Blastocyst quality and reproductive and perinatal outcomes: a multinational multicentre observational study.

STUDY QUESTION: Does the transfer of single low-grade blastocysts result in acceptable reproductive and perinatal outcomes compared to the transfer of single good-grade blastocysts? SUMMARY ANSWER: The transfer of single low-grade blastocysts resulted in a reduced live birth rate of around 30% (14% for very low-grade blastocysts) compared to 44% for single good-grade blastocysts, but does not lead to more adverse perinatal outcomes. WHAT IS KNOWN ALREADY: It is known that low-grade blastocysts can result in live births. However, the current studies are limited by relatively small sample sizes and single-centre designs. Furthermore, evidence on perinatal outcomes after transferring low-grade blastocysts is limited. STUDY DESIGN, SIZE, DURATION: We conducted a multi-centre, multi-national retrospective cohort study of 10 018 women undergoing 10 964 single blastocyst transfer cycles between 2009 and 2020 from 14 clinics across Australia, China, and New Zealand. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blastocysts were graded individually based on assessment of the morphology and development of the inner cell mass (ICM) and trophectoderm (TE), and were grouped into three quality categories: good- (AB, AB, or BA), moderate- (BB), and low-grade (grade C for ICM or TE) blastocysts. CC blastocysts were individually grouped as very low-grade blastocysts. Logistic regression with generalized estimating equation was used to analyse the association between blastocyst quality and live birth as well as other reproductive outcomes. Binomial, multinomial logistic, or linear regression was used to investigate the association between blastocyst quality and perinatal outcomes. Odds ratio (OR), adjusted OR (aOR), adjusted regression coefficient, and their 95% CIs are presented. Statistical significance was set at P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: There were 4386 good-grade blastocysts, 3735 moderate-grade blastocysts, and 2843 low-grade blastocysts were included in the analysis, for which the live birth rates were 44.4%, 38.6%, and 30.2%, respectively. Compared to good-grade blastocysts, the live birth rate of low-grade blastocysts was significantly lower (aOR of 0.48 (0.41-0.55)). Very low-grade blastocysts were associated with an even lower live birth rate (aOR 0.30 (0.18-0.52)) and their absolute live birth rate was 13.7%. There were 4132 singleton live births included in the analysis of perinatal outcomes. Compared with good-grade blastocysts, low-grade blastocysts had comparable preterm birth rates (<37 weeks, aOR 1.00 (0.65-1.54)), birthweight Z-scores (adjusted regression coefficient 0.02 (0.09-0.14)), and rates of very low birth weight (<1500 g, aOR 0.84 (0.22-3.25)), low birth weight (1500-2500 g, aOR 0.96 (0.56-1.65)), high birth weight (>4500 g, aOR 0.93 (0.37-2.32)), small for gestational age (aOR 1.63 (0.91-2.93)), and large for gestational age (aOR 1.28 (0.97-1.70)). LIMITATIONS, REASONS FOR CAUTION: Due to the nature of the retrospective design, residual confounding could not be excluded. In addition, the number of events for some perinatal outcomes was small. Between-operator and between-laboratory variations in blastocyst assessment were difficult to control. WIDER IMPLICATIONS OF THE FINDINGS: Patients undergoing IVF should be informed that low-grade blastocysts result in a lower live birth rate, however they do not increase the risk of adverse perinatal outcomes. Further research should focus on the criteria for embryos that should not be transferred and on the follow-up of long-term outcomes of offspring. STUDY FUNDING/COMPETING INTEREST(S): H.Z. is supported by a Monash Research Scholarship. B.W.J.M. is supported by a NHMRC Investigator grant (GNT1176437). R.W. is supported by an NHMRC Emerging Leadership Investigator grant (2009767). B.W.J.M. reports consultancy, travel support, and research funding from Merck. The other authors do not have competing interests to disclose. TRIAL REGISTRATION NUMBER: N/A.

What happens to abnormally fertilized embryos? A scoping review.

A dearth of evidence exists on embryos derived from oocytes without two pronuclei (2PN) or 'normal fertilization', i.e. embryos arising from non-pronuclear oocytes (0PN), mono-pronuclear oocytes (1PN) and tri-pronuclear oocytes (3PN). We searched the published literature on non-2PN oocytes and their clinical outcomes using a two-part collection strategy of relevant articles. A total of 33 articles were deemed eligible for the scoping review. A significant difference exists between potential development of oocytes with an abnormal number of pronuclei and those with 2PN in most studies; the abnormal pronuclei oocytes occur rarely and significant attrition occurs between day 1 and day 6, with corresponding reduction in chromosome integrity and clinical utility. Most recent studies describe outcomes of blastocysts derived from non-2PN oocytes, rather than cleavage stage embryo transfers. Compared with 2PN oocytes, blastocyst rates are lower in 1PN oocytes (68.3 versus 32.2%), with larger 1PN oocytes having better developmental potential compared with their smaller counterparts. Blastocysts from 1PN oocytes seem to have a slightly reduced implantation potential compared with those from 2PN blastocysts (33.3% versus 35.9%), with a reduced ongoing pregnancy rate (27.3% versus 28.1%). Live birth rates were only reported in 13 of the included studies. The comparators varied between studies, with live birth rates provided ranging from 0-66.7%, with two case reports (100%); this is a clear indication of the variability in practices and the significant heterogeneity of studies. A distinct lack of evidence exists on non-2PN oocytes; however, it seems that most abnormally fertilized oocytes that are non-viable will developmentally arrest in culture, and those that are viable can form viable pregnancies. Concerns remain about the outcome of pregnancies arising from the use of abnormally fertilized oocytes. Coupled with appropriate outcome measures, abnormally fertilized oocytes hold the potential to increase the pool of embryos eligible for transfer.

Assuring quality in assisted reproduction laboratories: assessing the performance of ART Compass - a digital art staff management platform.

PURPOSE: Staff management is the most cited ART/IVF laboratory inspection deficiency. Small ART/IVF clinics may be challenged to perform these activities by low staff volume; similarly, large ART/IVF networks may be challenged by high staff volume and large datasets. Here, we sought to investigate the performance of an automated, digital platform solution to manage this necessary task. METHODS: The ART Compass (ARTC) digital staff management platform was used to assess the clinical decision-making of ART laboratory staff. The survey modules presented standardized instructions to technologists and measured inter- and intra-technologist variability for subjective "clinical decision-making" type questions. Internal and external comparisons were achieved by providing technologists two answers: (1) a comparison to their own lab director and (2) to the most popular response collectively provided by all lab director level accounts. The platform is hosted on HIPAA compliant Amazon web servers, accessible via web browser and mobile applications for iOS (Apple) and Android mobile devices. RESULTS: Here, we investigated the performance of a digital staff management platform for single embryologist IVF practices and for three IVF lab networks (sites A, B, C) from 2020 to 2022. Embryology dish preparation survey results show variance among respondents in the following: PPE use, media volume, timing of oil overlay, and timing of moving prepared dishes to incubators. Surveying the perceived Gardner score and terms in use for early blastocysts reveals a lack of standardization of terminology and fair to poor agreement. We observed moderate inter-technologist agreement for ICM and TE grade (0.47 and 0.52, respectively). Lastly, the clinical decision of choice to freeze or discard an embryo revealed that agreement to freeze was highest for the top-quality embryos, and that some embryos can be highly contested, evenly split between choice to freeze or discard. CONCLUSIONS: We conclude that a digital platform is a novel and effective tool to automate, routinely monitor, and assure quality for staff-related parameters in ART and IVF laboratories. Use of a digital platform can increase regulatory compliance and provide actionable insight for quality assurance in both single embryologist practices and for large networks. Furthermore, clinical decision-making can be augmented with artificial intelligence integration.

Should we look for a low-grade threshold for blastocyst transfer? A scoping review.

Embryo quality is a key determinant of the success of IVF. Although the focus has been on selecting the best embryo for transfer, the classification of low-grade blastocysts (LGB) in existing scoring systems has received less attention. This is worrisome; embryo freezing allows optimal use of all created embryos, thus maximizing the cumulative live birth rate, which is arguably the most important outcome for infertile couples. A PubMed search was conducted in August 2020, using '((('poor-quality' OR 'poor quality') OR ('low-grade' OR 'low grade')) AND ('embryo' OR 'blastocyst')) AND ('pregnancy' OR 'live birth')'. This scoping review shows that LGB have similar euploidy and pregnancy success rates after implantation and have no adverse effects on pregnancy or perinatal outcomes. Evidence for pregnancy outcomes is lacking for different grades of LGB, with most studies clustering all LQB as one to compare with optimal blastocysts.

Fertility technologies and how to optimize laboratory performance to support the shortening of time to birth of a healthy singleton: a Delphi consensus.

PURPOSE: To explore how the assisted reproductive technology (ART) laboratories can be optimized and standardized to enhance embryo culture and selection, to bridge the gap between standard practice and the new concept of shortening time to healthy singleton birth. METHODS: A Delphi consensus was conducted (January to July 2018) to assess how the ART laboratory could be optimized, in conjunction with existing guidelines, to reduce the time to a healthy singleton birth. Eight experts plus the coordinator discussed and refined statements proposed by the coordinator. The statements were distributed via an online survey to 29 participants (including the eight experts from step 1), who voted on their agreement/disagreement with each statement. Consensus was reached if ≥ 66% of participants agreed/disagreed with a statement. If consensus was not achieved for any statement, that statement was revised and the process repeated until consensus was achieved. Details of statements achieving consensus were communicated to the participants. RESULTS: Consensus was achieved for all 13 statements, which underlined the need for professional guidelines and standardization of lab processes to increase laboratory competency and quality. The most important points identified were the improvement of embryo culture and embryo assessment to shorten time to live birth through the availability of more high-quality embryos, priority selection of the most viable embryos and improved cryosurvival. CONCLUSION: The efficiency of the ART laboratory can be improved through professional guidelines on standardized practices and optimized embryo culture environment, assessment, selection and cryopreservation methodologies, thereby reducing the time to a healthy singleton delivery.

Should we freeze it? Agreement on fate of borderline blastocysts is poor and does not improve with a modified blastocyst grading system.

STUDY QUESTION: What is the inter-observer agreement among embryologists for decision to freeze blastocysts of borderline morphology and can it be improved with a modified grading system? SUMMARY ANSWER: The inter-observer agreement among embryologists deciding whether to freeze blastocysts of marginal morphology was low and was not improved by a modified grading system. WHAT IS KNOWN ALREADY: While previous research on inter-observer variability on the decision of which embryo to transfer from a cohort of blastocysts is good, the impact of grading variability regarding decision to freeze borderline blastocysts has not been investigated. Agreement for inner cell mass (ICM) and trophectoderm (TE) grade is only fair, factors which contribute to the grade that influences decision to freeze. STUDY DESIGN, SIZE, DURATION: This was a prospective study involving 18 embryologists working at four different IVF clinics within a single organisation between January 2019 and July 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: All embryologists currently practicing blastocyst grading at a multi-site organisation were invited to participate. The survey was comprised of blastocyst images in three planes and asked (i) the likelihood of freezing and (ii) whether the blastocyst would be frozen based on visual assessment. Blastocysts varied by quality and were categorised as either top (n = 20), borderline (n = 60) or non-viable/degenerate quality (n = 20). A total of 1800 freeze decisions were assessed. To assess the impact of grading criteria on inter-observer agreement for decision to freeze, the survey was taken once when the embryologists used the Gardner criteria and again 6 months after transitioning to a modified Gardner criterion with four grades for ICM and TE. The fourth grade was introduced with the aim to promote higher levels of agreement for the clinical usability decision when the blastocyst was of marginal quality. MAIN RESULTS AND THE ROLE OF CHANCE: The inter-observer agreement for decision to freeze was near perfect (kappa 1.0) for top and non-viable/degenerate quality blastocysts, and this was not affected by the blastocysts grading criteria used (top quality; P = 0.330 and non-viable/degenerate quality; P = 0.18). In contrast, the cohort of borderline blastocysts received a mixed freeze rate (average 52.7%) during the first survey, indicative of blastocysts that showed uncertain viability and promoting significant disagreement for decision to freeze among the embryologists (kappa 0.304). After transitioning to a modified Gardner criteria with an additional grading tier, the average freeze rate increased (64.8%; P < 0.0001); however, the inter-observer agreement for decision to freeze was unchanged (kappa 0.301). Therefore, significant disagreement for decision to freeze among embryologists is an ongoing issue not resolved by the two grading criteria assessed here. LIMITATIONS, REASONS FOR CAUTION: Blastocyst assessment was performed from time-lapse images in three planes, rather than with a microscope in the laboratory. The inter-observer agreement for decision to freeze may be lower for embryologists working in different clinics with different grading protocols. WIDER IMPLICATIONS OF THE FINDINGS: The decision to freeze a blastocyst with borderline morphology is a common clinical issue that has the potential to arise for any patient during blastocyst culture. Disagreement for decision to freeze these blastocysts, and therefore clinical usability in frozen embryo transfer cycles, affects consistency in patient care due to a potential impact on cumulative live birth rates, as well as financial, emotional and time costs associated with the frozen embryo transfer cycles. We demonstrate significant disagreement for decision to freeze borderline blastocysts among embryologists using the same grading scheme within a large multisite organisation, a phenomenon which was not improved with a modified grading system. Decision-making around borderline embryos is an area requiring further research, especially as studies continue to demonstrate the reduced but modest live birth rates for low quality blastocysts (Grade C). These results provide support for emerging technology for embryo assessment, such as artificial intelligence. STUDY FUNDING/COMPETING INTEREST(S): None declared. TRIAL REGISTRATION NUMBER: Not applicable.

Between-laboratory reproducibility of time-lapse embryo selection using qualitative and quantitative parameters: a systematic review and meta-analysis.

PURPOSE: To investigate the between-laboratory reproducibility of embryo selection/deselection effectiveness using qualitative and quantitative time-lapse parameters. METHODS: A systematic search was performed on MEDLINE, EMBASE, and the Cochrane Library (up to February 2020) without restriction on date, language, document type, and publication status. Measuring outcomes included implantation, blastulation, good-quality blastocyst formation, and euploid blastocyst. RESULTS: We detected 6 retrospective cohort studies externally validating the first clinical time-lapse model (Meseguer) emphasizing quantitative parameters, of which 3 (including one involving 2 independent centers) were included for the pooled analysis. Receiver operating characteristics analysis showed reduced predictive power of the model when either including or not including sister clinic validation. Fifteen cohort studies evaluating qualitative parameters were included for meta-analysis, and the mean Newcastle-Ottawa Scale was 5.3. Overall, meta-analysis showed significantly adverse association between the presence of ≥ 1 cleavage abnormalities and embryo implantation rates (11 studies, n = 7266; RR = 0.39[0.28, 0.55]95% CI; I2 = 57%). Further analysis showed adverse impacts of direct cleavage (7 studies, n = 7065; RR = 0.28 [0.15, 0.54] 95% CI; I2 = 46%), reverse cleavage (2 studies, n = 3622; RR = 0.16 [0.03, 0.75] 95% CI; I2 = 0%), chaotic cleavage (2 studies, n = 3643; RR = 0.11 [0.02, 0.69] 95% CI; I2 = 24%), and multinucleation (5 studies, n = 2576; RR = 0.59 [0.50, 0.69] 95% CI; I2 = 0%), but not the < 6 intercellular contact points at the 4-cell stage (1 study, n = 185; RR = 0.17 [0.02, 1.15] 95% CI). CONCLUSIONS: Qualitative time-lapse parameters are reliably associated with embryo developmental potential among laboratories, whereas the reproducibility of time-lapse embryo selection model that emphasizes quantitative parameters may be compromised when externally applied.

Tracking quality: can embryology key performance indicators be used to identify clinically relevant shifts in pregnancy rate?

STUDY QUESTION: Can embryology key performance indicators (KPIs) detect shifts in laboratory performance that precede changes in clinical outcomes? SUMMARY ANSWER: Day 5 usable blastocyst rate (D5BUR) is an important embryology KPI that complements total usable blastocyst rate (TBUR) in its ability to rapidly identify adverse embryology outcomes, prior to changes in clinical outcomes. WHAT IS KNOWN ALREADY: The hypothesis that monitoring performance of an IVF laboratory using statistical process controls (SPCs) can act as an early warning signal of shifts in laboratory conditions is a hypothesis that requires validation. The Vienna consensus report recently defined KPIs for monitoring fresh IVF and ICSI cycles, but the effectiveness of using these KPIs for detecting clinically relevant shifts following changes in laboratory processes is unknown. STUDY DESIGN, SIZE, DURATION: A retrospective, multicentre, analysis of KPIs for 1971 fresh IVF and ICSI cycles during three consecutive 5-month periods (P1, P2 and P3) during which the culture medium was changed in the middle period. PARTICIPANTS/MATERIALS, SETTING, METHODS: ICSI fertilisation rate, IVF fertilisation rate, D5BUR, TBUR and clinical pregnancy rate (CPR) were tracked monthly and analysed for SPC using Shewhart control charts. Out-of-control KPIs were identified by warning (2-sigma) and control (3-sigma) limits. The effect of the laboratory culture medium change on embryology KPIs and cumulative CPR was investigated using a one-way ANOVA or Pearson Chi-squared test and logistic regression. MAIN RESULTS AND THE ROLE OF CHANCE: D5BUR decreased from 32 to 25% after the culture medium was changed, and the decrease was detected within 1 week after the change (P < 0.0001). D5BUR subsequently increased after a change back to the original medium. A decrease in CPR (51-36%) after the medium change was also observed but was not detected until 3 months after the shift in D5BUR (P = 0.0005). Overall, the change in culture medium independently influenced D5BUR, CPR and cumulative CPR. Importantly, TBUR (41%) was not affected by the change in culture medium, remaining within control limits for all three culture periods, indicating that the overall blastocyst rate alone may not sufficiently monitor embryology laboratory performance. LIMITATIONS REASONS FOR CAUTION: The statistical KPI monitoring system demonstrated by the current study may be less effective at identifying KPI shifts in smaller clinics with lower cycle volumes. Live-birth rate per cycle started was not included as a clinical KPI. WIDER IMPLICATIONS OF THE FINDINGS: This study demonstrates that statistical KPI monitoring systems have the potential to provide systematic, early detection of adverse outcomes in ART laboratories after planned or unexpected shifts in conditions. STUDY FUNDING/COMPETING INTEREST(S): No external funds were used for the study. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Should extended blastocyst culture include Day 7?

Extended culture to the blastocyst stage is widely practised, improving embryo selection and promoting single embryo transfer. Selection of useable blastocysts typically occurs on Days 5 and 6 of embryo culture. Embryos not suitable for transfer, biopsy or cryopreservation after Day 6 are routinely discarded. Some embryos develop at a slower rate, however, forming blastocysts on Day 7 of culture. Day 7 blastocysts can be viable, they can be of top morphological grade, euploid and result in a healthy live birth. Since ending culture on Day 6 is current practice in most clinics, viable Day 7 blastocysts may be prematurely discarded. Although Day 7 blastocysts make up only 5% of useable blastocysts, those which are suitable for cryopreservation or biopsy are clinically significant. Overall, culturing embryos an additional day increases the number of useable embryos per IVF cycle and provides further opportunity for pregnancy for patients, especially those who have only a few or low-quality blastocysts.

Improved detection of mineral oil toxicity using an extended mouse embryo assay.

PURPOSE: Successful in vitro fertilization (IVF) relies on sound laboratory methods and culture conditions which depend on sensitive quality control (QC) testing. This study aimed to improve the sensitivity of mouse embryo assays (MEA) for detection of mineral oil toxicity. METHODS: Five experiments were conducted to study modifications of the standard mouse embryo assay (MEA) in order to improve sensitivity using clinical grade mineral oil with known peroxide concentrations. Assessment of blastocyst development at either 96 h or in an extended MEA (eMEA) to 144 h was tested in each experiment. In experiment 1, ability to detect peroxides in oil was compared in the MEA, eMEA, and cell number at 96 h. In experiment 2, serial dilutions of peroxide in oil were used along with time-lapse imaging to compare sensitivity of the morphokinetic MEA to the eMEA. Culture conditions that may affect assay sensitivity were assessed in experiments 3-5, which examined the effect of group versus individual culture, oxygen concentration, and protein supplementation. RESULTS: Extended MEA and cell counts identified toxicity not detected by the routine endpoint of blastocyst rate at 96 h. The eMEA was fourfold more sensitive than the standard MEA, and this sensitivity was similar to the morphokinetic MEA. Group culture had a protective effect against toxicity, while oxygen concentration did not affect blastocyst development. Protein supplementation with HSA had a protective effect on blastocyst development in eMEA. CONCLUSIONS: The standard MEA used by manufacturers does not detect potentially lethal toxicity of peroxides in mineral oil. While group culture may mask toxicity, protein supplementation and oxygen concentration have minimal effect on assay sensitivity. The eMEA and time-lapse morphokinetic assessment are equally effective in detection of peroxide toxicity and thus provide manufacturers and end-users a simple process modification that can be readily adopted into an existing QC program.

The Aurora A-HP1γ pathway regulates gene expression and mitosis in cells from the sperm lineage.

BACKGROUND: HP1γ, a well-known regulator of gene expression, has been recently identified to be a target of Aurora A, a mitotic kinase which is important for both gametogenesis and embryogenesis. The purpose of this study was to define whether the Aurora A-HP1γ pathway supports cell division of gametes and/or early embryos, using western blot, immunofluorescence, immunohistochemistry, electron microscopy, shRNA-based knockdown, site-directed mutagenesis, and Affymetrix-based genome-wide expression profiles. RESULTS: We find that the form of HP1γ phosphorylated by Aurora A, P-Ser83 HP1γ, is a passenger protein, which localizes to the spermatozoa centriole and axoneme. In addition, disruption in this pathway causes centrosomal abnormalities and aberrations in cell division. Expression profiling of male germ cell lines demonstrates that HP1γ phosphorylation is critical for the regulation of mitosis-associated gene expression networks. In female gametes, we observe that P-Ser83-HP1γ is not present in meiotic centrosomes of M2 oocytes, but after syngamy, it becomes detectable during cleavage divisions, coinciding with early embryonic genome activation. CONCLUSIONS: These results support the idea that phosphorylation of HP1γ by Aurora A plays a role in the regulation of gene expression and mitotic cell division in cells from the sperm lineage and in early embryos. Combined, this data is relevant to better understanding the function of HP1γ in reproductive biology.

Air quality in the assisted reproduction laboratory: a mini-review.

Quality of air in the clinical embryology laboratory is considered critical for high in vitro fertilization (IVF) success rates, yet evidence for best practices is lacking. Predominantly anecdotal reports on relationships between air quality and IVF success rates have resulted in minimal authentic clinical laboratory guidelines or in recommendations that are based on industrial cleanroom particulate standards with little attention to chemical air filtration. As a result, a nascent industry of costly, specialized air handling equipment for IVF laboratories has emerged to provide air quality solutions that have not been clearly assessed or verified. Clinics are embracing such technology because their embryology laboratories have become epicenters of assisted reproductive technology as the practice of IVF has moved to blastocyst transfers and utilization of trophectoderm biopsy for preimplantation genetic testing (PGT). Thus, a laboratory's ability to culture, biopsy, and freeze blastocysts is a rate-limiting step that depends on technical proficiency and a supportive and stable culture environment based on a foundation of high-quality ambient air. This review aims to describe how evidence for the importance of air quality, in particular the role of volatile organic compounds (VOC), has resulted in an evolution of clinical practice that has arguably contributed to improved outcomes.

The impact of the protein stabilizer octanoic acid on embryonic development and fetal growth in a murine model.

PURPOSE: The purpose of this study was to determine the effect of the protein stabilizer octanoic acid on blastocyst development, implantation, and fetal growth in a murine model. METHODS: One-cell mouse embryos were collected and individually cultured in medium supplemented with recombinant human serum albumin for 96 h at 5 % oxygen in an EmbryoScope. Embryos were randomly allocated to four octanoic acid groups (0, 400, 800, or 1200 µM). Blastocyst development and cell cycle timings were calculated at 96 h of culture, and experiments were repeated in triplicate. Blastocysts were stained and fixed at 96 h for differential cell counts. Following 96 h of culture, blastocysts were transferred to recipients to determine implantation rates and fetal and placental weights. RESULTS: Blastocyst development, hatching rates, developmental kinetics, and total number of cells were negatively affected by octanoic acid at concentrations commonly used in human IVF. Implantation was not affected by octanoic acid but fetal and placental weights at 800 µM octanoic acid were increased relative to control. CONCLUSIONS: Octanoic acid, a standard additive to human protein supplements used in IVF, can have long-term negative effects on embryonic and fetal development. The use of octanoic acid for human embryo culture should be monitored and reduced.

Markers of cellular senescence are elevated in murine blastocysts cultured in vitro: molecular consequences of culture in atmospheric oxygen.

PURPOSE: We aimed to determine whether embryo culture induces markers of cellular senescence and whether these effects were dependent on culture conditions. METHODS: Murine blastocysts were derived in vitro and in vivo and assessed for 2 primary markers of senescence: senescence-associated ß-galactosidase (SA-ß-gal) and phosphorylated H2A.X (γ-H2A.X), the latter being a mark of DNA oxidative damage. Expression of senescence-associated genes p21, p16, and interleukin 6 (IL6) were also assessed. RESULTS: Compared with in vivo-derived blastocysts, in vitro embryos had high levels of SA-ß-gal, nuclear γ-H2A.X, and p21 mRNA expression, indicating that a senescence-like phenotype is induced by in vitro culture. To determine the role of culture conditions, we studied the effect of oxygen (5 % vs 20 %) and protein supplementation on senescence markers. Blastocysts in reduced oxygen (5 %) had low levels of both SA-ß-gal and γ-H2A.X compared with blastocysts cultured in ambient oxygen. Senescence markers also were reduced in the presence of protein, suggesting that antioxidant properties of protein reduce oxidative DNA damage in vitro. CONCLUSION: Elevated SA-ß-gal, γ-H2A.X, and p21 suggest that in vitro stress can induce a senescence-like phenotype. Reduced oxygen during embryo culture minimizes these effects, providing further evidence for potential adverse effects of culturing embryos at ambient oxygen concentrations.

Composition of protein supplements used for human embryo culture.

PURPOSE: To determine the composition of commercially available protein supplements for embryo culture media and test if differences in protein supplement composition are biologically relevant in a murine model. METHODS: Amino acid, organic acid, ion and metal content were determined for 6 protein supplements: recombinant human albumin (AlbIX), human serum albumin (HSA and Buminate), and three complex protein supplements (SSS, SPS, LGPS). To determine if differences in the composition of these supplements are biologically relevant, mouse one-cell embryos were collected and cultured for 120 hours in each protein supplement in Global media at 5 and 20 % oxygen in an EmbryoScope time-lapse incubator. The compositions of six protein supplements were analyzed for concentrations of 39 individual amino acids, organic acids, ions and elements. Blastocyst development and cell cycle timings were calculated at 96-hours of culture and the experiments were repeated in triplicate. Blastocyst gene expression was analyzed. RESULTS: Recombinant albumin had the fewest undefined components , the lowest concentration of elements detected, and resulted in high blastocyst development in both 5 and 20 % oxygen. Buminate, LGPS and SPS had high levels of transition metals whereas SSS had high concentrations of amino acids. Pre-compaction mouse embryo development was delayed relative to embryos in AlbIX for all supplements and blastocyst formation was reduced in Buminate, SPS and SSS. CONCLUSIONS: The composition of protein supplements are variable, consisting of previously undescribed components. High concentrations of pro-oxidant transition metals were most notable. Blastocyst development was protein dependent and showed an interaction with oxygen concentration and pro-oxidant supplements.

Diagnostic or prognostic? Decoding the role of embryo selection on in vitro fertilization treatment outcomes.

In this review, we take a fresh look at embryo assessment and selection methods from the perspective of diagnosis and prognosis. On the basis of a systematic search in the literature, we examined the evidence on the prognostic value of different embryo assessment methods, including morphological assessment, blastocyst culture, time-lapse imaging, artificial intelligence, and preimplantation genetic testing for aneuploidy.

Type of culture media does not affect embryo kinetics: a time-lapse analysis of sibling oocytes.

STUDY QUESTION: Are the morphokinetics of growing embryos affected by the type of culture media utilized? SUMMARY ANSWER: Morphokinetic parameters used for embryo selection are not affected between the two different concept culture media analyzed. WHAT IS KNOWN ALREADY: Studies on the effect of culture media on human embryos have focused on evaluating different in-house and commercially available media as well as comparing outcomes among different commercial media. Nonetheless, the evaluation of embryo development in these studies was based on static observations and very little is known from a dynamic point of view. STUDY DESIGN, SIZE, DURATION: Prospective cohort study, October 2010 and April 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: University-affiliated infertility center. Patients undergoing egg donation (n = 75) in which embryos were cultured with two different types of media in a time-lapse system. Embryo development was analyzed with time-lapse imaging for single step media (Global®) and sequential media (Sage® Cleavage). Variables studied included the timing to two cells (t2), three cells (t3), four cells (t4) and five cells (t5) as well as the length of the second cell cycle (cc2 = t3 - t2) and the synchrony in the division from two to four cells (s2 = t4 - t3). Implantation and clinical pregnancy rates were also analyzed. MAIN RESULTS AND THE ROLE OF CHANCE: No statistically significant differences were observed between the two media for all the variables analyzed. When analyzing the percentage of embryos falling within the optimal ranges proposed for s2, cc2 and t5, we did not find significant differences between the two media. Pregnancy and implantation rates were similar for the three types of transfers: 48.0% (CI 95% 28.4-67.6) and 42.0% (CI 95% 22.5-61.4) with Global media; 58.8% (CI 95% 35.4-82.2) and 38.2% (CI 95% 15.0-61.4) with Cleavage media; and 58.1% (CI 95% 40.7-75.4) and 37.1% (CI 95% 22.1-52.1) with mixed transferred, respectively. Multiple implantations (twins) were also similar among the three groups, with 24.0% (CI 95% 9.3-45.1) for transfers with embryos cultured in Global media, 17.6% (CI 95% 3.7-43.3) for transfers with embryos cultured in Cleavage media and 22.5% (CI 95% 9.5-41.0) with mixed transfers. LIMITATIONS, REASONS FOR CAUTION: The study was not powered to test differences in pregnancy rates between the two culture media, as this was not the hypothesis tested. Results are based on observations with embryos from oocyte donors and need to be repeated with embryos from infertile patients of different ages. WIDER IMPLICATIONS OF THE FINDINGS: The absence of differences in morphokinetics between two different media concepts validates the algorithm for embryo selection in diverse culture conditions. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was obtained for this study; it was solely funded by IVI. None of the authors have any economic affiliation with Unisense Fertilitech A/S but IVI is a minor shareholder in Unisense Fertilitech A/S.

Cdc20 is critical for meiosis I and fertility of female mice.

Chromosome missegregation in germ cells is an important cause of unexplained infertility, miscarriages, and congenital birth defects in humans. However, the molecular defects that lead to production of aneuploid gametes are largely unknown. Cdc20, the activating subunit of the anaphase-promoting complex/cyclosome (APC/C), initiates sister-chromatid separation by ordering the destruction of two key anaphase inhibitors, cyclin B1 and securin, at the transition from metaphase to anaphase. The physiological significance and full repertoire of functions of mammalian Cdc20 are unclear at present, mainly because of the essential nature of this protein in cell cycle progression. To bypass this problem we generated hypomorphic mice that express low amounts of Cdc20. These mice are healthy and have a normal lifespan, but females produce either no or very few offspring, despite normal folliculogenesis and fertilization rates. When mated with wild-type males, hypomorphic females yield nearly normal numbers of fertilized eggs, but as these embryos develop, they become malformed and rarely reach the blastocyst stage. In exploring the underlying mechanism, we uncover that the vast majority of these embryos have abnormal chromosome numbers, primarily due to chromosome lagging and chromosome misalignment during meiosis I in the oocyte. Furthermore, cyclin B1, cyclin A2, and securin are inefficiently degraded in metaphase I; and anaphase I onset is markedly delayed. These results demonstrate that the physiologically effective threshold level of Cdc20 is high for female meiosis I and identify Cdc20 hypomorphism as a mechanism for chromosome missegregation and formation of aneuploid gametes.

The in vitro fertilization laboratory: teamwork and teaming.

Delivery of fertility treatment involves both teamwork within a discipline as well as teaming across multiple work areas, such as nursing, administrative, laboratory, and clinical. In contrast to small autonomous centers, the in vitro fertilization (IVF) laboratory team in large clinics must function both as a team with many members and a constellation of teams to deliver seamless, safe, and effective patient-centered care. Although this review primarily focuses on teamwork within the IVF laboratory, which comprises clinical laboratory scientists and embryologists who perform both diagnostic and therapeutic procedures, it also discusses the laboratory's wider role with other teams of the IVF clinic, and the role of teaming (the ad hoc creation of multidisciplinary teams) to function highly and address critical issues.