Artículos

Cumulus matrix selection leads to isolation of spermatozoa with better motility, morphology, and lower DNA fragmentation

Maria Handzhiyska, Rumiana Ganeva, Dimitar Parvanov, Margarita Ruseva, Petar Eftimov, Vilyana Georgieva, Denitsa Velikova and Georgi Stamenov

Department of Research, Nadezhda Women’s Health Hospital, Sofi, Bulgaria; Department of Cell and Developmental Biology, Faculty of Biology, SU St. Climent Ohridski, Sofi, Bulgaria; Department of Andrology, Nadezhda Women’s Health Hospital, Sofi, Bulgaria; Department of Obstetrics and Gynecology, Nadezhda Women’s Health Hospital, Sofi, Bulgaria

Abstract: The objective of this study was to compare the motility, morphology, and levels of DNA fragmentation of spermatozoa subjected to conventional swim-up or cumulus matrix (CM) sperm selection. Semen samples were collected from 60 normozoospermic men at a private hospital between December 2021 and March 2022. After liquefaction, semen samples were separated into two portions – one part was subjected to conventional swim-up preparation, and the remaining spermatozoa were subjected to CM selection. The CM was obtained by mechanical isolation from healthy donor oocytes. Semen analysis and evaluation of sperm were performed according to the WHO 6th Edition Laboratory Manual and Kruger’s strict criteria, respectively. Sperm DNA fragmentation (SDF, %) of the two preparations was evaluated using the Halosperm G2 detection Kit (Halotech, Madrid, Spain). Wilcoxon rank-sum test
was used to compare the characteristics of spermatozoa obtained by the two preparations. Spermatozoa selected by CM showed signifiantly better rapidly progressive motility (43.5% vs 30.6%, respectively, P < 0.001), a higher percentage of morphologically normal forms (14.0% vs 9.0%, respectively, P < 0.05), and lower levels of SDF (26.0% vs 45.0%, P < 0.05)  compared to those prepared by conventional swim-up. Moreover, the incidence of multiple sperm defects was considerably lower in the samples that underwent CM selection compared to those that did not (30.0% vs 49.0%, respectively, P < 0.05).The selection by CM signifiantly increases sperm motility and reduces morphologically abnormal spermatozoa and DNA  ragmentation rates compared to the conventional swim-up preparation. The application of this selection technique may increase the chances of successful IVF outcomes.

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Reproduction & Fertility – https://doi.org/10.1530/RAF-23-0052

Received 24 July 2023, Accepted 25 April 2024, Available online 25 April 2024, Version of Record published 10 June 2024

Co-exposure to 55 endocrine-disrupting chemicals linking diminished sperm quality: Mixture effect, and the role of seminal plasma docosapentaenoic acid

Chang Gao, Nan Sun, Jinying Xie, Jiehao Li, Lin Tao, Lijuan Guo, Lan Shi, Xiaojin He, Xiaoting Shen, Hua Wang, Pan Yang, Adrian Covaci, Yichao Huang

Department of Toxicology, School of Public Health, Center for Big Data and Population Health of IHM, Key Laboratory of Environmental Toxicology of Anhui Higher
Education Institutes, MOE Key Laboratory of Population Health Across Life Cycle, Anhui Medical University, Hefei, China; Department of Public Health and Preventive Medicine, China Greater Bay Area Research Center of Environmental Health, School of Medicine, Jinan University, Guangzhou, China; NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract, Anhui Medical University, Hefei, Anhui Province, China; Reproductive Medicine Center, Department of Obstetrics and Gynecology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Reproductive Medicine Center, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China; Toxicological Center, University of Antwerp, Wilrijk, Belgium; Clinical Research Center, Suzhou Hospital of Anhui Medical University, Anhui Medical University, Suzhou, China.

Abstract: Isolated effects of single endocrine-disrupting chemicals (EDCs) on male reproductive health have been studied extensively, but their mixture effect remains unelucidated. Previous research has suggested that consuming diet enriched in omega-3 polyunsaturated fatty acids (PUFA) might be beneficial for reproductive health, whether omega-3 PUFA could moderate the effect of EDCs mixture on semen quality remains to be explored. In this study of 155 male recruited from a reproductive health center in China, we used targeted-exposomics to simultaneously measure 55 EDCs in the urine for exposure burden. Regression analyses were restricted to highly detected EDCs (≥55%, n = 34), and those with consistently elevated risk were further screened and brought into mixture effect models (Bisphenol A, ethyl paraben, methyl paraben [MeP], benzophenone-1 [BP1], benzophenone-3, mono(3-carboxypropyl) phthalate [MCPP]). Bayesian Kernel Machine Regression (BKMR) and quantile-based g-computation (QGC) models demonstrated that co-exposure to top-ranked EDCs was related to reduced sperm total (β = − 0.18, 95%CI: − 0.29 – − 0.07, P = 0.002) and progressive motility (β = − 0.27, 95%CI: − 0.43 – − 0.10, P = 0.002), but not to lower semen volume. BP1, MeP and MCPP were identified as the main
effect driver for deteriorated sperm motion parameters using mixture model analyses. Seminal plasma fatty acid profiling showed that high omega-3 PUFA status, notably elevated docosapentaenoic acid (DPA, C22:5n-3) status, moderated the association between MCPP and sperm motion parameters (total motility: β = 0.26, 95% CI: 0.01 – − 0.51, Pinteraction = 0.047; progressive motility: β = 0.64, 95%CI: 0.23 – 1.05, Pinteraction = 0.003). Coexposure to a range of EDCs is mainly associated with deteriorated sperm quality, but to a lesser extent on sperm
quantity, high seminal plasma DPA status might be protective against the effect. Our work emphasizes the importance of exposomic approach to assess chemical exposures and highlighted a new possible intervention target for mitigating the potential adverse effect of EDCs on semen quality.

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Environment International – https://repository.uantwerpen.be/docman/irua/49d3b2motoM1a
Received 27 January 2024; Received in revised form 5 March 2024; Accepted 7 March 2024

Analyse du sperme assistee par ordinateur : experience du Laboratoire d’Histologie Embryologie Cytogenetique et Biologie de la Reproduction du Centre Hospitalier Universitaire de Bogodogo avec le systeme SCA®

AWEH Adjongba Bruno; OUEDRAOGO Rakissida Alfred; OUEDRAOGO Rakiswende Alexis; HAMADOU Ibrahim; SANOU-LAMIEN Assita; OUEDRAOGO Aimé Sosthène; LOMPO Olga

 Service d’Histologie Embryologie Cytogénétique et Biologie de la Reproduction du Centre Hospitalier Universitaire de Bogodogo; Laboratoire d’Histo-Embryologie Cytogénétique et Pathologie Cellulaire « Pr Ag Moumouni Hassane » /Faculté des Sciences de la Santé/Université Abdou Moumouni de Niamey; Département des Sciences Fondamentales et Mixtes, UFR/SDS, Université Joseph KI-ZERBO, Ouagadougou, Burkina Faso

Résumé: La question de l’infertilité est de plus en plus préoccupante de nos jours. Dans la moitié des cas d’infertilité du couple, une origine masculine est évoquée. Le permogramme/spermocytogramme est l’un des premiers examens à être prescrit à l’homme dans la démarche diagnostique de l’infertilité masculine. C’est un examen de biologie médicale dont la réalisation comporte une grande part de subjectivité avec une importante variabilité inter-opérateur et inter-laboratoire. Les systèmes d’analyses automatisés du sperme dont le système SCA® (Sperm Class Analyzer) ont été développé afin de limiter ces variations. Ce système a révolutionné la pratique de cet examen de routine au sein des laboratoires
de biologie de la reproduction. Ce système offre des avantages dans le sens où il permet une analyse automatisée ou semi-automatisée, précise, fiable des paramètres du sperme. Bien que l’analyse soit quasiment automatique, le système offre la possibilité au biologiste de corriger manuellement les résultats des analyses.

Annales de l’Université Joseph KI-ZERBO
Réception : 30 / 01 / 2023 Révision : 20 / 03 / 2023 Acceptation : 22 / 01 / 2024

Association between self-reported mobile phone use and the semen quality of young men

Rita Rahban, Alfred Senn, Serge Nef, Martin Rӧӧsli

Swiss Centre for Applied Human Toxicology (SCAHT), University of Geneva, Geneva, Switzerland; Department of Genetic Medicine and Development, University of Geneva, Geneva, Switzerland; Department of Epidemiology and Public Health, Swiss Tropical and Public Health Institute, Allschwill, Switzerland; University of Basel, Basel, Switzerland

Objectives: To investigate the association between mobile phone exposure and semen parameters.

Design: A nationwide cross-sectional study.

Setting: Andrology laboratories in close proximity to 6 army recruitment centers.

Patients: In total, 2886 men from the general Swiss population, 18–22 years old, were recruited between 2005 and 2018 during military conscription.

Intervention: Participants delivered a semen sample and completed a questionnaire on health and lifestyle, including the number of hours they spent using their mobile phones and where they placed them when not in use.

Main Outcome Measures: Using logistic and multiple linear regression models, adjusted odds ratios and β coefficients were determined, respectively. The association between mobile phone exposure and semen parameters such as volume, sperm concentration, total sperm count (TSC), motility, and morphology was then evaluated.

Results: A total of 2759 men answered the question concerning their mobile phone use, and 2764 gave details on the position of their mobile phone when not in use. In the adjusted linear model, a higher frequency of mobile phone use (>20 times per day) was associated with a lower sperm concentration (adjusted β: −0.152; 95% confidence interval: −0.316; 0.011) and a lower TSC (adjusted β: −0.271; 95% confidence interval: −0.515; −0.027). In the adjusted logistic regression model, this translates to a 30% and 21% increased risk for sperm concentration and TSC to be below the World Health Organization reference values for fertile men, respectively. This inverse association was found to be more pronounced in the first study period (2005–2007) and gradually decreased with time (2008–2011 and 2012–2018). No consistent associations were observed between mobile phone use and sperm motility or sperm morphology. Keeping a mobile phone in the pants pocket was not found to be associated with lower semen parameters.

Conclusion: This large population-based study suggests that higher mobile phone use is associated with lower sperm concentration and TSC. The observed time trend of decreasing association is in line with the transition to new technologies and the corresponding decrease in mobile phone output power. Prospective studies with improved exposure assessment are needed to confirm whether the observed associations are causal.

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Fertility and Sterility – https://doi.org/10.1016/j.fertnstert.2023.09.009
Received June 29, 2022; revised September 11, 2023; accepted September 15, 2023; Published October 31, 2023

Oxido-Reduction Potential as a Method to Determine Oxidative Stress in Semen Samples

András Balló, Péter Czétány, Kinga Székvári Busznyákné, László Márk, Nóra Mike, Attila Török, Árpád Szántó, Gábor Máté

Pannon Reproduction Institute, 8300 Tapolca, Hungary; Urology Clinic, University of Pécs Clinical Centre, 7621 Pécs, Hungary; National Laboratory on Human Reproduction, University of Pécs, 7624 Pécs, Hungary; Department of Analytical Biochemistry, Institute of Biochemistry and Medical Chemistry, University of Pécs Medical School, 7624 Pécs, Hungary; MTA-PTE Human Reproduction Scientific Research Group, 7624 Pécs, Hungary; Szentágothai Research Centre, Department of Physiology, Medical School, University of Pécs, 7624 Pécs, Hungary.

Abstract: There are different estimates for the incidence of infertility. Its occurrence may vary from area to area, but on average, it affects 15% of couples and 10–12% of men worldwide. Many aspects of infertility can be linked to reactive oxygen species (ROS) and the process of oxidative stress (OS). The association between poor semen quality and OS is well known. Unfortunately, there is no accepted protocol for the diagnosis and treatment of OS in andrology. Oxido-reduction potential (ORP) measurement is a new method for determining the ratio between oxidant and antioxidant molecules. Currently, ORP measurement is one of the fastest and most user-friendly methods of andrological OS determination and our goals were to confirm published correlations between ORP values and sperm parameters, examine how sperm concentration influences these results, and investigate whether intracellular ROS formations are also manifested in the ORP values or not after artificial ROS induction. Intracellular ROS formations were induced by menadione (superoxide anion inducer), hydrogen peroxide, and tert-butyl hydroperoxide (lipid peroxidation inducer) treatments; sperm parameters like motility and viability were determined with an SCA Scope system, and ORP changes were recorded by the Mioxsys system. Significant correlations were noticed among the ORP, spermatozoa concentration, motility, progressive motility, and viability. Nevertheless, only the ORP value after normalization with the sperm count correlated with these parameters. Due to normalization, very low and very high sperm concentrations can give misleading results. The means of the non-normalized ORP values were almost the same. All of the applied treatments resulted in decreases in the viability, motility, and progressive motility, and interestingly, altered ORP levels were detected. In addition, it was determined that seminal plasma had a significant protective effect on spermatozoa. The elimination of seminal plasma caused higher sensitivity of spermatozoa against used OS inducers, and higher ORP levels and decreased viabilities and motilities were measured. The ORP level could be a good indicator of male OS; however, in cases of low and high sperm counts, its result can be misleading. Overall, the conclusion can be drawn that ORP determination is a suitable method for detecting intracellular ROS accumulation, but it has limitations that still need to be clarified.

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Int. J. Mol. Sci. 2023, 24(15), 11981; https://doi.org/10.3390/ijms241511981
Submission received: 19 June 2023 / Revised: 16 July 2023 / Accepted: 24 July 2023 / Published: 26 July 2023

New Approaches to Define The Functional Competency of Human Sperm Subpopulations and Its Relationship to Semen Quality

Shannen Keyer, Gerhard Van der Horst, Liana Maree

Department of Medical Bioscience, Faculty of Natural Science, University of the Western Cape, Bellville, South Africa

Abstract:

Background: This study aimed at comparing a comprehensive set of functional and structural sperm characteristics between sperm motility fractions and correlating results to the standard semen parameters. By grouping related variables, our objective was to establish the predictive power of semen parameters and whether they accurately reflect the functionality of sperm motility fractions or merely a small set of parameters within individual fractions.

Materials and Methods: In this non-invasive experimental study, donor semen samples (n=55) were separated via
double density gradient centrifugation, isolating a high (HM) and low motile (LM) sperm fraction. Fractions were evaluated for percentage vitality, chromatin integrity, mature spermatozoa, motility and kinematic parameters, hyperactivation, positive reactive oxygen species, intact mitochondrial membrane potential (MMP) and acrosome reaction.

Results: HM fractions had significantly (P<0.001) enhanced percentages of induced acrosome reaction (HM, 55.6 ± 14.3%, LM, 25.0 ± 16.5%), motility and kinematic parameters, hyperactivation, vitality (HM, 70.4 ± 9.7%, LM, 47.9 ± 10.3%), mitochondrial membrane intactness (HM, 67.2 ± 10.4%, LM, 44.7 ± 15.0%) and mature spermatozoa (HM, 83.4 ± 10.0%, LM, 64.6 ± 8.2%) with intact chromatin (HM, 80.5 ± 8.1%, LM, 71.3 ± 8.0%). Various sperm morphology abnormalities correlated with LM fractions’ grouped motility parameters (range, 0.46 to 0.51; range -0.4 to -0.75), whereas combined semen traits of total motility, progressive motility, viscosity and mucus penetration (MPT) correlated with HM fractions’ grouped motility parameters (range, 0.44 to 0.84).

Conclusion: Collectively, total and progressive motility, viscosity and MPT may represent a reliable grouping of semen parameters for predicting the quality of HM sperm fractions. Separating the same donor semen samples into two significantly diverse motility sperm fractions could be a potential model in mimicking the qualities of fertile and sub-fertile males’ sperm populations and used for future research on the improvement of sperm subpopulations from males with different fertility statuses.

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Int J Fertil Steril. 2022; 16(3): 140-151. – doi: 10.22074/IJFS.2021.531517.1132.
Received: 03/June/2021, Accepted: 13/December/2021

The influence of SARS-CoV-2 on semen parameters of infected infertile male in comparison with those that noninfected

Baida Rihan Ali, Saad A. Atiyah, Heba T. Yser, Ahmed M. Khelewe, Hameed N. Hameed

Department of Pathological Analysis, College of Science, University of Thi-Qar, Nasiriya, Iraq; Department of Microbiology, College of Medicine, University of Thi-Qar, Nasiriya, Iraq; Department of Biology, College of Pure Educational Science, University of Basrah, Basrah, Iraq; Thi-Qar Health Directorate, Ministry of Health/ Environment, Nasiriya, Iraq

Abstract:

Background: Coronavirus disease (COVID-19) is an infectious disease caused by SARS COV-2 that has spread globally, the virus can cause different pathological alterations in many organs, such as the lung, kidney, and testis. The study aimed to determine the effect of COVID-19 on the seminal fluid parameters of infected infertile males compared with those who are noninfected.

Methods: The study was performed in Al-Hussein Teaching Hospital during the period from September to November, 2021 and it involved 318 patients. The patients’ info included age, address, and vaccination. The sperm count, activity, and morphology were detected using Computer-assisted semen analysis CASA (Microptic-Spain) according to the WHO manual.

Results: There were high significant differences between the infertile males who were infected with COVID- 19 and those who were vaccinated (X2 = 12.509, p = 0.001). A high significant relation (p < 0.001) was recorded between types of infection severity and volume of semen (p < 0.001) and nonprogress life sperm (C) (p < 0.001). While significant differences were shown in the moderate progression sperm (B) (p = 0.012), and morphology (p = 0.02), respectively. High significant differences were reported between the types of infection severity (count of the sperm, presence of pus, B, C and D), (p < 0.001), while a significant difference was shown between severity types in relation to A and morphology of the sperms (p = 0.021 and 0.015), respectively.

Conclusion: The severity of COVID-19 has a significant impact on infertility and sperm parameters, particularly progression and sperm morphology, despite the fact that these parameters are unrelated to vaccination.

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J Clin Lab Anal. 2022;36:e24568. – https://doi.org/10.1002/jcla.24568
Received: 17 May 2022 / Revised: 8 June 2022 / Accepted: 9 June 202 / First published: 26 June 2022

Progesterone, Myo-Inositol, Dopamine and Prolactin Present in Follicular Fluid Have Differential Effects on Sperm Motility Subpopulations

Shannen Keyser, Gerhard van der Horst, Liana Maree

Comparative Spermatology Laboratory, Department of Medical Bioscience, Faculty of Natural Sciences, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa

Abstract: Considering the challenges surrounding causative factors in male infertility, rather than relying on standard semen analysis, the assessment of sperm subpopulations and functional characteristics essential for fertilization is paramount. Furthermore, the diagnostic value of sperm interactions with biological components in the female reproductive tract may improve our understanding of subfertility and provide applications in assisted reproductive techniques. We investigated the response of two sperm motility subpopulations (mimicking the functionality of potentially fertile and sub-fertile semen samples) to biological substances present in the female reproductive tract. Donor semen was separated via double density gradient centrifugation, isolated into high (HM) and low motile (LM) sperm subpopulations and incubated in human tubal fluid (HTF), capacitating HTF, HD-C medium, progesterone, myo-inositol, dopamine and prolactin. Treated subpopulations were evaluated for vitality, motility percentages and kinematic parameters, hyperactivation, positive reactive oxygen species (ROS), intact mitochondrial membrane potential (MMP) and acrosome reaction (AR). While all media had a significantly positive effect on the LM subpopulation, dopamine appeared to significantly improve both subpopulations’ functional characteristics. HD-C, progesterone and myo-inositol resulted in increased motility, kinematic and hyperactivation parameters, whereas prolactin and myo-inositol improved the LM subpopulations’ MMP intactness and reduced ROS. Furthermore, progesterone, myo-inositol and dopamine improved the HM subpopulations’ motility parameters and AR. Our results suggest that treatment of sub-fertile semen samples with biological substances present in follicular fluid might assist the development of new strategies for IVF treatment.

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Life 2021, 11(11), 1250; https://doi.org/10.3390/life11111250
Received: 19 October 2021 / Revised: 9 November 2021 / Accepted: 11 November 2021 / Published: 17 November 2021

Cementing the relationship between conventional and advanced semen parameters

Bashir M. Ayad, Ibukun P. Oyeyipo, Gerhard Van der Horst, Stefan S. Du Plessis

Department of Physiology, Faculty of Medicine, Misurata University, Misurata, Libya; Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa; Department of Physiology, College of Health Sciences, Osun State University, Osogbo, Nigeria; Department of Basic Medical Sciences, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai, United Arab Emirates

Abstract: 

Background: Affordable conventional semen analysis remains a fundamental procedure to be performed routinely during the diagnosis of male infertility. Advanced semen analyses provide valuable clinical insights in treatment-related decision-making, but these are highly expensive and lack universal standardization. This study aimed at determining the relationship between conventional semen parameters, measured with assistance of computer-aided sperm analysis (CASA), and a set of advanced semen tests. Basic semen analysis (n = 124) was performed according to the World Health Organization (WHO) guidelines. Sperm DNA fragmentation and intracellular superoxide (O2−•) levels were assessed by flow cytometry. Seminal plasma thiobarbituric acid reactive substances (TBARS) levels as well as superoxide dismutase (SOD) and catalase (CAT) activity were measured by spectrophotometry. Spearman’s rank correlation coefficient was used, with significance set at p < 0.05.

Results: Semen pH correlated negatively with TBARS (p < 0.01). The proportions of total and progressively motile as well as rapid spermatozoa correlated positively with CAT activity (p < 0.05). Sperm viability correlated negatively with both O2−• (p < 0.05) and DNA fragmentation (p = 0.01), while normal morphology correlated negatively with O2−• levels (p < 0.05) and positively with CAT activity (p < 0.05). Straight-line velocity (VCL) and average-path velocity (VAP) correlated negatively with both O2−• (p < 0.01) and TBARS (p < 0.01). Amplitude of lateral head displacement (ALH) correlated negatively with O2−• (p < 0.01) and DNA fragmentation (p < 0.01), while its correlation with SOD activity was positive (p < 0.05).

Conclusion: The results obtained from this study support the validity of some CASA parameters as sensitive indicators of changes in sperm oxidative status and DNA integrity. Predicting advanced from conventional parameters through the building of linear regression models should be considered for future studies.

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Middle East Fertil Soc J 26, 39 (2021) – https://doi.org/10.1186/s43043-021-00086-z
Received: 22 May 2021 / Accepted: 10 October 2021 / Published: 19 October 2021

Influence of Fruit and Vegetable Consumption on Antioxidant Status and Semen Quality: A Cross-Sectional Study in Adult Men

Dawid Madej, Dominika Granda, Dominika Granda, Ewa Sicinska, Ewa Sicinska, Joanna Kaluza, Joanna Kaluza

Department of Human Nutrition, Institute of Human Nutrition Sciences, Warsaw University of Life Sciences–SGGW, Warsaw, Poland

Abstract: The influence of fruit and vegetable consumption on semen quality by reducing oxidative stress is inconsistent. Thus, the association between the consumption of these products, antioxidant status, and semen quality was investigated in 90 men aged 18–40. The consumption of fruit and vegetables was collected using the 3-day food record method. Antioxidant status: total antioxidant capacity in semen (TAC-s) and blood (TAC-b), blood superoxide dismutase (SOD-b), glutathione reductase (GR-b), glutathione peroxidase (GPx-b), catalase (CAT-b) activity, and malondialdehyde concentration in blood (MDA-b) were measured. Sperm concentration, leukocytes in the ejaculate, vitality, motility, and sperm morphology were examined using computer-aided semen analysis (CASA). The consumption of fruit and vegetables was positively correlated with sperm concentration, vitality, motility, TAC-s, TAC-b, and SOD-b activity. The TAC-s and TAC-b were positively related to motility, TAC-s was inversely correlated with sperm tail defects. The SOD-b activity was positively correlated with vitality, motility, sperm morphology, and inversely with sperm tail defects and leukocytes in the ejaculate. Compared to the men in the first quartile of fruit and vegetable consumption (<318 g/day), those in the highest quartile (>734 g/day) had the highest sperm concentration, vitality, motility, TAC-s, TAC-b, GPx-b activity, and the lowest MDA-b concentration (based on multivariate regression models). A high consumption of fruit and vegetables may positively influence selected sperm quality parameters by improving the antioxidant status of semen and blood.

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Frontiers – https://doi.org/10.3389/fnut.2021.753843
15 October 2021

Decline in semen quality of North African men: a retrospective study of 20,958 sperm analyses of men from different North African countries tested in Tunisia over a period of 6 years (2013–2018)

Hatem Bahri, Mustapha Ben Khalifa, Maroua Ben Rhouma, Zied Abidi, Emna Abbassi, Khémais Ben Rhouma, Moncef Benkhalifa

HB Clinical Laboratory for Medical Analyses, Tunis, Tunisia; Research laboratory LR99ES11, Department of Biochemistry, La Rabta Hospital, Faculty of Medicine of Tunis, University of Tunis El Manar, Tunis, Tunisia; Laboratory of Integrated Physiology, Faculty of Science of Bizerte, University of Carthage, Tunisia; Reproductive Medicine, Reproductive Biology & Genetics, University Hospital & School of Medicine Jules Verne, Amiens, France; Peritox Laboratory, CURS, Picardie University Jules Verne, Amiens, France

Abstract: 

Background: According to numerous studies from around the world, semen quality seems to have declined dramatically in recent times. However, the data available on male fertility status and semen quality in North Africa are limited.

Aim: To investigate the status of semen quality in North-African men and to understand its variations.

Subjects & methods: 20,958 sperm-analyses (Spermogram – Spermocytogram) of North-African men (19–77 years old) consulting for infertility, performed in a private laboratory of medical analyses (Tunis, Tunisia) over a period of 6 years (2013–2018), were investigated. All patients had at least 1 year of unprotected intercourse with their partners before the test. Statistical analyses were performed using SPSS 22.0 software for windows.

Results: Libyan men presented a clear decline in all sperm parameters. A continuous decline in sperm morphology quality was shown in Tunisian and Algerian men. Mauritanian men presented a significant increase in sperm vitality with pseudo-stability in the rest of the sperm parameters during the whole study period.

Conclusion: North-African men presented remarkable decreases in their semen quality over the last decade. This data could confirm possible global common-causes that need to be identified in order to limit their negative impact on sperm quality, and consequently on male-fertility.

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Annals of Human Biology, 2021, Vol. 48, No. 4, 350-359 – https://doi.org/10.1080/03014460.2021.1957501
Received 15 December 2020 / Revised 23 May 2021 / Accepted 12 July 2021 / Published online: 07 Sep 2021

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

H Bahri, W Zidi, M Benkhalifa

HB laboratory, Andrology / Reproductive Biology, Tunis, Tunisia; La Rabta Hospital- Faculty of Medicine of Tunis- University of Tunis El Manar- Tunis- Tunisia., Research laboratory LR99ES11 Department of Biochemistry, Tunis, Tunisia

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 (<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 (>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<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 ≥15×106/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.

Human Reproduction, Volume 36, Issue Supplement_1, July 2021, deab130.042, https://doi.org/10.1093/humrep/deab130.042
Published: 06 August 2021

Improved sperm function in human sperm subpopulations: a model for studying subfertility

Shannen Keyser, Gerhard van der Horst, Liana Maree

Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa

Abstract: With many cases of male infertility being idiopathic or unknown, the necessity for in-depth functional and structural sperm analysis beyond routine basic semen analysis techniques has become crucial. Existence of subpopulations is often neglected in basic semen analysis thus sperm heterogeneity could interfere with an accurate evaluation of overall sperm quality. This study aimed to determine and compare various functional characteristics of two sperm subpopulations found within the same human semen samples before and after treatment with various capacitating media and chemicals. Normospermic donor semen samples were separated into two fractions via double density gradient centrifugation and further assessed for their sperm functional characteristics before and after treatment with capacitating media, progesterone (1.98, 3.96, 19.8 μM), and myoinositol (11 mM). High motile sperm fractions had significantly improved functional characteristics in contrast to the low motile fractions with a notable improvement in both sperm fractions’ functional characteristics after exposure to treatments. Selected sperm functional parameters in lower quality sperm subpopulations could be improved to the level of a higher quality sperm subpopulation, separated from the same semen samples. If the lower quality sperm subpopulation mimics the sperm population in subfertile males, this can be used as a model to study such subpopulations and possibly broaden the potential treatment regimens and sperm isolation procedures for ART.

Keyser S., van der Horst G., Maree L. (2021) Improved Sperm Function in Human Sperm Subpopulations: A Model for Studying Subfertility. In: Björndahl L., Flanagan J., Holmberg R., Kvist U. (eds) XIIIth International Symposium on Spermatology. Springer, Cham. https://doi.org/10.1007/978-3-030-66292-9_50
First Online: 23 July 2021

Proteomics and biomarker identification in improved sperm motility parameters after 4 h of ejaculatory abstinence

Dale M. Goss, Bashir Ayad, Maré Vlok, Suzél M. Hattingh, Gerhard van der Horst, Stefan S. du Plessis

Division of Medical Physiology, Stellenbosch University, Tygerberg, South Africa; Department of Biochemistry and LCMS-Central Analytical Facility, Stellenbosch University, Tygerberg, South Africa; Department of Basic Medical Sciences, College of Medicine, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai, UAE

Abstract: Various studies have sought to determine the optimal abstinence period after which semen samples should be collected, with many contradictory results reported. Several factors influence the semen microenvironment, and thus potentially sperm parameters. In this study, we focused on the secretions of the prostate, seminal vesicles and the epididymis. Semen samples were obtained from healthy normozoospermic males (n = 16) after 4 day and 4 h periods of ejaculatory abstinence (EA), and standard semen analysis was performed using computer-aided sperm analysis, whereas seminal plasma citric acid, neutral alpha-glucosidase and fructose concentrations were measured using assay kits. Proteomic analyses of seminal plasma proteins were performed using LC-MS/MS. There were significant decreases in total sperm count (P < 0.001), sperm concentration (P < 0.05) and semen volume (P < 0.05) after 4 h compared with 4 days ejaculatory abstinence. Furthermore, increases were observed in total sperm motility (P < 0.05), sperm progressive motility (P < 0.01), average path velocity (P < 0.001) and curvilinear velocity (P < 0.05) after a 4 h abstinence period, accompanied by significant reductions in citric acid (P < 0.05), neutral alpha-glucosidase (P < 0.01) and fructose (P < 0.01) concentrations. In addition, due to the decreased number of spermatozoa, these concentrations translated to a significant decrease in fructose (P < 0.05) per spermatozoon. A total of 2889 proteins were identified, with 24 proteins differentially expressed. These proteins have various roles in cellular processes and may influence the improvement of sperm motility after 4 h of EA. All these factors present in the seminal plasma indicate towards an intrinsic mechanism capitalising on alternative sources of energy for increased metabolic function and subsequent sperm motility.

Goss D.M., Ayad B., Vlok M., Hattingh S.M., van der Horst G., du Plessis S.S. (2021) Proteomics and Biomarker Identification in Improved Sperm Motility Parameters After 4 h of Ejaculatory Abstinence. In: Björndahl L., Flanagan J., Holmberg R., Kvist U. (eds) XIIIth International Symposium on Spermatology. Springer, Cham. https://doi.org/10.1007/978-3-030-66292-9_48
First Online: 23 July 2021

Seminal plasma metabolomics profiles following long (4-7 days) and short (2 hours) sexual abstinence periods

H. Alipour, R.K. Duus, R. Wimmer, F. Dardmeh, S.S. Du Plessis, N. Jørgensen, O.B. Christiansen, C. Hnida, H.I. Nielsen, G. Van Der Horst

Aalborg University, Department of Health Science and Technology, Biomedicine group, Aalborg, Denmark; Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark; Department of Basic Medical Sciences, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai, United Arab Emirates; Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa; University Department of Growth and Reproduction and International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, Copenhagen, Denmark; Aalborg University Hospital, Dept. of Obstetrics and Gynecology, Fertility unit, Aalborg, Denmark; Institute of Clinical Medicine, Aalborg University; University of the Western Cape, Department of Medical Biosciences, Cape Town, South Africa.

Abstract: 

Objective: Metabolomic profiling of seminal plasma has been suggested as a possible approach for a fast and non-invasive male infertility evaluation diagnosis. However, metabolomics profiles in normozoospermic men have not been thoroughly investigated, and the influence of ejaculation-abstinence has not been described. To provide interim reference values and find associations between the metabolomics profiles of human seminal plasma and length of ejaculation-abstinence period in normozoospermic men.

Study design: Semen samples collected after long (4-7 days) and short abstinence (2 hours) from 31 normozoospermic males were assessed for routine quality parameters before the seminal plasma was separated by centrifugation. Metabolomics profiles of the seminal plasma were then determined using untargeted Nuclear Magnetic Resonance Spectroscopy.

Results: In total, 30 metabolites were identified. Pyruvate showed a higher concentration, while fructose, acetate, choline, methanol, N-acetylglucosamine, O-acetylcarnitine, uridine, and sn-glycero-3-phosphocoline showed lower concentrations in samples collected after short abstinence (vs. long). All metabolites showed lower absolute amounts (volume x concentration) following shorter abstinence. However, the lower sperm concentration in samples collected after short abstinence resulted in higher absolute amounts of pyruvate and taurine per spermatozoa: pyruvate 1.92 (1.12-3.87) vs. 1.29 (0.83-2.62) (P<0.001) and taurine 0.58 (0.36-0.92) vs. 0.43 (0.28-0.95) (P<0.05) ng/106 spermatozoa. Simultaneously, there was a higher percentage of progressively motile spermatozoa in samples collected after the short abstinence.

Conclusion: The generally lower concentrations of seminal metabolites after short abstinence periods may be related to the shorter time available for secretion and collection of these metabolites by the accessory glands and the epididymides. The concomitant lower number of spermatozoa in the second ejaculate resulted in increased absolute amounts of pyruvate and taurine per spermatozoa, accompanied by increased spermatozoa motility in these samples. The simultaneous increase in percentages of motile spermatozoa and absolute amounts of pyruvate and taurine per spermatozoa after shorter abstinence might indicate that these two metabolites play a more critical role in sperm motility, which should be further investigated in future studies.

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European Journal of Obstetrics & Gynecology and Reproductive Biology; https://doi.org/10.1016/j.ejogrb.2021.07.024
Received Date: 29 March 2021 / Revised Date: 8 July 2021 / Accepted Date: 12 July 2021 / Published: July 14, 2021

The validity and reliability of computer-aided semen analyzers in performing semen analysis: a systematic review

Renata Finelli, Kristian Leisegang, Samhita Tumallapalli, Ralf Henkel, Ashok Agarwal

American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA; School of Natural Medicine, Faculty of Community and Health Sciences, University of the Western Cape, Bellville, South Africa; Case Western University, Cleveland, Ohio, USA; Department of Metabolism, Digestion and Reproduction, Imperial College London, London, UK; Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa; LogixX Pharma, Theale, Berkshire, UK

Abstract:

Background: Computer-aided sperm analyzers (CASA) are currently used worldwide for semen analysis. However, there are doubts about their reliability to fully substitute the human operator. Therefore, this study aimed to systematically review the current literature comparing results from semen evaluation by both CASA-based and manual approaches.

Methods: A systematic screening of the literature was performed based on the PRISMA guidelines and by searching on PubMed, Scopus, and Embase databases.

Results: A total of 14 studies were included. Our results showed a high degree of correlation for sperm concentration and motility when analysis was performed either manually or by using a CASA system. However, CASA results showed increased variability in low (<15 million/mL) and high (>60 million/mL) concentration specimens, while sperm motility assessment was inaccurate in samples with higher concentration or in the presence of non-sperm cells and debris. Morphology results showed the highest level of difference, due to the high amount of heterogeneity seen between the shapes of the spermatozoa either in one sample or across multiple samples from the same subject.

Conclusions: Overall, our study suggests CASA systems as a valid alternative for the evaluation of semen parameters in clinical practice, especially for sperm concentration and motility. However, further technological improvements are required before these devices can one day completely replace the human operator. Artificial intelligence-based CASA devices promise to offer higher efficiency of the analysis and improve the reliability of results.

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Translational Andrology and Urology; http://dx.doi.org/10.21037/tau-21-276
Submitted Apr 02, 2021. Accepted for publication May 27, 2021.

Comparison of commercially available chamber slides for computer-aided analysis of human sperm

Fereshteh Dardmeh, Mahmoud Heidari, Hiva Alipour

Laboratory of Regenerative Medicine, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark; Department of Biology, Islamic Azad University, Gorgan Branch, Gorgan, Iran

Abstract: Despite the increasing use of computer-aided sperm analysis (CASA) in clinical practice, there is still no golden standard for the type of slide to be used with these systems. Counting chamber depth and loading method, can profoundly influence motility and concentration estimates, thereby undermining the validity and accuracy of CASA. To contribute toward standardized sperm analysis, this study compared different commercially available capillary-filled slides including 10 and 20 µm deep Leja slides (Leja10 and Leja20); 10, 16 and 20 µm deep CellVision slides (CV10, CV16 and CV20); and drop-loaded slides including slide and coverslip (SCS) with a depth of 20.1 µm and the Makler chamber with a depth of 10 µm for sperm analysis when using CASA. The Sperm Class Analyzer (SCA) CASA system was used to assess concentration, motility, and detailed kinematic parameters of 20 normozoospermic human samples using the different chamber slides. Results were evaluated by the repeated measures ANOVA and Intraclass correlation coefficients. The Makler chamber showed significantly (P < 0.05) higher concentrations than other slides. However, there was no significant difference in the percentage of sperm in different motility groups among the slides. CV10, Leja10 and Makler showed significantly higher curvilinear-, average path- and straight-line velocity (VCL, VAP, VSL) values than other slides. In conclusion, despite the objectiveness of the assessments by CASA systems, there are still some discrepancies in the results of sperm concentration, motility and other kinematic parameters when using different commercially available slides. The possible negative influence of the sperm quality misdiagnosis on the selection of treatment strategy in a clinical setting, emphasizes the need for further standardization and quality control of the commercially available chamber slides for use with CASA. Furthermore, this study found more consistent results for capillary-filled chambers compared to drop-loaded slides, suggesting a superior method when using CASA.

Systems Biology in Reproductive Medicine – https://doi.org/10.1080/19396368.2020.1850907
Received 10 Jun 2020, Accepted 21 Oct 2020, Published online: 29 Dec 2020

Sperm DNA fragmentation is a novel biomarker for early pregnancy loss

Lesley Haddock, Stephen Gordon, Sheena E.M. Lewis, Peter Larsen, Amjad Shehata, Hassan Shehata

Examenlab Ltd, Unit 18A, Block K, Weavers Court Business Park, Linfield Road, Belfast BT12 5GH, UK; Urology at Epsom and St Helier University Hospitals NHS Trust, Dorking Rd, Epsom KT18 7EG, UK; Cryos International, Vesterbro Torv I, Aarhus 8000, Denmark; Centre for Reproductive Immunology and Pregnancy, Bramshott House, 137/139 High Street, Epsom KT19 8EH, UK; Maternal Medicine at Epsom and St Helier University Hospitals NHS Trust, Dorking Road, Epsom KT18 7EG, UK

Abstract:

Research question: Spontaneous pregnancy loss affects 10–15% of couples, with 1–2% suffering recurrent pregnancy loss and 50% of miscarriages remaining unexplained. Male genomic integrity is essential for healthy offspring, meaning sperm DNA quality may be important in maintaining a pregnancy. Does sperm DNA fragmentation measured by alkaline Comet assay act as a biomarker for early pregnancy loss?

Design: Sperm DNA fragmentation was measured by alkaline Comet test in 76 fertile donors and 217 men whose partners had recently experienced miscarriage. Couples were divided into five groups for analysis: one miscarriage after spontaneous conception; two or more miscarriages after spontaneous conception; one miscarriage after fertility treatment; two or more miscarriages after fertility treatment and biochemical pregnancy.

Results: Receiver operator characteristic curve analysis was used to determine ability of the average Comet score (ACS), low Comet score (LCS) and high Comet score (HCS) to diagnose miscarriage and develop clinical thresholds comparing men whose partners have miscarried with men with recently proven fertility. Male partners of women who had miscarried had higher sperm DNA damage (ACS 33.32 ± 0.57%) than fertile men (ACS 14.87 ± 0.66%; P < 0.001). Average Comet score, HCS and LCS all have promise as being highly predictive of sporadic and recurrent miscarriage using clinical thresholds from comparisons with fertile men’s spermatozoa: receiver operating characteristic curve AUC for ACS ≥26%, 0.965; LCS ≤70%, 0.969; HCS ≥2%, 0.883; P <0.0001.

Conclusions: Sperm DNA damage measured by the alkaline Comet has promise as a robust biomarker for sporadic and recurrent miscarriage after spontaneous or assisted conception, and may provide novel diagnoses and guidance for future fertility pathways.

Reproductive Biomedicine Online (RBMO) – DOI: https://doi.org/10.1016/j.rbmo.2020.09.016
Published: September 21, 2020

Microgravity effects on frozen human sperm samples

M. Boada, A. Perez-Poch, M. Ballester, S. García-Monclús, D. V. González, S. García, P. N. Barri & A. Veiga

Women’s Health Dexeus, Department of Obstetrics, Gynaecology and Reproduction, Hospital Universitari Dexeus, Avinguda Carles III 71-75, 08028, Barcelona, Spain; Universitat Politècnica de Catalunya, UPC BarcelonaTech, EEBE Campus Diagonal-Besòs, C. E. Maristany 16, 08019, Barcelona, Spain; Aeroclub Barcelona-Sabadell, Sabadell Airport, Carretera de Bellaterra s/n, 08205 Sabadell, Barcelona, Spain; Women’s Health Dexeus, Unit of Biostatistics, Avinguda Carles III 71-75, 08028, Barcelona, Spain; Barcelona Stem Cell Bank, Centre of Regenerative Medicine in Barcelona, Hospital Duran i Reynals, Gran Via de l’Hospitalet 199, 08908 Hospitalet de Llobregat, Barcelona, Spain.

Abstract:

Purpose: Microgravity has severe effects on cellular and molecular structures as well as on metabolic interactions. The aim of this study is to investigate the effects of microgravity (μg) exposure on human frozen sperm samples.

Methods: Sibling samples from 15 normozoospermic healthy donors were frozen using glycerol as cryoprotectant and analyzed under microgravity and ground conditions. Microgravity was obtained by parabolic flights using a CAP10B plane. The plane executed 20 parabolic maneuvers with a mean of 8.5 s of microgravity for each parabola.

Results: Frozen sperm samples preserved in cryostraws and stored in a secure and specific nitrogen vapor cryoshipper do not suffer significant alterations after μg exposure. Comparing the study group (μg) and the control group (1 g), similar results were obtained in the main parameters studied: sperm motility (M/ml) 13.72 ± 12.57 vs 13.03 ± 12.13 (− 0.69 95% CI [− 2.9; 1.52]), progressive a + b sperm motility (%) 21.83 ± 11.69 vs 22.54 ± 12.83 (0.03 95% CI [− 0.08; 0.15]), sperm vitality (%) 46.42 ± 10.81 vs 44.62 ± 9.34 (− 0.04 95% CI [− 0.13; 0.05]), morphologically normal spermatozoa (%) 7.03 ± 2.61 vs 8.09 ± 3.61 (0.12 95% CI [0.01; 0.24]), DNA sperm fragmentation by SCD (%) 13.33 ± 5.12 vs 13.88 ± 6.14 (0.03 95% CI [− 0.09; 0.16]), and apoptotic spermatozoa by MACS (%) 15.47 ± 15.04 vs 23.80 ± 23.63 (− 0.20 95% CI [− 0.66; 1.05]).

Conclusion: The lack of differences obtained between frozen samples exposed to μg and those maintained in ground conditions provides the possibility of considering the safe transport of human male gametes to space. Nevertheless, further research is needed to validate the results and to consider the possibility of creating a human sperm bank outside the Earth.

Journal of Assisted Reproduction and Genetics – DOI: 10.1007/s10815-020-01877-5
Received: 16 April 2020 / Accepted: 29 June 2020 / Published: 18 July 2020

The potential role of central obesity in male infertility: body mass index versus waist to hip ratio as they relate to selected semen parameters

Márton Keszthelyi, V. Anna Gyarmathy, András Kaposi and Zsolt Kopa

Department of Urology, Andrology Centre, Semmelweis University, Üllői út 78/b, Budapest, 1082 Hungary; EpiConsult LLC, 8 The Green, STE A, Dover, DE 19904 USA; Johns Hopkins Bloomberg School of Public Health, Baltimore, 615 N Wolfe St, Baltimore, MD 21205 USA; Department of Biophysics and Radiation Biology, Semmelweis University, Tűzoltó u. 37-47, Budapest, 1094 Hungary; Department of Urology, Andrology Centre, Semmelweis University, Üllői út 78/b, Budapest, 1082 Hungary

Abstract:

Background: Little is known about the potential role of central obesity among men. Our first aim was to confirm what is already known from prior research, namely that both BMI and WHR are inversely associated with selected semen parameters. Our second aim was to examine the potential role of central obesity by assessing if there was a difference between BMI and WHR regarding their relationships to these selected semen parameters.
Methods: In this cross-sectional study between January 2011 to January 2018, we analyzed semen samples from 1169 patients who visited an andrology clinic in Budapest for infertility reasons. Variables assessed were: body measurements (height, weight, waist circumference, and hip circumference), and the results of semen analysis (sperm concentration, total sperm count, progressive sperm motility, and normal sperm morphology).
Results: The mean height and weight were 180.6 cm and 87.3 kg, respectively – the mean BMI was 26.8. The mean waist and hip circumferences were 100.9 cm and 94.8 cm, respectively – the mean waist to hip ratio was 0.94. The mean sperm concentration, total sperm count, and percents of progressive motility and normal morphology were 48.7 M/ml, 165 million, 21.2, and 4.8%, respectively. Both BMI and WHR were significant correlates in all semen parameter regression models. When comparing the parameter estimates for BMI with those for WHR for each semen parameter, the parameter estimate for WHR was significantly lower (indicating a stronger negative association) than that for BMI for progressive motility and total sperm count, but not for normal morphology or concentration.
Conclusions: Our study is the first to examine, using a large patient sample, the potential role of central obesity by comparing the difference between BMI and WHR as they relate to selected semen parameters. Our findings indicate a potential role of central obesity for progressive motility and total sperm count, but not for normal morphology and concentration. Despite the limitations and the exploratory nature of this study, we can conclude that our results point to a potential role of central obesity in male infertility, but this finding should be confirmed and further explored in future research.

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BMC Public Health. 2020; 20: 307. – doi: 10.1186/s12889-020-8413-6
Received: 10 August 2019 Accepted: 26 February 2020 Published online 2020 Mar 12

Prevalence of genital Mycoplasma and response to eradication treatment in patients undergoing assisted reproductive techniques

Ernesto Veiga, Mercedes Treviño, Ana Belén Romay, Daniel Navarro, Rocío Trastoy, Manuel Macía

Human Assisted Reproduction Unit. University Clinical Hospital of Santiago de Compostela, Spain; Clinical Microbiology Unit. University Clinical Hospital of Santiago de Compostela, Spain

Abstract: 

OBJECTIVE: Several studies have reported greater success of fertilisation by ART in couples who were not infected by Ureaplasma. Increased semen quality and better results have also been observed in couples who were treated with antibiotics to eradicate the infection. The aim of this study was to determine the prevalence of genital mycoplasmas in urine samples from male partners enrolled in the Assisted Reproduction Program (ARP) in our healthcare area so that, positive cases can be treated prior to the use of ART in order to increase the quality of semen, improve the embryo implantation rates and minimize the risk of adverse effects during pregnancy.

METHODS: This study included couples enrolled in the ARP during 2016. Mycoplasma detection was made using real-time PCR. In positive cases, both members of the couple were treated with antibiotics until eradication of the microorganism. The antibiotics used were: azithromycin, doxycycline, levofloxacin, moxifloxacin, and clindamycin.

RESULTS: Of the 205 men studied, 33 were positive: Ureaplasma urealyticum 15.1%, Mycoplasma hominis 3.9%. Eradication treatment with azithromycin failed in 50% compared to 10.2% for doxycycline. Of the 5 cases treated with levofloxacin, only 2 achieved elimination of U. urealyticum.

CONCLUSIONS: We consider that genital mycoplasma routine screening could be useful in order to increase the quality of semen which could simplify the in vitro fertilisation procedures and raise the success rate of embryo implantation and pregnancy, especially when fast, sensitive and specific technics as real time PCR are used.

Rev Esp Quimioter.
Received: 17 March 2019; Revision Requested: 14 April 2019; Revision Received: 19 April 2019; Accepted: 14 May 2019

Computer‐aided sperm analysis, the new key player in routine sperm assessment

Benoit Schubert, Mélanie Badiou, André Force

Eurofins Biomnis – Institut Rhonalpin IVF Center, Clinique du Val d’Ouest – Medicentre, Ecully, France

Abstract: For sperm analysis, important inter-laboratory variations have been observed in manual analyses. In this study, a computer-aided sperm analysis (CASA) system was assessed versus manual technique, and specific software modifications were operated to fit the David’s classification already used in the laboratory. Four parameters were studied (concentration, motility, vitality and morphology), and at least 30 semen samples from 30 different patients have been tested. Manual and automated analyses were compared using a least-squares regression line analysis, Student’s t test, Bland-Altman plots and Passing-Bablok regressions. Repeatability was also assessed, and coefficients of variation (CV) were calculated. Both manual and automated methods gave similar results for sperm concentration (n = 150), motility (n = 30), vitality (n = 90) and morphology (n = 90). Repeatability always showed a decrease in the CV with automated analysis; for example in normal range of sperm values, CV for manual and CASA analyses were, respectively, 9.0% versus 4.4% for sperm concentration, 5.2% versus 4.1% for motility, 7.3% versus 4.2% for vitality and 11.4% versus 4.1% for morphology. All parameters were comparable between automated and manual analysis, and repeatability measures confirm the more reliable values of the SCA compared to those of manual analysis.

Andrologia – https://doi.org/10.1111/and.13417
Received 8 October 2018, Revised 17 May 2019, Accepted 3 June 2019

Evaluation of black glutinous rice (Oryza sativa L) extract as a novel nuclear stain for human sperm head assessment by microscopic examination

Sirinart Chomean, Tanawan Sukanto, Arreya Piemsup, Jirattikan Chaiya, Kolunya Saenguthai, Chollanot Kaset

Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University, Pathumthani; Thammasat Infertility Center and Medical laboratory, Thammasat University Hospital, Thammasat University, Pathumthani, Thailand

Abstract: 

Objective: To compare black rice (Oryza sativa L) extract with three different staining methods for human sperm head assessment.
Methods: Semen samples were collected from 34 volunteers. Four smears of each ejaculate were prepared for staining using the rapid Papanicolaou (PAP) stain, SpermBlue, DipQuick, and black rice extract. The percentage of defective sperm heads (mean±standard deviation) was compared.
Results: Black glutinous rice extract, a natural dye, was used instead of hematoxylin to stain the nuclei of the sperm heads. The percentage of defective sperm heads showed a significant difference between black rice extract and DipQuick (p=0.000). In contrast, black rice extract and rapid PAP showed no statistically significant difference (p=0.974). A strong correlation (r= 0.761) was found between the findings obtained using rapid PAP and black rice extract. In contrast, a weak correlation (r=0.248) was obtained between DipQuick and black rice extract for the percentage of defective sperm heads.
Conclusion: The results showed good agreement and a strong correlation between the rapid PAP and black rice extract stains. The advantages of black rice extract as a novel substitute for hematoxylin for nuclear staining include ease of preparation, local availability, and favorable nuclear staining properties. Further studies could also focus on comparing staining techniques in clinical samples

Full article here

Clinical and Experimental Reproductive Medicine – https://doi.org/10.5653/cerm.2019.46.2.60
Received: Feb 20, 2019 ∙ Revised: Apr 2, 2019 ∙ Accepted: Apr 2, 2019 ∙ Published: June 2019

Semen quality of young men in Switzerland: a nationwide cross‐sectional population‐based study

R. Rahban, L. Priskorn, A. Senn, E. Stettler, F. Galli, J. Vargas, M. Van den Bergh, A. Fusconi, R. Garlantezec, T. K. Jensen, L. Multigner, N. E. Skakkebæk, M. Germond, N. Jørgensen, S. Nef

Department of Genetic Medicine and Development, University of Geneva, Geneva, Switzerland; Department of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark; Swiss Armed Forces Joint Staff, Medical Services, Ittigen, Switzerland; National Institute for Cancer Epidemiology and Registration (NICER), Zurich, Switzerland; Centre de Procreation Medicalement Assist ee SA, Fertas SA et Fondation FA BER, Lausanne, Switzerland; Kinderwunschzentrum, Kantonspital Baden, Baden, Switzerland; Centro Cantonale di Fertilit
a, Ospedale di Locarno La Carit
a, Locarno, Switzerland;  and Inserm, EHESP, Irset (Institut de recherche en sante, environnement et travail) – UMR_S 1085, Universite de Rennes, Rennes, France

Abstract: 

Background: Sperm counts have been steadily decreasing over the past five decades with regional differences in the Western world. The reasons behind these trends are complex, but numerous insights indicate that environmental and lifestyle factors are important players.
Objective: To evaluate semen quality and male reproductive health in Switzerland.
Materials and methods: A nationwide cross‐sectional study was conducted on 2523 young men coming from all regions of Switzerland, recruited during military conscription. Semen volume, sperm concentration, motility, and morphology were analyzed. Anatomy of the genital area and testicular volume was recorded. Testicular cancer incidence rates in the general population were retrieved from Swiss regional registries.
Results: Median sperm concentration adjusted for period of sexual abstinence was 48 million/mL. Comparing with the 5th percentile of the WHO reference values for fertile men, 17% of men had sperm concentration below 15 million/mL, 25% had less than 40% motile spermatozoa, and 43% had less than 4% normal forms. Disparities in semen quality among geographic regions, urbanization rates, and linguistic areas were limited. A larger proportion of men with poor semen quality had been exposed in utero to maternal smoking. Furthermore, testicular cancer incidence rates in the Swiss general population increased significantly between 1980 and 2014.
Discussion: For the first time, a systematic sampling among young men has confirmed that semen quality is affected on a national level. The median sperm concentration measured is among the lowest observed in Europe. No specific geographical differences could be identified. Further studies are needed to determine to what extent the fertility of Swiss men is compromised and to evaluate the impact of environmental and lifestyle factors.
Conclusion: A significant proportion of Swiss young men display suboptimal semen quality with only 38% having sperm concentration, motility, and morphology values that met WHO semen reference criteria.

Full article here

ANDROLOGY – https://doi.org/10.1111/andr.12645
First published: 21 May 2019

Can the Sperm Class Analyser (SCA) CASA-Mot system for human sperm motility analysis reduce imprecision and operator subjectivity and improve semen analysis?

Chey Dearing, Channa Jayasena and Kevin Lindsay

School of Health & Sport Science and School of Nursing, Eastern Institute of Technology, Taradale Campus, Hawkes Bay, New Zealand; Andrology Laboratory, Hammersmith Hospital, Imperial College NHS Trust, London, UK; Andrology Laboratory, Hammersmith Hospital, Imperial College NHS Trust, London, UK

Abstract: Semen analysis (SA) is considered mandatory for suspected male infertility although its clinical value has recently become questionable. Sperm motility is an essential parameter for SA, but is limited by high measurement uncertainty, which includes operator subjectivity. Computer-assisted sperm analysis (CASA) can reduce measurement uncertainty compared with manual SA. The objective of this study was to determine whether the Sperm Class Analyser (SCA) CASA-Mot system could reduce specific components of sperm motility measurement uncertainty compared with the World Health Organization (WHO) manual method in a single laboratory undertaking routine diagnostic SA. The study examined: (i) operator subjectivity; (ii) precision, (iii) accuracy against internal and external quality standards; and (iv) a pilot sub-study examining the potential to predict an IVF fertilisation rate. Compared with the manual WHO method of SA on 4000 semen samples, SCA reduces but does not completely eliminate operator subjectivity. Study SCA and CASA-Mot are useful tools for well-trained staff that allow rapid, high-number sperm motility categorization with less analytical variance than the manual equivalent. Our initial data suggest that SCA motility may have superior predictive potential compared with the WHO manual method for predicating IVF fertilization.

Human Fertility – https://doi.org/10.1080/14647273.2019.1610581
Received 13 Dec 2018, Accepted 21 Mar 2019, Published online: 06 May 2019

Humanin levels in human seminal plasma and spermatozoa are related to sperm quality

M. Rao, Z. Wu, Y. Wen, R. Wang, S. Zhao, L. Tang

Department of Reproduction and Genetics, The First Affiliated Hospital of Kunming Medical University, Kunming, China

Abstract: 

Background: Humanin has reportedly been expressed in testis and spermatozoa, but no study has yet reported its presence in human seminal plasma (SP).

Objective: The aim of this study was to investigate the presence of humanin in human SP and to determine the correlation between humanin levels in SP/spermatozoa and sperm quality.

Materials and methods: Semen samples for SP/sperm humanin level measurement were collected from 164 patients who attended our andrology clinic for fertility evaluation. The localization of humanin in spermatozoa was evaluated using an immunofluorescence method, and SP/sperm humanin levels were measured with ELISA. Correlations between SP/sperm humanin levels and sperm parameters were analyzed.

Results: Humanin was expressed in the midpiece of the spermatozoa. Humanin concentrations in the SP ranged from 24.4 to 285.1 pg/mL, with a median of 89.7 pg/mL. The SP humanin concentrations in patients with normospermia were significantly higher than those in patients with oligospermia (p < 0.001), asthenospermia (p = 0.002), and oligoasthenospermia (p < 0.001). Spearman analysis showed a positive and significant correlation between SP humanin concentration and sperm concentration (r = 0.75, p < 0.001), and progressive sperm motility (r = 0.29, p < 0.001). Sperm humanin level was significantly and positively associated with progressive sperm motility (r = 0.70, p < 0.001). In addition, a significantly higher level of humanin was found in swim‐up spermatozoa than in non‐swim‐up spermatozoa (p = 0.03).

Conclusions: Seminal plasma and sperm humanin levels were significantly and positively correlated with sperm quality, especially sperm motility. Further studies of the origin of SP humanin and its role in spermatogenesis should be conducted.

Andrology – https://doi.org/10.1111/andr.12614
Received: 27-Aug-2018 / Revised: 26-Feb-20 19 / Accepted: 28-Feb-2019 / First published: 28 March 2019

Improved sperm motility after 4 h of ejaculatory abstinence: role of accessory sex gland secretions

Dale Goss, Bashir Ayad, Gerhard van der Horst, Bongekile Skosana and Stefan S. du Plessis

Division of Medical Physiology, Stellenbosch University, Private Bag X1, Matieland, 7602, Stellenbosch, South Africa

Abstract: Various studies have sought to determine the typical v. optimal abstinence period after which semen samples should be collected, with many contradictory results reported. Several factors influence the semen microenvironment, and thus sperm parameters. In this study we focused on the secretions of the prostate, seminal vesicles and the epididymis. Semen samples were obtained from healthy normozoospermic males (n = 16) after 4-day and 4-h periods of ejaculatory abstinence, and standard semen analysis was performed using computer-aided sperm analysis, whereas seminal plasma citric acid, neutral α-glucosidase and fructose concentrations were measured using assay kits. There were significant decreases in total sperm count (P < 0.001), sperm concentration (P < 0.05) and semen volume (P < 0.05) after 4 h compared with 4 days ejaculatory abstinence. Furthermore, increases were observed in total sperm motility (P < 0.05) and sperm progressive motility (P < 0.01) after a 4-h abstinence period, accompanied by significant reductions in citric acid (P < 0.05), α-glucosidase (P < 0.01) and fructose (P < 0.01) concentrations. In addition, due to the decreased number of spermatozoa, these concentrations translated to a significant decrease in fructose (P < 0.05) per spermatozoon, indicating an intrinsic mechanism capitalising on alternative sources of energy for increased metabolic function and subsequent sperm motility.

Reproduction, Fertility and Development – https://doi.org/10.1071/RD18135
Submitted: 11 April 2018  Accepted: 3 January 2019   Published online: 26 March 2019

Quantitative assessment of heavy metal effects on sperm function using computer-aided sperm analysis and cytotoxicity assays

Hardneck F, Israel G, Pool E, Maree L.

Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa

Abstract: One known environmental risk factor impacting on human reproduction is heavy metal pollution. Although some metals (e.g., Cu, Se and Zn) have protective effects on the male reproductive system in low doses, heavy metals can accumulate to toxic levels and result in poor semen quality and decreased sperm function. We investigated the effect of CuSO4 and CdCl2 (10, 50, 100 and 250 µg/ml or 500 µg/ml) on human sperm motility and vitality by using computer-aided sperm analysis (CASA) and two cytotoxicity assays (WST-1 and XTT). Several sperm motility parameters were significantly reduced after 5 hr of exposure to the highest concentrations of CuSO4 (250 µg/ml) and CdCl2 (500 µg/ml). The WST-1 assay also revealed significantly lower absorbance values for 50, 100 and 250 µg/ml CuSO4 and for 500 µg/ml CdCl2 ; however, no significant effect was seen with XTT. The calculated average IC50 value was 50.31±  4.34 µg/ml for CuSO4 and 392.32  ±76.79 µg/ml for CdCl2 . The effects of these metals were confirmed with MgCl2 , a positive control. This study provides threshold concentrations for the harmful effect of CuSO4 and CdCl2 on human spermatozoa and recommends the use of WST-1 as vitality assay in future in vitro studies.

DOI: 10.1111/and.13141

CRISPR-Cas9-mediated mutation revealed BSPH2 protein is dispensable for male fertility

Eskandari-Shahraki M, Prud’homme B, Manjunath P

Maisonneuve-Rosemont Hospital Research Centre, Montreal, Canada; Departments of Pharmacology and Physiology, Faculty of Medicine, University of Montreal, Montreal, Canada; Department of Medicine, Faculty of Medicine, University of Montreal, Montreal, Canada.

Abstract: Members of the Binder of SPerm (BSP) superfamily have been identified in both human and mouse epididymis. These proteins are known to bind sperm membrane and promote sperm capacitation. Studies suggest that BSPH2 might play a different role in sperm functions from its counterparts; however, the role of BSPH2 remains mainly unexplored. To investigate whether the absence of one member of the BSP family could affect fertility, mice lacking Bsph2 expression were generated using clustered regularly interspaced short palindromic repeats (CRISPR) associated 9 (Cas9) technology. Knockout (KO) male mice were mated with wild-type (WT) females, and the number and weight of the pups were determined. Sperm motility in WT and KO was assessed using sperm class analyzer (SCA). Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for protein identification. Fertility analysis of null Bsph2 mice did not reveal any phenotype. No differences were noticed on average litter size or average pup weight. Normal testis weight and morphology were observed in Bsph2+/- and Bsph2-/- compared to the WT. Quantitative polymerase chain reaction analyses revealed that Bsph1 messenger RNA expression was increased in mutant mice, whereas LC-MS/MS analysis displayed no increase in protein expression level. Taken together, we show the existence of redundant function for murine BSPH2 and the lack of BSPH2 itself does not lead to sterility.

DOI: 10.1002/mrd.23039

Sperm motility assessment using computer assisted semen analysis (CASA): a comparison of standard microscope slides and coverslips and the 20 µm MicroCell™

Callum Robinson, Peter Roberts, Phillip Matson

School of Medical and Health Sciences, Edith Cowan University, Joondalup, Western Australia 6027, Australia; Fertility North, Joondalup, Western Australia 6027, Australia

Abstract: Computer assisted semen analysis (CASA) uses instrumentation that makes precise measurements of sperm motility, but the values obtained can be influenced by a number of technical aspects. Motility of human sperm was measured using a Sperm Class Analyzer (Microptic S.L., Barcelona, Spain), and the effect of using different counting chamber/slide configurations was investigated. Results for 20μm MicroCell slides (Vitrolife Sweden AB, Göteborg, Sweden) were compared with microscope slides and 22mmx22mm coverslips loaded with either 5µl (CV.5µl) or 10µl (CV.10µl) semen. Operator-correction of readings for all slide configurations resulted in a significantly lower number of sperm assessed due to the elimination of non-sperm bodies. Following operator-correction, the MicroCell chamber and CV.10µl slide gave similar readings for both progressive motility and immotility for up to 5 minutes, whereas the CV.5µl had a progressive increase in immotile sperm. The interval to analysis was therefore standardised at 2 minutes prior to the measurement of kinetic parameters, and the MicroCell values were significantly different to the CV.10µl for curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL), and the CV.5µl for VAP. It is concluded that the same configuration be used within the same study, and that care should be taken when comparing different studies that have used different slide/chamber configurations.

J Reprod Stem Cell Biotechnol 7:1-7

Clinical application of the standardisation of computer-assisted sperm morphometry analysis with ASMA (assisted sperm morphometry analysis) using a semen system analyser SCA 5.4 (Sperm Class Analyzer, Microptic)

Clara Ramírez, José Ramón Alonso, Pedro Jiménez, Jordi Ramis, Josep María Gris, Carlos Aulesa

Unidad de Andrología, Laboratorios Clínicos, Hospital Universitario Vall d’Hebron, Barcelona, Spain; Servicio de Reproducción Asistida, Hospital Universitario Vall d’Hebron, Barcelona, Spain

Abstract

Introduction: Use is made of computer-assisted sperm morphometry analysis (ASMA) in order to analyse sub-populations as a function of several pathologies.

Material and methods: A total of 703 patients were classified into 7 pathology groups, and their sperm sub-populations were analysed. Pathology groups were classified into infertility, male factor stress (anxiety, insomnia and/or depression), diabetes mellitus type 1 or 2, varicocele, urogenital infections, oncological patients, and other endocrine diseases. Morphological analysis was performed using the semen analyser system SCA 5.4 (Sperm Class Analyzer) (Microptic S.L., Barcelona, Spain). Pre-treatment and staining method used has been previously described by this group.

Results: Automatic human sperm morphology showed that the study of sperm sub-populations has a clinical usefulness in the diagnosis of varicocele. Higher values of 30% elongated sperms had a sensitivity of 37% and a specificity of 79% (1.54 odds ratio with a 95% confidence interval from 1.05 to 2.26). Furthermore, groups of infertility, stress, and diabetes showed new sperm sub-populations which had not been described previously, so they could become a new diagnostic tool.

Discussion: The coefficients of variation of sperm morphology were decreased using automation, enabling the study sperm subpopulations in different pathologies. There is a diagnostic usefulness for varicocele. There could be a prediction to choose the best assisted reproductive technique for infertility patients. However, for the rest of groups new sperm subpopulations could be identified that could make it a diagnostic tool, but requires a higher casuistry to conclude the study.

https://doi.org/10.1016/j.medre.2017.11.001

Revisiting the relationship between the ejaculatory abstinence period and semen characteristics

Bashir M Ayad, Gerhard Van der Horst, Stefan S Du Plessis

Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.

Abstract:

Variation in the ejaculatory abstinence period suggested by different guidance bodies have resulted in a growing concern among researchers and clinicians over what the precise period of ejaculatory abstinence ought to be for an optimal semen sample. Several studies have thus been undertaken to examine the association between the length of sexual abstinence and semen characteristics. Not all studies, however, have arrived at the same conclusions. This study aims to review all existing literature published during the past few decades pertaining to the influence of ejaculatory abstinence on semen quality. For the purpose of this systematic review, all data related to sexual abstinence duration and seminal parameters were re-analysed to homogenize the current data. Thorough PubMed, MEDLINE and Google Scholar, a literature search was conducted using the keywords “sexual abstinence”, “ejaculatory abstinence”, “semen”, “spermatozoa”, “semen analysis”, “sperm parameters”, “motility”, “reactive oxygen species (ROS)” and “DNA fragmentation”. After carefully reviewing all the literature, 30 relevant papers, both written in English and published between January 1979 and December 2016, were included in this review. The weight of the evidence suggests that the decline in semen volume and sperm concentration with shorter abstinence periods is accompanied by a substantial improvement in sperm motility characteristics, especially progressive motility and velocity. Nevertheless, available data are insufficient to support definitive conclusions regarding the influence of the ejaculatory abstinence period on advanced semen parameters (ROS, DNA fragmentation and seminal plasma antioxidant capacity) and pregnancy rates. In conclusion, taking all data into account, shortening of the abstinence period may be beneficial to sperm quality. Furthermore, we recommend that the current guidelines regarding the prescribed abstinence period should be revisited.

doi:  10.22074/ijfs.2018.5192

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Effects of chronic musculoskeletal pain on fertility potential in lean and overweight male patients

Fereshteh Dardmeh, Hiva Alipour, Hans Ingolf Nielsen, Sten Rasmussen and Parisa Gazerani

Department of Health Science and Technology, Biomedicine Group, The Faculty of Medicine, Aalborg University, Aalborg, Denmark; SMI®, Department of Health Science and Technology, The Faculty of Medicine, Aalborg University, Aalborg, Denmark; Department of Clinical Medicine, The Faculty of Medicine, Aalborg University Hospital, Aalborg, Denmark

Abstract: Both chronic pain and obesity are known to affect reproductive hormone profiles in male patients. However, the effect of these conditions, alone or in combination, on male fertility potential has received less attention. 20 chronic musculoskeletal pain patients and 20 healthy controls were divided into lean and overweight subgroups according to their BMI. Current level of chronic pain (visual analogue scale) and pressure pain thresholds (PPTs) in 16 predefined sites, classically described and tested as painful points on the lower body, were measured. Levels of reproductive hormone and lipid profiles were assessed by ELISA. Sperm concentration and motility parameters were analyzed using a computer-aided sperm analysis system. Sperm concentration, progressive motility, and percentage of hyperactivated sperm were generally lower in the chronic pain patients in both lean and overweight groups. The overweight control and the lean chronic pain groups demonstrated a significantly lower percentage of progressively motile sperm compared with the lean control group, suggesting that musculoskeletal chronic pain may have a negative influence on sperm quality in lean patients. However, due to the potential great negative influence of obesity on the sperm parameters, it is difficult to propose if musculoskeletal chronic pain also influenced sperm quality in overweight patients. Further research in chronic pain patients is required to test this hypothesis.

Pain Research and Management – https://doi.org/10.1155/2017/4628627
Received 30 August 2017; Revised 3 November 2017; Accepted 13 November 2017; Published 24 December 2017

Association between obesity and sperm quality

G. A. Ramaraju, S. Teppala, K. Prathigudupu, M. Kalagara, S. Thota, M. Kota, R. Cheemakurthi

Center for Assisted Reproduction, Krishna IVF Clinic, Visakhapatnam, India

Summary:

There is awareness of likelihood of abnormal spermatozoa in obese men; however, results from previous studies are inconclusive. Advances in computer-aided sperm analysis (CASA) enable precise evaluation of sperm quality and include assessment of several parameters. We studied a retrospective cohort of 1285 men with CASA data from our infertility clinic during 2016. Obesity (BMI ≥30) was associated with lower (mean ± SE) volume (−0.28 ± 0.12, p-value = .04), sperm count (48.36 ± 16.51, p-value = .002), concentration (−15.83 ± 5.40, p-value = .01), progressive motility (−4.45 ± 1.92, p-value = .001), total motility (−5.50 ± 2.12, p-value = .002), average curve velocity (μm/s) (−2.09 ± 0.85, p-value = .001), average path velocity (μm/s) (−1.59 ± 0.75, p-value = .006), and higher per cent head defects (0.92 ± 0.81, p-value = .02), thin heads (1.12 ± 0.39, p-value = .007) and pyriform heads (1.36 ± 0.65, p-value = .02). Obese men were also more likely to have (odds ratio, 95% CI) oligospermia (1.67, 1.15–2.41, p-value = .007) and asthenospermia (1.82, 1.20–2.77, p-value = .005). This is the first report of abnormal sperm parameters in obese men based on CASA. Clinicians may need to factor in paternal obesity prior to assisted reproduction.

DOI: 10.1111/and.12888

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Short abstinence: A potential strategy for the improvement of sperm quality

Bashir M. Ayad, Gerhard Van der Horst, Stefan S. du Plessis

Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa

Abstract

Objective: To determine the effect of short (4 h) and long (4 days) abstinence periods on sperm quality based on functional and biochemical parameters in a population of normozoospermic men.

Methods: Two semen samples were collected in succession from potentially fertile, normozoospermic men (n = 100) after an abstinence period of 4 days and 4 h respectively. The mean values of semen volume, pH, viscosity, sperm concentration, percentage of total and progressively motile sperm, sperm kinematics/velocity, normal morphology, acrosome status, DNA fragmentation, intracellular superoxide (O2−•) levels and seminal antioxidant status were compared between the two abstinence duration groups.

Results: A significant increase in total and progressive motility and velocity parameter values were observed after short abstinence compared with long abstinence periods. Sperm DNA fragmentation and intracellular O2−• levels were not significantly different between the two abstinence periods. Despite the decrease in semen volume, sperm concentration and total sperm number after short abstinence periods, all mean values of the conventional semen parameters remained above the lower reference limits as reported by the WHO.

Conclusion: The data from this most comprehensive study of its kind challenges the generally accepted guidelines of the prolonged abstinence periods since the results show that 4 h of sexual abstinence yielded significantly better sperm samples from a functional point of view. Although this study was performed on normozoospermic men, future studies with infertile men might yield similar findings that could lead to employing short abstinence as a strategy to improve the outcome of ART and fertility preservation.

https://doi.org/10.1016/j.mefs.2017.07.005

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Not just the marriage of Figaro: but the marriage of WHO/ESHRE semen analysis criteria with sperm functionality

Gerhard van der Horst, Stefan S du Plessis

Department of Medical Bioscience, University of the Western Cape, Belville, South Africa; Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa

Abstract:

The authors present a critical review of the WHO5 (2010) manual of semen analysis and what it should be used for: The analysis of sperm quality and not analysis to predict fertility outcome per se. We show the strengths and shortcoming of WHO5 and then ask for a better “marriage” among these parameters and the outcome of sperm functionality and fertilization/live birth outcome. For many decades the basis of the WHO manual for semen analysis has not changed and we emphasize that sperm functionality testing has not really been considered/performed in the routine andrology laboratory. There is a need to first develop more objective and quantitative methodology such as computer-aided sperm analysis, to analyse sperm quality and sperm functionality that relates in many instances to fertilization/live birth outcome: 1) sperm cervical mucous penetration using computer aided sperm analysis (CASA), 2) endpoint of capacitation, hyperactivation as measured accurately by CASA, 3) acrosome reaction quantitatively, 4) chromatin maturity and DNA fragmentation quantitatively, 4) where possible oocyte binding tests (hemizona), 5) relationships of vitality and hypo-osmotic swelling test using modern technology 6) measurement of oxidative stress, 7) analysis of semen using proteomics (proteins that are importantly functionally expressed in seminal plasma) as well as 8) metabolomics representing a systematic study of the unique metabolic fingerprints (chemical) that specific cellular processes leave behind and inform us about function/dysfunction, 9) patient profile (obesity, smoking, age, stress, female cryptic choice, environment and many other patient characteristics) as important determinants in fertility outcome. We believe we can intelligently in the end construct a matrix which combine all these factors and others in the future that inform us about potential fertility outcome. But then realize WHO5/ESHRE current guidelines are not particularly informative in the above context.

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Improved sperm kinematics in semen samples collected after 2 h versus 4-7 days of ejaculation abstinence

Alipour H, Van Der Horst G, Christiansen OB, Dardmeh F, Jørgensen N, Nielsen HI, Hnida C

Biomedicine Group, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark; Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa; Department of Obstetrics and Gynecology, Aalborg University Hospital, Aalborg, Denmark; Department of Clinical Medicine, Aalborg University, Aalborg, Denmark; University Department of Growth and Reproduction and International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, Copenhagen, Denmark; Fertility Unit, Department of Obstetrics and Gynecology, Aalborg University Hospital, Aalborg, Denmark

Abstract

STUDY QUESTION:
Does a short abstinence period of only 2 h yield spermatozoa with better motility characteristics than samples collected after 4-7 days?

SUMMARY ANSWER:
Despite lower semen volume, sperm concentration, total sperm counts and total motile counts, higher percentages of motile spermatozoa with higher velocity and progressiveness were detected in samples obtained after 2 h.

WHAT IS KNOWN ALREADY:
Most studies that have assessed the effect of abstinence periods on sperm motility parameters in men with a sperm concentration below 15 million/ml have detected a higher percentage of motile spermatozoa in samples obtained after short abstinence periods. Studies of men with sperm concentrations above 15 million/ml have reported significantly decreased motile sperm counts after 24 h of abstinence compared with longer abstinence periods.

STUDY DESIGN, SIZE, DURATION:
This study had a controlled repeated-measures design based on semen samples from 43 male partners, in couples attending for IVF treatment, who had a sperm concentration above 15 million/ml. Data were collected between June 2014 and December 2015 in the Fertility Unit of Aalborg University Hospital (Aalborg, Denmark).

PARTICIPANTS/MATERIALS, SETTING, METHODS:
Participants provided a semen sample after 4-7 days of abstinence followed by another sample after only 2 h. For both ejaculates, sperm concentration, total sperm counts, motility groups and detailed kinematic parameters were assessed and compared by using the Sperm Class Analyzer (SCA) computer-aided sperm analysis system before and after density gradient selection. The laboratory’s local manual method (Makler chamber) was used for comparison.

MAIN RESULTS AND THE ROLE OF CHANCE:
The second raw ejaculate demonstrated lower semen volume (P < 0.0001), sperm concentration (P = 0.003) and sperm counts in all motility sub-groups (P < 0.001) but higher percentages of spermatozoa with higher velocity (P < 0.01), progressiveness (P < 0.001) and hyperactivation (P < 0.001), compared with the first raw ejaculate.

LIMITATIONS, REASONS FOR CAUTION:
The first ejaculate in this study was also used for the IVF/ICSI treatments and therefore only patients with a semen volume ≥2 ml and concentration ≥15 million/ml were included. Further validation in large prospective randomized controlled trials, more purposely directed at normozoospermic males with partners having problems conceiving when there appears to be no female factor, is needed to confirm the potential advantage of using a second semen sample in improving fertilization and pregnancy rates in assisted reproduction.

WIDER IMPLICATIONS OF THE FINDINGS:
Despite the significantly lower semen volume, sperm concentration and total sperm counts in all motility sub-groups, the significantly higher percentage of spermatozoa with better motility characteristics (velocity, progressiveness and hyperactivation) in the second ejaculate, may provide and allow for a simpler and more effective selection of higher quality spermatozoa. This could prove to be an advantage for ART procedures such as intracytoplasmic sperm injection where a large number of spermatozoa is not needed. It can also be speculated that pooling two consecutive ejaculates obtained after 4-7 days and after 2 h, could be an advantage for intrauterine insemination where a large number of motile spermatozoa are needed.

STUDY FUNDING/COMPETING INTEREST(S):
This study was supported by internal grants from the Department of Health Science and Technology, Faculty of Medicine, Aalborg University (Aalborg, Denmark). The SCA® was provided by a grant from ‘Ferring Pharmaceuticals’ to Aalborg University Hospital (H.I.N). G.V.D.H. is an external senior scientific consultant to Microptic S/L (Barcelona, Spain). H.A. has provided scientific input and presentations for Microptic S/L (Barcelona, Spain) on several occasions. All other authors declare no conflict of interest.

doi: 10.1093/humrep/dex101

Sperm Chromatin Dispersion Test before Sperm Preparation Is Predictive of Clinical Pregnancy in Cases of Unexplained Infertility Treated with Intrauterine Insemination and Induction with Clomiphene Citrate

Frank W. R. C. Vandekerckhove, Ilse De Croo, Jan Gerris, Etienne Vanden Abbeel and Petra De Sutter

Centre for Reproductive Medicine, University Hospital, Ghent, Belgium.

Background/aims: A large proportion of men with normal sperm results as analyzed using conventional techniques have fragmented DNA in their spermatozoa. We performed a prospective study to examine the incidence of DNA fragmentation in sperm in cases of couples with previously unexplained infertility and treated with intrauterine insemination. We evaluated whether there was any predictive value of DNA fragmentation for pregnancy outcome in such couples.

Methods: The percentage of DNA fragmentation and all classical variables to evaluate sperm before and after sperm treatment were determined. We studied the probable association between these results and pregnancy outcome in terms of clinical and ongoing pregnancy rate per started first cycle. We also assessed the optimal threshold level to diagnose DNA fragmentation in our center.

Results: When using threshold levels of 20, 25, and 30%, the occurrence of DNA fragmentation was 42.9, 33.3, and 28.6%, respectively. Receiver operating characteristic (ROC) analysis of all cases revealed an area under the curve of 80% to predict the clinical pregnancy rate per cycle from testing the sperm motility (a + b) before treatment. We failed to generate an ROC curve to estimate pregnancy outcome from the amount of DNA fragmentation before treatment. However, when selecting only those men with a pretreatment DNA fragmentation of at least 20%, the pretreatment result was statistically different between couples who achieved a clinical pregnancy and those who did not.

Conclusion: DNA fragmentation is often diagnosed in couples with unexplained infertility. Each center should evaluate the type of test it uses to detect DNA fragmentation in sperm and determine its own threshold values.

doi: 10.3389/fmed.2016.00063

Standardization of pretreatment and staining technique to perform automatic human sperm morphology with ASMA (assisted sperm morphometry analyses)

Clara Ramírez, José Ramón Alonso, Pedro Jiménez, Rocío Reyes, Jordi Ramis, Josep Maria Gris, Carlos Aulesa

Unidad de Andrología, Laboratorios Clínicos, Hospital Universitario Vall d’Hebron, Barcelona, Spain; Servicio de Reproducción Asistida, Hospital Universitario Vall d’Hebron, Barcelona, Spain

Abstract

Objective: Standardization ofpretreatment and staining technique to realizemorphological semen evaluation with computer-assisted sperm morphometry analysis system, with semen system analyzer SCA 5.4 (Sperm Class Analyzer, Microptic).

Material and methods: Morphological analysis was performed with semen system analyzer SCA 5.4 (Microptic SL, Barcelona, Spain). The staining method was a modification of Hemacolor kit (Merck). Between 60 and 125 semen samples were chosen randomly from our laboratory.

Results: Pretreatment of semen samples was a centrifugation for 5 minutes at 300 g, seminal plasma was rejected and the pellet was homogenized with .2 mL of the seminal plasma itself. Not change in sperm morphology have been found with this pretreatment. Staining was performed with Hemacolor kit (Merck) but with some modifications. Fixer has been buffered with phosphate buffer pH 7.2 at 10%, time recommended by the manufacturer has been reduced. Fixation for 5 seconds, 30 seconds with Eosin staining and 2 seconds with staining Azur. Finally it was washed for 30 seconds with pH 7.2 phosphate buffer, as indicated by the manufacturer. After the pretreatment and staining we have got reference values for our methodology.

Conclusions: An automation methodology to perform sperm morphology reduces coefficient variations of determination thereby we can increase its technical reliability and remove the subjectivity of the manual analysis. This standardization can be the first step to study the diagnostic value of advanced morphology in infertility and urological diseases.

DOI: 10.1016/j.androl.2016.05.002

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Ameliorative potentials of quercetin against cotinine-induced toxic effects on human spermatozoa

Dale Goss; Ibukun P. Oyeyipo; Bongekile T. Skosana; Bashir M. Ayad; Stefan S. du Plessis

Division of Medical Physiology. Faculty of Medicine and Health Sciences. Stellenbosch University. South Africa; Department of Physiology. College of Health Sciences. Osun State University. Osogbo. Osun State. Nigeria.

OBJECTIVES: Cotinine, the principal metabolite of nicotine found in smokers’ seminal plasma, has been shown to adversely affect sperm functionality while quercetin, a flavonoid with diverse properties is associated with several in vivo and in vitro health benefits. The aim of this study was to investigate the potential benefits of quercetin supplementation against damage caused by the by-products of tobacco smoke in human sperm cells.
METHODS: Washed human spermatozoa from 10 normozoospermic donors were treated with nutrient medium (control), quercetin (30 mmol/L) and cotinine (190 mg/mL, 300 ng/mL) with or without quercetin for 60 and 180 min incubation periods. Computer-aided sperm analysis was used to assess sperm motility while acrosomereacted cells were identified under a fluorescent microscope using fluorescein isothiocyanate-labelled Pisum Sativum Agglutinin as a probe, viability was assessed by
means of a dye exclusion staining technique (eosin/nigrosin) and oxidative stress by flow cytometry using dihydroethidium as a probe. Values were expressed as mean ± S.E.M. as compared by ANOVA.
RESULTS: Higher cotinine concentrations reduced the number of viable cells after 60 and 180 min of exposure while viability of cells was increased in the cotinine aliquots supplemented with quercetin after 180 min of exposure when compared with cotinine only treated group.
Conclusion: This study indicates that the ameliorating ability of quercetin on cotinineinduced decline in sperm function is associated with increased number of viable cells.

doi:10.1016/j.apjr.2016.03.005

Thyroxine is useful to improve sperm motility

Gabriela Ruth Mendeluk. Ph.D.; Mónica Rosales M.D.

Laboratory of Male Fertility. Hospital de Clínicas “José de San Martín”. Faculty of Pharmacy and Biochemistry. University of Buenos Aires. Buenos Aires. Argentina; Laboratory of Endocrinology. Hospital de Clínicas “José de San Martín”. Faculty of Pharmacy and Biochemistry. University of Buenos Aires. Buenos Aires. Argentina.

BACKGROUND: The aim of this study was to evaluate the non-genomic action of thyroxine on sperm kinetic and its probable use to improve sperm recovery after an enrichment method like “swim-up” in comparison to the available one pentoxifylline.
MATERIAL AND METHODS: A total of 50 patients consulting for infertility were recruited. Conventional sperm assay were performed according to WHO criteria- 2010. A CASA system was employed to assess kinetic parameters and concentration. The number of motile sperm recovered after the preparation technique was calculated.
RESULT(S): Addition of T4 (0.002ug/ml) to semen samples increased hypermotility at 20 min(control: 14.18 ± 5.1% Vs. 17.66 ± 8.88 % ; p<0.03; data expressed as mean ± SD )and remained unchanged after 40 min. Significant differences were found in the motile sperm recovered after “swim-up” (control: 8.93 x 106 ± 9.52 x 106 Vs. 17.20 x 106 ± 21.16 x106; p<0.03), achieving all the tested samples the desired threshold value for artificial insemination outcome, while addition of pentoxifylline increased the number of recovered sperm after swim-up in 60% of the studied cases. No synergism between treatments could be proved.
CONCLUSION(S): We are proposing a new and physiologic tool to improve artificial insemination. The discussion opens our minds to think in unknown pathways involved in sperm capacitation and gives innovative arguments to understand infertility.

Computer Aided Sperm Analysis, a useful tool to evaluate patient’s response to varicocelectomy

Julia I Ariagno, Gabriela R Mendeluk, María J Furlan, M Sardi, P Chenlo, Susana M Curi, Mercedes N Pugliese, Herberto E Repetto, Mariano Cohen

Departamento de Bioquímica Clínica, Laboratorio de Fertilidad Masculina, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires (CP1120), Argentina; Departamento de Urologia, Hospital de Clinicas “José de San Martín”, Universidad de Buenos Aires, Buenos Aires (CP 1120), Argentina

Pre-operative and post-operative sperm parameter values from infertile men with varicocele were analysed by computer-aided sperm analysis (CASA) to assess if sperm characteristics improved after varicocelectomy. Semen samples of men with proven fertility (n = 38) and men with varicocele-related infertility (n = 61) were also analysed. Conventional semen analysis was performed according to WHO (2010) criteria and a CASA system was employed to assess kinetic parameters and sperm concentration. Seminal parameters values in the fertile group were very far above from those of the patients, either before or after surgery. No significant improvement in the percentage normal sperm morphology (p=0.10), sperm concentration (p = 0.52), total sperm count (p = 0.76), subjective motility (%) (p = 0.97) nor kinematics (p = 0.30) was observed after varicocelectomy when all groups were compared. Neither was significant improvement found in percentage normal sperm morphology (p = 0.91), sperm concentration (p = 0.10), total sperm count (p = 0.89) or percentage motility (p = 0.77) after varicocelectomy in paired comparisons of pre-operative and post-operative data. Analysis of paired samples revealed that the total sperm count (0.01) and most sperm kinetic parameters: curvilinear velocity (p = 0.002), straight-line velocity (p = 0.0004), average path velocity (p = 0.0005), linearity (p = 0.02), and wobble (p = 0.006) improved after surgery. CASA offers the potential for accurate quantitative assessment of each patient’s response to varicocelectomy.

Asian Journal of Andrology.
Manuscript ID: AJA-5138

Evaluation of sperm motility using two cryoprotectants

Sandra Abalde Graña; Mª Esther Rendal Vázquez; Mariana García García; Mª Jesús López Piñón; Marcos Carballal Rodríguez; Catarina Barbero Cancelo; Rosario Olvido Fernández Mallo; Inma Míguez Torre; Teresa Bermúdez González; Sonia Pértega Díaz; Jacinto Sánchez Ibáñez

Unidad de Criobiología-Banco de Tejidos. CHUAC. A Coruña; Unidad de Fecundación in vitro. CHUAC. A Coruña; Unidad de Estadística. CHUAC. A Coruña

Infertility is a problem that affects a lot of couples. One of its causes is a decreased semen quality due to, for example, gonadotoxic treatments. The cryopreservation of human semen is the technique that allows sperm preserving and storing without losing their fertilizing capacity; being a fundamental tool in assisted reproduction. The aim of this study was to optimize the cryopreservation technique. To this end, a study carried out on samples of patients under study by fertility problems, in which two cryoprotectant media (SpermCryo™ All-round and CryoSperm™) and the execution or non-execution of an immersion of the samples in liquid nitrogen (before storage) were compared; and the effect of the time between ejaculation and the processing on the quality of the sample. Variations were studied with an automatic analyzer by performing pre- and post-thaw sperm motility tests.
The results show no difference between the two cryoprotectants media, but seems to have a tendency to obtain better postthaw mobility with either depending on sample characteristics. Moreover, the liquid nitrogen bath had no apparent effects on post-thaw results. However, we must highlight the importance of time in the processing of semen samples once liquefied, to avoid decreased sperm quality.
To improve post-thaw outcomes the key lies in the necessity to adjust the freezing protocol to the sample characteristics and a correct implementation of the protocol cryopreservation (selection and addition of cryoprotectant media…); favoring the management of infertility and the success of assisted reproduction techniques.

Rev. Iberoam. Fert Rep Hum, 2016

Computer Assisted Semen Analysis, an overcoming tool

Ariagno Julia I, Chenlo Patricia H, Mendeluk Gabriela R

Laboratorio de Fertilidad Masculina. Hospital de Clínicas “José de San Martín. Facultad de Farmacia Bioquímica. U.B.A., Argentina

Sperm motility is a key parameter while assessing semen quality. It provides information about sperm energy state, a requisite needed to achieve the active movement that enables the gamete to reach the oviduct site were fertilization takes place. The standard method to assess motility is subjective; it estimates speeds thus having a great inter-laboratory variation. In fact its use is against to the current tendency oriented to objectify all the biochemical studies.
Development of Assisted Computer Semen Analysis (CASA) has not only helped to increase the assessment accuracy and reliability, but has also provided much more information than that obtained by the conventional subjective method. However, certain factors (eg the operator, optics, software configuration, etc.) can affect the results obtained by the use of this technology. In accordance with current regulations we validated the CASA-SCA modules for mobility and concentration, before its use in the andrology clinic for evaluation of patient´s samples. Accuracy, method detection limit and reportable range were determined; a study on method comparison was done and the reference values were verified.
We want to highlight that CASA does not supplant the work of the expert who necessarily has to edit the images and who finally has to validate the clinical report. They can not predict semen sample «fertility». However, properly validated they provide detailed and accurate data useful for ejaculate characterization, exceeding the information that has so far been given by the subjective method.

SAEGRE

Nutrition and Reproductive Health: Sperm versus Erythrocyte Lipidomic Profile and -3 Intake

Gabriela Ruth Mendeluk, Mariano Isaac Cohen, Carla Ferreri, and Chryssostomos Chatgilialoglu

Laboratory of Male Fertility, Hospital de Cl´ınicas “Jos´e de SanMart´ın”, INFIBIOC, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, 5950-800 Buenos Aires, Argentina; Urology Division, Hospital de Cl´ınicas “Jos´e de SanMart´ın”, University of Buenos Aires, 5950-800 Buenos Aires, Argentina; Consiglio Nazionale delle Ricerche (CNR), Istituto per la Sintesi Organica e la Fotoreattivit`a (ISOF), 40129 Bologna, Italy; Institute of Nanoscience and Nanotechnology, National Center of Scientific Research “Demokritos”, Agia Paraskevi, 15310 Athens, Greece

Fatty acid analyses of sperm and erythrocyte cell membrane phospholipids in idiopathic infertile patients evidenced that erythrocyte contents of EPA, DHA, omega-6–omega-3 ratio and arachidonic acid provide a mathematical correspondence for the prediction of EPA level in sperm cells. The erythrocyte lipidomic profile of patients was significantly altered, with signatures of typical Western pattern dietary habits and no fish intake. A supplementation with nutritional levels of EPA and DHA and antioxidants was then performed for 3 months, with the follow-up of both erythrocyte and sperm cell membranes composition as well as conventional sperm parameters. Some significant changes were found in the lipidomic membrane profile of erythrocyte but not in sperm cells, which correspondently did not show significant parameter ameliorations. This is the first report indicating that membrane lipids of different tissues do not equally metabolize the fatty acid elements upon supplementation. Molecular diagnostic tools are necessary to understand the cell metabolic turnover and monitor the success of nutraceuticals for personalized treatments.

doi: 10.1155/2015/670526

Effect of environmental pH on sperm kinematic characteristics

H. Alipour, F. Dardmeh, M.C. Dissing, H.I. Nielsen

Laboratory of Reproductive biomedicine, Biomedicine Group, Department of Health Science and Technology, Faculty of Medicine, Aalborg University, Aalborg, Denmark; ORIGIO A/S, Maaloev, Denmark

Semen preparation medium have an important role in assisted reproduction techniques and their composition influences sperm binding and motility.
Some studies have assessed the influence of pH on sperm kinetics. However, no study to date has assessed the effect of environmental pH on subtle differences in the details of the sperm movement (kinematics) of human sperm provided by computerized sperm analysis systems. This study was designed to assess the effect of two different media pH levels on kinematic parameters of the human sperm.
Samples were prepared using the 40%/80% Pureception (Sage, USA) density gradient and resuspended in customized sperm culture media with pH levels of 7.9 and 8.3 (Origio, Denmark). Kinematic parameters of the sperm in both groups were analyzed using the Sperm Class Analyzer (Microptic S.L., Spain) at 0, 6 and 24 hours following the addition of media.
Results of this study illustrated a general insignificant decrease in the ratio of progressively motile and hyperactive sperm after 6 and 24 hours. However a significant difference between the test groups was observed in the curvilinear, straight line and Mean path velocity and Straightness after 6 and 24 hours. Linearity and Wobble showed significant difference after 24 hours.
This study demonstrated a difference in the sperm motion pattern and velocity in different environmental pH levels. Based on these findings, further investigations are required to elucidate knowledge about possible effect of marginal pH changes, affecting the sperm motion characteristics at different stages in the dynamic environment of the female reproductive tract.

Effect of short abstinence time on sperm motility parameters

H. Alipour, F. Dardmeh, G. Van Der Horst, G. Manoharan, A. Askeland, H.I. Nielsen

Laboratory of Reproductive Biomedicine, Biomedicine group, Dept. of Health Science and Technology, Aalborg University, Aalborg, Denmark; Department of Medical Biosciences, University of the Western Cape, Cape town, South Africa

The importance of establishing an optimal period of sexual abstinence has been an area of focus due to its influence on sperm quality. The ”WHO laboratory manual for the examination and processing of human semen” suggests an ejaculatory abstinence of 2-7 days before sperm collection. Several previous studies have evaluated the effect of abstinence period on sperm motility and presented controversial results, making the WHO recommendation debatable. However, no study to date has assessed the effect of a short (2 hours) abstinence period on sperm quality. The present study compared the sperm motility (as a biomarker of sperm quality) of samples collected after 2-7 days of abstinence with a consecutive ejaculation after 2 hours. The neat samples were analysed using the Sperm Class Analyzer (Microptic S.L., Spain) computer assisted sperm analysing system. The results displayed a significant increase in the ratio of progressively motile spermatozoa and a significant decrease in the percentage of immotile sperm after 2-hours of abstinence compared to a 2-7 day abstinence period.
Based on the results of this study, decreasing the abstinence time to as low as 2 hours could provide a better chance for the treatment of patients with male factor infertility. Further investigation is still required in order to compare the effect of abstinence time on additional sperm quality parameters.

Influence of two commercially available lubricants on sperm motility and kinematics

H.I. Nielsen, F. Dardmeh, K. Kannik, A.B.K. Jeppesen, L. Søresen, M.B. Hjelm, H. Alipour

Department of Health Science and Technology, Biomedicine Group, Faculty of Medicine, Aalborg University, Aalborg, Denmark

The use of lubricants is occasionally requested during intercourse and semen collection for assisted reproduction techniques. This might pose a problem because several lubricants have been reported to decrease sperm functional parameters, especially in regards to sperm motility. This study aimed to examine the influence of 2 commercially available personal lubricants, a water-based (Apotekets Glid) and a silicone-based (Klick Silk Glide), on human sperm motility. Semen samples were divided into three equal parts and added to the two lubricant test groups (Sperm Preparation Medium + 10 % lubricant) and a control group containing only medium. A detailed analysis of motility and different kinematic parameters was performed using the Sperm Class Analyzer® computer-aided sperm analysis system at 0, 0.5, 1, and 3 hours on the control and test groups.
The silicone-based lubricant demonstrated no significant difference compared to the control group. The water-based lubricant, however, had significantly lower fast progressive motility, total progressive motility, curvilinear velocity, straight line velocity, average path velocity, lateral head displacement, beat cross frequency, and hyperactivation compared to the control.
The results of this study indicate that the Klick Silk Glide silicone-based lubricant is less detrimental for sperm in terms of motility and kinematic parameters which have been related to pregnancy rates. Therefore, it can be suggested that when trying to conceive, both naturally and artificially, the Klick Silk Glide lubricant could have lower adverse effects on the sperm motility parameters compared to the water-based Apotekets Glid lubricant.

Comparison of different counting chambers using a computer-assisted semen analyzer

Nanni Peng, Xiangli Zou, and Lingwei Li

Reproductive Medical Center, People’s Hospital of Luohu District, Shenzhen, Guangdong, China

A routine computer-assisted sperm analysis is an important diagnostic test in the andrology laboratory. To evaluate the accuracy and precision of the  different types of counting chambers for human semen analysis in combination with a computer-assisted semen analyzer (CASA), a quality-control study that  compared human sperm analysis results obtained using different counting chambers (Makler chamber, disposable 8-cell GoldCyto chamber, disposable  4-cell Leja chamber, a plain glass slide, and a tissue culture dish cover with a 2424mm2 coverslip) in conjunction with the CASA systems was performed.  Significantly higher counts of sperm concentration were obtained from the reusable Makler chamber than from the other counting chambers. Sperm motility  from drop loaded counting chambers was significantly higher than that of capillary-loaded chambers. A plain glass slide and a tissue culture dish cover used  with a coverslip showed rather better performance in semen assessment. Disposable chambers are suitable for routine semen analysis with CASA in a  diagnostic andrology setting. With the proper workflow and quality control, a plain glass slide and the tissue culture dish cover are acceptable alternatives for  routine counting chambers with CASA as necessary. The type of counting chamber should be specified in test reports.

doi: 10.3109/19396368.2015.1063175

Fragmentation of sperm DNA using the TUNEL method

P.H. Chenlo, S.M. Curi, M.N. Pugliese, J.I. Ariagno, M. Sardi-Segovia, M.J. Furlan, H.E. Repetto, E. Zeitler, M. Cohen, G.R. Mendeluk

Departamento de Bioquímica Clínica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Hospital de Clínicas ‘‘José de San Martín’’, Buenos Aires, Argentina

Objectives: To establish the validity of the TUNEL assay in determining sperm DNA fragmentation, the relationship between the degree of fragmentation and the seminal parameters and the sample needed to conduct the test.
Material and methods: We used semen samples from healthy fertile men (n = 33), patients who consulted for infertility with a prescription for the TUNEL assay (n = 77) and patients with intracytoplasmic sperm injection failure (n = 20), analyzed according to the 2010 WHO. The TUNEL/propidium iodide test was performed by flow cytometry, on baseline and post-swim-up samples.
Results: The cutoff value for the TUNEL assay (ROC curves) was 26%, with a sensitivity and specificity of 85% and 89%, respectively. The pre-swim-up and post-swim-up medians of the results from the TUNEL assay showed no significant differences (17.0% vs. 12.9%, respectively). However, 39.1% of the samples showed a difference greater than 15 in absolute value between the results of the baseline and post-swim-up TUNEL assays. The linear correlation study of the morphology, mobility and vitality using the post-swim-up TUNEL assay showed a greater correlation than preselection, with significant results (r: −0.394, p < .0001; r: −0.461, p < .0001; r: −0.526, p < .0001).
Conclusions: The TUNEL assay is a valid test for clinical use. DNA fragmentation is a factor independent from traditional semen tests. We found a greater susceptibility to damage generated in the laboratory procedures in the samples with lower quality. The sample of choice for evaluating DNA fragmentation will depend on whether the clinician is treating a natural or assisted fertilization.

Actas Urol Esp. 2014;38(9):608-612

Photobiomodulation with light-emitting diodes improves sperm motility in men with asthenozoospermia

Ban Frangez H1, Frangez I, Verdenik I, Jansa V, Virant Klun I.

Reproductive Unit, Department of Obstetrics and Gynecology, University Medical Centre Ljubljana, Slajmerjeva 3, 1000, Ljubljana, Slovenia

Sperm motility is an important parameter of male fertility and depends on energy consumption. Photobiomodulation with light-emitting diode (LED) is known to stimulate respiratory chain in mitochondria of different mammalian cells. The aim of this research was to evaluate the effect of photobiomodulation with LED on sperm motility in infertile men with impaired sperm motility-asthenozoospermia. Thirty consecutive men with asthenozoospermia and normal sperm count who visited the infertility clinic of University Medial Centre Ljubljana between September 2011 and February 2012 were included in the study. Semen sample of each man was divided into five parts: one served as a non-treated (native) control and four parts were irradiated with LED of different wavelengths: (1) 850 nm, (2) 625, 660 and 850 nm, (3) 470 nm and (4) 625, 660 and 470 nm. The percentage of motile sperm and kinematic parameters were measured using a Sperm Class Analyser system following the WHO recommendations. In the non-treated semen samples, the average ratio of rapidly progressive sperms was 12 % and of immotile sperm 73 %. Treating with LED significantly increased the proportion of rapidly progressive sperm (mean differences were as follows: 2.83 (1.39-4.28), 3.33 (1.61-5.05), 4.50 (3.00-5.99) and 3.83 (2.31-5.36) for groups 1-4, respectively) and significantly decreased the ratio of immotile sperm (the mean differences and 95 % CI were as follows: 3.50 (1.30-5.70), 4.33 (2.15-6.51), 5.83 (3.81-7.86) and 5.50 (2.98-8.02) for groups 1-4, respectively). All differences were highly statistically significant. This finding confirmed that photobiomodulation using LED improved the sperm motility in asthenozoospermia regardless of the wavelength.

doi: 10.1007/s10103-014-1653-x

Obesity is associated with increased seminal insulin and leptin alongside reduced fertility parameters in a controlled male cohort

Leisegang K, Bouic PJ, Menkveld R, Henkel RR

Department of Medical Biosciences, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa

BACKGROUND: Obesity appears to be associated with male reproductive dysfunction and infertility, although this has been inconsistent and inconclusive. Insulin and leptin are known mediators and modulators of the hypothalamus-pituitary-testes axis, contributing to the regulation of male reproductive potential and overall wellbeing. These hormones are also present in semen influencing sperm functions. Although abdominal obesity is closely associated with insulin resistance (hyperinsulinaemia), hyperleptinaemia and glucose dysfunction, changes in seminal plasma concentrations of insulin, leptin and glucose in obese males has not previously been investigated.
METHODS: This small case controlled study assessed serum and seminal concentrations of insulin, leptin and glucose in obese (BMI > =30; n = 23) and non-obese (BMI < 30; n = 19) males. Following a detailed medical history and examination, participants meeting the inclusion criteria were entered for data analysis. Body parameters such as BMI, waist and hip circumference and the waist hip ratio were measured. Serum and semen samples were collected and assayed for insulin, leptin and glucose. Semen samples also underwent a standard semen analysis, with sperm mitochondrial membrane potential (MMP) and DNA fragmentation (DF).
RESULTS: Obesity was associated with increased serum and seminal insulin and leptin, with no significant difference in seminal glucose. Serum and seminal concentrations of insulin and leptin were positively correlated. Furthermore, obesity was associated with decreased sperm concentration, sperm vitality and increased MMP and DF, with a non-significant impact on motility and morphology.
CONCLUSIONS: Hyperinsulinaemia and hyperleptinaemia are associated with increased seminal insulin and leptin concentrations, which may negatively impact male reproductive function in obesity. Insulin was also found to be highly concentrated in the seminal plasma of both groups. This data will contribute to the contradictive information available in the literature on the impact of obesity and male reproduction.

doi: 10.1186/1477-7827-12-34

Increasing precision and reducing variation in sperm assessments using the Sperm Class Analyzer®

Rothman C, Sims CA, Shamonki J, Schiewe MC. 

California Cryobank (CCB), QC/R&D Lab, Los Angeles, CA 90025, USA.

Background
CCB endeavors to optimize precision of sperm count and motility calculations by reducing potential procedural sources of variation. The adaptation of real-time video imaging software has made Computer Assisted Semen Analysis (CASA) more versatile and practical for the end users. In fact, this technology allows for remote capturing of video images (i.e., satellite labs) to be processed at one central location, thus eliminating inter-technician/facility variation.
Objective
This prospective validation study aims to compare sperm concentration and motility determinations by manual (Makler chamber) or automated (Sperm Class Analyzer {SCA}, Microptics) methods.  Two different technicians (one per method) were used to eliminate technical bias.
Methods
50 specimens were acquired from men being evaluated as potential sperm donors.  Consent was obtained from all participants. Semen analysis was performed on each sample in concurrence with an IRB-approved, FDA-supported validation trial of the SCA unit. The standard manual evaluation of each sample consisted of duplicate or triplicate counting of 20 grids per analysis on a Maker chamber. For the SCA analysis we loaded Leja SC 20 disposable slides with 5 μl of sample per chamber and placed it on a standard phase contrast microscope. Using the SCA Motility program, at least 5 separate fields were captured using a 10X objective under green-filtered low light. Centralized regions from the opening to the base of the chamber were documented. The highest and lowest sperm concentration outliers were eliminated.
Results
The SCA-CASA system assessed an average of 848 sperm/analysis, while Makler chambers evaluated 83 sperm/analysis. The mean concentration of specimens analyzed with the Makler was 10.9% lower than those analyzed with the SCA (64±3.6 vs 71.8±2.3, x10⁶/ml). The manual method produced greater dispersion in motility estimates; total motility estimates were lower (48±5.4% vs 61±3.8%; 22% variation) and progressive motility estimates higher (32±4% vs 21±2%; 55% variation).
Discussion
The reproducibility of sperm count and motility calculation will always be subject to a degree of variation inherent to this biological product. Eliminating procedural variation and optimizing technician precision are important QC laboratory goals. A centralized SCA unit has the potential to provide superior evaluation and video documentation of specimens. Additional verification studies are needed to resolve why differences in motility exceed 20% differences in motility when compared to manual estimates.
Support
Microptic, Barcelona, Spain; ASPIRE IRB, LaMesa, CA.

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Role of zinc trafficking in male fertility: from germ to sperm

Foresta C, Garolla A, Cosci I, Menegazzo M, Ferigo M, Gandin V, De Toni L

Department of Medicine and Centre for Human Reproduction Pathology, University of Padova, Padova 35128, Italy

STUDY QUESTION: What are the dynamics of zinc (Zn) trafficking in sperm, at the testicular, epididymal and ejaculate levels?
SUMMARY ANSWER: Zn transporters are peculiarly expressed in the cells of the germ line and Zn uptake is maximal at the post-epididymal phase, where Zn is involved in the regulation of sperm functions.
WHAT IS KNOWN ALREADY: Zn is known to influence several phases of sperm life, from germ cell development to spermiation. Zn trafficking across the membrane is allowed by specific families of transporters known as the ZnTs, which are involved in effluent release, and the Zips, which mediate uptake.
STUDY DESIGN, SIZE, DURATION: We enrolled 10 normozoospermic healthy participants in an infertility survey programme, as well as 5 patients affected by testicular germ cell cancer, and 18 patients presenting with obstructive azoospermia, without mutations of the CFTR gene, and undergoing assisted reproductive technologies.
PARTICIPANTS/MATERIALS, SETTING, METHODS: The research study was performed at our University Clinic. Semen samples, or biopsies or fine needle aspirates from the testis or epididymis, were obtained from each of the participants. Protein expression of main members of the ZnT and Zip families of Zn transporters was examined in human testis and epididymis samples by immunofluorescence. Quantification of sperm Zn content was performed by flow cytometry, atomic absorption spectrometry (AA) and autometallography.
MAIN RESULTS AND THE ROLE OF CHANCE: Intratubular cells of the germ line displayed a high redundancy of Zip family members involved in Zn uptake, while ZnT transporters were more represented in epididymis. Testicular and epididymal spermatozoa contained less Zn than ejaculated spermatozoa (2.56 ± 0.51 and 12.58 ± 3.16 versus 40.48 ± 12.71 ng Zn/10(6)cells, respectively). Gain of hypermotility and acrosomal reaction were significantly linked to the loss of Zn content in ejaculated spermatozoa.
LIMITATIONS, REASONS FOR CAUTION: This was an ancillary study performed on a small cohort of normozoospermic subjects. Although these results clarify the Zn trafficking during different phases of sperm life, no conclusive information can be drawn about the fertilizing potential of sperm, and the overall pregnancy outcomes, after Zn supplementation.
WIDER IMPLICATIONS OF THE FINDINGS: Our data disclose the dynamics of Zn trafficking during over the sperm lifespan.
STUDY FUNDING/COMPETING INTEREST(S): No external funding was sought or obtained for this study. No conflict of interest is declared.

doi: 10.1093/humrep/deu075

Effect of Cissampelos capensis rhizome extract on human spermatozoa in vitro

Shalaweh SM1, Erasmus N, Weitz F, Henkel RR.

Department of Medical Biosciences, University of the Western Cape, Bellville, South Africa.

Cissampelos capensis is commonly known by the Afrikaans name ‘dawidjies’ or ‘dawidjieswortel’. C. capensis is the most important and best-known medicinal plant of the family Menispermaceae used by the Khoisan and other rural people in the western regions of South Africa. Among numerous other ailments, it is traditionally taken to treat male fertility problems. Yet, no studies have investigated the effects of this plant or its extracts on human spermatozoa. The aim of study was to investigate the effects of C. capensis extracts on sperm function. A total of 77 semen samples were collected. Spermatozoa were washed with HTF-BSA medium and incubated with different concentrations of C. capensis (0, 0.05, 0.5, 5, 50, 200 μg ml-1 ) for 1 h at 37 °C. Sperm motility, vitality, acrosome reaction, reactive oxygen species (ROS), capacitation, Annexin V binding, DNA fragmentation and mitochondrial membrane potential (Δψm ) were determined. While viability, Annexin V positivity and Δψm were not affected, the percentages of ROS-positive, TUNEL-positive, capacitated and hyperactivated spermatozoa increased significantly and dose-dependently. It is concluded that the alkaloids present in the extract of C. capansis rhizomes triggered sperm intrinsic superoxide production leading to sperm capacitation and DNA fragmentation.

doi: 10.1111/and.12264

Vérification de performances d’un système CASA Sperm Class Analyzer SCA

Nassima Tarhouchi, Julie Le Bachelet, Daniel Przyrowski, Olivier Moreau.

Laboratoire Bioatlantique AMP diagnostique

Le Sperm Class Analyzer (SCA® MICROPTIC) est un système de gestion et d’analyse des principaux paramètres du sperme humain. Le logiciel comprend différents modules: numération, mobilité, cytologie… Le laboratoire s’est intéressé au programme « motility » afin d’évaluer la numération et la mobilité des spermatozoïdes.
La vérification des méthodes est une confirmation par des preuves tangibles que les exigences, spécifiées par le laboratoire, ont été satisfaites et sont adaptées à l’utilisation prévue. Ce processus permet de vérifier les performances et de garantir la fiabilité des résultats de mesure obtenus dans notre laboratoire.
Conformément à la norme NF EN ISO 15189 : 2012, toutes performances analytiques doivent être préalablement vérifiées. C’est pourquoi le programme « motility » du SCA, a été vérifié en se basant sur des critères d’acceptation définis par le laboratoire, en ayant prit comme référence le guide de l’OMS : « WHO laboratory manual for the examination and processing of human semen, fifth edition ».

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Human semen cryopreservation: a sperm DNA fragmentation study with alkaline and neutral Comet assay

J. Ribas-Maynou, A. Fernandez-Encinas, A. Garcıa-Peiro, E. Prada, C. Abad, M. J. Amengual, J. Navarro and J. Benet

Departament de Biologia Cel
lular, Fisiologia i Immunologia, Universitat Aut
onoma de Barcelona, Bellaterra, Catalunya, Spain, Centro de Infertilidad Masculina y Analisis de Barcelona (CIMAB), Edifici Eureka, PBM5, Parc de Recerca de la UAB (PRUAB), Bellaterra, Spain, Servei de Ginecologia, Hospital Universitari Mutua de Terrassa, Terrassa, Spain, Servei d’Urologia, Corporacio Sanit
aria Parc Taulı, Institut Universitari Parc Taulı – UAB, Sabadell, Spain, and UDIAT, Centre Diagn
ostic, Corporacio Sanit
aria Parc Taulı, Institut Universitari Parc Taulı – UAB, Sabadell, Spain

Sperm cryopreservation is widely used for both research and reproduction purposes, but its effect on sperm DNA damage remains controversial. Sperm DNA fragmentation (SDF) has become an important biomarker to assess male infertility. In particular, the differentiation between single- and double-stranded DNA fragmentation (ssSDF and dsSDF) has clinical implications for male infertility where ssSDF is associated with reduced fertility, whereas dsSDF is associated with increased risk of miscarriage. In this study, semen samples from 30 human males have been analysed in both fresh and cryopreserved using the alkaline and neutral Comet assays.
Results show an increase of about 10% of ssSDF, assessed by the alkaline Comet assay, regardless of the male fertility status. Neutral Comet analysis of dsSDF does not show any statistical increase when comparing fresh and cryopreserved samples in any of the patient groups. Results support previous reports that oxidative stress is the major effector in DNA damage during sample cryopreservation, as, on one hand, ssSDF has previously been related to oxidative damage and, on the other hand, we have not found any effect on dsSDF. Therefore, there might be a slight risk of decreased fertility after using a freezed sample, but no evidence for increased miscarriage risk from cryopreserved spermatozoa should be expected.

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Validation of the Sperm Class Analyser CASA system for sperm counting in a busy diagnostic semen analysis laboratory

Dearing CG, Kilburn S, Lindsay KS.

Andrology Laboratory, Hammersmith Hospital, Imperial College NHS Trust , London , UK

Verification of reference values for semen analysis according to WHO 2010 in Buenos Aires

Susana Mercedes Curi, Patricia Haydee Chenlo, Mercedes Norma Pugliese, Julia Irene Ariagno, Herberto Ernesto Repetto, José Vazquez, Melba Sardi Segovia, Gabriela Ruth Mendeluk

Laboratorio de Fertilidad Masculina, Departamento de Bioquímica Clínica, INFIBIOC, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Córdoba 2351 (1120), Buenos Aires, Argentina.

WHO 2010 provided reference values for semen analysis; however clinical laboratories should verify them in healthy fertile males. In order to be able to transfer this reference interval to our unit, it was proposed to examine the available data in the Buenos Aires population. The range of data obtained in this population was also provided for the following: total number of progressively motile and morphologically normal spermatozoa in the ejaculate, interkinematic parameter values and functional tests, in an attempt to establish standardization in these parameters. The process of reference value verification was carried out according to C28-A2 CLSI (Clinical and Laboratory Standards Institute) guidelines; to that aim, twenty men born in Argentina who reside in Buenos Aires province or city were recruited to participate. They were of proven fertility in the last 12 months. Individuals with pathologies by andrological consultation were excluded. Samples were processed according to 2010 WHO. In Buenos Aires City, WHO reference values were confirmed whereas semen volume was not confirmed. The range of values for total progressive and morphologically normal spermatozoa as well as kinetic parameter values, enrichment by swim up and functional test results were established for the male fertile population studied in an attempt to contribute to the construction of future reference values.

Acta Bioquím Clín Latinoam 2014; 48 (4): 429-35

Sperm DNA fragmentation assay by sperm chromatin dispersion (SCD): correlation between DNA fragmentation and outcome of intracytoplasmic sperm injection

T. Sivanarayana; Ch. Ravi Krishna; G. Jaya Prakash; K. M. Krishna; K. Madan; G. Sudhakar; G. A. Rama Raju

Gynecology Division, Krishna IVF Clinic, Visakhapatnam, India; Embryology Research Group, Krishna IVF Clinic, Visakhapatnam, India; Genetics Department, Krishna IVF Clinic, Visakhapatnam, India; Department of Human Genetics, Andhra University, Visakhapatnam, India

Abstract

Purpose: The aim of the present study was to investigate the relationship between sperm DNA fragmentation index (sDFI) and outcome of intracytoplasmic sperm injection (ICSI).

Methods: All the patients were divided into two groups based on sperm DNA fragmentation analysis by the sperm chromatin dispersion (SCD) method. A total of 237 patients were in the DNA fragmentation normal group (sDFI ≤ 30 %), and 140 patients were in the DNA fragmentation abnormal group (sDFI ≥ 30 %). The relationship of sDFI with the outcome of ICSI was analyzed.

Results: A significant difference in semen parameters was observed between the DNA fragmentation normal and abnormal groups [count, motility and morphology (p < 0.05)]. However, no significant difference was seen between the number of oocytes retrieved and fertilization rates between the two groups, whereas the number of embryos progressed to day 3 and the blastocyst formation rate in the remaining embryos after transfer were significantly more in the DNA fragmentation normal group (p < 0.05). A significant negative correlation was noted between DFI values of more than 30 % and number of pregnancies and deliveries (p < 0.05). A higher DFI was also associated with increased abortion rates.

Conclusions: In the present study, sperm with DNA fragmentation showed a negative correlation with semen parameters. Further, sperm with damaged DNA have potential adverse effects on embryo progression, clinical pregnancy rate, and ongoing pregnancies.

doi: 10.1007/s12522-013-0168-7

A multi-regional study on new approaches to investigate the quality of human sperm – including DNA fragmentation, proteomics and metabolomics

Hiva Alipour, Ingolf Nielsen, Fereshteh Dardmeh, Gerhard Van der Horst

Aalborg University, Aalborg University Hospital, University of the Western Cape

Background: Preliminary data has also shown that there is less fragmented sperm in 2nd and 3rd ejaculates compared to first ones which could be a major factor in determining the pregnancy outcome. Assessing this factor objectively and relating it to other parameters in sperm quality in this study could result in new prediction criteria for the pregnancy outcome. Materials and Methods: As one of the goals, this study will focus on analyzing the seminal fluid from the semen sample using NMR and Mass Spectrometry in research for a correlation between the results of these methods and the DNA fragmentation, Kinetic parameters and finally implantation rates and pregnancy outcome. Results: As another goal in this study we plan to assess whether the second consecutive sample obtained within 12 hours of the first sample has a lower DNA fragmentation rate which will be very important in lowering the number of failed implantations and repeated abortions. Conclusion: Collecting and analyzing the samples from different locations (different countries and continents) using the SCA (Sperm Class Analyzer, Microptic, Spain) which has eliminated the inter technician variation and provides quantitative data and a means to assess samples at the exact same scale, will provide a comparison of the average sperm statistics and also the quality of consecutive sperm samples in these different locations. This would specially prove to be of utmost importance in determining and endorsing a global scale for the computer assisted sperm analysis results which seem to be taking over the old visual (subjective) analysis of sperm among the laboratories and clinics worldwide.

Conference: 14th Royan Congress on Reproductive Biomedicine, At Tehran, Iran, Volume: 7, Suppl. 1

Sperm DNA Fragmentation Assays Correlate with Sperm Abnormal Morphology and the Pregnancy Outcome

Adriana Fortunato, Rita Leo, Sofia Casale, Giuseppina Nacchia, Francesca Liguori, Elisabetta Tost

Animal Physiology and Evolution laboratory Stazione Zoologica Anton Dohrn, Naples, Italy; DF center, Piazza Municipio 4, 80138- Naples, Italy

Abstract: 

Purpose: A retrospective study of 89 hypo-fertile male patients attending for in vitro fertilization was undertaken in order to evaluate possible correlations among sperm DNA damage, sperm analysis parameters and pregnancies.

Methods: Sperm parameters (concentration, normal morphology and multiple anomalies) were evaluated according to the World Health Organization guidelines. DNA damages weresimultaneously evaluated on each sperm sample by i) sperm chromatin decondensation test; ii) sperm DNA fragmentation evaluated by Sperm Chromatin Dispersion and Halo sperm Kit; iii) sperm DNA fragmentation evaluated by the Terminal Uridine Nick-End Labelling procedure.

Results: Statistical analysis was performed by using analysis of variance and least squares regression. The sperm chromatin dispersion and fragmentation assays showed a statistically significant positive correlation (P=0.0017) in all the samples confirming the good efficacy of either of the two tests in detecting sperm DNA damage. Both the two test negatively correlated with normal sperm morphology (P=0.008), however only by using Halo sperm test we obtained a significant correlation with multiple sperm pathological morphologies (P=0.029) and an inverse correlation with pregnancies outcome (P=0.013). No correlation was detected among DNA damages, sperm concentration and chromatin decondensation.

Conclusions: These data suggest a similar efficacy of sperm chromatin dispersion and Terminal Uridine Nick-End Labelling in detecting sperm DNA integrity. Due to the higher sensitivity of Halo test, its prognostic role in the diagnosis of fertilizing ability of a semen sample and possible pregnancy rate is discussed.

See full article here

Journal of Fertilization – DOI: 10.4172/2375-4508.1000101
Received April 09, 2013; Accepted May 04, 2013; Published May 07, 2013

Study of human semen: implementation of an objective method

Patricia Haydee Chenlo, Julia Irene Ariagno, Mercedes Norma Pugliese, Herberto Ernesto Repetto, Lydia Melba Sardi Segovia, Gabriela Ruth Mendeluk, Susana Mercedes Curi

Departamento de Bioquímica Clínica. Facultad de Farmacia y Bioquímica-UBA, INFIBIOQ. Hospital de Clínicas “José de San Martín”. Bs. As, Argentina. Hospital de Clínicas “José de San Martín” – Av. Córdoba 2351 (1120) CABA. Argentina

The aim of this study was to standardize and validate a CASA system, SCA (Sperm Class Analyzer) for the parameters of sperm concentration and motility according to international standards. The type of speed for the classification of degrees and the proper depth of the camera were standardized. For the validation, accuracy, detection limit, measuring range and method comparison study were determined. High linear correlation was found in the classification of motion using curvilinear velocity or average speed (r=0.99, p<0.001), establishing the use of the first to obtain results comparable with other objectives. Likewise, the use of cameras 10 microns in depth was also standardized since better images are obtained for the analysis without affecting mobility. Average imprecision was 13.29%, 14.26% and 18.75% for counting, progressive and mobile grades respectively. High correlation was found with the manual method, both in concentration (r=0.97, p<0.0001), progressive mobiles (r=0.84, (p<0.001) and mobile grade a (r=0.82, p<0.0001). Linearity (r=0.99, p<0.001) was proved between 0.98 and 125 x 106 x 106 sperm/mL. It is concluded that the proposed method meets the requirements for use in the clinic, being image editing by a qualified operator indispensable.

Acta Bioquím Clín Latinoam 2013; 47 (1): 61-69

An improved experimental model for understanding the impact of sperm DNA fragmentation on human pregnancy following ICSI

Nuñez-Calonge R, Caballero P, López-Fernández C, Guijarro JA, Fernández JL, Johnston S, Gosálvez J

Clínica Tambre, Madrid, Spain

Using donor oocytes of proven fertility, the effect of sperm DNA fragmentation (SDF) and motility on reproductive success was examined in 70 couples undergoing ICSI. Both SDF and sperm motility were assessed at the time of sperm injection and using the same sperm sample that was processed for ICSI. While there was no difference in the fertilization rate, cleavage rate, embryo quality, or sperm motility between pregnant and nonpregnant couples, the SDF of nonpregnant couples (SDF = 23.9%) was higher than that of pregnant couples (SDF = 17.0%; U Mann-Whitney 347; P = .002). Using a combination of the sensitivity and specificity measures from the production of ROC (receiver-operating characteristic) curves and the Youden index, we determined a threshold SDF value for our data set of 17% for predicting pregnancy (77.8% sensitivity and 71.1% specificity). Our results suggest that proven donor oocytes in combination with SDF assessment at the time of sperm injection represent a useful experimental model for reducing the confounding influences of sperm DNA repair by the oocyte and iatrogenic sperm damage.

doi: 10.1177/1933719112459238

Effect of relaxin on human sperm functions

Ferlin A, Menegazzo M, Gianesello L, Selice R, Foresta C

University of Padova, Department of Histology, Microbiology and Medical Biotechnologies, Section of Clinical Pathology and Center for Male Gamete Cryopreservation, Padova, Italy

Relaxin is a circulating hormone with functions in pregnancy, parturition, and other aspects of female reproduction. It is also secreted from the prostate gland into the seminal fluid; however, the role of relaxin in male reproduction is debated. Studies conducted in the past have suggested possible actions on human spermatozoa, but the data were contrasting. Here, we show that the relaxin receptor RXFP1 (Relaxin Family Peptide Receptor 1) is expressed in human spermatozoa, and it mainly localizes in the astrodome. In vitro studies on human sperm demonstrated that this hormone attenuates the natural decline in sperm motility and maintains higher mitochondrial activity and lower apoptosis level. Furthermore, relaxin induced an increase in sperm hyperactivation, intracellular calcium and cAMP, and acrosome reaction. These effects were abolished by the use of the specific anti-RXFP1 antibody. Relaxin concentrations were low in the blood (x ± SD, 0.16 ± 0.03 nM) and very high in the seminal plasma (x ± SD, 10.3 ± 4.0 nM), confirming its secretion mainly by the prostate. Taken together, these data demonstrate that relaxin influences positively many sperm functions linked to fertilizing ability, and it preserves sperm functionality, with possible practical value in assisted reproduction techniques.

doi: 10.2164/jandrol.110.012625

Characterization of the kisspeptin system in human spermatozoa

Pinto FM, Cejudo-Román A, Ravina CG, Fernández-Sánchez M, Martín-Lozano D, Illanes M, Tena-Sempere M, Candenas ML

Instituto de Investigaciones Químicas, CSIC-Universidad de Sevilla, Spain

Kisspeptin, the product of the KISS1 gene, plays an essential role in the regulation of spermatogenesis acting primarily at the hypothalamic level of the gonadotropic axis. However, the presence of kisspeptin and its canonical receptor, KISS1R, in spermatozoa has not been explored nor the direct effects of kisspeptin on sperm function have been studied so far. In the present study, we analysed the expression of kisspeptin and its receptor in sperm cells by western blot and immunocytochemistry assays and evaluated the effects of exposure to kisspeptin on sperm intracellular Ca(2+) concentration,

Study for the diagnostic evaluation of the sperm DNA fragmentation index in a patient population of the fertility unit

Albert Blanco, Jordi Ramis, Carlos Aulesa, José Mª Gris

Laboratorio de Andrología, Unidad de Laboratorios Clínicos Vall d’Hebron, Barcelona. Servicio de Esterilidad Hospital Maternal Vall d’Hebron.

Summary:

Introduction: At present, parameters obtained via the common seminogram fail to provide complete information on the fertilizing potential of semen, as it is observed that between 10 to 15% of sterile males show a normal seminogram.

Objective: This study has verified the clinical utility of determination of the sperm DNA Fragmentation Index (DFI) as a diagnosis aid to male subfertility.

Design: 60 samples were processed from patients at the Fertility Unit, of which 42 patients showed proven infertility due to their clinical history, the remaining 18 were fertile males. The corresponding seminogram and FI were carried out using Halosperm® (Halotech D, Madrid) commercial equipment for the preparation of samples and a Sperm Class Analyzer (SCA) type Computer Assister Semen Assay® (CASA) for reading the sperm fragmentation.

Results: The FI sensitivity in diagnosis of subfertility was 54.8% and the specificity 66.7%, with a final diagnostic efficiency of 58.3%. The seminogram showed a sensitivity of 8.7% and a specificity of 50%, with diagnostic efficiency of 78.9%. The areas under the curve (AUC) are calculated from the ROC curves; it is shown that the FI presents a AUC=0.601 and the seminogram a AUC=0.679, which indicates that the seminogram has a higher diagnostic value.

Conclusions: Further to the data obtained, we reach the conclusion that the FI test does not appear to show a higher diagnostic value than the classic parameters of the seminogram. Likewise, we have observed that a pathological result in the FI can be a factor to consider for those couples who still have doubts about exploring other assisted reproductive techniques or Artificial Insemination by donor (AID).

Full article here

Revista Iberoamericana de Fertlidad y Reproducción Humana
Vol. 28 nº1 Enero-Febrero-Marzo 2011

Noninsulin-dependent diabetes mellitus: effects on sperm morphological and functional characteristics, nuclear DNA integrity and outcome of assisted reproductive technique

G. A. Rama Raju, G. Jaya Prakash, K. Murali Krishna, K. Madan, T. Siva Narayana & C. H. Ravi Krishna

Gynecology Division, Krishna IVF Clinic, Visakhapatnam, India; Embryology Research Group, Krishna IVF Clinic, Visakhapatnam, India; Genetic Department, Krishna IVF Clinic, Visakhapatnam, India

The aim of the study was to compare the semen characteristics and nuclear DNA fragmentation in spermatozoa of diabetic and nondiabetic men undergoing assisted reproduction and correlate them with pregnancy outcome. Semen characteristics and nuclear DNA fragmentation were analysed using computeraided semen analysis system and sperm chromatin dispersion assay (SCD), respectively. Spermatozoa from diabetic patients showed significantly lower progressive (Type A) motility (14.64 ± 9.60 versus 17.99 ± 11.51, P < 0.02) and increased nuclear DNA fragmentation (37.05 ± 12.68 versus 21.03 ± 10.13, P < 0.001). Furthermore, a positive correlation was observed in diabetic patients in terms of blastocyst formation rate (38.13% versus 55.46%, P < 0.001), pregnancy rate (28.57% versus 46.34%, P < 0.001) and miscarriage rate (50.0% versus 24.56%, P < 0.001). The higher percentage of sperm DNA damage because of oxidative stress seen in diabetic patients may be responsible for the poor embryonic development and pregnancy outcome in these individuals.

doi: 10.1111/j.1439-0272.2011.01213.x

CASA derived human sperm abnormalities: correlation with chromatin packing and DNA fragmentation

T. Sivanarayana, Ch. Ravi Krishna, G. Jaya Prakash, K. Murali Krishna, K. Madan, B. Sireesha Rani, G. Sudhakar, G. A. Rama Raju

Gynecology Division, Krishna IVF Clinic, Visakhapatnam, India; Embryology Research Group, Krishna IVF Clinic, Visakhapatnam, India; Genetics Department, Krishna IVF Clinic, Visakhapatnam, India; Department of Human Genetics, Andhra University, Visakhapatnam, India

Purpose The present study was undertaken to evaluate the effects of morphokinetic abnormalities of human spermatozoa on chromatin packing and DNA integrity and possible beneficial effects of sperm selection in ICSI.
Methods Semen samples from 1002 patients were analysed for morphology and motility using CASA. Protamine status and DNA fragmentation were analysed by chromomycin A3 staining and sperm chromatin dispersion assay respectively.
Results Sperms with elongated, thin, round, pyri, amorphous, micro and macro forms were significantly higher in teratozoospermic and oligoasthenoteratozoospermic groups. Significant difference in chromatin packing and DNA fragmentation index was observed in these abnormal groups compared with normal. Similarly significant correlation was also seen between abnormal motility parameters and DNA fragmentation index in asthenozoospermic group compared with normal.
Conclusions Specific abnormal morphological forms have higher incidence of chromatin packing abnormalities and DNA fragmentation. Using these sperms in ICSI might have an impact on fertilization, embryo development and abortion rates. These can be selectively avoided during ICSI procedure to improve ART outcome.

doi: 10.1007/s10815-012-9885-9

Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders

Gadea J, Molla M, Selles E, Marco MA, Garcia-Vazquez FA, Gardon JC

Dept. Physiology, University of Murcia, Murcia 30100, Spain

In this study, total glutathione content was determined in human spermatozoa before and after cryopreservation. Total GSH in fresh semen was 4.47±0.46nmol/10(8) cells. Following semen cryopreservation, GSH decreased to 1.62±0.13nmol/10(8) cells, a 64% reduction (p<0.01). This decrease in GSH content was associated with a decrease in sperm progressive motility (68% of reduction, p<0.01). Addition of 1mM GSH to the freezing extender increased the percentage of total motility and sperm viability. It also modified the motility pattern measured by CASA with changes in the straight-line and average path velocities and wobble of the curvilinear trajectory. Addition of GSH to the freezing media reduced spermatozoa ROS levels and increased the level of sulfhydryl groups on membrane proteins. Nevertheless, no effect of GSH addition on lipid membrane disorder or chromatin condensation was detected. Addition of 1 or 5mM GSH to the thawing media increased the percentage of motile and progressively motile spermatozoa, but no effect on viability was detected. In conclusion, the antioxidant defensive capacity of the GSH is severely altered by the freeze-thawing process. The addition of GSH to the freezing and thawing extender could be of partial and limited benefit in improving the function of frozen human spermatozoa.

doi: 10.1016/j.cryobiol.2010.12.001

Evaluation of sperm quality at a national level: Logistic, recruitment, analytical and statistical problems encountered in switzerland

Josefina Vargas, Roumen Parapanov, Marc Van Den Bergh, Eric Stettler, Pierre Crettaz, Alfred Senn, Marc Germond

Fondation Faber, Lausanne, Switzerland, Kantonspital Baden Ag, Fertilitätslabor, Switzerland, Swiss Army Medical Services, Switzerland, Federal Office of Public Health, Switzerland

Aim:
For decades, numerous studies have reported a decline in sperm quality among industrialised countries. Explanations for this decline point toward environmental factors acting on animals and humans. More recently, the detrimental role of endocrine disruptors during foetal development of the testicles has also been hypothesised. In order to test whether these effects are operating in Switzerland, a survey study among Swiss young men was initiated in 2005 and will cover within the next two years the entire country. The aim of this report is to present the differences in sperm quality observed so far in various geographic regions of the Switzerland.
Method:
One month before recruitment, all conscripts are informed about the study. When interested, the volunteers send a consent form and two questionnaires to the Swiss army physician in charge of supervising the study and warranting anonymity. Four recruiting centres (Lausanne, Rüti, Windisch, Monte Ceneri) are actively involved in the sample collection phase. At the end of their recruiting camp, volunteers are invited in a nearby laboratory for biological investigations. Sperm samples are analysed according to WHO recommendations. Sperm concentration, motility and morphology are measured using a computerised system (CASA, SCA Microptic, Spain).
Results:
Results are grouped according to a geographic stratification of Switzerland into (1) Plateau west and central, (2) Jura, (3) Alps, (4) Plateau north and east. Medians for sperm concentration were significantly lower in regions 3 and 4 compared to the two other regions, whereas sperm motility was less affected.
Conclusion:
The geographic differences observed in sperm quality throughout Switzerland are currently further investigated by enlarging the cohort and identifying possible explanations for such differences. A human biomonitoring is underway in order to identify the presence of various endocrine disruptors in serum and urine samples collected from the volunteers.

doi: 10.1016/S1472-6483(10)62403-0

Impact of spontaneous smoking cessation on sperm quality: case report

E. Prentki Santos, S. López-Costa, P. Chenlo, M. N. Pugliese, S. Curi, J. Ariagno, H. Repetto, M. Sardi, L. Palaoro & G. Mendeluk

Laboratory of Andrology and Male Fertility, Clinical Hospital, INFIBIOC, University of Buenos Aires, Argentina; Urology Division, Tobacco Cessation Program, Ramos Mejía Hospital, Government of the City of Buenos Aires, Argentina

We evaluated sperm quality after a 3-month smoking cessation programme by sperm analysis, objective sperm motility analysis, protein tyrosine phosphorylation in capacitating conditions and DNA fragmentation (TUNEL). Sperm analysis after smoking cessation revealed a distinctive improvement in sperm concentration, fast spermatozoa (‡35 lm/s), sperm vitality, percentage of spermatozoa recuperated after an enrichment technique and protein tyrosine phosphorylation. However, no changes were observed in the number of germinal cells in the ejaculate, sperm morphology and sperm DNA fragmentation. It is concluded that physicians should strongly advise their patients to quit smoking before undergoing medical treatment or assisted reproduction techniques to achieve pregnancy.

doi: 10.1111/j.1439-0272.2010.01089.x

Morphometric dimensions of the human sperm head depend on the staining method used

L. Maree, S.S. du Plessis, R. Menkveld and G. van der Horst

Department of Medical Biosciences, University of the Western Cape, Private Bag X17, Bellville 7530, South Africa. Division of Medical Physiology; Department of Biomedical Sciences, Stellenbosch University, PO Box 19063, Tygerberg 7505, South Africa; Andrology Laboratory, Department of Obstetrics and Gynaecology, Tygerberg Academic Hospital and Stellenbosch University, Tygerberg 7505, South Africa

BACKGROUND: Assessment of sperm morphology (including morphometry) is extensively used to determine one of the qualities of a semen sample and depends on the differential staining of spermatozoa. A staining technique should cause as little change to sperm dimensions and form as possible in order to reliably evaluate the morphometric features of the sperm. Various staining techniques have been employed, but only a few have been recommended by the World Health Organization and are amenable to automated sperm morphometry analysis. Our study was aimed at comparing the effect of three staining techniques [Papanicolaou (PAP), Rapidiff® (RD) and SpermBlue® (SB)] on human sperm head dimensions and to compare these with the head dimensions in fresh semen.
METHODS: Smears made from human semen samples (n = 24) were stained according to the three staining techniques and sperm head morphometry was assessed with the Sperm Class Analyzer. Head dimensions of fresh spermatozoa were measured with a digital calliper on a computer screen. The minimum number of spermatozoa to be analyzed to represent the sperm population and the degree of inter-laboratory variation were determined. Electron micrographs from the same semen samples were used to determine the actual acrosome coverage of the spermatozoa in the semen (n = 7) in order to verify the results of the automatic analyses.
RESULTS: The osmolality of human semen differs from that of the RD and PAP fixatives and stains, but is more similar to the SB fixative and stain. At least 100 spermatozoa should be analyzed to include a representative sample of the sperm population. RD caused sperm heads to swell, PAP caused them to shrink and SB had no significant effect on sperm head dimensions when compared with spermatozoa in fresh semen. Very little inter-laboratory variations were found. The percentage acrosome coverage was significantly different between the three staining techniques, as well as between the RD and PAP stains and the manual measurements obtained using the electron micrographs.
CONCLUSIONS: Different staining techniques change the morphometric dimensions of the human sperm head, probably due to the fact that either the fixatives or stains are not iso-osmotic in relation to human semen. Since these changes in sperm head dimensions are not uniform, care should be taken when selecting a staining technique. Ideally, stained spermatozoa should have dimensions as close to spermatozoa in fresh semen as possible, as was found with the SB staining method, resulting in accurate evaluations of sperm head morphometry.

doi: 10.1093/humrep/deq075

Variation of semen parameters in healthy medical students due to exam stress

Fanuel Lampiao

College of Medicine, Blantyre, Malawi

Aim
This study was aimed at investigating semen parameters that vary most in samples of healthy donors undergoing stressful examination period.
Methods
Samples were left to liquefy in an incubator at 37°C, 5% CO2 for 30 minutes before volume was measured. Concentration and motility parameters were measured by means of computer assisted semen analysis (CASA) using Sperm Class Analyzer® (Microptic S.L, Madrid, Spain).
Results
Sperm concentration was significantly decreased in samples donated close to the exam period as well as samples donated during the exam period when compared to samples donated at the beginning of the semester.
Conclusion
Stress levels of donors might prove to be clinically relevant and important when designing experiment protocols.

Malawi Med J. 2009 Dec; 21(4): 166–167.

Effects of H2O2 exposure on human sperm motility parameters, reactive oxygen species levels and nitric oxide levels

S. S. du Plessis, D. A. McAllister, A. Luu, J. Savia, A. Agarwal& F. Lampiao

Divison of Medical Physiology, University of Stellenbosch, Tygerberg, South Africa;Center for Reproductive Medicine, Glickman Urological and Kidney Institute, Cleveland Clinic, Cleveland, OH, USA

Research has revealed that reactive oxygen species (ROS) negatively affect sperm function, both in vivo and in vitro. Sperm preparation techniques for assisted reproductive technologies (ART) are potential causes for additional ROS production. This study aimed to correlate the concentration of exogenous H(2)O(2) with sperm motility parameters and intracellular ROS and nitric oxide (NO) levels to reiterate the importance of minimising ROS levels in ART. Human spermatozoa from 10 donors were incubated and exposed to different exogenous H(2)O(2) concentrations (0, 2.5, 7.5 and 15 mum). Subsequently, motility was determined using computer-aided semen analysis, while ROS (2,7-dichlorofluorescin diacetate) and NO (diaminofluorescein-2/diacetate) were analysed using fluorescence-activated cell sorting. Results showed that H(2)O(2) did affect the sperm parameters. Exogenous H(2)O(2) was detrimental to motility and resulted in a significant increase in overall ROS and NO levels. A significant increase in static cells was seen as well. It is important to elucidate the mechanisms between intracellular ROS levels with sperm motility parameters. While this experiment demonstrated a need to reduce exogenous ROS levels during ART, it did not illustrate the cause and effect relationship of intracellular ROS and NO levels with sperm motility. Further research needs to be conducted to define a pathological level of ROS.

doi: 10.1111/j.1439-0272.2009.00980.x

Functional polymorphism in H2BFWT-5’UTR is associated with susceptibility to male infertility

Lee J, Park HS, Kim HH, Yun YJ, Lee DR, Lee S

Department of Pharmacology, CHA Stem Cell Institute, School of Medicine, CHA University, Bundang-Gu, Seongnam-Si, Kyunggi-Do, Korea

H2B histone family, member W, testis-specific (H2BFWT) gene encodes a testis-specific histone that becomes incorporated into sperm chromatin. A male infertility-associated single nucleotide polymorphism (-9C > T) within the 5′ untranslated region (5’UTR) of the H2BFWT gene was identified by direct sequencing. Statistical association studies showed the polymorphism significantly associated with male infertility (n = 442, P = 0.0157), especially in non-azoospermia (n = 262, P = 0.018). Furthermore, this polymorphism is also associated with sperm parameters, especially sperm count (n = 164, P = 0.0127) and vitality (n = 164, P = 0.0076). We investigated how the genetic variant at 5’UTR confers susceptibility to non-azoospermia. Western blotting of His-tag H2BFWT revealed a difference at the translational level between -9T and the wild-type -9C in the absence of change at the transcriptional level. Reporter assays showed that this reducing translational change originated from an upstream open reading frame (uORF) generated by the -9C to -9T change. Finally, in vivo H2BFWT expression in sperm was significantly dependent on the -9C > T genotype from non-azoospermia (P = 0.0061). Therefore, this polymorphism could affect the translational efficiency of a quantitatively important histone protein by the uORF. Our data implicate H2BFWT as a susceptibility factor for male infertility, possibly with other genetic and environmental factors.

doi: 10.1111/j.1582-4934.2009.00830.x

Clinical implementation of the Halosperm® test kit in combination with SCA®

Kellie Williams, PhD and J. Kevin Thibodeaux, PhD, HCLD

Tulsa Fertility Center, 115 East 15th St., Tulsa OK 74119

Fertility Magazine, Volume 12, pages 66-68

Evaluation of automated system Sperm Class Analyzer (SCA) for semen analysis

Carlos Aulesa, M. Cabrera, R. Alonso, M. Benítez y M. Martínez

Unidad de Seminología, Laboratorios Clínicos, Ciutat Sanitària Vall d’Hebron, Barcelona, España

Objective
Evaluation of the efficiency of the New Sperm Class Analyzer® (SCA v.3.2.0) in sperm automated analysis for the calculation of the motility, concentration and morphology parameters using the latest CASA (Computer-Assisted Sperm Analysis) technology in software for spermiogram analysis.
Materials and methods
Analysis of 150 semen samples from the clinical areas of Fertility and Urology, 42 samples were analyzed with the SCA concentration module and 65 samples were analyzed with the SCA motility module using a disposable 10 micron deep Leja Chamber in both. At the same time the samples were analyzed by the manual method using the Neubauer Improved chamber for counting and slides with 22×22mm cover slip heated to 37°C with an average of 200 spermatozoa for motility, in order to test the precision, linearity and accuracy of SCA system compared with the manual method. The morphology analysis was evaluated using 50 pre-stained slides and evaluating 200 cells both manually by an ESHRE qualified technician and the SCA method and then calculating the correlation coefficient.
Results
The counting with SCA system compared with the manual method has shown a within-day imprecision of 10.13% with a range between 3.2% and 17.1% depending on the count level; correlation with the manual method was r=0.98 (P=0.001). The linearity of the SCA was shown to be linear between 0.5 million and 190 million sperm/ml. The motility module showed a higher within-day imprecision in counting WHO ¿type b¿ and ¿type c¿ spermatozoa (21.81%) than the ¿type a¿ and ¿type d¿, with an average value of 10.32%, also depending on the count rate. The reliability of the motility parameter was evaluated up to a of 120 million sperm/ml. The morphology module, with a customised configuration of parameters, had a significant positive correlation (r=0.899; P=0.0001) compared with the manual method.
Conclusions
The comparison of the concentration and motility of spermatozoa between the manual method and the SCA modules using 10 microns deep Leja chambers was positively evaluated. A series of premises are established for using the automated morphology performed on the Sperm Class Analyzer. The clinical usefulness of some of the new kinetic and morphometric parameters of sperm provided by the semen analyser is also established.

Laboratorio Clinico. 2009;02:8-16.