Vitrification preservation of good-quality blastocysts for more than 5 years reduces implantation and live birth rates.

STUDY QUESTION: Does vitrification cryopreservation of embryos for more than 5 years affect the pregnancy outcomes after frozen embryo transfer (FET)? SUMMARY ANSWER: Vitrification cryopreservation of good-quality blastocysts for more than 5 years is associated with a decrease in the implantation rate (IR) and live birth rate (LBR). WHAT IS KNOWN ALREADY: Previous studies have predominantly focused on embryos cryopreserved for relatively short durations (less than 5 years), yet the impact of extended cryopreservation duration on pregnancy outcomes remains a controversial issue. There is a relative scarcity of data regarding the efficacy and safety of storing embryos for 5 years or longer. STUDY DESIGN, SIZE, DURATION: This retrospective study involved 36 665 eligible vitrified-thawed embryo transfer cycles from 1 January 2016 to 31 December 2022, at a single fertility center in China. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were divided into three groups according to embryo storage time: Group 1 consisted of 31 565 cycles, with storage time of 0-2 years; Group 2 consisted of 4458 cycles, with a storage time of 2-5 years; and Group 3 included 642 cycles, with storage time exceeding 5 years. The main outcome measures were IR and LBR. Secondary outcome variables included rates of biochemical pregnancy, multiple pregnancy, ectopic pregnancy, and miscarriage, as well as neonatal outcomes. Reproductive outcomes were analyzed as binary variables. Multivariate logistic regression analysis was used to explore the effect of preservation time on pregnancy outcomes after correcting for confounding factors. In addition, we also assessed neonatal outcomes, such as large for gestational age (LGA) and small for gestational age (SGA). MAIN RESULTS AND THE ROLE OF CHANCE: IRs in the three groups (0-2, 2-5, and >5 years) were 37.37%, 39.03%, and 35.78%, respectively (P = 0.017), and LBRs in the three groups were 37.29%, 39.09%, and 34.91%, respectively (P = 0.028). After adjustment for potential confounding factors, compared with the 0-2 years storage group, prolonged embryo vitrification preservation time (2-5 years or >5 years) did not affect secondary outcomes such as rates of biochemical pregnancy, multiple pregnancy, ectopic pregnancy, and miscarriage (P > 0.05). But cryopreservation of embryos for more than 5 years reduced the IR (adjusted odds ratio (aOR) 0.82, 95% CI 0.69-0.97, P = 0.020) and LBR (aOR 0.76, 95% CI 0.64-0.91, P = 0.002). Multivariate stratified analysis also showed that prolonging the cryopreservation time of blastocysts (>5 years) reduced the IR (aOR 0.78, 95% CI 0.62-0.98, P = 0.033) and LBR (aOR 0.68, 95% CI 0.53-0.87, P = 0.002). However, no effect on cleavage embryos was observed (P > 0.05). We further conducted stratified analyses based on the number and quality of frozen blastocysts transferred, and the results showed that the FET results after transfers of good-quality blastocysts in the >5 years storage group were negatively affected. However, the storage time of non-good-quality blastocysts was not significantly associated with pregnancy outcomes. Regarding the neonatal outcomes (of singletons), embryo vitrification preservation time had no effect on preterm birth rates, fetal birth weight, or neonatal sex ratios. However, as the storage time increased, rates of SGA (5.60%, 4.10%, and 1.18%) decreased, while rates of LGA (5.22%, 6.75%, and 9.47%) increased (P < 0.05). After adjusting for confounding factors, the increase in LGA and the decrease in SGA were significantly correlated with the duration of storage time. LIMITATIONS, REASONS FOR CAUTION: This was a retrospective study using data from a single fertility center, even though the data had been adjusted, our findings still need to be validated in further studies. WIDER IMPLICATIONS OF THE FINDINGS: With the full implementation of the two-child policy in China, there may be more patients whose embryos have been frozen for a longer time in the future. Patients should be aware that the IR and LBR of blastocysts are negatively affected when the cryopreservation time is longer than 5 years. Couples may therefore consider shortening the time until FET treatment. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Nature Science Foundation of China (No. 82101672), Science and Technology Projects in Guangzhou (No. 2024A03J0180), General Guidance Program for Western Medicine of Guangzhou Municipal Health Commission (No. 20231A011096), and the Medical Key Discipline of Guangzhou (2021-2023). None of the authors have any conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

The gut microbial metabolite trimethylamine N-oxide aggravates GVHD by inducing M1 macrophage polarization in mice.

The diversity of the human microbiome heralds the difference of the impact that gut microbial metabolites exert on allogenic graft-versus-host (GVH) disease (GVHD), even though short-chain fatty acids and indole were demonstrated to reduce its severity. In this study, we dissected the role of choline-metabolized trimethylamine N-oxide (TMAO) in the GVHD process. Either TMAO or a high-choline diet enhanced the allogenic GVH reaction, whereas the analog of choline, 3,3-dimethyl-1-butanol reversed TMAO-induced GVHD severity. Interestingly, TMAO-induced alloreactive T-cell proliferation and differentiation into T-helper (Th) subtypes was seen in GVHD mice but not in in vitro cultures. We thus investigated the role of macrophage polarization, which was absent from the in vitro culture system. F4/80+CD11b+CD16/32+ M1 macrophage and signature genes, IL-1ß, IL-6, TNF-α, CXCL9, and CXCL10, were increased in TMAO-induced GVHD tissues and in TMAO-cultured bone marrow-derived macrophages (BMDMs). Inhibition of the NLRP3 inflammasome reversed TMAO-stimulated M1 features, indicating that NLRP3 is the key proteolytic activator involved in the macrophage's response to TMAO stimulation. Consistently, mitochondrial reactive oxygen species and enhanced NF-κB nuclear relocalization were investigated in TMAO-stimulated BMDMs. In vivo depletion of NLRP3 in GVHD recipients not only blocked M1 polarization but also reversed GVHD severity in the presence of TMAO treatment. In conclusion, our data revealed that TMAO-induced GVHD progression resulted from Th1 and Th17 differentiation, which is mediated by the polarized M1 macrophage requiring NLRP3 inflammasome activation. It provides the link among the host choline diet, microbial metabolites, and GVH reaction, shedding light on alleviating GVHD by controlling choline intake.

Toll-like receptor 4 protects against irradiation-induced hematopoietic injury by promoting granulopoiesis and alleviating marrow adipogenesis.

Irradiation induces severe damage in the hematopoietic system, which leads to bone marrow hyperplasia, pancytopenia, and aggravated tissue formation in bone marrow. Studies have shown that Toll-like receptor 4 (TLR4) has a protective effect against irradiation, but the underlying mechanism remains unclear. In this study, we used a TLR4 knockout (TLR4-/-) mouse irradiation model and found that the white blood cell and platelet counts in the peripheral blood of TLR4-/- mice recovered slowly after irradiation, with bone marrow hyperplasia and increased mortality. Additionally, we found that the proportion of CD11b+Gr1+ granulocytes in the peripheral blood and bone marrow of TLR4-/- mice was lower than that of wild-type mice after irradiation. Further, we found that the expression of NADPH Oxidases (NOXs) in the bone marrow was down-regulated after irradiation of TLR4-/- mice, and administration of the NOXs inhibitor VAS2870 reduced the proportion of CD11b+Gr1+ cells in the bone marrow and peripheral blood of wild-type mice after irradiation. Irradiation induced severe marrow adipocytes accumulation in TLR4-/- mice, TLR4 ligand lipopolysaccharide promoted proliferation and inhibited adipogenic differentiation of mesenchymal stromal cells. In summary, our data suggest that TLR4 promotes myeloid hyperplasia by up-regulating the expression of NOXs after irradiation, prohibits marrow adipogensis and increases the tolerance of mice to irradiation.

Cyclosporin A inhibits adipogenic differentiation and regulates immunomodulatory functions of murine mesenchymal stem cells.

Aplastic anemia (AA) is generally considered as an immune-mediated bone marrow failure syndrome. Several studies show that bone marrow mesenchymal stem cells (BM-MSCs), as key cellular components of the bone marrow microenvironment, are also involved in the pathogenic mechanism of AA. Cyclosporin A (CsA) is a classic immunosuppressive drug for AA, and it specifically inhibits mammalian T cells by preventing activation of transcription factors involved in cytokine gene expression. However, little is known about the effect of CsA on the BM-MSCs. In this study, murine BM-MSCs were stimulated in the presence of CsA. Further, we found that CsA could inhibit murine BM-MSC proliferation and promote BM-MSC apoptosis, what's more CsA could inhibit adipogenic differentiation. Our study also showed that CsA could inhibit interleukin-6 expression in BM-MSCs, while promoting programmed death-ligand 2 expression. In conclusion, our results proposed that CsA may exert an effect on regulating the bone marrow environment by influencing BM-MSCs, which have a beneficial effect on treating AA.

Effect of the co-culture of human bone marrow mesenchymal stromal cells with human umbilical vein endothelial cells in vitro.

Mesenchymal stem cells (MSCs) give origin to the marrow tromal environment that supports hematopoiesis. These cells present a wide range of differentiation potentials and a complex relationship with hematopoietic stem cells (HSCs) and endothelial cells. In addition to bone marrow (BM), MSCs can be obtained from other sites in the adult or the fetus. Recent studies have shown that cocultured endothelial cells and osteoblasts are mutually promotive in bone tissues repair. In this study, we observed the effects of coculture of endothelial cells and osteoblasts at different ratios on vasculogenesis and bone formation, and we found that angiogenic effect is more effective when endothelial cells are cocultured with osteoblasts at the ratio of 4:1, and osteogenic effect is more effective at the ratio of 1:4. It is concluded that the co-culture of human bone marrow mesenchymal stromal cells with human umbilical vein endothelial cells could be a promising culture system for bone tissue engineering applications.

Baicalin suppresses the migration and invasion of breast cancer cells via the TGF-ß/lncRNA-MALAT1/miR-200c signaling pathway.

Metastasis is the major cause of death and failure of cancer chemotherapy in patients with breast cancer (BC). Activation of TGF-ß/lncRNA-MALAT1/miR-200c has been reported to play an essential role during the metastasis of BC cells. The present study aimed to validate the suppression of BC-cell migration and invasion by baicalin and explore its regulatory effects on the TGF-ß/lncRNA-MALAT1/miR-200c signaling pathway. We found that baicalin treatment inhibited cell viability and migration and invasion. Mechanistically, baicalin treatment significantly downregulated the expression of TGF-ß, ZEB1, and N-cadherin and upregulated E-cadherin on both mRNA and protein levels. Additionally, baicalin treatment significantly downregulated the expression of lncRNA-MALAT1 and upregulated that of miR-200c. Collectively, baicalin significantly suppresses cell viability, migration, and invasion of BC cells possibly by regulating the TGF-ß/lncRNA-MALAT1/miR-200c pathway.

Monocytic myeloid-derived suppressive cells mitigate over-adipogenesis of bone marrow microenvironment in aplastic anemia by inhibiting CD8+ T cells.

Aplastic anemia (AA) is a blood disorder resulted from over-activated T-cell related hematopoietic failure, with the characterization of hypocellularity and enhanced adipogenic differentiation of mesenchymal stroma cells (MSCs) in bone marrow (BM). However, little is known about the relationship between immune imbalance and polarized adipogenic abnormity of BM microenvironment in this disease entity. In the present study, we differentiated BM-MSCs into osteoblastic or adipogenic lineages to mimic the osteo-adipogenic differentiation. Activated CD8+ T cells and interferon-γ (IFN-γ) were found to stimulate adipogenesis of BM-MSCs either in vitro or in vivo of AA mouse model. Interestingly, myeloid-derived suppressive cells (MDSCs), one of the immune-regulating populations, were decreased within BM of AA mice. We found that it was not CD11b+Ly6G+Ly6C- granulocytic-MDSCs (gMDSCs) but CD11b+Ly6G-Ly6C+ monocytic-MDSCs (mMDSCs) inhibiting both T cell proliferation and IFN-γ production via inducible nitric oxide synthetase (iNOS) pathway. Single-cell RNA-sequencing (scRNA-seq) of AA- and mMDSCs-treated murine BM cells revealed that mMDSCs transfusion could reconstitute BM hematopoietic progenitors by inhibiting T cells population and signature cytokines and decreasing immature Adipo-Cxcl12-abundant reticular cells within BM. Multi-injection of mMDSCs into AA mice reduced intra-BM T cells infiltration and suppressed BM adipogenesis, which subsequently restored the intra-BM immune balance and eventually prevented pancytopenia and hypo-hematopoiesis. In conclusion, adoptive transfusion of mMDSCs might be a novel immune-regulating strategy to treat AA, accounting for not only restoring the intra-BM immune balance but also improving stroma's multi-differentiating microenvironment.

Bioinformatics Analysis of Potential Therapeutic Targets and Prognostic Biomarkers amid CXC Chemokines in Ovarian Carcinoma Microenvironment.

BACKGROUND: Ovarian cancer (OC) is one of the leading lethal gynecologic cancers of women around the world. More than 70% of patients are diagnosed with stage III or IV with poor outcome. This is partly because of lacking early effective screening techniques and potential biomarkers of OC. CXC chemokines in tumor microenvironment (TME) and their interaction with relative receptors can excite the downstream signaling pathways to influence tumor progression. However, the role of CXC chemokines in OC has not been identified. METHODS: ONCOMINE, GEPIA, Kaplan-Meier plotter, cBioPortal, TIMER, Metascape, and LinkedOmics were applied in our study. RESULTS: The transcriptional levels of CXCL1/8/9/10/11/12/13/14/16/17 were significantly elevated while CXCL3 was obviously reduced in OC vs normal ovarian tissue. CXCL8/9/11/13 were correlated with clinic pathological stage. Patients with low expression of CXCL8/9/11/13 were associated with better prognosis. We also found that CXCL3 and CXC12 could be used as potential prognostic markers of OC through Kaplan-Meier plotter. Patients with high expression of CXCL3/12 had a significantly better prognosis. Their functions focus on locomotion, signaling, response to stimulus, undergoing the process of multiorganism, immune system, biological regulation, etc. The differentiated CXC chemokines mainly participate in cytokine-cytokine receptor interaction, chemokine signaling pathway, IL-17 signaling pathway, and toll-like receptor signaling pathway. Our results showed that CXC chemokines were highly correlated with infiltration of immune cells. The kinase targets of differentially expressed CXC chemokines are mainly in ATM, LYN, LCK, PLK1, FYN, CDK2, and ATR. CONCLUSIONS: Our results may provide a new insight for selecting precision biomarkers of targeted therapy of OC.

ZEB2 facilitates peritoneal metastasis by regulating the invasiveness and tumorigenesis of cancer stem-like cells in high-grade serous ovarian cancers.

Peritoneal metastasis is a common issue in the progression of high-grade serous ovarian cancers (HGSOCs), yet the underlying mechanism remains unconfirmed. We demonstrated that ZEB2, the transcription factor of epithelial-mesenchymal transition (EMT), was upregulated in ascites cells from HGSOC patients and in CD133+ cancer stem-like cells (CSLCs) from epithelial ovarian cancer (EOC) cell lines. SiRNA-mediated knockdown of ZEB2 in EOC cells decreased the percentage of CSLCs and reduced the colony forming potential, cell invasion capacity and expression of pluripotent genes Oct4 and Nanog. Inhibition of ZEB2 also induced cellular apoptosis and impacted the tumorigenicity of ovarian CSLCs. The mesenchymal markers N-cadherin and vimentin were downregulated, while the epithelial marker E-cadherin was upregulated after ZEB2 knockdown. MiR-200a, a molecule that downregulates ZEB2, had the opposite effect of ZEB2 expression in EOC-CSLCs. A retrospective study of 98 HGSOC patients on the relationship of ascites volume, pelvic and abdominal metastasis, International Federation of Gynecology and Obstetrics (FIGO) stage and the malignant involvement of abdominal organs and lymph nodes was performed. Patients with high expression of ZEB2 in tumour tissues had a higher metastasis rate and a poorer prognosis than those with low expression. The parameters of ZEB2 expression and ascites volume were strongly linked with the prognostic outcome of HGSOC patients and had higher hazard ratios. These findings illustrated that ZEB2 facilitates the invasive metastasis of EOC-CSLCs and can predict peritoneal metastasis and a poor prognosis in HGSOC patients.

Cost-effectiveness Analysis of CYP2D6*10 Pharmacogenetic Testing to Guide the Adjuvant Endocrine Therapy for Postmenopausal Women with Estrogen Receptor Positive Early Breast Cancer in China.

BACKGROUND: A CYP2D6 gene polymorphism is related to the effect of tamoxifen treatment in patient with estrogen-receptor positive (ER) positive breast cancer and CYP2D6*10 T/T can lead to a poor prognosis in Asian patients. Although one-off pharmacogenetic testing may optimize adjuvant endocrine therapy, testing prior to tamoxifen initiation incurs additional costs. AIM: We conducted a study to assess the cost-effectiveness of CYP2D6*10 pharmacogenetic testing to guide the adjuvant endocrine therapy compared with tamoxifen without CYP2D6*10 testing in China. METHODS: A semi-Markov model was developed to evaluate costs and health outcomes represented as quality adjusted life year (QALY) gained. Input data were obtained from the public literature. The results were expressed as incremental cost per QALY gained. A one-way deterministic sensitivity analysis explored the impact of uncertainty in the model parameters on results, and probabilistic uncertainty was assessed through a Monte Carlo probabilistic sensitivity analysis. RESULTS: In the base-case analysis, in the CYP2D6*10 testing and alternative adjuvant endocrine therapy group, the incremental total cost was US$17,966.95 and the incremental QALY was 3.582. Thus, the incremental cost-effectiveness ratio was US$5015.693 per QALY gained. Compared with a willingness-to-pay threshold of US$26,508/QALY in China, the CYP2D6*10 testing is the dominant strategy in postmenopausal women with ER-positive breast cancer in China, and the increased cost of genetic testing was completely worthwhile. The sensitivity analyses showed that the model we built was quite stable. CONCLUSION: From the perspective of the Chinese healthcare system, CYP2D6*10 pharmacogenetic testing was cost effective for postmenopausal women with ER-positive early breast cancer.

Key genes and integrated modules in hematopoietic differentiation of human embryonic stem cells: a comprehensive bioinformatic analysis.

BACKGROUND: The generation of hematopoietic stem cells (HSCs) and blood cells from human embryonic stem cells (hESCs) is a major goal for regenerative medicine; however, the differentiation mechanisms are largely undefined. Here, we aimed to identify the regulated genes and functional modules related to the early differentiation of the endothelial-to-hematopoietic transition (EHT) using comprehensive bioinformatics analyses. METHODS: Undifferentiated hESCs (hESC-H9), CD34+ cells from 10-day differentiated hESC-H9 cells, and CD34+ cells from umbilical cord cells were isolated and collected. Cells from these three groups were subjected to RNA extraction and microarray analysis by which differentially expressed genes (DEGs) and time-series profiles were analyzed by significance analysis of microarray (SAM) and short time-series expression miner (STEM) algorithms. Gene enrichment analysis was performed by ClusterProfiler Package in Rstudio, while a protein-protein interaction (PPI) network was constructed by search tool for the retrieval of interacting genes (STRING) and visualized in Cytoscape. Hub genes were further identified with the MCODE algorithm in Cytoscape. RESULTS: In the present study, we identified 11,262 DEGs and 16 time-series profiles that were enriched in biological processes of chromosome segregation, cell cycle, and leukocyte activation and differentiation, as well as hematopoiesis. Analysis using the MCODE algorithm further identified six integrated modules that might play an important role in the EHT process, including mitosis/cell cycle, mitochondrial process, splicing, ubiquitination, ribosome, and apoptosis. CONCLUSIONS: The study identified potential genes and integrated functional modules associated with the hematopoietic and endothelial differentiation of human ESCs.