In Vitro Fertilization Of Oocytes Research Articles

Hazardous impacts of silver nanoparticles on mouse oocyte maturation and fertilization and fetal development through induction of apoptotic processes.

Silver nanoparticles (AgNPs) are antibacterial materials widely used in numerous products and medical supplies. Previously, we showed that AgNPs trigger apoptotic processes in mouse blastocysts, leading to a decrease in cell viability and impairment of preimplantation and postimplantation embryonic development in vitro and in vivo. In the present study, we further investigated the hazardous effects of AgNPs on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent preimplantation and postimplantation development in vitro and in vivo. Data from in vitro experiments revealed that AgNPs impair mouse oocyte maturation, decrease IVF rates, and induce injury effects on subsequent embryonic development to a significant extent. In an animal model, intravenous injection of AgNPs (5 mg/kg body weight) led to a significant decrease in mouse oocyte maturation and IVF concomitant with impairment of early embryonic development in vivo. Importantly, pretreatment with N-acetylcysteine effectively prevented AgNP-triggered reactive oxygen species (ROS) production and apoptosis, clearly suggesting a critical role of ROS as an upstream initiator or key regulator of AgNP-induced hazardous effects on oocyte maturation and sequent embryonic development. Furthermore, preincubation of oocytes with Ac-DEVD-cho, a caspase-3-specific inhibitor, effectively prevented hazardous effects, highlighting the potential involvement of caspase-dependent apoptotic signaling cascades in AgNP-mediated events. Expression levels of p53 and p21 of blastocysts were upregulated upon preincubation of mouse oocytes with AgNPs. Our collective results imply that cell apoptosis in mouse blastocysts derived from the AgNP-pretreated oocytes via intracellular ROS generation, which is further mediated through p53-, p21-, and caspase-3-dependent regulatory mechanisms.

Live birth and multiple birth rates in US in vitro fertilization treatment using donor oocytes: a comparison of single-embryo transfer and double-embryo transfer.

To compare live birth rates (LBRs) and multiple birth rates (MBRs) between elective single-embryo transfer (eSET) and double-embryo transfer (DET) in donor oocyte in vitro fertilization (IVF) treatments in both a cycle-level and clinic-level analysis. Donor oocyte IVF treatments performed by US IVF clinics reporting to the Centers for Disease Control and Prevention in 2013-2014 were included in the analysis. Primary outcomes included LBR and MBR. Secondary outcomes included gestational age at delivery (GA) and birth weight (BW) of offspring. These outcomes were evaluated on an individual cycle level as well as on the clinic level. In multivariable models, LBR did not change significantly as clinics utilized eSET more frequently. MBR decreased significantly as utilization of eSET increased, from 39% MBR in clinics that utilized eSET 0-9% of the time to 7% MBR in clinics that used eSET 70% of the time (P < .0001). Mean BW and GA of IVF-conceived offspring both increased as clinics utilized eSET more frequently (2778 to 3185g [P < .0001] and 37.5 to 38.5weeks [P = .02] for clinics with the lowest and highest eSET utilization, respectively). US IVF clinics utilizing eSET with higher frequencies have clinically comparable LBRs and significantly lower MBRs than clinics with lower-frequency eSET utilization. Mean offspring BW and GA increased with higher eSET utilization, further confirming the improved safety of this practice.

A Proof-of-Concept Clinical Trial of A Single Luteal Use of Long-Acting Gonadotropin-Releasing Hormone Antagonist Degarelix in Controlled Ovarian Stimulation for In Vitro Fertilization: Long Antagonist Protocol.

A drawback of gonadotropin-releasing hormone (GnRH) antagonist protocols in in vitro fertilization (IVF) is that they have limited flexibility in cycle programming. This proof of concept study explored the efficacy of a single-dose, long-acting GnRH antagonist IVF protocol. Trial registration number is NCT03240159, retrospectively registered on March 08, 2017. The efficacy of a single-dose long-acting antagonist, degarelix, was explored initially in healthy donors and subsequently in infertile patients. In the first part, five healthy oocyte donors underwent ovarian stimulation with this new protocol: in the late luteal phase, at day 24, a bolus injection of degarelix was administered subcutaneously to control the LH surge in the follicular phase. Ovarian stimulation with gonadotropins was initiated subsequently from day 7 to day 10. End points were first to inhibit the LH surge later in the follicular phase and, second, to retrieve mature oocytes for IVF. In the second part, five infertile women received the same bolus injection of degarelix administered during the luteal phase at day 24. Different gonadotropin starting days (day 2 through day 8) were tested in order to observe possible differences in ovarian stimulation. In these infertile patients, fresh embryo transfers were performed to assess the pregnancy efficacy of this protocol on pregnancy outcomes and to address any possible negative effects on endometrium receptivity. In the first part of the study, all donors were effectively downregulated with a single luteal dose of 0.5 ml of degarelix for up to 22 days until the final oocyte maturation triggering day. Mature oocytes were retrieved after 36 h from all patients and all produced 2-7 blastocysts. In the second part, all five infertile patients achieved sufficient LH downregulation and completed ovarian stimulation without any LH surge. All patients (except one with freeze all strategy) had blastocysts transferred and pregnancy occurred in three out of five women. A single dose of the long-acting antagonist degarelix during the luteal phase appears to be effective in downregulating hypophysis during ovarian stimulation. This represents a possible new protocol for IVF, which should be further elucidated in RCTs.

80 Cumulus Cells Reflect the Success of IVF of Porcine Oocytes Treated with Dimethylthiourea

The aim of this study was to evaluate the effect of the antioxidant dimethylthiourea (DMTU) as a supplement during the collection and washing of cumulus–oocyte complexes (COC) on the apoptosis in cumulus cells (CC) and the competition for oocyte development in vitro. The COC were obtained by follicular aspiration. The aspiration and washing medium was TCM-199 with 10% porcine follicular fluid, 0.3 mM sodium pyruvate, and penicillin/streptomycin. Treated groups were supplemented with 2 or 20 mM DMTU. After 44 h of in vitro maturation (IVM), the percentage of nuclear maturation was determined by staining of the denuded oocytes with Hoechst 33442 (n = 357). After 18 h of in vitro fertilization (IVF) with 15°C refrigerated spermatozoa from boars of proven fertility, the suspected zygotes were stained with Hoechst 33342 to evaluate the percentages of sperm penetration (SP), monospermic penetration (MP), male pronucleus formation (MPN), and IVF efficiency (monospermic oocytes/total oocytes) (n = 260). In post-IVM CC, flow cytometry was performed after staining with annexin V-propidium iodide to evaluate apoptosis and viability (n = 90,000). Data were analysed with Flowing V 2.5.1 (http://flowingsoftware.btk.fi/). A comparison of proportion among categories was used (Fisher’s exact test), and significant differences were determined at P &lt; 0.05. No effects were observed in the percentages of nuclear maturation. Treatments with DMTU significantly increased the SP (Control: 35%; 2 mM: 72.9%; 20 mM: 67.8%) without modifying the MP. Although in both treatments, the amount of MPN was always greater than in the control, the percentages of MPN formation did not show significant differences. The 20 mM treatment significantly increased the efficiency of IVF (control: 22.2%; 2 mM: 31.9%; 20 mM: 45.7%). In the CC of the treated groups, a significant increase in viability (control: 7.4%; 2 mM: 12.2%; 20 mM: 14.7%) and a decrease in early apoptosis (control: 60.4%; 2 mM: 55.5%; 20 mM: 57.1%) were observed. Late apoptosis decreased in the 20 mM treatment (27.3%) compared with the other experimental groups (control: 31.3%; 2 mM: 31.2%). The addition of DMTU during COC collection and washing reduce the ROS-mediated apoptosis in CC and optimize the efficiency of IVF. Besides, CC quality reflects the effect of DMTU on oocytes, so viability and apoptosis analysis on CC constitutes an interesting noninvasive method to assess oocyte competence.

Pregnancy outcome of “delayed start” GnRH antagonist protocol versus GnRH antagonist protocol in poor responders: A clinical trial study

Background:Management of poor-responding patients is still major challenge in assisted reproductive techniques (ART). Delayed-start GnRH antagonist protocol is recommended to these patients, but little is known in this regards.Objective:The goal of this study was assessment of delayed-start GnRH antagonist protocol in poor responders, and in vitro fertilization (IVF) outcomes.Materials and Methods:This randomized clinical trial included sixty infertile women with Bologna criteria for ovarian poor responders who were candidate for IVF. In case group (n=30), delayed-start GnRH antagonist protocol administered estrogen priming followed by early follicular-phase GnRH antagonist treatment for 7 days before ovarian stimulation with gonadotropin. Control group (n=30) treated with estrogen priming antagonist protocol. Finally, endometrial thickness, the rates of oocytes maturation, , embryo formation, and pregnancy were compared between two groups. Results:Rates of implantation, chemical, clinical, and ongoing pregnancy in delayed-start cycles were higher although was not statistically significant. Endometrial thickness was significantly higher in case group. There were no statistically significant differences in the rates of oocyte maturation, embryo formation, and IVF outcomes between two groups.Conclusion:There is no significant difference between delayed-start GnRH antagonist protocol versus GnRH antagonist protocol.

Predicting the outcome of different protocols of invitro fertilization with anti-Muüllerian hormone levels in patients with polycystic ovary syndrome.

ObjectiveThis study evaluated associations of basal serum and follicular fluid (FF) anti-Muüllerian hormone (AMH) levels with in vitro fertilization (IVF) outcomes in polycystic ovary syndrome (PCOS) patients.MethodsThis prospective study included 179 consecutive women undergoing IVF, including 59 with PCOS and non-PCOS controls. Thirty PCOS cases had long gona-dotrophin-releasing hormone agonist (GnRH-a) and 29 had antagonist (GnRH-ant) protocols. Controls underwent conventional GnRH-a. Associations of basal serum and FF AMH levels with IVF outcomes were assessed.ResultsMedian serum and FF AMH levels, antral follicle count (AFC), oestradiol human chorionic gonadotropin injection day (peak E2), and retrieved oocyte numbers were higher in PCOS patients than in controls (all P < 0.01). Oocyte maturation and high-quality embryo rates were lower in PCOS patients than in controls (P < 0.01), but both groups had similar fertilization, implantation, clinical pregnancy, and newborn rates. Peak E2 was higher in GnRH-ant than in GnRH-a protocols (16.5 nmol/L vs. 12.1 nmol/L, P < 0.05). AMH levels were correlated with AFC in PCOS patients (P < 0.01). Peak E2 and FF AMH levels were independent predictors of oocyte number. Peak E2 predicted the fertilization rate.ConclusionSerum basal AMH levels are predictive of oocyte quantity, but not oocyte quality or IVF outcomes. Serum AMH, FF AMH, and outcomes are similar among protocols.

Significant heparin effect on bovine embryo development during sexed in vitro fertilization.

The aim of this study was to investigate the effect of different heparin concentrations in the course of sexed in vitro fertilization (IVF), on bovine embryonic development and development to term following embryo transfer (ET). With a total of 9156 oocytes for IVF, sorted as well as unsorted sperm from four bulls had different heparin requirements for achieving the highest rate of development in vitro. However, when optimal heparin concentrations were used (40 to 80 µg/ml), the performance of X-sorted sperm (0.3 × 106/ml/IVF droplet) from all four bulls, as judged by blastocyst development (Bulls A, B, C, and D: 25.2, 19.7, 25.1, and 9.8%, respectively), was significantly increased, and the blastocyst rate was comparable to that observed with unsorted sperm at certain heparin concentrations within the four bulls. We determined that near-optimal blastocyst development was possible with sorted sperm from all four bulls, when a heparin concentration of 40 µg/ml was used. Pregnancy rates at d 70 post ET ranged from 39.1 to 40.3% (P > 0.05), and the calving rates ranged from 34.4 to 35.1% (P > 0.05), when heparin was used at a concentration of 10 μg/ml (n = 236), 20 μg/ml (n = 189), and 40 μg/ml (n = 305), respectively. Our study demonstrates that, although the sorted sperm of different bulls performed optimally over a range of heparin concentrations, a generally accepted heparin concentration of 40 µg/ml can be set for sexed IVF. This improvement is beneficial when sexed embryo production by ovum pickup and IVF is an essential component of genetic breeding programs.

Producción de embriones de búfalo por fertilización in vitro luego de la maduración de los ovocitos durante el transporte prolongado

El búfalo &lt;i&gt;(Bubalus bubalis)&lt;/i&gt; es una especie con excelente adaptación a sectores inundables. El mejoramiento genético a través de superovulación y transferencia embrionaria ha tenido escasos resultados debido a dificultades en la detección de celo, pobre respuesta ovárica y limitada recuperación de embriones post-lavaje. La técnica de fertilización in vitro de embriones (FIV) es una biotecnología de gran impacto en el progreso genético. El objetivo del presente trabajo fue estudiar los eventos tempranos de la FIV, analizando la tasa de maduración y desarrollo embrionario post-fertilización de ovocitos madurados in vitro (IVM) durante el transporte. Ovocitos bovinos y bubalinos fueron obtenidos por punción folicular de ovarios post-mortem e IVM durante el transporte por un período de 18 h. Se realizó la FIV con toros de fertilidad comprobada, con una concentración en microgotas de inseminación de 3-4 x 106 espermatozoides motiles/ml por un período de 6 horas. Los embriones fueron cultivados en medio oviductal sintético SOFaa en incubadora gaseada y ambiente humidificado a 38,5ºC durante 9 días. Se evaluaron las tasas de IVM, clivaje (día 2 post-fertilización) y blastocisto (días 7 a 9). Los resultados fueron analizados estadísticamente utilizando Fischer’s Exact Test (p&lt;0,05). No se observaron diferencias significativas en la tasa de maduración de ovocitos bubalinos de buena calidad respecto al control sin transporte (72 vs 88%), pero se registró una reducción significativa en la maduración de los ovocitos bubalinos de mala calidad (35%). Asimismo, se lograron producir los primeros embriones bubalinos luego de FIV, aunque las tasas de clivaje (34 vs 70 y 78%) y blastocisto (3 vs 27 y 31%) fueron significativamente menores en búfalos que en bovinos con y sin transporte, respectivamente. Los datos del presente trabajo constituirían el primer informe de FIV en búfalos y producción in vitro de embriones luego de IVM de ovocitos durante el transporte.

Effect of hexavalent chromium-treated sperm on in vitro fertilization and embryo development.

Hexavalent chromium (Cr(VI)) is an environmental contaminant that is associated with reproductive abnormalities in both humans and animals. In the present study, we evaluated the cytotoxic effect of Cr(VI) on sperm function and subsequent embryo development after in vitro fertilization (IVF). Sperm obtained from BDF1 male mice were treated with potassium dichromate (0, 3.125, 6.25, 12.5, 25, or 50 μM) for 3 h. Cr(VI) significantly decreased sperm viability and acrosome reaction with increasing dose. These Cr(VI)-treated sperms were further used for IVF of oocytes obtained from BDF1 female mice. Results showed that Cr(VI)-treated sperm caused a significant reduction in IVF success, higher developmental arrest at the two-cell stage of embryos, and delayed blastocyst formation with increasing dose. In particular, most blastocysts from the Cr(VI)-treated sperm resulted in hatching failure as well as decreased inner cell mass and trophectoderm (TE). Furthermore, blastocysts obtained from Cr(VI)-treated sperm showed lower expression of not only TE-associated genes (eomes, cdx2, and krt8) but also pluripotent marker genes (sox2, pou5f1, and klf4) that are responsible for further embryo development of blastocyst embryos. The results of our current study showed that Cr(VI)-treated sperm had negative effects on oocyte fertilization and subsequent embryo development.

The growth and reproduction performance of TALEN-mediated β-lactoglobulin-knockout bucks.

With the technological development of several engineered endonucleases (EENs), such as zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and CRISPR/Cas9, gene targeting by homologous recombination has been efficiently improved to generate site-specifically genetically modified livestock. However, few studies have been done to investigate the health and fertility of these animals. The purpose of the present study is to investigate if gene targeting events and a recloning procedure would affect the production traits of EEN-mediated gene targeted bucks. TALEN-mediated β-lactoglobulin (BLG) gene mono-allelic knockout (BLG (+/-)) goats and bi-allelic knockout (BLG (-/-)) buck produced by using sequential gene targeting combined with recloning in fibroblasts from BLG (+/-) buck were used to evaluate their health and fertility. Birth weight and postnatal growth of BLG (+/-) bucks were similar to the wild-type goats. None of the parameters for both fresh and frozen-thawed semen quality were significantly different in BLG (+/-) or BLG (-/-) bucks compared to their corresponding comparators. In vitro fertilization (IVF) test revealed that the proportion of IVF oocytes developing to the blastocyst stage was identical among BLG (+/-), BLG (-/-) and wild-type bucks. Conception rates of artificial insemination were respectively 42.3, 38.0 and 42.6% for frozen-thawed semen from the BLG (+/-), BLG (-/-) and wild-type bucks. In addition, germline transmission of the targeted BLG modification was in accordance with Mendelian rules. These results demonstrated that the analyzed growth and reproductive traits were not impacted by targeting BLG gene and recloning, implicating the potential for dairy goat breeding of BLG (+/-) and BLG (-/-) bucks.

Anti-laminin-1 antibodies in serum and follicular fluid of women with Hashimoto’s thyroiditis undergoing in vitro fertilization

The aim of this study is to evaluate the presence of anti-laminin-1 antibodies (aLN-1) in sera and follicular fluid (FF) of infertile women affected by Hashimoto's thyroiditis (HT) undergoing in vitro fertilization (IVF) and its impact on oocyte maturation and IVF outcome. aLN-1 were measured by a home-made enzyme linked immunosorbent assay (ELISA) in: (1) sera and FF from 44 infertile women affected by HT (HTIW) with tubal factor or male factor as primary cause of infertility; (2) in sera and FF from 28 infertile women without HT, with tubal factor or male factor as cause of infertility (infertile controls-ICTR); and (3) in sera from 50 fertile women (FW). aLN-1 serum levels were significantly higher in HTIW when compared with both fertile women and ICTR (P <0.001and P <0.01, respectively). Assuming as cutoff the 99th percentile of values obtained in sera of FW, 43.2% of HTIW and 3.6% of ICTR were aLN-1 positive (P = 0.0001). Also aLN-1 detected in FF from HTIW were significantly higher in comparison with those found in FF of ICTR (P = 0.006). In HTIW, metaphase II oocyte count showed inverse correlation with both serum and FF aLN-1 levels (r = 0.34, P = 0.02 and r = 0.33, P = 0.03, respectively). Implantation and pregnancy rates were significantly lower in HTIW (7.9% and 9.1%, respectively) when compared with ICTR (23% and 31.1%, respectively) (P = 0.015 and P = 0.03, respectively). Our results demonstrated for the first time the presence of aLN-1 in a relevant percentage of HTIW and suggest that these auto-antibodies may impair IVF outcome.

The effect of amniotic membrane stem cells as donor nucleus on gene expression in reconstructed bovine oocytes.

Nuclear reprogramming of a differentiated cell in somatic cell nuclear transfer (SCNT) is a major concern in cloning procedures. Indeed, the nucleus of the donor cell often fails to express the genes which are a prerequisite for normal early embryo development. This study was aimed to evaluate the developmental competence and the expression pattern of some reprogramming related genes in bovine cloned embryos reconstructed with amniotic membrane stem cells (AMSCs) in comparison with those reconstructed with mesenchymal stem cells (MSCs) and adult fibroblasts (AF) as well as with in vitro fertilized (IVF) oocytes. In vitro matured abattoir-derived oocytes were considered as recipients and a hand-made cloning technique was employed for oocyte enucleation and nuclear transfer (NT) procedures. The expression pattern of genes involved in self-renewal and pluripotency (POU5F1, SOX2, NANOG), imprinting (IGF2, IGF2R), DNA methylation (DNMT1, DNMT3A), histone deacetylation (HDAC2), and apoptosis (BAX, BCL2) were evaluated in NT and IVF derived embryos. Despite the insignificant difference in cleavage rate between reconstructed and IVF oocytes, the blastocyst rate in the IVF group was higher than that of other groups. Among reconstructed oocytes, a higher blastocysts rate was observed in MSC-NT and AMSCs-NT derived embryos that were significantly higher than AF-NT derived ones. There were more similarities in the expression pattern of pluripotency and epigenetic modification genes between MSC-NT and IVF derived blastocysts compared with other groups. In conclusion, considering developmental competence, AMSCs, as alternative donors in SCNT procedure, like MSCs, were prone to have more advantage compared with AF.

Apoptotic effects on maturation of mouse oocytes, fertilization and fetal development by puerarin

Previously we identified puerarin, an isoflavone compound, as a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to retardation of embryonic development and cell viability. In the current study, we investigated whether puerarin exerts deleterious effects on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, puerarin caused significant impairment of these processes in vitro. Pre-incubation of oocytes with puerarin during in vitro maturation led to increased post-implantation embryo resorption and decreased mouse fetal weight. In an in vivo animal model, intravenous injection with or without puerarin (1, 3 and 5 mg/kg body weight/day) for 4 days caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, pre-incubation of oocytes with a caspase-3-specific inhibitor effectively blocked puerarin-triggered deleterious effects, clearly implying that embryonic injury induced by puerarin is mediated by a caspase-dependent apoptotic mechanism. These results clearly demonstrate that puerarin has deleterious effects on mouse oocyte maturation, fertilization and subsequent embryonic development in vitro and in vivo.

Comprehensive elucidation of amino acid profile in human follicular fluid and plasma of in vitro fertilization patients

The assessment of oocyte quality in human in vitro fertilization (IVF) is in focus of intensive studies. Follicular fluid (FF) constitutes the major medium for the developing oocyte and therefore it is a perfect target to search for the biomarkers of female infertility. The objective of this study was to compare the amino acid (AA) profile of human FF and plasma (PL) (from 96 IVF patients) and to examine if the AA composition is related to oocyte quality, IVF results and women’s infertility. In both biological fluids, Gln, Gly and Ala appeared as dominant AAs. Most of AAs are more abundant in PL; the exceptions are Glu, Thr, Ala and Gly, which are higher in FF and Gln, Arg and Phe, the contents of which are similar in both biological fluids. Ser in FF and Met and Phe in PL were detected as potential biomarkers as their content varied depending on the IVF outcome. Significant differences were also detected between the groups of different infertility reasons. Our results suggest that intra-follicular AAs might reflect the condition of the preovulatory follicle and together with PL, AAs can be used to characterize the infertility etiology and oocyte quality related to IVF outcome.

Total levels, localization patterns, and proportions of sperm exhibiting phospholipase C zeta are significantly correlated with fertilization rates after intracytoplasmic sperm injection

To study the relationship of total levels, localization patterns, and proportions of sperm exhibiting phospholipase C zeta, with fertilization rates after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Laboratory study; controls vs. patients after IVF (n = 27) or ICSI (n = 17) treatment. Fertility center. A total of 44 semen samples, subjected to either IVF or ICSI treatment. Oocyte collection, ICSI or IVF, determination of sperm concentration and motility, and immunocytochemical analyses of phospholipase C zeta (PLCζ). None. Percentages of sperm exhibiting PLCζ. Significant positive correlation between ICSI fertilization rates and total levels, localization patterns, and the proportion (percentage) of sperm exhibiting PLCζ. Total levels, localization patterns, and the proportion of sperm exhibiting PLCζ are correlated with fertilization rates for ICSI, but not for IVF. Evaluating total levels, localization patterns, and proportions of PLCζ may represent a useful diagnostic tool for clinical purposes in men for whom IVF is not advised or has previously failed. This clinical study further supports the fundamental role of PLCζ in the oocyte activation process.

Multiple aster formation is frequently observed in bovine oocytes retrieved from 1-day stored ovaries.

We have recently reported that multiple aster formation after in vitro fertilization (IVF) was one of the factors that negatively affected the developmental competence of vitrified-warmed bovine matured oocytes, and that short-term culture of the post-warm oocytes with an inhibitor of Rho-associated coiled-coil kinase (ROCK) suppressed the multiple aster formation and improved the blastocyst yield. The present study was conducted to investigate whether increased multiple aster formation following IVF was involved in impaired developmental competence of stored ovary-derived bovine oocytes. Oocytes retrieved from 1-day stored ovaries had lower developmental potential to day 8 blastocysts when compared with those from fresh ovaries (37 versus 63%). Immunostaining of α-tubulin 10 h post-IVF revealed that a higher incidence of multiple aster formation occurred in oocytes retrieved from stored ovaries than from fresh ovaries (31 versus 15%). Treatment of post-in vitro maturated (post-IVM) oocytes with ROCK inhibitor for 2 h significantly suppressed the incidence of multiple aster formation (10 versus 32% in the control group). However, the suppression effect of ROCK inhibitor on multiple aster formation in IVM/IVF oocytes did not improve blastocyst yield from stored ovary-derived oocytes (41 versus 37% in the control group). These results suggested that the higher incidence of multiple aster formation by bovine ovary storage was not responsible for the decreased developmental competence of IVF oocytes.

Apoptotic effects of dillapiole on maturation of mouse oocytes, fertilization and fetal development

Previously, we reported that dillapiole, a phenylpropanoid with antileishmanial, anti-inflammatory, antifungal and acaricidal activities, is a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to impaired embryonic development and cell viability. In the current study, we investigated the deleterious effects of dillapiole on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, dillapiole induced significant impairment of mouse oocyte maturation, decrease in the IVF rate and inhibition of subsequent embryonic development in vitro. Pre-incubation of oocytes with dillapiole during in vitro maturation led to an increase in post-implantation embryo resorption and decrease in mouse fetal weight. In an in vivo animal model, 2.5, 5 or 10 μM dillapiole provided in drinking water caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, pre-incubation of oocytes with a caspase-3-specific inhibitor effectively blocked dillapiole-triggered deleterious effects, clearly implying that embryonic injury induced by dillapiole is mediated via a caspase-dependent apoptotic mechanism. To the best of our knowledge, this is the first study to establish the impact of dillapiole on maturation of mouse oocytes, fertilization and sequential embryonic development.

Effects of ochratoxin a on mouse oocyte maturation and fertilization, and apoptosis during fetal development.

We previously reported that ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, and is a risk factor for abnormal embryonic development. More specifically, OTA triggers apoptotic processes in the inner cell mass of mouse blastocysts, decreasing cell viability and embryonic development. In the current study, we investigated the deleterious effects of OTA on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre- and postimplantation development both in vitro and in vivo. Notably, OTA significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with OTA during in vitro maturation increased postimplantation embryonic resorption and decreased mouse fetal weight. In an in vivo animal model, provision of 1-10 μM OTA in the drinking water or intravenous injection of 1 or 2 mg/kg body weight of OTA decreased oocyte maturation and IVF, and had deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase-3-specific inhibitor effectively blocked these OTA-triggered deleterious effects, suggesting that the embryonic injury induced by OTA is mediated via a caspase-dependent apoptotic mechanism. Furthermore, OTA upregulated the levels of p53 and p21 in blastocyst cells derived from OTA-pretreated oocytes, indicating that such cells undergo apoptosis via p53-, p21-, and caspase-3-dependent regulatory mechanisms. This could have deleterious effects on embryonic implantation and fetal survival rates, as seen in our animal models. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 724-735, 2016.

In vitro development of secondary follicles from pre-pubertal and adult goats cultured in two-dimensional or three-dimensional systems

The aim of this study was to evaluate the influence of two-dimensional (2D) and three-dimensional (3D) alginate culture systems on in vitro development of pre-antral caprine follicles. In addition, the influence of the reproductive age of the ovary donor on the in vitro culture success was investigated. Pre-antral follicles from pre-pubertal or adult goats were isolated and cultured directly on a plastic surface (2D) or encapsulated in an alginate-based matrix (3D). After 18 days, the oocytes underwent in vitro maturation (IVM) and in vitro fertilization (IVF) to produce embryos. The 3D system showed higher rates of follicle survival, lower rates of oocyte extrusion, and a greater number of recovered oocytes for IVM and IVF (P < 0.05). Only pre-antral follicles from adult animals produced MII oocytes and embryos. The estradiol concentrations increased from day 2 to day 12 of culture in all groups tested (P < 0.05). Conversely, progesterone concentrations were lower in 3D-cultured follicles than in 2D-cultured follicles, with differences on days 2 and 6 of culture (P < 0.05). We provide compelling evidence that a 2D or 3D alginate in vitro culture system offers a promising approach to achieving full in vitro development of caprine pre-antral follicles to produce mature oocytes that are capable of fertilization and viable embryos.

Pregnancy outcomes decline in recipients over age 44: an analysis of 27,959 fresh donor oocyte in vitro fertilization cycles from the Society for Assisted Reproductive Technology

To use a large and recent national registry to provide an updated report on the effect of recipient age on the outcome of donor oocyte in vitro fertilization (IVF) cycles. Retrospective cohort study. United States national registry for assisted reproductive technology. Recipients of donor oocyte treatment cycles between 2008 and 2010, with cycles segregated into five age cohorts: ≤34, 35 to 39, 40 to 44, 45 to 49, and ≥50 years. None. Implantation, clinical pregnancy, live-birth, and miscarriage rates. In donor oocyte IVF cycles, all age cohorts ≤39 years had similar rates of implantation, clinical pregnancy, and live birth when compared with the 40- to 44-year-old reference group. Patients in the two oldest age groups (45 to 49, ≥50 years) experienced statistically significantly lower rates of implantation, clinical pregnancy, and live birth compared with the reference group. Additionally, all outcomes in the ≥50-year-old group were statistically significantly worse than the 45- to 49-year-old group, demonstrating progressive decline with advancing age. Recent national registry data suggest that donor oocyte recipients have stable rates of pregnancy outcomes before age 45, after which there is a small but steady and significant decline.