Objective To explore the alterations in macrophage polarization and the expression of decorin (DCN) protein in the decidua of patients with missed abortion (MA), as well as to elucidate the regulatory effect of DCN on macrophage polarization. Methods Flow cytometry was employed to assess the polarization ratio of decidual macrophages in MA, recurrent spontaneous abortion (RSA) and normal pregnancy (NP); The expression and localization of DCN and hypoxia-inducible factor 1α (HIF-1α) in decidua and villi were assessed by immunohistochemistry staining, while their protein levels were measured by Western blot. Primary trophoblasts and peripheral blood mononuclear cell (PBMC)-derived macrophages were isolated and cultured. ELISA was conducted to quantify DCN levels in the culture supernatant of primary trophoblast and PBMC-derived macrophages. Additionally, flow cytometry was applied to evaluate the polarization ratio of PBMC-derived macrophages. Immunofluorescence cytochemical staining was conducted to examine HIF-1α expression in macrophages. Western blot was performed to detect the expression of proteins related to the gene associated with retinoid-IFN-induced mortality 19 (GRIM-19)/signal transducer and activator of transcription 3 (STAT3)/HIF-1α signaling pathway in macrophages. Results The polarization ratio of M1 macrophages in the decidua of abortion patients was significantly higher than that of NP, whereas the ratio of M2 macrophages was significantly lower. The expression of DCN and HIF-1α protein were significantly evaluated in abortion patients compared to NP. The supernatant DCN content and HIF-1α protein expression of primary trophoblast and PBMC-derived macrophages cultured under 10 mL/L O₂ for 24 hours were markedly increased compared to cells treated with 210 mL/L O₂. Compared with the PBS group, the proportion of M1 macrophage and GRIM-19 protein expression were significantly reduced in the DCN group, while phosphorylated STAT3 (p-STAT3) and HIF-1α protein expression were significantly increased. Conclusion The expression of DCN in decidua and villi of MA is higher than that of NP. DCN exhibits an inhibitory effect on the M1 polarization of PBMCs-derived macrophages, which is likely mediated through the GRIM-19/STAT3/HIF-1α signaling pathway.
Humans ; Female ; Decidua/metabolism* ; Macrophages/cytology* ; Decorin/genetics* ; Pregnancy ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Adult ; Leukocytes, Mononuclear/cytology* ; Abortion, Missed/genetics* ; STAT3 Transcription Factor/metabolism* ; Cells, Cultured ; Young Adult

PURPOSE: The retear rate of rotator cuff (RC) after surgery is high, and the rapid and functional enthesis regeneration remains a challenge. Whether acellular amniotic membrane (AAM) helps to promote the healing of tendon to bone and which treatment is better are both unclear. The study aims to investigate the effect of AAM on the healing of RC and the best treatment for RC repair. METHODS: Thirty-three Sprague Dawley rats underwent RC transection and repair using microsurgical techniques and were randomly divided into the suturing repair only (SRO) group (n = 11), the AAM overlaying (AOL) group (n = 11), and the AAM interposition (AIP) group (n = 11), respectively. Rats were sacrificed at 4 weeks, then examined by subsequent micro-CT, and evaluated by histologic and biomechanical tests. The statistical analyses of one-way ANOVA or Kruskal-Wallis test were performed using with SPSS 23.0. A p < 0.05 was considered a significant difference. RESULTS: AAM being intervened between tendon and bone (AIP group) or overlaid over tendon to bone junction (AOL group) in a rat model, promoted enthesis regeneration, increased new bone and cartilage generation, and improved collagen arrangement and biomechanical properties in comparison with suturing repair only (SRO group) (AOL vs. SRO, p < 0.001, p = 0.004, p = 0.003; AIP vs. SRO, p < 0.001, p < 0.001, p < 0.001). Compared with the AOL group, the AIP group had better results in micro-CT evaluation, histological score, and biomechanical testing (p = 0 0.039, p = 0.011, p = 0.003, respectively). CONCLUSION In the RC repair model, AAM enhanced regeneration of the tendon to bone junction. This regeneration was more effective when the AAM was intervened at the tendon to bone interface than overlaid above the tendon to bone junction.
Animals ; Rats, Sprague-Dawley ; Rotator Cuff Injuries/surgery* ; Amnion/transplantation* ; Rats ; Wound Healing ; Rotator Cuff/surgery* ; Male ; X-Ray Microtomography ; Tendons/surgery* ; Biomechanical Phenomena

Pericytes are multifunctional mural cells that surround the abluminal wall of endothelial cells and are associated with vascular development, vascular permeability, and angiogenesis. Additionally, pericytes demonstrate stem cell-like properties and contribute to neuroinflammatory processes. Pericytes have been extensively studied in the central nervous system. However, specific mechanisms underlying its involvement in various physiological and pathological conditions, especially in erectile dysfunction (ED), remain poorly understood. Advancements in in vitro and in vitro techniques, such as single-cell RNA sequencing, are expanding our understanding of pericytes. Recent studies have shown that pericyte dysfunction is considered an important factor in the pathogenesis of vascular and neurological ED. Therefore, this study aims to analyze the specific role of pericytes in ED, focusing on diabetic and neurogenic ED. This article provides a comprehensive review of research findings on PubMed from 2000 to 2023, concerning pericyte dysfunction in the process of ED, offering valuable insights, and suggesting directions for further research.
Pericytes/physiology* ; Humans ; Male ; Penis/innervation* ; Erectile Dysfunction/physiopathology* ; Nerve Regeneration/physiology* ; Neovascularization, Physiologic/physiology* ; Animals ; Angiogenesis

The onset of pregnancy is marked by the formation of a zygote, while the culmination of gestation is manifested by the delivery of a fetus. Meanwhile, a successful pregnancy entails a meticulously coordinated sequence of events from embryo implantation to sustained decidualization of the uterus to placental development and childbirth. The decidual reaction, a pivotal process occurring within the endometrium during pregnancy, is finely regulated by sex steroids and cytokines. Notably, fibroblast growth factors (FGFs), particularly FGF2, play a critical role in this physiological cascade. Dysregulated FGF expression may trigger inadequate decidualization, precipitating a spectrum of adverse pregnancy outcomes, including preeclampsia, recurrent implantation failure, and miscarriage. Furthermore, the human decidua, distinct from most mammalian species and similar to great apes, undergoes regular cycles of formation and shedding, independent of the presence of the embryo in the endometrium. This process is also tightly controlled by various FGFs. In this review, we comprehensively compare diverse research decidualization models, delineate the trend of endometrial FGFs during the menstrual cycle, and provide a synopsis of endometrial diseases triggered by FGF dysregulation.
Humans ; Female ; Pregnancy ; Decidua/physiology* ; Animals ; Endometrium/physiology* ; Fibroblast Growth Factors/metabolism* ; Embryo Implantation ; Menstrual Cycle/physiology*

The regulatory processes in developmental biology research are significantly influenced by long non-coding RNAs (lncRNAs). However, the dynamics of lncRNA expression during human tooth development remain poorly understood. In this research, we examined the lncRNAs present in the dental epithelium (DE) and dental mesenchyme (DM) at the late bud, cap, and early bell stages of human fetal tooth development through bulk RNA sequencing. Developmental regulators co-expressed with neighboring lncRNAs were significantly enriched in odontogenesis. Specific lncRNAs expressed in the DE and DM, such as PANCR, MIR205HG, DLX6-AS1, and DNM3OS, were identified through a combination of bulk RNA sequencing and single-cell analysis. Further subcluster analysis revealed lncRNAs specifically expressed in important regions of the tooth germ, such as the inner enamel epithelium and coronal dental papilla (CDP). Functionally, we demonstrated that CDP-specific DLX6-AS1 enhanced odontoblastic differentiation in human tooth germ mesenchymal cells and dental pulp stem cells. These findings suggest that lncRNAs could serve as valuable cell markers for tooth development and potential therapeutic targets for tooth regeneration.
Humans ; RNA, Long Noncoding/metabolism* ; Odontogenesis/genetics* ; Tooth Germ/embryology* ; Cell Differentiation ; Gene Expression Regulation, Developmental ; Mesoderm/metabolism* ; Tooth/embryology* ; Gene Expression Profiling ; Sequence Analysis, RNA ; Dental Pulp/cytology*

Positional information plays a crucial role in embryonic pattern formation, yet its role in tooth development remains unexplored. In this study, we investigated the regional specification of lingual and buccal dental mesenchyme during tooth development. Tooth germs at the cap stage were dissected from mouse mandibles, and their lingual and buccal mesenchymal regions were separated for bulk RNA sequencing. Gene ontology analysis revealed that odontogenesis, pattern specification, and proliferation-related genes were enriched in the lingual mesenchyme, whereas stem cell development, mesenchymal differentiation, neural crest differentiation, and regeneration-related genes were predominant in the buccal mesenchyme. Reaggregation experiments using Wnt1^creERT/+; R26R^tdT/+ and WT mouse models demonstrated that lingual mesenchyme contributes to tooth formation, while buccal mesenchyme primarily supports surrounding tissues. Furthermore, only the lingual part of tooth germs exhibited odontogenic potential when cultured in vitro and transplanted under the kidney capsule. Bulk RNA transcriptomic analysis further validated the regional specification of the lingual and buccal mesenchyme. These findings provide novel insights into the molecular basis of positional information in tooth development and pattern formation.
Animals ; Mice ; Odontogenesis/genetics* ; Tooth Germ/cytology* ; Mesoderm/cytology* ; Cell Differentiation ; Mesenchymal Stem Cells ; Tooth/embryology*

Trophoblast cells serve as the foundation for placental development. We analyzed published multiomics sequencing data and found that trophoblast cells highly expressed RRS1 compared to primitive endoderm and epiblast. We used HTR-8/SVneo cells for further investigation, and Western blot and immunofluorescence staining confirmed that HTR-8/SVneo cells highly expressed RRS1. RRS1 was successfully knocked down in HTR-8/SVneo cells using siRNA. Using IncuCyte S3 live-cell analysis system based on continuous live-cell imaging and real-time data, we observed that proliferation, migration, and invasion abilities were all significantly decreased in RRS1-knockdown cells. RNA-seq revealed that knockdown of RRS1 affected the gene transcription, and upregulated pathways in extracellular matrix organization, DNA damage response, and intrinsic apoptotic signaling, downregulated pathways in embryo implantation, trophoblast cell migration, and wound healing. Differentially expressed genes were enriched in diseases related to placental development. Consistent with these findings, human chorionic villus samples collected from spontaneous abortion cases exhibited significantly reduced RRS1 expression compared to normal controls. Our results highlight the functional importance of RRS1 in human trophoblasts and suggest that its deficiency contributes to early pregnancy loss.
Humans ; Trophoblasts/physiology* ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Female ; Pregnancy ; Abortion, Spontaneous/metabolism* ; Cell Line ; Placentation/genetics*

OBJECTIVES

To validate a method in detecting SARS-CoV-2 via RT-qPCR in pregnant and non-pregnant samples other than nasopharyngeal swabs and/or oropharyngeal swabs such as cervical, rectal, amniotic fluid, placental, umbilical cord blood, and breastmilk.

We performed a validation experiment using MGI easy extraction kits and BGI PCR kits on non-conventional specimens, including cervical, rectal, amniotic fluid, placental, umbilical cord blood, and breastmilk to detect and confirm the presence of SARS-CoV-2. In addition, we tested the validated method on 572 purposively sampled field-collected non-conventional specimens from a cohort of 109 unvaccinated pregnant and 47 unvaccinated non-pregnant women to assess which candidate non-conventional maternal- and fetal-associated specimens may contribute to maternal-fetal viral vertical transmission.

Positive detection of SARS-CoV-2 viral RNA in non-conventional specimens was demonstrated and verified. Of the 572 non-conventional samples tested, 1.8% (10/572) were positively validated by RT-qPCR for SARS-CoV-2 in the maternal-associated specimens particularly the rectal (5), placental (1), and cervical (4) swabs among six pregnant and four non-pregnant individuals. In contrast, no SARS-CoV-2 viral RNA was detected in fetal-associated specimens.

The results of the validation study may serve as an additional diagnostic screening layer to support maternal-child care. Furthermore, viral detection in these non-conventional maternal specimens may also be utilized to provide guidance in the clinical management of neonates, and pregnant women during delivery.

Accumulating studies have demonstrated that non-coding RNAs (ncRNAs), functioning as important regulators of transcription and translation, are involved in the establishment and maintenance of pregnancy, especially the maternal immune adaptation process. The endometrial stromal cells (ESCs), trophoblast cells, and decidua immune cells that reside at the maternal-fetal interface are thought to play significant roles in normal pregnancy and pregnancy-associated diseases. Here, we reviewed the up-to-date evidence on how microRNA, long non-coding RNA, and circular RNA regulate ESCs, trophoblast cells, and immune cells and discussed the potential applications of these ncRNAs as diagnostic and therapeutic markers in pregnancy complications.
Pregnancy ; Female ; Humans ; MicroRNAs/genetics* ; RNA, Long Noncoding/genetics* ; RNA, Circular/genetics* ; Trophoblasts ; Pregnancy Complications/genetics*

To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.
Animals ; Mice ; Mice, Knockout ; CRISPR-Cas Systems ; Blood Pressure ; Gene Knockout Techniques ; Zygote