Oocyte Morphology Research Articles

Molecular Typing of Eimeria ahsata and E. crandallis Isolated From Slaughterhouse Wastewater.

Molecular Typing of Eimeria ahsata and E. crandallis Isolated From Slaughterhouse Wastewater.

In vitro culture of oocytes and granulosa cells collected from normal, obese, emaciated and metabolically stressed ewes

The present study was undertaken to investigate the oocyte morphology, its fertilizing capacity and granulosa cell functions in ewes (obese, normal, metabolic stressed and emaciated). Ewes (Ovis aries) of approximately 3 years of age (Bellary breed) from a local village were screened, chosen and categorized into a) normal b) obese but not metabolically stressed, c) Emaciated but not metabolically stressed d) Metabolically stressed based on body condition scoring and blood markers. Oocytes and granulosa cells were collected from ovaries of the ewes of all categories after slaughter and were classified into good (oocytes with more than three layers of cumulus cells and homogenous ooplasm), fair (oocytes one or two layers of cumulus cells and homogenous ooplasm) and poor (denuded oocytes or with dark ooplasm). The good and fair quality oocytes were in vitro matured and cultured with fresh semen present and the fertilization, cleavage and blastocyst development were observed. The granulosa cells were cultured for evaluation of metabolic activity by use of the MTT assay, and cell viability, cell number as well as estrogen and progesterone production were assessed. It was observed that the good and fair quality oocytes had greater metabolic activity when collected from normal and obese ewes compared with those from emaciated and metabolically stressed ewes. No significant difference was observed in oocyte quality and maturation amongst the oocytes collected from normal and obese ewes. The cleavage and blastocyst production rates were different for the various body condition classifications and when ranked were: normal>obese>metabolically stressed>emaciated. Lesser metabolic activity was observed in granulosa cells obtained from ovaries of emaciated ewes. However, no changes were observed in viability and cell number of granulosa cells obtained from ewes with the different body condition categories. Estrogen and progesterone production from cultured granulosa cells were not different in normal and obese ewes. Estrogen and progesterone secretions were less from granulosa cells recovered from metabolically stressed and emaciated ewes. The results suggested that oocyte morphology, fertilizing capacity and granulosa cell growth were dependent on body condition and feeding status of the animals.

북방전복 Haliotis discus hannai 난자형성과정 동안 난모세포의 형태 및 계측형질 변화

북방전복 Haliotis discus hannai 난자형성과정 동안 난모세포의 형태 및 계측형질 변화

Ameliorative Effect of Grape Seed Proanthocyanidin Extract on Cadmium-Induced Meiosis Inhibition During Oogenesis in Chicken Embryos.

Cadmium (Cd) is an environmental endocrine disruptor that has toxic effects on the female reproductive system. Here the ameliorative effect of grape seed proanthocyanidin extract (GSPE) on Cd-induced meiosis inhibition during oogenesis was explored. As compared with controls, chicken embryos exposed to Cd (3 µg/egg) displayed a changed oocyte morphology, decreased number of meiotic germ cells, and decreased expression of the meiotic marker protein γH2AX. Real time RT-PCR also revealed a significant down-regulation in the mRNA expressions of various meiosis-specific markers (Stra8, Spo11, Scp3, and Dmc1) together with those of Raldh2, a retinoic acid (RA) synthetase, and of the receptors (RARα and RARβ). In addition, exposure to Cd increased the production of H2 O2 and malondialdehyde in the ovaries and caused a corresponding reduction in glutathione and superoxide dismutase. Simultaneous supplementation of GSPE (150 µg/egg) markedly alleviated the aforementioned Cd-induced embryotoxic effects by upregulating meiosis-related proteins and gene expressions and restoring the antioxidative level. Collectively, the findings provided novel insights into the underlying mechanism of Cd-induced meiosis inhibition and indicated that GSPE might potentially ameliorate related reproductive disorders.

Sirt6 depletion causes spindle defects and chromosome misalignment during meiosis of mouse oocyte

Sirt6, a member of the sirtuin family of NAD-dependent protein deacetylases, has been implicated in multiple biological processes. However, the roles of Sirt6 in meiosis have not been addressed. In the present study, by employing knockdown analysis in mouse oocytes, we evaluated the effects of Sirt6 on meiotic apparatus. We found that specific depletion of Sirt6 results in disruption of spindle morphology and chromosome alignment in oocytes. Consistent with this observation, incidence of aneuploidy is also markedly increased in Sirt6-depleted oocytes. Furthermore, confocal scanning showed that kinetochore-microtubule interaction, an important mechanism controlling chromosome segregation, is severely impaired in metaphase oocytes following Sirt6 knockdown. Unexpectedly, we discovered that Sirt6 modulates the acetylation status of histone H4K16 as their knockdown specifically induces the hyperacetylation of H4K16 in oocytes, which may be associated with the defective phenotypes described above via altering kinetochore function. Altogether, our data reveal a novel function of Sirt6 during oocyte meiosis and indicate a pathway regulating meiotic apparatus.

Correlation between oocyte morphology and the embryo aneuploidy rate in IVF cycles

Purpose: To evaluate the aneuploidy rates of 13, 18, and 21 and the X and Y chromosomes in embryos from patients with morphologically normal oocytes and different oocyte dysmorphisms.Methods: This prospective cohort study included 84 patients treated with in vitro fertilization (IVF) at a single academic center. The patients were divided into the following three groups: group 1 – women with cytoplasmic dysmorphisms (n = 28), group 2 – women with extracytoplasmic dysmorphisms (n = 28), and group 3 – women with morphologically normal oocytes (n = 28). One blastomere from each embryo was analyzed for aneuploidies of chromosomes 13, 18, 21, X, and Y.Results: The highest prevalence of aneuploid embryos was observed in the group 1 (68.4%) followed by the group 2 (38.9%) and the group 3 (31.3%) (р < 0.0001). The adjusted OR for receiving an aneuploid embryo in the case of cytoplasmic dysmorphism was 3.6 (95% CI = 1.8; 7.2), in the case of extracytoplasmic dysmorphisms – 1.3 (95% CI = 0.7; 2.1).Conclusions: Women with morphological oocyte abnormalities are at risk for developing aneuploid embryos during IVF cycles. We recommend that woman with cytoplasmic oocyte dysmorphisms receive additional genetic counseling to define the indications for the genetic screening of embryos.

Dynamic Morphology of the Human Ooplasm and Subsequent Embryonic Development Analyzed by Time-Lapse Cinematography

It has been reported that oocyte dysmorphisms, such as the appearance of a cytoplasmic vacuole, granularity, and deformed shape, are strongly associated with poor embryonic development. In this study, we analyzed the dynamics of cytoplasmic dysmorphisms of oocytes and zygotes using time-lapse cinematography (TLC) that we had originally developed. Mature oocytes (n=1,601) were classified into two groups: good quality oocytes (GQO), which indicated oocytes without any defects; and poor quality oocytes (PQO), oocytes with one or more defect. Of the 1601 oocytes, 74.1% were GQO and the remaining were PQO, which were evaluated by three senior embryologists. The rate of unfertilized oocytes was significantly lower in PQO. GQO also showed a higher development rate to good quality embryos than PQO. The time required for embryonic development in GQO was significantly less than that in PQO (P<0.01). In the zygotes (n=144), 11.4% showed vacuole-like phenomenon (VLP) in the cytoplasm. The VLP appeared in the peripheral cytoplasm after extrusion of the second polar body and moved synchronously as the mPN and fPN migrated toward the central cytoplasm. The VLP size gradually increased and remained after the first cleavage. The rate of multinucleated blastomere (MNB) formation was significantly higher in embryos with VLP than in those without VLP. The appearance of VLP was closely associated with aberrant embryonic development, particularly MNB formation. Our results suggest that the dynamic morphology of the cytoplasm in oocytes and zygotes influences subsequent embryonic development and viability, suggesting that the assessment of oocyte morphology could be a useful parameter in the prediction of embryonic development.

Effect of oocyte dysmorphisms on intracytoplasmic sperm injection cycle outcomes in normal ovarian responders.

The aim of this study was to identify the influence of oocyte dysmorphisms on clinical outcomes after intracytoplasmic sperm injection cycle in normal responders. In the prospective study, morphology of 1999 metaphase II oocytes retrieved from 316 intracytoplasmic sperm injection cycles was evaluated from March 2011 to March 2013 at Royan Institute. Controlled ovarian stimulation was performed by long standard agonist protocol. Oocyte morphology was assessed before sperm injection by one embryologist. The associations between fertilization rate, embryo quality and the independent variables were analyzed using odds ratio (OR) calculated with unconditional logistic regression test. From all retrieved oocytes, 1543 (77.1%) showed at least one morphologic aberration. Presence of cytoplasmic vacuoles and high cytoplasmic viscosity were associated with a significant decrease in the fertilization rate (OR: 0.5; P = 0.004 and OR: 0.6; P = 0.03, respectively). The results showed that oocyte morphology did not affect embryo quality. The number of gonadotrophin injections used showed a direct relation with presence of large perivitelline space. No significant difference was observed among four groups (women with total normal morphologic oocytes [group 1], women with total extracytoplasmic dysmorphic oocytes [group 2], women with total cytoplasmic dysmorphic oocytes [group 3] and women with total oocytes containing multiple dysmorphic features [group 4]) in terms of implantation and clinical pregnancy rates. Metaphase II oocyte morphology had minor impacts on fertilization rate, pronuclear morphology and embryo quality in women with normal ovarian response.

Effect of Acrylamide on Oocyte Nuclear Maturation and Cumulus Cells Apoptosis in Mouse In Vitro.

Acrylamide (ACR) is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus–oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus–oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro.

Folliculogenesis and Oocyte Quality in Awassi Sheep

Female reproductive performance of Awassi ewe, the predominant breed of fat‐tail sheep of Lebanon are not well studied; we aimed to evaluate oocyte morphology and quality. 36 Awassi ovaries (Exp 1) from local slaughterhouses were collected from May to October and Follicles (&gt;3 mm) aspirated for oocyte morphology evaluation. Furthermore, 10 Awassi ewes at the Lebanese Agricultural Research Institute (Exp 2) were monitored via ultrasonography from May to July to determine dominant and subordinate follicles; ovaries and ooyctes collected, and evaluated following slaughter. AMH, ZAR‐1 and BMP15 gene expression amplified using qRTPCR with SYBR and normalized to 18S rRNA. Results showed (exp 1) that 68% of collected oocytes were medium vs. 9% large oocytes, and 60% of Awassi oocytes satisfied more than 3 criteria of good quality, more frequently in October, but detected as early as May. Folliculogenesis in Awassi ewes (exp 2) showed an estrous cycle of 17±3 days with 2 waves and a dominant follicle of 4±0.9 mm. Of the dominant follicles, only 24.4% contained morphologically good vs. 17.8% bad oocytes. In the search for an oocyte quality marker, AMH mRNA was high in small and dominant follicles, but not subordinate; BMP‐15 was down‐regulated in small oocytes while ZAR‐1 did not differ among different quality oocytes. Therefore, these genes can be used to distinguish between different quality oocytes.&#x0D; This research was supported by a grant from the LCNRS and NDU

Oocyte zona pellucida dysmorphology is associated with diminished in-vitro fertilization success.

BackgroundAlthough irregularities in human zona pellucida (ZP) morphology are well described, there is scant literature on the clinical significance of ZP dysmorphology. We, therefore, designed a retrospective cohort trial of ZP dysmorphology to assess the clinical significance of ZP dysmorphology and its affect on IVF outcome. Over the same time period a random sample of 77 cycles of 77 subjects with all normal oocyte morphology were selected as controls.MethodsBetween July 2006 and December 2010, all fresh non-donor cases performed at a university hospital IVF center were assessed for ZP dysmorphology. ZP dysmorphology included extracytoplasmic abnormalities (dark ZP and large perivitelline space) and oocyte shape (oval or irregularly shaped ZP). 136 IVF cycles from 119 subjects were identified where a majority of oocytes displayed ZP dysmorphology. Over the same time period a random sample of 77 cycles of 77 subjects with all normal oocyte morphology were selected as controls. IVF prognostic and outcome parameters were compared between the patients with dysmorphic and normal oocytes.Results136/1710 (8.0%) cycles of fresh non-donor IVF displayed predominant ZP dysmorphology. Dysmorphic and normal oocytes showed no difference in the oocyte quality predictors such as FSH (6.03+/−2.5 vs. 6.8+/−2.3 IU/L), or AMH (2.5+/−2.0 vs. 2.30+/−1.5 ng/ml levels). ZP dysmorphology was associated with markedly diminished clinical pregnancy rates (44% vs. 70%; RR:0.62 [0.48, 0.80]; p = 0.0002), implantation rates (.17 vs. .36; IRR: 0.48 [0.34, 0.68]; p < 0.0001) and live birth rates as compared to non dysmorphic oocytes (29% vs. 52%; RR:0.55 [0.39, 0.79]; p = 0.001).ConclusionsZP dysmorphology is associated with markedly diminished pregnancy and implantation rates in IVF. The poorer outcome appears to be independent of the usual markers of ovarian reserve.

A correlative study of oocytes morphology with fertilization, cleavage, embryo quality and implantation rates after intra cytoplasmic sperm injection

Introduction: Non-invasive selection of developmentally human oocytes may increase the overall efficiency of human assisted reproduction. Morphologic abnormalities in the oocyte are relevant for determining its developmental fate. The objective is to evaluate the influence of MII oocyte morphology on intra cytoplasmic sperm injection (ICSI) outcomes. Material and Methods: 132 patients undergoing ICSI cycles and having female factors of infertility and unexplained infertility. Couples having male factors of infertility were excluded. A total of 1200 oocytes were retrieved from 132 ICSI cycles, of which 1056 MII oocytes were evaluated. The criteria for morphological evaluations were: (i) Normal MII oocytes showing clear cytoplasm with uniform texture and homogenous fine granularity, a round or ovoid first polar body with a smooth surface, and perivitelline space of normal size. (ii) MII oocytes with extra cytoplasmic abnormalities (first polar body and perivitelline space abnormalities). (iii) MII oocytes with cytoplasmic abnormalities (dark cytoplasm, granular cytoplasm, inclusion body and presents of vacuoles). (iv) MII oocytes with combined abnormalities. Result: From 1056 MII oocytes, 180 (17.04%) had normal morphology while 876 (82.95%) had at least one demonstrable morphological abnormality. Cytoplasmic abnormalities were observed in 516 (58.9%) of the oocytes. Extra cytoplasmic abnormalities were observed in 104 (11.87%) while combined abnormalities were responsible for the remaining 256 (29.22%). There were no significant differences in fertilization, cleavage, and embryo quality between the groups but there was a highly significant difference in implantation rate which was higher in the group of normal oocytes morphology than abnormal oocytes morphology, oocytes with cytoplasmic, extracytoplasmic and combined abnormality 11.11%, 7.33%, 9.03%, 2.3%, and 4.34% respectively. Conclusion: MII oocyte morphology did not affect fertilization, cleavage, and embryo quality, but affecting implantation rate.

Fish egg size: a characteristic determined by parental care

Fish egg size: a characteristic determined by parental care

Endometriosis Affects Oocyte Morphology in Intracytoplasmic Sperm Injection Cycles?

To identify associations between presence of endometriosis and oocyte defects, embryo developmental potential, and cycle outcomes. This study looked into the impact of endometriosis on oocyte and embryo quality, and blastocyst formation probability. Endometriosis was also correlated with cycle characteristics. In order to avoid age-related bias, in the first analysis only patients aged 36 years or younger were included, and the cycles were split into endometriosis infertility cycles (n=431; 3172 oocytes) and other cycles (n=2510; 24480 oocytes). The number of retrieved oocytes (10.6±21.2 vs. 14.6±21.1, P<0.001), oocyte yield (68.1±20.0% vs. 70.6±19.6%, P=0.015), and embryos obtained (6.1±4.43 vs. 7.8±5.12, P<0.001) were lower among patients with endometriosis. Implantation rates (28.1%±38.9% vs. 33.9±42.7, P<0.001) were lower among patients with endometriosis, but fertilization, pregnancy, miscarriage and cycle cancelation rates were not different. There was a significant increase in the incidence of extra-cytoplasmic, but not intra-cytoplasmic, oocyte defects among patients with endometriosis. The quality of embryos (45.3% vs. 47.3%, P=0.037) collected from patients with endometriosis was lower, but blastocyst formation rates were unaltered. A possible explanation for the lower implantation rates seen in patients with endometriosis is the poorer quality of the oocytes and embryos observed in this group of patients.

306 EFFECT OF DIFFERENT IN VITRO MATURATION MEDIA ON DEVELOPMENTAL POTENTIAL OF GOAT OOCYTES ALREADY FOUND DENUDED AT COLLECTION

A grade classification (I, II, and III) based on the number of cumulus layers and oocyte morphology is currently used by many laboratories. Oocytes found denuded at collection (grade III) are considered not suitable and routinely discarded. Thus, if a particular strategy could be applied to their use in labour-intensive processes, such as ovum pickup, it would be a benefit. This experiment was performed to examine the effect of IVM medium composition to produce goat embryos in vitro using oocytes already found denuded at collection (DOC). In total, 411 DOC and 141 intact COC (control treatment) obtained by slaughterhouse ovaries were analysed in 4 replicates. The maturation medium consisted in TCM199 supplemented either with (1) 10 ng mL–1 of EGF and 100 µM cysteamine (simplified); (2) 10 ng mL–1 of EGF, 5 IU mL–1 of hCG, 10 IU mL–1 of eCG, 19 ng mL–1 of IGF-1, 2.2 ng mL–1 of FGF, 5 µg mL–1 of ITS, 90 µg mL–1 of l-cystein, 0.1 mM β-mercaptoethanol, 75 µg mL–1 of vitamin C, 720 µg mL–1 of glycine, 0.1 mg mL–1 of glutamine, and 110 µg mL–1 of pyruvate (semidefined); or (3) 10% fetal calf serum (FCS), 100 µM cysteamine, and 50 ng mL–1 of oFSH (complex). Both DOC and COC were subjected to IVM, IVF, and IVD as previously described (Souza-Fabjan et al. 2014, Theriogenology 81, 1021–31). The COC were matured only in simplified medium. On Day 8, all expanded blastocysts were fixed and stained with Hoechst for cell counting. Statistical analysis was performed using all tests with a significant interval of 95%. All variables were compared among treatments using ANOVA and SNK test. The results are described as mean per replicate ± s.e.m. No significant differences were found in simplified, semidefined, or complex medium, respectively, in cleavage rate (52 ± 7.5, 60 ± 9.4, or 51 ± 15.0%), blastocyst from cleaved (36 ± 3.9, 39 ± 9.3, or 41 ± 4.8%), blastocyst from initial DOC (19 ± 5.0, 23 ± 8.1, or 21 ± 3.3%), hatching rate (55 ± 22.9, 55 ± 15.9, or 52 ± 14.8%), or total blastomeres number (184 ± 12.6, 179 ± 12.4, or 190 ± 13.8). The control COC showed no significant differences to any DOC treatment on cleavage (77 ± 3.4%) and blastocyst from cleaved (60 ± 2.2%). However, the blastocyst rate from initial COC was higher (46 ± 0.5%; P &lt; 0.05) than all DOC treatments. Even though the blastocyst yield was lower than COC (~21 v. 46%), it is reasonable to affirm that it is possible to produce embryos from oocytes that would not be utilised, which may represent additional number of embryos. The blastocyst cell numbers in COC (192 ± 13.7) were similar (P &gt; 0.05) to DOC, indicating that the goat embryos produced were of good quality. In conclusion, the inclusion of more complex substances in IVM media did not increase the development rate of DOC and, therefore, more simple IVM media could be used for this purpose. Finally, the goat embryos produced had satisfactorily number of blastomeres, demonstrating that the in vitro development step is able to generate good quality embryos from grade III oocytes. Therefore, some oocytes that in general would be discarded will develop to blastocysts and may represent benefits, especially after ovum pick-up from genetically valuable goats.

297 EFFECT OF CUMULUS CELL REMOVAL DURING IN VITRO MATURATION OF PORCINE CUMULUS–OOCYTE COMPLEXES ON THE APOPTOTIC STATUS AND MEIOTIC PROGRESSION OF THE OOCYTES

This study was undertaken to examine the apoptotic status and meiotic progression of oocytes from small (SF) and medium follicles (MF) when the oocytes were denuded from cumulus cells (CC) before, during and after culture for in vitro maturation (IVM). Cumulus-oocyte complexes (COC) were aspirated from SF (0.5–2 mm in diameter) or MF (3–6 mm in diameter) of slaughtered prepubertal gilt ovaries. Only COC with a good morphology of the surrounding cumulus cells were cultured for IVM in modified porcine oocyte medium supplemented with 50 µM β-mercaptoethanol, 1 mM dibutyryl c-AMP, 10 IU mL–1 of eCG, and 10 IU mL–1 of hCG for 20 h at 39°C and 5% CO2 in air and then continued culture in the absence of dibutyryl c-AMP, eCG, and hCG in the same medium for another 24 h. Before and 20 h after the start of IVM culture, some of the oocytes were denuded of CC and the oocytes continued the IVM culture. After IVM culture, oocyte viability and meiotic progression were examined by the annexin V/PI viability assay and DAPI staining. Statistical analyses of 5 replicate data were performed with a 2-way ANOVA and a Tukey's multiple comparisons test. Before IVM culture, there was no significant difference between the viability of SF and MF oocytes, but the incidence of oocytes at the GV0 stage was higher in specimens from SF than MF (24.8 v. 3.3%), and that of oocytes at the GVI stage was the opposite (57.8 in MF v. 22.7% in SF). After IVM culture, apoptotic status of oocytes was only affected by the decumulation timing. The percentage of normal live oocytes was significantly higher when CC were removed after 20 and 44 h of IVM in both SF (39.7 and 39.3 v. 17.7%) and MF (45.4 and 37 v. 22.2%). The incidence of early and late apoptotic oocytes was significantly higher when the CC were removed before IVM culture in both SF (74.3 and 7.4%) and MF (69.4 and 6.7%). The incidence of mature live oocytes was significantly affected by both the origin of COC and the decumulation timing. Although the percentage of mature oocytes was higher in MF, maturation rates were significantly higher when oocytes were denuded at 20 h of IVM culture (SF 65.4%, MF 83.1%) as compared at 0 (SF 27.9%, MF 32.3%) and 44 h (SF 41%, MF 68.5%). However, the percentage of oocytes with normal spindle morphology was significantly higher when oocytes were denuded at 44 h of IVM culture (SF 70.6%, MF 91.5%) than 20 h (SF 66.8%, MF 73%). In summary, regardless of COC from SF and MF, removal of CC at 20 h of IVM culture seems to promote meiotic progression of the oocytes to the MII stage, but factor(s) from or communication with CC during the latter half of IVM culture may be needed to obtain a normal spindle morphology in mature oocytes.

252 BIOCHEMICAL SERUM PROFILE AND LAPAROSCOPIC OVUM PICKUP CORRELATION IN ANGLO NUBIAN GOATS FED WITH DIFFERENT FAT SOURCES

The aim of the present study was to evaluate the correlations between biochemical serum profile with total number of aspirated follicles (AF), oocytes recovered (OR), number of corpus lutea (CL), oocyte morphology quality, and oocyte in vitro maturation (IVM) rate. Eighteen goats were randomly divided into 3 groups based on treatment diets, with different fat sources [soy oil (SG, n = 6), linseed (LG, n = 6), and Megalac® (MG, n = 6)], formulated with 40% concentrate, 60% corn silage, of which 4% was ether extract from fat sources. The animals were submitted to a 15-day adaptation period and then a 70-day experimental period. The laparoscopic ovum pickup (LOPU) was performed after 36 h of an ovarian superstimulation protocol [FSH (80 mg) + eCG (300 IU)] on Day 42 (LOPU1) and Day 70 (LOPU2) of the trial. The number of AF, and the presence and number of CL were evaluated during the LOPU procedure. Blood samples were taken at 14 days, 7 days, and just before each LOPU, and grouped as P1 (mean of blood samples results obtained before LOPU1) and P2 (LOPU2); serum metabolites analysed were total cholesterol, triglycerides, aspartate aminotransferase (AST), creatine kinase, and plasma glucose. The recovered oocytes were classified by morphological appearance, and only G1 (homogenous ooplasma and at least 3 layers of cumulus cells), G2 (homogenous ooplasma and at least 1 complete layers of cumulus cells), and G3 (homogenous ooplasma and incomplete layers or no cumulus cells) were submitted to IVM. The oocytes were incubated in maturation media (TCM 199 supplemented with 10% heat inactivated goat serum) at 38.5°C and 5% CO2 for 27 h. Then, oocytes were subsequently fixed and stained on Hoechst 33342 and analysed using a fluorescent microscope for nuclear maturation. The IVM and analysis were performed for each individual. Pearson correlation test (P &lt; 0.05) was applied in R® software between serum metabolites and LOPU variables. The triglycerides, creatine kinase, and glucose were not correlated with any LOPU variables. The total cholesterol was negatively correlated with G1, G2, and CL, and positively correlated with OR and G3 with determination coefficients (R) of –0.28, –0.38, –0.24, 0.25, and 0.44, respectively. The AST were negatively correlated with AF, G1, G2, and IVM, with R of –0.11, –0.43, –0.13, and –0.16, respectively. In conclusion, these results are suggestive that high serum levels of cholesterol and AST may interfere with oocyte quality, indicating a negative hyperlipidemia effect on reproductive efficiency as reported in cows.Financial support was provided by CNPq and FAPESP.

Biological and hematological responses of Biomphalaria alexandrina to mycobiosynthsis silver nanoparticles.

Silver nanoparticles (AgPNs) extracts were prepared from seven Seven fungal isolates were evaluated through measuring their toxicity against Biomphalaria alexandrina snails. The effects of the two promising Paecilomyces variotii and Aspergillus niger AgNPs sublethal concentrations (LC10 & LC25) on the levels of steroid sex hormones, liver enzymes, total protein, lipids, albumhin, glucose, total and differential count of hemocytes and morphology of hemocytes, oocytes and sperms were studied in this work. The short period of snails' exposure (24h) to the two fungal AgNPs resulted in significant decrease in the levels of progesterone in B. alexandrina. The level of testosterone hormone showed significant increase in snails exposed to P. variotii AgNPs while no significant change was recorded at the exposure to A. niger AgNPs. Also, estradiol hormone concentration increased significantly in this investigation with the increase of the concentration of the two tested compounds. In addition, significant elevation in ALT, AST and Alkaline phosphatase was recorded. The total number of the hematocytes increased significantly by 17.4-47.8%. Snails' granulocytes were reduced by 19.1-43.8%, while hyalinocytes increased by 63.6-354.5%. The exposure of B. alexandrina to LC25 of both P. variotii and A. niger AgNPs showed apoptotic hemolymph cells, fragmented, vacuolated and degenerated cytoplasm, shrunken nucleus and phagocytosis in the light microscopy photographs of the hemocytes. Besides, the photographs showed also, abnormal nuclear division, degeneration and large fat vacuoles in the cytoplasm and swallowed atretic oocytes. Also, the photographs showed dead sperm head separated from its tail, other sperms showed abnormal swallowed head with severely nodded tail, dead sperms with wrinkled tails, hyperplasia and necrotic sperm heads led to overlapping of tails. In conclusion, applying the biosynthesized compounds which led to destruction of blood cells (the immune system), ova and sperms (the reproductive system) of snails is an important effective step to control schistosomiasis.

Human embryos from overweight and obese women display phenotypic and metabolic abnormalities.

Is the developmental timing and metabolic regulation disrupted in embryos from overweight or obese women? Oocytes from overweight or obese women are smaller than those from women of healthy weight, yet post-fertilization they reach the morula stage faster and, as blastocysts, show reduced glucose consumption and elevated endogenous triglyceride levels. Female overweight and obesity is associated with infertility. Moreover, being overweight or obese around conception may have significant consequences for the unborn child, since there are widely acknowledged links between events occurring during early development and the incidence of a number of adult disorders. We have performed a retrospective, observational analysis of oocyte size and the subsequent developmental kinetics of 218 oocytes from 29 consecutive women attending for ICSI treatment and have related time to reach key developmental stages to maternal bodyweight. In addition, we have measured non-invasively the metabolic activity of 150 IVF/ICSI embryos from a further 29 consecutive women who donated their surplus embryos to research, and have related the data retrospectively to their body mass index (BMI). In a clinical IVF setting, we compared oocyte morphology and developmental kinetics of supernumerary embryos collected from overweight and obese women, with a BMI in excess of 25 kg/m(2) to those from women of healthy weight. A Primovision Time-Lapse system was used to measure developmental kinetics and the non-invasive COnsumption/RElese of glucose, pyruvate, amino acids and lactate were measured on spent droplets of culture medium. Total triglyceride levels within individual embryos were also determined. Human oocytes from women presenting for fertility treatment with a BMI exceeding 25 kg/m(2) are smaller (R(2) = -0.45; P = 0.001) and therefore less likely to complete development post-fertilization (P < 0.001). Those embryos that do develop reach the morula stage faster than embryos from women of a BMI < 25 kg/m(2) (<0.001) and the resulting blastocysts contain fewer cells notably in the trophectoderm (P = 0.01). The resulting blastocysts also have reduced glucose consumption (R(2) = -0.61; P = 0.001), modified amino acid metabolism and increased levels of endogenous triglyceride (t = 4.11, P < 0.001). Our data further indicate that these differences are independent of male BMI. Although statistical power has been achieved, this is a retrospective study and relatively small due to the scarcity of human embryos available for research. Consequently, subanalysis of overweight and obese was not possible based on the sample size. The analysis has been performed on supernumerary embryos, originating from a single IVF unit and not selected for use in treatment. Thus, it was not possible to speculate how representative the findings would be of the better quality embryos transferred or frozen for each patient. The data indicate that a high BMI of women at conception is associated with distinct phenotypic changes in the embryo during the preimplantation period, highlighting the importance of prepregnancy body weight in optimizing the chances of fertility and safeguarding maternal and offspring health. These changes to the metabolic fingerprint of human embryos which are most likely a legacy of the ovarian conditions under which the oocyte has matured may reduce the chances of conception for overweight women and provide good evidence that the metabolic profile of the early embryo is set by sub-optimal conditions around the time of conception. The observed changes could indicate long-term implications for the health of the offspring of overweight and obese women. This study was funded by the Hull IVF Unit Charitable Trust and the Hull York Medical School. There are no conflict of interests.

Flow cytometric assessment of morphology, viability, and production of reactive oxygen species of Crassostrea gigas oocytes. Application to Toxic dinoflagellate (Alexandrium minutum) exposure

The Pacific oyster Crassostrea gigas accounts for a large part of shellfish aquaculture production worldwide. Aspects of morphological and functional characteristics of oyster oocytes remain poorly documented, and traditional techniques, such as microscopic observations of shape or fertilization rate, are time and space consuming. The purpose of this study was to assess for the first time viability and reactive oxygen species (ROS) production of Pacific oyster oocytes using flow cytometry (FCM) and to apply this method to determine oocyte responses to in vitro exposure to the toxic dinoflagellate Alexandrium minutum. A culture of A. minutum caused a significant increase in oocyte ROS production, which gradually increased with the age of the culture, but viability was not affected. Effect of the supernatant of the same A. minutum culture did not cause any significant modifications of oocyte morphology, viability, or ROS level. This study confirmed that some oocyte cellular characteristics can be assessed using FCM techniques.