Application of amphiregulin in IVM culture of immature human oocytes and pre-insemination culture for COCs in IVF cycles.

Background: Amphiregulin (AR) is a growth factor that resembles the epidermal growth factor (EGF) and serves various functions in different cells. However, no systematic studies or reports on the role of AR in human oocytes have currently been performed or reported. This study aimed to explore the role of AR in human immature oocytes during in vitro maturation (IVM) and in vitro fertilization (IVF) in achieving better embryonic development and to provide a basis for the development of a pre-insemination culture medium specific for cumulus oocyte complexes (COCs). Methods: First, we examined the concentration of AR in the follicular fluid (FF) of patients who underwent routine IVF and explored the correlation between AR levels and oocyte maturation and subsequent embryonic development. Second, AR was added to the IVM medium to culture immature oocytes and investigate whether AR could improve the effects of IVM. Finally, we pioneered the use of a fertilization medium supplemented with AR for the pre-insemination culture of COCs to explore whether the involvement of AR can promote the maturation and fertilization of IVF oocytes, as well as subsequent embryonic development. Results: A total of 609 FF samples were examined, and a positive correlation between AR levels and blastocyst formation was observed. In our IVM study, the development potential and IVM rate of immature oocytes, as well as the fertilization rate of IVM oocytes in the AR-added groups, were ameliorated significantly compared to the control group (All P < 0.05). Only the IVM-50 group had a significantly higher blastocyst formation rate than the control group (P < 0.05). In the final IVF study, the maturation, fertilization, high-quality embryo, blastocyst formation, and high-quality blastocyst rates of the AR-added group were significantly higher than those of the control group (All P < 0.05). Conclusion: AR levels in the FF positively correlated with blastocyst formation, and AR involvement in pre-insemination cultures of COCs can effectively improve laboratory outcomes in IVF. Furthermore, AR can directly promote the in vitro maturation and developmental potential of human immature oocytes at an optimal concentration of 50 ng/ml.

Multiple collapses of blastocysts after full blastocyst formation is an independent risk factor for aneuploidy - a study based on AI and manual validation.

BACKGROUND: The occurrence of blastocyst collapse may become an indicator of preimplantation embryo quality assessment. It has been reported that collapsing blastocysts can lead to higher rates of aneuploidy and poorer clinical outcomes, but more large-scale studies are needed to explore this relationship. This study explored the characteristics of blastocyst collapse identified and quantified by artificial intelligence and explored the associations between blastocyst collapse and embryo ploidy, morphological quality, and clinical outcomes. METHODS: This observational study included data from 3288 biopsied blastocysts in 1071 time-lapse preimplantation genetic testing cycles performed between January 2019 and February 2023 at a single academic fertility center. All transferred blastocysts are euploid blastocysts. The artificial intelligence recognized blastocyst collapse in time-lapse microscopy videos and then registered the collapsing times, and the start time, the recovery duration, the shrinkage percentage of each collapse. The effects of blastocyst collapse and embryo ploidy, pregnancy, live birth, miscarriage, and embryo quality were studied using available data from 1196 euploid embryos and 1300 aneuploid embryos. RESULTS: 5.6% of blastocysts collapsed at least once only before the full blastocyst formation (tB), 19.4% collapsed at least once only after tB, and 3.1% collapsed both before and after tB. Multiple collapses of blastocysts after tB (times ≥ 2) are associated with higher aneuploid rates (54.6%, P > 0.05; 70.5%, P < 0.001; 72.5%, P = 0.004; and 71.4%, P = 0.049 in blastocysts collapsed 1, 2, 3 or ≥ 4 times), which remained significant after adjustment for confounders (OR = 2.597, 95% CI 1.464-4.607, P = 0.001). Analysis of the aneuploid embryos showed a higher ratio of collapses and multiple collapses after tB in monosomies and embryos with subchromosomal deletion of segmental nature (P < 0.001). Blastocyst collapse was associated with delayed embryonic development and declined blastocyst quality. There is no significant difference in pregnancy and live birth rates between collapsing and non-collapsing blastocysts. CONCLUSIONS: Blastocyst collapse is common during blastocyst development. This study underlined that multiple blastocyst collapses after tB may be an independent risk factor for aneuploidy which should be taken into account by clinicians and embryologists when selecting blastocysts for transfer.

Clinical effectiveness and safety of time-lapse imaging systems for embryo incubation and selection in in-vitro fertilisation treatment (TILT): a multicentre, three-parallel-group, double-blind, randomised controlled trial.

BACKGROUND: Time-lapse imaging systems for embryo incubation and selection might improve outcomes of in-vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) treatment due to undisturbed embryo culture conditions, improved embryo selection, or both. However, the benefit remains uncertain. We aimed to evaluate the effectiveness of time-lapse imaging systems providing undisturbed culture and embryo selection, and time-lapse imaging systems providing only undisturbed culture, and compared each with standard care without time-lapse imaging. METHODS: We conducted a multicentre, three-parallel-group, double-blind, randomised controlled trial in participants undergoing IVF or ICSI at seven IVF centres in the UK and Hong Kong. Embryologists randomly assigned participants using a web-based system, stratified by clinic in a 1:1:1 ratio to the time-lapse imaging system for undisturbed culture and embryo selection (time-lapse imaging group), time-lapse imaging system for undisturbed culture alone (undisturbed culture group), and standard care without time-lapse imaging (control group). Women were required to be aged 18-42 years and men (ie, their partners) 18 years or older. Couples had to be receiving their first, second, or third IVF or ICSI treatment and could not participate if using donor gametes. Participants and trial staff were masked to group assignment, embryologists were not. The primary outcome was live birth. We performed analyses using the intention-to-treat principle and reported the main analysis in participants with primary outcome data available (full analysis set). The trial is registered on the International Trials Registry (ISRCTN17792989) and is now closed. FINDINGS: 1575 participants were randomly assigned to treatment groups (525 participants per group) between June 21, 2018, and Sept 30, 2022. The live birth rates were 33·7% (175/520) in the time-lapse imaging group, 36·6% (189/516) in the undisturbed culture group, and 33·0% (172/522) in the standard care group. The adjusted odds ratio was 1·04 (97·5% CI 0·73 to 1·47) for time-lapse imaging arm versus control and 1·20 (0·85 to 1·70) for undisturbed culture versus control. The risk reduction for the absolute difference was 0·7 percentage points (97·5% CI -5·85 to 7·25) between the time-lapse imaging and standard care groups and 3·6 percentage points (-3·02 to 10·22) between the undisturbed culture and standard care groups. 79 serious adverse events unrelated to the trial were reported (n=28 in time-lapse imaging, n=27 in undisturbed culture, and n=24 in standard care). INTERPRETATION: In women undergoing IVF or ICSI treatment, the use of time-lapse imaging systems for embryo culture and selection does not significantly increase the odds of live birth compared with standard care without time-lapse imaging. FUNDING: Barts Charity, Pharmasure Pharmaceuticals, Hong Kong OG Trust Fund, Hong Kong Health and Medical Research Fund, Hong Kong Matching Fund.

[Application of the blastomere count variations "skip value" in the embryo AI assessment].

Objective: To explore the correlation between blastomere count variations "skip value" which extracted from by time-lapse technology (TLT) combined with artificial intelligence (AI) and morphological features of in vitro fertilization (IVF) embryo, and to test its feasibility in clinical applications. Methods: This study was a diagnostic experiment (AI reassessment of embryo transferred patients), a total of 6 545 embryos from 1 226 patients who underwent IVF at the Women and Children's Hospital of Chongqing Medical University from December 2020 to December 2021 were retrospectively analyzed, of which 2 869 embryos were attempted to cultured to blastocyst stage by TLT. The embryo dynamic map (EDM) was drawn by Embryo Viewer, a TLT recording software, based on embryo developmental kinetics. The self-developed AI embryo evaluation software identified and recorded the number of cleavages in real time during embryonic development, and compared with the EDM, the correlation between the skip value formed by the change of cleavage sphere counts and the outcomes of the embryos was analyzed. The correlation among skip value, morphological score of embryo, implantation rate and live birth rate were performed by Spearman and step-up logistic regression. The receiver operating characteristic (ROC) curve was selected for reporting there relationship of skip value and morphology. Finally, predicting power of skip value for implantation and live birth rate were performed by ROC analysis. Results: The total skip values extracted from the blastomere count of embryos (72 hours post-fertilization) were negatively correlated with abnormal cleavage, blastocyst formation rate, day 3 (D3)-cell score, uneven size and fragmentation (the ß values were -0.268, -0.116, -0.213, -0.159 and -0.222, respectively; all P<0.001); positively correlated with D3-cell number (ß=0.034; P<0.001); negatively correlated with blastocyst formation rate and implantation rate (OR=0.97, 95%CI: 0.93-0.99, P=0.034; OR=0.96, 95%CI: 0.93-0.98, P=0.044). The power of predicting implantation were similar between the order selection of skip values and traditional morphology criteria [area under curve (AUC): 0.679 vs 0.620]. Live birth rate were negatively correlated with female age (OR=0.91, 95%CI: 0.88-0.93; P<0.001), D3 general score (OR=0.77, 95%CI: 0.59-0.99; P=0.045) and order selection of skip values (OR=0.98, 95%CI: 0.96-0.99; P=0.038), while positively correlated with retrieved oocyte number and endometrial thickness in embryo transferred (OR=1.08, 95%CI:1.05-1.11, P<0.001; OR=1.09, 95%CI:1.06-0.12, P<0.001, respectively) from multivariate regression analysis, and the power of predicting live birth was 0.666 for AUC. Conclusions: The skip value and its order form is a systematic quantification of embryo development, correlated with embryo developmental quality and clinical outcome. It could be an addition parameter for embryo culture and selection.

Optimal ICSI timing on immature oocytes for low prognosis patients under the POSEIDON classification.

BACKGROUND: The optimal timing of performing ICSI on immature oocytes for POSEIDON patients is still unknown to get better early embryonic development outcomes. The purpose of this study was to implore the most appropriate time to carry out ICSI on in vitro maturation GV and MI oocytes for POSEIDON patients. METHODS: Two hundred thirty-nine immature oocytes from 163 POSEIDON patients were prospectively performed ICSI at different timings: P-ICSI (ICSI was performed on in vitro matured oocytes 4-6 h after the first polar body extrusion, N = 81), R-ICSI (ICSI was performed on in vitro matured oocytes less than 4 h after the first polar body extrusion, N = 80), and E-ICSI (ICSI was performed on in vitro matured oocytes the next day after oocytes retrieval, N = 78). Fertilization and embryonic development outcomes were collected and statistically analyzed. Mitochondria distribution of cytoplasm of in vitro matured oocytes with different time cultures after the first polar body (PB1) extrusion was stained. RESULTS: Compared to the E-ICSI group, more day 3 embryos from P-ICSI became blastocysts after sequential culture though without statistical significance (OR = 3.71, 95% CI: 0.94-14.63, P = 0.061). Compared to the E-ICSI group, more embryos from both P-ICSI and R-ICSI groups were clinically used with statistical significance (OR = 5.67, 95% CI: 2.24-14.35, P = 0.000 for P-ICSI embryos; OR = 3.23, 95% CI: 1.23-8.45, P = 0.017 for R-ICSI embryos). Compared to the E-ICSI group, transferred embryos from P-ICSI and R-ICSI had a higher implantation rate though without statistical significance (35.3% for P-ICSI embryos; 9.1% or R-ICSI embryos and 0% for E-ICSI embryos, P = 0.050). Among the three group, there were most healthy babies delivered from the P-ICSI group (5, 1 and 0 for P-ICSI, R-ICSI and E-ICSI respectively). The mitochondria in the cytoplasm of in vitro matured oocytes with a less than 4 h and 4-6 h culture after PB1 extrusion presented semiperipheral and diffused distribution patterns, respectively. CONCLUSIONS: Our results revealed P-ICSI (ICSI was performed on in vitro matured oocytes 4-6 h after the first polar body extrusion) provided the most efficient method to utilize the immaturation oocytes basing on embryos utilization and live birth outcome for low prognosis patients under the POSEIDON classification. The mitochondria distribution of the in vitro matured oocytes' cytoplasm from P-ICSI varied that from R-ICSI.

In Vitro Culture of Mammalian Embryos: Is There Room for Improvement?

Preimplantation embryo culture, pivotal in assisted reproductive technology (ART), has lagged in innovation compared to embryo selection advancements. This review examines the persisting gap between in vivo and in vitro embryo development, emphasizing the need for improved culture conditions. While in humans this gap is hardly estimated, animal models, particularly bovines, reveal clear disparities in developmental competence, cryotolerance, pregnancy and live birth rates between in vitro-produced (IVP) and in vivo-derived (IVD) embryos. Molecular analyses unveil distinct differences in morphology, metabolism, and genomic stability, underscoring the need for refining culture conditions for better ART outcomes. To this end, a deeper comprehension of oviduct physiology and embryo transport is crucial for grasping embryo-maternal interactions' mechanisms. Research on autocrine and paracrine factors, and extracellular vesicles in embryo-maternal tract interactions, elucidates vital communication networks for successful implantation and pregnancy. In vitro, confinement, and embryo density are key factors to boost embryo development. Advanced dynamic culture systems mimicking fluid mechanical stimulation in the oviduct, through vibration, tilting, and microfluidic methods, and the use of innovative softer substrates, hold promise for optimizing in vitro embryo development.

The biological characteristics of long cell-free DNA in spent embryos culture medium as noninvasive biomarker in in-vitro embryo selection.

An improved understanding of the cfDNA fragmentomics has proved it as a promising biomarker in clinical applications. However, biological characteristics of cfDNA in spent embryos culture medium (SECM) remain unsolved obstacles before the application in non-invasive in-vitro embryo selection. In this study, we developed a Tn5 transposase and ligase integrated dual-library construction sequencing strategy (TDual-Seq) and revealed the fragmentomic profile of cfDNA of all sizes in early embryonic development. The detected ratio of long cfDNA (>500 bp) was improved from 4.23 % by traditional NGS to 12.80 % by TDual-Seq. End motif analysis showed long cfDNA molecules have a more dominance of fragmentation intracellularly in apoptotic cells with higher predominance of G-end, while shorter cfDNA undergo fragmentation process both intracellularly and extracellularly. Moreover, the mutational pattern of cfDNA and the correlated GO biological process were well differentiated in cleavage and blastocyst embryos. Finally, we developed a multiparametric index (TQI) that employs the fragmentomic profiles of cfDNA, and achieved an area under the ROC curve of 0.927 in screening top quality embryos. TDual-Seq strategy has facilitated characterizing the fragmentomic profile of cfDNA of all sizes in SECM, which are served as a class of non-invasive biomarkers in the evaluation of embryo quality in in-vitro fertilization. And this improved strategy has opened up potential clinical utilities of long cfDNA analysis.

Expression profile of viability and stress response genes as a result of resveratrol supplementation in vitrified and in vitro produced cattle embryos.

BACKGROUND: Resveratrol, a potent antioxidant, is known to induce the up-regulation of the internal antioxidant system. Therefore, it holds promise as a method to mitigate cryopreservation-induced injuries in bovine oocytes and embryos. This study aimed to (i) assess the enhancement in the quality of in vitro produced bovine embryos following resveratrol supplementation and (ii) monitor changes in the expression of genes associated with oxidative stress (GPX4, SOD, CPT2, NFE2L2), mitochondrial function (ATP5ME), endoplasmic reticulum function (ATF6), and embryo quality (OCT4, DNMT1, CASP3, ELOVL5). METHODS AND RESULTS: Three groups of in vitro bovine embryos were cultured with varying concentrations of resveratrol (0.01, 0.001, and 0.0001 µM), with a fourth group serving as a control. Following the vitrification process, embryos were categorized as either good or poor quality. Blastocysts were then preserved at - 80 °C for RNA isolation, followed by qRT-PCR analysis of selected genes. The low concentrations of resveratrol (0.001 µM, P < 0.05 and 0.0001 µM, P < 0.01) significantly improved the blastocyst rate compared to the control group. Moreover, the proportion of good quality vitrified embryos increased significantly (P < 0.05) in the groups treated with 0.001 and 0.0001 µM resveratrol compared to the control group. Analysis of gene expression showed a significant increase in OCT4 and DNMT1 transcripts in both good and poor-quality embryos treated with resveratrol compared to untreated embryos. Additionally, CASP3 expression was decreased in treated good embryos compared to control embryos. Furthermore, ELOVL5 and ATF6 transcripts were down-regulated in treated good embryos compared to the control group. Regarding antioxidant-related genes, GPX4, SOD, and CPT2 transcripts increased in the treated embryos, while NFE2L2 mRNA decreased in treated good embryos compared to the control group. CONCLUSIONS: Resveratrol supplementation at low concentrations effectively mitigated oxidative stress and enhanced the cryotolerance of embryos by modulating the expression of genes involved in oxidative stress response.

Individually Cultured Bovine Zygotes Successfully Develop to the Blastocyst Stage in an Extremely Confined Environment.

The possibility of detecting the developmental competence of individually cultured embryos through analysis of spent media is a major current trend in an ART setting. However, individual embryo culture is detrimental compared with high-density group culture due to the reduced concentration of putative embryotropins. The main aim of this study was to identify an individual culture system that is not detrimental over high-density group culture in the bovine model. Blastocyst rates and competence were investigated in a conventional (GC) group, semi-confined group (MG), and individual culture (MS) in a commercial microwell device. Main findings showed that: (1) individual embryos can be continuously cultured for 7 days in ~70 nL microwells (MS) without detrimental effects compared with the GC and MG; (2) MS and MG blastocysts had a reduced number of TUNEL-positive cells compared to GC blastocysts; (3) though blastocyst mean cell numbers, mitochondrial activity, and lipid content were not different among the three culture conditions, MS blastocysts had a higher frequency of small-sized lipid droplets and a reduced mean droplet diameter compared with GC and MG blastocysts. Overall, findings open the way to optimize the development and competence of single embryos in an ART setting.

A novel method for gas mixing and distribution in multi-chamber embryo incubators.

BACKGROUND: High-quality control of the gas environment in incubators is crucial for in vitro embryo development, which requires high accuracy, fast recovery, and low gas consumption. OBJECTIVE: In this study, we propose a novel gas mixing and distribution system and method as an alternative solution for multi-chamber embryo incubators. METHODS: The system-based embryo incubator enables a controllable gas circulation process and a quantitative supply of CO2 and N2. To determine the optimal parameters for the mixing time and flow rate of the circulated gases, we conducted contrast experiments on the system-based incubator. To evaluate the performance of the gas system in the incubator, we conducted tests under four different initial conditions, simulating various practical application scenarios. Furthermore, we performed a mouse embryo assay to assess the system's effectiveness. RESULTS: The results show that the system achieved a gas concentration accuracy of ± 0.2% (volume fraction) after stabilization, a minimum recovery time of 5 minutes, an average consumption of 8.9 L/d for N2 and 0.83 L/d for CO2 during routine operation, and a blastocyst rate exceeding 90% observed after 96 hours of culture in the incubator. CONCLUSION: The system and method demonstrate a significant advantage in terms of low gas consumption compared to existing incubators, while still maintaining high accuracy and fast recovery.

Establishing a 3D-cultured system based on alginate-hydrogel embedding benefits the in vitro maturation of porcine Oocytes.

The in vitro maturation (IVM) quality of oocytes is directly related to the subsequent developmental potential of embryos and a fundamental of in vitro embryo production. However, conventional IVM methods fail to maintain the gap-junction intercellular communication (GJIC) between cumulus-oocyte complexes (COCs), which leads to insufficient oocyte maturation. Herein, we investigated the effects of three different three-dimensional (3D) culture methods on oocyte development in vitro, optimized of the alginate-hydrogel embedding method, and assessed the effects of the alginate-hydrogel embedding method on subsequent embryonic developmental potential of oocytes after IVM and parthenogenetic activation (PA). The results showed that Matrigel embedding and alginate-hydrogel embedding benefited the embryonic developmental potential of oocytes after IVM and PA. With the further optimization of alginate-hydrogel embedding, including crosslinking and decrosslinking of parameters, we established a 3D culture system that can significantly increase oocyte maturation and the blastocyst rate of embryos after PA (27.2 ± 1.5 vs 36.7 ± 2.8, P < 0.05). This 3D culture system produced oocytes with markedly increased mitochondrial intensity and membrane potential, which reduced the abnormalities of spindle formation and cortical granule distribution. The alginate-hydrogel embedding system can also remarkably enhance the GJIC between COCs. In summary, based on alginate-hydrogel embedding, we established a 3D culture system that can improve the IVM quality of porcine oocytes, possibly by enhancing GJIC.

Melatonin application during cryopreservation improves the development and clinical outcomes of human vitrified-warmed oocytes.

In this clinical study, we investigated the potential of melatonin (MT) supplementation in the freeze-thaw medium used for cryopreserved human oocytes. In total, 152 patients who underwent in vitro fertilization between January 2020 and December 2022 were included and categorized into different groups as follows: the donor group, comprising 108 patients who donated their oocytes, with 34 patients using a vitrification and warming medium supplemented with MT (D-MT subgroup) and 74 patients using conventional medium without MT (D-0 subgroup); and the autologous group, comprising 38 patients who used their own oocytes, with 19 patients using medium supplemented with MT (A-MT subgroup) and 19 patients using medium without MT (A-0 subgroup). After thawing, the surviving oocytes in the D-MT and A-MT subgroups and D-0 and A-0 subgroups were cultured in a fertilization media with and without 10-9 MMT for 2.5 h, respectively, followed by intracytoplasmic sperm injection insemination, embryo culture, and transfer. The survival, cleavage, high-quality embryo, clinical pregnancy, ongoing pregnancy, and implantation rates were significantly higher in the D-MT subgroup than in the D-0 subgroup (all P < 0.05). Similarly, the survival, fertilization, high-quality embryo, and high-quality blastocyst rates were significantly higher in the A-MT subgroup than in the A-0 subgroup (all P < 0.05). These findings indicate that MT addition during cryopreservation can enhance the development of vitrified-warmed human oocytes and improve clinical outcomes.

Successful pregnancy with intracytoplasmic sperm injection after bacterial contamination of embryo culture in in vitro fertilization: a case report.

BACKGROUND: Bacterial infection of embryo culture medium is rare but may be detrimental. The main source of embryo culture contamination is semen. Assisted reproduction centers currently lack consensus regarding the methods for preventing and managing embryo culture infection. In our recent case, a successful pregnancy was achieved with intracytoplasmic sperm injection after failed conventional in vitro fertilization owing to bacterial contamination. CASE PRESENTATION: We present a case report of two consecutive in vitro fertilization-intracytoplasmic sperm injection cycles with photo and video documentation of the bacterial growth. A 36-year-old Hungarian woman and her 37-year-old Hungarian partner came to our department. They had two normal births followed by 2 years of infertility. The major causes of infertility were a closed fallopian tube and asthenozoospermia. Bacterial infection of the embryo culture medium was observed during in vitro fertilization and all oocytes degenerated. The source was found to be the semen. To prevent contamination, intracytoplasmic sperm injection was used for fertilization in the subsequent cycle. Intracytoplasmic bacterial proliferation was observed in one of the three fertilized eggs, but two good-quality embryos were successfully obtained. The transfer of one embryo resulted in a successful pregnancy and a healthy newborn was delivered. CONCLUSION: Intracytoplasmic sperm injection may be offered to couples who fail conventional in vitro fertilization treatment owing to bacteriospermia, as it seems to prevent infection of the embryo culture. Even if bacterial contamination appears, our case encourages us to continue treatment. Nevertheless, the development of new management guidelines for the prevention and management of bacterial contamination is essential.

Embryo development and live birth in women with one previously failed full IVF/ICSI cycle.

PURPOSE: To evaluate the embryological and pregnancy outcomes of women who failed in their first IVF treatment if they attempted a second cycle. METHODS: For evaluating the embryological outcomes, the study cohort included 1,227 women who failed to obtain a live birth after the initial IVF cycle from September 2018 to August 2021 and returned for a second attempt. To evaluate reproductive outcomes including live birth rates (LBRs), 1227 women who returned for a second attempt were compared with 13,195 women undergoing their first oocyte retrieval with blastocyst culture attempted during the same study period. RESULTS: In women who had a second cycle, the median number of oocyte retrieved (11 vs 9), fertilized oocytes (7 vs 5), usable embryos (6 vs 4) and blastocysts (3 vs 1) was higher in the second cycle compared to the first cycle (All p < 0.001). Blastocyst formation rates were significantly increased from 33% in the first cycle to 50% in the second cycle across the age group (p < 0.001). However, the primary transfer LBRs were significantly lower in the second cycle than that in the initial cycle (40.82% versus 51.79%, aOR: 0.74 [0.65, 0.84]). LBRs in the second cycle were 42.26%, 42.68%, 25.49% and 16.22% in women aged < 35, 35-37, 38-40, and > 40 years. CONCLUSION: There was a notable enhancement in laboratory outcomes following the second attempt in women whose initial IVF cycles were unsuccessful. However, the uncertainty inherent in the successful implantation and the consequent progression to live birth remains a significant challenge.

Temperature fluctuations during embryo transfer can be mitigated by optimizing transfer protocol.

RESEARCH QUESTION: What impact do variations in embryo transfer catheter loading and movement procedures have on temperature and pH fluctuations during embryo transfer? DESIGN: Mock embryo transfers were conducted to test the impact of air flow/movement, use of catheter coverings, and the type of workstation used for catheter loading on catheter temperature. A thermocouple probe inserted into the tip of the outer catheter or taped to the exterior of the inner catheter recorded temperature within the catheter every 5 s from time of mock embryo loading (TL) to 60 s (TL + 60 s) or from the start of transit (TT). Fluctuations in culture medium pH in embryo transfer dishes were monitored. RESULTS: The rate of cooling during transit was faster (all P < 0.05) when catheters were uncovered compared with all covering methods tested. This resulted in a lower catheter temperature at TL + 20 s (28.43 ± 0.30 °C) compared with catheters covered by plastic tubing (31.4 ± 0.30 °C), paper (31.0 ± 0.26 °C) or paper + thumb (31.1 ± 0.78 °C; all P ≤ 0.05). Temperature was maintained more effectively when catheters were loaded in a crib compared with a heated stage, until initiation of transit, when the rate of temperature decrease was similar. Culture medium pH increased more rapidly when embryo transfer dishes remained on a heated stage during the procedure compared with in an open crib. CONCLUSIONS: Temperature loss during the embryo transfer procedure can be mitigated by reducing the transit time and using catheter coverings. Use of a crib for catheter loading only improved temperature stability while the catheter remained in the crib, not during transit, and reduced pH fluctuations during the procedure.

The utility of human two plus one small pronucleated zygotes (2.1PN) based on clinical outcomes and the focused ploidy analysis.

PURPOSE: Are human embryos arising from two plus one small pronucleated zygotes, called 2.1 pronuclei (PN), clinically useful? METHODS: In a retrospective embryo cohort study and prospective experimental study, a total of 287 cycles in which at least one 2.1PN was identified in the fertilization check were included. Embryonic development and clinical outcome were compared for the 1395 2PN zygotes and 304 2.1PN zygotes that were siblings. All embryos were individually cultured in time-lapse systems. Twenty-five 2.1PN-derived blastocysts, donated for research, were used in focused single-nucleotide variant ploidy analysis to identify the distribution pattern of heterozygosity. RESULTS: The average diameter of PN was 24.9 ± 2.4 µm for large PN and 10.2 ± 2.4 µm for small PN; 79.9% of small PN was derived from female pronuclei. Blastocyst formation rate and good-quality blastocyst rate were significantly lower with 2.1PN embryos than with 2PN embryos (40.0% vs. 57.7%, 21.4% vs. 33.5%, respectively). A total of 13 embryos derived from 2.1PN were transferred, and three healthy babies were born. In ploidy constitutions of trophectoderm (TE), 2.1PN-derived blastocyst TE was shown to be mostly diploid (95.8%, 23/24), and only one blastocyst showed triploid. CONCLUSIONS: It was suggested that 2.1PN embryos have lower embryonic developmental potential than 2PN embryos, but most of the 2.1PN were diploid, indicating that they are likely to be clinically usable. It is recommended to perform embryo transfer following a combination of PGT-A and ploidy analysis.

Effect of time post warming to embryo transfer on human blastocyst metabolism and pregnancy outcome.

PURPOSE: This study is aiming to test whether variation in post warming culture time impacts blastocyst metabolism or pregnancy outcome. METHODS: In this single center retrospective cohort study, outcomes of 11,520 single frozen embryo transfer (FET) cycles were analyzed from January 2015 to December 2020. Patient treatments included both natural and programmed cycles. Time categories were determined using the time between blastocyst warming and embryo transfer: 0 (0- <1h), 1 (1-<2h), 2 (2-<3h), 3(3-<4h), 4 (4-<5), 5 (5-<6), 6 (6-<7) and 7 (7-8h). Non-invasive metabolic imaging of discarded human blastocysts for up to 10h was also performed using Fluorescence lifetime imaging microscopy (FLIM) to examine for metabolic perturbations during culture. RESULTS: The mean age of patients across all time categories were comparable (35.6 ± 3.9). Live birth rates (38-52%) and miscarriage rate (5-11%) were not statistically different across post-warming culture time. When assessing pregnancy outcomes based on the use of PGT-A, miscarriage and live birth rates were not statistically different across culture hours in both PGT-A and non-PGT cycles. Further metabolic analysis of blastocysts for the duration of 10h of culture post warming, revealed minimal metabolic changes of embryos in culture. CONCLUSION: Overall, our results show that differences in the time of post warming culture have no significant impact on miscarriage or live birth rate for frozen embryo transfers. This information can be beneficial for clinical practices with either minimal staffing or a high number of patient cases.

Development and validation of an automated robotic system for preparation of embryo culture dishes.

OBJECTIVE: To study the development and clinical validation of the ART Pipetting Robot for the IVF Laboratory (APRIL), a liquid-handling robot customized for the precise preparation of microdroplet culture dishes in the field of in vitro fertilization (IVF). DESIGN: A prospective randomized study conducted at an academic IVF center comparing mouse and human embryo outcomes and quantitative measures of accuracy in embryo dishes prepared using APRIL compared with standard manual preparation. SETTING: Academic IVF center. SUBJECTS: The study involved the assessment of the automated culture dish preparation system, APRIL, compared with manual preparation methods in the context of IVF treatment. INTERVENTION: ART Pipetting Robot for the IVF Laboratory is an enclosed liquid-handling robot equipped with custom three-dimensional-printed adapters and designed to dispense embryo culture media and mineral oil into microdroplet culture dishes. MAIN OUTCOME MEASURES: The study evaluated the precision and consistency of APRIL in culture dish preparation by looking at droplet mass, pH of prepared media droplets, and mouse and human embryo development rates. Clinical implementation was assessed by comparing embryo development and outcomes in dishes prepared by APRIL and human embryologists. RESULTS: Compared with embryo culture dishes prepared using standard manual procedures, embryo culture dishes prepared using APRIL demonstrated a greater than 10-fold improvement in consistency (coefficient of variation, 0.46% vs. 6%-7%), maintained optimal pH levels (pH range, 7.281-7.33 vs. 7.275-7.311), and had a greater mouse embryo blastocyst rate (100% vs. 90%-91%). Human embryos cultured in dishes prepared by APRIL had a higher rate of development on days 3 (92.4% vs. 82.6%) and 5 (19.75% vs. 15.57%), and a total number of usable embryos (50.3% vs. 46.1%) compared with manually prepared dishes, although the last two outcomes did not reach statistical significance. CONCLUSION: The results suggest that the use of an automated robotic system for preparation of embryo culture dishes may improve accuracy and outcome measures while reducing the need for trained laboratory personnel to prepare the dishes manually.

Influence of the storage conditions of embryo culture media on mouse development.

The culture of preimplantation embryos in vitro is an important method for human and mouse reproductive technology. This study aims to investigate the influence of different conditions of culture media on the preimplantation stage of mouse embryos cultured in vitro, and monitor the post-implantation development of new mice after embryo transfer to surrogate females. We demonstrated here that mouse embryos cultured in vitro in fresh M16, KSOM, Global, and HTF embryo culture media from one cell to the blastocyst stage and the subsequent embryo transfer to surrogate females are able to proceed through post-implantation development and, after birth, develop into healthy mice. However, culture of embryos in differently aged media shows various (often unpredictable) results. To find the optimal storage conditions of culture media, we suggest that the freezing and long-term storage of these media at - 80°C will not influence the quality of the media. To test this hypothesis, we grew embryos from one cell to blastocysts in vitro in the selected media after thawing and subsequently transferring them to surrogate females. Embryo culture in these four media after thawing does not affect preimplantation and postnatal mouse development. Thus, we have shown that storage of embryo culture media at low temperature (- 80°C) does not impact the quality of the media, and subsequently, it can be used for the culture of embryos for the full preimplantation period, the same as in fresh media.