Objective To study the gene expression profiles of megakaryocytes(MKs) from human cord blood CD34+ cells in vitro expanded and to understand megakaryopoiesis at the molecular level. Methods CD34+ cells were isolated using density gradient centrifugation and magnetic activated cell sorting. The cells were cultured and stimulated with recombinant human TPO ( 100 ng/ml). After 12 days, the MKs fraction was separated using an anti-CD41 monoclonal antibody by immunomagnetic sorting. The gene expression profiles of MKs, non-MKs as well as meg-01 cells were studied by gene chip assay. THBSI, HOX A9,β-actin, lL-8,Annexin A6, FGF-8 were selected to validate the gene chip results by RT-PCR. Results A total of 116 genes between MKs and non-MKs cells were significantly different, 52 genes were up-regulated and 64 genes were down-regulated. In addition, 158 genes between MKs and meg-01 cells were significantly different, 71 genes were up-regulated and 87 genes were down-regulated. THBSI showed higher expression in MKs than in non-MKs. HOXA9 showed lower expression in MKs than in non-MKs. The expression of β-actin did not show any significant difference in MKs and non-MKs. IL-8 showed higher expression in MKs than in meg-01 cells, while ANXA6 showed lower expression in MKs than in meg-01 cells. The expression of FGF-8 did not show any significant difference between MKs and meg-01 cells. Conclusions MKs, non-MKs and meg-01 cells show different gene expression profiles. The regulatory genes include stress response genes,immune related genes, DNA synthesis and repair genes, metabolism genes, pro-onco genes and tumor suppressor genes.

Objective To investigate the effects on pregnancy outcome and neonate by artificial shrinkage by microsucting the fluid of expanded blastocysts before vitrification using glass micropipette (GMP).Methods From Jan.2006 to Dec.2009, 342 vitrified-thawed blastocyst cycles from patients that performed in vitro fertilization-embryo transfer (IVF-ET) or intracyteplasmic sperm injection ( ICSI ) were enrolled in this study in Reproductive Medicine center, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region.Three hundred and fourteen cycles of expanded blastocysts were artificially shranked by microsucting blastocoelic fluid with a micro-needle before vitrification as artificial shrinkage group, in the mean time, 28 cycles without artificial shrinkage were chosed as control group.The survival rate, implantation rate, clinical pregnancy rate and transfer canceled rate were compared between artificial shrinkage group and control group.Among pregnant women, the miscarriage rate, live birth rate, congenital birth defect rate, neonatal weight and gestational age were compared with those of fresh embryo transfers in 520 cycles.Results The blastocyst survival rate, implantation rate and clinical pregnancy rate were 95.3%(403/423), 38.0% ( 153/403), 44.6% (140/314) in artificial shrinkage group and 64.3 % (27/42),7.4% (2/27), 7.1% (2/28) in control group, respectively, which reached statistical difference (P＜0.05).The transfer canceled rate was 0 in artificial shrinkage group and 25.0% (7/28) in control group, which also reached statistical difference ( P ＜ 0.05 ).Among pregnant patients, the miscarriage rate of 18.2% (10/55), live birth rate of 80.0% (44/55), gestational age of (38.2 ± 1.3) weeks, congenital birth defect rate of 2.1% (1/47), birth weight of newborns of (2989 ±640) gram in artificial shrinkage group were not significantly different with 17.5% (91/520), 74.0% (385/520), (37.9 ±2.3) weeks,1.7% (8/479) and (2856±640) gramin fresh embryo transfer group (P＞0.05).Conclusion Artificial shrinkage by microsucting blastocoelic fluid with a micro-needle before vitrification significantly improved the vitrification effects of expanded blastocyst and no distinct increasing rate of neonates congenital anomality were observed.

Objective To detect fetal RhCcEe genotype from fetal DNA in maternal plasma for noninvasive prenatal diagnosis.Methods DNA from maternal plasma sample was extracted by use of QIAamp DNA Kit. The existence of fetal DNA was confirmed by amplified fetal SRY gene. The fetal RhCcEe gene was amplified by polymerase chain reaction (PCR) from 30 pregnant maternal plasma. The results of fetal RhCcEe genotype were evaluated retrospectively by the serologic analysis of infant and pregnant woman RhCcEe phenotype.Results Among the 30 samples, 13 were the same phenotypes between mother and infant, 17 were different. When mother phenotypes were RhCC, cc, EE and ee homozygous, the deleted allele gene can be successfully amplified from mother plasma.Conclusion Noninvasive fetal RhCcEe genotyping is reliable. When the mother was homogyzous, genotyping the fetal CcEe alleles was very significant and useful for HDN (hemolysis disease of newborn) diagnosis and therapy.

0.05),but the numbers of CD41+ cells and platelets were increased significantly(P

Objective To investigate the effect of blastocyst quality on the strategy of single blastocyst transfer in frozen-thawed cycles. Methods A retrospective analysis was performed in Reproductive Medicine Center, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region on clinical data of single frozen-thawed blastocyst transfer cycles from January 2008 to December 2013. All cycles were divided into four groups (AA, AB/BA, BB, BC/CB) according to the blastocyst score, then the clinical outcomes were compared between groups. And on this basis, the clinical outcomes were further explored when the group of outcomes with single blastocyst transfer wasn′t ideal, which would diverted to transfer two blastocyst. Results In single frozen blastocyst transfer cycles, the clinical pregnancy rate of each group with the blastocyst scored AA, AB/BA, BB, BC/CB were 61.4%(470/765), 51.2%(330/645), 40.5%(407/1 005), 22.9%(60/262), live births rate in each group were 52.2%(399/765), 41.2%(266/645), 30.4%(306/1 005), 13.7%(36/262), and the abortion rate were 13.6%(64/470), 16.7%(55/330), 21.4%(87/407), 35.0%(21/60), separately. This showed that the clinical pregnancy rate and live births rate decreased significantly with the decline of blastocyst quality (P<0.01), but the abortion rate showed significant upward trend (P<0.01). When single blastocyst scored≥BB grade transferred, an acceptable clinical pregnancy rate (>40%) and live births rate (>30%) could be obtained, however, the clinical pregnancy rate of 22.9% and live births rate of 13.7%could only be acquired when blastocyst scored BC/CB only transferred one embryo, which significant lower than those of each group scored ≥BB grade (P<0.01). So, after that, the blastocyst scored BC/CB were further divided into two groups (single blastocyst transferred versus two blastocyst transferred) to investigate, then the result showed that the clinical pregnancy rate [22.9%versus 38.5%(67/174), P<0.01] and live births rate [13.7%versus 30.5%(16/67), P<0.01] were significantly increased in the group of two blastocyst transferred compared with the group of one blastocyst transferred, and the abortion rate was also significantly decreased from 35.0%to 17.9%(12/67;P<0.05). So when two blastocyst scored BC/CB were transferred, the clinical outcomes were similar to the group of one blastocyst scored BB transferred (P>0.05). Conclusions Of single blastocyst transfer in frozen-thawed cycles, the clinical pregnancy rate and liver births rate showed significant upward trend, but the abortion rate showed significant downward trend, with the decline of blastocyst quality. When the blastocyst scored ≥BB grade, the single blastocyst transfer could be considered to be performed.

BACKGROUND: Revascularization is a challenge for the tissue-engineered bone carrying cells after implanted into human body. Previous studies have found that tanshinol can improve the functions of endothelial progenitor cells and exert vascular protective effects.OBJECTIVE: To prepare the β-calcium phosphate (β-TCP) scaffold with tanshinol coating, and to observe its cytocompatibility.METHODS: The β-TCP scaffolds coated with 10-7, 10-6 and 10-5 mol of tanshinol were constructed by negative pressure absorption method. The distribution of tanshinol coating on the scaffold was observed using scanning electron microscopy,and the inner ingredients were analyzed by infrared spectrum. Human endothelial progenitor cells (hEPCs) were cultured in the extracts of β-TCP and β-TCP scaffolds with 10-7, 10-6, 10-5 mol of tanshinol coatings, respectively. The cell proliferation was detected at 2, 4, 6, 8 and 10 days of culture; the levels of nitric oxide and vascular endothelial growth factor in the supernatants were detected at 1, 7 and 14 days of culture; the lumen formation on the matrigel was observed after 14-day culture. hEPCs were respectively seeded onto the β-TCP and β-TCP scaffolds with different dosages of tanshinol coating,and then the cell growth was observed under scanning electron microscope at 7 days.RESULTS AND CONCLUSION: The tanshinol coating evenly distributed on the inner surface of the pores, and its crystalline structure became dense with dosage increasing. Infrared spectrum analysis revealed no changes in the characteristic absorption peak of tanshinol and TCP in the scaffold. The β-TCP scaffolds with tanshinol coating could promote the proliferation of hEPCs, especially the scaffolds with 10-6 and 10-5 mol tanshinol coating. Compared with the β-TCP scaffold, the scaffolds with 10-6 and 10-5 mol tanshinol coating significantly upregulated the nitric oxide level at 14 days of culture, and significantly increased the level of vascular endothelial growth factor at 7 and 14 days of culture (P <0.05). Although it could be found in all β-TCP scaffolds with tanshinol coating, the lumen formation was the maturest in the scaffold with 10-5 mol tanshinol coating. These results suggest the β-TCP scaffolds with tanshinol coating can promote the proliferation and endothelial differentiation of hEPCs, and hold a good cytocompatibility.

Objective To evaluate the potential differences in serum proteomic profiles between patients with esophageal squamous cell carcinoma(ESCC)and precancerous lesions in order to establish proteomic pattern model for diagnosis of ESCC and precancerous lesions in high risk area,and to investigate its value in screening ESCC.Methods The serum and endoscopic biopsy samples were obtained from 38 normal controls,63 patients with atypical hyperplasia(class Ⅰ 26 cases,class Ⅱ 26 cases,class Ⅲ 11 cases)and 36 patients with advanced esophageal carcinoma.The serum proteomic patterns were examined using surface enhanced laser desorption/ionization time of flight mass spectrometry(SELDI-TOF-MS)and CM10 protein chip.The data was analyzed and disease diagnostic models were established using support vector machine(SVM).The diagnostic model was evaluated and validated by leave one cross validation.Results ①The diagnostic model could differentiate advanced esophageal carcinoma from normal controls with a specificity of 89.47%and a sensitivity of 83.33%.②The results delivered 92.31%,80.77% and 90.91%specificity,and 80.56%,83.33%and 94.44%sensitivity for discrimination of atypical hyperplasia Ⅰ,Ⅱand Ⅲ,respectively,using diagnostic models.③Four(4291,5644,5664,8775)m/z peaks observed repeatedly using diagnostic models.Conclusions The SELDI-TOF-MS and SVM provide a new approach for discrimination of ESCC and precancerous lesions in high risk area.Four(4291,5644,5664,8775)m/z peaks may considered as potential biomarkers which related to the ESCC and esophageal precancerous lesions.

The aim of this study was to report a successful pregnancy using completely immotile frozen-thawed spermatozoa selected by laser. A single laser shot was used to detect the presence of viable immotile spermatozoa in fresh and frozen-thawed testicular spermatozoa. The viability rate was 55.8% after the laser detection, and cryopreservation was carried out immediately. The thawing test was performed on the day of oocyte pick-up, and no motile sperm were observed after extending the culture for another 4 hours, while a survival rate of 39.8% was detected using the laser. In all, five mature oocytes were injected, resulting in four cases of normal fertilization (80%) on day 1. Further, two high-quality day 3 embryos were transferred, which resulted in a singleton pregnancy. Our study demonstrates that completely immotile spermatozoa are worth cryopreserving for further intracytoplasmic sperm injection, which provides a new insight into male fertility preservation in cases of completely immotile spermatozoa.
Cryopreservation ; Embryonic Structures ; Fertility Preservation ; Fertilization ; Humans ; Male ; Oocytes ; Pregnancy* ; Sperm Injections, Intracytoplasmic ; Spermatozoa* ; Survival Rate

A viable spermatozoon is a prerequisite for fertilization in intracytoplasmic sperm injection (ICSI). Thus, it is crucial to select viable but immotile spermatozoa on the day of ICSI. We report conflicting results in the identification of viable but immotile spermatozoa between the eosin-nigrosin staining and the laser test, which resulted in confusion for embryologists during assisted reproductive technology (ART). Three patients’ semen samples that showed no motile spermatozoa are described in this report. To identify viable spermatozoa, we used both the eosin-nigrosin test and the laser test for each sample, and repeated the semen analysis twice in each patient. Viable but immotile spermatozoa selected by the laser test were used for ICSI. Viable spermatozoa were detected by both the eosin-nigrosin and laser tests in two patients (case 1, 95.00% vs. 24.21% and 92.68% vs. 22.22%; case 2, 41.18% vs. 23.48% and 39.81% vs. 22.52%), indicating consistent results between the two methods. In the third patient, the eosin-nigrosin test yielded viability rates of 20.75% and 19.14%, while the result of the laser test was 0%. Thus, testicular aspiration was performed to collect viable sperm from this patient. Normal fertilization was achieved after the injection of viable but immotile spermatozoa selected from these patients by the laser test, resulting in the birth of two healthy babies. Our study documents a case where the eosin-nigrosin test showed a limitation in identifying viable but immotile spermatozoa for ART, while the laser test may overcome this limitation. Larger samples may be required to corroborate the clinical value of the laser test.
Fertilization ; Humans ; Parturition ; Reproductive Techniques, Assisted ; Semen ; Semen Analysis ; Sperm Injections, Intracytoplasmic ; Spermatozoa

Osteoarthritis (OA) is a common chronic debilitating disease among middle-aged and old people,which can occur in the hip,knee,ankle,spine and other joints,but it is most commonly seen in the knee.The clinical manifestations of the knee osteoarthritis (KOA) include pain,swelling,stiffness,joint deformity,seriously affectting the quality of life.Mechanical and metabolic factors have been shown to play roles in the initiation and progression of this disease,resulting in a slow,progressive pathological process,and the normal balance of anabolic and catabolic activities of the chondrocytes has been disrupted.The mechanical factors are the joint imbalance caused by cartilage degeneration and ligament damage,and biochemical factors are mainly the changes of the joint microenvironment caused by the dysregulation of chondrocytes and synovitis.Infrapatellar fat pad (IPFP)is situated in the lower part of patella and femoral condyle,between tibia condyle and the patellar ligament,and it is an intracapsular but extrasynovial elastic fiber adipose tissue.Researches have shown that besides the synovial membrane,ligament,cartilage and bone,IPFP may play an important role in the onset and progression of KOA and knee pain.IPFP has long been regarded as a structural fatty tissue without metabolic reactions,thus often been neglected,what's more,to get a clear vision in knee surgery,IPFP is often partially or totally resected,but recently owning to its potential biological mechanics,endocrine function,which can produce a variety of inflammatory cytokines,chemokines,rich in nerve fiber structure and IPFP-adipose derived stem cells,more and more scholars pay attention to the IPFP.IPFP may play a protective role in the early stage of KOA by cushioning shock,stabilizing lubrication,but this article emphatically explain how IPFP play a desctructive role in the initiation and progressionof KOA through leptin,adiponectin,and many other adipocytokines,or inflammatory mediators,so as to get further understanding of KOA,and discuss whether IPFP should be resected or not in knee surgery,providing a new method to the prevention and treatment of KOA.