Allelic variants of the estrogen receptor genes and frailty phenotype in postmenopausal women.

OBJECTIVE: The prevalence of frailty has been related to menopause. Our main objective was to investigate whether single nucleotide polymorphisms (SNPs) of the estrogen receptor (ER) ERα and ERß genes were related to the frailty phenotype in a population of community-dwelling postmenopausal women. METHODS: A cross-sectional study was performed in which we selected five SNPs, three in the ERα gene and two in the ERß. Linear regression was used to estimate the percentage of phenotypic variance after adjusting for confounding variables. RESULTS: A total of 470 women (mean ± standard deviation age 63.83 ± 8.16 years) were included, of whom 137 women were frail. The SNP rs3798577 of the ERα gene was the only variant associated with frailty, but this significance faded in the multivariant analysis. Body mass index (p = 0.012), number of comorbidities (0 vs. ≥2, p = 0.002) and two reproductive variables, number of miscarriages (none vs. ≥2, p = 0.036) and of childbirths (one vs. ≥3, p = 0.008), were independently related to frailty. CONCLUSION: The five SNPs of the ERα and ERß genes tested were not correlated with frailty. Other SNPs of the ER warrant analysis to clarify whether variance in the gene response affects frailty status.

The impact of coffee on health.

OBJECTIVE: Coffee is a beverage used worldwide. It includes a wide array of components that can have potential implication on health. We have reviewed publications on the impact of coffee on a series of health outcomes. METHODS: Articles published between January 1990 and December 2012 were selected after crossing coffee or caffeine with a list of keywords representative of the most relevant health areas potentially affected by coffee intake. RESULTS: Caffeine, chlorogenic acids and diterpenes are important components of coffee. Tolerance often acts as a modulator of the biological actions of coffee. There is a significant impact of coffee on the cardiovascular system, and on the metabolism of carbohydrates and lipids. Contrary to previous beliefs, the various forms of arterial cardiovascular disease, arrhythmia or heart insufficiency seem unaffected by coffee intake. Coffee is associated with a reduction in the incidence of diabetes and liver disease. Protection seems to exist also for Parkinson's disease among the neurological disorders, while its potential as an osteoporosis risk factor is under debate. Its effect on cancer risk depends on the tissue concerned, although it appears to favor risk reduction. Coffee consumption seems to reduce mortality. CONCLUSION: The information gathered in recent years has generated a new concept of coffee, one which does not match the common belief that coffee is mostly harmful. This view is further supported by the discovery of a series of phyto-components with a beneficial profile. Reasonable optimism needs to be tempered, however, by the insufficiency of the clinical data, which in most cases stem from observational studies.

Endometrial response to concurrent treatment with vaginal progesterone and transdermal estradiol.

ABSTRACT Objective To describe the effect of the intermittent administration of vaginal progesterone and a low-dose estradiol patch on endometrial stability, as assessed by the rate of amenorrhea and endometrial stimulation. Methods This was an open study in which 64 moderately symptomatic, postmenopausal women were treated in the outpatient clinic of our University Hospital for different intervals up to 1 year. The treatment consisted of a combination of patches delivering 25 µg/day estradiol and intravaginal pills containing 100 mg of micronized progesterone. Patches and pills were administered concomitantly in a twice-a-week protocol. The endometrial response was assessed by endovaginal ultrasound completed with suction biopsy when required. Results Both cumulative amenorrhea and no-bleeding rates increased progressively and reached 88.9% and 100.0%, respectively, by the 12th month. Isolated or repetitive episodes of bleeding, bleeding and spotting, or only spotting were reported by three, four, and 12 women, respectively. Endometrial thickness remained unaltered. Endometrium was atrophic in the seven women in whom a biopsy was performed. Conclusion The substantially reduced progestogen load determined by this combination achieved an acceptable incidence of spotting or bleeding when associated with a low estrogenic dose. There was no apparent endometrial stimulation. Additional studies are required to confirm this observation.

Gene-gene interaction between CD40 and CD40L reduces bone mineral density and increases osteoporosis risk in women.

SUMMARY: We have analysed the association of single-nucleotide polymorphisms (SNPs) in CD40 and CD40L genes with bone mineral density (BMD) in our women. Results showed that women with TT genotype for rs1883832 (CD40) and for rs1126535 (CD40L) SNPs displayed reduced BMD and increased risk for osteopenia/osteoporosis. Our data notwithstanding, the results need to be replicated. INTRODUCTION: Recent data have revealed that the CD40/CD40L system can be implicated in bone metabolism regulation. Moreover, we previously demonstrated that rs1883832 in the CD40 gene was significantly associated with BMD and osteoporosis risk. The objective of the present work was to determine whether polymorphisms in CD40 and CD40L genes are associated with BMD and osteoporosis risk. METHODS: We conducted an association study of BMD values with SNPs in CD40 and CD40L genes in a population of 811 women of which 693 and 711 had femoral neck (FN) and lumbar spine (LS) densitometric studies, respectively. RESULTS: Women with the TT genotype for rs1883832 (CD40) showed a reduction in FN-BMD (P = 0.005) and LS-BMD (P = 0.020) when compared with women with the CC/CT genotype. Moreover, we found that rs1126535 (CD40L) was significantly associated with LS-BMD so that women with the TT genotype displayed lower BMD (P = 0.014) than did women with the CC/CT genotype. Interestingly, we have found a strong interaction between polymorphisms in these genes. Thus, women with the TT genotype for both rs1883832 and rs1126535 SNPs (TT + TT women) showed a lower age-adjusted BMD (Z-score) for FN (P = 0.0007) and LS (0.007) after adjusting by years since menopause, body mass index, smoking and menopausal status, densitometer type, hormone replacement therapy (HRT) use and HRT duration and after making the Bonferroni adjustment for multiple comparisons than did the remaining women. Logistic regression analysis adjusted by these covariates showed that TT + TT women had increased risk for FN (odds ratio (OR) = 2.76; P = 0.006) and LS (OR = 2.39; P = 0.020) osteopenia or osteoporosis than did the other women. CONCLUSIONS: Our results suggest that interaction between genetic variants in the CD40 and CD40L genes exerts a role on BMD regulation. Further studies, which we welcome, are needed to replicate these data in other populations.

Progestogens reduce thromboxane production by cultured human endothelial cells.

OBJECTIVES: Progestogens have been poorly studied concerning their roles in endothelial physiology. Prostanoids are vasoactive compounds, such as thromboxane A2, a potent vasoconstrictor, and prostacyclin, a vasodilator. We examined the effects of two progestogens used clinically, progesterone and medroxyprogesterone acetate, on thromboxane A2 production by cultured human umbilical vein endothelial cells (HUVEC) and investigated the role of progesterone receptors and the enzymes involved in production of thromboxane A2 and prostacyclin. METHODS: Cells were exposed to 1-100 nmol/l of either progesterone or medroxyprogesterone acetate, and thromboxane A2 production was measured in culture medium by enzyme immunoassay. Gene expression of prostacyclin synthase and thromboxane synthase was analyzed by quantitative real-time polymerase chain reaction. Expression of prostacyclin synthase protein was analyzed by Western blot. RESULTS: Both progestogens decreased thromboxane A2 release after 24 h. Protein and gene expression of prostacyclin synthase were increased after exposure to both progestogens, without changes in thromboxane synthase expression. These effects induced by progestogens were mediated through progesterone receptors, since they were decreased in the presence of the progesterone receptor antagonist RU486. The cyclo-oxygenase-1 selective inhibitor reduced thromboxane release. CONCLUSION: Progesterone and medroxyprogesterone acetate decreased HUVEC thromboxane release in a progesterone receptor-dependent manner, without changes in thromboxane synthase expression and enhanced prostacyclin synthase gene and protein expression.

Selective estrogen receptor modulators and risk for coronary heart disease.

Coronary heart disease (CHD) is the leading cause of death in women in most countries. Atherosclerosis is the main biological process determining CHD. Clinical data support the notion that CHD is sensitive to estrogens, but debate exists concerning the effects of the hormone on atherosclerosis and its complications. Selective estrogen receptor modulators (SERMs) are compounds capable of binding the estrogen receptor to induce a functional profile distinct from estrogens. The possibility that SERMs may shift the estrogenic balance on cardiovascular risk towards a more beneficial profile has generated interest in recent years. There is considerable information on the effects of SERMs on distinct areas that are crucial in atherogenesis. The complexity derived from the diversity of variables affecting their mechanism of action plus the differences between compounds make it difficult to delineate one uniform trend for SERMs. The present picture, nonetheless, is one where SERMs seem less powerful than estrogens in atherosclerosis protection, but more gentle with advanced forms of the disease. The recent publication of the Raloxifene Use for The Heart (RUTH) study has confirmed a neutral effect for raloxifene. Prothrombotic states may favor occlusive thrombi at sites occupied by atheromatous plaques. Platelet activation has received attention as an important determinant of arterial thrombogenesis. Although still sparse, available evidence globally suggests neutral or beneficial effects for SERMs.

Alterations in the phenotype and function of immune cells in ovariectomy-induced osteopenic mice.

BACKGROUND: Within the last few years, much evidence has been presented on the involvement of the immune system in certain types of bone loss, such as activated T cells in rheumatoid arthritis and in periodontitis. Estrogen deficiency induces bone loss; however, how this deficiency affects the immune system has not been sufficiently studied. METHODS: To evaluate the effects of estrogen withdrawal on the status and functionality of the immune system, mice were ovariectomized or sham-operated, and 5 weeks after surgery, when osteopenia had developed, several parameters were analysed in spleen and in bone marrow. We analysed bone turnover, cell phenotype by flow cytometry, cell function by cell proliferation assays, and the expression of several genes related to the process. RESULTS: Five weeks after ovariectomy, augmented osteoclastogenesis persisted in the bone marrow. In addition, the ovariectomized mice had more B-cells and CD3+ T-cells expressing the receptor activator of NF-kappaB ligand (CD3+/RANKL+). The ovariectomized mice had lower serum alkaline phosphatase activity, a normal amount of T cells, lower percentages of CD11b+ and CD51+ cells in the bone marrow, and a lower serum interferon-gamma level compared with sham-operated controls. CONCLUSIONS: The data suggest that, 5 weeks after ovariectomy, bone turnover remains imbalanced, with increased osteoclastogenesis and a decreased rate of bone formation. Moreover, there is an increase in B-cell formation, with normal and decreased percentages of T cells and myelomonocytic cells (CD11b+), respectively, in the bone marrow. Decreased serum interferon-gamma levels could be involved in the increased osteoclastogenesis found in the present work.

Progestogens stimulate prostacyclin production by human endothelial cells.

BACKGROUND: The effects of progestogens on endothelial physiology are poorly studied. Prostacyclin is a potent vasodilator synthesized by two isoforms of cyclooxygenase (COX) in endothelium. We examined the effects of two clinically used progestogens, progesterone and medroxyprogesterone acetate (MPA), on prostacyclin production by cultured human umbilical vein endothelial cells (HUVEC) and the possible role of progesterone receptors and both COX enzymes. METHODS: Cells were exposed to 1-100 nmol/l of either progesterone or MPA and prostacyclin production was measured in culture medium. RESULTS: Both progestogens significantly increased prostacyclin release in a time- and dose-dependent manner, being higher than control after 24 h. Progesterone and MPA, both at 10 nmol/l, increased mRNA expression and protein content of both COX. All these effects were mediated through progesterone receptor activation, since they were abolished by treatment of cells with the progesterone receptor antagonist RU-486. Selective inhibitors of COX-1 and -2 (SC-560 and NS-398 respectively) reduced basal prostacyclin release, and eliminated increased production in response to progestogens. In combination with estradiol, progestogens had an additive effect without eliminating estradiol-induced prostacyclin production. CONCLUSIONS: Our results support the hypothesis that progesterone and MPA increased HUVEC prostacyclin production in a progesterone receptor-dependent manner, by enhancing COX-1 and COX-2 expression and activities.

Bone turnover markers and PTH levels in surgical versus natural menopause.

In order to assess similarities and differences in women that suffer surgical versus natural menopause, a series of bone, clinical, and biochemical parameters was assayed in a clinical sample of 35 women with surgical menopause and 112 women with natural menopause. Biochemical parameters included hormones [parathyroid hormone (PTH) and the sex steroids estradiol and testosterone] and several markers of bone turnover measured in urine (N-telopeptide and calcium/creatinine ratio) or serum (osteocalcin, total alkaline phosphatase, total and ionic calcium, phosphate, and magnesium). In addition to type of menopause, women were divided by years since menopause (ysm 2). To detect differences and relationships between variables, ANOVA, ANCOVA, and linear regression analyses were used. Only N-telopeptide, one resorption marker, was significantly affected by the variable years since menopause 2 ( P <0.01), but not by type of menopause. The age-corrected level of PTH was significantly decreased in the surgical menopause group ( P < 0.05). In conclusion, type of menopause did not impose significant differences in bone turnover markers. PTH, one powerful resorption hormone, was diminished in surgical menopause.

The effect of oral hormone replacement therapy on lipoprotein profile, resistance of LDL to oxidation and LDL particle size.

OBJECTIVES: To disclose if oral estradiol (E(2)), alone or in combination with natural progesterone (P) or medroxyprogesterone acetate (MPA), may modify the oxidizability of low density lipoprotein (LDL), and if the effect is achieved at physiological dosages. LDL oxidizability was assessed by the resistance to oxidation by copper and by the particle size profile, since small particles have increased oxidation susceptibility. METHODS: Thirty-three women received two consecutive, two-month length doses of 1 and 2 mg/day of oral E(2). They were then randomly assigned to a fourteen-day treatment of 2 mg/day E(2) plus either 300 mg/day P or 5 mg/day MPA. A parallel group of experiments was performed on a pool of baseline plasma, where hormones were added at the desired concentration. Lipoprotein levels, resistance of LDL to oxidation, and LDL particle diameter, were measured at baseline and after each treatment. RESULTS: Estradiol reduced LDL levels and increased high density lipoprotein (HDL) and triglycerides. P abolished these changes, whereas MPA only reversed the increase of HDL. Estradiol protected LDL from oxidation in a dose-dependent manner, although only at pharmacological concentrations (1 microM or higher). Both P and MPA were inert at either physiological or pharmacological concentrations. The size of the LDL particles remained unaffected except under MPA, in which it was reduced. CONCLUSIONS: Estradiol has a protective effect against LDL oxidation, although only at pharmacological dosages. P and MPA did not limit the E(2) action. The size of the LDL particles remained unaltered after each E(2) dose, but MPA, and not P, was associated with a diminution.

Do human concepti have the potential to enter into diapause?

Although there is no direct evidence as to whether human concepti have the potential to enter into diapause before implantation, the possibility that human concepti may be capable of following this developmental pathway if exposed to an appropriate environment cannot be ruled out. Direct evidence remains elusive because of the ethical restraints associated with research activities within this area of knowledge. If conceptus diapause has evolved in primates and persists at the present time despite its apparent limited or no adaptive advantage, artificial induction of diapause in humans may have clinical implications for increasing: (i) the viability of concepti after biopsy, freezing-thawing or any other experimental procedure that tends to decrease cell numbers or division rate of concepti; and (ii) the relatively low implantation rates obtained at the present time after uterine transfer of human concepti fertilized in vitro. Furthermore, conceptus diapause may be a good paradigm to understand the interplay between the different genetic/molecular components of both the conceptus and endometrium at implantation.

Effect of intracellular Ca2+ chelation with the acetoxymethyl ester-derived form of bis(o-aminophenoxy)ethane-N,N,N,N',N'-tetraacetic acid on meiotic division and chromosomal segregation in mouse oocytes.

PURPOSE: Our purpose was to ascertain the effect of intracellular Ca2+ chelation on the chromosomal distribution and segregation of mouse oocytes during maturation in vitro. METHODS: Germinal vesicle oocytes were loaded with the acetoxymethyl ester-derived form of bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Chromosomal distribution and segregation of control and BAPTA-AM-treated metaphase II (MII) oocytes were evaluated at 16 hr, and intracellular ATP content at 0, 1, and 16 hr after BAPTA-AM loading. RESULTS: BAPTA-AM treatment decreased (P < or = 0.05) the potential for in vitro maturation, increased (P < or = 0.0001) the percentage of oocytes displaying an abnormal distribution of metaphase II chromosomes in the meiosis II spindle and aneuploidy, and decreased (P < or = 0.005) the ATP content at 0, 1, and 16 hr of culture compared to the control groups. CONCLUSIONS: These findings raise some concern about any other condition/drug that may directly or indirectly decrease the intracellular Ca2+ concentration in human oocytes.

Two-year prospective, randomized trial comparing an innovative twice-a-week progestin regimen with a continuous combined regimen as postmenopausal hormone therapy.

OBJECTIVE: To compare compliance, symptom control, bleeding patterns, endometrial response, and lipid changes in postmenopausal women treated with transdermal E2 and a regimen of either intermittent or continuous dosing of progestin. DESIGN: Randomized, prospective study. SETTING: Menopausal Outpatient Clinic at an academic tertiary care hospital. PATIENT(S): One hundred women who had reached menopause naturally and had been amenorrheic for at least 1 year. Fifty women were randomly assigned to receive each regimen. INTERVENTION(S): All patients received 50-microg E2 patches and medroxyprogesterone acetate, either 5 mg twice per week or 2.5 mg daily. The bleeding pattern was registered in diary cards. Endometrial status was assessed by vaginal ultrasound and endometrial biopsy. Lipid levels were measured by ELISA. MAIN OUTCOME MEASURE(S): The number of patients who dropped out of the study and the number of days each patient reported spotting or bleeding were recorded. Endometrial thickness was measured and histologic examination of the endometrial tissue was performed. Plasma levels of total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol were measured. RESULT(S): Fourteen women dropped out of the intermittent dosing group and 13 dropped out of the continuous dosing group. Irregular spotting or bleeding occurred at a similar rate in both groups. Biopsies performed at the end of the study showed adequate endometrial stabilization in both groups. No significant changes in lipid levels were detected with either regimen. CONCLUSION(S): Both regimens were similarly effective. The high rate of atrophic endometria suggests the possibility of further reduction of the progestin dose.

Effect of menopause and different combined estradiol-progestin regimens on basal and growth hormone-releasing hormone-stimulated serum growth hormone, insulin-like growth factor-1, insulin-like growth factor binding protein (IGFBP)-1, and IGFBP-3 levels.

OBJECTIVE: To determine the effects of menopause and three different formulations of E2 plus medroxyprogesterone acetate on serum concentrations of basal and growth hormone-releasing hormone (GHRH)-stimulated growth hormone (GH), insulin-like growth factor-1 (IGF-1), insulin-like growth factor binding protein (IGFBP)-1, IGFBP-3, insulin, and C peptide. DESIGN: Prospective, controlled trial. SETTING: Menopausal outpatient clinic at an academic tertiary care hospital. PATIENT(S): Nineteen postmenopausal women with different menopausal ages. Seventeen premenopausal women were included as controls. INTERVENTION(S): Oral estrogen (E2 valerate, 2 mg/d) or transdermal estrogen (50-microg or 100-microg E2 patch) was administered for 8 weeks. Medroxyprogesterone acetate (5 mg/d) was administered during weeks 3, 4, 7, and 8 of each protocol. Blood samples were collected before treatment and after the completion of each protocol from postmenopausal women, and on cycle days 6-8 from premenopausal women. MAIN OUTCOME MEASURE(S): Levels of GH, IGF-1, IGFBP-1, IGFBP-3, insulin, and C peptide. RESULT(S): Basal GH levels were negatively correlated with age in premenopausal women but not in postmenopausal women. The area under the GHRH-induced GH curve decreased in older postmenopausal women after the oral estrogen protocol. Levels of IGF-1 diminished after the oral E2 protocol in postmenopausal women. CONCLUSION(S): The administration of oral, but not transdermal, E2 plus medroxyprogesterone acetate at the usual clinical doses used in postmenopausal women decreased IGF-1 levels and the response of GH to GHRH in older women. No substantial changes were detected in IGFBP-1, IGFBP-3, insulin, or C peptide levels.

Origin and ploidy of multipronuclear zygotes.

Recently, several authors have proposed strategies for correction of triploidy based on the removal of the extra pronucleus at the zygote stage. In the present bioassay, the following were analysed: (1) the different factors that can induce the formation of multipronuclear zygotes in mammals; (2) the different morphological patterns established according to the number of pronuclei and polar bodies that can be observed at the zygote stage and used to distinguish the origin of multipronuclear zygotes; and (3) the pattern of chromosomal segregation during the first mitotic division and ploidy status of the resulting preimplantation embryos. Such an analysis shows that the morphological criterion of counting the number of pronuclei and polar bodies can be misleading and should not be used for ascertaining the origin of tripronuclear zygotes. In addition, although monospermic digynic zygotes have a single sperm centromere, which likely organizes a single bipolar spindle during the first mitotic division, more data on chromosomal distribution and segregation of digynic tripronuclear zygotes should be collected before strategies for correction of triploidy are implemented in humans.

The heparin-glutathione test: an alternative to the hypo-osmotic swelling test to select viable sperm for intracytoplasmic sperm injection.

OBJECTIVE: To evaluate the heparin-glutathione test (HEGLUT) for the selection of viable sperm for intracytoplasmic sperm injection (ICSI). DESIGN: A prospective study. SETTING: Department of Pediatrics, Obstetrics and Gynecology, University of Valencia and Instituto Valenciano de Infertilidad. PATIENT(S): Semen samples from healthy donors and patients with infertility. INTERVENTION(S): Sperm samples were kept in culture for different periods in Ham's F-10 medium supplemented or not supplemented with heparin, reduced glutathione (GSH), or a heparin-GSH mixture. Control and heparin-GSH-treated spermatozoa were injected into hamster oocytes. The HEGLUT and ICSI were performed. MAIN OUTCOME MEASURE(S): Sperm nuclear decondensation, progressive and nonprogressive motility, and male pronucleus formation. RESULT(S): The maximum proportion of sperm nuclear decondensation (28.7%+/-2.1% versus 2.6%+/-0.5% in the control group) was reached after 60 minutes of incubation in the presence of a heparin-GSH mixture. Differences in the percentages of progressive and nonprogressive motility among treatments and times of incubation, although statistically significant, were biologically negligible. No statistically significant differences were observed in the rate of sperm head decondensation (8.2% [4/49] versus 11.1% [6/54]) and male pronucleus formation (18.4% [9/49] versus 22.2% [12/541) after the injection of control and treated spermatozoa into hamster oocytes. CONCLUSION(S): The HEGLUT may offer an alternative to the hypo-osmotic swelling test for the selection of viable sperm for ICSI.

Two distinct two-step ranks of progesterone stimulation after three different levels of oestrogen priming. Effect on induction of luteinizing hormone surges in young and climacteric women.

Age and menopausal status were evaluated as potential modulators of the progesterone action in the initiation of the mid-cycle luteinizing hormone (LH) surge in women. Three distinct levels of oestradiol priming were used, in combination with two different two-step ranks of progesterone stimulation (10/25 mg and 25/50 mg i.m. injections of progesterone in oil, over 2 consecutive days) in two groups of women, ten premenopausals, aged between 18 and 25 years, and 14 postmenopausals, aged between 48 and 57 years. The low, moderate and high levels of oestradiol priming were defined in the premenopausal group by days 5 and 9 of the cycle, and 0.4 mg transdermal oestradiol applied in the early follicular phase respectively. The corresponding situation in the postmenopausal women was defined by the absence of treatment, 0.1 mg transdermal oestradiol, and 0.4 mg transdermal oestradiol respectively. The oestradiol patches were maintained for 5 days, and the first progesterone dose administered on day 3 of treatment. Unambiguous LH surges, detected in serum and urine, were restricted to the protocols using 0.4 mg oestradiol in both groups, with an onset soon after progesterone administration. The surge was higher in the postmenopausal group in serum and urine. The percentage LH increase above baseline, however, was higher in the premenopausal women. The dose of progesterone did not result in any changes in pituitary LH release. Therefore, the oestradiol threshold for the progesterone stimulatory effect on LH release was similar in both groups. The postmenopausal women did not yield defective LH surges when adequately primed with oestradiol and progesterone.

Antioxidant therapy counteracts the disturbing effects of diamide and maternal ageing on meiotic division and chromosomal segregation in mouse oocytes.

This study aims (i) to ascertain whether oxidative-stress-induced disturbances in chromosomal distribution in the metaphase-II spindle of mouse oocytes can be counteracted by supplementing culture medium with antioxidants; and (ii) to determine whether supplemental intake of antioxidants neutralizes the disturbing effects of maternal ageing on segregation of chromosomes during the first meiotic division and distribution of chromosomes in the metaphase-II spindle. (i): Germinal vesicle oocytes from unstimulated 10-12 week old mice were matured in vitro in the presence or absence of diamide and/or dithiothreitol. Metaphase-II oocytes were fixed and stained with 4',6-diamidino-2-phenylindole (DAPI) to detect abnormalities in chromosomal distribution. The percentage of oocytes arrested in metaphase I (12.9% vs 28.4%; P < or = 0.05) or with a telophase-I chromosome configuration (0.0% vs 8.2%; P < or = 0.0005) was decreased in diamide-DTT-treated oocytes when compared to diamide-treated oocytes. (ii): Mice were fed, from the first day of weaning until their death, a diet supplemented or not with an antioxidant mixture of vitamin C and vitamin E. Ovulated oocytes were fixed and stained with DAPI or C-banded for chromosome analysis. The percentage of abnormal (chromosome scattering and nulloploidy) or asynchronous (anaphase I or telophase I) oocytes was 2.7-fold higher in controls than in females fed an antioxidant diet (24.4% vs 8.9%, P < or = 0.05). Furthermore, the percentage of aneuploidy (2.2% vs 0.0%; P < or = 0.01) and diploidy (5.8% vs 1.7%; P < or = 0.05) was significantly higher in controls than in females fed an antioxidant diet. These findings support Tarin's oxidative stress hypothesis of aneuploidy and have clinical implications for preventing both laboratory-induced and maternal-age-associated aneuploidy in human beings.

Dithiothreitol prevents age-associated decrease in oocyte/conceptus viability in vitro.

The present study was designed to ascertain whether the negative effects on reproductive potential of post-ovulatory ageing in vitro of oocytes can be prevented by antioxidant therapy. Mouse metaphase II (MII) oocytes were aged in vitro for 12 h prior to insemination in the presence of varying concentrations of L-ascorbic acid, 6-methoxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), L-cystine dihydrochloride, ethylenediaminetetraacetic acid (EDTA), beta-mercaptoethanol and DL-dithiothreitol (DTT). In-vitro ageing of oocytes was associated with lower fertilization rate, higher proportion of concepti exhibiting cellular fragmentation at 24 h post-insemination and lower percentage of concepti reaching the blastocyst stage. Ascorbic acid, Trolox and EDTA had no effect on cellular fragmentation or potential of oocytes for development. However, the probability of an oocyte reaching the blastocyst stage was decreased (P < or = or = 0.05) in oocytes incubated in the presence of L-cystine (50 and 500 microM) and beta-mercaptoethanol (5, 50 and 500 microM) when compared to control aged oocytes. Age-associated cellular fragmentation at 24 h post-insemination was partially prevented (P < or = 0.05) by incubating oocytes in the presence of beta-mercaptoethanol (500 microM). DTT (50 and 500 microM) increased (P < or = 0.05) fertilization rate and number of cells at 81 h post-insemination to levels similar to those exhibited by control oocytes. Furthermore, both age-associated fragmentation at 24 h post-insemination (P < or = 0.05) and decreased potential of oocytes for development to the blastocyst stage (P < or = 0.05) were prevented, at least in part, by culturing oocytes in the presence of DTT (50 microM). Although the mechanism by which DTT exerts its beneficial effects on aged oocytes remains to be elucidated, it may protect oocytes by preventing oxidation of free thiol groups and/or altering a redox-independent signalling pathway that mediates cellular fragmentation and death.

Dietary antioxidant supplementation did not affect declining sperm function with age in the mouse but did increase head abnormalities and reduced sperm production.

The present study aims to ascertain whether dietary administration of a mixture of vitamins C and E may prevent the negative effects of paternal age on male fertility variables in the mouse. Experimental males were fed a standard diet supplemented with either a low or high dose of vitamins C and E. Oocytes enclosed in cumulus masses were inseminated using a 2 x 2 factorial design established according to whether males were young (3-4 months of age) or aged (22-24 months of age) and whether they were fed a control or antioxidant diet. Aged males showed a significant decrease in number of spermatozoa/mg epididymis when compared to young males. Dietary supplementation with low doses of vitamins C and E did not have any effect on sperm quality, fertilization and conceptus development in vitro. However, high doses of dietary vitamins C and E decreased the number of spermatozoa/mg epididymis, and increased the percentage of spermatozoa with misshapen heads distended in the distal part. These findings suggest that dietary supplementation with pharmacological doses of vitamins C and E may disturb spermatogenesis. The mechanism involved may be associated with either the antioxidant properties of vitamins C and E or their inhibitory action on steroidogenesis by Leydig and/or Sertoli cells.