497 RBMO VOLUME 38 ISSUE 4 2019

ARTICLE

Assessment of the calcium releasing machinery

in oocytes that failed to fertilize after
conventional ICSI and assisted oocyte activation
BIOGRAPHY
Minerva Ferrer-Buitrago graduated with a BSc in Biochemistry from the University of
Valencia (Spain) and holds a PhD in Health Sciences from the University of Ghent
(Belgium). Minerva developed her career as a clinical embryologist at CREA Valencia
(Spain), where she currently coordinates research and training programmes for clinical
embryologists.
1 1 1
Minerva Ferrer-Buitrago , Davina Bonte , Lien Dhaenens ,
2 1 1 1,
Sanne Vermorgen , Yuechao Lu , Petra De Sutter , Björn Heindryckx *

KEY MESSAGE
2+ 2+
Oocyte Ca analysis can be used to reveal or refute Ca releasing deficiencies in oocytes experiencing
2+
fertilization failure after ICSI which, in some cases, cannot be overcome by Ca ionophores during
assisted oocyte activation.
ABSTRACT
Research question: Can oocyte-related activation deficiencies be evaluated in oocytes that failed to fertilize
after intracytoplasmic sperm injection (ICSI) combined with assisted oocyte activation (AOA)?
Design: Evaluation of the spindle–chromosome complexes and intracellular distribution of inositol trisphosphate
type 1 receptors (IP3R1) in in-vitro matured (IVM) and failed-to-fertilize oocytes from patients undergoing AOA.
2+ 2+
Assessment of the oocyte-related Ca releasing capacity in response to Ca ionophores and sperm microinjection
in oocytes that failed to fertilize after ICSI or ICSI-AOA.
Results: IVM oocytes from patients undergoing conventional ICSI (control) and ICSI-AOA (study group) revealed a similar
normalcy of spindle–chromosome complexes and distribution patterns of IP3R1. Failed-to-fertilize oocytes from both groups
showed significant differences in proportion of normal or abnormal spindle–chromosome complex conformations. However,
migration of IP3R1 was identified in a higher proportion of failed-to-fertilize oocytes after ICSI-AOA than after conventional
ICSI. It was further observed that oocytes which failed to fertilize, either after ICSI or ICSI-AOA, mostly retain their capacity
2+
to respond to stimuli such as exposure to Ca ionophores or to sperm microinjection.
Conclusions: Evaluation of spindle–chromosome normalcy and distribution of IP3R1 does not help identify the
2+ 2+
presence of Ca releasing deficiencies in these oocytes. However, oocyte Ca analysis adds value in identifying
2+
Ca releasing incapacity of oocytes that failed to fertilize after ICSI or ICSI-AOA. Some patients experiencing
2+
fertilization failure after ICSI-AOA present with a suspected activation deficiency downstream of the Ca machinery,
2+
which cannot be overcome by ICSI-AOA based on the use of Ca ionophores.
1 
Ghent -Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University KEYWORDS
Hospital, Ghent, Belgium Assisted oocyte activation
2 
Ghent University (UGent Honours Programme in Life Sciences), Ghent, Belgium Calcium
Fertilization failure
© 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. E-mail address: bjorn.heindryckx@UGent.be (B Heindryckx). https://doi.org/10.1016/j. ICSI
rbmo.2018.12.035 1472-6483/© 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. Intracytoplasmic sperm injection
Declaration: The authors report no financial or commercial conflicts of interest.
498 RBMO VOLUME 38 ISSUE 4 2019
O INTRDUCTION
ocyte activation is a Ca -
2+
dependent process
(Horner and Wolfner,
2008), which initiates upon
gamete
fusion. The sperm delivers the sperm-
related activation factor phospholipase
C zeta (PLCζ) (Swann and Lai, 2016),
which hydrolyses the non-plasma
membrane bound phosphatidylinositol
bisphosphate (PIP2) (Yu et al., 2012)
into two products: diacylglycerol (DAG),
which initiates a signalling pathway via
protein kinase C (PKC) (Halet, 2004)
and inositol trisphosphate (IP3), which
2+
further stimulates the discharge of Ca
from the endoplasmic reticulum through
its cognate receptor (IP3R1) present in
the ooplasm (Jellerette
et al., 2000). The production of IP3
further stimulates the coordination of
2+
Ca releasing machinery of the oocyte
(Martin-Romero et al., 2007; Wakai et
al., 2013), which ensures the
2+
maintenance of long-term Ca
oscillations (Whitaker, 2006). Thus, both
gametes, sperm and oocyte, play
pivotal roles during the process of
oocyte activation, which is necessary for
a successful fertilization. Hence,
deficiencies observed either in the
testis-specific factor PLCζ or in the Ca
2+
releasing mechanisms such as IP3R1,
are likely to contribute to fertilization
failures (Yeste et al., 2017).
Experiencing fertilization failure after
treatment with assisted reproductive
technology is an intensely distressing
event, which leaves couples with little
perspective for future treatments
(Verhaak et al., 2005). To improve the
chances of treatment success after
experiencing recurrent fertilization
failures, it is crucial to identify the
associated gamete origin. Gamete
donation is sometimes proposed as
an option for patients experiencing
fertilization failure after intracytoplasmic
sperm injection (ICSI); however, some
couples might not be willing to accept
gamete donation as a first alternative.
When an oocyte activation deficiency
is involved, assisted oocyte activation
(AOA) may be proposed as a treatment
to overcome fertilization failures after
ICSI (Ebner et al., 2015). The AOA
protocols that are currently in use consist
of mediating a replacement of enough
2+
intracytoplasmic Ca required for
fertilization, commonly induced by the
2+
use of Ca ionophores.
As the availability of human oocytes that
are donated for research purposes is
limited (Araki et al., 2004; Rybouchkin et
al., 1995), the development of heterologous
ICSI has been used to determine the
activation potential of human spermatozoa
(Talansky et al., 1991; Yazawa et al., 2000).
For instance, the mouse oocyte activation
test (MOAT) is a diagnostic tool to evaluate
the activation potential of spermatozoa, by
injecting human spermatozoa into mouse
oocytes (Rybouchkin et al., 1995). The
MOAT result is based on the percentage of
2-cell embryos observed 24 h post-ICSI
(Heindryckx et al., 2005), and allows the
classification of spermatozoa into three
categories: low (MOAT 1, ≤20%),
moderate (MOAT 2, 21–84%) and normal
(MOAT 3, ≥85%) activation potential,
in comparison to a normal fertilizing control
spermatozoa. In line with these
observations, the effect of AOA for
overcoming failed fertilization would be
more pronounced in cases showing
2+
deficient cytoplasmic Ca changes, thus
when the presence of sperm-related
activation deficiencies is revealed by the
MOAT. Indeed, AOA may help in
overcoming fertilization failure particularly
in cases with severe sperm-related oocyte
activation deficiencies (MOAT 1), such as
patients with globozoospermia (Kuentz et
al., 2013; Yassine et al.,
2014) or PLCζ mutation carriers (Kashir et
al., 2012). Interestingly, AOA also improved
fertilization rates in patients with moderate
(MOAT 2) or normal (MOAT 3) sperm
activation capacity (Vanden Meerschaut et
al., 2012). In the latter group, an oocyte-
related activation deficiency was
suspected, showing a lower benefit of AOA
than in the MOAT group 1 and 2 (Vanden
Meerschaut et al., 2012) in terms of
pregnancy outcome. More recently, the
2+
analysis of Ca oscillatory responses
induced into mouse oocytes by human
spermatozoa was shown to be more
sensitive in identifying sperm-related
oocyte activation deficiencies than the
MOAT (Vanden Meerschaut et al., 2013). It
should be noted that despite similarities in
the mechanisms of action of PLCζ (Coward
et al., 2011; Cox et al., 2002), human PLCζ
has greater potency to activate mouse
oocytes than mouse PLCζ (Nomikos et al.,
2014). Hence, it was postulated that
spermatozoa from some patients, who
showed normal activation capacity after the
MOAT, may not be able to induce oocyte
activation in human oocytes. To overcome
this, human oocyte
2+
Ca analysis (H-OCA) has recently been
developed as a homologous ICSI test for
2+
determining the specific Ca pattern
elicited by human spermatozoa in spare
human oocytes. As a result, the H-OCA has
been shown to be more accurate than the
2+
mouse oocyte Ca analysis (M-OCA) or
MOAT in revealing sperm-related activation
deficiencies (Ferrer-Buitrago et al., 2018b).
Still, the scarcity of human oocytes donated
for research constrains the sole
applicability of H-OCA for diagnosing the
origin of ICSI failures.
It has been shown that human failed-to-
fertilize oocytes retain the capacity to
2+
generate and maintain Ca signalling
activity in response to activation stimuli
days after ICSI (Swann et al., 2012;
Tesarik et al., 1995). Because Ca
2+
oscillations were observed after the
exogenous injections of PLCζ cRNA
into failed-to-fertilize oocytes, it is worth
2+
considering that the Ca machinery
responsible for supporting oocyte
activation may remain functional for a
prolonged period after ICSI. Indeed,
earlier findings showed that IP3R1 are
still present in the oocyte hours after
fertilization (Goud et al., 1999).
In the present study, three different
strategies were assessed to reveal
whether an oocyte-related factor was
involved in patients previously
experiencing fertilization failure after
ICSI, who further underwent ICSI - AOA
cycles. In the first instance, the
normalcy of the spindle–chromosome
complex configurations and the
distribution of IP3R1 in in-vitro matured
metaphase II (IVM- MII) and failed-to-
fertilize oocytes from patients
undergoing ICSI- AOA cycles were
evaluated. These data were compared
with results obtained in IVM-MII and
failed-to-fertilize oocytes from patients
undergoing conventional ICSI with
normal fertilization rates (control group).
In a second set of experiments, the
2+
functionality of the Ca releasing
machinery in failed-to-fertilize oocytes
2+
was evaluated, by assessing the Ca
releasing capacity in response to an
additional exposure to ionomycin or to a
second sperm microinjection.
MATERIALS AND METHODS
Ethical approval
This study was approved by the Ethics
Committee of Ghent University Hospital
under reference 2014/1309.

Study population and participants
The present study included patients
presenting at the authors’ centre with
previous failed or low fertilization
(<33.3%) (Vanden Meerschaut et al.,
2013) after ICSI, who further experienced
fertilization failure after ICSI-AOA at the
centre. It is worth noting that clinic policy
is that sperm samples from patients
experiencing total or low fertilization
failure are analysed by MOAT before
receiving medical counselling oriented to
ICSI-AOA in a subsequent treatment
(TABLE 1).
Study design
FIGURE 1 shows the experimental design
of the present study. The oocyte-
related origin of fertilization failure was
evaluated in oocytes from patients
experiencing fertilization failure
after ICSI-AOA (study group) and
patients with normal fertilization after
conventional ICSI (control group). The
first proposed strategy comprises
assessment of the oocyte activation
potential using immunodetection
to visualize spindle–chromosome
complexes and IP3R1 distribution in
both groups of patients. The analysis
was performed using two sources of
oocytes: in-vitro matured metaphase II
(IVM -MII) and failed- to-fertilize oocytes
from both the study and the control
2+
groups. To evaluate the Ca releasing
capacity of oocytes that failed to fertilize
2+
after ICSI-AOA, direct exposure to Ca
ionophores (strategy 2) or sperm
microinjection (strategy 3) was
TABLE 1  PATIENTS INCLUDED IN THE PRESENT STUDY
Previous ICSI cycles
Patient n cycles n MII oocytes / n 2PN (fertiliza-
tion rate; %)
a 1 4/12 (33.3)
b 2 0/13 (0.0)
c 3 2/81 (2.4)
d 1 2/7 (28.7)
e 2 2/27(7.4)
f 4 0/30 (0.0)
g 3 0/29 (0.0)
h 2 1/15 (6.7)
i 1 1/18 (5.5)
j 3 2/27 (7.4)
All patients experienced low or failed fertilization in previous ICSI cycles. MOAT was performed in all patients before undergoing further ICSI-AOA treatment. Failed-to-ferti-
2+
lize oocytes after ICSI-AOA were analysed for Ca releasing capacity by direct exposure to ionomycin (patients a to e) or by sperm microinjection (patients f to j).
2PN = two-pronuclear; ICSI = intracytoplasmic sperm injection; MII = metaphase II; MOAT = mouse oocyte activation test.
performed in failed-to-fertilize oocytes
from patients experiencing fertilization
failure (study group) and normal
fertilization (control group).
Source and culture of human oocytes
Human oocytes were obtained from
patients undergoing conventional ICSI and
ICSI-AOA treatments at Ghent University
Hospital between October 2014 and August
2016. Immature oocytes were discarded for
ICSI and donated for research after a
written informed consent was obtained from
all patients. Oocytes were collected at one
of the two following stages of maturation: (i)
in-vitro matured oocytes retrieved at
prophase I (germinal vesicle) or (ii)
metaphase I
(MI). To allow in-vitro maturation (IVM),
oocytes at germinal vesicle stage were
cultured for 24 h in medium 199 (M199)
supplemented with growth factors and
hormones, as described elsewhere
(Nikiforaki et al., 2014). MI oocytes were
cultured in Cook Cleavage Medium
(Cook Ireland Ltd) for 3 h or 24 h, until
the presence of a second polar body
was observed. Failed-to-fertilize oocytes
were collected once there was definitely
no sign of the presence of pronuclei
formation and/or embryo cleavage.
Sperm preparation for ICSI
Sperm selection was performed by a
swim-up method (Mortimer and
Mortimer, 1992). Briefly, thawed
spermatozoa were washed in
Gamete Buffer (Cook Ireland Ltd) by
centrifugation at 1200 rpm for 10 min.
MOAT group (%)
3 (93)
2 (83)
2 (84)
3 (96)
2 (24)
3 (85)
2 (72)
2 (72)
3 (87)
3 (96)
RBMO VOLUME 38 ISSUE 4 2019 499
Supernatant was discarded and 500 µl of
fresh washing medium was gently added to
avoid pellet disruption. Samples were left
for 30 min at room temperature and the
supernatant fraction, containing motile
spermatozoa, was recovered and further
washed by centrifugation
at 1200 rpm for 10 min. Samples were
concentrated in a final volume of 50–
100 µl. Sperm cells of samples showing
a low sperm count (<5 million sperm/ml)
were manually selected under an
inverted light microscope after the first
washing step.
Intracytoplasmic sperm injection
Human oocytes were microinjected
following a standard ICSI protocol, by
which the zona pellucida and the
oolemma are perforated with a 30°
angled spiked needle (Humagen,
Origio; CooperSurgical, Trumbull, CT,
USA). ICSI was performed on a heated
stage (37°C).
Assisted oocyte activation
AOA was performed using an ionomycin-
based protocol. The procedure involved the
injection of a spermatozoa into
the oocyte together with CaCl2. Thirty
minutes after ICSI, the oocytes were
exposed to 10 μmol/l ionomycin (Sigma,
I9657) dissolved in Cook Cleavage
Medium for 10 min, followed by
extensive washing. A second exposure
to ionomycin for 10 min was applied 30
min later. Finally, oocytes were
extensively washed and cultured in CC
at 37°C, 6% CO2 and 5% O2.
ICSI-AOA cycles
n cycles n MII oocytes / n 2PN
(fertilization rate; %)
3 6/19 (31.6)
1 4/13 (30.8)
1 12/15 (80.0)
1 0/4 (0.0)
1 3/8 (37.5)
2 0/8 (0.0)
1 4/16 (25.0)
2 3/16 (18.8)
1 0/11 (0.0)
1 0/4 (0.0)
500 RBMO VOLUME 38 ISSUE 4 2019
FIGURE 1  Schematic representation of the three strategies followed in the present study. AOA = assisted oocyte activation; GV = germinal vesicle;
ICSI = intracytoplasmic sperm injection; IP3R1 = inositol trisphosphate receptor type 1; MI = metaphase I; MII = metaphase II.
Immunodetection of spindle–
chromosome complexes and IP3R1 IVM
oocytes were fixed in a microtubule-
stabilizing buffer, as previously described
(Heindryckx et al., 2011). Briefly, IVM
oocytes were fixed and extracted in a
microtubule-stabilizing buffer (0.1 mmol/l
PIPES, 5 μmol/l MgCl2, 2.5 μmol/l EGTA,
0.01% aprotinin, 1 μmol/l dithiothreitol,
50% deuterium oxide, 1  ×  10
–12
mol/l
taxol, 0.1% Triton X-100 and 3% formalin)
for 30 min at 37°C. Following three
sessions of intensive washing (15 min),
oocytes were subsequently stored at 4°C in
PBS with azide until fluorescence staining
was performed. At staining, IVM oocytes
were incubated overnight at 4°C with a 1/1
mixture of mouse monoclonal anti-α and β-
tubulin (1/200) and/or a primary antibody
(1/1000) against the IP3R1 in mouse
oocytes (rabbit, polyclonal, KU Leuven)
(Parys
et al., 1995). After washing (3  ×  15 min),
samples were treated with the secondary
antibodies Alexa Fluor 488 goat anti-
mouse IgG (H+L) (Molecular Probes)
(1/200) and/or CY3 donkey anti-rabbit IgG
(H+L) (Jackson ImmunoResearch) (1/500)
for 1 h, followed by extended washing (3
 ×  15 min). Meanwhile, the negative
controls were treated
without primary antibody but with the
secondary antibodies alone. In addition,
chromosomes were stained with
Hoechst 33258 for 30 min. Finally, the
oocytes were mounted in Mowiol
containing 0.01% phenylenediamine
and imaged using a laser scanning
confocal microscope.
Evaluation of the normalcy of the
spindle–chromosome complexes
and IP3R1 distribution
To evaluate the spindle configuration and
chromosome alignment of IVM-MII
oocytes, the categorization previously
published elsewhere was used
(Combelles et al., 2010). Briefly, spindles
were classified as normal (bipolar) or
abnormal (mono- or multi-polar) (FIGURE 2).
If the spindle was absent in the oocyte,
but polymerized tubulin was detected
throughout the cytoplasm, oocytes were
classified into the category of
‘polymerized tubulin’. The chromosomes
were classified as normal when they were
aligned forming MII plates in the
equatorial region of the spindle, or
abnormal when more than three
chromosomes were misaligned and away
from the equatorial region or dispersed
with all chromosomes located
throughout the spindle (FIGURE 2). Oocytes
showing DNA distributed into forming
pronuclei were classified as ‘pronuclei-like’
oocytes. Regarding the IP3R1 pattern,
three different types of distribution were
described (FIGURE 2) based on observations
previously published by Goud et al. (1999).
Briefly, a cortical location was defined as
Class I; a uniform redistribution with
reduced intensity of the fluorescence signal
at the cortex was used to describe Class II;
and a central clustering of IP3R1, identified
by the increase of fluorescence signal
surrounding the resulting pronuclei,
was defined as Class III.
2+
Oocyte preparation for Ca imaging
Before ionomycin exposure or ICSI, failed-
to-fertilize oocytes were prepared following
the methodology described elsewhere
(Nikiforaki et al., 2014). Briefly, failed-to-
fertilize oocytes were incubated in Cook
Cleavage Medium containing fura-PE3-AM
(Teflabs, TX, US) at
7.5  ×  10
–6
mol/l under standard culture
conditions (37°C and 6% CO2 and 5% O2)
for 30 min and were afterwards
extensively washed in correspondent
culture media prior to microinjection with
human spermatozoa.
 RBMO VOLUME 38 ISSUE 4 2019 501

FIGURE 2  Study of tubulin/chromosome complexes and IP3R1 distribution in oocytes from patients undergoing conventional ICSI and ICSI-AOA. a.
Spindles (tubulin, green) were classified as normal (bipolar) or abnormal (mono- or multi-polar). If polymerized tubulin was detected throughout the
cytoplasm, oocytes were classified as ‘polymerized tubulin’. b. Chromosomes (DNA, blue) were classified as normal when they were aligned,
forming a metaphase II plate in the equatorial region of the spindle, or abnormal when more than three chromosomes were misaligned and away
from the equatorial region or dispersed with all chromosomes located throughout the spindle. Oocytes showing DNA distributed into forming
pronuclei were classified as ‘pronuclei-like’ oocytes. c. IP3R1 (red) distribution was categorized into three different classes: Class I: a cortical
location, Class II: a uniform redistribution with reduced intensity of the fluorescence signal at the cortex, Class III: an increase of the fluorescence
signal surrounding the resulting pronuclei.

2+
Ca imaging
2+
To test the efficacy of Ca ionophores,
failed-to-fertilize oocytes were exposed
to 10 μmol/l ionomycin (Sigma, I9657),
dissolved in Cook Cleavage Medium.
For ICSI, failed-to-fertilize oocytes
were placed 30 min after sperm
microinjection in culture medium in a
glass-bottomed dish (MatTek Corp.,
Ashland, MA, USA) covered with
paraffin oil (Irvine Scientific, USA). For
both tests, oocytes were individually
monitored under an inverted
epifluorescence microscope (Olympus
IX71, Olympus Soft Imaging Solutions
GmbH, Belgium) equipped to stabilize
culture conditions at 37°C and 6%
CO2 (OKO Labs, Olympus
GmbH, Belgium) with a 10 × objective
and a filter switch (Lambda DG-4 filter
switch, Sutter Instrument Company,
Novato, CA, USA) to provide excitation
alternating between 340 and 380 nm.
Data acquisition and analysis
2+
Ca records were transferred to
Clampfit 10.2 software (Axon
Laboratories, Molecular Devices UK
Ltd) for further analysis. Baseline
drifting was adjusted before retrieving
values for amplitude (A, value at
maximum increase in fluorescence
intensity per peak) expressed in
arbitrary units (AU). The product of the
mean amplitude (A) and total number
2+
of Ca spikes during the recording
period (F) (A  ×  F), and the area under
the curve (AUC, in AU), were used to
score the activation potential for each
sperm sample.
Statistical analyses
Statistical Package for the Social
Sciences (SPSS® Statistics 24, IBM
Corp., NY, USA) was used to analyse
amplitudes and AUC, applying a
Kruskal–Wallis test for independent
samples. Amplitude and AUC data are
presented as SEM. Cross-tabulations
were used to compare the frequencies of
the immunostaining outcomes, using
Fisher's exact test. The threshold for
significance was set at <0.05.
502 RBMO VOLUME 38 ISSUE 4 2019

TABLE 2  EVALUATION OF SPINDLE–CHROMOSOME COMPLEXES AND DISTRIBUTION OF IP3R1

IVM oocytes from patients undergoing conventional ICSI (n = 14) or ICSI-AOA (n = 17)
Chromosome alignment Normal Abnormal Pronuclei-like
Conventional ICSI 2 12 0
ICSI-AOA 3 12 2
Spindle configuration Normal Abnormal Polymerized tubulin
Conventional ICSI 5 9 0
ICSI-AOA 4 11 2
IP3R1 distribution Class I Class II Class III
Conventional ICSI 10 4 0
ICSI-AOA 8 7 2
Failed-to-fertilize oocytes after conventional ICSI (n = 21) or ICSI-AOA (n = 21)
Chromosome alignment Normal Abnormal Pronuclei-like
Conventional ICSI 10 11 0
ICSI-AOA 4 13 4
Spindle configuration Normal Abnormal Polymerized tubulin
Conventional ICSI 13 8 0
ICSI-AOA 5 12 4
IP3R1 distribution Class I Class II Class III
Conventional ICSI 8 9 4
ICSI-AOA 1 a 6
14
Chromosome alignment is classified as normal or abnormal based on the absence or the presence of lagging chromosomes at the metaphase plate, respectively. When
the DNA was observed as forming pronuclei, the oocytes were classified as pronuclei-like. Spindles were evaluated based on the tubulin distribution. Barrel-shaped
spindles were classified as normal or abnormal based on the presence or absence of two poles, respectively. When spindle was absent, and the tubulin was polymerized
throughout the cytoplasm, the oocytes were classified as polymerized tubulin. The distribution of IP3R1 was identified as Class I (cortical), Class II (uniform) and Class III
(clustered and central localization).
AOA = assisted oocyte activation; ICSI = intracytoplasmic sperm injection; IP3R1 = inositol-triphosphate type 1 receptors; IVM = in-vitro matured.
a 
Significance level <0.05.

RESULTS
Strategy 1. Evaluation of spindle–
chromosome complex configuration
and distribution of IP3R1
Results obtained using IVM oocytes
The spindle–chromosome complexes and
the intracellular distribution of IP3R1 were
analysed in a total of 17 IVM-MII oocytes
from patients undergoing ICSI-AOA (study
group) and 14 IVM-MII oocytes from
patients undergoing conventional ICSI
(control group) (TABLE 2).
The results revealed that IVM-MII oocytes
from patients undergoing ICSI-AOA
oocytes did not present a higher
incidence of abnormal spindle
configurations than IVM-MII oocytes from
patients undergoing conventional ICSI
(TABLE 2). Next, IVM-MII oocytes from
patients undergoing conventional ICSI
showed a similar higher proportion of
oocytes with IP3R1 concentrated at the
cortex (Class I) than oocytes from
patients undergoing ICSI-AOA. Class III
oocytes were observed in similar lower
proportions in both groups (TABLE 2).
Overall, these observations
correspond to the pattern expected in
normal MII oocytes.
Results obtained using failed-
to-fertilize oocytes
The methodology described above was
used to evaluate spindle–chromosome
complexes and the distribution of IP3R1 in
failed-to-fertilize oocytes after ICSI-AOA
(study group; n = 21) and conventional ICSI
(control group; n = 21). Failed-to-fertilize
oocytes after ICSI-AOA showed a higher
incidence of abnormal spindle–
chromosome complexes compared with
failed-to-fertilize oocytes after conventional
ICSI (TABLE 2), although the difference was
not statistically significant. Moreover, 4 out
of 21 failed-to-fertilize oocytes from the
ICSI-AOA group were observed to show the
DNA in a pronuclear-like configuration. The
distribution of IP3R1 was also examined.
Whereas failed-to-fertilize oocytes after
conventional ICSI (control) showed that
IP3R1 patterns were similarly distributed
between Class I (n = 8, cortical) and
uniform (n = 9, Class II) failed-to-fertilize
oocytes after ICSI-AOA mainly presented
with a uniform pattern (n = 14, Class
II, P < 0.05). A Class III pattern, which
denotes a migration of the IP3R1
towards a central location, was
observed in 4 out of 21 failed-to-fertilize
oocytes after conventional ICSI, as well
as in 6 out of 21 oocytes that failed to
fertilize after ICSI-AOA (TABLE 2).
Strategy 2. Assessment of the
capacity of failed-to -fertilize oocytes
to respond to a second treatment with
ionomycin
To test whether oocytes that failed to
fertilize after ICSI-AOA were capable of
responding to the applied AOA
2+
treatment, the cytoplasmic Ca increase
associated with direct exposure to
ionomycin was determined (FIGURE 3) in a
total of 16 failed-to-fertilize oocytes, from
5 patients (patient a to patient e) who
underwent ICSI-AOA (TABLES 1 and 3). All
patients had a previous history of
 RBMO VOLUME 38 ISSUE 4 2019 503

2+
FIGURE 3  Representative Ca responses from failed-to-fertilize oocytes after direct exposure to ionomycin (a, b) and sperm microinjection (c, d).
2+ 2+
Each line represents one oocyte. As a normal response, an increase in intracellular Ca is observed, either as a single Ca transient induced by
2+
ionomycin (a) or displaying Ca oscillations induced by sperm microinjection (c). Abnormal responses reflect low changes or the total absence of
2+
Ca increase (b, d).
fertilization failure after conventional activation potential, MOAT group 3), oocytes exposed to a second treatment
ICSI, with an average fertilization rate except for patient e, who showed a with ionomycin showed an abrupt surge
of 7% (TABLE 1). Of these, 4 of 5 patients low capacity to activate mouse oocytes 2+
of cytoplasmic Ca during the 10 min
showed high MOAT values (normal (TABLE 1). It was observed that 81% of the period of exposure. Interestingly, 3 out of
4 oocytes from patient e did not respond
2+
TABLE 3  ASSESSMENT OF CA RELEASING CAPACITY OF FAILED-TO- to the effect of ionomycin. Furthermore,
FERTILIZE OOCYTES IN RESPONSE TO (A) IONOMYCIN AND (B) SPERM 2+
the amplitude of the Ca peak (A) as
MICROINJECTION
well as AUC values were measured and
(a) Failed-to-fertilize oocytes after ICSI-AOA with second direct
compared with control oocytes (failed-to-
exposure to ionomycin
2+
fertilize oocytes after conventional ICSI).
Patient Failed-to-fertilize oocytes with Ca peaks after Failed ICSI-AOA oocytes (study group)
ionomycin exposure 24 h ICSI
showed lower values of A (n = 13, 1.18 ±
a 4/4 0.34 AU) and AUC (n = 13, 1.17 ± 0.7
b 3/3 AU) than control oocytes (n = 3, A = 1.7
± 0.05 AU; AUC = 2.00 ± 0.3 AU),
c 3/3
however these results were not
d 2/2 significantly different, probably due to
e 1/4 small numbers. It should be noted that
oocytes that did not respond to the effect
(b) Failed-to-fertilize oocytes after ICSI-AOA with second sperm microinjection
2+ 2+
of ionomycin were not considered to
Patients Failed-to-fertilize oocytes with Ca peaks / n Donated IVM oocytes with Ca calculate mean values of A and AUC.
failed-to-fertilize oocytes 40 h ICSI peaks / total n IVM donated oocytes
f 2/4 d 4/4

g 2/2 p – Strategy 3. Assessment of the

capacity of failed-to-fertilize oocytes
h 0/2 p 0/10
to respond to a second sperm
i 0/3 p 1/15
microinjection
j 2/3 p – 2+
Patterns of Ca oscillations after the
For the study of couple f, failed-to-fertilize oocytes were injected with donor's spermatozoa with normal activation microinjection of control spermatozoa
capacity (superscript d). For the study of patients g to j, failed-to-fertilize oocytes were injected with partner's
into control failed-to-fertilize oocytes
spermatozoa (superscript p).
(oocytes from patients with normal
AOA = assisted oocyte activation; ICSI = intracytoplasmic sperm injection; IVM = in-vitro matured.
fertilization rates, which failed to fertilize
504 RBMO VOLUME 38 ISSUE 4 2019
after conventional ICSI) (FIGURE 3) were
first determined. To evaluate the oocyte
activation capacity, the product of
2+
the amplitude of the Ca peak (A)
2+
and total number of Ca oscillations
(F) during a recording period of 10 h was
used. As a result, it was observed that
control failed-to-fertilize oocytes showed
similar A  ×  F values as control vitrified
IVM oocytes used in a previous study (A
 ×  F = 7.28 AU versus A  ×  F = 6.18 AU)
(Ferrer-Buitrago
et al., 2018b). Hence, this strategy was
also used to study the oocyte activation
capacity of failed-to-fertilize oocytes
from patients undergoing ICSI-AOA
in the authors’ centre. In a first case
(patient f), spermatozoa with proven
normal fertilization (control) were used to
inject failed-to-fertilize oocytes from a
patient who experienced total fertilization
failure after ICSI-AOA. This couple (f)
had 4 out of 5 failed-to-fertilize oocytes
that survived 40 h post-ICSI. From the
four surviving oocytes, two displayed a
2+
normal Ca oscillatory activity, with both
2+
oocytes showing five Ca spikes during
the 10 h measurement (A  ×  F = 6.13
AU). To discern whether the negative
response to ICSI-AOA could be
associated with the inability of the
2+
spermatozoa to stimulate Ca
oscillatory activities, the H-OCA (Ferrer-
Buitrago et al., 2018b) was performed
with donor oocytes using patient's
spermatozoa. The spermatozoa proved
2+
capable of initiating long-lasting Ca
responses in three out of three IVM-MII
donor oocytes
(A  ×  F = 23.7 AU). It should be noted
that in both the assays described
above, A  ×  F values were calculated in
a low number of oocytes. However,
these outcomes identified that oocytes
and spermatozoa from couple f were
capable of initiating and maintaining the
2+
Ca spiking activity compatible with
fertilization.
2+
In the following cases the specific Ca
patterns observed in failed-to-fertilize
oocytes after ICSI-AOA induced directly by
the partner's spermatozoa were assessed.
In two out of the four cases studied
2+
(patients g and j), Ca oscillations were
observed 40 h post - ICSI-AOA, with a
product of A  ×  F of 8.44 and 12.1 AU,
respectively. In contrast to these results,
spermatozoa and oocytes from patients h
2+
and i displayed a total absence of Ca
oscillations
40 h after ICSI-AOA (TABLE 3). In these
two cases, H-OCA was also performed
(patient's spermatozoa with IVM-MII donor
oocytes) to confirm or refute the incapability
2+
of the spermatozoa to stimulate Ca
release. H-OCA revealed a total absence of
2+
Ca oscillations in IVM-MII donor oocytes
injected with spermatozoa from patient h.
Likewise, spermatozoa from patient i was
2+
incapable of eliciting a normal Ca
response in IVM-MII donor oocytes, with
only 1 out of 15 oocytes showing a single
2+
Ca spike during the 10 h measurement.
DISCUSSION
The present study describes further
exploration of the origin of fertilization
failure based on oocytes that failed to
fertilize after conventional ICSI and ICSI-
AOA. It has been shown that fertilization
failure after ICSI still occurs in
approximately 3–5% of all ICSI cycles
(Bhattacharya et al., 2013). Remarkably,
the use of AOA technology offers
couples experiencing fertilization failure
the chance to use their own gametes.
Several studies show that the efficiency
of AOA is more evident in cases showing
sperm-related oocyte activation
deficiencies (Ebner et al., 2012; Ferrer-
Buitrago et al., 2018b; Kuentz et al.,
2013). Hence, identifying the gamete
origin responsible for the fertilization
failure is of major importance in
orientating a couple's decision towards
future treatments, especially when
seeking gamete donation.
The authors’ centre has an established
programme for investigating cases
experiencing failed fertilization after
ICSI (Heindryckx et al., 2005; Vanden
Meerschaut et al., 2013). Spermatozoa
involved in recurrent fertilization failures
after conventional ICSI is first evaluated
by the MOAT. This initial classification
allows patients to undergo ICSI-AOA
with a diagnosis on the sperm activation
potential, with patients who showed low
activation rates in mouse oocytes (MOAT
1 and 2) expecting favourable responses
to AOA treatment (Heindryckx et al.,
2008). In contrast, in MOAT group 3
patients, the presence of an oocyte-
related activation deficiency is suspected,
taking into account a lower pregnancy
rate based on previous experience
(Vanden Meerschaut et al., 2012). Earlier
studies revealed that oocyte activation
failure is associated with factors either
of maternal or parental origin, with
failed-to-fertilize oocytes showing
defects in cytoplasmic components
(Combelles et al., 2010; Rawe et al., 2000)
and defective or abnormal spindle–
chromosome complexes or altered
chromosome numbers (Racowsky et al.,
1997; Rosenbusch, 2000). In
line with these observations, we first
evaluated whether oocytes from patients
undergoing ICSI-AOA treatment, with
previous history of fertilization failure
after conventional ICSI, showed a higher
incidence of abnormal spindle–
chromosome configurations. We did not
identify differences in the proportion of
abnormal spindle configurations when
IVM-MII oocytes from patients
undergoing ICSI-AOA were compared
with oocytes from patients undergoing
conventional ICSI. This outcome might
reflect a similar oocyte quality in both the
study groups. Moreover, when using the
same methodology to evaluate failed-to-
fertilize oocytes after the treatment, we
also observed that oocytes from
the study group (ICSI-AOA) showed a
similar incidence of abnormal spindle–
chromosome complexes than oocytes that
failed-to-fertilize after conventional ICSI. It
is worth mentioning that AOA is applied
during the progress of the second meiotic
division, in which the oocyte will undergo
meiotic spindle dynamics and as a result,
chromosome segregation. Therefore, it
2+
has been suggested that differing Ca
signals, other than the physiological Ca
2+
oscillations, could impair the correct
chromosome segregation (Yeste et al.,
2016). Recent studies have investigated
whether the use of AOA would induce
errors during oocyte meiotic division,
which can be associated with later embryo
aneuploidies. However, the use of
ionomycin was not associated with an
increased number of chromosomal
abnormalities after ICSI-AOA treatment
(Deemeh et al., 2015). Moreover, the
2+
effect of Ca ionophores did not prove to
increase the incidence of meiotic errors of
maternal origin, proved in a reduced
number of human oocytes that were
exposed to the effect of calcimycin
(A23187) (Capalbo et al., 2016).
According to these findings, we suspect
that the proportion of abnormal spindle–
chromosome complexes that we observed
in our study might be associated with an
overall lower oocyte quality in these
patients, irrespective of the use of
conventional ICSI or ICSI-AOA. However,
further investigations in a higher number
of oocytes are needed to confirm the
safety of AOA during the meiotic
progression.

Furthermore, to assess the effect of the
2+
ICSI-AOA treatment on the Ca releasing
machinery, we evaluated the patterns
of distribution of IP3R1 in IVM-MII and
failed-to-fertilize oocytes after ICSI-AOA
and conventional ICSI. We observed that a
high proportion of failed-to-fertilize oocytes
after ICSI-AOA displayed a uniform
distribution of IP3R1 throughout the
ooplasm (Class II) or central IP3R1
clustering (Class III), patterns that reflect
the migration of the IP3R1 from cortex
towards the resulting pronuclei in response
to the activation stimuli. Despite the fact
that these observations may indicate
initiation of the activation process, the
absence of pronuclei formation lead us to
interpret that these oocytes were incapable
of accomplishing meiosis, probably due to
2+
an insufficient amount of the Ca to
complete oocyte activation (Tóth et al.,
2006). Our data show that the evaluation of
cytoplasmic components, such as spindle–
chromosome complexes and IP3R1
distribution patterns, are useful to evaluate
features associated with oocyte quality.
However, in our experience, this strategy is
weak at discerning whether oocyte
or sperm factors were responsible for
fertilization failure after ICSI or ICSI-AOA.
In a second set of experiments, we
evaluated whether fertilization failure after
ICSI-AOA could be associated with the
2+
inability of the oocytes to respond to Ca
ionophores during AOA treatment. We
observed that the majority of the oocytes
[13 out of 16; TABLE 3(a)] were able to
respond to ionomycin 24 h after the initial
sperm microinjection. Overall, we observed
that performing additional exposures to
2+
Ca ionophores may yield information
about the efficacy of AOA in mediating
2+
intracytoplasmic Ca increases, either
from culture media or for the internal
cellular stores. Our analysis identified
oocyte-related deficiencies in one couple
(patients e), who previously showed a
reduced MOAT value (24%) and for whom
fertilization rates remained low after ICSI-
AOA.
The exposure to ionomycin after failed
ICSI-AOA revealed that 3 out of 4 failed-
to-fertilize oocytes did not manifest
2+
normal intracellular Ca increases, an
observation that supports the presence
of an additional oocyte-related activation
deficiency factor causing suboptimal
fertilization. Moreover, for this couple,
ICSI-AOA treatment resulted in two
embryos being transferred, and a
clinical pregnancy, which unfortunately
ended as a mid-trimester loss.
Interestingly, previous observations
showed that failed-to-fertilize oocytes
2+
can retain their Ca releasing capacity
several hours after the treatment
(Swann et al., 2012; Tesarik et al., 1995).
Hence, we further evaluated whether
2+
the analysis of the Ca oscillatory
activity after sperm injection of failed-
to-fertilize oocytes after ICSI-AOA
could more accurately identify the
presence of oocyte-related activation
deficiencies. Failed-to-fertilize oocytes
after conventional ICSI (control) showed
normal capacity of eliciting long-lasting
2+
Ca oscillations 40 h post-ICSI (with
control sperm with proven normal
fertilization capacity). In support of
previous findings (Swann et al., 2012;
Tesarik et al., 1995), the current
observations validated the use of failed-
to-fertilize oocytes to detect the capacity
2+
of oocytes to support Ca activity, even
40 h after the initial ICSI. In one couple,
sperm with proven normal fertilization
capacity (control) was injected in failed-
to-fertilize oocytes after ICSI-AOA. We
further refuted the presence of sperm-
related activation deficiency by observing
2+
normal Ca patterns after the H-OCA
using donated IVM-MII oocytes. Overall,
we reasoned that this couple may
suffer from oocyte-related deficiencies
affecting mechanisms downstream of
2+
the Ca oscillatory signalling, which
cannot be overcome by the use of Ca
2+
ionophores. However, we did not have
any of the patient's oocytes available to
further confirm this.
2+
Hereafter, we decided to study Ca
patterns in cases experiencing failed
fertilization after ICSI-AOA after
microinjecting failed-to-fertilize oocytes
with partner's sperm 40 h after the initial
ICSI-AOA, and once pronuclei formation
and/or embryo cleavage are no longer
observed. We predetermined a strategy
that in case of observing absence or
2+
abnormally low Ca activity, we would
proceed to perform H-OCA (donated IVM
oocytes microinjected with patient's
spermatozoa) to confirm or refute the
presence of sperm-related activation
deficiencies. A total of two out of four
patients showed that the spermatozoa had
2+
the ability to initiate normal Ca release
in failed-to-fertilize oocytes after ICSI-
AOA. In light of these results, we would
suggest performing an additional test by
which we would inject donor spermatozoa
into patient's oocytes resulting from a
following cycle. In this way, we could
confirm or refute
RBMO VOLUME 38 ISSUE 4 2019 505
the presence of oocyte-related factor
accounting for previous failed ICSI and
ICSI-AOA treatment. Otherwise, these
cases are candidates to participate in
oocyte donation programmes because
spermatozoa were shown to be able to
2+
initiate Ca oscillatory responses, but
ICSI-AOA treatments were not capable of
restoring fertilization. We believe that
2+
mechanisms other than the series of Ca
2+
channels and Ca pumps involved in
2+
generating and maintaining the Ca
oscillatory activity (Ferrer-Buitrago
et al., 2018a; Yeste et al., 2016) might
be impairing the progress of oocyte
activation in these cases. Interestingly,
one of these two couples decided to opt
for oocyte donation in the fertility centre
of origin, which resulted in the birth of a
healthy singleton.
On the contrary, two other patients
2+
showed a total absence of Ca spikes
when partner's spermatozoa were
injected in failed-to-fertilize oocytes
after ICSI-AOA. We investigated further
whether the spermatozoa were capable
of activating IVM-MII control human
oocytes by H-OCA. Indeed, the H-OCA
revealed that IVM-MII control oocytes
injected with spermatozoa from patients
h and i showed a total absence or very
2+
low incidence of Ca oscillations.
These results indicate the presence
of sperm-related activation deficiency.
The question remains whether an
additional oocyte-related activation
deficiency was involved in causing failed
fertilization in these couples. In line with
the previous comment, the confirmation
of the presence of oocyte-related factors
by the injection with donor spermatozoa
into patient's oocytes would be
recommended. Interestingly, whereas
ICSI-AOA failed to overcome fertilization
failure in a second attempt by patient i,
AOA restored fertilization and
subsequent embryo development in 3
out of 16 oocytes when couple h
underwent ICSI-AOA and resulted in the
live birth of a singleton.
The present study shows that elucidating
the origin of fertilization failures is a
puzzling challenge, taking into account that
speculative conclusions do not always
satisfy patients’ needs and expectations. In
our experience, the MOAT is a valuable
method to perform an initial triage of
patients experiencing fertilization failures.
Hence, patients can undergo AOA
treatments knowing about the presence of
sperm-related (MOAT 1 and 2) or
506 RBMO VOLUME 38 ISSUE 4 2019
suspected oocyte-related activation
deficiencies (MOAT 3). The MOAT is of
added value, as it can predict favourable
responses to AOA treatments, most likely in
cases with a low capacity for activating
mouse oocytes (MOAT 1 and lower rates of
MOAT 2). When failed fertilization after
ICSI-AOA still occurs, we showed that
failed-to-fertilize oocytes can be used
to assess the oocyte-related activation
capacity. From the three strategies
proposed in this paper, we believe that
2+
performing Ca pattern analysis after
donor or partners’ sperm microinjection
yields the most valuable information to
orientate couples towards a following
2+
treatment. The presence of Ca spiking
activity will indicate that the spermatozoa is
capable of initiating oocyte activation.
Furthermore, despite the oocyte showing
its efficiency in supporting long-lasting
2+
Ca oscillations, failed fertilization
2+
indicates that the Ca response probably
failed to be integrated during the activation
process, as a consequence impeding the
successful release of the meiotic arrest.
Our diagnostic strategy continues with the
application of H-OCA in cases whose
failed-to-fertilize oocytes further failed to
respond to the 40 h ICSI. H-OCA will
confirm or refute the presence of sperm-
related activation deficiencies. Patients
whose spermatozoa showed an inability to
activate human oocytes will require the
application of AOA, irrespective of the use
of their own or donated oocytes. Overall,
our study reflects the complexity of
unravelling the aetiology of fertilization
failure, in support of favourable responses
to AOA treatments. Further investigations
are still needed to develop therapies
for cases with oocyte-related activation
deficiencies, because, to date, the only
further treatment option for these cases
is seeking oocyte donation.
ACKNOWLEDGEMENTS
The authors would like to acknowledge
Professor Jan Parys (KU Leuven,
Department for Cellular and Molecular
Medicine, Leuven, Belgium) for kindly
providing the primary antibody against
IP3R1. We would also like to thank
Dr Dimitra Nikiforaki and the IVF lab
team for their collaboration during the
collection of human oocytes.
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