Midpiece Defects Research Articles

Human papillomavirus and male infertility correlation analysis following World Health Organization 2021 guidelines

Male infertility is a complex issue influenced by multiple environmental and pathological factors. In this context, the impact of Human papillomavirus (HPV) infection on male fertility remains controversial. The introduction of new WHO 2021 evaluation criteria, included in the 6th ed. of Laboratory Manual for the examination and processing of human semen, i.e. DNA fragmentation index (DFI), slow and rapid progressive motility, could provide additional information about this correlation. 121 semen samples of male partners of HPV-positive women attending In Vitro Fertilization (IVF) were evaluated following WHO 2021 and HPV-DNA test. Comparing HPV-negative and positive samples for rapid and slow progressive motility showed significantly different results (p = 0.0018, p = 0.0004), contrary to what was observed for total progressive motility. Regarding sperm DFI, only high-risk HPV infections affected DNA integrity. In addition, the correlation between the different semen parameters revealed a significant correlation between midpiece morphological defects and rapid progressive motility in the HPV-positive group (rho = 0.43, p = 0.0006). In conclusion, WHO 2021 provides additional information regarding HPV’s impact on seminal parameters. The correlation between HPV positivity, midpiece defects and a higher rapid progressive motility opens new research perspectives that may help unravel the issues surrounding the role of HPV in compromising sperm quality.

Effect of Breed and Season in Buck Semen Cryopreservation: The Portuguese Animal Germplasm Bank.

The aims of this study were to characterize the semen as well as the influence of breed, season, and semen processing on spermatozoa (SPZ) traits of four native Portuguese goat breeds used for the bank of Portuguese animal germplasm (BPAG). A total of 1017 ejaculates from Serrana (n = 30), Bravia (n = 15), Charnequeira (n = 11), and Preta de Montezinho (n = 3) bucks were collected between 2004 and 2020 at (EZN-INIAV; 39° N) during the whole year under natural conditions. All the fresh and cryopreserved (-196 °C) semen was evaluated and stored in the BPAG. Bravia bucks (the smallest breed) produced less (p < 0.05) volume of ejaculate than all the other breeds, which was higher during the full breeding season (September-January; p < 0.05), regarding all the other breeds. Contrarily, in general, SPZ concentration was lower during September-January, but total SPZ per ejaculate remained similar (p > 0.05) during May-August and September-January in Serrana bucks. The SPZ viability and SPZ midpiece defects were slightly influenced by breed and SPZ head defects by season (lowest % in February-April; p < 0.05). On the contrary, the freezing-thawing cycle strongly influenced (p < 0.01) all SPZ traits. The correlation coefficients of these traits between fresh and thawed SPZ were low (up to 0.33; p < 0.01), highlighting the importance of semen processing in semen cryopreservation. We conclude that breed and season had a relevant effect on ejaculate traits, but it was much less evident for the studied SPZ traits. These native goats can serve as semen donors throughout the year, under natural conditions.

High percentage of midpiece defects in Brangus bull sperm with no reduction in sperm kinematics.

The study investigated midpiece defects in sperm from a 5-year-old Brangus bull with a high rate of semen batch rejection, due to morphologically abnormal sperm, with no reduction in sperm kinematics. A comprehensive evaluation was conducted over a 16-month period, involving 28 ejaculates. Notably, despite the high proportion of midpiece defects (average 37.73%, from 3% to 58%), the study revealed stable sperm production, with no discernible differences in the kinematic data before and after cryopreservation. Electron microscopy identified discontinuities in the mitochondrial sheath, characteristic of midpiece aplasia (MPA). The anomalies were attributed to be of genetic origin, as other predisposing factors were absent. Additionally, the electron microscopy unveiled plasma membrane defects, vacuoles and chromatin decondensation, consistent with previous findings linking acrosome abnormalities with midpiece defects. The findings underscored the necessity of conducting thorough laboratory evaluations before releasing cryopreserved semen for commercialization. Despite substantial morphological alterations, the initial semen evaluation data indicated acceptable levels of sperm kinematics, emphasizing the resilience of sperm production to severe morphological changes. This case report serves as a contribution to the understanding of midpiece defects in bull sperm, emphasizing the need for meticulous evaluation and quality control in semen processing and commercialization.

Association of Endogenous Seminal L-Carnitine Levels with Post-Thaw Semen Parameters in Humans

Cryopreservation of semen is a useful tool for male fertility preservation. Some evidence for a beneficial effect of L-carnitine supplementation of freezing media on cryopreserved semen samples has been reported. Here, we examined the association of endogenous levels of seminal L-carnitine with post-thaw semen parameters. We also investigated the effect of freezing medium supplemented with L-carnitine on sperm characteristics, related to endogenous seminal L-carnitine levels. Semen analyses were performed on 125 fresh samples, and after standard cryopreservation and with L-carnitine as a supplement. Participants were categorized into two groups based on the median levels of endogenous seminal L-carnitine: low L-carnitine (≤38.8 µg/ml) and high L-carnitine (&gt;38.8 µg/ml). After standard cryopreservation, semen samples with high L-carnitine levels showed higher rapid progressive, progressive and total sperm motility and a reduced seminal static oxidation–reduction potential (ORP) level than samples with low L-carnitine levels. Only in post-thaw samples with low L-carnitine levels, there was an increase in the amount of sperm neck midpiece defects, compared to the fresh samples. Cryopreservation with L-carnitine had the most beneficial effect on rapid progressive sperm motility in samples with high endogenous L-carnitine levels. In conclusion, L-carnitine has a beneficial impact on sperm characteristics in post-thaw samples both as an endogenous component in seminal plasma and as a supplement in the freezing medium, by improving sperm motility and reducing seminal oxidative stress.

P-020 Association between male age and the frequency of sperm abnormalities

Abstract Study question Does advancing male age influence the frequency of occurrence of sperm abnormalities? Summary answer Advancing male age was associated with increased incidence of certain head and tail defects – elongated head, nuclear vacuoles, and coiled tail. What is known already Evaluation of male reproductive potential is based mainly on sperm motility and sperm morphology. Morphological sperm features serve as a reliable indicator in predicting the fertilizing capacity of sperm. Nowadays, there are various sperm selection techniques based on sperm morphology. However, advancing male age has been associated with decrease in semen quality. Therefore, the aim of this study was to examine the effect of aging on the frequency of occurrence of sperm abnormalities. Study design, size, duration This retrospective study includes 3980 men aged between 15 and 72 years, with a mean of 36 years. Semen samples were collected between October 2015 and December 2022 in a private in-vitro hospital. Men were divided into three groups according to their age: 1) from 15 to 25 years (n = 144), 2) from 25 to 45 years (n = 3447), and 3) over 45 years (n = 393). Participants/materials, setting, methods Sperm morphology was evaluated according to the Kruger’s strict criteria. Totally, 23 types of abnormalities were determined. Head defects included small, large, amorphous, elongated, round, pear-shaped, double, detached head, acephalic cells, small or large acrosomal areas, and spermatozoa without acrosome. Midpiece defects included thick, thin, bent, asymmetric midpiece and cytoplasmic droplets. Tail defects included short, coiled, and double tail. Acrosomal vacuoles and nuclear vacuoles were also evaluated. Statistics: ANOVA, followed by LSD post-hoc test. Main results and the role of chance The results are presented as mean±SD. Among the studied population the percentage of normozoospermic men was 44%, while the percentage of teratozoospermic semen samples was 56%. The total mean frequencies of occurrence of head, midpiece and tail abnormalities were 85.9±7.9%, 34.1±12.8% and 4.7±5.1%, respectively. Only 3 (13%) of the analysed 23 types of sperm abnormalities were significantly more common with advanced male age – elongated head, nuclear vacuoles, and coiled tail.The frequency of occurrence of elongated head differed significantly between the age groups 1-2, and 1-3: 11.38±10.1% vs. 13.79±11.58%, p = 0.015, and 11.38±10.1% vs. 14.4±11. 8%, p = 0.008, respectively. No difference in the incidence of this defect was observed between groups 2 and 3 (p &amp;gt; 0.05).Furthermore, the frequency of nuclear vacuoles significantly increased with age as observed in the following age group comparisons 1-2, 1-3, and 2-3: 4.28±4.93% vs. 5.4±6.1%, p = 0.04, 4.28±4.93% vs. 6.48±7.89%, p &amp;lt; 0.001, and 5.4±6.1% vs. 6.48±7.89%, р=0.001, respectively.Finally, coiled tail was observed more frequently in age groups 2 and 3 compared to age group 1: 13.79±11.58% vs. 11.38±10.1%, p &amp;lt; 0.001, and 14.4±11. 8% vs. 11.38±10.1%, p &amp;lt; 0.001, respectively. Limitations, reasons for caution This is a single-institution retrospective study. Wider implications of the findings The findings of this study showed that aging is associated with an increased percentage of several sperm abnormalities: spermatozoa with elongated head, nuclear vacuoles, and coiled tail. Further studies are required to clarify the negative impact of aging on spermatogenesis leading to morphological defects. Trial registration number NA

How Well Do Semen Analysis Parameters Correlate with Sperm DNA Fragmentation? A Retrospective Study from 2567 Semen Samples Analyzed by the Halosperm Test.

Sperm DNA fragmentation (SDF) levels have been measured in the workup for in vitro fertilization (IVF) at PIVET since 2007, with the Halosperm test having replaced the previous sperm chromatin structure assay (SCSA) since 2013. Of 2624 semen samples analyzed for the Halosperm test, 57 were excluded as the sperm concentration was <5 million/mL, a level too low for accurate testing, leaving 2567 samples for assessment within this study. The SDF rates were categorized in 5 sperm DNA fragmentation indices (DFI), ranging from <5% to levels >30%, and these categories were correlated with the respective semen analysis profiles and two clinical parameters, namely the age of the male and the ejaculatory abstinence period prior to the sample. The results showed a significant correlation with male age (r = 0.088; p < 0.0001), the abstinence period (r = 0.076; p = 0.0001), and the semen volume (r 0.063; p = 0.001), meaning an adversely high SDF was associated with advanced age, prolonged abstinence, and raised semen volume parameters. There was a significant negative correlation with sperm morphology (r = -0.074; p = 0.0001), progressive motility (r = -0.257; p < 0.0001), and semen pH (r = -0.066; p < 0.001), meaning these semen anomalies were associated with high SDF values. With respect to abnormal morphology, sperm tail defects had a positive correlation (r = 0.096; p < 0.0001) while midpiece defects showed a negative correlation (r = -0.057; p = 0.004), meaning that tail defects are most likely to associate with adverse DFI values. With respect to motility patterns, the poorer patterns showed a positive correlation with increased DFI, namely C pattern (r = 0.055; p = 0.005) and D pattern (r = 0.253; p < 0.0001). These results imply that raised DFI reflects poor sperm quality and should be investigated in clinical trials involving IVF and the consideration of intracytoplasmic sperm injection (ICSI).

New Mutations in DNHD1 Cause Multiple Morphological Abnormalities of the Sperm Flagella.

Male infertility is a common and complex disease and presents as a wide range of heterogeneous phenotypes. Multiple morphological abnormalities of the sperm flagellum (MMAF) phenotype is a peculiar condition of extreme morphological sperm defects characterized by a mosaic of sperm flagellum defects to a total asthenozoospermia. At this time, about 40 genes were associated with the MMAF phenotype. However, mutation prevalence for most genes remains individually low and about half of individuals remain without diagnosis, encouraging us to pursue the effort to identify new mutations and genes. In the present study, an a cohort of 167 MMAF patients was analyzed using whole-exome sequencing, and we identified three unrelated patients with new pathogenic mutations in DNHD1, a new gene recently associated with MMAF. Immunofluorescence experiments showed that DNHD1 was totally absent from sperm cells from DNHD1 patients, supporting the deleterious effect of the identified mutations. Transmission electron microscopy reveals severe flagellum abnormalities of sperm cells from one mutated patient, which appeared completely disorganized with the absence of the central pair and midpiece defects with a shortened and misshapen mitochondrial sheath. Immunostaining of IFT20 was not altered in mutated patients, suggesting that IFT may be not affected by DNHD1 mutations. Our data confirmed the importance of DNHD1 for the function and structural integrity of the sperm flagellum. Overall, this study definitively consolidated its involvement in MMAF phenotype on a second independent cohort and enriched the mutational spectrum of the DNHD1 gene.

Impact of chronological ageing on semen parameters in southern Indian men visiting infertility centre

Objective: To investigate the association between age and semen parameters among male partners of subfertile couples. Methods: This retrospective study analyzed the semen of 1 523 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&lt;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 &gt;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&gt;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.

Male Infertility Coexists with Decreased Sperm Genomic Integrity and Oxidative Stress in Semen Irrespective of Leukocytospermia.

Our research was designed to verify the relationship between male infertility, basic semen characteristics (with respect to detailed sperm morphology), sperm DNA fragmentation (SDF), oxidation-reduction potential in semen (ORP), and leukocytospermia. The obtained results showed that infertile groups (with or without leukocytospermia) had significantly lower basic semen characteristics and higher SDF, raw ORP, and static ORP (sORP) than fertile controls. The thresholds of 13% SDF (AUC = 0.733) and 1.40 sORP (AUC = 0.857) were predictive values for discriminating infertile from fertile men. In infertile groups, a higher prevalence and risk for >13% SDF and >1.40 sORP were revealed. Unexpectedly, leukocytospermic subjects had lower sORP, prevalence, and risk for >1.40 sORP than leukocytospermic-negative men. These groups did not differ in SDF and raw ORP. Both SDF and sORP negatively correlated with basic semen parameters but positively correlated with sperm head and midpiece defects. sORP positively correlated with sperm tail defects, immature sperm cells with excess residual cytoplasm, and SDF. In turn, raw ORP negatively correlated with sperm count but positively correlated with SDF and sORP. These findings indicate that (1) there is a relationship between male infertility, SDF, and OS in semen; (2) in infertile men, there is a clinically significant risk of SDF and OS irrespective of leukocytospermia; and (3) the assessment of SDF and oxidative stress should be independent of leukocytospermia.

Biallelic loss-of-function mutations in SEPTIN4 (C17ORF47), encoding a conserved annulus protein, cause thin midpiece spermatozoa and male infertility in humans.

Asthenoteratozoospermia is the primary cause of infertility in humans. However, the genetic etiology remains largely unknown for those suffering from severe asthenoteratozoospermia caused by thin midpiece defects. In this study, we identified two biallelic loss-of-function variants of SEPTIN4 (previously SEPT4) (Patient 1: c.A721T, p.R241* and Patient 2: c.C205T, p.R69*) in two unrelated individuals from two consanguineous Chinese families. SEPT4 is a conserved annulus protein that is critical for male fertility and the structural integrity of the sperm midpiece in mice. SEPT4 mutations disrupted the formation of SEPT-based annulus and localization of SEPTIN subunits in sperms from patients. The ultrastructural analysis demonstrated striking thin midpiece spermatozoa defects owing to annulus loss and disorganized mitochondrial sheath. Immunofluorescence and immunoblotting analyses of the mitochondrial sheath proteins TOMM20 and HSP60 further indicated that the distribution and abundance of mitochondria were impaired in men harboring biallelic SEPT4 variants. Additionally, we found that the precise localization of SLC26A8, a testis-specific anion transporter that colocalizes with SEPT4 at the sperm annulus, was missing without SEPT4. Moreover, the patient achieved a good pregnancy outcome following intracytoplasmic sperm injection. Overall, our study demonstrated for the first time that SEPT4 variants that induced thin midpiece spermatozoa defects were directly associated with human asthenoteratozoospermia.

EFFECT OF VITAMIN E SUPPLEMENTATION ON SPERM MORPHOLOGY AND SEMEN CHARACTERISTICS OF RABBIT BUCKS

Twenty mixed breed rabbit bucks aged between 6–8 weeks with an average weight of 861g were randomly assigned to four (4) dietary treatments with five (5) bucks per treatment in a Completely Randomized Design to determine the effect of feeding graded levels of Vitamin E on sperm morphology and semen characteristics of rabbit bucks. The rabbits received graded levels of vitamin E/kg feed (0mg/kg, 20 mg/kg, 40 mg/kg and 60 mg/kg for treatments 1, 2, 3 and 4, respectively). Feed and water were given to the rabbit bucks ad libitum during the experiment. The semen collected was taken to laboratory for sperm morphology and semen characteristics and the data collected were analysed using general linear model procedure of statistical analysis system (SAS). The results revealed that the mean number of normal cells, detached head and mid piece defect were not significantly (P&gt;0.05) different across the treatments. However, the percent abnormal defects percent of free tail and coiled tail were significantly (P&lt;0.05) lower for rabbits fed 20 and 60 mg/kg and the least bent tail was obtained in 60 mg/kg diet. The results for semen characteristics had significant (P&lt;0.05) differences in all the parameters except for semen volume and pH. The results showed that those fed 60 mg/kg of vitamin E diet gave significantly (P&lt;0.05) higher sperm motility, sperm concentration, live ratio and low dead ratio (87.50 %, 232x106, 87.50 % and 12.00 %) respectively. Therefore, supplementation of vitamin E at 60mg/kg in the diet of rabbit bucks is recommended

Nanowater enhances cryoprotective properties of glycerol-containing extenders used for ram semen freezing: A preliminary study spanning laboratory testing

Abstract It has been suggested that nanowater (NW-water declustered in the cold plasma generator and characterized by a low freezing point and high diffusivity) could improve ram semen quality after freezing in glycerol-containing extenders. Eighteen ejaculates from six Olkuska rams were divided into six equal portions each, and then diluted (800×106 spermatozoa/ml) and frozen in the fructose-skimmed milk-egg yolk Kareta extenders containing 3% or 7% of glycerol. The extenders were prepared with deionized water (DW-3% and DW-7%) or NW declustered for 15 min (NW15’) or 30 min (NW30’). Post-thaw sperm motility, proportions of sperm defects and percentages of apoptotic, necrotic, and live spermatozoa were determined. The proportion of spermatozoa with midpiece defects was lower (P&lt;0.05) in NW15’-3% compared with DW-3%. Sperm progressive motility was greater (P&lt;0.05) for spermatozoa cryopreserved in both NW30’ (NW30’-3%/7%) extenders compared with their respective controls (DW30’-3%/7%). The proportion of necrotic spermatozoa 1 h after thawing was lower (P&lt;0.05) in NW30’-7% compared with DW-7%, whereas the proportion of live cells detected immediately and 1 h after thawing was greater (P&lt;0.05) in NW30’-7% than in DW-7%. In summary, NW enhanced cryoprotective effects of glycerol-containing extenders with an increase in sperm viability being greater with 7% than 3% of glycerol. Different declustering times appear to alter NW properties. These observations merit future studies of the utility of NW for semen cryopreservation in rams and other mammalian species. The specific mechanisms whereby NW ameliorates the quality of frozen-thawed ram spermatozoa remain to be elucidated.

Biallelic mutations in ARMC12 cause asthenozoospermia and multiple midpiece defects in humans and mice

BackgroundAsthenozoospermia is a major factor contributing to male infertility. The mitochondrial sheath (MS), an important organelle in the midpiece of spermatozoa, is crucial to sperm motility. ARMC12 is a mitochondrial...

Cover, Volume 43, Issue 3

The cover image is based on the Research Article Novel biallelic mutations in SLC26A8 cause severe asthenozoospermia in humans owing to midpiece defects: Insights into a putative dominant genetic disease by Xiaojin He et al., https://doi.org/10.1002/humu.24322.

Novel biallelic mutations inSLC26A8cause severe asthenozoospermia in humans owing to midpiece defects: Insights into a putative dominant genetic disease

To investigate the genetic cause of male infertility characterized by severe asthenozoospermia, two unrelated infertile men with severe asthenozoospermia from nonconsanguineous Chinese families were enrolled, and whole exome sequencing were performed to identify the potential pathogenic mutations. Novel compound heterozygous mutations (NK062 III-1: c.290T>C, p.Leu97Pro; c.1664delT, p.Ile555Thrfs*11/NK038 III-1: c.212G>T, p.Arg71Leu; c.290T>C, p.Leu97Pro) in SLC26A8 were identified. All mutations were inherited from their heterozygous parents and are predicted to be disease-causing by sorts intolerant from tolerant, PolyPhen-2, Mutation Taster, and Combined Annotation Dependent Depletion. In silico mutant SLC26A8 models predict that mutations p.Leu97Pro and p.Arg71Leu cause changes in the α-helix, which may result in functional defects in the protein. Notably, heterozygous male carriers of each mutation in both families were able to reproduce naturally, which is inconsistent with previous reports. Ultrastructural analysis revealed severe asthenozoospermia associated with absence of the mitochondrial sheath and annulus in spermatozoa from both the probands, and both structural defects were verified by HSP60 and SEPT4 immunofluorescence analysis. SLC26A8 levels were significantly reduced in spermatozoa from patients harboring biallelic SLC26A8 mutations, and both patients achieved good prognosis following intracytoplasmic sperm injection. Our findings indicate that mutations in SLC26A8 could manifest as a recessive genetic cause of severe asthenozoospermia and male infertility.

P–049 The presence of double heads is associated with increased frequency of the other sperm abnormalities

Abstract Study question Is there an association between the presence of spermatozoa with double heads and the other sperm abnormalities in human semen? Summary answer Patients with double-headed spermatozoa had a significantly increased percentage of morphological abnormalities (head, midpiece and tail defects). What is known already The morphological evaluation of spermatozoa has a prognostic value for successful IVF procedure. It has been proven that certain morphological defects have a negative impact on fertilization, embryo quality, and pregnancy outcome in in-vitro fertilization cycles. Sperm abnormalities, such as double head, double tail and thin midpiece are rarely observed. However, their effect on the other sperm defects has not been well studied yet. The aim of this study was to examine the effect of the presence of double-headed spermatozoa on the frequency of occurrence of the other sperm defects. Study design, size, duration This retrospective study includes 2140 men aged between 18 and 73 years, with a mean of 36 years. It was conducted at Nadezhda Women’s Health Hospital, Bulgaria between October 2015 and August 2020. A comparative analysis was performed between semen samples with and without double-headed spermatozoa and the other sperm abnormalities, as well as the percentage of morphologically normal forms. Participants/materials, setting, methods Morphological analysis was performed according to the Kruger’s strict criteria. Totally 23 types of abnormalities were determined: head defects (small, large, amorphous, elongated, round, pear-shaped, double, acephalic, detached head, small and large acrosomal areas and spermatozoa without acrosome), midpiece defects (thick, bent, asymmetric, thin midpiece and cytoplasmic droplets), tail defects (stumped, coiled and double tail), acrosomal vacuoles, nuclear vacuoles and multiple defects. Statistics: Mann-Whitney U-test and T-test; P ≤ 0.05. Main results and the role of chance Presence of double-headed spermatozoa was observed in 12.62% (270/2140) of the studied samples. In these patients the frequency of occurrence of double-headed spermatozoa ranged between 1% and 29% with a mean of 0.41%±1.71%. Men with double-headed spermatozoa had significantly higher percentage of spermatozoa with small heads (24.51%±22.65%, P = 0.04), round heads (11.69%±10.13%, P &amp;lt; 0.01), nuclear vacuoles (10.64%±5.25%, P &amp;lt; 0.01), sperm without acrosome (9.76%±8.61%, P = 0.05), asymmetric midpiece (4.73%±3.96%, P &amp;lt; 0.05), bent midpiece (8.9%±7.22%, P &amp;lt; 0.01), thin midpiece (2.13%±4.44%, P &amp;lt; 0.01), double tail (1.78%±0.8%, P &amp;lt; 0.01), detached head (1.98%±1.42%, P &amp;lt; 0.01), stumped tail (6.03%±5.19%, P = 0.02), and cytoplasmic droplets (8.86%±5.02%, P &amp;lt; 0.01) compared to the patients without double-headed spermatozoa. Moreover, the percentage of sperm with multiple defects in the double-headed group was significantly higher (35.53%±29.91%, P &amp;lt; 0.01), while the percentage of normal forms was significantly lower (2.93%±3.64%, P &amp;lt; 0.01) compared to the patients without double heads. Limitations, reasons for caution In this study unequal sample sized groups were compared. We also need to investigate whether the obtained results will be confirmed in patients with certain pathological states, such as oligozoospermia, teratozoospermia, and asthenozoospermia. Wider implications of the findings: The present study revealed that the presence of double-headed spermatozoa in the ejaculate is related to an increased frequency of the other semen abnormalities. The double-headed spermatozoa could be used as an indicator for the total morphological quality of human spermatozoa Trial registration number Not applicable

P–007 Effect of swim-up on sperm morphology

Abstract Study question To evaluate the effect of swim-up on the percentage of certain morphological defects in the semen population Summary answer Swim-up preparation led to significantly lower percentage of spermatozoa with cytoplasmic droplets, thick neck and also multiple defects. What is known already Swim-up is routinely used sperm preparation technique in ART practice. It is widely known that swim-up enhances sperm quality in terms of motility and sperm morphology. However, the effect of swim-up on the frequency of occurrence of the specific sperm morphological abnormalities is still missing. Study design, size, duration This observational study involved 30 teratozoospermic patients of Nadezhda Women’s Health hospital between December 2020 and January 2021. Sperm morphology was evaluated before and after swim-up preparation. Participants/materials, setting, methods Native semen was liquefied and was subjected to swim up. Semen analysis performed according to WHO 2010. Native semen and swim up samples from the same men were subjected to Kruger strict morphological evaluation. The analyzed sperm morphological defects included: head defects (large, small, tapered, pyriform, round, amorphous and double heads); midpiece defects (bent, asymmetrical, thin, thick, presence of cytoplasmic droplet); tail defects (short, hairpin, bent, coiled tail and terminal droplet) and multiple defects. Main results and the role of chance Wilcoxon paired test showed that the percentage of morphologically normal spermatozoa was significantly higher in the swim-up samples in comparison to the native semen (8.5±4.2% vs 4.9±3.2%, p &amp;lt; 0.05). In addition, the percentage of spermatozoa bearing multiple defects was found to be significantly lower in the swim-up samples than in the native semen (25.8±11.6% vs 37.0±15.0%, p &amp;lt; 0.05). Two specific sperm morphological defects were found to be significantly lower after swim-up preparation: the presence of cytoplasmic droplets (6.0±1.0% vs 8.6±1.5%, p &amp;lt; 0.05) and the thick neck (9.7±5.5% vs 12.8±5.8%, p &amp;lt; 0.05). No significant different were observed in the other morphological defects between swim up samples and native semen (p &amp;gt; 0.05). Limitations, reasons for caution Results obtained from this study need to be confirmed by larger group of samples. Wider implications of the findings: Our study showed a significant reduction of certain midpiece defects after swim-up. The observed selection of spermatozoa without thick necks and cytoplasmic droplets explains the effectiveness of swim-up on ART. In addition, the obtained results can serve as a guide for future validation of new sperm preparation techniques. Trial registration number Not applicable

P–043 Six years’ retrospective statistical study (2013 – 2018) investigating the impact of Sperm DNA fragmentation on sperm analysis parameters

Abstract Study question What is the relationship between Sperm DNA fragmentation (SDF) levels and sperm analysis (Spermocytogramme) parameters results? Summary answer SDF level of patients with pathological spermocytogramme presents negative correlations to total spermatozoa mobility, vitality and concentration, and positive correlation to sperm morphology defects. What is known already The relationship between SDF and Sperm analysis parameters and especially sperm morphology needs to be more studied since few studies over the last years were focused on this relationship. However, abnormalities in these two parameters are considered as the most important biological indicators of male infertility. The pathogenesis of Teratozoospermia (&amp;lt;4% morphologically normal sperm cells according to WHO 2010) is continuously increasing over the last decade according to several studies. In addition, SDF is also increasing over the years because of several factors such as pollution, stress and lifestyle changing. Study design, size, duration Retrospective study including 331 infertile patients undergoing SDF-index testing with Spermocytogramme from January 2013 – December 2018. Patients divided into two groups: 143 patients with normal-Spermocytogramme and 188 patients with pathological-Spermocytogramme. Each group includes patients with abnormal SDF levels (&amp;gt;30%). Statistical analyzes were performed using SPSS22.0 for Windows-software. Kolmogorov–Smirnov-test for normality analysis and comparisons by Student-t-test or Mann–Whitney U-test, as appropriate. Pearson/Spearman’ tests for correlations were used as appropriate, P-value&amp;lt;0.05 was considered as significant. Participants/materials, setting, methods 143 patients with normal Spermocytogramme (2.8% abnormal-SDF) vs 188 patients with pathological Spermocytogramme (10.6% abnormal-SDF). WHO–2010 instructions for sperm-analysis were used through Makler®-counting-chamber (Sefi-Medical Instrument Ltd) for sperm-concentration and motility-determination using Sperm-class-analyzer-software (CASA-system (Microptic®)) to detect sperm abnormalities. Normozoospermia was determined when sperm progressive-motility is ≥ 32%, sperm-concentration ≥15x106/mL, and sperm-morphology ≥4%. “Diff-Quick” staining-method for the coloration of the fixed-sperm-slides was used for Sperm-morphology analysis. GoldCyto Sperm®Kit (Goldcyto Biotech corp.) was used to analyze SDF. Main results and the role of chance SDF is significantly higher in pathological spermocytogramme’ patients than in normal spermocytogramme’ patients (17.02 ± 11.88 vs 12.16 ± 9.58 respectively). In patients with pathological spermocytogramme, SDF is negatively correlated to Progressive sperm motility (r= –0.137; p = 0.042), Total sperm motility (r= –0.153; p = 0.036), vitality (r=–0.140; p = 0.048) and concentration (r=–0.195; p = 0.007). In the other hand, SDF presented positive correlation with teratozoospermia and especially with sperm midpiece defects (r = 0.171; p = 0.02). However, SDF did not present any correlation with age, testosterone levels and total ejaculated sperm volume. However the latter was positively correlated to spermatozoa midpiece and head defects (r = 0.156; p = 0.034; r = 0.203; p = 0.006, respectively). These results are in accordance with García-Ferreyra et al. (2014) who found that men with abnormal spermatozoa morphology showed high levels of DNA fragmentation, Sá et al. (2015) who confirmed that semen with lower concentration, motility and morphology have higher levels of SDF and showed that sperm head staining patterns are correlated with the degree of SDF. In addition, recently the study of Jakubik-Uljaszstudy et al. (2020) could confirms our results when it concluded that detailed sperm structural defects coexist with abnormal nuclear sperm DNA dispersion and that men with teratozoospermia may have a higher risk for sperm DNA damage. Limitations, reasons for caution Our study is a retrospective statistical investigation that included patients attending to the laboratory for fertility diagnosis after a period of infertility. Meta-analyzes studies in addition to more prospective-randomized-controlled-trials with couples undergoing assisted-reproductive-treatments and in comparison with fertile men are needed to confirm the relationship between SDF and spermocytogramme defects. Wider implications of the findings: These results should interest andrologists, reproductive science fundamentalists and embryologists who want to improve the investigations on the origin of infertility especially when it comes from male side. Trial registration number Not applicable

Papanicolaou-Stained Cytosmear Preparations in the Evaluation of Leucocytospermia: A Tertiary Centre Experience and Assessment of Utility.

Objectives Leucocytospermia is a rare cause of infertility with a variable incidence in infertile men. In many andrology laboratories, semen analysis is primarily centered on analyzing basic parameters (sperm count, motility, and viability). We examined the role of cytomorphological analysis on Papanicolaou (PAP)-stained smears in the work up of male infertility, with special reference to leukocytospermia and assessed the morphological features of sperms in these cases. Materials and Methods All cases signed out as “leukocytospermia” between November 2017 and January 2018 were evaluated. Cases showing pus cells (≥ 1/5hpf) on cytosmear evaluation were also analyzed. Parameters obtained on modified Neubauer’s chamber, wet mount preparations, and autoanalyzer SQA-IIC-P (Medical Electronic Systems, Israel) were tabulated. PAP-stained smears were examined to assess morphological defects and other findings, if any. Results Out of 348 semen samples, 6 (1.72%) were diagnosed as leukocytospermia, all displaying pus cells on cytomorphological evaluation. Five cases having white blood cells < 1 × 10 9 /L were analyzed, which displayed the presence of pus cells (≥ 1/5 hpf) on cytosmear preparations (1.43%). Nine cases (81.8%) showed reduced motile sperm concentration, functional sperm concentration, and sperm motility index. Morphological evaluation of the PAP-stained smears showed mid-piece defects in 18.1% cases. Conclusion Diligent cytomorphological evaluation of semen is essential to diagnose leukocytospermia, assess morphological defects, and serve as a quality control measure.

Melatonin-Caffeine Combination Modulates Gamma Radiation-induced Sperm Malformations in C57BL/6 Male Mice at Sublethal Dose of Gamma Radiation.

Aims:The aim of this study was to assess the protective effect of the melatonin–caffeine combination against γ radiation-induced alterations in the morphological characteristics of sperms.Settings and Design:C57BL/6 male mice (n = 30) were randomly divided into five groups: control, radiation (2 Gy), melatonin (100 mg/kg body wt.) + radiation (2 Gy), caffeine (30 mg/kg body wt.) + radiation (2 Gy), melatonin–caffeine (100–30 mg/kg body wt.) + radiation (2 Gy).Materials and Methods:All the mice were sacrificed 24 h postirradiation, and cauda epididymis was collected. In this study, sperm concentration along with any abnormality in their morphology (amorphous heads, pinheads, hookless, coiled tails, midpiece defect, and tail-less) was observed in the control and treatment group of animals.Results:Radiation exposure (2 Gy) considerably decreases the sperm count when compared with the control group. However, pretreatment with melatonin and melatonin–caffeine combination before gamma irradiation increased the sperm count (P < 0.05), but with caffeine alone could not produce a significant difference. The higher rate of abnormal sperms was observed in the γ-irradiated mice when compared with the control group (P < 0.05). Besides, the numbers of sperm that are hookless and coiled tails were significantly increased after irradiation. Melatonin significantly reduced the number of sperm with amorphous heads and coiled tails. Melatonin–caffeine combination further reduced sperm malformations when compared with the melatonin + 2 Gy radiation and caffeine + 2 Gy radiation group.Conclusions:This study suggests that caffeine exerts a protective effect when given in combination with melatonin against gamma irradiation in sperms of C57BL/6 mice and could be a potent combination for the development of radioprotector.