Carin Huyser

The effect of pulsed 900-MHz GSM mobile phone radiation on the acrosome reaction, head morphometry and zona binding of human spermatozoa.

Several recent studies have indicated that radiofrequency electromagnetic fields (RF-EMF) have an adverse effect on human sperm quality, which could translate into an effect on fertilization potential. This study evaluated the effect of RF-EMF on sperm-specific characteristics to assess the fertilizing competence of sperm. Highly motile human spermatozoa were exposed for 1 h to 900-MHz mobile phone radiation at a specific absorption rate 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 using computer-assisted sperm analysis. 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%) was reported among exposed sperm compared with unexposed controls. Sperm-zona binding was assessed directly after exposure using the hemizona assay. 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 although 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 fertilization potential.

In vitro effect of pulsed 900 MHz GSM radiation on mitochondrial membrane potential and motility of human spermatozoa

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.

Mobile Phone Radiation Does Not Induce Pro-apoptosis Effects in Human Spermatozoa

Abstract 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 apoptosis-related properties in human spermatozoa. Ejaculated, density-purified, 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.

Microbial flora in semen during in vitro fertilization

Semen samples from 183 consecutive unselected men participating in an in vitro fertilization program were retrospectively studied to determine the bacterial and fungal contamination rate before and after antibiotic treatment. To ascertain the influence of semen preparation (wash and swim-up method) on the incidence of microorganisms, semen from 102 male patients was studied before and after swimup. Antimicrobial treatment by, prescription of antibiotics decreased the incidence of pathogens by 16.3% (P<0.0001). Semen processing was more effective by ridding 57.4% of semen samples of microbial contaminants (P<0.0001). When infection of culture media was observed during routine microscopy, all infected oocytes were degenerated, without evidence of fertilization or pronuclei.

The detection of blood contamination in human follicular fluid

Purpose:The aim of this study was to compare the reliability of the methods conventionally used to identify low levels of blood contamination in human follicular fluid (hFF) as applicable in the clinical environment.Methods:Follicular fluid (n=339) and plasma samples (n=20) were collected from patients (n=138) attending the Centre for Fertility Studies, HF Verwoerd Hospital, University of Pretoria, South Africa. hFF blood contamination was assessed by means of (a) visual inspection, (b) hematocrit (Hct), (c) spectrophotometric analysis, (d) spectrophotometric hemoglobin kit, and (e) Combur-9-test urine sticks.Results:(1) Neither hematocrit nor spectrophotometry provided reliable detection at low levels of blood contamination. (2) Visual inspection presented with a better discriminatory ability than either Hct or spectrophotometry. (3) Combur-9-test sticks identified up to 50% of blood-contaminated fluids. (4) Spectrophotometrically determined hemoglobin levels presented with weak discriminatory abilities for detecting blood-contaminated fluids.Conclusions:Visual inspection as performed in this study provides a fast and relatively reliable method for the determination of blood-contaminated hFFs. In a laboratory environment, however, it would be recommended that a combination of visual inspection, Hct, and spectrophotometric evaluation be employed for the selection of blood-free fluids.

Elimination of bacteria from human semen during sperm preparation using density gradient centrifugation with a novel tube insert

The occurrence of bacteria in sperm samples intended for in vitro fertilisation can compromise the outcome of assisted reproductive techniques. Effective semen processing procedures should therefore be implemented to remove bacteria from semen. Unfortunately, technique failure does occur whereby bacteria can be found in processed sperm preparations. To improve the effectiveness of semen processing, a novel centrifuge tube insert was developed to facilitate the layering of density gradients and semen, and to prohibit the re‐infection of purified sperm pellets. The purpose of this study was to: (i) determine the prevalence and type of bacteria present in semen of patients participating in the Unit’s assisted reproduction program and (ii) evaluate the effectiveness of density gradient centrifugation with the novel tube insert, for the elimination of bacteria and yeast from spiked human semen samples. A survey in 2007–2010 indicated that 50% of semen samples were found to have positive bacterial cultures. Semen processing by means of density gradient centrifugation with the novel tube insert eliminated significantly more in vitro derived (spiked) bacteria and yeast from semen compared to processing without the insert (P < 0.004). Therefore, it is highly recommended that the centrifuge tube insert, ProInsert™, be incorporated into assisted reproductive programs.

Interleukin-1β, interleukin-6, and growth hormone levels in human follicular fluid

PurposeTo investigate possible relationships of interleukin-1Β (IL-1Β, interleukin-6 (IL-6), and growth hormone (GH) with biochemical variables in human follicular fluid (FF) and selected in vitro fertilization (IVF) parameters.MethodsA total of 67 FF samples (n=67 patients undergoing oocyte retrieval for IVF) was evaluated. IL-1Β, IL-6, GH, hLH, FSH, PRL, hCG, testosterone, total protein, fibrinogen, sialic acid, α1-antitrypsin, plasminogen levels, and spectrophotometric absorbance at 458 nm were analyzed for selected FF. IL-6 and GH levels of serum and FF samples were also compared (n=23).ResultsImmunoreactive levels of IL-1Β, IL-6, and GH were detected in all FF samples. A positive correlation existed for IL-6 (r=0.5069, P=0.0161 when serum-to-FF levels were compared (concentration ratio, 1∶1.857). Smaller-volume follicles (<4 ml) were associated with high IL-1Β levels (P=0.0229, and an additional tendency of IL-1Β to decrease with increasing embryo cleavage and scoring was observed. With the exception of a weak positive correlation between follicular IL-1Β and testosterone levels (r=0.3128, P=0.025, no other relationship with biochemical variables or IVF parameters (etiology, e.g., endometriosis) could be implicated.ConclusionsSubstantially higher IL-6 levels occurred in FF compared to serum, thus supporting intrafollicular production. Interleukin- 1Β,IL-6, and GH levels in FF are, however, unsuitable markers for in vitro fertilization outcome.

Large volume cryoprotectant‐free vitrification: an alternative to conventional cryopreservation for human spermatozoa

Vitrification is a simple and cost‐effective method for the storage of human spermatozoa without the use of conventional cryoprotectants, by plunging the sperm suspension directly into liquid nitrogen. As a result, solidification of living cells without the formation of ice crystals is achieved during cooling. This study aimed to compare cryoprotectant‐free vitrification to conventional cryopreservation protocols. Semen samples (n = 35) were collected from patients seeking diagnostic assistance at the Reproductive and Endocrine Unit at Steve Biko Academic Hospital. Samples were processed using a discontinuous density‐gradient centrifugation method. Washed samples were split into two aliquots and cryopreserved either by means of cryoprotectant‐free vitrification (sucrose + 1% albumin) or conventional slow freezing (TEST‐yolk buffer). Post‐thawing, the sperm motion parameters, mitochondrial membrane potential (Δψm) and DNA fragmentation were compared between the two groups. No significant differences were observed in the sperm motility parameters (P > 0.05). Significantly higher percentages of Δψm (11.99% ± 4.326% versus 6.58% ± 1.026%; P < 0.001) and lower percentages of DNA fragmentation (2.79% ± 1.017% versus 3.86% ± 1.38%; P < 0.01) were observed when comparing cryoprotectant‐free vitrification to conventional cryopreservation. Cryoprotectant‐free vitrification is a rapid and promising alternative to conventional methods resulting in good‐quality spermatozoa post‐thaw.

Influence of temperature and sperm preparation on the quality of spermatozoa.

This study investigated the effects of long-term (24h) in-vitro sperm incubation at room temperature (RT; 23°C) versus testis temperature (35°C) on various sperm-quality parameters. Semen samples (n=41) were prepared both by density-gradient centrifugation (DGC) and the swim-up technique in order to compare the influence of sperm preparation on sperm quality after incubation. Progressive motility and morphology were significantly higher after incubation at RT compared with 35°C (P<0.001 and P<0.01, respectively). The proportions of acrosome-reacted, apoptotic and dead spermatozoa were significantly lower in samples incubated for 24h at RT compared with 35°C (P<0.001, P=0.01 and P<0.001, respectively). The number of motile, morphologically normal, non-acrosome-reacted and nonapoptotic spermatozoa recovered after sperm preparation was significantly higher in DGC compared with swim-up samples (P<0.001). However, spermatozoa prepared by swim-up showed better survival after incubation compared with DGC-prepared spermatozoa, especially when incubated at 35°C. In conclusion, this study indicates a significantly better and longer preservation of sperm quality when incubation is performed at RT. These findings may convince laboratories to change the routinely used sperm storage conditions in order to maximize the quality of the prepared sperm sample.

Comparison between propidium iodide and 7-amino-actinomycin-D for viability assessment during flow cytometric analyses of the human sperm acrosome

Evaluation of the acrosome reaction can shed light on the fertilising competence of spermatozoa. To eliminate false‐positive results when evaluating the acrosome status of human sperm cells, two viability probes propidium iodide (PI) and 7‐amino‐actinomycin D (7‐AAD) were compared for their ability to stain nonviable cells post‐fixation and permeabilisation. Both the mean fluorescence and % dead cells differed significantly with time (P < 0.0001). Unlike PI, 7‐AAD did not leach from cells and fluorescence remained stable for up to 4 h. Furthermore, 7‐AAD proved to be a proficient marker to exclude dead sperm cells during flow cytometric evaluation of ionophore‐induced acrosome reaction.