P-568 Correlation of ploidy analysis in two types of zygotes with 3 pronuclei (3PN) observed: same size 3PNs and one micronucleus 3PNs
Authors: Escriba M , Benavent M , Miret C , Teruel J , Garcia A , Gonzalez D , De la Fuente E , Rodrigo L , Crespo J , Rubio C 
Published: July 2024

Abstract

Study question
Do all zygotes with three observed pronuclei have an actual triploid chromosomal complement?

Summary answer
Not all zygotes with 3 pronuclei observed are triploid, most of embryos with two even pronuclei and one smaller micronucleus are diploid.

What is known already
In the in vitro fertilization (IVF) setting, normal oocyte fertilisation is commonly confirmed 16–19 hours after conventional insemination or intracytoplasmic sperm injection (ICSI) with the appearance of two pronuclei (2PN).
The presence three pronuclei (3PN) shows an abnormal oocyte fertilization. The incidence of 3PN zygotes in IVF is between 5.0%- 8.1% in conventional insemination and between 2.0%- 6.2% in ICSI. 3PN fertilized embryos are commonly discarded due to the suspected increased risk of a triploid chromosomal complement.
However, no definitive genetic evidence has been obtained to show that all 3PN zygotes are chromosomally abnormal.

Study design, size, duration
An Ethics Committee for Investigation accredited this prospective observational study performed in a private centre between June 2023 and January 2024.
120 3PN zygotes from 113 cycles were cultivated until blastocyst stage. 46 were biopsied to clarify ploidy and euploidy status.
3PNs were divided into 2 groups based on the size of their pronuclei: Micro Triploid (MT) (smallest pronucleus &lt;70% of the diameter of the largest pronucleus) and Pure Triploid (PT) (smallest &gt;70% of the largest).

Participants/materials, setting, methods
Patients with 1 or more 3PN embryo were offered to participate, culturing and assessing their 3PNs. If 3PNs reached blastocyst stage, they were biopsied and analysed using Next-Generation Sequencing strategy with Single Nucleotide Polymorphisms technology without the need for parental samples, to assess ploidy and aneuploidy status.
Measures of the pronuclei size were taken to sort the 3PNs in both groups. Comparisons between these groups regarding blastocyst rates, ploidy status and aneuploidy rates were assessed.

Main results and the role of chance
120 3PNs zygotes were identified in 1054 cycles, representing 1.9% of all fertilized embryos.
The blastocyst rate (BR) for overall 3PNs was 38.3%, while the BR for 2PNs on those 113 cycles was 58.3%, showing significant differences (p &lt; 0.001).
3PN zygotes were classified into the MT and PT groups described above. The MT group had 40.4% BR, and the PT group had 37.0% BR. No significant differences were found.
Of the 46 biopsied embryos, 37 were informative (80.4%), 17 for the MT group and 20 for the PT group. The diploidy rate for the overall 3PNs was 37.8% (n = 14). The MT group had 82.4% (n = 14) of diploidy rate, while the PT group had 0% diploid rate (n = 0), showing significant differences between them. (p &lt; 0.001).
The overall euploidy rate for 3PN zygotes was 28.6% (n = 12) (36.2 mean age), which was similar to the euploidy rate (30.3%) for our overall 2PNs biopsied during 2023 (38.2 mean age). No differences found.
Out of the 14 diploid 3PNs, 28.6% (n = 4) were euploid, showing no differences with our 2023 euploidy rate. Two diploid 3PNs were mosaic, resulting in a total of 6 embryos that could potentially be transferred.

Limitations, reasons for caution
This study is limited by the number of triploid embryos recruited. As blastocyst rate for 3PNs population is lower than observed in the 2PN population the number of biopsied embryos is reduced.
Euploidy rates for 3PNs and 2PNs are very similar, but the mean age differs by 2 years.

Wider implications of the findings
It is very important to distinguish between both types of 3PNs observed: PT are not worthy to keep and analyse, while MT embryos are very likely to be diploid, and it is worthwhile to culture and analyse them.
Patients with few embryos available in particular could benefit from this study.

Trial registration number
not applicable