Low fetal fraction in obese women at first trimester cell-free DNA based prenatal screening is not accompanied by differences in total cell-free DNA

Abstract

Objective

Reasons for first trimester non-invasive prenatal screening (NIPS) test failure in obese women remain elusive. As dilution from maternal sources may be explanatory, we determined the relationship between obesity, fetal fraction (FF), and total cfDNA using our NIPS platform.

Methods

We assessed differences in first trimester (≤14 weeks) FF, indeterminate rate, and total cfDNA between obese (n=518) and normal weight women (n=237) after exclusion of confounders (anticoagulation, autoimmunity, aneuploidy) and controlling for covariates.

Results

FF was lower, and the indeterminate rate higher, in obese compared to controls (9.2% ± 4.4 vs. 12.5% ± 4.5, p<0.001 and 8.4 vs. 1.7%, p<0.001, respectively), but total cfDNA was not different (92.0 vs. 82.1 pg/uL, p=0.10). For each week, the FF remained lower in obese women (all p<0.01) but did not increase across the first trimester for either group. Obesity increased the likelihood of indeterminate result (OR 6.1, 95% CI 2.5, 14.8; p<0.001) and maternal BMI correlated with FF (β −0.27, 95% CI −0.3, −0.22; p<0.001), but not with total cfDNA (β 0.49, 95% CI −0.55, 1.53; p=0.3).

Conclusions

First trimester obese women have persistently low FF and higher indeterminate rates, without differences in total cfDNA, suggesting placental-specific mechanisms versus dilution from maternal sources as a potential etiology.

INTRODUCTION

Non-invasive prenatal screening via analysis of cell-free DNA (cfDNA) in maternal plasma is increasingly used as a prenatal screening tool in both high and low risk populations.^1–3 The proportion of cfDNA derived from the placenta, commonly known as the “fetal fraction”, is detected in maternal plasma as early as 10–11 weeks gestation² and increases with advancing gestation, improving test characteristics.^4–6 As the fetal fraction may be vulnerable to various factors, there is no singular ideal gestational age to minimize variability across patients, although, it is most commonly performed in the first trimester.³ As a placental derivative, the fetal fraction has also been posited as a potential biomarker for the prediction of adverse pregnancy outcomes,^7–10 which may be most relevant in high-risk cohorts, such as obese gravidas.

It is well established that a higher rate of an indeterminate result occurs in the obese population, likely due to low fetal fractions, as lower fetal fractions are reported in obese women across all gestational ages.^3,11,12 This is problematic as aneuploidies are over-represented in the setting of indeterminate results due to low fetal fraction^13–15, potentially leading to both more invasive procedures and missed diagnoses in this population. Delaying cfDNA-based non-invasive prenatal screening (NIPS) in obese women has been proposed by some,¹¹ but discouraged by others.¹² Certainly, this strategy decreases its utility as a first trimester screen and may limit pregnancy decision-making. DNA sequencing methods may also play a role, with next-generation methods likely performing better at lower fetal fraction thresholds.^12,16 Understanding mechanisms underlying these differences in the obese population is of great importance. Limited studies to date suggest dilution from maternal sources of cfDNA, such as excess adipose tissue, as a possible explanation for lower fetal fraction in this high risk cohort, however, these studies have not included first trimester samples, when NIPS is most commonly performed.^17,18 Currently available data regarding indeterminate results and fetal fraction values in the obese population is also limited by the use of varied platforms, inconsistent fetal fraction thresholds, limited obstetric characterization, and lack of incorporation of fetal sex.^{5,6,11,12,19,20}

We sought to determine the relationship between maternal obesity and first trimester NIPS test characteristics, including fetal fraction, indeterminate result rate, and total cfDNA, after exclusion of factors known to influence these test characteristics and with the incorporation of important covariates. We utilized a clinically validated in-house developed low-pass whole genome platform for all samples, coupled with comprehensive maternal medical and obstetrical data. We hypothesized that compared to normal weight women, obese women will have lower fetal fractions at each week of gestation in the first trimester and a less pronounced rise in fetal fraction across the first trimester. Correspondingly, obesity will be associated with an increased likelihood of an indeterminate result. Lastly, we hypothesized that first trimester total cfDNA concentrations will be higher in obese women compared to normal weight women.

METHODS

This is a single institution retrospective cohort study using data from NIPS performed between May 2017 and December 2019. Since May 2017, the University of Washington has clinically used an internally developed and validated NIPS assay that employs low-pass whole genome sequencing of cfDNA extracted from maternal plasma to provide a high-quality screen for autosomal and sex chromosome aneuploidies.

Study Population

All patients with singleton gestations who completed first trimester NIPS through the University of Washington between the study period were eligible for inclusion (n=1520). Given the intent of prenatal screening, we restricted our primary analysis to first trimester samples (≤14 weeks) for which body mass index (BMI) and fetal fraction were available at the time of NIPS draw (n=1105). We excluded those with suspected fetal aneuploidy on NIPS, those on anticoagulation (enoxaparin or heparin) at the time of NIPS draw, and those with autoimmune disease, as these factors can affect the fetal fraction,^14,21–24 resulting in a final study population of n=1032 (Figure 1). We defined obesity as BMI ≥30kg/m² and normal weight as BMI <25kg/m². We excluded overweight women (BMI 25.0–29.9kg/m², n=277) from our primary analysis comparing fetal fraction, indeterminate result rate, and total cfDNA concentration to avoid confounding in this middle category. As we included BMI as a continuous variable for regression analyses, all participants were included in these analyses. Given the in-house nature of our platform, we have access to detailed test characteristics as well as medical, obstetric, and neonatal data, allowing for review of any notable cases. Any such cases, particularly related to abnormal test results, were reconciled by CML and RS. Fetal sex was confirmed via obstetric/neonatal records. Institutional Review Board approval was obtained from the University of Washington (STUDY00005540). Study data was collected and managed using REDCap electronic data capture tools hosted at the University of Washington.^25,26

Figure 1:

Flowchart of hierarchical exclusions to identify the study population.

Sample collection and quantification of cfDNA

Whole blood from Streck (BCT1) tubes was centrifuged and plasma was isolated as per the package insert. Streck tubes are cell-stabilizing and have been shown to be reliable up to 7 days post-collection and tubes are stored at room temperature. Once plasma is separated, cfDNA is immediately extracted using the QIAsymphony Circulating DNA Kit. Total cfDNA concentration was measured by Qubit fluorometry, a highly sensitive method for quantification of intact double-stranded DNA (pg/uL). The Agilent TapeStation workflow, an automated electrophoresis system, was used to assess the size and integrity of the DNA and functions as a critical quality control step throughout next-generation library preparation, hybridization capture, and sample pooling before sequencing.

Sequencing and quantification of fetal fraction

Library preparation is initiated within 24 hour post-extraction of cfDNA. Following library preparation with KAPA HyperPrep for adapter and index ligation followed by Agencourt AMPureXP purification and amplification, libraries were pooled using an equimolar strategy. Libraries were sequenced using an Illumina NextSeq 500 High Output 75 cycle kit with a 37bp paired-end read configuration. On average, each cfDNA sample was sequenced to a depth of approximately 20 million paired-end reads, corresponding to an average genome depth of 0.5X. Reads were aligned to the human reference genome (hg19) with Bowtie (version 1.1.2), and run metrics are calculated with Picard (version 1.141). The placental-derived fraction for each sample is calculated either by the percent of reads that align to the Y chromosome, or a custom bioinformatic algorithm based on the aggregate length distribution in sequencing reads for samples in which the Y chromosome is not present (i.e. female fetuses).²⁷ An indeterminate result was noted if the fetal fraction was <4%. Failures due to technical issues were purposely excluded from the indeterminate group as these were related to collection or processing errors.

Statistical Analysis

Our primary outcome was to compare the fetal fraction and total cfDNA concentration between obese and normal weight women in the first trimester. Subsequently, we evaluated the differences in these two parameters for each week of the first trimester, along with differences in total cfDNA concentration for differing BMI categories (normal weight, overweight, and class I, II, and III obesity). Categorical variables were compared used ∑², Fisher’s exact, or Mann-Whitney tests as appropriate. Continuous variables were tested for normality and were compared using the t-test or Kruskal-Wallis test as appropriate. ANOVA was used to compare the change in fetal fraction across the first trimester in normal weight and obese populations separately. A nonparametric test for trend was used to compare the change in total cfDNA concentration across the first trimester given non-normality of the data. Multivariable logistic regression was performed to determine the likelihood of an indeterminate result in obese women, while controlling for gestational age draw, maternal age at delivery, gravidity, and fetal sex. Linear regression analyses were performed to determine the association between maternal BMI at the time of sample collection and both fetal fraction and total cfDNA concentration, controlling for the relevant covariates listed above. Because the fetal fraction is more reliably estimated in pregnancies with male fetuses, we repeated all of our fetal fraction analyses to include only those with pregnancies with male fetuses. STATA/IC 16.1 (Statacorp, College Station, TX) was used for all statistical analyses.

RESULTS

Of the 1032 NIPS results that met our inclusion criteria, n=518 were from normal weight, n=277 from overweight, and n=237 from obese women. As expected, the obese population was older, had a higher BMI, higher gravidity, and advanced maternal age as the more common indication for NIPS (all p<0.01) [Table 1]. There were no differences with respect to maternal race, use of assisted reproductive technologies, or fetal sex between groups. The obese and overweight populations had NIPS drawn at a slightly later gestational age compared to normal weight women (12.2 vs. 12.0 weeks, p<0.001) [Table 1].

Table 1:

Study population demographics for normal weight, overweight, and obese women from first trimester samples.

	Normal Weight (n=518)	Overweight (n=277)	Obese (n=237)	p-value
BMI at sample collection (kg/m2)a	22.2 ± 1.8	27.0 ± 1.3	35.8 ± 4.9	<0.001
Maternal age at delivery (years)a	35.2 ± 4.0	35.9 ± 4.1	36.5 ± 4.4	<0.001
White raceb	335 (64.7)	187 (67.5)	154 (65.0)	0.71
Median gravidityc	2 (1–3)	2 (1–4)	3 (2–5)	<0.001
Assisted reproductive technologyb	39 (7.5)	23 (8.3)	21 (8.9)	0.81
Indication for NIPSd
Advanced maternal age	325 (63.5)	194 (71.1)	190 (80.2)	<0.001
First line screening	167 (32.5)	68 (24.9)	38 (16.0)
Abnormal serum screening	0 (0.0)	0 (0.0)	1 (0.42)
Ultrasound abnormality	15 (2.9)	8 (2.9)	5 (2.1)
Other	6 (1.2)	3 (1.1)	3 (1.3)
Gestational age at sample collection (weeks)a	12.0 ± 0.9	12.2 ± 0.9	12.2 ± 0.8	<0.001
Female fetal sexb	209 (44.2)	113 (46.5)	86 (43.8)	0.90

Data are mean ± standard deviation, n (%), or median (interquartile range).

BMI, body mass index

NIPS, non-invasive prenatal screening

cfDNA, cell-free DNA

^a,One-way analysis of variance

^b,Chi-square test

^c,Kruskal-Wallis equality-of-populations rank test

^d,Fisher’s exact test

The mean fetal fraction was significantly lower in obese compared to normal weight women (9.2% ± 4.4 vs. 12.5% ± 4.5, p<0.001), and correspondingly, the rate of indeterminate result was higher in the obese population (8.4% vs. 1.7%, p<0.001) [Table 2]. In some cases (n=9 normal weight, n=18 obese), repeat NIPS was drawn after an initial indeterminate result (mean interval 1.2 weeks for normal weight and 2.1 weeks for the obese population). The rate of persistent indeterminate results was high and not significantly different between groups (22.2% normal weight vs. 50.0% obese, p=0.23). Total cfDNA concentration did not differ between groups (median 82.1 pg/uL for normal weight vs. 92.0 pg/uL for the obese population, p=0.10) [Table 2]. There was one notable outlier in our normal weight group with a total cfDNA concentration of 1380 pg/uL. Review of this chart, including medical and delivery data, did not identify any obvious reason for this increased value. Exclusion of this outlier did not change our results (p=0.09).

Table 2:

Differences in NIPS test characteristics between normal weight and obese individuals

	Normal Weight (n=518)	Obese (n=237)	p-value
Fetal fraction (%)a	12.5 ± 4.5	9.2 ± 4.4	<0.001
Indeterminate resultb	9 (1.7)	20 (8.4)	<0.001
Total cfDNA concentration (pg/uL)c	82.1 (61.3 – 112.0)	92.0 (66.3 – 122.0)	0.10

Data are mean ± standard deviation, n (%), or median (interquartile range).

NIPS, non-invasive prenatal screening

cfDNA, cell-free DNA

^a,Two-sample t-test

^b,Chi-square test

^c,Kruskal-Wallis equality-of-populations rank test

We then investigated the differences in fetal fraction and total cfDNA concentration in each week of the first trimester between the obese and normal weight populations. After 10 weeks gestation, the mean fetal fraction remained significantly lower in each week for obese compared to normal weight women (Table 3, Figure 2). Interestingly, the fetal fraction did not significantly change with each advancing week in the first trimester for both the normal weight (p=0.21) and the obese population (p=0.59). Conversely, the median total cfDNA concentration did not differ between normal and obese women for each week of the first trimester and did not increase across the first trimester for each group individually (Table 4, Figure 3). As differences in the obese population may not be appreciated with grouping all obese women together, we investigated total cfDNA concentration for normal weight, overweight, class I, II, and III obese women separately. Again, no difference in total cfDNA concentration was noted between groups (Table 5). After exclusion of the previously identified outlier in the normal weight group, and another outlier in the overweight group (with a cfDNA concentration of 1570 pg/uL), our results were unchanged (p=0.09).

Table 3:

Mean fetal fraction for each week of the first trimester among normal weight and obese women.

	≤10w0d (n=19)	10w1d–11w0d (n=96)	11w1d–12w0d (n=215)	12w1d–13w0d (n=336)	13w1d–14w0d (n=89)	p-value
Normal weight	10.9 ± 4.1	11.7 ± 4.3	12.7 ± 4.6	12.8 ± 4.5	12.4 ± 4.8	0.21b
Obese	9.0 ± 8.4	8.1 ± 4.5	8.8 ± 4.4	9.5 ± 4.5	9.6 ± 4.0	0.59b
p-value	0.69a	<0.001a	<0.001a	<0.001a	0.008a	--

Data are mean ± standard deviation.

^a,Kruskal-Wallis equality-of-populations rank test

^b,ANOVA

Figure 2:

Change in the fetal fraction for each first trimester week in normal weight and obese women.

Data are mean ± SD

Table 4:

Total cell-free DNA concentration (pg/uL) for each week of the first trimester among normal weight and obese women.

	≤10w0d (n=19)	10w1d–11w0d (n=91)	11w1d–12w0d (n=207)	12w1d–13w0d (n=322)	13w1d–14w0d (n=82)	p-value
Normal weight	88.3 (48.4 – 109.0)	81.7 (65.2 – 107.0)	79.7 (59.5 – 114.5)	81.9 (61.7 – 117.0)	87.5 (67.3 – 112.0)	0.43b
Obese	108.3 (67.7 – 149.0)	91.3 (72.0 – 112.0)	97.6 (61.1 – 125.0)	91.5 (62.4 – 122.5)	93.0 (69.0 – 116.0)	0.71b
p-value	0.59a	0.34a	0.24a	0.56a	0.72a	---

Data are median (interquartile range).

^a,Kruskal-Wallis equality-of-populations rank test

^b,Non-parametric test of trend for ranks

Figure 3:

Change in the total cfDNA concentration for each first trimester week in normal weight and obese women

Data are median (interquartile range)

cfDNA, cell-free DNA

ns, not significant

Table 5:

Total cell-free DNA concentration (pg/uL) by BMI category for the entire study population.

	BMI (kg/m2)
	< 25.0 (n=491)	25.0 – 29.9 (n=265)	30.0 – 34.9 (n=120)	35.0 – 39.9 (n=70)	≥ 40.0 (n=40)
Total cfDNA concentration (pg/uL)	82.1 (61.5 – 112.0)	82.5 (59.6 – 116.0)	95.9 (69.4 – 122.5)	74.2 (54.7 – 111)	99.5 (80.2 –130.0)
p-value	0.09a

Data are median (interquartile range).

BMI, body mass index

cfDNA, cell-free DNA

^a,Non-parametric test of trend for ranks

Logistic regression analysis revealed that maternal obesity was significantly associated with an increased likelihood of a first trimester indeterminate NIPS result (OR 6.1, 95% CI 2.4, 14.8; p<0.001) after controlling for gestational age at sample collection, maternal age at delivery, gravidity, and fetal sex. Linear regression revealed a significant negative correlation between increasing maternal BMI and fetal fraction (β −0.27, 95% CI −0.3, −0.22; p<0.001), after controlling for the above listed covariates (Figure 4). No correlation was noted between maternal BMI and total cfDNA concentration in either the unadjusted (β 0.33, 95% CI −0.56, 1.22, p=0.46) [Figure 5] or adjusted models (β 0.49, 95% CI −0.55, 1.53; p=0.3).

Figure 4:

Linear regression illustrating the relationship between fetal fraction and maternal BMI at the time of sample collection (n=1032).

BMI, body mass index

Figure 5:

Linear regression illustrating the relationship between total cfDNA concentration and maternal BMI at the time of sample collection (n=1032).

cfDNA, cell-free DNA

BMI, body mass index

Analysis of our data with inclusion of only male fetuses revealed no differences in our findings (both univariate and multivariate regression), with notably even more discrete differences between groups with respect to fetal fraction and continued lack of difference between groups with respect to total cfDNA concentration (data not shown).

COMMENT

Compared to normal weight women, obese women had significantly lower fetal fractions and a higher rate of indeterminate result on first trimester NIPS using our low-pass whole genome sequencing platform. The likelihood of an indeterminate result was associated with obesity, and this relationship persisted when controlling for relevant covariates. Additionally, after 10 weeks, in each week of the first trimester, obese women had lower fetal fractions compared to normal weight women. The fetal fraction, however, did not appreciably increase with each advancing week in the first trimester for both obese and normal weight women. Repeat NIPS testing following an indeterminate result revealed persistently high indeterminate rates in both groups. Concordantly, increasing maternal BMI was significantly negatively associated with fetal fraction. Importantly we found no differences in total cfDNA concentration between normal and obese women in the first trimester and maternal BMI did not correlate with total cfDNA concentration.

Our results confirm those by others: that fetal fraction on NIPS is lower in the setting of increasing maternal weight.^{11,12,17,28–30} Our results, along with others,¹² suggests that delaying NIPS draw within the first trimester in obese women may not be an effective strategy to increase the likelihood of a determinate result by avoiding test failure due to an insufficient fetal fraction. It is likely that delaying until later in pregnancy will overcome the low fetal fractions seen in the first trimester, however, this practice may limit reproductive options for these women. The high rates of repeat indeterminate results in our population is similar to those seen by others,¹² and is consistent with our finding that fetal fraction does not appreciably change in obese women. Importantly, we found that cfDNA concentrations did not differ between groups, suggesting that the phenomenon of low first trimester fetal fraction in obese women may not be solely explained by dilution from excess maternal sources of cfDNA.

We add to a growing body of literature solidifying the importance of maternal weight on NIPS test performance via its effect on the fetal fraction. The exact etiology of a lower fetal fraction in obese women is not known. Dilution from maternal sources of cfDNA has been proposed by several investigators, however, these studies are notably different from ours, most importantly with respect to the gestational age at sample collection. Vora et al studied those undergoing scheduled Cesarean delivery at term, and although they noted a trend towards increased total cfDNA (measured via real time quantitative PCR amplification of glyceradehyde-3-phosphate dehydrogenase, G6PDH) with higher pre-gravid BMI in the obese population (n=16), this trend was not significant.¹⁷ A prior study with large numbers (n=406), again using qPCR-based techniques to measure total cfDNA, noted that maternal BMI correlated with total cfDNA in the second and third trimesters, but did not include any first trimester samples.¹⁸ Additionally, the mean BMI for their second trimester study population was 24.1 kg/m², with the majority of their population having a BMI<30kg/m². For evaluation of the placental-derived fraction (i.e. fetal fraction), both of these studies included only male fetuses due to reliance on amplification of Y-specific genes for calculations. Canick et al evaluated differences in fetal fraction and total cfDNA in a dataset of over 1400 women and although a trend towards higher total cfDNA (reported as DNA copies/mL) was noted with advancing maternal weight, this study grouped approximately 50% first trimester and 50% second trimester samples.³⁰ Notably, this latter study was able to include both male and female fetuses due to reliance on methylation-based DNA discrimination for determination of the fetal fraction. Studies to date confirm that increasing maternal weight is associated with lower fetal fractions, but additional research regarding contributing mechanisms is necessary.

Findings in this study, in conjunction with others, further drives the impetus to determine the optimal testing platform and timing of prenatal screening in obese gravidas. Results from our in-house developed and clinically utilized platform suggests that obese women with failed NIPS due to low fetal fraction may not benefit from repeat testing in the first trimester given minimal increase in fetal fraction with advancing gestation. Secondly, persistent indeterminate results remain high in obese women, further suggesting a low yield with this approach. Larger studies are needed to answer these critically important questions.

Our total cfDNA concentration results are in contrast to the few other studies evaluating this parameter,^17,18 which have suggested that maternal sources of plasma cfDNA in obese women lead to a decrease in the fraction contributed by the placenta. Given notable differences in the trimester of evaluation, it is plausible that increased adipose cell death, thought to be a cause at least towards the end of pregnancy,³¹ may not be at play in the first trimester, and that the maternal contribution from adipose tissue turnover may overwhelm the rise in the placental contribution in the second/third trimester. Our results suggest that alternative or additional factors may contribute to a low first trimester fetal fraction in obese women, such as altered placental pathology. Our results using only those samples with male fetuses is consistent with this given the directly measured placental contribution. This is also in keeping with studies demonstrating the value of first trimester fetal fraction in predicting later adverse pregnancy outcomes related to placental dysfunction, such as preeclampsia and intrauterine growth restriction, after accounting for maternal BMI.^7,8 Additional larger studies of first trimester NIPS results, most relevant for prenatal screening, are needed to more completely elucidate mechanisms for the unique test characteristics in obese women.

Our study utilized a single validated platform in current clinical use with a uniform fetal fraction cutoff for interpretation (≥4%), removing any issues related to test performance of differing platforms. Unlike other reports of the influence of maternal weight on fetal fraction, we restricted our analysis to only include cases from the first trimester given that this is the most common period for NIPS. Due to the in-house nature of our testing platform, we have access to detailed laboratory and obstetric data, thus we were able to reconcile all cases which resulted in a “no-call”. Our designation of a result as being indeterminate solely includes those due to low fetal fraction and excludes any other potential etiologies for a “no-call” result. Our approach to exclude cases with suspected aneuploidy on NIPS, those on anticoagulation at the time of NIPS draw, and those with autoimmune disease avoids potential confounders known to influence the fetal fraction. Additionally, in line with contemporary research regulatory guidelines, we included fetal sex as a biological variable in all regression analyses and did not exclude any pregnancies based on fetal sex.^32,33 We were well suited to delineate obesity as an important moderator of fetal fraction given our study population size and the breadth of representation of maternal weights in our cohort. Indeed, 23% (237/1032) of our entire study population was obese. Comparisons were further strengthened by exclusion of overweight gravidas in our primary analyses so as to clearly evaluate the implications of our exposure of interest (obesity) compared to normal controls. With respect to our total cfDNA results, we may be underpowered to detect differences between groups, leading to a possible type II error. Additionally, the cfDNA concentration values at each time point were highly variable, suggesting likely multifactorial contributing mechanisms. Although regression analysis of BMI and total cfDNA approached significance after removing two outliers, review of detailed medical records at the time of draw (and for the entire pregnancy) along with review by our laboratory personnel for any sample anomalies were unrevealing, suggesting that these may be representations of the normal variance in total maternal plasma cfDNA (of note, both of these cases had normal fetal fractions of 12 and 13%). There are several inter-individual and intra-individual differences in cfDNA concentrations that do not reflect pathologic conditions, such as post-exercise.^34–36 Nonetheless, the significant differences we observe at the population level remain notable. Unfortunately, sample size calculations in this setting are fraught with error as no published values for normal maternal first trimester cfDNA concentrations are available.

Interpretation of our results is limited by the retrospective nature of our study. Importantly, our study does not confirm mechanisms contributing to low fetal fraction in obese women, but suggests that dilution from maternal sources of cfDNA is not primarily responsible for the low fetal fractions noted in the first trimester and that multifactorial processes may be at play. We encourage others with access to larger sample sizes of first trimester NIPS test characteristics to evaluate total cfDNA and its relationship to maternal BMI after excluding relevant confounding cases and controlling for important covariates.

In summary, we found that maternal obesity is associated with an increased likelihood of an indeterminate result due to low fetal fraction on first trimester NIPS compared to normal weight women. The fetal fraction did not appreciably increase with each advancing week in the first trimester, for both normal weight and obese women, and the rate of persistent indeterminate result on repeat assessment remained high for both groups. Increasing maternal BMI was associated with decreasing fetal fraction, however, it was not associated with changes in the total cfDNA concentration in the first trimester, suggesting that dilution from maternal sources is unlikely a major contributor to this finding. Further studies, specifically those focused on mechanisms, are necessary to better understand the effect of maternal BMI on first trimester NIPS test characteristics.

BULLETED STATEMENTS.

A. What’s already known about this topic?

• Maternal obesity influences cfDNA based non-invasive prenatal screening test performance.

• Dilution from maternal sources of cfDNA may explain this phenomenon, however, data restricted to the first trimester is lacking.

What does this study add?

• Fetal fraction remains low throughout the first trimester in obese women.

• First trimester total cfDNA does not differ between obese and normal weight women, suggesting additional or alternative mechanisms to dilution from maternal cfDNA sources as an explanation for the low fetal fraction.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.