Little is known about the sensitivity of NIPT for detection of CPM. Brison et al (2018) found evidence that NIPT is more sensitive for the detection of placental mosaicism due to the observation of a higher proportion of mosaicism for the common aneuploidies with NIPT as compared to conventional karyotyping.1 In contrast, Benn et al2 showed a significantly lower sensitivity for the detection of rare autosomal aneuploidies (RATs), mostly involved in CPM, for NIPT (0.32%) vs CVS (0.41%). The use of study cohorts with probably different a priori risk figures for CPM, may explain the conflicting results of both studies. Moreover, whereas placental studies shed some light on the correlation between cytogenetic results of CVS and those from term placentae, little is known about how cytogenetic results of NIPT relate to those from CVS and placenta. Papers on placental cytogenetic studies after NIPT are rare and amniocentesis is generally the preferred technique for confirmatory diagnostic testing after an abnormal NIPT result. Potential detection of chromosomal mosaicism in CV, which may require an undesired second invasive procedure for clarification of the fetal karyotype, may discredit CVS. However, if both cell layers of CV (CTB and mesenchymal core [MC]) are investigated separately, thus enabling differentiation between their respective chromosomal constitution, the risk of a confirmatory amniocentesis after CVS is predicted to be low for the common trisomies (eg, trisomy 21 (2%), trisomy 18 (4%), and trisomy 13 (8%‐22%)3, 4). On the contrary, if NIPT indicates another trisomy, CPM is the most likely reason for this result. In such cases CVS for confirmation is only recommended for RATs that are mostly involved in CPM type 1, like trisomy 3, 7, 8, 9, 20.3 In all other cases, amniocentesis is indeed the preferred confirmatory test. Hence little is known about the representation of the placental chromosomal constitution in the cfDNA fraction in cases of CPM and about the sensitivity of NIPT to detect it. It is assumed that the entire placental trophoblast sheds cfDNA into the maternal circulation and that a CPM restricted to smaller placental areas may be detected by NIPT and missed by CVS.1 However, as far as we know, there are no studies comparing NIPT, CVS and placenta cytogenetic data. Based on four cases with normal CVS results after genome‐wide (gw) NIPT revealed a RAT, we show evidence that NIPT is better able to detect (low‐level) placental mosaicism involving the cytotrophoblast than CVS.

Genome‐wide NIPT was performed as part of the Dutch Trident 2 study (Trident = Trial by Dutch laboratories for Evaluation of NIPT), using shallow massively parallel sequencing and WISECONDOR for analysis.5 The four cases presented here involved one case of trisomy 5 and trisomy 7 and three cases of trisomy 8. According to our local protocol, a CVS was recommended, which was performed transabdominally in all cases. Cytogenetic investigations of first trimester CV were performed with SNP array (Illumina Infinium GSA + MD‐24 v1.0 BeadChip genotyping array) on DNA isolated from the CTB and MC that were separated as described previously.6 Maternal genomic DNA was investigated as well to exclude a maternal origin of the chromosomal aberration. In all four cases, a normal result was achieved in CV (both CTB and MC) and maternal blood. The test characteristics of NIPT (gestational age (GA), fetal fraction (FF) (SeqFF)7 and z‐score (chromosome‐wide aneuploidy test [CWAT]8) and CVS (GA and amount of CV) are shown in Table 1. Since maternal genomic DNA was normal in all cases, a diagnosis of CPM was most likely, despite normal CV results. After birth, we collected the placentae and performed cytogenetic analysis of four CV biopsies from four quadrants, with methods described for first trimester CV (Table 1). In all cases, the chromosomal aberration was confirmed in the term placenta. In two cases, it was present only in one of four biopsies, involving a 100% trisomy 5 and trisomy 7 in case 1 (Figure 1), but a very low level mosaic in case 2. The presence of only 10% abnormal cells in one biopsy in case 2 was sufficient to lead to an abnormal NIPT‐result. However, sampling of only 4 × 1 cm³ biopsies does not exclude higher levels of trisomic cells elsewhere in the placenta. In cases 3 and 4, a 100% trisomy was present in two of the four biopsies, while first trimester CV showed normal results, confirming the nonrepresentativity of first trimester CV for the placenta as a whole, as illustrated in the past. Our study shows a higher sensitivity of NIPT for detection of CPM involving the cytotrophoblast as compared to CVS.

A striking observation is that whereas the results in placental studies of cases 3 and 4 were comparable, z‐scores of the NIPT were much higher in case 4 (32.6) as compared to case 3 (9.5). This may be partly due to a higher FF (11.6% in case 4 vs 7.5% in case 3), however, other factors may be involved that can explain this difference. Firstly, only 4 biopsies of 1 cm³ were investigated leaving the largest part of the placenta uninvestigated, which may contain much higher levels of trisomic cells in case 4 as compared to case 3. Secondly, it is also possible that apoptotic activity in the affected placental parts in case 4 is much higher than in case 3, leading to a higher trisomic cfDNA fraction. In order to get more insight into the representativeness of NIPT for the placenta anomaly, further studies are necessary and preferably should involve more than four placental biopsies.

In three of the four cases, all involving CPM type 1 with the chromosome aberration restricted to the CTB, children without congenital anomalies and with appropriate birth weights were born, as can be expected for this CPM type. Clinical outcome data are shown in Table 1. In case 3 with the chromosome aberration present in both cell layers, a premature delivery due to a rupture of membranes occurred at 20 1/7 weeks. Unfortunately, no cord blood could be obtained and therefore, a fetal trisomy 8 could not be excluded.

CPM is associated with an increased risk for preterm birth, small for gestational age newborns, and adverse pregnancy outcomes.9 This association especially exists for CPM type 3, mostly of meiotic origin, in which both CTB and MC of first trimester CV are affected, often with high percentages of abnormal cells, and less for CPM type 1 (only CTB affected) and type 2 (only MC affected).9 Discrimination of the various types of CPM is only possible when both the CTB and MC of CV are investigated. When both cell layers are affected with high levels of abnormal cells, while the fetus is chromosomally normal, proper clinical follow‐up investigations like expert ultrasound can be recommended. However, if potential CPM is detected with NIPT, no differentiation between CPM type 1 and 3 is possible since only the CTB is investigated with NIPT. Also, little is known about the extent of the distribution of abnormal cells over the placenta when NIPT reveals CPM. Moreover, as shown in this paper, NIPT seems to be very sensitive for detection of CPM, even if restricted to a small area of the placenta, with probably less clinical consequences. This all complicates predictions on the clinical relevance of CPM when detected with NIPT. Further research is necessary in order to learn to differentiate clinically relevant CPM from benign CPM. Recently, Pertile et al (2017) and Brison et al (2018) found an association between trisomic fraction and pregnancy outcome. When a trisomic fraction as compared to fetal fraction was low, pregnancy outcome was favorable and if trisomic fraction was high there was an increased risk for adverse outcome such as miscarriage, intrauterine fetal death, intrauterine growth retardation1, 10 This shows that the trisomic fraction may be a good indicator for aneuploidy‐load in the placenta, and its calculation probably may improve clinical guidance of the pregnancy.1

In conclusion, the present study shows that NIPT seems to be more sensitive than CVS for the detection of CPM involving the cytotrophoblast. However, the ability of NIPT to detect a low level mosaic restricted to a small placental part will probably be dependent on the FF. This study also gives more insight into the representation of CPM in the cfDNA fraction of maternal blood. However, more studies are necessary to understand the correlation between NIPT z‐scores/trisomic fraction and level and distribution of mosaicism in the placenta in order to learn to predict the clinical consequences of CPM, when detected with NIPT.

关于 NIPT 检测 CPM 的敏感性知之甚少。Brison 等人 (2018) 发现证据表明 NIPT 对胎盘嵌合的检测更敏感，因为与常规核型分析相比，NIPT 观察到的常见非整倍体的嵌合比例更高。1相比之下，本恩等人2NIPT (0.32%) 与 CVS (0.41%) 在检测罕见常染色体非整倍体 (RAT)（主要与 CPM 相关）方面的灵敏度显着降低。使用具有可能不同的 CPM 先验风险数据的研究队列，可以解释这两项研究的相互矛盾的结果。此外，虽然胎盘研究揭示了 CVS 细胞遗传学结果与足月胎盘细胞遗传学结果之间的相关性，但对 NIPT 细胞遗传学结果与 CVS 和胎盘细胞遗传学结果之间的关系知之甚少。关于 NIPT 后胎盘细胞遗传学研究的论文很少见，羊膜穿刺术通常是 NIPT 结果异常后进行确认性诊断测试的首选技术。潜在检测 CV 中的染色体嵌合体，这可能需要不希望的第二次侵入性程序来澄清胎儿核型，可能会诋毁 CVS。然而，如果分别研究 CV 的两个细胞层（CTB 和间充质核心 [MC]），从而能够区分它们各自的染色体构成，对于常见的三体性（例如， 21 三体 (2%)、18 三体 (4%) 和 13 三体 (8%‐22%)3、4）。相反，如果 NIPT 指示另一个三体性，则 CPM 是导致此结果的最可能原因。在这种情况下，仅建议对主要涉及 CPM 1 型的 RAT（如 3、7、8、9、20 三体）进行 CVS 确认。3在所有其他情况下，羊膜穿刺术确实是首选的确认试验。因此，对于 CPM 情况下 cfDNA 部分中胎盘染色体构成的表示以及 NIPT 检测它的敏感性知之甚少。假设整个胎盘滋养层将 cfDNA 释放到母体循环中，并且限制在较小胎盘区域的 CPM 可能会被 NIPT 检测到，而被 CVS 忽略。1然而，据我们所知，目前还没有比较NIPT、CVS和胎盘细胞遗传学数据的研究。基于在全基因组 (gw) NIPT 显示 RAT 后具有正常 CVS 结果的四个病例，我们显示证据表明 NIPT 能够比 CVS 更好地检测涉及细胞滋养层的（低水平）胎盘嵌合。

全基因组 NIPT 是荷兰三叉戟 2 研究（Trident = 荷兰实验室评估 NIPT 的试验）的一部分，使用浅层大规模平行测序和 WISECONDOR 进行分析。5这里介绍的 4 例病例涉及 1 例 5 三体和 7 三体以及 3 例 8 三体。根据我们当地的方案，建议进行 CVS，所有病例均经腹进行。使用 SNP 阵列（Illumina Infinium GSA + MD-24 v1.0 BeadChip 基因分型阵列）对从 CTB 和 MC 分离的 DNA 进行细胞遗传学研究，这些 DNA 如前所述分离。6还研究了母体基因组 DNA，以排除染色体畸变的母体起源。在所有四种情况下，CV（CTB 和 MC）和母血的结果均正常。NIPT（胎龄 (GA)、胎儿分数 (FF) (SeqFF) 7和 z 分数（全染色体非整倍体测试 [CWAT] 8）的测试特征) 和 CVS（GA 和 CV 量）如表 1 所示。由于母体基因组 DNA 在所有病例中均正常，因此尽管 CV 结果正常，但最有可能诊断为 CPM。出生后，我们收集了胎盘并对来自四个象限的四个 CV 活检进行了细胞遗传学分析，方法描述了早期妊娠 CV（表 1）。在所有情况下，染色体畸变在胎盘一词中得到证实。在两种情况下，它仅出现在四次活检中的一次中，在病例 1 中涉及 100% 的 5 三体和 7 三体（图 1），但在病例 2 中存在非常低水平的镶嵌。仅存在 10% 的异常细胞病例 2 中的一次活检足以导致 NIPT 结果异常。然而，取样仅 4 × 1 cm ³活组织检查不排除胎盘其他地方更高水平的三体细胞。在案例 3 和案例 4 中，四次活检中有两次出现 100% 三体性，而孕早期 CV 显示正常结果，证实了孕早期 CV 对整个胎盘的非代表性，如过去所示。我们的研究表明，与 CVS 相比，NIPT 检测涉及细胞滋养层的 CPM 具有更高的灵敏度。

一个惊人的观察结果是，虽然案例 3 和案例 4 的胎盘研究结果具有可比性，但案例 4 的 NIPT 的 z 分数 (32.6) 与案例 3 (9.5) 相比要高得多。这可能部分是由于较高的 FF（案例 4 中的 11.6% 与案例 3 中的 7.5%），但是，可能涉及其他可以解释这种差异的因素。首先，只有 4 个 1 cm ^{3 的}活组织检查调查了胎盘的最大部分未调查，与案例 3 相比，案例 4 中可能包含更高水平的三体细胞。 其次，案例 4 中受影响胎盘部分的凋亡活性也可能远高于在情况 3 中，导致更高的三体 cfDNA 分数。为了更深入地了解 NIPT 对胎盘异常的代表性，进一步的研究是必要的，最好应该包括四个以上的胎盘活检。

在四个案例中的三个，都涉及 CPM 1 型，染色体畸变仅限于 CTB，没有先天性异常且出生体重适当的儿童出生，这对于这种 CPM 类型是可以预期的。临床结果数据显示在表1中。在两个细胞层中都存在染色体畸变的情况3中，由于胎膜破裂导致的早产发生在20 1/7周。不幸的是，无法获得脐带血，因此不能排除胎儿 8 三体性。

CPM 与早产、小于胎龄新生儿和不良妊娠结局的风险增加有关。9这种关联尤其存在于 CPM 3 型，主要是减数分裂起源，其中早期妊娠 CV 的 CTB 和 MC 都受到影响，通常异常细胞百分比很高，而 CPM 1 型（仅 CTB 受影响）和 2 型则较少（仅 MC 受影响）。9只有在调查 CV 的 CTB 和 MC 时，才能区分各种类型的 CPM。当两个细胞层都受到高水平异常细胞的影响，而胎儿染色体正常时，建议进行适当的临床随访检查，如专家超声检查。然而，如果使用 NIPT 检测到潜在的 CPM，则不可能区分 CPM 类型 1 和 3，因为使用 NIPT 仅调查 CTB。此外，当 NIPT 显示 CPM 时，对胎盘上异常细胞分布的程度知之甚少。此外，如本文所示，NIPT 似乎对 CPM 的检测非常敏感，即使仅限于胎盘的一小部分区域，临床后果可能较少。当用 NIPT 检测到时，这一切都使对 CPM 临床相关性的预测变得复杂。为了学会区分临床相关的 CPM 和良性 CPM，进一步的研究是必要的。最近，Pertile 等人 (2017) 和 Brison 等人 (2018) 发现三体分数与妊娠结局之间存在关联。当三体分数与胎儿分数相比较低时，妊娠结果是有利的，如果三体分数较高，则不良结果的风险会增加，例如流产、宫内胎儿死亡、宫内发育迟缓1, 10这表明三体分数可能是胎盘中非整倍体负荷的良好指标，其计算可能会改善妊娠的临床指导。1

总之，本研究表明 NIPT 似乎比 CVS 对检测涉及细胞滋养层的 CPM 更敏感。然而，NIPT 检测仅限于小胎盘部分的低水平马赛克的能力可能取决于 FF。这项研究还让我们更深入地了解 CPM 在母血 cfDNA 部分中的表现。然而，需要更多的研究来了解 NIPT z 分数/三体分数与胎盘中嵌合水平和分布之间的相关性，以便学习预测 CPM 的临床后果，当用 NIPT 检测时。