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Background
In vitro fertilization with coculture has been utilized in animal in vitro
embryo culture systems for over 30 years and more recently in some clinical
human in vitro fertilization laboratories as well. The basic concept involves
growing embryos in a culture medium on top of a proliferating monolayer of
cells such as fallopian tube cells or cells from the lining of the uterus
called endometrial cells.
The idea is that these cells, which are sometimes referred to as "feeder"
cells or "helper" cells, will stimulate development of the embryos by removing
toxins from the medium, adding growth factors, or some other beneficial
effect. Some studies have demonstrated improved pregnancy rates and delivery
rates with utilization of coculture for human in vitro fertilization.
Why don't all IVF centers use coculture?
There are several reasons that coculture is not currently more widely used
for human IVF:
1. Coculture involves a lot of tedious work in the laboratory which leads to
additional expense.
2. Most IVF labs are not experienced with culture of cells other than eggs,
sperm, and embryos. Although culturing cells from the endometrium or
fallopian tube is not extremely difficult, it does involve learning some new
techniques.
3. There is not universal agreement that coculture is necessary to provide
optimal pregnancy rates from human in vitro fertilization.
4. Another issue is that depending on the source of cells used for the
coculture there may be concerns about transmission of infectious diseases
such as viruses from the cell line to the developing embryos. To date there
have been no reported cases of viral transmission to a human fetus.
Non-autologous cell lines should be screened for infectious diseases prior
to use in human embryo coculture.
3 embryos with bovine tubal cell coculture
Embryos look fuzzy because plane of focus is on the tubal cells
Who might be most likely to benefit from coculture?
Coculture is usually not applied universally to all cases in an IVF program.
It is usually reserved for use in the "poor prognosis" patients. Studies
have suggested that these patients can benefit the most from IVF with
coculture. Examples of poor prognosis patients include women over 40, women
with previous IVF failures, women with elevated FSH (follicle stimulating
hormone) levels, and women who respond poorly to ovarian stimulation with
gonadotropins.
The debate regarding coculture
In general there are two schools of thought in this area.
One school says that coculture can be of a benefit for some patients
undergoing in vitro fertilization. The philosophy here is that we do not
need to know the exact mechanism of the benefit of coculture, or exactly how
standard in vitro culture systems are deficient - what we want is to help the
couple get their baby.
The other school says coculture is a crutch that masks the real problem which
is sub-optimal in vitro embryo culture systems. These people would prefer to
use very pure and carefully defined media in order to maximize the culture
environment. They believe that this can yield an equally high pregnancy rate
without the use of coculture.
What kinds of cells are used for coculture?
Clinical in vitro fertilization programs that are utilizing coculture for
their human IVF generally use one of three cell types. However, there are
numerous other cell lines that have been successfully utilized as well.
The cell lines most often used are fallopian tube cells which can be from
either human or animal origin, endometrial cells from the lining of the
uterus, or Vero cells which are from an immortalized cell line derived from
African Green Monkey kidney cells. Cumulus cells from around the egg with or
without granulosa cells from the walls of the ovarian follicles where the
eggs develop can also be used for coculture.
Our center is currently performing coculture in selected cases. According to
the preferences of the couple, we use either endometrial cells from the
woman's own uterine lining, or fallopian tube cells from a cow (bovine tubal
cells) for coculture with the embryos.
How is coculture done?
Tubal cell monolayer as used for coculture
Phase contrast microscopy with Hoffman optics
Most commonly the eggs and sperm are mixed together on the day of egg
retrieval without the coculture cells. The next morning, after identification
of the fertilized eggs (called zygotes), these embryos are then transferred
on to the coculture cells which have been prepared several days in advance.
The embryos are then cultured with the helper cells until the time of embryo
transfer. This is usually two more days of culture.
Coculture with blastocyst transfer
Mouse embryos from coculture
Hatching blastocyst at bottom left
Completely hatched blastocyst at upper right
Another potential application of coculture for human in vitro fertilization
programs is that of culturing embryos to the blastocyst stage and then
performing blastocyst transfer. This allows selection of embryos that have
been able to survive through the early cleavage stages of the first five days
after fertilization. It is generally very difficult to get good numbers of
high quality blastocysts when culturing in defined medium (no coculture).
This technique can allow transfer of fewer embryos while still maintaining an
excellent pregnancy rate. For example, some programs have cultured embryos
to blastocyst stage and had very good pregnancy rates resulting from transfer
of only two blastocysts. This would greatly reduce the risk of high order
multiple pregnancy that is seen in some programs transferring higher numbers
of embryos.
Several published studies have demonstrated improved pregnancy rates and
delivery rates with utilization of coculture for human IVF. Coculture has
also been shown to produce a higher proportion of embryos that develop to the
blastocyst stage. One study showed that coculture resulted in a 68%
blastocyst formation rate and a 50% pregnancy rate per transfer in patients
with several previous IVF failures (R. Schillaci, et al., Human Reproduction,
Volume 9, p. 1131-1135, 1994). Also, transfer of "leftover" cocultured
blastocysts that had been cryopreserved (frozen) resulted in very good
delivery rates. In one program with 563 thawed blastocyst transfer cycles,
the pregnancy rate was 26% per transfer for hormonally controlled cycles,
and 13% per transfer for "natural" cycles (R. Kaufmann, et al., Fertility and
Sterility, Volume 64, p. 1125-1129, 1995). In another study, 101 cycles of
coculture with blastocyst transfer for couples with multiple previous IVF
failures resulted in a pregnancy rate of 29% per retrieval and 37% per
transfer. This was accomplished with a maximum of 3 blastocysts transferred
per cycle (F. Olivennes, et al., Human Reproduction, Volume 9, p. 2367-2373,
1994).
The future of coculture
Further research is needed in order to define exactly which patients would be
benefited by coculture. Also, the coculture technique itself may be able to
be further modified such that in vitro embryonic development is even better
than what can be achieved with current technology. For example, many aspects
of the coculture technique could be altered, such as using a different cell
line, a different medium, smaller droplets for culture, changing the medium
more frequently, or other changes. By varying the usual coculture techniques,
we might obtain a further improvement in embryonic development over what is
currently possible.
A practical problem with research in this area is that studies using variations
on standard techniques are relatively easy to perform using animal embryos,
but studies using human embryos are problematic to set up and implement.
Results from coculture studies done with animal embryos will not necessarily
be applicable to IVF with human embryos.
Much has been learned about coculture both for animal in vitro embryo culture
and for in vitro culture in the human as well. Studies continue to attempt
to discover exactly how coculture improves embryonic development. If the
cells make certain products that stimulate development of healthier embryos,
these products might be able to be produced commercially and added to
conventional culture media.
It is possible that pure and exactly defined chemical media might someday be
so improved as compared to what is now in use that coculture would not offer
any increase in pregnancy rates, even for poor prognosis patients. However,
we do not appear to be at that point today. Further coculture research is
needed.
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