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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.

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.

Background: Some epidemiological evidence tentatively suggests that children born to older parents may have lower intellectual development and maturity than children whose parents are younger. This study aims to analyse the long-term effects of delayed motherhood in mice on postnatal development and behavioural traits later in life., Methods: Hybrid females, either at the age of 10 weeks or 51 weeks, were individually housed with a randomly selected 12-14 week old hybrid male. After a postweaning resting period of 1 week, dams were caged again with a new randomly selected 12-14 week old male. This sequence of events was repeated until old females reached the end of their reproductive life., Results: Delayed motherhood in mice not only had negative effects on reproductive potential but also on preweaning development of offspring as evidenced by higher mortality, retarded sensorimotor integration and lower body weights as well as on behavioural traits of young adult offspring including decreased spontaneous motor activity, lower step-through latencies in the retention trial of a passive avoidance behaviour test, and no changes in escape latencies throughout five daily sessions in a Morris water maze test., Conclusion: Advanced maternal age at conception may influence preweaning development and learning capacity of offspring in the mouse model.

This article aims to throw light on the controversial topic of whether women have a 'heat' period within their menstrual cycle. The majority of publications in this field report, in addition to a periovulatory peak, no changes at all or even rises in male- and female-initiated sexual activity, woman's sexual desire, autosexual activity and sexual arousability, and interpersonal sexual activities during the mid-follicular and late luteal phases. The lack of a distinct pattern of women's sexual behaviour across the menstrual cycle may be explained by the interplay between cyclical endocrine fluctuations and many psychological, social, cultural and environmental factors, as well as the methodological shortcomings associated with menstrual cycle research. However, studies focused on cycling changes in women's olfactory and visual perception show that, in comparison with women at other phases of the menstrual cycle, women at mid-cycle exhibit increased sexual motivation that biases recognition performance towards objects with a sexual meaning, evaluate the unattractive sweat substance androstenone as more pleasant, and display enhanced preference for the odour and face shape of masculinized, physically attractive and symmetric men. On the other hand, men find the scent of women at mid-cycle more pleasant and sexually attractive than during the luteal phase.

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.

The present study aims to define, characterize and compare the long-term effects on offspring of delayed parenthood. Data published so far on this topic show that maternal and paternal ageing may affect offspring by different mechanisms. Delayed motherhood is characterized by increased probability of obstetric complications and/or fetal and perinatal problems which, in turn, may increase the risks of mortality and morbidity in newborns and later life. Furthermore, maternal ageing is distinguished by a decreased ratio of male to female infants and higher odds of conceiving a trisomic child and/or an individual suffering from mitochondrial DNA disorders. In contrast, delayed fatherhood is associated with higher risks of conceiving an individual suffering from new inheritable-mutation disorders. The different pattern of disease in offspring associated with maternal and paternal ageing may be explained, among other factors, by the fact that (i) oocytes of middle-aged women may suffer oxidative stress because their mitochondria produce higher amounts of reactive oxygen species; (ii) diplotene oocytes and to a lesser extent metaphase I and II oocytes have an efficient DNA repair system which is essentially independent of maternal age; and (iii) mitochondria are transmitted to the next generation along the matrilineal line. Moreover, (i) the activities of antioxidant enzymes within the seminal plasma and spermatozoa from older men may be reduced and so spermatozoa may be more vulnerable to mutational changes than spermatozoa from younger men; and (ii) late spermatids, and immature and mature spermatozoa do not have a DNA repair system.

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.

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.

The inheritance, replication and perpetuation of the sperm centriole in the early human embryo are reported. Both normal monospermic and abnormal dispermic embryos (n = 127) were examined by transmission electron microscopy. Centrioles were traced from fertilization to the hatching blastocyst stage. The sperm proximal centriole is introduced into the oocyte at fertilization and remains attached to the expanding spermhead during sperm nuclear decondensation, as it forms the male pronucleus. A sperm aster is initially formed after the centriole duplicates at the pronuclear stage. At syngamy, centrioles occupy a pivotal position on opposite spindle poles, when the first mitotic figure is formed. Bipolar spindles were found in the majority of embryos, while tripolar spindles were seen in four dispermic embryos at syngamy. Two single centrioles were detected at two poles of two tripolar spindles, while two additional centrioles were located on the sides of a bipolar spindle of a dispermic embryo. Sperm tails were detected near spindle poles at syngamy and in later embryos. Typical centrioles showing the characteristic pin-wheel organization of nine triplets of microtubules were evident. During centriolar replication, the daughter centriole grows laterally from the parent and gradually acquires pericentriolar material (PCM). The two centrioles are surrounded by a halo of electron-dense PCM, which nucleates microtubules, thus making it a typical centrosome. The usual alignment of diplosomes at right angles to each other was maintained. Centrioles were detected at all stages of embryonic cleavage from the 1-cell through 8-cell stages, right up to the hatching blastocyst stage. They were closely associated with nuclei at interphase, when they were often replicating, and were prominently located at spindle poles during the first four cell cycles. In blastocysts, they were detected in trophoblast, embryoblast and endoderm cells respectively. It is evident that the sperm centrosome is the functional active centrosome in human, while the female is inactive but may contribute some centrosomal material to the zygote centrosome. It is very likely that the paternal centriole is the ancestor of the centrioles in fetal and adult somatic cells.

The present study aims to ascertain whether sex selection may be inadvertently performed in human in-vitro fertilization (IVF) and embryo transfer (IVF-embryo transfer) programmes when selecting for high quality embryos (those with the fastest cleaving rates and/or the best morphology) at the fresh transfer cycle. All patients entering into the study were treated with gonadotrophins after pituitary suppression with gonadotrophin-releasing hormone agonists (GnRHa) and had intrauterine embryo transfer on day 2 post-insemination. These patients were retrospectively divided into three groups according to whether the difference in mean number of cells between embryos transferred and all embryos available for transfer in a given cycle was less than (negative selection), equal to (no selection) or greater (positive selection) than zero. In cycles resulting in singleton births, the sex ratio of the resulting babies was significantly (P < or = 0.005) shifted toward the female (88.8%) and to the male (90.0%) in the negative and positive selection groups respectively. No shift in sex ratio was observed in cycles resulting in multiple births. Maternal age was another independent factor affecting sex ratio at birth. Sex ratio was significantly (P < or = 0.05) skewed in favour of males (62.7%) and females (71.4%) in women < 35 and > or = 35 years of age respectively. Maternal age, number of embryos transferred and the event of selecting or not selecting the slowest cleaving embryos for transfer were entered automatically in a three-group discriminant model for distinguishing cycles resulting in only boys, both boys and girls, and only girls. These data suggest that (i) sex selection may be inadvertently performed in IVF-embryo transfer programmes when selecting for high quality embryos at the fresh transfer cycles; (ii) human endometria may be favourable, indifferent or hostile to either fast cleaving or slow cleaving embryos depending on maternal age; and (iii) "natural' sex selection may be performed for social, psychological or medical reasons.

A general model is put forward to explain the mechanism by which age-associated aneuploidies are produced. This is based on the free radical theory of ageing, which assumes a rise in oxidative stress with age. It is proposed that determination of indicators of oxidative stress in oocytes from various sources could be a first step in the testing of this hypothesis.

This review aims to analyse and compare the results to date of subzonal insemination (SUZI), partial zona dissection (PZD) and intracytoplasmic sperm injection (ICSI) to evaluate critically whether it is now possible to replace SUZI and PZD by ICSI. It appears that ICSI is a much more efficient assisted reproduction technique than SUZI and PZD for resolving cases of severe male infertility and/or repeated failure of conventional in-vitro fertilization (IVF). For ICSI compared with SUZI and PZD, fertilization (49.4, 17.7 and 16.8% respectively), percentage of patients reaching embryo transfer (91.0, 55.1 and 23.3% respectively), percentage of transfers performed with two or three embryos (83.3% ICSI and 39.3% SUZI), pregnancy rate per embryo replacement (28.2, 18.7 and 16.5% respectively) and pregnancy rate per oocyte retrieval (24.8, 10.3 and 3.8% respectively) are all improved. In addition, cases of severely impaired semen characteristics, which were condemned to infertility for life with conventional IVF, SUZI or PZD, can now be treated and resolved efficiently with ICSI.

There is conflicting clinical evidence suggesting a positive role for gonadotrophin-releasing hormone analogues (GnRHa) on implantation in humans. This potential effect was evaluated in this study taking the oocyte donation programme as a model. Patients were randomly allocated into one of the two treatment groups: group I received simultaneous treatment with GnRHa and steroids, and group II only received exogenous steroid replacement. An analysis of the donors and semen quality showed similarity between recipient groups. There was no significant difference between groups in the number and quality of embryos replaced, clinical pregnancy and implantation rates. In summary, using a model in which the endometrium can be analysed independently of the embryos, the results suggest that GnRHa are neither effective nor detrimental for embryo implantation in humans.

The present study aimed to evaluate whether ascorbate, a reactive oxygen species (ROS) scavenger, can improve fertilization and development of human embryos in vitro when added to the simple salt solution human tubal fluid (HTF) or the complex tissue culture medium Ham's F-10, which contains iron and copper in its formulation. Human oocytes, spermatozoa and embryos from 83 infertile IVF couples were randomly allocated and cultured in the presence or absence of 62.5 microM ascorbate in HTF medium (39 couples) or Ham's F-10 medium (44 couples). No significant effect of ascorbate on fertilization, number of cells and embryo grade per embryo on days 2 and 3 after insemination, or percentage of embryos showing developmental block on day 3 (those embryos that were still at the 2-cell stage) was observed when data were analysed together or divided into several groups according to the cause of infertility, quality of semen sample used for insemination and women's age in either of the two media tested. Despite these results, a positive effect of ascorbate on fertilization and embryo development in vitro cannot be totally ruled out until the effects of other, non-physiological concentrations of ascorbate and longer-term embryo cultures (to the blastocyst stage) have been tested.

A retrospective analysis of our in-vitro fertilization (IVF) and oocyte donation programmes was carried out in order to gain clinical knowledge of the factors involved in the aetiology of the endometriosis-associated infertility. Comparison between the IVF outcomes from 96 cycles in 78 patients with tubal infertility and from 96 cycles in 59 women with endometriosis indicates that endometriosis patients have a poor IVF outcome in terms of reduced pregnancy rate per cycle (P < 0.0004), reduced pregnancy rate per transfer (P < 0.002), and reduced implantation rate (P < 0.003). The analysis of patients undergoing oocyte donation for different reasons, including low response with or without endometriosis, showed that patients with this disease have the same chances of implantation and pregnancy as other recipients when the oocytes came from donors without known endometriosis. However, when the results of oocyte donation were classified according to the origin of the oocytes donated, patients who received embryos derived from endometriotic ovaries showed a significantly (P < 0.05) reduced implantation rate as compared to the remaining groups. Taken together, all these observations suggest that infertility in endometriosis patients may be related to alterations within the oocyte, which in turn result in embryos with decreased ability to implant.

The purpose of the present study was to analyse daily measurements of human chorionic gonadotrophin (HCG) in in-vitro fertilization (IVF) cycles and to reproduce the effects of HCG in vitro using human granulosa-luteinized cells from the same patients. The study population consisted of nine women undergoing IVF because of tubal infertility in whom blood was drawn every 24 h from the day of the ovulatory dose of HCG (10,000 IU) until 6 days after ovum pick-up. Granulosa-luteal cells from the follicular aspirates were collected and cultured in vitro up to 6 days in the presence of increasing concentrations (0, 0.01, 0.1, 1.0 and 100.0 IU/ml) of HCG. Serum progesterone and HCG in vivo as well as progesterone accumulation in vitro on days 2, 4 and 6, were the main outcome measures. Maximum HCG concentrations (0.25 IU/ml) were reached the day before ovum pick-up, and continuously decreased until day 6 after ovum retrieval. HCG did not stimulate progesterone production in vitro at any dose tested until day 6 after ovum pick-up. Then, 0.01 IU/ml resulted significantly (P < 0.05) stimulatory compared to controls, while 1.0 IU/ml was inhibitory (P < 0.05). It is concluded that HCG supplementation in an IVF cycle is unnecessary until day 6 after ovum pick-up. On day 6, progesterone production is stimulated with very low concentrations of HCG.

Evidence has accumulated in mammals suggesting a positive role for epidermal growth factor (EGF) as an inducer of oocyte maturation. The potential use of EGF as inducer of cytoplasmic and nuclear maturation was tested in women with > 10 oocytes retrieved in in-vitro fertilization (IVF), since we have previously observed that such oocytes are immature. Oocytes from 17 high responders were randomly allocated to one of the three treatment groups upon retrieval: control receiving no EGF (n = 93), 1.0 ng/ml EGF (n = 92) and 10.0 ng/ml EGF (n = 77) for 6 h before insemination. The rates of fertilization were respectively 54.6, 59.0, and 46.1%, suggesting that EGF is not effective at this maturational stage after this length of exposure. Embryo development was further analysed by the appearance of the embryos under the dissecting microscope and the number of blastomeres developed 48 h after insemination. No difference between groups was observed considering the number of blastomeres developed. However, embryos derived from oocytes treated with 10 ng/ml EGF displayed a worse appearance under the microscope. It is concluded that a 6 h incubation with EGF does not seem to affect cytoplasmic maturation in oocytes obtained after gonadotrophin treatment, as ascertained by the rate of fertilization following oocyte insemination.

Regression analysis of the proportion of unfertilized oocytes on the number of oocytes retrieved per patient was applied to three different ovarian stimulation protocols in order to establish the relativity of the concept 'high responder to gonadotrophins' for in-vitro fertilization (IVF) patients. After fitting the data to the model: probit(proportion of unfertilized oocytes) + 5 = intercept + B Log10(oocytes retrieved per patient), women with a number of oocytes retrieved greater than or equal to the value necessary to obtain 50% fertilization were defined as high responders. Exogenous gonadotrophin stimulation which was commenced after complete suppression of ovarian activity by a gonadotrophin-releasing hormone analogue (GnRHa) (long protocol) resulted in a significantly higher number of oocytes retrieved (10.15) to obtain 50% fertilization compared to a short protocol (6.84) (exogenous stimulation began 2 days after the GnRHa administration). Women stimulated without use of a GnRHa showed an intermediate response. Implantation, pregnancy and miscarriage rates showed no difference between low-moderate and high responders. These results demonstrate the relativity of the concept 'high responder to gonadotrophins' and indicate that the drawback of low fertilization in high responders could be balanced by the high number of oocytes retrieved per patient (and available embryos for transfer) and the selection of the best embryos for transfer.

Several reports have shown a relatively high incidence of chromosome anomalies in human inseminated-unfertilized oocytes from infertile women. In the present study, cytogenetic analysis was attempted in 73 human oocytes from 17 fertile women in order to establish the incidence of chromosome anomalies in the fertile population and to compare this with the incidence in inseminated-unfertilized oocytes from infertile women. Of 56 oocytes that could be analysed, 42 oocytes were haploid, 12 were hypohaploid and two exhibited fragmented chromosomes. The low rate of chromosome anomalies (3.6%) found in this population suggests that there is natural selection at fertilization against diploid oocytes and oocytes with fragmented chromosomes. This result also questions the high incidence of chromosome anomalies found in previous reports using inseminated-unfertilized oocytes.