应用全基因组扩增技术对α地中海贫血进行植入前遗传学诊断的效果分析

目的研究α地中海贫血患者囊胚活检后先进行全基因组扩增后再应用荧光PCR进行诊断的效果。方法回顾分析本中心2015年1月至2016年11月α地中海贫血PGD周期资料,共139个周期,对比分析卵裂球活检后直接应用荧光PCR进行诊断(诊断方法一)与囊胚活检后先进行全基因组扩增后再应用荧光PCR进行诊断(诊断方法二)的结果。结果两组在女方年龄、获卵数、正常受精数均无统计学差异(P0.05),但方法二(7.61±3.05)的平均活检个数显著小于方法一(9.48±3.66)(P0.05)。两种方法的正常胚胎率(35.22%vs.25.87%)、杂合子胚胎率(30.11%vs.50.19%)、异常胚胎率(30.11%vs.23.94%)、诊断失败率(16.21%vs.2.81%)有统计学差异(P0.05),临床妊娠率(50.00%vs.52.17%)无统计学差异(P0.05)。结论囊胚活检后先进行全基因组扩增后再应用荧光PCR进行诊断的效果明显好于卵裂球活检后直接应用荧光PCR进行诊断。     

在β地中海贫血着床前遗传学诊断中应用多重置换扩增进行预处理的临床分析

目的分析应用多重置换扩增技术(multiple displacement amplification,MDA)进行全基因组扩增的预处理是否影响β地中海贫血着床前遗传学诊断(preimplantation genetic diagnosis,PGD)的准确效能。方法回顾性地分析2009年1月至2013年6月,因双方均为β地中海贫血携带者而行PGD治疗的周期资料,其中34个周期采用多重巢式聚合酶链反应(PCR)结合反向斑点杂交技术对单细胞进行诊断,另有38个周期行MDA进行全基因组扩增的预处理后,再结合反向斑点杂交技术进行诊断。结果两组患者在年龄、获卵数等实验室指标上无统计学差异。MDA组未检出(扩增失败)率为9.79%,低于行巢式PCR组的15.24%,而杂合子率46.33%则略高,但两种方法在诊断结果上并无统计学差异。结论应用MDA技术进行全基因组的预扩增可有效增加检测模板,实现多位点及多种疾病的诊断,而且不影响β地中海贫血地贫基因的诊断效能。     

新发染色体异常对罗氏易位和相互易位携带者植入前遗传学诊断的影响分析

目的探讨植入前遗传学诊断(PGD)中新发染色体异常对易位携带者胚胎诊断结果和临床结局的影响。方法对27个周期的罗氏易位和64个周期的相互易位携带者的囊胚采用二代测序(NGS)行PGD。结果罗氏易位和相互易位携带者的囊胚PGD结果显示,34.1%(139/408)的胚胎存在新发染色体异常;罗氏易位和相互易位各自的数据显示,罗氏易位携带者的完全新发染色体异常率为31.7%(40/126),数值上高于相互易位携带者的18.4%(52/282),但差异无统计学意义(P0.05)。罗氏易位和相互易位携带者分别有70.4%(19/27)和54.7%(35/64)的PGD周期检出完全新发染色体异常囊胚;临床结局显示分别有7.4%(2/27)的罗氏易位PGD周期和18.8%(12/64)的相互易位PGD周期由于新发染色体异常导致无正常结果的胚胎可供移植。结论罗氏易位和相互易位携带者的囊胚PGD均不同程度的面临新发染色体异常的影响。相比于相互易位携带者,罗氏易位携带者中完全新发染色体异常的囊胚检出率更高,但由于罗氏易位携带者可用的正常或易位携带胚胎率更高,实际其PGD周期受到的影响要小于相互易位。     

胚胎植入前遗传学诊断的临床应用分析

目的回顾性分析胚胎植入前遗传学诊断(PGD)临床病例,为PGD技术的临床应用提供参考。方法收集2013年11月至2017年12月在本院生殖中心进行PGD的病例资料136例,按照不同临床适应证进行数据统计、总结分析。PGD采用二代测序技术(NGS)进行。结果 136例PGD,172个取卵周期中,染色体病116例,8个病种;单基因病20例,8个病种。全部采用囊胚期活检,活检474个胚胎,活检成功率100%,全基因组扩增成功率95.6%,有434个胚胎获得明确诊断,胚胎正常或携带者率50.9%(221/434)。98例患者进行了115个周期的解冻移植,均采用单囊胚移植,胚胎复苏率100%,76例获临床妊娠(66.1%),4例流产(5.3%),2例多胎妊娠(2.6%),2例宫外孕(2.6%),出生31个男婴和30个女婴。结论 NGS技术可精准检出染色体异常和致病基因突变,有效筛选可用胚胎,阻断遗传疾病的传递。PGD后单囊胚解冻移植可获得理想的临床结局。     

雌激素受体亚型ER-α和ER-β在前列腺癌组织中表达的研究

目的探讨雌激素受体ER-α和ER-β在前列腺癌(PCa)发病及恶性进展中的作用,为PCa的预防、靶向治疗和预后提供理论依据。方法收集山东省潍坊市中医院和上海交通大学附属第一人民医院的42例PCa和20例正常前列腺组织标本,采用免疫组织化学(S-P法)染色,观察前列腺组织标本中的ER-α和ER-β阳性表达情况,分析ER-α和ER-β与前列腺癌的相关性。结果 ER-α在PCa和正常前列腺上皮组织中的阳性表达率分别为4.8%和15.0%,差异无显著性意义(P0.05);ER-α在PCa和正常前列腺间质组织中的阳性表达率分别为64.3%和30.0%,差异有显著性意义(P0.05)。ER-β在PCa和正常前列腺上皮组织中的阳性表达率分别为31.0%和60.0%,差异有显著意义(P0.05);ER-β在PCa和正常前列腺间质组织中的阳性表达率分别为19.0%和30.0%,差异无显著意义(P0.05)。ER-α的阳性表达与PCa患者的年龄无显著意义(P0.05),与PSA、Gleason评分呈正相关(P0.05)。ER-β的阳性表达与PCa患者的年龄及PSA水平无显著意义(P0.05),而与Gleason评分呈负相关(P0.05)。结论 ER-α在前列腺组织中的表达以间质细胞为主,而ER-β主要表达于上皮细胞。ER-α对前列腺的增殖、恶变可能起到促进作用,而ER-β可能作为一种保护性受体对PCa的发病及恶性进展发挥抑制作用。     

异常形态精子对植入前胚胎发育和妊娠结局的影响

目的探讨异常形态精子(畸形率≥98%)对植入前胚胎发育及妊娠结局的影响。方法采用回顾性队列研究,分析2017年1~12月在唐都医院妇产科生殖医学中心行ART助孕的2419例患者临床资料,根据异常形态精子分为3组,即IVF对照组(畸形率≤96%,n=2129)、IVF实验组(畸形率≥98%,n=90)和ICSI实验组(畸形率≥98%,n=200)。比较3组间植入前受精失败率(受精率<30%)、正常受精率、可用胚胎率等胚胎发育参数和着床率、临床妊娠率、流产率及活产率等妊娠结局的差异。结果(1)胚胎发育结果:组间比较,IVF实验组受精失败率显著高于IVF对照组(P<0.05),ICSI实验组的受精失败率为0;ICSI实验组正常受精率显著高于IVF对照组和IVF实验组(P<0.05);IVF实验组可用胚胎率显著低于IVF对照组和ICSI实验组(P<0.05)。(2)妊娠结局:单因素分析结果显示,与IVF对照组、ICSI实验组相比,IVF实验组的着床率、临床妊娠率、流产发生率和活产率差异均有统计学意义(P<0.05);IVF对照组和ICSI实验组组间妊娠结局指标比较均无显著性差异(P>0.05)。(3)Logistic多因素分析显示:IVF实验组的受精失败风险显著高于IVF对照组(P=0.002),可用胚胎率、活产率显著低于IVF对照组(P=0.002);ICSI实验组的正常受精率显著高于IVF对照组(P=0.05)。结论对于活力正常、但异常形态精子率≥98%的患者,采用ICSI授精方式,能降低受精失败风险,提高正常受精率和可用胚胎率,同时提高妊娠率和活产率并降低流产发生率。     

Y染色体异常不育症患者胚胎植入前遗传学诊断36例临床分析

目的探讨使用荧光原位杂交(FISH)方法对Y染色体异常男性不育患者进行胚胎植入前遗传学诊断(PGD)的临床意义.方法36对不孕夫妇采用控制性促超排卵和卵胞浆内单精子注射(ICSI),受精后第3天行胚胎活检、卵裂球固定和FISH检测,第4或第5天选择核型为XX的女性胚胎移植,怀孕16周时,进行羊水复检.结果36例患者,每例患者均进行了一个ICSI-PGD周期,共36个周期,获卵总数580个.成熟卵子473个,正常受精398个,受精率为84.14%(398/473);胚胎形成率99.50%(396/398);优质胚胎率29.55%(117/396);实施活检胚胎281个,胚胎活检成功率为100%(281/281);卵裂球固定率为100%(281/281),其中12个卵裂球无细胞核;有细胞核卵裂球的FISH杂交成功率为100%(269/269).FISH杂交结果显示,核型为XX的女性胚胎数为38.29%(103/269);核型为XY的男性胚胎数为26.77%(72/269);非整倍性异常胚胎数为34.94%(94/269).共移植52个女性胚胎,着床率38.46%(20/52);临床17例妊娠,妊娠率为47.22%.8例行染色体G显带核型分析及FISH复查,结果显示均为女性.目前已有12例正常女婴成功分娩.结论应用ICSI结合PGD治疗Y染色体异常男性不育症患者,选择正常女性胚胎植入母体子宫,是解决此类患者不育问题、同时避免Y染色体异常患儿出生的有效手段.     

α地中海贫血孕妇妊娠期血红蛋白、平均红细胞体积及平均红细胞血红蛋白量变化相关临床分析

目的了解不同类型α地中海贫血(Thalassemia)孕妇妊娠早中晚期血红蛋白(hemoglobin;haemoglobin,Hb)、平均红细胞体积(erythrocyte mean corpuscular volume,MCV)、平均红细胞血红蛋白量(mean corpuscular hemoglobin,MCH),并探讨上述指标的变化。方法选取2013年7月至2014年10月在我院产检的α地贫孕妇263例,分为中间型组、轻型组、静止型组,对照组随机选取周期健康孕妇100例。记录孕早中晚期Hb、MCV、MCH值。结果在同一孕期,中间型组、轻型组、静止型组及对照组的Hb值和MCV值依次显著上升(P0.05);中间型组MCV值显著高于轻型组,两者均低于静止型组及对照组(P0.05),而轻型组、静止型组和对照组的MCH值依次显著上升(P0.05)。结论中间型及轻型α地中海贫血孕妇Hb下降较明显,应注意监测;α地中海贫血孕妇MCV及MCH值出现变化,均低于正常值,将Hb、MCV、MCH联合进行地中海贫血筛查可提高准确性。     

改良式脾脏次全切除术治疗地中海贫血综合症26例报告

目的 探讨改良式脾脏次全切除术对治疗地中海贫血综合征的疗效。方法 自2000年1月至2004年6月对我科行改良性脾脏次全切除术的26例地中海贫血综合征患者进行回顾性分析。结果 1例极重度贫血合并心功能不全患者,术后因心功能衰竭死亡。3例出现肺部感染,经抗感染治疗后治愈。其余患者未出现腹腔出血,膈下积液,膈下脓肿及切口感染等并发症。术后随访19例,随访率73％,随访时间6～50个月平均21个月。术后3个月复查血常规,发现RBC,WBC,及PLT均有明显改善。5例反复出现肺部感染,但无凶险性感染。10例仍需间歇输血。结论 改良式脾脏次全切除术适应症为严重地中海贫血综合征及巨脾并有脾功能亢进。本手术能起到减少血细胞在脾脏中的破坏,使部分病人在随访期内不需输血,而部分病人可减少输血次数和输血量。同时,能减少脾脏切除后引起免疫功能的下降。     

合并海洋性贫血的尿毒症患者肾移植的临床观察

目的探讨海洋性贫血对尿毒症患者肾移植效果的影响。方法为46例合并海洋性贫血的尿毒症患者施行。肾移植（海洋性贫血组），其中α海洋性贫血26例，β海洋性贫血20例，观察患者术后移植。肾功能恢复延迟（DGF）和排斥反应的发生率以及贫血的纠正情况，对于移植。肾功能恢复正常者，记录其。肾功能恢复正常的时间，并测定血肌酐值。以同期施行的131例。肾移植（均伴有程度不等的贫血，但非海洋性贫血）为对照。结果海洋性贫血组DGF的发生率为26．1％，对照组为23．7％，二者比较，差异无统计学意义。术后6个月，人、肾均存活，且未失访的患者，海洋性贫血组有39例，对照组有109例，6个月内，海洋性贫血组30．8％发生排斥反应，对照组32．1％发生排斥反应，两组比较，差异无统计学意义；海洋性贫血组的血肌酐值为（121±20）μmol／L，对照组为（128±33）μmol／L，两组比较，差异无统计学意义；海洋性贫血组79．5％的贫血得到纠正，对照组76．1％的贫血得到纠正，两组比较，差异无统计学意义。结论合并海洋性贫血的尿毒症患者可接受肾移植治疗，临床效果与不合并该病者相仿。     

An unusual presacral mass: Extramedullary hematopoiesis

Presacral masses are a rare finding in the adult patient, confronting the physician with diagnostic and therapeutic challenges. We present an unusual case of a symptomatic presacral mass caused by extramedullary hematopoietic tissue in a thalassemic patient and review the unique aspects of this entity. No financial support has been granted for the work described in this article and no involvement exists with any organization with an interest in the subject matter.     

Circulating osteoprotegerin and receptor activator of NF-κB ligand system in patients with β-thalassemia major

Osteoporosis represents an important cause of morbidity in patients with β-thalassemia major, and its etiology is multifactorial. Thus, the aim of this study was to characterize the possible role of the osteoprotegerin (OPG) and receptor activator of the NF-κB ligand (RANKL) system in thalassemia-related bone loss. Serum concentrations of OPG, soluble RANKL (s-RANKL), markers of bone turnover, and lumbar spine bone mineral density (BMD) were measured in random samples of males (n = 29; mean age ± SEM, 24.26 ± 1.29 years; range, 13–41 years) and females (n = 31; age, 24.59 ± 0.95 years; range, 12–34 years) with β-thalassemia major and in 30 healthy age-, height-, and weight-matched subjects. Thalassemic patients had significantly lower levels of OPG compared with controls (2.54 ± 0.12 vs. 3.25 ± 0.122, respectively; P < 0.05) and higher, albeit not statistically significantly, serum levels of s-RANKL (0.350 ± 0.03 vs. 0.295 ± 0.046, respectively; P < 0.05). s-RANKL correlated negatively with age (r = −0.3, P < 0.05), and OPG correlated positively with the duration of the interval between the onset of transfusions and chelation therapy (r = 0.52, P < 0.001). Regarding markers of bone metabolism, plasma values of osteocalcin correlated positively with s-RANKL (r = 0.40, P < 0.05) and negatively with OPG/s-RANKL ratio (r = −0.55, P < 0.01). In multiple regression analysis only cross-linked N-teleopeptide of type I collagen (NTX) significantly accounted for BMD. Although the OPG/RANKL system may have some clinical usefulness as a marker of bone turnover in β-thalassemia, conventional markers of bone turnover more accurately represent changes in the BMD of these patients.     

Renal tubular function in children with beta-thalassemia minor

Background: beta-thalassemia minor is a common heterozygous haemoglobinopathy that is characterized by both microcytosis and hypochromia. It requires no treatment. It has been postulated that low-grade haemolysis, tubular iron deposition and toxins derived from erythrocytes might cause renal tubular damage in adult patients with beta-thalassemia minor. Our aim was to investigate the renal tubular functions in children with beta-thalassemia minor and to determine its possible harmful effects. Methods: The study was conducted on 32 children (14 female and 18 male) at the age of 5.8 +/- 3.1 years (range 2-14 years) with beta-thalassemia minor. The patients were classified as anaemic (haemoglobin (Hb) 11 g/dL) (Group 2, n = 18). A control group was formed with 18 healthy children whose ages and sexes match those in other groups (Group 3, n = 18). Fractional excretion of sodium (FE(Na), %), fractional excretion of magnesium (FE(Mg), %), fractional excretion of uric acid (FE(UA), %) and tubular phosphorus reabsorption (TPR,%) were calculated with standard formulas. Urinary calcium excretion (mg/kg per 24 h), zinc (Zn) (microg/dL), glucosuria (mg/dL), beta-2 microglobulin (mg/dL) and N-acetyl-beta-D-glycosaminidase (NAG, U/mmol creatinine) levels were measured through biochemical methods. Results: There was no statistically significant difference among the three groups in terms of the results of FE(Na) (%), FE(Mg) (%), FE(UA) (%), TPR (%), calciuria (mg/kg per 24 h), NAG, urine Zn, proteinuria, glucosuria or urine beta- 2 microglobulin levels (P > 0.05). Conclusion: On the contrary of children with beta-thalassemia major, renal tubular dysfunction has not been determined in children with beta-thalassemia minor in the present study.     

Renal function in children with β-thalassemia major and thalassemia intermedia

In beta-thalassemia, profound anemia and severe hemosiderosis cause functional and physiological abnormalities in various organ systems. In recent years, there have been few published studies demonstrating proteinuria, aminoaciduria, low urine osmolality, and excess secretion of the tubular damage markers, such as urinary N-acetyl-D-glucosaminidase (U(NAG)) and beta2 microglobulin, in patients with thalassemia. The object of this study was to analyze renal tubular and glomerular function in pediatric patients with beta-thalassemia and to correlate the renal findings to iron overload. Thirty-seven patients with beta-thalassemia major and 11 with thalassemia intermedia were studied. Twelve children without iron metabolism disorders or renal diseases served as a control group. No difference in blood urea nitrogen (BUN), serum creatinine, creatinine clearance, electrolytes, fractional excretion of sodium and potassium, and tubular phosphorus reabsorption was found. Serum uric acid was equal in the two groups, but its urine excretion was significantly higher in the thalassemic group. U(NAG) and U(NAG) to creatinine ratio (U(NAG/CR)) were elevated in all patients with thalassemia compared with the control group (p < 0.001) and were directly correlated to the amount of transfused iron but not to actual ferritin level. We found that renal tubular function is impaired in children with beta- thalassemia major and intermedia. It is not known whether these functional abnormalities would have any long-term effects on the patients. Further studies are needed, and means of preventing these disturbances should be sought.     

Renal function in pediatric patients with β-thalassemia major

In patients with β-thalassemia major, the most important cause of mortality and morbidity is organ failure due to deposits of iron.. In this study, the nature of the kidney injury and possible pathogenetic factors were investigated. Seventy children with β-thalassemia major and 14 age and sex-matched healthy children were involved in the study. Blood and timed urine samples were obtained for hematological and biochemical tests. The mean values of blood urea nitrogen (BUN), serum creatinine, creatinine clearance, serum sodium, urine osmolality, fractional excretion of sodium, potassium, and uric acid were not statistically different between the groups. Serum levels of potassium, phosphorus, and uric acid and the urine volume, high urinary protein to creatinine (U_P/Cr), urinary N-acetyl-β-d-glucosaminidase to creatinine (U_NAG/Cr), and urinary malondialdehyde to creatinine, (U_MDA/Cr) and the tubular phosphate reabsorption (TRP) values were statistically different between two groups (P<0.05). Increased serum levels of potassium, phosphorus, and uric acid in the patient group were attributed to the rapid erythrocyte turnover. The presence of high U_P/Cr, U_NAG/Cr and U_MDA/Cr ratios shows that in these patients with proximal renal tubular damage may be secondary to oxidative lipid peroxidation mediated by the iron overload. Received: 30 September 1999 / Revised: 19 May 2000 / Accepted: 22 May 2000     

Quantitative ultrasound of bone and clodronate effects in thalassemia-induced osteoporosis

Osteoporosis in -thalassemia major has emerged as a topic of interest since optimized transfusion regimens have increased life expectancy and quality in these patients. Although the pathogenesis of thalassemic osteopathy is multifactorial, the evidence of an increased resorption phase suggests that the use of antiresorptive drugs such as bisphosphonates can be considered a valuable therapeutic strategy to reduce bone turnover and the risk of fragility fractures. We compared the effects of long-term cyclical clodronate therapy (300mg intravenous infusion every 3 weeks for 2 years) and of an active placebo (calcium 1 vitamin D) on bone mass and bone turnover in 30 male patients with -thalassemia major. We also tested the possibility of using quantitative ultrasound (QUS) for assessing bone involvement in thalassemic osteopenia and in monitoring the response to antiresorptive therapy. Broadband ultrasound attenuation (BUA) was significantly reduced in patients with -thalassemia major as compared to healthy controls. In calcium and vitamin D-treated patients, a significant decline in spine, femoral, and total body areal bone density was observed. In the patients given intravenous clodronate we measured a substantial stability of bone mass, which was not significantly changed at the end of the study. The urinary excretion of deoxypyridinoline (a marker of bone resorption) showed a progressive significant decline throughout the study period in clodronate-treated patients. No significant change was observed in BUA values in both groups of patients. These results indicate that intermittent intravenous clodronate administration was not able to increase areal bone density in our thalassemic patients. Moreover, this is the first study to have assessed the usefulness of broadband ultrasound measurements in -thalassemia major.     

Vasopressor hormone response following mesenteric traction during major abdominal surgery

Background : We investigated the vasopressor hormone response following mesenteric traction (MT) with hypotension due to prostacyclin (PGI₂) release in patients undergoing abdominal surgery with a combined general and epidural anesthesia. Methods : In a prospective, randomized, placebo-controlled study we administered 400 mg ibuprofen (i.v.) in 42 patients scheduled for abdominal surgery. General anesthesia was combined with epidural anesthesia (T4-L1). Before as well as 5, 15, 30, 45, and 90 min after MT we recorded plasma osmolality, hemodynamics and measured 6-keto-PGFlα (stabile metabolite of PGI₂), TXB₂ (stabile metabolite of thromboxane A₂) active renin, and arginine vasopressin (AVP) plasma concentrations by radioimmunoassay. Catecholamine levels were assessed by high-pressure liquid chromatography (HPLC) with electrochemical detection. Results : Following MT, arterial hypotension occurred along with a substantial PGI₂ release. This was completely abolished by ibuprofen administration. Although plasma levels of 6-keto-PGF_1α (1133 (708) vs. 60 (3) ng/L, median (median absolute deviation), P=0.0001, placebo vs. ibuprofen) remained significantly elevated, blood pressure was restored within 30 min after MT in the placebo group. At the same point in time plasma concentrations of TXB² (164 (87) vs. 58 (1) ng/L, P=0.0001), epinephrine (46 (33) vs. 14 (6) ng/L, P=0.001), AVP (41 ± (18) vs. 12 (7) ng/L, P=0.0004), and active renin (27 (12) vs. 12 (4) ng/L, P = 0.001) were significantly higher in placebo-treated patients. Conclusion : Under combined general and epidural anesthesia arterial hypotension following MT due to endogenous PGI₂ release is associated with enhanced release of AVP, active renin, epinephrine and thromboxane A₂, presumably contributing to hemodynamic stability within 30 min after MT.