Jennifer L. Eaton

Embryo culture media and neonatal birthweight following IVF

BACKGROUND Infants conceived from IVF are at increased risk for low birthweight. Animal studies suggest that embryo culture medium influences birthweight but it is unknown whether this association exists in humans. This study examines the relationship between culture medium and birthweight following IVF. METHODS We identified all IVF cycles with start dates between 1 January 1999 and 31 December 2008 that used autologous oocytes with resulting embryos cultured in G1.3, Global or G1.5 medium. The population was restricted to singleton deliveries following Day 3, fresh single embryo transfer, or twin deliveries following Day 3, fresh double embryo transfer, at a gestational age of ≥ 34 weeks. Only the first cycle during the study period was included for each woman. Women were excluded if the number of gestational sacs on ultrasound differed from the number of infants born. Variables were evaluated with the χ²-test or analysis of variance. Multiple linear regressions controlled for potential confounders. RESULTS Of the 198 women with singleton deliveries, 102 embryos were cultured in G1.3, 53 in Global and 43 in G1.5 medium. Of the 303 twin deliveries, 172 pairs of embryos were cultured in G1.3, 58 in Global and 73 in G1.5 medium. No significant association between culture medium and birthweight was observed, even when controlling for potential confounders. CONCLUSIONS This retrospective study demonstrated no significant association between embryo culture medium and birthweight following IVF. Although our careful selection of patients minimized the influence of potential confounders, further research is required to elucidate this issue with larger numbers of patients.

Amniocytes can serve a dual function as a source of iPS cells and feeder layers

Clinical barriers to stem-cell therapy include the need for efficient derivation of histocompatible stem cells and the zoonotic risk inherent to human stem-cell xenoculture on mouse feeder cells. We describe a system for efficiently deriving induced pluripotent stem (iPS) cells from human and mouse amniocytes, and for maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are thus autologous to a single donor. Moreover, the use of human feeder cells reduces the risk of zoonosis. Generation of iPS cells using retroviral vectors from short- or long-term cultured human and mouse amniocytes using four factors, or two factors in mouse, occurs in 5–7 days with 0.5% efficiency. This efficiency is greater than that reported for mouse and human fibroblasts using similar viral infection approaches, and does not appear to result from selective reprogramming of Oct4+ or c-Kit+ amniocyte subpopulations. Derivation of amniocyte-derived iPS (AdiPS) cell colonies, which express pluripotency markers and exhibit appropriate microarray expression and DNA methylation properties, was facilitated by live immunostaining. AdiPS cells also generate embryoid bodies in vitro and teratomas in vivo. Furthermore, mouse and human amniocytes can serve as feeder layers for iPS cells and for mouse and human embryonic stem (ES) cells. Thus, human amniocytes provide an efficient source of autologous iPS cells and, as feeder cells, can also maintain iPS and ES cell pluripotency without the safety concerns associated with xenoculture.

Pregnancy outcomes decline with increasing body mass index: analysis of 239,127 fresh autologous in vitro fertilization cycles from the 2008-2010 Society for Assisted Reproductive Technology registry.

OBJECTIVE To examine the effect of body mass index (BMI) on IVF outcomes in fresh autologous cycles. DESIGN Retrospective cohort study. SETTING Not applicable. PATIENT(S) A total of 239,127 fresh IVF cycles from the 2008-2010 Society for Assisted Reproductive Technology registry were stratified into cohorts based on World Health Organization BMI guidelines. Cycles reporting normal BMI (18.5-24.9 kg/m(2)) were used as the reference group (REF). Subanalyses were performed on cycles reporting purely polycystic ovary syndrome (PCOS)-related infertility and those with purely male-factor infertility (34,137 and 89,354 cycles, respectively). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Implantation rate, clinical pregnancy rate, pregnancy loss rate, and live birth rate. RESULT(S) Success rates and adjusted odds ratios (ORs) with 95% confidence intervals (CIs) for all pregnancy outcomes were most favorable in cohorts with low and normal BMIs and progressively worsened as BMI increased. Obesity also had a negative impact on IVF outcomes in cycles performed for PCOS and male-factor infertility, although it did not always reach statistical significance. CONCLUSION(S) Success rates in fresh autologous cycles, including those done for specifically PCOS or male-factor infertility, are highest in those with low and normal BMIs. Furthermore, there is a progressive and statistically significant worsening of outcomes in groups with higher BMIs. More research is needed to determine the causes and extent of the influence of BMI on IVF success rates in other patient populations.

Assessment of day-3 morphology and euploidy for individual chromosomes in embryos that develop to the blastocyst stage.

OBJECTIVE To examine the relationship between day-3 morphology and euploidy for individual chromosomes in embryos that develop to the blastocyst stage by day 5. DESIGN Retrospective cohort study. SETTING Boston IVF, a large university-affiliated reproductive medicine practice. PATIENT(S) Ninety-nine patients undergoing their first preimplantation genetic screening (PGS) cycle between January 1 and December 31, 2006. INTERVENTION(S) In vitro fertilization (IVF) and preimplantation genetic screening (PGS). MAIN OUTCOME MEASURE(S) Prevalence of euploidy for chromosomes X, Y, 8, 13, 14, 15, 16, 17, 18, 20, 21, and 22 in day-3 high implantation potential (HIP) versus non-HIP embryos that grew to day-5 blastocysts. RESULT(S) Seven hundred three embryos from 99 cycles in 99 patients underwent PGS. Three hundred sixty-four (52%) embryos from 88 cycles in 88 patients developed to the blastocyst stage by day 5. High implantation potential embryos were more likely to be euploid for chromosomes X/Y, 8, 15, 16, 18, and 22 compared with non-HIP embryos, with similar trends for chromosomes 14 and 17. There were no statistically significant differences between HIP and non-HIP embryos in euploidy prevalence for chromosomes 13, 20, and 21. CONCLUSION(S) Our data suggest that PGS may detect potentially viable but detrimental chromosomal abnormalities that are not detected by embryo morphology alone.

Increased AKT or MEK1/2 Activity Influences Progesterone Receptor Levels and Localization in Endometriosis

CONTEXT Endometriosis is characterized by progesterone resistance and hyperactivity of the AKT and MAPK pathways. Kinases can cause posttranslational modifications of the progesterone receptor (PR) to influence cellular localization and protein stability. OBJECTIVE The objective of this study was to determine whether the increased AKT or MAPK kinase-1/2 (MEK1/2) activity observed in endometriotic stromal cells (OSIS) from ovarian endometriomas influences levels of PR protein. In turn, the effects of inhibiting AKT or MEK1/2 in the presence of the progestin R5020 on cell viability were investigated. RESULTS Inhibiting AKT with MK-2206 or MEK1/2 with U0126 for 24 hours in the absence of R5020 increased total and nuclear PRA and PRB protein levels in OSIS but not in eutopic endometrial stromal cells from disease-free patients from disease-free patients. MK-2206 and R5020 decreased OSIS viability and increased apoptosis. Trends toward decreased volumes of sc grafted endometriosis tissues were demonstrated with MK-2206 and progesterone. CONCLUSIONS Inhibition of AKT or MEK1/2 increased total and nuclear PR protein in OSIS. MK-2206 and R5020 decreased OSIS viability and increased apoptosis. The AKT and MAPK pathways may be potential molecular targets for the treatment of endometriosis.

Pregnancy outcomes decline with increasing recipient body mass index: an analysis of 22,317 fresh donor/recipient cycles from the 2008–2010 Society for Assisted Reproductive Technology Clinic Outcome Reporting System registry

OBJECTIVE To examine the effect of recipient body mass index (BMI) on IVF outcomes in fresh donor oocyte cycles. DESIGN Retrospective cohort study. SETTING Not applicable. PATIENT(S) A total of 22,317 donor oocyte cycles from the 2008-2010 Society for Assisted Reproductive Technology Clinic Outcome Reporting System registry were stratified into cohorts based on World Health Organization BMI guidelines. Cycles reporting normal recipient BMI (18.5-24.9) were used as the reference group. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Implantation rate, clinical pregnancy rate (PR), pregnancy loss rate, live birth rate. RESULT(S) Success rates and adjusted odds ratios with 95% confidence intervals for all pregnancy outcomes were most favorable in cohorts of recipients with low and normal BMI, but progressively worsened as BMI increased. CONCLUSION(S) Success rates in recipient cycles are highest in those with low and normal BMI. Furthermore, there is a progressive and statistically significant worsening of outcomes in groups with higher BMI with respect to clinical pregnancy and live birth rate.

Fertility and infertility in rheumatoid arthritis.

Purpose of reviewDespite decades of evidence suggesting that women with rheumatoid arthritis (RA) have fewer children than their healthy peers, this information is not widely known among clinicians. The causes of decreased fertility in this population have been largely unexplored, but likely revolve around altered inflammation, increased age when conception is attempted, limited sexual function, and possibly effects of medications on ovarian function. Recent findingsSeveral large Scandinavian cohorts and a cohort study in the United States demonstrate that women with RA have smaller families and are slower to conceive compared with other women. Personal choice to limit family size plays some role, as does infertility. Sexual function in women with RA may be hampered by pain and fatigue, perhaps decreasing the opportunity for conception. Finally, data about the role of NSAIDs in preventing ovulation suggest that continued use of these medications may hinder conception. SummaryInfertility in women with RA is an under-recognized, but remarkably common phenomenon. Although research continues into the underlying causes, physicians can discuss this topic and refer women to reproductive endocrinology when needed, thereby helping patients to build the families that they desire.

Ectopic pregnancy rate increases with the number of retrieved oocytes in autologous in vitro fertilization with non-tubal infertility but not donor/recipient cycles: an analysis of 109,140 clinical pregnancies from the Society for Assisted Reproductive Technology registry

OBJECTIVE To study the impact of controlled ovarian stimulation on ectopic pregnancy (EP) rate as a function of the number of oocytes retrieved, using donor IVF cycles as a control. DESIGN Retrospective cohort study using a large national database. SETTING Not applicable. PATIENT(S) Data from 109,140 cycles from the 2008-2010 SART registry, including 91,504 autologous cycles and 17,636 donor cycles in patients with non-tubal infertility. INTERVENTION(S) Varying amounts of oocytes retrieved in autologous and donor IVF. MAIN OUTCOME MEASURE(S) Ectopic pregnancy rates. RESULT(S) In autologous cycles, the EP rate significantly increased as oocyte yield increased. This association was not found in oocyte recipients. CONCLUSION(S) In autologous IVF cycles, increasing oocyte yield is correlated with a significantly increased EP rate. This association is not found in oocyte recipients, indicating that the increased EP rate may be due to the supraphysiologic hormone levels achieved with controlled ovarian hyperstimulation.

A large network of interconnected signaling pathways in human ovarian follicles is supported by the gene expression activity of the granulosa cells.

Human follicular fluid (hFF), as an extra oocyte microenvironment, is essential to the biological processes of oocyte development. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 426 proteins as consistently present in hFF from different participants. According to our gene chip data, the granulosa cells in the follicle locally produce 235 of these proteins. These data suggest that the granulosa cells actively participate in the follicular development by synthesizing important molecules to support the activity of pathways that are essential to oocyte development and genomic preservation. The computational Ingenuity Pathway Analysis (IPA) suggests that the identified proteins have well-established functions in the pathways of steroidogenesis, cell-to-cell signaling and interaction, molecular transport, the antioxidative system, interleukin 1 (IL-1) and IL-6 signaling, liver X receptor/retinoid X receptor (LXR/RXR) activation, and the interconnective insulin-like growth factor and lipid metabolism networks. The hFF peptide composition is likely to serve not only the inflammatory follicular state as has been previously suggested; rather, it is a highly diverse and multifunctional environment with several interconnected pathways. These results provide us with important knowledge related to the environment in which the oocyte develops as well as the molecular basis for controlling the process independently of blood supply.

Pax6- and Six3-Mediated Induction of Lens Cell Fate in Mouse and Human ES Cells

Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.