P-249 Exploring the possible impact of single-step culture media refreshment on embryo ploidy: A prospective sibling oocyte study
Authors: Elkhatib I , Nogueira D , Bayram A , Abdala A , Ferracuti V , Melado L , Kalafat E , Ata M B , Lawrenz B , Fatemi H 
Published: June 2023

Abstract

Study question
Does refreshing the media on day 3 of development improve rates of blastocyst utilization and euploidy rate?

Summary answer
Refreshing single-step culture medium on day 3 of embryo development does not improve blastocyst euploidy rate, neither does it improve embryo utilization rate.

What is known already
The impact of culture conditions on blastocyst development and aneuploidy is not fully understood. Single-step culture media provide embryos with all the nutrients required throughout the development. The debated practice till today is whether refreshing media during the seven days of culture is required to compensate for possible, even subtle, osmolarity changes, and to replenish nutrients and exchange metabolites, alleviating embryos from a possible toxic environment. Up to date, no study has investigated the effect of using the same type of culture media, refreshing it or not, in a sibling oocyte manner, on blastocyst developmental quality and embryo ploidy.

Study design, size, duration
A prospective sibling oocyte study, conducted between July 2021 and December 2022 in a tertiary IVF center. A total of 1549 metaphase II (MII) oocytes from 93 PGT-A cycles were randomized between the 2 groups. Outcomes were defined as euploid rate, mosaicism rate and blastocyst utilization rate.

Participants/materials, setting, methods
Patients undergoing ICSI and PGT-A with females aged 18-40yrs and BMI&lt;35kg/m2 who signed the informed consent. MII oocytes were randomized between Group 1(GI): embryos were moved into a newly calibrated culture media only on day 3, 67-69 hours post ICSI (hpi), and Group 2(GII): only on day 5, 114-118hpi. ICSI was performed by the same operator for both groups. Fertilization check was done 18-20hpi and no further assessment was done till day 5.

Main results and the role of chance
Patients and cycle characteristics presented as median (IQR) were female age 31yrs (28-34yrs), BMI 26.5 kg/m2 (23-28.6 kg/m2), AMH 3.4ng/ml (2.1-5.9ng/ml), cumulus-oocyte complex (COC) 21 (14-26) and oocyte maturation rate 0.8 (0.7-0.9). In GI, 775 MIIs and in GII, 774 MIIs were inseminated by ICSI. Fertilization rates were similar in both groups (77.2% vs 78.7%, mean difference: -1.5%, 95% CI: -5.9% to 2.9%, GI vs GII, respectively, P = 0.499). No significant differences were observed in day 5 blastulation rates between groups (66.3% vs 63.9%, mean difference: 2.4%, 95% CI: -3.7% to 8.5%, G1 vs G2, respectively, P = 0.436). Blastocyst utilization rate (blastocysts biopsied per 2PN) were similar between the groups (52.6% vs 50.7%, mean difference: 1.8%, 95% CI: -4.4% to 8.0%, G1 vs G2, respectively, P = 0.567). PGT-A outcomes were defined as euploid, aneuploid or mosaic aneuploid. Overall euploid rates were similar between GI and GII (57.86% vs 59.5%, p = 0.68, respectively), as well as mosaicism rates (7.7% vs 6.8%, p = 0.67, respectively). The rate of blastocysts biopsied on day 5,6 or 7 was comparable between both GI and GII (p = 0.62, 0.83 and 0.59, respectively).

Limitations, reasons for caution
While the outcome of this study did not show improvement in euploid rate or blastocyst utilization rate by refreshing the culture medium on day 3, the results may not be reproducible in other laboratories applying different protocols since culture conditions and protocols may differ among laboratories.

Wider implications of the findings
The fact that there are no significant differences in blastulation and euploid rates between the groups, supports the stability of culture conditions without the need for media refreshment and allows for reducing embryo manipulation, workload and costs adhered by culture medium refreshment on day 3.

Trial registration number
NCT04969575