P–261 The human embryo following biopsy on day5 vs day3: Implantation, cytoskeleton, ultrastructure and effects of endometrial damage/inflammation on receptivity as revealed by scanning electron microscopy
Authors: Chatzimeletiou A , Sioga A , Nikas G , Petrogiannis N , Panagiotidis Y , Prapa M , Patrikiou A , Papanikolaou K , Zervakakou G , Kolibianakis E , Tarlatzis B , Grimbizis G 
Published: August 2021

Abstract

Study question
Are there any differences in implantation, cytoskeleton and ultrastructure of embryos biopsied on day5 vs day3 and how endometrial damage/inflammation may affect receptivity and implantation?

Summary answer
No differences are observed in implantation rates but vitrification following day5 biopsy led to more cytoskeletal/ultrastructural anomalies. Infections and epithelial damage severely affected endometrial receptivity.

What is known already
Successful implantation is dependent on the correct synchronization of the window of implantantation with the transfer of chromosomally/genetically normal embryos, in a well prepared receptive endometrium. This is the first study to examine the effects of day5 vs day3 embryo biopsy by comparing implantation/pregnancy rates and by analysing cytoskeleton using Confocal Laser Scanning Microscopy (CLSM), and ultrastructure by Transmission Electron Microscopy (TEM). In addition, Scanning Electron Microscopy (SEM) was used on endometrial biopsies to assess possible uterine pathologies/inflammation that may be responsible for the failed implantation after PGT-A/M and if subsequent treatment can increase implantation/pregnancy rates in succeeding PGT-A/M cycles.

Study design, size, duration
470 embryos were biopsied on day5 for PGT-A (n = 152–37 cycles) or on day3 for PGT-A (n = 162–29 cycles) and PGT-M (n = 156–22 cycles). Following transfer of normal embryos, spare embryos, rejected for transfer following day5 or day3 biopsy were processed for Cytoskeletal analysis (n = 30 fresh day3 biopsied, n = 30 day5 biopsied/vitrified) or TEM (n = 20 fresh day3 biopsied, n = 20 day5 biopsied/vitrified). Also, patients with a –ve hCG test, underwent endometrial biopsy to detect infection/inflammation and assess receptivity.

Participants/materials, setting, methods
Cytoskeletal analysis was performed by embryo immunostaining with α-tubulin, γ-tubulin, acetylated-tubulin antibodies and DAPI or/ PI 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. Endometrial biopsies were fixed in gluteraldehyde solution and processed for SEM using standard methods. The study was conducted in an academic hospital with an IVF/PGD laboratory and 3 private IVF Units.

Main results and the role of chance
162 embryos were biopsied on day 3 for PGT-A (29 cycles), 10 cycles had no normal embryos for transfer, 30 normal embryos were transferred in 19 cycles leading to 12/19 (63.2%) +vehCG/ET and 11/19 (57.9%) Ongoing pregnancy rate/ET. 156 embryos were biopsied on day 3 for PGT-M (22 cycles), 2 cycles had no normal embryos for transfer, 34 normal or carrier embryos were transferred in 20 cycles leading to 15/20 (75.0%) +vehCG/ET and 13/20 Ongoing pregnancy rate/ET (65.0%). 152 embryos were biopsied on day 5 for PGT-A (37 cycles), 8 cycles had no normal embryos for transfer, 34 normal embryos were transferred in 29 cycles leading to 18/29 (62.1%) +vehCG/ET and 16/29 (55.2%) Ongoing pregnancy rate /ET. Analysis of endometrial biopsies with SEM revealed bacterial infections, inflammation and epithelial damage. So far, 33.3% of patients who received intracavitary infusions-antibiotic treatment per os achieved a +vehCG/ET in their next PGT-A/M cycle. Cytoskeletal analysis showed that the majority of spindles examined in both day3 and day5 biopsied embryos were normal (85/114(74.6%) and 87/137(63.5). However vitrification following day5 biopsy led to more cytoskeletal/ultrastructural anomalies which included multipolar/abnormally shaped spindles, chromosome-bridging, chromosome-lagging and more vacuoles, lipofuscins. and distension of mitochondria.

Limitations, reasons for caution
Patients undergoing PGT-A have various aetiologies (heterogeneous group). The embryos used for cytoskeletal and ultrastructural analysis in this study were all diagnosed with either chromosomal abnormalities or single gene defects following PGT-A or PGT-M.
Wider implications of the findings: This is the first study to compare implantation/pregnancy rates, cytoskeleton and ultrastructure of day5 vs day3 biopsied embryos. The similarities observed in implantation/pregnancy rates, and the limited ultrastuctural and cytoskeletal anomalies identified confirm the procedures’ safety and indicate in certain cases endometrial factors/inflammation responsible for failed implantation following PGT-A/M.

Trial registration number
Not applicable