DNA无创产前检测及彩色多普勒超声检查在高危孕妇胎儿 染色体异常筛查中的应用价值

目的探讨DNA无创产前检测(NIPT)及彩色多普勒超声(简称彩超)检查在高危孕妇胎儿染色体异常筛查中的应用价值。方法选取2020年1月至2022年12月于该院接受产前检查的5862例高危孕妇作为研究对象,均接受NIPT、彩超检查,以羊水穿刺结果或分娩结局作为诊断胎儿染色体异常的金标准,比较NIPT、彩超检查及二者联合检查对高危孕妇胎儿染色体异常的诊断效能。结果5862例高危孕妇中共检出167例胎儿染色体异常,检出率为2.85%。167例胎儿染色体异常中胎儿染色体数目异常161例,构成比为96.41%;胎儿染色体结构异常6例,构成比为3.59%。彩超检查共诊断出119例孕妇胎儿染色体异常,经一致性分析,彩超检查诊断胎儿染色体异常的灵敏度为0.713,特异度为0.884,准确率为87.96%,Kappa=0.215,P<0.05。NIPT共诊断出133例孕妇胎儿染色体异常,经一致性分析,NIPT诊断胎儿染色体异常的灵敏度为0.796,特异度为0.945,准确率为94.05%,Kappa=0.408,P<0.05。彩超检查联合NIPT共诊断出158例孕妇胎儿染色体异常,经一致性分析,二者联合检查诊断胎儿染色体异常的灵敏度为0.946,特异度为0.986,准确率为98.50%,Kappa=0.775,P<0.05。结论NIPT与彩超检查用于筛查高危孕妇胎儿染色体异常均具有一定价值,二者联合检查可获得更高的灵敏度、特异度和准确率,能有效降低漏诊及误诊风险。

不同病情乙型肝炎患者HBV-DNA定量、两对半检测结果差异

目的分析乙型肝炎患者乙型肝炎病毒(HBV)-DNA定量检测与两对半检测结果的相关性,并观察不同病情HBV-DNA阳性率和两对半检测结果差异。方法选取江苏省泰兴市人民医院2021年4月至2023年4月收治的375例乙型肝炎患者病例为研究资料。比较不同乙肝两对半检测[乙肝表面抗原(HBsAg)、乙肝表面抗体(HBsAb)、乙肝e抗原(HBeAg)、乙肝e抗体(HBeAb)、乙肝核心抗体(HBcAb)]结果患者HBV-DNA定量检测结果差异,将患者根据HBV-DNA定量检测结果分为阳性组和阴性组,比较乙肝两对半结果差异,使用Pearson相关性分析;将患者根据疾病类型分为肝炎组、肝硬化组和肝癌组,比较三组患者乙肝两对半和HBV-DNA定量检测结果差异。结果不同乙肝两对半类型患者HBV-DNA阳性率比较,差异有统计学意义(P<0.05);HBV-DNA阳性组患者HBsAg、HBeAg水平高于HBV-DNA阴性组,HBsAb、HBeAb、HBcAb水平低于HBV-DNA阴性组,差异有统计学意义(P<0.05);HBsAg、HBeAg水平与HBV-DNA呈正相关(P<0.05),HBsAb、HBeAb、HBcAb水平与HBV-DNA呈负相关(P<0.05);不同疾病类型患者乙肝两对半类型HBV-DNA阳性率比较,差异有统计学意义(P<0.05),不同疾病类型患者HBV-DNA定量检测结果比较,差异有统计学意义(P<0.05)。结论乙型肝炎患者HBV-DNA定量检测与两对半检测结果有相关性,不同乙肝两对半类型间及不同疾病类型间HBV-DNA均有差异,HBV-DNA定量检测联合两对半检测可较好地反映病毒复制和疾病进展情况,为临床治疗提供指导。

Fecal microbial biomarkers combined with multi-target stool DNA test improve diagnostic accuracy for colorectal cancer

BACKGROUND Colorectal cancer(CRC)is a major global health burden.The current diagnostic tests have shortcomings of being invasive and low accuracy.AIM To explore the combination of intestinal microbiome composition and multi-target stool DNA(MT-sDNA)test in the diagnosis of CRC.METHODS We assessed the performance of the MT-sDNA test based on a hospital clinical trial.The intestinal microbiota was tested using 16S rRNA gene sequencing.This case-control study enrolled 54 CRC patients and 51 healthy controls.We identified biomarkers of bacterial structure,analyzed the relationship between different tumor markers and the relative abundance of related flora components,and distinguished CRC patients from healthy subjects by the linear discriminant analysis effect size,redundancy analysis,and random forest analysis.RESULTS MT-sDNA was associated with Bacteroides.MT-sDNA and carcinoembryonic antigen(CEA)were positively correlated with the existence of Parabacteroides,and alpha-fetoprotein(AFP)was positively associated with Faecalibacterium and Megamonas.In the random forest model,the existence of Streptococcus,Escherichia,Chitinophaga,Parasutterella,Lachnospira,and Romboutsia can distinguish CRC from health controls.The diagnostic accuracy of MT-sDNA combined with the six genera and CEA in the diagnosis of CRC was 97.1%,with a sensitivity and specificity of 98.1%and 92.3%,respectively.CONCLUSION There is a positive correlation of MT-sDNA,CEA,and AFP with intestinal microbiome.Eight biomarkers including six genera of gut microbiota,MT-sDNA,and CEA showed a prominent sensitivity and specificity for CRC prediction,which could be used as a non-invasive method for improving the diagnostic accuracy for this malignancy.

Clinical-DNA Correlates of Anxiety in Patients with Ehlers-Danlos Syndrome

Introduction: Anxiety disorders have a lifetime prevalence of 34% with a similar level of heritability (31%) yet lack objective markers that could differentiate patients with underlying conditions. Up to 60%-70% of patients with Ehlers-Danlos syndrome have anxiety that meets criteria of generalized anxiety disorder, their clinical-DNA findings worth examining as biomarkers for patients with generalized anxiety. Method: Of the 1899 patients diagnosed with Ehlers-Danlos syndrome, 1261 were systematically evaluated for 80 history and 40 physical findings and separated into 826 who reported anxiety and 435 who did not. The most consistently reported or management-directing 60 of these clinical findings were, along with variations in genes relevant to these disorders, examined for association with anxiety. Results: Among the 30 anxiety- associated findings judged most predictive of Ehlers-Danlos syndrome in patients with anxiety were expected ones of adrenergic stimulation (difficulty concentrating-87% frequency and 1.26 anxiety/no anxiety ratio;chronic fatigue-84%, 1.17;sleep issues 69%, 1.52 that are criteria for generalized anxiety disorder) or of cholinergic suppression (e.g., frequent nausea 64%, 1.26). Less associated but more discriminating for underlying disease were those reflective of neuromuscular impact (e.g., chronic daily headaches 76%, 1.12);hypermobility (e.g., awareness of flexibility 72%, 1.03), or skin changes (e.g., elasticity around jaw 71%, 1.06). Anxiety-associated DNA variants included 54 of 88 in collagen type I/V/VII/IX genes, 14 of 16 in sodium channel SCN9A/10A/ 11A genes, 59 of 85 in POLG/MT-DNA genes, and 21 of 28 in profilaggrin- FLG genes that respectively impacted tissue laxity, sensory neural, autonomic-mitochondrial, and autonomic-inflammatory functions. Conclusion: Analysis of pathogenetic mechanisms in Ehlers-Danlos syndrome selected some 50 clinical-DNA findings useful for its diagnosis in those with generalized anxiety disorders.

NT超声检测结合无创DNA检测用于高龄孕妇产前筛查的价值分析

目的探讨胎儿颈项透明层(NT)超声检测结合无创DNA检测用于高龄孕妇产前筛查的应用价值。方法选取本院486例进行产检、分娩的高龄单胎孕妇的临床资料,根据孕妇选择产前筛查方法不同,将其分为NT组(n=127例)和联合组(n=359例)。NT组采用NT超声检测,联合组采用NT超声检测结合无创DNA检测,对比两组胎儿染色体疾病的检出率和漏诊率。结果以羊水穿刺检测结果为金标准,NT组胎儿染色体疾病的检出率为66.67%,联合组胎儿染色体疾病的检出率为80.00%,联合组的检出率高于NT组;NT组漏诊率为33.33%,联合组漏诊率为20.00%,联合组的检测漏诊率低于NT组。结论NT超声检测结合无创DNA检测对高龄孕妇产前筛查有效,胎儿染色体疾病的检出率高,漏诊率低。

游离DNA最新研究进展及法医学应用展望

近年来,随着DNA提取和检测技术的不断进步,游离DNA(cell-free DNA,cfDNA)已经在生命科学领域得到了广泛应用,在法医学鉴定领域中的潜在应用价值也越来越明显。本文回顾了cfDNA概念、形成机制与分类等,并阐述了cfDNA在法医学现场接触检材的个体识别和无创产前亲缘关系鉴定应用中的最新研究进展,同时总结了cfDNA在损伤推断中的应用潜力,并探讨了常用cfDNA分析方法和技术的优缺点及应用展望,为cfDNA在法医学领域的广泛应用提供新思路。

HPV DNA、HPV E6/E7蛋白和TCT在宫颈上皮内瘤变及宫颈癌筛查中的价值

目的 探讨人乳头瘤病毒(human papilloma virus,HPV)DNA、HPV E6/E7蛋白和液基薄层细胞学检查(thin-prep cytology test,TCT)在宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)及宫颈癌筛查中的价值。方法 选取2021年7月至2022年6月于诸暨市人民医院妇科接受早期宫颈癌筛查的成年女性190例为研究对象,分别进行HPV DNA、HPVE6/E7蛋白及TCT检测,并进一步行阴道镜活检检查。比较不同病变患者的HPVDNA、HPVE6/E7蛋白和TCT对高级别病变的诊断效能。结果 CIN3及宫颈癌患者的HPV DNA、HPV E6/E7蛋白、TCT检查及三者联合检测的阳性率均显著高于宫颈炎患者(P<0.05),宫颈癌患者的HPV DNA、HPV E6/E7蛋白、TCT检查及三者联合检测的阳性率均显著高于CIN1患者(P<0.05)。CIN2+患者的HPV DNA、HPV E6/E7蛋白、TCT及三者联合检测的阳性率显著高于CIN1-患者。HPVDNA、HPVE6/E7蛋白、TCT三者联合诊断高级别病变的敏感度、特异性、阳性预测值和阴性预测值分别为90.80%、30.10%、52.32%、79.48%。结论 HPV DNA、HPV E6/E7蛋白及TCT可作为筛查宫颈癌和癌前病变的手段,且三者联合检测的敏感度最高。

无创DNA检查在单项超声软指标异常胎儿染色体筛查中的临床意义

目的:探究无创DNA检查(non-invasive DNA,NIPT)在单项超声软指标(ultrasound soft index,USM)异常胎儿染色体筛查中的临床意义。方法:选取2022年1月至2023年8月于郑州四六〇医院进行NIPT的174例单项USM阳性孕妇进行回顾性研究,分析其USM及NIPT结果,以临床最终结果为“金标准”,分析NIPT诊断结果及诊断效能,进一步分析不同检查结果的妊娠结局情况。结果:174例单项USM异常孕妇中,NIPT提示161例低风险,13例高风险,提示高风险包括2例性染色体数目异常,18-三体、13-三体各1例,21-三体9例;核型分析检测提示9例21-三体,1例47、XYY,18-三体、13-三体各1例,其中1例NIPT提示性染色体数目异常,患者核型分析检测结果提示正常;NIPT诊断灵敏度为85.71%,准确度为98.28%,漏诊率为15.38%;NIPT提示161例低风险,另有1位NIPT提示高风险患者经核型分析结果显示其染色体未出现明显异常现象,共计162例,其中18例在其他医院分娩,4例患者失访,其他孕妇均在郑州四六〇医院妇产科行产检、分娩,随访结果显示新生儿无明显异常;NIPT提示14例高风险孕妇中,XYY染色体数目异常、核型正常孕妇选择足月分娩,21-三体、13-三体、18-三体核型异常孕妇均选择引产。结论:NIPT在单项USM异常孕妇中具有较高的诊断灵敏度、准确度,漏诊率相对较低,可为临床及时筛查单项USM异常孕妇胎儿异常,降低流产风险提供可靠依据。

HR-HPV DNA联合TCT检查在子宫颈癌前病变筛查中的应用价值

目的 分析高危型人乳头瘤病毒基因(HR-HPV DNA)联合液基细胞学(TCT)检查在子宫颈癌前病变筛查中的应用价值。方法 选取2022年1月至12月于我院妇科门诊接受子宫颈癌前病变筛查的1 374例妇女作为研究对象。所有研究对象均接受TCT、HR-HPV DNA及组织病理学检查。以组织病理学检查结果为金标准,分析TCT、HR-HPV DNA单独及联合检查结果并比较诊断效能。结果 病理学检查结果显示,1 374例妇女中阳性184例,检出率为13.39%。1 374例妇女中,TCT检查检出180例阳性患者,阳性检出率为13.10%;HR-HPV DNA检查检出195例阳性患者,阳性检出率为14.19%;TCT与HR-HPV DNA联合检查检出191例阳性患者,阳性检出率为13.90%。TCT与HR-HPV DNA联合检查的诊断灵敏度、特异度、准确度、阳性预测值、阴性预测值高于HR-HPV DNA检查(P<0.05);TCT与HR-HPV DNA联合检查的诊断灵敏度、准确度、阴性预测值高于TCT检查(P<0.05);TCT、HR-HPV DNA联合检查与TCT检查的诊断特异度、阳性预测值比较,差异无统计学意义(P>0.05)。结论 HR-HPV DNA与TCT联合检查应用于子宫颈癌前病变筛查中可提高效能,为医师早期诊断提供准确参考依据,值得推广。

入侵植物一年蓬基因组DNA提取方法优化

一年蓬为菊科飞蓬属植物,是分布较广、危害较重的一种外来入侵杂草。为优选该植物基因组DNA的提取方法,本研究采取四因素三水平正交试验设计,对一年蓬基因组DNA的十六烷基三甲基溴化铵(CTAB)提取方法进行优化,并与常规的植物基因组试剂盒法进行对比。结果表明,一年蓬基因组DNA较优的提取方法为改良CTAB法,即一年蓬的干燥叶片经CTAB-free去除多糖,并加入β-巯基乙醇防止酚氧化,用氯仿∶异戊醇(24∶1)抽提3次。改良CTAB法提取的一年蓬基因组DNA质量较高,微量分光光度计检测的A_(260)/A_(280)数值接近1.8~2.0,且凝胶电泳DNA条带清晰,无明显拖带。该方法为该植物的遗传多样性和入侵机制等研究提供参考。

结直肠癌患者血浆Septin9 DNA甲基化联合FOBT检测的临床应用价值

目的:探讨血浆Septin9 DNA甲基化及粪便隐血试验(FOBT)在结直肠癌(CRC)诊断中的应用价值。方法:回顾性收集2017年6月至2022年1月我院收治并经病理检查确诊的101例结直肠良性腺瘤患者、209例结直肠癌患者分别作为良性腺瘤组和结直肠癌组,选取同期在我院进行体检的98例健康人群作为正常对照组,比较三组一般资料及血浆Septin9 DNA甲基化及FOBT阳性情况,比较不同病理特征、不同临床分期结直肠癌患者血浆Septin9 DNA甲基化、FOBT阳性情况,采用受试者工作曲线(ROC)评估血浆Septin9 DNA甲基化、FOBT单项及联合检测对结直肠癌的诊断价值。结果:三组血浆Septin9 DNA甲基化、FOBT阳性率比较,结直肠癌组高于良性腺瘤组及正常对照组,良性腺瘤组高于正常对照组(P<0.05);肿瘤低分化、淋巴结转移、脉管/神经侵犯的结直肠癌患者血浆Septin9 DNA甲基化和FOBT阳性率高于肿瘤高分化、中分化及未发生淋巴结转移、未发生脉管/神经侵犯的结直肠癌患者(P<0.05);随着结直肠癌TNM临床分期升高,血浆Septin9 DNA甲基化及FOBT阳性率升高,差异具有统计学意义(P<0.05);血浆Septin9 DNA甲基化和FOBT联合诊断结直肠癌的敏感度、曲线下面积(AUC)(87.08%、0.885)均高于两者单独诊断(59.33%、0.744和52.63%、0.643,P<0.05);血浆Septin9 DNA甲基化诊断结直肠癌的特异度、阳性预测值和阴性预测值分别为89.45%、85.52%和67.68%;FOBT分别为76.38%、70.06%和60.56%,两者联合诊断分别为89.95%、90.10%和86.89%。联合诊断优于单项诊断。结论:随着结直肠病变恶性程度的增加,血浆Septin9 DNA甲基化和FOBT阳性率升高,血浆Septin9 DNA甲基化、FOBT联合检测对结直肠癌具有较高的诊断价值,可作为结直肠癌的实验室诊断指标。

基于胎儿游离DNA的地中海贫血无创产前诊断的研究进展

重型地中海贫血(地贫)目前尚无有效治疗措施,通过产前诊断阻止重型患儿出生是国内外公认的首选预防措施。目前,地贫的产前诊断仍依赖于有创的方法,即绒毛活检、羊水穿刺和脐静脉血穿刺,存在一定的宫内感染和流产风险,安全、无创、快速和准确的地贫产前诊断方法对高发区意义重大。孕妇外周血胎儿游离DNA(cffDNA)的发现,下一代测序(NGS)的快速发展与应用,使无创产前DNA检测(NIPT)成为现实。目前,利用cffDNA检测胎儿13、18和21号染色体非整倍体的NIPT技术已经成熟应用于临床,但地贫的NIPT仍处于研究探索阶段。文章主要基于cffDNA和NGS的地贫NIPT研究进展进行综述。

无创产前筛查在双胎妊娠染色体非整倍体筛查中的应用及胎儿游离DNA浓度分析

目的评估无创产前筛查(non-invasive prenatal testing,NIPT)在双胎妊娠染色体非整倍体的应用价值,并对胎儿游离DNA浓度进行分析。方法收集2018年1月至2023年5月在广州市花都区妇幼保健院接受NIPT检测的双胎样本617例(辅助生殖双胎284例,自然妊娠双胎333例)为研究对象,同期12688例单胎妊娠样本作为对照,采用高通量测序方法进行检测,对染色体非整倍体高风险样本行核型分析,根据Y染色体唯一比对条数对男胎进行胎儿游离DNA浓度计算,使用基于浅深度的母亲血浆DNA测序方法(SeqFF)对女胎进行胎儿游离DNA浓度计算。结果双胎妊娠检出2例T21和1例T18,未检出T13,T21、T18的阳性预测值分别为100%、0,阴性预测值均为100%;单胎妊娠T21、T18、T13的阳性预测值分别为88.89%、45.45%、25%,阴性预测值均为100%。双胎妊娠胎儿游离DNA浓度为10.48%,较单胎妊娠(11.35%)稍低;双胎妊娠首次建库成功率为99.03%,较单胎妊娠(99.99%)低,差异有统计学意义(χ^(2)=104.105,P=0.00)。结论NIPT在双胎妊娠染色体非整倍体筛查有一定应用价值,T21筛查效率高,胎儿游离DNA浓度影响NIPT检测的成败和结果准确性。

山东威海地区孕妇无创产前DNA检测结果分析

目的探讨孕妇无创产前DNA检测(NIPT)的临床应用价值。方法选取在我院行NIPT检测10604名孕妇作为研究对象,根据孕妇送检原因的不同,分为年龄高风险组(A组)、产前筛查高风险组(B组)、产前筛查临界风险组(C组)、超声软指标异常组(D组)和直接选择NIPT组(E组),并对NIPT高风险孕妇进行羊膜腔穿刺及胎儿染色体核型分析。结果10604名孕妇检测出NIPT高风险175例,阳性检出率为1.65%,其中120例继续行介入性产前诊断,检出异常核型80例,阳性预测值为66.67%。其中A组、B组、C组、D组、E组NIPT检测出高风险例数分别为54,38,64,6,13例,阳性检出率分别为1.80%、4.82%、1.12%、5.94%、1.30%;A组、B组、C组、D组、E组NIPT高风险孕妇继续行介入性产前诊断的孕妇编入新组A1组、B1组、C1组、D1组、E1组,检出异常核型分别为35,15,22,3,5例,阳性预测值分别为79.6%、53.6%、61.1%、100.0%、55.6%。结论NIPT作为新兴检测技术广泛应用于孕妇产前筛查中,但存在假阳性(误诊)和假阴性(漏诊)的可能性,NIPT高风险孕妇仍需进行介入性产前诊断进一步确诊。

乙肝五项、前S1抗原与HBV-DNA联合检测的临床应用

目的:探讨乙肝五项、前S1抗原和HBV-DNA在肝炎诊断的价值;方法:收集上海市松江区中心医院2022年7月1日-8月31日检测乙肝五项的标本3364例,分析其乙肝五项、前S1抗原的检出率、HBV-DNA的载量,另外收集上海市松江区中心医院2022年3月1日-2022年8月31日HBV-DNA高载量标本46例,中载量标本54例,低载量标本76例,对照组标本52例,统计并分析其ALT、AST、GGT含量;结果:3364例标本中HBSAg阳性率为9.3%,检出率较高的模式分别为全阴、25(+)、2(+)、245(+)、145(+)、5(+);在主要的乙肝五项型别中,只有145(+)和135(+)型别检测到HBV-DNA,135(+)检出率高于145(+);前S1抗原与HBeAg检出率不同;不同DNA载量的四个组别ALT、AST、GGT差别均有统计学意义,四个组别ALT、AST、GGT不完全相同;结论:乙肝五项、前S1抗原和HBV-DNA联合检测对乙肝的诊断有重要的意义。

Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

AIM:To compare the clinical performance of a real-time PCR assay with the COBAS Amplicor Hepatitis B Virus (HBV) Monitor test for quantitation of HBV DNA in serum samples. METHODS: The reference sera of the Chinese National Institute for the Control of Pharmaceutical and Biological Products and the National Center for Clinical Laboratories of China, and 158 clinical serum samples were used in this study. The linearity, accuracy, reproducibility, assay time, and costs of the real-time PCR were evaluated and compared with those of the Cobas Amplicor test. RESULTS: The intra-assay and inter-assay variations of the real-time PCR ranged from 0.3% to 3.8% and 1.4% to 8.1%, respectively. The HBV DNA levels measured by the real-time PCR correlated very well with those obtained with the COBAS Amplicor test (r = 0.948). The real-time PCR HBV DNA kit was much cheaper and had a wider dynamic range. CONCLUSION: The real-time PCR assay is an excellent tool for monitoring of HBV DNA levels in patients with chronic hepatitis B.

Patient perceptions of stool DNA testing for pan-digestive cancer screening:A survey questionnaire

AIM:To explore patient interest in a potential multiorgan stool-DNA test(MUST)for pan-digestive cancer screening.METHODS:A questionnaire was designed and mailed to 1200 randomly-selected patients from the Mayo Clinic registry.The 29-item survey questionnaire included items related to demographics,knowledge of digestive cancers,personal and family history of cancer,personal concern of cancer,colorectal cancer(CRC)screening behavior,interest in MUST,importance of test features in a cancer screening tool,and comparison of MUST with available CRC screening tests.All responses were summarized descriptively.χ2 and Rank Sum Test were used for categorical and continuous variables,respectively.RESULTS:Completed surveys were returned by 434(29%aged 50-59,37%60-69,34%70-79,52%women).Most participants(98%)responded they would use MUST.In order of importance,respondents rated multicancer detection,absence of bowel preparation,safety and noninvasiveness as most attractive characteristics.For CRC screening,MUST was preferred over colorectal-only stool-DNA testing(53%),occult blood testing(75%),colonoscopy(84%),sigmoidoscopy(91%),and barium enema(95%),P<0.0001 for each.Among those not previously screened,most(96%)indicated they would use MUST if available.Respondents were confident in their ability to follow instructions to perform MUST(98%).Only 9%of respondents indicated that fear of finding cancer was a concern with MUST,and only 3%indicated unpleasantness of stool sampling as a potential barrier.CONCLUSION:Patients are receptive to the concept of MUST,preferred MUST over conventional CRC screening modalities and valued its potential feature of multi-cancer detection.

Multiple z-Score Based Method for Noninvasive Prenatal Test Using Cell-Free DNA in Maternal Plasma

Objective: To improve the detecting accuracy of chromosomal aneuploidy of fetus by non-invasive prenatal testing (NIPT) using next generation sequencing data of pregnant women’s cell-free DNA. Methods: We proposed the multi-Z method which uses 21 z-scores for each autosomal chromosome to detect aneuploidy of the chromosome, while the conventional NIPT method uses only one z-score. To do this, mapped read numbers of a certain chromosome were normalized by those of the other 21 chromosomes. Average and standard deviation (SD), which are used for calculating z-score of each sample, were obtained with normalized values between all autosomal chromosomes of control samples. In this way, multiple z-scores can be calculated for 21 autosomal chromosomes except oneself. Results: Multi-Z method showed 100% sensitivity and specificity for 187 samples sequenced to 3 M reads while the conventional NIPT method showed 95.1% specificity. Similarly, for 216 samples sequenced to 1 M reads, Multi-Z method showed 100% sensitivity and 95.6% specificity and the conventional NIPT method showed a result of 75.1% specificity. Conclusion: Multi-Z method showed higher accuracy and robust results than the conventional method even at low coverage reads.

Two approaches for calculating female fetal DNA fraction in noninvasive prenatal testing based on size analysis of maternal DNA fragments

The concentration of cell-free fetal DNA fragments should be detected before noninvasive prenatal testing(NIPT).The fetal DNA molecules have significant clinical potential in determining the overall performance of NIPT and clinical interpretation.It is important to measure fetal DNA fraction before NIPT.However,there is still little research on how to calculate the concentration of female fetuses.Two estimation approaches were proposed to calculate fetal DNA fraction,including the fragments size-based approach,aneuploid-based approach,which are all approaches based on chromosome segments.Based on high-throughput sequencing data,two approaches to calculate the DNA fraction of male fetuses were tested and obtained the experiment values,which were close to the actual values.The correlation coefficient of fragments size-based approach was 0.9243(P<0.0001)and the aneuploid-based approach reached 0.9339(P<0.0001).We calculated the concentration of female fetuses and obtained remarkable experimental results.We came up with two approaches for calculating the fetal DNA fraction of female fetuses.It provides an important theoretical basis for the detection of female fetal concentration in future clinical diagnosis.

Determination of double-and single-stranded DNA breaks in bovine sperm is predictive of their fertilizing capacity

Background:The analysis of chromatin integrity has become an important determinant of sperm quality.In frozenthawed bovine sperm,neither the sequence of post-thaw injury events nor the dynamics of different types of sperm DNA breaks are well understood.The aim of the present work was to describe such sperm degradation aftermath focusing on DNA damage dynamics,and to assess if this parameter can predict pregnancy rates in cattle.Results:A total of 75 cryopreserved ejaculates from 25 Holstein bulls were evaluated at two post-thawing periods(0-2 h and 2-4 h),analyzing global and double-stranded DNA damage through alkaline and neutral Comet assays,chromatin deprotamination and decondensation,sperm motility,viability,acrosomal status,and intracellular levels of total ROS,superoxides and calcium.Insemination of 59,605 females was conducted using sperm from the same bulls,thus obtaining the non-return to estrus rates after 90 d(NRR).Results showed an increased rate of double-stranded breaks in the first period(0-2 h:1.29±1.01%/h vs.2-4 h:0.13±1.37%/h;P<0.01),whereas the rate of sperm with moderate+high single-stranded breaks was higher in the second period(0-2 h:3.52±7.77%/h vs.2-4h:21.06±11.69%/h;P<0.0001).Regarding sperm physiology,viability decrease rate was different between the two periods(0-2 h:-4.49±1.79%/h vs.2-4 h:-2.50±3.39%/h;P=0.032),but the progressive motility decrease rate was constant throughout post-thawing incubation(0-2 h:-4.70±3.42%/h vs.2-4 h:-1.89±2.97%/h;P>0.05).Finally,whereas no correlations between bull fertility and any dynamic parameter were found,there were correlations between the NRR and the basal percentage of highly-damaged sperm assessed with the alkaline Comet(Rs=-0.563,P=0.003),between NRR and basal progressive motility(Rs=0.511,P=0.009),and between NRR and sperm with high ROS at 4 h post-thaw(Rs=0.564,P=0.003).Conclusion:The statistically significant correlations found between intracellular ROS,sperm viability,sperm motility,DNA damage and chromatin deprotamination suggested a sequence of events all driven by oxidative stress,where viability and motility would be affected first and sperm chromatin would be altered at a later stage,thus suggesting that bovine sperm should be used for fertilization within 2 h post-thaw.Fertility correlations supported that the assessment of global DNA damage through the Comet assay may help predict bull fertility.