To study specific micronutrients, the most direct approach is to use archived prenatal biological specimens from birth cohorts followed up for schizophrenia. The first large birth cohort studies to collect and archive biological specimens from pregnant women were the Child Health and Development Study (CHDS) and the National Collaborative Perinatal Project.60–62
These were contemporaneous studies initiated in the 1950s in the United States. Decades later, the archived sera from both these cohorts are being used to study prenatal nutrients and offspring risk of schizophrenia. The strategy is that of a nested case-control design, ie, the cases of schizophrenia in the cohort are ascertained and are compared with a control group selected from the same cohort.2
Thus, the serologic study can be efficiently completed using a few hundred subjects rather than the many thousands enrolled in the original cohort.
Although these 2 cohorts were the first to archive prenatal specimens, many other cohorts established in later years have done so. We anticipate, therefore, that this strategy will be widely applied because these other cohorts pass through the age of risk for schizophrenia.63,64
A prototype for this approach is the Prenatal Determinants of Schizophrenia (PDS) study60
(for a detailed description see PDS design article). The cohort members in the PDS study were derived from the CHDS.65
During 1959–1966, the CHDS recruited virtually all pregnant women receiving obstetric care from the Kaiser Permanente Medical Care Plan, Northern California Region (KPMCP) in Alameda County, CA. Their liveborn offspring (N
044) were automatically enrolled in KPMCP. Comprehensive data were prospectively collected from maternal medical records, maternal interviews, and other sources. Approximately 30% of the population of the county received their health care by KPMCP. KPMCP membership was diverse; racially, educationally, and occupationally, it was similar to the employed population of the Bay Area of California at the time, although there was underrepresentation of the extremes of income.66
The at-risk cohort comprised the 12
094 live births who were members of KPMCP between January 1, 1981 (the year in which computerized registries became available), and December 31, 1997.60
Following exclusion of subjects who did not receive a maternal interview including important demographic and lifestyle factors, and random selection of one subject per family in order to eliminate nonindependent observations, the final cohort consisted of 7796 subjects.
Potential cases of schizophrenia and other SSDs were identified by a screening procedure involving computerized record linkages between CHDS and KPMCP identifiers from inpatient and outpatient registries based on diagnoses of International Classification of Diseases, Ninth Revision,
295–299, and by a pharmacy registry, based on prescriptions for antipsychotics. Psychiatric and medical records were then reviewed for evidence of psychotic symptoms by an experienced, board-certified research psychiatrist. Subjects who screened in for psychotic illnesses were administered the Diagnostic Interview for Genetic Studies (DIGS),67
and Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition,
diagnoses were assigned by consensus of 3 experienced research psychiatrists. Potential cases not interviewed were diagnosed by chart review by experienced clinicians, and all chart diagnoses were confirmed by a research psychiatrist. This protocol resulted in 71 total SSD cases, 44 of whom received the DIGS and 27 of whom were diagnosed by chart review.
In the CHDS, a wealth of data were collected on prenatal, perinatal, and neonatal exposures, and a subsample were followed up through childhood. A maternal interview included a detailed reproductive history; health-related behaviors in mother and father; lifestyle habits, including smoking, alcohol, and weight before and during pregnancy; and detailed sociodemographic information. Detailed data on medical conditions and prescribed medications were abstracted from obstetric and medical records for all enrolled mothers. Extensive data on labor and delivery records were abstracted. Additional data included blood groups and placental morphology.
As noted earlier, a remarkable feature of the study that has made it possible to test hypotheses regarding specific prenatal micronutrient deficiencies was the availability of archived maternal sera, which were drawn during pregnancy, frozen, and archived for the past 40 years. Prenatal sera are available in virtually all pregnancies in the cohort for later gestation and for about 40% during the first trimester. The PDS study is well suited for both cohort and nested case-control designs. The latter design is particularly appropriate for serologic analyses, given the prohibitive costs and logistics involved with analyses of prenatal sera on a large cohort. In the nested case-control design, the controls for each case were selected to represent the population at risk at the time the case was ascertained. Matching criteria for controls included membership in KPMCP, date of birth, sex, number of maternal serum samples drawn during the index pregnancy, and timing of the first maternal blood draw during the index pregnancy. Matching for time of KPMCP membership ensured that controls represented the population at risk at the time of case ascertainment. Matching on birth date ensured control of potential confounding by season of birth. Gender was matched to allow for assessment of different effects for men and women. Matching on maternal blood draws was conducted to permit sufficient and comparable data for serologic analyses.
Serologic analyses are presently underway to examine the micronutrients described earlier. Both the potential and the limitations of this approach are illustrated by a recent study from our group. hcy levels are a reliable marker of serum folate, which is not measurable in archived sera. hcy has long been known to increase folate deficiency states secondary to the critical role of folate in the methylation of hcy and its conversion to methionine.
Using the archived sera of the PDS cohort, we found that elevated hcy in the first trimester was associated with a nearly 2-fold increase in risk of schizophrenia. We had only a small number of subjects with available first trimester sera, however, and the finding fell well short of statistical significance. Moreover, in these subjects, the sera were generally drawn during the latter period of the first trimester, approximately 2 months or longer after the periconceptional period. Thus, the first trimester results were inconclusive, though suggested that further studies with a larger number of cases with first trimester samples might substantiate the folate deficiency hypothesis.
The sample size did offer sufficient power to test the hypothesis that elevated third trimester hcy was associated with risk of schizophrenia68
because maternal archived serum specimens drawn during late pregnancy were available for virtually all cohort members. We found that subjects with elevated third trimester hcy, defined as the highest tertile of the distribution, was associated with a significant, greater than 2-fold increased risk of schizophrenia in the offspring; the findings persisted following adjustment for several potential confounders including maternal education, race, smoking, and age.
The result for third trimester hcy does not necessarily support an effect of folate deficiency in early gestation. At physiologic glycine concentrations, hcy has NMDA receptor antagonist properties, and dysfunction of the NMDA receptor has been implicated in schizophrenia.69–71
Moreover, perinatal administration of phencyclidine, a known NMDA receptor antagonist, induces a disruption in synaptogenesis, prepulse inhibition, and working memory.72
hcy is also known to cause placental vasculopathy through several mechanisms,73–75
which might lead to fetal hypoxia, a putative risk factor for schizophrenia.52
We had therefore hypothesized that elevated third trimester hcy would be associated with risk of schizophrenia, based on the late gestational effects of NMDA receptor blockade on endophenotypes of schizophrenia and on the increase in placental blood flow during the third trimester. Because this is the first time that elevated prenatal hcy has been associated with schizophrenia, this finding requires replication in an independent sample.