P-544 Viability assessment using fluorescent markers, ultrastructure and implantation of human embryos following blastocyst biopsy using three different laser systems
Authors: Chatzimeletiou A , Sioga A , Petrogiannis N , Panagiotidis Y , Filippa M , Kolibianakis E , Handyside A , Nicolaides K , Grimbizis G 
Published: July 2024

Abstract

Study question
Are there any differences in viability, ultrastructure and implantation of human blastocysts biopsied using three different laser systems?

Summary answer
No statistically significant differences are observed in viability, ultrastructure and implantation of human blastocysts biopsied using three different laser systems

What is known already
Most studies in human embryos concentrate on assessing the success of biopsy/vitrification on survival rates and clinical outcomes following transfer. Our previous published studies examined in addition to the survival rates, the cytoskeleton, ultrastructure and viability of cleavage and blastocyst stage biopsied embryos, using a specific laser system, emphasizing differences between the two methods and alerting on effective strategies to minimise potential laser induced damage. This is the first study to investigate the safety of 3 different laser systems for blastocyst biopsy by comparing implantation rates and by analysing cell viability using Fluorescence Microscopy and ultrastructure by Transmission Electron Microscopy(TEM).

Study design, size, duration
405 human blastocysts were biopsied on day 5 for PGT-A or PGT-SR or PGT-M using the Saturn laser (n = 145-58 cycles) or the Octax laser (n = 132-55 cycles) or the Lykos laser (n = 128-46 cycles). Following transfer of normal embryos, spare embryos, rejected for transfer due to being diagnosed as abnormal were processed following vitrification/warming for viability analysis (n = 30 Saturn laser, n = 30 Octax laser, n = 30 Lykos laser) or TEM (n = 20 Saturn laser, n = 20 Octax laser, n = 20 Lykos laser).

Participants/materials, setting, methods
The study was conducted in an academic hospital with an IVF/PGT laboratory, 2 private IVF Units and an IVF Unit of a Naval hospital. Blastocyst viability after biopsy/vitrification/warming was assessed using two fluorescent markers: carboxyfluorescein-diacetate succinimidylester (CFSE-viable cells) and propidium iodide (PI–damaged cells) in combination with DAPI to visualise DNA. TEM analysis was carried out following embryo fixation in gluteraldehyde, incubation in osmium, aqueous uranyl acetate, dehydration through ethanol series, and immersion in Epon.

Main results and the role of chance
The implantation rates (+ve hCG / ET) were not statistically different among the 3 laser systems (64.4% -Saturn, 63.4%- Octax, 64.7%- Lykos p &gt; 0.05). Viability staining with CFSE/PI following vitrification/warming revealed that all biopsied blastocysts had damaged cells at the position of cutting (range 4-8cells) and additional PI stained cells in other parts (range 2-15), possibly due to damage sustained during vitrification/warming. The majority of embryos across the 3 groups had ≤7% PI stained damaged cells (73.3% Saturn, 73.3% Octax, 76.7% Lykos), indicating no statistically significant difference in blastocyst viability among the 3 laser systems (p &gt; 0.05). No difference was also observed in the incidence of lysed biopsied TE cells or the PGT results. TEM analysis revealed similar ultrastracture among the 3 groups, as documented by the presence of normal organelles’ structures including smooth/rough endoplasmic reticulum, Golgi apparatus, mature mitochondria with cisternae, zona and microvilli. Multivesicular bodies, residual bodies, lipofuscins and autophagic vacuoles were also evident and indicative of elimination and rescue process. Excessive accumulation of glycogen granules and distension of mitochondria were more prominent in Grade B blastocysts compared to Grade A blastocysts, which may indicate a ‘block’ of glycogenolysis secondary to the mitochondrial alterations, suggesting changes in normal embryo metabolism.

Limitations, reasons for caution
The biopsied blastocysts used for viability and ultrastructural analysis in this study were all diagnosed with either chromosomal abnormalities or single gene defects following PGT-A or PGT-SR or PGT-M.

Wider implications of the findings
The similarities observed in implantation rates, the minimal levels of cell damage and the embryo quality dependent limited ultrastuctural anomalies identified confirm the safety of all three laser systems and emphasize the blastocysts’ ability to initiate elimination/rescue processes in their attempt to fully recover from ‘traumas’ sustained during biopsy/vitrification.

Trial registration number
not applicable