Rheumatoid arthritis (RA) usually improves or even remits during pregnancy, while a disease flare regularly occurs within 3–4 months postpartum (1, 2). Arthritis improvement during pregnancy with active disease postpartum similarly has been reported in patients with juvenile idiopathic arthritis (JIA) (3, 4). Yet, little is known about the mechanisms involved in this clinically important phenomenon. In 2 previous studies, fetal–maternal HLA class II disparity was found to correlate with pregnancy-induced improvement in RA (5, 6). As a result of advances in techniques for prenatal diagnosis, it has recently been established that fetal cells and cell-free DNA routinely traffic into the maternal circulation during normal pregnancy (7, 8). Circulating fetal DNA is found in maternal serum/plasma in the first trimester of normal gestation, and the concentration of fetal DNA increases during the course of pregnancy (8). Rapid removal of fetal DNA occurs following parturition (9). Thus, recent studies of normal pregnancy, when considered along with previous observations of improvement in RA, led us to ask whether changes in serum levels of fetal DNA correlate with alterations in arthritis activity during and after pregnancy. Fetal DNA in the serum of pregnant RA patients was measured by real-time quantitative polymerase chain reaction (PCR), targeting unshared, paternally transmitted HLA sequences, a Y chromosome–specific sequence (DYS14), or an insertion sequence within the glutathione S-transferase M1 (GSTM1) gene.
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- PATIENTS AND METHODS
Improvement or remission of arthritis occurred during pregnancy in 15 patients with adult-onset RA and in all 6 patients with JIA. These patients were assigned to group A. Among these women, 62% had experienced at least some improvement by the end of the first trimester. Once improvement took place, it was sustained or increased further during the course of gestation. Disease recurrence was observed in 90% of patients in group A by the third to fourth month after delivery. Four RA patients with active disease throughout pregnancy, including 1 who had disease onset within the first trimester, were assigned to group B, and the disease in these patients remained active postpartum. Consistent with previous reports (2), most women who had experienced improvement during previous pregnancies improved during the index study pregnancies, with 1 exception. Levels of fetal DNA were evaluated in each patient over the course of the study pregnancy and postpartum (i.e., each patient as her own reference), with an additional evaluation that compared all of group A with all of group B.
Levels of circulating fetal DNA in serum increased with advancing gestation (Figure 1), reaching a median of 24 gE/ml (range 0–334) in the first trimester, 61 gE/ml (range 0–689) in the second trimester, and 199 gE/ml (range 0–2,576) in the third trimester. Clearance of serum fetal DNA was observed in the postpartum period (median 0; range 0–166). Serum fetal DNA levels were significantly higher during the third trimester in group A (median 270 gE/ml; range 12–2,576) as compared with group B (median 78 gE/ml; range 0–224) (P < 0.001), with fetal DNA detectable in 100% of study subjects from group A and 50% of subjects from group B. No qualitatively or quantitatively significant differences in serum fetal DNA were identified between the 2 groups within 3 months after delivery.
Figure 1. Changes in serum fetal DNA levels during and after pregnancy in patients with arthritis. Each line represents 1 patient.
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Overall, there was an inverse relationship between the serum fetal DNA level and arthritis disease activity. As fetal DNA quantities doubled, the likelihood of arthritis improvement increased 1.2-fold (95% confidence interval 1.1–1.3; P < 0.001). There was an increased likelihood of improvement with increasing levels of serum fetal DNA (without suggestion of a specific threshold). Results did not differ between patients with adult-onset RA and those with juvenile-onset arthritis (nor for the 2 adult patients who had definite RA according to the 1958 criteria but not the 1987 revised criteria). Results were not substantially altered by adjustment for the potential confounders, as described in Patients and Methods.
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- PATIENTS AND METHODS
Fetal DNA has not previously been studied in women with RA during pregnancy. In the current study, we found a significant inverse correlation between arthritis activity and the serum fetal DNA concentration over the course of pregnancy and postpartum. Arthritis improved in 79% of patients with adult-onset disease and in all women with JIA, over a time course similar to the time course of rising serum fetal DNA levels, which increased as pregnancy progressed and dropped to undetectable or very low levels after delivery, coincident with arthritis recurrence. In the third trimester, serum levels of fetal DNA were significantly higher in patients with arthritis improvement as compared with those with active disease.
A number of hypotheses have been proposed to explain arthritis improvement during pregnancy, including increased pregnancy-associated levels of serum α2-glycoprotein (16), elevated levels of sex hormones (e.g., estrogen, progesterone) (17), and a shift in cytokine production from a Th1 to a Th2 profile (18). In 2 prior studies, fetal–maternal HLA class II disparity was associated with the pregnancy-induced amelioration of RA (5, 6). No association was described in another study (19). However, when identifying the pregnancy-induced amelioration of RA, it is important to compare disease during pregnancy with that prior to pregnancy, whereas this study did not include the requirement that a woman have active arthritis in the 6 months prior to conception. (The use of “no change” as a disease activity category can result in identical classification of a patient whose disease was in remission prior to pregnancy and remained in remission and a patient who had active disease and continued to have active disease during pregnancy.)
Recent findings in studies of normal pregnancy, when considered along with the results of the current study and previous studies of RA, led us to a hypothesis for the mechanism by which pregnancy induces improvement in RA. Whether serum fetal DNA has any direct biologic effects is unknown, and although a direct effect remains possible given reports of horizontal transfer of DNA in other conditions (20), in our hypothesis, fetal DNA measured in serum is an indicator of other processes, as described further. Villous trophoblast apoptosis (a normal physiologic phenomenon during placental growth) results in extrusion of a large quantity of apoptotic fetal trophoblasts into the maternal circulation, which is thought to be the most important source of serum fetal DNA (7). Although trophoblasts do not express HLA class II antigens, HLA–DR protein has recently been identified within the cytoplasm (21). Thus, increasing levels of serum cell-free fetal DNA may reflect a progressive release of fetal HLA class II alloantigens from cell lysis as pregnancy progresses. In addition, not all fetal DNA in serum is cell-free, since some derives from fetal cells in the process of apoptosis (14, 22).
The uptake and cross-presentation of soluble fetal paternally transmitted HLA peptides and/or peptides packaged within apoptotic fetal cells (23) by maternal antigen-presenting cells, such as dendritic cells, is likely to have immunologic effects that may reasonably be anticipated to also affect autoimmunity in RA patients, for example by induction of regulatory T cells or by altering the maternal peripheral T cell repertoire. After delivery, the maternal T cell repertoire would be expected to return to the prepregnancy “baseline” due to lack of a source of apoptotic fetal cells, coincident with the relapse of arthritis. In a recent experimental study, persistence of allogeneic donor microchimeric cells was found to be essential for maintenance of the unresponsiveness of host T cells by clonal deletion of the donor-specific T cell repertoire (24). Therefore, whether intact fetal cells could modulate RA disease activity during pregnancy is of additional interest.
The current study has a number of limitations. First, most women develop RA in their postreproductive years, (25), and as in prior prospective studies of RA and pregnancy, the number of study subjects is modest. Second, some study subjects took medications during gestation, and whether the medications influence levels of serum fetal DNA is unknown. To the extent that we could examine this possibility, prednisone did not appear to have an effect on serum fetal DNA levels (4 patients in group A took prednisone and 2 in group B took it during the third trimester of their pregnancies). Third, most of our quantitative assays are based on HLA differences, so we were not able to evaluate the additional question of whether fetal–maternal HLA class II compatibility influences levels of fetal DNA in the mother. Finally, our study subjects were identified when they were already pregnant and prepregnancy serum samples were not available, thus, the results could possibly be confounded by persisting fetal DNA from a previous pregnancy. However, serum fetal DNA is rapidly cleared after pregnancy (9), and a previous study described persistent fetal cells among PBMCs but not in serum (14). We did not observe any difference in the results according to the number of prior pregnancies.
In summary, the serum fetal DNA concentration increased throughout gestation and was effectively cleared after delivery, with an inverse correlation observed between changes in fetal DNA levels and arthritis activity. Whether the dynamic changes in fetal DNA reflect the potential for immune modulation of maternal arthritis, are a result of disease activity changes, or are not causally related cannot be determined from these studies. If the changes reflect immune modulation, further studies could generate new therapeutic strategies for RA.