Clinical Application of Double Ovulation Stimulation in Patients with Diminished Ovarian Reserve and Asynchronous Follicular Development Undergoing Assisted Reproduction Technology.

OBJECTIVE: This study aimed to compare the clinical effects of double ovulation stimulation (DouStim) applied during the follicular and luteal phases with the antagonist protocol in patients with diminished ovarian reserve (DOR) and asynchronous follicular development undergoing assisted reproductive technology (ART). METHODS: The clinical data of patients with DOR and asynchronous follicular development receiving ART from January 2020 to December 2021 were retrospectively analyzed. The patients were divided into two groups according to their ovulation stimulation protocol: DouStim group (n=30) and antagonist group (n=62). Assisted reproduction and clinical pregnancy outcomes were compared between the two groups. RESULTS: In the DouStim group, the number of oocytes retrieved, metaphase II (MII) oocytes, two-pronuclei (2PN), day 3 (D3) embryos, D3 high-quality embryos as well as blastocyst formation, implantation, and human chorionic gonadotropin-positive rates were significantly greater than those in the antagonist group (all P<0.05). No significant differences were found in MII, fertilization, or continued pregnancy rates at the first frozen embryo transfer (FET), in-vitro fertilization (IVF) cancellation, or early medical abortion rates between the groups (all P>0.05). Except for the early medical abortion rate, the DouStim group generally had favorable outcomes. In the DouStim group, the dosage and duration of gonadotropin and the fertilization rate were significantly greater in the first ovulation stimulation induction than in the second ovulation stimulation induction (P<0.05). CONCLUSION: The DouStim protocol efficiently and economically obtained more mature oocytes and high-quality embryos for patients with DOR and asynchronous follicular development.

Distribution and identification of Mycobacterium tuberculosis lineage in Kashgar prefecture.

OBJECTIVES: Kashgar prefecture is an important transportation and trade hub with a high incidence of tuberculosis. The following study analyzed the composition and differences in Mycobacterium tuberculosis (M.tb) lineage and specific tags to distinguish the lineage of the M.tb in Kashgar prefecture, thus providing a basis for the classification and diagnosis of tuberculosis in this area. METHODS: Whole-genome sequencing (WGS) of 161 M.tb clinical strains was performed. The phylogenetic tree was constructed using Maximum Likelihood (ML) based on single nucleotide polymorphisms (SNPs) and verified through principal component analysis (PCA). The composition structure of M.tb in different regions was analyzed by combining geographic information. RESULTS: M.tb clinical strains were composed of lineage 2 (73/161, 45.34%), lineage 3 (52/161, 32.30%) and lineage 4 (36/161, 22.36%). Moreover, the 3 lineages were subdivided into 11 sublineages, among which lineage 2 included lineage 2.2.2/Asia Ancestral 1 (9/73, 12.33%), lineage 2.2.1-Asia Ancestral 2 (9/73, 12.33%), lineage 2.2.1-Asia Ancestral 3 (18/73, 24.66%), and lineage 2.2.1-Modern Beijing (39/73, 53.42%). Lineage 3 included lineage 3.2 (14/52, 26.92%) and lineage 3.3 (38/52, 73.08%), while lineage 4 included lineage 4.1 (3/36, 8.33%), lineage 4.2 (2/36, 5.66%), lineage 4.4.2 (1/36, 2.78%), lineage 4.5 (28/36, 77.78%) and lineage 4.8 (2/36, 5.66%), all of which were consistent with the PCA results. One hundred thirty-six markers were proposed for discriminating known circulating strains. Reconstruction of a phylogenetic tree using the 136 SNPs resulted in a tree with the same number of delineated clades. Based on geographical location analysis, the composition of Lineage 2 in Kashgar prefecture (45.34%) was lower compared to other regions in China (54.35%-90.27%), while the composition of Lineage 3 (32.30%) was much higher than in other regions of China (0.92%-2.01%), but lower compared to the bordering Pakistan (70.40%). CONCLUSION: Three lineages were identified in M.tb clinical strains from Kashgar prefecture, with 136 branch-specific SNP. Kashgar borders with countries that have a high incidence of tuberculosis, such as Pakistan and India, which results in a large difference between the M.tb lineage and sublineage distribution in this region and other provinces of China.

[Delayed decompression for cauda equina syndrome secondary to lumbar disc herniation: long-term follow-up results].

OBJECTIVE: To assess the impact of delayed decompression on long-term neurological and bladder function recovery in patients with cauda equina syndrome (CES) secondary to lumbar disc herniation (LDH). METHODS: The clinical data of 35 patients receiving delayed decompression surgery for CES secondary to LDH were reviewed. The bladder empty function, bowel control, sexual ability and neurological functions of the lower limbs were evaluated after the operation, and the urodynamic changes were assessed in 6 patients with urodynamic data before and after the operation. RESULTS: Surgical decompression was performed at 4.1∓3.9 weeks in 12 patients with complete CES and at 5.5∓7.6 weeks in 23 patients with incomplete CES after the onset of symptoms. The patients were followed up for a mean of 43.0∓28.9 months (3-110 months). In the 23 patients with incomplete CES, 19 obtained full recovery, 4 had slight sensory alterations in the saddle area or the lower limbs. In the 12 patients with complete CES, 2 had full recovery, 4 reported slight sensory alterations in the saddle area or the lower limbs (including 2 with occasional constipation); 6 still had sense deficit in the saddle area and difficulties in bladder or bowl emptying, but they all reported significant improvements compared to the condition before operation. Urodynamic analysis in the 6 patients with pre- and postoperative urodynamic data showed increased abdominal pressure when voiding with significantly reduced residual urine in all the 6 patients; 4 patients with abnormal first desire volume before operation reported recovery after the operation. CONCLUSION: Patients with LDH-induced CES who missed the chance of early decompression can still expect favorable functional recovery in the long term. The improvement of bladder function following decompression is probably a result of recovery of bladder sensation and the compensation by increased intra-abdominal pressure. The key strategy to promote bladder function recovery in these patients is to promote the detrusor recovery.

Distribution and expression characteristics of triterpenoids and OSC genes in white birch (Betula platyphylla suk.).

Betulin and oleanolic acids (pentacyclic triterpenoid secondary metabolites) have broad pharmacological activities and can be potentially used for the development of anti-cancer and anti-AIDS drugs. In this study, we detected the accumulation and the distribution characteristics of betulin and oleanolic acid in various organs of white birch at different ages. We also determined the expression of 4 OSC genes (LUS, ß-AS, CAS1 and CAS2) involved in the triterpenoid synthesis pathways by real time RT-PCR. The result showed that the 1-year old birch can synthesize betulin and oleanolic acid. In addition, betulin and oleanolic acids were mainly distributed in the bark, while the content in the root skin and leaf was very low. The content of betulin and oleanolic acid in birch varied in different seasons. The content of betulin and oleanolic acid and their corresponding LUS and ß-AS gene expression were very low in 1-year old birch. With increasing age of birch, betulin content was increased, while oleanolic acid was decreased. Similar changes were also observed for their corresponding synthesis genes LUS and ß-AS. In the leaf of 1-year old plant, the highest expression of CAS1 and CAS2 occurred at end of September, while expression of LUS and the ß-AS was low from June to October. In the stem skin,high expression of ß-AS and the LUS genes occurred from the end of July to September. In the root, high expression of the ß-AS gene was observed at the end of October. These results indicated that triterpenoid gene expression was similar to the triterpene accumulation. Expression of LUS gene and ß-AS gene in birch with different ages were corresponding to the betulinic and oleanolic acid accumulation. Expression of CAS1 and CAS2 genes were elevated with increasing age of birch. This study provides molecular mechanisms of triterpenes synthesis in birch plants.

Human amniotic fluid-derived stem cells can differentiate into hepatocyte-like cells in vitro and in vivo.

Although human amniotic fluid is an attractive source of multipotent stem cells, the potential of amniotic fluid stem cells (AFSCs) to differentiate into hepatic cells has not been extensively evaluated. In this study, we examined whether human AFSCs can differentiate into a hepatic cell lineage in vitro and in vivo. After being treated with cytokines (fibroblast growth factor 4, basic fibroblast growth factor, hepatocyte growth factor, and oncostatin), AFSCs developed a morphology similar to that of hepatocytes. RT-PCR and immunofluorescence analysis showed that the treated AFSCs expressed the hepatocyte-specific markers albumin, cytokeratin 18, and alpha-fetoprotein. The differentiated cells also developed hepatocyte-specific functions, i.e., they secreted albumin, absorbed indocyanine green, and stored glycogen. When transplanted into CCl(4)-injured immunodeficient mice, undifferentiated AFSCs were integrated into the liver tissue, and they expressed markers characteristic of mature human hepatocytes. Although integration of AFSCs into the liver was limited (0.1-0.3% of hepatocytes), histological analysis showed that the recipient mice recovered more rapidly from CCl(4) injury than CCl(4)-injured mice that did not receive AFSCs. AFSCs can differentiate into hepatocyte-like cells in vitro and in vivo and can represent an easily accessible source of progenitor cells for hepatocyte regeneration and liver cell transplantation.

[Predictive value of fluorescence in situ hybridization in patients with bladder cancer].

OBJECTIVE: To assess the value of fluorescence in situ hybridization (FISH) in the diagnosis of bladder cancer. METHODS: Urine samples from 100 patients suspected of having bladder cancer were collected before cystoscopy for immediate urine cytology and FISH analysis. The criteria for FISH abnormality were determined by evaluating the urine specimens from 20 subjects without urogenital neoplasm. RESULTS: The overall sensitivity of cytology and FISH was 43.2% and 82.4%, and their specificity was 92.3% and 88.5%, with diagnostic concordance rate of 56.0% and 84.0%, respectively. The differences between FISH and cytology showed statistical significance in the sensitivity, diagnostic concordance rate, non-muscle-invasive cancer and primary cancer. CONCLUSION: The sensitivity and efficiency of FISH in the detection of bladder cancer are superior to those of cytology, especially for prophase cancer.

Synthesis, structures, and solution dynamics of palladium complexes of quinoline-functionalized N-heterocyclic carbenes.

A new type of quinoline-functionalized palladium N-heterocyclic carbene (NHC) complexes has been synthesized via silver transmetallation. The quinoline moiety was either directly attached to the imidazole ring or linked to it by a methylene group. NHCs with a methylene linker tend to form trans biscarbene complexes in the reaction of Pd(COD)Cl2, while NHCs without any linker form chelating NHC-quinoline (NHC-N) complexes. These two types of carbenes also react with [Pd(allyl)Cl]2 to give monodentate NHC palladium eta(3)-allyl chlorides [Pd(NHC)(allyl)Cl]. Fluxionality in the NMR time scale was observed for most complexes, and the origin of their dynamic behaviors was discussed for each type of structure. For [Pd(NHC)(allyl)Cl] with a relatively small wing tip group of the NHC, the fluxionality (selective line-broadening of (1)H NMR signals) is caused by selective eta(3)-eta(1)-eta(3) allyl isomerization. For NHC with a bulkier (t)Bu group, a different line-broadening pattern was observed and was ascribed to partially hindered Pd-C(carbene) bond rotation. For cationic chelating complexes [Pd(NHC-N)(allyl)]BF4, the dynamic exchange process likely originates from a dissociative boat-to-boat inversion of 7-membered palladacycles. Activation parameters were measured for this process. Crystal structures were reported for representative complexes in each category.

Long-lived radical cations as model compounds for the reactive one-electron oxidation product of vitamin E.

Heterocyclic compounds with structures similar to vitamin E, but without the hydroxyl hydrogen atom, were synthesized and their electrochemical behavior examined in acetonitrile solutions and as solids in aqueous solutions of varying pH by attaching the compounds to the surface of a glassy carbon electrode. Compound 1, containing a fully methylated aromatic ring was found to be the most long-lived following one-electron oxidation, with its radical cation (1+*) surviving in acidic aqueous solutions and able to be isolated as a salt, 1+*(SbF6-), when reacted with NOSbF6 in CH3CN. Electrochemical, UV-vis and FTIR experiments on 1+*, in addition to the results from theoretical calculations, indicated that the electrochemical, electronic and structural properties of 1+* are very similar to those of the radical cation of vitamin E.

Variable scan rate cyclic voltammetry and theoretical studies on tocopherol (vitamin E) model compounds.

Variable scan rate (0.1-500 V s(-1)) cyclic voltammetry experiments were performed on a series of model tocopherol (vitamin E) compounds with differing degrees of methyl substitution around the aromatic (phenolic) ring. alpha-Tocopherol, with a fully methylated aromatic ring, produced stable phenoxonium cations upon oxidation in CH3CN, and was modeled via an ECE mechanism (where "E" represents an electron transfer and "C" a chemical step). Compounds with less methyl substitution around the aromatic ring were more reactive following oxidation, and formed additional oxidation products (hemiketals and p-quinones), and were modeled according to a more complicated ECECC mechanism. The equilibrium and rate constants associated with the chemical steps were estimated by digital simulations of the variable scan rate data over a range of temperatures ( T = 253-313 K) in acetonitrile containing 0.5 M Bu4NPF6 as the supporting electrolyte. The relative lifetimes of the phenoxonium cations of tocol and the tocopherols were compared with theoretical results obtained from molecular orbital calculations.

Investigation into phenoxonium cations produced during the electrochemical oxidation of chroman-6-ol and dihydrobenzofuran-5-ol substituted compounds.

A series of chroman-6-ol and dihydrobenzofuran-5-ol based compounds with structures similar to vitamin E were examined by cyclic voltammetry and controlled potential electrolysis. The compounds displayed characteristic voltammetric features that enabled their electrochemical behavior to be interpreted in relation to the oxidation mechanism for vitamin E. The electrochemical experiments indicated the presence of several oxidized species: cation radicals, phenoxyl radicals, phenoxonium ions, hemiketals, and p-quinones, whose lifetimes varied depending on the extent of methylation of the aromatic ring (R(1), R(2), R(3)) and the nature of substituents R(4) and R(5).

[Establishment and characteristics of clonal human embryonic stem cell lines].

OBJECTIVE: To establish clonal human embryonic stem cell lines and investigate their biological characteristics. METHODS: Cells were derived from one inner cell mass of human blastocyst, multiplied for 20 passages, and then dissociated into single cell suspension by digestion with 0.5% trypsin. Single cell was picked up and plated into individual well of a 96-well plate containing feeder-layers directly under a dissection microscope. The outgrowth clones were passed by treatment with collagenase. Surface markers were detected by cytochemistry and histoimmunochemistry. Karyotypes were tested using standard G-banding techniques. The pluripotency was analyzed by inoculating cells into severe combined immunodeficient (SCID) mice. RESULTS: Two clonal human embryonic stem cell lines were established. Cells of these two lines possess the characteristics and differentiating potencies: normal 46 XX karyotypes; expressing a series of surface markers such as: alkaline phosphotase, stage-specific embryonic antigen (SSEA)-4, tumor recognition antigen (TRA)-1-60, TRA-1-81 etc; and forming teratomas comprising derivatives of three embryonic germ layers such as neural tissue, cartilage, squamous epithelium and columnar epithelium when injected into SCID mice. CONCLUSIONS: The two single cell-cloned human embryonic stem (hES) cell lines were derived in our laboratory. The cells possess stable biological characteristics of undifferentiated hES cells.

[Influence of cryoprotectant and cooling rate in vitrification method on the spindles of rabbit oocytes].

OBJECTIVE: To investigate the influence of cryoprotectants and cooling rates in vitrification method on the spindles of rabbit M II oocytes. METHODS: Rabbit oocytes were verified by using cryoloop with ethylene glycol (EG) singly or EG combined with dimethyl sulphoxide (DMSO) as cryoprotectants, and cooled by taking oocytes directly into liquid nitrogen or by vitrification machine. After frozen rabbit oocytes thawed, the microtubulin and chromosome of the spindles were fixation and stained by immunofluorescent method. Confocal microscope was used to reveal spindle configuration. RESULTS: In the two protocols of single EG used and EG combined with DMSO, the spindles were severely injured. But in protocol of EG combined with DMSO and at ultra-rapid cooling rate, the normal configuration of spindle rate of thawed rabbit oocytes was similar to that of the control group. CONCLUSION: The protocol of EG combined with DMSO as cryoprotectants and with extremely high cooling rate by vitrification machine can produce the best effect on conservation of spindle configuration in vitrification of rabbit oocytes.

[Human embryonic stem cells forming embryoid bodies comprising multiple types of cells].

OBJECTIVE: To investigate the pluripotency of human embryonic stem cells in vitro. METHODS: Human embryonic stem cells were cultured in suspension without feeder layers and bFGF in medium. mRNAs of different types of cells were analyzed by RT-PCR. RESULTS: When Human embryonic stem cells were cultured in suspension without feeder layers and bFGF in medium, they formed floating aggregates termed embryoid bodies (EBs), in which many precursors and functional cells were detected by RT-PCR, such as neural and islet precursors, neurons, insulin-secreting cells,glucagon-secreting cells and liver cells et al. CONCLUSION: Human embryonic stem cells are able to form embryoid bodies naturally in vitro which express specific markers of many types of precursors and mature functional cells.

[Establishment of a single cell-cloned mouse embryonic stem cell line].

OBJECTIVE: To establish embryonic stem cells culture system and methods. METHODS: Inner cell masses were isolated from blastocysts of 3.5 day-old 129SvJ mice by immunosurgery, seeded onto gamma-irradiated mouse fibroblasts feeder layer. The outgrowths were passed by digestion with trypsin. Surface markers were identified by cytochemistry and immunohistochemistry. Karyotype was tested with standard G-banding technique. Differential potency was tested both in vivo and in vitro. RESULTS: One single cell cloned mouse embryonic stem cell line G(11) was established, which had normal 40XY karyotype and proliferated rigorously for a long time, expressed surface markers such as AP and SSEA-1, and was able to differentiate into many cell types both in vivo and in vitro. CONCLUSION: Mouse embryonic stem cell culture system has been established in our laboratory.