Maternal physiology and blastocyst morphology are correlated with an inherent difference in peri-implantation human embryo development.

OBJECTIVE: To determine what patient and embryo characteristics are correlated with the developmental potential of the peri-implantation embryo. DESIGN: Retrospective study. SETTING: Research laboratory. PATIENTS: Six hundred fifty-one cryopreserved human blastocysts donated for research with informed patient consent. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Blastocyst attachment to fibronectin-coated plates, trophectoderm outgrowth area, epiblast cell number, total cell number, human chorionic gonadotropin secretion. RESULTS: Patients' body mass index, age, follicle-stimulating hormone: luteinizing hormone ratio on menstrual cycle day 3, antral follicle count on menstrual cycle day 3, antimüllerian hormone level on menstrual cycle day 3, and blastocyst morphological grade were correlated with peri-implantation development outcomes. After controlling for good-quality morphological grades, blastocysts from patients of advanced maternal age developed fewer epiblast cells than blastocysts from younger patients. CONCLUSIONS: Extended embryo culture during the peri-implantation period mirrors several disparities in fertility treatment outcome that we see clinically, including those from patients with advanced maternal age, high body mass index, and low ovarian reserve and from embryos with lower-quality morphological grades. This model system may be useful by providing an alternative or more sensitive endpoint assessment in studying patient, clinical, or laboratory factors that may influence preimplantation embryo developmental potential.

Fused blastocysts as a consequence of group embryo culture: observations, complications, and potential solutions.

How embryos are cultured, in groups or individually, can influence their development and have other unforeseen impacts on subsequent assisted reproductive technologies. Although a group culture of embryos improves the blastocyst formation rates, this can create conditions wherein separate blastocysts may fuse. This fusion of 2 blastocysts can create unique logistic issues for embryo biopsy and genetic analysis. New culture approaches have emerged to facilitate individual embryo culture without losing the benefit of the group culture approach. Unique culture dishes and adjustments of laboratory culture/embryo handling protocols offer possible solutions to minimize or avoid blastocyst fusion.

Comparison of electronic versus manual witnessing of procedures within the in vitro fertilization laboratory: impact on timing and efficiency.

OBJECTIVE: To evaluate the impact of an electronic witnessing system (EWS) on witnessing standard operating procedures and to assess embryologist perceptions of the EWS. DESIGN: Prospective cohort study. SETTING: Private in vitro fertilization laboratory network. PATIENTS: None. INTERVENTIONS: None. MAIN OUTCOME MEASURES: The time difference between manual and electronic double-witnessing procedures, and embryologist perceptions of the EWS. RESULTS: From 342 witnessing times analyzed (114 EWS, 114 manual, and 114 interruptions to witnesses), the EWS reduced mean (SD) total witnessing time (in seconds) by 91.5 (23.6) for intracytoplasmic sperm injection, 62.0 (17.9) for Day 3 embryo assessment, 58.3 (18.9) for fresh embryo transfer, and 59.4 (13.3) for frozen embryo transfer. This time reduction significantly decreased the overall time required for double-witnessing by 3.1- to 5.2-fold. A survey with 50 embryologists within the laboratory network indicated that most embryologists considered the EWS to improve sample traceability (78.3%), reduce errors in labeling issues (80.4%), and reduce the risk of sample mismatch errors by minimizing disruptions (60.9%). Furthermore, 82.6% thought that visual completion of the EWS dashboard provided peace of mind when leaving work and 84.8% were more confident knowing that all procedures were completed according to the EWS. CONCLUSIONS: An EWS can improve laboratory efficiency by significantly decreasing the time required for witnessing procedures and by minimizing interruptions. The EWS was well perceived by embryologists and laboratory managers and enhanced their confidence and peace of mind with regard to witnessing compliance and safety/accuracy.

Absence of SARS-CoV-2 (COVID-19 virus) within the IVF laboratory using strict patient screening and safety criteria.

RESEARCH QUESTION: Is there a risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral exposure and potential cross-contamination from follicular fluid, culture media and vitrification solution within the IVF laboratory using strict patient screening and safety measures? DESIGN: This was a prospective clinical study. All women undergoing transvaginal oocyte retrieval were required to have a negative SARS-CoV-2 RNA test 3-5 days prior to the procedure. Male partners were not tested. All cases used intracytoplasmic sperm injection (ICSI). The first tube of follicular fluid aspirated during oocyte retrieval, drops of media following removal of the embryos on day 5, and vitrification solution after blastocyst cryopreservation were analysed for SARS-CoV-2 RNA. RESULTS: In total, medium from 61 patients, vitrification solution from 200 patients and follicular fluid from 300 patients was analysed. All samples were negative for SARS-CoV-2 viral RNA. CONCLUSIONS: With stringent safety protocols in place, including testing of women and symptom-based screening of men, the presence of SARS-CoV-2 RNA was not detected in follicular fluid, medium or vitrification solution. This work demonstrates the possibility of implementing a rapid laboratory screening assay for SARS-CoV-2 and has implications for safe laboratory operations, including cryostorage recommendations.

Evaluation of the TMRW vapor phase cryostorage platform using reproductive specimens and in vitro extended human embryo culture.

OBJECTIVE: To assess the impact of shipment and storage of sperm, oocytes, and blastocysts in vapor phase nitrogen compared with static storage in liquid phase nitrogen. DESIGN: Prospective cohort-matched study. SETTING: Multiple in vitro fertilization laboratories in an in vitro fertilization network. PATIENT(S): Fifty-eight human embryos, 32 human oocytes, 15 units of bovine semen. INTERVENTION(S): Vapor vs. liquid nitrogen. MAIN OUTCOME MEASURE(S): The postwarming survival of oocytes, sperm, and blastocysts, and the developmental potential of blastocysts during in vitro extended culture. RESULT(S): Custom-designed labware, for use with the TMRW platform, enables continuous temperature monitoring during shipment and/or storage in the vapor phase robotic storage system. The highest temperature recorded for specimens shipped to a domestic laboratory was -180.2 °C with a mean ± SD of -190.4 ± 0.5 °C during shipment and -181.1 ± 0.6 °C during storage. Likewise, specimens shipped internationally had a high of -180.2 °C with a mean ± SD of -193.5 ± 0.6 °C during shipment and -181.2 ± 0.7 °C during storage. Results from the extended culture assays have revealed no deleterious effect of shipment and storage in nitrogen vapor. The viability of mammalian gametes and embryos was equivalent between the vapor phase and liquid phase storage. CONCLUSION(S): The evaluated system did not have any deleterious effects on the postwarming survival of sperm, oocytes, and blastocysts. The postwarming developmental potential of human blastocysts during in vitro extended culture was unaffected by storage and handling in the vapor phase nitrogen TMRW platform when compared with static liquid phase nitrogen storage. Our results suggest that the vapor phase cryostorage platform is a safe system to handle and store reproductive specimens for human assisted reproductive technology.

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

RESEARCH QUESTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Controversies in ART: can the IVF laboratory influence preimplantation embryo aneuploidy?

Published reports have indicated that rates of preimplantation embryo aneuploidy in a control donor population may vary between IVF centres. This suggests that location-specific conditions, in the clinic, IVF or genetics laboratory, may be influencing the chromosome dynamics or diagnosis. More recent reports suggest that rates of embryo mosaicism, representing mitotic errors, may vary between IVF centres. This would suggest perhaps a laboratory-controlled variable is influencing mitotic cell division during preimplantation embryo development. Various IVF laboratory-controlled factors may be impacting chromosome separation and segregation. Variables including type of culture media, pH, temperature, osmolality and oxygen concentration could all be possible factors influencing embryo aneuploidy. Furthermore, laboratory techniques, method of insemination, laser use or handling of biopsied cells may also influence genetic results. These IVF laboratory variables will be reviewed and literature suggesting a possible link to mitotic aneuploidy/mosaicism discussed.

Controversies in ART: considerations and risks for uninterrupted embryo culture.

With new time-lapse incubators, IVF laboratories have increased use of single step media, often used in an uninterrupted approach. This simplifies the culture process for embryologists, may help reduce costs, offers the potential to reduce cell handling and associated harmful environmental stressors, and improves embryo quality and outcomes. One could argue, however, that optimized quality control and culture conditions are required to implement uninterrupted culture successfully. Without impeccable laboratory conditions and oversight, while trying to reduce harmful environmental stress, the laboratory could be imparting stress. Factors such as medium evaporation and associated osmolality and pH increases, as well as volatile organic compound accumulation, could offset any advantage of reduced dish or embryo handling. When implementing uninterrupted embryo culture, attention must be paid to incubator humidity, amount, quality and type of oil used, medium formulation and protein quality, as well as laboratory air and gas supply quality, and volatile organic compound content.

Forty years of IVF.

This monograph, written by the pioneers of IVF and reproductive medicine, celebrates the history, achievements, and medical advancements made over the last 40 years in this rapidly growing field.

Live-cell quantification and comparison of mammalian oocyte cytosolic lipid content between species, during development, and in relation to body composition using nonlinear vibrational microscopy.

Cytosolic lipids participate in the growth, development, and overall health of mammalian oocytes including many roles in cellular homeostasis. Significant emphasis has been placed on the study of lipids as a dynamic organelle, which in turn requires the development of tools and techniques to quantitate and compare how lipid content relates to cellular structure, function, and normalcy. Objectives of this study were to determine if nonlinear vibrational microscopy (e.g., coherent anti-Stokes Raman scattering or CARS microscopy) could be used for live-cell imaging to quantify and compare lipid content in mammalian oocytes during development and in relation to body composition; and compare its efficacy to methods involving cellular fixation and staining protocols. Results of this study demonstrate that CARS is able to identify lipids in live mammalian oocytes, and there exists quantifiable and consistent differences in percent lipid composition across ooctyes of different species, developmental stages, and in relation to body composition. Such a method of live-cell lipid quantification has (i) experimental power in basic cell biology, (ii) practical utility for identifying developmental predictive biomarkers while advancing biology-based oocyte/embryo selection, and (iii) ability to yield rationally supporting technology for decision-making in rodents, domestic species, and human assisted reproduction and/or fertility preservation.

Optimizing the culture environment and embryo manipulation to help maintain embryo developmental potential.

With increased use of comprehensive chromosome screening (CCS), the question remains as to why some practices do not experience the same high levels of clinical success after implementation of the approach. Indeed, the debate surrounding the efficacy and usefulness of blastocyst biopsy and CCS continues. Importantly, several variables impact the success of an assisted reproductive technology cycle. Transfer of a euploid embryo is but one factor in an intricate system that requires numerous steps to occur successfully. Certainly, the culture environment and the manipulations of the embryo during its time in the laboratory can impact its reproductive potential. Environmental stressors ranging from culture media to culture conditions and even culture platform can impact biochemical, metabolic, and epigenetic patterns that can affect the developing cell independent of chromosome number. Furthermore, accompanying procedures, such as biopsy and vitrification, are complex and, when performed improperly, can negatively impact embryo quality. These are areas that likely still carry room for improvement within the IVF laboratory.

Antimüllerian hormone as a predictor of good-quality supernumerary blastocyst cryopreservation among women with levels <1 ng/mL versus 1-4 ng/mL.

OBJECTIVE: To determine whether antimüllerian hormone (AMH) levels predict the availability of good-quality supernumerary blastocysts for cryopreservation. DESIGN: Retrospective study. SETTING: Two fertility centers. PATIENT(S): First fresh IVF cycles (n = 247) grouped as follows: 40 women <35 year old with AMH <1 ng/mL and 77 women with AMH 1-4 ng/mL; 62 women ≥35 year old with AMH <1 ng/mL, and 68 women with AMH 1-4 ng/mL. INTERVENTION(S): AMH level measured before IVF with ovarian stimulation protocols based on patient age and AMH level, including short gonadotropin-releasing hormone (GnRH) agonist, GnRH antagonist, or GnRH agonist microdose flare; supernumerary good-quality blastocysts cryopreserved on days 5 or 6 after retrieval. MAIN OUTCOME MEASURES(S): Supernumerary good-quality blastocysts for cryopreservation in relation to AMH levels. RESULT(S): Among women <35 years of age, there was a statistically significant difference in the number of patients with supernumerary good-quality blastocysts for cryopreservation between the groups with AMH <1 ng/mL and AMH 1-4 ng/mL (30.0% vs. 58.4%) when adjusted for age. Among women ≥35 years of age, there was a statistically significant difference in the number of patients with supernumerary good-quality blastocyst cryopreservation between groups with AMH <1 ng/mL and AMH 1-4 ng/mL (16.1% vs. 42.6%), when adjusted for age. CONCLUSION(S): Low AMH levels are associated with a statistically significantly lower likelihood of blastocysts for cryopreservation as compared with higher AMH levels. This effect was seen among women both <35 and ≥35 years of age. Patient counseling should include realistic expectations for the probability of good-quality supernumerary blastocysts available for cryopreservation.

Optimal human embryo culture.

A large contributor to success during in vitro fertilization (IVF) lies in the processes occurring within the IVF laboratory. These processes make up the "culture system." This system entails numerous procedures and technical steps that must be optimized to produce a competent embryo. Notably, variations exist between programs that include differences in patient population, clinical stimulation, and other factors. Thus, a single "optimal" culture system to be utilized between all laboratories is likely not feasible. Rather, laboratory procedures should be optimized based on an individual laboratory's performance. That being said, within the scientific literature, there are key components, approaches, and techniques within the culture system that have been shown to be superior to alternatives. These key components important in improving embryo culture are discussed.

Decisions for the IVF laboratory: comparative analysis of embryo culture incubators.

Incubators in the IVF laboratory play a pivotal role in providing a stable and appropriate culture environment required for optimizing embryo development and clinical outcomes. With technological advances, several types of incubators are now available and careful consideration is required for selection. Examination of variables, such as recovery/stabilization of temperature, gas atmosphere and humidity, as well as understanding various approaches utilized by each device to regulate these variables, is critical. Additionally, a comprehensive examination of clinical studies that compare various incubators may provide insight into their efficacy. Other factors, both technical and practical, must also be considered when selecting an incubator. Importantly, proper management, including patient volume and workflow, is paramount in optimizing function of any incubator, regardless of the technology incorporated. This review highlights incubator function and reviews key environmental variables controlled and the technology utilized in various units. Additionally, existing comparative studies focused on incubator recovery and clinical outcomes are critically analysed. Finally, strategies employed for incubator management, as well as future potential incubator improvements are discussed. While existing reports indicate that smaller benchtop/topload incubators provide faster recovery of environmental variables, there is no clear advantage of any particular incubator based on clinical outcomes.

Peering beneath the surface: novel imaging techniques to noninvasively select gametes and embryos for ART.

Embryo imaging has long been a critical tool for in vitro fertilization laboratories, aiding in morphological assessment of embryos, which remains the primary tool for embryo selection. With the recent emergence of clinically applicable real-time imaging systems to assess embryo morphokinetics, a renewed interest has emerged regarding noninvasive methods to assess gamete and embryo development as a means of inferring quality. Several studies exist that utilize novel imaging techniques to visualize or quantify intracellular components of gametes and embryos with the intent of correlating localization of organelles or molecular constitution with quality or outcome. However, the safety of these approaches varies due to the potential detrimental impact of light exposure or other variables. Along with complexity of equipment and cost, these drawbacks currently limit clinical application of these novel microscopes and imaging techniques. However, as evidenced by clinical incorporation of some real-time imaging devices as well as use of polarized microscopy, some of these imaging approaches may prove to be useful. This review summarizes the existing literature on novel imaging approaches utilized to examine gametes and embryos. Refinement of some of these imaging systems may permit clinical application and serve as a means to offer new, noninvasive selection tools to improve outcomes for various assisted reproductive technology procedures.

Could time-lapse embryo imaging reduce the need for biopsy and PGS?

PURPOSE: To review relevant studies examining the relationship between embryo morpho-kinetics and aneuploidy. METHODS: Search of Pubmed and Medline using relevant keywords pertaining to morphology, morphokinetics and embryonic aneuploidy, as well as examination of various reference lists and conference proceedings. RESULTS: An abundance of publications, both preliminary and peer-reviewed, have emerged regarding the usefulness of time-lapse imaging in tracking embryo development and improving embryo selection. Recently, these publications have explored ability to not only predict blastocyst formation and implantation, but also the ability to detect embryonic chromosomal aneuploidy. Of the two peer-reviewed retrospective studies on morpho-kinetics and embryonic aneuploidy, one demonstrates that early cleavage timings can indicate chromosomal complement, while the other demonstrates that key events following the maternal-zygotic transition can be markers of aneuploidy. A recent paper also demonstrates improved outcomes following IVF using a selection algorithm to identify embryos at "low risk" of chromosomal abnormalities. However, the predictive nature of these events and timings is far from ideal. Additionally, results may be dependent upon the day of biopsy and method utilized for chromosomal assessment. CONCLUSION: With continued effort, the combination of multiple morphologic endpoint assessments and developmental timings and refinement of modeling systems may improve the predictive ability to determine embryonic aneuploidy. This may help select a subset of embryos that are less likely to carry chromosomal abnormalities and improve assisted reproductive outcomes. However, embryo biopsy, followed by preimplantation genetic screening/comprehensive chromosomal screening still remains the most reliable method to assess chromosomal complement of preimplantation embryos.

Oocyte cryopreservation: searching for novel improvement strategies.

PURPOSE: To highlight emerging techniques aimed at improving oocyte cryopreservation. METHODS: Review of available and relevant literature through Pubmed and Medline searches. RESULTS: Oocyte cryopreservation is an increasingly common procedure utilized for assisted reproduction and may benefit several patient populations. Therefore, improving efficiency is paramount in realizing the tremendous promise of this approach. However, in addition to numerous studies looking to improve oocyte cryopreservation efficacy via examination of variables involved with protocol methodology, such as type/concentration of cryoprotectant (CPA), type of storage device, or cooling/warming rates, there are more novel approaches for improvement. These alternate approaches include utilizing different the stages of oocytes, examining alteration of basal media and buffer composition, optimizing CPA exchange protocols and device loading through use of automated technology, as well as examination/manipulation of oocyte cellular composition to improve cryotolerance. Finally, elucidating more accurate or insightful indicators of "success" is crucial for continued improvement of oocyte cryopreservation. CONCLUSION: Oocyte cryopreservation has improved dramatically in recent years and is receiving widespread clinical use. Novel approaches to further improve success, as well as improved methods to assess this success will aid in continued improvement.