D.L. Walker

Vitrification versus programmable rate freezing of late stage murine embryos: a randomized comparison prior to application in clinical IVF

A prospective randomized trial was performed to compare post-thaw development of murine blastocysts following programmable rate freezing and two methods of vitrification. Frozen 2-cell murine embryos (n = 429) thawed and cultured for 48 h, were randomly allocated by stage of development into four groups: control (not refrozen), programmable rate freezing (PR) in 0.25 ml straws, vitrification in flexible micropipettes by immersion in super-cooled (VSC) liquid nitrogen (LN2), and vitrification in flexible micropipettes by immersion in LN2 (VLN). Survival, developmental stage progression, presence or absence of an inner cell mass (ICM), and cell counts were recorded 24 h post-thaw. All measured outcomes were different between embryos from the control group and all freezing methods. Controlled-rate freezing resulted in the lowest total cell counts and fewest embryos with a distinct ICM. A higher percentage of embryos survived 24 h post-thaw, progressed to more advanced developmental stages and had higher total cell counts after VLN compared with PR. Moreover, fewer embryos, frozen by either PR or VSC, contained a detectable ICM compared with VLN. These data demonstrate that vitrification may be a better method for freezing murine blastocysts than PR, and may prove to be a superior method for freezing human blastocysts.

Washing mineral oil reduces contaminants and embryotoxicity

OBJECTIVE To determine if washing improves the quality of mineral oil used for embryo culture. DESIGN A 2 × 3 factorial experimental study. SETTING University hospital-based infertility center. ANIMAL(S) Mice. INTERVENTION(S) The chemical nature of contaminants present in two lots of mineral oil was determined. Effect of washing on toxicity and amount of toxin present in media was determined. MAIN OUTCOME MEASURE(S) The effect of washing was determined by a quality control bioassay or by directly determining the level of contaminant in oil-conditioned culture media. RESULT(S) Water, culture media, and media plus albumin were equally effective in reducing toxicity and concentration of toxin. Temperature did not affect washing results. Peroxide, aldehydes, and alkenals were present in one lot of oil, and Triton X-100 was identified in the other lot. Washed oil containing peroxide passed the one-cell mouse embryo bioassay, and washing reduced the amount of Triton X-100 by 25%. CONCLUSION(S) Mineral oil is the least defined component used for in vitro fertilization and embryo culture; therefore, it is important to determine if washing oil is beneficial. This study provides clear evidence that washing reduces toxicity of mineral oil.

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.

Potential of inner cell mass outgrowth and amino acid turnover as markers of quality in the in vitro fertilization laboratory

OBJECTIVE To compare sensitivity of inner cell mass (ICM) outgrowth assay and analysis of culture media amino acid turnover with the sensitivity of the human sperm motility assay (HSMA) and murine embryo assay (MEA) for detection of formaldehyde toxicity. DESIGN Prospective in vitro study. SETTING University hospital-based infertility center. ANIMAL(S) Murine embryos. INTERVENTION(S) The HSMA, MEA, and ICM outgrowth assays were performed with media containing 0-64-μM concentrations of formaldehyde. These assays were compared with dynamics of amino acid turnover in culture media. MAIN OUTCOME MEASURE(S) The lowest concentration of formaldehyde in culture media detected by each quality control assay. RESULT(S) Sperm forward progression, but not motility, detected formaldehyde at a concentration of 32 μM. Sperm motility index identified formaldehyde toxicity at 64 μM, whereas blastocyst rates in the MEA were affected at 32 μM formaldehyde. Evaluation of ICM using outgrowth and grade detected 16 μM formaldehyde. Leucine turnover in culture media detected 64 μM formaldehyde in the amino acid assay. CONCLUSION(S) Inner cell mass outgrowth is a more sensitive bioassay than MEA and HSMA for the detection of formaldehyde in culture media. Amino acid metabolism may also provide a sensitive quality control measure for detection of formaldehyde.

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.

Equivalent blastocyst rates after freezing murine embryos in Cryo Bio System high security or standard instruments-medicine-veterinarian straws.

OBJECTIVE To validate the Cryo Bio System (CBS) straw in our current cryopreservation system before using it in clinical practice. DESIGN A prospective comparison of blastocyst development rates in 278 murine embryos after refreezing and thawing at the two-cell stage against the standard Instruments-Medicine-Veterinarian (IMV) straw used in our cryopreservation program. SETTING Private IVF laboratory. PATIENT(S) No human subjects or material was used in this study. INTERVENTION(S) Frozen two-cell murine embryos were thawed and randomized into three treatments [1] refreezing in the CBS straws, [2] refreezing in IMV 0.25-mL straws, and [3] control embryos remaining in culture without refreezing. Embryos were refrozen using identical cryoprotectants and identical programmed controlled-rate freezers. After cryopreservation, straws were held in liquid nitrogen for a brief period before thawing and continued culture. MAIN OUTCOME MEASURE(S) Postthaw murine blastocyst development rate. RESULT(S) When the manufacturers filling and loading protocol was used for the CBS straw there was no significant difference in the blastocyst development rate between CBS (75.0%) and IMV (76.4%) straws. CONCLUSION(S) The CBS straw may be a viable and potentially safer alternative for cryopreservation of human embryos, particularly for patients with known infections.

The effect of repetition rate on air-conducted ocular vestibular evoked myogenic potentials (oVEMPs)

PURPOSE Ocular vestibular evoked myogenic potentials (oVEMPs) are used to describe utricular/superior vestibular nerve function; however, optimal recording parameters have not been fully established. This study investigated the effect of repetition rate on air-conducted oVEMPs. METHOD Ten healthy adults were evaluated using 500-Hz tone bursts (4-ms duration, Blackman gating, 122 dB pSPL). Four repetition rates were used (1.6, 4.8, 8.3, and 26.6 Hz) and resulting oVEMP response presence, amplitude, amplitude asymmetry, and n1/p1 latency were assessed. RESULTS Response presence was significantly reduced for 26.6 Hz using monaural stimulation and for 8.3 Hz and 26.6 Hz for binaural stimulation. For monaural stimulation using 1.6, 4.8, and 8.3 Hz, no significant differences were noted for amplitude or latency. Responses obtained using binaural stimulation demonstrated a significant effect of rate on amplitude, with 8.3 Hz producing significantly reduced amplitude. Binaural amplitudes were significantly larger than monaural contralateral responses but with reduced response presence. No significant differences were noted for latency or amplitude asymmetry. CONCLUSION Using repetition rates of approximately 5 Hz or less may produce more consistent oVEMP response presence with minimal effects on amplitude for monaural or binaural recordings.

Cryosystem assessment by glucose uptake of murine blastocysts

Glucose uptake was used as a measure of metabolic activity and implantation potential to compare vitrification and slow freezing in a prospective randomized trial using murine blastocysts. Frozen 2-cell embryos (n = 132) thawed and cultured for 48 h to the blastocyst stage were randomly divided into four groups: (i) control - not refrozen; (ii) slow freezing using a programmed rate (PR); (iii) vitrification by super-cooled (VSC) liquid nitrogen; and (iv) vitrification in liquid nitrogen (VLN). Upon re-thawing, embryos were cultured individually for 24 h to determine glucose uptake non-invasively. Morphological assessments included total cell counts and inner cell mass (ICM) detection following immunosurgery. Mean glucose uptake was lower for each treatment (PR and VSC, 4.3 pmol/embryo per h; VLN, 4.9 pmol/embryo per h) versus controls (6.8 pmol/embryo per h). PR and VSC embryos had fewer cells (57.4 +/- 24.2 and 64.1 +/- 31.5) versus controls (85.7 +/- 26.2), and fewer embryos containing a detectable ICM (42.9 and 61.8%) compared with controls (88.2%). The only difference between control and VLN embryos was absolute glucose uptake, although in both treatments glucose uptake was increased from embryos with an ICM compared with those without. Glucose uptake appears to be a sensitive, non-invasive method to validate cryopreservation protocols.