Figure 1. Biopsy of a human embryo. The holding pipette on the left abuts the zona pellucida of the embryo. The biopsy pipette on the right is inside the opening in the zona pellucida. One cell is inside the pipette (1).

​​INTRODUCTION

The importance of aneuploidy screening in assisted reproduction has gained popularity since the introduction of preimplantation genetic testing (PGT) at the beginning of the 1990s (2). PGT for aneuploidies screening (PGT-A) is recommended for couples experiencing recurrent pregnancy loss and implantation failure, since aneuploidies could be the reason behind these issues. Aneuploidy rate increases with maternal age (3), which is why PGT-A is also offered to women above 37 years old. A few years ago, infertility was only attributed to women, but now it is well documented that male factor plays also an important role (4), and so, aneuploidies in sperm or chromosome alterations as a result of defective meiosis in sperm are too indications to perform PGT. Different techniques have been developed in order to select euploid embryos for transfer. Initially, fluorescent in situ hybridization (FISH) was the only technique to test for aneuploidy, but it only allowed for the assessment of some chromosomes. Subsequently, Comparative Genomic Hybridization array (CGHa) and most recently Next-Generation Sequencing (NGS) enable the detection of cytogenetic changes, including every chromosome in the complement.

In order to perform PGT, embryo biopsying is needed. Different techniques are available to collect the required sample: biopsy of polar bodies or embryo at either cleavage or blastocyst stage are the most extended, yet invasive methodologies. A new approach for non-invasive PGT (NIPGT) is reviewed below: the analysis of cell-free DNA from embryo culture.

CURRENT PGT TECHNIQUES

POLAR BODY (PB) BIOPSY

First polar body and second polar body are indicative of oocyte maturation (oogenesis). Once the primary oocyte completes the first meiotic division, first polar body is produced, whereas the second one is only formed if fertilization is achieved (as a result of the completion of the second meiotic division). During oocyte and embryo development polar bodies degenerate after being extruded. However, they can be collected during an in vitro fertilization cycle, either sequentially or simultaneously (Figure 2).

Figure 2. Laser-assisted polar body (PB) biopsy. Prior to biopsy the oocyte is carefully positioned with the first PB in focus. (a) An opening of approximately 20 μm is formed in the zona pellucida (ZP) by two laser shots (14 ms pulse duration). (b) The biopsy pipette can be easily introduced through the opening for aspiration of the PB. (c) Finally, the PB is aspirated through the pipette. A skilled operator can carry out the whole procedure in less than 1 min. Modified from (5).
​It is important to point out that both PBs contain only maternal genetic material. Thus, paternal contribution cannot be analyzed, which is the main limitation of this technique. Nevertheless, PB biopsy is less invasive than other methodologies, and as such it avoids ethical, religious and legal problems related with embryo manipulation.

EMBRYO BIOPSY AT CLEAVAGE STAGE

Three days after fertilization, the embryo reaches cleavage stage. At this point, a normal embryo should contain around 16 individually distinguishable cells known as blastomeres. Biopsy at cleavage stage implies the removal of one or two blastomeres. Similarly to PB biopsy, a hole in the zona pellucida (ZP) is created using either a laser pulse or acid Tyrode’s solution (Figure 3). The blastomeres are pulled away using a biopsy pipette (6) and collected into a microtube. The genetic material of the blastomere(s) is then amplified, and the result is considered representative of the embryo genome. In 1990 Hardy et al concluded that this practice does not compromise in vitro development (7). Biopsy of cleavage stage embryos was performed in approximately 90% of all reported PGT cases in 2012 (8), but it does not allow to distinguish mosaic embryos (9). These are embryos with two or more cell lines containing both euploid and aneuploid cells. Mosaic embryos can be considered for transfer following specific recommendations (Preimplantation Genetic Diagnosis International Society (PGDIS)).

Figure 3. Cleavage-stage embryo biopsy. (a) Cleavage-stage embryo ready to be biopsied. (b) The embryo is immobilized with a holding pipette and one blastomere is selected. (c-d) The nucleus-containing targeted blastomere is gently aspirated into the pipette (6).

EMBRYO BIOPSY AT BLASTOCYST STAGE

The embryo reaches blastocyst stage on day 5 of embryonic development or day 6 after fertilization. Membrane ion transporters and channels, such as Na+/K+ pump, are activated and so fluid is accumulated in the blastocoel (Blastocoel Fluid, BF). This process is termed cavitation, and two structures are at this point differentiated: the inner cell mass (ICM), which will eventually form the embryo, and the trophectoderm (TE), which will give rise to the placenta and associated tissues.

Since the first blastocyst biopsies until now, PGT cases have increased on Day 5 or Day 6 (D5/D6) embryos. This consists in making a small hole, between 30 to 35 µm, in the ZP with the ICM positioned either at 8 o’clock or 11 o’clock, this is, away from the laser. This provides direct access to TE cells, so they can be biopsied with no damage to the ICM (10).

Similarly to PB and cleavage stage embryo biopsy, the ZP can be breached by mechanical or physical methods, laser being the most common one (although more cells are removed by this procedure ~5-10 cells). Furthermore, it can be performed at two different times (10, 11):

  • The first possibility would be opening the hole on the third day after fertilization and the biopsy on the fifth-sixth day (Figure 4a).
  • The second option would be to make both the hole and the biopsy on the fifth-sixth day after fertilization (Figure 4b).

Not only D5 biopsy does not compromise embryo viability, but it also enables a high pregnancy rate (60-69.2%, approximately). This methodology allows to reduce both technical errors and the risk of mistakenly detecting mosaicism, because it implies the removal of 5-10 cells (12, 13).

Figure 4. Two different stages to make a hole in the zona pellucida (ZP). (a) The hole is made on D3 and by D5, as the blastocyst expands, and either trophectoderm (TE) cells (a1) or inner cell mass (ICM) and TE cells (a2) will protrude from the ZP. (B) Both hole and biopsy are carried out on day 5 or 6. Modified from (11).

​NON-INVASIVE PGT

EVOLUTION OF PGT TOWARDS NON-INVASIVE PGT (NIPGT)

Although embryo biopsy is considered to be a safe procedure, other less invasive methods are being investigated as an alternative to provide lesser manipulation of the embryo. Recent studies have shown that BF and culture medium could contain small amounts of cell-free embryonic DNA, which may be used for PGT (14, 15, 16).

Currently, there are three ongoing research lines on the topic:

  • Blastocoel fluid
  • Culture medium
  • Blastocoel fluid and culture medium

WHAT IS THE ORIGIN OF BF-DNA?

The origin of BF-DNA is not entirely clear, but certain studies indicate it may arise from cells undergoing apoptosis during blastulation as part of normal development, lysed cells or even discarded abnormal cells (17). As discussed later, several studies have investigated whether such DNA may correspond to DNA from TE (see the following sections “Blastocoel fluid”, “Cell free DNA on the spent culture media” and “Blastocoel fluid and culture”).

BLASTOCOEL FLUID (BF)

In 2012, a study published by Alessandro et al (18) detected a series of metabolites in the BF. The methodology employed, referred to as blastocentesis, consisted in immobilizing expanded blastocysts using a holding pipette in a plate without culture medium in order to avoid contamination, and then using an intracytoplasmic sperm injection (ICSI) pipette to aspirate the BF for analysis (Figure 5). Blastocentesis is nowadays a routine methodology employed by professionals in some laboratories to collapse blastocysts before vitrification.

Figure 5. Biopsy of blastocoel fluid. (A) The intracytoplasmic sperm injection (ICSI) pipette is inserted inside the embryo blastocoel. (B) The blastocoel fluid (BF) is aspirated through the ICSI pipette. (C) BF-aspirated embryo collapses (19).

From these findings, Pallini and collaborators performed the blastocentesis technique in order to find DNA in BF (14). This was the first study exploring the presence of genetic material in BF, and DNA was detected in 90% of the samples. Furthermore, the authors used the DNA found to confirm the sex of embryos as well as to detect aneuploidies.

Several investigations have been conducted with the aim of detecting DNA in BF samples. First of all, DNA from BF or TE is amplified and then analyzed. In these studies, rate of amplification failure in TE samples was found to be lower than 2%; however, amplification of DNA from BF was harder, with higher failure rate than DNA from TE (Table 1).

Table 1. DNA amplification from blastocoel fluid (BF). Different studies detected DNA from blastocoel fluid and obtained amplification rate lower than that from trophectoderm (TE) samples.
Even so, the quantity of DNA obtained from BF was similar to that obtained from a single blastomere (14, 22). Conversely, Li and co-authors have recently reported that DNA extracted from BF is insufficient for amplification and sequencing (25). Table 2 exhibits a relation of studies with concordant and discordant results between BF and TE samples, respectively (Table 2).
Table 2. Different studies indicated concordance between blastocoel fluid (BF) and trophectoderm (TE) cells, which are in contrast with the second column, showing studies that reported discrepancies in the results. Consequently, further investigations will be in order.

CELL-FREE DNA ON SPENT CULTURE MEDIA

The presence of embryonic free cells in the spent embryo culture have been shown by several studies. The origin of cell-free DNA is still under investigation, but the most likely source is remnants of apoptotic embryo cells (17). A study by Stigliani et al about mitochondrial DNA content in embryo culture medium showed that 99% of embryos at day 2 an day 3 were accompanied by free DNA in their spent culture media (15). The authors additionally reported two important things: (i) a larger amount of free DNA in the medium of bad-quality cleavage embryos, and (ii) higher mitochondrial/genomic DNA ratios in spent medium were associated with successful implantation outcomes.

Since the discovery of the presence of free DNA in embryo culture media, some researchers have focused on evaluating the concordance between cytogenetic results of invasive techniques and the analysis of free DNA. In order to accomplish such goal, embryos are cultured individually and researchers collect a microdrop of media after 3, 4 or 5 days of culture. Simultaneously, embryo biopsy is performed and both samples are analyzed. Results reported suggested that cell-free DNA analysis is a promising option for NIPGT. Feichtinger et al (26) found a 72.2% concordance between results from PB biopsies and culture media using CGHa. Xu et al (27) in turn analyzed the genetic complement of free DNA from culture media, as well as DNA from TE biopsies by NGS. Their results showed a specificity of 84% (false positives) and a sensitivity of 88.2% (false negatives). However, this technique would need further refinement, since more recent findings have suggested that spent culture media contains not only embryonic, but also maternal DNA, which is a confounding factor. In fact, Vera-Rodriguez and collaborators (17) reported a concordance of only 30.4% between culture media DNA and TE DNA when analyzed by NGS. This could be attributed to maternal contamination, as well as to the inclusion of mosaic embryos in the study, which may actually hinder reliable diagnosis.

Such big differences between findings may be attributed to the lack of a standardized protocol to develop NIPGT. Culture systems and conditions, culture volume, as well as DNA amplification methodology, differ between research groups, and so it becomes necessary to find reproducible conditions to verify the consistency of such results.

EMBRYO CULTURE MEDIUM AND BLASTOCOEL FLUID (ECB)

In 2018, preliminary studies were carried out to combine DNA from blastocoel fluid and culture medium (ECB) and, thereby increase embryonic DNA amount to improve accuracy and reliability of the non-invasive preimplantation genetic screening. Fully expanded blastocysts were collapsed with a laser (16) or a laser was used to breach the ZP (25), thus obtaining enough DNA from the mix-up of spent culture medium and BF.

Data reported by Li et al (25) after amplification and sequencing of DNA from both ECB and TE have shown different aneuploidy chromosomal patterns detected in approximately 50% of the cases. Conversely, a 100% of embryonic DNA has been successfully amplified by the team of Kuznyetsov and collaborators (16). These authors have shown the concordance rate between TE biopsy and the combination of BF and culture medium to be 87.5% for whole chromosome copy number.

Other sources of DNA must be carefully considered in order to analyze data, since foreign DNA may eventually reach the media. Contamination with non-embryonic DNA (either maternal or paternal DNA) (17, 21, 28) or degraded DNA fragments from culture medium needs to be avoided. One way to minimise such contamination is by transferring embryos to fresh medium on day 4, which poses no particular problem if the goal does not include to measure other molecules such as metabolites, for instance.

COMPARISON BETWEEN INVASIVE PGT AND NON-INVASIVE PGT

As we have already explained, there are different methodologies to get a small sample of the embryo in order to perform PGT. Table 3 summarizes advantages and disadvantages of each one of them. Polar body biopsy is minimally invasive and avoids ethical and legal issues, but only allows to analyze maternal genetic complement. On the other hand, biopsy at day 3 and at day 5 are more invasive but enable the detection of the whole genetic complement. Non invasive techniques such as blastocoel fluid, spent media culture or ECB are still under development, but promises minimal manipulation.
Table 3. Advantages and dissadvantages of each methodology.

​CONCLUSIONS

Further research is still necessary to improve NIPGT until reliable consistent results are obtained, as well as efficiency concordance and similar amplification failure as that of current PGT. However, before implementation of NIPGT, several aspects must be kept in mind:

  • DNA from BF fluid and/or culture medium must be representative of the embryo; in other words, collected DNA cannot include that from abnormal cells (17, 21, 28).
  • Methods used to isolate embryonic DNA must be optimized, avoiding potential contamination from maternal (cumulus cells) or paternal (sperm cells after IVF) DNA (17, 21, 29).
  • NIPGT accessibility should become more accessible, thus providing higher resolution at a lower cost.
  • Technical improvements are still needed in order to obtain similar amounts of DNA across samples.

Given current available data, blastocoel fluid, culture spent media or both are potential candidates to become the next sources of embryonic DNA. This could eventually revolutionize the horizons of PGT and the achievable clinical outcomes. Technical hindrances are, as usual, in the way of improving and implementing actual routine applications of new methodologies. But, these are exciting and challenging times, and non-invasive PGT may be one step closer to becoming a reality.

REFERENCES

  1. Swanson A, Strawn E, Lau E, Bick D Preimplantation genetic diagnosis: Technology and clinical applications. WMJ: official publication of the State Medical Society of Wisconsin. 2007;106: 145-151. 
  2. Handyside AH, Kontogianni EH, Hardy K, Winston RM. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature. 1990;344(6268):768-770. 
  3. Hassold T, Hunt P. Maternal age and chromosomally abnormal pregnancies: what we know and what we wish we knew. Curr Opin Pediatr. 2009;21(6):703-708. 
  4. Colaco S, Sakkas D. Paternal factors contributing to embryo quality. J Assist Reprod Genet. 2018;doi: 10.1007/s10815-018-1304-4 [Epub ahead of print]. 
  5. Montag M, van der Ven K, Dorn C, van der Ven H. Outcome of laser-assisted polar body biopsy and aneuploidy testing Montag, Reprod BioMedOnline , 2004;9(4) , 425 – 429.
  6. Piyamongkol W, Vutyavanich T, Piyamongkol S, Wells D, Kunaviktikul C, Tongsong T, Chaovisitsaree S, Saetung R, Sanguansermsri T. A successful strategy for Preimplantation Genetic Diagnosis of beta-thalassemia and simultaneous detection of Down’s syndrome using multiplex fluorescent PCR. J Med Assoc Thai. 2006;89(7):918-927.
  7. Hardy K,  Martin K,  Leese H,  Winston R,  Handyside A. Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage, Hum Reprod , 1990;5:708-714.
  8. Thornhill A.R. Cleavage-Stage Embryo Biopsy. In: Nagy Z., Varghese A., Agarwal A. (eds) Practical Manual of In Vitro Fertilization. Springer, New York, NY; 2012.
  9. Gleicher  N, Vidali A,  Braverman  J,  Kushnir VA,  Barad DH, Hudson C, Wu YG, Wang Q, Zhang L, Albertini DF. Accuracy of preimplantation genetic screening (PGS) is compromised by degree of mosaicism of human embryos. Reprod Biol Endocrinol. 2016;14:54.
  10. Bustamante-Aragonés A, Fernández E, Peciña A, Rueda J, Ramos C, Giménez C, et al. Guía de buenas prácticas en diagnóstico genético preimplantacional. Medicina Reproductiva y Embriología Clínica. 2016;3(2):104–111. 
  11. Cimadomo D, Capalbo A, Ubaldi FM, Scarica C, Palagiano A, Canipari R, et al. The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis. Biomed Res Int. 2016;(4):1-10
  12. McArthur SJ, Leigh D, Marshall JT, de Boer KA, Jansen RPS. Pregnancies and live births after trophectoderm biopsy and preimplantation genetic testing of human blastocysts. Fertil Steril. 2005;84(6):1628–36.
  13. Fragouli E, Wells D. Current status and future prospects of noninvasive preimplantation genetic testing for aneuploidy. Fertil Steril. 2018;110(3):408–9.
  14. Palini S, Galluzzi L, De Stefani S, Bianchi M, Wells D, Magnani M, et al. Genomic DNA in human blastocoele fluid. Reprod Biomed Online. 2013;26(6):603–10. 
  15. Stigliani S, Anserini P, Venturini PL, Scaruffi P. Mitochondrial DNA content in embryo cultura medium is significantly associated with human embryo fragmentation. Hum Reprod. 2013;28(10):2652-2660.
  16. Kuznyetsov V, Madjunkova S, Antes R, Abramov R, Motamedi G, Ibarrientos Z, et al. Evaluation of a novel non-invasive preimplantation genetic screening approach. PLOS ONE. 2018;13(5):e0197262. 
  17. Vera-Rodriguez M, Diez-Juan A, Jimenez-Almazan J, Martinez S, Navarro R, Peinado V, et al. Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development. Hum Reprod. 2018;33(4):745–756.
  18. D’Alessandro A, Federica G, Palini S, Bulletti C, Zolla L. A mass spectrometry-based targeted metabolomics strategy of human blastocoele fluid: a promising tool in fertility research. Mol Biosyst. 2012;8(4):953–958.
  19. Tobler KJ, Zhao Y, Ross R, Benner AT, Xu X, Du L, et al. Blastocoel fluid from differentiated blastocysts harbors embryonic genomic material capable of a whole-genome deoxyribonucleic acid amplification and comprehensive chromosome microarray analysis. Fertil Steril. 2015;104(2):418–425.
  20. Magli MC, Pomante A, Cafueri G, Valerio M, Crippa A, Ferraretti AP, et al. Preimplantation genetic testing: polar bodies, blastomeres, trophectoderm cells, or blastocoelic fluid? Fertil Steril. 2016;105(3):676–683.
  21. Capalbo A, Romanelli V, Patassini C, Poli M, Girardi L, Giancani A, et al. Diagnostic efficacy of blastocoel fluid and spent media as sources of DNA for preimplantation genetic testing in standard clinical conditions. Fertil Steril. 2018;110(5):870–879.
  22. Zhang Y, Li N, Wang L, Sun H, Ma M, Wang H, et al. Molecular analysis of DNA in blastocoele fluid using next-generation sequencing. J Assist Reprod Genet. 2016;33(5):637–645.
  23. Gianaroli L, Magli MC, Pomante A, Crivello AM, Cafueri G, Valerio M, et al. Blastocentesis: a source of DNA for preimplantation genetic testing. Results from a pilot study. Fertil Steril. 2014;102(6):1692–1699.
  24. Tšuiko O, Zhigalina DI, Jatsenko T, Skryabin NA, Kanbekova OR, Artyukhova VG, et al. Karyotype of the blastocoel fluid demonstrates low concordance with both trophectoderm and inner cell mass. Fertil Steril. 2018;109(6):1127–1134.
  25. Li P, Song Z, Yao Y, Huang T, Mao R, Huang J, et al. Preimplantation Genetic Screening with Spent Culture Medium/Blastocoel Fluid for in Vitro Fertilization. Sci Rep. 2018;8(1):9275. 
  26. Feichtinger M, Vaccari E, Carli L, Wallner E, Mädel U, Figl K, et al. Non-invasive preimplantation genetic screening using array comparative genomic hybridization on spent culture media: a proof-of-concept pilot study. Reprod Biomed Online. 2017;34(6):583-589.
  27. Xu J, Fang R, Chen L, Chen D, Xiao JP, Yang W, et al. Noninvasive chromosome screening  of human embryos by genome sequencing of embryo culture medium for in vitro fertilization. Proc Natl Acad Sci U S A. 2016;113(42):11907-11912.
  28. Cohen J, Grudzinskas G, Johnson MH. Embryonic DNA sampling without biopsy: the beginnings of non-invasive PGD? Reprod Biomed Online. 2013;26(6):520–521.
  29. Hammond ER, Shelling AN, Cree LM. Nuclear and mitochondrial DNA in blastocoele fluid and embryo culture medium: evidence and potential clinical use. Hum Reprod. 2016;31(8):1653–1661.