In Depth

The IN DEPTH section of WiSSPers will include one to two detailed articles about current topics related to stillbirth and stillbirth evaluation. If there are any topics you would like to see addressed in future issues, we would like to hear from you.

Fetal-Maternal Hemorrhage and Stillbirth

Richard M. Pauli, M.D., Ph.D.

Introduction
Fetal-maternal hemorrhage (FMH) has been of considerable interest and importance to obstetricians for decades: leakage of fetal cells into the maternal circulation is the mechanism through which Rh sensitization arises. In addition, more recent data have shown that when large volumes of fetal blood are lost in this way, then serious and potentially fatal fetal or neonatal outcomes can result. The Wisconsin Stillbirth Service Program to this point has not recommended routine assessment for FMH as a possible cause of stillbirth. Literature review was undertaken in order to determine if this policy should be changed.

Leakage of fetal red blood cells can begin anytime from the mid-first trimester onward. It presumably results from a breach in the integrity of the placental circulation. As pregnancy continues, more and more women will show evidence of fetal red cells in their circulation so that by term about 50% will have detectable fetal cells. Most of these, however, are very small leaks; total fetal blood volume lost in this way is 2 milliliters or less in 96-98% of pregnancies.

Association between fetal-maternal hemorrhage and stillbirth
Small leaks are not implicated in intrauterine death. However, massive FMH (generally defined as 30 ml or more of fetal blood loss) is found in increased frequency in stillbirth and probably causes a significant number of these deaths. Fetal-maternal transfusion of 30 ml occurs only in about 0.3% of otherwise uncomplicated pregnancies. In contrast, series in the literature from which frequencies in stillborns can be derived yield estimates of 2.5%, 3.%, 4.5% and 4.6% for similar large hemorrhages. While none of these estimates comes from a truly unbiased prospective and unselected sample, it still is likely that around one in every 50 stillbirths is associated with and is presumably caused by FMH. How large a hemorrhage must be to cause intrauterine death is unknown. Based on the available data, a best guess is that loss of 25% of fetal blood volume should be considered presumptive evidence for causality.

Detection of Fetal-Maternal Hemorrhage
The standard method of detecting FMH is the Kleihauer-Betke test. This takes advantage

of the differential resistance of fetal hemoglobin to acid. A standard blood smear is prepared from mother's blood. An acid bath is then used which removes all adult hemoglobin but does not remove fetal hemoglobin. Subsequent staining makes fetal cells (containing fetal hemoglobin) rose-pink while adults cells are only seen as 'ghosts'. A large number of cells (e.g. 5000) are counted under the microscope and a ratio of fetal to maternal cells generated.

Comparison with other more expensive or high-tech methods (e.g. flow cytometry) has shown that the Kleihauer-Betke method is just as accurate. Background counting errors can result in estimates of as much as 5 ml fetal blood loss when there is none, but standard methods available in most laboratories should never result in a false positive for large FMH.

Estimation of Severity of Hemorrhage
To determine if a positive test for FMH should be considered as the likely cause of fetal death, the percent of total fetal blood volume lost should be calculated. Such a calculation uses the following correction factors: fetal red cells are 122% the size of adult red blood cells; 92% of fetal red cells are detected by the Kleihauer-Betke test on average; maternal red cell volume near term averages about 1800 ml; average fetal hematocrit is about 50%; fetal blood volume is about 150 ml per kilogram of body weight. Combining all of these then means that:

Percent Fetal Blood = Fetal Cells x 1800 x 1.22 x 100
Volume Lost Maternal Cells 92
x 2 x 100
150 x fetal wt in kg

Or, to make things lots simpler,

Percent Fetal Blood = Fetal Cells x 3200 ÷ fetal wt

Volume Lost Maternal Cells in kg

So, for example, if the Kleihauer-Betke shows that 200 of 5000 cells counted are fetal andthe fetus weighs 2.0 kg, then the estimate of percent blood volume loss would be:

200/4800 x 3200 ÷ 2.0, or 66%.

Probably less than 20% volume loss is enough to cause death if it happens all at once. On the other hand, much larger volumes can be lost over a long period and the fetus can compensate. Unfortunately there is no straightforward way to know whether one is dealing with acute or chronic hemorrhage. This makes determination of whether a hemorrhage is or is not causal more problematic.

Detection Problems
In general fetal blood cells have the same life expectancy in the maternal circulation as the mother's cells. So usually it isn't crucial to sample within some very short period of time after recognition of fetal death. However, if the mother and fetus are ABO incompatible, then the fetal cells will disappear from the mother's circulation very quickly and an underestimate of the size of a FMH will be made.

Lots of concern has been raised in the literature concerning false positives when sampling is done after delivery. In general this is not a problem. Delivery does result in higher frequency of detection of micro-hemorrhages but this should not confound interpretation of FMH as a possible cause of stillbirth. It is not necessary to draw the sample before induction, onset of labor, delivery, placental delivery etc. despite what some published literature purports. However, if caesarean section is to be used, failure to draw the sample prior to that will result in a 2% false positive rate.

Finally, anything which causes persistence of fetal hemoglobin in maternal blood cells will make interpretation much trickier. Certain hemoglobinopathies, the most common of which is sickle cell trait, do this. Overall, somewhere around 1-3% of the time this could result in false interpretation.

Antecedent Risk Factors
If specific risk factors were clearly linked to FMH then these could be used to be selective in which stillbirths would be tested. The American College of Obstetrics and Gynecology has generated such a list. Unfortunately many of the items aren't applicable to stillborns, many are not well supported by studies in the literature and in general are poorly predictive. Those risk factors which are, by literature review, correlated with increasing risk of massive FMH and which could be of relevance in assessing stillborns include: maternal trauma; placental abruption; placental tumors; third trimester amniocentesis; fetal hydrops; pale fetal organs; antecedent sinusoidal fetal heart tracing; twinning. If one elected to selectively test, then certainly all pregnancies with one or more of these features should lead to completing a Kleihauer-Betke test. Unfortunately, even then more than half of etiologically significant hemorrhages will remain undetected.

Cost
Approximate cost of a Kleihauer-Betke test is about $20-40. Assuming that one in every 50 stillbirths is secondary to FMH, then each such diagnosis currently being missed would cost about $1,500.

Utility
Knowing the FMH is the likely cause of a stillbirth allows for reassurance to be provided to the parents since recurrence is extremely rare (and, in fact, those reported in the literature probably don't exceed the number expected by chance), and to care providers (since it is rarely an avoidable event). Certainly subsequent pregnancies could be monitored with Kleihauer-Betke testing, although given how rare are recurrences this may not be justified. If done and if monitoring does detect recurrent FMH a variety of strategies are available for intervention.

Conclusion and Recommendations
Massive FMH may be the cause of around 1 in every 50 stillbirths. No antecedent historical or clinical features allows sufficient selection, so that with any selectivity a large proportion of FMH will remain undetected. Cost is modest. The information gained can be of substantial importance. Therefore, we recommend --

•Stillbirth assessment should, in all instances, incorporate testing of maternal blood for evidence of massive fetal-maternal hemorrhage.

•Blood drawing can be done pre- or postpartum at the convenience of the care provider and the mother; only if caesarean section is anticipated is it important to draw the sample prior to delivery.

•Standard Kleihauer-Betke testing in any experienced laboratory is sufficient.

•Tests which show 25% or more of estimated fetal blood volume lost should be viewed as probably causal of the fetal death.

•In those with positive tests, followup testing (at a postpartum check) should be done to rule out the possibility of a false positive because of a process (e.g. sickle cell trait) in the mother which causes persistent elevation of fetal hemoglobin.

We invite your comments on these recommendations. Pending those comments, we anticipate incorporating these recom-mendations into the WiSSP protocol in the near future.

Further Reading
Fliegner JRH, Fortune DW, Barrie JU (1987):

Occult fetomaternal haemorrhage as a cause of fetal mortality and morbidity. Aust NZ obstet gynaecol 27:158-161.

Laube DW, Schauberger CW (1982): Fetomaternal bleeding as a cause for "unexplained" fetal death. Obstet gynecol 60:649-651.

Marions L, Thomassen P (1991): Six cases of massive feto-maternal bleeding causing intra-uterine fetal death. Acta obstet gynecol Scand 70:85-88.

Owen J, Stedman CM, Tucker TL(1989): Comparison of predelivery versus postdelivery Kleihauer-Betke stains in cases of fetal death. Am j obstet gynecol 161:663-666.

*Sebring ES, Polesky HF (1990): Fetomaternal hemorrhage: incidence, risk factors, time of occurrence, and clinical effects. Transfusion 30:344-357.

[* = of particularly high quality or particularly useful] Copies of these and other relevant articles are available for personal use by request from WiSSP.]

LOWER MESODERMAL DEFECTS

Richard M. Pauli, M.D., Ph.D.

The principle function of the Wisconsin Stillbirth Service Program is to provide information and help directly to families who have experienced stillbirth and to the health personnel involved in those families' care. In addition, however, another important part of WiSSP's mission is to increase our general understanding of stillbirth. In fact, WiSSP provides a unique opportunity to identify and document previously unrecognized causes of intrauterine death. In order to identify previously unrecognized recurring patterns of malformations in stillborn babies one must have a large series of uniformly evaluated infants. To assess the importance of any particular process that is identified that series must be unbiased and nonselective in its 'sampling' of the entire population of stillborns. Because WiSSP has been involved in the assessment of around 1300 infants and, because its community based format results in assessment of a large and largely unbiased proportion of stillborns, it fulfills those needs well. This article summarizes one of the processes which has been identified through WiSSP as a relatively common cause of stillbirth.

What is the Lower Mesodermal Defect Sequence?
Over the ten years of WiSSP's existence, a group of infants has been evaluated, members of which share a series of birth defects involving the urinary, genital, lower gastrointestinal and axial skeletal system. In fact, 17 babies have been identified with what we have called the Lower Mesodermal Defects Sequence.

The Lower Mesodermal Defects Sequence is a recurrent clustering of specific anomalies. Often, because of obstruction of urine outflow, these babies will have huge abdomens. In addition, many will have marked internal structural abnormalities of the kidneys (absence, hypoplasia or malmigration), ureters (absence or stenosis), bladder (absent or small or, alternatively, markedly enlarged because of obstruction of urine outflow), urethra (absent or blind), internal genitalia (particularly abnormalities of the uterus and fallopian tubes in females), gonads (sometimes absent) and lumbosacral spine (hemivertebrae, sacral agenesis), and usually have an imperforate anus. Often there is either external genital ambiguity or complete absence of the external genitals. Such babies present a confusing picture of 'multiple congenital anomalies', which, however, can be fully explained.

Not all affected infants have all of these features. However, phenotypic analysis and statistical methods show that, nevertheless, they all do appear to have the same underlying process.

How does the Lower Mesodermal Defects Sequence arise?
We think that all of these problems arise together because all of the affected parts

derive from what is termed the intraembryonic mesoderm. Early in embryonic life three tissue types develop -- ectoderm, mesoderm and endoderm. The mesoderm can be further topologically divided of which one part is the 'lower' (i.e. below the umbilicus) portion. It appears that damage to this lower portion of the mesoderm early in gestation (perhaps by the third or fourth week after conception) results in failure of development and other abnormalities of the organs that are usually derived from this portion of the mesoderm. The accompanying diagram shows how these effects are embryologically linked.

Why wasn't this recognized a long time ago?
In a sense, it was. Instances which we feel are examples of Lower Mesodermal Defects were reported beginning more than 30 years ago. However, because in these early publications attention was focused on a single organ system and/or the populations which were looked at were small, no one previously realized that there was this single, common, recognizable explanation for these anomalies.

What causes the Lower Mesodermal Defects Sequence?
So far we aren't certain. Limited information from families who have had a baby with this disorder suggests that it is a non-genetic process. Probably it arises secondary to a disruption of the tissues of the lower mesoderm shortly after those tissues are formed. Thus far no specific environmental trigger of this disruption has been found.

How common is the Lower Mesodermal Defects Sequence?
More than 1% (one in every 89) of assessed stillborns were found to have features consistent with this diagnosis. The Lower Mesodermal Defects Sequence, then, accounts for more than 4% of fetal diagnoses generated through WiSSP. One can roughly extrapolate to the frequency overall with which Lower Mesodermal Defects arise. Since about one in 115 pregnancies which progress to 20 weeks gestation or more ends in fetal death, then the proportion of such pregnancies resulting both in the Lower Mesodermal Defects Sequence and in stillbirth would be (1/89 x 1/115) or about 1 in 10,000. Since some affected infants are born alive the total population frequency of this sequence is certainly greater than 1 in 10,000. Compared with many other syndromes and birth defects this is a rather common process.

Why is recognition of this process important?
First, many families find some solace in knowing that there is a specific recognizable cause for their baby's death rather than just 'multiple congenital anomalies'. Furthermore, Lower Mesodermal Defects can be viewed as a single birth defect (with many secondary manifestations) rather than a seemingly overwhelming number of things that were 'wrong' with their baby. Families are often comforted in knowing that this is a disruption and that, prior to the accident which resulted in such a disruption of the lower mesoderm, their baby was perfect. Families can be assured that no environmental cause has been identified and therefore certainly no easily recognized event which they may be worried about can possibly be linked to these problems in their baby; that is, it was nothing they did or didn't do that resulted in this process and in their baby's death. Finally, it appears that this diagnosis implies little or no risk for recurrence; such reassuring information could not be provided if the diagnosis remained just 'multiple congenital anomalies.'

On a different level, recognition of this specific process is one small step in the further understanding of why babies die.