Single-cell and spatial transcriptomics reveal alterations in trophoblasts at invasion sites and disturbed myometrial immune microenvironment in placenta accreta spectrum disorders.

BACKGROUND: Placenta accreta spectrum disorders (PAS) are a severe complication characterized by abnormal trophoblast invasion into the myometrium. The underlying mechanisms of PAS involve a complex interplay of various cell types and molecular pathways. Despite its significance, both the characteristics and intricate mechanisms of this condition remain poorly understood. METHODS: Spatial transcriptomics (ST) and single-cell RNA sequencing (scRNA-seq), were performed on the tissue samples from four PAS patients, including invasive tissues (ST, n = 3; scRNA-seq, n = 4), non-invasive normal placenta samples (ST, n = 1; scRNA-seq, n = 2). Three healthy term pregnant women provided normal myometrium samples (ST, n = 1; scRNA-seq, n = 2). ST analysis characterized the spatial expression landscape, and scRNA-seq was used to identify specific cellular components in PAS. Immunofluorescence staining was conducted to validate the findings. RESULTS: ST slices distinctly showed the myometrium in PAS was invaded by three subpopulations of trophoblast cells, extravillous trophoblast cells, cytotrophoblasts, and syncytiotrophoblasts, especially extravillous trophoblast cells. The pathways enriched by genes in trophoblasts, smooth muscle cells (SMC), and immune cells of PAS were mainly associated with immune and inflammation. We identified elevated expression of the angiogenesis-stimulating gene PTK2, alongside the cell proliferation-enhancing gene EGFR, within the trophoblasts of PAS group. Trophoblasts mainly contributed the enhancement of HLA-G and EBI3 signaling, which is crucial in establishing immune escape. Meanwhile, SMC regions in PAS exhibited upregulation of immunomodulatory markers such as CD274, HAVCR2, and IDO1, with CD274 expression experimentally verified to be increased in the invasive SMC areas of the PAS group. CONCLUSIONS: This study provided information of cellular composition and spatial organization in PAS at single-cell and spatial level. The dysregulated expression of genes in PAS revealed a complex interplay between enhanced immune escape in trophoblasts and immune tolerance in SMCs during invasion in PAS. These findings will enhance our understanding of PAS pathogenesis for developing potential therapeutic strategies.

Exploring myometrial microenvironment changes at the single-cell level from nonpregnant to term pregnant states.

The microenvironment and cell populations within the myometrium play crucial roles in maintaining uterine structural integrity and protecting the fetus during pregnancy. However, the specific changes occurring at the single-cell level in the human myometrium between nonpregnant (NP) and term pregnant (TP) states remain unexplored. In this study, we used single-cell RNA sequencing (scRNA-Seq) and spatial transcriptomics (ST) to construct a transcriptomic atlas of individual cells in the myometrium of NP and TP women. Integrated analysis of scRNA-Seq and ST data revealed spatially distinct transcriptional characteristics and examined cell-to-cell communication patterns based on ligand-receptor interactions. We identified and categorized 87,845 high-quality individual cells into 12 populations from scRNA-Seq data of 12 human myometrium tissues. Our findings demonstrated alterations in the proportions of five subpopulations of smooth muscle cells in TP. Moreover, an increase in monocytic cells, particularly M2 macrophages, was observed in TP myometrium samples, suggesting their involvement in the anti-inflammatory response. This study provides unprecedented single-cell resolution of the NP and TP myometrium, offering new insights into myometrial remodeling during pregnancy.NEW & NOTEWORTHY Using single-cell RNA sequencing and spatial transcriptomics, the myometrium was examined at the single-cell level during pregnancy. We identified spatially distinct cell populations and observed alterations in smooth muscle cells and increased M2 macrophages in term pregnant women. These findings offer unprecedented insights into myometrial remodeling and the anti-inflammatory response during pregnancy. The study advances our understanding of pregnancy-related myometrial changes.

Thrombomodulin (TM), thrombin-antithrombin complex (TAT), plasmin-α2-plasmininhibitor complex (PIC), and tissue plasminogen activator-inhibitor complex (t-PAIC) assessment of fibrinolytic activity in postpartum hemorrhage: a retrospective comparative cohort study.

Background: Detecting the changes of coagulation function in the early stage of postpartum hemorrhage (PPH), which is the leading cause of maternal death, is the key to treatment this serious disease, however, there is less effective assessment methods. Thrombomodulin (TM), thrombin-antithrombin complex (TAT), plasmin-α2-plasmininhibitor complex (PIC), and tissue plasminogen activator-inhibitor complex (t-PAIC) are the new direct indicators for coagulation and fibrinolysis, and considered sensitive molecular markers of the fibrinolytic system changing. The aim of this study was to investigate the changes of these 4 new indicators in the early stage of PPH. Methods: We retrospectively reviewed the new coagulate indicators, TM, TAT, PIC, and t-PAIC, obtained from PPH patients at Guangzhou Women and Children's Medical Center from January 2021 to July 2021. According to the amount of blood loss, the patients were divided into 3 groups: Mild group (blood loss <1,500 mL, n=17), Severe group (blood loss ≥1,500 mL, n=24); another 12 women who did not experience PPH were selected as the Normal group (n=12). The four indicators were measured at the time of PPH happened, or immediately when baby was born in the Normal group, and evaluated for the prediction of PPH. Results: The t-PAIC level of the Severe group was significantly lower than the other 2 groups (Normal group vs. Mild group vs. Severe group: 13.9±2.0 vs. 9.4±1.8 vs. 7.5±0.9, P=0.020), and the PIC level was the highest (Normal group vs. Mild group vs. Severe group: 1.1±0.2 vs. 2.7±0.8 vs. 2.9±0.6, P=0.012). There was no significant difference in TM and TAT among the 3 groups. The odds ratio (OR) value of t-PAIC was 0.822, 95% confidence interval (CI): 0.697-0.970, P=0.020. The area under the curve (AUC) of PIC was 0.5, and t-PAIC was 0.775, the cut-off point was 6.295, the specificity was 75%, and the sensitivity was 75%. Conclusions: With the increasing of blood loss, t-PAIC decreased gradually, and is associated with severe PPH. This indicator may be a new predictor of PPH, and should be used in the early period of PPH for treatment.

Predicting poor outcomes and the need for surgical treatment in neonates with meconium peritonitis.

OBJECTIVE: The objective of this study is to determine factors associated with poor outcomes and the need for surgical treatment in neonates with meconium peritonitis (MP). METHODS: We evaluated the association between prenatal ultrasound features, maternal characteristics, and the likelihood of surgery, mortality, and serious morbidity in 49 neonates with a prenatal diagnosis of MP, who were born in Guangzhou Women and Children's Medical Center between January 2011 and December 2016. RESULTS: Thirty of 49 neonates (61.2%) required surgical treatment, and 17 (34.7%) had a poor outcome. Independent predictors of need for surgical treatment were polyhydramnios, maternal intrahepatic cholestasis of pregnancy (associated with lower risk), and persistence of peritoneal fluid. The model correctly predicted 70.0% of the neonates who required surgery (at a 10% false-positive rate; area under the curve [AUC]: 0.86 [95% CI, 0.75-0.97]). For poor outcomes, independent predictors were low gestational age at birth, persistence of peritoneal fluid, and polyhydramnios. For the latter, the model only achieved a detection rate of 52.9% (10% false-positive rate, AUC: 0.82 [95% CI, 0.70-0.94]). CONCLUSIONS: A combination of prenatal ultrasound features and maternal characteristics correctly predicted 70.0% the need for neonatal surgery. Prediction of poor outcome-based prenatal ultrasound features and gestational age did not perform well.

Dexmedetomidine Reduces Diabetic Neuropathy Pain in Rats through the Wnt 10a/ß-Catenin Signaling Pathway.

Diabetic neuropathy pain (DNP), a spontaneous pain with hyperalgesia and allodynia, greatly compromises patients' quality of life. Our previous study suggested that dexmedetomidine (DEX) can relieve hyperalgesia in rats by inhibiting inflammation and apoptosis at the level of the spinal cord. In the present study, we aimed to evaluate the role of Wnt 10a/ß-catenin signaling in DEX-induced alleviation of DNP in rats. Forty-eight rats were randomly allocated to four groups (n=12/group): control, DNP, DEX, and yohimbine groups. The DNP model was established by streptozotocin (STZ) injection. The effects of DEX with or without the α 2 adrenergic antagonist yohimbine were assessed by behavior tests (mechanical withdrawal threshold and thermal withdrawal latency). Spinal cord tissue was evaluated by immunofluorescence staining of astrocytes as well as for Wnt 10a and ß-catenin expression, western blot analysis of Wnt 10a and ß-catenin expression, and enzyme-linked immunosorbent assay measurement of proinflammatory cytokines (tumor necrosis factor-α and interleukin-1ß). Rats with STZ-induced DNP had a decreased pain threshold, activated astrocytes, increased expression of Wnt 10a and ß-catenin, and increased levels of proinflammatory cytokines compared to the control group, and these effects were ameliorated by treatment with DEX. Yohimbine administration partly abolished the protective effects of DEX in the DNP model rats. In conclusion, DEX alleviated DNP in rats by inhibiting inflammation and astrocyte activation, which may be attributed to downregulation of the Wnt 10a/ß-catenin signaling pathway.

The Therapeutic Effect of Dexmedetomidine on Rat Diabetic Neuropathy Pain and the Mechanism.

Diabetic neuropathy pain (DNP) is a common chronic complication of diabetes characterized by spontaneous pain, hyperalgesia and allodynia. Dexmedetomidine is a selective α2 adrenergic agonist that relieves sympathetic nervous tension and reduces the release of glutamate. Thus, it is possible that dexmedetomidine may relieve DNP as well. In this study, we examined the effect of dexmedetomidine on DNP in the presence or absence of the α2 adrenergic antagonist yohimbine in rats utilizing a streptozotocin (STZ)-induced diabetes as a model of DNP. To examine DNP, we examined behavior using the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) tests, and microglia and astrocyte activation was examined by immunofluorescence staining. The levels of pro-inflammatory cytokine tumor necrosis factor (TNF)-α and interleukin (IL)-1ß in the spinal cord were measured by enzyme-linked immunosorbent assay (ELISA). Cell apoptosis in spinal cord was examined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Glutamate production in caudal lumbar was measured by HPLC. We found that STZ-treated rats had decreased pain threshold, elevated activation of microglia but not astrocytes, increased level of pro-inflammatory cytokines, increased apoptosis and glutamate production compared to control animals, and these effects were ameliorated by dexmedetomidine treatment. Pretreatment of yohimbine abolished almost all of the protective effects of dexmedetomidine except for glutamate production. IN CONCLUSION: our data confirmed that dexmedetomidine can relieve hyperalgesia in diabetic neuropathy pain, and protect spinal cord cells from apoptotic death. The mechanism may be related to dexmedetomidine-mediated inhibition of microglia activation, reduction of inflammatory reaction in the spinal cord, and suppression of glutamate production.

IL-12 induced the generation of IL-21- and IFN-γ-co-expressing poly-functional CD4+ T cells from human naive CD4+ T cells.

Interleukine-12 is critical for the differentiation of Th1 cells and can improve the development of Th1 cells with Tfh cell features in mouse model. Human effector CD4(+) T cells also exhibit poly-functionality by co-expressing IL-21 and IFN-γ. However, the effects of IL-12 on regulating generation of human IL-21- and IFN-γ-expressing CD4(+) T cells are still incompletely understood. Our studies found that IL-12 but not IL-21 could induce the differentiation of human naive CD4(+) T cells into multi-cytokine expressing CD4(+) T cells in vitro, which co-expressed IL-21 and IFN-γ with or without IL-2 and TNF-α. At early stage of differentiation, addition of excess exogenous IFN-γ could increase the generation of IL-21- and IFN-γ-expressing CD4(+) T cells, furthermore, anti-IFN-γ depressed the percentage of poly-functional CD4(+) T cells. Phenotypically, IL-21(+)IFN-γ(+)CD4(+) T cells exhibited more characteristic features about both of Th1 and Tfh cells than IL-21 or IFN-γ single-expressing CD4(+) T cells. Mechamistically, IL-12 modulated the differentiation of IL-21(+)IFN-γ(+)CD4(+) T cells from naive CD4(+) T cells via the pathways of STAT-1/4, T-bet and BCL(-)6. Different from naive CD4(+) T cells, IL-12 increasing the generation of IL-21(+)IFN-γ(+)CD4(+) T cells from memory CD4(+) T cells was only involved in STAT-4 pathway but not STAT-1. Poly-functional CD4(+) T cells were contributed to generation and progress of varies diseases and our studies provide basic theoretics for the designs of vaccine and therapies of diseases.

Association of biochemical hyperandrogenism with type 2 diabetes and obesity in Chinese women with polycystic ovary syndrome.

OBJECTIVE: To evaluate the effect of hyperandrogenism on metabolic disorders among patients with polycystic ovary syndrome (PCOS) diagnosed using the Rotterdam criteria. METHODS: A retrospective analysis of the clinical records of 883 women with PCOS and 717 premenopausal controls identified from the general population. RESULTS: A total of 686 (77.7%) patients were classified with PCOS based on National Institutes of Health (NIH) criteria, and 164 out of 197 (83.2%) additional patients had no hyperandrogenism. Women with normal androgen levels exhibited lower frequencies of obesity, type 2 diabetes, acanthosis nigricans, genetic history of diabetes, and elevated Matsuda index compared with hyperandrogenic patients. Hyperandrogenemia, but not hirsutism, was independently associated with the risk for type 2 diabetes (odds ratio [OR] 5.7; P=0.028) and obesity (OR 1.7; P=0.005) among Chinese patients with PCOS. CONCLUSIONS: Hyperandrogenemia is associated with type 2 diabetes and obesity in Chinese women with PCOS and should be considered at first-line management of hyperandrogenism and infertility due to PCOS.

[Effect of anti-Müllerian hormone on P450 aromatase mRNA expression in cultured human luteinized granulose cells].

OBJECTIVE: To investigate the effect of anti-Müllerian hormone (AMH) on hormone secretion and P450 aromatase mRNA expression from cultured human luteinized granulosa cells. METHODS: Human luteinized granulose cells were derived from 10 patients treated by in vitro fertilization-embryo transplantation (IVF-ET) in the Second Affiliated Hospital of Sun Yat-sen University from June to December 2006. Granulose cells were divided into group A, B, C, D, E depending on different concentration of AMH, testosterone group and blank group. 1x10(-7) mol/L testosterone and 1, 5, 10, 20, 50 microg/L AMH were added into the culture medium of group A, B, C, D and E. 1x10(-7) mol/L testosterone was added into the culture medium of testosterone group while no other ingredient was added into the medium of blank group. Estrogen levels in supernates were measured at 24, 48, 72 hours after cell incubation. RT-PCR was performed to detect the P450 aromatase mRNA expression in group B, C, D, E and testosterone group at 72 hours after cell incubation. RESULTS: (1) Estrogen levels in supernates of granulose cell culture at 24, 48, 72 hours were (8.529+/-0.381)x10(4), (10.977+/-0.436)x10(4), (13.309+/-0.506)x10(4) pmol/L in group A, (7.027+/-0.276)x10(4), (9.167+/-0.300)x10(4), (10.794+/-0.555)x10(4) pmol/L in group B, (6.039+/-0.226)x10(4), (7.585+/-0.548)x10(4), (8.797+/-0.518)x10(4) pmol/L in group C, (5.118+/-0.460)x10(4), (5.716+/-0.496)x10(4), (6.205+/-0.667)x10(4) pmol/L in group D, (4.932+/-0.148)x10(4), (5.323+/-0.184)x10(4), (5.629+/-0.212)x10(4) pmol/L in group E. When compared with blank group [(0.001+/-0.001)x10(4), (0.006+/-0.003)x10(4), (0.029+/-0.011)x10(4) pmol/L], the statistical differences were observed in group A, B, C, D, E (P<0.01); when compared with testosterone group [(8.418+/-0.569)x10(4), (10.841+/-0.689)x10(4), (13.301+/-0.637)x10(4) pmol/L], the statistical differences were observed in group B, C, D and E (P<0.01); statistical differences were also observed in group C, D and E when compared with group B, and also group D and E when compared with group C (P<0.01). No significant difference was observed between group D and E (P>0.05). In group A, B, C, D, E and testosterone group, the estrogen levels at 24 hours after cell culture were significantly lower than those at 48 and 72 hours (P<0.01); statistical difference was observed between estrogen levels at 48 and 72 hours (P<0.01). No significant difference was observed among 24, 48 and 72 hours in blank group (P>0.05). (2) Relative ratios of intensity of P450 aromatase/beta-actin at 72 hours of cell culture in group B, C, D and E were 0.6148+/-0.0046, 0.5156+/-0.0012, 0.4698+/-0.0027 and 0.4282+/-0.0017, respectively, which were statistically lower than that in testosterone group (0.8224+/-0.0021, P<0.01); statistical differences were also observed in group C, D and E when compared with group B, and also group D and E when compared with group C (P<0.01). No significant difference was observed between group D and E (P>0.05). CONCLUSION: It is suggested that AMH might affect estrogen synthesis by inhibiting P450 aromatose activity so that lead to hyperandrogenism microenvironment in local ovary.

Low prevalence of the metabolic syndrome but high occurrence of various metabolic disorders in Chinese women with polycystic ovary syndrome.

OBJECTIVE: Variations in the prevalence of metabolic syndrome (MetS) among women with polycystic ovary syndrome (PCOS) in different races were reported. We sought to report this prevalence and its components in Chinese women with PCOS and compared these characteristics with healthy controls. DESIGN: Anthropometric measurements and biochemical parameters were evaluated in 578 PCOS patients diagnosed by the Rotterdam criteria and 281 age- and body mass index (BMI)-matched controls. International Diabetes Federation criteria for MetS were used. RESULTS: The prevalence of MetS was 16.8% in this study, and 60.7% of patients displayed at least one component of MetS. Among the patients, the rates of dyslipidemia, impaired fasting glucose, and elevated blood pressure were 41.6, 19.8, and 16.1% respectively; the rates of these corresponding components in age- and BMI-matched controls were 14.6, 5.3, and 5.7% respectively. In PCOS patients, the prevalence of MetS was 0.0, 3.9, 20.2, and 51.1% for four different BMI groups respectively; the prevalence of MetS was 7.3, 14.9, 24.2, and 42.4% in the four age groups respectively. Nearly 90% of patients diagnosed with MetS belonged to overweight and obese groups. BMI and age rather than free testosterone, free androgen index, fasting insulin, or sex hormone-binding globulin were included in formulation for predicting MetS according to multivariable logistic regression. CONCLUSIONS: Low prevalence of MetS but high occurrence of various metabolic disorders was found in women with PCOS compared with age- and BMI-matched controls in this study. BMI and age appeared to contribute more to developing MetS than other parameters associated with insulin resistance or hyperandrogenism.