Clinical significance of the low normal sperm morphology value as proposed in the fifth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen

The very low cut-off value for sperm morphology of 4% morphologically normal spermatozoa, as proposed in the new edition of the World Health Organization （WHO） manual on semen analysis, is in agreement with recently published values and reflects the trend of a decline in reported mean values for normal sperm morphology. The reduced value for morphologically normal spermatozoa over the years may be due to several factors. The first is the introduction of strict criteria for the evaluation of sperm morphology. Other reasons may include the introduction of additional criteria for sperm morphology abnormalities and the suggested decrease in semen parameters because of increasing negative environmental influences. Although on its own the newly proposed very low normal value may not provide the strong predictive value for a males＇ fertility potential, as originally reported for sperm morphology evaluated according to strict criteria, a good predictive value can still be obtained if the holistic, strict approach for sperm morphology evaluation is followed together with additional sperm morphology parameters now available, because certain morphology patterns and sperm abnormalities are now known to be of strong prognostic value. In addition, better international standardization of the technical methodology, consensus on the interpretation of sperm morphology evaluation criteria and standardized international external quality control （EQC） schemes, are of utmost importance to maintain the good predictive value of sperm morphology.

Measurement and significance of sperm morphology

The measurement or evaluation and clinical significance of human sperm morphology has always been and still is a controversial aspect of the semen analysis for the determination of a male＇s fertility potential. In this review the background of the development of the evaluation criteria for sperm morphology will be discussed. Aspects of criticism on the strict criteria definition and use of the criteria for sperm morphology evaluation will be discussed as well as possible reasons for the decline in normal sperm morphology values and how we can compromise for this phenomenon resulting in the very low normal reference value as published in the 2010 WHO manual for the Examination and Processing of Human Semen. One of the possible solutions may be to give more attention to a limited number of abnormal sperm morphology categories and the inclusion of sperm morphology patterns. It is concluded in this review that if done correctly and with care and with strict application of existing guidelines as outlined in the 2010 WHO manual, sperm morphology measurement still has a very important role to play in the clinical evaluation of male fertility potential.

Semen analysis with regard to and functional aspects sperm number, sperm morphology

The new World Health Organization （WHO） Manual for Semen Analysis contains several improvements. One is that the 20 million spermatozoa per mL paradigm has been ousted in favour of proper calculations of lower reference limits for semen from men, whose partners had a time-to-pregnancy of 12 months or less. The recommendation to grade the progressive motility as described in the third and fourth editions of the WHO manual was not evidence-based, and WHO was therefore motivated to abandon it. However, the new recommendation is not evidence-based either, and it is difficult to understand the rational for the new assessment. It may have been a compromise to avoid returning to the rather robust system recommended in the first edition （1980）. The unconditional recommendation of the ＇Tygerberg strict criteria＇ is not evidence-based, and seems to be the result of an unfortunate bias in the composition of the Committee in favour of individuals known to support the ＇strict criteria＇ method. This recommendation will have negative effects on the develop- ment ofandrology as a scientific field. Given the importance of the WHO manual, it is unfortunate that the recommenda- tions for such important variables, as motility and morphology, lack evidence-based support.

Effect of lead on survival, locomotion and sperm morphology of Asian earthworm, Pheretima guillelmi

To provide basic toxicity data for formulating risk characterization benchmarks, the effects of lead on survival, locomotion, and sperm morphology were investigated in the Asian earthworm Pheretima guillelmi. The LC50 of P. guiUelmi for 7 and 14 d were 4285＋339 mg/kg and 3207＋248 mg/kg, which shows P. guillelmi can tolerate a higher concentration of lead nitrate. The average weight of the surviving earthworms decreased at concentration of 2800 mg Pb/kg soil, and the locomotor ability of earthworms exposed to a range of soil Pb concentrations showed a general decrease with increasing Pb concentrations. We also presented data depicting the sperm morphology of earthworms, which shows potential as a sensitive biomarker for measuring the effects of heavy metal on reproduction.

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.

No difference in high-magnification morphology and hyaluronic acid binding in the selection of euploid spermatozoa with intact DNA

In this study, we compared conventional sperm selection with high-magnification morphology based on the motile sperm organellar morphology examination （MSOME） criteria, and hyaluronic acid （HA） binding for sperm chromosome aneuploidy and DNA fragmentation rates. Semen from 50 severe male factor cases was processed through density gradient centrifugation, and subjected to sperm selection by using the conventional method （control）, high magnification at x6650 or HA binding. Aneuploidy was detected by fluorescence in situ hybridization with probes for chromosomes 13, 18, 21, X and Y, and DNA fragmentation by the terminal deoxynucleotidyl transferase dUTP nick end labelling （TUNEL） method. Spermatozoa selected under high-magnification had a lower DNA fragmentation rate （2.6% vs. 1.7%; P=0.032）, with no significant difference in aneuploidy rate （0.8% vs0.7%; P=0.583）, than those selected by the HA binding method. Spermatozoa selected by both methods had much lower aneuploidy and DNA fragmentation rate than the controls （7% aneuploidy and 26.8% DNA fragmentation rates, respectively）. In the high-magnification group, the aneuploidy rate was lower when the best spermatozoa were selected than when only the second-best spermatozoa were available for selection, but the DNA fragmentation rate was not different. In conclusion, sperm selection under high magnification was more effective than under HA binding in selecting spermatozoa with low DNA fragmentation rate, but the small difference （0.9%） might not be clinically meaningful. Both methods were better than the conventional method of sperm selection.

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.

Semen analysis and sperm function testing

Despite controversy regarding the clinical value of semen analysis, male fertility investigation still relies on a standardized analysis of the semen parameters. This is especially true for infertility clinics in both developing and developed countries. Other optional tests or sophisticated technologies have not been widely applied. The current review addresses important changes in the analysis of semen as described in the new World Health Organization （WHO） manual for semen analysis. The most important change in the manual is the use of evidence-based publications as references to determine cutoff values for normality. Apart from the above mentioned changes, the initial evaluation and handling methods remain, in most instances, the same as in previous editions. Furthermore, the review evaluates the importance of quality control in andrology with emphasis on the evaluation of sperm morphology. WHO sperm morphology training programmes for Sub-Saharan countries were initiated at Tygerberg Hospital in 1995. The external qualitY control programme has ensured that the majority of participants have maintained their morphological reading skills acquired during initial training. This review reports on current sperm functional tests, such as the induced acrosome reaction, and sperm-zona pellucida binding assays, as well as the impact of sperm quality in terms of DNA integrity, and the relationship of sperm function tests to sperm morphology.

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.

The reversibility of sperm quality after discontinuing nandrolone decanoate in adult male rats

Aim： To investigate the reversibility of the effect of nandrolone decanoate （ND） on sperm parameters after discontinuing the drug. Methods： Three groups of rats received peanut oil （control）, low and high doses of ND for 14 weeks, respectively. Each group was divided into subgroups A and B, in which rats were killed 14 and 28 weeks after initiating the injection, respectively. Results： Sperm count and motile sperm fraction were decreased in the subgroups A and B that received low and high doses of ND in comparison with the controls A and B, respectively. The sperm count and motile sperm fraction increased in the subgroups B that received low and high doses of ND in comparison with their corresponding subgroups A. The number of normal morphology sperm was decreased significantly in subgroups A receiving low and high doses of ND in comparison with the control subgroup A. However, this parameter was not decreased in subgroups B receiving low and high doses in comparison with the control subgroup B. The normal sperm morphology did not show any significant differences in the subgroups B in comparison with their corresponding subgroups A. Conclusion： The 14-week injection of low and high doses of ND decreases sperm quality and quantity in rats. These parameters were improved after discontinuing ND, but not recovered completely even when they are left untreated for 14 weeks.

UNE COLORATION SIMPLE ET RAPIDE DU FROTTIS DU SPERME

Objective To introduce a rapid simple staining method for sperm morphology.Methods Liquifiedsemen was on the glass, fixed by methanol, treated with phosphonic acid buffer, stained by A, B staining solution separately, and rinsed by running water. Results After staining, the whole sper-matozoid was very clear. The cephalic zone of spermatozoid head , acrosome area , was colored pink; its caudal zone colored violet red or violet blue. The body and tail of spermatozoid colored pink or light blue. Normal and abnormal morphology of spermatozoid could be differentiated easily. The sloughed spermatogenic cells and white blood cells could be differentiated also. Conclusion This rapid staining method of semen smear can produce the same effect of other usual staining method , but the staining time is shorter and the procedure is simpler.

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.

Normal reference ranges for semen quality and their relations to fecundity

Several recent studies have shown that the fecundity of a man decreases progressively with sperm concentrations below 40 million spermatozoa per mL. Therefore, it is unfortunate that the new World Health Organization guidelines for semen analysis recommend lowering the lower cutoff value for normal sperm concentration from 20 to 15 million spermatozoa per mL. As a result large groups of subfertile men across the world may not receive appropriate andrological help in the future.

Practical semen analysis： from A to Z

Accurate semen analysis is critical for decisions about patient care, as well as for studies addressing overall changes in semen quality, contraceptive efficacy and effects of toxicant exposure. The standardization of semen analysis is very difficult for many reasons, including the use of subjective techniques with no standards for comparison, poor technician training, problems with proficiency testing and a reluctance to change techniques. The World Health Organization （WHO） Semen handbook （2010） offers a vastly improved set of standardized procedures, all at a level of detail that will preclude most misinterpretations. However, there is a limit to what can be learned from words and pictures alone. A WHO- produced DVD that offers complete demonstrations of each technique along with quality assurance standards for motility, morphology and concentration assessments would enhance the effectiveness of the manual. However, neither the manual nor a DVD will help unless there is general acknowledgement of the critical need to standardize techniques and rigorously pursue quality control to ensure that laboratories actually perform techniques ＇according to WHO＇ instead of merely reporting that they have done so. Unless improvements are made, patient results will continue to be compromised and comparison between studies and laboratories will have limited merit.

Effect of age on quality of fresh and frozen-thawed semenin White Italian ganders

<abstract>Aim: To comparatively evaluate the fresh semen quality of 1, 2 and 3-yr-old White Italian ganders (Anser anser L.) and the susceptibility of spermatozoa to freezing-thawing procedure. Methods: Semen was collected by dorso-abdominal massage every 2 days^3 days from three groups of ganders: 1-yr-old (n=11), 2-yr-old (n=7) and 3-yr-old (n=9). In the pooled fresh semen samples, the following parameters were evaluated: the ejaculate volume, the blood or fecal contamination and the motility, concentration and morphology of spermatozoa. Sperm motility and morphology were evaluated in the frozen-thawed semen. Semen diluted with EK extender was frozen in straws in a computerized freezing unit with 6 % dimethyl-formamide to -140 癈 at a rate 60 癈/min and then transferred into the LN2 container. Straws with semen were thawed in a water bath at 60 癈. Results: The ejaculate volume decreased with the age (0.21 mL for 1-yr-old, 0.18 mL for 2-yr-old and 0.14 mL for 3-yr-old ganders); the sperm concentration increased with the age (327xl06mL-1 for 1-yr-old, 431xl06mL-1 for 2-yr-old and 547xl06mL-1 for 3-yr-old ganders); the number of live - normal sperm was significantly (P<0.01) lower in the 1-yr-old than that in the 2- and 3-yr-old ganders (26.61 %, 41.54 %and 35.9 %, respectively). The percentage of normal cells survived the freezing-thawing process was 37.7 %, 43.3 % and 40.9 % for 1-, 2- and 3-yr-old ganders, respectively. Conclusion: Freezing and thawing processes more significantly (P<0.01) affected the motility, viability and morphology of spermatozoa in semen of 1-yr-old ganders in comparison with older males.

Seasonal variation in semen quality of swamp buffalo bulls （Bubalus bubalis） in Thailand

Aim： To test the hypothesis that season affects the semen quality of swamp buffalo （Bubalus bubalis） bulls used for artificial insemination （AI） under tropical conditions in Thailand, as it does in Bos taurus and Bos indicus. Methods： Clinical and andrological examinations, and monitoring of semen production and quality were carried out on five mature, healthy swamp buffalo AI bulls in Thailand from July 2004 to the end of June 2005. Sperm output, motility, morphology and plasma membrane integrity （PMI） were compared between three seasons of the year （rainy, i.e. July-October; winter, i.e. November-February; and summer, i.e. March-June） with distinct ambient temperature and humidity. Results： All bulls were diagnosed as clinically healthy and with good libido throughout the study. Ejaculate volume, pH, sperm concentration, total sperm number and initial sperm motility did not differ between seasons, whereas PMI and the relative proportion of morphologically normal spermatozoa were highest in summer and lowest in winter （P 〈 0.05）. Buffalo age, week of collection and season influenced sperm morphology （P 〈 0.05-0.001）. Among morphological abnormalities, only proportions of tail defects were affected by season, being highest in the rainy season and lowest in summer （P 〈 0.001）. In conclusion, climatic changes did not seem to largely affect semen sperm output or viability. Although the proportions of PMI and tail abnormalities were affected by season, they were always below what is considered unacceptable for AI bull sires. Conclusion： Seasonal changes did not appear to cause deleterious changes in sperm quality in swamp buffalo AI-sires in tropical Thailand.

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.