Placental Measurements Workshop 

November 3-4, 2003
Holiday Inn Select
Bethesda, MD

This meeting was held in conjunction with the National Children’s Study, which is led by a consortium of federal agency partners: the U.S. Department of Health and Human Services (including the National Institute of Child Health and Human Development [NICHD] and the National Institute of Environmental Health Sciences [NIEHS], two parts of the National Institutes of Health, and the Centers for Disease Control and Prevention [CDC]) and the U.S. Environmental Protection Agency (EPA).

Introduction

Moderators for the Workshop were Kenneth Schoendorf, M.D., M.P.H., CDC, DHHS, and Catherine Spong, M.D., NICHD, NIH, DHHS. Dr. Schoendorf explained that the purpose of the meeting was to discuss the ways in which pathologic analysis of placentas and related materials can contribute to the National Children’s Study (Study). He then provided an overview of the Study. Dr. Spong stated that participants would be asked how sampling placentas, membranes, and cords could help answer Study questions.

General Placental Measurements

Participants discussed numerous methods to obtain and measure the placentas of women enrolled in the Study. The suggested methods included:

• Trim the membranes and cord, weigh the placenta, and compare to the expected weight for that period of gestation.
• Take photos of the top, bottom, and a representative transmural slice of the placenta.
• Fresh freeze a percentage of the placentas because some of the needed studies cannot be done on formalin-preserved specimens.
• Examine the placenta to help identify the cause of stillbirth or preterm delivery.

Maternal Infection and Inflammation

Workshop participants made numerous suggestions on ways to evaluate maternal infection and inflammation using placental evaluations, including:

• Obtain standard placental measurements such as:
  • Shape of the placenta (round-oval, not round, or other)
  • Cord insertion to the closest placental edge
  • Chorionic disk dimensions
  • Placental thickness
  • Placental weight.
• Weigh the placenta on site, take pictures, and perform a standard histologic examination to obtain information on the duration and intensity of infection.
• Culture and stain each placenta for organisms and freeze for DNA, to identify a constellation of microorganisms that cause damage. All cases with abnormalities should receive full evaluation, as should a random sample from "normal" infants.
• Try to obtain full evaluations on all 100,000 Study placentas.
• Freeze amniotic fluid to answer questions related to cytokines. Maternal cytokines must be examined to evaluate extrauterine infections in the mother and pregnancy outcomes.
• Collect cytokine information from placental samples to prevent the need to collect cytokine levels on every subject.
• Obtain histology to learn whether inflammation is present. For example, 15 percent of full-term infants with cerebral palsy have villitis, and this can be analyzed with current techniques.
• Store samples in a variety of ways to allow future identification of currently unknown infectious agents.

One of the Workshop participants stated that measuring placental weight is not sufficient, and she presented a model showing how placental volume moderates the placental weight/birth weight ratio. She suggested that collecting stereological random samples could be used to help understand when glucose metabolism starts to change the intrauterine environment in a way that can increase cardiovascular demands on the heart by changing villous capillarization. Other recommendations concerning maternal glucose metabolism included:

• Use placental material to validate or clarify the relationship between the clinical staging of maternal diabetes and its effects.
• Note that capillaries are increased in diabetic placentas and chronically reduced in other placentas such as those associated with abnormally poor vascularization of the retina.
• Recognize that placental tissue in formalin is sufficient to answer many questions, but frozen tissue is required for markers.
• Note that infants with small placentas have an increased number of nucleated red blood cells, and these need to be assessed.

More sophisticated measurements of the placenta can be used to study maternal glucose metabolism. Utero-placental blood flow needs to be assessed, but it is not clear how to do so. Doppler was suggested as a possible solution, but a participant argued against its use, noting that uterine arteries have a waveform but not necessarily low pressure except in the intervillous space.

A participant suggested said that the placenta might provide information that can help identify which gestational diabetics will have stillbirths. This information could be used to determine whether a baby needs to be delivered early.

Chemical Exposures

The group discussed using placental measurements to evaluate chemical exposures. Suggestions included:

• Conduct chromatography, molecular analysis, and other tests of chemical and toxin exposure on frozen placental tissue or fluids and use fixed or embedded tissue for histological examination. Ideally, tissue would be snap frozen, placed on dry ice, and transferred to a -70° freezer.
• Researchers have identified a relationship between exposure to polychlorinated biphenyls (PCBs) and other chemicals and neurodevelopment; many other toxins might be found to have an impact over the course of the Study. Some toxins, especially PCBs, are concentrated in breast milk and have more effect through breast milk than through placental transmission.
• For chemical exposures and toxins, save as many different tissues and fluids as possible, because the locations of compound concentrations vary tremendously. For example, some heavy metals are concentrated in the placenta, whereas others are equally concentrated in the mother, placenta, and fetus. Breast milk may be important for identifying ongoing exposures.
• According to the literature, dose is not the only important factor; whether an exposure is short- or long-term plays a major role. Also, method of exposure (for example, transdermal versus inhalational exposure) should be assessed.
• Collect the infant’s urine and meconium at delivery.
• Collect mothers’ urine to document exposures during pregnancy, and then examine the placenta for indications of these exposures.
• Use information from placentas to establish baseline normal levels. This information would be very useful to evaluate the impact of future exposures. Because the effects of low-dose pesticides are not always apparent early in life, having samples available would be useful if it becomes necessary to determine whether a cohort was exposed to a chemical.
• Recognize that umbilical cords are good sources of fetal tissue; the placenta provides both maternal and fetal tissue.

Maternal Stress

Sources of maternal stress and their impacts on pregnancy were discussed. Participants expressed opinions about the effects of continuous stress throughout pregnancy versus acute stress in and around the time of delivery. Several participants listed potential uses for placental bed biopsies:

• Little is known about the causes of preeclampsia or of intrauterine growth restriction (IUGR) without preeclampsia, and placental samples could be used to study the reasons for preterm deliveries and preeclampsia. Because IUGR is heterogeneous, studying placental bed biopsies could make it possible to separate subgroups with specific etiologies.
• Many of the same cytokine factors are operative in preeclampsia and other perfusion states. Placental bed biopsies could be used to compare a population with preeclampsia without infection to a population with both preeclampsia and infection to determine whether their actions are synergistic or additive.
• Soluble vascular endothelial growth factor (VEGF) in the maternal circulation could be a marker for adverse events. Having the full length of the vessel would be useful, as it is not known whether soluble VEGF is produced by the placenta or an invasive trophoblast. But soluble VEGF should only be analyzed in patients who develop preeclampsia and matched controls.
• Placental bed biopsies are useful in studying inflammation, abnormal growth of the placenta, and the effect of therapeutic steroid use by mothers.

A participant noted that some Study hypotheses address cortisol and norepinephrine. He recommended taking villous samples to assess corticotropin-releasing hormone levels and membrane samples for type 1 enzymes. A final recommendation was the need to evaluate the use of steroids in preterm labor.

Maternal Nutrition

It was noted that nutritional stresses in pregnancy may result in malformations or decreases in growth of the fetus or in inflammation or adverse effects of inflammation. Investigators could study the placenta to determine concentrations of micronutrients, vitamins, and minerals. Pregnancy-induced hypertension (PIH) is associated with micronutrient or nutrient deficiencies, but the reason is not known. A participant explained that information is not currently available on the association of certain lesions in the placenta and certain micronutrients. The Study will provide an excellent opportunity to make such correlations. Suggestions included:

• Evaluating tissue explants is critical because cellular function and mechanisms must be examined in culture.
• Because the placenta is probably important in processing nutrients to regulate the maternal and fetal systems, studying it with both fixed and frozen tissue is vital.

Explants are more appropriate for addressing a specific question at the time that they are collected because they are useful for only a limited time. It was noted that explants are very expensive.

Other Topics

Dr. Spong asked participants to mention any topics not covered in the Workshop that could be addressed through perinatal pathology in the Study. Suggestions included:

• Adding a hypothesis on maternal vasculopathy, PIH, IUGR, preterm birth, and hypoxic-ischemic encephalopathy (HIE). A participant noted that HIE and cerebral palsy are difficult to study because they are so rare. Therefore, a large-scale study will provide a unique opportunity to obtain a sufficient number of samples.
• Evaluating recurrent-loss patients could be done with perinatal pathology.
• Diagnosing chronic villitis
• Increasing knowledge about the effects of adverse pregnancy outcomes on the life of the child.

Dr. Spong presented a summary of logistical issues related to collecting and processing specimens for the Study and asked for feedback.

• For histology, a stem-to-stern section of normal and obviously abnormal tissue is needed to examine morphology.
• RNA and DNA in the placenta are relatively stable for up to 2 days, so processing need not be immediate. One suggestion was to obtain a morphology fixative section and then remove three samples for freezing.
• A complete placental examination cannot always be conducted on site. A participant suggested taking a single slice through the center, taking photographs, putting the specimen into a plastic bag, adding fixative, putting the specimen into the refrigerator, and then sending it to the pathologist. The pathologist can then take photos of the sections, make hematoxylin and eosin sections, and send the specimens to a tertiary center for storage.
• The pathology needed for the Study cannot interfere with the traditional clinical pathologic examination needed for many of the Study participants.
• A section of the cord should not be taken at the insertion slice if the section is needed for diagnostic pathology.
• The frozen tissues needed include fetal plate, a portion from the mid-zone, and a portion of decidual chorionic villous interface, with one section taken from the margin closest to the zone of rupture.
• Specimens can be preserved in the refrigerator for some time before freezing; Study nurses will not need to be available around-the-clock.
• Explants should be handled immediately and only at certain centers. However, a frozen sample should be taken from every placenta.
• A standardized gross and microscopic examination performed on every placenta under a protocol put together by experts like those in this Workshop would accomplish a great deal.
• Obtaining frozen tissues to study inflammation requires very little additional time and effort.
• Developing a template for gross examination of placentas in different facilities could help ensure proper collection and preparation of all samples.

Wrap-Up and Next Steps

Dr. Schoendorf asked each participant to share one last comment. They included:

• Much more than routine histology can be done on slides with fixed tissue, such as protein, DNA, and RNA analyses.
• Including placental pathology is very important for the Study.
• A hierarchical protocol should be developed in which the least expensive, most basic tasks are included in the Study, and more expensive steps are included if funding is available.
• In all future studies of this kind, experts in perinatal and placental pathology should be involved in developing Study parameters.
• Frozen samples can be obtained without compromising the ability to conduct a clinical examination of the placenta.
• Collecting all of the placentas and obtaining the same samples in the same way from all of them is important.
• Causal pathways involved in all of the Study hypotheses involve the placenta as a mediator or "fellow traveler."
• If the Study were to result in the identification of a way to prevent preterm delivery, HIE, or cerebral palsy, billions of dollars could be saved that would easily justify the Study.
• Preeclampsia should be addressed in the Study.
• Histology of the placenta should be a top priority.
• Many of the techniques discussed during this Workshop are complicated and expensive, but in the context of the Study, the incremental cost of obtaining the placentas and doing the studies mentioned would be small. It would be a "crime, waste, and tragedy" not to obtain as many placentas as possible.

Drs. Schoendorf and Spong stated that they and their colleagues would develop a report that will be shared with Workshop participants. In addition, a journal manuscript would be developed based on the Workshop discussions. The manuscript will address the benefits of perinatal pathology in understanding pregnancy and long-term outcomes. Workshop participants were asked to submit the references cited in the discussions to Dr. Spong or Dr. Schoendorf.

Participants

Ona Marie Faye-Peterson, M.D., University of Alabama,  Birmingham
Marjorie Grafe, M.D., Ph.D., Oregon Health and Science University
M. Halit Pinar, M.D., Brown University Medical School and Affiliated Hospitals
Edwina J. Popek, D.O., Baylor College of Medicine
Raymond W. Redline, M.D., University Hospital of Cleveland
Beverly Rogers, M.D., University of Texas Southwestern Medical School
Joanne Rutgers, M.D., Long Beach Memorial Medical Center
Carolyn Salafia, M.D., M.S., Columbia University
Kenneth C. Schoendorf, M.D., M.P.H., National Center for Health Statistics, CDC, DHHS
Catherine Y. Spong, M.D., NICHD, NIH, DHHS
John M. Thorp, Jr., M.D., University of North Carolina
Marian Willinger, Ph.D., NICHD, NIH, DHHS
Christopher M. Zahn, M.D., Uniformed Services University of the Health Sciences