OBJECTIVE: Existing data indicate that assisted reproductive technologies (ART) may result in alterations in genomic methylation at specific loci, as evidenced by animal models and an increased incidence of imprinting disorders in children conceived via ART. We have previously presented preliminary evidence demonstrating that ART conditions may also result in global alterations of genomic methylation in murine blastocysts. We now present microarray data to further test this hypothesis. DESIGN: Mouse model, DNA microarray analysis MATERIALS AND METHODS: C57/BL6 female mice were crossed with C3H male mice to obtain F1 hybrid embryos. For ART conditions, female mice were injected with 5U of PMSG followed 48 hours later with 5U of hCG. Mice were then mated and the resulting embryos were harvested on E0.5 and cultured in HTF media until E3.5. For the control group, female mice were mated in estrus without superovulation and the resulting embryos were flushed from the oviducts at E3.5. Genomic DNA was prepared from morphologically normal blastocysts by standard methods for 4 ART embryos and 4 control embryos. DNA was digested with a methylation sensitive restriction enzyme and then ligated to double-stranded linkers. PCR with a biotinylated primer was then performed. Amplified DNA fragments were purified via streptavidin paramagnetic particles and then re-amplified using multiple strand displacement whole genome amplification. The microarray contained 2 to 3 oligonucleotides for each expected DNA sequence from the original digest of chromosome 7. Eight hybridizations were performed with color reversal. The first 4 experiments compared control embryos to control embryos; the second 4 experiments compared control to ART embryos. RESULTS: Genomic DNA from ART blastocysts exhibited a similar degree of genomic methylation as controls. CONCLUSIONS: These results indicate that the aberrant DNA methylation observed under ART conditions are not a global phenomenon but may be present only a certain, highly sensitive loci.

OBJECTIVE: To evaluate bleeding patterns, endometrial thickness and patient satisfaction with a levonorgesterol intrauterine device (L-IUD) vs. a copper IUD (C-IUD). DESIGN: Single blind prospective randomized trial, patient survey, bleeding diaries and ultrasound MATERIALS AND METHODS: This is a preliminary sub-group analysis of patient experience in a trial designed to assess the effect of IUDs on coagulation. Women aged 18-52 were randomized to the L-IUD or the C-IUD. Bleeding diaries were completed for one cycle prior to IUD insertion and again 2-4 months post-IUD insertion. A patient satisfaction survey was conducted 4 months after insertion. Follicular phase endometrial thickness was assessed via transvaginal ultrasound prior to IUD insertion and 4 months post-insertion. RESULTS: Eleven patients were randomized to the C-IUD and 7 were randomized to the L-IUD. The most common reasons for choosing the IUD were desire for contraception that was long-term (94%), reversible (83%), effective (72%), didn't require remembering (72%), and avoided systemic hormones (50%). Patients randomized to the C-IUD had an increase in number of days of spotting (1.45 vs. 3.54, p=0.02) and heavy bleeding (0.91 vs. 1.82, p=0.04) compared while patients randomized to the L-IUD had a decrease in days of heavy bleeding (1.86 vs. 0.57, p=0.01) compared to pre-insertion. Comparing the two groups post-IUD insertion, patients randomized to the L-IUD had significantly fewer days of heavy bleeding compared to patients randomized to the C-IUD (p=0.0002). Other bleeding parameters (spotting, light, moderate, and heavy bleeding) were unchanged. Endometrial thickness was significantly reduced 4 months post-IUD insertion in the L-IUD group compared to the C-IUD group (4.74 vs. 3.17 mm, p=0.03). Patient satisfaction averaged 4.4 on a 5 point scale and was not different between groups. CONCLUSIONS: The C-IUD resulted in increased menstrual flow and the L-IUD resulted in decreased flow and endometrial thickness. Patient satisfaction was high for both groups.

OBJECTIVE: Existing data indicate that assisted reproductive technologies (ART) may result in alterations in genomic methylation at specific loci, as evidenced by an increased incidence of imprinting disorders in children conceived via in vitro fertilization (IVF). Our objective was to test the hypothesis that ART conditions also result in widespread global alterations of genomic methylation in pre-implantation blastocysts. DESIGN: Mouse model of ART, DNA analysis of single blastocysts. MATERIALS AND METHODS: C57/BL6 female mice were crossed with C3H male mice to obtain F1 hybrid embryos. For ART conditions, female mice were injected with 5 units of pregnant mare's serum (PMSG) followed 48 hours later with 5 units of human chorionic gonadotropin (hCG). Mice were then mated and the resulting embryos were harvested on E0.5 and cultured in Human Tubal Fluid (HTF) media until E3.5. For the control group, female mice were mated in estrus without superovulation and the resulting embryos were flushed from the oviducts at E3.5. Morphologically normal blastocysts were chosen for analysis. Genomic DNA was prepared from individual blastocysts by standard methods for 7 ART embryos and 5 control embryos. Methylation-sensitive amplified representations were prepared using a novel method that relies on restriction enzyme digest followed by a two-stage amplification procedure as follows: Genomic DNA was digested with a methylation sensitive restriction enzyme and then ligated to double-stranded linkers. PCR with a biotinylated primer corresponding to the linker was then performed. Amplified DNA fragments were purified by binding to streptavidin paramagnetic particles and then re-amplified using multiple strand displacement whole genome amplification. The genomic representations of DNA amplified by this method depend on the location and degree of methylation present in the original sample. A qualitative assessment of the differences between methylation-sensitive representations of ART vs. control embryos was performed by amplification using rare cutting restriction enzymes. The number and size of amplified DNA fragments was visualized on 2% agarose gels. RESULTS: Genomic DNA from E3.5 ART embryos exhibited substantially less genomic methylation than controls. CONCLUSIONS: Methylation differences resulting from ART are likely to be large and to impact the entire genome. In the future, we plan to test this hypothesis through the use of microarray analysis of amplified representations.

ObjectiveTo determine if a parallel, perpendicular or oblique alignment of the pronuclei and polar bodies in day 1 pronuclear zygotes is correlated with day 3 embryo quality.DesignRetrospective cohort analysis.Materials and methodsAn evaluation of all embryos conceived via controlled ovarian hyperstimulation and IVF in 2006 at the University of Vermont was performed. Cycles using both standard IVF and IVF with intracystoplasmic sperm injection were included. Day 1 pronuclear zygotes were examined and an assessment of the orientation of the long axis of the pronuclei to the polar bodies was recorded. Embryos whose growth arrested prior to day 3 were excluded from the analysis. Embryo quality was assessed on day 3 based on cell number and embryo grade. Day 3 embryo grade was based on percent fragmentation (FG) with grade A embryos having 0–10% FG, grade B having 10–20% FG, grade C having 20–30% FG and grade D having >30% FG. Multiple regression analysis was used to evaluate the relationship between day 1 pronuclei/polar body alignment and day 3 embryo quality. Because there is no gold standard for day 3 embryo quality, 3 different multiple regression models were used. The first model considered only day 3 embryo grade. The second model considered day 3 embryo cell number, with 8 cells representing the highest quality embryos and embryos containing more or fewer than 8 cells representing progressively lower quality. The third model considered qualitative scores, with "high quality" embryos having a score of 7–10A, "medium quality" embryos having a score of 7–10B, >10A or <7A, and "poor quality" embryos having a score of <7B, >10B or any C or D grade embryos.Results82 patient cycles containing 364 embryos were included in the analysis. All 3 multiple regression models demonstrated a statistically significant correlation between pronuclei/polar body alignment and day 3 embryo quality. An oblique alignment was associated with the highest quality, followed by a parallel alignment and a perpendicular alignment was associated with the poorest embryo quality (all P<0.001).ConclusionsAn oblique alignment of the pronuclei and polar bodies in day 1 pronuclear zygotes is associated with high day 3 embryo quality and a perpendicular alignment is associated with poor embryo quality. The finding that pronuclei and polar body alignment is strongly associated with day 3 embryo quality indicates that it may also be associated with implantation potential. ObjectiveTo determine if a parallel, perpendicular or oblique alignment of the pronuclei and polar bodies in day 1 pronuclear zygotes is correlated with day 3 embryo quality. To determine if a parallel, perpendicular or oblique alignment of the pronuclei and polar bodies in day 1 pronuclear zygotes is correlated with day 3 embryo quality. DesignRetrospective cohort analysis. Retrospective cohort analysis. Materials and methodsAn evaluation of all embryos conceived via controlled ovarian hyperstimulation and IVF in 2006 at the University of Vermont was performed. Cycles using both standard IVF and IVF with intracystoplasmic sperm injection were included. Day 1 pronuclear zygotes were examined and an assessment of the orientation of the long axis of the pronuclei to the polar bodies was recorded. Embryos whose growth arrested prior to day 3 were excluded from the analysis. Embryo quality was assessed on day 3 based on cell number and embryo grade. Day 3 embryo grade was based on percent fragmentation (FG) with grade A embryos having 0–10% FG, grade B having 10–20% FG, grade C having 20–30% FG and grade D having >30% FG. Multiple regression analysis was used to evaluate the relationship between day 1 pronuclei/polar body alignment and day 3 embryo quality. Because there is no gold standard for day 3 embryo quality, 3 different multiple regression models were used. The first model considered only day 3 embryo grade. The second model considered day 3 embryo cell number, with 8 cells representing the highest quality embryos and embryos containing more or fewer than 8 cells representing progressively lower quality. The third model considered qualitative scores, with "high quality" embryos having a score of 7–10A, "medium quality" embryos having a score of 7–10B, >10A or <7A, and "poor quality" embryos having a score of <7B, >10B or any C or D grade embryos. An evaluation of all embryos conceived via controlled ovarian hyperstimulation and IVF in 2006 at the University of Vermont was performed. Cycles using both standard IVF and IVF with intracystoplasmic sperm injection were included. Day 1 pronuclear zygotes were examined and an assessment of the orientation of the long axis of the pronuclei to the polar bodies was recorded. Embryos whose growth arrested prior to day 3 were excluded from the analysis. Embryo quality was assessed on day 3 based on cell number and embryo grade. Day 3 embryo grade was based on percent fragmentation (FG) with grade A embryos having 0–10% FG, grade B having 10–20% FG, grade C having 20–30% FG and grade D having >30% FG. Multiple regression analysis was used to evaluate the relationship between day 1 pronuclei/polar body alignment and day 3 embryo quality. Because there is no gold standard for day 3 embryo quality, 3 different multiple regression models were used. The first model considered only day 3 embryo grade. The second model considered day 3 embryo cell number, with 8 cells representing the highest quality embryos and embryos containing more or fewer than 8 cells representing progressively lower quality. The third model considered qualitative scores, with "high quality" embryos having a score of 7–10A, "medium quality" embryos having a score of 7–10B, >10A or <7A, and "poor quality" embryos having a score of <7B, >10B or any C or D grade embryos. Results82 patient cycles containing 364 embryos were included in the analysis. All 3 multiple regression models demonstrated a statistically significant correlation between pronuclei/polar body alignment and day 3 embryo quality. An oblique alignment was associated with the highest quality, followed by a parallel alignment and a perpendicular alignment was associated with the poorest embryo quality (all P<0.001). 82 patient cycles containing 364 embryos were included in the analysis. All 3 multiple regression models demonstrated a statistically significant correlation between pronuclei/polar body alignment and day 3 embryo quality. An oblique alignment was associated with the highest quality, followed by a parallel alignment and a perpendicular alignment was associated with the poorest embryo quality (all P<0.001). ConclusionsAn oblique alignment of the pronuclei and polar bodies in day 1 pronuclear zygotes is associated with high day 3 embryo quality and a perpendicular alignment is associated with poor embryo quality. The finding that pronuclei and polar body alignment is strongly associated with day 3 embryo quality indicates that it may also be associated with implantation potential. An oblique alignment of the pronuclei and polar bodies in day 1 pronuclear zygotes is associated with high day 3 embryo quality and a perpendicular alignment is associated with poor embryo quality. The finding that pronuclei and polar body alignment is strongly associated with day 3 embryo quality indicates that it may also be associated with implantation potential.

BACKGROUND Cigarette smoking is widely believed to be associated with decreased fecundity in naturally conceiving populations; however, the effect of female smoking on pregnancy outcomes in patients undergoing IVF is unclear. METHODS A retrospective analysis of 389 consecutive patients undergoing first cycle IVF was performed. Outcomes of peak estradiol (E(2)) levels, log mean ovarian volume, number of oocytes retrieved, oocyte maturity in ICSI, fertilization rate, cleavage rate, embryo quality, percentage of high-quality embryos, pregnancy and live birth were assessed in patients reported as never smokers, past smokers and current smokers. Potential confounding variables evaluated included day 3 FSH, number of oocytes retrieved, embryo quality, caffeine and alcohol consumption. The population was also stratified by female age (<35 and >or=35 years). RESULTS A total of 9.3% of our patients reported current smoking and 12.1% reported a history of smoking. Smoking status did not significantly affect pregnancy outcome, live birth rate or any other indicated outcome. CONCLUSIONS A total of 21.4% of IVF patients in this study had past or present exposure to cigarette smoking with no measurable effect on IVF outcome.