To assess the value of deep learning in selecting the optimal embryo for in vitro fertilization, a multicenter, randomized, double-blind, noninferiority parallel-group trial was conducted across 14 in vitro fertilization clinics in Australia and Europe. Women under 42 years of age with at least two early-stage blastocysts on day 5 were randomized to either the control arm, using standard morphological assessment, or the study arm, employing a deep learning algorithm, intelligent Data Analysis Score (iDAScore), for embryo selection. The primary endpoint was a clinical pregnancy rate with a noninferiority margin of 5%. The trial included 1,066 patients (533 in the iDAScore group and 533 in the morphology group). The iDAScore group exhibited a clinical pregnancy rate of 46.5% (248 of 533 patients), compared to 48.2% (257 of 533 patients) in the morphology arm (risk difference -1.7%; 95% confidence interval -7.7, 4.3; P = 0.62). This study was not able to demonstrate noninferiority of deep learning for clinical pregnancy rate when compared to standard morphology and a predefined prioritization scheme. Australian New Zealand Clinical Trials Registry (ANZCTR) registration: 379161. A randomized controlled trial evaluating the selection of a single blastocyst for transfer by deep learning did not demonstrate noninferiority in clinical pregnancy rates when compared to trained embryologists using standard morphology criteria.

Research question: Do cell numbers and degree of fragmentation in cleavage-stage embryos, assessed manually, correlate with evaluations made by deep learning algorithm model iDAScore v2.0?Design: Retrospective observational study (n = 5040 embryos; 1786 treatments) conducted at two Swedish assisted reproductive technology centres between 2016 and 2021. Fresh single embryo transfer was carried out on days 2 or 3 after fertilization. Embryo evaluation using iDAScore v2.0 was compared with manual assessment of numbers of cells and grade of fragmentation, analysed by video sequences.Results: Data from embryos transferred on days 2 and 3 showed that having three or fewer cells compared with four or fewer cells on day 2, and six or fewer cells versus seven to eight cells on day 3, correlated significantly with a difference in iDAScore (medians 2.4 versus 4.0 and 2.6 versus 4.6 respectively; both P < 0.001). The iDAScore for 0-10% fragmentation was significantly higher compared with the groups with higher fragmentation (P < 0.001). When combining cell numbers and fragmentation, iDAScore values decreased as fragmentation increased, regardless of cell number. iDAScore discriminated between embryos that resulted in live birth or no live birth (AUC of 0.627 and 0.607), compared with the morphological model (AUC of 0.618 and 0.585) for day 2 and day 3, respectively. Conclusions: The iDAScore v2.0 values correlated significantly with cell numbers and fragmentation scored manually for cleavage-stage embryos on days 2 and 3. iDAScore had some predictive value for live birth, conditional that embryo selection was based on morphology.

Abstract Study question Does the embryo evaluation model iDAScore v2.0 correlate with manually assessed cell numbers and fragmentation grade in early-stage embryos? Summary answer iDAScore v2.0 significantly correlated with morphological assessment of preimplantation embryos on days 2 and 3. Correlation between cell numbers and iDAScore decreased with increasing fragmentation. What is known already Time-lapse technology, including continuous documentation of embryo development, has enabled the implementation of more automated scoring systems, based on images and video sequences. iDAScore (ranging from 1-9.9) is a fully automated annotation-free scoring system based on deep learning and has been shown to discriminate between implanting and non-implanting embryos to a similar extent as KIDScore, which required manual annotations. iDAScore v2.0 also demonstrated a good agreement between predicted probabilities and observed implantation rates. However, not much is known regarding the correlation between specific morphological day 2 and day 3 variables and iDAScore v2.0. Study design, size, duration Retrospective observational study from two ART centers, together performing > 2000 OPU per year. The study includes all normally fertilized embryos (n = 5378) from 1792 treatments between 2016 and 2021 where fresh single embryo transfer was performed on day 2 or day 3 post fertilization. The association of iDAScore v2.0 with manual assessment of cell numbers and grade of fragmentation on day 2 (treatments = 762, embryos = 2170) and day 3 (treatments = 1030, embryos = 3208) was analysed using video sequences. Participants/materials, setting, methods Inclusion criteria were treatment cycles with own (autologous) oocytes and embryo transfer on day 2 or 3. Exclusion criteria were cycles with double embryo transfers or PGT cycles. Clinical and laboratory processes were performed according to standard procedures. Time-lapse culture and embryo annotations were performed using the Embryoscope+® with GTL® culture medium (Vitrolife, Gothenburg, Sweden). Wilcoxon rank sum test was used to compare iDAScores between groups. Main results and the role of chance Day 2: For the number of cells, iDAScore differed between embryos having < = 3 cells compared to > = 4 cells (medians: 2.3 vs. 4.0, p < 0.001). The median iDAScores for fragmentation grade 0%; <20%; <50%; > 50% were 4.7; 3.9; 2.6 and 2.1, respectively. Furthermore, 0% was significantly different from each of the other groups (p < 0.001). When cell numbers and fragmentation grade were combined, it was seen that having < = 3 cells versus > = 4 was significantly correlated to iDAScore when the fragmentation grade was scored as 0%, but this association decreased as the fragmentation grade increased. Day 3: A similar pattern was seen for day 3 morphology analyses. It was found that having < = 5 cells vs. > = 6 cells correlated to a significant difference in iDAScore (medians: 2.5 vs. 4.4, p < 0.001). The median iDAScores for fragmentation grade 0%; <20%; <50%; > 50% were 5.6; 3.8; 2.6 and 2.3, respectively. Furthermore, 0% was significantly different from each of the other groups (p < 0.001). When combining cell numbers and fragmentation grade in the same analysis, the association of cell numbers to iDAScore decreased as fragmentation increased. Limitations, reasons for caution The present study is of retrospective design, with analysis of data from two clinics, and the results may not apply in a wider context. In addition, only two morphological variables, cell numbers and fragmentation, were included. Wider implications of the findings Our results show that there is a strong correlation between iDAScore v2.0 and the number of cells and the fragmentation grade of the embryo, both for each individual variable and when combined. This indicates that iDAScore v2.0 identifies some of the well-established morphological evaluations for cleavage stage transfers. Trial registration number Not applicable

Study question Does the embryo evaluation model iDAScore v2.0 correlate with manually assessed cell numbers and fragmentation grade in early-stage embryos? Summary answer iDAScore v2.0 significantly correlated with morphological assessment of preimplantation embryos on days 2 and 3. Correlation between cell numbers and iDAScore decreased with increasing fragmentation. What is known already Time-lapse technology, including continuous documentation of embryo development, has enabled the implementation of more automated scoring systems, based on images and video sequences. iDAScore (ranging from 1-9.9) is a fully automated annotation-free scoring system based on deep learning and has been shown to discriminate between implanting and non-implanting embryos to a similar extent as KIDScore, which required manual annotations. iDAScore v2.0 also demonstrated a good agreement between predicted probabilities and observed implantation rates. However, not much is known regarding the correlation between specific morphological day 2 and day 3 variables and iDAScore v2.0. Study design, size, duration Retrospective observational study from two ART centers, together performing > 2000 OPU per year. The study includes all normally fertilized embryos (n = 5378) from 1792 treatments between 2016 and 2021 where fresh single embryo transfer was performed on day 2 or day 3 post fertilization. The association of iDAScore v2.0 with manual assessment of cell numbers and grade of fragmentation on day 2 (treatments = 762, embryos = 2170) and day 3 (treatments = 1030, embryos = 3208) was analysed using video sequences. Participants/materials, setting, methods Inclusion criteria were treatment cycles with own (autologous) oocytes and embryo transfer on day 2 or 3. Exclusion criteria were cycles with double embryo transfers or PGT cycles. Clinical and laboratory processes were performed according to standard procedures. Time-lapse culture and embryo annotations were performed using the Embryoscope+® with GTL® culture medium (Vitrolife, Gothenburg, Sweden). Wilcoxon rank sum test was used to compare iDAScores between groups. Main results and the role of chance Day 2: For the number of cells, iDAScore differed between embryos having < = 3 cells compared to > = 4 cells (medians: 2.3 vs. 4.0, p < 0.001). The median iDAScores for fragmentation grade 0%; <20%; <50%; > 50% were 4.7; 3.9; 2.6 and 2.1, respectively. Furthermore, 0% was significantly different from each of the other groups (p < 0.001). When cell numbers and fragmentation grade were combined, it was seen that having < = 3 cells versus > = 4 was significantly correlated to iDAScore when the fragmentation grade was scored as 0%, but this association decreased as the fragmentation grade increased. Day 3: A similar pattern was seen for day 3 morphology analyses. It was found that having < = 5 cells vs. > = 6 cells correlated to a significant difference in iDAScore (medians: 2.5 vs. 4.4, p < 0.001). The median iDAScores for fragmentation grade 0%; <20%; <50%; > 50% were 5.6; 3.8; 2.6 and 2.3, respectively. Furthermore, 0% was significantly different from each of the other groups (p < 0.001). When combining cell numbers and fragmentation grade in the same analysis, the association of cell numbers to iDAScore decreased as fragmentation increased. Limitations, reasons for caution The present study is of retrospective design, with analysis of data from two clinics, and the results may not apply in a wider context. In addition, only two morphological variables, cell numbers and fragmentation, were included. Wider implications of the findings Our results show that there is a strong correlation between iDAScore v2.0 and the number of cells and the fragmentation grade of the embryo, both for each individual variable and when combined. This indicates that iDAScore v2.0 identifies some of the well-established morphological evaluations for cleavage stage transfers. Trial registration number Not applicable

Abstract Study question Can iDAScore v2.0 predict likelihood of development into a utilizable blastocyst from time-lapse images captured during the first two or three days of embryo development? Summary answer iDAScore v2.0 predicts utilizable blastocyst formation from time-lapse images of early embryo development. Specific score thresholds can be established to ensure high specificity of prediction. What is known already Treatment efficacy would benefit from a reliable method to predict the potential of early embryos to develop into utilizable blastocysts and select fresh IVF treatment cycles suitable for blastocyst culture. Reports indicate that time-lapse images captured during the first few days of development and analyzed by deep learning methods may be an accurate approach. A fully automated AI model, iDAScore, is reported to be more accurate than routine morphology and morphokinetics for prediction of implantation. However, the applicability of iDAScore calculated on day 2 and 3 as a tool to predict blastocyst development has yet to be tested. Study design, size, duration This is a retrospective multicenter cohort study including time-lapse videos of 9111 embryos cultured to blastocyst stage, from 1283 fresh IVF treatments between 2016-2021 at two ART clinics together performing > 2000 OPU per year. Images captured during the first days of culture were used to calculate iDAScores (at 48 and 66 hours post insemination) and determine the ability to discriminate embryos likely to develop into utilizable blastocysts according to Gardner score of 3BB or more. Participants/materials, setting, methods Patients were required to be undergoing IVF treatment using their own (autologous) oocytes whereby all zygotes were cultured up to the blastocyst stage in GTL® culture medium using the Embryoscope+® time-lapse system (Vitrolife, Gothenburg, Sweden). At least two experienced embryologists graded blastocysts on day 5/6, selecting utilizable blastocysts for transfer/vitrification. To evaluate the performance of iDAScore to predict development into utilizable blastocysts, sensitivity, specificity, and area under ROC curve (AUC) values were calculated. Main results and the role of chance The blastocyst utilization rate was 43.8% (3990 utilizable blastocysts of 9111 day 2/3 embryos developing from 2PN zygotes). Prediction of utilizable blastocysts obtained AUCs of 0.768 (0.759 - 0.778) and 0.782 (0.773 - 0.791) for day 2 and day 3, respectively. These results suggest IDAScore prediction performed slightly better when calculated on day 3 than on day 2. The ability to discriminate between utilizable and non-utilizable blastocysts at different thresholds of iDAScore on day 2 and on day 3, suggests that embryos should have a minimum iDAscore of 5 on day 2 to obtain a high specificity of 89%, and a threshold of 6 on day 3 to obtain a high specificity of 88%. Lower sensitivities were observed for these potential thresholds, 35% on day 2 and 41% on day 3. Limitations, reasons for caution This is a retrospective analysis where most cycles have at least five zygotes that supported subsequent blastocyst development, limiting relevance for patients with fewer zygotes. IDAScore is developed to predict implantation potential, not utilizable blastocyst, thus could be an underestimation of the potential performance of a model for utilization prediction. Wider implications of the findings The iDAScore v2.0 may provide valuable information for embryologists and be useful as a prognostic tool to select patients with high chance to obtain blastocysts based on day2/3 time-lapse images and thereby may achieve similar live birth rate per transfer with no need for extended culture. Trial registration number Not applicable

Abstract STUDY QUESTION Can use of a commercially available time-lapse algorithm for Day 5 blastocyst selection improve pregnancy rates compared with morphology alone? SUMMARY ANSWER The use of a time-lapse selection model to choose blastocysts for fresh single embryo transfer on Day 5 did not improve ongoing pregnancy rate compared to morphology alone. WHAT IS KNOWN ALREADY Evidence from time-lapse monitoring suggests correlations between timing of key developmental events and embryo viability. No good quality evidence exists to support improved pregnancy rates following time-lapse selection. STUDY DESIGN, SIZE, DURATION A prospective multicenter randomized controlled trial including 776 randomized patients was performed between 2018 and 2021. Patients with at least two good quality blastocysts on Day 5 were allocated by a computer randomization program in a proportion of 1:1 into either the control group, whereby single blastocysts were selected for transfer by morphology alone, or the intervention group whereby final selection was decided by a commercially available time-lapse model. The embryologists at the time of blastocyst morphological scoring were blinded to which study group the patients would be randomized, and the physician and patients were blind to which group they were allocated until after the primary outcome was known. The primary outcome was number of ongoing pregnancies in the two groups. PARTICIPANTS/MATERIALS, SETTING, METHODS From 10 Nordic IVF clinics, 776 patients with a minimum of two good quality blastocysts on Day 5 (D5) were randomized into one of the two study groups. A commercial time-lapse model decided the final selection of blastocysts for 387 patients in the intervention (time-lapse) group, and blastocysts with the highest morphological score were transferred for 389 patients in the control group. Only single embryo transfers in fresh cycles were performed. MAIN RESULTS AND THE ROLE OF CHANCE In the full analysis set, the ongoing pregnancy rate for the time-lapse group was 47.4% (175/369) and 48.1% (181/376) in the control group. No statistically significant difference was found between the two groups: mean difference −0.7% (95% CI −8.2, 6.7, P = 0.90). Pregnancy rate (60.2% versus 59.0%, mean difference 1.1%, 95% CI −6.2, 8.4, P = 0.81) and early pregnancy loss (21.2% versus 18.5%, mean difference 2.7%, 95% CI −5.2, 10.6, P = 0.55) were the same for the time-lapse and the control group. Subgroup analyses showed that patient and treatment characteristics did not significantly affect the commercial time-lapse model D5 performance. In the time-lapse group, the choice of best blastocyst changed on 42% of occasions (154/369, 95% CI 36.9, 47.2) after the algorithm was applied, and this rate was similar for most treatment clinics. LIMITATIONS, REASONS FOR CAUTION During 2020, the patient recruitment rate slowed down at participating clinics owing to coronavirus disease-19 restrictions, so the target sample size was not achieved as planned and it was decided to stop the trial prematurely. The study only investigated embryo selection at the blastocyst stage on D5 in fresh IVF transfer cycles. In addition, only blastocysts of good morphological quality were considered for transfer, limiting the number of embryos for selection in both groups: also, it could be argued that this manual preselection of blastocysts limits the theoretical selection power of time-lapse, as well as restricting the results mainly to a good prognosis patient group. Most patients were aimed for blastocyst stage transfer when a minimum of five zygotes were available for extended culture. Finally, the primary clinical outcome evaluated was pregnancy to only 6–8 weeks. WIDER IMPLICATIONS OF THE FINDINGS The study suggests that time-lapse selection with a commercially available time-lapse model does not increase chance of ongoing pregnancy after single blastocyst transfer on Day 5 compared to morphology alone. STUDY FUNDING/COMPETING INTEREST(S) The study was financed by a grant from the Swedish state under the ALF-agreement between the Swedish government and the county councils (ALFGBG-723141). Vitrolife supported the study with embryo culture dishes and culture media. During the study period, T.H. changed his employment from Livio AB to Vitrolife AB. All other authors have no conflicts of interests to disclose. TRIAL REGISTRATION NUMBER ClinicalTrials.gov registration number NCT03445923. TRIAL REGISTRATION DATE 26 February 2018. DATE OF FIRST PATIENT’S ENROLMENT 11 June 2018.

PURPOSE:miRNAs have been suggested as biomarkers of embryo viability; however, findings from preliminary studies are divergent. Furthermore, the presence of other types of small RNA molecules remains to be investigated. The purpose of this study was to perform a comprehensive analysis of small non-coding RNA levels in spent and unconditioned embryo culture media, along with miRNA levels in blastocoelic fluid samples from human embryos.METHODS:miRNAs in unconditioned culture medium from 3 different manufacturers, along with miRNA from day 5 conditioned culture medium, control medium, and corresponding blastocoel fluid from 10 human blastocysts were analyzed with array-based q-PCR analysis. Subsequently, deep sequencing of total and small RNA in day 5 spent culture medium from 5 human blastocysts and corresponding controls was performed.RESULTS:In spite of using state-of-the-art sensitive detection methods, no miRNAs were found to be reliably present in the spent culture medium or the blastocoel fluid. Ct values were above the recommended limit for detection in the array-based analysis, a finding that was confirmed by deep sequencing. The majority of miRNAs identified by deep sequencing were expressed in all samples including control media and seem to originate from sources other than conditioned IVF media.CONCLUSIONS:Our findings question the use of miRNAs as a reliable biomarker and highlight the need for a critical methodological approach in miRNA studies. Interestingly, tiRNA fragments appear to be overexpressed in conditioned IVF media samples and could potentially be a novel biomarker worthy of investigation.

Globally, IVF patients are routinely offered and charged for a selection of adjunct treatments and tests or 'add-ons' that they are told may improve their chance of a live birth, despite there being no clinical evidence supporting the efficacy of the add-on. Any new IVF technology claiming to improve live birth rates (LBR) should, in most cases, first be tested in an appropriate animal model, then in clinical trials, to ensure safety, and finally in a randomized controlled trial (RCT) to provide high-quality evidence that the procedure is safe and effective. Only then should the technique be considered as 'routine' and only when applied to the similar patient population as those studied in the RCT. Even then, further pediatric and long-term follow-up studies will need to be undertaken to examine the long-term safety of the procedure. Alarmingly, there are currently numerous examples where adjunct treatments are used in the absence of evidence-based medicine and often at an additional fee. In some cases, when RCTs have shown the technique to be ineffective, it is eventually withdrawn from the clinic. In this paper, we discuss some of the adjunct treatments currently being offered globally in IVF laboratories, including embryo glue and adherence compounds, sperm DNA fragmentation, time-lapse imaging, preimplantation genetic screening, mitochondria DNA load measurement and assisted hatching. We examine the evidence for their safety and efficacy in increasing LBRs. We conclude that robust studies are needed to confirm the safety and efficacy of any adjunct treatment or test before they are offered routinely to IVF patients.

AIM To investigate whether human embryonic stem cells (hESCs) could be made to attach, grow and differentiate on a human Descemet’s membrane (DM). METHODS Spontaneously differentiated hESCs were transferred onto a human corneal button with the endothelial layer removed using ocular sticks. The cells were cultured on a DM for up to 15 d. The genetically engineered hESC line expressed green fluorescent protein, which facilitated identification during the culture experiments, tissue preparation, and analysis. To detect any differentiation into human corneal endothelial-like cells, we analysed the transplanted cells by immunohistochemistry using specific antibodies. RESULTS We found transplanted cells form a single layer of cells with a hexagonal shape in the periphery of the DM. The majority of the cells were negative for octamer-binding transcription factor 4 but positive for paired box 6 protein, sodium potassium adenosine triphosphatase (NaKATPase), and Zona Occludens protein 1. In four of the 18 trials, the transplanted cells were found to express CK3, which indicates that the stem cells differentiated into corneal epithelial cells in these cases. CONCLUSION It is possible to get cells originating from hESCs to become established on a human DM, where they grow and differentiate into corneal endothelial-like cells in vitro.

Sir, Ensuring and monitoring the health of children conceived through IVF is paramount, of that there can be no compromise. Hence, we found it perplexing to read the paper by Kleijkers et al. (2016) on the effects of culture media and birthweight. This is not the first randomized controlled trial (RCT), being predated by others, including the prospective trial by Carrasco et al. (2013) who reported the same weight for G5 (3213 g) and Cook (3154 g) media, but did not identify either as resulting in low birthweight. Similarly, the Kleijkers paper does not mention studies such as that of De Vos et al. (2015) whose data on birthweight are in line with those of Carrasco et al., nor the data of many the retrospective studies on birthweights, including Eskild et al. (2013) who reported a mean birthweight of 3444 g following culture in G5 medium. A recent study by Maas et al. (2016) including 6265 singleton live births showed no impact of media over an 18-year period. In fact, they concluded that the fresh versus frozen birthweights seemed was the only significant factor impacting IVF singleton birthweight. Of significance, this study was not powered for any conclusions about birthweight; the primary endpoint was live birth rate. Further, the birthweights of twins in both media in the Kleijkers study are the same, questioning how one medium can differentially affect the growth trajectory of singletons. It is therefore curious to speculate about why the birthweight difference of 158 g, restricted to singletons, was selected for comment when most outcomes favoured G5. It is apparent from Table 4 that the foetal growth trajectories of the two groups were not different, which suggests that the difference in birthweight is related to the higher rate of births <37 weeks in the G5 group. The isolated effect on gestationally mediated reduction in birthweight for singletons is consistent with residual confounding by medical interventions, e.g. induction of labour. The analysis is less than ideal in other regard as they adjusted for twinship, but not for clustering within site, which are clearly very different. They could also have adjusted for source and concentration of albumin, day of transfer, number of embryos transferred and cryopreservation, but did not. This would require more power, but the randomization is not presented by site, so it is not possible from the presented data to know if randomization was effective for potential confounders. The study was therefore underpowered for the task. Of further concern to us was not only the large number of variables in this trial, but the use of the medium HTF, a medium that became obsolete at the turn of the century due to its lack of amino acids, and which in our view, has no future place in human ART. From our perspective, the use of this medium in a trial is completely unacceptable, and explains why only 15% of embryos, from patients with an average age of 37, implanted in this group. What also becomes apparent from the data set is that significantly more patients required a second and third ovarian hyperstimulation in the HTF group (Supplementary data, Table 1); a patient-related risk that is completely ignored. Finally, the statement that hyaluronan was a new inclusion of the G media is erroneous given that it has been in all G series media since 2000. The companion paper by Sunde et al. (2016) subsequently argues that the differences in culture media formulations evoke the ‘Barker hypothesis’, now better known as the ‘Developmental Origins of Health and Disease (DOHaD)’, which proposes that birthweight (more accurately leanness at birth for gestational age), is an indicator of subsequent adult metabolic health. Initially controversial when proposed, the hypothesis has since received worldwide confirmation and acceptance. This includes many epidemiological studies in humans and experimental animal studies that demonstrate the peri-conception period is a sensitive time, where mechanisms, most likely epigenetic in nature, operate that determine fetal growth. However, we argue that the Kleijkers paper does not prove that differences in birthweight are induced by differences in media formulation, primarily because the Kleijker et al. paper is so highly confounded as an RCT, hence the relatively small difference in birthweight could readily be due to the myriad of confounding factors. Centrally against the DOHaD argument, the incidence of small for gestational age (SGA) infants did not differ between the groups. There are important independent long-term developmental effects of birthweight, SGA and prematurity. Unfortunately, these factors are conflated in the reported RCT. We agree with Sunde et al. (2016) that current media manufacturers should be required to inform all end-users of formulation changes. We also agree with Sunde et al.’s position that more appropriately conducted RCTs are required. We argue that a debate on disclosure of formulation should be held with media manufacturers, so their point of view is represented and, therefore, the debate is balanced. However, raising concerns about how differences in formulations are linked to long-term adult health outcomes on the basis of this one insufficient RCT is premature.

Sir, We read with interest the paper by Groenewoud et al. (2016), recently published in Human Reproduction. The authors performed an ambitious multi-centre trial aimed at evaluating whether the artificial cycle is inferior, or equal to, the modified natural cycle for cryopreservedthawed embryo transfer. The trial is a huge undertaking and is performed according to high academic standards. However, we were surprised to see that the authors report very low success rates from cryopreservation/thawing cycles. According to the most recent European Infertility Monitoring (EIM) report (Kupka et al., 2016), deliveries per thawing cycle on a national level are reported to lie between 10.0 and 25.4%. Thus, the results reported in the paper are below the lowest national levels reported in the EIM. Additionally, the artificial cycle, which according to the paper is not inferior to the modified natural cycle, showed live birth rates at 8.8% per cycle. In any single clinic, this would lead to immediate analysis of results and procedures; immediate cessation of activities would be recommended until the reason for the low delivery rate had been identified. When results in a clinical programme are sub-optimal, immediate action must be considered. In a large multi-centre study, the situation may perhaps be considered to be more complex. Feedback on clinical results is slow and any corrective action may be difficult to achieve. At the time of publication however, any such problems should be addressed. Additionally, we infer from the data that the rate of miscarriage (not explicitly reported in the paper) seems alarmingly high. The overall rate of pregnancy loss in the study is 38.5% according to the paper as 66 of the clinical pregnancies did not lead to a live birth. This is, in our opinion, another indication of a severe problem in the cryopreservation/thawing programme. Optimal cryopreservation methods give low rates of miscarriage and much higher pregnancy rates can be expected in a successful cryopreservation/thawing programme (Edgar and Gook, 2012; Ahlström et al., 2013). Here also, a comment is motivated. The methods reported in the paper also give rise to some questions that need clarification:

Within the past few years the morphological evaluation of in vitro fertilized embryos has been extended to include continuous surveillance, enabled by the introduction of time-lapse incubators developed specifically for IVF treatment. As a result time-lapse monitoring has been implemented in many clinics worldwide. The proposed benefits compared with culture in a standard incubator and fixed time-point evaluation are uninterrupted culture, a flexible workflow in the laboratory, and improved embryo selection. The latter is based on the reasonable assumption that more frequent observations will provide substantially more information on the relationship between development, timing, and embryo viability. Several retrospective studies have confirmed a relationship between time-lapse parameters and embryo viability evaluated by developmental competence, aneuploidy, and clinical pregnancy. Furthermore a much anticipated randomized study has shown improved pregnancy rates (PRs) after culture in a time-lapse incubator combined with selection using a hierarchical time-lapse selection model. At present this is the only randomized study on possible benefits of time lapse in human embryology. Strict evidence may still seem too weak to introduce time lapse in routine clinical setting. This aim of this review is therefore to perform a balanced discussion of the evidence for time-lapse monitoring. (C) 2015 by American Society for Reproductive Medicine.

OBJECTIVE:To study whether a culture medium that allows undisturbed culture supports human embryo development to the blastocyst stage equivalently to a well-established sequential media. DESIGN:Randomized, double-blinded sibling trial. SETTING:Independent in vitro fertilization (IVF) clinics. PATIENT(S):One hundred twenty-eight patients, with 1,356 zygotes randomized into two study arms. INTERVENTION(S):Embryos randomly allocated into two study arms to compare embryo development on a time-lapse system using a single-step medium or sequential media. MAIN OUTCOME MEASURE(S):Percentage of good-quality blastocysts on day 5. RESULT(S):Percentage of day 5 good-quality blastocysts was 21.1% (standard deviation [SD] ± 21.6%) and 22.2% (SD ± 22.1%) in the single-step time-lapse medium (G-TL) and the sequential media (G-1/G-2) groups, respectively. The mean difference (-1.2; 95% CI, -6.0; 3.6) between the two media systems for the primary end point was less than the noninferiority margin of -8%. There was a statistically significantly lower number of good-quality embryos on day 3 in the G-TL group [50.7% (SD ± 30.6%) vs. 60.8% (SD ± 30.7%)]. Four out of the 11 measured morphokinetic parameters were statistically significantly different for the two media used. The mean levels of ammonium concentration in the media at the end of the culture period was statistically significantly lower in the G-TL group as compared with the G-2 group. CONCLUSION(S):We have shown that a single-step culture medium supports blastocyst development equivalently to established sequential media. The ammonium concentrations were lower in the single-step media, and the measured morphokinetic parameters were modified somewhat. CLINICAL TRIAL REGISTRATION NUMBER:NCT01939626.

PURPOSE:The aim of this study was to investigate whether cells originating from human embryonic stem cells (hESCs) could be successfully transplanted onto a partially wounded human cornea. A second aim was to study the ability of the transplanted cells to differentiate into corneal epithelial-like cells.METHODS:Spontaneously, differentiated hESCs were transplanted onto a human corneal button (without limbus) with the epithelial layer partially removed. The cells were cultured on Bowman's membrane for up to 9 days, and the culture dynamics documented in a time-lapse system. As the transplanted cells originated from a genetically engineered hESC line, they all expressed green fluorescent protein, which facilitated their identification during the culture experiments, tissue preparation and analysis. To detect any differentiation into human corneal epithelial-like cells, we analysed the transplanted cells by immunohistochemistry using antibodies specific for CK3, CK15 and PAX6.RESULTS:The transplanted cells established and expanded on Bowman's membrane, forming a 1-4 cell layer surrounded by host corneal epithelial cells. Expression of the corneal marker PAX6 appeared 3 days after transplantation, and after 6 days, the cells were expressing both PAX6 and CK3.CONCLUSION:This shows that it is possible to transplant cells originating from hESCs onto Bowman's membrane with the epithelial layer partially removed and to get these cells to establish, grow and differentiate into corneal epithelial-like cells in vitro.

Near-infrared (NIR) spectroscopic metabolomic profiling of spent embryo-culture media has been used to calculate a viability score for individual embryos. These scores have been found to correlate to the reproductive potential of cleavage-stage embryos. In this study, 137 spent blastocyst media samples were collected after single-embryo transfer and analysed by NIR spectroscopy to generate an algorithm and calculate viability scores. To blindly validate the algorithm development process, another algorithm was trained on 47 preselected samples from clinic 1 and then used to predict the outcome of 42 samples from clinic 2. The overall pregnancy rate from the two clinical sites was 50.4%. A positive correlation (R(2)=0.82, P=0.03) was observed with the increasing viability score quintiles and their associated implantation rates. Cross-validation of an algorithm generated from NIR analysis of media samples at one clinical setting blindly was shown to predict implantation potential of blastocysts cultured at another clinic in a different culture media and culture volume. This study demonstrates that metabolomic profiling by NIR spectroscopic analysis of day-5 spent embryo-culture media can predict the implantation potential of blastocysts. Furthermore, this method may not be restricted to a specific set of culturing conditions. The successes of IVF treatment cycles are in part limited by the ability to select the best single embryo from a cohort of patient embryos for transfer back to the woman. Routine procedures of embryo selection are based on morphology, including cell number and size, and the timing of cell division. These methods are favoured because they are quick and easy to assess. Human embryos are grown in culture solutions, which are specific for their stage of development. Recent studies analysing the culture solution in which the embryo are grown, by near infrared (NIR) spectroscopic analysis, have been able to predict if an embryo will implant or not. As culture conditions often vary between IVF laboratories the questions remained if the NIR technique could be used to independently predict the implantation potential of an embryo cultured at one laboratory using an algorithm trained on embryos at a second clinic, a so-called cross-validation. The results of this study show that NIR spectroscopy can predict the ability of embryos to implant even when grown in different IVF laboratories and in two different culture solutions. This information supports the idea that NIR spectroscopy can be used globally not relying on specific culture conditions or media.

Assessment of embryo viability is a key component of in vitro fertilization (IVF) and currently relies largely on embryo morphology and cleavage rate. In this study, we used receiver operating characteristic (ROC) analysis to compare the Viability Score (generated by metabolomic profiling of spent embryo culture media using near infrared (NIR) spectroscopy) to morphologic grading for predicting pregnancy in women undergoing single embryo transfer (SET) on day 5.A total of 198 spent embryo culture media samples were collected in four IVF centers located in the USA, Europe and Australia. First, 137 samples (training set) were analyzed by NIR to develop an algorithm that generates a Viability Score predictive of pregnancy for each sample. Next, 61 samples (validation set) were analyzed by observers blinded to embryo morphology and IVF outcome, using the Day 5 algorithm generated with the training set. Pregnancy was defined as fetal cardiac activity (FCA) at 12 weeks of gestation.The Area Under the Curve (AUC) was greater for the metabolomic Viability Score compared to Morphology [Training set: 0.75 versus 0.55, p = 0.0011; Validation set: 0.68 versus 0.50, P = 0.021], and for a Composite score (obtained using a model combining Viability Score with morphologic grading), compared to morphology alone [0.74 versus 0.50, p = 0.004].Our findings suggest that Viability Score alone or in combination with morphologic grading has the potential to be a better classifier for pregnancy outcome than morphology alone in women undergoing SET on day 5.