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CHAPTER 3.1
a CEROS (Hamilton-Thorne Inc., Beverly, USA) CASA system using two different settings (CASA1 and
CASA2, respectively; see further). Settings for CASA analysis were obtained from established semen
processing laboratories as routinely applied to fresh, cooled and frozen-thawed semen samples.
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The major settings used for CASA1 were straightness (STR) threshold for progressive motility:
75%; average path velocity (VAP) threshold for progressive motility 50 µm/s; VAP threshold for static
cells: 20 µm/s (Loomis and Graham, 2008). These settings were first obtained after a personal
communication (Loomis P., personal communication) and settings for cell detection were adapted
for optimal sperm recognition. The major settings used for CASA2 were STR threshold for
progressive motility: 50%; VAP threshold for progressive motility 30 µm/s; VAP threshold for static
cells: 15 µm/s, settings for cell detection are listed as well (Waite et al., 2008).
Six µL of diluted semen was loaded into a 20 µm Leja counting chamber (Leja, Nieuw-Vennep,
The Netherlands), placed on a minitherm stage warmer (Hamilton-Thorne Inc., Beverly, USA) during
the analysis, to maintain the sample at 37.5°C. Five different fields were analyzed 4 times to obtain a
total of 20 fields in order to obtain a minimum of 1500 cells analyzed. For both CASA settings this
was done twice per sample (T1 and T2).
distributed).
The parameters analyzed were percentage total motility (TM), PM and PMS (normally
To assess repeatability each sample was analyzed twice within one minute. First, it was
tested whether the parameters changed over time (T1 vs. T2), using a paired-samples t-test. Second,
scatter plots were produced, where the value obtained at T1 was plotted against the value obtained
at T2. The line of perfect agreement (hatched line at 45° and intercept zero) was integrated in each
plot as comparison to the regression line to fit the data (Arunvipas et al., 2003). Third, Bland and
Altman plots were produced. This method plots the difference of the paired measurements (y-axis)
against their mean (x-axis) (Bland and Altman, 1986). Finally, for each parameter, the coefficient of
variation (CV) was calculated per sample and reported as mean CV over all samples.
To assess agreement, the values obtained at T1 were used for comparison, and observations
obtained with CASA1 were compared with these obtained with CASA2. Statistical analysis was done
as described for repeatability using a paired samples t-test, scatter plots and Bland and Altman plots.
Statistical analysis was done using SPSS (version 17.0, SPSS Inc., Chicago, Illinois, USA).