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.

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.

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.

Combined laparoscopic and thoracoscopic repair of adult right-sided Bochdalek hernia with massive liver prolapse: A case report

BACKGROUND A Bochdalek hernia(BH)is a congenital diaphragmatic hernia that often develops in the neonatal period.BH typically occurs on the left side of the diaphragm.A right-sided BH in an adult is rare.CASE SUMMARY A 45-year-old man was referred to our hospital because of an abnormal shadow seen on chest radiography during a medical check-up.A chest radiograph showed elevation of the right hemidiaphragm.Computed tomography showed prolapse of multiple intraabdominal organs into the right thoracic cavity,corresponding to a right-sided BH.The herniated contents included the stomach,transverse colon,and left lobe of the liver.The left lobe of the liver was enlarged,particularly the medial segment.Laparoscopic surgery was performed.However,the left lobe of the liver was completely trapped in the thoracic cavity.Therefore,thoracoscopic manipulation had to be performed to return the liver to the abdominal cavity.The hernia was repaired with interrupted nonabsorbable sutures and reinforced with mesh.CONCLUSION Combined laparoscopic and thoracoscopic surgery was successfully performed for right-sided BH with massive liver prolapse and abnormal liver morphology.

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.

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.

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.

Aberrant protamination in sperm correlates to anomalous nuclear and cytoplasmic architectures in infertile males with sperm dysmorphology

Aberrant sperm protamination is linked to sperm dysmorphology and nuclear chromatin condensation.Yet,its effects on sperm cytoplasmic maturation remain largely unexplored.The relationships of protamines,sperm morphology,DNA damage,and cytoplasmic remodeling were illustrated in this study to provide fresh perspectives on the mechanisms of male infertility.A total of 205 infertile males were allocated into 5 groups according to the percentage of spermatozoa exhibiting abnormal morphology within their samples.Sperm concentration,motility,abnormal sperm morphology,cytoplasmic droplets(CDs),and excess residual cytoplasm(ERC)were analyzed according to the World Health Organization manual(2010).Sperm nuclear vacuoles(NVs)were determined by propidium iodide(PI)staining.Sperm protamine expressions(P1 and P2)were detected by western blot.DNA damage was measured by acridine orange test(AOT)to calculate the proportion of sperm with single-strand DNA breaks(SSBs).Our data showed that sperm concentration and motility in infertile males significantly decreased with the severity of abnormal sperm morphology(both P<0.01).P1 level,P1/P2 ratio,and SSB rate increased with the severity of sperm dysmorphology,whilst the P2 level decreased(all P<O.01).NVs,CDs,and ERC were more common in males with sperm dysmorphology and positively correlated with the SSB rate(all P<O.01).The relationships between the SSB rate and the P1/P2 ratio were also significant(P<0.01).Aberrant protamination may cause sperm dysmorphology and compromise male fertility by impairing sperm's nucleus and cytoplasm maturation,with the P1/P2 ratio potentially serving as a valuable indicator of sperm quality and male fertility.