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D K Gardner

Publications and source records attributed to D K Gardner.

At least 55 records · Page 3Linked to original sources
PubMed1998

Intracellular pH of the preimplantation mouse embryo: effects of extracellular pH and weak acids.

Although intracellular pH (pHi), is a regulator of numerous biological processes, it has received relatively little attention with regard to the physiology of the mammalian preimplantation embryo. Interestingly, there is some controversy as to whether the early embryo can recover from an acid load. The significance of this is that two constituents of mouse embryo culture media are pyruvate and lactate. These carboxylic acids are utilised by the early mouse embryo for energy production. However, as weak acids, pyruvate and lactate may induce perturbations in the pHi and thus alter the physiology of the embryo. The aims of this study were therefore to measure the pHi of the mouse preimplantation embryo and to determine the effect of lactate on pHi at different developmental stages. The pHi was measured using the ratio-metric fluorophore carboxy-seminaphthorhodafluor-1-acetoxymethylester (SNARF-1) in conjunction with confocal microscopy. The pHi increased significantly with development from the zygote to the morula stage. Furthermore, at concentrations greater than 5 mM, lactate caused the pHi of the zygote to become significantly more acidic. It was demonstrated that facilitative transport in association with a smaller passive component was responsible for the movement of lactate into the zygote. Metabolic studies revealed that, through their acidifying effect, weak acids caused a reduction in glycolytic activity in the early embryo. In contrast, the pHi of the compacted embryo remained unchanged by the presence of lactate in the external media. Furthermore, incubation with weak acids did not affect the rate of glycolysis in the morula. These data suggest that, by the generation of a transporting epithelium at compaction, the embryo develops the ability to regulate pHi against an acid load.

Acids↗
PubMed1998

Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers.

OBJECTIVE: To determine whether the transfer of blastocysts on day 5, developed in sequential culture media, resulted in an increase in implantation rate compared with embryos transferred on day 3. DESIGN: Comparative study of embryo culture regimes. SETTING: Private practice assisted reproductive technology center. PATIENT(S): Twenty-three patients undergoing routine IVF cycles. INTERVENTION(S): Culture of embryos to day 3 in either standard culture conditions or a serum-free chemically defined medium. One hundred one embryos were subsequently cultured from day 3 to day 5 in a second serum-free medium specifically designed to support development of the blastocyst. MAIN OUTCOME MEASURE(S): Embryo cell number and quality on day 3. Blastocyst development on day 5. Implantation rate (determined by fetal heart) and ongoing pregnancy rate (PR). RESULT(S): Implantation rates for embryos transferred at the blastocyst stage of development were twice that observed for embryos transferred on day 3, around the eight-cell stage. Significantly more embryos were required for transfer on day 3, compared with day 5, to establish similar PRs. CONCLUSION(S): Viable human blastocysts can be obtained in sequential culture media in the absence of coculture and serum. Transfer of blastocysts in IVF will facilitate high PRs while limiting the number of embryos transferred and therefore minimizes the risk of multiple gestation.

Adult↗
PubMed1998

Human embryo viability: what determines developmental potential, and can it be assessed?

The understanding of the embryo's nutrient requirements and physiology has led to the development of more physiological culture media, capable of supporting acceptable levels of human blastocyst development in vitro. The success of such media can be attributed to catering to the embryo's changing nutrient requirements, while minimizing culture-induced stress, thereby facilitating normal cell function. Most important, blastocysts derived from such sequential culture systems have a high viability. The ability to identify those blastocysts from within a given cohort which have the highest developmental potential will lead to further increases in implantation and pregnancy rate. Such an approach should ultimately lead to the routine transfer of a single blastocyst in a given IVF cycle, while being able to maintain a high pregnancy rate.

Blastocyst↗
PubMed1998

Evolution of a culture protocol for successful blastocyst development and pregnancy.

A cell-free culture system was designed for human embryo development to the blastocyst stage by testing a range of culture conditions in a series of protocols. The culture system that was evolved has a brief 1 h exposure to spermatozoa and then culture of the pronucleate zygote for 2 days in IVF-50 medium. Two or three embryos were cultured together in 20 microl microdrops of medium under oil. Embryos were then regrouped and two or three at a similar stage were cultured together in 50 microl microdrops of Gardner's G2 medium under oil from days 3 to 5. Embryos were transferred to fresh G2 medium on day 5 and cultured for a further 1 or 2 days (day 6 or 7). No serum was used in any of the cultures. The embryo transfer medium and G2 medium were supplemented with human serum albumin. The zonae of all blastocysts to be transferred to patients were completely removed enzymatically. Using this protocol, 52% of zygotes developed to blastocysts and 34 out of 35 patients treated received 82 blastocysts and 11 morulae on day 5 or 6. Twenty-one fetal sacs with positive heartbeats (23% implantation rate) were detected in 13 ongoing pregnancies (38% pregnancy rate/transfer or 37%/patient treated). We anticipate that further improvements in embryo development and the selection of viable embryos can be achieved using this simple and effective culture system.

Adult↗
PubMed1998

A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization.

The effectiveness of blastocyst culture and transfer in human in-vitro fertilization (IVF) was evaluated in a prospective randomized trial in patients having a moderate to good response to gonadotrophin stimulation. Embryos were transferred either on day 3 after culture to around the 8-cell stage in Ham's F-10 medium supplemented with fetal cord serum, or on day 5 after culture to the blastocyst stage in the sequential serum-free media G 1.2 and G 2.2. The pregnancy rates after transfer on day 3 or day 5 were equivalent, 66 and 71% respectively; however, significantly more embryos were transferred on day 3 (3.7) than on day 5 (2.2). The number of blastocysts transferred did not affect the implantation rate, and pregnancy rates when either two or three blastocysts were transferred were 68 and 87% respectively. The implantation rate of the blastocysts (50.5% fetal heart beat) was significantly higher compared to the cleavage stage embryos transferred on day 3 (30.1%). The percentage of blastocyst development was not affected by the number of 2-pronuclear embryos, or by maternal age. Irrespective of the number of blastocysts formed, pregnancy rates were similar. Furthermore, the pregnancy rate following blastocyst transfer in patients with 10 or more follicles at the time of human chorionic gonadotrophin administration was not affected by patient age. More than 60% of patients having blastocyst culture and transfer had supernumerary embryos for cryopreservation. The establishment of a pregnancy following thaw and transfer confirmed the viability of cryopreserved blastocysts cultured in the absence of serum or co-culture. The ability to transfer just two blastocysts while maintaining high pregnancy rates will therefore help to eliminate high order multiple gestations and improve the overall efficiency of human IVF.

Adult↗
PubMed1998

Intracellular pH of the mouse preimplantation embryo: amino acids act as buffers of intracellular pH.

The inclusion of specific amino acids in conventional culture media has been shown to enhance mammalian embryo development in vitro. Amino acids have been shown to confer their benefits to the preimplantation embryo in a number of different ways. However, their ability to buffer intracellular pH (pHi) has not been investigated. Thus, the aim of this study was to determine if amino acids regulate pHi in the mouse preimplantation embryo. pHi was determined using carboxy-seminaphthorhodafluor-1 (SNARF-1) and confocal microscopy. Incubation with 5,5-dimethyl-2,4-oxazol-idinedione (DMO), a non-metabolizable weak acid, resulted in a significant intracellular acidification in the zygote, 2-, 4- and 8-16-cell embryo. However, in the presence of groups of amino acids, the degree of acidification due to DMO was markedly reduced in the mouse embryo up to the 4-cell stage. Specifically, non-essential amino acids and glutamine had the greatest capacity to buffer pHi in the early embryo. The ability of amino acids to buffer pHi was not apparent from the 8-16-cell stage onwards. In contrast to the precompacted embryo, the morula did not undergo a significant decrease in pHi until exposed to DMO concentrations > or = 10 mM in the absence of amino acids. This may be due to the generation of a permeability seal during compaction, thus enabling the morula to regulate its own pHi. This regulatory ability could either be reversed by causing the morula to decompact, or created by inducing premature compaction in the 8-16-cell embryo. Data presented in this study indicate that amino acids act as buffers of pHi in the early embryo and play a key role in regulating cell physiology. Further evidence for this was provided by the result that only those embryos cultured in 30 mM DMO in the presence of non-essential amino acids and 1 mM glutamine did not block at the 2-cell stage, but grew on to develop into expanded blastocysts.

Amino Acids↗
PubMed1998

Amino acids and vitamins prevent culture-induced metabolic perturbations and associated loss of viability of mouse blastocysts.

Culture of in-vivo-developed mouse blastocysts in a simple culture medium based on a balanced salt solution supplemented with carbohydrates for 3 h significantly perturbed embryo metabolism. Maximal perturbation occurred after just 6 h of culture. Similarly, culture of rat blastocysts in a simple culture medium for 3 h also resulted in perturbed metabolism. Cultured mouse and rat blastocysts both had an abnormally elevated rate of glycolysis of approximately 100% after culture (P < 0.05). Rates of pyruvate oxidation by mouse blastocysts were also significantly reduced after culture in a simple medium for 6 h (P < 0.01). Furthermore, the developmental competence of mouse blastocysts after transfer was significantly reduced by just 6 h of culture in a simple medium (P < 0.05). Addition of Eagle's amino acids or vitamins to the culture medium reduced the perturbation of both the glycolytic activity and oxidative capacity of cultured mouse blastocysts and acted in synergy to further the inhibition. Importantly, culture with amino acids and vitamins prevented any loss of viability of mouse blastocysts after culture for 6 h. It can be concluded that the mouse blastocyst is sensitive to its environment and that culture-induced stress results in the loss of normal cellular function, as manifested in this case by an abnormal pattern of glucose utilization and loss of viability.

Amino Acids↗
PubMed1998

Culture of viable human blastocysts in defined sequential serum-free media.

In human in-vitro fertilization (IVF), embryos are routinely transferred to the uterus on either day 2 or day 3 of development, resulting in a 10-15% implantation rate. However, in other mammalian species, the transfer of cleavage stage embryos, which normally reside in the oviduct, to the uterus results in a significantly lower implantation rate compared with blastocysts. It is therefore proposed that, in order to increase implantation rates in human IVF, one has to move to extended culture and transfer at the blastocyst stage. The transfer of blastocysts will not only help synchronize the embryo with the female tract but will facilitate the identification of those embryos with little or no developmental potential. In order to culture viable blastocysts it is important to use more than one culture medium to cater for the changing requirements of the preimplantation embryo as it develops and differentiates. If sequential culture media are not used, one can obtain blastocysts but their resultant viability is low. The use of sequential serum-free media in human IVF has resulted in > 50% of embryos becoming blastocysts with an implantation rate of approximately 50%. Further advances in human embryo culture should come from the replacement of protein with the glycosaminoglycan hyaluronate, which is more suitable than albumin in supporting implantation in the mouse, and which will eliminate biological variation and possible contamination from blood products. With the routine culture of human blastocysts will come the introduction of non-invasive tests of embryo viability, capable of identifying those blastocysts most likely to develop from a given cohort. As the implantation rate of blastocysts is higher than that of the cleavage stage embryo, fewer embryos will be required for transfer in order to establish a successful pregnancy, thereby reducing the number of multiple gestations and increasing the overall efficiency of human IVF.

Animals↗
PubMed1998

Development of serum-free media for the culture and transfer of human blastocysts.

The culture and transfer of the blastocyst stage embryo has several advantages for assisted reproduction in the human. However, due to inadequacies of present culture conditions in human in-vitro fertilization (IVF), embryos are routinely transferred to the uterus on either day 2 or day 3 of development around the 4- to 8-cell stage, with resultant implantation rates of only 10-25%. In other mammalian species the transfer of cleavage stage embryos, which normally reside in the oviduct, results in a significantly lower implantation rate compared with the transfer of blastocysts. Extended culture of human embryos in vitro will help to identify those embryos with little, if any, developmental potential. It is therefore plausible that the blastocyst has an intrinsically higher viability than the cleavage stage embryo. It has now been shown in human IVF that sequential serum-free media can support > 50% blastocyst development, with an implantation rate per blastocysts of 50%, double that obtained for cleavage stage embryos. As the implantation rate of the blastocyst is higher than the cleavage stage embryo, fewer blastocysts are required for transfer. The development of completely defined embryo culture media may prove feasible by the replacement of protein with the glycosaminoglycan hyaluronate. Hyaluronate, which is protein-free, is more suitable than albumin in supporting implantation in the mouse, and can eliminate the biological variation inherent when using protein and the potential for contamination when using blood products such as albumin.

Animals↗
PubMed1998

Reactivating tammar wallaby blastocysts oxidize glucose.

Metabolic reactivation of the tammar blastocyst appears to be characterized by a change in the pathway of glucose metabolism rather than an absolute increase in substrate uptake. The switch in type of metabolism used was examined to gain information on the timing and physiology of blastocyst reactivation. Fluorescent and radioisotope techniques were used sequentially to determine the activity of pathways of glucose metabolism by individual wallaby blastocysts during diapause and 3, 4, 5, 6, 7, 8, and 10 days after removal of pouch young (RPY). Maternal endometrial and luteal cell metabolism and circulating hormone levels were measured and correlated with blastocyst activity. Observed differences between rates of blastocyst reactivation could be explained by variation in the maternal response between animals. While blastocysts recovered 4 days after RPY oxidized more glucose compared with Day 0 blastocysts (p < 0.05), rates of glycolysis did not change until Day 10. Blastocysts recovered between 4 and 10 days after RPY oxidized a significantly greater percentage of the glucose taken up (p < 0.01). The reduced ATP:ADP ratio within blastocysts recovered 3 days after RPY (p < 0.05) indicates that conditions are suitable for blastocysts to undergo a metabolic switch from glycolytic to oxidative metabolism of glucose on Day 4 after RPY. The increased oxidation results in greater ATP production, which plausibly fuels the increased energy requirements of wallaby blastocysts during the early stages of reactivation.

Adenosine Diphosphate↗
PubMed1997

Modifications made to culture medium by bovine oviduct epithelial cells: changes to carbohydrates stimulate bovine embryo development.

Co-culture remains a common method to support the development of bovine embryos, derived from IVM/IVF procedures. However, the mechanism by which somatic cells confer their benefit to the developing embryo remains undetermined. This study therefore analysed the changes made to the culture medium TCM-199, used in bovine embryo co-culture systems, by somatic cells and determined the effects of specific changes in medium composition on bovine embryo development in culture. Bovine oviduct epithelial (BOE), Buffalo rat liver (BRL) and fibroblast (3T3) cells were compared. The concentrations of glucose, L-lactate, pyruvate, amino acids, NH4+, H+ and the gas tensions of O2 and CO2 were measured in TCM-199 supplemented with 10% fetal calf serum (FCS) prior to and directly following 48 h incubation periods with each cell type. All three somatic cell types modified the carbohydrate composition of the media in a similar manner with the greatest changes made by the BOE cells. Notable alterations were an increase in the levels of L-lactate and pyruvate and a reduction in glucose concentration, which in the case of the BOE cells, fell from 5.55 mM to 2.67 mM. In order to determine the relevance of such changes in carbohydrate concentrations on bovine embryo development, modifications were made to carbohydrate levels in synthetic oviduct fluid (SOF) medium and their effect on blastocyst development in vitro assessed. In SOF medium supplemented with amino acids and BSA (SOFaa), significantly more zygotes developed to the blastocyst stage (64%; P < 0.01) than in SOFaa medium with the concentrations of glucose, D/L-lactate and pyruvate equivalent to those in TCM-199 (11%). Interestingly, when the levels of carbohydrates in SOFaa mimicked those present in TCM-199 following a 48 h incubation with BOE cells, 57% of zygotes reached the blastocyst stage. This improvement was ascribed to the reduction in glucose and increases in D/L-lactate and pyruvate concentrations in the culture system. Results from this study demonstrate that BOE cells create an environment favourable to embryonic development. The analysis of media samples by enzymatic methods meant that only the biologically active L-isomer of lactate was quantified. However, in SOFaa, both the L-isomer and inactive D-isomer are present in equimolar amounts. As such, culture media in which D/L-lactate syrup is used actually contain only 50% biologically active lactate meaning that all D/L-lactate concentrations are reported at twice the effective concentration. Therefore the effect of D/L-lactate concentration on blastocyst development was subsequently determined in this study. Blastocyst development was poor (24-36%) until the total D/L-lactate was present in the culture system at concentrations equal to or greater than 0.82 mM. However, blastocyst cell numbers remained low (60.1 +/- 6.9 - 78.5 +/- 6.6) until a total D/L-lactate concentration of 3.3 mM. This data reinforces that embryo morphological appearance is not sensitive enough to be used as the sole criterion for assessing embryo development.

3T3 Cells↗
PubMed1997

Nonessential amino acids and glutamine decrease the time of the first three cleavage divisions and increase compaction of mouse zygotes in vitro.

PURPOSE: The objective of this study was to determine the effect of supplementing embryo culture media with amino acids on the duration of the first three cell cycles of mouse zygotes in vitro. METHODS: Zygotes were cultured in the presence of different groups of amino acids and cleavage assessed every 30 min. RESULTS: Culture of zygotes with Eagle's nonessential amino acids and glutamine significantly reduced the time of cleavage divisions to the eight-cell stage compared to culture without amino acids. Beneficial effects of amino acids were found to be cumulative over time. Nonessential amino acids and glutamine also increased the percentage of eight-cells that compacted after 57 hr of culture compared to embryos in medium devoid of amino acids. CONCLUSIONS: The present data suggest that media for the development of cleavage-stage embryos, such as in clinical IVF, should be supplemented with Eagle's nonessential amino acids and glutamine.

Amino Acids↗
PubMed1997

Postpartum mood disorders: clinical perspectives.

Mood disorders are common in women. A prepregnancy personal history of mood disorder (bipolar or major depression), premenstrual syndrome, or (possibly) postpartum blues places a woman at high risk for a postpartum exacerbation of symptoms. Untreated or unrecognized postpartum mood disorders can lead to serious psychologic and social consequences, in some cases even leading to suicide or infanticide. Women at risk for postpartum mood disorders need to be referred for psychiatric consultation before pregnancy and parturition. Informed, professional collaboration offers the best opportunities for prevention, as well as the earliest recognition and treatment of emergent symptoms.

Affective Disorders, Psychotic↗
PubMed1997

Differential regulation of mouse embryo development and viability by amino acids.

The amino acid requirements of the preimplantation mouse embryo in culture changes as development proceeds from the zygote to the blastocyst stage. Eagle's non-essential amino acids and glutamine significantly increased cleavage rates during the first four cell cycles, while Eagle's essential amino acids without glutamine did not confer any benefit to embryo development before the eight-cell stage. After the eight-cell stage, non-essential amino acids and glutamine no longer stimulated cleavage rates but significantly increased blastocoel development and blastocyst hatching. In contrast, after the eight-cell stage essential amino acids increased cleavage rates as well as stimulating development of the inner cell mass of the resultant blastocysts. Fetal development after transfer of blastocysts was also significantly increased by culture with essential amino acids from the eight-cell stage. Consequently highest rates of development in vitro and viability after transfer were achieved when embryos were cultured with non-essential amino acids and glutamine to the eight-cell stage followed by development to the blastocyst stage in the presence of all 20 amino acids. Analysis of the parameters measured revealed a significant relationship between number of blastocyst cells and inner cell mass development with viability after transfer. Blastocyst formation and hatching could not be used to assess subsequent developmental potential.

Amino Acids↗
PubMed1996

Carbohydrate uptake by quiescent and reactivated mouse blastocysts.

The mouse, Mus musculus, can maintain blastocysts in embryonic diapause in the uterus while suckling young. This study used microfluorimetry to simultaneously examine glucose and pyruvate uptake by quiescent blastocysts, and at four hourly intervals after administration of the reactivating stimulus, oestradiol-17 beta. Following the non-invasive analysis of energy metabolism, blastocysts were incubated in colcemid (0.2 mg/ml), and mitotic activity determined. Mitoses and cell numbers in reactivated embryos increased significantly within 8 and 12 hours, respectively, after oestradiol-17 beta administration, compared to those of diapause (control) blastocysts (0.5 +/- 0.1 vs. 0.22 +/- 0.03 mitoses/embryo; P < 0.05, and 141.8 +/- 1.5 vs. 133.8 +/- 2.4 cells/ embryo; P < 0.05). Similarly, pyruvate uptake by reactivating blastocysts (9.3 +/- 1.1) was significantly higher than controls (5.8 +/- 0.8 pmol/embryo/hour; P < 0.05), within 4 hours of oestradiol-17 beta, but by 16 hours after oestradiol-17 beta administration, pyruvate uptake by reactivating blastocysts was no longer significantly different from the delayed controls. In contrast, significant differences in glucose uptake between the reactivated and control groups were not evident until 16 hours after oestradiol-17 beta (reactivating, 14.9 +/- 1.5; control, 10.6 +/- 1.7 pmol/embryo/hour; P < 0.05). These results demonstrate that pyruvate rather than glucose could supply the additional energy required during the first 12 hours of reactivation in the mouse, but from 16 hours after injection of the reactivating stimulus oestradiol-17 beta, glucose is the predominant energy source.

Animals↗
PubMed1996

Nutrient uptake and utilization can be used to select viable day 7 bovine blastocysts after cryopreservation.

The ability of individual bovine blastocysts to survive freezing and thawing procedures was assessed by measuring glucose and pyruvate uptake and lactate production immediately before and after cryopreservation. Using glucose and pyruvate uptake and lactate production it was not possible to determine, prior to freezing, which blastocysts would be viable after thawing. However, in the 5 hr immediately after thawing, those blastocysts which expanded their blastocoel had significantly greater glucose and pyruvate uptake and lactate production (P < 0.01) than those embryos which failed to develop after a 14 hr overnight incubation. Interestingly, after thawing, two distinct populations of blastocysts existed with respect to glucose uptake and lactate production, indicating that it is possible to identify those blastocysts immediately after thawing which will reexpand. In contrast, there was a considerable degree of overlap in pyruvate uptakes between the viable and nonviable groups of embryos, indicating that this parameter could not be used to select viable embryos after thawing. There was an increase in the calculated oxidation of carbohydrates after thawing, consistent with a partial uncoupling of the inner mitochondrial membrane. In conclusion, glucose uptake and lactate production can be used to select prospectively viable blastocysts immediately after thawing, indicating that glycolysis is a major energy-generating pathway for the embryo at this time.

Animals↗
PubMed1996

Environment of the preimplantation human embryo in vivo: metabolite analysis of oviduct and uterine fluids and metabolism of cumulus cells.

OBJECTIVE: To determine the levels of metabolites surrounding the human oocyte and embryo in vivo. DESIGN: Oviduct and uterine fluids were collected throughout the menstrual cycle. Cumulus cells were collected at oocyte retrieval and their production of metabolites was assessed. Samples were analyzed for pyruvate, lactate, and glucose by microfluorimetry. PATIENTS: Luminal fluids were collected from naturally cycling patients at the time of routine clinical investigation. Patient consent and hospital ethics approval were obtained for this study. RESULTS: Pyruvate in the oviduct did not vary with the day of cycle, the mean value was 0.24 mM. Lactate and glucose concentrations varied with the day of cycle; lactate increasing from 4.87 mM in the follicular phase to 10.50 mM at the time of ovulation, whereas glucose decreased from 3.11 mM in the follicular phase to 0.50 mM midcycle and subsequently increased to 2.32 mM in the luteal phase. The concentrations of pyruvate, lactate, and glucose in uterine fluid remained constant throughout the cycle (0.10, 5.87, and 3.15 mM, respectively). All metabolite concentrations in uterine fluid were significantly different from those in the oviduct midcycle. Cumulus cells readily consumed glucose in vitro, with lactate being the major metabolite produced. CONCLUSION: These data indicate that lactate and glucose concentrations in the oviduct change with day of cycle and that the human embryo is exposed to different metabolite concentrations as it passes along the tract. Furthermore, cumulus cells readily consume glucose, producing lactate. Therefore, the early human embryo is exposed to low glucose and high lactate levels in vivo.

Body Fluids↗