Scientific Papers

What proportion of couples with a history of recurrent pregnancy loss and with a balanced rearrangement in one parent can potentially be identified through cell-free DNA genotyping? | Molecular Cytogenetics

Balanced (reciprocal) translocations are reported in a significant proportion of couples with RPL. Although it is thought that Robertsonian translocations are common, we showed that reciprocal translations with unique breakpoints (68.2% of all aberrations) are more often detected. Therefore a genome-wide analysis needs to be performed to detect those unique structural variants. Although new molecular methods are emerging, at this moment the only widely implemented technique to detect balanced translocations and inversions in clinical settings is laborious karyotyping. To ensure the efficiency of testing, clinicians attempted to select couples for karyotyping. However, a recent review found insufficient evidence for a difference in the frequency of abnormal karyotyping results between women with two and three or more pregnancy losses [12]. As karyotyping is a laborious method, we searched for an alternative high throughput method to identify potential carriers after recurrent miscarriages have been diagnosed, ideally a method that could also be used for prenatal screening in future pregnancies.

Our study shows that the vast majority of couples carrying a structural chromosome aberration could potentially be identified if cfDNA genotyping (genome-wide NIPT) was employed at the time of pregnancy loss after 8 weeks of pregnancy: 98.7% of couples carried a balanced aberration potentially causing at least one imbalance larger than 10 Mb. However, although adjacent 1 and 2 segregation patterns are most common, in clinical settings, while assessing individual patients’ elibility for the NIPT test one needs to take also 3:1 mal segregation into account. Especially if an acrocentric chromosome is involved, it may produce smaller imbalance than the typical adjacent segregation pattern.

The feasibility to detect unbalanced translocations through cfDNA (NIPT testing) has already been shown by several authors [7, 8, 13, 14]. However, a risk of missing an unbalanced aberration due to NIPT limitations such as too low fetal fraction (sometimes due to high BMI) and limited resolution should be included in the pre-test counseling if NIPT was offered for this purpose.

So far, there have been only a few studies that investigated cell-free DNA testing in nonviable gestations. Several studies showed that cell-free fetal DNA persists in the maternal plasma when the gestation remains in situ, possibly as a result of continuous placental apoptosis after fetal demise [15,16,17]. Therefore, blood sampling for cfDNA genotyping at that point is a good alternative for identifying the chromosomal aberrations causing pregnancy loss.

Additional benefits of NIPT testing in miscarriage

Another argument for performing NIPT in case of a miscarriage is the fact that it would not only lead to the identification of potential balanced aberration carriers, but it will also identify other chromosomal causes of early miscarriage. Chromosomal abnormalities are found in ca. 50% of fetal deaths at less than 20 weeks of gestation and 6–15% at more than 20 weeks of gestation [18,19,20,21]. The American Society of Reproductive Medicine recommends cytogenetic evaluation of the products of conception in women with two or three spontaneous miscarriages [22]. Still, so far there is no such recommendation in the Netherlands. Detecting the cause of fetal loss may not only prevent other unnecessary testing in case of RPL [14], but it will probably also positively contribute to the psychological coping process after RPL [23]. It has been shown that an abnormal embryonic karyotype is the most frequent cause of recurrent miscarriage and the true unsolved cases can be limited to about 25% of the couples with RPL [24]. Peng et al. recently showed that in comparison to current practices of cytogenetic testing of products of conception (POCs), cfDNA testing allowed not only for a lower cost per patient but for better sample accessibility as well: blood sampling for cfDNA test immediately after diagnosing a non-viable pregnancy is possible. In addition, using cfDNA can potentially reduce the number of patients undergoing unnecessary workups resulting in overall cost savings [14]. The study of Peng and colleagues showed that the inclusion of cfDNA testing is a cost-effective approach to guide RPL workup and not only to identify balanced translocation carriers.

NIPT in future pregnancies

Finally, an additional advantage is that a cfDNA test (NIPT) can be employed in future pregnancies as an alternative to invasive testing, when a couple was shown to carry a balanced chromosome aberration that NIPT can detect if the fetus is unbalanced. We have previously shown that unbalanced translocations are detectable in maternal plasma cfDNA, and assessed that imbalances larger than 10 Mb should be detectable if the fetal fraction is sufficiently high [8].

Genome-wide NIPT testing in future pregnancies could be recommended to couples with RPL to investigate the presence of unbalanced translocation/inversion, even if testing of a previous pregnancy loss failed or cfDNA test was unavailable or failed due to too low fetal fraction. Additionally, it should be noted that the NIPT test is a high throughput technique where patient samples can be run in multiplex reactions, whereas parental karyotyping is still a rather laborious process, resulting in more expensive hands-on time per patient than in case of NIPT analysis. We anticipate that testing the pregnancy loss with NIPT instead of directly karyotyping both partners in cases of RPL might be more (cost)efficient [14]. It will allow selecting only those parents at risk for being a carrier instead of karyotyping all parents with RPL. Moreover, it will help to diagnose the etiology of pregnancy loss in more cases [25]. However, it should be noted that high throughput SNP microarray testing in POC is the method of choice if the miscarriage tissue is available and of desired quality.

Limitations of the NIPT approach

The conclusions of this study are based on the theoretical assessment of the size of the chromosomal segments, irrespective of viability of the different unbalanced patterns Many segregation products will have very large imbalances that would be lethal and not reach the gestational age when NIPT can be performed. The cfDNA test depends on the moment of the blood collection and the fetal cfDNA fraction present. It should be drawn as soon as fetal death is diagnosed when the pregnancy is still in situ. Otherwise, some patients may be too late for blood collection. Some patients who experience a miscarriage before 8 weeks of gestation may not benefit from the NIPT approach as before 7 weeks of gestation the fetal fraction of cfDNA is more likely to be insufficient [16]. Although most spontaneous miscarriages are detected between 8 and 13 weeks of gestation, couples with recurrent miscarriages are very aware of becoming pregnant and early monitoring can lead to earlier detection of the miscarriage [16]; therefore, blood can be drawn at the moment of diagnosis before the products of conception are discharged [5, 26]. Colley and colleagues have shown that in 66% of samples before 7 weeks of gestation their cfDNA test provided successful diagnosis [16]. cfDNA testing cannot replace karyotyping or microarray in fetal tissue, however it has potential to detect the cause of miscarriage in couples with RPL. Potentially in about 6% of patients a retention of the trophoblastic tissue can be observed [27], however, to our knowledge there are no reports on usefulness of cfDNA in such cases. In this study, we analyzed only the size of the theoretical, potential imbalances and the possibility of its detection assuming the blood sample contains enough fetal cfDNA. Large prospective cohort studies are necessary to establish how many patients cannot benefit from the cfDNA test, either because the sampling is performed too late or the miscarriage occurred before 8 weeks of pregnancy and insufficient fetal fraction did not allow to achieve NIPT results.

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