Vitrification Times Research Articles

P-114 The timing from post-insemination to blastocyst vitrification correlates with ploidy and gender

Abstract Study question Which morphology parameter of blastocysts has the strongest correlation with the euploid rate? Summary answer The vitrification times post-insemination and trophectoderm grade (TE) are more predictive parameters for euploid blastocysts, and female blastocysts require less time for vitrification. What is known already In current ART, the use of preimplantation genetic tests for aneuploidy (PGT-A) has increased by 2 to 25 percent in different countries. PGT-A analyses have helped patients achieve an increased pregnancy rate, choose the gender of the embryo and prevent miscarriages. Moreover, the potential for blastocyst euploid is strongly correlated with the developmental speed and morphology grades. However, for patients without PGT-A, the criteria for selecting transferred blastocysts are mostly based on morphology and the day of the blastocyst. Therefore, it is essential to clarify which parameter is more predictive of euploid embryos during elective FET cycles. Study design, size, duration This retrospective analysis included 713 blastocysts from 417 patients treated at the Ojinmed IVF Center, Mongolia, between May 2019 and June 2023. The morphology parameters (morphology grade, blastocyst formation time, vitrification time and inner diameter) were compared between the PGT-A results and the control results. Blastocyst quality was evaluated with the Gardner and Schoolcraft grading system, and the inner diameter of the blastocyst was measured. Participants/materials, setting, methods All patients underwent a clomiphene-based minimal ovarian stimulation protocol or drug-free natural cycle of IVF treatment. When the blastocyst diameter exceeded 160 μm, a trophectoderm (TE) biopsy was taken, and the blastocyst was immediately vitrified using the Cryotop method. The time of vitrification and formation of the blastocyst were recorded from the insemination. The patients were analyzed using high-resolution NGS PGT-A (10 Mb resolution for copy number variations and 30%-70% for mosaicism). Main results and the role of chance The PGT-A analysis reported euploid, aneuploid, and mosaic rates. Euploid, aneuploid, and mosaic rates were significantly correlated with blastocyst formation time (114.78 ± 4.49 h, 116.12 ± 6.42 h and 116.33 ± 6.87 h, p = 0.003) and vitrification blastocyst time (126.65 ± 9.8 h, 131.57 ± 10.8 h and 130.44 ± 11.19 h, p < 0.001), along with women’s age (34.9 ± 4.32, 38.14 ± 3.77, and 35.84 ± 4.5, p < 0.001). In logistic regression analysis, aneuploid rates were significantly lower in blastocysts with A and B grade of ICM (OR 0.36, CI 95% 0.24-0.32, p < 0.01 and OR 0.45, CI 95% 0.32-0.63, p < 0.01) and A and B grade of TE (OR 0.20, CI 95% 0.11-0.37, p < 0.01 and OR 0.20, CI 95% 0.25-0.48, p < 0.01) compared to C grades, respectively. The AUCs for prediction of euploid were maternal age 0.66 (95% CI 0.62-0.70), TE grade 0.64 (95% CI 0.57-0.64), blastocyst vitrification time 0.62 (95% CI 0.58-0.66), ICM grade 0.60 (95% CI 0.61-0.68), blastocyst formation time 0.55 (95% CI 0.51-0.59) and inner diameter 0.46 (95% CI 0.42-0.5). Although blastocyst vitrification time is correlated with blastocyst gender (female blastocyst 127.9 ± 10.25h vs male blastocyst 129.68 ± 10.84h, p = 0.02). Limitations, reasons for caution The results of the retrospective study are limited to data from a single IVF center and conventional incubators were used in this study. Wider implications of the findings These findings might help non PGT-A patients in deciding on embryo selection for transfer. The vitrification time and TE grade of blastocysts are strongly correlated with euploid rate than the ICM, inner diameter and formation time. Also, female blastocysts take less time to vitrification time than male blastocysts. Trial registration number *

P-141 Elevating human oocyte vitrification and warming to a new level: fast, efficient, and effective

Abstract Study question Due to the rising trend for oocyte cryopreservation and increasing demand, there is an urgency to make oocyte vitrification and warming protocols more efficient. Summary answer Oocyte vitrification and warming can be done successfully in 4 minutes, reducing the time of commonly used current protocols by up to 25 minutes. What is known already Vitrification and warming of oocytes is widely used in many IVF clinics worldwide and routinely utilized as a dominant technique for fertility preservation and freezing of donor as well as autologous oocytes in many clinical settings. Outcome data using cryopreserved oocytes is comparable to fresh oocytes. However, vitrification and warming of human oocytes can be time consuming. Usually, vitrification of oocytes is done in about 11-16 minutes, and warming requires another 11 minutes. Also, it is a common practice to load not more than 2 oocytes per carrier at time of vitrification. Study design, size, duration From November 2022 to November 2023, 504 germinal vesicles (GV) and 207 metaphase I (MI) oocytes, which did not convert into mature oocytes at day of oocyte retrieval, were vitrified in Vit Kit Freeze (FujiFilm Irvine Scientific, USA), and RapidVit Oocyte (Vitrolife, Sweden). Their survival rates were compared, and as an additional viability indicator, post-warming developmental capabilities were evaluated after 24 and 48hrs of culture in Global High Protein (LifeGlobal, USA). Participants/materials, setting, methods A group of 4 GV or MI oocytes was vitrified at room temperature for 1 minute in Equilibration solution (ES) and 1 minute in Vitrification solution (VS), loaded on a S-Cryolock (Biotech, USA) and then plunged into liquid nitrogen. Warming was done at 37ºC in 0.5M sucrose for 1 minute and Wash solution (WS) for another minute. Post warming, GV and MI oocytes were cultured for developmental evaluation. Statistical significance was defined as P < 0.05. Main results and the role of chance There was no statistically significant difference in the survival rate of GV oocytes between Irvine (209/219; 95.4%), and Vitrolife media (270/285; 94.7%; P = 0.72). The survival of MI oocytes was also comparable and not significantly different between both media; Irvine: 103/106; 97.2%, vs Vitrolife: 96/101; 95.0%; P = 0.43). Because of the similar survival rates of oocytes in both groups, for the developmental potential and conversion rates, oocytes of both groups were combined. Of the 504 GV, 479 survived (95.0%) and after 24hrs of culture, 441 converted to MI (92.1%). After a culture of 48hrs, 56.5% (249/441) converted to metaphase II (MII) oocytes. Combining the vitrified and warmed MI oocytes from both groups, 96.1% (199/207) survived the warming, and after 24hrs of culture, 168 (84.4%) converted to MII oocytes. Vitrification and warming of oocytes at different developmental stages can be done with a short exposure time to hypertonic (ES/VS) and hypotonic (0.5M sucrose) solutions in a total of 4 minutes. Limitations, reasons for caution Immature oocytes were used as study material to evaluate a new protocol with significantly reduced vitrification and warming times. At this moment we cannot provide data on fertilization, embryo development, and blastocyst formation. Wider implications of the findings Oocytes are exposed to less suboptimal temperatures during dehydration and rehydration and help to maintain their developmental potential. Loading 4 oocytes per carrier combined with the decrease in time resulted in increased efficiency. Warming in 0.5M instead of 1M sucrose contributes positively to survival and allows earlier resumption of functionality. Trial registration number not applicable

P-369 DuoStim could be a proper option for women seeking fertility preservation for non-medical reasons

Abstract Study question Is Duostim a suitable option for women who wish to preserve fertility for social reasons? Summary answer Providing the option of DuoStim is an efficient way to expand the cohort of vitrified oocytes in women seeking fertility preservation for non-medical reasons. What is known already A growing number of reproductive-age women are choosing to postpone motherhood, giving rise to the concept of reproductive autonomy. Numerous strategies aim to refine ovarian stimulation protocols. DuoStim, entailing dual ovarian stimulations and retrievals within one menstrual cycle, proves advantageous for those with diminished ovarian reserve or facing cancer. To our knowledge, this is the first study offering DuoStim for social preservation. Study design, size, duration Retrospective controlled case-control study conducted with paired follicular phase stimulation (FPS) and luteal phase stimulation (LPS) derived cohorts of oocytes collected in the same ovarian cycle (DuoStim). The study included 160 patients aged 36.85 ± 3.3 years old, who underwent DuoStim for social egg freezing from October 2018 to September 2023. The primary outcome was the number of MII oocytes retrieved per cycle and secondary outcomes included stimulation duration and gonadotropin dose. Participants/materials, setting, methods FPS and LPS were performed using the same daily dose of recombinant gonadotropins in an antagonist protocol. Oocyte retrieval was performed 36 hours after triggering with gonadotropin-releasing hormone agonist triptorelin acetate (0,2 mg) after the FPS. Luteal phase stimulation protocol began on the 5th day after ovarian puncture using dual trigger, followed by a second oocyte retrieval. Mature oocytes were vitrified 2 hours later following the Kitazato® protocol. Main results and the role of chance Stimulation duration and gonadotropin dose were lower during Follicular Phase Stimulation (FPS) vs. Luteal Phase Stimulation (LPS) (10.8 ± 1.5 days vs. 13.0 ± 2.3, P < 0.05 and 2,487.9 ± 580.8 vs. 3,264.0 ± 942.7, P < 0.05). The number of retrieved oocytes was comparable between the two groups (4.78 ± 3.0 vs. 5.32 ± 3.7; P = 0.15). The mean number of MII oocytes acquired in the LPS group exceeded that in the FPS group (4.16 ± 3.0 vs. 3.58 ± 2.3; P = 0.054), indicating a tendency towards more MII oocytes in the LPS group without achieving statistical significance. A total of 110 patients (69.29%) obtained the same or a greater number of MII during the LPS compared to the FPS. Limitations, reasons for caution Although DuoStim is acknowledged as an option to obtain a greater number of eggs in certain situations, the evidence supporting its utility in social fertility preservation is still limited. It is crucial to recognize the inherent biases in observational retrospective studies. Wider implications of the findings DuoStim could be an advantage for women who live abroad or those who dispose of short periods of time for oocyte vitrification, also potentially reducing the drop-out rate. Trial registration number not applicable

O-198 The decline in semen quality parameters enhances mosaicism in trophectoderm biopsy of human blastocysts

Abstract Study question Do poor sperm parameters impact chromosomal analysis compared to sperm with normal parameters? Summary answer Fertilization with more than two abnormal patterns, including severe oligoasthenoteratozoospermia sperm, may increase the rate of embryo mosaicism. What is known already Sperm cells constitute the genetic half of the embryo, and the male factor plays a crucial role in embryo chromosome integrity. While PGT-A is widely used to enhance IVF outcomes, the influence of male factors on embryo genetics remains underexplored. We aimed to determine the effect of semen profile on the outcome of PGT-A cycles performed at the blastocyst stage. Study design, size, duration A single-center retrospective study conducted between January 2019 and July 2023 analyzed 609 blastocysts with Next Generation Sequencing (NGS) of trophectoderm (TE) biopsies. Four study groups were formed based on sperm parameters: normozoospermia (N), moderate male factor (MMF), severe oligoasthenoteratozoospermia (s-OAT), obstructive azoospermia (OA). Participants/materials, setting, methods Patients treated with a minimal ovarian stimulation protocol. PGT-A cycles were divided into three paternal age groups. OUTCOMES: Euploidy, aneuploidy, mosaicism rates; blast formation time; good-quality blastocyst rates. METHODS: Multivariate logistic analysis, Chi-squared tests, and STATA 12.1 statistical software. Main results and the role of chance In the 609 TE biopsied blastocysts, euploidy rate was 49.9%, aneuploidy rate was 31.5%, and mosaicism rate was 18.6%. Paternal age ≥43 increased aneuploidy risk compared with <35 (40.1% vs 26.1%, p < 0.05). The euploidy rate and aneuploidy rate were no different in MMF, s-OAT, and OA compared with N (44.8%, 40.6%, and 42.1% vs. 54.2%; 36.8%, 25.0%, and 31.6% vs. 30.5%). Mosaicism rates were significantly difference between sperm parameter groups (N 17.1%, MMF 18.4%, s-OAT 34.4% and OA 26.3%, p < 0.05). In logistic regression analysis adjusted for confounders, severe oligoasthenoteratozoospermia showed 2.5 times higher mosaicism rate (aOR 2.58; 95% CI 1.08-5.08, p = 0.027). The good-quality blastocyst rate was significantly reduced in the OA compared with N, MMF, and s-OAT (21.1% vs 43.4%, 39.2% and 37.5%, p < 0.05). No impact of semen parameters on blastocyst formation time and vitrification time outcomes was observed. Limitations, reasons for caution Retrospective, single-center study. Excludes DNA fragmentation index and TESE results, impacting sperm quality evaluation. Wider implications of the findings Severe oligoasthenoteratozoospermia is positively associated with mosaicism rate. Poor sperm parameters lead to mosaic embryos. Couples undergoing PGT-A for male factors should discuss potential outcomes. Patients with abnormal semen parameters should consider pretreatment for improved semen quality. Results useful for genetic counseling and clinical decision-making. Trial registration number None

Interactions of hydrophilic birch wood (Betula pendulaRoth) extractives with adhesives for load-bearing timber structures

This study investigated how water-soluble extractives of silver birch (Betula pendulaRoth) wood interact with the most common adhesives used for producing engineered wood products (EWPs), i.e., melamine-urea-formaldehyde (MUF) and one-component polyurethanes (PUR). Therefore, the extractives were characterized via various chromatographic techniques and mass spectrometry. The effects of extractives on the curing kinetics were investigated using rheometry. The impact of extractives on the chemical and mechanical properties of the cured adhesives was investigated with FTIR spectrophotometry and tensile stress-strain measurements, respectively. Moreover, a comparative study on the shear strength of birch wood with and without extractives was performed.The organic fraction of the water extractives of birch mainly consisted of phenolic glycosides, carboxylic (fatty) acids, and saccharides. The extractives decelerated the curing process of MUF, as observed in a rheological small-angle oscillatory shear experiment using wood substrates as the lower plate. This was indicated by a reduced gel time from 5 to 3.8 h on pristine vs. extracted wood. When measuring MUF on extracted wood, this deceleration was also reached by adding ∼1% (W/W) of the isolated extractives into the MUF resin. Similar experiments with PUR showed a slight acceleration when dispersing extract into the adhesive, indicated by a reduction of vitrification time of −0.9 ± 0.3 h per percent of extractives added. However, measurements with pure PUR on pristine and extracted wood showed no significant difference in curing kinetics due to the extractives’ limited mobility and solubility in PUR. Both PUR and MUF cured adhesive films showed a reduced stiffness, elastic stress limit and tensile strength in uniaxial tensile stress-strain measurements of adhesive films upon increasing the amount of hydrophilic birch extractives concentration. Comparative standard tensile shear strength measurements on pristine and extracted wood indicate a 7–19% strength increase due to extraction when bonding with MUF, while the bond strength of PUR bond lines was less affected by the extraction procedure.

P-411 Cumulative live-birth rate after oocyte vitrification for fertility preservation (FP) in oncologic or benign conditions: a retrospective comparative monocentric study

Abstract Study question Does cumulative livebirth-rate (CLBR) after reutilization of vitrified oocytes differ between patients having undergone fertility preservation (FP) for oncologic (onco-FP) reason or benign conditions (benign-FP)? Summary answer Although CLBR per woman was lower in the onco-FP group, there was no statistically significant association in multivariate analysis between onco-FP and CLBR. What is known already Recent ESHRE guidelines reported oocyte vitrification after controlled ovarian hyperstimulation (COH) as an established option for FP. Although age at the time of oocyte vitrification remains the main predictive factor of success, several lines of evidence suggest that the use of COH or in vitro maturation (IVM), as well as the indication of FP, in particular the type of disease, may influence outcomes after devitrification. The present investigation aimed at clarifying this issue. Study design, size, duration Observational comparative, monocentric retrospective study including all patients having reused, between January 2014 and December 2021, their oocytes vitrified for oncologic or benign conditions between 2013 and 2021. Women having undergone FP for non-medical indication were excluded. Participants/materials, setting, methods Among the 2201 patients having vitrified their oocytes, 94 (4.7%) returned for reutilization. The primary objective was the comparison of CLBR between onco-FP (n = 48) and benign-FP (n = 46) groups. A logistic model was performed. Factors associated with the CLBR in univariate analysis were included in a multivariate model. A secondary analysis was performed, comparing the benign-FP group and 2 groups of onco-FP according to the use of COH (Onco-COH, n = 25) or in-vitro maturation (Onco-IVM, n = 23). Main results and the role of chance Overall, in comparison with benign FP, women with malignant diseases were younger (median [IQR]: 34.0 y [31.0;37.0] vs. 36.5 y [33.2;38.0], p = 0.04) and had fewer oocytes vitrified (median [IQR]: 6.0 [3.0;9.2] vs.15.5 [6.2;18.0]), p < 0.001). The CLBR in onco-FP and benign-FP groups were 14.6% (7/48), and 32.6% (15/46), respectively. In univariate analysis, the CLBR was significantly lower in the onco-FP group when compared with the benign-FP group: (OR [95%CI]: 0.35 [0.12;0.94], p = 0.04). However, this difference in the CLBR did not reach significance after multivariate analysis (OR [95%CI]: 0.38 [0.09;1.51], p = 0.18). As expected, age at the time of oocyte vitrification was negatively associated with CLBR (OR [95%CI]: 0.80 [0.67;0.92], p = 0.005), while the number of oocytes inseminated was positively related to CLBR (OR [95%CI]: 1.22 [1.08;1.40], p = 0.002). Unlike onco-COH, onco-IVM was associated with a significant decrease in CLBR when compared to benign-FP (only COH), in univariate analysis: 8.7% (OR [95%CI]: 0.20 [0.03;0.80], p = 0.04), without significance after multivariate analysis (OR [95%CI]: 0.37 [0.04;2.16], p = 0.29).The livebirth rate (LBR) per oocyte was 2.1% in the onco-IVM subgroup (3 babies for 141 oocytes thawed), 2.7% in the onco-COH subgroup (7 babies for 260 oocytes thawed) and 3.7% (18 babies for 479 oocytes thawed) in the benign-FP group. Limitations, reasons for caution The small size of our population was probably responsible for a lack of statistical power. The retrospective and monocentric nature of our study may also be a weakness. Furthermore, LBR per oocyte was analyzed without age-adjustment. Wider implications of the findings This study provides actual data on chances of achieving a livebirth after oocyte vitrification, according to the use of a COH or IVM and the context of FP, oncologic or benign conditions. Trial registration number not applicable

P-702 Secretion of beta-human chorionic gonadotropin (b-hCG) by post-implantation embryos differs according to different chromosomal status.

Abstract Study question To evaluate secretion of b-hCG hormone by human post-implantation embryos cultured in-vitro up to day 10 according to their chromosomal status. Summary answer Secreted b-hCG levels differs among embryos with different chromosomal load, being severely diminished in monosomic embryos when compared either to euploid or trisomic embryos. What is known already Exploring early post-implantation embryo development is subject of growing interest because errors in correct embryo development at this period might cause early pregnancy losses or development of pathologies affecting postnatal health. Down's Syndrome (DS) is one of the most common examples of chromosomal supernumerary (trisomy 21; T21) aneuploidy that leads to live birth but associated to pathology in postnatal life while monosomy of chromosome 21 (M21) is more likely to cause embryo developmental arrest. Furthermore, conclusions extracted from our previous studies put forward the idea of different gene and metabolite expression according to different chromosomal load (Sanchez-Ribas et al., 2019). Study design, size, duration This is an exploratory comparative study which includes a total of 33 supernumerary science-donated human blastocysts on day 6 from in-vitro fertilization cycles followed by preimplantation genetic test for aneuploidies. Euploid (n = 10), pure T21 (n = 13) and pure M21 (n = 10) were included in the study. All day 6 blastocyst included were good-quality embryos and were initiating hatching at the time of vitrification. Embryos degenerated during culture were not used for b-hCG quantifications. Participants/materials, setting, methods Euploid, T21 and M21 day blastocysts were thawed and cultured in-vitro until day 10. Embryos were cultured in culture medium specifically designed to support post-implantation embryo development. Each day of culture, half volume of culture media was replaced by fresh media. Levels of b-hCG in conditioned embryo media were quantified by ELISA immunoassay at day 7, 9, and 10. Results are expressed as mean ± standard deviation. Embryo morphology was tracked by bright-field images. Main results and the role of chance All euploid and T21 embryos thawed managed to hatch completely and expand the blastocoel cavity, while 40% of M21 embryos failed to hatch being arrested inside the zona pellucida on day 7. On day 7, mean concentration of b-hCG secreted did not differ among euploid, T21 and M21 embryos (37,29±6,27; 36,05±6,61 and 27,16±9,95 ng/mL respectively). Interestingly, euploid and T21 embryos reported a progressive time-dependent increase in b-hCG levels along post-implantation in-vitro culture while secreted levels of b-hCG by M21 embryos showed a decreasing trend, reporting diminished b-hCG secretion at the end of the culture (day 10) compared to day 7 (14,86±2,57 vs 27,16±9,95 ng/mL; p < 0.05). No significant differences in b-hCG secretions were observed between euploid and T21 embryos neither on day 9 d.p.f. (61,05±8,47 and 56,08±8,10 ng/mL respectively) nor on day 10 (74,88±6,36 and 71,75±7,01 ng/mL respectively). However, b-hCG secreted by M21 embryos on day 9. and day 10 (27,11±11,45 and 14,86±2,57 ng/mL respectively) were significantly reduced (p < 0.01) compared with euploid or T21 embryos. Besides, while all euploid and T21 embryos developed until day 10, 16,67% of expanded M21 embryos degenerated before day 9. Limitations, reasons for caution This research focused only on embryo development, no maternal tissues were considered. Further proteins should be investigated to reveal other pathways implicated in healthy embryo development and not only trophoblast proliferation. Wider implications of the findings Monosomic embryos, associated to implantation failures, secrete lower b-hCG when compared to either euploid or trisomic embryos, which show similar behavior. Besides, b-hCG in culture media could become a non-invasive marker to track embryo progression along post-implantation stages and detect embryos with increased likelihood of developmental arrest or early miscarriage. Trial registration number Not Applicable

P-140 Relationship between re-expansion rate of vitrified blastocysts during recovery culture post-warming and pregnancy rate after single blastocyst transfers

Abstract Study question What is the relationship between re-expansion rate of vitrified-warmed blastocysts and pregnancy rate after single blastocyst transfers (SVBT)? Summary answer Re-expansion rate of vitrified-warmed blastocysts was higher with increasing the number of TEs and yielded higher pregnancy rate after SVBT. What is known already The increased use of blastocyst transfer has emphasized the importance of selecting high-quality embryos. Pre-vitrification morphological grading: Gardner’s criteria or time-lapse imaging of embryo morpho kinetics and developmental speed, is established a tool for selection of high-quality blastocysts. The recovery culture of vitrified blastocysts post-warming is performed before embryo transfer. Embryos that have dehydrated and shrunk during vitrification are restored to their pre-vitrification state during the recovery culture. Developmental competence in vivo of re-expanded blastocysts after thawing was higher than that of non-expanded blastocysts, but it is not clear the relationship between the re-expansion rate and the pregnancy rate, quantitatively. Study design, size, duration We analyzed 1034 vitrified-warmed blastocysts in 758 patients from July 2017 to June 2021. All embryos were monitored by a time-lapse system (EmbryoScope+, Vitrolife). Expanded blastocysts which reached the blastocoel cavity of 160 µm or more with obvious ICM were vitrified and cryopreserved. The blastocysts were transferred in the following cycle or later. Fetal heartbeat was detected as the pregnancy confirmation. Participants/materials, setting, methods The re-expansion rate was defined as the percentage of re-expanded blastocyst diameter after the recovery culture to the blastocyst diameter at the time of vitrification. The time of recovery culture was about 3 hours. The re-expansion rate and pregnancy rate were analyzed by logistic regression analysis. We also examined the association between re-expansion rate and the number of trophectoderm cells at equatorial region (eTE) or iDAScore: the parameters for selecting high-quality embryos. Main results and the role of chance Logistic regression analysis showed a significant relationship between re-expansion rate and pregnancy rate (P < 0.0001), with an AUC of 0.58 and a cutoff value of 88.5%. Pregnancy rates in re-expansion rates ≥ 88.5% were significantly higher than those in < 88.5% (51.7 vs. 37.4%). Therefore, we classified re-expansion rates ≥ 88.5% as positive and < 88.5% as negative, and performed logistic regression analysis of women age, vitrification time of blastocysts, eTE, and iDASCore. The results showed that women age, vitrification time, and iDAScore all did not differ significantly from the re-expansion rate. The number of eTE was significantly higher with increasing re-expansion rates (P < 0.0001), with an AUC of 0.58 and a cutoff value of 7.5. Limitations, reasons for caution We used only the expanded blastocysts with 160 µm or more in diameter and obvious ICM for vitrification. In addition, this study was retrospective, and further research is needed to confirm the reproducibility of the study in other clinics with different vitrification protocols and patient populations. Wider implications of the findings Quantitative analysis of re-expansion rate of vitrified blastocysts indicated that higher re-expansion rate (≥ 88.5%) had high pregnancy rate. It is useful to exploring the influence factors of re-expansion for producing high-quality embryos. Here, we found that the number of eTE is as an important factor for re-expansion. Trial registration number not applicable

P-722 Impact of blastocyst quality and post-thaw embryo culture factors in non-invasive PGT-A informativity on vitrified blastocysts.

Abstract Study question What are the most important factor/s involved in the informativity of non-invasive PGT-A (niPGT-A) of vitrified blastocysts? Summary answer The day of vitrification and post-thaw time in culture of vitrified blastocysts are the most important factors for niPGT-A informativity, without correlation with blastocyst quality. What is known already niPGT-A is a technique that allows the non-invasive study of the chromosomal content of blastocysts. The informativity and concordance rates for fresh (Rubio, 2020) and frozen-thawed embryos (Garcia-Pascual, 2022) have been related with the time in culture and the day of media collection. However, there is a need to understand the optimal conditions needed to obtain optimal informativity for frozen-thawed vitrified blastocyst undergoing niPGT-A. Study design, size, duration Prospective study including 135 spent blastocyst media (SBM) and the corresponding blastocyst samples from thawed day-5 and day-6 blastocysts from PGT-A (73) and niPGT-A (62) cycles collected from January 2021 until March 2022. Blastocysts were discarded under IRB approval. The embryos were divided into 3 groups depending on the day of development and the time in culture after thawing: group-1, day-5 blastocysts cultured 8h; group-2, day-5 blastocysts cultured 24h and group-3, day-6 blastocysts cultured 8h. Participants/materials, setting, methods Media and blastocysts were obtained from IVF patients (29-42 years-old). After thawing, each blastocyst was washed to remove remaining cumulus cells and individually transferred to a 10 μL droplet of fresh media or to an EmbryoScope dish with 20 μL per well. SBM and blastocysts were collected, frozen and analysed by Next Generation Sequencing. Correlation between concordance rates (overall agreement between SBM and blastocyst samples), informativity, embryo quality and time in culture were evaluated. Main results and the role of chance All the embryos had a good embryo quality (BB or greater using Gardner Criteria) but different expansion degree. Informativity was significantly lower when day-5 embryos were cultured for only 8 hours (group 1), regardless of the degree of expansion (expansion degree 3, 28.57%; degree 4, 64.29% and degree 5-6, 57.14%, p < 0.0005). The concordance rates between SBM and whole blastocysts were very high and no significant different in relation to the expansion degree when comparing day-5 embryos cultured for 8 hours (group 1, 90.5%) with 24 hours (group 2, 93.6%) or with day-6 embryos cultured for 8 hours (group 3, 92.3%). The data suggest that the important factor impacting the informativity rate is the day of vitrification, and the time in culture after thawing, but not the expansion degree of the embryos. Regarding the concordance rates, there is no impact of the time in culture nor the expansion degree. Limitations, reasons for caution The main limitation of this study is that all the embryos analysed had fair or good quality but different expansion degrees. Wider implications of the findings The high concordance rates observed in frozen blastocysts opens the possibility of expanding the use of niPGT-A to frozen blastocyst without an impact of the expansion degree in the results. Trial registration number Not applicable

Oocyte vitrification for oncological and social reasons.

The aim of this review is to present information related to oocyte cryopreservation, and particularly oocyte vitrification, performed to preserve fertility in oncologic and social indications. The success rates of oocyte cryopreservation have increased with the widespread use of the vitrification technique and are currently similar to those of in vitro fertilization performed with fresh oocytes. Vitrification is the most successful technique for oocyte cryopreservation. The most important factors that influence the success rate are the patient's age at the time of vitrification and the number of mature oocytes frozen. Thus, live birth rates differ for each age depending on the number of oocytes thawed and the freezing method. The American Society of Reproductive Medicine and the American Society of Clinical Oncology recommend presenting the option of oocyte cryopreservation for fertility preservation in cancer patients. Besides cancer patients, use of oocyte vitrification is increasing in women who wish to postpone pregnancy age and to have reproductive freedom with the development of the cryopreservation technique and the achievement of pregnancy rates similar to the use of fresh oocytes. Patients are provided consultancy service in terms of indication, the success rates by age, and the total number of oocytes frozen. It should be emphasized that this procedure is not a type of insurance policy for fertility, especially in elective oocyte cryopreservation.

Miscibility, thermal degradation and rheological analysis of epoxy/MABS blends.

The effect of the addition of the methyl methacrylate acrylonitrile butadiene styrene (MABS) copolymer on the miscibility, thermal degradation and rheological properties of epoxy systems is described. Epoxy resin/MABS blends containing 5, 10, 15 and 20 phr MABS were prepared using the solution mixing technique. Homogenous blends obtained using this technique have undergone a polymerization reaction induced phase separation process by the introduction of the curing agent 4,4'-diaminodiphenyl sulfone (DDS). The isothermal rheology at four different temperatures, 150, 160, 170 and 180 °C, was used to examine the effect of MABS on the gelation and vitrification time. The evolution of storage modulus, loss modulus and tan delta was found to be closely related to the evolution of complex phase separation. The increase in the complex viscosity during curing was determined by in situ rheometry and theoretically analysed by fitting with the Williams-Landell-Ferry equation. An exponential increase in complex viscosity was observed, which was induced by cross-linking. The variation of Tg before and after curing was studied using DSC analysis and dynamic kinetic modeling of the curing process was carried out by utilizing dynamic DSC scans. Thermal stability studies of completely cured epoxy/MABS blends using thermogravimetric analysis revealed that all the blends and neat epoxy exhibited single step degradation. Thermal degradation kinetics was calculated using the Coats Redfern equation.

The ability of blastocysts to re-expand after trophectoderm (TE) biopsy using different biopsy techniques

Background and Aim: At the time of vitrification, the re-expansion of blastocyst can be graded as fully expanded (FE), partially expanded (PE) or collapsed (CL) blastocyst. A study showed that FE and PE blastocysts resulted in higher implantation than the CL blastocysts (Chen et al., 2017). This prospective study investigates the ability of the blastocysts to re-expand after TE biopsy using different biopsy techniques.

P-141 The good, the bad and the ugly – The fate of collapsed blastocysts in frozen embryo transfer

Abstract Study question What is the clinical outcome of collapsed blastocysts without signals of re-expansion in frozen embryo transfers (FET)? Summary answer FET of non-re-expanded blastocysts after thawing showed a live-birth rate in 11.5% of the cases. Embryos vitrified on day 5 revealed the best results. What is known already Embryo viability is crucial for FET to be successful. Since the introduction of vitrification techniques, outcomes have improved substantially and are even comparable to fresh transfers. Nevertheless, some indicators may help predict the outcome of frozen-thawed blastocysts. Blastocoele re-expansion after thawing has been widely studied and is a strong predictor of clinical pregnancy outcome. However, accurate data on non-re-expanded blastocysts after FET is still not available. Hence, this study aims to evaluate the clinical outcome of non-re-expanded blastocyst after FET. Study design, size, duration The retrospective study was designed and conducted at the Kinderwunsch Institut Schenk GmbH (Dobl, Austria). Data was collected from FET performed between 2017 and 2021. In total, 104 FET cycles of collapsed blastocysts were analysed. All the transfers performed were single embryo transfers (SET). Patient’s average age was of 33.6 years at the time of embryo vitrification. Embryos were excluded if they showed more fragmentation than before vitrification. Participants/materials, setting, methods Patients were divided into two groups following the time of blastocyst cryopreservation: day 5 (44 SET) and day 6 (60 SET); which was dependant on the time of blastocoel expansion. They were further divided according to the trophectoderm quality: good (42 SET) and poor (62 SET) and the overall embryo quality, considering trophectoderm and inner cell mass (ICM): good (37 SET) and poor (67 SET). In both cases quality was assessed before vitrification. Main results and the role of chance A total of 104 collapsed blastocysts were transferred (SET). 16.3% of the patients had a positive biochemical pregnancy. 11.5% of the pregnancies resulted in a live birth and 4.8% of the pregnancies ended in abortion. FET with day 5 non-re-expanded blastocysts after thawing showed better results than day 6 thawed blastocysts regarding biochemical pregnancy (29.5% vs 6.6%, p = 0.02) and live birth rate (20.4% vs 5%, p = 0.017). Good embryo quality before vitrification showed a trend for better clinical outcome, however, results were not significant. A good trophectoderm quality before vitrification revealed a live birth rate of 16.7% compared to 8.1% in poor trophectoderm quality. Regarding overall embryo quality (trophectoderm and ICM quality) the results were 14.3% deliveries for good quality blastocysts vs 7.6% for poor quality blastocysts. Limitations, reasons for caution The sample size may be seen as a study limitation. However, statistically significant results were obtained. Nonetheless, results should be confirmed with a bigger sample size. Wider implications of the findings The present study revealed a lower chance of live birth for non-re-expanded blastocysts after thawing, which should nevertheless not be overlooked; specially in cases with good quality embryos frozen and thawed on day 5. Transfer of collapsed embryos after thawing would be recommended taking into consideration the study results. Trial registration number Not Applicable

Confinement Effects on the Spatially Inhomogeneous Dynamics in Metallic Glass Films.

This work develops the elastically collective nonlinear Langevin equation theory to investigate, for the first time, the glassy dynamics in capped metallic glass thin films. Finite-size effects on the spatial gradient of structural relaxation time and glass transition temperature (Tg) are calculated at different temperatures and vitrification criteria. Molecular dynamics is significantly slowed down near rough solid surfaces, and the dynamics at location far from the interfaces is sped up. In thick films, the mobility gradient normalized by the bulk value obeys the double-exponential form since interference effects between two surfaces are weak. Reducing the film thickness induces a strong dynamic coupling between two surfaces and flattens the relaxation gradient. The normalized gradient of the glass transition temperature is independent of vitrification time scale criterion and can be fitted by a superposition function as the films are not ultrathin. The local fragility is found to remain unchanged with location. This finding suggests that one can use Angell plots of bulk relaxation time and the Tg spatial gradient to characterize glassy dynamics in metallic glass films. Our computational results agree well with experimental data and simulation.

Determination of the formulation and curing conditions of thermosetting epoxy resins for optimizing their properties and future use in gelcasting process

AbstractThis work investigates the suitability of epoxy resins for possible use in an industrial gelcasting process. Two different formulations are studied using complementary experimental techniques. Both reactive formulations are based on a difunctional epoxy prepolymer (aliphatic BDGE or aromatic RDGE) mixed with a polyamine hardener (TEPA). The combination of analytical chemistry (NMR and FTIR spectroscopy) and calorimetric measurements (DSC) are used to determine the optimal epoxy/amine ratio for each reactive formulation. The gelation and vitrification times are investigated by dynamic rheometry through kinetic analyses over a large range of fixed temperature. It is found that the RDGE‐TEPA mixture is more reactive than BDGE‐TEPA with shorter curing starting times. The thermomechanical analyses performed on both optimized formulations show that the RDGE‐TEPA material presents better performances after curing (Tg close to 80°C) than the BDGE‐TEPA system (Tg lower than ambient temperature). Then, the effects of the cooling cycle on the thermomechanical profile of each polymer are investigated. The RDGE‐TEPA formulation is found to be much more subject to the formation of internal stresses created during the cross‐linking and cooling step than the BDGE‐TEPA material. Considering all these criteria, the BDGE‐TEPA system is ultimately more suitable for the gelcasting process than RDGE‐TEPA.

Semi-automated versus manual embryo vitrification: inter-operator variability, time-saving, and clinical outcomes.

Does semi-automated vitrification have lower inter-operator variability and better clinical outcomes than manual vitrification? Retrospective analyses of 282 patients whose embryos had been cryopreserved, manually with Irvine®-CBS® (MV) or semi-automatically vitrified with the GAVI® method (AV) (from November 2017 to September 2020). Both techniques were performed during the same period by 5 operators. Inter-operator variability was statistically analyzed between operators who performed the vitrification and those who performed the warming process to compare the intact survival rate (% embryos with 100% intact blastomeres) and the positive survival rate (at least 50% intact blastomeres). Additionally, the complete vitrification time was assessed for the 2 techniques according to the number of vitrified embryos. Manual vitrification involved warming 338 embryos in 266 cycles for 181 couples compared to 212 embryos in 162 AV cycles for 101 patients. The positive survival rate was higher (p < 0.05) after MV (96%; 323/338) than after AV (90%; 191/212). The intact survival rate (86 vs 84%) and the clinical pregnancy rate (27 vs 22%) were not significantly different between MV and AV. Regarding the inter-operator variability, no significant difference in positive and intact survival rate was evident between the 5 technicians, neither by vitrification nor by warming steps with MV and AV. Concerning time-saving, the MV technique proved to be quicker than AV (minus 11 ± 9min). Manual vitrification exhibited favorable total survival rates and was more time efficient, while both MV and AV cooling and warming treatments showed little operator variability.

The Impact of Embryo Storage Time on Pregnancy and Perinatal Outcomes and the Time Limit of Vitrification: A Retrospective Cohort Study.

BackgroundThe technique of embryo cryopreservation has been increasingly applied in clinical settings. However, there has been a concern about the safety and efficacy of long-term freezing of embryos. Therefore, the aim of this study was to evaluate whether storage time of vitrification had any effects on pregnancy as well as perinatal outcomes, further, to explore the appropriate time limit of vitrification.MethodsThe study included women who underwent at least one frozen-thawed cycle with single embryo transfer between January 1st, 2016 and September 30th, 2019. Patients were assigned into 3 groups according to the storage time (<3 months, 3-12 months and >12 months) to evaluate the impact of embryo storage time on pregnancy and perinatal outcomes. To further investigate the time limit of vitrification, propensity score matching was used to compare the primary outcomes of patients with storage time of 1-3 years, 3-5 years, and >5 years to those stored for ≤1 year.ResultsA total of 9806 frozen-thawed embryo transfer cycles were included in our study. After adjustment for confounding variables, no significant differences were found in pregnancy outcomes among groups. However, postponement of transfer increased the risks of large for gestational age and placenta previa. In addition, after propensity score matching, 171 cycles with storage time >5 years were matched with those ≤1 year, both the clinical pregnancy rate and live birth rate decreased significantly when the storage time exceeded 5 years.ConclusionsThe duration of vitrification did not significantly affect the pregnancy outcomes within 5 years period. However, the clinical pregnancy rate and live birth rate both decreased significantly when the duration of vitrification exceeded 5 years. It is worth noting that the conclusion was drawn from a small sample study after propensity score matching and should be treated with caution. In addition, the cycles were from different time periods, which could have an impact on the results.

Thermal shrinkage reveals the feasibility of pulse-delay photocuring technique

ObjectivesTo resolve the feasibility of the pulse-delay photocuring technique as a clinical strategy for reducing the detrimental polymerization stress induced in dental composites during the photocuring process. MethodsModel dental composites with high and low-filler contents were cured with the pulse-delay photocuring technique using different combinations of photocuring variables (irradiance, exposure time, and delay time). Irradiance used ranged from 0.1W/cm2 to 4W/cm2. The exposure time of the first pulse varied from 0.2s to 27.2s and the delay times ranged from 10s to 120s. The radiant exposure was varied from 4J/cm2 to 20J/cm2. A cantilever-beam based instrument (NIST Standards Reference Instrument 6005) was used to implement the photocuring technique for the measurement of the polymerization properties (the degree of monomer conversion, polymerization stress induced due to shrinkage, and temperature change due to the reaction exotherm and curing light absorbance) simultaneously in real-time. These properties were compared with those obtained using the conventional photocuring technique (i.e., using a constant irradiance for a fixed exposure time, a uniform exposure). ResultsThere exists a minimum radiant exposure, such that a reduction in the polymerization stress can be achieved without sacrificing the degree of monomer conversion by using the pulse-delay over the conventional photocuring technique. More specifically, stress reductions of up to 19% and 32% was observed with the pulse-delay when compared with the conventional photocuring technique at an irradiance of 0.5W/cm2 and 4W/cm2, respectively. The reduction occurred when the exposure time of the first pulse was greater than, but closer to, the gelation time (i.e., lower than the vitrification time) of the composite, regardless of the delay time used. Lower thermal shrinkage (contraction) during the post-curing time, rather than the stress relaxation during the delay time or lower degree of monomer conversion as claimed in the literature, is the cause of the reduction in the polymerization stress. SignificanceThe study clarifies a long-standing confusion and controversy on the applicability of the pulse-delay photocuring technique for reducing the polymerization stress and promotes its potential clinical success for dental restorative composites.

P–454 Oocyte vitrification for fertility preservation in women with benign gynecological disease: French national clinical guidelines with a modified Delphi consensus process

Abstract Study question Is there consensual clinical practices about fertility preservation (FP) for benign gynecological diseases BGD)? Summary answer A consensus study using the modified Delphi method identified 28 consensual practices concerning oocyte vitrification for fertility preservation in women with benign gynecological disease. What is known already Clinical Practical international guidelines are still published in oncology for offering standardized information and care for adults and children with cancer. Recently, the ESHRE Female Fertility Preservation Guideline Development Group published recommendations for healthcare professionals involved in fertility preservation for post-pubertal women and transgender adolescents and young adults. However, benign gynecological indications weren’t distinctly individualized of malignant conditions. There’s a lack of large cohort studies assessing the risks and outcome of FP for benign gynecological diseases. Healthcare professionals need consensus for defining the “good” indications of FP for benign gynecological diseases that could impair fertility. Study design, size, duration A steering group composed by 14 healthcare professionals and a patient representative with lived experience of endometriosis identified 42 potential practices concerning fertility preservation for benign gynecological disease. Then, 114 key stakeholders including various healthcare professionals (n = 108) and patient representatives(n = 6) were asked to answer at two rounds of a modified Delphi via an online survey from February to September 2020. Participants/materials, setting, methods Participants had to score 42 items for the first round and 31 for the second round using a nine-point Likert scale. These statements were distributed into five categories: Information to deliver to age-reproductive women with a BGD (n = 9), technical aspect of fertility preservation for BGD (n = 6), indications of FP for endometriosis (n = 13), indications of FP for none-endometriosis BGD (n = 10), idiopathic diminished ovarian reserve in the absence of gynecologic and endocrinologic diseases (n = 4). Main results and the role of chance Survey response of stakeholders was 75% (86 out of 114) for the round 1 and 87% (75 out of 86) for the round 2. Consensus recommendations were achieved for 28 items, and no consensus between stakeholders was achieved in the remaining items. Stakeholders rated the importance of an age-specific information concerning the risk of diminished ovarian reserve after surgery and the necessity to inform about the benefice/ risk balance of oocyte vitrification, in particular about the chance of live-birth according to the age at the time of oocyte vitrification. They endorsed oocyte vitrification as the reference FP technique for those benign indications. Experts rejected to determine lower and upper age limits in women for fertility preservation. FP shouldn’t be offered in rAFS stages I and II endometriosis without endometriomas. Limitations, reasons for caution Experts were only French native speakers from France, and Belgium. It would have been interesting to conduct this survey with experts from other continents. Wider implications of the findings: At our knowledge, we present here the first guideline s focusing on FP in women with BGD, following a designed scientific Delphi procedure. These guidelines could be useful for gynecologists to better inform women with benign gynecological diseases about the indication or not to offer a FP procedure. Trial registration number Not applicable

Factors associated with vitrification-warming survival in 6167 euploid blastocysts

To understand the clinical factors associated with embryo survival after vitrification in a cohort of human blastocysts screened by preimplantation genetic testing for aneuploidy (PGT-A). Patient demographic, embryo, and cycle characteristics associated with failed euploid blastocyst survival were compared in a cohort of women (n = 6167) who underwent IVF-PGT-A. Compared to those that survived warming, vitrified euploid embryos that failed to survive after warming came from IVF cycles with significantly higher estradiol levels at time of surge (2754.8 ± 1390.2 vs. 2523.1 ± 1190.6pg/mL, p = 0.03), number of oocytes retrieved (19.6 ± 10.7 vs. 17.5 ± 9.8, p = 0.005), and basal antral follicle count (BAFC) (15.3 ± 8.5 vs. 13.9 ± 7.2, p = 0.05). Euploid embryos were less likely to survive warming if they came from cycles before 2015 (24.6% vs. 13.2%, p < 0.001), were cryopreserved on day 7 versus day 5 or 6 (9.1% vs. 3.0%, p < 0.001), underwent two trophectoderm biopsies (6.9% vs. 2.3%, p < 0.001), had a grade C inner cell mass (15.4% vs. 7.7%, p < 0.001), or were fully hatched (41.1% vs. 12.2%, p < 0.001). In the multivariate model, which controlled for relevant confounders, the association between decreased survival and increased BAFC, year of IVF cycle, double trophectoderm biopsy, and fully hatched blastocysts remained statistically significant. Euploid embryos that are fully hatched at time of vitrification, come from patients with high ovarian reserve, or require repeat trophectoderm biopsy are less likely to survive vitrification-warming. Our results provide a framework for reproductive counseling and offer realistic expectations to patients about the number of embryos needed to achieve family building goals.