A novel homozygous frameshift variant in DNAH8 causes multiple morphological abnormalities of the sperm flagella in a consanguineous Pakistani family

Multiple morphological abnormalities of the sperm flagella(MMAF)is a severe form of asthenozoospermia categorized by immotile spermatozoa with abnormal flagella in ejaculate.Whole-exome sequencing(WES)is used to detect pathogenic variants in patients with MMAF.In this study,a novel homozygous frameshift variant(c.6158_6159insT)in dynein axonemal heavy chain 8(DNAH8)from two infertile brothers with MMAF in a consanguineous Pakistani family was identified by WES.Reverse transcription-polymerase chain reaction(RT-PCR)confirmed DNAH8 mRNA decay in these patients with the DNAH8 mutation.Hematoxylin–eosin staining and transmission electron microscopy revealed highly divergent morphology and ultrastructure of sperm flagella in these patients.Furthermore,an immunofluorescence assay showed the absence of DNAH8 and a reduction in its associated protein DNAH17 in the patients'spermatozoa.Collectively,our study expands the phenotypic spectrum of patients with DNAH8-related MMAF worldwide.

Absence of murine CFAP61 causes male infertility due to multiple morphological abnormalities of the flagella

Impaired flagellar development and impaired motility of sperm is a cause of infertility in males. Several genes, including those of the AKAP, CCDC, CFAP, and DNAH families, among others, are involved in the‘‘multiple morphological abnormalities of the flagella"(MMAF) phenotype;these are the most common causes of male infertility. The Cilia-and flagella-associated protein(CFAP) family includes six members reported to cause MMAF phenotypes: CFAP43, CFAP44, CFAP69, CFAP65, CFAP70, and CFAP251. Here, we found that cilia-and flagella-associated protein 61(Cfap61) is highly expressed specifically in murine testes and show that the Cfap61-knockout male mice demonstrate MMAF phenotype, including sperm with short, coiled, and irregular flagella. Deletion of Cfap61 resulted in severe morphological and behavior abnormalities in sperm, reduced total sperm counts, impaired sperm motility, and led to male infertility.Notably, absence of Cfap61 impaired sperm flagella ultrastructural abnormalities on account of numerous distortions in multiple flagellum components. Immunostaining experiments in wild-type mice and healthy adult humans indicated that Cfap61 is initially localized at the neck of sperm, where it potentially functions in flagellum formation, and is later localized to the midpiece of the sperm. Thus, our study provides compelling evidence that dysregulation of Cfap61 affects sperm flagellum development and induces male infertility in mice. Further investigations of the CFAP61 gene in humans alongside clinical evidence showing MMAF phenotype in humans should contribute to our understanding of developmental processes underlying sperm flagellum formation and the pathogenic mechanisms that cause male infertility.

Novel mutation in ODF2 causes multiple morphological abnormalities of the sperm flagella in an infertile male

Numerous genes have been associated with multiple morphological abnormalities of the sperm flagella(MMAF),which cause severe asthenozoospermia and lead to male infertility,while the causes of approximately 50%of MMAF cases remain unclear.To reveal the genetic causes of MMAF in an infertile patient,whole-exome sequencing was performed to screen for pathogenic genes,and electron microscope was used to reveal the sperm flagellar ultrastructure.A novel heterozygous missense mutation in the outer dense fiber protein 2(ODF2)gene was detected,which was inherited from the patient’s mother and predicted to be potentially damaging.Transmission electron microscopy revealed that the outer dense fibers were defective in the patient’s sperm tail,which was similar to that of the reported heterozygous Odf2 mutation mouse.Immunostaining of ODF2 showed severe ODF2 expression defects in the patient’s sperm.Therefore,it was concluded that the heterozygous mutation in ODF2 caused MMAF in this case.To evaluate the possibility of assisted reproductive technology(ART)treatment for this patient,intracytoplasmic sperm injection(ICSI)was performed,with the help of a hypo-osmotic swelling test and laser-assisted immotile sperm selection(LAISS)for available sperm screening,and artificial oocyte activation with ionomycin was applied to improve the fertilization rate.Four ICSI cycles were performed,and live birth was achieved in the LAISS-applied cycle,suggesting that LAISS would be valuable in ART treatment for MMAF.

Novel biallelic loss-of-function mutations in CFAP43 cause multiple morphological abnormalities of the sperm flagellum in Pakistani families

Multiple morphological abnormalities of the sperm flagella(MMAF)is a specific type of asthenoteratozoospermia,presenting with multiple morphological anomalies in spermatozoa,such as absent,bent,coiled,short,or irregular caliber flagella.Previous genetic studies revealed pathogenic mutations in genes encoding cilia and flagella-associated proteins(CFAPs;e.g.,CFAP43,CFAP44,CFAP65,CFAP69,CFAP70,and CFAP251)responsible for the MMAF phenotype in infertile men from different ethnic groups.However,none of them have been identified in infertile Pakistani males with MMAF.In the current study,two Pakistani families with MMAF patients were recruited.Whole-exome sequencing(WES)of patients and their parents was performed.WES analysis reflected novel biallelic loss-of-function mutations in CFAP43 in both families(Family 1:ENST00000357060.3,p.Arg300Lysfs*22 and p.Thr526Serfs*43 in a compound heterozygous state;Family 2:ENST00000357060.3,p.Thr526Serfs*43 in a homozygous state).Sanger sequencing further confirmed that these mutations were segregated recessively in the families with the MMAF phenotype.Semiquantitative reverse-transcriptase polymerase chain reaction(qRT-PCR)was carried out to detect the effect of the mutation on mRNA of the affected gene.Previous research demonstrated that biallelic loss-of-function mutations in CFAP43 accounted for the majority of all CFAP43-mutant MMAF patients.To the best of our knowledge,this is the first study to report CFAP43 biallelic loss-of-function mutations in a Pakistani population with the MMAF phenotype.This study will help researchers and clinicians to understand the genetic etiology of MMAF better.

Gene-knockout by iSTOP enables rapid reproductive disease modeling and phenotyping in germ cells of the founder generation

Cytosine base editing achieves C·G-to-T·A substitutions and can convert four codons(CAA/CAG/CGA/TGG)into STOP-codons(induction of STOP-codons,iSTOP)to knock out genes with reduced mosaicism.iSTOP enables direct phenotyping in founders’somatic cells,but it remains unknown whether this works in founders’germ cells so as to rapidly reveal novel genes for fertility.Here,we initially establish that iSTOP in mouse zygotes enables functional characterization of known genes in founders’germ cells:Cfap43-iSTOP male founders manifest expected sperm features resembling human“multiple morphological abnormalities of the flagella”syndrome(i.e.,MMAF-like features),while oocytes of Zp3-iSTOP female founders have no zona pellucida.We further illustrate iSTOP’s utility for dissecting the functions of unknown genes with Ccdc183,observing MMAF-like features and male infertility in Ccdc183-iSTOP founders,phenotypes concordant with those of Ccdc183-KO offspring.We ultimately establish that CCDC183 is essential for sperm morphogenesis through regulating the assembly of outer dynein arms and participating in the intra-flagellar transport.Our study demonstrates iSTOP as an efficient tool for direct reproductive disease modeling and phenotyping in germ cells of the founder generation,and rapidly reveals the essentiality of Ccdc183 in fertility,thus providing a time-saving approach for validating genetic defects(like nonsense mutations)for human infertility.

In sight on multiple morphological abno rmalities of sperm flagella in male infertility:what is new?

The syndrome of multiple morphological abnormalities of the sperm flagella(MMAF)is a specific kind of asthenoteratozoospermia with a mosaic of flagellar morphological abnormalities(absent,short,bent,coiled,and irregular flagella).MMAF was proposed in 2014 and has attracted increasing attention;however,it has not been clearly understood.In this review,we elucidate the definition of MMAF from a systematical view,the difference between MMAF and other conditions with asthenoteratozoospermia or asthenozoospermia(such as primary mitochondrial sheath defects and primary ciliary dyskinesia),the knowledge regarding its etiological mechanism and related genetic findings,and the clinical significance of MMAF for intracytoplasmic sperm injection and genetic coun sell ng.This review provides the basic kno wledge for MMAF and puts forward some suggestions for further investigations.

特殊类型畸精子症行卵细胞胞质内单精子注射的治疗结局分析

目的:特殊类型畸精子症是男性不育中罕见病和单基因遗传疾病,本文探讨其卵细胞胞质内单精子注射(ICSI)的治疗结局。方法:回顾性分析2011年1月至2021年1月于江苏省人民医院生殖医学中心实施ICSI治疗的特殊类型畸精子症患者,根据精子形态分为三组(圆头精子症、无头精子症和精子鞭毛多发形态异常),三组患者基因检测均发现致病或可能致病的基因突变。比较三组患者间的临床和实验室数据(年龄、BMI、精子参数、成熟卵子数、受精率、优质胚胎率、临床妊娠率、活产率和自然流产率)差异。结果:三组共34对患者夫妇,三组患者间年龄,BMI,MII卵子数差别无统计学意义(P>0.05),圆头精子症的精子浓度和精子活力显著高于其他两组(P<0.01),圆头精子症组的受精率显著低于其他两组(P<0.01)。三组共34个取卵周期,其中4个取卵周期未形成可移植胚胎,形成可移植胚胎的30个取卵周期在第一个胚胎移植周期中获得22例临床妊娠,最终活产20例,自然流产2例。无头精子症和精子鞭毛多发形态异常两组的临床妊娠率和活产率显著高于圆头精子症组(P<0.01),三组间自然流产率没有统计学差异(P>0.05)。结论:圆头精子症患者实施人工卵母细胞激活依然有部分患者完全不受精或受精率低下。精子鞭毛多发形态异常和无头精子症患者可以通过ICSI取得相对较好的妊娠结局。

精子发生障碍的遗传学研究进展

育龄人群中约15%的夫妻被不孕不育困扰,其中男方因素导致的不孕不育约占50%。男性不育通常由精子发生障碍导致,呈现为少、弱、畸形精子症,最严重的是无精子症。本文以精子发生障碍为主线,重点综述了非梗阻性无精子症和畸形精子症的遗传学病因研究。近年来,随着高通量芯片和测序技术的快速发展,无精子症和畸形精子症的遗传学因素得以深入的揭示与解析。围绕无精子症,全基因组关联研究与高通量测序研究揭示了一批非梗阻性无精子症的风险位点和致病基因;围绕畸形精子症,全外显子测序等研究鉴定了一系列致病基因,极大地丰富了精子鞭毛多发性形态异常等精子畸形的遗传学病因。大量致病基因的发现,促进了男性不育病理机制的阐明。全面而深入地了解精子发生障碍中的遗传因素,对男性不育的诊断、临床治疗和遗传咨询具有重要的意义。

CFAP65基因突变导致精子鞭毛多发形态异常的遗传学初步研究

目的:探寻精子鞭毛多发形态异常(MMAF)可能的致病基因。方法:通过对1例典型的MMAF患者进行全外显子组测序(WES),分析可能的致病基因;运用扫描电镜和透射电镜观察MMAF患者精液样本,明确其鞭毛超微结构特点;通过精子免疫荧光技术分析cilia and flagella-associated protein 65(CFAP65)在精子发生过程中的表达模式。结果:该例患者存在CFAP65基因的一个纯合致病性突变c.2675G>A(p.Trp892*);扫描电镜发现该患者精子具有典型的MMAF特征,即表现为无尾,折尾,卷尾,短尾或不规则尾巴;透射电镜发现患者精子鞭毛"9+2"结构缺失和紊乱:精子鞭毛纤维鞘组装异常,伴有中心微管缺失和动力蛋白臂缺失。细胞免疫荧光提示该CFAP65基因在小鼠各级生殖细胞均有表达。结论:CFAP65基因参与了精子鞭毛结构的组装,其突变可引起MMAF表型而导致男性不育。

外层致密纤维在Akap4基因缺陷致小鼠精子尾部多种形态异常中的作用

目的:探讨外层致密纤维(outer dense fobres,ODF)在Akap4基因缺陷引起小鼠精子尾部多种形态异常中的作用。方法:通过基因编辑技术构建Akap4基因缺陷小鼠模型。实验分为2组:成年Akap4基因缺陷雄鼠为实验组(n=7),成年野生型雄鼠为对照组(n=7),比较2组小鼠的体重和睾丸重量;采用计算机辅助精液分析(computer aided sperm analysis,CASA)检测精子活动力,改良巴氏染色(modified pap staining)检测精子形态学,扫描及透射电镜观察精子超微结构,免疫荧光检测精子尾部蛋白表达及定位,睾丸切片HE及PAS染色检测睾丸生精功能,透射电镜观察曲精细管超微结构。结果:Akap4基因缺陷小鼠精子总活动力为8.81%,明显低于野生型小鼠(P<0.01),且无正常形态精子,尾部缩短与尾部卷曲比例达91.18%,明显高于野生型小鼠(P<0.01),睾丸重量、睾丸生精功能、精子数量2组比较差异无统计学意义(P>0.01);Akap4基因缺陷精子的纤维鞘纵柱缺失,横肋柱结构部分残留,主段的3和8号ODF排列紊乱,与ODF2蛋白定位结果相符;睾丸中精子纤维鞘发育障碍,无正常纤维鞘结构形成,但未见异常膨大的精子尾部。结论:Akap4基因缺陷使纤维鞘横肋发育不良,纵肋的缺失引起3和8号ODF分离,"9+2"微管结构紊乱,使主段管腔异常膨大,导致小鼠精子尾部多种形态异常。

Coiled-coil domain-containing 38 is required for acrosome biogenesis and fibrous sheath assembly in mice

During spermiogenesis,haploid spermatids undergo dramatic morphological changes to form slender sperm flagella and cap-like acrosomes,which are required for successful fertilization.Severe deformities in flagella cause a male infertility syndrome,multiple morphological abnormalities of the flagella(MMAF),while acrosomal hypoplasia in some cases leads to sub-optimal embryonic developmental potential.However,evidence regarding the occurrence of acrosomal hypoplasia in MMAF is limited.Here,we report the generation of base-edited mice knocked out for coiled-coil domain-containing 38(Ccdc38)via inducing a nonsense mutation and find that the males are infertile.The Ccdc38-KO sperm display acrosomal hypoplasia and typical MMAF phenotypes.We find that the acrosomal membrane is loosely anchored to the nucleus and fibrous sheaths are disorganized in Ccdc38-KO sperm.Further analyses reveal that Ccdc38 knockout causes a decreased level of TEKT3,a protein associated with acrosome biogenesis,in testes and an aberrant distribution of TEKT3 in sperm.We finally show that intracytoplasmic sperm injection overcomes Ccdc38-related infertility.Our study thus reveals a previously unknown role for CCDC38 in acrosome biogenesis and provides additional evidence for the occurrence of acrosomal hypoplasia in MMAF.

精子鞭毛轴丝结构相关基因异常与弱精子症

精子鞭毛轴丝是精子运动的主要动力来源,参与鞭毛组装和运动调控的基因变异可导致精子活力降低,从而引起弱精子症(asthenozoospermia,ASZ)。常见的弱精子症包括两大类:(1)精子鞭毛在光学显微镜下无明显畸形,(2)精子鞭毛多发形态异常(multiple morphological abnormalities of the sperm flagella,MMAF)。弱精子症主要由轴丝组分编码基因变异所致,在过去的十年里,在揭示致病基因方面取得了显著进展。在MMAF的遗传研究领域,中国和法国是两个涉及比较广的国家。通过系统文献检索和Meta分析中国和法国关于MMAF的基因变异研究,纳入1796名不育男性参与者,结果表明,在中国的弱精子症患者中,DNAH1基因的突变比例显著高于法国(OR=4.97,95%CI=[1.70;14.49],P<0.01)。而CFAP43、CFAP44、CFAP251等基因在两国间未显示显著性差异(P>0.05)。这一发现为理解弱精子症的遗传变异的多样性奠定了基础。

DNAH1基因突变引起精子鞭毛多发形态异常的不育患者辅助生殖助孕结局分析

目的探讨DNAH1基因突变引起的精子鞭毛多发形态异常(multiple morphological abnormalities of the sperm flagella,MMAF)不育患者行卵胞质内单精子注射(intracytoplasmic sperm injection,ICSI)助孕后的临床结局。方法回顾性队列研究分析2018年2月至2020年1月期间在河南省人民医院生殖中心就诊的39例MMAF不育患者的临床资料和基因检测结果,12例由DNAH1突变引起的MMAF患者为DNAH1阳性组,27例未提示DNAH1突变的MMAF患者为DNAH1阴性组,选择同一时期男女双方年龄匹配进行ICSI助孕治疗的100例精子形态正常的男性不育症患者作为对照组,观察并分析3组不育夫妇进行辅助生殖助孕的治疗结局。结果39例MMAF患者均行全外显子组测序检测,其中12例患者检测到DNAH1基因突变,分别为10例复合杂合突变和2例纯合突变,另27例患者未检测到目前已知的引起MMAF的基因突变。3组患者夫妇均行ICSI助孕治疗,DNAH1阳性组、DNAH1阴性组和对照组在获卵数和MII卵子数上的差异均有统计学意义[(17.08±5.32)枚、(9.59±3.98)枚和(10.44±6.33)枚,P=0.001;(14.58±5.18)枚、(6.78±3.38)枚和(8.32±5.31)枚,P<0.001],在胚胎种植率、临床妊娠率、早期流产率和活产率上的差异均无统计学意义(均P>0.05)。12例由DNAH1突变引起的不育患者夫妇共接受12个取卵周期,形成第3天胚胎79枚,首次新鲜胚胎或复融胚胎移植共12次,获得10个亲生子代。结论对于由DNAH1基因突变引起的MMAF患者,ICSI助孕可以帮助其生育亲生子代,且有较高的临床妊娠率和活产率。

5例精子鞭毛多发形态异常患者的非整倍体率与卵胞质内单精子注射的临床结局研究

目的评估精子鞭毛多发形态异常(multiple morphological abnormalities of the sperm flagella,MMAF)患者的精子非整倍体率与卵胞质内单精子注射(intracytoplasmic sperm injection,ICSI)的临床结局关系。方法本研究在2017年1月至2018年6月期间共收集到来自郑州大学第二附属医院生殖中心的5例MMAF患者及10例精液参数正常的可育对照,通过巴氏染色和电子显微镜观察患者精子的形态及超微结构特征,采用荧光原位杂交技术(FISH)检测患者精子非整倍体率,最后对患者行ICSI助孕并观察其临床结局。结果患者精液中存在大量缺失的、短的、弯的、卷曲的和不规则的精子鞭毛,且鞭毛轴丝中央微管缺失;患者精子非整倍体率与正常相比差异无统计学意义(P>0.05);5对MMAF患者夫妇经过7个ICSI周期,均实现临床妊娠,其中活产3例,自然流产2例。结论MMAF患者精子鞭毛存在严重形态和超微结构异常,但患者较低的精子非整倍体率提示MMAF患者进行ICSI治疗具有较好的临床结局。

精子鞭毛多发形态异常的临床诊断

精子鞭毛多发性形态异常(MMAF)是一种严重的精子鞭毛畸形,典型MMAF患者精子尾部出现缺失、短、卷曲、弯折和不规则等,从而导致精子活力严重不足;透射电镜观察鞭毛横断面出现中心微管缺失、纤维鞘和外层致密纤维结构紊乱、动力蛋白臂缺失等超微结构缺陷。MMAF的遗传学病因尚未完全明确,是目前该疾病临床与转化研究所关注的重点。MMAF的临床诊断缺乏统一标准和流程。本综述重点讨论了其诊断策略,为临床MMAF的诊疗提供参考。

两例精子鞭毛多发形态异常男性不育症患者的DNAH1基因变异分析

目的对2例由严重弱精子症导致原发性男性不育患者进行临床和遗传学分析,明确其可能的致病原因。方法提取患者及父母外周血基因组DNA,采用全外显子组测序技术对患者进行基因变异分析,并对疑似致病变异进行Sanger测序验证和致病性分析。结果全外显子测序显示例1 DNAH1基因存在c.2016T>G(p.Y672X)和c.6017T>G(p.V2006G)复合杂合变异;例2 DNAH1基因存在c.2610G>A(p.W870X)纯合变异,分别遗传自父亲和母亲。按照美国医学遗传学会与基因组学学会遗传变异分类标准与指南,DNAH1基因c.2016T>G(p.Y672X)和c.2610G>A(p.W870X)变异均为致病(PVS1+PM2+PM3+PP3)。结论2例患者可能均为DNAH1基因变异导致的精子鞭毛多发形态异常,进而引起原发性男性不育。