doi:10.1111/jog.12916 J. Obstet. Gynaecol. Res. Vol. 42, No.

4: 417–421, April 2016

Comparison of normal and abnormal fertilization of in vitro-

matured human oocyte according to insemination method

Ju Hee Park1, Byung Chul Jee2,3 and Seok Hyun Kim3,4

1
M fertility Center, 3Department of Obstetrics and Gynecology, Seoul National University College of Medicine, 4Department of
Obstetrics and Gynecology, Seoul National University Hospital, Seoul, and 2Department of Obstetrics and Gynecology, Seoul National
University Bundang Hospital, Seongnam, Korea

Abstract
Aim: Our purpose was to compare the normal fertilization rate, multi-pronuclei (PN) formation rate, and embry-
onic development of in vitro-matured oocytes between conventional insemination and intracytoplasmic sperm
injection (ICSI).
Methods: A total of 213 stimulated in vitro fertilization (IVF) cycles were selected, in which at least one immature
oocyte was obtained (from 2010 to 2014). Immature oocytes were assigned to germinal vesicle (GV)-stage or meta-
phase I (MI)-stage oocyte groups. Cycles with obligatory ICSI due to male-factor infertility were excluded. Cycles
were divided into two groups according to fertilization method: there were 97 cycles with conventional insemina-
tion and 116 cycles with ICSI. After in vitro maturation of 324 GV-stage oocytes and 341 MI-stage oocytes, the fer-
tilization rate, multi-PN formation rate, and embryonic development were compared according to the fertilization
method.
Results: The normal fertilization rate was similar in the conventional insemination and the ICSI both in
GV-derived and MI-derived oocytes. Both fertilization methods resulted in a similar multi-PN formation rate in
GV-derived oocytes; however, in MI-derived oocytes, the multi-PN formation rate was zero with ICSI and this
was significantly lower than that with conventional insemination (9.6%, P = 0.001).
Conclusion: In non-male-factor infertility, ICSI should be considered when MI oocytes are matured.
Key words: fertilization, immature oocyte, intracytoplasmic sperm injection.

Introduction we have routinely performed conventional insemination

In most laboratories, intracytoplasmic sperm injection
(ICSI) is the standard procedure for fertilization of
in vitro-matured oocytes. This practice was developed
mainly based on two comparative studies where the fer-
tilization rate (FR) of in vitro-matured oocytes obtained
from unstimulated in vitro fertilization (IVF) cycles al-
most doubled by using the ICSI method.1,2 However,
we previously demonstrated a similar FR between con-
ventional insemination and the ICSI method when ger-
minal vesicle (GV)-stage oocytes obtained from a
stimulated IVF cycle were matured in vitro.3 Since then,
for fertilization of in vitro-matured oocytes obtained
from stimulated IVF cycles.
There has been an increase in ICSI use in the IVF
population even without male-factor infertility.4
Although controversy exists, ICSI has been commonly
used in couples with low FR, repetitive implantation
failure, and in several cases of female infertility
(advanced maternal age, presence of endometrioma)
or oocyte factor (low oocyte yield, poor quality of
oocyte).5,6 Thus, a significant portion of in vitro-matured
oocytes are being fertilized by the ICSI method in
our center.

Received: June 30 2015.

Accepted: October 31 2015.
Correspondence: Dr Byung Chul Jee, Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 166 Gumi-ro,
Bundang-gu, Sungnam 463-707, Korea. Email: blasto@snubh.org

© 2016 Japan Society of Obstetrics and Gynecology 417

J. H. Park et al.
A similar FR between conventional insemination and
ICSI in our previous study suggests that a GV-stage
oocyte has no substantial zona hardening during
in vitro maturation, if they are obtained from a stimulated
IVF cycle. Our previous findings are different from
common belief; exposure of oocytes to artificial conditions
of in vitro culture seems to be implicated in zona hardening,
resulting in low FR by conventional insemination.7
However, our previous study had some limitations:
only a relatively small number of GV-stage oocytes
was included and we focused on normal FR only.
Metaphase I (MI) oocytes generally show a faster mat-
uration and a higher maturation rate than GV oocytes,8
thus MI oocytes are less exposed to in vitro culture and
this may affect the extent of zona hardening. In respect
to zona behavior, multi-pronuclei (PN) formation is
another indication for zona normalcy.9 One of the
consequences of conventional insemination in denuded
oocytes is polyspermy, which may cause zygotes with
3PN or more. However, we did not investigate the PN
formation rate in our previous study.
In the present study, we retrospectively compared the
normal FR, multi-PN formation rate, and subsequent
embryonic development of in vitro-matured oocytes
between conventional insemination and ICSI in stimu-
lated IVF cycles. We also analyzed the fertilization and
embryonic development rate of in vivo-matured oocytes
according to insemination method.
Methods
This retrospective study included 213 stimulated fresh
IVF cycles, where at least one GV stage or MI oocyte
was obtained. This study was approved by the Institu-
tional Review Board of the Seoul National University
Bundang Hospital (B-1506-302-124). All cycles were
planned to transfer embryos on day 3 and performed
during a period from 2010 to 2014 at the Seoul National
University Bundang Hospital. Cycles with obligatory
ICSI due to male-factor infertility were excluded. Cycles
were divided into two groups according to fertilization
method: there were 97 cycles with conventional insemi-
nation and 116 cycles with ICSI.
Our indications for ICSI in non-male-factor infertility
were as previously described6: (i) ≤20% FR in a prior con-
ventional insemination cycle; (ii) repetitive implantation
failure ≥ 3 times; (iii) advanced maternal age (≥40 years);
(iv) presence of endometrioma on the day of oocyte
retrieval; (v) low oocyte yield (number of oocytes ≤ 3);
or (vi) poor-quality oocytes that included blood clots
418
attached to the cumulus oophorus at the time of
retrieval. The decision to perform ICSI was at the discre-
tion of the attending physician and embryologist.
Ovarian stimulation was performed using recombi-
nant follicle-stimulation hormone (rFSH; Gonal-F;
Serono) under the gonadotrophin-releasing hormone
(GnRH) antagonist protocol or the long GnRH agonist
protocol. After two or more follicles reached a diameter
≥18 mm, 250 μg of recombinant human chorionic gonad-
otrophin (hCG; Ovidrel; Serono) was injected. The
oocyte was retrieved 36 h after the hCG injection.
The cumulus-oocyte complexes (COC) were collected
and the maturity was assessed according to the presence
or absence of a GV or the first polar body (PB) by
inverted microscope (×200). Usually, oocyte maturity
could be easily identified under stereomicroscope on
the basis of cumulus pattern. In situations with unclear
maturity due to dark COC or blood clots, the oocytes
were denuded by using 85 IU/mL hyaluronidase (Cook)
and mechanical pipetting. Immature oocytes were de-
fined by the absence of the first PB and then classified
as GV-stage or MI-stage depending on visible GV.
Isolated GVand/or MI stage oocytes were then cultured
in maturation medium (Cook-BL, Cook) supplemented
with rFSH 75 mIU/mL (Serono), rhCG 0.5 IU/mL
(Serono) and rEGF 10 ng/mL (Invitrogen). All oocytes
were cultured in 1 mL of maturation medium for up
to 48 h in an atmosphere of 5% CO2 and 95% air with
high humidity. After in vitro maturation, they were
stripped with 80 IU/mL hyaluronidase and mechanical
pipetting until completely denuded of their cumulus
cells. Maturation was considered when they had the
first PB. The matured oocytes were then fertilized by
the conventional method or ICSI. Normal fertilization
was confirmed when two distinct PN and a second PB
were present 16–18 h later. The fertilized oocytes were
maintained in the culture medium (Sydney IVF cleav-
age medium; Cook).
The main outcome measures were the normal FR
(2PN per matured oocyte), multi-PN formation rate
(3PN or more per matured oocyte), cleavage rate per
2PN, and embryo grade at day 3. The quality of embryos
was evaluated by morphological criteria based on the
fragmentation degree and the regularity of blastomeres
on day 3 after fertilization. The embryo grades were as
follows: A, 0% anucleate fragments, regularity of blasto-
meres, and no apparent morphologic abnormalities; B,
<20% anucleate fragments, regularity of blastomeres,
and no apparent morphologic abnormalities; C,
20–50% anucleate fragments, irregularity of blastomeres,
and no apparent morphologic abnormalities; and D,
© 2016 Japan Society of Obstetrics and Gynecology
 Fertilization of in vitro-matured human oocyte

>50% anucleate fragments, irregularity of blastomeres,
and apparent morphologic abnormalities.
All statistical analyses were performed using SPSS 18.
The data were analyzed using the Student’s t-test or
χ 2-test as indicated. The result was considered significant
when the P-value was <0.05.
Results
The mean age of women was significantly higher in ICSI
cycles (Table 1). This was mainly caused by a higher
proportion of age factor (≥40 years) and/or decreased
ovarian reserve in ICSI cycles.
Among 213 stimulated IVF cycles, 324 GV-stage
oocytes, 341 MI-stage oocytes, and 691 in vivo-matured
oocytes were obtained. Low MII recovery rate (51%)
was observed, but this was not real oocyte maturity in
our center, because cycles where mature oocytes only
were obtained were excluded from this study.
As shown in Table 2, the overall maturation rate of
immature oocytes was almost doubled in MI oocytes
(82.1%, 280/341) when compared with GV oocytes
(42.9%, 139/324) (P < 0.001). The normal FR was similar
between conventional insemination and the ICSI
method both in GV-derived and MI-derived oocytes.
Both insemination methods resulted in a similar multi-
PN formation rate in GV-derived oocytes; however, in
MI-derived oocytes, the multi-PN rate was zero with
ICSI and this was significantly lower than that with
conventional insemination (9.6%, P < 0.001). The
embryo cleavage rate and embryo grade or development
(≥6-cell) at day 3 was similar in the conventional insem-
ination and ICSI both in GV- and MI-derived oocytes.

Table 1 Basal characteristics of non-male-factor IVF cycles according to fertilization method

Conventional IVF ICSI P
Cycles 97 116
Age (years) 34.4 ± 3.4 37.0 ± 4.4 <0.05
Infertility factors
Tubal/peritoneal 31 (32.0%) 24 (20.7%) NS
Unexplained 24 (24.7%) 23 (19.8%) NS
Ovulatory 14 (14.4%) 8 (6.9%) NS
Endometriosis 10 (10.3%) 22 (19.0%) NS
Uterine 13 (13.4%) 5 (4.3%) <0.05
DOR or old age† 5 (5.2%) 34 (29.3%) <0.05
Mean ± standard deviation. †Older than 40 years. DOR, decreased ovarian reserve; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertiliza-
tion; NS, not significant.

Table 2 Fertilization and embryonic developmental outcomes of immature or in vivo-matured oocytes according to fertilization
method
GV-stage oocyte MI-stage oocyte In vivo-matured oocyte
Conv ICSI Conv ICSI Conv ICSI
Cycles 66 72 90 94 93 96
Oocyte 176 148 186 155 422 269
Matured (%/oocyte) 76 (43.2%) 63 (42.6%) 156 (83.9%) 124 (80.0%) – –
2PN (%/mature) 45 (59.2%) 41 (65.1%) 117 (75.0%) 102 (82.3%) 343 (81.3%) 241 (89.6%)*
Multi-PN (%/mature) 2 (2.6%) 1 (1.6%) 15 (9.6%) 0 (0%)** 43 (10.2%) 2 (2.1%)**
Cleaved at D3 (%/2PN) 40 (88.9%) 31 (75.6%) 110 (94.1%) 93 (91.2%) 326 (95.1%) 234 (97.1%)
Embryo grade at D3
A 7 5 22 22 119 80
B 15 12 40 33 128 81
(%A+B/cleaved) (55.0%) (54.9%) (56.4%) (59.2%) (75.8%) (68.8%)
C 16 10 34 30 59 60
D 2 4 14 8 20 13
Cleaved ≥6-cell at D3 15 10 68 60 272 192
(%/cleaved) (37.5%) (32.3%) (61.9%) (64.6%) (83.5%) (82.1%)
*P < 0.005 and **P < 0.001 when compared between conventional insemination and ICSI group. Conv, conventional insemination; D3, day 3; GV,
germinal vesicle; ICSI, intracytoplasmic sperm injection; MI, metaphase I; PN, pronucleus.

© 2016 Japan Society of Obstetrics and Gynecology 419

J. H. Park et al.
In in vivo-matured oocytes, ICSI resulted in a
significantly higher normal FR and a significantly lower
multi-PN formation rate than those with conventional
insemination (Table 2). However, embryo cleavage rate
and embryo grade or development (≥6-cell) at day 3
was similar in conventional insemination and ICSI
methods.
Discussion
In the present study, normal FR was similar in conven-
tional insemination and ICSI in GV-derived oocytes. This
finding is consistent with our previous study.3 Here, we
have shown a similar multi-PN formation rate in
GV-derived oocytes. Interestingly, MI-derived oocytes
showed a similar normal FR in the two fertilization
methods but the multi-PN formation rate was signifi-
cantly lower with ICSI (0%) than with conventional
insemination (9.6%). Conventional insemination in MI-
derived oocytes yielded frequent multi-PN formation,
which was much higher than conventional insemination
of GV-derived oocytes (2.6%). This finding indicates that
MI-derived oocytes are prone to allow polyspermy, thus
showing abnormal zona behavior. Therefore, two kinds
of immature oocytes seem to have different zona charac-
teristics when they are matured in vitro.
In vitro maturation has been a practice of intentionally
retrieving immature oocytes from small antral follicles
and maturing them in vitro. However, after ovarian stim-
ulation, some of the retrieved oocytes are often imma-
ture. In spite of adequate ovarian stimulation,
immature oocytes were more frequently retrieved in a
GnRH antagonist cycle than in a GnRH agonist cycle.10
This may be the consequence of less follicular synchroni-
zation in a GnRH antagonist cycle.11
Immature oocytes obtained from stimulated cycles
can be used as surplus oocytes by maturing in vitro,
which is termed as ‘rescue in vitro maturation.’12 Based
on the assumption that oocyte maturity is a prerequisite
for obtaining normal fertilization, attempts have been
made to mature GV and MI oocytes in vitro.12 Despite
the use of various culture techniques and oocytes from
different treatment procedures, in vitro-matured
oocytes show commonly lower FR compared with
in vivo-matured oocytes.13,14 A relatively higher matura-
tion and FR of immature oocytes was observed in this
study. Although the FR of GV-derived oocytes (59.2%)
was significantly lower than that of in vivo-matured
oocytes (81.3%), the FR of MI-derived oocytes (75%)
was similar to that of in vivo-matured oocytes, even by
420
conventional insemination. We previously reported a
relatively higher FR (68.4%) in GV-derived oocytes.3
We think that the key point was medium for in vitro
maturation. We have used commercial Cook-BL
medium, supplemented with rFSH 75 mIU/mL, rhCG
0.5 IU/mL and rEGF 10 ng/mL. Efforts should be con-
tinued to find optimal culture conditions for immature
oocytes in clinical perspectives.
In the present study, the overall maturation rate was
almost doubled in MI oocytes when compared with
GV oocytes. This finding is consistent with our previous
report.8 In in vivo-matured oocytes, ICSI resulted in a
significantly higher normal FR and significantly lower
multi-PN formation rate than conventional insemina-
tion. This finding is also consistent with our recent
report6 and this supports the benefit of ICSI in the
non-male-factor IVF population.
The main limitation of this study was that the basal
characteristics of infertile women could not be controlled.
Various infertility etiologies and various indications of
ICSI were included. The mean age of women in the ICSI
group was higher, thus further age-adjusted study is
needed. The absence of pregnancy outcomes from use
of immature oocytes is another limitation. Usually, the re-
sultant embryos seldom participated in embryo transfer,
and moreover, it was extremely rare that only embryos
derived from immature oocytes were transferred.
In summary, both fertilization methods could be applied
in GV-derived oocytes, but ICSI is favored in MI-derived
oocytes because ICSI could prevent multi-PN formation.
Further investigations are required to determine the charac-
teristics of zona pellucida in in vitro-matured oocytes.
Acknowledgments
This work was supported by a grant (A120043) from the
Korea Health Care Technology R&D Project, Ministry of
Health and Welfare, Korea.
Disclosure
We hereby declare that we have no conflict of interest
and have nothing to disclose.
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