Methods and Materials
Study population. Data were analyzed from a single hospital in
Shenzhen, a Southern Chinese city, in accordance with the relevant
guidelines and regulations as set out by the approving body, the Ethics
Committee, Shenzhen Baoan Women’s and Children’s Hospital, Jinan
University (IRB:LLSC 2018-08-01). All the study participants donated a
5ml venous blood after gave a written informed consent. All
participants’ names and other identifying information were removed in
all sections of this manuscript.
From September to November 2018, we recruited at random 72 fertile women
undergoing pre-pregnancy physical examinations at our hospital, where
the rate of response was >95%. The subjects were between
the ages of 20 and 40 years old, and were all local Han Chinese and
long-term residents in Shenzhen, China. With the exception of four
persons who suffered from uterine fibroids, recurrent miscarriage,
oligomenorrhea and cervicitis, 68 healthy women were included in this
study. After obtaining the informed consent, information of the subjects
such as age, height, weight, AAM, menstrual cycle and period, and
reproductive history were collected by trained nurses through a
questionnaire survey, and 5ml venous blood was collected for flow
cytometry analyses. Indicators including peripheral blood red blood cell
(RBC), white blood cell (WBC) and hemoglobin (Hb) were obtained from the
results of physical examination.
Follow-up of pregnancy status. The pregnancy status of the
subjects were followed-up by interview until December 2019, which was
verified by serum human chorionic gonadotropin (HCG) and ultrasound, and
the first day of last menstrual period (LMP) was recorded. Among of
these subjects, there were 35 pregnancies. Each pregnant woman was
scheduled to come back to our hospital for antepartum examinations
according to our hospital routine methods. Moreover, each pregnancy
early signs of miscarriage was timely detected and treated by our
hospital, and 7 ones were maintained to birth (defined as threatened
miscarriage); However another 8 ones were aborted (defined as
spontaneous miscarriage, with the exception of chromosome abnormality,
congenital deformity of the fetuses, and other secondary diseases of the
gravidas and fetuses). The information on weight gain at early
pregnancy, thyroid stimulating hormone (TSH), free T4 (FT4) and
thyroperoxidase antibodies (TPO-ab) at early pregnancy, and blood
pressure, fast glucose, triglyceride (TG), cholesterol (CHOL), and HbA1c
during mid-trimester pregnancy was collected from medical records of
antepartum examinations.
Flow cytometry analyses. Pre-pregnancy immune functions were
tested according to our routine methods(21). Briefly, peripheral white
cells were isolated from whole blood at pre-pregnancy physical
examinations by adding FACS LYSING SOLUTION (BD Biosciences, USA). Then
the precipitates were collected by centrifugation. After washed twice by
phosphate-buffered saline (PBS, sigma, USA), and cells were labelled
according to our published paper(21). Antibodies for APC-H7-conjugated
anti-CD3; PE-Cy7-conjugated anti-CD4 and anti-CD28; FITC-conjugated
anti-CD45RA; PE-conjugated anti-CD25; BV421-conjugated anti-CD56,
anti-CD127; BV510-conjugated anti-CD8; Alexa Fluor 647-conjugated
anti-CCR7 and anti-CXCR5; Alexa Fluor 488-conjugated anti-CD183, and
BV421-conjugated anti- CD194 were purchased from BD Biosciences.
Samples were run on a BD LSR Fortessa (BD Biosciences) at Shuangzhi
Purui Medical Laboratory Co., Ltd. (China), and the data was analyzed
using FlowJo 10.1 software (Tree Star Inc, USA).
Data Collection of public GWAS studies. The published GWAS data
for AAM and Tfh cells were collected from UK Biobank
(http://www.nealelab.is/blog/2017/9/11/details-and-considerations-of-the-uk-biobank-gwas )
and TwinsUK(22), respectively.
Statistical analysis and public GWAS data analysis. The SPSS
25.0 statistical software (SAS Institute Inc., Chicago, IL, USA) was
used to process and analyze the data. Data were presented as n ,
proportion, median and min/max. We first performed association analyses
between immune functions and AAM by using Spearman correlation analyses.
Manny-Whitney U tests were then used to compare immune cells between
healthy pregnancies and threatened or spontaneous miscarriages. Spearman
correlation analyses also were used to analyze the associations between
Tfh cells and the various biological indexes during pregnancy. Lastly,
we used the Kaplan-Meier estimate and Cox regression analyses to study
the associations between Tfh cells and miscarriages. The relative risk
(RR) and its 95% confidence interval (CI) were calculated. All tests
were two sided, and the level of significance was set at 0.05.
Statistical figures were produced by using GraphPad Prism8.0 (GraphPad
Software, USA).
To test the genetic association of Tfh cells and AAM, MR-Base software
(http://www.mrbase.org/ ) were used for two sample MR test
according to the method as described(23). Briefly, inverse-variance
weighted approach, weighted median method, MR-Egger method, and single
mode and weighted mode method were used for MR tests as a web
application. Then we performed a Causal direction test to measure MR
causal estimates. Lastly, a leave-one-out analysis was also performed to
assess whether the MR estimate is driven or biased by a single SNP.