Mundhir T. Ridha-Albarzanchi

In vitro effects of cilostazol, a phosphodiesterase 3A inhibitor, on mouse oocyte maturation and morphology

Inhibition of phosphodiesterase 3A (PDE3A) in oocytes has been reported to arrest oocyte maturation and to increase intra‐oocyte cyclic adenosine monophosphate levels. Although many PDE3A inhibitors have been found to arrest oocyte maturation in different species, including humans, the most commonly prescribed PDE3A inhibitor named cilostazol (CLZ) has not yet been fully evaluated in reproduction. The present study was designed to investigate the potential inhibitory effects of CLZ on oocyte maturation and morphology in vitro. Antral oocytes were recovered from hyperstimulated mice and allocated to 10 different CLZ concentrations (0.00–67.66 μmol/L). Oocytes were then assessed after 24 and 48 h of incubation for maturation and morphology. Some of the evaluated CLZ concentrations (1.06–4.23 μmol/L) were made similar to those observed in human clinical trials. CLZ arrested oocyte maturation at the germinal vesicle (GV) stage at concentrations as low as 1.06 μmol/L (P < 0.0001). A selective degenerative impact of CLZ targeting arrested oocytes at the GV stage was observed during 24 h of incubation (r = −0.781, P < 0.0001). This was not the case with non‐arrested oocytes (r = −0.082, P = 0.64). Such degenerative impact was dose‐dependent (P < 0.0001), suggesting a role for cyclic adenosine monophosphate in this degenerative process. The degenerated oocytes were of distorted oolema or fragmented cytoplasm. Based on the experiments, it is concluded that CLZ can inhibit oocyte maturation in vitro, at concentrations similar to those observed in humans taking CLZ, and under such conditions the prolonged maintenance of oocytes at the GV stage is harmful.

Cilostazol blocks pregnancy in naturally cycling swine: An animal model.

AIMS Cilostazol (CLZ) is an FDA approved therapeutic that is indicated for patients with intermittent claudication disease. CLZ is a selective inhibitor for phosphodiesterase 3A (PDE3A); an enzyme that controls oocyte maturation in many mammals including humans. Recently, CLZ has been reported to block pregnancy and oocyte maturation in mice. The objective of the present work was to evaluate the potential non-steroidal contraceptive capacity of CLZ using a more advanced translational model for humans. MAIN METHODS Three groups of naturally cycling sows were treated orally with 0, 100, or 200mg CLZ, twice a day (bid), for 6days before estrus and continued for three days after estrus. Each sow was mated by one of two proven fertile boars on alternate days during estrus. KEY FINDINGS CLZ dose of 100mg, bid, completely blocked pregnancy in sows when compared to control sows (P<0.01). However, the 200mg dose of CLZ, bid, failed to significantly block pregnancy in pigs. No significant differences were observed in heart rates of treated and control animals. Re-mating of the previously treated sows exhibited normal pregnancies and litter sizes. SIGNIFICANCE This study shows that CLZ is capable of producing a reversible non-steroidal contraceptive effect without adverse effects on the heart rate in pigs. The observed contraceptive effect of CLZ was at doses similar to those indicated to humans. This FDA approved agent, for treatment of patients with intermittent claudication, may have an additional therapeutic effect as a non-steroidal contraceptive agent. Cilostazol merits further evaluation in women and might be useful for controlling the population of homeless animals.

Improvement in in vitro fertilization outcome following in vivo synchronization of oocyte maturation in mice

Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2–4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients.

Improvement in pregnancy outcomes in couples with immunologically male infertility undergoing prednisolone treatment and conventional in vitro fertilization preceded by sperm penetration assay: a randomized controlled trial

PurposeAnti-sperm antibodies (ASA) in men impair not only sperm motility but also fertilization and conception. However, utilization of corticosteroids to suppress ASA has shown variable pregnancy outcomes. This controversy is also extended to include the usefulness of conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in treatments of men with ASA. This study was therefore designed to define factors contributing to these inconsistent results.MethodsInfertile men having ASA (n = 241) were randomly assigned for treatment with or without prednisolone for three cycles each of 21 days of their partner’s menstrual cycles. Control and treated men underwent then human sperm penetration assay (SPA), of hamster oocytes, to diagnose men with impaired sperm fusogenic capacity. Men with positive or negative SPA results were admitted to conventional IVF or ICSI programs, respectively.ResultsTreated patients had improved sperm motility and progressive motility when compared to control patients (P < 0.001). Fertilization (P = 0.04), embryo cleavage (P = 0.01), and chemical (P = 0.02) and clinical (P = 0.04) pregnancy rates were higher in treated patients than in control patients undergoing conventional IVF but not ICSI cycles.ConclusionsMen with ASA may also have compromised sperm fusogenic capacity, which can mask the clinical significance of corticosteroids. Corticosteroid administration in men with ASA, but without compromised sperm fusogenic capacity, improves conventional IVF but not ICSI outcomes; the reason being that ICSI bypasses issues of compromised fusogenic capacity. Inclusion of SPA in infertility clinics that offer both conventional IVF and ICSI services may be useful to identify which patients with ASA benefit from corticosteroid treatments.

Improvements in oocyte competence in superovulated mice following treatment with cilostazol: Ovulation of immature oocytes with high developmental rates

Graphical abstract Figure. No Caption available. ABSTRACT Exogenous administration of superovulatory hormones negatively affects oocyte competence in mammals. Phosphodiesterase 3A inhibitors were found to improve competence of oocytes matured in vitro in several species, including humans. This study was therefore designed to define oocyte maturation synchronization and competence, in vivo, using superovulated mice treated with cilostazol, a selective phosphodiesterase 3A inhibitor. Swiss Webster mice were superovulated and treated orally with 7.5 mg cilostazol once or twice to result in ovulation of immature oocytes at the metaphase I (MI) or germinal vesicle (GV) stage, respectively. Control immature oocytes were recovered from preovulatory follicles of superovulated mice not treated with cilostazol. Treated GV oocytes had significantly higher rates of synchronized and advanced chromatin configuration and cortical granule distribution than did control GV oocytes. Treated GV oocytes had a moderate increase in cAMP levels and consequently higher rates of meiotic maturation, IVF, and blastocyst formation than did control GV oocytes (P < 0.0001). Treated MI oocytes had higher rates of normal spindles and chromosomes aligned at the metaphase plate than did control MI oocytes (P < 0.003). Treated mice ovulating MI oocytes produced litter sizes larger than those observed in control mice ovulating mature oocytes (P < 0.002). This study reveals that synchronization of oocyte maturation in superovulated mice improves oocyte development and competence. The capability of cilostazol, a clinically approved medication, to improve mouse oocyte competence suggests the potential benefit of including this compound in ovarian hyperstimulation programs to improve in vitro fertilization outcomes in infertile women.

353 EFFECTS OF CULTURE DURATION AND CUMULUS CELL REMOVAL ON CANINE OOCYTE IN VITRO MATURATION AND FERTILIZATION USING VASAL SPERM

The objective of this study was to test the hypothesis that in vitro maturation (IVM) and fertilization (IVF) rates of canine oocytes could be improved by increasing culture duration or decreasing/increasing cumulus cell contact with the oocytes when using sperm retrieved from the vas deferens. The canine oocyte is ovulated at the germinal vesicle stage, and maturation of the oocyte occurs in the oviduct and requires up to five days. Therefore, an increase in the culture duration may cause an increase in oocyte nuclear maturation. Canine ovaries and testes were collected from a local clinic, placed in warm saline solution, and transported to the laboratory. Two distinct experiments were carried out, one involving IVM (M-II) after cumulus cell removal at 72 h and 96 h for nuclear maturation evaluation, and the second experiment the same but continued up to IVF. The oocytes were recovered from the ovaries by mincing them in warm Medium-199 with Hanks salts, L-glutamine, and HEPES (GIBCO, Grand Island, NY, USA; Invitrogen Co., Carlsbad, CA, USA). Canine oocytes with a dark cytoplasm and at least 2 layers of cumulus cells were cultured in Medium-199 supplemented with Earles salts, 2200 mg mL−1 sodium bicarbonate, 25 mM HEPES, 2 mM sodium pyruvate, 5 µg mL−1 progesterone, 100 ng mL−1 epidermal growth factor, 10 IU mL−1 human chorionic gonadotropin (HCG), 5 µg mL−1 insulin, 0.50 mM epinephrine, 10p estrus bitch serum, 0.01 mM nonessential amino acids, and 20 µg mL−1 gentamicin. The oocytes were cultured for 72, 96, 120, or 144 h at 38.5°C in 5p CO2 in humidified air. The cumulus cells were removed after either a 72- or 96-h culture period. For the first 48 h, the cumulus–oocyte complexes were cultured in the modified Medium-199 containing 10 IU mL−1 HCG and then cultured in the same medium free of HCG. The oocytes were denuded by pipetting, stained with Hoechst 33342, and examined for nuclear maturation. ANOVA was used for statistical analysis of the data. The IVM rate (MII) was significantly higher (P 0.05). The sperm motility index (SMI e motility percentage × sperm activity grade) was significantly higher in sperm retrieved from the vas deferens (vasal sperm) compared to epididymal and testicular sperm (259 vs. 95 and 19.2, respectively, P 0.05). Sperm penetration was significantly higher at 96 h compared to 72 h, and the number of sperm heads inside the ooplasm was 3.2 for the 72 h group vs. 4.8 for the 96 h group (P < 0.05). In conclusion, increasing the IVM culture period beyond 72 h did not increase the oocyte maturation rates, and increasing the culture time to 96 h without cumulus cells present increased the rate of sperm penetration.

370 BILATERAL DIFFERENTIAL TESTICULAR BIOPSIES IMPROVE TESE AND ICSI OUTCOMES IN NON-OBSTRUCTIVE AND OBSTRUCTIVE AZOOSPERMIC INFERTILE PATIENTS

The goal of the present study was to evaluate the clinical significance of bilateral differential testicular biopsies (BDTB) to improve testicular sperm extraction (TESE) and ICSI outcomes in non-obstructive azoospermic (NOA) men. The male patients were divided into an obstructive azoospermic group (OAG, n = 40) and an NOA group (n = 50). The women in both groups had normal ovulatory cycles and reproductive hormone concentrations. BDTB were taken from upper, middle, and lower zones of the testes. At least 50 transverse histologic sections of seminiferous tubules were examined in each biopsy. The FSH, LH, and prolactin concentrations were significantly higher in the NOA group compared to the OAG (18.3 vs. 6.2 mIU mL-1, 8.5 vs. 4.1 mIU mL-1, and 9.6 vs. 6.4 ng mL-1, respectively; P 0.05). The TESE-ICSI rates were 75.9p (117/154) in the OAG and 59.5p (106/178) in the NOA group (P 0.05. The clinical pregnancy rate per embryo transfer was 52.5p in the OAG and 40p in the NOA group (P > 0.05). It was concluded from the results of this study that both the BDTB and TESE were useful diagnostic and prognostic predictors of successful sperm retrieval for ICSI treatment in the non-obstructive and obstructive azoospermic male patients. The clinical embryo implantation rates were not significantly different between both groups (NOA group and OAG) which may indicate that the embryos of both groups have similar implantation potentials. Bilateral differential testicular biopsies were found to be positively correlated with the TESE and ICSI outcomes.