Viability assessment using fluorescent markers and ultrastructure of human biopsied embryos vitrified in open and closed systems.

RESEARCH QUESTION: Are there any differences in viability and ultrastructure amongst embryos biopsied on Day 5 versus Day 3 following vitrification in open and closed systems and compared to fresh embryos? DESIGN: One hundred human embryos (40 blastocysts biopsied on Day 5 and subsequently vitrified in open or closed systems and 60 Day 3 biopsied embryos that developed to blastocysts but were rejected for transfer following preimplantation genetic testing for monogenic/single gene defects and for aneuploidies were either treated fresh [n = 20] or vitrified [n = 40] in open or closed systems) and following warming and culture for 4 h were subjected to viability staining with carboxyfluorescein-diacetate succinimidylester/propidium iodide or processed for transmission electron microscopy. RESULTS: No statistically significant differences were observed in the viability of human biopsied embryos following vitrification in open and closed systems. Compared to fresh embryos, vitrified ones had a higher incidence of damage (propidium iodide-stained cells) irrespective of the vitrification method (P = 0.005). These damaged cells were more prominent in Day 5 biopsied blastocysts and mainly located at the position of cutting. Characteristic lipofuscin droplets (representative of apoptosis) and a higher number of vacuoles and distension of mitochondria were also more evident in vitrified embryos, although this was not statistically assessed. CONCLUSIONS: Vitrification in open and closed systems does not adversely affect the viability and ultrastructure of Day 5 and Day 3 biopsied embryos as revealed by the minimal yet statistically significant cell damage observed. This damage may be compensated by the embryos, which in their attempt to fully recover following vitrification, potentially enable 'rescue' processes to eliminate it.

Focus on time-lapse analysis: blastocyst collapse and morphometric assessment as new features of embryo viability.

The main goal of assisted reproductive technology (ART) is to achieve a healthy singleton live birth after the transfer of one embryo. A major objective of IVF scientists has always been to use adequate criteria for selecting the embryo for transfer according to its implantation potential. Indeed, embryo quality is usually assessed by evaluating visual morphology, which relies on the removal of the embryo from the incubator and might include inter- and intra-evaluator variation among embryologists. Recently, an advancement in embryo culture has taken place with the introduction of a new type of incubator with an integrated time-lapse monitoring system, which enables embryologists to analyse the dynamic events of embryo development from fertilization to blastocyst formation. This novel practice is rapidly growing and has been used in many IVF centres worldwide. Therefore, the main aim of this review is to present the benefits of time-lapse monitoring in a modern embryology laboratory; in particular, we discuss blastocyst collapse and morphometric blastocyst assessment, and analyse their association with embryo viability and implantation potential. In addition, we highlight preliminary studies involving artificial intelligence and machine learning models as non-invasive markers of clinical pregnancy.

Incidence, Origin, and Predictive Model for the Detection and Clinical Management of Segmental Aneuploidies in Human Embryos.

Despite next-generation sequencing, which now allows for the accurate detection of segmental aneuploidies from in vitro fertilization embryo biopsies, the origin and characteristics of these aneuploidies are still relatively unknown. Using a multifocal biopsy approach (four trophectoderms [TEs] and one inner cell mass [ICM] analyzed per blastocyst; n = 390), we determine the origin of the aneuploidy and the diagnostic predictive value of segmental aneuploidy detection in TE biopsies toward the ICM's chromosomal constitution. Contrary to the prevalent meiotic origin of whole-chromosome aneuploidies, we show that sub-chromosomal abnormalities in human blastocysts arise from mitotic errors in around 70% of cases. As a consequence, the positive-predictive value toward ICM configuration was significantly lower for segmental as compared to whole-chromosome aneuploidies (70.8% versus 97.18%, respectively). In order to enhance the clinical utility of reporting segmental findings in clinical TE biopsies, we have developed and clinically verified a risk stratification model based on a second TE biopsy confirmation and segmental length; this model can significantly improve the prediction of aneuploidy risk in the ICM in over 86% of clinical cases enrolled. In conclusion, we provide evidence of the predominant mitotic origin of segmental aneuploidies in preimplantation embryos and develop a risk stratification model that can help post-test genetic counseling and that facilitates the decision-making process on clinical utilization of these embryos.

BOARD INVITED REVIEW: Post-transfer consequences of in vitro-produced embryos in cattle.

In vitro embryo production (IVP) in cattle has gained worldwide interest in recent years, but the efficiency of using IVP embryos for calf production is far from optimal. This review will examine the pregnancy retention rates of IVP embryos and explore causes for pregnancy failures. Based on work completed over the past 25 yr, only 27% of cattle receiving IVP embryos will produce a live calf. Approximately 60% of these pregnancies fail during the first 6 wk of gestation. When compared with embryos generated by superovulation, pregnancy rates are 10% to 40% lower for cattle carrying IVP embryos, exemplifying that IVP embryos are consistently less competent than in vivo-generated embryos. Several abnormalities have been observed in the morphology of IVP conceptuses. After transfer, IVP embryos are less likely to undergo conceptus elongation, have reduced embryonic disk diameter, and have compromised yolk sac development. Marginal binucleate cell development, cotyledon development, and placental vascularization have also been documented, and these abnormalities are associated with altered fetal growth trajectories. Additionally, in vitro culture conditions increase the risk of large offspring syndrome. Further work is needed to decipher how the embryo culture environment alters post-transfer embryo development and survival. The risk of these neonatal disorders has been reduced by the use of serum-free synthetic oviductal fluid media formations and culture in low oxygen tension. However, alterations are still evident in IVP oocyte and embryo transcript abundances, timing of embryonic cleavage events and blastulation, incidence of aneuploidy, and embryonic methylation status. The inclusion of oviductal and uterine-derived embryokines in culture media is being examined as one way to improve the competency of IVP embryos. To conclude, the evidence presented herein clearly shows that bovine IVP systems still must be refined to make it an economical technology in cattle production systems. However, the current shortcomings do not negate its current value for certain embryo production needs and for investigating early embryonic development in cattle.

Noninvasive embryo assessment technique based on buoyancy and its association with embryo survival after cryopreservation.

Embryo cryopreservation offers many benefits by allowing genetic preservation, genetic screening, cost reduction, global embryo transport and single embryo transfer. However, freezing of embryos decreases embryo viability, as intracellular ice crystal formation often damages embryos. Success rates of frozen embryo transfer are expected to be 15-20% less than fresh embryo transfer. We have developed a noninvasive embryo assessment technique (NEAT) which enables us to predict embryo viability based on buoyancy. The purpose of this research was twofold. First was to determine if a NEAT, through a specific gravity device can detect embryo survival of cryopreservation. Second, it was to relate embryo buoyancy to embryo viability for establishing pregnancies in sheep. Blastocysts descent times were measured on one-hundred sixty-nine mice blastocysts before cryopreservation, according to standard protocol and post-thawing blastocysts descent times were measured again. There was a significant difference in blastocyst post-thaw descent times with NEAT in those blastocysts which demonstrated viability from those that did not (P < 0.05). This suggests NEAT is successful in determining blastocysts viability in cryopreserved mice blastocysts. At a commercial ovine facility, NEAT was performed on fourteen frozen and thawed ovine blastocysts. Blastocysts of similar descent times were paired and transferred into recipient ewes as twins. Pregnancy was later confirmed by blood test and multiple gestation outcomes were determined at lambing. Six of seven recipient ewes were pregnant and all pregnant ewes delivered lambs without complication. Four ewes delivered twin lambs and two ewes delivered singletons, which totals ten of the fourteen (71%) blastocysts surviving to term. This pregnancy rate is comparable to expected to pregnancy rates in a commercial setting. The blastocysts which did not establish pregnancy demonstrated less buoyancy versus those blastocysts which established pregnancies which survived to term (P < 0.05). These results suggest NEAT can identify which blastocysts survive cryopreservation, thus significantly reduce the transfer of non-viable embryos. Further studies on a larger scale commercial setting will evaluate the efficacy of NEAT.

An in vitro assessment of the value of sucrose supplementation to warming media for the vitrification of bovine embryos.

　　BACKGROUND: In order to thaw slow-cooled bovine embryos it is standard practice to draw out permeating cryoprotectants by passing embryos through successively decreasing osmotic solutions. However, recently it has been suggested that sucrose may not be needed in the warming media. OBJECTIVE: The aim of this experiment was to compare the effect of warming media prepared with or without the inclusion of sucrose on the survival and hatching capacity of vitrified in vitro-derived bovine embryos. MATERIALS AND METHODS: Expanded blastocysts were produced in vitro and vitrified. Vitrified embryos were warmed either successively through 0.5, 0.3 and 0.2 M sucrose solutions ('stepwise'), or by placing directly into the blastocyst solution without the addition of sucrose ('direct'). A total of 93 expanded blastocysts were assigned randomly to two treatment groups, respectively. RESULTS: The re-expansion rates of vitrified embryos warmed after 24h in vitro culture were similar between the two groups (46/46, 100%; 46/47, 97.9%). From those vitrified embryos that expanded at 24 h there was also no significant difference in hatching rates after 48 h in vitro culture (42/46, 91.3%; 40/46, 87.0%). CONCLUSION: The findings indicate that stepwise warming through sucrose solutions is not required for continued embryo development. Hence, a more time-efficient warming method for vitrified embryos may be followed when conducting cattle embryo transfers.

Assisted reproduction with gametes and embryos: what research is needed and fundable?

Principles for selecting future research projects include interests of investigators, fundability, potential applications, ethical considerations, being able to formulate testable hypotheses and choosing the best models, including selection of the most appropriate species. The following 10 areas of assisted reproduction seem especially appropriate for further research: efficacious capacitation of bovine spermatozoa in vitro; improved in vitro bovine oocyte maturation; decreasing variability and increasing efficacy of bovine superovulation; improved fertility of sexed semen; improving equine IVF; improving cryopreservation of rooster spermatozoa; understanding differences between males in success of sperm cryopreservation and reasons for success in competitive fertilisation; mechanisms of reprogramming somatic cell nuclei after nuclear transfer; regulation of differentiation of ovarian primordial follicles; and means by which spermatozoa maintain fertility during storage in the epididymis. Issues are species specific for several of these topics, in most cases because the biology is species specific.

Morphological assessment of embryo viability.

Morphological assessment is discussed in the context of significant literature at all stages of in vitro development, beginning with the oocyte and culminating at the blastocyst stage. Current evidence is used to debate the inclusion of commonly observed morphological features in grading schemes. The biological rationale behind observed phenomena such as multinucleation and fragmentation are also explored. Current limitations as well as technological advancements that increase our ability to assess viability are highlighted. Particular attention is paid to the relationship between developmental timing and assessment schemes. Failure to standardize assessment timing and inclusion criteria is glaring weaknesses of the literature that currently make consensus unattainable. Mounting evidence suggests that the future of static assessment is very likely to be influenced by information gathered from preimplantation genetic screening and other invasive techniques as well as from continuous monitoring tools such as time lapse.

Assessment of histiotrophic nutrition using fluorescent probes.

Histiotrophic nutrition is a process whereby the rodent visceral yolk sac (VYS) internalizes exogenous macromolecules, degrades them, and sends the degradation products to the embryo for use in de novo macromolecular biosynthesis. This process is important for embryonic development during early gestation prior to the formation of the functional placenta. Quantification and visualization of histiotrophic nutrition can be accomplished using fluorescent tracer molecules such as fluorescein isothiocyanate-conjugated albumin (FITC-albumin) that can be visualized using fluorescent microscopy and quantified using fluorescent spectroscopy. The methods are simple and can provide complementary functional and structural information in studies of the effects of embryotoxicants on yolk sac function.

Assessing the mechanical energy costs of various tissue reshaping mechanisms.

Early-stage embryos must reshape the tissues of which they are made into organs and other life-sustaining structures; and if non-mammalian embryos fail to complete these tasks before the energy provided by their yolk runs out, they die. The aim of this study is to use a cell-level computational model to investigate the energetic cost of a variety of mechanisms that can drive an in-plane reshaping pattern known as convergent extension--a motif in which a tissue narrows in one in-plane direction and expands in another. Mechanisms considered include oriented lamellipodia, directed mitosis, stress fibers, and anisotropic external tension. Both isolated patches of tissue and actively contracting tissues that deform adjacent passive areas are considered. The cell-level finite element model used here assumes that the cell membrane and its associated proteins generate a net tension γ along each cell-cell interface and that the cytoplasm and its embedded networks and structures have an effective viscosity µ. Work costs are based exclusively on mechanical considerations such as edge lengths and tensions, and because a traditional mechanical efficiency cannot be calculated, mechanisms are compared on the basis of the work they must do to the tissue to cause a specified rate of in-plane reshaping. Although the model contains a number of simplifications compared to real embryonic tissues, it is able to show that the work requirements for tissue reshaping by mitoses and by lamellipodia are of the same order. Lamellipodia are energetically most effective when their tensions are approximately twice as large as the interfacial tensions in the surrounding cells. The model also shows that stress fibers or other direct stretch or compression mechanisms are at least five times more efficient for tissue reshaping than are mitoses or lamellipodia and that the work needed to deform a typical cellular tissue is more than thirty times greater than if it did not contain cell boundaries. Collectively, these findings indicate that common tissue reshaping mechanisms have mechanical efficiencies of less than one percent and that mechanical efficiency is not the primary determinant of which mechanism(s) an embryo uses to reshape its tissues.

Time-lapse monitoring as a tool for clinical embryo assessment.

As elective transfer of a single embryo (eSET) becomes increasingly accepted, the need to improve implantation rates becomes crucial. Selecting the most competent embryo therefore constitutes a major challenge in assisted reproductive technology. Embryo morphology and developmental stage at given time points are closely correlated with developmental competence and assessment of morphological parameters at discrete inspection points thus remains the preferred way of evaluating embryonic potential. Lately, more attention has been given to the assessment of dynamic embryo development as a tool for evaluating embryonic potential. The introduction of time-lapse equipment approved for use on human embryos offers novel clinical opportunities for continuous monitoring of embryos, enabling flexible evaluation of known morphological parameters and potentially introducing new dynamic markers of viability. Due to lack of larger, randomized clinical studies it remains to be elucidated whether embryo selection using dynamic parameters improves clinical outcome and which parameters are of significance. Before such randomized controlled studies are organized, the most promising parameters to evaluate must be identified. This mini-review summarizes the current knowledge about dynamic markers of viability and discusses the potential clinical role of time-lapse analysis in embryo assessment and selection.

Serum unconjugated bisphenol A concentrations in men may influence embryo quality indicators during in vitro fertilization.

Here we assess bisphenol A (BPA) in couples undergoing in vitro fertilization (IVF) and indicators of embryo quality; embryo cell number (ECN) and embryo fragmentation score (EFS). Twenty-seven couples provided serum on the day of oocyte retrieval. Unconjugated BPA was measured by HPLC with Coularray detection. Odds ratios (OR) were generated using ordinal logistic regression including female and male BPA concentrations, age and race, and day of embryo transfer for ECN. Inverse associations are suggested for male BPA with ECN (OR=0.70, P=0.069), and EFS (OR=0.54, P=0.009), but not for women. Male BPA exposure may affect embryo quality during IVF.

Toxic trace metals and embryo quality indicators during in vitro fertilization (IVF).

Trace exposures to the toxic metals mercury (Hg), cadmium (Cd) and lead (Pb) may interfere with in vitro fertilization (IVF). The aim of this study is to explore biologically plausible hypotheses concerning associations between metals and embryo quality indicators during IVF. For 24 female patients, a multivariable ordinal logistic regression model suggests a 75% reduction in the odds for higher embryo cell cleavage per µg/dL increase in blood Pb (adjusted odds ratio (aOR) 0.25, 95% confidence interval (CI) 0.07-0.86). For 15 male partners, each µg/L increase in blood Hg (aOR 0.60, 95% CI 0.45-0.79) and µg/dL increase in blood Pb (aOR 0.58, 95% CI 0.37-0.91) is associated with a decrease in the analogous odds. Embryo fragmentation is reduced by higher blood Hg (aOR 0.85, 95% CI 0.72-1.00), but increased by higher blood Pb (aOR 1.47, 95% CI 1.11-1.94) in men. The magnitude of these suggested effects warrants confirmation in a larger study.

Noninvasive bovine oocyte quality assessment: possibilities of a single oocyte culture.

Although bovine embryos are routinely produced in vitro for several decades, there still exists a critical need for techniques to accurately predict the oocyte's developmental competence in a noninvasive way, before the in vitro embryo production procedure. In this review, several noninvasive methods to evaluate oocyte quality are discussed, such as morphological assessment of the cumulus oocyte complex and the use of brilliant cresyl blue. Because an individual oocyte and embryo culture method can possibly generate additional insights into the factors that determine oocyte quality, the second part of this review summarizes the state of the art of bovine single oocyte culture. The optimization of individual in vitro embryo production can obviously accelerate the quest for better noninvasive oocyte quality markers, because more information about the oocyte's requirements and intrinsic quality will be revealed. Although each step of in vitro culture has to be re-examined in light of the hampered production of single embryos, the reward at the end will be substantial. Individual scored oocytes will be traceable along the in vitro embryo production procedure and the final blastocyst outcome can be linked to the original oocyte quality and follicular environment without the bias caused by simultaneously developing embryos.

A more cost effective and rapid high percentage germ-line transmitting chimeric mouse generation procedure via microinjection of 2-cell, 4-cell, and 8-cell embryos with ES and iPS cells.

The long-standing traditional method of delivering embryonic stem (ES) cells adjacent to the inner cell mass (ICM) of blastocysts to generate chimeras improved with the advent of laser- or Piezo assisted 8-cell embryo microinjection. Building on this technology but omitting either the laser or the Piezo to penetrate the zona pellucida and making use of earlier embryonic stages (2-cell and 4-cell), we were able to significantly speed up and economize our ES cell microinjection and chimera production throughput. We demonstrate here that embryonic (ES) and induced pluripotent stem (iPS) cells can stay fully pluripotent when delivered into 2-cell- and 4-cell-stage embryos, long before they would naturally be incorporated into the ICM of a blastocyst (E3.5) and give rise to high percentage and germline transmitting chimeras.

Assessment of day-3 morphology and euploidy for individual chromosomes in embryos that develop to the blastocyst stage.

OBJECTIVE: To examine the relationship between day-3 morphology and euploidy for individual chromosomes in embryos that develop to the blastocyst stage by day 5. DESIGN: Retrospective cohort study. SETTING: Boston IVF, a large university-affiliated reproductive medicine practice. PATIENT(S): Ninety-nine patients undergoing their first preimplantation genetic screening (PGS) cycle between January 1 and December 31, 2006. INTERVENTION(S): In vitro fertilization (IVF) and preimplantation genetic screening (PGS). MAIN OUTCOME MEASURE(S): Prevalence of euploidy for chromosomes X, Y, 8, 13, 14, 15, 16, 17, 18, 20, 21, and 22 in day-3 high implantation potential (HIP) versus non-HIP embryos that grew to day-5 blastocysts. RESULT(S): Seven hundred three embryos from 99 cycles in 99 patients underwent PGS. Three hundred sixty-four (52%) embryos from 88 cycles in 88 patients developed to the blastocyst stage by day 5. High implantation potential embryos were more likely to be euploid for chromosomes X/Y, 8, 15, 16, 18, and 22 compared with non-HIP embryos, with similar trends for chromosomes 14 and 17. There were no statistically significant differences between HIP and non-HIP embryos in euploidy prevalence for chromosomes 13, 20, and 21. CONCLUSION(S): Our data suggest that PGS may detect potentially viable but detrimental chromosomal abnormalities that are not detected by embryo morphology alone.

Symposium: innovative techniques in human embryo viability assessment. Oocyte assessment and embryo viability prediction: birefringence imaging.

Embryo viability is a key element for success in assisted reproduction. Since the beginning of the era of assisted reproduction treatment, embryo viability was mostly considered to be a function of developmental progression during the preimplantation phase. In the last decade, several morphological criteria of oocytes and embryos were evaluated with regard to their potential for predicting embryo viability. The introduction of polarization light microscopy systems in assisted reproduction has enabled the detection of structures within oocytes that possess a natural birefringence. Birefringence imaging of the meiotic spindle and the zona pellucida in living animal and human oocytes represents a new approach in the assessment of oocyte and embryo viability. The technique was applied in several studies to select oocytes in order to improve treatment success. This review will summarize the present knowledge of birefringence imaging. The various applications in basic and clinical research as well as in clinical treatment will be presented, especially with regard to their effect on assisted reproduction.

Symposium: innovative techniques in human embryo viability assessment. Human oocyte respiration-rate measurement--potential to improve oocyte and embryo selection?

Oocyte and embryo selection are not highly successful, with fewer than 10% of oocytes in assisted reproduction resulting in a delivery. Techniques for oocyte and embryo selection rely on highly subjective morphology assessment, with few true quantitative techniques available. One aspect of oocyte health that could be considered is the ability to produce ATP through respiration. Using a non-invasive technology, the respiration rates of individual human oocytes were recorded in an attempt to correlate respiration and oocyte health with probable subsequent development. Oocytes used were either immature or mature, non-fertilized oocytes from a clinical assisted reproduction programme. Differences in respiration rates between oocytes within a cohort and between cohorts of oocytes were recorded. The differences between cohorts reflected many of the currently known differences in oocyte health, related to age and FSH concentrations. However, within a cohort, differences between oocytes were observed, with some having high rates and others low. Oocytes with respiration rates of between 0.48 and 0.55 nl O(2)/h were viable, with lower rates consistent with lack of continued in-vitro maturation or atresia. This technology may have a future in the clinical laboratory as a predictor of oocyte health and ability to develop into an embryo with greater potential of delivery.

Use of comprehensive chromosomal screening for embryo assessment: microarrays and CGH.

One of the most important factors influencing embryo viability is chromosome imbalance (aneuploidy). Embryos derived from aneuploid gametes have little potential for forming a viable pregnancy, but cannot be distinguished from normal embryos using standard morphological evaluation. For more than a decade, preimplantation genetic screening (PGS) has been used to assist in the identification of aneuploid embryos. However, current strategies, based upon cell biopsy followed by fluorescent in situhybridization, allow less than half of the chromosomes to be screened. In this review, we discuss methods that overcome the limitations of earlier PGS strategies and provide screening of the entire chromosome complement in oocytes and embryos. In recent months, there has been a rapid growth in the number of PGS cycles utilizing one such method, comparative genomic hybridization (CGH). Data from IVF cycles utilizing CGH must be considered preliminary, but appear to indicate a dramatic increase in embryo implantation following comprehensive chromosomal screening. It is expected that methods based upon microarrays will yield similar clinical results and may be sufficiently rapid to permit comprehensive screening without the need for embryo cryopreservation. Some microarray platforms also offer the advantage of embryo fingerprinting and the potential for combined aneuploidy and single gene disorder diagnosis. However, more data concerning accuracy and further reductions in the price of tests will be necessary before microarrays can be widely applied.