The effect of follicular fluid hormones on oocyte recovery after ovarian stimulation: FSH level predicts oocyte recovery.

BACKGROUND: Ovarian stimulation for assisted reproductive technology (ART) overcomes the physiologic process to develop a single dominant follicle. However, following stimulation, egg recovery rates are not 100%. The objective of this study is to determine if the follicular fluid hormonal environment is associated with oocyte recovery. METHODS: This is a prospective study involving patients undergoing ART by standard ovarian stimulation protocols at an urban academic medical center. A total of 143 follicular fluid aspirates were collected from 80 patients. Concentrations of FSH, hCG, estradiol, progesterone, testosterone and prolactin were determined. A multivariable regression analysis was used to investigate the relationship between the follicular fluid hormones and oocyte recovery. RESULTS: Intrafollicular FSH was significantly associated with oocyte recovery after adjustment for hCG (Adjusted odds ratio (AOR) = 1.21, 95%CI 1.03-1.42). The hCG concentration alone, in the range tested, did not impact the odds of oocyte recovery (AOR = 0.99, 95%CI 0.93-1.07). Estradiol was significantly associated with oocyte recovery (AOR = 0.98, 95% CI 0.96-0.99). After adjustment for progesterone, the strength of association between FSH and oocyte recovery increased (AOR = 1.84, 95%CI 1.45-2.34). CONCLUSION: The relationship between FSH and oocyte recovery is significant and appears to work through mechanisms independent of the sex hormones. FSH may be important for the physiologic event of separation of the cumulus-oocyte complex from the follicle wall, thereby influencing oocyte recovery. Current methods for inducing the final stages of oocyte maturation, with hCG administration alone, may not be optimal. Modifications of treatment protocols utilizing additional FSH may enhance oocyte recovery.

A quantitative assessment of follicle size on oocyte developmental competence.

OBJECTIVE: To quantitatively assess the impact of follicle size on oocyte maturation, fertilization, and embryo quality. DESIGN: Prospective study. SETTING: Academic medical center. PATIENT(S): Couples undergoing ovarian stimulation and in vitro fertilization (IVF). INTERVENTION(S): A total of 235 cycles were monitored prospectively, and 2934 oocytes were collected from five groups of follicle size. Repeated measures multivariate analyses were used to compare the smaller follicle sizes with the lead follicle. MAIN OUTCOME MEASURE(S): Oocyte maturation, fertilization, and embryo quality. RESULT(S): Compared with the lead follicular group (>18 mm), the odds of a mature oocyte from a 16 to 18 mm size follicle were 37% and declined progressively with each size. The odds of fertilization of oocytes from follicles 16 to 18 mm in size was 28% less than the lead group and decreased with each size. The rate of polyspermy with conventional insemination was increased for the smaller follicular groups (adjusted odds ratio = 2.37). Follicle size did not predict embryo cell number, but embryos from smaller follicles had a statistically significantly higher fragmentation compared with the lead group. CONCLUSION(S): The lead follicular group was most likely to have a mature oocyte that was capable of fertilization and best suited for development into a high-quality embryo. The smaller follicles were capable of producing metaphase II oocytes that could fertilize, but at rates approaching only 60% that of the lead follicular group.

Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos.

In vitro culture (IVC) of preimplantation mouse embryos is associated with changes in gene expression. It is however, not known if the method of fertilization affects the global pattern of gene expression. We compared gene expression and development of mouse blastocysts produced by in vitro fertilization (IVF) versus blastocysts fertilized in vivo and cultured in vitro from the zygote stage (IVC) versus control blastocysts flushed out of the uterus on post coital day 3.5. The global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. It appears that each method of fertilization has a unique pattern of gene expression and development. Embryos cultured in vitro had a reduction in the number of trophoblastic cells (IVF 33.5 cells, IVC 39.9 cells, and 49.6 cells in the in vivo group) and, to a lesser degree, of inner cell mass cells (12.8, 11.7, and 13.8 respectively). The inner cell mass nuclei were larger after culture in vitro (140 microm(2), 113 microm(2), and 86 microm(2) respectively). Although a high number of genes (1912) was statistically different in the IVF cohort when compared with the in vivo control embryos, the magnitude of the changes in gene expression were low and only a minority of genes (29 genes) was changed more than fourfold. Surprisingly, IVF embryos were different from IVC embryos (3058 genes were statistically different, but only three changed more than fourfold). Proliferation, apoptosis, and morphogenetic pathways are the most common pathways altered after IVC. Overall, IVF and embryo culture have a profound effect on gene expression pattern and phenotype of mouse preimplantation embryos.

Methylenetetrahydrofolate reductase (MTHFR) is associated with ovarian follicular activity.

OBJECTIVE: Polymorphisms in the MTHFR gene have been associated with decreased cell division and apoptosis. This finding led us to evaluate whether MTHFR polymorphisms were associated with follicular growth within the ovary. More specifically, we investigated the effect of the two common polymorphisms C677T and A1298C in our population of women undergoing ovarian stimulation. DESIGN: Prospective cohort study. SETTING: Academic medical center. PATIENT(S): Two hundred twenty-three women undergoing ovarian stimulation. INTERVENTION(S): The DNA from patients was genotyped at the MTHFR C677T and A1298C polymorphisms. MAIN OUTCOME MEASURE(S): Day 3 FSH, E(2), antral follicle count, amount of gonadotropin used, the number of follicles >13 mm, E(2) on the day of hCG administration, and oocyte number. RESULT(S): Women with the variant MTHFR 1298 C allele had significantly higher basal FSH levels, and after ovarian stimulation, produced fewer follicles >13 mm, had lower E(2) levels on the day of hCG administration, and required more ampules of gonadotropin hormone during treatment. Women with the variant MTHFR 677 T allele demonstrated no significant differences. CONCLUSION(S): The MTHFR A1298C polymorphism, but not the C677T polymorphism, is associated with higher basal FSH levels and may be a determinant of response to ovarian stimulation. These findings make a compelling case for the MTHFR A1298C polymorphism to modulate folliculogenesis.

Effects of oxygen tension on gene expression in preimplantation mouse embryos.

OBJECTIVE: To compare the effects of atmospheric and physiologic oxygen concentrations on the global patterns of gene expression during mouse preimplantation development. DESIGN: Comparative analysis of in vivo-produced and in vitro-produced embryos. SETTING: Research laboratory. PATIENT(S): None. INTERVENTION(S): Control embryos at the blastocyst stage that developed in vivo were collected from uteri. Experimental embryos were obtained at the zygote stage and cultured to the blastocyst stage in Whitten's medium or KSOM medium with amino acids under 20% oxygen (atmospheric) or 5% oxygen (physiologic). MAIN OUTCOME MEASURE(S): Embryo development, cell number, and gene expression assayed by microarray technology. RESULT(S): Low (physiologic) oxygen concentration is associated with faster embryo development and increased cell number. In addition, there are marked perturbations in the global pattern of gene expression, as assessed by oligonucleotide microarray, after culture in 20% oxygen as compared with 5% oxygen. CONCLUSION(S): Culture in low oxygen is associated with fewer perturbations in the global pattern of gene expression and more closely resembles that of the in vivo control embryos. These findings provide rationale for culturing human embryos in the presence of 5%, rather than 20%, oxygen.

Oocyte degeneration after intracytoplasmic sperm injection: a multivariate analysis to assess its importance as a laboratory or clinical marker.

OBJECTIVE: Oocyte degeneration has historically been associated with the intracytoplasmic (ICSI) technique. We sought to determine whether oocyte degeneration rates were associated with the technician performing the procedure, the baseline characteristics of the patient, and/or ovarian stimulation variables. We also evaluated whether the degeneration rate could serve as a surrogate marker for implantation potential. DESIGN: Cohort study. SETTING: Academic medical center. PATIENT(S): Couples undergoing ICSI. INTERVENTION(S): Six thousand six hundred fifty-three injected oocytes were analyzed to determine whether the degeneration rate was technician dependent. Two hundred thirty first-entry down-regulated cycles were examined to identify predictors associated with oocyte degeneration. Multivariate analyses were performed using generalized linear model routines. MAIN OUTCOME MEASURE(S): Oocyte degeneration rates and implantation rates. RESULT(S): Neither the ICSI technician nor the stripping technician was associated with the oocyte degeneration rate. However, the day 3 FSH, number of mature oocytes retrieved, and E2 levels on the day of hCG were significant independent predictors of degeneration rate. Physician-adjustable ovarian stimulation variables were not associated with the degeneration rate. The degeneration rate did not appear to be associated with the implantation rate. CONCLUSION(S): These data suggest that oocyte degeneration is not technician or physician dependent. Degeneration is likely a function of the inherent oocyte quality in women who underwent ovarian stimulation. However, the remaining cohort of retrieved oocytes appears to be unaffected by virtue of an uncompromised implantation rate.

Day 2 transfer improves pregnancy outcome in in vitro fertilization cycles with few available embryos.

OBJECTIVE: Delaying ET to day 3 to optimize embryo selection is well accepted. However, in cases where there are not enough embryos to perform selection, it is not clear whether there is a difference in clinical outcomes with the day of ET. DESIGN: Cohort study. SETTING: Academic medical center. PATIENT(S): Two hundred forty-two fresh IVF/intracytoplasmic sperm injection (ICSI) cycles from 2002-2004, where all generated embryos were transferred irrespective of quality because of an extremely low number of available embryos. INTERVENTION(S): In time period 1, ET was on day 3. In time period 2, ET was on day 2. MAIN OUTCOME MEASURE(S): Patient response to stimulation was analyzed along with pregnancy outcome and implantation rate. RESULT(S): Miscarriage rates were decreased, and ongoing pregnancy rates were increased with a day 2 ET in patients <40 years of age. CONCLUSION(S): In women <40 years of age, the day of transfer is a significant predictor of clinical outcome in cases in which a low number of embryos are available for transfer. The evidence suggests that limiting embryo culture to only 2 days reduces the incidence of miscarriage and increases ongoing pregnancy rates.

Triploidy formation after intracytoplasmic sperm injection may be a surrogate marker for implantation.

OBJECTIVE: Triploidy after intracytoplasmic sperm injection (ICSI) is due mostly to retention of the second polar body. Our interest was to determine the predictors of triploidy and to determine whether the presence of triploidy can serve as a surrogate marker of implantation for the remaining cohort of zygotes. DESIGN: Cohort study. SETTING: Academic research center. PATIENT(S): Infertile couples undergoing IVF/ICSI. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Triploid zygote (3PN) rate, implantation rate. RESULT(S): The 3PN rate is a significant predictor of implantation rate for the remaining cohort of zygotes. The starting and total dose of gonadotropins administered and the total days of stimulation are independent predictors of the 3PN rate. CONCLUSION(S): In couples with a normal semen analysis undergoing IVF/ICSI, the 3PN rate may serve as a surrogate marker of oocyte quality and may be altered by adjusting the stimulation protocol.

The unique transcriptome through day 3 of human preimplantation development.

Successful human development is dependent upon a cascade of events following fertilization. Unfortunately, knowledge of these critical events in humans is remarkably incomplete. Although hundreds of thousands of human embryos are cultured yearly at infertility centers worldwide, the vast majority fail to develop in culture or following transfer to the uterus. In this study, we sought to characterize global patterns of gene expression in individual, normal embryos during the first three days of embryonic life using microarrays; we then compared gene expression between normally growing and growth-arrested embryos using quantitative PCR. Our results documented several novel findings. First, we found that a complex pattern of gene expression exists; most genes that are transcriptionally modulated during the first three days following fertilization are not upregulated, as was previously thought, but are downregulated. Second, we observed that the majority of genes exhibiting differential expression during preimplantation development are of unknown identity and/or function. Third, we show that embryonic transcriptional programs are clearly established by day 3 following fertilization, even in embryos that arrested prematurely with 2-, 3- or 4-cells. This indicates that failure to activate transcription is not associated with the majority of human preimplantation embryo loss. Finally, taken together, these results provide the first global analysis of the human preimplantation embryo transcriptome, and demonstrate that RNA can be amplified from single oocytes and embryos for analysis by cDNA microarray technology, thus lending credence to additional studies of genetic regulation in these cell types, as well as in other small biological samples.