A statistical investigation of parameters associated with low cell-free fetal DNA fraction in maternal plasma for noninvasive prenatal testing

Figures & data

Figure 1. Flow chart of study population. It is necessary to point out that the influence of gestational age on RLFF was compared by dividing RLFF into two groups: RLFF1 (gestational age at first NIPT) and RLFF2 (gestational age at second NIPT). RLFF1 and RLFF2 represent the same population at different gestational stages for NIPT.

Figure 2. Boxplots and stacked bars of factors associated with LFF and RLFF. (A) Distribution of BMI across all 63,883 subjects in the three groups. (B) Distribution of gestational age across all 63,883 subjects in the four groups. RLFF1 (gestational age at first NIPT) and RLFF2 (gestational age at second NIPT). (C) Distribution of maternal age across all 63,883 subjects in the three groups. (D) Ratio of twin pregnancy cross all 63,883 subjects in the three groups. (E) Ratio of IVF across all 63,883 subjects in the three groups. All measurement data (BMI, gestational age, and maternal age) are represented by median and quartile intervals (M (Q1–Q3)) (Table 1). **p < 0.01; ****p < 0.0001; NS, no statistical significance.

Table 1. Dataset summary of factors associated with LFF in 63,883 subjects undergoing NIPT in the study population.

	Control (n = 63,605)	LFF (n = 197)	RLFF (n = 81)	Z/X^2	p
BMI at NIPT (kg/m^2)^†	22.43 (20.55–24.76)	25.71 (22.96-28.66)	24.54 (22.23–28.26)	139.20	<0.0001
Gestational age at NIPT (days)	130 (119–140)	126 (111-134)	122 (108.5-130.5)/ 133 (120–142)	38.72	<0.0001
Maternal age at NIPT (years)	29 (26–33)	29 (26–33)	33 (29–35.5)	23.44	<0.0001
Twin pregnancy (%)	2093 / (3.3%)	21 / (10.7%)	9 / (11.1%)	48.51	<0.0001
IVF (%)	2977 / (4.7%)	23 / (11.7%)	20 / (21.3%)	93.10	<0.0001

Note: ^†All measurement data (BMI, gestational age, and maternal age) are represented by median and quartile intervals (M (Q1–Q3)).

Table 2. Transformed measurement data (BMI, gestational age, maternal age) into hierarchical data.

BMI (kg/m^2)	<18.50	18.50–24.99	25.00–29.99	30.00–34.99	35.00–40.00	>40.00
Control (n = 63,605)	3815 (1)	45,264 (2)	12,213 (3)	1902 (4)	284 (5)	127 (6)
LFF (n = 197)	3 (1)	89 (2)	73 (3)	24(4)	7 (5)	1 (6)
RLFF (n = 81)	2 (1)	41 (2)	26 (3)	9 (4)	1 (5)	2 (6)
Gestational age (days)	<80	80–99	100–119	120–139	140–160	>160
Control (n = 63,605)	1 (1)	5844 (2)	10,669 (3)	30,695 (4)	10,634 (5)	5762 (6)
LFF (n = 197)	0 (1)	27 (2)	42(3)	94 (4)	29 (5)	5 (6)
RLFF (n = 81)	0 (1)	10 (2)	28 (3)	37 (4)	5 (5)	1 (6)
Maternal age (years)	<20	20–25	26–30	31–35	36–40	>40
Control (n = 63,605)	1020 (1)	11,616 (2)	25,645 (3)	17,559 (4)	7082 (5)	683 (6)
LFF (n = 197)	4 (1)	34 (2)	80 (3)	52 (4)	24 (5)	3 (6)
RLFF (n = 81)	1 (1)	4 (2)	26 (3)	30 (4)	17 (5)	3 (6)

Table 3. Multiple logistic regression analysis of factors associated with LFF and RLFF.

Outcome: 0 vs. 1	Control (n = 63,605) vs. (LFF + RLFF, n = 278)			LFF (n = 197) vs. RLFF (n = 81)
	OR	p-Value	95% CI	OR	p-Value	95% CI
BMI^#	2.18	<0.0001	(1.94–2.45)	0.97	NS	(0.70–1.33)
Gestational age	0.76	<0.0001	(0.67–0.87)	0.89	NS	(0.65–1.22)
Maternal age	1.01	NS	(0.89–1.15)	1.54	0.0023	(1.17–2.05)
Twin pregnancy (0, 1)^†	1.62	0.0353	(1.02–2.52)	0.58	NS	(0.21–1.48)
IVF (0, 1)^‡	2.68	<0.0001	(1.82–3.86)	2.55	0.016	(1.19–5.54)

Note: ^#BMI, gestational age, and maternal age data were transferred into six incremental level (see Table 2).

^† After removing the 859 twins of IVF from the three groups, for control vs. (LFF + RLFF): twin pregnancy (OR = 1.95, p = 0.026) and IVF (OR = 2.94, p < 0.0001), for LFF vs. RLFF: twin pregnancy (OR = 1.50, P = NS) and IVF (OR = 4.07, p = 0.0016). OR of other factors remain similar.

^‡ After removing the factor (twin pregnancy) from the multiple logistic regression, for control vs. (LFF + RLFF): IVF (OR = 3.13, p < 0.0001), for LFF vs. RLFF: IVF (OR = 2.16, p = 0.03). OR of other factors remain similar.

Figure 3. Boxplots of serological markers and pregnancy outcomes associated with LFF and RLFF. (A) Distribution of β-hCG in the three groups. (B) Distribution of PAPP-A in the three groups. (C) Distribution of gestational age at delivery in the three groups. (D) Distribution of birth weight at delivery in the three groups. All measurement data (β-hCG, PAPP-A, Gestational age at delivery, Birth weight at delivery) are represented by median and quartile intervals (M (Q1–Q3)) (Table 4). *p < 0.05; **p < 0.01; ****p < 0.0001; NS, no statistical significance.

Table 4. Serological markers and pregnancy outcome of the LFF and RLFF cases.

	Control	LFF	RLFF	Z	p
β-hCG (MOM)^†	1.19 (0.60–2.12) (n = 314)	0.59 (0.39–1.09) (n = 127)	0.79 (0.46–1.63) (n = 51)	48.41	<0.0001
PAPP-A (MOM)^‡	1.01 (0.68–1.37) (n = 234)	0.87 (0.55–1.21) (n = 83)	0.94 (0.66–1.38) (n = 25)	5.44	NS
Gestational age at delivery (weeks)	39.3 (38.4–39.9) (n = 241)	38.9 (38.1–39.7) (n = 137)	38.6 (37.9–39.1) (n = 56)	16.60	<0.0001
Birth weight at delivery (kg)	3.33 (2.99–3.60) (n = 241)	3.36 (3.04–3.72) (n = 137)	3.28 (2.95–3.65) (n = 56)	2.10	NS

Note: ^†MOM means multiples of the median of this gestational age. ^‡All measurement data (β-hCG, PAPP-A, gestational age at delivery, birth weight at delivery) are represented by median and quartile intervals (M (Q1–Q3)).

Table 5. Outcome of prenatal diagnosis and maternal diseases of the LFF and RLFF cases during pregnancy.

	Cytogenetic abnormalities^‡		Scarred uterus^†, #	Diabetes^†	Hypertens-ion^†	Thrombo-philia^†	Autoimmune diseases^†	Inflammation^†	No diseases^†
LFF (n = 181,16 lost to follow-up)		1 trisomy18	31/181 (17.1%)	40/181 (22.1%)	8/181 (4.4%)	13/181 (7.2%)	7/181 (3.9%)	17/181 (9.4%)	90/181 (49.7%)
RLFF (n = 78, 3 lost to follow-up)		1 trisomy21 1 del22q11.21 1 46, r(Y) (p11.31q11.2)	18/78 (23.1%)	18/78 (23.1%)	8/78 (10.3%)	4/78 (5.1%)	7/78 (9.0%)	12/78 (15.4%)	22/78 (28.2%)
p-Value			0.26	0.86	0.07	0.54	0.1	0.16	0.0013

Note: ^†The data in the column is represented as: the number of subjects with this disease in the group/total number of this group (percentage of the disease in this group). All the diseases listed in Table 5 are associate with LFF. Subjects with thrombophilia were undergoing treatment, but we are not sure about the medication and whether it was used when the NIPT was performed. The types of diseases in autoimmune diseases include Hashimoto’s thyroiditis, ankylosing spondylitis, systemic lupus erythematosus, and connective tissue disorders. The types of diseases in inflammation include active hepatitis, mastitis, fungous vaginitis, and acute pharyngitis, which may increase the number of white blood cells in blood.

^‡ Prenatal diagnosis for genetic testing (karyotyping and chromosomal microarray analysis) was performed by patient request on 1 of 197 LFF cases and 12 of 81 RLFF cases.

^# Among the individuals in the LFF and RLFF groups who had a history of a scarred uterus, their median maternal age and quartile intervals were 34 (30–36) years.

Data availability statement

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