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Original Article

Noninvasive Prenatal Testing for Fetal XXY Aneuploidies Among Pregnancies in Beijing of China

Chang, Jia-Zhen1; Qi, Qing-Wei2; Zhou, Xi-Ya2; Jiang, Yu-Lin2; Hao, Na2; Liu, Jun-Tao2,∗

Editor(s): Shi, Dan-Dan

Author Information
doi: 10.1097/FM9.0000000000000056
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Abstract

Introduction

As one of the most common sex chromosome aneuploidies (SCAs), Klinefelter Syndrome has a prevalence rate of about 1/660–1/450 in the male fetus of live birth.1,2 The typical karyotype of Klinefelter Syndrome is 47, XXY. Patients often refer to hospitals due to growth retardation and abnormal adolescent development or infertility.3,4 Klinefelter Syndrome have a wide variety of clinical manifestations: some patients are affected only mildly or even asymptomatic, while others develop serious signs and symptoms, such as mental retardation, oligo-atheno-spermia, hypogonadism,5 neurodevelopmental anomalies, language barriers, and other related secondary phenotypes like fifth-finger clinodactyly, pes planus, and increased length of extremities.6 In infancy, boys with Klinefelter Syndrome may show hypotonia, slightly asymmetrical motor patterns, and growth retardation.7 Since fetuses with Klinefelter Syndrome often lack typical prenatal ultrasound manifestations, and some born affected boys seem asymptomatic, many patients are undiagnosed. However, because of insufficient testosterone generating, replacement therapy is needed since age of 11–12 years, which could be conductive to the neurocognitive development of adolescent with Klinefelter syndrome.8,9 Therefore, an early diagnosis will benefit the treatment and prognosis of young patients.

Since Lo found cell-free fetal DNA in maternal serum in 1997, noninvasive prenatal testing (NIPT) has rapidly entered the medical field in a variety of countries for prenatal screening of fetal chromosomal aneuploid.10 By sequencing the cell-free DNA (cfDNA) in maternal peripheral blood, NIPT could determine whether the fetus is high-risk for aneuploid. In current clinical practice, NIPT is mostly used to screen for fetal chromosome 21, 18, and 13 aneuploid, and sometimes SCAs.11 Although NIPT results are considered to be highly accurate for chromosome 21, 18, and 13 aneuploidy, slightly lower sensitivities and specificities have been consistently reported for SCAs in prospective studies.12–14 With the wide application of NIPT, more and more fetuses with Klinefelter Syndrome might be able to be revealed in the prenatal stage. The purpose of this study was to evaluate the screening performance of NIPT based on high-throughput massively parallel sequencing (MPS) technology for the fetal XXY aneuploidy among pregnancies in Beijing of China, and to evaluate the benefits and limitations.

Materials and methods

Sample collection

This study was a retrospective study, which recruited consecutive pregnant women who attended the Peking Union Medical College Hospital, Beijing, China, for NIPT from January 1, 2016 to December 31, 2019. The inclusion criteria: (1) pregnant women aged 20–50 years; (2) gestational age ≥12 weeks. The exclusion criteria: (1) fetal structural abnormalities or soft indexes discovered by ultrasound; (2) history of blood transfusion, stem cell therapy, immunotherapy, or transplant within a year; (3) those with known malignant tumors; (4) either one of a couple with known chromosomal abnormalities; (5) those with history of birth defects. A total of 26 913 pregnant women were recruited in this study. All the testing data and clinical records were collected and analyzed. This study was approved by the Institutional Review Board of the Peking Union Medical College Hospital with the protocol number of S-K1179, and a waiver of written patient informed consent was obtained.

NIPT

A sample of whole blood (10 mL) was collected from pregnant women into ethylene diamine tetraacetic acid anticoagulant vacutainers. Plasma was separated by centrifugation 10 minutes at 1 600×g and 10 minutes at 16 000×g, respectively within 48 hours. Maternal plasma layer and leukocyte layer were reserved. After cfDNA extraction from 1 mL plasma using DNA extraction and purification kit (R0011, Berry Genomics Corporation, Hangzhou, China), the concentration of total cfDNA was measured by Qubit 3.0 fluorometer. If the concentration of total cfDNA is qualified (reference range 0.05–0.70 ng/μL), DNA library would be constructed using Library Preparation and Purification Kit (R0022, Berry Genomics Corporation). Library concentration was determined by quantitative fluorescence polymerase chain reaction. Illumina NextSeq CN 500 sequencing platform was used for MPS, and the sequencing data was mapped to human genome reference sequence (hg19, NCBI build36). The mapping results were analyzed by Bambni Test system. Chromosome z-scores were used to determine whether a fetal was high-risk for aneuploid, with reference range −3 < z < 3. For each sample, fetal fraction ≥4% was required, otherwise, it would be considered that there was not sufficient fetal data to analyze.

Criteria for potential XXY samples

According to the calculation method proposed by Liang in 2013, the z-scores of chromosome X and Y should have a linear relationship with each other for the male samples since the copy numbers of sex chromosomes were proportional to the percentage of fetal DNA in the maternal plasma. We plotted the z-scores of chromosome X against chromosome Y to find the outliers fell within a certain range, which were potential XXY samples.15

Recommendations for potential XXY pregnancies

For potential XXY pregnancies, invasive prenatal diagnosis (IPD) (amniocentesis for example) was always recommended for validation. If declined, detailed medical records were taken after fully informed consent, and routine prenatal examinations were continued.

IPD

Genetic counseling was provided before undertaking IPD. After signing the consent, amniocentesis was performed under ultrasound guidance at 17–22 gestational weeks. Chromosome karyotyping analysis (G banding, 300–400) and fluorescence in situ hybridization analysis (F01001-01, Beijing GP Medical Technologies, Ltd., Beijing, China) were performed according to standard protocols.

Confirmation of maternal karyotypes by genomic DNA sequencing

The maternal leukocyte layer was used to extract maternal DNA and to construct the library. MPS was performed on Illumina NextSeq CN 500 sequencing platform, and the sequencing data were mapped to human genome reference sequence (hg19, NCBI build36). The gain or loss of chromosome regions was detected via the fused lasso algorithm.16

Following-up

The following-up was mainly through telephone calling, record inquiring, and other methods.

The clinical process of NIPT and IPD for fetal XXY is shown in Figure 1.

Figure 1
Figure 1:
The clinical process of NIPT and IPD for fetal XXY. IPD: Invasive prenatal diagnosis; NIPT: Noninvasive prenatal testing; SCA: Sex chromosome aneuploidy.

Results

General situation

Of the 26 913 pregnancies who accepted NIPT, 34 was suspected for fetal XXY (aged: (30 ± 8) years, gestational weeks: (16 ± 4) weeks), accounting for 0.13% (34/26 913).

Choices for potential XXY pregnancies

All pregnant women underwent NIPT received post-test genetic counseling. After discussion focused on the uncertain accuracy and limitations of NIPT as well as the potential phenotypes of Klinefelter Syndrome, the majority (30/34) of the potential XXY pregnancies received IPD for validation, and the rest (4/34) declined.

Outcomes of IPD and maternal DNA sequencing

Of the 30 cases received IPD, 19 were confirmed fetal 47, XXY and 11 were recognized as false positive, with a positive predictive value (PPV) of 63.33% (19/30). In the 11 false positive cases, one were verified maternal SCA. In the four refused cases, one were verified maternal SCA. The possibility of accidental discovery for maternal SCA is 5.88% (2/34).

Following-up

Among the 19 cases of confirmed fetal XXY, 14 elected pregnancy termination, and the rest five led to live birth. Four of the five babies (1 month, 5 months, 1 year, 1.5 years old, respectively) did not show clinical symptoms by now, but the eldest one, who was 2 years old at then, showed developmental delay, mental retardation, severe autism, verbal disability, fine motor disorders, and digestive problems. The boy has been taking pertinency training since the age of one. All the five families joined the XXY supporting organizations and showed enough basic knowledge about Klinefelter Syndrome. Special attention and more love were paid to these babies in these families.

For the 11 cases confirmed normal fetal karyotypes and four cases who declined IPD, all led to live birth and no apparent birth defects were observed by now.

In the 26 879 cases with low-risk for fetal XXY, there were no cases found to have fetal XXY by now. Based on all the data in this study, the detection rate (DR) of XXY screening was 100.00% (19/19) and the false-positive rate (FPR) was 0.04% (11/26 890). However, the limitation of these evaluation estimates is that they may not reflect the true DR and FPR because not all participants had karyotyping results (and it would not be ethically justifiable to karyotype every newborns).

Detailed information of NIPT, cytogenetic assessment, maternal DNA sequencing, and pregnancy outcomes are shown in Table 1.

Table 1
Table 1:
Detailed information of NIPT, cytogenetic assessment, maternal DNA sequencing, and pregnancy outcomes.

Discussion

Mostly, the parents of patients with Klinefelter Syndrome have normal karyotypes. There is no definite conclusion about the cause of Klinefelter Syndrome. Studies have shown that couples with advanced ages (such as those over 35 years old) are more likely to conceive fetuses with Klinefelter Syndrome than younger ones.3,17,18 47, XXY can arise from either de novo mutations during meiosis of oogenesis, spermatogenesis, or from mitosis after fertilization.19 Statistics have shown that in patients with 47, XXY, the probability of abnormalities caused by maternal origin is about 55%, slightly higher than that caused by paternal origin (45%).3 Currently, prenatal screening involves maternal serum biochemical screening tests and ultrasound examination; however, none of these is designed to detect XXY directly.20 For the past 30 years, XXY has been detected as an incidental finding during prenatal karyotyping.21 However, NIPT is different from traditional screening. By sequencing of maternal plasma, it allows direct prediction of fetal XXY.22,23 Because of the complex issues, routine prenatal screening has not yet been suggested, even though NIPT could provide useful information about fetal XXY.24–26 In this study, the results of NIPT were compared with karyotyping of amniotic fluid cells, the golden standard for fetal aneuploidy diagnosis. The comparison helps us to investigate the screening performance of NIPT for fetal XXY, and to find other factors that may have an influence on the accuracy.

The screening performance

NIPT for XXY is more complicated than for autosomal aneuploidy and can be confounded by a variety of different maternal and fetal biological phenomena. The American College of Obstetricians and Gynecologists, The International Society of Prenatal Diagnosis, and The National Society of Genetic Counselors recommend that NIPT be routinely used for common trisomy screening, and be used for XXY and other SCAs screening when requested.27 It is reported that the sensitivity and specificity of NIPT for SCAs were 91.0% and 99.6%, respectively, and the PPVs, which varied slightly, were about 20%–40% and FPRs were 0.0%–0.1%.11,28–31

In this NIPT cohort, the PPV based on the information of karyotyping was calculated to be 63.33%. However, four cases declined IPD and no karyotype information was available. Also, there might be undetected patients due to the mild phenotype of Klinefelter Syndrome. Based on the incomplete clinical follow-up information in this cohort, the screening performance cannot be accurate calculated. However, a range of possible DR, PPV, and FPR can be determined. For example, if there are no false-negative cases and the 11 known discordant cases were the only false-positive cases, the overall DR, PPV, and FPR would be 100.00% (19/19), 67.65% (23/34), and 0.04% (11/26 890), respectively. Alternatively, if all four cases refused IPD were also false-positive, the overall DR, PPV, and FPR would be would be 100.00% (19/19), 55.88% (19/34), and 0.06% (15/26 894), respectively. And if there are false-negative cases, the DR would be reduced and the FPR would have a small increase. Thus, although the true DR for XXY screening is difficult to estimate, the true PPV in this cohort ranges between 55.88% and 67.65%, and the true FPR is about 0.04%–0.06%, or a little higher. This data is consistent with literature reports. It seems the performance is not as good as for Trisomy 21, 18, and 13, but it provided possibility for prenatal screening for fetal XXY.

Factors that may influence the accuracy of XXY screening

As we know, cfDNA in the maternal circulation is derived from both maternal and placental tissues, so there are several factors that may influence the accuracy of XXY screening by NIPT, like confined placental mosaicism (CPM) and maternal SCAs.

CPM

Although not observed in this study, it is already known that CPM occurs in about 1% of pregnancies.32,33 Since the fetal-derived cfDNA in the maternal circulation mainly results from cytotrophoblast apoptosis, it is the placenta rather than fetus itself that being detected by NIPT. In a CPM, the degree of mosaicism will impact the performance of the screen because it would reduce the effective fetal fraction.34–36 This means that CPM has a significant impact on NITP results and that explains for one reason why false positive/negative cases of NIPT are inevitable.

Maternal factors

Moreover, NIPT does not distinguish placental from maternal cfDNA when testing, so maternal biological factors such as maternal somatic mosaicism, undiagnosed maternal SCA, and maternal copy number variations could influence the accuracy of NIPT.37 It has been reported that maternal SCAs can be present in apparently healthy women. For example, the incidence of 47, XXX is about 45/100 000, and most of the patients have no obvious clinical abnormalities except for a relatively tall figure.38 About up to 90% of women with 47, XXX may not be aware of their chromosomal abnormalities.38 However, the maternal XXX could mislead NIPT to suggest high-risk of fetal XXY. In our study, in the 34 cases with high-risk of fetal XXY indicated by NIPT, two were found to have maternal SCAs.

Genome characteristics and technical limitations

There are 1 098 genes on chromosome X and 78 genes on chromosome Y, of which 58 are homologous, and located on both chromosome X and Y. As the sequencing length is only 36 bases, it is difficult for accurate mapping between chromosome X and Y. The incorrect reads mapping might also be one of the reasons for the low PPV of NIPT for XXY.39,40

Based on the above information, when applying NIPT for fetal XXY screening, patients should be counseled about the benefits and limitations.

Potential benefits of NIPT for XXY

Mitigating the risk of miscarriage

The known risk of miscarriage of IPD is about 0.2%–0.3%, which is not a high risk, but many women are uncomfortable with the idea that any percentage of risk could be wagered against the pregnancy.41 Therefore, the most obvious benefit of NIPT for XXY screening is that it is noninvasive for the fetus, which could effectively mitigate the risk of miscarriage.

Eliminates the risk of diagnostic odyssey

Patients of Klinefelter Syndrome show significant clinical heterogeneity, which made it difficult to accurately identify them as early as possible. But prenatal screening could identify the potential XXY fetuses before they begin to generate clinical symptoms and even before they are born, which may effectively improve diagnostic efficiency. In this study, with a PPV of 63.33%, we believe that NIPT could be a potential method for XXY prenatal screening.

Leaving adequate time for preparation and intervention

The biggest benefit of prenatal screening is that it may affect the expectations of pregnant couples for their children. A comprehensive genetic counseling would help the couples to understand the future development of their babies and to choose whether to take pregnancy intervention. With fully understand of the future development of neurological, psychological, physical, and reproductive conditions of children with Klinefelter Syndrome in the prenatal period, parents would have enough time to prepare for the arrival of unusual babies. Moreover, clinical intervention measures can be taken before the occurrence of related symptoms to help improve the development conditions, such as learning disabilities, dysgraphia, and social anxiety based on language competence. This would be far more effective than symptomatic treatment. In this study, for the 14 families who could not accept the possibility to have children with Klinefelter Syndrome, they elected pregnancy termination in time. For the five families who decided to reserve the confirmed XXY babies, they thought that the greatest benefit of this early identification was being able to prepare for the changes associated with Klinefelter Syndrome. All of them had enrolled their child in early intervention services sooner than they would have otherwise without a prenatal screening. Four younger babies who did not show any clinical symptoms by now would be brought to pediatrician at a certain frequency to have developmental assessments. The one who showed developmental problems has been taking pertinency training since the age of one. They were prepared with necessary knowledge and willing to pay special attention and more love to these babies.

Limitations of NIPT for XXY

NIPT is not a diagnosis but a screening

NIPT can have false-positive and false-negative cases. The PPV of NIPT for an affected fetus with XXY was 63.33% in our population. Especially, the incidence of mosaicism of both maternal and fetal origin should be considered as one of the important limitations of NIPT.42 IPD is needed for high-risk cases and residual risk still exists in low-risk cases.

Challenges to genetic counseling

Although NIPT makes it possible for prenatal screening for XXY, it also raises great challenges for genetic counseling. Approximately 10%–15% of Klinefelter Syndrome cases are mosaic. The heterogeneity of clinical phenotypes and the possibility of mosaicism increases the complexity of genetic counseling.32,43 We can never tell the pregnant couples definitely what would happen to their babies. In our five born boys with Klinefelter Syndrome, four did not show any clinical symptoms by now, while one showed developmental delay, mental retardation, severe autism, verbal disability, fine motor disorders, and digestive problems. No one being able to predict the conditions of prognosis precisely, it is of great difficult for the couples to make decisions.

Pressures on pregnant couples

Prenatal screening for XXY may put more extra pressure on the pregnant couples. While most couples did not elect pregnancy termination directly based on NIPT but accepted IPD for further confirmation, this whole process increased anxiety during their pregnancy. In our 34 cases with potential XXY predicted by NIPT, the pregnant couples expressed varied degrees of tension and anxiety. Even after being proved to be false positive, the anxiety of some couples lasted until after delivery.

Pre-and post-testing genetic consulting

Taking all the benefits and limitations into account, the pre-and post-testing genetic consulting is of great importance. Pregnant women should be informed that NIPT for XXY screening is optional before testing. They should be given a chance to think about the possible effects that may bring to them. It should be emphasized that NIPT is a screening test that divides pregnant women into high-risk and low-risk groups. The benefits and limitations above should be informed in detail. For those who get high-risk results for fetal XXY, IPD for conformation is strongly recommended before any decisions made to have pregnancy termination, while for those with low-risk results, routine inspection should be continued to reduce the residual risk. For cases with discordance between NIPT and fetal karyotyping, maternal DNA sequencing would help to identify the cause of false positive/false negative. For pregnant women with known SCAs, NIPT is not a good option.

Pregnancy choices

For pregnancies confirmed fetal XXY, the percentage chose to elect pregnancy termination was 70% reported by Shaffer investigation in 2006, which was lower than that of autosomal trisomy, but higher than other SCAs such as 47, XYY.44 According to our following-up of pregnancy outcomes, 14 of the confirmed XXY pregnancies elected pregnancy termination, with a percentage of 73.68% (14/19), which was consistent with reported data. Factors that influence pregnant women's choices include faith, cultural, family background, and consulting tendency. This highlight the critical need for comprehensive counseling provided by a multidisciplinary team having specific knowledge about the possible features and prognosis of the fetuses.45

Study limitations

Study limitations include its small size and our inability to calculate the false negative rates of NIPT detection for XXY, for the reason that many patients with Klinefelter Syndrome do not show significant clinical abnormalities in their childhood, and they may not be able to confirm their SCAs until puberty or encountering fertility requirements.

Conclusions

In this study, NIPT could be used as a potential method for XXY screening, although the accuracy needs to be improved. Detailed and comprehensive genetic counseling is required before testing. The benefits and limitations should be explained to pregnant couples and they should be aware that NIPT is not diagnostic but screening. For those who received high-risk results, it is increasingly important to take IPD for confirmation before any decision is made about pregnancy termination, and for those who received low-risk results, residual risks should also be reminded. For cases with discordance between NIPT and fetal karyotyping, maternal DNA sequencing will help to identify the cause of false positive/false negative.

Acknowledgments

The authors thank members of the hospital for their assistance in collecting the plasma samples and performing karyotyping. The authors are grateful to all pregnant women for their participation in this study.

Funding

None.

Author Contributions

Jia-Zhen Chang and Jun-Tao Liu had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Jia-Zhen Chang contributed to the data analysis and manuscript drafting with support from Qing-Wei Qi, Xi-Ya Zhou, and Yu-Lin Jiang. Jia-Zhen Chang and Na Hao carried out the experiment. Jun-Tao Liu contributed to conception or study design and final approval of the version to be published.

Conflicts of Interest

None.

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Keywords:

Noninvasive prenatal testing; Sex chromosome aneuploidies; Screening; XXY

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