Extended Embryo Culture Research Articles

Embryotoxic impact of Zika virus in a rhesus macaque in vitro implantation model†.

Zika virus (ZIKV) infection is associated with adverse pregnancy outcomes in humans, and infection in the first trimester can lead to miscarriage and stillbirth. Vertical and sexual transmissions of ZIKV have been demonstrated, yet the impact of infection during the initial stages of pregnancy remains unexplored. Here we defined the impact of ZIKV on early embryonic and placental development with a rhesus macaque model. During in vitro fertilization (IVF), macaque gametes were inoculated with a physiologically relevant dose of 5.48log10 plaque-forming units (PFU) of Zika virus/H.sapiens-tc/PUR/2015/PRVABC59_v3c2. Exposure at fertilization did not alter blastocyst formation rates compared to controls. To determine the impact of ZIKV exposure at implantation, hatched blastocysts were incubated with 3.26log10, 4.26log10, or 5.26log10 PFU, or not exposed to ZIKV, followed by extended embryo culture for 10days. ZIKV exposure negatively impacted attachment, growth, and survival in comparison to controls, with exposure to 5.26log10 PFU ZIKV resulting in embryonic degeneration by day 2. Embryonic secretion of pregnancy hormones was lower in ZIKV-exposed embryos. Increasing levels of infectious virus were detected in the culture media post-exposure, suggesting that the trophectoderm is susceptible to productive ZIKV infection. These results demonstrate that ZIKV exposure severely impacts the zona-free blastocyst, whereas exposure at the time of fertilization does not hinder blastocyst formation. Overall, early stages of pregnancy may be profoundly sensitive to infection and pregnancy loss, and the negative impact of ZIKV infection on pregnancy outcomes may be underestimated.

Effect of extended embryo culture after thawing on clinical pregnancy rate

Journals Journal of Men’s Health Frontiers in Bioscience-Elite Frontiers in Bioscience-Scholar Frontiers in Bioscience-Landmark Journal of Integrative Neuroscience Reviews in Cardiovascular Medicine Journal of Molecular and Clinical Medicine European Journal of Gynaecological Oncology Clinical and Experimental Obstetrics & Gynecology Resources For Authors For Reviewers For Editors Article Processing Charges Open Access Editorial Policies Publishing Ethic Copyright & License Digital Archive Privacy Policy Advertising Policies Peer Review Policy About Overview Management Team Contact Us Search Submit Home About Overview Aims & Scope Editorial Board Reviewer Board Indexing & Archiving Journal Awards Conferences Articles Forthcoming Issue Current Issue All Issues Special Issues Special Issues Edit a Special Issue Sections For Authors Submit Instructions for Authors Article Processing Charge Editorial Process Editorial Office Article Article Special Issue Advanced Section All sections Countries | Regions Countries | Regions Article Types Article Types Year — Volume Issue Pages — Search

Impact of a controlled culture temperature gradient on mouse embryo development and morphokinetics.

Temperature control within the IVF laboratory is an important aspect of a quality control system, helping to reduce environmental stress and ensure good-quality embryo development. Temperature fluctuations are probably more common than expected and the optimal temperature for embryo culture is not known. Modern incubators offer the opportunity to examine the impact of culture temperature on preimplantation embryo development while controlling for other variables within the system. The purpose of this study was to examine the effect of a range of temperatures during extended embryo culture on resulting mouse embryo development and morphokinetic timings. Using a single time-lapse incubator with six individual chambers, frozen-thawed one-cell mouse embryos were cultured individually at temperatures adjusted by 0.5°C between chambers to cover the range of 35.0-37.5°C. Resulting blastocyst formation and embryo morphokinetic timings were recorded and compared. Changes in culture temperature had a significant impact on mouse blastocyst development and morphokinetic timings (P<0.05). Under the conditions used in this study, blastocyst development was best at 37.0°C. Mouse preimplantation embryo mitotic cell divisions were generally slower at cooler temperatures and accelerated as the temperature increased from 35.0°C to 37.5°C. Incubator culture temperature must be carefully controlled, as even slight variations of 0.5°C in the temperature used for extended embryo culture can have significant impacts on embryo development and mitotic cell divisions. These data have potential implications for application of universal morphokinetic selection algorithms and may help explain differences in mitotic errors/embryo mosaicism between laboratories.

Perinatal and maternal outcome after vitrification of blastocysts: a Nordic study in singletons from the CoNARTaS group.

Is transfer of vitrified blastocysts associated with higher perinatal and maternal risks compared with slow-frozen cleavage stage embryos and fresh blastocysts? Transfer of vitrified blastocysts is associated with a higher risk of preterm birth (PTB) when compared with slow-frozen cleavage stage embryos and with a higher risk of a large baby, hypertensive disorders in pregnancy (HDPs) and postpartum hemorrhage (PPH) but a lower risk of placenta previa when compared with fresh blastocysts. Transfer of frozen-thawed embryos (FETs) plays a central role in modern fertility treatment, limiting the risk of ovarian hyperstimulation syndrome and multiple pregnancies. Following FET, several studies report a lower risk of PTB, low birth weight (LBW) and small for gestational age (SGA) yet a higher risk of fetal macrosomia and large for gestational age (LGA) compared with fresh embryos. In recent years, the introduction of new freezing techniques has increased treatment success. The slow-freeze technique combined with cleavage stage transfer has been replaced by vitrification and blastocyst transfer. Only few studies have compared perinatal and maternal outcomes after vitrification and slow-freeze and mainly in cleavage stage embryos, with most studies indicating similar outcomes in the two groups. Studies on perinatal and maternal outcomes following vitrified blastocysts are limited. This registry-based cohort study includes singletons born after frozen-thawed and fresh transfers following the introduction of vitrification in Sweden and Denmark, in 2002 and 2009, respectively. The study includes 3650 children born after transfer of vitrified blastocysts, 8123 children born after transfer of slow-frozen cleavage stage embryos and 4469 children born after transfer of fresh blastocysts during 2002-2015. Perinatal and maternal outcomes in singletons born after vitrified blastocyst transfer were compared with singletons born after slow-frozen cleavage stage transfer and singletons born after fresh blastocyst transfer. Main outcomes included PTB, LBW, macrosomia, HDP and placenta previa. Data were obtained from the CoNARTaS (Committee of Nordic ART and Safety) group. Based on national registries in Sweden, Finland, Denmark and Norway, the CoNARTaS cohort includes all children born after ART treatment in public and private clinics 1984-2015. Outcomes were assessed with logistic multivariable regression analysis, adjusting for the country and year of birth, maternal age, body mass index, parity, smoking, parental educational level, fertilisation method (IVF/ICSI), single embryo transfer, number of gestational sacs and the child's sex. A higher risk of PTB (<37weeks) was noted in the vitrified blastocyst group compared with the slow-frozen cleavage stage group (adjusted odds ratio, aOR [95% CI], 1.33 [1.09-1.62]). No significant differences were observed for LBW (<2500g), SGA, macrosomia (≥4500g) and LGA when comparing the vitrified blastocyst with the slow-frozen cleavage stage group. For maternal outcomes, no significant difference was seen in the risk of HDP, placenta previa, placental abruption and PPH in the vitrified blastocyst versus the slow frozen cleavage stage group, although the precision was limited.When comparing vitrified and fresh blastocysts, we found higher risks of macrosomia (≥4500g) aOR 1.77 [1.35-2.31] and LGA aOR 1.48 [1.18-1.84]. Further, the risks of HDP aOR 1.47 [1.19-1.81] and PPH aOR 1.68 [1.39-2.03] were higher in singletons born after vitrified compared with fresh blastocyst transfer while the risks of SGA aOR 0.58 [0.44-0.78] and placenta previa aOR 0.35 [0.25-0.48] were lower. Since vitrification was introduced simultaneously with blastocyst transfer in Sweden and Denmark, it was not possible to explore the effect of vitrification per se in this study. The results from the change of strategy to vitrification of blastocysts are reassuring, indicating that the freezing technique per se has no major influence on the perinatal and maternal outcomes. The higher risk of PTB may be related to the extended embryo culture rather than vitrification. The study is part of the ReproUnion Collaborative study, co-financed by the European Union, Interreg V ÖKS. The study was also financed by grants from the Swedish state under the agreement between the Swedish government and the county councils, the ALF agreement (LUA/ALF 70940), Hjalmar Svensson Research Foundation and NordForsk (project 71 450). There are no conflicts of interest to declare. ISRCTN11780826.

Blastulation and good blastulation between days 5 and 6 of extended embryo culture

Blastulation and good blastulation between days 5 and 6 of extended embryo culture

Perinatal outcomes in singleton live births after fresh blastocyst-stage embryo transfer: a retrospective analysis of 67 147 IVF/ICSI cycles.

Are perinatal outcomes different between singleton live births conceived from fresh blastocyst transfer and those following the transfer of fresh cleavage-stage embryos? Fresh blastocyst transfer does not increase risks of preterm birth (PTB), low/high birth weight or congenital anomaly and does not alter the sex ratio at birth or prejudice the chance of having a healthy baby. Extended embryo culture is currently considered the best option for embryo selection, but concerns have been raised about increased risks of preterm delivery and large-for-gestational-age (LGA) babies. We conducted a retrospective cohort study based on data from the Human Fertilisation and Embryology Authority (HFEA) anonymised and cycle-based dataset in the UK between 1999 and 2011. Baseline characteristics were compared between in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI) blastocyst-stage and cleavage-stage embryo transfer cycles using the χ2 test for categorical/dichotomised covariates and the Mann-Whitney test for continuous covariates. Statistical significance was set at <0.005. Poisson regression and multinomial logistic regression were used to establish relationships between perinatal outcomes and blastocyst-stage embryo transfer or cleavage-stage embryo transfer. Risk ratios (RRs), adjusted risk ratios (aRRs) and their 99.5% confidence intervals (CIs) were calculated as a measure of strength of associations. Results were adjusted for clinically relevant covariates. A sub-group analysis included women undergoing their first IVF/ICSI treatment. The level of significance was set at <0.05, and 95% CIs were calculated in the sub-group analysis. Of a total of 67 147 IVF/ICSI cycles, 11 152 involved blastocyst-stage embryo(s) and 55 995 involved cleavage-stage embryo(s). The two groups were comparable with regards to the risk of PTB (aRR, 1.00; 99.5% CI, 0.79-1.25), very-preterm birth (VPTB) (aRR, 1.00; 99.5% CI, 0.63-1.54), very-low birth weight (VLBW) (aRR, 0.84; 99.5% CI, 0.53-1.34), low birth weight (LBW) (aRR, 0.92; 99.5% CI, 0.73-1.16), high birth weight (HBW) (aRR, 0.94; 99.5% CI, 0.75-1.18) and very-high birth weight (VHBW) (aRR, 1.05; 99.5% CI, 0.66-1.65). The risk of congenital anomaly was 16% higher in the blastocyst-stage group than in the cleavage-stage group, but this was not statistically significant (aRR, 1.16; 99.5% CI, 0.90-1.49). The chance of having a healthy baby (born at term, with a normal birth weight and no congenital anomalies) was not altered by extended culture (aRR, 1.00; 99.5% CI, 0.93-1.07). Extended culture was associated with a marginal increase in the chance having a male baby in the main cycle-based analysis (aRR, 1.04; 99.5% CI, 1.01-1.09) but not in the sub-group analysis of women undergoing their first cycle of treatment (aRR, 1.04; 95% CI, 1.00-1.08). In the sub-group analysis, the risk of congenital anomalies was significantly higher after blastocyst-stage embryo transfer (aRR, 1.42; 95% CI, 1.12-1.81). This study is limited by the use of observational data and inability to adjust for key confounders, such as maternal smoking status and body mass index (BMI), which were not recorded in the HFEA dataset. As the main analysis was cycle-based and we were unable to link cycles within women undergoing more than one IVF/ICSI cycle, we undertook a sub-group analysis on women undergoing their first treatment cycle. Our findings should reassure women undergoing blastocyst-stage embryo transfer. For the first time, we have shown that babies born after blastocyst transfer have a similar chance of being healthy as those born after cleavage-stage embryos transfer. The research activity of Dr Nicola Marconi was funded by the scholarship 'A. Griffini-J. Miglierina', Fondazione Comunitaria del Varesotto, Provincia di Varese, Italy. The authors do not have any competing interests to disclose. N/A.

Optimising the Outcome of Embryo Transfer

In vitro fertilisation (IVF) is a complex procedure, the success of which is dependent on several factors at every step of the process. Despite major advances, successful implantation rates in IVF remain low. Aside from the status of the embryo and endometrium, embryo transfer (ET) plays a major role in implantation. There are numerous variables in ET that are causative factors for IVF success. In this article, the authors discuss whether the stage at which (cleavage versus blastocyst) ET occurs; a fresh or frozen ET; and the technique of ET affects the results of an assisted reproductive technology cycle. Blastocysts had higher implantation potential than cleavage-stage embryos and it was also observed that extended embryo culture was not related to increased adverse obstetric and perinatal outcome. Though freezing has several advantages over fresh cycles, one must remember that evidence is still lacking for its use in all patients. Elective cryopreservation of all embryos with transfer in subsequent frozen ET cycles may be requited in cases at risk of developing ovarian hyperstimulation syndrome, women undergoing preimplantation genetic screening or preimplantation genetic diagnosis for genetic analysis, polycystic ovarian syndrome patients, and those who have high progesterone levels on the day of human chorionic gonadotropin, but to date it is debatable whether a freeze-all strategy will benefit normal and poor responders. For an optimal ET technique, the use of soft catheters and performing the process under ultrasound guidance will improve results by making it less traumatic, standardised across centres, and more technically precise.

30. LIVE BIRTHS FOLLOWING DAY 7 BLASTOCYST TRANSFER AFTER PREIMPLANTATION GENETIC TESTING FOR ANEUPLOIDY (PGT-A)

Introduction Extended culture to blastocyst stage is being adopted in many IVF clinics, with the aim to improve embryo selection and promoting single embryo transfer. Selection of these blastocysts usually occurs up to Day 6 of embryo culture. However, a study ( Su et al., 2016 ) has revealed that Day 7 blastocysts can be euploid and can result in healthy live births. In this study, we evaluate the euploidy rate of Day 7 blastocysts and the clinical outcome of transferring these euploid Day 7 vitrified-warmed blastocysts in Alpha Fertility Centre. Materials & Methods Forty-five (45) patients had utilisable blastocysts only on Day 7 of culture between Aug-2015 and Nov-2018. A total of 58 blastocysts which were at least fair graded (Gardner, 1999) were biopsied on Day 7 and analysed using Next-Generation Sequencing (Life Technologies, USA). Vitrification was done after biopsy using Cryotec method (Cryotec, Japan). Clinical outcome was assessed following the transfer of euploid blastocysts. All successful pregnancies were monitored until delivery. Results The mean maternal age was 36.0 (age range:22-44). Of the 58 blastocysts analysed, 46.6% (27/58) blastocysts were euploid, 39.6% (23/58) blastocysts were aneuploid and 13.8% (8/58) blastocysts were mosaic. Subsequently, 10 patients had frozen blastocyst transfer: 9 had single blastocyst transfer (SBT); 1 had double blastocysts transfer (DBT). All blastocysts survived intact post-warmed. The clinical pregnancy rate was 70% (7/10) with an implantation rate of 63.6% (7/11). Two (2) of these patients each delivered a healthy baby weighing 2.81 kg and 3.08 kg respectively, while 1 pregnancy is ongoing at 30+3 weeks of gestation at the time of writing. Unfortunately, the remaining 4 pregnancies resulted in miscarriages at 8+1, 8+2, 7+5 and 9+4 weeks of gestation respectively. Conclusions Our study suggests that Day 7 blastocysts can indeed be euploid and yield healthy live births albeit the seemingly higher miscarriage rate. Therefore, extended culture of embryos to Day 7 should be considered as a standard practice in IVF laboratories for patients with no utilisable blastocyst on Day 5 or Day 6 of culture.

The impact of IVF on birthweight from 1991 to 2015: a cross-sectional study.

Has birthweight (BW) changed over time among IVF-conceived singletons? Singleton BW has increased markedly over the past 25 years. IVF conceived singletons have had a higher incidence of low BW compared to spontaneously conceived singletons, and this has raised concerns over long-term increased risks of cardio-metabolic disease. However, few causal links between IVF procedures and BW have been robustly established, and few studies have examined whether BW has changed over time as IVF techniques have developed. A total of 2780 live born singletons conceived via IVF or ICSI treated in the reproductive medicine department of a single publicly funded tertiary care centre between 1991 and 2015 were included in this retrospective study. The primary outcome measure was singleton BW adjusted for gestational age, maternal parity and child gender. Multivariable linear regression models were used to estimate the associations between patient prognostic factors and IVF treatment procedures with adjusted BW. All singletons conceived at the centre following IVF/ICSI using the mother's own oocytes, and non-donated fresh or frozen/thawed embryos with complete electronic data records, were investigated. Available electronic records were retrieved from the Human Fertilization and Embryology Authority for dataset collation. Multiple linear regression analysis was used to evaluate associations between IVF treatment parameters and BW, after adjusting for the year of treatment and patient characteristics and pregnancy factors. In the primary multivariable model, singleton BW increased by 7.4 g per year (95% CI: 3.2-11.6 g, P = 0.001), an increase of close to 180 g throughout the 25-year period after accounting for gestational age, maternal parity, child gender, IVF treatment parameters, patient prognostic characteristics and pregnancy factors. Fresh and frozen embryo transfer-conceived singletons showed a similar increase in BW. Frozen/thawed embryo transfer conceived singletons were on average 53 g heavier than their fresh embryo conceived counterparts (95% CI: 3.7-103.3 g, P = 0.035). The independent variables included in the study were limited to those that have been consistently recorded and stored electronically over the past two decades. There has been a progressive BW increase in IVF singletons over time in one large centre with consistent treatment eligibility criteria. Such a change is not seen in the general population of live born singletons in the UK or other developed countries, and seems to be specific to this IVF population. This may be a reflection of changes in practice such as undisturbed extended embryo culture to the blastocyst stage, optimized commercial culture media composition, single embryo transfer and ICSI. Moreover, singletons conceived from frozen/thawed embryos had higher birth weights when compared to their fresh embryo transfer counterparts. The causal pathway is unknown; however, it could be due to the impact on embryos of the freeze/thaw process, self-selection of embryos from couples who produce a surplus of embryos, and/or embryo replacement into a more receptive maternal environment. This work was supported by the EU FP7 project grant, EpiHealthNet (FP7-PEOPLE-2012-ITN-317146). The authors have no competing interests to declare. N/A.

Extended in vitro culture of human embryos demonstrates the complex nature of diagnosing chromosomal mosaicism from a single trophectoderm biopsy.

What is the accuracy of preimplantation genetic testing for aneuploidies (PGT-A) when considering human peri-implantation outcomes in vitro? The probability of accurately diagnosing an embryo as abnormal was 100%, while the proportion of euploid embryos classified as clinically suitable was 61.9%, yet if structural and mosaic abnormalities were not considered accuracy increased to 100%, with a 0% false positive and false negative rate. Embryo aneuploidy is associated with implantation failure and early pregnancy loss. However, a proportion of blastocysts are mosaic, containing chromosomally distinct cell populations. Diagnosing chromosomal mosaicism remains a significant challenge for PGT-A. Although mosaic embryos may lead to healthy live births, they are also associated with poorer clinical outcomes. Moreover, the direct effects of mosaicism on early pregnancy remain unknown. Recently, developed in vitro systems allow extended embryo culture for up to 14 days providing a unique opportunity for modelling chromosomal instability during human peri-implantation development. A total of 80 embryos were cultured to either 8 (n = 7) or 12 days post-fertilisation (dpf; n = 73). Of these, 54 were PGT-A blastocysts, donated to research following an abnormal (n = 37) or mosaic (n = 17) diagnosis. The remaining 26 were supernumerary blastocysts, obtained from standard assisted reproductive technology (ART) cycles. These embryos underwent trophectoderm (TE) biopsy prior to extended culture. We applied established culture protocols to generate embryo outgrowths. Outgrowth viability was assessed based on careful morphological evaluation. Nine outgrowths were further separated into two or more portions corresponding to inner cell mass (ICM) and TE-derived lineages. A total of 45 embryos were selected for next generation sequencing (NGS) at 8 or 12 dpf. We correlated TE biopsy profiles to both culture outcomes and the chromosomal status of the embryos during later development. Of the 73 embryos cultured to 12 dpf, 51% remained viable, while 49% detached between 8 and 12 dpf. Viable, Day 12 outgrowths were predominately generated from euploid blastocysts and those diagnosed with trisomies, duplications or mosaic aberrations. Conversely, monosomies, deletions and more complex chromosomal constitutions significantly impaired in vitro development to 12 dpf (10% vs. 77%, P < 0.0001). When compared to the original biopsy, we determined 100% concordance for uniform numerical aneuploidies, both in whole outgrowths and in the ICM and TE-derived outgrowth portions. However, uniform structural variants were not always confirmed later in development. Moreover, a high proportion of embryos originally diagnosed as mosaic remained viable at 12 dpf (58%). Of these, 71% were euploid, with normal profiles observed in both ICM and TE-derived lineages. Based on our validation data, we determine a 0% false negative and 18.5% false positive error rate when diagnosing mosaicism. Overall, our findings demonstrate a diagnostic accuracy of 80% in the context of PGT-A. Nevertheless, if structural and mosaic abnormalities are not considered, accuracy increases to 100%, with a 0% false positive and false negative rate. The inherent limitations of extended in vitro culture, particularly when modelling critical developmental milestones, warrant careful interpretation. Our findings echo current prenatal testing data and support the high clinical predictive value of PGT-A for diagnosing uniform numerical aneuploidies, as well as euploid chromosomal constitutions. However, distinguishing technical bias from biological variability will remain a challenge, inherently limiting the accuracy of a single TE biopsy for diagnosing mosaicism. This research is funded by the Ghent University Special Research Fund (BOF01D08114) awarded to M.P., the Research Foundation-Flanders (FWO.KAN.0005.01) research grant awarded to B.H. and De Snoo-van't Hoogerhuijs Stichting awarded to S.M.C.d.S.L. We thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests. N/A.

Worldwide decline of IVF birth rates and its probable causes.

With steadily improving pregnancy and live birth rates, IVF over approximately the first two and a half decades evolved into a highly successful treatment for female and male infertility, reaching peak live birth rates by 2001–2002. Plateauing rates, thereafter, actually started declining in most regions of the world. We here report worldwide IVF live birth rates between 2004 and 2016, defined as live births per fresh IVF/ICSI cycle started, and how the introduction of certain practice add-ons in timing was associated with changes in these live birth rates. We also attempted to define how rapid worldwide ‘industrialization’ (transition from a private practice model to an investor-driven industry) and ‘commoditization’ in IVF practice (primary competitive emphasis on revenue rather than IVF outcomes) affected IVF outcomes. The data presented here are based on published regional registry data from governments and/or specialty societies, covering the USA, Canada, the UK, Australia/New Zealand (combined), Latin America (as a block) and Japan. Changes in live birth rates were associated with introduction of new IVF practices, including mild stimulation, elective single embryo transfer (eSET), PGS (now renamed preimplantation genetic testing for aneuploidy), all-freeze cycles and embryo banking. Profound negative associations were observed with mild stimulation, extended embryo culture to blastocyst and eSET in Japan, Australia/New Zealand and Canada but to milder degrees also elsewhere. Effects of ‘industrialization’ suggested rising utilization of add-ons (‘commoditization’), increased IVF costs, reduced live birth rates and poorer patient satisfaction. Over the past decade and a half, IVF, therefore, has increasingly disappointed outcome expectations. Remarkably, neither the profession nor the public have paid attention to this development which, therefore, also has gone unexplained. It now urgently calls for evidence-based explanations.

Frozen versus fresh single blastocyst transfer in ovulatory women: a multicentre, randomised controlled trial

Elective single embryo transfer (eSET) has been increasingly advocated, but concerns about the lower pregnancy rate after reducing the number of embryos transferred have encouraged transfer of multiple embryos. Extended embryo culture combined with electively freezing all embryos and undertaking a deferred frozen embryo transfer might increase pregnancy rate after eSET. We aimed to establish whether elective frozen single blastocyst transfer improved singleton livebirth rate compared with fresh single blastocyst transfer. This multicentre, non-blinded, randomised controlled trial was undertaken in 21 academic fertility centres in China. 1650 women with regular menstrual cycles undergoing their first cycle of in-vitro fertilisation were enrolled from Aug 1, 2016, to June 3, 2017. Eligible women were randomly assigned to either fresh or frozen single blastocyst transfer. The randomisation sequence was computer generated, with block sizes of two, four, or six, stratified by study site. For those assigned to frozen blastocyst transfer, all blastocysts were cryopreserved and a delayed frozen-thawed single blastocyst transfer was done. The primary outcome was singleton livebirth rate. Analysis was by intention to treat. This trial is registered at the Chinese Clinical Trial Registry, number ChiCTR-IOR-14005405. 825 women were assigned to each group and included in analyses. Frozen single blastocyst transfer resulted in higher rates of singleton livebirth than did fresh single blastocyst transfer (416 [50%] vs 329 [40%]; relative risk [RR] 1·26, 95% CI 1·14-1·41, p<0·0001). The risks of moderate or severe ovarian hyperstimulation syndrome (four of 825 [0·5%] in frozen single blastocyst transfer vs nine of 825 [1·1%] in fresh single blastocyst transfer; p=0·16), pregnancy loss (134 of 583 [23·0%] vs 124 of 481 [25·8%]; p=0·29), other obstetric complications, and neonatal morbidity were similar between the two groups. Frozen single blastocyst transfer was associated with a higher risk of pre-eclampsia (16 of 512 [3·1%] vs four of 401 [1·0%]; RR 3·13, 95% CI 1·06-9·30, p=0·029). Frozen single blastocyst transfer resulted in a higher singleton livebirth rate than did fresh single blastocyst transfer in ovulatory women with good prognosis. The increased risk of pre-eclampsia after frozen blastocyst transfer warrants further studies. The National Key Research and Development Program of China.

Risk factors for monozygotic twinning after in vitro fertilization: a systematic review and meta-analysis

To establish the risk factors for monozygotic twin (MZT) and monochorionic twin (MCT) pregnancies after invitro fertilization (IVF). Systematic review and meta-analysis. Not applicable. Women who achieved MZT and non-MZT pregnancies through IVF. Systematic search of Medline from January 1995 to October 2018 with cross-checking of references from relevant articles in English. Possible risk factors for MZT or MCT pregnancies after IVF, comprising extended embryo culture, insemination method (conventional IVF and intracytoplasmic sperm injection [ICSI]), embryo biopsy for preimplantation genetic testing for aneuploidies or for monogenic/single-gene defects (PGT-A or PGT-M) programs, assisted hatching (AH), oocytes donation, female age, and embryo cryopreservation. A total of 40 studies were included. Blastocyst transfer compared with cleavage-stage embryo transfer, and female age <35years were associated with a statistically significant increase in the MZT and MCT pregnancy rate after IVF: (23 studies, OR 2.16, 95% CI, 1.74-2.68, I2=78%; 4 studies, OR 1.29; 95% CI, 1.03-1.62, I2=62%; and 3 studies, OR 1.90, 95% CI, 1.21-2.98, I2=59%; 2 studies, OR 2.34; 95% CI, 1.69-3.23, I2=0, respectively). Conventional IVF compared with ICSI and assisted hatching were associated with a statistically significantly increased risk of MZT pregnancy (9 studies, OR 1.19, 95% CI, 1.04-1.35, I2=0; 16 studies, OR 1.17, 95% CI, 1.09-1.27, I2=29%, respectively). Embryo biopsy for PGT-A or PGT-M, embryo cryopreservation, and oocytes donation were not associated with MZT pregnancies after IVF. Blastocyst transfer is associated with an increased risk of both MZT and MCT pregnancies after IVF. Further evidence is needed to clarify the impact of female age, insemination method and AH on the investigated outcomes.

Characterisation of bovine embryos following prolonged culture in embryonic stem cell medium containing leukaemia inhibitory factor.

In order to help elucidate the process of epiblast and trophoblast cell differentiation in bovine embryos invitro, we attempted to develop a suitable culture medium to allow extended embryo culture. Day 7 bovine blastocysts developed in conventional medium were cultured further in embryonic stem cell medium with or without leukaemia inhibitory factor (LIF) until Day 23. At Day 14, the expression of octamer-binding transcription factor 3/4 (OCT3/4) and VIMENTIN was significantly higher in embryos cultured with than without LIF, but embryonic disc formation was not observed. Although expression of SRY (sex determining region Y)-box 17 (SOX17) mRNA was significantly lower in Day 14 embryos cultured with and without LIF than in invivo embryos, hypoblast cells formed just inside the trophoblast cells of the invitro-cultured embryos. On Day 23, expression of placental lactogen (PL) and prolactin-related protein 1 (PRP1) was not affected by LIF in invitro-cultured embryos, levels of both genes were significantly lower in the invitro than invivo embryos. Similar to invivo embryos, binucleate cell clusters seen in Day 23invitro-cultured embryos were composed of PL-negative and -positive cells. These results suggest that our culture system partially reproduced the differentiation process of trophoblast cells invivo.

Extended embryo culture and ploidy of morphologically normal embryos assessed by next gen sequencing PGS: a single center retrospective study

To analyze embryo culture metrics with regard to absolute measures (chromosome ploidy by NGS-PGS, age of oocyte) and trends (embryo age, embryo grade, egg donor recipient). A single - center, retrospective, observational study. Oocyte retrieval was performed 34 hours after trigger under ultrasound guidance. After retrieval, the oocytes were denuded mechanically by pipetting with cumulase (Origio, enzyme activity 40-120 U/ml). ICSI was performed on all metaphase II oocytes. Fertilized oocytes were cultured in microdroplets of culture media (Vitrolife) overlayed with Ovoil (Vitrolife), gassed with a blood gas mixture (WestAir Gases, 5% oxygen, 7.2% carbon dioxide, balanced nitrogen), until the day of assisted hatching (day 3 of embryo culture). Assisted hatching was performed and embryos were subsequently transferred to fresh culture medium. Embryos were assessed for blastocyst development, biopsy and cryopreservation criteria on days 5, 6, and 7. Blastocysts that met the criteria, either good (distinct inner cell mass (ICM)/numerous trophectoderm (TE) cells) or fair (small ICM/some TE cells) were biopsied and cryopreserved. All genetic analysis was performed by Ovation Fertility Genetics (Las Vegas) using Next Gen Sequencing. 2557 blastocysts from 257 cycles were analyzed in the present study. A total of 1179 on Day 5, 1289 on day 6, and 89 blastocysts at day 7 were biopsied and analyzed by NGS -PGS. On day 5 of culture 64% of embryos tested were euploid, 34% aneuploid. On day 6 of culture 49% of embryos were euploid and 49% aneuploid, and day 7 of culture, 46% of embryos tested were euploid and 52% were aneuploid. The chi-square statistic is 58.1 and is significant at p < .01, indicating that ploidy and length of time in culture are dependent variables. Our optimized culture system routinely yields blastocysts that are good and fair quality on day 7, which did not meet our biopsy and cryopreservation criteria on day 5 or 6 of culture. In the present study, we retrospectively analyzed PGS data from day 5, 6, and 7 blastocysts derived from IVF patients in our clinic during 2017- 2018, and analyzed trends in embryo development and ploidy. Our results (46% euploid blastson day 7) agree with the small, but growing body of evidence that suggests that extended embryo culture to day 7 is clinically important for patients who do not have day 5 and/or day 6 blastocysts. Additionally, we believe for patients with day 5 and/or day 6 blastocysts, day 7 culture may still be necessary, considering the time and costs of IVF cycles, particularly when constructing an IVF cycle with donor eggs.

The risk of birth defects among children born after vitrified blastocyst transfers and those born after fresh and vitrified cleavage-stage embryo transfers.

To explore the risk of birth defects among children born after vitrified blastocyst transfers and those born after fresh and vitrified cleavage-stage embryo transfers. A retrospective cohort study was conducted including infants born after fresh and vitrified day 3 embryo transfers and those born after vitrified day 5 or 6 blastocyst transfers from January 2005 through December 2016. The outcome measures included any birth defect, multiple birth defects and 13 individual categories of birth defects. Any birth defect occurred in 1.15% of infants born after fresh day 3 embryo transfers, 1.75% of infants born after vitrified day 3 embryo transfers, 1.60% of infants born after vitrified day 5 blastocyst transfers and 1.10% of infants born after vitrified day 6 blastocyst transfers. There was no difference in the risk of birth defects between vitrified blastocyst-stage transfers and vitrified cleavage-stage transfers (including day 5 vs. day 3 and day 6 vs. day 3) among all births or in only singletons or twins. For infants born after cleavage-stage embryo transfers at day 3, there was no difference in the risk of birth defects between fresh embryo transfers and vitrified embryo transfers among all births or in only singletons or twins. Transfer of vitrified day 5 or 6 blastocysts does not increase the risk of birth defects compared with vitrified day 3 embryos. However, randomized control trials and follow-up studies of the long-term outcome of children born after blastocyst-stage transfers are needed to confirm the clinical safety of extending embryo culture to the blastocyst stage.

Elevated incidence of monozygotic twinning is associated with extended embryo culture, but not with zona pellucida manipulation or freeze-thaw procedure

To identify the incidence and risk factors associated with IVF-conceived monozygotic twinning (MZT). Retrospective study. Academic hospital. A total of 3,463 women with clinical pregnancies between January 2014 and February 2015 were analyzed. None. The measures were the incidence of MZT based on the number of embryos that were replaced, type of insemination method (conventional IVF or intracytoplasmic sperm injection [ICSI]), with or without the use of assisted hatching (AH), and day of embryo transferred in fresh and frozen cycles. Ninety-three women (2.69%) with MZT were observed. No statistically significant differences were observed in the cycle parameters of fresh or frozen cycles between MZT and other non-MZT pregnancies. Specific IVF procedures or techniques, such as the number of embryo replaced, zona pellucida manipulation (ICSI and AH), and freeze-thaw procedure, did not significantly increase the rate of MZT, except for the day of embryo transferred. Compared with day 3 transferred, day 4 and 5/6 transferred showed an increased probability of MZT (odds ratio [OR], 2.73; 95% confidence interval [CI], 1.16-6.42 for day 4 transferred and OR, 3.68; 95% CI, 2.29-5.93 for day 5/6 transferred). Extended culture (advanced embryo stage) in fresh and frozen cycles appeared to be associated with increased rates of MZT. The effect of the number of embryos transferred, ICSI and AH, and freeze-thaw procedures on the risk for MZT was not demonstrated.

Randomized controlled trial of low (5%) versus ultralow (2%) oxygen for extended culture using bipronucleate and tripronucleate human preimplantation embryos

To determine whether [1] exposure of embryos to 5% oxygen (O2) from day 1 (D1) to D3, and then to 2% O2 from D3 to D5, improves total blastocyst yield, as compared with continuous exposure to 5% O2; and [2] extended culture in 2% O2 alters key metabolic processes and O2-regulated gene expression in human preimplantation embryos. Randomized controlled trial. Academic medical center. Bipronucleate and tripronucleate embryos donated for research. On D1, sibling zygotes were randomized to culture in 5% O2 from D1 to D5 (n = 102; "5% group") or 5% O2 from D1 to D3, then 2% O2 from D3 to D5 (n = 101, "2% group"). Developmental stage and grade; D5 total cell counts; mass spectrometry of spent media; quantitative polymerase chain reaction of 21 genes in inner cell mass and trophectoderm. Among cleaved embryos (n = 176, 87%), those in the 2% group were less likely to arrest at the cleavage stage on D5 (34 of 87, 39.1%) compared with the 5% group (52 of 89, 58.4%) (adjusted odds ratio 0.38, 95% confidence interval 0.18-0.80). Those in the 2% group were more likely to blastulate (35 of 87, 40.2%) than those in the 5% group (20 of 89, 22.5%) (adjusted odds ratio 2.55, 95% confidence interval 1.27-5.12). Culture in 2% O2 was associated with significantly fewer cells in early and advanced blastocysts, alteration in relative abundances of anabolic amino acids and metabolites involved in redox homeostasis, and differential expression of MUC1 in trophectoderm. These findings provide foundational evidence for future investigation of 2% O2 as the preferred O2 tension for extended culture of human embryos.

The use of low oxygen concentration during embryo culture seems not to affect blastocyst aneuploidy rate

Introduction: Trofoectoderm biopsy requires extended embryo culture until blastocyst stage. Culture conditions such as using low oxygen concentrations (~5%) can be determinant for embryo developmental viability.

Abstracts of the 22st Annual Congress of the SBRA, Brasilia/DF, 01-04 August 2018.

ObjectiveTo compare the results of in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles in women with good prognosis, when they were electively transferred, a blastocyst stage embryo (group 1) versus a blastocyst stage and a mórula stage embryo (group 2).Methods35 transfers from January/2014 to January/2018 were included, these occurred in na assisted reproduction center. Selection criteria: Patients in first or second treatment for IVF/ICSI less than 35 years old, with body mass index <30kg/m2 who used their own oocytes with normal ovarian reserve (baseline of antral follicles ≥11) and recipients without age restriction. Cases of severe male fator (spermatic concentration below 1million/ml) were excluded. The transfer was realized on fifth day of embryonic development. Luteal phase support was performed with micronized progesterone 600mg/day vaginally and estradiol 6mg/day orally, started the day after eggs collection. The receivers used 6mg/day of estradiol valerate orally, beginning on first day of menstrual cycle and 600mg/day of micronized progesterone, vaginally, for five days before embryo transfer. Were evaluated: mean age of women under 35 years who used their own oocytes and donors; pregnancy rate (βHCG positive); pregnancy loss rate and ongoing pregnancy rate (fetus alive at 12th week of pregnancy).ResultsBetween group 1 (n=24) and 2 (n=11), mean age of women under 35 who used their own oocytes were 33(±1.6) and 31(±1.8) years old and for donors were 34(±0.5) and 29(±0) years old, for both groups, respectively. Pregnancy rate was 50% (12/24) for group 1 and 18% (02/11) for group 2 (p=0.02). Pregnancy loss rate was 17% (02/12) for group 1 and 50% (01/02) for group 2 (p=0.07). Ongoing pregnancy rate was 42% (10/24) for group 1and 9% (01/11) for group 2 (p=0.01).ConclusionThe analysis of our data shows that transfer another embryo with a slower development with a blastocyst, with the intention of increase pregnancy rates, can actually worsen the results. There is a worldwide trend about extending embryo culture by fifth or sixth day, then these data should be validated in a larger number of cases. The reason for an embryo with slower development impact on development of another embryo with normal development should be evaluated. We emphasize that the results were extracted from cases with good prognosis and can not be extrapolated to other cases.