Deanne Feil

Human cumulus cell gene expression as a biomarker of pregnancy outcome after single embryo transfer

OBJECTIVE To identify the cumulus cell gene expression associated with oocyte developmental competence, specifically live birth, after single ET (SET) assisted reproductive technology. DESIGN Retrospective gene expression analysis in human cumulus cells from oocytes that established a pregnancy resulting in live birth versus no pregnancy after SET. SETTING Independent IVF clinic and research institute. PATIENT(S) Women undergoing IVF/intracytoplasmic sperm injection with SET. INTERVENTION(S) Quantitative reverse-transcriptase-polymerase chain reaction analysis was performed on cumulus masses collected before insemination. Oocytes and embryos were cultured and transferred independently in 38 patients undergoing elective SET. Paired cumulus samples from oocytes that developed into high- versus low-grade embryos also were compared. MAIN OUTCOME MEASURE(S) Gene expression profiles of metabolic (ALDOA, LDHA, PFKP, PKM2), signaling (AHR, GREM1, PTGS2, STS), extracellular matrix (HAS2, PTX3, TNFAIP6, VCAN), and loading control GAPDH in individual cumulus masses. RESULT(S) VCAN and PTGS2 mRNA expression was significantly higher in cumulus cells from oocytes yielding a pregnancy resulting in a live birth, while PTX3 mRNA expression trended toward higher expression in pregnant samples. Cumulus cell levels of VCAN, GREM1, and PFKP correlated with birth weight in patients at 38 weeks of gestation. No genes correlated with clinical embryo morphology scores. CONCLUSION(S) Cumulus cell VCAN, PTGS2, GREM1, and PFKP expression may identify oocytes with high developmental potential, leading to enhanced implantation rates and greater developmental capacity throughout gestation.

Effect of culturing mouse embryos under different oxygen concentrations on subsequent fetal and placental development.

The oxygen concentration used during embryo culture can influence embryo development and quality. Reducing the oxygen concentration in the atmosphere to 2% during post‐compaction culture of mouse embryos perturbs embryonic gene expression. This study examined the effect of culturing mouse embryos under different oxygen concentrations on subsequent fetal and placental development. Embryos were cultured from the zygote to morula stage under 7% oxygen, followed by 20, 7 or 2% oxygen to the blastocyst stage. Cultured and in vivo developed blastocysts were transferred into pseudopregnant recipients. Fetal and placental outcomes were analysed at day 18 of pregnancy. Implantation rate was not influenced by embryo culture conditions, but resorption rates were increased in embryos cultured under 2% oxygen, compared with 7% oxygen. Day 18 fetal weights were reduced following culture under 2%, compared with 7 or 20% oxygen, or in vivo development. Placental weight was not influenced by culture conditions. No differences in the proportion of junctional or labyrinthine exchange regions within the placenta or the morphometry of the labyrinthine region were detected. Surface density (surface area/gram labyrinth) of trophoblast available for exchange was reduced in placentas developed from embryos cultured under 2% oxygen, compared with 7% oxygen. Placental gene expression of Slc2a1, Slc2a3, Igf2, Igf2r and H19 was not influenced by oxygen conditions during embryo culture. Thus, exposure to 2% oxygen during post‐compaction pre‐implantation embryo development has adverse consequences for fetal development in the mouse. Oxygen is a significant component of the embryonic environment and reductions in oxygen availability can influence both embryonic gene expression and subsequent fetal development.

Utilization of endogenous fatty acid stores for energy production in bovine preimplantation embryos

Although current embryo culture media are based on carbohydrate metabolism of embryos, little is known about metabolism of endogenous lipids. L-carnitine is a β-oxidation cofactor absent in most culture media. The objective was to investigate the influence of L-carnitine supplementation on bovine embryo development. Abattoir-derived bovine cumulus oocyte complexes were cultured and fertilized. Post-fertilization, presumptive zygotes were transferred into a basic cleavage medium ± carbohydrates (glucose, lactate and pyruvate) ± 5 mm L-carnitine and cultured for 4 days in vitro. In the absence of carbohydrates during culture, embryos arrested at the 2- and 4-cell stages. Remarkably, +L-carnitine increased development to the morula stage compared to +carbohydrates alone (P < 0.001). The beneficial effects of L-carnitine were further demonstrated by inclusion of carbohydrates, with 14-fold more embryos reaching the morula stage after culture in the +carbohydrates +L-carnitine group compared to the +carbohydrates group (P < 0.05). Whereas there was a trend for +L-carnitine to increase ATP (P = 0.09), ADP levels were higher and ATP: ADP ratio were 1.9-fold lower (main effect, P < 0.05) compared to embryos cultured in -L-carnitine. Therefore, we inferred that +L-carnitine embryos were more metabolically active, with higher rates of ATP-ADP conversion. In conclusion, L-carnitine supplementation supported precompaction embryo development and there was an additive effect of +L-carnitine +carbohydrates on early embryo development, most likely through increased β-oxidation within embryos.

Day 4 embryo selection is equal to Day 5 using a new embryo scoring system validated in single embryo transfers

BACKGROUND Single embryo transfers (SETs) require the most viable embryo from a cohort to be selected. Day 4 embryos may provide a selection advantage similar to blastocyst transfer as embryos are replaced post-embryonic genome activation and into the uterus where they would normally reside. However, there is currently no adequate morphological system to assess Day 4 embryos. Therefore, we developed an assessment system for Day 4 embryos and retrospectively assessed Day 4 SET success rates compared with Day 5 SETs. METHODS Embryos (n = 996 after IVF or ICSI) were assessed on Day 4 of development, prior to implementing Day 4 embryo transfers, to obtain a descriptive range of embryo morphologies observed at this time point. A morphological scoring system was developed from this. All patients having an SET after extended culture (Day 4, n = 124 or Day 5, n = 193), at Repromed, Adelaide, Australia, during June 2006-January 2007 were analysed for pregnancy outcome. Ongoing pregnancy was determined by fetal cardiac activity at 6-8 weeks after oocyte collection. RESULTS Day 4 and 5 SETs resulted in similar ongoing pregnancy rates of 38.7% and 32.1%, respectively. CONCLUSIONS A Day 4 scoring system was successfully developed and implemented. Day 4 SETs were found to be a viable option or alternative to Day 5 SETs with no difference in pregnancy rates.

Effect of varying glucose and glucosamine concentration in vitro on mouse oocyte maturation and developmental competence

The effects of hyper- and hypo-glycaemic conditions during the in vitro maturation of mouse cumulus-oocyte complexes on developmental competence were examined, with an emphasis on the role of the hexosamine biosynthesis pathway. A low (1 mM) glucose concentration achieved optimal oocyte competence (3-fold higher blastocyst development rate compared with high (30 mM) glucose, P<0.05). In addition, glucose supplementation during only the first hour after release from the follicle was necessary and sufficient to support oocyte maturation and embryo development to the blastocyst stage. Glucosamine (a known hyperglycaemic mimetic and specific activator of the hexosamine pathway) was able to substitute for glucose during this first hour, indicating that flux through the hexosamine pathway is essential for oocyte competence. In the absence of glucose throughout the maturation period, glucosamine was not able to increase developmental competence, and at higher concentrations (2.5 and 5 mM) had a detrimental effect on MII and blastocyst development rates, compared with controls (P<0.05). These experiments underscore the importance of glucose metabolic pathways during in vitro maturation and support the concept that excess flux through the hexosamine pathway has detrimental consequences.

92 UTILISATION OF ENDOGENOUS FATTY ACID STORES FOR ENERGY PRODUCTION IN BOVINE PRE-IMPLANTATION EMBRYOS

Current embryo culture media are based on the carbohydrate metabolism of embryos. However, little is known about the metabolism of endogenous lipids. This is surprising given the high intracellular lipid densities of embryos of some species and the potential for ATP production via β-oxidation. L-carnitine is a β-oxidation co-factor that is absent in most culture media. The aim of this study is to investigate the influence of carnitine supplementation ± carbohydrates on bovine embryo development. Abattoir-derived cattle cumulus–oocyte complexes were cultured and fertilised (Sutton-McDowall et al. 2006 Biol. Reprod. 74, 881–888). Post-fertilisation (24 h), presumptive zygotes were transferred into an amino acid-free cleavage media ± carbohydrates (glucose, lactate and pyruvate) ±5 mM carnitine and cultured for 4 days. The absence of carbohydrates during culture resulted in embryos arresting at the 2- and 4-cell stages. Remarkably, +carnitine significantly increased development to the morula stage compared with +carbohydrates alone (20.4 ± 3% vs 4.7 ± 2.5% morula development; P < 0.001). The combination of carbohydrates and carnitine supplementation further improved embryo development, with 14-fold more embryos reaching the morula stage after culture in the +carbohydrates +carnitine group compared with the +carbohydrates group (+carbohydrates = 3.1 ± 1.9 vs +carbohydrates +carnitine = 43.8 ± 9.1% morula development; P < 0.05). The beneficial effects of carnitine supplementation on embryo development were reversed when embryos were cultured in presence of etomoxir, a non-reversible inhibitor of the rate-limiting enzyme of β-oxidation (development to 8-cell stage; +carnitine = 33.9 ± 8% vs +carnitine +etomoxir = 19.2 ± 4.9%; P < 0.05). Intracellular lipid content of embryos +carnitine was determined by culturing presumptive zygotes in media -carbohydrates ± carnitine for 24 h. Lipid content of embryos was determined by measuring BODIPY 493/503 dye fluorescence. Carnitine supplementation reduced fluorescence intensity 1.8-fold (P < 0.001). Adenosine triphosphate and ATP:ADP levels were measured in embryos after 24 h of culture ± carbohydrates ± carnitine. While there was a trend for +carnitine to increase ATP levels (P = 0.09), ADP levels were higher and ATP:ADP ratio were 1.9-fold lower (main effect, P < 0.05) compared with embryos cultured in –carnitine. This indicates +carnitine embryos were more metabolically active, with higher rates of ATP-ADP conversion. We have shown carnitine supplementation supports pre-compaction embryo development and there is an additive effect of +carnitine +carbohydrate on early embryo development. This is most likely through increased β-oxidation levels within embryos. Current disparities between in vivo and in vitro embryo production, in particular increased lipid content (Romek et al. 2010 Theriogenology 74, 265–276) and decreased developmental potential of in vitro-produced embryos, may be an artefact resulting from limited lipid oxidation in vitro.

Mitochondrial fatty acid transport enzyme deficiency—implications for in vitro fertilization

OBJECTIVE To describe the individualized clinical and embryologic care required by a patient with adult-onset carnitine palmitoyl transferase II deficiency during IVF treatment and in a subsequent pregnancy. DESIGN A case study. SETTING(S) A private fertility unit and public obstetric hospital. PATIENT(S) A patient with a rare mutation in the mitochondrial fatty acid transport enzyme, carnitine palmitoyl transferase II. INTERVENTION(S) The patient received (IVF) fertility treatment and pregnancy care. MAIN OUTCOME MEASURE(S) Pregnancy and delivery of a healthy child. RESULT(S) This patient conceived on her first cycle of IVF and went on to deliver a healthy child. CONCLUSION(S) Patients with adult-onset carnitine palmitoyl transferase II deficiency can have successful pregnancy outcomes after IVF, provided clinical and embryologic care is optimized.