Impact of the number of retrieved oocytes on IVF outcomes: oocyte maturation, fertilization, embryo quality and implantation rate.

The process of oocyte retrieval represents a key phase during the cycles of in vitro fertilization (IVF). It involves controlled ovarian stimulation to retrieve the highest number of oocytes possible. According to many previous studies, the higher the number of oocytes the higher the chances of obtaining embryos for multiple transfers. In this study, in total, 1987 patients were retrospectively reviewed to investigate the correlations between the number of retrieved oocytes and the subsequent IVF outcomes. Patients were divided into three groups according to the number of retrieved oocytes (Group 1: ≤5 oocytes; Group 2: 6-15 oocytes; Group 3: ≥15 oocytes). The results showed a significant negative correlation between oocyte number and maturation rate as well as fertilization rate. However, a significant positive correlation was found between oocyte number and the blastulation rate. The implantation rate after fresh embryo transfers was higher in group 2 (6-15 oocytes) compared with group 1 (≤5 oocytes). According to our findings, we conclude that oocyte numbers between 6 and 15 oocytes can result in the highest chances of positive IVF outcomes in terms of embryo quality and fresh embryo transfers with lower risks of ovarian hyperstimulation.

Antioxidant activities and lipid peroxidation status in human follicular fluid: age-dependent change.

Maternal age is a significant factor influencing in vitro fertilization (IVF) outcomes. Oxidative stress (OS) is one of the major causes of age-related cellular and molecular damage. The purpose of this work was to investigate the correlation between maternal age with intrafollicular antioxidants and OS markers in follicular fluid (FF), and also to determine the OS status in patients of advanced age. This study was a prospective study including 201 women undergoing IVF whose age was between 24 and 45 years old. FF samples were obtained from mature follicles at the time of oocyte retrieval. After treatment of FF, lipid peroxidation levels (MDA) and enzyme activities such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione (GSH) level were evaluated using spectrophotometry. The results indicated that the age cutoff point for increasing the MDA level was fixed at 37 years, allowing the study to be differentiated into two age groups. Group I included patients whose age was less than 37 years, and group II included patients whose age was greater than or equal 37 years. Statistical analysis revealed that MDA and GSH levels and GR activity were significantly higher in group II compared with group I. The SOD and CAT activities were significantly less in group II compared with group I. We concluded that from 37 years old a reproductive ageing was accompanied by a change in the antioxidant pattern in FF that impaired reactive oxygen species scavenging efficiency.

Reactive oxygen species in reproduction: harmful, essential or both?

The process of embryonic development is crucial and radically influences preimplantation embryo competence. It involves oocyte maturation, fertilization, cell division and blastulation and is characterized by different key phases that have major influences on embryo quality. Each stage of the process of preimplantation embryonic development is led by important signalling pathways that include very many regulatory molecules, such as primary and secondary messengers. Many studies, both in vivo and in vitro, have shown the importance of the contribution of reactive oxygen species (ROS) as important second messengers in embryo development. ROS may originate from embryo metabolism and/or oocyte/embryo surroundings, and their effect on embryonic development is highly variable, depending on the needs of the embryo at each stage of development and on their environment (in vivo or under in vitro culture conditions). Other studies have also shown the deleterious effects of ROS in embryo development, when cellular tissue production overwhelms antioxidant production, leading to oxidative stress. This stress is known to be the cause of many cellular alterations, such as protein, lipid, and DNA damage. Considering that the same ROS level can have a deleterious effect on the fertilizing oocyte or embryo at certain stages, and a positive effect at another stage of the development process, further studies need to be carried out to determine the rate of ROS that benefits the embryo and from what rate it starts to be harmful, this measured at each key phase of embryonic development.

Competitive enzyme-linked immunosorbent assay using monoclonal antibodies to the Brucella melitensis BP26 protein to evaluate antibody responses in infected and B. melitensis Rev.1 vaccinated sheep.

Competitive enzyme-linked immunosorbent assay (C-ELISA) was performed using 15 monoclonal antibodies (MAbs), specific for Brucella BP26 (previously also called CP28), a periplasmic protein antigen, to investigate antibody responses in naturally and B. melitensis H38 experimentally infected and B. melitensis Rev.1 vaccinated sheep. The antigen preparation consisted of cytosoluble protein extract (CPE) of B. melitensis B115. By combining the C-ELISA results of several MAbs, a high percentage of naturally infected animals were detected which showed different status in the current conventional diagnostic tests. Indeed, 90% of sheep which were positive in the conventional bacteriological and serological tests were positive in C-ELISA. 72% of the bacteriologically negative but serologically and delayed type hypersensitivity positive sheep were also positive in the C-ELISA. Moreover, 79% of the bacteriologically and serologically negative sheep but delayed type hypersensitivity positive were also detected by C-ELISA. Thus, these results confirmed the importance of BP26 as a frequently recognized target of the humoral immune response of infected sheep. The 8 B. melitensis H38 experimentally infected sheep showed various degrees of antibody responses at the 90th day after infection, which was delayed in comparison to that against O-polysaccharide (O-PS). Of the 15 MAbs tested, only one MAb was weakly inhibited (20 to 35% inhibition) by 56% of negative control sera. Furthermore, no antibody response against BP26 was detected in B. melitensis Rev.1 vaccinated sheep. Results of the C-ELISA with the 15 MAbs showed individual variability of the antibody responses against BP26. Thus, it is suggested that several epitopes of BP26 are of interest for diagnosis of B. melitensis infection in sheep.

Production and characterisation of monoclonal antibodies to Brucella melitensis cytosoluble proteins that are able to differentiate antibody responses of infected sheep from Rev. 1 vaccinated sheep.

Monoclonal antibodies (MAbs) were produced to Brucella melitensis cytosoluble proteins (CP) with apparent molecular masses of 12, 24 and 28 kDa (CP12, CP24 and CP28) which were previously shown by immunoblotting to differentiate antibody responses of infected sheep from those of B. melitensis strain Rev. 1 vaccinated sheep. These MAbs were derived from mice infected with virulent smooth (S) B. melitensis strain H38. Most MAbs obtained were directed to CP28, which indicated (as was shown in infected sheep) that this protein was also highly immunogenic in mice. A large number of MAbs that showed reactivity to CP in ELISA but did not show reactivity in immunoblotting of CP were also obtained and might recognise conformational epitopes of these proteins. MAbs were used to localise CP12, CP24 and CP28. None of the MAbs reacted with whole B. melitensis cells in ELISA but showed reactivity with sonicated bacteria in ELISA, which indicated an internal localisation of these proteins. Among several B. melitensis B115 subcellular fractions tested, the anti-CP12 and anti-CP28 MAbs reacted essentially with the CP extract (CPE) in both ELISA and immunoblotting, whereas the anti-CP24 MAbs reacted with both CPE and cell envelope fraction (CEF)-although with lower intensity to the latter fraction. The internal localisation of these proteins was confirmed by immuno-electron microscopy of thin-sectioned B. melitensis B115 or B. melitensis 16M cells. Immunogold labelling was mainly observed in the cytoplasm and, consequently, CP12, CP24 and CP28 are probably cytoplasmic proteins. Immunoblotting of whole cell lysates with the MAbs also showed the presence of these proteins in all Brucella species and biovars, including the vaccine strains B. melitensis Rev. 1 and B. abortus B19. The use of these MAbs should help further study of antibody responses in sheep and other hosts and may be of considerable value for developing new diagnostic tests for ovine brucellosis.

Cloning, nucleotide sequence, and expression of the Brucella melitensis bp26 gene coding for a protein immunogenic in infected sheep.

We have previously identified a Brucella melitensis 28 kDa cytosoluble protein (CP28) which was highly immunogenic in infected sheep and which in addition made possible the serological differentiation between infected and B. melitensis Rev. 1 vaccinated sheep. Monoclonal antibodies against CP28 were used to screen a B. melitensis 16M genomic library and to clone the corresponding gene. DNA sequencing of the gene encoding CP28 of B. melitensis 16M revealed that it was nearly identical to that of the recently published bp26 gene of Brucella abortus vaccine strain S19 coding for a periplasmic protein. The differences between the B. melitensis 16M gene and that of B. abortus S19 consisted of single nucleotide substitutions, one or two codon deletions, one codon addition, and most importantly a 21-bp deletion. The corresponding region of B. abortus S19 contains two 10-bp direct repeats which could have been involved in the genesis of the deletion. Expression of the B. melitensis 16M bp26 gene in Escherichia coli studied by the use of the monoclonal antibodies showed the same characteristics as reported for the B. abortus S19 bp26 gene, i.e. the presence of a higher molecular mass preprotein and a lower molecular mass band which probably corresponds to the mature protein exported to the periplasm. Immunoblotting performed with sera from either naturally infected or B. melitensis H38 experimentally infected sheep confirmed the importance of the B. melitensis CP28/BP26 protein as diagnostic antigen.

Enzyme-linked immunosorbent assay with partially purified cytosoluble 28-kilodalton protein for serological differentiation between Brucella melitensis-infected and B. melitensis Rev.1-vaccinated sheep.

The problem of differentiating sheep infected with Brucella melitensis from those vaccinated or exposed to cross-reaching organisms has not been resolved by conventional serological tests or through the use of the smooth lipopolysaccharide in primary binding assays. We therefore analyzed sera from ewes experimentally infected with B. melitensis H38, from ewes naturally infected with B. melitensis, and from B. melitensis Rev.1-vaccinated ewes by enzyme-linked immunosorbent assay with three antigenic fractions: O polysaccharide, a cytosoluble protein extract (CPE) from the rough strain B. melitensis B115, and a partially purified cytosoluble protein of 28 kDa (CP28) from the CPE. Immunoglobulin G anti-O polysaccharide and anti-CPE responses were detected in all groups of animals tested (Rev.1 vaccinated and B. melitensis infected). However, false-positive reactions with CPE occurred with sera from Brucella-free ewes. The use of partially purified CP28 abolished these false-positive reactions. Furthermore, no immunoglobulin G antibodies against CP28 were detected in sera from vaccinated ewes, whereas 80% (8 of 10) of ewes experimentally infected with B. melitensis H38 and 89% (25 of 28) of naturally infected ewes showed various degrees of anti-CP28 reactivity (absorbance values of between 0.5 and 2.5). The results obtained with CP28 showed the potential usefulness of this antigen to permit the detection of B. melitensis-infected ewes and their differentiation from B. melitensis Rev.1-vaccinated ones.

Overproduction of the Brucella melitensis heat shock protein DnaK in Escherichia coli and its localization by use of specific monoclonal antibodies in B. melitensis cells and fractions.

The Brucella melitensis dnaK gene was amplified by the polymerase chain reaction using primers chosen according to the published sequence of B. ovis and cloned in multiple copy plasmids enabling expression under the control of the Plac promoter. Monoclonal antibodies (mAb) obtained by immunizing mice with B. melitensis B115 cell wall (CW) fraction or by infecting mice with virulent B. melitensis strain H38 and recognizing a 73-kDa band in immunoblotting of the B. melitensis CW fraction reacted with the cloned dnaK gene product and were thus shown to be specific for the heat shock protein DnaK. The anti-Dnak protein mAbs did not react with Escherichia coli control cells or cell lysates and could therefore be specific to Brucella DnaK protein epitopes. These mAbs were further used to study overproduction of the DnaK protein. B. melitensis DnaK overproduction in E. coli resulted in a defect in cell septation and formation of cell filaments. Immunogold labelling with the mAbs and electron microscopy localized the DnaK protein inside as well as outside the E. coli cells, probably resulting from lysis due to toxicity of the overproduced DnaK protein. These results indicated that overproduction of the B. melitensis DnaK protein in E. coli had similar physiological consequences as that of E. coli overproduced in E. coli. The DnaK protein localization in B. melitensis cells was essentially cytoplasmic, as shown by immunoelectron microscopy. Heat shock treatment of these cells resulted in increased binding of mAbs and labelling in the cytoplasm. However, in subcellular fractions the DnaK protein was predominantly found in the cell envelope fraction of B. melitensis, which could perhaps be due to interaction of the DnaK protein with membrane proteins.

Identification of sero-reactive Brucella melitensis cytosoluble proteins which discriminate between antibodies elicited by infection and Rev.1 vaccination in sheep.

Distinction between Brucella melitensis infected and vaccinated sheep is needed to fully achieve ovine brucellosis eradication in several countries. For this purpose, we probed immunoblots of cytosoluble protein extract (CPE) of the rough (R) B. melitensis strain B115 with sera of Brucella-free, naturally infected, B. melitensis H38 experimentally infected and B. melitensis Rev.1 vaccinated sheep to identify immunogenic Brucella cytosoluble proteins which may lead to the development of more useful diagnostic tests and which may eventually differentiate vaccinated sheep from infected sheep. Brucella-free sheep sera showed IgM antibody reactivity to protein bands between 19 and 92 kDa. The use of conjugate specific for sheep IgG avoided non specific reactivity to all these bands except to the 39 and 50 kDa bands which were still detected in some Brucella-free sheep sera. In sera of B. melitensis H38 experimentally infected sheep, a specific IgG antibody response was observed against proteins of molecular masses of 19, 24, 28, 32, 39, 50 and 54 kDa. These proteins were also variably detected by IgG of sera from naturally infected sheep. Other proteins of 10, 12, 23, 36, 38, 42, 46, 68, 80 and 92 kDa were also detected by the latter sera. In sera from B. melitensis Rev.1 vaccinated sheep, an IgG antibody response was only observed against proteins with molecular masses of 39 and 50 kDa. These results suggest that the 19, 24, 28, 32 and 54 kDa proteins (the first recognized after experimental infection and that provoked a consistent humoral response during natural infection) could be interesting to develop serological tests for differentiating B. melitensis infection from B. melitensis Rev.1 vaccination.

Antibody response to Brucella melitensis outer membrane antigens in naturally infected and Rev1 vaccinated sheep.

Sera from Brucella infected and B. melitensis Rev1 vaccinated sheep were analysed by immunoblotting using the cell envelope fraction (CEF) of B. melitensis B115. The CEF of B. melitensis B115 was analysed using a bank of monoclonal antibodies. The fraction consisted mainly of S-LPS like molecules, R-LPS and outer membrane proteins (OMPs) of molecular masses of 10, 16.5, 19, 25-27, 31-34, 36-38, 73 and 89 kDa. Immunoblot analysis indicates that the antibody response in infected sheep was mainly directed against the major OMPs of 25-27, 31-34, 36-38 kDa, against 55 to 62, 70-73 and 89 to 94 kDa proteins associated with the CEF and, against S-LPS like molecules. Some infected sheep reacted with antigens of molecular mass lower than 20 kDa. Sera from vaccinated sheep reacted only with OMPs of 36-38, 60, 70-73 and 89 kDa. The major 25-27 and 31-34 kDa OMPs and proteins below 20 kDa were only detected by the sera of infected sheep. These differences may be due to the persistence of the field infection also reflected by the fact that antibody response against O-polysaccharide (O-PS), as measured by enzyme-linked immunosorbent assay (ELISA), was more intense in infected sheep than in vaccinated ones. These results also indicate that these OMPs could be useful to differentiate B. melitensis infection from B. melitensis Rev.1 vaccination in sheep.