Association Between Serum Glycated Hemoglobin Levels at Early Gestation and the Risk of Subsequent Pregnancy Loss in Pregnant Women Without Diabetes Mellitus: Prospective Cohort Study

Background: As a severe morbidity during pregnancy, the etiology of spontaneous pregnancy loss (SPL) remains largely unknown. Serum glycated hemoglobin (HbA 1c ) level is an established predictor of SPL risk among women with diabetes, but little is known about whether such an association exists among pregnant women without diabetes when glycemic levels are within the normal range. Objective: This study aimed to quantify the association between maternal HbA 1c levels in early pregnancy and subsequent SPL risk in a cohort of pregnant women without diabetes. Methods: This prospective cohort study involved 10,773 pregnant women without diabetes enrolled at their first antenatal care visit at a hospital’s early pregnancy clinic from March 2016 to December 2018 in Shanghai, China. HbA 1c and fasting blood glucose (FBG) levels were examined at enrollment. Participants with diabetes before or pregnancy or those diagnosed with gestational diabetes were excluded. Diagnosis of SPL, defined as fetal death occurring before 28 gestational weeks, was derived from medical records and confirmed via telephone interviews. We used generalized linear models to quantify the associations of continuous and dichotomized maternal HbA 1c levels with SPL risk and reported crude and adjusted risk ratios (RRs) and 95% CIs. A restricted cubic spline (RCS) regression model was used to assess the potential nonlinear dose-response relationship. Adjusted covariates included maternal age, education level, preconception BMI, gestational weeks, gravidity, history of adverse pregnancy outcomes, family history of diabetes, folic acid supplementation, and smoking and drinking during the periconception period. Results: In total, 273 (2.5%) SPL cases occurred. Every 0.5% increase in HbA 1c levels was linearly associated with a 23% increase in SPL risk (adjusted RR [aRR] 1.23; 95% CI 1.01-1.50). The RCS model revealed that this association was linear ( P =.77 for the nonlinearity test). Analyses based on dichotomized HbA 1c levels showed a significantly increased risk of SPL when HbA 1c levels were ≥ 5.9% (aRR 1.67; 95% CI 0.67-3.67), and the significance threshold was ≥ 5.6% (aRR 1.60; 95% CI 1.01-2.54). Sensitivity analyses showed similar results when including the participants with missing SPL records or HbA 1c data. Linear


Introduction
Spontaneous pregnancy loss (SPL), also known as spontaneous abortion, is one of the serious morbidities during pregnancy and precedes an increased risk of reduced fertility, long-term depression, and anxiety among pregnant women [1].As the definition of SPL varies between countries and international organizations, estimates of the prevalence of SPL vary among previous studies [2].Data from a large nationally representative survey from the United States showed that approximately 20% of clinically recognized pregnancies ended in SPL (including stillbirths and ectopic or tubal pregnancies) during the whole gestation period [3].Pregnancy losses from the first antenatal care visit to that at 28 weeks' gestation are recorded in practice in China [4,5].A nationwide study with 6.4 million medical records of Chinese pregnant women reported 2.8% of pregnancy losses before 28 weeks of gestation [6].To date, the mechanism underlying the etiology of SPL remains largely unknown, and over 50% of women with SPL have no identified risk factors [7,8].
Poor glycemic control during pregnancy is an established independent predictor for adverse pregnancy outcomes [9].Serum glycated hemoglobin (HbA 1c ) levels are conventionally used for monitoring blood glucose control [10].According to the American Diabetes Association's (ADA's) clinical practice guidelines, individuals with HbA 1c levels within 5.7%-6.4% and ≥6.5% are classified as having prediabetes and diabetes, respectively, in the general population, and HbA 1c levels of 5.9% and higher in pregnant women are considered an early indicator of abnormal glucose metabolism and a higher risk of adverse pregnancy and neonatal outcomes [11].Previous studies have reported associations of HbA 1c levels of pregnant women with insulin-dependent diabetes and SPL risk [12][13][14].However, little is known about these associations in pregnant women without diabetes.
An HbA 1c test at the first prenatal visit has been recommended for those at risk of developing gestational diabetes only, including having obesity and having a family history of gestational diabetes, but not yet for the general population of pregnant women [11,15,16].In this prospective cohort study, we aimed to evaluate whether HbA 1c levels in pregnant women without diabetes are associated with a subsequent SPL risk.

Study Population
Pregnant women included in this study were a subcohort of the ongoing Shanghai Preconception Cohort Study (SPCC; ClinicalTrials.govNCT02737644) [17], who were enrolled between March 2016 and December 2018 from one of the study sites-a tertiary maternity hospital where HbA 1c and fasting blood glucose (FBG) levels were routinely examined at the first antenatal visit for all pregnant women.This maternity hospital is one of the largest delivery hospitals (with >20,000 births per year) during the study period and accounts for over 20% of the annual deliveries in the city.Participants were enrolled at their first antenatal visit at early pregnancy clinics, and each woman had only one medical record for this study.Biochemistry and SPL diagnosis data were extracted from the hospital's electronic medical record system.Among all pregnant women during the study period (N=13,129), 10,773 were eligible for the primary analysis after the exclusion of those who met any one of the following criteria: missing medical records after the first antenatal visit, missing information regarding HbA 1c or FBG levels at entry, having received artificial abortions, self-reported diabetes before pregnancy (ie, an HbA 1c level of ≥6.5% or FBG level of ≥7.0 mmol/L at the first antenatal visit), or having received insulin treatment during pregnancy or taking oral hypoglycemic drugs before or during pregnancy.We defined this group as pregnant women without diabetes.

Ethical Considerations
Ethics approval for this study was sought from the institutional Ethics Committee of the Children's Hospital of Fudan University (2016-49).Written informed consent was obtained from all participants before recruitment.All data were anonymously analyzed.

Exposures and Covariates
We treated the fasting HbA 1c levels in early pregnancy as the main exposure in this study.Given that FBG levels are also an index of glycemic control, both HbA 1c and FBG levels were abstracted from the medical records for analyses.According to the routine practice of the hospital where this subcohort was recruited, HbA 1c and FBG levels were measured in 2 hours using a venous blood sample after overnight fasting (>8 hours of fasting) at their first antenatal care visit.Venous blood for the HbA 1c test was collected in an EDTA-containing tube and determined using high-performance liquid chromatography (Bio-Rad) in the hospital's certified standard clinical examination center following standard protocols.
A series of variables regarding known or suspected risk factors for SPL were considered covariates in the association analysis [18].As described elsewhere, demographic characteristics and pregnancy history of the participants upon enrollment were collected through a prespecified standard self-administered questionnaire and an interview with the obstetric nurse during the first antenatal visit.Maternal preconception BMI (pre-BMI) was calculated using self-reported measures of prepregnancy body weight and categorized as normal weight or overweight.We used both the Chinese standard (≥24 kg/m 2 ) and the international standard (≥25 kg/m 2 ) to define the overweight status [19,20].We defined a family history of diabetes as having at least 1 first-degree relative diagnosed with diabetes.Smoking exposure and alcohol drinking were defined as smoking cigarettes or having been exposed to second-hand cigarette smoke, and as consuming any alcoholic beverages within 3 months before or during the current pregnancy, respectively.Folic acid supplementation (FAS) was defined as having a regular intake of pure folic acid tablets or multivitamins containing folic acid before or during early pregnancy.Gestational weeks at enrollment were routinely determined by the last menstruation period and confirmed through a routine ultrasonographic examination.Participants were defined as having a history of adverse pregnancy outcomes if they had abortions, preterm delivery, stillbirths, or ectopic pregnancy in previous pregnancies.

Outcomes
The recorded SPL cases from the hospital's electronic medical system were fetal deaths occurring before 28 gestational weeks in accordance with the Chinese clinical guidelines [4].Stillbirth (fetal deaths after 28 gestational weeks), and artificial abortions due to ectopic pregnancies, molar pregnancies, or any clinically recognized disorders were not considered SPL in this study.Trained staff verified these diagnoses through a personal telephone interview with pregnant women or their husbands before the analysis.

Statistical Analysis
Continuous variables were reported as mean (SD) values for a normal or approximate normal distribution and median (IQR) values for a skewed distribution, and 2-tailed unpaired Student t tests or Mann-Whitney U tests were used for comparisons between the SPL and non-SPL group, respectively.Normality was visually inspected using frequency histograms.Categorical variables were summarized as frequencies and percentages, and chi-square tests were used for the group comparisons.
Our primary aim was to investigate the associations of HbA 1c and FBG levels as continuous variables with SPL risk.We used generalized linear models with binomial family and log link functions treating HbA 1c (rescaled through dividing by 0.5) and FBG levels as continuous variables to estimate crude and adjusted risk ratios (aRRs) and 95% CIs.A 0.5% absolute increment was chosen for the HbA 1c level because it reflects a clinically important change [21].Considering that a HbA 1c level of 5.9% in early pregnancy has been suggested as the cutoff for identifying women at increased risk of adverse pregnancy outcomes [11], we further assessed the associations based on dichotomous HbA 1c (coded as 1 for a HbA 1c level of ≥5.9% and 0 for an HbA 1c level of <5.9%).We used a restricted cubic spline (RCS) regression model with 3 knots (5th, 50th, and 95th percentile levels) fitted in R (rms package) to assess the potential nonlinear dose-response relationship of HbA 1c levels with SPL risk.Adjusted covariates included maternal age, education level, pre-BMI, gestational week at HbA 1c examination, gravidity, history of adverse pregnancy outcomes, family history of diabetes, FAS, and exposure to smoking and drinking during the periconception period.
We conducted an exploratory analysis to investigate the HbA 1c cutoff indicating pregnant women at an increased risk of SPL by using a series of regression models using HbA 1c levels from the median level of the study population (5.1%) to 6.0% in a 0.1% interval without adjustment for multiple testing.
As there were 2147 participants-1360 (10.5%) with missing medical records after the first antenatal visit and 787 (6.1%) with missing HbA 1c data-we conducted 2 sensitivity analyses to test the robustness of the main results after multiple imputation with chained equations based on a missing at random assumption.First, we added 1360 pregnant women with missing SPL data after imputation (sensitivity analysis 1: n=10,773+1360).Second, we included 787 pregnant women with missing HbA 1c levels at enrollment after imputation (sensitivity analysis 2: n=10,773+787).To ensure that our main results are free from potential bias from unmeasured confounders, we further repeated the primary association analyses within subgroups with a potentially low risk of SPL, including pregnant women younger than 35 years, without overweight, a family history of diabetes, a history of adverse pregnancy outcomes, or smoking or alcohol drinking exposures.Statistical analyses were performed by Stata (version 16.0; StataCorp) and R package (version 3.6.1;The R Foundation), and all statistical tests were 2-sided at a significance level of .05.A post hoc power analysis was performed for the main association analyses of continuous HbA 1c levels with SPL risk, which revealed that with the current sample size, associations with a risk ratio (RR) lower than 0.84 or greater than 1.19 for continuous exposures of interest will ensure a power of ≥80% at an α level of 5%.

Characteristics of the Study Population
A total of 10,773 eligible pregnant women were included in the main analysis (Figure 1), with a mean age of 30.5 (SD 4.0) years and an average of 10.8 (SD 1.7) weeks of gestation at enrollment (Table 1).The majority of participants (n=9866, 91.6%) had a college or higher level of education, 4394 (40.9%) had more than 1 gravidity, 2056 (19.1%) had overweight before pregnancy, and 3874 (36.3%) had a family history of diabetes.Smoking and alcohol consumption were reported by 1043 (9.8%) and 857 (8.0%) pregnant women, respectively.Besides, 2817 (26.4%) pregnant women had a history of adverse pregnancy outcomes.The distribution of HbA 1c and FBG levels approximately met a normal distribution, with a mean value of 5.09% (SD 0.30%) and 4.49 (SD 0.78) mmol/L, respectively.The 2 markers were weakly correlated (r=.19;P<.001) and overlapped in quartiles (Figure S1 in Multimedia Appendix 1).We summarized the characteristics of the 10,773 eligible pregnant women and 2147 women with missing medical records or HbA 1c data (Table S1 in Multimedia Appendix 1), which were overall similar.e Data for gestational weeks at enrollment were missing for 60 out of 10,773 (0.6%), 2 out of 273 (0.7%), and 58 out of 10,500 (0.5%) pregnant women in the total sample, SPL group, and non-SPL group, respectively.f FAS: folic acid supplementation.g FBG: fasting blood glucose.h FBG levels were ≥6.1% for 29 out of 10,773 (0.3%), 1 out of 273 (0.4%), and 28 out of 10,500 (0.3%) pregnant women in the total sample, SPL group, and non-SPL group, respectively (P=.75).i HbA 1c : serum glycated hemoglobin.
The incidence rate of SPL was 2.5% among 10,773 pregnant women in this study.Compared to pregnant women without SPL, those with SPL were 1.6 years older (mean ages of the SPL and non-SPL groups were 32.0, SD 4.4 vs 30.4,SD 3.9 years, respectively) during pregnancy, were more likely to have overweight before pregnancy (23.8% vs 19.0%), and had higher levels of HbA 1c (5.15% SD 0.33% vs 5.09% SD 0.30%; albeit in the clinically normal range) and FBG (4.64, SD 0.39 vs 4.48, SD 0.47 mmol/L) at the first antenatal visit.

Associations Between Maternal HbA1c and FBG Levels and SPL Risk
As shown in Table 2, maternal HbA 1c levels showed significant positive associations with SPL risk with an unadjusted RR of 1.34 (95% CI 1.10-1.63)per 0.5% increase in HbA 1c levels, and the association remained significant after adjusting for covariates (aRR 1.23, 95% CI 1.01-1.50;P=.04).The RCS model showed a linear association of SPL risk with increasing HbA 1c levels through the whole range of HbA 1c values (P=.77 for the nonlinearity test; Figure 2).No significant association was found between a HbA 1c level of ≥5.9% and SPL risk after adjusting for covariates.Every 1 mmol/L increment in maternal FBG levels was associated with a >2-fold higher risk of SPL (aRR 2.12, 95% CI 1.61-2.80;P<.001).

Sensitivity and Subgroup Analyses
In the first sensitivity analysis including 1360 pregnant women with missing medical diagnosis records, the association between HbA 1c levels and SPL risk did not substantially change (aRR per 0.5% increment 1.25, 95% CI 1.02-1.53;P=.03; Table 2).Similar results were also observed in the second sensitivity analysis upon including pregnant women with missing data on HbA 1c levels (aRR per 0.5% increment 1.25, 95% CI 1.01-1.55;P=.04).
Our exploratory analyses showed that the strength of the associations increased markedly from below 1.2 to 1.6 at an HbA 1c level of 5.6% (aRR 1.60; 95% CI 1.01-2.54;P=.048) and increased further at higher cutoff levels, although significance was not achieved at HbA 1c levels of 5.9% and 6.0% (Figure S2 in Multimedia Appendix 1).In further subgroup analyses including low-SPL risk populations without overweight, alcohol drinking, a family history of diabetes, and a history of adverse pregnancy outcomes, the associations of every 0.5% increase in HbA 1c levels with SPL risk remained very similar compared to those in the main analysis (aRR 1.32, 95% CI 1.07-1.63;aRR 1.18, 95% CI 1.01-1.38;aRR 1.24, 95% CI 1.03-1.48;and aRR 1.73, 95% CI 1.26-2.36;respectively; Table S2 in Multimedia Appendix 1).

Principal Findings
In this large prospective cohort study, we provide solid evidence that among pregnant women without diabetes, HbA 1c levels in early gestation within the clinically normal range were associated with an increased risk of SPL in a linear dose-response manner.Although far below the recommended threshold for diagnosing gestational diabetes, HbA 1c levels may indicate an increased subsequent SPL risk in pregnant women in general.Our findings in a prospective cohort are novel and deepen our understanding of the important pathophysiologic role of impaired maternal glycemic metabolism in the development of SPL.

Comparison With Prior Work
Several studies have investigated the adverse effect of elevated HbA 1c levels on SPL risk only among pregnant women with diabetes and have reported conflicting findings [12][13][14]22].A case-control study including 432 control women and 386 women with type 1 diabetes found that the rate of SPL did not significantly differ between the 2 groups (16.1% vs 16.2%, respectively) [12].However, another case-control study among women with type 1 diabetes reported that elevated HbA 1c levels were associated with SPL risk [13].Another cohort study of 573 women with type 1 diabetes observed a linear association between HbA 1c levels and the risk of adverse pregnancy outcomes including SPL when HbA 1c levels were >7.0%[14].This is the first prospective cohort study demonstrating the link between maternal HbA 1c levels in early pregnancy and SPL risk among pregnant women without diabetes.These associations were still significant in the population at a low risk of SPL.We also observed a highly overlapped distribution of FBG levels among HbA 1c categories, and consistent associations with SPL were also observed with regard to FBG levels.However, regarding their clinical application, FBG level is a less ideal marker than HbA 1c level because the former is less stable, requires a fasting state for examination, and has relatively greater intraindividual variability [23].Our findings suggest that attention to glycemic control should not be limited to pregnant women with diabetes but should also include those with high HbA 1c levels within the clinical range during early gestation for the related increased risk of SPL.

Research Implications
Our study addresses 2 important issues with important clinical and research implications.First, our findings expand on the literature on risk factors for SPL, an adverse pregnancy outcome whose modifiable determinants remain poorly understood.Second, we address a risk factor that is known to be modifiable through lifestyle and pharmacological interventions.Measurement of HbA 1c levels during pregnancy is conventionally used to monitor glycemic control in pregnant women with diabetes [24]; this marker is superior to FBG levels, mainly for its greater stability but lesser variability among individuals and its nonrequirement of fasting [23].Given the special physiological status of pregnancy, the target HbA 1c level for ideal glycemic control remains inconclusive.In China, based on recommendations for the general population, an HbA 1c level less than 6.5% is recommended to indicate ideal glycemic control before conception [25].An HbA 1c level of ≥5.9% is recommended by the ADA's guidelines as an indicator for screening pregnant women with a higher risk of preeclampsia, macrosomia, shoulder dystocia, and perinatal death [11].Our findings add new evidence for the linear relationship between glucose metabolism markers and SPL risk when HbA 1c levels are clinically normal [11], and the magnitudes of the associations are even stronger in those exposed to smoking or those who have a history of adverse pregnancy outcomes.
The mechanism underlying the association between maternal HbA 1c levels and SPL in women without diabetes remains unclear.Animal studies found that poor glycemic control may facilitate premature programmed cell death of key progenitor cells of the blastocyst and promote pregnancy loss [26].In addition, poor glycemic control may interfere with implantation by inhibiting trophectoderm differentiation and increasing oxidative stress, thus affecting the expression of critical genes essential for embryogenesis [27,28].Further population studies exploring the biological mechanisms underlying the association between HbA 1c levels and SPL in pregnant women without diabetes are warranted.
The findings of our exploratory analysis are clinically relevant, indicating that pregnant women with HbA 1c levels above 5.6% need attention for owing to a potentially increased risk of SPL.Given that HbA 1c levels are 0.5%-0.6%lower in early pregnancy than in nonpregnant women [29], HbA 1c levels over 5.6% at early gestation may reflect an elevated HbA 1c status that occurred before or at pregnancy (ie, prediabetes status), which accounts for over 13% of the population [6].Another large cohort study and systematic review have reported associations between an HbA 1c level of <6.4% with increased severe maternal morbidity and subsequent gestational diabetes mellitus [30,31].Taken together, based on the aforementioned evidence and our findings, we propose that monitoring of HbA 1c levels in early pregnancy is necessary general pregnant women, and HbA 1c levels exceeding 5.6% might be considered an indicator of high risk for SPL and additional medical care.

Strengths and Limitations
The merits of our study include the prospective nature of the data, the large sample size, and the consistent results from robust analyses, which make our findings convincing.However, several limitations exist.First, residual confounding, such as uterine abnormalities, chromosomal abnormalities, antiphospholipid syndrome, and thyroid disorders, cannot be completely ruled out due to the nature of observational studies.Data on important biomarkers such as insulin levels during early gestation were not available.Second, in the exploratory analyses, we did not correct for multiplicity.Prospective multicenter cohort studies investigating whether a maternal HbA 1c level of 5.6% in early pregnancy could indicate an increased risk of SPL are warranted in the future.Third, compared with the rate of clinically registered pregnant women ending in SPL (11%-20%), the SPL rate in this cohort was much lower.Given the nature of the study population, which was recruited at their first antenatal care visit (with a median of 10 weeks of gestation), SPL occurred earlier than our observation was inevitably missed, selection bias exists, and our findings are not generalizable to the entire general population.The study population we selected, comprising pregnant women without diabetes, may include some cases of mild gestational diabetes diagnosed midgestation and without insulin treatment.Moreover, potential biases such as outcome misclassification could not be ruled out from the association analyses based on imputation.

Conclusions
This study is the first to document that maternal HbA 1c levels in early pregnancy are associated with the subsequent risk of SPL in a dose-response manner among pregnant women without diabetes.Our findings support the need to monitor HbA 1c levels for identifying high risk of subsequent SPL in pregnant women in general and expand on the growing literature linking overall metabolic health to reproductive and pregnancy health among otherwise healthy women.

Figure 2 .
Figure 2. Restricted cubic spline plots for the association between maternal serum glycated hemoglobin (HbA 1c ) levels in early pregnancy with spontaneous pregnancy loss (SPL) risk.

Table 1 .
Baseline characteristics of study population at the first antenatal care visit.
c Not available.d N/A: not applicable.

Table 2 .
Associations between maternal HbA 1c a and FBG b levels and SPL c risk during early pregnancy.

Sensitivity analysis 2 (including 787 pregnant women with missing HbA 1c levels based on imputation)
Adjusted for age, pre-BMI, gestational weeks, education, gravidity, history of abnormal pregnancy, family history of diabetes, folic acid supplementation, and drinking and smoking status.
a HbA 1c : serum glycated hemoglobin.bFBG: fasting blood glucose.c SPL: spontaneous pregnancy loss.d e N/A: not applicable.