ObjectiveTo detect the proportion of splenic follicular helper T （Tfh） cells and their functionally related cytokine expression levels in the incomplete embryo implantation disorder （EID） model mice， and to explore the immunological mechanism of Tfh in infertility caused by embryo implantation disorder. MethodsSixteen female Kunming mice were randomly divided into two groups， with eight mice in each group. On day 4 of pregnancy， an incomplete EID mouse model was established by oral gavage of mifepristone suspension， while an equal volume of saline was administered to the control group. On day 8 of pregnancy， the mice were euthanized. Flow cytometry was used to detect the levels of Tfh cells in the spleen lymphocytes of both incomplete EID mice and normal control mice. qRT-PCR was performed to measure the mRNA levels of B-cell lymphoma 6 （Bcl-6） and C-X-C chemokine receptor type 5 protein （CXCR5） in the spleen lymphocytes of both groups. Western blot was employed to assess the protein expression levels of Bcl-6 and CXCR5 in the spleen lymphocytes of both groups. Serum levels of interleukin-4 （IL-4）， IL-6， and IL-21 were measured by ELISA. Immunohistochemistry （IHC） was used to observe the expression levels of progesterone receptor （PR）， Bcl-6， and CXCR5 proteins in the uterine endometrial tissue of mice in both groups. ResultsIncomplete-type EID mice had a reduced number of embryo implantation points and reduced endometrial PR expression. Flow assay results showed that the proportion of CD4⁺CXCR5⁺Tfh cells in splenic lymphocytes of incomplete-type EID mice was significantly higher than that of normal controls （P<0.05）. Compared with the normal control group， Bcl-6 and CXCR5 mRNA levels and protein levels were elevated in splenic lymphocytes of incomplete EID mice， with statistically significant differences （P<0.05）； serum IL-4， IL-6， and IL-21 levels were elevated in incomplete EID mice， and Bcl-6 and CXCR5 proteins in the endometrium were significantly elevated （P<0.05）. ConclusionThe increase of Tfh cells and their associated cytokines Bcl-6 and CXCR5 is associated with the development of incomplete EID， and may be involved in the development of female immune infertility.

Objective:Haracterization of follicular helper T cells(Tfh)cells and related molecules early onset in premature ovarian insufficiency(POI)based on a mouse model of POI.Methods:After acclimatization feeding of 30 BALB/c mice were selected,mice were subjected to vaginal exfoliative cytology from 08:00 onwards every day for 10 consecutive days under light microscope obser-vation,20 healthy female mice with regular estrous cycle were screened out and randomly divided into model(POI)group and healthy control(HC)group according to random number table method,different chemical interventions were given and processed and tissue sampling was done on the 21st day.Every morning from the beginning to the end of the modeling period,the mice were subjected to cell smears of vaginal secretions,which were stained with Giemsa's stain and then observed under a light microscope.After the mice were sacrificed,bilateral ovarian tissues were taken and ovarian paraffin sections were made,and the tissues were stained with HE to observe pathological changes in ovaries of mice.Immunohistochemistry was used to detect expressions of ovarian Tfh-related molecules CXCR5 and BCL-6;flow assay was used to detect frequency of CD4+CXCR5+Tfh cells and CD4+ICOS+Tfh cells in spleens of mice;ELISA was used to detect levels of IL-6 and IL-21 in splenic tissues of mice;qRT-PCR was used to detect mRNA levels of CXCR5 and BCL-6 in mice;correlation analysis was performed to correlate frequencies of CD4+CXCR5+Tfh and CD4+ICOS+Tfh cells with levels of IL-6 and IL-21 in spleens of mice in both groups.Results:Pathological changes in ovarian tissue of mice in POI group:primordial folli-cles and growing follicles decreased,while atretic follicles increased.BCL-6 and CXCR5 were highly expressed in ovarian tissue of mice in POI group.Frequency of CD4+CXCR5+Tfh and CD4+ICOS+Tfh cells in spleen of POI mice was higher than that in HC group.Levels of IL-6 and IL-21 in spleens of mice in POI group were significantly higher than those in HC group,and were positively correlated with the frequency of CD4+CXCR5+Tfh and CD4+ICOS+Tfh cells in spleen of mice.Conclusion:In POI mouse model,Tfh cells are highly expressed and the level of Tfh-related molecules changes,which provides basic reference for studying pathogenesis of POI.

Objective:Haracterization of follicular helper T cells(Tfh)cells and related molecules early onset in premature ovarian insufficiency(POI)based on a mouse model of POI.Methods:After acclimatization feeding of 30 BALB/c mice were selected,mice were subjected to vaginal exfoliative cytology from 08:00 onwards every day for 10 consecutive days under light microscope obser-vation,20 healthy female mice with regular estrous cycle were screened out and randomly divided into model(POI)group and healthy control(HC)group according to random number table method,different chemical interventions were given and processed and tissue sampling was done on the 21st day.Every morning from the beginning to the end of the modeling period,the mice were subjected to cell smears of vaginal secretions,which were stained with Giemsa's stain and then observed under a light microscope.After the mice were sacrificed,bilateral ovarian tissues were taken and ovarian paraffin sections were made,and the tissues were stained with HE to observe pathological changes in ovaries of mice.Immunohistochemistry was used to detect expressions of ovarian Tfh-related molecules CXCR5 and BCL-6;flow assay was used to detect frequency of CD4+CXCR5+Tfh cells and CD4+ICOS+Tfh cells in spleens of mice;ELISA was used to detect levels of IL-6 and IL-21 in splenic tissues of mice;qRT-PCR was used to detect mRNA levels of CXCR5 and BCL-6 in mice;correlation analysis was performed to correlate frequencies of CD4+CXCR5+Tfh and CD4+ICOS+Tfh cells with levels of IL-6 and IL-21 in spleens of mice in both groups.Results:Pathological changes in ovarian tissue of mice in POI group:primordial folli-cles and growing follicles decreased,while atretic follicles increased.BCL-6 and CXCR5 were highly expressed in ovarian tissue of mice in POI group.Frequency of CD4+CXCR5+Tfh and CD4+ICOS+Tfh cells in spleen of POI mice was higher than that in HC group.Levels of IL-6 and IL-21 in spleens of mice in POI group were significantly higher than those in HC group,and were positively correlated with the frequency of CD4+CXCR5+Tfh and CD4+ICOS+Tfh cells in spleen of mice.Conclusion:In POI mouse model,Tfh cells are highly expressed and the level of Tfh-related molecules changes,which provides basic reference for studying pathogenesis of POI.

Objective:To investigate the changes in follicular helper T cells(Tfh)and related cytokines in pe-ripheral blood and endometrial tissue of patients with unexplained recurrent implantation failure(URIF),and evalu-ate their predictive value for URIF.Methods:Sixty-four patients with URIF who visited the Reproductive Medicine Centre of the First Affiliated Hospital of Xinjiang Medical University from December 2020 to June 2022 were select-ed as the study group(URIF group),and 61 healthy women of childbearing age who visited our centre for male in-fertility in the same period were selected as the healthy control group(HC group).GSE microarrays of patients with recurrent implant failure(RIF)were collected in the Gene Expression Omnibus(GEO)database,and the pro-portion of different immune cells was identified using the CIBERSORT algorithm,and immune cell infiltration analy-sis and visualisation processing were performed using the Cibersort.R package.Peripheral blood samples and endometrial tissue samples were collected from the two groups of patients,flow cytometry was used to detect the proportion of Tfh cells in peripheral blood,enzyme-linked immunosorbent assay(ELISA)was used to detect serum levels of IL-4,IL-6,and IL-21,and immunohistochemical staining was used to detect the expression of CXCR5,Bcl-6,and IL-21 in endometrial tissue.Factors associated with URIF were analysed using multifactorial Logistic re-gression analysis.The predictive value of individual and combined tests for each indicator for URIF was analysed using the operating characteristic curve(ROC).The Drug Therapy Target Database(TTD)was used to predict potential therapeutic agents for URIF targeting IL-6 and IL-21,respectively.Results:The results of immune cell in-filtration in the GSE dataset in the GEO database showed that compared with normal controls,the numbers of ac-tivated memory CD4+T cells,Tfh,regulatory T cells(Treg),inactive macrophages,and resting dendritic cells were increased in the endometrial tissues of the patients with RIF(P＜0.05).The number of initial B cells,yδT cells,M2-type macrophages and activated dendritic cells were all decreased(P＜0.05).The levels of peripheral blood Tfh,IL-6 and IL-21 in the URIF group were all significantly higher than those in the HC group,and the difference was statistically significant(P＜0.05).Immunohistochemical staining results suggested that the expression of CX-CR5,Bcl-6 and IL-21 in the endometrium of patients in the URIF group was increased compared with that in the HC group(P＜0.05).Multivariate Logistic regression analysis showed that IL-6,IL-2,and Tfh were independent risk factors for the occurrence of URIF(OR＞1,P＜0.05).The area under the curve(AUC=0.974)of peripheral blood Tfh combined with IL-6 and IL-21 for diagnosis of URIF was higher than that predicted by each index alone,according to ROC analysis(P＜0.05).Five drugs targeting IL-6 or IL-21 with potential therapeutic effects on URIF were detected and screened through the TTD database.Conclusions:The levels of Tfh,IL-6 and IL-21 in the pe-ripheral blood of patients with URIF were abnormally elevated,and CXCR5,Bcl-6 and IL-21 were abnormally ex-pressed in the endometrium of patients with URIF,suggesting that Tfh cells and their cytokines are closely related to the occurrence and development of URIF.The combined prediction of these indicators has good diagnostic val-ue and may serve as a therapeutic target.

Objective:To explore the role of methyltransferase-like 3 (METTL3) on endometrial decidualization.Methods:Bioinformatics was used to analyze the expression of 26 N6-methyladenosine (m6A) regulators in recurrent implantation failure (RIF), recurrent spontaneous abortion (RSA) and the decidualization of endometrial stromal cells. Using Medroxyprogesterone and Dibutyryl-cAMP to induce human endometrial stromal cell s (HESCs)decidualization, we examined changes in the expression of total m6A, METTL3, Forkhead framing protein O1 (FOXO1), and decidualization markers insulin-like growth factor binding protein 1 (IGFBP1) and prolactin in the control and decidualization group. Constructing HESC models with METTL3 interference using lentivirus, we used CCK-8 and Edu to detect proliferation in the negative control and knockdown METTL3 group. Lentiviral transfection was successfully followed by induction of decidualization, and we examined changes in the expression of FOXO1 and decidualization markers.Results:The results of bioinformatics analysis showed that the expression of METTL3 was significantly downregulated in the endometrium of patients with RIF and RSA ( P=0.014, P=0.016), while it was significantly upregulated in the endometrium with decidualization ( P=0.029). In vitro, induction of HESCs decidualization increased in the expression levels of total m6A, METTL3, and FOXO1 proteins in the decidualization group ( P=0.015, P=0.016, P=0.004). Knocking down METTL3 resulted in a decrease in FOXO1 protein expression in HESCs ( P=0.009). The CCK-8 and Edu results showed that the proliferation of HESCs was inhibited after knocking down METTL3. After inducing the knockdown of METTL3 in vitro, there was no statistically significant difference in the expression levels of IGFBP1 mRNA, PRL mRNA, and FOXO1 protein in HESCs compared with the negative control group (all P>0.05). Conclusion:In endometrial stromal cells, METTL3 can regulate the expression of FOXO1, promote the expression of IGFBP1 and PRL, and affect the decidualization of endometrial cells.

Objective:To explore the role of methyltransferase-like 3 (METTL3) on endometrial decidualization.Methods:Bioinformatics was used to analyze the expression of 26 N6-methyladenosine (m6A) regulators in recurrent implantation failure (RIF), recurrent spontaneous abortion (RSA) and the decidualization of endometrial stromal cells. Using Medroxyprogesterone and Dibutyryl-cAMP to induce human endometrial stromal cell s (HESCs)decidualization, we examined changes in the expression of total m6A, METTL3, Forkhead framing protein O1 (FOXO1), and decidualization markers insulin-like growth factor binding protein 1 (IGFBP1) and prolactin in the control and decidualization group. Constructing HESC models with METTL3 interference using lentivirus, we used CCK-8 and Edu to detect proliferation in the negative control and knockdown METTL3 group. Lentiviral transfection was successfully followed by induction of decidualization, and we examined changes in the expression of FOXO1 and decidualization markers.Results:The results of bioinformatics analysis showed that the expression of METTL3 was significantly downregulated in the endometrium of patients with RIF and RSA ( P=0.014, P=0.016), while it was significantly upregulated in the endometrium with decidualization ( P=0.029). In vitro, induction of HESCs decidualization increased in the expression levels of total m6A, METTL3, and FOXO1 proteins in the decidualization group ( P=0.015, P=0.016, P=0.004). Knocking down METTL3 resulted in a decrease in FOXO1 protein expression in HESCs ( P=0.009). The CCK-8 and Edu results showed that the proliferation of HESCs was inhibited after knocking down METTL3. After inducing the knockdown of METTL3 in vitro, there was no statistically significant difference in the expression levels of IGFBP1 mRNA, PRL mRNA, and FOXO1 protein in HESCs compared with the negative control group (all P>0.05). Conclusion:In endometrial stromal cells, METTL3 can regulate the expression of FOXO1, promote the expression of IGFBP1 and PRL, and affect the decidualization of endometrial cells.

Objective:To investigate the changes in follicular helper T cells(Tfh)and related cytokines in pe-ripheral blood and endometrial tissue of patients with unexplained recurrent implantation failure(URIF),and evalu-ate their predictive value for URIF.Methods:Sixty-four patients with URIF who visited the Reproductive Medicine Centre of the First Affiliated Hospital of Xinjiang Medical University from December 2020 to June 2022 were select-ed as the study group(URIF group),and 61 healthy women of childbearing age who visited our centre for male in-fertility in the same period were selected as the healthy control group(HC group).GSE microarrays of patients with recurrent implant failure(RIF)were collected in the Gene Expression Omnibus(GEO)database,and the pro-portion of different immune cells was identified using the CIBERSORT algorithm,and immune cell infiltration analy-sis and visualisation processing were performed using the Cibersort.R package.Peripheral blood samples and endometrial tissue samples were collected from the two groups of patients,flow cytometry was used to detect the proportion of Tfh cells in peripheral blood,enzyme-linked immunosorbent assay(ELISA)was used to detect serum levels of IL-4,IL-6,and IL-21,and immunohistochemical staining was used to detect the expression of CXCR5,Bcl-6,and IL-21 in endometrial tissue.Factors associated with URIF were analysed using multifactorial Logistic re-gression analysis.The predictive value of individual and combined tests for each indicator for URIF was analysed using the operating characteristic curve(ROC).The Drug Therapy Target Database(TTD)was used to predict potential therapeutic agents for URIF targeting IL-6 and IL-21,respectively.Results:The results of immune cell in-filtration in the GSE dataset in the GEO database showed that compared with normal controls,the numbers of ac-tivated memory CD4+T cells,Tfh,regulatory T cells(Treg),inactive macrophages,and resting dendritic cells were increased in the endometrial tissues of the patients with RIF(P＜0.05).The number of initial B cells,yδT cells,M2-type macrophages and activated dendritic cells were all decreased(P＜0.05).The levels of peripheral blood Tfh,IL-6 and IL-21 in the URIF group were all significantly higher than those in the HC group,and the difference was statistically significant(P＜0.05).Immunohistochemical staining results suggested that the expression of CX-CR5,Bcl-6 and IL-21 in the endometrium of patients in the URIF group was increased compared with that in the HC group(P＜0.05).Multivariate Logistic regression analysis showed that IL-6,IL-2,and Tfh were independent risk factors for the occurrence of URIF(OR＞1,P＜0.05).The area under the curve(AUC=0.974)of peripheral blood Tfh combined with IL-6 and IL-21 for diagnosis of URIF was higher than that predicted by each index alone,according to ROC analysis(P＜0.05).Five drugs targeting IL-6 or IL-21 with potential therapeutic effects on URIF were detected and screened through the TTD database.Conclusions:The levels of Tfh,IL-6 and IL-21 in the pe-ripheral blood of patients with URIF were abnormally elevated,and CXCR5,Bcl-6 and IL-21 were abnormally ex-pressed in the endometrium of patients with URIF,suggesting that Tfh cells and their cytokines are closely related to the occurrence and development of URIF.The combined prediction of these indicators has good diagnostic val-ue and may serve as a therapeutic target.

With the elevating prevalence and comorbidity rate of chronic diseases,the burden of disease treatment for patients is increasing and quality of life is declining. Recently, the minimal disruptive medicine(MDM)has attracted more attention and its positive impact has been recognized. In this article we review the research progress in the clinical effects of MDM on chronic disease patients,which would promote the exploration and implementation of scientific and effective treatment strategies for general practice in China.

Objective : To investigate the expression of macrophage migration inhibitory factor (MIF) and its effect on autophagy and insulin resistance of ovarian granulosa cells in polycystic ovary syndrome ( PCOS) . Methods: Follicular fluid and ovarian granulosa cells were collected from 40 PCOS patients ( n = 40) and non PCOS patients (control group, n = 20) . PCOS included patients with IR (PCOS with IR, n = 20) and patients without IR (PCOS without IR, n = 20) . The expression of MIF in follicular fluid was detected by ELISA . The ratio of autophagy vacuoles to microtubule⁃associated protein light chain Ⅱ( LC3 Ⅱ) /microtubule⁃associated protein light chain Ⅰ( LC3 Ⅰ)in granulosa cells was observed by transmission electron microscopy and Western blot . Human ovarian granule cell line KNG was cultured in vitro and CCK⁃8 was used to detect the effects of different concentrations of MIF on granule cell activity . KNG cells were divided into four groups: normal culture group ( NC group), MIF group, chloroquine group (CQ group) and MIF + CQ group . The effects of MIF on the expression of autophagy related proteins LC3, autophagy related genes 7(Atg7), ubiquitin binding protein(p62), and insulin signaling pathway related proteins insulin receptor substrate⁃1(ISR⁃1), glucose transporter 4(GLUT4), protein kinase B(Akt) phosphorylation were observed by Western blot, and the effect of MIF on glucose uptake ability of granulosa cells was detected by glucose uptake test . Results : Compared with the control group or PCOS without IR group, MIF expression in follicular fluid and autophagy level of granulosa cells in PCOS with IR group increased (P < 0. 05 or P < 0. 01); In vitro experiments showed that MIF could significantly inhibit the cellular activity of KNG in granulocytes in a concentration⁃dependent manner, in which 100 ng/ml MIF was selected for subsequent relevant experiments; Compared with NC group, LC3Ⅱ/LC3I ratio and Atg7 protein in MIF group and MIF + CQ group increased (P < 0. 05), while p62 protein, IRS⁃1 protein, Akt phosphorylation level, GLUT4 protein expression level and glucose uptake ability decreased ( P < 0. 05 ), while the above autophagy markers in MIF + CQ group were significantly higher than those in MIF group (P < 0. 05), and the protein related to insulin signal transduction and glucose uptake increased (P < 0. 05) . Conclusion MIF may promote IR development in PCOS patients by up⁃regulating the autophagy level of granulosa cells, while inhibiting the autophagy of granulosa cells can improve MIF⁃induced IR.

Regulatory T (Treg) cells are a subtype of lymphocytes with immune suppressive function, and are abundantly expressed on ovarian and endometrium. They have the functions of regulating follicular development, suppressing immune response, promoting maternal-fetal immune tolerance and maintaining early pregnancy during reproduction. The abnormal expressing of Tregs is closely related to the occurrence and development of female reproductive diseases, and it is expected to become a new diagnostic marker and therapeutic target in reproductive medicine.