Screening and follow-up results of neonate medium-chain acyl-CoA dehydrogenase deficiency in Zibo, Shandong province.

OBJECTIVE: To analyze the incidence, phenotype, genotype and prognosis of neonatal medium-chain acyl-CoA dehydrogenase deficiency (MCADD) in Zibo city of Shandong province. METHODS: A total of 241 297 neonates were screened for MCADD in Zibo city of Shandong province from November 2013 to January 2022. Non-derivatized tandem mass spectrometry was used to detect blood free carnitine and acylcarnitine profiles in neonatal screening. Neonates with octanoylcarnitine (C8)≥0.25 µmol/L, or combined with C8/decanoylcarnitine (C10)≥1.5 were recalled, and second-generation high-throughput sequencing was performed for genetic diagnosis. RESULTS: Among 241 297 neonates, 6 cases of MCADD were screened, including 2 boys and 4 girls, with an incidence of 1/40 216. Two mutation sites of ACADM gene were identified in all MCADD infants, and 12 mutation with 8 types were detected in total. The hot spot mutations were c.449_452del (p.T150Rfs*4) and c.387+1delG， and exon 11 c.1076C>T (p.A359V) was a newly detected mutation. No phenotype-genotype correlation was found. One case died on day 4 after birth; 5 cases were followed up for 2 to 60 months, none of them received special diet treatment. The growth and intellectual development of the surviving cases were normal, and no abnormality was found in routine biochemical indicators. CONCLUSIONS: The incidence of MCADD in Zibo city seems to be higher than that in other areas in China. The ACADM gene mutations c.449_452del (p.T150Rfs*4) and c.387+1delG are common, and a new mutation c.1076C>T (p.A359V) has been detected. No phenotype-genotype correlation has been found. Early diagonsis and treatment are effective measures to reduce poor prognosis.

MiR-520h inhibits viability and facilitates apoptosis of KGN cells through modulating IL6R and the JAK/STAT pathway.

This study embarked on the assessment regarding the function and mechanism of miR-520h/IL6R axis in polycystic ovary syndrome (PCOS). Specifically, we analyzed the differential expression of IL6R in PCOS samples and normal samples based on the GEO database, and then verified IL6R expression in KGN cells (Human granulosa-like tumor cell line) using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and western blot. MiRNA targeting IL6R was predicted by bioinformatics analysis and verified by luciferase reporter assay, qRT-PCR and western blot. KGN cells were transfected with miR-520h inhibitor and si-IL6R, and then the cell viability and apoptosis were detected by cell counting kit-8 (CCK-8) and flow cytometry assays. Additionally, western blot was applied to examine the expressions of cell cycle-, apoptosis-, and JAK / STAT pathway-related proteins. IL6R was highly expressed in PCOS and KGN cells, and IL6R silencing inhibited the viability, while promoting the apoptosis of KGN cells. Importantly, miR-520h directly targeted IL6R and inhibited IL6R expression. Moreover, downregulation of miR-520h enhanced the cell viability, impeded the cell apoptosis, upregulated the expressions of CDK2, CCNB1, Bcl-2, activated JAK/STAT pathway and downregulated Bax expression in KGN cells. Of note, knockdown of IL6R can reverse the biological functions of miR-520h in KGN cells. Collectively, miR-520h hindered the proliferation and promoted the apoptosis of KGN cells via targeting IL6R to inhibit the development of PCOS, and these effects were possibly realized by JAK/STAT pathway. However, the effect of miR-520h in the progression of PCOS need to further study in the GCs.

[Self-made metal freezing plate for cryopreservation of human epididymal sperm: An experimental study].

OBJECTIVE: To study the effect of a new human sperm freezing method on the sperm recovery rate and search for an optimal method for cryopreservation of human epididymal sperm. METHODS: We collected semen samples from 76 men with obstructive azoospermia by percutaneous epididymal sperm aspiration and divided each sample into two parts to be cryopreserved with a self-made metal freezing plate (the experimental group) or by slow freezing (the control group), respectively. We measured the percentage of progressively motile sperm (PMS) with the computer-assisted semen analysis system and compared the membrane function, DNA fragmentation index (DFI), acrosin activity and morphological abnormality of the sperm between the two groups before and after cryopreservation. RESULTS: After thawing, both the percentages of PMS and hypotonically swollen sperm were significantly higher in the experimental than in the control group (ï¼»12.0 ± 7.5ï¼½% vs ï¼»8.0 ± 5.1ï¼½%, P < 0.05; ï¼»22.0 ± 17.5ï¼½% vs ï¼»18.0 ± 20.5ï¼½%, P < 0.05), though both decreased in comparison with the pre-freezing parameters (ï¼»20.7 ± 8.8ï¼½% and ï¼»30.0 ± 13.5ï¼½%) (P < 0.05). The sperm acrosin activity was remarkably higher in the experimental than in the control group after thawing (ï¼»75.2 ± 9.5ï¼½ vs ï¼»55.7 ± 8.3ï¼½ µIU/106sperm, P < 0.05), though decreased as compared with the baseline (ï¼»120.0 ± 10.5ï¼½ µIU/106 sperm, P < 0.05). No statistically significant differences were observed between the experimental and the control groups after thawing in the percentage of morphologically abnormal sperm (ï¼»98.7 ± 8.8ï¼½% vs ï¼»98.5±9.2ï¼½%, P > 0.05) or sperm DFI ï¼»38.2 ± 8.5ï¼½% vs ï¼»39.5 ± 10.2ï¼½%, P > 0.05), though both markedly elevated in comparison with the pre-freezing parameters (ï¼»97.2 ± 9.5ï¼½% and ï¼»30.8 ± 9.7ï¼½%) (P < 0.05). The post-thaw recovery rate of sperm was significantly higher in the experimental than in the control group (ï¼»65.2 ± 12.0ï¼½% vs ï¼»52.3 ± 18.0ï¼½%, P < 0.05). CONCLUSIONS: The self-made metal freezing plate, with its advantages of low cost, high efficiency, and easy operation, can be used as an effective method for cryopreservation of human sperm to achieve a high post-thaw sperm recovery rate, progressive sperm motility, and sperm acrosin activity.

The expression of cytokines IFN-γ, IL-4, IL-17A, and TGF-ß1 in peripheral blood and follicular fluid of patients testing positive for anti-thyroid autoantibodies and its influence on in vitro fertilization and embryo transfer pregnancy outcomes.

The aim of this work was to study the expression of the cytokines IFN-γ, IL-4, IL-17 A, and TGF-ß1 in peripheral blood and follicular fluid (FF) of patients positive for antithyroid autoantibodies (ATA+) with normal thyroid gland function and the influence of these autoantibodies on in vitro fertilization and embryo transfer (IVF-ET) pregnancy outcomes. Nineteen patients were in the ATA+ group, and 27 patients tested negative for anti-thyroid autoantibody (ATA-). Blood samples were drawn from the two groups of patients on the oocyte retrieval day and the 5th and 14th days of transplantation; in addition, FF was extracted on the oocyte retrieval day from both groups of patients and tested through enzyme-linked immunosorbent assay (ELISA) for IFN-γ, IL-4, IL-17 A, and TGF-ß1. For the ATA+ group, the concentration of IFN-γ increased whereas the concentration of TGF-ß1 decreased in peripheral blood on the oocyte retrieval day (p < .05); the concentration of IL-4 decreased in peripheral blood on the 5th and 14th days of transplantation for the ATA+ group (p < .05); further, the concentration of IL-17 A increased whereas that of TGF-ß1 decreased in FF (p < .05). The ratio of IL-17 A/TGF-ß1 in the ATA+ group significantly increased in FF and peripheral blood on the oocyte retrieval day and the 14th day of transplantation (p < .05). The ratio of IL-17 A/TGF-ß1 in FF of the pregnant patients was significantly lower than in the non-pregnant patients (p < .05). The findings suggested that the ratio between pro-inflammatory and anti-inflammatory cytokines was adversely affected; therefore, adverse pregnancy outcomes of patients with ATA+ undergoing IVF-ET treatment may be attributed to immunological mechanisms.

[F10 gene knock-down mediated by RNA interference induces apoptosis of KLE cells].

OBJECTIVE: To study the effect of short double-stranded RNA (dsRNA) on F10 gene expression in KLE cells and the effect of F10 knock-down on KLE cell apoptosis. METHODS: The short dsRNA specifically targeting F10 gene prepared by in vitro transcription was transfected into KLE cells via lipofectamine 2000. The expression of F10 mRNA in the transfected KLE cells was detected by real-time quantitative RT-PCR, and the apoptosis of the cells was assayed by flow cytometry. RESULTS: Real-time quantitative RT-PCR demonstrated that transfection of the KLE cells with the short dsRNA induced effective knock-down of F10 gene, and transfection of the cells with 20 nmol/L dsRNA for 48 h decreased the expression of F10 mRNA by 83%. Compared with the control, the apoptosis index of the transfected KLE cells increased from 0.36% to 8.91%. CONCLUSION: F10 gene in KLE cells can be specifically knocked-down with dsRNA prepared by in vitro transcription, and the down-regulation of F10 gene induces apoptosis of KLE cells.

[Pituitary down-regulation and ovulation stimulation with gradually increased gonadotropin in controlled ovarian hyperstimulation].

OBJECTIVE: To evaluate the efficacy of ovulation stimulation protocol with gradual increment of gonadotropin in women with high ovarian response. METHODS: A retrospective study was conducted between june 2005 and April 2006 in 70 women undergoing controlled ovarian hyperstimulation. The clinical outcomes of the women using gradual increment protocol were compared with those of women receiving other ovulation-stimulating protocols. RESULTS: The mean number of large follicles (>or=14 mm) and retrieved oocytes on the day of retrival was significantly lower, but the duration of stimulation was significantly longer in the gradual increment group than in the control group. The rate of follicular puncture was also higher in the former group. The clinical pregnancy rate, total gonadotropin dosage, cancellation rate and incidence of ovarian hyperstimulation syndrome were similar for the two groups. CONCLUSION: Ovulation stimulation protocol with gradually increased gonadotropin may provide a promising alternative for controlled ovarian hyperstimulation in women with a strong ovarian response.