2004年 · 第117卷第04期

Background Clinical programs for preventing β-thalassemia are presently based on prospective carrier screening and prenatal diagnosis. This paper report an achievement of a pregnancy with unaffected embryos using in vitro fertilization and embryo transfer (IVF-ET), in combination with preimplantation genetic diagnosis (PGD), for a couple at risk of having children with p-thalassemia. Methods A couple carrying different thalassemia mutations, both a codon 41-42 mutation and the IVS II 654 mutation, received standard IVF treatment, with intracytoplasmic sperm injection, embryo biopsiy, single cell polymerase chain reaction (PCR) and DNA analysis. Only unaffected or carrier embryos were transferred to the uterine cavity. After confirmation of pregnancy, a prenatal diagnosis was performed. Results Of a total of 13 embryos analyzed for β-globin mutations, PGD indicated that 2 were normal, 3 were affected, and 6 were carriers. Diagnosis could not be made in the other 2 embryos. Three embryos were transferred to the uterus on the third day after oocyte retrieval. Ultrasonography revealed a twin pregnancy with one blighted ovum. The prenatal genetic diagnosis revealed that both fetuses were unaffected, and two healthy boys were born, confirming the results of PGD. Conclusions We developed a single-cell based primer extension preamplification (PEP)-PCR assay for the detection of p-thalassemia mutations. The assays were efficient and accurate at all stages of the procedure, and resulted in the birth of PGD-confirmed β-thalassemia free children in China. PEP was used here in PGD for p-thalassemia. Chin Med J 2004; 117(4): 483-487

Background Insulin-like growth factor binding protein-1 (IGFBP-1), which is a carrier of Insulin-like growth factors (IGFs) regulates the fetal development by working as an active factor controlling the combination of IGFs with their receptors. This study was designed to investigate the relationship between IGFBP-1 and human embryonic development during weeks 6 -10 of gestation. Methods A total of 44 pregnant women with singleton pregnancy were divided into two groups; one with abnormal embryo development (n=32) and the other with normal embryo development (n=12). Enzyme-linked immunosorbent assay (ELISA) was employed to detect IGFBP-1 levels in maternal serum and decidual tissue. The expression of IGFBP-1 mRNA in deciduas was examined by reverse transcription polymerase chain reaction (RT-PCR) technique. Results The level of IGFBP-1 protein in maternal serum was significantly higher in the abnormal group [(125.36 ± 47. 93) μg/ml] than in the normal group [(70. 72 ±21. 21) μg/ml]. Both of IGFBP-1 and IGFBP-1 mRNA in deciduas were higher in abnormal group [(1.60 ±1.39) μg/ml and 1.66 ±1. 64, respectively] than in the normal group [(0. 35 ±0. 23) μg/ml and 0. 40 ±0. 20,respectively]. The level of IGFBP-1 in maternal serum was positively correlated with IGFBP-1 mRNA(r=0.90, P<0.05) and IGFBP-1 protein (r=0. 92, P<0. 05) in decidual tissue. Conclusions During weeks 6-10 of gestation, abnormal embryonic development is correlated with elevated IGFBP-1. The level of IGFBP-1 in maternal serum is related to the concentrations of IGFBP-1 mRNA and IGFBP-1 in decidual tissue. The IGFBP-1 level in maternal serum may be used as a predictive marker to evaluate embryonic development. Chin Med J 2004; 117(4): 488-491

Background DNA analysis has shown a lack of significant compatibility between couples affected by unexplained recurrent spontaneous abortion (URSA) compared with normal fertile couples,8although one study that made use of a PCR-sequence-specific oligonucleotide (SSO) method did observe evidence of significant compatibility in the HLA-DQA1 and DQB1 alleles between patients and aborted fetuses.9 This study was designed to investigate whether URSA were associated with particular DQ alleles or promoter alleles. Methods Thirty-two patients with URSA and 54 women who had had at least one successful pregnancy were included in this study. HLA-DQ genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The HLA-DQB1 promoter was detected by the SSO and sequence-specific primer (SSP) methods. The DQA1, DQB1, and DQB1 promoter (QBP) gene frequencies in the patients were compared with the gene frequencies in normal controls. The data were analyzed statistically with the χ2 and Fisher's exact tests. Results The results showed that the frequency of DQB1 * 0604/0605 was significantly higher and the frequency of DQB1 * 0501/0502 was significantly lower in the patient group as compared with the normal controls. In addition, the frequencies of the DQA1 * 01-DQB1 * 0604/0605 and QBP6. 2-DQB1 * 0604/0605 haplotypes were overrepresented in the patients relative to the controls. Our results did not show any differences between URSA patients and the controls with regard to DQA1 and QBP allele frequencies. Conclusions Our data suggest that URSA is associated with the HLA-DQB1 coding region, and is not associated with its upstream regulatory region. The DQB1 * 0604/0605, DQA1 * 01-DQB1 * 0604/0605, and QBP6. 2-DQB1 * 0604/0605 haplotypes may confer susceptibility to URSA, while the DQB1 * 0501/0502 allele may protect women from URSA. Chin Med J 2004; 117(4): 492-497

Background It is believed that estrogen plays pivotal roles in the regulation of follicle/oocyte maturation and oocyte fertilizability. It is also involved in the functional preparation of the fallopian tubes for subsequent gamete interaction, in early embryonic development occurring in the tubal microenvironment, and in the preparation of the uterus for implantation. This study was designed to determine whether estrogen is required for follicular and embryonic development. Methods The biosynthesis of estrogen was blocked by a daily injection of the aromatase inhibitor, Arimidex, at a dose of 100 μg/d, using 3-4 week old C57B6 F1 female mice. Injections were continued for 3 days in experiment 1 (n=10) and for 5 days in experiment 2 (n=23). Mice in the control group (n=27) were given the same amount of saline. Exogenous gonadotrophin [7. 5 IU pregnant mare serum gonadotrophin (PMSG)] was administered to induce follicular growth and development on the second day. In experiment 1, we tested estrogen and progesterone levels and examined ovary morphology two days later. In experiment 2, 47 hours after PMSG injection, 5 IU human chorionic gonadotropin (hCG) was given and two female mice were then caged with a male mouse overnight. Two days later, we measured estrogen and progesterone levels. We then removed the embryos, cultured them, and examined embryonic development every 24 hours for 3 days. Results Before hCG injection, estrogen levels in mice from the Arimidex group were suppressed by 94%, and progesterone levels were suppressed by 75%. There was no difference between the two groups in mean number of total follicles found per animal (30.4 follicles/animal in the control group and 27 follicles/animal in the Arimidex group). Two days after hCG injection, estrogen levels in the Arimidex group were significantly lower than that in the control group (P <0.01), while progesterone levels were not significantly lower (P> 0. 05) . The rate of development of embryos, morulae, blastocysts, and hatching blastocysts was not significantly different between the two groups (P=0. 20, 0.10, 0. 44, and 0. 38, respectively). Conclusions In the present study, by depriving mice of normal estrogen support, we have been able to rule out the absolute need for rising levels of estrogen for the completion of the follicular maturation process and the development of embryos in vitro. Chin Med J 2004; 117(4):498-502

Background Chromosomal aberrations are the major cause of pre- and post-implantation embryo wastage and some studies suggest that half of all human conceptions have a chromosomal abnormality. A chromosomal aberration in human sperms is also one of the causes of failure of in vitro fertilization. This study was designed to ascertain whether chromosomal aneuploidy in spermatozoa is a risk factor for male infertility. Methods Twelve infertile men were divided into two groups: 10 with oligoasthenoteratozoospermia(OAT, Group A) and two with a normal semen analysis (Group B). Two normal healthy sperm donors acted as controls (Group C). We used fluorescence in situ hybridization (FISH) and probes for chromosomes X, Y and 18 to determine the frequency of aneuploidy. Results The frequencies of spermatozoa disomy for chromosomes X, Y and 18 were 0.30%and 0. 30%, respectively, in Group B. The percentages were not significantly different from those of Group C (0.15%and 0.16%). The frequencies of nullisomy for chromosomes X, Y and 18 were 0.15%and 0 for Group B, and 0 and 0.15%for Group C (P>0. 05). In Group A, the incidences of disomy were 1.13% and 0. 96% and the frequencies of nullisomy were 1.13% and 1. 60%. In these three groups, the incidences of diploidy were 0.60%, 1.00%, and 0.30%, respectively. Both the frequencies of disomic and nullisomic spermatozoa for chromosomes X, Y, and 18 and of diploid spermatozoa were significantly higher in Group A than in Groups B and C. The estimated total aneuploidy rates in the sperm from the three groups were 42.44%, 6.05%, and 2.59%,respectively. Conclusion These results indicate that chromosomal aneuploidy in spermatozoa may be a risk factor for infertility. Chin Med J 2004; 117(4): 503-506

Background Although great advances in techniques for noninvasive prenatal diagnosis using fetal cells from maternal peripheral blood have achieved, current technology does not meet the demands required for clinical use. In this study, we aimed to establish reliable methods for the gene analysis of fetal cells from maternal peripheral blood. Methods Primed extension preamplification (PEP) -polymerase chain reaction (PCR), multiple primed in situ labeling (PRINS), and nested PCR were individually applied to detect the sex determining region Y (SRY) gene in single fetal cells collected from maternal peripheral blood. Results The sensitivity and specificity of the detection of the SRY gene by PEP-PCR were 97. 39%(149/153) and 99.17%(119/120), respectively. The sensitivity and specificity of PRINS were 97. 56%(40/41) and 100%(35/35), respectively. The sensitivity and specificity of nested-PCR were 80. 00%(24/30) and 87. 50%(14/16), respectively. Conclusions PEP-PCR and PRINS are reliable techniques for the gene analysis of single fetal cells from maternal peripheral blood because of their high sensitivity and specificity. PEP-PCR and PRINS can be used as standard methods of noninvasive prenatal diagnosis using single fetal cells from maternal peripheral blood. Chin Med J 2004; 117(4): 507-510

Background This study was designed to evaluate the relationship between high-density lipoprotein cholesterol (HDL-C) level and acute myocardial infarction (AMI) and coronary heart disease (CHD)death and to explore the protective effect of HDL against CHD in the elderly Chinese. Methods Started from 1986, 1211 retirees (92% males) were enrolled consecutively and studied prospectively. The average starting age was 70 ±9 years, and that at the end of the study was 80 ±9years. During the follow-up study, all the participants received yearly physical examination and blood chemistry survey from 1986 -2000. The average duration of the follow up study was 11.2 years. The end point of this study was either attacks of AMI or death due to CHD and other causes. CHD risk factors were screened by logistic regression analysis. According to their HDL-C levels, cases were divided into low (<1. 03 mmol/L), medium (or normal, 1. 03 -1.56 mmol/L) and high (>1.56mmol/L) level groups, the differences in incidence of AMI and CHD death in each group were analyzed. Results The cumulative attacks of acute coronary syndrome (mostly AMI) were 214 cases, including 89 cases of coronary death and 308 death caused by other diseases during the follow up study. AMI occurrence and CHD death in normal HDL-C group were lower than those in the low HDL-C group by 40%and 53%; and those in the high HDL-C group were lower than in the normal group by 56%and 50%, respectively. Statistical analysis on normal lipid cases (411 cases, total cholesterol <5.17mmol/L, triglyceride <1. 69 mmol/L) revealed that the cases at low HDL-C level had similar rates of AMI events and CHD mortality as those of the entire group (including hyperlipidemia);however, AMI attacks and CHD deaths decreased significantly at the normal and high HDL-C levels. The results demonstrated that the protective effect of HDL against coronary artery disease is more prominent in people with low lipid level. Conclusion Low HDL is an important independent risk factor for AMI attacks and CHD death in the elderly; high HDL has significant protective effect against coronary artery disease. Chin Med J 2004; 117(4): 511-515

Background Right ventricular apical pacing has been reported to reduce cardiac performance. But there are few reports on the effects of dual chamber (DDD) pacing on cardiac function compared to sinus rhythm. In this study, we evaluated the effects of right atrial and ventricular DDD pacing on cardiac function and ventricular contraction synchrony using equilibrium radionuclide angiography. Methods Ten patients implanted with a right atrial and ventricular DDD pacemaker underwent equilibrium radionuclide angiography. The scintigraphic data were obtained during sinus rhythm and pacing rhythm. Cardiac function parameters were obtained semimanually. Phase analysis was used to study the ventricular activation sequence and ventricular synchrony. Results The left ventricular 1/3 ejection fraction decreased significantly during pacing compared with that during sinus rhythm[(23.4 ±6. 1)% vs (27.7 ±4.5)%, P=0.01]. Regional ejection fraction also decreased during pacing, although the difference was not statistically significant. Phase analysis showed that the right ventricle was activated earlier than the left ventricle during pacing, and that the phase shift was significantly greater during pacing than that during sinus rhythm [64.13° ±16.80o vs 52.88° ±9.26°, P=0.007]. The activation of both ventricles occurred simultaneously during sinus rhythm, with the activation sequence from proximal septum or base of left ventricle to apex. The earliest activation during pacing occurred at the right ventricular apex, and subsequently spread to the base and left ventricle. Conclusion Right atrial and ventricular DDD pacing impairs left ventricular systolic function and ventricular synchrony. Chin Med J 2004; 117(4): 516-520

Background The purpose of this study was to assess the morphological changes and physiological function of coronary arteries in patients presenting with chest pain but having normal coronary angiograms, using intravascular ultrasound imaging (IVUS) and intracoronary Doppler (ICD) flow measurements, in order to elucidate the mechanism of syndrome X. Methods A total of 126 patients [67 males, 59 females, mean age (53.1 ±13.0) years] who experienced chest pain but had normal coronary angiograms were included in this study. ICD flow measurements of the left anterior descending coronary artery (LAD) were performed using a Cardiometrics FloMap II system. Coronary flow velocity reserve (CFVR) was defined as the ratio of the average peak velocity during hyperemia to that at baseline, induced by an intracoronary bolus injection of 18 μg adenosine. A 3. 2F or 2. 9F 30 MHz mechanical rotating ultrasound catheter (CVIS, Boston Scientific) or a 3. OF 20MHz electronic ultrasound catheter (Endosonics) was used for IVUS. Results The mean CFVR value of the LAD was 2. 71 ±0.74. Reduction of CFVR (<3.0) was found in 82 of 126 (65.1%) patients. IVUS images of the LAD were available for 109 patients. Plaque formation was detected in 76/109 (69. 7%) patients. Based on the presence or absence of plaque formation as well as the reduction or non-reduction of CFVR, patients were divided into four groups; Group I (n=10), normal IVUS findings and normal CFVR; Group II (n=23), normal IVUS findings with reduction in CFVR; Group III (n=29), IVUS evidence of plaque formation but normal CFVR; and Group IV (n=47), IVUS evidence of plaque formation with reduction in CFVR. Conclusion This study shows the important clinical value of a combination of IVUS and ICD in diagnosing patients with angiographically normal coronary arteries. Only 10%of patients studied(Group I) were found to be truly free of coronary disease, while 20%of patients (Group II) would be diagnosed as suffering from syndrome X. Chin Med J 2004; 117(4): 521-527

Background Some studies have confirmed that the right ventricular walls of most rodents, such as canines and humans, have evident transient outward potassium current (lto1) heterogeneity, and this heterogeneity is closely related to J point elevation, J wave formation, and some ventricular tachycardias such as ventricular fibrillations caused by Brugada syndrome. This study is designed to investigate transmural electrical heterogeneity of the canine right ventricle during repolarization (phase 1) from the viewpoint of 4-aminopyridine sensitive and calcium-independent lto1. Methods Adult canine single right ventricular epicardial (Epi) cells, mid-myocardial (M) cells, and endocardial (Endo) cells were enzymatically dissociated. Whole cell voltage-clamp recordings were made to compare the lto1 values of the three cell types. Results At 37℃ and using 0.2 Hz and + 70 mV depolarizing test potentials, the average peak lto1values of Epi cells and M cells averaged (4070 ±1720) pA and (3540 ±1840) pA, respectively. The activated and inactivated Epi and M cells kinetic processes were in accordance with the Boltzmann distribution. Compared with lto1 in Epi cells and M cells, the average peak lto1 in Endo cells was very low, averaged (470 ±130) pA. Conclusions These results suggest that there are evident differences and potent gradients in lto1between the three cardiac cell types, especially between Epi and Endo cells. These differences are among the prominent manifestations of right ventricular electrical heterogeneity, and may form an important ionic basis and prerequisite for some malignant arrhythmias in the right ventricle, including those arising from Brugada syndrome and other diseases. Chin Med J 2004; 117(4): 528-531