The advent of Intracytoplasmic sperm injection
(ICSI) was a fundamental and effective approach
in domain of male infertility treatment (
Considering the role of paternal DNA in development of sperm, selection of intact sperm for
ICSI is of paramount importance (
An ideal sperm separation technique should
present the following characteristic: i. remove abnormal and dead spermatozoa, leukocytes and
bacteria, ii. abolish toxicants or bioactive factors
[reactive oxygen species (ROS) and apoptosis], iii.
be applicable to oligospermic samples, iv. avoid of
provoking un-physiological ROS, v. separate live
and motile sperm, vi. select functional sperm with
intact DNA and membrane and vii. be a fast, simple
and cost-effective procedure (
Recently, different novel sperm selection procedures have been proposed, for further details, see
the reviews by Nasr-Esfahani et al. (
Hypo-osmotic swelling test (HOST),based on its
subtypes, has been proposed as a simple, safe, and
repeatable method in order to identify live and intact spermatozoa (
Recently, Stanger et al. (
Previously, some studies reported a positive correlation between percentages of viable sperm, assessed by HOST,with sperm parameters, sperm
zona-free hamster ovum penetration assay and in
vitro fertilization (IVF) outcomes (
This case-control study received the approval of the Institutional Review Board of Isfahan Fertility and Infertility Center (IFIC) and Royan Institute. Sixteen semen samples of normozospermic men were collected from individuals attending the Andrology Unit of the Isfahan Fertility and Infertility Center, after signing a written informed consent document.
Sixteen semen samples were collected and each
sample divided into two portions as follows: one
portionwas washed with Ham’s buffer and used
to assess sperm parameters according to World
Health Organization (WHO)-2010, while the other
portion was washed with Ham’s buffer. Then, 100
µl of the washed latter portion was diluted with
1 ml of hypo-osmotic swelling solution (Ham’s
medium: sterile purified H2
O; v/v) at 37˚C for five
minutes. Immediately, percentages of different
patterns of HOST (a-g) were counted according to
WHO criteria (
Accordingly, a-pattern was considered as nonviable sperm, while from b-pattern to g-patterns were considered as different degrees of alivesperm. Then, each HOS sample was put on a slide and evaluated for the following parameters: morphology, protamine deficiency, early and late apoptosis by Papanicoulau staining, chromomycin A3 (CMA3) staining, Annexin V staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.
After HOST procedure, 20 µl of each sample was
placed on a slide. Slides were manually stained by
Papanicoulau staining according to WHO (
Two hundred spermatozoa were counted for each sample and percentages of head and acrosome abnormalities were determined. Considering sperm tail curling during hypo osmotic swelling test and its effect on morphology of sperm tail and neck, we avoided reporting these abnormalities.
After HOST procedure, 20 μl of each washed
samples were fixed with paraformaldehyde and
Carnoy’s solution for TUNEL assay and CMA3
staining, respectively, as described by Nasr-Esfahani et al. and Bassiri et al. For Annexin V staining, spermatozoa did not required to be fixed, so
stainingwas carried out according to Bassiri et al.
For each case, 200 spermatozoa were assessed
randomly, and percentages of DNA fragmentation
and CMA3-positive sperm were recorded (
Logistic regression analysis was used to obtain odds ratio (OR) for spermatozoa with abnormal head morphology and abnormal acrosome, then the obtained results were compared in each HOST subtypes with a-pattern sperm because this type of spermatozoa, as the worst type, was considered as reference group. Pearson correlation test was used to assess the correlations between parameters. Also, Statistical Package for the Social Studies (SPSS 11.5; Chicago, USA) software was carried out to analyze statistical data.
The mean sperm concentration was 69.46 ± 9.07×106 spz/ml with a minimum and maximum of 7×106 and 151.30×106 spz/ml, mean sperm motility was 48.22 % ± 4.57 with a minimum and maximum of 40 to 70%, as well as mean sperm abnormal morphology was 85% ± 1.5 with a minimum and maximum 76 to 98%.
Hence, other patterns of HOS were compared
with this reference group, and obtained percentage of abnormal sperm head morphology were
presented as OR.The ORs for abnormal sperm
head morphology of each HOST grades were as
follows: 0.54, p=0.09 (b-sperm); 0.38, p=0.03 (csperm); 0.15, p=0.00 (d-sperm); 0.66, p=0.29 (e-
sperm); 0.62, p=0.35 (f-sperm); and 2.54, p=0.05
The ORs of abnormal sperm head morphology and abnormal acrosomefor HOST grade.
The ORs for abnormal acrosome in spermatozoa
of each HOST grade compared to a-sperm were
as follows: 0.78, p=0.2 (b-sperm); 0.61, p=0.1 (csperm); 0.07, p=0.01 (d-sperm); 0.69, p=0.06 (esperm); 0.57, p=0.05 (f-sperm); and 1.21, p=0.18
correlations between assessed parameters revealed a negative significant correlation
between sperm concentration with protamine deficiency (r=-0.57; p=0.03) and percentages of apoptotic sperm (r=-0.664; p=0.01). In addition, a negative significant correlation was observed between
percentage of sperm motility and DNA fragmentation (r=-0.56; p=0.01), while a positive significant
correlation was observed between percentage of
protamine deficiency and abnormal sperm morphology (r=0.59; p=0.02). Percentages of apoptotic
sperm also showed a positive significant correlation with protamine deficiency (r=0.669; p=0.009)
Correlation between of conventional sperm parameters and percentage of DNA fragmentation, protamine deficiency and apoptotic sperm
|Group||Sperm concentration (106)||Sperm motility %||Abnormal morphology %||Apoptotic sperm %||DNA fragmentation %||Protamine deficiency %|
*; Show significantly different for p<0.05 , and **; for p<0.01.
Correlation between of conventional sperm parameters, percentage of DNA fragmentation, protamine deficiency and apoptotic sperm with different patterns of HOST
|Parameters||HOST a||HOST b||HOST c||HOST d||HOST e||HOST f||HOST g|
*; Show significantly different for p<0.05 , and **; for p<0.01.
The structural and functional integrity of sperm
plasma membrane is critical for the capability of
spermatozoa for fertilization process. The most commonly tests for assessment of membrane integrity
are eosin and HOST (
In this study, we did not observe a relation between sperm morphology with DNA fragmentation, despite a strong correlation between sperm
morphology and protamine deficiency. In this
regard, some authors suggested DNA fragmentation in spermatozoa with normal morphology is
a proper selection for ICSI. Indeed, Avendan˜o et
al. reported selecting a DNA fragmented sperm
with normal morphology substantially increase in
sub-fertile and infertile individuals (
The significant correlation between sperm concentration and DNA fragmentation with d-sperm
suggest that percentage of d-sperm increases with
sperm concentration, and the degree of sperm
DNA fragmentation reduces in this type of semen
samples. It is also interesting to note, the coefficient of correlations from (+0.5) decreases with increased tail swelling, and reaches a negative value
(-0.3) in g-sperm. This suggests that there might be
mechanism underlying the relation between motility and HOST patterns, which remains to be identified. One possible explanation for this observation
might be functional distribution of Na+/K+ and Na+/H+ pumps in different patterns of spermatozoa. In
reality, hypotonic resistance which is considered
as a better term than HOST reflects the functions
of these pumps in sperm plasma membrane (
It is likely that hyper distribution of these pumps might account for minimal swelling in b-sperm, while Na+/K+ ATPase dysfunction might account for g-sperm. ing-sperm. Therefore, optimal distribution of these pumps might be considered for best sperm, indicating that other functional properties in these spermatozoa are optimal, and this might be the reason for minimal sperm DNA fragmentation, apoptosis, proper histone/protamine exchange, etc. However, this hypothesis needs further evaluation.
The results of this study suggest that there is a relation between sperm integrity and different HOST patterns. It is important to note, sperm membrane integrity can be relateddirectly to ROS production, but integrity of sperm membrane should not be considered as a direct indicator of DNA integrity, whereas DNA fragmentation can be observed even in sperm with normal morphology. The results of correlations in this study, specially the negative significant correlation between d-sperm and DNA fragmentation, verify the findings of the pervious study that d- and c-sperm should be selected for HOST-ICSI, while insemination of g-sperm should be avoided.