Production of Live Offspring from Vitrified-Warmed Oocytes Collected at Metaphase I Stage
Production of Live Offspring from Vitrified- Warmed Oocytes Collected at Metaphase I Stage
Ching-Chien Chang 0 1
Wei-Fang Chang 1
Jie Xu 1
An-Sheng Cheng 1 3
Chia-Chun Chang 1
Zsolt Peter Nagy 0 1
Cho-Chen Yang 1
Shih-Torng Ding 1 2
Li-Ying Sung 1
0 Reproductive Biology Associates , Atlanta , Georgia , United States of America, 2 Institute of Biotechnology, National Taiwan University , Taipei, Taiwan , 3 Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center , Ann Arbor, Michigan , United States of America
1 Editor: Xiuchun Tian, University of Connecticut , UNITED STATES
2 Department of Animal Science and Technology, National Taiwan University , Taipei, Taiwan , 7 Agricultural Biotechnology Research Center , Academia Sinica, Taipei, Taiwan , 8 Animal Resource Center, National Taiwan University , Taipei , Taiwan
3 Department of Medicinal Plant Development, Yupintang Traditional Chinese Medicine Foundation, Kaohsiung City, Taiwan, 5 Department of Animal Science, National Chiayi University , Chiayi City , Taiwan
Vitrification of matured oocytes is widely adopted in human clinics and animal research laboratories. Cryopreservation of immature oocytes, particularly those at metaphase I (MI), remains a challenge. In the present work, mouse MI oocytes denuded of cumulus cells were vitrified and warmed (V/W) either prior to (V/W-BEFORE-IVM, n = 562) or after (V/WAFTER-IVM, n = 664) in vitro maturation (IVM). Derivative metaphase II (MII) oocytes were then used for intracytoplasmic sperm injection (ICSI). In the control groups, in vivo matured MII oocytes were used freshly (FRESH-MII, n = 517) or after V/W (MII-V/W, n = 617). In vitro and in vivo developmental competencies were compared among groups. Satisfactory blastocyst rates were achieved in V/W-BEFORE-IVM (27.5%) and V/W-AFTER-IVM (32.4%) groups, albeit as expected still lower than those from fresh-MII (56.1%) or MII-V/W (45.6%) oocytes. Similarly, the term development rates from V/W-BEFORE-IVM and V/W-AFTERIVM were 12.4% and 16.7% respectively, acceptable but lower than those of the fresh-MII (41.2%) and MII-V/W (23.3%) groups. These data demonstrate that oocytes collected at MI stage are amenable to V/W, which can be performed before or after IVM with acceptable development rates including production of healthy pups. These findings provide useful knowledge to researchers and clinical practitioners for preservation and use of the other-
Data Availability Statement; All relevant data are within the paper
Funding: This study was supported by grants from
Yupintan Traditional Chinese Medicine Foundation
(Kaohsiung, Taiwan, R.O.C.) and the National Taiwan
University, Taiwan, R.O.C., Grant #
NTU-CDG105R7885 to LYS and 103R4000 to WFC. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared
that no competing interests exist.
wise discarded MI oocytes.
Conventional slow freezing and vitrification are common methods to preserve mammalian
gametes and embryos. Vitrification, defined as the solidification of a solution at low
temperature by extreme elevation in viscosity during cooling without ice crystallization, has
been increasingly used in the past two decades. The glass state formed during vitrification has
the same ionic and molecular distribution as the liquid phase, thus avoiding both chemical and
mechanical damage to gametes and embryos. Importantly, vitrification has been proven
effective on cryopreserving matured female gametes, often leading to minor compromise of the
fertility and developmental competency; consequently it is widely adopted in human IVF clinics
] and in animal research laboratories [
While vitrification of MII oocytes is reliable, it is noted that a working protocol to vitrify
immature oocytes remains to be developed. Notably, 15–30% oocytes retrieved in a routine
human IVF cycle are immature and discarded [
] due to lack of such protocols. One
important parameter is the timing of vitrification with regard to IVM: (1) V/W is conducted
prior to IVM; or (2) IVM is conducted before V/W. It is argued that the maturation status may
render differential cryotolerance to the oocytes . Most previous studies used germinal
vesicle (GV) stage oocytes to do the comparisons [
]. The effects of vitrification timing on MI
oocytes have not been evaluated, and there are no reports on production of live offspring using
oocytes V/W at MI stage.
In the present work, we conducted V/W on MI oocytes at two time points: (1) before IVM;
and (2) after IVM (Fig 1). Derivative MII oocytes from both groups, named
V/W-BEFORE-IVM and V/W-AFTER-IVM respectively, were subjected for fertilization. Blastocyst rates
and term rates after embryo transfer (ET) were used to evaluate the in vitro and in vivo
developmental competencies of these oocytes, in comparison to the two control groups, fresh MII
(FRESH-MII) or vitrified-warmed MII (MII-V/W) oocytes.
Material and Methods
All animal maintenance, care and use procedures were reviewed and approved by the
Institutional Animal Care and Use Committee (NTU-103-EL-47) of National Taiwan University,
Taiwan. The 8–12 weeks-old B6D2F1 hybrid mice used as sperm and oocyte donors for ICSI were
from C57BL/6 (B6) females breed with DBA/2 males, the female CD1 mice were used as
Groups of oocytes
A schematic illustration of the oocyte groups is shown in Fig 1. In FRESH-MII Group, in vivo
matured (IVO) MII oocytes were harvested from the oviducts 15 h after the hCG trigger, and
were injected with a spermatozoa without any vitrification. In MII-V/W Group, IVO MII
oocytes were harvested from the oviducts 15 h after the hCG trigger, and were immediately
vitrified, stored in liquid nitrogen until they were warmed (or interchangeably referred to as
“thawed” in this manuscript) for ICSI at a later time. In V/W-AFTER-IVM Group, cumulus
cells were removed from MI oocytes, which were collected 6–7 h post hCG trigger. After
another 8 h of in vitro maturation in KSOM+AA medium, the oocytes with an extruded PB
(presumably MII) were selected, vitrified and stored, before they were warmed at a later date
for ICSI. In V/W-BEFORE-IVM Group, MI oocytes were collected 6–7 h after the hCG trigger,
denuded of cumulus cells, vitrified and stored before they were warmed at a later date, in vitro
matured for another 8 h in KSOM+AA medium; oocytes with an extruded PB (presumably
MII) were selected for ICSI. In V/W-BEFORE-IVM and V/W-AFTER-IVM groups, cumulus
cells were removed to simulate clinical applications in human ICSI practices in which
(undetermined) immature oocytes are denuded of cumulus cells upon retrieval.
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Fig 1. Schematic illustration of treatments in FRESH-MII (Control 1), VW-MII (Control 2), V/W-AFTER-IVM, and V/W-BEFORE-IVM groups. The
duration of oocyte maturation was 15 hours in all groups, which used hCG trigger as a set point. In FRESH-MII group, the freshly collected IVO MII oocytes
were injected with a spermatozoa without any vitrification. In VW-MII Group, IVO MII oocytes were harvested 15 h after the hCG trigger from the oviducts,
and those IVO MII oocytes were vitrified for later ICSI. In V/W-AFTER-IVM Group, cumulus cells were removed from MI oocytes, which collected 6–7 h
after the hCG trigger from ovaries. After another 8 h of in vitro maturation, the oocytes with a PB extruded (IVM MI-II oocytes) were vitrified for later ICSI. In
V/W-BEFORE-IVM Group, cumulus cells removed MI oocytes, which collected 6–7 h after the hCG trigger from ovaries, were vitrified for storage. After
oocyte warming, the MI oocytes were in vitro matured for another 8 h, and the oocytes with a PB extruded (IVM MI-II) were regarded as mature oocytes for
Collection of metaphase II and metaphase I oocytes
In vivo matured metaphase II (IVO MII) oocytes. B6D2F1 female mice were subjected
to the following hormone priming protocol: superovulation was induced with 5 IU of equine
chorionic gonadotrophin (eCG) followed 48 h later with 5 IU of human chorionic
gonadotrophin (hCG). Oocytes at the MII stage were harvested 15 h after the hCG trigger from oviducts
and denuded of cumulus cells by 2-min exposure to 0.1 mg/mL of hyaluronidase at 37°C with
gentle pipetting (Fig 2A). Oocytes with an extruded PB (Fig 2B) were used for ICSI either
freshly (FRESH-MII), or after vitrify-warming (VW-MII).
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Fig 2. Collection of metaphase oocytes at different time points. (A) MII COCs were harvested 15 hours after hCG trigger from
oviducts. (B) The cumulus cells were removed from MII COCs. (C) MI COCs were harvested 6–7 hours after hCG trigger from ovaries. (D)
The cumulus cells were removed from MI COCs. Scale bar = 50 μm.
V/W-AFTER-IVM oocytes. B6D2F1 female mice were subjected to the following
hormone priming protocol: superovulation was induced with 5 IU of equine chorionic
gonadotrophin (eCG) followed 48 h later with 5 IU of human chorionic gonadotrophin (hCG). Oocytes
at the MI stage (Fig 2C) were collected 6–7 h after the hCG trigger from ovaries and removed
cumulus cells by 2 min exposure to 0.1 mg/mL of hyaluronidase at 37°C with pipetting (Fig
2D). After another 8 h of culture in vitro, oocytes with an extruded PB were vitrified (V/
W-AFTER-IVM) and stored before they were warmed on a later date for ICSI.
V/W-BEFORE-IVM oocytes. B6D2F1 female mice were subjected to the following
hormone priming protocol: superovulation was induced with 5 IU of equine chorionic
gonadotrophin (eCG) followed 48 h later with 5 IU of human chorionic gonadotrophin (hCG).
Oocytes at the MI stage were collected 6–7 h after the hCG trigger from ovaries and
removed cumulus cells by 2 min exposure to 0.1 mg/mL of hyaluronidase at 37°C with
pipetting (Fig 2D). The denuded MI oocytes were vitrified for storage. After warming,
the MI oocytes were in vitro matured for another 8 h, and oocytes with an extruded PB
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Vitrification of oocytes
The basal medium used for oocyte cryopreservation was HEPES-buffered human tubal fluid
(HTF) supplemented with 20% (v/v) fetal bovine serum (FBS; SH0070.03; Hyclone, Logan,
UT, USA). The denuded oocytes were vitrified by the minimum volume cooling method as
described previously [
]. Briefly, the oocytes were equilibrated in equilibration medium
[basal medium with 7.5% (v/v) ethylene glycol and 7.5% (v/v) dimethylsulphoxide (DMSO)] at
room temperature for 5 min. The oocytes were then transferred into the vitrification medium
[basal medium with 15% (v/v) ethylene glycol, 15% (v/v) DMSO, and 0.5 mol/L sucrose] at
room temperature (RT) for 45–60 sec. The cryoprotectant-treated oocytes were placed onto a
fine polypropylene strip (Cryotop1, Kitazato BioPharma Co., Fuji, Shizuoka, Japan). The
polypropylene strip carrying the oocytes was then submerged immediately into liquid nitrogen
and was ready for storage.
The polypropylene strip with vitrified oocytes was immersed directly into 3.5 mL of thawing
solution [HEPES buffered HTF with 20% (v/v) FBS and 1.0 mol/L sucrose] at 37°C for 1 min.
Oocytes were then picked up and transferred into 1.0mL of the dilution solution
[HEPES-buffered HTF with 20% (v/v) FBS and 0.5 mol/L sucrose] for 3 min at RT. The oocytes were
subsequently washed in 1.0mL washing solution [HEPES-buffered HTF with 20% (v/v) FBS] for 10
min at RT. Finally, the oocytes were incubated in KSOM+AA medium (Specialty Media,
Phillipsburg, NJ, USA) allow recover for at least 1h before sperm injection.
Intracytoplasmic sperm injection
ICSI was performed as described by Kishigami et al [
]. Briefly, mature spermatozoa were
obtained from the epididymides of 8–12 weeks old B6D2F1 male. A cauda epididymis was
removed and placed into a 1.5-ml polypropylene centrifuge tube contents 500 ml of KSOM
+AA medium, then incubated for 25~30 min at 37°C to allow the spermatozoa to swam into
the medium. A small drop (about 2μl) of sperm suspension from upper part was mixed with
about 10 μl of HEPES-CZB medium containing 12% (w/v) polyvinylpyrrolidone (PVP; 360
kDa) in a micromanipulation chamber. To increase the survival rate after sperm injection, the
oocytes were incubated in calcium-free CZB medium containing 5 mM strontium for 20 min
before ICSI [
]. Following activation of oocytes, the head of a spermatozoon was injected into
the oocyte by using a pipette with an inner diameter of 6–8 μm assisted by piezo-drill pulses.
Injected oocytes were left for 10–15 min on the stage of the microscope at room temperature,
and then further cultured in KSOM+AA medium for 4 days at 37°C in 5% CO2 humidified air.
Cleavage and blastocyst rates were recorded 1 day and 4 days post culture.
The embryos that developed to the blastocyst stage were transferred into the uteri of day 2.5
pseudopregnant CD1 females mated with vasectomized males. The live pups were obtained
through a natural birth from recipient mothers around 17 days later following transfer and
raised by recipient mothers.
Percentages were transformed using arcsin transformation. Percentage transformed data and
other numbers were analyzed by ANOVA and means compared by Fisher’s LSD using
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Graphpad Prism (v6.02, Graphpad Software, Inc., La Jolla, CA). Significant differences were
defined as P<0.05 ( ).
Oocyte survival and in vitro embryo development after warming
We assigned oocytes in four treatment groups (Table 1). In FRESH-MII Group, a total of 517
IVO MII oocytes were collected and freshly subjected to ICSI. In MII-V/W Group, a total of
617 IVO MII oocytes were collected and vitrified. In V/W-AFTER-IVM Group, 790 MI stage
oocytes were retrieved, out of which 664 matured (84.1%) after 8 h of in vitro culture. A total
589 out of 617 from MII-V/W Group (95.3%) and 644 out of 664 from V/W-AFTER-IVM
Group (96.8%) MII oocytes survived after warming (MII-V/W Group vs. V/W-AFTER-IVM
Group; NS; Table 1; Fig 3). In V/W-BEFORE-IVM Group, 818 MI oocytes were retrieved and
vitrified, the MI oocyte survival rate was 92.4% (756/818) post vitrification/warming, and a
total of 562 out of 818 starting MI oocytes were matured (68.7%) after 8 h of in vitro culture,
significantly lower than that achieved in the V/W-AFTER-IVM Group (P<0.05). In
vitrification groups, only viable and mature MII oocytes were selected for ICSI.
To gain knowledge of the combinational effects of IVM and V/W on MI oocytes, we
compared the ratio of number of viable MII oocytes over the starting number of MI oocytes. The
overall efficiency was higher in V/W-AFTER-IVM than in V/W-BEFORE-IVM (81.5 vs.
68.7%; P<0.05; Table 1), suggesting that MII oocytes have better tolerance for vitrification
than MI oocytes do.
MII oocytes in all groups were subjected to ICSI using sperm from B6D2F1 males. Survival
rates post ICSI and embryo cleavage rates were checked after 24 h of ICSI. To allow
comparison among these four groups, we used total available MII oocyte number as the denominator
to calculate the rates (Table 1).
The survival rate post ICSI was highest in the FRESH-MII Group (P<0.05). No significant
difference was found among the remaining three groups (Table 1; 56.4% vs. 50.7% vs. 45.9%; NS).
The order of subsequent embryo cleavage rates, from the highest to the lowest, FRESH-MII,
MII-V/W, V/W-AFTER-IVM, and V/W-BEFORE-IVM (Fig 3; Table 1; 72.8% vs. 55.8% vs.
48.3%vs. 44.6%; p<0.05).
On Day 4 post ICSI, blastocyst formation was assessed (Fig 3, Table 1). The blastocyst
formation rates were highest in FRESH-MII (56.1%), moderate in MII-V/W and V/
No. of MI
of per MI
No. of No. of
available MII oocytes
oocytes (% survived
of per MI post
oocyte) warming (%
of per MII
Percentage data were arcsine transformed and subjected to Tukey's multiple comparisons test using Graphpad Prism (v6.02).
a, b, c different superscripts within the same column indicates statistical difference (P<0.05).
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Fig 3. Preimplantation embryo development of FRESH-MII, VW-MII, V/W-AFTER-IVM and V/W-BEFORE-IVM oocytes following ICSI. (A) FRESH-MII
oocytes. (B) MII-V/W oocytes after warming. (C) V/W-AFTER-IVM oocytes. (D) V/W-BEFORE-IVM oocytes. (E) Cleavage stage embryos derived from
FRESH-MII oocytes. (F) Cleavage stage embryos derived from VW-MII oocytes. (G) Cleavage stage embryos derived from V/W-AFTER-IVM oocytes. (H)
Cleavage stage embryos derived from V/W-BEFORE-IVM oocytes. (I) Blastocyst stage embryos derived from Fresh-MII oocytes. (J) Blastocyst stage
embryos derived from VW-MII oocytes. (K) Blastocyst stage embryos derived from V/W-AFTER-IVM oocytes. (L) Blastocyst stage embryos derived from V/
W-BEFORE-IVM oocytes. Scale bar = 100 μm.
W-AFTER-IVM (45.6% vs. 32.4%), and lowest in V/W-BEFORE-IVM (27.5%). As expected,
the efficiencies of blastocyst formation in FRESH-MII Group was significantly higher than in
the MII-V/W Group (56.1% vs. 45.6%, P<0.05; group FRESH-MII Group vs. MII-V/W
Group), indicating V/W procedure adversely affected the capability of subsequent
development. Further, the blastocyst rate was higher in MII-V/W (45.6%) than in V/W-AFTER-IVM
(32.4%), confirming that in vivo matured oocytes possess higher developmental competency
than in vitro matured ones.
a, b different superscripts within the same column indicates statistical difference (P<0.05).
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No. of recipient used
No. of pregnancy (%)
No. of embryos transferred No. of offspring born(%)
Fig 4. In vivo development of Fresh-MII, VW-MII, V/W-AFTER-IVM and V/W-BEFORE-IVM oocytes following ICSI. (A) 21 days-old offspring
derived from Fresh-MII oocytes. (B) 17 days-old offspring derived from VW-MII oocytes. (C) 17 days-old offspring derived from V/W-AFTER-IVM
oocytes. (D) 13 days-old offspring derived from V/W-BEFORE-IVM oocytes. Sources of both oocytes and sperms were derived from B6D2F1
strain; hence, the coat color of the ICSI pups (B6D2F2) showed various colors with gray, brown and black.
Among in vitro matured groups, maturation prior to V/W (IVM-BEFORE-V/W) appeared
to yield higher blastocyst rates than maturation post V/W (IVM-AFTER-V/W) (32.4% vs.
27.5%); however such difference is not statistically significant (P>0.05) when calculated based
on the number of viable MII oocytes used for ICSI.
The term development rates after embryo transfer
We next performed embryo transfer to evaluate the in vivo developmental competency of
embryos derived using oocytes of all four groups (Table 2). A total of 80 blastocysts were
transferred to 8 recipient mice in FRESH-MII Group, resulting in 7 pregnancies and 33 term pups.
In MII-V/W Group, 90 blastocysts were transferred to 7 recipient mice, of which 5 became
pregnant, delivering 21 term pups. In V/W-AFTER-IVM Group, 132 blastocysts were
transferred to 9 recipient mice, of which 7 became pregnant and 22 pups were born. In V/
W-BEFORE-IVM Group, 89 blastocysts were transferred to 6 recipient mice, of which 4
became pregnant and 11 pups were born (Fig 4).
We calculated the birth rates as the ratio of number of term pups over total embryos
transferred. Birth rates were significantly higher in Fresh-MII Group (41.2%) than in the other three
V/W groups (12.4–23.3%) (P<0.05), indicating that the influence of V/W is not limited till
blastocyst stage but continue onto the in vivo embryo development.
Among the three V/W groups, the term rate was highest in MII-V/W Group (23.3%),
followed by V/W-AFTER-IVM Group (16.7%), and V/W-BEFORE-IVM Group (12.4%), with no
statistical difference among them (P>0.05).
Notably, satisfactory birth rates (12.4–16.7%) were achieved in both V/W-AFTER-IVM and
V/W-BEFORE-IVM Groups. All offspring appear normal and healthy (Fig 4). These findings
demonstrate the feasibility of using MI oocytes for production of live healthy offspring.
Oocyte vitrification is of basic and practical importance given the scarcity of female gametes in
animals and in humans. We previously reported successful vitrification of MII and immature
MI oocytes in mice, and derived embryonic stem cells (ntESCs) by nuclear transfer using the
MII-V/W and V/W-BEFORE-IVM oocytes [
] but we didn’t examine their in vivo
developmental capacities. To date, efficient vitrification of MII oocytes in a number of mammalian
species including mice, rabbit, bovine, porcine, and humans has been achieved using a number of
variants of vitrification protocols such as electro microscope grids [
], open pulled straw [
], drops into liquid nitrogen, gel loading tips [
]. Vitrification of
immature oocytes, however, remains to be a challenge. Development of an effective protocol to
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cryopreserve immature oocytes will significantly expand the available materials for fertility and
developmental biology research, as well as for human IVF applications. In a routine cycle of
controlled ovarian hyper-stimulation for ICSI, approximately 15–30% of retrieved oocytes are
immature (i.e. MI & GV oocytes) [
]. These immature oocytes are excluded from almost
all clinical uses, representing a substantial waste of the female gametes.
The present work looked at the effects of vitrification on MI oocytes, both in vitro and in
vivo. Satisfactory blastocyst rates and term rates were obtained using embryos derived from MI
oocytes that were subjected to combinational treatments of IVM and V/W. To our knowledge,
this is the first study reporting the generation of live offspring using V/W mouse IVM oocytes
collected at MI stage. Our findings have several practical implications: (1) MI oocytes can be
used for production of live healthy offspring, provided they are properly in vitro matured and
V/W. (2) V/W can be performed before or after IVM. Together the present work demonstrates
that by combining V/W and IVM, MI oocytes can be “rescued” for fertilization, resulting in
satisfactory term development.
The effects of companion cumulus cells
The blastocyst rate was lower in the V/W-AFTER-IVM group (32.4%) than in the MII-V/W
group (45.6%) (Table 1), despite that the oocytes in both groups were V/W at MII stage. The
compromised developmental competency of the V/W-AFTER-IVM group oocytes are likely
caused by multiple factors. First, these oocytes were matured in vitro whereas the MII-V/W ones
were in vivo matured. Second, perhaps more significantly, the oocytes in the V/W-AFTER-IVM
group were denuded of cumulus cells upon retrieval (to determine their maturation status).
The oocyte communicates with and modifies its surroundings via direct physical contact
with cumulus cells [
]. With the known roles of cumulus cells for optimal oocyte maturation,
the lack of cumulus cells is one of many limitations in any IVM culture system using leftover
oocytes from ICSI. The foundation of this relationship lies partly in highly specialized
oocytecumulus cell contacts called trans-zonal projections (TZP) that are established at the onset of
]. It is known that removal of the cumulus oophorus before IVM can be
detrimental to oocyte quality and subsequent embryo development [
]. Without the TZP
between companion cumulus cells and oocytes, the denuded MI oocytes undergoing IVM are
thus in a compromised micro-environment which is essential for complete oocyte maturation.
The effects of oocyte stage at vitrification
A number of groups have reported effects of cryopreservation of GV stage oocytes. The effects
of slow freezing are controversial: two studies showed that slow freezing at GV stage had better
outcome than at post-IVM MII stage [
], while several other studies reported low
maturation rates when slow freezing at the GV stage was performed [
18, 19, 33
]. Vitrification at GV
stage was reported to negatively affect maturation rates [
The present work looked at effects of V/W and IVM on oocytes collected at the MI stage. It
appears that V/W have similar impacts on the survival of MI oocytes (based on morphology
observation), as compared to MII stage oocytes. As shown in Table 1, the oocyte survival rate
was 95.3% for MII-V/W group, similar to that in the V/W-BEFORE-IVM group (92.4%) and
that in the V/W-AFTER-IVM group (96.8%). Given that MI oocytes have similar surface to
volume (S/V) ratio and cytoskeleton organization to those of MII oocytes, it is not surprising
that when the same V/W protocols (i.e. same cooling rates and cryoprotectants) were applied,
similarly high survival rates were obtained.
On the other hand, our data show that after V/W, the maturation rate dropped significantly
(P<0.0001, Chi square test) to 74.3% (total matured/total survived after V/W, based V/
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W-BEFORE-IVM group data) vs. 84.1% in the V/W-AFTER-IVM group (Table 1), indicating
that the V/W treatment negatively affected the IVM process. It remains to be tested what
factors contributed to the difference. One possibility is the oocyte membrane permeability to the
cryoprotectants may have changed along the maturation process, with MI oocytes having
lower tolerance to cryoinjuries, consequently leading to comprised IVM outcome.
Nevertheless, many healthy live offspring were produced in either group. Researchers can choose which
protocol to use based on their preference and work context (e.g. available manpower, etc.).
The present work demonstrates that MI oocytes are amenable to V/W. We show that
combinational treatments of IVM and V/W on MI oocytes resulted in acceptable blastocyst
development rates, and importantly, healthy offspring. These findings provide confidence and
flexibility to researchers and clinical practitioners to store and use the otherwise discarded MI
Conceived and designed the experiments: Ching-CC JX LYS. Performed the experiments:
WFC LYS Chia-CC. Analyzed the data: WFC Ching-CC ZPN CCY STD LYS. Contributed
reagents/materials/analysis tools: LYS ASC. Wrote the paper: Ching-CC JX LYS.
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