Narges Farahi, MD

EVIDENCE SUMMARY

To evaluate the longstanding belief that a short IPI after miscarriage is associated with adverse outcomes in subsequent pregnancies, a 2017 systematic review and meta-­analysis of 16 studies (3 randomized controlled trials [RCTs] and 13 retrospective cohort studies) with a total of more than 1 million patients compared IPIs shorter and longer than 6 months (miscarriage was defined as any pregnancy loss ­before 24 weeks).¹ The meta-analysis included 10 of the studies (2 RCTs and 8 cohort studies), with a total of 977,972 women and excluded 6 studies because of insufficient data. The outcomes investigated were recurrent miscarriage, preterm birth, stillbirth, pre-eclampsia, and low birthweight in the pregnancy following miscarriage.

Only 1 study reported the specific gestational age of the index miscarriage at 8.6 ± 2.8 weeks.² All studies adjusted data for age, and some considered other confounders, such as race, smoking status, and body mass index (BMI).

Women included in the meta-analysis were from Asia, Europe, South America, and the United States and had a history of at least 1 miscarriage.¹ A study of 257,908 subjects (Conde-Agudelo) also included women with a history of induced abortion from Latin American countries, where abortion is illegal, and made no distinction between spontaneous and induced abortions in those data sets.³ Women with a history of illegal abortion could be at greater risk of subsequent miscarriage than women who underwent a legally performed abortion.

IPI shorter than 6 months carries fewer risks

Excluding the Conde-Agudelo study, women with an IPI < 6 months, compared with > 6 months, had lower risks of subsequent miscarriage (7 studies, 46,313 women; risk ratio [RR] = 0.82; 95% confidence interval [CI], 0.78-0.86) and preterm delivery (7 studies, 60,772 women; RR = 0.79; 95% CI, 0.75-0.83); a higher rate of live births (4 studies, 44,586 women; RR = 1.06; 95% CI, 1.01-1.11); and no increase in stillbirths (4 studies, 44,586 women; RR = 0.88; 95% CI, 0.76-1.02), low birthweight (4 studies, 284,222 women; RR = 1.05; 95% CI, 0.48-2.29) or pre-eclampsia (5 studies, 284,899 women; RR = 0.95; 95% CI, 0.88-1.02) in the subsequent pregnancy.

Including the Conde-Agudelo study, the risk of preterm delivery was the same in women with an IPI < 6 months and > 6 months (8 studies, 318,880 women; RR = 0.93; 95% CI, 0.58-1.48).¹ Four of the 10 studies evaluated the risk of miscarriage with an IPI < 3 months compared with > 3 months and found either no difference or a lower risk of subsequent miscarriage.^2,4-6

IPI shorter than 3 months has lowest risk of all

A 2017 prospective cohort study examined the association between IPI length and risk of recurrent miscarriage in 514 women who had experienced recent miscarriage (defined as spontaneous pregnancy loss before 20 weeks of gestation).⁷ Average gestational age at the time of initial miscarriage wasn’t reported. Study participants were 30 years of age on average and predominantly white (76.8%); 12.3% were black.

The authors compared IPIs of < 3 months, 3 to 6 months, and > 18 months with IPIs of 6 to 18 months, which correlates with the IPIs recommended by the World Health Organization (WHO).⁸ They adjusted for maternal age, race, parity, BMI, and education. An IPI < 3 months was associated with the lowest risk of subsequent miscarriage (7.3% compared with 22.1%; adjusted hazard ratio = 0.33; 95% CI, 0.16-0.71). Women with IPIs of 3 to 6 months and > 18 months didn’t experience statistically significant differences in subsequent miscarriage rates compared with IPIs of 6 to 18 months.⁷

Continue to: But a short IPI after second-trimester loss increases risk of miscarriage

By including all miscarriages, the meta-analysis effectively examined IPI after first-trimester loss because first-trimester loss occurs far more frequently than does second-trimester loss.¹ A retrospective cohort study of Australian women, not included in the meta-analysis, assessed 4290 patients with a second-trimester pregnancy loss to specifically examine the association between IPI and risk of recurrent pregnancy loss.⁹

After a pregnancy loss at 14 to 19 weeks, women with an IPI < 3 months, compared with an IPI of 9 to 12 months, had an increased risk of recurrent pregnancy loss (21.9 vs 11.3%; P < .001). Women with an IPI > 9 to 12 months had rates of pregnancy loss similar to an IPI of 3 to 6 months (RR = 1.24; 95% CI, 0.89-1.7) and 6 to 9 months (RR = 1.02; 95% CI, 0.7-1.5). Women who experienced an initial loss at 20 to 23 weeks, for unclear reasons, showed no evidence that the IPI affected the risk of subsequent loss.

Short IPI may be linked to anxiety in first trimester of next pregnancy

A large cohort study of 20,308 pregnant Chinese women, including 1495 with a previous miscarriage, explored the mental health impact of IPI after miscarriage compared with no miscarriage.¹⁰ Investigators used the Self-Rating Anxiety Scale to evaluate anxiety and the Center for Epidemiologic Studies Depression Scale to evaluate depression.

An interpregnancy interval of < 6 months after miscarriage is associated with a higher live birth rate in the subsequent pregnancy than a longer IPI.

Women with an IPI of < 7 months after miscarriage were more likely to experience anxiety symptoms in the subsequent pregnancy than were women with no previous miscarriage (adjusted odds ratio [AOR] = 2.76; 95% CI, 1.4-5.5), whereas women with a history of miscarriage and IPI > 6 months weren’t. Women with IPIs < 7 months and 7 to 12 months, compared with women who had no miscarriage, had an increased risk of depression (AOR = 2.5; 95% CI, 1.4-4.5, and AOR = 2.6; 95% CI, 1.3-5.2, respectively). Women with an IPI > 12 months had no increased risk of depression compared with women with no history of miscarriage.

The odds ratios were adjusted for age, education, BMI, income, and place of residence. The higher rates of depression and anxiety didn’t persist beyond the first trimester of the subsequent pregnancy.

Continue to: RECOMMENDATIONS

RECOMMENDATIONS

The American College of Obstetricians and Gynecologists’ Practice Bulletin on Early Pregnancy Loss states that no quality data exist to support delaying conception after early pregnancy loss (defined as loss of an intrauterine pregnancy in the first trimester) to prevent subsequent pregnancy loss or other pregnancy complications.¹¹

WHO recommends a minimum IPI of at least 6 months after a spontaneous or elective abortion. This recommendation is based on a single multi-center cohort study in Latin America that included women with both spontaneous and induced abortions.⁸

Editor’s takeaway

High-quality evidence now shows that shorter IPIs after first-trimester miscarriages result in safe subsequent pregnancies. However, some concern remains about second-trimester miscarriages and maternal mental health following a shorter IPI, based on lower-quality evidence.

EVIDENCE SUMMARY

To evaluate the longstanding belief that a short IPI after miscarriage is associated with adverse outcomes in subsequent pregnancies, a 2017 systematic review and meta-­analysis of 16 studies (3 randomized controlled trials [RCTs] and 13 retrospective cohort studies) with a total of more than 1 million patients compared IPIs shorter and longer than 6 months (miscarriage was defined as any pregnancy loss ­before 24 weeks).¹ The meta-analysis included 10 of the studies (2 RCTs and 8 cohort studies), with a total of 977,972 women and excluded 6 studies because of insufficient data. The outcomes investigated were recurrent miscarriage, preterm birth, stillbirth, pre-eclampsia, and low birthweight in the pregnancy following miscarriage.

Only 1 study reported the specific gestational age of the index miscarriage at 8.6 ± 2.8 weeks.² All studies adjusted data for age, and some considered other confounders, such as race, smoking status, and body mass index (BMI).

Women included in the meta-analysis were from Asia, Europe, South America, and the United States and had a history of at least 1 miscarriage.¹ A study of 257,908 subjects (Conde-Agudelo) also included women with a history of induced abortion from Latin American countries, where abortion is illegal, and made no distinction between spontaneous and induced abortions in those data sets.³ Women with a history of illegal abortion could be at greater risk of subsequent miscarriage than women who underwent a legally performed abortion.

IPI shorter than 6 months carries fewer risks

Excluding the Conde-Agudelo study, women with an IPI < 6 months, compared with > 6 months, had lower risks of subsequent miscarriage (7 studies, 46,313 women; risk ratio [RR] = 0.82; 95% confidence interval [CI], 0.78-0.86) and preterm delivery (7 studies, 60,772 women; RR = 0.79; 95% CI, 0.75-0.83); a higher rate of live births (4 studies, 44,586 women; RR = 1.06; 95% CI, 1.01-1.11); and no increase in stillbirths (4 studies, 44,586 women; RR = 0.88; 95% CI, 0.76-1.02), low birthweight (4 studies, 284,222 women; RR = 1.05; 95% CI, 0.48-2.29) or pre-eclampsia (5 studies, 284,899 women; RR = 0.95; 95% CI, 0.88-1.02) in the subsequent pregnancy.

Including the Conde-Agudelo study, the risk of preterm delivery was the same in women with an IPI < 6 months and > 6 months (8 studies, 318,880 women; RR = 0.93; 95% CI, 0.58-1.48).¹ Four of the 10 studies evaluated the risk of miscarriage with an IPI < 3 months compared with > 3 months and found either no difference or a lower risk of subsequent miscarriage.^2,4-6

IPI shorter than 3 months has lowest risk of all

A 2017 prospective cohort study examined the association between IPI length and risk of recurrent miscarriage in 514 women who had experienced recent miscarriage (defined as spontaneous pregnancy loss before 20 weeks of gestation).⁷ Average gestational age at the time of initial miscarriage wasn’t reported. Study participants were 30 years of age on average and predominantly white (76.8%); 12.3% were black.

The authors compared IPIs of < 3 months, 3 to 6 months, and > 18 months with IPIs of 6 to 18 months, which correlates with the IPIs recommended by the World Health Organization (WHO).⁸ They adjusted for maternal age, race, parity, BMI, and education. An IPI < 3 months was associated with the lowest risk of subsequent miscarriage (7.3% compared with 22.1%; adjusted hazard ratio = 0.33; 95% CI, 0.16-0.71). Women with IPIs of 3 to 6 months and > 18 months didn’t experience statistically significant differences in subsequent miscarriage rates compared with IPIs of 6 to 18 months.⁷

Continue to: But a short IPI after second-trimester loss increases risk of miscarriage

By including all miscarriages, the meta-analysis effectively examined IPI after first-trimester loss because first-trimester loss occurs far more frequently than does second-trimester loss.¹ A retrospective cohort study of Australian women, not included in the meta-analysis, assessed 4290 patients with a second-trimester pregnancy loss to specifically examine the association between IPI and risk of recurrent pregnancy loss.⁹

After a pregnancy loss at 14 to 19 weeks, women with an IPI < 3 months, compared with an IPI of 9 to 12 months, had an increased risk of recurrent pregnancy loss (21.9 vs 11.3%; P < .001). Women with an IPI > 9 to 12 months had rates of pregnancy loss similar to an IPI of 3 to 6 months (RR = 1.24; 95% CI, 0.89-1.7) and 6 to 9 months (RR = 1.02; 95% CI, 0.7-1.5). Women who experienced an initial loss at 20 to 23 weeks, for unclear reasons, showed no evidence that the IPI affected the risk of subsequent loss.

Short IPI may be linked to anxiety in first trimester of next pregnancy

A large cohort study of 20,308 pregnant Chinese women, including 1495 with a previous miscarriage, explored the mental health impact of IPI after miscarriage compared with no miscarriage.¹⁰ Investigators used the Self-Rating Anxiety Scale to evaluate anxiety and the Center for Epidemiologic Studies Depression Scale to evaluate depression.

An interpregnancy interval of < 6 months after miscarriage is associated with a higher live birth rate in the subsequent pregnancy than a longer IPI.

Women with an IPI of < 7 months after miscarriage were more likely to experience anxiety symptoms in the subsequent pregnancy than were women with no previous miscarriage (adjusted odds ratio [AOR] = 2.76; 95% CI, 1.4-5.5), whereas women with a history of miscarriage and IPI > 6 months weren’t. Women with IPIs < 7 months and 7 to 12 months, compared with women who had no miscarriage, had an increased risk of depression (AOR = 2.5; 95% CI, 1.4-4.5, and AOR = 2.6; 95% CI, 1.3-5.2, respectively). Women with an IPI > 12 months had no increased risk of depression compared with women with no history of miscarriage.

The odds ratios were adjusted for age, education, BMI, income, and place of residence. The higher rates of depression and anxiety didn’t persist beyond the first trimester of the subsequent pregnancy.

Continue to: RECOMMENDATIONS

RECOMMENDATIONS

The American College of Obstetricians and Gynecologists’ Practice Bulletin on Early Pregnancy Loss states that no quality data exist to support delaying conception after early pregnancy loss (defined as loss of an intrauterine pregnancy in the first trimester) to prevent subsequent pregnancy loss or other pregnancy complications.¹¹

WHO recommends a minimum IPI of at least 6 months after a spontaneous or elective abortion. This recommendation is based on a single multi-center cohort study in Latin America that included women with both spontaneous and induced abortions.⁸

Editor’s takeaway

High-quality evidence now shows that shorter IPIs after first-trimester miscarriages result in safe subsequent pregnancies. However, some concern remains about second-trimester miscarriages and maternal mental health following a shorter IPI, based on lower-quality evidence.

EVIDENCE SUMMARY

To evaluate the longstanding belief that a short IPI after miscarriage is associated with adverse outcomes in subsequent pregnancies, a 2017 systematic review and meta-­analysis of 16 studies (3 randomized controlled trials [RCTs] and 13 retrospective cohort studies) with a total of more than 1 million patients compared IPIs shorter and longer than 6 months (miscarriage was defined as any pregnancy loss ­before 24 weeks).¹ The meta-analysis included 10 of the studies (2 RCTs and 8 cohort studies), with a total of 977,972 women and excluded 6 studies because of insufficient data. The outcomes investigated were recurrent miscarriage, preterm birth, stillbirth, pre-eclampsia, and low birthweight in the pregnancy following miscarriage.

Only 1 study reported the specific gestational age of the index miscarriage at 8.6 ± 2.8 weeks.² All studies adjusted data for age, and some considered other confounders, such as race, smoking status, and body mass index (BMI).

Women included in the meta-analysis were from Asia, Europe, South America, and the United States and had a history of at least 1 miscarriage.¹ A study of 257,908 subjects (Conde-Agudelo) also included women with a history of induced abortion from Latin American countries, where abortion is illegal, and made no distinction between spontaneous and induced abortions in those data sets.³ Women with a history of illegal abortion could be at greater risk of subsequent miscarriage than women who underwent a legally performed abortion.

IPI shorter than 6 months carries fewer risks

Excluding the Conde-Agudelo study, women with an IPI < 6 months, compared with > 6 months, had lower risks of subsequent miscarriage (7 studies, 46,313 women; risk ratio [RR] = 0.82; 95% confidence interval [CI], 0.78-0.86) and preterm delivery (7 studies, 60,772 women; RR = 0.79; 95% CI, 0.75-0.83); a higher rate of live births (4 studies, 44,586 women; RR = 1.06; 95% CI, 1.01-1.11); and no increase in stillbirths (4 studies, 44,586 women; RR = 0.88; 95% CI, 0.76-1.02), low birthweight (4 studies, 284,222 women; RR = 1.05; 95% CI, 0.48-2.29) or pre-eclampsia (5 studies, 284,899 women; RR = 0.95; 95% CI, 0.88-1.02) in the subsequent pregnancy.

Including the Conde-Agudelo study, the risk of preterm delivery was the same in women with an IPI < 6 months and > 6 months (8 studies, 318,880 women; RR = 0.93; 95% CI, 0.58-1.48).¹ Four of the 10 studies evaluated the risk of miscarriage with an IPI < 3 months compared with > 3 months and found either no difference or a lower risk of subsequent miscarriage.^2,4-6

IPI shorter than 3 months has lowest risk of all

A 2017 prospective cohort study examined the association between IPI length and risk of recurrent miscarriage in 514 women who had experienced recent miscarriage (defined as spontaneous pregnancy loss before 20 weeks of gestation).⁷ Average gestational age at the time of initial miscarriage wasn’t reported. Study participants were 30 years of age on average and predominantly white (76.8%); 12.3% were black.

The authors compared IPIs of < 3 months, 3 to 6 months, and > 18 months with IPIs of 6 to 18 months, which correlates with the IPIs recommended by the World Health Organization (WHO).⁸ They adjusted for maternal age, race, parity, BMI, and education. An IPI < 3 months was associated with the lowest risk of subsequent miscarriage (7.3% compared with 22.1%; adjusted hazard ratio = 0.33; 95% CI, 0.16-0.71). Women with IPIs of 3 to 6 months and > 18 months didn’t experience statistically significant differences in subsequent miscarriage rates compared with IPIs of 6 to 18 months.⁷

Continue to: But a short IPI after second-trimester loss increases risk of miscarriage

By including all miscarriages, the meta-analysis effectively examined IPI after first-trimester loss because first-trimester loss occurs far more frequently than does second-trimester loss.¹ A retrospective cohort study of Australian women, not included in the meta-analysis, assessed 4290 patients with a second-trimester pregnancy loss to specifically examine the association between IPI and risk of recurrent pregnancy loss.⁹

After a pregnancy loss at 14 to 19 weeks, women with an IPI < 3 months, compared with an IPI of 9 to 12 months, had an increased risk of recurrent pregnancy loss (21.9 vs 11.3%; P < .001). Women with an IPI > 9 to 12 months had rates of pregnancy loss similar to an IPI of 3 to 6 months (RR = 1.24; 95% CI, 0.89-1.7) and 6 to 9 months (RR = 1.02; 95% CI, 0.7-1.5). Women who experienced an initial loss at 20 to 23 weeks, for unclear reasons, showed no evidence that the IPI affected the risk of subsequent loss.

Short IPI may be linked to anxiety in first trimester of next pregnancy

A large cohort study of 20,308 pregnant Chinese women, including 1495 with a previous miscarriage, explored the mental health impact of IPI after miscarriage compared with no miscarriage.¹⁰ Investigators used the Self-Rating Anxiety Scale to evaluate anxiety and the Center for Epidemiologic Studies Depression Scale to evaluate depression.

An interpregnancy interval of < 6 months after miscarriage is associated with a higher live birth rate in the subsequent pregnancy than a longer IPI.

Women with an IPI of < 7 months after miscarriage were more likely to experience anxiety symptoms in the subsequent pregnancy than were women with no previous miscarriage (adjusted odds ratio [AOR] = 2.76; 95% CI, 1.4-5.5), whereas women with a history of miscarriage and IPI > 6 months weren’t. Women with IPIs < 7 months and 7 to 12 months, compared with women who had no miscarriage, had an increased risk of depression (AOR = 2.5; 95% CI, 1.4-4.5, and AOR = 2.6; 95% CI, 1.3-5.2, respectively). Women with an IPI > 12 months had no increased risk of depression compared with women with no history of miscarriage.

The odds ratios were adjusted for age, education, BMI, income, and place of residence. The higher rates of depression and anxiety didn’t persist beyond the first trimester of the subsequent pregnancy.

Continue to: RECOMMENDATIONS

RECOMMENDATIONS

The American College of Obstetricians and Gynecologists’ Practice Bulletin on Early Pregnancy Loss states that no quality data exist to support delaying conception after early pregnancy loss (defined as loss of an intrauterine pregnancy in the first trimester) to prevent subsequent pregnancy loss or other pregnancy complications.¹¹

WHO recommends a minimum IPI of at least 6 months after a spontaneous or elective abortion. This recommendation is based on a single multi-center cohort study in Latin America that included women with both spontaneous and induced abortions.⁸

Editor’s takeaway

High-quality evidence now shows that shorter IPIs after first-trimester miscarriages result in safe subsequent pregnancies. However, some concern remains about second-trimester miscarriages and maternal mental health following a shorter IPI, based on lower-quality evidence.

PRACTICE CHANGER
Discuss cell-free DNA testing when offering fetal aneuploidy screening to pregnant women.^1,2

Strength of recommendation
A: Based on multiple large, multicenter cohort studies.^1,2

A 28-year-old woman (gravida 2, para 1001) at 10 weeks’ gestation presents to your clinic for a routine first-trimester prenatal visit. Her first child has no known chromosomal abnormalities, and she has no family history of aneuploidy. She asks you which tests are available to screen her fetus for chromosomal abnormalities.

Pregnant women have traditionally been offered some combination of serum biomarkers and nuchal translucency to assess the risk for fetal aneuploidy. Cell-free DNA testing (cfDNA) is a form of noninvasive prenatal testing that uses maternal serum samples to conduct massively parallel sequencing of cell-free fetal DNA fragments.

It has been offered to pregnant women as a screening test to detect fetal chromosomal abnormalities since 2011, after multiple clinical studies found high sensitivities, specificities, and negative predictive values (NPVs) for detecting aneuploidy.^3-6 However, until 2015, practice guidelines from the American Congress of Obstetricians and Gynecologists (ACOG) recommended that standard aneuploidy screening or diagnostic testing be offered to all pregnant women and cfDNA be reserved for women with pregnancies at high risk for aneuploidy (strength of recommendation: B).⁷

CARE (Comparison of Aneuploidy Risk Evaluation) and NEXT (Noninvasive Examination of Trisomy) are two large studies that compared cfDNA and standard aneuploidy screening methods in pregnant women at low risk for fetal aneuploidy. Based on new data from these and other studies, ACOG and the Society for Maternal-Fetal Medicine (SMFM) released a new consensus statement in June 2015 that addressed the use of cfDNA in the general obstetric population. The two groups still recommend conventional first- and second-trimester screening by serum chemical biomarkers and nuchal translucency as the firstline approach for low-risk women who want to pursue aneuploidy screening; however, they also recommend that the risks and benefits of cfDNA be discussed with all patients.⁸

Continue for study summaries >>

STUDY SUMMARIES
CARE was a prospective, blinded, multicenter (21 US sites across 14 states) study that compared the aneuploidy detection rates of ­cfDNA to those of standard screening. Standard aneuploidy screening included assays of first- or second-trimester serum biomarkers with or without fetal nuchal translucency measurement.

This study enrolled 2,042 pregnant patients ages 18 to 49 (mean, 29.6) with singleton pregnancies. The population was racially and ethnically diverse (65% white, 22% black, 11% Hispanic, 7% Asian). This study included women with diabetes, thyroid disorders, and other comorbidities. cfDNA testing was done on 1,909 maternal blood samples for trisomy 21 and 1,905 for trisomy 18.

cfDNA and standard aneuploidy screening results were compared to pregnancy outcomes. The presence of aneuploidy was determined by physician-documented newborn physical exam (97%) or karyotype analysis (3%). In both live and nonlive births, the incidence of trisomy 21 was 5 of 1,909 cases (0.3%) and the incidence of trisomy 18 was 2 of 1,905 cases (0.1%).

The NPV of cfDNA in this study was 100% (95% confidence interval, 99.8%-100%) for both trisomy 21 and trisomy 18. The positive predictive value (PPV) was higher with cfDNA compared to standard screening (45.5% vs 4.2% for trisomy 21 and 40% vs 8.3% for trisomy 18). This means that approximately 1 in 25 women with a positive standard aneuploidy screen actually has aneuploidy. In contrast, nearly 1 in 2 women with a positive cfDNA result has aneuploidy.

Similarly, false-positive rates with cfDNA were significantly lower than those with standard screening. For trisomy 21, the cfDNA false-positive rate was 0.3% compared to 3.6% for standard screening (P < .001); for trisomy 18, the cfDNA false-positive rate was 0.2% compared to 0.6% for standard screening (P = .03).

NEXT was a prospective, blinded cohort study that compared cfDNA testing with standard first-trimester screening (with measurements of nuchal translucency and serum biochemical analysis) in a routine prenatal population at 35 centers in six countries.

This study enrolled 18,955 women ages 18 to 48 (mean, 31) who underwent traditional first-trimester screening and cfDNA testing. Eligible patients included pregnant women with a singleton pregnancy with a gestational age between 10 and 14.3 weeks. Prenatal screening results were compared to newborn outcomes using a documented newborn physical examination and, if performed, results of genetic testing. For women who had a miscarriage or stillbirth or chose to terminate the pregnancy, outcomes were determined by diagnostic genetic testing.

The primary outcome was the area under the receiver-operating-characteristic (ROC) curve for trisomy 21. Area under the ROC curve is a measure of a diagnostic test’s accuracy that plots sensitivity against 1 – specificity; < .700 is considered a poor test, whereas 1.00 is a perfect test. A secondary analysis evaluated cfDNA testing in low-risk women (ages < 35).

The area under the ROC curve was 0.999 for cfDNA compared with 0.958 for standard screening (P = .001). For diagnosis of trisomy 21, cfDNA had a higher PPV than standard testing (80.9% vs 3.4%; P < .001) and a lower false-positive rate (0.06% vs 5.4%; P < .001). These findings were consistent in the secondary analysis of low-risk women.

Both the CARE and NEXT trials also evaluated cfDNA testing versus standard screening for diagnosis of trisomy 13 and 18 and found higher PPVs and lower false-positive rates for cfDNA, compared with traditional screening.

WHAT’S NEW
Previously, cfDNA was recommended only for women with high-risk pregnancies. The new data demonstrate that cfDNA has substantially better PPVs and lower false-positive rates than standard fetal aneuploidy screening for the general obstetric population.

So while conventional screening tests remain the most appropriate methods for aneuploidy detection in the general obstetric population, according to ACOG and SMFM, the two groups now recommend that all screening options—including cfDNA—be discussed with every woman. Any woman may choose cfDNA but should be counseled about the risks and benefits.⁸

Continue for caveats >>

CAVEATS
Both the CARE and NEXT studies had limitations. They compared cfDNA testing with first- or second-trimester screening and did not evaluate integrated screening methods (sequential first- and second-trimester biomarkers plus first-trimester nuchal translucency), which have a slightly higher sensitivity and specificity than first-trimester screening alone.

Multiple companies offer cfDNA, and the test is not subject to FDA approval. The CARE and NEXT studies used tests from companies that provided funding for these studies and employ several of the study authors.

Although cfDNA has increased specificity compared to standard screening, there have been case reports of false-negative results. Further testing has shown that such false-negative results could be caused by mosaicism in either the fetus and/or placenta, vanishing twins, or maternal malig­nancies.^8-10

In the CARE and NEXT trials, cfDNA produced no results in 0.9% and 3% of women, respectively. Patients for whom cfDNA testing yields no results have higher rates of aneuploidy, and therefore require further diagnostic testing.

Because the prevalence of aneuploidy is lower in the general obstetric population than it is among women whose pregnancies are at high risk for aneuploidy, the PPV of cfDNA testing is also lower in the general obstetric population. This means that there are more false-positive results for women at lower risk for aneuploidy. Therefore, it is imperative that women with positive cfDNA tests receive follow-up diagnostic testing, such as chorionic villus sampling or amniocentesis, before making a decision about termination.

All commercially available cfDNA tests have high sensitivity and specificity for trisomy 21, 18, and 13. Some offer testing for sex chromosome abnormalities and microdeletions. However, current cfDNA testing methods are unable to detect up to 17% of other clinically significant chromosomal abnormalities,¹¹ and cfDNA cannot detect neural tube or ventral wall defects. Therefore, ACOG and SMFM recommend that women who choose cfDNA as their ­aneuploidy screening method also be offered maternal serum alpha-fetoprotein or ultrasound evaluation.

Continue for challenges to implementation >>

CHALLENGES TO IMPLEMENTATION
cfDNA testing is validated only for singleton pregnancies. Clinicians should obtain a baseline fetal ultrasound to confirm the number of fetuses, gestational age, and viability before ordering cfDNA to ensure it is the most appropriate screening test. This may add to the overall number of early pregnancy ultrasounds conducted.

Counseling patients about aneuploidy screening options is time-consuming and requires discussion of the limitations of each screening method and caution that a negative cfDNA result does not guarantee an unaffected fetus, nor does a positive result guarantee an affected fetus. However, aneuploidy screening is well within the scope of care for family practice clinicians who provide prenatal care, and referral to genetic specialists is not necessary or recommended.

Some patients may request cfDNA in order to facilitate earlier identification of fetal sex. In such cases, clinicians should advise patients that cfDNA testing also assesses trisomy risk. Patients who do not wish to assess their risk for aneuploidy should not receive cfDNA testing.

Finally, while cfDNA is routinely recommended for women with pregnancies considered at high risk for aneuploidy, many insurance companies do not cover the cost of cfDNA for women with low-risk pregnancies, and the test may cost up to $1,700.¹² The overall cost-effectiveness of cfDNA for aneuploidy screening in low-risk women is unknown.

References
1. Bianchi DW, Parker RL, Wentworth J, et al; CARE Study Group. DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med. 2014;370:799-808.
2. Norton ME, Jacobsson B, Swamy GK, et al. Cell-free DNA analysis for noninvasive examination of trisomy. N Engl J Med. 2015;372: 1589-1597.
3. Chiu RW, Akolekar R, Zheng YW, et al. Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ. 2011; 342:c7401.
4. Ehrich M, Deciu C, Zwiefelhofer T, et al. Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting. Am J Obstet Gynecol. 2011;204:205.e1-11.
5. Bianchi DW, Platt LD, Goldberg JD, et al; MatERNal BLood IS Source to Accurately diagnose fetal aneuploidy (MELISSA) Study Group. Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol. 2012;119:890-901.
6. Norton ME, Brar H, Weiss J, et al. Non-invasive chromosomal evaluation (NICE) study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18. Am J Obstet Gynecol. 2012;207: 137.e1-e8.
7. American College of Obstetricians and Gynecologists Committee on Genetics. Committee Opinion No. 545: Noninvasive prenatal testing for fetal aneuploidy. Obstet Gynecol. 2012;120:1532-1534.
8. American College of Obstetricians and Gynecologists Committee on Genetics. Committee Opinion No. 640: Cell-free DNA screening for fetal aneuploidy. Obstet Gynecol. 2015;126:e31-e37.
9. Wang Y, Zhu J, Chen Y, et al. Two cases of placental T21 mosaicism: challenging the detection limits of non-invasive prenatal testing. Prenat Diagn. 2013;33:1207-1210.
10. Choi H, Lau TK, Jiang FM, et al. Fetal aneuploidy screening by maternal plasma DNA sequencing: ‘false positive’ due to confined placental mosaicism. Prenat Diagn. 2013; 33:198-200.
11. Norton ME, Jelliffe-Pawlowski LL, Currier RJ. Chromosome abnormalities detected by current prenatal screening and noninvasive prenatal testing. Obstet Gynecol. 2014;124:979-986.
12. Agarwal A, Sayres LC, Cho MK, et al. Commercial landscape of noninvasive prenatal testing in the United States. Prenat Diagn. 2013;33:521-531.

ACKNOWLEDGEMENT
The PURLs Surveillance System was supported in part by Grant Number UL1RR024999 from the National Center For Research Resources, a Clinical Translational Science Award to the University of Chicago. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center For Research Resources or the National Institutes of Health.

Copyright © 2016. The Family Physicians Inquiries Network. All rights reserved.

Reprinted with permission from the Family Physicians Inquiries Network and The Journal of Family Practice. 2016;65(1):49-52.

PRACTICE CHANGER
Discuss cell-free DNA testing when offering fetal aneuploidy screening to pregnant women.^1,2

Strength of recommendation
A: Based on multiple large, multicenter cohort studies.^1,2

A 28-year-old woman (gravida 2, para 1001) at 10 weeks’ gestation presents to your clinic for a routine first-trimester prenatal visit. Her first child has no known chromosomal abnormalities, and she has no family history of aneuploidy. She asks you which tests are available to screen her fetus for chromosomal abnormalities.

Pregnant women have traditionally been offered some combination of serum biomarkers and nuchal translucency to assess the risk for fetal aneuploidy. Cell-free DNA testing (cfDNA) is a form of noninvasive prenatal testing that uses maternal serum samples to conduct massively parallel sequencing of cell-free fetal DNA fragments.

It has been offered to pregnant women as a screening test to detect fetal chromosomal abnormalities since 2011, after multiple clinical studies found high sensitivities, specificities, and negative predictive values (NPVs) for detecting aneuploidy.^3-6 However, until 2015, practice guidelines from the American Congress of Obstetricians and Gynecologists (ACOG) recommended that standard aneuploidy screening or diagnostic testing be offered to all pregnant women and cfDNA be reserved for women with pregnancies at high risk for aneuploidy (strength of recommendation: B).⁷

CARE (Comparison of Aneuploidy Risk Evaluation) and NEXT (Noninvasive Examination of Trisomy) are two large studies that compared cfDNA and standard aneuploidy screening methods in pregnant women at low risk for fetal aneuploidy. Based on new data from these and other studies, ACOG and the Society for Maternal-Fetal Medicine (SMFM) released a new consensus statement in June 2015 that addressed the use of cfDNA in the general obstetric population. The two groups still recommend conventional first- and second-trimester screening by serum chemical biomarkers and nuchal translucency as the firstline approach for low-risk women who want to pursue aneuploidy screening; however, they also recommend that the risks and benefits of cfDNA be discussed with all patients.⁸

Continue for study summaries >>

STUDY SUMMARIES
CARE was a prospective, blinded, multicenter (21 US sites across 14 states) study that compared the aneuploidy detection rates of ­cfDNA to those of standard screening. Standard aneuploidy screening included assays of first- or second-trimester serum biomarkers with or without fetal nuchal translucency measurement.

This study enrolled 2,042 pregnant patients ages 18 to 49 (mean, 29.6) with singleton pregnancies. The population was racially and ethnically diverse (65% white, 22% black, 11% Hispanic, 7% Asian). This study included women with diabetes, thyroid disorders, and other comorbidities. cfDNA testing was done on 1,909 maternal blood samples for trisomy 21 and 1,905 for trisomy 18.

cfDNA and standard aneuploidy screening results were compared to pregnancy outcomes. The presence of aneuploidy was determined by physician-documented newborn physical exam (97%) or karyotype analysis (3%). In both live and nonlive births, the incidence of trisomy 21 was 5 of 1,909 cases (0.3%) and the incidence of trisomy 18 was 2 of 1,905 cases (0.1%).

The NPV of cfDNA in this study was 100% (95% confidence interval, 99.8%-100%) for both trisomy 21 and trisomy 18. The positive predictive value (PPV) was higher with cfDNA compared to standard screening (45.5% vs 4.2% for trisomy 21 and 40% vs 8.3% for trisomy 18). This means that approximately 1 in 25 women with a positive standard aneuploidy screen actually has aneuploidy. In contrast, nearly 1 in 2 women with a positive cfDNA result has aneuploidy.

Similarly, false-positive rates with cfDNA were significantly lower than those with standard screening. For trisomy 21, the cfDNA false-positive rate was 0.3% compared to 3.6% for standard screening (P < .001); for trisomy 18, the cfDNA false-positive rate was 0.2% compared to 0.6% for standard screening (P = .03).

NEXT was a prospective, blinded cohort study that compared cfDNA testing with standard first-trimester screening (with measurements of nuchal translucency and serum biochemical analysis) in a routine prenatal population at 35 centers in six countries.

This study enrolled 18,955 women ages 18 to 48 (mean, 31) who underwent traditional first-trimester screening and cfDNA testing. Eligible patients included pregnant women with a singleton pregnancy with a gestational age between 10 and 14.3 weeks. Prenatal screening results were compared to newborn outcomes using a documented newborn physical examination and, if performed, results of genetic testing. For women who had a miscarriage or stillbirth or chose to terminate the pregnancy, outcomes were determined by diagnostic genetic testing.

The primary outcome was the area under the receiver-operating-characteristic (ROC) curve for trisomy 21. Area under the ROC curve is a measure of a diagnostic test’s accuracy that plots sensitivity against 1 – specificity; < .700 is considered a poor test, whereas 1.00 is a perfect test. A secondary analysis evaluated cfDNA testing in low-risk women (ages < 35).

The area under the ROC curve was 0.999 for cfDNA compared with 0.958 for standard screening (P = .001). For diagnosis of trisomy 21, cfDNA had a higher PPV than standard testing (80.9% vs 3.4%; P < .001) and a lower false-positive rate (0.06% vs 5.4%; P < .001). These findings were consistent in the secondary analysis of low-risk women.

Both the CARE and NEXT trials also evaluated cfDNA testing versus standard screening for diagnosis of trisomy 13 and 18 and found higher PPVs and lower false-positive rates for cfDNA, compared with traditional screening.

WHAT’S NEW
Previously, cfDNA was recommended only for women with high-risk pregnancies. The new data demonstrate that cfDNA has substantially better PPVs and lower false-positive rates than standard fetal aneuploidy screening for the general obstetric population.

So while conventional screening tests remain the most appropriate methods for aneuploidy detection in the general obstetric population, according to ACOG and SMFM, the two groups now recommend that all screening options—including cfDNA—be discussed with every woman. Any woman may choose cfDNA but should be counseled about the risks and benefits.⁸

Continue for caveats >>

CAVEATS
Both the CARE and NEXT studies had limitations. They compared cfDNA testing with first- or second-trimester screening and did not evaluate integrated screening methods (sequential first- and second-trimester biomarkers plus first-trimester nuchal translucency), which have a slightly higher sensitivity and specificity than first-trimester screening alone.

Multiple companies offer cfDNA, and the test is not subject to FDA approval. The CARE and NEXT studies used tests from companies that provided funding for these studies and employ several of the study authors.

Although cfDNA has increased specificity compared to standard screening, there have been case reports of false-negative results. Further testing has shown that such false-negative results could be caused by mosaicism in either the fetus and/or placenta, vanishing twins, or maternal malig­nancies.^8-10

In the CARE and NEXT trials, cfDNA produced no results in 0.9% and 3% of women, respectively. Patients for whom cfDNA testing yields no results have higher rates of aneuploidy, and therefore require further diagnostic testing.

Because the prevalence of aneuploidy is lower in the general obstetric population than it is among women whose pregnancies are at high risk for aneuploidy, the PPV of cfDNA testing is also lower in the general obstetric population. This means that there are more false-positive results for women at lower risk for aneuploidy. Therefore, it is imperative that women with positive cfDNA tests receive follow-up diagnostic testing, such as chorionic villus sampling or amniocentesis, before making a decision about termination.

All commercially available cfDNA tests have high sensitivity and specificity for trisomy 21, 18, and 13. Some offer testing for sex chromosome abnormalities and microdeletions. However, current cfDNA testing methods are unable to detect up to 17% of other clinically significant chromosomal abnormalities,¹¹ and cfDNA cannot detect neural tube or ventral wall defects. Therefore, ACOG and SMFM recommend that women who choose cfDNA as their ­aneuploidy screening method also be offered maternal serum alpha-fetoprotein or ultrasound evaluation.

Continue for challenges to implementation >>

CHALLENGES TO IMPLEMENTATION
cfDNA testing is validated only for singleton pregnancies. Clinicians should obtain a baseline fetal ultrasound to confirm the number of fetuses, gestational age, and viability before ordering cfDNA to ensure it is the most appropriate screening test. This may add to the overall number of early pregnancy ultrasounds conducted.

Counseling patients about aneuploidy screening options is time-consuming and requires discussion of the limitations of each screening method and caution that a negative cfDNA result does not guarantee an unaffected fetus, nor does a positive result guarantee an affected fetus. However, aneuploidy screening is well within the scope of care for family practice clinicians who provide prenatal care, and referral to genetic specialists is not necessary or recommended.

Some patients may request cfDNA in order to facilitate earlier identification of fetal sex. In such cases, clinicians should advise patients that cfDNA testing also assesses trisomy risk. Patients who do not wish to assess their risk for aneuploidy should not receive cfDNA testing.

Finally, while cfDNA is routinely recommended for women with pregnancies considered at high risk for aneuploidy, many insurance companies do not cover the cost of cfDNA for women with low-risk pregnancies, and the test may cost up to $1,700.¹² The overall cost-effectiveness of cfDNA for aneuploidy screening in low-risk women is unknown.

References
1. Bianchi DW, Parker RL, Wentworth J, et al; CARE Study Group. DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med. 2014;370:799-808.
2. Norton ME, Jacobsson B, Swamy GK, et al. Cell-free DNA analysis for noninvasive examination of trisomy. N Engl J Med. 2015;372: 1589-1597.
3. Chiu RW, Akolekar R, Zheng YW, et al. Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ. 2011; 342:c7401.
4. Ehrich M, Deciu C, Zwiefelhofer T, et al. Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting. Am J Obstet Gynecol. 2011;204:205.e1-11.
5. Bianchi DW, Platt LD, Goldberg JD, et al; MatERNal BLood IS Source to Accurately diagnose fetal aneuploidy (MELISSA) Study Group. Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol. 2012;119:890-901.
6. Norton ME, Brar H, Weiss J, et al. Non-invasive chromosomal evaluation (NICE) study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18. Am J Obstet Gynecol. 2012;207: 137.e1-e8.
7. American College of Obstetricians and Gynecologists Committee on Genetics. Committee Opinion No. 545: Noninvasive prenatal testing for fetal aneuploidy. Obstet Gynecol. 2012;120:1532-1534.
8. American College of Obstetricians and Gynecologists Committee on Genetics. Committee Opinion No. 640: Cell-free DNA screening for fetal aneuploidy. Obstet Gynecol. 2015;126:e31-e37.
9. Wang Y, Zhu J, Chen Y, et al. Two cases of placental T21 mosaicism: challenging the detection limits of non-invasive prenatal testing. Prenat Diagn. 2013;33:1207-1210.
10. Choi H, Lau TK, Jiang FM, et al. Fetal aneuploidy screening by maternal plasma DNA sequencing: ‘false positive’ due to confined placental mosaicism. Prenat Diagn. 2013; 33:198-200.
11. Norton ME, Jelliffe-Pawlowski LL, Currier RJ. Chromosome abnormalities detected by current prenatal screening and noninvasive prenatal testing. Obstet Gynecol. 2014;124:979-986.
12. Agarwal A, Sayres LC, Cho MK, et al. Commercial landscape of noninvasive prenatal testing in the United States. Prenat Diagn. 2013;33:521-531.

ACKNOWLEDGEMENT
The PURLs Surveillance System was supported in part by Grant Number UL1RR024999 from the National Center For Research Resources, a Clinical Translational Science Award to the University of Chicago. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center For Research Resources or the National Institutes of Health.

Copyright © 2016. The Family Physicians Inquiries Network. All rights reserved.

Reprinted with permission from the Family Physicians Inquiries Network and The Journal of Family Practice. 2016;65(1):49-52.

PRACTICE CHANGER
Discuss cell-free DNA testing when offering fetal aneuploidy screening to pregnant women.^1,2

Strength of recommendation
A: Based on multiple large, multicenter cohort studies.^1,2

A 28-year-old woman (gravida 2, para 1001) at 10 weeks’ gestation presents to your clinic for a routine first-trimester prenatal visit. Her first child has no known chromosomal abnormalities, and she has no family history of aneuploidy. She asks you which tests are available to screen her fetus for chromosomal abnormalities.

Pregnant women have traditionally been offered some combination of serum biomarkers and nuchal translucency to assess the risk for fetal aneuploidy. Cell-free DNA testing (cfDNA) is a form of noninvasive prenatal testing that uses maternal serum samples to conduct massively parallel sequencing of cell-free fetal DNA fragments.

It has been offered to pregnant women as a screening test to detect fetal chromosomal abnormalities since 2011, after multiple clinical studies found high sensitivities, specificities, and negative predictive values (NPVs) for detecting aneuploidy.^3-6 However, until 2015, practice guidelines from the American Congress of Obstetricians and Gynecologists (ACOG) recommended that standard aneuploidy screening or diagnostic testing be offered to all pregnant women and cfDNA be reserved for women with pregnancies at high risk for aneuploidy (strength of recommendation: B).⁷

CARE (Comparison of Aneuploidy Risk Evaluation) and NEXT (Noninvasive Examination of Trisomy) are two large studies that compared cfDNA and standard aneuploidy screening methods in pregnant women at low risk for fetal aneuploidy. Based on new data from these and other studies, ACOG and the Society for Maternal-Fetal Medicine (SMFM) released a new consensus statement in June 2015 that addressed the use of cfDNA in the general obstetric population. The two groups still recommend conventional first- and second-trimester screening by serum chemical biomarkers and nuchal translucency as the firstline approach for low-risk women who want to pursue aneuploidy screening; however, they also recommend that the risks and benefits of cfDNA be discussed with all patients.⁸

Continue for study summaries >>

STUDY SUMMARIES
CARE was a prospective, blinded, multicenter (21 US sites across 14 states) study that compared the aneuploidy detection rates of ­cfDNA to those of standard screening. Standard aneuploidy screening included assays of first- or second-trimester serum biomarkers with or without fetal nuchal translucency measurement.

This study enrolled 2,042 pregnant patients ages 18 to 49 (mean, 29.6) with singleton pregnancies. The population was racially and ethnically diverse (65% white, 22% black, 11% Hispanic, 7% Asian). This study included women with diabetes, thyroid disorders, and other comorbidities. cfDNA testing was done on 1,909 maternal blood samples for trisomy 21 and 1,905 for trisomy 18.

cfDNA and standard aneuploidy screening results were compared to pregnancy outcomes. The presence of aneuploidy was determined by physician-documented newborn physical exam (97%) or karyotype analysis (3%). In both live and nonlive births, the incidence of trisomy 21 was 5 of 1,909 cases (0.3%) and the incidence of trisomy 18 was 2 of 1,905 cases (0.1%).

The NPV of cfDNA in this study was 100% (95% confidence interval, 99.8%-100%) for both trisomy 21 and trisomy 18. The positive predictive value (PPV) was higher with cfDNA compared to standard screening (45.5% vs 4.2% for trisomy 21 and 40% vs 8.3% for trisomy 18). This means that approximately 1 in 25 women with a positive standard aneuploidy screen actually has aneuploidy. In contrast, nearly 1 in 2 women with a positive cfDNA result has aneuploidy.

Similarly, false-positive rates with cfDNA were significantly lower than those with standard screening. For trisomy 21, the cfDNA false-positive rate was 0.3% compared to 3.6% for standard screening (P < .001); for trisomy 18, the cfDNA false-positive rate was 0.2% compared to 0.6% for standard screening (P = .03).

NEXT was a prospective, blinded cohort study that compared cfDNA testing with standard first-trimester screening (with measurements of nuchal translucency and serum biochemical analysis) in a routine prenatal population at 35 centers in six countries.

This study enrolled 18,955 women ages 18 to 48 (mean, 31) who underwent traditional first-trimester screening and cfDNA testing. Eligible patients included pregnant women with a singleton pregnancy with a gestational age between 10 and 14.3 weeks. Prenatal screening results were compared to newborn outcomes using a documented newborn physical examination and, if performed, results of genetic testing. For women who had a miscarriage or stillbirth or chose to terminate the pregnancy, outcomes were determined by diagnostic genetic testing.

The primary outcome was the area under the receiver-operating-characteristic (ROC) curve for trisomy 21. Area under the ROC curve is a measure of a diagnostic test’s accuracy that plots sensitivity against 1 – specificity; < .700 is considered a poor test, whereas 1.00 is a perfect test. A secondary analysis evaluated cfDNA testing in low-risk women (ages < 35).

The area under the ROC curve was 0.999 for cfDNA compared with 0.958 for standard screening (P = .001). For diagnosis of trisomy 21, cfDNA had a higher PPV than standard testing (80.9% vs 3.4%; P < .001) and a lower false-positive rate (0.06% vs 5.4%; P < .001). These findings were consistent in the secondary analysis of low-risk women.

Both the CARE and NEXT trials also evaluated cfDNA testing versus standard screening for diagnosis of trisomy 13 and 18 and found higher PPVs and lower false-positive rates for cfDNA, compared with traditional screening.

WHAT’S NEW
Previously, cfDNA was recommended only for women with high-risk pregnancies. The new data demonstrate that cfDNA has substantially better PPVs and lower false-positive rates than standard fetal aneuploidy screening for the general obstetric population.

So while conventional screening tests remain the most appropriate methods for aneuploidy detection in the general obstetric population, according to ACOG and SMFM, the two groups now recommend that all screening options—including cfDNA—be discussed with every woman. Any woman may choose cfDNA but should be counseled about the risks and benefits.⁸

Continue for caveats >>

CAVEATS
Both the CARE and NEXT studies had limitations. They compared cfDNA testing with first- or second-trimester screening and did not evaluate integrated screening methods (sequential first- and second-trimester biomarkers plus first-trimester nuchal translucency), which have a slightly higher sensitivity and specificity than first-trimester screening alone.

Multiple companies offer cfDNA, and the test is not subject to FDA approval. The CARE and NEXT studies used tests from companies that provided funding for these studies and employ several of the study authors.

Although cfDNA has increased specificity compared to standard screening, there have been case reports of false-negative results. Further testing has shown that such false-negative results could be caused by mosaicism in either the fetus and/or placenta, vanishing twins, or maternal malig­nancies.^8-10

In the CARE and NEXT trials, cfDNA produced no results in 0.9% and 3% of women, respectively. Patients for whom cfDNA testing yields no results have higher rates of aneuploidy, and therefore require further diagnostic testing.

Because the prevalence of aneuploidy is lower in the general obstetric population than it is among women whose pregnancies are at high risk for aneuploidy, the PPV of cfDNA testing is also lower in the general obstetric population. This means that there are more false-positive results for women at lower risk for aneuploidy. Therefore, it is imperative that women with positive cfDNA tests receive follow-up diagnostic testing, such as chorionic villus sampling or amniocentesis, before making a decision about termination.

All commercially available cfDNA tests have high sensitivity and specificity for trisomy 21, 18, and 13. Some offer testing for sex chromosome abnormalities and microdeletions. However, current cfDNA testing methods are unable to detect up to 17% of other clinically significant chromosomal abnormalities,¹¹ and cfDNA cannot detect neural tube or ventral wall defects. Therefore, ACOG and SMFM recommend that women who choose cfDNA as their ­aneuploidy screening method also be offered maternal serum alpha-fetoprotein or ultrasound evaluation.

Continue for challenges to implementation >>

CHALLENGES TO IMPLEMENTATION
cfDNA testing is validated only for singleton pregnancies. Clinicians should obtain a baseline fetal ultrasound to confirm the number of fetuses, gestational age, and viability before ordering cfDNA to ensure it is the most appropriate screening test. This may add to the overall number of early pregnancy ultrasounds conducted.

Counseling patients about aneuploidy screening options is time-consuming and requires discussion of the limitations of each screening method and caution that a negative cfDNA result does not guarantee an unaffected fetus, nor does a positive result guarantee an affected fetus. However, aneuploidy screening is well within the scope of care for family practice clinicians who provide prenatal care, and referral to genetic specialists is not necessary or recommended.

Some patients may request cfDNA in order to facilitate earlier identification of fetal sex. In such cases, clinicians should advise patients that cfDNA testing also assesses trisomy risk. Patients who do not wish to assess their risk for aneuploidy should not receive cfDNA testing.

Finally, while cfDNA is routinely recommended for women with pregnancies considered at high risk for aneuploidy, many insurance companies do not cover the cost of cfDNA for women with low-risk pregnancies, and the test may cost up to $1,700.¹² The overall cost-effectiveness of cfDNA for aneuploidy screening in low-risk women is unknown.

References
1. Bianchi DW, Parker RL, Wentworth J, et al; CARE Study Group. DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med. 2014;370:799-808.
2. Norton ME, Jacobsson B, Swamy GK, et al. Cell-free DNA analysis for noninvasive examination of trisomy. N Engl J Med. 2015;372: 1589-1597.
3. Chiu RW, Akolekar R, Zheng YW, et al. Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ. 2011; 342:c7401.
4. Ehrich M, Deciu C, Zwiefelhofer T, et al. Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting. Am J Obstet Gynecol. 2011;204:205.e1-11.
5. Bianchi DW, Platt LD, Goldberg JD, et al; MatERNal BLood IS Source to Accurately diagnose fetal aneuploidy (MELISSA) Study Group. Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol. 2012;119:890-901.
6. Norton ME, Brar H, Weiss J, et al. Non-invasive chromosomal evaluation (NICE) study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18. Am J Obstet Gynecol. 2012;207: 137.e1-e8.
7. American College of Obstetricians and Gynecologists Committee on Genetics. Committee Opinion No. 545: Noninvasive prenatal testing for fetal aneuploidy. Obstet Gynecol. 2012;120:1532-1534.
8. American College of Obstetricians and Gynecologists Committee on Genetics. Committee Opinion No. 640: Cell-free DNA screening for fetal aneuploidy. Obstet Gynecol. 2015;126:e31-e37.
9. Wang Y, Zhu J, Chen Y, et al. Two cases of placental T21 mosaicism: challenging the detection limits of non-invasive prenatal testing. Prenat Diagn. 2013;33:1207-1210.
10. Choi H, Lau TK, Jiang FM, et al. Fetal aneuploidy screening by maternal plasma DNA sequencing: ‘false positive’ due to confined placental mosaicism. Prenat Diagn. 2013; 33:198-200.
11. Norton ME, Jelliffe-Pawlowski LL, Currier RJ. Chromosome abnormalities detected by current prenatal screening and noninvasive prenatal testing. Obstet Gynecol. 2014;124:979-986.
12. Agarwal A, Sayres LC, Cho MK, et al. Commercial landscape of noninvasive prenatal testing in the United States. Prenat Diagn. 2013;33:521-531.

ACKNOWLEDGEMENT
The PURLs Surveillance System was supported in part by Grant Number UL1RR024999 from the National Center For Research Resources, a Clinical Translational Science Award to the University of Chicago. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center For Research Resources or the National Institutes of Health.

Copyright © 2016. The Family Physicians Inquiries Network. All rights reserved.

Reprinted with permission from the Family Physicians Inquiries Network and The Journal of Family Practice. 2016;65(1):49-52.

Illustrative case

A 28-year-old gravida 2, para 1001 at 10 weeks gestation presents to your clinic for a routine first-trimester prenatal visit. Her first child has no known chromosomal abnormalities and she has no family history of aneuploidy. She asks you which tests are available to screen her fetus for chromosomal abnormalities.

Pregnant women have traditionally been offered some combination of serum biomarkers and nuchal translucency to assess the risk of fetal aneuploidy. Cell-free DNA testing (cfDNA) is a form of noninvasive prenatal testing that uses maternal serum samples to conduct massively parallel sequencing of cell-free fetal DNA fragments. It has been offered to pregnant women as a screening test to detect fetal chromosomal abnormalities since 2011 after multiple clinical studies found high sensitivities, specificities, and negative predictive values (NPVs) for detecting aneuploidy.^3-6 However until 2015, practice guidelines from the American Congress of Obstetricians and Gynecologists (ACOG) recommended that standard aneuploidy screening or diagnostic testing be offered to all pregnant women and cfDNA be reserved for women with pregnancies at high risk for aneuploidy (strength of recommendation: B).⁷

CARE (Comparison of Aneuploidy Risk Evaluation) and NEXT (Noninvasive Examination of Trisomy) are 2 large studies that compared cfDNA and standard aneuploidy screening methods in pregnant women at low risk for fetal aneuploidy. Based on new data from these and other studies, ACOG and the Society for Maternal-Fetal Medicine (SMFM) released a new consensus statement in June 2015 that addressed the use of cfDNA in the general obstetric population. The 2 groups still recommend conventional first- and second-trimester screening by serum chemical biomarkers and nuchal translucency as the first-line approach for low-risk women who want to pursue aneuploidy screening; however, they also recommend that the risks and benefits of cfDNA should be discussed with all patients.⁸

STUDY SUMMARIES

CARE was a prospective, blinded, multicenter (21 US sites across 14 states) study that compared the aneuploidy detection rates of cfDNA to those of standard screening. Standard aneuploidy screening included assays of first- or second-trimester serum biomarkers with or without fetal nuchal translucency measurement.

This study enrolled 2042 pregnant patients ages 18 to 49 (mean: 29.6 years) with singleton pregnancies. The population was racially and ethnically diverse (65% white, 22% black, 11% Hispanic, 7% Asian). This study included women with diabetes mellitus, thyroid disorders, and other comorbidities. cfDNA testing was done on 1909 maternal blood samples for trisomy 21 and 1905 for trisomy 18.

cfDNA and standard aneuploidy screening results were compared to pregnancy outcomes. The presence of aneuploidy was determined by physician-documented newborn physical exam (97%) or karyotype analysis (3%). In both live and non-live births, the incidence of trisomy 21 was 5 of 1909 cases (0.3%) and the incidence of trisomy 18 was 2 of 1905 cases (0.1%).

The NPV of cfDNA in this study was 100% (95% confidence interval, 99.8%-100%) for both trisomy 21 and trisomy 18. The positive predictive value (PPV) was higher with cfDNA compared to standard screening (45.5% vs 4.2% for trisomy 21 and 40% vs 8.3% for trisomy 18). This means that approximately 1 in 25 women with a positive standard aneuploidy screen actually has aneuploidy. In contrast, nearly one in 2 women with a positive cfDNA result has aneuploidy.

Similarly, false positive rates with cfDNA were significantly lower than those with standard screening. For trisomy 21, the cfDNA false positive rate was 0.3% compared to 3.6% for standard screening (P<.001); for trisomy 18, the cfDNA false positive rate was 0.2% compared to 0.6% for standard screening (P=.03).

NEXT was a prospective, blinded cohort study that compared cfDNA testing with standard first-trimester screening (with measurements of nuchal translucency and serum biochemical analysis) in a routine prenatal population at 35 centers in 6 countries.

This study enrolled 18,955 women ages 18 to 48 (mean: 31 years) who underwent traditional first-trimester screening and cfDNA testing. Eligible patients included pregnant women with a singleton pregnancy with a gestational age between 10 and 14.3 weeks. Prenatal screening results were compared to newborn outcomes using a documented newborn physical examination and, if performed, results of genetic testing. For women who had a miscarriage or stillbirth or chose to terminate the pregnancy, outcomes were determined by diagnostic genetic testing.

The primary outcome was the area under the receiver-operating-characteristic (ROC) curve for trisomy 21. Area under the ROC curve is a measure of a diagnostic test’s accuracy that plots sensitivity against 1-specificity; <.700 is considered a poor test, whereas 1.00 is a perfect test. A secondary analysis evaluated cfDNA testing in low-risk women (ages <35 years).

cfDNA can't detect neural tube or ventral wall defects, so women who choose this method should be offered maternal serum alpha-fetoprotein or ultrasound evaluation.

The area under the ROC curve was 0.999 for cfDNA compared with 0.958 for standard screening (P=.001). For diagnosis of trisomy 21, cfDNA had a higher PPV than standard testing (80.9% vs 3.4%; P<.001) and a lower false positive rate (0.06% vs 5.4%; P<.001). These findings were consistent in the secondary analysis of low-risk women.

Both the CARE and NEXT trials also evaluated cfDNA testing vs standard screening for diagnosis of trisomy 13 and 18 and found higher PPVs and lower false positive rates for cfDNA compared with traditional screening.

WHAT'S NEW

Previously, cfDNA was recommended only for women with high-risk pregnancies. The new data demonstrate that cfDNA has substantially better PPVs and lower false positive rates than standard fetal aneuploidy screening for the general obstetrical population.

So while conventional screening tests remain the most appropriate methods for aneuploidy detection in the general obstetrical population, according to ACOG and SMFM, the 2 groups now recommend that all screening options—including cfDNA—be discussed with every woman. Any woman may choose cfDNA but should be counseled about the risks and benefits.⁸

CAVEATS

Both the CARE and NEXT studies had limitations. They compared cfDNA testing with first- or second-trimester screening and did not evaluate integrated screening methods (sequential first- and second-trimester biomarkers plus first-trimester nuchal translucency), which have a slightly higher sensitivity and specificity than first-trimester screening alone.

Multiple companies offer cfDNA, and the test is not subject to Food and Drug Administration approval. The CARE and NEXT studies used tests from companies that provided funding for these studies and employ several of the study authors.

Although cfDNA has increased specificity compared to standard screening, there have been case reports of false negative results. Further testing has shown that such false negative results could be caused by mosaicism in either the fetus and/or placenta, vanishing twins, or maternal malignancies.^8-10

In the CARE and NEXT trials, cfDNA produced no results in 0.9% and 3% of women, respectively. Patients for whom cfDNA testing yields no results have higher rates of aneuploidy, and therefore require further diagnostic testing.

Many insurance companies do not yet cover cfDNA for women with low-risk pregnancies, and the test may cost up to $1,700.

Because the prevalence of aneuploidy is lower in the general obstetric population than it is among women whose pregnancies are at high risk for aneuploidy, the PPV of cfDNA testing is also lower in the general obstetric population. This means that there are more false positive results for women at lower risk for aneuploidy. Therefore, it is imperative that women with positive cfDNA tests receive follow-up diagnostic testing such as chorionic villus sampling or amniocentesis before making a decision about termination.

All commercially available cfDNA tests have high sensitivity and specificity for trisomy 21, 18, and 13. Some offer testing for sex chromosome abnormalities and microdeletions. However, current cfDNA testing methods are unable to detect up to 17% of other clinically significant chromosomal abnormalities,¹¹ and cfDNA cannot detect neural tube or ventral wall defects. Therefore, ACOG and SMFM recommend that women who choose cfDNA as their aneuploidy screening method should also be offered maternal serum alpha-fetoprotein or ultrasound evaluation.

CHALLENGES TO IMPLEMENTATION

cfDNA testing is validated only for singleton pregnancies. Physicians should obtain a baseline fetal ultrasound to confirm the number of fetuses, gestational age, and viability before ordering cfDNA to ensure it is the most appropriate screening test. This may add to the overall number of early pregnancy ultrasounds conducted.

Counseling patients about aneuploidy screening options is time-consuming, and requires discussion of the limitations of each screening method and caution that a negative cfDNA result does not guarantee an unaffected fetus, nor does a positive result guarantee an affected fetus. However, aneuploidy screening is well within the scope of care for family physicians who provide prenatal care, and referral to genetic specialists is not necessary or recommended.

Some patients may request cfDNA in order to facilitate earlier identification of fetal sex. In such cases, physicians should advise patients that cfDNA testing also assesses trisomy risk. Patients who do not wish to assess their risk for aneuploidy should not receive cfDNA testing.

Finally, while cfDNA is routinely recommended for women with pregnancies considered at high risk for aneuploidy, many insurance companies do not cover the cost of cfDNA for women with low-risk pregnancies, and the test may cost up to $1,700.¹² The overall cost-effectiveness of cfDNA for aneuploidy screening in low-risk women is unknown.

ACKNOWLEDGEMENT 
The PURLs Surveillance System was supported in part by Grant Number UL1RR024999 from the National Center For Research Resources, a Clinical Translational Science Award to the University of Chicago. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center For Research Resources or the National Institutes of Health.

Illustrative case

A 28-year-old gravida 2, para 1001 at 10 weeks gestation presents to your clinic for a routine first-trimester prenatal visit. Her first child has no known chromosomal abnormalities and she has no family history of aneuploidy. She asks you which tests are available to screen her fetus for chromosomal abnormalities.

Pregnant women have traditionally been offered some combination of serum biomarkers and nuchal translucency to assess the risk of fetal aneuploidy. Cell-free DNA testing (cfDNA) is a form of noninvasive prenatal testing that uses maternal serum samples to conduct massively parallel sequencing of cell-free fetal DNA fragments. It has been offered to pregnant women as a screening test to detect fetal chromosomal abnormalities since 2011 after multiple clinical studies found high sensitivities, specificities, and negative predictive values (NPVs) for detecting aneuploidy.^3-6 However until 2015, practice guidelines from the American Congress of Obstetricians and Gynecologists (ACOG) recommended that standard aneuploidy screening or diagnostic testing be offered to all pregnant women and cfDNA be reserved for women with pregnancies at high risk for aneuploidy (strength of recommendation: B).⁷

CARE (Comparison of Aneuploidy Risk Evaluation) and NEXT (Noninvasive Examination of Trisomy) are 2 large studies that compared cfDNA and standard aneuploidy screening methods in pregnant women at low risk for fetal aneuploidy. Based on new data from these and other studies, ACOG and the Society for Maternal-Fetal Medicine (SMFM) released a new consensus statement in June 2015 that addressed the use of cfDNA in the general obstetric population. The 2 groups still recommend conventional first- and second-trimester screening by serum chemical biomarkers and nuchal translucency as the first-line approach for low-risk women who want to pursue aneuploidy screening; however, they also recommend that the risks and benefits of cfDNA should be discussed with all patients.⁸

STUDY SUMMARIES

CARE was a prospective, blinded, multicenter (21 US sites across 14 states) study that compared the aneuploidy detection rates of cfDNA to those of standard screening. Standard aneuploidy screening included assays of first- or second-trimester serum biomarkers with or without fetal nuchal translucency measurement.

This study enrolled 2042 pregnant patients ages 18 to 49 (mean: 29.6 years) with singleton pregnancies. The population was racially and ethnically diverse (65% white, 22% black, 11% Hispanic, 7% Asian). This study included women with diabetes mellitus, thyroid disorders, and other comorbidities. cfDNA testing was done on 1909 maternal blood samples for trisomy 21 and 1905 for trisomy 18.

cfDNA and standard aneuploidy screening results were compared to pregnancy outcomes. The presence of aneuploidy was determined by physician-documented newborn physical exam (97%) or karyotype analysis (3%). In both live and non-live births, the incidence of trisomy 21 was 5 of 1909 cases (0.3%) and the incidence of trisomy 18 was 2 of 1905 cases (0.1%).

The NPV of cfDNA in this study was 100% (95% confidence interval, 99.8%-100%) for both trisomy 21 and trisomy 18. The positive predictive value (PPV) was higher with cfDNA compared to standard screening (45.5% vs 4.2% for trisomy 21 and 40% vs 8.3% for trisomy 18). This means that approximately 1 in 25 women with a positive standard aneuploidy screen actually has aneuploidy. In contrast, nearly one in 2 women with a positive cfDNA result has aneuploidy.

Similarly, false positive rates with cfDNA were significantly lower than those with standard screening. For trisomy 21, the cfDNA false positive rate was 0.3% compared to 3.6% for standard screening (P<.001); for trisomy 18, the cfDNA false positive rate was 0.2% compared to 0.6% for standard screening (P=.03).

NEXT was a prospective, blinded cohort study that compared cfDNA testing with standard first-trimester screening (with measurements of nuchal translucency and serum biochemical analysis) in a routine prenatal population at 35 centers in 6 countries.

This study enrolled 18,955 women ages 18 to 48 (mean: 31 years) who underwent traditional first-trimester screening and cfDNA testing. Eligible patients included pregnant women with a singleton pregnancy with a gestational age between 10 and 14.3 weeks. Prenatal screening results were compared to newborn outcomes using a documented newborn physical examination and, if performed, results of genetic testing. For women who had a miscarriage or stillbirth or chose to terminate the pregnancy, outcomes were determined by diagnostic genetic testing.

The primary outcome was the area under the receiver-operating-characteristic (ROC) curve for trisomy 21. Area under the ROC curve is a measure of a diagnostic test’s accuracy that plots sensitivity against 1-specificity; <.700 is considered a poor test, whereas 1.00 is a perfect test. A secondary analysis evaluated cfDNA testing in low-risk women (ages <35 years).

cfDNA can't detect neural tube or ventral wall defects, so women who choose this method should be offered maternal serum alpha-fetoprotein or ultrasound evaluation.

The area under the ROC curve was 0.999 for cfDNA compared with 0.958 for standard screening (P=.001). For diagnosis of trisomy 21, cfDNA had a higher PPV than standard testing (80.9% vs 3.4%; P<.001) and a lower false positive rate (0.06% vs 5.4%; P<.001). These findings were consistent in the secondary analysis of low-risk women.

Both the CARE and NEXT trials also evaluated cfDNA testing vs standard screening for diagnosis of trisomy 13 and 18 and found higher PPVs and lower false positive rates for cfDNA compared with traditional screening.

WHAT'S NEW

Previously, cfDNA was recommended only for women with high-risk pregnancies. The new data demonstrate that cfDNA has substantially better PPVs and lower false positive rates than standard fetal aneuploidy screening for the general obstetrical population.

So while conventional screening tests remain the most appropriate methods for aneuploidy detection in the general obstetrical population, according to ACOG and SMFM, the 2 groups now recommend that all screening options—including cfDNA—be discussed with every woman. Any woman may choose cfDNA but should be counseled about the risks and benefits.⁸

CAVEATS

Both the CARE and NEXT studies had limitations. They compared cfDNA testing with first- or second-trimester screening and did not evaluate integrated screening methods (sequential first- and second-trimester biomarkers plus first-trimester nuchal translucency), which have a slightly higher sensitivity and specificity than first-trimester screening alone.

Multiple companies offer cfDNA, and the test is not subject to Food and Drug Administration approval. The CARE and NEXT studies used tests from companies that provided funding for these studies and employ several of the study authors.

Although cfDNA has increased specificity compared to standard screening, there have been case reports of false negative results. Further testing has shown that such false negative results could be caused by mosaicism in either the fetus and/or placenta, vanishing twins, or maternal malignancies.^8-10

In the CARE and NEXT trials, cfDNA produced no results in 0.9% and 3% of women, respectively. Patients for whom cfDNA testing yields no results have higher rates of aneuploidy, and therefore require further diagnostic testing.

Many insurance companies do not yet cover cfDNA for women with low-risk pregnancies, and the test may cost up to $1,700.

Because the prevalence of aneuploidy is lower in the general obstetric population than it is among women whose pregnancies are at high risk for aneuploidy, the PPV of cfDNA testing is also lower in the general obstetric population. This means that there are more false positive results for women at lower risk for aneuploidy. Therefore, it is imperative that women with positive cfDNA tests receive follow-up diagnostic testing such as chorionic villus sampling or amniocentesis before making a decision about termination.

All commercially available cfDNA tests have high sensitivity and specificity for trisomy 21, 18, and 13. Some offer testing for sex chromosome abnormalities and microdeletions. However, current cfDNA testing methods are unable to detect up to 17% of other clinically significant chromosomal abnormalities,¹¹ and cfDNA cannot detect neural tube or ventral wall defects. Therefore, ACOG and SMFM recommend that women who choose cfDNA as their aneuploidy screening method should also be offered maternal serum alpha-fetoprotein or ultrasound evaluation.

CHALLENGES TO IMPLEMENTATION

cfDNA testing is validated only for singleton pregnancies. Physicians should obtain a baseline fetal ultrasound to confirm the number of fetuses, gestational age, and viability before ordering cfDNA to ensure it is the most appropriate screening test. This may add to the overall number of early pregnancy ultrasounds conducted.

Counseling patients about aneuploidy screening options is time-consuming, and requires discussion of the limitations of each screening method and caution that a negative cfDNA result does not guarantee an unaffected fetus, nor does a positive result guarantee an affected fetus. However, aneuploidy screening is well within the scope of care for family physicians who provide prenatal care, and referral to genetic specialists is not necessary or recommended.

Some patients may request cfDNA in order to facilitate earlier identification of fetal sex. In such cases, physicians should advise patients that cfDNA testing also assesses trisomy risk. Patients who do not wish to assess their risk for aneuploidy should not receive cfDNA testing.

Finally, while cfDNA is routinely recommended for women with pregnancies considered at high risk for aneuploidy, many insurance companies do not cover the cost of cfDNA for women with low-risk pregnancies, and the test may cost up to $1,700.¹² The overall cost-effectiveness of cfDNA for aneuploidy screening in low-risk women is unknown.

ACKNOWLEDGEMENT 
The PURLs Surveillance System was supported in part by Grant Number UL1RR024999 from the National Center For Research Resources, a Clinical Translational Science Award to the University of Chicago. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center For Research Resources or the National Institutes of Health.

Illustrative case

A 28-year-old gravida 2, para 1001 at 10 weeks gestation presents to your clinic for a routine first-trimester prenatal visit. Her first child has no known chromosomal abnormalities and she has no family history of aneuploidy. She asks you which tests are available to screen her fetus for chromosomal abnormalities.

Pregnant women have traditionally been offered some combination of serum biomarkers and nuchal translucency to assess the risk of fetal aneuploidy. Cell-free DNA testing (cfDNA) is a form of noninvasive prenatal testing that uses maternal serum samples to conduct massively parallel sequencing of cell-free fetal DNA fragments. It has been offered to pregnant women as a screening test to detect fetal chromosomal abnormalities since 2011 after multiple clinical studies found high sensitivities, specificities, and negative predictive values (NPVs) for detecting aneuploidy.^3-6 However until 2015, practice guidelines from the American Congress of Obstetricians and Gynecologists (ACOG) recommended that standard aneuploidy screening or diagnostic testing be offered to all pregnant women and cfDNA be reserved for women with pregnancies at high risk for aneuploidy (strength of recommendation: B).⁷

CARE (Comparison of Aneuploidy Risk Evaluation) and NEXT (Noninvasive Examination of Trisomy) are 2 large studies that compared cfDNA and standard aneuploidy screening methods in pregnant women at low risk for fetal aneuploidy. Based on new data from these and other studies, ACOG and the Society for Maternal-Fetal Medicine (SMFM) released a new consensus statement in June 2015 that addressed the use of cfDNA in the general obstetric population. The 2 groups still recommend conventional first- and second-trimester screening by serum chemical biomarkers and nuchal translucency as the first-line approach for low-risk women who want to pursue aneuploidy screening; however, they also recommend that the risks and benefits of cfDNA should be discussed with all patients.⁸

STUDY SUMMARIES

CARE was a prospective, blinded, multicenter (21 US sites across 14 states) study that compared the aneuploidy detection rates of cfDNA to those of standard screening. Standard aneuploidy screening included assays of first- or second-trimester serum biomarkers with or without fetal nuchal translucency measurement.

This study enrolled 2042 pregnant patients ages 18 to 49 (mean: 29.6 years) with singleton pregnancies. The population was racially and ethnically diverse (65% white, 22% black, 11% Hispanic, 7% Asian). This study included women with diabetes mellitus, thyroid disorders, and other comorbidities. cfDNA testing was done on 1909 maternal blood samples for trisomy 21 and 1905 for trisomy 18.

cfDNA and standard aneuploidy screening results were compared to pregnancy outcomes. The presence of aneuploidy was determined by physician-documented newborn physical exam (97%) or karyotype analysis (3%). In both live and non-live births, the incidence of trisomy 21 was 5 of 1909 cases (0.3%) and the incidence of trisomy 18 was 2 of 1905 cases (0.1%).

The NPV of cfDNA in this study was 100% (95% confidence interval, 99.8%-100%) for both trisomy 21 and trisomy 18. The positive predictive value (PPV) was higher with cfDNA compared to standard screening (45.5% vs 4.2% for trisomy 21 and 40% vs 8.3% for trisomy 18). This means that approximately 1 in 25 women with a positive standard aneuploidy screen actually has aneuploidy. In contrast, nearly one in 2 women with a positive cfDNA result has aneuploidy.

Similarly, false positive rates with cfDNA were significantly lower than those with standard screening. For trisomy 21, the cfDNA false positive rate was 0.3% compared to 3.6% for standard screening (P<.001); for trisomy 18, the cfDNA false positive rate was 0.2% compared to 0.6% for standard screening (P=.03).

NEXT was a prospective, blinded cohort study that compared cfDNA testing with standard first-trimester screening (with measurements of nuchal translucency and serum biochemical analysis) in a routine prenatal population at 35 centers in 6 countries.

This study enrolled 18,955 women ages 18 to 48 (mean: 31 years) who underwent traditional first-trimester screening and cfDNA testing. Eligible patients included pregnant women with a singleton pregnancy with a gestational age between 10 and 14.3 weeks. Prenatal screening results were compared to newborn outcomes using a documented newborn physical examination and, if performed, results of genetic testing. For women who had a miscarriage or stillbirth or chose to terminate the pregnancy, outcomes were determined by diagnostic genetic testing.

The primary outcome was the area under the receiver-operating-characteristic (ROC) curve for trisomy 21. Area under the ROC curve is a measure of a diagnostic test’s accuracy that plots sensitivity against 1-specificity; <.700 is considered a poor test, whereas 1.00 is a perfect test. A secondary analysis evaluated cfDNA testing in low-risk women (ages <35 years).

cfDNA can't detect neural tube or ventral wall defects, so women who choose this method should be offered maternal serum alpha-fetoprotein or ultrasound evaluation.

The area under the ROC curve was 0.999 for cfDNA compared with 0.958 for standard screening (P=.001). For diagnosis of trisomy 21, cfDNA had a higher PPV than standard testing (80.9% vs 3.4%; P<.001) and a lower false positive rate (0.06% vs 5.4%; P<.001). These findings were consistent in the secondary analysis of low-risk women.

Both the CARE and NEXT trials also evaluated cfDNA testing vs standard screening for diagnosis of trisomy 13 and 18 and found higher PPVs and lower false positive rates for cfDNA compared with traditional screening.

WHAT'S NEW

Previously, cfDNA was recommended only for women with high-risk pregnancies. The new data demonstrate that cfDNA has substantially better PPVs and lower false positive rates than standard fetal aneuploidy screening for the general obstetrical population.

So while conventional screening tests remain the most appropriate methods for aneuploidy detection in the general obstetrical population, according to ACOG and SMFM, the 2 groups now recommend that all screening options—including cfDNA—be discussed with every woman. Any woman may choose cfDNA but should be counseled about the risks and benefits.⁸

CAVEATS

Both the CARE and NEXT studies had limitations. They compared cfDNA testing with first- or second-trimester screening and did not evaluate integrated screening methods (sequential first- and second-trimester biomarkers plus first-trimester nuchal translucency), which have a slightly higher sensitivity and specificity than first-trimester screening alone.

Multiple companies offer cfDNA, and the test is not subject to Food and Drug Administration approval. The CARE and NEXT studies used tests from companies that provided funding for these studies and employ several of the study authors.

Although cfDNA has increased specificity compared to standard screening, there have been case reports of false negative results. Further testing has shown that such false negative results could be caused by mosaicism in either the fetus and/or placenta, vanishing twins, or maternal malignancies.^8-10

In the CARE and NEXT trials, cfDNA produced no results in 0.9% and 3% of women, respectively. Patients for whom cfDNA testing yields no results have higher rates of aneuploidy, and therefore require further diagnostic testing.

Many insurance companies do not yet cover cfDNA for women with low-risk pregnancies, and the test may cost up to $1,700.

Because the prevalence of aneuploidy is lower in the general obstetric population than it is among women whose pregnancies are at high risk for aneuploidy, the PPV of cfDNA testing is also lower in the general obstetric population. This means that there are more false positive results for women at lower risk for aneuploidy. Therefore, it is imperative that women with positive cfDNA tests receive follow-up diagnostic testing such as chorionic villus sampling or amniocentesis before making a decision about termination.

All commercially available cfDNA tests have high sensitivity and specificity for trisomy 21, 18, and 13. Some offer testing for sex chromosome abnormalities and microdeletions. However, current cfDNA testing methods are unable to detect up to 17% of other clinically significant chromosomal abnormalities,¹¹ and cfDNA cannot detect neural tube or ventral wall defects. Therefore, ACOG and SMFM recommend that women who choose cfDNA as their aneuploidy screening method should also be offered maternal serum alpha-fetoprotein or ultrasound evaluation.

CHALLENGES TO IMPLEMENTATION

cfDNA testing is validated only for singleton pregnancies. Physicians should obtain a baseline fetal ultrasound to confirm the number of fetuses, gestational age, and viability before ordering cfDNA to ensure it is the most appropriate screening test. This may add to the overall number of early pregnancy ultrasounds conducted.

Counseling patients about aneuploidy screening options is time-consuming, and requires discussion of the limitations of each screening method and caution that a negative cfDNA result does not guarantee an unaffected fetus, nor does a positive result guarantee an affected fetus. However, aneuploidy screening is well within the scope of care for family physicians who provide prenatal care, and referral to genetic specialists is not necessary or recommended.

Some patients may request cfDNA in order to facilitate earlier identification of fetal sex. In such cases, physicians should advise patients that cfDNA testing also assesses trisomy risk. Patients who do not wish to assess their risk for aneuploidy should not receive cfDNA testing.

Finally, while cfDNA is routinely recommended for women with pregnancies considered at high risk for aneuploidy, many insurance companies do not cover the cost of cfDNA for women with low-risk pregnancies, and the test may cost up to $1,700.¹² The overall cost-effectiveness of cfDNA for aneuploidy screening in low-risk women is unknown.

ACKNOWLEDGEMENT 
The PURLs Surveillance System was supported in part by Grant Number UL1RR024999 from the National Center For Research Resources, a Clinical Translational Science Award to the University of Chicago. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center For Research Resources or the National Institutes of Health.