J Assist Reprod Genet (2017) 34:33–41

DOI 10.1007/s10815-016-0823-0

GAMETE BIOLOGY

The combination of calcium ionophore A23187 and GM-CSF can

safely salvage aged human unfertilized oocytes after ICSI
Konstantinos A. Economou 1 & Dimitra Christopikou 1 & Erika Tsorva 1 & Stephen Davies 1 &
Minas Mastrominas 1 & Haris Cazlaris 1 & Michael Koutsilieris 2 &
Panagoula Angelogianni 2 & Dimitris Loutradis 3

Received: 13 April 2016 / Accepted: 27 September 2016 / Published online: 14 October 2016
# Springer Science+Business Media New York 2016

Abstract Results Oocytes unfertilized after ICSI can be activated with

Purpose Artificial oocyte activation using calcium iono- the calcium ionophore A23187 to show two pronuclei and two
phores and enhancement of embryonic developmental poten- polar bodies. Addition of rhGM-CSF in the culture medium of
tial by the granulocyte-macrophage colony-stimulating factor A23187-activated oocytes enhances their cleaving and blastu-
(GM-CSF) have already been reported. In this study, we eval- lation potential and results in more euploid blastocysts com-
uated the synergistic effect of these two methods on aged pared to the culture medium alone.
human unfertilized oocytes after intracytoplasmic sperm in- Conclusions This study shows that activating post-ICSI aged
jection (ICSI). Then, we cultured the resulting embryos to the human unfertilized oocytes with a combination of a calcium
blastocyst stage and screened them for chromosomal abnor- ionophore and a cytokine can produce good-morphology eu-
malities, to assess the safety of this protocol. ploid blastocysts.
Methods Aged human oocytes deemed unfertilized after ICSI
were activated, either by briefly applying the calcium iono- Keywords Calcium ionophore A23187 . GM-CSF . Artificial
phore A23187 alone (group A) or by briefly applying the oocyte activation . aCGH . ICSI . Fertilization failure
ionophore and then supplementing the culture medium with
recombinant human GM-CSF (rhGM-CSF) (group B). Next,
the development was monitored in a time-lapse incubator sys- Introduction
tem, and ploidy was analyzed by array comparative genomic
hybridization (aCGH), after whole embryo biopsy and whole Fertilization is one of the major events in in vitro fertilization
genome amplification. Differences between oocytes and (IVF). In cases of severe male factor infertility, intracytoplasmic
resulting embryos in both groups were evaluated statistically. sperm injection (ICSI) has revolutionized treatment [1]. Total
fertilization failure has been reported to occur in 1–3 % of ICSI
Capsule The exposure of human unfertilized oocytes 18 h after ICSI to a cycles [2, 3] with devastating psychological results for the cou-
combination of calcium ionophore A23187 and the GM-CSF cytokine
can safely salvage the oocytes, resulting in the development of good-
ple [4]. For patients, the psychological burden is equivalent to
morphology, euploid blastocysts, with potential clinical use. that experienced after low fertilization post ICSI. In such cases,
repeating the ICSI procedure may result in fertilization failure
* Konstantinos A. Economou again, despite normal semen parameters and satisfactory ovar-
keconomou@gmail.com ian response [3, 5]. The mechanism underlying post-ICSI fer-
tilization failure (PIFF) is linked to failure of oocyte activation
1
Embryogenesis, Assisted Reproduction Unit, 49 Kifissias Avenue
after the injection [6]. Sperm-derived phospholipase C-ζ
and Ziridi Street, 151 23 Maroussi, Athens, Greece (PLCζ) has been characterized as an important physiological
2
Department of Physiology, School of Medicine, National and
agent of oocyte activation since 2002 [7]. The phos-
Kapodistrian University of Athens, Athens, Greece phatidylinositol pathway triggered by PLCζ is responsible for
3
First Department of Obstetrics and Gynaecology, School of
calcium release from the oocyte’s endoplasmic reticulum,
Medicine, Alexandra University Hospital, National and Kapodistrian resulting in oocyte activation and subsequent fertilization, with
University of Athens, Athens, Greece characteristic Ca 2+ flux oscillations [8]. The role and
34 J Assist Reprod Genet (2017) 34:33–41

mechanism of PLCζ and other factors in oocyte activation are There is recent evidence that AOA does not cause a wide-
still under investigation [9–11]. spread increase in chromosome segregation errors during mei-
In PIFF cases, different methods have been applied to osis [44], thereby indicating that this method may be safe for
achieve artificial oocyte activation (AOA): activation using an clinical use. The same observation applies to GM-CSF sup-
electric current [12–14], modifications of ICSI techniques [5, plementation in culture media. There was only one previous
15], and the use of chemical agents, such as strontium chloride study, based on fluorescence in situ hybridization (FISH) anal-
[16], puromycin [17], and Ca2+ ionophores, with calcium ion- ysis of only seven chromosomes [38], indicating that GM-
ophore A23187 being the most widely used [8, 18–23]. CSF has no effect on the chromosomal constitution of human
The use of calcium ionophore A23187 has been found to embryos.
salvage oocyte activation failure cases, leading to pregnancies For this study, we applied a modified AOA protocol,
in cycles with diminished oocyte reserves [24], although this in which we combined exposure to calcium ionophore
is still a matter of discussion [25]. Delivery of healthy babies A23187 with supplementation of the culture medium
has been reported in severe male factor IVF cycles [18–20, with GM-CSF, to activate human oocytes deemed unfer-
26–28] and in normozoospermic IVF cycles [21]. tilized 18 h post ICSI. We compared the results with the
Furthermore, A23187 treatment improved embryo develop- standard AOA protocol where calcium ionophore
ment and IVF outcome in couples with embryos showing A23187 alone was used. We hypothesized that GM-
developmental problems in previous IVF attempts [29]. CSF may exert its effects on activated oocytes and on
Granulocyte-macrophage colony-stimulating factor (GM- the resulting embryos. Our primary goal was to evaluate
CSF) has been found to improve embryo developmental po- the synergistic effect of both of these stimuli on activated
tential in IVF. GM-CSF is a hematopoietic cytokine that was oocytes and subsequent embryonic development; our sec-
initially identified as a derivative of activated T lymphocytes, ondary goal was to examine the safety of this modified
playing a role in the proliferation and differentiation of myeloid protocol on oocytes and developing embryos, by means
hematopoietic cells [30]. In humans, GM-CSF expression has of array comparative genomic hybridization (aCGH)
been identified in different sites of the female reproductive analysis of the full chromosomal complement of the
tract, such as endometrial tissue [31], fallopian tubes [32], pla- resulting embryos.
centa and trophoblastic cells [33, 34], theca cells around large
follicles, ovarian luteal cells [35], and follicular fluid [36].
In human embryos, the GM-CSF receptor is expressed from Materials and methods
the fertilized oocyte to the blastocyst stage [37]. In vitro culture
of human preimplantation embryos in culture media containing Study design
2 ng/ml of recombinant human GM-CSF (rhGM-CSF) has led
to an improved and accelerated embryonic development, an The present study is a prospective randomized trial. We in-
increase of the number of early-cleavage embryos that develop cluded couples presenting for ICSI treatment in our clinic.
to blastocyst, an increase of blastomere survival, an increase of Pools of post-ICSI unfertilized oocytes were randomly allo-
inner cell mass viability, a decrease in the number of apoptotic cated, by flipping a coin, into two groups: group A (expo-
nuclei, an increased hatching rate of blastocysts in vitro, and sure to calcium ionophore A23187 alone, n = 75) and group
generally, an enhancement of human blastocyst development B (exposure to calcium ionophore A23187 + GM-CSF sup-
in vitro [37–40]. Based on the above, GM-CSF has been con- plementation, n = 65). We used only unfertilized oocytes
sidered as a cytokine involved in human preimplantation em- showing the first polar body and no apparent pronucleus in
bryo development and has been highlighted as a major maternal the cytoplasm upon fertilization assessment by time-lapse
determinant of pregnancy outcome [41]. Recent studies con- imaging. Target characteristics of oocytes and produced em-
firm that GM-CSF supplementation of culture media enhances bryos in both groups were statistically analyzed, and the
the implantation rate and pregnancy rate in cases of repeated chromosomal constitution of the embryos was evaluated by
IVF failures [42] and this is also seen in a randomized clinical aCGH analysis. Patient consent and ethical approval were
trial [43]. obtained as described below in the BCompliance with ethical
While there are several studies highlighting the value of standards^ section. The experimental workflow is illustrated
calcium ionophore A23187 for AOA immediately after or in Fig. 1.
within a short time post ICSI, there are only two studies on
its use in salvaging aged human unfertilized oocytes, at either Patients
20 h post ICSI (combination of A23187 and puromycin) [17]
or 3 days post ICSI [16]. So far, only one live birth after All participating patients consented to donate unfertilized
activation of 1-day-old unfertilized oocytes with calcium ion- oocytes for inclusion in this study. These oocytes were
ophore A23187 has been reported [22]. collected from 66 controlled ovarian stimulation cycles.
J Assist Reprod Genet (2017) 34:33–41 35

Total oocytes
pronuclei in an oocyte during fertilization assessment, the
n=140 time-lapse video was cross-examined by a second embryolo-
gist and, if any doubt persisted, the oocyte was manually
checked outside the time-lapse system, under a high-power
Group A Group B
inverted microscope.
(A23187 alone) (A23187 + GM-CSF)
n=75 n=65
Oocyte activation with calcium ionophore A23187
and culture with rhGM-CSF
Activation rate assessment Activation rate assessment
(2PN + 1PN + DC) and (2PN + 1PN + DC) and
Cleavage rate assessment Cleavage rate assessment Oocyte activation was performed according to the following
(2PN activated oocytes) (2PN activated oocytes)
protocol: unfertilized oocytes in both groups were batch-
cultured in 50 μl drops of human tubal fluid medium
Culture of embryos to day 5 and Culture of embryos to day 5 and
assessment of their assessment of their
(Modified HTF, Irvine Scientific, Santa Ana, CA, USA) sup-
quality characteristics and quality characteristics and plemented with 10 % serum substitute supplement (SSS;
chromosomal status chromosomal status
Irvine Scientific, Santa Ana, CA, USA), containing 5 μM
Fig. 1 Schematic representation of the experimental workflow (2PN two calcium ionophore A23187 (Sigma, St. Louis, MO, USA;
pronuclei, 1PN one pronucleus, DC direct cleavage) stored at 1 mg/ml in dimethyl sulfoxide (DMSO), at
−20 °C). The drops were placed under tissue culture oil
Oocytes in group A derived from 30 couples with an av- (Sage In-Vitro Fertilization, Inc., Trumbull, CT, USA) in the
erage female age of 35.83 ± 5.10 years (median 37 years) dark and incubated at 37 °C for 10 min. Then, oocytes were
and an average male age of 39.83 ± 6.10 years (median thoroughly washed through five drops of fresh HTF medium
39.5 years). Oocytes in group B derived from 36 couples supplemented with 10 % SSS, previously equilibrated at
with an average female age of 36.61 ± 4.75 years (median 37 °C. Oocytes in group A were cultured in single 29.8 μl
37 years) and an average male age of 40.47 ± 5.45 years drops of culture medium (Sage 1-Step™, Origio a/s, Måløv,
(median 40 years). The number of treatment cycles per Denmark), in EmbryoSlide® (Vitrolife, Gothenburg, Sweden)
ICSI indication is shown in Table 1. culture dishes, under 1.4 ml of tissue culture oil (Sage In-Vitro
Fertilization, Inc., Trumbull, CT, USA). After activation with
Ovarian stimulation, oocyte collection, and fertilization A23187, oocytes in group B were cultured in 29.8 μl drops of
culture medium containing 2 ng/ml of rhGM-CSF (R&D
Ovarian stimulation was performed with the GnRH antagonist Systems, Minneapolis, MN, USA). The cytokine was stored
protocol, and oocytes were retrieved by ultrasound-guided as a stock solution at 2 μg/ml in Dulbecco’s Phosphate Buffer
transvaginal aspiration 36 h after 5000 IU β-human chorionic Saline (Gibco®) containing 0.1 % SSS, at −20 °C. Oocytes in
gonadotropin (β-hCG) administration. group B were cultured in the same way in the time-lapse
Standard ICSI procedure was performed [1]. After ICSI, system.
the injected oocytes were cultured in single drops of culture The time-lapse images were examined 18 h later for signs
media (Sage 1-Step™, Origio a/s, Måløv, Denmark) under oil of activation. Oocytes in either group, showing one or two
(Sage In-Vitro Fertilization, Inc., Trumbull, CT, USA) and pronuclei (PN) with one or two PBs (bipronucleate zygotes
observed by time-lapse imaging (EmbryoScope® time-lapse may rarely show only one PB due to degeneration or heavy
incubator, Vitrolife, Gothenburg, Sweden). fragmentation of the first PB) or undergoing direct cleavage
Oocytes having extruded the first polar body (PB), but after treatment with calcium ionophore A23187, were deemed
showing no pronuclei 18 h after ICSI, were deemed unfertil- activated; these were further cultured until developmental ar-
ized and would normally be discarded; instead, we used them rest in the time-lapse incubator, in order to assess embryo
for our study. In the event of doubt about the appearance of quality. Oocyte activation rate, cleavage rate, embryo quality,
development to day 5, and blastulation rate were calculated
and compared between the two groups. Embryo quality of
Table 1 Number of cleavage-stage embryos was categorized into four grades
treatment cycles per ICSI ICSI indications
(A–D) as follows: grade A, no fragmentation present and
indication
Oligozoospermia 28 equally sized blastomeres; grade B, <20 % fragmentation
Asthenoteratozoospermia 34 with similarly sized blastomeres; grade C, 20–50 % fragmen-
Obstructive azoospermia 2 tation with unequally sized blastomeres; and grade D, >50 %
Antisperm antibodies 1 fragmentation with unequally sized blastomeres [17].
Unexplained infertility 1 Blastocyst-stage embryo quality was assessed and categorized
according to a well-defined scoring system [45], assessing the
36 J Assist Reprod Genet (2017) 34:33–41

extent of blastocoel expansion, and the inner cell mass and Statistical analysis
trophectoderm quality. Grade A and B cleavage-stage embry-
os and grade 4AA, 4AB, 4BA, and 4BB blastocysts were The activation rate, cleavage rate, high embryo quality rate,
considered high quality embryos. day 5 development rate, and blastulation rate were compared
between groups A and B by using Pearson’s chi-square test
(two-tailed) or Fisher’s exact test (two-tailed) as appropriate
Whole embryo biopsy and preparation for aCGH analysis (depending on the expected cell count). All p values in the
BResults^ section below are chi-square test values (except
All embryos analyzed by aCGH were morula and blastocyst- those marked [F] for Fisher’s exact test). SPSS Statistics
stage embryos from either group A or B, derived from two (Statistics Package for Social Sciences, version 17) was used
pronuclei (2PN) or from direct cleavage, 16–18 h after activa- for all statistical analyses. A p value of <0.05 was considered
tion with calcium ionophore A23187. statistically significant.
In order to avoid any residual contamination from cumulus
or corona cells, the zona pellucida of all analyzed embryos
was removed by micromanipulation, using a non-contact laser Results
system (Saturn, Research Instruments Ltd., Cornwall, UK).
Holding the embryo firmly by gentle suction with a holding The effect of calcium ionophore A23187 on oocyte
pipette (Research Instruments Ltd., Cornwall, UK) at 9 activation of unfertilized oocytes after ICSI
o’clock, the zona pellucida was completely cut from 12 to 6
o’clock clockwise using the laser, and the embryo was re- In the present study, a total of 140 oocytes deemed unfertilized
moved from the remaining zona pellucida (whole embryo after ICSI by monitoring in the time-lapse system were ran-
biopsy) by gentle suction using a biopsy pipette (The Pipette domly allocated in either group A or B (see Fig. 1). In group
Company (TPC), Thebarton, South Australia). The procedure A, 75 oocytes were treated with calcium ionophore A23187.
was performed in microdrops of buffered culture medium In group B, 65 oocytes were initially treated with A23187 as
(Modified HTF, Irvine Scientific, Santa Ana, CA, USA) under in group A and then cultured in medium supplemented with
tissue culture oil (Sage In-Vitro Fertilization, Inc., Trumbull, rhGM-CSF. In group A, 32/75 oocytes were activated
CT, USA). (42.66 %), of which 19 showed 2PN/two polar bodies (2PB)
The denuded embryos were then transferred in the mini- (59.37 %), 8 showed 1PN/2PB (25.00 %), and 5 were directly
mum possible volume of medium (less than 0.5 μl) to 0.2-ml cleaved (15.62 %). In group B, 35/65 oocytes were activated
PCR tubes containing 2 μl phosphate-buffered saline (53.84 %), of which 20 showed 2PN/2PB (one activated oo-
(BlueGnome, Cambridge, UK) in a sterile hood. A pulled cyte showed two distinct pronuclei and three satellite
sterilized micropipette was used for this transfer, and embryos micronuclei) (57.14 %), 4 showed 1PN/2PB (11.43 %), and
were observed under a stereomicroscope throughout the entire 11 were directly cleaved (31.42 %). No differences were ob-
transfer procedure. A different sterile micropipette was used to served (see Fig. 2) in the activation rate between the two
collect a negative wash control drop. groups (p = 0.187), nor was any statistically significant differ-
ence observed between the 2PN/2PB rate (p = 0.853), the
1PN/2PB rate (p = 0.148), and the directly cleaving embryos
Whole genome amplification and aCGH analysis (p = 0.130). The cleavage rate of the 2PN/2PB activated oo-
cytes 24–48 h post activation was 15/19 (78.94 %) in group A
Whole genome amplification (SurePlex, Illumina, Inc.) and and 20/20 (100 %) in group B, which was statistically signif-
aCGH (24sure V3) analysis of morulae and blastocysts was icant (p = 0.047 [F]) in favor of group B (Fig. 2). There was no
carried out according to the manufacturer’s instructions and statistical significance in the cleavage rate of 1PN/2PB oo-
as previously described [46]. The slides were scanned using cytes between the two groups (p = 0.333 [F], data not shown).
a 10-μm laser scanner (Innopsys 710A; Innopsys S.A.,
Carbonne, France), and the images analyzed with the The effect of rhGM-CSF on embryo quality
BlueFuse Multi software (BlueGnome, Cambridge, UK). and developmental potential of the embryos produced
The aCGH ratio plots of each embryo were analyzed for after activation with A23187
gains and losses by at least two independent assessors, to
determine the euploid or aneuploid status of each embryo. After activation with calcium ionophore A23187, the oocytes
The chromosomal status of an embryo showing three to four were further cultured in the time-lapse system, either alone
aneuploidies was defined as complex; embryos showing (group A) or in the presence of rhGM-CSF (group B), for
more than four aneuploidies were defined as complex 5 days after PN appearance or after direct cleavage. In group
chaotic. A, 6/32 (18.75 %) activated oocytes resulted in high quality
J Assist Reprod Genet (2017) 34:33–41 37

Fig. 2 Effect of A23187 (group

b
A) and combination of A23187 Group A (A23187)
and rhGM-CSF (group B) on Group B (A23187 + GM-CSF)
oocyte activation and subsequent 100%
100%
embryonic development (p > 0.05
(a); p < 0.05 (b–e))

79%
80%

a c

Percentage
60% 57%
54%
e

43% d
40%
40%
32%

19%
20%
13%

6%

0%
Activation Rate Cleavage Rate High Quality Day 5 Development Blastulation Rate
2PN/2PB Embryos
AOA Characteristics

embryos, whereas in group B, 20/35 (57.14 %) activated oo- abnormal. Two of the 2PN day 5 embryos in group A showed
cytes resulted in high quality embryos; this difference was a double aneuploidy, while the third had complex chromo-
found to be highly significant (p = 0.001, see Fig. 2). In group somal abnormalities (chaotic embryo). The directly cleaving
A, 5/6 (83.33 %) of the top quality embryos derived from the embryo in group A also showed a single aneuploidy (trisomy
2PN/2PB activated group, while the sixth high quality embryo 22).
derived from a directly cleaving activated oocyte (16.66 %). In group B, five of the 2PN-stage embryos were found to
In group B, 11/20 (55.00 %) top quality embryos derived from be chromosomally normal for all 23 pairs of chromosomes
2PN/2PB embryos, 8/20 (40.00 %) derived from directly (autosomes and sex chromosomes). All five embryos pro-
cleaving embryos, and 1/20 (5.00 %) from 1PN/2PB embryos. duced good quality blastocysts on day 5. The rest of the
In group A, 4/32 embryos (2 blastocysts and 2 morulae; 2PN-stage and directly cleaving embryos were found to be
12.5 %) bypassed the day 3 developmental arrest stage and chromosomally abnormal, with six embryos showing single
reached the day 5 stage, while in group B, 14/35 embryos (11 aneuploidies, three embryos showing complex chromosomal
blastocysts and 3 morulae; 40.00 %) reached the day 5 stage. abnormalities (two of them being chaotic with more than four
This difference was also found to be statistically significant chromosomes abnormal), one embryo showing complex ab-
(p = 0.011, see Fig. 2). In group A, 2/32 (6.25 %) embryos normalities, and two embryos being chaotic. No chromosom-
reached the blastocyst stage on day 5, while in group B, 11/35 ally normal embryo that derived from directly cleaving em-
(31.42 %) embryos reached the blastocyst stage 5 days after bryos was identified in either group.
activation; this was found to be highly significant (p = 0.009, As regards to the 2PN-stage embryos, it is obvious
see Fig. 2). that while none of the 2PN-stage embryos that reached
the day 5 stage were chromosomally normal (0/3–0 %)
Array CGH chromosomal analysis of day 5 embryos in group A, 5/8 (62.5 %) of the 2PN-stage embryos that
in the two groups reached the day 5 stage in group B were chromosomally
normal.
In group A, 4 embryos (2 blastocysts and 2 morulae) that Of the analyzed embryos in group A, one was confirmed as
reached the day 5 stage were analyzed by aCGH to determine male and one as female and two showed a sex chromosome
their chromosomal status; in group B, 14 embryos (11 blasto- abnormality; in group B, five embryos were male, seven were
cysts and 3 morulae) were also analyzed. female, and two showed a sex chromosome abnormality.
As summarized in Table 2, the analysis of day 5 embryos in Three of the male embryos in group B were among those with
group A showed that all four embryos were chromosomally a normal chromosomal status.
38 J Assist Reprod Genet (2017) 34:33–41

Table 2 Characteristics and

aCGH results of day 5 embryos Group Embryo no. Activation status Embryo quality Sex aCGH outcome
created in groups A and B
A 1 2PN Blastocyst, 4CC Turner (XO) Mon 7, Mon X
A 2 2PN Morula Male Mon 6, Tri 15
A 3 2PN Morula Klinefelter (XXY) Complex Chaotic
A 4 DC Blastocyst, 4BA Female Tri 22
B 1 2PN Blastocyst, 4AB Female 46,XX
B 2 2PNa Blastocyst, 4AA Male 46,XY
B 3 2PN Blastocyst, 4AA Male 46,XY
B 4 2PN Blastocyst, 4BB Female 46,XX
B 5 2PN Blastocyst, 4BB Male 46,XY
B 6 2PN Blastocyst, 3CC Triple X (XXX) Complex
B 7 2PN Morula Male Complex chaotic
B 8 2PN Morula Female Mon 6, Mon 22
B 9 DC Blastocyst, 4BB Turner (XO) Mon 8, Mon 10
B 10 DC Morula Female Complex chaotic
B 11 DC Blastocyst, 4BA Female Mon 18, Mon 19
B 12 DC Blastocyst, 3CB Male Mon 19, Tri 20
B 13 DC Blastocyst, 5AA Female Mon 19
B 14 DC Blastocyst, 5AA Female Tri 20

DC direct cleavage, 2PN two pronuclei, Mon monosomy, Tri trisomy

a
This embryo (B2) showed two distinct pronuclei and three satellite micronuclei

Discussion difference in favor of group B in the cleavage rate of 2PN/

2PB activated oocytes, in high quality embryo formation rate,
An unexpectedly low fertilization rate, or complete PIFF, is in the potential of embryos produced to develop to the day 5
perhaps the worst case scenario for couples undergoing stage, and in blastulation rate. These results suggest that
assisted reproductive treatment. An effective treatment for rhGM-CSF may significantly improve the developmental po-
cases of previous partial or complete PIFF with the use of tential not only of embryos derived from conventional ICSI
calcium ionophore A23187 for artificial oocyte activation but also of embryos produced after AOA with calcium iono-
(AOA) has already been established [20]. This technique has phore A23187 of 18-h aged unfertilized oocytes after ICSI.
been reported to result in improved fertilization rates [18, 47], Nevertheless, the activation rate for both groups is lower than
embryonic developmental potential [8], pregnancy rates [48, the one reported in a previous study [17], and this could be
49], and live birth rates [20, 21]. However, the safety of the explained by the higher mean age of our patients.
AOA technique has been questioned [50, 51]. The present study is also the first one where the full chro-
The present study is the first to examine the effect of AOA mosomal complement of human embryos derived from AOA
using the calcium ionophore A23187 in combination with of aged oocytes is analyzed by array comparative genomic
rhGM-CSF supplementation on 18-h aged human unfertilized hybridization (aCGH). A secondary endpoint of our study
oocytes after ICSI. Our primary endpoint was to apply a mod- was indeed to examine the safety of the modified protocol.
ified AOA protocol on aged human unfertilized oocytes 18 h In the majority of AOA studies, the calcium ionophore
after ICSI, using calcium ionophore A23187 and then cultur- A23187 is applied a short time after ICSI [18, 20, 21, 29], in
ing the embryos produced with rhGM-CSF. The goal was to cases of previously known partial or complete PIFF. Failed
compare this protocol to standard AOA, in which A23187 oocyte activation after ICSI can be foreseen to a certain extent,
alone is applied to activate the oocytes artificially, and then by assessing the relative expression of PLCζ protein [52].
embryos are cultured in a standard medium. However, complete or near-complete fertilization failure can-
We cultured the oocytes with the calcium ionophore A23187 not be predicted for all fresh cycles in the assisted reproduc-
under oil at 37 °C, as per standard IVF laboratory practice. This tion clinic and the cause of PIFF may be only examined a
may introduce a limitation, since the ionophore is soluble in oil posteriori [53]. This limits the use of calcium ionophore
and could therefore partly leak out of the medium; however, given A23187 in AOA only to known cases with previous fertiliza-
the short incubation time (10 min), this depletion seems unlikely. tion problems. The ideal strategy would be to develop a pro-
The comparison of the two study groups showed no differ- tocol that would allow AOA and salvage of unfertilized oo-
ence in the oocyte activation rate. We report a significant cytes 18 h after ICSI, should partial or complete fertilization
J Assist Reprod Genet (2017) 34:33–41 39

failure occur. However, this may only have a clinical value if activated unfertilized oocytes after ICSI, as has been previous-
the procedure is safe. Indeed, most of the studies on AOA ly reported [17]. Our results indicate that AOA in combination
safety have been conducted in cases where the AOA stimulus with rhGM-CSF supplementation can lead to the safe activa-
was applied during or immediately after ICSI [54, 55]. tion of unfertilized oocytes and to the development of chro-
Furthermore, if the procedure proves to be safe by appropriate mosomally healthy blastocysts on day 5.
studies, it may lead to yet another general source of human In conclusion, we applied and compared two AOA proto-
embryonic stem cell lines [56] for therapeutic purposes. cols on 18-h aged human unfertilized oocytes after ICSI: a
We found that only embryos derived from group B were traditional protocol, in which oocytes were briefly treated with
euploid, while no day 5 embryos were chromosomally normal calcium ionophore A23187 alone, and a modified protocol, in
in group A. The presence of euploid embryos in group B only which oocytes were cultured with rhGM-CSF after activation
should be considered as a random finding, due to the low with A23187. By using time-lapse and aCGH technologies,
number of group A embryos reaching the desired develop- we demonstrated that activating 18-h aged unfertilized oo-
mental stage on day 5, in order to be analyzed by aCGH. On cytes by the combination of A23187 and rhGM-CSF can lead
the other hand, rhGM-CSF may have exerted a direct positive to the development of 2PN/2PB embryos showing a high
effect. It is well known that mitotic chromosomal errors are a cleavage rate and having the potential to develop into euploid,
common aneuploidy source in all developmental stages of good-morphology blastocysts on day 5. If our findings are
human preimplantation embryos [57–59]. Cytokinesis errors confirmed by further and larger-scale studies, blastocysts de-
are a common cause for mitotic aneuploidies [60–64]. Failed rived from aged unfertilized oocytes salvaged with the tech-
or asymmetric cytokinesis results in the formation of binucle- nique described above could be used clinically for embryo
ated cells, tetraploidy or spindle pole abnormalities, and chro- transfer, in cases of unexpected partial or complete post-ICSI
mosomal chaos [65]. The supplementation of the culture me- fertilization failure.
dium with rhGM-CSF, a cytokine that promotes proper cell
division and cytokinesis in general, may have contributed in Acknowledgments The authors wish to thank Mr. Christos
limiting or eliminating such errors, therefore yielding a higher Karamalegos for the help with statistical analysis and Ms. Maria
rate of euploid embryos in group B. This GM-CSF effect on Papadopoulou for collecting the patient consent forms.
alleviating chromosomal errors needs to be examined further.
We were able to accurately identify the artificially activated Compliance with ethical standards
oocytes that produced two pronuclei, and to distinguish them
from the directly cleaving ones, by observing the culture in the Conflict of interest The authors declare that they have no conflict of
interest.
time-lapse system. Among the 2PN/2PB euploid embryos in
group B, we identified embryo B2 (see Table 2), which Ethical approval This article does not contain any studies with human
showed two distinct pronuclei and three satellite micronuclei participants or animals performed by any of the authors.
after activation. It is known that aCGH technology cannot The study was approved by the institutional research ethics commit-
detect polyploidy, resulting in an estimated 0.2 % missed ab- tee, and then it was submitted to and obtained authorization by the
Hellenic National Authority of Medically Assisted Reproduction (autho-
normalities [66]. Despite the theoretical chance of embryo B2 rization ref. 134/15), as required by national legislation for research use of
being uniformly polyploid due to the presence of the human embryos not destined to embryo transfer.
micronuclei, the distinctive male sex of the embryo leads us
to consider that it is euploid. Informed consent All patients who donated oocytes and embryos to be
In addition, only the day 5 embryos in group B that devel- included in the study signed consent forms, after receiving exhaustive
oped from 2PN/2PB artificially activated oocytes were found to information on the purpose of the research carried out, on the procedures
to be used, on the possibility to withdraw their consent at any time up to the
be chromosomally normal by aCGH analysis, while no embry- first actual use of each oocyte or embryo in an experiment, and on the legal
os developed from oocytes activated by direct cleavage were and ethical obligation of the authors to destroy all embryos after comple-
euploid, despite their good morphology on day 5 (Table 2). tion of the experiments, without attempting to transfer them in vivo.
Some of the directly cleaving embryos in group B may have
resulted from the cytokinetic effect of GM-CSF itself. In a non-
AOA study [67], direct cleavage has been correlated to aneu-
ploidy and our study confirms this finding as well.
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