Blastocyst development and pregnancies after IVF of mature oocytes retrieved from unstimulated patients with PCOS after in-vivo HCG priming: Case report
Blastocyst development and pregnancies after IVF of mature oocytes retrieved from unstimulated patients with PCOS after in-vivo HCG priming
Weon-Young Son 0
San-Hyun Yoon 0 1
Suk-Won Lee 0
Yong Ko 1
Hye-Gyun Yoon 0
Jin-Ho Lim 0
0 Maria Infertility Hospital
1 Department of Animal Science, Korea University , Seoul , Korea
A major side-effect of controlled ovarian stimulation (COS) in patients with polycystic ovarian syndrome (PCOS) is the risk of ovarian hyperstimulation syndrome (OHSS). In-vitro maturation (IVM) of immature oocytes represents a potential alternative for the fertility treatment of these patients. Two patients at high risk of OHSS were primed with 10 000 IU HCG 36 h before oocyte retrieval. After retrieval, oocyte maturity was evaluated. Oocytes considered to be mature at the time of collection were inseminated by IVF or ICSI, and the resulting embryos were cultured to blastocysts. Transfer of these blastocysts resulted in pregnancy in both patients. Immature oocytes were cultured in YS medium supplemented with 30% human follicular fluid, 1 IU/ml rFSH, 10 IU/ml HCG and 10 ng/ml epidermal growth factor (rhEGF). After in-vitro maturation of the oocytes, ICSI was performed. Two and five expanded blastocysts were obtained after 5 day culture and were cryopreserved. This report indicates that mature oocytes can be collected at the time of retrieval using only in-vivo HCG priming in women with PCOS, and clinical pregnancy can be established by transfer of blastocysts derived from the mature oocytes. This approach opens a potential for a new dimension in the management of patients with PCOS.
Controlled ovarian stimulation (COS) is used to achieve
multifollicular recruitment, enabling an increased number of
embryos to be transferred. However, there are disadvantages
associated with COS in the case of patients with polycystic
ovarian syndrome (PCOS). The major side-effect of
superovulation in patients with PCOS is ovarian hyperstimulation
(Cobo et al., 1999)
. Thus, in-vitro
maturation (IVM) of immature oocytes from OHSS patients would
be an attractive option to eliminate this problem.
Knowledge regarding the IVM of immature human oocytes
and its clinical application has been accumulated during the
past couple of years. Fertilization, embryo development and
term pregnancies of IVM oocytes have been reported in
(Nagy et al., 1996)
, natural cycles
et al., 1997)
and PCO patients
(Trounson et al., 1994)
The efficiency of current IVM techniques is suboptimal in
terms of obtaining the number of mature oocytes by natural
cycle as compared with that by COS cycles
(Barnes et al.,
1995; Russell et al., 1997)
. In addition, the quality of oocytes
is poor after IVM, showing frequently retarded cleavage and
blockage of development
(Barnes et al., 1996; Trounson et al.,
, which may be a significant factor in the low pregnancy
rates achieved. So far, only one case of blastocyst embryo
transfer and pregnancy derived from immature oocytes
collected from unstimulated ovaries has been reported
et al., 1995)
. Although the number of IVM cycles was small,
recent studies have shown improved pregnancy rates per
(Mikkelsen et al., 1999; Chain et al., 2000)
Chian et al. reported that higher rates of oocyte maturation
and pregnancies were achieved in patients with PCOS by HCG
priming (Chian et al., 2000). However, they reported that the
pregnancies were achieved by transfer of day 2 or day 3
embryos derived from IVM of immature oocytes.
Here we report that mature oocytes can also be retrieved after
HCG priming in women with PCOS in an IVM programme.
The oocytes can undergo normal fertilization and blastocyst
development, and pregnancies can be established by
Two patients with PCOS presented with anovulation, polycystic
ovaries visible on ultrasound, elevated serum testosterone
concentrations, 10 IU/ml LH in serum on menstrual cycle
day 2 and a minimum 3 year history of infertility. The patients
had a high risk of OHSS. Before the study, approval was
obtained from the Institutional Review Board of the Maria
A 27 year old woman with PCOS failed to become pregnant
after three cycles of conventional IVF treatment over the past
3 years. To initiate each treatment cycle, the patient received
i.m. injections of progesterone (Progestin; Samil
Pharmacology, Seoul, Korea). Withdrawal bleeding occurred on 3 days
after the last dose. Ovarian follicle development was monitored
by transvaginal ultrasonography (Aloka, Tokyo, Japan)
beginning on cycle day 3. The patient was given 10 000 IU of HCG
(IVF-C; LG Chemical, Seoul, Korea) subcutaneously on cycle
day 9 based on the cycle length and the endometrial thickness.
After 36 h, oocytes were aspirated with a 19 gauge aspiration
needle (Cook, Eight Mile Plains, Queensland, Australia) under
the guidance of transvaginal ultrasound. A portable aspiration
pump was used with a pressure between 80 and 100 mmHg.
The aspirates were collected in tubes containing prewarmed
heparinized Ham?s F-10 medium supplemented with
bicarbonate, HEPES and 0.3% bovine serum albumin. Oocytes were
isolated by washing the follicular aspirate through a filter
(70 mm mesh, Falcon 1060; Becton Dickinson, NJ, USA). In
order to remove erythrocytes and small cellular debris, the
filtrate was further washed with Ham?s F-10 medium by
vigorous pipetting using 10 ml serological pipette. The retained
cells were then resuspended in the medium and the oocytes
were isolated under a stereomicroscope. All oocyte handling
procedures were conducted in a mini-chamber under 5% CO2
atmosphere at 37?C. At the time of oocyte collection, oocytes
with a germinal vesicle (GV) were transferred to maturation
medium for culture and oocytes without GV were denuded of
cumulus cells with 0.003% hyaluronidase (Sigma Chemical
Co., St Louis, MO, USA) and mechanical pipetting. Oocytes
without an intact GV were defined as metaphase I (MI) and
oocytes with a first polar body extrusion were identified as
metaphase II (MII).
The IVM medium consisted of YS medium
(Yoon et al.,
supplemented with 30% human follicular fluid (hFF),
1 IU/ml rFSH, 10 IU/ml HCG and 10 ng/ml recombinant
human epidermal growth factor (rhEGF). The hFF was prepared
as the method reported by Chi et al.
(Chi et al., 1998)
were cultured in IVM medium at 37?C in an atmosphere of
5% CO2, 5% O2 and 90% N2. Nuclear maturation was assessed
under the dissecting microscope.
Conventional IVF or intracytoplasmic sperm injection (ICSI)
was used to fertilize the mature oocytes. Fertilization was
assessed 19 h after insemination to detect the appearance of
two distinct pronuclei and two polar bodies. Zygotes were
cocultured with cumulus cells in 10 ? l YS medium supplemented
with 10% hFF. The cumulus cells for co-culture were prepared
Pregnancies after blastocyst transfer in unstimulated PCOS patients
using the method reported by Yoon et al.
(Yoon et al., 2001a)
Blastocysts on day 5 were transferred to the patients, and the
remaining embryos were cultured until day 7. Those embryos
that developed to expanded blastocyst stage were cryopreserved
by Me?ne?zo et al?s method
(Me?ne?zo et al., 1992)
For the preparation of the endometrium, the patients were
also given 10 000 IU HCG at the time of oocyte recovery.
Oestrogen valerate 6 mg (Progynova; Schering, Berlin,
Germany) was administered daily from the day of oocyte
retrieval. Progestin 100 mg was administered daily from 1 day
after oocyte retrieval. Both medications were continued until
a fetal heartbeat was positively identified.
A total of 36 oocytes were retrieved; 18 oocytes were at
MII stage, six were MI stage and 12 were GV stage. MII stage
oocytes were inseminated with conventional IVF (n 11) or
ICSI (n 7). Thirteen oocytes [7/11 (64%) from IVF and
6/7 (86%) from ICSI] were fertilized. Three blastocysts (one
middle expanding blastocyst and two early blastocysts) were
observed at day 5 and were transferred to the patient. On the
day of embryo transfer, the endometrial thickness was 10 mm
on transvaginal ultrasonogram. Two weeks after embryo
transfer, serum ?-HCG concentration was 649.3 IU/ml, and 4
weeks after embryo transfer, an ongoing intrauterine twin
pregnancy was observed using transvaginal ultrasonography.
Immature oocytes (MI 6, GV 12) at the time of oocyte
retrieval were transferred to maturation medium for culture,
11 oocytes reached metaphase II stage 24 h later. After
ICSI, eight oocytes (72.7%) were fertilized. Two expanding
blastocysts observed at day 5 after insemination were
A 32 year old woman with PCOS and a 4 year history of
infertility had failed to become pregnant after six cycles
of conventional IVF. To initiate each treatment cycle, the
patient received IM injections of progesterone (Progestin;
Samil Pharmacology, Seoul, Korea). Withdrawal bleeding
occurred on 2 days after the last dose. The patient was given
10 000 IU of HCG (IVF-C; LG Chemical) s.c. on cycle day
13 based on the cycle length and the endometrial thickness.
Oocyte retrieval was performed on day 15. Three MII stage
and 29 GV stage oocytes were retrieved from both ovaries.
Three MII oocytes were inseminated with conventional IVF
using the husband?s sperm. Three oocytes were fertilized
(100%). All embryos developed to blastocysts on day 5 (one
middle expanding blastocyst and two early blastocysts) and
were transferred to the patient. The endometrial thickness was
10 mm on the day of embryo transfer. Two weeks after
embryo transfer, serum ?-HCG concentration was 165 IU/ml,
and 4 weeks after embryo transfer, a pregnancy with single
fetal heartbeat was confirmed by ultrasonography. Out of
29 GV oocytes, 12 and 11 (total 23) reached metaphase II
stage after 24 and 48 h of incubation in the IVM medium
respectively. Twenty-two oocytes (96%) were fertilized after
ICSI, and five expanding blastocysts were observed at day 5
after insemination, and were cryopreserved.
This study demonstrates that mature oocytes retrieved from
women with PCOS following only HCG priming can undergo
fertilization and development, and that the transfer of resulting
blastocysts can establish pregnancies.
YS medium was designed to support the development of
the human zygote to the blastocyst stage under the cumulus
cell co-culture system
(Yoon et al., 2001a,b)
. The medium is
without glucose and contains vitamins, amino acids and taurine
(Yoon et al., 2001a,b)
. We routinely use YS medium for
blastocyst culture and have achieved an acceptable pregnancy
rate for 6 years
(Yoon et al., 2001a; Yoon et al., 2001b)
Therefore, the zygotes derived from mature oocytes at the
time of oocyte collection in IVM/F?embryo transfer cycles
were also cultured to blastocysts in YS medium under the
cumulus cell co-culture. In addition, we obtained 23.3%
(7/30) of expanded blastocyst in embryos derived from IVM/
IVF of immature oocytes collected from two patients. This
strongly suggests that our IVM culture system is acceptable.
One of the important factors regulating the number and
quality of oocytes maturing in vitro is the culture conditions
used for IVM. The composition of most media used for human
IVM is based on experiences with other mammalian species.
Cekleniak et al. observed a higher maturation rate of immature
oocytes in P1 medium without glucose than with TCM-199
(Cekleniak et al., 2001)
. We used YS
medium without glucose for culture of the human immature
oocytes. We supplemented gonadotrophins into IVM medium
at a 1:10 ratio of rFSH:HCG, as this ratio of gonadotrophins
was found to improve embryonic developmental competence
(Anderiez et al., 2000). In addition, EGF, a growth factor that
demonstrated increasing maturation of immature oocytes, was
added to the IVM medium. In our IVM/IVC culture system,
we obtained 72.3% (34/47) of maturation and 23.3% (7/30)
expanded blastocyst formation in two patients. High blastocyst
rate (23.3%) from embryos derived from immature oocytes
was obtained in this report. This indicates that the present
IVM and IVC system may be very good, even though the
numbers of patients were too small to draw significant
For preparation of endometrium, we induced luteinization
with HCG potentially to enhance the uterine and embryo
synchrony, as suggested previously
(Barnes et al., 1995)
administrations of HCG were given to the patient at 36 h
before and at the time of oocyte recovery. This may aid the
collection of metaphase II oocytes and the synchronizing of
embryo development with the endometrium.
To the best of our knowledge, only one case report has
demonstrated thus far that early blastocyst stage and pregnancy
can be obtained from immature oocytes recovered from a
(Barnes et al., 1995)
. This is the first report to
obtain mature oocytes by HCG priming and also establish a
pregnancy by transfer of blastocysts derived from mature
oocytes. This approach potentiality opens a new dimension in
the management of patients with PCOS.
The authors are grateful to Wei-Hua Wang and Li Meng, Division of
Reproductive Medicine and Infertility, Department of Obstetrics and
Gynecology, Brown University School of Medicine, Rhode Island,
USA, for their critical review of the manuscript.
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Received on June 8 , 2001; accepted on September 17 , 2001