Association of maternal risk factors with fetal aneuploidy and the accuracy of prenatal aneuploidy screening: a correlation analysis based on 12,186 karyotype reports

Fetal aneuploidy is often associated with disease and developmental abnormalities that severely affect the health and quality of life of patients and place a heavy burden on families and society, most notably T21, T18, T13 and SCAs. Current prenatal primary screening follows the sequential screening risk assessment method proposed in 1999 [1], which combines NT scan, ultrasound and serological screening in early/mid pregnancy to provide clinicians with as much information as possible. NIPT is a method of sequencing free fetal DNA fragments in maternal plasma using massively parallel sequencing technology, which provides excellent detection of common fetal aneuploidies [2]. Clear risk factors and effective screening strategies can effectively prevent fetal aneuploidy, reduce the incidence of birth defects and improve the quality of the population. This study was a retrospective analysis of 12,186 karyotype reports. Maternal risk factors for fetal aneuploidy were investigated and the clinical value of prenatal aneuploidy screening was assessed.

Our results support an association between maternal age and fetal aneuploidy. It is well known that AMA (maternal age over 35 years) increases the risk of fetal aneuploidy [3, 4] and we also found that AMA is more likely to be associated with T13 and SCAs. It is important to note that T21 is the earliest identified and most easily understood human autosomal aberration and is the most easily identified in prenatal aneuploidy screening. Therefore, in actual clinical practice, many embryos or fetuses with T21 do not undergo chromosomal diagnosis. Therefore, our results do not prove that T13 and SCAs are more common in AMA than T21. Furthermore, the present study shows that adolescent pregnancies (maternal age < 20 years) seem to be riskier than AMA, especially in T13. Some studies have reported an association between adolescent pregnancies and adverse pregnancy outcomes, including preterm birth, miscarriage, high neonatal mortality, high infant mortality, high under-five mortality and fetal growth restriction [5‐7], and other studies have reported that births to young mothers may be a marker of low fertility [8].

It was very difficult to collect samples under the age of 20 in this study as the legal age of marriage for women in mainland China is 20 and the principle of patient autonomy is respected in the consultation process. Therefore, we could not collect a larger amount of samples of teenage pregnancies. Also, we cannot force all pregnant women to undergo karyotyping of fetus or aborted tissue. This is a retrospective cohort study and we cannot intervene or give guidance beyond the principles of treatment. However, at least we observed some positive results in all samples that reported karyotyping. WHO estimates that adolescent pregnancies account for 11% of all births worldwide, with more than 95% of these occurring in developing countries [9]. This study provides a sound theoretical basis for reducing adolescent pregnancy, improving reproductive health and implementing public health interventions in low- and middle-income countries.

There is increasing evidence of an association between maternal risk factors and aneuploidy pregnancy loss [10, 11] and this study also supports an association between a history of specific medical conditions and fetal aneuploidy. Of the special medical histories collected, a history of fetal malformations was associated with a higher risk of aneuploidy and was more likely to have T13 and T18. A history of RSA was associated with a slightly higher risk of T18 than T13, whereas a history of IVF-ET was more likely to have T18 and SCAs. This finding is more easily explained by the fact that embryos with severe chromosomal abnormalities tend to stop growing early in pregnancy, whereas fetuses with chromosomal abnormalities but who are barely viable often have a variety of malformations or disorders [12, 13].

Notably, the findings of increased risk of fetal aneuploidy with IVF appear to be inconsistent with previous reports [14‐16]. We conducted further follow-up and found that although fetal malformations or RSA could not be diagnosed prior to IVF, there must have been some specific reasons for choosing IVF, such as older maternal age, biochemical pregnancy or only one case of embryonic arrest, and untested aborted tissue. In addition to the IVF process itself, ovarian stimulation and cryopreservation are potential factors contributing to fetal aneuploidy [17, 18] and we must acknowledge that the aforementioned confounding factors were not controlled for in this study and it was difficult for us to do so. There is no doubt that the rapid development of ART has solved many fertility problems. However, as the technology has become more widespread, it has been found that most couples are more likely to achieve a normal chromosomal pregnancy without ART [19]. Although PGT-A can accurately detect most chromosomal abnormalities in embryos, we cannot force all couples undergoing IVF-ET to undergo PGT, which would have socio-economic and reproductive ethical implications. At the same time, there is still an urgent need for PGT to overcome some technical problems, such as the low efficiency of chimerism detection [20].

Prior to the discovery and popularization of NIPT, sequential screening by ultrasound combined with serology was the most effective screening method for prenatal chromosomal abnormalities. In this study, the TPR and NPV of primary screening were 73.24 and 98.23%, respectively, essentially achieving the goal of identifying normal karyotypes. For experimental results where specific medical history improved the accuracy of primary screening, we analyzed them in the context of practical clinical work and found that there was a greater degree of diagnostic suspicion bias. For example, sonographers will perform more detailed examinations on pregnant women with a specific medical history, leading to more reliable results.

Further combination of primary screening and NIPT methods has yielded greater health economic benefits [21, 22]. NIPT had high sensitivity and specificity in this study, with high PPV in T21 (89.92%) and T18 (69.77%) and moderate PPV in T13 (53.49%) and SCAs (43.24%), which is generally consistent with previous reports [23‐26]. Apart from aneuploidy, NIPT has also made some breakthroughs in the diagnosis of chromosomal abnormalities such as monogenic diseases and copy number variants [27‐30]. In conclusion, NIPT is of great social and economic value in accurately screening fetuses with trisomy and SCAs, thereby improving reproductive health [21, 31].

Due to the extremely low levels of cffDNA, the accuracy of NIPT analysis is highly dependent on the presence of sufficient cffDNA in the sample [32]. In this study, maternal age, early gestational age at NIPT testing or a history of IVF-ET were found to reduce the accuracy of the test, all of which were directly related to the cffDNA ratio in the peripheral blood. The cffDNA ratio has been reported to be negatively correlated with maternal age and positively correlated with gestational age [33, 34]. In addition, one study showed that cffDNA was reduced in ART patients compared to natural pregnancies and that cf. fdna was more significantly reduced in ART pregnancies after fresh ET than after frozen ET [35]. Recent studies have also found a significantly higher false positive rate in the ART population, especially for T13 and SCAs [36]. In addition to prenatal aneuploidy screening, the NIPT technique, based on plasma cell-free DNA testing, has been applied to cancer diagnosis and the assessment of immune rejection after transplantation [37].

Maternal occult tumors have been shown to cause NIPT results to be inconsistent with karyotype reports [38]. This raises concerns about the 121 NIPT results in our sample that were inconsistent with the karyotype report, although we did not follow up pregnant women with cancer. Primary care physicians should emphasize the significance and importance of cancer screening to pregnant women. Furthermore, NIPT has limited ability to identify sex and screen for chromosomal abnormalities in multiple pregnancies, and the efficiency of the test is inconsistent. Therefore, greater caution should be exercised in interpreting NIPT results in women with multiple pregnancies [39, 40].

To further improve the accuracy of NIPT in prenatal aneuploidy screening, many scholars have explored various aspects of bioinformatics technology and software engineering to improve the technology [41, 42]. Domestic scholars have found that cffRNA is more stable in maternal circulation [43]. Although NIPT has many clear advantages, the introduction of NIPT into routine antenatal care in many countries has also raised a number of ethical issues [31]. The debate has centered on the impact of prenatal aneuploidy screening on pregnancy outcomes and the fact that equal access to healthcare for every pregnant woman is a core principle in areas of self-paying NIPT [44, 45]. Therefore, the need for high-quality pregnancy counselling and a well-developed process for prenatal aneuploidy screening challenges the quality of maternal health services in each country [46, 47].

Finally, some limitations of this study need to be explained. Studies have shown an association between paternally inherited risk factors and adverse pregnancy outcomes [48]. Paternal information was not collected in this study, which may have confounded the results to some extent. The results of this study are not representative of the overall level of screening in mainland China. The service area mainly covers the whole of southern and northern Jiangsu province and parts of Anhui province. This is one of the most developed and modernized regions in mainland China. Again, the results of this study are not representative of the incidence of fetal aneuploidy in the region. The sample is influenced by the actual clinical practice and guidelines for prenatal aneuploidy screening and does not represent the findings of the whole or randomly selected population in the region.

Our results reveal more details about maternal risk factors that influence fetal aneuploidy: ①Pregnant patients with maternal age below 20 years had higher risk of aneuploidy, especially in T13; ②A history of fetal malformations is more risky than RSA, with the former more likely to have T13 and the latter more likely to have T18. In addition, we conducted a clinical evaluation of prenatal aneuploidy screening: ③Primary screening essentially achieves the goal of identifying a normal karyotype, and NIPT can accurately screen for fetal aneuploidy; ④A number of maternal risk factors may influence the accuracy of NIPT diagnosis, including older age, premature testing, or a history of IVF-ET.