The relationship between DNA fragmentation and the intensity of morphologically abnormal human spermatozoa

Objective:To determine the relationship between teratozoospermia and sperm DNA fragmentation(SDF)in the human ejaculate.Methods:This retrospective study included 100 normozoospermic men as a control cohort(abnormal forms>14%),210 patients with a high level of abnormal forms(≤4%)and 65 patients presenting with a moderate level of abnormal forms(>4%to≤14%)based on the World Health Organization definitions.Sperm morphology was assessed using bright field microscopy.Sperm DNA fragmentation was assessed using the sperm chromatin dispersion assay.Non-parametric analyses were conducted to examine the relationship between abnormal sperm morphology and sperm DNA fragmentation;receiver operating characteristic(ROC)analyses were conducted to assess sensitivity and specificity of this relationship.Results:A correlation analysis revealed that the higher the proportion of abnormal spermatozoa in the ejaculate,the higher the level of SDF(Spearman's Rho=-0.230;P<0.001).Significant differences in the proportion of SDF were found when all cohorts were compared(P<0.001);these significant differences were also retained when the different cohorts were compared pairwise.ROC analysis showed a moderate but significant predictive value for SDF to differentiate patients with different levels of teratozoospemia.Conclusions:Although analysis of a more continuous range of values for teratozoospermia would help further clarify any causal relationship with SDF,there is clearly a synergistic or coincident affiliation between these variables that needs to be acknowledged by the clinician when interpreting the spermiogram.

Modified strict sperm morphology threshold aids in the clinical selection of conventional in vitro fertilization(IVF)or intracytoplasmic sperm injection(ICSI)

For infertility treatment,the selection of in vitro fertilization(IVF)or intracytoplasmic sperm injection(ICSI)is decided by multiplying indicators(including fallopian tube factors,semen count,and semen motility),except for sperm morphology.In this study,we conducted a retrospective analysis,from implantation to birth,over a period of 5 years.A total of 1873 couples with primary or secondary fallopian tube factors and an increased defective sperm morphology rate(DSMR)were divided into different groups to receive IVF or ICSI cycles.By comparing the outcomes,we found that the F1 group(DSMR<96%,IVF group 1)had higher cleavage rate,biochemical pregnancy rate,clinical pregnancy rate,and live birth rate than the F3 group(DSMR>98%,IVF group 3;P<0.05).In contrast,there was no significant difference in the ICSI subgroups.Furthermore,a comparison of the outcomes between IVF and ICSI showed that the S3 group(DSMR>98%,ICSI group 3)had higher cleavage rate(P<0.001),biochemical pregnancy rate(P<0.05),clinical pregnancy rate(P<0.05)and live birth rate(P<0.05)than the F3 group.However,the ICSI subgroup had a lower two pronuclei fertilization rate than the IVF subgroup(P<0.05).Our data suggest that the sperm morphology should also be considered when selecting IVF or ICSI combined with other semen parameters before the first assisted reproductive technologies(ART)cycle,especially for males with severe sperm defects.

Sperm collection in Black-legged Kittiwakes and characterization of sperm velocity and morphology

Background:Collecting and studying live sperm is central to many important fields of biology.Yet,a simple method to collect live sperm is lacking in wild seabird species.Here,we describe a non?invasive method to collect viable sperm samples based on a simple massage technique applied to male Black?legged Kittiwakes(Rissa tridactyla).Methods:We studied a colony breeding at Kongsfjorden,Svalbard and successfully obtained sperm samples from 32 males.With a subset of samples(n = 12 males),we compared the suitability of several extenders(0.9% NaCl,PBS,Earle's balance salt solution,Dulbecco's modified Eagle medium) in maintaining sperm alive long enough for analyses.With another 18 ejaculates,we conducted computer assisted sperm analyses using the CASA plugin for ImageJ.We provide details about the settings to be used for such analyses.Lastly,droplets from 20 ejaculates were smeared on glass slides and preserved with formalin to characterize sperm morphology in terms of total sperm length,sperm head length,midpiece length and flagellum length,and percentage of abnormal sperm.Results:With this method and under field conditions,we were able to obtain sufficient amounts of live sperm to assess traits related to sperm quality(e.g.sperm morphology,percentage of motile sperm,sperm velocity).We found that two extenders,Earle's balanced salt solution and Dulbecco modified Eagle's medium,yielded similarly good results.Additionally,we investigated whether specific behaviours were associated with successful sperm collection and whether sperm collection success depended on how long before laying sperm collection was attempted.Finally,we provide mean values for sperm morphology,sperm swimming ability and percentage of motile sperm,which may prove useful for future comparative analyses,and we report high levels of sperm abnormality and within?ejaculate variation in sperm morphology.Conclusions:We discuss the high percentage of abnormal sperm and high within?ejaculate variation in sperm morphology in light of sperm competition theory and conclude that these figures are likely due to relaxed post?cop?ulatory sexual selection,kittiwakes being strictly monogamous.Finally,we suggest that this method could be applied to other seabird species sharing similar ecology.

Ameliorative effect of Punica granatum on sperm parameters in rats exposed to mobile radioelectromagnetic radiation

Objective:To study the effect of mobile emitted radio frequency electromagnetic radiation(RF-EMR)on sperm parameters(sperm count,viability,motility,progressivity,and morphology)and ameliorative effect of pomegranate juice in rats.Methods:Thirty male Sprague Dawley rats were divided into five groups(n=6 in each group).GroupⅠwas neither exposed to RF-EMR nor given pomegranate juice,groupⅡ,ⅢandⅣwere exposed to mobile emitted RF-EMR for 60 min/day for 90 days.After 90-day exposure to RF-EMR,groupⅢwas supplemented with pomegranate juice for 90 days(1 mL/day)and groupⅣwas allowed to recover for 90 days without supplementation of pomegranate juice.GroupⅤwas supplemented with pomegranate juice for 90 days without exposure to RF-EMR.At the end of intervention,epididymal sperm parameters(sperm count,viability,motility,progressivity,and morphology)were measured.Results:The microscopic examination of sperm parameters such as sperm count,sperm viability,sperm motility,progressivity were significantly decreased in groupⅡ(the mobile RF-EMR group)compared to groupⅠ(the control group)(P<0.05).In addition,sperm morphology was also significantly altered(abnormal)in groupⅡcompared to groupⅠ(P<0.05).However,the sperm parameters including sperm morphology were significantly altered in groupⅢ(mobile RF-EMR+pomegranate juice)compared to groupⅡ(P<0.05).The sperm parameters including sperm morphology were not significantly altered in groupⅣ(the mobile RF-EMR recovery group)compared to groupⅡ(P>0.05).The sperm parameters were non-significantly increased in group V(the pomegranate juice group)compared to groupⅠ(P>0.05).Conclusions:Mobile RF-EMR exposure reduces the sperm parameters,which,however,can be reversed by treatment with pomegranate juice,indicating that pomegranate juice can be used as a nutritional supplement to improve sperm quality.

Impact of chronological ageing on semen parameters in southern Indian men visiting infertility centre:A retrospective study

Objective:To investigate the association between age and semen parameters among male partners of subfertile couples.Methods:This retrospective study analyzed the semen of 1523 infertile men aged 26 to 50 years.Data were extracted from GarbhaGudi IVF Centre database from January 2019 to September 2020.The basic semen parameters were interpreted according to the WHO manual 2021,6th edition.Semen parameters in different age groups were compared.Results:Total and progressive motile sperms were significantly higher in the age group of 26-30 years compared to other age groups(P<0.05).Normal sperm count was significantly higher in the age group of 26-30 years compared to the age groups of 41-45 years and>46 years(P=0.001).However,sperm head defects,neck and midpiece defects,tail defects,and cytoplasmic droplets showed statistically insignificant difference in all the age groups(P>0.05).Semen viscosity showed no statistical difference in all the age groups compared to the reference age group of 26 to 30 years.Conclusions:Higher age can lead to a significant decrease in normal sperms and motility in subfertile men.Hence,male partner age should be considered as one of the major determining factors for reproductive outcomes.

A Preliminary Study on the Evaluation of Human Sperm Head Morphology with a Domestic Digital Holographic Image System

The head of sperm was imaged with domestic digital holographic microscopy(DHM),and then the quantitative three-dimensional size information of normal sperm and teratozoospermic sperm was compared and analyzed.DHM sperm imaging and repeated quantitative evaluation were used to determine the morphology of the sperm head in two patients with teratozoospermia and four volunteers with normal semen parameters.Sixty and 139 sperm of teratozoospermia patients and normal people were photographed by digital hologram,respectively.The differences in head height and width were compared and statistically analyzed.The sperm head height of the teratozoospermia group was 3.06±1.66μm,which was significantly lower than that of the normal sperm group(4.54±1.60μm,p<0.01),but there was no significant difference in the head width between the two groups.Compared with the traditional two-dimensional optical microscope observation method,the DHM system can provide three-dimensional quantitative information for the sperm head and thus may help in the comprehensive clinical evaluation of the sperm head structure.

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.

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.

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.

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.

The influence of Mycoplasma species on human and canine semen quality:a review

Mycoplasma species(spp.)are bacteria that are difficult to detect.Currently,the polymerase chain reaction(PCR)is considered the most effective diagnostic tool to detect these microorganisms in both human and veterinary medicine.There are 13 known species of human Mycoplasma and 15 species of canine Mycoplasma.Owing to the difficulties in identifying the individual species of Mycoplasma,there is a lack of information regarding which species are saprophytic and which are pathogenic.The prevalence of the individual species is also unknown.In addition,in both humans and dogs,the results of some studies on the impact of Mycoplasma are conflicting.The presence of Mycoplasma spp.on the epithelium of reproductive tract is often associated with infertility,although they are also detected in healthy individuals.The occurrence of Mycoplasma spp.is more common in dogs(even 89%)than in humans(1.3%–4%).This is probably because the pH of a dog's genital is more conducive to the growth of Mycoplasma spp.than that of humans.Phylogenetically,human and canine Mycoplasma are related,and majority of them belong to the same taxonomic group.Furthermore,40%of canine Mycoplasma spp.are placed in common clusters with those of human.This suggests that species from the same cluster can play a similar role in the canine and human reproductive tracts.This review summarizes the current state of knowledge about the impact of Mycoplasma on canine and human male fertility as well as the prospects of further development in this field.

The effect of age and abstinence time on semen quality:a retrospective study

This study analyzed the effects of male age and abstinence time on semen quality and explored the best abstinence time for Chinese males among different age groups.Semen parameters,including sperm kinetics,morphology,and DNA fragmentation index(DFI),were reviewed from 2952 men.Samples were divided into six age groups(≤25 years,26–30 years,31–35 years,36–40 years,41–45 years,and>45 years)and were divided into six groups according to different abstinence time(2 days,3 days,4 days,5 days,6 days,and 7 days).The differences in semen quality between the groups were compared,and the effect of age and abstinence time on semen quality was analyzed.Significant differences were observed in semen volume,progressive motility(PR),and DFI among the age groups(all P<0.05),and no significant differences were observed in sperm morphological parameters(all P>0.05).There were significant differences in semen volume,PR,and DFI among different abstinence time groups(all P<0.05)and no significant differences in sperm morphological parameters(all P>0.05).Pearson analysis showed that male age and abstinence time were both significantly correlated with sperm kinetics and DFI(both P<0.05),while no significant correlation was found with sperm morphological parameters(all P>0.05).The box plots and histograms of men’s age,abstinence time,and semen quality show that most semen quality parameters differ significantly between the 2 days and 7 days abstinence groups and other groups at different ages.Except for the sperm morphology parameters,sperm kinetic parameters and sperm DFI are linearly related to male age and abstinence time.