In 1997, after Professor Dannie’sLo of the University of Central Hong Kong published “A significant proportion of cell-freefetal DNA (cffDNA) in the plasma of pregnant women” in Oxford University, related research sprang up and gradually became the academic field of prenataldiagnosis. prenataldiagnosis (PND) has become an academic discipline. In recent years, with the rapid development of nextgeneration sequencing (NGS) instruments, non-invasive prenatal testing (NIPT) has been formally applied in clinical practice as one of the prenatal testing methods for fetal chromosomal abnormalities. Traditionally, the detection of chromosomal abnormalities in fetuses has been done in two steps: prenatal screening and prenatal diagnosis. Prenatal screening involves riskassessment of fetal chromosomal abnormalities in early or mid-pregnancy using non-invasive methods such as maternal biochemistrymarkers and fetal ultrasound markers. For pregnant women with high risk of chromosomal abnormalities, the second stage of prenatal diagnosis is recommended, i.e. amniocentesis or chorionicvillussampling (CVS), which is an invasive test to confirm the normal karyotype of the fetus. The generally accepted prenatal screening has a sensitivity of about 85% and a specificity of 95%; that is, 15% of fetal chromosomal abnormalities are undetectable; and nearly 5% of cases should undergo further invasive diagnostics that may lead to abortion and infection. Nearly 5% of the cases should undergo further invasive prenatal diagnosis that may lead to abortion and infection. NIPT is a non-invasive method of collecting maternal blood and applying NGS to directly analyze plasma DNA from fetal genetic material (rather than indirect biometabolic markers) for common fetal chromosomal abnormalities, including trisomy 21, trisomy 18 and trisomy 13. The sensitivity of NIPT is up to 98% and the specificity is over 99%; therefore, NIPT has been recognized as a progressive alternative to the traditional two-stage fetal chromosomal abnormality testing from prenatal screening to prenatal diagnosis, and is known as a diagnostic level screening test. The basic principle of NIPT is to compare incomplete free DNA fragments (averaging 50-200 bases) in the peripheral plasma of pregnant women, mainly from their own tissue cells and, to a lesser extent, from apoptosis of placental trophoblast cells, with a large database of 36-base nucleotide sequences already established in the NGS instrument, using massivelyparallel The number or frequency of DNA fragments from different chromosomal locations (without distinguishing whether the DNA fragments are from pregnant women or placenta) is calculated and then analyzed in a logical and statistical manner to determine whether the DNA fragments of a specific chromosomal origin exceed the standard. A chromosomal aneuploidy is considered to be a Zscore of more than 3 or normalizedchromosomevalues (NCVs) of more than 4, using the more commonly used analysis procedures. The cellular free fetal DNA fragments in NIPT are mainly from placental cells. During early pregnancy, once the placenta is formed, the trophoblast cells continue to metabolize and die, and DNA fragments continue to flow into the maternal circulation. The half-life of free fetal DNA fragments is only 16 min, and no free fetal DNA fragments from this pregnancy can be found 2 h after delivery. The fetalfraction of cellular free DNA fragments in maternal plasma averaged 10.2% at 10 weeks of gestation, then increased slowly at a rate of 0.11% per week until 20 weeks of gestation, when the fetalfraction increased again at a more pronounced rate. For an accurate NIPT test, the fetalfraction of free DNA fragments in maternal plasma must be at least 4%, which means that most pregnant women can undergo NIPT at 10 weeks of gestation, giving NIPT the advantage of early prenatal testing compared to other prenatal screening or prenatal diagnosis. In addition to the number of weeks of gestation, maternal weight, ethnicity, serum markers, smoking, and karyotype are also factors that affect the fetal ratio, and thus directly or indirectly affect the sensitivity and specificity of the NIPT test. In addition, since the main source of maternal plasma fetal cell free DNA fragments in the NIPT test is the placenta; theoretically, this test should be similar to the chorionic villus sampling in prenatal diagnosis, and therefore a falsepositive or falsenegative karyotype report, similar to the chorionic villus sampling, may occur. Preliminary statistics show that the false positive rate of NIPT is about 0.1-0.2%. However, it is unknown what proportion of the inconsistency between NIPT results and fetal karyotype results can be attributed to the statistical analysis scheme of the operators in terms of attribution between chromosomal haploid (euploidy) and chromosomal aneuploidy, and what proportion can be attributed to differences in their genetic interpretation due to the sensitivity of MPS sequencing. Confined placental mosaicism (CPM) is often considered to be one of the causes of false positive NIPT. Approximately 1% to 2% of first-trimester placentas present with CPM status. In some case reports, CPM detected by chorionic villus sampling is considered to be associated with false positive NIPT results.Mennuti et al. presented two cases of CPM associated with NIPT results for trisomy 13, in one of which only trisomy 13 was later detected by chorionic villus sampling; in the other case, chorionic villus sampling showed 46,XX,+13, der(13,13) (q10;q10), while being consistent with NIPT results. Hall et al. suggested that in the case of NIPT results suggestive of chromosome 13 trisomy, interphase nuclear fluorescence in situ hybridization (FISH) and conventional chromosome culture analysis showed chromosome 13 trisomy chimerism (47,XY,+13/46,XY). In this case, the fetus showed growth retardation at birth, but did not show signs of chromosome 13 trisomy, and the newborn blood chromosome analysis showed a normal karyotype, 46,XY; the placental chromosome examination showed abnormalities in two of the four tested sites, so it was a chromosome 13 trisomy chimerism. Another interesting case is uni-parental diploidy (UPD), in which the same pair of chromosomes is preserved from one of the two parents due to the disomicrescue mechanism. In a case of NIPT presenting with chromosome 21 trisomy, QF-PCR at 14 weeks of gestation was performed to examine seven short tandem repeats (STR) on chromosome 21, which were found to be from the mother’s chromosome 21 UPD. UPD, and the other three tests were for chromosome 21 trisomy. CPM is often reported in combination with fetal growth retardation; therefore, once CPM is suspected or confirmed in this one case after NIPT testing, a detailed serial fetal ultrasound should be scheduled prenatally for fetal biophysiological assessment, and after birth for ongoing weight and growth curve assessment.CPM is also often associated with chromosomal preservation mechanisms leading to a UPD scenario, and thus The UPD karyotype is one of the scenarios that should be considered when the NIPT test results are inconsistent with the fetal karyotype. In addition, in cases where NIPT test results are not reflected in the conventional karyotype, or where the results are inconsistent, microarray testing may further solve this mystery. In addition to the three major chromosomal trisomies, other chromosomes can also detect CPM conditions. In a large study of pregnant women in a Chinese population, one case of NIPT was suggestive of multiple chromosomal aneuploidy: 47,XXY, chromosome 21 trisomy and chromosome 17 trisomy. In another case, NIPT results suggested trisomy 22; after delivery, the newborn showed normal chromosome 22 double in cord blood karyotype and chromosome 22 trisomy in three sites of placental karyotype. After birth, the fetus showed growth retardation, but there were no abnormal signs of chromosomal abnormalities. When inconsistent results of NIPT and fetal karyotype occur, maternal physiological factors should be considered as well. Such conditions include maternal chromosomal aneuploidy, which usually occurs with sex chromosome abnormalities, or conditions with intrinsic genetic alterations, such as solidtumor. The first case report of maternal sex chromosome aneuploidy (SCA) occurred in a normal 25-year-old pregnant woman with NIPT results suggestive of X-chromosome trisomy. Further amniotic fluid karyotyping showed a normal 46,XX and the neonate was born with a normal presentation. Further follow-up of the maternal blood karyotype revealed a full 47,XXX, an example of the considerable variability in the expression of sex chromosome aneuploidy. In another case of a 44-year-old woman with a sex chromosome abnormality chimerism, the NIPT test results showed an abnormal X chromosome ratio that did not match the X chromosome ratio of a previously diagnosed 45,X case at this testing center. Further karyotyping of the pregnant woman revealed that she was 45,X/46,XX and the newborn was born with a normal karyotype. Wang et al. developed a rapid karyotype analysis using the MPS method while testing maternal leukocytes to assess maternal sex karyotype chimerism when NIPT results were inconsistent with SCA. Of the 181 cases that tested positive for SCA by NIPT, further analysis revealed that 16 (8.6%) were due to maternal X-chromosome chimerism. It can be speculated that SCA, especially chimeric, can lead to inconsistent results with the fetus in NIPT testing, which may be underestimated in clinical practice. Application of NIPT for interpretation as SCA should provide adequate discussion prior to testing and complete genetic counseling after testing. Another source of DNA that may produce interference and lead to inconsistent NIPT results is the maternal parenchymal tumor. In the actual case presented, NIPT was performed at 13 and 17 weeks of gestation, and the results suggested chromosome 13 trisomy and chromosome 18 monosomy, and the fetal amniotic fluid karyotype showed normal 46,XY. The case was examined after delivery for pelvic pain and metastatic neuroendocrine malignancy was found in the pelvic cavity, which was later diagnosed as smallcellcarcinoma of vaginal origin. Fluorescence in situ heterozygous staining of the cancer cells revealed that most of the cancer cells (80%) had higher fluorescence intensity on chromosome 13 than on chromosome 18, which is consistent with the results of NIPT. It is not clear what proportion of cancers in pregnant women will be reported with abnormal NIPT, but the possibility of multiple chromosome aneuploidy on NIPT should not be overlooked in clinical practice. There are few reported cases of false-negative results due to inconsistent karyotypes of NIPT and chromosomes, but in a few reported cases, it may be related to improper handling of the test, fetal ratio of free DNA fragments in maternal plasma, or chimerism. Currently, the clinical and commercialization mechanisms of NIPT are developing rapidly and it has gradually become one of the routine prenatal tests performed by obstetricians. higher than the actual incidence. In addition, the NIPT test unit did not provide quantitative analysis data to the clinical end for regression analysis of the background of false positive and false negative cases. In the future, the application of NIPT may be extended from the current high-risk group to all low-risk groups, and it is expected that the false-positive status of the NIPT test will be more significant in this scenario. One of the possible solutions is to jointly establish a “Non-invasive Prenatal Testing Case Registration and Tracking System” with the testing and clinical partners. Through this registration and tracking system, NIPT positive cases are compared with karyotype results, while NIPT negative cases are compared with clinical results through information provided by clinicians and birth notification records. The registration tracking system also reveals the false-positive and false-negative rates of NIPT through surveys and analyses. For confirmed false-positive NIPT cases, further CPM, UPD and gene microarray tests can be performed. The data from the unit analysis, the fetal ratio of free DNA fragments in maternal plasma, and the collection of maternal and neonatal data can be used to further investigate the true causes of false-positive NIPT and to improve the sensitivity and specificity of NIPT testing.