David K. Gardner

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.

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 Hams 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.

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.

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.

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.

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 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.

Ammonium Induces Aberrant Blastocyst Differentiation, Metabolism, pH Regulation, Gene Expression and Subsequently Alters Fetal Development in the Mouse

Abstract 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°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 μM 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.

Handbook of in Vitro Fertilization

Historical Perspective of IVF-Carl Wood and Alan O. TrounsonIVF patient preparation-Carl Wood and Gillian GravesEndocrinology of IVF-Talia Eldar-Geva, Ilan Calderon, Vivien MacLachlan and David HealyOocyte Retrieval-Luk rombauts and Carl WoodIn vitro maturation and developmental comptetence of human primary oocytes-Frank BarnesAssessment of the male and preparation of sperm for ARTs-Gordon Baker, D.Y. Liu and Harold BourneIn Vitro Fertilization-Ariff Bongso and Alan TrounsonAssisted Fertilization-Andre Van SteirteghamEmbryo Development-Ariff Bongso and David GardnerCoCulture of Embryos with Somatic Helper Cells-Denny Sakkas, Ariff Bongso and David GardnerEmbryo Culture Systems-David Gardner and Michelle LaneMicromanipulation as a clinical tool-Jacques Cohen, Steen Willadsen, Tim Schimmel and Jacob LevronPreimplantation genetic diagnosis of numerical abormalities using FISH-Santiago MunneChromosomal Analysis of Development-Ariff BongsoAssessment of Embryo Metabolism and Viability-David Gardner and Henry J. LeeseCryopreservation of Oocytes and Embryos-Jill Shaw, Apichart Oranratnachai and Alan O. TrounsonTransplantation and cryopreservation of ovarian tissue-Jillian Shaw, Carl Wood and Alan O. TrounsonUltrastructure of Human Gamete, Fertilization and Embryo Development-A. Henry SathananthanUltrastructure of ICSI-A. Henry Sathananthan and Alan TrounsonOocyte and Embryo Donation-John Leeton and Alan O. TrounsonUterine receptivity and embryo transfer-Peter Rogers and John LeetonEthical Considerations of IVF and Human Embryo Research-Karen DawsonFuture developments in IVF and related technologies-Alan O. Trounson and Carl WoodIndex

Mammalian embryo culture in the absence of serum or somatic cell support

Development of the mammalian preimplantation embryo in culture is associated with retarded cleavage rates (Bowman and McLaren, 1970; Streffer et aI., 1980), reduction ofviability (Lane and Gardner, 1992) or even developmental arrest (Goddard and Pratt, 1983; Gandolfi and Moor, 1987), culminating in reduced pregnancy rates in clinical IVF and assisted breeding programs in domestic animals. The development of more suitable culture conditions for the mammalian preimplantation embryo has therefore been the focus of extensive research over the past decade.