COGEN Position Statement on Chromosomal Mosaicism Detected in Preimplantation Blastocyst Biopsies

BACKGROUND

The occurrence of aneuploidy (an incomplete or abnormal chromosome copy number) in human embryos is a natural phenomenon in embryos conceived either in vivo or following the use of in vitro fertilization (IVF) technology; this has been understood since the early 1980s^1,2. Alterations from the euploid (complete) chromosome copy number can occur at any age, but the incidence of aneuploidy increases with maternal age (>90% risk in women over 40 years old and approximately 50% risk in women age <35). Aneuploidy is the largest single cause of miscarriage³, and thus the ability to evaluate chromosome copy number prior to embryo transfer has been a clinical imperative of the IVF community for many years. In recent years, screening methods known as preimplantation genetic diagnosis for aneuploidy (PGD-A) or preimplantation genetic screening (PGS) have been developed to accurately assess the copy number of all 24 chromosomes from a single or multiple cell(s) biopsied from human preimplantation embryos.

However, since 1983 and the introduction of chorionic villus sampling (CVS), a discrepancy between the chromosomal makeup of the placenta and the fetus has been documented in approximately 1-2% of ongoing pregnancies; this is known as "confined placental mosaicism" (CPM) ⁴. Since trophectoderm biopsy is now widely viewed as the preferred method of aneuploidy assessment, these samples also manifest mosaicism in a small but significant percent of embryos, known as "embryonic mosaicism". During the 2016 COGEN Congress (Barcelona, September 22-25, 2016) and in the following Position Statement of PGDIS⁵, the issue of embryonic mosaicism in human blastocysts was discussed at length. The conclusions thereof are presented in this position statement in order to develop the best approach to this phenomenon in clinical practice.

In embryonic mosaicism, two or more genetically distinct cell lineages are present, typically one with a chromosome abnormality and the other possessing a normal chromosome constitution. Embryonic mosaicism may originate in the pre- implantation embryo through mitotic errors, or by anaphase lag. Mitotic errors that occur in earlier divisions result in embryos with a great percentage of abnormal cells.

The clinical significance of mosaicism in preimplantation embryos has become highlighted since the shift of clinical PGS toward blastocyst biopsy, vitrification, and diagnosis by high-resolution next generation sequencing (NGS). Data presented at the 2016 PGDIS conference (Bologna, May 8-11, 2016; www.pgdis.org) demonstrated that 10-20% of human blastocysts present with mosaicism when analyzed on a validated NGS platform. The presence of mosaicism in human blastocysts has been extensively reported over the years, but it is unknown if it is the same phenomenon as observed in CPM detected through CVS. There is some evidence of variation in level of mosaicism among blastocyst biopsy samples obtained from different IVF clinics, suggesting a potential iatrogenic origin distinct from mosaicism of biological significance. Technical variables affecting the quality of biopsy, or of downstream NGS procedures, may impact the significance and clinical implication of mosaic results. To date the scientific community agrees that <20% or >80% abnormal cells from a trophoblast biopsy cannot differentiate between mosaicism and ‘technical noise’. In support of the PGDIS Positon Statement (http://www.pgdis.org/docs/newsletter_071816.html), we make the following recommendations.

GOOD PRACTICE RECOMMENDATIONS

For reliable detection of biological mosaicism, a minimum of 5 cells (but, importantly, <10) should be biopsied, with as little cell damage as possible. If the biopsy utilizes a laser, the contact points should be minimal and preferably at cell junctions. Additionally, a validated, high-resolution NGS platform should be utilized. Diagnostic and clinical dilemmas arise when mosaicism consists of one normal euploid cell line and one aneuploid line (monosomic or trisomic). We believe these complexities can be managed with good practice recommendations and appropriate genetic counseling.

1. PREFERED TRANSFER OF EUPLOID NON-MOSAIC EMBRYOS
   1. Non-mosaic euploid embryos should preferentially be transferred whenever available.
   2. Fully aneuploid embryos (greater than 70% abnormal cells, for the purposes of clinical practice) should not be transferred, as they may result in adverse obstetric or pediatric outcomes.
   3. If a non-mosaic euploid embryo is not available, another IVF cycle with appropriate preimplantation testing should be offered.
2. TRANSFERRING MOSAIC EMBRYOS IN ABSENCE OF ALTERNATIVES
   1. If non-mosaic euploid embryos are not available and there is no option for undergoing another IVF cycle, a mosaic embryo may be considered for transfer. In such circumstances, the following constitutes good practice.
      • Embryos with lower levels (20-40%) of mosaic aneuploidy are preferential to those with higher levels (40-70%).
      • For embryos with higher levels of mosaicism in the trophectoderm, there exists a higher probability that aneuploidy is present in the inner cell mass. (ICM). Such embryos will likely implant less and miscarry more.
   2. In the case of complex mosaicism (mosaicism observed across multiple chromosomes), transfer is not recommended.
3. RECOMMENDATIONS FOR PRIORITIZING MOSAIC EMBRYOS FOR TRANSFER
   1. If a decision is made to transfer a non-complex, low-level mosaic embryo, one can prioritize selection based on the specific chromosome involved.
   2. Embryos mosaic for trisomies capable of live born viability (chromosomes 13, 18, 21, 22) are of lowest priority.
   3. Embryos mosaic for trisomies associated with uniparental disomy (chromosomes 14, 15) are low priority.
   4. Embryos mosaic for trisomies associated with intrauterine growth retardation (chromosomes 2, 7, 16) are low priority.
   5. Mosaicism involving chromosomes 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 17, 19, 20, have not been associated with the aforementioned adverse outcomes; only adverse outcomes have been observed when mosaicism is present in the fetus.
   6. Mosaic monosomies seem to implant with a similar incidence to mosaic trisomies. They may contain trisomic cell lines, and should be considered to have similar risk as their counterpart trisomies.

• PATIENT REVIEW
  1. It is self-evident that special care is required with regard to counselling patients, and appropriate professional guidance at the highest level must be available.
  2. Equally, all parties must be reassured that the signing of informed consent for embryo transfer is undertaken after such counselling and demonstrable understanding by the patient.
• FOLLOW UP AFTER TRANSFER OF MOSAIC EMBRYOS
  1. If ongoing pregnancy is established after transfer of a mosaic embryo, the developing fetus should be retested for aneuploidy by prenatal diagnosis. Amniocentesis is recommended over CVS as the mosaicism persists in the trophoblast.
  2. Reproductive outcomes of mosaic embryo transfers should be conveyed to the PGS laboratory performing the original diagnosis, enabling data collection to refine and improve practice recommendations.
  3. And where possible a follow-up on mosaic data should be obtained from the live birth

CONCLUSIONS

1. Aneuploid embryos exist at a high incidence in human conception; it is therefore incumbent on IVF practitioners to try to detect and avoid the transfer and cryopreservation of such embryos using PGS technology
2. The use of PGS technology has been shown to improve the incidence of live birth, whilst, importantly, also reducing multiple pregnancy by the transfer of fewer, but viable embryos⁶
3. Mosaicism is a natural occurrence and therefore is present in trophoblast biopsy of a few but significant number of embryos
4. It is equally important to provide patients with advanced counselling on this issue as well as essential to have access to appropriate genetic counselling should mosaicism occur following biopsy
5. The purpose of this COGEN Consensus Statement on Mosaicism is to underscore the need for PGS in IVF practice whilst supporting, with some modification, the appropriate PGDIS Statement on the recommendations for clinical practice
6. And finally, but most importantly, best practice in all aspects of medicine will evolve over time, and whilst we all endeavor to maximize the most efficient route to a healthy birth for our patients in a single attempt, that which we agree to be the best approach today will inevitably modify as knowledge increases.

References

1. R.R. Angell, R.J. Aitken, P.F. van Look, M.A. Lumsden, A.A. Templeton, Chromosome abnormalities in human embryos after in vitro fertilization, Nature 303 (1983), 336–338.
2. R.R. Angell, A.A. Templeton, R.J. Aitken, Chromosome studies in human in vitro fertilization, Hum. Genet. 72 (1986) 333–339
3. T. Hassold, P. Hunt, To err (meiotically) is human: the genesis of human aneuploidy, Nat. Rev. Genet. 2 (2001) 280–291
4. Kalousek D.K and Dill F.J. Chromosomal mosaicism confined to the placenta in human conceptions. Science. (1983), 12;221(4611):665-7
5. http://www.pgdis.org/docs/newsletter_071816.html
6. Forman EJ, Hong KH, Ferry KM, Tao X, Taylor D, Levy B, et al. In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fertil Steril 2013;100:100–7