O-119 Time-lapse comprehensive portrait of cytoplasmic strings appearing during blastulation: prevalence and implications for embryo assessment.
Authors: Marconetto A , Innocenti F , Saturno G , Taggi M , Chiappetta V , Albricci L , Maggiulli R , Soscia D , Casciani V , De Falco F , Coticchio G , Ahlström A , Ubaldi F M , Rienzi L , Cimadomo D 
Published: June 2023

Abstract

Study question
What are the prevalence and implications of cytoplasmic strings (Cyt-S) in preimplantation development after blastulation?

Summary answer
Cyt-S are common in human embryos and are associated with faster blastocyst development, larger expansion, and better morphological quality.

What is known already
Cyt-S are dynamic cellular projections connecting inner-cell-mass (ICM) and trophectoderm during blastocyst expansion. Their prevalence in human embryos in vitro has been estimated 44-93%. Currently, their role is unknown. Studies in animal models suggested they might be involved in ICM-to-trophectoderm communication or cell fate-related events. Nevertheless, comprehensive descriptions and clear definitions, evidence-based hypotheses about their function, and data about their clinical implications are lacking, limited or controversial. Here we leveraged time-lapse microscopy, artificial intelligence, and chromosomal testing to comprehensively portray these common features of blastulation and expansion processes.

Study design, size, duration
Observational study involving 124 PGT-A cycles in EmbryoScope (Vitrolife) with ≥1 blastocyst (N = 315) between May-2013 and November-2014. Timings from tSB to biopsy (t-biopsy, i.e., blastocyst full-expansion) in hours-post-insemination (hpi) and embryo area (including zona-pellucida in µm2) were automatically annotated through an AI-based software (CHLOE™, Fairtility). One senior embryologist annotated Cyt-S presence, number, timings, and type (thick cell-to-cell connections and/or threads), and blastocyst collapses (i.e., reduction of embryo area preventing the discrimination of ICM from trophectoderm).

Participants/materials, setting, methods
ICSI, continuous blastocyst culture (day 5-7) in time-lapse incubators, trophectoderm biopsy without zona-pellucida drilling on fully-expanded blastocysts, and qPCR to assess non-mosaic full-chromosome aneuploidies were all conducted. Blastocyst morphological quality was defined according to Gardner’s schemes as excellent (AA), good (AB,BA), average (BB,AC,CA) and poor (CC,BC,CB). Only vitrified-warmed euploid single-embryo-transfers were performed. Along with developmental timings, extent of expansion and morphological quality, we also assessed euploidy and live-birth rates in blastocysts with and without Cyt-S.

Main results and the role of chance
94.4% of the patients (N = 117/124) had ≥1 embryo with Cyt-S (mean:2.2±1.6, range:0-9). 86% of all blastocysts analyzed (N = 271/315) had ≥1 Cyt-S (3.5±2.1, 1-13; duration of the longest Cyt-S: 5.8±3.4 hours, 0.5-20.9). Overall, we analyzed 937 Cyt-S detected at 117.3±11.8 hpi (85.7-160.5) and lasting 3.8±2.7 hours (0.2-20.9). The first Cyt-S of each embryo lasted longer (4.4±3.2 hours) compared to the following (≈3 hours or less). Cyt-S were mostly threads (N = 133/271, 54.2%) or thick cell-to-cell connections becoming threads (N = 382/937, 40.8%) than connections of constant diameter (N = 47/937, 5%). Of the 271 embryos with ≥1 Cyt-S, 71.9% showed these projections only during early expansion processes after tSB (N = 195; 3.0±1.6, 1-10), 7.7% only after collapses (N = 21; 2.6±1.5, 1-6), and 20.3% both in early expansion and after collapses (N = 55; 2.3±1.3 and 3.2±2.4, 1-11 respectively). Poor- versus excellent-quality blastocysts (N = 54/75, 72% versus N = 120/129, 93%, multivariate-OR=0.32, 95%CI 0.11-0.92, adjusted-p=0.03), t-biopsy (multivariate-OR=0.97, 95%CI 0.95-0.99, p = 0.05) and blastocyst area at t-biopsy (multivariate-OR=1.1, 95%CI 1.01-1.18, p = 0.02) were all associated with the prevalence of embryos with Cyt-S. Lastly, while euploidy rates were comparable between blastocysts with and without Cyt-S, an association instead was shown with live-birth-rates per vitrified-warmed euploid transfers (N = 34/77, 44.2% versus N = 0/10, 0%; p &lt; 0.01), although these data are very preliminary.

Limitations, reasons for caution
Some Cyt-S after collapses might correspond to connections already present before these events. Larger datasets are required to confirm putative associations between Cyt-S and clinical outcomes. Only associations could be reported here, but not causations/consequences. Specific basic research studies are required to this end

Wider implications of the findings
Embryos with ≥1 Cyt-S were faster in reaching all blastocyst timings (tSB to t-biopsy) and to expand, achieved a larger area at t-biopsy and had a better morphology. These dynamic features of blastocyst expansion might be physiological and represent biomarkers of embryo quality. Nevertheless, their function is yet unknown

Trial registration number
not applicable