Michelle Lane

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.

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.

Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality

Oocyte quality is a key limiting factor in female fertility, yet we have a poor understanding of what constitutes oocyte quality or the mechanisms governing it. The ovarian follicular microenvironment and maternal signals, mediated primarily through granulosa cells (GCs) and cumulus cells (CCs), are responsible for nurturing oocyte growth, development and the gradual acquisition of oocyte developmental competence. However, oocyte-GC/CC communication is bidirectional with the oocyte secreting potent growth factors that act locally to direct the differentiation and function of CCs. Two important oocyte-secreted factors (OSFs) are growth-differentiation factor 9 and bone morphogenetic protein 15, which activate signaling pathways in CCs to regulate key genes and cellular processes required for CC differentiation and for CCs to maintain their distinctive phenotype. Hence, oocytes appear to tightly control their neighboring somatic cells, directing them to perform functions required for appropriate development of the oocyte. This oocyte-CC regulatory loop and the capacity of oocytes to regulate their own microenvironment by OSFs may constitute important components of oocyte quality. In support of this notion, it has recently been demonstrated that supplementing oocyte in vitro maturation (IVM) media with exogenous OSFs improves oocyte developmental potential, as evidenced by enhanced pre- and post-implantation embryo development. This new perspective on oocyte-CC interactions is improving our knowledge of the processes regulating oocyte quality, which is likely to have a number of applications, including improving the efficiency of clinical IVM and thereby providing new options for the treatment of infertility.

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.

Paternal obesity initiates metabolic disturbances in two generations of mice with incomplete penetrance to the F2 generation and alters the transcriptional profile of testis and sperm microRNA content

Obesity is highly prevalent, and its incidence is increasing. The previous study showing a major effect of paternal obesity on metabolic health of offspring is confounded by comorbidity with diabetes. Therefore, we investigated the effect of diet‐induced paternal obesity, in the absence of diabetes, on the metabolic health of two resultant generations and the molecular profiles of the testes and sperm. Founder (F0) male C57BL6 mice were fed either a high‐fat diet (HFD) or a control diet (CD); n = 10/diet for a period of 10 wk. Testis expression of mRNA/microRNAs was analyzed by microarray and qPCR and sperm microRNA abundance by qPCR Two subsequent generations were generated by mating F0 and then F1 mice to CD mice, and their metabolic health was investigated. All mice, other than F0 males, were maintained on a CD. HFD feeding induced paternal obesity with a 21% increase in adiposity, but not overt diabetes, and initiated intergenerational transmission of obesity and insulin resistance in two generations of offspring. This distinct phenotypic constellation is either partially or fully transmitted to both female and male F1 offspring and further transmitted through both parental lineages to the F2 generation, with a heightened effect on female F1 offspring (+67% in adiposity) and their F2 sons (+24% in adiposity). Founder male obesity altered the testes expression of 414 mRNAs by microarray and 11 microRNAs by qPCR, concomitant with alterations in sperm microRNA content and a 25% reduction in global methylation of germ cell DNA Diet‐induced paternal obesity modulates sperm microRNA content and germ cell methylation status, which are potential signals that program offspring health and initiate the transmission of obesity and impaired metabolic health to future generations. This study implicates paternal obesity in the transgenerational amplification of obesity and type 2 diabetes in humans.—Fullston, T., Ohlsson Teague, E. M. C., Palmer, N. O., DeBlasio, M. J., Mitchell, M., Corbett, M., Print, C. G., Owens, J. A., Lane, M., Paternal obesity initiates metabolic disturbances in two generations of mice with incomplete penetrance to the F2 generation and alters the transcriptional profile of testis and sperm microRNA content. FASEBJ. 27, 4226‐4243 (2013). www.fasebj.org

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.

Obese Women Exhibit Differences in Ovarian Metabolites, Hormones, and Gene Expression Compared with Moderate-Weight Women

CONTEXT Obese women experience longer times to conception, even if they are young and cycling regularly, which is suggestive of alterations in ovarian function during the periconceptual period. OBJECTIVE This study sought to determine whether there are alterations in the preovulatory follicular environment that are likely to influence oocyte developmental competence. DESIGN, SETTING, AND PARTICIPANTS Women attending a private infertility clinic were categorized into body mass index (BMI) groups of moderate (n = 33; BMI 20-24.9 kg/m(2)), overweight (n = 31; BMI 25-29.9 kg/m(2)), and obese (n =32; BMI >or=30 kg/m(2)). INTERVENTION For each patient, follicular fluid was recovered from single follicles at oocyte retrieval, granulosa cells were pooled from multiple follicular aspirates and cumulus cells were pooled after separation from the oocytes. MAIN OUTCOME MEASURES Follicle fluid was assayed for hormones and metabolites. Granulosa and cumulus cells were analyzed for mRNA expression of insulin signaling components (IRS-2 and Glut4), glucose-regulated genes (ChREBP, ACC, and FAS) and insulin-regulated genes (SREBP-1, CD36, and SR-BI) associated with obesity/insulin resistance. RESULTS Increasing BMI was associated with increased follicular fluid insulin (P < 0.001), lactate (P = 0.01), triglycerides (P = 0.0003), and C-reactive protein (P < 0.0001) as well as decreased SHBG (P = 0.001). IRS-2, Glut4, ChREBP, and SREBP exhibited cell-type-specific expression but were not affected by BMI. CD36 and SRBI mRNA were modestly altered in granulosa cells of obese compared with moderate-weight women. CONCLUSIONS Obese women exhibit an altered ovarian follicular environment, particularly increased metabolite, C-reactive protein, and androgen activity levels, which may be associated with poorer reproductive outcomes typically observed in these patients.

Containerless vitrification of mammalian oocytes and embryos

Adapting a proven method for flash-cooling protein crystals to the cryopreservation of live cells.

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.