P-157 Improved embryo development and clinical outcome using bicarbonate buffer as the oocyte holding medium during Intracytoplasmic sperm injection (ICSI) compared to MOPS buffer
Authors: Mendola R , Walmsley R , Schimmel T , Sagerer E , Mullen C , Russell H , Angle M , Garrisi J 
Published: June 2023

Abstract

Study question
Does bicarbonate buffer used as the oocyte holding medium during ICSI result in improved embryo development and clinical outcome compared to the zwitterionic buffer MOPS?

Summary answer
Pre-equilibrated bicarbonate buffer is acceptable for ICSI use in atmospheric conditions, and results in superior sibling-oocyte cohort study outcomes compared to ICSI with MOPS buffer.

What is known already
Maintaining proper pH is of the utmost importance during IVF micromanipulation procedures. However, currently used zwitterionic buffers, HEPES and MOPS, may significantly influence intrinsic biological mechanisms outside pH maintenance, impacting oocyte competence, embryo development, and clinical outcome. Zwitterionic buffer influence is of concern during ICSI because piercing the oolemma allows the influx of the surrounding buffer into the oocyte (Mendola unpublished data, 2022). Bicarbonate is the physiological buffer for pH maintenance in all mammals providing numerous benefits for cellular development.

Study design, size, duration
An iSTAT handheld blood analyzer was used to analyze the pH shift of pre-equilibrated bicarbonate buffer dishes to determine the acceptablity for use during ICSI in atmospheric conditions. A clinical sibling-oocyte cohort study consisting of 115 patients and 1,997 oocytes (1,016 bicarbonate/981 MOPS) was analyzed to compare the embryo development and clinical outcomes of bicarbonate buffer vs. MOPS as the oocyte-holding medium during ICSI. Morphokinetic time points for all PGT-A tested embryos were also compared.

Participants/materials, setting, methods
We analyzed the pH shift of pre-equilibrated bicarbonate buffer in atmospheric conditions using 50x9mm culture dishes (Falcon, cat#351006) comparing light and high viscosity paraffin oil overlay, OvOil and Heavy OvOil (Vitrolife), after 5-minute, 10-minute, and 15-minute intervals. We analyzed all data points in triplicate. We analyzed the sibling-oocyte cohort study comparing fertilization rate, abnormal fertilization rate, useable blastocyst rate, mosaicism rate, euploidy/aneuploidy rate, morphokinetic time points, pregnancy rate, and ongoing pregnancy rate.

Main results and the role of chance
Pre-equilibrated (6.5%CO2/5.0%O2/7.301pH) bicarbonate buffer in 50 × 9mm ICSI culture dishes, with 5.0mL of OvOil overlay, maintained proper pH (7.301/7.328/7.354pH) for the time required to complete the ICSI procedure in atmospheric conditions (0min/5min/10min). The use of the high viscosity paraffin oil overlay, Heavy OvOil, reduced pH drift of the bicarbonate buffer ICSI dishes (7.301/7.323/7.342pH). In this clinical sibling-oocyte cohort study, ICSI was performed in pre-equilibrated bicarbonate buffer culture drops (6µL) in the 50x9mm dishes with 5.0mL Heavy OvOil overlay.
Utilization of bicarbonate buffer as the oocyte-holding medium during ICSI is superior to MOPS buffer. Bicarbonate buffer used with ICSI resulted in higher fertilization rates (85.4% vs. 78.8%, P&lt;0.0001), higher useable blastocyst rate per ICSI oocyte (50.6% vs. 46.2%, P&lt;0.05), lower abnormal fertilization rate (4.5% vs. 6.7%, P&lt;0.05), and a lower whole chromosome mosaicism rate (4.2% vs. 8.2%, P&lt;0.03) compared to the MOPS buffer. Pregnancy rates were higher in the bicarbonate group compared to MOPS but not statistically significant. Morphokinetic analysis revealed a slight delay in all MOPS developmental time points compared to bicarbonate, but only statistically significant in the time to compaction (P&lt;0.03).

Limitations, reasons for caution
During this study, different genetic testing companies, with differing mosaicism thresholds and decisions on included ploidy results, were used for PGT-A analysis of patient samples.

Wider implications of the findings
We determined that pre-equilibrated bicarbonate buffer can be used for ICSI in atmospheric conditions, therefore avoiding the potential detrimental effect of zwitterionic buffer after influx into the oocyte from the ICSI procedure. Bicarbonate buffer used during ICSI was determined to be superior to MOPS buffer.

Trial registration number
Not applicable