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

Successful cloning through somatic cell nuclear transfer (SCNT) faces significant challenges due to epigenetic obstacles. Recent studies have highlighted the roles of H3K4me3 and H3K27me3 as potential contributors to these obstacles. However, the underlying mechanisms remain largely unclear. In this study, we generated genome-wide maps of H3K4me3 and H3K27me3 in mouse pre-implantation NT embryos. Our analysis revealed that aberrantly over-represented broad H3K4me3 domain and H3K27me3 signal lead to increased bivalent marks at gene promoters in NT embryos compared with naturally fertilized (NF) embryos at the 2-cell stage, which may link to relatively low levels of H3K36me3 in NT 2-cell embryos. Notably, the overexpression of Setd2, a H3K36me3 methyltransferase, successfully restored multiple epigenetic marks, including H3K36me3, H3K4me3, and H3K27me3. In addition, it reinstated the expression levels of ZGA-related genes by reestablishing H3K36me3 at gene body regions, which excluded H3K27me3 from bivalent promoters, ultimately improving cloning efficiency. These findings highlight the excessive bivalent state at gene promoters as a potent barrier and emphasize the removal of these barriers as a promising approach for achieving higher cloning efficiency.
Animals ; Mice ; Histone-Lysine N-Methyltransferase/biosynthesis* ; Histones/genetics* ; Nuclear Transfer Techniques ; Female ; Gene Expression Regulation, Developmental ; Promoter Regions, Genetic ; Epigenesis, Genetic ; Embryo, Mammalian/metabolism*

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*

OBJECTIVE: To assess the value of single sperm sequencing in preimplantation genetic testing for monogenic disease (PGT-M). METHODS: A Chinese couple with two children whom had died of Spinal muscular atrophy (SMA) and attended the Jiangxi Provincial Maternal and Child Health Care Hospital in June 2020 was selected as the subject. Eleven single sperm samples were isolated by mechanical immobilization and subjected to whole genome amplification. Real-time PCR and Sanger sequencing were used to detect the SMN1 variants in the single sperm samples. Genomic DNA of the wife, her parents and the husband, as well as one single sperm sample harboring the SMN1 variant and two single sperm samples without the variant were used for the linkage analysis. Targeted capture and high-throughput sequencing were carried out to test 100 single nucleotide polymorphisms distributed within 2 Mb up- and downstream the variant site. The haplotypes linked with the SMN1 variants were determined by linkage analysis. Blastocyst embryos were harvested after fertilizing by intracytoplasmic sperm injection. Cells from the trophoblasts of each embryo were biopsied and subjected to whole genome amplification and targeted capture and high-throughput sequencing to determine their carrier status. Chromosomal aneuploidy of wild-type embryos was excluded. An euploid embryo of high quality was transferred. Amniotic fluid sample was taken at 18 weeks of gestation to confirm the status of the fetus. RESULTS: Genetic testing showed that the couple both had deletion of exons 7 ~ 8 of the SMN1 gene. The wife has inherited the deletion from her father, while the husband was de novo. The haplotypes of the husband were successfully constructed by single sperm sequencing. Preimplantation genetic testing has indicated that 5 embryos had harbored the heterozygous variant, 4 embryos were of the wild type, among which 3 were euploid. Prenatal diagnosis during the second trimester of pregnancy has confirmed that the fetus did not carry the deletion. CONCLUSION By single sperm sequencing and PGT-M, the birth of further affected child has been successfully avoided.
Humans ; Pregnancy ; Female ; Child ; Male ; Preimplantation Diagnosis ; East Asian People ; Semen ; Genetic Testing ; Muscular Atrophy, Spinal/genetics* ; Aneuploidy ; Blastocyst/pathology* ; High-Throughput Nucleotide Sequencing ; Spermatozoa

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*

Humans ; Consensus ; Blastocyst ; Embryonic Development ; Fertilization in Vitro

Pregnancy ; Female ; Humans ; Placenta ; Nuclear Transfer Techniques ; Embryo, Mammalian ; Cell Nucleus

Objective: To investigate the effect of embryo quality at different developmental stages on the secondary sex ratio (SSR) of single live birth neonates. Methods: Data for patients with singleton live births after embryo transferred between January 2016 and January 2022 were retrospectively analyzed. The effect of embryo quality at different development stages on the SSR of 11 713 singleton live births were investigated. The association of SSR and embryo quality at different development stages was examined in univariate analysis and in a multivariate logistic regression model, after adjustment for confounders, using two models (Ⅰ and Ⅱ). Results: The age of both male and female, body mass index of both male and female, basal follicle stimulating hormone and estradiol, smoking of male, methods of insemination, methods of sperm extraction, types of transfer cycle and the number of embryo transferred were not related with SSR (all P>0.05). After adjustment for confounders, the probability of a male live birth was higher after transfer of good-quality blastula than after transfer of poorer-quality blastula (model Ⅰ: aOR=0.73, 95%CI: 0.65-0.82, P<0.001; model Ⅱ: aOR=0.73, 95%CI: 0.65-0.82, P<0.001). The quality of cleavage stage embryo was not associated with SSR (model Ⅰ: aOR=0.99, 95%CI: 0.87-1.13, P=0.937; model Ⅱ: aOR=0.99, 95%CI: 0.87-1.13, P=0.899). Conclusions: The SSR of singleton live births after embryo transfer is not correlated with the quality of cleavage stage embryo, but is correlated with the quality of blastula. Good-quality blastula transfer is more likely to result in a male live birth.
Infant, Newborn ; Pregnancy ; Humans ; Male ; Female ; Live Birth ; Retrospective Studies ; Sex Ratio ; Semen ; Blastocyst

OBJECTIVE: To compare the prevalence of chromosomal aneuploidies and pregnancy outcomes of D5 and D6 blastocysts subjected to preimplantation genetic testing for aneuploidy (PGT-A). METHODS: Clinical and laboratory data of 268 couples who underwent PGT-A at the Reproductive Center of the First Affiliated Hospital of Zhengzhou University from September 2018 to September 2020 were collected. The prevalence of chromosomal aneuploidies and pregnancy outcomes of D5/D6 biopsied blastocysts were compared. RESULTS: Compared with D6 blastocysts, the euploidy rate of D5 blastocysts was significantly higher (49.1% vs. 41.1%, P = 0.001 1), whilst their aneuploidy rate was significantly lower (50.9% vs. 58.9%, P = 0.001 1). The rate of numerical abnormalities of D6 blastocysts was significantly higher than that of D5 blastocysts (27.9% vs. 20.2%, P = 0.000 5). For patients under 35 years old, the euploidy rate of D5 blastocysts was significantly higher than that of D6 blastocysts (53.8% vs. 44.3%, P = 0.001), whilst the numerical abnormality rate was significantly lower (16.3% vs. 23.9%, P = 0.001). For both D5 and D6 blastocysts, the euploidy rates for patients <= 35 were significantly higher than those for > 35. The elder group had the lowest rates for aneuploidies and live births. Compared with those receiving D6 blastocysts transplantation, the pregnancy rate, implantation rate and live birth rate for those receiving thawed D5 blastocysts transplantation were significantly higher (60.2% vs.37.0%, P = 0.000 3; 59.1% vs.37.0%, P = 0.000 6; 47.7% vs. 28.3%, P = 0.002). CONCLUSION For patients undergoing PGT-A, the chromosomal euploidy rate for D5 blastocysts is higher than that for D6 blastocysts, and the clinical outcome of D5 blastocysts with normal signal is better than that of D6 blastocysts. Elder patients have a higher rate of aneuploidies.
Female ; Pregnancy ; Humans ; Aged ; Adult ; Pregnancy Outcome ; Aneuploidy ; Blastocyst ; Genetic Testing ; Laboratories

OBJECTIVE: To assess the value of using flat-sided culture tubes for preparing chromosomes through chorionic villi (CV) and amniotic fluid (AF) cell cultures during prenatal diagnosis. METHODS: From February to March 2020, 157 CV samples and 147 AF samples subjected to prenatal diagnosis at the Maternal and Child Health Care Hospital of Guangxi Zhuang Autonomous Region were selected as the study subjects. For each sample, one flat-sided tube and one flask culture were set up by following the standard protocols. The methods were evaluated by comparing the cell growth, experimental process, quality of chromosome preparation and costs. RESULTS: The success rates for the culturing of CV and AF samples by the flat-sided culture tube method were 97.45% (153/157) and 97.96% (144/147), respectively. By contrast, the success rates for the conventional flask method were 98.72% (155/157) for CV and 98.64% (145/147) for AF samples. No significant difference was found between the two methods (P > 0.05). The average harvest time required by the flat-sided culture tube method was 8.45 days for CV and 9.43 days for AF cultures, whilst the average harvest time for conventional flask method was 9.05 days and 9.54 days, respectively. The flat-sided culture tube method for CV had required significantly shorter average harvest time than the conventional method (P < 0.001). No statistical significant difference was found in the average harvest time for AF by the two methods (P > 0.05). The conventional culturing method had required three containers with two sample transfers. By contrast, the flat-sided culture tube method was carried out in one tube without any sample transfer. The average total amount of medium used was 3.91 mL for each flat-sided culture tube and 6.26 mL for each conventional flask. CONCLUSION The flat-sided culture tube method can provide a simple, cost-effective and error-reducing procedure for the CV and AF samples culture during prenatal diagnosis.
Child ; Female ; Pregnancy ; Humans ; China ; Prenatal Diagnosis ; Chorionic Villi Sampling ; Amniotic Fluid ; Cell Proliferation