Human semen can be air-dried prior to testing for sperm DNA fragmentation with the Halosperm® G2 kit

Objective: To explore a method of semen storage prior to assessment of sperm DNA fragmentation. Methods: This study examined a simplified alternative of air-drying semen on a microscope slide and reconstituting in seminal plasma prior to assessment of sperm DNA fragmentation using the halosperm® G2 kit. Results: It showed that semen could be air-dried and stored overnight at room temperature with no detrimental effect on DNA quality. A significant correlation between results existed for 20 semen samples both air-dried and snap-frozen in liquid nitrogen (r=0.982, P=0.000). A mean difference between the results of only −1.98% confirmed the effectiveness of air-drying compared to snap-freezing. Conclusions: Future studies to refine this technique are required on the effect of extrinsic factors such as the choice of reconstituting medium, and stability over an extended time-frame at different temperatures

The relationship between the halosperm assay and semen analysis performed according to the 4th and 5th Editions of the World Health Organization guidelines

Background: As a standard reference to evaluate male factor infertility, the majority of fertility laboratories use the 4th or 5th Editions of the World Health Organization’s semen analysis guidelines. Following the release of the 5th Edition, debate over its legitimacy has resulted in some laboratories using the 4th and others the 5th Edition. DNA integrity tests have been shown to be a valuable adjunct to semen analysis and have subsequently been adopted by many fertility laboratories. This study explored the prevalence of samples with high DNA fragmentation levels according to semen analysis categories using both the 4th and the 5th Edition reference ranges. Materials and Methods: The study included 905 consecutive semen samples from 863 infertile couples attending a fertility clinic. A semen analysis was conducted according to both the 4th and 5th Edition guidelines published by the World Health Organization. DNA damage was assessed using the Halosperm G2 test kit and expressed as a percentage DNA fragmentation level. Results: Alongside both the World Health Organization 4th and 5th Edition semen analysis criteria abnormal DNA fragmentation levels were more common in abnormal semen samples however elevated DNA fragmentation levels were also found in normal semen samples using the same criteria. Of the samples that were graded as normozoospermic according to the 5th Edition guidelines 16% were deemed to have elevated DNA fragmentation levels compared to 11.7% graded by the 4th Edition guidelines. The number of normozoospermic samples, graded according to the 5th Edition guidelines was significantly higher (n=697) than when the same samples were graded according to 4th Edition guidelines (n=385) (p=0.001). A significant proportion of samples with an abnormal DNA fragmentation level corresponding to the World Health Organisation 4th and 5th Edition criteria were evident in normozoospermic (

The fate of frozen human embryos when transferred either on the day ofthawing or after overnight culture

Objective: To study the performance of thawed zygotes and cleavage stage embryos transferred either on the day of thaw or after overnight culture. Methods: A retrospective study of 864 frozen embryo transfer cycles. Cryosurvival rates per thawed embryo and implantation rates were analysed for embryos frozen on Day 1, Day 2 or Day 3 relative to oocyte collection (Day 0) and transferred on the day of thaw or after overnight culture, together with clinical pregnancy rates and prevalence of multiple gestations. Results: Survival of Day 3 embryos was significantly lower than those frozen on Day 1 (P=0.017) or Day 2 (P=0.015). Following overnight culture, resumption of mitosis of zygotes was more frequent than Day 2 (P=0.000) which are in turn higher than Day 3 (P=0.000) embryos. The implantation rate for Day 2 embryos dividing overnight was significantly higher than those that did not divide for women (P=0.001) but not those women (P=0.055). There were no differences in the implantation rates for those dividing or not after culture, for embryos frozen on Day 3 for women (P=0.254) or (P=0.403). Conclusions: Later cleavage stage post-thaw embryos survive and resume mitosis less frequently compared to earlier stages. Embryos not resuming mitosis after culture overnight can implant, particularly Day 3 embryos, suggesting that they can further increase the cumulative pregnancy rate per oocyte collection and that discarding them is wasteful. Overnight culture is best used for logistical reasons rather than a strategy to improve pregnancy rates

Internal Quality Control for the Direct MAR IgG Test: A Simple and Effective Method Using Spiked Seminal Plasma

Objective: To introduce a system using spiked seminal plasma for the QC of the MAR direct test, and to facilitate the determination of assay variability. Methods: A simple quality control (QC) system for use in the direct MAR test was developed using samples prepared by adding serum to antibody-negative centrifuged seminal plasma to obtain optimal binding, and storing 0.4 mL aliquots at −20 °C in straws. The serum was either from vasectomised men (positive control) or an antibody-negative woman (negative control). QC samples were thawed and mixed 1:1 with donor semen and pre-incubated for 1 hr at 37 °C, and tested for sperm antibodies using the direct method of the MarScreen IgG kit (Fertility Technology Resources, USA). Two batches of controls were prepared and one of these was run on each day. Results: The negative controls invariably gave binding of less than 5%, whereas the two positive controls had binding (mean ± sd) of 89.5% ± 6.2 % (coefficient of variation [CV] = 7.0%) and (97.2 ± 2.5) (CV = 2.6%). Conclusions: In summary, spiked seminal plasma incubated with whole semen can be used as a QC sample in the direct MAR IgG test