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Objective To investigate the role of member septin family(SEPT12)in human spermatogenesis and its influence on sperm motility and sperm ultrastructure. Methods Whole exome sequencing(WES) was performed on peripheral blood DNA extracted from 375 patients with asthenoteratozoospermia, and a patient with idiopathic infertility carrying compound heterozygous mutation ofSEPT12was screened out. Sanger sequencing was performed to verify the mutation, and co-segregation analysis was performed in the family. The morphological abnormalities of sperm were analyzed by hematoxylin-eosin(HE) staining and scanning electron microscopy(SEM), and the ultrastructural defects of sperm were analyzed by transmission electron microscopy(TEM). Then the effects of the mutation on the level and position of the protein and the changes of the location and level of the defect structure markers were analyzed by Western blot and immune-fluorescence(IF). Results The compound heterozygous mutations c. C332A(p. T111K) and c. 406＿416 del TGCTCGTATTG(p.q136 VFS*39) in theSEPT12gene were screened and identified in a patient with asthenoteratozoospermia. The mutations were verified by Sanger sequencing, which was consistent with the co-segregation genetic pattern of the family. The mutations resulted in loss of protein expression, decreased sperm motility and sperm morphological deformities, mainly including short tail, curly tail and irregular sperm head. The ultrastructure of sperm showed that the annulus between the mid-piece and the principle-piece was missing, the acrosome membrane of sperm head fell off and the nucleus contained vacuoles. In the mid-piece of sperm flagella, the arrangement of mitochondrial sheath was disordered, most of flagella axoneme central pair was absent, microtubules doublet was missing or disordered, and some radical spoke was absent. By Western blot and IF, the marker proteins of related structural components were detected, and the results showed that the level of SEPT4 protein decreased, SEPT6 protein unchanged, acrosomal related proteins ACTL7A and ACROSIN protein missing, and the expression levels of mitochondrial and axoneme related proteins TOMM20, SPAG6 and RSPH3 protein significantly decreased. Conclusion The deletion ofSEPT12protein caused bySEPT12gene mutation leads to the deletion of the annulus between the mid-piece and the principle-piece, and the abnormal assembly of sperm acrosome, mitochondrial sheath and flagella.

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Objective To investigate the impact of positive antinuclear antibody(ANA) and subsequent intervention therapy on the assisted reproductive technology outcomes among the patients experiencing recurrent implantation failure(RIF). Methods A retrospective study was conducted on 344 RIF patients. Based on ANA test results, the patients were divided into ANA-positive group(294 cases) and negative control group(50 cases). The ANA-positive group was further divided into a low titer group(214 cases) and a high titer group(80 cases). Comparative statistical analyses such as the Wilcoxon rank-sum test, Mann-Whitney U test, Kruskal-Wallis test and chi-square test, etc. were employed to evaluate differences in general clinical data, embryo-related parameters, and pregnancy outcomes between the positive and negative groups. The impact of ANA on the assisted reproductive outcomes of patients with recurrent implantation failure was analyzed, and the outcomes of ANA-positive patients after intervention therapy were also analyzed. Results Notably, the clinical pregnancy rates of patients in the ANA-positive low titer group and high titer group were significantly lower than those in the negative control group(P<0.001). Similarly, the rates of fertilization and cleavage of oocytes in ANA-positive patients were also significantly lower than those in the negative control group(P<0.05). For patients who did not achieve pregnancy after embryo transfer due to ANA positivity, immunomodulatory therapy significantly improved both the clinical pregnancy rate and cumulative clinical pregnancy rate(P<0.05). Conclusion Compared with the negative control group, the clinical pregnancy rates decrease in both ANA-positive low titer subgroup and high titer subgroup. However, clinical intervention therapy enhances the single-cycle clinical pregnancy and cumulative pregnancy rates among ANA-positive patients, indicating that ANA positivity is an important factor in RIF. Immunomodulatory therapy is an effective measure to improve recurrent implantation failure among ANA-positive patients.

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Objective To examines the correlation between lifestyle factors and semen quality among sperm donors at Anhui human sperm bank.Methods Demographic and lifestyle data were collected from 1,222 volunteers who donated sperm between January 2021 and December 2023,and their association with semen quality was analyzed.Results Univariate chi-square analysis revealed significant associations between several lifestyle factors and abnormal semen parameters(P<0.05),including non-student status,frequent masturbation,short-term abstinence,low exercise frequency,frequent staying up late,smoking,drinking,and short sleep duration.Moreover,multivariate logistic regression analysis demonstrated that non-student status,longer abstinence time,and insufficient sleep were linked to abnormal semen volume.Additionally,abstinence time,exercise frequency,staying up late,smoking,and sleep duration were significantly correlated with abnormal semen concentration and sperm motility(P<0.05).Conclusion Analysis reveals a close relationship between semen quality and volunteers' lifestyles,including factors such as abstinence time,staying up late,sleep duration,smoking,drinking,and exercise frequency.

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Objective To analyze the characteristics of males with autologous sperm preservation(ASP) in Anhui human sperm bank, and to explore the future direction of ASP in human sperm bank. Methods The basic information of males applied for ASP in Anhui human sperm bank from January 2019 to December 2023 was retrospectively analyzed. Results During this period, there were 424 males applied for ASP. 93.40%(396/424) came from Anhui Province, of which 46.46%(197/424) came from Hefei.The age range of them was 15 to 59 years old. 66.04%(280/424) had a college degree or above. 23.11%(98/424) were employees of public institutions or enterprises. 26.89%(114/424) were unmarried and 89.39%(379/424) were childless. 67.45%(286/424) patients applied for ASP because of assisted reproductive technology treatment. 15.33%(65/424) patients did it due to tumors, among which testicular cancer, lymphoma, leukemia and seminoma were the main reasons.A total of 1 163 semen samples were saved, and 53 males had used their sperm. Conclusion Only a few people applied for ASP, and the characteristics of males with ASP can be used to further strengthen publicity for key groups, especially cancer patients, so as to benefit more people with autologous sperm preservation.

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Objective To study the effect of changes in estradiol(E2) levels on the reproductive outcomes of patients with polycystic ovary syndrome(PCOS) undergoing controlled ovarian stimulation(COS) with gonadotropin-releasing hormone antagonist(GnRH-ant) protocol. Methods A retrospective study was conducted involving 338 patients with polycystic ovary syndrome(PCOS) infertility who underwent GnRH-ant protocol for controlled ovarian hyperstimulation followed byin vitrofertilization(IVF) or intracytoplasmic sperm injection(ICSI), and subsequently underwent their first frozen-thawed embryo transfer(FET). The clinical data of these patients were analyzed.Patients were grouped based on the changes in serum E2levels on the first and fourth day of GnRH-ant administration(blood samples collected before GnRH-ant injection): the E2elevation group(Group A, E2value increased more than 30%, 165 cases), the E2stable group(Group B, E2rate of change was within-30%～30%, 162 cases), and the E2decline group(E2value declined more than 30%, 11 cases, not included in statistical analysis due to small sample size).The differences in demographic characteristics, ovulation induction outcomes, embryo outcomes and clinical pregnancy-related indicators were analyzed between Groups A and B. Results There were no statistically significant differences in basic information such as age, duration of infertility, body mass index(BMI), basal endocrine levels, and anti-Müllerian hormone(AMH) levels between groups A and B. Regarding embryo characteristics, there were no statistically significant differences in fertilization method and number of transferred embryos between groups A and B. However, group A had a higher total number of retrieved oocytes, normal fertilization rate, number of high-quality embryos, and rate of high-quality embryos compared to group B, with statistically significant differences(P<0.05). In terms of clinical pregnancy outcomes, there were no statistically significant differences in the incidence of severe ovarian hyperstimulation syndrome(OHSS) between groups A and B. However, group A had higher rates of clinical pregnancy, implantation, and live birth compared to group B, with statistically significant differences. Group A also had a lower rate of early miscarriage compared to group B, with statistically significant differences. Conclusion Choosing the GnRH-ant protocol for IVF/ICSI-FET in PCOS patients, if the blood E2level increases by more than 30% after 4 days of adding the antagonist(blood sample collected before administering GnRH-ant), the clinical pregnancy outcome will be better.

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Objective To compare the differences in clinical outcomes of ovulation induction with highly purified human menopausal gonadotropin(HP-hMG, Menopur) versus recombinant human follicle stimulating hormone(rFSH, Gonal-F) in normal ovarian responder patients treated with the GnRH-antagonist protocol. Methods Patients treated with the GnRH-antagonist protocol were retrospectively analyzed and divided by gonadotropin(Gn) usage into HP-hMG group and rFSH group. The basic characteristics, ovulation induction method, laboratory indexes, and clinical outcomes were compared between the two groups. Results A total of 614 normal ovarian responder patients were enrolled in the study, with 216 in the HP-hMG group and 398 in the rFSH group. There was no significant difference inPlevels and LH levels on the hCG trigger day between the two groups. However, the E2levels on the hCG trigger day were significantly lower in the HP-hMG group(P<0.001). Moreover, the HP-hMG group had a higher biochemical pregnancy rate(69.23%vs63.51%) and clinical pregnancy rate(66.67%vs59.46%) compared to the rFSH group.The incidence of OHSS in the HP-hMG group(2.31%vs3.02%) showed a decreasing trend compared to the rFSH group. Conclusion Compared to rFSH, HP-hMG offers distinct advantages in reducing the incidence of OHSS and improving pregnancy outcomes in normal ovarian responder patients undergoing pituitary suppression with the GnRH-antagonist protocol.

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Objective To investigate the expression and clinical value of γδT cells and related inflammatory factors in endometrial polyps(EP).Methods Subjects were patients with infertility due to EP(EP group,n=57) and infertility due to non-uterine factors(Control group,n=44).EP tissues and normal endometrial tissues were taken,respectively.The general characteristics of the two groups were evaluated by independent samples T-test or Wilcoxon rank sum test.The expressions of CD3 and TCRγδ in proliferative endometrium and endometrial polyps were observed by immunofluorescence,and the expressions of IL-17 and IFN-γ in tissue homogenate were detected by ELISA.Uni-variable linear regression was used to analyze the correlation between the number of γδT cells and the expression level of IL-17 and IFN-γ.Results Both the number of γδT cells and he levels of IL-17 and IFN-γsignificantly increased in endometrial polyp tissues,and the relationship between is linear(all P<0.05).Conclusion γδT cells are associated with EP among infertile women,which may lead to embryo implantation failure by affecting the expression of inflammatory factors and regulating the imbalance of cell subsets.

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Objective To investigate the clinical effects and pregnancy outcomes of using luteal phase long protocol and GnRH antagonist protocol in patients with polycystic ovary syndrome(PCOS) who have failed their first GnRH antagonist protocol therapy. Methods The clinical data of 163 PCOS patients who underwent IVF/ICSI-ET were retrieved. After the failure of their first GnRH antagonist protocol treatment, they were divided into two groups in the second controlled ovarian hyperstimulation(COH) cycle: Luteal phase long protocol group(n=95) and GnRH antagonist protocol group(n=68). A retrospective analysis and comparison of basic clinical data, clinical and laboratory indicators, and pregnancy outcomes between two groups were conducted. Results (1) There was no statistically significant difference in basic clinical indicators between two group except LH.(2) Compared the first and second cycle treatments of patients in the luteal phase long protocol group, the initiation dose of gonadotropin(Gn), total number of Gn days, total Gn usage, estradiol(E2) on the day of hCG injection, number of retrieved eggs, oocyte maturation rate, 2PN fertilization rate, 2PN cleavage rate, blastocyst formation rate, high-quality blastocyst formation rate, and moderate to severe OHSS rate were significantly higher than those in the first GnRH antagonist cycle(P<0.05). The GnRH antagonist protocol group also showed similar improvements.(3) The comparison of the second COH cycle between two groups showed that the total number of Gn days, total Gn usage, and total Gn cost in the luteal phase long protocol group were significantly higher(P<0.05), while the E2and LH on the day of hCG injection, and the maturation rate of eggs were significantly lower than those in the GnRH antagonist protocol group(P<0.05). However, there was no statistically significant difference in the number of retrieved eggs, 2PN fertilization, 2PN cleavage, blastocyst formation rate, high-quality blastocyst formation rate, and OHSS rate between the two groups;(4) The comparison of fresh transplantation cycles for the second COH cycle between the two groups showed that the luteal phase long protocol fresh transplantation rate, implantation rate, clinical pregnancy rate, and live birth rate were slightly higher than those of the GnRH antagonist protocol group, but the difference was not statistically significant. Comparing the outcomes of pregnancy following the initial frozen-thawed embryo transfer(FET) between two groups, the biochemical pregnancy rate and clinical pregnancy rate of the GnRH antagonist protocol group were higher than those of the luteal phase long protocol group(P<0.05). However, no significant statistical variations were found in implantation rate, live birth rate, neonatal gestational age, and birth weight. Conclusion For PCOS patients who fail the first GnRH antagonist protocol, an appropriate increase in the initiating dose and usage of Gn can achieve satisfactory pregnancy outcomes with both protocols. Compared with change to a luteal phase long protocol, reusing the GnRH antagonist protocol still maintains its long-standing advantages, such as shorter total Gn days, lower costs, and better patient compliance.

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Objective To compare the early embryonic developmental potential and clinical outcomes of oocytes matured in vivo and those matured by modified in vitro maturation(LVM) technology during the same controlled ovarian hyperstimulation(COH) cycle,and to explore the clinical application of melatonin-supplemented IVM technology.Methods 159 patients were recruited into the study.920 mature oocytes were collected during their COH cycles processed for conventional IVF/ICSI protocols,while 1 283 immature oocytes from the same cycles were matured in a melatonin-supplemented IVM medium before ICSI was performed.A retrospective analysis was conducted to compare the impact of conventional assisted reproductive technology and improved IVM technology on the outcomes of assisted reproductive therapy and pregnancy outcomes.Results Compared with mature oocytes collected from COH cycles treated with conventional IVF/ICSI,oocytes promoted by improved melatonin-supplemented IVM technology had a lower rate of high-quality blastocyst formation.However,after embryo transfer,there was no significant difference in the clinical outcomes of mature oocytes obtained through two methods,including clinical pregnancy rate,full-term birth rate,neonatal length,and neonatal Apgar score.Conclusion The application of melatonin-supplemented IVM significantly increases the utilization of immature oocytes collected from COH cycles,improving the pregnancy outcomes of patients assisted by assisted reproductive technology.

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Objective To investigate the effect of growth hormone(GH) pretreatment on the improvement of euploid and pregnancy outcome. Methods A prospective analysis was conducted on 134 patients undergoing preimplantation genetic testing for aneuploidy(PGT-A), among whom 30 patients were self-controlled and 104 patients were inter-group controlled. According to whether GH was added, the patients were divided into GH pretreatment group and GH non-pretreatment group. GH pretreatment included subcutaneous injection of GH 2U/day for 4-6 weeks before the start of gonadotropin(Gn), and the dose was doubled on the day of Gn until the trigger day. GH non-pretreatment meant no GH treatment, GH pretreatment was given when the previous PGT-A cycle failed within one year when the PGT-A was performed again, forming the self-control group.The basic situation, blastocyst situation and pregnancy outcome were compared between the groups by inter-group and self-control. Results No matter in the group control or self-controlled group, the endometrial thickness on the day of HCG, ovarian sensitivity index(OSI), number of oocytes obtained, MII oocytes, 2PN number, 2PN fertilization rate, available oocyte rate, number of biopsy blastocysts, number of euploid blastocysts, euploid blastocyst rate, and at least one euploid rate significantly increased after GH pretreatment, with statistically significant differences(P<0.05). The total amount of Gn, Gn days, number of mosaic blastocysts, and mosaic blastocyst rate were not significantly changed after GH pretreatment, with no statistically significant differences. The implantation rate and clinical pregnancy rate increased after GH pretreatment, but with no statistically significant differences. Conclusion GH pretreatment can significantly improve the number and rate of euploid embryos in patients undergoing PGT-A, and has a tendency to improve pregnancy outcome.

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Objective To provide an animal model for studying the effect of TDO2 on the function of macrophages on the occurrence and development of diseases by constructing macrophage-specific tryptophan, 2, 3-dioxygenase(TDO2) gene knockout mice. Methods TDO2flox/floxLyz2-iCre+mice were constructed based on Cre/LoxP system. The genotypes of mice were identified by PCR amplification and agarose gel electrophoresis.Western blot and immunofluorescence were used to verify the effect of TDO2 knockdown in mouse macrophages. The spontaneous lesions in major tissues and organ were observed by HE stainings. Results The results of genotype identification showed that the mice with only one band at 407 bp or 408 bp for the flox amplification product and one band at 543 bp for the Cre amplification product were TDO2flox/floxLyz2-iCre+mice. Western blot results showed that TDO2 expression in bone marrow-derived macrophages(BMDMs) of TDO2flox/floxLyz2-iCre+mice decreased compared with TDO2flox/floxmice(P<0.01). Immunofluorescence results showed that TDO2 expression in peritoneal macrophages and BMDMs of TDO2flox/floxLyz2-iCre+mice decreased compared with TDO2flox/floxmice. HE staining showed no significant differences in cell morphology in the liver, brain, kidney and spleen tissues of TDO2flox/floxLyz2-iCre+mice compared to TDO2flox/floxmice. Conclusion TDO2flox/floxLyz2-iCre+mice is successfully constructed, providing a more precise experimental animal model for subsequent in-depth study of the role and mechanism of TDO2-regulated macrophage activation in disease.

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Objective To construct a knockdown recombinant adeno-associated virus(AAV-shCTSK) targeting the mouse Cathepsin K(CTSK)gene and assess the knockdown efficiency of AAV-shCTSK in mice, and to investigate its impact on lipid storage within adipose tissue. Methods ShRNA primers specific to both the negative control(NC) and CTSK were designed, annealed, and integrated into the backbone vector. Following clone selection and sequencing for validation, recombinant plasmids were purified. Adeno-associated viral vectors, along with packaging and helper plasmids, were co-transfected into 293T cells using the transfection reagent PEI for adeno-associated viral packaging and amplification.The resultant AAV-shNC and AAV-shCTSK were injected into the epididymal adipose tissue of miceviain situ injection. Two weeks post-injection, the expression of CTSK protein was evaluated through immunoblotting assay and the size of intracellular lipid droplets in mouse adipose tissue was detected by HE staining. Results Successful acquisition of AAV-shNC and AAV-shCTSK adeno-associated viruses was achieved. In mice subjected to in situ injection of AAV-shCTSK, effective knockdown of CTSK in adipose tissue was confirmed, accompanied by a significant reduction in the size of white adipocytes. Conclusion A knockdown adeno-associated virus targeting CTSK in mice is successfully constructed, and CTSK knockdown in adipose tissue leads to a notable decrease in lipid content.

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Objective To investigate the effect of palmitoleic acid(POA) on pyroptosis of cardiomyocytes after hypoxia/reoxygenation-induced injury in the human myocardium. Methods The experiment comprised a control group(Control, normal culture), a hypoxia/reoxygenation group(HR), a palmitoleic acid-treated group(HR+POA), and an anhydrous ethanol control group(HR+ET). Cardiomyocytes viability was assessed using CCK-8 assay, and the level of pyroptosis of cardiomyocytes was measured through the double staining with Hoechst33342/PI and LDH assay. ELISA was employed to detect the release of inflammatory factors IL-1β and IL-18 in the cell culture supernatant. qRT-PCR and Western blot were utilized to determine the relative expression levels of mRNA and protein of pyroptosis-related genes, namely NLRP3, ASC, Caspase-1, GSDMD, IL-1β and IL-18, respectively. Results CCK-8 assay showed that the survival of hypoxic/reoxygenated cardiomyocytes increased with the addition of POA at concentrations ranging from 25 to 100 μmol/L, as compared to the HR group; a hypoxia/reoxygenation model of cardiomyocyte was established. The expression of protein and mRNA increased in NLRP3, ASC, Cleaved caspase-1, GSDMD-N, IL-Iβ and IL-18vsthe control group(P<0.05), the positive percentage of Hoechst33342/PI staining in cardiomyocytes increased significantly(P<0.05), the release of LDH, IL-Iβ, and IL-18 increased(P<0.05). After intervention with 100 μmol/L POA, the protein and mRNA expression levels of NLRP3, ASC, Cleaved caspase-1, GSDMD-N, IL-Iβ, and IL-18 were significantly reduced in the HR+POA group vs HR+ET group(P<0.05).The positive percentage of Hoechst33342/PI staining in cardiomyocytes decreased significantly, and the levels of LDH, IL-Iβ and IL-18 significantly decreased(P<0.05). Conclusion Palmitoleic acid may alleviate hypoxia/reoxygenation-induced injury of cardiomyocytes by inhibiting pyroptosis and inflammatory response after hypoxia/reoxygenation in human myocardium.

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Objective To screen Cuproptosis genes and characteristic genes for differential prognosis in acute myeloid leukemia(AML) and explore their prognosis in AML as well as their biological roles and correlations in the immune and tumor microenvironment. Methods AML clinical, transcriptome, genomic, and copy number data were downloaded from three major databases, TCGA, GEO, and UCSC, and Cuproptosis genes were collected from published studies. From the perspective of multiomics, the effects of Cuproptosis gene and characteristic gene on survival, immunity, tumor microenvironment, stem cell correlation and drug sensitivity were studied by various bioinformatics methods, meta-analysis and secondary typing. Results One Cuproptosis gene was identified as a differential prognostic gene in AML and five characteristic genes were identified as influencing the prognosis of AML patients by influencing Cuproptosis, and a prognostic model was established. The differential genes were mainly concentrated in mitochondrial activity, REDOX enzyme and energy metabolism. In terms of immunity, macrophage M0, neutrophils, activated memory CD4 T cells and Tregs were positively correlated with risk score, while macrophage M2, resting mast cells, immature CD4 T cells, helper follicular T cells and memory B cells were negatively correlated with risk score. In terms of tumor microenvironment, the immune cell score of the low-risk group was lower than that of the high-risk group, and in the total score, the tumor microenvironment score of the low-risk group was also lower than that of the high-risk group, indicating that the tumor purity of the high-risk group was lower than that of the low-risk group. However, there was no significant association between stem cells in the high-risk and low-risk groups, and a total of 14 drugs were found to be sensitive to treat AML. Conclusion Cuproptosis gene and characteristic gene are closely related to immune and tumor microenvironment in AML by constructing a prognostic model of AML.

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Objective To explore the molecular mechanism of N6-methyladenosine(m6A) methylation mediated by methyltransferase 3(METTL3) in regulating lipopolysaccharide(LPS)-induced endothelial cell permeability changes. Methods Human umbilical vein endothelial cells(HUVECs) were culturedin vitro. HUVECs were treated with LPS 50, 125, 250, 500, 1 000, 2 000 ng/ml for 24 h. METTL3 mRNA expression was detected by Real-time PCR. After HUVECs were intervened with 500 ng/ml for 24 h, the methylation level of m6A was detected, and cell permeability was measured by cell permeability test. Real-time PCR and Western blot were used to detect mRNA and protein expression of intercellular junction proteins(Claudin-5, Occludin and VE-caherin). METTL3 overexpressed stable cell lines were constructed to measure the changes of m6A methylation level and permeability of endothelial cells during METTL3 overexpression. Results Compared to the control group, LPS inhibited the expression of HUVECs METTL3 mRNA, decreased the methylation of m6A, increased the cell permeability, and decreased the mRNA and protein expression of intercellular junction proteins(Claudin-5, Occludin and VE-Caherin). When METTL3 was overexpressed, the m6A methylation levels of endothelial cells were enhanced, and the increase of endothelial cell permeability induced by LPS was reversed. Conclusion METTL3-mediated m6A methylation can improve the permeability of endothelial cells induced by sepsis.

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Objective To investigate whether miR-19a interacts with insulin-like growth factor 2 receptors(IGF-2R) in infantile hemangiomas(IHs) and affects the proliferation, migration, and adipogenesis of hemangioma stem cells(HemSCs). Methods HemSCs were isolated, screened and cultured from IH specimens. IGF-2R expression in HemSCs was identified using immunohistochemistry. HemSCs transfected with miR-19a mimics and inhibitors were subjected to CCK-8, wound healing, Transwell, qRT-PCR, and Western blot analyses. Results Compared with the control, the proliferation and migration rate of HemSCs treated with miR-19a inhibitors were significantly increased, and overexpression of miR-19a significantly inhibited IGF-2 induced cell migration and proliferation(P<0.05). Conclusion MiR-19a may inhibit HemSCs proliferation, migration, and adipogenesis by targeting IGF-2R.

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Objective To investigate the effects of hypothyroidism and drug intervention on the cognitive function of rat offspring through the establishment of a rat model of experimental hypothyroidism in pregnancy. Methods A total of 20 SD female rats were randomly divided into control group(CON group) and hypothyroid group(PTU group).The hypothyroid model was established by propylthiouracil(PTU), the thyroid hormone levels of female mice were detected by electrochemiluminescence immunoassay, and the differences between the two groups were compared. After successful modeling, the male mice were mated in cages, and the hypothyroid group was randomly divided into no intervention group(group I), first trimester intervention group(group II) and second and third trimester intervention group(group III). The Morris water maze(MWM) experiment was used to test the learning and memory ability of the rats. The morphological structure of hippocampal neurons, the expression of nucleoprotein(NeuN) and synapse-associated protein(SYN) in mature neurons were observed by hematoxylin-eosin staining(HE), Nissl staining and immunohistochemistry. Results (1) Compared with the CON group, the female mice in the PTU group had a significant increase in TSH and a significant decrease in FT4(P<0.05).(2) In the positioning navigation test, the evasion latency of the pups in each group was gradually shortened. On the 5th day, the incubation period of group I was significantly longer than that of groups CON, II, III(P<0.05). There was little change between groups II and III and CON groups(P>0.05).(3) The residence time of group II was significantly different from that in group I during the space exploration stage(P<0.05). There was a significant difference between the number of pups crossing the platform in group I and groups CON, II and III(P<0.05).(4) There was no significant difference between HE staining and Nissl staining in hippocampal tissues of rats in each group. However, compared with the CON group, the average absorbance of NeuN and SYN proteins in the hippocampus of mice in groups I, II and III was significantly reduced(P<0.05). Conclusion Hypothyroidism will have adverse effects on the cognitive function and hippocampal neuron development of pregnant rats, and the effects of intervention on the cognitive function of offspring at different stages of pregnancy are different, the earlier the intervention, the smaller the damage to cognitive function.

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Objective To investigate the killing effect of Rhodium nanoparticles loaded composite hydrogel NPN + Rh-PEG NPs(NRP) on pancreatic cancer BxPC-3 cells. Methods Block copolymers were synthesized using atom transfer radical polymerization(ATRP), followed by the synthesis of PEG-modified Rhodium nanoparticles through an aqueous method. A premixed solution was prepared by ultrasonication and then heated to synthesize the composite hydrogel NRP loaded with nanoparticles. It was then characterized and its catalytic properties were verified. The morphology of Rhodium nanoparticles and the composite hydrogel NRP was characterized by transmission electron microscopy and scanning electron microscopy. The thermal imaging instrument was used to detect the photothermal properties of the composite hydrogel NRP, and then the growth inhibitory effect on BxPC-3 cells was observed using the MTT and live-dead staining methods. Finally, its biological safety was verified using MTT and blood compatibility testing. Results Rh-PEG with a particle size of about 10 nm was successfully prepared. The composite hydrogel showed porous structure under cryo scanning electron microscope, and Rhodium was evenly distributed in the composite hydrogel. Under the irradiation of 808 nm near-infrared light(NIR) with a laser power of 1 W/cm2, the ability of 80 μg/ml NRP to generate reactive oxygen species(ROS) was 19.6 times that of pure hydrogel(NPN)(P<0.05). Under light conditions, the catalytic decomposition rate of hydrogen peroxide was as high as 96.8%. Under the irradiation of 808 nm NIR with a laser power of 1 W/cm2, the temperature of 80 μg/ml NRP could rise to 58.9 ℃ within 5 minutes. MTT results showed that the survival rate of BxPC-3 cells was the lowest, only 14.8%, after 40 μg/ml NRP was irradiated with 1 W/cm2of 808 nm NIR. The results of live dead cell staining proved that the cell killing effect of 40 μg/ml NRP under light irradiation was stronger than that without 808 nm NIR irradiation. Conclusion The composite hydrogel NRP uniformly loaded with Rhodium nanoparticles can effectively enhance the killing effect on pancreatic cancer BxPC-3 cells.

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Objective To explore the mechanism by which total flavones of rhododendron(TFR) protect against cerebral ischemia-reperfusion(I/R) injury by inhibiting the TNF-α/caspase-8/caspase-3 signaling pathway. Methods The middle cerebral artery occlusion(MCAO) method was used to establish the rat I/R model. Rats were randomly divided into Sham surgery, MCAO, and post-I/R intervention with TFR 200 mg/kg(TFR 200 mg/kg) groups. After establishing the MCAO rat model, rats in the TFR 200 mg/kg group were administered TFR(200 mg/kg) solution for 14 consecutive days following I/R injury surgery. Hematoxylin-Eosin(HE) staining was used to observe neurological function scoring, cerebral blood flow assessment, histological examination of brain tissue, assay kits were used to detect lactate dehydrogenase(LDH) and neuron-specific enolase(NSE) activities in rat serum. ELISA assay kits was used to measure interleukin-1(IL-1) and interleukin-6(IL-6) levels, and Western blot and immunohistochemistry were conducted to detect the expression levels of cleaved caspase-3, caspase-8, and TNF-α proteins in rat brain tissue 14 days post-surgery. Results After cerebral ischemia-reperfusion treatment, MCAO resulted in abnormal neurological function in rats, significantly increased neurological function scoring index, obvious changes in cerebral tissue histomorphology and cerebral blood flow, significant upregulation of cleaved caspase-3, caspase-8, and TNF-α protein expression levels in brain tissue, and significant elevation of LDH, NSE, IL-1, and IL-6 levels in serum. Rats in the TFR 200 mg/kg group showed significantly reduced neurological function scoring, significant improvement in cerebral tissue pathological damage, decreased expression levels of cleaved caspase-3, caspase-8, and TNF-α proteins in brain tissue, as well as decreased levels of LDH, NSE, IL-1, and IL-6 in serum. Conclusion TFR may alleviate cerebral ischemic hypoxic injury by inhibiting the TNF-α/caspase-8/caspase-3 signaling pathway.

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Objective To investigate the role of Taraxasterol(TAR) on ferroptosis in chondrocytes induced by Erastin. Methods The C28/I2 chondrocyte line was treated with Erastin to construct the ferroptosis model of chondrocytes in vitro and the experiments were divided into Control, Erastin, TAR,and TAR + Erastin groups.Cell viability was detected by the CCK-8 assay.Cytotoxicity was detected by the lactate dehydrogenase(LDH) kit and the Calcein/PI cytokinesis kit.Flow cytometry was used to detect lipid reactive oxygen species(ROS).The intracellular glutathione(GSH) content was detected by GSH kit.Mitochondrial membrane potential was detected by JC-1 staining and RH123 staining.ACSL4 and GPX4 protein expression and the key indicators of ferroptosis were detected by Western blot. Results TAR restored the decreased cell viability of C28/I2 chondrocytes induced by Erastin treatment as well as reduced Erastin-induced cytotoxicity(P<0.01).Compared with the control group, the level of intracellular lipid ROS increased(P<0.01) and the content of GSH decreased(P<0.01) after treatment with Erastin, while TAR could reduce the production of lipid ROS(P<0.01) and increase the content of GSH(P<0.01).TAR restored mitochondrial membrane potential in C28/I2 chondrocytes ferroptosis, decreased ACSL4 protein expression(P<0.01) and increased GPX4 protein expression(P<0.01).In addition, TAR restored the reduced cell viability caused by IL-1β treatment. Conclusion TAR can inhibit Erastin induced ferroptosis in C28/I2 chondrocytes, which may be related to the regulation of ACSL4 and GPX4 protein expression.

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Objective To construct nomograms using data extracted from the surveillance, epidemiology, and end results(SEER) program database to predict overall survival(OS) and cancer-specific survival(CSS) for patients with choroidal melanoma(CM), and to evaluate the epidemiological characteristics, survival periods, and prognostic factors of CM patients. Methods Data on patients diagnosed with CM from 2010 to 2020 were extracted from the SEER database. The included patients were randomly divided into a training set(n=1,841) and a validation set(n=789) at a 7:3 ratio. Univariate Cox regression analysis was conducted in the training set, followed by incorporation into a multivariate Cox proportional hazards regression model. Independent influencing factors were screened in the multivariate Cox regression model to construct nomograms predicting 3-year and 5-year OS and CSS for CM. Decision curve analysis(DCA) was used to assess the clinical utility of the prediction models by quantifying the net benefit of the nomograms in decision-making support, and comparisons were made with the SEER stage model. Individual risk scores were obtained based on the established nomograms. Results A total of 2,630 patients were included in the study. The results indicated that gender, age, liver metastasis, surgery, radiotherapy, and chemotherapy were independent risk factors affecting OS in CM patients. Age, liver metastasis, surgery, and chemotherapy were independent risk factors affecting CSS in CM patients. The nomograms for 3-year and 5-year OS and CSS showed strong discriminative ability. Furthermore, in the validation set for OS and CSS, DCA indicated that the nomograms had good clinical potential. Kaplan-Meier(K-M) curves demonstrated that in both the training and validation sets, patients in the high-risk group had significantly lower OS and CSS rates compared to those in the low-risk group. Conclusion Age, liver metastasis, surgery, and chemotherapy are common predictors of OS and CSS in CM patients. A relatively comprehensive and accurate prognostic nomogram model based on the SEER database has been established. After calibration and further refinement, this nomogram model can be applied clinically to guide the treatment and prognosis of patients.

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Objective To characterize the genetic and functional heterogeneity of peripheral blood mononuclear cells(PBMC) in Takayasu arteritis(TAK) based on single cell nuclear transcriptome sequencing(snRNA-seq) technology and to elucidate the immune mechanisms that may be involved. Methods A total of 6 PBMC specimens were collected from patients who were diagnosed with TAK and had not received drug treatment recently and were admitted to the Second Affiliated Hospital of Anhui Medical University and the Second Affiliated Hospital of Wannan Medical College as the disease group(TAK group). During the same period, PBMC specimens were collected from patients who visited the hospital′s physical examination center. Six cases of PBMC in the population were in the normal group(NC group). Based on the snRNA-seq workflow, library construction and sequencing were performed, and R3.6.2 software was used to carry out PCA and tSNE analysis on the off-machine data from the two groups of PBMC sample pools for detection of gene expression in mononuclear cells. Results The relative proportion and content of immune cells in PBMC of TAK patients had changed; highly cloned TCR could match the amino acid sequence of integrin beta-3 with high homology; T cell-mediated interferon gamma and TNFα pathways were significantly up-regulated. The proportion of TNFRSF13B+ memory B cells increased significantly.Monocytes consistently formed the largest number of ligand-receptor interactions of all cell types. Conclusion During the occurrence and development of TAK, PBMC are involved in complex regulation, and a variety of immune and inflammatory factors are abnormally expressed, among which monocytes play an important role in the interaction of PBMC.

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Objective To evaluate the angiogenesis of decidua in spontaneous abortion patients and to investigate the change of thyroid hormone receptor signal expression in decidua in miscarriage patients.Methods The decidua of pregnant women was collected.Patients with miscarriage were taken as the experimental group and healthy pregnant women who underwent abortion by social factors were taken as the control group.The nuclear translocation of thyroid hormone receptor α(THR-α) and THR-β in decidua tissues was detected by Western blot method,and the distribution of THR-α and THR-β in decidua tissues was determined by immunohistochemistry.The expression of Deiodinase 2(DIO2) in decidua tissues was determined by Western blot.The distribution of DIO2 in decidua tissues was determined by immunofluorescence and the fluorescence intensity was measured.The decidual vessel density was determined by immunohistochemistry using CD34 as an endothelial marker.The protein expression of vascular endothelial growth factor receptor(VEGFR-1) was detected by Western blot.Results There was no significant difference in age,gestation days,TSH,T3,T4 and other basic data between the control group and the abortion group.The results of Western blot showed that the expression levels of THR-α and THR-β protein decreased,and the results of immunohistochemical staining of decidual tissues showed that the nuclear translocation of decidual thyroid hormone receptor in patients with spontaneous abortion decreased(P<0.05).Western blotting results showed that the expression of DIO2 protein in the abortion group was lower than that in the control group,suggesting that thyroid hormone metabolism in decidua of spontaneous abortion pregnant women was affected.The results of immunofluorescence test showed that the fluorescence intensity of DIO2 in the abortion group was weaker than that in the control group(P<0.05).Immunohistochemical CD34 labeling of decidual small blood vessels showed that the number of CD34+blood vessels decreased in the abortion group with statistical difference(P<0.05).Western blot results showed that there was no significant change in VEGFR-1 between the two groups,and the difference between the two groups was not statistically significant.Conclusion The abnormal signal of thyroid hormone receptors in decidua may decrease decidua angiogenesis and participate in spontaneous abortion.

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Objective To investigate the correlation between the methylenetetrahydrofolate reductase(MTHFR)C677Tpolymorphism and disease in the course of Alzheimer′s disease(AD),as well as whether whether it is affected byAPOEgene. Methods A total of 74 AD patients, 85 aMCI patients and 81 healthy controls(HC) were included. The levels of serum homocysteine(Hcy), folate, and vitamin B12, as well as the genotypes ofMTHFR C677TandAPOE, were determined. Logistic regression analysis was conducted to explore the relationship betweenMTHFR C677Tpolymorphism and the risk of AD and aMCI, as well as in different APOE ε4 subgroups. Results Compared with HC group, the serum Hcy levels in AD group and aMCI group were significantly higher(P<0.001,P<0.001), while serum folate levels in aMCI group was significantly lower(P=0.017). The serum folate level was significantly lower(P=0.038) in individuals with theMTHFR TTgenotype compared to those withCCandCTgenotypes, while the serum Hcy level was significantly higher(P=0.002). Regression analysis showed that theMTHFR TTgenotype might increase the risk of aMCI in the subgroup ofAPOE ε4non-carriers(OR=3.670, 95%CI=1.077-12.509,P=0.038), but not inAPOE ε4carriers. Conclusion MTHFR C677Tpolymorphism plays an important role in Hcy metabolism, which leads to increased serum Hcy levels and decreased folate levels. InAPOE ε4non-carriers, theMTHFR TTgenotype may increase the risk of aMCI.

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Objective To measure the anatomical parameters of the first maxillary molars in children of different age groups and evaluate the age-related changes in dental crowns. Methods A retrospective analysis was conducted on cone beam computed tomography(CBCT) images of 4-8-year-old children. NNT software was used to analyze multiple important indicators of maxillary first molar. Results A total of 308 first maxillary molars, including 154 pediatric patients, were evaluated in this study. The thickness of the pulp apex H1(left,P=0.01; right,P=0.02) and the thickness of the pulp chamber floor H3(left and rightP<0.01) were positively correlated with age, while the height of the pulp cavity H2(left and rightP<0.01) and the height of the palate tip D1(leftP=0.003, rightP=0.002) showed a negative correlation with age. There was no significant correlation between the height of the buccal tip and age(P>0.05). There were significant differences in H1 and H3 between the 4-year-old and 5-year-old age groups between the 8-year-old age group(P<0.05), as well as significant differences in H2 and D1 between the 4-year-old and 5-year-old between the 6-year-old, 7-year-old and 8-year-old age groups(P<0.05). Conclusion The age-related changes in the crowns of the first maxillary molars are important references for the clinical treatment, and can be accurately measured through CBCT data.

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C57/B6J mice were fixed in traditional fixative(4% formaldehyde solution, the same below) and novel fixative respectively, and the routine procedure of paraffin section preparation was performed, as well as the novel fixative was validated in HE-stained paraffin sections of the eyes of mice and human embryos in the age-related macular degeneration(AMD) model. Compared with the traditional fixative, the quality of the paraffin sections treated with the novel fixative was better than that of the traditional fixative, and successfully simulated the changes of the retinal layers of AMD mice at different time periods. The novel fixative is feasible for the preparation of high-quality retinal paraffin sections, which lays the foundation for subsequent studies on retinal paraffin sections.