P-264 Analysis of embryo development based on time of pronuclei appearance (tPNa) and aspects of overcoming delayed pronuclei appearance through morphokinetic patterns
Authors: Jung N  H , Jeong S  H , Kim M  J , Jeong H  J , Kim M  H , Lee H  S , Chung M  K 
Published: July 2024

Abstract

Study question
How much delay in the appearance of 2PN can be considered normal when predicting embryo transfer potential?

Summary answer
Embryos with tPNa &lt;13hpi post-insemination develop into good-quality blastocysts; embryos with tPNa ≥13hpi develop blastocysts by shortening the time of tPNf-tPNa.

What is known already
Oocytes generally exhibit 2PNs at 17–20 hours, with pronuclei fading (time of pronuclei fading; tPNf) at 23–25 hours and developing into two cells (time of 2-cell division; t2) at 25–33 hours. Fertilization confirmation is typically performed 17–18 hours after insemination. However, a few oocytes with no visible PN (0PN) at the time of fertilization confirmation develop morphologically normal blastocysts, ultimately leading to pregnancy. The failure to identify PNs can be attributed to two scenarios: rapid fading or delayed appearance. Notably, there is still a lack of research on the normal range for delayed PN appearance.

Study design, size, duration
This study was conducted with 2153 embryos obtained from 390 intracytoplasmic sperm injection cycles (August 2021 to June 2023). All embryos were incubated for 5 days using a time-lapse system (EmbryoScopeTM+, Vitrolife). The blastocyst development rate and morphokinetic parameters according to the tPNa of embryos were analyzed using KIDScore D5 v3 and iDAScore v2.0 (VTH server+, Vitrolife). Clinical pregnancy was also analyzed.

Participants/materials, setting, methods
Morphokinetic parameters were analyzed from time of second polar body (tPB2) ∼ time of expanded blastocyst (tEB). The times taken for PN to appear after the second polar body release (tPNa–tPB2), for PN to fade (tPNf–tPNa) and for 2-cell division (t2–tPNf) were calculated and compared. Blastocysts were graded using the Gardner system, a grade of BB or higher divided into good quality blastocysts (GQ-BL).

Main results and the role of chance
tPNa was observed as 8.09±2.11hpi [1.93hpi∼32.46hpi; &lt;5hpi (n = 49), 5∼6hpi (n = 474), 7∼8hpi (n = 1220), 9∼10hpi (n = 266), 11∼12hpi (n = 91), 13∼14hpi (n = 29), 15∼20hpi (n = 18) and &gt;20hpi (n = 6)]. The rate of blastocysts was highest at 5∼6hpi (64.98%) and significantly lower at 9∼10hpi (54.14%), 11∼12hpi (42.86%) and 13∼14hpi (31.03%) (p &lt; 0.005). Similarly, the rate of GQ-BL was also highest at 5∼6hpi (29.11%) and significantly lower at 9∼10hpi (19.17%), 11∼12hpi (10.99%) and 13∼14hpi (3.45%) (p &lt; 0.005). No embryos developed into GQ-BL at 15∼20hpi, and no embryos developed into blastocysts at &gt; 20hpi. The iDAScore was significantly different at &lt; 13hpi and ≥13hpi (6.00±1.86 vs. 4.24±2.26, p &lt; 0.005). Similarly, KIDScore D5 showed the same patterns (6.33±1.93 vs. 4.05±1.66, p &lt; 0.005). At ≥ 13hpi, no blastocysts led to pregnancy.
Morphokinetic parameters were analyzed to identify the factors influencing the development of blastocysts. The analysis revealed that tPNf-tPNa tended to gradually become shorter with delayed tPNa. Notably, there were significant differences in tPNf-tPNa between &lt;13hpi and ≥13hpi (16.37±4.10 vs. 13.72±6.28, p &lt; 0.005). At ≥ 13hpi, tPNf-tPNa was shorter in blastocysts than in cases of cleavage arrest (10.68±5.74 vs. 15.28±5.97, p &lt; 0.05). It was particularly observed that tPNf-tPNa of GQ-BL in ≥ 13hpi was 7.90hpi, and t2 was not significantly different from blastocysts and GQ-BL in &lt; 13hpi (25.06 vs. 25.66±3.18 vs. 25.19±2.97).

Limitations, reasons for caution
More data are needed for conclusive pregnancy results due to the small number of samples. Additionally, the maturation of oocytes had yet to be considered; further detailed studies related to oocyte maturity are needed.

Wider implications of the findings
Embryos with a tPNa of &lt; 13hpi develop into GQ-BL. Embryos with delayed tPNa tend to overcome this by shortening the tPNf-tPNa, resulting in develop into blastocysts. Predicting blastocyst development can be achieved by considering factors such as tPNa∼t2. This can aid in improving the selection of embryos for cleavage-stage ET.

Trial registration number
N/A