Received: 2 October 2018 Revised: 31 January 2019 Accepted: 1 February 2019

DOI: 10.1002/pd.5436

ORIGINAL ARTICLE

Effects of medication intake in early pregnancy on the fetal

fraction of cell‐free DNA testing

Maggie Kuhlmann‐Capek | Giuseppe Chiossi | Prapti Singh | Luis Monsivais |

Violetta Lozovyy | Lauren Gallagher | Nathan Kirsch | Elizabeth Florence |

Victoria Petruzzi | Jeffrey Chang | Sofia Buenaventura | Paul Walden | Benjamin Gardner |

Mary Munn | Maged Costantine

Department of Obstetrics and Gynecology,

The University of Texas Medical Branch at
Galveston, Galveston, TX, USA
Correspondence
Maggie Kuhlmann‐Capek, Department of
Obstetrics and Gynecology, The University of
Texas Medical Branch at Galveston, 301
University Blvd., Galveston, TX 77555‐0587,
USA.
Email: makuhlma@utmb.edu
Abstract
Objectives: To determine the association between medications intake in early
pregnancy and variation in the fetal fraction (FF) in pregnant women undergoing
cell‐free DNA (cfDNA) testing.
Methods: We performed a retrospective cohort study of women (n = 1051) under-
going cfDNA testing at an academic center. The exposed group included women tak-
ing medications (n = 400; 38.1%), while the nonexposed group consisted of women
taking no medications (n = 651; 61.9%). Our primary outcome was FF. We performed
univariate and multivariate analyses as appropriate.
Results: The FFs were 8.8% (6.6‐12.1), 8.7% (6.3‐11.6), and 7.7% (5.1‐9.3) among
women taking 0, 1, and two or more medications, respectively (P < 0.01). Using mul-
tivariable linear mixed effects model, the mean FF was significantly lower among
those taking two or more medications compared with the nonexposed group. FF
was directly correlated with gestational age at the time of cfDNA testing and
inversely correlated with maternal obesity. Exposure to metformin was associated
with 1.8% (0.2‐3.4) lower mean FF when compared with the nonexposed group
(P = 0.02). Obesity and intake of two or more medications were associated with
higher hazard ratio of having a low FF less than 4%.
Conclusions: Exposure to metformin or two or more medications was associated
with decreased FF, and obesity is associated with delay in achieving adequate FF per-
centage. These findings should be considered while counseling patients on test
limitations.

1 | I N T RO D U CT I O N has a number of limitations that may complicate interpretation. The

Cell‐free DNA (cfDNA) is a novel screening method for fetal aneu-
ploidy that, despite increased sensitivity to detect common trisomies,
The abstract was presented as an oral presentation at the Society of Reproductive Investiga-
tion Annual Meeting in San Diego, California, March 8‐12, 2018.
most frequent reason for test failure, which occurs in approximately
1% to 8% of cases,1,2 depending on the testing platform utilized, is a
low fetal fraction (FF). FF is the proportion of cfDNA arising from
the placenta, and it represents the ratio between fetal genetic material
and the expected number of copies of a given chromosome per
genome in a mixture of DNA from a euploid mother and trisomic

Prenatal Diagnosis. 2019;39:361–368. wileyonlinelibrary.com/journal/pd © 2019 John Wiley & Sons, Ltd. 361
362
fetus. The relationship between maternal and fetal DNA in a given
sample can be expressed as either 3FF + 2(1‐FF) or as 2 + FF, per
genome.2 The lower the FF, the harder it is to discriminate aneu-
ploidies. For example, in the case of detecting trisomy 21, if the FF
is 10%, the test can differentiate between 2.10% for an affected fetus
and 2.00% for an unaffected fetus, whereas if the FF is 2%, the test
has to capture a difference between 2.02% from 2.00%, a significantly
narrower margin of error.
Several maternal and fetal characteristics have been reported to
affect FF, such as maternal age, body mass index (BMI), race, cigarette
smoking, assisted conception, fetal crown‐rump length, and the pres-
ence of trisomy 13 and 18.3-6 A recent report7 suggests that low‐
molecular‐weight heparin (LMWH) may affect trophoblast apoptosis,
leading to lower FF and possibly test failure. We hypothesized that
some other medications may have similar mechanisms and cause sim-
ilar changes in the FF.
Medication use in pregnancy is on the rise. Over the past three
decades, first‐trimester use of prescription medications has increased
by more than 60%, and the use of four or more medications has tri-
8
pled. In 2008, approximately 50% of women reported taking at least
8
one medication during their pregnancy. More importantly, medication
use by pregnant women is also increasing in the first trimester, with
the average number of medications used in a given pregnancy increas-
ing by approximately 63% from 1976 to 1978 to 2006 to 2008. This is
concerning, as the first trimester is a critical period for fetal organo-
genesis and placental development.9 Little is known regarding the
effects of medications on FF. As the average age of pregnant women
10
increases, the incidence of comorbid conditions requiring medical
treatment such as hypertension and diabetes is expected to rise
accordingly, possibly explaining the significant increase in the use of
medications in pregnancy. Therefore, the aim of this study is to deter-
mine whether medications use at the time of obtaining the cfDNA test
is correlated with changes in the FF distribution, as well as the preva-
lence of low FF, defined as less than or equal to 4%.
2 | METHODS
We performed a retrospective cohort study of women presenting for
ultrasound exam or genetic counseling between March 2012 and
2017, at the University of Texas Medical Branch, Galveston, Texas,
who underwent a blood test for cfDNA prenatal testing (Panorama
by Natera). The study was approved by the Institution Review Board
(#17‐0184).
All patients who underwent a cfDNA testing were eligible for
screening. We excluded women with multifetal gestations and those
whose FFs were not reported. If a patient had more than one preg-
nancy during the study period, only the first was included in this
analysis.
The exposed group included patients who were receiving any med-
ication at the time of testing. The nonexposed group included patients
who were not taking any medications at the time of testing (with the
exception of prenatal vitamins). No matching was done. Our primary
KUHLMANN‐CAPEK ET AL.
What is already known about this
topic?
• Several factors have been reported to affect the fetal
fraction of cell‐free DNA noninvasive prenatal testing.
While there are reports that heparin may affect the
fetal fraction and may lead to false‐negative results for
Down syndrome, there are sparse data from larger
studies on the effect of medications intake in pregnancy
on fetal fraction.
W h a t d o e s t hi s st u d y a d d ?
• This study investigates heparin, as well as other
commonly used classes of medications, in regard to
their effect on fetal fraction at the time of noninvasive
prenatal testing. We report that metformin may have a
significant effect on the fetal fraction. We also confirm
previous findings regarding the effects of obesity and
gestational age at testing on fetal fraction.
outcome was the cfDNA FF as reported (%) on the test result. Our
major secondary outcome was low FF defined as FF < 4%. Although
individual testing platforms may be validated at different FFs, we
chose 4%, as this is a commonly used threshold. Such laboratory
thresholds serve to improve testing accuracy in the interpretation of
results for this screening tool. We reviewed patients' electronic medi-
cal records and collected data regarding maternal baseline characteris-
tics (age, parity, ethnicity, BMI, and gestational age at time of testing),
cfDNA indications and results (including the FF), medications used at
the time of or prior to the cfDNA testing, and birth outcomes. Of note,
there were no discrepancies (false negatives or false positives)
between cfDNA testing results and presence of aneuploidy at birth.
Samples were collected in tubes provided with the Panorama
testing kits, and collection procedure was uniform throughout the study
period. Single nucleotide polymorphisms (SNP)‐based analysis was
performed for all samples as per the test guidelines by Natera. The nature
of our patient population (age, ethnicity, average BMI, presence of
medical comorbidities, etc) was consistent throughout the study period.
3 | STATISTICAL ANALYSIS
The initial analysis was performed for all medications combined.
Prespecified analyses were done for classes of medications thought
to affect trophoblast apoptosis and thus the FF. FF was analyzed as
a continuous and as a categorical variable. One‐way analysis on ranks
and chi‐square tests were used as appropriate to evaluate univariate
differences in the study population according to medication exposure
at the time of cfDNA testing.
KUHLMANN‐CAPEK ET AL. 363

Linear mixed‐effects model was used to describe how the mean FF
changes over time, as women whose initial cell cfDNA resulted in low
FF may have repeated the test later in pregnancy, and assessments at
different time points in the same pregnancy are correlated. In a multi-
variate analysis, we investigated how the gestational age at time of
cfDNA testing, maternal age, obesity (BMI ≥ 30 kg/m2),
race/ethnicity, and prescription drugs may affect mean FF. Interaction
between maternal obesity and number of prescribed medications was
also tested. The fixed effects account for the mean responses of the
model at different time points (ie, gestational age at cfDNA), which
are shared across all subjects. As each subject contributed only one
pregnancy, the random effects account for pregnancy‐specific (longi-
tudinal) correlations. Ignoring the correlation within pregnancy‐
specific observations can lead to incorrect conclusions, such as inaccu-
rate standard error estimates in fixed effects and false results from
hypothesis tests. Random intercept models were considered at the
individual level.
Multivariate Cox proportional hazards models were used to esti-
mate the association between maternal characteristics and the hazard
to have a low FF. This type of modeling demonstrates the incidence or
hazard rate, which is the number of new adverse events per popula-
tion at‐risk per unit time. It is a semiparametric method, which
gestations, and three times among 10 women, while only one woman
was tested four times.
The indications for cfDNA testing included advanced maternal age
(288; 27.4%), previous abnormal aneuploidy screening (115; 11%),
abnormal ultrasound findings (98; 9.3%), family history of aneuploidy
(11; 1%), previous child with aneuploidy (5; 0.5%), or others/multiple
indications (534; 50.8%).
Table 1 lists the medications patients were prescribed at the time
of cfDNA testing. Two hundred sixty‐eight women were receiving
only one medication, 132 were on two or more medications, and
651 were not on any medication. The mean (±SD) age of the subjects
enrolled in our study was 30.9 ± 6.1 years; 332 (32.2%) were nullipa-
rous; 450 (42.8%) were obese with BMI ≥ 30 kg/m2. The mean FF for
the entire cohort was 9.4 ± 4.6%, while the mean gestational age at
cfDNA testing was 15.5 ± 5 weeks gestation. Table 2 presents the
patients' characteristics according to medication exposure. Women
taking medications were more likely to be obese, Caucasian, and to
have had cfDNA testing earlier in pregnancy. The FF was 8.8% (95%
CI, 6.6‐12.1) among those not taking any medications compared with
8.7% (6.3‐11.6) and 7.7% (5.1‐9.3) among those taking one or two or
TABLE 1 Medications used by patients at the time of cfDNA testing

assumes a particular probability model to represent survival times Number (%) of Women
and is therefore more robust than parametric methods. It can accom- Pharmacological Agent Taking the Medication
modate both discrete and continuous measures of event times, as well ASA 23 (4.8)
as time‐dependent covariates.11 Multivariate Laplace regression was NSAIDS other than ASA 2 (0.4)
used to study the median gestational age at which cfDNA reaches
Plavix 1 (0.2)
FF ≥ 4%. Laplace regression was introduced in the biostatistical liter-
Heparin (LMWH and UGH) 16 (3.3)
ature as a method of evaluating conditional quantiles of a potentially
Metformin 25 (5.1)
censored outcome. The method has been presented in the epidemio-
logic literature as a percentile‐based approach for the analysis of time‐ Insulin 30 (6.2)

to‐event data. In survival analysis, the outcome is represented by a Glyburide 15 (3.1)

time variable that is typically skewed and censored. In this situation, GLP1 agonist 1 (0.2)
modeling the mean of the outcome would not only introduce the Progesterone (im, vaginal) 34 (7)
discussed limitation but also typically require data extrapolation PPI, anti H2, antiacid 36 (7.4)
beyond the range of the observed data. The estimation of mean sur-
Antiemetics 90 (18.5)
vival is commonly computed by assuming a parametric distribution
Antihistaminic 47 (9.7)
for the unobserved part of the survival function, although it is not easy
Narcotics 8 (1.6)
to capture the shape of the survival function after the end of follow‐
Muscle relaxant 11 (2.3)
up.12,13 If cfDNA was tested multiple times on the same patient, only
the first assessment was used for the Cox proportional hazard models Antispasmodic 2 (0.4)

and the Laplace regression. P < 0.05 was considered statistically signif- Antibiotics 2 (0.4)
icant. Statistical analyses were performed using the Stata 14.2 Immunomodulators 2 (0.4)
(StataCorp, College Station, Texas). Chemotherapy 2 (0.4)
Antiepileptic 13 (2.7)
Antiviral 3 (0.6)
4 | RESULTS
Anticholesterol 2 (0.4)
Thyroid (levothyroxine, methimazole, PTU) 46 (9.5)
Out of 1186 women, we excluded 11 patients because their cfDNA
tests results did not include FF, 16 because of twin gestations, and Antidepressants, mood stabilizers, 75 (15.4)
antipsychotic
three subjects who had spontaneous pregnancy reduction from twins
Antihypertensive 52 (4.4)
to singleton, leaving data from 1156 patients available for analysis.
cfDNA was tested once among 1051 pregnancies, twice in 94 Abbreviation: cfDNA, cell‐free DNA.
364 KUHLMANN‐CAPEK ET AL.

TABLE 2 Univariate analysis: Patients' characteristics according to exposure

No medications 1 Medication ≥ 2 Medications

(n 651) (n 268) (n 132) p

Age, years 30.8 (26‐36) 30.9 (26‐36) 31.8 (28‐36) 0.3a

Gestational age, weeks 14 (12‐20) 13 (11‐19) 12 (11‐18) <0.01a
FF (%) 8.8 (6.6‐12.1) 8.7 (6.3‐11.6) 7.7 (5.1‐9.3) <0.01a
FF < 4% 29 (4.5%) 16 (6%) 14 (10.6%) 0.03b
Race/ethnicity
Caucasian 275 (43.3%) 134 (50%) 80 (60.6%) <0.01b
Hispanic 200 (30.7%) 91 (34%) 37 (28%)
Black 78 (12%) 35 (13%) 16 (12.1%)
Asian 33 (5.1%) 8 (3%) 8 (6.1%)
Nulliparous 191 (31.4%) 95 (35.4%) 39 (29.5%) 0.4b
Obesity 260 (40%) 113 (42.1%) 76 (57.8%) <0.01b

Data presented as median (IQ range) or frequency (%); obesity defined as BMI ≥ kg/m2. Abbreviation: FF, fetal fraction.
a
One‐way Analysis of Variance on Ranks;
b
Chi Square.

more medications, respectively (p < 0.01; Table 2, Figure 1).
Multivariate linear mixed effects model revealed that mean FF was
directly correlated with gestational age at the time of cfDNA testing
and inversely correlated with maternal obesity (Table 3). Mean FF
was 2.7% (95% CI, 2.3‐3.2) lower among obese when compared with
non‐obese women, as it averaged 6% (95% CI, 5.4‐6.7) among
mothers with BMI < 30 as opposed to 3.3% (95% CI, 2.5%‐4.2%)
among those with BMI greater than or equal to 30 (p < 0.01). FF
increased by 0.3% (95% CI, 0.2‐0.35) with every 1‐week increase in
the gestational age when cfDNA was tested (p < 0.01). When com-
pared with the nonexposed group, the mean FF was significantly
lower among pregnant mothers taking two or more prescription drugs
(3.3%; 95% CI, 2.5‐4.2% vs 5.4%; 95% CI, 4.6‐6.3; p = 0.02). No signif-
icant interaction was found between maternal obesity and prescribed

FIGURE 1 Fetal fraction according to time

of cfDNA and number of medications
exposure. Graphical representation of the
data point: Smoothing was obtained using a
locally weighted least square regression of FF
on gestational age____ 0 medications (704
observations).… … . 1 Medication (302
observations).‐ ‐ ‐ ‐ ‐ ≥2 Medications (144
observations).
KUHLMANN‐CAPEK ET AL. 365

TABLE 3 Multivariate linear mixed effect modes investigating factors affecting mean FF (%)

ASA vs No Heparin vs No Metformin vs No

Entire Cohort Medications Medications Medications
(n = 1150) (n = 619) (n = 613) (n = 627)

Obesity −2.7 (−3.2‐2.3) <0.01 −3.1 (−3.7‐2.4) <0.01 −3 (−3.7‐2.3) <0.01 −3 (−3.7‐2.4) <0.01
Gestational age 0.3 (0.2‐0.35) <0.01 0.3 (0.2‐0.3) <0.01 0.3 (0.2‐0.3) <0.01 0.3 (0.2‐0.3) <0.01
Medications
0 §
1 0.1 (−0.2‐0.5) 0.5
≥2 −0.6 (−1.2‐0.1) 0.02
ASA −0.6 (−2.4‐1.1) 0.5
Heparina −0.4 (−1.8‐0.9) 0.5
Metformin −1.8 (−3.4‐0.2) 0.02

Linear mixed effects models with random intercept at the individual level: Fixed effects coefficients indicate the increase (positive value) or decrease (neg-
ative value) in the mean FF value for a unit change in continuous covariates or when compared with the reference group (§) in case of categorical covariates.
The random effects covariance matrix was assessed for each model, and it was found to be 16.4 when the entire cohort was studied, while 17.7, 17.9, and
18 when the model respectively investigated aspirin, heparin, or metformin. Abbreviation: FF, fetal fraction.
a
Heparin includes women exposed to either UFH or LMWH.

medications. When pharmacological agents were investigated individ-
ually, exposure to metformin was associated with 1.8% (95% CI, 0.2‐
3.4; p = 0.02) lower mean FF when compared with subjects not taking
any prescription drugs. Treatment with aspirin or heparin (either
unfractionated or low molecular weight) did not affect FF.
We then investigated the determinants of FF above 4%. Multivar-
iate Cox proportional hazard model revealed a significantly higher
Hazard ratio (HR) for FF less than 4% among obese women (HR 9.7;
95% CI, 4.1‐22.6; p < 0.01), and women taking two or more prescrip-
tion drugs (HR 2.3; 95% CI, 1.2‐4.4; p < 0.01; Table 4). As FF is gesta-
tional age‐dependent, we investigated the potential factors that
could affect the median gestational age at which cfDNA collection would
lead to FF values greater than or equal to 4%. Multivariate Laplace
regression suggested that maternal obesity delays by 5 weeks (95% CI,
3.6‐6.3), the gestational age at which such cfDNA threshold is reached:
The median gestational for cfDNA with FF ≥ 4% was 13 weeks (95%
CI, 12.3‐13.6) for women with BMI < 30 as opposed to 18 weeks (95%
CI, 16.6‐19.3) for obese women (p < 0.01; Table 4, Figure 2). The same
model also revealed that women taking two or more prescription
drugs developed FF ≥ 4% 1.6 weeks (95% CI, 0.7‐2.4) earlier than
women taking only prenatal vitamins (p < 0.01; Table 4).
5 | DISCUSSION
Noninvasive prenatal testing using cfDNA is a widely used aneuploidy
screening test in pregnancy. However, despite its widespread use,
there are limited data about conditions, which may affect the reliability
of results, particularly the FF. Our study provides evidence that sup-
ports previously reported data that gestational age and obesity signif-
icantly impact the FF. We also found that metformin may negatively
impact the FF. In addition to metformin, our research shows trends,
which imply that other medications, such as aspirin and heparin, may
also impact FF, but the low numbers of patients in our study who were
taking these medications of interest may have limited our ability to
show significance. This lack of statistical significance, presumably

TABLE 4 Multivariate Cox regression investigating factors associated with FF < 4% and multivariate Laplace regression determining covariates
associated with median gestational age at cfDNA testing leading to FF ≥ 4%

Likelihood of FF < 4% Median time to FF ≥ 4%

Multivariate Cox regression Multivariate Laplace regression
Hazard ratio Coefficient

Obesity 9.7 (4.1‐22.6) < 0.01 5 (3.6‐6.3) <0.01

Medications
0 § §
1 1.5 (0.8‐2.8) 0.1 −0.4 (−1.2‐0.4) 0.3
≥2 2.3 (1.2‐4.4) < 0.01 −1.6 (−2.4‐0.7) <0.01

Multivariate Cox regression: HR > 1 indicate an increased likelihood, while HR < 1 indicated a decreased likelihood of achieving FF < 4% at cfDNA testing,
when compared with the reference group (§) for categorical covariates. Multivariate Laplace Regression: Coefficients indicate an increase (positive value) or
a decrease (negative value) in the median gestational age when cfDNA reaches values ≥4% for a unit change when compared with the reference group (§)
in case of categorical covariates.
366
secondary to limited sample size, is in contrast with previously pub-
lished literature, which demonstrated a significant negative effect with
low‐molecular‐weight heparin.
A recently published case report showed that a patient had a false‐
negative cfDNA testing for trisomy 21 (with FF 1.6%) while she was
receiving low‐molecular‐weight heparin. A repeat cfDNA while patient
was off low‐molecular‐weight heparin was high risk for trisomy 21
with FF 5.76%.14 The authors concluded that the reason for the
false‐negative result was the use of low‐molecular‐weight heparin,
which is hypothesized to reduce trophoblast apoptosis and enhance
survival. Other cases report low‐FF and cfDNA failures in patients tak-
ing aspirin, prednisolone, and low‐molecular‐weight heparin.15,16
Women using low‐molecular‐weight heparin have smaller DNA frag-
ments and higher proportion of guanine‐cytosine base pairs. This cor-
relates with higher content of chromosome 18, which may lead to
false‐positive testing, and lower content of chromosomes 21 and 13,
which may lead to false‐negative testing. Low‐molecular‐weight hepa-
rin is used for prophylaxis and treatment of venous thromboembolism
and conditions, which may predispose an individual to developing
venous thromboembolism in pregnancy, such as antiphospholipid anti-
body syndrome and inherited thrombophilias. We hypothesized that
other medications may cause similar changes to the ones observed
with low‐molecular‐weight heparin. However, for most of these, there
are little to no data, which address the effects of these medications on
FF levels and the potential mechanisms by which they may affect
these levels. Aspirin is a nonsteroidal anti‐inflammatory drug, which
is used clinically for treatment of pain and fever, as well as for its anti-
platelet properties for women with cardiovascular disease. In addition,
many pregnant women with risk factors for preeclampsia take aspirin
to reduce their risk of developing this disease. Metformin is a
KUHLMANN‐CAPEK ET AL.
FIGURE 2 Gestational age at fetal fraction
(FF) ≥ 4% according to maternal obesity.
Graphical representation of gestational age (x‐
axis) at which the FF (y‐axis) reaches 4% in
non‐obese (solid line) and obese women
(dotted line)
biguanide, which works by decreasing gluconeogenesis and increasing
peripheral insulin sensitivity. This drug is useful in the treatment of
diabetes and polycystic ovarian syndrome. Insulin is a naturally occur-
ring substance in the human body, used to control the level of glucose
in the blood, but there are many different forms of synthetic insulin,
which, when administered to diabetic patients, may affect the body
differently than that which is produced by the pancreas. Steroids are
used as anti‐inflammatory agents in a wide variety of conditions,
including asthma exacerbations and lupus.
Our study has both strengths and limitations. The sample size is one
of the largest to date on this subject; however, it may not have been
large enough to identify differences with individual medications. All of
the cfDNA analyzed and included in this study were done using the Pan-
orama test by Natera, which allows consistency in sample handling,
since the sampling methods and procedures can differ between individ-
ual tests. However, this also means that these results may or may not be
applicable to testing done by other platforms. Additional strengths
include a diverse patient demographic, which enhances the generaliz-
ability of our results, and the availability of data on most of the babies
at time of delivery, which allowed us to unequivocally verify the results
of the screening test. While we reported on the rates of re‐draws in our
study, we did not analyze the effect of time between re‐draws or FF at
each interval. We did not perform a formal power analysis or included
the effect of coefficient of variation of the assay into that analysis.
Other weaknesses of this study include the possibility of confounding
data, low numbers of patients using the medications of interest, and
an inability to separate the effect of the medication from that of the dis-
ease process itself. The patients in our study who were taking metfor-
min were diabetics. Therefore, it is plausible that the effects of
metformin on FF may have been secondary to the disease itself. This
KUHLMANN‐CAPEK ET AL.
is an important distinction, and until there is a plausible proposed mech-
anism by which medication, rather than disease process, mechanistically
impacts FF, care must be taken when discussing the effects of either
factor. While metformin has been shown to reduce the secretion of sol-
uble fms‐like tyrosine kinase 1 and soluble endoglin secretion from vil-
lous cytotrophoblast cells, and preterm preeclamptic placental villous
explants,17 we cannot speculate based on our data the exact molecular
mechanisms by which metformin (or other medications) may affect FF,
as we did not perform corresponding lab work to inform such assertions.
Moreover, while Natera states that results of the Panorama cfDNA test
are validated at FF ≥ 2.8%,13 Cuckle2 reports that 1.6% to 6.4% repre-
sent the low FF range depending on the company/platform used, and
Revello1 states that up to 8% is considered low FF. For this study, we
used 4% as this the most commonly used cutoff in other publications,
which provides a basis for comparison. Of note, we studied FF not only
as a categorical/binary variable but also as a continuous variable.
The relationship between maternal and fetal DNA in a given sample
can be expressed as either 3FF + 2(1‐FF), or as 2 + FF, per genome.2 The
lower the FF, the harder it is to discriminate aneuploidies. The specific
cfDNA test utilized in this analysis (Panorama by Natera) analyzes
single‐nucleotide polymorphisms. Of note, this specific methodology
for estimating FF is associated with a relatively high rate of uninterpret-
able results, compared with other methods.18 Y chromosome‐based
methods for determining FF are useful if the fetus is male, as theY chro-
mosome is an unambiguous indication of fetal DNA. There are also
methods, which have been validated independent of fetal gender, such
as the SeqFF and SANEFALCON methods. SeqFF is a multivariate
model, which contains two regression models. SANEFALCON deter-
mines FF through the distribution of reads, which have been mapped
around nucleosome positions on autosomal chromosomes.19
An interesting outcome of our study was the Laplace regression,
which actually projected that women exposed to two or more medica-
tions would achieve a higher FF at an earlier time point, which
contradicted what was determined with previous models. We believe
this can be explained by recognizing that the previous models investi-
gated mean FF and the hazard ratio of having a low FF, while Laplace
regression investigates time to event. We do not investigate factors
associated with a specific event but with the time in which the said
event occurs. The univariate analysis in Table 2 shows that women
exposed to two or more medications had Non‐invasive prenatal test-
ing (NIPT) significantly earlier that all the others. This may be the rea-
son why, in the Laplace model, which studies the factors associated
with the time when FF becomes greater than or equal to 4%, exposure
to two or more medication is associated with earlier gestational ages—
all the women taking two or more medications had early testing.
The Kaplan‐Mayer estimator (Figure 1) provides a way in which
providers might apply an understanding of the effects of maternal
obesity on FF. This model suggests that maternal obesity delays the
gestational age at which the threshold FF to achieve an interpretable
result is reached. According to this model, the median gestational
age to achieve FF greater than or equal to 4% in obese women was
18 weeks. In non‐obese women, the median gestational age to
achieve FF greater than or equal to 4% was 13 weeks. Of note, this
367
gestational age is different from other published data, which show a
median FF of approximately 10% by 10 to 13 weeks' gestation in all
women.20,21 Along with consideration of other factors, which may
affect FF, providers can begin to understand the likelihood of achiev-
ing an interpretable result and counsel patients accordingly. This data
suggest that if mothers are obese, NIPT may need to be ordered at a
later gestational age to decrease the chance of low FF.
In conclusion, our study confirms the evidence that gestational age
and obesity correlate with significant changes in FF. We also found
that metformin intake may be associated with lower FF. It is impera-
tive that, as the scope of testing utilizing cfDNA continues to expand,
we conduct further research to understand how different factors in
the prenatal condition might impact results. This also further highlights
the importance of considering testing using cfDNA to be a screening
test only and that women should have definitive testing to confirm
any suspected anomalies.
CONFLIC T OF INT E RE ST
The authors report no conflict of interest and no sources of support.
ORCID
Maggie Kuhlmann‐Capek https://orcid.org/0000-0002-7632-121X
RE FE RE NC ES
1. Revello R, Sarno L, Ispas A, Akolekar R, Nicolaides KH. Screening for
trisomies by cell‐free DNA testing of maternal blood: consequences
of a failed result. Ultrasound Obstet Gynecol. 2016;47(6):698‐704.
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How to cite this article: Kuhlmann‐Capek M, Chiossi G, Singh
P, et al. Effects of medication intake in early pregnancy on the
fetal fraction of cell‐free DNA testing. Prenatal Diagnosis.
2019;39:361–368. https://doi.org/10.1002/pd.5436