Physiologic ICSI: Hyaluronic acid (HA) favors

selection of spermatozoa without DNA fragmentation

and with normal nucleus, resulting in improvement of
embryo quality
Lodovico Parmegiani, B.Sc., Graciela Estela Cognigni, M.D., Silvia Bernardi, B.Sc., Enzo Troilo, B.Sc.,
Walter Ciampaglia, M.D., and Marco Filicori, M.D.
Reproductive Medicine Unit, GynePro Medical Centers, Bologna, Italy

Objective: To evaluate the role of hyaluronic acid (HA) for sperm selection before intracytoplasmic sperm injection (ICSI).
Design: Three prospective studies.
Setting: Private assisted reproduction center in Italy.
Patient(s): Study 1: 20 men. Study 2: 15 men. Study 3: 206 couples treated with ICSI on a limited number of
oocytes per patient (13) in accordance with Italian IVF law.
Intervention(s): Study 1: determination of sperm DNA fragmentation of HA-bound spermatozoa versus spermatozoa in polyvinylpyrrolidone (PVP). Study 2: assessment of nuclear morphology of HA-bound spermatozoa
versus spermatozoa in PVP. Study 3: randomized study comparing conventional PVP-ICSI to ICSI in which the
spermatozoa are selected for their capacity to bind to HA (HA-ICSI).
Main Outcome Measure(s): Study 1: sperm DNA fragmentation rate. Study 2: sperm nucleus normalcy rate
according to motile sperm organellar morphology examination criteria. Study 3: fertilization, embryo quality
and development, and implantation and pregnancy.
Result(s): Spematozoa bound to HA show a significant reduction in DNA fragmentation (study 1) and a significant
improvement in nucleus normalcy (study 2) compared with spermatozoa immersed in PVP. Furthermore, injection
of HA-bound spermatozoa (HA-ICSI) significantly improves embryo quality and development (study 3).
Conclusion(s): Hyaluronic acid may optimize ICSI outcome by favoring selection of spermatozoa without DNA
fragmentation and with normal nucleus. Furthermore, HA may also be used to speed up the selection of spermatozoa with normal nucleus during intracytoplasmic morphologically selected sperm injection (IMSI). (Fertil
Steril 2010;93:598604. 2010 by American Society for Reproductive Medicine.)
Key Words: Hyaluronic acid selection, physiologic ICSI, sperm selection, HA-ICSI, sperm DNA fragmentation,
MSOME, IMSI, PICSI

In the natural human fertilization process, hyaluronic acid

(HA) seems to play a pivotal role in physiological sperm
selection. In fact, HA is normally present in the extracellular
matrix (ECM) of cumulus oophorus surrounding the oocyte.
The ECM is a formidable barrier that only mature spermatozoa that have extruded their specific receptors to bind to and
digest HA, can overcome to reach and penetrate the zona
pellucida and fertilize the oocyte.
Spermatozoa that are able to bind permanently to HA
in vitro are mature and have completed the spermiogenetic
process of plasma membrane remodeling, cytoplasmic extrusion, and nuclear maturity (1); these mature spermatozoa
Received December 22, 2008; revised February 19, 2009; accepted
March 4, 2009; published online April 25, 2009.
L.P. has nothing to disclose. G.E.C. has nothing to disclose. S.B. has
nothing to disclose. E.T. has nothing to disclose. W.C. has nothing to
disclose. M.F. has nothing to disclose.
Reprint requests: Lodovico Parmegiani, B.Sc., Reproductive Medicine
Unit, GynePro Medical Centers, GynePro Medical, Via T. Cremona, 8,
40137 Bologna, Italy (FAX: 39 051 441 135; E-mail: l.parmegiani@
gynepro.it).

598

have a high density of HA receptors. In fact, in normal

sperm maturation, elongating spermatids undergo cytoplasmic extrusion and plasma membrane remodeling, leading to
the formation of binding sites for HA and oocyte zona pellucida. In contrast, immature spermatozoa with deficient
plasma membrane remodeling are not able to bind to HA
(2); in spermatozoa with arrested/diminished maturity, there
is a deficiency in the zona pellucida and HA binding sites.
Immature spermatozoa also show a higher retention of
creatine kinase and other cytoplasmic enzymes, increased
levels of lipid peroxidation and consequent DNA fragmentation, and an abnormal sperm morphology (3). Furthermore, spermatozoa with arrested maturity have low heat
shock protein HspA2 expression, which causes meiotic
defects and could be related to chromosomal aneuploidies.
This may explain the fact that the frequency of chromosomal
aneuploidies is reduced 5.4-fold in HA-bound mature spermatozoa compared with non HA-bound spermatozoa (4).
The selection of ideal spermatozoa before intracytoplasmic sperm injection (ICSI) could help to optimize the
outcome of the treatment, because the injection of

Fertility and Sterility Vol. 93, No. 2, January 15, 2010

Copyright 2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

0015-0282/10/$36.00
doi:10.1016/j.fertnstert.2009.03.033

spermatozoa with damaged DNA, for example, may lead to

an increased risk of pregnancy loss (5). Effective sperm selection becomes especially critical when a limited number of
oocytes are available for injection or when insemination of
no more than three gametes is allowed by law, as in Italy (6).
The aim of the present study was to better define the role of
HA for selection of spermatozoa with normal chromatin content to optimize ICSI outcome, particularly when injecting
a limited number of oocytes.
MATERIALS AND METHODS
All these studies were conducted in the Reproductive Medicine Unit, GynePro Medical Centers, Bologna, Italy. The procedure was approved by the Institutional Review Board in
this center. All patients were informed of the procedure,
and written consent was obtained from each.
Study 1: Determination of Sperm DNA Fragmentation
Sperm DNA fragmentation of 20 patients was studied. Sperm
count was carried out 30 minutes after ejaculation. Each
semen sample was divided into two aliquots: One aliquot
was analyzed for DNA fragmentation (group 1) and the other
aliquot was treated via swim-up as described elsewhere (7).
After swim-up, the semen sample was subsequently divided
into three aliquots: The first aliquot underwent DNA fragmentation analysis (group 2), but one of the remaining two
aliquots was put into a polyvinylpyrrolidone (PVP) droplet
(PVP Clinical Grade; MediCult, Jyllinge, Denmark) and
the other into an HA-containing medium droplet (Sperm
Slow; MediCult), both on a Petri dish under oil (Liquid Paraffin; MediCult). Spermatozoa were collected from PVP with
an injecting pipette (ICSI Micropipettes; Humagen Fertility
Diagnostics, Charlottesville, VA) as for conventional ICSI
procedure (8), whereas only the spermatozoa bound to HA
(after 15 minutes of incubation at 37 C) were collected
with injecting pipette from the HA-containing medium droplet. DNA fragmentation of spermatozoa collected from PVP
(group 3) and HA (group 3) was analyzed. Thus, DNA fragmentation rate was observed as follows:
30 minutes after ejaculation (group 1: initial semen sperm).
After sperm treatment via swim-up (group 2: spermatozoa
after Swim-Up).
After recovery in PVP by an injecting pipette (group 3:
spermatozoa in PVP).
After recovery of HA-bound spermatozoa by an injecting
pipette (group 4: spermatozoa bound to HA).
Determination of DNA fragmentation was performed with
sperm chromatin dispersion (SCD) test using a ready-to-use
kit (Halosperm kit; Indas Laboratories, Madrid, Spain). The
basis of the SCD test lies in the differential response offered
by the nuclei of spermatozoa with fragmented DNA
compared with those with their DNA intact (9): When
DNA is intact, the controlled denaturation of the DNA,
followed by the extraction of the nuclear proteins, gives
rise to partially deproteinized nucleoids in which the DNA
Fertility and Sterility

loops expand, forming large halos of chromatin dispersion.

The spermatozoa nucleoids whose DNA is fragmented do
not develop a large dispersion halo. After SCD procedure
the spermatozoafixed on agarose-coated test-slides
were stained and observed at 40 magnification using an
inverted microscope (TE 2000 U; Nikon Instruments Italia,
Firenze, Italy) equipped with digital imaging software for
measurements and count (NIS Elements BR; Nikon Instruments Italia). Five hundred spermatozoa were analyzed per
each test slide in groups 1 and 2, and 200 spermatozoa per
test slide in groups 3 and 4 (owing to the lower number of
spermatozoa collectable with the injecting pipette). DNA
fragmentation rate was calculated as: number of spermatozoa
without halo on total number of spermatozoa observed 
100.
Study 2: Sperm Nucleus Normalcy Assessment
This study was carried out on 15 patients. Spermatozoa were
first treated with a two-layer density gradient system (Sil
Select, FertiPro, Beernem, Belgium) (10). Then one aliquot
of prepared sperm suspension was put into a PVP (PVP Clinical Grade, MediCult) droplet and another aliquot into an
HA-containing medium (Sperm Slow, MediCult) droplet,
both on a glass-bottom Petri dish (WillCo-dish; WillCo
Wells, Amsterdam, The Netherlands) under oil (Liquid Paraffin, MediCult). The nucleus normalcy rate of spermatozoa in
PVP and of spermatozoa bound to HA (after 15 minutes of
incubation at 37 C) was analyzed.
The nucleus normalcy was assessed in real time according
to motile sperm organelle morphology examination
(MSOME) criteria defined by Bartoov et al. (11). The examination was performed using an inverted light microscope (TE
2000 U; Nikon Instruments Italia) equipped with high-power
Nomarski optics enhanced by digital imaging to achieve
a magnification up to 6,300. According to MSOME criteria,
normally shaped sperm nucleus is smooth, symmetric, and
with oval configuration. Average lengths and widths (SD)
must be 4.75  0.28 mm and 3.28  0.20 mm, respectively. Nuclear chromatin content is considered to be abnormal if sperm
head contains one or more vacuoles (diameter of 0.78  0.18
mm) that occupy more than 4% of the normal nuclear area. To
be considered morphologically normal, a sperm nucleus has to
have both normal shape and normal chromatin content (12). In
this study, for rapid evaluation of nuclear normalcy, a fixed,
transparent, celluloid form of a sperm nucleus fitting MSOME
criteria for length and width was superimposed on the examined cell: the nuclear shape was considered to be abnormal if
it differed in length or width by two standard deviations
from the normal mean axis values; vacuoles were examined
using a similar celluloid form (12). Furthermore, spermatozoa
were then measured for nuclear length, width, and vacuoles
with digital imaging software (NIS Elements BR, Nikon
Instruments Italia). One hundred spermatozoa per patient
were observed and measured in each group. The nucleus normalcy rate was calculated as number of spermatozoa with normal nucleus on total number of spermatozoa analyzed  100.
599

Study 3: PVP-ICSI Versus HA-ICSI

This study was carried out from March 2004 to December
2005. In this period, 232 ICSI treatments were randomly carried out with PVP (PVP Clinical Grade; MediCult) to reduce
sperm motility (group PVP-ICSI) or with HA-containing
product (Sperm Slow; MediCult) for sperm selection (group
HA-ICSI). Inclusion criteria for semen parameters of the
male partner were: presence of ejaculate motile spermatozoa
with total sperm number R1  106 and sperm motility R5%.
The randomization process was conducted with sealed envelopes, assigning 130 subjects to each treatment group. At the
end of the period assigned for the study (December 2005),
107 treatments were carried out on 94 women for the
PVP-ICSI group and 125 treatments on 112 women for the
HA-ICSI group, respectively.
Controlled ovarian stimulation was achieved using gonadotropin-releasing hormone analogues in combination with
a graded gonadotropin administration (13). Oocyte retrieval
was performed 35 hours after ovulation induction with either
5,000 or 10,000 IU of hCG. After retrieval, oocytes were
cultured at 37 C in an atmosphere of 5% CO2 before the
complete removal of cumulus mass and corona cells by enzymatic digestion of recombinant hyaluronidase (SynVitro
Cumulase; MediCult) and by gentle mechanical aspiration
with plastic pipettes (Stripper Tips; MidAtlantic Diagnostic,
Mount Laurel, NJ). The denuded oocytes were then evaluated
to assess their nuclear maturation stage. The oocytes that had
released the first polar body (metaphase II [MII]) underwent
a strict selection by morphologic features (zona pellucida
thickness, perivitelline space size, oocyte shape, cytoplasm
color and granularity, presence of vacuoles and first polar
body morphology) under an inverted microscope with Hoffman modulation contrast (TE 2000 U; Nikon Instruments
Italia). The oocytes classified as high quality were those
which were colorless and of regular shape, with regular
zona pellucida and small perivitelline space without debris,
homogeneous cytoplasm, and no vacuoles or granulations
(1416). Among the high-quality oocytes, the presence
of an intact, round, or ovoid polar body with smooth surface
was considered to be a selection criterion (17). After decumulation and quality evaluation, the three best available MII
oocytes were inseminated by ICSI, in accordance with the
Italian law regulating assisted reproductive technology (6).
The supernumerary MII oocytes reaching our own high
quality standards were cryopreserved for later use.
Conventional PVP-ICSI procedure was executed as
described elsewhere (8). For HA-ICSI, spermatozoa were
selected for their ability to bind to HA: A 2-mL droplet
with suspension of spermatozoa was connected with a pipette
tip to a 5-mL droplet of HA-containing medium (Sperm Slow;
MediCult) and allowed to incubate for 15 minutes at 37 C
under oil (Liquid Paraffin; MediCult). Spermatozoa bound
to HA in the junction zone of the two droplets were selected
and easily detached by injecting pipette (ICSI Micropipette;
Humagen Fertility Diagnostics) and subsequently injected
into oocytes.
600

Parmegiani et al.

Sperm selection with hyaluronic acid

Fertilization and embryo development were examined by

inverted microscope. Embryos were graded 15, with grade
1 assigned to the best-quality embryos containing equally
sized symmetric blastomers with no fragmentation, according to the criteria previously described by Veeck (18). The
embryo development rating (EDR) as described by Cummins
et al. (19) was calculated to define the growth rate of transfered embryos. The formula for calculating the EDR was as
follows: EDR (TE/TO)  100 (TE time expected; TO
time observed). The ideal EDR is 100: this value is
obtained when a hypothetic normally growing embryo is
at the two-cell stage at 33.6 hours, at the four-cell stage at
45.5 hours, and at eight-cell stage at 56.4 hours. Embryo
transfer was carried out 2 (day 2) or 3 (day 3) days after
ICSI. Clinical pregnancy was defined as the presence of
a gestational sac with or without fetal heart beat at ultrasound
examination, two weeks after positive hCG testing.
Statistical Analysis
Continuous variables are presented as mean and standard
error (SE). Categoric variables are presented as percentage.
Normality of distribution of continuous variables was
assessed with a Kolmogorov-Smirnov test (with Lillefor
correction). Between-group differences of normally distributed continuous variables were assessed with parametric
statistic (Student t test), whereas nonparametric statistics
(Mann-Whitney rank sum test) were used when the normality
test was not passed. Between-group differences in noncontinuous variables were assessed using the c2 method with Yates
correction if needed or with Fisher exact test. Difference was
considered to be significant when the P value was < .05.
RESULTS
Study 1: Determination of Sperm DNA Fragmentation
Mean patient age was 38.7  1.0 years (median 39 years).
Mean total motile sperm number was 39.4 (7.3)  106;
12 patients were normozoospermic (R20  106/mL spermatozoa) and 8 oligozoospermic (<20  106/mL spermatozoa)
according to the World Health Organization (WHO) criteria
(20).
Sperm DNA fragmentation rate was significantly lower
(P%.001) after swim-up (11.0%, group 2) compared with
initial semen sperm (16.5%, group 1). Spermatozoa collected
in PVP (group 3) showed the same DNA fragmentation rate
as spermatozoa of group 2 (spermatozoa after swim-up).
The HA-bound spermatozoa (group 4) had the lowest fragmentation rate (5.3%; P%.001 vs. groups 1, 2, and 3), resulting in a threefold reduction compared with initial semen
sperm (Table 1).
Study 2: Sperm Nucleus Normalcy Assessment
Mean patient age was 39.5  1.2 years (median 40 years).
Mean total motile sperm number was 42.6 (9.1)  106;
nine patients were normozoospermic (R20  106/mL
Vol. 93, No. 2, January 15, 2010

TABLE 1
Study 1: Determination of sperm DNA fragmentation.
Group 1:
Initial semen sperm

Group 2:
Spermatozoa
after swim-up

Group 3:
Spermatozoa
in PVP

Group 4:
Spermatozoa
bound to HA

1,655

1,100

442

214

10,000

10,000

4,000

4,000

16.5%a

11.0%b

11.0%c

5.3%d

Spermatozoa with
fragmented DNA
Total no. of spermatozoa
analyzed
DNA fragmentation rate

Note: HA hyaluronic acid; PVP polyvinylpyrrolidone.

a
P%.001 versus groups 2, 3, and 4.
b
P%.001 versus group 4.
c
P%.001 versus group 4.
Parmegiani. Sperm selection with hyaluronic acid. Fertil Steril 2010.

spermatozoa) and six oligozoospermic (<20  106/mL spermatozoa) according to the WHO criteria (20).

was seen in the HA-ICSI group, where sperm selection was

carried out with HA-containing product (Table 3).

Nucleus normalcy rate according to MSOME criteria was

significantly higher (P0.013) in HA-bound spermatozoa
than in spermatozoa in PVP (14.5% vs. 11.0%; Table 2).

DISCUSSION
The selection of ideal spermatozoa before injection may
optimize the outcome of ICSI treatments. It has been demonstrated that sperm dimension and shape, when observed with
conventional magnification for ICSI (40), are not reliable
attributes for prediction of chromatin integrity or the absence
or presence of numerical chromosomal aberrations (21). The
injection of aneuploid spermatozoa may be the cause of an
increased incidence of sex chromosome aberrations in ICSI
offspring (22, 23). Furthermore, oocyte fertilization with
spermatozoa with damaged DNA may lead to an increased
risk of pregnancy loss (5). Sperm selection becomes critical
when a limited number of oocytes are available for injection,
as in Italy where insemination of no more than three gametes
is allowed by law (6).

Study 3: PVP-ICSI Versus HA-ICSI

Mean male age was 40.4  0.6 years (median 40 years) for
the PVP-ICSI group and 40.7  0.4 years (median 40 years)
for the HA-ICSI group. Mean total motile sperm number was
10.6 (2.1)  106 in the PVP-ICSI group and 10.4 (1.9) 
106 the HA-ICSI group. Mean female age was 37.1  0.4
years (median 37 years) for the PVP-ICSI group and of
37.5  0.4 years (median 38 years) for the HA-ICSI group.
Mean number of injected oocytes was 2.6  0.1 in both
groups. The best-quality embryo rate (grade 1) in the HAICSI group was significantly higher (35.8%; P.046) than
in the PVP-ICSI group (24.1%). The EDR was significantly
higher (95.0  0.8) in the HA-ICSI group than in the PVPICSI group (84.0  1.1; P% 0.001). In addition, although
the differences were not statistically significant, a trend toward better fertilization, pregnancy, and implantation rates

Given that HA has a natural sperm-selective function

during human fertilization, a method for in vitro selection
of mature spermatozoa based on sperm-HA binding can be
effective (1, 4). The HA-bound spermatozoa are easily

TABLE 2
Study 2: Sperm nucleus normalcy (MSOME criteria).

Spermatozoa with normal nucleus

Total no. of spermatozoa measured
Nucleus normalcy rate

Spermatozoa in PVP

Spermatozoa bound to HA

165
1,500
11.0%a

218
1,500
14.5%a

Note: MSOME motile sperm organelle morphology examination; other abbreviations as in Table 1.
a
P .013, PVP versus HA.
Parmegiani. Sperm selection with hyaluronic acid. Fertil Steril 2010.

Fertility and Sterility

601

TABLE 3
Study 3: PVP-ICSI versus HA-ICSI.

No. treatments
Mean female age  SE at oocyte retrieval
Fertilized oocytes (%)
Grade 1 embryos (%)
Mean embryo development rating  SE
No. of embryo transfers
Clinical pregnancy rate per transfer (%)
Implantations (%)
Abortions (%)
No. of live births

PVP-ICSI

HA-ICSI

107
37.1  0.4
236/275 (85.8)
55/228 (24.1)a
84.0  1.1c
105
22/105 (20.9)
23/226 (10.2)
4/22 (18.2)
19

125
37.5  0.4
304/332 (91.6)
101/282 (35.8)b
95.0  0.8d
125
31/125 (24.8)
35/282 (12.4)
6/31 (19.3)
29

Note: ICSI intracytoplasmic sperm injection; other abbreviations as in Table 1.

a,b
P .046, PVP-ICSI versus HA-ICSI.
c,d
P%.001, PVP-ICSI versus HA-ICSI.
Parmegiani. Sperm selection with hyaluronic acid. Fertil Steril 2010.

recovered by an injecting pipette, so this method may at least

represent a physiologic alternative for slowing sperm motility
before ICSI, avoiding any potential damaging effect of synthetic plastic PVP (2426). Polyvinylpyrrolidone is routinely
used to reduce sperm motility during ICSI procedure in the
majority of assisted reproduction centers. It has been demonstrated that injection of spermatozoa recovered from HAcontaining products had no negative effects on post-injection
zygote development (27, 28). Furthermore, a selection
method based on sperm-HA binding seems to be useful in
reducing the potential genetic complications and adverse
public health effects of ICSI (4). Currently, two ready-touse systems specially designed for sperm-HA binding selection are available: a plastic culture dish with microdots of HA
hydrogel attached to the bottom interior of the dish (PICSI
Sperm Selection Device; MidAtlantic Diagnostic) or
a viscous medium containing HA (Sperm Slow; MediCult).
Both systems have received the CE mark, indicating their conformity with health and safety requirements in the European
Economic Area; the PICSI system has also been cleared for
marketing by the U.S. Food and Drug Administration. These
systems, in which the spermatozoa are selected for their
capacity to bind to HA (HA-ICSI), allow the execution of
a more physiologic ICSI than conventional PVP-ICSI (8).
The purpose of the present study was to better define the
role of HA for selection of spermatozoa with a normal chromatin content, and to assess the effect of their injection when
performing a physiologic HA-ICSI on a limited number of
oocytes.
The role of HA in selection of spermatozoa with intact
DNA was assessed in study 1 (Table 1), where HA-bound
spermatozoa showed a significant twofold reduction in the
fragmentation rate compared with spermatozoa recovered
from PVP (and a threefold reduction compared with the basal
sample). Only 5.3% of HA-bound spermatozoa showed DNA
602

Parmegiani et al.

Sperm selection with hyaluronic acid

fragmentation. This observation confirms the findings of

Huszar et al. that observed that almost all HA-bound spermatozoa are devoid of DNA fragmentation (2) and persistent
histones (1), which are correlated to DNA chain breakage
(29). It is interesting to observe that, in the present study,
spermatozoa collected in PVP showed the same DNA fragmentation rate as spermatozoa after swim-up, demonstrating
that PVP has no selective function. It should be remembered,
however, that basal sperm DNA fragmentation rate was significantly reduced after swim-up, confirming that this simple
semen treatment improves the percentage of spermatozoa
with normal chromatin structure, as previously demonstrated
by Spano et al. (30). For the assessment of sperm DNA fragmentation, we decided to perform the SCD test. We believe
that for the small number of spermatozoa collectable with
injecting pipette this was the most easily executable test.
Furthermore, the SCD test is a simple and cost-effective
procedure, showing similar predictive values to other chromatin assays for DNA fragmentation (31). In a recent study,
a negative correlation between sperm-HA binding with DNA
fragmentation (performed via SCD test) was observed,
validating further the hypothesis that sperm with DNA
fragmentation have a lower potential to bind to HA (32).
A new method of unstained, real-time, high-magnification
MSOME has been developed by Bartoov et al. (12). This
examination was performed using an inverted light microscope equipped with high-power Nomarski optics enhanced
by digital imaging to achieve a magnification up to 6,300.
Application of this method to patients undergoing ICSI demonstrated that fine morphologic integrity of the human sperm
nuclei is an important parameter associated with pregnancy
rate (12, 33). In fact, ICSI outcome was significantly
improved by the exclusive microinjection into the oocyte of
spermatozoa with a strictly defined morphologically normal
nucleus in couples with previous ICSI failures (3336); this
Vol. 93, No. 2, January 15, 2010

modified ICSI procedure was named intracytoplasmic

morphologically selected sperm injection (IMSI) (33). In
the present study 2, the sperm nucleus normalcy rate (according to MSOME criteria) of spermatozoa in PVP and of
spermatozoa bound to HA was analyzed. We found that
nucleus normalcy rate was significantly higher in HA-bound
spermatozoa than in spermatozoa in PVP (Table 2). Based on
this finding, it could be hypothesized that HA may help to
select a subpopulation of spermatozoa with normal nucleus,
thus speeding up the time-consuming IMSI procedure: in
fact, selecting a normal spermatozoon according to MSOME
criteria may require 60120 minutes (34).
In study 3 we analyzed 232 ICSI treatments performed on
a limited number of oocytes (1 to 3) per patient, as limited
by IVF law in Italy, with the obligation to reimplant all available embryos. The overall results in terms of fertilization,
cleavage, pregnancy, implantation, and abortion rates were
similar to other ICSI studies carried out on women with
a mean age of 37 years under the same law (6). In the present
study we compared conventional PVP-ICSI to ICSI in which
the spermatozoa were selected for their capacity to bind to
HA (HA-ICSI), and we observed a trend toward better fertilization, pregnancy, and implantation in the HA-ICSI group
(Table 3). The same positive trendwhen injecting HA-bound
spermatozoawas observed by Menezo et al. comparing 110
PVP-ICSI and 92 HA-ICSI treatments (37). Furthermore, a statistically significant improvement in fertilization rate and
embryo quality and a reduction in the number of miscarriages
were observed by Worrilow et al. performing PICSI versus
conventional ICSI in a study of 240 patients (38). A possible
explanation for the lack of improvements in abortion rate
with the HA-selected sperm is due to the Italian legal obligation to reimplant all available embryos. Otherwise, in another
small study on HA-ICSI (18 treatments) no differences in
fertilization, pregnancy, and implantation rates were observed
compared with PVP-ICSI (39). All of these studies were
meeting reports. Recently, Nasr-Esfahani et al. (32) have
published a study (performed on 50 couples) observing
a higher fertilization rate when injecting oocytes with HA-selected spermatozoa. To our knowledge, the present study is the
widest prospective-randomized study comparing conventional
PVP-ICSI and HA-ICSI presented for publication. This study
revealed that injection of HA-bound spermatozoa (HA-ICSI)
determines a statistically significant improvement in embryo
quality and development (Table 3) when performing ICSI on
a limited number of oocytes (between 1 and 3). This finding
confirms the positive effect of HA sperm selection on ICSI
outcome observed by other authors (32, 37, 38).
The present studies have demonstrated that spermatozoa
bound to HA show a significant reduction in DNA fragmentation and nuclear anomalies compared with spermatozoa
recovered from PVP, and their injection determines improvement of embryo quality after ICSI. We can conclude that, by
favoring selection of spermatozoa with intact DNA and
normal nucleus, HA may optimize ICSI outcome. If further
studies confirm these beneficial effects on ICSI outcome,
Fertility and Sterility

HA could be considered as the recommended choice for

sperm physiologic selection before ICSI because of its
potential capacity to reduce genetic complications and for
its total lack of toxicity. Furthermore, HA may also be used
to speed up the selection of spermatozoa with normal nucleus
during the IMSI procedure.
Acknowledgments: The authors thank Ms. Maggie Baigent and Dr. Lars
Johansson for revising the manuscript. The authors also thank Alessandra
Arnone, B.Sc., and Eros Nikitos, B.Sc. (GynePro Medical Centers, Bologna,
Italy) for their valuable collaboration in the collection of the data.

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Vol. 93, No. 2, January 15, 2010