The association between fetal derived MC and later development of MS, as suggested by our findings, is consistent with other studies examining this relationship.13
Our results are also similar to those reported for scleroderma in which the presence of rare allogeneic cells has been documented.14
However, the levels of MC observed in our study exceed that of others,13
particularly in subjects that were reportedly nulliparous.15,16
We can only speculate that alternative methodologies and reporting strategies in part account for this difference. More specifically, this may be attributable to the higher levels of DNA analyzed in our study. Given the limited sample size, we are, however, reluctant to overstate this finding.
The data from our study are based on 1,500,000 inputs for each sample in which approximately 1–12 male allogeneic microchimeric cells were detected in a positive subject. This is distinctly different from the comparatively robust levels of MC encountered in transfusion-associated MC in which the allogeneic, minor population may occupy as much as 1–4% of circulating host leukocyte burden. Sustained high-level MC following transfusion has been repeatedly described in subjects transfused following severe traumatic injury.17–19
The clinical significance of a quantitative difference between transfusion-associated MC and fetal associated MC is still not known.
Our study also draws attention to the deficiencies inherent to MC evaluation using Y-chromosome based platforms. This followed the unexpected finding in which a similar proportion of MS+
females displayed male MC independent of a known history of male pregnancy. Eleven of 24 (~46%) of MS+ females without known pregnancy tested positive for MC. Although counterintuitive, similar unexpected findings have been reported in other studies,15,16
albeit at lower levels than that encountered in our study. This underscores that self-reported negative pregnancy history may not definitively exclude a history of pregnancy: up to 30% of pregnancies end in early fetus loss with up to 14% being occult and clinically unrecognized.20
It has further been suggested these early miscarriages may indeed convey greater risk to develop persistent fetal MC.21
Other sources of potential error were also addressed: spurious reporting and coding errors were primarily excluded upon audit of the results. PCR contamination, another necessary consideration, was deemed unlikely as both positive and negative controls were run in parallel with the assays. Furthermore, in the event that amplicon contamination occurs, it tends to be at a quantitatively higher level than that encountered in our study and is typically uniform across samples.22
One would not expect intermittent, exceedingly low-level contamination while still preserving the ability to discriminate between experimental groups such as MS and non-MS subjects as observed in the current study. Assay function and reliability is critical to MC analysis and our group has consequently studied different aspects of sample viability with concurrent PCR contamination in view of MC detection. The outlined results do not appear consistent with any known form of amplicon contamination.23
We have also conducted extensive technical validation of MC PCR assay performance, which includes both rigorous spiking studies, as well as direct sequencing to definitively verify the identity of reaction products.24,25
Although the unexpected findings were therefore postulated to be real, we employed a biological control in order to validate the results. Female neonatal cord bloods were used for this purpose, representing an ideal control having never been pregnant. Although still subject to trafficking of cells from the mother, maternal cells will elude capture by a Y-chromosome based assay. There does remain the rare possibility of an undetected or resorbed male twin contributing cells; this is, however, considered unlikely. There also remains the theoretical possibility of intergenerational chimerism whereby trafficking of cells from a prior pregnancy into the mother could lead to downstream exchange with the new fetus as reflected in the associated cord blood.26,27
The rationale for including both qualitative and quantitative results (see ) is to present a balanced interpretation of the data. Simply reporting as positive vs. negative neglects a grey area where subjects test positive, but are near a threshold for positivity. Although categorized as being chimeric, these cases are more likely attributable to non-specific amplification and background noise. This was evident in the CBUs: despite selection of these samples as the closest approximation to a biological control, results demonstrate there were still qualitative positives. However, the quantitative data (number of positive wells) derived in this study demonstrated a more plausible negative interpretation, i.e., results approached an absolute negative, both in the proportion of samples affected when compared with the nulliparous MS-siblings, and also the observed rate of positivity (). The latter was not significantly different from zero, and also significantly lower than corresponding rates in the two microchimerism groups. Findings in the current study also emphasize the inherent limitations of using sex chromosome probes in evaluation of MC; this has lead to utilization of alternative platforms using HLA-based and Non HLA-Insertion-deletion (Indel) panels to impart greater precision for this purpose.
Results from our analyses raise the question of whether MS confers a higher risk of fetal loss. The literature asserts the contrary: MS confers neither increased risk of fetal loss nor other pregnancy related complication.28–30
Pregnancy is also associated with clinical improvement while disease relapse is frequently evident in the post-partum period. The age of MS onset was earlier in subjects that reported never having been pregnant. It is possible, however, that a diagnosis of MS may have influenced a decision to pursue pregnancy, at least in this small group of individuals.
In summary, these pilot results, while bound by certain limitations, do suggest that low-level MC is associated with MS. Prospective study of a larger subject population, using greater input of genomic DNA with serial blood samplings of subjects is needed. In addition, confirmation of MC by alternative assays is important. Of note the HLA-DR and InDel assays have already achieved remarkable results in the setting of transfusion associated MC.9,25
Through targeting selective, informative alleles expressed on a panel of somatic chromosomes, these assays both bypass the gender restriction of the Y-chromosome based assays as well as avoid the associated problems of a sex-chromosome based probe as illustrated by this study.9,25
Finally, it is important to note that blood may not be the ideal target tissue in which to evaluate MC; rather it is a tissue of convenience for both the present study as well as other studies seeking to gauge tissue MC. In view of these pilot findings, spinal fluid and affected neural tissue (brain) may provide a more representative sample for examination of MC. If MC is common in MS and involves target tissues specific to the disease, these findings could unravel new conceptual models for future investigation of MS.