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Biomedical subjects

D K Gardner

Publications and source records attributed to D K Gardner.

At least 37 records · Page 2Linked to original sources
PubMed2000

Embryo nutrition and energy metabolism and its relationship to embryo growth, differentiation, and viability.

Over the past decade there has been a resurgence of interest in the culture media used in clinical in vitro fertilization. Unfortunately, during this time more confusion than consensus appears to have developed regarding the composition of these media. In order to facilitate a clearer understanding of this field, it is important to understand the role of specific medium components and how their use is regulated by the embryo. The roles of the key nutrients glucose, pyruvate, lactate, and amino acids during the preimplantation period have therefore been presented. Analysis of how the embryo regulates the utilization of such nutrients has led to a clearer understanding of the embryo's requirements during the dynamic period of preimplantation development. From such information, sequential culture media have been developed along with novel noninvasive tests of embryonic viability. It is proposed that continued studies on the human embryo will lead to further improvements in embryo culture conditions and the optimization of viability assays, culminating in the ability to transfer single embryos for the majority of, if not all patients.

Amino Acids↗
PubMed2000

A single medium supports development of bovine embryos throughout maturation, fertilization and culture.

Oocytes and embryos are typically exposed sequentially to varying culture media in standard in-vitro protocols. Expenditures of energy may be required following each medium change to adjust to the changing environment. Therefore, a single base medium was evaluated for its ability to support in-vitro maturation, fertilization and pre-implantation development (IVM/F/C) of bovine oocytes and embryos. Four treatments were examined: a standard maturation [tissue culture medium (TCM) 199 with bovine calf serum (BCS)], fertilization (modified Tyrode's medium with albumin, lactate and pyruvate) and culture (hamster embryo culture medium/TCM with BCS) system (control) and three synthetic oviductal fluid (SOF) treatments; maturation in SOF with bovine serum albumin (SOFBSA), SOF with bovine calf serum (SOFBCS) or the control maturation medium (TCM199 with BCS; SOF199), followed by fertilization and culture in SOF medium. The percentage of total inseminated oocytes successfully developing to the morula and blastocyst stage did not differ (P > 0. 05) between treatments (control, 30.5 +/- 3.5; SOFBSA, 24.6 +/- 3.2; SOFBCS, 22.4 +/- 4.7; SOF199, 27.3 +/- 3.2). Embryos cultured in SOFBCS (92.1 +/- 6.4) had significantly higher cell numbers (P < 0. 05) than those cultured in control (74.8 +/- 4.8) and SOFBSA (71.6 +/- 6.6) but not SOF199 (81.2 +/- 6.8). In conclusion, a single medium can be used successfully throughout maturation, fertilization and pre-implantation embryo development. Moreover, inclusion of serum during maturation in the single medium system resulted in significantly greater cell numbers, possibly reflecting increased quality of the embryos produced.

Animals↗
PubMed2000

Lactate regulates pyruvate uptake and metabolism in the preimplantation mouse embryo.

This study was an investigation of the interaction of lactate on pyruvate and glucose metabolism in the early mouse embryo. Pyruvate uptake and metabolism by mouse embryos were significantly affected by increasing the lactate concentration in the culture medium. In contrast, glucose uptake was not affected by lactate in the culture medium. At the zygote stage, the percentage of pyruvate taken up and oxidized was significantly reduced in the presence of increasing lactate, while at the blastocyst stage, increasing the lactate concentration increased the percentage of pyruvate oxidized. Lactate oxidation was determined to be 3-fold higher (when lactate was present at 20 mM) at the blastocyst stage compared to the zygote. Analysis of the kinetics of lactate dehydrogenase (LDH) determined that while the V(max) of LDH was higher at the zygote stage, the K(m) of LDH was identical for both stages of development, confirming that the LDH isozyme was the same. Furthermore, the activity of LDH isolated from both stages was reduced by 40% in the presence of 20 mM lactate. The observed differences in lactate metabolism between the zygote and blastocyst must therefore be attributed to in situ regulation of LDH. Activity of isolated LDH was found to be affected by nicotinamide adenine dinucleotide(+) (NAD(+)) concentration. In the presence of increasing concentrations of lactate, zygotes exhibited an increase in autofluorescence consistent with a depletion of NAD(+) in the cytosol. No increase was observed for later-stage embryos. Therefore it is proposed that the differences in pyruvate and lactate metabolism at the different stages of development are due to differences in the in situ regulation of LDH by cytosolic redox potential.

Animals↗
PubMed2000

Culture and transfer of viable blastocysts: a feasible proposition for human IVF.

In spite of the numerous advances in the field of human assisted reproductive technologies (ART) over the past 20 years, a rate-limiting factor in the overall efficiency of the procedure (the implantation rate) has remained at 10-30%. The development of sequential media has led to the ability to culture routinely the human embryo to the viable blastocyst stage. Transfer of such blastocysts has resulted in a significant increase in implantation rates. Increases in implantation rates following blastocyst transfer have been reported for specific groups of patients culminating in the elimination of high order multiple gestations. Of greater significance, however, is that the introduction of blastocyst transfer to all patients entering infertility clinics is associated with an overall increase in implantation and pregnancy rates. Blastocysts derived from the use of sequential media are readily cryopreserved and produce high implantation rates after transfer. Using a model to account for both total embryo utilization per cycle (transferred plus cryopreserved) and implantation rate, it has been calculated that extended embryo culture and blastocyst transfer is approximately 20% more efficient than the transfer of cleavage stage embryos on day 3. Furthermore, as the score of the blastocysts obtained using sequential media is directly related to implantation and pregnancy rates, it is possible to determine which patients should be offered a single blastocyst transfer, thereby addressing the issue of twins conceived through ART.

Adult↗
PubMed1999

Mouse embryos used as a bioassay to determine control of marsupial embryonic diapause.

Mouse blastocysts appear to be under direct inhibition from the uterine environment, whereas no evidence of direct inhibition during diapause in the tammar wallaby has been observed. Normally developing (day 4) and quiescent mouse blastocysts were incubated for up to 12 hr in media supplemented with BSA, wallaby plasma, wallaby day 0 (day of removal of pouch young; RPY), day 5, or day 10 endometrial exudates at a concentration of 2 mg/ml of protein, and analyzed for rates of carbohydrate metabolism using fluorescence and radioisotopes. Rates of glucose uptake and lactate production by day 4 blastocysts increase after incubation with day 10 and day 5 wallaby exudates compared with rates by blastocysts incubated in BSA. Pyruvate uptake increased after 8 hr irrespective of incubation media, except for embryos incubated in day 0 exudate, which maintained levels significantly lower than BSA-incubated embryos. Quiescent mouse embryos displayed a high ATP/ADP ratio during diapause (1.06 +/- 0.24) which decreased after 4 hr incubation in all media (0.42 +/- 0.05; P < 0.01) but embryos incubated in day 0 exudate media remained at a significantly higher level than embryos incubated in BSA. These results indicate that quiescent tammar endometrial exudate is not capable of initiating diapause in mouse embryos at the concentration used, but is able to slow the rate of reactivation of quiescent blastocysts. Importantly, reactivated wallaby exudate increases mouse blastocyst glucose metabolism and lactate production. It is possible that the quiescent tammar endometrial environment has an inhibitory factor necessary to maintain diapause in the tammar blastocyst.

Animals↗
PubMed1999

In vitro development and nutrient uptake by embryos derived from oocytes of pre-pubertal and adult cows.

The present study compared the developmental potential and uptake of nutrients by embryos from pre-pubertal and adult cows. Oocytes retrieved from ovaries of 5 to 7 month old calves and adult cows were matured and fertilized in vitro. Embryos were cultured in SOFaa to the blastocyst stage (7 days post-insemination). At successive stages of development, rates of glucose and pyruvate uptake were measured non-invasively by microfluorescence for individual embryos. Fertilization was equivalent in embryos from pre-pubertal and adult cows (P > 0.05), however development to blastocyst was significantly lower in embryos from pre-pubertal cows (9.8% versus 33.7%, respectively; P < 0.05). Total blastocyst cell number was not different between pre-pubertal and adult material (P > 0.05). Glucose uptake was exponential (pre-pubertal, r = 0.82; adult, r = 0. 82; P < 0.05), with an increase in uptake beyond the 8- to 16-cell stage. Glucose uptake was significantly lower in embryos from pre-pubertal cows at the 2- to 4-cell stages (1.5 versus 3.0 pmoles/embryo/hr; P < 0.05), but was equivalent to the adult cow at all other stages of development (P > 0.05). Pyruvate uptake was low until the blastocyst stage. Pyruvate uptake by embryos from pre-pubertal cows was significantly different to adult cows at the 1-cell stage (2.7 versus 4.6 pmoles/embryo/hr, respectively; P < 0. 05) and 2- to 4-cell stages (4.9 versus 3.6 pmoles/embryo/hr, respectively; P < 0.05). Pyruvate uptake was equivalent in the two groups in the later stages of development (P > 0.05). Perturbations in the uptake of nutrients by embryos from pre-pubertal cows were most likely due to the presence of a high proportion of developmentally incompetent embryos. Further, embryos from pre-pubertal cows that did develop to the blastocyst were as viable as blastocysts from adult cows with respect to nutrient uptakes and total cell number.

Animals↗
PubMed1999

Metabolism of glucose, pyruvate, and glutamine during the maturation of oocytes derived from pre-pubertal and adult cows.

The aim of the study was to compare the energy metabolism of oocytes from pre-pubertal (2 to 3 months) and adult cows during maturation, to identify the cause of poor developmental potential in many pre-pubertal oocytes. The metabolism of [5-(3)H] glucose, [2-(14)C] pyruvate, and [G-(3)H] glutamine was measured at 0 hr, 12 hr, and 24 hr maturation. Oxidative metabolism was important during maturation of oocytes from both pre-pubertal and adult cows, with pyruvate metabolism peaking at 12 hr and glutamine metabolism increasing linearly and peaking at 24 hr. Peak oxidative metabolism was significantly lower in oocytes from pre-pubertal animals, for both pyruvate and glutamine (P < 0.05). Glucose metabolism increased significantly during oocyte maturation in both groups (0hr to 24 hr). Glucose metabolism was significantly lower in oocytes from pre-pubertal cows at 12 hr (P < 0.05). Oocytes from pre-pubertal animals were significantly smaller than oocytes from adult cows at 0 hr, 12 hr, and 24 hr maturation (P < 0.05). When metabolic rates were corrected for oocyte volume, there were no significant differences in substrate metabolism between oocytes from pre-pubertal and adult cows. There was however, a delay in the increase in glucose metabolism in pre-pubertal oocytes 0 hr to 12 hr maturation. Germinal vesicle breakdown was slower in oocytes from pre-pubertal animals with more oocytes still at the germinal vesicle stage approximately 5 hr post-aspiration, compared to oocytes from adult cows (P < 0.05). By 24 hr, development to metaphase II was equivalent for pre-pubertal and adult oocytes. This study identified differences in energy metabolism, oocyte size, and meiotic progression between the oocytes from pre-pubertal and adult cows that may account for the poor developmental potential of many pre-pubertal oocytes.

Animals↗
PubMed1999

Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs.

OBJECTIVE: To determine whether previously described advanced blastocyst development and high implantation rates are confirmed in an expanded multicenter trial. DESIGN: Retrospective review. SETTING: Two private assisted reproductive technology units. PATIENT(S): One hundred seventy-four patients who underwent blastocyst culture and transfer. INTERVENTION(S): Culture of all pronucleate embryos in sequential media to the blastocyst stage (day 5) followed by ET. MAIN OUTCOME MEASURE(S): The number and percentage of blastocysts developed, implantation rates, pregnancy rates, and parameters that affected outcome were analyzed. RESULT(S): Only 3 of 174 patients failed to achieve blastocyst-stage ET. The mean blastocyst development rate was 48%. The ongoing pregnancy rate was 66.3% per oocyte retrieval, with a mean (+/-SE) of 2.2 +/- 0.05 blastocysts transferred and an implantation rate of 48% per blastocyst transferred. CONCLUSION(S): Blastocyst culture and transfer is an effective means of treating patients who respond well to gonadotropins. High pregnancy rates can be accomplished with low numbers of embryos transferred. Patients who failed to achieve ET were rare.

Adult↗
PubMed1999

Introduction of blastocyst culture and transfer for all patients in an in vitro fertilization program.

OBJECTIVE: To evaluate the nonselective application of extended embryo culture on the outcome of IVF. DESIGN: Retrospective analysis. SETTING: Private practice assisted reproductive technology center. PATIENT(S): Seven hundred ninety nonselected patients undergoing IVF with controlled ovarian stimulation. INTERVENTION(S): For day 3 ET, multicell embryos were cultured in human tubal fluid medium and 12% synthetic serum substitute. For day 5 ET, embryos were cultured for 48 hours in S1 medium and then for 48 hours in S2 medium. MAIN OUTCOME MEASURE(S): Implantation rate (determined by total no. of visualized gestational sacs), ongoing pregnancy rate, and number of embryos available for ET. RESULT(S): Respective day 3 and day 5 implantation rates for patients aged <35 years (29.5% and 38.9%), patients aged 35-39 years (20.7% and 28.2%), and all patients combined (23.3% and 32.4%) were statistically significantly different. Significantly more embryos were transferred on day 3 than on day 5 for patients aged <35 years (2.9 vs. 2.4), patients aged 35-39 years (3.1 vs. 2.6), and all patients combined (3.0 vs. 2.5). The difference in ongoing pregnancy rates per retrieval was statistically significant for day 3 compared with day 5 transfers for all patients combined (35.9% vs. 43.8%). Cancellation rates for transfer after retrieval increased significantly for day 3 compared with day 5 transfer (2.9% vs 6.7%). CONCLUSION(S): These results demonstrate the feasibility of using extended embryo culture in a nonselective manner for couples undergoing IVF. Overall, extended embryo culture was associated with a significant increase in pregnancy rates and implantation rates and a significant decrease in the number of embryos transferred. The rate of multiple implantation among patients aged <35 years warrants consideration of single blastocyst transfers for this group.

Adult↗
PubMed1999

Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique.

OBJECTIVE: To vitrify mouse and human blastocysts with use of the cryoloop procedure and to assess subsequent development. DESIGN: Controlled study of vitrification of mouse and human blastocysts. SETTING: Research department of a private assisted reproductive technology unit. PATIENT(S): Blastocysts that were not suitable to be frozen were donated from patients. INTERVENTION(S): Culture of pronucleate embryos in sequential media to the blastocyst stage. MAIN OUTCOME MEASURE(S): Survival of the vitrification procedure was assessed by reexpansion, hatching, and outgrowth in culture. In addition, the viability of mouse blastocysts was assessed after transfer to pseudopregnant recipients. RESULT(S): Vitrification of mouse blastocysts did not affect the ability to reexpand, hatch, or outgrow in culture. Furthermore, implantation rates and fetal development were equivalent for nonfrozen and vitrified blastocysts. Vitrified human blastocysts were able to hatch and outgrow in culture at rates similar to nonfrozen controls. CONCLUSION(S): Cryoloop vitrification was able to cryopreserve mouse and human blastocysts without any reduction in the ability to reexpand and hatch in culture. Furthermore, viability was not reduced by the cryoloop vitrification of mouse blastocysts.

Animals↗
PubMed1999

Fetal development after transfer is increased by replacing protein with the glycosaminoglycan hyaluronan for mouse embryo culture and transfer.

The effect of macromolecules on mouse embryo development and viability after culture in sequential media was investigated. It was found that high rates of viable blastocysts could be obtained in the absence of any macromolecule. Blastocyst cell numbers were increased when bovine serum albumin was present in the culture medium, although this benefit was not manifest after blastocyst transfer. Rather, the highest rates of implantation and fetal development after blastocyst transfer were observed when hyaluronan was the macromolecule in the culture media. Subsequent analysis revealed that the beneficial effects of hyaluronan were due to its presence in the transfer medium. As the highest cell numbers and hatching rates obtained in this study occurred when both serum albumin and hyaluronan were present in the same medium, it is proposed that embryo culture media should contain both serum albumin and hyaluronan, while the transfer medium need only contain hyaluronan.

Animals↗
PubMed1999

Temporal and differential effects of amino acids on bovine embryo development in culture.

The aim of the study was to determine the amino acid requirements of the in vitro-produced bovine embryo as it develops from the zygote to the blastocyst, using a two-step culture system. When added to synthetic oviduct fluid (SOF) for the first 72-h culture, Eagle's nonessential amino acids and glutamine (NeGln) significantly increased development to the 8- to 16-cell stage (Day 4 postinsemination [pi]) and subsequent blastocyst development (Day 7 pi). Glutamine alone during the first 72-h culture did not stimulate development to the 8- to 16-cell stage (p > 0.05); however, the removal of glutamine from NeGln reduced the stimulatory effects of the nonessential amino acids. Replacing glutamine with betaine (an organic osmolyte) in NeGln did not stimulate development to the 8- to 16-cell stage compared to culture in SOF, but it did improve subsequent blastocyst development, indicating an osmolytic function of glutamine during the first 72-h culture. The addition of Eagle's essential amino acids and glutamine to SOF, or to medium already containing nonessential amino acids and glutamine for the first 72-h culture, did not affect cleavage to the 8- to 16-cell stage or subsequent blastocyst development (p > 0.05). Beyond Day 4 pi, culture with 20aa (nonessential and essential amino acids and glutamine) increased blastocyst development, total cell number, and the number of cells in both the trophectoderm and inner cell mass, compared to culture with other groups of amino acids (p < 0.05). Substituting betaine for glutamine in 20aa reduced blastocyst formation, indicating a non-osmolytic function of glutamine during the second 72-h culture. Further, there was a significant negative correlation between the concentration of essential amino acids (quarter, half, or single strength) and embryo development during both the first 72-h and second 72-h culture (p < 0.01), indicating that the concentration of essential amino acids was too high during culture of the bovine embryo. This study identified the temporal and differential effects of amino acids during development of the bovine embryo from the zygote to the blastocyst.

Amino Acids↗
PubMed1999

Culture and transfer of human blastocysts.

The transfer of the human embryo at the blastocyst stage during an in-vitro fertilization procedure is a way of increasing implantation rates. This, in turn, means that significantly fewer embryos are required to be transferred in order to establish a successful pregnancy. The result of this is that high order multiple gestations are eliminated, while maintaining high pregnancy rates, in in-vitro fertilization.

Blastocyst↗
PubMed1999

Reactivating tammar wallaby blastocysts oxidize fatty acids and amino acids.

The tammar wallaby, Macropus eugenii, has a ruminant-like digestive system which may make a significant concentration of amino acids and fatty acids available to the blastocyst via uterine fluids. Fluorescent and radioisotope analyses were performed to determine the rate of glutamine and palmitate use by blastocysts recovered on day 0, 3, 4, 5 and 10 after reactivation induced by removal of pouch young (RPY). Between day 0 and 4 glutamine uptake increased from 15.6 +/- 6.6 to 36.1 +/- 2.7 pmol per embryo h-1 (P < 0.01) and ammonium production increased from 8.2 +/- 4.3 to 26.6 +/- 3.0 pmol per embryo h-1 (P < 0.01). Glutamine oxidation did not increase until day 10 after RPY (P < 0.01), but the percentage of glutamine oxidized increased from 4.5 +/- 3.1% during diapause to 31.2 +/- 12.6% (P < 0.01) by day 5 after RPY and increased further to 51.0 +/- 15.8% (P < 0.01) by day 10 after RPY. Palmitate oxidation also increased from 0.3 +/- 0.1 by day 0 blastocysts to 3.8 +/- 1.7 pmol per embryo h-1 (P < 0.01) by day 4 blastocysts. This increase provides a greater potential for ATP production, possibly to supply increased demand due to the coincident resumption of mitoses. The ATP:ADP ratio within blastocysts had reduced by the time of the first measurement at day 3 (0.5 +/- 0.2 pmol per embryo h-1; P < 0.01) compared with day 0 blastocysts (1.4 +/- 0.3 pmol per embryo h-1). It is likely that metabolism of amino acids and fatty acids contributes to the energy supply during reactivation of tammar wallaby blastocysts after embryonic diapause.

Adenosine Diphosphate↗
PubMed1999

Development of serum-free culture systems for the ruminant embryo and subsequent assessment of embryo viability.

The mammalian embryo undergoes considerable changes in its physiology and energy metabolism as it proceeds from the zygote to the blastocyst stage. Complete development of the mammalian zygote in vitro was restricted to a few strains of mice and their F1 hybrids for many years, as the ruminant embryo arrested development at the 8- to 16-cell stage. The introduction of co-culture of ruminant embryos with somatic cells in the mid-1980s helped to alleviate this in vitro induced arrest. However, such culture systems required the use of complex tissue culture media and serum. Serum has subsequently been shown to induce several abnormalities during embryo development in culture and has been associated with the production of offspring with significantly greater birth weights than normal, leading to both difficulties in pregnancy management and an unacceptable frequency of neonatal death. Resurgence of interest in mammalian embryo physiology has culminated in the formulation of defined embryo culture media, capable of supporting a high percentage of viable blastocyst development in vitro. Optimum embryo development in culture has been shown to take place not in one, but two or more media, each designed to cater for the changing requirements and metabolism of the embryo as it develops. The development of viability assays to identify those embryos with the highest developmental potential will further increase the efficiency of embryo transfer procedures. Assays based upon nutrient uptake and subsequent utilization make promising candidates.

Animals↗
PubMed1998

Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture.

Along with the transition from maternal to embryonic genome control the mammalian preimplantation embryo undergoes significant changes in its physiology during development. Concomitant with these changes are altering patterns of nutrient uptake and differences in the subsequent fate of such nutrients. The most significant nutrients to the developing mammalian preimplantation embryo are carbohydrates and amino acids, which serve not only to provide energy but also to maintain embryo function by preventing cellular stress induced by suboptimal culture conditions in vitro. It is subsequently proposed that optimal development of the mammalian embryo in culture requires the use of two or more media, each designed to cater for the changing requirements of the embryo. Importantly, culture conditions that maintain the early embryo are not ideal for the embryo post-compaction, and conditions that support excellent development and differentiation of the blastocyst can actually be inhibitory to the zygote. A marker of in vitro-induced cellular stress to the embryo is the relative activity of the metabolic pathways used to generate energy for development. Quantification of embryo energy metabolism may therefore serve as a valuable marker of embryo development and viability.

Animals↗