Abstract Several recent studies have indicated that radiofrequency electromagnetic fields (RFEMF) have an adverse effect on human sperm quality, which could translate to an effect on fertilization potential. The present study evaluated the effect of RF-EMF on spermspecific characteristics in order to assess the fertilizing competence of sperm. Highly motile human spermatozoa, were exposed for one hour to 900 MHz mobile phone radiation at a specific absorption rate (SAR) of 2.0 W/kg and examined at various times after exposure. The acrosome reaction was evaluated using flow cytometry. The radiation did not affect sperm propensity for the acrosome reaction. Morphometric parameters were assessed by computer assisted sperm analysis (CASA). Significant reduction in sperm head area (9.2 ± 0.7 μm2 vs. 18.8 ± 1.4 μm2) and acrosome percentage of the head area (21.5 ± 4% vs. 35.5 ± 11.4%) were reported among exposed sperm compared with unexposed controls. Sperm–zona binding was assessed directly after exposure using the hemizona assay (HZA). The mean number of zona-bound sperm of the test hemizona and controls was 22.8 ± 12.4 and 31.8 ± 12.8 (p<0.05), respectively. This study concludes that while RF-EMF exposure did not adversely affect the acrosome reaction, it had a significant effect on sperm morphometry. In addition a significant decrease in sperm binding to the hemizona was observed. These results could indicate a significant effect of RF-EMF on sperm fertilisation potential.

Recent reports suggest that mobile phone radiation may diminish male fertility. However, the effects of this radiation on human spermatozoa are largely unknown. The present study examined effects of the radiation on induction of apoptosisrelated properties in human spermatozoa. Ejaculated, densitypurified, highly motile human spermatozoa were exposed to mobile phone radiation at specific absorption rates (SARs) of 2.0 and 5.7 W/kg. At various times after exposure, flow cytometry was used to examine caspase 3 activity, externalization of phosphatidylserine (PS), induction of DNA strand breaks, and generation of reactive oxygen species. Mobile phone radiation had no statistically significant effect on any of the parameters studied. This suggests that the impairment of fertility reported in some studies was not caused by the induction of apoptosis in spermatozoa.

Ejaculated, density purified, human spermatozoa were exposed to 900 MHz GSM mobile phone radiation at two specific absorption rate levels (SAR 2.0 and 5.7 W/kg) and examined at various time points post exposure. Change in sperm mitochondrial membrane potential was analyzed using flow cytometry. Sperm motility was determined by computer assisted sperm analysis (CASA). There was no effect of 900MHz GSM radiation on mitochondrial membrane potential. This was also the case for all kinematic parameters assessed at SAR of 2.0 W/kg. However, two kinematic parameters (VSL and BCF) were statistically significantly altered after the exposure at SAR 5.7 W/kg. Effects seen cannot be ascribed to heating, as the temperature did not increase by more than 0.3ºC. A thorough investigation at lower SAR levels is required to determine the extent of the influence of RF-EMF on human sperm motility.

Abstract Ejaculated, density purified, human spermatozoa were exposed to pulsed 900 MHz GSM mobile phone radiation at two specific absorption rate levels (SAR 2.0 and 5.7 W/kg) and compared with controls over time. Change in sperm mitochondrial membrane potential was analysed using flow cytometry. Sperm motility was determined by computer assisted sperm analysis (CASA). There was no effect of pulsed 900 MHz GSM radiation on mitochondrial membrane potential. This was also the case for all kinematic parameters assessed at a SAR of 2.0 W/kg. However, over time, the two kinematic parameters straight line velocity (VSL) and beat-cross frequency (BCF) were significantly impaired (P<0.05) after the exposure at SAR 5.7 W/kg and no exposure by time interaction was present. This result should not be ascribed to thermal effects, due to the cooling methods employed in the RF chamber and temperature control within the incubator.

Thesis (MMed)--Stellenbosch University, 2105. / ENGLISH ABSTRACT: Intracytoplasmic sperm injection (ICSI), as well as other micromanipulation assisted reproductive technology methods, such as physiologic ICSI (PICSI) and intracytoplasmic morphologically selected sperm injection (IMSI), are routinely used in many fertility laboratories around the world. An integral part of these methods is the manipulation of spermatozoa in preparation of the injection into the oocyte. It is common practice to place prepared spermatozoa in a viscous holding medium to facilitate the handling, manipulation and slowdown of spermatozoon movement during the immobilization and injection processes of ICSI. The possible effect of these holding mediums on basic semen parameters, as well as the sperm deoxyribonucleic acid (DNA) and structural integrity of spermatozoa, is of importance. Hamilton Thorne IVOS® developed an automated software solution for live sperm morphology evaluation under high magnification, called IMSI StrictTM. It combines Tygerberg Strict Criteria morphological classification of human spermatozoa with motile sperm organelle morphology examination (MSOME) and provides software-based categorization. The IMSI StrictTM software was developed to aid in the IMSI spermatozoon selection process that enables objective classification of spermatozoa to remove inter-technician variation. For good optics and spermatozoon evaluation in IMSI StrictTM, spermatozoa need to be moving very slowly or be immotile, but still viable. This can be achieved by placing spermatozoa in a viscous holding medium, either polyvinylpyrrolidone (PVP) or SpermSlowTM, sometimes for a substantial time period. Before marketing the clinical use of IMSI StrictTM, the possible toxicity or deleterious effect of PVP and SpermSlowTM on spermatozoa needs to be excluded. The primary objective of this study was to evaluate the effect of PVP and SpermSlowTM on human spermatozoa after different exposure times using a viability stain, CASA motility and kinetic parameters, chromatin packaging analysis (CMA3 staining analysis) and DNA fragmentation analysis (TUNEL analysis). The secondary objective was to evaluate the effect of PVP and SpermSlowTM on human spermatozoa‟s ultrastructure with Transmission Electron Microscopy. This prospective analytical study was conducted at Drs Aevitas Fertility Clinic (Vincent Pallotti Hospital, Cape Town, South Africa) as well as the Fertility Unit at Tygerberg Hospital (Cape Town, South Africa) between July 2013 and October 2014. A total of 90 separate (no duplication) semen samples were analysed for the quantitative analysis (primary objective) and 1 sample for the descriptive analysis (secondary objective). Results showed that although PVP and SpermSlowTM treated sperm outcomes often differed significantly after typical statistical analysis, clinically these two mediums were shown to be equivalent (using a specific statistical test for equivalence) for the tested outcomes. PVP and SpermSlowTM had no detrimental effect clinically on sperm viability, motility parameters, chromatin packaging and DNA fragmentation rate. The secondary investigation indicated that SpermSlowTM might exert a disintegrating effect on various sperm membranes, and as a secondary consequence of the eventual necrotic process, alteration of chromatin and cytoskeletal components. PVP medium on the other hand did not show these disintegrating effects. This finding needs to be further investigated since only one semen sample was evaluated. Based on this study‟s results, either PVP or SpermSlowTM can be used for IMSI StrictTM purposes. However, the study did not include the technical aspects of the usage of PVP and SpermSlowTM. / AFRIKAANSE OPSOMMING: Intrasitoplasmiese sperm inspuiting (ICSI), sowel as ander mikro-manipulasie voortplantings tegnieke, soos fisiologiese ICSI (PICSI) en intrasitoplasmiese morfologies geselekteerde sperm inspuiting (IMSI), word in baie fertiliteitsklinieke regoor die wêreld gebruik. 'n Integrale deel van hierdie metodes is die manipulasie van spermatosoa ter voorbereiding van die inspuitproses. Dit is algemeen om voorbereide spermatosoa in 'n viskose medium te plaas om die hantering, manipulasie en vertraging van spermatosoön beweging tydens die immobilisasie en inspuitproses van ICSI te fasiliteer. Die effek van hierdie mediums op basiese semenparameters, sowel as die sperm deoksiribonukleïensuur (DNS) en strukturele integriteit van spermatosoa, is van belang. Hamilton Thorne IVOS® het 'n sagteware oplossing, IMSI StrictTM, vir lewende sperm morfologie evaluering onder hoë vergroting ontwikkel. Hierdie sagteware bied sagteware-gebaseerde morfologiese klassifikasie deur die Tygerberg streng kriteria morfologiese klassifikasie met beweeglike spermorganel morfologie ondersoek (MSOME) te kombineer. Die IMSI StrictTM sagteware is ontwikkel om die objektiewe klassifikasie van spermatosoa vir IMSI spermatosoön seleksie moontlik te maak. Spermatosoa moet baie stadig beweeg of immotiel, maar steeds lewensvatbaar wees om goeie optika en spermatosoön evaluering vir IMSI StrictTM te verseker. Dit sal bereik kan word deur spermatosoa in 'n viskose medium, hetsy PVP (“polyvinylpyrrolidone”) of SpermSlowTM, vir 'n aansienlike tydperk te inkubeer. Voordat IMSI StrictTM vir kliniese gebruik bemark kan word moet die moontlike toksisiteit of nadelige effek van PVP en SpermSlowTM op spermatosoa uitgesluit word. Die primêre doel van hierdie studie was om die effek van PVP en SpermSlowTM op menslike spermatosoa na verskillende inkubasie tye te evalueer deur ʼn lewensvatbaarheid kleuring toets, twee sperm DNS toetse (CMA3 en TUNEL) en rekenaar geëvalueerde sperm beweeglikheid toetse te gebruik. Die sekondêre doel was om die effek van PVP en SpermSlowTM op menslike spermatosoa se ultrastruktuur deur middel van Transmissie Elektronmikroskopie te evalueer. Hierdie studie is by Drs Aevitas Fertiliteitskliniek (Vincent Pallotti Hospitaal, Kaapstad, Suid-Afrika) sowel as die Fertiliteitseenheid by Tygerberg Hospitaal (Kaapstad, Suid-Afrika) tussen Julie 2013 en Oktober 2014 uitgevoer. 'n Totaal van 90 semenmonsters vir die kwantitatiewe analise (primêre doel) en een vir die beskrywende analise (sekondêre doel) is ontleed. Resultate het getoon dat alhoewel PVP en SpermSlowTM geïnkubeerde spermuitkomste dikwels na ʼn tipiese statistiese analise betekenisvol verskil, hierdie twee mediums vir die geëvalueerde uitkomste klinies ekwivalent (bepaal deur middel van spesifieke statistiese toetse vir ekwivalensie) is. Die mediums het ook nie klinies 'n nadelige effek op sperm lewensvatbaarheid, beweeglikheid parameters, chromatien verpakking en DNS fragmentasie koers getoon nie. Die sekondêre ondersoek het getoon dat SpermSlowTM hoofsaaklik 'n effek van disintegrasie op verskeie spermmembrane getoon het. Hierdie nekrotiese proses kan lei tot verandering van chromatien en sitoskelet komponente. PVP medium het egter nie dieselfde disintegrerende effek getoon nie. Hierdie bevinding moet egter verder ondersoek word, aangesien slegs een semenmonster geëvalueer is. Alhoewel hierdie studie nie die tegniese aspekte van die gebruik van PVP en SpermSlowTM geëvalueer het nie, kan aanbeveel word dat óf PVP óf SpermSlowTM op grond van geëvalueerde uitkomste tydens die IMSI StrictTM sperm seleksie proses gebruik word.

Several studies have highlighted the possibility that radio-frequency electromagnetic fields (RF-EMF) used in mobile phone technology could influence DNA integrity of male germ cells as well as sperm motility. Current knowledge concerning the influence of RF-EMF on male germ cells is extremely limited. In the present study the hypothesis that 900 MHz GSM radiation could induce the activation of stress response in human spermatozoa was investigated. Ejaculated, density purified, human spermatozoa from donors were exposed to 900 MHz GSM mobile phone radiation at specific absorption rate (SAR) levels of 2.0 and 5.7 W/kg and examined at various time points post exposure. Sperm motility and morphology were evaluated by computer-aided sperm analysis (CASA). The ability of RF-EMF exposed sperm to undergo the acrosome reaction was evaluated by flow cytometry. Sperm binding to the zona pellucida of human oocytes was determined by the hemi-zona (HZA) assay. Apoptotic markers, phosphatidylserine (PS) externalization, change in mitochondrial membrane potential (Δψm), reactive oxygen species (ROS) generation, caspase activation and DNA fragmentation were analysed using flow cytometry. Heat shock protein (Hsp) 27 and 70 expression and activity were analyzed using specific antibodies with flow cytometry and Western blot methods. Stress fibre stabilization (F-actin polymerization) was visualized using fluorescent dye labelled phalloidin. No effect was seen on kinematic parameters assessed at SAR 2.0 W/kg, however straight line velocity (VSL) and beat cross frequency (BCF) were significantly altered after exposure at SAR 5.7 W/kg. Sperm shrinkage (decrease in surface area) was observed at both exposure levels. RF-EMF did not influence exposed spermatozoa’s ability to undergo the acrosome reaction. A significant decrease in sperm-zona binding was observed at both exposure levels. RF radiation did not have an effect on any apoptotic markers. ROS generation increased significantly with an increase in SAR (5.7 W/kg). RF-EMF did not induce a stress response in exposed sperm (no activation of Hsp70 and 27 activity). These results cannot be ascribed to heating, as the temperature did not increase by more than 0.2 - 0.3ºC during exposure. The decrease in sperm-zona binding is the result of an alternative non-stress inducible pathway. This study should be replicated at lower SAR levels that would simulate the radiation absorption from carrying the cell phone in a pocket close to the testes. / Thesis (PhD (Reproductive Biology))--University of Pretoria, 2008. / Obstetrics and Gynaecology / unrestricted

Introduction: Cryopreservation of human sperm is considered a routine practice in assisted reproduction laboratories. Semen samples are mainly cryopreserved as a back-up for procedures, donor sperm, and validation of samples from human immunodeficiency virus-positive patients. Human immunodeficiency virus semen samples generally result in a low yield of purified spermatozoa after decontamination. These samples need to be cryopreserved for later use. Unlike conventional cryopreservation, vitrification does not use harmful cryoprotectants, thereby potentially reducing sperm damage. Vitrification is not yet common practice for sperm cryopreservation in assisted reproduction. The aim of this study was to establish the feasibility of utilising vitrification as an alternative to current conventional cryopreservation of spermatozoa. Methods: Semen samples were collected from human immunodeficiency virus-negative patients seeking diagnostic assistance from the unit. All samples were processed according to the unit’s standard protocol. For Study 1A (n=10) washed samples were divided and cryopreserved using three different cryopreservation media, and two different freezing protocols. In Study 1B (n=15), washed samples were divided and preserved using cryoprotectant-free vitrification in 100 μl, 300 μl and 500 μl volumes. For Study 2 (n=35) washed samples were split and cryopreserved using cryoprotectant-free vitrification (utilizing the volume that resulted in the highest quality spermatozoa in Study 1B) and conventional slow freezing (using the medium and protocol that resulted in superior quality spermatozoa in Study 1A). Post thawing, motility and kinetic parameters (Studies 1 and 2), viability (Study 1), mitochondrial membrane potential (Study 2), and DNA fragmentation (Study 2) of the two groups were compared. vi Results: Study 1A indicated that cryopreserving spermatozoa using Freezing Medium resulted in the highest quality spermatozoa with regards to motility and viability (p<0.05). Comparing the two preservation protocols, no conclusion could be reached on which protocol yielded superior results (p>0.05). The RBL freezing method is shorter, simpler and requires less equipment, and was therefore deemed the preferred method. Study 1B showed that the larger vitrification volumes (300 μl and 500 μl) yielded better spermatozoa in terms of motility and viability (p<0.05). No significant difference was observed with respect to the 300 μl and 500 μl vitrification volume groups. For practical reasons, 300 μl volumes will provide sufficient sperm for any procedure and, the intermediate volume ensures that more than one straw can be preserved. Study 2 found that cryoprotectant-free vitrification resulted in spermatozoa with significantly higher mitochondrial membrane potential and significantly lower apoptosis post thawing (p<0.05). Discussion: Conventional cryopreservation methods may compromise various sperm parameters and final yield. In this study, cryopreservation and cryoprotectant-free vitrification had equivalent outcomes with respect to sperm motility. However, the latter method yielded superior results in terms of ΔΨ and DNA sperm fragmentation. In conclusion, vitrification is an easy, rapid and more affordable technique that requires no special equipment. Using vitrification for purified sperm samples of patients could potentially result in a better post thaw quality for ART procedures. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Obstetrics and Gynaecology / unrestricted

Orientador: Wilma De Grava Kempinas / Resumo: A infertilidade afeta aproximadamente 15% dos casais em idade reprodutiva, sendo o homem responsável por cerca da metade dos casos. A diminuição da fertilidade masculina é devido a uma variedade de condições que incluem malformações congênitas, distúrbios genéticos e endócrinos, doenças infecciosas e inflamatórias, disfunção sexual e estilo de vida. A proteína espermática SP22 (sperm protein, 22kDa) tem demonstrado ser um biomarcador de fertilidade masculina, visto que, em ratos, está bem estabelecido que sua concentração, em extratos de espermatozoides da cauda epididimária, se correlaciona com a fertilidade desses gametas. No entanto, para incorporar essa proteína como um biomarcador em estudos epidemiológicos e clínicos são necessários dados quantitativos em humanos. Assim, o presente estudo teve por objetivos padronizar a técnica de quantificação e imunolocalização da proteína espermática SP22, investigar possíveis correlações entre parâmetros seminais convencionais e funcionais, de homens férteis e inférteis, com a concentração dessa proteína e avaliar o seu potencial preditivo como um biomarcador, não invasivo, de fertilidade. Para tanto foram realizados dois estudos observacionais: um do tipo caso-controle e outro do tipo seccional. O estudo caso-controle foi desenvolvido em amostras seminais de 44 voluntários da cidade de Botucatu e região, com idade entre 20 a 50 anos, que foram agrupados de acordo com a fertilidade: férteis (um ou mais filhos) e inférteis. Já o estu... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Infertility affects approximately 15% of couples in reproductive age and 50% of the cases are directly related to man. The decrease in male fertility is due to several conditions including congenital malformations, genetic and endocrine disorders, infectious and inflammatory diseases, sexual dysfunction and lifestyle. Sperm protein SP22 (22kDa) has been shown to be a male fertility biomarker since, in rats, it is well established that its concentration, in sperm extractions from the epididymal tail, correlates with the fertility of these gametes. However, to incorporate this protein as a fertility biomarker in epidemiological and clinical studies, quantitative data are required in humans. Thus, the present study aimed to standardize the SP22 quantification and immunolocalization technique, investigate possible correlations among conventional and functional seminal parameters of fertile and infertile men with the concentration of this protein and to evaluate its predictive potential as a non-invasive fertility biomarker. Therefore, two observational studies were performed: one case-control type and the other sectional type. The case-control study was carried out on 44 volunteers’s seminal samples from Botucatu city and region, aged between 20 and 50 years, who were placed according to fertility: fertile (one or more children) and infertile. The sectional study was carried out on 12 patients’s attendend by the Human Reproduction Center of the Botucatu Medical School – UNESP, al... (Complete abstract click electronic access below) / Doutor

<p>Prostasomes are extracellularly occurring organelles which are secreted in human semen by the prostate gland. Prostasomes have several known biological activities, but their physiological function is still unclear. In this thesis some new aspects were studied on the biological role of the prostasomes. </p><p>The motility-stimulatory effect of prostasomes on cryopreserved spermatozoa was further studied by supplementing the swim-up medium with seminal prostasomes, and with prostasomes purified from a PC-3 prostate cancer cell line (PC-3 prostasomes), on fresh spermatozoa. The recovery of motile spermatozoa after swim-up increased by 50% when the swim-up medium was supplemented with prostasomes. The PC-3 prostasomes bore a functional resemblance to seminal prostasomes as regards various expressions of sperm motility promotion. </p><p>Prostasomes proved to have potent antibacterial effects. The effects were not strictly confined to Bacillus megaterium since a few other bacteria were also sensitive. The high percentage of patients with anti-prostasome antibodies showed that prostasomes could be one of the major targets for antisperm antibodies (ASA). The results demonstrate that ASA in serum of infertile men and women recognise prostasomes as antigens, and that polyclonal antibodies raised against prostasomes agglutinate human spermatozoa. This suggests that prostasomes contribute at least partly to immunological infertility. Three types of prostasomes (seminal-, native- and metastasis-derived prostasomes) demonstrated similarities regarding a high cholesterol/phospholipid ratio and some marker enzymes. The conclusion is that prostasomes have a common and exclusive prostatic origin in man and that they are internalised in storage vesicles of the secretory cells and released in toto by an ordinary exocytotic event.</p>

Prostasomes are extracellularly occurring organelles which are secreted in human semen by the prostate gland. Prostasomes have several known biological activities, but their physiological function is still unclear. In this thesis some new aspects were studied on the biological role of the prostasomes. The motility-stimulatory effect of prostasomes on cryopreserved spermatozoa was further studied by supplementing the swim-up medium with seminal prostasomes, and with prostasomes purified from a PC-3 prostate cancer cell line (PC-3 prostasomes), on fresh spermatozoa. The recovery of motile spermatozoa after swim-up increased by 50% when the swim-up medium was supplemented with prostasomes. The PC-3 prostasomes bore a functional resemblance to seminal prostasomes as regards various expressions of sperm motility promotion. Prostasomes proved to have potent antibacterial effects. The effects were not strictly confined to Bacillus megaterium since a few other bacteria were also sensitive. The high percentage of patients with anti-prostasome antibodies showed that prostasomes could be one of the major targets for antisperm antibodies (ASA). The results demonstrate that ASA in serum of infertile men and women recognise prostasomes as antigens, and that polyclonal antibodies raised against prostasomes agglutinate human spermatozoa. This suggests that prostasomes contribute at least partly to immunological infertility. Three types of prostasomes (seminal-, native- and metastasis-derived prostasomes) demonstrated similarities regarding a high cholesterol/phospholipid ratio and some marker enzymes. The conclusion is that prostasomes have a common and exclusive prostatic origin in man and that they are internalised in storage vesicles of the secretory cells and released in toto by an ordinary exocytotic event.