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 preparation and culture of washed human sperm: a comparison of a suite of protein-free media with media containing human serum albumin

Objective To compare two suites of culture media (one with HSA and one protein-free (PF) supplemented with methylcellulose) for washing human sperm in IVF. Methods Semen samples (n = 41) underwent parallel density gradient preparation using PF or HSA-supplemented culture medium and subsequent yield, survival, morphology and motility were compared. Results The PF medium resulted in a significantly higher sperm yield (P \u3c 0.0001), but similar sperm morphology (P = 0.822) and 24-h survival (P = 0.11). There was, however, a lower percentage of progressively motile sperm (P \u3c 0.0001) and a higher proportion of sperm demonstrating non-progressive motility (P \u3c 0.0001) in the PF medium when observed on a Makler Chamber, apparently an artefact as a similar sperm motility index was measured using a Sperm Quality Analyser (P = 0.83). Attachment of sperm in PF medium to the glass chamber reduced with time and any differences had disappeared after 6 min on the counting chamber. Conclusion These results support the use of PF media supplemented with methylcellulose as an alternative to HSA, although a modification to the manufacturer\u27s protocol of 6-min pre-incubation before assessing sperm motility must be used. Further studies should investigate the function of such sperm prepared in PF medium

The measurement of oestradiol, progesterone, LH, FSH and hCG for assisted reproduction: A comparison of the Siemens Centaur CP and Roche e411 automated analysers

Objective: To compare the results of two automated analysers by measuring reproductive hormones using the same quality control. Methods: Results obtained by the Roche Cobas e411 automated analyser in a specialised fertility clinic were compared to the Siemens Centaur CP for the reproductive hormones oestradiol, progesterone, LH, FSH and hCG. Results: Commercially-available quality control (QC) samples showed significant differences between the two assays for all five hormones at one or more levels. In clinical samples, the range of concentrations encountered was similar to the QC samples for LH and FSH but much higher for oestradiol, progesterone, and hCG showing the limitations of such QC samples in a specialised fertility setting when they are intended for general pathology use. There was a high degree of correlation for all hormones (all r\u3e0.985) and a gradient close to 1 for all, except for hCG when the Siemens analyser read ≥1 000 IU/L (r=1.209) and this is reflected in a large mean bias (-2 647.9 IU/L) and coefficient of repeatability (11 690.0 IU/L) when using a Bland-Altman plot. Conclusions: Despite an overall agreement between the two assay platforms for progesterone, LH and FSH, small differences between the two analysers in the concentrations of oestradiol and hCG as encountered in natural ovarian cycles or at the time of pregnancy testing may require a redefinition of clinical cut-offs

Serum concentrations of the biomarkers CA125, CA15-3, PSA and PAPP-A in early pregnancy

Blood samples were collected serially from 14 women with healthy pregnancies, beginning in gestational week 4 at the time of a positive pregnancy test through to the identification of a foetal heart by ultrasound. Six biomarkers were measured in the serum retrospectively including two reproductive hormones (progesterone and human chorionic gonadotrophin (hCG)) and four additional biomarkers (prostate-specific antigen (PSA), cancer antigen 15-3 (CA15-3), cancer antigen 125 (CA125) and pregnancy-associated plasma protein A (PAPP-A)) measured on a Siemens Centaur XP and a Roche Cobas e411 automated analysers, respectively. The progesterone and hCG results were unremarkable following established patterns, but distinctive patterns of change were seen in the other four biomarkers during this period of early pregnancy. PAPP-A and PSA levels rose steadily as the pregnancies progressed, while CA125 levels rose until week 5.5 and then returned to baseline values. CA15-3 serum concentrations were observed to drop as the pregnancies progressed. These biomarker results suggest further investigation is warranted to allow a non-invasive correlation of the biomarkers with the various stages of embryogenesis, implantation and placentation

Longitudinal changes in thyroid hormones during conception cycles and early pregnancy

Objective: The aim of this study was to characterize changes in free triiodothyronine (fT3), free thyroxine (fT4) and thyroid-stimulating hormone (TSH) during the follicular and luteal phase and during subsequent early pregnancy in individual women. Method: TPOAb negative women with a viable pregnancy (n=49) had fT3, fT4 and TSH measured longitudinally in serum samples at baseline/non-pregnant (gestation week 0), ovulation (gestation week 2), mid-luteal phase (gestation week 3) and twice weekly from gestation weeks 4 to 6.5. Patient groups received in their conception cycle either no medication (n=13), low ovarian stimulation, (n=17) or controlled ovarian hyperstimulation (COH) for IVF treatment (n=19). Results: Women receiving COH had a transient drop in TSH at the time of ovulation followed by a peak at midluteal (p=0.024). Levels of fT3 and fT4 at each gestation week were not significantly different between the treatment groups, whereas TSH levels were significantly higher at all gestation weeks (p=0.036) in the COH group compared to the natural and low stimulation groups. There were significant changes in thyroid function once pregnancy was established (gestation week 4) through to gestation week 6.5, with a gradual decrease in serum fT3 (r=-0.104, p=0.030) and TSH (r=-0.123 p=0.031), whilst fT4 levels remained constant. 3 women (6.1%) had TSH levels \u3e4.0 mU/L during their pregnancy although these were isolated measurements. Conclusion: Thyroid hormones in individual women did not remain constant but showed discrete changes. TSH was significantly lower at time of ovulation in women who received high doses of ovarian stimulation medication for IVF, and was higher throughout pregnancy than for the other groups. Serum fT3 and TSH decreased significantly during early pregnancy irrespective of medication given in the conception cycle

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

Serum concentrations of the biomarkers CA125, CA15-3, CA72-4, tPSA and PAPP-A in natural and stimulated ovarian cycles

Objective: Biomarkers associated with cancer screening (CA125, CA15-3, CA72-4, total prostate specific antigen [tPSA]) and the monitoring of pregnancy (pregnancy associated plasma protein-A [PAPP-A]) were measured during natural and stimulated ovarian cycles in disease-free non-pregnant women to determine if they could reflect normal events relating to ovulation and/or endometrial changes. Methods: A total of 73 blood samples (10 women) collected throughout the natural menstrual cycle, and 64 blood samples (11 women) taken during stimulated ovarian cycles, were analysed on the Roche Cobas e411 automated analyser. Results: Detectable levels of tPSA were measured in at least one point in the cycle in 6/10 of women in the natural cycle and 10/11 of women in stimulated cycles, and CA72-4 was detected in only 12/21 women tested. Concentrations of CA125, tPSA, CA15-3 and CA72-4 showed no significant difference between the natural and stimulated ovarian cycle groups. On average the mean PAPP-A of the natural group was (2.41±0.58) mIU/L higher than the stimulated group (t=4.10, P\u3c 0.001). CA125 and CA15-3 results were both significantly influenced by the stage of the cycle (P\u3c0.0001), whereas tPSA and PAPP-A concentrations revealed no significant changes (P≥0.65). CA72-4 was not affected by the stage of the cycle nor ovarian stimulation. Conclusion: Ovarian stimulation reduced serum PAPP-A levels, CA125 and CA15-3 levels were generally unaffected by ovarian stimulation but displayed cyclical changes throughout both natural and stimulated cycles, whilst tPSA and CA72-4 were not affected by the stage of the cycle or ovarian stimulation

The Influence of Sample Volume Applied to the Makler Sperm Counting Chamber Upon the Measured Concentration of Latex Beads: A Multi-Centre Study

Objective: To undertake a multi-centre study to maximize the number of Makler chambers used. Methods: A total of 15 laboratories participated with 31 Makler chambers. A suspension of latex beads was prepared to a concentration of 20 millions per milliliter, and 0.5 mL aliquots distributed to each participating laboratory. They measured the concentration on their Makler chamber(s) used for routine semen analysis by adding 3, 4, 5, 7 and 10 μL volumes of bead suspension to the chamber. Results: There was no difference in within-chamber analysis of the bead concentration according to the volume of bead suspension applied within the range of 3–10 μL (F4,14=2.634, P=0.056). However, the between-chamber effects were significantly different (F30,124=4.937, P=0.000), and 24/31 (77.5%) chambers tested had an average bias\u3e10% compared to the target bead concentration. Conclusions: A volume of 3–10 μL added to Makler counting chambers does not influence the concentration measured of latex beads, but the between-chamber variability and positive bias seen would suggest that other sources of error are present which are yet to be identified