P–104 Assessment of sperm motility according to WHO classification using convolutional neural networks

Study question Following 6thWHO (2021), we analysed the correlation between DNA fragmentation index (DFI), Human papillomavirus (HPV), and seminal parameters, highlighting slow and rapid progressive motility alterations. Summary answer DFI rates and seminal parameters correlated with rapid, slow, and progressive motility. However, HPV-positivity caused the loss of association between DFI and slow progressive motility. What is known already HPV detection in semen samples has long opened an investigation into its influence on male infertility. Some studies indicate that HPV can affect sperm quality and DFI, while others have failed to find any correlation. With reference to 2010 WHO guidelines, our latest work highlighted how HPV positivity significantly impairs progressive motility, morphology, and immotile sperm rate. Since the latest 2021 WHO guidelines included the evaluation of slow and rapid progressive motility and DFI, we analysed if these new parameters and the other conventional parameters could be altered by HPV infection. Study design, size, duration From August 2021 to December 2022, 121 semen samples were collected from male partners of HPV-positive women attending in vitro fertilization (IVF). Every specimen underwent DFI evaluation, analysis of seminal parameters, and HPV test. Participants/materials, setting, methods Seminal samples were collected by masturbation after 3-5 days of sexual abstinence. The inclusion criteria were as follows: no other sexually transmitted infections, no genetic diseases, and no inflammatory disorders. Sperm concentration, morphology, non-progressive and immotile sperms, and both slow and rapid progressive motility were evaluated according to WHO 2021 guidelines. DFI analysis was assessed by sperm chromatin dispersion test (SCD), while HPV-DNA detection was performed using InnoLipa HPV Genotyping Extra II (Fujirebio, Tokyo, Japan). Main results and the role of chance Of the 121 semen samples tested, 60 (49.6%) were HPV-positive and 61 (50.4%) were HPV-negative. DFI rates showed a significant negative correlation with rapid progressive motility in both groups and a positive correlation with slow progressive motility in the HPV-negative group. Conversely, the significance of the correlation between DFI and slow progressive motility was completely lost in HPV-positive patients. Sperm concentration, normal forms and immotile spermatozoa percentages were correlated with both motility parameters in the HPV-negative group. Similar results were observed in HPV-positive samples, except for the normal form rate, which was not associated with slow progressive motility. In addition, the same samples displayed a negative correlation between non-progressive motility and rapid progressive motility, absent in HPV-negative samples. Significant associations were found also for the derived parameter of progressive motility, which was correlated with DFI, sperm concentration, immotile sperm, and normal forms rate in both groups. The results suggest how high DFI rates, in the presence or absence of HPV infection, could affect reproductive health through a consistent impairment of spermatozoa motility. In particular, the distinction of slow and rapid progressive motility by WHO 2021 allows a deeper understanding of the possible correlations between DFI, semen parameters and HPV infection. Limitations, reasons for caution This is a preliminary study characterized by a small number of samples. Therefore, confirmation of these findings requires the enlargement of the patient cohort, which is already taking place. Wider implications of the findings Our results highlight how the introduction of the new WHO 2021 evaluation criteria, i.e. DFI, and slow and rapid progressive motility, provides additional information about sperm quality and the impact of HPV infection on it. Trial registration number Not applicable

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.

Acute Effects of Blood Transfusion on Spermatogenesis and Pituitary Gonadal axis in Eugonadal Males with Beta Thalassemia Major (pilot study)

Prostate-specific antigen (PSA) is important for semen liquefaction and sperm motility. Anti-PSA antibodies may lead to immune infertility. The present study was conducted to evaluate the impact of seminal anti-PSA antibodies on semen parameters in fertile and infertile men. A cross-sectional analytic study was conducted on 105 fertile men (≥21-50years) having biological children (within last 2years) with normal semen analysis as controls and 105 infertile men with abnormal semen analysis as cases. All semen samples were cryopreserved till 210 samples were collected, followed by estimation of anti-PSA antibodies using an indirect enzyme-linked immunosorbent assay technique. Mean±standard deviation (s.d.) age of 210 participants was 30.0±4.65years. Mean±s.d. levels of seminal anti-PSA antibodies in infertile men were 27.82±102.19ng/mL and in fertile men -30.45±49.49ng/mL (P=0.001). A significant negative correlation was observed between anti-PSA antibody levels and sperm concentration (P=0.013), rapid progressive motility (P=0.001), slow progressive motility (P=0.006), progressive sperm motility (P=0.001), and normal morphology (P=0.001), and significant positive correlation was observed with immotile sperms (P=0.001). The overall accuracy of anti-PSA antibody for differentiating infertile from fertile men was 63.33%. Seminal anti-PSA antibodies were significantly correlated with semen parameters in fertile and infertile men with an accuracy of 63.33%. A negative correlation was observed between antibody levels and progressive sperm motility. Seminal anti-PSA antibodies can be used as a biomarker for male infertility assessment.

To determine whether semen quality in Slovenians has changed over 14 years (1983-96), we analysed retrospectively the semen of 2343 healthy men with a normal spermiogram, who were partners of women with tubal infertility included in the IVF-ET programme. Age at semen collection, duration of sexual abstinence, semen volume, sperm concentration, total sperm count, percentage of spermatozoa with progressive motility, and normal morphology were determined. Multiple regression analysis was used to assess the changes in sperm characteristics according to the year of semen collection, year of the man's birth and the duration of sexual abstinence. Semen volume, sperm concentration, sperm count and total sperm motility did not change between 1983 and 1996, whereas between 1988 and 1996 rapid progressive sperm motility decreased by 0.95% per year (p < 0.0001). Semen volume, sperm concentration, and sperm count increased with duration of sexual abstinence. After adjustment for the year of semen collection and duration of sexual abstinence, multiple regression analysis showed that sperm concentration decreased by 0.67% per each successive year of birth (p = 0.03). Thus the sperm concentration decreased from 87.6 x 10(6)/mL in men born in the 1940s to 77.3 x 10(6)/mL in those born between 1956 and 1960. After 1960, sperm concentration was found to increase. In 2343 healthy men, no decline in semen quality, except in rapid progressive motility, was observed in the study period. Lower sperm concentration was found among men born between 1950 and 1960. This could be related to worse socio-economic status, stress or negative environmental factors in this time period.

Semen analysis is central in infertility investigation. Manual assessment of sperm motility according to the WHO recommendations is the golden standard, and extensive training is a requirement for accurate and reproducible results. Deep convolutional neural networks (DCNN) are especially suitable for image classification. In this study, we evaluated the performance of the DCNN ResNet-50 in predicting the proportion of sperm in the WHO motility categories. Two models were evaluated using tenfold cross-validation with 65 video recordings of wet semen preparations from an external quality assessment programme for semen analysis. The corresponding manually assessed data was obtained from several of the reference laboratories, and the mean values were used for training of the DCNN models. One model was trained to predict the three categories progressive motility, non-progressive motility, and immotile spermatozoa. Another model was used in predicting four categories, where progressive motility was differentiated into rapid and slow. The resulting average mean absolute error (MAE) was 0.05 and 0.07, and the average ZeroR baseline was 0.09 and 0.10 for the three-category and the four-category model, respectively. Manual and DCNN-predicted motility was compared by Pearson’s correlation coefficient and by difference plots. The strongest correlation between the mean manually assessed values and DCNN-predicted motility was observed for % progressively motile spermatozoa (Pearson’s r = 0.88, p < 0.001) and % immotile spermatozoa (r = 0.89, p < 0.001). For rapid progressive motility, the correlation was moderate (Pearson’s r = 0.673, p < 0.001). The median difference between manual and predicted progressive motility was 0 and 2 for immotile spermatozoa. The largest bias was observed at high and low percentages of progressive and immotile spermatozoa. The DCNN-predicted value was within the range of the interlaboratory variation of the results for most of the samples. In conclusion, DCNN models were able to predict the proportion of spermatozoa into the WHO motility categories with significantly lower error than the baseline. The best correlation between the manual and the DCNN-predicted motility values was found for the categories progressive and immotile. Of note, there was considerable variation between the mean motility values obtained for each category by the reference laboratories, especially for rapid progressive motility, which impacts the training of the DCNN models.

The effect of various air pollution and participants' age on semen quality in southern Taiwan

Effects of Tyrosine Kinase Inhibitors On Spermatogenesis and Pituitary Gonadal Axis in Males with Chronic Myeloid Leukemia

Acute effects of blood transfusion on pituitary gonadal axis and sperm parameters in adolescents and young men with thalassemia major: a pilot study

A live motile sperm sorting device (LensHooke® CA0) developed to prevent the deleterious effects of centrifugation was evaluated comparatively with conventional density-gradient centrifugation (DGC) and microfluidic-based device (Zymot) in sperm selection. Semen samples from 239 men were collected. CA0 under different incubation intervals (5, 10, 30, and 60min) and temperatures (20, 25, and 37℃) was conducted. The sperm quality in CA0-, DGC-, and Zymot-processed samples was then comparatively evaluated. Semen parameters included concentration, motility, morphology, motion kinematics, DNA fragmentation index (DFI), and the rate of acrosome-reacted sperm (AR). Total motility and motile sperm concentration increased in a time- and temperature-dependent manner and the total motility peaked for 30min at 37℃. In paired analysis, CA0 showed significantly higher total motility (94.0%), progressive motility (90.8%), rapid progressive motility (83.6%), normal morphology (10.3%), and lower DFI (2.4%) and AR (4.7%) than the other two methods in normozoospermic samples (all p < 0.05). For non-normozoospermic samples, CA0 had significantly better results than the other two methods (total motility 89.2%, progressive motility 80.4%, rapid progressive motility 74.2%, normal morphology 8.5%, DFI 4.0%, and AR 4.0%; all p < 0.05). CA0 yielded spermatozoa with enhanced sperm fertilization potentials; DFI was minimized in samples processed by CA0. CA0 was effective for both normal and abnormal semen samples due to its consistent selection efficiency.

From the European Academy of Andrology. Italian pilot study for an external quality control scheme in semen analysis and antisperm antibiotics detection.

Study question Is paternal age associated with lower mitochondrial activity in fresh and cryopreserved semen? Summary answer Paternal age does not appear to severely compromise sperm mitochondrial activity in fresh semen but determines higher mitochondrial vulnerability to sperm cryopreservation. What is known already The impact of paternal age on male fertility has not been fully clarified although growing evidence suggests age-related loss of sperm quality. Mitochondrial activity has been recognized as a reliable marker of sperm functionality, reflecting motility, vitality and fertilization competence. Sperm cryopreservation has been largely utilized in fertility preservation and ART schemes, despite its potential harm on cell membranes including those of the mitochondria. The influence of advanced paternal age (APA) on sperm vulnerability to cryopreservation-induced cell damage is still not known. Study design, size, duration Twenty three normospermic patients, 11 of which ≤ 35 (non-APA) and 12 ≥ 42 years old (APA), provided semen samples by masturbation after 1-5 days of abstinence, between April and August of 2022. A 250µl semen aliquot was frozen. Sperm concentration, motility, vitality, morphology and mitochondrial functionality were compared in fresh semen from non-APA vs. APA patients, while motility and mitochondrial functionality were compared in thawed semen from both groups with the two-tailed Student t-test. Participants/materials, setting, methods Fresh and thawed semen samples were provided by patients under evaluation for couple infertility treatment in our fertility center. Semen was cryopreserved with CryoSperm (Origio) and analyzed after rapid thawing and washing/dilution in Sperm Preparation Medium (Origio). Sperm vitality was assessed with VitalScreen (FertiPro) and mitochondrial functionality was evaluated with MitoTracker Red CMXRos (Cell Signalling Technology), through the percentage of stained cells and fluorescence intensity measured in 75 spermatozoa per patient with the ImageJ software. Main results and the role of chance Mean ages for non-APA and APA patients were 32.6 and 45.3 years, respectively. Age groups did not differ for any of the parameters assessed in fresh semen (volume: 2.3±1.1 vs. 2.6±1.2mL; concentration: 58±32 vs. 47±20 x106/mL; rapid progressive motility 5±5 vs. 5±8%; slow progressive motility 40±10 vs. 36±10%; non-progressive motility 10±5 vs. 10±3%; immotile: 44±9 vs. 49±13%; normal morphology: 6±3 vs. 5±1%; vitality: 71±8 vs. 66±15%, for non-APA and APA, respectively). However, non-APA patients presented a higher percentage of spermatozoa with rapid progressive motility (5±7 vs. 0%; p = 0.04) and a lower percentage of immotile spermatozoa (76±12 vs. 88±9%; p = 0.02) after thawing. Regarding mitochondrial functionality, no differences were observed in fresh semen from different age groups [93±11 vs. 92±7% stained cells; 3.33±1.57 vs. 3.35±1.49 (fluorescence arbitrary units), for non-APA and APA, respectively). In cryopreserved semen, however, although age groups did not differ for the percentage of stained spermatozoa (92±10 vs. 90±5%), fluorescence intensity was higher in spermatozoa from non-APA patients (2.53±1.63 vs. 1.76±0.54; p &amp;lt; 0.01). Limitations, reasons for caution Our study is limited by the potential interference of confounding factors not equally distributed in age groups. The conclusions from this study must be confirmed in other patient populations with different race/genetics and habits. Wider implications of the findings Our study sheds light on the impact of paternal age on sperm quality, a topic highly relevant to reproductive medicine still not fully understood. We provide evidence that APA is associated with higher vulnerability of mitochondria to the stress induced by cryopreservation and its metabolic consequences. Trial registration number not applicable

BackgroundThere is clinical evidence to show that sperm DNA damage could be a marker of sperm quality and extensive data exist on the relationship between DNA damage and male fertility status. Detecting such damage in sperm could provide new elements besides semen parameters in diagnosing male infertility. We aimed to assess sperm DNA fragmentation and oxidation and to study the association between these two markers, routine semen parameters and malondialdehyde formation.MethodsSemen samples from 55 men attending the Histology-Embryology Laboratory of Sfax Faculty of Medicine, Tunisia, for semen investigations were analysed for sperm DNA fragmentation and oxidation using flow cytometry. The Sperm was also assessed spectrophotometrically for malondialdehyde formation.ResultsWithin the studied group, 21 patients were nonasthenozoospermic (sperm motility ≥ 50%) and 34 patients were considered asthenozoospermic (sperm motility < 50%). A positive correlation was found between sperm DNA fragmentation and oxidation (p = 0.01; r = 0.33). We also found a negative correlation between sperm DNA fragmentation and some sperm parameters: total motility (p = 0.001; r = -0.43), rapid progressive motility (type a motility) (p = 0.04; r = -0.27), slow progressive motility (type b motility) (p = 0.03; r = -0.28), and vitality (p < 0.001; r = -0.65). Sperm DNA fragmentation was positively correlated with coiled tail (p = 0.01; r = 0.34). The two parameters that were found to be correlated with oxidative DNA damage were leucocytes concentrations (p = 0.01; r = 0.38) and broken neck (p = 0.02; r = 0.29). Sperm MDA levels were negatively correlated with sperm concentration (p < 0.001; r = -0.57), total motility (p = 0.01; r = -0.35) and type a motility (p = 0.03; r = -0.32); but not correlated with DNA fragmentation and DNA oxidation.ConclusionsOur results support the evidence that oxidative stress plays a key role in inducing DNA damage; but nuclear alterations and malondialdehyde don't seem to be synchronous.

Although the effect of maternal age on fertility is well known, it is unclear whether paternal age also affects fertility. The aim of this retrospective study was to establish an association between the age of the individuals from Medellin, Colombia with semen volume, rapid progressive motility (a), total progressive motility (a + b) and concentration. We evaluated semen volume using a graduated tube, progressive motility using light microscopy (40×) and sperm concentration using a Makler Chamber. Semen samples were grouped according to age into three arbitrary groups (≤ to 30 years; between 31 and 39 years; and ≥ to 40 years). The semen volume, rapid progressive motility (a) and total progressive motility (a + b), concentration and total sperm count were found to be inversely related to age (p < 0.05). The reduction in semen parameters of 1364 men attending an andrology center was associated with increasing age of the individuals.

To study the effect of supplementing biotin to sperm preparation medium on the motility of frozen-thawed spermatozoa. Semen samples of men attending the University infertility clinic (n = 105) were cryopreserved using glycerol-egg yolk-citrate buffered cryoprotective medium in liquid nitrogen. After a period of two weeks, the semen samples were thawed and the motile spermatozoa were extracted by swim-up technique using Earle's balanced salt solution (EBSS) medium supplemented with either biotin (10 nM) or pentoxifylline (1 mM). The post-wash motility was observed up to 4 h after incubation. Both biotin and pentoxifylline supplementation resulted in significant increase in total motility (p < 0.05), progressive motility (p < 0.001) and rapid progressive motility (p < 0.05 v/s biotin and p < 0.01 v/s pentoxifylline) compared to the control at 1 h post-incubation period. Significantly higher percentage of total (p < 0.01, p < 0.05 in biotin and pentoxifylline respectively), progressive (p < 0.001) and rapid progressive motilities (p < 0.01) were observed in these two groups even at 2 h compared to the control. In the control group at 4 h after incubation, ~11% decline in total motility and ~8% decline in progressive motility was observed. However, in both biotin and pentoxifylline group the motility was significantly higher than control (p < 0.001). No significant difference in the motility was observed between biotin and pentoxifylline groups at any of the time intervals studied. Biotin can enhance the sperm motility and prolong the survival of frozen-thawed semen samples which may have potential benefit in assisted reproductive technology field.