1 INTRODUCTION

Women with advanced maternal age (AMA) refer to pregnant women older than 35 years. The number of women with AMA has increased year by year worldwide. The proportion of women with AMA in China rose from 10.1% in 20111 to 20.5% in 2016.2 For decades, AMA has been a leading indicator of invasive prenatal testing. In recent years, many pregnant women with AMA are willing to accept non-invasive prenatal testing (NIPT) that can identify the most common fetal aneuploidies (trisomy 21, trisomy 18, and trisomy 13), and sex chromosome aneuploidies (SCAs)3; however, there are still a large number of women with AMA opting for invasive testing.

Conventional karyotyping can identify the majority of fetal chromosome abnormalities, at a resolution of greater than 10 Mb.4 With the implementation of chromosomal microarray analysis (CMA), many chromosomal abnormalities that were undetectable by karyotyping were found in either prenatal or postnatal testing.5-7 Microdeletions and microduplications have been reported in approximately 6% of pregnancies with a structural fetal abnormality and approximately 1.7% of other high-risk pregnancies.8, 9 Wapner et al9, 10 suggest that all pregnant women, regardless of age, would equally benefit from invasive testing by fetal microarray analysis. Previous reports support CMA for all women undergoing prenatal invasive testing regardless of the presence or absence of fetal abnormality. It is well known that AMA women are at increased risk for aneuploidy due to non-disjunction, while non-AMA pregnancies have a higher risk for submicroscopic pathogenic aberrations than for Down syndrome.11 Therefore, AMA women without fetal ultrasound abnormalities (UA) always express hesitation about performing CMA. Other concerns include the theoretical risk of detecting variants of unknown clinical significance (VOUS), and variants in susceptibility loci (SL),12, 13 as well as the higher cost of CMA compared to that of karyotyping. The purpose of this study was to evaluate the efficiency of CMA in the prenatal diagnosis of AMA women with or without UA, to provide further practical evidence for pre-testing consultation.

2 MATERIALS AND METHODS

3 RESULTS

4 DISCUSSION

There have been various reports on the efficacy of CMA in high-risk (mainly USA) and low-risk populations.15, 16 However, in the population of AMA women, comparison between pregnancy with and without ultrasound findings was rarely reported. The purpose of this study was to investigate the necessity to perform CMA in women with AMA undergoing invasive prenatal testing.

We found significant differences in gestational age at diagnosis, types of specimens, and pregnancy outcomes between the two experimental groups in this study. Amniotic fluid was the predominant specimen in group A, whereas the proportion of cord blood in group B was significantly higher. This was closely related to the gestational age at the time of diagnosis. Majority of pregnant women in group A readily preferred amniocenteses because compared with choriocentesis and cordocentesis, amniocentesis is more acceptable. Accordingly, they underwent amniocentesis at the proper time. Ultrasonic structure screening is generally conducted during the 20th and 24th gestational week, and amniocentesis is generally performed before 24 weeks of gestation. Therefore, several pregnant women may be detected with fetal ultrasound problems at a later gestational age after amniotic fluid testing. This was the circumstance for case 15. The patient made the decision to terminate the pregnancy, taking into account the CMA results and subsequent ultrasound findings. This further indicates the importance of offering CMA when performing amniocentesis, that is, to avoid later cordocentesis and save time for the patient as well.

As expected, trisomy was the most frequent abnormal findings in this study. Chromosomal microarray analysis showed a detection rate of 0.9% for submicroscopic findings with clinical significance in the group of AMA without UA, lower compared with 1.0%-2.1% reported in other literatures.17-20 It has been reported that incremental diagnostic yield of CMA for fetuses with UA ranged from 5.2% to 10%, fetuses with ultrasound structural abnormalities ranged from 3.1% to 7.9%.16 In our study, the value was 2.6% and 3.6%, respectively, this can be explained by the great proportion of aneuploidies caused by AMA women's basic risk for aneuploidy. The clinical significant findings in group B is more frequent than that in group A, but submicroscopic findings in the two groups showed no statistic difference. This may further imply that AMA women could be recommended for CMA testing just as UA group. Notably, clinically significant submicroscopic findings in group A were mostly associated with neurological or mental dysplasia. A susceptibility locus (SL) for neurodevelopmental disorders is considered the most common finding in pregnancies without UA.20 They may be categorized as pathogenic/likely pathogenic findings in spite of their incomplete penetrance and variable expressivity, even inheritance from normal parents.21-23 SL is generally associated with autism, learning and speech problems, mild intellectual disability (ID), or developmental delay (DD). It is well established that a second hit plays a vital role in causing an abnormal phenotype.24 In our study, there were four cases harboring SL: 15q11.2 deletion (case 15) and 16p13.11 duplication (case 16) in group A; 15q11.2 deletion (case 30), 16p13.11 deletion (case 31) in group B. The 15q11.2 microdeletion was considered to be responsible for 15q11.2 BP1-BP2 microdeletion syndrome. It was previously recognized as a VOUS but now is considered as likely pathogenic/pathogenic variants in many reports.25, 26 The penetration of the variants was estimated to be 10.4%,27 and the prevalence in patients with mental retardation, ID, and/or multiple congenital anomalies was about two to fourfold of that in control population.27-29 It is well known that being rich in low copy repeats (LCRs) increases the occurrence of non-allelic homologous recombination resulting in duplications or deletions. This is particular for chromosome 16. Nearly 9.89% of chromosome 16 consists of LCRs. The 16p13.11 locus is a genomic hotspot encompasses a core set of eight protein-coding genes, including NDE1, the strongest candidate gene for the neurodevelopmental phenotypes. 16p13.11 deletions are considered as pathogenic for its association with multiple phenotypic manifestations, including neurodevelopmental phenotypes such as autism, epilepsy, physical dysmorphisms, and other congenital anomalies.30-33 16p13.11 microduplication syndrome was the most commonly detected microduplication syndrome in a postnatal cohort with unexplained DD, ID, and autistic spectrum disorder (ASD)34; the penetration was estimated to be 10.6%. As shown in our study, fetuses with SL can be found in ultrasound normal or soft marker group, although they were live born with normal phenotype, long-time follow-up is crucial to assess the actual risks for neurodevelopmental disorders.35

Chromosomal microarray analysis is of great value not only for detecting submicroscopic anomalies but also cytogenetic abnormalities with unknown origins, such as small supernumerary marker chromosomes (sSMC), whose clinical phenotype and prognosis depend on the chromosomal origin. sSMC were reported to be associated with AMA.36 Identification of the nature and source of sSMC is the most important for pregnancy guidance. In the current study, 8 cases with de novo sSMC were diagnosed in both the groups (0.14%), 4 in each; this figure is higher than that reported previously.37, 38 Finally, one case in each group was confirmed by CMA as having no change in the genetic material, and the pregnancy was continued.

In fact, an increasing number of women has gradually accepted this relatively high-cost test and self-financed testing. Other barriers for consultants to offer CMA lie in interpreting the VOUS results and patient-related concerns such as anxiety.39 The VOUS rate detected by CMA shows considerable variability.40, 41 In present study, VOUS was all detected from UA group and had a relatively lower detection rate (0.4%) than other literatures, this may be explained by the limited case number. From a patient's point of view, they want to exclude as many abnormalities as possible to ensure that the fetus is healthy. Whether to choose CMA mainly depends on pre-test counseling. As shown in this study and previous literature, while improving the detection rate of submicroscopic abnormalities in pregnancies of AMA, CMA also yields uncertain results which are unavoidable in prenatal testing for any other indications. Therefore, research should be focused on how the results should be managed, and how to provide reasonable consultation, rather than trying to avoid the uncertain results. A reasonable consultation is inseparable from the accumulation of experience, and prenatal findings combined with long-term postnatal follow-up are essential means to gaining experience. It is only by this method that we can provide more evidence for consultation, thus release the burdens on genetic consultants and patients.

In conclusion, CMA has a higher detection rate of chromosomal abnormality than conventional karyotyping, either in AMA women with or without UA. As for the population of AMA women, CMA should be available to all pregnancies undergoing invasive prenatal testing, regardless of ultrasound results.

ETHICAL APPROVAL

The present study was approved by the Protection of Human Ethics Committee of Fujian Provincial Maternity and Children's Hospital, affiliated Hospital of Fujian Medical University. Written informed consent for participation was received for all patients.