Use of FTA® classic cards for epigenetic analysis of sperm DNA.

FTA® technologies provide the most reliable method for DNA extraction. Although FTA technologies have been widely used for genetic analysis, there is no literature on their use for epigenetic analysis yet. We present for the first time, a simple method for quantitative methylation assessment based on sperm cells stored on Whatman FTA classic cards. Specifically, elution of seminal DNA from FTA classic cards was successfully tested with an elution buffer and an incubation step in a thermocycler. The eluted DNA was bisulfite converted, amplified by PCR, and a region of interest was pyrosequenced.

Gonadotropin-releasing hormone antagonists increase follicular fluid insulin-like growth factor-I and vascular endothelial growth factor during ovarian stimulation cycles.

The aim of the present study was to investigate the effect of gonadotropin-releasing hormone (GnRH) antagonists (GnRH-ant) on follicular fluid (FF) insulin-like growth factor-I (IGF-I) and FF vascular endothelial growth factor (VEGF) levels. Sixty women undergoing assisted reproduction were randomized and assigned to two different GnRH analog regimens: GnRH agonist (GnRH-a) and GnRH-ant. FF VEGF and FF IGF-I concentrations were significantly increased in the patients treated with GnRH-ant (p < 0.001). In the same patients we observed a statistically significant reduction in serum luteinizing hormone (LH) and estradiol (E2) levels (p < 0.001 and p < 0.05, respectively), FF E2 and FF androstenedione levels (p < 0.05 and p < 0.001, respectively), as well as a reduction in the number of pregnancies although this was not statistically significant. In the GnRH-ant group, FF VEGF levels were positively correlated with FF IGF-I levels, and both were negatively correlated with serum LH levels. The increase in FF IGF-I and FF VEGF levels in women treated with GnRH-ant could be explained by a deleterious follicular environment in response to profound suppression of LH and E2 levels.

Ovarian response and pregnancy outcome related to midfollicular LH levels in down-regulated women undergoing assisted reproduction.

OBJECTIVE: To assess ovarian response and pregnancy outcome related to midfollicular luteinizing hormone (LH) levels in normogonadotropic women down-regulated with gonadotropin-releasing hormone-agonist (GnRH-a) (daily and depot) during an assisted reproduction cycle. STUDY DESIGN: In a prospective randomized trial, 50 women were down-regulated with leuprorelin, 0.5 mg/d subcutaneously (group A) and 50 with leuprorelin, 3.75 mg depot (group B), prior to ovarian stimulation with recombinant follicle-stimulating hormone (FSH). Midfollicular serum LH levels < or = 0.5 mIU/mL (subgroups A1 and B1) versus > 0.5 mIU/mL (subgroups A2 and B2) were considered. A comparative analysis was carried out on the clinical effects (duration of stimulation, total FSH dose, number of oocytes retrieved and pregnancy rate). RESULTS: Maximum estradiol levels and pregnancy rates were higher in subgroups A2 and B2, with LH > 0.5 mIU/mL. No differences were observed in the cumulative amount of gonadotropin used, number of oocytes retrieved or fertilization rate. CONCLUSION: When FSH only is used for ovarian stimulation, very low LH serum concentrations in normogonadotropic women profoundly suppressed with GnRH-a, especially if given as a depot, may adversely affect in vitro fertilization outcome.

Clinical and endocrine effects of ovulation induction with FSH and hCG supplementation in low responders in the midfollicular phase. A pilot study.

OBJECTIVE: To assess the endocrine and clinical effects of luteinizing hormone (LH) activity supplementation administered in the midfollicular phase during controlled ovarian hyperstimulation to poor responders who were candidates for in vitro fertilization (IVF)--embryo transfer. STUDY DESIGN: Prospective, controlled, nonrandomized trial with historical controls. Twenty-five IVF patients who had shown a poor response to standard, long-protocol GnRH-a and FSH only in a preceding cycle (cycle A), were stimulated in the next cycle after six months with hCG supplementation (50 I.U. subcutaneously daily) starting on day 7 during standard, long-protocol GnRH-a and FSH (cycle B). The comparative analysis of clinical effects (duration of stimulation, total highly purified (HP)-FSH dose, number of oocytes retrieved and pregnancy rate) and endocrine responses (serum E2, follicular E2 and androstenedione levels) were determined between cycles A and B. RESULTS: Maximum serum E2 levels and clinical pregnancy rate were higher in cycle B, with hCG supplementation. Also, the follicular E2 and androstenedione levels were higher in cycle B. No differences were noted between cycles as regards the duration of stimulation, total HP-FSH dose and number of oocytes retrieved. CONCLUSION: LH activity supplementation in the midfollicular phase yields favorable pregnancy results in low responders. This may be due to enhanced release of follicular precursors for greater synthesis of E2.