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The purpose of this study was to determine if morphometric parameters that can be measured quantitatively using a time-lapse embryo incubator are associated with aneuploidy. Embryos cultured in a time-lapse incubator and assessed with preimplantation genetic testing for aneuploidy (PGT-A) were analyzed retrospectively. Morphokinetic analysis included timing of cell divisions. Quantitative morphometric measurements included the distance between the second and first polar body, zona pellucida thickness at the pronuclear stage and at the 2-cell stage, and blastomere area at the 2- and 4-cell stages. Symmetry at the 2-cell stage was determined by percent difference between blastomeres; symmetry at the 4-cell stage was the percent difference between the smallest and largest blastomeres. Maternal age, blastocyst grade and day of biopsy were recorded. Euploid embryo characteristics were compared to aneuploid embryos. A receiver operating characteristic (ROC) curve was used to evaluate cell symmetry as a predictor of aneuploidy. Embryos (n = 182) from 21 patients (age 22–43; median = 34) were analyzed. Of the 182 embryos, 45% were euploid. Euploid and aneuploid embryos had similar morphokinetics and morphometry across many measures. As expected, age and blastocyst grade were associated with embryo ploidy. It was notable that, additionally, symmetry at the 4-cell stage (27% vs 31%, p = 0.01) was also associated with embryo ploidy. The optimized cutoff from the ROC curve to predict aneuploidy was determined to be 21%. Embryos with > 21% asymmetry at the 4-cell stage had high rates of aneuploidy while morphokinetic parameters were similar. In conclusion, this suggests that embryo selection models using time-lapse parameters would improve if they incorporate cleavage-stage morphometrics.

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.

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 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.

Sperm DNA integrity is important for fertility, however the incidence of high levels of DNA fragmentation (DNA fragmentation index (DFI)30%) is not well described. In 2011, our clinics implemented guidelines for sperm DNA fragmentation (SDF) testing based on risk factors using the sperm chromatin structure assay (SCSA). The aim of this retrospective study is to characterise SDF and associated factors (age, semen parameters, smoking status and BMI) for sub-fertile males (n = 1082) and sperm donors (n = 234). The average DFI was 12.1 ± 9.8%. The distribution of men with low, moderate and high SDF (15, 15-30 and30%) was 74.8%, 19.4% and 5.8%, respectively. Men with high DFI were older (45 ± 9.5 vs 38 ± 6.7) and had lower percentage of motile sperm (38.8 ± 16.1% vs 55.3 ± 15.8%) than men with normal DFI. Over 17% of the men in the quartile with the highest age and lowest motility had a high DFI (30%), compared to a high DFI rate of 2-4% for the other 3 quartiles. Repeat testing following lifestyle interventions was available for 29 couples where the men had high initial DFI (35 ± 9.5%). Of these men, 71.4% had a decrease of DFI into the moderate or low range. This study shows that SDF testing can be targeted based on age and sperm motility, thereby reducing unnecessary testing. Furthermore, we provide evidence that lifestyle modifications can reduce DNA fragmentation in men with high DFI.

Study question What is the live birth rate after single, low-grade blastocyst (LGB) transfer? Summary answer The live birth rate for LGBs is 28%, ranging between 15–31% for the different inner cell mass (ICM) and trophectoderm (TE) subgroups of LGBs. What is known already Live birth rates following LGB transfer are varied and have been reported to be in the range of 5–39%. However, these estimates are inaccurate as studies investigating live birth rates following LGB transfer are inherently limited by sample size (n = 10–440 for LGB transfers) due to LGBs being ranked last for transfer. Further, these studies are heterogenous with varied LGB definitions and design. Collating LGB live birth data from multiple clinics is warranted to obtain sufficient numbers of LGB transfers to establish reliable live birth rates, and to allow for delineation of different LGB subgroups, including blastocyst age and female age. Study design, size, duration We performed a multicentre, multinational retrospective cohort study in 9 IVF centres in China and New Zealand from 2012 to 2019. We studied the outcome of 6966 single blastocyst transfer cycles on days 5–7 (fresh and frozen) according to blastocyst grade, including 875 transfers from LGBs ( Participants/materials, setting, methods The main outcome measured was live birth rate. Blastocysts were grouped according to quality grade: good-grade blastocysts (GGBs; n = 3849, aa, ab and ba), moderate-grade blastocysts (MGBs; n = 2242, bb) and LGBs (n = 875, ac, ca, bc, cb and cc) and live birth rates compared using the Pearson Chi-squared test. A logistic regression analysis explored the relationship between blastocyst grade and live birth after adjustment for the confounders: clinic, female age, expansion stage, and blastocyst age. Main results and the role of chance The live birth rates for GGBs, MGBs and LGBs were 45%, 36% and 28% respectively (p Limitations, reasons for caution Despite the large multicentre design of the study, analyses of transfers occurring within the smallest subsets of the LGB group were limited by sample size. The study was not randomised and had a retrospective character. Wider implications of the findings: LGBs maintain satisfactory live birth rates (averaging 28%) in the general IVF population. Even those in the lowest grading tier maintain modest live birth rates (15%; cc). It is recommended that LGBs not be universally discarded, and instead considered for subsequent frozen embryo transfer to maximize cumulative live birth rates. Trial registration number Not applicable

The purpose of this study was to determine if morphometric parameters that can be measured quantitatively using a time-lapse embryo incubator are associated with aneuploidy. Embryos cultured in a time-lapse incubator and assessed with preimplantation genetic testing for aneuploidy (PGT-A) were analyzed retrospectively. Morphokinetic analysis included timing of cell divisions. Quantitative morphometric measurements included the distance between the second and first polar body, zona pellucida thickness at the pronuclear stage and at the 2-cell stage, and blastomere area at the 2- and 4-cell stages. Symmetry at the 2-cell stage was determined by percent difference between blastomeres; symmetry at the 4-cell stage was the percent difference between the smallest and largest blastomeres. Maternal age, blastocyst grade and day of biopsy were recorded. Euploid embryo characteristics were compared to aneuploid embryos. A receiver operating characteristic (ROC) curve was used to evaluate cell symmetry as a predictor of aneuploidy. Embryos (n = 182) from 21 patients (age 22-43; median = 34) were analyzed. Of the 182 embryos, 45% were euploid. Euploid and aneuploid embryos had similar morphokinetics and morphometry across many measures. As expected, age and blastocyst grade were associated with embryo ploidy. It was notable that, additionally, symmetry at the 4-cell stage (27% vs 31%, p = 0.01) was also associated with embryo ploidy. The optimized cutoff from the ROC curve to predict aneuploidy was determined to be 21%. Embryos with 21% asymmetry at the 4-cell stage had high rates of aneuploidy while morphokinetic parameters were similar. In conclusion, this suggests that embryo selection models using time-lapse parameters would improve if they incorporate cleavage-stage morphometrics.

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.

RESEARCH QUESTION: To investigate whether patient factors influence the decision to freeze a blastocyst with low implantation potential. DESIGN: This experimental study assessed 170 practicing embryologists from a variety of countries who were recruited via an online survey. Participants were currently practicing embryologists, who grade blastocysts as part of this role. The survey presented decision-making ‘vignettes’ to participants. These included specific patient information, as well as an image of an expanded blastocyst that was of borderline quality for inner cell mass and trophectoderm, for which the embryologist selected whether or not to freeze. High/low maternal age, the presence/absence of other top quality blastocysts, and the presence/absence of previously unsuccessful IVF cycles were systematically varied within the patient information in a 2 × 2 × 2 design. Participants reported how likely they would be to freeze a particular blastocyst on a scale of 1 (Extremely Unlikely) to 7 (Extremely Likely), and whether or not they would ultimately freeze each blastocyst (Yes or No). RESULTS: Lower maternal age, no other high-quality blastocysts within the cohort, and multiple unsuccessful IVF cycles were associated with greater likelihood of recommending to freeze (P

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.

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); I(2) = 57%). Further analysis showed adverse impacts of direct cleavage (7 studies, n = 7065; RR = 0.28 [0.15, 0.54] (95% CI); I(2) = 46%), reverse cleavage (2 studies, n = 3622; RR = 0.16 [0.03, 0.75] (95% CI); I(2) = 0%), chaotic cleavage (2 studies, n = 3643; RR = 0.11 [0.02, 0.69] (95% CI); I(2) = 24%), and multinucleation (5 studies, n = 2576; RR = 0.59 [0.50, 0.69] (95% CI); I(2) = 0%), but not the

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. 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. 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. 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.

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.

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.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.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.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.

What are appropriate performance indicators (PIs) for ART laboratories for use in monitoring 'fresh' IVF and ICSI cycles?Minimum performance (competence) levels and aspirational (benchmark) values were recommended for a total of 19 indicators, including 12 key PIs (KPIs), five PIs and two reference indicators (RIs).PIs are necessary for systematic monitoring of the laboratory and an important element within the Quality Management System. However, there are no established PIs for ART laboratories and there is very little evidence on the topic.This is the report of a 2-day consensus meeting of expert professionals. As a starting point for the discussion, two surveys were organized to collect information on indicators used in IVF laboratories. During the meeting, the results of the surveys, scientific evidence (where available), and personal clinical experience where integrated into presentations by experts on specific topics. After presentation, each proposed indicator was discussed until consensus was reached within the panel.Expert professionals representing different countries and settings convened in the consensus meeting.The paper is divided in two parts: the workshop report and the recommendations of the expert panel. The second part reflects the discussion on each of the indicators, with the agreed definition, competence level and benchmark value for each of the 19 indicators, including 12 KPIs, 5 PIs and 2 RIs.The KPIs are mainly based on expert opinion. Future research may warrant an update of the recommended KPIs, their definition and the competence level and benchmark values.Based on the information presented, each ART laboratory should select its own set of KPIs founded on laboratory organization, and processes.The consensus meeting and writing of the paper was supported by funds from ESHRE and Alpha. Alpha gratefully acknowledges the following organizations for their financial support, through the provision of unrestricted educational grants: Global Fertility Alliance, Merck, Origio and Vitrolife. There are no conflicts of interest to disclose.

Are the morphokinetics of growing embryos affected by the type of culture media utilized?Morphokinetic parameters used for embryo selection are not affected between the two different concept culture media analyzed.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.Prospective cohort study, October 2010 and April 2011.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.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.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.The absence of differences in morphokinetics between two different media concepts validates the algorithm for embryo selection in diverse culture conditions.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.

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.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.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.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 quality of in vitro culture conditions is a key component of a successful clinical embryology laboratory. Many, but not all, supplies used in the embryology laboratory are screened by the supplier with a bioassay. Embryology laboratories use a variety of approaches to verify the quality of mineral oil, protein, and disposables before clinical use; however, a best practice has not been determined. Some laboratories test every supply, even those already screened by the supplier, whereas other laboratories perform as little testing as possible. Despite screening by the supplier, recent reports of embryo toxicity, specifically with mineral oil, highlight that the integrity of the supply system has gaps. This review describes current bioassay quality control testing and discusses how it applies to screening of products with documented lot-to-lot variation.

To assess sperm morphology with Tygerberg (strict) and World Health Organization (WHO) 3rd criteria for intrauterine inseminations (IUI) between two eras to determine if there was a difference in pregnancy rates.Retrospective study.Academic institution.127 couples with 290 IUI treatments during 1996-97 (era 1) and 133 couples with 317 IUI treatments during 2005-06 (era 2).None.Pregnancy rates per cycle and couple.Average sperm morphology was higher in era 1 than era 2 for both WHO 3rd (37 ± 13% vs. 23 ± 10%) and strict criteria (8.0 ± 5.0% vs. 4.0 ± 3.0%). Pregnancy rates per cycle were 5.9% versus 19.8% in era 1 and 16.7% versus 19.3% in era 2 for couples with WHO 3rd morphology30% or ≥30%, respectively. Pregnancy rates per cycle were 2.7% versus 15.0% in era 1 and 13.3% versus 14.7% in era 2 for couples with strict morphology ≤4% or4%, respectively.There was a strong relationship between morphology and IUI outcome in era 1 that was not present in era 2. These results suggest that classification drift increased the percentage of men diagnosed with teratozoospermia and resulted in a loss of predictive value.

Fat tissue, frequently the largest organ in humans, is at the nexus of mechanisms involved in longevity and age-related metabolic dysfunction. Fat distribution and function change dramatically throughout life. Obesity is associated with accelerated onset of diseases common in old age, while fat ablation and certain mutations affecting fat increase life span. Fat cells turn over throughout the life span. Fat cell progenitors, preadipocytes, are abundant, closely related to macrophages, and dysdifferentiate in old age, switching into a pro-inflammatory, tissue-remodeling, senescent-like state. Other mesenchymal progenitors also can acquire a pro-inflammatory, adipocyte-like phenotype with aging. We propose a hypothetical model in which cellular stress and preadipocyte overutilization with aging induce cellular senescence, leading to impaired adipogenesis, failure to sequester lipotoxic fatty acids, inflammatory cytokine and chemokine generation, and innate and adaptive immune response activation. These pro-inflammatory processes may amplify each other and have systemic consequences. This model is consistent with recent concepts about cellular senescence as a stress-responsive, adaptive phenotype that develops through multiple stages, including major metabolic and secretory readjustments, which can spread from cell to cell and can occur at any point during life. Senescence could be an alternative cell fate that develops in response to injury or metabolic dysfunction and might occur in nondividing as well as dividing cells. Consistent with this, a senescent-like state can develop in preadipocytes and fat cells from young obese individuals. Senescent, pro-inflammatory cells in fat could have profound clinical consequences because of the large size of the fat organ and its central metabolic role.

Objective To determine the role of saline infusion sonography (SIS) in uterine evaluation before a frozen embryo transfer (FET) cycle. Design Retrospective cohort analysis. Setting University hospital. Patient(s) Thirty-six patients who had uterine evaluation by SIS before FET cycle. Intervention(s) The SIS was performed in the follicular phase of the menstrual cycle before the actual FET cycle. Main Outcome Measure(s) The SIS findings, clinical pregnancy rate (PR), ongoing PR, and correlation between positive SIS findings with and without subsequent treatment and pregnancy outcome. Result(s) Positive SIS findings were found in 11/36 patients (30.5%), which included uterine septum (9.0%), endometrial polyp (45.4%), intramural fibroid with normal cavity (9.0%), cystic endometrial changes (9.0%), cervical stenosis (18.1%), and calcification with normal cavity (9.0%). The overall clinical PR in all groups was 51.4%, with an ongoing PR of 45.7%. Patients with positive SIS finding who underwent subsequent hysteroscopic correction (7/11) had a clinical PR of 85.7% as compared to 54.1% in patients with normal uterine cavity (24/35). Patients with positive SIS findings and no operative hysteroscopy (4/11) had a clinical PR of 50% and pregnancy loss rate of 100%. Conclusion(s) Obtaining an SIS before the FET cycle can be helpful in the detection of uterine abnormalities. If time since uterine evaluation has been more than 1–2 years, performing an SIS is recommended as subsequent correction of the anomalies may improve FET outcome.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12861-015-0073-x) contains supplementary material, which is available to authorized users.

Objective To determine compositions of commercial single-step culture media and test with a murine model whether differences in composition are biologically relevant. Design Experimental laboratory study. Setting University-based laboratory. Animal(s) Inbred female mice were superovulated and mated with outbred male mice. Intervention(s) Amino acid, organic acid, and ions content were determined for single-step culture media: CSC, Global, G-TL, and 1-Step. To determine whether differences in composition of these media are biologically relevant, mouse one-cell embryos were cultured for 96 hours in each culture media at 5% and 20% oxygen in a time-lapse incubator. Main Outcome Measure(s) Compositions of four culture media were analyzed for concentrations of 30 amino acids, organic acids, and ions. Blastocysts at 96 hours of culture and cell cycle timings were calculated, and experiments were repeated in triplicate. Result(s) Of the more than 30 analytes, concentrations of glucose, lactate, pyruvate, amino acids, phosphate, calcium, and magnesium varied in concentrations. Mouse embryos were differentially affected by oxygen in G-TL and 1-Step. Conclusion(s) Four single-step culture media have compositions that vary notably in pyruvate, lactate, and amino acids. Blastocyst development was affected by culture media and its interaction with oxygen concentration.

We aimed to determine whether embryo culture induces markers of cellular senescence and whether these effects were dependent on culture conditions. Murine blastocysts were derived in vitro and in vivo and assessed for 2 primary markers of senescence: senescence-associated β-galactosidase (SA-β-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. Compared with in vivo–derived blastocysts, in vitro embryos had high levels of SA-β-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-β-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. Elevated SA-β-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.

This chapter focuses on the critical aspects of building a laboratory in a private practice setting as part of an assisted reproductive technology (ART) clinic. Knowledge of these factors is essential for ART professionals planning a new facility or redesigning an existing one.

To screen human serum albumin (HSA) preparations for toxicity and investigate causes of variation.Experimental laboratory study.University-based laboratory.FVB and CF1 mice crossed to create embryos used in experiments.Mouse embryo assay performed with 5% or 15% HSA (100 mg/mL albumin) from three samples from three separate manufacturers (A, B, C).Blastocyst rates calculated at 96 hours of culture (experiments repeated in triplicate).The HSA preparations were desalted to remove stabilizers added during HSA processing, then mass spectrometry was used to determine the relative variation in stabilizer concentrations; the effect of the stabilizer octanoic acid on embryo development was tested. At 5% HSA, all samples had blastocyst rates ≥ 70%; at 15% HSA, the blastocyst rates for samples B and C were50%. Desalting did not affect sample B but did improve the blastocyst rates of sample C. Mass spectrometry revealed high levels of octanoic acid in sample C compared with sample A. The addition of octanoic acid to sample A produced toxicity similar to sample C.The stabilizer octanoic acid varies by lot and inhibits embryo development. Because octanoic acid is known to cause disruptions in mitochondrial bioenergetics, reduce intracellular pH, and induce oxidative damage in peripheral tissues, its use in embryo culture should be monitored and limited.

Mineral oil overlay microdrop is commonly used during in vitro fertilization (IVF) procedures. Though mineral oil appears homogeneous, it is an undefined product that can vary in quality. Here, we describe the history, chemistry, processing, and optimal use of mineral oil for IVF and embryo culture.

Objective To compare sensitivity of inner cell mass (ICM) outgrowth assay and analysis of culture media amino acid turnover with the sensitivity of the human sperm motility assay (HSMA) and murine embryo assay (MEA) for detection of formaldehyde toxicity. Design Prospective in vitro study. Setting University hospital–based infertility center. Animal(s) Murine embryos. Intervention(s) The HSMA, MEA, and ICM outgrowth assays were performed with media containing 0–64-μM concentrations of formaldehyde. These assays were compared with dynamics of amino acid turnover in culture media. Main Outcome Measure(s) The lowest concentration of formaldehyde in culture media detected by each quality control assay. Result(s) Sperm forward progression, but not motility, detected formaldehyde at a concentration of 32 μM. Sperm motility index identified formaldehyde toxicity at 64 μM, whereas blastocyst rates in the MEA were affected at 32 μM formaldehyde. Evaluation of ICM using outgrowth and grade detected 16 μM formaldehyde. Leucine turnover in culture media detected 64 μM formaldehyde in the amino acid assay. Conclusion(s) Inner cell mass outgrowth is a more sensitive bioassay than MEA and HSMA for the detection of formaldehyde in culture media. Amino acid metabolism may also provide a sensitive quality control measure for detection of formaldehyde.

Mineral oil overlay microdrop is commonly used during in vitro fertilization (IVF) procedures. Though mineral oil appears homogeneous, it is an undefined product that can vary in quality. Here, we describe the history, chemistry, processing, and optimal use of mineral oil for IVF and embryo culture.

Human chorionic gonadotropin (hCG) is a heteromeric glycoprotein hormone with a molecular mass of ca. 38 000. The carbohydrate side chains terminate with sialic acid and account for roughly 30% of the mass of the hormone. Glycoforms of hCG have been routinely identified with conventional methods of isoelectric focusing or chromatofocusing and exhibit varied bioactivity. In the present report, high-performance capillary electrophoresis (HPCE) was used to separate the glycoforms of hCG and its subunits. Optimal conditions for obtaining near-baseline resolution of the glycoforms were 25 mM borate, pH 8.8 containing 5 mM diaminopropane. The samples were separated in a 100 cm fused-silica capillary with an internal diameter of 50 μm at 25 kV and 28°C. In its native form, hCG migrated in less than 50 min as 8 distinct, highly resolved peaks. In the absence of diaminopropane, hCG migrated as a single, broad peak. When analyzed individually, the α subunit separated into four peaks and the β subunit resolved as seven peaks. The two subunits could also be separated when the heterodimer was incubated in 0.25% trifluoroacetic acid for 1 h prior to injection into the capillary. To illustrate the potential clinical application of this technique, four different sources of hCG were analyzed. The number of different isoforms was constant among the four samples; however, the relative concentration (amounts) of the isoforms varied. These results illustrate the potential utility of HPCE in the clinical diagnostic analysis of hCG microheterogeneity.

Of the estimated 1.5 million men and women who were diagnosed as having cancer in 2010, approximately 10% are younger than 45 years. For these individuals, cancer treatment can be lifesaving but can permanently affect reproductive capacity. The American Society of Clinical Oncology has recommended that oncologists discuss the possibility of infertility with reproductive-age cancer patients and offer referral for fertility preservation consultation and therapy. Fertility preservation is an emerging field that offers treatment aimed at protecting future reproductive ability for individuals with cancer or other serious illnesses. Although fertility preservation strategies vary by patient age and sex, many allow patients to store gametes or reproductive tissues for potential future use to create offspring. As an emerging discipline, many questions remain about the role of fertility preservation. We performed a MEDLINE search from 1950 to June 2010 using the following MeSH terms: amenorrhea; antineoplastic agents; ovarian failure; premature; infertility, female; fertility preservation; infertility, male; adolescent and cancer; child and cancer; cryopreservation; and reproductive technologies, assisted. Studies considered for inclusion included those written in English and published before June 2010.

Synthetic peptides are valuable tools for determining sites of interaction between hormones and their receptors. We have learned much about linear receptor binding regions of the glycoprotein hormone human choriogonadotropin (hCG) using synthetic peptides corresponding to its primary sequence. Of paramount importance in any study using synthetic peptides as a tool to investigate protein structure are an efficient means of peptide purification and a definitive measure of peptide purity and composition. Purification is easily achieved for all but the most hydrophobic peptides using preparative reverse-phase HPLC. Of the methods available for analysis of peptide purity, mass spectrometry is perhaps the most useful and often most rapid approach. Other essential components of studies involving synthetic peptides and hormone binding are reproducible hormone labeling, receptor preparations, and bioassays. The ability of peptides to compete with hCG for binding to specific receptors is tested in radioreceptor binding assays and bioassays with transformed Leydig cell lines. These cells express the lutropin receptor that is coupled to a measurable endpoint such as cAMP or steroid production. The conditions for these assays are optimized for rapid and accurate measurement of peptide activity. Since the three-dimensional structure of hCG is not known, a systematic approach to the identification of potential receptor binding sites is advocated. First, a comprehensive analysis using synthetic overlapping peptides of uniform length that span the entire sequence of the α-subunit is employed. This approach is an effective means for surveying the entire subunit for receptor binding sites. Next, the boundaries of the active regions are delimited by a series of nested peptides sequentially shortened in length. The importance of each residue within the delimited binding regions is then studied using a series of peptides containing single alanine substitutions. Finally, modifications to enhance activity of the synthetic peptides are further made on the basis of data from alanine substitution studies, circular dichroic analysis, and molecular modeling. These studies provide valuable information to aid in the design of synthetic hormone analogs and for further analysis of the structure–function of hCG via site-directed mutagenesis.