EFFICACY OF GENETIC TESTING IN CASES OF AMBIGUOUS GENITALIA DETECTED ON PRENATAL! ULTRASOUND ! ! ! by! EVELYN ROSE! CRAWFORD ! ! Submitted in partial fulfillment of !the requirements for the degree of Master of! Science ! ! ! Thesis Advisor: Larisa! Baumanis, MS ! ! Genetic Counseling! Department !of Genetics CASE WESTERN RESERVE! UNIVERSITY ! ! ! ! ! ! ! August, 2014 ! ! CASE WESTERN RESERVE! UNIVERSITY SCHOOL OF GRADUATE! STUDIES We hereby approve! the thesis of: Evelyn Rose! Crawford candidate for the degree of !Master of Science degree.* ! ! Larisa Baumanis, MS (Committee Chair) ! Anne Matthews, RN, PhD ! Noam Lazebnik, MD ! Aditi Parikh, MD ! Sara Debanne, PhD ! ! ! ! Date of Defense June 20, 2014 ! ! ! ! ! *We also certify that written approval has been obtained for any proprietary material

contained therein

2 ! ! ! TABLE OF CONTENTS List of Tables 4 List of Figures 5 Acknowledgements 6 Abstract 7 Introduction 8 Purpose of Study & Specific Aims 10 Background 11 Detection of Ambiguous Genitalia on Prenatal Ultrasound 11 Current use of Genetic testing in determining a specific diagnosis 13 The Importance of Prenatal Diagnosis in Cases of Ambiguous Genitalia 18 Significance for genetic counselors 19 Conclusions 20 Methodology 22 Systematic Review of the Literature 22 Chart Review 27 Algorithm & Analysis 29 Results 31 Analysis 52 Discussion 55 Appendix I: First Review Matrix Organization and summary of literature review articles 62 Appendix II: Second Review Matrix Organization and summary of case studies from the literature review 78 Appendix III: Third Review Matrix Organization and summary of chart review cases 94 References 102

3 LIST OF TABLES ! !Table 1: Keyword Combinations for Literature Search 22 !Table 2: Example First Review Matrix 25 !Table 3: Example Second Review Matrix 25 !Table 4: Protocol Key 26 !Table 5: Example Third Review Matrix 29 Table 6: Imaging characteristics to differentiate cloacal exstrophy, bladder !exstrophy and cloacal malformation (Calvo-Garcia et al. 2013) 40 !Table 7: All Literature Review Protocols 44 !Table 8: Confidence Intervals 46 !Table 9: All Chart Review Protocols 48 Table 10: Cases from chart review for example application of proposed algorithm 52

4 ! ! ! ! LIST OF FIGURES Figure 1: Flow chart of pre and postnatal diagnosis made !in each case reviewed by Cheikhelard et al. (2000) 12 Figure 2: Algorithm for the diagnostic work-up of a !fetus with ambiguous genitalia (Pajkt, Petersen & Chitty, 2008) 14 Figure 3: Algorithm for the diagnostic work-up of a !fetus with ambiguous genitalia (Chitty et al. 2012) 15 Figure 4: Flow chart summarizing review of literature !and identification of articles relevant to the current study 31 Figure 5: Flow chart summary of chart review and !identification of cases relevant to the current study 47 Figure 6: Algorithmic recommendations from synthesis of the literature 51

5 ! ! ! ACKNOWLEDGEMENTS! Larisa Baumanis, LGC Noam Lazebnik, MD Anne Matthews, PhD Aditi Parikh, MD Sara Debanne, PhD Elizabeth Ruzga, CNM, DNP Rebecca Darrah, PhD University Hospitals Medical Records Department ! !

6 ! ! Efficacy of Genetic Testing in Cases of Ambiguous Genitalia Detected on Prenatal Ultrasound! ! Abstract! ! By EVELYN ROSE! CRAWFORD This study assessed the accuracy of genetic testing in identifying a diagnosis prenatally for ambiguous genitalia through a systematic review of published case studies and cases of prenatally suspected ambiguous genitalia identified from University

Hospitals Case Medical Center (UHCMC) ultrasound database. A total of 28 articles

(2006-2014) met inclusion criteria. The UHCMC chart review yielded 39 cases from

2006 to 2014. Cases analyzed from the literature demonstrated that regardless of which protocol was used, a prenatal etiological diagnosis of ambiguous genitalia was correctly diagnosed in 60% of cases. From these data, an algorithmic testing guideline was generated. Cases from the chart review that had prenatal and postnatal records were used to assess the efficiency of the proposed algorithm of identifying the correct etiological diagnosis and was successful in 10 of 15 cases (66.6%), with 5 cases being unclear as to whether a diagnosis could be reached using the proposed algorithm. !

7 ! INTRODUCTION Genetic testing along with ultrasound imaging can aid clinicians in determining a specific diagnosis in cases of prenatally identified ambiguous genitalia. However, because the range of available genetic tests has grown rapidly, it is unclear how to apply current genetic testing strategies in these cases.

Literature on the topic of ambiguous genitalia diagnosed prenatally can be difficult to interpret when these cases are presented as single, isolated case studies and because the definition of ambiguous genitalia varies. In this study, ambiguous genitalia are defined as external genitalia that are undifferentiated or indistinct. The incidence of ambiguous genitalia is estimated to be 1 in 50,000 to 1 in 70,000 individuals (Abu-

Rustum & Chaaban 2009).

In the majority of pregnancies, ultrasound technology and maternal serum screening are the primary tools used to screen for fetal abnormalities. Despite the impressive detail of current ultrasound technology, the images produced may not provide enough information to reach an etiological diagnosis, defined as the nature or cause, of the ambiguous genitalia. Thus, the information from genetic testing, along with the images produced by ultrasound, offers important information that can aid in the diagnosis.

Pajkrt et al. (2009) and Chitty et al. (2012) have published algorithmic guidelines based on retrospective chart reviews for the effective application of genetic testing in cases of prenatal ambiguous genitalia. However, these algorithms may not be widely applicable within the United States because they are based on research conducted in the

8 Netherlands where the use of genetic tests such as cell free fetal DNA (cffDNA) have different European standards of use. Adam et al. (2012), conducted a retrospective chart review in the United States, resulting in recommendations including use of a multidisciplinary team, detailed ultrasound anatomy scan and karyotype for the management of all cases of prenatally identified ambiguous genitalia. Adam and colleagues (2012) offer valuable information about the use of genetic testing in the prenatal management of ambiguous genitalia, but their study is only representative of one hospital’s data and no statistical interpretation of testing strategies was included in their analysis. Rather than presenting an algorithm for testing, the authors presented a written analysis of the “pros and cons” of the testing approaches that they used, such as ultrasound, karyotype and targeted testing for single gene disorders. An algorithm for effectively utilizing genetic testing in cases of prenatally detected ambiguous genitalia that may be more applicable within the United States has yet to be presented.

This study draws on published case studies as well as unpublished cases of prenatally suspected ambiguous genitalia from the MacDonald Women’s Hospital,

University Hospitals Case Medical Center ultrasound database to present widely applicable recommendations for genetic testing as an algorithm that can be used in the management of prenatally identified ambiguous genitalia and evaluates the success of the algorithm using real-life cases. !

9 PURPOSE OF STUDY

The purpose of this descriptive study is to conduct a systematic review of the literature and chart review regarding the accuracy of genetic testing in identifying a specific diagnosis for prenatally suspected ambiguous genitalia and, based on this review, propose an algorithm that can be used in the management of prenatally suspected ambiguous genitalia.

SPECIFIC AIMS

1. Organize and centralize the current published data regarding ambiguous genitalia identified on ultrasound, testing performed in these cases, and outcome information through a systematic review of the literature.

2. Identify cases of suspected ambiguous genitalia from the prenatal ultrasound database at MacDonald Women’s Hospital, University Hospitals Case Medical Center (UHCMC) and review patient data, testing performed, and outcome information, if possible, from medical charts.

3. Compare and assess consistency in data across the published literature and UHCMC chart review data identified in Aims 1 and 2.

4. Propose an algorithm for testing based on analysis of data in aim 3.

5. Validate the ability of the algorithm to reach a diagnosis using cases identified in aim

2.

10 BACKGROUND !Detection of Ambiguous Genitalia on Prenatal Ultrasound While maternal serum screening along with ultrasound offers risk assessment for chromosome abnormalities, ultrasound is the sole screening technique for identifying physical defects for the majority of pregnancies. Ultrasound has been proven to be a reliable and accurate method for noninvasive prenatal gender assignment in pregnancies without abnormalities. However, ultrasound does not appear to be as reliable in detecting the presence of ambiguous genitalia.

Cheikhelard et al. (2000) conducted the only study with the specific purpose of assessing the accuracy and clinical implications of the prenatal diagnosis of abnormal genitalia. Of the 53 cases reviewed, 10 cases presented with abnormal genitalia at birth with no prenatal indication of an abnormality. The remaining 43 cases had a prenatal indication of abnormal genitalia; however 9/43 (20.9%) of these cases appeared normal at birth. Thirty-four cases had both prenatal detection and presented with ambiguous genitalia at birth and 10 had no prenatal diagnosis of ambiguous genitalia but had abnormal genitalia at birth, indicating a detection rate of 79.1% (Fig. 1). In reviewing their collected data, the researchers concluded that the accuracy of ultrasound in detecting ambiguous genitalia appeared to depend on the genetic sex, particularly in cases of pseudohermaphroditism (Cheikhelard et al. 2000). Male pseudohermaphroditism was defined as “posterior hypospadias with chordee and scrotal anomaly with or without cryptorchidism” and female pseudohermaphroditism was defined as “clitoromegaly with or without vulvar or vaginal anomalies” (Cheikhelard et al. 2000). The term genetic sex

11 refers to the biological sex as indicated by the sex chromosomes as opposed to phenotypic sex as indicated by an individual’s appearance.

While the study by Cheikhelard et al. has its limitations, 13 years after publication, it remains one of the only studies to look specifically at the detection rate of genital abnormalities by ultrasound. Although the study’s data may not be generalizable to other institutions, it demonstrates that the accuracy of detection in regard to genital abnormalities appears to be less than that of normal fetal sex determination.

Three-Dimensional (3D) ultrasound provides a more detailed image then traditional two-dimensional (2D) ultrasound. However, it remains unclear whether a more detailed image increases the rate of accurate prenatal diagnosis in cases of ambiguous genitalia (Abu-Rustum and Chaaban 2009; Verwoerd- Dikkeboom et al. 2008).

12 Ultimately, imaging alone can never be 100% accurate in cases of prenatally suspected genital abnormalities because the technology relies on interpretation.

Current Use of Prenatal Genetic Testing in Cases of Ambiguous Genitalia

Both 2D and 3D ultrasound provide imaging methodology for prenatal cases of ambiguous genitalia as well as detailed anatomical information potentially leading to a diagnosis. However, the information generated by imaging technology is limited and, in order to avoid misdiagnosis, should be interpreted in the larger context provided by genetic testing results or hormonal assays. There is fairly robust literature available regarding the utilization of current genetic testing strategies in the context of prenatally diagnosed ambiguous genitalia, and it is summarized here along with inconsistencies between recommended testing approaches within the literature.

In 2008, Pajkrt, Petersen & Chitty conducted a retrospective review of fetal medical records along with ultrasound images. Based on their review of 24 cases, an algorithm for genetic testing starting with detailed ultrasound exam followed by a diagnostic workup (Fig. 2) including cell free fetal DNA (cffDNA) for genotypic sex confirmation was proposed. !

13 ! Recently, the above algorithm was adapted for a 2012 review also conducted in the Netherlands (Fig. 3) (Chitty et al., 2012). The review featured the prenatal presentation and management of disorders of sexual development (DSD) and an overview of the sonographic appearance of normal genitalia.

14

The generalizability of these algorithms presented may be limited because they are derived from cases managed in only one hospital. Additionally, in the Netherlands, the United Kingdom and other European countries, non-invasive prenatal testing such as cffDNA is now standard of care for determining fetal sex in pregnancies at-risk for X- linked genetic disorders and congenital adrenal hyperplasia despite debate over use of cffDNA as a diagnostic tool (Chitty et al. 2012). In particular, cffDNA has been used to reduce the risk of miscarriage due to invasive testing while also allowing better management of steroid therapy in pregnancies at-risk for congenital adrenal hyperplasia by providing an early detection method for fetal sex (Chitty et al. 2012).

15 On the other hand, cffDNA use in the United States is typically offered only to those women who have been identified as having a higher risk for having a child with aneuploidy and has not been utilized as a reliable way to determine fetal genetic sex. In the United States, a joint committee from the American College of Obstetrics and

Gynecology and the Society for Maternal-Fetal Medicine published guidelines for the clinical use of non-invasive prenatal testing for fetal aneuploidy: cffDNA (American

College of Obstetricians and Gynecologists 2012). The committee guidelines state that because cffDNA has primarily been evaluated for women at high-risk for having a child with aneuploidy, cffDNA should not be used as part of routine prenatal laboratory assessment. In addition, while cffDNA sensitivity for determining fetal gender is very good, ranging between 96-99.9% (Nicolaides et al., 2013), standard of care still considers it to be a screening tool, thus, cffDNA is not recommended for the determination of genetic sex (an individual’s sex indicated by their chromosome composition). Moreover, the algorithms cannot be widely applied in the United States as cffDNA has only recently become clinically available and use of cffDNA in the clinical setting varies.

While Pajkrt et al. (2008) and Chitty et al. (2012) have published the sole algorithmic recommendations for genetic testing in cases of ambiguous genitalia, Adam and colleagues (Adam et al. 2012) undertook a medical chart review of prenatal diagnostic evaluation of presumed ambiguous genitalia and the most common postnatal outcomes. The researchers reviewed the records from Seattle Children’s Hospital from the last 17 years for cases where a prenatal diagnosis of a disorder of sexual development

(DSD), including cases of ambiguous genitalia, was suspected in order to assess what

16 prenatal diagnostic workup was undertaken and the postnatal outcome, including whether a postnatal genetic diagnosis was confirmed.

Based solely on the descriptive statistics obtained from their study, Adam and colleagues recommended that those prenatal tests that rule out conditions that lead to multiple anomalies and/or cognitive disability should take priority (Adam et al. 2012).

While the Adam et al. (2012) study does not present an algorithm, it does present valuable information towards the management of these cases in the United States that can be used to suggest an applicable testing protocol.

The recommendations provided by Adam et al. share many similarities with the algorithms proposed by both Pajkrt et al. (2008) and Chitty et al. (2012). All three studies recommend starting an investigation of the fetus with a detailed anatomical scan.

However, the authors vary on when to determine fetal genetic sex and what test to use.

The first algorithm (Pajkrt et al. 2008) proposes the use of cffDNA for those cases where ambiguous genitalia appear isolated and growth appears normal and for those cases where ambiguous genitalia occurs with renal anomalies, whereas the second algorithm proposes the use of cffDNA in all cases of ambiguous genitalia (Chitty et al. 2012).

Meanwhile, from the review presented by Adam et al. (2012), these authors recommend a full karyotype in all cases where ambiguous genitalia is present as a way to detect fetal sex and rule out chromosomal abnormalities. This current study aims to propose an algorithm based on the cases presented in all three papers as well as other published cases published to make recommendations for testing that may be more generalizable in clinical practice.

17 The Importance of Prenatal Diagnosis in Cases of Ambiguous Genitalia

Accurately determining fetal sex is not only of utmost importance to new parents, but many syndromes associated with ambiguous genitalia present significant health risks to the newborn. For example, one of the more common causes of ambiguous genitalia is congenital adrenal hyperplasia (CAH) in which individuals may have a deficiency of aldosterone, which can lead to salt-wasting, a potentially life threatening crisis (Murphy et al. 2011); thus determining an diagnosis promptly is very important in developing a plan for long term management. If aldosterone deficiency is mild and not detected in infancy, the individual may present later with premature pubarche or hyperandrogenism in females. In addition, individuals with CAH with over virilization can experience glucocorticoid deficiency and excessive androgen secretion, both of which have an effect on growth and development (Deaton, M., Glorioso, J. E. & McLead, D., 1999).

The identification of ambiguous genitalia is also important to make, as it can be part of a larger syndrome, which may have other associated symptoms and anomalies. If it is possible to identify a syndrome during pregnancy, management options may change and postnatal evaluation and treatment can potentially improve outcome. For instance, individuals with Smith-Lemli-Opitz syndrome may present with ambiguous genitalia, but may also have less obvious findings such as heart and lung malformations. Although most cases of syndromic ambiguous genitalia present with other anomalies that help to create a “larger picture,” diagnosis of a syndromic etiology when ambiguous genitalia appears isolated can guide evaluation and treatment and influence the options available to parents regarding continuation and management of the pregnancy.

18 In addition to the medical aspects of an identified diagnosis, ambiguous genitalia in the newborn presents an extremely difficult situation for parents, as the sex of the baby is usually the first question asked after birth. Even with sex assignment, corrective surgery and hormone replacement treatment, there is no guarantee that individuals raised as a specific gender will later identify as such. This was demonstrated by researchers who studied the impact of the change regarding sex assignment for infants with 5α-reductase 2 deficiency and 17β hydroxysteroid dehydrogenase-3 deficiency (Cohen-Kettenis, 2005).

The study reported that many of these individuals who had been raised as females based on presentation of external genitalia, identified as males later after puberty (Cohen-

Kettenis, 2005). Identifying a diagnosis before birth has the potential to reduce this period of uncertainty for parents and can allow time for preparation for the medical and social needs of their newborn.

In summary, prenatally noted ambiguous genitalia may have a variety of causes, some of which may have severe clinical presentations. When ambiguous genitalia are noted on prenatal ultrasound, the identification of a specific diagnosis can have a dramatic impact on newborn evaluation and treatment. Additionally, accurate prenatal diagnosis allows time for parents to prepare for the otherwise unforeseen medical needs of a child born with ambiguous genitalia and make informed decisions regarding medical management (Pajkrt et al., 2008). !Significance for genetic counseling In the presence of ambiguous genitalia, both accuracy of detection and identifying a specific diagnosis or etiology complicate communication between healthcare providers

19 and parents in regard to providing clear and accurate information. With recent developments in genetic testing, genetic counselors play an essential role as part of the multidisciplinary team necessary to manage a case where ambiguous genitalia are present. Genetic counselors, in consultation with physicians, often discuss recommendations about specific genetic testing including educating parents about the tests, risks involved, and possible outcomes of such testing. As trained counselors, genetic counselors are in a unique position to aid physician communication with parents in order to help facilitate decision-making regarding medical management.

It is anticipated that the results of this study will aid genetic counselors’ testing recommendations by making the current data regarding cases of prenatally detected ambiguous genitalia easily interpretable, and by suggesting an algorithmic approach as to how best to utilize testing options. The review of the literature will provide a synthesis of previous testing approaches and the outcome of these approaches. Genetic counselors will be able to more efficiently find relatable individual cases and an appropriate testing protocol to guide recommendations for individuals cases based on which genetic tests have been informative in the past in identifying an etiological diagnosis. Ultimately, being able to identify similar individual cases as well as how genetic testing has been applied in a larger context will help guide genetic counselors in their approach in cases of ambiguous genitalia. !Conclusions Ultrasound is a reliable non-invasive method of determining fetal gender in typical cases without genital anomalies; however, ultrasound is less reliable at detecting

20 ambiguous genitalia. In cases where ambiguous genitalia have been confirmed, 3D ultrasound might offer a more detailed image of the developing fetus. Because 2D/3D imaging has its limitations, it may not provide enough data for clinicians to reach an accurate diagnosis. Recent developments in genetic testing now offer additional information that gives context to ultrasound imagery. New data on the use of genetic tests in cases of ambiguous genitalia are now available, including two proposed algorithms for how best to utilize new testing options. The algorithms proposed only represent data from one institution in the Netherlands in which they were originally developed, and therefore may not be generalizable to other countries, such as the United States, where tests such as cffDNA have only recently become available and clinical use varies. In the last year, a review of management of cases of prenatally detected ambiguous genitalia was published

(Adam et al. 2012). Recommendations regarding the use of genetic testing were made, but statistical analysis of their collected data were not employed, nor was there a proposed algorithm. This study aims to use the information published in the above articles in combination with other published case studies as well as cases from

MacDonald Hospital University Hospitals Case Medical Center (UHCMC) in order to review current recommendations for the use of genetic testing in cases of prenatally diagnosed ambiguous genitalia and propose an algorithm for how to effectively use this testing approach. !

21 METHODOLOGY! To address the proposed study aims, a systematic review of the literature was conducted along with an additional chart review. The chart review included cases with prenatally suspected ambiguous genitalia identified in the MacDonald Women’s Hospital,

University Hospitals Case Medical Center’s (UHCMC) prenatal ultrasound database. !Systematic Review of the Literature The systematic review of the literature addressed Aim 1 by organizing and centralizing the current published literature to make the data more easily accessible. The literature review focused on identifying cases of prenatally detected ambiguous genitalia and the use of ultrasound and genetic testing to prenatally identify the etiology of the genital ambiguity. Literature published between 2000 to 2014 was reviewed as the quality of ultrasound imaging has improved significantly and the availability and range of genetic testing has steadily grown in the past decade. The review included studies that met the following criteria:

1. Identified ambiguous genitalia in the differential diagnosis 2. Mentioned ambiguous genitalia or disorders of sexual development 3. Were published in a peer-reviewed journal ! 4. Were published in the English language Databases PubMed and Web of Science were searched using the following key word combinations:

Table 1: Keyword Combinations for Literature Search

Term PubMed Term Web of Science

Ambiguous Genitalia 567 Ambiguous Genitalia 942

22 Table 1: Keyword Combinations for Literature Search

Term PubMed Term Web of Science

Disorders of Sexual Disorders of Sexual 8666 132 Development (MeSH) Development

("Disorders of Sex Disorders of Sexual Development"[Mesh]) 107 Development AND 4 AND "Ultrasonography, ultrasonography* Prenatal"[Mesh]

("Ultrasonography"[Me sh]) AND "Disorders of Disorders of Sexual 200 3 Sex Development AND Prenatal Development"[Mesh]

Total 9540 Total 1081 ! ! ! References identified during the search were imported into EndNote, a software tool designed for managing bibliographies, and duplicate references were eliminated.

Each reference was grouped into one of three categories based on likely relevance of title:

1. Title appeared relevant to research topic 2. Unknown relevance of title ! 3. Title did not appear relevant to research topic Once sorted into the above categories, articles with titles that appeared relevant to the research topic and articles with titles of unknown relevance were evaluated by abstract. If the abstract was unavailable, the articles were evaluated by full text. If the abstract did not specifically indicate the gestational age at which a patient presented or, in the case of neonates, any prenatal concerns, full text was used to clarify if the case were

23 one in which a suspicion of prenatal ambiguous genitalia was indicated. The articles were then sorted into the following categories:

1. Appropriate ! 2. Inappropriate Articles placed in the “Appropriate” category describe cases of prenatally identified ambiguous genitalia. Articles placed in the “Inappropriate” category did not describe cases of prenatally identified ambiguous genitalia or did not meet the inclusion criteria. In addition, literature focused on cases of prenatally detected ambiguous genitalia as part of a familial study, twin gestations or the effects of prenatal administration of dexamethasone for suspected or confirmed cases of Congenital Adrenal

Hyperplasia were placed in the “Inappropriate” category. This categorization attempted to avoid biased detection based on previous family history, limited ability to compare testing protocols in twin gestations with singleton gestations and the influence of treatment on the course of detection and testing.

The review of the literature utilized an adaptation of the Matrix Method (Garrard

2011). The Matrix Method involves the creation of a matrix to organize and interpret the data. The first review matrix (Appendix I) organized data from the literature into rows containing the identifying information about the document and columns containing different topics used to break down and summarize the data. ! ! ! ! !

24 Table 2: Example of First Review Matrix: Summary of 28 articles identified from the !review of the literature.

! To analyze the use of prenatal genetic testing in cases of ambiguous genitalia in the literature, a second review matrix (Appendix II) consolidated data regarding aspects of individual case studies. Each row of the second review matrix contained a single case from the published literature. Columns contained the following information about each case: study from which data were collected, gestational age at the time of ambiguous genitalia noted on ultrasound, all findings on prenatal ultrasound, prenatal testing, prenatal etiological diagnosis, and postnatal etiological diagnosis.

Table 3: Example of Second Review Matrix: Summary of 89 case studies from the 28 articles identified in the literature review

! The matrix also contained a column for an assigned testing protocol code. Each test was assigned a letter or number, i.e. Test A = karyotype, Test B = maternal serum hormone assay, etc (Table 4). Each protocol was presented as an order of letters and numbers, indicating which tests were performed and in what order. For those cases in which the order of testing was not specified, an order assignment was estimated based on other cases from the specific study.

25 Table 4: Protocol Code Key Code Letter Test

A 2D Ultrasound

B 3D Ultrasound

C MRI

D 4D Ultrasound

E Fetoscopy

F Amniocentesis

G CVS

H Karyotype

I FISH for SRY

J 17-Hydroxyprogesterone in amniotic fluid

K 7-Dehydrocholesterol in Amniotic fluid

L SRY Sequencing

M Androgen Receptor Sequencing

N Doppler Studies

O Cord Blood Sampling

P CGH Microarray

Q FGFR3 Sequencing

R First Trimester Screen

S Triple Test/Quad Screen

T Maternal Serum Estriol

U Maternal Serum AFP

V Thoracocentesis

W Echocardiogram

X FISH for TUPLE

Y iSpace imaging system

Z 4-Androstenedione in amniotic fluid

1 Viral studies on Amniotic Fluid

2 AFP in amniotic fluid

3 FISH for STS

26 ! Chart Review The chart review addressed both Aims 2 and 3 by providing information from an additional institution and comparing the data obtained from the UHCMC chart review with the studies identified in Aim 1, thus adding unpublished data to the current body of literature. The student researcher worked with Dr. Noam Lazebnik, an obstetrician and faculty member, in the Department of OB/GYN at UHCMC’s MacDonald Women’s

Hospital, to identify appropriate cases of prenatally suspected ambiguous genitalia in the database and obtain prenatal and postnatal medical charts.

Cases of suspected ambiguous genitalia were identified in the UHCMC

MacDonald Women’s Center prenatal ultrasound database. The current UHCMC

MacDonald Women’s Center ultrasound database is currently maintained using

Viewpoint software. The Viewpoint database was established in 2006 and therefore the cases identified for this project are those entered into the database starting January 2006 to present (March 2014). Originally the chart review intended to include data from all charts identified between January 2000 and March 2014; however, due to limited access to the archived records, data from charts between January 2006 to March 2014 were extracted. The chart review took place between March 2014 and May 2014. This study was approved by University Hopitals Case Medical Center Institutional Review Board

Internal Review Board.

In this study, ambiguous genitalia are defined as external genitalia that are undifferentiated or indistinct. “Hypospadias” and “clitoral hypertrophy” are often used when describing genital anomalies on prenatal ultrasound, as indicated by Verwoerd-

27 Dikkeboom et al. (2008) who used all three terms in their exploration of 2D, 3D and holographic imaging technology on cases of prenatal ambiguous genitalia. Thus, when searching the UHCMC prenatal ultrasound database, all three terms were used.

All ultrasound reports were reviewed in each identified case in order to confirm suspicious findings. Cases of twin pregnancies were excluded from the study due to limited comparison between interpretations of prenatal test results with singleton pregnancies. Charts for each of these cases were compiled for review. Electronic medical records were also reviewed in each case as many of the electronic records for those cases with paper charts contained additional information relevant to the study, such as the delivery record. For those cases in which delivery at a University Hospitals facility was confirmed, the baby’s chart were compiled by linking the mother’s information to the baby’s date of birth in the delivery record.

Using the same format as the second review matrix, the data extracted from the charts of each case was imported into a third matrix (Appendix III). Each row contained data from a single chart and the columns contained the following information about each case: year of birth, initial method of identification, gestational age, findings on prenatal ultrasound, prenatal testing, prenatal diagnosis, postnatal testing and postnatal diagnosis.

Each case was assigned a number at random in order to maintain confidentiality of personal medical information. Using the same protocol coding for literature cases, each case from the chart review was assigned a series of letters and numbers representing the order in which prenatal tests were performed.

28 Table 5: Example of Third Review Matrix: Summary of cases identified from chart review

! !Algorithm Following a descriptive analysis of the review of the literature in Aim 1 and the protocol data generated from Aims 1 and 2, an algorithm summarizing recommendations on how to use genetic testing in cases of prenatal ambiguous genitalia was generated (see results). !Analysis In order to better understand current clinical testing in cases of ambiguous genitalia, the protocol codes assigned to each case study from the literature were used to calculate the number of total protocols used, the most commonly used protocols, and the success rate of each protocol. The success rate of a protocol was defined as a percentage, that is, how often the protocol was able to correctly confirm a final diagnosis. Analysis of the protocol data involved the calculation of confidence intervals for the most commonly used protocols as well as for the success rate of all cases regardless of the protocol.

The same method of protocol assessment was applied to the cases generated from the chart review. The chart review data also allowed for the calculation of a detection rate for ultrasound for comparison to the data presented in the background. The detection rate was defined as the rate at which a genital anomaly was confirmed on postnatal exam after prenatal suspicion of ambiguous genitalia.

29 Using identified cases from the chart review that provided both prenatal, delivery, and postnatal exam records, the study’s proposed algorithm was analyzed for a potential success rate. The success rate was defined as the rate at which the proposed algorithm would have determined the documented final diagnosis based on the ultrasound findings in each case. Final diagnosis may indicate an underlying genetic diagnosis, such as congenital adrenal hyperplasia, or may indicate an isolated genital anomaly. For each case from the chart review, the algorithm testing recommendations were used to determine if, based on the reported ultrasound findings, the final diagnosis provided in the chart would have been correctly identified. ! ! ! ! ! !

30 ! RESULTS !Review of the Literature The search of the PubMed and Web of Science databases yielded a total 10,621 articles meeting the criteria for review, with duplicates removed by the EndNote software, the articles to be reviewed totaled 9,382. The following flow chart demonstrates the number of articles sorted into each category following the early outlined methodology.

Of the 32 articles identified as appropriate for the study, 28 presented at least one case of prenatally detected ambiguous genitalia. These 28 articles were used to compile the First Review Matrix for the literature synthesis (see Appendix I).

The literature analysis presents an overview of testing recommendations in cases of prenatally identified ambiguous genitalia. The term “management” refers to the testing

31 recommendations for each case. In total, the articles reviewed were categorized into 5 major foci: general prenatal management of ambiguous genitalia (6), followed by prenatal management of ambiguous genitalia in association with: cloacal anomalies (9) chromosome abnormalities (7), micro deletions and syndromes (4), and anomalies of the urinary tract, including hypospadias (2). In order to simplify testing recommendations, the cases presented in these articles were discussed as cases of isolated ambiguous genitalia or cases of ambiguous genitalia with additional anomalies. Articles discussing the general prenatal management of prenatally suspected ambiguous genitalia all refer to isolated cases. !Prenatal management of isolated ambiguous genitalia !1. Imaging Studies General management of ambiguous genitalia begins with the identification of the anomaly by ultrasound. There are multiple tools for imaging: 2D ultrasound, 3D ultrasound, Magnetic Resonance Imaging (MRI) and less common 4D, holographic imaging systems. Despite inconsistencies documented between different imagery methods, more advanced imaging systems offer better depth perception than traditional

2D ultrasound, which can be useful for distinguishing micropenis or clitoromegally from labia minora because depth helps estimate the size and exact position of external structures and gives insight that protruding structures can sometimes be normal labia

(Verwoerd- Dikkeboom et al. 2008).

As noted in the background to this study, interpretation of advanced imagery should be within the context of additional test results (Abu-rustumm & Chaaban 2009).

32 This notion was supported by additional texts identified within the literature review. In summary, while investigation of ambiguous genitalia should always begin with a detailed ultrasound examination of fetal anatomy, the interpretation of such images can be more informative in the context of additional information (Abu-Rustuum et al. 2009, Adam et al. 2012; Pakjrt et al. 2008; Pinhas-Hamiel et al. 2002; Katorza, E., Pinhas-Hamiel, O.,

Mazkereth, R., Gilboa, Y. & Achiron, R. 2009).

In addition to interpreting single images, guidelines for the evaluation of ambiguous genitalia on prenatal ultrasound should include longitudinal ultrasound examinations with a focus on external genital appearance to assess changes over the course of the pregnancy (Katorza et al. 2009). Because of the potential changes in the size and structure of reproductive organs change throughout pregnancy, an ultrasound performed at 13-14 weeks is not a good indicator of subsequent abnormalities (Pinhas-

Hamiel et al. 2002). !2. Karyotype, cffDNA and FISH There is a consensus on the use of ultrasound in the assessment of ambiguous genitalia; however, the use of genetic testing to identify chromosome abnormalities is more controversial. The authors’ of the reviewed literature disagreed as to when it is appropriate to use cffDNA or karyotype to determine fetal gender. This disagreement appears to stem from various recommendations as to how to implement cffDNA between institutions and countries and as to whether cffDNA can be considered a diagnostic test or a screening tool. In the United States, American College of Obstetricians and

Gynecologists (ACOG) guidelines, published in 2012, designate cffDNA as a screening

33 tool and it is recommended only for women with an increased risk of having children with chromosome abnormalities. If the testing returns positive for one of the specific conditions (ie trisomy 21, trisomy 18, trisomy 13, Turner syndrome, Triple X syndrome or Klinefelter syndrome) tested for, amniocentesis and karyotype is recommended to confirm chromosome abnormality; as positive predictive value is not 100%. While cffDNA can report genetic sex with between 96 and greater than 99.9% accuracy, the ability of the test to report gender is also not 100% and cffDNA only provides information limited to the likelihood of select chromosome abnormalities. cffDNA does not provide information on all chromosome abnormalities.

Based on their interpretation of the protocols recorded in each case, a study conducted in the United States concluded that karyotype and fluorescence in-situ hybridization (FISH) for the presence of SRY, the sex determining region on the Y chromosome, were the most informative tests performed prenatally in cases where ambiguous genitalia or sexual discordance appeared isolated (Adam et al. 2012). Katorza et al. (2009) supported Adam et al.’s conclusions and asserted that guidelines for the evaluation of ambiguous genitalia noted on prenatal ultrasound should include chromosome analysis.

In contrast, Pajkrt et al. (2008) conducted a study in the Netherlands from which the use of cffDNA was recommended for fetal sex and the use of fetal karyotype in the context of specific findings on ultrasound. Pajkrt and colleagues concluded that if ambiguous genitalia appears isolated on ultrasound and fetal growth is within normal limits, cffDNA is sufficient for determination of fetal sex (Pajkrt et al. 2008); however, in

34 cases where ambiguous genitalia appears isolated in the context of intra-uterine growth restriction (IUGR), fetal karyotype was warranted (Pajkrt et al. 2008). The researchers also recommended a karyotype analysis be done when ambiguous genitalia is noted in the presence of multiple anomalies. However, if the associated anomalies are renal, the researchers recommended the use of cffDNA for determination of fetal sex and emphasize the need to rule out bladder exstrophy or a cloacal anomaly (Pajkrt et al.

2008).

While the researchers appear to agree that karyotype analysis should always be considered in cases of multiple anomalies because they may present with a wide spectrum of anomalies, when looking more broadly at the cases described in the literature, chromosome abnormalities cannot be ruled out in cases of seemingly isolated genital anomalies either. For instance, three cases of distinct chromosome abnormalities,

45, X/69, XY mosaicism (Quigley et al. 2005), 46, XX/46, XY mosaicism (Chen et al.

2005) and 9p- (Vialard et al. 2002), all presented with suspicion for isolated ambiguous genitalia on prenatal ultrasound.

The decision to do an invasive test for a karyotype ultimately lies with the parents and will influence testing protocols. However, in cases of suspected ambiguous genitalia, it is clear that even in cases without additional anomalies, the possibility of an underlying chromosome abnormality that may not be detected by cffDNA exists, and that in the interest of determining a diagnosis in a time sensitive manner, chromosome analysis by karyotype should be offered.

3. Hormone Analysis and congenital adrenal hyperplasia (CAH)

35 ! Similar to the recommendations for karyotype, the various researchers did not converge on a direct recommendation regarding the use of hormone studies in cases of prenatal ambiguous genitalia. Katorza et al. (2009) recommended the use of hormonal assays on amniotic fluid and maternal urine in all cases of prenatal ambiguous genitalia; however, Pajkrt et al. (2008) recommended the use of hormone assays only in specific situations, such as in cases of isolated ambiguous genitalia in a fetus with cffDNA confirmed female sex, but not in a fetus with isolated ambiguous genitalia which has been identified on cffDNA to be male.

A normal karyotype does not rule out the possibility of a chromosomal microdeletion, microduplication or other syndrome. The identification of a syndrome can provide a more accurate prognosis and recurrence risk. Thus, if the differential diagnosis includes a particular genetic syndrome, testing to rule out that specific syndrome, such as a hormonal analysis, should be considered.

For instance, although it can present with additional findings, CAH should be considered in cases of isolated ambiguous genitalia in a confirmed fetus with confirmed

46, XX karyotype (Saada et al. 2004). Although CAH can be present without these features, there are three distinct characteristics that can indicate the presence of CAH prenatally: maternal virilization, bone malformations, such as bowed femurs or craniosynostosis, on prenatal ultrasound and distinct pattern of steroid metabolites

(Reisch et al. 2013).

36 Maternal virilization, a classic sign of CAH during pregnancy, does not consistently present and appears to be independent of a particular genotype or sex of the fetus. Overall approximately 7 of 20 (35%) mothers with affected pregnancies showed signs of virilization during their affected pregnancy (Reisch et al. 2013). Similarly, skeletal abnormalities may or may not be seen prenatally in fetuses affected with CAH and findings on ultrasound can range from normal appearance to multiple visible anomalies.

A consistent way to diagnose CAH prenatally is through the use of hormonal studies. If amniotic fluid is unavailable for hormone assay, a maternal urine steroid profile can be monitored after 12 weeks of gestation for a potential steroid metabolite pattern indicative of CAH. Low levels of estriol in both maternal serum and urine can indicate the presence of CAH; however, low estriol is a non-specific marker. EpialloPD, another steroid metabolite in maternal urine has also been used to indicate CAH because it replaces estriol as the major terminal product of fetal adrenal steroid synthesis in CAH.

Together, the ratio of estriol to EpialloPD in maternal urine can be used to document

CAH prenatally (Reisch et al. 2013).

In addition to hormonal studies, particular attention should be paid to the fetal adrenal glands in suspected cases of CAH. The fetal adrenal glands become visible in the early second trimester and are often abnormally shaped. In particular, the fetal adrenal glands appear triangular in shape, are retroperitoneal paraspinous in location and appear to have caps on the upper renal poles. In addition, on ultrasound the adrenal cortex appears large and hyperechoic while the adrenal medulla appears thin and, relative to the

37 adrenal cortex, more hyperechoic. Other, relatively more rare disorders, such as adrenal hemorrhage and virilizing cortical tumors, are also associated with similar adrenal gland presentation on ultrasound (Saada et al. 2004). However, evaluation of fetal adrenal gland on prenatal ultrasound can provide evidence towards a diagnosis of CAH. !4. Partial Androgen Insensitivity Syndrome While limited information is available from the literature on prenatal diagnosis of partial androgen insensitivity syndrome (PAIS). However, in cases of isolated ambiguous genitalia with a fetus with confirmed 46, XY karyotype PAIS should be considered by androgen receptor sequencing (Onderglu et al. 2012). Prenatal diagnosis of PAIS can be made through sequencing of the androgen receptor gene. !Prenatal management of ambiguous genitalia in association with additional anomalies Determining that the suspected genitalia is most likely a solely physical anomaly without an underlying genetic or hormonal component can only be accomplished through a testing approach that excludes these alternative possibilities. When the karyotype, hormonal assays, and additional testing, such as single gene tests or microarray, yield normal results, a specific etiology or diagnosis is aided by further interpretation of multiple anomalies. Karyotype should be conducted in all cases of multiple anomalies to identify possible chromosome abnormalities that can present with overlapping features. !1. Microdeletions The single case of 1p32-31 microdeletion syndrome presented in the literature review reinforces that in the instance of a fetus with multiple birth defects, if karyotype appears normal, this does not exclude a microduplication or microdeletion syndrome.

38 Microarray technology can be implemented prenatally and may serve as a useful broad approach to cases with multiple anomalies and normal karyotypes. CGH microarray on uncultured amniocytes obtained by amniocentesis in late gestation is an efficient and rapid tool for diagnosis of subtle chromosome anomalies, such as microdeletion. (Chen et al. 2011) !2. OEIS OEIS should be suspected with two characteristic findings on ultrasound: an abdominal wall defect and inability to visualize the fetal bladder without anhydramnios or oligohydramnios (Calvo-Garcia et al. 2013; Goto et al. 2008). Abdominal wall defects associated with OEIS are typically on the midline and the umbilical cord may appear more inferior in placement (Tonni et al. 2011). Another characteristic ultrasound marker of OEIS is known as the “Elephant’s trunk” sign for the appearance of the prolapsed terminal ileum into two exstrophied hemibladders (Goto et al. 2008; Tiblad et al. 2008 ;

Tonni et al. 2011; Witters et al. 2012). Spinal defects are also a hallmark features of OEIS and, if found in conjunction with abdominal wall defect and inability to visual bladder, should strengthen suspicion of OEIS (Tiblad et al. 2008).

The more anomalies detected, the more difficult it may be to distinguish OEIS from other conditions (Tiblad et al. 2008). Other features of OEIS complex can include: persistent cloacal membrane, lumber mylomenigocele, ambiguous genitalia, limb defects, renal anomalies, gastrointestinal anomalies, craniofacial features, and cardiac defects

(Calvo-Garcia et al. 2008; Tiblad et al. 2008; Tonni et al. 2001). Thus, a fetal

39 echocardiogram is mandatory to assess potential cardiac involvement because there is a known association (Tonni et al. 2001).

When OEIS is suspected, it is necessary to distinguish between a cloacal malformation, abdominal wall defect and bladder exstrophy on prenatal ultrasound as the presentations can appear similar (Witters et al. 2012; Calvo-Garcia et al. 2008; Goto et al.

2008). Calvo-Garcia et al. (2008) present a chart for distinguishing the two on prenatal ultrasound, which is provided below (Table 6).

As classic OEIS complex includes anomalies of the spine with or without open neural tube defect, maternal serum α-fetal-protein (AFP) may provide additional evidence suspect of OEIS; however, maternal serum AFP is elevated in approximately 40% of

40 OEIS (Tonni et al. 2001). Thus, this screening tool is unreliable, as a normal result does not rule out spinal anomalies (Tiblad et al. 2008).

While a constellation of anomalies on prenatal ultrasound can indicate when to suspect OEIS complex, magnetic resonance imaging (MRI) can provide additional evidence of OEIS when a normal bladder is not visualized on prenatal ultrasound by distinguishing between internal organ structures (Calvo-Garcia et al. 2008; Tonni et al.

2001, Goto et al. 2008). Ideal timing for MRI study is at approximately 21 weeks gestation (Calvo-Garcia et al. 2008). Findings associated with OEIS on MRI can include: protuberant abdomino-pelvic contour, absence of meconium filled rectum and colon, genitourinary and spinal malformations and a thin walled anterior cyst protruding through intra umbilical pelvic wall (Calvo-Garcia et al. 2008). !3. Cloacal anomalies Cases of cloacal anomalies as a cause of ambiguous genitalia usually present with a normal karyotype. Although the presentation of cloacal anomalies is a wide spectrum, the three predominant features appear to be genital abnormalities along with heart and spinal defects. If there is a suspicion of a cloacal anomaly, serial ultrasounds are recommended. MRI can help distinguish internal fetal organs, such as fetal bladder from uterus, and potentially confirm suspicions of cloacal or urinary tract anomalies (Calvo-

Garcia et al. 2008; Rios et al 2012; Pauleta et al. 2010; Lee et al. 2012). MRI can also identify the presence of a meconium filled colon, an absence of which is suggestive of cloacal anomalies (Witters et al. 2004). In all cases in which a malformation of the

41 urinary tract is suspected, management should include consultation with a pediatric urologist (Warne et al 2002; Witters et al. 2004). !4. Urinary Tract Anomaly Along with cloacal anomalies, anomalies of the urinary tract should also be considered in cases, which present with a cystic pelvic mass. The single case study in this literature review of ambiguous genitalia in association with persistent urogenital sinus demonstrates that the presentation of these particular anomalies can overlap with cloacal anomalies (Pauleta et al. 2010). MRI offered better visualization of the anomalies and allowed the researchers to assess anorectal integrity, as the findings in this case overlap with other urogenital and anorectal malformations (Pauleta et al. 2010). Thus again, MRI can provide insight into internal fetal organs and help to distinguish between cloacal or urinary tract anomalies (Rios et al 2012; Pauleta et al. 2010; Lee et al. 2012). !5. Hypospadias Hypospadias is often categorized separately, but is an anomaly of the urinary tract. 2D ultrasound is considered the best approach for detecting hypospadias. An anatomy scan should include a careful evaluation of the urinary tract, as 40% of fetuses with hypospadias have upper urinary tract anomalies (Bamberg, C., Brauer, M.,

Degenhardt, P., Szekessy, D. & Henrich, W. 2010). Hypospadias can also be associated with cleft lip and palate, congenital heart defects, neural tube defects, and anorectal malformations. Hypospadias can also have an underlying aneuploidy etiology and specific syndromes such as Fraser syndrome and Smith Lemli Opitz, reinforcing the need

42 to eliminate possible genetic and hormonal etiologies of the anomaly before deciding that the anomaly is isolated.

Protocols and Current Practice

In addition to reviewing the literature for recommended testing approaches in cases of prenatally suspected ambiguous genitalia, data were also collect on what testing approaches are currently in use.

In the Literature

A total of 27 unique protocols were used among the 89 cases extracted from the literature (Table 7). In this study, a suspected prenatal diagnosis refers to an etiological or specific diagnosis, such as a chromosome anomaly or congenital adrenal hyperplasia, not including isolated anomalies, as opposed to a description of the specific anomaly. In order to determine the number of cases in which a suspected prenatal etiological diagnosis was confirmed, the number of cases in which a suspected prenatal diagnosis was provided was necessary for comparison to the final, postnatal, diagnosis.

43

The most common protocol among the cases from the literature was 2D ultrasound followed by amniocentesis for chromosome analysis via karyotype (AFH).

Thirty of the 89 (33.7%) total cases used this protocol. Of the 30 cases, 11 (36.7%) provided a suspected or prenatal diagnosis and 6 of these 11 (54.5%) were confirmed to be correct via prenatal testing, on postnatal exam, or during additional testing after birth.

The second most common protocol was the sole use of 2D ultrasound (A), which was used in 20 of 89 (22.5%) cases. Eight of 20 (40%) provided a prenatally suspected or prenatal diagnosis. Four of these 8 (50%) cases confirmed the prenatally suspected or prenatal diagnosis to be correct. The third most common protocol was applied in 6 of 89

(6.7%) of cases. This protocol consisted of 2D ultrasound, amniocentesis for karyotype

44 analysis and FISH for SRY (AFHI). Four of 6 (66.7%) provided a specific suspected prenatal diagnosis, with 2 (50%) of these having the suspected prenatal diagnosis confirmed correct. When all 89 cases are combined regardless of protocol, 48 of 89

(54%) provided suspected prenatal diagnoses and total number of correctly identified suspected prenatal diagnoses was 31 of 48 (63.3%).

Due to the variety of protocols in the literature, as well as the relatively small number of cases, a statistical test to identify significant differences among protocols in a suspected prenatal diagnosis being confirmed as correct would not provide meaningful information. This rate of correct prenatal diagnosis for the three most common protocols and the rate for all protocols combined appears to be approximately 60%. Confidence intervals for the proportions were generated for the two most common protocols, AFH and A, as well as for all combined protocols. The small sample sizes for other protocols preclude the computation of meaningful confidence intervals for them.

The proportion of cases with correct suspected prenatal diagnosis for AFH and A is 0.5455 and 0.5000 respectively and the corresponding 95% confidence intervals are

(0.2799 - 0.7875) and (0.2152 - 0.7848) respectively. These two intervals have substantial overlap, indicating the lack of statistically significant differences between the two protocols. The proportion of cases with correct suspected prenatal diagnosis for all protocols combined is 0.6458, and the corresponding 95% confidence interval is (0.5040

- 0.7661). This result supports the statement that, from those cases examined from the literature, regardless of a particular protocol, a prenatally suspected etiological diagnosis

45 in cases of ambiguous genitalia will be confirmed as correct approximately 60% of the time.

Table 8: Confidence Intervals by Protocol

Proportion with correct 95% confidence interval for prenatal diagnosis the proportion

AFH 0.5455 0.2799 - 0.7875

A 0.5000 0.2152 - 0.7848

Overall 0.6458 0.5040 - 0.7661 ! Chart Review

Forty-nine total cases were identified in the electronic Viewpoint database as having “ambiguous genitalia,” “hypospadias” or “clitoral hypertrophy.” Of the 49 cases,

39 were singleton pregnancies with suspicion for genital anomalies noted on prenatal ultrasound during pregnancy. Charts for each of these 39 cases were compiled for review.

Three cases had no medical charts and many of the electronic records for those cases with paper charts contained additional information relevant to the study, such as the delivery record. Of the 39 cases, 18 delivered at University Hospitals. Of these 18 cases, postnatal charts were available for 15 cases (Figure 5).

46

A total of 17 unique protocols were used among the 39 cases extracted from the charts. The most common protocol utilized was 2D ultrasound in combination with second trimester maternal serum screening (quad screen)(AS). Nine of the 39 (23.1%) total cases used this protocol. Of the 9 cases, 1 (11.1%) had provided a suspected prenatal diagnosis. The single suspected prenatal diagnosis was incorrect, as it did not match the provided final diagnosis.

The second most common protocol was the sole use of 2D ultrasound (A), which was used in 7 of 39 (17.9%) cases. One of 7 (14.3%) provided a suspected prenatal diagnosis, which was confirmed to be incorrect. The third most common protocol was applied in 5 of 39 (12.8%) cases. This protocol consisted of 2D ultrasound in combination with maternal serum first trimester screening (AR). None of the 5 provided a suspected prenatal diagnosis. When all 39 cases are combined regardless of protocol, 8

47 of 39 (20.5%) provided suspected prenatal diagnoses. The total number of correct prenatal diagnoses was 3 of 9 (33.3%). Overview of all 17 protocols is provided below

(Table 9).

Recommendations and Algorithm

The results of the literature provided detailed recommendations based on a variety of studies and individuals cases. However, when evaluated by testing protocols in individuals cases, no one protocol appeared to provide an advantage for determining a prenatal diagnosis over another. The same is true for the testing protocols used in the cases identified for the chart review. For this reason, an algorithm (figure 6) was

48 developed based on the review of the literature, rather than the statistical data provided from evaluation of the various testing protocols.

To summarize the recommendations, a karyotype and detailed anatomical survey should be offered in all cases of suspected ambiguous genitalia. While cffDNA may provide an accurate fetal genetic sex, cffDNA is not diagnostic and provides limited scope as to assessment of chromosome abnormalities. If chromosome analysis returns a normal karyotype, the confirmed fetal genetic sex dictates the approach to additional testing. While karyotype is available beginning at 10 weeks gestation via chorionic villus sampling and beginning at 16 weeks gestation via amniocentesis, an informative detailed anatomy scan can only be conducted at or after 18 weeks gestation, thus it is important to consider timing when considering the likelihood of suspected ambiguous genitalia. If a karyotype was obtained by chorionic-villus sampling, amniocentesis may be performed for hormone assay. Maternal serum estriol and maternal serum steroid profile may be performed.

If amniocentesis is performed, amniotic fluid should be reserved for hormone assay. Hormone assay of amniotic fluid should include, but is not limited to: 17- hydroxyprogesterone, testosterone, andostendione, 11-deoxycholesterol, 7- dehydrocholesterol, cholesterol, 17-α-hydroxylase, 20-lyase, and 21-hydroxylase. These metabolites can rule out the possibility of CAH in the case of a fetus with a female karyotype with ambiguous genitalia or Smith Lemli Opitz (SLO) in the case of a fetus with a male karyotype. Ruling out SLO is important as the condition is also associated with congenital heart defects, which may not have previously been detected. In a fetus

49 with a male karyotype with seemingly isolated ambiguous genitalia without a chromosome abnormality and a normal steroid profile, consider androgen receptor sequencing. In a female with seemingly isolated ambiguous genitalia and a normal karyotype and steroid profile, consider potential exposures, such as medications or maternal tumors.

In cases of ambiguous genitalia and multiple anomalies, if the karyotype and steroid profile are normal and the anomalies are not suggestive of a particular syndrome, consider a microarray on amniocytes, as a normal karyotype does not rule out the possibility of a micro deletion or micro duplication. The algorithm below (figure 6) summarizes these concluding testing recommendations in cases of prenatally suspected ambiguous genitalia based on the synthesis of the literature in this study.

50

Figure 6: Algorithmic recommendations from synthesis of the literature.

51 ANALYSIS ! Of the 15 cases from the UHCMC database with full pre and postnatal records, the algorithm from this study would have provided an etiology or correct diagnosis for 10 cases, giving the algorithm and overall success rate of 66.6%. Cases in which the final diagnosis was normal male or female without genital anomalies on postnatal exam were included in the “success rate” of the algorithm if, when the algorithm was applied, the results ruled out the possibly of a chromosome abnormality or larger syndrome and suggested the likelihood of an isolated anomaly, if any anomaly existed at all, given the documented detection rate of ambiguous genitalia on ultrasound. Thus, if there was an isolated genital malformation noted at birth or the genitals were normal at birth and the algorithm had suggested either of these findings, the algorithm was considered to be

“successful”. Of the remaining 5 cases (33.3%) it is unclear as to whether the algorithm would have provided an etiology or diagnosis as the postnatal chart did not include this information.

Table 10: Cases from chart review for example application of proposed algorithm.

Case Ultrasound Final Diagnosis Would the proposed algorithm have findings provided a prenatal etiological diagnosis? 1 SGA, Male with bilateral Unknown - Would have suggested cardiomegaly supernumerary 5th microarray for multiple anomalies, if , possible digits and normal would suggest giving hypospadias cardiomegaly consideration to exposures or syndromes

52 2 Increase NT, Male with sacral Unknown - Would have suggested Hydronephro dimples, bilateral microarray for multiple anomalies, if sis, risk for reflux, patent anus normal would suggest giving macrocephaly consideration to exposures, isolation or , possible syndromes hypospadias 3 Possible Normal male Yes - Would have ended with likely hypospadias, isolated anomaly if present oligohydram nios 4 Possible Normal Male Yes - Would have ended with likely hypospadias isolated anomaly if present

5 Possible Male with genital Yes - would have ended with likely ambiguous anomaly and heart isolated anomaly if present genitlia murmur 6 Possible Normal Male Yes - Would have ended with likely hypospadias isolated anomaly if present or epispadias 7 Possible Normal Female Yes - Would have ended with likely ambiguous isolated anomaly if present genitalia 8 IUGR, Male with 10p.15.3 Yes - would have suggested Ambiguous duplication microarray which would have genitalia identified microduplication 9 Possible Male with Yes - would have suggested ambiguous delXp22.3 and microarray for multiple anomalies genitalia del3p14.1 which would have identified micro deletions 10 Possible Male with Yes - Would have ended with likely ambiguous hydrocele isolated anomaly if present genitalia 11 Bilateral Male with Unknown - Would have suggested hydronephros ureteropelvic microarray for multiple anomalies, if is, SUA, junction normal would suggest giving polyhydramn obstruction, sacral consideration to exposures, isolated ios, dimple and anomaly or syndromes hypospadias chordee

53 12 Bilateral Male with bilateral Unknown - Would have suggested clubfeet, club feet, caput and microarray for multiple anomalies, if polyhydramn hypospadias normal would suggest giving ios, possible consideration to exposures, isolation or hypospadias syndromes 13 Possible Premature male Yes - Would have ended with likely hypospadias with no reported isolated anomaly if present abrnomalities 14 Thickened Male with Unknown - Would have suggested NT, short hydrocele microarray for multiple anomalies, if femur, normal would suggest giving possible consideration to exposures, isolation or hypospadias syndromes 15 Possible Normal Female Yes - Would have ended with likely clitoral isolated anomaly if present hypertrophy ! ! ! ! !

54 ! ! DISCUSSION! The data in this study indicate that the presented algorithm has the potential to improve the rate of prenatal diagnosis in cases of prenatally suspected ambiguous genitalia. Applying the algorithm to real case examples from the chart review demonstrates its potential to increase the rate of correct prenatal diagnoses among cases with suspected ambiguous genitalia. The algorithm is designed to indicate a likely isolated anomaly or absence of anomaly through elimination of possible underlying genetic and hormonal etiology, and increased the rate of successful prenatal diagnosis from 33.3% percent to 66.6%. Although analysis did not directly apply the algorithm to the cases in the literature, the success rate of the proposed algorithm from the chart review cases (66.6%) suggested a slightly higher success rate than what was noted in the literature (60%). Direct application of the algorithm would be needed to determine whether it would actually increase success rate of accurate prenatal diagnoses for the cases in the literature.

Strengths

A strength of this study includes focusing solely on cases of ambiguous genitalia, unlike many of the studies presented in the literature review, which combine cases of ambiguous genitalia and sex reversal. While the initial approach to such cases may be similar by use of karyotype to determine genetic sex, once the genetic sex has been determined and the possibility of a chromosome abnormality has been ruled out, the

55 approach to testing begins to differ between when ambiguous genitalia is suspected versus sex reversal by differential diagnosis.

In addition, using real case examples in combination with a database of published cases makes the algorithm more widely applicable. Unlike the previously published testing algorithms, the proposed algorithm complies with ACOG and other American societal guidelines (ACOG 2012). Although the goal of creating the algorithm was to make a more uniform approach to prenatal cases of ambiguous genitalia within the

United States, the data are drawn from the literature published internationally and may be applicable in other countries as well.

The algorithm’s strengths also include the inclusion of the most recent testing methods, such as microarray. Prenatal use of microarray was included in this algorithm following the identification of its use in a single case study in the literature (Chen et al

2011). Although the use of microarray in prenatal cases has not been available until more recently, this case study demonstrates its increased application by this technology. The use of microarray was not discussed in the most recently published algorithm, which was published approximately one year after the single case study included in this review

(Chitty et al. 2012). The inclusion of microarray technology in this study does limit comparison to previously presented recommendations, as the technology may not have been available.

In addition to incorporating new technology that may not have previously been available for study, the algorithm presented in this study provides recommendations towards distinguishing physical anomalies associated with ambiguous genitalia, such as a

56 cloacal anomaly, from an abnormality of the urinary tract, that often do not present with an underlying genetic or hormonal etiology. Identifying the likelihood of ambiguous genitalia in association with one of these physical anomalies may require a different intervention and change of prognosis.

The focus of the proposed algorithm was to determine the most efficient means of determining a prenatal diagnosis in cases of ambiguous genitalia. In doing so, a recommendation for chromosome analysis via invasive testing to obtain a karyotype was included as the first step in the algorithm because it provides information about chromosome abnormalities that would not be made on non-invasive procedure (i.e., non- invasive prenatal screening), and is diagnostic for genetic sex. While cases from the literature support the placement of karyotype analysis at the beginning of the algorithm, many prospective parents may hesitate to consider an invasive test as both chorionic villus sampling and amniocentesis for karyotype carry a risk for pregnancy complications, including miscarriage. In addition, data from the chart review suggest a detection rate that is less than ideal (46.7%); thus, the suggestion of starting the analysis with an invasive procedure both strengthens the efficiency of the algorithm while limiting its clinical application. Clinicians utilizing cffDNA to predict fetal sex instead of a full karyotype analysis should note that not only is the possibility of a chromosome abnormality still present, but that further use of the algorithm may be misleading and lead to unnecessary tests.

Relevance to Genetic Counseling

57 Genetic counselors often rely on professional society guidelines in their recommendations for prenatal testing. Genetic counselors can benefit from the data presented in this study in the following ways. The algorithm provides a standardized approach when ambiguous genitalia is suspected on their prenatal ultrasound, which is thought to increase the rate of reaching a correct prenatal diagnosis. This study does; however, demonstrate that the correct diagnosis of ambiguous genitalia on prenatal ultrasound in 100% of cases is impossible to achieve because ultrasound, unlike additional testing such as hormone assays, relies heavily on interpretation. Thus the inclusion of additional testing is beneficial for genetic counselors to consider in an effort provide a more accurate risk assessment. Not only does the algorithm show step-by-step recommendations for a testing approach, but the algorithm has been evaluated with real life cases that allow counselors to provide an estimated rate of success (66.6%) for either identifying a correct diagnosis or ruling out the likelihood of a syndromic etiology and suggesting an isolated anomaly or no abnormality at all, to the best of our ability prenatally. This algorithm can aid in reducing uncertainty for prospective parents by eliminating possible diagnoses with a specific prognosis.

Genetic counselors using this statistic with prospective parents are reminded to add the disclaimer that the success rate in the analysis is defined as determining a diagnosis or eliminating the possibility of underlying genetic or hormonal causes and assessing the likelihood that the anomaly is an isolated physical anomaly or in fact, the genitalia are normal, given the limitations of detection rate on prenatal ultrasound. The testing approach will always be limited by the nature of prenatal genetic counseling and

58 the inability to fully rule out all syndromes prenatally as is true of other symptoms, such as developmental delay, which may not present until well after birth. Ultrasound as a tool is heavily influenced by a number of variables and relies heavily on interpretation and therefore will never be able to consistently determine if an anomaly is truly present.

Limitations

This study is limited by the available published literature. Information regarding prenatal exposures, previous pregnancy history or demographic information was not provided for many of the cases reviewed. Although pregnancies with known family history were excluded in order to avoid detection bias, if that information was not provided in the context of the information regarding the specific pregnancy, cases with family history of ambiguous genitalia cannot be entirely ruled out.

This study was also limited in the number of available cases. Thirty-nine cases were identified within the ultrasound database for review; however there was no guarantee that because these women had ultrasounds performed at UHCMC that they delivered within the same system. Thus, only 15 cases had postnatal charts for confirmation of genital anomalies at birth. In addition, the number of cases with ambiguous genitalia identified on postnatal exam without being suspected prenatally is unknown, limiting the interpretation of sensitivity and specificity of ultrasound detection.

Due to limited availability of the archived ultrasound data, the time frame of the chart review did not match the time frame of the literature. Thus, the overlap between the literature and the charts reviewed limits interpretation. In particular, potentially with access to the archived ultrasound database, the number of charts to review would have

59 been more comparable to those reviewed from the literature adding to the strength of comparing the rate of prenatal etiological diagnosis with postnatal etiological diagnosis.

The scope of this study was also limited to the prenatal and neonatal periods. This study did not collect additional information regarding the development of the children born who had a suspicious prenatal ultrasound finding identified. Thus, one of this study’s limitations is the lack of data regarding long-term follow up in cases of prenatal suspected ambiguous genitalia. Although an etiology may not have been listed on a postnatal exam, this does not exclude the possibility of later developmental concerns and/ or a specific diagnosis being determined at a later date.

Conclusions

In conclusion, analysis of the protocol data from the review of the literature and the chart review did not indicate an advantage of one protocol from another in determining a prenatal diagnosis when ambiguous genitalia was suspected on prenatal ultrasound. Due to the limitations of the protocol data, an algorithmic testing protocol was developed primarily using the literature review. In addition to providing an estimated detection rate in cases of prenatally suspected ambiguous genitalia, the chart review data was also applied to the proposed algorithm in order to assess its potential for successfully identifying a diagnosis. Assessing the use of the algorithm with real case examples from the chart review demonstrated the potential for an increased rate of correct prenatal diagnoses when the algorithm was applied to the same cases. The primary limitations of this study include the ability to interpret the data due to the number of cases and incomplete information, limitations in the literature data, the inconsistent time frame of

60 the literature review and the chart review and the lack of long term follow up in each case.

This study adds to the foundation of available literature on testing in cases of prenatally suspected ambiguous genitalia. Future studies can build off of the review presented here by assessing the application of the proposed algorithm in future cases of prenatally suspected cases of ambiguous genitalia. A study focusing specifically on the application of the algorithm and post natal follow up could provide the increased number of cases needed to better evaluate the application of the proposed testing approach. In addition, future prospective studies evaluating the clinical applicability of the proposed algorithm in theory would also be able to provide stronger statistics towards determining a true detection rate for the presence of ambiguous genitalia on prenatal ultrasound. ! ! ! !

61 ! ! APPENDIX I: First Review Matrix Organization and summary of literature review articles

62 63 64 65

66 67

68 69 70 71 72 73 74 !

75 ! 76 ! 77 APPENDIX II: Second Review Matrix Organization and summary of case studies from the literature review

78 79

80 81

82 83

84 85

86

87

88 89 90

91

92 93 ! APPENDIX III: Third Review Matrix Organization and summary of chart review cases

94 95 96

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