RESUMO
BACKGROUND: Melatonin is a hormone produced by the pineal gland and it has antioxidant properties. AIM: This study aimed to evaluate the effects of melatonin on assisted reproductive technologies through a systematic review and a meta-analysis. MATERIALS AND METHODS: Search strategies were used in PubMed and in other databases covering the last 15 years. After screening for eligibility, 17 articles were selected for the systematic review. For the meta-analysis statistics, two groups were formed, the treatment group (with melatonin) and the control group (without melatonin) for various assisted reproduction outcomes. RESULTS: The main results were that no statistical differences were found concerning the clinical pregnancy outcome (p = 0.64), but there was a statistical difference with respect to Mature Oocytes (MII) (p = 0.001), antral follicle count (p = 0.0002), and the fertilization rate (p ≤ 0.0001). CONCLUSIONS: Melatonin had beneficial effects such as the improvement in the fertilization rate, although the authors did not obtain significance in the clinical pregnancy rate.
Assuntos
Melatonina , Taxa de Gravidez , Melatonina/uso terapêutico , Melatonina/farmacologia , Humanos , Feminino , Gravidez , Técnicas de Reprodução Assistida , Antioxidantes/farmacologia , Fertilização in vitro/métodos , Fertilização in vitro/efeitos dos fármacos , Resultado da Gravidez , Fertilização/efeitos dos fármacos , Fertilização/fisiologiaRESUMO
This study evaluated the effects of oocyte meiosis inhibitors roscovitine (ROS) and butyrolactone I (BL-I) on in vitro production of bovine embryos. Bovine oocytes were maintained in pre in vitro maturation (pre-IVM) with 25 µM ROS or 100 µM BL-I for 24 h to delay meiosis and for 24 h in in vitro maturation (IVM). Following this treatment, the nuclear maturation index was evaluated. All embryos degenerated following this procedure. In the second set of experiments, oocytes were maintained for 6 or 12 h in pre-IVM with the following three treatments: ROS (25 µM or 12.5 µM), BL-I (100 µM or 50 µM) or a combination of both drugs (6.25 µM ROS and 12.5 µM BL-I). Oocytes were cultivated for 18 or 12 h in IVM. When a meiosis-inducing agent was used during pre-IVM for 24 h, more degenerated oocytes were observed at the end of the IVM period. This effect decreased when the meiotic blocking period was reduced to 6 or 12 h. No significant differences were observed in the blastocyst production rate of oocytes in pre-IVM for 6 h with ROS, BL-I, or ROS + BL-I compared with that of the control group (P > 0.05). However, inhibition of oocytes for 12 h resulted in decreased embryo production compared with that in the controls (P < 0.05). There was no difference in the post-vitrification embryo re-expansion rate between the study groups, showing that the meiotic inhibition for 6 or 12 h did not alter the embryo cryopreservation process.
Assuntos
4-Butirolactona/análogos & derivados , Blastocisto/citologia , Embrião de Mamíferos/citologia , Fertilização in vitro/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos , Meiose , Oócitos/citologia , Roscovitina/farmacologia , 4-Butirolactona/farmacologia , Animais , Blastocisto/efeitos dos fármacos , Bovinos , Embrião de Mamíferos/efeitos dos fármacos , Feminino , Oócitos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologiaRESUMO
PURPOSE: Oocyte maturation is a complex process involving nuclear and cytoplasmic modulations, during which oocytes acquire their ability to become fertilized and support embryonic development. The oocyte is apparently "primed" for maturation during its development in the dominant follicle. As bovine oocytes immediately resume meiosis when cultured, it was hypothesized that delaying resumption of meiosis with cyclic nucleotide modulators before in vitro maturation (IVM) would allow the oocytes to acquire improved developmental competence. METHODS: We tested the Simulated Physiological Oocyte Maturation (SPOM) system that uses forskolin and 3-isobutyl-1-methylxanthine for 2 h prior to IVM against two different systems of conventional IVM (Con-IVM). We evaluated the ultrastructure of matured oocytes and blastocysts and also assessed the expression of 96 genes related to embryo quality in the blastocysts. RESULTS: In summary, the SPOM system resulted in lower blastocyst rates than both Con-IVM systems (30 ± 9.1 vs. 35 ± 8.7; 29 ± 2.6 vs. 38 ± 2.8). Mature SPOM oocytes had significantly increased volume and number of vesicles, reduced volume and surface density of large smooth endoplasmic reticulum clusters, and lower number of mitochondria than Con-IVM oocytes. SPOM blastocysts showed only subtle differences with parallel undulations of adjacent trophectoderm plasma membranes and peripherally localized ribosomes in cells of the inner cell mass compared with Con-IVM blastocysts. SPOM blastocysts, however, displayed significant downregulation of genes related to embryonic developmental potential when compared to Con-IVM blastocysts. CONCLUSIONS: Our results show that the use of the current version of the SPOM system may have adverse effects on oocytes and blastocysts calling for optimized protocols for improving oocyte competence.
Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Fertilização in vitro/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos , Oócitos/efeitos dos fármacos , 1-Metil-3-Isobutilxantina/administração & dosagem , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/patologia , Bovinos , Colforsina/administração & dosagem , Células do Cúmulo/efeitos dos fármacos , Feminino , Meiose/genética , Oócitos/crescimento & desenvolvimento , Oócitos/patologia , Oogênese/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Gravidez , Ribossomos/efeitos dos fármacosRESUMO
Polycystic ovary syndrome (PCOS) affects 6-10% of women and could be considered one of the most common endocrine alterations in women of reproductive age. The syndrome is characterized by several hormonal and metabolic alterations, including insulin resistance and hyperandrogenism, which play a severe detrimental role in the patient's fertility. We aimed to offer an overview about drug metabolism in the PCOS population. Nevertheless, we did not find any study that directly compared drug metabolism between PCOS and healthy women. We therefore decided to summarize briefly how hormonal and insulin sensitizer drugs act differently in healthy and PCOS women, who show altered steroidogenesis by theca cells and metabolic imbalance, focusing especially on assisted reproductive techniques. To date, data about drug metabolism in the PCOS population appears to be extremely limited. This important gap could have significant implications for therapeutic approaches and future perspectives: the dosage of drugs commonly used for the treatment of PCOS women should be tailored according to each patient's characteristics; we should implement new clinical trials in order to identify the best pharmacologic strategy for PCOS patients undergoing in vitro fertilization (IVF); it would be advisable to create an international expert panel to investigate the drug metabolism in the PCOS population.
Assuntos
Androgênios/metabolismo , Androgênios/uso terapêutico , Fertilização in vitro/efeitos dos fármacos , Síndrome do Ovário Policístico/tratamento farmacológico , Síndrome do Ovário Policístico/fisiopatologia , Adulto , Feminino , HumanosRESUMO
The increased use of cannabis as a therapeutic drug in recent years has raised some concerns due to its potential effects on reproductive health. With regards to the male, the endocannabinoid system is involved in the spermatogenesis and in the sperm function. The chronic use of tetrahidrocannabinol (THC) has been associated with sperm anomalies, decreased sperm motility and structural changes in the testis. However, whether THC affects sperms ability to fertilize and to generate embryos remains unclear. The aim of this study was to evaluate this effect using a mice model of THC chronic treatment. For this purpose, a chronic treatment with THC was carried out. Mice were randomly allocated into two groups: an experimental group treated with a daily dose of 10â¯mg/kg-body weight THC for a period of 30â¯days and a control group treated with a vehicle. The THC-mice cortex showed a significant decrease of mRNA of Cnr1 compared to control-mice while, in the testis, the expression of Cnr1 was not affected. The weight of testis and epididymis and the histological analysis did not show any change between groups. On the other hand, no changes were observed in the sperm motility or the sperm concentration. The chronic use of THC did not generate any methylation change in the three CpG regions of Cnn1 analysed, neither in the brain nor in the embryos generated by in vitro fertilization (IVF). Finally, the embryo production by IVF was no different using spermatozoa from both THC and control mice. This work contradicts the belief that THC consumption has a negative effect on male reproductive processes.
Assuntos
Dronabinol/toxicidade , Espermatozoides/efeitos dos fármacos , Testículo/efeitos dos fármacos , Animais , Apoptose , Peso Corporal/efeitos dos fármacos , Encéfalo/metabolismo , Dronabinol/farmacocinética , Embrião de Mamíferos/metabolismo , Epididimo/anatomia & histologia , Epigênese Genética , Fertilização in vitro/efeitos dos fármacos , Masculino , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Regiões Promotoras Genéticas , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Contagem de Espermatozoides , Motilidade dos Espermatozoides/efeitos dos fármacos , Testículo/anatomia & histologia , Testículo/metabolismoRESUMO
The aim of the present study was to evaluate the effect of frutalin (FTL) on in vitro maturation (IVM), and fertilization (IVF) of pig oocytes. In the Experiment 1, cumulus-oocyte complexes (COCs) were submitted to IVM in maturation medium alone or supplemented with different FTL concentration (0.6, 6 and 60⯵g/mL), or 0.3⯵g/mL doxorubicin (DXR). After IVM, some oocytes were evaluated for chromatin configuration, and the remaining oocytes were submitted to in vitro fertilization. In Experiment 2, matured oocytes were fertilized in IVF medium alone (control) or in presence of different FTL concentration (0.6, 6 and 60⯵g/mL), or 0.3⯵g/mL DXR. After 18â¯h post fertilization, the endpoints penetration rate, monospermy, spermatozoa per oocyte, and the IVF efficiency were evaluated in both experiments. In Experiment 1, 6 and 60⯵g/mL FTL, as well as DXR increased (Pâ¯<â¯0.05) the rate of oocytes with abnormal chromatin configuration when compared to oocyte matured in control medium alone or supplemented with 0.6⯵g/mL FTL. The percentage of meiotic resumption in oocytes cultured with 60⯵g/mL FTL or DXR was less (Pâ¯<â¯0.05) than in the other treatments. Moreover, oocytes matured with 6 or 60⯵g/mL FTL and DXR had a lesser IVM efficiency when compared to those matured with 0.6⯵g/mL FTL or in control medium. Additionally, there was a greater (Pâ¯<â¯0.05) with culture in a medium containing 6⯵g/mL FTL for the rate of partenogenetically activated oocytes when compared with the other treatments. Culturing of COCs during IVM in a medium containing 6 or 60 FTL resulted in a lesser (Pâ¯<â¯0.05) sperm penetration and spermatozoa/oocyte rates when compared to other treatments, and IVF efficiency was less (Pâ¯<â¯0.05) than that in control medium alone or with a medium containing 0.6⯵g/mL FTL. In Experiment 2, culturing in a medium containing 0.6⯵g/mL FTL resulted in greater (Pâ¯<â¯0.05) monospermy and IVF efficiency rates when compared to culturing in the control medium. In addition, culturing in a medium with 6 and 60⯵g/mL FTL resulted in a lesser (Pâ¯<â¯0.05) spermatozoa penetration, sperm/oocyte rates and IVF efficiency, although there were greater (Pâ¯<â¯0.05) monospermy rates. In conclusion, culturing in a medium containing 0.6⯵g/mL FTL resulted in lesser spermatozoa penetration rates and number of spermatozoa/oocyte increasing the IVF efficiency without harmful effects. Use of a greater concentration of FTL in the medium has toxic effects during oocyte maturation and results in a reduced IVF efficiency.
Assuntos
Fertilização in vitro/veterinária , Galectinas/farmacologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Suínos , Animais , Relação Dose-Resposta a Droga , Fertilização in vitro/efeitos dos fármacos , Galectinas/administração & dosagem , Oócitos/fisiologiaRESUMO
This study was conducted to optimize the cryopreservation of epididymal bison sperm harvested in the field. In the first experiment, epididymal bison sperm were treated with or without seminal plasma (n = 6) and cooled to 5 °C over 2 hours. In a separate experiment, glycerol was added at different times and sperm was held at 5 °C for different periods of time before cryopreservation (n = 11). In addition, epididymal sperm frozen with and without seminal plasma (n = 6) and after 4, 24, and 48 hours (n = 5) of equilibration at 5 °C, were evaluated for their in vitro fertilizing ability. Post-thaw motility of bison epididymal sperm was similar when cryopreserved with or without seminal plasma or when glycerol was added at either 0, 4, 24, or 48 hours before freezing (P > 0.05). However, sperm incubated at 5 °C for 24 hours before freezing exhibited higher percentages of motile sperm (44% vs. 35% for 4 hours or 48 hours, P < 0.05). Fertilization rates of bison oocytes were not different for any treatments. Chilling the whole epididymis for 24 or 48 hours resulted in complete loss of sperm viability. In conclusion, bison epididymal sperm can be chilled outside of the epididymis for at least 48 hours before cryopreservation without compromising post-thaw sperm motility providing flexibility for technicians performing field collections.
Assuntos
Bison/fisiologia , Criopreservação/veterinária , Crioprotetores/farmacologia , Epididimo/fisiologia , Glicerol/farmacologia , Refrigeração/veterinária , Preservação do Sêmen/veterinária , Animais , Fertilização in vitro/efeitos dos fármacos , Masculino , Sêmen , Preservação do Sêmen/métodos , Manejo de Espécimes/métodos , Manejo de Espécimes/veterinária , Motilidade dos EspermatozoidesRESUMO
The aim of the present study was to evaluate the effects of sperm motility enhancers and different IVF times on cleavage, polyspermy, blastocyst formation, embryo quality and hatching ability. In Experiment 1, sex-sorted X chromosome-bearing Bos taurus spermatozoa were incubated for 30min before 18h fertilisation with hyperactivating factors, namely 10mM caffeine (CA), 5mM theophylline (TH), 10mM caffeine and 5mM theophylline (CA+TH); and untreated spermatozoa (control). In Experiment 2, matured B. taurus oocytes were fertilised using a short (8h) or standard (18h) fertilisation length, comparing two different fertilisation media, namely synthetic oviducal fluid (SOF) fertilisation medium (SOF-FERT) and M199 fertilisation medium (M199-FERT). Cleavage and blastocyst formation rates were significantly higher in the CA+TH group (77% and 27%, respectively) compared with the control group (71% and 21%, respectively). Cleavage rates and blastocyst formation were significantly lower for the shortest fertilisation time (8h) in M199-FERT medium (42% and 12%, respectively). The SOF-FERT medium with an 8h fertilisation time resulted in the highest cleavage rates and blastocyst formation (74% and 29%, respectively). The SOF-FERT medium produced the highest embryo quality (50% Grade 1) and hatching rate (66%). Motility enhancers did not affect polyspermy rates, whereas polyspermy was affected when fertilisation length was extended from 8h (3%) to 18h (9%) and in M199-FERT (14%) compared with SOF-FERT (6%). We conclude that adding the motility enhancers CA and TH to sex sorted spermatozoa and Tyrode's albumin lactate pyruvate (TALP)-Sperm can improve cleavage and embryo development rates without increasing polyspermy. In addition, shortening the oocyte-sperm coincubation time (8h) resulted in similar overall embryo performance rates compared with the prolonged (18h) interval.
Assuntos
Cafeína/farmacologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Bovinos , Separação Celular , Feminino , Fertilização in vitro/efeitos dos fármacos , Fertilização in vitro/métodos , Fertilização in vitro/veterinária , Citometria de Fluxo , MasculinoRESUMO
The aim of this study was to evaluate the use of antifreeze protein type III (AFP III) into vitrification medium on meiotic spindle morphology of in vitro matured bovine oocytes as well as the fertilization and blastocyst rates. Mature cumulus-oocyte complexes (COC) were distributed in four groups: control (untreated), vitrified without supplementation (AFP0) or supplemented with 500 (AFP500) or 1000 ng/mL (AFP1000) into vitrification solutions. Samples from each group were used to analyze the organization of meiotic spindle by confocal microscopy and the remaining COC were submitted to in vitro fertilization and culture for eight days. Control group exhibited only 15% of abnormal spindle. However, the spindle morphology was affected in all vitrified groups regardless to AFP concentration: 75.8%, 76.1% and 69.2% (P > 0.05) for AFP0, AFP500 and AFP1000, respectively. Similar cleavage rate was obtained among the vitrified groups (AFP0 = 17.9%, AFP500 = 16.9% and AFP1000 = 17.8%), but lower (P < 0.05) compared with control group (68.7%). At Day 5 of culture, embryo production rate of AFP500 (30.8%) and AFP1000 (25.0%) were similar to control group (49.4%). However, at Day 8 of culture, AFP0, AFP500 and AFP1000 groups exhibited lower (P < 0.05) blastocyst rates (10.0%, 3.8% and 9.4%, respectively) when compared to control (41.1%). In conclusion, AFP III did not preserve meiotic spindle organization against the cryoinjuries. However, the use of AFP III improved embryo development at Day 5 of culture, although this effect was not maintained up to the blastocyst formation.
Assuntos
Proteínas Anticongelantes/farmacologia , Criopreservação/métodos , Crioprotetores/farmacologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos , Vitrificação , Animais , Blastocisto/efeitos dos fármacos , Bovinos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilização in vitro/efeitos dos fármacos , Fertilização in vitro/métodos , Microscopia Confocal , Oócitos/metabolismoRESUMO
UNLABELLED: The present study evaluates the possible antifertility effect of aqueous crude extract (OBACE) of Echeveria gibbiflora, a plant that belongs to the crassulaceae family, used in traditional Mexican medicine as a vaginal post coital rinse to prevent pregnancy and shown to have an immobilization/agglutination effect on sperm of different mammal species. We evaluated the effect of OBACE on functional parameters of mouse sperm, such as viability, capacitation, and acrosome reaction. In addition, due to the high concentrations of calcium bis-(hydrogen-1-malate) hexahydrate [Ca (C4H5O5)2â¢6H2O] present in this plant extract, we evaluated its effect on Ca(2+) influx in mouse sperm under capacitating conditions. Moreover, we determined the acute toxicity of OBACE and its in vivo effect in mouse sperm motility administering a single daily dose of 50 and 100 mg/kg during seven days, intraperitoneally. The sperm viability was not affected by the presence of different concentrations of OBACE, however, the capacitation and acrosome reaction suffered a significant decrease in a concentration-dependent manner, coinciding with the reduction of Ca(2+) influx. Furthermore, OBACE displayed an LD50 of 3,784.42 mg/kg and can be classified as a low toxic substance. Also, in vivo OBACE showed an inhibition of total and progressive motility on mouse sperm alongside a significant decrease of motility kinematic parameters and IVF rates. The results confirm the antifertility effect of this plant used in Mexican folk medicine. Further study on OBACE as a possible contraceptive treatment is warranted because of its activity and low in vivo toxicity. ABBREVIATIONS: ALH: lateral amplitude; AP: acid phosphatase; BCF: beat frequency; BSA: bovine serum albumine; CTC: chlortetracycline; FDA: fluorescein diacetate; Fura-2 AM: fura-2-acetoxymethyl ester; HIV: human immunodeficiency virus; IVF: in vitro fertilization; OBACE: aqueous crude extract of Echeveria gibbiflora; PI: propidum iodide; SN: supernatant; VAP: average path velocity; VCL: track speed; VSL: straight line velocity.
Assuntos
Anticoncepcionais/farmacologia , Crassulaceae/química , Extratos Vegetais/farmacologia , Espermatozoides/efeitos dos fármacos , Reação Acrossômica/efeitos dos fármacos , Animais , Fertilização in vitro/efeitos dos fármacos , Masculino , Medicina Tradicional , México , Camundongos , Capacitação Espermática/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacosRESUMO
STUDY QUESTION: Does ulipristal acetate (UPA), a selective progesterone receptor modulator used for emergency contraception (EC), interfere with fertilization or early embryo development in vitro and in vivo? SUMMARY ANSWER: At doses similar to those used for EC, UPA does not affect mouse gamete transport, fertilization or embryo development. WHAT IS KNOWN ALREADY: UPA acts as an emergency contraceptive mainly by inhibiting or delaying ovulation. However, there is little information regarding its effects on post-ovulatory events preceding implantation. STUDY DESIGN, SIZE, DURATION: This was an in vitro and in vivo experimental study involving the use of mouse gametes and embryos from at least three animals in each set of experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: For in vitro fertilization experiments, mouse epididymal spermatozoa capacitated in the presence of different concentrations of UPA (0-1000 ng/ml) were used to inseminate cumulus-intact or cumulus-free eggs in the presence or absence of UPA during gamete co-incubation, and the percentage of fertilized eggs was determined. For in vivo fertilization experiments, superovulated females caged with proven fertile males were injected with UPA (40 mg/kg) or vehicle just before or just after mating and the percentage of fertilized eggs recovered from the ampulla was determined. To investigate the effect of UPA on embryo development, zygotes were recovered from mated females, cultured in the presence of UPA (1000 ng/ml) for 4 days and the progression of embryo development was monitored daily. MAIN RESULTS AND THE ROLE OF CHANCE: In vitro studies revealed that the presence of UPA during capacitation and/or gamete co-incubation does not affect fertilization. Whereas the in vivo administration of UPA at the same time as hCG injection produced a decrease in the number of eggs ovulated compared with controls (vehicle injected animals, P < 0.05), no effects on fertilization were observed when UPA was administered shortly before or after mating. No differences were observed in either the percentage of cleaved embryos or the cleavage speed when UPA was present during in vitro embryo culture. LIMITATIONS, REASONS FOR CAUTION: Considering the ethical and technical limitations inherent to the use of human gametes for fertilization studies, the mouse model was used as an approach for exploring the potential effects of UPA on in vivo sperm transport and fertilization. Nevertheless, the extrapolation of these results to humans requires further investigation. WIDER IMPLICATIONS OF THE FINDINGS: This study presents new evidence on the lack of effect of UPA on gamete interaction and embryo development, providing new insights into the mechanism of action of UPA as an emergency contraceptive method with potential clinical implications. These new findings could contribute to increase the acceptability and proper use of UPA as an emergency contraceptive method. STUDY FUNDING/COMPETING INTERESTS: This study was partially supported by a National Agency of Scientific and Technological Promotion (ANPCyT), Argentina grants PICT 2011-061 to D.J.C. and PICT 2011-2023 to P.S.C. None of the authors has any competing interests to declare.
Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Fertilização in vitro/efeitos dos fármacos , Norpregnadienos/farmacologia , Receptores de Progesterona/efeitos dos fármacos , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Animais , Anticoncepção Pós-Coito , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Fibroblast growth factor (FGF10) acts at the cumulus oocyte complex, increasing the expression of cumulus cell expansion-related genes and oocyte competency genes. We tested the hypothesis that addition of FGF10 to the maturation medium improves oocyte maturation, decreases the percentage of apoptotic oocytes and increases development to the blastocyst stage while increasing the relative abundance of developmentally important genes (COX2, CDX2 and PLAC8). In all experiments, oocytes were matured for 22 h in TCM-199 supplemented with 0, 2.5, 10 or 50 ng/ml FGF10. In Experiment 1, after maturation, oocytes were stained with Hoechst to evaluate meiosis progression (metaphase I, intermediary phases and extrusion of the first polar body) and submitted to the TUNEL assay to evaluate apoptosis. In Experiment 2, oocytes were fertilized and cultured to the blastocyst stage. Blastocysts were frozen for analysis of COX2, CDX2 and PLAC8 relative abundance. In Experiment 1, 2.5 ng/ml FGF10 increased (p < 0.05) the percentage of oocytes with extrusion of the first polar body (35%) compared to 0, 10 and 50 ng/ml FGF10 (21, 14 and 12%, respectively) and FGF10 decreased the percentage of oocytes that were TUNEL positive in all doses studied. In Experiment 2, there was no difference in the percentage of oocytes becoming blastocysts between treatments and control. Real-time RT-PCR showed a tendency of 50 ng/ml FGF10 to increase the relative abundance of COX2 and PLAC8 and of 10 ng/ml FGF10 to increase CDX2. In conclusion, the addition of FGF10 to the oocyte maturation medium improves oocyte maturation in vitro, decreases the percentage of apoptotic oocytes and tends to increase the relative abundance of developmentally important genes.
Assuntos
Apoptose/efeitos dos fármacos , Bovinos , Desenvolvimento Embrionário/efeitos dos fármacos , Fator 10 de Crescimento de Fibroblastos/farmacologia , Meiose/efeitos dos fármacos , Oócitos/citologia , Animais , Blastocisto/química , Blastocisto/fisiologia , Fator de Transcrição CDX2 , Meios de Cultura , Células do Cúmulo/fisiologia , Ciclo-Oxigenase 2/genética , Técnicas de Cultura Embrionária/veterinária , Feminino , Fertilização in vitro/efeitos dos fármacos , Fator 10 de Crescimento de Fibroblastos/administração & dosagem , Genes Controladores do Desenvolvimento , Proteínas de Homeodomínio/genética , Marcação In Situ das Extremidades Cortadas , Técnicas de Maturação in Vitro de Oócitos , Oócitos/química , Proteínas da Gravidez/genética , RNA Mensageiro/análise , Transativadores/genéticaRESUMO
Os androgênios, por agirem de forma positiva no desenvolvimento folicular, estão sendo atualmente utilizados na reprodução humana assistida como uma alternativa para melhorar a resposta ovariana de mulheres consideradas más respondedoras. Esta revisão sistemática avalia o efeito do desidroepiandrosterona (DHEA) na resposta à estimulação ovariana de mulheres más respondedoras submetidas às técnicas de reprodução assistida. Os artigos para este estudo foram pesquisados no PubMed e publicados entre 1999 e 2013. Vinte e sete artigos foram avaliados e 18 deles foram selecionados, incluindo estudos experimentais e observacionais. O DHEA foi associado a um maior número de folículos recrutados, de oócitos selecionados e melhor qualidade embrionária, à diminuição do risco de aneuploidias e à maior taxa de gravidez clínica e nascidos vivos. Apesar de o DHEA apresentar efeito positivo na resposta ovariana de mulheres más respondedoras, os resultados obtidos foram pouco consistentes. Mais estudos controlados e randomizados devem ser realizados antes de se implantar o DHEA de rotina no tratamento de más respondedoras submetidas à reprodução humana assistida.(AU)
Androgens are currently being used in assisted human reproduction as an alternative to improve ovarian response of women considered poor responder by acting positively in follicular development. This systematic review evaluates the effects of dehydroepiandrosterone (DHEA) in response to ovarian stimulation of poor responder women undergoing assisted reproductive techniques. All articles for this study were searched in PubMed and published between 1999 and 2013. Twenty seven articles were evaluated and 18 of them were selected, including experimental and observational studies. DHEA was associated with a greater number of follicles, oocyte selected and better embryo quality, the decreased risk of aneuploidy and higher rates of clinical pregnancy and live birth. Although DHEA has positive effect on the ovarian response of poor responder women, the results were inconsistent. More randomized controlled trials should be conducted before using DHEA in routine treatment of poor responders undergoing assisted reproduction.(AU)
Assuntos
Humanos , Feminino , Gravidez , Indução da Ovulação/métodos , Fertilização in vitro/efeitos dos fármacos , Desidroepiandrosterona/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Bases de Dados Bibliográficas , Infertilidade Feminina/tratamento farmacológicoRESUMO
The objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) on transgene expression efficiency and on mosaic expression patterns of IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated with transgene. The DNA damage induced by the transgene or cell cycle inhibitors was measured by detection of phosphorylated histone H2AX foci presence (marker of DNA double-stranded breaks). Cloning of egfp blastomeres was included to determine continuity of expression after additional rounds of cellular division. The pCX-EGFP [enhanced green fluorescent protein gene (EGFP) under the chimeric cytomegalovirus IE-chicken-ß-actin enhancer promoter control] gene plasmid (50 ng/µL) was injected alone (linear or circular exogenous DNA, leDNA and ceDNA, respectively) or associated with ooplasmic vesicles (leDNA-v or ceDNA-v). The effects of 2 mm DMAP or 1 µm DhL for 6 h (from 15 to 21 h post IVF) was evaluated for groups injected with vesicles. The DMAP increased (P < 0.05) egfp homogenous expression relative to transgene alone (21%, 18%, and 11% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively) and also increased (P < 0.05) the phosphorylated histone H2AX foci area. Expression of egfp was higher (P < 0.05) for linear than for circular pCX-EGFP, and egfp blastocyst rates were higher (P < 0.05) for groups injected with linear transgene coincubated with vesicles than for linear transgene alone (95%, 77%, 84%, and 52% for leDNA-v + DMAP, leDNA-v + DhL, leDNA-v, and leDNA, respectively). Moreover, DMAP tended to improve egfp blastocysts rates for both circular and linear transgenes. Based on fluorescent in situ hybridization (FISH) analysis, there was evidence of integration in egfp embryos. Finally, clones derived from leDNA-v + DMAP had the highest egfp expression rates (96%, 65%, and 65% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively). Transgenesis by cytoplasmic injection of leDNA-v + DMAP is a promising alternative for transgenic animal production.
Assuntos
Animais Geneticamente Modificados , Bovinos/embriologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Clonagem de Organismos/métodos , Fertilização in vitro , Inibidores de Proteínas Quinases/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Bovinos/genética , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos , Feminino , Fertilização in vitro/efeitos dos fármacos , Fertilização in vitro/veterinária , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Lactonas/farmacologia , Masculino , Sesquiterpenos/farmacologia , Transgenes/genéticaRESUMO
In this work, we have investigated the role of the bovine sperm proteasome during in vitro fertilisation (IVF) and the acrosome reaction (AR). Motile spermatozoa, obtained by a swim-up method in Sperm-Talp medium, were capacitated for 3.5 h and incubated in the presence or absence of the specific proteasome inhibitor epoxomicin for 30 and 60 min. Then, the spermatozoa were co-incubated with mature bovine cumulus oocytes and after 48 h the cleavage rate of inseminated oocytes was evaluated. In addition, we evaluated the participation of the sperm proteasome during the progesterone-induced AR. Capacitated spermatozoa were incubated for 30 min with or without epoxomicin, then progesterone was added and the ARs were evaluated using the dual fluorescent staining technique 'Hoechst and chlortetracycline'. The results indicate that the proteasome inhibitor decreased the cleavage rate of oocytes inseminated with treated spermatozoa. In addition, acrosomal exocytosis levels were statistically significantly higher in the samples treated with the AR inducer progesterone than in control samples in the absence of the inducer. However, the progesterone-induced AR was significantly reduced by previous treatment of the spermatozoa with epoxomicin (P < 0.001). These observations indicate that the bovine sperm proteasome participates in the IVF and AR processes.
Assuntos
Reação Acrossômica/fisiologia , Bovinos , Fertilização in vitro/veterinária , Complexo de Endopeptidases do Proteassoma/fisiologia , Espermatozoides/enzimologia , Reação Acrossômica/efeitos dos fármacos , Animais , Inibidores Enzimáticos/farmacologia , Fertilização in vitro/efeitos dos fármacos , Masculino , Oligopeptídeos/farmacologia , Inibidores de Proteassoma , Capacitação EspermáticaRESUMO
Aiming to achieve the ideal time of ovum pick-up (OPU) for in vitro embryo production (IVP) in crossbred heifers, two Latin square design studies investigated the effect of ovarian follicular wave synchronization with estradiol benzoate (EB) and progestins. For each experiment, crossbred heifers stage of estrous cycle was synchronized either with a norgestomet ear implant (Experiment 1) or a progesterone intravaginal device (Experiment 2) for 7d, followed by the administration of 150microg d-cloprostenol. On Day 7, all follicles >3mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14d. Each experiment had three treatment groups. In Experiment 1 (n=12), heifers in Group 2X had their follicles aspirated twice a week and those in Groups 1X and 1X-EB were submitted to OPU once a week for a period of 28d. Heifers from Group 1X-EB also received 2mg EB i.m. immediately after each OPU session. In Experiment 2 (n=11), animals from Group 0EB did not receive EB while heifers in Groups 2EB and 5EB received 2 and 5mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28d. Therefore, in both experiments, four OPU sessions were performed in heifers aspirated once a week and in Experiment 1, eight OPU sessions were done in heifers aspirated twice a week. Additionally, during the 7-d period following follicular aspiration, ovarian ultrasonography examinations were conducted to measure diameter of the largest follicle and blood samples were collected for FSH quantification by RIA. In Experiment 1, all viable oocytes recovered were in vitro matured and fertilized. Results indicated that while progestin and EB altered follicular wave patterns, this treatment did not prevent establishment of follicular dominance on the ovaries of heifers during OPU at 7-d intervals. Furthermore, the proposed stage of follicular wave synchronization strategies did not improve the number and quality of the recovered oocytes, or the number of in vitro produced embryos.
Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Estradiol/análogos & derivados , Sincronização do Estro/fisiologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Progestinas/farmacologia , Animais , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fase de Clivagem do Zigoto/efeitos dos fármacos , Dinoprosta/administração & dosagem , Implantes de Medicamento/administração & dosagem , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/fisiologia , Estradiol/farmacologia , Estradiol/uso terapêutico , Sincronização do Estro/efeitos dos fármacos , Feminino , Fertilização in vitro/efeitos dos fármacos , Injeções Intramusculares , Recuperação de Oócitos/métodos , Recuperação de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Folículo Ovariano/diagnóstico por imagem , Pregnenodionas/administração & dosagem , Pregnenodionas/farmacologia , Progestinas/uso terapêutico , Controle de Qualidade , UltrassonografiaRESUMO
OBJECTIVE: To evaluate effects of pre- and/or postnatal exposure to ambient fine particulate matter on fertilization, embryo development, and cell lineage segregation in preimplantation blastocysts using the IVF mouse model. DESIGN: Animal model. SETTING: Academic institution. ANIMAL(S): Six-week-old, superovulated mice. INTERVENTION(S): Pre- and postnatal exposure to filtered air (FA-FA), filtered-ambient air (FA-AA), or ambient air (AA-AA) in exposure chambers 24 hours a day for 9 weeks. MAIN OUTCOME MEASURE(S): Gestation length, litter size, sex ratio, ovarian response to superovulation, fertilization rate, embryo development, blastocyst and hatching rates, total cell count, and proportion of cell allocation to inner-cell mass (ICM) and trophectoderm (TE). RESULT(S): Gestation length, litter size and birth weight, live-birth index, and sex ratio were similar among exposure groups. Ovarian response was not affected by the exposure protocol. A multivariate effect for pre- and/or postnatal exposure to ambient fine particulate matter on IVF, embryo development, and blastocyst differential staining was found. Cell counts in ICM and ICM/TE ratios in blastocysts produced in the FA-FA protocol were significantly higher than in blastocysts produced in the FA-AA and AA-AA protocols. No difference in total cell count was observed among groups. CONCLUSION(S): Our study suggests that exposure to ambient fine particulate matter may negatively affect female reproductive health by disrupting the lineage specification at the blastocyst stage without interfering in early development of the mouse embryo.
Assuntos
Linhagem da Célula/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Fertilização in vitro/efeitos dos fármacos , Material Particulado/farmacologia , Animais , Células Cultivadas , Feminino , Exposição por Inalação/efeitos adversos , Masculino , Exposição Materna/efeitos adversos , Camundongos , Indução da Ovulação , Tamanho da Partícula , Material Particulado/toxicidade , Gravidez , Taxa de Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Reprodução/efeitos dos fármacosRESUMO
Despite the probable inhibitory effects of GnRH analogues on ovarian steroidogenesis in vitro, their association with assisted reproduction protocols shows favorable results. This suggests that there are important differences in the behaviors of these drugs when administered in vivo versus in vitro. To clarify these differences, this study was designed to analyze the effect of leuprolide acetate (LA) on ovarian steroidogenesis in women undergoing In Vitro Fertilization (IVF). A prospective, randomized open label study was conducted on 14 women (26-35 years): seven receiving only gonadotrophins (Group 1) and seven receiving gonadotrophin plus LA at 1mg/day (Group 2). The LA in vivo effect was determined with serum and follicular fluid (FF) samples and via luteinized granulosa cell cultivation (GCC), where cells were obtained during oocyte retrieval after ovarian hyperstimulation. In vitro analysis was performed via addition of LA to GCC only for Group 1 (without LA) at progressively higher concentrations (0, 10(-12), 10(-9) and 10(-6)M). In vivo, the main observation was a reduction in androgen production in Group 2, represented by lower androstenedione production in FF (G1=6479+/-3458; G2=3021+/-1119 ng/ml; p=0.04) and a lower testosterone peak in GC at 96h (G1=0.64+/-0.12 ng/ml; G2=0.50+/-0.19 ng/ml; P=0.02), but a higher fertilization rate (G1=67%; G2=83%; p=0.009). In vitro, testosterone, estradiol and progesterone were also reduced by LA, even though this reduction occurred for progesterone only at the highest LA dosage (10(-6)M; 606.0+/-114.3 ng/ml versus 1524.0+/-246.5 ng/ml; p=0.02). Results show that LA reduces ovarian steroidogenesis in vivo by essentially inhibiting androgen synthesis; whereas, in vitro, ovarian steroidogenesis is reduced overall.
Assuntos
Fármacos para a Fertilidade Feminina/uso terapêutico , Fertilização in vitro/efeitos dos fármacos , Infertilidade/tratamento farmacológico , Leuprolida/uso terapêutico , Ovário/efeitos dos fármacos , Esteroides/biossíntese , Adulto , Células Cultivadas , Combinação de Medicamentos , Feminino , Líquido Folicular/citologia , Líquido Folicular/efeitos dos fármacos , Líquido Folicular/metabolismo , Hormônio Liberador de Gonadotropina , Gonadotropinas/farmacologia , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Humanos , Técnicas In Vitro , Infertilidade/metabolismo , Ovário/metabolismo , Estudos Prospectivos , Esteroides/sangueRESUMO
Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 x 10(5) frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.
Assuntos
Anticorpos/farmacologia , Bovinos/fisiologia , Fertilização/fisiologia , Oviductos/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Zona Pelúcida/metabolismo , Animais , Western Blotting/veterinária , Bovinos/embriologia , Feminino , Fertilização in vitro/efeitos dos fármacos , Fertilização in vitro/veterinária , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Imuno-Histoquímica/veterinária , Oxirredutases Intramoleculares/imunologia , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/imunologia , Lipocalinas/metabolismo , Masculino , Osteopontina/imunologia , Osteopontina/metabolismo , Gravidez , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Zona Pelúcida/fisiologiaRESUMO
The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO(2) in air) for 1h in the following treatments either 500 microL of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1h either FM or FM with alpha L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) alpha L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) alpha L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 x 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha L-PGDS: (1) 26.4+/-3.0; (2) 25.6+/-3.0; (3) 59.7+/-3.0; (4) 56.4+/-3.0; and (5) 57.1+/-3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P<0.05) compared with the control: (1) 89.2+/-2.0%; (2) 87.5+/-2.0%; (3) 19.4+/-2.0%; (4) 27.2+/-3.1%; and (5) 14.1+/-3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization.