Search PubMed⌕ Search

Biomedical subjects

David K Gardner

Publications and source records attributed to David K Gardner.

36 records · Page 2Linked to original sources
PubMed2005

Ex vivo early embryo development and effects on gene expression and imprinting.

The environment to which the mammalian embryo is exposed during the preimplantation period of development has a profound effect on the physiology and viability of the conceptus. It has been demonstrated that conditions that alter gene expression, and in some instances the imprinting status of specific genes, have all previously been shown to adversely affect cell physiology. Thus, questions are raised regarding the aetiology of abnormal gene expression and altered imprinting patterns, and whether problems can be averted by using more physiological culture conditions. It is also of note that the sensitivity of the embryo to its surroundings decreases as development proceeds. Post compaction, environmental conditions have a lesser effect on gene function. This, therefore, has implications regarding the conditions used for IVF and the culture of the cleavage stage embryo. The developmental competence of the oocyte also impacts gene expression in the embryo, and therefore superovulation has been implicated in abnormal methylation and imprinting in the resultant embryo. Furthermore, the genetics and dietary status of the mother have a profound impact on embryo development and gene expression. The significance of specific animal models for human assisted reproductive technologies (ART) is questioned, given that most cattle data have been obtained from in vitro-matured oocytes and that genes imprinted in domestic and laboratory animals are not necessarily imprinted in the human. Patients treated with ART have fertility problems, which in turn may predispose their gametes or embryos to greater sensitivities to the process of ART. Whether this is from the drugs involved in the ovulation induction or from the IVF, intracytoplasmic sperm injection or culture procedures themselves remains to be determined. Alternatively, it may be that epigenetic alterations are associated with infertility and symptoms are subsequently revealed through ART. Whatever the aetiology, continued long-term monitoring of the children conceived through ART is warranted.

Animals↗
PubMed2005

Heterogeneity and oxidation status of commercial human albumin preparations in clinical use.

OBJECTIVE: Human serum albumin is indicated for the treatment of shock, acute restoration of blood volume, and in hypoalbuminemia. Conflicting reports are found in the literature for the clinical safety and efficacy of human serum albumin administration to critically ill patients. We sought to analyze various commercially available albumin preparations for common, posttranslational modifications. DESIGN: Analysis of six commercially available albumin preparations for clinical use. SETTING: Trauma research laboratory. SUBJECTS: Commercially available human serum albumin preparations and healthy volunteers. INTERVENTIONS: Six commercially available human serum albumin preparations were analyzed by high-performance liquid chromatography. The presence of various posttranslational modifications was identified by positive electrospray ionization, time-of-flight mass spectrometry. Three different lots from three preparations were also analyzed to assess variability within lots from the same manufacturer. Also, for the purpose of comparison, human serum albumin was analyzed in the plasma of healthy volunteers. MEASUREMENTS AND MAIN RESULTS: The six human serum albumin preparations analyzed contained a high percentage (57.2 +/- 3.3%) of bound Cys34 (oxidation of cysteine in position 34 on the human serum albumin molecule) in comparison to the plasma human serum albumin from healthy volunteers (22.9 +/- 4.8%). Lot-to-lot variability in native human serum albumin ranged between 4.8% and 11.2% in three separate commercial albumins. Significant differences existed among the various commercial preparations in other posttranslational modifications of albumin. CONCLUSIONS: Human serum albumin species with a bound Cys34 account for a large percentage of the composition of human serum albumin preparations used for the treatment of critically ill patients. Also, the variability within lots from the same manufacturer is significant. Consequences of the administration of these oxidized forms of human serum albumin to critically ill patients warrants further investigation.

Chromatography, High Pressure Liquid↗
PubMed2005

Noninvasive methods to assess embryo quality.

PURPOSE OF REVIEW: The present review examines recent publications concerned with the issue of the number of embryos to replace in an in-vitro fertilization cycle and in doing so concentrates on the proposed selection methods that may allow us to transfer one or two embryos. RECENT FINDINGS: A number of techniques that will allow us to ascertain the health and viability of the embryo are discussed. These range from methods to assess embryo morphology to those that require more complex forms of technology. Logically the use of the simple methodology is more abundant. However, there is accumulating evidence that some of the more complex assessment techniques may be ready for a more general use. SUMMARY: In performing embryo selection we need to develop processes that will be extremely critical, allowing us to limit the number of embryos replaced without detriment to pregnancy rates.

Cell Nucleus↗
PubMed2005

Metabolic markers of developmental competence for in vitro-matured mouse oocytes.

In vitro maturation of oocytes has enormous potential in assisted reproductive technology, but its use has been limited due to insufficient knowledge of oocyte physiology during this dynamic period and lack of an adequate maturation system. The aim of this study was to characterize the metabolic profiles of three groups of oocytes throughout maturation: cumulus-oocyte complexes (COCs), denuded oocytes, and denuded oocytes co-cultured with cumulus cells. Mouse oocytes were collected from 28-day-old unstimulated females and matured in a defined medium. Oocytes were matured individually and transferred into fresh 0.5 microl drops of medium at 4 h intervals until 16 h. Ultramicrofluorimetry was used to quantitate carbohydrate consumption from and metabolite release into the medium. Glucose consumption and lactate production of COCs increased (P < 0.001) over the maturation interval (0-16 h). Glucose consumption by COCs that subsequently fertilized was higher between 8-12 h of maturation than by COCs that did not fertilize (38 versus 29 pmol/COC per h, respectively; P < 0.01). Lactate production by COCs that subsequently fertilized was higher between 8-16 h of maturation, than by oocytes that did not fertilize (8-12 h, 66 versus 46 pmol/COC per h, P < 0.01; 12-16 h, 56 versus 40 pmol/COC per h, respectively; P < 0.05). These data indicate that the final hours of maturation may hold a unique marker of oocyte competence, as during this time fertilizable COCs take up more glucose and produce more lactate than those not subsequently fertilized.

Animals↗
PubMed2005

A proteomic analysis of mammalian preimplantation embryonic development.

Genetic studies on the mammalian preimplantation embryo are providing a wealth of information regarding gene expression. However, changes in the transcriptome do not always reflect cellular function or the complexity and diversity of the mammalian proteome with post-translational modifications or protein-protein interactions. To elucidate embryonic cellular function, a detailed understanding at the protein level is necessary. The aim of this study was to generate protein profiles of mammalian embryos throughout development, and to investigate the effects of oxygen concentration on the embryonic proteome. A protocol was developed to analyse small groups of embryos (n = 5) by time-of-flight mass spectrometry. F1 mice zygotes were cultured in G1/G2 sequential media with recombinant albumin (2.5 mg/ml) in 6% CO(2) and O(2) concentrations of either 5% or 20%. In vivo-developed embryos were flushed from the reproductive tract (day 4). Protein profiles were generated for all embryonic samples and statistical analysis revealed 32 potential proteins/biomarkers with significant changes (P < 0.05). Embryos generated under 5% O(2) more closely resembled in vivo-developed embryos. Under 20% O(2) conditions, embryos showed down-regulation of 10 proteins/biomarkers (masses between 4 to 20 kDa) (P < 0.05) confirming the pathological effects of oxygen during embryonic development. These data demonstrate for the first time the complexity of the mammalian preimplantation proteome. The unique protein profiles of in vivo-developed embryos and a panel of selected biomarkers represent optimal cellular function, against which comparisons can be made to facilitate improvements in mammalian assisted reproduction techniques procedures.

Animals↗
PubMed2004

Single blastocyst transfer: a prospective randomized trial.

OBJECTIVE: To determine the efficacy of single blastocyst transfer. DESIGN: Prospective randomized trial. SETTING: Private assisted reproductive technology unit. PATIENT(S): Forty-eight women undergoing IVF-embryo transfer with day 3 FSH 12 mm in diameter on day of hCG administration. INTERVENTION(S): Embryo culture to the blastocyst stage in sequential media G1/G2 followed by transfer of either one or two blastocysts. MAIN OUTCOME MEASURE(S): Implantation rate, ongoing pregnancy rate, and twinning. RESULT(S): The transfer of a single blastocyst resulted in an implantation and ongoing pregnancy rate of 60.9% with no twins. The transfer of two blastocysts resulted in an implantation rate of 56%, an ongoing pregnancy rate of 76% with a 47.4% incidence of twins. CONCLUSION(S): Single blastocyst transfer is an effective method of eliminating multiple births while maintaining high pregnancy rates in this selected group of patients.

Adult↗
PubMed2004

Extent of nuclear DNA damage in ejaculated spermatozoa impacts on blastocyst development after in vitro fertilization.

OBJECTIVE: To determine whether the extent of ongoing apoptotic cell death measured as the presence of DNA strand breaks in spermatozoa affects embryo development to the blastocyst stage in IVF. DESIGN: A prospective comparative study. SETTING: A university IVF clinic and a private IVF clinic. PATIENT(S): Men (n = 49) undergoing infertility treatment with IVF. INTERVENTION(S): After density gradient centrifugation preparation, part of the sperm sample was used for infertility treatment, and the rest was fixed in paraformaldehyde. Strand breaks in DNA that are indicative of apoptosis were detected by the in situ DNA nick end labeling (TUNEL) technique. A total of 15,000 spermatozoa from each sample were evaluated for TUNEL reactivity by flow cytometry. MAIN OUTCOME MEASURE(S): Percentage of ejaculated spermatozoa with DNA strand breaks indicative of apoptosis, blastocyst development rate, and pregnancy rate. RESULT(S): Blastocyst development showed a significant negative correlation with percentage TUNEL positivity in spermatozoa. When 20% was used as a cutoff for TUNEL positivity in sperm samples, the percentage of blastocyst development was 50% higher in the <20% TUNEL-positivity group (n = 27) compared with those with >/=20% TUNEL positivity (n = 22; 44.7% blastocyst development vs. 29.8%). Clinical pregnancy rates in these two groups were 52% vs. 44%, respectively. CONCLUSION(S): The extent of nuclear DNA fragmentation in prepared ejaculated spermatozoa used in IVF negatively correlates with blastocyst development. A larger series of patients needs to be assessed to determine whether this paternal effect on blastocyst development may also affect pregnancy outcome.

Apoptosis↗
PubMed2004

Oestradiol, cyclodextrin-encapsulated 17beta-oestradiol and the oestradiol solubilizer 2-hydroxypropyl-beta-cyclodextrin all impair preimplantation mouse embryo development.

The aim of this study was to examine the effects of 2-hydroxypropyl-beta-cyclodextrin (HbetaC) used as a solubilizer for oestradiol, 17beta-oestradiol (ethanol soluble) and HbetaC-encapsulated-17beta-oestradiol on mouse embryo development in vitro. HbetaC had no effect on day 3 development. In contrast, blastocyst development and blastocyst cell number were significantly reduced in the presence of 10(-4) mol/l solubilizer equivalent, but not at lower concentrations. The proportion of compacted embryos was significantly reduced with 10(-4) mol/l 17beta-oestradiol. No blastocysts were formed at 10(-4) mol/l concentration of 17beta-oestradiol, although the rate of blastocyst formation did not differ at lower concentrations. Blastocyst cell number was significantly decreased compared with controls at 10(-5) mol/l 17beta-oestradiol. The dose-response using HbetaC-encapsulated-17beta-oestradiol revealed that at 17beta-oestradiol concentrations of 10(-4) and 10(-5) mol/l, blastocyst development was significantly reduced. Blastocyst cell number was significantly reduced compared with controls for all concentrations of HbetaC-encapsulated-17beta-oestradiol. Exposure of embryos to 17beta-oestradiol (10(-4) mol/l) reduced blastocyst development on days 4 and 5 significantly in cultures initiated at the zygote, 2-cell and 8-cell, but not the morulae, stages of development. Trophectoderm, ICM and blastocyst cell numbers as well as percentage ICM development were reduced significantly, regardless of the stage of development. Therefore, 17beta-oestradiol does compromise embryo development.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed2003

Ammonium induces aberrant blastocyst differentiation, metabolism, pH regulation, gene expression and subsequently alters fetal development in the mouse.

The presence of ammonium in the culture medium has significant detrimental effects on the regulation of embryo physiology and genetics. Ammonium levels build up linearly over time in the culture medium when media containing amino acids are incubated at 37 degrees C. Ammonium in the culture media significantly reduces blastocyst cell number, decreases inner cell mass development, increases apoptosis, perturbs metabolism, impairs the ability of embryos to regulate intracellular pH, and alters the expression of the imprinted gene H19. In contrast, the rate of blastocyst development and blastocyst morphology appear to be normal. The transfer of blastocysts exposed to ammonium results in a significant reduction in the ability to establish a pregnancy. Furthermore, of those embryos that manage to implant, fetal growth is significantly impaired. Embryos exposed to 300 microM ammonium are retarded by 1.5 days developmentally at Day 15 of pregnancy. It is therefore essential that culture conditions for mammalian embryos are designed to minimize the buildup of ammonium to prevent abnormalities in embryo physiology, genetic regulation, pregnancy, and fetal development.

Animals↗
PubMed2003

Cryo-survival and development of bovine blastocysts are enhanced by culture with recombinant albumin and hyaluronan.

Recombinant albumin can be used to supplement culture medium for the maturation and fertilization of bovine oocytes and subsequent embryo development to the blastocyst stage. Recombinant albumin was able to support blastocyst development at rates equivalent to that of bovine serum albumin (BSA) supplemented media. Supplementation of media containing recombinant albumin and citrate stimulated blastocyst expansion. Culture with recombinant albumin and citrate significantly increased the ability of the resultant blastocysts to re-expand and hatch following cryopreservation. The further addition of the glycosaminoglycan hyaluronan to the culture medium containing either BSA or recombinant albumin also increased the ability of blastocysts to survive cryopreservation. Inclusion of recombinant albumin and hyaluronan in culture media facilitates the development of physiological defined culture conditions. For bovine embryos this has implications for both research and commercial applications where defined reproducible conditions are desirable.

Albumins↗
PubMed2003

Changing the start temperature and cooling rate in a slow-freezing protocol increases human blastocyst viability.

OBJECTIVE: To determine the effect of start temperature and cooling rate of a slow freezing protocol on human blastocyst viability. DESIGN: Controlled-rate freezing of human blastocysts using different start temperatures and cooling rates. SETTING: Private assisted reproductive technology unit. PATIENT(S): Patients donated with consent cryopreserved pronucleate embryos. INTERVENTION(S): Culture of thawed pronucleate embryos in G III series media, containing hyaluronan, followed by cryopreservation of 36 blastocysts with subsequent noninvasive analysis of embryo metabolism. MAIN OUTCOME MEASURE(S): Pyruvate and glucose consumption and blastocyst reexpansion and quality. RESULT(S): Glucose consumption and blastocyst reexpansion after thaw were significantly higher when a start temperature of -6 degrees C and a cooling rate of 0.5 degrees C/min to -32 degrees C were used compared with a start temperature of 20 degrees C and a cooling rate of 2 degrees C to -6 degrees C, followed by cooling at 0.3 degrees C to -35 degrees C. Pyruvate uptake after thaw was not affected by the freezing procedure. Clinical use of the lower start temperature and quicker cooling rate, combined with culture in hyaluronan-based media, has led to the establishment of a 30% implantation rate. CONCLUSION(S): Human embryos cultured to the blastocyst stage in hyaluronan-based sequential media are readily cryopreserved and maintain their viability after thaw.

Blastocyst↗
PubMed2003

Use of G1.2/G2.2 media for commercial bovine embryo culture: equivalent development and pregnancy rates compared to co-culture.

The expanded application of commercial bovine IVM, IVF, and IVC systems is dependent on the ability to produce embryos in culture that are capable of producing normal pregnancies. Because serum containing culture systems can induce neonatal and fetal problems there exists a definite need for a serum-free culture system that produces viable blastocysts. This study demonstrated that the physiological sequential media system G1.2/G2.2 could produce bovine blastocysts at rates equivalent to co-culture. Additionally, these blastocysts had equivalent or increased cell numbers and inner cell mass development. Blastocysts produced in the G1.2/G2.2 culture system produced pregnancies following both fresh transfer and cryopreservation at equivalent rates to co-culture. Finally, this study demonstrated that the media system G1.2/G2.2 could be used in a commercial OPU transfer program without any loss in the numbers of blastocysts produced or the numbers of pregnancies resulting following transfer from either fresh or cryopreserved blastocysts.

Animals↗
PubMed2003

Towards a single embryo transfer.

The delivery of a single, healthy child is the desired outcome of human assisted reproduction techniques. To attain this goal, there is an increasing movement toward single embryo transfer. The question is, therefore, at what stage to transfer the human embryo back to the uterus? Maximal implantation rates reported to date have come from the transfer of blastocysts (70% fetal heart rate). In any given cycle of treatment the probability of conceiving a child will be further increased by the ability to cryopreserve those embryos not transferred. It is therefore proposed that the transfer of a single blastocyst is the best treatment for most patients, given the high implantation rates of fresh transfers, and that it is now possible to cryopreserve supernumerary blastocysts effectively. The next decision is how to culture the human embryo to the blastocyst stage. The use of sequential culture media, designed not only to allow for changes in nutrient requirements and metabolism as development proceeds, but also to minimize intracellular trauma, can facilitate the development of highly viable blastocysts. Sequential culture media have been evaluated against a single-step culture system. It has been shown that sequential media (G1/G2) produce more viable blastocysts than those embryos cultured in a single medium formulation (simplex optimized medium with elevated potassium and with amino acids, KSOM(AA)) throughout the preimplantation period. Furthermore, even if KSOM(AA) is used for embryo culture, it is essential that the medium be renewed after 48 h to alleviate the toxicity associated with ammonium build-up. Of great significance, embryos cultured in sequential media G1 and G2 have the same rate of development as embryos developed in vivo.

Animals↗
PubMed2002

Vitrification of human blastocysts using the cryoloop method: successful clinical application and birth of offspring.

PURPOSE: The purpose was to investigate vitrification of human blastocysts using the cryoloop. METHODS: Thirteen couples elected to have blastocysts cryopreserved via vitrification (N = 54 blastocysts). Embryos were exposed to two vitrification solutions, pipetted onto the cryoloop then plunged into liquid nitrogen. On the day of embryo replacement, blastocysts were warmed by passage through three dilution media, and rinsed into culture medium. RESULTS: Four couples returned for cryopreserved embryo replacement. Fifteen blastocysts were warmed; all 15 (15/15; 100%) were recovered. Fifteen (15/15; 100%) blastocysts were deemed viable, and 13 were replaced. Two replacements did not result in pregnancy; one resulted in clinical pregnancy with a blighted ovum; one resulted in clinical pregnancy with the live, term birth of a healthy male infant. Two of 13 embryos implanted yielding an implantation rate of 15.4%. CONCLUSIONS: These preliminary data suggest that blastocysts can be successfully vitrified using the cryoloop method.

Blastocyst↗
PubMed2002

Addition of ascorbate during cryopreservation stimulates subsequent embryo development.

BACKGROUND: Embryo development following cryopreservation is reduced compared with fresh embryos. One of the traumas that cryopreservation imparts on embryos is an increase in oxidative stress. Therefore, this study investigated the effects of the addition of the antioxidant ascorbate to the cryopreservation solutions on subsequent embryo development. METHODS: Mouse embryos at the 2-cell and blastocyst stages were either slow-frozen or vitrified in solutions containing either no ascorbate or 0.1 or 0.5 mmol/l ascorbate. The effects on the levels of hydrogen peroxide and subsequent embryo development and physiology were assessed. RESULTS: Addition of ascorbate to the cryopreservation solutions reduced the levels of hydrogen peroxide in embryos. Furthermore, addition of 0.1 mmol/l ascorbate significantly enhanced inner cell mass development in blastocysts. Embryos cryopreserved with ascorbate had significantly lower levels of lactate dehydrogenase leakage, and increased rates of metabolism compared with those cryopreserved in the absence of ascorbate. The benefits of ascorbate were significantly greater in embryos that were slow-frozen compared with those that were vitrified. CONCLUSIONS: These data indicate that the addition of 0.1 mmol/l ascorbate to the cryopreservation solutions for the mammalian embryo would be of significant value.

Animals↗
PubMedDate not supplied

Physiology and culture of the human blastocyst.

The human embryo undergoes many changes in physiology during the first 4 days of life as it develops and differentiates from a fertilized oocyte to the blastocyst stage. Concomitantly, the embryo is exposed to gradients of nutrients within the female reproductive tract and exhibits changes in its own nutrient requirements and utilization. Determining the nature of such nutrient gradients in the female tract and the changing requirements of the embryo has facilitated the formulation of stage-specific culture media designed to support embryo development throughout the preimplantation period. Resultant implantation rates attained with the culture and transfer of human blastocysts are higher than those associated with the transfer of cleavage stage embryos to the uterus. Such increases in implantation rates have facilitated the establishment of high pregnancy rates while reducing the number of embryos transferred. With the introduction of new scoring systems for the blastocyst and the non-invasive assessment of metabolic activity of individual embryos, it should be possible to move to single blastocyst transfer for the majority of patients.

Blastocyst↗