Human Reproduction vol 11 DO 7 pp 1525-1528, 1996
Effect of different co-culture systems in early human
embryo development
HX-Feng1-2-5, X.H.Wen2, TAmet 3 and S.C.Presser4
'Department of Urology, The University of Iowa, 200 Hawkins
Drive, 3 RCP, Iowa City, IA 52242-1089, USA, 2Laboratones of
Embryo Technology, Department of Biological Sciences, University
of Agriculture and Animal Sciences, Changchum 130062, People's
Republic of China, 3Department of Biochemistry, Molecular
Biology and Cell Biology, Northwestern University, Evanston, PL
60208-3500 and Reproductive Center, The New Margaret Hague
Women's Health Institute, Secaucus, NJ 07094, USA
'To whom correspondence should be addressed
The objective of this study was to examine the effects of
different culture systems on the development of early
human embryos in vitro. A total of 460 fertilized oocytes
from 82 cycles of patients were transferred into one of
four systems: (1) into droplets of Ham's F10 medium +
12% normal human serum (NHS); (2) co-cultured on a
human granulosa monolayer; (3) co-cultured with bovine
oviductal epithelial cells (BOEC); or (4) co-cultured with
bovine uterine epithelial cells (BUEC). The percentage
of cleavage and the morphological appearance of
embryos were recorded daily for 72 h in each system
using an inverted phase-contrast microscope. The results
showed that the proportions of the fertilized oocytes which
developed to the four-cell stage 48 h after retrieval
were, by culture system: (1) 70% (84/120); (2) 74% (85/
115); (3) 78% (91/117); and (4) 76% (82/108). At 72 h after
retrieval, the proportions of the eight-cell stage were, by
culture system: (1) 45% (38/84); (2) 62% (53/85); (3) 75%
(68/91); and (4) 70% (57/82). We concluded that a higher
proportion of fertilized oocytes developed to embryos at
the eight-cell stage in systems 2, 3 and 4 than in system 1.
This indicates the beneficial effect of co-culture of human
embryos with granulosa cell, BOEC and BUEC monolayers,
which may be due to various factors.
Key words: coculture/embryo culture/human fertilization
in vitro
Introduction
The optimal embryo culture medium for in-vitro fertilization
(TVF) and embryo transfer has not been established. Attempts
to improve in-vitro culture conditions by changes in media
formulations and supplements have met with little success
despite stringent quality control on embryo culture media
components and instruments. However, recent evidence
suggests that co-culture of human embryos with various cellular
monolayers results in increased rates of embryo development,
© European Society for Human ReproducUon and Embryology
decreased fragmentation and improvement in implantation and
pregnancy rates in IVF and embryo transfer (Wiemer et al,
1989; M6n6zo et al, 1990; Bongso et al., 1991; M6nezo et al.,
1992; Gregory et al., 1994; Mansour et al, 1994; Tucker
et al., 1994; Freeman et al, 1995). These beneficial impacts
on FVF outcome are supported by a substantial body of
evidence from animal studies (Thibodeaux et al, 1992; Bongso
et al, 1993; Catt, 1994; Feng et al, 1994a; Leppens and
Sakkas, 1995). Although this evidence suggests a positive
effect of co-culture cells upon IVF, the rate of development
has been lower when the embryos have been cultured in vitro
as compared with in-vivo development Further improvements
are therefore needed if the in-vitro maturation and fertilization
of oocytes and the in-vitro culture of the resulting embryos
are to be used more reliably for research and clinical purposes.
The present study was carried out to evaluate the effects of
four in-vitro systems on the development of early human
embryos and to establish an in-vitro model.
Materials and methods
Oocyte collection and fertilization in vitro
This experiment was done using 460 fertilized oocytes from 82 cycles
of patients undergoing IVF for various reasons. Ovarian follicle
stimulation was achieved by using gonadotrophin-releasing hormone
agonist (GnRHa)/human menopausal gonadotrophin (HMG) (Serono
Lab Inc, Randolph, MA, USA) and human chononic gonadotrophin
(HCG) (Schein Pharmaceutical Inc., Florham Park, NJ, USA). Following retrieval, oocytes were washed three tunes in Ham's F10 medium
(Gibco, Gaithersburg, MD, USA) supplemented with 12% normal
human serum (NHS) (Sigma, St Louis, MO, USA) and then placed
individually in 100 uJ droplets of the same medium at 10 oocytes
per droplet under sterile paraffin oil in a Petn dish (Falcon plastic
#3001: Becton Dickinson, Lincoln Park, USA) Insemination was
performed by adding 4.5 X 104 motile spermatozoa per oocyte Spermatazoa and oocytes were co-cultured in a humidified atmosphere of
5% CO7 in air at 37°C.
Establishment of co-culture system
The oviduct and utenne epithelial cells were isolated and maintained for culture by a modified procedure reported previously
(Thibodeaux et aL, 1992; Feng et al, 1994a) The oviduct and uterus
that were ipsilateral to ovaries containing a corpus haemorrhagicum
were collected from healthy bovines and placed in 50 ml conical
tubes containing phosphate buffered saline (PBS), supplemented with
100 IU penicillin and 100 (ig streptomycin/ml, and then cooled on
crushed ice in a thermos flask They were immediately transported
to the laboratory for tissue processing Excess tissue was removed
from the oviduct and the uterus prior to opening. The lumens of
oviduct and uterus were cut open longitudinally with sterile scissors
and cut into sections approximately 10 mm long, which were nnsed
1525
H-L.Feng et aL
three times with pre-incubated PBS/EDTA and then incubated in
35X10 mm Petri dishes containing PBS/EDTA (0 76% EDTA in
PBS) at 37°C for 2 h. After incubation, the epithelial sheets or cell
pellets were extracted from the lumens into a 35 mm Petn dish by
means of small stenle forceps The clumps of cells were further
broken down by repeated aspiration through an 18-gauge needle
attached to a 1.0 ml syringe containing 1 ml of EDTA/PBS. The
resulting suspension of clumps of epithelial cells were transferred to
15 ml conical tube containing 10 ml of TCM 199 (Earle's Salts'
Gibco, Life Technologies, Grand Island, USA) and washed by
centrifugation. Harvested cells were resuspended in fresh TCM-199
and washed two or three times and then resuspended in TCM 199
with 10% FBS supplemented with 100 units of penicillin and 100 u.g
of streptomycin/ml. These cell isolation techniques provided sufficient
cells from a single oviduct and uterus to seed four-well tissue plates
(Nunc, Roskilde, Denmark) which were cultured at 37°C in 5% CO2
in humidified air. At 36-48 h after the start of the culture, the medium
and unattached cells were removed, and 0.4—0.5 ml of fresh TCM
199 plus 10% FBS was added. The culture medium was replaced at
48 h intervals. Confluent monolayers with actively dividing cells
formed within 3—4 days and were utilized for co-culture.
Human granulosa cells for co-culture were collected from the
follicular aspirates by procedures previously published (Feng et aL,
1994a, Freeman et aL, 1995). These cells were pooled and washed
three times in PBS containing 2 mg bovine serum albumin (BSA)
per ml (B-PBS). The cells were then dispersed mechanically by
pipetting them repeatedly with a 1 ml pipette until all the cell clumps
had been reduced to a suspension of single cells. This cell suspension
was washed with B-PBS, and the cells were resuspended in Ham's
F10 medium containing 12% NHS and dispensed directly into fourwell culture plates and incubated for 24—36 h. This resulted in an
actively growing monolayer covering - 5 0 % of the culture areas.
Residual blood cells and unattached cells were removed by gentle
flushing of culture medium over the cell surface with a transfer
pipette followed by two rinses with culture medium Fresh medium
was added to the culture for 2-3 days before they were used.
In a second method of cultunng the granulosa cells, the proliferating
monolayer of cells from the original oocytes during maturation culture
was left undisturbed Fresh medium was added to the droplet at
intervals of 36-48 h.
In-vitro culture
At 17-20 h after insemination, the cumulus cells were removed
mechanically from the oocytes by using a finely drawn fire-polished
pipette. Individual marked oviductal and utenne cell monolayers were
washed three times with Ham's F10 medium plus 12% NHS prior to
being covered with a final 0.5 ml aliquot of medium ready for
embryo co-culture. Following equilibration, the fertilized oocytes
were randomly allocated to one of four systems (10-15/drop or
well): (1) into 100 (il droplets of Ham's F10 medium plus 12% NHS;
(2) co-cultured with human granulosa cell monolayer, (3) co-cultured
with bovine oviductal epithelial cells (BOEC); or (4) co-cultured with
bovine uterine epithelial cell (BUEC). All systems were cultured at
37°C in 5% CO2 and 100% humidity. Each medium was replaced
at 36—48 h intervals. The percentage of cleavage and the morphological appearance criteria of embryos were recorded daily for 72 h
in the four systems using an inverted phase-contrast microscope.
Statistical analysis
The significance of difference between the treatment groups was
established by %2 analysis. Significance was defined as P < 0.05.
1526
Table L Development of early human embryo in different culture systems
Culture system
1
2
3
4
Fertilized oocytes
120
115
117
108
Embryos developed m vitro (%)
Four-cell 48 h
Eight-cell 72 h
84(70)
85 (74)
91 (78)
82 (76)
38 (45)*-*
53 (62) b
68 (15f
57 (70)c
•Percentages with different superscripts differ, P < 0 05
Results
Characteristics of granulosa, oviductal and uterine epithelial
cell cultures
The granulosa, oviductal and uterine epithelial cells were
observed to be quite different in morphology when placed in
culture. The oviductal cells were composed of small clumps
of spherical, cibated cells which, over the initial 24 h in
culture, either dispersed into single cells or formed ciliated
vesicles that never became attached. Attachment occurred to
only a small extent during this initial penod of 24 h. During
the second period of 24 h, larger groups of oviductal epithelial
cells were observed to attach and spread over the plastic
surface of the wells, resulting m the formation of a 50-60%
monolayer. After 72 h in culture, the oviductal monolayers
could be separated into two morphologically distinct cell
populations consisting of islands of small, oblong, ciliated
cells separated by tracts of more slender secretory type cells.
By contrast, the granulosa and uterine epithelial cells consisted
of morphologically homogeneous squamous or cuboidal type
cells which progressed to form a 40-50% monolayer after
48 h in culture. Confluent monolayers with actively dividing
cells formed within 3—4 days in three co-culture systems and
were utilized for co-culture.
Development offertilized oocytes in different culture systems
in vitro
In this experiment, 460 pronuclear oocytes with excellent
morphology from 82 cycles were chosen and randomly allocated to one of four culture systems.
The experimental results are summarized in Table I. The
proportions of the fertilized oocytes which developed from the
pronuclear stage to the four-cell stage did not differ significantly
between the four culture systems. However, in system 3 or 4
a higher proportion of the fertilized oocytes developed to the
eight-cell stage than in either system 1 or 2
Discussion
The results indicate that the proportions of fertilized oocytes
which developed from the pronuclear stage to the four-cell
stage did not differ significantly between the four culture
systems. However, the percentages of embryos that reached
the eight-cell stage 72 h after retrieval were significantly higher
when co-cultured than when cultured alone. These results
agree well with those reported previously by Mansour et al.
(1994), who found that co-culture of human oocytes with their
Co-culture of early human embryos
cumulus cells significantly increased the percentage of embryos
that reached the eight-cell stage 72 h after retrieval compared
with control groups.
Cultunng the embryos an additional day in vitro would have
several advantages. Freeman et al. (1995) reported that the
implantation rate per embryo transferred was increased, in a
clinical trial, when all embryos were co-cultured and transferred
3 days after oocyte retrieval (24.3%), compared with the
preliminary study (10 3%) where a combination of co-cultured
and conventionally cultured embryos were transferred 2 days
after oocyte retrieval. One possible explanation for this increase
is that cultunng the embryos an extra day in vitro before
embryo transfer allowed the selection of embryos for transfer
that were not delayed in growth. Some embryos appeared
unfragmented and to be developing at the correct growth rate
on day 2, only to show increased fragmentation or slower
development by day 3 (Freeman et al, 1995). Dokras et al.
(1993) demonstrated that the development of individual
embryos to the blastocyst stage is poorly associated with the
grade of the embryo on day 2. Extending the in-vitro culture
period by an additional day also lessens the disparity between
the time that embryos would naturally move from the Fallopian
tube into the uterus. In addition, this could enhance the
possibility of embryo selection and pre-implantation genetic
diagnosis; thus embryos with chromosomal or genetic abnormalities and/or polyspermia may be excluded (Plachot et al,
1987). Another advantage of extending in-vitro culture time
includes the increased potential for successful embryo cryopreservation. This is in contrast to the pronuclear-stage zygote
in which damage would completely destroy the cell or to the
two-cell embryo where damage to one cell would destroy 50%
of the embryo and reduce its developmental capacity (Trounson
and Jones, 1993).
Many in-vitro culture studies have been designed to avoid
this loss of viability and to overcome the so called 'embryo
developmental arrest' or 'blocks to development' in vitro.
These blocks, which are attributed to artifacts of the culture
environment, occur at stages of development characteristic for
each species. For example, pig embryos cultured from the
one- or two-cell stages typically block at the four-cell stage,
while cattle, goat and sheep embryos blocked at the eight to
16-cell stages. Rodent embryos (other than those from a few
inbred mouse strains) are blocked at the two-cell and later
stages, whereas horse and human are blocked at the four-cell
stages (Tesarik, 1989; Feng, 1994b; Bavister, 1995). These
blocks are usually observed at the approximate time of genomic
activation or occur at the stage of embryo transit from the
oviduct to the uterus. Moreover, it is now clear that co-culture
systems can effectively support development through the
characteristic stage of in-vitro developmental block (Rexroad,
1989; Feng et al, 1994a). Our results demonstrating that the
co-culture systems can overcome the four-cell stage block
in vitro are, consistent with these findings.
Regardless of the developmental outcome, co-culture of
embryos with other types of cells enhanced the frequency and
duration of survival in vitro compared with that of embryos
cultured alone. Co-culture with various cell types has been
used extensively to improve development of early embryos
from a variety of species, including goats, cattle, horse,
(Thibodeaux and Godke, 1992), mouse (Leppens and Sakkas,
1995) and rhesus monkeys (Goodeaux et al, 1990), and in
particular for human FVF early embryos (Bongso et al, 1992;
Mansour et al, 1994; Freeman et al, 1995; Tucker et al,
1995; Ben-Chetrit et al, 1996). However, a substantial number
of other reports have demonstrated no overt or statistically
significant improvement in early embryogenesis (Van Blerkom,
1993; Plachot et al., 1993) or clime pregnancy rates (Sakkas
et al, 1994). These arguments have been well summarized by
Bavister (1992, 1995). We have confirmed in the present study
that the positive influence of embryo co-culture systems is
extended to FVF early human embryos.
The biological basis of this phenomenon is not fully
understood. Possible functions of co-culture cells include: (i)
detoxifying die culture medium, for example by chelation of
heavy metal ions; (ii) reducing the concentration of normal
constituents of the medium, such as glucose, that inhibit
embryo development; (lii) secretion of factors into the medium
that stimulate embryos or enhance the maternal embryonic
genome shift and improve cell organelle structure, such as (a)
nutrients and substrates, including amino acids or pyruvate, or
(b) proteins or 'growth factor' (Gandolfi et al, 1989 a,b); (IV)
stabilization of the physio-chemical conditions, such as pH,
O2/CO2 concentrations, or the culture medium (Bongso et al,
1993); or (v) a combination of several of these possible
mechanisms. We suggest either that embryos not exposed to
the co-culture environment during early cleavage may be
unable to overcome damage that was incurred earlier, or that
co-cultured embryos obtain certain beneficial factors for normal
development in vitro.
The beneficial effects of co-culture in support of the early
embryo development is observed in a variety of diverse cell
types, including cumulus and granulosa cells, oviduct and
uterine cell monolayers, chicken skin cell monolayers, liver
cell monolayer, mouse testicular cell monolayers and monkey
kidney cells (Goto et al, 1988; Bavister, 1995). This observation suggests that at least part of the influence of co-culture
on early development is a very general one that may reflect
secretion into the medium of a pool of 'embryotrophic' factors
that are not cell-type specific, or might simply consist of a
common ability of these cell types to purify the culture
environment by acting as a sink for the removal of deletenous
culture elements. However, there is a definite cell type-specific
influence supenmposed upon this more general effect (Watson
et al, 1994). This concept is in agreement with the results of
our present study.
It has been suggested that cumulus and granulosa cells may
have a supportive effect through cell to cell interactions and
selective transport of nutrients regained from the culture
medium to the embryos. In addition, they have a capacity for
steroid synthesis (Mansour et al, 1994). The present study
indicates that co-culture with BOEC (system 3) and BEUC
(system 4) is more efficient than co-culture with human
granulosa cells (system 2) in enhancing the development of
embryos to the eight-cell stage at 72 h after retrieval. It is
possible that systems 3 and 4 are more effective in the
production of embryotrophic factors) or in the prohferation
1527
H.L.Feng et aL
of cell numbers. Recently, it has been dembnstrated that
oviductal and uterine cells may produce more specific glycoproteins, free amino acids, lactate, and growth factors such
as insulin-like growth factor (IGF), binding protein (BP),
leukaemia inhibiting factor (LIF), colony-stimulating-1 (CSF1), interleukin (IL)-l and platelet-derived growth factor
(PDGF) (Bongso et al., 1993; Moreau et al., 1995). Although
monolayers of both oviductal and uterine epithelial cells can
secrete embryotrophic factor(s), it is possible that the different
cell types may produce different beneficial factors or different
amounts of specific factors). In addition, BOEC may promote
the metabolism and development of the embryo by virture of
their ciliary action which provides an in-vitro environment for
early-stage human embryos more similar to the in-vivo situation. This may be the reason why system 3 is more efficient
than the other two co-culture systems.
In conclusion, human embryos may require additional
embryotrophic factor(s) not present in simple culture media
for continued development of an increased proportion of
embryos beyond the second to third day after fertilization. Coculture with monolayers of granulosa cells, BOEC and BUEC
can enhance the development of early human embryos and
possibly overcome a block to development. This method
may also be used to culture embryos for an extra day to
reduce asynchrony between the embryo and uterus, as well as
to enhance the possibility of embryo selection and preimplantation genetic diagnosis. Collectively, these findings
may provide useful information enabling human assisted
reproduction programmes to develop the optimum culture
system to increase the quality of human embryos available for
replacement.
Acknowledgements
Appreciation is extended to Drs Jay I.Sandlow, Amy Sparks, and
Alexander Sandra for their expert contributions to this manuscript, and
to Ms Kristina Gaunt for preparing and editing the final manuscript.
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Received on February 20, 1996, accepted on May 8, 1996