P-165 Artificial removal of the zona pellucida at the pronuclear stage in patients with poor quality embryos with severe fragmentation
Authors: Mae R , Mizuta S , Matsubayashi H , Ishikawa T 
Published: June 2023

Abstract

Study question
Is artificial removal of the zona pellucida (ZP) at the pronuclear stage effective in patients with poor quality embryos with severe fragmentation?

Summary answer
Artificial removal of the ZP at the pronuclear stage significantly improved culture results in patients with poor quality embryos with severe fragmentation.

What is known already
In 2020, Yumoto et al. first reported that artificial removal of the ZP (ZP-free) at the pronuclear stage decreased the rate of fragmentation and improved the culture outcomes of 3 pronuclei (3PN) embryos reaching to the good blastocysts. Therefore, the ZP may not always be necessary for normal embryonic development after the pronuclear stage. The same group presented at the 37th ESHRE meeting in 2021, that the blastocyst transfer after ZP-free embryo of 2 pronuclei (2PN) resulted in normal pregnancy in two patients with poor-quality embryos resulting in live births.

Study design, size, duration
This study included 211 of 2PN embryos from 46 cycles for 30 couples obtained between July 2021 and December 2022, whose previous oocyte retrieval cycle showed massive cytoplasmic fragmentation at the first cleavage confirmed by a time-laps incubator (TLI). Average age of women was 38.8years. The embryos with artificial removal of the ZP at the pronuclear stage were cultured in a TLI up to 7 days. All blastocysts were cryopreserved for future embryo transfer cycles.

Participants/materials, setting, methods
All participants provided written informed consent, and Institutional Review Board approval was obtained. Artificial removal of the ZP at the pronuclear stage was performed by following procedure. 1) Embryos were placed in 0.125M sucrose-containing HEPES for cytoplasm shrinking. 2) The ZP without adhesion was removed with a laser system. 3) Ooplasms were completely separated from ZP by blowing the medium to adhesion point like a jet car wash. 4) ZP-free embryo was obtained.

Main results and the role of chance
Day 3 embryos were divided into 3 groups using the Veeck’s classification: 8 cell embryos with grade 2 or higher were defined as good, embryos with grade 4 or 5 regardless of the number of cells were defined as poor, and others were defined as fair. We compared the culture results of ZP-free cycle and previous oocyte retrieval cycle (ZP-intact). For day 3 embryos, good, fair and poor grade were 19.4% (41/211), 62.6% (132/211) and 18.0% (38/211) in ZP-free embryos compared with 2.9% (15/561), 37.4% (210/561), and 59.9% (336/561) in the past, respectively (p &lt; 0.001). Good-quality blastocysts were defined as 3BB or more on day 5 by the Gardner’s classification. As to the blastocysts, blastocysts development and good-quality blastocysts rates were 45.0% (95/211) and 27.0% (57/211) in ZP-free embryos compared with 23.0% (112/488) and 6.8% (33/488) in the past, respectively (p &lt; 0.001). Of these, 30 embryo transfers were performed resulting in 11 clinical pregnancies. We had 3 live births without any congenital anomalies and one miscarriage. One was delivered at 33 weeks of gestation with caesarian section, one mother had gestational diabetes.

Limitations, reasons for caution
This study was retrospective study without any control group and was conducted at only single fertility center. It is necessary to conduct sibling oocytes study to demonstrate the effectiveness of ZP-free culture, and to create criteria who are benefit from ZP-free culture. Moreover, safety of ZP-free culture should be evaluated.

Wider implications of the findings
ZP-free culture reduced fragments and improved culture outcomes in patients with poor quality embryos with severe fragmentation. ZP-free culture might bring light to the patients who have difficulty obtaining good-quality embryos with severe fragmentation.

Trial registration number
Not Applicable