Blastocyst Re-expansion Research Articles

P-133 Impaired blastocyst viability induced by vitrification during re-expansion after trophectoderm biopsy can be prevented by artificial shrinkage before vitrification

Abstract Study question Does the duration between trophectoderm biopsy and vitrification or blastocyst re-expansion affect blastocyst adhesion and outgrowth competence? Summary answer Outgrowth was impacted when blastocysts were vitrified during re-expansion, especially at 2 h post-biopsy; however, this can be mitigated using artificial shrinkage pre-vitrification. What is known already Biopsied blastocysts are usually vitrified until the results of preimplantation genetic testing for aneuploidy are available. The viability of biopsied blastocysts after vitrification is affected by various factors; in fact, a rodent study reported that the duration from biopsy to vitrification and re-expansion at vitrification affected subsequent developmental competence. The vitrification of biopsied blastocysts immediately post-biopsy is recommended in the clinical setting. However, in some cases, embryologists cannot perform vitrification immediately after a biopsy because of other procedures. In this case, blastocysts start re-expanding, which requires embryologists to wait for the completion of the blastocyst re-expansion. Study design, size, duration Human blastocysts donated for research by consenting couples were randomly allocated into biopsied (n = 175) and non-biopsied (control; n = 50) groups. The re-expansion degree (%) at vitrification was calculated using the following formula: [1- (distance of perivitelline cavity at vitrification)/(distance of perivitelline cavity post-biopsy)] × 100. The blastocysts were categorized according to expansion degree: I, ≤0%; II, 1–49%; III, 50–99%; and IV, ≥100%. Participants/materials, setting, methods Biopsied and control blastocysts were vitrified 0–3 h post-biopsy. Some blastocysts were artificially shrunken in hypertonic solution before the vitrification. The blastocysts were warmed and plated on fibronectin-coated dishes and cultured for 96 h to assess the competence for blastocyst adhesion and outgrowth. The correlations of outgrowth outcomes with vitrification timing, re-expansion degree, and artificial shrinkage were retrospectively analysed. Main results and the role of chance No significant intergroup difference was observed in blastocyst adhesion rate. The multivariate linear regression analysis showed that the blastocyst outgrowth area was significantly smaller when the blastocysts were vitrified at 2 h post-biopsy (P = 0.0103). Re-expansion degree was positively correlated with duration after biopsy (Spearman’s rank correlation coefficient, 0.6921; P < 0.0001). The proportion of expansion in group III was significantly higher at 2 h post-biopsy (P < 0.0001). On multivariate linear regression analysis, the blastocyst outgrowth area was significantly smaller in group III (P = 0.0134). The outgrowth area of blastocysts vitrified at 2 h post-biopsy was significantly increased by artificial shrinkage before vitrification (P = 0.0025) and comparable with those of blastocysts vitrified at 0, 1, and 3 h post-biopsy. Multivariate linear regression analysis also demonstrated the effectiveness of artificial shrinkage for blastocysts vitrified at 2 h post-biopsy and for blastocysts for which re-expansion was initiated at vitrification. Limitations, reasons for caution Since this study used discarded vitrified blastocysts, double vitrification was performed. This was an experimental study; therefore, clinical studies are required to validate our findings. Wider implications of the findings The blastocysts are recommended to be vitrified before the initiation of re-expansion, which complicates the laboratory workflow. Our data demonstrate that blastocysts that expand post-biopsy can be vitrified after artificial shrinkage without decreasing their implantation competence. Our findings may contribute to improving the day-to-day practice in embryology laboratories. Trial registration number Not Applicable

P-263 Assisted hatching, embryo quality, and re-expansion: Exploring their impact on frozen embryo transfer outcomes and predictive factors for implantation

Abstract Study question Do post-warming assisted hatching, embryo re-expansion grade and quality affect clinical pregnancy rates in frozen embryo transfer cycles? Summary answer Assisted hatching (AH) improves clinical outcomes in poor-quality embryos that struggle to hatch on their own. Embryo re-expansion grade and quality are predictive for implantation. What is known already In recent years, the use of frozen embryo transfer has grown considerably. Pre-vitrification morphological classification is a well-established method for assessing embryo quality and predicting treatment outcomes. However, there is limited evidence regarding the prognostic value of blastocyst characteristics after thawing. Embryo vitrification has revolutionized assisted reproductive technology. However, this technique can stiffen the embryo’s zona pellucida, making hatching and implantation in the uterus more challenging. To facilitate these processes, AH is performed in laboratories. While research has been conducted on AH’s efficacy, controversy remains, and it is not yet clear when it is beneficial for embryos. Study design, size, duration Retrospective observational study of 486 patients undergoing single embryo cryotransfers between January 2022 and December 2023. Patients were divided into two groups: AH-NO (n = 246) and AH-YES (n = 240). In the AH-YES group, a 1/4 opening of the zona pellucida was performed using laser shots (Octax-NaviLase®) after thawing. Images of embryos cultured for at least 4 hours (x = 5.23 hours) in a time-lapse incubator (EmbryoScope+™, Vitrolife) were analysed from the time of thawing until the time of transfer. Participants/materials, setting, methods Blastocyst morphology was assessed on two categories: good quality (GQ) embryos (expansion 4-6, inner cell mass (ICM) and trophectoderm (TE) A/B) and poor quality (BQ) embryos (expansion 3-6, ICM and TE C) according to the Gardner-Schoolcraft classification. Blastocyst re-expansion was evaluated in three categories: fully re-expanded (F), partially re-expanded, 30-70% re-expansion (P) and unre-expanded/collapsed (N) embryos following thawing before transfer. Statistical analysis was performed using the Chi-squared test. Main results and the role of chance In the comparative analysis of clinical data regarding whether or not AH was performed after thawing, there were no significant differences in implantation rates (AH-YES: 58.33%, AH-NO: 58.54%, p > 0.05) or miscarriage rates (14.39% vs 15.97%, p > 0.05) between the two groups. When comparing by embryo quality, there were no differences in implantation rates of good-quality embryos AH-YES: 60.59%(n = 216) vs AH-NO: 60.34%(n = 237). However, in poor-quality embryos, there was an improvement in the implantation rate in those in which AH had been performed, AH-YES: 41.67%(n = 24) vs AH-NO: 11,11%(n = 9), although no statistical difference was observed, likely due to the sample size. When comparing by degree of re-expansion, there were significant differences (p < 0.05) in implantation rates of completely re-expanded embryos AH-YES: 61.31% (n = 168) vs AH-NO: 62.93% (n = 205), partially re-expanded embryos AH-YES: 56.26% (n = 54) vs AH-NO 39.29% (n = 28), and unre-expanded or collapsed embryos AH-YES: 23.53% (n = 17) vs AH-NO: 33.33% (n = 12). Limitations, reasons for caution The study is limited by its retrospective nature and the low number of poor-quality embryos transferred as they are the last option for transfer. Additionally, it is common to transfer more than one poor-quality embryo to increase the chances of pregnancy, therefore losing implantation track. Wider implications of the findings Assisted hatching (AH) could benefit poor-quality embryos, and the degree of re-expansion before embryo transfer could be a predictor of implantation. Trial registration number not applicable

P-153 Optimizing clinical outcomes in single vitrified-thawed blastocyst transfer cycles: impact of blastocyst re-expansion status

Abstract Study question is the blastocyst re-expansion status according to the thawing-transfer time interval related to the clinical outcomes in a single frozen-thawed blastocyst transfer cycles? Summary answer The post-thawing blastocyst shrinkage is linked to lower clinical outcomes, and minimizing the time between thawing and transfer optimizes outcomes especially for shrunken blastocysts. What is known already • A reduced ART outcome was observed with the use of shrunken post-warming blastocysts (Ito et al., 2023) • The spontaneous blastocyst shrinkage observed during in vitro embryonic development is considered as a negative prognosis factor for implantation (Sciorio et al., 2020) • The blastocyst’s re-expansion level after thawing proves to be the best post-thaw parameter for the prediction of live birth (The Vienna consensus., 2017. Ahlstrom et al., 2013). • The fast blastocele re-expansion seems to have a predictive value for implantation in frozen-thawed blastocyst transfer cycle (Shu et al., 2009). Study design, size, duration This was a retrospective study of 577 single frozen-thawed blastocyst transfer cycles from January 2022 to April 2023. Participants/materials, setting, methods Only B4 and B5 blastocyst quality obtained in day-5 were included in this study. The morphological characteristics were evaluated: after thawing (T0) and at Transfer time (TR). Blastocysts with 50% reduction of blastocele were classified “Shrunken”, the rest are “re-expanded”. and categorized into Group-1: re-expanded at T0 (n = 173); Group-2: shrunken at T0 and re-expanded at TR (n = 135); Group-3: shrunken at T0 and TR (n = 199). Statistical tests: chi-square, T-test and Mann-Whitney. (Statistical significance p < 0.05*). Main results and the role of chance Clinical outcomes of re-expanded blastocysts comparing to shrunken blastocysts at TR were respectively 42.21% versus 32.71% for pregnancy rate (PR) (p < 0.007),39.61% versus 30.48% for implantation rate (IR) (p < 0.02) and 35.71% versus 22.30% for life birth rate (LBR) (p < 0.004), a significantly higher outcomes were observed for re-expanded blastocysts. The miscarriage rate decreased from 25.00% to 9.23% (p < 0.001). The transfer of shrunken blastocysts in an interval of time within 1 hour or less comparing to more than 1 hour between T0 and TR showed the following results: PR = 40.82% versus 28.07% (p < 0.032), IR = 39.81% to 25.10%, (p < 0.02) and LBR = 26.53% versus 19.88% (p < 0.2). a significantly higher clinical outcomes were observed for shrunken blastocysts transferred within 1 hour or less. Group analysis: the PR, IR and LBR significantly increased for Group-1 comparing to Group 2. For Group-3, shrunken blastocysts at TR had the lower outcomes. (respectively p = 0.003; 0.017; 0.0002) Limitations, reasons for caution It is an ongoing retrospective observational study. Blastocysts with a low quality (B3 or less) were excluded from this study how limit our ability to extend the findings to low morphology degrees not covered by this research. Wider implications of the findings The management of frozen blastocyst transfers in IVF laboratories have to take into consideration in addition to the classic morphological evaluation, the blastocoele re-expansion state and the time interval between thawing and transfer to optimize the transfer outcomes. Trial registration number not applicable

Blastocyst re-expansion 1 hour after biopsy: an independent predictor of live birth

Blastocyst re-expansion 1 hour after biopsy: an independent predictor of live birth

Human recombinant interleukin-6 improves the morphological quality of cryopreserved in vitro produced bovine blastocysts

This work explored whether a well-characterized recombinant human interleukin-6 (hIL6) protein will influence in vitro produced (IVP) bovine embryo development and survival after cryopreservation. Cumulus oocyte complexes were collected from abattoir derived ovaries, matured for 24 h, and fertilized using pooled semen from Holstein bulls. Embryos were treated with 0, 25, 50, or 100 ng/mL hIL6 on day 5 post-fertilization. An increase in ICM cell numbers was observed in each hIL6 treatment, with the lowest hIL6 treatment having the same magnitude of response as the middle and highest hIL6 concentration. No effects on TE cell numbers were observed. The second study involved cryopreserving (via slow freezing) of hIL6-treated blastocysts, then examining post-thaw blastocyst survival by incubating for 24 h in the absence of hIL6 treatments. Blastocyst re-expansion and hatching rates were unaffected by any of the IL6 treatments, however, increases in both ICM and TE cell numbers were detected at 24 h post-thawing in blastocysts exposed to 100 ng/mL hIL6 but not lower concentrations before freezing. A reduction in the percentage of TUNEL-positive TE cells was observed after thawing in blastocysts exposed to 25, 50 and 100 ng/mL hIL6 before cryopreservation. No treatment-dependent changes in TUNEL-positive ICM cells were observed. In summary, hIL6 supplementation improves ICM cell numbers in bovine blastocysts to a degree that is commensurate with what has been observed when using bovine recombinant IL6. This positive effect of hIL6 on ICM cell numbers is maintained after freezing and thawing, and a novel improvement in post-thaw TE cell numbers occur in hIL6 treated embryos. This positive effect on TE cell numbers is attributed, at least in part, to an hIL6-dependent reduction in TE cell apoptosis.

Live birth outcomes following single-step blastocyst warming technique - optimizing efficiency without impacting live birth rates.

To evaluate the impact of a single-step (SS) warming versus standard warming (SW) protocol on the survival/expansion of vitrified blastocysts and their clinical outcomes post-frozen embryo transfer (FET). Retrospective analysis was performed on 200 vitrified/warmed research blastocysts equally divided amongst two thawing protocols utilizing the Fujifilm Warming NX kits (Fujifilm, CA). SW utilized the standard 14-minute manufacturer's guidelines. SS protocol required only a one-minute immersion in thaw solution (TS) before the embryos were transferred to culture media. A time-interrupted study was performed evaluating 752 FETs (SW: 376 FETs, SS 376 FETs) between April 2021-December 2022 at a single academic fertility clinic in Boston, Massachusetts. Embryologic, clinical pregnancy, and live birth outcomes were assessed using generalized estimated equation (GEE) models, which accounted for potential confounders. There was 100% survival for all blastocysts (n = 952 embryos) with no differences in blastocyst re-expansion regardless of PGT status. Adjusted analysis showed no differences in implantation, clinical pregnancy, spontaneous abortion, or biochemical pregnancy rate. A higher odds of multiple gestation [AdjOR(95%CI) 1.06 (1.01, 1.11), p = 0.019] were noted, even when adjusting for number of embryos transferred [AdjOR(95%CI) 1.05 (1.01, 1.10)]. Live birth outcomes showed no differences in live birth rates or birthweight at delivery. The study found equivalent outcomes for SS and SW in all parameters except for a slight rise in the rate of multiple gestations. The results suggest that SS warming is an efficient, viable alternative to SW, reducing thaw times without adverse effects on live birth rates or neonatal birth weights.

Preimplantation Genetic Testing: Personal Views Regarding the Invasiveness of Trophectoderm Biopsy and Risks on Embryos Development “An Operators Survey”

This study aims to assess the Trophectoderm (TE) biopsy practice in Jordan in terms of the following effectiveness parameters: timing of zona breaching, risk of inner cell mass herniation if zona breaching was done on day 3, timing of TE sampling, method of biopsy (pulling or flicking), number of laser pulses, assessment of embryo survival after biopsy, and degeneration rate. An online cross-sectional survey was conducted in November 2022. The collected data presented the perception of embryologists (>10 years experience) about the difficulty of the technique and the awareness of the risks it imposes on embryonic development. Potential predictors of embryologists’ awareness of the risks of trophectoderm biopsy in preimplantation genetic testing (PGT) and procedure difficulty were investigated. 125 embryologists were eligible, and 72 (57.6%) adequately filled the questionnaire, of which 51 (70.8%) perceived the procedure as moderately difficult. However, 8 (11.1%) embryologists perceive it as very difficult. Regarding the preferred time of zona breaching, 39 (54.2%) of embryologists perform zona breaching on day 5 of embryonic life. 68% claim they primarily use flicking when performing TE biopsy. Moreover, 33 (45.8%) of the 72 surveyed embryologists claimed they use 2-3 laser pulses, and 56 (77.8%) claimed it takes 2 to 3 minutes to finish the procedure. Regarding the embryologists’ awareness of the risk of Inner Cell Mass (ICM) herniation, most embryologists 46 (64%) believed there is a moderate risk if zona breaching is done on day 3. 23 (32%) acknowledge the procedure as having a low risk for embryonal development. 29 (40.3%) of embryologists assess survival by checking the re-expansion of the biopsied blastocyst after 2 hours, while 18% check blastocyst re-expansion after 15 minutes. 39 (54.2%) claimed that the incidence of degeneration rate post-TE biopsy is rare. TE biopsy strategy is one of the most promising biopsy techniques in PGT. Most embryologists in Jordan perceive the procedure as moderately difficult due to the technical considerations involved in performing the optimum TE biopsy.

Evaluation of blastocyst re-expansion, quality in relation to storage temperature, and sexing using blastocoel fluid after manual perforation with a hand-held needle involving in vivo produced equine embryos

The present study was designed to evaluate equine blastocyst re-expansion rate, quality, and sex following perforation of the blastocoel, collection of blastocoel fluid (BF), and PCR amplification of free DNA. Experiment 1 tested the feasibility of the BF sample collection with a hand-held, small-gauged needle (26g) and subsequent PCR amplification of the TSP-Y gene for males and AMEL-Y gene for males and AMEL-X gene for females. Experiment 2 tested the application of the technique. Equine embryos were collected via uterine flushes 8d after ovulation. Thereafter, embryos (n = 19) were initially assessed and transferred to a 50 μL droplet of holding medium in which the blastocoel was manually perforated as in Experiment 1. Within 1 min of detecting a diameter decrease or collapse, the entire volume of each droplet of medium was collected and stored at −20 °C until PCR. In Experiment 1, amplification of the TSP-Y gene was positive for males at 60% (9/15) and negative for females at 40% (6/15). In Experiment 2, a total of 42 embryos were randomly assigned to a collapsed embryo (CE) or intact embryo (IE) groups and stored at room temperature (RT, 25 °C) or cold temperature (CT, 5 °C) for 24h as follows: 1) CERT, n = 11; 2) CECT n = 11; 3) IERT, n = 10; and 4) IECT, n = 10. After 24h, embryo diameter and quality were reassessed. For all collapsed embryos (n = 19), blastocoel fluid was subjected to double PCR amplification of the TSPY gene with blood from adult male and female horses as controls. Positive gene amplification indicated 57.9% (11/19) of embryos were male and negative amplification indicated 31.6% (6/19) of embryos were female. Relative to the least diameter (0%) after perforation of collapsed embryos or fullest diameter (100%) of intact embryos at T0, percentage change in diameter and quality Grade 1 or 2 embryos after 24h of storage for all groups were, respectively: 31.2% and 54% for CERT group, 28.2% and 0% for CECT group, 25.9% and 100% for IERT group, 4.3% and 80% for IECT group, respectively. Thus, needle-induced leakage and collapse of the blastocoel at T0 resulted in a high rate of blastocyst re-expansion (69%) with many embryos (54%) achieving good quality at T24 with potential for transfer as either male or female embryos. For both collapsed and intact embryos, it was observed that storage for 24h at room temperature (25 °C) was associated with improved embryo growth and morphological quality compared to storage at cold temperature (5 °C).

Blastocoel fluid aspiration improves vitrification outcomes and produces similar sexing results of in vitro-produced cattle embryos compared to microblade biopsy

The potential applications of in vitro-produced (IVP) cattle embryos are significantly enhanced when combined with genotype selection and cryopreservation techniques. While trophectoderm (TE) biopsies are frequently used for genotyping, cell-free DNA (cfDNA) found in blastocoele fluid (BF) arises as a less-invasive method. Moreover, the blastocoel collapse produced by BF aspiration could be beneficial for embryo cryotolerance. This study was conducted to test the BF as a source of cell free-DNA (cfDNA) and to compare the BF to the TE biopsy in terms of sexing efficiency/accuracy, embryo survival and gene expression after vitrification/warming. IVP day 7 expanded blastocysts were artificially collapsed by aspiration of BF (VIT-Collapsed) or biopsied (VIT-Biopsied). After sample collection, embryos were vitrified/warmed by the Cryotop method and individually cultured in vitro. Intact fresh non-vitrified and vitrified/warmed blastocysts served as Fresh Control and VIT-Control, respectively. After sex identification of BF or TE biopsies and the corresponding surviving embryos, amplification efficiency and sexing accuracy were assessed. There were no differences between the BF and TE biopsy samples in terms of sexing accuracy or efficiency. Although all vitrified groups showed lower post-warming re-expansion rates (p < 0.05), the blastocyst re-expansion rates in the VIT-Collapsed group were comparable to those in the Fresh Control group whereas biopsied blastocysts showed the lowest (p < 0.05) re-expansion rates. VIT-Collapsed blastocysts had hatching rates that were comparable to those of Fresh Control blastocysts but significantly higher than those of the other vitrification treatments. Proapoptotic gene BAX was overexpressed in VIT-Biopsied embryos, whereas BCL2 transcripts were more abundant in the VIT-Collapsed group. On the other hand, VIT-Biopsied embryos showed altered ATP1B1- and AQP3-mRNA levels. The analysis of the cfDNA present in the BF is an efficient, minimally invasive approach to sex IVP cattle embryos. Besides, the artificial collapse of blastocoel prior to vitrification resulted in higher re-expansion and hatching ability than when embryos were vitrified after being biopsied.

Influences of Supplementing Selective Members of the Interleukin-6 Cytokine Family on Bovine Oocyte Competency.

This work explored whether supplementing selective members of the interleukin-6 (IL6) cytokine family during in vitro bovine oocyte maturation affects maturation success, cumulus-oocyte complex (COC) gene expression, fertilization success, and embryo development potential. Human recombinant proteins for IL6, IL11, and leukemia inhibitory factor (LIF) were supplemented to COCs during the maturation period, then fertilization and embryo culture commenced without further cytokine supplementation. The first study determined that none of these cytokines influenced the rate that oocytes achieved arrest at meiosis II. The second study identified that LIF and IL11 supplementation increases AREG transcript abundance. Supplementation with IL6 supplementation did not affect AREG abundance but reduced HAS2 transcript abundance. Several other transcriptional markers of oocyte competency were not affected by any of the cytokines. The third study determined that supplementing these cytokines during maturation did not influence fertilization success, but either LIF or IL11 supplementation increased blastocyst development. No effect of IL6 supplementation on subsequent blastocyst development was detected. The fourth experiment explored whether each cytokine treatment affects the post-thaw survivability of cryopreserved IVP blastocysts. None of the cytokines supplemented during oocyte maturation produced any positive effects on post-thaw blastocyst re-expansion and hatching. In conclusion, these outcomes implicate IL11 and LIF as potentially useful supplements for improving bovine oocyte competency.

The Timing of Vitrification After Biopsy

The blastocysts are often vitrified after trophectoderm biopsy to secure the time for transportation to external facilities and diagnosis. Previous studies reported that the time interval from the biopsy to vitrification affected the viability of biopsied blastocysts after warming. Furthermore, the degree of blastocyst re-expansion at vitrification was associated with the survival rate after warming. In this presentation, I will share the summary of previous studies, the results of our recent study, and the optimal timing of vitrification after biopsy.

Using blastocyst re-expansion rate for deciding when to warm a new blastocyst for single vitrified–warmed blastocyst transfer

Research questionCan predictive post-warm parameters that support the decision to transfer a warmed blastocyst or to warm another blastocyst be identified in women with multiple frozen–vitrified blastocysts? DesignRetrospective single-centre observational cohort analysis. A total of 1092 single vitrified–warmed blastocyst transfers (SVBT) with known Gardner score, maternal age and live birth were used to develop live birth prediction models based on logistic regression, including post-warm re-expansion parameters. Time-lapse incubation was used for pre-vitrification and post-warm embryo culture. A dataset of 558 SVBT with the same inclusion criteria was used to validate the model, but with known clinical pregnancy outcome instead of live birth outcome. ResultsThree different logistic regression models were developed for predicting live birth based on post-warm blastocyst re-expansion. Different post-warm assessment times indicated that a 2-h post-warm culture period was optimal for live birth prediction (model 1). Adjusting for pre-vitrification Gardner score (model 2) and in combination with maternal age (model 3) further increased predictability (area under the curve [AUC] = 0.623, 0.633, 0.666, respectively). Model validation gave an AUC of 0.617, 0.609 and 0.624, respectively. The false negative rate and true negative rate for model 3 were 2.0 and 10.1 in the development dataset and 3.5 and 8.0 in the validation dataset. ConclusionsClinical application of a simple model based on 2 h of post-warm re-expansion data, pre-vitrification Gardner score and maternal age can support a standardized approach for deciding if warming another blastocyst may increase the likelihood of live birth in SVBT.

O-163 The influence of aquaporin 3 on dynamics of cavitation, implantation and re-expansion after vitrification/warming

Abstract Study question We wished to investigate whether aquaporin 3 (AQP3) is involved in trophectoderm-related events: cavity formation and maintenance, implantation and re-expansion after the vitrification/warming process. Summary answer AQP3 regulates blastocoel formation and expansion. It is also required for implantation (as assessed by in vitro implantation assay) and maintaining developmental potential after vitrification. What is known already During blastocoel expansion blastocyst pulsates, i.e. contracts and re-expands in an oscillatory way. Recent studies utilizing a mouse model have shown, that the dynamics of cavitation and biomechanical properties of the trophectoderm are intertwined, and our results suggest, that it may, to a certain extent, reflect the developmental capabilities of blastocysts. Moreover, the dynamics of blastocyst re-expansion after vitrification/warming is also suggested to be a promising biomarker of embryo quality. As AQP3 is involved in water influx and transportation of small molecules (such as cryoprotectants) through the epithelial layer, it is a good candidate for a regulator of blastocoel expansion. Study design, size, duration We analyzed 142 mouse blastocysts to correlate the dynamics of their cavitation with their ability to implant in vitro (an outgrowth assay). We investigated the expression of Aqp3 throughout the preimplantation development. We tested how inhibition of AQP3 affects the dynamics of cavitation (n = 37; control n = 118) and formation of outgrowths (n = 77; control n = 81). We vitrified blastocysts with a limited activity of AQP3 (n = 41; control n = 38) and investigated their ability to re-expand and implant in vitro. Participants/materials, setting, methods The dynamics of cavitation was assessed by time-lapse imaging. After filming, mouse blastocysts were cultured for additional 4 days to test their ability to form outgrowths. Inhibition of AQP3 was performed by adding 100µM DFP00173 (a selective AQP3 inhibitor) to the culture medium. AQP3-inhibited blastocysts were analyzed for the dynamics of cavitation and outgrowth formation. Additionally, embryos with a limited activity of AQP3 were vitrified, warmed and then the outgrowth assay was performed. Main results and the role of chance Blastocysts unable to implant in vitro properly (i.e. forming too small outgrowths or not forming outgrowths at all) contract longer and more frequently than embryos that form correct outgrowths. Moreover, in such blastocysts, we observed shorter and weaker re-expansion phases. As AQP3 may be one of the regulators of cavitation, we followed its expression in embryos and showed that Aqp3 mRNA level increases from the 2-cell to the morula stage. AQP3 protein can be found in the cytoplasm during cleavage and translocates to basolateral membranes in trophectodermal cells in blastocysts. We revealed that inhibition of AQP3 resulted in delayed cavity formation. Moreover, inhibition of AQP3 significantly weakened the blastocyst’s ability to (re-)expand and also extended the duration and decreased frequency of the blastocoel contractions. Importantly, changes in the duration of contractions and amplitude of re-expansions were similar to those observed in blastocysts not able to implant in vitro properly. This accords with our observation that blastocysts with a limited activity of AQP3 are mostly unable to create outgrowths. Finally, we proved that inhibition of AQP3 strongly weakened re-expansion of the cavity after vitrification/warming. Furthermore, such blastocysts had a significantly lower ability to implant in vitro after vitrification. Limitations, reasons for caution We examined the role of AQP3 in blastocoel (re)-expansion and the relationship between cavitation dynamics and embryo’s ability to implant only in mice, so additional studies on other mammalian species, including humans, are needed. Moreover, our in vitro implantation assay does not fully reflect the complexity of implantation in vivo. Wider implications of the findings Our data indicate, that AQP3 regulates the dynamics of blastocoel expansion in mouse embryos. Impaired dynamics of cavitation, unsuccessful implantation or vitrification failures might be a result of decreased AQP3 activity, possibly not only in a mouse but also in other mammalian species, including humans. Trial registration number not applicable

Equine blastocyst re-expansion rate, quality, and sex following embryonic collapse using needle perforation of blastocoel and subsequent PCR amplification of blastocoel DNA

Collapse of equine blastocysts following needle perforation of the blastocoel prior to vitrification (Ferris et al., Journal of Equine Veterinary Science. 2016; 100:64-65) and collection and amplification of the free DNA blastocoel fluid may be used for sexing embryos (Herrera et al., Theriogenology. 2015; 83:415-420). The present study aimed to evaluate equine blastocyst re-expansion rate, quality, and sex following manual perforation of the blastocoel and PCR amplification of blastocoel fluid DNA. Embryos (n=19) were collected on day 8 after ovulation, assessed initially and transferred to a 50µL droplet of holding medium in which the blastocoel was manually perforated with a 26g hypodermic needle. Within one minute of detecting a diameter decrease or collapse, the entire volume of each droplet of medium was collected and stored at -20°C until PCR. Some of the collapsed embryos (n=11) were moved and immersed in 2mL of holding medium and cultured in isothermal boxes at 25°C. After 24h, embryo size and quality were reassessed. Post-culture, collapsed blastocysts had partially re-expanded to their initial diameter with a mean expansion rate of 68.8%. Quality of re-expanded embryos was Grade 2 (good, 6/11), Grade 3 (poor, 4/11), and Grade 4 (degenerated, 1/11) (McKinnon,Squires, J. Am. Vet Med. Assoc. 1988; 192:401-406). Non-perforated embryos (n=10) used as controls were initially Grade 1 (excellent, 8/10) and Grade 2 (2/10) and, after 24h of culture, Grade 1 (9/10) and Grade 2 (1/10) with a 25.9% growth rate. Blastocoel fluid from all collapsed embryos (n=19) was subjected to double PCR amplification of the male TSPY gene with blood of an adult male and female horse used as controls. Gene amplification indicated 57.9% (11/19) of embryos were male and non-amplification indicated 31.6% (6/19) of embryos were female. Gene amplification was inconclusive for 10.5% (2/19) of the embryos even though they were completely collapsed. Thus, needle-induced leakage and collapse of the blastocoel resulted in a high rate of blastocysts re-expansion (69%) with many embryos (54%) achieving good quality and potential for transfer as either male or female embryos. In summary, while the number of potentially transferable embryos that recovered from collapse was less than in the control group, the success of these preliminary results warrants further development to decrease damage and increase the culture re-expansion rate and quality of transferable male or female embryos for field application in an equine embryo transfer program.

Molecular phenotypes of bovine blastocyst derived from in vitro-matured oocyte supplemented with PAPP-A.

Insulin-like growth factor-1 (IGF-1) regulates cellular lipid content, whereas pregnancy-associated plasma protein-A (PAPP-A) increases IGF-1 bioavailability. Using in vitro-matured cumulus-oocyte complexes, we aimed to evaluate the impact of PAPP-A on the blastocyst lipid content, embryo cryotolerance and embryonic transcriptional profile. We determined that PAPP-A did not affect the lipid content of oocytes, blastocysts, or blastocyst yield (P > 0.05). However, PAPP-A modulated the embryo transcriptional profiles by downregulating PPARGC1A and AKR1B1, which are related to lipid metabolism; CASP9, a pro-apoptotic gene; and IFN-τ, a marker of embryo quality (P < 0.05). Furthermore, the use of PAPP-A improved blastocyst re-expansion in the first 3h of culture after vitrification (P < 0.05). Although PAPP-A did not affect the blastocyst lipid content or embryo production, we suggest that embryonic transcriptional modulation could contribute to maintain the balance in embryo lipid metabolism. Furthermore, PAPP-A's approach seems to control key intracellular pathways that improve post-cryopreservation development of blastocysts.

Development and validation of a nomogram for predicting ongoing pregnancy in single vitrified-warmed blastocyst embryo transfer cycles.

The global adoption of the "freeze-all strategy" has led to a continuous increase in utilization of single vitrified-warmed blastocyst embryo transfer (SVBT) owing to its clinical effectiveness. Accurate prediction of clinical pregnancy is crucial from a patient-centered perspective. However, this remains challenging, with inherent limitations due to the absence of precise and user-friendly prediction tools. Thus, this study primarily aimed to develop and assess a nomogram based on quantitative clinical data to optimize the efficacy of personalized prognosis assessment. We conducted a retrospective cohort analysis of ongoing pregnancy data from 658 patients with infertility who underwent SVBT at our center between October 17, 2017, and December 18, 2021. Patients were randomly assigned to the training (n=461) or validation (n=197) cohort for nomogram development and testing, respectively. A nomogram was constructed using the results of the multivariable logistic regression (MLR), which included clinical covariates that were assessed for their association with ongoing pregnancy. The MLR identified eight significant variables that independently predicted ongoing pregnancy outcomes in the study population. These predictors encompassed maternal physiology, including maternal age at oocyte retrieval and serum anti-Müllerian hormone levels; uterine factors, such as adenomyosis; and various embryo assessment parameters, including the number of fertilized embryos, blastocyst morphology, blastulation day, blastocyst re-expansion speed, and presence of embryo string. The area under the receiver operating characteristic curve in our prediction model was 0.675 (95% confidence interval [CI], 0.622-0.729) and 0.656 (95% CI, 0.573-0.739) in the training and validation cohorts, respectively, indicating good discrimination performance in both cohorts. Our individualized nomogram is a practical and user-friendly tool that can provide accurate and useful SVBT information for patients and clinicians. By offering this model to patients, clinical stakeholders can alleviate uncertainty and confusion about fertility treatment options and enhance patients' confidence in making informed decisions.

Clinical Usability of Embryo Development Using a Combined Qualitative and Quantitative Approach in a Single Vitrified-Warmed Blastocyst Transfer: Assessment of Pre-Vitrified Blastocyst Diameter and Post-Warmed Blastocyst Re-Expansion Speed

Improving the safety and efficacy of assisted reproductive technology programs has been a continuous challenge. Traditionally, morphological grading has been used for embryo selection. However, only a few studies have assessed the morphokinetic variables and morphological dynamics of blastocysts. In the present study, we aimed to perform a quantitative analysis of blastocyst diameter and re-expansion speed. This in-depth morphokinetic evaluation can correlate with currently observed pregnancy outcomes. In total, 658 single vitrified-warmed blastocyst transfer cycles were performed between October 2017 and December 2021, which were divided into four groups according to the pre-vitrified blastocyst diameter. After warming, the groups were subdivided according to the blastocyst re-expansion speed. These quantitative measurements were performed using a time-lapse system. Both diameter and speed are essential in determining the blastocyst quality, while age, day of freezing, and blastocyst quality are crucial from a clinical perspective. The application of both quantitative (diameter and speed) and qualitative (blastocyst quality scores) parameters can help evaluate the clinical usability of blastocysts. This method can prove useful for embryologists in counseling their patients and determining pregnancy patient-oriented strategies.

Predicting implantation of vitrified-warmed blastocysts: Artificial Intelligence applied to post-warming dynamics

Background and Aim: Post-warmed blastocyst dynamics was described by Coello et al., in 2017. They found a correlation between blastocyst re-expansion and implantation rate and developed a hierarchical model for implantation prediction. In our study, we evaluated the post-warming dynamics to predict the implantation potential of vitrified-warmed blastocysts by using Artificial Neural Networks (ANNs) based on artificial intelligence.

P-193 Using blastocyst re-expansion rate in the decision-making when to warm a new blastocyst for optimal single vitrified-warmed blastocyst transfer

Abstract Study question Can post-warmed blastocyst re-expansion rate be used to identify embryos with low likelihood of live birth (LB) in single vitrified blastocyst transfer (SVBT)? Summary answer A statistical model using blastocyst re-expansion rate and Gardner score can be used to identify blastocysts that do not result in live birth. What is known already The ability to identify the most viable embryo with greatest chance of implantation and LB is crucial. As embryo development is a dynamic process, research has recently focused on finding the most predictive morphokinetic parameters to incorporate in the assessment of which embryos to select for transfer. Time-lapse systems allow continuous monitoring of the embryo while cultured uninterruptedly. Several studies have found that the degree of blastocoele expansion and re-expansion are significant parameters for prediction of LB. Furthermore, re-expansion rate has been demonstrated to correlate with pregnancy outcomes in SVBT. Study design, size, duration This study is a retrospective cohort study including a total of 931 SVBT´s at the Fertility Clinic at Horsens Regional Hospital in a 2-year period from 2019-2020. Prior to vitrification, laser assisted collapsing was performed on all blastocysts with an expansion grade ≤ 4, and grade 5 after individual assessment. Only blastocysts with data on both pre-vitrification Gardner score, LB outcome and at least two hours of post-warmed time-lapse data were included in the analysis. Participants/materials, setting, methods Using a time lapse system, both fresh and post-vitrification embryo developmental parameters were evaluated during culturing in an EmbryoScope (Vitrolife, Denmark). Re-expansion rate was calculated as the change in blastocyst area within the first two hours. Logistic regression models were used to estimate the relation between LB and pre- and post-vitrification variables. Based on model predictions, the diagnostic performance was evaluated at thresholds corresponding to a 1%, 2% and 5% false negative rate (FNR). Main results and the role of chance Of the 931 SVBT´s, a total of 388 resulted in a LB (41.7%). Using the initial blastocyst size and the two-hour re-expansion rate yielded a model with an area under the ROC curve (AUC) of 0.61 (95% bootstrap CI: 0.57-0.65) Adding pre-vitrification Gardner score to the model increased the AUC to 0.62 (95% bootstrap CI: 0.59-0.66). This model was used to calculate the true negative rate (TNR), being the proportion of transferred blastocysts that would not result in LB at each threshold. At a FNR threshold of 2%, the model predicts that out of 50 discarded blastocysts, 7 would have resulted in a LB and 43 would not have resulted in LB. In summary, the ratio of erroneously discarded embryos vs. correctly discarded embryos would be approximately 1:6 using a 2% FNR. Limitations, reasons for caution The statistical model was developed and tested on the same dataset with a risk of overfitting. Any potential clinical application of the model requires testing and validation on an independent dataset. Furthermore, clinical procedures and in particular laser collapse may bias the results. Wider implications of the findings Being able to identify blastocysts with a low likelihood of a LB allows for the possibility to thaw a new blastocyst. In a clinical setting, this could lead to a reduced time to pregnancy and LB in women who have multiple vitrified blastocysts. Trial registration number 1-16-02-583-20

Standardization of Post-Vitrification Human Blastocyst Expansion as a Tool for Implantation Prediction.

The increased use of vitrified blastocysts has encouraged the development of various criteria for selecting the embryo most likely to implant. Post-thaw assessment methods and timetables vary among investigators. We investigated the predictive value of well-defined measurements of human blastocyst re-expansion, following a fixed incubation period. Post-thaw measurements were taken exactly at 0 and 120 ± 15 min. Minimum and maximum cross-sectional axes were measured. Three groups were defined: Group 1: embryos that continued to shrink by 10 µm or more; group 2: embryos that ranged from −9 to +9 µm; and group 3: re-expansion of 10 µm or more. Patient and morphokinetic data were collected and integrated into the analysis. A total of 115 cases were included. The clinical pregnancy rate for group 1 was 18.9%; group 2, 27%; and group 3, 51.2% (p = 0.007). Pre-thaw morphologic grading and morphokinetic scores of the study groups did not reveal differences. p-values were 0.17 for the pre-thaw morphologic score, 0.54 for KID3, and 0.37 for KID5. The patients’ demographic and clinical data were similar. The clinical pregnancy rate correlated with the degree of thawed blastocyst re-expansion measured 2 h after incubation. This standardized measure is suggested as a tool to predict the potential of treatment success before embryo transfer.