Is it time for genetic PN testing?

Is it time for genetic PN testing?

In this article, David Chrimes, PhD, discusses the argument for genetic PN checking which could increase the chances of finding more embryos to transfer, increasing the live birth rate per cycle.

Since the beginning of IVF, morphological assessments of embryo development have been implemented and defined checkpoints established, and these remain a fundamental element of embryology today.

These checkpoints now include well-defined criteria such as the Gardner scale for blastocyst grading. The first of these key checkpoints remains the 16-19 hours post-insemination check when zygotes are visually assessed for the appearance of two pro-nuclei, indicating successful fertilization has occurred.

Pronuclear fertilization checks, commonly shortened to PN checks, ensure not only that fertilization has occurred but also the presence of 0PN, 1PN, or 3PN, which indicates both failed fertilization or abnormal fertilization.

1PN indicates a possible haploid embryo (one set of 23 chromosomes, half the number of a normal 46-chromosome cell), and 3PN indicating a potential triploid embryo (69 chromosomes).

Haploid and triploid embryos can produce unwanted events, such as implantation leading to miscarriage with triploid, or molar pregnancies. As well as being useful in screening out haploid and triploid embryos, PN checking can also indicate how many embryos may continue to grow to blastocyst and therefore be considered for implantation.

This is a key piece of early post-insemination information for patients during their IVF cycle. What labs then do with a PN fertilization check for failed zygotes varies. In some ART labs, these embryos are discarded and in others, they are cultured alongside 2PNs.

PN Checking Challenges

Assessing embryos for PN at a single time point may not be as simple or accurate as we think. PN may appear at slightly different times, especially if IVF rather than ICSI is used for insemination. The embryos in a cycle may have to be checked several times over the expected time period of PN formation to accurately assess each embryo.

Even then there are potential complications to accurate PN scoring; PN may be faint, appear or fade asynchronously, micro PN may be apparent, and PN may be vertically stacked making identification of 2PN difficult.

Using ICSI and time-lapse incubators may improve the determination of PN due to the more precise timing of insemination and PN formation after ICSI, as well as better visualization over the expected time period of the formation of PN with the TLI, compared to static microscopy.

How accurate is PN checking?

Our CooperGenomics internal data indicates a 3% triploidy rate in biopsies analyzed with our PGTaiSM 2.0 Plus test. The vast majority of these embryos were presumably screened by PN checking, therefore indicating a 3% false-negative rate for PN checking. Putting this simply, this means that ~3% of embryos scored as 2PN by morphological checks are 3PN by the latest PGT-A technology. Is this a figure we could help improve for the benefit of patients?

This also opens up a question about the false-negative rate. What is the genetic nature of embryos labeled 0PN, 1PN, and 3PN when PN checking? How accurately are these reported?

There is an increasing amount of data available that shows embryos scored as 0PN or 1PN, if they grow to blastocyst, can be diploid (with 46 chromosomes) containing maternal and paternal contributions. These are often-called bi-paternal diploids, or what we mostly call euploids. Hence, PN fertility checks have a measurable false-positive rate, and traditional PN checking incorrectly scores embryos suitable for consideration to be implanted. Again, we must ask if this occurs at a level we and our patients deem acceptable.

A recent very large study from Li et al1 looked at the outcomes of 0PN, 1PN, and 2PN embryos grown to the blastocyst stage. This outstanding study looked at the outcomes of 435 0PN, 281 1PN, and 13,167 2PN embryos that were supplementary to fresh transferred embryos in over 100,000 IVF cycles and then vitrified for future use.

The live birth rate (LBR) per embryo transfer of the 0PN embryos was remarkably similar to that of the 2PN embryos in the study (35.6% 0PN versus 35.1% 2PN), while the 1PN embryos tended to be slightly worse at 27.4%.

There was also a higher miscarriage rate in pregnancies resulting from 1PN transferred embryos (33.62% 1PN versus 23.84 2PN), and there were some indications of a lower birth weight after the transfer of 0PN embryos.

2PN or not 2PN?

While there is evidence that 0PN and 1PN embryos can produce healthy live births, are we still at risk of implanting chromosomally aberrant embryos if we use them? Hence, do we need a further test that can identify haploid embryos (with 23 chromosomes) or triploid embryos (with 69 chromosomes)?

Recent advances in preimplantation genetic testing for aneuploidy (PGT-A) allow 0PN and 1PN embryos to be biopsied at the blastocyst stage, then, using parental cheek brush DNA and these new PGT-A tests, we can differentiate between haploid/triploid embryos and euploid embryos.2

Our CooperGenomics PGTai 2.0 Plus test uses parental DNA samples (Plus refers to the extra parental DNA) and counts single nucleotide polymorphisms (SNPs) to determine the of DNA in the embryo, not just the number of chromosomes in the embryo.

Counting SNPs, we can see otherwise invisible things (so-called ‘copy neutrals’) such as haploid embryos and all forms of triploidy embryos, unlike most other PGT-A tests on the market. If we only see maternal DNA, we know that the embryo is haploid (with 23 chromosomes), or if we see more maternal than paternal DNA, we know the embryo is female triploid (chromosomes).

Has this Genetic PN Checking approach been tested before?

Destouni et al 20182, used an SNP-based PGT-A technology similar to PGTai 2.0 Plus to look at 0PN and 1PN embryos in PGT-Monogenic (formerly called PGD) patients. They found that 75% of 0PN embryos grown to blastocyst stage were euploid, and 43% of 1PN embryos were euploid with the remainder either being haploid, triploid, or uniparental.

Although approximately 77% of blastocyst biopsies in this study found to be euploid (bi-parental diploid) were from 2PN scored embryos, 17% were from 0PN scored embryos and 6% were from 1PN scored embryos. Euploid 1PN and 0PN embryos were transferred and resulted in live births.

Using the latest developments in PGT-A, such as PGTai 2.0 Pluswe can identify embryos visually graded as 0PN, IPN, or 3PN that are most suitable for transfer, having both 46 chromosomes and an equal contribution from each parent.

How can these developments be used in practice?

Clearly, in any IVF cycle, embryos scored as 2PN, and preferably tested with PGT-A to avoid triploid embryos, should be transferred in preference to 0PN and 1PN embryos.

However, if we culture 0PN and 1PN scored embryos alongside 2PN to find euploid embryos, we could increase the chances of finding more embryos to transfer.  This would in turn increase the live birth rate per cycle.


1Ming Li, Huang J, Zhuang X, Lin S, Dang Y, Wang Y, Liu D, Li R, Liu P, Qiao J Obstetric and neonatal outcomes after the transfer of vitrified-warmed blastocysts developing from nonpronuclear and monopronuclear zygotes; a retrospective cohort study. Fertility and Sterility (2021) Vol 115(1) P110-117

2Destouni A, Dimitriadou E, Masset H, Debrock S, Melotte C, Van Den Bogaert K, Esteki M Z, Ding J, Voet T, Denayer E, de Ravel T, Legius E, Meuleman C, Peeraer K, Vermeesch J R Genome-wide haplotyping embryos developing from 0PN and 1PN zygotes increases transferrable embryos in PGT-M. Human Reproduction 33(12):2302-2311.

About the author

Dr Chrimes is a PhD molecular geneticist with 14 yrs experience in molecular diagnostics. After working at various UK University and Research Institutes in the early 2000s, he joined BlueGnome in Jan 2005.

Dr Chrimes was instrumental in delivering state of the art copy number genomics analysis solutions to the clinical market including the CytoChip and BlueFuse Multi visualization software. Many of these advances were made through the management of research collaborations with leading hospitals in both the UK and USA. In 2008, BlueGnome launched the 24sure product line for aneuploidy testing of embryos and Dr Chrimes used his extensive experience of genomics data analysis to drive the innovation in embryo genomic data to aid interpretation of the sometimes complex data. The result of this innovation is that the vast majority of global PGS is performed using 24sure/VeriSeq PGS and in 2012 BlueGnome was sold to Illumina.

Dr Chrimes was Associate Director in the Illumina Reproductive Health Group where he continued to innovate genomics analysis including embryo analysis, targeted genomics disease panels through to whole genome sequencing analysis.

In 2016 Dr Chrimes started at Genesis Genetics to help drive advances in their embryo testing and after the acquisition in April 2016 of Genesis Genetics by Cooper Surgical Industries (USA), Dr Chrimes took on a global role as Director of Global Genomics Business Development. This role ensures Dr Chrimes is both driving innovation in genomics testing and also gaining regular insights to the challenges faced by IVF clinics today.

David Chrimes PhD

Director of Global Genomics Business Development, CooperSurgical

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