Mating induces early transcriptional response in the rat endosalpinx: the role of TNF and RA.

During mating, males provide not only the spermatozoa to fertilize the oocyte but also other stimuli that are essential for initiating and maintaining the reproductive programme in females. In the mammalian oviduct, mating regulates sperm storage, egg transport, fertilization, early embryonic development, and oestradiol metabolism. However, the main molecules underlying these processes are poorly understood. Using microarray analyses, we identified 58 genes that were either induced or repressed by mating in the endosalpinx at 3 h post-stimulus. RT-qPCR confirmed that mating downregulated the expression of the Oas1h and Prim1 genes and upregulated the expression of the Ceacam1, Chad, Chst10, Slc5a3 and Slc26a4 genes. The functional category 'cell-to-cell signalling and interaction' was over-represented in this gene list. Network modelling identified TNF and all-trans retinoic acid (RA) as upstream regulators of the mating-induced transcriptional response, which was confirmed by intraoviductal injection of TNF or RA in unmated rats. It partially mimicked the transcriptional effect of mating in the rat endosalpinx. Furthermore, mating decreased RA levels in oviductal fluid, and RA-receptor-gamma (RARG) exhibited a nuclear location in oviductal epithelium in both unmated and mated rats, indicating RA-RARG transcriptional activity. In conclusion, the early transcriptional response regulated by mating in the rat endosalpinx is mediated by TNF and RA. These signalling molecules regulate a cohort of genes involved in 'cell-to-cell signalling and interactions' and merit further studies to understand the specific processes activated in the endosalpinx to sustain the events that occur in the mammalian oviduct early after mating.

Effect of single post-ovulatory administration of mifepristone (RU486) on transcript profile during the receptive period in human endometrium.

Progesterone regulates uterine function during the luteal phase and is essential for the acquisition of endometrial receptivity. The objective of the present study was to identify endometrial transcripts whose expression is altered during the window of implantation after the administration of 200 mg of the antiprogestin mifepristone, 48 h after the LH peak (LH+2, LH+0=LH peak), and to determine the relationship of these transcripts with those regulated during the acquisition of receptivity. Endometrial samples were obtained in LH+7 from seven women of proven fertility, each one contributing with one cycle treated with placebo and another with mifepristone. Additionally, endometrial samples were obtained in LH+2 and LH+7 during a single untreated spontaneous cycle from seven normal fertile women as a reference. DNA microarrays were used to identify transcripts significantly regulated (defined as ≥ 2.0-fold change with false discovery rate below 1% using t-test) with the administration of mifepristone vs placebo, or during the transition from pre-receptive to receptive (LH+2 vs LH+7). Approximately 2000 transcripts were significantly regulated in both comparisons (mifepristone vs placebo and LH+2 vs LH+7), but only 777 of them were coincident and displayed opposite regulation except for 25. The mRNA level for eight selected genes regulated by mifepristone was confirmed by real-time RT-PCR. We conclude that not all changes in endometrial transcript levels occurring in the transition from LH+2 to LH+7 seem to be regulated by the progesterone receptor and ∼ 37% of the genes whose transcript levels changed by effect of mifepristone could be associated with the acquisition of receptivity.

Endometrial gene expression reveals compromised progesterone signaling in women refractory to embryo implantation.

BACKGROUND: Endometrial function is essential for embryo implantation. The aim of this study was to analyze gene expression profiles from individual endometrial samples obtained from women with repeated implantation failure after IVF in oocyte donation programs. METHODS: Seventeen volunteers were recruited: women who had previously participated as recipients in oocyte donation cycles and repeatedly exhibited implantation failure (Group A, study group, n = 5) or had at least one successful cycle (Group B, control group, n = 6) and spontaneously fertile women (Group C, normal fertility group, n = 6). An endometrial cycle was induced with exogenous estradiol (E2) and progesterone (P) and an endometrial sample was collected on the seventh day of P treatment. RESULTS: Transcriptome analysis showed 82 genes with consistent differential gene expression when comparing A vs. B and A vs. C. One hundred transcripts differentially expressed in group A vs. B have been shown to be regulated by P, suggesting compromised P signaling in the endometrium. The P receptor (PR) mutation PROGINS was not detected in women from group A. Semi-quantitation of immunoreactive PRA/B, PRB and Sp1 (a transcription factor related to P signaling) in paraffin-embedded endometrial sections, did not show statistically significant differences amongst groups. However immunostaining glycodelin was significantly decreased in endometrial samples from group A. CONCLUSIONS: We conclude that some cases of repeated implantation failure could be associated with an aberrant gene expression profile. Compromised P signaling might be the underlying mechanism for such endometrial gene expression deregulation in women with repeated implantation failure.

In vitro assessment of some sperm function following exposure to levonorgestrel in human fallopian tubes.

BACKGROUND: The mechanism of action of levonorgestrel (LNG) as emergency contraception (EC) remains a subject of debate and its effect on sperm function has been only partially explained. The aim of this study was to assess whether LNG at a similar dose to those found in serum following oral intake for EC could affect spermatozoa when exposed to human fallopian tubes in vitro. METHODS: Fifteen mini-laparotomies were performed, the side on which ovulation occurred was recorded, and both tubes were removed and perfused with a suspension containing 1 × 10(6) motile spermatozoa, with or without LNG. Following 4-hour incubation, the tubes were sectioned to separate the isthmus and the ampulla. Each segment was flushed and the material was evaluated to quantify the number of motile sperm, the number of spermatozoa adhering to the oviductal epithelium and the acrosome reaction (AR) rate. RESULTS: The addition of LNG did not significantly alter the number of recovered motile spermatozoa either at the isthmus or at the ampulla, nor did it have any effect on the number of recovered spermatozoa adhered to the human tubal epithelium. Furthermore, LNG did not affect the AR rate. No significant differences were found even when the side on which ovulation occurred was taken into account. CONCLUSIONS: In a similar dose to that observed in serum following oral intake for EC, LNG had no effect on the number of motile spermatozoa recovered from the human fallopian tubes in vitro, on their adhesion to the tubal epithelium, distribution or AR rate. The possible effect of LNG as EC on sperm function remains poorly understood.

Bioinformatic detection of E47, E2F1 and SREBP1 transcription factors as potential regulators of genes associated to acquisition of endometrial receptivity.

BACKGROUND: The endometrium is a dynamic tissue whose changes are driven by the ovarian steroidal hormones. Its main function is to provide an adequate substrate for embryo implantation. Using microarray technology, several reports have provided the gene expression patterns of human endometrial tissue during the window of implantation. However it is required that biological connections be made across these genomic datasets to take full advantage of them. The objective of this work was to perform a research synthesis of available gene expression profiles related to acquisition of endometrial receptivity for embryo implantation, in order to gain insights into its molecular basis and regulation. METHODS: Gene expression datasets were intersected to determine a consensus endometrial receptivity transcript list (CERTL). For this cluster of genes we determined their functional annotations using available web-based databases. In addition, promoter sequences were analyzed to identify putative transcription factor binding sites using bioinformatics tools and determined over-represented features. RESULTS: We found 40 up- and 21 down-regulated transcripts in the CERTL. Those more consistently increased were C4BPA, SPP1, APOD, CD55, CFD, CLDN4, DKK1, ID4, IL15 and MAP3K5 whereas the more consistently decreased were OLFM1, CCNB1, CRABP2, EDN3, FGFR1, MSX1 and MSX2. Functional annotation of CERTL showed it was enriched with transcripts related to the immune response, complement activation and cell cycle regulation. Promoter sequence analysis of genes revealed that DNA binding sites for E47, E2F1 and SREBP1 transcription factors were the most consistently over-represented and in both up- and down-regulated genes during the window of implantation. CONCLUSIONS: Our research synthesis allowed organizing and mining high throughput data to explore endometrial receptivity and focus future research efforts on specific genes and pathways. The discovery of possible new transcription factors orchestrating the CERTL opens new alternatives for understanding gene expression regulation in uterine function.

Participation of the oviductal s100 calcium binding protein G in the genomic effect of estradiol that accelerates oviductal embryo transport in mated rats.

BACKGROUND: Mating changes the mechanism by which E2 regulates oviductal egg transport, from a non-genomic to a genomic mode. Previously, we found that E2 increased the expression of several genes in the oviduct of mated rats, but not in unmated rats. Among the transcripts that increased its level by E2 only in mated rats was the one coding for an s100 calcium binding protein G (s100 g) whose functional role in the oviduct is unknown. METHODS: Herein, we investigated the participation of s100 g on the E2 genomic effect that accelerates oviductal transport in mated rats. Thus, we determined the effect of E2 on the mRNA and protein level of s100 g in the oviduct of mated and unmated rats. Then, we explored the effect of E2 on egg transport in unmated and mated rats under conditions in which s100 g protein was knockdown in the oviduct by a morpholino oligonucleotide against s100 g (s100 g-MO). In addition, the localization of s100 g in the oviduct of mated and unmated rats following treatment with E2 was also examined. RESULTS: Expression of s100 g mRNA progressively increased at 3-24 h after E2 treatment in the oviduct of mated rats while in unmated rats s100 g increased only at 12 and 24 hours. Oviductal s100 g protein increased 6 h following E2 and continued elevated at 12 and 24 h in mated rats, whereas in unmated rats s100 g protein increased at the same time points as its transcript. Administration of a morpholino oligonucleotide against s100 g transcript blocked the effect of E2 on egg transport in mated, but not in unmated rats. Finally, immunoreactivity of s100 g was observed only in epithelial cells of the oviducts of mated and unmated rats and it was unchanged after E2 treatment. CONCLUSIONS: Mating affects the kinetic of E2-induced expression of s100 g although it not changed the cellular localization of s100 g in the oviduct after E2 . On the other hand, s100 g is a functional component of E2 genomic effect that accelerates egg transport. These findings show a physiological involvement of s100 g in the rat oviduct.

Suppression of follicular rupture with meloxicam, a cyclooxygenase-2 inhibitor: potential for emergency contraception.

BACKGROUND: There is evidence that cyclooxygenase-2 (COX-2) inhibitors can prevent or delay follicular rupture. COX-2 inhibitors, such as meloxicam, may offer advantages over emergency contraception with levonorgestrel, such as extending the therapeutic window for up to 24 h. We assessed the effect of meloxicam administered in the late follicular phase upon ovulation in women. MATERIALS AND METHODS: This was a single center, double blind, crossover study designed to assess the effects in 27 eligible women (18-40 years old, surgically sterilized with regular menstrual cycles) of meloxicam, 15 or 30 mg/day, administered orally for five consecutive days during the late follicular phase, starting when the leading follicle reached 18 mm diameter. Volunteers underwent two treatment cycles separated by one resting cycle, with randomization to dose sequence. Main outcomes were follicular rupture; serum LH, progesterone and estradiol (E2) levels; and incidence of adverse events. RESULTS: Twenty-two volunteers completed the study. There were no differences between meloxicam doses in menstrual cycle length. Dysfunctional ovulation was observed in 11/22 (50%) cycles treated with 15 mg/day and 20/22 (90.9%) cycles with 30 mg/day (P = 0.0068). All women had normal luteal phase progesterone levels; mean maximal values +/- SEM were 42 +/- 4.1 and 46.8 +/- 2.6 nmol/l for 15 and 30 mg/day groups, respectively. There were no serious adverse events, and no changes in LH and E2 levels or in cycle length. CONCLUSIONS: Meloxicam 30 mg given for five consecutive days in the late follicular phase is safe, effective and may be an alternative form of emergency contraception.

A non-genomic signaling pathway shut down by mating changes the estradiol-induced gene expression profile in the rat oviduct.

Estradiol (E(2)) accelerates oviductal egg transport through intraoviductal non-genomic pathways in unmated rats and through genomic pathways in mated rats. This shift in pathways has been designated as intracellular path shifting (IPS), and represents a novel and hitherto unrecognized effect of mating on the female reproductive tract. We had reported previously that IPS involves shutting down the E(2) non-genomic pathway up- and downstream of 2-methoxyestradiol. Here, we evaluated whether IPS involves changes in the genomic pathway too. Using microarray analysis, we found that a common group of genes changed its expression in response to E(2) in unmated and mated rats, indicating that an E(2) genomic signaling pathway is present before and after mating; however, a group of genes decreased its expression only in mated rats and another group of genes increased its expression only in unmated rats. We evaluated the possibility that this difference is a consequence of an E(2) non-genomic signaling pathway present in unmated rats, but not in mated rats. Mating shuts down this E(2) non-genomic signaling pathway up- and downstream of cAMP production. The Star level is increased by E(2) in unmated rats, but not in mated rats. This is blocked by the antagonist of estrogen receptor ICI 182 780, the adenylyl cyclase inhibitor SQ 22536, and the catechol-O-methyltransferase inhibitor, OR 486. These results indicate that the E(2)-induced gene expression profile in the rat oviduct differs before and after mating, and this difference is probably mediated by an E(2) non-genomic signaling pathway operating on gene expression only in unmated rats.

Mating changes the subcellular distribution and the functionality of estrogen receptors in the rat oviduct.

BACKGROUND: Mating changes the mode of action of 17beta-estradiol (E2) to accelerate oviductal egg transport from a nongenomic to a genomic mode, although in both pathways estrogen receptors (ER) are required. This change was designated as intracellular path shifting (IPS). METHODS: Herein, we examined the subcellular distribution of ESR1 and ESR2 (formerly known as ER-alpha and ER-beta) in oviductal epithelial cells of rats on day 1 of cycle (C1) or pregnancy (P1) using immunoelectron microscopy for ESR1 and ESR2. The effect of mating on intraoviductal ESR1 or ESR2 signaling was then explored comparing the expression of E2-target genes c-fos, brain creatine kinase (Ckb) and calbindin 9 kDa (s100g) in rats on C1 or P1 treated with selective agonists for ESR1 (PPT) or ESR2 (DPN). The effect of ER agonists on egg transport was also evaluated on C1 or P1 rats. RESULTS: Receptor immunoreactivity was associated with the nucleus, cytoplasm and plasma membrane of the epithelial cells. Mating affected the subcellular distribution of both receptors as well as the response to E2. In C1 and P1 rats, PPT increased Ckb while both agonists increased c-fos. DPN increased Ckb and s100g only in C1 and P1 rats, respectively. PPT accelerated egg transport in both groups and DPN accelerated egg transport only in C1 rats. CONCLUSION: Estrogen receptors present a subcellular distribution compatible with E2 genomic and nongenomic signaling in the oviductal epithelial cells of C1 and P1 although IPS occurs independently of changes in the distribution of ESR1 and ESR2 in the oviductal epithelial cells. Mating affected intraoviductal ER-signaling and induced loss of functional involvement of ESR2 on E2-induced accelerated egg transport. These findings reveal a profound influence on the ER signaling pathways exerted by mating in the oviduct.

Differences in the endometrial transcript profile during the receptive period between women who were refractory to implantation and those who achieved pregnancy.

BACKGROUND: Gene expression profiling of normal receptive endometrium has been characterized, but intrinsic defects in endometrial gene expression associated with implantation failure have not been reported. METHODS: Women who had previously participated as recipients in oocyte donation cycles and repeatedly exhibited implantation failure (Group A, study group) or had at least one successful cycle (Group B, control group) and spontaneously fertile women (Group C, normal fertility group) were recruited. All were treated with exogenous estradiol and progesterone to induce an endometrial cycle, and an endometrial biopsy was taken on the seventh day of progesterone administration. RNA from each sample was analysed by cDNA microarrays to identify differentially expressed genes between groups. RESULTS: 63 transcripts were differentially expressed (>or=2-fold) between Groups A and B, of which 16 were subjected to real time RT-PCR. Eleven of these were significantly decreased in Group A with regard to Groups B and C. Among the dysregulated genes were MMP-7, CXCR4, PAEP and C4BPA. CONCLUSIONS: Repeated implantation failure in some oocyte recipients is associated with an intrinsic defect in the expression of multiple genes in their endometrium. Significantly decreased levels of several transcripts in endometria without manifest abnormalities is demonstrated for the first time and shown to be associated with implantation failure.

Copper-T intrauterine device and levonorgestrel intrauterine system: biological bases of their mechanism of action.

All intrauterine devices (IUDs) that have been tested experimentally or clinically induce a local inflammatory reaction of the endometrium whose cellular and humoral components are expressed in the tissue and the fluid filling the uterine cavity. Depending on the reproductive strategy of the species considered and the anatomical features and physiologic mechanisms that characterize their reproductive system, the secondary consequences of this foreign body reaction can be very localized within the uterus, as in the rabbit, or widespread throughout the entire genital tract as in women or even systemic as in some farm animals. Levonorgestrel released from an IUD causes some systemic effects, but local effects such as glandular atrophy and stromal decidualization, in addition to the foreign body reaction, are dominant. Copper ions released from an IUD enhance the inflammatory response and reach concentrations in the luminal fluids of the genital tract that are toxic for spermatozoa. In the human, the entire genital tract appears affected due to luminal transmission of the noxa that accumulates in the uterine lumen. This affects the function and viability of gametes, decreasing the rate of fertilization and lowering the chances of survival of any embryo that may be formed, before it reaches the uterus. The bulk of the data indicate that if any embryos are formed in the chronic presence of an IUD, it happens at a much lower rate than in non-IUD users. The common belief that the usual mechanism of action of IUDs in women is destruction of embryos in the uterus is not supported by empirical evidence.

Effectiveness of levonorgestrel emergency contraception given before or after ovulation--a pilot study.

BACKGROUND: Although widely used, the mechanisms of action of the levonorgestrel emergency contraceptive pill (LNG ECP) are still unclear. There are increasing data to indicate that LNG is particularly effective as an ECP by interrupting follicular development and ovulation. An important outstanding question is whether it has any effect on fertilization or implantation. METHOD: Ninety-nine women participated; they were recruited at the time they presented with a request for emergency contraception. All women took LNG 1.5 mg in a single dose during the clinic consultation. A blood sample was taken immediately prior to ingestion of the ECP for estimation of serum LH, estradiol and progesterone levels to calculate the day of ovulation. The specimens were analyzed in a single batch. Based on these endocrine data, we estimated the timing of ovulation to be within a +/-24-h period with an accuracy of around 80%. Women were followed up 4-6 weeks later to ascertain pregnancy status. The effectiveness of ECP when taken before and after ovulation was determined. RESULTS: Three women became pregnant despite taking the ECP (pregnancy rate, 3.0%). All three women who became pregnant had unprotected intercourse between Days -1 and 0 and took the ECP on Day +2, based on endocrine data. Day 0 was taken as ovulation day. Among 17 women who had intercourse in the fertile period of the cycle and took the ECP after ovulation occurred (on Days +1 to +2), we could have expected three or four pregnancies; three were observed. Among 34 women who had intercourse on Days -5 to -2 of the fertile period and took ECP before or on the day of ovulation, four pregnancies could have been expected, but none were observed. We found major discrepancies between women's self-report of stage of the cycle and the dating calculation based on endocrine data. CONCLUSION: These data are supportive of the concept that the LNG ECP has little or no effect on postovulation events but is highly effective when taken before ovulation.

Pharmacokinetic study to compare the absorption and tolerability of two doses of levonorgestrel following single vaginal administration of levonorgestrel in Carraguard gel: a new formulation for "dual protection" contraception.

OBJECTIVE: The study was conducted to assess levonorgestrel (LNG) serum levels achieved after a single administration of two different doses of Carraguard vaginal gel containing LNG (CARRA/LNG), designed for use as microbicide and contraceptive for potential dual protection. MATERIALS AND METHODS: This was a randomized double-blind pharmacokinetic study conducted in 12 subjects enrolled at two centers. Each subject received a single vaginal administration of CARRA/LNG containing either 0.75 or 1.5 mg LNG per 4 mL of gel on Days 10-12 of the menstrual cycle. LNG serum levels were measured at 0, 1, 2, 4, 8 and 12 h after administration and for the following 7 days. LH and progesterone (for a preliminary evaluation of effect on the ovarian function) as well as SHBG were measured in the daily samples. RESULTS: Serum LNG maximum concentrations (Cmax) were 14.1+/-2.1 and 11.7+/-2.7 nmol/L and Tmax was 12.0 and 6.0 h for the low and high dose, respectively, with large intersubject variability within the first 48 h. Mean levels at 96 h were 10% of Cmax. Differences in AUC between both doses were not statistically significant. SHBG levels decreased approximately 25% by Day 4 after administration. Luteal activity was observed in 3/6 and 5/6 of the subjects in the low- and high-dose group, respectively. CONCLUSION: This study demonstrates that the CARRA/LNG gel can sustain elevated serum levels of the contraceptive steroid for up to 96 h after a single application. The serum levels attained with the 0.75-mg formulation are in the range expected to perturb the ovulatory process as observed in some subjects. The lack of correlation between the administered dose and serum concentrations of the steroid may be related to a rate-limiting absorption of LNG from the vaginal mucosa. The results reported here suggest that the CARRA/LNG formulation has good potential to become a dual-protection method, possibly preventing conception and sexually transmitted infections.

Effect of a single vaginal administration of levonorgestrel in Carraguard gel on the ovulatory process: a potential candidate for "dual protection" emergency contraception.

OBJECTIVE: The study was conducted to evaluate the effect of Carraguard vaginal gel containing 0.75 mg of levonorgestrel (CARRA/LNG gel) administered in a single dose at different stages of follicle development over subsequent follicle rupture and hormonal levels. METHOD: Randomized, blinded, cross-over study comparing the effects of a single administration of CARRA/LNG gel or Carraguard (CARRA) gel. Twenty-four healthy women were enrolled in two centers. The gels were administered when the follicle had reached diameters of 12-14, 15-17 and > or =18 mm in eight women each. Volunteers were followed for one treatment, one washout cycle and a second treatment cycle. Follicle rupture or nonrupture was assessed by transvaginal ultrasound. Luteinizing hormone, estradiol and progesterone levels were measured daily for 5 days following treatment, and three times per week until menses. RESULTS: No follicular rupture within the 5-day period following administration was observed in 74% and 30% of the CARRA/LNG and CARRA gel treatment cycles, respectively, while ovulation was documented in 4% and 61%, respectively. The overall proportion of cycles with lack of follicular rupture or ovulatory dysfunction (follicle rupture preceded by an inadequate LH surge) was 96% for CARRA/LNG and 39% in the CARRA gel cycles. CONCLUSION: Single vaginal administration of 0.75 mg LNG in CARRA gel in the late follicular phase is effective for interfering with the ovulatory process.

Effect of oestradiol and progesterone on the instant and directional velocity of microsphere movements in the rat oviduct: gap junctions mediate the kinetic effect of oestradiol.

The oviducal transport of eggs to the uterus normally takes 72-96 h in the rat, but this is reduced to less than 20 h after a single injection of oestradiol (E2). This accelerated transport is associated with an increased frequency of pendular movements in the isthmic segment of the oviduct, with increased levels of the gap junction (GJ) component Connexin (Cx) 43, and is antagonised by progesterone (P). In the present study, we investigated the effect of these hormones on the instant and directional velocity of pendular movements and the role of the GJ and its Cx43 component in the kinetic response of the oviduct to E2 and P. Using microspheres as egg surrogates, microsphere instant velocity (MIV) was measured following treatment with E2, P or P + E2, which accelerate or delay egg transport. Microspheres were delivered into the oviduct of rats on Day 1 of pregnancy and their movement within the isthmic segment was recorded. Oestrogen increased MIV with faster movement towards the uterus. After P or P + E2, MIV was similar to that in the control group. Two GJ uncouplers, namely 18 alpha- and 18 beta-glycyrrhetinic acid, blocked the effect of E2 on MIV. Connexin 43 mRNA levels increased over that seen in control with all treatments. In conclusion, the effects of E2 on MIV resulted in faster movements that produced accelerated egg transport towards the uterus. Gap junctions are probably involved as smooth muscle synchronisers in this kinetic effect of E2, but the opposing effects of E2 and P are not exerted at the level of Cx43 transcription.

Inositol triphosphate participates in an oestradiol nongenomic signalling pathway involved in accelerated oviductal transport in cycling rats.

Oestradiol (E(2)) accelerates oviductal transport of oocytes in cycling rats through a nongenomic pathway that involves the cAMP-PKA signalling cascade. Here we examined the role of the inositol triphosphate (IP3) and mitogen-activated protein kinase (MAPK) signalling cascades in this nongenomic pathway. Oestrous rats were injected with E(2) s.c. and intrabursally (i.b) with the selective inhibitors of phospholipase C (PLC) ET-18-OCH(3) or MAPK PD98059. The number of eggs in the oviduct assessed 24 h later showed that ET-18-OCH(3) blocked E(2)-induced egg transport acceleration, whereas PD98059 had no effect. Other oestrous rats were treated with E(2) s.c. and 1, 3 or 6 h later oviducts were excised and the levels of IP3 and phosphorylated MAPK p44/42 (activated) were determined by radioreceptor assay and western blot, respectively. Oestradiol administration increased IP3 level at 1 and 6 h after treatment, whereas activated MAPK p44/42 level was unchanged. Finally, we explored whether cAMP-PKA and PLC-IP3 signalling cascades are coupled. Inhibition of adenylyl cyclase by i.b. injection of SQ 22536 blocked the increase of IP3 levels induced by E(2), while inhibition of PLC by ET-18-OCH(3) had no effect on E(2)-induced PKA activity. Furthermore, activation of adenylyl cyclase by Forskolin increased oviductal IP3 levels. Thus, activation of PLC-IP3 by E(2) requires previous stimulation of cAMP-PKA. We conclude that the nongenomic pathway utilised by E(2) to accelerate oviductal transport of oocytes in cycling rats involves successive activation of the cAMP-PKA and PLC-IP3 signalling cascades and does not require activation of MAPK. These findings clearly illustrate a non-genomic pathway triggered by E(2) that regulates a complex physiologic process accomplished by an entire organ.

Pain from copper intrauterine device insertion: randomized trial of prophylactic ibuprofen.

OBJECTIVES: This study was undertaken to determine whether 400 mg of prophylactic ibuprofen can alleviate pain from insertion of an intrauterine device (IUD) and to measure level of pain with improved techniques. STUDY DESIGN: We conducted a randomized, double-blind, placebo-controlled trial of 2019 first-time IUD users: 1008 women received placebo and 1011 women received 400 mg of ibuprofen. Participants took the single tablet at least 45 minutes before IUD insertion. Immediately after insertion, participants recorded level of pain by using a 10-cm visual analog scale, with the value of 10 meaning "worst imaginable pain." RESULTS: Median level of pain was 1.0 for both ibuprofen and placebo participants; rank test statistics confirmed no difference. Some subgroups of women experienced higher pain (eg, nulliparous women), but ibuprofen still had no important impact on level of pain. CONCLUSION: Even among first-time users, pain from IUD insertion is generally low. Prophylactic ibuprofen as used in this protocol does not reduce IUD insertion pain.

Emergency contraception - a human rights issue.

Emergency contraception is the only resource that women can use to avoid becoming pregnant after having sexual intercourse without contraceptive protection. It could be a powerful means to prevent unwanted pregnancies and their devastating consequences for women's health, social wellbeing and life project, and for the unwanted child, if all people had ample access to good quality information, education and services for sexual and reproductive health. In spite of the preventive medicine value of emergency contraception, conservative sectors oppose its availability, appealing to moral values that are not universally shared in pluralistic societies. Excluding the only contraceptive that can be used after intercourse because some consider the mechanism of action to be unacceptable would mean restricting the right of choice of others, and imposing one particular belief or set of values on all members of the community, thus violating the freedom of conscience. Authorities have a moral obligation to protect human rights.

Extended use of a progesterone-releasing vaginal ring in nursing women: a phase II clinical trial.

AIM: This study evaluates the performance of extended use of a progesterone (P)-releasing vaginal ring (PVR) in nursing women. METHOD: An open-label, noncomparative study on the safety and contraceptive efficacy of PVR replaced every 4 months of use (instead of 3 months) in 192 PVR acceptors. PVR use was initiated at day 59+/-2 (mean+/-SD) postpartum and continued until weaning or completing the use of three PVRs. RESULTS: Plasma P levels attained with the ring decreased from 17+/-1 to 14+/-1 nmol/L (mean+/-SE) from the third to the fourth month of use. These levels are still over the critical level of 10 nmol/L required for contraceptive protection. One pregnancy occurred in the third month of use of the second ring in 1998 woman-months of exposure. Extended use of the ring did not appear to affect breast-feeding performance or the rate of infant growth, and lactational amenorrhea was prolonged. No differences in the characteristics of bleeding between the third and fourth month of ring use were observed. CONCLUSION: The results indicate that use of the PVR for 4 months represents a safe and effective contraceptive for nursing women.

Contraceptive vaginal rings releasing Nestorone and ethinylestradiol: a 1-year dose-finding trial.

In a multicenter 1-year trial of contraceptive vaginal rings (rings) involving 150 women, three dose combinations of the progestin Nestorone (NES) and ethinylestradiol (EE) were compared with respect to effectiveness, safety and acceptability. Mean in vitro drug release rates for the three doses were 150 and 15, 150 and 20 and 200 and 15 microg/day of NES and EE, respectively. Each ring remained in situ for 21 days, removed for 7 days and then reinserted for a total of 13 cycles of use. We studied ring performance with respect to pregnancy and other termination events, adverse events, the extent of ovulation inhibition, serum drug levels and bleeding control. We also assessed the rings' effects on the vagina using a standardized colposcopy procedure. Seventy-two percent of the women completed the 1-year (> or = 350 days) study. In studied cycles, luteal activity (progesterone > or = 10 nmol/L) was noted in 17%, 7% and 12% of subjects with monitored cycles at the 150/15, 150/20 and 200/15 doses, respectively (p = .34). Two pregnancies occurred, both in subjects using the 200/15 microg/day ring. Breakthrough bleeding during ring use averaged about 2 days/year and breakthrough bleeding and spotting averaged about 7 days/year. In the entire trial, only two women discontinued because of bleeding problems. Medical conditions, chiefly vaginal problems, personal reasons and device loss or repeated expulsion were the principal reasons given for study discontinuation. Vaginal and cervical colposcopy, conducted with standardized techniques and standardized interpretations, revealed no elevated event incidence attributable to ring use. Clinical performance and adverse event profiles indicate that each of these 1-year NES/EE rings, used on a 21-day-in and 7-day-out regimen, provided women effective, acceptable and safe long-acting contraception under their own control.