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.

ObjectiveTo establish a new noninvasive， simple， and convenient clinical predictive model by identifying independent predictive factors for rebleeding after endoscopic therapy in cirrhotic patients with esophagogastric variceal bleeding （EGVB）， and to provide a basis for individualized risk assessment and development of clinical intervention strategies. MethodsCirrhotic patients with EGVB who were diagnosed and treated in The First Affiliated Hospital of Xi’an Medical University from September 2018 to October 2023 were enrolled as subjects， and according to whether the patient experienced rebleeding within 1 year after endoscopic therapy， they were divided into rebleeding group with 93 patients and non-rebleeding group with 84 patients. Clinical data were collected and analyzed. The independent samples t-test was used for comparison of normally distributed continuous data between two groups， and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups； the chi-square test was used for comparison of categorical data between two groups. A Logistic model was established based on the results of the univariate and multivariate analyses， and the receiver operating characteristic （ROC） curve and the area under the ROC curve （AUC） were used to assess the accuracy of the model. R software was used to visualize the model by plotting a nomogram， and the Bootstrap method was used for internal validation of the model. ResultsThe multivariate analysis showed that red blood cell count （RBC）， cholinesterase （ChE）， alkaline phosphatase （ALP）， albumin （Alb）， thrombin time （TT）， portal vein trunk diameter， sequential therapy， and primary prevention were independent predictive factors for rebleeding. Based on the results of the multivariate analysis， a logistic model was established as logit（P）=-0.805-1.978×（RBC）+0.001×（ChE）-0.020×（ALP）-0.314×（Alb）+0.567×（TT）+0.428×（portal vein trunk diameter）-2.303×［sequential therapy （yes=1， no=0）］-2.368×［primary prevention （yes=1， no=0）］. The logistic model （AUC=0.928， 95% confidence interval ［CI］： 0.893—0.964， P<0.001） had a better performance in predicting rebleeding than MELD score （AUC=0.603， 95%CI： 0.520—0.687， P=0.003）， Child-Pugh class （AUC=0.650， 95%CI： 0.578—0.722， P=0.001）， and FIB-4 index （AUC=0.587， 95%CI： 0.503—0.671， P=0.045）. The model had an optimal cut-off value of 0.607， a sensitivity of 0.817， and a specificity of 0.817. Internal validation confirmed that the model had good predictive performance and accuracy. ConclusionSequential therapy， implementation of primary prevention， an increase in RBC， and an increase in Alb are protective factors against rebleeding， while prolonged TT and widened main portal vein diameter are risk factors. The logistic model based on these independent predictive factors can predict rebleeding and thus holds promise for clinical application.

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 establish an oxidative stress injury model by using hydrogen peroxide (H 2O 2) to induce human ovarian granulosa cells COV434. Methods:Human ovarian granulosa cells line COV434 were randomly divided into 6 groups, control group was not treated, H 2O 2 groups were treated with H 2O 2 of 200 μmol/L, 400 μmol/L, 600 μmol/L, 800 μmol/L and 1 000 μmol/L for 0.5 h, 1 h, 2 h, 4 h and 6 h, respectively, and the cell viability was determined by CCK-8 method. The follow-up experiments were treated with different concentrations of H 2O 2 for 1 h. β-galactosidase staining was used to determine the degree of cell senescence. DCFH-DA fluorescence staining was determined by flow cytometry, and the level of reactive oxygen species (ROS) in cells was determined. JC-1 staining was used to determine the mitochondrial membrane potential of cells. Western blotting was used to determine the expression levels of apoptosis-related proteins Caspase-3 and Caspase-9. After the successful establishment of the model, in order to verify the usability of the cell model, the cells were pretreated with the antioxidant vitamin E for 12 h, followed by the addition of H 2O 2 for intervention, and the ROS level and mitochondrial membrane potential were measured. Results:The cell viability of the 200 μmol/L and 400 μmol/L groups decreased first and then increased compared with the control, and tended to be stable after 1 h of intervention, and there was no significant difference in cell viability at each time point (all P>0.05). When the concentration of H 2O 2 increased to 600 μmol/L, the cell viability gradually decreased with the treatment time and tended to stabilize after 1 h, and decreased significantly to nearly 50% ( P<0.001). When the concentration of H 2O 2 continued to increase to 800 μmol/L and 1 000 μmol/L, the cell viability gradually decreased with the treatment time and stabilized after 1 h, and decreased to less than 10% (all P<0.001). When the concentration of H 2O 2 was 200 μmol/L and 400 μmol/L, there was no significant difference in the ratio of β-galactosidase-positive cells and the relative ROS intensity after 1 h compared with the control (all P>0.05). When the concentration of H 2O 2 increased to 600 μmol/L, 800 μmol/L and 1 000 μmol/L, the ratio of β-galactosidase-positive cells and the relative ROS intensity increased significantly (β-galactosidase staining: P=0.011 at 600 μmol/L, P=0.003 at 800 μmol/L, P=0.005 at 1 000 μmol/L; the relative ROS intensity: P=0.002 at 600 μmol/L, P<0.001 at 800 μmol/L and 1 000 μmol/L). Compared with the control, the mitochondrial membrane potential of cells decreased gradually after intervention with different concentrations of H 2O 2, and was negatively correlated with H 2O 2 concentration (all P<0.001). There was no difference in the expression of Cleaved-Caspase-3 in the H 2O 2 group at 200 μmol/L compared with the control, and the expression was significantly increased at 400 μmol/L, 600 μmol/L, 800 μmol/L and 1 000 μmol/L (all P<0.001). The expressions of Caspase-3 and Caspase-9 were significantly increased in all H 2O 2 treated groups (all P<0.001). Compared with the model group, the relative ROS intensity of the vitamin E group was significantly reduced ( P=0.009), and the mitochondrial membrane potential was significantly increased ( P<0.001), but it could not be restored to the level of the control. Conclusion:Using 600 μmol/L H 2O 2 to continuously treat COV434 cells for 1 h can quickly establish a stable and effective oxidative stress injury model of human ovarian granulosa cells.

Objective:To investigate the distribution of CGG repeat numbers in the FMR1 gene among reproductive-age women in China, providing data reference for carrier screening and genetic counseling of Fragile X syndrome. Methods:This cross-sectional study recruited 16 610 reproductive-age women from 12 medical institutions between July 2022 and October 2023. Peripheral venous blood samples (3 mL) were collected, and genomic DNA was extracted. The number of CGG repeats in the FMR1 gene was determined using the triplet-primed polymerase chain reaction (TP-PCR) combined with capillary electrophoresis technology. Statistical analyses were performed to assess the prevalence and distribution of CGG repeat expansions. Results:Among 16 610 women of childbearing age, 5 684 (34.220%) women had the same number of CGG repeats in the two alleles of FMR1 gene, and 10 926 (65.780%) women had different numbers of repeats in the two alleles. Among the 33 220 FMR1 alleles in 16 610 women of reproductive age, the most common CGG repeat numbers were 29 [48.645% (16 160/33 220)] and 30 [26.276% (8 729/33 220)], while the most frequent CGG genotype was CGG 29/29 [24.726% (4 107/16 610)]. The CGG repeat numbers of FMR1 gene were normal in 16 498 women (99.326%). Among the 112 women (0.674%) with CGG repeat abnormities, 96 (0.578%) women were classified as intermediate carriers, 15 (0.090%) as premutation carriers, and 1 (0.006%) as a full mutation carrier, whose CGG genotype was (36, >200). Conclusion:In the general reproductive-age female population in China, the normal CGG repeat numbers of the FMR1 gene account for 99.326%, while the intermediate carrier rate is 0.578%, and the combined carrier rate of the premutation and full mutation types is 0.096%.

Objective:To carry out carrier screening among people of childbearing age, detect the pathogenic genes of monogenic genetic diseases and analyze the carrier status of pathogenic variants, so as to provide fertility guidance and intervention measures for high-risk families.Methods:From August 2022 to August 2023, 1 533 families of childbearing age who met the criteria were recruited in the Chinese PLA General Hospital, including a total of 3 044 subjects. According to the standard enrollment procedure, 223 genes (197 autosomal recessive genes and 26 X-linked genes) of the subjects were tested. According to the screening results, genetic counseling and fertility guidance were provided to the subjects. Invasive prenatal diagnosis was performed for high-risk couples (both couples being carriers of the same autosomal recessive disease gene or the woman was a carrier of X-linked disease gene), and their pregnancy pattern, outcome and offspring phenotype were followed up.Results:(1) A total of 3 044 cases from 1 511 couples and women of childbearing age from 22 families were included for carrier screening. Totally 1 503 families chose simultaneous screening and 30 families chose sequential screening out of the 1 533 families. Among the 3 044 subjects, 1 603 individuals carried at least one pathogenic or likely pathogenic variant, and the overall carrier rate was 52.66% (1 603/3 044). A total of 2 292 pathogenic or likely pathogenic variants were detected, and 0.75 variants (2 292/3 044) were detected per capita. (2) The three genes with the highest carrier rates were GJB2 (8.67%, 264/3 044), CYP21A2 (3.19%, 97/3 044) and PAH (3.09%, 94/3 044). There were 32 genes with a carrier rate ≥1/200, 17 genes with a carrier rate ≥1/100, and 7 genes with a carrier rate ≥1/50. (3) Thirty-eight high-risk families were identified. After excluding G6PD gene mutation, there were 33 high-risk families, of which 25 couples were carriers of the same autosomal recessive gene, 9 women were carriers of X-linked gene, and 1 family was double high-risk couple with both autosomal recessive and X-linked gene. After further excluding the GJB2 c.109G>A mutation, 21 high-risk families were identified. Preimplantation genetic testing for monogenic disease was performed in 12 families after genetic counseling. Prenatal diagnosis was completed in 4 out of 5 high-risk families who conceived naturally. Two fetuses carried the parental variants and terminated the pregnancy, one fetus did not carry the parental variants but was induced due to trisomy 21 syndrome, and one fetus was a carrier of congenital disorders of glycosylation type 1a.Conclusions:Carrier screening effectively identifies high-risk genetic disease families and provides reproductive guidance to prevent the birth of affected children. However, establishing multidisciplinary team is essential for managing complex cases. Implementation should prioritize prenatal institutions with genetic counseling or diagnostic expertise for monogenic disorders or established referral networks.

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 establish an oxidative stress injury model by using hydrogen peroxide (H 2O 2) to induce human ovarian granulosa cells COV434. Methods:Human ovarian granulosa cells line COV434 were randomly divided into 6 groups, control group was not treated, H 2O 2 groups were treated with H 2O 2 of 200 μmol/L, 400 μmol/L, 600 μmol/L, 800 μmol/L and 1 000 μmol/L for 0.5 h, 1 h, 2 h, 4 h and 6 h, respectively, and the cell viability was determined by CCK-8 method. The follow-up experiments were treated with different concentrations of H 2O 2 for 1 h. β-galactosidase staining was used to determine the degree of cell senescence. DCFH-DA fluorescence staining was determined by flow cytometry, and the level of reactive oxygen species (ROS) in cells was determined. JC-1 staining was used to determine the mitochondrial membrane potential of cells. Western blotting was used to determine the expression levels of apoptosis-related proteins Caspase-3 and Caspase-9. After the successful establishment of the model, in order to verify the usability of the cell model, the cells were pretreated with the antioxidant vitamin E for 12 h, followed by the addition of H 2O 2 for intervention, and the ROS level and mitochondrial membrane potential were measured. Results:The cell viability of the 200 μmol/L and 400 μmol/L groups decreased first and then increased compared with the control, and tended to be stable after 1 h of intervention, and there was no significant difference in cell viability at each time point (all P>0.05). When the concentration of H 2O 2 increased to 600 μmol/L, the cell viability gradually decreased with the treatment time and tended to stabilize after 1 h, and decreased significantly to nearly 50% ( P<0.001). When the concentration of H 2O 2 continued to increase to 800 μmol/L and 1 000 μmol/L, the cell viability gradually decreased with the treatment time and stabilized after 1 h, and decreased to less than 10% (all P<0.001). When the concentration of H 2O 2 was 200 μmol/L and 400 μmol/L, there was no significant difference in the ratio of β-galactosidase-positive cells and the relative ROS intensity after 1 h compared with the control (all P>0.05). When the concentration of H 2O 2 increased to 600 μmol/L, 800 μmol/L and 1 000 μmol/L, the ratio of β-galactosidase-positive cells and the relative ROS intensity increased significantly (β-galactosidase staining: P=0.011 at 600 μmol/L, P=0.003 at 800 μmol/L, P=0.005 at 1 000 μmol/L; the relative ROS intensity: P=0.002 at 600 μmol/L, P<0.001 at 800 μmol/L and 1 000 μmol/L). Compared with the control, the mitochondrial membrane potential of cells decreased gradually after intervention with different concentrations of H 2O 2, and was negatively correlated with H 2O 2 concentration (all P<0.001). There was no difference in the expression of Cleaved-Caspase-3 in the H 2O 2 group at 200 μmol/L compared with the control, and the expression was significantly increased at 400 μmol/L, 600 μmol/L, 800 μmol/L and 1 000 μmol/L (all P<0.001). The expressions of Caspase-3 and Caspase-9 were significantly increased in all H 2O 2 treated groups (all P<0.001). Compared with the model group, the relative ROS intensity of the vitamin E group was significantly reduced ( P=0.009), and the mitochondrial membrane potential was significantly increased ( P<0.001), but it could not be restored to the level of the control. Conclusion:Using 600 μmol/L H 2O 2 to continuously treat COV434 cells for 1 h can quickly establish a stable and effective oxidative stress injury model of human ovarian granulosa cells.

Objective:To investigate the distribution of CGG repeat numbers in the FMR1 gene among reproductive-age women in China, providing data reference for carrier screening and genetic counseling of Fragile X syndrome. Methods:This cross-sectional study recruited 16 610 reproductive-age women from 12 medical institutions between July 2022 and October 2023. Peripheral venous blood samples (3 mL) were collected, and genomic DNA was extracted. The number of CGG repeats in the FMR1 gene was determined using the triplet-primed polymerase chain reaction (TP-PCR) combined with capillary electrophoresis technology. Statistical analyses were performed to assess the prevalence and distribution of CGG repeat expansions. Results:Among 16 610 women of childbearing age, 5 684 (34.220%) women had the same number of CGG repeats in the two alleles of FMR1 gene, and 10 926 (65.780%) women had different numbers of repeats in the two alleles. Among the 33 220 FMR1 alleles in 16 610 women of reproductive age, the most common CGG repeat numbers were 29 [48.645% (16 160/33 220)] and 30 [26.276% (8 729/33 220)], while the most frequent CGG genotype was CGG 29/29 [24.726% (4 107/16 610)]. The CGG repeat numbers of FMR1 gene were normal in 16 498 women (99.326%). Among the 112 women (0.674%) with CGG repeat abnormities, 96 (0.578%) women were classified as intermediate carriers, 15 (0.090%) as premutation carriers, and 1 (0.006%) as a full mutation carrier, whose CGG genotype was (36, >200). Conclusion:In the general reproductive-age female population in China, the normal CGG repeat numbers of the FMR1 gene account for 99.326%, while the intermediate carrier rate is 0.578%, and the combined carrier rate of the premutation and full mutation types is 0.096%.

Objective:To investigate the clinical and genetic variation characteristics of a child with autosomal dominant lateral temporal lobe epilepsy caused by de novo variation of the MICAL1 gene. Methods:Clinical data of the patient with autosomal dominant lateral temporal lobe epilepsy caused by MICAL1 gene variation diagnosed in Children′s Hospital of Soochow University in August 2019 were collected. The whole exome sequencing was performed on the core members of the family, and the characteristics of gene variations were analyzed. Results:The proband, a 10 years and 5 months old boy, was admitted to the hospital because of "intermittent convulsions for 7 years". The clinical manifestations included focal or generalized tonic-clonic seizures and hearing aura, with normal language and intellectual development. No abnormalities were found in the T 1 and fluid attenuated inversion recovery sequences of the cranial 3.0 T magnetic resonance imaging and 3D thin-slice magnetic resonance imaging.Long-range video electroencephalogram showed the distribution of spinous and slow spinous waves in the left frontal and temporal areas. The results of whole exome gene sequencing in the core family members showed heterozygous de novo missense variation in the MICAL1 gene of the proband (NM_022765): c.763G>T(exon6)(p.Val255Leu) that had not been reported. According to American College of Medical Genetics and Genomics and Association for Molecular Pathology guidelines (2015), the mutation was considered potentially pathogenic. The application of antiepileptic drugs was effective in controlling epileptic seizures. Conclusions:Auditory symptoms are main clinical manifestations for the child with autosomal dominant lateral temporal lobe epilepsy. Antiepileptic drugs can effectively control epileptic seizures of the child, and the MICAL1 gene c.763G>T (p.Val255Leu) mutation is the genetic cause of the proband.