Embryonic Membranes Research Articles

Sorption of PFOS onto polystyrene microplastics potentiates synergistic toxic effects during zebrafish embryogenesis and neurodevelopment

Microplastics (MPs) have become an emerging anthropogenic pollutant, and their ability to sorb contaminants potentially enhances the threats to the ecosystem. Only a few studies are available to understand the combined effects of microplastics and other pollutants. The present study investigated the sorption of perfluorooctane sulfonic acid (PFOS) onto polystyrene microplastics (PS-MPs) at varying concentrations, using molecular dynamics simulation (MDS) to preliminarily explore the adsorption behavior. The MDS results revealed negative interaction energies between PFOS and PS-MPs, underscoring PS-MPs' role as a potential adsorbent for PFOS in an aqueous solution. Thereafter, zebrafish embryos were employed to explore the toxic effects of combined exposure to PS-MPs and PFOS. Fluorescence and Scanning Electron Microscopy (SEM) suggested PS-MP accumulation individually and in combination with PFOS on the embryonic chorion membrane. As a result, the exposed group showed increased inner pore size of the chorionic membrane and accelerated heartbeat, indicating hypoxic conditions and hindered gaseous exchange. PS-MPs aggravated the toxicity of PFOS during larval development manifested by delayed hatching rate, increased mortality, and malformation rate. Additionally, increased ROS accumulation and altered antioxidant enzymatic status were observed in all the exposed groups suggesting perturbation of the redox state. Additionally, co-exposure of zebrafish larvae to PS-MPs and PFOS resulted in an abrupt behavioral response, which decreased AChE activity and altered neurotransmitter levels. Taken together, our results emphasize that PS-MPs can act as a potential vector for PFOS, exerting synergistic toxic effects in the aquatic environment, and hence their health risks cannot be ignored.

An in vivo tumour organoid model based on the chick embryonic chorioallantoic membrane mimics key characteristics of the patient tissue: a proof-of-concept study

BackgroundPatient-derived tumour organoids (PDOs) are highly advanced in vitro models for disease modelling, yet they lack vascularisation. To overcome this shortcoming, organoids can be inoculated onto the chorioallantoic membrane (CAM); the highly vascularised, not innervated extraembryonic membrane of fertilised chicken eggs. Therefore, we aimed to (1) establish a CAM patient-derived xenograft (PDX) model based on PDOs generated from the liver metastasis of a colorectal cancer (CRC) patient and (2) to evaluate the translational pipeline (patient – in vitro PDOs – in vivo CAM-PDX) regarding morphology, histopathology, expression of C-X-C chemokine receptor type 4 (CXCR4), and radiotracer uptake patterns.ResultsThe main liver metastasis of the CRC patient exhibited high 2-[18F]FDG uptake and moderate and focal [68Ga]Ga-Pentixafor accumulation in the peripheral part of the metastasis. Inoculation of PDOs derived from this region onto the CAM resulted in large, highly viable, and extensively vascularised xenografts, as demonstrated immunohistochemically and confirmed by high 2-[18F]FDG uptake. The xenografts showed striking histomorphological similarity to the patient’s liver metastasis. The moderate expression of CXCR4 was maintained in ovo and was concordant with the expression levels of the patient’s sample and in vitro PDOs. Following in vitro re-culturing of CAM-PDXs, growth, and [68Ga]Ga-Pentixafor uptake were unaltered compared to PDOs before transplantation onto the CAM. Although [68Ga]Ga-Pentixafor was taken up into CAM-PDXs, the uptake in the baseline and blocking group were comparable and there was only a trend towards blocking.ConclusionsWe successfully established an in vivo CAM-PDX model based on CRC PDOs. The histomorphological features and target protein expression of the original patient’s tissue were mirrored in the in vitro PDOs, and particularly in the in vivo CAM-PDXs. The [68Ga]Ga-Pentixafor uptake patterns were comparable between in vitro, in ovo and clinical data and 2-[18F]FDG was avidly taken up in the patient’s liver metastasis and CAM-PDXs. We thus propose the CAM-PDX model as an alternative in vivo model with promising translational value for CRC patients.Graphical

Preparation of PBAT microplastics and their potential toxicity to zebrafish embryos and juveniles

The extensive use of traditional non-biodegradable plastics results in the generation of microplastics (MPs), forming a new pollutant that can pose significant environmental risks. Biodegradable plastics (BP) possess degradation properties and can partially replace conventional plastics, thereby reducing pollution. However, further investigation is needed into the toxicity of biodegradable microplastics (BMPs) on aquatic organisms. This study explores the toxic effects of PBAT microplastics (PBAT-BMPs) and microplastics produced from degradable PBAT/TPS (thermoplastic starch) composite film (PBAT/TPS-BMPs) on zebrafish embryos. Our findings indicate that the presence of microplastics on the embryo's surface increases with higher BMPs concentration. Nonetheless, PBAT-BMPs tend to aggregate and are blocked by the embryonic membrane, thus diminishing their toxic effects on the embryo. Acute toxicity experiments revealed that 30 mg/L of PBAT-BMPs significantly reduced the survival rate of zebrafish embryos, whereas PBAT/TPS-BMPs had a lesser effect on survival. Both types of BMPs influenced the hatching rate of the embryos, leading to prolonged incubation periods. Additionally, both types of BMPs impacted the locomotor behavior of zebrafish larvae, causing an increase in larval locomotor speed. However, these BMPs had little impact on larval body development and heartbeat behavior. Fluorescent microplastic tracer experiments demonstrated that PBAT-BMPs persisted in juvenile fish for at least 144 h and were difficult to metabolize and excrete. Our study aims to gain a better understanding of the potential effects of BMPs on aquatic ecosystems and biological health, as well as to propose effective strategies for reducing environmental pollution and protecting organisms.

O-213 Analysis of risk factors for monozygotic twins following in vitro fertilization-embryo transfer

Abstract Study question To investigate the risk factors associated with the incidence of monozygotic twinning（MZT）following in vitro fertilization and embryo transfer (IVF-ET). Summary answer MZT following IVF-ET may be associated with a reduced endometrial thickness on the day of embryo transfer and a decrease in estradiol levels post-transfer. What is known already MZT pregnancy arises from the division of a single fertilized ovum, resulting in offspring with identical genetic material. These twins exhibit markedly higher rates of complications compared to dizygotic counterparts, including increased incidences of miscarriage, congenital anomalies, intrauterine fetal demise, growth restriction, and preterm delivery. The underlying biological processes leading to MZT remain elusive. Presently, scholarly discourse centers around two predominant models and one hypothesis: the zygote division model, the embryonic membrane fusion model, and the over-mature gametes hypothesis, respectively. IVF-ET has been implicated in the heightened occurrence of MZT, suggesting a potential iatrogenic element to this phenomenon. Study design, size, duration A retrospective cohort study has been conducted. We analyzed 582 IVF-ET cycles at a large teaching hospital from January 2007 to December 2022, including 291 clinically confirmed MZTs, and 291 matched non-MZTs. Participants/materials, setting, methods A total of 582 participants, 291 in both MZT group and non-MZT group. There were 146 fresh embryo transfer cycles and 145 frozen-thawed cycles in the MZT group. Each cycle type was matched on a 1:1 basis by age and embryo transfer date with a control group that did not result in MZT following IVF-ET. We examined the disparities between the MZT group and the non-MZT group, and performed the Logistics regression analysis. Main results and the role of chance Significant differences were observed in the MZT group compared to the non-MZT group in the fresh embryo transfer cycle, particularly regarding estradiol (E2) and progesterone (P) levels on day 7 post-transfer, E2/P ratios on day 14 post-transfer, and endometrial thickness on the day of transfer (p<0.05). Logistic regression analysis indicated a correlation between reduced endometrial thickness on the transfer day and the occurrence of MZT. During the frozen-thawed cycle, significant differences were noted in E2/P ratios, E2 levels on day 7 post-transfer, and endometrial thickness on embryo transfer day (p<0.05). Logistic regression analysis suggested that lower E2 levels on day 7 post-transfer and reduced endometrial thickness on embryo transfer day were predictive of MZT. Additional potential influencing factors included the duration of the participants’ menstrual cycle, the protocol of ovulation induction, and the duration of embryo culture in vitro. Limitations, reasons for caution Primarily, the retrospective design may introduce bias, notably due to the potential absence of comprehensive data. Secondly, the study is constrained by the sample size, with 146 fresh cycles and 145 frozen cycles, which may not provide a fully representative statistical power. Wider implications of the findings The incidence of MZT may implicate signaling pathways at the maternal-fetal interface, with early embryonic split potentially serving as a compensatory mechanism in response to suboptimal estrogen levels or diminished endometrial thickness, thus enhancing embryonic viability. Trial registration number not applicable

Effect of aripiprazole on neural tube development in early chick embryos

IntroductionAripiprazole (ARI) is a recently developed antipsychotic medication that belongs to the second generation of antipsychotics. The literature has contradictory information regarding ARI, which has been classified as pregnant use category C by the FDA. Methods125 pathogen-free fertilized eggs were incubated for 28 h and divided into five groups of 25 eggs each (including the control group), and 18 eggs with intact integrity were selected from each group. After the experimental groups were divided, ARI was administered subblastodermally with a Hamilton micro-injector at 4 different doses (1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg). At the 48th hour of incubation, all eggs were hatched and embryos were removed from the embryonic membranes. And then morphologic (position of the neural tube (open or closed), crown-rump length, number of somites, embryological development status), histopathologic (apoptosis (caspase 3), cell proliferation (PCNA), in situ recognition of DNA breaks (tunnel)), genetic (BRE gene expression) analyzes were performed. ResultsAccording to the results of the morphological analysis, when the frequency of neural tube patency was evaluated among the experimental groups, a statistically significant difference was determined between the control group and all groups (p < 0.001). In addition, the mean crown-rump length and somite number of the embryos decreased in a dose-dependent manner compared to the control group. It was determined that mRNA levels of the BRE gene decreased in embryos exposed to ARI compared to the control group (p < 0.001). ConclusionMorphologically, histopathologically, and genetically, aripiprazole exposure delayed neurogenesis and development in early chick embryos. These findings suggest its use in pregnant women may be teratogenic. We note that these results are preliminary for pregnant women, but they should be expanded and studied with additional and other samples.

Effects of Nicotine on Chicken Embryo Development: A Review

Background: Studies have shown that 22.3% of the world’s population use tobacco and nicotine exposure during pregnancy remains a concern in embryonic development. Cigarette smoke contains several toxic and carcinogenic chemicals and has been known to cause pregnancy complications including premature births, low birthweights, and stillbirths. Purpose: This review aims to study nicotine exposure in chicken embryo development comprehensively. Methods: PubMed, Centers for Disease Control and Prevention (CDC), and Antpedia sites were used to search for studies using chicken embryos as a model. Studies that reported findings on nicotine&amp;apos;s effects on various developmental processes were considered for this review. Conclusion: In total, 55 published articles were included in this review to discuss findings of nicotine-induced alterations during chicken embryo development. Findings have shown that nicotine affects angiogenesis, and embryonic and chorioallantoic membrane (CAM) growth by inhibiting cell proliferation. Nicotine affects brain and cerebellar cortex development by suppressing tumor protein p53 reactions. Nicotine also causes abnormal axial rotation and incomplete formation and closure of neural tubes. The compounds like green tree extract, vitamin C, and folic acid can reduce the effects of nicotine to suppress femur growth, decrease the contractility of cardiomyocytes, and reduce survival rates. These compounds are not protective measures to completely overcome the teratogenic effects of nicotine.

Reversine inhibits proliferation and induces apoptosis of human osteosarcoma cells through targeting MEK1

Reversine, or 2-(4-morpholinoanilino)-6-cyclohexylaminopurine, is a 2,6-disubstituted purine derivative. This small molecule shows anti-tumor potential by playing a central role in the inhibition of several kinases related to cell cycle regulation and cytokinesis. In this study, systematic review demonstrated the feasibility and pharmacological mechanism of anti-tumor effect of reversine. Firstly, we grafted MNNG/HOS, U-2 OS, MG-63 osteosarcoma cell aggregates onto chicken embryonic chorioallantoic membrane (CAM) to examine the tumor volume of these grafts after reversine treatment. Following culture, reversine inhibited the growth of osteosarcoma cell aggregates on CAM significantly. In vitro experiment, reversine suppressed osteosarcoma cell viability, colony formation, proliferation, and induced apoptosis and cell cycle arrest at G0-G1 phase. Scratch wound assay demonstrated that reversine restrained cell migration. Reversine increased the protein expression of E-cadherin. The mRNA expression of Rac1, RhoA, CDC42, PTK2, PXN, N-cadherin, Vimentin in MNNG/HOS, U-2 OS and MG-63 cells were suppressed and PTEN increased after reversine treatment. Network pharmacology prediction, molecular docking and systematic review revealed MEK1 can be used as an effective target for reversine to inhibit osteosarcoma. Western blot results show the regulation of MEK1 and ERK1/2 by reversine was not consistent in different osteosarcoma cell lines, but we found that reversine significantly inhibited the protein expression of MEK1 in MNNG/HOS, U-2 OS and MG-63. All these suggested that reversine can exert its anti-tumor effect by targeting the expression of MEK1.

Adiponectin and visfatin expression profile in extra-embryonic annexes and role during embryo development in layer and broiler chickens

Some evidence showed differences between layer and broiler embryo development. We recently showed that two adipokines, adiponectin and visfatin are expressed in the extra embryonic membranes and fluids. However, their role in the embryo development is unknown. Thus, our objectives were 1. to compare the expression of AdipoQ and its receptors AdipoR1 and AdipoR2 and visfatin in extra-embryonic annexes in broiler and layer breeders during the embryo development and 2. to investigate the role of two adipokines in embryo development in both broiler and layer breed after in ovo injection of blocking antibodies against chicken adiponectin or visfatin. We found that adiponectin, AdipoR1, AdipoR2 and visfatin were mainly more expressed in the allantoic that in amniotic membranes. In addition, these expressions increased according the stage of embryo development. We observed a higher expression in layer than in broiler of AdipoQ in allantoic membranes at ED14 and ED18, of AdipoR1 and AdipoR2 in both allantoic and amniotic membranes at ED7 and ED14 and of visfatin only in allantoic membrane from ED7 to ED18. AdipoQ and visfatin were absent in amniotic fluid at ED7 but present at ED14 or ED18 where higher concentrations were detected in layer than in broiler. Interestingly, we showed a strong positive correlation between Adipo and visfatin concentration in amniotic fluid and the body weight of embryo in both breeds. However, after in ovo injection of Adipo antibodies we did not observe any effect on the embryo mortality whereas injection of visfatin antibodies increased in a dose dependent manner the embryo mortality in both breeds. Taken together, Adipo and visfatin are higher expressed in layer than broiler in extra-embryonic membranes and amniotic fluid. Our data suggest also that visfatin could be a main regulator of embryo development.

Effect of Zuclopenthixol Acetate on Neural Tube Development in Early Chick Embryos

Objective: Neural tube defects are one of the congenital malformations of the central nervous system. Although the factors that cause the development of neural tube defects and their mechanisms of action are still not clearly explained, genetic predisposition, drug use and some environmental factors are thought to play a role. In this study, it was aimed to investigate the effects of zuclopenthixol acetate (ZA) on neural tube development in a chick embryo model.&#x0D; Methods: Fourty specific pathogen-free (SPF) eggs were used in the study. The eggs were incubated for 28 hours and divided into four groups of 10 eggs each. At the end of the 28th hours, saline was injected to the control group, while ZA was administered subblastodermically to the experimental groups in 3 different doses (0.7, 1.4, 2.1 mg/kg). At the end of the 48th hours, all the eggs were opened and the embryos were dissected from the embryonic membranes and evaluated morphologically and histopathologically.&#x0D; Results: When the study groups were evaluated according to the neural tube positions (open or closed), it was found that the neural tube patency increased depending on the ZA dose, which was statistically significant (p &lt; 0.05). In addition, morphological developments of embryos were evaluated. Compared to the control group, a statistically significant decrease was observed in the mean somite numbers in all ZA-treated groups, while a significant decrease was found in the mean cranio-caudal length only in the high-dose group.&#x0D; Conclusion: In this study, it was observed that neural tube and morphological development were adversely affected in the groups treated with ZA in the chick embryo model. It was shown that neural tube closure defects in embryos increased in direct proportion with ZA doses. However, we believe that it will not be possible to fully adapt the results of this study, which was carried out in the chick embryo model, to humans and that more comprehensive research should be conducted.

Перспективи внутрішньоутробного лікування запальних змін евентрованих органів при гастрошизисі у плодів (огляд літератури)

Gastroschisis (GS) - is one of the most severe defects of newborns, which is characterized by congenital eventration of the abdominal organs outside the anterior abdominal wall into the amniotic fluid, due to a through defect of the anterior abdominal wall. The defect is adjacent to the normal, unaltered umbilical cord, usually to the right of the umbilicus, the umbilical ring is split, the eventrated organs are not covered by embryonic membranes or their remains. The frequency of gastroschisis is 0.31-4.72 per 10,000 births. Although, with the development of modern surgical approaches over the past 15 years, the mortality of newborns with gastroschisisis dynamically decreasing, however, this pathology remains a significant problem in neonatal and pediatric surgery, as it requires early surgery, associated with a significant risk of disability in children due to short bowel syndrome, abdominal cancer and recurrent esophageal disease, adhesive intestinal obstruction, etc. Purpose - to analyze, according to the literature, the prospects for the use of cellular technologies for the in utero treatment of inflammatory changes in the enteric organs in fetal GS in experimental conditions. The main reason for the unsatisfactory results of GS treatment is the pathological changes of the eventrated organs and their consequences. Therefore one of the ways to reduce mortality in GS is intrauterine treatment and the prevention of inflammatory changes of the eventrated organs in the fetuses. Modern regenerative medicine offers several new approaches for the treatment of fetal malformations, including the use of cell technology. Stem cells, from various sources, are one of the alternative therapies that can inhibit inflammatory processes in tissues, activate endogenous reparative mechanisms, and, ultimately, together with the implementation of preventive measures, reduce perinatal mortality and disability. The availability of domestic cell drugs, the development of technologies for their transplantation, and methods of delivery of pregnant women with GS in the fetus, with proven clinical efficacy, will significantly improve the comprehensive treatment of patients with congenital malformations, which is significant social and economic importance. Therefore, the study of regenerative potential and assessment of the prospects for the use of stem cells in obstetrics and perinatal medicine from various sources is of great scientific and practical interest. No conflict of interests was declared by the authors.

Expression of chemerin and its receptors in extra-embryonic annexes and role of chemerin and its GPR1 receptor in embryo development in layer and broiler hens

Intensive genetic selection of broiler breeders and layer hens resulted in differences in the mechanisms of growth and also in cell metabolism during embryogenesis. Previous research has shown that an adipokine named chemerin and one of these receptors, CMKLR1 were potentially involved in broiler embryo development. Here, our objectives were 1. to compare the expression of chemerin and its receptors CMKLR1, GPR1 and CCRL2 and chemerin concentration in extra-embryonic annexes (allantoic and amniotic membranes and fluids and plasma) in broiler and layer fertile eggs during the development (embryonic day (ED) 7, 14 and 18) by RT-qPCR and specific chicken ELISA and 2. to investigate the role of chemerin and one of its receptors GPR1 in embryo development after in ovo injections of neutralizing antibodies against chicken chemerin and GPR1. We found that chemerin expression in amniotic membranes was higher in layer than broiler eggs at ED7 and ED14 whereas the expression of the three receptors was higher in layer than broiler in the allantoic membranes at ED14 and ED18. Chemerin concentration was more important in layer than broiler at ED14 and ED18 in amniotic liquid and at all the studied stages in blood plasma. We also showed positive correlation between amniotic chemerin concentration and chemerin amniotic membrane expression, chemerin plasma concentration and embryo body weight in both breeds. Finally, in ovo injection of chicken chemerin and GPR1 neutralizing antibodies increased embryo mortality in both layer and broiler eggs. Taken together, even if chemerin concentration and chemerin system expression in embryonic membranes are mainly higher expressed in layer than in broiler, chemerin potentially through GPR1 could promote embryo development in both breeds.

A critical role of the endothelial S-phase kinase-associated protein 2/phosphatase and tensin homologue axis inangiogenesis and psoriasis.

Psoriasis is a common chronic skin disorder. Pathologically, it features abnormal epidermal proliferation, infiltrating inflammatory cells and increased angiogenesis in the dermis. Aberrant expression of E3 ubiquitin ligase and a dysregulated protein ubiquitination system are implicated in the pathogenesis of psoriasis. To examine the potential role of S-phase kinase-associated protein 2 (Skp2), an E3 ligase and oncogene, in psoriasis. Gene expression and protein levels were evaluated with quantitative reverse transcriptase polymerase chain reaction, Western blotting, immunohistochemistry and immunofluorescence staining of skin samples from patients with psoriasis vulgaris and an imiquimod (IMQ)-induced mouse model, as well as from cultured endothelial cells (ECs). Protein interaction, substrate ubiquitination and degradation were examined using co-immunoprecipitation, Western blotting and a cycloheximide chase assay in human umbilical vein ECs. Angiogenesis was measured in vitro using human dermal microvascular ECs (HDMECs) for BrdU incorporation, migration and tube formation. In vivo angiogenesis assays included chick embryonic chorioallantoic membrane, the Matrigel plug assay and quantification of vasculature in the mouse lesions. Skp2 gene global knockout (KO) mice and endothelial-specific conditional KO mice were used. Skp2 was increased in skin samples from patients with psoriasis and IMQ-induced mouse lesions. Immunofluorescent double staining indicated a close association of Skp2 expression with excessive vascularity in the lesional dermal papillae. In HDMECs, Skp2 overexpression was enhanced, whereas Skp2 knockdown inhibited EC proliferation, migration and tube-like structure formation. Mechanistically, phosphatase and tensin homologue (PTEN), which suppresses the phosphoinositide 3-kinase/Akt pathway, was identified to be a novel substrate for Skp2-mediated ubiquitination. A selective inhibitor of Skp2 (C1) or Skp2 small interfering RNA significantly reduced vascular endothelial growth factor-triggered PTEN ubiquitination and degradation. In addition, Skp2-mediated ubiquitination depended on the phosphorylation of PTEN by glycogen synthase kinase 3β. In the mouse model, Skp2 gene deficiency alleviated IMQ-induced psoriasis. Importantly, tamoxifen-induced endothelial-specific Skp2 KO mice developed significantly ameliorated psoriasis with diminished angiogenesis of papillae. Furthermore, topical use of the Skp2 inhibitor C1 effectively prevented the experimental psoriasis. The Skp2/PTEN axis may play an important role in psoriasis-associated angiogenesis. Thus, targeting Skp2-driven angiogenesis may be a potential approach to treating psoriasis.

Molecular detection and phylogenic characterization of Neospora caninum in naturally infected sheep in Alborz and Qazvin provinces, the north of the central region of Iran.

Neospora caninum is a protozoan coccidian parasite that can act as a cause of abortion in sheep. The aim of this study was to investigate the presence of this parasitic agent and its role in causing abortion in sheep of Iran. Between June 2019 and February 2022, 100 samples [brain (n = 39), placenta (n = 8), embryonic membrane (n = 7), cotyledon (n = 7), umbilical cord (n = 2), homogenate mixture of tissues (heart, liver, spleen and digestive track) (n = 37)] that were collected following the necropsies of 39 aborted ovine fetuses from different parts of the Alborz and Qazvin provinces, the north of the central region of Iran were employed for DNA extraction. Nc-5 was selected as the target gene sequence for amplification of DNA by using four pairs of primers in two semi-nested PCR. Samples considered positive for the presence of the NC-5 gene were examined to further confirm the presence of the ITS1 gene. Sequence of NC-5 gene was detected from the 27 tissue samples of 23 aborted ovine fetuses. The ITS1 gene sequence was detected in all of the 27 tissue samples that were positive for the NC-5 gene analysis. Brain tissue was the most studied tissue, and the highest number of positive cases was observed in this tissue. The present study updated the situation of ovine neosporosis in the central region of Iran and confirmed the presence of the N. caninum among sheep flocks' abortion.

Cell-Cell and Cell-Matrix Interactions at the Presumptive Stem Cell Niche of the Chick Corneal Limbus.

(1) Background: Owing to its ready availability and ease of acquisition, developing chick corneal tissue has long been used for research purposes. Here, we seek to ascertain the three-dimensional microanatomy and spatiotemporal interrelationships of the cells (epithelial and stromal), extracellular matrix, and vasculature at the corneo-scleral limbus as the site of the corneal stem cell niche of the chicken eye. (2) Methods: The limbus of developing (i.e., embryonic days (E) 16 and 18, just prior to hatch) and mature chicken eyes was imaged using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and the volume electron microscopy technique, serial-block face SEM (SBF-SEM), the latter technique allowing us to generate three-dimensional reconstructions from data sets of up to 1000 serial images; (3) Results: Data revealed that miniature limbal undulations of the embryonic basement membrane, akin to Palisades of Vogt (PoV), matured into distinct invaginations of epithelial cells that extended proximally into a vascularized limbal stroma. Basal limbal epithelial cells, moreover, occasionally exhibited a high nuclear:cytoplasmic ratio, which is a characteristic feature of stem cells. SBF-SEM identified direct cell-cell associations between corneal epithelial and stromal cells at the base of structures akin to limbal crypts (LCs), with cord-like projections of extracellular matrix extending from the basal epithelial lamina into the subjacent stroma, where they made direct contact with stomal cells in the immature limbus. (4) Conclusion: Similarities with human tissue suggest that the corneal limbus of the mature chicken eye is likely the site of a corneal stem cell niche. The ability to study embryonic corneas pre-hatch, where we see characteristic niche-like features emerge, thus provides an opportunity to chart the development of the limbal stem cell niche of the cornea.

Reappraisal of the lateral mesorectum and its clinical importance

The mesentery has been defined as a double fold of the peritoneum connecting some regions of the intestine to the posterior abdominal wall. It emerges from the superior mesenteric root region and fans out to span the intestine from the duodenum to the rectum. The mesorectal is a continuation of the intraperitoneal mesentery in the pelvic cavity. The lateral structure of the rectum is complex and the traditional view calls it the lateral ligament of the rectal. However, this structure could be called the lateral mesorectum from the perspective of embryonic development and membrane anatomy. The lateral mesorectum is the bridge of the vessels, lymphatic, and nerves between the rectum and the pelvic wall. It anchors the rectum to the lateral pelvic wall and is the anatomical basis of lateral lymph node metastasis in low rectal cancer. Meanwhile, it is important to identify the lateral mesorectum and its surrounding structure to radically resect the tumor and protect the pelvic autonomic nerve during the total mesorectal excision procedure.

Isolation of a Population of Cells Co-Expressing Markers of Embryonic Stem Cells and Mesenchymal Stem Cells from the Rudimentary Uterine Horn of a Patient with Uterine Aplasia

More than 50% cells isolated from the endometrial cavity scraping and the myometrium of the rudimentary horn of an underdeveloped uterus removed from a patient with uterine aplasia and maintained under culturing conditions normal for mesenchymal stem cells (MSC) expressed embryonic transcription factors Oct4 and Nanog, embryonic cell membrane sialyl glycolipid SSEA4, and MSC markers. After 2-3 passages, the cells lost the expression of the early embryogenesis markers, but retained MSC markers. The presence of dormant stem cells in the underdeveloped endometrium and in the uterus indicates that this tissue has a regenerative potential that can be activated and used for completion of organ morphogenesis. This task requires the development of methods of early diagnosis of morphogenesis impairment and tools for safe reactivation of the ontogenesis.

Comparative evaluation of two commercial pregnancy-associated glycoproteins tests for early detection of pregnancy in dairy cattle

The aim of this study was to determine and compare the diagnostic accuracy of two pregnancy-associated glycoproteins tests (IDEXX on-farm pregnancy test [OFPT] and IDEXX rapid visual pregnancy test [RVPT]) for early pregnancy diagnosis in dairy cattle. Blood samples were collected from Holstein cows (n = 317) by coccygeal venipuncture 28–31 days after artificial insemination (AI). The OFPT and RVPT were performed on the farm within 2 h after blood collection using whole blood or blood serum. Transrectal ultrasonography (USG) was performed for pregnancy diagnosis on day 32 post-AI as a gold standard. One-hundred fourteen cows were diagnosed as pregnant and 203 were determined to be nonpregnant. Furthermore, embryonic mortality was detected in four of the nonpregnant animals, based on fragmented/dispersed embryonic membranes and the absence of heartbeat. Sixteen and 14 false positive results (13 jointly for both tests) were obtained for the RVPT and OFPT, respectively. Three false negative results were obtained for the RVPT. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy for the OFPT were 100%, 93.1%, 89.1%, 100% and 95.6%, and for RVPT were 97.4%, 92.1%, 87.4%, 98.4% and 94.0%, respectively. The ability of both tests to distinguish between pregnant and open cows was very good (AUC of both tests above 0.9). The OFPT and RVPT almost perfectly agreed with each other. According to McNemar's analysis, false positive results have been caused difference between the two pregnancy tests and USG. In conclusion, both the RVPT and OFPT proved to be reliable and practical methods for pregnancy diagnosis 28–31 days after AI in dairy cows. However, the results of both pregnancy tests were affected by the occurrence of embryonic mortality around the time of their employment.

A REVIEW OF THE MATERNAL AND FETAL OUTCOMES IN PATIENTS PRESENTING WITH PREMATURE RUPTURE OF MEMBRANE

Background: Premature rupture of membrane (PROM) is defined as rupture of membrane before 37 completedweeks. This study aims to evaluate the maternal and fetal outcomes after the recommended treatment for PPROMand to observe maternal and fetal effects after that recommended treatment.Objectives : Determining probable difficulties, safeguarding the health of the mother and fetus throughout pregnancy,and comprehending the development and function of the amnion and chorion are among the goals of studying maternaland fetal outcomes in early embryonic membrane formation.Study design : A observational StudyDuration and place of study: This observational research was conducted at the Mardan Medical ComplexHospital’s Gynecology and Obstetrics Department. In this research, 100 PPROM patients were enrolled betweenJuly 2018 and August 2019Method: In 2019, this research was carried out at the Gynae A Ward of the Mardan Medical Complex Hospital. For our study, 100 PPROM patients were included. We tracked maternal and fetal parameters throughout ourtrial, from the beginning of PROM until delivery, including the manner of delivery, infant weight, Apgar score, andpostnatal conditions for both the mother and the newborn.Results: Thirty of the one hundred patients we included for our research had PPROM far from the term, whereasseventy patients had PPROM close to time. The delay was lower for PPROM closer to the time. Ten individualsdeveloped LSCS, while ninety of the 100 women had vaginal births. While 80 patients remained stable over thecourse of the research, 20 individuals had maternal morbidity. Five stillbirths occurred among the approximately 95live births. Twenty babies suffered from newborn septicemia and RDS.Conclusion: Intensive monitoring during labour and postpartum NICU care are necessary for the birth of a pretermbaby. Management is feasible only at tertiary care institutions with superior NICU facilities. Senior obstruction shoulddecide on prudent management and induction.Keywords: Intraventricular Hemorrhage (IVH), Neonatal Death (NND), Neonatal Sepsis (NNS), PretermPremature Rupture of Membrane (PPROM), (NICU)

Reproductive toxicity and cross-generational effect of polyethylene microplastics in Paramisgurnus dabryanus

Pollution of microplastics (MPs) has become a global environmental issue due to the difficulty in its degradation and may cause unexpected ecological effects. Nevertheless, little is known about the potential effects of MPs on reproduction toxicity in aquatic species. In this study, adult loach (Paramisgurnus dabryanus, F0 generation) were exposed to two concentrations (1 and 10 mg/L) of polyethylene MPs (PE-MPs) for 15 or 30 days, and the toxic effects in parental loach and the offspring (F1 generation) were examined. Our results showed that PE-MPs exposure could change the indicators content of antioxidant system in the brain, liver, and gonad. PE-MPs can accumulate in the gonads, disrupt the transcription of HPG-axis related genes, alter sex hormone levels, increase cell apoptosis and gonadal pathological lesions, lead to the damage of biological characteristics of semen, and affect the reproduction in F0 generation. PE-MPs remaining in the parental gonads can be transferred to the F1 generation embryos and accumulated on the embryonic chorionic membrane, increasing mortality and malformation rates, accelerating hatching time, and decreasing hatching rate and body length. These results suggest that PE-MPs leads to a potential adverse influence on reproduction and serious impacts on population sustainability. This work provides a new perspective into the effects of MPs on reproductive damage and cross-generational effects in teleost fish, which have implications in fields of freshwater ecology and environmental toxicology.

Toxic Effects and Mechanism of Pefloxacin on the Early Life Stage of Zebrafish (Danio rerio)

Pefloxacin (PFLX) is a type of fluoroquinolone (FQ) that has been widely used in aquaculture and animal husbandry. It is also a new kind of pseudo-persistent pollutant, which has some toxic effects. In this study, zebrafish (Danio rerio) embryos were used as exposure models to analyze the toxicity of PFLX. The results showed that PFLX was absorbed on the outer membrane surface at the beginning, and then a few PFLX slowly entered the cytoplasm. The adsorption complied with the Langmuir isotherm adsorption model. The surface morphology of the embryonic membrane was changed. PFLX exposure had no effect on fetal autonomous movement, but it significantly inhibited the embryo hatching rate. After PFLX exposure, the embryo and larval survival rates showed a clear dose–effect relationship. Though PFLX exposure did not cause teratogenic toxicity, it still affected cardiovascular function. The potential toxicity mechanism and developmental toxicity of PFLX to zebrafish were revealed. These results could be important for studies concerning FQ toxicology.