Cytoplasmic strings in human blastocysts: hypotheses of their role and implications for embryo selection.

STUDY QUESTION: What are the implications of the presence cytoplasmic strings (Cyt-S) and their quantity and dynamics for the pre-implantation development of human blastocysts? 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 and trophectoderm (TE) cells, that can be observed during blastocyst expansion. Their prevalence in human embryos has been estimated to be between 44% and 93%. Data relevant to their clinical implications and role in development are lacking, limited, or controversial. STUDY DESIGN, SIZE, DURATION: Retrospective study conducted at a single IVF center between May 2013 and November 2014 and involving 124 pre-implantation genetic testing for aneuploidy cycles in a time-lapse incubator with ≥1 blastocyst biopsied and vitrified (N = 370 embryos assessed). These cycles resulted in 87 vitrified-warmed single-euploid blastocyst transfers. PARTICIPANTS/MATERIALS, SETTING, METHODS: ICSI, continuous blastocyst culture (Days 5-7), TE biopsy of fully expanded blastocysts without Day 3 zona pellucida drilling, qPCR to assess uniform full-chromosome aneuploidies, and vitrification were all performed. Only vitrified-warmed euploid single-embryo-transfers were conducted. Blastocyst morphological quality was defined according to Gardner's criteria. The AI-based software CHLOE™ (Fairtility) automatically registered timings from time of starting blastulation (tSB) to biopsy (t-biopsy, i.e. blastocyst full-expansion) as hours-post-insemination (hpi), embryo area (including zona pellucida in µm2), and spontaneous blastocyst collapses. One senior embryologist manually annotated Cyt-S presence, quantity, timings, and type (thick cell-to-cell connections and/or threads). All significant associations were confirmed through regression analyses. All couples', cycles', and embryos' main features were also tested for associations with Cyt-S presence, quantity, and dynamics. MAIN RESULTS AND THE ROLE OF CHANCE: About 94.3% of the patients (N = 117/124) had ≥1 embryo with Cyt-S. Out of a total of 370 blastocysts, 55 degenerated between blastulation and full-expansion (N = 55/370, 14.9%). The degeneration rate among embryos with ≥1 Cyt-S was 10.8% (N = 33/304), significantly lower than that of embryos without Cyt-S (33.3%, N = 22/66, P < 0.01). Of the remaining 315 viable blastocysts analyzed, 86% (N = 271/315; P < 0.01) had ≥1 Cyt-S, on average 3.5 ± 2.1 per embryo ranging 1-13. The first Cyt-S per viable embryo appeared at 115.3 ± 12.5 hpi (85.7-157.7), corresponding to 10.5 ± 5.8 h (0.5-31) after tSB. Overall, we analyzed 937 Cyt-S showing a mean duration of 3.8 ± 2.7 h (0.3-20.9). Cyt-S were mostly threads (N = 508/937, 54.2%) or thick cell-to-cell connections becoming threads (N = 382/937, 40.8%) than thick bridges (N = 47/937, 5.0%). The presence and quantity of Cyt-S were significantly associated with developmentally faster (on average 6-12 h faster) and more expanded (on average 2700 µm2-larger blastocyst's area at t-biopsy) embryos. Also, the presence and duration of Cyt-S were associated with better morphology. Lastly, while euploidy rates were comparable between blastocysts with and without Cyt-S, all euploid blastocysts transferred from the latter group failed to implant (N = 10). LIMITATIONS, REASONS FOR CAUTION: Cyt-S presence and dynamics were assessed manually on seven focal planes from video frames recorded every 15 min. The patients included were mostly of advanced maternal age. Only associations could be reported, but no causations/consequences. Lastly, larger datasets are required to better assess Cyt-S associations with clinical outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Cyt-S are common during human blastocyst expansion, suggesting their physiological implication in this process. Their presence, quantity and dynamics mirror embryo viability, and morphological quality, yet their role is still unknown. Future basic science studies are encouraged to finally describe Cyt-S molecular nature and biophysical properties, and Artificial Intelligence tools should aid these studies by incorporating Cyt-S assessment. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

The importance of cytoplasmic strings during early human embryonic development.

Objectives: During human in vitro fertilisation (IVF) treatments, embryologists attempt to select the most viable embryos for embryo transfer (ET). Previously, embryos were evaluated based on light microscopic morphological parameters. However, this is currently accomplished by morphokinetic analysis of time-lapse recordings. This technique provides us the opportunity to observe cytoplasmic strings at the blastocyst stage. The aim of this work was to examine the relationship between the presence of cytoplasmic strings (CS) and the embryo viability in human in vitro fertilised embryos. Study design: Herein, we present an evaluation of the morphokinetic data on the development of embryos obtained during IVF treatments performed at the Division of Assisted Reproduction between December 2020 and March 2021. The dynamics of embryo development, embryo morphology, and morphokinetic scores generated by a time-lapse system were compared between the presence of cytoplasmic strings (CS+) and their absence (CS-) at the blastocyst stage. Results: The development of 208 embryos from 78 patients was examined. Moreover, 81.2% of the embryos had CS in the blastocyst stage; 77% of CS existed in embryos created by conventional IVF, while 86% of CS existed in embryos fertilised by intracytoplasmic sperm injection (ICSI) (p = 0.08). A greater number of CS+ embryos developed into a higher quality blastocyst (52.1% vs. 20.5%, p = 0.02). The morphokinetic score values characterising the development of embryos, such as Known Implantation Data Score (KIDScore) and Intelligent Data Analysis (iDAScore), were higher in CS+ groups (KID: 6.1 ± 2.1 vs. 4.7 ± 2.07; iDA: 8.0 ± 1.9 vs. 6.8 ± 2.3, p < 0.01). The dynamics of the early embryo development were similar between the two groups; however, CS+ embryos reached the blastocyst stage significantly earlier (tB: 103.9 h vs. tB: 107.6 h; p = 0.001). Conclusion: Based on our results, the number of embryos with cytoplasmic strings was higher than that without cytoplasmic strings, and its presence is not related to the fertilisation method. These embryos reached the blastocyst stage earlier, and their morphokinetic (KIDScore and iDAScore) parameters were better. All these results suggest that the presence of CS indicates higher embryo viability. The examination of this feature may help us make decisions about the embryos with higher implantation potential.

Inter- and Intra-Observer Agreement Between Embryologists for Cytoplasmic String Assessment in Day 5/6 Human Blastocysts.

To investigate inter- and intra-observer agreement in the assessment of cytoplasmic string (CS) by embryologists on day 5/6 human blastocysts using the EmbryoViewer software. This was a prospective study involving five embryologists working between 2019 and 2020. Inter-observer agreement was calculated using assessments performed on 104 day 5/6 blastocysts regarding the presence, number, and location of CS and CS vesicle activity using timelapse videos. Intra-observer agreement was calculated when the same embryologists repeated the observations after a month's break. Inter- and intra-observer agreement was assessed using Fleiss' kappa coefficient and the intra-class correlation coefficient (ICC). The inter-observer agreement on the presence of CS (kappa: 0.477, 95% CI: 0.301-0.639) and their vesicles (kappa: 0.494, 95% CI: 0.345-0.643) was moderate, while the specific characteristics of CS assessment ranged from fair to moderate (kappa scores between: 0.157 and 0.563). The intra-observer agreement indicated an improvement on the level of agreement (kappa scores between: 0.162 and 0.795) compared to the inter-observer agreement. This study has shown a moderate level of inter- and intra-observer agreement when assessing day 5/6 human blastocysts for the presence of CS and their vesicles. When the specific characteristics of CS assessment occurred (such as the number of CS/vesicles) a slight to moderate level of agreement was seen among the embryologists. Agreement of specific characteristics of CS was not optimal, suggesting the need for further training using specifically designed CS quality assurance programme (QAP) modules, to determine if inter- and intra-observer agreement can be improved.

The presence of cytoplasmic strings in human blastocysts is associated with the probability of clinical pregnancy with fetal heart.

PURPOSE: Is the presence of cytoplasmic strings (CS) in human blastocysts associated with the probability of clinical pregnancy with fetal heart (CPFH) after transfer. METHODS: This case-control study involved 300 single blastocyst transfers. 150 of these resulted in a CPFH (cases) while 150 did not (controls). All embryos were cultured in Embryoscope+ and AI software (IVY) was used to select the blastocyst with the highest score from the cohort for transfer. An embryologist, blind to the transfer outcome, recorded the CS number, location, and duration of their activity. RESULTS: There was a significant difference in the number of blastocysts that contained CS, with 97.3% of women's blastocysts resulting in +CPFH containing the CS compared to 88.7% of blastocysts in women who did not have a pregnancy (p = 0.007, OR; 4.67, CI 95% 1.5-14.2). CS appeared 2.4 h earlier in embryo development in the +CPFH group compared to their negative counterparts (p = 0.007). There was a significant difference in the average number of CS/blastocyst with a higher number being present in those that achieved a clinical pregnancy (mean: 6.2, SD 2.9) compared to those that did not (mean: 4.6, SD 3.0) (p ≤ 0.0001). There was a significant increase in the number of vesicles seen traveling along the CS with more seen in the blastocysts resulting in a +CPFH (mean: 4.3 SD 2.1) compared to those in the -CPFH group (mean: 3.1, SD 2.1). CONCLUSION: This study has shown that the presence of cytoplasmic strings in human blastocysts is associated with the probability of clinical pregnancy with fetal heart.

Time-lapse imaging of cytoplasmic strings at the blastocyst stage suggests their association with spontaneous blastocoel collapse.

RESEARCH QUESTION: To study the origin and temporal behaviour of cytoplasmic strings spanning the blastocoel (main objective) and their influence on treatment outcome (secondary objective). DESIGN: This retrospective analysis of prospectively collected data was set up in a university medical centre. Patients who either underwent fresh (n = 95) or vitrified-warmed (n = 55) single blastocyst transfer were included. Time-lapse sequences of in-vitro developed blastocysts were screened for the presence of cytoplasmic strings. Pregnancies in string-positive and string-negative transfers were followed up to live birth. RESULTS: A total of 387 blastocysts were obtained in the fresh cycles of 100 patients, corresponding to a blastocyst formation rate of 62.4%. Cytoplasmic strings were first detected around full stage (108.5 ± 6.4 h) in 170 blastocysts (43.9%). The number of strings varied (range: 1-7) and the duration of visibility was 5.2 ± 3.5 h. The occurrence of cytoplasmic strings was significantly associated with the presence of blastocoelic collapses (P < 0.001) but not with any of the annotated morphokinetic parameters. Live birth and neonatal outcome were the same for both string-positive and string-negative pregnancies. Moreover, collapses did not affect treatment outcome. CONCLUSION: Time-lapse analysis of cytoplasmic strings at the blastocyst stage revealed that this morphological feature was not a negative predictor as previously reported. Although physiologically normal, at least some of the cytoplasmic strings are an artefact, possibly associated with blastocoelic collapses.