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.

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.

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.

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.

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.

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.

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'.

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.

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.

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.

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.

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).

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).

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.

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.

Long-term storage of mouse spermatozoa after evaporative drying.

To determine if mouse spermatozoa could be preserved long-term without using liquid nitrogen, mouse spermatozoa in trehalose-EGTA solution were partially evaporatively dried under nitrogen gas (5 min at flow rate10 l/min) and stored for 1 week and 5 months at 4, -20, and -80 degrees C before intracytoplasmic sperm injection. Fertilization rates were neither different with spermatozoa stored at 4, -20, or -80 degrees C for 1 week or 1, 3, and 5 months respectively, nor blastocyst formation rates with spermatozoa stored for 1 week and 1 month. However, spermatozoa stored at 4 and -20 degrees C for 3 months resulted in fewer blastocysts (35.

Mouse sperm desiccated and stored in trehalose medium without freezing.

Mouse sperm with and without trehalose were desiccated under nitrogen gas and stored at 4 degrees C and 22 degrees C. After rehydration, sperm were injected into oocytes using intracytoplasmic sperm injection and embryonic development was followed. Sperm were dried for 5.

Discrepancies between the effects of glutamine in cultures of preimplantation mouse embryos.

A review of the literature shows divergent differences between laboratories of the effects of glutamine in mouse preimplantation embryo culture media. One laboratory reported several cases of exencephaly, which was attributed to ammonia produced by the breakdown of glutamine. Two other laboratories have found no such effects.

Enhanced effect of glycyl-L-glutamine on mouse preimplantation embryos in vitro.

A comparison of the effects of replacing L -glutamine with either glycyl-L-glutamine or alanyl-L-glutamine in a KSOM-type medium on the development of mouse preimplantation embryos in vitro has been made. Alanyl-L-glutamine has no significant effect on the rates of blastocyst formation, onset or completion of hatching, and on the numbers of inner cell mass and trophectoderm cells that develop. Glycyl-L-glutamine has no effect on the rate of blastocyst formation; it stimulates slightly the onset of hatching, but significantly increases the numbers of inner cell mass and trophectoderm cells that develop.

Chemically defined media and the culture of mammalian preimplantation embryos: historical perspective and current issues.

Considerable advances in media development for the culture of preimplantation mammalian embryos have been made since mouse embryos were first cultured and successfully transferred to foster mothers. The purpose of this review is to detail the history of the development of chemically defined media for the culture of preimplantation embryos. Two approaches have been used to determine the composition of chemically defined media: the 'back-to-nature' approach and 'let the embryo choose' or empirical optimization approach.

Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotection.

The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years.