ORIGINAL ARTICLE Embryology
Assisted oocyte activation is not
beneficial for all patients with a
suspected oocyte-related activation
F. Vanden Meerschaut*, D. Nikiforaki, S. De Gheselle, V. Dullaerts,
E. Van den Abbeel, J. Gerris, B. Heindryckx, and P. De Sutter
Department for Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium
*Correspondence address. De Pintelaan 185, B-9000 Ghent, Belgium. Tel: +32(0)9-332-02-89; E-mail: firstname.lastname@example.org
Submitted on October 4, 2011; resubmitted on December 30, 2011; accepted on February 8, 2012
background: Despite the success of ICSI, total fertilization failure (TFF) still occurs in 1–3% of all ICSI cycles. ICSI followed by assisted
oocyte activation (ICSI-AOA) can restore fertilization, most efficiently in cases of sperm-related fertilization deficiency. The indication for
ICSI-AOA is less obvious when the capacity of the sperm to activate oocytes is considered normal, as proved by a heterologous ICSI
model, such as the mouse oocyte activation test (MOAT). In this study, we verified whether ICSI-AOA is beneficial for patients in
whom an oocyte-related activation deficiency is suspected.
methods: A prospective study was conducted including patients presenting with a history of TFF or low fertilization (LF) following con-
ventional ICSI in our centre (in-house cases, n ¼ 2) or elsewhere (out-house cases, n ¼ 12). In all cases a sperm deficiency was refuted by the
MOAT. In a next treatment cycle, ICSI-AOA was performed on half of the sibling metaphase II oocytes and conventional ICSI on the rest
(‘split ICSI-AOA cycle’). The main outcome parameters were fertilization, pregnancy and live birth rates.
results: Overall, ICSI-AOA was able to improve fertilization rates in couples with a suspected oocyte-related fertilization problem, with
a mean fertilization rate of 74.2% following ICSI-AOA compared with 43.5% following conventional ICSI (P , 0.001). Cumulative pregnancy
rate and live birth rate per cycle were 35.7 and 14.3%, respectively. Considering the out-house patients only, fertilization rates with ICSI-AOA
were higher in couples with previous TFF than with conventional ICSI (P , 0.001). Interestingly, for out-house patients who had experienced
low, but not zero, fertilization elsewhere, ICSI-AOA could not enhance the fertilization rate. For the two in-house patients, both suffering from
previous LF following conventional ICSI, the ICSI-AOA procedure enhanced the mean fertilization rate (25 versus 75%, respectively).
conclusions: For patients with a suspected oocyte-related activation deficiency, as diagnosed by a heterologuous ICSI model, the
indication for ICSI-AOA still remains debatable. Our data show that ICSI-AOA is very efficient in patients with a suspected oocyte-
related activation deficiency and previous TFF after conventional ICSI. In contrast, when there was a history of LF in another centre, one
should be careful and test the efficiency of ICSI-AOA on half of the sibling oocytes, because ICSI-AOA is not always beneficial for patients
with previous LF and a suspected oocyte-related activation deficiency. For these patients, a split ICSI-AOA cycle using sibling oocytes can
help to distinguish between a molecular oocyte-related activation deficiency and a previous technical or other biological failure. Moreover,
this split ICSI-AOA strategy enables us to set the appropriate strategy for future treatment cycles. Further research with larger groups of
patients is now required.
Key words: failed fertilization / oocyte activation deficiency / assisted oocyte activation / ionophore / mouse oocyte activation test
ICSI is used in two-thirds of the artificial reproduction technology
(ART) cycles in European fertility clinics (Mouzon et al., 2010). ICSI
was developed in the early 1990s and millions of couples suffering
from severe male infertility or previously failed IVF conceived by this
technique. On average, ICSI leads to fertilization rates of approximate-
ly 70–80% (Palermo et al., 2009). Unfortunately, total fertilization
& The Author 2012. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Human Reproduction, Vol.27, No.7 pp. 1977–1984, 2012
Advanced Access publication on May 2, 2012doi:10.1093/humrep/des097
by guest on October 26, 2015
failure (TFF) still occurs in ?1–3% of all ICSI cycles and can recur in
subsequent cycles (Flaherty et al., 1995; Esfandiari et al., 2005). Failure
of oocyte activation is considered to be the main cause of fertilization
failure following conventional ICSI (Liu et al., 1995; Flaherty et al.,
1998; Rawe et al., 2000). Less common causes include failed sperm
head decondensation, premature sperm chromatin condensation,
spindle defects or sperm aster defects and incorrect sperm injection
(Swain and Pool, 2008). Furthermore, fertilization failure can be
caused by technical problems, a limited availability of mature or mor-
phologically normal oocytes, a lack of motile spermatozoa or severe
forms of teratozoospermia, such as globozoospermia (Yanagida
et al., 2004; Dam et al., 2007).
When a mammalian oocyte is activated, the first observed cytoplas-
mic event is an increase in intracellular calcium concentration. The
initial calcium increase (trigger) starts within a few minutes of
sperm–oocyte fusion in IVF, whereas following ICSI this trigger is pro-
voked immediately during the injection of the sperm by the artificial
calcium influx from the surrounding injection medium (Tesarik et al.,
2000). This trigger is followed by a typical pattern of calcium oscilla-
tions which are crucial for normal fertilization and further embryo de-
velopment (Ducibella et al., 2002; Parrington et al, 2007; Kashir et al.,
2010). Substantial evidence suggests that these calcium oscillations are
the result of the release of the sperm phospholipase C zeta (PLCz)
into the oocyte cytosol (Saunders et al., 2002; Kashir et al., 2010).
During final oocyte maturation, the ability to generate calcium oscilla-
tions is developed within the oocyte. This means that successful fertil-
ization also depends on the inherent quality and cytoplasmic maturity
of the oocyte (Ajduk et al., 2008). Hence, both the sperm and the
oocyte play a role in the oocyte activation mechanism, fertilization
and further embryo development: the sperm by supplying the
protein PLCz and the oocyte by its responsiveness to PLCz and down-
stream molecular pathways (Tesarik and Mendoza, 1999).
In our centre, a diagnostic test was developed to distinguish sperm-
from oocyte-related causes of fertilization failure (Rybouchkin et al.,
1996). The mouse oocyte activation test (MOAT) is offered to
patients with TFF or low fertilization (LF) following conventional
ICSI. Test sperm is injected by heterologous ICSI into mouse
oocytes, followed by assessment of the percentage of oocyte activa-
tion. The MOAT enabled the classification of patients into three
groups (Heindryckx et al., 2008). Patients with an oocyte activation
percentage of ,20%, being the upper limit of the negative control,
are classified as MOAT Group 1 patients. In these cases, a sperm-
related activation deficiency is extremely likely. In contrast, when
85% or more mouse oocytes are activated, being the lower limit of
the positive control, patients are classified as MOAT Group 3 patients.
In this group, a sperm deficiency can be excluded. Less distinct are
MOAT Group 2 patients, in which 21–84% of the mouse oocytes are
activated, pointing to either a sperm or an unknown deficiency.
Recently, there has been growing interest in the use of ICSI in com-
bination with assisted oocyte activation (ICSI-AOA) in patients suffering
from previous TFF or LF following conventional ICSI (Nasr-Esfahani
et al., 2010). Several activation agents, such as calcium ionophore,
strontium ions and electrical pulses, are efficient in restoring fertilization
and pregnancy rates by artificially provoking calcium rises in the cyto-
plasm of the oocyte (Yanagida et al., 1999, 2006; Eldar-Geva et al.,
2003; Chi et al., 2004; Heindryckx et al., 2005). Others reported the
use of a mechanically modified ICSI technique to overcome activation
failure (Tesarik et al. 2002; Ebner et al., 2004). Chemical activation
using the ionophore ionomycin is the most commonly used method
for AOA, resulting in high fertilization rates (Nasr-Esfahani et al., 2010).
In our centre, ICSI-AOA is performed using ionomycin, which
enables us to restore fertilization rates to normal in most patients
with a history of previously failed or LF (Heindryckx et al., 2005,
2008). The current study focuses on the patients with a normal
MOAT result, namely patients in MOAT Group 3, who are presenting
with TFF or LF probably caused by an oocyte-related activation pro-
blems. Our former policy was to perform ICSI-AOA on all available
oocytes following the MOAT in most of these patients. Nevertheless,
pregnancy results were lower in this group compared with patients in
whom a sperm activation deficiency (MOAT Group 1) or a diminished
oocyte activation capacity (MOAT Group 2) was found (Heindryckx
et al., 2008). To our knowledge, ‘split activation’ on part of the avail-
able metaphase II (MII) oocytes was firstly mentioned by Yanagida
(2004) in a review regarding fertilization failure following ICSI. Yana-
gida stated that ‘split activation’ could be performed when fertilization
failure is anticipated but did not discussed the validity of this method
since, at that time, no data were available on the subject. Therefore,
since 2006, we changed our policy for patients in MOAT Group 3 to
verify their real benefit of ICSI-AOA. We strongly advised convention-
al ICSI and split ICSI-AOA on sibling oocytes. The majority of patients
in MOAT Group 3 are referred to our centre because of a history of LF
or TFF following conventional ICSI elsewhere. By the split ICSI-AOA
policy, we aim to distinguish between a previous coincidental LF
rate or a real oocyte-related activation deficiency, in order to assess
the necessity of AOA in these patients. According to the fertilization
outcome of the split ICSI-AOA cycle, either conventional ICSI or
ICSI-AOA will be advised on all oocytes in the next treatment cycle.
The primary objective of this prospective case series is thus to
provide further insight into the efficiency of ICSI-AOA compared
with conventional ICSI in patients with a suspected oocyte-related
activation deficiency. Furthermore, we present a diagnostic and
therapeutic approach based on the results of the split ICSI-AOA
cycle for this rare but challenging group of patients.
Materials and Methods
Inclusion and exclusion criteria for patients
Couples with a history of failed or LF following conventional ICSI in whom
the MOAT revealed a possible oocyte-related activation deficiency
(MOAT result .84%) were eligible to be included in this prospective
case series. Patients were divided into two separate groups: the out-house
patients (referred patients) and in-house patients (own patients). All
patients were treated in our centre from January 2006 until December
2011. Controlled ovarian hyperstimulation was performed in all included
patients using the short GnRH agonist protocol. This protocol consisted
of daily injections of triptorelin (0.1 mg) starting on Day 3 of the cycle, fol-
lowed by gonadotrophin stimulation (112.5–300 IU) from Day 5 of the
cycle. Gonadotrophin stimulation was performed with recombinant FSH
or HMG. In each case ICSI-AOA was performed on half of the MII
oocytes, if six or more MII oocytes were available. Patients with less
than six MII oocytes available at retrieval were excluded from the analysis,
because in such cases the agreement was to perform ICSI-AOA on all
of their available MII oocytes. Main end-points were fertilization rate,
pregnancy rate and live birth rate.
Meerschaut et al.
by guest on October 26, 2015
MII mouse oocytes were collected from B6D2 F1 hybrid female mice after
ovulation induction by 10 IU pregnant mare’s serum gonadotrophin
(PMSG, Folligonw, Intervet, Boxmeer, The Netherlands), followed
46–48 h later by 10 IU hCG (Chorulonw, Intervet, Boxmeer, The Neth-
erlands). MII oocytes were collected 13–14 h following hCG adminis-
tration. Culture and handling media were potassium simplex optimized
medium (KSOM) and KSOM-HEPES respectively, both containing
0.2 mmol/l glucose and supplemented with 0.4% w/v bovine serum
albumin (BSAw, MP Biochemicals, Asse-Relegem, Belgium). Cumulus
cells were removed by a brief exposure to 200 IU hyaluronidase/ml.
Injection of immobilized frozen-thawed spermatozoa using piezo pulses
was performed at 15–178C in KSOM-HEPES supplemented with 20%
fetal bovine serum (FBSw, Gibco BRL, Carlsbad, USA). Four experimental
groups were set up: (i) ICSI with patients spermatozoa; (ii) ICSI with
donated spermatozoawith proven
control); (iii) sham ICSI (negative control) and (iv) non-manipulated
oocytes (medium control) to exclude spontaneous parthenogenetic
activation. Next, the oocytes were put in culture and 1 day later the
percentage of oocyte activation was determined by examining the
number of 2-cell embryos versus the number of surviving injected oocytes.
AOA and outcome
When six or more MII oocytes were available, ICSI-AOA was performed
on half of the sibling MII oocytes. When an odd number of oocytes was
available, ICSI-AOA was chosen for half plus one of the oocytes.
ICSI-AOA was performed at least 6 h after oocyte retrieval, as previously
described by Heindryckx et al. (2008). Briefly, a spermatozoon was
injected into the oocyte using conventional ICSI together with a small
amount of 0.1 mol/l CaCl2 (corresponding to the diameter of the
oocyte). Thereafter, the oocytes were incubated for 30 min at 378C in
a 6% CO2 air atmosphere. Next, the oocytes were exposed to a
calcium ionophore (10-mM ionomycin, I9657, Sigma-Aldrich, Bornem,
Belgium) for 10 min. Following this ionophore exposure, the oocytes
were washed with Cook Cleavage medium (Cook Ireland Ltd, Limerick,
Ireland) and were incubated again. After another 30 min, the calcium iono-
phore treatment was repeated for 10 min. Finally, the oocytes were
washed and incubated overnight. Fertilization was evaluated 16 h after
the ICSI procedure. Normal fertilized oocytes showed two pronuclei
and two polar bodies. The fertilization rate was defined by the number
of normally fertilized oocytes compared with the total number of injected
and surviving MII oocytes. Embryo transfer was carried out on Day 2 or
3. Pregnancy rate was defined by the number of clinical pregnancies (ges-
tational sac with fetal heartbeat at 6–7 weeks) as a proportion of the
number of fresh cycles. Birth rates were defined as the number of live
born babies compared with the proportion of clinical pregnancies.
The student’s t-test, x2test or Fisher’s exact test were used for statistical
analysis where appropriate using the Statistical Package for the Social
Sciences (SPSSwStatistics 19, IBM Corp., NY, USA). A P-value , 0.05
was considered statistically significant.
MOAT results and inclusion of patients
From January 2006 until December 2011, 74 MOATs were performed
in our centre. MOAT Group 1 (sperm deficiency confirmed) accounted
for 17.6% of the results and MOAT Group 2 (sperm deficiency sus-
pected) accounted for 16.2%. MOAT Group 3 (no sperm deficiency)
was confirmed in 66.2% of the cases. The reasons for exclusion
were the renouncement of any further treatment, signing up for
oocyte donation and conventional ICSI or ICSI-AOA on all of the
available oocytes (the latter options were taken at the request of
the patient; see Fig. 1).
Demographics, fertility history and semen
The mean age of the included women was 31.6+3.73 years. The age
of the two in-house patients was 30.0 and 30.2 years. The out-house
patients were split into two groups regarding (i) a history of TFF
(n ¼ 5) or (ii) a history of LF (n ¼ 7) following conventional ICSI.
The mean age in the TFF group was 33.4+3.05 years compared
with 31.3+4.23 years in the LF group (P ¼ 0.365). Thirteen of the
included women were of Caucasian descent and one couple
was Arab. Semen characteristics and fertility treatment history are
presented in Table I.
Overall fertilization and pregnancy rates
Following conventional ICSI 43.5% of the oocytes were fertilized,
whereas following ICSI-AOA the fertilization rate increased to
74.2% (P , 0.001). In total, 14 split ICSI-AOA cycles were performed
in this study population. As a result of these split ICSI-AOA cycles, five
pregnancies were achieved (overall pregnancy rate: 35.7%), of which
two ended in the birth of a healthy singleton (overall live birth rate:
14.3%) and one pregnancy is still ongoing. The fertilization history,
results and outcome are presented in Table II.
In-house cases with a history of LF rates
Two in-house cases (Cases 1 and 2) are included in this study (Supple-
mentary Table SI). Both cases showed LF in previous ICSI cycles.
ICSI-AOA was shown to be beneficial, with a mean fertilization rate
of 75.0% for ICSI-AOA compared with 25.0% for conventional ICSI
Figure 1 Overview of the MOAT results from January 2006 until
December 2011 and subsequent inclusion of study patients.
Indication for assisted oocyte activation
by guest on October 26, 2015
on sibling oocytes (P , 0.01). In one patient (Case 2) an ongoing
pregnancy was achieved and a healthy singleton was born.
Out-house cases with a history of TFF
Five out-house patients (Cases 3–7) consulted our centre with a history
of TFF in one or more ICSI cycles (Supplementary Table SII). Overall,
ICSI-AOA was beneficial, with a mean fertilization rate of 72.7% (24/
33) for ICSI-AOA compared with 25.0% (7/28) for conventional ICSI
(P , 0.001). One of these patients had a miscarriage following the
transfer of a single ICSI-AOA embryo.
Out-house patients with a history of LF rates
Seven out-house patients (Cases 8–14) were referred to our centre
with a history of LF in one or more ICSI cycles (Supplementary
Table SIII). The previous mean fertilization rate was 21.4%. Overall,
ICSI-AOA was not beneficial, with a mean oocyte fertilization rate
of 75.0% (39/52) compared with 60.4% (29/48) for conventional
ICSI (P ¼ 0.118). Three patients became pregnant during this split
ICSI-AOA cycle. One pregnancy ended in a miscarriage, one resulted
in the birth of a healthy singleton and one pregnancy is still ongoing.
Diagnostic and therapeutic algorithm
Figure 2 represents a diagnostic and therapeutic algorithm based on
the outcomes of the above described groups. From 2003 until May
2011, the mean fertilization rate of 13136 ICSI cycles registered in
our centre was 73.7+22.72%. The 5th, 25th, 50th and 75th percen-
tiles were 33.3, 62.5, 76.92 and 90.0%, respectively. Based on this, in
our centre, abnormal LF is now considered to be a recurrent fertiliza-
tion rate of 33.3% or less. A MOAT is thus proposed when the fertil-
ization rate after ICSI is ,33.3%. According to the result of the
MOAT, patients are classified into three MOAT groups. For MOAT
Groups 1 and 2, ICSI-AOA is performed on all oocytes. For patients
in MOAT Group 3 with a history of TFF, ICSI-AOA is performed on
all oocytes. For patients in MOAT Group 3 with a history of LF, a
‘split ICSI-AOA cycle’ is proposed. According to the fertilization
results of this split cycle, the decision is made to perform conventional
ICSI or ICSI-AOA on all available oocytes in future cycles.
ICSI-AOA has proved to be a very efficient technique in cases of
recurrent TFF or LF rates after ICSI (Nasr-Esfahani et al., 2010).
The MOAT is a diagnostic tool which helps to predict the usefulness
of ICSI-AOA in a subsequent cycle and helps to diagnose sperm-
related activation deficiencies (Rybouchkin et al., 1996; Yanagida
et al., 1999; Tesarik et al., 2002; Araki et al., 2004; Heindyckx et al.,
2005, 2008). ICSI-AOA with ionophore or electrical pulses is success-
ful in restoring fertilization and pregnancy rates by manipulating the
intracellular calcium concentration (Yanagida et al., 1999; Eldar-Geva
et al. 2003; Murase et al., 2004; Heindryckx et al., 2005). To our
knowledge, this is the first study to test the efficiency of ICSI-AOA
compared with conventional ICSI on sibling oocytes of patients with
a possible oocyte-related activation deficiency, as diagnosed by the
MOAT, namely patients in MOAT Group 3. As this technique is still
considered to be experimental, research to assess the beneficial
In-house cases (both LF)
Case 1 21.106
Case 3 14.106
Case 10 1.106
Table I Semen characteristics and treatment history of the included patients.
Semen characteristics at MOAT Sperm concentration
A 1 B (%)
Sperm morphology% normalTreatment history
IUI, IVF, ICSI
IUI, IVF, ICSI
IUI, IVF, ICSI
IUI, IVF, ICSI
TFF, total fertilization failure after previous conventional ICSI; LF, low fertilization after previous conventional ICSI (considered to be 33.3% or less, based on the 5th percentile of mean
IUI, intrauterine insemination;
MOAT, mouse oocyte activation test.
Meerschaut et al.
by guest on October 26, 2015
effect of, and necessity for, ICSI-AOA in this specific patient group
remains highly important.
Owing to practical considerations, the MOAT was performed using
frozen-thawed human sperm. Cryopreservation is widely known to
raise impaired sperm motility and decrease fertilization rates through
detrimental effects on membranes, acrosomal structure and acrosin
activity (Cross and Hanks, 1991). Nevertheless, we consider our
MOAT results to be reliable because only motile spermatozoa with
the best morphology were used for heterologous ICSI. Moreover,
data on fertilization and pregnancy rates after ICSI comparing fresh
and frozen-thawed human ejaculated spermatozoa are reassuring:
no statically significant differences were found between fresh and
frozen-thawed sperm in fertilization rates (Kuczyn ´ski et al., 2001). Fur-
thermore, we did not find any differences in calcium releasing ability
after heterologous ICSI when fresh sperm were compared with
frozen-thawed sperm from the same patient (unpublished data).
The mean age of the 14 included women in this study is 31.6 years.
The age did not differ between the in-house compared with the out-
house cases, neither did the age differ between the out-house group
with TTF in their history compared with the out-house group with
LF in their history. Several authors have reported on the age-related
decline in the success of IVF/ICSI cycles. Most likely this lies in the
progressively diminished ovarian reserve, with a decreasing quantity
and quality of oocytes (Broekmans et al., 2007; van Loendersloot
et al., 2010). However, in this relatively young study population, it is
plausible that age did not yet influence oocyte quality.
Two in-house patients with previous LF rates were included in this
study. It is clearly shown that ICSI-AOA was clinically and statistically
(albeit for only two patients) beneficial compared with conventional
ICSI for both cases. This emphasizes the presence of a real
oocyte-related activation deficiency in these in-house patients, rather
than a previous technical failure. Unfortunately, in Case 1, although
ICSI-AOA was efficient in improving the fertilization rate, neither the
split ICSI-AOA cycle nor two subsequent 100% ICSI-AOA cycles
led to a pregnancy. Even after several oocyte donation cycles, no
ongoing pregnancy was achieved. So, it is clear that this patient
suffers from severe underlying female factor infertility, and whether
Table II Fertilization and pregnancy rates before and after the split ICSI-AOA cycle.
Fertilization rate before
Split cycle fertilization ratea
Live birth ratec
Source of embryos
transferred leading to
In-house LF (n ¼ 2)
29.3 (19.2 and 37.5) (17/58)
75.0 (57.1–88.9) (12/16)*
25.0 (14.3–33.3) (4/16)*
1× mixed DETd
Out-house TFF (n ¼ 5)
0 (0–0) (0/54)
72.7 (28.6–90.9) (24/33)†
25.0 (0.0–100) (7/28)†
1× AOA SETd
Out-house LF (n ¼ 7)
21.4 (6.3–35.1) (27/126)
75.0 (40.0–100) (39/52)‡
60.4 (33.3–80.0) (29/48)‡
2× mixed DET, 1× AOA SET
17.7 (0–37.5) (44/248)
74.2 (28.6–100) (75/101)$
43.5 (0.0–100) (40/92)$
AOA, assisted oocyte activation.
aValues are percentage (range) (number of normally fertilized oocytes/number of injected metaphase II oocytes).
bValues are percentage (number of clinical pregnancies/number of fresh cycles).
cValues are percentage (number of live births/number of fresh cycles).
dMixed DET ¼ double embryo transfer of one ICSI-AOA embryo and one conventional ICSI embryo; AOA SET ¼ single embryo transfer of an ICSI-AOA embryo.
eOne pregnancy is still ongoing.
*P , 0.01 (Pearson x2test).
†P , 0.001 (Pearson x2test).
‡P ¼ 0.118 (Pearson x2test).
$P , 0.001 (Pearson x2test).
Figure 2 Diagnostic and therapeutic approach of patients with pre-
vious total fertilization failure or LF rates following conventional ICSI.
Indication for assisted oocyte activation
by guest on October 26, 2015
this infertility is strictly oocyte related or not remains debatable. Case
2 did also benefit from ICSI-AOA according to fertilization rate,
although not as explicitly as compared with Case 1. In Case 2, a struc-
tural oocyte defect lies at the basis of the history of the LF: as
described by Heindryckx et al. (2008), a spindle defect was suspected
owing to (i) the absence of spindle visualization using polarized micros-
copy and (ii) commonly seen formation of multiple pronucleii and
multinucleated embryos following IVF, conventional ICSI as well as
ICSI-AOA. Previous studies on unfertilized oocytes in IVF cycles
have revealed the presence of abnormal spindle and interphase micro-
tubules, indicating that deficiencies in ooplasmic components may be a
cause of failed fertilization (Kovacic and Vlaisavljevic, 2000; Rawe et al.,
2000). A very interesting case was recently reported by Combelles
et al. (2010). In this case, no fertilization was seen following conven-
tional ICSI. Subsequently, heterologuous ICSI showed a normal
ability of the husband’s sperm to activate mouse oocytes. Next,
ICSI-AOA was performed on all oocytes after ICSI with half of the
sperm from the husband and half from a donor. None of the injected
oocytes in either group was fertilized. Thus, in contrast to both our
in-house patients, the oocyte-related activation failure in the case
described by Combelles et al. (2010) could not be rescued by
ICSI-AOA. Analysis of the cytoskeletal and chromatin organization
of the unfertilized oocytes revealed severe cytoplasmic abnormalities
across the cohort of oocytes. It is obvious that further research is
necessary to find other treatment options for such patients.
The patients from the out-house TFF group showed a significant
benefit from ICSI-AOA over conventional ICSI. This underlines the
probability of an oocyte-related activation deficiency of unknown
source in this group, which can be rescued by ICSI-AOA. It is well
known that nuclear as well as cytoplasmic maturation is crucial for
oocytes before being able to respond properly to the sperm PLCz
during the fertilization process (Swain and Pool, 2008). Thus, success-
ful fertilization depends also on the inherent quality of the oocyte,
which correlates strongly with successful oocyte maturation. The
ability to generate calcium oscillations requires several cytoplasmic
changes: reorganization of the endoplasmic reticulum (ER), an increase
in the number of inositol-3-phosphate receptors (IP3Rs), changes in
the biochemical properties of the IP3Rs (sensitivity to IP3), an increase
in the concentration of Ca2+ions stored in ER and redistribution of
Ca2+-binding ER proteins (Goud et al., 1999; Goud et al., 2002;
Ajduk et al., 2008; Vanderheyden et al., 2009). As our ICSI-AOA
protocol using CaCl2 and ionomycin artificially provokes some
calcium rises in the oocyte cytoplasm, it is likely that an underlying
cytoplasmic defect (related to the Ca2+-releasing machinery of the
oocyte) is the reason for the previous TFF in the out-house patients,
rather than a structural cytoskeletal-related or nuclear defect.
In contrast to the out-house TFF group, the patients from the out-
house LF group showed no significant benefit from ICSI-AOA. More-
over, Cases 8, 9 and 13 had pregnancies, four miscarriages and one
ongoing pregnancy, with the same male partner before being referred
to our centre. Interestingly, all but one of the out-house LF group had
acceptable fertilization rates in our centre (considering the .33.3%
threshold for normal fertilization) when conventional ICSI was
applied as part of the split cycle. This underlines even more that the
indication for ICSI-AOA in this group should not be made implicitly
and that a split cycle could be advised instead of 100% AOA. The
aim of the split ICSI-AOA cycle policy was thus to distinguish
between a molecular oocyte-related activation deficiency from a pre-
vious technical or other biological failure. Nevertheless, the absence of
benefit from ICSI-AOA in this group leads to the assumption that
either a previous technical (and neither an oocyte nor a sperm defi-
ciency) or an unidentified temporary biological failure could have led
to LF rates elsewhere. Although ICSI is considered to be a routine
technique, it remains one of the most demanding techniques for
embryologists to master. It is not unusual for any embryologist to ex-
perience a dip in performance following the acquisition of proficiency
in the technique. A comprehensive investigation of factors that influ-
ence fertilization rates after ICSI, found that the ICSI embryologist
conducting the procedure was a significant predictor of success,
while laboratory conditions, such as incubators or storage of eggs
individually versus grouped, did not affect the fertilization rates
(Shen et al., 2003).
The factors leading to fertilization failure are complex and may
involve either sperm- or oocyte-related activation deficiencies, which
can be indicated by the MOAT result and overcome with ICSI-AOA
(Heindryckx et al., 2008). It is important to keep in mind that the
human PLCz has a higher activation potential compared with mouse
PLCz on mouse oocytes (Cox et al., 2002; Ito et al., 2008). The
latter needs to be considered when interpreting a MOAT result,
because the activation rate in mouse oocytes cannot be extrapolated
as such to human oocytes. Furthermore, factors leading to fertilization
failure may also relate to cycle-specific parameters and the number
and quality of the mature oocytes, as well as the availability of
normal motile sperm (Liu et al., 1995; Flaherty et al., 1998, Kovacic
and Vlaisavljevic, 2000, Esfandiari et al., 2005, Swain and Pool, 2008).
Although ICSI-AOA is considered a very efficient technique to over-
come fertilization failure, this is not true for all cases of fertilization
failure. A previous study has showed that AOA is more efficient to
overcome sperm-related activation deficiencies, as diagnosed by the
MOAT, rather than suspected oocyte-related activation deficiencies
(Heindryckx et al., 2008). In the study of Heindryckx et al., (2008),
patients from all three MOAT groups were included and the main
treatment strategy was 100% ICSI-AOA. Since in this patient series
only one MOAT group 3 couple received split ICSI-AOA, the validity
of the split ICSI-AOA strategy could not be investigated. Nevertheless,
it was demonstrated that although fertilization rates could be restored
by ICSI-AOA for all MOAT groups, the clinical pregnancy rates in
MOAT Group 3 patients were lower compared with MOAT Group 1
and 2 patients (17 versus 34 and 43%, respectively). The latter is con-
firmed by the current study, with a mean pregnancy rate and birth rate
of 35.7 and 14.3%, respectively.
In order to increase the efficiency of AOA in some subgroups of
patients (for example, with extreme oligoasthenoteratozoospermia
or when an oocyte factor is responsible for the failed ICSI), injection
of PLCz cRNA or recombinant PLCz protein might offer a better
alternative as these provide a more physiological stimulus than iono-
phore. Furthermore, the use of ionophore is still experimental
because of insufficient knowledge about the potential cytotoxic,
teratogenic and mutagenic effects on embryos and offspring. In previ-
ous animal research, no adverse effects of ionomycin on in-vitro or
in-vivo mouse embryo development were noticed, giving arguments
in favour of the use of ionomycin (Heytens et al., 2008). On the
Meerschaut et al.
by guest on October 26, 2015
contrary, no long-term follow-up studies of children born after
ICSI-AOA using ionomycin are yet available. Therefore, ICSI-AOA
should not be performed without a proper indication in patients
with previous LF, nor should it be used as an ultimate resort when
other ARTs have failed. In this respect, it is advisable that patients
that might benefit from a treatment with AOA after previously
having TFF or LF after ICSI, first seek a diagnostic test, such as the
MOAT, to be able to confirm whether a sperm-borne activation de-
ficiency is present (Rybouchkin et al., 1996; Araki et al., 2004).
Recent studies have applied ionophore treatment or electrical stimu-
lation for infertile patients with no obvious indication, because in
these patients the fertilization and pregnancy rates were comparable
with or without AOA (Mansour et al., 2008; Borges et al., 2009b).
In one study, AOA was even applied in the first cycle ICSI attempt
(Borges et al., 2009a).
The primary objective of this follow-up study was to provide further
insight in the necessity of ICSI-AOA in patients in MOAT Group 3 and
its efficiency with respect to fertilization and pregnancy rates. The low
sample number and heterogeneity of the study population might inter-
fere with the predictive value of this study. Nevertheless, we are
dealing with a very rare but challenging group of patients, for which
we hope this study can set guidelines. The proposed diagnostic and
therapeutic algorithm based on the outcomes of this study enables
the evidence-based counseling of couples suffering from TFF or LF
after ICSI. A MOAT, or similar heterologous ICSI test, should
always be recommended when the previous ICSI fertilization rate
was ,33.3% before ICSI-AOA is considered. This threshold is
chosen because 33.3% is the 5th percentile of the fertilization rate
after ICSI in our centre. Next, if the MOAT shows a normal result,
any further treatment should depend on the fertility history. If the pre-
vious fertilization rate was 0%, ICSI-AOA is advisable on all available
MII oocytes. In contrast, if the previous fertilization rate was
,33.3%, but not zero, the best strategy is to perform ICSI-AOA on
half of the sibling oocytes to distinguish between a previous coinciden-
tal LF rate or a real oocyte-related activation deficiency. When a
future cycle is necessary, AOA should be performed on all or none
of the available MII oocytes according to the fertilization results of
the split cycle. Finally, if any structural oocyte defect is suspected, non-
invasive spindle evaluation on fresh MII oocytes by means of polarized
light microscopy or invasive spindle evaluation on spare (MI or unfer-
tilized) oocytes by means of immunostaining should be considered.
Thus, taking into consideration the history of the couple, a split
ICSI-AOA cycle on sibling oocytes, together with additional diagnostic
approaches, such as spindle evaluation of the oocytes, is essential to
select the proper therapeutic strategy in these cases. Based on the
limited data available, we were able to claim that the fertility history
of the patients in MOAT Group 3 plays a critical role in choosing the
appropriate diagnostic and therapeutic approach. Furthermore, it
was shown that ICSI-AOA is not beneficial for all patients with a sus-
pected oocyte-related activation deficiency. As ICSI-AOA is still an
elaborate and experimental technique, it should only be considered
in case of a well-diagnosed indication.
Supplementary data are available at http://humrep.oxfordjournals.
F.V.M. analysed the data, interpreted the results and wrote the manu-
script; D.N. contributed to interpreting the results; S.D, V.D. and B.H.
performed the assisted oocyte activation; E.V.A and J.G. revised the
manuscript; B.H. and P.D.S. designed the study, revised the manu-
script and approved the final draft.
F.V.M. is the holder of an aspirant clinical research mandate by the
Flemish Foundation of Scientific Research (FWO-Vlaanderen). P.D.S.
is the holder of a fundamental clinical research mandate by the
same Flemish foundation of Scientific Research (FWO-Vlaanderen).
Conflict of interest
Ajduk A, Malagocki A, Maleszewski M. Cytoplasmic maturation of
mammalian oocytes: development of a mechanism responsible for
sperm-induced Ca2+ oscillations. Reprod Biol 2008;8:3–22.
Araki Y, Yoshizawa M, Abe H, Murase Y, Araki Y. Use of mouse oocytes
to evaluate the ability of human sperm to activate oocytes after failure of
Borges E Jr, de Almeida Ferreira Braga DP, de Sousa Bonetti TC,
Iaconelli A Jr, Franco JG Jr. Artificial oocyte activation using calcium
ionophore in ICSI cycles with spermatozoa from different sources.
Reprod Biomed Online 2009a;18:45–52.
Borges E Jr, de Almeida Ferreira Braga DP, de Sousa Bonetti TC,
Iaconelli A Jr, Franco JG Jr. Artificial oocyte activation with calcium
ionophore A23187 in intracytoplasmic sperm injection cycles using
surgically retrieved spermatozoa. Fertil Steril 2009b;92:131–136.
Broekmans FJ, Knauff EA, te Velde ER, Macklon NS, Fauser BC. Female
reproductive ageing: current knowledge and future trends. Trends
Endocrinol Metab 2007;18:58–65.
Chi HJ, Koo JJ, Song SJ, Lee JY, Chang SS. Successful fertilization and
pregnancy after intracytoplasmic sperm injection and oocyte activation
with calcium ionophore in a normozoospermic patient with extremely
low fertilization rates in intracytoplasmic sperm injection cycles. Fertil
Combelles CM, Morozumi K, Yanagimachi R, Zhu L, Fox JH, Racowsky C.
Diagnosing cellular defects in an unexplained case of total fertilization
failure. Hum Reprod 2010;25:1666–1671.
Cox LJ, Larman MG, Saunders CM, Hashimoto K, Swann K, Lai FA. Sperm
phospholipase Czeta from humans and cynomolgus monkeys triggers
Ca2+ oscillations, activation and development of mouse oocytes.
Cross NL, Hanks SE. Effects of cryopreservation on human sperm
acrosomes. Hum Reprod 1991;6:1279–1283.
Dam AH, Feenstra I, Westphal JR, Ramos L, van Golde RJ, Kremer JA.
Globozoospermia revisited. Hum Reprod Update 2007;13:63–75.
Ducibella T, Huneau D, Angelichio E, Xu Z, Schultz RM, Kopf GS,
Fissore R, Madoux S, Ozil JP. Egg-to-embryo transition is driven by
differential responses to Ca(2+) oscillation number. Dev Biol 2002;
sperm injection. Zygote 2004;
Indication for assisted oocyte activation
by guest on October 26, 2015
Ebner T, Moser M, Sommergruber M, Jesacher K, Tews G. Complete
oocyte activation failure after ICSI can be overcome by a modified
injection technique. Hum Reprod 2004;19:1837–1841.
Eldar-Geva T, Brooks B, Margalioth EJ, Zylber-Haran E, Gal M, Silber SJ.
Successful pregnancy and delivery after calcium ionophore oocyte
activation in a normozoospermic patient with previous repeated failed
fertilization after intracytoplasmic sperm injection. Fertil Steril 2003;
Esfandiari N, Javed MH, Gotlieb L, Casper RF. Complete failed fertilization
after intracytoplasmic sperm injection—analysis of 10 years’ data. Int J
Fertil Womens Med 2005;50:187–192.
Flaherty SP, Payne D, Swann NJ, Mattews CD. Aetiology of failed and
abnormal fertilization after intracytoplasmic sperm injection. Hum
Flaherty SP, Payne D, Matthews CD. Fertilization failures and abnormal
fertilization after intracytoplasmic sperm injection. Hum Reprod 1998;
Goud PT, Goud AP, Van Oostveldt P, Dhont M. Presence and dynamic
redistribution of type I inositol 1,4,5-trisphosphate receptors in
human oocytes and embryos during in-vitro maturation, fertilization
and early cleavage divisions. Mol Hum Reprod 1999;5:441–51.
Goud PT, Goud AP, Leybaert L, Van Oostveldt P, Mikoshiba K,
Diamond MP, Dhont M. Inositol 1,4,5-trisphosphate receptor function
in human oocytes: calcium responses and oocyte activation-related
phenomena induced by photolytic release of InsP3are blocked by a
specific antibody to the type I receptor. Mol Hum Reprod 2002;
Heindryckx B, Van der Elst J, De Sutter P, Dhont M. Treatment option for
sperm- or oocyte-related fertilization failure: assisted oocyte activation
following diagnosticheterologous ICSI.HumReprod2005;20:2237–2241.
Heindryckx B, De Gheselle S, Gerris J, Dhont M, De Sutter P. Efficiency of
assisted oocyte activation as a solution for failed intracytoplasmic sperm
injection. Reprod Biomed Online 2008;17:662–668.
Heytens E, Soleimani R, Lierman S, De Meester S, Gerris J, Dhont M, Van
der Elst J, De Sutter P. Reprod Biomed Online 2008;17:764–771.
Ito M, Shikano T, Oda S, Horiguchi T, Tanimoto S, Awaji T, Mitani H,
Miyazaki S. Difference in Ca2+ oscillation-inducing activity and
nuclear translocation ability of PLCZ1, an egg-activating sperm factor
candidate, between mouse, rat, human, and medaka fish. Biol Reprod
Kashir J, Heindryckx B, Jones C, De Sutter P, Parrington J, Coward K.
Oocyte activation, phospholipase C zeta and human infertility. Hum
Reprod Update 2010;16:690–703.
Kovacic B, Vlaisavljevic V. Configurationofmaternalandpaternalchromatin
and pertaining microtubules in human oocytes failing to fertilize after
intracytoplasmic sperm injection. Mol Reprod Dev 2000;55:197–204.
Kuczyn ´ski W, Dhont M, Grygoruk C, Grochowski D, Wołczyn ´ski S,
Szamatowicz M. The outcome of intracytoplasmic injection of fresh
randomized study. Hum Reprod 2001;16:2109–2113.
Liu J, Nagy Z, Joris H, Tournaye H, Smitz J, Camus M, Devroey P, Van
Steirteghem A. Analysis of 76 total fertilization failure cycles out of
2732 intracytoplasmic sperm injection cycles. Hum Reprod 1995;
Mansour R, Fahmy I, Tawab NA, Kamal A, El-Demery Y, Aboulghar M,
Serour G. Electrical activation of oocytes after intracytoplasmic
sperm injection: a controlled randomized study. Fertil Steril 2009;
Mouzon J, Goossens V, Bhattacharya S, Castilla JA, Ferraretti AP, Korsak V,
Kupka M, Nygren KG, Nyboe Andersen A. Assisted reproductive
technology in Europe, 2006: results generated from European
registers by ESHRE. European IVF-monitoring (EIM) Consortium, for
the European Society of Human Reproduction and Embryology
(ESHRE). Hum Reprod 2010;25:1851–1862.
Murase Y, Araki Y, Mizuno S, Kawaguchi C, Naito M, Yoshizawa M,
Araki Y. Pregnancy following chemical activation of oocytes in a
couple with repeated failure of fertilization using ICSI: case report.
Hum Reprod 2004;19:1604–1607.
Nasr-Esfahani MH, Deemeh MR, Tavalaee M. Artificial oocyte activation
and intracytoplasmic sperm injection. Fertil Steril 2010;94:520–526.
Palermo GD, Neri QV, Takeuchi T, Rosenwaks Z. ICSI: where we have
been and where we are going. Semin Reprod Med 2009;27:191–201.
Parrington J, Davis LC, Galione A, Wessel G. Flipping the switch: how a
sperm activates the egg at fertilization. Dev Dyn 2007;236:2027–2038.
Rawe VY, Olmedo SB, Nodar FN, Doncel GD, Acosta AA, Vitullo AD.
Cytoskeletal organization defects and abortive activation in human
oocytes after IVF and ICSI failure. Mol Hum Reprod 2000;6:510–516.
Rybouchkin A, Dozortsev D, Pelinck MJ, De Sutter P, Dhont M. Analysis of
the oocyte activation capacity and chromosomal complement of
roundheaded human spermatozoa by their injection into mouse
oocytes. Human Reprod 1996;11:2170–2175.
Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM,
Swann K, Lai FA. PLC zeta: a sperm-specific trigger of Ca(2+)
oscillations in eggs and embryo development. Development 2002;
Shen S, Khabani A, Klein N, Battaglia D. Statistical analysis of factors
affecting fertilization rates and clinical outcome associated with
intracytoplasmic sperm injection. Fertil Steril 2003;79:355–360.
Swain JE, Pool TB. ART failure: oocyte contributions to unsuccessful
fertilization. Hum Reprod Update 2008;14:431–446.
Tesarik J, Mendoza C. In vitro fertilization by intracytoplasmic sperm
injection. Bioessays 1999;21:791–801.
Tesarik J, Mendoza C, Greco E. The activity (calcium oscillator?)
responsible for human oocyte activation after injection with round
spermatids is associated with spermatid nuclei. Fertil Steril 2000;
Tesarik J, Rienzi L, Ubaldi F, Mendoza C, Greco E. Use of a modified
intracytoplasmic sperm injection technique to overcome sperm-borne
and oocyte-borne oocyte activation failures. Fertil Steril 2002;
Vanderheyden V, Wakai T, Bultynck G, De Smedt H, Parys JB, Fissore RA.
Regulation of inositol 1,4,5-trisphosphate receptor type 1 function
during oocyte maturation by MPM-2 phosphorylation. Cell Calcium
van Loendersloot LL, van Wely M, Limpens J, Bossuyt PMM, Repping S,
van der Veen F. Predictive factors in in vitro fertilization (IVF): a
systematic review and meta-analysis. Human Reprod Update 2010;
Yanagida K. Complete fertilization failure in ICSI. Hum Cell 2004;
Yanagida K, Katayose H, Yazawa H, Kimura Y, Sato A, Yanagimachi H,
Yanagimachi R. Successful fertilization and pregnancy following ICSI
and electrical oocyte activation. Hum Reprod 1999;14:1307–1311.
Yanagida K, Morozumi K, Katayose H, Hayashi S, Sato A. Successful
pregnancy after ICSI with strontium oocyte activation in low rates of
fertilization. Reprod Biomed Online 2006;13:801–806.
Meerschaut et al.
by guest on October 26, 2015