Cytogenetic prenatal diagnosis on chorionic villi (CV) can be complicated by the detection of “chromosomal mosaicism.” This is one of the main issues of first-trimester cytogenetic prenatal diagnosis as it can involve different types of chromosomal abnormalities, and the prediction of the fetal involvement is challenging because the detected abnormal mosaic cell line is not necessarily extended to fetal tissues. In addition, because the cell-free fetal DNA that is targeted by the new technologies for fetal aneuploidy risk assessment is mainly derived from the CV cells, the same challenges related to chromosomal mosaicism can be transferred into this new clinical field. This review illustrates the phenomenon of fetoplacental mosaicism, the management of prenatal diagnosis cases complicated by the detection of such a biological phenomenon, and the implications of its presence for the management of high-risk cfDNA testing results for fetal aneuploidies.
Highlights
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Fetoplacental chromosomal mosaicism is one of the main issues in the first-trimester cytogenetic prenatal diagnosis on CV.
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It has implications on the interpretation and management of cfDNA testing results.
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Provided risk figures can be utilized as guidance during the post-test counseling after the detection of a mosaicism in CV and during pre- and post-test cfDNA testing counseling.
Introduction
First-trimester cytogenetic prenatal diagnosis on chorionic villi (CV) can be complicated by the detection of the presence of two or more cell lines with different karyotypes derived from a single zygote. This condition is known as “chromosomal mosaicism” . This biological phenomenon is observed in approximately 2% of the analyzed CV samples and can involve different types of chromosomal abnormalities (numerical and/or structural abnormalities and typically trisomies). In addition, the abnormal mosaic cell line detected in CV cytogenetic analysis is not necessarily extended to fetal tissues; consequently, the prediction of the fetal involvement is challenging .
The aim of this review is to
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illustrate prenatal diagnosis by CV cytogenetic analysis;
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describe how chromosomal mosaicism can arise during fetoplacental development and why this biological phenomenon is a common finding during the cytogenetic analysis of CV;
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provide biological motivations for follow-up amniocentesis after the detection of mosaic in CV;
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present the overall frequencies of the six different combinations of fetoplacental mosaicisms after the sequential analysis of the amniocytes in cases with mosaic in CV;
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report the predicted risks of fetal confirmation when different combinations of placental layers are detected to be affected by different types of abnormal cell lines;
- vi.
discuss the implications of the presence of a fetoplacental mosaicism on cfDNA testing result;
- vii.
provide the algorithms for the use of cytogenetic analysis of CV and confirmatory amniocentesis in prenatal diagnosis and of cfDNA testing.
Mechanisms of formation of chromosomal mosaicism
Chromosomal mosaicism is a mitotic phenomenon generated by two principal mechanisms, both involving a postzygotic event:
A) the postzygotic mitotic nondisjunction in a somatic cell of an euploid conceptus: this mitotic error generates two daughter cells, one with 47 chromosomes (trisomic) and the other one with 45 chromosomes (monosomic). After this event, the chromosomal mosaicism starts with three different cell lines: the original euploid and the two generated trisomic and monosomic ones. However, they quickly reduce to a two-cell-line mosaicism as autosomal monosomy is almost universally lethal during cell development. For sex chromosomes, all three cell lines are instead tolerated as monosomy X is less subjected to negative selection ;
B) the postzygotic mitotic trisomy rescue after a meiotic nondisjunction: this mechanism is a biological attempt to “recover” a trisomic conceptus generated after the fertilization of an abnormal disomic gamete, usually an oocyte, by a normal sperm. This rescue is performed through the loss of one supernumerary chromosome in a somatic cell, restoring a disomic cell line .
Embryo-fetal tissue differentiation begins in the early postfertilization stages; the fetus itself is likely to be derived from only a small subset (3 out of 64) of the blastocyst progenitor cells. The remaining cells are believed to give rise to extraembryonic structures . The distribution of the different cell lines (the normal and abnormal ones) in the fetus and the placenta is dependent not only on the type of chromosome mechanism but also on its timing and embryo-fetal localization: if it occurs before the embryological separation between fetal and extrafetal compartments, the chromosomal mosaicism will likely be generalized to both the placenta and fetus; if it occurs after this stage, it will likely be confined to only one of them .
Cytogenetic analysis of CV and confirmatory sequential amniocentesis: when and why
During the conventional cytogenetic analysis of CV, both placental layers are usually analyzed: direct preparation (or short-term incubation) and long-term culture (LTC) allow the karyotyping of the cytotrophoblast and of the mesenchymal core, respectively. The cytotrophoblast is an epithelial tissue localized immediately under the syncitiotrophoblast, which is the most external multinucleated CV layer. The mesenchyme is the stromal tissue located in the core of the CV containing the capillary endothelial cells. The most accredited hypothesis on their embryological origin is that the cytotrophoblast derives from a trophoblastic precursor generated immediately after the first division of the initial fertilized egg and the mesenchyme from the yolk sac derived from hypoblast of the inner cell mass .
During CV analysis, the biopsy is divided into two aliquots after careful inspection under an inverted microscope: from this point, the two specimens will take two different routes of analysis. The karyotype results coming from each protocol will be then unified in the final report. Direct preparation is performed after simple incubation of the native CV. The cytotrophoblast cells are in a phase of active mitotic division, so only a short incubation (of 24 to 48 h) is needed to visualize metaphase spreads. Chromosome preparations are obtained after a short treatment with 60% aqueous acetic acid solution. This treatment dissociates the cells of the villus, thus freeing the spontaneous metaphases that can be scored for karyotyping. As the cytotrophoblast is not cultivated, this analysis is not susceptible to maternal cell contamination due to the growth in culture of maternal decidua cells that can remain adherent to the seeded CV fragments . The LTC starts with enzymatic treatments with pronase and collagenase to disaggregate the external layers of the CV and free the stromal cells for their cultivation. After 5 to 10 days of cell culture, the mesenchymal cells are available for karyotype analysis . Both the initial fresh or stored CV biopsy and the cultivated mesenchymal cells are excellent sources of consistent amounts of DNA for supplementary molecular testing (rapid molecular tests such as quantitative fluorescent-PCR and prenatal BACs-on-Beads, chromosomal microarray, or sequencing).
After the cytogenetic analysis of the metaphase spreads obtained from the two different protocols/placental layers, the results are reported and interpreted consequently. If both are normal or homogenously abnormal, they are reported as such, and no other cytogenetic investigations are performed. If an abnormal cell line is detected together with a normal 46,XX/XY one, three different types of mosaicism can be recognized, depending on the combination of the tissues containing the affected cells: type I identifies a condition where the abnormal cell line is detected in cytotrophoblast only, type II in mesenchyme only, and type III in both layers ( Figure 1 ).
As the exact embryo-fetal developmental stage in which the mitotic error occurs cannot be established with certainty with karyotyping, the retrieval of a mosaic in CV does not necessarily imply a fetal involvement (true fetal mosaicism, TFM) as it could be restricted to the placenta (confined placental mosaicism, CPM). For this reason, in this situation, a confirmatory karyotype on amniocytes is recommended to discriminate a generalized mosaicism (placenta and fetus affected by the abnormal cell line) from a confined mosaicism (only the placenta affected but not the fetus). Amniotic fluid sampling is considered the standard procedure of confirmation because it provides cells for cytogenetic analysis coming mainly from fetal anatomical districts including the urogenital tract, the respiratory apparatus, and the epithelial system representing different embryological layers . The amniocytes are seeded and cultivated under appropriate conditions for 8-10 days and analyzed for the determination of the fetal karyotype .
Overall frequencies of fetoplacental mosaicisms and risk of TFM
In addition to the clinical reassessment with ultrasound before confirmatory amniocentesis, women shall be counseled about the likelihood that the specific abnormal cell line in CV could also be extended to the fetus (TFM). Herein we present an update of TOMA laboratory survey regarding the risk of TFM that now includes 67030 CV samples; part of it was progressively reported in previous papers . The likelihood of fetal involvement for each individual chromosome abnormality in relation to its specific distribution in CV layers was retrospectively calculated from this survey by extrapolating the largest collection of mosaic CV samples characterized by the sequential cytogenetic analysis of the cytotrophoblast, mesenchyme, and amniocytes.
Out of the 67030 consecutive cytogenetic diagnoses analyzing both placental layers in all samples, 1457 mosaic CV were detected (2.17%). Of them, in 1100 performed a confirmatory amniocentesis that was analyzed by the TOMA laboratory: a TFM was identified in 13.5% of the cases (n=148). These TFMs can be classified into three different types of fetoplacental discordances, all affecting both the fetus and placenta with different CV tissue combinations: TFM IV (1.55%; n=17) and TFM V (5.36%; n=59) with an abnormal cytotrophoblast and abnormal mesenchyme only, respectively; TFM VI (6.55%; n=72) with both placental layers having the abnormal cell line.
In the remaining 952 samples (86.5%), a CPM was identified because amniocytes’ karyotype was normal. Similar to TFMs, the CPMs also can be classified into three different types of fetoplacental discordances, with three different abnormal CV tissue combinations: CPM I (35.73%; n=393) and CPM II (40.45%; n=445) with an abnormal cytotrophoblast and abnormal mesenchyme only, respectively; CPM III (10.36%; n=114) with both placental layers having the abnormal cell line ( Table 1 ).
| Involved embryo-fetal compartments | Type of fetoplacental mosaicism | Corresponding combination of affected tissues | Frequency % (n/total n) | ||
|---|---|---|---|---|---|
| Cytotrophoblast | Mesenchyme | Amniotic fluid | |||
| Placenta only | CPM I | Abnormal | Normal | Normal | 35.73% (393/1100) |
| CPM II | Normal | Abnormal | Normal | 40.45% (445/1100) | |
| CPM III | Abnormal | Abnormal | Normal | 10.36% (114/1100) | |
| Placenta and fetus | TFM IV | Abnormal | Normal | Abnormal | 1.55% (17/1100) |
| TFM V | Normal | Abnormal | Abnormal | 5.36% (59/1100) | |
| TFM VI | Abnormal | Abnormal | Abnormal | 6.55% (72/1100) | |
TFM IV is the rarest type of fetoplacental mosaicism, and to the best of our knowledge, it has been identified and reported only by our group, with 17 cases, and by Battaglia et al. . These 17 TFM IV cases included clinically relevant chromosome abnormalities: 1 case of trisomy (T) 13, 1 case of T21, 1 case of multiple trisomies, 8 cases of monosomy X, 1 case of triple X, 4 cases of small supernumerary marker chromosomes (sSMCs), and 1 case of complex structural chromosome rearrangement ( Table 1 ). Although rare, the detection of the TFM IV cases supports the value of karyotyping the cytotrophoblast, which, however, has been abandoned by many labs worldwide, mainly for practical organizational laboratory needs . Nevertheless, the information collected by the present and other surveys in which the cytotrophoblast is routinely analyzed is a valuable resource not only for the accurate prediction of the fetal involvement after the detection of a mosaic in CV but also for the interpretation of the discordant results provided by the most recent revolutionary prenatal test: the noninvasive testing based on quantification of the “fetal” cell-free circulating DNA in the maternal plasma (also known as cfDNA testing or noninvasive prenatal testing/screening, NIPT/S). The cfDNA fragments targeted by these tests are not derived from the fetus itself but from the apoptosis of the cytotrophoblast . This topic is extensively discussed in the following paragraph (e.g., fetoplacental mosaicisms and the implications on cfDNA testing).
Regarding prenatal diagnosis by CV cytogenetic analysis, the prediction of the fetal confirmation risks in cases with mosaic in CV can be extrapolated from the collected CV experience, not only for the different combinations of the affected placental tissues ( Table 2 ) but also for each individual chromosome abnormality affecting the placental tissue/s with different distributions ( Table 3 ).
