Optimal Embryo Selection: The Irreplaceable Role of the Embryologist in an Age of Advancing Technology.

Background: Time-lapse incubators allow for ongoing evaluation of embryos without culture condition disruption. The use of time-lapse incubation has been shown to improve outcomes either by improving overall conditions or providing additional information to aid in embryo selection for transfer. Time-lapse incubators can also utilise morphokinetic models to rank embryos based on morphokinetic parameters. We sought to compare a morphokinetic model for embryo comparison to traditional morphologic evaluation. Aims: The aim of the study is to compare a morphokinetic model for embryo comparison to traditional morphologic evaluation. Settings and Design: This is a retrospective cohort design. Materials and Methods: Embryos cultured in a time-lapse culture system that had traditional morphologic evaluation, morphokinetic modelling and known live birth outcomes were included in this study. Embryos with unknown competence were excluded, including when two embryos were transferred with a single live birth resulted. Statistical Analysis Used: Receiver operating characteristic (ROC) curves were determined for both the morphologic analysis and the morphokinetic model on culture day 3 and day 5. Using the ROC-determined cutoff that optimised both sensitivity and specificity, a binary outcome for each test was analysed using agreement statistics to determine if one method of embryo evaluation was superior to the other. Results: Morphological and morphokinetic grading were both predictive of embryo competence on days 3 and 5. However, on day 3, morphologic grading was superior to morphokinetic grading with area under the curve (AUC) of 0.66 (P < 0.001) and 0.58 (P = 0.009), respectively. Contrarily, on day 5, the morphokinetic model had a higher AUC of 0.65 (P = 0.03) compared to the morphologic grading, AUC 0.56 (P = 0.02). Conclusion: Traditional morphology was noted to be a better diagnostic tool (higher AUC) on culture day 3 while a morphokinetic model was superior on day 5.

Embryo Blastomere Exclusion Identified in a Time-Lapse Culture System Is Associated with Embryo Ploidy.

This study examined blastomere exclusion which is seen during embryo development and could represent imperfect cell division or a mechanism of aneuploidy correction. This was a retrospective cohort study which included embryos cultured in a time-lapse incubator undergoing preimplantation genetic testing for aneuploidy (PGT-A) with trophectoderm biopsy. Embryos were evaluated for blastomere exclusion early in development, late in development, both, or neither. Blastomere exclusion was compared to embryo ploidy. Embryos with no blastomere exclusion had an aneuploidy rate of 52.9%, while embryos displaying blastomere exclusion at any stage had an aneuploidy rate of 68.5% (p < .001). Early blastomere exclusion was not significantly associated with an increased aneuploidy risk (59.2% vs. 52.9% in no blastomere exclusions; p = 0.22). However, embryos with late blastomere exclusion were significantly more likely to be aneuploid, compared to embryos with no blastomere exclusions (77.5% vs. 52.9%; p < 0.001) as were embryos with both early + late blastomere exclusions (71.2% vs. 52.9%; p < 0.001). Upon restricting the analysis to aneuploid embryos, the presence of any blastomere exclusion was not significantly associated with complex aneuploidy, defined as 2 more affected chromosomes (43.9% vs. 38.7%; p = 0.28). However, the proportion with adverse embryo genetics significantly increased with the timing of blastomere exclusion (38.7%, 37%, 45.5%, and 50% for none, early, late, and early + late; p = 0.043). Late blastomere exclusion or a combination of both early + late blastomere exclusion was associated with an increased risk of aneuploid embryo genetics. Embryo selection using time-lapse culture systems should incorporate these findings when untested embryos are transferred.

Comparison of live birth rates after IVF-embryo transfer with and without preimplantation genetic testing for aneuploidies.

RESEARCH QUESTION: Does the use of preimplantation genetic testing for aneuploidies (PGT-A) result in higher live birth rates when compared with both fresh and frozen embryo transfers (FET) not utilizing PGT-A? DESIGN: Retrospective cohort study at a single tertiary centre using inverse probability of treatment weighting (IPTW) to adjust for differences in baseline characteristics between groups. RESULTS: A total of 107 FET using PGT-A from 74 patients, along with 321 fresh and 286 FET not using PGT-A from 381 patients met the inclusion criteria for this study. In the IPTW-adjusted analysis of transfer-level data, PGT-A transfers resulted in a significantly higher live birth rate when compared with both non-PGT-A fresh (49.5% versus 38.6%, P = 0.036) and FET (50.6% versus 35.8%, P = 0.016). When data were analysed per retrieval level, the live birth rate was similar and acceptably high with or without PGT-A (63.7% versus 52.3%, P = 0.09). CONCLUSION: When comparing PGT-A to non-PGT-A fresh and FET, PGT-A embryo transfers have a significantly higher live birth rate. However, this difference did not persist at a per-retrieval level. Further investigation is needed to understand in what scenarios PGT-A has clinical significance and whether differences in the number of available embryos for transfer negates the benefit of PGT-A.

Consult and procedure incidence outcomes following establishment of a fertility preservation program for children with cancer.

PURPOSE: Fertility is a quality of life outcome adversely affected by cancer therapy. Many childhood cancer patients, however, are not offered options to preserve their fertility. Providers acknowledge difficulty discussing impaired fertility to patients due to lack of knowledge of available options. Our objective was to review the impact of a pediatric multidisciplinary fertility preservation program on providers' fertility preservation counseling and discussion of options. METHODS: A retrospective medical chart review was conducted for pediatric cancer patients prior to and following program establishment. Fertility preservation discussions, consults, and incidence were noted. Following filtering and stratification, 198 and 237 patients were seen prior to and following program establishment, respectively. RESULTS: Following program establishment, provider-patient discussions of impaired fertility (p = 0.007), fertility preservation consults (p = 0.01), and incidence of fertility preservation procedures (p < 0.001) increased among patients. Furthermore, the number of patients who received fertility preservation consults after receiving gonadotoxic treatment decreased (p < 0.001). This trend was particularly noted in pre-pubertal and female patients, for whom fertility preservation options are limited without an established program. CONCLUSION: The establishment of a formal program greatly improved access to fertility preservation consults and procedures in children with cancer.

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

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

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

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

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

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

Composition of protein supplements used for human embryo culture.

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

Variability in protein quality used for embryo culture: embryotoxicity of the stabilizer octanoic acid.

OBJECTIVE: To screen human serum albumin (HSA) preparations for toxicity and investigate causes of variation. DESIGN: Experimental laboratory study. SETTING: University-based laboratory. ANIMAL(S): FVB and CF1 mice crossed to create embryos used in experiments. INTERVENTION(S): Mouse embryo assay performed with 5% or 15% HSA (100 mg/mL albumin) from three samples from three separate manufacturers (A, B, C). MAIN OUTCOME MEASURE(S): Blastocyst rates calculated at 96 hours of culture (experiments repeated in triplicate). RESULT(S): The HSA preparations were desalted to remove stabilizers added during HSA processing, then mass spectrometry was used to determine the relative variation in stabilizer concentrations; the effect of the stabilizer octanoic acid on embryo development was tested. At 5% HSA, all samples had blastocyst rates ≥ 70%; at 15% HSA, the blastocyst rates for samples B and C were <50%. Desalting did not affect sample B but did improve the blastocyst rates of sample C. Mass spectrometry revealed high levels of octanoic acid in sample C compared with sample A. The addition of octanoic acid to sample A produced toxicity similar to sample C. CONCLUSION(S): The stabilizer octanoic acid varies by lot and inhibits embryo development. Because octanoic acid is known to cause disruptions in mitochondrial bioenergetics, reduce intracellular pH, and induce oxidative damage in peripheral tissues, its use in embryo culture should be monitored and limited.

Mouse strain and quality control testing: improved sensitivity of the mouse embryo assay with embryos from outbred mice.

OBJECTIVE: To determine the relative sensitivities of embryos from different strains of mice to in vitro stress. DESIGN: Laboratory experiment with embryos from different mouse strains. SETTING: University hospital-based fertility clinic. ANIMAL(S): Mice. INTERVENTION(S): Fresh one-cell embryos from outbred (CF1), inbred (FVB), F1 hybrid (B6/CBA), and cryopreserved F2 hybrid embryos (bcl/B6 × B6/bcl) compared in a mouse embryo assay (MEA) using six doses of each of three in vitro stressors: cumene hydroperoxide in mineral oil, Triton X-100 (TX-100) in media, and hyperosmolality. MAIN OUTCOME MEASURE(S): Blastocyst rate at 96 hours. RESULT(S): All studies were conducted in triplicate; data were analyzed with chi-square analysis based on fitting a logistic regression model. Both cumene hydroperoxide and Triton X-100 affected blastocyst formation in the outbred strain at concentrations that were less than half of the concentration that affected the other strains. The total number of cells was affected by the treatments in all strains. CONCLUSION(S): Outbred CF1 embryos are genetically diverse and more sensitive to toxins than either inbred or hybrid mouse embryos. Outbred embryos provide an additional tool for effective quality-control testing.

Peroxides in mineral oil used for in vitro fertilization: defining limits of standard quality control assays.

PURPOSE: To determine the relative sensitivities of the 1 and 2-cell mouse embryo assays (MEA) and the human sperm motility assay (HSMA) for peroxides in mineral oil. The effect of peroxide on blastocyst cell number and apoptosis was also studied. METHODS: One and two-cell MEA and HSMA were performed using mineral oil containing cumene hydroperoxide (CH). RESULTS: The 1-cell MEA was twice as sensitive as the 2-cell MEA and 20-times more sensitive than the HSMA for CH in mineral oil. The sensitivity of the 1-cell MEA doubled when embryos were cultured individually versus group culture. CH decreased blastocyst cell number in a dose dependent manner. CONCLUSIONS: Individually cultured 1-cell embryos had the highest sensitivity for peroxides in mineral oil. Current quality control assays, including group cultured murine embryos and human sperm motility, have limited sensitivity for peroxides in mineral oil and may not detect levels of peroxides that cause sub-lethal cellular damage.

Parthenogenic activation of surplus in vitro-matured human oocytes: a tool for validation of oocyte cryopreservation.

Controlled-rate frozen and vitrified mature human oocytes were thawed or warmed and parthenogenically activated using ionomycin and 6-dimethylaminopurine to assess pronuclear development compared with fresh controls. This chemical activation model provides a useful tool for laboratories to assess proficiency before offering oocyte cryopreservation to their patients.

Preimplantation genetic screening in a case of recurrent trisomy 21 offspring.

OBJECTIVE: To describe a unique case of recurrent aneuploidy and the use of preimplantation genetic screening (PGS). DESIGN: Case report. SETTING: Midwest academic medical center. PATIENT(S): A 36-year-old woman with two trisomy 21 offspring. INTERVENTION(S): Preimplantation genetic screening. MAIN OUTCOME MEASURE(S): Karyotype of embryos, liveborn eukaryotic infant. RESULT(S): Preimplantation genetic screening was performed on three cryopreserved embryos, followed by a two-embryo transfer yielding a eukaryotic infant. CONCLUSION(S): Preimplantation genetic screening may prove to be useful as a diagnostic tool to help ensure a euploid pregnancy when termination is not a viable option for a couple.

Efficient isothermal amplification of the entire genome from single cells.

Preimplantation genetic diagnosis for single gene disorders is usually performed using polymerase chain reaction (PCR)-based methodologies modified for use in single cells. At present, single cell PCR tests require costly and time-consuming development and validation of highly sensitive amplification strategies to cover a growing number of mutations responsible for genetic disease. Whole-genome amplification (WGA) provides an opportunity to amplify the genome from a single blastomere to a level at which multiple tests can be performed on the same cell. Early WGA methods (primer extension preamplification and degenerate oligonucleotide-primed PCR) have not proved sufficiently accurate and reliable for routine clinical use. However, WGA using multiple displacement amplification (MDA) offers approx 5 million-fold amplification with fidelity, apparently sidestepping the limitation of a single cell, and is sufficient for use in most off-the-shelf molecular tests. This chapter describes an optimized MDA protocol for the preparation of genomic DNA from single fibroblasts.