GUEST CORNER

Research into the Causes of Stillbirth

Leslie Biesecker, M.D.

Couples who experience the stillbirth of a child have to deal with many issues surrounding their grief. An important component of the grief process for many couples is gaining an understanding of the cause of the loss. Unfortunately, the cause for the loss in many cases remains unknown. The Wisconsin Stillbirth Service Program has made strides in improving causal diagnosis of children who are stillborn. In spite of this improvement, more needs to be learned, since more than half of couples still have no certain medical explanation for the loss they have experienced. Diagnoses can be important for determining accurate recurrence risks for individual couples, and for improving medical care for all couples. To improve this situation, medical research must try to understand the cause for more of these losses.

To help accomplish this, Dr. Pauli and I have agreed to start a research project to attempt to find the cause for more of these losses. The project is an extension of one of the known causes of stillbirth — chromosomal abnormalities.

Chromosomes are the structures in the cell nucleus that contain the genetic information in the form of DNA. As detailed elsewhere in this issue of WiSSPers, chromosome abnormalities can cause fetal loss. They may result from extra or missing chromosomes. Or, chromosomes can break and a person can have a piece of a chromosome missing. Or, these small pieces of broken chromosome can reattach to another chromosome, which can cause part of a chromosome to be out of balance.

The current, standard technique for detecting chromosome problems is to stain the chromosomes and view them under the microscope. In this way, missing, extra, or broken chromosomes can be identified. This technique is very useful in evaluating the cause of fetal loss and has led to a diagnosis in about 5-10% of all fetal deaths. Unfortunately, the microscope can only detect fairly large chromosome abnormalities. We believe that smaller, undetected chromosome abnormalities may cause other fetal losses. These are being looked for using molecular techniques that can identify exceedingly small pieces of missing or abnormally duplicated chromosomal material.

Another category of chromosomal problems is what are called "parent of origin effects." There are 46 chromosomes in 23 pairs. One member of each of the pairs is usually inherited from each parent. We now know that there are some children who inherited both members of a single chromosome pair from one parent and no member of that pair from the other parent. For some chromosomes this causes no difficulty, but for others this inheritance pattern causes abnormalities in the child. Importantly, there are mice that have been altered so that they often produce offspring with this pattern of chromosome inheritance. For some mouse chromosomes, no pups are ever liveborn, suggesting that, in mice at least, this inheritance pattern may cause intrauterine death.

To study these potential causes of fetal loss my laboratory has developed an experimental diagnostic test. This test looks for the inheritance of chromosomes from each parent. Using genetic "markers" we can check at a molecular level to see if an individual has inherited one member of each chromosome pair from each parent. In addition, we can use these same markers to see if any pieces of chromosome are out of balance. To do this we will ask for fetal tissue from stillborn infants and a blood sample from each parent.

Genetic markers are used as indicators of chromosome problems. Distributed throughout the chromosomes are stretches of DNA that vary in length among individuals and usually between the two chromosomes of each pair that each individual carries. To check for a chromosome alteration at a given spot on the chromosomes, we choose a marker that sits on that spot of the chromosome. The marker segment of the DNA is amplified with radioactivity in the laboratory until we have enough copies of the amplified marker to see with x-ray film. When we see the marker on the film, we can determine the length of the piece of DNA that we amplified. It is the length of the stretches of marker DNA that we use to check for chromosome abnormalities. So, for example, we might find, for one marker on a particular chromosome pair:

In this example, the mother, father and child each have 2 markers, one from each copy of the chromosome being evaluated. In addition, it can be seen from the marker lengths that the child inherited marker ‘92’ from the mother and marker ‘90’ from the father. We can therefore assume that the child has two chromosomes in the region of this marker and that the child received one chromosome from the mother and one from the father (that is, a normal result).

An example of an abnormality of chromosomal inheritance would be if the child had a marker result of ’92,92' (actually we can not distinguish ’92,92' from a single ‘92’ in a marker test). This result would suggest that the child received one or two copies of the chromosome with marker length ‘92’ from his mother and none from his father. This could be a cause of abnormalities in the child. It could be lethal and cause intrauterine death, just like in the mouse model.

The difficult part of this research is that many markers need to be checked to look at all chromosome segments. Furthermore, not every marker is as informative as in this example. For instance, if the parents share a marker length (by coincidence) it will be difficult or impossible to determine the origin of the child’s markers. If the mother is ’92,92' and the father is ’92,94', very little information will be accessible about the child, especially if the child’s result is, for example, ‘92’. Again, since we can not discriminate between ’92,92' and a single ‘92’ by marker testing, the child could actually have any of the following:

1. The child got a ‘92’ from mom and a ‘92’ from dad (normal);
2. The child got either one or two ‘92’s from mom and none from dad;
3. The child got two ‘92’s from mom and also one ‘92’ from dad (since we also can not tell two ‘92’s from three ‘92’s);
4. The child got one or two ‘92’s from dad and none from mom, etc.

The first result is by far the most likely for any given marker. However, to be sure, we have to select another marker from the same area of the chromosome and repeat the test in order to rule out the abnormal possibilities. For this reason this research is labor intensive and takes a long time.

This is a research project and not a part of the routine assessment of a stillborn. Some couples may be asked by a counselor or physician to participate in this project if they meet certain eligibility criteria. There is a formal consent process for the study that explains both the risks and potential benefits of participation. We hope that assessment of sufficient numbers of unexplained stillbirths will tell us whether tiny chromosome aberrations and/or parent of origin effects are important in understanding the causes of intrauterine death in humans.