Evaluation of nausea and vomiting in the first trimester on the risk of adverse birth outcomes and the contribution of genetic polymorphisms: a pilot prospective study

To evaluate the impact of Nausea and Vomiting in Pregnancy (NVP) on the risk of Preterm Birth (PTB) and Low Birth Weight (LBW), and explore the effect of genetic polymorphisms on the severity of NVP. A prospective study was conducted. Participants’ experience of NVP prior to 12 gestational weeks were evaluated by a Pregnancy-Unique Quantification of Emesis and Nausea (PUQE) scale. 11 Single Nucleotide Polymorphisms (SNPs) loci located in growth differentiation factor 15 (GDF15) and leucine-rich repeat containing 25 (LRRC25) gene of chr19p13.11 and intergenic region of chr4q12 were genotyped, which were implicated as genetic risk factors for NVP. Logistic regression models were applied to determine the effect of NVP in the first trimester on the risk of PTB and LBW, and genetic polymorphisms on the risk of NVP. Among 413 pregnant women, the incidence of nausea and vomiting was 85.5% (n = 353) in the first trimester, including 38.7% (n = 160) mild vomiting, 42.6% (n = 176) moderate vomiting and 4.1% (n = 17) severe vomiting. 33 were PTB, 20 were LBW. Compared with pregnant women without NVP, women with mild, moderate or severe NVP in the first trimester were not associated with the risk of PTB and LBW. Besides, the polymorphisms of 11 SNPs loci were not associated with the risk of NVP. Our study indicated that symptoms of nausea and vomiting in the first trimester were not significantly associated with PTB and LBW, and there were also no associations between GDF15 and LRRC25 polymorphisms and NVP.


Introduction
Nausea and vomiting in pregnancy (NVP) is the most common clinical complaints in the first trimester of pregnancy, which affects 50-90% of pregnant women [1,2].NVP usually starts at approximately 6-8 weeks of gestation and generally subsiding between 16-20 weeks [1,3], and up to 10% of women have the symptoms that can be prolonged until the time of delivery [4].Symptoms ranges from mild to the extremely severe form, also known as hyperemesis gravidarum (HG) which occurs in 0.5-2% of all pregnancies [5].NVP have substantial negative impacts on the overall quality of life of pregnant women, including mental and physical health problems [5,6], especially HG, which is the leading cause of hospitalization in the first trimester [7].
The underlying pathogenesis of NVP is poorly understood, but posited to be multifactorial, involving genetic, endocrine, gastrointestinal, environmental, and psychosocial factors [1,4].One research have reported that sisters with HG have a 17-fold increased risk of having HG themselves [25].A twin study estimated the heritability for presence of NVP as high as 73% [26].A genomewide association study (GWAS) provided insights into the genetic risk factors contributing to HG, which showed that two loci, chr19p13.11and chr4q12, were associated with the increased risk of HG [27].Later research revealed that SNP polymorphism of GDF15 gene located in chr19p13.11was associated with an eight-fold higher risk of recurrence of HG [28].Subsequently, further evidences were provided by another study for the role of GDF15 and IGFBP7 gene located in chr4q12 in the pathogenesis of HG [29].However, no study has probed the relationship between the genetic susceptibility and NVP in the Chinese pregnant women.
In the present study, Chinese pregnant women were categorized into four different groups according to the severity of NVP.We first evaluated the association between NVP and pregnancy outcomes including preterm birth and low birth weight, then explored the association between NVP and polymorphisms in 13 SNP loci located in the GDF15 and LRRC25 gene of chr19p13.11and the intergenic region of chr4q12 in Chinese pregnant women for the first time.

Study population and epidemiological data collection
This prospective cohort study was launched in October 2019 and all participants were recruited from the people's hospital of Leshan.Pregnant women who were attending their first antenatal appointment between 6and 12 weeks of gestation were eligible to participate if they planned to attend perinatal care and deliver at this hospital.Each participant completed the self-administered questionnaire under faceto-face instruction from a trained investigator at the time of her first antenatal examination.The standard questionnaire comprised eight parts, including parental social demographics, maternal reproductive history, maternal illness and drug use history, living environment, living habits, maternal diet and nutrition, and sleep quality.Maternal blood samples (4 ml) were also collected in EDTA during venipuncture, then centrifuged into plasma and blood cells, and stored in the − 80 °C refrigerator, respectively.As of February 2022, a total of 756 pregnant women have participated in the study.
Pregnancies resulting in two or multiple births, induced labor or miscarriage, and fetuses with birth defects were excluded.In addition, pregnant women who have not yet delivered and have no pregnancy outcomes also were excluded.Gestational age was calculated from the first day of the last menstrual period and ultrasound examination.The definition of preterm birth and low birth weight were gestational age of < 37 completed weeks (< 259 days) and < 2500 g (a birth weight of less than 2.5 kg), respectively.

Categories of NVP
Each participant was asked to complete a modified version of the Pregnancy-Unique Quantification of Emesis and Nausea (PUQE) scale developed in 2018 by American College of Obstetricians and Gynecologists (ACOG) [30] before 12 weeks of gestation.The total score from the modified PUQE index ranges from 3 to 15 points, with a higher score indicating more severe NVP.The participants were categorized into one of four different groups according to the composite sum of the PUQE category scores: normal group if the score was 3 points of lower (≤ 3 points), mild NVP if between 4 and 6 points, moderate NVP if between 7 and 12 points, and severe NVP if the score was 13 points or higher (≥ 13 points).

DNA extraction and genotyping
Genomic DNA was extracted from peripheral blood cells with magnetic bead method (BioTeke, Wuxi, China) according to the recommended protocol.SNPs in the GDF15 and LRRC25 gene of chr19p13.11and the intergenic region of chr4q12 were selected based on the following principal criteria: (1) an association with diseases in previous studies or expression level of the protein (2) a minor allele frequency > 0.05 in Han Chinese.In total, 11 SNPs were selected.The SNPs were genotyped using next generation sequencing iGeneTech Biotechnologies Inc. (Beijing, China).More detailed information about the studied genetic variants and genotyping is presented in Supplementary Appendix F, Table S1.
For quality-control assessment, genotyping was repeated in 10% of samples, and the consistency rate was 100%.

Statistical analysis
Covariates included maternal age (Age at the last menstrual period, < 35 years, ≥ 35 years), maternal ethnicity (Han, others), maternal education level (Senior high school or below, University or above), maternal pre-pregnancy body mass index (ppBMI) (BMI = weight/(height × height) (kg/m 2 ) before known pregnancy, ≤ 18.5, 18.5-24, ≥ 24), intended pregnancy (Whether the pregnancy was planned, Yes or No), parity (Nullipara, Multipara), abnormal pregnancy history (Whether the pregnant woman had a spontaneous abortion, induced abortion, stillbirth, or birth defect, Yes or No), parental smoking or ETS exposure (Whether either/ both of the parents smoked during the three months prior to the first trimester, Yes or No), maternal alcohol consumption (Whether the mother drank during the first trimester, Yes or No), folic acid supplements (Whether supplemented with folic acid during the first trimester, Yes or No).Chi-square statistics was used to evaluate differences in covariates between the cases and controls.
Relative ratio (RR) was calculated by unconditional logistic regression analysis to evaluate the association between NVP and preterm birth or low birth weight using the software SAS version 8.02 (SAS Institute, Cary, North Carolina, USA).
Hardy-Weinberg equilibrium was assessed in the controls using Plink software (http:// pngu.mgh.harva rd.edu/ ~purce ll/ plink/).Unconditional logistic regression analysis was performed to investigate the association between individual genetic polymorphisms and NVP using Plink software.
All analyses were adjusted for covariates/potential confounders.False discovery rate (FDR) correction of multiple hypothesis testing was performed.Two-sided P < 0.05 was considered statistically significant.

Descriptive characteristics of the study participants
According to the inclusion and exclusion criteria, 413 pregnant women were available for the analysis, including 373 normal controls, 33 PTB, and 20 LBW.The baseline characteristics of the participants are presented in Table 1.The differences between the control group and PTB group were statistically significant for maternal age, parity, abnormal pregnancy history.The variables were not different between control group and LBW group.

Association between NVP and the risk of PTB as well as LBW
Among the 413 pregnant women analyzed, the incidence of nausea and vomiting was 85.5% (n = 353) in the first trimester.The numbers of normal, mild, moderate and severe NVP were 60, 160, 176, 17, respectively.There were 33 cases of PTB and 20 cases of LBW.NVP distribution in control and case groups, and the RR values between NVP severity and preterm birth are presented in Table2, the results of grouping by sex of newborn are shown in Supplementary Appendix F, Table S2.When compared with pregnant women without NVP, women with mild or moderate or severe NVP were not associated with the risk of PTB, neither in male infants or female infants.Similarly, no significant association was found between NVP and LBW.

Association between gene polymorphisms and the risk of NVP
The genotype distribution for polymorphisms of GDF15, LRRC25 and one intergenic region in control subjects were all consistent with the Hardy-Weinberg equilibrium  hypothesis (P > 0.05, Supplementary Appendix F, Table S3).Table 3 shows the association between single gene loci polymorphisms and the risk of NVP severity when assuming various genetic models.However, none of SNP polymorphisms was significantly associated with the risk of NVP severity in all comparison models.

Discussion
Previous studies lacks consensus about whether nausea or vomiting pregnancy increases the risk of PTB and LBW.In this prospective study, we did not observe an association between NVP in the first trimester and adverse birth outcomes including preterm birth as well as low birth weight, which was similar to the results of a China study showing no differences of PTB and LBW between women with and without severe vomiting [23].Another observational cohort study in rural Nepal reported no significant association between symptoms of nausea, vomiting and poor appetite during the first trimester and PTB [13].
A cohort study in Norwegian showed that NVP was not associated with PTB, but associated with LBW [19].A Hungarian Case-Control study reported no statistical difference between studied groups for low birth weight, but reduced risk for preterm birth among mothers with severe NVP [18].These two results were partially similar to our findings.Whereas our results were inconsistent with the findings of two recent China studies, one showed that vomiting in the first trimester increased the risk for PTB [10], another observed that male infants born to women with NVP at the first trimester and no NVP at the second trimester were more likely to have LBW [12].Besides, some studies reported an association between NVP and increased risk of PTB [9,11], or LBW [9,11,14].However, other studies reported lower risk of PTB [17,18], or LBW [19] in pregnant women who experienced NVP.
The discrepancies across studies may be due to the heterogeneity of populations.
For example, a large cohort study which was conducted among Chinese women found an increased risk of PTB in women who experienced vomiting in the first trimester [10], a study from Hungarian indicated a significant association of vomiting with a decreased risk of PTB [18], no association was found between maternal vomiting and PTB in a study form USA [31].The conflicting results can likely be explained by differences in classification of NVP and time of exposure.Some studies classified NVP into four categories: no NVP, mild, moderate, and severe NVP [17].Some studies classified NVP into three categories: no NVP, nausea only, and nausea/vomiting [19,32].Some studies classified NVP into two categories: no NVP, and NVP [11,13], or no NVP, and vomiting only [10,14].Some studies evaluated the effects of NVP exposure throughout the pregnancy [9,11,19], others only assessed the effects of NVP in the first trimester [10,14,17], and still others analyzed effects of NVP in the first, second, and third trimesters [13,14], respectively.Other possible reasons for inconsistent results may also be due to differences in sample size.The statistically significant associations between NVP and pregnancy outcomes were commonly found in studies with larger sample sizes [10,[17][18][19], while no associations were observed in studies with smaller sample sizes [31,32], including the current study.
There are different speculations about the underlying pathophysiology of the association between NVP in pregnancy and birth outcomes.One hypothesis speculate that from an evolutionary adaptation perspective, nausea and vomiting in pregnancy are thought to a defensive strategy in terms of expelling harmful foods, or teratogenic substances such as infective microorganisms [1,20,21,33,34], and lead to changes in the maternal dietary intake [35], this can lead to positive effects on birth outcome.However, the common hypothesis about the harmful effect of NVP propose that daily maternal nausea and vomiting may also lead to suboptimal maternal nutrition, which may subsequently result in suboptimal fetal nutrition and developmental adaptations [36].Moreover, NVP may also affect maternal and fetal physiology through dehydration and the modulation of stress-related risk factors [37].In addition, nausea and vomiting in pregnancy have been associated with pregnancy complications such as anemia [38], preeclampsia [23,38,39], eclampsia [38], which are risk factors for adverse birth outcome.
This observational cohort study also showed that there were no significant association between 11 SNPs (9 SNPs in GDF15 gene, 1 SNP in LRRC25 gene, 1 SNP in intergenic region of chr4q12) and the risk of NVP.GDF15 gene encodes a TGF-β superfamily member that is expressed at its highest levels in the trophoblast cells of the placenta [40], and is believed to suppress production of proinflammatory cytokines to facilitate placentation and maintain pregnancy [41].The statistically significant association of GDF15 gene polymorphisms with NVP was first reported in a genome-wide association study [27].One study suggested that the SNP rs16982345 in GDF15 gene was associated with recurrence risk of hyperemesis gravidarum (HG), and might play a role in non-familial HG [28].However, in our study, nine SNPs in GDF15 gene, including rs3787023, rs1058587, rs105510 which are in high linkage disequilibrium (LD) with the rs16982345, were not associated with the risk of NVP, whether mild, moderate, or severe NVP.In addition, a genome-wide association study showed that the SNP rs45543339 in LRRC25 gene was associated with the decreased risk of HG, SNP rs4865234 in chr4q12 was associated with the increased risk of HG [27].But no significant association between these two SNPs and NVP of different degrees in our study.Currently, there have been very few association studies on SNPs polymorphism and NVP, and our results are inconsistent with others.However, our sample size was relatively small, especially when NVP was grouped, and the sample size of severe NVP was even smaller, so our statistical power was relatively poor.More studies are needed to explore the genetic susceptibility of NVP in different populations and provide scientific evidence for individualized counseling and therapy.The present study features several strengths.First, it was conducted among pregnant women, and not limited to HG patients, which allowed us to explore the association between mild or moderate NVP and pregnancy outcome.Second, NVP was measured using the modified PUQE and evaluated in the early pregnancy, so this was relatively objective and had a small recall bias.Third, this was the first study on the relationship between genetic susceptibility and NVP in Chinese pregnant women.However, our results should be considered alongside the limitations mentioned below.For one thing, the relatively small sample size in the present study might limit the statistical power and weaken potential associations.Further studies with a larger sample size are required to confirm or refute our findings.Furthermore, we only analyzed the effects of NVP in the first trimester on preterm birth and low birth weight, and did not continuously observe the effects of NVP in the second and third trimesters.In addition, severe NVP may be treated pharmacologically.We cannot rule out that these treatments could confound the association between NVP and PTB and LBW through pharmacological pathways.
Our study indicated that maternal nausea and vomiting in early pregnancy was not associated with adverse birth outcomes, including PTB and LBW, and the polymorphisms of GDF15 and LRRC25 were not associated with the risk of NVP.Further studies are needed in similar settings to determine possible underlying mechanisms involved and effects on future offspring health.

Table 1
Descriptive characteristics of the participants *Of the 20 low birth weight, 13 were also preterm birth a The exposure was defined from the 3 months before pregnancy to the first trimester

Table 2
Association between NVP and Preterm birth and low birth weight *Adjusted for maternal age, maternal race, maternal education level, pre-pregnancy BMI, intended pregnancy, parity, abnormal pregnancy history, parental smoking or ETS exposure, maternal alcohol consumption, folic acid supplements

Table 3
Association between SNP polymorphisms and NVP