Genetic testing in recurrent pregnancy loss: when and which

About 50% of sporadic miscarriages are caused by genetic abnormalities, which can be divided into chromosomal or monogenic.
Chromosomal abnormalities are absolutely the most frequent cause of miscarriage and can concern both the number of chromosomes (aneuploidies) or their structure (structural abnormalities). According to a recent literature review carried out by Papas et al. (2021), autosomal trisomies due to meiotic nondisjunction during gametogenesis are the most frequent cause, being present in about 60% of cases. Among these, chromosome 16 is the most involved in early miscarriages, while chromosomes 21, 18 and 13 in the late ones. Among monosomies, the ones involving the X chromosome are the most frequent, being present in about 20% of cases. Uniparental diploidy is fairly rare, while polyploidies are present in about 20% of cases.

For this reason, it is necessary to make some observations: first of all, most of these genetic abnormalities are de novo, that is, they are identified for the first time in the embryo or fetus and have not been inherited from either parent. In a very low percentage of recurrent pregnancy loss (4-12%), one of the parents is a carrier of a balanced chromosomal abnormality (almost always a translocation or an inversion) and similarly, these structural abnormalities are identified 5-10 times greater in couples with RPL than in the general poplution. Although the identification of balanced chromosomal abnormalities in the parents may be one of the main contributing factors to the history of recurrent miscarriage, the latter cannot be considered the sole cause. This hypothesis is supported by the fact that the identification of chromosomal abnormalities in the products of conception occurs with the same frequency both in cases of spontaneous abortion and in cases of recurrent abortion (PMID: 32417200).

Other less common genetic factors associated with RPL are placental confined mosaicism, skewed X inactivation, copy number variations and monogenic diseases.

When we talk about genetic testing in RPL, it is necessary to elucidate the starting material. In some cases, it is preferable to conduct the genetic test directly on the abortive material, also called “product of conception” (POC). In other cases, or when the POC is not available, it is possible to do the genetic test on the parents.

Among the various types of genetic tests that are used in the context of RPL we find:

1. Karyotype. The karyotype remains one of the gold standard techniques used in the case of RPL. It can be done both on POC, to identify any chromosomal abnormalities, and on parents, to confirm/deny the presence of unbalanced rearrangements. The karyotype also has some technical limitations: first of all, it requires the use of live cells that must be kept in culture (which can fail in about 30% of cases) and secondly, there is a high risk of maternal contamination (which can lead to false negatives).
2. POC Chromosomal microarray (CMA). Unlike karyotype, this technique uses DNA as starting material and does not require the use of cell cultures. Furthermore, it allows to distinguish DNA of maternal origin and consequently to reduce false negatives. It thus allows having a higher diagnostic yield. One of the main limitations is that it does not allow the identification of balanced rearrangements.
3. Other techniques such as MLPA and FISH, which are rarely applied, following the clinical evaluation of the specific case.

There is absolutely no classification that makes one technique better than the other. Indeed, each has its own advantages and disadvantages. Sure, karyotype has long been the gold standard for genetic analysis on POC, but in recent years the trend is shifting towards CMA. The latter certainly has a greater sensitivity, which however does not always translate into greater effectiveness. Just think of those cases in which a variant is identified to which it is difficult to assign a clinical interpretation (variants of uncertain clinical significance), which could even cause an unnecessary psychological obstacle from a prognostic point of view.

The use of genetic testing on POC in cases of sporadic miscarriage and RPL is widely debated in the scientific community. According to the European Society of Human Reproduction and Embryology (ESHRE) 2018 guidelines on RPL, genetic testing of POC is not recommended in routine testing, but can be conducted for explanatory purposes and if possible through CMA. Also the parental karyotype, which is not recommended in routine analysis, can be conducted following a personal risk assessment.

However, numerous studies give conflicting information in this regard. For example, according to Frikha and colleagues (2021) it is important to conduct parental cytogenetic analysis in the case of RPL, starting from the second time, to establish the diagnostic procedure and the best therapeutic approach. Or again, according to Papas and colleagues, it is important to carry out the genetic analysis on POC starting from the second miscarriage, not so much because this can influence the therapeutic process, but because knowing the reason for the miscarriage can be of great emotional comfort for parents. In contrast, according to Smits and colleagues (2020), routine POC testing for chromosomal abnormalities should not be performed due to the low probability of recurrence of the same chromosomal aberration and the absence of clinical benefit.