RT carriers are phenotypically normal, but they are known to be at increased risk of repeated miscarriages compared with the general population estimated at about 15% of pregnancies, and also resulting in the birth of a child with congenital anomalies or a mental retardation 19. Preimplantation Genetic Testing (PGT) is therefore a solution for RT carriers. Although the advent of new molecular technologies provided an accurate PGT-A analysis, FISH technique is widely used in PGT-SR and is considered as the technique of choice for structural abnormality detections in embryonic samples 10,14,20-24. The objective of our study was to minimize the risk of transferring a viable unbalanced embryo by adopting a reliable FISH strategy.
Our approach was based on a comparison of the efficiency of two FISH probe strategies. During PGT-SR attempts, similar rates of balanced embryos were observed with two versus three FISH probes used (40.4% and 40.5% respectively). As mentioned, embryos were classified as normal/balanced if the two nuclei showed two distinct signals for each probe tested. We showed clearly that an addition of a third paracentromeric probe didn’t improve the rate of balanced embryos and consequently embryo transfer rates. Several reports had suggested that centromeric probes were tending to give inconclusive results and were more prone to be involved in No Result (NR) than locus specific probes 9. These observations were confirmed by Mir et al. (2010) 10. Moreover, when one or two blastomere cells were analyzed, embryos showed similar confirmation rates of balanced embryos either with two or three probes. The biopsy of two cells did not increase the accuracy of FISH technique. Our results confirm these reported data 10. The authors revealed similar rates of diagnosis between Day 3 and Day 5 when one or two blastomeres had been analyzed on Day 3 and thus, they concluded, that biopsy of two blastomeres did not increase the accuracy of FISH technique.
On the basis of ESHRE recommendations 7,14, balanced embryos are consistently and reliably detected when probes set contain sufficient probes to detect all expected unbalanced forms of rearranged chromosomes. In this study, we showed that two-FISH probes allowed the detection of all imbalances. The addition of the third probe was not essential for PGT-SR analysis and detection of imbalances (Table 2), but mainly to strengthen physician interpretations. Given the results achieved through this study, we might conclude that an accurate diagnosis may be performed on RT carriers with a set of two subtelomeric FISH probes as well as a diagnosis performed with a set of three FISH probes. A single cell biopsy is adequate and will not compromise the ’confidence,’ of the result given by the 2 probes strategy.
During this study, we also observed that rates of mosaicism were ranged from 19.7% to 34.4%. Reassuringly, mosaicism rates performed at a cleavage stage were similar to the intervals cited in many previous studies (24% to 53%) in embryos at risk of Robertsonian translocation 20,25. We thoroughly compared the incidence of mosaic chromosomal abnormalities according to FISH probe strategies. Interestingly, unlike the category of balanced embryos, mosaic embryo results showed different rates (p <0.001) according to FISH probe strategies. Mosaic embryo rates were raised when FISH were performed with 2 probes compared to results obtained with a third paracentromeric probe. Inversely, the rate of unbalanced embryos decreased when diagnosis was performed with two than three FISH probes. We supposed that the addition of a third probe could disturb signal FISH interpretation. Mosaic embryos were more and more considered mainly for couples without euploid embryos 3,12,13,26. Concern about transferring mosaic embryos arises from the apprehension that it could lead to abnormal pregnancies, although the following study showed that transfers of mosaic embryos resulted into healthy live births 27, negative or biochemical pregnancies. Other recent studies by means of NGS based PGT-A performed an analysis of mosaic blastocyst transfers resulting in ongoing pregnancies and lead to babies that are healthy by routine examination 3,12,13,26. However, pregnancy rates were directly related to the degree (low and high) and/or type (single or multiple segmental mosaics, whole chromosome mosaics, complex mosaics) of mosaicism. This has undoubtedly added a complexity to the PGT interpretation. Victor et al. (2019) Published that embryos with a single segmental mosaic fared better than the other types affected by one or two whole chromosomes, multiple segmental gains/losses, or complex mosaics 13. Even though authors described that cells harboring monosomies and trisomies result in similar clinical outcomes, we note that future larger studies are needed to refute or corroborate these conclusions. Through the present findings, we sought to propose an approach for mosaic embryo transfers resulting from Robertsonian translocations inspired by Grati et al. (2018) conclusions 28. Our strategy of mosaic embryos transfer after a biopsy on Day 3 is based on risk evaluation to give a birth of an abnormal child. To this end, we established a classification according to chromosomal monosomies and trisomies embryo status. Mosaic euploid/monosomy embryos involving acrocentric chromosomes (13, 14, 15, 21, 22) could be considered for transfer with a lower risk of viability followed by a mosaic euploid/trisomy embryos involving chromosomes 14, 15, 22. Mosaic embryo euploid/trisomies (chromosomes: 13, 21) are the lowest priority for the transfer because it may lead to a viable affected birth (figure 1). Contrary to Grati’s recommendations, we consider that the risk related to uniparental disomies (UPD) of chromosomes 14, 15 should be excluded. A recent study performed in 1747 UPD, demonstrated that the risk of UPD in a fetus carrying an inherited Robertsonian translocation was estimated ~0.06%, which was significantly lower than previous estimations 29.
To conclude, many diagnostic laboratories are still performing PGT-SR analysis with FISH probe strategies. This choice is explained by local regulations of certain countries. Further, the controversial contribution of the new technologies (NGS) in terms of costs and gains discouraged laboratories. Through the present study undertaken on the largest cohort of male and female RT carriers, a reliable and accurate FISH probe strategy could be proposed to PGT-SR laboratories to manage RT carrier attempts. This analysis allowed to demonstrate that an increase of FISH probes number from two to three didn’t improve balanced embryo rates carried out on one or two biopsied cells but reveals a significant mosaic embryo number (34.4 % versus 19.7%).
To improve the possibility of pregnancy in absence of balanced embryos obtained, we propounded a strategy of mosaic embryo transfer after a Day 3 FISH analysis. This possibility should be considered after detailed counseling to ensure that the patient is aware of risks and potential benefits is of utmost importance to ensure informed decision-making by patients.
In addition to the considerations of embryo selection, it is essential that genetic counseling include a discussion about prenatal testing benefits and limitations. A prospective study on mosaic embryo transfer is currently discussed as part of a metacentric research project. Outcomes of pregnancies achieved following a mosaic embryo transfer will be decisive to confirm our proposition.