Zhibai dihuang pill (ZBDH) exhibits therapeutic effects on idiopathic central sexual precocity in rats by modulating the gut microflora.

To reveal the role of gut microbiota (GM) in the occurrence and development of idiopathic central precocious puberty (ICPP) using 16S rDNA sequencing and bioinformatics analysis. The Danazol-induced ICPP model was successfully constructed in this study. ZBDH and GnRHa treatments could effectively inhibit ICPP in rats, as manifested by the delayed vaginal opening time, reduced weight, decreased uterine organ coefficient, and decreased uterine wall thickness and corpus luteum number, as well as remarkably reduced serum hormone (LH, FSH, and E2) levels. According to 16S rDNA sequencing analysis results, there was no significant difference in the GM community diversity across different groups; however, the composition of the microbial community and the abundance of the dominant microbial community were dramatically different among groups. ZBDH and GnRHa treatments could effectively reduce the abundance of Muribaculateae and Lactobacillus and promote Prevotella abundance. ZBDH and GnRHa were effective in treating Danazol-induced ICPP model rats. The therapeutic effects of ZBDH and GnRHa could be related to the changes in GM in rats.

CD24+ decidual stromal cells: a novel heterogeneous population with impaired regulatory T cell induction and potential association with recurrent miscarriage.

OBJECTIVE: To explore the heterogeneity of CD24+ decidual stromal cells (DSCs) in patients with recurrent miscarriages (RMs). DESIGN: We have discerned that the expression of CD24 serves to differentiate two stable and functionally distinct lineages of DSCs. The heterogeneity of CD24+ DSCs has been scrutinized, encompassing variances in stromal markers, transcriptional profiles, metabolic activity, and immune regulation. SETTING: Department of Reproductive Immunology, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University; Shanghai Institute of Immunity and Infection, Chinese Academy of Science. PATIENTS: A total of 129 early decidual samples were obtained, comprising 36 from healthy donors and 93 from patients with RMs. Blood samples were collected before the surgical procedure. Paraffin-embedded segments from 20 decidual samples of patients with RMs were obtained. INTERVENTIONS: None. MAIN OUTCOME MEASURES: The flow cytometry was used to quantify the expression of CD24+ DSCs in both healthy donors and patients with RMs, although it also evaluated the cellular heterogeneity. To ascertain the transcriptomic profiles of CD24+ DSCs by reanalyzing our single-cell transcriptomic data. Additionally, to measure the metabolomic activity of CD24+ DSCs from patients with RMs, ultraperformance liquid chromatography-mass spectrometry was employed. Through the implementation of a coculture system, we unraveled the role of CD24+ DSCs in immune regulation. RESULTS: Patients with RMs exhibit a notable enrichment of CD24+ DSCs, revealing a pronounced heterogeneity characterized by variations in stromal markers and transcriptional profiles. The heightened enrichment of CD24+ DSCs may play a pivotal role in triggering decidual inflammation and dysfunction in decidualization. Furthermore, CD24+ DSCs showed diverse metabolic activities and impeded the induction of naïve CD4+ T cells into regulatory T cells through the abundant secretion of 3-hydroxyisovaleric acid. Finally, our investigations have revealed that intraperitoneal administration of 3-hydroxyisovaleric acid in mouse models can elevate the risk of RM. CONCLUSION: We have successfully identified a disease-associated subset of CD24+ decidual stromal cells that could potentially contribute to the development of RM through the impairment of decidual immune tolerance. Targeting these specific CD24+ DSCs might hold promising prospects for therapeutic interventions in the clinical management of RM.

The association of APOH and NCF1 polymorphisms on susceptibility to recurrent pregnancy loss in women with antiphospholipid syndrome.

BACKGROUND: Recurrent pregnancy loss (RPL) is the main manifestation of pathological pregnancy in antiphospholipid syndrome (APS) women. The immune state plays a significant role in the occurrence/development of APS and RPL susceptibility, but there is little research on genetic factors. METHOD: Previous studies have described the important role of APOH and NCF1 in APS and pregnancy. To explore the association of APOH and NCF1 gene variants with RPL susceptibility in APS patients, we collected and analyzed 871 controls, 182 APS and RPL, and 231 RPL patients. Four single nucleotide polymorphisms (SNPs) (rs1801690, rs52797880, and rs8178847 of APOH and rs201802880 of NCF1) were selected and genotyped. RESULTS: We found rs1801690 (p = 0.001, p = 0.003), rs52797880 (p = 8.73e-04, p = 0.001), and rs8178847 (p = 0.001, p = 0.001) of APOH and rs201802880 (p = 3.77e-26, p = 1.31e-26) of NCF1 showed significant differences between APS and RPL patients and controls in allelic and genotype frequencies respectively. Moreover, rs1801690, rs52797880, and rs8178847 showed strong linkage disequilibrium. Especially, our results revealed a complete linkage disequilibrium (D' = 1) between rs52797880 and rs8178847. Furthermore, higher serum TP (total protein) level was described in APOH rs1801690 CG/GG (p = 0.007), rs52797880 AG/GG (p = 0.033), and rs8178847 CT/TT (p = 0.033), while the higher frequency of positive serum ACA-IgM was found in NCF1 rs201802880 GA (p = 0.017) in APS and RPL patients. CONCLUSION: Rs1801690, rs52797880, and rs8178847 of APOH and rs201802880 of NCF1 were associated with RPL susceptibility in APS patients.

Single-cell profiling reveals mechanisms of uncontrolled inflammation and glycolysis in decidual stromal cell subtypes in recurrent miscarriage.

STUDY QUESTION: Do distinct subpopulations of decidual stromal cells (DSCs) exist and if so, are given subpopulations enriched in recurrent miscarriage (RM)? SUMMARY ANSWER: Three subpopulations of DSCs were identified from which inflammatory DSCs (iDSCs) and glycolytic DSCs (glyDSCs) are significantly enriched in RM, with implicated roles in driving decidual inflammation and immune dysregulation. WHAT IS KNOWN ALREADY: DSCs play crucial roles in establishing and maintaining a successful pregnancy; dysfunction of DSCs has been considered as one of the key reasons for the development of RM. STUDY DESIGN, SIZE, DURATION: We collected 15 early decidual samples from five healthy donors (HDs) and ten RM patients to perform single-cell RNA sequencing (scRNA-seq). A total of 43 RM patients and 37 HDs were enrolled in the validation cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS: Non-immune cells and immune cells of decidual tissues were sorted by flow cytometry to perform scRNA-seq. We used tissue microarrays (TMA) to validate three distinct subpopulations of DSCs. The expression of inflammatory and glycolytic proteins by DSCs was validated by immunohistochemistry (IHC) and multiplex immunohistochemistry (mIHC). Different subsets of decidual NK (dNK) cells and macrophages were also validated by multicolor flow cytometry and mIHC. Cell ligand-receptor and spatial analyses between DSCs and immune cells were analyzed by mIHC. MAIN RESULTS AND THE ROLE OF CHANCE: We classify the DSCs into three subtypes based on scRNA-seq data: myofibroblastic (myDSCs), inflammatory (iDSCs) and glycolytic (glyDSCs), with the latter two being significantly enriched in RM patients. The distribution patterns of DSC subtypes in the RM and HD groups were validated by mIHC. Single-cell analyses indicate that the differentiation of iDSCs and glyDSCs may be coupled with the degrees of hypoxia. Consequently, we propose a pathological model in which a vicious circle is formed and fueled by hypoxic stress, uncontrolled inflammation and aberrant glycolysis. Furthermore, our results show that the inflammatory SPP1+ macrophages and CD18+ dNK cells are preferentially increased in the decidua of RM patients. Cell ligand-receptor and mIHC spatial analyses uncovered close interactions between pathogenic DSCs and inflammatory SPP1+ macrophages and CD18+ NK cells in RM patients. LARGE SCALE DATA: The raw single-cell sequence data reported in this paper were deposited at the National Omics Data Encyclopedia (www.biosino.org), under the accession number OEP002901. LIMITATIONS, REASONS FOR CAUTION: The number of decidual samples for scRNA-seq was limited and in-depth functional studies on DSCs are warranted in future studies. WIDER IMPLICATIONS OF THE FINDINGS: Identification of three DSC subpopulations opens new avenues for further investigation of their roles in RM patients. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Strategic Priority Research Program (No. XDB29030302), Frontier Science Key Research Project (QYZDB-SSW-SMC036), Chinese Academy of Sciences; National Key Research and Development Program of China (2021YFE0200600), National Natural Science Foundation of China (No. 31770960), Shanghai Municipal Science and Technology Major Project (No. 2019SHZDZX02, HS2021SHZX001), and Shanghai Committee of Science and Technology (17411967800). All authors report no conflict of interest.

IQUB deficiency causes male infertility by affecting the activity of p-ERK1/2/RSPH3.

STUDY QUESTION: Can new genetic factors responsible for male infertility be identified, especially for those characterized by asthenospermia despite normal sperm morphology? SUMMARY ANSWER: We identified the novel pathogenetic gene IQ motif and ubiquitin-like domain-containing (IQUB) as responsible for male infertility characterized by asthenospermia, involving sperm radial spoke defects. WHAT IS KNOWN ALREADY: To date, only a few genes have been found to be responsible for asthenospermia with normal sperm morphology. Iqub, encoding the IQUB protein, is highly and specifically expressed in murine testes and interacts with the proteins radial spoke head 3 (RSPH3), CEP295 N-terminal like (CEP295NL or DDC8), glutathione S-transferase mu 1 (GSTM1) and outer dense fiber of sperm tails 1 (ODF1) in the yeast two-hybrid system. STUDY DESIGN, SIZE, DURATION: The IQUB variant was identified by whole-exome sequencing in a cohort of 126 male infertility patients with typical asthenospermia recruited between 2015 and 2020. Knockout (KO) and knockin (KI) mouse models, scanning and transmission electron microscopy (TEM), and other functional assays were performed, between 2019 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: The IQUB variant was identified by whole-exome sequencing and confirmed by Sanger sequencing. Iqub KO and KI mice were constructed to mimic the phenotype of the affected individual. After recapitulating the phenotype of human male infertility, scanning and TEM were performed to check the ultrastructure of the sperm. Western blot and co-immunoprecipitation were performed to clarify the pathological mechanism of the IQUB variant. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a homozygous nonsense IQUB variant (NM_001282855.2:c.942T> G(p.Tyr314*)) from an infertile male. Iqub KO and KI mice mimicked the infertility phenotype and confirmed IQUB to be the pathogenetic gene. Scanning and TEM showed that sperm of both the mouse models and the affected individual had radial spoke defects. The functional assay suggested that IQUB may recruit calmodulin in lower Ca2+ environments to facilitate the normal assembly of radial spokes by inhibiting the activity of RSPH3/p-ERK1/2 (a nontypical AKAP (A-Kinase Anchoring Protein) forming by RSPH3 and phosphorylation of extracellular signal-regulated kinase 1 and 2 (p-ERK1/2)). LIMITATIONS, REASONS FOR CAUTION: Additional cases are needed to confirm the genetic contribution of IQUB variants to male infertility. In addition, because no IQUB antibody is available for immunofluorescence and the polyclonal antibody we generated was only effective in western blotting, immunostaining for IQUB was not performed in this study. Therefore, this study lacks direct in vivo proof to confirm the effect of the variant on IQUB protein level. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest a causal relation between IQUB variants and male infertility owing to asthenospermia, and partly clarify the pathological mechanism of IQUB variants. This expands our knowledge of the genes involved in human sperm asthenospermia and potentially provides a new genetic marker for male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Development Program of China (2021YFC2700100), the National Natural Science Foundation of China (32130029, 82171643, 81971450, 82001538, and 81971382) and the Guangdong Science and Technology Department Guangdong-Hong Kong-Macao Joint Innovation Project (2020A0505140003). There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

The peripheral and decidual immune cell profiles in women with recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) affects 1-2% of couples of reproductive age. Immunological analysis of the immune status in RPL patients might contribute to the diagnosis and treatment of RPL. However, the exact immune cell composition in RPL patients is still unclear. Here, we used flow cytometry to investigate the immune cell profiles of peripheral blood and decidual tissue of women who experienced RPL. We divided peripheral immune cells into 14 major subgroups, and the percentages of T, natural killer T (NKT)-like and B cells in peripheral blood were increased in RPL patients. The decidual immune cells were classified into 14 major subpopulations and the percentages of decidual T, NKT-like cells and CD11chi Mφ were increased, while those of CD56hi decidual NK cells and CD11clo Mφ were decreased in RPL patients. The spearmen correlation analysis showed that the proportion of peripheral and decidual immune cells did not show significant correlations with occurrences of previous miscarriages. By using flow cytometry, we depicted the global peripheral and decidual immune landscape in RPL patients. The abnormalities of peripheral and decidual immune cells may be involved in RPL, but the correlations with the number of previous miscarriages need further verification.

Research progress on ANXA5 in recurrent pregnancy loss.

The incidence of recurrent pregnancy loss (RPL) in fertile women ranges from 1% to 5%, of which about 50% of them are due to unknown causes. The possible pathogenesis of RPL is an urgent problem to be solved in the clinical. Mutations or polymorphisms of certain genes in the coagulation mechanism are associated with susceptibility to thrombotic diseases and are one of the main reasons for the occurrence of RPL. Among them, the ANXA5 gene was newly studied and some single nucleotide polymorphisms (SNPs) in the promoter region of ANXA5 have been reported to be associated with RPL in multiple races. In this review, we summarized the research progress on the correlation between the SNPs in ANXA5 and RPL, hoping to provide some valuable guidance for the future studies.

Assessment of the Levels of Peripheral Lymphocytes Subpopulation in Healthy Women of Childbearing Age: a Prospective Cohort Study.

BACKGROUND: To evaluate changes in the levels of the peripheral lymphocyte subpopulation (PBLS) during the menstrual cycle in healthy women of childbearing age (HWOBA), for establishing a normal reference range. METHODS: Flow cytometric analysis was performed for the assessment of the levels of PBLS during one menstrual cycle in HWOBA. Moreover, ovulation was monitored. The multivariable logistic regression models were used to evaluate the effects of age and body mass index (BMI) on PBLS. RESULTS: The levels of PBLS including CD3+, CD3+CD4+, CD3+CD8+, CD19+, CD16+CD56+ lymphocytes and ratio of Th1/Th2 cells, were relatively stable during the menstrual cycle. However, each increment in age (year) was proportional to a decrease (14%) in the level of CD8+ T cells and an increase (10%) in the level of CD19+ B cells (p < 0.05). Increased BMI was inversely proportional to the level of CD16+CD56+ natural killer (NK) cells. Thus, the normal reference intervals for PBLS in HWOBA were established. CONCLUSIONS: The levels of PBLS can be used for daily monitoring of immune status in HWOBA. Notably, age and BMI may affect PBLS level. The normal reference ranges for PBLS levels can provide a basis for the research and treatment of recurrent pregnancy loss.

Hyperglycemia affects global 5-methylcytosine and 5-hydroxymethylcytosine in blood genomic DNA through upregulation of SIRT6 and TETs.

BACKGROUND: Accumulating evidence suggests that epigenetic changes play key roles in the pathogenesis of type 2 diabetes mellitus (T2DM). However, the dynamic regulation of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in diabetic peripheral blood DNA remains to be elucidated. RESULTS: We collected fasting blood samples (104 patients and 108 healthy controls) and glucose-stimulated blood samples at different time points (11 patients and 5 healthy controls underwent oral glucose tolerance test (OGTT)), as well as blood samples from six couples of diabetic and control rats. A HPLC-MS/MS system was used for quantifying global 5mC and 5hmC in genomic DNA from white blood cells (WBCs), and qPCR was performed for detecting mRNA expression of SIRT6 and TETs. We found that global 5mC decreased, while global 5hmC increased in both patients and diabetic rats, with lower 5mC being a risk factor of T2DM (OR = 0.524, 95%CI 0.402-0.683, p = 1.64 × 10-6). The OGTT data from patients showed that 5mC declined within 1 h and then returned to the fasting status at 2 h, while 5hmC rose from 0.5 h to 3 h with increasing glucose. However, the similar patterns were not found in the controls. The mRNA expression of TET2, TET3, and SIRT6 was upregulated in patients (p = 0.012, p = 0.026, and p = 0.035, respectively). The similar results were observed in diabetic OGTT and rats. Correlation analysis indicated that SIRT6 was positively correlated with TET2 in humans (r = 0.277, p < 0.001) and rats (r = 0.942, p < 0.001), in addition to a correlation between glucose and SIRT6 (r = 0.162, p = 0.045) and TET2 (r = 0.174, p = 0.036). CONCLUSIONS: Hyperglycemia appeared to promote the mRNA expression of SIRT6 and TETs, which in turn might cause the dynamic changes of 5mC and 5hmC in WBCs from T2DM patients.

B-RCA revealed circulating miR-33a/b associates with serum cholesterol in type 2 diabetes patients at high risk of ASCVD.

AIMS: Type 2 diabetes (T2D) is a complex metabolic disease with high incidence throughout the world. Dyslipidemia is the leading cause of atherosclerotic cardiovascular diseases (ASCVD) in T2D patients. hsa-miR-33 (miR-33) serves as a regulator in lipid metabolism. We hypothesized that blood miR-33 associates with serum lipids in T2D patients at high risk of ASCVD events. METHODS: We developed a branched rolling circle amplification (B-RCA) method and assessed its sensitivity and specificity with miR-33a/b standards by traditional TaqMan assay. Circulating miR-33a/b level was then determined with B-RCA in 30 T2D patients at high risk for developing ASCVD and 33 healthy controls. Pearson correlation coefficient was used to evaluate the correlation between circulating miR-33a/b and serum cholesterol. RESULTS: Compared with TaqMan assay, B-RCA method showed a similar specificity and a 100-fold higher sensitivity for miR-33a detection. Circulating miR-33a/b level is positively correlated with serum total cholesterol (TC) (r = 0.364, p = 0.048) and low-density lipoprotein cholesterol (LDL-C) (r = 0.383, p = 0.037) in T2D patients at high risk for developing ASCVD. CONCLUSIONS: Our B-RCA method provided an alternative strategy with specificity and high sensitivity for circulating miRNAs detection, and the results demonstrated that miR-33a/b might play an important role in cholesterol regulation.