Embryos undergoing rapid cell division after conception require adequate levels of folate to develop properly, and folate insufficiency has been associated with a variety of reproductive problems including NTD and CL&P (Kirke et al., 1993
; O'Neill, 1998
; George et al., 2002
; Wilcox et al., 2007
). In preliminary studies, antibodies to the folate receptor have been associated with both of these birth defects (Rothenberg et al., 2004
; Bliek et al., 2006
; Cabrera et al., 2008
). In our study, mothers with plasma that inhibited folic acid from binding to FRα had an increased risk of NTDs in their offspring. Despite the very small number of NTD cases, this association was significant before and after adjusting for assay variation. We saw no similar increase in risk for oral facial clefts.
Previous studies of maternal autoantibodies to folate receptors have found large increases in risks of NTDs [odds ratio (OR) 27, 95% CI 3.8–190] and CL/P (OR 11, 95% CI 1.4–81) despite very small sample sizes (Rothenberg et al., 2004
; Bliek et al., 2006
). Table includes a summary of all previous studies of folate receptor antibodies and birth defects, which used blood samples collected during pregnancy as well as at varying lengths of time after pregnancy. The assays performed in these studies also varied in the source of folate receptor (bovine, human placental or human recombinant) and used different assays for antibodies that bind to the receptor or inhibit the binding of folic acid to the receptor.
Previous studies of folate receptor antibodies and birth defects.
We assessed several maternal factors for their associations with inhibited folic acid binding to FRα. Smoking and folic acid supplementation are both associated with risk of CL/P and consistent with a previous study in Norway (Wilcox et al., 2007
); however, these factors were not associated with inhibited folic acid binding to FRα. In fact the strongest correlation with maternal age was in the opposite direction from a previous non-significant finding of older age in mothers with antibodies (Bliek et al., 2006
). While plates of samples processed concurrently had similar distribution of inhibition levels, the large differences between the two groups of plates increased the overall variation. This may have decreased our power to detect modest associations with biologically plausible environmental exposures.
The assays in this study used recombinant human FRα rather than placental or bovine-derived folate receptor. Antibodies to both human placental FR and bovine folate-binding protein (FBP) have been reported to be more common in mothers of NTD cases (Cabrera et al., 2008
). No association with oral facial clefts was seen in the only prior study using the recombinant folate receptor (Bille et al., 2010
), and we feel that the recombinant source of folate receptor is more consistent than the human placental preparations previously used. High homology between human folate receptor and bovine FBP may lead to antibodies that do not bind specifically to the human form of the receptor, and could potentially lead to assay cross-reactivity with antibodies to bovine FBP from milk exposure. Carriers of FR-blocking autoantibodies have been reported to have higher levels of milk intake (Berrocal-Zaragoza et al., 2009b
) but we found no such correlation. Unfortunately there is no crystal structure for human FRα, and the actual epitope is unknown, which complicates the prediction of antibody response.
The majority of NTDs in Norway are detected at the 17-week ultrasound, which is the obstetric visit at which mothers enrolled in MoBa. Most pregnancies which are affected by an NTD in Norway are terminated and would not be included in the MBR, where we identified our cases. If a woman terminated or considered terminating the pregnancy she may be less likely to enroll in MoBa, which is designed to follow the children's development over many years: this presumably explains the much lower than expected number of NTD cases ascertained.
Selection bias may impact our results when considering who enrolled in MoBa and which case mothers had 17-week blood samples available. A study of the MoBa cohort found a self-selection bias for prevalence estimates of 23 exposures and outcomes, but the eight exposure-outcome associations studied were not biased (Nilsen et al., 2009
). Of the 85 cases of CL/P we identified in version 3 of the MoBa database, 72 had 17-week samples available (85%). Of the 38 cases of CPO, 27 had samples (71%); while only 11 of 20 NTD cases (55%) had available samples. Control samples were selected randomly from 17-week samples stored on the same plate as the case samples to match time and processing at the Biobank.
It is not known how levels of antibodies that bind to FRα might fluctuate before, during, and after pregnancy. A study of fertility found that levels of inhibited binding to the folate receptor varied (around the assay detection limit) over several measured cycles of attempted conception, with preconception samples from subfertile cases more likely than controls to have positive folate receptor antibody titers (Berrocal-Zaragoza et al., 2009a
Many previous studies have relied on maternal blood samples which were collected months or years after the delivery of the index pregnancy (Table ). We used blood samples collected at around 17 weeks gestation (~15 weeks post-conception)—a time-point closer to the critical window for closure of the neural tube (Week 4 post-conception) and the lip and palate (Weeks 5–10 post-conception). Our association with NTDs but not oral clefts is consistent with two previous studies that only used samples taken during pregnancy (Cabrera et al., 2008
; Bille et al., 2010
), but conflicts with two studies that only used samples taken after the pregnancy (Bliek et al., 2006
; Molloy et al., 2009
). However, these studies also used different assays in different laboratories (Table ).
The direction of causation in this study is not clear. We cannot determine if maternal inhibition of folic acid binding to FRα leads to an increased risk of having a child with an NTD, or if the pregnancy affected by an NTD causes the maternal serum to develop antibodies that inhibit folic acid binding to FRα (for example, by maternal exposure to fetal neuronal tissue). It would be necessary to collect maternal blood before pregnancy to clarify this and to determine the utility of measuring inhibited folic acid binding to folate receptor as a preconception clinical screening tool.
In conclusion, mothers of NTD cases in the Norwegian Mother and Child Cohort Study had more inhibition of folic acid binding to FRα in 17-week gestation plasma samples but no such association was seen for mothers of oral facial clefts. Parental demographics and prenatal exposures were not associated with inhibited folic acid binding to FRα. Given similar findings with respect to high maternal titers of blocking antibodies among women carrying NTD pregnancies in cohorts in both NY, USA and CA, USA, it appears that these blocking antibodies may play a role in the etiologic pathway of NTDs (Rothenberg et al., 2004
; Cabrera et al., 2008
). Larger sample sizes will be required to determine whether environmental factors, such as smoking, can contribute to the development of these folate receptor-blocking antibodies.