Cell-Free DNA Screening Positive for Monosomy X: Clinical Evaluation and Management of Suspected Maternal or Fetal Turner Syndrome

Abstract

Initially provided as an alternate to evaluation of serum analytes and nuchal translucency for the evaluation of pregnancies at high-risk of Trisomy 21, cell-free DNA (cfDNA) screening for fetal aneuploidy, also referred to as non-invasive prenatal screening (NIPS), can now also screen for fetal sex chromosome anomalies (SCAs) such as monosomy X as early as 9 to 10 weeks of gestation. Early identification of Turner syndrome, a SCA resulting from the complete or partial absence of the second X chromosome, allows for medical interventions such as optimizing obstetrical outcomes, hormone replacement therapy, fertility protection and support as well improved neurocognitive outcomes. However, cfDNA screening for SCAs and monosomy X in particular is associated with high false positive rates and low positive predictive value. A cfDNA result positive for monosomy X may represent fetal TS, maternal TS, or confined placental mosaicism. A positive screen for monosomy X with discordant results of diagnostic fetal karyotype presents unique interpretation and management challenges due to potential implications for previously unrecognized maternal Turner syndrome (TS). The current international consensus clinical practice guidelines for the care of individuals with TS throughout the lifespan do not specifically address management of individuals with a cfDNA screen positive for monosomy X. The objective of this manuscript is to provide context and expert-driven recommendations for maternal and/or fetal evaluation and management when cfDNA screening is positive for monosomy X. We highlight unique challenges of cfDNA screening that is incidentally positive for monosomy X, present recommendations for determining if the result is a true positive and discuss when diagnosis of TS is applicable to the fetus or the mother. While we defer the subsequent management of confirmed TS to the clinical practice guidelines, we highlight unique considerations for these individuals initially identified through cfDNA screening.

Condensation:

Cell-free DNA (cfDNA) screening positive for monosomy X can represent fetal or maternal Turner syndrome. This article provides recommendations for the management of pregnancies complicated by cfDNA screening positive for monosomy X.

Introduction

Initially provided as an alternate to evaluation of serum analytes and nuchal translucency for the evaluation of pregnancies at high-risk of Trisomy 21, cell-free DNA (cfDNA) screening for fetal aneuploidy, also referred to as non-invasive prenatal screening (NIPS), can now also screen for fetal sex chromosome anomalies (SCAs) [1–6]. However, new challenges have emerged regarding false positive screening results for SCAs such as monosomy X. The peripheral blood sample taken for cfDNA screening contains both maternal and placental (trophoblast) cfDNA, the latter serving as a proxy for fetal DNA [7, 8]. While false positive results may be due to twin demise, other etiologies include confined placental mosaicism (CPM) with aneuploid placenta/euploid fetus, maternal malignancy, or maternal aneuploidy [9–12]. In comparison to a positive predictive value (PPV) of cfDNA screening for Trisomy 21 of >85% [13, 14], the PPV for monosomy X is consistently lower (pooled estimates of ~26%)[15–17]. PPV represents the likelihood that a fetus is affected by monosomy X if cfDNA screening is positive. Important explanations as to why cfDNA has a poorer performance in screening for monosomy X include lack of uniform algorithms and cutoffs for SCA screening results at different laboratories [18]and lack of uniform reporting of mosaic karyotypes, i.e. the inclusion of only nonmosaic karyotypes in calculating true positive and false positive results [19]. A lower population incidence of monosomy X [20, 21], unknown maternal mosaicism and structural abnormalities of the X chromosome [9, 15], and CPM [22] can also result in a lower PPV. Mosaicism refers to the presence of two or more cell lines of distinct genotypes in an individual derived from a single zygote [23]. Because most circulating cfDNA in the peripheral blood is maternal in origin [24], maternal mosaicism can affect the accuracy of cfDNA screening for fetal SCAs. Maternal X chromosome aneuploidy and mosaicism account for 9 to 24% of discordant NIPS results for SCAs [9, 15, 25]. Lack of available clinical data and incomplete cytogenetic testing following positive cfDNA screen for monosomy X may also result in a lower PPV of NIPS for SCA [16, 26]. Pregnant women may choose not to undergo confirmatory diagnosis prenatally or postnatally in the absence of phenotypic manifestations of SCAs [27, 28]. In addition, difficulty in obtaining placental tissue for cytogenetic analysis limits the ability to accurately account for CPM as a cause of discordant cfDNA results[16].

A positive screen for monosomy X with discordant results of diagnostic fetal karyotype presents unique interpretation and management challenges due to potential implications for previously unrecognized maternal Turner syndrome (TS). TS, an SCA resulting from the complete or partial absence of the second sex chromosome, occurs in 1 out of 2500 individuals assigned female sex at birth[29] and is characterized by a variable phenotypic spectrum including cardiac and renal abnormalities, short stature, primary ovarian insufficiency (POI), autoimmune conditions and neuropsychosocial challenges [30]. Although 40–50% of women with TS have a karyotype of monosomy X, the remainder have a large variation in karyotype including mosaic compositions [30–32]. In individuals under 50 years of age with at least one associated phenotypic finding, 5% has been used as the lower limit for monosomy X that defines TS [32–34]. Observational studies show that clinically recognizable TS phenotypes such as smaller height are associated with at least 8% mosaicism in adult women [35]. However, the threshold of mosaicism associated with adverse obstetrical outcomes remains unknown [36]. Women with TS and mosaic karyotype such as 45,X/46,XX have a higher rate of spontaneous menarche and pregnancy [33, 37, 38]. Although these individuals have been reported as having a “milder” phenotype with less severe congenital heart disease [32, 39], their risk of cardiovascular and endocrine complications is higher than the general population [40]. An increased rate of preeclampsia and an approximate 2% risk of pregnancy-associated death and aortic dissection has been reported in women with TS and pregnancy achieved with oocyte donation [41, 42]. However, the risk of pregnancy-induced hypertensive disease and aortic dissection in women with TS and spontaneous pregnancies is unclear. In a U.S. cohort of 276 adults with cytogenetically proven TS, there were no cases of pregnancy-induced hypertension or aortic dissection in the 5 women with spontaneous pregnancies [43]. Whether the prevalence of obstetrical complications and the optimal management of women with the secondary finding of maternal X mosaicism during cfDNA screening for fetal aneuploidy resembles that of women with mosaic TS and spontaneous pregnancy remains unknown.

Early identification of SCA such as TS allows for medical interventions such as hormone replacement therapy, fertility protection and support as well improved neurocognitive outcomes [44, 45]. The current international consensus clinical practice guidelines for the care of individuals with TS throughout the lifespan (TS Guidelines)[30] do not specifically address management of individuals with a cfDNA screen positive for monosomy X. The objective of this manuscript is to provide context and expert-driven recommendations for maternal and/or fetal workup and management when cfDNA screening results are positive for monosomy X. While we defer to the TS Guidelines whenever relevant[30], we highlight unique considerations for individuals incidentally identified to have TS through cfDNA screening.

Positive cfDNA screen for monosomy X: Now What?

The initial question when cfDNA screening is positive for monosomy X is whether this result is a true positive (fetus has TS) or a false positive (Figure 1). Ideally, both possibilities would have been disclosed through appropriate genetic counseling prior to cfDNA screening as recommended both by the American College of Obstetricians and Gynecologists (ACOG) as well as the American College of Medical Genetics and Genomics (ACMG)[7, 46]. Genetic counseling should also be provided after a positive cfDNA result and should include implications of a true and false positive result as well as the individualized estimated likelihood of these scenarios. Despite ACOG and ACMG recommendations, there is a lack of consistency in the clinical care of individuals with NIPS positive for monosomy X. In an anonymous cross-sectional survey of 176 genetic counselors, although the majority offered prenatal diagnostic testing (>88%) and anatomy ultrasound (~90%), the percent consistently offering maternal karyotype (22–52%) and postnatal evaluation varied (28–87%) [47]. The ACMG recommends that laboratories providing cfDNA screening report the detection rate, clinical specificity, PPV and negative predictive value (NPV) for each SCA to assist with clinical interpretation of the results. If these metrics are not provided on the laboratory report, clinicians may estimate the PPV utilizing an online calculator at www.perinatalquality.org/vendors/nsgc/nipt/¹.

Figure 1: ALGORITHM FOR EVALUATION OF CELL-FREE DNA TEST POSITIVE FOR MONOSOMY X (TURNER SYNDROME).

Abbreviations: CVS, chorionic villus sampling; FISH, Fluorescent In-situ hybridization. MFM, Maternal Fetal Medicine

* Consider if both fetal and maternal evaluation negative for monosomy X.

** Lack of standard guidelines.

As a positive NIPS for monosomy X may not correlate with the true fetal genotype, diagnostic testing should be advised whether or not pregnancy termination would be considered. Confirmatory diagnostic testing can be undertaken via chorionic villus sampling (CVS) or amniocentesis. Results from CVS should be interpreted with caution because CPM, which occurs in up to 2% of placental tissue analysis [48–50], is the most common cause of false positive NIPS results [51] CVS involves sampling of the 3 components of chorionic villi (syncytiotrophoblast, cytotrophoblast and mesenchymal core). Mosaic cytogenetic abnormalities have been reported in upwards of 87% of CPM [50]. Culturing cells from the mesenchymal core is a preferred method of avoiding CPM because mosaicism confined to the cytotrophoblast with normal mesenchymal core culture is almost never associated with true fetal mosaicism [52, 53]. Amniocentesis also avoids potential CPM and is required if CVS results suggest mosaicism [18]. The higher PPV from CVS (55%) than amniocentesis (17%) after cfDNA screen positive for monosomy X in a recent study was likely due to the clinical preference to proceed with CVS in the presence of fetal anomalies detected on early ultrasound[54] as well as the classification of placental mosaicism in the setting of a euploid fetus as determined by amniocentesis as a true positive cfDNA screen. The frequency of mosaicism for monosomy X on CVS, which can be performed earlier in pregnancy, is reportedly much higher (59%) than that of Trisomy 21 (2%) [55]and would result in a recommendation for confirmatory amniocentesis[55]. As such, in the absence of sonographic findings suggestive of TS (Figure 2), deferring confirmatory testing until amniocentesis can be performed at 15 weeks of gestation may be considered [56]. If cfDNA screening is confirmed as a true positive and the fetal karyotype is consistent with a diagnosis of TS, recommendations for prenatal and postnatal management are informed by the TS Guidelines [30]. If the fetal karyotype is normal, evaluation for other causes false positives such as maternal TS should be pursued.

Figure 2. Intrauterine sonographic findings of Turner syndrome^71,72.

In some situations, fetal diagnostic testing may be declined even if recommended. CVS and amniocentesis are associated with a low risk of procedure-related fetal loss of 1 in 455 and 1 in 900 respectively [57]. If fetal ultrasound findings are suggestive of TS (Figure 2) and pregnancy termination would not be considered, fetal TS can be presumed and definitive testing deferred until delivery. Alternatively, if the cfDNA report indicates monosomy X is likely maternal in origin (reported by some laboratories utilizing single nucleotide polymorphism methods)[58] or the maternal phenotype is suggestive of mosaic TS (e.g. short stature, cardiac or renal anomalies, history of infertility with pregnancy conceived using an egg donor), maternal genetic testing can be performed with the understanding that confirmation of maternal monosomy X does not completely rule out the possibility of fetal TS. In the future, newer noninvasive prenatal screening technologies, including cell-based approaches that isolate rare circulating fetal cells, may be able to differentiate fetal from maternal SCA [59].

Although both mosaic TS and variants have been reported following a positive cfDNA test, the sensitivity of cfDNA screening to identify mosaic or variant TS is unknown[60]. When diagnostic testing following a positive cfDNA screen for monosomy X is being pursued, testing should ideally identify both low level aneuploidy (including mosaicism) as well as structural chromosomal defects. Depending on laboratory protocols, monosomy X less than 6 to 15% may not be reported when assessing 20 to 50 metaphase cells to determine karyotype[61]. If clinical suspicion for TS remains high, FISH (fluorescence in situ hybridization) for sex chromosome probes on interphase cells can identify lower levels of mosaicism[32, 62].

Suspected or confirmed fetal Turner syndrome:

A large proportion of fetuses with monosomy X are known to undergo spontaneous abortions in the first and second trimester, with only 1% surviving to term[63]. Prior to cfDNA, fetal TS was identified on products of conception from spontaneous abortions or in pregnancies with abnormal ultrasound findings (Figure 2). However, the absence of features such as cystic hygroma does not rule out fetal TS, as nearly one-third of all fetuses with TS may not have ultrasound findings[64, 65].

Genetic counseling for suspected or confirmed fetal TS is ideally conducted by a multidisciplinary team familiar with the phenotypic spectrum and TS Guidelines [30]. There is considerable disagreement among different specialists (obstetricians, pediatricians, and geneticists) regarding termination decisions and termination rates vary considerably depending on counseling[66]. A multidisciplinary team approach to counseling consisting at minimum of a geneticist and a pediatric endocrinologist is essential to provide a balanced and accurate view of the diagnosis and prognostic expectations. Other specialists such as cardiologists may be helpful if cardiac anomalies are detected by ultrasound or fetal echocardiogram. Although the postnatal clinical course cannot be definitively predicted based on karyotype alone, the specific karyotype and sonographic findings can provide guidance for individualized prenatal counseling. The absence of any TS ultrasound findings has been associated with fewer postnatal phenotypic features and a higher proportion of mosaic karyotypes[67].

Early diagnosis of TS improves quality of life by allowing proactive monitoring and timely interventions for the associated comorbidities. However, additional research is needed to delineate the natural history of individuals identified through cfDNA screening with no obvious TS stigmata. Although we acknowledge the potential for over-medicalization and stigmatization that may accompany an incidental genetic diagnosis, currently there is sparse evidence to suggest more limited surveillance for these individuals[68, 69].

Prenatal evaluation and management for suspected or confirmed prenatal Turner syndrome

If fetal TS is suspected or confirmed, the patient should be referred to a maternal fetal medicine (MFM) specialist. Fetal ultrasound should be performed at ~11 to 14 weeks gestation with particular attention to cardiac and renal anatomy, as well as the presence of nuchal or generalized edema [70, 71]. Although some TS-associated congenital heart disease (CHD) such as isolated bicuspid aortic valve, partial anomalous pulmonary venous return or mild coarctation of aorta may not be detected by fetal echocardiography, more severe CHD such as critical coarctation of the aorta and hypoplastic left heart syndrome can be detected by fetal echocardiography. Prenatal detection of CHD can identify the potential risk of hemodynamic compromise and/or need for urgent postnatal cardiac interventions [72]. TS is also associated with small for gestational age (SGA)[73, 74] and ultrasound monitoring of fetal growth is recommended to optimize outcomes. Prenatal counseling and planning for perinatal management should have a multidisciplinary team approach, including but not limited to pediatric cardiology and neonatology.

Postnatal management for suspected or confirmed prenatal Turner syndrome

A detailed review of postnatal management of TS (Figure 3) is described in detail in the TS Guidelines [30] and is beyond the scope of this article. One key point is that a 30-cell karyotype should be completed on cord blood or postnatal peripheral blood even if prenatal CVS or amniocentesis was completed[30, 75]. FISH may also be considered to identify lower-level mosaicism that may not be detectable by routine karyotype and identifying Y chromosome material that could change clinical management such as consideration for prophylactic gonadectomy to decrease the risk of gonadal tumors[61, 62].

Figure 3: Postnatal evaluation in prenatally diagnosed Turner syndrome^49,53,73.

In addition, infants with TS have a higher risk of neonatal hypoglycemia compared with the general population[76], and feeding difficulties and failure to thrive in infancy are also common[77]. Proactive support of breastfeeding with lactation services and monitoring for inadequate feeding and/or weight gain is recommended. Infants should be referred to multidisciplinary TS specialty clinics, when available, to provide coordinated care. Finally, information on TS advocacy organizations and parent support groups can be valuable for families.

Suspected or Confirmed Maternal Turner Syndrome

Maternal X-chromosome aneuploidy is an important cause of a false positive cfDNA[9]. In a study of single-nucleotide polymorphism- based NIPS, maternal chromosomal microarray confirmed X chromosome abnormalities in 100/106 cases with a PPV of 94% [58]. While mosaic or nonmosaic 45,X was confirmed in 66% of cases with suspected maternal abnormality, in this study the diagnosis of maternal TS after NIPS positive for monosomy X was only 0.02% [58]. The clinical significance of maternal monosomy X found incidentally during cfDNA screening for fetal aneuploidy remains unknown. There are no standard guidelines for the medical management of these women as their risk of obstetrical complications such as aortic dissection and gynecologic complications such as primary ovarian insufficiency, which are increased in women with a known diagnosis of TS prior to pregnancy, is unclear. Maternal complications of TS include liver enzyme abnormalities, thyroid dysfunction, gestational diabetes, hypertension, pre-eclampsia, cardiovascular events such as aortic dissection, and death [42, 78, 79]. Fetal complications include ischemic placental disease, fetal growth restriction, intrauterine fetal demise, premature birth, and fetal congenital anomalies[42, 78, 80]. The TS Guidelines and the American Heart Association have published extensive recommendations for the management of pregnancy in individuals with TS[30, 72] (Figure 4). Implementation of these TS-specific recommendations led to mitigation of maternal cardiovascular risks and premature delivery[79]. Given the potential maternal implications in pregnancy, diagnostic testing for TS should be promptly performed if fetal TS is not identified or maternal TS is suspected. Of note, an abnormal maternal karyotype including mosaicism for TS does not rule out a fetal diagnosis of TS[52].

Figure 4: Recommended management of pregnancy in individuals with Turner syndrome^22,49.

Acknowledging the lack of a specific cutoff of X mosaicism that precludes pregnancy risk, we propose that women with >5% monosomy X mosaicism undergo a comprehensive TS evaluation including a maternal echocardiogram[30, 72]. If maternal TS features are present and testing is normal or inconclusive (<5% monosomy X detected), testing of a second tissue type such as a buccal swab should be considered[30].

Antepartum evaluation and management for suspected or confirmed maternal Turner syndrome

After diagnosis, the patient should be promptly referred to a MFM specialist and a cardiologist with expertise in congenital heart disease for appropriate evaluation and management[30, 72]. Evaluations should include a renal ultrasound and laboratory assessment of thyroid and liver function, and hemoglobin A1c or fasting glucose in addition to a routine evaluation for gestational diabetes[30, 81]. The recommended cardiac workup includes electrocardiogram, transthoracic echocardiogram (TTE), and cardiac magnetic resonance imaging (MRI) for determination of the aortic size index (ASI)[72] as well as to detect thoracic aortic aneurysms, coarctation, anomalous pulmonary veins, or shunts that frequently co-exist with left sided heart lesions but may be missed by echocardiography[82]. Of greatest concern is the risk of maternal death resulting from aortic dissection in pregnant women with TS, which has been reported to be as high as 2.2%[41, 42]. The aortic size index (ASI), calculated by dividing the ascending aortic diameter by body surface area, is the primary parameter used to assess the risk of aortic dissection (AoD) in individuals with TS[72]. In individuals with TS > 15 years of age, an ASI ≥ 2.5 cm/m², or an ASI 2.0 to 2.5 cm/m² with associated risk factors for AoD (systemic hypertension, a 45,X peripheral blood karyotype, bicuspid aortic valve, elongation of transverse aorta, coarctation of the aorta, previous AoD, or previous aortic surgery) is associated with an increased risk for peripartum AoD. Strict blood pressure control, serial imaging, and even prophylactic surgery, if the aortic diameter rapidly increases, are recommended to prevent maternal mortality from aortic dissection [30, 72, 81].

Up to half of women with TS develop significant depression or anxiety at some point in their lifetime[83, 84]. Proactively addressing this known risk by evaluating and providing resources for mental health care is ideal.

Postpartum management for suspected or confirmed maternal Turner syndrome

Following delivery, a coordinated, multidisciplinary approach to the care of individuals with TS in accordance with TS Guidelines remains essential[30]. A cardiac evaluation including an echocardiogram within 1 month following delivery for individuals with an ASI > 2.0 cm/m ² or any risk factors for AoD is recommended [72, 81]. As all women with TS, including those with spontaneous pregnancy, are at risk of POI, continuing gynecologic care to monitor and treat POI is recommended. Referral to additional specialists including endocrinology for screening and management of thyroid dysfunction, diabetes, and bone health, and audiology/otolaryngology to evaluate for hearing loss, should be followed as per the TS Guidelines[30, 85].

Conclusion:

cfDNA screening positive for monosomy X may represent CPM, fetal or maternal TS. Prospective study of the clinical phenotype and outcomes for individuals with monosomy X identified by cfDNA screening is needed to guide future management recommendations.