Evidence of Paraoxonases 1, 2, and 3 Expression in Human Ovarian Granulosa Cells.

Increasing evidence suggests that the antioxidant paraoxonase proteins, PON1, PON2, and PON3, have a role in reproduction and may be synthesized by ovarian cells. The aim of this work was to investigate whether human ovarian granulosa cells (GC) express paraoxonases 1, 2, and 3 (PON1, PON2, and PON3) at both the transcriptional and protein levels. Cells were purified from follicle samples of women undergoing ovarian stimulation at oocyte retrieval. We analyzed mRNA by polymerase chain reaction using specific primers for the different variants and quantified the proteins by Western blot using commercially available human recombinant PON proteins as standards. The protein subcellular distribution was determined by immunofluorescence and confocal microscopy and the cell cycles by flow cytometry. Thymidine was used for cellular synchronization at G1/S. Human hepatoma HepG2 and immortalized granulosa COV434 cell lines were used to optimize methodologies. mRNAs from PON1, the two variants of PON2, and PON3 were detected in GC. The cells actively secreted PON1 and PON3, as evidenced by the protein detection in the incubation medium. PON1 and PON3 were mainly distributed in the cytoplasm and notably in the nucleus, while PON2 colocalized with mitochondria. Subcellular nucleo-cytoplasmic distribution of PON1 was associated with the cell cycle. This is the first evidence describing the presence of mRNAs and proteins of the three members of the PON family in human ovarian GC. This study provides the basis of further research to understand the role of these proteins in GC, which will contribute to a better understanding of the reproduction process.

Screening of Natural Stilbene Oligomers from Vitis vinifera for Anticancer Activity on Human Hepatocellular Carcinoma Cells.

The characterization of bioactive resveratrol oligomers extracted from Vitis vinifera canes has been recently reported. Here, we screened six of these compounds (ampelopsin A, trans-ε-viniferin, hopeaphenol, isohopeaphenol, R2-viniferin, and R-viniferin) for their cytotoxic activity to human hepatocellular carcinoma (HCC) cell lines p53 wild-type HepG2 and p53-null Hep3B. The cytotoxic efficacy depended on the cell line. R2-viniferin was the most toxic stilbene in HepG2, with inhibitory concentration 50 (IC50) of 9.7 ± 0.4 µM at 72 h, 3-fold lower than for resveratrol, while Hep3B was less sensitive (IC50 of 47.8 ± 2.8 µM). By contrast, hopeaphenol (IC50 of 13.1 ± 4.1 µM) and isohopeaphenol (IC50 of 26.0 ± 3.0 µM) were more toxic to Hep3B. Due to these results, and because it did not exert a large cytotoxicity in HH4 non-transformed hepatocytes, R2-viniferin was selected to investigate its mechanism of action in HepG2. The stilbene tended to arrest cell cycle at G2/M, and it also increased intracellular reactive oxygen species (ROS), caspase 3 activity, and the ratio of Bax/Bcl-2 proteins, indicative of apoptosis. The distinctive toxicity of R2-viniferin on HepG2 encourages research into the underlying mechanism to develop the oligostilbene as a therapeutic agent against HCC with a particular genetic background.

Oocytes of women who are obese or overweight have lower levels of n-3 polyunsaturated fatty acids compared with oocytes of women with normal weight.

OBJECTIVE: To ascertain whether the oocytes of women who are obese or overweight have a different fatty acid (FA) profile than women with normal weight. DESIGN: Prospective case-control study. SETTING: Two IVF centers. PATIENT(S): A total of 205 women undergoing IVF and intracytoplasmic sperm injection (ICSI) were included in the study, totaling 922 oocytes. INTERVENTION(S): The unfertilized and the immature oocytes from the women who underwent IVF/ICSI were subjected to FA analysis with capillary gas chromatography. Women were classified according their body mass index (BMI) as normal, overweight, or obese. Germinal vesicle oocytes, metaphase I oocytes, and unfertilized metaphase II oocytes were analyzed separately. MAIN OUTCOME MEASURE(S): Fatty acid profile. RESULT(S): A very different oocyte FA pattern was observed for each BMI. Women with normal weight had higher levels of saturated FAs, and lower levels of monosaturated FAs. Women who were obese had lower levels of n-3 polyunsaturated FA, and the lowest n-6:n-3 ratios. Regarding specific FAs, docosahexaenoic acid levels were lower in women with normal weight than in those who are overweight, and in women who are overweight than in those who are obese. The opposite occurred with eicosapentaenoic acid, with the highest levels in women who have normal weight followed by those who are overweight and lower levels in those women who were obese. When FA analysis was restricted to a subset of oocytes, many of these differences persisted. CONCLUSION(S): Our study shows that oocytes from women who are obese or overweight have a different FA composition. This difference in levels could be related to the IVF poor outcome in these women. Therefore, this different composition could suggest that offspring of women who are obese or overweight have an unfavorable milieu even before conception.

A comprehensive serum lipidome profiling of amyotrophic lateral sclerosis.

Objective: To perform a comprehensive lipid profiling to evaluate potential lipid metabolic differences between patients with amyotrophic lateral sclerosis (ALS) and controls, and to provide a more profound understanding of the metabolic abnormalities in ALS. Methods: Twenty patients with ALS and 20 healthy controls were enrolled in a cross-sectional study. Untargeted lipidomics profiling in fasting serum samples were performed by optimized UPLC-MS platforms for broad lipidome coverage. Datasets were analyzed by univariate and a variety of multivariate procedures. Results: We provide the most comprehensive blood lipid profiling of ALS to date, with a total of 416 lipids measured. Univariate analysis showed that 28 individual lipid features and two lipid classes, triacylglycerides and oxidized fatty acids (FAs), were altered in patients with ALS, although none of these changes remained significant after multiple comparison adjustment. Most of these changes remained constant after removing from the analysis individuals treated with lipid-lowering drugs. The non-supervised principal component analysis did not identify any lipid clustering of patients with ALS and controls. Despite this, we performed a variety of linear and non-linear supervised multivariate models to select the most reliable features that discriminate the lipid profile of patients with ALS from controls. These were the monounsaturated FAs C24:1n-9 and C14:1, the triglyceride TG(51:4) and the sphingomyelin SM(36:2). Conclusions: Peripheral alterations of lipid metabolism are poorly defined in ALS, triacylglycerides and certain types of FAs could contribute to the different lipid profile of patients with ALS. These findings should be validated in an independent cohort.

Metabolic adaptations in spontaneously immortalized PGC-1α knock-out mouse embryonic fibroblasts increase their oncogenic potential.

PGC-1α controls, to a large extent, the capacity of cells to respond to changing nutritional requirements and energetic demands. The key role of metabolic reprogramming in tumor development has highlighted the potential role of PGC-1α in cancer. To investigate how loss of PGC-1α activity in primary cells impacts the oncogenic characteristics of spontaneously immortalized cells, and the mechanisms involved, we used the classic 3T3 protocol to generate spontaneously immortalized mouse embryonic fibroblasts (iMEFs) from wild-type (WT) and PGC-1α knockout (KO) mice and analyzed their oncogenic potential in vivo and in vitro. We found that PGC-1α KO iMEFs formed larger and more proliferative primary tumors than WT counterparts, and fostered the formation of lung metastasis by B16 melanoma cells. These characteristics were associated with the reduced capacity of KO iMEFs to respond to cell contact inhibition, in addition to an increased ability to form colonies in soft agar, an enhanced migratory capacity, and a reduced growth factor dependence. The mechanistic basis of this phenotype is likely associated with the observed higher levels of nuclear ß-catenin and c-myc in KO iMEFs. Evaluation of the metabolic adaptations of the immortalized cell lines identified a decrease in oxidative metabolism and an increase in glycolytic flux in KO iMEFs, which were also more dependent on glutamine for their survival. Furthermore, glucose oxidation and tricarboxylic acid cycle forward flux were reduced in KO iMEF, resulting in the induction of compensatory anaplerotic pathways. Indeed, analysis of amino acid and lipid patterns supported the efficient use of tricarboxylic acid cycle intermediates to synthesize lipids and proteins to support elevated cell growth rates. All these characteristics have been observed in aggressive tumors and support a tumor suppressor role for PGC-1α, restraining metabolic adaptations in cancer.

Ovarian stimulated cycles reduce protection of follicular fluid against free radicals.

Controlled ovarian hyperstimulation cycle with exogenous gonadotropins (COH) is associated with clinical complications. The aim of this work was to determine whether COH alters the physiological antioxidant status of follicular fluid in women with no reproductive dysfunction, compared to the natural cycle (NC). In this longitudinal study, forty-one women (oocyte donors) consecutively underwent NC and COH. Follicular fluid was collected at oocyte retrieval and different redox biomarkers were determined: total antioxidant activity (TAA), oxygen radical absorbance capacity (ORAC), nitric oxide, α- and γ-tocopherol, the fatty acid composition, activities of superoxide dismutase, catalase, total and Se-dependent glutathione peroxidases, and the antioxidant paraoxonase (PON) family. Results showed that TAA (1.70 ±â€¯0.03 mM versus 1.86 ±â€¯0.03 mM, p < 0.05), α-tocopherol (4.37 ±â€¯0.26 µM versus 5.74 ±â€¯0.30 µM, p < 0.05), PON1 paraoxonase (245 ±â€¯24 nmol/min/ml versus 272 ±â€¯27 nmol/min/ml, p < 0.05), PON1 arylesterase (87.2 ±â€¯4.6 µmol/min/ml versus 99.3 ±â€¯4.8 µmol/min/ml, p < 0.05), and PON3 simvastatinase (13.48 ±â€¯0.52 nmol/min/ml versus 16.29 ±â€¯0.72 nmol/min/ml, p < 0.001) were significantly lower in COH versus NC. Fatty acids from COH were more saturated, increasing palmitate and decreasing the n-6 and total polyunsaturated fatty acids (PUFAs). Docosahexaenoic acid also increased (p < 0.05). Results suggest that COH could lead to premature ovarian aging and provide new insights into the possible prevention of the adverse effects of ovarian hyperstimulation by directing therapeutic applications to the maintenance of the redox balance and fatty acid status, with special attention to paraoxonase proteins and docosahexaenoic acid.

Enkephalinase activity is modified and correlates with fatty acids in frontal cortex depending on fish, olive or coconut oil used in the diet.

OBJECTIVE: Enkephalins are neuropeptides involved in functions such as pain modulation and/ or cognitive processes. It has been reported that dietary fat modifies enkephalins in the brain. Since enkephalins are hydrolyzed by enkephalinases, the study of the influence of dietary fats, differing in their degree of saturation, on brain fatty acids content and enkephalinase activity is important to understand its regulatory role on neuropeptides under different type of diets. METHODS: We analyzed enkephalinase activity, assayed with alanine-ß-naphthylamide as sub-strate, in frontal cortex of adult male rats fed diets supplemented with fish oil, olive oil or coconut oil, which markedly differed in the saturation of their fatty acids. RESULTS: Rats fed a diet enriched with coconut oil had lower soluble enkephalinase activity than the group fed olive oil (p<0.01) and fish oil (p<0.05) whereas rats fed a diet enriched with fish oil had lower membrane-bound enkephalinase activity than the group fed with olive (p<0.001) or coconut oil (p<0.05). Significant negative correlations were observed between certain fatty acids and enkephalinase activities in the groups fed with olive and coconut oils. No correlations were observed in the group fed with fish oil. CONCLUSIONS: Dietary fat modifies enkephalinase activity in the frontal cortex depending on the degree of saturation of the used oil. It is postulated that the functions, in which enkephalins are involved, such as pain modulation or cognitive functions, may also be affected according to the type of oil used in the diet.

Lower follicular n-3 polyunsaturated fatty acid levels are associated with a better response to ovarian stimulation.

OBJECTIVES: To analyze in detail the fatty acid (FA) composition of follicular fluid (FF) from two-sized follicles at oocyte retrieval and to determine associations of the FAs from large follicles with the woman's age and the response to ovarian stimulation. DESIGN: Observational study. SETTING: University and fertility clinic. PATIENTS: Sixty-four women (age 19-46), consisting of unfertile patients and oocyte donors, undergoing controlled ovarian stimulation. INTERVENTIONS: None. MAIN OUTCOME MEASURE(S): FF from small (< 12 mm) and large (≥ 18 mm) follicles was collected at oocyte retrieval. FAs by gas chromatography-flame ionization detection. RESULT: Thirty-two FAs with chain lengths ranging from 14 to 25 carbons were identified. There was a readjustment in FA distribution as follicle size increased, raising very long-chain saturated FAs, nervonic (24:1n-9), arachidonic (20:4n-6), and n-3 polyunsaturated FAs (PUFA, P < 0.001), the latter mainly due to an increase in docosahexaenoic acid (22:6n-3, DHA). In large follicles, double bond and peroxidizability indices and total n-3 PUFA, particularly DHA, correlated positively with the woman's age and negatively with the number of total and mature oocytes, total and top-quality embryos, and fertilization rate. CONCLUSIONS: We have described 32 FAs in ovarian FF, of which 16 changed their distribution with follicle size. The results also indicate that lower n-3 PUFA levels in large follicles, which are associated with younger women, predict a better response to ovarian stimulation based on the recovery of total and mature oocytes, total and top-quality embryos, and fertilization rate per cycle. KEY MESSAGE: The fatty acid profile of ovarian FF changes as the follicle grows and lower n-3 PUFA levels in large follicles, associated with younger women, predict a better response to ovarian stimulation.

Analysis of Protein Oxidative Modifications in Follicular Fluid from Fertile Women: Natural Versus Stimulated Cycles.

Oxidative stress is associated with obstetric complications during ovarian hyperstimulation in women undergoing in vitro fertilization. The follicular fluid contains high levels of proteins, which are the main targets of free radicals. The aim of this work was to determine specific biomarkers of non-enzymatic oxidative modifications of proteins from follicular fluid in vivo, and the effect of ovarian stimulation with gonadotropins on these biomarkers. For this purpose, 27 fertile women underwent both a natural and a stimulated cycle. The biomarkers, glutamic semialdehyde (GSA), aminoadipic semialdehyde (AASA), Nε-(carboxymethyl)lysine (CML), and Nε-(carboxyethyl)lysine (CEL), were measured by gas-liquid chromatography coupled to mass spectrometry. Results showed that follicular fluid contained products of protein modifications by direct metal-catalyzed oxidation (GSA and AASA), glycoxidation (CML and CEL), and lipoxidation (CML). GSA was the most abundant biomarker (91.5%). The levels of CML amounted to 6% of the total lesions and were higher than AASA (1.3%) and CEL (1.2%). In the natural cycle, CEL was significantly lower (p < 0.05) than in the stimulated cycle, suggesting that natural cycles are more protected against protein glycoxidation. These findings are the basis for further research to elucidate the possible relevance of this follicular biomarker of advanced glycation end product in fertility programs.

Unraveling the in vitro antitumor activity of Vismia baccifera against HepG2: role of hydrogen peroxide.

Currently natural products derived from plants are receiving huge attention because of their antitumor activities. In previous work we reported that an aqueous leaf extract of Vismia baccifera induced toxicity in HepG2. The present study focuses on the mechanisms of the cytotoxic actions induced by the extract. Results showed that V. baccifera was innocuous in non-transformed human HH4 hepatocytes. In HepG2 it caused deregulation of antioxidant status (increasing superoxide dismutase expression and decreasing glutathione levels and glutathione peroxidase activity) and accumulation of reactive oxygen species, particularly hydrogen peroxide. The extract induced a) cell cycle arrest at G2/M phase, b) phosphorylation of ATM (protein kinase ataxia-telangiectasia mutated) and γH2AX (γ-histone family 2A variant), c) caspase-3 activation, and e) deregulation of the Bax/Bcl family, increasing pro-apoptotic proteins. ATM did not seem to be involved in γH2AX activation. Co-incubation with catalase prevented the alterations elicited by V. baccifera in HepG2. Taking together, these results indicate that hydrogen peroxide mediates the HepG2 cytotoxic response and provide evidence for more in-depth studies of the signaling involved.

Paraoxonase activities in human follicular fluid: role in follicular maturation.

The paraoxonases (PONs) are antioxidant enzymes associated with beneficial effects against several diseases and some exposures. Little is known, however, about the role of PONs in human reproduction. This work was conducted to investigate whether any association existed between the activities of the PON enzymes (1, 2, and 3) with the follicular size and fertility parameters in assisted reproduction. The study included 100 subfertile women (patients) and 55 proven fertile women (oocyte donors), all undergoing an ovarian stimulation cycle. Follicular fluid from small (diameter <12 mm) and large (diameter ≥18 mm) follicles was collected from each woman. The PONs were quantified in follicular fluid by immunoblotting. PON1 arylesterase and paraoxonase, PON2 methyl paraoxonase and PON3 simvastatinase activities from both donors and patients were significantly higher (P < 0.001) in follicular fluid from large follicles compared with small ones. In large follicles, PON3 activity was significantly higher (P < 0.01) in donors compared with patients. Follicular fluid PON1 arylesterase and paraoxonase activity was positively correlated with the number of retrieved oocytes in donors. This study shows an increase in the activities of PONs with follicle size, thus providing indirect evidence for the role of PONs in follicle maturation.

Influence of oxygen partial pressure on the characteristics of human hepatocarcinoma cells.

Most of the in vitro studies using liver cell lines have been performed under atmospheric oxygen partial pressure (21% O2). However, the oxygen concentrations in the liver and cancer cells are far from this value. In the present study, we have evaluated the influence of oxygen on 1) the tumor cell lines features (growth, steady-state ROS levels, GSH content, activities of antioxidant enzymes, p66 Shc and SOD expressions, metalloproteinases secretion, migration, invasion, and adhesion) of human hepatocellular carcinoma cell lines, and b) the response of the cells to an oxidant stimulus (aqueous leaf extract of the V. baccifera plant species). For this purpose, three hepatocarcinoma cell lines with different p53 status, HepG2 (wild-type), Huh7 (mutated), and Hep3B (deleted), were cultured (6-30 days) under atmospheric (21%) and more physiological (8%) pO2. Results showed that after long-term culturing at 8% versus 21% O2, the cellular proliferation rate and the steady-state levels of mitochondrial O2- were unaffected. However, the intracellular basal ROS levels were higher independently of the characteristics of the cell line. Moreover, the lower pO2 was associated with lower glutathione content, the induction of p66 Shc and Mn-SOD proteins, and increased SOD activity only in HepG2. This cell line also showed a higher migration rate, secretion of active metalloproteinases, and a faster invasion. HepG2 cells were more resistant to the oxidative stress induced by V. baccifera. Results suggest that the long-term culturing of human hepatoma cells at a low, more physiological pO2 induces antioxidant adaptations that could be mediated by p53, and may alter the cellular response to a subsequent oxidant challenge. Data support the necessity of validating outcomes from studies performed with hepatoma cell cultures under ambient O2.

Glutathione S-transferase activity in follicular fluid from women undergoing ovarian stimulation: role in maturation.

Female infertility involves an emotional impact for the woman, often leading to a state of anxiety and low self-esteem. The assisted reproduction techniques (ART) are used to overcome the problem of infertility. In a first step of the in vitro fertilization therapy women are subjected to an ovarian stimulation protocol to obtain mature oocytes, which will result in competent oocytes necessary for fertilization to occur. Ovarian stimulation, however, subjects the women to a high physical and psychological stress, thus being essential to improve ART and to find biomarkers of dysfunction and fertility. GSH is an important antioxidant, and is also used in detoxification reactions, catalysed by glutathione S-transferases (GST). In the present work, we have investigated the involvement of GST in follicular maturation. Patients with fertility problems and oocyte donors were recruited for the study. From each woman follicles at two stages of maturation were extracted at the preovulatory stage. Follicular fluid was separated from the oocyte by centrifugation and used as the enzyme source. GST activity was determined based on its conjugation with 3,4-dichloronitrobenzene and the assay was adapted to a 96-well microplate reader. The absorbance was represented against the incubation time and the curves were adjusted to linearity (R(2)>0.990). Results showed that in both donors and patients GST activity was significantly lower in mature oocytes compared to small ones. These results suggest that GST may play a role in the follicle maturation by detoxifying xenobiotics, thus contributing to the normal development of the oocyte. Supported by FIS/FEDER (PI11/02559), Gobierno Vasco (Dep. Educación, Universiades e Investigación, IT687-13), and UPV/EHU (CLUMBER UFI11/20 and PES13/58). The work was approved by the Ethics Committee of the UPV/EHU (CEISH/96/2011/RUIZLARREA), and performed according to the UPV/EHU and IVI-Bilbao agreement (Ref. 2012/01).

Characterization of the paraoxonase system in follicular fluid of women subjected to an ovarian stimulation cycle.

Reactive oxygen species (ROS) and antioxidants are involved in the regulation of reproductive processes. Previous studies in infertile women undergoing an ovarian stimulation cycle have suggested a possible role for ROS in the occurrence of conception in In Vitro Fertilization. In this context the control of the redox balance of follicular fluid becomes essential for reproduction, so that the presence of enzymes with antioxidant activities, such as the paraoxonase (PON) system, would play a role in maintaining this balance. The objective of this work was a) to characterize the paraoxonase system in follicular fluid of women undergoing a controlled ovarian stimulation cycle, analysing the associated PON1, PON2, and PON3 activities, and b) to study the possible involvement of the PON system in follicular maturation. The enzyme activities were quantified in follicular fluid from large and small follicles from women undergoing an ovarian stimulation cycle in the IVI-Bilbao clinic. PON activities were quantified using spectrophotometric and HPLC techniques. Statistical comparisons were performed using the Student's t-test for paired data. Results indicate that follicular fluid presents paraoxonase activities which are detectable by the methods developed in this study. PON activities were associated with follicular maturation, suggesting that the PON system plays a role in oocyte maturation. This work was supported by research grants from the Ministry of Health and Consumption (FIS/FEDER PI11/02559), the Basque Country Government (Dep. Education, Universities and Research ref. IT687-13, and DCIT ref. S-PE13UN063), and UPV/EHU (CLUMBER UFI11/20 and PES13/58). The work was approved by the Ethics Committee of the UPV/EHU (CEISH/96/2011/RUIZLARREA), and performed according to the UPV/EHU and IVI-Bilbao agreement (Ref. 2012/01).

Piper and Vismia species from Colombian Amazonia differentially affect cell proliferation of hepatocarcinoma cells.

There is an increasing interest to identify plant-derived natural products with antitumor activities. In this work, we have studied the effects of aqueous leaf extracts from Amazonian Vismia and Piper species on human hepatocarcinoma cell toxicity. Results showed that, depending on the cell type, the plants displayed differential effects; thus, Vismia baccifera induced the selective killing of HepG2, while increasing cell growth of PLC-PRF and SK-HEP-1. In contrast, these two last cell lines were sensitive to the toxicity by Piper krukoffii and Piper putumayoense, while the Piperaceae did not affect HepG2 growth. All the extracts induced cytotoxicity to rat hepatoma McA-RH7777, but were innocuous (V. baccifera at concentrations < 75 µg/mL) or even protected cells from basal death (P. putumayoense) in primary cultures of rat hepatocytes. In every case, cytotoxicity was accompanied by an intracellular accumulation of reactive oxygen species (ROS). These results provide evidence for the anticancer activities of the studied plants on specific cell lines and suggest that cell killing could be mediated by ROS, thus involving mechanisms independent of the plants free radical scavenging activities. Results also support the use of these extracts of the Vismia and Piper genera with opposite effects as a model system to study the mechanisms of the antitumoral activity against different types of hepatocarcinoma.

Lipid oxidation inhibitory effects and phenolic composition of aqueous extracts from medicinal plants of Colombian Amazonia.

Diverse plants of ethnobotanic interest in Amazonia are commonly used in traditional medicine. We determined the antioxidant potential against lipid peroxidation, the antimicrobial activity, and the polyphenol composition of several Amazonian plants (Brownea rosademonte, Piper glandulosissimum, Piper krukoffii, Piper putumayoense, Solanum grandiflorum, and Vismia baccifera). Extracts from the plant leaf, bark, and stem were prepared as aqueous infusions, as used in folk medicine, and added to rat liver microsomes exposed to iron. The polyphenolic composition was detected by reverse-phase HPLC coupled to diode-array detector and MS/MS analysis. The antimicrobial activity was tested by the spot-on-a-lawn method against several indicator microorganisms. All the extracts inhibited lipid oxidation, except the P. glandulosissimum stem. The plant extracts exhibiting high antioxidant potential (V. baccifera and B. rosademonte) contained high levels of flavanols (particularly, catechin and epicatechin). By contrast, S. grandiflorum leaf, which exhibited very low antioxidant activity, was rich in hydroxycinnamic acids. None of the extracts showed antimicrobial activity. This study demonstrates for the first time the presence of bioactive polyphenolic compounds in several Amazonian plants, and highlights the importance of flavanols as major phenolic contributors to antioxidant activity.

Glutathione peroxidase activity in seminal plasma and its relationship to classical sperm parameters and in vitro fertilization-intracytoplasmic sperm injection outcome.

OBJECTIVE: To relate the glutathione peroxidase (GPX) activity level in human seminal plasma with standard semen parameters and spermatozoa fertilization potential in terms of fertilization and pregnancy rates in an IVF program. DESIGN: Prospective study. SETTING: Human Reproduction Unit at Cruces Hospital (Vizcaya, Spain). PATIENT(S): Three hundred consecutive males from infertile couples participating in the IVF program. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Analysis of GPX activity in seminal plasma by spectrophotometry. RESULT(S): GPX activity in seminal plasma was significantly lower in patients with abnormal sperm as assessed by 1999 and 2010 World Health Organization (WHO) criteria, compared with normozoospermic individuals. There was a more significant decrease in those samples with severe sperm pathologies. GPX values were significantly lower in samples with severe asthenozoospermia, oligozoospermia, and teratozoospermia compared with normal samples. However, there was no correlation between GPX activity in seminal plasma in IVF patients and fertilization rates or pregnancy outcome. CONCLUSION(S): Although seminal plasma GPX activity was related to semen quality according to WHO parameters, such an association was not found with IVF-intracytoplasmic sperm injection (ICSI) outcome, presumably because of the well-known ability of IVF-ICSI procedures to overcome sperm deficiencies in the fertilization process.

Involvement of G-463A MPO gene polymorphism in the response of postmenopausal women to hormone therapy.

OBJECTIVE: The aims of this work were to determine (1) the effects of estrogen plus progestogen therapy (EPT) and raloxifene on oxidative stress and cardiovascular risk biomarkers in postmenopausal women and (2) the involvement of the functional G-463A polymorphism of the myeloperoxidase (MPO) gene in the therapy responses. METHODS: Postmenopausal women (45-55 y old) were assigned to three groups receiving (1) EPT (continuous 50 µg transdermal estradiol daily and 200 mg/d micronized progesterone orally the first 14 d of each month; n = 21), (2) raloxifene (60 mg daily; n = 17), and (3) no treatment (control; n = 21). Blood and urine samples were taken before and after 6 months of therapy. Measurements were serum lipid profile, C-reactive intercellular adhesion molecule 1 (ICAM-1), α-tocopherol, γ-tocopherol, uric acid, total antioxidant activity (TAA), malondialdehyde, and urinary 1,4-dihydroxynonane-mercapturic acid (the major urinary 4-hydroxynonenal metabolite). The G-463A MPO polymorphism was analyzed by polymerase chain reaction and restriction fragment length polymorphism. RESULTS: EPT significantly decreased TAA and the levels of ICAM-1, not modifying other cardiovascular risk or oxidative stress markers. The raloxifene and control groups experienced no modifications in oxidative stress or endothelial dysfunction markers. The MPO genotype specifically influenced the outcomes in the EPT group. Thus, TAA decreased significantly in GG (high-expression genotype) homozygotes, whereas ICAM-1 levels were reduced in A allele carriers. CONCLUSIONS: EPT exerted a negative action on the serum oxidant/antioxidant balance in the MPO GG homozygotes and a positive effect on the ICAM-1 endothelial dysfunction marker in carriers of the low-expression A allele. This observation provides evidence of the importance of this polymorphism in the response to EPT.

Ala16Val SOD2 polymorphism is associated with higher pregnancy rates in in vitro fertilization cycles.

OBJECTIVE: To investigate whether the Ala16Val polymorphism in the SOD2 gene, encoding for mitochondrial manganese superoxide dismutase (SOD), is associated with [1] infertility and [2] the pregnancy rate (PR) in IVF cycles. DESIGN: Prospective case-control study. SETTING: Public university and public university hospital. PATIENT(S): A total of 362 newborns (nonselected population) and 148 infertile women undergoing an IVF cycle, from which 44 became pregnant and 104 did not. INTERVENTION(S): Blood samples extracted from the patients and newborn umbilical cord. MAIN OUTCOME MEASURE(S): Genotype and allele distribution of the Ala16Val polymorphism in the SOD2 gene using the tetra-primer amplification refractory mutation system-polymerase chain reaction (PCR). RESULT(S): The polymorphism distribution of the subfertile women was similar to that of a nonselected population. The SOD2 Ala allele frequency was 49% both in controls and IVF patients. In IVF population the Ala/Ala SOD2 genotype was 25%, with a 28% Val/Val homozygous. In contrast, the Ala/Ala genotype was associated with higher PRs in IVF (47% in Ala/Ala vs. 23% in no Ala/Ala). A multivariate logistic regression analysis revealed that the Ala/Ala genotype was an independent predictor of pregnancy (odds ratio [OR] = 3.29), followed by the number of transferred embryos (OR = 2.37) and age (OR = 0.84). CONCLUSION(S): The Ala/Ala SOD2 genotype is a significant independent predictor of the occurrence of pregnancy in IVF. Data also support a role for antioxidant defense, particularly in the mitochondria, in conception in IVF.

Serum oxidizability and antioxidant status in patients undergoing in vitro fertilization.

OBJECTIVE: To evaluate the serum oxidizability and antioxidant status in women undergoing an in vitro fertilization (IVF) cycle and to assess the possible relationship of the oxidizability indexes with the pregnancy rate. DESIGN: Prospective, longitudinal study. SETTING: Public university and public university hospital. PATIENT(S): Systematically recruited cohort of 125 women undergoing either IVF or intracytoplasmic sperm injection (ICSI). INTERVENTION(S): Serum samples were collected before the beginning of the use of gonadotropins (basal) and the day of human chorionic gonadotropin (hCG) administration (final) during an IVF cycle. MAIN OUTCOME MEASURE(S): The Cu2+-induced serum oxidation in terms of the oxidation rate in the lag (Vlag) and propagation (Vmax) phases and the time at which the oxidation rate is maximal (tmax), and measurements of serum total antioxidant activity (TAA), tocopherol, hydrophilic antioxidants, malondialdehyde, and nitric oxide. RESULT(S): Albumin, urate, bilirubin, alpha-tocopherol and gamma-tocopherol, TAA, and tmax statistically significantly decreased after the IVF cycle. Conception cycles were associated with a serum more prone to oxidation compared with nonconception cycles. In multivariate logistic regression analysis, the difference (final-basal) of the oxidation index Vlag (OR 1.394) and the body mass index (OR 0.785) were independent predictors of pregnancy. CONCLUSION(S): Treatment with IVF induces the production of reactive oxygen species (ROS), which is reflected in a serum less protected against oxidation. The results also suggest a role for ROS in the occurrence of conception in IVF.