Results
Patient characteristics. Sixty-eight healthy Chinese Han women
for pre-pregnancy examination were included in this study, as described
in our previous report(21). Table S1 showed in summary the
demographic characteristics of the 68 subjects. The mean age of the
subjects was (29.9 ± 3.9) years, where 36 (52.9%) were younger than 30
years. The Body Mass Index (BMI) of 59 subjects (86.8%) was below 24
kg/m2. The average AAM was (14 ± 1.6) years old, where
26 (38.2%) experienced AAM below the age of 14 years. The average
menstrual cycle was 30 days, where 10 females (14.7%) had abnormal
cycles, among of which two subjects (2.9%) had short menstrual cycles
(less than 24 days) and 8 ones (11.8%) had prolonged menstruation (more
than 35 days). In addition, 40 females (58.8%) had at least one
gravidities, and 33 ones (48.5%) were parous. Twenty (29.4%) had one
miscarriage, and nine (13.2%) have had more than two miscarriage.
There were 35 pregnancies after 15 months follow-up. Among of these
subjects, the body weight of 10 ones (41.2%) increased more than 19.98
kg at early pregnancy. And the median of TSH, FT4 and TPO-ab of
pregnancy subjects at early pregnancy were 1.9 mIU/ml, 10.4 pmol/L, and
32.1 IU/ml, respectively. During the mid-trimester pregnancy, the median
of fast glucose, triglyceride, cholesterol , HbA1c, and
systolic/diastolic blood pressure was 4.5 mmol/L, 2.1 mmol/L, 6.1
mmol/L, and 117/66 mmHg, respectively.
In these 35 gravidas, there were 20 healthy pregnancies, 7 threatened
miscarriages and 8 spontaneous miscarriages. No chromosome abnormality
and congenital deformity of the fetuses was found in these miscarriages.
Early AAM is associated with pre-pregnancy Tfh1/Tfh2 ratio. We
analyzed the correlations between AAM and T cells, γδ T cells and their
subsets according to the method as described(21). No significant
correlations was found between AAM and CD3+, double
positive T (CD4+CD8+) and double
negative (CD4-CD8-) T cells, T
helper cells (CD4+, also known as Th cells) and
subsets Th1/2/17, and cytotoxic T cells (CD8+, also known as Tc cells)
and subsets Tc1/2/17 (all P values >0.05). At the
same time, no significant correlations were found between AAM and
CD4/CD8 memory cells such as Naïve, central memory (CM), effect memory
(EM), and EM CD45RA+ (EMRA). No significant correlations were found
between AAM and γδ T cells either (all P values >
0.05).
Among T follicular helper cells
(CD3+CD4+CXCR5+,
also known as Tfh cells), Tfh1
(CD4+CXCR5+CXCR3+CCR4-)
were significantly negatively correlated with AAM (r =-0.273,P =0.027), wherever Tfh2
(CD4+CXCR5+CXCR3-CCR4+)
were positively associated with AAM (Spearman r =0.342,P =0.005), as shown in Figure 1B. Further, the Tfh1/Tfh2
ratio also showed significantly negative correlated with AAM (Spearmanr =-0.283, P =0.019). There was no discernible correlation
between Tfh17
(CD4+CXCR5+CXCR3-CCR4-CCR6+)
and AAM (Spearman r =-0.137, P =0.265).
Pre-pregnancy Tfh1/Tfh2 ratio is associated with TSH at early
pregnancy. The pregnancy status of the subjects were followed-up by
interview until December 2019. After a 15-month follow-up, there were 35
pregnancies, including 20 healthy pregnancies, 7 threatened miscarriages
and 8 spontaneous miscarriages.
We analyzed the correlation between pre-pregnancy Tfh1/Tfh2 ratio and
some physical examination indexes during pregnancy. We found that the
ratios of Tfh1/Tfh2 at pre-pregnancy examinations were significantly
positively associated with TSH at early pregnancy (Spearmanr =0.367, P =0.046, Figure 2 ).
However, there no significant associations between pre-pregnancy
Tfh1/Tfh2 ratio and either of the following indexes: weight gain, FT4
and TPO-ab at early pregnancy; and BMI, blood pressure, fast glucose,
triglyceride, cholesterol, and HbA1c during mid-trimester pregnancy (allP values >0.05, as shown in Table S2 ).
Pre-pregnancy Tfh1/Tfh2 ratio was associated with an increased
risk and a shorter time to spontaneous miscarriage. Consistent with
previous reports(10), the average AAM of
threatened miscarriages and
spontaneous miscarriages was both early than healthy pregnancies (bothP values <0.05, Table S3 ).
Therefore, we analyzed the relationship between pre-pregnancy Tfh1/Tfh2
ratio and miscarriages. Firstly, Manny-Whitney U tests were used to
compare immune cells between healthy pregnancies and threatened or
spontaneous miscarriages. We found
that Tfh1/Tfh2 ratio was higher in spontaneous miscarriages than the
ratio in healthy pregnancies (median 0.288 vs. 0.179, Manny-Whitney
U=-2.288, P =0.021, Figure s1 ), but not in threatened
miscarriages.
We further studied the association of spontaneous miscarriage rates with
the Tfh1/Tfh2 ratio and AAM during the study period in 35 gravidas. Rate
of spontaneous miscarriages were analyzed by the Kaplan-Meier estimate
and Cox regression analyses. The log-rank test was used to compare
spontaneous miscarriages curves. We found that the women with
AAM<14 years had a higher risk of spontaneous miscarriages
(adjusted RR=17.28, 95% CI=2.04-146.75, P =0.009, Table
1 ), and a shorter spontaneous miscarriage time (45 days vs. 185 days,
log-rank P =0.036, Figure 3A ). The adjusted confounding
factors included age, previous times of spontaneous abortion and BMI.
Moreover, the women with the Tfh1/Tfh2 ratio≥0.209 (median) also had a
higher risk of spontaneous miscarriages (adjusted RR=12.25, 95%
CI=1.72-87.46, P =0.013, Table 1 ) and a short time to the
spontaneous miscarriage (42 days vs. 115 days, log-rank P =0.038,Figure 3B ). Our results implied a possible logical connection
with earlier AAM, higher pre-pregnancy Tfh1/Tfh2 ratio and spontaneous
miscarriage.
However, no distinguishable association between mothers’ menarche age
and the gestational weeks and birth weight of newborns were found in
this study.
Genetic factors of AAM contributes to Tfh cells. To test the
causal relationship and shared genetic factors between Tfh cells and
AAM, we performed two sample MR tests by using publicly available
genetic summary data of Genome-wide association studies (GWAS) from
176008 subjects in UK Biobank
(http://www.nealelab.is/blog/2017/9/11/details-and-considerations-of-the-uk-biobank-gwas )
and 497 female participants from the UK Adult Twin Register (TwinsUK)
(22) in MR-base(24).
At a GWAS threshold of statistical significance
(P <5×10-8; linkage disequilibriumR 2<0.1), we found that 84 AAM-related
SNPs were significantly associated with PD1- naïve Tfh
cells by Weighted median method (β=0.38±0.19, P =0.040,Figure S2 ) and by Weighted mode (β=0.62±0.31, P =0.045).
Moreover, these SNPs can explain 19% of variance in
PD1- Tfh naïve cells (DirectionalityP =4.28×10-10). However, there were no
significant causal relationship between AAM and two other types of naïve
Tfh cells (CCR4-CXCR3- and
CCR4+CXCR3+), CD4+Tfh cells and CD8+ Tfh cells in MR-base (All Pvalues > 0.05).
In MR-Base, there were another two AAM database: ReproGen consortium
studies(25) and MRC-IEU consortium studies
(http://www.ewascatalog.org ). However, there were no significant
association between AAM and Tfh cells by using GWAS Data from ReproGen
and MRC-IEU consortium studies. These results might due to the small
samples with outcomes in TwinsUK dataset, and the genetic background of
TwinsUK might be more different to ReproGen and MRC-IEU consortium
studies than to UK Biobank.
We further performed a leave-one-out analysis to assess whether the MR
estimate is driven or biased by a single SNP. Using this method, we
found that rs1516883 and rs1933801 were the major share genetic factors
between AAM and PD-1- naïve Tfh cells (also shown asFigure S2 ). These two SNPs were located in chromosome region
9p12 (LINC01505, TAL2 and TMEM38B ) and near geneLIN28B respectively, both are reported AAM loci(25-28).