Low cryoprotectant concentration rapid vitrification of mouse oocytes and embryos.

Vitrification of mammalian oocytes and embryos is typically a two-step procedure involving two solutions of increasing concentrations of cryoprotectants. In the present study, we report a simple vitrification protocol that uses low cryoprotectant concentration and a single medium (LCSM). This medium, along with the traditional high concentration two media (HCTM) protocol, was used to vitrify mouse oocytes, zygotes, and blastocysts using silica capillary, cryotop, cryolock, and 0.25 ml straws. Survival rates, two-cell rates, and blastocyst formation rates were compared for oocytes and zygotes vitrified using both protocols. Results show that the LCSM protocol was as good as or better than the traditional HCTM protocol for vitrifying mouse MII oocytes and zygotes using silica capillary, cryotop, and cryolock. On the other hand, for blastocysts, only silica capillary using LCSM had comparable results with the traditional HCTM protocol while cryolock and cryotop had significantly lower percentages of re-expanded and hatched blastocysts. Collapsing blastocysts prior to vitrification or longer duration for better cryoprotectant distribution in multicellular embryos may improve the outcome. In conclusion, the LCSM protocol, with one medium of much lower cryoprotectant concentrations and shorter equilibration time, reduces exposure to cryoprotectant toxicity while improves efficiency, consistency and reliability for mammalian oocyte and embryo preservation.

Cryopreservation of human spermatozoa with minimal non-permeable cryoprotectant.

Cryopreservation of human spermatozoa is a commonly used technique in assisted reproduction, however freezing low concentrations of sperm while maintaining adequate post-thaw motility remains a challenge. In an effort to optimize post-thaw motility yields, low volumes of human sperm were frozen in polyimide-coated fused silica micro-capillaries using 0.065 M, 0.125 M, 0.25 M, or 0.5 M trehalose as the only cryoprotectant. Micro-capillaries were either initially incubated in liquid nitrogen vapor before plunging into liquid nitrogen, or directly plunged into liquid nitrogen. Post thaw sperm counts and motility were estimated. Spermatozoa that were initially incubated in liquid nitrogen vapor had greater post thaw motility than those plunged immediately into liquid nitrogen independent of trehalose concentration. The protective effect of 0.125 M d-glucose, 3-O-methyl-d-glucopyranose, trehalose, sucrose, raffinose, or stachyose were evaluated individually. Trehalose and sucrose were the most effective cryoprotectants, recovering 69.0% and 68.9% of initial sperm motility, respectively.

Live pups from evaporatively dried mouse sperm stored at ambient temperature for up to 2 years.

The purpose of this study is to develop a mouse sperm preservation method based on evaporative drying. Mouse sperm were evaporatively dried and stored at 4°C and ambient temperature for 3 months to 2 years. Upon rehydration, a single sperm was injected into a mature oocyte to develop into a blastocyst after culture or a live birth after embryo transfer to a recipient female. For the samples stored at 4°C for 3, 6, 12, 18, and 24 months, the blastocyst formation rate was 61.5%, 49.1%, 31.5%, 32.2%, and 41.4%, respectively. The blastocyst rate for those stored at ambient temperature (∼22°C) for 3, 6, 12, and 18 months was 57.8%, 36.2%, 33.6%, and 34.4%, respectively. Fifteen, eight and three live pups were produced from sperm stored at room temperature for 12, 18, and 24 months, respectively. This is the first report of live offspring produced from dried mouse sperm stored at ambient temperature for up to 2 years. Based on these results, we suggest that evaporative drying is a potentially useful method for the routine preservation of mouse sperm.

Localization of parathyroid hormone-related protein in the preimplantation mouse embryo is associated with events of blastocyst hatching.

PURPOSE: To determine the pattern of expression of parathyroid hormone-related protein (PTHrP) and its receptor, parathyroid hormone receptor 1 (PTHR1), in mouse embryos in different stages of preimplantation development. METHODS: Embryos were cultured from the pronuclear zygote stage and harvested as 2-cell, 4-cell and 8-cell embryos, morulae and blastocysts. RT-PCR was carried out on mRNAs of these and of trophoblast outgrowths for detection of PTHrP and PTHR1. Whole mounted embryos intact or stripped of zonae pellucidae were immunofluorescently stained for PTHrP and PTH receptor and observed with confocal microscopy. RESULTS: PTHrP mRNA was present in the pronuclear zygote, not present in 2-cell, 4-cell and uncompacted 8-cell embryos, present in the 8-cell compacting embryo, and not detected in 16-cell morulae or blastocysts. The mRNA was present in trophoblasts growing on fibronectin beds. mRNA for PTHR1 was detected in the pronuclear zygote, then undetected until the compacted 8-cell stage and thereafter. PTH receptor protein was observed in 2-cell embryos, morulae and in the inner cell mass and trophectoderm of blastocysts. PTHrP was observed dispersed in the cytoplasm of 2-cell, 4-cell and uncompacted 8-cell embryos, and in distinct foci near the nuclei of morulae. In blastocysts, PTHrP appeared on the apical surface of only trophoblast cells which had extruded from the zona pellucida. Fully hatched blastocysts expressed the protein on the apical side of all trophoblasts. When morulae were prematurely stripped of their zonae, PTHrP was observed on the embryos' outer surface. CONCLUSIONS: PTHrP protein is expressed throughout early embryo development, and its receptor PTHR1 is expressed from the morula stage. Embryo hatching is associated with translocation of PTHrP to the apical plasma membrane of trophoblasts. PTHrP may thus have autocrine effects on the developing blastocyst.

Human preimplantation embryo development in vitro: a morphological assessment of sibling zygotes cultured in a single medium or in sequential media.

A comparison was made of the development of human zygotes in either a one-step (Global® medium) or two-step culture system (Quinn's Advantage®). A total of 257 normally fertilized 2PN zygotes from 28 patients were used in the study. The study was broken down into two parts: the first concerned the development of embryos from Days 1 to 3 in Global® medium and Quinn's Advantage® cleavage medium; the second consisted of a comparison of the development of embryos from Day 3 to 5/6 in Global® medium and Quinn's Advantage® blastocyst medium. There were no significant differences between the two culture media with respect to embryo quality throughout the preimplantation phase of human embryo development as determined by the extent and variability of the cell counts, fragmentation, and nucleation. A difference was noted in the blastomere symmetry of Day 2 embryos in the two media, but was no longer apparent on examination of Day 3 embryos. No differences were noted in the rates of blastocyst development, inner cell mass (ICM), and trophectoderm (TE) scores in the two culture media. Finally, no significant differences were noted with either the proportion of blastocysts chosen for transfer or cryopreservation (vitrification). The findings support the view that two-step sequential media protocols are sufficient but not necessary to support the complete in vitro development of human preimplantation embryos.

3D multi-isotope imaging mass spectrometry reveals penetration of 18O-trehalose in mouse sperm nucleus.

The prevalence of genetically engineered mice in medical research has led to ever increasing storage costs. Trehalose has a significant beneficial effect in preserving the developmental potential of mouse sperm following partial desiccation and storage at temperatures above freezing. Using multi-isotope imaging mass spectrometry, we are able to image and measure trehalose in individual spermatozoa. We provide the first evidence that trehalose penetrates the nucleus of a mammalian cell, permitting tolerance to desiccation. These results have broad implications for long-term storage of mammalian cells.

IVF and embryo transfer: historical origin and development.

IVF and embryo transfer for the treatment of human infertility has now resulted in the birth of over 4 million babies. The technique did not arise as a quantum event but was built on the efforts of many earlier workers in the fields of reproductive endocrinology and development. One should remember the famous saying of Isaac Newton: 'If I have seen further than most, it is because I have stood on the shoulder's of giants'. Ethical and moral issues have always arisen when investigators study early mammalian development, particularly human development. This paper documents these earlier studies and also draws attention to the ethical and moral arguments that inevitably arose.

Mutant mice derived by ICSI of evaporatively dried spermatozoa exhibit expected phenotype.

Apolipoprotein E (Apoe)-deficient knockout mice were used to test the hypothesis that mutant mice preserved as evaporatively dried (ED) spermatozoa, stored at -80 °C for 6 months, and then recovered by ICSI will exhibit the same phenotype as before preservation. The birth rate of mice recovered by ICSI of evaporatively dried spermatozoa was lower than that of fresh spermatozoa (17.5 vs 38.0%). Progeny of mice preserved using evaporatively dried spermatozoa were reproductively sound. From these, the second generation of mice produced by natural mating showed lesions typical of APOE deficiency, including severe hypercholesterolemia, hypertriglyceridemia, markedly increased plasma low-density lipoprotein level, and extensive and severe atherosclerotic lesions in the aorta. We conclude that the expected phenotype caused by an induced genetic mutation can be faithfully recapitulated and sustained in subsequent generations of mice preserved and stored as ED spermatozoa and recovered using ICSI. Because it is simpler, faster, and cheaper than conventional (cryopreservation) and nonconventional (freeze-drying) preservation procedures, evaporative drying is a viable, cost-effective, and efficient method for preserving and storing valuable mutant mouse strains.

Preservation of mouse sperm by convective drying and storing in 3-O-methyl-D-glucose.

With the fast advancement in the genetics and bio-medical fields, the vast number of valuable transgenic and rare genetic mouse models need to be preserved. Preservation of mouse sperm by convective drying and subsequent storing at above freezing temperatures could dramatically reduce the cost and facilitate shipping. Mouse sperm were convectively dried under nitrogen gas in the Na-EGTA solution containing 100 mmol/L 3-O-methyl-D-glucose and stored in LiCl sorption jars (Relative Humidity, RH, 12%) at 4°C and 22°C for up to one year. The functionality of these sperm samples after storage was tested by intracytoplasmic injection into mouse oocytes. The percentages of blastocysts produced from sperm stored at 4°C for 1, 2, 3, 6, and 12 months were 62.6%, 53.4%, 39.6%, 33.3%, and 30.4%, respectively, while those stored at 22°C for 1, 2, and 3 months were 28.8%, 26.6%, and 12.2%, respectively. Transfer of 38 two- to four-cell embryos from sperm stored at 4°C for 1 year produced two live pups while 59 two- to four-cell embryos from sperm stored at 22°C for 3 months also produced two live pups. Although all the pups looked healthy at 3 weeks of age, normality of offspring produced using convectively dried sperm needs further investigation. The percentages of blastocyst from sperm stored in the higher relative humidity conditions of NaBr and MgCl(2) jars and driest condition of P(2)O(5) jars at 4°C and 22°C were all lower. A simple method of mouse sperm preservation is demonstrated. Three-O-methyl-D-glucose, a metabolically inactive derivative of glucose, offers significant protection for dried mouse sperm at above freezing temperatures without the need for poration of cell membrane.

National collection of embryo morphology data into Society for Assisted Reproductive Technology Clinic Outcomes Reporting System: associations among day 3 cell number, fragmentation and blastomere asymmetry, and live birth rate.

OBJECTIVE: To evaluate the validity of collecting day 3 embryo morphology variables into the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System (SART CORS). DESIGN: Retrospective. SETTING: National database-SART CORS. PATIENT(S): Fresh autologous assisted reproductive technology (ART) cycles from 2006-2007 in which embryos were transferred singly (n=1,020) or in pairs (n=6,508) and embryo morphology was collected. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Relationship between live birth, maternal age, and morphology of transferred day 3 embryos as defined by cell number, fragmentation, and blastomere symmetry. Logistic multiple regressions and receiver operating characteristic curve analyses were applied to determine specificity and sensitivity for correctly classifying embryos as either failures or successes. RESULT(S): Live birth rate was positively associated with increasing cell number up to eight cells (<6 cells: 2.9%; 6 cells: 9.6%; 7 cells: 15.5%; 8 cells: 24.3%; and >8 cells: 16.2%), but was negatively associated with maternal age, increasing fragmentation, and asymmetry scores. An area under the receiver operating curve of 0.753 (95% confidence interval 0.740-0.766) was derived, with a sensitivity of 45.0%, a specificity of 83.2%, and 76.4% of embryos being correctly classified with a cutoff probability of 0.3. CONCLUSION(S): This analysis provides support for the validity of collecting morphology fields for day 3 embryos into SART CORS. Standardization of morphology collections will assist in controlling for embryo quality in future database analyses.

Ultra-rapid vitrification of mouse oocytes in low cryoprotectant concentrations.

The ideal cryopreservation protocol would combine the benefits of slow freezing with the benefits of vitrification. This report describes a method for the ultra-rapid vitrification of oocytes using slush nitrogen in quartz capillaries. The approach minimizes the thermal mass of the vitrification vessel by using open microcapillaries made of highly conductive quartz and achieves cooling rates of 250,000 degrees C/min. The process of vitrification can be optimized by maximizing the rate at which the sample is cooled, which allows for the use of lower cryoprotectant concentrations. Mouse oocytes can be successfully vitrified using 1.5 mol/l 1,2-propanediol and 0.5 mol/l trehalose and achieve survival rates of 90.0%(36/40). Fertilization and blastocyst formation rates of vitrified-warmed and fresh oocytes were not significantly different. A total of 120 blastocysts from each of the vitrified-warmed and fresh oocytes were transferred to surrogate mothers and 23 and 27 offspring were born respectively. All offspring in both groups were healthy, grew and bred normally and gave rise to a second generation of pups. Thus, an ultra-rapid vitrification technique has been developed for mouse oocytes that uses low concentrations of cryoprotectants and slush nitrogen in quartz capillaries, which combines the benefits of slow freezing and vitrification.

Evaporative drying of mouse spermatozoa.

Mouse spermatozoa can be evaporatively dried under a stream of nitrogen gas at ambient temperature and stored for 5 months at -80 degrees C. The method as it stands at present is available for practical use, although its value would be greatly increased if the spermatozoa could be stored at ambient temperature. The need for further research is stressed.

Is there an advantage in scoring early embryos on more than one day?

BACKGROUND: This study was undertaken to determine what characteristics should be recorded on which days to build a predictive model for selection of Day 3 embryos. METHODS: Embryos failing to form a clinical sac or that formed a viable fetus (to > or =12 weeks), and transferred singly (n = 269) or in pairs (n = 1326) were scored for early cleavage and pronuclear status on Day 1, and cell number, fragmentation, and symmetry on Days 2 and 3, with number of nuclei per blastomere also recorded on Day 2. Seven candidate models were identified using a priori clinical knowledge and univariate analyses. Each model was fit on a training-set and evaluated on a test-set with resampling, with discrimination assessed using the area under the ROC curve (AUC) and calibration assessed using the Hosmer-Lemeshow statistics. RESULTS: Models built using Day 1, 2 or 3 scores independently on the 30 resampled data sets showed that Day 1 evaluations provided the poorest predictive value (median AUC = 0.683 versus 0.729 and 0.725, for Day 2 and 3). Combining information from Day 1, 2 and 3 marginally improved discrimination (median AUC = 0.737). Using the final Day 3 model fitted on the whole dataset, the median AUC was 0.732 (95% CI, 0.700-0.764), and 68.6% of embryos would be correctly classified with a cutoff probability equal to 0.3. CONCLUSIONS: Day 2 or Day 3 evaluations alone are sufficient for morphological selection of cleavage stage embryos. The derived regression coefficients can be used prospectively in an algorithm to rank embryos for selection.

Further optimization of mouse spermatozoa evaporative drying techniques.

It has been shown in the past that mouse spermatozoa could be dried under a stream of nitrogen gas at ambient temperature and stored at 4 degrees C or 22 degrees C for up to 3 months and was capable of generating live-born offspring. In previous desiccation work, dried sperm were stored in a vacuum-sealed plastic bag placed in a vacuum-packed Mylar bag. However, dried specimens stored in this way often lost moisture, particularly in samples stored at higher temperatures (22 degrees C) compared to lower temperatures (4 degrees C). The present report describes a method which minimizes this water loss from the dried sperm samples. Its use is described in a preliminary study on the effect of supplementing the trehalose with glycerol. The results have demonstrated that mouse sperm can be stored at 4 degrees C over saturated NaBr without the uptake of water which occurs when they are stored in Mylar packages. In addition, we were able to get some survival of sperm (9-15%) at room temperature storage after 3 months. The addition of glycerol to trehalose had little effect on the survival of dried mouse sperm stored over NaBr for 1 and 3 months.

Choosing a culture medium: making informed choices.

OBJECTIVE: To analyze critically the reasons justifying the choice of two-step protocols requiring two media for the culture of human preimplantation embryos from the zygote to the blastocyst. DESIGN: Literature review. RESULT(S): Two types of protocol are used for the culture of human preimplantation embryos from the zygote to the blastocyst, using either one medium (one-step protocol) or two media of different composition (two-step protocol). Two-step protocols are the most widely used, largely because all but one of the commercially available protocols are of this type. The reasons for the adoption of two-step protocols are described and critically analyzed. They are based on considerations of the functions of glucose, ethylenediaminetetraacetic acid (EDTA), glutamine, and amino acids that are included in the media. A reappraisal of the reasons for selecting two-step protocols is important because recent animal experiments and clinical observations have raised doubts as to whether the more complex, two-step protocols have any advantage over one-step protocols. The analyses show that all of conclusions reached should be considered equivocal. CONCLUSION(S): Clinical embryologists should evaluate the justification for selecting two-step protocols for the culture of human preimplantation embryos from the zygote to the blastocyst.

Rotationally oscillating drill (Ros-Drill) for mouse ICSI without using mercury.

Intracytoplasmic sperm injection (ICSI) is an important assisted reproductive technology (ART). Due to deployment difficulties and low efficiency of the earlier (conventional) version of ICSI, especially in the mouse, a piezo-assisted ICSI technique had evolved as a popular ART methodology in recent years. An important and remaining problem with this technique, however, is that it requires small amounts of mercury to stabilize the pipette tip when piezoelectric force pulses are applied. To eliminate this problem we developed and tested a completely different and mercury-free technology, called the "Ros-Drill" (rotationally oscillating drill). The technique uses microprocessor-controlled rotational oscillations on a spiked micropipette without mercury or piezo. Preliminary experimental results show that this new microinjection technology gives high survival rate (>70% of the injected oocytes) and fertilization rate (>80% of the survived oocytes), and blastocyst formation rates in early trials (approximately 50% of the survived oocytes). Blastocysts created by Ros-Drill ICSI were transferred into the uteruses of pseudopregnant surrogate mothers and healthy pups were born and weaned. The Ros-Drill ICSI technique is automated and therefore; it requires a very short preliminary training for the specialists, as evidenced in many successful biological trials. These advantages of Ros-Drill ICSI over conventional and piezo-assisted ICSI are clearly demonstrated and it appears to have resolved an important problem in reproductive biology.

Phenotypic analysis of C57BL/6J and FVB/NJ mice generated using evaporatively dried spermatozoa.

Combination of evaporative drying and frozen storage at -80 degrees C has been used successfully to preserve hybrid B6D2F1 mouse spermatozoa. To determine whether this method can be applied equally well to inbred mice, spermatozoa of C57BL/6J and FVB/ NJ mice were evaporatively dried and stored for 1 mo at -80 degrees C before being used for intracytoplasmic sperm injection (ICSI) to produce live offspring. After weaning, 1 male and 1 female mouse from each litter were randomly selected at 8 wk of age for natural mating to produce live offspring. Results showed that spermatozoa from both inbred strains that had been evaporatively dried and subsequently stored at -80 degrees C could be used successfully to derive live, healthy, and reproductively sound offspring by ICSI. No significant differences were found in embryo transfer rate (number of pups born/number of embryos transferred), litter size, weaning rate, body weight, number of pathologic lesions, and amount of contamination by pathogens of mice produced by ICSI using evaporatively dried spermatozoa compared with mice produced by natural mating or by ICSI using fresh (that is, nonpreserved) spermatozoa. Progeny produced by mating mice generated from ICSI using evaporatively dried spermatozoa were normal. Therefore, spermatozoa from inbred mouse strains C57BL/6J and FVB/NJ can be preserved successfully after evaporative drying and frozen storage at -80 degrees C.