J.R. Fredrickson

Composition of protein supplements used for human embryo culture

PurposeTo 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.MethodsAmino 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.ResultsRecombinant 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.ConclusionsThe 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.

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

PurposeTo 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.MethodsOne and two-cell MEA and HSMA were performed using mineral oil containing cumene hydroperoxide (CH).ResultsThe 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.ConclusionsIndividually 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.

Advances in quality control: mouse embryo morphokinetics are sensitive markers of in vitro stress

STUDY QUESTION Can time-lapse analysis of cell division timings [morphokinetics (MK)] in mouse embryos detect toxins at concentrations that do not affect blastocyst formation? SUMMARY ANSWER An MK algorithm enhances assay sensitivity while providing results 24–48 h sooner than the traditional mouse embryo assay (MEA). WHAT IS KNOWN ALREADY Current quality control testing methodology is sensitive but further improvements are needed to assure optimal culture conditions. MKs of embryo development may detect small variations in culture conditions. STUDY DESIGN Cross sectional—control versus treatment. Mouse embryo development kinetics of 466 embryos were analyzed according to exposure to various concentrations of toxins and toxic mineral oil. MATERIALS, SETTING, METHODS Cryopreserved 1-cell embryos from F1 hybrid mice were cultured with cumene hydroperoxide (CH) (0, 2, 4, 6 and 8 µM) and Triton X-100 (TX-100; 0, 0.0008, 0.0012, 0.0016 and 0.002%). Using the Embryoscope, time-lapse images were obtained every 20 min for 120 h in seven focal planes. End-points were timing and pattern of cell division and embryo development. The blastocyst rate (BR) was defined as the percentage of embryos that developed to the expanded blastocyst stage within 96 h. MAIN RESULTS AND THE ROLE OF CHANCE BR was not affected for embryos cultured in the three lowest concentrations of CH and the four lowest concentrations of TX-100. In contrast, a unique MK model detected all concentrations tested (P < 0.05). The MK model identified toxicity in two lots of toxic mineral oil that did not affect BR (P < 0.05). LIMITATIONS, REASONS FOR CAUTION A limited number of toxins were used so that the results may not apply to all potential embryo toxins. A larger sample size may also demonstrate other statistically significant developmental kinetic parameters. WIDER IMPLICATIONS OF THE FINDINGS MKs in mouse embryos are a sensitive and efficient method for quality control testing of in vitro culture conditions. BR, the end-point of traditional quality control assays, did not detect sublethal concentrations of toxins in the culture milieu in our study. This study demonstrates that temporal variation at key developmental stages reflects the quality of the culture environment. An MEA that incorporates MK will provide enhanced sensitivity and faster turn-around times. STUDY FUNDING/COMPETING INTEREST(S) The study was supported by Mayo Clinic Department of Obstetrics and Gynecology Small Grant Program. The authors have no competing interests to declare.

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.

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

BackgroundHP1γ, 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.ResultsWe 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.ConclusionsThese 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.

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.

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.

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.