Abstract Background Atrial size strongly influences atrial fibrillation (AF) development, maintenance, and therapeutic outcome, including pharmacological cardioversion. Despite this, preclinical research relies heavily on models considerably smaller than the human atrium, such as cell-based and small-animal models. Alternatively, large-animal models—though closer in size—are costly, ethically challenging, and limited by species-specific differences. Consequently, there is a need for real-size models of human AF that enable therapeutic testing at a patient-relevant scale, encompassing intermediate and enlarged atrial sizes. Purpose To develop a full human atrium–sized model of AF, establishing a new experimental scale for preclinical therapeutic testing regarding improved pharmacological cardioversion. Methods Monolayers of conditionally immortalized human atrial myocytes (hiAM) were created in three sizes: standard in vitro size (9.5 cm²), young adult human atrium-sized (55 cm²) and enlarged human atrium-sized (152 cm²). Hereafter, these model sizes are referred to as small, intermediate and enlarged, respectively. To visualize electrical activity of these monolayers and characterize their electrophysiological features, we performed optical voltage mapping. For arrhythmia studies, re-entrant circuits were induced by tachypacing. Arrhythmia complexity was quantified by the number of concurring circuits, which cores were tracked to assess their trajectories. Subsequent pharmacological testing during arrhythmias included flecainide, and flecainide + ibutilide. Results Upon 1 Hz electrical pacing all monolayers showed homogeneous wave propagation and similar wavelength between sizes. Tachypacing-induced re-entrant circuits showed a clear size-dependent increase in arrhythmia complexity and circuit movement, while dominant frequency declined with increasing size (Figure 1). Subsequent therapeutic testing revealed size-dependent drug effects. Vehicle infusion (0.1% DMSO) had no effect on dominant frequency and arrhythmia complexity, and arrhythmias persisted in all models except for one termination in the small model. Yet, Flecainide (3 and 10 µM) reduced dominant frequency and arrhythmia complexity across all models. However, termination efficacy decreased with increasing size, with arrhythmia termination in the enlarged model occurring only at the higher dose. In contrast, combined treatment with 3 µM flecainide and 20 nM ibutilide enhanced re-entry termination in human-atrium sized models. Notably, this combination did terminate arrhythmias in the enlarged model—an effect not observed with 3 µM Flecainide alone—demonstrating synergistic antiarrhythmic effects. Conclusion Using human atrium-sized in vitro modelling of AF we demonstrate that size matters in the study of both arrhythmia dynamics and pharmacological responses, enabling identification of synergistic drugs as an improved pharmacological cardioversion at an enlarged atrial size.Figure 1For image description, please refer to the figure legend and surrounding text.

The increasing use of assisted reproduction technologies (ART), including ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI), has improved the efficient use and accessibility of genetically superior bloodlines in equine breeding. However, only half of transferred embryos result in a foal. As in humans, chromosomal and genome-wide aneuploidy plays an important role in equine pregnancy loss (Lawson et al., 2024). Selective transfer of genetically normal embryos based on biopsy analysis may increase success rates. We analyzed 42 biopsies from 41 transferable embryos originating from 3 laboratories using whole-genome amplification (REPLI-g Single Cell) followed by 5 × sequencing. Samples included crude biopsies (n = 22), lysed biopsies (n = 3), and half lysates (n = 17). Data were evaluated using chromosome- and genome-wide coverage metrics, log-R ratio plots, regions of homozygosity, and B-allele frequency profiles. Limited cell input frequently resulted in noisy coverage profiles, complicating interpretation. Mean data quality (visually scored: 1 [best] to 4 [worst]) was significantly lower for half lysates (2.8) compared with crude biopsies (1.6) and whole lysates (1.0) (Kruskal–Wallis, P < 0.001). DNA concentration was lower for half lysates (621.8 ng/µL) compared with crude (783.0) and whole lysates (776.7) (ANOVA, P < 0.001). Mean coverage did not differ between groups (6.5; 6.9; 8.1; P = 0.32). Fourteen embryos (34%) showed genome-wide aneuploidy. One high-quality embryo demonstrated a tetraploid profile with a deletion of chromosome 18. The remaining cases were detected in low-quality samples and require confirmation. These suggested haploid or uniparental diploid states (n = 3), one of which additionally showed a duplication of chromosome 3 and deletions of chromosomes 14 and 21, or triploid states (n = 10), including one with a suspected duplication of chromosome 12 and one with a suspected deletion of chromosome 18. Inferred aneuploidy states in low-quality samples will be validated using genome-wide haplotyping where familial references are available. While 5 × sequencing identified euploidy and chromosomal and genome-wide aneuploidy in high-quality samples, reduced data quality limited reliable clinical detection. Pregnancy outcomes are being monitored to assess clinical relevance (n = 5 transfers).

As shown previously, the “speed of in vitro embryo development” (i.e., the interval between intracytoplasmic sperm injection (ICSI) and blastocyst formation) is associated with pregnancy rate, early pregnancy loss (EPL), foaling rate, and the foal’s sex (Lazzari et al., 2020; Claes et al., 2020; Lewis et al., 2023). While fast in vitro embryo development (d 6 to 8 post ICSI) has been associated with improved outcomes and a higher proportion of male offspring (Claes et al., 2020), the relationship of slow in vitro embryo development (d 9 to 10 post ICSI) with foaling rates and foal sex is less explored. The aim of the study was to examine whether delayed in vitro embryo development is associated with decreased foaling rates and a female-biased sex. Blastocysts (n = 179) were produced on d 7 to 10 after ICSI (d 7 [n = 17], −8 [n = 73], −9 [n = 51], −10 [n = 38]) of in vitro-matured oocytes from Warmblood mares (n = 22) (3–21 yr), and transferred either fresh (n = 35) or vitrified-warmed (n = 144) into recipient mares on d 4 after ovulation. Pregnancy, EPL (loss between d 14 and d 42), foaling rate, and foal sex were recorded and compared using multivariable generalized linear mixed-effects logistic regression models, correcting for year of IVP (2022–2024) and donor mare age. Day of blastocyst formation was not significantly associated with initial pregnancy rates (d 14; P > 0.05), but slow in vitro embryo development showed increased odds of EPL (d 7, 8, 9, and 10; 0%, 5.7%, 11.1%, and 20.0%, respectively; P < 0.001) and a lower likelihood of foaling (d 7, 8, 9, and 10; 88.2%, 64%, 60.8%, and 50%, respectively; P < 0.001). The odds ratio for foaling was 0.162 for transfer of d 10 IVP blastocysts compared with d 7 (P < 0.05). Finally, the odds of obtaining a filly rather than a colt were 5.6-fold higher for d 10 IVP blastocysts compared with d 7 (P < 0.05). No other pairwise comparisons were statistically significant (P > 0.05). In conclusion, slow in vitro embryo development is associated with decreased foaling rates and a female-biased sex ratio. Although transfer of d 10 IVP blastocysts was associated with lower foaling rates, outcomes were still acceptable (50% foaling rate) with a higher proportion of female offspring.

BACKGROUND:While fast in vitro embryo development has been associated with improved outcomes, the relationships of slow in vitro embryo development and vitrified versus fresh in vitro produced (IVP) embryo transfer to foaling percentage and foal sex are less explored. OBJECTIVES:To determine the relationships of (1) day of blastocyst formation (D7-11 after intracytoplasmic sperm injection (ICSI)) and (2) vitrification and warming method (one- and three-step) versus fresh transfer with pregnancy, early pregnancy loss (EPL), foaling percentage and foal sex. STUDY DESIGN:Retrospective clinical study. METHODS:Blastocysts (n = 201) were collected on days 7-11 after ICSI of in vitro matured oocytes from Warmblood mares and transferred either fresh or vitrified-warmed into recipient mares on day 4 after ovulation. Pregnancy, EPL, foaling percentage, and foal sex were compared using multivariable generalised linear mixed-effects logistic regression models. RESULTS:Day of blastocyst formation was significantly associated with pregnancy outcome at D14 (p = 0.048), D42 (p = 0.046) and foaling percentage (p < 0.001). The odds of foaling decreased with developmental age, with significantly lower odds after D11 blastocyst transfer (OR = 0.0045, 95% CI 0.00034-0.061; p < 0.0001). Slow embryo development was associated with a significantly higher proportion of female offspring. The odds of obtaining a filly versus a colt were 5.6-fold higher for D10 blastocysts than D7 (95% CI 1.14-25.0; p = 0.034). Transfer of fresh versus vitrified-warmed blastocysts, using a one- or three-step protocol, did not significantly affect pregnancy or foaling outcome. MAIN LIMITATIONS:Small sample size per day of blastocyst formation. CONCLUSIONS:Slow in vitro embryo development is associated with decreased pregnancy and foaling outcomes following transfer of D11 IVP blastocysts. Transfer of D10 IVP blastocysts yields acceptable foaling percentages and is associated with a higher proportion of female offspring. Vitrification is effective for preserving equine embryos and allows the use of a simplified one step warming protocol.

When performing ovum pick-up (OPU) followed by intracytoplasmic sperm injection (ICSI) in horses, the goal is to obtain multiple high-quality embryos to maximize the chance of healthy foals. In human medicine, earlier cleavage and blastocyst formation, assessed by time-lapse, are associated with improved implantation and live birth rates. In the horse, we demonstrated that ICSI with capacitated spermatozoa yields higher blastocyst rates than control ICSI (69.0% vs. 45.9% blastocysts/cleaved; P = 0.03), but potential effects on embryo quality remain to be determined. The present study hypothesized that embryos produced using capacitated spermatozoa develop faster than those made with control spermatozoa. A total of 67 mature oocytes (MII) were fertilized by piezo-driven ICSI using the same frozen-thawed semen batch that was either incubated in capacitating conditions (CAP) or not (NC). For NC, spermatozoa were washed twice in G-MOPS immediately before ICSI (3 min at 400 × g). For CAP, spermatozoa were selected using a VetCount microfluidics device (MotilityCount ApS, Denmark) and incubated for 10 h in FERT-TALP-PHE in 5% CO2. Three stallions of proven fertility were used, with 2 replicates per stallion (24 MII for stallion J, 23 MII for stallion C, and 20 MII for stallion V). Injected oocytes were cultured in a time-lapse incubator (Geri, Genea Biomedix, Sydney) in Global Medium (Cooper Surgical, Venlo, the Netherlands) with 10% FBS for 5 d followed by DMEM/F-12 with 10% FBS in 6% CO2, 5% O2, and 89% N2. Cleavage and blastocyst rates were analyzed using binary logistic regression, and timing of first cleavage and blastocyst formation using ANOVA (P < 0.05). Cleavage rates were higher following ICSI with CAP compared with NC (88.6% [31/35] vs. 65.6% [21/32]; P = 0.03). Blastocyst rates per injected oocyte were also increased by CAP (42.9% [15/35] vs. 21.9% [7/32]; P = 0.02). Timing of first cleavage did not differ between groups (P = 0.30), whereas blastocyst formation occurred earlier in CAP embryos (148.9 ± 0.5 h) compared with NC (158.5 ± 0.6 h; P = 0.04). Stallion identity significantly affected the overall cleavage (P = 0.03) and blastocyst rates (P = 0.048). In conclusion, ICSI using capacitated spermatozoa improves embryo development rates and accelerates blastocyst formation, suggesting enhanced embryo quality.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council (Starting grant 716509) to D.A. Pijnappels Background Recently it was demonstrated how the heart itself could be enabled to quickly restore its rhythm by realizing a biologically-integrated cardiac defibrillator (BioICD) through modification and subsequent expression of ion channels in cardiomyocytes [1]. By incorporating these frequency-dependent depolarizing ion channels, abnormal cardiac rhythm could be rapidly detected and terminated to restore sinus rhythm in a fully biological and shock-free manner. However, from a translational point of view, it remains unclear how such rhythm restoration can be realized via ion channel gene therapy. Purpose To explore and understand the importance of the distribution and number of BioICD-expressing cardiomyocytes in realizing fully biological restoration of cardiac rhythm. Methods To this purpose, two different realistic gene therapy configurations, i.e. those corresponding to systemic and local transgene delivery, were tested in a digital twin of human ventricular cardiac monolayers. In the systemic delivery group, BioICD-expressing cells were uniformly distributed over the tissue at different total expression percentages. In the local delivery group, circular areas were populated with BioICD-expressing cells, randomly arranged in a Gaussian distribution. Spiral waves were initiated in both groups and studied for 10 seconds. An additional set of simulations for the systemic delivery group involved an adapted BioICD ion channel with a slower opening and faster closing rate. Results Upon comparing both gene therapy methods, the systemic approach showed a gradually increasing arrhythmia termination chance with increasing BioICD-expression percentage, while local delivery did not result in termination. Instead, local delivery resulted in islands of ionic heterogeneity, causing attraction and anchoring of the spiral waves in a size and distance-dependent manner. Building on the results of the systemic approach, different ion channel parameters were tried, which resulted in normal rhythm being restored in all cases for >50% BioICD expressing cells. Time till termination was inversely related to the percentage, resulting in only 4.3s and 2.5s for 50% and 100%, respectively. Regarding termination, it was observed that conduction blocks appeared throughout the tissue and subsequently connected to force arrhythmic waves to terminate, while this process remained incomplete in the <50% groups. Conclusion This study reveals that wide-spread distribution of BioICD-expressing cardiomyocytes is required for the realization of fully biological self-restoration of cardiac rhythm, of which the efficiency is ion-channel-dynamics- and dosage-dependent. Local expression, however, results in stabilization of spiral wave activity. Further exploration of this emerging concept of biological cardioversion may not only expand our understanding of cardiac arrhythmias, but also pave the way to breakthrough advances in arrhythmia management.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council Starting Grant and Consolidator Grant to Daniël A Pijnappels Background The membrane potential (Vm) in cardiomyocytes fulfills essential regulatory roles in various biological functions, including not only action potential (AP) propagation and mechanical contraction, but also homeostatic regulation. In-depth studies into these roles have been severely hampered by the lack of research methods allowing full control over Vm, especially in multicellular cardiac preparations. Purpose We aimed to unlock new research possibilities by the development of an experimental system (APqr) capable of full Vm control, including instantaneous modulation of AP morphology on a multicellular levelin the multicellular setting. Methods Monolayers of immortalized human atrial myocytes (hiAMs, n=7) were genetically modified to express the blue light-activatable cation channel Cheriff and the red light-sensitive inward chloride pump Jaws for depolarizing and re- or hyperpolarizing effects, respectively. Real-time Vm readout was obtained by patch clamp electrophysiology. Deviations of Vm from reference values were calculated by a custom closed-coop controller. A 470-nm and a 617-nm LED were modulated by the controller in a Vm deviation-dependent manner, selectively activating Cheriff or Jaws. Electrical disturbances were introduced by application of 4-aminopyridien (4AP, 200 µM) or a preprogrammed blue-light pulse creating abnormal cation influx during the repolarization phase of the hiAM AP. Results Average AP durations at 90% repolarization (APD90) increased by 82.4 ms following 4AP application and by 362.5 ms in the presence of light-induced abnormal cation influx compared to control conditions. APqr reduced these APD90 differences to 1.9 ms and 4.6 ms on average, with Vm deviations less than 2.5 mV in 74.7% and 94.4% of the time, respectively. APqr could also be applied for the enforcement of arbitrary AP shapes. In these experiments, reference Vm values consisted of APs recorded from hiAM monolayers exposed to drugs with APD-prolonging (4AP) or shortening (carbachol) effects. APqr was able to enforce these reference APs on hiAM monolayers with high accuracy, with Vm deviations less than 2.5 mV in 95.8% and 94.4% of the time, respectively. Conclusions APqr preserves AP morphologies in the presence of electrical perturbations of different origin without any prior knowledge of the disturbance and enforces arbitrary AP morphologies with high accuracy in an immediate and self-regulatory manner. Collectively, these results set the stage for the refinement and application of opto-electronic control systems to enable in-depth investigation into the regulatory roles of the membrane potential in health and disease.

In different species, embryonic aneuploidies and genome-wide errors are a major cause of developmental failure. The increasing number of equine embryos being produced worldwide provides the opportunity to characterize and rank or select embryos based on their genetic profile prior to transfer. Here, we explored the possibility of generic, genome-wide preimplantation genetic testing concurrently for aneuploidies (PGT-A) and monogenic (PGT-M) traits and diseases in the horse, meanwhile assessing the incidence and spectrum of chromosomal and genome-wide errors in in vitro-produced equine embryos. To this end, over 70,000 single nucleotide polymorphism (SNP) positions were genotyped in 14 trophectoderm biopsies and corresponding biopsied blastocysts, and in 26 individual blastomeres from six arrested cleavage-stage embryos. Subsequently, concurrent genome-wide copy number detection and haplotyping by haplarithmisis was performed and the presence of aneuploidies and genome-wide errors and the inherited parental haplotypes for four common disease-associated genes with high carrier frequency in different horse breeds (GBE1, PLOD1, B3GALNT2, MUTYH), and for one color coat-associated gene (STX17) were compared in biopsy-blastocyst combinations. The euploid (n = 12) or fully aneuploid (n = 2) state and the inherited parental haplotypes for 42/45 loci of interest of the biopsied blastocysts were predicted by the biopsy samples in all successfully analyzed biopsy-blastocyst combinations (n = 9). Two biopsies showed a loss of maternal chromosome 28 and 31, respectively, which were confirmed in the corresponding blastocysts. In one of those biopsies, additional complex aneuploidies not present in the blastocyst were found. Five out of six arrested embryos contained chromosomal and/or genome-wide errors in most of their blastomeres, demonstrating their contribution to equine embryonic arrest in vitro. The application of the described PGT strategy would allow to select equine embryos devoid of genetic errors and pathogenetic variants, and with the variants of interest, which will improve foaling rate and horse quality. We believe this approach will be a gamechanger in horse breeding.

Abstract Background Recently it was demonstrated how the heart itself could be enabled to quickly restore its rhythm by realizing a biologically-integrated cardiac defibrillator (BioICD) through modification and subsequent expression of ion channels in cardiomyocytes [1]. By incorporating these frequency-dependent depolarizing ion channels, abnormal cardiac rhythm could be rapidly detected and terminated to restore sinus rhythm in a fully biological and shock-free manner. However, from a translational point of view, it remains unclear how such rhythm restoration can be realized via ion channel gene therapy. Purpose To explore and understand the importance of the distribution and number of BioICD-expressing cardiomyocytes in realizing fully biological restoration of cardiac rhythm. Methods To this purpose, two different realistic gene therapy configurations, i.e. those corresponding to systemic and local transgene delivery, were tested in a digital twin of human ventricular cardiac monolayers. For the systemic delivery group, BioICD-expressing cells were homogeneously distributed over the tissue with fixed total expression percentage. For the local delivery group, circular areas were given BioICD-expressing cells, randomly patterned in a Gaussian distribution. In both groups spiral waves were initiated and subsequently studied for 10 seconds. For systemic delivery, an additional set of simulations was performed for an adapted BioICD ion channel with a slower opening and faster closing rate. Results Upon comparing both gene therapy methods, the systemic approach showed a gradually increasing arrhythmia termination chance with increasing BioICD-expression percentage, while local delivery did not result in termination. Instead, local delivery resulted in islands of ionic heterogeneity, causing attraction and anchoring of the spiral waves in a size and distance-dependent manner. Building on the results of the systemic approach, different ion channel parameters were tried, which resulted in normal rhythm being restored in all cases for >50% BioICD expressing cells. Time till termination was inversely related to the percentage, resulting in only 4.3s and 2.5s for 50% and 100%, respectively. Regarding termination, it was observed that conduction blocks appeared throughout the tissue and subsequently connected to force arrhythmic waves to terminate, while this process remained incomplete in the <50% groups. Conclusion This study reveals that wide-spread distribution of BioICD-expressing cardiomyocytes is required for the realization of fully biological self-restoration of cardiac rhythm, of which the efficiency is ion-channel-dynamics- and dosage-dependent. Local expression, however, results in stabilization of spiral wave activity. Further exploration of this emerging concept of biological cardioversion may not only expand our understanding of cardiac arrhythmias, but also pave the way to breakthrough advances in arrhythmia management.

Abstract Background There is currently a binary view on cardiac arrhythmias: (1) arrhythmias are present and thereby affect the atria or ventricles as a whole, or (2) they are not present and therefore the heart is in sinus rhythm. However, complex fractionated atrial electrograms (CFAEs) under sinus rhythm (SR) are observed in patients with atrial fibrillation (AF), raising the question whether both cardiac states (arrhythmia and SR) could coexist. Although CFAEs in AF patients have been associated with functional and structural heterogeneities (e.g. dense fibrotic regions), the underlying mechanisms of fractionation under SR remain incompletely understood. Purpose To test the hypothesis that an arrhythmia can exist locally with the majority of cardiac tissue in sinus rhythm, made possible through dense local fibrotic regions forming a small electrically isolated re-entrant circuit that can "trap" excitation waves, which can only be "released" under dynamic tissue changes at an isthmus connected to the bulk of the tissue. Methods By harnessing the unique possibilities of light-gated depolarizing ion channels (CatCh) to precisely control in vitro cardiac excitability in time and space, complemented with advanced computational modelling of whole human atria, we explored the geometry of such an electrically isolated circuit and assessed its clinical relevance. Results Optical mapping studies, in monolayers of CatCh-activated neonatal rat atrial cardiomyocytes (n=8), revealed that re-entry can be established and trapped by creating an electrically isolated pathway with a bulk-connecting isthmus causing source-sink mismatch. A tachyarrhythmia was shown to exist locally with SR prevailing in the bulk of the monolayer. Next, conditions were found under which re-entry could escape this pathway, thereby converting a local dormant arrhythmic source into an active driver with global impact. Escape could be established by overcoming the source-sink mismatch through widening of the isthmus or a reduction of the gap junctional coupling. In a digital twin of the human atria, it was revealed that the conditions for "trapped re-entry" and its release can be realized as well. Unipolar pseudo-electrograms derived from these complementary computational 3D studies showed CFAEs at the site of "trapped re-entry" in coexistence with normal electrograms of SR in the bulk of the atria. Upon release of the re-entry, acute arrhythmia onset occurred, affecting the complete atria as evidenced by wave front and electrogram visualization. Conclusion Through the concept of "trapped re-entry", we not only present a new mechanism of acute manifestation of focal tachyarrhythmias, but also provide novel insight into the origin of fractionated electrograms. This insight may provide new rationales for treatment of cardiac arrhythmias, especially for ablation by site-directed targeting.Trapped re-entry in vitro and in silico

Abstract Background Cardiomyocytes maintain a membrane potential (Vm), which fulfills essential regulatory roles in various biological functions, including not only action potential (AP) propagation and mechanical contraction, but also homeostatic regulation. In-depth studies into these roles have been severely hampered by the lack of research methods allowing full control over Vm, especially in multicellular cardiac preparations. Purpose We aimed to unlock new research possibilities by the development of an experimental system (APqr) capable of full Vm control, including instantaneous modulation of AP morphology on a multicellular level. Methods Immortalized human atrial myocytes (hiAMs) grown in monolayer format (n=7) were genetically modified to express the blue light-activatable cation channel Cheriff and the red light-sensitive inward chloride pump Jaws for depolarizing and hyperpolarizing effects, respectively. Real-time Vm readout was obtained by patch clamp electrophysiology. Dynamic Vm control was achieved by a custom closed-coop controller calculating Vm deviations from reference values. A 470-nm and a 617-nm LED were modulated by the controller in a Vm deviation-dependent manner, selectively activating Cheriff and Jaws. To explore the feasibility of AP restoration, electrical disturbances were introduced by non-selective potassium channel blockade (4-aminopyridine, 4AP, 200 µM) or by a preprogrammed blue-light pulse creating abnormal cation influx during the repolarization phase of the hiAM AP. Results 4AP induced a 82.4 ± 27.8 ms increase of AP duration at 90% repolarization (APD90) values in hiAM monolayers. Using APs recorded under control conditions (CTL) as reference, the APqr maneuver reduced this APD90 difference to 1.9 ± 3.1 ms. Similarly, APqr suppressed the effects of light-induced abnormal cation influx on APD90 (CTL: 342.9 ± 51.7 ms, blue-light pulse: 700.8 ± 64.5 ms, APqr: 338.3 ± 68 ms) effectively, with Vm deviation less than 2.5 mV in 96.7% of the time. APqr could also be applied for the enforcement of arbitrary AP shapes. In these experiments, the Vm references consisted of AP shapes of hiAM monolayers exposed to drugs with APD-prolonging (4AP) or shortening (carbachol) effects. APqr was able to enforce these reference APs, that were distinctly different from the endogenous AP shapes, on hiAM monolayers with great accuracy, with Vm deviations less than 2.5 mV in 95.8% and 94.4% of the time, respectively. Conclusions APqr restores AP morphologies in the presence of electrical perturbations of different origin without any prior knowledge about the disturbance and enforces arbitrary AP morphologies with high accuracy in an immediate and self-regulatory manner. Collectively, these results set the stage for in-depth investigations into the regulatory roles of Vm in healthy and diseased cardiomyocytes via the application of such real-time opto-electronic control systems.Real-time (re)shaping of cardiac APs

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

In addition to fulfilling many breeders’ curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that can be directly performed on few embryonic cells, and to test the effect of a needle aspiration biopsy on the viability of the in vitro-produced embryo. To this end, a multiplex quantitative real-time PCR (qPCR) assay with dual-labelled probes was designed to allow the simultaneous generation of both male-specific and control fragments in a single closed-tube reaction, avoiding potential sample loss or contamination. To improve sensitivity, multicopy and polymeric genes were chosen to be specifically amplified, i.e., eight copies of Y-chromosomal ETSTY5 as male-specific and four autosomal UBC monomers as control fragment. Specificity was enhanced by the equine-specific character of ETSTY5 and by using dual-labelled probes. The assay was optimised with equine male and female genomic DNA and demonstrated a 100% accuracy and a >95% qPCR efficiency down to 10 pg of DNA. The assay was subsequently applied to determine the sex of 44 in vitro-produced embryos, collecting trophectoderm biopsies by means of a needle aspiration biopsy and herniating cells. Of all trophectoderm biopsies and herniating cell samples (n = 54), 87% could be diagnosed. Assay results were validated on a second sample obtained from the biopsied embryo (n = 18) or, by ultrasound-based sex determination of the foetus (n = 7) following the transfer of the biopsied embryo to a recipient mare, with about half of the embryos being fillies and colts. The needle aspiration biopsy procedure did not impair initial pregnancy rate or early pregnancy losses as compared to non-biopsied embryos. In conclusion, we report a safe, reliable, fast, and cost-effective assay for equine sex determination which was validated for the sex determination of in vitro-produced embryos based on few embryonic cells, and needle aspiration biopsy did not impair the embryo's viability. The assay and safe biopsy strategy hold potential for other applications.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): This study was supported by the European Research Council (Starting grant 716509) to D.A. Pijnappels. Background Recently it was demonstrated how the heart itself could be enabled to quickly restore its rhythm by realizing a biologically-integrated cardiac defibrillator (BioICD) through modification and subsequent expression of ion channels in cardiomyocytes [1]. By incorporating these frequency-dependent depolarizing ion channels, abnormal cardiac rhythm could be rapidly detected and terminated to restore sinus rhythm in a fully biological and shock-free manner. However, from a translational point of view, it remains unclear how such rhythm restoration can be realized via ion channel gene therapy. Purpose To explore and understand the importance of the distribution and number of BioICD-expressing cardiomyocytes in realizing fully biological restoration of cardiac rhythm. Methods To this purpose, two different realistic gene therapy configurations, i.e. those corresponding to systemic and local transgene delivery, were tested in a digital twin of human ventricular cardiac monolayers. For the systemic delivery group, BioICD-expressing cells were homogeneously distributed over the tissue with fixed total expression percentage. For the local delivery group, circular areas were given BioICD-expressing cells, randomly patterned in a Gaussian distribution. In both groups spiral waves were initiated and subsequently studied for 10 seconds. For systemic delivery, an additional set of simulations was performed for an adapted BioICD ion channel with a slower opening and faster closing rate. Results Upon comparing both gene therapy methods, the systemic approach showed a gradually increasing arrhythmia termination chance with increasing BioICD-expression percentage, while local delivery never resulted in termination. Instead, local delivery resulted in islands of ionic heterogeneity, causing attraction and anchoring of the spiral waves in a size and distance-dependent manner. Building on the results of the systemic approach, different ion channel parameters were tried, which resulted in normal rhythm being restored in all cases for >50% BioICD expressing cells. Time till termination was inversely related to the percentage, resulting in only 4.3s and 2.5s for 50% and 100%, respectively. Regarding termination, it was observed that conduction blocks appeared throughout the tissue and subsequently connected to force arrhythmic waves to terminate, while this process remained incomplete in the <50% groups. Conclusion This study reveals that wide-spread distribution of BioICD-expressing cardiomyocytes is required for the realization of fully biological self-restoration of cardiac rhythm, of which the efficiency is ion-channel-dynamics- and dosage-dependent. Local expression, however, results in stabilization of spiral wave activity. Further exploration of this emerging concept of biological cardioversion may not only expand our understanding of cardiac arrhythmias, but also pave the way to breakthrough advances in arrhythmia management.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

In vitro embryo production has been rapidly evolved in horses, since the immature oocytes could be viably held overnight and shipped for intracytoplasmic sperm injection (ICSI). However, in facilities where ovum pick-up (OPU) and ICSI are performed in situ, direct in vitro maturation (IVM) can be performed. In a recent study, holding of oocytes followed by extended IVM (36h) resulted in increased embryo development compared to direct IVM for 26 to 28h. Nevertheless, the effect of holding was not evaluated separately in a clinical ovum pick-up (OPU)-ICSI program. Therefore, the current study aimed to determine whether holding oocytes prior IVM could affect their developmental competence. Data from 253 OPU-ICSI sessions at Ghent University (136 mares and 54 stallions) were analysed retrospectively. Collected oocytes were submitted to direct IVM (DM:1152) (TCM-199 Earl's salts (Gibco) containing 10% (v/v) FBS (Gibco), 9.4 µg/mL follicle-stimulating hormone, and 1.88 µg/mL luteinizing hormone (Stimufol, Reprobiol, Ouffet, Belgium)) or kept in commercial holding medium (HM:1371) (Emcare, Agtech, Zulte, Belgium) at room temperature for 17–24 h in a 5 mL Falcon® Test Tube (Corning Europe, Lasne, Belgium) prior to IVM. After an average of 28.4 h of IVM (DM: 28.2; HM: 28.6), oocytes with a polar body werefertilized by piezo-drill ICSI. The zygotes were cultured in DMEM/F-12 (Gibco) with 10% (v/v) FBS under oil (38.2°C, 5% O2, 5% CO2, and 90% N2). Cleavage was evaluated 2 or 3 days after ICSI, and blastocyst development was monitored daily from day six up to day 13. For statistical analysis, generalized linear model were used to test the effect of holding on maturation, cleavage, and blastocyst rate. Always, the oocyte/ zygote was considered as the unit of interest, and results are expressed as least square means ± standard error. No significant differences were observed between treatments for maturation (DM: 64.9±1.4%; HM: 67.0±1.3; p=0.3), cleavage (DM: 62.7±1.9%; HM: 66.8±1.6; p=0.1), and blastocyst rates (DM: 19.0±1.4%; HM: 17.0±1.2; p=0.3). Likewise, comparable kinetics of embryo development were obtained with 9.08 days in average for DM and 9.33 for HM (p = 0.08). In conclusion, oocyte holding prior to IVM did not affect developmental competence when an average IVM duration of 28h was maintained. Therefore, the observed increased embryo development rates in HM vs DM reported previously primarily resulted from an extended maturation time rather than from the inclusion of an oocyte holding step. Other variables, such as the type of sperm, the effect of the mare, the time of the year could be included in further analyses.

Abstract Funding Acknowledgements Type of funding sources: None. Background Deep learning is increasingly used in modern biomedical research and applications due to the substantial availability of large clinical datasets. These approaches are invaluable in tasks involving noisy imaging data, such as tumour segmentation in histological images. In cardiology, a deep learning approach could be helpful in real-time tracking of the sources of arrhythmia, i.e. electrical rotational activity in the heart. However, the existing optical or electrophysiological recordings that could be used for training such a model are recorded under highly variable conditions and are not always annotated, thereby requiring data augmentation. Purpose To use deep neural networks trained on synthetic data to obtain concise (low dimensional) representations of noisy optical mapping recordings of cardiac arrhythmias and rapidly locate spiral wave centres. Methods To overcome the lack of experimental training data, a digital twin of a neonatal rat ventricular cardiomyocyte monolayer was used to create a large synthetic training dataset of noiseless spiral wave recordings. Spiral wave centres were detected and labelled by making use of classical algorithms which are proven to work well on noiseless data. After labelling the centres, noise was added to the spiral wave recordings to simulate realistic experimental measurements. Subsequently, these data were fed into three different deep learning architectures: 1) a variational auto-encoder (VAE) to denoise optical mapping recordings of cardiac arrhythmias in an unsupervised manner, 2) a convolutional neural network (CNN) to detect the spiral centres, and 3) a combination of both to denoise the recording and detect centres simultaneously. Results After training on synthetic datasets, each architecture could accurately predict what it was designed for (noiseless wave fronts, spiral centres including chirality, or both) on both synthetic and experimental data. These spiral centre detection results were compared with 5 classical methods of denoising and spiral centre detection for accuracy and speed. Our method was as accurate as the best performing yet slow classical algorithm, which can only detect centres after observing a full rotation cycle (~300ms). At the same time, it was as fast as the fastest classical method, needing only 30ms after enabling the algorithm to detect spiral wave centres. This allows quasi-real-time tracking of arrhythmic sources. Conclusion This study reveals that modern deep learning strategies in combination with synthetic simulation datasets can be used on experimental measurements to aid in the development of new technologies, here applied to the detection of spiral wave centres in optical mapping recordings of cardiac arrhythmias. Given the combination of speed and accuracy at which these algorithms produce results, further exploration and refinement may improve the identification of targets for catheter ablation, thereby potentially improving the outcome.