In-vitro maturation of human oocytes:
before or after vitrification?
Giovanna Fasano & Isabelle Demeestere & Yvon Englert
Received: 18 January 2012 /Accepted: 16 March 2012
#Springer Science+Business Media, LLC 2012
Purpose This study aims to determine if in-vitro maturation
(IVM) of human immature oocytes should be performed
before or after vitrification.
Methods A total of 184 immature oocytes were randomly
divided into two different groups: 100 were vitrified at
metaphase II (MII) stage 24 h-48 h after IVM (group 1)
and 84 were immediately vitrified at germinal vesicle (GV)
or metaphase I (MI) stages and in vitro matured after warm-
ing (group 2).
Results Survival rate after warming was similar in both
groups (86.9% versus 84.5%). However, oocyte maturation
rate per collected oocyte was significantly higher for
oocytes matured before vitrification (group 1, 46%) than
for oocytes vitrified before IVM (group 2, 23.8%) (p<0.01).
Consequently, the number of MII oocytes inseminated per
oocyte collected was significantly higher for group 1 (40%)
than for group 2 (23.8%) (p<0.05).
Conclusion IVM procedure is more efficient when it is
performed before oocyte vitrification.
The development of an effective cryopreservation program
for immature oocytes may have a major impact on in vitro
fertilization (IVF) clinical practice, especially to preserve the
fertility of women who will be treated with cytotoxic drugs
[1,2] orfor women withpolycystic ovarian syndrome(PCOS)
at risk of ovarian hyper-stimulation syndrome (OHSS) [3–5].
Moreover, for cancer patients, transplantation of cryopre-
served ovarian tissue is not always conceivable because of
the risk of retransmission of the disease due to the presence of
neoplasic cells in the tissue. In these cases, combining IVM
and vitrifcation offers a novel approach for fertility preserva-
tion, especially when the risk and the delay due to classical
gonadotrophin stimulation beforecollecting mature oocytes is
not indicated, as in advanced breast cancer patients .
Healthy infants have been born following IVM [7–9].
However, improving the IVM success rate remains an im-
portant challenge to obtain better efficiency for fertility
preservation. Moreover, vitrification is a widely applied
and highly successful approach for cryopreservation in re-
productive biology, including storage of human oocytes
[10–12]. Recently, studies reported high morphological sur-
vival rates after vitrification of mature oocytes, with in vitro
embryo development, implantation and pregnancy rates
comparable to those achieved with fresh oocytes [13–15].
The timing to vitrify the oocytes for long-term storage may
however significantly affect the efficiency of the IVM pro-
cess. Importantly, the maturation stage at which oocytes are
vitrified may impact the efficiency of the IVM process. We
previously showed that there was no significant difference in
Capsule The adequate timing for in vitro maturation was evaluated by
comparing maturation and survival rates of oocyte vitrified before or
after the procedure.
G. Fasano:I. Demeestere:Y. Englert
Research Laboratory on Human Reproduction, Medicine Faculty,
Université Libre de Bruxelles (ULB), Belgium,
G. Fasano (*)
Fertility Clinic, Department of Obstetrics and Gynaecology,
Erasme Hospital, Free University of Brussels, Belgium,
Route de Lennik 808,
Brussels 1070 Belgium
J Assist Reprod Genet
survival rates between oocytes vitrified at GV and oocytes
vitrified at MI (86.3% vs 76.5%, respectively). Oocytes vitri-
fied at GV stage gave, however, significantly worse results of
in vitro maturation (6.8%) than the oocytes vitrified at MI
stage (43.2%) (p<0.01) .
In the present study, we assessed the efficiency of IVM
before and after vitrification of human immature oocytes.
Materials and methods
Experimental design and patients
Between May 2009 and February 2010, a total of 184
immature oocytes at the GV or MI stages were obtained
from 83 patients who underwent an intracytoplasmic sperm
injection (ICSI) cycle. All MII oocytes retrieved were used
for patients’ treatments, while immature oocytes, according
to a previously prepared randomization list established on a
weekly basis, were randomly allocated to one of the groups
and vitrified after in-vitro maturation (group 1) or immedi-
ately (group 2) (Fig. 1).
All patients included were thoroughly informed and
signed written consent forms. The study was accepted by
the ULB University Ethical Committee, by the Erasme
Hospital Ethical Committee and by the Belgian Federal
Research and Scientific Committee.
Stimulation protocol and oocyte retrieval procedures
Pituitary down regulation was initiated with either
Gonadotrophin-releasing hormone (GnRH) agonist (Buserelin
acetate: Suprefact® spray; Hoechst Inc., Germany or Triptor-
elin: Decapeptyl®; Ipsen, France) or GnRH antagonist
(Cetrorelix: Cetrotide®, Merck-Serono, Geneva, Switzerland)
administration. Urinary gonadotrophins (hMG) (Menopu®r,
Organon Inc., The Netherlands) or recombinant follicle-
stimulating hormone (FSH) (Gonal F®, Merck-Serono, Ge-
neva, Switzerland) were added to stimulate the ovary until
at least 3 follicles reached a diameter of 17 mm. Ovulation
was induced by the administration of urinary human chori-
onic gonadotrophin (hCG) (Pregnyl®, Organon Inc., The
Netherlands) or recombinant hCG (Ovitrelle®, Merck-
Serono, Geneva, Switzerland). Oocyte retrieval was per-
formed through vaginal puncture under ultrasound guidance
36 h later. Oocyte collection and laboratory procedures are
described elsewhere . After the oocyte collection, the
oocyte-cumulus complexes were denuded using finely
drawn pipettes (170–130 μm) following 1 min of exposure
to 80 IU/ml hyaluronidase solution (Sigma Aldrich SrL,
UK.). The MII oocytes obtained after ovarian stimulation
were used for clinical ICSI cycles, and the GV and MI
stage oocytes available as discarded material for research
were used for this study.
Oocytes were vitrified in the medium 199 (M-199) based
solutions according to the manufacturer’s instructions of the
vitrification Vit Kit®-Freeze (Irvine Scientific, California).
First equilibration step was performed in 7.5% ethylene
glycol (EG) and 7.5% dimethyl sulphoxide (DMSO) solu-
tion at room temperature for 7 min before transferring the
oocyte in the vitrification solution containing 15% EG, 15%
DMSO and 0.5 mol/l sucrose for 30 s. The oocytes were
then loaded on in vitrification high security straws (VHS,
CryoBioSystem, France) in a volume of <1 μl, and imme-
diately submerged into liquid nitrogen.
During warming, the cryoprotectants were removed
according to the manufacturer’s instructions of the devitri-
fication kit Vit Kit®-Thaw (Irvine Scientific, California) by
sequentially washing the oocytes in four different M-199
based solution containing respectively 1.0 M (mol/l) sucrose
(1 min at 37°C), 0.5 mol/l sucrose (4 min at room temper-
ature) and M-199 alone (6 min at room temperature).
After warming, all the oocytes were cultured at 37°C
in a humidified atmosphere of 5%CO2-5%O2-90%N2in
the fertilization medium (Cook, Australia) and checked
after 1 h for survival. Post-warming survival rate was
assessed using morphological criteria, indicated by ab-
sence of overt cell degeneration, elongated shape, thick
or distorted zona, expanded perivitelline space and dark
pronounced cytoplasm. Subsequent oocytes competence
was assessed using their ability to mature in vitro and to
Sibling GV and MI oocytes
n = 100
n = 84
Evaluation of fertilisation, cleavage and blastocyst rate
Evaluation of survival and ivm rate
Fig. 1 Experimental design used to test the efficiency of vitrification
before or after in vitro maturation (IVM) of oocytes collected at
germinal vesicle (GV) and metaphase I (MI) stages
J Assist Reprod Genet
In vitro maturation, IVF and embryo culture
Oocytes were placed in a commercial IVM medium (Sage,
Copper Surgical, USA) supplemented with 0.075 IU/ml
FSH and 0.075 IU/ml LH according to the manufacturer’s
instructions for 24-48 h.
Matured oocytes were inseminated by ICSI using do-
nor sperm. Oocytes were individually cultured in micro-
drops of fertilization medium under paraffin mineral oil
(Cook, Australia) at 37°C in 5%CO2. Fertilization was
assessed 16-18 h after ICSI and success measured by the
appearance of two pronuclei and two polar bodies. Em-
bryos were cultured for 3 days in cleavage medium
(Cook, Australia) and the quality was evaluated daily by
scoring fragmentation, number and appearance of blasto-
meres. Embryos were cultured in blastocyst medium
(Cook, Australia) until day 5 to evaluate their develop-
Statistical analyses were performed using Chi-square or Non
parametric Mann–Whitney Test as appropriate. Values
p<0.05 indicated statistical significance.
100 oocytes (68 GV and 32 MI) were included in vitro
matured before vitrification group (group 1), while 84
oocytes were included in the vitrified before IVM group
(52 GVand 32 MI) (group 2). The proportion of GVand MI
stages is similar between groups.
Survival rate after warming, IVM, fertilization rate after
ICSI and developmental rate of vitrified oocytes were
assessed (Table 1). Survival rate after warming was similar
in both groups (86.9% versus 84.5% for groups 1 and 2,
respectively) and between GV and MI stage (84.2% versus
86.6% for GV and MI, respectively). In group 1, of the 46/
100 MII obtained, 26/32 were from MI and 20/68 were from
GV stage; in group 2, of the 20/71 MII obtained, 20/29 were
from MI and 0/42 GV stage. These results confirm our
previous study  providing evidence that survival rate is
similar between GVand MI stage and that MI stage mature
better that GV stage (p<0.001). However, oocyte matura-
tion rate per collected oocyte was significantly higher for
oocytes matured before vitrification (group 1, 46%) than for
oocytes vitrified before IVM (group 2, 23.8%) (p<0.01).
Fertilization rate per injected and per included oocyte
(52.5% and 21% for group 1 and 45% and 11% for the
group 2, respectively) as well as cleavage rate per fertilised
and per injected oocyte (52.4% and 27.5% for group 1
versus 55.5% and 25% for the group 2, respectively) were
similar for both groups. Around half of the embryos reached
four cells stages in both groups, but no blastocysts were
obtained. In conclusion, the study shows that the IVM
procedure is more efficient when it is performed before
Oocyte maturation is a lengthy process, during which the
oocyte acquires the competence to be fertilized and to un-
dergo embryogenesis. Significant progress has been made to
improve pregnancy and implantation rates with in-vitro
matured oocytes , but the mechanisms regulating early
folliculogenesis and oocyte maturation in the human are still
poorly understood . Further research remains necessary
to address the mechanism of oocyte maturation in order to
refine culture conditions and improve the implantation rate
of in vitro matured oocytes. Many factors affecting in vitro
maturation rate have been reported. It has been
Table 1 Survival and in vitro
maturation (IVM) rate, fertiliza-
tion and cleavage rate of vitrified
oocytes after or before in vitro-
IVM before vitrification
IVM after vitrification
Oocytes included (n)
Oocytes vitrified (n)
Oocytes survived (n)
In vitro matured oocytes/oocytes included
Mature oocytes inseminated by ICSI/
Fertilization rate/oocytes injected
Fertilization rate/oocytes included
Cleaved embryos/oocytes fertilised
Cleaved embryos/oocytes injected
100 (68 GVand 32 MI)
84 (52 GVand 32MI)
J Assist Reprod Genet
demonstrated that priming of ovarian immature oocytes with
follicle-stimulating hormone or human chorionic gonadotro-
phin prior to immature oocyte retrieval improves oocyte
maturation rates and embryo quality as well as pregnancy
rates in infertile women with polycystic ovaries or polycys-
tic ovary syndrome [20,21]. The size of the follicles may
also be important for the subsequent embryonic develop-
ment, while the developmental competence of oocytes de-
rived from the small antral follicles is not adversely affected
by the presence of a dominant follicle . Finally, in vitro
oocyte maturation is affected by the presence of cumulus
cells, the composition of IVM media and the culture con-
ditions [23–25]. Despite the fact that implantation rate per
transferred embryo using in vitro maturated oocyte doesn’t
yet reach results obtained after classical IVF , this
technique is already offered to patients at high risk of
developing hyperstimulation syndrome or for fertility
On the other hand, vitrification has strongly improved the
efficiency of oocytes cryopreservation technique and the
survival rate is similar whatever the oocytes maturation
stage (GV or MII) . However, cryopreserving immature
oocytes required in vitro maturation after warming. As
shown in previous studies, the cryopreservation procedures
may have detrimental effects on the maturation capacity
[27,28]. Developmental failure of cryopreserved oocytes is
related to critical disturbances of various cell components,
such as the chromosome segregation apparatus, the intracel-
lular calcium signalling system, and the cytoskeleton .
Cao et al. confirmed these findings reporting that, despite
the spectacular improvement of both survival rate and oo-
cyte ultrastructure at warming after vitrification, oocyte
maturation rates are significantly reduced when oocytes
were vitrified at immature GV stage followed by IVM
(50.8%) in comparison with the group first in vitro matured
and then vitrified (70.4%) . After ICSI, there was no
difference in the fertilization (62.1% versus 58.8%), cleav-
age (69.5% versus 67.5%) and blastocyst development rates
between these two groups. In this study, immature oocytes
were obtained from women with PCOS who underwent
IVM treatment. The patients were primed with clomiphene
citrate and hMG starting on day 3 of their menstrual cycle.
When the size of the leading follicles in the ovaries reached
8–10 mm in diameter as observed by ultrasound scan, hCG
was given and retrieval immature oocytes was performed
36 h later. The different starting material may account for
the better IVM rate obtained in this study. Our study has
been performed with a less optimal material (failed-matured
oocytes) and results are not comparable as those obtained in
PCOS patients. Recently, Asimakopoulos et al. 
reported the efficiency of vitrified immature human oocytes
before undergoing in vitro maturation, fertilization, and
embryo development. For two women (34 and 36 years
old) undergoing IVF cycle, immature oocytes were vitrified.
The patients were treated using a gonadotrophin agonist
protocol with hMG as ovarian stimulation. Final maturation
was induced with hCG. In the first case, 17 oocytes were
retrieved: six at MII, six at GV stage, and five with multiple
vacuoles or abnormal shape. ICSI was performed with the
six MII oocytes and the GV oocytes were vitrified. One
week later, the GV oocytes were successfully thawed and
placed in IVM media. Four oocytes reached metaphase II
after 22 h, one oocyte after 38 h, and the sixth oocyte after
48 h. Meanwhile, the woman became pregnant from the
transfer of the three fresh embryos; therefore, the embryos
produced by GV oocytes remain in storage. In the second
case, only three oocytes were retrieved: one at MI and two at
GV stage and they were vitrified. Two week later, the
oocytes were thawed; two of them survived (one MI and
one GV) and were placed in IVM media. After 23 h, both
oocytes reached MII stage and were fertilized by ICSI. At
that time, the woman was not prepared for embryo transfer
and the embryos were vitrified. Two months later, both
embryos were successfully thawed and transferred. There
was no pregnancy. These cases demonstrate that vitrified
immature oocytes can undergo post-thaw in vitro maturation
and fertilization and that the produced embryos are capable
to undergo vitrification and thawing. The authors however
did not compare the efficiency of this strategy with the
vitrification of in vitro matured oocytes. On the other hand,
Zhang et al.  suggested that, as cryopreservation meth-
od, vitrification of immature oocytes better preserves the
microtubule organization and reduces the cytoskeletal spin-
dle damage. In another recent study, Versieren et al. 
reported that cryopreserved GV oocytes showed decreased
and delayed maturation after warming but activation poten-
tial was not affected. In this study, oocytes at different
developmental stages of maturation (GV, MI, or MII) and
oocytes that failed to fertilize after IVF or ICSI were cry-
opreserved by slow freezing. Warmed oocytes were artifi-
cially activated to verify activation potential and compared
with oocytes that were not cryopreserved. They concluded
that immature oocytes should be cryopreserved after in vitro
maturation. Here, we confirmed that the number of matured
oocytes available for IVF is higher when vitrification is
performed before IVM than after IVM. However, fertiliza-
tion and cleavage rates are similar whether the oocytes are
vitrified before or after IVM. Furthermore, we showed that
the vitrification procedure has a negative impact on oocyte
maturation competence after warming. Whatever the timing,
MI oocytes are more competent to reach MII stage than GV
oocytes, suggesting that the first step of maturation is critical
and particularly affected by vitrification. The poor develop-
mental competence to blastocyst stage of the matured
oocytes suggests that vitrification affects the oocyte at both
GV and MI stage compared to MII stage when it is
J Assist Reprod Genet
combined with IVM. Nevertheless, discarded oocytes,
failed-matured in vivo coming from stimulated patients in
ICSI cycle, were used for this study. Despite the fact that
these denuded oocytes are not fully developmentally com-
petent, they constitute a very interesting and useful source of
human material, limiting the randomization of patients’
oocytes for studies. As suggested by several authors, imma-
ture oocytes from stimulated cycles can be matured in vitro
creating a potential source of human oocytes for experimen-
tal research despite their inferior quality [34,35]. Additional
studies are required, however, to confirm our results, to
improve the oocyte developmental competence after IVM
and vitrification procedures and to explore new methods in
human oocyte cryopreservation techniques.
In conclusion, immature oocytes should be vitrified at the
MII stage following IVM because oocyte maturation rates
were significantly reduced when oocytes were vitrified at
immature stage followed by IVM. Vitrification of in-vitro
matured oocytes may be an important additional option to
preserve fertility for many patients especially women who
will be treated with cytotoxic drugs or women at risk of
hyper-stimulation ovarian syndrome.
grant from the Fonds National Recherche Scientifique (FNRS) of
Belgium, and an unconditional grant from Merck Pharmaceuticals.
The study was supported by an unconditional
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