Significant differences in efficiency between two commonly used ionophore solutions for assisted oocyte activation (AOA): a prospective comparison of ionomycin and A23187.

PURPOSE: Despite the success of ICSI in treating severe male factor infertile patients, total fertilization failure (FF) still occurs in around 1-3% of ICSI cycles. To overcome FF, the use of calcium ionophores has been proposed to induce oocyte activation and restore fertilization rates. However, assisted oocyte activation (AOA) protocols and ionophores vary between laboratories, and the morphokinetic development underlying AOA remains understudied. METHODS: A prospective single-center cohort study involving 81 in vitro matured metaphase-II oocytes from 66 oocyte donation cycles artificially activated by A23187 (GM508 CultActive, Gynemed) (n=42) or ionomycin (n=39). Parthenogenesis was induced, and morphokinetic parameters (tPNa, tPNf, t2-t8, tSB, and tB) were compared between the 2 study groups and a control group comprising 39 2PN-zygotes from standard ICSI cycles. RESULTS: Ionomycin treatment resulted in higher activation rates compared to A23187 (38.5% vs 23.8%, p=0.15). Importantly, none of the A23187-activated parthenotes formed blastocysts. When evaluating the morphokinetic dynamics between the two ionophores, we found that tPNa and tPNf were significantly delayed in the group treated by A23187 (11.84 vs 5.31, p=0.002 and 50.15 vs 29.69, p=0.005, respectively). t2 was significantly delayed in A23187-activated parthenotes when compared to the double heterologous control embryo group. In contrast, the morphokinetic development of ionomycin-activated parthenotes was comparable to control embryos (p>0.05). CONCLUSION: Our results suggest that A23187 leads to lower oocyte activation rates and profoundly affects morphokinetic timings and preimplantation development in parthenotes. Despite our limited sample size and low parthenote competence, standardization and further optimization of AOA protocols may allow wider use and improved outcomes for FF cycles.

Applied causal inference methods for sequential mediators.

BACKGROUND: Mediation analysis aims at estimating to what extent the effect of an exposure on an outcome is explained by a set of mediators on the causal pathway between the exposure and the outcome. The total effect of the exposure on the outcome can be decomposed into an indirect effect, i.e. the effect explained by the mediators jointly, and a direct effect, i.e. the effect unexplained by the mediators. However finer decompositions are possible in presence of independent or sequential mediators. METHODS: We review four statistical methods to analyse multiple sequential mediators, the inverse odds ratio weighting approach, the inverse probability weighting approach, the imputation approach and the extended imputation approach. These approaches are compared and implemented using a case-study with the aim to investigate the mediating role of adverse reproductive outcomes and infant respiratory infections in the effect of maternal pregnancy mental health on infant wheezing in the Ninfea birth cohort. RESULTS: Using the inverse odds ratio weighting approach, the direct effect of maternal depression or anxiety in pregnancy is equal to a 59% (95% CI: 27%,94%) increased prevalence of infant wheezing and the mediated effect through adverse reproductive outcomes is equal to a 3% (95% CI: -6%,12%) increased prevalence of infant wheezing. When including infant lower respiratory infections in the mediation pathway, the direct effect decreases to 57% (95% CI: 25%,92%) and the indirect effect increases to 5% (95% CI: -5%,15%). The estimates of the effects obtained using the weighting and the imputation approaches are similar. The extended imputation approach suggests that the small joint indirect effect through adverse reproductive outcomes and lower respiratory infections is due entirely to the contribution of infant lower respiratory infections, and not to an increased prevalence of adverse reproductive outcomes. CONCLUSIONS: The four methods revealed similar results of small mediating role of adverse reproductive outcomes and early respiratory tract infections in the effect of maternal pregnancy mental health on infant wheezing. The choice of the method depends on what is the effect of main interest, the type of the variables involved in the analysis (binary, categorical, count or continuous) and the confidence in specifying the models for the exposure, the mediators and the outcome.

Optical properties of copper helical nanostructures: the effect of thickness on the SPR peak position.

In this study, we have investigated the effect of thickness on the structural and optical properties of copper (Cu) helical nanostructures. Thin films with thicknesses of 160 nm, 280 nm, 450 nm, and 780 nm were obtained by e-beam glancing angle deposition. The morphology and the microstructure were studied by field emission scanning electron microscopy, x-ray diffraction and transmission electron microscopy, while for the optical analysis measurements spectroscopic ellipsometry was used. The results show that the deposited structures are porous with nanometer-sized crystallites preferentially oriented along (111) planes, as well as that the diameter of the helices increases with thickness. Detailed analyses of optical properties have demonstrated that the dielectric function of Cu structures is greatly influenced by the films thicknesses. With increasing thickness from 160 nm to 780 nm, the surface plasmon resonance peak was shifted from 1.31 eV to 1.05 eV, which was correlated with the growth mechanism and the size of deposited nanostructures.

Comparative analysis of mouse and human preimplantation development following POU5F1 CRISPR/Cas9 targeting reveals interspecies differences.

STUDY QUESTION: What is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model? SUMMARY ANSWER: POU5F1 is required for successful development of mouse and human embryos to the blastocyst stage as knockout embryos exhibited a significantly lower blastocyst formation rate, accompanied by lack of inner cell mass (ICM) formation. WHAT IS KNOWN ALREADY: Clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR-Cas9) has previously been used to examine the role of POU5F1 during human preimplantation development. The reported POU5F1-targeted blastocysts always retained POU5F1 expression in at least one cell, because of incomplete CRISPR-Cas9 editing. The question remains of whether the inability to obtain fully edited POU5F1-targeted blastocysts in human results from incomplete editing or the actual inability of these embryos to reach the blastocyst stage. STUDY DESIGN, SIZE, DURATION: The efficiency of CRISPR-Cas9 to induce targeted gene mutations was first optimized in the mouse model. Two CRISPR-Cas9 delivery methods were compared in the B6D2F1 strain: S-phase injection (zygote stage) (n = 135) versus metaphase II-phase (M-phase) injection (oocyte stage) (n = 23). Four control groups were included: non-injected media-control zygotes (n = 43)/oocytes (n = 48); sham-injected zygotes (n = 45)/oocytes (n = 47); Cas9-protein injected zygotes (n = 23); and Cas9 protein and scrambled guide RNA (gRNA)-injected zygotes (n = 27). Immunofluorescence analysis was performed in Pou5f1-targeted zygotes (n = 37), media control zygotes (n = 19), and sham-injected zygotes (n = 15). To assess the capacity of Pou5f1-null embryos to develop further in vitro, additional groups of Pou5f1-targeted zygotes (n = 29) and media control zygotes (n = 30) were cultured to postimplantation stages (8.5 dpf). Aiming to identify differences in developmental capacity of Pou5f1-null embryos attributed to strain variation, zygotes from a second mouse strain-B6CBA (n = 52) were targeted. Overall, the optimized methodology was applied in human oocytes following IVM (metaphase II stage) (n = 101). The control group consisted of intracytoplasmically sperm injected (ICSI) IVM oocytes (n = 33). Immunofluorescence analysis was performed in human CRISPR-injected (n = 10) and media control (n = 9) human embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS: A gRNA-Cas9 protein mixture targeting exon 2 of Pou5f1/POU5F1 was microinjected in mouse oocytes/zygotes or human IVM oocytes. Reconstructed embryos were cultured for 4 days (mouse) or 6.5 days (human) in sequential culture media. An additional group of mouse-targeted zygotes was cultured to postimplantation stages. Embryonic development was assessed daily, with detailed scoring at late blastocyst stage. Genomic editing was assessed by immunofluorescence analysis and next-generation sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Genomic analysis in mouse revealed very high editing efficiencies with 95% of the S-Phase and 100% of the M-Phase embryos containing genetic modifications, of which 89.47% in the S-Phase and 84.21% in the M-Phase group were fully edited. The developmental capacity was significantly compromised as only 46.88% embryos in the S-Phase and 19.05% in the M-Phase group reached the blastocyst stage, compared to 86.36% in control M-Phase and 90.24% in control S-Phase groups, respectively. Immunofluorescence analysis confirmed the loss of Pou5f1 expression and downregulation of the primitive marker SRY-Box transcription factor (Sox17). Our experiments confirmed the requirement of Pou5f1 expression for blastocyst development in the second B6CBA strain. Altogether, our data obtained in mouse reveal that Pou5f1 expression is essential for development to the blastocyst stage. M-Phase injection in human IVM oocytes (n = 101) similarly resulted in 88.37% of the POU5F1-targeted embryos being successfully edited. The developmental capacity of generated embryos was compromised from the eight-cell stage onwards. Only 4.55% of the microinjected embryos reached the late blastocyst stage and the embryos exhibited complete absence of ICM and an irregular trophectoderm cell layer. Loss of POU5F1 expression resulted in absence of SOX17 expression, as in mouse. Interestingly, genetic mosaicism was eliminated in a subset of targeted human embryos (9 out of 38), three of which developed into blastocysts. LIMITATIONS, REASONS FOR CAUTION: One of the major hurdles of CRISPR-Cas9 germline genome editing is the occurrence of mosaicism, which may complicate phenotypic analysis and interpretation of developmental behavior of the injected embryos. Furthermore, in this study, spare IVM human oocytes were used, which may not recapitulate the developmental behavior of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Comparison of developmental competency following CRISPR-Cas-mediated gene targeting in mouse and human may be influenced by the selected mouse strain. Gene targeting by CRISPR-Cas9 is subject to variable targeting efficiencies. Therefore, striving to reduce mosaicism can provide novel molecular insights into mouse and human embryogenesis. STUDY FUNDING/COMPETING INTEREST(S): The research was funded by the Ghent University Hospital and Ghent University and supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Germline nuclear transfer in mice may rescue poor embryo development associated with advanced maternal age and early embryo arrest.

STUDY QUESTION: Can pronuclear transfer (PNT) or maternal spindle transfer (ST) be applied to overcome poor embryo development associated with advanced maternal age or early embryo arrest in a mouse model? SUMMARY ANSWER: Both PNT and ST may have the potential to restore embryonic developmental potential in a mouse model of reproductive ageing and embryonic developmental arrest. WHAT IS KNOWN ALREADY: Germline nuclear transfer (NT) techniques, such as PNT and ST, are currently being applied in humans to prevent the transmission of mitochondrial diseases. Yet, there is also growing interest in the translational use of NT for treating infertility and improving IVF outcomes. Nevertheless, direct scientific evidence to support such applications is currently lacking. Moreover, it remains unclear which infertility indications may benefit from these novel assisted reproductive technologies. STUDY DESIGN, SIZE, DURATION: We applied two mouse models to investigate the potential of germline NT for overcoming infertility. Firstly, we used a model of female reproductive ageing (B6D2F1 mice, n = 155), with ages ranging from 6 to 8 weeks (young), 56 (aged) to 70 weeks (very-aged), corresponding to a maternal age of <30, ∼36 and ∼45 years in humans, respectively. Secondly, we used NZB/OlaHsd female mice (7-14 weeks, n = 107), as a model of early embryo arrest. This mouse strain exhibits a high degree of two-cell block. Metaphase II (MII) oocytes and zygotes were retrieved following superovulation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian reserve was assessed by histological analysis in the reproductive-aged mice. Mitochondrial membrane potential (△Ψm) was measured by JC-1 staining in MII oocytes, while spindle-chromosomal morphology was examined by confocal microscopy. Reciprocal ST and PNT were performed by transferring the meiotic spindle or pronuclei (PN) from unfertilised or fertilised oocytes (after ICSI) to enucleated oocytes or zygotes between aged or very-aged and young mice. Similarly, NT was also conducted between NZB/OlaHsd (embryo arrest) and B6D2F1 (non-arrest control) mice. Finally, the effect of cytoplasmic transfer (CT) was examined by injecting a small volume (∼5%) of cytoplasm from the oocytes/zygotes of young (B6D2F1) mice to the oocytes/zygotes of aged or very-aged mice or embryo-arrest mice. Overall, embryonic developmental rates of the reconstituted PNT (n = 572), ST (n = 633) and CT (n = 336) embryos were assessed to evaluate the efficiency of these techniques. Finally, chromosomal profiles of individual NT-generated blastocysts were evaluated using next generation sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Compared to young mice, the ovarian reserve in aged and very-aged mice was severely diminished, reflected by a lower number of ovarian follicles and a reduced number of ovulated oocytes (P < 0.001). Furthermore, we reveal that the average △Ψm in both aged and very-aged mouse oocytes was significantly reduced compared to young mouse oocytes (P < 0.001). In contrast, the average △Ψm in ST-reconstructed oocytes (very-aged spindle and young cytoplast) was improved in comparison to very-aged mouse oocytes (P < 0.001). In addition, MII oocytes from aged and very-aged mice exhibited a higher rate of abnormalities in spindle assembly (P < 0.05), and significantly lower fertilisation (60.7% and 45.3%) and blastocyst formation rates (51.4% and 38.5%) following ICSI compared to young mouse oocytes (89.7% and 87.3%) (P < 0.001). Remarkably, PNT from zygotes obtained from aged or very-aged mice to young counterparts significantly improved blastocyst formation rates (74.6% and 69.2%, respectively) (P < 0.05). Similarly, both fertilisation and blastocyst rates were significantly increased after ST between aged and young mice followed by ICSI (P < 0.05). However, we observed no improvement in embryo development rates when performing ST from very-aged to young mouse oocytes following ICSI (P > 0.05). In the second series of experiments, we primarily confirmed that the majority (61.8%) of in vivo zygotes obtained from NZB/OlaHsd mice displayed two-cell block during in vitro culture, coinciding with a significantly reduced blastocyst formation rate compared to the B6D2F1 mice (13.5% vs. 90.7%; P < 0.001). Notably, following the transfer of PN from the embryo-arrest (NZB/OlaHsd) zygotes to enucleated non-arrest (B6D2F1) counterparts, most reconstructed zygotes developed beyond the two-cell stage, leading to a significantly increased blastocyst formation rate (89.7%) (P < 0.001). Similar findings were obtained after implementing ST between NZB/OlaHsd and B6D2F1 mice, followed by ICSI. Conversely, the use of CT did not improve embryo development in reproductive-age mice nor in the embryo-arrest mouse model (P > 0.05). Surprisingly, chromosomal analysis revealed that euploidy rates in PNT and ST blastocysts generated following the transfer of very-aged PN to young cytoplasts and very-aged spindles to young cytoplasts were comparable to ICSI controls (with young mouse oocytes). A high euploidy rate was also observed in the blastocysts obtained from either PNT or ST between young mice. Conversely, the transfer of young PN and young spindles into very-aged cytoplasts led to a higher rate of chromosomal abnormalities in both PNT and ST blastocysts. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The limited number of blastocysts analysed warrants careful interpretation. Furthermore, our observations should be cautiously extrapolated to humans given the inherent differences between mice and women in regards to various biological processes, including centrosome inheritance. The findings suggest that ST or PNT procedures may be able to avoid aneuploidies generated during embryo development, but they are not likely to correct aneuploidies already present in some aged MII oocytes. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first study to evaluate the potential of PNT and ST in the context of advanced maternal age and embryonic developmental arrest in a mouse model. Our data suggest that PNT, and to a lesser extent ST, may represent a novel reproductive strategy to restore embryo development for these indications. STUDY FUNDING/COMPETING INTEREST(S): M.T. is supported by grants from the China Scholarship Council (CSC) (Grant no. 201506160059) and the Special Research Fund from Ghent University (Bijzonder Onderzoeksfonds, BOF) (Grant no. 01SC2916 and no. 01SC9518). This research is also supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051017N, G051516N and G1507816N). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Double trouble: myocardial infarction with non-obstructive coronary arteries as a presentation of Hughes syndrome in monozygotic twins.

Myocardial infarction with non-obstructive coronary arteries (MINOCA) is a recently described, clinically significant entity, with prevalence rates ranging from 1% to 14% and a mean of 6% of all patients with myocardial infarction. Antiphospholipid syndrome (APS; Hughes syndrome) is characterized by the presence of antiphospholipid antibodies associated with thrombosis (arterial and/or venous) and/or pregnancy morbidity and could be the cause of MINOCA. Data on genetic predisposition to APS are scarce. The present study describes a unique case of monozygotic twin brothers who, at a young age, developed the same clinical presentation of APS. The diagnosis of APS was later confirmed, along with a diagnosis of systemic lupus erythematosus in one brother.

Comparative analysis of different nuclear transfer techniques to prevent the transmission of mitochondrial DNA variants.

Prevention of mitochondrial DNA (mtDNA) diseases may currently be possible using germline nuclear transfer (NT). However, scientific evidence to compare efficiency of different NT techniques to overcome mtDNA diseases is lacking. Here, we performed four types of NT, including first or second polar body transfer (PB1/2T), maternal spindle transfer (ST) and pronuclear transfer (PNT), using NZB/OlaHsd and B6D2F1 mouse models. Embryo development was assessed following NT, and mtDNA carry-over levels were measured by next generation sequencing (NGS). Moreover, we explored two novel protocols (PB2T-a and PB2T-b) to optimize PB2T using mouse and human oocytes. Chromosomal profiles of NT-generated blastocysts were evaluated using NGS. In mouse, our findings reveal that only PB2T-b successfully leads to blastocysts. There were comparable blastocyst rates among PB1T, PB2T-b, ST and PNT embryos. Furthermore, PB1T and PB2T-b had lower mtDNA carry-over levels than ST and PNT. After extrapolation of novel PB2T-b to human in vitro matured (IVM) oocytes and in vivo matured oocytes with smooth endoplasmic reticulum aggregate (SERa) oocytes, the reconstituted embryos successfully developed to blastocysts at a comparable rate to ICSI controls. PB2T-b embryos generated from IVM oocytes showed a similar euploidy rate to ICSI controls. Nevertheless, our mouse model with non-mutated mtDNAs is different from a mixture of pathogenic and non-pathogenic mtDNAs in a human scenario. Novel PB2T-b requires further optimization to improve blastocyst rates in human. Although more work is required to elucidate efficiency and safety of NT, our study suggests that PBT may have the potential to prevent mtDNA disease transmission.

Extended in vitro culture of human embryos demonstrates the complex nature of diagnosing chromosomal mosaicism from a single trophectoderm biopsy.

STUDY QUESTION: What is the accuracy of preimplantation genetic testing for aneuploidies (PGT-A) when considering human peri-implantation outcomes in vitro? STUDY ANSWER: The probability of accurately diagnosing an embryo as abnormal was 100%, while the proportion of euploid embryos classified as clinically suitable was 61.9%, yet if structural and mosaic abnormalities were not considered accuracy increased to 100%, with a 0% false positive and false negative rate. WHAT IS ALREADY KNOWN: Embryo aneuploidy is associated with implantation failure and early pregnancy loss. However, a proportion of blastocysts are mosaic, containing chromosomally distinct cell populations. Diagnosing chromosomal mosaicism remains a significant challenge for PGT-A. Although mosaic embryos may lead to healthy live births, they are also associated with poorer clinical outcomes. Moreover, the direct effects of mosaicism on early pregnancy remain unknown. Recently, developed in vitro systems allow extended embryo culture for up to 14 days providing a unique opportunity for modelling chromosomal instability during human peri-implantation development. STUDY DESIGN, SIZE, DURATION: A total of 80 embryos were cultured to either 8 (n = 7) or 12 days post-fertilisation (dpf; n = 73). Of these, 54 were PGT-A blastocysts, donated to research following an abnormal (n = 37) or mosaic (n = 17) diagnosis. The remaining 26 were supernumerary blastocysts, obtained from standard assisted reproductive technology (ART) cycles. These embryos underwent trophectoderm (TE) biopsy prior to extended culture. PARTICIPANTS/MATERIALS, SETTING, METHODS: We applied established culture protocols to generate embryo outgrowths. Outgrowth viability was assessed based on careful morphological evaluation. Nine outgrowths were further separated into two or more portions corresponding to inner cell mass (ICM) and TE-derived lineages. A total of 45 embryos were selected for next generation sequencing (NGS) at 8 or 12 dpf. We correlated TE biopsy profiles to both culture outcomes and the chromosomal status of the embryos during later development. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 73 embryos cultured to 12 dpf, 51% remained viable, while 49% detached between 8 and 12 dpf. Viable, Day 12 outgrowths were predominately generated from euploid blastocysts and those diagnosed with trisomies, duplications or mosaic aberrations. Conversely, monosomies, deletions and more complex chromosomal constitutions significantly impaired in vitro development to 12 dpf (10% vs. 77%, P < 0.0001). When compared to the original biopsy, we determined 100% concordance for uniform numerical aneuploidies, both in whole outgrowths and in the ICM and TE-derived outgrowth portions. However, uniform structural variants were not always confirmed later in development. Moreover, a high proportion of embryos originally diagnosed as mosaic remained viable at 12 dpf (58%). Of these, 71% were euploid, with normal profiles observed in both ICM and TE-derived lineages. Based on our validation data, we determine a 0% false negative and 18.5% false positive error rate when diagnosing mosaicism. Overall, our findings demonstrate a diagnostic accuracy of 80% in the context of PGT-A. Nevertheless, if structural and mosaic abnormalities are not considered, accuracy increases to 100%, with a 0% false positive and false negative rate. LIMITATIONS REASONS FOR CAUTION: The inherent limitations of extended in vitro culture, particularly when modelling critical developmental milestones, warrant careful interpretation. WIDER IMPLICATIONS OF THE FINDINGS: Our findings echo current prenatal testing data and support the high clinical predictive value of PGT-A for diagnosing uniform numerical aneuploidies, as well as euploid chromosomal constitutions. However, distinguishing technical bias from biological variability will remain a challenge, inherently limiting the accuracy of a single TE biopsy for diagnosing mosaicism. STUDY FUNDING, COMPETING INTEREST(S): This research is funded by the Ghent University Special Research Fund (BOF01D08114) awarded to M.P., the Research Foundation-Flanders (FWO.KAN.0005.01) research grant awarded to B.H. and De Snoo-van't Hoogerhuijs Stichting awarded to S.M.C.d.S.L. We thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Calcium influx differentially regulates migration velocity and directedness in response to electric field application.

Neural precursor cells (NPCs) respond to externally applied direct current electrical fields (DCEFs) by undergoing rapid and directed migration toward the cathode in a process known as galvanotaxis. It is unknown if the underlying mechanisms of galvanotactic migration is common to non-electrosensitive cells and if so, how NPCs and other galvanotactic cells sense and transduce electrical fields into cellular motility. In this study, we show that distinct aspects of NPC galvanotactic migration: motility (quantified through |velocity|) and directedness, are differentially regulated by calcium. We use low-Ca2+ culture conditions; an intracellular Ca2+ chelator; and voltage gated calcium channel (VGCC) inhibitors to specific channels expressed on NPCs, to demonstrate the role of Ca2+ influx in DCEF-induced NPC migration. Consistent with existing literature, we show Ca2+ is involved in F-actin polymerization that lengthens NPC membrane protrusions necessary for cellular motility. However, inhibiting Ca2+ results in reduced velocity but has no effect on DCEF-induced directedness. This dissociation between velocity and directedness reveal that these migration parameters can be independently regulated, thus suggesting a parallel process of sensing DCEFs by NPCs.

Intracellular localisation of platelet-activating factor during mammalian embryo development in vitro: a comparison of cattle, mouse and human.

Platelet-activating factor (PAF) is a well-known marker for embryo quality and viability. For the first time, we describe an intracellular localisation of PAF in oocytes and embryos of cattle, mice and humans. We showed that PAF is represented in the nucleus, a signal that was lost upon nuclear envelope breakdown. This process was confirmed by treating the embryos with nocodazole, a spindle-disrupting agent that, as such, arrests the embryo in mitosis, and by microinjecting a PAF-specific antibody in bovine MII oocytes. The latter resulted in the absence of nuclear PAF in the pronuclei of the zygote and reduced further developmental potential. Previous research indicates that PAF is released and taken up from the culture medium by preimplantation embryos invitro, in which bovine serum albumin (BSA) serves as a crucial carrier molecule. In the present study we demonstrated that nuclear PAF does not originate from an extracellular source because embryos cultured in polyvinylpyrrolidone or BSA showed similar levels of PAF in their nuclei. Instead, our experiments indicate that cytosolic phospholipase A2 (cPLA2) is likely to be involved in the intracellular production of PAF, because treatment with arachidonyl trifluoromethyl ketone (AACOCF3), a specific cPLA2 inhibitor, clearly lowered PAF levels in the nuclei of bovine embryos.

Genome-wide meta-analysis identifies novel loci associated with free triiodothyronine and thyroid-stimulating hormone.

PURPOSE: Thyroid hormones are essential for the normal function of almost all human tissues, and have critical roles in metabolism, differentiation and growth. Free triiodothyronine (fT3), free thyroxine (fT4) and thyroid-stimulating hormone (TSH) levels are under strong genetic influence; however, most of the heritability is yet unexplained. METHODS: In order to identify novel loci associated with fT3, fT4 and TSH serum levels we performed a genome-wide meta-analysis of 7 411 206 polymorphisms in up to 1731 euthyroid individuals from three Croatian cohorts from Dalmatia region: two genetically isolated island populations and one mainland population. Additionally, we also performed a bivariate analysis of fT3 and fT4 levels. RESULTS: The EPHB2 gene variant rs67142165 reached genome-wide significance for association with fT3 plasma levels (P = 9.27 × 10-9) and its significance was confirmed in bivariate analysis (P = 9.72 × 10-9). We also found a genome-wide significant association for variant rs13037502 upstream of the PTPN1 gene and TSH plasma levels (P = 1.67 × 10-8). CONCLUSION: We identified a first genome-wide significant variant associated with fT3 plasma levels, as well as a novel locus associated with TSH plasma levels. These findings are biologically relevant and enrich our knowledge about the genetic basis of pituitary-thyroid axis function.

Transcriptional landscape changes during human embryonic stem cell derivation.

STUDY QUESTION: What are the transcriptional changes occurring during the human embryonic stem cell (hESC) derivation process, from the inner cell mass (ICM) to post-ICM intermediate stage (PICMI) to hESC stage, that have downstream effects on pluripotency states and differentiation? SUMMARY ANSWER: We reveal that although the PICMI is transcriptionally similar to the hESC profile and distinct from ICM, it exhibits upregulation of primordial germ cell (PGC) markers, dependence on leukemia inhibitory factor (LIF) signaling, upregulation of naïve pluripotency-specific signaling networks and appears to be an intermediate switching point from naïve to primed pluripotency. WHAT IS KNOWN ALREADY: It is currently known that the PICMI exhibits markers of early and late-epiblast stage. It is suggested that hESCs acquire primed pluripotency features due to the upregulation of post-implantation genes in the PICMI which renders them predisposed towards differentiation cues. Despite this current knowledge, the transcriptional landscape changes during hESC derivation from ICM to hESC and the effect of PICMI on pluripotent state is still not well defined. STUDY DESIGN, SIZE, DURATION: To gain insight into the signaling mechanisms that may govern the ICM to PICMI to hESC transition, comparative RNA sequencing (RNA-seq) analysis was performed on preimplantation ICMs, PICMIs and hESCs in biological and technical triplicates (n = 3). PARTICIPANTS/MATERIALS, SETTING, AND METHODS: Primed hESCs (XX) were maintained in feeder-free culture conditions on Matrigel for two passages and approximately 50 cells were collected in biological and technical triplicates (n = 3). For ICM sample collection, Day 3, frozen-thawed human embryos were cultured up to day five blastocyst stage and only good quality blastocysts were subjected to laser-assisted micromanipulation for ICM collection (n = 3). Next, day six expanded blastocysts were cultured on mouse embryonic fibroblasts and manual dissection was performed on the PICMI outgrowths between post-plating Day 6 and Day 10 (n = 3). Sequencing of these samples was performed on NextSeq500 and statistical analysis was performed using edgeR (false discovery rate (FDR) < 0.05). MAIN RESULTS AND THE ROLE OF CHANCE: Comparative RNA-seq data analysis revealed that 634 and 560 protein-coding genes were significantly up and downregulated in hESCs compared to ICM (FDR < 0.05), respectively. Upon ICM to PICMI transition, 471 genes were expressed significantly higher in the PICMI compared to ICM, while 296 genes were elevated in the ICM alone (FDR < 0.05). Principle component analysis showed that the ICM was completely distinct from the PICMI and hESCs while the latter two clustered in close proximity to each other. Increased expression of E-CADHERIN1 (CDH1) in ICM and intermediate levels in the PICMI was observed, while CDH2 was higher in hESCs, suggesting a role of extracellular matrix components in facilitating pluripotency transition during hESC derivation. The PICMI also showed regulation of naïve-specific LIF and bone morphogenetic protein signaling, differential regulation of primed pluripotency-specific fibroblast growth factor and NODAL signaling pathway components, upregulation of phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway (PI3K/AKT/mTORC), as well as predisposition towards the germ cell lineage, further confirmed by gene ontology analysis. Hence, the data suggest that the PICMI may serve as an intermediate pluripotency stage which, when subjected to an appropriate culture niche, could aid in enhancing naïve hESC derivation and germ cell differentiation efficiency. LARGE-SCALE DATA: Gene Expression Omnibus (GEO) Accession number GSE119378. LIMITATIONS, REASONS FOR CAUTION: Owing to the limitation in sample availability, the sex of ICM and PICMI have not been taken into consideration. Obtaining cells from the ICM and maintaining them in culture is not feasible as it will hamper the formation of PICMI and hESC derivation. Single-cell quantitative real-time PCR on low ICM and PICMI cell numbers, although challenging due to limited availability of human embryos, will be advantageous to further corroborate the RNA-seq data on transcriptional changes during hESC derivation process. WIDER IMPLICATIONS OF THE FINDINGS: We elucidate the dynamics of transcriptional network changes from the naïve ICM to the intermediate PICMI stage and finally the primed hESC lines. We provide an in-depth understanding of the PICMI and its role in conferring the type of pluripotent state which may have important downstream effects on differentiation, specifically towards the PGC lineage. This knowledge contributes to our limited understanding of the true nature of the human pluripotent state in vitro. STUDY FUNDING/COMPETING INTEREST(S): This research is supported by the Concerted Research Actions funding from Bijzonder Onderzoeksfonds University Ghent (BOF GOA 01G01112).The authors declare no conflict of interest.

Familial tauopathy with P364S MAPT mutation: clinical course, neuropathology and ultrastructure of neuronal tau inclusions.

AIMS: This report presents the clinical course, neuropathology and ultrastructure of neuronal tau inclusions of four Slovene relatives with P364S MAPT mutation. METHODS: The clinical history of three out of four P364S MAPT mutation carriers was taken. After formalin fixation, thorough sampling of the central nervous system was followed by paraffin embedding, H&E, Gallyas, Bielschowsky and immunostaining with AT8, anti-3R, anti-4R tau, anti-amyloid-ß, anti-TDP43 and anti-alpha-synuclein antibodies. The distribution and density of different types of neuronal tau inclusions were semiquantitatively assessed. In addition, the ultrastructure of neuronal tau inclusions was analysed. RESULTS: Macroscopic examination of the brains was unremarkable. Microscopically, neuronal tau inclusions of almost all known types were widespread and distributed fairly uniformly in all cases. Pick bodies and swollen neurones were found in only one family member. Mutant tau was composed of 3R and 4R isoforms, with a slight predominance of 3R tau. Composite neuronal tau inclusion (CNTI), found in all four relatives, was a hallmark of the P364S MAPT mutation. CNTI showed compartmental differences in H&E and Gallyas staining, tau isoforms immunolabelling and ultrastructure, displaying fuzzy fibrils in the core and paired twisted tubules at the periphery. CONCLUSIONS: P364S MAPT mutation is characterized clinically by a variable combination of frontotemporal dementia, parkinsonism and motor neurone disease of short duration, and neuropathologically by a widespread uniform distribution of all known neuronal tau inclusions in one family member. Two-compartment CNTI is a unique characteristic of the P364S MAPT mutation.

Chromosomal mosaicism in human blastocysts: the ultimate challenge of preimplantation genetic testing?

STUDY QUESTION: To what extent does a trophectoderm (TE) biopsy reliably reflect the chromosomal constitution of the inner cell mass (ICM) in human blastocysts? SUMMARY ANSWER: Concordance between TE and ICM was established in 62.1% of the embryos analysed. WHAT IS KNOWN ALREADY: Next generation sequencing (NGS) platforms have recently been optimised for preimplantation genetic testing for aneuploidies (PGT-A). However, higher sensitivity has led to an increase in reports of chromosomal mosaicism within a single TE biopsy. This has raised substantial controversy surrounding the prevalence of mosaicism in human blastocysts and the clinical implications of heterogeneity between the TE and ICM. STUDY DESIGN, SIZE, DURATION: To define the distribution and rate of mosaicism in human blastocysts, we assessed chromosomal profiles of the ICM and multiple TE portions obtained from the same embryo. We evaluated donated embryos with an unknown chromosomal profile (n = 34), as well as PGT-A blastocysts, previously diagnosed as abnormal or mosaic (n = 24). Our intra-embryo comparison included a total of 232 samples, obtained from 58 embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS: Four embryo samples, including the ICM and three distinct TE portions, were acquired from good quality blastocysts by micromanipulation. Whole genome amplification (WGA), followed by NGS was performed on all embryo segments. Profiles were compared between samples from the same embryo, while the results from pretested blastocysts were further correlated to the original report. The embryos investigated in our untested group were obtained from good prognosis patients (n = 25), with maternal age ranging from 23 to 39 years. For the pretested embryo group, maternal age ranged from 23 to 40 years (n = 18). MAIN RESULTS AND THE ROLE OF CHANCE: We uncover chromosomal mosaicism, involving both numerical and structural aberrations, in up to 37.9% of the blastocysts analysed. Within the untested group, the overall concordance between the ICM and all TE portions was 55.9%. A normal ICM was detected in 20.6% of blastocysts for which at least one TE portion showed a chromosomal aberration. Conversely, 17.6% of embryos presented with mosaic or uniform abnormalities within the ICM, while showing normal or mosaic TE profiles. For the pretested blastocysts, the overall concordance between the ICM and all TE samples was 70.8%. However, 50% of embryos previously diagnosed with mosaicism did not confirm the original diagnosis. Notably, 31.3% of embryos with a mosaic aberration reported in the original TE biopsy, revealed a euploid profile in the ICM and all three TE samples. Taken together, concordance between the ICM and all TE portions was established in 62.1% of blastocysts, across both embryo groups. Finally, we could not observe a significant effect of age on embryo mosaicism (P = 0.101 untested group; P = 0.7309 pretested group). Similarly, ICM and TE quality were not found to affect the occurrence of chromosomal mosaicism (P = 0.718 and P = 0.462 untested group; P = 1.000 and P = 0.2885 pretested group). LARGE SCALE DATA: All data that support the findings of this study are available online in Vivar (http://cmgg.be/vivar) upon request. LIMITATIONS, REASONS FOR CAUTION: Evaluating biological variation in some instances remains challenging. The technological limitations of sampling mitotic errors that lead to mosaicism, as well as WGA artefacts, warrant careful interpretation. WIDER IMPLICATIONS OF THE FINDINGS: Our results highlight the complex nature of genetic (in)stability during early ontogenesis and indicate that blastocysts harbour a higher rate of chromosomal mosaicism than may have been anticipated. Moreover, our findings reveal an overall high diagnostic sensitivity and relatively low specificity in the context of PGT-A. This suggests that a considerable proportion of embryos are potentially being classified as clinically unsuitable. Ultimately, more precise quantification will benefit the clinical management of embryo mosaicism. STUDY FUNDING/COMPETING INTEREST(S): M.P. is supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF01D08114). J.T. and L.D. are supported by the agency for innovation through science (131673, 141441). B.H. and this research are supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF15/GOA/011). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Rabies as a threat to wildlife.

The impact of infectious disease may become progressively more harmful to a species' survival as a wild population approaches an 'extinction vortex'. This risk is especially relevant for pathogens that spread rapidly and result in high mortality rates. Rabies, a virus that infects many mammalian species, can be efficiently transmitted through infected saliva, and is fatal without prior vaccination or rapid post-exposure prophylaxis (in humans). The authors conducted an extensive literature review to identify all wild mammal species reported to have been infected with rabies virus. They found reports of infection in 190 mammalian species, including 16 with elevated risk of extinction and two for which rabies is a direct conservation threat: the Ethiopian wolf (Canis simensis) and the African wild dog (Lycaon pictus). This paper discusses selected examples in which rabies has contributed to the population decline of a species of conservation concern. In addition, the authors note the importance of the transmission of rabies virus (RABV) from domestic dogs to wildlife, and the many challenges associated with the vaccination of wild animals. With this in mind, they present potential solutions to reduce the burden of rabies on wildlife. Once stable control of RABV is achieved in domestic dogs, remaining rabies threats to wildlife conservation can be addressed more effectively.

L'impact des maladies infectieuses peut constituer une menace croissante pour la survie d'espèces animales sauvages dès lors que leurs populations sont entraînées dans la « spirale de l'extinction ¼. Ce risque se pose plus particulièrement lorsqu'il s'agit d'agents pathogènes qui se propagent rapidement et induisent un taux de mortalité élevé. Le virus de la rage affecte un grand nombre d'espèces de mammifères et se transmet facilement par contact avec de la salive infectée ; l'infection virale entraîne la mort en l'absence d'une vaccination préalable ou, chez l'être humain, d'une prophylaxie post-exposition administrée rapidement. Les auteurs ont procédé à un examen exhaustif de la littérature afin d'inventorier les espèces de mammifères sauvages chez qui l'infection rabique a été rapportée. Des cas ont été notifiés chez 190 espèces de mammifères, dont 16 présentant un risque élevé d'extinction et deux directement menacées d'extinction en raison de la rage : le loup d'Abyssinie (Canis simensis) et le lycaon (Lycaon pictus). Les auteurs apportent des précisions sur un nombre choisi d'espèces vulnérables ou en danger dont le déclin des populations est en partie imputé à la rage. En outre, ils soulignent l'importance de la transmission du virus de la rage des chiens domestiques aux animaux sauvages et décrivent les nombreuses difficultés liées à la vaccination de la faune sauvage. Ces éléments établis, ils présentent quelques solutions envisageables pour réduire le fardeau de la rage dans la faune sauvage. Une fois le virus de la rage contrôlé de manière pérenne chez le chien domestique il sera possible de lutter plus efficacement contre les autres menaces que la rage fait peser sur la conservation de la faune.

Una enfermedad infecciosa puede tener efectos cada vez más dañinos en la supervivencia de una especie a medida que una población silvestre se va aproximando a un «vórtice de extinción¼. Este riesgo tiene especial importancia en el caso de patógenos que se propagan con rapidez y causan elevadas tasas de mortalidad. La rabia, enfermedad provocada por un virus que infecta a muchas especies de mamíferos y puede transmitirse eficazmente a través de saliva infectada, resulta letal en ausencia de vacunación previa o de rápidas medidas de profilaxis tras la exposición (en el ser humano). Los autores realizaron un amplio estudio bibliográfico para determinar todas aquellas especies de mamíferos silvestres en que se hubiera descrito una infección por el virus de la rabia. Encontraron infecciones descritas en 190 especies de mamíferos, de las que 16 presentan un elevado riesgo de extinción y dos cuya conservación se ve directamente amenazada por la rabia: el lobo etíope (Canis simensis) y el licaón, o perro salvaje africano (Lycaon pictus). Los autores exponen una serie de ejemplos en los que la rabia ha contribuido al declive demográfico de una especie cuya pervivencia está en mayor o menor peligro. Los autores señalan además la importancia que reviste la transmisión del virus de la rabia de los perros domésticos a la fauna silvestre y los numerosos problemas que presenta la vacunación de los animales silvestres. Teniendo presente esta dificultad, exponen posibles soluciones para reducir la carga de rabia en la fauna silvestre. Una vez se logre estabilizar el control del virus rábico en el perro doméstico, será posible combatir más eficazmente la amenaza que representa para la conservación de las especies silvestres.

Insulin-like growth factor I and risk of epithelial invasive ovarian cancer by tumour characteristics: results from the EPIC cohort.

BACKGROUND: Prospective studies on insulin-like growth factor I (IGF-I) and epithelial ovarian cancer (EOC) risk are inconclusive. Data suggest risk associations vary by tumour characteristics. METHODS: We conducted a nested case-control study in the European Prospective Investigation into Cancer and Nutrition (EPIC) to evaluate IGF-I concentrations and EOC risk by tumour characteristics (n=565 cases). Multivariable conditional logistic regression models were used to estimate associations. RESULTS: We observed no association between IGF-I and EOC overall or by tumour characteristics. CONCLUSIONS: In the largest prospective study to date was no association between IGF-I and EOC risk. Pre-diagnostic serum IGF-I concentrations may not influence EOC risk.

CXCL10 induces the recruitment of monocyte-derived macrophages into kidney, which aggravate puromycin aminonucleoside nephrosis.

The mechanism responsible for trafficking of monocyte-derived macrophages into kidney in the puromycin aminonucleoside model of nephrotic syndrome in rats (PAN-NS), and the significance of this infiltration, remain largely unknown. CXCL10, a chemokine secreted in many T helper type 1 (Th1) inflammatory diseases, exhibits important roles in trafficking of monocytes and activated T cells. We hypothesized that induction of circulating interferon (IFN)-γ and glomerular tumour necrosis factor (TNF)-α during PAN-NS would stimulate the release of CXCL10 by podocytes, leading to infiltration of activated immune cells and greater glomerular injury. We found that serum IFN-γ, glomerular Cxcl10 mRNA and intra- and peri-glomerular macrophage infiltration were induced strongly during the late acute phase of PAN-NS in Wistar rats, but not in nude (Foxn1(rnu/rnu) ) rats lacking functional effector T lymphocytes. Wistar rats also developed significantly greater proteinuria than nude rats, which could be abolished by macrophage depletion. Stimulation of cultured podocytes with both IFN-γ and TNF-α markedly induced the expression of Cxcl10 mRNA and CXCL10 secretion. Together, these data support our hypothesis that increased circulating IFN-γ and glomerular TNF-α induce synergistically the production and secretion of CXCL10 by podocytes, attracting activated macrophages into kidney tissue. The study also suggests that IFN-γ, secreted from Th1 lymphocytes, may prime proinflammatory macrophages that consequently aggravate renal injury.

Exploring the determinants of fracture risk among individuals with spinal cord injury.

UNLABELLED: In this cross-sectional study, we found that areal bone mineral density (aBMD) at the knee and specific tibia bone geometry variables are associated with fragility fractures in men and women with chronic spinal cord injury (SCI). INTRODUCTION: Low aBMD of the hip and knee regions have been associated with fractures among individuals with chronic motor complete SCI; however, it is unclear whether these variables can be used to identify those at risk of fracture. In this cross-sectional study, we examined whether BMD and geometry measures are associated with lower extremity fragility fractures in individuals with chronic SCI. METHODS: Adults with chronic [duration of injury ≥ 2 years] traumatic SCI (C1-L1 American Spinal Cord Injury Association Impairment Scale A-D) reported post injury lower extremity fragility fractures. Dual-energy X-ray absorptiometry (DXA) was used to measure aBMD of the hip, distal femur, and proximal tibia regions, while bone geometry at the tibia was assessed using peripheral quantitative computed tomography (pQCT). Logistic regression and univariate analyses were used to identify whether clinical characteristics or bone geometry variables were associated with fractures. RESULTS: Seventy individuals with SCI [mean age (standard deviation [SD]), 48.8 (11.5); 20 females] reported 19 fragility fractures. Individuals without fractures had significantly greater aBMD of the hip and knee regions and indices of bone geometry. Every SD decrease in aBMD of the distal femur and proximal tibia, trabecular volumetric bone mineral density, and polar moment of inertia was associated with fracture prevalence after adjusting for motor complete injury (odds ratio ranged from 3.2 to 6.1). CONCLUSION: Low knee aBMD and suboptimal bone geometry are significantly associated with fractures. Prospective studies are necessary to confirm the bone parameters reported to predict fracture risk in individuals with low bone mass and chronic SCI.

Use of screening to recruitment ratios as a tool for planning and implementing spinal cord injury rehabilitation research.

STUDY DESIGN: Descriptive report. OBJECTIVES: To describe screening to recruitment (S:R) ratios and discuss their use for planning and implementing research among individuals with spinal cord injury (SCI) . SETTING: Toronto, Ontario, Canada. METHODS: We calculated S:R ratios for SCI research by study methodology and nature of the exposure/intervention for 25 studies previously conducted in a tertiary SCI rehabilitation facility. Study methodologies included ten randomized controlled trials (RCTs), nine cohort studies and six panel studies. Exposures included seven rehabilitation interventions, and three drug studies, ten telephone interviews/chart abstractions (TI/CA) and five surveys. A S:R ratio was calculated for each study methodology, and exposure type, by dividing the number of consenting individuals who underwent screening by the number of eligible recruited participants enrolled in the study. RESULTS: In terms of design, RCTs had the highest median S:R ratio (3:1), followed by cohort studies (2:1) and panel studies (2:1). In terms of intervention type, drug studies had the largest median S:R ratio (5:1), followed in descending order by rehabilitation studies (2:1), TI/CAs studies (2:1) and surveys (2:1). CONCLUSIONS: Reported S:R ratios varied substantially with study methodology and the associated study intervention exposure. Awareness of S:R ratios may assist researchers in estimating recruitment timelines, personnel needs and study budgets for a required sample size based on the planned study methodology and intended study exposure. We advocate for the routine reporting of S:R ratios to inform the success of future SCI research.

International spinal cord injury upper extremity basic data set.

OBJECTIVE: To develop an International Spinal Cord Injury (SCI) Upper Extremity Basic Data Set as part of the International SCI Data Sets, which facilitates consistent collection and reporting of basic upper extremity findings in the SCI population. SETTING: International. METHODS: A first draft of a SCI Upper Extremity Data Set was developed by an international working group. This was reviewed by many different organisations, societies and individuals over several months. A final version was created. VARIABLES: The final version of the International SCI Upper Extremity Data Set contains variables related to basic hand-upper extremity function, use of assistive devices, SCI-related complications to upper extremity function and upper extremity/hand reconstructive surgery. Instructions for data collection and the data collection form are freely available on the ISCoS website (www.iscos.org.uk). CONCLUSION: The International SCI Upper Extremity Basic Data Set will facilitate consistent collection and reporting of basic upper extremity findings in the SCI population.