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In vitro fertilization with coculture

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We no longer offer coculture

With recent improvements in IVF culture media and techniques, our IVF pregnancy rates (without coculture) have improved dramatically. Therefore, we no longer see a need for coculture.

Endometrial Coculture IVF
IVF embryos on endometrial cell coculture using cells from the mother's own uterine lining
These day 3 embryos are being "cocultured" with the endometrium cells
Endometrial cells are in the background - forming a monolayer on the bottom of the culture flask

Human embryos on tubal cells
3 human embryos in coculture on a proliferating monolayer of tubal cells
The bare plastic bottom of the culture dish is seen between the cells

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 new techniques.

There is not universal agreement that coculture is necessary to provide optimal pregnancy rates from human in vitro fertilization. Another issue is that depending on the source of cells used for the coculture there are 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.

Bovine tubal coculture for ivf
3 embryos with bovine tubal cell coculture
Embryos look fuzzy because plane of focus is on the tubal cells

More detail of tubal cells
More detail of tubal cells and one embryo

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 was performing coculture in the past for selected cases using endometrial cells from the woman's own uterine lining for coculture with the embryos.

How is coculture done?

Tubal cell monolayer for coculture
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

Hatching blastocysts on coculture
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.

There are many published studies on coculture in the medical literature. Some are listed here. Many other studies are referenced in these articles.

Bongso, A., Ng, S., Fong, C., and Ratnam, S. (1991) Cocultures: a new lead in embryo quality improvement for assisted reproduction. Fertil. Steril., 56, 179-191.

Menezo, Y., Hazout, A., Dumont, M., Herbaut, N., and Nicollet, B. (1992) Coculture of embryos on Vero cells and transfer of blastocysts in humans. Hum. Reprod., 7, 101-106.

Morgan, K., Wiemer, K., Steuerwald, N., Hoffman, D., Maxson, W., and Godke, R. (1995) Use of videocinematography to assess morphological qualities of conventionally cultured and cocultured embryos. Hum. Reprod., 10, 2371-2376.

Sherbahn R., Frasor, J., Radwanska, E., Binor, Z., Wood-Molo, M., Hibner, M., Mack, S., Rawlins, R.G. (1996) Comparison of mouse embryo development in open and microdrop coculture systems. Hum. Reprod., 11, 2223-2229. Click here to read the abstract of this article

Wiemer, K.E., Hoffman, D.I., Maxson, W.S., Eager, S., Muhlberger, B., Fiore, I., and Cuervo, M. (1993) Embryonic morphology and rate of implantation of human embryos following co-culture on bovine oviductal epithelial cells. Hum. Reprod., 8, 97-101.

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