Placental Dysfunction in Assisted Reproductive Pregnancies: Perinatal, Neonatal and Adult Life Outcomes

Obstetric and newborn outcomes of assisted reproductive technology (ART) pregnancies are associated with significative prevalence of maternal and neonatal adverse health conditions, such as cardiovascular and metabolic diseases. These data are interpreted as anomalies in placentation involving a dysregulation of several molecular factors and pathways. It is not clear which extent of the observed placental alterations are the result of ART and which originate from infertility itself. These two aspects probably act synergically for the final obstetric risk. Data show that mechanisms of inappropriate trophoblast invasion and consequent altered vascular remodeling sustain several clinical conditions, leading to obstetric and perinatal risks often found in ART pregnancies, such as preeclampsia, fetal growth restriction and placenta previa or accreta. The roles of factors such as VEGF, GATA3, PIGF, sFLT-1, sEndoglin, EGFL7, melatonin and of ART conditions, such as short or long embryo cultures, trophectoderm biopsy, embryo cryopreservation, and supraphysiologic endometrium preparation, are discussed. Inflammatory local conditions and epigenetic influence on embryos of ART procedures are important research topics since they may have important consequences on obstetric risk. Prevention and treatment of these conditions represent new frontiers for clinicians and biologists involved in ART, and synergic actions with researchers at molecular levels are advocated.

C. elegans PEZO-1 is a mechanosensitive ion channel involved in food sensation

PIEZO channels are force sensors essential for physiological processes, including baroreception and proprioception. The Caenorhabditis elegans genome encodes an orthologue gene of the Piezo family, pezo-1, which is expressed in several tissues, including the pharynx. This myogenic pump is an essential component of the C. elegans alimentary canal, whose contraction and relaxation are modulated by mechanical stimulation elicited by food content. Whether pezo-1 encodes a mechanosensitive ion channel and contributes to pharyngeal function remains unknown. Here, we leverage genome editing, genetics, microfluidics, and electropharyngeogram recording to establish that pezo-1 is expressed in the pharynx, including in a proprioceptive-like neuron, and regulates pharyngeal function. Knockout (KO) and gain-of-function (GOF) mutants reveal that pezo-1 is involved in fine-tuning pharyngeal pumping frequency, as well as sensing osmolarity and food mechanical properties. Using pressure-clamp experiments in primary C. elegans embryo cultures, we determine that pezo-1 KO cells do not display mechanosensitive currents, whereas cells expressing wild-type or GOF PEZO-1 exhibit mechanosensitivity. Moreover, infecting the Spodoptera frugiperda cell line with a baculovirus containing the G-isoform of pezo-1 (among the longest isoforms) demonstrates that pezo-1 encodes a mechanosensitive channel. Our findings reveal that pezo-1 is a mechanosensitive ion channel that regulates food sensation in worms.

Live Fluorescence Imaging of F-Actin Organization in Chick Whole Embryo Cultures Using Sir-Actin

Morphogenesis is a continuous process of pattern formation so complex that it requires in vivo monitoring for better understanding. Changes in tissue shape are initiated at the cellular level, where dynamic intracellular F-actin networks determine the shape and motility of cells, influence differentiation and cytokinesis and mediate mechanical signaling. Here, we stain F-actin with the fluorogenic probe SiR-actin for live fluorescence imaging of whole chick embryos. We found that 50 nM SiR-actin in the culture medium is a safe and effective concentration for this purpose, as it provides high labeling density without inducing morphological malformations.

Effect of melatonin on the clinical outcome of patients with repeated cycles after failed cycles of in vitro fertilization and intracytoplasmic sperm injection

Background: Melatonin (MT), a powerful antioxidant, can effectively ameliorate the in vitro development of animal embryos, but few studies have been performed on human embryos. Therefore, we investigated whether the application of MT in embryo cultures can improve embryonic development and clinical outcomes of patients with repeated cycles after failed in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles.Methods: Human immature oocytes from controlled ovarian hyperstimulation cycles were collected for in vitro maturation culture and ICSI fertilization. The obtained embryos were cultured in vitro in medium containing 0, 10-11, 10-9, 10-7 or 10-5 M MT, and 10-9 M was determined to be the optimal concentration. Subsequently, 140 patients who experienced failed IVF/ICSI cycles underwent 140 cycles of embryo culture in vitro with medium containing 10-9 M MT. High-quality blastocysts were collected and cryopreserved for three months before vitrified-warmed embryo transfer. These culture cycles served as the experimental (10-9 M) group. The control (0 M) group comprised previous failed cycles. The patients were further divided into subgroups of 1, 2 or ≥3 failed cycles. The fertilization and embryo development statuses were compared.Results: The fertilization, cleavage, high-quality embryo, blastocyst, and high-quality blastocyst rates of the 10-9 M group were significantly higher than those of the 0 M group (87.7% vs. 83.6%, p <0.01; 94.1% vs. 90.5%, p <0.01; 58.3% vs. 43.8%, 51.1% vs. 41.8%, 43.4% vs. 22.9%, all p <0.0001). To date, a total of 50 vitrified-warmed cycle transfers were performed in the 10-9 M group and the implantation rate, biochemical pregnancy rate and clinical pregnancy rate were significantly higher than those in the 0 M group (65.6% vs. 9.7%, p <0.0001; 64.0% vs. 12.5%, p <0.0001; 40.0% vs. 11.7%, p <0.0001). Two healthy infants were delivered successfully and the other 18 women who achieved clinical pregnancy also had good examination indexes.Conclusion: The application of MT to embryo cultures in vitro improved embryonic development in patients with repeated cycles after failed IVF/ICSI cycles and had good clinical outcomes. The optimal concentration of MT was 10-9 M.Trial registration: Name in the registry: Effect of melatonin on the clinical outcome of patients with repeated cycles after failed cycles of in vitro fertilization and intracytoplasmic sperm injection; registration number: ChiCTR2100045552; date of registration: April 19, 2021(retrospectively registered); URL of trial registry record: www.medresman.org.cn.

C. elegans PEZO-1 is a Mechanosensitive Channel Involved in Food Sensation

PIEZO channels are force sensors essential for physiological processes including baroreception and proprioception. The Caenorhabditis elegans genome encodes an ortholog gene of the Piezo family, pezo-1, expressed in several tissues including the pharynx. This myogenic pump is an essential component of the C. elegans alimentary canal whose contraction and relaxation are modulated by mechanical stimulation elicited by food content. Whether pezo-1 encodes a mechanosensitive channel and contributes to pharyngeal function remains unknown. Here, we leverage genome editing, genetics, microfluidics, and electropharyngeogram recordings to establish that pezo-1 is expressed in the pharynx, including a proprioceptive-like neuron, and regulates pharyngeal function. Knockout (KO) and gain-of-function (GOF) mutants reveal that pezo-1 is involved in fine-tuning pharyngeal pumping frequency, sensing osmolarity, and food quality. Using pressure-clamp experiments in primary C. elegans embryo cultures, we determine that pezo-1 KO cells do not display mechanosensitive currents, whereas cells expressing wild-type or GOF PEZO-1 exhibit mechanosensitivity. Moreover, infecting the Spodoptera frugiperda cell line with a baculovirus containing the pezo-1 isoform G (among the longest isoforms) demonstrates that pezo-1 encodes a mechanosensitive channel. Our findings reveal that pezo-1 is a mechanosensitive ion channel that regulates food sensation in worms.

Retinoic Acid Signaling Plays a Crucial Role in Excessive Caffeine Intake-Disturbed Apoptosis and Differentiation of Myogenic Progenitors

Whether or not the process of somitogenesis and myogenesis is affected by excessive caffeine intake still remains ambiguous. In this study, we first showed that caffeine treatment results in chest wall deformities and simultaneously reduced mRNA expressions of genes involved in myogenesis in the developing chicken embryos. We then used embryo cultures to assess in further detail how caffeine exposure affects the earliest steps of myogenesis, and we demonstrated that the caffeine treatment suppressed somitogenesis of chicken embryos by interfering with the expressions of crucial genes modulating apoptosis, proliferation, and differentiation of myogenic progenitors in differentiating somites. These phenotypes were abrogated by a retinoic acid (RA) antagonist in embryo cultures, even at low caffeine doses in C2C12 cells, implying that excess RA levels are responsible for these phenotypes in cells and possibly in vivo. These findings highlight that excessive caffeine exposure is negatively involved in regulating the development of myogenic progenitors through interfering with RA signaling. The RA somitogenesis/myogenesis pathway might be directly impacted by caffeine signaling rather than reflecting an indirect effect of the toxicity of excess caffeine dosage.

Homozygous Transgenic Barley (Hordeum vulgare L.) Plants by Anther Culture

Production of homozygous lines derived from transgenic plants is one of the important steps for phenotyping and genotyping transgenic progeny. The selection of homozygous plants is a tedious process that can be significantly shortened by androgenesis, cultivation of anthers, or isolated microspores. Doubled haploid (DH) production achieves complete homozygosity in one generation. We obtained transgenic homozygous DH lines from six different transgenic events by using anther culture. Anthers were isolated from T0 transgenic primary regenerants and cultivated in vitro. The ploidy level was determined in green regenerants. At least half of the 2n green plants were transgenic, and their progeny were shown to carry the transgene. The process of dihaploidization did not affect the expression of the transgene. Embryo cultures were used to reduce the time to seed of the next generation. The application of these methods enables rapid evaluation of transgenic lines for gene function studies and trait evaluation.

A new concept in minimally invasive embryo transfer

Considerable variation in embryo transfer (ET) catheter types, diverging opinions on their quality and functionality, complications following the insertion of catheters, low efficiency of the application of ET methods in humans, and their widely varying efficiency in animals demonstrate the need to improve ET methods and to look for new types of catheters. Such an opportunity is offered by the introduction of catheters made of new-generation biomaterials. This study was aimed to introduce a new generation of biomaterials into reproductive biotechnology. New-generation materials were compared with materials that have been used for many years, and the functionality of newly produced catheters was compared in vivo. Five types of biomaterials were tested: polycaprolactone (PCL), dibutyryl chitin (DBC), polypropylene (PP), polyethylene (PE) and polylactide (PLA). The study was carried out in two stages. Firstly, the basic utility parameters such as geometric stability, surface structure and catheter resistance were evaluated. Subsequently, the biocompatibility of selected biomaterials in embryo cultures was examined, and the development potential of the obtained blastocysts was evaluated. In the second stage, in in vivo with live animals, the biomaterials were tested for biocompatibility and the obtained catheters were examined for their ET functionality. Efficiency with the use of the newly produced catheters was determined, the quality of the blastocysts obtained after embryo culture in the uterus was assessed, and oviducts were subjected to histopathological examination after embryo transfer. Of the tested biomaterials, only polyethylene (PE) showed adequate biological and material properties and proved suitable for production of ET catheters.

Control of sinus venous valve and sinoatrial node development by endocardial NOTCH1

Aims Sinus venous valve (SVV) and sinoatrial node (SAN) develop together at the sinoatrial junction during embryogenesis. SVV ensures unidirectional cardiac input and SAN generates sinus rhythmic contraction, respectively; both functions are essential for embryonic survival. We aim to reveal the potential role of endocardial NOTCH signalling in SVV and SAN formation. Methods and results We specifically deleted Notch1 in the endocardium using an Nfatc1Cre line. This deletion resulted in underdeveloped SVV and SAN, associated with reduced expression of T-box transcription factors, Tbx5 andTbx18, which are essential for the formation of SVV and SAN. The deletion also led to decreased expression of Wnt2 in myocardium of SVV and SAN. WNT2 treatment was able to rescue the growth defect of SVV and SAN resulted from the Notch1 deletion in whole embryo cultures. Furthermore, the Notch1 deletion reduced the expression of Nrg1 in the SVV myocardium and supplement of NRG1 restored the growth of SVV in cultured Notch1 knockout embryos. Conclusion Our findings support that endocardial NOTCH1 controls the development of SVV and SAN by coordinating myocardial WNT and NRG1 signalling functions.