非梗阻性无精子症的遗传学基础及研究进展

非梗阻性无精子症(non-obstructive azoospermia,NOA)是导致男性不育的重要原因,影响着约0.6%的男性或10%的不育男性.NOA是一种由多因素引起的具有高度遗传异质性和表型异质性的复杂疾病,其中遗传学病因包括染色体异常、Y染色体微缺失、基因突变以及表观遗传修饰等.目前临床上针对NOA患者的遗传学检测,还仅限于结合附睾和睾丸穿刺活检的核型分析及Y染色体微缺失检测,而且一直缺乏理想的治疗方案.因此,深入解析NOA的具体分子机理,对阐明NOA的病因、提高男性不育的临床诊断和治疗具有重要意义.本综述将从NOA的遗传学基础、NOA的病理特征、临床诊断及治疗等方面进行系统的探讨.     

经皮附睾穿刺取精术在男性不育症诊疗中的应用(附58例报告)

目的:探讨经皮附睾穿刺取精术在男性梗阻性无精子症患者的不育诊治中的应用价值.方法:对58例临床诊断为无精子症的患者,用模型法测量睾丸体积,化学发光法测定血清性激素水平,用7号蝶形针头穿刺附睾头,同时抽吸附睾液.结果:58例无精子症患者中,31例附睾液中可见精子,其中睾丸体积正常者为27例,睾丸体积偏小者为4例;血清FSH水平正常者为28例,血清FSH水平增高者为3例.27例未见精子者,其中睾丸体积正常者为15例,睾丸体积偏小者为12例;血清FSH正常者为17例,血清FSH增高者为10例.结果显示睾丸体积正常的患者,穿刺成功率明显高于睾丸体积偏小者,差异有显著性(P<0.05);血清FSH水平正常的患者,穿刺成功率明显高于FSH水平增高者,差异有显著性(P<0.05).结论:经皮附睾穿刺取精术可简便、快速地鉴别梗阻性和非梗阻性无精子症,也是严重不育症患者获取精子的理想方法.     

基于高通量转录组测序技术的无精子症患者睾丸组织比较转录组分析

本研究探讨不同程度无精子症患者睾丸组织的转录组差异,了解差异表达基因(DEG)在功能、分类和代谢通路的不同,揭示无精子症患者精子发生分子机制,为促进男性不育研究的发展提供理论基础.选取1份非梗阻性无精子症和4份梗阻性无精子症患者睾丸组织样品(从无精子到有精子),进行RNA提取和文库构建,利用Illumina HiSeqTM2500高通量测序,构建无精子症患者睾丸组织转录组文库,并用生物信息学方法进行分析.结果发现,样品比对基因组数据库的平均比对率为94.38%,共检测2 242个属于预测新的蛋白质编码基因的转录本.得到差异表达基因统计结果为:NOA vs. OA1基因上调8 045,下调1 150;OA1 vs. OA2基因上调1 538,下调420;OA2 vs. OA3基因上调1 275,下调1 690;OA3 vs. OA4基因上调1 834,下调1 853.比较5例无精子症睾丸组织的差异基因KEGG,主要富集在RNA降解通路、基底细胞瘤通路、癌通路、黑色素生成通路和调节干细胞多能性等信号通路. PRM1、PRM2、TNP1、UBXN6、CXCL16、NUPR2、CCDC136和...     

特发性少精子症和无精子症与Pygo2基因蛋白编码区SNPs的相关性

男性不育常伴随精子数量减少。Pygo2基因在染色质重塑的伸长精细胞中表达, 其功能受损会导致精子形成阻滞和精子生成减少而引发不育。文章旨在检测引起人特发性少精子症和无精子症的Pygo2基因突变。从77例正常生育力男性和195例特发性少精子症和无精子症患者静脉血提取DNA, 采用聚合酶链式反应-测序方法对Pygo2基因3个蛋白质编码区进行测序对比, 非同义单核苷酸多态性(Single nucleotide polymorphisms, SNPs)位点分别用SIFT、Polyphen-2和 Mutation Taster软件进行诱发蛋白质结构和表型改变的检测和分析。结果表明, 195例患者中, 178例(30例轻度或中度少精子症, 57例重度少精子症和91例无精子症)基因序列分析报告完好, 无精子症中3例患者分别在2个位点(rs61758740, rs141722381)发生了非同义突变SNPs, 重度少精子症中1例患者在位点rs61758741发生了非同义突变, 3个突变位点在SNPs基因数据库都已有报道, 轻度或中度少精子症患者以及正常生育力男性中不存在SNPs。rs61758740可使PYGO2蛋白第141位蛋氨酸(M)变为异亮氨酸(I), rs61758741使PYGO2蛋白第261位碱性赖氨酸(K)变为酸性谷氨酸(E), rs141722381使PYGO2蛋白第240位亲水侧链天冬酰胺(N)变为疏水侧链异亮氨酸(I)。软件分析表明, 在所发现的3个SNP非同义突变位点中, rs141722381引起的单个氨基酸改变会导致PYGO2蛋白空间结构破坏和诱发相关疾病。因此, Pygo2基因蛋白质编码序列区SNPs可能是特发性少精子症和无精子症的诱发因素之一, 导致男性不育。     

氧化应激诱导的自噬与隐睾症及畸形精子症的相关性研究

为了分析氧化应激诱导的自噬与隐睾症及畸形精子症的相关性,首先采用高通量的qPCR array技术检测44个自噬相关基因在隐睾症患者和生育力正常男性的睾丸组织中的表达差异,筛选出11个表达差异显著的自噬相关基因;然后用不同浓度过氧化氢(hydrogen peroxide, H_2O_2)分别处理小鼠精原细胞系GC-1 spg和睾丸支持细胞系TM4,通过3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT)]分析H_2O_2处理对细胞增殖活性的影响,并采用实时荧光定量PCR (real-time quantitative PCR, qRT-PCR)检测11个差异表达的自噬相关基因在细胞氧化应激条件下的表达;最后利用GEO (Gene Expression Omnibus)数据库分析自噬相关基因与畸形精子症的关系。通过综合比较发现, BCL2L1、EIF2AK3在隐睾症、畸形精子症及氧化应激过程中均表达上调,表明这两个基因与男性不育的发生有关,推测其作用机制可能是通过响应氧化应激信号来实现。     

少弱精子症患者精子候选差异蛋白Trx 2、TrxR 1的验证及在少精、弱精子症中的表达研究

目的:根据TMT技术筛选少弱精子症患者精子差异蛋白的结果,选取硫氧还蛋白2(thioredoxin 2,Trx 2)、硫氧还蛋白还原酶1(thioredoxin reductase 1,TrxR 1)进行验证,探讨二者在少精、弱精和少弱精子症中的表达变化及其意义。方法:收集105例少精子症组(O组)、150例弱精子症组(A组)、50例少弱精子症组(OA组)和106例正常精液男性(N组)精液,分离出精子,对少弱精子症进行串联质谱标签(Tandem Mass Tag,TMT)技术蛋白质组学分析,根据少弱精子症组的精子差异蛋白结果选取Trx 2、TrxR 1,通过免疫荧光和免疫印迹方法检测其在O组、A组、OA组的表达情况。结果:TMT技术蛋白质组学结果显示Trx 2为上调差异蛋白(为N组的1.31倍),TrxR 1为下调差异蛋白(为N组的0.82倍)。免疫荧光和免疫印迹结果显示O组、A组、OA组Trx 2表达显著高于N组(P0.05),O组、OA组TrxR 1的表达显著低于N组(P0.05)。二者在OA组的结果与蛋白质组学结果一致。结论:Trx 2、TrxR 1可能在少精、弱精及少弱精子症的发生中起着重要的作用,并有望成为少弱精子症患者精子的候选标志物及治疗靶点。     

POLG1外显子1、3、4、7突变与弱精子症的相关性及其对mtDNA和4977 bp缺失的影响

为了探索POLG1外显子1、3、4、7突变与弱精子症的相关性及对mtDNA序列突变和4 977 bp缺失的影响,按WHO标准收集了120例弱精子症和101例精子活力正常的精液标本,经PCR测序分析POLG1外显子1、3、4、7突变,继而测序检测9例外显子4 c.948 GA突变的弱精子症标本、9例无c.948 GA突变的弱精子症标本和9例正常对照标本的mtDNA全序列,利用巢式PCR技术分析9例c.948 GA突变标本、9例无c.948 GA突变的弱精子症标本和9例对照标本的4 977 bp缺失。结果显示:在120例弱精子症中发现POLG1外显子4 c.948 GA突变9例(7.5%),显著高于对照组(0%,P0.05)。c.948 GA突变组mtDNA全序中突变率与对照组比无统计学差异(P0.05)。作者关注的两组中,突变数有差异的位点累积突变频次突变组显著高于对照组(P0.05),但与无c.948 GA突变的弱精子症标本的累积突变频次比较无统计学意义;突变组mtDNA 4 977 bp缺失率(7/9,77.8%)显著高于对照组(2/9,22.2%,P0.05)和无c.948 GA突变的弱精子症组(2/9,22.2%,P0.05)。以上结果提示,弱精子症的发生可能与POLG1 c.948 GA突变有相关性,弱精子症线粒体DNA某些位点的累积突变率增高,但可能不是POLG1 c.948 GA突变引起;c.948 GA突变可能会增加mtDNA 4 977 bp缺失,从而影响精子线粒体功能,导致精子活动力下降。     

POLG1外显子1、3、4、7突变与弱精子症的相关性及其对mtDNA和4977bp缺失的影响

为了探索POLG1外显子1、3、4、7突变与弱精子症的相关性及对mtDNA序列突变和4977bp缺失的影响,按WHO标准收集了120例弱精子症和101例精子活力正常的精液标本,经PCR测序分析POLG1外显子1、3、4、7突变,继而测序检测9例外显子4c．948G〉A突变的弱精子症标本、9例无C．948G〉A突变的弱精子症标本和9例正常对照标本的mtDNA全序列,利用巢式PCR技术分析94Pie．948G〉A突变标本、9例无C．948G〉A突变的弱精子症标本和9例对照标本的4977bp缺失。结果显示：在120例弱精子症中发现P说G,外显子4c．948G〉A突变9例（7．5％）,显著高于对照组（0％,P〈0．05）。c．948G〉A突变组mtDNA全序中突变率与对照组比无统计学差异俨〉0．05）。作者关注的两组中,突变数有差异的位点累积突变频次突变组显著高于对照组俨〈0．05）,但与无C．948G〉A突变的弱精子症标本的累积突变频次比较无统计学意义;突变组mtDNA4977bp缺失率（7／9,77．8％）显著高于对照组（2／9,22．2％,P〈0．05）和无c．948G〉A突变的弱精子症组（2／9,22．2％,P〈0．05）。以上结果提示,弱精子症的发生可能与POLG,c．948G〉A突变有相关性,弱精子症线粒体DNA某些位点的累积突变率增高,但可能不是POLGlc．948G〉A突变引起;c．948G〉A突变可能会增加mtDNA4977bp缺失,从而影响精子线粒体功能,导致精子活动力下降。     

急性梗阻性化脓性胆管炎患者血清Presepsin、IAP、HMGB1与经皮肝穿刺胆道引流术治疗预后的关系

摘要 目的：探讨急性梗阻性化脓性胆管炎（AOSC）患者血清前蛋白酶（Presepsin）、免疫抑制酸性蛋白（IAP）、高迁移率族蛋白B1（HMGB1）与经皮肝穿刺胆道引流术（PTCD）治疗预后的关系。方法：选取2021年1月～2023年1月保定市第二中心医院收治220例接受PTCD治疗的AOSC患者纳入AOSC组,另选取同期体检健康志愿者88名纳入对照组。根据AOSC患者PTCD术后住院期间是否死亡分为存活组36例和死亡组184例。检测血清Presepsin、IAP、HMGB1水平。采用多因素Logistic回归模型分析影响AOSC患者PTCD治疗预后的因素,受试者工作特征（ROC）曲线分析血清Presepsin、IAP、HMGB1水平对AOSC患者PTCD术后死亡的预测价值。结果：AOSC组血清Presepsin、IAP、HMGB1水平高于对照组（P＜0.05）。220例AOSC患者PTCD术后住院期间死亡率为16.36%（36/220）。多因素Logistic回归分析显示,发病至手术时间≥3 d、术后24 h胆汁引流量＜300 mL、Presepsin升高、IAP升高、HMGB1升高为影响AOSC患者PTCD治疗预后的独立危险因素（P＜0.05）。ROC曲线分析显示,血清Presepsin、IAP、HMGB1水平联合预测AOSC患者PTCD术后死亡的曲线下面积为0.920,大于单一指标预测。结论：AOSC患者血清Presepsin、IAP、HMGB1水平升高,是PTCD治疗预后的独立危险因素。血清Presepsin、IAP、HMGB1水平联合预测AOSC患者PTCD治疗预后的价值较高。     

经皮肝穿刺胆道引流术与逆行胰腺胆管造影术对结石性梗阻性黄疸患者的疗效比较研究

目的:比较经皮肝穿刺胆道引流术(PTCD)与逆行胰腺胆管造影术(ERCP)对结石性梗阻性黄疸患者的治疗效果。方法:选取海军军医大学第三附属医院东方肝胆外科医院于2016年3月～2018年4月间收治的结石性梗阻性黄疸患者80例。按照介入治疗术式的异同将患者分为ERCP组(n=40,给予ERCP治疗)和PTCD组(n=40,给予PTCD治疗),记录两组手术时间、术中出血量、住院费用、住院时间、治疗成功率、黄疸缓解率、并发症发生情况,比较两组术前、术后1 d、术后7 d肝功能指标情况。结果:两组患者手术时间、术中出血量、治疗成功率、黄疸缓解率比较差异无统计学意义(P0.05),ERCP患者住院费用少于PTCD组患者,住院时间亦短于PTCD组患者(P0.05)。两组患者术后1 d、术后7 d丙氨酸转氨酶(ALT)、总胆红素(TBIL)、直接胆红素(DBIL)水平均较术前降低,且两组患者术后7 d上述指标水平低于术后1 d(P0.05),ERCP组术后1 d、术后7 d ALT、TBIL、DBIL水平与PTCD组比较差异无统计学意义(P0.05)。两组患者术后并发症总发生率比较差异无统计学意义(P0.05)。结论:PTCD、ERCP治疗结石性梗阻性黄疸,均能有效改善患者临床症状和肝功能,且手术安全性相当,但ERCP可明显减少住院时间和住院费用。     

Successful microdissection testicular sperm extraction for men with non-obstructive azoospermia

Non-obstructive azoospermia (NOA) is the most severe form of male infertility, defined by lack of spermatozoa in the ejaculate caused by impaired spermatogenesis. The chance of biological fatherhood of these men has been improved since the introduction of microdissection testicular sperm extraction (MD-TESE) combined with intracytoplasmic sperm injection. A thorough patient evaluation preoperatively is essential to recognize any underlying conditions, and to assist in patient counseling on the sperm recovery rate and pregnancy results. This review article summarizes the present data on MD-TESE to reach optimal results is treating men with NOA.     

Follicle-stimulating hormone may predict sperm retrieval rate and guide surgical approach in patients with non-obstructive azoospermia

Testicular sperm aspiration- (TESA) or micro-dissection testicular sperm extraction- (MD-TESE) combined intracytoplasmic sperm injection (ICSI) was the only option for non-obstructive azoospermia (NOA) patients to have a biological offspring and they had different success rates in sperm retrieval. Our study aimed to find predictor(s) for predicting the sperm retrieval rate (SRR) in NOAs and guide clinicians in choosing different surgical approaches, TESA or MD-TESE for NOAs. 294 NOAs who had undergone TESA or MD-TESE were divided into TESA group and MD-TESE group. Depending on sperm retrieval, each group was divided into two subgroups: successful subgroups and failure subgroups. They respectively were 24 cases and 131 cases, 53 cases and 86 cases. Clinical data, including body mass index (BMI), testicular volume, and serum hormone levels, were analyzed in a retrospective manner. The results showed that follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels and SRR were lower in TESA group as compared to these in MD-TESE group, while testicular volume was higher (P < 0.05). The surgical approach of sperm retrieval significantly affected the SRR (P < 0.05). In TESA subgroups, testicular volume, FSH and LH differed significantly (P < 0.05). In MD-TESE subgroups, the level of FSH and LH differed significantly between both groups (P < 0.05). Using logistics regression, we found a negative correlation (β=-0.083) between FSH and the SRR in TESA group but a positive correlation (β = 0.064) in MD-TESE group (P < 0.05). In conclusion, serum FSH level can predict the SRR of NOAs and guide the clinicians while selecting the suitable surgery approach for NOAs.     

Altered microRNA expression in patients with non-obstructive azoospermia

Background  

MicroRNAs (miRNAs), a class of small non-coding RNA molecules, are indicated to play essential roles in spermatogenesis. However, little is known about the expression patterns or function of miRNAs in human testes involved in infertility.     

HLA-DPB1*04:01 allele is associated with non-obstructive azoospermia in Japanese patients

Azoospermia is defined by absence of sperm in the semen and can either be caused by obstruction of the seminal tract (obstructive azoospermia) or by defects in spermatogenesis (non-obstructive azoospermia, NOA). Previous studies reported that specific alleles and single nucleotide polymorphisms (SNPs) in the human leukocyte antigen (HLA) region were associated with NOA in East Asians. We attempt to expand upon previous findings by genotyping more HLA genes and to replicate SNP associations by focusing on Japanese NOA patients. HLA typing of six genes (HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1) was done on 355 NOA patients using SSO-Luminex assay while genotyping of two previously reported SNPs (rs498422 and rs3129878) was done on 443 patients and 544 fertile males using TaqMan assay. Association between the HLA alleles and SNP with NOA was assessed with Chi squared and logistic regression tests. We found that HLA-DPB1*04:01 [corrected p value, P _c 7.13 × 10^?6; odds ratio (OR) 2.52], DRB1*13:02 (P _c 4.93 × 10^?4, OR 1.97), DQB1*06:04 (P _c 8.94 × 10^?4, OR 1.91) and rs3129878 (p value 3.98 × 10^?4; OR 1.32) showed significant association with NOA, however, these loci are in linkage disequilibrium with each other. The conditional logistic regression tests showed that DPB1*04:01 is independently associated with NOA, confirming the involvement of the HLA region in the etiology of NOA in Japanese patients.     

Analysis of seminal plasma from patients with non-obstructive azoospermia and identification of candidate biomarkers of male infertility

Infertility affects approximately 15% of couples with equivalent male and female contribution. Absence of sperm in semen, referred to as azoospermia, accounts for 5-20% of male infertility cases and can result from pretesticular azoospermia, non-obstructive azoospermia (NOA), and obstructive azoospermia (OA). The current clinical methods of differentiating NOA cases from OA ones are indeterminate and often require surgical intervention for a conclusive diagnosis. We catalogued 2048 proteins in seminal plasma from men presented with NOA. Using spectral-counting, we compared the NOA proteome to our previously published proteomes of fertile control men and postvasectomy (PV) men and identified proteins at differential abundance levels among these clinical groups. To verify spectral counting ratios for candidate proteins, extracted ion current (XIC) intensities were also used to calculate abundance ratios. The Pearson correlation coefficient between spectral counting and XIC ratios for the Control-NOA and NOA-PV data sets is 0.83 and 0.80, respectively. Proteins that showed inconsistent spectral counting and XIC ratios were removed from analysis. There are 34 proteins elevated in Control relative to NOA, 18 decreased in Control relative to NOA, 59 elevated in NOA relative to PV, and 16 decreased in NOA relative to PV. Many of these proteins have expression in the testis and the epididymis and are linked to fertility. Some of these proteins may be useful as noninvasive biomarkers in discriminating NOA cases from OA.     

Chromosomal evaluation in a group of Tunisian patients with non-obstructive azoospermia and severe oligozoospermia attending a Tunisian cytogenetic department

Male infertility is the cause in half of all childless partnerships. Numerous factors contribute to male infertility, including chromosomal aberrations and gene defects. Few data exist regarding the association of these chromosomal aberrations with male infertility in Arab and North African populations. We therefore aimed to evaluate the frequency of chromosomal aberrations in a sample of 476 infertile men with non-obstructive azoospermia (n = 328) or severe oligozoospermia (n = 148) referred for routine cytogenetic analysis to the department of cytogenetics of the Pasteur Institute of Tunis. The overall incidence of chromosomal abnormalities was about 10.9%. Out of the 52 patients with abnormal cytogenetic findings, sex chromosome abnormalities were observed in 42 (80.7%) including Klinefelter syndrome in 37 (71%). Structural chromosome abnormalities involving autosomes (19.2%) and sex chromosomes were detected in 11 infertile men. Abnormal findings were more prevalent in the azoospermia group (14.02%) than in the severe oligozoospermia group (4.05%). The high frequency of chromosomal alterations in our series highlights the need for efficient genetic testing in infertile men, as results may help to determine the prognosis, as well as the choice of an assisted reproduction technique. Moreover, a genetic investigation could minimize the risk of transmitting genetic abnormalities to future generations.     

Influence of inbreeding on reproductive performance, ejaculate quality and testicular volume in the dog

The influence of fast freezing and thawing on bovine embryos at different stages of development was investigated. A total of 20 day-7 embryos and 37 day-8 embryos were thawed and classified morphologically before being transferred nonsurgically to synchronized recipients. Ninety percent (18 20 ) of the day 7 embryos (late morulae and blastocysts) were classified as transferable and a pregnancy rate of 52.9% (9 17 ) was obtained with these embryos. Seventy eight percent (29 37 ) of the day 8 embryos (expanded blastocysts) were classified as transferable, but only 24.1% (7 29 ) of these embryos resulted in pregnancy. The best pregnancy rate was obtained with the blastocysts of day 7 (5 8 or 62.5%), which compares favorably with that of freshly collected embryos. It is suggested that the low pregnancy rate found in the day 8 embryos is related to ultrastructural damages of the desmosomes and tonofilaments within the trophoblast layer, which results in a disturbance of the normal hatching process.     

Increased growth hormone responses to growth hormone releasing hormone and thyrotropin releasing hormone in patients with metastatic testicular cancer

In 16 patients with metastatic testicular cancer and 10 age matched male control subjects growth hormone (GH) responses to growth hormone releasing hormone (GHRH; 1 microgram/kg body weight iv.) and thyrotropin releasing hormone (TRH; 200 micrograms iv.) were measured. Basal GH levels and GH levels following stimulation with GHRH or TRH were significantly increased in cancer patients compared to control subjects. 9 patients with testicular cancer were studied both in the stage of metastatic disease and after they had reached a complete remission. In complete remission GH responses to GHRH tended to decrease but the differences did not reach statistical significance. Our data suggest an alteration of hypothalamic and/or pituitary regulation of GH secretion in patients with metastatic testicular cancer.     

Meiotic studies of infertile men in case of non-obstructive azoospermia with normal karyotype and no microdeleted Y-chromosome precise the clinical couple management

To identify meiotic criteria for infertility management in non-obstructive azoospermic men, a prospective and multicentric study was organized in Andrological Departments of Paris (France), Roma (Italy) and Budapest (Hungary). In 117 non-obstructive azoospermic men with normal karyotype and no Y-chromosome microdeletion, histology and meiotic studies on bilateral bipolar testicular biopsies were done. Histologically, 40 patients (34%) presented spermatocyte or spermatid arrest, 39 (33%) hypospermatogenesis whereas no meiotic cell could be observed in the remaining patients (33%). Cytogenetically, meiotic figures could only be obtained from the two first histological groups. Meiotic abnormalities were observed in a total of 44 patients (37.6%) including nine patients (7.7%) with severe class I and class IIB anomalies and 19 patients (16.2%) with class IIC environmentally linked meiotic abnormalities. These results provided essential clues for an accurate clinical management. For patients with no meiotic figures and patients with class I and class IIB anomalies, an hormonal stimulation is illusory and a sperm gift should be directly proposed. An hormonal stimulation should be proposed to all the other patients, either directly or following the treatment of the testicular microenvironment for the patients presenting class IIC anomalies. The genetic risk and possibility of prenatal chromosomal analysis in case of pregnancy should be clearly exposed to all the couples in all the cases where type IIA, III or IV anomalies are present. This therapeutical strategy has been applied to all the patients in our series.