Document Type : Original Article
1 Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran;Department of Biology, Damghan Branch, Islamic Azad University, Damghan
2 Isfahan Fertility and Infertility Center, Isfahan, Iran
3 Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
4 Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran;Isfahan Fertility and Infertility Center, Isfahan, Iran
5 Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran
Although intra-cytoplasmic sperm injection (ICSI)
has revolutionized the treatment of male infertility, its safety is still debatable. During ICSI, motile
sperm with normal morphology are visually selected for insemination of an oocyte (
Because the ICSI procedure bypasses natural sperm selection barriers and increases the
chance of insemination of a defective or DNA
damaged sperm in severe male infertility cases,
it is believed that insemination of these sperm
should be avoided. The consequences of insemination of damaged sperm not only result in
reduced ICSI outcomes in terms of fertilization,
blastocyst formation, implantation, pregnancy
and live birth rates, however there may be other
consequences such as an increased cancer rate
after birth that have yet to be determined (
To avoid insemination of defective sperm, researchers in the field of sperm biology have focused on novel sperm selection procedures (for
more detail see Nasr-Esfahani et al. and Said
and Land) (
HOST was initially introduced as a functional
assay for infertility diagnosis by Jeyendran et
Pattern a or a-sperm have no expansion and are considered nonviable and nonfunctional. In patterns b-sperm to g-sperm, volume expansion indicates different degrees of integrity and functionality of the sperm’s membrane.
Recently, Stanger et al. have assessed DNA
integrity of different sperm patterns and showed
that d-, e-, and f-sperm are associated with minimal DNA damage (
The aim of this study was to evaluate, for the first time, the potential of HOST in a clinical setting. In the treatment group we divided sibling oocytes into two subgroups, i. oocytes inseminated by routine ICSI procedure following density gradient centrifugation (DGC group) and ii. oocytes inseminated by sperm chosen according to their HOST pattern following DGC processing (HOST group). The control group consisted of couples undergoing routine ICSI. The clinical outcomes of this group (control) following embryo transfer were compared with the clinical outcomes of couples who received embryo transfer solely from the HOST group.
In this preliminary prospective clinical trial
study, semen samples were obtained from 102
infertile individuals who referred to the Isfahan
Fertility and Infertility Center for ICSI procedures from 2011 to 2012. All couples were informed about the study and consent forms were
signed by all participants. Semen samples were
collected by individuals through the process of
masturbation, after three or four days of abstinence, on the day of oocyte retrieval. Routine
semen analysis was carried out by light microscopy according to World Health Organization
(WHO) criteria (
Infertile couples were informed about the
study and grouped according to their preference
for inclusion in either the treatment or control
We recruited couples into the study until the treatment group reached 39 couples based on a sample size formula constructed according to a 95% confidence coefficient and 80% power of study.
Couples with primary male infertility included in this study had at least two abnormal semen parameters according to the WHO-2010. Those with less than six normal appearing, mature metaphase II oocytes were excluded. We excluded couples over the age of 40 years from the study.
Study design. Hypo-osmotic swelling test (HOST) and Density gradient centrifugation (DGC).
Routine density gradient centrifugation (DGC) and novel hypo-osmotic swelling test (HOST) sperm selection procedures.
We performed sperm processing, super ovulation, the ICSI procedure and embryo culture
according to our previous study (
We assessed embryo quality three days post-oocyte retrieval according to Nasr-Esfahani et al. using
a three-point scoring system and taking into consideration the following parameters: absence of fragmentation and/or fragmentation rate less than 25%
of the embryonic surface; equality of blastomere
size and shape; and the number of cells (greater or
less than eight). We compared the chemical pregnancy, clinical pregnancy, implantation and abortion
rates between treatment and control groups (
Embryologists who divided the oocytes into two groups, scored the embryos and followed the cycle outcomes were blinded to treatment assignments.
In order to evaluate the correlation between DNA
fragmentation and fertilization in sibling oocytes
(DGC vs. HOST group), we evaluated DNA fragmentation by terminal deoxynucleotidyl transferase
mediated dUTP nick end labeling (TUNEL) in the
treatment semen samples according to Tavalaee et al.
The mean, range of variables, Pearson’s corre- lation coefficient, student’s t test (paired and independent sample) and chi-square test were performed using the Statistical Package for the Social Studies (SPSS 11.5; Chicago, IL, USA) software for correlation analyses and a comparison of the results between different procedures. Values are presented as mean ± SEM in the Results and tables. P<0.05 was considered to be significant.
Descriptive analysis of sperm parameters and characteristics of couples in the treatment and control groups are presented in table 1. These parameters were similar between the two groups, except for female age which was higher in the control group.
Mean semen parameters and couples’ characteristics between treatment and control groups.
Values are mean ± SE. Common letters represent significant difference (p < 0.05).
According to table 2, the fertilization rates were
67.9 ± 3.5 (DGC), 68.1 ± 2.9 (HOST) and 66.9 ± 2.6
(control); percentages of good quality embryos were
28.9 ± 4.2 (DGC), 42 ± 5.7 (HOST) and 31.4 ± 2.8
(control); percentage of moderate quality embryos
were 40.3 ± 4.9 (DGC), 34.5 ± 5.6 (HOST) and 46.1
± 2.8 (control); and the percentage of poor quality embryos were 30.6 ± 3.8 (DGC), 23.4 ± 4.9
(HOST) and 22.1 ± 2.8 (control). The fertilization
rate in the HOST group was insignificantly higher
compared to the other two groups. There were a
significantly higher percentage of good quality
embryos (p=0.03) in the HOST group compared to
the sibling oocytes in the DGC group (
Percentage of fertilization and embryo quality between hypo-osmotic swelling test (HOST), density gradient centrifugation (DGC) and control groups
Values are mean ± SE. Common letters represent significant difference (p<0.05).
Comparison of fertilization and good embryo quality rates between treatment and control groups. Asterisk: Significant difference at p<0.05.
Embryo selection for transfer was based on the availability of good quality embryos. Therefore out of 39 couples in the treatment group, 25 received embryos from the HOST group and the remaining 14 couples received embryos from both the DGC and/or HOST groups. We compared implantation, pregnancy, and abortion rates between treatment group couples who received embryos from the HOST procedure with the control group.
We also assessed the percentage of DNA fragmentation by TUNEL in sperm with normal or abnormal morphology. According to the results, there was a significant correlation between the percentage of TUNEL negative sperm with normal morphology and fertilization rate in DGC group (r=0.4, p=0.00), but not in the HOST group (r=0.1, p=0.4).
The chemical pregnancy rate percentages were evaluated in the treatment and control groups. Out
of 25 infertile individuals in the treatment group,
12 (48%) became pregnant. In the control group
13 out of 58 (22.4%) became pregnant, which was
significant (p=0.02). In addition, the clinical pregnancy rate was 34.7% (8 out of 23) for the treatment group compared to 21.0% (12 out of 57) for
the control group which was insignificantly higher
Clinical outcomes between treatment and control group
|Groups||Chemical pregnancy ß-hCG+/No. ET||Clinical pregnancy Heart beat/No.ET||Implantation rate No. Sac/No. ET||Abortion rate|
|48% (12/25)||34.7% (8/23)||21.7% (13/60)||0% (0/8)|
|22.4% (13/58)||21% (12/57)||9.3% (15/160)||0% (0/15)|
In conventional ICSI sperm selection is based
on motility and morphology, which does not alleviate the likelihood of inseminating sperm that
have subtle defects (
Spermolemma, the sperm’s outer layer, can become damaged when exposed to non physiological conditions, toxicants such as reactive oxygen
species (ROS) or by internal factors that induce
apoptosis. Under these circumstances the genome
integrity is also affected. Therefore, integrity of
the sperm’s membrane may be taken as an index
for cellular integrity (
In this study, we divided the oocytes from each
couple into two subgroups, where one-half were
inseminated by a routine sperm selection procedure and the other half underwent insemination
using the HOST procedure (
Implantation rates percentages also significantly differed between the HOST (21.7%) and control (9.3%) groups. The implantation rate in the control group was lower than the rates commonly reported in the literature and obtained in our center. This was likely due to the type of patients (severe male infertility) chosen for this study. Clinical pregnancies were also higher in the HOST (34.7%) compared to the control (21%) group, but this difference was insignificant.
Recently, Avendaño et al. have demonstrated
that ICSI outcomes did not show a significant relationship with DNA fragmentation in whole semen samples, rather a significant correlation was
observed when the degree of DNA fragmentation
was evaluated in sperm that had normal morphology in each sample. Therefore, they have concluded that "the evaluation of DNA integrity in
morphologically normal spermatozoa after sperm
selection is a better approach to examine sperm
DNA fragmentation and any potential impact on
ICSI procedure" (
Based on the results of previous and current studies, sperm selection based on HOST patterns has
been shown to improve sperm quality including
chromatin integrity, a low level of apoptosis, and
sperm morphology (
Although the basis of these observations, according to the literature, have been mainly attributed to sperm motility and viability, today we know
these observations could be attributed to sperm
functionality or quality. Therefore the HOST pattern selects sperm based on functionality rather
than sperm viability. In our previous study we have
shown that the percentage of d-sperm or d-HOST
pattern sperm had a significant positive correlation
with sperm concentration and a negative correlation with sperm DNA fragmentation. A retrospective look at our previous data has revealed that
with a higher percentage of HOST positive sperm
in a sample, there would be a lower percentage of
g-pattern sperm with higher DNA fragmentation.
By contrast, in this type of sample, the percentage of d-pattern sperm which shows lower rates of
DNA fragmentation is higher (
By taking into consideration the limitation of this study, which was patient preference, therefore the results of this preliminary study and previous study suggested that selection of sperm according to the HOST pattern might potentially improve ICSI outcomes. Despite the improvement in this study and due to the limitations (blinded and randomization) and shortcomings (slightly higher female age in the control group and longer duration of infertility), we have proposed that additional clinical trials are necessary to verify these outcomes.