Daniela Nogueira

Meiotic Arrest In Vitro by Phosphodiesterase 3-Inhibitor Enhances Maturation Capacity of Human Oocytes and Allows Subsequent Embryonic Development

Abstract Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. We aimed to evaluate the quality of in vitro-matured, germinal vesicle (GV)-stage human oocytes following a prematuration culture (PMC) with a meiotic arrester, phosphodiesterase 3-inhibitor (PDE3-I). Follicles (diameter, 6–12 mm) were retrieved 34–36 h post-hCG administration from informed, consenting patients who had undergone controlled ovarian stimulation. Cumulus-enclosed oocytes (CEOs) presenting moderate expansion or full compaction were placed in PMC with the PDE3-I, Org9935, for 24 or 48 h. Subsequently, oocytes were removed from PMC, denuded of cumulus cells, matured in vitro, and fertilized, and the resulting embryos were cultured. In the presence of PDE3-I, approximately 98% of the oocytes were arrested at the GV stage. Following PDE3-I removal, oocytes acquired a higher maturation rate than oocytes that were immediately denuded of cumulus cells after retrieval and in vitro matured (67% vs. 46%, P = 0.01). In controls, immature CEOs retrieved with moderate expansion reached higher maturation rates compared to fully compacted CEOs, but in PMC groups, high values of maturation were achieved for both morphological classes of CEOs. No effect of PMC on fertilization was observed. A 24-h PMC period proved to be the most effective in preserving embryonic integrity. Similar proportions of nuclear abnormalities were observed in embryos of all in vitro groups. In summary, PMC with the specific PDE3-I had a beneficial effect on human CEOs by enhancing maturation, benefiting mainly the fully compacted CEOs. This resulted in an increased yield of mature oocytes available for insemination without compromising embryonic development. These results suggest that applying an inhibitor to control the rate of nuclear maturity by regulating intraoocyte PDE3 activity may allow the synchronization of nuclear and ooplasmic maturation.

Nuclear status and cytogenetics of embryos derived from in vitro–matured oocytes

OBJECTIVE To analyze embryos after in vitro maturation by investigating their nuclear status and cytogenetic constitution. DESIGN Prospective randomized laboratory study. SETTING Reproductive medicine unit in an academic hospital. PATIENT(S) Patients with male and tubal factor infertility undergoing fertility treatment. INTERVENTION(S) Denuded immature oocytes (n = 75) were matured in vitro for 24-30 hours, and intracytoplasmic sperm injection was performed 30 hours after oocyte retrieval. Fluorescence in situ hybridization was performed on the produced embryos. MAIN OUTCOME MEASURE(S) Blastomere content of the total embryo. RESULT(S) The in vitro-matured oocytes showed a similar fertilization rate as the in vivo-matured oocytes, but with a higher incidence of noncleavage (21.0%). In addition, 26.7% of these embryos arrested at the first mitotic division. Thirty embryos were processed for fluorescence in situ hybridization; only 6.7% had all mononuclear blastomeres, 30.0% had at least one binuclear blastomere, 43.3% had at least one multinuclear blastomere, and 56.6% contained anuclear cells. The chromosomal constitution was analyzed in 14 embryos, and chromosomal anomalies were found in 11 (78.5%). CONCLUSION(S) Germinal vesicle oocytes retrieved from superovulated patients and cultured in vitro for a short time had the ability to resume meiosis and achieve fertilization. However, arrest of embryo development was common. These embryos showed a high incidence of multinuclear blastomeres and aneuploidy, suggesting abnormal cytokinesis or genetic abnormalities.

Human Oocytes Reversibly Arrested in Prophase I by Phosphodiesterase Type 3 Inhibitor In Vitro

Abstract This study addresses the role of cAMP hydrolytic isoenzyme phosphodiesterase type 3 (PDE 3) modulation on human oocyte maturation in vitro. Presence of phosphodiesterase type 3 A (PDE 3A) mRNA was confirmed in human germinal vesicle-stage (GV) oocytes. Making use of a selective PDE 3 inhibitor, Org 9935 (10 μM), oocytes retrieved from immature follicles were arrested in prophase I with a high efficiency for up to 72 h. Cumulus oocyte complexes (COCs) were retrieved in the follicular phase of the cycle before or after exposure to endogenous LH or hCG administration in vivo and randomly distributed into maturation medium with or without the PDE 3 inhibitor. Previous exposure of small follicles to LH activity in vivo had no influence on the arresting capacity of the PDE 3 inhibitor. Reversal from pharmacological arrest leads to a progression through meiosis in a normal time frame with formation of a well-aligned metaphase plate. Ultrastructure analysis of COC derived from follicles between 8 and 12 mm showed that the induced extension of prophase I arrest in vitro resulted in cytoplasm changes but not in apparent nuclear changes during culture.

Effect of phosphodiesterase type 3 inhibitor on developmental competence of immature mouse oocytes in vitro

Abstract In vitro use of arresters of meiosis could improve cytoplasmic maturation of immature oocytes by controlling the period of prophase I. Phosphodiesterases (PDE) are responsible for the breakdown and concomitant inactivation of the cyclic nucleotides cAMP and cGMP and are implicated in the regulation of oocyte meiotic maturation. Selective inhibitors of phosphodiesterase type 3 (PDE3) prevent meiotic resumption of mammalian oocytes. This study evaluated the impact of meiosis arrest by PDE3 inhibitor, Org 9935, on developmental competence of geminal vesicle (GV)-stage oocytes from small antral follicles. Cumulus-oocyte complexes (COC), retrieved from antral follicles 24 h after eCG exposure and cultured in the presence of PDE3 inhibitor (10 μM) for an additional 24 h, remained arrested in the meiotic prophase. The GV configuration of oocytes before and after the arrest by PDE3 inhibitor was examined. After the period of meiosis arrest, a significantly increased proportion of oocytes had acquired a nucleolus surrounded by a condensed chromatin rim at the GV, which is a morphological correlate of transcriptional repression. Removal of inhibitor resulted in 90.6% ± 8.3% of oocytes with the first polar body extruded. Fertilization was significantly improved in oocytes that had been arrested compared with oocytes collected 24 h after eCG and undergoing in vitro maturation immediately. Embryonic preimplantation and live offspring rates of arrested oocytes were higher, although not significantly, than those of nonarrested oocytes. These results suggest that a temporal block of meiosis by PDE3 inhibitor promotes developmental competence of mice oocytes retrieved from small antral follicles.

Assessment of a new in vitro maturation system for mouse and human cumulus-enclosed oocytes: three-dimensional prematuration culture in the presence of a phosphodiesterase 3-inhibitor

BACKGROUND Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. In the present study, the quality of mouse and human cumulus-enclosed oocytes (CEOs) was examined after a two-step culture consisting of a three-dimensional prematuration culture (3D-PMC), followed by in vitro maturation (IVM). METHODS Mouse and human CEOs were embedded in an extracellular matrix (collagen-gel Type I). The gels containing the CEOs were cultured in medium with a phosphodiesterase 3-inhibitor (PDE3-I; cilostamide 1 microM) for 24 h. Afterwards, CEOs were removed from the gel and washed away from inhibitor then underwent IVM. The optimal concentration of collagen (diluted 1:2 versus not-diluted) was first determined in the mouse model. Cytoplasmic maturation after IVM of human and mouse oocytes was assessed in relation to fertilization and embryonic developmental capacity. RESULTS The diluted form of collagen was better for supporting the structure of the expanding CEOs and meiotic competence of the oocytes. Electron microscopy in combination with Lucifer Yellow dye coupling assay revealed that oocyte-cumulus cell connections could be preserved during 3D-PMC. Percentages of mouse 2-cell embryos after IVF were higher in the 3D-PMC group compared with in vitro controls and 2D-PMC oocytes, but lower compared with in vivo controls. In the human model, percentages of polar body-extruded oocytes were significantly higher in the 3D-PMC group compared with conventionally matured oocytes. The 3D-PMC also had a beneficial effect on embryonic development on Day 3 post-ICSI. CONCLUSIONS Applying a 3D-PMC in the presence of a PDE3-I preserves oocyte-cumulus cell connections and influences oocyte developmental capacity.

Effect of temporary nuclear arrest by phosphodiesterase 3-inhibitor on morphological and functional aspects of in vitro matured mouse oocytes.

The present study aimed to analyze detailed morphological and functional characteristics of mouse in vitro matured oocytes after a pre‐maturation culture (PMC) by temporary nuclear arrest with the specific phosphodiesterase 3‐inhibitor (PDE3‐I) Cilostamide. In a first experiment the lowest effective dose of Cilostamide was determined. Cumulus–oocyte complexes (COCs), isolated from small antral follicles, were exposed to different concentrations of Cilostamide (ranging from 0 (control) to10 µM) for 24 hr. Afterwards, oocytes were removed from PDE3‐I‐containing medium and underwent in vitro maturation (IVM) for 16–18 hr. A concentration of 1 µM Cilostamide was the lowest effective dose for maximum level of inhibition and reversibility of meiosis inhibition. This concentration was used in further experiments to evaluate oocyte quality following IVM in relation to different parameters: kinetics of meiotic progression, metaphase II (MII) spindle morphology, aneuploidy rate, fertilization, and embryonic developmental rates. The results were compared to nonarrested (in vitro control) and in vivo matured oocytes (in vivo control). Following withdrawal of the inhibitor, the progression of meiosis was more synchronous and accelerated in arrested when compared to nonarrested oocytes. A PMC resulted in a significant increase in the number of oocytes constituting a MII spindle of normal morphology. None of the oocytes exposed to PDE3‐I showed numerical chromosome alterations. In addition, fertilization and embryonic developmental rates were higher in the PMC group compared to in vitro controls, but lower than in vivo controls. These results provide evidence that induced nuclear arrest by PDE3‐I is a safe and reliable method to improve oocyte quality after IVM. Mol. Reprod. Dev. 75: 1021–1030, 2007.

Immature oocyte in-vitro maturation: clinical aspects

The development of immature oocyte collection techniques for in-vitro maturation (IVM), combined with novel culture techniques, opens new possibilities for assisted reproductive technology. Optimization of clinical management of IVM cycles will enhance pregnancy outcome, so that IVM might become an effective alternative assisted reproduction treatment for infertile patients irrespective of the cause of infertility. Parameters such as age and baseline antral follicular count are predictive of outcome and should be used as selection criteria for IVM treatment. Women with polycystic ovary disease and normo-ovulatory patients at risk of developing ovarian hyperstimulation syndrome might benefit from earlier retrieval of oocytes followed by IVM and embryo transfer. HCG priming before oocyte retrieval seems beneficial in terms of oocyte yield and maturational competence, and may increase the harvest of mature oocytes and lead to better endometrial synchronization with the developing embryo. The timing of aspiration may be crucial in IVM and selection criteria for follicle size at aspiration need defining prospectively for infertility type. Finer calibre aspiration needles and low aspiration pressure yield more oocytes. A combination of natural cycle IVF with IVM is a promising, mild and inexpensive assisted reproduction treatment, widely accessible the infertile population.

Avoidance of multiple pregnancies after ovulation induction by supernumerary preovulatory follicular reduction

OBJECTIVE To evaluate the effect of supernumerary preovulatory follicular reduction as an approach to avoid multiple pregnancies in ovulation induction or superovulation cycles. DESIGN Retrospective study. SETTING Tertiary referral center. PATIENT(S) In 26 cycles, 24 patients underwent ovulation induction or superovulation with either clomiphene citrate or hMG. INTERVENTION(S) Selective follicle aspiration was performed before hCG administration. MAIN OUTCOME MEASURE(S) Clinical pregnancy rate and numbers of multiple pregnancies. RESULT(S) A mean number of 4.5 follicles with a diameter > or =15 mm and a mean number of 4.5 follicles with a diameter < or =14 mm were observed before hCG administration. A mean number of 2.3 follicles with a diameter > or =15 mm and a mean number of 1.8 follicles with a diameter < or =14 mm were aspirated before the hCG administration. Seven singleton pregnancies (26.9% per cycle) ensued from the treatment. CONCLUSION(S) Aspiration of supernumerary follicles after ovulation induction or superovulation seems to be a valid approach to avoid multiple pregnancies without affecting pregnancy rate.

Supplemented tissue culture medium 199 is a better medium for in vitro maturation of oocytes from women with polycystic ovary syndrome women than human tubal fluid

OBJECTIVE To compare oocyte maturation, fertilization and cleavage rates, and embryonic developmental quality after culture of human immature oocytes from polycystic ovary syndrome (PCOS) patients in human tubal fluid (HTF) or tissue culture medium (TCM) 199. DESIGN Prospective, randomized, controlled trial. SETTING University hospital. PATIENT(S) Thirteen women undergoing 23 in vitro maturation cycles, from whom 119 oocytes were retrieved. INTERVENTION(S) Cumulus-enclosed germinal vesicle-stage oocytes matured in TCM-199-supplemented or HTF-supplemented media. MAIN OUTCOME MEASURE(S) Oocyte maturation and fertilization rates, embryonic developmental quality. RESULT(S) Significant differences were observed between TCM 199 and HTF regarding maturation rate (82% vs. 56.9%), fertilization rate (70% vs. 39.4%), and embryo quality (81.3% vs. 41.7%). CONCLUSION(S) Human tubal fluid medium, although widely used for embryo fertilization and maintenance in IVF techniques, is not an appropriate medium for the maturation of oocytes obtained from PCOS patients in nonstimulated cycles.

Effects of Chilling on Structural Aspects of Early Preantral Mouse Follicles

Abstract Chilling injury is one of the major limiting factors for achieving optimal cryopreservation of gametes. This study aimed to determine potential chilling-induced damage on several structural aspects of early preantral mouse follicles. Mechanically isolated intact early preantral follicles (type 3b-4) were exposed to 0°C for 1, 5, 10, or 30 min. Control and chilled follicles were analyzed by confocal microscopy after staining for tubulin, F-actin, and chromatin, and by electron microscopy. Chilling for only 1 min was sufficient to cause depolymerization of microtubules in the oocyte and the surrounding granulosa cell layer as evidenced by a substantial decrease in fluorescence intensity after antitubulin labeling. Cooling for longer periods caused alterations in microtubule organization in the follicle-enclosed oocyte. These alterations included the loss of interphase microtubules, concomitant with the formation of perinuclear or cortical microtubule asters and sometimes a complete disappearance of microtubules. The extent of microtubule modification was related to the time of chilling, but was fully reversible after rewarming follicles at 37°C for 1 h. Chilling had only minor effects on the actin-containing elements located predominantly in the oocyte cortex and the transzonal projections. Ultrastructural analysis confirmed that oocyte-somatic cell interactions were present. There was no influence on the chromatin configuration within the follicle-enclosed oocyte. These results indicate that mouse follicles are relatively tolerant to direct chilling injury and, as a consequence, are able to withstand the cooling-warming steps during conventional cryopreservation procedures.