Biomedical subjects

D K Gardner

Publications and source records attributed to D K Gardner.

At least 19 recordsLinked to original sources

PubMed2001

Substrate utilization in porcine embryos cultured in NCSU23 and G1.2/G2.2 sequential culture media.

Embryo metabolism is an indicator of viability and, therefore, efficiency of the culture medium. Currently, little is known regarding porcine embryo metabolism. The objective of our study was to evaluate glucose and pyruvate uptake and lactate production in porcine embryos cultured in two different media systems. Oocytes were matured and fertilized according to standard protocols. Embryos were allocated randomly into two culture treatments, NCSU23 medium or G1.2/G2.2 sequential culture media 6-8 h post-insemination (hpi). Embryo substrate utilization was measured at the two-cell (24-30 hpi), 8-cell (80 hpi), morula (120 hpi), and blastocyst (144 hpi) stages using ultramicrofluorimetry. Glucose uptake was higher (P < 0.05) in two-cell embryos cultured in G1.2 than in NCSU23 medium (4.54 +/- 0.71, 2.16 +/- 0.87 pmol/embryo/h, respectively). Embryos cultured in G1.2/G2.2 produced significantly more lactate than those in NCSU23 at the eight-cell stage (9.41 +/- 0.71, 4.42 +/- 0.95 pmol/embryo/hr, respectively) as well as the morula stage (11.03 +/- 2.31, 6.29 +/- 0.77 pmol/embryo/hr, respectively). Pyruvate uptake was higher (P < 0.05) in morula cultured in G1.2/G2.2 versus NCSU23 (22.59 +/- 3.92, 11.29 +/- 1.57 pmol/embryo/h, respectively). Lactate production was greater (P < 0.05) in blastocysts cultured in G1.2/G2.2 (38.13 +/- 15.94 pmol/embryo/h) than blastocysts cultured in NCSU23 (8.46 +/- 2.38 pmol/embryo/h). Pyruvate uptake was also greater in blastocysts cultured in G1.2/G2.2 (24.3 +/- 11.04) than those in NCSU23 (11.30 +/- 2.70). When cultured in NCSU23 medium, two- and eight-cell embryos utilized less glucose than morulae and blastocysts, and two-cell embryos produced less lactate than blastocysts (P < 0.05). In G1.2/G2.2 media, two-cells took up less pyruvate than morulae or blastocysts, while blastocysts produced more lactate and utilized more glucose than two-cell, eight-cell and morula stage embryos (P < 0.05). As in other species, glycolysis appears to be the primary metabolic pathway in post-compaction stage porcine embryos. Culture medium composition affects not only substrate uptake, but also metabolic pathways by which these substrates are utilized in porcine embryos at several developmental stages.

Animals↗

PubMed2001

Vitrification of mouse oocytes using a nylon loop.

Cryopreservation of mouse oocytes was improved by the use of ultra-rapid vitrification using a nylon loop of 0.5 mm diameter. Oocytes that were vitrified using the loop survived at high rates and were fertilized following a small hole being made in the zona pellucida (69.8%) and developed to the blastocyst stage in culture (67.4%) at similar rates to that of oocytes that were not cryopreserved. Blastocysts resulting from oocytes vitrified using the nylon loop had similar development of the inner cell mass and trophectoderm as blastocysts from non-cryopreserved oocytes. In contrast, oocytes that were cryopreserved using a slow-freezing protocol where most of the Na+ is replaced with choline had lower rates of fertilization (39.5%), reduced development to the blastocyst stage (25.7%), and blastocysts had reduced development of the inner cell mass. Blastocysts derived from oocytes that were vitrified with the nylon loop were able to implant (88.0%) and develop into fetuses (56.5%) at significantly higher rates compared to blastocysts derived from oocytes that were slow-frozen (52.4 and 26.2%, respectively). Vitrification of mouse oocytes using the nylon loop results in the retention of viability of the oocytes and subsequent embryos.

Animals↗

PubMed2001

Assessment of metabolism of equine morulae and blastocysts.

Nutrient uptakes and metabolite production by equine morula and blastocyst stage embryos were determined by non-invasive microfluorometry. Equine morula took up equal amounts of both pyruvate and glucose. However, at the early blastocyst there was a small increase in glucose uptake and, by the expanded blastocyst stage, glucose was the predominant nutrient. Expanded blastocysts took up five times more glucose than pyruvate. Expanded blastocysts exhibited an exponential increase in glucose uptake and lactate production with respect to both diameter and surface area. As less than 50% of the glucose was accounted for by lactate production, the equine blastocyst appears to have a significant capacity to oxidize glucose. Embryos with a higher morphological grade consumed more nutrients than those with a poorer morphology. However, there was a large range in nutrient consumption within the highest grade blastocysts. This suggests that nutrient uptake may be useful as a viability marker of equine blastocysts.

Animals↗

PubMed2001

Inhibiting 3-phosphoglycerate kinase by EDTA stimulates the development of the cleavage stage mouse embryo.

Addition of EDTA to the medium significantly enhances mouse embryo development in culture. Embryos cultured in the absence of EDTA exhibit abnormal increases in glycolytic activity that result in reduced development. Culture with EDTA was able to prevent this increase in glycolysis and, therefore, maintain developmental competence. EDTA was shown to inhibit the activity of the glycolytic enzyme, 3-phosphoglycerate kinase. Additionally, the effect of EDTA on maintaining high rates of embryo development in culture could be mimicked by the addition of Cibacron blue, an inhibitor of 3-phosphoglycerate kinase. The inhibition of 3-phosphoglycerate kinase by EDTA could be overcome by the addition of exogenous magnesium, indicating that the effect of EDTA was to reduce the availability of this co-factor to the glycolytic kinases. Embryos cultured with EDTA had significantly lower levels of intracellular magnesium compared to embryos cultured without EDTA. Therefore, the effect of EDTA appears to be as a chelator of divalent cations such as magnesium, that are required for normal activity of kinases such as 3-phosphoglycerate kinase.

Animals↗

PubMed2001

Embryo transfer: techniques and variables affecting success.

OBJECTIVE: To review the literature on the variables affecting embryo transfer success or failure and to define technical factors associated with optimal outcome. DESIGN: Literature review. RESULTS: Avoidance of blood, mucus, bacterial contamination, excessive uterine contractions, and trauma to the endometrium is associated with optimal pregnancy and implantation rates after transcervical embryo transfer. A trial transfer, ultrasonographic guidance, and use of "soft" catheters appear to facilitate successful embryo transfer. CONCLUSION: An understanding of the variables associated with embryo transfer success together with adherence to techniques shown to facilitate atraumatic embryo transfer will enhance the efficiency of IVF by maximizing embryo implantation.

Blastocyst↗

PubMed2001

Noninvasive assessment of human embryo nutrient consumption as a measure of developmental potential.

OBJECTIVE: To determine the relationship between blastocyst development and morphology and embryo metabolism. DESIGN: Noninvasive assessment of carbohydrate uptake and ammonium production by individual embryos. SETTING: Private assisted reproductive technology unit. PATIENT(S): Patients donated, with consent, cryopreserved pronucleate embryos and noncryopreserved blastocysts. INTERVENTION(S): Culture of 60 thawed pronucleate embryos in sequential media to the blastocyst stage with concomitant noninvasive analysis of embryo metabolism and analysis of 13 blastocysts from noncryopreserved embryos. MAIN OUTCOME MEASURE(S): Pyruvate and glucose consumption as well as blastocyst formation and quality. RESULT(S): Pyruvate and glucose uptakes on day 4 were significantly higher by embryos that went on to form blastocysts than by embryos that failed to develop to the blastocyst stage. Glucose uptakes were greatest in those blastocysts of highest grade, whereas pyruvate uptakes were similar irrespective of blastocyst grade, indicating that glucose is the more important nutrient for the human blastocyst. Among blastocysts of the same grade from the same patient, there was considerable spread of glucose consumption, indicating that glucose consumption may be of use in identifying blastocysts for transfer. Ammonium production by individual embryos was also measured, reflecting amino acid transamination and use by the human embryo. CONCLUSION(S): The ability to identify in culture the embryo with the highest developmental potential will facilitate the move to single-embryo transfers.

Blastocyst↗

PubMed2001

Effect of essential amino acids on mouse embryo viability and ammonium production.

PURPOSE: To examine the effect of essential amino acids concentrations on mouse embryo development. METHODS: Mouse embryos were cultured in medium with different concentrations of essential amino acids and development to the blastocyst stage and viability assessed. Ammonium production resulting from medium breakdown and amino acid metabolism by embryos were also assessed. RESULTS: Reducing the essential amino acid concentration significantly increased blastocyst development and cell numbers. Lowering the essential amino acid concentration decreased ammonium production in the medium. CONCLUSIONS: Culture media for the development of preimplantation embryos should have a reduced essential amino acid concentration to facilitate embryo development.

Amino Acids, Essential↗

PubMed2001

Blastocyst versus day 2 or 3 transfer.

The formulation of new sequential culture media, capable of supporting the development of viable human blastocysts, has reopened the discussion regarding the best day for embryo transfer following in vitro fertilization (IVF). Although several laboratories have reported overall increases in implantation rate and IVF efficiency following the transfer of blastocysts, others have failed to observe any benefit from extended culture. While culture conditions for the mammalian embryo undoubtedly have improved significantly over the past few years, relatively little attention has been paid to the quality of oocytes derived from ovarian hyperstimulation or the quality and receptivity of the endometrium following such hormonal regimes. It appears that differences in controlled ovarian hyperstimulation are among the major factors determining embryo quality and subsequent implantation. This therefore has confounded comparisons between different laboratories. In spite of this there are a growing number of reports demonstrating that the advantages of extended culture and blastocyst transfer, such as increased implantation rates, are not limited to specific groups of patients or specific etiologies. Rather, blastocyst transfer may be of benefit to the majority if not all patients attending for IVF.

Blastocyst↗

PubMed2001

Extended embryo culture in human assisted reproduction treatments.

In order to evaluate the niche of extended embryo culture in an IVF programme, retrospective analysis of non-selected IVF patients, who underwent ovarian stimulation from April 1998 to June 1999 in a single private practice assisted reproductive technology centre, was performed. Embryos were cultured for 48 h in S1/G1.2 medium followed by 48 to 72 h of culture in S2/G2.2 to day 5 or day 6. Only fertilized oocytes exhibiting two pronuclei from donor and non-donor IVF and intracytoplasmic sperm injection (ICSI) cases were examined to determine the relationship between embryo cell number on day 3 and subsequent rate of blastocyst formation. Results indicated that a proportional relationship existed between the number of blastomeres present in day 3 embryos and the rate of blastocyst formation. Fifty-four per cent of embryos that had six cells on day 3 formed blastocysts, while 76% of those embryos with eight cells formed blastocysts. Blastocyst development did not increase further when embryos had more than eight cells on day 3, indicating that embryos with greater cell numbers on day 3 are not always predictive of a greater likelihood of blastocyst formation. Fertilized oocytes exhibiting two pronuclei from donors produced significantly more blastocysts (67%) than those from IVF patients (52%; P < 0.01), and had a significantly higher implantation rate (54%) compared with IVF patients (30%; P < 0.01). Furthermore, blastocyst cryopreservation resulted in significantly higher implantation rates than cryopreserved cleavage stage embryos (P < 0.001).

Blastocyst↗

PubMed2000

EDTA stimulates cleavage stage bovine embryo development in culture but inhibits blastocyst development and differentiation.

Culture of bovine zygotes in medium SOFaa supplemented with 100 microM EDTA significantly increased cleavage rates during the first 72 hr of development compared to development in SOFaa. However, continued culture in the presence of EDTA for a further 72 hr (total of 6 days of culture) resulted in significantly reduced development to the morulae/blastocyst and blastocyst stages compared to culture without EDTA. Highest rates of development to the morulae/blastocyst stage (56.5%) and to the blastocyst stage (43.2%) were achieved when zygotes were cultured for 72 hr with EDTA before transfer to medium SOFaa without EDTA. Resultant blastocysts also had significantly increased blastocyst cell number and ICM cell number compared to those cultured without EDTA in the first 72 hr. EDTA was shown to inhibit glycolytic activity of the cleavage stage embryo, thereby preventing the premature stimulation of glycolysis and enhancing development. However, EDTA should not be used for the later stage embryo as the inhibition of glycolysis reduces energy production at the blastocyst stage and significantly inhibits inner cell mass development.

Animals↗

PubMed2000

Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer.

OBJECTIVE: To determine the relationship between blastocyst score and pregnancy outcome. DESIGN: Retrospective review of blastocyst transfer in an IVF clinic. SETTING: Private assisted reproductive technology unit. PATIENT(S): 107 patients undergoing blastocyst culture and transfer of two embryos. INTERVENTION(S): Culture of all pronucleate embryos in sequential media to the blastocyst stage (day 5), followed by transfer of two blastocysts. MAIN OUTCOME MEASURE(S): Implantation rates, pregnancy rates, and twinning were analyzed. RESULT(S): When a patient received two top-scoring blastocysts (64% of patients), implantation and pregnancy rates were 70% and 87%, respectively. The twinning rate in this group was 61%. When only one top-quality blastocyst was available for transfer (21% of patients), the implantation and pregnancy rates were 50% and 70%. The twinning rate for this group was 50%. In contrast, when only low-scoring blastocysts were available for transfer (15% of patients), implantation and pregnancy rates were 28% and 44%, and the twinning rate was 29%. No monozygotic twins were observed in this group of patients. CONCLUSION(S): The ability to transfer one high-scoring blastocyst should lead to pregnancy rates greater than 60%, without the complication of twins.

Adult↗

PubMed2000

Blastocyst culture and transfer increases the efficiency of oocyte donation.

OBJECTIVE: To determine the impact of blastocyst transfer on an oocyte donation program. DESIGN: Retrospective review of embryo transfer in an IVF clinic. SETTING: Private assisted reproductive technology unit. PATIENT(S): Two hundred and twenty nine patients undergoing oocyte donation. INTERVENTION(S): Culture of pronucleate embryos to either day 3 or day 5 followed by embryo transfer. MAIN OUTCOME MEASURE(S): Implantation rates, pregnancy rates, and multiple gestations were analyzed. RESULT(S): Implantation rates and pregnancy rates were significantly increased by moving to extended embryo culture and transfer on day 5. After day 3 transfers, implantation and pregnancy rates were 47.1% and 75%, respectively. In contrast, on day 5 these rates were increased to 65.8% and 87.6%. Concomitantly, there were significantly fewer embryos transferred on day 5 (2.1) compared to day 3 (3.2). CONCLUSION(S): Blastocyst transfer is a highly effective treatment for patients who receive donor oocytes, allowing excellent pregnancy rates while significantly reducing the incidence of high-order multiple gestations.

Blastocyst↗

PubMed2000

Regulation of ionic homeostasis by mammalian embryos.

Control and regulation of cellular homeostasis are essential for normal embryo development and maintenance of viability. By understanding the role of ionic homeostasis in normal cell development and homeostatic control by the developing embryo, it is possible to develop culture systems that minimize cellular stress and therefore maintain embryo viability. This article discusses the regulation of intracellular levels of protons (pHi), calcium, magnesium, and phosphate in mammalian embryos.

Animals↗

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↗