Discussion
Main Findings In this study, we found that higher Tfh1/Tfh2
ratio before pregnancy was seen in females with early AAM, and this
increased Tfh1/Tfh2 ratio was significant with higher TSH level at early
pregnancy. Moreover, the aberrant Tfh1/Tfh2 ratio was also related to a
higher risk and a shorter time to spontaneous miscarriage. Lastly, The
contribution of early AAM on hallmarks of Tfh cells was further
validated by a two sample MR test.
Interpretation Tfh cells are differentiated from Naïve CD4
cells, and is required for B cell development(29). They interact with
antigen-specific B cells in germinal center (GC), and promote somatic
hypermutation, class switch recombination and affinity maturation of B
cells(30). In addition, Tfh cells also facilitate the differentiation of
GC B cells, transforming them into memory B cells and long-lived plasma
cells(31). The hallmarks of the Tfh cells will lead to the uncontrolled
activation of B cells, thus produce a large amount of antibodies(32).
There were three subsets of Tfh cells in peripheral blood,
Tfh1(CXCR3+CCR6-), Tfh2(CXCR3-CCR6-) and Tfh17 (CXCR3-CCR6+)
cells28,29. Among of them, Tfh1 cells are major
regulators of proinflammatory stress, and can strengthen the immune
reactions(33); While Tfh2 cells induce the differentiation of naïve B
cells into plasmablasts(34). Therefore, increase in Tfh1/Tfh2 ratio
usually implicates an inflammation.
To the best of our knowledge, the present study revealed the association
of early AAM with Tfh cells in women for the first time. The present
study showed that genetic factors of AAM contributes to the variances of
PD1- naïve Tfh cells. Compared to
PD1+ Tfh cells, PD1- naïve Tfh cells
are less proliferative and produce less cytokines(35). It implied that
early AAM will impair Tfh progenitor cells, thus aberrant Tfh1/Tfh2
ratio and unregulated humoral immunity. In shared genetic factors of AAM
and Tfh cells, LIN28B encodes a key repressor of let-7 microRNA
biogenesis and cell pluripotency(36). Transgenic Lin28b mice
demonstrate altered pubertal growth(37). Moreover, LIN28B/let-7 pathway
influences Naïve CD4 cells survival(38) and B cell development(39).
let-7 is also aberrantly expressed in the embryonic chorion tissue of
spontaneous abortion(40). Therefore, it is biological plausible thatLIN28B involves the etiology of early AAM, aberrant Tfh cell
level, and miscarriages. For chromosome region 9p12, it includes three
AAM-related genes LINC01505 (26), TAL2 (27) andTMEM38B (28). Among of them, TAL2 is crucial for
thymocyte development(41), and chromosome translocation involving this
gene occurs in T cell acute lymphoblastic leukemia(42). Therefore,
chromosome region 9p12 is linked to both AAM and immunity. Taken
together above reports, our findings on the association of AAM and Tfh
cells are supported by genetic evidences. Additionally, the longer
hormone exposure and obesity, which are produced by early AAM(13), both
are found to play pivotal roles in the function of Tfh cells in recent
years(43-45). Thus, our findings on the relationship between early AAM
and aberrant Tfh cells function are credible.
Another important finding of this study is the association between
pre-pregnancy Tfh1/Tfh2 ratio and spontaneous miscarriage. Because
humoral immunity plays a pivotal role in miscarriages(46), imbalance of
pro-inflammatory and anti-inflammatory cytokines may lead to impairment
of pregnancy(47). The relationship between Tfh cells and spontaneous
miscarriage is biological plausible. Different types of Tfh cells can
secrete different antibodies: Tfh1 cells secrete IFN-γ, which can
trigger to produce IgG2α, then cause abortion; Tfh2 secrete IL-4, which
can trigger to produce IgG 1 and IgE, and was down-regulated in abortion
women(48); Tfh17 secrete IL-17, which triggers IgA(49, 50). It can be
concluded that the balance of Tfh1/Tfh2 is one of the important immune
factors for abortion. When Tfh1 is dominant, the proportion of IFN-γ
increases and IL-4 decreases, which will lead to abortion. Therefore, It
is credible that Tfh cells, especially Tfh1 cells, mediates the process
of early AAM related miscarriages, which was found in this study.
In this study, we also found that the pre-pregnancy ratio of Tfh1/Tfh2
was associated with TSH at early pregnancy. Thyroid diseases in women of
childbearing age are mainly caused by autoimmune diseases(51).
Autoimmune thyroid disease (AITD), including autoimmune hypothyroidism
(Hashimoto’s thyroiditis, HT) and autoimmune hyperthyroidism (Graves’
disease, GD). It has been found that the percentage of Tfh cells is
higher in HT patients and Tfh cells exist in the thyroid, suggesting
that Tfh cells drive autoimmunity and participate in the
pathogenesis(52). Abnormal immune microenvironment results in increased
expression of CXCR5 in thyroid tissue, which leads to immune response of
Tfh cells and the development of AITD(53). The disorder of thyroid
function in pregnancy can cause hyperthyroidism or incomplete thyroid
function. Women with hyperthyroidism during pregnancy are more likely to
have obstetric complications such as miscarriage, premature birth,
abruptio placentae, thyroid store, and heart failure or adverse neonatal
outcomes such as prematurity, LBW, intrauterine growth restriction
(IUGR) and still birth than normal women(17). Some studies have pointed
out that even in healthy women without thyroid dysfunction, the risk of
miscarriage, fetal death or neonatal death increases as TSH levels
rise(54). Thus, we infer that the changes of the pre-pregnancy ratio of
Tfh1/Tfh2immune function before pregnancy will affect the thyroid
function during pregnancy, thus affecting the outcome of pregnancy.
Because early AAM also relates to many diseases, such as cardiovascular
diseases(5), autoimmune diseases(7) and cancers(55), our study may hold
potential to initiate new avenues of research.
Strengths and Limitations The most strength of the present
study is using multiple analyses to show the mediation of Tfh cells in
the process of early AAM-induced subsequent pregnancy events. First, we
performed Spearman correlation analyses to study the relationship
between AAM and Tfh cells at the pre-pregnancy examinations. Second, we
used Manny-Whitney U tests, Spearman correlation analyses, Kaplan-Meier
estimate and Cox regression analyses to investigate the association
between AAM-related pregnancy indexes by a follow-up study. The
pregnancy indexes included pregnancy status, thyroid function at early
pregnancy, and metabolic indexes at mid pregnancy, gestational weeks and
birth weight of newborns. Last but not the least, we validated the
association between early AAM and aberrant function of Tfh cell by a
two-sample MR test. Therefore, this preliminary work may provide a
proof-of-concept for evaluating the role of Tfh cells in early
AAM-related reproductive events.
The present study has some limitations: First, the pilot study was
conducted in a small Chinese population, while the genetic data that
support its findings were collected from two consortiums that were of
European origin. The generalizability of our findings needs to be
confirmed. Second, other productive events such as ectopic pregnancies
cannot be analyzed because of the limited sample size. Third, many women
have improved their awareness of pregnancy examination, such as blood
glucose and blood pressure during pregnancy, which have been well
controlled. Forth, there were only 497 females in TwinsUK. To avoid
overestimate of locus-specific effects from GWAS data(56), the genetic
contribution of Tfh cells to spontaneous miscarriages was not analyzed
in this study.
Conclusion The present study found that early AAM may produce
hallmarks of Tfh cells, then induce subsequent reproductive events, such
as miscarriages and higher TSH levels. Our results may provide a new
concept on the etiology of the AAM-related reproductive events. Larger
population studies and functional studies are warranted.