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.

Metabolic imaging of human embryos is predictive of ploidy status but is not associated with clinical pregnancy outcomes: a pilot trial.

STUDY QUESTION: Does fluorescence lifetime imaging microscopy (FLIM)-based metabolic imaging assessment of human blastocysts prior to frozen transfer correlate with pregnancy outcomes? SUMMARY ANSWER: FLIM failed to distinguish consistent patterns in mitochondrial metabolism between blastocysts leading to pregnancy compared to those that did not. WHAT IS KNOWN ALREADY: FLIM measurements provide quantitative information on NAD(P)H and flavin adenine dinucleotide (FAD+) concentrations. The metabolism of embryos has long been linked to their viability, suggesting the potential utility of metabolic measurements to aid in selection. STUDY DESIGN, SIZE, DURATION: This was a pilot trial enrolling 121 IVF couples who consented to have their frozen blastocyst measured using non-invasive metabolic imaging. After being warmed, 105 couples' good-quality blastocysts underwent a 6-min scan in a controlled temperature and gas environment. FLIM-assessed blastocysts were then transferred without any intervention in management. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eight metabolic parameters were obtained from each blastocyst (4 for NAD(P)H and 4 for FAD): short and long fluorescence lifetime, fluorescence intensity, and fraction of the molecule engaged with enzyme. The redox ratio (intensity of NAD(P)H)/(intensity of FAD) was also calculated. FLIM data were combined with known metadata and analyzed to quantify the ability of metabolic imaging to differentiate embryos that resulted in pregnancy from embryos that did not. De-identified discarded aneuploid human embryos (n = 158) were also measured to quantify correlations with ploidy status and other factors. Statistical comparisons were performed using logistic regression and receiver operating characteristic (ROC) curves with 5-fold cross-validation averaged over 100 repeats with random sampling. AUC values were used to quantify the ability to distinguish between classes. MAIN RESULTS AND THE ROLE OF CHANCE: No metabolic imaging parameters showed significant differences between good-quality blastocysts resulting in pregnancy versus those that did not. A logistic regression using metabolic data and metadata produced an ROC AUC of 0.58. In contrast, robust AUCs were obtained when classifying other factors such as comparison of Day 5 (n = 64) versus Day 6 (n = 41) blastocysts (AUC = 0.78), inner cell mass versus trophectoderm (n = 105: AUC = 0.88) and aneuploid (n = 158) versus euploid and positive pregnancy embryos (n = 108) (AUC = 0.82). LIMITATIONS, REASONS FOR CAUTION: The study protocol did not select which embryo to transfer and the cohort of 105 included blastocysts were all high quality. The study was also limited in number of participants and study sites. Increased power and performing the trial in more sites may have provided a stronger conclusion regarding the merits of the use of FLIM clinically. WIDER IMPLICATIONS OF THE FINDINGS: FLIM failed to distinguish consistent patterns in mitochondrial metabolism between good-quality blastocysts leading to pregnancy compared to those that did not. Blastocyst ploidy status was, however, highly distinguishable. In addition, embryo regions and embryo day were consistently revealed by FLIM. While metabolic imaging detects mitochondrial metabolic features in human blastocysts, this pilot trial indicates it does not have the potential to serve as an effective embryo viability detection tool. This may be because mitochondrial metabolism plays an alternative role post-implantation. STUDY FUNDING/COMPETING INTEREST(S): This study was sponsored by Optiva Fertility, Inc. Boston IVF contributed to the clinical site and services. Becker Hickl, GmbH, provided the FLIM system on loan. T.S. was the founder and held stock in Optiva Fertility, Inc., and D.S. and E.S. had options with Optiva Fertility, Inc., during this study. TRIAL REGISTRATION NUMBER: The study was approved by WCG Connexus IRB (Study Number 1298156).

Day after rescue ICSI: eliminating total fertilization failure after conventional IVF with high live birth rates following cryopreserved blastocyst transfer.

STUDY QUESTION: What is the impact of day after rescue ICSI (r-ICSI) on success of fresh and frozen embryo transfers? SUMMARY ANSWER: The use of r-ICSI can virtually allay fears of total fertilization failure (TFF) after conventional IVF (C-IVF) and achieve high live birth rates after frozen blastocyst transfer. WHAT IS KNOWN ALREADY: More infertility clinics have resorted to the use of ICSI in place of C-IVF in IVF treatment owing to fear of TFF or a low fertilization rate. r-ICSI has been attempted either on the day of IVF or the day after. Day after r-ICSI has proved unsuccessful in the past. STUDY DESIGN, SIZE, DURATION: A retrospective data analysis was performed of 16 608 qualifying cases between April 2010 and July 2021 conducted at a single private academically affiliated fertility clinic. PARTICIPANTS/MATERIALS, SETTING, METHODS: r-ICSI was performed principally on patients with >4 metaphase II oocytes, showing no signs of fertilization 18 h after C-IVF. C-IVF was performed on patients who had >4 million total motile sperm after preparation. r-ICSI was then performed 18-24 h after insemination, using the sperm sample from the previous day. r-ICSI fertilization rates, cryopreservation of cleavage and blastocysts embryos, and pregnancy rates after fresh or frozen transfer were then assessed. MAIN RESULTS AND THE ROLE OF CHANCE: r-ICSI was performed on 377 patients (2.3% of eligible retrieval cycles) who had a mean (±SD) female and male age of 35.9 ± 4.5 and 38.1 ± 9.1 years, respectively. A total of 5459 oocytes were initially retrieved. Of the oocytes undergoing r-ICSI, 2389 (49.5%) fertilized normally, and 205 (54.4%) patients underwent a fresh embryo transfer. The live birth rates were 23/186 (12.3%) for fresh cleavage and 5/19 (26.3%) for fresh blastocyst stage transfers. In 145 cycles a blastocyst was frozen, and 137 transfers were performed with a 64/137 (46.7%) live birth rate. Of the 377 cycles receiving r-ICSI only, 25 of the qualifying cases failed to have any fertilization, reducing TFF to 25/16 608 (0.15%). LIMITATIONS, REASONS FOR CAUTION: This was a single-center retrospective study on a specific subset of patients, which may limit its generalizability to other clinics. WIDER IMPLICATIONS OF THE FINDINGS: r-ICSI allows a second opportunity to fertilize oocytes despite poor initial outcomes. Patients who had a frozen blastocyst transfer achieved high live birth rates, indicating that a resynchronization of the embryo with the endometrium can optimize r-ICSI cases. r-ICSI allays fears of TFF when using C-IVF, providing evidence that the overuse of ICSI in patients without male factor may not be warranted. STUDY FUNDING/COMPETING INTEREST(S): The study was internally funded by Boston IVF. The authors declare that they have no conflict of interest in relation to the data published in the article. TRIAL REGISTRATION NUMBER: N/A.

Multicenter prospective study of concordance between embryonic cell-free DNA and trophectoderm biopsies from 1301 human blastocysts.

BACKGROUND: The recent identification of embryonic cell-free DNA in spent blastocyst media has opened a new era of possibilities for noninvasive embryo aneuploidy testing in assisted reproductive technologies. Yet, previous studies assessing a limited number of embryos reported variable concordance between embryonic cell-free DNA and trophectoderm biopsies, thus questioning the validity of this approach. OBJECTIVE: This study aimed to evaluate the concordance and reproducibility of testing embryonic cell-free DNA vs trophectoderm DNA obtained from the same embryo in a large sample of human blastocysts and to assess the contribution of the inner cell mass and trophectoderm to embryonic cell-free DNA released to the culture media. STUDY DESIGN: This is an interim analysis of a prospective, observational study among 8 in vitro fertilization centers in 4 continents to assess consistency between noninvasive embryo aneuploidy testing of embryonic cell-free DNA and conventional trophectoderm biopsy. The analysis included 1301 day-6/7 blastocysts obtained in 406 in vitro fertilization cycles from 371 patients aged 20-44 years undergoing preimplantation genetic testing for aneuploidy. Fresh oocytes underwent intracytoplasmic sperm injection or in vitro fertilization. No previous assisted hatching or vitrification was allowed before media collection. Individual spent blastocyst medium was collected from embryos cultured at least 40 hours from day 4. After media collection, conventional preimplantation genetic testing for aneuploidy, comprising trophectoderm biopsy and blastocyst vitrification, was performed. Embryonic cell-free DNA was analyzed blindly after embryo transfer. Inner cell mass and trophectoderm biopsies were also performed in a subset of 81 aneuploid blastocysts donated for research. RESULTS: Embryonic cell-free DNA analyses were 78.2% (866/1108) concordant with the corresponding trophectoderm biopsies. No significant differences were detected among centers ranging from 72.5% to 86.3%. Concordance rates exceeded 86% when all defined steps in the culture laboratory were controlled to minimize the impact of maternal and operator contamination. Sensitivity per center ranged from 76.5% to 91.3% and specificity from 64.7% to 93.3%. The false-negative rate was 8.3% (92/1108), and false-positive rate was 12.4% (137/1108). The 2 fertilization techniques provided similar sensitivity (80.9% vs 87.9%) and specificity (78.6% vs 69.9%). Multivariate analysis did not reveal any bias from patient clinical background, ovarian stimulation protocols, culture conditions, or embryo quality on testing accuracy of concordance. Moreover, concordances of embryonic cell-free DNA with trophectoderm and inner cell mass suggest that the embryonic cell-free DNA originates from both compartments of the human embryo. CONCLUSION: Noninvasive analysis of embryonic cell-free DNA in spent blastocyst culture media demonstrates high concordance with trophectoderm biopsy results in this large multicenter series. A noninvasive approach for prioritizing embryo euploidy offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population.