Study on the Mechanism of Üstikuddus Sherbiti in Ischemic Cerebrovascular Diseases: Based on Network Pharmacology.

This paper aims to study the potential biological mechanism of Üstikuddus Sherbiti (ÜS) in the treatment of ischemic cerebrovascular diseases (ICVD) by the network pharmacology method. Traditional Chinese Medicine Systems Pharmacology (TCMSP) database was used to obtain effective constituents of ÜS by screening eligible oral utilization, drug similarity, and blood-brain barrier permeability threshold. By drug target prediction and stroke treatment target mining, 2 target data sets were analyzed to find intersection targets and the corresponding constituents were used as active constituents. An active constituent target network and an effective constituent target network were constructed by using Cytoscape 3.7.2 software. Degree parameters of the effective constituent target network were analyzed to find important effective constituents and targets. Through protein-protein interaction (PPI) analysis/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, potential signaling pathways of ÜS in ischemic stroke were found out. AutoDock was used for molecular docking verification. A total of 90 active constituents of ÜS were screened out. There were 10 active constituents against ICVD, including quercetin, luteolin, kaempferol, and naringenin, and 10 important targets for anticerebral ischemia, namely, PIK3CA, APP, PIK3R1, MAPK1, MAPK3, AKT1, PRKCD, Fyn, RAC1, and NF-κB1. Based on the protein interaction network, the important targets of ÜS were significantly enriched in PI3K-Akt signaling pathway, neuroactive ligand-receptor interaction pathway, Ras signaling pathway, etc. ÜS in ICVD has characteristics like multiple targets, multiple approaches, and multiple pathways. Results of molecular docking showed that the active components in ICVD had a good binding ability with the key targets. Its main biological mechanism may be related to the PI3K-Akt and Ras-MAPK centered signaling pathway. Our study demonstrated that ÜS exerted the effect of treating ICVD by regulating multiple targets and multiple channels with multiple components through the method of network pharmacology and molecular docking.

The predictive value of urinary kidney injury molecular-1 for long-term graft function in kidney transplant patients: a prospective study.

BACKGROUND: Monitoring allograft function during the early stages is crucial, and therefore requires biomarkers more sensitive than serum creatinine (Scr). Kidney injury molecular-1 (KIM-1) is a potent biomarker; however, disparities exist in the literature concerning its predictive value in allograft function. Therefore, this study aimed to evaluate its predictive value for the long-term prognosis of kidney transplantation patients. METHODS: A prospective study with a cohort comprising 160 patients scheduled for kidney transplantation was conducted to evaluate the predictive power of urinary KIM-1 (uKIM-1) and other renal ischemia-reperfusion biomarkers including urinary L-type fatty acid binding protein (uL-FABP), urinary N-acetyl-ß-D glucosaminidase (uNAG), and urinary neutrophil gelatinase-related lipoprotein (uNGAL) for allograft prognosis. RESULTS: One hundred and forty kidney recipients who were admitted to our hospital between September 2014 and December 2017 with a median follow-up of 30.3 months were included. Thirty-seven recipients had functional delayed graft function (fDGF) in the first week post transplantation, and 42 recipients had progressed to allograft dysfunction [estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2] by the end of the study, while nine recipients deteriorated into allograft loss (defined by the initiation of dialysis). The levels of uKIM-1 in the fDGF group were higher than those in the immediate graft function (IGF) recipients (P<0.05) at 0 hour post transplantation [5.885 (4.420-7.913) vs. 4.605 (3.417-5.653) ng/mmol], and on the first day post transplantation [5.569 (4.181-6.722) vs. 4.002 (3.222-6.488) ng/mmol]. The levels of uL-FABP in the fDGF group were also higher than those in the IGF group at 0 hour post transplantation (89.818±39.332 vs. 69.187±37.926 µg/mmol) and on the third day post transplantation [77.835 (60.368-100.678) vs. 66.841 (28.815-89.783) µg/mmol]. Multivariate Cox regression analysis demonstrated that recipients with higher uKIM-1 levels on the first day post transplantation had a 23.5% increase in the risk of developing fDGF and a 27.3% increase in the risk of prolonged renal allograft dysfunction. CONCLUSIONS: uKIM-1 on the first day post transplantation can predict short-term graft function and is a potent biomarker for the long-term prognosis of graft function.

[Micro-dissection testicular sperm extraction for non-obstructive azoospermia patients with the history of secondary testicular injury].

Objective: To investigate the value of micro- dissection testicular sperm extraction (micro-TESE) in the treatment of non-obstructive azoospermia (NOA) in patients with the history of secondary testicular injury. METHODS: Totally, 121 NOA patients with the history of secondary testicular injury underwent micro-TESE in our hospital from September 2014 to December 2017. We analyzed the correlation of the sperm retrieval rate with the causes of testicular injury and compared the outcomes of the ICSI cycles with the sperm retrieved from the NOA males by micro-TESE (the micro-TESE group) and those with the sperm ejaculated from severe oligospermia patients (sperm concentration <1×106/ml, the ejaculate group). Comparisons were also made between the two groups in the female age, two-pronucleus (2PN) fertilization rate, transferrable embryos on day 3 (D3), D3 high- quality embryos, D14 blood HCG positive rate, embryo implantation rate, and clinical pregnancy rate. RESULTS: Testicular sperm were successfully retrieved by micro-TESE in 86.0% of the patients (104/121), of whom 98.4% had the history of orchitis, 75.5% had been treated surgically for cryptorchidism, and 63.6% had received chemo- or radiotherapy. No statistically significant differences were observed between the micro-TESE and ejaculate groups in the 2PN fertilization rate (59.4% vs 69.3%, P > 0.05), D14 blood HCG positive rate (44.6% vs 57.9%, P > 0.05), embryo implantation rate (31.8 %% vs 32.6%, P > 0.05) and clinical pregnancy rate (41.5% vs 48.7%, P > 0.05). However, the rate D3 transferrable embryos was significantly lower in the micro-TESE than in the ejaculate group (40.5% vs 52.2%,P < 0.05), and so was that of D3 high-quality embryos (32.5% vs 42.1%, P < 0.05). CONCLUSIONS: Micro-TESE can be applied as the first choice for NOA patients with the history of secondary testicular injury, but more effective strategies are to be explored for the improvement of ICSI outcomes with the sperm retrieved by micro- TESE.

Comparison of amino acid metabolism in frozen-thawed and fresh early-stage human embryos.

AIMS: To compare the amino acid differences of changes of frozen-thawed early-stage human embryos and fresh cultured early-stage human embryos. MATERIAL AND METHODS: Discarded embryos and their in vitro culture medium of patients who underwent in vitro fertilization-embryo transfer (IVF-ET) at the Research Center for Reproductive Medicine, the Sixth Affiliated Hospital of Sun Yat-sen University, from September 2010 to April 2011 were collected. Amino acid levels were determined by high performance liquid chromatography. RESULTS: The amino acid differences of changes in the culture medium of fresh embryos (661.50 µmol/L) were significantly higher than in the medium of post-thawed embryos (232.00 µmol/L) at 0.5 h (P < 0.001). At 1 and 2 h, no significant difference of change was found in all amino acids. Differences in the concentration of amino acids between post-thawed embryos and blank control medium were already present beginning at 1 h. CONCLUSIONS: The level of amino acid metabolism of frozen-thawed early-stage human embryos has already recovered from the state of metabolic stagnation during cryopreservation at 1 h of incubation after thawing, and the amino acid metabolism level at that time approximates that in fresh embryos before freezing. This may be established as the optimal embryo transfer time in IVF-ET.

[Analysis of associated factors for remission and relapse in proliferative and membranous lupus nephritis patients: a 4-year follow-up study].

OBJECTIVE: To explore the related factors of remission and relapse in lupus nephritis (LN) patients. METHODS: A retrospective study was conducted for proliferation and membrane LN patients diagnosed from 2003 to 2010. Their clinical, laboratory and pathological parameters were collected. According to the response to treatment, they were divided into 3 groups of complete remission (CR), partial remission (PR) and no response (NR). Those in remission were divided into 2 groups of relapsing and non-relapsing during maintenance period. Associated factors for remission and relapse were analyzed. RESULTS: (1) Among a total of 105 patients, there were 99 females and 6 males with an average follow-up period of (51 ± 30) months. Eighty-six patients achieved remission after 6-month treatment. (2) The outcomes were CR (n = 36), PR (n = 50) and NR (n = 19). Proteinuria in PR group was higher than that in CR group (4.7 (3.1-7.6) vs 1.7 (1.4-3.8), P < 0.01) while proteinuria of CR group was lower than that of NR group (1.7 (1.4-3.8) vs 3.0 (2.3-5.9), P < 0.01). Serum albumin level of CR group was significantly higher than those of PR (30.6 (27.8-34.6) vs 22.4 (19.3-29.4), P < 0.01) and NR groups (30.6 (27.8-34.6) vs 23.1 (18.9-28.6), P < 0.01). Serum creatinine was significantly higher in NR group than those of CR (128.9 (69.9-184.3) vs 58.1 (53.0-70.9), P < 0.01) and PR group (128.9 (69.9-184.3) vs 67.5 (53.5-129.1), P < 0.05). Acute index (AI) and chronic index (CI) were lower in CR group than those of PR and NR groups. (3) A total of 86 cases achieved remission (CR/PR) while 20 cases (23.3%) had relapse. During the maintenance period, the relapse rate was higher in the group on prednisone alone than those on combined therapy of prednisone plus immunosuppressant (P < 0.05). Sixty patients (90.9%) in non-relapse group and 12 cases (60.0%) in recurrence group had good compliance. CONCLUSIONS: Initial proteinuria, serum creatinine, serum albumin, estimated glomerular filtration rate and AI were related with remission of induction period. Prednisone-alone therapy is an independent risk factor for relapse during maintenance period. Poor compliance of patients may be one of the risk factors for relapse during maintenance period. It may be useful to maintain sustained remission and reduce relapse in LN patients by improving their compliance and using steroids plus immunosuppressant during maintenance period.

[Change and significance of growth differentiation factor 9 and bone morphogenetic protein expression during oocyte maturation in polycystic ovary syndrome patients with ovarian stimulation].

OBJECTIVE: To investigate the expression pattern and significance of two important oocyte-secreted factors: growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) during oocyte maturation in women with polycystic ovary syndrome (PCOS) and infertile women due to husband factors. METHODS: Total of 25 oocytes [9 at germinal vesicle GV stage, 9 at MI stage and 7 at MII stage] were obtained from 12 patients with PCOS and 82 oocytes (29 at GV stage, 26 at MI stage and 27 at MIIstage) were from 56 controls. The nested quantitative real time (RT) PCR was used to detect the abundance of GDF9 and BMP15 mRNA in each oocyte. RESULTS: (1) The expression level of GDF9 mRNA at the GV stage, MI stage and MII stage in PCOS group were 44.8 (4.2 - 529.0), 27.6 (9.8 - 172.7) and 49.0 (0.2 - 65.9) respectively, the expression in were 149.9 (55.4 - 387.9), 29.9 (2.5 - 205.8) and 657.8 (149.4 - 1376.2) in control group, respectively. The expression of GDF9 mRNA at MII stage was significantly lower in PCOS group than in controls (P < 0.01), however, the differences didn't reach statistical significant at GV or MI stage between the two groups (P > 0.05). The expression of GDF9 mRNA displayed some changes at different maturation stage in controls (P < 0.05, P < 0.01), however, the expression didn't demonstrate any dynamic changes in PCOS group (P > 0.05). (2) The expression level BMP15 mRNA at the GV stage, MI stage and MII stage in PCOS group were 0.1 (0.1 - 22.0), 3.2 (0.6 - 55.0) and 6.4 (3.2 - 8.5), respectively, the expression were 41.6 (6.5 - 96.1), 4.0 (2.0 - 10.4) and 49.7 (2.3 - 139.5) in control group, respectively. The expression of BMP15 mRNA at GV stage was significantly lower in PCOS group than in controls (P < 0.01), however, the differences were not significant at MI or MII stage between the two groups (P > 0.05). The expression of BMP15 mRNA also displayed some changes at different maturation stage in controls (P < 0.05), however, the level didn't demonstrate any dynamic changes in PCOS group (P > 0.05). CONCLUSION: It was suggested that the low expression of oocyte secreted factors in mature oocytes from PCOS patients might be associated with impaired oocyte quality and developmental competence in PCOS.

[Analysis of incidence and risk factor in hospitalized patients with acute kidney injury].

OBJECTIVE: To investigate the epidemiology and the risk factors of acute kidney injury (AKI) in hospitalized patients in order to help clinicians better understand and prevent AKI. METHODS: All patients hospitalized in Renji Hospital of Shanghai Jiao Tong University, which is a three-level General Hospital in Shanghai, during January to December of 2008 were screened by Lab Administration Network. Study group was comprised of the patients with full clinical data of AKI, as defined by Acute Kidney Injury Network (AKIN). The incidence, etiology and distribution characteristics of hospitalized patients with AKI were retrospectively analyzed. Logistic regression analysis was used to investigate the risk factors in severity of AKI. RESULTS: Nine hundred and thirty-four patients suffering from AKI for 1 001 episodes were enrolled. The incidence of AKI in hospitalized patients was 2.4% (934/38 734). The ratio of male to female was 1.88:1. The mean age was (60.82 ± 16.94) years old. Higher incidence was seen with an increase in age. Three hundred and thirty-one(35.4%) patients with AKI were found in medical department, 592(63.4%) patients in surgical department and 11(1.2%) patients in department of gynecologic and obstetrics. Analysis of the causes of AKI showed that pre-AKI accounted for 52.0%, followed by renal parenchyma AKI (44.7%) and postrenal AKI (3.3%). The most common reason for AKI was acute tubular necrosis (ATN, 37.5%), followed by absolute (33.6%) and relative inadequacy of blood volume (13.4%). Multivariate logistic regression analysis showed that chronic kidney disease (CKD) [odds ratio (OR)=2.085, 95% confidence interval (95%CI): 1.536-2.830,P<0.01], renal injurious drugs (OR=1.438, 95%CI: 1.087-1.901 ,P<0.05), and failure of organs other than kidney (OR=1.327, 95%CI: 1.014-1.737,P<0.05) were independent risk factors for stage II-III AKI. CONCLUSION: AKI is one of the most common clinical syndromes in hospitalized patients. With the increase of age, the incidence increases gradually. The most common reasons for hospitalized AKI are pre-AKI and ATN. CKD, renal injurious drugs and failure of other organs are independent risk factors of medium to serious AKI.

Abnormal expression of growth differentiation factor 9 and bone morphogenetic protein 15 in stimulated oocytes during maturation from women with polycystic ovary syndrome.

OBJECTIVE: To investigate the expression pattern of two oocyte-secreted factors-growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BM15)-during oocyte maturation in women with polycystic ovary syndrome (PCOS) and in controls and to evaluate the expression differences in oocytes between the two groups. DESIGN: Case-control study. SETTING: University-affiliated hospital. PATIENT(S): Twenty-five oocytes were obtained from 12 patients with PCOS and 82 oocytes from 56 controls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The abundance of GDF9 and BMP15 mRNA in oocytes of the germinal vesicle (GV), metaphase I (MI), and MII stage. RESULT(S): The expression of GDF9 and BMP15 mRNA displayed dynamic changes during oocyte maturation in controls after ovarian stimulation, with a decline at the MI stage and an increase to the peak at the MII stage. However, their expression in oocytes from patients with PCOS demonstrated a reduced state without any dynamic change. CONCLUSION(S): The results suggest that the expression of GDF9 and BMP15 in oocytes from patients with PCOS cannot reach the normal level even after ovarian stimulation and that the expression pattern is abnormal during oocyte maturation, which may be associated with impaired oocyte quality and developmental competence in PCOS.

[Effects of Astragalus on expression of renal angiopoietin receptor Tie-2 in diabetic rats].

OBJECTIVE: To study the expression of angiopoietin receptor Tie-2 in the renal tissue of diabetic rats and the effects of Astragalus. METHODS: SD rats were randomly divided into normal control group, diabetes group and Astragalus-treated group. The expression of receptor Tie-2 in the renal tissue was assessed by using real-time quantitative polymerase chain reaction and immunohistochemical method. RESULTS: Glomerule Tie-2 protein expression was significantly elevated in the diabetes group as compared with the normal control group (P<0.01). Glomerule Tie-2 protein expression in the Astragalus-treated group was decreased as compared with the diabetes group (P<0.01). CONCLUSION: Tie-2 may play an important role in the pathogenesis of the early stage diabetic renal injury. The reno-protection effect of Astragalus may be mediated by down-regulating the expression of Tie-2 in the kidney tissue of diabetic rats.

[Meiotic spindle location and embryo development of in vivo and in vitro matured human oocytes].

OBJECTIVE: To analyze the relationship between meiotic spindle location and embryo developmental potential of in vivo and in vitro matured human oocytes. METHODS: One hundred and thirty-four in vivo matured oocytes and 45 in vitro matured oocytes were observed with polscope at the time of intracytoplasm sperm injection (ICSI). RESULTS: Meiotic spindle was detected in 83.6% (112/134) and 82.2% (37/45) in in vivo and in vitro matured oocytes respectively. In vivo matured oocytes which showed a minimal angle (0-5 degrees ) between the meiotic spindle and the first polar body had a higher fertilization rate (93.3%) than the others. The frequency of the oocytes which had a 0-5 degrees spindle angle in in vivo and in vitro matured oocytes was 22.4% and 17.8%, respectively, and that of oocytes which had a 6 degrees - 45 degrees, 46 degrees-90 degrees and > 90 degrees spindle angle was 55.2% vs 51.1%, 3.0% vs 8.9%, and 3.0% vs 4.4%. No significant difference was found between them. No relationship was found between the position of meiotic spindle and embryo quality. CONCLUSIONS: There is some relationship between the angle of the meiotic spindle with the first polar body and fertilization rate. No significant difference is found in the position of the meiotic spindle between in vivo and in vitro matured human oocytes.

[Study on the value assessment of various screening programs regarding cervical cancer screening strategy in the rural areas of China].

OBJECTIVE: To evaluate the application value and feasibility of various cervical screening methods and to explore a rapid and efficient cervical cancer screening program for the women in the rural areas of China. METHODS: We sequentially conducted human papillomavirus (HPV) DNA test by hybrid capture-2 (hc2) with cervical cells, liquid-based thinprep cytology test (TCT), visual inspection with acetic acid (VIA), visual inspection with iodine (VILI), colposcopy respectively for the 2499 married women between 30 and 49 years from Xiushui county of Jiangxi province. All the detection methods were performed independently under double-blind design. Women who were diagnosed positive for having any VIA,VILI and colposcopy inspection or for those women who were diagnosed negative for VIA, VILI and colposcopy but with positive result of HPV or TCT test underwent cervical biopsy directly and endocervical curettage (ECC)when necessary. We performed cervical biopsy endocervical curettage within two weeks to observe the sensitive (SE), specificity (SP), negative predict value (NPV) and positive predict value (PPV) of these detection methods when used alone or combined each other, including HPV test, TCT inspection, VIA, VILI, and colposcopy, the pathological diagnosises of cervical tissue were confirmed by IARC (International Agency for Research on Cancer) while the cytological findings were underegone through the updated program of TBS (The Bethesda System) in 2001. RESULTS: A total of 2499 women underwent the screening and found 443 women who were diagnosed as HPV positive, 337 women with abnormal cervical cytology and 27 women with ASC-H, 157 cases with ASCUS; 103 cases with HSIL, 49 cases with LSIL and 1 cervical cancer. According to the pathological findings. There were 181 women diagnosed as cervical intraepithelial neoplasia (CIN) or cervical cancer, including 81 cases with CIN1 37 cases with CIN2,60 case cervical cancer. The sensitivity rates of HPV, TCT, HPV+ TCT, VIA, VILI, VIA+VILI and colposcopy were 96.67%, 89.47%, 97.98%, 56.57%, 36.36%, 63.64% and 39.39%, and the specificity rates were 85.00%, 96.91%, 86.97%, 94.60%, 96.23%, 92.97% and 98.14% respectively. CONCLUSION: HPV + TCT seemed to be more sensitive than other screening methods in the cervical cancer screening program.

[Influence of the depth of embryo transfer on pregnancy outcome in in vitro fertilization-embryo transfer].

OBJECTIVE: To detect whether the depth of embryo transfer has influence on pregnancy outcome in in vitro fertilization-embryo transfer (IVF-ET). METHODS: The distance between the high echogenic transfer dot and the fundal endometrium was measured under guidance of transabdominal ultrasound. The average distance 0.75 cm was used to divide patients into two groups. Group 1 included 44 patients with a distance > 0.75 cm, while group 2 had 48 patients with a distance

[Sperm sex chromosome analysis and preimplantation genetic diagnosis of patients with sex chromosome anomalies].

OBJECTIVE: To investigate the constitution of abnormal spermatozoa from patients with sex chromosome anomalies. METHODS: Triple color fluorescence in situ hybridization (FISH) was used to determine the sex chromosome constitution of spermatozoa from three patients with sex chromosome anomalies (case 1:46,XY/47,XXY, case 2:45,XO/46,X,Yqh-, case 3:47,XXY). The preimplantation genetic diagnosis (PGD) was performed to case 2. RESULTS: An increased ratio (2.05 vs 1) of X-bearing to Y-bearing spermatozoa was only observed in case 2, who also had an increased incidence of total abnormal spermatozoa (29.71%). An increased incidence of total abnormal spermatozoa (4.91%) was also observed in case 3. Among the constitution of abnormal spermatozoa, case 2 had the increased proportions of XY18 disomy, O18 monosomy and XO monosomy, while case 3 had an increase proportion of XY18 disomy (1.87%). PGD was performed to case 2 and one embryo with XX1818 was selected for implanting. CONCLUSION: Using FISH to detect the sperm sex chromosomes in patients with sex chromosome anomalies can provide the useful information to evaluate the risk of sex chromosome anomalies in preimplantation embryos.

[Clinical analysis of intracytoplasmic sperm injection in patients with previous fertilization failure after conventional IVF].

OBJECTIVE: To analyze the clinical outcome of intracytoplasmic sperm injection (ICSI) in patients with previous fertilization failure after conventional IVF. METHODS: Data from 20 ICSI cases (22 ICSI cycles) with previous complete failure of fertilization or with fertilization rate < or = 20% between January 2002 and December 2004 were retrospectively analyzed. The control group consisted of 100 consecutive ICSI cycles for male factor infertility in the same period. RESULTS: The fertilization rate dramatically increased from 5.4% after conventional IVF to 76.9% after ICSI treatment (chi-squared = 264.66, P < 0.001). However, the fertilization rate in the subgroup with previous low fertilization was significantly lower than those in the control and in the subgroup without previous fertilization (67.9% vs 77.5%, 67.9% vs 84.2%). Compared with the control group, the subgroup without previous fertilization had a higher pregnancy rate and implantation rate, but only the difference in the implantation rate was statistically significant (40.5% vs 18.9%). CONCLUSION: ICSI can overcome previous fertilization failure with conventional in vitro fertilization and thus improve the clinical outcome.

[Outcome of repeated epididymal sperm aspiration or testicular sperm extraction in azoospermic patients].

OBJECTIVE: To review the outcome of repeated percutaneous sperm aspiration (PESA) and testicular sperm extraction (TESE) for intracytoplasmic sperm injection (ICSI). METHODS: Forty-three cycles of 31 cases of azoospermic patients which underwent at least two PESA or TESE for ICSI from January 2001 to December 2002 were collected. The sperm retrieval, fertilization, implantation and clinical pregnancy were analyzed. RESULTS: Twenty-four cases underwent PESA and 7 cases underwent TESE. There were not any complications in these patients. Compared with the first cycle of 154 cases, the fertilization rate, implantation rate and clinical pregnancy rate were 78.39% vs 73.64%, 19.68% vs 18.38% and 34.88% vs 37.91%, respectively(P > 0.05). CONCLUSIONS: Repeated PESA or TESE is safe and well tolerated in azoospermic patients. Compared with the first cycle, the differences of repeated PESA or TESE cycles in fertilization rate, implantation rate and clinical pregnancy rate were not statistically significant.

[Preimplantation genetic diagnosis for beta-thalassemia using whole genome amplification].

OBJECTIVE: To achieve pregnancy with unaffected embryo using in vitro fertilization and embryo transfer (IVF-ET) and preimplantation genetic diagnosis(PGD) for the couples at risk of having children with beta-thalassemia. METHODS: A couple carrying different thalassemia mutations of codon 41/42 and codon IVS2 position 654 received standard IVF treatment and intracytoplasmic sperm injection, embryo biopsy, single cell polymerase chain reaction and DNA analyses, and only the unaffected or carrier embryos were transferred to uterus. Pregnancy confirmation, and prenatal diagnosis were done at 20 week's gestation. RESULTS: A total of 13 embryos were analyzed in the IVF cycle. PGD indicated that 2 were normal 18.1 , 3 were affected 27.3 , and 6 were carriers 54.5 ; diagnosis was not possible in 2. Three embryos were transferred to uterus on the third day after oocyte retrieval. Ultrasonography showed twin pregnancy with one blighted ovum. The prenatal diagnoses revealed that both fetuses were unaffected, one normal baby and one carrier were born. CONCLUSION: These studies represent the successful application of PGD for beta-thalassemia in China.

[Clinical application of a modified pronuclear stage scoring system following conventional in-vitro fertilization].

OBJECTIVE: To assess the clinical value of pronuclear stage scoring system in human conventional in vitro fertilization-embryo transfer (IVF-ET) program. METHODS: A modified pronuclear scoring system was used to score zygotes 16 - 18 hours after insemination during conventional IVF. RESULTS: A total of 178 IVF cycles were recruited in this study. Cycles with mean pronuclear score (sum of pronuclear scores of all transferred embryos divided by total number of transferred embryos) > or = 13 had significant high pregnancy and implantation rate, as compared to those with less than 13 (49.1% and 26.5% Vs 29.4% and 15.0% respectively). Although the pregnancy, implantation rate and multiple pregnancy rate tend to increase with the increasing number of zygotes with full pronuclear score (= 15) among transferred embryos, no significant difference could be found (P > 0.05). When all embryos were analyzed, 77.2% zygotes with pronuclear score > or = 11 could be transferred or cryopreserved, while those with pronuclear score less than 11 decreased to 46.1%. CONCLUSIONS: Pronuclear stage scoring system provides additional parameter for embryo selection. However, pronuclear morphology could only partly represent embryonic viability. Combination with the scoring of both pronuclear stage and cleavage stage embryos for selecting transferred embryos is indispensable.

[Preimplantation genetic diagnosis for beta-thalassemia].

OBJECTIVE: To investigate the effect of in vitro fertilization and embryo transfer (IVF-ET) and preimplantation genetic diagnosis (PGD) for the couples at risk of having children with beta-thalassemia. METHODS: Four couples carrying different thalassemia mutations received standard IVF treatment. Embryo biopsy was conducted. Single blastomeres were genotyped by a protocol involving primer extension preamplification, nested polymerase chain reaction and reverse dot-blot analysis. Only the unaffected embryos were transferred to the uterus. RESULTS: A total of 97 oocytes were retrieved from the four female carriers. Among them, 83% showed two pronuclei. Embryo biopsy was performed on 47 of these embryos. The amplification efficiency was 84.8%. The average ADO rate was 14.9%. Ten unaffected embryos were transferred. A twin pregnancy with one blighted ovum was confirmed at 7 weeks' gestation by ultrasonography and one normal baby and one carrier of thalassemia mutation were born finally. CONCLUSION: This unaffected pregnancy resulting from PGD for beta-thalassemia demonstrates that PGD technique can be a powerful diagnostic tool for couples carrying beta-thalassemia mutations who desire a healthy child and wish to avoid abortion of an affected fetus.

[Successful preimplantation genetic diagnosis for beta-thalassemia using primer extension preamplification].

OBJECTIVE: To achieve preimplantation genetic diagnosis (PGD) of the couples at risk of having children with beta-thalassemia, as an alternative to prenatal diagnosis. METHODS: Two couples carrying different thalassemia mutations of codon 41/42 and codon intervening sequence 2 position 654 received standard in vitro fertilization treatment and intracytoplasmic sperm injection, embryo biopsy and the whole genome was amplified by primer extension preamplification (PEP). Nested polymerase chain reaction was then used to amplify two mutation sites separately. Both were detected by reverse dot-blot. RESULTS: A total of 35 oocytes were retrieved from the two patients. Among them, 87% showed two pronuclei, and embryo biopsy was performed on 16 of these embryos and 25 blastomeres were obtained. The amplification efficacy was 84%. The genotype study of non-transferred and surplus embryos showed 15% of allele drop-out rate. Five embryos were transferred to the uterus of both patients. One pregnancy achieved, resulted in live healthy twin births, which confirmed the results of PGD. CONCLUSIONS: This unaffected pregnancy resulting from PGD by PEP for beta-thalassemia demonstrates that this technique can be a effective diagnostic tool for carrier couples who desire a healthy child.

[Sex determination of human preimplantation embryo using nested polymerase chain reaction].

OBJECTIVE: Using nested polymerase chain reaction (PCR) to perform preimplantation gender diagnosis. METHODS: One (or two) lymphocyte and blastomere (n=50/group) were collected and prepared under the following conditions: (1) water only (H(2)O); (2) freeze-thaw liquid nitrogen, then boiling; (3) potassium hydroxide/dithiotheriol, heated to 65 degree centigrade, followed by acid neutralization (KOH). Cells were analyzed by PCR using nested primers amplification with amelogenin gene. RESULTS: The amplification rate and allele dropout (ADO) rate for male lymphocytes by the three methods were 83%, 94%, 95% and 24%, 12%, 4%, respectively. Using two cells per reaction did not increase the amplification rate for the KOH method. CONCLUSION: The KOH method for DNA preparation is superior to the other methods evaluated. Dual blastomere biopsy and independent blastomere analysis may improve preimplantation diagnostic reliability.