Document Type : Original Article
Authors
1 Department of Reproduction and Development, Reproductive Biomedicine Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran;Payame Noor University, Isfahan, Iran
2 Department of Reproduction and Development, Reproductive Biomedicine Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran
3 Department of Reproduction and Development, Reproductive Biomedicine Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran;Isfahan Fertility and Infertility Center, Isfahan, Iran
4 4Islamic Azad University-Khorasgan Branch, Isfahan, Iran
5 Department of Reproduction and Development, Reproductive Biomedicine Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran;Isfahan Fertility and Infertility Center, Isfahan, Iran;5Department of Embryolo
Abstract
Keywords
Daily exposure to a number of common chemicals
in the home and workplace is one of the main
reasons for infertility. Both environmental and
other causes have made infertility a pandemic phenomenon.
Therefore, understanding the precise
molecular mechanisms underlying infertility may
provide useful hints for its prevention and treatment
(
Assessment of male infertility based upon semen
analysis as described by the World Health Organization
(WHO) guidelines only provides quantitative
and qualitative analysis rather than mechanistic
or functional information (
One of the main factors affecting the susceptibility
of sperm to undergo denaturation and inevitably
DNA damage is the proper replacement of histones
with protamine during chromatin compaction in
the process of spermiogenesis (
In a previous study, we standardized the CMA3
staining procedure to indirectly assess semen sample
protamination by flow cytometry. Our study determined
that sperm could be assessed as fixed or
unfixed. The fluorescence microscopic procedure
utilized washed sperm samples that were fixed
prior to assessment. According to our research;
both the sperm concentration and duration of exposure
should be constant. Thus, we have proposed
that a concentration of 2 million sperm should be
stained with CMA3 solution for an optimal time
of 60 minutes. Fixed samples can be stained and
assessed later (
A study of the literature has revealed that semen
in addition to somatic and germ cells may contain
round structures like sperm head, in different
dimensions, and are observable via light microscopy.
They are apoptotic bodies which have been
named merocyanine 540 (M540) bodies, since they
promptly stain with M540 and contain no DNA or
small quantities of fragmented DNA (
These properties partially locate these M540bodies
in the same region of flow cytometric dot plot (forward
and side scatter) that contain sperm samples.
Therefore, if M540 bodies are not excluded from
flow cytometric analysis the results will be underestimated
(
CMA3 is a flurochrome that can be assessed by both fluorescence detectors 2 (FL-2) and 3 (FL-3) and to our knowledge, the choice of detector used when staining with CMA3 remains to be determined. Therefore, the aim of this study is to evaluate the percentage of CMA3 by PI staining with the purpose of excluding M540 bodies as they prevent underestimation of the percentage of CMA3 positivity by flow cytometery.
Semen samples were collected from 104 infertile
men who referred to the Andrology Unit of
the Isfahan Fertility and Infertility Center. This
experimental study was approved by Royan Institutional
Review Board. Informed consent forms
were signed by all the individuals who provided
semen samples for this study. Samples were initially
assessed according to WHO criteria (
For measurement of M540 bodies, 15μl from M540 (2.09 μM; Sigma, USA) and Yo-Pro-1 (0.01 μM; Sigma, USA) were added to the sperm suspension and incubated at room temperature for 20 minutes in the dark. For sample analysis, a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a 488 nm laser was chosen and 10000 apparently sperm-specific events were calculated. A forward and side scatter (FSC and SSC) gate was used to select single sperm from debris and aggregates. Fluorescence compensation was set by unstained sperm and separately stained with Yo-Pro-1 and M540. Green fluorescence from Yo-Pro-1 and red fluorescence from M540 were collected in fluorescence detector 1 (FL-1) with a 530/30 nm band-pass filter and FL-2 with a 585/42 nanometer (nm) band-pass filter, respectively. Data were analyzed using BD Cell Quest Pro and WinMDI 2.9 software.
CMA3 staining was assessed according to a previously
described procedure for CMA3 flow cytometry
and fluorescence microscopy (
The excitation range of CMA3 has been reported
to be between ~350 and ~500nm (maximum excitation
limitation: 430-457 nm). In this study we
use a 488 nm laser, which is at the end of the excitation
range of CMA3 that allowed us to obtain detectable
emissions in both FL-2 and FL-3. Therefore,
we recommend the results to be repeated on a
flow cytometer equipped with a laser to emit light
at around 445 nm (between 430 to 457 nm) to excite
CMA3 (
Following assessment of CMA3 by flow cytometry, the number of CMA3 positive sperm in 10000 counted cells was defined. This number was divided by the number of PI positive cells in the 10000 counted cells, multiplied by 10000 cells with the assumption that all the counted CMA3 positive cells were sperm. For example, the number of CMA3 positive cells was 30% or 3000 in 10000 counted cells and the number of PI positive cells was 95% or 9500 cells in the 10000 counted cells. Therefore, the true number of CMA3 positive cells would be 3160 rather than 3000 cells per 10000 counted and the true percentage of CMA3 would be 31.6% instead of 30%.
Results are expressed as means ± SEM. For statistical analysis, coefficients of correlation and paired t-tests were carried out using the Statistical Package for the Social Studies (SPSS, version. 11.5, Chicago, IL, USA) software to compare results between different groups. A probability of p<0.05 was considered statistically significant.
Figure 1A shows the plot of M540 and Y1 staining
in semen samples (upper left are apoptotic bodies,
or the M540 positive, and Y1 negative) while Figure
1B shows the PI histogram of the same sample (PI
negative population). We assessed the correlation
between the percentage of PI negative cells and the
percentage of M540 bodies in ten semen samples
where a strong correlation was observed (r=+0.95,
p<0.001,
Following the above conclusion, 104 samples were simultaneously assessed for CMA3 staining by fluorescence microscopy and flow cytometry by FL-2 and FL-3. Of the samples, 86 were processed simultaneously by DGC and then assessed for CMA3 staining. Both the processed and neat samples have been assessed for the percentage of PI positive or negative cells. Figure 2 shows that in the DGC procedure, the percent of the CMA3 positive sperm decreased significantly with fluorescence microscopy. However assessment by flow cytometry revealed an increase in the percentage of CMA3 positive cells post-DGC, when FL-2 was used and a decrease in the percentage of CMA3 positive cells when FL-3 was implemented, irrespective of apoptotic bodies (with and without PI). These values were only significantly different when FL-2 was implemented without taking into account the M540 bodies.
(A) Dot plot of M540 versus Yo-Pro-1 staining and (B) PI histogram in a semen sample. (C). Correlation between the percentage of PI negative cells and M540 positive/ Yo-Pro-1 negative. (D) Dot plot of M540 versus Yo-Pro-1 staining and (E) PI histogram in a processed sample by DGC. (F) Comparison of the percentage of PI negative cells and M540 positive/ Yo-Pro-1 negative before and after processing. Common letters are significantly different at p<0.05.
Comparison of the percentage of sperm with CMA3 positivity by fluorescence microscopy and flow cytometry using fluorescence detectors 2 or 3 (FL-2 and FL-3) in semen and DGC. Common letters are significantly different at p<0.05.
Figures 3A and B show the correlations of percent
of CMA3 positivity as assessed by fluorescence
microscopy and flow cytometry using FL-2
and FL-3. A significant weak correlation was observed
between fluorescent microscopy and flow
cytometry using FL-2 (r=0.320; p=0.006) and FL-3
(r=0.273; p=0.048). However, a strong significant
correlation was observed between FL-2 and FL-3
(r=0.819 and p<0.001,
In addition, a comparison of the mean percent of
CMA3 between the slide method and flow cytometry
using FL-2 or FL-3 showed that both flow cytometric
measurements were significantly lower than
the slide method. However, the mean FL-3 measurement
was closer to the slide method (
Correlations of the percentage of CMA3 positivity assessed by fluorescence microscopy and flow cytometry using fluorescence detector 2 or 3 (FL-2 and FL-3).
Comparison of the percentage of sperm with CMA3 positivity between the slide method and flow cytometry using fluorescence detectors 2 or 3 (FL-2 and FL-3). Common letters are significantly different at p<0.05.
The presence of over 1500 reports in the literature
emphasizes the evaluation of protamine content
by indirect CMA3 staining. Except for our recent
report on standardization and implementation of
CMA3 staining by flow cytometry in sperm (
The most common procedure for the assessment
of M540 bodies is the use of merocyanine along
with Y1 staining (
The results of this study also revealed that the
percentage CMA3 positive sperm obtained by the
slide method or microscopic procedure, despite
accounting for M540 bodies, were still significantly
greater than the flow cytometric method,
irrespective of assessment by FL-2 or FL-3 (
In flow cytometry the mean values of CMA3 obtained, excluding M540 bodies, are significantly greater in FL-3 than FL-2. Despite a significant correlation between the CMA3 assessment by FL-2 and FL-3, figure 3A and B show a wider range of CMA3 in FL-3 than FL-2; this may suggest that variations within a heterogeneous sample are more assessable in FL-3. In addition, as shown in this study and well accepted in the literature, one expects a reduction in the percentage of CMA3 positive sperm following DGC, even though no significance has been observed with FL-3, in contrast to FL2. Although we could not account for this difference between FL-2 and FL-3, despite the use of the same semen samples for these assessments, one possible explanation could be that FL-2 does not provide the optimal wavelength for assessment of CMA3.Therefore, variations within samples are more observable in FL-3.
In considering the evaluation of CMA3 for assessment
of protamine deficiency, we propose the assessment
of CMA3 by flow cytometry using FL-3
and staining for PI to exclude M540 bodies, especially
in samples with oligo-astheno-teratospermia
or astheno-tratospermia where the presence of these
bodies has been reported to be high (