references was processed across two study sites with the Agilent OnePGT solution. Libraries were sequenced on an Illumina NextSeq500 system, and data were analysed with Agilent Alissa OnePGT software. The embedded PGT-M pipeline utilises the principles of haplarithmisis to deduce haplotype inheritance whereas both the PGT-A and PGT-SR pipelines are based upon read-count analysis in order to evaluate embryonic ploidy. Concordance analysis was performed for both analysis strategies against the reference PGT method. MAIN RESULTS AND THE ROLE OF CHANCE: PGT-M analysis was performed on 189 samples. For nine samples, the data quality was too poor to analyse further, and for 20 samples, no result could be obtained mainly due to biological limitations of the haplotyping approach, such as co-localisation of meiotic crossover events and nullisomy for the chromosome of interest. For the remaining 160 samples, 100% concordance was obtained between OnePGT and the reference PGT-M method. Equally for PGT-SR, 100% concordance for all 36 embryos tested was demonstrated. Moreover, with embryos originally analysed for PGT-M or PGT-SR for which genome-wide copy-number reference data were available, 100% concordance was shown for whole chromosome copy-number calls (PGT-A). LIMITATIONS, REASONS FOR CAUTION: Inherent to haplotyping methodologies, processing of additional family members is still required. Biological limitations caused inconclusive results in 10% of cases. WIDER IMPLICATIONS OF THE FINDINGS: Employment of OnePGT for PGT-M, PGT-SR, PGT-A or combined as comprehensive PGT offers a scalable platform, which is inherently generic and thereby, eliminates the need for family-specific design and optimisation. It can be considered as both an improvement and complement to the current methodologies for PGT. STUDY FUNDING/COMPETING INTEREST(S): Agilent Technologies, the KU Leuven (C1/018 to J.R.V. and T.V.) and the Horizon 2020 WIDENLIFE (692065 to J.R.V. and T.V). H.M. is supported by the Research Foundation Flanders (FWO, 11A7119N). M.Z.E, J.R.V. and T.V. are co-inventors on patent applications: ZL910050-PCT/EP2011/060211- WO/2011/157846 'Methods for haplotyping single cells' and ZL913096-PCT/EP2014/068315 'Haplotyping and copy-number typing using polymorphic variant allelic frequencies'. T.V. and J.R.V. are co-inventors on patent application: ZL912076-PCT/EP2013/070858 'High-throughput genotyping by sequencing'. Haplarithmisis ('Haplotyping and copy-number typing using polymorphic variant allelic frequencies') has been licensed to Agilent Technologies. The following patents are pending for OnePGT: US2016275239, AU2014345516, CA2928013, CN105874081, EP3066213 and WO2015067796. OnePGT is a registered trademark. D.L., J.T. and R.L.R. report personal fees during the conduct of the study and outside the submitted work from Agilent Technologies. S.H. and K.O.F. report personal fees and other during the conduct of the study and outside the submitted work from Agilent Technologies. J.A. reports personal fees and other during the conduct of the study from Agilent Technologies and personal fees from Agilent Technologies and UZ Leuven outside the submitted work. B.D. reports grants from IWT/VLAIO, personal fees during the conduct of the study from Agilent Technologies and personal fees and other outside the submitted work from Agilent Technologies. In addition, B.D. has a patent 20160275239 - Genetic Analysis Method pending. The remaining authors have no conflicts of interest.

Genome-wide haplotyping embryos developing from 0PN and 1PN zygotes increases transferrable embryos in PGT-M.

STUDY QUESTION: Can genome-wide haplotyping increase success following preimplantation genetic testing for a monogenic disorder (PGT-M) by including zygotes with absence of pronuclei (0PN) or the presence of only one pronucleus (1PN)? SUMMARY ANSWER: Genome-wide haplotyping 0PNs and 1PNs increases the number of PGT-M cycles reaching embryo transfer (ET) by 81% and the live-birth rate by 75%. WHAT IS KNOWN ALREADY: Although a significant subset of 0PN and 1PN zygotes can develop into balanced, diploid and developmentally competent embryos, they are usually discarded because parental diploidy detection is not part of the routine work-up of PGT-M. STUDY DESIGN, SIZE, DURATION: This prospective cohort study evaluated the pronuclear number in 2229 zygotes from 2337 injected metaphase II (MII) oocytes in 268 cycles. PGT-M for 0PN and 1PN embryos developing into Day 5/6 blastocysts with adequate quality for vitrification was performed in 42 of the 268 cycles (15.7%). In these 42 cycles, we genome-wide haplotyped 216 good quality embryos corresponding to 49 0PNs, 15 1PNs and 152 2PNs. The reported outcomes include parental contribution to embryonic ploidy, embryonic aneuploidy, genetic diagnosis for the monogenic disorder, cycles reaching ETs, pregnancy and live birth rates (LBR) for unaffected offspring. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blastomere DNA was whole-genome amplified and hybridized on the Illumina Human CytoSNP12V2.1.1 BeadChip arrays. Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the embryonic genome architecture. Bi-parental, unaffected embryos were transferred regardless of their initial zygotic PN score. MAIN RESULTS AND THE ROLE OF CHANCE: A staggering 75.51% of 0PN and 42.86% of 1PN blastocysts are diploid bi-parental allowing accurate genetic diagnosis for the monogenic disorder. In total, 31% (13/42) of the PGT-M cycles reached ET or could repeat ET with an unaffected 0PN or 1PN embryo. The LBR per initiated cycle increased from 9.52 to 16.67%. LIMITATIONS, REASONS FOR CAUTION: The clinical efficacy of the routine inclusion of 0PN and 1PN zygotes in PGT-M cycles should be confirmed in larger cohorts from multicenter studies. WIDER IMPLICATIONS OF THE FINDINGS: Genome-wide haplotyping allows the inclusion of 0PN and 1PN embryos and subsequently increases the cycles reaching ET following PGT-M and potentially PGT for aneuploidy (PGT-A) and chromosomal structural rearrangements (PGT-SR). Establishing measures of clinical efficacy could lead to an update of the ESHRE guidelines which advise against the use of these zygotes. STUDY FUNDING/COMPETING INTEREST(S): SymBioSys (PFV/10/016 and C1/018 to J.R.V. and T.V.), the Horizon 2020 WIDENLIFE: 692065 to J.R.V., T.V., E.D., A.D. and M.Z.E. M.Z.E., T.V. and J.R.V. co-invented haplarithmisis ('Haplotyping and copy-number typing using polymorphic variant allelic frequencies'), which has been licensed to Agilent Technologies. H.M. is fully supported by the (FWO) (ZKD1543-ASP/16). The authors have no competing interests to declare.

Genome stability of bovine in vivo-conceived cleavage-stage embryos is higher compared to in vitro-produced embryos.

STUDY QUESTION: Is the rate and nature of chromosome instability (CIN) similar between bovine in vivo-derived and in vitro-cultured cleavage-stage embryos? SUMMARY ANSWER: There is a major difference regarding chromosome stability of in vivo-derived and in vitro-cultured embryos, as CIN is significantly lower in in vivo-derived cleavage-stage embryos compared to in vitro-cultured embryos. WHAT IS KNOWN ALREADY: CIN is common during in vitro embryogenesis and is associated with early embryonic loss in humans, but the stability of in vivo-conceived cleavage-stage embryos remains largely unknown. STUDY DESIGN, SIZE, DURATION: Because human in vivo preimplantation embryos are not accessible, bovine (Bos taurus) embryos were used to study CIN in vivo. Five young, healthy, cycling Holstein Friesian heifers were used to analyze single blastomeres of in vivo embryos, in vitro embryos produced by ovum pick up with ovarian stimulation (OPU-IVF), and in vitro embryos produced from in vitro matured oocytes retrieved without ovarian stimulation (IVM-IVF). PARTICIPANTS/MATERIALS, SETTING, METHODS: Single blastomeres were isolated from embryos, whole-genome amplified and hybridized on Illumina BovineHD BeadChip arrays together with the bulk DNA from the donor cows (mothers) and the bull (father). DNA was also obtained from the parents of the bull and from the parents of the cows (paternal and maternal grandparents, respectively). Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the genomic architecture of 171 single bovine blastomeres of 16 in vivo, 13 OPU-IVF and 13 IVM-IVF embryos. MAIN RESULTS AND THE ROLE OF CHANCE: The genomic stability of single blastomeres in both of the in vitro-cultured embryo cohorts was severely compromised (P < 0.0001), and the frequency of whole chromosome or segmental aberrations was higher in embryos produced in vitro than in embryos derived in vivo. Only 18.8% of in vivo-derived embryos contained at least one blastomere with chromosomal anomalies, compared to 69.2% of OPU-IVF embryos (P < 0.01) and 84.6% of IVM-IVF embryos (P < 0.001). LARGE SCALE DATA: Genotyping data obtained in this study has been submitted to NCBI Gene Expression Omnibus (GEO; accession number GSE95358). LIMITATIONS REASONS FOR CAUTION: There were two main limitations of the study. First, animal models may not always reflect the nature of human embryogenesis, although the use of an animal model to investigate CIN was unavoidable in our study. Second, a limited number of embryos were obtained, therefore more studies are warranted to corroborate the findings. WIDER IMPLICATIONS OF THE FINDINGS: Although CIN is also present in in vivo-developed embryos, in vitro procedures exacerbate chromosomal abnormalities during early embryo development. Hence, the present study highlights that IVF treatment compromises embryo viability and should be applied with care. Additionally, our results encourage to refine and improve in vitro culture conditions and assisted reproduction technologies. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Agency for Innovation by Science and Technology (IWT) (TBM-090878 to J.R.V. and T.V.), the Research Foundation Flanders (FWO; G.A093.11 N to T.V. and J.R.V. and G.0392.14 N to A.V.S. and J.R.V.), the European Union's FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, SARM, EU324509 to J.R.V., T.V., O.T, A.D., A.S. and A.K.) and Horizon 2020 innovation programme (WIDENLIFE, 692065 to J.R.V., O.T., T.V., A.K. and A.S.). M.Z.E., J.R.V. and T.V. are co-inventors on a patent application ZL913096-PCT/EP2014/068315-WO/2015/028576 ('Haplotyping and copy-number typing using polymorphic variant allelic frequencies'), licensed to Cartagenia (Agilent Technologies).

How can zygotes segregate entire parental genomes into distinct blastomeres? The zygote metaphase revisited.

Zygote cytokinesis produces two symmetric blastomeres, which contain one copy of each parental genome. Contrary to this dogma, we recently discovered that mammalian zygotes can spontaneously segregate entire parental genomes into different blastomeres and coined this novel form of genome segregation heterogoneic division. The molecular mechanisms underlying the emergence of blastomeres with different parental genomes during the first mitotic cycle remain to be elucidated. Here, we speculate on which parental genome asymmetries could provide a mechanistic foundation for these remarkable zygote divisions. In reviewing the field and considering our findings, we revisit the architecture of the first zygotic metaphase by invoking asymmetric interactions between the mitotic spindle and the parental kinetochores. We also speculate on how asynchronous parental cell cycles can be a source of heterogoneic zygote divisions through the formation of parental genome private spindles.

Zygotes segregate entire parental genomes in distinct blastomere lineages causing cleavage-stage chimerism and mixoploidy.

Dramatic genome dynamics, such as chromosome instability, contribute to the remarkable genomic heterogeneity among the blastomeres comprising a single embryo during human preimplantation development. This heterogeneity, when compatible with life, manifests as constitutional mosaicism, chimerism, and mixoploidy in live-born individuals. Chimerism and mixoploidy are defined by the presence of cell lineages with different parental genomes or different ploidy states in a single individual, respectively. Our knowledge of their mechanistic origin results from indirect observations, often when the cell lineages have been subject to rigorous selective pressure during development. Here, we applied haplarithmisis to infer the haplotypes and the copy number of parental genomes in 116 single blastomeres comprising entire preimplantation bovine embryos (n = 23) following in vitro fertilization. We not only demonstrate that chromosome instability is conserved between bovine and human cleavage embryos, but we also discovered that zygotes can spontaneously segregate entire parental genomes into different cell lineages during the first post-zygotic cleavage division. Parental genome segregation was not exclusively triggered by abnormal fertilizations leading to triploid zygotes, but also normally fertilized zygotes can spontaneously segregate entire parental genomes into different cell lineages during cleavage of the zygote. We coin the term "heterogoneic division" to indicate the events leading to noncanonical zygotic cytokinesis, segregating the parental genomes into distinct cell lineages. Persistence of those cell lines during development is a likely cause of chimerism and mixoploidy in mammals.

Complex preimplantation genetic diagnosis for beta-thalassaemia, sideroblastic anaemia, and human leukocyte antigen (HLA)-typing.

Preimplantation genetic diagnosis (PGD) to select histocompatible siblings to facilitate curative haematopoeitic stem-cell transplantation (HSCT) is now an acceptable option in the absence of an available human leukocyte antigen (HLA) compatible donor. We describe a case where the couple who requested HLA-PGD, were both carriers of two serious haematological diseases, beta-thalassaemia and sideroblastic anaemia. Their daughter, affected with sideroblastic anaemia, was programmed to have HSCT. A multiplex-fluorescent-touchdown-PCR protocol was optimized for the simultaneous amplification of: the two HBB-gene mutated regions (c.118C> T, c.25-26delAA), four short tandem repeats (STRs) in chr11p15.5 linked to the HBB gene, the SLC25A38 gene mutation (c.726C > T), two STRs in chr3p22.1 linked to the SLC25A38 gene, plus eleven informative STRs for HLA-haplotyping (chr6p22.1-21.3). This was followed by real-time nested PCR and high-resolution melting analysis (HRMA) for the detection of HBB and SLC25A38 gene mutations, as well as the analysis of all STRs on an automatic genetic analyzer (sequencer). The couple completed four clinical in vitro fertilization (IVF)/PGD cycles. At least one matched unaffected embryo was identified and transferred in each cycle. A twin pregnancy was established in the fourth PGD cycle and genotyping results at all loci were confirmed by prenatal diagnosis. Two healthy baby girls were delivered at week 38 of pregnancy. The need to exclude two familial disorders for HLA-PGD is rarely encountered. The methodological approach described here is fast, accurate, clinically-validated, and of relatively low cost.

Prenatal diagnosis for CF using High Resolution Melting Analysis and simultaneous haplotype analysis through QF-PCR.

BACKGROUND: High Resolution Melting (HRM) Analysis is a validated, robust, low-cost, high throughput CF screening method. Here, we report the development and retrospective evaluation of the diagnostic value of a novel multiplex HRM, genotyping and haplotyping method for CF prenatal diagnosis (generic HRM/haplotyping). METHODS: 80 study samples from 20 carrier couples referred for PND (whole blood in EDTA and CVS or amniotic fluid) were genotyped retrospectively using the suggested protocol. RESULTS: All DNA samples (variable sources, extraction methods and unknown concentrations) were successfully amplified by the 1st and 2nd round PCR. The Se, Sp, NPV and PPV for the generic HRM/haplotyping method are calculated at 100%. CONCLUSIONS: This generic protocol for PND using HRM, facilitates the simultaneous analysis of DNA samples from various sources in a fast, robust and efficient way. It can be easily adapted and applied for any genetic condition.

A generic, flexible protocol for preimplantation human leukocyte antigen typing alone or in combination with a monogenic disease, for rapid case work-up and application.

Human leukocyte antigen (HLA) typing of in vitro fertilization (IVF) embryos, aims to establish a pregnancy that is HLA compatible with an affected sibling who requires hematopoietic stem cell transplantation (HSCT). It can be performed with or without preimplantation genetic diagnosis (PGD) for exclusion of a single-gene disorder (SGD) and it is a multistep, technically challenging procedure at every stage. Our purpose was to address the difficulties of genetic analysis by developing a fast, reliable and accurate PGD-HLA protocol, to simplify patient work-up and PGD application, while providing high flexibility for combination with any SGD. Requests included PGD-HLA for ß-thalassemia (ß-thal)/sickle cell disease (most common request), Diamond-Blackfan anemia (DBA), chronic granulomatous disease (CGD) and preimplantation-HLA typing only. For HLA haplotyping, we selected a panel of 26 short tandem repeats (STRs) distributed across the entire HLA locus, following PGD guidelines. When required, mutation detection was performed by both a direct and indirect approach. To support concurrent SGD exclusion and HLA typing, a one-step, single-tube, multiplex fluorescent touchdown-polymerase chain reaction (PCR) was optimized. The described touchdown-PCR was successfully applied for all PGD-HLA protocols. Eight clinical cycles were performed with a diagnosis achieved for 94.7% of amplified biopsied blastomeres. Embryo transfer took place in six cycles, with two pregnancies achieved and two healthy female infants (from a twin pregnancy) born so far. Our protocol enables HLA typing in a single PCR, reducing the risk of contamination and the cost, and providing faster results. It requires minimum optimization before clinical application, irrespective of the SGD involved, decreasing the waiting time from referral to treatment for all PGD-HLA cases.

Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study.

Preimplantation genetic diagnosis (PGD) for monogenic disorders currently involves polymerase chain reaction (PCR)-based methods, which must be robust, sensitive and highly accurate, precluding misdiagnosis. Twelve adverse misdiagnoses reported to the ESHRE PGD-Consortium are likely an underestimate. This retrospective study, involving six PGD centres, assessed the validity of PCR-based PGD through reanalysis of untransferred embryos from monogenic-PGD cycles. Data were collected on the genotype concordance at PGD and follow-up from 940 untransferred embryos, including details on the parameters of PGD cycles: category of monogenic disease, embryo morphology, embryo biopsy and genotype assay strategy. To determine the validity of PCR-based PGD, the sensitivity (Se), specificity (Sp) and diagnostic accuracy were calculated. Stratified analyses were also conducted to assess the influence of the parameters above on the validity of PCR-based PGD. The analysis of overall data showed that 93.7% of embryos had been correctly classified at the time of PGD, with Se of 99.2% and Sp of 80.9%. The stratified analyses found that diagnostic accuracy is statistically significantly higher when PGD is performed on two cells versus one cell (P=0.001). Se was significantly higher when multiplex protocols versus singleplex protocols were applied (P=0.005), as well as for PGD applied on cells from good compared with poor morphology embryos (P=0.032). Morphology, however, did not affect diagnostic accuracy. Multiplex PCR-based methods on one cell, are as robust as those on two cells regarding false negative rate, which is the most important criteria for clinical PGD applications. Overall, this study demonstrates the validity, robustness and high diagnostic value of PCR-based PGD.

RASSF1A in maternal plasma as a molecular marker of preeclampsia.

OBJECTIVES: This study aimed to quantitate cell free (cf) and cell free fetal (cff) DNA in maternal plasma by determining RASSF1A levels before and after enzyme digestion in women who subsequently developed preeclampsia (PE) and compare them with uncomplicated pregnancies. METHODS: Twenty-four samples from pregnant women who developed PE and 48 samples from women with uncomplicated pregnancies were analysed. Blood samples were obtained at 11-13 weeks. cfDNA was determined by quantifying RASSF1A using qRT-PCR. A second qRT-PCR was performed following methylation-sensitive enzyme digestion by BstUI, to quantitate hypermethylated RASSF1A sequences of fetal origin. ACTB gene was used as control to confirm complete enzyme digestion. RESULTS: cfDNA and cffDNA levels were significantly increased in women who developed PE as compared with uncomplicated pregnancies (median cfDNA: 9402 vs 2698, median cffDNA: 934.5 vs 62, respectively). Following operating characteristic curve analysis, cut-off values of 7486 Εq/mL for cfDNA and 512 Εq/mL for cffDNA were chosen, which provided a sensitivity of 75% and 100% and specificity of 98% and 100%, respectively, to identify women at risk for PE. CONCLUSIONS: The study demonstrates potential use of cfDNA and cffDNA in maternal plasma as markers for the early prediction of women at risk for PE.

Psychomotor development of children born after preimplantation genetic diagnosis and parental stress evaluation.

BACKGROUND: The increasing number of children conceived following preimplantation genetic diagnosis (PGD) necessitates the evaluation of their motor and cognitive development. The primary study objective was to evaluate the physical, developmental, and neurological outcome of children born after PGD in Greece. In addition, the secondary study objective was to compare the stress levels regarding parental roles between parents of PGD children and those of naturally conceived children. METHODS: A cross-sectional study design was applied. The study population consisted of 31 children (aged 2 months to 7.5 years) born after PGD analysis and their parents. The developmental evaluation of children included a detailed physical evaluation and cognitive assessment with the Bayley Scales of Infant Development. The parent stress index was applied to evaluate comparative parental stress levels between those parents of PGD children and those of naturally conceived healthy children. RESULTS: High rates of caesarean deliveries, increased incidence of prematurity, multiples and low-birth weight were observed among the 31 PGD children. Overall, 24 of the 31 PGD children had cognitive skills within normal range [general developmental quotient (GDQ): 86-115], while 6 children had lower levels of cognitive skills (GDQ<85). With regard to parental stress, PGD parents reported lower levels of parenting stress as compared to parents of naturally conceived children (P<0.01). CONCLUSIONS: The enhanced frequency of poor cognitive and motor skills as well as low parental stress necessitates early detection and intervention for developmental delays among PGD children.