OBJECTIVE: To develop a quality consistency evaluation strategy for traditional Chinese medicine (TCM) granules using sugar free Yangwei Granules as a model drug, and demonstrate the effectiveness of the developed method. METHODS: The strategy integrates several methods including, HPLC fingerprint and physical fingerprint methods analyze the similarity in chemical and physical properties of the TCM granule samples. Near-infrared (NIR) spectroscopy with principal components cluster analysis method is used to monitor normal operating conditions (NOC) samples accurately and to identify different types of abnormal operating conditions (AOC) samples, particularly those that deviate from the normal range. RESULTS: The combined use of HPLC fingerprint and physical fingerprint provides insights into the chemical and physical properties of the samples. NIR spectroscopy, combined with principal components cluster analysis, achieves high accuracy in monitoring NOC samples and identifying AOC samples without misjudgment. The approach proves useful as a complementary method in cases where HPLC fingerprint and physical fingerprint alone lack sufficient resolution. CONCLUSION This study establishes the feasibility and utility of the integrated approach for assessing the quality consistency of TCM granules. The strategy shows a high degree of generalization and holds significant importance for enhancing the quality control processes of TCM granules.

Objective:To analyze the clinical characteristics and genetic basis of a male patient with primary infertility caused by Acephalic spermatozoa syndrome.Methods:A patient who had presented at the Henan Provincial People′s Hospital on October 1, 2022 was selected as the study subject. Clinical data and results of laboratory exams and sperm electron microscopy were collected. The patient was subjected to whole exome sequencing (WES), and candidate variants were verified by Sanger sequencing and pathogenicity analysis.Results:WES revealed that the patient has harbored compound heterozygous variants of the PMFBP1 gene, namely c. 853del (p.Ala285Leufs*24) and c. 1276A>T (p.Lys426X), which were both unreported previously. Sanger sequencing suggested that the c. 853del (p.Ala285Leufs*24) variant has derived from his deceased mother, whilst the c. 1276A>T (p.Lys426X) variant has derived from his father. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were classified as pathogenic (PVS1+ PM2_Supporting+ PP4). Conclusion:The compound heterozygous variants of the PMFBP1 gene probably underlay the Acephalic spermatozoa syndrome in this patient. The discovery of the novel variants has also enriched the mutational spectrum of Acephalic spermatozoa syndrome.

Objective To summarize the medication law of TCM in the treatment of chronic bronchitis;To provide reference for clinical medication.Methods Medical records of patients with chronic bronchitis who were hospitalized in the Respiratory Department of the First Affiliated Hospital of Henan University of Chinese Medicine from January 1,2016 to December 31,2021 were extracted based on HIS electronic medical record data.After screening,the TCM prescriptions used by patients with chronic bronchitis were input into Excel 2019 to establish a database.Based on the software Lantern 5.0,the latent structure model was learned,hidden variables and explicit variables were obtained,and the model was interpreted.SPSS Modeler 18.0 was used to establish model points with Apriori algorithm for Chinese materia medica with a frequency greater than 6%,to obtain the association rules between drugs,and to analyze the medication law of TCM in treating chronic bronchitis.Results A total of 3 410 cases were included,involving 423 kinds of Chinese materia medica,with a cumulative frequency of 82 766 times.Among them,109 kinds of Chinese materia medica with a frequency of>6 % had a cumulative frequency of 69 845 times.The top five commonly used medicines were Fritillariae Cirrhosae Bulbus,Poria,Atractyodis Macrocephalae Rhizoma,Asteris Radix et Rhizoma,Citri Reticulatae Pericarpium,mainly with medicines of reducing cough and phlegm,antiasthmatic medicine,tonifying deficiency,clearing heat,relieving superficies,promoting blood circulation and removing blood stasis.The medicinal properties were warming,cold and mild,and the main tastes were bitter,sweet and pungent,and the meridians were mainly lung,spleen,liver and stomach meridians.Through analysis of latent structure,49 hidden variables and 149 hidden classes were obtained.Combined with professional knowledge,10 comprehensive clustering models and 21 core formulas were deduced,such as Sangbaipi Decoction,Xuefu Zhuyu Decoction,Xiaoqinglong Decoction,Erchen Decoction,Shashen Maidong Decoction,Liuwei Dihuang Pills,Yinqiao Powder,Zhisou Powder,Yupingfeng Powder,Xuefu Zhuyu Decoction combined with Daotan Decoction,etc.It was concluded that the chronic bronchitis syndrome included phlegm-heat stagnation lung syndrome,qi stagnation blood stasis syndrome,cold fluid attacking lung syndrome,phlegm-dampness accumulation lung syndrome,lung qi and yin deficiency syndrome,kidney yin deficiency syndrome,wind heat attacking lung syndrome,wind cold attacking lung syndrome,lung qi and spleen deficiency syndrome,phlegm stasis interjunction syndrome.A total of 41 strong association rules were screened in the analysis of association rules,including 5 strong association rules for two and 36 strong association rules for three.The high confidence rules were Saposheikovize Radix + Angelicae Sinensis Radix →Atractyodis Macrocephalae Rhizoma,Saposheikovize Radix + Codonopsis Radix → Atractyodis Macrocephalae Rhizoma,Codonopsis Radix + Citri Reticulatae Pericarpium → Atractyodis Macrocephalae Rhizoma;the higher degree of improvement were Bupleuri Radix + Mori Cortex → Scutellariae Radix,Perillae Fructus + Belamcandae Rhizoma → Fritillariae Cirrhosae Bulbus,Armeniacae Semen Amarum + Pinelliae Rhizoma → Citri Reticulatae Pericarpium,etc.Conclusion In the treatment of chronic bronchitis,TCM is mainly used to reduce phlegm,relieve cough and asthma,and the method of promoting blood circulation and removing blood stasis is commonly used to help eliminate phlegm.In addition,TCM pays attention to the application of methods such as tonifying lung and securing the exterior,invigorating spleen and benefiting qi.

Objective:To investigate the clinical outcomes of infertility patients with multiple morphological abnormalities of the sperm flagella (MMAF) caused by DNAH1 gene mutation after intracytoplasmic sperm injection (ICSI). Methods:A retrospective cohort study analyzed the clinical data and genetic test results of 39 MMAF infertility patients who were treated in the Center for Reproductive Medicine of Henan Provincial People's Hospital from February 2018 to January 2020. Twelve MMAF patients caused by DNAH1 mutations were acted as DNAH1 positive group and 27 MMAF patients with no DNAH1 mutations were acted as DNAH1 negative group. Totally 100 cases of infertility patients with normal sperm morphology and their spouses who were age-matched by both men and women for ICSI during the same period were selected as control group. The outcomes of assisted pregnancy treatment in the three groups were analyzed. Results:All 39 MMAF patients underwent whole-exome sequencing. Among them, 12 patients had DNAH1 gene mutations, 10 cases of compound heterozygous mutations and 2 cases of homozygous mutations, and the other 27 cases were not detected the currently known DNAH1 mutations. The patients of three groups were treated with ICSI, and the differences in the number of oocytes obtained and the number of M II oocytes in the DNAH1 gene positive group, DNAH1 gene negative group and control group were statistically significant (17.08±5.32, 9.59±3.98, 10.44±6.33, P=0.001; 14.58±5.18, 6.78±3.38, 8.32±5.31, P<0.001). There were no statistically significant differences in the embryo implantation rate, the clinical pregnancy rate, the embryo miscarriage rate and the live birth rate (all P>0.05). Among them, 12 couples of male infertility caused by DNAH1 mutation received a total of 12 cycles of oocyte extraction, forming 79 day 3 embryos, 12 times of the first fresh or frozen embryo transplantation, and 10 biological offspring were obtained. Conclusion:For patients with MMAF caused by DNAH1 gene mutation, ICSI can help them to give birth to their own offspring, and has a higher clinical pregnancy rate and live birth rate.

Objective:To investigate the clinical outcomes of infertility patients with multiple morphological abnormalities of the sperm flagella (MMAF) caused by DNAH1 gene mutation after intracytoplasmic sperm injection (ICSI). Methods:A retrospective cohort study analyzed the clinical data and genetic test results of 39 MMAF infertility patients who were treated in the Center for Reproductive Medicine of Henan Provincial People's Hospital from February 2018 to January 2020. Twelve MMAF patients caused by DNAH1 mutations were acted as DNAH1 positive group and 27 MMAF patients with no DNAH1 mutations were acted as DNAH1 negative group. Totally 100 cases of infertility patients with normal sperm morphology and their spouses who were age-matched by both men and women for ICSI during the same period were selected as control group. The outcomes of assisted pregnancy treatment in the three groups were analyzed. Results:All 39 MMAF patients underwent whole-exome sequencing. Among them, 12 patients had DNAH1 gene mutations, 10 cases of compound heterozygous mutations and 2 cases of homozygous mutations, and the other 27 cases were not detected the currently known DNAH1 mutations. The patients of three groups were treated with ICSI, and the differences in the number of oocytes obtained and the number of M II oocytes in the DNAH1 gene positive group, DNAH1 gene negative group and control group were statistically significant (17.08±5.32, 9.59±3.98, 10.44±6.33, P=0.001; 14.58±5.18, 6.78±3.38, 8.32±5.31, P<0.001). There were no statistically significant differences in the embryo implantation rate, the clinical pregnancy rate, the embryo miscarriage rate and the live birth rate (all P>0.05). Among them, 12 couples of male infertility caused by DNAH1 mutation received a total of 12 cycles of oocyte extraction, forming 79 day 3 embryos, 12 times of the first fresh or frozen embryo transplantation, and 10 biological offspring were obtained. Conclusion:For patients with MMAF caused by DNAH1 gene mutation, ICSI can help them to give birth to their own offspring, and has a higher clinical pregnancy rate and live birth rate.

Objective:To explore the effect of dibutyl phthalate (DBP) on the rat testis Leydig cell apoptosis by AMP activated protein kinase/mammalian rapamycin target protein (AMPK/mTOR) signaling pathway.Methods:Rats with reproductive function impairment were divided into model (DBP) group of 17 rats, model+AMPK inhibitor [DBP+compound C (CC)] group of 17 rats, model+AMPK agonist [DBP+metformin (MF)] group of 17 rats, DBP+AMPK inhibitor+agonist (DBP+CC+MF) group of 17 rats by body mass ranking grouping method. Another 11 rats were taken as the blank group. The blank group and DBP group were intraperitoneally injected with the same amount of normal saline, while DBP+CC group and DBP+MF group were intraperitoneally injected with 20 mg/kg CC and 200 mg/kg MF respectively, and DBP+CC+MF group was intraperitoneally injected with CC and MF once a day for 4 weeks. Luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone (T) were measured by radioimmunoassay. Sperm quality was analyzed by automatic sperm quality analysis system. Leydig cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and flow cytometry. The expressions of AMPK, mTOR, Caspase 3 mRNA and protein, p-AMPK and p-mTOR protein were detected by RT-PCR and Western blotting. Results:The serum level of FSH in DBP+MF group [(9.88±0.67) U/L] increased, while that in DBP+CC group [(6.82±0.60) U/L] decreased compared with DBP group [(9.07±0.52) U/L] (all P<0.001). The serum LH, T levels and sperm concentration, percentage of (a+b) grade sperm in DBP+MF group [(3.97±0.70) U/L, (2.96±0.11) mg/L, (13.15±2.63)×10 6/mL, (22.20±4.13)%], DBP+CC group [(6.52±0.71) U/L, (4.48±0.15) mg/L, (25.47±2.18)×10 6/mL, (45.60±4.78)%] increased compared with DBP group [(4.51±0.75) U/L, (3.25±0.11) mg/L, (16.46±3.40)×10 6/mL, (25.43±4.36)%] (DBP group vs. DBP+MF group PLH=0.038, the other all P<0.001). HE staining showed that the structure of testis in blank group was normal. In DBP group and DBP+CC+MF group, the epithelial cells of seminiferous tubules atrophied and twisted in irregular shape, and the disease became serious in DBP+MF group, and there were a lot of vacuoles around the nucleus. The number of apoptosis, p-AMPK/AMPK protein relative expression and Caspase 3 mRNA and protein relative expression of Leydig cells in DBP+MF group (286.60±30.17, 0.95±0.08, 2.17±0.18, 1.23±0.10) increased, and DBP+CC group (88.00±21.34, 0.42±0.04, 1.35±0.15, 0.54±0.06) decreased compared with those in DBP group (142.40±26.78, 0.70±0.07, 1.85±0.14, 0.80±0.09, all P<0.001). Compared with DBP group (0.45±0.06), the p-mTOR/mTOR of DBP+MF group (0.23±0.04) decreased, and the p-mTOR/mTOR of DBP+CC group (0.84±0.07) increased (all P<0.001). Conclusion:DBP can damage the reproductive system of rats and increase the apoptosis rate of Leydig cells, which may be related to AMPK activation and mTOR inhibition.

Objective:To investigate the effects of different partial deletions in azoospermia factor (AZF) locus of Y-chromosome on the clinical outcome of severe oligoasthenozoospermia patients by intracytoplasmic sperm injection (ICSI).Methods:A retrospective cohort study was conducted on the patients undergoing high-throughput sequencing for Y chromosome microdeletion screening and ICSI treatment in Reproductive Medicine Center of Henan Provincial People's Hospital from December 2017 to July 2020. According to whether carrying AZFc microdeletions or not, the patients were divided into the AZFc-deletion group and control group. And AZFc-deletion group was divided into 3 subgroups, b2/b3 deletion, b2/b4 deletion and gr/gr deletions subgroup, by the types of partial deletion.Results:The day 3 (D3) available embryo rate, the high-quality embryo rate, and the blastocyst formation rate in patients with AZFc deletion were statistically lower than those in control group [70.4% (556/790) vs. 78.5% (2867/3651), P<0.001; 24.7% (199/807) vs. 34.3% (1284/3747), P<0.001; 51.7% (277/536) vs. 58.0% (1540/2592), P=0.007], and there were no statistical differences in implantation rate, clinical pregnancy rate, live birth rate during transplantation cycle between the two groups (all P>0.05). The AZFc b2/b3 deletion subgroup had no significant differences in D3 available embryo rate, high-quality embryo rate, blastocyst formation rate, implantation rate, clinical pregnancy rate and live birth rate, compared with control group (all P>0.05). The rate of high-quality embryos in patients with the b2/b4 deletion subgroup [23.2% (32/138)] was lower than that of control group ( P=0.004), but there were no statistical differences in D3 available embryo rate, blastocyst formation rate, implantation rate, clinical pregnancy rate and live birth rate (all P>0.05). The D3 available embryo rate [71.6% (280/391)], the high-quality embryo rate [20.8% (84/403)] and the blastocyst formation rate [48.7% (133/273)] in patients of gr/gr deletion subgroup were significantly lower than those in control group ( P=0.002, P<0.001, P<0.001), but there were no statistical differences in implantation rate, clinical pregnancy rate and live birth rate (all P>0.05). Conclusion:AZFc b2/b3 deletion and b2/b4 deletion in the AZFc locus of Y chromosome have no significant effect on embryonic development and pregnancy outcome in patients with severe oligoasthenozoospermia undergoing ICSI. Gr/gr deletion has most adverse effect on embryonic development but no effect on pregnancy outcome.

Objective:To explore the effect of dibutyl phthalate (DBP) on the rat testis Leydig cell apoptosis by AMP activated protein kinase/mammalian rapamycin target protein (AMPK/mTOR) signaling pathway.Methods:Rats with reproductive function impairment were divided into model (DBP) group of 17 rats, model+AMPK inhibitor [DBP+compound C (CC)] group of 17 rats, model+AMPK agonist [DBP+metformin (MF)] group of 17 rats, DBP+AMPK inhibitor+agonist (DBP+CC+MF) group of 17 rats by body mass ranking grouping method. Another 11 rats were taken as the blank group. The blank group and DBP group were intraperitoneally injected with the same amount of normal saline, while DBP+CC group and DBP+MF group were intraperitoneally injected with 20 mg/kg CC and 200 mg/kg MF respectively, and DBP+CC+MF group was intraperitoneally injected with CC and MF once a day for 4 weeks. Luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone (T) were measured by radioimmunoassay. Sperm quality was analyzed by automatic sperm quality analysis system. Leydig cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and flow cytometry. The expressions of AMPK, mTOR, Caspase 3 mRNA and protein, p-AMPK and p-mTOR protein were detected by RT-PCR and Western blotting. Results:The serum level of FSH in DBP+MF group [(9.88±0.67) U/L] increased, while that in DBP+CC group [(6.82±0.60) U/L] decreased compared with DBP group [(9.07±0.52) U/L] (all P<0.001). The serum LH, T levels and sperm concentration, percentage of (a+b) grade sperm in DBP+MF group [(3.97±0.70) U/L, (2.96±0.11) mg/L, (13.15±2.63)×10 6/mL, (22.20±4.13)%], DBP+CC group [(6.52±0.71) U/L, (4.48±0.15) mg/L, (25.47±2.18)×10 6/mL, (45.60±4.78)%] increased compared with DBP group [(4.51±0.75) U/L, (3.25±0.11) mg/L, (16.46±3.40)×10 6/mL, (25.43±4.36)%] (DBP group vs. DBP+MF group PLH=0.038, the other all P<0.001). HE staining showed that the structure of testis in blank group was normal. In DBP group and DBP+CC+MF group, the epithelial cells of seminiferous tubules atrophied and twisted in irregular shape, and the disease became serious in DBP+MF group, and there were a lot of vacuoles around the nucleus. The number of apoptosis, p-AMPK/AMPK protein relative expression and Caspase 3 mRNA and protein relative expression of Leydig cells in DBP+MF group (286.60±30.17, 0.95±0.08, 2.17±0.18, 1.23±0.10) increased, and DBP+CC group (88.00±21.34, 0.42±0.04, 1.35±0.15, 0.54±0.06) decreased compared with those in DBP group (142.40±26.78, 0.70±0.07, 1.85±0.14, 0.80±0.09, all P<0.001). Compared with DBP group (0.45±0.06), the p-mTOR/mTOR of DBP+MF group (0.23±0.04) decreased, and the p-mTOR/mTOR of DBP+CC group (0.84±0.07) increased (all P<0.001). Conclusion:DBP can damage the reproductive system of rats and increase the apoptosis rate of Leydig cells, which may be related to AMPK activation and mTOR inhibition.

Objective:To investigate the effects of different partial deletions in azoospermia factor (AZF) locus of Y-chromosome on the clinical outcome of severe oligoasthenozoospermia patients by intracytoplasmic sperm injection (ICSI).Methods:A retrospective cohort study was conducted on the patients undergoing high-throughput sequencing for Y chromosome microdeletion screening and ICSI treatment in Reproductive Medicine Center of Henan Provincial People's Hospital from December 2017 to July 2020. According to whether carrying AZFc microdeletions or not, the patients were divided into the AZFc-deletion group and control group. And AZFc-deletion group was divided into 3 subgroups, b2/b3 deletion, b2/b4 deletion and gr/gr deletions subgroup, by the types of partial deletion.Results:The day 3 (D3) available embryo rate, the high-quality embryo rate, and the blastocyst formation rate in patients with AZFc deletion were statistically lower than those in control group [70.4% (556/790) vs. 78.5% (2867/3651), P<0.001; 24.7% (199/807) vs. 34.3% (1284/3747), P<0.001; 51.7% (277/536) vs. 58.0% (1540/2592), P=0.007], and there were no statistical differences in implantation rate, clinical pregnancy rate, live birth rate during transplantation cycle between the two groups (all P>0.05). The AZFc b2/b3 deletion subgroup had no significant differences in D3 available embryo rate, high-quality embryo rate, blastocyst formation rate, implantation rate, clinical pregnancy rate and live birth rate, compared with control group (all P>0.05). The rate of high-quality embryos in patients with the b2/b4 deletion subgroup [23.2% (32/138)] was lower than that of control group ( P=0.004), but there were no statistical differences in D3 available embryo rate, blastocyst formation rate, implantation rate, clinical pregnancy rate and live birth rate (all P>0.05). The D3 available embryo rate [71.6% (280/391)], the high-quality embryo rate [20.8% (84/403)] and the blastocyst formation rate [48.7% (133/273)] in patients of gr/gr deletion subgroup were significantly lower than those in control group ( P=0.002, P<0.001, P<0.001), but there were no statistical differences in implantation rate, clinical pregnancy rate and live birth rate (all P>0.05). Conclusion:AZFc b2/b3 deletion and b2/b4 deletion in the AZFc locus of Y chromosome have no significant effect on embryonic development and pregnancy outcome in patients with severe oligoasthenozoospermia undergoing ICSI. Gr/gr deletion has most adverse effect on embryonic development but no effect on pregnancy outcome.

OBJECTIVE: To explore the clinical feature and gene variant for two cases of primary male infertility caused by severe asthenospermia and to analyze the etiology of the disease. METHODS: Genomic DNA of peripheral blood samples of patients and their parents was extracted and gene variant analysis of the patients was conducted by using whole exome sequencing. Suspected pathogenic variant was verified by Sanger sequencing and pathogenic analysis. RESULTS: Whole exome sequencing showed that the DNAH1 gene of patient 1 had two heterozygous variants of c.2016T>G(p.Y672X) and c.6017T>G (p.V2006G). The DNAH1 gene of patient 2 had a homozygous variant of c.2610G>A(p.W870X), which were inherited from his father and mother, respectively. According to American College of Medical Genetics and Genomics standards and guidelines, the c.2016T>G (p.Y672X) and c.2610G>A (p.W870X) varaints of DNAH1 gene were predicted to be pathogenic (PVS1+PM2+PM3+PP3). CONCLUSION The two patients of multiple morphological abnormalities of the sperm flagella may be caused by DNAH1 gene variant, which has resulted in primary male infertility.
Dyneins/genetics* ; Genomics ; Humans ; Infertility, Male/genetics* ; Male ; Mutation ; Sperm Tail/pathology* ; Whole Exome Sequencing