Objective To further explore the mechanism by which bone morphogenetic protein 4(BMP-4)might be involved in hematopoietic differentiation of the yolk sac.We observed the expression of BMP-4,CD34,CD133 and tyrosine kinase receptors(KDR) in the blood island of the yolk sac at embryonic 3 to 8 weeks.Methods Gene expression was analyzed by RT-PCR and the presence of BMP-4,CD34,CD133 and KDR proteins was confirmed by immunohistochemistry in 57 human embryos.Results In the human yolk sac,we found that BMP-4 was expressed at high levels from the 16th day to the 7th week,and decreased quickly after week 7.The results showed that KDR,CD133 and CD34 largely appeared on the 21st and 30th day,then increased at the 6th week,and decreased quickly after week 7.Furthermore,Ihh,SCl,GATA-1,GATA-2 and PU.1 mRNAs showed that PU.1 was not expressed on the 16th day;however,other factors were expressed all the time.Conclusion The distribution of BMP-4,KDR,CD34,CD133 and transcription factors expression highly suggested that BMP-4 was secreted from the yolk sac which might exert its effects on the specification of human hemangioblast and hematopoietic stem cells from the embryonic mesoderm in vivo through transcription factors.

Objective To assess sperm chromatin structure assay (SCSA) and sperm chromatin dispersion test (SCD) for DNA fragmentation evaluation in human infertility, and the correlation between these two methods. Methods We used SCSA and SCD assays to detect DNA fragmentation in sperm from 134 infertile men. The correlation of SCSA and SCD assays was analyzed. The sperm DNA fragmentation index (DFI) was divided into 3 groups (≤15%DFI, >15~≤30%DFI and>30%DFI), and the difference between SCSA and SCD assays was assessed. Results The SCSA assay was strongly correlated with the SCD assay for sperm DNA fragmentation (r=0.915, P<0.001). There was no significant difference between>15~ ≤30%DFI and>30%DFI groups. However, SCD showed higher levels of DNA fragmentation than that measured by SCSA for≤15%DFI group (13.50 4.82 vs 9.79 2.60, P<0.001). Conclusion There is a strong positive correlation between SCSA and SCD assays in detection of DNA fragmentation. SCD assay showed higher levels of DNA fragmentation than that measured by SCSA for≤15%DFI group.

Objective To explore the electrophysiological charaterstics of upper extremities nerves on the patients with Hirayama disease (HD),amyotrophic lateral sclerosis (ALS),and distal cervical spondylotic amyotrophy (DCSA).Methods The data of electrophysiological examination of the upper limbs of 87 patients with HD,83 with ALS and 28 with DCSA were reviewed retrospectively.Seventy-two patients with HD among 87 had unilateral upper limb's amyotrophy and the other 15 ones had bilateral amyotrophy.There were 30 patients had unilater upper limb's amyotrophy and 53 ones had bilateral amyotrophy from the group of patients with ALS; 20 patients with DSCA were affected unilaterally and 8 ones were bilaterally affected.Results Compound muscle action potential (CMAP) evoked by ulnar stimulation had a lower ampititude compared with that evoked by median stimulation in HD patients.In ALS cases that was just the opposite.However,the CMAPs were similar in DCSA cases.The mean ratio of CMAP amplitude by ulnar stimulation to by median stimulation was 0.58±0.40 in HD group; 2.28±1.25 in ALS and 1.31±0.63 in DCSA.The differences in the three groups were statistical significance.The U/M CMAP ratio was less than 0.6in 62 patients with HD,3 with ALS and 1 with DCSA,and more than 1.7 in 73 cases (57 ALS,12 HD and 4 DCSA).Conduction velocities (CV) of the sensory and motor nerves,the amplitude of the sensory nerve action potential in bilateral limbs,and the CMAP amplitude of the unaffected limb were normal in all cases.Conclusion This study could concluded that the severity of amyotropy in hypothenar mucles were higher than that in thenal muscles in patients with HD; there was just opposite in ALS cases and similar in DSCA.

Objective To investigate the correlation between seminal plasma hepatitis B virus (HBV) DNA copy and semen parameters and sperm DNA fragmentation (SDF).Methods The seminal plasma HBV-DNA was detected by the real-time PCR in 148 infertility males,and those with serum HBV-DNA above (positive) or below (negative) 5.0 × 102U/ml were analyzed respectively by semen parameters,sperm morphology and sperm DNA fragmentation (SDF).Results Of 148 male,60 (40.5％) were seminal plasma HBV-DNA positive,and of 60 positive patients,56 (93.3％) were serum hepatitis B e antigen(HBeAg) positive,which was higher than those of seminal plasma HBV-DNA negative males (31cases,35.2％).Serum HBeAg and HBV-DNA in seminal plasma HBV-DNA positive patients were 845.7(0.2 ～ 1455.0) S/CO and (1.7 ± 1.1) × 108U/ml,which were higher than those of HBV-DNA negative patients [HBeAg:0.1 (0.1 ～ 1374.0) S/CO;HBV-DNA:(2.3 ± 1.1) × 107 U/ml,P ＜ 0.01].Seminal plasma HBV-DNA positive patients exhibited lower semen volume,sperm concentration,the percentage of forward moving sperm and less normal morphology compared to HBV-DNA negative patients [(2.44±1.2)mlvs.(3.07±1.3)ml,(66.8±49.1) ×106/mlvs.(87.1 ±65.4) ×106/ml,(54.3± 16.1)％ vs.(59.1 ±15.3)％,(3.77 ±2.8)％ vs.(6.15 ±4.2)％,P＜0.05].The number of patients with teratozoospermia was significantly higher in seminal plasma HBV-DNA positive patients (56.7％ versus 34.1％,(P ＜ 0.01).The SDF in seminal plasma HBV-DNA positive patients was(18.1 ± 12.3)％,while it was(14.4 ± 8.4)％ in negative patients,and the difference of SDF in these two groups was significantly (t =2.197,P ＜ 0.05).Conclusion Seminal plasma HBV-DNA positive could affect the semen parameters,sperm morphology and SDF.

Objective To investigate the effects of inhalation anesthetics on human sperm motility and capacitation in vitro. Methods Sperm samples were obtained from normal adults and prepared with discontinuous percoll gradient centrifugation technique. The samples were incubated for 5 h in an airtight glass container filledwith 5% CO2-95% air at 37 ℃ with or without sevoflurane (SEV 2%, 4% ) or isoflurane (ISO 1.1%, 2.2% ).Then human sperm motility was examined in vitro at 37℃ and analyzed by the computer-assisted sperm analysis (CASA), including sperm motility (a + b)%, curvilinear velocity (VCL), straight line velocity (VSL), averagepath velocity (VAP) and amplitude of lateral head displacement (ALH). The capacitation effect was assessed by using the chlortetracycline (CTC) staining and phase-contract microscopy. Results 2% and 4% SEV significantly reduced (a + b)% , VCL, VSL and VAP in a dose-dependent manner, while only 4% SEV significantly decreased ALH and the capacitation ability of the sperm compared with control group. 2.2% ISO significantly decreased ( a + b)%, VCL, VSL and VAP compared with control and 1.1% ISO group. The capacitation ability of the sperm was significantly decreased by 1.1% and 2.2% ISO as compared with control group. Conclusion Sevoflurane and isoflurane have significant inhibitory effects on human sperm motility and capacitation in a dose-dependent manner. Sevoflurane has stronger inhibitory effect than isoflurane.

OBJECTIVETo investigate variation of sperm DNA fragmentation index (DFI) in male partners of infertile couples.

METHODSA total of 539 males between April 2009 and April 2012 were analyzed. At least one repeated routine semen analysis and sperm DNA fragmentation test were performed for each sample by sperm chromatin dispersion (SCD) analysis following World Health Organization guidelines. Coefficient of variation (CV) for DFI was calculated.

RESULTSRespectively, 1, 2, 3 and 4 repeated SCD analyses were carried out on 473, 59, 6 and 1 semen samples. The median interval between the first and repeated SCD measurements was 3.0 (1.0-11.0) months. For the first tested samples, the between-sample coefficient of variation (CVB) for DFI was 71.2%. A significant difference has been found between DFI of the first measurement and DFI of repeated measurement in 0.5 to 3 months, 3 to 12 months and 12 to 34 months (P< 0.01). Compared with the first test, 26.3% of males were on both sides of the cut-off point of 18%. The median within-subject coefficient of variation (CVw) for DFI of 539 men was 26.0% (12.6%-42.8%). And the median CVw DFI was significantly lower compared with CVw of sperm count, concentration, progressive motility and normal morphology (P< 0.01). Significant correlations were found between the CVw DFI and sperm count, concentration and interval among the samples (P< 0.05).

CONCLUSIONDFI of male partners for infertile couples is a parameter with substantial variation, repeated SCD measurements are therefore recommended.

Adult ; DNA Damage ; DNA Fragmentation ; Female ; Humans ; Infertility, Male ; genetics ; Male ; Middle Aged ; Sperm Count ; Spermatozoa ; metabolism ; Young Adult

OBJECTIVETo assess the diagnostic value of sperm DNA fragmentation (SDF) for male infertility.

METHODSTwo hundred and ninety-nine males attending infertility clinic were classified into 157 primary infertile cases and 142 fertile controls. Semen analysis was performed as recommended by the World Health Organization (WHO). SDF was assessed by sperm chromatin dispersion (SCD) assay, and the results were expressed as DNA fragmentation index (DFI).

RESULTSThe DFI was significantly higher in infertile males than that in fertile controls [(17.1± 9.3)% vs. (14.2± 9.0)%](P< 0.01). No significant difference was detected in the age of male and female partners, seminal volume, sperm count, motility and morphology between infertile males and fertile controls (P> 0.05). The area under the receiver operating characteristic curve (AUC) was 0.861 [95% confidence interval (CI)= 0.814-0.907] for 15.1% of SDF. The threshold level of 15.1% was derived as cut-off value to discriminate infertile men from fertile controls. By this threshold, specificity was 88.2% and sensitivity was 81.8%. The 299 men were divided into group A (n= 120) with DFI≥ 15.1% and group B (n= 179) with DFI< 15.1% based on the cut-off value. The percentage of infertile men in group A was significantly higher than that in group B (79.2% vs. 34.6%) (P< 0.01). The odds ratio (OR) for infertility in the two groups was 7.2 (95%CI= 4.2-12.3).

CONCLUSIONSperms with high-level of DNA fragmentation can impair male fertility. DFI can be used as a good diagnostic marker for male infertility.

Adolescent ; Adult ; DNA ; metabolism ; DNA Fragmentation ; Female ; Humans ; Infertility, Male ; diagnosis ; genetics ; Male ; Spermatozoa ; metabolism ; Young Adult

OBJECTIVETo identify whether there is significant changes between the cervical neutral F-waves and cervical flexion F-waves in the patients with Hirayama disease.

METHODSThis study was performed on 25 normal subjects and 22 male patients with identified Hirayama disease (age: 15 to 44 years; height: 165 to 183 cm; duration: 6 to 240 months) between May 2010 and March 2014. Both cervical flexion F-wave (cervical flexion 45 °, 30 minutes) and conventional F-waves to median nerve stimulation and to ulnar nerve stimulation were performed in all subjects bilaterally.

RESULTSwere analyzed by t-test or Fisher exact probability.

RESULTSIn the normal subjects, all measurements of the bilateral F-waves didn't have any difference between the cervical flexion position and the cervical neutral position. On the cervical neutral position, the persistence (t = 5.209, P = 0.000), average latencies (t = 4.731, P = 0.022) and minimal latencies (t = 23.843, P = 0.006) of ulnar F-wave on the symptomatic heavier side from the patients with identified Hirayama disease were significantly lower or longer than those from the normal subjects, and the repeat F-waves were found in 3 patients (13.6%). On the symptomatic lighter side, the ulnar F-waves only had lower persistence (t = 22.306, P = 0.001) along with 5 repeat F-waves. Only lower persistence were found in the median F-wave on the both side (higher side t = 23.696, P = 0.000; lighter side t = 23.998, P = 0.000), along with 5 (22.7%) repeat F-waves on the symptomatic heavier side and 6 (27.3%) ones on the symptomatic lighter side. After cervical flexion maintaining 30 minutes, the increased maximal amplitudes (t = -2.552, P = 0.019), average amplitudes (t = -3.322, P = 0.003), duration (t = -3.323, P = 0.00), persistence (t = -2.604, P = 0.017) and frequency of repeat F-waves (9/22, 41%) (P = 0.044) were found on the symptomatic heavier side of ulnar F-wave, and 5 of 10 absent ulnar F-wave on the cervical neutral position were also recover. The median F-wave on the symptomatic heavier side mainly had increased maximal amplitude (t = -3.847, P = 0.001), average amplitudes (t = -2.188, P = 0.040) and persistence (t = -2.421, P = 0.025), and 1 of 6 absent median F-wave on the cervical neutral position were also recover after cervical flexion.

CONCLUSIONThe cervical flexion F-waves have significant regular changes compared to the cervical neutral F-waves in patients with Hirayama diseases, especially maximal and average amplitudes of F-waves.

Adolescent ; Adult ; Humans ; Male ; Neck ; Range of Motion, Articular ; Spinal Muscular Atrophies of Childhood ; physiopathology ; Ulnar Nerve ; Young Adult

Objective　To establish Huashan diagnostic criteria and clinical classification system for Hirayama disease.Methods　Retrospective analysis 359 cases of puberty onset,upper extremity muscle atrophy as main clinical manifestations,and complete clinical data from September 2007 to August 2018.There were 348 males and 11 females(31.6:1 male and female)in this group.The average age of onset was 16.7±2.2 years,the average age of visits was 19.2±4.5 years,and the average duration of treatment was 29.3±45.4 months.Descriptive study of the clinical manifestations,radiologic and neurophysiological findings of this group of patients,the Huashan clinical diagnostic criteria of Hirayama disease were established by including 100% of the clinical manifestations,imaging and neurophysiological findings.According to the following parameters,the clinical classification system of Hirayama disease was proposed.The parameters specifically included:the muscle atrophy involves the upper limbs,whether the quadriplegia was active or hyperactive,the Babinski sign positive and other pyramidal tract damage,whether it was accompanied by sensory dysfunction such as upper limb numbness,muscle atrophy location,and the progress of clinical symptoms or electrophysiological examination within 6 months.Thirty patients were randomly selected from the above 359 cases.Four orthopedic surgeons who were not involved in the classification system completed the clinical classification within the specified time.The Kappa value was used for the credibility evaluation.Results　The Huashan diagnostic criteria of Hirayama disease included clinical manifestations,imaging examinations and neurophysiological examinations.The main diagnostic indicators were:1)occult onset puberty,more common in men;2)localized muscle atrophy and weakness in the upper extremities;3)compared with the cervical neutral MRI,the MRI of cervical flexion showed that spinal cord was significantly shift forward and the anterior spinal cord was narrowed or disappeared.4)MRI T2 weighting of the cervical flexion showed cyst-wall separation behind the spinal cord;5)Neurophysiological examination showed that the affected muscles were neurogenic damage.6)The affected parts are limited to the middle and lower neck segment.At the same time,it was necessary to combine imaging and neurophysiological manifestations to distinguish cervical spondylosis with upper limb muscle atrophy and motor neuron disease.According to the clinical characteristics of different patients,Hirayama disease can be divided into type I-III.Type I:72.1%,one-sided upper limb or one upper limb-based hand inner muscle and forearm muscle atrophy.According to whether progress of symptoms or electrophysiological examination was seen in the past 6 months,type I can be divided into:Ia.stable period.Regular follow-up assessment was recommended.If the disease progressed,to wear a cervical collar was suggested;surgery could be done if necessary;Ib.progression period,it was recommended to use a cervical collar,and regularly evaluate,if patients could not wear cervical collar for long,it was recommended to operate.Type II:14.2%,unilateral upper limb or one upper limb-based hand inner muscle and forearm muscle atrophy with pyramidal tract injury.Surgery was recommended.Type II:13.7%,atypical Hirayama disease,including upper limb proximal muscle atrophy,symmetrical double upper limb muscle atrophy,and sensory disturbances associated with upper limb numbness.Wear a cervical collar,and follow-up and assess closely,and choose surgical treatment if necessary.The credibility evaluation showed that the average Kappa value of the classification was 0.732(0.688-0.834),which is a basic credibility.Conclusion　The Huashan diagnostic criteria of Hirayama disease was conducive to the early diagnosis.The clinical classification system of Hirayama disease has good credibility and good clinical intervention guidance value.

Objective To explore the role of S1 root stimulation H-reflex in evaluating the efficacy of lumbar disc hernia-tion (LDH). Methods 95 LDH patients (55 males, 40 females) who had underwent discectomy for the lumber herniated discs were recruited in this research from January 2014 to January 2016. The average was (40.5±6.7) years, ranged 17-60 years. The vi-sual analogue scale (VAS) scores and the MOS item short from health survey (SF-36) scale was evaluated in preoperative, day 7, 3 month and 1 year after operation, respectively. Meanwhile, the S1 H-reflex and routine H-reflex were taken before operation and 1 year of postoperation. Results The subjects were divided into two groups according to the results of the preoperative S1 H-reflex:①60 patients with S1 present group, the H-M interval was significantly longer (8.1±1.2) ms, 47 of those with routine H-reflex and the latency was prolonged (31.8 ± 2.5) ms, 13 of those with absent routine H-reflex. 32 among 60 patients H-M interval was short-en before operation (7.8 ± 1.0) ms in 1 year follow-up, and there was statistically significant (P=0.001);the latency of conventional H-reflex was shorter than that of preoperation (28.5 ± 2.3) ms in 20 patients, there was statistical difference (P=0.023);the H-re-flexes were detected in the 6 patients from that 13 with absent routine H-reflex.②35 patients absent group, of which 30 cases of conventional H reflex disappeared, only 5 had normal routine H-reflex and the latency was prolonged (31.2 ± 3.0) ms. There were no H-wave patterns detected in 18 patients with H-reflexed one year later, and there was no significant difference in the H-reflex latency (31.0 ± 3.1) ms. All patient's VAS scores and SF-36 were significantly improved from preoperation to postoperation. Both scores were no difference between two groups in 7 d of post surgery. The mean VAS score of two groups: 3 months (1.7 ± 1.0) points, (2.1 ± 1.2) points (t=2.618, P=0.010), and 1 year (1.3 ± 0.9) points, (1.8 ± 1.1)( t=3.311, P=0.002). SF-36 in two groups:3 months (28.9 ± 5.6) points, (33.2 ± 5.5) points ( t=-2.670, P=0.008), 1 year (23.2 ± 6.2), (30.2 ± 5.6) (t=-3.012, P=0.001). Conclusion The patients with LDH had detected S1 H-reflex before surgery which indicated the minor leisure in intravertebral nerves, so that nerve can get better recovery and functional score of postoperation, it could objectively evaluate the efficacy of LDH surgery.