High pregnancy and implantation rates can be obtained using Magnetic-Activated Cell Sorting (MACS) to selection spermatozoa in patients with high levels of spermatic DNA fragmentation

Abstract

Purpose: DNA fragmentation in spermatozoa has been associated with poor semen quality, low fertilization
rates, impaired preimplantation development, and high miscarriage rates. The objective of this study was to evaluate
the effects of the use of non-apoptotic MACS-selected spermatozoa in ICSI cycles, compared to those ICSI cycles
in patients with normal sperm DNA fragmentation.
Methods: A total of 134 cycles of ICSI were included in the study. The study group consisted of patients with
high DNA fragmentation and their spermatozoa were selected by MACS (n=57); and the control group, of patients
with normal DNA fragmentation and their spermatozoa were selected by classic morphological characteristics. The
fertilization rate, cleavage rate, embryo quality, pregnancy rate (PR), implantation rate (IR) and miscarriage rate
(MR) were compared between groups.
Results: There was no difference in the fertilization rate (74.5 and 76.5%), cleavage rate and good embryo
quality at Day 3 (98.3 and 89.1%; 88.4 and 83.7%) or blastocyst formation rate (50.8 and 41.1%) for the study and
control groups, respectively. PRs, IRs and MRs were similar for the study group compared to the control group (PR:
63.2 versus 45.5%; IR: 37.4 versus 28.1%; MR: 8.3 versus 17.1). The women were distributed into three groups:
<35 years, 35-39 years and ≥ 40 years. Pregnancy, implantation and miscarriage rates were similar in the three
evaluated groups (P: NS).
Conclusions: By selecting non-apoptotic sperm by MACS, we can achieve very acceptable pregnancy and
implantation rates; being

Full text

García-Ferreyra et al., JFIV Reprod Med Genet 2014, 2:4
http://dx.doi.org/10.4172/2375-4508.1000133
Research Article
Journal of Fertilization : In Vitro, IVF-Worldwide,
Reproductive Medicine, Genitics & Stem Cell Biology
Volume 2 • Issue 4 • 1000133
JFIV Reprod Med Genet
ISSN: 2375-4508 JFIV, an open access journal
Keywords: Sperm DNA fragmentation; SCD test; ICSI; Pregnancy
rate; Implantation rate
Introduction
Semen quality is frequently used as an indirect measure of male
infertility. Ejaculate volume, sperm concentration, motility and
morphology, determined according to World Health Organisation
(WHO) standards, are the most important parameters evaluated in
infertility centers as a part of routine semen analysis. However, these
traditional criteria provide little indication of possible nuclear DNA
damage.
Normal sperm genetic material is required for a successful
fertilization, as well as for further embryo and fetal development in
order to produce healthy ospring. Sperm DNA contributes half of
the ospring´s genomic material and abnormal DNA can lead to
derangements in the reproductive process. ere is now good evidence
that shows that sperm DNA and chromatin damage are associated with
male infertility and reduced natural conception rates [1-3].
In the last years, the integrity of sperm DNA is being recognized
as a new parameter of semen quality and a marker of male infertility
[4,5]. Nevertheless, DNA integrity assessment is not being carried out
as a routine part of semen analysis in the clinical laboratory [6]. Sperm
DNA fragmentation can be caused by apoptosis in the seminiferous
tubule epithelium, defects in chromatin remodeling during the process
of spermiogenesis, oxygen radical-induced DNA damage during
sperm migration from the seminiferous tubules to the epididymis,
the activation of sperm caspases and endonucleases, damage induced
by chemotherapy and radiotherapy, and the eect of environmental
toxicants [7]. In humans, high levels of sperm nuclear DNA damage
have been related to low fertility potential, failure to obtain blastocysts,
blockage in embryo development aer embryo implantation,
increased risk of recurrent miscarriages, reduced chances of successful
implantation, and negative eects on the health of the ospring [8-11].
Magnetic-Activated Cell Sorting (MACS) is an excellent tool for
selecting the desired cells or sperms out of a mixed cell population on
membrane surface markers [12]. Externalization of phosphatidylserine
(PS) to the outer membrane of sperm is considered early sign of
apoptosis. Annexin V is a protein with a molecular weight of 35 KDa that
has high anity to PS in the presence of physiological concentrations
of Ca+2 and is unable to pass through intact sperm membranes; thus,
annexin V binding by a sperm cell indicates that its membrane integrity
has been compromised [13,14]. MACS using annexin V conjugated
with magnetic microspheres, which are exposed to a magnetic eld in
an anity column, can separate apoptotic from non-apoptotic sperm.
*Corresponding author: Javier García-Ferreyra, FERTILAB Laboratory of
Assisted Reproduction, Av. San Felipe 1017, Lima 11, Peru, Tel: +51-1-471-1111;
E-mail: jgarciaf@fertilab.pe
Received August 13, 2014; Accepted October 29, 2014; Published November
06, 2014
Citation: García-Ferreyra J, Villegas L, Romero R, Zavala P, Hilario R, et al. (2014)
High Pregnancy and Implantation Rates Can Be Obtained Using Magnetic-Activat-
ed Cell Sorting (MACS) to Selection Spermatozoa in Patients with High Levels of
Spermatic DNA Fragmentation. JFIV Reprod Med Genet 2: 133. doi:10.4172/2375-
4508.1000133
Copyright: © 2014 García-Ferreyra J, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided
the original author and source are credited.
Abstract
Purpose: DNA fragmentation in spermatozoa has been associated with poor semen quality, low fertilization
rates, impaired preimplantation development, and high miscarriage rates. The objective of this study was to evaluate
the effects of the use of non-apoptotic MACS-selected spermatozoa in ICSI cycles, compared to those ICSI cycles
in patients with normal sperm DNA fragmentation.
Methods: A total of 134 cycles of ICSI were included in the study. The study group consisted of patients with
high DNA fragmentation and their spermatozoa were selected by MACS (n=57); and the control group, of patients
with normal DNA fragmentation and their spermatozoa were selected by classic morphological characteristics. The
fertilization rate, cleavage rate, embryo quality, pregnancy rate (PR), implantation rate (IR) and miscarriage rate
(MR) were compared between groups.
Results: There was no difference in the fertilization rate (74.5 and 76.5%), cleavage rate and good embryo
quality at Day 3 (98.3 and 89.1%; 88.4 and 83.7%) or blastocyst formation rate (50.8 and 41.1%) for the study and
control groups, respectively. PRs, IRs and MRs were similar for the study group compared to the control group (PR:
63.2 versus 45.5%; IR: 37.4 versus 28.1%; MR: 8.3 versus 17.1). The women were distributed into three groups:
<35 years, 35-39 years and ≥ 40 years. Pregnancy, implantation and miscarriage rates were similar in the three
evaluated groups (P: NS).
Conclusions: By selecting non-apoptotic sperm by MACS, we can achieve very acceptable pregnancy and
implantation rates; being a good option for couples with high sperm DNA fragmentation and repeated assisted
reproduction failures.
High Pregnancy and Implantation Rates Can Be Obtained Using
Magnetic-Activated Cell Sorting (MACS) to Selection Spermatozoa in
Patients with High Levels of Spermatic DNA Fragmentation
Javier García-Ferreyra1*, Lucy Villegas1, Rocío Romero2, Patricia Zavala2, Roly Hilario2 and Julio Dueñas-Chacón1,2
1FERTILAB Laboratory of Assisted Reproduction, Lima, Peru
2PROCREAR Fertility Center, Lima, Peru

Page 2 of 6
Volume 2 • Issue 4 • 1000133
JFIV Reprod Med Genet
ISSN: 2375-4508 JFIV, an open access journal
Citation: García-Ferreyra J, Villegas L, Romero R, Zavala P, Hilario R, et al. (2014) High Pregnancy and Implantation Rates Can Be Obtained Using
Magnetic-Activated Cell Sorting (MACS) to Selection Spermatozoa in Patients with High Levels of Spermatic DNA Fragmentation. JFIV
Reprod Med Genet 2: 133. doi:10.4172/2375-4508.1000133
Several studies reported an improvement in fertilization rates
[15,16] and embryo quality [17-19] because the best sperms were
selected using MACS compared to standard selection methods. On the
other hand, Gil et al. [20] showed that the implantation and miscarriage
rated did not vary between MACS or standard sperm selection methods;
however, they did observe an improvement in pregnancy rates.
In this study, we evaluated the eects of using MACS as a sperm
selection technique in patients with high DNA fragmentation compared
with patients with normal DNA fragmentation and determine the
ecacy of such technique in the hopes of improving ART success rates.
Materials and Methods
Patients
In this study, we analyzed 134 cycles of ICSI that were done
between March 2012 and April 2014 at FERTILAB Laboratory of
Assisted Reproduction (Lima, Peru), all approved by the Institutional
Review Board (IRB) and the corresponding Ethics Committee. Written
consent was obtained from all patients and their partners included in
this study. e study group was made up of patients with high sperm
DNA fragmentation (n = 57) and their wife’s oocytes were injected
with non-apoptotic spermatozoa selected using MACS and; the control
group, of patients with normal sperm DNA fragmentation (n = 77) and
their wife’s oocytes were injected with spermatozoa selected by classic
morphological characteristics.
Sperm DNA fragmentation assessment
Prior to the hormonal stimulation, sperm DNA fragmentation
values were evaluated with the Sperm Chromatin Dispersion (SCD)
test [21] using Halosperm® Kit (Halotech Dna, Spain). Briey, sperm
samples from each patient, containing aer dilution or concentration
not <5 million and not >10 million spermatozoa per milliliter, were
used. e kit contains aliquots of agarose gel in Eppendorf tubes. Each
semen sample was processed aer the agarose gelled (from immersion
in a water bath at 90ºC for 5 min). When the Eppendorf tubes reached
a temperature of 37ºC (5 min at 37ºC in a dry atmosphere), 25 µL
of sperm were added and gently mixed. Twenty microliters of this
mixture were placed on precoated slides and covered with 22 x 22-mm
coverslide. e slides were maintained at 4ºC for 5 minutes to produce
a microgel containing embedded spermatozoa. e coverslides were
gently removed, and the slides were immersed in a previously prepared
acid solution (80 µL of HCl added to 10 mL of distilled water) for 7
minutes. Aer removal from this solution, the slides were incubated
for 25 minutes in 10 mL of lysing solution (provided in the Halosperm
kit). Aer rinsing in distilled water, the slides were dehydrated for 2
minutes in three concentrations of alcohol (70%, 90% and 100% vol)
for 2 minutes each and either were stored (storage was possible several
months in optimal conditions) or were processed immediately with
staining solution for 10 minutes with continuous airow. Staining
was performed with 1:1 (vol/vol) by using Wright’s solution (Merck,
Darmstadt, Germany) and phosphate-buered saline solution (Merck).
e slides were rinsed in tap water, allowed to dry at room temperature,
processed for upright or inverted bright-eld microscopy at x100, and
covered with 22 x 22 coverslide. Operators scored ≥500 spermatozoa
for each patient according to the patterns established by Fernández et
al. [21]. Strong staining is preferred to visualize the dispersed DNA
loop halos. Removal of sperm nuclear proteins results in nucleoids with
a central core and a peripheral halo of dispersed DNA loops. e sperm
tails remain preserved. e acid treatment produces DNA unwinding
that is restricted in those nuclei with high levels of DNA strand breakage.
Aer the subsequent lysis, sperm nuclei with fragmented DNA produce
very small or no halos of dispersed DNA. However, nuclei without
DNA fragmentation released their DNA loops to form large halos.
Ovarian stimulation and oocyte collection
e menstrual cycles of patients were stimulated using recombinant
FSH (Gonal®, Merck Serono laboratories, Peru) and HMG (Menopure®,
Ferring Pharmaceutical, Peru) according to the stimulation protocols
previously established and starting on day 2 of the menstrual cycle
until when at least three follicles reached ~18 mm in diameter. e
oocyte pickup was performed by vaginal ultrasound 36 h aer the
intramuscular application of Human Chorionic Gonadotropin, hCG
(Pregnyl®, Ferring Pharmaceutical, Peru). For the procedure, the patient
was under general anesthesia with 200 mg of Propofol iv (Diprivan® 1%
P/V; AstraZeneca Laboratories, UK).
During the follicular aspiration procedure, the oocytes were
recovered in Global®-HEPES-buered medium (IV Fonline, Canada)
supplemented with 10% vol/vol Serum Substitute Supplement (SSS;
Irvine Scientic, USA). Aer retrieval, cumulus-oocyte complexes
were manually trimmed of excess cumulus cells and cultured in ~200
µL drops of Global®-Fertilization medium (IV Fonline, Canada) plus
10% SSS under oil at 37°C and an atmosphere containing 6% CO2, 5%
O2 and 89% N2 for 5 hours before the ICSI procedure.
Seminal samples and sperm non-apoptotic selection by MACS
Study group sperm cells were prepared by density gradient
centrifugation aer MACS of non-apoptotic spermatozoa. Control
group sperm cells were prepared by density gradient centrifugation
without magnetic sorting. On the day of the ICSI procedure, all
patients’ partners collected the semen samples by masturbation in
aseptic conditions into sterile cups aer 3-5 days of sexual abstinence.
Concentration, progressive motility and morphology from spermatozoa
were assessed aer semen liquefaction for 30 min at room temperature
according to World Health Organization criteria (2010). Motile
spermatozoa were separated from the seminal plasma by centrifugation
through 1.0 mL 95% and 45% Isolate gradients (Irvine Scientic,
USA). e pellet was washed once by centrifugation for 5 min, and was
resuspended in HEPES-buered Global medium + 10% SSS for ICSI.
For magnetic selection, spermatozoa were incubated with annexin
V-conjugated microbeads (Miltenyi Biotec, GmbH, Bergisch Gladbach,
Germany) for 15 min at room temperature. One hundred microliters of
microbeads were used for each 10 million separated cells. e sperm/
microbead suspension was loaded in a separation column containing
iron globes, which was tted in a magnetic eld (Mini MACS; Miltenyi
Biotec). e fraction composed of apoptotic spermatozoa was retained
in the separation column, whereas the fraction with intact membranes
was eluted through the column and was collected as non-apoptotic
spermatozoa.
ICSI, fertilization and embryo culture
In every patient, all oocytes in metaphase II were injected 5 hours
aer aspiration according to methods previously described [22]. Aer
the ICSI procedure (day 0), all injected oocytes were cultured at 37°C in
an atmosphere of 6% CO2, 5% O2 and 89% N2.
e fertilization was evaluated 16-18 hours post injection by
presence of two pronuclei (day 1). e zygotes were individually
cultured under mineral oil, in 10-µL droplets of Global® medium (IVF
online, Canada) supplemented with 10% vol/vol SSS from day 1 to day
3. On day 3, the embryos were moved to fresh 10-µL droplets of Global®
medium + 10% SSS and cultured 2 days more up to the transfer day in
blastocyst stage.

Page 3 of 6
Volume 2 • Issue 4 • 1000133
JFIV Reprod Med Genet
ISSN: 2375-4508 JFIV, an open access journal
Citation: García-Ferreyra J, Villegas L, Romero R, Zavala P, Hilario R, et al. (2014) High Pregnancy and Implantation Rates Can Be Obtained Using
Magnetic-Activated Cell Sorting (MACS) to Selection Spermatozoa in Patients with High Levels of Spermatic DNA Fragmentation. JFIV
Reprod Med Genet 2: 133. doi:10.4172/2375-4508.1000133
On day 3 the embryos were evaluated for cell number,
fragmentation, and multinucleation. Good quality day 3 embryos were
dened as those with 6-8 cells and ≤ 10% of fragmentation. Good
quality blastocysts were dened as having an inner cell mass (ICM)
and trophoectoderm type A or B [23]. e ICM score was evaluated
as follow: type A = compact area, many cells present; type B = cells are
loosely grouped. e trophoectoderm was scored as follows: type A =
many cells forming a tight epithelial network of cells; type B = few cells
forming a loose network of cells.
Embryos were transferred on day 5 in all patients using an Emtrac
embryo transfer catheter (Gynétics Medical Products, Lommel,
Belgium) that had been previously washed with culture medium. e
catheter was completely lled with culture medium and the blastocysts
lled in the last 10 µL of the catheter. All transfers were performed
according to the methods previously described by Mansour [24]. e
blastocysts that were not transferred were cryopreserved or discarded
according to their morphology.
Pregnancy determinations
e biochemical pregnancy was assessed 14 days aer the embryo
transfer by measuring the Human Chorionic Gonadotropin beta
subunit (hCG-b) in blood. e clinical pregnancy was determined
by transvaginal ultrasonography to detect gestational sacs and fetal
heartbeats at approximately 21 and 28 days aer transfer, respectively.
Statistical analysis
Statistical analysis was carried out using the statistic package Stata
10 (StataCorp, College Station, TX). Data are represented as Mean ±
SD. Group comparisons were made using the χ2 test and Student’s t-test.
It was considered a statistical signicant dierence when P <0.05.
e normal fertilization rate was calculated from the number of
zygotes with two pronuclei of ICSI and divided by the number of oocytes
injected by 100. e cleavage rate was calculated from the number of
embryo with ≥ 6 cells at day 3 and divided by the total number of zygotes
by 100. e rate of implantation was calculated dividing the number of
gestational sacs observed by ultrasound at the 21 st day post transfer
divided by the total number of embryos transferred by 100. e rate
of clinic pregnancy was calculated from the number of patients with at
least one gestational sac divided by the total embryo transfers by 100.
e miscarriage rate was dened as the number of pregnancies with
total loss of gestational sacs before the 20 weeks of gestation between
the numbers of pregnancies by 100.
Results
e mean days of stimulation were similar in the study and control
groups (8.10 ± 1.08 versus 8.02 ± 1.32 days; P: NS). e women of the
study group compared with the control group had signicantly lower
mean rFSH treatment (1292.21 ± 291.71 versus 1386.19 ± 313.21; IU/l;
data not shown). Women and their partners from the study group were
signicantly older than that those from the control group (P <0.05;
Table 1). Furthermore, the study group had higher percentages of DNA
fragmentation compared to the control group (P <0.05). Both evaluated
groups had similar sperm concentration, progressive motility and
morphology (P: NS; Table 1).
Results of laboratory and clinical outcomes obtained from the
study group (high DNA Fragmentation) and the control group (normal
DNA fragmentation) are shown in (Table 2). A total of 588 and 672
oocytes were collected from women of study and control groups,
respectively. Four hundred and eighty-six and ve hundred and
seventy-eight oocytes from de study and control groups, respectively,
were inseminated. e normal fertilization (2PN) was similar in both
evaluated groups (study group: 74.5% versus control group: 76.5%).
ere was no dierence in the cleavage rate and good embryo quality at
day 3 between groups. Blastocyst development rates were similar (50.8%
and 41.1%, respectively) for the study and control groups. In addition,
embryos reaching the blastocyst stage were morphologically similar in
both groups. In the study group, a total of 107 embryos were transferred
to 57 patients with a mean of 1.88 embryos. In the control group, a
total of 146 embryos were transferred to 77 patients with a mean of
1.90 embryos. ere was no signicant dierence in the clinical PR per
transfer in the evaluated groups: 63.2% in the study group and 45.5% in
the control group. Implantation and miscarriage rates were similar in
both groups (Figure 1). e percentages of single and twin pregnancies
were similar between both studied groups.
e total number of cycles was allocated to three age groups: <35;
35-39 and ≥ 40 years old (Table 3). e pregnancy, implantation and
miscarriage rates were similar in each age group in both the high DNA
fragmentation (study group) and normal DNA fragmentation (control
group). Our data showed that pregnancy and implantation rate is
similar with advancing age in the study group.
Study Group Control Group
No. cycles 57 77
Female age (years) (Mean ± SD) 34.64 ± 4.55*32.76 ± 4.21
Male age (years) (Mean ± SD) 42.24 ± 7.76*38.38 ± 5.79
Sperm DNA fragmentation (%) 38.21 ± 11.33*18.76 ± 9.07
Sperm concentration (x 106/mL) 77.65 ± 41.29 67.53 ± 35.57
Progressive motility (%) 23.63 ± 11.22 21.60 ± 11.58
Sperm morphology (%) 5.01 ± 4.85 6.67 ± 6.84
*P<0.05 compared to the control group
Table 1: Comparison of seminal results in the study and control groups.
Study Group Control Group
Cycles 57 77
Total number of oocytes 588 672
Total number of injected oocytes 486 578
Total number of fertilized oocytes (2PN) (%) 362 (74.5) 442 (76.5)
Cleavage rate of embryo at day 3 (%) 98.3 89.1
Number of cells at day 3 (Mean ± SD) 7.19 ± 0.88 7.24 ± 0.87
Good quality embryos at day 3 (%) 88.4 83.7
Blastocyst development (%) 50.8 41.1
Good quality blastocysts (%) 71.9 69.3
Full blastocyst (%) 35.3 43.5
Expanded blastocyst (%) 60.9 54.2
Hatching blastocyst (%) 3.8 2.3
Total number of embryo transferred/patient
(Mean ± SD) 107 146
(1.88 ± 0.33) (1.90 ± 0.38)
Pregnancy rate (%) 63.2 45.5
Implantation rate (%) 37.4 28.1
Single pregnancies (%) 88.9 82.9
Twin pregnancies (%) 11.1 17.1
Miscarriages (%) 8.3 17.1
Biochemical pregnancy rate (%) 0 0
P: NS
Table 2: Comparison of laboratory results and clinical outcomes between both
evaluated groups.

Page 4 of 6
Volume 2 • Issue 4 • 1000133
JFIV Reprod Med Genet
ISSN: 2375-4508 JFIV, an open access journal
Citation: García-Ferreyra J, Villegas L, Romero R, Zavala P, Hilario R, et al. (2014) High Pregnancy and Implantation Rates Can Be Obtained Using
Magnetic-Activated Cell Sorting (MACS) to Selection Spermatozoa in Patients with High Levels of Spermatic DNA Fragmentation. JFIV
Reprod Med Genet 2: 133. doi:10.4172/2375-4508.1000133
Discussion
Sperm chromatin is a well-organized, compact, crystalline
structure, consisting of haploid DNA and heterogeneous proteins. Its
highly condensed and insoluble nature plays a protective role during
the transfer of the paternal genetic information through the male and
female reproductive tracts, adjusting to the extremely limited volume of
the sperm nucleus [25,26].
Apoptosis is an ongoing physiological phenomenon that maintains
the number of germ cells within the supportive capacity of the Sertoli
cells [27]. is process includes a cascade of events such as interruption
of membrane phospholipid asymmetry, condensation and destruction
of the chromatin, compaction of cytoplasmic organelles, reduced
mitochondrial transmembrane potential, mitochondrial release of
cytochrome c, production of reactive oxygen species, expansion of the
endoplasmic reticulum, and a diminishing in cell volume [28]. Among
these events, translocation of phosphatidylserine (PS), as an early event
of the execution phase of apoptosis, is considered one of the signals
for specic recognition and removal of apoptotic cells by phagocytosis
[29]. In men, increased rates of externalized PS are associated with
decreased sperm motility, morphology or concentration in ejaculated
semen [30], and its occurrence is reported to be higher in semen
samples from infertile men compared with fertile men [31]. Based on
PS externalization to the outer membrane, and using magnetic cell
sorting using annexin V (used as an apoptotic marker) conjugated with
microbeads, apoptotic sperm can be separated from non-apoptotic
sperm [32].
Sperm DNA fragmentation may exert its eect at dierent stages
of the reproductive procedure, beginning from the pre-implantation
development of the embryo to achievement and sustaining of
pregnancy and nally the creation of healthy ospring. Several studies
demonstrated the impact of sperm DNA damage and its correlation
with clinical endpoints including fertilization rates, embryonic
development, implantation, pregnancy and miscarriage rates [33-35].
Negative correlation has been associated between fertilization
results with the presence of high levels of sperm DNA fragmentation
[9,36-39]. However, if the type and extent of DNA damage can be
balanced by the reparative ability of the oocyte, it is possible to achieve
fertilization even in the presence of elevated sperm DNA fragmentation
rates [10,40,41]. Additionally, in ICSI, the natural selection barriers are
bypassed entirely and fertilization with highly DNA-fragmented sperm
is possible. Although this damage may also be repaired in the oocyte,
excessive damage may potentially result in early reproductive failures
[42]. During the 4 to 8 cell stage, when the paternal genome is switched
on, development of the embryo is denitely aected by sperm DNA
integrity. en, apoptosis and fragmentation will be present within
the embryo and subsequently there is some diculty in reaching to
blastocyst stage [26,43]. An inverse relationship has been reported
between the likelihood of achieving pregnancy either by natural
intercourse, intrauterine insemination or by application of ART and
the presence of high sperm DNA fragmentation levels [11,40,44,45].
In the present study, MACS was utilized for the selection of non-
apoptotic sperm in men with high DNA fragmentation levels, and these
selected spermatozoa were used in an ICSI procedure. e results have
shown the feasibility and eciency of MACS to select non-apoptotic
spermatozoa. e data obtained demonstrated similar fertilization
rates, preimplantation embryo development and clinical outcomes in
those patients with high sperm DNA fragmentation treated with MACS
compared with those patients with normal sperm DNA fragmentation.
ese ndings are very important because they show that the utilization
of selected spermatozoa favors normal pregnancy and implantation
rates. Furthermore, the use of this technique is a good choice for
couples with repeated assisted reproduction failures in which sperm
apoptosis is present.
ere are multiple applications for MACS technology in the male
reproduction. MACS, in combination with anti-CD45 microbeads,
have been used repeatedly with great success to eliminate leukocytes
from seminal uid [46,47]. Also, non-apoptotic sperm obtained
through MACS can be used for insemination in infertile men couples
with autoimmune male infertility [48,49]. Additionally, MACS has
been employed to facilitate the analysis of distinctive homogeneous
spermatogenic cell populations by overcoming the heterogeneity of
somatic and germ cells within the testicular tissue [50,51]; and to select
a high proportion of sperm with normal morphology and signicantly
lower sperm deformity index [52].
In regards to the application of MACS in ART, our study showed
an improvement in the fertilization, embryo quality, pregnancy and
implantation rates in those patients with high levels of fragmented
spermatic DNA. Several authors previously reported similar results in
fertilization rates [15,16,53], embryo quality [17,18], pregnancy rates
[18,20,53-55], and additionally healthy infants who are born with
normal neonatal assessments [53,56-58].
Cleavage rate, cleavage stage embryo morphology, cytoplasmic
fragmentation and multinucleation have been shown to be important
markers of embryo quality and viability that may be observed over time
during in vitro culture. Aer the advent of extended embryo culturing,
higher IRs have been reported because of better embryo selection
compared with earlier developmental stages and because of better
synchronization between the embryo developmental stage and uterine
environment.
Our observations demonstrate that MACS is a exible, fast and
simple cell sorting system for separation of cells; and employing
annexin V microbeads can eectively remove apoptotic sperm.
0
10
20
30
40
50
60
70
Pregnacy rate Implantaon rate Miscarriage rate
Study group
Control group
P: NS
Figure 1: Pregnancy, Implantation and Miscarriage rates
Study Group Control Group
Age (years) n PR (%) IM (%) MR (%) n PR (%) IR (%) MR (%)
<35 36 66.7 38.9 8.3 53 49.1 31 19.2
35-39 15 53.3 29.6 0 19 36.8 21.1 14.3
≥ 40 6 66.7 50 25 5 40 25 50
PR: Pregnancy rate; IM: Implantation rate; MR: Miscarriage rate
P: NS
Table 3: Clinical outcomes in the study and the control group according the age
of patients.

Page 5 of 6
Volume 2 • Issue 4 • 1000133
JFIV Reprod Med Genet
ISSN: 2375-4508 JFIV, an open access journal
Citation: García-Ferreyra J, Villegas L, Romero R, Zavala P, Hilario R, et al. (2014) High Pregnancy and Implantation Rates Can Be Obtained Using
Magnetic-Activated Cell Sorting (MACS) to Selection Spermatozoa in Patients with High Levels of Spermatic DNA Fragmentation. JFIV
Reprod Med Genet 2: 133. doi:10.4172/2375-4508.1000133
Hence, sperm cells prepared by MACS have high motility, viability,
morphology, and display reduced apoptosis manifestations, including
DNA fragmentation. Additionally, by selecting non-apoptotic sperm by
MACS, we can achieve satisfactory pregnancy and implantation rates,
rendering the procedure a good option for couples with high sperm
DNA fragmentation and repeated assisted reproduction failures.
Finally, our data demonstrated the importance of adequate
diagnosis and sperm selection pre ART when high levels of sperm DNA
damage are observed. Consequently, we suggest that further research
and well-designed prospective studies are carried out, in which all
variables are controlled, in order to reveal more advantages regarding
the utilization of MACS in ART.
References
1. Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, et al. (1999) Utility of
the sperm chromatin structure assay as a diagnostic and prognosis tool in the
human fertility clinic. Hum Reprod 14:1039-1049.
2. Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cardelli E, et al. (2000) Sperm
chromatin damage impairs human fertility. The Danish First Pregnancy Planner
Study Team. Fertil Steril 73:43-50.
3. Giwercman A, Lindstedt L, Larsson M, Bungum M, Spano M, et al. (2010)
Sperm chromatin structure assay as an independent predictor of fertility in vivo:
a case-control study. Int J Androl 33:221-227.
4. Agarwal A, Said TM (2003) Role of sperm chromatin abnormalities and DNA
damage in male infertility. Hum Reprod Update 9:331-345.
5. Sakkas D, Manicardi GC, Bizzaro D (2003) Sperm nuclear damage in
the human. In: Robaire B, Hales BF (Eds.), Advances in male mediated
developmental toxicity. Kluwer Academic/Premium Publishers, New York.
6. De Jonge C (2002) The clinical value of sperm nuclear DNA assessment. Hum
Fertil 5:51-53.
7. Sakkas D, Alvarez JG (2010) Sperm DNA fragmentation: mechanisms of origin,
impact on reproductive outcome, and analysis. Fertil Steril 93:1027-1036.
8. Seli E, Gardner DK, Schoolcraft WB, Moffatt O, Sakkas D (2004) Extent of
nuclear DNA damage in ejaculated spermatozoa impacts on blastocyst
development after in vitro fertilization. Fertil Steril 82:378-383.
9. Borini A, Tarozzi N, Bizzaro D, Bonu MA, Fava L, et al. (2006) Sperm DNA
fragmentation: paternal effect on early post-implantation embryo development
in ART. Hum Reprod 21:2876-2881.
10. Benchaib M, Lornage J, Mazoyer C, Lejeune H, Salle B, et al. (2007) Sperm
deoxyribonucleic acid fragmentation as a prognostic indicator of assisted
reproductive technology outcome. Fertil Steril 87:93-100.
11. Bungum M, Humaidan P, Axmon A, Spano M, Bungum L, et al. (2007) Sperm
DNA integrity assessment in prediction of assisted reproduction technology
outcome. Hum Reprod 22:174-179.
12. Said T, Agarwal A, Grunewald S, Rasch M, Baumann T, et al. (2006) Selection
of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction
outcomes: an in-vitro model. Biol Reprod 74:530-537.
13. Van Heerde WI, de Groot PG, Reutelingsperger CP (1995) The complexity of
the phospholipid binding protein annexin V. Thromb Haemost 73:172-179.
14. Glander HJ, Schaller J (1999) Binding of annexin V to plasma membranes of
human spermatozoa: a rapid assay for detection of membrane changes after
cryostorage. Mol Hum Reprod 5:109-115.
15. Grunewald S, Reinhardt M, Blumenauer V, Said TM, Agarwal A, et al.
(2009) Increased sperm chromatin decondensation in selected nonapoptotic
spermatozoa of patients with male infertility. Fertil Steril 92:572-577.
16. Romary L, Meseguer M, Garcia-Herrero S, Pellicer A, Garrido N (2010)
Magnetic activated sorting selection (MACS) of nonapoptotic sperm (NAS)
improves pregnancy rates in homologous intrauterine insemination (IUI).
Preliminar data. Fertil Steril 94:S14.
17. Said TM, Agarwal A, Sharma RK, Thomas AJ, Sikka SC (2005) Impact of sperm
morphology on DNA damage caused by oxidative stress induced by beta-
nicotinamide adenine dinucleotide phosphate. Fertil Steril 83:95-103.
18. Dirican EK, Özgün OD, Akarsu S, Özge E, Mukaddes U, et al. (2008) Clinical
outcome of magnetic activated cell sorting of non-apoptotic spermatozoa before
density gradient centrifugation for assisted reproduction. J Assist Reprod Genet
25:375-381.
19. Alvares Sedó C, Uriondo H, Lavolpe M, Noblia F, Papier S, et al. (2010) Clinical
outcome using non-apoptotic sperm selection for ICSI procedures: report of 1
year experience. Fertil Steril 94:Suppl 4S232.
20. Gil M, Sar-Shalom V, Melendez Sivira Y, Carreras R, Checa MA (2013) Sperm
selection using magnetic activated cell sorting (MACS) in assisted reproduction:
a systematic review and meta-analysis. J Assist Reprod Genet 30:479-485.
21. Fernández JL, Muriel L, Rivero MT, Goyanes V, Vasquez R, et al. (2003) The
sperm chromatin dispersion test: a simple method for the determination of
sperm DNA fragmentation. J Androl 24:59-66.
22. García J, Noriega Hoces L, Gonzales GF (2007) Sperm chromatin stability
and its relationship with fertilization rate after Intracytoplasmic Sperm Injection
(ICSI) in an assisted reproduction program. J Assist Reprod Genet 24:587-593.
23. Gardner DK, Cshoolcraft WB (1999) In vitro culture of human blastocysts. In:
Jansen R and Mortimer D (Eds.), Towards reproductive certainly: infertility and
genetics beyond 1999. Parthenon press, UK.
24. Mansour R (2005) Minimizing embryo expulsion after embryo transfer: a
randomized controlled study. Hum Reprod 20:170-174.
25. Fraser L (2004) Structural damage to nuclear DNA in mammalian spermatozoa:
its evaluation techniques and relationship with male infertility. Pol J Vet Sci
7:311-321.
26. Acharyya S, Kanjilal S, Bhattacharyya AK (2005) Does human sperm nuclear
DNA integrity affect embryo quality?. Indian J Exp Biol 43:1016-1022.
27. Sinha Hikim AP, Swerdloff RS (1999) Hormonal and genetic control of germ cell
apoptosis in the testis. Rev Reprod 4:38-47.
28. Arends MJ, Wyllie AH (1991) Apoptosis: mechanisms and roles in pathology. Int
Rev Exp Pathol 32:223-254.
29. Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, et
al. (1995) Early redistribution of plasma membrane phosphatidylserine is
a general feature of apoptosis regardless of initiating stimulus: inhibition by
overexpression of Bcl-2 and Ab1. J Exp Med 182:1545-1556.
30. Zhang HB, Lu SM, Ma CY, Wang L, Li X, et al. (2008) Early apoptotic changes
in himan spermatozoa and their relationships with conventional semen
parameters and sperm DNA fragmentation. Asian J Androl 10:227-235.
31. Paasch U, Grunewald S, Fitzl G, Glander HJ (2003) Deterioration of plasma
membrane is associated with activated caspases in human spermatozoa. J
Androl 24:246-252.
32. Oosterhuis GJ, Mulder AB, Kalsbeek-Batenburg E, Lambalk CB, Schoemaker
J, et al. (2000) Measuring apoptosis in human spermatozoa: a biological assay
for semen quality?. Fertil Steril 74:245-250.
33. Karydis S, Asimakopoulos B, Papadopoulos N, Vakalopoulos I, Al-Hasani S, et
al. (2005) ICSI outcome is not associated with the incidence of spermatozoa
with abnormal chromatin condensation. In Vivo 19:921-925.
34. Lewis SE, Agbaje I, Alvarez J (2008) Sperm DNA tests as useful adjuncts to
semen analysis. Syst Biol Reprod Med 54:111-125.
35. Aitken RJ, Koppers AJ (2011) Apoptosis and DNA damage in human
spermatozoa. Asian J Androl 13:36-42.
36. Vicari E, Perdichizzi A, De Palma A, Burrello N, D’Agata R, et al. (2002)
Globozoospermia is associated with chromatin structure abnormalities: case
report. Hum Reprod 17:2128-2133.
37. Virro MR, Larson-Cook KL, Evenson DP (2004) Sperm chromatin structure
assay (SCSA) parameters are related to fertilization, blastocyst development
and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm
injection cycles. Fertil Steril 81:1289-1295.
38. Huang CC, Lin DP, Tsao HM, Cheng TC, Liu CH, et al. (2005) Sperm DNA
fragmentation negatively correlates with velocity and fertilization rates, but
might not affect pregnancy rates. Fertil Steril 84:130-140.
39. Brugnon F, Van Assche E, Verheyen G, Sion B, Boucher D, et al. (2006) Study
of two markers of apoptosis and meiotic segregation in ejaculated sperm of
chromosomal translocation carrier patients. Hum Reprod 21:685-693.
40. Collins JA, Barnhart KY, Schlegel PN (2008) Do sperm DNA integrity tests
predict pregnancy with in vitro fertilization?. Fertil Steril 89:823-831.

Page 6 of 6
Volume 2 • Issue 4 • 1000133
JFIV Reprod Med Genet
ISSN: 2375-4508 JFIV, an open access journal
Citation: García-Ferreyra J, Villegas L, Romero R, Zavala P, Hilario R, et al. (2014) High Pregnancy and Implantation Rates Can Be Obtained Using
Magnetic-Activated Cell Sorting (MACS) to Selection Spermatozoa in Patients with High Levels of Spermatic DNA Fragmentation. JFIV
Reprod Med Genet 2: 133. doi:10.4172/2375-4508.1000133
41. Lin MH, Kuo-Kuang Lee R, Li SH, Lu CH, Sun FJ, et al. (2008) Sperm chromatin
structure assay parameters are not related to fertilization rates, embryo quality,
and pregnancy rates in in vitro fertilization and intracytoplasmic sperm injection,
but might be related to spontaneous abortion rates. Fertil Steril 90:352-359.
42. Zini A, Jamal W, Cowan L, Al-hathal N (2011) Is sperm dna damage associated
with IVF embryo quality? A systematic review. J Assist Reprod Genet 28:391-
397.
43. Spano M, Seli E, Bizzaro D, Manicardi GC, Sakkas D (2005) The signicance of
sperm nuclear DNA strand break son reproductive outcome. Curr Opin Obstet
Gynecol 17:255-260.
44. Muriel L, Garrido N, Fernández JL, Remohi J, Pellicer A, et al. (2006) Value
of the sperm deoxyribonucleic acid fragmentation level, as measured by the
sperm chromatin dispersion test, in the outcome of in vitro fertilization and
intracytoplasmic sperm injection. Fertil Steril 85:371-383.
45. Zini A, Sigman M (2009) Are tests of sperm DNA damage clinically useful? Pros
and cons. J Androl 30:219-229.
46. Krausz C, West K, Buckingham D, Aitken RJ (1992) Development of a
technique for monitoring the contamination of human semen samples with
leukocytes. Fertil Steril 57:1317-1325.
47. Hipler UC, Schreiber G, Wollina U (1998) Reactive oxygen species in human
semen: investigations and measurements. Arch Androl 40:67-78.
48. Foresta C, Varotto A, Caretto A (1990) Immunomagnetic method to select
human sperm without sperm surface-bound autoantibodies in male autoimmune
infertility. Arch Androl 24:221-225.
49. Vigano P, Fusi FM, Brigante C, Busacca M, Vignali M (1991) Immunomagnetic
separation of antibody-labelled from antibody-free human spermatozoa as a
treatment for immunologic infertility. A preliminary report. Andrologia 23:367-
371.
50. von Schonfeldt V, Krishnamurthy H, Foppiani L, Schlatt S (1999) Magnetic
cell sorting is a fast and effective method of enriching viable spermatogonia
from Djungarian hamster, mouse, and marmoset monkey testes. Biol Reprod
61:582-589.
51. van der Wee KS, Johnson EW, Dirami G, Dym TM, Hofmann MC (2001)
Immunomagnetic isolation and long-term culture of mouse type A
spermatogonia. J Androl 22:696-704.
52. Aziz N, Said T, Paasch U, Agarwal A (2007) The relationship between human
sperm apoptosis, morphology and the sperm deformity index. Hum Reprod
22:1413-1419.
53. Van Thillo G, Guidobono M, Young E, Ruiz Jorro MR, Vila M, et al. (2011)
Biological safety and live births after selection of nonapoptotic spermatozoa
during ICSI. Fertil Steril 96 Suppl 3:S160-S161.
54. Buzzi J, Valcarcel A, Lombardi E, Oses R, Rawe V, et al. (2010) Magnetic
activated cell sorting (MACS) improves oocytes donation results associated to
severe male factor infertility. Hum Reprod 25 suppl 1:i118-52.
55. Young E, De Caro R, Marconi G, Lombardi E, Young E, Tiveron M (2010)
Reproductive outcome using Annexin V columns for nonapoptotic sperm
selection. Hum Reprod 25 Suppl 1:i6-9.
56. Polak De Fried E, Denaday F. (2010) Single and twin ongoing pregnancy in
two cases of previous ART failure after ICSI performed with sperm sorted using
annexin V microbeads. Fertil Steril 94:351.e15-8.
57. Rawe V, Uriondo Boudri H, Alvares Sedó C, Carro M, Papier S, et al. (2010)
Healthy baby born after reduction of sperm DNA fragmentation using cell
sorting before ICSI. Rep Biomed Online 20:320-323.
58. Vilela M, Tiveron M, Lombardi C, Viglierchio MI, Marconi G, et al. (2011)
Implantation potential after vitrication of embryos obtained using magnetic
active sperm sorting combined with annexin V during ICSI. Hum Reprod 26
Suppl 1:i200.
Citation: García-Ferreyra J, Villegas L, Romero R, Zavala P, Hilario R, et al.
(2014) High Pregnancy and Implantation Rates Can Be Obtained Using Mag-
netic-Activated Cell Sorting (MACS) to Selection Spermatozoa in Patients with
High Levels of Spermatic DNA Fragmentation. JFIV Reprod Med Genet 2: 133.
doi:10.4172/2375-4508.1000133
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