O-064 Can artificial removal of the zona pellucida with severe fragmentation improve culture and pregnancy outcomes?
Authors: Mizuta S , Mae R , Maekawa T , Matsubayashi H , Takeuchi T , Ishikawa T 
Published: July 2024

Abstract

Study question
Can artificial removal of the zona pellucida (ZP-free) in patients whose embryos had severe fragmentation improve culture and pregnancy outcomes?

Summary answer
In patients with severe fragmentation, blastulation and live birth rate were improved by ZP-free procedure, and 14 live babieshave been obtained so far.

What is known already
In 2020, Yumoto et al. first reported that ZP-free procedure at the pronuclear stage decreased the rate of fragmentation and improved blastulation of 3 pronuclei embryos. 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 transfer of blastocyst derived from 2 pronuclei embryos (2PN) after ZP-free procedure resulted in normal pregnancy and live births in two patients who had had poor-quality embryos only before ZP-free procedure. We also presented culture outcomes improved by ZP-free procedure at the 2023 ESHRE meeting.

Study design, size, duration
This study included 571 embryos derived from 2PN on 118 cycles in 61 couples obtained between July 2021 and November 2023, whose previous oocyte retrieval cycle had shown massive cytoplasmic fragmentation at the first cleavage confirmed by a time-laps incubator (TLI) in two institutes. Mean age of women was 40.0±3.8 years. Culture and pregnancy outcomes after blastocyst transfer were compared with 1272 embryos derived from 2PN in the past 247 cycles without ZP-free procedure (ZP-intact).

Participants/materials, setting, methods
All participants provided written informed consent, and IRB approval was obtained. Embryos were placed in 0.125M sucrose-containing HEPES for cytoplasm shrinking at the pronuclear stage. The ZP was partially removed in non-adhesive areas with a laser system. Ooplasms were completely separated from ZP by blowing the medium to adhesion point like a jet car wash. The ZP-free embryos were cultured in TLI up to 7 days, followed by blastocyst cryopreservation for future embryo transfer cycles.

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 outcomes of ZP-free cycle and previous ZP-intact cycle. For day 3 embryos, good, fair and poor grade were 15.6%, 55.3% and 29.1% in ZP-free embryos compared with 3.7%, 42.5%, and 53.8% in ZP-intact embryos, respectively (p &lt; 0.001). As to the blastocysts, blastocysts development and good-quality blastocysts (G3BB or more on day 5 by the Gardner’s classification) rates were 36.9% and 19.4% in ZP-free embryos compared with 23.1% and 6.0% in ZP-intact embryos, respectively (p &lt; 0.001). Clinical pregnancy rates for ZP-free and ZP-intact blastocyst transfer were 36.1% (22/61) and 15.0% (17/113), miscarriage rates were 18.2% (4/22) and 82.4% (14/17), and ongoing pregnancy rates were 29.5% (18/61) and 2.7% (3/113), respectively, with statistical significance after ZPF procedure (P &lt; 0.01). Fourteen live babies have given birth by single blastocyst transfer with ZP-free procedure so far.

Limitations, reasons for caution
This study was retrospective study without any control group and was conducted at only two fertility centers. It is necessary to conduct sibling oocytes study or extensive randomized controlled trial to create criteria for patients who are benefit from ZP-free procedure. Moreover, safety of ZP-free culture should be evaluated.

Wider implications of the findings
ZP-free procedure in patients with severe fragmentated embryos at the pronuclear stage, reduced fragmentation and improved blastocyst development and clinical outcomes. We suggest that ZP-free embryos could be proposed as a new treatment option for patients who have had poor quality embryos with severe fragmentation.

Trial registration number
Not applicable