Microchimerism in women with recurrent miscarriage

Abstract

Miscarriage is the most common pregnancy complication, and recurrent miscarriage (3 or more consecutive pregnancy losses) affects 1–5% of couples. Maternal-fetal exchange and the persistence of exchanged material as microchimerism appears to be disrupted in complicated pregnancies. We recently conducted a longitudinal cohort study of microchimerism in women with recurrent miscarriage. Our initial data raise multiple questions that require further investigation. Here, we review our data from this recent study and provide additional information regarding microchimerism in the granulocyte cell layer. This area of investigation offers a unique window into early reproductive events, and future related studies have the potential to identify novel therapeutic approaches and insights into human evolution.

Miscarriage occurs in as many as 50–70% of all human conceptions and in 15–20% of clinically recognized pregnancies.^1-4 Recurrent miscarriage (RM), defined as 3 or more consecutive pregnancy losses, affects approximately 1–5% of couples.⁵ Despite advancements in understanding underlying causes of RM, etiology remains undetermined in about 50% of cases.^5,6 Dysfunctional immune interactions may underlie a proportion of RM,^7,8 as evidenced by an increased risk of RM in women with some autoimmune diseases,⁹ as well as by differential risk according to maternal-paternal immunogenetic relationships.^10-12

In normal pregnancy, one of the ways in which maternal-fetal interaction occurs is through the exchange of cells. Maternal-fetal exchange results in microchimerism, defined as small amounts of foreign cells or DNA detectable in a genetically distinct individual. Microchimerism originally acquired in pregnancy can durably persist for decades, is associated with later-life health benefits and risks, and can be considered in a multigenerational context.¹³ An adult woman (or “proband”) acquired microchimerism from her own mother (“mother-of-the-proband,” or MP) when she herself was a fetus. This “graft,” acquired during fetal immune system development, contributes to aspects of immune tolerance,¹⁴ can remain in her system into adulthood, and represents a preexisting inhabitant as she experiences pregnancy herself.¹⁵ During subsequent pregnancies, new fetal sources of microchimerism are acquired and may interact with the previously acquired MP microchimerism.¹⁶

To explore microchimerism in the setting of RM, we recently conducted a prospective cohort study to evaluate MP microchimerism in women with unexplained RM, both prior to conception and longitudinally across pregnancy.¹⁷ We also explored whether women with RM who become pregnant have fetal microchimerism during pregnancy and whether there is any correlation of microchimerism with subsequent pregnancy outcome.

We studied women with idiopathic primary RM before conception and longitudinally throughout the subsequent pregnancy(ies). Subjects had 3 or more documented consecutive miscarriages of less than 20 weeks gestation, unexplained after comprehensive clinical evaluation,^18,19 with the same reproductive partner. An informative euploid karyotype result (46,XX or 46,XY) in at least one miscarriage was required, with no prior pregnancy reaching 20 weeks gestation. Women were excluded if they were a twin, had received a prior blood transfusion, had a history of autoimmune disease, prior elective pregnancy termination and inability or refusal to give written informed consent. MP microchimerism prior to conception in RM subjects was compared to a group of controls derived from a population of healthy women without reproductive complications.

Peripheral blood samples drawn prior to conception from RM subjects and controls were studied, and RM subjects with a subsequent pregnancy were also followed longitudinally throughout the subsequent pregnancy(ies). Samples from family members were collected for genotyping to identify non-shared polymorphisms in HLA or other genes,²⁰ allowing customized application of a specific quantitative PCR assay to identify and measure microchimerism from the MP or fetus.²¹ From each sample, Ficoll-purified peripheral blood mononuclear cells (PBMC) were collected, DNA was extracted, and microchimerism was measured by quantitative PCR and reported as the number of genome equivalents (gEq) of microchimerism per 100,000 total gEq. For some samples, the erythrocyte/granulocyte cell layer was collected²² and the erythrocytes lysed for subsequent isolation of DNA from the granulocyte layer (GL) and microchimerism testing.

Overall, 107 PBMC samples from 23 RM subjects were studied for microchimerism. Maternal age and race were similar among women with RM and controls. As expected, women with RM had higher gravidity (median [IQI]: 4 [3–5] vs. 1 [0–2]; p < 0.0001 by Wilcoxon rank sum) and lower parity (median [IQI]: 0 [0–0] vs. 1 [0–2]; p < 0.0001 by Wilcoxon rank sum) than the uncomplicated controls. Of those with available preconception samples, MP microchimerism in PBMC was detected in 1/16 (6.3%) of RM probands and 6/31 (19.4%) of control probands; OR for detection after adjustment for total number of cell equivalents tested 0.30 (95% CI 0.03–3.02).

Of the 23 total RM probands, 14 were followed in one (n = 13 ) or two (n = 1) subsequent pregnancies. Of the 15 pregnancies, 11 resulted in a birth and 4 in another miscarriage. The preconception and longitudinal results of microchimerism testing in PBMC in RM subjects, as reported in the original study, are provided in Figure 1, along with limited results of microchimerism testing in GL in some subjects. Of potential interest, 7 GL samples from 2 probands with RM who went on to have a subsequent miscarriage were tested for MP microchimerism, and 6 (86%) were found to be positive. In addition, while direct comparison of microchimerism concentrations in PBMC versus GL may be confounded by the possibility that some cell-free DNA could be included in the GL, somewhat higher concentrations were observed within the GL than typically seen in PBMC. Among those samples with detectable microchimerism, the former ranged from 1 to 142 gEq (median 33 gEq), and the latter 1 to 52 gEq (median 1gEq) of microchimerism per 100,000 total gEq.

Figure 1.

Microchimerism detection in PBMC and GL in preconception and longitudinal samples (during and after pregnancy) according to pregnancy outcome. Results are summarized for all samples from different timepoints with the number of individual probands indicated in brackets (probands often contributed more than one blood sample from different timepoints). While our primary focus was MP microchimerism, limited results for fetal microchimerism are also included; because fetal tissue was rarely obtained after miscarriage, only 2 pregnancies from one proband were tested in this category.

Overall, our exploratory study of MP microchimerism in RM demonstrates microchimerism detection in a proband's PBMC and GL. Though our sample size precludes drawing conclusions, results point to a number of interesting questions, including: Are there particular cell populations in which MP microchimerism is concentrated? Is MP microchimerism in PBMC diminished prior to conception in women with RM compared with control women? Is MP microchimerism in GL more commonly detected in women with RM who go on to have another miscarriage? If observations regarding MP microchimerism prove to be true, one hypothesis might be that MP microchimerism in specific adaptive immune cell subsets can facilitate implantation, while a shift toward its presence in innate cell types could interfere with implantation.

As our understanding of the implications of maternal-fetal exchange during pregnancy improves, one area of interest is the role of microchimerism in facilitating normal obstetric outcomes. From both immunologic and evolutionary perspectives, the impact of previously acquired grafts on newly acquired cell populations is of particular interest. During fetal life, maternal cells are acquired and influence the development of fetal regulatory T cells, resulting in durable tolerable to non-inherited maternal antigens.^14,23 In normal pregnancy, cells that a woman acquired in fetal life from her own mother (MP microchimerism) are detectable in circulation, particularly during the third trimester, and are absent in preeclampsia, a pregnancy disorder thought by some to reflect maternal immune dysfunction.¹⁵ Our previously reported data suggest that in the PBMC layer, prior to conception MP microchimerism may also be diminished in women with RM compared with controls.¹⁷ On the other hand, MP microchimerism in the GL may be more frequently detectable in RM, especially in those experiencing recurrent miscarriage. It is possible that MP microchimerism plays an active role in, or reflects, the capacity for maternal adaptation to pregnancy. Further investigation is needed to delineate these relationships.

From an evolutionary perspective, competition between microchimeric “grafts” in the mother informs hypothesis generation in regard to fetal microchimerism.^24,25 Fetal cells acquired by a woman during a prior pregnancy may impact her subsequent pregnancies, which may have particular relevance for women with RM. Future studies are needed to explore the potential for “graft” vs “graft” interactions among the different sources of microchimerism that can be harbored by women and the impact of these interactions on longterm health.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

The authors acknowledge funding from the National Institutes of Health: AI072547, HD01264, and HD067221.