Measures of Maternal and Fetal Infection and Inflammation Workshop 

 

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).

Welcome and Introductions
Co-Chairs: William J. Rodriguez, M.D., Ph.D., Center for Drug Evaluation and Research, U.S. Food and Drug Administration, and Robert H. Yolken, M.D., School of Medicine, Johns Hopkins University Medical Institutions

Dr. Yolken and Dr. Rodriguez welcomed the participants and briefly highlighted some of the key information that would be covered during the workshop.

Dr. Yolken presented the scientific background and goals of the workshop.

• Common neuropsychiatric diseases of childhood and early adulthood are complex disorders that involve the interaction of environmental and genetic factors.
• Some of these environmental factors are interacting during fetal development and early life.
• The best way to study the effect of environmental factors is through a prospective cohort study of a large number of pregnant women and their offspring.
• The goal of the workshop is to advise the Study about how best to use its resources to study the common neuropsychiatric disorders of infancy, childhood, adolescence, and early adulthood.
• Target disorders include cerebral palsy, autism, schizophrenia, mental retardation, and mood and behavioral disorders.

Kenneth C. Schoendorf, M.D., M.P.H., National Center for Health Statistics, CDC, DHHS, thanked the participants and the co-chairs. He gave a slide presentation about the Study that addressed its history, background, and attributes. He reviewed the following aspects of the Study:

• Priority outcomes
  • Adverse pregnancy outcomes
  • Neurobehavioral development
  • Injury
  • Asthma
  • Obesity and physical development
• Priority exposures
  • The physical environment
  • Chemical exposure
  • Biologic environment
  • Psychosocial environment
  • Genetics
• Relevant hypotheses, assumptions, and projected timelines.

Dr. Schoendorf said that the workshop would consider:

• Optimal and feasible measurements of maternal infection and inflammation
• Potential assessment of fetal infection and inflammation or fetal response to maternal infection and inflammation
• Measurements, not merits of hypotheses.

Lessons from the Collaborative Perinatal Project (CPP) Study: What Should Be Done Differently

CPP Trial: Study Design
Janet Hardy, M.D., C.M., Johns Hopkins University

Dr. Hardy began by saying that she was honored to be invited to present at the workshop and that the CPP had occupied much of her professional life. She provided handouts but did not use slides.

• The CPP was an ambitious, state-of-the-art effort to look at perinatal loss, based on the Lilienfeld hypothesis of a continuum of reproductive casualty. The Study has its roots in the CPP.
• In the 1950s, there was wide concern about high rates of perinatal and infant mortality. Handicapping conditions were estimated at that time to affect more than 20 million children and adults in the United States. Cerebral palsy was a substantial problem, and CPP actually began as the Cerebral Palsy Project.
• Congress provided funding to establish the National Institute of Neurological Diseases and Blindness and gave the Institute responsibility for the CPP.
• At the time, there was rudimentary neonatal care, and high-risk obstetrical care had not yet been heard of. The CPP led to the development of neonatal care units and the development of new subspecialties. Dr. Hardy said that she would look to the Study as a chance to develop new facilities, new concepts, and new methods as a byproduct of the Study.
• The CPP was unique in its study design. There was only one way the study could be organized—by using major medical centers at 12 universities around the country. While each site had unique characteristics, the study ended up with a good cross-section of the population. Enrollment began in 1959 after a pretest year. Mothers were enrolled at the first prenatal visit.
• The CPP collected serological specimens, cord blood, and placental specimens. Some of the specimens are still being used today.
• Live-born children were followed from birth, with repeated physical, neurological, psychological, and other testing done through age 7. In about half of the centers, there were also detailed assessments of language, hearing, and speech.
• Data flowed from the field to a central core, where the data were processed and stored on computers. While use of computer storage was a breakthough at the time, the Study will be able to capitalize on the enormous advances in technology developed in the intervening years.
• There was much frustration stemming from both internal and external problems. External problems included a controversy in professional circles regarding the wisdom of doing such a huge study, and many were opposed to it. This opposition had a long-term detrimental effect on the study and eventually led to shutting the study down before its data were thoroughly examined. Several committees were involved, and the continuing availability of funding was a constant worry. There was also disagreement within NIH regarding the Institute receiving the money, and the various committees also diverted money from the study’s budget, a further detriment.
• Internal problems had to do with the governance of the study. The roles and responsibilities of the various parties (advisory committee, director, and investigators) were not clearly delineated in advance, and planning was not adequate. Dr. Hardy stressed that, for the Study, transparency and careful planning are essential.
• In the CPP, governance of the study was divided. Project directors were responsible for details of planning, while the advisory committee addressed broad matters. Committees of experts assisted the project directors in designing study components. The Perinatal Research Branch (PRB) of the Institute had coordinating responsibility as well as responsibility for data storage and analysis.
• There was tension between the PRB and the project directors regarding publications. There was no plan for the publication of study results until about 1970, and there were no visible products for a long time. It was difficult to maintain public and congressional interest in and support for the study, and as a result, interest and support dwindled over time. Eventually, a publications committee was created. Dr. Hardy urged that the Study clearly identify major products that will result from the Study.
• The CPP was shut down and the data were handed over to an outside contractor, which had little experience with data of this sort. The data also were not in a user-friendly form. Mark Klebanoff, M.D., NICHD, NIH, DHHS, developed a summary file that provided the only usable opportunity to access the data. In the past few years, the data have been revisited, and John Lawlor, Johns Hopkins Bloomberg School of Public Health, has created a user-friendly, usable dataset.
• General achievements of the CPP include more than 400 publications. Intangible gains were far more important, such as the development of new subspecialties and new knowledge that ended unnecessary treatment of initial febrile seizures, with associated health care cost savings.

Recommendations: First, regarding the Study design, Dr. Hardy urged that the Study begin enrollment no later than the mother’s first prenatal visit. While a 21-year follow-up period is good, follow-up should extend even further. Outcomes can be delayed for many years. The Barker hypothesis, which has enormous importance for the Study, holds that the development of coronary heart disease and other midlife diseases relate to fetal life.

Second, the social environment in infancy and childhood is as important-or even more so-than the biologic environment and merits close attention. The CPP data suggested that early parenting is very important. The mother’s responsiveness to the child is significant for drug abuse, teen pregnancy, and mental health.

Questions and Comments:

• Dr. Rodriguez asked about the repository and the surviving specimens. Dr. Yolken and Dr. Hardy commented that Dr. Klebanoff and others at NICHD were "heroes," as they saved the samples from being destroyed.
• A participant asked how the Study might handle the enrollment of women who do not get early prenatal care. Dr. Hardy replied that a study design different from that of the CPP might be better able to achieve early enrollment of women. She gave an example of a study that used a city with a representative population and followed the population over time.
• John Lawlor commented that the context of the CPP was very important to what happened with the data in the end. The National Children’s Study is probably going to accumulate the largest amount of data ever collected, and it is important to maximize use of the data. Experience with the CPP tells us what not to do.
• Mark Cosentino, D.P.M., Ph.D., Science Applications International Corporation (SAIC), said that how the data goes in is important as well as how efficient it is to get the data out. The Study will need to consider how the data will be linked, sorted, and used in the future.
• Phillip G. Nelson, M.D., Ph.D., NICHD, NIH, DHHS, commented about his wife’s frustration with the CPP data and the difficulty in getting answers to questions. The Study will need to have good communication and planning regarding the data, and a tight relationship will be needed between data collection/input and data use.
• Dr. Klebanoff said that the CPP data were on punch cards. The study had collected a lot of free-text, handwritten notes, and these were preserved. This was fortunate because collecting this type of data was out of favor for a long time.
• Dr. Cosentino commented about the need for standardization of data. He suggested that the Study include in the RFP a requirement for potential contractors to address how they would deal with issues of future data needs.

CPP Trial: Psychiatric Disease
Fuller Torrey, M.D., Stanley Medical Research Institute

Dr. Torrey presented details about the CPP, described psychiatric studies using CPP data, and discussed lessons learned relevant to the Study.

• The CPP registered 53,043 births from 1959 through 1966. Twelve university hospital centers were involved.
• The study was designed as a prospective investigation with extended follow-up of the children. It was intended to allow associations between perinatal events and neurological defects in children to be established.
• The CPP collected pregnancy data, delivery data, neonatal data, and data obtained at intervals through age 7, including physical, neurological, psychological, motor, and other assessments.
  • 4 months: physical and neurological
  • 8 months: psychological and motor tests
  • 1 year: physical and neurological
  • 3 years: speech, language, and growth
  • 4 years: psychological and growth assessment
  • 7 years: neurological, psychological, vision, and IQ.
• There are full 7-year datasets available on approximately 40,000 children.
• Key people involved were Dr. Klebanoff; Karin Nelson, M.D., National Institute of Neurological Disorders and Stroke (NINDS), NIH, DHHS; Paul Nichols, Ph.D., NINDS, NIH, DHHS; Dr. Hardy, Johns Hopkins University; John Sever, M.D., Children’s National Medical Center, Washington, DC; and John Lawlor, Johns Hopkins University.
• There is an online list of publications compiled by Matthew Longnecker available at: http://dir.niehs.nih.gov/direb/cpp/pubs_cpp2.htm.
• Dr. Torrey’s study identified 15 autistic children, matched them with controls, and found that bleeding during pregnancy was associated with "infantile autism." At that time, it was thought that schizophrenia was either not a real illness or was caused by "refrigerator mothers."
• Dr. Torrey gave an example of data from the CPP that proved useful years after being collected. After an article about head circumference and autism was published recently in JAMA, Dr. Torrey was able to go back to the data and found that autistic children did indeed have normal head circumferences at birth. Autism is associated with disproportionate growth in general, not just of the head. There is now a paper in press on this topic.

Recommendations: Dr. Torrey discussed lessons learned from the CPP in several areas and made recommendations for the Study. These included:

• Long-term planning
  • Include young researchers (ideally those with local ties who won’s move away).
  • Create a designated line of succession to plan for future use of the data.
  • Consider the aging issue.
• Consent issues. These will be much more complex in the Study due to the Health Information Portability and Accountability Act (HIPAA) and will likely become even more difficult over time.
• Cooperation among centers. This was a big problem in the CPP.
• Storage of serological and other specimens. This was also a problem in the CPP, and specimens were nearly lost.
• Authorship issue. Authorship problems prevented timely analysis and publication of some data from the CPP. With timely publication of results, the CPP might have received the long-term funding it needed.

Questions and Comments:

• Dr. Hardy commented that some CPP project directors were very young, had no clout in their universities, and had difficulties with compliance with data collection as a result. Dr. Yolken said that today, with multisite trials, there are standards that have to be met, and incentives and other measures are used to ensure that the data are collected.
• There was some discussion about unusual samples, pathology data, and where the brains of children who died are currently stored. Much of the pathological data were kept at Hershey. Dr. Hardy said that the brains were sent to Walter Reed Army Medical Center.

CPP Trial: Analysis of Stored Blood Samples
Robert H. Yolken, M.D., Johns Hopkins University Medical Institutions

Dr. Yolken discussed various issues related to the CPP study and its findings.

• Human psychiatric diseases are complex disorders that involve gene-environment interactions. Many laboratories are studying the genetic components of these interactions. This has led to the discovery of many genes, each of which has a relatively small effect.
• Few studies examine environmental factors that might increase these effects. The main limitation to the study of environmental factors is the availability of informative biological samples collected from carefully evaluated cohorts.
• CPP studies of infection and neuropsychiatric diseases addressed the target diseases schizophrenia, autism, and mental retardation. Methods included:
  • Identification of cases and controls
  • CPP database
  • Follow-up studies
  • Retrieval of serum samples from repository
  • Testing for antibodies and other factors
• Financial support for these studies came from the Stanley Medical Research Institute.

Dr. Yolken discussed Enzyme Immune Assays (ELISA) and the applicability of ELISA to cohort studies.

• Advantages of ELISA are that it is a mature technology; there are multiple sources of reagents and supplies; the assays are reproducible and adaptable to automation; a small sample size can be used for antibodies; and ELISA is relatively insensitive to matrix effects.
• Limitations of ELISA are imprecise timing of infection, moderate sensitivity, and the need for a larger sample size for cytokines.
• ELISA tests performed in the CPP included testing for immunoglobulins (IgG, IgM, IgA, and albumin); antibodies (HSV-1/2, CMV, EV, influenza A/B, and others); cytokines (including IL2, IL6, IL8, and TNF-alpha); and thyroid, Vitamin D, and Vitamin A tests.

Dr. Yolken presented information about studies of infection and neuropsychiatric diseases, including studies that addressed the role of maternal infections in schizophrenia, autism, and mental retardation.

Schizophrenia Studies: Investigators identified and interviewed offspring of CPP mothers with psychiatric diagnoses. Controls were derived from the CPP database, interviewed, and found to be free of disease. Serum samples originally obtained from mothers of cases and controls and infants (cords) were retrieved from the repository and tested for total immunoglobulins, specific antibodies, and cytokines. Levels of cases were compared with those of controls using a multivariate analysis employing demographic variables. Dr. Yolken presented two slides showing results of the tests.

The schizophrenia studies have found that:

• Risk of schizophrenia in offspring is associated with increased maternal levels at term of total IgG and IgM, IgG antibodies to HSV-2, TNF-alpha, IgM antibodies to toxoplasma gondii, and IgG antibodies to influenza viruses.
• Risk of schizophrenia is not associated with maternal levels of vitamin D.

Autism Study: This study involved the cases identified by Dr. Torrey and matching controls (five per case) obtained from the CPP database. Serum samples were obtained from the repository from preterm mothers, term mothers, and cords. The samples were tested for total immunoglobulins and antibodies. Dr. Yolken presented three slides showing findings of this study.

Infection and Mild to Moderate Mental Retardation: Serum was obtained from mothers of the entire Baltimore Pathways to Adulthood (PAS) cohort at term and tested for antibodies to infectious agents. This study found that antibody levels correlated with IQ below the fifth percentile without other etiologies for syndromes, meningitis, birth trauma, and extreme prematurity. The risk of low IQ at age 7 was associated with maternal antibodies at birth, with greatest relative risk for influenza B. Influenza B has been shown to cause alterations in fetal brain development.

Recommendations: Dr. Yolken recommended that the Study:

• Collect and store maternal and neonatal biological samples (blood and other samples, that is, urine, saliva, respiratory, and stool samples)
• Collect and store samples throughout childhood
• Monitor effects of storage in real time
• Collect cognitive and behavioral data
• Monitor efficacy of specimen and data collection and retrieval
• Encourage the development of assay methods that use small sample amounts.

Collaborators for the infections and psychiatric diseases studies included researchers from Johns Hopkins, the Harvard School of Public Health, the Stanley Medical Research Institute, and NICHD.

Use of Archived Blood Samples: Dr. Yolken discussed effects of storage.

• Mean levels of IgG, IgM, and cytokines were similar to those measured in contemporary cohorts.
• Levels of IgG were stable during pregnancy.
• High correlation was seen among preterm maternal, term maternal, and cord levels of IgG.
• Fewer data were available for other analytes.

Questions and Comments:

• A participant asked whether Dr. Yolken had looked at subjects with dual diagnoses. Dr. Yolken replied that they did not have the data because data collection ended at age 7. However, there is a need to study disease data together.
• Dr. Hardy commented about the 1957-1958 influenza A epidemic in Baltimore. The study found that maternal infection with influenza was associated with lower birthweight but not congenital anomalies. She also noted that inadequate test results are related to various difficulties in pregnancy and afterwards.
• There was a question about freezing and thawing of the samples and whether any of the samples had never been thawed. Dr. Klebanoff replied that there were samples that had not been thawed and said that a "hashmark" method was used to note when a sample was thawed. This information is computerized now. Dr. Yolken said they the CPP stuck to measures that would remain relatively stable.
• Dr. Cosentino spoke about sentinel—samples extra samples that are stored. The National Children’s Study RFP should call for a problem solving arrangement for sample storage issues. All vials tracked should be linked to "parent" vials.
• Dr. Rodriguez stated that he was concerned about agreement between laboratories. There needs to be standardization and validation between laboratories.
• Dr. Schoendorf commented that these issues are being considered for the Study, and he agreed that data issues must be addressed up front.

Neurodevelopment and Childhood Psychiatric Diseases

Maternal Immunologic Disorders and Childhood Autism
Lisa Croen, Ph.D., Kaiser Permanente Division of Research

Dr. Croen presented results from an ongoing study examining maternal autoimmune diseases, asthma, and allergy around the time of pregnancy and diagnoses of autism in children. She also discussed a plan for a newly funded study looking for prenatal and neonatal biologic markers for autism. Judith Grether, Ph.D., California Department of Health Services, is collaborating on the study.

Dr. Croen listed the core features of autism, which include:

• Communication impairments
• Social interaction impairments
• Restricted, repetitive, stereotyped behaviors and interests.

She discussed immune function in autism. Immune function abnormalities are widely reported, but findings remain largely unreplicated. Immune system disturbances include:

• Changed number/activity of immune cells
• Alterations in ratio of Th1 and Th2 cell subsets
• Decreased plasma complement component C4b
• Cytokine and serum immunoglobulin imbalance
• Decreased response to T-cell mitogens
• Increased humoral immune responses.

Autoimmunity in autism is evident in increased frequency of autoantibody production. Polymorphisms in genes are implicated in autoimmune disorders. Studies that have found a family history of autoimmune diseases in those with autism have had small sample sizes and have relied on self-reported autoimmune disease histories.

Dr. Croen’s study objective was to investigate the association between a history of maternal autoimmune diseases, asthma, and allergies around the time of pregnancy and subsequent diagnoses of autism in children. The study was a case-control study in Kaiser Permanente (KP) Northern California that included 135,352 births between January 1995 and June 1999 of children who remained in the health plan for at least 2 years. Cases (407) had been diagnosed with autism spectrum disorder (ASD), as recorded in outpatient databases. The children were between age 3 and 7 when identified. Controls (2,095) had no ASD diagnoses and were randomly sampled and matched on birth year, gender, and birth hospital.

Maternal diseases, diagnosed within a 4-year time period corresponding to date of delivery (DOD) +/- 2 years and as recorded in inpatient and outpatient databases, included 44 autoimmune diseases, asthma, and allergies (allergic rhinitis, conjunctivitis, atopic eczema, angioedema, anaphylaxis, and urticaria).

Data on covariates were drawn from KP databases and birth certificate files and included:

• Maternal characteristics
  • Age, education, race/ethnicity, parity, country of birth, medication use during pregnancy
• Child characteristics
  • Gender, birth weight, gestational age, plurality, birth order, number of autism-affected siblings.

Results: The study found no significant difference in the frequency of any maternal autoimmune disease between ASD cases and controls. For specific autoimmune diseases, significant increases in frequency occurred in cases for psoriasis and type I diabetes. However, once adjusted for maternal age, education, plurality, and maternal race/ethnicity, only the association with psoriasis remained. Asthma and allergy were more common in mothers of children with ASD, and there were greater differences between cases and controls than with autoimmune diseases. No significant differences were observed when adjusted odds ratios and 9 percent confidence intervals were calculated for autism and all maternal autoimmune diseases. Dr. Croen hopes to follow the women to determine diagnosis of autoimmune diseases later in life.

A significant association between maternal asthma and autism persisted after adjusting for maternal age, education, race/ethnicity, and plurality. With maternal allergies, the highest association was seen in the second trimester of pregnancy. Regarding maternal medication use, there was not a large effect. The study also identified families with only one child with ASD, no ASD children, or with multiple ASD children and looked at the frequency of maternal disease in those groups. For maternal autoimmunity, there was no trend in frequency across family types. But for maternal asthma and allergies, the study found an increased diagnosis of disease in families with multiple ASD children.

Dr. Croen discussed potential biologic mechanisms. Increased levels of maternal antibodies can cross the placenta and disrupt fetal neurodevelopment by cross-reacting with fetal brain antigens via molecular mimicry. Altered levels of circulating cytokines or related substances in maternal serum can cross the placenta and directly affect fetal neurons and glia. Shared environmental risk factors and shared genetic susceptibility are also likely mechanisms.

Dr. Croen described a new study, Prenatal and Neonatal Biologic Markers for Autism, which is a case-control study funded by the National Institute of Mental Health (NIMH). It will include 100 children with autism, 100 children with idiopathic mental retardation, and 100 general population controls in Orange County, California, born between July 2000 and September 2001. The study will use archived maternal serum-alpha-fetoprotein specimens from maternal prenatal screening and newborn screening. It will perform maternal serum assays for autoantibodies, cytokines, immunoglobulins, viral agents, neuropeptides, hormones, and metals. The study will also use neonatal bloodspot assays for T4 and TSH, and with additional grant funding, for autoantibodies, IgG levels, antibodies to specific infectious agents, and polymorphisms in cytokine genes.

Recommendations: Dr. Croen made the following recommendations for maternal and child data collection in the National Children’s Study.

• Maternal data collection
  • Blood specimens from first trimester, second trimester, and third trimester, and post-partum
  • Prenatal and labor/delivery medical record abstraction
  • Illness and medication use diary during pregnancy/breast feeding
  • Placental pathology
  • Breast milk specimens
• Child data collection
  • Blood specimens from newborn period, first year, second year, and third year, and post-diagnosis
  • Neonatal and pediatric medical record abstraction
  • Illness and medication use diary during the first three years of life
  • Fever and behavioral change diary during the first three years of life
  • Developmental/behavioral screens at appropriate ages.

Questions and Comments:

• Dr. Cosentino asked whether Dr. Croen was recommending commercial multiplex assays. Dr. Croen said she would defer the question to a lab expert.
• Dr. Torrey asked about the hypothesis and whether they have looked at data in reverse, whether an infection in the child affects the mother. Dr. Croen said no, but she would like to do that.
• Dr. Rodriguez asked about collecting data on alternative medicine use and suggested that looking at intake of herbal teas and other alternative medicines would be useful.
• Dr. Yolken asked about statistical power with measuring 30 different things. Dr. Croen said she was not a statistician, and that it is important to look at each thing individually.
• Dr. Grether commented about medical records documentation of autoimmune diseases in the study and said that the Study might want to take a different approach. She also said that, with the bloodspot studies of protein markers, they looked at as many variables as they could, given the small amount of specimen. Looking at internal consistency gave them confidence.
• Stefan C. Dombrowski, Ph.D., M.B.A., Rider University, commented that new computer modeling techniques can find unexpected relationships between variables. Expertise in statistical analysis can help drive some questions.

Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections
Susan E. Swedo, M.D., National Institute of Mental Health, NIH, DHHS

Dr. Swedo began with a case study of a child with sudden onset of obsessive-compulsive disorder (OCD). The child developed tics, bed-wetting, hand-washing, and other unusual behaviors. The child was diagnosed with otitis media and treated with amoxicillin, and all symptoms resolved within a week.

Criteria for pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) include:

• Presence of OCD and/or tic disorder
  • Must meet lifetime diagnostic criteria for OCD and/or tic disorder (DSM-III-R or DSM-IV)
  • DSM-IV diagnostic criteria for OCD include obsessions (recurrent and persistent thoughts, impulses, or images) or compulsions (repetitive ritualized behaviors) that are recognized as excessive or unreasonable (children are exempt from this criterion) and cause marked distress, take more than 1 hour a day, or interfere with normal routine, occupation, academics, or social activities
  • OCD symptoms in pediatric patients include obsessions (such as concerns about contamination, safety, right/wrong, intrusive thoughts) and compulsions (such as ordering/arranging, symmetry, hoarding/collecting, and repeating rituals)
  • Diagnosis is complicated by barriers to recognition, including embarrassment, secrecy, and the presence of more prominent comorbid symptoms
  • Screening tools include the Children’s Yale-Brown Obsessive Compulsive Scale (CY-BOCS) and the Multidimensional Anxiety Scale for Children (MASC)
  • Diagnostic criteria for tic disorder include:
    • Tourette disorder: Both multiple motor tics and at least one vocal tic present for more than 1 year without a 3-month, tic-free interval
    • Chronic motor or vocal tic disorder: Motor or vocal tics (but not both) are present for more than 1 year without a 3-month, tic-free interval
    • Transient tic disorder: Single or multiple motor or vocal tics present for at least 4 weeks, but for no longer than 12 consecutive months
  • Barriers to recognition of tic disorder include situation-specificity, transience, not being aware of tics or not recognizing them as abnormal, and the ability of the child to suppress the tics
• Prepubertal onset
  • Obsessive-compulsive and/or tic symptoms must be present prior to the onset of puberty
• Episodic course of symptom severity
  • Clinical course characterized by an abrupt onset of neuropsychiatric symptoms followed by an episodic course of near complete remissions and sudden acute exacerbations
• Association with neurological abnormalities
  • Neuropsychiatric symptom onset and exacerbations preceded by group A streptococcal (GABHS) infections
• Temporal relationship between symptom exacerbations and streptococcal infections
  • Motoric hyperactivity, choreiform movements, or other neurologic abnormalities present during neuropsychiatric symptom onset and exacerbations.

Dr. Swedo presented data showing severity of symptoms and antistreptolysin O antibody (ASO) titers over time for PANDAS and non-PANDAS. Children with symptoms can be classified as consistent, possibly consistent, or inconsistent with the PANDAS subgroup classification.

She discussed the etiology of PANDAS and described a model of pathogenesis involving a susceptible host and abnormal immune response, resulting in central nervous system and clinical manifestations. She discussed a number of relevant studies, host susceptibility as shown by increased familial rates of OCD and tics and rates of rheumatic fever, and the abnormal immune response seen in local, regional, and systemic responses, including identification of antineuronal antibodies and cytokine abnormalities.

Recommendations: Dr. Swedo listed the following questions that could be answered in the Study:

• Age-related differences in prevalence and incidence of OCD and other anxiety disorders, tics and movement disorders
  • Genetic vulnerabilities
  • Environmental triggers
• Etiopathogenesis of childhood-onset versus later-onset cases of OCD and related disorders.

Potential problems with assessment include:

• OCD—disorder of secrecy
• Tics—difficult to diagnose by history
• Anxiety—reports influenced by parental psychopathology
• PANDAS—episodic course; subclinical cases most common; not established as accepted diagnostic group.

Opportunities include:

• No previous large-scale epidemiologic studies for OCD or tic disorders
• Possible diagnostic test for PANDAS
• Genetic studies to determine etiology
• Identification of common immunologic abnormalities

Questions and Comments:

• William Andrews, M.D., Ph.D., University of Alabama, Birmingham, asked whether it is "the host or the bug," and whether there were animal models to study these disorders. Dr. Swedo replied that there has been a large gap between progress made in virology and bacteriology, and an animal model for rheumatic carditis is being tested.
• Dr. Rodriguez said that investigators were using MRI in adults post exposure to psychotropic agents and finding enlarged basal ganglia. He asked whether this has been done with children. Dr. Swedo replied that basal ganglia enlargement in seen in severely ill children.
• Dr. Yolken said that this is the age group where there is the biggest gap and it is a great opportunity for the Study. The Study should get data from the child, not just the parent. School records, such as handwriting samples, would also be valuable, and better ways to analyze such samples will most likely be developed.

Cytokines and HPA Axis Hormones in the Etiology of Schizophrenia
Brad Pearce, Ph.D., Emory University

Dr. Pearce described evidence that schizophrenia is a neurodevelopmental disorder, including:

• Exposure to gestational and perinatal infections, and obstetrical complications (OC)
• Birth in winter season and urban area
• Dermatoglyphic abnormalities
• Minor physical anomalies
• Neuromotor abnormalities (for example, involuntary movements)
• Delays in early developmental milestones
• Social impairment (such as preference for solitary play).

Dr. Pearce listed "culprits," including cytomegalovirus, herpes simplex virus, rubella, and others, and outcomes including epilepsy, mental retardation, hearing loss, blindness, learning disabilities, cerebral palsy, and hydrocephalus. Cytomegalovirus can cause mental retardation, but is responsible for only a small proportion of cases.

Schizophrenia probably has multiple antecedents. Obstetrical complications are associated with schizophrenia, as shown by a recent meta-analysis. Neurodevelopment insults include prenatal stress and infection.

Dr. Pearce discussed a study looking at high-risk women with psychotic disorders; the study measures cytokines and hormones in maternal/fetal circulation. Another study examines high-risk children with schizotypal personality disorder (SPD) and uses immunohormonal measures. Animal studies look for brain and neuroimmune abnormalities following experimental infections.

Dr. Pearce discussed cytokine (IL-1, IL-6, and TNF) interactions with the developing brain and listed proposed effects:

• Disrupt growth factors
• Amplify excitotoxicity
• Damage by free radicals
• Induction of apoptosis
• Alter synapse formation
• Disrupt blood-brain barrier.

He displayed a diagram showing the HPA axis response to stress with the release of cortisol, and suggested that cortisol may help "program" fetal neurocircuitry. There is evidence of cortisol resistance in up to 40 percent of schizophrenics, and hypothalamic-pituitary-adrenal (HPA) axis dysfunction is associated with poor outcomes. Key questions are whether immune and hormonal abnormalities predate schizophrenia diagnosis and whether these abnormalities date back to the womb. These are questions that can be addressed by the Study. Detectable prenatal or neonatal factors include cytokines, growth factors, hormones, and immune responses/susceptible genes. These measures could be correlated with premorbid risk indicators.

Some questions are:

• Do schizophrenic women who become pregnant have more infections and complications, and do they have an exaggerated immune or HPA axis response to infections or obstetric complications?
  • Most studies indicate an increased rate of obstetrical complications in schizophrenic women, and their children are at increased risk of schizophrenia. However, better definitions of obstetrical complications are needed as well as better control of confounds. Another question is whether infection, immune activation, or HPA axis dysregulation predict transition to schizophrenia among high-risk adolescents.
• Does infection, immune activation, or HPA axis dysregulation predict transition to schizophrenia among high-risk adolescents?
  • Looking at SPD (Axis II), 42 percent transition to an axis I psychotic disorder by age 20.

Dr. Pearce suggested identifying youths with SPD and obtaining serial samples for viral serology, cytokines, and HPA axis and sex hormones. He also discussed animal models that show that the consequences of infections during neurodevelopment might be delayed.

Types of genes possibly related in infectious causes of schizophrenia include those involved in:

• Virus entry and cell susceptibility (for example, virus receptors)
• Immune responses (for example, HLA, regulators of T or B cell repertoires, and cytokine promoters)
• Immunoactive hormone production (for example, cortisol and TSH)
• Maternal-fetal interactions (for example, placenta molecules)
• Brain vulnerability (for example, blood-brain barrier)
• Neural compensation (neurotrophic/growth factors).

Recommendations: Dr. Pearce made the following recommendations for the Study:

• Standardize recording of obstetric complications (fetal blood gases).
• Do genotyping of mother and child for polymorphisms in cytokine genes, hormone and binding proteins, and their receptors. There are many candidates: IL-1, IL-4R, IL-6, IL10, TNF-alpha, IFN-gamma, TGF-B1, CRH, CBG, and GR. Consider a high throughput multiplex (SNP chip, SNapshot).
• Do serial plasma collection (once per trimester, and maternal and cord blood at delivery) for serology, proinflammatory and anti-inflammatory cytokines, hormones, autoantibodies, and proteomics. Store PBMCs; these allow for genetic analysis, HLA typing, microarray (mRNA) analysis, and functional immunology (for example, memory T-cells).
• Do extra collection during significant prenatal or neonatal infection or other identified complication. Include acute and convalescent titers.
• Include representation of women with severe mental illness, including schizophrenia.
• Correlate immunohormonal responses to obstetric complications and immunohormonal genotyping with child attainment of developmental milestones, motor functioning, and minor physical anomalies. Ethical issues, such as hand printing large number of subjects and stigmatization, will have to be considered.
• Focus on children who develop preschizophrenic personality disorders (for example, SPD). Obtain serial plasma and PBMCs (every 6 months) for serology, cytokines, hormone measures, proteomics, and microarrays. Follow-up to determine biological predictors of transition to schizophrenia.

Questions and Comments:

• Dr. Yolken commented about the issue of oversampling versus a cohort study. The CPP was a cohort study and the same data were collected on all participants. He said that he felt it would be difficult to obtain acute and convalescent samples. He asked how often testing should be done if the same tests were done on all participants.
• Dr. Pearce said that considering the amount of data that will be collected in the Study, he felt that the Study would be remiss not to identify subpopulations, for example, those with SPD, to study more intensively.
• Dr. Cosentino commented about how this would affect the repository; logistically, he expects that there would be problems. Studying a subpopulation in an ancillary study would be better, as the participants could be tracked separately. Dr. Hardy said that about 100 cases in the rubella epidemic were identified and studied in an ancillary study.
• Dr. Klebanoff mentioned that there are risks of having a restrictive study core and doing ancillary studies. Also, there are informed consent issues with enrolling women with mental illness, and he wondered if these could be overcome. Dr. Pearce said that it is easier with medicated patients than with unmedicated patients. Dr. Yolken said that the Study would need to be sure that women with mental illness are not excluded from the Study, but there may not be a need to oversample them. Diana Schendel, Ph.D., National Center on Birth Defects and Development Disabilities, CDC, DHHS, added that there are ethical issues related to children with preschizophrenic personality disorders. Dr. Pearce agreed and said that confidentiality is essential.
• Dr. Swedo asked how the Study would enroll teenagers and women with mental illness, since they do not tend to get early prenatal care. Dr. Schoendorf explained that the sampling strategy has not yet been determined. Dr. Schendel said that it would be interesting to do simultaneous cohorts. Dr. Yolken said that practically, the Study must decide what it can get. For example, antibodies can be measured in saliva, and that is an easier way to collect the samples. Less intrusive measures are needed.
• Dr. Croen asked about follow-up of the effects of pregnancy on mothers’ future health in the Study. Dr. Schoendorf replied that there is interest in that; however, children’s health outcomes must be the priority. Dr. Hardy commented that two good reasons to follow the mothers are that the mothers can help to locate the young adults and can validate historical data provided by their children.
• There was discussion about methods of preserving serum samples. There are difficult technical questions concerning preservatives. Multiple methods of preservation could be used for different samples. Dr. Schendel suggested encouraging and supporting ongoing research on methodologies and other scientific questions to provide feedback for assay development, and Dr. Yolken said this could be done through the existing pilot study program. Dr. Schendel stressed the need for ongoing research.

Placental Markers: Maternal Infection II

Maternal Immune Response Changes Over Time During Pregnancy
Diana Schendel, Ph.D., National Center on Birth Defects and Development Disabilities, CDC, DHHS

Dr. Schendel presented research performed for a Ph.D. dissertation by Anjali Deshpande, Emory University, titled "Maternal Immune System Changes in Term Pregnancy." The data came from a Danish cohort.

Not a great deal is known about the maternal immune system in pregnancy. What is known is based largely on animal models, and includes:

• Wegmann’s paradigm
  • Shift from Th1 (cell-mediated) immunity to Th2 (humoral) immunity to inhibit maternal rejection of fetus
  • Likely oversimplification: both Th1 and Th2 cytokines secreted in early pregnancy
• Cytokines’ role in labor
  • ↑ IL-6, IL-1beta, TNF-alpha in term labor.

Methodological issues and knowledge gaps include:

• Cross-sectional study designs
• Small sample sizes
• Timing of measurement (preceding/in labor)
• Compartment of measurement: AF, serum/plasma
• Type of assay: bioassay, immunoassay
• Single/multiple analytes
• Consideration/control of other factors.

She described a study to investigate the natural history of multiple select immune markers in term pregnancy that addressed:

• Pro-inflammatory cytokines
• IL-1beta (Th1)
• TNF-alpha (Th1)
• IL-6 (Th2)
• Marker for INF-gamma (cell mediated immunity)
• Neopterin.

The research was a nested case-control study, using a 1992-1994 cohort from the catchment area of Odense University Hospital (65 percent of county deliveries). The cohort was 2,927 women (81 percent of those eligible). There were 84 preterm delivery cases, 224 gestational controls matched to PTD cases on gestational age at PTD delivery, and 300 delivery controls.

Serum samples were obtained at enrollment for all, in pregnancy (gestational controls), and at delivery for all. There were 125 gestational controls with serum samples drawn 3 times in pregnancy. Immune marker measurements were made for cytokines and neopterin using commercially available assays. The sample included 125 singleton term deliveries with mean age of 28 years.

Results showed that most assays were successful and median values were detected. Dr. Schendel presented a series of scatterplots and discussed the results. Significant factors affecting change in concentration of analytes during pregnancy included:

• All markers:
  • Gestational age (continuous/categorical)
  • Enrollment levels of other markers
  • Subject-specific mean effects
• TNF-alpha, neopterin:
  • Reproductive history variations-gravidity, parity, previous induced abortion
• Neopterin:
  • Maternal age.

Unfortunately, there were no anti-inflammatory measures and no prepregnancy baseline measures.

In summary, based on serum measurements, the findings were:

• All markers
  • Detectable at some point-early in pregnancy and/or at delivery
  • Absolute increase in concentration over course of pregnancy
  • Significant rate of change with gestational age
  • Mean levels influenced by early levels of one or another marker
  • Significant inter-individual variation in mean levels
• By marker
  • Pattern of increase differed
  • Early increase, then level: IL-1beta, TNF-alpha
  • Increase throughout: neopterin, IL-6
  • Dramatic increase in labor: IL-6
• Influence of previous reproductive history, maternal age.

Dr. Schendel discussed implications for future biomarker studies. It is part of normal pregnancy for markers to increase. The future search for inflammatory markers of adverse outcomes may need to consider:

• Multiple markers; influence of other marker levels on marker of interest
• Marker-specific patterns of change in pregnancy
  • Timing of measurement effects observed levels
• Deviation from longitudinal pattern of change, absolute level
• Maternal prepregnancy factors: reproductive history, age
• Significant individual variation in overall mean levels even in normal pregnancy (need to further investigate whether subject-specific patterns of change are important).

Recommendations: Dr. Schendel made the following recommendations for the Study:

• Multiple sample collection
• Laboratory methods development
  • Accommodate multiple analyte analysis (multiplex assays)
  • Accommodate small sample volumes (microanalytic techniques)
  • High sensitivity
  • Experimental techniques of today may be standards of tomorrow
• Enrollment
  • Prepregnancy baseline levels desirable (added justification for prepregnancy enrollment effort)
    • Post-natal sampling may be an alternative if "return" to baseline
  • Enroll subsequent pregnancies to assess effects of previous pregnancy history on current pregnancy
• Analysis
  • Interaction of other markers on marker of interest relative to outcome
    • Don’t limit focus to marker-outcome relationship
    • Data reduction:
      • Reduce number of markers
      • Develop marker profile
  • Early products of the Study:
    • Analysis and publication of "normal pregnancy" changes and interactions among immune markers, hormones, neurotrophic factors-against which pathological changes associated with adverse outcomes can be compared.

Questions and Comments:

• Dr. Yolken asked how many samples should be taken during pregnancy. Dr. Schendel said that in the Danish cohort study, there were only two samples, and the Study should definitely obtain more than two. Dr. Andrews said that blood is usually drawn several times during prenatal care, which is an opportunity to collect samples without requiring separate samples. It is often possible to get three or four samples during pregnancy.
• Dr. Pearce said that cytokine profiles were a good idea instead of looking at individual markers-they are not independent markers.
• Dr. Yolken said that the Study must be able to validate new tests. Test samples from individuals who do not remain in the Study will be valuable for developing new technologies.

The Relationship Between Bacterial Infection and Preterm Birth
William Andrews, M.D., Ph.D., University of Alabama, Birmingham

Dr. Andrews said that his role was to put into perspective the relationship between bacterial infection and preterm birth in order to understand how the fetus is exposed. He said that approximately 12 percent of births are preterm births, and preterm births account for 75 percent of perinatal mortality and 50 percent of long-term neurological handicaps. Most of the mortality, as shown in a graph showing a survival curve, occurs in the very preterm babies.

Dr. Andrews listed preterm birth outcome improvement strategies, including:

• Corticosteroids
• TRH, phenobarbitol, vitamin K
• GBS prevention
• Newborn asphyxia prevention
• Neonatal intensive care
• Regionalized access to care
• Antibiotics in preterm premature rupture of membranes (PROM).

He showed data indicating that while survival rates of low birthweight infants at the University of Alabama, Birmingham (UAB) have been rising, mean birthweight has been declining. Today, survival rates for a baby born at 1,000 grams or more is over 90 percent. Preterm birth prevention strategies include:

• Prenatal care
• Psychosocial support
• Nutrition
• Pharmacological intervention
• Anti-smoking programs
• Anti-drug programs
• Home uterine activity monitoring.

Dr. Andrews discussed the incidence and etiology of preterm birth in the United States. Preterm birth rates have been gradually increasing, and preterm birth is more common in African Americans. Most preterm births are due to spontaneous onset of labor or spontaneous PROM; about 20 percent of preterm births are medically indicated births due to maternal medical complications or obstetrical complications. Dr. Andrews displayed a graphic image of a fetus illustrating the prevailing theory-the Romero hypothesis of aberration of bacteria in the vagina or cervix causing a cascade of events leading to preterm delivery.

At the UAB, chorioamnion colonization was seen in 40 percent of spontaneous preterm births, a rate 3 times higher than in indicated preterm births. For 446 maternal-infant pairs, 64 percent were spontaneous preterm births and 37 percent were indicated preterm births. Overall results were that 51 percent had acute histologic chorioamnionitis and 54 percent had a positive culture for at least one organism. Dr. Andrews pointed out that these infections are in asymptomatic women, so there is no way for the physician to know that colonization is present.

Conclusions were that:

• Acute histologic chorioamnionitis is 11 to 33 times more common among women with a spontaneous compared to an indicated preterm birth
• Microbial chorioamnion invasion is 3 to 4 times more common in women with a spontaneous compared to an indicated preterm birth.

These data provide compelling support for the hypothesis that chorioamnion infection/inflammation play a substantial role in spontaneous but not indicated preterm births.

Future directions include:

• Better markers of preterm birth
• Ecosystem microbiology
• Maintenance of healthy ecosystem
• Pathophysiology
• Role of immune system
• Hypo-responsive
• Hyper-responsive
• Gene-environment interactions
• Antibiotic treatment
• Immunomodulation treatment
• Effect on child
  • Neonatal outcome
  • Neurodevelopment.

Data needed include:

• Placental histology
• Cultures
• Biomarkers
• Clinical outcomes
• Genetic studies
• Environmental exposures.

Dr. Andrews discussed future directions for laboratories (real time studies and archive) with respect to placenta/umbilical cord specimens, amniotic fluid, blood, and other specimens. Future directions for clinical work include the need for detailed, standardized data on pregnancy, the neonatal course, long-term development, parents, the environment, and other factors.

Questions and Comments:

• Dr. Yolken said that there are data from the CPP study that have not been analyzed. This does raise questions about standardization of histology and cultures. Dr. Andrews said that few have the expertise to do this. One problem is how to tell what is being compared. Perinatal pathologists can do this. Definitions need to be clear; for example, what does neonatal sepsis mean? A data collection sheet is needed for data abstraction.
• Dr. Schoendorf asked about correlation between IL-6 in different specimens. Dr. Andrews said that was hard to answer. Not many studies have tried to compare analytes across samples.
• There was a question about the role of viruses. Dr. Andrews said that this is almost completely unstudied, but the potential role of viral exposures may be very important. Bacterial infections may set up an environment in which viruses can flourish.
• Dr. Pearce asked about the disparities among African Americans versus other racial groups with regard to preterm birth. Dr. Andrews said they have looked at this in several ways, but so far have found nothing. The disparity remains to be explained. More work is needed in this area, and currently cytokine polymorphisms are being examined.

Placental Inflammation and Fetal Origins of (Childhood and) Adult Disease
Carolyn Salafia, M.D., M.S., Columbia University

Dr. Salafia discussed the following issues in placental inflammation and fetal origins of adult disease (FOAD):

• What to measure?
  • Sampling and reliability
• How to measure?
  • Scoring and validity
• Analytic methods for histology items
  • Scales, EFA
• Tissue sampling protocol issues.

Dr. Salafia displayed a graphic image of three major ways that the mother can affect FOAD via the placenta. Maternal systemic processes could:

• Pass across the placenta and have a direct effect on the fetus, with no measurable effect on the placenta.
• Have direct effects on both the fetus and the placenta. There may be evidence of a placental effect that is equivalent to or a mirror image of the fetal injury, or the maternal process may leave a more poorly defined marker.
• Have direct effect only on the placenta, which then would have a direct effect on fetal anatomy or physiology.

Dr. Salafia discussed the effects of measurement error, which can lead to led to attenuation of correlations with placental variables. There are issues with gross measurement of the placenta, and standard measures of placental dimensions are poor. Long-standing infections and chronic inflammation can be clinically "silent." Concerning histology, there are three questions: What can you sample? Why do you want to? How should you sample?

Dr. Salafia discussed aspects of acute infection (AI) and chronic inflammation. AI is complex and multifaceted, and it is important to understand the route by which infectious agents reach the fetus, such as ascending transcervical infection. The tissue sampling scheme can distinguish the routes of infection, and an ideal sampling strategy would distinguish between ascent close to delivery and remote ascent and chronic upper general tract infection. She discussed options for tissue sampling, the adequacy of sampling strategies, and inconsistency in sampling.

Goals for the Study’s AI protocols should be to:

• Distinguish routes of infection
• Distinguish anatomic and person-specific aspects of response
• Capitalize on in vivo orientation of tissues
• Sample enough to get a "big picture" of the multifaceted process of acute placental inflammation
• Use replicate samples
• Use replicate histology items
• Use highly intercorrelated scores
  • Redundancy is essential to reliability.

The Study would want to choose diagnoses that had evidence suggesting they could be diagnosed reliably. Dr. Salafia discussed the use of scales to increase power, reliability, and validity. She reviewed protocol issues, including:

• Should all placentas be grossed equally?
  • Digital photography makes this plausible.
• Should all placentas be sampled equally?
  • Pro: For low frequency lesions, one error is significant.
  • Con: The Study would end up with a large number of negative tissues.
  • Block-and-hold?

In summary, the acute inflammatory response varies by:

• Person (mother versus the fetus)
• Route of infection
• Gestational age of the fetus
• Location
• Aspect of acute inflammation studied.

Other considerations are that:

• Reliability and validity will vary with the sample. Reliability requires replicate measures and multiple intercorrelated histology item scores. Validity requires criteria for judging the predictive value of any histology scoring item or set of items (separately or in a scale).
• Cost-benefit ratios must balance sampling and diagnostic yield.

Recommendations. Dr. Salafia made the following technical recommendations for the Study:

• Digitally photograph all placentas
  • Fetal surface
  • 7-surface Cavalieri method
• Front-load samples by block-and-hold
  • Samples labeled by tissue and relative intrauterine position
  • Multiple samples
• Cut a simple screen of samples.

She also made these histological recommendations:

• Protocols must be geared to the low lesion prevalence of a birth cohort.
  • Redundancy of samples, histology items, and intercorrelated items
• "Consensus" is not "validity"
• Histology items are reflections of a latent unmeasurable construct.
  • Scales
  • Scales not restricted to strictly AI lesions
  • EFA, CFA.

Regarding centralization, she recommended:

• Local:
  • Gross measurement photograph captured locally.
  • Anatomically specified sampling
• Central:
  • Quality assurance (QA) on gross digital image analyses
  • Specification of samples to be cut
  • QA on histology scoring
  • Data analyses.

Questions and Comments:

• Dr. Yolken said that this was an important set of issues and asked about digital imaging at the microscopic level. Dr. Salafia said that this was important and added that her grant is going to develop a Web-based data dictionary.

Effects of Cytokines on Brain Development
John Gilmore, M.D., University of North Carolina at Chapel Hill

Dr. Gilmore discussed schizophrenia as a neurodevelopmental disorder with prenatal/perinatal origins. The disorder is associated with pregnancy and birth complications, subtle childhood neurodevelopmental abnormalities, brain abnormalities on MRI present at first episode, and neuropathologic findings consistent with neurodevelopmental abnormalities.

He described studies of maternal infection and risk for schizophrenia and the association between infection and other disorders such as cerebral palsy, autism, and neural tube defects. The hypothesis is that these associations are a general phenomenon related to infection-not the specific agent; the outcome is dependent on the timing of the infection in relation to neurodevelopmental events and genetic vulnerabilities; and schizophrenia is on the more subtle end of the spectrum neuropathologically.

Dr. Gilmore discussed inflammatory cytokines as a mechanism, cited a number of studies showing associations between cytokines and schizophrenia, and presented a series of slides with data from rat studies showing the neuropathology of schizophrenia, including effects on the cortex, thalamus, and hippocampus. Conclusions were that:

• Inflammatory cytokines typically generated in response to infection can inhibit the development of dendrites in cortical neurons.
• This offers a tangible link between prenatal exposure to infection and the cortical neuropathology observed in schizophrenia.
• Especially relevant is the lowest concentration tested, in which 100 U TNF-alpha and the combination of IL-1beta and TNF-alpha caused significant decreases in dendrite development while having no effect on overall neuron survival.
• Prenatal exposure to lipopolysaccharide (LPS) decreases cortical synapse number in vivo.

Dr. Gilmore presented additional animal study data, citations, and the following conclusions:

• Maternal infection (LPS and Poly I:C) alters cytokine and neurotrophic factor expression in the maternal-fetal unit and the fetal and neonatal brain.
• This represents a mechanism through which maternal infection can change brain development, especially dendrite and synapse development, and increase risk for schizophrenia and other neurodevelopmental disorders.
• There is no clear relationship between peripheral and CNS levels of these cytokines or neurotrophic factors.

Dr. Gilmore discussed future directions, including:

• What is the mechanism of action?
• Role of glia
• Other potential mechanisms
  • Neuron proliferation, migration, cell type determination, programmed cell death
• Gene-environment interactions.

Recommendations: Thoughts for the Study included:

• It is critical to determine the timing of the exposure, especially with relation to brain development.
• Multiple assessments in pregnancy are needed; biomarkers may be transiently elevated.
• Ultrasound measures of brain size and head circumference will be useful.

Questions and Comments:

• Dr. Klebanoff said that head circumference is the easiest measure to obtain. Dr. Yolken said that the number of measurements needs to be considered.
• In response to a participant question, Dr. Gilmore said that it was not clear if the placenta makes factors that affect the developing brain.
• Dr. Salafia asked about the level of brain development in rats and whether exposure correlates with midtrimester in humans. Dr. Gilmore replied that his data suggest not. There is a downregulation of fetal expression.
• Dr. Andrews commented that many macrophages and cells can produce cytokines. Maternal cytokines probably do get to the fetus. Dr. Salafia mentioned two in vitro perfusion studies in which the placenta was a barrier.
• Dr. Rodriguez asked whether it is possible to inject an animal and see if the substance makes it into the placenta. Dr. Gilmore said that some studies have indicated that cytokines can cross the placenta. Dr. Yolken summarized that there is clearly a need to study humans and better understand these processes.

Gestational Maternal Fever and the Possible Association with Psychological and Behavioral Outcomes in Children
Stefan Dombrowski, Ph.D., M.B.A., Rider University

Dr. Dombrowski discussed the hyperthermia literature and said that animal studies have shown that hyperthermia is very teratogenic, and human studies have shown some associations between first trimester hyperthermia and adverse effects such as spina bifida and cerebral palsy. The influenza virus is of particular interest to researchers, and research has shown that infection in the second trimester with influenza is associated with adverse outcomes such as schizophrenia and autism. The second trimester is a period of brain growth and vulnerability.

Dr. Dombrowski suggested that fever and/or an associated response of fever (for example, direct cytokine effects or heat shock response) may disrupt these processes, resulting in adverse psychological/behavioral outcomes. Dr. Dombrowski and colleagues investigated the impact of maternal fever during the second and third trimester of pregnancy on the psychological, behavioral, and academic development of offspring at various stages, including infancy, age 5, and age 12. They found significant evidence that elevated temperature during the first trimester of pregnancy produces adverse physical outcomes and congenital defects, and some evidence that fever during the latter half of pregnancy may contribute to psychological and/or behavioral outcomes.

Dr. Dombrowski speculated that the heat shock response may interfere with central nervous system processes or there may be an autoimmune response to heat shock proteins. He discussed a number of research questions that remain to be answered.

In a handout, Dr. Dombrowski summarized his hypothesis: Gestational fever/hyperthermia during pregnancy can have an impact on the development of the central nervous system (CNS) of the fetus that leads to adverse psychological/behavioral outcomes in offspring. It is important to be mindful of the complex and overlapping processes of gestational CNS development and whether there might be a window of vulnerability depending upon the timing, magnitude, and duration of a febrile perturbation.

Recommendations: Dr. Dombrowski made the following recommendations for the Study:

• Monthly monitoring at a clinic that would include temperature readings and blood sampling (to ascertain heat shock protein levels) for subsequent analysis.
• Twice weekly temperature self monitoring by pregnant women.
• When expectant mothers experience an elevated temperature (for example, 38.3 C/101 F or 38.9 C/102 F or greater), they should report to an assigned clinic for additional blood sampling to ascertain heat shock protein levels (and, if possible, endogenous pyrogen levels).
• During a febrile episode, the expectant mother’s temperature should be monitored every 2 hours until fever subsides. The goal will be to obtain magnitude and duration data. The onset and remission dates and times of fever should also be recorded. A febrile episode lasting longer than 3-5 days should warrant further medical investigation.
• A related issue is: What should be done to palliate fever?

Questions and Comments:

• Dr. Yolken commented that self monitoring raises issues of quality control, but it can be done.
• A participant said that it needed to be sorted out whether fever leads to cytokines, or whether it is due to environmental impact. Dr. Dombrowski agreed that there are many confounds.
• Dr. Croen mentioned some anecdotal reports that fever helps behavior in autism. Another participant said that this was confounded and that improved behavior could be a general illness effect. Dr. Yolken stressed that it is most important that one makes the right measures in the first place. Dr. Rodriguez commented that in older populations, fever can cause psychosis.
• Dr. Swedo said that self monitoring of temperature twice a week would not be often enough. The Study will have to consider subject burden; however, daily recording at home can actually be easier for people to do than less frequent recording.

Dr. Yolken asked the group to think about how many blood samples would be tolerated by participants.

Measurement of Markers Relevant to Inflammation and Brain Development

Analysis of Neonatal Proteins Related to Brain Development
Judith Grether, Ph.D., California Department of Health Services

Dr. Grether discussed newborn screening. Reasons for testing in the newborn period are that this period is relatively early in the pathogenic sequence. It is before the cascade of secondary effects, before treatment for a disorder, and before consequences of behaviors. She described the California newborn screening program, which archived neonatal blood and provides a statewide system for identifying children with autism and other developmental disabilities. A number of cerebral palsy and autism studies have been done using the blood samples.

The cerebral palsy research showed that there was an inflammatory process going on in the children with cerebral palsy. A subsequent study in preterm children without cerebral palsy showed that some infants had increased levels of inflammatory cytokines and chemokines; however, there were no differences between cases and controls.

The autism study looked at children with autism, children with mental retardation without autism, children with cerebral palsy, and a control group. Levels of proteins were found not to be associated with day of blood draw, gestational age, birthweight, sex, race/ethnicity, and whether the birth was twin or singleton. The researchers believe that inflammatory and coagulation processes have important effects, and there is evidence from elsewhere that is supportive. Other neonatal bloodspot studies have looked at IgG antibodies and candidate genes for cerebral palsy and autism.

Strengths of the California newborn screening system are:

• Stability of analytes on filter paper
• Relatively easy and inexpensive to archive
• Newborn screening specimens for 100 percent of newborns
• Good source of DNA on large populations.

Limitations are that:

• Technology has not caught up with questions regarding proteins.
• There is a small amount of specimen, and biologic activity cannot be measured with these specimens.
• Metals cannot be measured.
• For some analyses, contamination is a big consideration.
• The specimens are not routinely archived under frozen conditions and made available to researchers.

Recommendations: Dr. Grether discussed considerations for the Study:

• Technology will catch up, so it is important to create archives.
• For immunoassay studies, different mediums, technologies, and antibodies may yield different answers.
• New discoveries could lead to routine newborn screening.
• At minimum, the Study should obtain neonatal filter paper specimens on all children for comparisons within individuals over time
• Maternal specimens are needed for comparison to their newborns.
• Measure digit ratios of second and fourth fingers (lengths) as a window into mid-gestational hormonal environment.
• Meconium should be obtained for metals and other exogenous gestational exposures.
• The Study should consider obtaining other specimens including baby teeth, first baby hair, and toenails; this would require standardized protocols and parent compliance.

Questions and Comments:

• Dr. Andrews said that this is an important body of work that introduced concepts of microanalytical technology to the field. Measuring panels of analytes may be more valuable than measuring single analytes. He said the negative findings with preterm babies were surprising and asked about timing of the bloodspot specimens. Dr. Grether said that the time interval has shortened because of women leaving the hospital sooner after giving birth, but they have not seen differences with different intervals. Dr. Yolken suggested that the Study should get the samples as late as possible, because cord blood would also be available.
• There was a question about the percentage of cord blood that is the mother’s. Dr. Andrews responded that cord blood is fetal blood, but it must be collected properly to avoid contamination. Another issue is whether the sample is venous, arterial, or mixed blood. Standardization would be needed regarding collection. Dr. Andrews commented further about amount of blood in samples and the fact that filter paper bloodspots require a very small amount of blood. Dr. Yolken said that it would be important to get both cord blood and bloodspot samples and added that levels of maternal blood IgM and IgA antibodies were generally not detectable in cord blood samples collected in the CPP.
• There was discussion about the type of filter paper used. It will be important for the Study to use the best paper available and to use the same type of paper consistently.
• Digital imaging was described as a useful technology, but another participant said that there are disadvantages to it as well. There are storage and quality control matters to consider. The Study could fine-tune the sampling methods.

Analysis of Cytokines from Human Samples
Philip G. Nelson, M.D., Ph.D., NICHD, NIH, DHHS

Dr. Nelson discussed the determination of cytokines and brain derived neurotrophic factor (BDNF) in dried blood spot (DBS) eluates from neonates. The Study will have to bring together samples from many labs into a master data set, and it will be important to avoid problems with differences in sampling. He discussed the standard ELISA assay method and the new Luminex technology, which produces color-coded microsphere sets for multiple analytes. He suggested that the Study should use a broad range of analytes. Dr. Nelson displayed a series of charts comparing cytokine test results with standard curves, showing the sensitivity of the measures. It is important to demonstrate that one is measuring what one thinks one is measuring.

Dr. Nelson displayed tables showing results using different methods. One table showed cytokine values (pg/ml) for autistic and healthy newborns, with values corrected for dilution factor. No differences between autistic and healthy children for IL-1beta, IL-6, IL-8, and vascular endothelial growth factor were seen. Another table displayed results for autistic and healthy newborns with regard to BDNF; again, there were no differences between methods.

Recommendations: Dr. Nelson made the following recommendations for the Study:

• Provide support for sample handling research (for example, regarding stabilization of samples). There will be difficult choices to make.
• Consider methods development, for example, spectroscopic methods. More specific methodologies are needed.
• Use an analyte standard repository for quality control. Results will be dependent on what is used as the standard. It will be very important to have standards when multiple labs/sites are collecting the Study samples.

Questions and Comments:

• Dr. Yolken stressed the need for a "gold standard" for multiple labs and added that a stable source of reagents for all assays is needed. Regarding serum versus plasma, he thinks the Study will end up with plasma for most measures-filter paper samples, anticoagulated. Dr. Nelson said that could be sources of errors with plasma.
• Dr. Gilmore said they had looked at stabilization of BDNF using different methods and did a study of cord blood. There was a significant decrease of BDNF in cord blood over 24 hours. Dr. Nelson said that there are differences in the ways different analytes behave.
• Dr. Yolken said that something could be put in the samples to stop all these activities and changes. There is a standard way to minimize changes over time in storage. Dr. Pearce commented about binding proteins and hormones and said that knowledge of total and free amounts would be needed.
• Dr. Andrews agreed that there is a need for standards for sample collection and storage. He said that it would not be possible to get the same reagents from even a single source for 20 years. Standardization is needed at the point of measurement. Dr. Yolken said that a standard, blinded set of samples could be sent out to the labs. It will be essential for the Study to have a set of absolute, stable standards.

Intrapartum Fever: Antepartum and Intrapartum Contributions
Laura M. Goetzl, M.D., M.P.H., Baylor College of Medicine

Dr. Goetzl presented data showing that the rate of intrapartum fever rose from 0.6 percent to 11 percent after the introduction of epidural analgesia. There were 420,000 excess fever cases, and roughly 97 percent of fever in term nulliparas is associated with epidural analgesia. The study also found that those with intrapartum fever had higher IL-6 levels prior to labor. Pre-labor IL-6 levels had no association with gestational age, birthweight, or placental IL-6. Dr. Goetzl also displayed a table of multivariate risk factors for cerebral palsy at term.

Recommendations: Dr. Goetzl made the following recommendations for the Study:

• Consider term blood samples
• Use uniform maternal temperature ascertainment
• Record epidural analgesia as a continuous variable rather than dichotomous
• Consider assessment of progesterone levels.

Questions and Comments:

• Dr. Klebanoff said the data are hard to pull from charts after the fact and asked about digitally recorded data. Dr. Goetzl agreed that it is difficult, but the anesthesia record provides information on epidural analgesia. Electronic medical records may not be compatible.
• Dr. Andrews asked whether the presence of a catheter or use of injection might be a factor. Dr. Goetzl said that doses of anesthetic did not have much effect. Dr Andrews commented that in a subgroup of women who also get a tubal ligation, the catheter is left in place, and usually it is required that there be no fever before going to the operating room. Dr. Goetzl said that it seems there is some interaction with the labor process, perhaps via the placenta.
• In response to a question from Dr. Rodriguez, Dr. Goetzl said that rates of fever were similar to other studies with different intervals of temperature ascertainment.

The Genetics of Autism
Jennifer Stone, B.S., University of California, Los Angeles

Ms. Stone presented a brief overview of autism and possible causes. She discussed evidence for a genetic component from twin studies and other research. Idiopathic autism is complex and genetically heterogeneous; this confounds and obscures genetic analysis. There is a profound gender bias in autism-it is at least four times more common in males.

Her research hypothesis was that the genetic predisposition in multiplex families containing an affected female is different from genetic predisposition in multiplex families only containing affected males. The study design was a sample from 257 families with 2 or more affected children, drawn from the Autism Genetic Resource Exchange (AGRE) sample. Study families were divided into those with only boys and those that contained girls.

Ms. Stone described the gender environment:

• It is well established that male and female brains develop, are structured, and function differently.
• It is also well established that males and females exhibit differences in immune response and response to stress.
• There is a need to start including and acknowledging gender differences in study designs and analyses.

Recommendations: Ms. Stone provided a list of things for the Study to think about:

• High density genetic markers on each sample, such as a 100K Affy SNP chip or other new technology
• Inclusion of family members (father and siblings)
• Collecting as much clinical data as possible on as many people (family members) as possible (it is not known which data will prove to be important 30 years from now)
• Expression arrays-use placental tissue and all parts of fetus tissue from miscarriages
• The need for large and efficient data storage and computing capability to handle the vast amounts of genetic and expression data
• The need for trained statisticians and bioinformaticists to analyze the data
• Web-based data access
• A user friendly and regularly and correctly updated database (essential).

Questions and Comments:

• Dr. Yolken commented that good family histories are hard to get, and there are ethical issues. Dr. Croen said that in California, they were having success in obtaining biological specimens from family members, but obtaining them prospectively would be much more difficult.
• A participant asked whether there was a need to go further back in the mother’s pedigree. Ms. Stone said she did not think that would be necessary; a case-control transmission test should be sufficient.
• Dr. Schoendorf commented that the genetic component of planning for the Study was relatively underdeveloped, but the Study is clearly well positioned to look at genes. There is disagreement as to whether the Study can do gene discovery. Dr. Yolken said that the main thing is to get the samples and family data that will be needed.
• Dr. Schendel commented about having satellite studies so that not all data would have to be collected on every Study participant, thus reducing subject burden. Dr. Yolken said that the Study would have certain "core" measures and that sites could apply to do other measures.
• Dr. Pearce commented that ownership of the data was a legal quagmire, for example, related to fathers. There was discussion about obtaining consent from both parents at enrollment. In some areas, institutional review boards require consent from both parents for studies on the fetus.

CPP Trial: Data Collection and Analysis
John Lawlor, Johns Hopkins University Bloomberg School of Public Health

Mr. Lawlor did not use a slide presentation but provided a handout. He urged the Study planners not to repeat mistakes made in the CPP trial. The CPP data were not user friendly and so were not used to the fullest extent possible. The Study will produce ten times as much data as the CPP did, and it is important that Study data be user friendly for maximize use.

Mr. Lawlor offered considerations for the Study related to:

• Parsimony in data versions: distribute rectangular or relational databases?
• Redundancy in description and documentation: learning and long-term benefits
• Unit: family/house/mother? Pregnancy? Product(s)? Live or dead-what is a child?
• Data manipulation and statistics more complex (GEE/mixed models for full use)
• Ongoing study: revisions of datasets/documentation, "devil in the details," and staff changes
• More resources necessary as a proportion of the total for documentation
• Audience-those supported who can easily use the products?
• Philosophy and resources of support and distribution remain same; technology changes.

Recommendations: Mr. Lawlor made the following recommendations for the Study:

• Anticipate and prevent problems now related to publications and data distribution.
• Data access and deliverables must be clearly addressed in the design phase.
• The Study should consider who gives what to whom by what means/in what form and when.
• Flexibility will be needed to meet changing needs and technology.
• A mechanism/body to resolve disputes should be established before the Study begins.
• Practice distribution on the CPP datasets inexpensively via the Internet.
• Analyze CPP as a valid contribution to science and a planning tool for the Study.
• Digitize as much as possible (CPP booklets).

Collection and Storage of Specimens
Mark Cosentino, D.P.M., Ph.D., Science Applications International Corporation

Dr. Cosentino used overheads rather than a slide presentation.

Recommendations: Dr. Cosentino began with recommendations for the Study:

• Work backwards-start from the test.
• Do not collect everything-focus.
• Choose/design a flexible database able to handle all types of data collected.
• Place sentinel samples in the collection for "real time" monitoring of labile analytes.
• Cull the specimens.
• Blend technology with testing.

Regarding working backwards, Dr. Cosentino provided the following details:

• Identify the test.
• Determine the needed sample for the test.
• Determine aliquot size and storage conditions.
• Determine the quantity of specimen to collect and store.
• Select products and procedures for high throughput processing. Automate if possible.
• Compile all tests to be done and determine if specimen sharing can occur.
• Link specimen types and quantity to storage temperature and disaster prevention.
• Factor in the number of collection visits and number of individuals donating.
• Storage should be stratified-active versus archived specimens. Archived specimens should be protected and should not leave the repository collection.

Dr. Cosentino said that label design is a big issue, and labels should contain two pieces of information only-a randomized number that cannot be linked to identification information and a 2-D barcode.

Specimen stability and storage issues include:

• Saliva-dried or frozen
• PBMCs-cryopreserved or pellet
• Whole blood-cryoprotectant added or naked
• Buffy coat-cryoprotectant added or naked.

Repository database criteria include:

• Upgradeable/expandable language
• Historical inventory
• Linkable/interfaceable-results, clinical data, and shipping
• Real-time
• Web-based yet secure
• User friendly.

Summary and Recommendations

Dr. Rodriguez reviewed the key questions for data collection in the Study related to infection and inflammation, including:

• What to collect? Specific tests/analyses/data
• Why is the information necessary?
• Who to collect from?
• When to collect? Timing and number of specimens
• How to collect? Special issues for specific tests.

He mentioned the lessons learned from the CPP as discussed by Dr. Hardy, including the need for a publications plan. He briefly discussed his experience with a study that followed families for 5 years and fed back information to the families about disease. It was most difficult to obtain samples from the fathers, and 20 percent of the families disappeared during the study. He summarized other thoughts from the workshop, including the need for standardization of testing and the use of animal models to help focus research.

Dr. Croen commented that it is difficult to anticipate what could and should be reported back to families. Dr. Schoendorf said that in the National Health and Examination Survey, more disclosure was thought best, but sometimes there may be no interpretation of results given. Dr. Rodriguez said that when no explanation is provided, the individuals must work with their doctors to understand results. Dr. Swedo said that in her study, they are not allowed to share results from uncertified labs. Consent forms must spell this out.

Dr. Schoendorf discussed some general issues that the Study must address. Regarding publications and data ownership, the Study has no specific policies in place yet and the Study design and the sites are also not defined yet. In his opinion, all data should be publicly available in an accessible format as quickly as possible. There should be a limited time for the principal investigators to have exclusive use of the data (although this obviously does not apply to any ancillary studies).

Another theme addressed by Dr. Schoendorf was methods development studies over the course of the Study, which has been recognized as important. Continued input is needed. He said that the intent of the workshop had been to come up with recommendations related to the issues listed by Dr. Rodriguez. He said that Dr. Yolken would give the workshop participants a charge-homework-to consider answers to these questions and come back with recommendations.

Dr. Schoendorf described a potential model for deciding who to collect from (the entire Study population or a selected subset) versus when the data analysis would be done (at the time of collection or later). Some considerations for prioritization of testing include:

• Participant burden or invasiveness
• Logistics of collection, preparation, and storage
• Economic cost
• Alternate samples (instead of blood)
  • Questionnaires
  • Urine
  • Saliva
  • Cervical secretions
  • Fetal cells in maternal circulation
  • Other
• How important the test and data are for the hypothesis.

Dr. Dombrowski said this was consistent with what he was thinking. He will provide some more detailed recommendations concerning collection and storage of data on fever.

Dr. Andrews raised the issue of the infrastructure of the Study and said that decisions about sample collection should be done in the context of the Study infrastructure. Dr. Schoendorf responded that while the planning of the Study has been as open and inclusive as possible, it has been difficult. The structure seems to be moving toward a contract-driven study with a centralized data/coordinating center and multiple sites across the United States. It is likely that academic medical centers will be involved. There might be a core protocol designed by the Study staff, and then when the sites are chosen, the protocol can be refined and ancillary studies added.

Dr. Andrews said that it would be difficult to add detail to recommendations without knowing what types of centers would be involved and what their capabilities were. Dr. Yolken suggested reading Dr. Hardy’s article and said that the group could decide what the most important scientific issues were and make recommendations about what the Study should collect without knowing the Study structure. Dr. Schoendorf agreed and asked that participants begin with the research questions to make their recommendations.

Dr. Schendel suggested that a template be provided for workshop participants to fill in with specific recommendations. Dr. Yolken agreed and said that a template would be provided for that purpose.

Dr. Swedo said that it would help to know the framework (for example, if there are going to be annual exams). Dr. Yolken said that participants could suggest things that are not practical to do now. Dr. Rodriguez commented that there are other Working Groups whose input will be coordinated with the input from this workshop.

Dr. Yolken asked presenters to let him know if they prefer that their slide presentations not be included in the workshop report. He said that he would e-mail a template to participants and anticipates a 100 percent response. He thanked everyone again for participating in the workshop and adjourned the meeting.

Participants

John Alexander, M.D., Center for Drug Evaluation and Research, U.S. Food and Drug Administration
William W. Andrews, M.D., Ph.D., University of Alabama, Birmingham
Mark Cosentino, D.P.M., Ph.D., Science Applications International Corporation
Lisa Croen, Ph.D., Kaiser Permanente
Stefan C. Dombrowski, Ph.D., M.B.A., Rider University
John H. Gilmore, M.D., University of North Carolina, Chapel Hill
Laura M. Goetzl, M.D., M.P.H., Baylor College of Medicine
Judith Grether, Ph.D., California Department of Health Services
Janet Hardy, M.D., C.M., Johns Hopkins University
Mark A. Klebanoff, M.D., M.P.H., NICHD, NIH, DHHS
Thea Kuddo, M.D., NICHD, NIH, DHHS
John Lawlor, Johns Hopkins University
Glenn B. Mannheim, M.D., Center for Drug Evaluation and Research, U.S. Food and Drug Administration
Phillip G. Nelson, M.D., Ph.D., NICHD, NIH, DHHS
Craig J. Newschaffer, Ph.D., Johns Hopkins University
Neha Patel, M.P.H., RTI International
Brad Pearce, Ph.D., Emory University
William J. Rodriguez, M.D., Ph.D., Center for Drug Evaluation and Research, U.S. Food and Drug Administration
Carolyn Salafia, M.D., M.S., Columbia University
Diana E. Schendel, Ph.D., National Center on Birth Defects and Developmental Disabilities, CDC, DHHS
Kenneth C. Schoendorf, M.D., M.P.H., National Center for Health Statistics, CDC, DHHS
Eun Young Song, NICHD, NIH, DHHS
Jennifer Stone, B.S., University of California, Los Angeles
Susan E. Swedo, M.D., National Institute of Mental Health, NIH, DHHS
Tracey W. Thomas, Ph.D., National Center for Environmental Assessment, EPA
E. Fuller Torrey, M.D., Stanley Medical Research Institute
Robert H. Yolken, M.D., Johns Hopkins University Medical Institutions

Presenter slides are available upon request from Kenneth C. Schoendorf, M.D., M.P.H., National Center for Health Statistics, CDC, DHHS.