Chair, Stanford University School of Medicine - Obstetrics & Gynecology (1999 - Present)
Dr. Polan's research has centered around ovarian function during both the follicular and luteal phases. Studies of steroidogenesis, LH receptor synthesis, and the involvement of the plasminogen activator system in ovarian events have been performed. In addition, this laboratory has investigated the synthesis and secretion of interleukin-1 by human peripheral monocytes as well as by human granulosa-luteal cells isolated from IVF patients. More recently this laboratory has focused on the role of IL-1, IL-1ra, and IL-1 type 1 receptor, as well as collagenase and TIMPs, in the process of embryonic implantation. Studies with IL-1 receptor antagonist have documented its ability to prevent implantation in the mouse model.
To investigate the expression of transforming growth interacting factor (TGIF), a Smad transcriptional corepressor, in leiomyoma and matched myometrial tissue samples and the effect of TGIF overexpression in myometrial cells.Experimental study.Tertiary university hospital.Uterine leiomyoma and myometrial tissues from 16 patients.None.The distribution of TGIF in leiomyoma and myometrial tissues by immunohistochemistry stain, mRNA, and protein expression levels by real-time quantitative polymerase chain-reaction (QPCR) and Western blot. Transcriptional regulation of TGIF in myometrial cells with overexpressed TGIF.Although TGIF is present in the smooth muscle cells of the leiomyoma and the myometrium, it is not found in the extracellular matrix. The TGIF mRNA and protein expressions were statistically significantly higher in the leiomyoma compared with the matched, unaffected myometrial tissues in both phases of the menstrual cycle. There were no differences in mRNA or protein expression throughout the menstrual cycle. Overexpression of TGIF protein in myometrial cells statistically significantly suppressed up-regulation of plasminogen activator inhibitor (PAI-1) induced by TGF-beta1 treatment.Expression of TGIF is increased in leiomyoma compared with myometrium. This increase in TGIF expression is not affected by endogenous ovarian hormones. Thus, TGIF is a potential repressor of TGF-beta pathways in myometrial cells.
View details for DOI 10.1016/j.fertnstert.2009.05.001
View details for Web of Science ID 000280407900038
View details for PubMedID 19524896
View details for PubMedCentralID PMC2888713
The goal of this study was to evaluate the first formal counseling program for obstetric fistula patients in Eritrea.To evaluate the impact of the counseling program, clients were interviewed both before pre-operative counseling and again after post-operative counseling. A questionnaire was used in the interviews to assess women's knowledge about fistula, self-esteem, and their behavioral intentions for health maintenance and social reintegration following surgical repair. In addition, two focus groups were conducted with a total of 19 clients assessing their experiences with the surgical care and counseling.Data from the questionnaires revealed significant improvements in women's knowledge about fistula, self-esteem, and behavioral intentions following counseling. Focus group data also supported increased knowledge and self-esteem.Evaluation of the short-term impact of an initial formal counseling program for fistula patients in sub-Saharan Africa affirmed the positive effects that such a program has for fistula patients, with increased knowledge about the causes of fistula, fistula prevention and enhanced self-esteem.Culturally appropriate counseling can be incorporated into services for surgical repair of obstetric fistula in low-resource settings and has the potential to improve the physical and mental well-being of women undergoing fistula repair.
View details for DOI 10.1016/j.pec.2009.11.010
View details for Web of Science ID 000280384500020
View details for PubMedID 20034756
We investigated whether the expression of alpha-1 antitrypsin (ATT), neutrophil elastase (NE), and lysyl oxidase-like protein 1 (LOXL-1) vary within the vagina in subjects with pelvic organ prolapse (POP).Biopsies were obtained from the anterior and posterior vaginal wall of 22 women with POP (> or =stage 2 by POP-Q). The subjects were grouped by the most prominent defect: cystocele, cystocele plus uterine prolapse, and rectocele. Comparative real-time PCR, Western blotting, and NE enzyme activity assay were performed.The ratio of anterior and posterior vaginal wall ATT, NE, and LOXL-1 expression varied between individuals within the same defect group.ATT, NE, and LOXl-1 expression was variable among different biopsy sites in the vagina. No consistent pattern was present when the subjects were grouped by the most prominent defect. We recommend careful consideration of biopsy sites in future studies on POP to enhance reproducibility of data.
View details for DOI 10.1007/s00192-009-0905-y
View details for Web of Science ID 000272796900006
View details for PubMedID 19763368
View details for PubMedCentralID PMC2835807
We investigated the effect of relaxin on extracellular matrix remodeling in cultured myometrial compared with leiomyoma smooth muscle cells. Relaxin increases elastase activity and protease inhibitor expressions in myometrial smooth muscle cells, but no effect was observed in leiomyoma cells.
View details for DOI 10.1016/j.fertnstert.2008.03.043
View details for Web of Science ID 000265132600018
View details for PubMedID 18554597
View details for PubMedCentralID PMC2728012
The objective of this study is to compare relaxin's effect on transforming growth factor (TGF)- beta1 and latent TGF-beta1-binding protein (LTBP-1) in vaginal fibroblasts from women with stress urinary incontinence (SUI) to continent women (controls) in both phases of the menstrual cycle. Fibroblasts were treated with relaxin. TGF-beta1 levels were measured by enzyme-linked immunosorbent assay. LTBP-1 expression was evaluated by Western blot. In the proliferative phase, total TGF-beta1 level in the supernatant, cells, and extracellular matrix (ECM) of SUI fibroblasts decreased with increasing relaxin concentration (P < .05). Active TGF-beta1 levels increased at a low concentration of relaxin (P < .05) in the supernatant but decreased in the ECM of SUI fibroblasts at high concentration (P < .05). In the secretory phase, total TGF-beta1 levels decreased with relaxin treatment (P < .05) in the supernatant, cells and ECM of both women with SUI and controls. Relaxin decreased the levels of total and active TGF-beta1 in the ECM isolated from SUI vaginal fibroblasts.
View details for DOI 10.1177/1933719108315299
View details for Web of Science ID 000255410900012
View details for PubMedID 18421026
Loss of mechanical stability of the urethra and bladder is thought to be important in the development of stress urinary incontinence (SUI). The vaginal wall is the main supporting tissue for pelvic organs and changes in components of supporting tissues are known to be involved in the pathophysiology of SUI.We evaluated changes in expression of alpha2-macroglobulin (alpha2-M), a protease inhibitor, in vaginal wall tissues from premenopausal women (aged 42-45 years) with SUI (n = 28) compared with menstrual cycle-matched continent women (controls, n = 29). The distribution of alpha2-M in vaginal wall tissues and fibroblasts was analysed by immunohistochemistry and immunofluorescence. Expression levels of alpha2-M mRNA and protein was determined by relative real-time quantitative PCR and enzyme-linked immunosorbent assay, respectively. Protease inhibition was measured to assess bioactivity.Vaginal wall tissues do express alpha2-M. Expression of alpha2-M mRNA and protein was significantly higher in tissues from controls compared to women with SUI in both proliferative and secretory phases (P < 0.05). Protease inhibitory activity of alpha2-M was significantly higher in tissues from controls compared to women with SUI in the secretory phase (P < 0.05), but we found no difference in the proliferative phase between groups. alpha2-M protein level was lower in the proliferative phase than the secretory phase in both controls and SUI patients, while for alpha2-M mRNA this was found only in controls.Decreased expression of alpha2-M mRNA and protein and protease inhibitory activity in the vaginal wall tissues of women with SUI may contribute to the development of SUI.
View details for DOI 10.1093/humrep/dem370
View details for Web of Science ID 000252544300023
View details for PubMedID 18077315
CD9 mRNA and protein expression levels in mouse slow frozen-rapid thawed oocytes were compared with those in fresh oocytes by using comparative quantitative real time reverse transcription-PCR and semiquantitative Western blot, respectively. The expression levels of both CD9 mRNA and protein in the frozen oocytes were significantly lower than those found in the fresh oocytes.
View details for DOI 10.1016/j.fertnstert.2006.11.130
View details for Web of Science ID 000248716000044
View details for PubMedID 17307168
To investigate changes in mRNA and protein levels of biglycan (BGN), decorin (DCN) and fibromodulin (FMOD) in vaginal wall tissue from women with stress urinary incontinence (SUI) compared to menstrual-cycle matched continent women.We determined mRNA expressions of BGN, DCN and FMOD by quantitative real-time PCR. They were localized in vaginal wall tissue by immunohistochemistry. We performed western blot analysis to examine protein expression.BGN, DCN and FMOD co-localized with collagen and elastin in the extracellular matrix (ECM) of vaginal wall tissue from both groups. The mRNA expression of FMOD was significantly lower in cases versus controls in the proliferative phase (P = 0.03). DCN mRNA expression in cases was higher in the proliferative (P = 0.05) and secretory phases (P = 0.02) versus controls. BGN mRNA expression showed no significant differences in either phase. Protein expression of FMOD in cases was lower in the proliferative phase versus controls (six out of nine pairs), whereas DCN and BGN protein expression in the secretory phase in cases was higher (seven out of nine pairs).BGN, DCN and FMOD expressions in vaginal wall tissue differ in women with SUI and are hormonally modulated. Differences in small proteoglycans may contribute to the altered pelvic floor connective tissues found in these women.
View details for DOI 10.1093/humrep/dem039
View details for Web of Science ID 000247470700033
View details for PubMedID 17395685
We compared latent TGF-ss binding protein-1 (LTBP-1) and fibrillin-1 (FBN-1) expression in leiomyomata and myometrium, correlated with leiomyomata size. We studied in vivo and in vitro effects of ovarian steroids using matched leiomyomata and myometrium samples from both phases of the menstrual cycle. Leiomyomata were divided into small (
View details for PubMedID 17360930
This article presents findings from qualitative interviews with women seeking medical care for obstetric fistula in Eritrea. The interviews were designed to inform programme design for the prevention and treatment of obstetric fistula. Interviews were conducted with 11 new fistula repair patients, 15 women returning for follow-up for their fistula repairs, and five accompanying family members at Massawa Hospital in the Northern Red Sea Zone of Eritrea during November-December 2004. The women described long delays in accessing emergency obstetric care due to delayed recognition of the seriousness of the problem and lack of transportation from remote villages. Follow-up patients described improvements in their conditions, but many continued to have problems with incontinence and sexual health. Both new and returning patients lacked specific information about their condition, what to expect in terms of treatment and recovery, and how to care for themselves. The findings point to a need for community mobilization and education on safe motherhood for prevention of fistula, as well as for improved information, counselling, follow-up, and social services for women who develop obstetric fistulas.
View details for DOI 10.1080/17441690600648728
View details for PubMedID 19280388
Altered elastin metabolism is implicated in pelvic floor disorders. We studied neutrophil elastase (NE) and matrix metalloproteinase (MMP) activities in vaginal tissues from premenopausal women with stress urinary incontinence (SUI).Elastase and NE activities in vaginal tissues were assessed. Protein and mRNA expressions were determined by RT-PCR and Western blot. Total elastin and collagen contents were evaluated. To compare the relative elastolytic effect of NE and MMP-2, we used their respective antibodies to immunoprecipitate these proteins from vaginal fibroblast extracts prior to assessing elastase activity.Elastase activity in vaginal wall tissues was significantly higher in the secretory compared to the proliferative phase. NE mRNA and protein expressions were similar between control and SUI tissues from the secretory phase. However, NE activity in the SUI tissues was higher compared to control tissues. The mRNA expression of alpha-1 antitrypsin (ATT) was higher in control tissues from the proliferative phase compared to those from the secretory phase, while no difference was observed in SUI tissues between either phase. Protein expression of the active form of ATT was decreased in SUI tissues compared to controls during the secretory phase. Anti-NE antibody reduced total elastase activity by 60-70%, compared to less than 20% reduction with anti-MMP-2 antibody.During the secretory phase, elastolytic activity is increased in pelvic tissues from women with SUI, through an increase in NE activity and a concurrent decrease in ATT expression. The serine protease, NE, appears to be a more significant modulator of elastase activity compared to MMP-2.
View details for DOI 10.1002/nau.20347
View details for Web of Science ID 000245557300023
View details for PubMedID 17089373
To evaluate differential expression of transforming growth factor (TGF-beta1), latent transforming factor-binding proteins (LTBP-1, LTBP-2) and elastin microfibril components (fibrillin-1 and fibrillin-2) in vaginal tissue from women with stress urinary incontinence (SUI).In this case-control study, vaginal tissue from women in both phases of the menstrual cycle was obtained. Messenger RNA (mRNA) expressions of LTBP-1, LTBP-2, fibrillin-1, fibrillin-2 and TGF-beta1 were determined by relative real-time quantification PCR. Tissue localization was analysed by immunohistochemistry, and semiquantitative protein expression was evaluated by Western blot analysis.Vaginal wall fibroblasts synthesized all proteins tested. LTBP-1, LTBP-2 and TGF-beta1 co-localized with elastin microfibrils, fibrillin-1 and fibrillin-2 in the extracellular matrix. LTBP-1 mRNA and protein expressions were higher in control versus women affected with SUI in the proliferative phase (P = 0.04), while in the secretory phase, mRNA expression in cases was higher (P = 0.04). Fibrillin-1 mRNA was higher in women affected by SUI versus controls in both phases, but no statistical differences in fibrillin-1 protein expression were observed between the two groups in either phase. LTBP-2 and TGF-beta1 mRNA expressions showed the same trends as LTBP-1.LTBP-1, LTBP-2, TGF-beta1, fibrillin-1, and fibrillin-2 expressions are hormonally regulated in vaginal wall fibroblasts and differ in women affected by SUI when compared to controls. These data suggest a mechanism to regulate TGF-beta1 activity in pelvic connective tissue.
View details for DOI 10.1093/humrep/dei485
View details for Web of Science ID 000236818600026
View details for PubMedID 16452154
The pathophysiology of pelvic floor dysfunction resulting in stress urinary incontinence (SUI) in women is complex. Evidence suggests that there is also a genetic predisposition towards SUI. We sought to identify differentially expressed genes involved in extracellular matrix (ECM) metabolism in vaginal tissues from women with SUI in the secretory phase of menses compared with asymptomatic women.Tissue samples were taken from the periurethral vaginal wall of five pairs of premenopausal, age-matched SUI and continent women and subjected to microarray analysis using the GeneChip Human Genome U133 oligonucleotide chip set.Extensive statistical analyses generated a list of 79 differentially expressed genes. Elafin, keratin 16, collagen type XVII and plakophilin 1 were consistently identified as up-regulated ECM genes. Elafin, a serine protease inhibitor involved in the elastin degradation pathway and wound healing, was expressed in pelvic fibroblasts and confirmed by Western blot, quantitative competitive PCR and immunofluorescence cell staining.Genes involved in elastin metabolism were differentially expressed in vaginal tissue from women with SUI, suggesting that elastin remodelling may be important in the molecular aetiology of SUI.
View details for DOI 10.1093/humrep/dei276
View details for Web of Science ID 000233846700003
View details for PubMedID 16126751
To determine the impact of nutritional supplementation on female fertility.A double blind, placebo-controlled study of the effects of FertilityBlend for Women, a proprietary nutritional supplement containing chasteberry, green tea, L-arginine, vitamins (including folate) and minerals, on progesterone level, basal body temperature, menstrual cycle length, pregnancy rate and side-effects.Ninety-three (93) women, aged 24-42 years, who had tried unsuccessfully to conceive for six to 36 months, completed the study. After three months, the FertilityBlend (FB) group (N = 53) demonstrated a trend toward increased mean mid-luteal progesterone (P(ml)), but among women with basal pretreatment P(ml) < 9 ng/ml, the increase in progesterone was highly significant. The average number of days with luteal-phase basal temperatures over 98 degrees F increased significantly in the FB group. Both short and long cycles (< 27 days or > 32 days pretreatment) were normalized in the FB group. The placebo group (N = 40) did not show any significant changes in these parameters. After three months, 14 of the 53 women in the FB group were pregnant (26%) compared to four of the 40 women in the placebo group (10%; p = 0.01). Three additional women conceived after six months on FB (32%). No significant side-effects were noted.Nutritional supplements could provide an alternative or adjunct to conventional fertility therapies.
View details for PubMedID 17211965
To evaluate the incidence of and demographic characteristics associated with obstetric fistula in Eritrea. To determine the outcomes of surgical repair of complex fistula in Eritrea by a visiting surgical team.A surgical team comprising expert gynecologic surgeons traveled to Eritrea in September 2004. We evaluated 50 patients with genitourinary fistula and performed surgical repairs of these fistulas on 37 women via both vaginal and abdominal approaches. Demographic and basic medical data were obtained at the time of evaluation, and follow-up questionnaires were completed at 4 weeks postoperative.The majority of the women had fistulas related to obstructed labor at their first pregnancy unattended by any healthcare professional. The average duration of labor was 3 days, and more than half had resulted in stillbirths. The rate of successful repair in women with primary vesicovaginal fistulas (VVF) was 63%, and that in women with recurrent vesicovaginal fistulas was 61%. Two women required urinary diversion procedures because of the severity of the damage to the genital tract. Urethral reconstruction in women with urethrovaginal fistulas (UVFs) was successfully accomplished in 77% of patients. The rate of successful repair of rectovaginal fistulas (RVFs) was 87%.We have demonstrated that a team of specialized surgeons can successfully accomplish surgical procedures and repairs of very complex urinary tract fistulas in a very short mission to a resource-poor nation.
View details for Web of Science ID 000234347900007
View details for PubMedID 16313211
The purpose of this study was to investigate the effect of relaxin on extracellular matrix protein expression in pelvic fibroblasts that were cultured from women with stress urinary incontinence compared with asymptomatic control subjects.Periurethral vaginal wall fibroblasts from premenopausal women with stress urinary incontinence and continent women (in both the proliferative and secretory phase of the menstrual cycle) were stimulated with increasing concentrations of relaxin (0-500 ng/mL). The supernatant was sampled for matrix metalloproteinase-2 and -9 by zymography. Tissue inhibitors of metalloproteinase-1 and -2 and alpha-1 antitrypsin were evaluated with Western blot. Total elastase activity was measured by generation of free amino groups from succinylated elastin. Increasing concentrations of alpha-1 antitrypsin were added to cell lysate to evaluate total elastase activity inhibition.Proliferative-phase stress urinary incontinence fibroblasts demonstrated an increase in matrix metalloproteinase-2 and no change in matrix metalloproteinase-9 and tissue inhibitors of metalloproteinase-1 and -2 expressions with increasing relaxin concentrations. Cells from control subjects showed increased expression of matrix metalloproteinase-2 and -9, but no change in tissue inhibitors of metalloproteinases. Secretory-phase stress urinary incontinence fibroblasts showed no response in matrix metalloproteinase or tissue inhibitors of metalloproteinase expressions with relaxin stimulation. Secretory-phase control fibroblasts reacted by increasing matrix metalloproteinase-2 and -9 and tissue inhibitors of metalloproteinase-2. With respect to total elastase activity and alpha-1 antitrypsin expression, increasing doses of relaxin appear to increase elastolytic activity in stress urinary incontinence cells by decreasing the expression of alpha-1 antitrypsin in proliferative phase cells or increasing the total elastase activity in secretory phase cells. Fibroblast total elastase activity was inhibited by increasing concentrations of alpha-1 antitrypsin.Elastase activity appears to be increased in relaxin-stimulated stress urinary incontinence fibroblasts by either decreased inhibitor (alpha-1 antitrypsin) production or increased elastase activity.
View details for DOI 10.1016/j.ajog.2004.11.027
View details for Web of Science ID 000229384100057
View details for PubMedID 15902165
To investigate the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the development of preimplantation mouse embryos.Mouse 2-cell embryos were collected and cultured in P-1 medium supplemented with GM-CSF at different concentrations. Using reverse transcription-polymerase chain reaction, expression Bcl-2 and Bax mRNA in blastocyst were evaluated in the GM-CSF group and control group. Apoptosis detection was performed using the in situ apoptosis detection kit in mouse blastocysts. The statistical significance of the data was analyzed using t-test and chi-square test.The development of blastocyst increased to 89% in the addition of GM-CSF (0.125 ng/mL), compared to controlled group (80%). The number of cells staining for apoptosis was lower in GM-CSF group than that in the control group. Bcl-2 expression was found to be upregulated in blastocysts in the GM-CSF supplemented group compared to the control group.These results suggest that GM-CSF might be an important regulator in embryo development.
View details for DOI 10.1007/s10815-005-0817-9
View details for Web of Science ID 000227837900006
View details for PubMedID 15807219
To determine whether ArginMax (The Daily Wellness Co., Sunnyvale, CA) or the Panax ginseng extract it contains has any estrogenic activity. ArginMax for Women, a nutritional supplement for optimization of sexual health, contains L-arginine, ginseng, ginkgo, damiana, multivitamins, and minerals.A human endometrial adenocarcinoma cell line, Ishikawa, which contains an alkaline phosphatase (AP) enzyme sensitive to estrogen stimulation, was used in a bioassay to determine whether Panax ginseng or ArginMax contained estrogenic components.Neither ArginMax nor Panax ginseng stimulated AP at any of the concentrations tested.No estrogenic activity was evident in the sample of Panax ginseng extract tested or in a sample of ArginMax containing this extract in combination with other ingredients.
View details for Web of Science ID 000221764200010
View details for PubMedID 15186659
To determine the impact of nutritional supplementation on optimization of reproductive health in women.A double-blind, placebo-controlled pilot study was initiated to determine the effects of FertilityBlend (Daily Wellness Co., Sunnyvale, California), a proprietary nutritional supplement containing chasteberry and green tea extracts, L-arginine, vitamins (including folate) and minerals. Changes in progesterone level, basal body temperature, menstrual cycle, pregnancy rate and side effects were monitored.Thirty women aged 24-46 years who had tried unsuccessfully to conceive for 6-36 months completed the study. After 3 months, the supplement group (n = 15) demonstrated a trend toward an increase in mean midluteal phase progesterone level (from 8.2 to 12.8 ng/mL, P = .08) and a significant increase in the average number of days in the cycle with basal temperatures >37 degrees C during the luteal phase (6.8-9.7 days, P = .04). The placebo group (n = 15) did not show any notable changes after treatment in any of the parameters studied. After 5 months, 5 of the 15 women in the supplement group were pregnant (33%), and none of the 15 women in the placebo group were (P <.01). No significant side effects were noted.Nutritional supplementation may provide an attractive alternative or complement to conventional fertility therapy.
View details for Web of Science ID 000220953500007
View details for PubMedID 15134155
The percentage of women faculty at the professor level has remained at approximately 11%. The medical community could benefit from knowing what is required to attract, retain, and advance women in the academy.The Committee on Women in Medicine and Science at Stanford University School of Medicine was charged with improving career success and well-being of women faculty. In 2001-02, a survey instrument including both needs and perceived school climate was sent to 309 women faculty. Responses were analyzed using one-way analyses of variance with Tukey follow-up tests.A total of 163 (53%) faculty responded. The highest ranked needs were a flexible work environment without negative consequences for women with young children (mean = 4.37 on a five-point scale); a three-month sabbatical from clinical and administrative duties (mean = 4.15); departmental mentoring for academic career development (mean = 4.13); and school/departmental administrative secretarial support for grant and manuscript preparation (mean = 4.11). Climate data obtained in 2002, compared to data from similar surveys in 1994 and 1995, showed a nonsignificant decrease in mean ratings for sexual harassment, gender discrimination, and gender insensitivity in the intervening years. Mean ratings for positive climate and cohesion increased between 1994 and 1995 but remained stable from 1995 to 2002. Results of the survey were presented to the dean, faculty, and staff of the medical school.Women faculty members were able to clearly indicate specific interventions that would improve their career success and sense of well-being. Since administrators are committed to serious consideration of these recommendations, this was a key step in advancing women's careers in academic medicine at Stanford.
View details for Web of Science ID 000222831600007
View details for PubMedID 15044163
This article (1) reviews the decades-long history of short-term dosing regimens delaying the onset of expected, spontaneous menses or withdrawal bleeding in oral contraceptive users up to 20 days; (2) outlines treatment schedules that suppress menstrual bleeding for several months; and (3) evaluates the recently approved extended dosing regimen of 3 months' duration. For single-term postponement of normal menses, estrogen-progestogen combinations can be employed, starting about 7 days after ovulation. Oral contraceptive users can skip the 7-day pill-free period and continue with the active pills in the next package. The main focus of this review is the development of extended dosing schedules that result in cycles lasting 7 weeks up to several months and reduce the number of periods of bleeding and menstrual discomfort. Recently a dosing schedule was introduced into clinical use consisting of ethinyl estradiol, 30 microg, plus levonorgestrel, 150 microg/d, for 84 days, followed by 7 days of placebo. The pregnancy rate was < 1% for compliant women and 1.5% for all participants. A monophasic 21 + 7-day combination using the same daily doses had slightly higher pregnancy rates. The discontinuation rate for unscheduled bleeding and spotting was higher with extended dosing than with the conventional, 21 + 7 schedule.
View details for Web of Science ID 000220526600005
View details for PubMedID 15098885
Weakening of pelvic supportive tissues is thought to be a contributing etiology in female pelvic floor disorders such as stress urinary incontinence and/or pelvic organ prolapse (SUI/POP). Since elastin modulates the mechanical properties of supportive tissues, we examined elastase activity in vaginal tissue from women with pelvic floor dysfunction compared to asymptomatic controls, by comparing overall elastase activity, human neutrophil elastase, cathepsin K, and alpha-1 antitrypsin (a serine protease inhibitor) mRNA and protein levels.Full-thickness peri-urethral vaginal wall tissues were collected from age and menstrual-phase matched SUI/POP and control women at the time of pelvic surgery. Elastolytic activity in the homogenized tissue was determined by the generation of amino groups from succinylated elastin. To quantify mRNA levels of each protein, quantitative competitive-PCR and confirmatory Western blot analyses were performed on the samples for human neutrophil elastase, cathepsin K, and alpha-1 antitrypsin.The mean elastolytic activity in vaginal tissues from the SUI/POP group was similar to that in the control group. With respect to the proteolytic enzymes, neither human neutrophil elastase nor cathepsin K differed between the two groups. However, alpha-1 antitrypsin mRNA and protein levels were significantly decreased in tissues from affected women.A significant decrease in alpha-1 antitrypsin expression was seen in tissues from women with SUI/POP compared to controls. This data suggest that altered elastin metabolism may contribute to the connective tissue alterations observed in pelvic floor dysfunction. Future investigations are warranted to help define the role of elastin turnover in pelvic floor dysfunction.
View details for DOI 10.1002/nau.20012
View details for Web of Science ID 000220085600008
View details for PubMedID 14983422
This study was designed to assess female sexual arousal by using a combination of physiologic measures and self-reported level of arousal.Twenty subjects viewed a 23-minute sequence of randomly ordered relaxation and erotic tapes, both with and without auditory stimulus. The physiologic parameters of vaginal blood flow, galvanic skin resistance, respiration, pulse, and blood pressure, as well as self-reported level of arousal, were simultaneously recorded and correlated with video segments.An academic teaching hospital.The 20 subjects (mean age +/- SD: 24.9 +/- 3.0 years) included Caucasian (10), Hispanic (2), Asian-American (4), and African-American (4) women. All women were screened for normal sexual function with the Female Sexual Function Index (FSFI) and with the Beck Anxiety Inventory and Beck Depression Inventory.Randomly ordered sequences of erotic and relaxation tapes with and without sound.Physiologic and behavioral data, as well as subjective arousal rating, were acquired. The resulting set of multichannel data was correlated with erotic segments and analyzed for sound vs. no sound and time to maximal physiologic arousal.Four independent variables were found to have beta values that were significantly different from 0: respiration (mean = -0.239, SD = 0.177, range = -0.55-0.09, t = -6.04), VPP (mean = 0.158, SD = 0.37, range = -0.48-0.80, t = 1.91), rVPP (mean = 0.161, SD = 0.35, range = -0.537-0.686, t = 2.075), and erotic marker (mean = 0.582, SD = 0.191, range = 0.16-0.85, t = 13.6). Neither heart rate nor galvanic skin resistance beta values approached significance. Respiration period was correlated negatively with arousal rating, indicating that subjects breathed faster when aroused. Auditory stimuli during erotic segments did not increase subjective arousal, and for both subjective arousal rating as well as VPP measurement, maximal response occurred within 2 minutes.Simultaneous measurement of vaginal blood flow, respiration, pulse, and a variable accounting for the onset and offset of erotic video segments accounts for approximately 50% of the variance in predicting subjective female arousal. Regardless of the presence or absence of audio input, 2 minutes was the average minimum time required to reach maximal arousal in young, sexually functional women.
View details for DOI 10.1016/S0015-0282(03)02210-6
View details for Web of Science ID 000187284200029
View details for PubMedID 14667887
To examine human embryos at various stages of preimplantation development for simultaneous expression of matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs).mRNAs of specific MMPs and TIMPs were examined in single human embryos, at different stages of preimplantation development, by reverse transcription-polymerase chain reaction (RT-PCR). Single embryo immunohistochemistry was applied to examine the protein expression.University-affiliated IVF-ET program.Couples, attending the university-affiliated IVF-ET program, electing to donate poor prognosis embryos with anomalous morphology.None.The expression of MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 in preimplantation embryos.The MMP-2 mRNA was expressed consistently during development from one-cell to blastocyst stage. The TIMP-1 and TIMP-3 mRNAs were detected in embryos at all stages; however, in the later preimplantation developmental stages, an increasing proportion of embryos expressing TIMP-1 and TIMP-3 mRNA were noted. The MMP-1, MMP-9, and TIMP-2 mRNAs were detected in only a minority of human embryos studied. Immunohistochemistry showed MMP-1 and TIMP-1 protein expression in preimplantation embryos.The existence of MMP and TIMP mRNA expression in human preimplantation embryos argues for a role for these metalloproteinases and their inhibitors during the process of implantation in humans.
View details for DOI 10.1016/S0015-0282(03)00782-9
View details for Web of Science ID 000185603100008
View details for PubMedID 14505747
To gain a comprehensive view of the gene expression and regulation involved in uterine leiomyomata and matched normal myometrium using oligonucleotide microarray-based hybridization analysis.Retrospective analyses of tissue obtained in a prospective randomized clinical study.Academic institution.Seven patients with leiomyomata scheduled for surgery during the proliferative phase.Seven paired samples of leiomyomata and adjacent myometrium were obtained from patients undergoing hysterectomy.The total RNA extracted from leiomyomata and myometrium was used for gene expression profiling of 6800 human genes using high-density oligonucleotide microarrays. In addition, reverse transcriptase-semiquantitative polymerase chain reaction and immunohistochemistry were used to validate tumor-specific gene expression.A comparison of expression patterns in each paired sample revealed 68 genes significantly up- or down-regulated in each paired tissue sample, of which 23 genes showed increased expression and 45 showed decreased expression in leiomyomata compared with normal myometrium. Cluster analysis supported the relevance of these candidate genes for distinguishing between normal myometrium and leiomyomata biologic activity.Expression profiling of uterine leiomyomata using high-density oligonucleotide microarrays yields signature patterns that reflect the distinctive differences between normal human myometrium and leiomyomata during the proliferative phase. These observations suggest that a number of genes are involved in the tumorigenesis of leiomyomata.
View details for DOI 10.1016/S0015-0282(03)00730-1
View details for Web of Science ID 000184634700007
View details for PubMedID 12909484
The purpose of this study was to investigate the effect of increasing estrogen concentrations on metalloproteinase and tissue inhibitors of metalloproteinase protein expressions in cultured pelvic fibroblasts that were obtained from continent and incontinent women.Periurethral vaginal wall tissues were taken from four stress incontinent and three continent premenopausal women who underwent gynecologic surgery for benign indications. Protein was extracted from these tissues, and Western blot analysis was performed to document that fibroblasts from continent and incontinent women differed with respect to metalloproteinase and tissue inhibitors of metalloproteinase production. One age-matched tissue pair was prepared for fibroblast culture. Cells were cultured with increasing concentrations of estradiol (0-500 pg/mL). Extracellular metalloproteinase and tissue inhibitors of metalloproteinase were assessed semiquantitatively with Western blotting.Periurethral vaginal tissues from incontinent women expressed less tissue inhibitors of metalloproteinase when compared with tissue from the control subjects; there was no difference in the expression of cleaved, active metalloproteinase protein. Tissue inhibitors of metalloproteinase expression from fibroblasts of continent women significantly increased with increasing estradiol concentrations (0-100 pg/mL, P <.05). No significant dose response was seen in fibroblasts from an incontinent woman. Metalloproteinase expression was not altered by increasing estradiol concentrations in fibroblasts from either continent or incontinent women.This preliminary in vitro study suggested that, in fibroblasts that were derived from the continent woman, tissue inhibitors of metalloproteinase protein production increases with increasing estrogen levels and that, in stress incontinent fibroblasts, no similar increase occurs. Neither group demonstrated a change in metalloproteinase production in response to varying estrogen levels, which suggests that estrogen may inhibit collagen degradation in continent women by increasing tissue inhibitors of metalloproteinase production but exerts a reduced inhibitory effect on collagenolysis in women with stress urinary incontinence.
View details for DOI 10.1067/mob.2003.378
View details for Web of Science ID 000184296400011
View details for PubMedID 12861139
The mechanical stability of the genitourinary tract is dependent on intact collagen fibers that support the bladder neck, urethra, and pelvic organs. We hypothesize that genetic differences in collagen metabolism may contribute to stress urinary incontinence. Because sex hormones have substantial influence on the female lower urinary tract throughout adult life, we investigated the gene expression of vaginal tissue of women with stress incontinence compared with women with no stress incontinence in the proliferative phase of the menstrual cycle.Quantitative competitive polymerase chain reaction was used to verify that the gene expressions were similar between periurethral vaginal tissue and pelvic ligamentous tissue. Labeled complementary RNA was obtained from periurethral vaginal tissue in five pairs of age- and menstrual phase-matched, premenopausal women with and without stress urinary incontinence. The vaginal tissues were then hybridized on HuGeneFL arrays that contained probes representing 6800 full-length human genes. The Student t test and Mann-Whitney ranking were used independently to select candidates with probability values <.05. Hierarchical clustering analysis was performed on the selected candidates to assess the ability of these genes to discriminate between normal and affected individuals.Tissue inhibitor of metalloproteinases-1 and estrogen receptor-alpha messenger RNA expressions were found to be similar between uterosacral ligament and periurethral vaginal tissue in six participants. Of the 90 candidate genes that were identified, 62 genes were up-regulated and 28 were down-regulated in the stress urinary incontinence group. Genes that were involved in extracellular matrix activity in the up-regulated group include transforming growth factor-beta3, laminin, and collagen type VI. Down-regulated genes that may participate in collagen metabolism include laminin-related protein, collagen XVII, serine/threonine protein kinase, type II interleukin-1 receptor, and platelet-derived growth factor-associated protein.In this preliminary study, we identified differential gene expressions that may contribute to extracellular matrix remodeling in pelvic tissue from women with stress urinary incontinence in the proliferative phase versus continent control subjects. The alteration in expression of these candidate genes suggests that they should be targets for further investigation.
View details for DOI 10.1067/mob.2003.373
View details for Web of Science ID 000184296400016
View details for PubMedID 12861144
Medical students at Stanford University established an elective lecture series in reproductive health to teach 10 women's health competencies to preclinical medical students. In the second year, student organizers implemented interactive components to improve the number of competencies students learned. Study Design: We surveyed students from the first two years this series was offered to assess their preferred modes of learning and the number of competencies students perceived they had gained. Students were asked to self-assess their learning of key reproductive health competencies taught in the course.We identified 4 factors associated with statistically significant improvements in the number of competencies students learned, according to self-assessment.Students who felt they learned more competencies agreed that they were active participants in the course, that their preferred style of learning was matched by the course, and that they had received academic credit for the course. Furthermore, students who attended an innovative reproductive health fair organized by students participating in the course perceived that they had learned significantly more competencies. We expect these self-assessed improvements to correlate with increased demonstration of the competencies as students progress to the clinical level.
View details for DOI 10.1067/mob.2002.127363
View details for Web of Science ID 000178345700011
View details for PubMedID 12235437
Our aim was to determine the impact of freezing, thawing, and subcutaneous transplantation on follicular development in grafted mouse ovaries.The mice were divided into 3 groups: control (group 1), frozen-thawed grafting (group 2), and frozen-thawed grafting with human menopausal gonadotropin injection (group 3). After freezing and thawing, the ovaries were transplanted into the subcutaneous tissue. Two weeks after transplantation, grafted ovaries and blood samples were collected.Ovaries from group 3 contained significantly more follicles (246 +/- 43 follicles) than group 2(P <.05). The pattern and intensity of Cx37 immunohistochemical staining was similar in all groups. Follicle-stimulating hormone concentrations were significantly decreased in group 2 after ovarian grafting.In mice, gonadotropin treatment before subcutaneous grafting improved the survival of growing follicles. Subcutaneous ovarian transplantation may restore ovarian function and could obviate many of the problems that are related to ovarian banking for humans.
View details for DOI 10.1067/mob.2002.123606
View details for Web of Science ID 000177661200029
View details for PubMedID 12193927
Phospholipase A(2) (PLA(2)) and cyclooxygenase (COX) are two key enzymes in PG synthesis; the latter has two forms, COX-1 and COX-2. mRNA was extracted from single preimplantation embryos and examined for PLA(2), COX-1, and COX-2 gene expression by RT-PCR to investigate whether PLA(2) and COX genes are expressed in human preimplantation conceptuses from zygote to blastocyst stage and to compare COX-1 and COX-2 gene expression within the same stage of embryonic development. Expression of PLA(2), COX-1, and COX-2 was detected in 48, 37, and 45%, respectively, of total embryos examined. COX-1 was expressed in approximately 66% of early human preimplantation embryos from zygote to two-cell stage, whereas COX-2 was expressed in about 58% of later stage embryos from eight-cell to blastocyst stage (P < 0.05). Furthermore, COX-2 mRNA and protein were localized to trophectoderm in blastocyst stage embryos. In conclusion, PLA(2), COX-1, and COX-2 are expressed during early human embryonic development and may contribute to the production of PGs such as PGE(2) in human embryogenesis. COX-1 and COX-2 are differentially expressed, with COX-2 being primarily expressed by trophectoderm in late-stage human preimplantation embryos, which may promote embryonic differentiation and implantation.
View details for Web of Science ID 000176241000033
View details for PubMedID 12050227
Despite the brain's central role in sexual function, little is known about relationships between brain activation and sexual response. In this study, we employed functional MRI (fMRI) to examine relationships between brain activation and sexual arousal in a group of young, healthy, heterosexual males. Each subject was exposed to two sequences of video material consisting of explicitly erotic (E), relaxing (R) and sports (S) segments in an unpredictable order. Data on penile turgidity was collected using a custom-built pneumatic pressure cuff. Both traditional block analyses using contrasts between sexually arousing and non-arousing video clips and a regression using penile turgidity as the covariate of interest were performed. In both types of analyses, contrast images were computed for each subject and these images were subsequently used in a random effects analysis. Strong activations specifically associated with penile turgidity were observed in the right subinsular region including the claustrum, left caudate and putamen, right middle occipital/ middle temporal gyri, bilateral cingulate gyrus and right sensorimotor and pre-motor regions. Smaller, but significant activation was observed in the right hypothalamus. Few significant activations were found in the block analyses. Implications of the findings are discussed. Our study demonstrates the feasibility of examining brain activation/sexual response relationships in an fMRI environment and reveals a number of brain structures whose activation is time-locked to sexual arousal.
View details for Web of Science ID 000175626900010
View details for PubMedID 11960892
The aim of this study was to investigate quantitative mRNA expression of matrix metalloproteinases MMP-1, MMP-2, MMP-9, and their inhibitors, the tissue inhibitors of metalloproteinases TIMP-1, TIMP-2 and TIMP-3, in vaginal wall tissue from women with stress urinary incontinence compared to continent controls. Vaginal wall tissues were obtained from 7 women with stress urinary incontinence/severe pelvic prolapse and 15 continent controls. RNA was then extracted and quantified. Quantitative competitive reverse transcription (QC-RT-PCR) was carried out with oligonucleotide primers to quantify MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2 and TIMP-3 mRNA expression. Stress continent women demonstrated a significant decrease in TIMP-1 and mRNA expression (P = 0.03). There was no difference in TIMP-2, TIMP-3, MMP-2 or MMP-9 mRNA expression between stress incontinent women and controls. However, MMP-1 mRNA expression was significantly increased (P = 0.05) in the incontinent group and the MMP-1/TIMP-1 ratio (P = 0.04) was consistent with increased collagen degradation in the stress incontinence. Stress incontinent women demonstrated an increase in MMP-1 mRNA expression and a decrease in the inhibitor TIMP-1 mRNA expression. Both these findings are consistent with increased collagen breakdown as a pathologic etiology of incontinence.
View details for Web of Science ID 000175825400004
View details for PubMedID 12054187
The interleukin-1 (IL-1) system plays an integral role in local intercellular interactions during implantation. In addition, the plasminogen activator system, especially urokinase plasminogen activator (u-PA), plasminogen activator inhibitor (PAI-1), and u-PA receptor (u-PAR), are crucial during embryo implantation. Decidualization and implantation are complex processes dependent upon several proteases, including u-PA, and IL-1 is known to affect PA activity in several cell types. We investigated the role of IL-1beta in regulating u-PA, PAI-1, u-PAR, and soluble u-PAR messenger ribonucleic acid (mRNA) expression in cultured human endometrial stromal cells using quantitative competitive PCR. For confirmation of the mRNA data, we measured PAI-1 and u-PAR protein by enzyme-linked immunosorbent assay. Confluent stromal cell cultures treated with progesterone and estradiol for 9 days were stimulated with IL-1beta, and IL-1beta plus IL-1beta antibody for an additional 24 h. Total RNA was extracted, reverse transcribed, and coamplified using quantitative and competitive PCR with internal standards. IL-1beta increased PAI-1, u-PAR, and soluble u-PAR expression in a dose-dependent manner, and this result was reversed by anti-IL-1beta antibody treatment. u-PA mRNA expression was not dependent on IL-1beta. These results suggest that IL-1 may be important in regulating PAI-1 and u-PAR during stromal cell decidualization before implantation.
View details for Web of Science ID 000167512000061
View details for PubMedID 11238529
The interleukin (IL)-1 system is a major regulator of local cellular interactions during embryonic implantation. Because IL-1beta and IL receptor antagonist (IL-1ra) are both expressed in human endometrium, we hypothesized that an appropriate ratio of IL-1beta to IL-1ra might favor the process of embryo implantation. Therefore, we investigated IL-1 regulation of the quantitative ratio of IL-1beta/IL-1ra messenger RNA (mRNA) expression in human endometrial stromal cells using quantitative competitive PCR, as well as intracellular protein expression after stromal cell solubilization. Confluent stromal cell cultures were stimulated with human IL-1beta (0-1000 IU/mL) for 24 h. After 24 h, total RNA was extracted, reverse transcribed, and coamplified by PCR with a defined amount of internal standard. The quantitative ratio was determined by the density of target to the internal standard. After culture with IL-1beta for 24 and 48 h, stromal cells were solubilized, and the intracellular protein levels of IL-1beta and IL-1ra were measured by enzyme-linked immunosorbent assay. The IL-1beta and IL-1ra mRNA were both up-regulated, and IL-1R tI mRNA was down-regulated, by IL-1beta in a dose-dependent manner. The quantitative ratio of IL-1beta to IL-1ra mRNA was constant with the presence of increasing concentrations of IL-1beta (1-1000 IU/mL). IL-1beta and IL-1ra protein was not detected in conditioned media of cultures before addition of IL-1beta. IL-1beta and IL-1ra protein levels increased with increasing amounts of IL-1beta after solubilization of stromal cells. The IL-1beta was detectable after 12 h of culture, in comparison with IL-1ra, which was detectable after 24 h of IL-1beta stimulation. These results suggest that IL-1 may play a crucial role in embryo-maternal interaction by regulating stromal cell expression of IL-1beta and IL-1ra, resulting in an appropriate ratio during the process of embryonic implantation.
View details for Web of Science ID 000167512000068
View details for PubMedID 11238536
To detect the expression of vascular endothelial growth factor (VEGF) mRNA and/or secretion of VEGF protein by human preimplantation embryos.Human preimplantation embryos not suitable for uterine transfer were examined for beta-actin and VEGF mRNA expression. Culture media from normally fertilized and developing preimplantation embryos were assessed for VEGF protein secretion.Clinics and academic research laboratories at the Departments of Obstetrics and Gynecology at the Stanford University, Palo Alto, California and the Heinrich-Heine-University, Düsseldorf, Germany.Couples undergoing IVF by intracytoplasmic sperm injection for various reasons.Six unfertilized oocytes and 33 pathologically fertilized (tripronucleic, 3PN) preimplantation embryos were examined for VEGF mRNA expression, and 16 embryos were examined for VEGF protein secretion.Embryonic expression of VEGF mRNA and VEGF protein as determined by reverse transcription (RT)/nested polymerase chain reaction (PCR) and ELISA.VEGF mRNA and protein could not be detected in unfertilized oocytes. However, 30/33 preimplantation embryos did express VEGF mRNA (11/12 10-to-16-cell embryos, 3/4 morulae, 11/12 early blastocysts, 5/5 hatched blastocysts). The VEGF protein level was below the sensitivity of the ELISA.Production of VEGF may give the embryo the ability to induce neoangiogenesis at the implantation site, thus creating an environment necessary for its survival.
View details for Web of Science ID 000165897300027
View details for PubMedID 11119754
The advent of assisted reproductive techniques such as intracytoplasmic sperm injection has markedly reduced the problem of unsuccessful fertilization in modern IVF. However, pregnancy rates and 'take-home-baby' rates remain unsatisfactorily low. Attempts to overcome low pregnancy rates by transferring a larger number of embryos to the mother often result in multiple pregnancies. The preimplantation embryo synthesizes several proteins that may signal its presence to the maternal system, and the interaction between the embryo and the endometrium is controlled, at least in part, by cytokines and growth factors. However, little is known about the interactions between the embryonic and maternal proteins. A better understanding of normal preimplantation embryo development may lead to improved in vitro culture conditions and higher pregnancy rates. This review gives an overview of the current knowledge of the embryonic factors produced during the preimplantation period. The development of the interleukin 1 system for screening human preimplantation embryos is also discussed. Current biochemical embryonic screening procedures are highly experimental, but increasing knowledge of the physiology of embryonic development might enable these screening procedures to be used to identify embryos that are capable of successful implantation.
View details for Web of Science ID 000086913800017
View details for PubMedID 10889844
Previous studies have established the presence of an extrahypothalamic GnRH in a variety of tissues. GnRH receptor is known to be present in the placenta, which produces and secretes the decapeptide from the very early stages of placentation. We hypothesized that GnRH may play a role in the preimplantation development of embryos. To examine this hypothesis, we assessed GnRH and GnRH receptor messenger RNA (mRNA; RT-PCR) and protein expression (Immunohistochemistry) in preimplantation murine embryos at various developmental stages. Furthermore, preimplantation murine embryos were cultured with GnRH agonist and antagonist in vitro to assess the influence of GnRH analogs on embryo development. GnRH is expressed in the developing mouse embryo from morula to hatching blastocyst stages at the mRNA and protein levels. GnRH receptor mRNA is also present in the developing embryos studied. Preimplantation embryonic development was significantly enhanced by incubation with increasing concentrations of GnRH agonist and is significantly decreased by GnRH antagonist compared with that in the control group. Moreover, GnRH antagonist (5 and 10 microM) was able to completely block embryo development. The deleterious effect of GnRH antagonist on embryo development was reversed by increasing concentrations of the agonist, as determined by the number of embryos reaching the blastocyst stage.
View details for Web of Science ID 000081536400039
View details for PubMedID 10433230
Gonadotrophin-releasing hormone (GnRH) regulates gonadotrophin biosynthesis and release in the anterior pituitary via specific receptors. Extrapituitary expression and action of GnRH have been demonstrated in several species. A possible role for GnRH in preimplantation embryonic development, endometrial preparation, and the implantation process has been previously suggested. Moreover, the presence of an immunoreactive GnRH in preimplantation embryos has been demonstrated in different species; however, there are no data for human embryos. We postulate that in humans GnRH may play a role in preimplantation embryonic development as well as in the implantation process. To examine this hypothesis, we assessed GnRH and GnRH-receptor mRNA and protein expression in human preimplantation embryos with three pronuclei. GnRH is expressed in peri-implantation human embryos at both the mRNA and protein level. GnRH-receptor mRNA is also present in the embryos studied. Immunohistochemical localization of GnRH showed intense staining in all the blastomeres at morula stage as well as in the trophectoderm and inner cell mass of the blastocysts. The results of the present study challenge the widely held view that GnRH has a predominantly central action, and suggests a pathway to describe a local role for the GnRH system in successful preimplantation embryonic development and implantation.
View details for Web of Science ID 000079092600008
View details for PubMedID 10333357
Early human trophoblast shows dramatic invasive properties during early pregnancy. The simultaneous synthesis of matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) in both human trophoblast and decidual membranes suggests that their controlled and balanced expression is crucial for the rapid matrix remodeling and controlled invasion during early pregnancy. Recently, we have described the presence of an extrahypothalamic GnRH immunologically, biologically and chemically identical to the hypothalamic hormone in periimplantation human embryos. Moreover, the production of this decapeptide by the human trophoblast during the early stages of placentation is well documented. TIMP-1 and -3 messenger ribonucleic acid (mRNA) expression in cultured stromal cells and protein secretion into the medium were significantly decreased by GnRH agonist compared to that in control groups. Moreover, expression of TIMP-1 was affected to a greater extent than that of TIMP-3. GnRH antagonist ablated the down-regulation of TIMPs by the GnRH agonist. MMP-9 mRNA expression was not detected in the control groups or in the groups treated with GnRH analogs. Our results provide evidence that trophoblastic GnRH may play an important role in placental tissue organization and in the early embryo-maternal dialogue by enhancing trophoblast invasion through the specific inhibition of TIMPs.
View details for Web of Science ID 000078464000042
View details for PubMedID 10022430
GnRH is one of the paracrine/autocrine regulators of hCG secretion produced by the human trophoblast during pregnancy. We hypothesized that GnRH may play a role in the embryonic/endometrial dialogue during early implantation. To examine this hypothesis, we assessed GnRH and GnRH-receptor mRNA and protein expression in human endometrium throughout the menstrual cycle of premenopausal fertile patients. Quantitation of the mRNA was performed by reverse transcription (RT)-competitive polymerase chain reaction (PCR) in the presence of a competitive cDNA fragment. RT-PCR revealed that unfractioned endometrium and isolated endometrial stromal and epithelial cells express GnRH and GnRH-receptor mRNA throughout all phases of the menstrual cycle. Quantitative PCR showed a dynamic pattern in the GnRH mRNA expression throughout the cycle, with a significant increase (p < 0.05) in the secretory phase as compared to the proliferative phase. Furthermore, quantitative competitive PCR of isolated glandular and stromal cells showed higher mRNA levels (p < 0.05) in the luteal phase in both compartments. GnRH immunostaining was localized in all major compartments, with the most intense staining during the luteal phase. On the basis of these data, we suggest that during reproductive life, endometrial GnRH may play a paracrine/autocrine role in the early stages of implantation by modulating embryonic trophoblastic secretion of hCG.
View details for Web of Science ID 000075594900028
View details for PubMedID 9716567
Gathering knowledge about the molecular events during preimplantation development is one of the most important challenges in in-vitro fertilization (IVF). The interleukin-1 (IL-1) system has been shown to be intimately involved in embryonic implantation. The aim of our study was to detect the major components of the IL-1 system in single blastomeres from human preimplantation embryos and to relate our findings to the further development of the biopsied embryos in vitro. Single blastomeres were removed from morphologically normal embryos obtained from dipronuclear zygotes and examined by reverse transcription (RT)-nested polymerase chain reaction (PCR). Expression of beta-actin (external standard), IL-1beta, IL-1 receptor antagonist (IL-1ra) and IL-1 receptor (IL-1R) type I mRNA were related to embryonic development and IVF outcome. Blastomeres from 12 embryos were examined: beta-actin and IL-1R type I mRNA were detected in all blastomeres (100%) whereas IL-1beta could be detected in only nine of the blastomeres (75%). IL-1ra was expressed in only two (17%) of the blastomeres and those were simultaneously positive for IL-1beta. Both IL-1ra positive embryos were arrested in development before reaching blastocyst stage. Five embryos (three of them IL-1beta mRNA positive and two IL-1beta mRNA negative) were transferred as blastocysts; none of the transfers resulted in a pregnancy. We postulate that embryos expressing IL-1ra mRNA in a detectable amount appear more likely to be arrested in early developmental stages.
View details for Web of Science ID 000075642500036
View details for PubMedID 9756297
To investigate the protein expression of GnRH in the endometrium of fertile patients throughout the menstrual cycle.Prospective longitudinal study.Department of Gynecology and Obstetrics, Reproductive Immunology Laboratory, Stanford University Medical Center.Twenty-two fertile premenopausal women submitted to laparoscopic surgery for benign gynecologic indications. None of the 22 women had endometriosis or pelvic inflammatory disease.An endometrial biopsy specimen using the Novak curette was obtained at the time of surgery.Protein expression and localization from unfractioned endometrial tissue was analyzed by immunohistochemistry.Gonadotropin-releasing hormone is expressed at the protein level in both the endometrial stroma and epithelium throughout the entire menstrual cycle of fertile women. Immunostaining in the human epithelium reached maximal levels in the midluteal phase and was elevated in the stroma throughout the entire luteal phase.Our results demonstrate the presence of GnRH in the human endometrium at the protein level throughout the entire menstrual cycle of fertile women, with an increase in the luteal phase compared with the preovulatory endometrium.
View details for Web of Science ID 000074479600018
View details for PubMedID 9660429
Interleukin-1 (IL-1) is expressed in human endometrium and has been shown to play an integral role in local cellular interactions during implantation. In addition, the matrix metalloproteinase (MMP) and its inhibitor, the tissue inhibitor of metalloproteinase (TIMP), are crucial during implantation, mediating in vitro trophoblast penetration, and are regulated by several cytokines expressed by trophoblast cells. We have investigated the roles of IL-1 beta and transforming growth factor-beta (TGF beta) in regulating TIMP-1, TIMP-3, and 92-kDa type IV collagenase messenger ribonucleic acid (mRNA) expression in human endometrial stromal cells using quantitative competitive PCR. Confluent stromal cell cultures treated with progesterone and estradiol for 9 days were stimulated with IL-1 beta, IL-1 beta plus anti-IL-1 beta antibody, TGF beta, and TGF beta plus anti-TGF beta antibody for an additional 24 h. Competitive complementary DNA fragments were constructed by deletion of a defined fragment from each of the target complementary DNA sequences and coamplified in quantitative competitive PCR as an internal standard. TIMP-1 and TIMP-3, but not 92-kDa type IV collagenase mRNA, were expressed in stromal cells. The 92-kDa type IV collagenase mRNA was only expressed after stimulation with IL-1 beta. IL-1 beta both augmented 92-kDa type IV collagenase mRNA expression and decreased TIMP-1 and TIMP-3 mRNA expression in a dose-dependent manner. Conversely, TGF beta augmented TIMP-1 and TIMP-3 mRNA expression, but did not affect 92-kDa type IV collagenase expression. IL-1 and TGF beta-mediated changes were both neutralized by specific antibodies. These results provide indirect evidence that IL-1 and TGF beta may play crucial roles at the embryo-maternal interface during trophoblast invasion by regulating stromal cell expression of TIMP-1, TIMP-3, and 92-kDa type IV collagenase, all of which are known to be important in trophoblast invasion.
View details for Web of Science ID 000073535300055
View details for PubMedID 9589682
Gaining knowledge about the physiological timetable of gene expression during preimplantation embryo development is crucial, and a better understanding of cytokine and growth factor expression in early embryonic development could lead to improved in vitro culture conditions and enhance in vitro fertilization implantation rates. Our aim was to detect the patterns and levels of two messenger ribonucleic acids [mRNAs; beta-actin and interleukin-1 receptor type I (IL-1R tI)] in single human blastomeres by RT-nested PCR and to compare possible variations in the gene expression both between different embryos and in multiple blastomeres within the same embryo. Single blastomeres from nine human tripronucleic preimplantation embryos were examined by one round of RT and two rounds of nested competitive PCR. Beta-actin mRNA was detected in each blastomere, and IL-1R tI mRNA was found in 72% of the blastomeres examined. Beta-actin was expressed at a level of 511-12185 molecules of complementary DNA/blastomere, and IL-1R tI was expressed at a level of 2-290 molecules of complementary DNA/blastomere. Our results suggest that the mRNA pattern of an embryo cannot be reliably quantitated from the mRNA pattern of a single blastomere and therefore imply limitations for the use of this method for preimplantation diagnosis.
View details for Web of Science ID 000072403500044
View details for PubMedID 9506755
Embryos cocultured with Vero cells display enhanced development in vitro. This could be due to an interaction between the embryo and cellular layer mediated by paracrine cytokines. The interleukin-1 (IL-1) system is composed of two IL-1 agonists, IL-1 receptor antagonist (IL-1ra), and two major IL-1 receptors (IL-1R tII). In this study, we measured Vero cell expression of IL-1 system mRNAs with reverse transcription polymerase chain reaction (RT-PCR) and validated the results with an immunohistochemistry study. RT-PCR revealed beta-actin and IL-1R tI mRNA expression in Vero cell cocultures without detectable IL-1 beta and IL-1ra mRNA expression. To determine the effect of IL-1 beta on IL-1R tI mRNA expression in Vero cells, quantitative competitive PCR methodology was developed. A competitive cDNA fragment was coamplified as an internal standard with the target cDNA sequence of IL-1R tI, showing a 50% decrease in Vero cell IL-1R tI cDNA cultured in the presence of IL-1 beta (100 IU/mI) compared to control Vero cell cultures (62.5 fg vs. 125 fg). Down-regulation of IL-1R tI mRNA by IL-1 beta is dose-dependent, with increasing concentrations (0-1000) IU/ml) of IL-1 beta producing progressive attenuation of IL-1R tI expression. Treatment with anti-IL-1 beta antibody ablate the inhibitory effect of IL-1 beta (100 IU/ml) on IL-1R tI mRNA expression. Immunostaining confirmed the presence of IL-1r tI protein in Vero cells. These results demonstrate the presence of IL-1 R tI in Vero cell monolayers and regulation of Il-1R tI mRNA by IL-1 beta.
View details for Web of Science ID A1997XX46000014
View details for PubMedID 9314581
The interleukin-1 (IL-1) system has been shown to play an important role in human and murine embryo implantation. Recent studies have documented immunohistochemical evidence of interleukin-1 beta (IL--1 beta), interleukin-1 receptor antagonist (IL-1ra) and interleukin-1 receptor type I (IL-1R tI) in human preimplantation embryos and protein levels of interleukin-1 alpha (IL-1 alpha), IL-1 beta and IL1ra in human preimplantation embryo culture fluid have been correlated with successful implantation and pregnancy. Our aim in this study was to detect IL-1 beta, Il-1ra and Il-1R tI mRNA in single preimplantation mouse embryos and to describe the frequency of positive mRNA-expression at different developmental stages. B6C3F1-mice, 12 weeks old were pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-stimulated and mated. Animals were sacrificed at day 0.5, and zygotes were flushed from the tubes and cultured in HAMs-F10 medium. 2-cell- (2C-), 8-cell- (8C-), morula- (M-), early blastocyst- (EB-) and hatching blastocyst- (HB-) stage embryos were examined by one round of reverse transcriptase (RT) followed by two rounds of polymerase chain reaction (PCR) carried out on individual mouse embryos for beta-actin (internal standard), IL-1 beta, IL-1ra and IL-1R tI-mRNAs. The frequencies of positive mRNA-expressions were as follows (2C/8C/M/EB/HB); beta-actin: 91/96/100/100/98%; IL-1b: 0/0/2.5/6.25/19; IL-1ra; 0/5/30/41/74% and IL-1R tI: 0/0/10/20/25%. The incidence of IL-1ra mRNA expression increased with developmental stage. IL-1ra mRNA seems to be expressed in a very high percentage (74%) of embryos near the time of implantation, whereas the percentage of IL-1 beta-mRNA positive embryos is surprisingly low (19%).
View details for Web of Science ID A1997XT94400001
View details for PubMedID 9292778
In this study, we report a total of 292 mouse embryos cultured on Vero cell monolayers and 77 embryos cultured in medium alone at different preimplantation stages examined individually for embryonic mRNA of beta-actin, interleukin-1beta (IL-1beta), interleukin-1 receptor antagonist (icIL-1ra) and interleukin-1 receptor type I (IL-1RtI) using reverse transcription and two-step polymerase chain reaction (RT-PCR). The rates of blastocyst formation and blastocyst hatching were both significantly higher in embryos co-cultured with Vero cells in comparison with the embryos cultured in control medium (81.2 +/- 2.6 versus 42.2 +/- 3.7%, P < 0.001; 75.6 +/- 2.7 versus 19.2 +/- 6.2%, P < 0.001 respectively). We have identified a similar pattern of interleukin-1 family embryonic mRNA transcripts expressed from the compact morula stage through to hatching blastocyst in both control and Vero cell cultured embryos with significantly increased icIL-1ra transcript at hatching blastocyst stage (P < 0.05, P < 0.001 respectively). There was a significant increase in IL-1beta mRNA transcripts of embryos at hatching blastocyst stage compared to compact morula stage in Vero cell cultured embryos (P < 0.05). These findings support the hypothesis that the IL-1 system is an important factor in embryo-maternal molecular communication during the implantation process.
View details for Web of Science ID A1997XV35600035
View details for PubMedID 9262292
Implantation biology is now at a stage where experimental science will be very productive in answering basic questions about the ability of an embryo to implant. The advancement of our knowledge of cytokines and growth factors has been critically important in fuelling the recent new understanding of embryo implantation. Specifically, our increased knowledge of the interleukin (IL)-1 system, as well as leukaemia-inhibiting factor (LIF), epidermal growth factor and colony-stimulating factor-1, and the availability of recombinant protein, specific antibodies and knockout mice, have led to a more detailed outline of implantation events. LIF and IL-1 are the two systems where recent advances have suggested their importance in implantation events. Recently, LIF has been shown in mice to be an endometrial requirement for implantation and embryo development. Although LIF is a pleiotropic molecule, with many interactions in multiple body tissues, in the uterus, concentrations are elevated on day 4 of pregnancy. Experiments with knockout mice have shown the requirement for endometrial LIF for successful implantation. The IL-1 system, consisting of two agonists (IL-1 alpha and IL-1 beta), two receptors (IL-1R types I and II) and the homologous IL-1 receptor antagonist (ra), has also been studied. Knowledge that the embryo secretes IL-1 suggested the interaction between embryonic IL-1 and endometrial receptor, which has been shown to occur. IL-1R type I is plentiful on endometrial epithelial cells and appears to interact with embryonically secreted IL-1 beta to favour implantation. Such implantation events in vivo in mice are blocked by the introduction of large quantities of IL-1ra, consistent with the hypothesis that appropriate interactions between agonist and receptor at the level of the endometrial surface are a requisite for successful implantation. As more specific information on each cytokine or growth factor system comes to light, more complete information on the multiple molecular steps of implantation will become apparent. However, it is clear that no single cytokine or growth factor will be able to explain the complicated events of embryo implantation. Such an important necessary phenomenon has multiple redundancies. The interactions between cytokines and growth factors are becoming increasingly apparent and will need more experimental evidence before a full understanding of implantation is available.
View details for Web of Science ID A1995TT19500004
View details for PubMedID 8745298
This study investigated whether gonadal steroids modulate the expression of the cytokine Interleukin-1 receptor antagonist in monocytes. Human male peripheral monocytes were isolated and cultured in serum free media with serially diluted concentrations of estradiol and progesterone. mRNA expressions with increasing steroid concentrations were compared by reverse transcription-polymerase chain reaction for intracellular and secretory interleukin-1 receptor antagonist specific primers and glyceraldehyde 3-phosphate dehydrogenase primers. Monocyte expression of secretory Interleukin-1 receptor antagonist mRNA was significantly elevated in the presence of normal physiological levels of estradiol (10(-11) M) and progesterone (10(-8) M), while expression was suppressed by higher concentrations of steroids. Intracellular receptor antagonist was also detected. This study is the first to describe the dose related response of cytokine interleukin-1 receptor antagonist to gonadal steroids.
View details for Web of Science ID A1995QQ91400040
View details for PubMedID 7726847
To investigate the regulation of messenger ribonucleic acid (mRNA) levels of interleukin-1 beta (IL-1 beta), interleukin-1 (IL-1) receptor type 1, and plasminogen activator (PA) inhibitor-1 and -2 in cumulus cells, granulosa-luteal cells, and macrophage-depleted granulosa-luteal cells obtained from human preovulatory follicles.Prospective longitudinal study. SETTING, PATIENTS: Patients undergoing assisted reproductive technologies (ART) in the Department of Gynecology and Obstetrics, Stanford University, Stanford, California.Cumulus cells and granulosa-luteal cells were collected by ultrasound-guided transvaginal aspiration at the time of ART.Northern blot analysis of mRNA levels of IL-1 beta, IL-1 receptor type 1, PA inhibitor-1 and -2 in cumulus cells, granulosa-luteal cells and macrophage-depleted granulosa-luteal cells, and indirect immunocytochemical analysis of the IL-1 system and macrophages in granulosa-luteal cell preparations were performed.Interleukin-1 beta mRNA levels in uncultured cumulus cells were less than those of uncultured granulosa-luteal cells with no differences in IL-1 receptor type 1 mRNA levels between these two cell types. Granulosa-luteal cell IL-1 receptor type 1 mRNA levels were expressed constitutively throughout 24 hours of culture with no effect by hCG, whereas IL-1 beta mRNA levels increased within 6 hours, and then remained elevated for 24 hours with no effect by hCG. Interleukin-1 beta significantly increased granulosa-luteal cell mRNA levels of IL-1 beta (over twofold), IL-1 receptor type 1 (over twofold), PA inhibitor-1 (approximately 1.4-fold), and PA inhibitor-2 (approximately 1.6-fold). In contrast, IL-1 beta had no effect on IL-1 beta and IL-1 receptor type 1 mRNA levels in macrophage-depleted granulosa-luteal cells. Granulosa-luteal cells, not macrophages, account for the majority of the immunocytochemical staining for IL-1 beta and IL-1 receptor type 1 in follicular aspirates.These studies suggest that the IL-1 system is regulated in human granulosa-luteal cells during the periovulatory period. Furthermore, the augmentation of PA inhibitor-1 and -2 mRNA levels by IL-1 beta suggests a potential role for IL-1 beta in remodeling of the human ovary during the periovulatory period.
View details for Web of Science ID A1994PJ40500015
View details for PubMedID 7926084
Indirect evidence has implicated the interleukin-1 (IL-1) system in ovulation. Thus, the ability of IL-1 beta to induce ovulation in rat and rabbit perfused ovaries has been demonstrated. In the present study, the involvement of the IL-1 system in ovulation was directly tested in vivo, in the rat model. For this purpose, the natural inhibitor of the IL-1 system, interleukin-1 receptor antagonist (IL-1ra), was administered locally by use of an intrabursal injection route. Twenty-six-day-old Sprague-Dawley rats received injections of eCG (10 IU), followed 56 h later by hCG (15 IU). IL-1ra (75 micrograms/bursa) was administered locally into the periovarian sac, 6 h (n = 5), 2 h (n = 11), and 0 h (n = 5) before hCG administration. Control animals (n = 10) received injections of the same volume (50 microliters) of vehicle (PBS). IL-1ra administered locally into the periovarian sac inhibited ovulation from the treated ovary, reaching 40% inhibition (p < 0.05) when injected 2 h prior to hCG, as compared to the untreated contralateral ovary (6 +/- 1.4 ova vs. 10 +/- 1.8 ova) and PBS-injected control ovaries (6 +/- 1.4 ova vs. 8.2 +/- 0.7). Injection of IL-1ra 6 h before or concomitantly with hCG did not affect the ovulation rate. Internucleosomal DNA fragmentation was evaluated by 3' end-labeling and autoradiography for detecting apoptotic changes. No difference in DNA fragmentation was found between treated and untreated ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1994PG80500011
View details for PubMedID 7819447
The purpose of this study was to determine whether cells acquired from individual human preovulatory follicles undergo apoptosis (physiologic cell death) and, if so, to correlate the degree of apoptosis with characteristics of the follicles or the oocytes derived from the follicles.We devised a sensitive nonradioactive method for detecting apoptotic DNA fragmentation in small numbers of cells derived from rat atretic follicles and follicular aspirates of patients undergoing assisted reproductive technologies.Using this method, apoptotic DNA was detected in rat atretic follicles, with optimal detection at 10-100 ng. Furthermore, apoptotic DNA was detected in some, but not all individual human follicular aspirates from several patients, and was found in follicles that produced oocytes that fertilized and developed into embryos.Apoptosis occurs in cells from human ovarian preovulatory follicles and may be a normal physiologic process of the follicle during luteinization.
View details for Web of Science ID A1994QG06600009
View details for PubMedID 9419787
Cytokines are important in reproduction. Interleukin-1, an established immune mediator, is one of the best-characterized members of the cytokine family. We describe what is known about the interactions between the interleukin-1 system and the hypothalamic-pituitary-gonadal, the hypothalamic pituitary-adrenal, and the hypothalamic-pituitary-thyroid axes. We also review the ovarian role of the interleukin-1 system. This cytokine has an immense and, as yet, imperfectly understood effect on the human reproductive tract. Clearly the immune system has a potential autocrine, paracrine, and endocrine role in regulating human reproductive events such as ovulation, luteinization, and implantation.
View details for Web of Science ID A1994NM91800001
View details for PubMedID 8048225
Interleukin-1 receptor type I, IL-1 beta, IL-1 receptor antagonist, and human macrophages were immunohistochemically localized in the villous trophoblast, maternal-trophoblast interphase, and maternal decidua during early human implantation. Immunostaining for IL-1 receptor type I was present in the syncytiotrophoblast and hyperplastic endometrial glands in the maternal decidua. Immunoreactive IL-1 beta was present in the villous cytotrophoblast, syncytiotrophoblast, intermediate trophoblast, and maternal stromal decidual cells. IL-1 receptor antagonist staining was observed in the glandular endometrium of the maternal decidua and in isolated cells located inside the chorionic villi, intervillous space, and maternal decidua. Mature human macrophages, as defined by both CD/68+ and HAM56+, were present in the chorionic villi, maternal blood of intervillous space, and maternal decidua. Co-localization studies demonstrated that macrophages in all of the reported locations also stained for immunoreactive IL-1 beta. Our results show the shared presence in maternal and embryonic tissues of this receptor-agonist-antagonist system during early human implantation. This finding supports an autocrine/paracrine role for the IL-1 system in human implantation.
View details for Web of Science ID A1994NF02100011
View details for PubMedID 8157710
Because we hypothesize that the interleukin-1 (IL-1) system may be important in the dialogue between mother and embryo during the implantation process, we have analyzed the effect of IL-1 beta, a secretory product of the human embryo and human endometrium, on the mRNA and protein levels of IL-1 receptor type I (IL-1R tI) in the human endometrium. For this purpose, endometrial epithelial cells (EEC) and stromal cells (ESC) were isolated and cultured with progesterone (3.18 micrograms/mL) and epidermal growth factor (20 ng/mL) for 8 days in the presence or absence of hrIL-1 beta (20 pg/mL). EEC from proliferative and secretory endometrium expressed high levels of IL-1R tI mRNA compared to ESC, and these levels were not modulated by IL-1 beta. However, prostaglandin E2 levels peaked on day 4 in EEC treated with progesterone, epidermal growth factor, and IL-1 beta (208.7 +/- 92 ng/10(7) cells), whereas no prostaglandin E2 was detectable in cells not treated with IL-1 beta, indicating that these cells responded to IL-1 beta. With regard to ESC from secretory endometrium, IL-1 beta increased its own receptor mRNA levels (4 +/- 0.5-fold increase) after 8 days in culture. However, when ESC were isolated from proliferative endometrium, an up-regulation of IL-1R tI (3.5 +/- 0.5-fold increase) was observed on days 6 and 8 of culture regardless of the presence or absence of IL-1 beta. Immunoreactive IL-1R tI was identified in cultured EEC and ESC, and patterns similar to those of mRNA were observed. The constitutive presence of IL-1R tI in EEC, which was not affected by IL-1 beta, and the up-regulation of IL-1R tI mRNA by its ligand IL-1 beta in ESC isolated during the luteal phase suggest a role for the IL-1 system in human implantation.
View details for Web of Science ID A1994MZ30700028
View details for PubMedID 8126141
We have investigated the relevance of interleukin-1 receptor type I (IL-1R tI) in the implantation process in vivo in a murine model. Indirect immunofluorescence experiments demonstrate that IL-1R tI is located in mouse endometrial lumenal epithelium with increased intensity in the periimplantation period, whereas IL-1 beta staining is located in the mouse placenta. PMSG/human CG (hCG)-stimulated and mated 12-week-old B6C3F-1 female mice were randomly allocated to three groups: A, control noninjected; B, buffer-injected animals; and C, animals injected ip with 20 micrograms recombinant human IL-1 receptor antagonist (rhIL-1ra) every 12 h beginning on pregnancy day 3. Injections were continued until day 9, and animals were killed 12 h after the last injection. Pregnancy rates in the three groups were: noninjected, 58.8% (10 of 17); buffer-injected, 73.7% (14 of 19); rhIL-1ra-injected, 6.7% (1 of 15), P = 0.0001155, Fisher exact test. To rule out the possibility that pregnancy failure was due to an embryotoxic effect of rhIL-1ra, 2-cell mouse embryos (n = 276) were flushed from the same group of animals used for in vivo experiments and cultured with increasing concentrations of rhIL-1ra: 0 microgram/ml (n = 91), 1 microgram/ml (n = 36), 50 micrograms/ml (n = 36), 100 micrograms/ml (n = 52), and 200 micrograms/ml (n = 61) rhIL-1ra. The percentages of 2-cell mouse embryos reaching the blastocyst stage after 72 h in culture were 85.7%, 91.6%, 94.4%, 96%, and 85.2%, respectively. We further cultured these blastocysts for 5 days on fibronectin-coated plates with or without 200 micrograms/ml rhIL-1ra. In both groups, hatching, attachment to fibronectin, outgrowth, and migration were documented to be similar. Furthermore, our longitudinal morphological study of embryonic implantation in control and rhIL-1ra-injected mice shows that the blockade of IL-1R tI interferes with the attachment of mouse blastocysts to maternal endometrium in vivo. In summary, we demonstrate that blockade of maternal endometrial IL-1R tI with IL-1ra prevents implantation in the mouse by interfering with embryonic attachment, without adverse effects on blastocyst formation, hatching, fibronectin attachment, outgrowth, and migration in vitro.
View details for Web of Science ID A1994MV44300002
View details for PubMedID 8299552
The distribution of immunoreactive interleukin-1 receptor type I (IL-1R tI), IL-1 alpha, and IL-1 beta, and of macrophages, was investigated immunohistochemically in the mouse ovary during follicular growth, ovulation, and luteinization. For this purpose, an indirect immunofluorescence technique, using specific monoclonal antibodies against mouse IL-1R tI, mouse IL-1 alpha, IL-1 beta, and macrophage antigens (CD11b/CD18) was used with sections of paraffin-embedded ovaries from eCG and eCG/hCG-treated 12-wk-old B6C3F-1 female mice. During follicular development, IL-1 alpha, IL-1 beta, and IL-1R tI staining were confined to the theca-interstitial layer of growing follicles with one remarkable exception. Intense IL-1R tI still staining was present in the cytoplasm and plasma membrane of the murine oocyte. During ovulation, IL-1 alpha and IL-1 beta were still confined to the theca layer, but faint IL-1R tI staining was initiated in cumulus cells and in granulosa cells just before follicle rupture. Immediately after follicle rupture, granulosa cells stained positive for IL-1R tI, IL-1 alpha, and IL-1 beta. During luteinization, granulosa-luteal cells of the corpus luteum demonstrated strong IL-1R tI, IL-1 alpha, and IL-1 beta staining. Macrophages were detected in the theca layer and stroma, but never within the follicle before ovulation. Immediately after ovulation, there was a rapid entry of macrophages into the follicle, and macrophages were also present inside the corpus luteum. Our morphological results support a possible autocrine-paracrine role of the mouse ovarian IL-1 system in ovulation and luteinization.
View details for Web of Science ID A1994MW52100030
View details for PubMedID 8142562
Previous studies in the human suggest that the interleukin-1 (IL-1) system, may be an important paracrine/autocrine mediator in local intercellular interaction in endometrial tissue. In this study we have determined that IL-1 receptor type I (IL-1R tI) is expressed at the messenger RNA (mRNA) and protein levels in glandular cells and its ligand, IL-1 beta has been localized by immunohistochemical methods in endothelial cells and isolated stromal cells in the human endometrium throughout the menstrual cycle. IL-1R tI mRNA was detected in glandular epithelium using both specific complementary DNA and complementary RNA 32P-labeled probes. Human glandular epithelium contains a 5.1-kilobase mRNA transcript throughout the complete menstrual cycle. Quantitative densitometric analysis of slot blot hybridization signals shows an increase of IL-1R tI mRNA in both early and mid-late secretory phases in comparison with the proliferative phase (P < 0.05). IL-1R tI protein was localized in endometrial glandular epithelial cells using both indirect immunofluorescence and avidin-biotin-peroxidase methods. However, more intense staining for IL-1R tI was observed in lumenal epithelial cells compared with the staining present deep in the endometrial glands. Using the same methods, IL-1 beta was detected in endothelial cells of spiral vessels and isolated stromal cells throughout the menstrual cycle, and an increased staining from proliferative to secretory phase was observed. The detection of IL-1R tI in the human endometrial epithelium and its ligand, IL-1 beta, in isolated stromal cells and endothelial cells, is another example of possible communication between the immune and reproductive systems with special relevance to human implantation.
View details for Web of Science ID A1993LR71900047
View details for PubMedID 8345061
To investigate the messenger ribonucleic acid (mRNA) expression of interleukin-1 (IL-1) type I receptor in the endometrial tissue of normal patients during the menstrual cycle.Prospective longitudinal study.Department of Obstetrics and Gynecology, Stanford University Medical Center, Stanford, California.Twenty fertile women between 19 and 41 years of age underwent hysterectomy for benign reasons (n = 9) and laparoscopy for tubal ligation (n = 11). In all cases, endometriosis was not visualized.Endometrial biopsy using the Novak curette was obtained at the time of surgery.Total RNA extracted from unfractioned endometrial tissue was analyzed on Northern blots by using specific complementary deoxyribonucleic acid probes.We found IL-1 type I receptor mRNA expression in endometrial tissue throughout the entire menstrual cycle. However, IL-1 type I receptor mRNA levels were significantly higher during both early and late luteal phases than follicular and midluteal phases.Our results demonstrate the presence of the IL-1 system in the human endometrium and that the receptor is regulated throughout the menstrual cycle with a 4.1-fold increased expression of the IL-1 receptor gene in the early luteal phase compared with preovulatory endometrium.
View details for Web of Science ID A1993KV24900014
View details for PubMedID 8458498
Plasminogen activators and their inhibitors have been implicated in the process of fibrinolysis, tissue remodeling, and ovulation. Epidermal growth factor (EGF), a paracrine hormone found in the human ovary, increases plasminogen activator (PA) activity and the gene expression of PA and plasminogen activator inhibitor (PAI) in human endothelial cells and human cell lines. Gonadotropins also increase PA activity and gene expression in rat preovulatory granulosa cells. We have now analyzed the gene expression of PAI-1 and PAI-2 in uncultured human cumulus cells (CC), uncultured granulosa-luteal cells (GLC), and cultured GLC obtained from preovulatory follicles of patients undergoing assisted reproductive technologies. We also studied the effects of hCG and EGF on PAI-1 and PAI-2 mRNA levels in cultured GLC; GLC were cultured in serum-free medium for various times within 24 h with or without hCG and for 6 h with or without hCG, EGF, or EGF plus hCG. Total RNAs from CC and GLC were extracted, and blot hybridizations with 32P-labeled PAI-1, PAI-2, or 28S ribosomal RNA cDNA probes were performed. Both CC and GLC expressed PAI-1 and PAI-2 genes. In GLC, steady state levels of PAI-1 mRNA levels steadily increased within 24 h of culture, whereas PAI-2 levels peaked at 6 h of culture. PAI-1 mRNA levels were not affected by hCG or EGF at 6 h of culture, but PAI-2 mRNA levels were significantly increased by EGF at 6 h of culture. These studies demonstrate that human GLC PAI-1 and PAI-2 mRNA levels are differentially regulated and suggest that EGF may be involved in modulation of the human ovarian PA system during the periovulatory period.
View details for Web of Science ID A1993KM44500048
View details for PubMedID 8432798