Human Reproduction vol.10 no. 12 pp.3243-3247, 1995
CASE REPORT
Blastocyst development and birth after in-vitro
maturation of human primary oocytes, intracytoplasmic
sperm injection and assisted hatching
Frank L.Barnes, Amanda Crombie2,
David K.Gardner, Annette Kausche,
Orly Lacham-Kaplan, Anne-Maria Suikkari2,
Jennifer Tiglias, Carl Wood1 and Alan O.Trounson3
Institute for Reproduction and Development and 'Department of
Obstetrics and Gynaecology, Monash University, Monash Medical
Centre, 246 Clayton Road, Clayton, Victoria 3168 and 2Monash
IVF Pty Ltd, Epworth Hospital, Erin Street, Richmond, Victoria
3121, Australia
3
To whom correspondence should be addressed
Immature oocyte recovery followed by in-vitro oocyte
maturation and in-vitro fertilization is a promising new
technology for the treatment of human infertility. The
technology is attractive to potential oocyte donors and
infertile couples because of its reduced treatment intervention. Immature oocytes were recovered by ultrasoundguided transvaginal follicular aspiration. Oocytes were
matured in vitro for 36-48 h followed by intracytoplasmic
sperm injection (ICSI). Embryos were cultured in vitro
for 3 or 5 days before replacement. Assisted hatching was
performed on a day 5 blastocyst stage embryo. Embryo
and uterine synchrony were potentially enhanced by luteinization of the dominant follicle at the time of immature
oocyte recovery. Mature oocyte and embryo production
from immature oocyte recovery were similar to the previous
IVF results of the patients. A blastocyst stage embryo,
produced as a result of in-vitro maturation, ICSI, in-vitro
culture and assisted hatching, resulted in the birth of a
healthy baby girl at 39 weeks of gestation.
Key words: assisted hatching/human IVF/in-vitro oocyte
maturation/intracytoplasmic sperm injection
Introduction
The activation of primary oocytes when liberated from the
ovarian follicle has been recognized for a very long time
(Pincus and Enzmann, 1935; Pincus and Saunders, 1939) and
was originally considered as potentially the most appropriate way to obtain mature oocytes for in-vitro fertilization
(IVF) techniques (Edwards, 1965). However, the completion
of nuclear maturation in vitro was not necessarily indicative
of oocyte developmental competence unless the oocyte was
cultured within the intact follicle in vitro (Moor and Trounson,
1977). Progress in the technique of in-vitro maturation has more
recently led to the demonstration of complete developmental
© Oxford University Press
competence in mice (Schroeder and Eppig, 1984) and a
range of other animal species (Trounson, 1992). The in-vitro
maturation of primary oocytes is widely used in cattle to
enhance the efficiency of embryo production of subfertile
females (Looney et al., 1994; Trounson et al, 1994a). The
use of in-vitro matured oocytes in the treatment of human
infertility is promising. There are recent reports of
pregnancies and live births resulting from in-vitro matured
oocytes recovered from ovariectomy specimens for donation
and from infertile polycystic ovary syndrome (PCOS) patients
(Cha et al, 1991; Trounson et al, 199-"1..). With the development of procedures to collect primary oocytes followed by
in-vitro maturation and IVF, it is now feasible to consider its
clinical application in human infertility.
The most successful use of in-vitro matured oocytes has
been in an oocyte donation programme (Cha et al., 1991). The
successful application of immature oocyte recovery in routine
IVF, with the infertile couple producing the gametes as well
as the female partner becoming the embryo transfer recipient,
remains a challenging one. Here we describe conditions that
have resulted in the birth of a healthy baby after the recovery
of immature oocytes from an infertile couple. The serial linkage
of immature oocyte collection, in-vitro oocyte maturation,
intracytoplasmic sperm injection (ICSI), in-vitro culture,
assisted hatching and improved embryo and uterine synchrony
provides a basis for the implementation of immature oocyte
collection in the treatment of human infertility.
Case report
Patient case history and treatment
A couple presented for an infertility evaluation in 1990. The
wife had undergone surgery in 1983 for pelvic inflammatory
disease. Left Fallopian tube damage was evident and was
treated by left cuff salpingostomy. Further surgery in 1989
resulted in a left salpingectomy. Adhesions to the right ovary
and Fallopian tube were noted and a patency test revealed no
spill of dye through the right Fallopian tube. The patient was
also diagnosed as having polycystic ovaries based on the
ultrasonographic polycystic appearance of the ovaries (>10
follicles of 2 to 8 mm in diameter on both ovaries) and
irregular menstrual cycles between 28 and 56 days. The
husband had previously undergone a vasectomy and subsequent
reversal in 1985.
Three attempts using standard IVF techniques failed to
produce a sustained pregnancy. Reduced fertilization was
3243
F.L.Barnes et al.
Table I. Patient treatment history by conventional in-vitro fertilization
Cycle
No. of mature
oocytes recovered
No. of fertilized
oocytes (%)
No. of cleaved
embryos (%)
No. of embryos
transferred
No. of embryos
frozen
1
2
3
21
4
13
16(76)
1(25)
1(8)
8(38)
1 (25)
1 (8)
3
la
1
5
0
0
a
Resulted in pregnancy, aborted at 8 weeks.
observed in IVF cycles 2 and 3 (Table I). Standard [150 000
spermatozoa per oocyte in 1 ml of human tubal fluid (HTF)
medium (Quinn et al., 1985) in 5 ml Falcon culture tubes]
and microdrop (500 000 spermatozoa per oocyte in 20 (i.1 drops
of HTF medium with 10% heat-treated patient's serum under
oil) insemination procedures in cycles 2 and 3 respectively
failed to improve fertilization results. Cycle 2 produced a
pregnancy, but spontaneous abortion occurred during week 8
of pregnancy. The patient did not wish to receive any further
treatment with fertility drugs, and after consultation with the
patient review committee the couple agreed to participate in
an ongoing study examining the in-vitro maturation of oocytes
from polycystic ovary patients with subsequent fertilization
and anticipated embryo replacement.
Oocyte retrieval, maturation, fertilization and embryo culture
No ovarian stimulation with fertility drugs or hormones was
used for immature oocyte recovery. Oocytes were recovered
by ultrasound-guided transvaginal follicular aspiration on
days 10-12 of a spontaneous menstrual cycle. Follicles
between 2 and 10 mm in diameter were identified vaginally
with an Acuson 128/XP4, 5 MHz transvaginal ultrasound
(Acuson, Melbourne, Australia) and aspirated using a specially
designed aspiration needle (Cook Australia, Brisbane,
Queensland, Australia) and technique (Trounson et al., 1994b).
Oocytes were collected in 10 ml culture tubes (#2001
Falcon; Becton Dickinson, Lincoln Park, NJ, USA) containing
2 ml warm buffered HTF medium. The recovery and isolation
of oocytes were performed by washing the follicular aspirate
through an Em-Con embryo concentrating filter (Immuno
Systems, Inc., Spring Valley, WI, USA). Samples were rinsed
with Dulbecco's phosphate-buffered saline (D-PBS; Gibco
BRL, Life Technologies, Inc., Grand Island, NY, USA) with
2% heat-treated fetal calf serum (FCS; PA Biologicals Co.
Pty Ltd, Sydney, New South Wales, Australia) to remove
erythrocytes. The filter retentant was poured into 100X15 mm
Petri dishes (Nunclon, Roskilde, Denmark) and oocyte cumulus
masses were identified using a stereo-dissecting microscope.
The immaturity of oocytes was determined by cumulus compaction and/or the presence or absence of a germinal vesicle.
All oocyte handling procedures were conducted on warm
stages and plates at 37°C. Cumulus oocyte complexes and
cumulus-denuded oocytes were washed in D-PBS with 2%
FCS, and then in maturation medium. Maturation medium
consisted of TCM199 (Sigma, St Louis. MO. USA) supplemented with 10% FCS, 0.075 lU/ml recombinant human
follicle-stimulating hormone (FSH; Gonal-F; Serono, French
Forest, New South Wales, Australia), 0.5 IU/ml human chor3244
ionic gonadotrophin (HCG; Profasi; Serono), 0.29 mM pyruvate, 0.05 mg/ml penicillin and 0.075 mg/ml streptomycin.
Immature oocytes were cultured in pre-equilibrated 50 |il
drops of maturation medium under silicon oil (Dow Corning
200/500 cs; BDH Laboratory Suppliers, Poole, UK) in
60X15 mm Petri dishes (#1007; Falcon) for 36-46 h at 37°C
in 5% CO2 in air. The cultures were maintained in a sealed
glass desiccator.
In immature oocyte cycle 1 (Table II), mature oocytes were
inseminated individually with Percoll-separated (45 and 90%
gradients; Pharmacia LKB Biotechnology AB, Uppsala,
Sweden) spermatozoa in 25 u.1 drops of HTF medium under
silicon oil, with 10% heat-treated patient's serum and a final
sperm concentration of 1.6X106 spermatozoa/ml. Oocytes were
inseminated at 36 h after the onset of maturation and assessed
for fertilization 14 h after insemination. Fertilization was
determined by the appearance of two distinct pronuclei and
two polar bodies.
In immature oocyte collection cycles 2 and 3, ICSI was
performed (Palermo etai, 1992). Mature oocytes were denuded
of cumulus using finely drawn glass pipettes 46 h after the
onset of maturation following a 1 min exposure to 8 IU/ml
sheep testes hyaluronidase (Sigma) in D-PBS + 4 mg/ml
bovine serum albumin (BSA; Miles Pentex Inc., Diagnostics
Division, Kankakee, IL, USA) and allowed to 'rest' for 3060 min in HTF medium with 10% patient's serum at 37°C
maintained in a gas atmosphere of 5% CO2 in air. ICSI
spermatozoa were prepared by mini-Percoll separation (45 and
90% gradients) at 1800 g for 20 min. After Percoll separation,
the 90% layer was washed in HTF with 24 mg/ml BSA at
1800 g for 5 min. Following the wash procedure, the supernatant was removed and the sperm pellet overlain with 0.251.00 ml HTF, with 24 mg/ml BSA and spermatozoa allowed
to swim-up for 2-3 h at 37°C in 5% CO 2 in air (1.0 ml was
used for high concentration sperm suspensions and as little as
0.25 ml were used for low concentration sperm suspensions).
Prior to ICSI, the swim-up supernatant was washed with DPBS with 4 mg/ml BSA and resuspended at a final concentration
of 1X106 spermatozoa/ml. Mature oocytes were added to a
100 nl drop of spermatozoa in D-PBS + 4 mg/ml BSA in a
specially prepared microinjection chamber (Trounson and
Sathananthan, 1993) for ICSI. Embryos were placed immediately into culture upon completion of ICSI. Fertilization was
assessed 19-21 h after ICSI for the appearance of two distinct
pronuclei and two polar bodies.
In immature oocyte collection cycle 1, no embryos were
produced. In immature oocyte collection cycle 2 (Table II),
single embryos were cultured in HTF medium as described
In-vitro maturation of human primary oocytes
Table II. Patient treatment history by immature oocyte collection
Cycle
No. of immature
oocytes recovered
No. of mature
oocytes (%)
Fertilization
procedure
No. of fertilized
oocytes
No. of embryos
developed
No. of embryos
transferred
1
2
3
19
20
13
9(47)
11 (55)
10 (77)
IVF3
lCSIb
ICSI
0
5 (45%)
8 (80%)
0
5
6
0
3
lc
IVF = in-vitro fertilization.
"Insemination in microdrops.
b
Intracytoplasmic sperm injection (ICSI).
c
Birth of a healthy baby.
Table III. Composition of Gardner's Gl and G2 media
Component
Gl medium
G2 medium
85.16
NaCl (mM)
85.16
KCI (mM)
5.5
5.5
0.5
NaH-,PO4-2H-,O (mM)
0.5
1.8
1.8
CaCfr2H,0 ("mM)
1.0
1.0
MgSO4-7H,O (mM)
25.0
25.0
NaHCO3 (mM)
Sodium lactate (60% syrup) (mM) 21.0(10.5 L-isomer) 11.74 (5.87 L-isomer)
Sodium pyruvate (mM)
0.32
0.10
Glucose (mM)
0.50
3 15
Glutamine (mM)
1.0
1.0
Taunne (mM)
0.1
00
Non-essential amino acids
All
All
Essential amino acids
All
None
Ethylenediaminetetraacetic acid (mM) 0.1
0
Bovine serum albumin (g/1)
2.0
2.0
Penicillin (g/1)
0.06
0.06
Streptomycin (g/1)
0.05
0.05
Phenol red (g/1)
0.01
0.01
above in 25 (il drops under silicon oil. In immature oocyte
collection cycle 3, single embryos were cultured either in
1 ml HTF medium in 5 ml Falcon culture tubes or Gardner's
Gl medium (Table III) containing non-essential amino acids
at concentrations in Eagle's minimum essential medium (Eagle,
1959) in 20 (0.1 drops under silicon oil. Embryo cultures were
maintained at 37°C in glass desiccators purged with 5% CO2,
5% O2 and 90% N2. Embryos were assessed for cleavage 68
h after ICSI; in immature oocyte collection cycle 3, they were
transferred to fresh equilibrated HTF medium or Gardner's
G2 medium containing both essential and non-essential amino
acids (Eagle, 1959), until 110 h after ICSI and re-evaluated.
In immature oocyte collection cycle 3, the zona pellucida of
an early blastocyst stage embryo was partially digested with
acidified HTF medium (pH 2.4) using the micromanipulation
techniques described by Cohen et al. (1992) for the assisted
hatching of embryos from the zona.
There was no dominant follicle present on the ovaries at
the time of oocyte recovery in immature oocyte collection
cycles 1 and 2, and plasma oestradiol concentrations were 327
and 192 pmol/1 respectively. Hormone replacement therapy
consisted of 4 mg/day oestradiol valerate (Progonova; Schering
AG, Alexandria, NSW, Australia), commencing at the time of
oocyte recovery, and progesterone vaginal pessaries, 300 mg/
day, commencing -48 h later at the time of insemination.
Endometrial assessment at embryo transfer in immature oocyte
collection cycle 2 revealed a 5 mm proliferative endometrium.
In immature oocyte collection cycle 3, a dominant follicle of
18 mm in diameter was present at the time of oocyte recovery
on cycle day 12 and the plasma oestradiol concentration was
391 pmol/1. It was left intact in an attempt to improve
the likelihood of corpus luteum formation and endometrial
conversion to secretory type. At the time of oocyte recovery,
1000 IU HCG were administered, followed by progesterone
pessaries, 300 mg/day, 48 h later. Ultrasound assessment
revealed a corpus luteum and a secretory endometrium of 9 mm at the time of embryo transfer. Pregnancy
was determined by rising plasma serum HCG concentrations
and a fetal heart beat confirmed by ultrasound at 6 weeks of
gestation.
Results
Oocyte and embryo production were similar when different
treatment modalities were examined (gonadotrophin-stimulated
versus immature oocyte collection cycles, Tables I and II).
The frequencies of mature oocytes from the gonadotrophinstimulated cycles and the immature oocyte collection cycles
were similar (12.6 and 10 oocytes respectively). Similarly, a
mean of 3.3 embryos was produced in the gonadotrophinstimulated cycles and a mean of 3.7 embryos was produced
from the immature oocyte collection cycles. In immature
oocyte collection cycle 3, six embryos cleaved to the 2- to
5-cell stage by 68 h after ICSI. Three embryos were transferred to fresh medium and cultured to 110 h. One early
blastocyst stage embryo was produced in Gardner's G1/G2
media. After assisted hatching, it was transferred to the patient's
uterus and resulted in the birth of a healthy baby girl of mass
2995 g at 39 weeks of gestation.
Discussion
In immature oocyte collection cycle 3, considerations were
made which may have affected its success in achieving a
pregnancy. These considerations included the acquisition of
developmental competence of the in-vitro matured oocytes,
ICSI to increase fertilization rates and the development of
embryos, assisted hatching and an improvement of the synchrony between embryo development and uterine receptivity
by the transfer of later stage embryos. The selection of a
developmentally competent oocyte or embryo was achieved
by allowing embryo cleavage to progress to the blastocyst
stage in vitro. Blastocyst stage development was achieved in
3245
F.L.Barnes el al.
a cell- and serum-free medium (Gardner's G1/G2 media).
To our knowledge this is the first report of its kind using
in-vitro matured human oocytes. In animals, the developmental
competence of oocytes may be influenced by the maturation
medium, the follicle size and the presence and nature of
associated follicle cells, and it may determine the ability of
oocytes to develop into blastocyst stage embryos in vitro
(Sirard et al., 1988; Eppig et al., 1992; Pavlok et al, 1992).
While it has been demonstrated that follicle size affects meiotic
competence in the human (Tsuji et al., 1985), further research
is required to determine the importance of other factors in
human oocyte maturation.
It has been documented in animals that qualitative changes
occur in the zona pellucida during oocyte maturation in vitro
which may reduce the fertilization rates (Choi et al., 1987).
These changes are often referred to as zona hardening. Similar
changes in the zona pellucida have been described for human
oocytes when exposed to certain cryoprotectants (Pickering
et al., 1991). It has been suggested that different patient
aetiologies, such as PCOS, patients with high basal FSH
concentrations or patients with multiple failed IVF attempts,
may have a thickened zona pellucida or induced changes in
the structure of the zona pellucida leading to zona hardening
(Cohen et al., 1992; Wiemer et al., 1994). Zona hardening
would be impossible to identify and inherent in in-vitro matured
oocytes, whereas oocytes and embryos with thickened zona
are more easily distinguished. To circumvent these obstacles,
we decided to use ICSI and assisted hatching (Cohen et al.,
1992; Palermo et al., 1992).
To potentially enhance the uterine and embryo synchrony,
the dominant follicle was left intact during immature oocyte
collection and luteinization was induced with HCG. The
administration of HCG to the patient at the time of oocyte
recovery and the initiation of in-vitro maturation may aid the
synchronizing of oocyte maturation and embryo development
with the endometrium. Preliminary evidence in our laboratory
has shown that the oocyte is rarely recovered from the leading
follicles in excess of 10 mm3. When attempting to recover
oocytes from these larger follicles, significant numbers of
mural granulosa cells are often washed from the follicle. This
may account for a poor luteal and subsequent endometrial
response in some immature oocyte collection patients.
The preceding report is the second birth resulting from the
maturation of immature oocytes from this laboratory (Trounson
et al., 1994b). Whether or not the serial linkage of the rather
invasive laboratory procedures enhanced the developmental
probability of immature oocytes must be viewed with speculation. Clearly, this report demonstrates that immature oocytes
may be fertilized by ICSI and develop to the blastocyst stage
in vitro, and subsequently resulted in the birth of a healthy
baby girl. Current studies are under way to evaluate the
relative significance of the described procedural modifications
compared with those first reported (Trounson et al., 1994b).
There are several advantages to the clinical application of
the immature oocyte collection procedure. Patients need not
undergo ovarian hyperstimulation and are therefore not at risk
for developing ovarian hyperstimulation syndrome and other
undesired consequences involving fertility drugs (Trounson
3246
et al., 1994b). Patient management and discomfort are reduced
significantly because there is no need for multiple injections
of fertility drugs and there is a minimal requirement for blood
tests and sonographic scans of the ovaries to monitor ovarian
stimulation. This should result in substantial cost savings for
fertility drugs and patient monitoring. These, as well as
other potential benefits, make immature oocyte recovery more
appealing to potential oocyte donors and patients desiring
reduced treatment intervention.
Acknowledgements
This study was supported by grants from Monash IVF and IVF
America Inc., Purchase, New York, NY, USA.
References
Cha, K.Y., Koo, J.J., Ko, J.J., Choi, D.H., Han, S.Y. and Yoon, T.K. (1991)
Pregnancy after in vitro fertilization of human folhcular oocytes collected
from non-stimulated cycles, their culture in viiro and their transfer in a
donor oocyte program. Fertil. Sterti, 55, 109-113.
Choi,T.S., Mon, M., Kohmoto, K. and Shoda, Y. (1987) Beneficial effect of
serum on the fertilizability of mouse oocytes matured in vitro. J. Reprod.
Fertil., 79, 565-568.
Cohen, J., Alikani, M., Trowbridge, J. and Rosenwaks, J. (1992) Implantation
enhancement by selective assisted hatching using zona drilling of human
embryos with poor prognosis. Hum. Reprod., 7, 685-691.
Eagle, H. (1959) Amino acid metabolism in mammalian cell cultures. Science,
130, 432^37.
Edwards, R.G. (1965) Maturation in vitro of mouse, sheep, cow, pig, rhesus
monkey and human ovarian oocytes. Nature, 102, 493—497.
Eppig, J.J., Schroeder, A.C. and O'Brien, M.J. (1992) Developmental capacity
of mouse oocytes matured in vitro: effects of gonadotrophic stimulation,
follicular origin, and oocyte size. J. Reprod. Fertil., 95, 119-127.
Looney. C.R., Lindsay, B.R., Gonseth, C.L. and Johnson, D.L. (1994)
Commercial aspects of oocyte retrieval and in vitro fertilization (IVF) for
embryo production in problem cow. Theriogenology, 41, 67-72.
Moor, R.M. and Trounson, A.O. (1977) Hormonal and follicular factors
affecting maturation of sheep oocytes in vitro and their subsequent
developmental capacity. J. Reprod. Fertil., 49, 101-109.
Palermo, P., Joris, H., Devroey, P. and Van Steirteghem, A.C. (1992)
Pregnancies after intracytoplasmic injection of single spermatozoon into an
oocyte. Lancet, 340, 17-18.
Pavlok, A , Lucas-Hahn, A. and Niemann, H. (1992) Fertilization and
developmental competence of bovine oocytes derived from different
categories of antral follicles. Mol. Reprod. Dev., 31, 63-67.
Pickering, S.J., Braude, PR. and Johnson, M.H. (1991) Cryoprotection of
human oocytes: inappropriate exposure to DMSO reduces fertilization rates.
Hum. Reprod., 6, 142-143.
Pincus, G. and Enzmann, E.V. (1935) The comparative behaviour of
mammalian eggs in vivo and in vitro. I. The activation of ovarian eggs. J.
Exp. Med., 62, 655-675.
Pincus, G. andSaunders. B. (1939) The comparative behaviour of mammalian
eggs m vivo and in vitro. IV. The maturation of human ovarian ova. Anat.
Rec, 75, 537-545.
Quinn. P.. Kerin, J.F. and Warnes, G.M. (1985) Improved pregnancy rate in
human in vitro fertilization with the use of a medium based on the
composition of human tubal fluid. Fertil. Steril., 44, 493-498.
Schroeder, A.C. and Eppig. J.J. (1984) The developmental capacity of mouse
oocytes that matured spontaneously in vitro is normal. Dev. Biol., 102,
493^97.
Sirard, M.A., Parish, J.J., Ware, C.B., Leibfried-Rutledge.M.L. and First, N.L.
(1988) The culture of bovine oocytes to obtain developmentally competent
embryos. Biol. Reprod., 39, 546-552.
Trounson, A.O. (1992) The production of ruminant embryos in vitro. Anim.
Reprod. Sci., 28, 125-137.
Trounson, A. and Sathananthan. A.H. (1993) Fertilization using
micromanipulation techniques. In Trounson.A. and Gardner.D.K. (eds).
Handbook of In Vitro Fertilization. CRC Press, Boca Raton, FL, USA,
pp. 131-150.
Trounson, A., Pushett. D., MacLellan, L.J., Lewis. I. and Gardner. D.K.
In-vitro maturation of human primary oocytes
(1994a) Current status of IVM/IVF and embryo culture in humans and
farm animals. Theriogenology. 41, 57-66.
Trounson, A.O., Wood, C. and Kausche, A. (1994b) In vitro maturation and
the fertilization and developmental competence of oocytes recovered from
untreated polycystic ovarian patients. Fertil. Steril., 62, 353-362.
Tsuji, K., Sowa, M and Nakano, R. (1985) Relationship between human
oocyte maturation and different follicular sizes. Biol. Reprod., 32, 413—417.
Wiemer, K.E., Hu, Y., Cuervo. M., Genetis. P. and Leibowitz, D. (1994) The
combination of coculture and selective assisted hatching: results from their
clinical application. Fertil. Steril., 61, 105-110.
Received on June 20, 1995; accepted on August 31, 1995
3247