RESUMO
OBJECTIVE: To test the developmental competence of oocytes in a nonhCG-triggered in vitro maturation (IVM) system when oocyte-cumulus complexes (OCC) are retrieved from antral follicles with a diameter of <6 mm. DESIGN: Prospective cohort study. SETTING: Tertiary university-based referral center. PATIENT(S): From January 2010 to September 2011, 121 patients with polycystic ovaries/polycystic ovary syndrome underwent 239 IVM cycles in total. In 58 of these cycles (44 patients), all antral follicles had a diameter of <6 mm on the day of oocyte retrieval. INTERVENTION(S): NonhCG-triggered IVM of oocytes, fresh or vitrified/warmed embryo transfer (ET). MAIN OUTCOME MEASURE(S): Oocyte diameter, maturation rate, fertilization rate, embryo development and morphology, implantation rate, clinical pregnancy rate, ongoing pregnancy rate. RESULT(S): Oocyte retrieval yielded 16.7 OCC/cycle, and 50.8% of oocytes completed IVM. The mean oocyte diameter increased from 108.8 ± 4.3 µm to 111.9 ± 4.1 µm after IVM. Mean fertilization rate was 63.7%, and 45.4% of 2-pronuclei oocytes developed into a morphologically good-quality embryo on day 3 after intracytoplasmic sperm injection. Fresh ET resulted in two ongoing pregnancies (2/37; 5.4%). Deferred vitrified-warmed ET led to an ongoing pregnancy rate of 34.6% (9/24). Three healthy babies were born and eight pregnancies were still ongoing. CONCLUSION(S): Oocytes retrieved from follicles with a diameter of <6 mm grow during a 40-hour IVM culture can acquire full competence in vitro, as illustrated by their development into healthy offspring. Endometrial quality appears to be a crucial determinant of pregnancy after nonhCG-triggered IVM.
Assuntos
Técnicas de Maturação in Vitro de Oócitos/métodos , Recuperação de Oócitos/métodos , Oócitos/crescimento & desenvolvimento , Folículo Ovariano/crescimento & desenvolvimento , Síndrome do Ovário Policístico/terapia , Adulto , Tamanho Celular , Transferência Embrionária/métodos , Feminino , Humanos , Recém-Nascido , Oócitos/patologia , Folículo Ovariano/patologia , Síndrome do Ovário Policístico/patologia , Gravidez , Taxa de Gravidez/tendênciasRESUMO
PURPOSE: The in-vitro environment influences oocyte competence and gene expression in cumulus cells and oocytes. Effects of culturing under non-attachment conditions and varying follicle exposure to FSH were investigated at the mRNA level and on oocyte developmental capacity. METHODS: Quantitative PCR analysis of Gdf9, Mater, Nmp2 (in oocytes), Lhcgr and Amh (in cumulus cells), and oocyte developmental competence after in vitro follicle culture were evaluated. RESULTS: Follicle survival (98.7%) and polar body rate (94%) were similar for all conditions. Estradiol and progesterone production were significantly lower in non-attachment follicles (10-fold and 3-fold, respectively). Under non-attachment conditions, a higher two-cell rate (69.9%) and total blastocyst yield (48.5%) were obtained and, by decreasing FSH levels during culture, Lhcgr transcripts were significantly reduced to levels similar to in-vivo. Levels of oocyte-specific transcripts were not significantly influenced by in-vitro conditions. CONCLUSION: Non-attachment conditions influence follicle steroid secretory capacity and, together with dynamic FSH doses, positively influence cumulus cell gene expression and oocyte developmental competence.
Assuntos
Hormônio Foliculoestimulante/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Animais , Técnicas de Cultura de Células , Células do Cúmulo/citologia , Células do Cúmulo/metabolismo , Feminino , Fertilização in vitro , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Receptores do LH/genética , Receptores do LH/metabolismoRESUMO
OBJECTIVE: To compare clinical outcomes of fresh embryo transfer (ET) and vitrified-warmed ET in an artificial endometrial priming cycle in patients with polycystic ovaries (PCO) or polycystic ovary syndrome (PCOS) who underwent oocyte in vitro maturation (IVM) in non-hCG-primed cycles. DESIGN: Prospective cohort study. SETTING: University-based tertiary referral center. PATIENT(S): Thirty-nine consecutive patients <37 years old with PCO or PCOS, who underwent 73 cycles of immature oocyte retrieval. INTERVENTION(S): Immature oocyte collection after ovarian stimulation with a cumulative dose of 450 IU uFSH or highly purified hMG, but without hCG priming. IVM of oocytes followed by ET if endometrium thickness ≥ 6 mm. Embryo vitrification at the cleavage stage. ET in an artificial cycle. MAIN OUTCOME MEASURE(S): Implantation rate (IR) and clinical pregnancy rate (CPR). RESULT(S): Fresh ET after IVM resulted in an IR of 6.9% (5/72) per ET and a CPR of 9.4% (5/53). ET of vitrified-warmed IVM embryos in an artificial cycle resulted in significantly better outcomes (IR 21.9% [7/32] and CPR 31.8% [7/22] per ET). CONCLUSION(S): A non-hCG-primed IVM system in PCO or PCOS performs poorly when embryos are transfered in a fresh cycle. Transfer of vitrified-warmed IVM embryos in an artificial cycle leads to significantly improved clinical outcomes. These data illustrate that IVM embryos in PCO or PCOS have good survival rates and suggest that hCG may be needed to support endometrial receptivity in the fresh IVM cycle.
Assuntos
Gonadotropina Coriônica , Recuperação de Oócitos/métodos , Oócitos/crescimento & desenvolvimento , Síndrome do Ovário Policístico/fisiopatologia , Adulto , Estudos de Coortes , Feminino , Humanos , Síndrome do Ovário Policístico/complicações , Gravidez , Taxa de Gravidez/tendências , Estudos Prospectivos , Resultado do Tratamento , Adulto JovemRESUMO
The phosphodiesterase (PDE) family is a group of enzymes that catalyzes the transformation of cyclic nucleotides into 5' nucleotides. Based on rodents, the current mammalian model of PDE distribution in the ovarian follicle predicts Pde3a in the oocyte and Pde4d in the somatic cells. Using bovine as an experimental model, the present results showed that PDE3 was the predominant PDE activity in oocytes. However, cumulus cell cAMP-PDE activity was predominantly resistant to inhibition by 3-isobutyl-methylxantine, indicating PDE8 activity (60% of total PDE activity) and a minor role for PDE4 (<5%). A total of 20% of total oocyte PDE activity was also attributed to PDE8. The PDE activity measurements in mural granulosa cells from 2 to 6 mm in diameter suggest the presence of PDE4 and PDE8. In granulosa cells from follicles >10 mm, total PDE and PDE8 activities along with PDE8A protein level were increased compared with smaller follicles. The RT-PCR experiments showed that cumulus cells expressed PDE8A, PDE8B, and PDE10A. Western blot experiments showed PDE8A, PDE8B, and PDE4D proteins in mural granulosa cells and cumulus-oocyte complexes. PDE8 inhibition using dipyridamole in a dose-dependent manner increased cAMP levels in the cumulus-oocyte complexes and delayed oocyte nuclear maturation. These results are the first to demonstrate the functional presence of PDE8 in the mammalian ovarian follicle. This challenges the recently described cell-specific expression of cAMP-PDEs in the ovarian follicle and the notion that PDE4 is the predominant granulosa/cumulus cell PDE. These findings have implications for our understanding of hormonal regulation of folliculogenesis and the potential application of PDE inhibitors as novel contraceptives.