精子DNA完整性及对辅助生殖技术治疗结局的影响

精子遗传物质(包括核DNA和线粒体DNA)易受各种因素影响发生损伤,其损伤机制可能涉及染色质组装异常、氧化应激、凋亡等。精子DNA完整性是成功受精和胚胎发育的基本保证,通过检测精子DNA损伤,评估精子质量,选择优质精子用于临床,可提高辅助生殖技术(ART)成功率,减少技术失败引起的经济、社会和情感问题。本综述主要探讨男性因素不育患者精子DNA完整性及其损伤的可能机制,以及对ART治疗结局的影响。     

中药生精散对精子DNA完整性的影响及其在IVF-ET治疗中的临床疗效

目的:观察中药生精散对不育患者精子DNA完整性的影响及其在IVF-ET治疗中的临床疗效。方法:因少、弱精子症行IVF/ICSI-ET治疗的患者49例,随机分为中药组(22例,服用生精散治疗)和对照组(27例,未经生精散治疗)。观察比较中药组和对照组精液常规分析(精子密度、活率、活力)、DNA碎片指数(DNA fragmentation index,DFI)、受精率、卵裂率、种植率及临床妊娠率。结果:中药治疗后精子活率及活力显著提高(P<0.05),且中药组DFI值较治疗前及对照组明显下降(P<0.05)。中药组胚胎种植率(31.11%)及临床妊娠率(45.45%)明显高于对照组(13.73%和18.51%)(P<0.05)。结论:中药生精散可通过降低不育患者精子DNA损伤程度,改善精子质量,提高IVF-ET的治疗效果。     

低渗介导抽提牛精子线粒体DNA

目的:建立一种通过低渗介导的简便、有效的抽提冷冻牛精子线粒体DNA(mtDNA)的方法。方法:低渗处理牛精子,结合去污剂破膜释放精子线粒体,饱和酚去蛋白,无水乙醇沉淀DNA,设计线粒体特异性引物进行PCR分析以及通过NlaⅢ酶切对抽提获得的mtDNA作酶切鉴定,并以水解法作对照。结果:低渗法获得的mtDNA的PCR产物电泳分析和酶切鉴定都清楚地显示与预期相符的目的条带,而对照组水解法抽提冷冻牛精子mtDNA存在稳定性差及重复性差等缺点。结论:低渗介导是一种抽提精子mtDNA的有效方法。     

精子DNA完整性的检测及其在辅助生殖技术中的作用

辅助生殖技术(ART)中,卵胞浆内单精子注射(ICSI)的出现开创了治疗男性不育的新局面,同时使得精子DNA完整性倍受人们关注。精子DNA完整性对自然受孕、人工授精、胚胎、胎儿、婴儿乃至成人的发育至关重要。研究表明精子DNA完整性是优于常规精液分析的独立参数,与ART结局有很高的相关性。因此精子DNA完整性检测对于ART有很重要的意义。目前有多种方法检测精子DNA完整性。     

影响精子DNA损伤因素研究进展

精子DNA损伤是引起不育的原因之一,同时可以增加遗传缺陷风险,因此对精子DNA损伤的研究已成为生殖医学的热点之一。精子DNA检测在评价男性不育患者时越来越受到重视。精子中的DNA包括核DNA和线粒体DNA,它们均易受损伤,损伤机制可能有氧化应激、精子染色质组装缺陷、异常凋亡。精子DNA损伤与很多因素有关,如化学治疗和放射治疗、高龄和生活方式、睾丸温度升高、吸烟与生殖道炎症、环境毒素、精索静脉曲张、激素等,它是多因素共同作用的结果。     

精子DNA损伤对生殖结局的影响及临床意义

精子DNA易受各种因素影响而被损伤,人类精子DNA损伤的机制,包括在精子生成过程及在生殖道转运过程中的损伤。其损伤机制可能涉及精子发生过程中染色质组装异常,精细管上皮细胞凋亡,精子由生精管到附睾迁移过程中的氧化作用等。目前用于检测精子DNA损伤的方法及决定精子DNA损伤的预测价值在诊断和治疗不孕症中有参考意义。越来越多的研究表明,精子DNA损伤影响受精和胚胎发育,最终导致不孕不育、流产、死产、子代智力低下和染色体疾病。与传统检测方法相比,检测精子DNA的损伤可能更有临床意义。     

柠檬酸合成酶对卵巢癌线粒体DNA相对拷贝数的影响

目的:探讨柠檬酸合成酶(CS)对卵巢癌线粒体DNA(mtDNA)相对拷贝数的影响。方法:Real-time PCR法检测卵巢良性肿瘤组织、卵巢癌组织及人正常卵巢上皮细胞(HOSE)和卵巢癌细胞株SKOV3、A2780中CS mRNA水平,Western blot法检测标本中CS蛋白水平。siRNA干扰SKOV3和A2780中CS表达,Real-time PCR检测干扰前后mtDNA相对拷贝数(mtND2/HBB)和线粒体转录因子A(TFAM)的变化,Western blot法检测p-ERK和p-p38水平。结果:卵巢癌组织中CS、mtDNA相对拷贝数(mtND2/HBB)和TFAM水平高于良性卵巢肿瘤组织;SKOV3和A2780细胞株中CS高于正常卵巢上皮细胞。siRNA干扰SKOV3、A2780中CS后,mtDNA相对拷贝数和TFAM水平降低,p-ERK和p-p38也降低。结论:CS在卵巢癌组织和卵巢癌细胞株中呈高表达,其水平影响卵巢癌细胞中mtDNA相对拷贝数,其机制可能与ERK/p38通路相关。     

精子核DNA完整性测定方案的优化及其在辅助生殖技术中的应用价值

目的:对现有精子核DNA完整性测定方案进行优化,并探讨其在辅助生殖技术(ART)中的应用价值。方法:选择2021年1月1日至2022年12月8日于江西中医药大学附属生殖医院就诊的拟接受体外受精-胚胎移植(IVF-ET)助孕的194对夫妇作为研究对象。采集男方的精液作为对照组(n=194),同一精液经双层密度梯度离心法优化处理后精子混合液作为观察组(n=194)。根据精子核DNA碎片率(DFI)测定结果将对照组及观察组各分为3个亚组,对照A组和观察A组：DFI<15%,对照B组和观察B组：DFI 15%～30%,对照C组和观察C组：DFI≥30%。对观察组与对照组的DFI值及各亚组间的助孕及妊娠情况进行比较。结果:(1)观察组DFI明显低于对照组,差异有统计学意义[(13.55±10.17)%vs.(18.56±11.54)%,P<0.05]。(2)6个亚组间的受精率、卵裂率及优胚率两两比较,差异无统计学意义(P>0.05)。(3)6个亚组的妊娠率和着床率比较,差异有统计学意义(P<0.05);其中,对照A组、对照B组、观察A组、观察B组4组的临床妊娠率(均在65...     

不明原因重复性自然流产妇女配偶精子DNA完整性

目的:探讨精子DNA完整性与重复性自然流产(RSA)的关系。方法:85例不明原因RSA妇女配偶(RSA组)和50例已生育的成年健康男性(对照组)的精液,应用精子染色质扩散实验(SCD)检测精子DNA完整性。将RSA组根据1年后怀孕结果分为3个亚组:怀孕组(30例)、流产组(26例)、未孕组(29例)。结果:RSA妇女配偶DNA损伤精子的百分率(14.6±6.9)%与对照组的(12.9±3.8)%相比无统计学差异(P0.05)。DNA损伤精子百分率大于20%视为精子DNA完整性异常,则有17.6%的RSA患者配偶的精子DNA完整性异常,6%的正常生育男性精子DNA完整性异常,但差异无统计学意义(P0.05)。怀孕组、流产组、未孕组配偶的DNA损伤精子百分率分别为(12.4±5.3)%,(14.6±6.5)%和(16.8±8.1)%,未孕组与对照组比较,差异有统计学意义(P0.05),怀孕组、流产组与对照组比较,差异无统计学意义(P0.05)。结论:精子DNA完整性异常与RSA继发不育有关,有必要筛查RSA患者配偶的精子DNA完整性。     

受精后精子线粒体的命运

人们普遍认为线粒体基因的遗传方式为母系遗传,精子线粒体在各种动物胚胎中的命运在很大程度上取决于核基因组与线粒体基因组间的相容性,而与精子线粒体相关的有些细胞组分被观察到能够在受精过程中得到传递,并且父系线粒体的遗传也在患者和种间杂交中被发现。然而用ICSI与IVF产生的正常后代中并没有发现父系mtDNA,反而在异常胚胎中检测到父系mtDNA.。本文通过对多种动物(如人、牛、小鼠和大鼠)精子线粒体在受精后的生物学特征进行总结,分析了其消失的可能机制及产生这种结果的原因。     

Influence of microsurgical varicocelectomy on human sperm mitochondrial DNA copy number: a pilot study

Background

There is good evidence to show that varicocele repair can improve conventional sperm parameters, as well as, sperm DNA integrity, in infertile men with a clinical varicocele.

Objective

To examine the effect of varicocelectomy on sperm quality, specifically, sperm nuclear chromatin integrity and sperm mitochondrial DNA (mtDNA) copy number.

Design, Setting, and Participants

A prospective study done between March 2007 and January 2008. We evaluated a consecutive series of infertile men (n = 14) presenting to Ovo clinic with one year or more history of infertility, a clinically palpable varicocele and poor motility (<25 % rapid progressive and <50 % progressive).

Surgical Procedure

Microsurgical sub-inguinal varicocelectomy.

Outcome Measurements and Statistical Analysis

Conventional sperm parameters, sperm mtDNA copy number (by real time PCR) and sperm chromatin structure assay (SCSA) parameters (%DFI,% HDS) before and 4 months after microsurgical varicocelectomy.

Results and Limitations

Sperm concentration and SCSA parameters (%DFI and %HDS) improved significantly after surgery (P < 0.05). Sperm mitochondrial DNA copy number decreased significantly after surgery (27 ± 30 to 9 ± 6 copies per sperm, respectively, P = 0.032). There was a significant negative correlation between mitochondrial DNA copy number and sperm motility (r = − 0.71, P = 0.002).

Conclusion

These findings support the concept that correction of a varicocele can improve spermatogenesis and sperm function, as mitochondrial DNA copy number has been suggested to reflect the efficiency of spermatogenesis and has been inversely related to sperm motility.     

Genetic variations related to maternal whole blood mitochondrial DNA copy number: a genome-wide and candidate gene study

We conducted genome-wide (GWAS) and candidate gene association studies of maternal mitochondrial DNA copy number. Maternal peripheral blood was collected during labor and delivery admission from 471 participants of a placental abruption case-control study conducted in Lima, Peru. Single nucleotide polymorphism (SNP) genotyping was performed using the Illumina Cardio-Metabo Chip. Whole blood mitochondrial DNA (mtDNA) copy number was measured using qRT-PCR techniques. We evaluated 119,629 SNPs in the GWAS and 161 SNPs (in 29 mitochondrial biogenesis and oxidative phosphorylation genes) in the candidate association study. Top hits from GWAS and the candidate gene study were selected to compute weighted genetic risk scores (wGRS). Linear regression models were used to calculate effect size estimates and related nominal p values. The top hit in our GWAS was chr19:51063065 in FOXA3 (empirical p values?=?2.20e???6). A total of 134 SNPs had p values?p values?=?6.32e???6) and chr19:51083059 in MYPOP (p values?=?3.23e???5). In the candidate association study, several SNPs in PPARG, PRKCA, SP1 and THRB were associated with mtDNA copy number (p values?β?=?0.49, 95% CI:0.38–0.60, p?相似文献   

Altitude can alter the mtDNA copy number and nDNA integrity in sperm

Objective  

Mitochondria are factories for energy production and genetic alterations in mtDNA will directly impact OXPHOS function. The copy number of mtDNA (i.e., the number of mtDNA per spermatozoon) is one of the major mitochondrial genetic features. Besides mtDNA copy number, the change of either mtDNA or nDNA integrity is another important factor causing asthenospermia, or poor sperm motility in infertile men. In this study, we investigated the mtDNA copy number and the integrities of mtDNA and nDNA respectively in semen samples from different donors at 5,300 m altitudes.     

Modern human sperm freezing: Effect on DNA,chromatin and acrosome integrity

Objective

Presence of vitrification method in sperm freezing and the introduction of solid surface vitrification beside rapid freezing in vapour, opens an easy and safe way to help infertility centres. While the effects of cryopreservation on motility, morphology and viability of sperm are documented, the question of the probable alteration of sperm DNA, chromatin and acrosome integrity after freezing and thawing procedures in different methods is still controversial.

Sperm mitochondrial DNA depletion in men with asthenospermia

OBJECTIVE: To determine the content of sperm mitochondrial DNA (mtDNA) in patients with asthenospermia or with poor sperm motility. DESIGN: Analysis of the content of mtDNA as the ratio between the amount of mtDNA and nuclear DNA by using a new concurrent polymerase chain reaction. SETTING: University hospital infertility center. PATIENT(S): Eighty-six men who sought infertility therapy. INTERVENTION(S): Moving characteristics of sperm were examined with a computer-assisted semen analyzer. MAIN OUTCOME MEASURE(S): Sperm samples were classified into two groups, one with asthenospermia and the other with normal moving characteristics. The content of mtDNA in sperm was determined by polymerase chain reaction. We analyzed the mitochondrial mass by MitoTracker Green staining and analyzed DNA content with propidium iodide staining by flow cytometry. RESULT(S): A decrease in sperm mtDNA content was detected in patients with asthenospermia or with poor sperm motility (<20% motility). The relative mtDNA contents in the asthenospermia and normal groups were 7.2 +/- 1.3 (mean +/- SD, n = 23) and 74.1 +/- 2.0 (n = 29), respectively. Lower intensities of propidium iodide staining were detected in patients with asthenospermia or poor motility, but there was no significant difference in MitoTracker Green staining between the sperm with different motility. CONCLUSION(S): We suggest that mtDNA content may serve as a useful indicator of sperm quality and that mtDNA depletion may play an important role in the pathophysiology of some male infertility.     

Cell-free DNA test for pathogenic copy number variations: A retrospective study

ObjectiveTo evaluate the detection rate (DR) by prenatal cell-free DNA test for pathogenic copy number variations (CNVs)＞2 Mb among pregnancies with fetal ultrasound abnormalities.Materials and methodsThis was a retrospective study on 29 pregnant women with fetuses diagnosed as microdeletion/microduplication syndromes by prenatal chromosome microarray analysis (CMA). Cell-free DNA from the maternal plasma was sequenced on the NextSeq CN500 sequencer. The quality standard of unique map reads in a single sample was greater than 10 M and only gains and losses of more than 2 Mb were reported.ResultsA total of 24 CNVs were identified by cell-free DNA test among the 21 fetuses with pathogenic CNVs identified by prenatal CMA, including 20 consistent CNVs and 4 inconsistent CNVs. Overall, the DR of cell-free DNA test for pathogenic CNVs ＞2 Mb was 69%. Microdeletions or microduplications at 22q11.2 were the most common CNVs, with a DR of 4/5 (80%) and 3/4 (75%) respectively.ConclusionCell-free DNA test exhibited a moderate DR for pathogenic CNVs ＞2 Mb among fetuses with ultrasound abnormalities. Cell-free DNA test could provide an opportunity for early screening before the appearance of abnormalities on fetal ultrasound, while further clinical data and cost-effectiveness assessment are needed.