The association between fetal fraction and pregnancy-related complications among Chinese population.

To examine the association of fetal fraction with a wide spectrum of pregnancy-related complications among Chinese population, we carried out a single-institution retrospective cohort study of women with negative Noninvasive prenatal testing (NIPT) results and singleton pregnancies between May 2018 and May 2020. Indicators of pregnancy-related complications were examined individually, including preterm birth, low birth weight, hypertensive disorders of pregnancy, gestational diabetes, oligohydramnios and intrahepatic cholestasis. We evaluated disease odds ratios (ORs) and 95% confidence intervals (CIs), after controlling for potential confounders including body mass index (BMI), maternal age and gestational week at NIPT. A total of 3534 women were included in our analyses. Women with fetal fraction<15.15% had increased risk of gestational hypertension (OR 4.41, CI [1.65, 12.45]) and oligohydramnios (OR 2.26, CI [1.33, 3.80]) compared to women with fetal fraction≥15.15%. No significant associations with fetal fraction were found for preterm birth, low birth weight, gestational diabetes, and intrahepatic cholestasis. In Summary, fetal fraction is inversely associated with the risk of gestational hypertension and oligohydramnios.

Introduction

Pregnancy-related complications are the leading cause of the morbidity and mortality of pregnancy. Nearly one fifth of all pregnant women are hospitalized before delivery due to complications [1]. Common pregnancy-related complications include hypertensive disorders of pregnancy (HDP) and gestational diabetes [2-4]. In addition, other complications such as intrahepatic cholestasis are associated with some specific adverse pregnancy outcomes including preterm birth and pregnancy loss [5]. It is desirable to identify patients at risk for pregnancy-related complications before the onset of symptoms to better manage diseases and reduce mortality.

Fetal cell-free DNA (cfDNA) is a candidate biomarker for pregnancy related complications. Cell free DNA in the blood plasma of pregnant women is composed of DNA fragments mainly from the mother herself, but also from the fetus. Fetal cfDNA contributes approximately 10–20% of the total cfDNA [6]. Fetal cfDNA is released into the maternal circulation via apoptosis of placental villous trophoblasts throughout gestation [7]. Noninvasive prenatal testing (NIPT) has become a standard screening test for trisomy 21, 18, 13 and other selected chromosomal abnormalities since its introduction into the clinic in the early 21st century [8]. With the development and commercial availability of NIPT, fetal and maternal cfDNA can be sequenced efficiently and cost-effectively through massively parallel DNA sequencing (MPS). Other than its utility in testing the chromosomal abnormalities of the fetus, NIPT sequencing data can also give reliable fetal fraction estimation in the maternal plasma, defined as the proportion of fetal cfDNA among the total amount of cfDNA, which usually exceeds 4% beginning at 10 weeks’ gestation and peaks between 10 and 21 weeks of gestation [7].

Previous studies have investigated the relationship between fetal fraction and various pregnancy complications including HDP [9-13], gestational diabetes [14], and preterm birth [10–12, 15]. However, the significance and directionality of these associations were inconsistent. In addition, most of previous studies were conducted in European and American populations. Studies in East Asians, especially in Chinese populations are limited. Thus, combing the rich clinical information and sequencing data from a large single-center NIPT study, we can assess the relationship between fetal fraction and the subsequent development of pregnancy-related complications in Chinese women.

Materials and methods

Study design and data collection

We performed a single-institution retrospective cohort study of all women that underwent NIPT screening from May 2018 to May 2020 at Mianyang People’s Hospital. Women were recruited without any prior indication of genetic diseases or fetal structural abnormalities, following the guideline by the National Health and Family Planning Commission of China (http://www.nhc.gov.cn/ewebeditor/uploadfile/2016/11/20161111103703265.docx). Genetic counseling was provided before the NIPT test and when returning the NIPT result. Only women who had singleton pregnancies and showed no chromosomal aneuploidies from NIPT and with valid fetal fraction estimation and pregnancy complications information were included. Further exclusion criteria were: (1) women with multiple pregnancies; (2) women with missing information on whether singleton or multiple pregnancy; (3) women with missing fetal fraction information; (4) women with a history of chronic hypertension or diabetes.

All NIPT sequencing was performed by the same laboratory (Mianyang People’s Hospital). The cell-free DNA extraction, library construction, sequencing and bioinformatics analysis were performed using protocols as described in a previous study [16]. In brief, the BioelectronSeq 4000 sequencing instrument and the Sequencing Reaction Universal Kit (CapitalBio, Dongguan, People’s Republic of China) were used for MPS of plasma cfDNA fragments. Sequencing reads were filtered and aligned to the human reference genome (hg19). The fetal fraction was estimated based on the different fragment length distributions between maternal and fetal cfDNA. Fetal cfDNA fragment tend to have a higher proportion of short plasma DNA fragments (∼130–140 bp; region A) and a lower proportion of long plasma DNA fragments (∼155–175 bp; region B). Locally weighted scatterplot smoothing (LOESS) regression was applied to fit the fetal fraction against reads ratio in features A and B. Male fetuses with fetal fraction estimated from Y chromosome reads were used to train the model parameters [17].

The demographic characteristics and clinical information on pregnancy-related complications were extracted from electronic medical records. Body mass index (BMI) was calculated from the height and weight data recorded at the time of NIPT test. Parity was dichotomized to either nulliparous or multiparous. Written informed consent was obtained from all participating women and the study received approval from the Institutional Review Board at Mianyang People’s Hospital (No. 20201008).

The outcomes of interest were pregnancy-related complications, including low birth weight (<2500g), preterm birth (<37 weeks), gestational diabetes, hypertensive disorders of pregnancy (HDP), oligohydramnios, and intrahepatic cholestasis. HDP include gestational hypertension and preeclampsia. Gestational hypertension was defined as having a systolic blood pressure greater than 140 mmHg or diastolic blood pressure greater than 90 mmHg after 20 weeks of gestation without the evidence of proteinuria, and the blood pressure returning to normal within 12 weeks after the delivery. Preeclampsia was defined as meeting the aforementioned blood pressure criteria plus any of the following complications after the 20th week of pregnancy: proteinuria greater than or equal to 300 mg per 24-hour urine collection; a low platelet count; impaired liver function; signs of kidney problems indicated by the serum creatinine level; pulmonary edema and new-onset cerebral or visual disturbances. Gestational diabetes was defined as diabetes firstly seen in a pregnant woman who had no diabetes before the pregnancy, and was diagnosed by an initial glucose challenge test followed by a 75g oral glucose tolerance test. Intrahepatic cholestasis of pregnancy was defined as severe itchiness and increased serum bile acid concentrations. Oligohydramnios was defined as a single deepest vertical pocket (DVP) of less than 2cm on ultrasound examination at 28–40 weeks.

We analyzed the variable of interest, fetal fraction, as both continuous and dichotomous variables. Since the directionality of the associations between fetal fraction and pregnancy complications were conflicting from previous studies, we firstly treated fetal fraction as a continuous variable to determine the significance and directionality of the relationships. We then carried out analyses treating fetal fraction as a dichotomous variable. A threshold of low or high fetal fraction was defined based on its distribution within the cohort, the results from the associations between continuous fetal fraction and the outcomes of interest, as well as existing literatures [11].

Statistical analysis

Baseline characteristics were compared between the two groups of dichotomous fetal fraction. We compared categorical data using chi-square test and continuous data using student t-test. We calculated odds ratios (ORs) and 95% confidence intervals (CIs) using multivariable logistic regression to determine the associations between fetal fraction and pregnancy-related complications, controlling for gestational age at NIPT, maternal age and BMI. Each pregnancy complication was examined individually. We firstly assessed the association between fetal fraction with HDP, combining gestational hypertension and preeclampsia. We also evaluated the relationships of fetal fraction with gestational hypertension and preeclampsia separately, to show which specific disease contributes more to the fetal fraction-HDP association. For all association tests with each complication, only normal samples were included in the control group.

We hypothesized that relationships of fetal fraction with pregnancy complications could be different for women undergoing NIPT in different trimesters of pregnancy. To examine this hypothesis, we performed stratified analyses based on gestational age at NIPT: first trimester (10–14.9 weeks), second trimester (15 and 22 weeks) and third trimester (>22 weeks) in order to find the optimal time to perform the NIPT screening tests. All analyses were conducted using R version 4.0.3 (R Core Team, 2020). A P<0.05 from the association test was considered statistically significant.

Results

Basic characteristics of study participants

There are 3834 women who underwent NIPT at Mianyang People’s Hospital between May 2018 and May 2020. Twenty-six women had NIPT chromosomal abnormality (a positive rate of 0.68%), which were excluded from the study. We also excluded 8 women with a history of chronic hypertension or diabetes. We finally obtained 3534 cases in the study after further excluding 266 patients without NIPT results, with multiple pregnancy and with missing information on whether singleton or multiple pregnancy. Details regarding the excluded cases were shown in Fig 1.

Fig 1

Flow chart defining the study population.

The flow chart illustrates the filtering steps applied in the study. NIPT, noninvasive prenatal testing.

Fetal fractions within the study population ranged from 4.32% to 50.38%, with a mean value of 18.48% and a standard deviation of 5.28%. The dichotomization cut-off point for fetal fraction was set to the 25th percentile, namely 15.15%. The choice of the cut-off point is based on the results from the distributions of fetal fraction stratified by the traits showing significant associations with continuous fetal fraction, which is mentioned below, as well as previous literatures [11]. The 25th percentile cut-off is also widely used in stratifications in statistical analysis. Baseline characteristics of the two groups of dichotomous fetal fraction are shown in Table 1. We found statistically significant differences between the two groups regarding maternal BMI, gestational age at delivery, and method of delivery. This is not surprising given that associations of fetal fraction with gestational age and maternal BMI are well established in previous publications [18, 19]. However, the distributions of maternal age and gestational age at NIPT, and the percentages of nulliparous and IVF-ET pregnancies were similar between the two groups of pregnant women with the dichotomous fetal fraction.

Table 1

Characteristics of pregnant women with low fetal fraction compared with women with normal fetal fraction.

	Fetal fraction≥25th percentile (N = 2650)	Fetal fraction<25th percentile (N = 884)	P value
Age (mean (SD))	28.51 (3.94)	28.67 (4.15)	0.297
Gestational age at NIPT (mean (SD))	17.77 (2.79)	17.64 (2.61)	0.217
Parity, n (%)
multiparous	984 (37.1%)	335 (37.9%)	0.714
nulliparous	1666 (62.9%)	549 (62.1%)
IVF-ET pregnancy, n (%)
no	2604 (98.3%)	868 (98.5%)	0.765
yes	45 (1.7%)	13 (1.5%)
BMI (mean (SD))	21.89 (2.90)	23.30 (3.52)	<0.001
Gestational age at delivery, (mean (SD))	39.44 (1.03)	39.34 (1.13)	0.010
Method of delivery, n (%)
cesarean	1313 (49.5%)	476 (53.8%)	0.030
normal	1337 (50.5%)	408 (46.2%)

NIPT, noninvasive prenatal testing; IVF-ET, In Vitro Fertilization and Embryo Transfer; BMI, body mass index

The association of fetal fraction with pregnancy-related complications

Table 2 shows associations between continuous fetal fractions and the pregnancy outcomes. The increase of fetal fraction had significant protective effects on hypertensive disorder of pregnancy (HDP), intrahepatic cholestasis and oligohydramnios. When dividing HDP into gestational hypertension and preeclampsia, a significant association was only observed for gestational hypertension. After adjusting for BMI, maternal age and gestational age at NIPT, only the association with gestational hypertension remained significant (P = 0.0484) (Table 2). For every one percent increase of fetal fraction, the odds of gestational hypertension decreased by a factor of 0.89 (OR = 0.89, 95% CI = [0.78, 0.99]). We found no association between the continuous fetal fraction and risks of low birth weight, preterm birth, and gestational diabetes. The fetal fraction distributions comparing women with gestational hypertension versus normal women, women with oligohydramnios versus normal women (S1 and S2 Figs) reveal that the maximum segregation of distribution occurs around the 25th percentile (fetal fraction = 15.15%).

Table 2

Relationships between fetal fraction (continuous variable) and pregnancy-related complications.

Disease (N)	Crude OR (95% CI)	P value	Adjusted OR a (95% CI)	P value
Low birth weight (N = 78)	1.00 (0.96, 1.04)	0.883	1.01 (0.95, 1.07)	0.788
Preterm birth (N = 64)	1.01 (0.96, 1.05)	0.706	1.05 (0.98, 1.11)	0.169
HDP (N = 71)	0.93 (0.89, 0.98)	0.00764	0.92 (0.85, 1.00)	0.0524
Gestational Hypertension (N = 29)	0.89 (0.81, 0.96)	0.00591	0.89 (0.78, 0.99)	0.0484
Preeclampsia (N = 42)	0.96 (0.90, 1.02)	0.21	0.95 (0.85, 1.05)	0.380
Gestational diabetes (N = 459)	0.98 (0.96, 1.00)	0.0857	1.00 (0.98, 1.03)	0.814
Intrahepatic cholestasis (N = 148)	0.96 (0.93, 0.99)	0.0149	0.97 (0.93, 1.02)	0.292
Oligohydramnios (N = 113)	0.95 (0.92, 0.99)	0.0205	0.96 (0.90, 1.01)	0.0922

a. Adjusted for body mass index (BMI), gestational age at NIPT and maternal age

NIPT, noninvasive prenatal testing; HDP, Hypertensive disorder of pregnancy; OR, odds ratio; CI, confidence interval

The associations of the dichotomous fetal fractions (<25th percentile and ≥25th percentile) with pregnancy outcomes are presented in Table 3. We found significant associations of fetal fraction with the risk of HDP and oligohydramnios. For HDP, when considering gestational hypertension and preeclampsia as separate diseases, both were significantly associated with fetal fraction. When adjusted for BMI, maternal age and gestational age at NIPT, the associations with HDP and oligohydramnios remained significant. Compared to pregnant women with fetal fraction≥15.15%, women with fetal fraction<15.15% were 2.49 times more likely to have HDP (OR = 2.49, 95% CI = [1.22, 5.02], P = 0.0111) during pregnancy (Table 3). However, similar to the analyses using continuous fetal fraction, only the association with gestational hypertension remained significant after controlling for potential confounders (P = 0.00343), and the association with preeclampsia became insignificant (P = 0.467). For women with fetal fraction<15.15%, we saw 3.41 times increase in the odds of getting gestational hypertension, compared to women with fetal fraction≥15.15% (OR = 4.41, 95% CI = [1.65, 12.45]). The odds ratio of oligohydramnios for pregnancy women in fetal fraction<15.15% group compared with the fetal fraction≥15.15% group was 2.26 (95% CI = [1.33, 3.80], P = 0.0022), after adjusting for BMI, maternal age and gestational age at NIPT (Table 3). For other traits, we found that fetal fraction was not statistically significantly associated with low birth weight, preterm birth, gestational diabetes and intrahepatic cholestasis.

Table 3

Relationship between fetal fraction (Fetal fraction<25th percentile vs. fetal fraction≥25th percentile ) and pregnancy-related complications.

Disease (N)	Crude OR (95% CI)	P value	Adjusted OR b (95% CI)	P value
Low birth weight (N = 78)	1.11 (0.65, 1.81)	0.695	0.89 (0.35, 1.93)	0.775
Preterm birth (N = 64)	1.48 (0.85, 2.47)	0.148	1.30 (0.52, 2.94)	0.546
HDP (N = 71)	2.65 (1.64, 4.27)	<0.001	2.49 (1.22, 5.02)	0.0111
Gestational Hypertension (N = 29)	3.76 (1.80, 8.01)	<0.001	4.41 (1.65, 12.45)	0.00343
Preeclampsia (N = 42)	2.08 (1.09, 3.85)	0.0218	1.45 (0.50, 3.86)	0.467
Gestational diabetes (N = 459)	1.12 (0.89, 1.40)	0.341	0.82 (0.56, 1.19)	0.310
Intrahepatic cholestasis (N = 148)	1.33 (0.92, 1.91)	0.121	1.25 (0.71, 2.10)	0.419
Oligohydramnios (N = 113)	1.95 (1.31, 2.87)	0.000845	2.26 (1.33, 3.80)	0.0022

a. Fetal fraction≥25th percentile as reference group

b. Adjusted for body mass index (BMI), gestational age at NIPT and maternal age

HDP, Hypertensive disorder of pregnancy; OR, odds ratio; CI, confidence interval

Subgroup analyses

Previous studies showed that the patterns of associations were different when women performed NIPT tests at different trimesters [9, 15]. Thus, we conducted stratified analyses of the associations between fetal fraction and pregnancy complications according to gestational age at NIPT. Ninety-one pregnant women underwent NIPT during the first trimester, three thousand thirty-seven women during the second trimester and four hundred six women during the third trimester (Fig 1). For women who underwent NIPT during the first trimester (<15 weeks), only the trait of gestational diabetes had enough samples (n = 13) for the data analysis. All outcomes of interest had enough observations among the cohort including women who underwent NIPT during the second trimester (15 and 22 weeks). For women who underwent NIPT during the third trimester (>22 weeks), only 2 traits: gestational diabetes (n = 50) and intrahepatic cholestasis (n = 19) had enough samples for the data analysis.

After adjusting for BMI and maternal age, women in the fetal fraction<25th percentile group during the second trimester were significantly more likely to develop HDP and oligohydramnios than women in the fetal fraction≥25th percentile group (Table 4). Women with fetal fraction <15.15% had more than 2 times the odds of having HDP compared to women whose fetal fraction with ≥15.15% (OR = 2.19, 95%CI = 1.03–4.58, P = 0.0386). Specifically, the odds ratio of gestational hypertension for pregnant women in fetal fraction<15.15% group compared with fetal fraction≥15.15% group was 3.32 (95%CI = 1.16–9.76, P = 0.0242, Table 4). However, fetal fraction at the second trimester showed no significant association with preeclampsia after controlling for potential confounders (Table 4). At second trimester, women with fetal fraction<15.15% showed 1.13 times increase in the odds of getting oligohydramnios compared to women with fetal fraction≥15.15% after controlling for BMI and maternal age (OR = 2.13, 95%CI = 1.20–3.70, P = 0.008, Table 4). For women undergoing NIPT during the first and the third trimester, no significant associations were found between fetal fraction and pregnancy-related complications (Table 4).

Table 4

Relationship between fetal fraction (Fetal fraction<25th percentile vs. fetal fraction≥25th percentile ) and pregnancy-related complications stratified by gestational age at NIPT.

Disease (N)	Crude OR (95% CI)	P value	Adjusted OR b (95% CI)	P value
First Trimester
Gestational diabetes (N = 13)	1.44(0.34, 5.35)	0.592	0.61(0.0065, 25.43)	0.799
Second Trimester
Low birth weight (N = 65)	1.13(0.64, 1.93)	0.658	0.67(0.22, 1.62)	0.412
Preterm birth (N = 52)	1.87(1.04, 3.26)	0.0304	1.78(0.69, 4.24)	0.210
HDP (N = 63)	2.69(1.62, 4.47)	0.000127	2.19(1.03, 4.58)	0.0386
Gestational Hypertension (N = 26)	3.45(1.58, 7.65)	0.00181	3.32(1.16, 9.76)	0.0242
Preeclampsia (N = 37)	2.26(1.15, 4.35)	0.0156	1.56(0.53, 4.25)	0.393
Gestational diabetes (N = 396)	1.10(0.85, 1.40)	0.466	0.86(0.57, 1.27)	0.447
Intrahepatic cholestasis (N = 126)	1.33(0.89, 1.95)	0.158	1.21(0.67, 2.10)	0.508
Oligohydramnios (N = 101)	1.57(1.02, 2.38)	0.0371	2.13(1.20, 3.70)	0.008
Third Trimester
Gestational diabetes (N = 50)	1.22 (0.57, 2.47)	0.586	0.40 (0.06, 1.50)	0.235
Intrahepatic cholestasis (N = 19)	1.38 (0.43, 3.82)	0.553	0.79 (0.040, 5.42)	0.839

a. Fetal fraction≥25th percentile as reference group

b. Adjusted for body mass index (BMI) and maternal age

NIPT, noninvasive prenatal testing; HDP, Hypertensive disorder of pregnancy; OR, odds ratio; CI, confidence interval

Discussion

In this retrospective cohort study, we observed significant inverse associations of fetal fraction with the risk of hypertensive disorders of pregnancy (HDP) and oligohydramnios after adjusting for potential confounders. The odds of having HDP for women with fetal fraction<15.15% was nearly 3 folds compared to women with fetal fraction≥15.15%. When considering gestational hypertension alone, the odds ratio increased to a value greater than 4. However, when considering eclampsia alone, the association was no more significant. Also, for pregnant women with fetal fraction<15.15%, they were 2.26 times more likely to have oligohydramnios, compared to pregnant women with fetal fraction≥15.15%. We found no associations of fetal fraction with the subsequent development of low birth weight, preterm birth, gestational diabetes and intrahepatic cholestasis.

We observed a significant association of fetal fraction with the subsequent development of oligohydramnios, which contradicted early data from a smaller study [11]. Limited sample size of the previous study may explain the difference, since their study only included 25 oligohydramnios cases in total [11]. Although the exact cause of oligohydramnios is complicated and still unclear, we speculated that subtle placental structural abnormalities could potentially cause oligohydramnios. This could also potentially reduce the release of fetal cfDNA into mother’s blood, resulting in low fetal fraction of cfDNA. The association between fetal fraction and oligohydramnios has significant potential clinical value. Oligohydramnios increases the risk of neonatal deaths, stillbirths and low birth weight [20]. Because of the association between oligohydramnios and neonatal abnormalities, surveillance by ultrasound is often recommended to women at risk as early as possible, and when needed, pregnant women are admitted to hospitals for frequent screening and to monitor the signs of preterm labor [21]. A low fetal fraction result in the first trimester or early second trimester may help clinicians identify women who warrant frequent ultrasound screening for the amount of amniotic fluid.

Our findings are in accordance with the results from several previous studies that found lower fetal fraction being associated with a higher risk of HDP. For instance, Gerson et al. found statistically significant association of HDP with lower fetal fraction measured in either the first or the second trimester NIPT in their prospectively enrolled cohort of 639 women [11]. Also, Suzumori et al. found fetal cell-free DNA fraction sampled between 10 and 20 weeks’ gestation were associated with HDP in a cohort of more than 5000 Japanese pregnant women [13]. However, in the study by Shook at al., no association of HDP with fetal fraction in the first trimester was found [10]. This is probably due to different definition of fetal fraction cut-offs, since they compared high fetal fraction (≥95th percentile) and normal fetal fraction (between the 5th and 95th percentiles). When we tested gestational hypertension and preeclampsia as separate outcomes of interests, our results are inconsistent with previous studies. Gerson at al. and Kim et al. did not find significant relationship between fetal cell-free DNA or fetal fraction in the first or second trimester and the risk of getting gestational hypertension [11, 22]. In terms of preeclampsia, the results from different studies were inconclusive. We did not find any statistically significant association between fetal fraction and preeclampsia in our cohort. The results agree with the findings of Gerson et al., which showed no association between preeclampsia and low fetal fraction measured in the first or second trimester NIPT screening [11]. Prior studies by Thurik et al., Silver et al. and Poon et al. also found no association of preeclampsia with absolute levels of fetal cfDNA in the first trimester and the development of preeclampsia [23-25]. However, the results from studies by Alberry at al., Yu et al. and Muñoz-Hernández et al. indicated that significantly elevated levels of fetal cfDNA were associated with a risk of preeclampsia [26-28]. These studies did not adequately control for potential confounders including BMI and a history of chronic hypertension which have been shown to independently affect the association. The discrepancy may also result from the heterogeneity of the definition of preeclampsia, and the severe form of preeclampsia is more likely to be associated with fetal cfDNA [11]. We speculate that the pathophysiologic differences in women with preeclampsia and those with gestational hypertension could lead to different association results. Early placental arteriolopathy are more prevalent in women with preeclampsia [29]. The apoptosis process involved could potentially raise the fetal fraction of cfDNA [30]. There are findings indicating the racial differences in the incidence rate of preeclampsia [31, 32], but how this links to the genetic or environmental causes is still an open question. Finding the exact cause of preeclampsia is still a big scientific challenge. Further studies are merited to find the mechanisms behind the associations of fetal fraction with the different categories of HDP.

Previous reports have suggested that absolute values of cfDNA or fetal fraction do not have prognostic value for preterm birth prediction [10, 11, 15, 25], and our study substantiated those findings. However, some other studies found significant association between fetal fraction or fetal cfDNA and the risk of preterm birth. The study from Dugoff et al. reported a significant association between fetal fraction and preterm birth only among pregnant women who took NIPT in the second trimester of gestation and no significant association among those who took NIPT in the first trimester [15]. In addition, Jakobsen et al. found that high levels of fetal fraction (≥95th percentile) was significantly associated with a higher risk of preterm birth [33]. It has been hypothesized that fetal cfDNA can be pro-inflammatory, which elicits the inflammation-parturition cascade and causes preterm birth [34]. However, in vivo studies provided conflicting results. Van Boeckel et al. found that inflammation did not change the amount of fetal cfDNA, and fetal cfDNA was not pro-inflammatory. Thus, fetal cfDNA was unlikely to be a cause of inflammation or preterm birth [35]. On the contrary, the study from Gomez-Lopez et al. showed that fetal cfDNA increased before preterm birth that were induced by systemic inflammation, but not before intra-amniotic inflammation-induced preterm birth [36]. Both observational studies and animal models indicated the controversial relationship between fetal fraction and preterm birth.

We observed no associations of fetal fraction with the risk of gestational diabetes and low birth weight. Our findings support the results established from previous studies [12, 14]. Our findings regarding the association between fetal fraction and intrahepatic cholestasis contradicted the early data implicating high total cfDNA in intrahepatic cholestasis cases [5]. One explanation for this difference is that they were using the total cfDNA and did not discriminate the fetal and maternal cfDNA, thus it was not the same as the fetal fraction.

Our study has several strengths. We used a large cohort of more than 3500 women who underwent routine NIPT from a single institution. The sample size in our study was 5–10 times larger than previous studies investigating similar outcomes of interest, which improved the robustness and accuracy of the estimation of associations. In addition, there is significant variation on the definition of fetal fraction and so far, standardization of measuring fetal fraction is still not available. Different screening products using different quantitation methods likely introduce variability of fetal fraction levels [37]. As all NIPT in our study were performed at the same laboratory and used the same screening kit, we could then minimize the possibility for variation due to technical differences. In addition, we excluded patients with a history of chronic hypertension and controlled for potential confounders such as BMI, maternal age and gestational age at NIPT, which made our results more robust. Prior studies have indicated that the associations between fetal fraction and pregnancy complications were different across races [12]. To our knowledge, our study is one of the first investigating the associations between fetal fraction and a wide spectrum of pregnancy-related complications among Chinese population. Existing studies were mainly done in western populations. Thus, this study can help us gain a more complete understanding of the associations of cfDNA fetal fraction with the pregnancy complications in a diverse ethnic background.

This study has a few limitations. We intended to assess whether the relationships of fetal fraction with pregnancy-related complications were different for women who undertook NIPT in different pregnancy trimesters. While our study had a relatively large sample size, pregnant women enrolled in our study mainly had NIPT in the second trimester. Thus, we did not have sufficient statistical power to test the associations for women undergoing NIPT in the first and the third trimester. Another limitation is that we could not exclude the possibility of other hidden residual confounding, even though we carefully controlled for a series of known confounders including a history of chronic hypertension, BMI, maternal age and gestational age at NIPT.

In summary, the results of this retrospective cohort study show that women with a fetal fraction<15.15% are at increased risk for gestational hypertension and oligohydramnios after controlling for BMI, maternal age and gestational age at NIPT. Other than the purpose of screening for fetal chromosomal abnormalities, NIPT could also provide cfDNA fetal fraction estimation that could potentially guide the screening of women at high risk of certain pregnancy-related complications.

The distribution of fetal fraction grouped by the gestational hypertension status.

Density plot of fetal fraction from gestational hypertension samples and normal samples in 2 different colors. Dotted lines represent the mean values of fetal fraction from the 2 groups.

(TIF)

Click here for additional data file.

The distribution of fetal fraction grouped by the oligohydramnios status.

Density plot of fetal fraction from oligohydramnios samples and normal samples in 2 different colors. Dotted lines represent the mean values of fetal fraction from the 2 groups.

(TIF)

Click here for additional data file.

(CSV)

Click here for additional data file.

14 Apr 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Badri Padhukasahasram

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Competing Interests section:

I have read the journal's policy and the authors of this manuscript have the following competing interests: YZ, KZ, and XM are employees of GenomCan Inc.. DZ is an employee of Chengdu CapitalBio Technology Co., Ltd.. The remaining authors do not have any conflicts of interest to disclose.

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Additional Editor Comment:

Dear Dr.Yin,

The reviews of the manuscript "The association between fetal fraction and pregnancy-related complications among Chinese population" are now available. Based on the comments provided by 2 expert reviewers, I conclude that the article requires major revisions. Reviewers note the value of a large study of this size conducted for the first time for Chinese populations. More methodological and analysis details, and improvements to language and grammar will significantly improve this manuscript.

There are also additional queries and details requested by reviewers for choices made in study design such as gestational age.

I look forward to a revised manuscript that addresses all the major and minor comments from the reviewers.

Sincerely,

Dr. Badri Padhukasahasram

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Manuscript ID PONE-D-21-33442 entitled "The association between fetal fraction and pregnancy-related complications among Chinese population"

Dear Authors,

My comments are followings:

1. Are there any indications for performing NIPT in this study? Do you provide genetic counseling for those who wish to do so?

2. I think the DISEASE in line 116 should be DISORDER.

3. Why did you perform NIPT around 17 weeks of gestation instead of 10-14 weeks?

4. Please indicate in the Materials and methods section at which gestational week the diagnosis of oligohydramnios was made.

5. Could you list the total number of NIPT performed, the positive rate, and the unreportable results rate in the figure?

6. It is speculated that the difference in the pathogenesis of HDP and preeclampsia affects the results of FF. What would you think about placental remodeling in early gestational age?

7. How do you think the FF tends to be lower in pregnant women with oligohydramnios?

8. Do you think there is a background of racial differences or genetic/environmental influences that cause preeclampsia?

9. I would notice a number of errors of English, so that the manuscript should be checked by native speakers of English.

Reviewer #2: The manuscript entitled “The association between fetal fraction and pregnancy-related complications among Chinese population” by Jiang et al. investigates the relationship between fetal fraction and a variety of pregnancy related complications. As stated by the authors, the primary novelty of the work is the fact that few studies of this size have been conducted on the Chinese population, although similar studies have been conducted in European populations. The primary finding of the study was an association between low fetal fraction and increased risk of HDP. An association between low fetal fraction and gestational hypertension was also demonstrated. The odds ratio was larger when stratifying the fetal fraction population by the 25th percentile (15.15%) as opposed to treating fetal fraction as a continuous variable in the logistic regression models.

Although many of the findings in this manuscript have been previously reported, there is value in confirming these findings in a large Chinese population. When a logistic regression model was fit controlling for BMI, gestational age, and maternal age; fetal fraction was only marginally significant (P=0.048) and the confidence interval for the odds ratio nearly covered 1.0. The significance and odds ratio improved dramatically when fetal fraction was dichotomized using the 25th percentile, but no explanation is provided for why this cutoff was chosen. The paper may benefit from more figures showing a comparison of the fetal fraction distributions in the gestation hypertension and normal populations.

The authors also did not provide enough details on some of the methods. The fetal fraction calculation was described very loosely. Either more detail should be provided or a reference to a paper than employed a similar method should be provided. It also wasn’t clear how the control group was defined for the statistical analysis. When evaluating the relationship between gestational hypertension and fetal fraction, were all other diagnoses included? Or only the gestational hypertension samples and the normal samples?

The data was provided, but some more detail on how to analyze it to recreate the data in the paper would be useful. For example, it was not clear how to reproduce the data filtering from the flow chart provided. A list of issues is provided below.

• Figure 1 states there are 3808 total individuals, but the provided data set has 3800 rows.

• Figure 1 states that 226 samples were excluded due to Not singleton pregnancy, however the data has 228 samples with twin == 2.

• Patients with history of hypertension (7) and diabetes (1) are not indicated. Perhaps this explains the difference between the 3808 and 3800?

Finally, there were many grammatical errors in the manuscript. I have tried to address most of them below. Some errors that were made more than once include the unnecessary use of personal pronouns (we, our, etc) and using an incorrect tense.

Line 52: “leading causes” should be “leading cause.” Also, the statement is fairly obvious. “Have been” usually implies some passage of time. Consider: “Pregnancy related complications are the leading cause”.

Lines 53-54: Change “due to the complications” to “due to complications”

Lines 54-56: Consider rephrasing. Start the sentence with: “Common pregnancy related complications include…”

Line 61: cfDNA is a candidate biomarker for pregnancy related complications.

Lines 61-62: Cell free DNA is composed of DNA fragments released into the blood plasma.

Lines 64-66 :Fetal cfDNA is released into the maternal circulation via apoptosis of placental villous trophoblasts throughout gestation.

Line 69: Remove we: “… NIPT, fetal and maternal cfDNA can be sequenced…”

Lines 76: Replace “the links of fetal fraction with” with “the relationship between fetal fraction and”

Line 78: Consider replacing conflicting with inconsistent.

Line 90: Replace countries with populations or perhaps cohorts.

Line 80: Replace “were limited” with “are limited”

Line 88: Replace undergoing with that underwent

Line 89 : Replace receiving with received

Line 99: add “the” before instrument and reagents

Line 101: MPS already defined as an acronym on line 70

Lines 102-105: The method for estimating fetal fraction is not adequately described. Simply writing “estimated using different length distributions” is too vague. Either provide more detail, or provide a reference to a previous method. The reads ratio has not been defined.

Line 103: Add fragment before length

Line 104: Male fetus should be plural

Line 114: Replace “Our” with “The”

Line 126: who did not have..

Line 132: Replace “our” with “the”

Lines 136-137: Please elaborate on how the dichotomous threshold was determined from the FF distribution in the cohort.

Line 148: Unclear what “fetal fraction as a whole” means. If you are not referring to fetal fraction, but rather the combined set of gestational hypertension and preeclampsia, then please clarify.

Lines 148: Replace “Then we went further and” with “We also”

Line 153: replace “our” with “this” and remove “also”

Line 155: Replace “proper” with “optimal.”

Lines 151-155: You discuss stratifying by trimester and identifying the best trimester for testing. Was this done separately for each pregnancy complication? This sentence comes directly after a discussion of hypertensive disease, so it’s unclear if it is just for that complication or for all. If it is for all, consider starting a new paragraph.

Line 162. Generally, numbers greater than 1,000 are written as numbers

Line 162: Replace “our institution” with the institution name.

Line 170: Replace “For our population, the fetal fraction ranged from” with “The fetal fraction within the study population ranged from”

Line 171: Standard deviation needs % units.

Lines 172-175: This sentence is very hard to follow. It has too many commas and it isn’t clear what the subject of the sentence is. If the sentence is meant to describe how the FF cutoff was established, as verb is missing. For example: “The identification of the cut-off point was based”

Lines 176-178: The relationship between FF, gestational age, and BMI is well established. It may be worth mentioning this.

Line 195: add “a”: “For a one percent…”

Line 272: Change significantly to significant

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

29 May 2022

We would like to thank the editor and reviewers for their suggestions.

Please find our responses below.

To the editor:

1. New manuscript file format was changed to meet the PLOS ONE style.

2. We updated the competing of interests as suggested by the editor:

Competing interests:

YZ, KZ, and XM are employees of GenomCan Inc.. DZ is an employee of Chengdu CapitalBio Technology Co., Ltd.. The remaining authors do not have any conflicts of interest to disclose. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

3. Captions for Supporting Information files were added at the end of manuscript.

We added 2 supplementary figures and captions are provided at the end.

“S1 Fig. The distribution of fetal fraction grouped by the gestational hypertension status. Density plot of fetal fraction from gestational hypertension samples and normal samples in 2 different colors. Dotted lines represent the mean values of fetal fraction from the 2 groups.

S2 Fig. The distribution of fetal fraction grouped by the oligohydramnios status. Density plot of fetal fraction from oligohydramnios samples and normal samples in 2 different colors. Dotted lines represent the mean values of fetal fraction from the 2 groups.”

Reviewer 1:

1. Are there any indications for performing NIPT in this study? Do you provide genetic counseling for those who wish to do so?

Apologies that we didn't make the inclusion criteria very clear.

This study is based on a screening test following the inclusion guideline by National Health and Family Planning Commission of the People's Republic of China. [Technical specifications for noninvasive prenatal screening and diagnosis using cell-free fetal DNA. (in Chinese) http://www.nhc.gov.cn/ewebeditor/uploadfile/2016/11/20161111103703265.docx ]. No indications required for the test, but conditions listed by the guideline were excluded.

In this study, NIPT positive samples were excluded, and patients with history of chronic hypertension or diabetes were excluded. And genetic counseling was provided before taking the NIPT test and when returning positive NIPT results. We have changed the description in materials and methods section accordingly.

" We performed a single-institution retrospective cohort study of all women that underwent NIPT screening from May 2018 to May 2020 at Mianyang People’s Hospital. Women were recruited without any prior indication of genetic diseases or fetal structural abnormalities, following the guideline by the National Health and Family Planning Commission of China (http://www.nhc.gov.cn/ewebeditor/uploadfile/2016/11/20161111103703265.docx). Genetic counseling was provided before the NIPT test and when returning the NIPT result. Only women who had singleton pregnancies and showed no chromosomal aneuploidies from NIPT and with valid fetal fraction estimation and pregnancy complications information were included. Further exclusion criteria were: (1) women with multiple pregnancies; (2) women with missing information on whether singleton or multiple pregnancy; (3) women with missing fetal fraction information; (4) women with a history of chronic hypertension or diabetes."

2. Why did you perform NIPT around 17 weeks of gestation instead of 10-14 weeks?

As per the China Government guideline listed above, NIPT should be taken from 12+0-22+6 weeks. And most patients came for genetic consulting during first pregnancy visit around 12 weeks, then came for the NIPT test during next hospital visit, which is usually 5-6 weeks later, which lead to a mean of ~17 weeks.

3. Please indicate in the Materials and methods section at which gestational week the diagnosis of oligohydramnios was made.

We have added the gestational week in the methods section.

"Oligohydramnios was defined a single deepest vertical pocket (DVP) less than 2cm on ultrasound examination at 28-40 weeks."

4. Could you list the total number of NIPT performed, the positive rate, and the unreportable results rate in the figure?

We fixed Fig 1 to reflect the whole filtering process and the numbers.

“There are 3834 women who underwent NIPT at Mianyang People’s Hospital between May 2018 and May 2020. Twenty-six women had NIPT chromosomal abnormality (a positive rate of 0.68%), which were excluded from the study. We also excluded 8 women with a history of chronic hypertension or diabetes. We finally obtained 3534 cases in the study after further excluding 266 patients without NIPT results, with multiple pregnancy and with missing information on whether singleton or multiple pregnancy. Details regarding the excluded cases were shown in Fig 1.”

5. It is speculated that the difference in the pathogenesis of HDP and preeclampsia affects the results of FF. What would you think about placental remodeling in early gestational age?

Thanks for the suggestion. We added the following text in the discussion.

“We speculate that the pathophysiologic differences in women with preeclampsia and those with gestational hypertension could lead to different association results. Early placental arteriolopathy are more prevalent in women with preeclampsia[29]. The apoptosis process involved could potentially raise the fetal fraction of cfDNA[30].”

6. How do you think the FF tends to be lower in pregnant women with oligohydramnios?

We added the following discussion.

“Although the exact cause of oligohydramnios is complicated and still unclear, we speculated that subtle placental structural abnormalities could potentially cause oligohydramnios. This could also potentially reduce the release of fetal cfDNA into mother's blood, resulting in low fetal fraction of cfDNA.”

7. Do you think there is a background of racial differences or genetic/environmental influences that cause preeclampsia?

Thanks for pointing this out. We included a bit discussion on this topic in the updated manuscript.

“There are findings indicating the racial differences in the incidence rate of preeclampsia[31,32], but how this links to the genetic or environmental causes is still an open question. Finding the exact cause of preeclampsia is still a big scientific challenge. Further studies are merited to find the mechanisms behind the associations of fetal fraction with the different categories of HDP.”

Reviewer 2:

1. The significance and odds ratio improved dramatically when fetal fraction was dichotomized using the 25th percentile, but no explanation is provided for why this cutoff was chosen.

We explicitly explained our choice in 2 places in the revision.

Lines 179-184:

“The dichotomization cut-off point for fetal fraction was at the 25th percentile, namely 15.15%. The choice of the 25th percentile cut-off point is based on the results from the distributions of fetal fraction stratified by the traits showing significant associations with continuous fetal fraction, which is mentioned below, as well as the existing literature [11]. The 25th percentile cut-off is also widely used in stratifications in statistical analysis.”

Lines 208-211:

“The fetal fraction distributions comparing women with gestational hypertension versus normal women, women with oligohydramnios versus normal women (S1 and S2 Figs.) reveal that the maximum segregation of distribution occurs around the 25th percentile (fetal fraction = 15.15%).”

2. The paper may benefit from more figures showing a comparison of the fetal fraction distributions in the gestation hypertension and normal populations.

“The fetal fraction distributions comparing women with gestational hypertension versus normal women, women with oligohydramnios versus normal women (S1 and S2 Figs.) revealed that the maximum segregation of distribution occurs around the 25th percentile (fetal fraction = 15.15%).”

3. The fetal fraction calculation was described very loosely. Either more detail should be provided or a reference to a paper than employed a similar method should be provided.

“The fetal fraction was estimated based on the different fragment length distributions between maternal and fetal cfDNA. Fetal cfDNA fragment tend to have a higher proportion of short plasma DNA fragments (∼130–140 bp; region A) and a lower proportion of long plasma DNA fragments (∼155–175 bp; region B). Locally weighted scatterplot smoothing (LOESS) regression was applied to fit the fetal fraction against reads ratio in features A and B. Male fetuses with fetal fraction estimated from Y chromosome reads were used to train the model parameters[17].”

4. How the control group was defined for the statistical analysis. When evaluating the relationship between gestational hypertension and fetal fraction, were all other diagnoses included? Or only the gestational hypertension samples and the normal samples?

We added a clarification in the method section:

“For all association tests with each complication, only normal samples were included in the control group.”

5. The data was provided, but some more detail on how to analyze it to recreate the data in the paper would be useful. For example, it was not clear how to reproduce the data filtering from the flow chart provided. A list of issues is provided below.

• Figure 1 states there are 3808 total individuals, but the provided data set has 3800 rows.

• Figure 1 states that 226 samples were excluded due to Not singleton pregnancy, however the data has 228 samples with twin == 2.

• Patients with history of hypertension (7) and diabetes (1) are not indicated. Perhaps this explains the difference between the 3808 and 3800?

We made the filtering steps more specific in the revised Fig 1.

We excluded 8 patients with a history of chronic diseases (7 with hypertension and 1 with diabetes) in the dataset provided.

We firstly excluded patients whose NIPT results were not available. Then, we excluded patients without singleton pregnancies. There were two patients with twin pregnancies as well as missing NIPT results. Thus, it shows the 2-case difference in calculation of 226 vs 228.

We fixed all grammar errors in the manuscript as suggested by the reviewer and other errors we identified.

Line 52: “leading causes” should be “leading cause.” Also, the statement is fairly obvious. “Have been” usually implies some passage of time. Consider: “Pregnancy related complications are the leading cause”.

“Pregnancy-related complications are the leading cause of the morbidity and mortality of pregnancy.”

Lines 53-54: Change “due to the complications” to “due to complications”

“Nearly one fifth of all pregnant women are hospitalized before delivery due to complications [1]. ”

Lines 54-56: Consider rephrasing. Start the sentence with: “Common pregnancy related complications include...”

“Common pregnancy-related complications include hypertensive disorders of pregnancy (HDP) and gestational diabetes [2-4].”

Line 61: cfDNA is a candidate biomarker for pregnancy related complications.

“Fetal cell-free DNA (cfDNA) is a candidate biomarker for pregnancy related complications.”

Lines 61-62: Cell free DNA is composed of DNA fragments released into the blood plasma.

“Cell free DNA in the blood plasma of pregnant women is composed of DNA fragments mainly from the mother herself, but also from the fetus.”

Lines 64-66 : Fetal cfDNA is released into the maternal circulation via apoptosis of placental villous trophoblasts throughout gestation.

“Fetal cfDNA is released into the maternal circulation via apoptosis of placental villous trophoblasts throughout gestation [7].”

Line 69: Remove we: “... NIPT, fetal and maternal cfDNA can be sequenced...”

“With the development and commercial availability of NIPT, fetal and maternal cfDNA can be sequenced efficiently and cost-effectively through massively parallel DNA sequencing (MPS).”

Lines 76: Replace “the links of fetal fraction with” with “the relationship between fetal fraction and” Line 78: Consider replacing conflicting with inconsistent.

“Previous studies have investigated the relationship between fetal fraction and various pregnancy complications including HDP [9-13], gestational diabetes [14], and preterm birth [10-12,15].”

Line 78: Consider replacing conflicting with inconsistent.

“However, the significance and directionality of these associations were inconsistent.”

Line 90: Replace countries with populations or perhaps cohorts.

“However, the significance and directionality of these associations were inconsistent. In addition, most of previous studies were conducted in European and American populations.”

Line 80: Replace “were limited” with “are limited”

“Studies in East Asians, especially in Chinese populations are limited.”

Line 88: Replace undergoing with that underwent
Line 89 : Replace receiving with received

“We performed a single-institution retrospective cohort study of all women that underwent NIPT screening from May 2018 to May 2020 at Mianyang People’s Hospital. Women were recruited without any prior indication of genetic diseases or fetal structural abnormalities, following the guideline by the National Health and Family Planning Commission of China (http://www.nhc.gov.cn/ewebeditor/uploadfile/2016/11/20161111103703265.docx). Genetic counseling was provided before the NIPT test and when returning the NIPT result. Only women who had singleton pregnancies and showed no chromosomal aneuploidies from NIPT and with valid fetal fraction estimation and pregnancy complications information were included. Further exclusion criteria were:”

Line 99: add “the” before instrument and reagents

Line 101: MPS already defined as an acronym on line 70

“In brief, the BioelectronSeq 4000 sequencing instrument and the Sequencing Reaction Universal Kit (CapitalBio, Dongguan, People's Republic of China) were used for MPS of plasma cfDNA fragments.”

Lines 102-105: The method for estimating fetal fraction is not adequately described. Simply writing “estimated using different length distributions” is too vague. Either provide more detail, or provide a reference to a previous method. The reads ratio has not been defined.

“The fetal fraction was estimated based on the different fragment length distributions between maternal and fetal cfDNA. Fetal cfDNA fragment tend to have a higher proportion of short plasma DNA fragments (∼130–140 bp; region A) and a lower proportion of long plasma DNA fragments (∼155–175 bp; region B). Locally weighted scatterplot smoothing (LOESS) regression was applied to fit the fetal fraction against reads ratio in features A and B. Male fetuses with fetal fraction estimated from Y chromosome reads were used to train the model parameters[17].”

Line 103: Add fragment before length

“The fetal fraction was estimated based on the different fragment length distributions between maternal and fetal cfDNA.”

Line 104: Male fetus should be plural

“Male fetuses with fetal fraction estimated from Y chromosome reads were used to train the model parameters[17].”

Line 114: Replace “Our” with “The”

“The outcomes of interest were pregnancy-related complications,”

Line 126: who did not have..

“Gestational diabetes was defined as diabetes firstly seen in a pregnant woman who had no diabetes before the pregnancy”

Line 132: Replace “our” with “the”

“We analyzed the variable of interest, fetal fraction, as both continuous and dichotomous variables.”

Lines 136-137: Please elaborate on how the dichotomous threshold was determined from the FF distribution in the cohort.

We explicitly explained our choice in 2 places in the revision.

Lines 179-184:

“The dichotomization cut-off point for fetal fraction was at the 25th percentile, namely 15.15%. The choice of the 25th percentile cut-off point is based on the results from the distributions of fetal fraction stratified by the traits showing significant associations with continuous fetal fraction, which is mentioned below, as well as the existing literature [11]. The 25th percentile cut-off is also widely used in stratifications in statistical analysis.”

Lines 208-211:

“The fetal fraction distributions comparing women with gestational hypertension versus normal women, women with oligohydramnios versus normal women (S1 and S2 Figs.) reveal that the maximum segregation of distribution occurs around the 25th percentile (fetal fraction = 15.15%).”

Line 148: Unclear what “fetal fraction as a whole” means. If you are not referring to fetal fraction, but rather the combined set of gestational hypertension and preeclampsia, then please clarify.

Yes. It means HDP.

“We firstly assessed the association between fetal fraction with HDP, combining gestational hypertension and preeclampsia.”

Lines 148: Replace “Then we went further and” with “We also”

“We also evaluated the relationships of fetal fraction with gestational hypertension and preeclampsia separately,”

Line 153: replace “our” with “this” and remove “also”

Line 155: Replace “proper” with “optimal.”

“To examine this hypothesis, we performed stratified analyses based on gestational age at NIPT: first trimester (10-14.9 weeks), second trimester (15 and 22 weeks) and third trimester (>22 weeks) in order to find the optimal time to perform the NIPT screening tests.”

Lines 151-155: You discuss stratifying by trimester and identifying the best trimester for testing. Was this done separately for each pregnancy complication? This sentence comes directly after a discussion of hypertensive disease, so it’s unclear if it is just for that complication or for all. If it is for all, consider starting a new paragraph.

It is for all traits. We started as a new paragraph.

Line 162. Generally, numbers greater than 1,000 are written as numbers

Line 162: Replace “our institution” with the institution name.

“There are 3834 women who underwent NIPT at Mianyang People’s Hospital between May 2018 and May 2020. Twenty-six women had NIPT chromosomal abnormality (a positive rate of 0.68%), which were excluded from the study. We also excluded 8 women with a history of chronic hypertension or diabetes. We finally obtained 3534 cases in the study after further excluding 266 patients without NIPT results, with multiple pregnancy and with missing information on whether singleton or multiple pregnancy.”

Line 170: Replace “For our population, the fetal fraction ranged from” with “The fetal fraction within the study population ranged from”

Line 171: Standard deviation needs % units.

“Fetal fractions within the study population ranged from 4.32% to 50.38%, with a mean value of 18.48% and a standard deviation of 5.28%.”

Lines 172-175: This sentence is very hard to follow. It has too many commas and it isn’t clear what the subject of the sentence is. If the sentence is meant to describe how the FF cutoff was established, as verb is missing. For example: “The identification of the cut-off point was based”

“The choice of the cut-off point is based on the results from the distributions of fetal fraction stratified by the traits showing significant associations with continuous fetal fraction, which is mentioned below, as well as previous literatures [11]. The 25th percentile cut-off is also widely used in stratifications in statistical analysis.”

Lines 176-178: The relationship between FF, gestational age, and BMI is well established. It may be worth mentioning this.

“We found statistically significant differences between the two groups regarding maternal BMI, gestational age at delivery, and method of delivery. This is not surprising given that associations of fetal fraction with gestational age and maternal BMI are well established in previous publications [18,19].”

Line 195: add “a”: “For a one percent…”

“For every one percent increase of fetal fraction,”

Line 272: Change significantly to significant

“In this retrospective cohort study, we observed significant inverse associations of fetal fraction with the risk of hypertensive disorders of pregnancy (HDP) and oligohydramnios after adjusting for potential confounders.”

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

27 Jun 2022

The association between fetal fraction and pregnancy-related complications among Chinese population

PONE-D-21-33442R1

Dear Dr. Yin,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Badri Padhukasahasram

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors have responded to my comments in their entirety. I have no additional comments to make.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

**********

4 Jul 2022

PONE-D-21-33442R1

The association between fetal fraction and pregnancy-related complications among Chinese population

Dear Dr. Yin:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Badri Padhukasahasram

Academic Editor

PLOS ONE