Antioxidant supplementation reduces endometriosis-related pelvic pain in humans.

We previously suggested that women with endometriosis have increased oxidative stress in the peritoneal cavity. To assess whether antioxidant supplementation would ameliorate endometriosis-associated symptoms, we performed a randomized, placebo-controlled trial of antioxidant vitamins (vitamins E and C) in women with pelvic pain and endometriosis. Fifty-nine women, ages 19 to 41 years, with pelvic pain and history of endometriosis or infertility were recruited for this study. Patients were randomly assigned to 2 groups: vitamin E (1200 IU) and vitamin C (1000 mg) combination or placebo daily for 8 weeks before surgery. Pain scales were administered at baseline and biweekly. Inflammatory markers were measured in the peritoneal fluid obtained from both groups of patients at the end of therapy. Our results indicated that after treatment with antioxidants, chronic pain ("everyday pain") improved in 43% of patients in the antioxidant treatment group (P = 0.0055) compared with the placebo group. In the same group, dysmenorrhea ("pain associated with menstruation") and dyspareunia ("pain with sex") decreased in 37% and 24% patients, respectively. In the placebo group, dysmenorrhea-associated pain decreased in 4 patients and no change was seen in chronic pain or dyspareunia. There was a significant decrease in peritoneal fluid inflammatory markers, regulated upon activation, normal T-cell expressed and secreted (P ≤ 0.002), interleukin-6 (P ≤ 0.056), and monocyte chemotactic protein-1 (P ≤ 0.016) after antioxidant therapy compared with patients not taking antioxidants. The results of this clinical trial show that administration of antioxidants reduces chronic pelvic pain in women with endometriosis and inflammatory markers in the peritoneal fluid.

The mouse gamete adhesin, SED1, is expressed on the surface of acrosome-intact human sperm.

OBJECTIVE: To determine whether SED1, a protein secreted by the mouse epididymis that coats sperm and participates in sperm adhesion to the zona pellucida, is present on human sperm and in human epididymal tissue. DESIGN: SED1 expression was analyzed by immunoblot and indirect immunofluorescence assays. SETTING: Academic clinical and research laboratories. PATIENT(S): Human breast milk was donated. Unused semen was donated by men presenting for semen analysis or in vitro fertilization (IVF). Cadaveric epididymal tissue was obtained from the institutional body donor program. INTERVENTION(S): Human milk fat globule membranes and human seminal plasma proteins were analyzed by immunoblot. Human sperm and epididymis were analyzed by indirect immunofluorescence microscopy. Acrosomal status was determined by staining with fluorescein isothiocyanate-Pisum sativum agglutinin. MAIN OUTCOME MEASURE(S): Immunoblot and indirect immunofluorescence assays. RESULT(S): Human SED1 is recognized by two different polyclonal anti-SED1 antisera. SED1 is localized to the plasma membrane of human sperm overlying the intact acrosome. In acrosome-reacted sperm, SED1 is localized to the equatorial segment. SED1 is expressed by the epithelium of the anterior caput epididymis. CONCLUSION(S): SED1 is expressed on the surface of acrosome-intact human sperm and in the anterior caput of the human epididymis, similar to that seen in mouse.

Ovulation induction in a poor responder with panhypopituitarism: a case report and review of the literature.

BACKGROUND: Most women with panhypopituitarism will undergo successful ovulation induction with gonadotropin therapy. Few proven treatment options exist for those who respond poorly to such therapy. A poor response may indicate diminished ovarian reserve, or reflect a deficiency of other key components for ovarian function. CASE: A 31-year-old female with panhypopituitarism and a poor response to gonadotropin therapy took growth hormone (GH) replacement for 4 months prior to restarting gonadotropins. When the serum level of insulin-like growth factor-I normalized, she began ovulation induction with gonadotropins with transdermal estradiol. After 63 days of gonadotropin therapy, she had a leading follicle of 18 mm, followed by follicles of 16.5 mm and 15.5 mm. The serum estradiol was 796 pg/ml, and human chorionic gonadotropin was administered. The patient conceived with timed intercourse. A prior attempt at ovulation induction with gonadotropins alone failed to produce follicular development. CONCLUSION: Prolonged gonadotropin treatment may be necessary to achieve ovulation and avoid the misdiagnosis of ovarian failure. Co-treatment with GH and estrogen may improve the follicular response in a poor responder with panhypopituitarism.

Glycodelin mediates the increase in vascular endothelial growth factor in response to oxidative stress in the endometrium.

OBJECTIVE: We hypothesized that glycodelin stimulates vascular endothelial growth factor (VEGF) expression in response to oxidative stress. STUDY DESIGN: EM42 (human endometrial epithelial cell line) and primary endometrial epithelial cells were subjected to oxidative stress with minimally oxidized low density lipoprotein (mLDL). Cells were also incubated with no LDL (control) or native LDL (nLDL). Each condition was incubated with and without glycodelin antibody. Glycodelin and VEGF protein and messenger RNA (mRNA) levels were analyzed. Primary cells were cultured with glycodelin peptide to evaluate the effect on VEGF protein and mRNA. RESULTS: Glycodelin and VEGF protein and mRNA were higher for cells grown with mLDL (P < .05), while glycodelin antibody attenuated the increase in VEGF protein (P < .01). Glycodelin peptide increased VEGF mRNA and protein (P < .05). CONCLUSION: Glycodelin may act as an autocrine factor within endometriotic implants to increase VEGF expression during oxidative stress.

Effect of glycodelin on the production of vascular endothelial growth factor in cumulus cells.

Glycodelin modulates vascular endothelial growth factor (VEGF) production in cumulus cells in vitro. Patients with normal gonadotropin responses who were undergoing IVF demonstrated increased VEGF production to glycodelin, whereas poor responders had a decreased response to glycodelin.

RU486 inhibits expression of lysophosphatidic acid induced glycodelin.

OBJECTIVE: This study was undertaken to provide evidence for the mode of action of RU486 on glycodelin produced in K562 cells. To show that histiocytes may be a source of glycodelin in leiomyoma. STUDY DESIGN: With the use of K562, a leukemia cell line, the effect of lysophosphatidic acid (LPA), RU486, antioxidants, and ZK112,993 on glycodelin protein and gene expression was studied. Immunocytochemistry for glycodelin and HAM-56 (macrophage) was performed on leiomyoma and myometrium. RESULTS: Incubation of K562 cells with LPA, progesterone, ZK112,933 and RU486 significantly induced the expression of glycodelin protein and messenger RNA. The addition of RU486 to LPA activated cells markedly reduced expression of glycodelin. Addition of ZK112,993, an antiprogestin without antioxidant properties, to LPA activated cells did not reduce glycodelin. Histiocytes in leiomyoma and myometrium co-localize with glycodelin. CONCLUSION: RU486, partly acting as an antioxidant, markedly reduces LPA stimulated glycodelin production. Histiocytes in leiomyoma and myometrium immunostain for glycodelin and suggests a source for glycodelin in leiomyoma.

The presence of endometrial cells in the peritoneal cavity enhances monocyte recruitment and induces inflammatory cytokines in mice: implications for endometriosis.

OBJECTIVE: To determine the inflammatory response in the peritoneal cavity by the presence of endometrial cells and the role of the mesothelium. DESIGN: In vivo study using mice. SETTING: University research laboratory. ANIMAL(S): Female Swiss Webster mice, 8 to 10 weeks old. INTERVENTION(S): Homogenous mouse endometrial epithelial and stromal cells were injected intraperitoneally. Peritoneal lavage and mesothelium were collected 4 to 72 hours after the administration. MAIN OUTCOME MEASURE(S): We determined the number of peritoneal macrophages, and the production and gene expression of monocyte chemotactic protein-1 (MCP-1/JE), interleukin 1alpha (IL-1alpha), and interleukin 6 (IL-6). RESULT(S): The intraperitoneal administration of endometrial cells increased the number of peritoneal macrophages, production of MCP-l, IL-1alpha, and IL-6, and expression of mesothelial MCP-1/JE, IL-1alpha, and IL-6 genes in recipient mice. CONCLUSION(S): These results suggest that retrograde menstruation could account for the increased presence of inflammatory mediators in the peritoneal cavity of women with endometriosis. The mesothelium could play an active role in endometriosis in addition to providing an attachment stratum for the endometrial cells.

Presence of endometrial epithelial cells in the peritoneal cavity and the mesothelial inflammatory response.

OBJECTIVE: To determine the contribution of endometrial cells in the development of endometriosis. Specifically the response of the mesothelium to endometrial cells in the production of monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), and IL-8 was studied. DESIGN: In vitro study. SETTING: University Research Laboratory. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Cellular MCP-1, IL-6 secretion and MCP-1, and IL-6 and IL-8 messenger RNA expression were evaluated by ELISA and reverse transcription-polymerase chain reaction (RT-PCR) assay. RESULT(S): The mesothelial cells produced more MCP-1 and IL-6 than endometrial epithelial and stromal cells. Mesothelial cells cultured in the presence of endometrial epithelial cells produced even greater levels of MCP-1 and IL-6 than those cultured in the presence of stromal cells or cultured alone. The MCP-1, IL-6, and IL-8 mRNA expression also increased when mesothelial cells were co-cultured with endometrial epithelial cells. CONCLUSION(S): The results suggest that endometrial epithelial cells may be important in evoking the inflammatory reaction in the peritoneal cavity during retrograde menstruation and that mesothelial cells may play an important role in the chemotaxis of monocytes and in the inflammatory process during the development of endometriosis.

Induction of monocyte chemotactic protein-1 in peritoneal mesothelial and endometrial cells by oxidized low-density lipoprotein and peritoneal fluid from women with endometriosis.

OBJECTIVE: To elucidate the effect of oxidized low-density lipoprotein (LDL) and peritoneal fluid of women with endometriosis on monocyte chemotactic protein-1 (MCP-1) production by peritoneal mesothelial cells and endometrial cells. DESIGN: In vitro study. SETTING: University medical center. PATIENT(S): Five women undergoing surgery for pelvic pain, infertility, or endometriosis; five women without endometriosis who were undergoing tubal ligation were the controls. INTERVENTION(S): Mesothelial cells and endometrial cells in culture were treated with oxidized LDL and peritoneal fluid from control and endometriosis patients, then MCP-1 levels were measured. MAIN OUTCOME MEASURE(S): ELISA was used to measure MCP-1 in the culture supernatants exposed to oxidized LDL and peritoneal fluid from control and endometriosis patients. Cellular MCP-1 messenger RNA expression was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) assay. RESULT(S): Treatment with oxidized LDL caused an increase in accumulation of immunoreactive MCP-1 in the medium of cultured mesothelial and endometrial cells (primary endometrial stromal cells and endometrial cell line EM42). The mesothelial cells secreted more MCP-1 than did endometrial cells under the culture condition. The EM42 cells cultured in the presence of peritoneal fluid from endometriosis patients secreted more MCP-1 than those cultured with peritoneal fluid from normal women. However, no differences were found in MCP-1 levels in the supernatant of endometrial stromal cells cultured with peritoneal fluid. CONCLUSION(S): This is the first report of MCP-1 expression in mesothelial cells induced by oxidized LDL, and provides direct evidence of inflammatory action of peritoneal fluid of women with endometriosis.

Glycodelin levels in uterine flushings and in plasma of patients with leiomyomas and polyps: implications for implantation.

BACKGROUND: Glycodelin, a glycoprotein, is present in both blood plasma and uterine flushings. It has been implicated in the process of implantation and angiogenesis. During the secretory phase, progesterone secretion is related to glycodelin production. METHODS AND RESULTS: We obtained uterine flushings, prospectively, from 47 infertile patients during the proliferative phase. Patients were recruited from our university practice. Transvaginal ultrasound and sonohysterography permitted the stratification of patients into control, leiomyoma or polyp groups. Total plasma and uterine flushing glycodelin was measured with enzyme-linked immunosorbent assay. Blood was also analysed for progesterone. Uterine flushing glycodelin levels were significantly increased in patients with polyps when compared with controls. An increase in uterine flushing glycodelin levels was noted in patients with leiomyomas compared with controls, though not statistically significant. Plasma glycodelin levels were significantly increased in patients with leiomyomas and polyps when separately compared with controls. There was a significant relationship between plasma glycodelin production and progesterone levels in patients with polyps. CONCLUSIONS: Leiomyomas and polyps are growing tumours and thus produce significant plasma glycodelin levels. Uterine glycodelin flushings are elevated in patients with both polyps and leiomyomas. Elevated glycodelin levels in the follicular and peri-ovulatory period may impair fertilization and implantation.

Clinical aspects of endometriosis.

Endometriosis is one of the most commonly encountered gynecologic diseases requiring medical and/or surgical therapy. It is a leading cause of hysterectomy in the United States and has significant associated morbidity. The most frequent symptoms of genital tract endometriosis are dysmenorrhea, dyspareunia, chronic pelvic pain, and infertility. Endometriosis occurs in the pelvis, most commonly the ovaries and the dependent areas covered with peritoneum. Diagnosis requires surgical intervention and is usually made by laparoscopy. In women being evaluated for pelvic pain, the diagnosis of endometriosis is made frequently (40-60%) and varies with the population being studied. Infertility and endometriosis have long been associated. Although women with infertility may have pelvic pain, subfertility (20-30%) can be the only presenting symptom. In asymptomatic women, the diagnosis of endometriosis ranges from 2% to 22% of reproductive-age women. Its true incidence and natural history remain to be clarified. Endometriosis is a significant public health issue because of the large number of women it affects and the significant morbidity associated with this disease.

Macrophages, oxidation, and endometriosis.

Retrograde menstruation has been suggested to be the cause for the presence of endometrial cells in the peritoneal cavity. However, little is known about the events that lead to the adhesion and growth of these cells that ultimately result in endometriosis, considering the fact that the disease occurs only in certain women despite the common occurrence of retrograde menstruation in most women. We postulate that, in normal women, the endometrial cells and tissue that arrive in the peritoneal cavity during menstruation are effectively removed by macrophages that are chemoattracted and become resident tissue macrophages in the peritoneal cavity. In contrast, the peritoneal macrophages in women with endometriosis are nonadherent and ineffectively scavenged, resulting in the sustained presence and growth of the endometrial cells. We also postulate that the peritoneal fluid is not a passive reservoir of the factors secreted by cells of the peritoneum, but actively promotes endometriosis. The peritoneal fluid is rich in lipoproteins, particularly low-density lipoprotein, which generates oxidized lipid components in a macrophage-rich inflammatory milieu. The oxidants exacerbate the growth of endometriosis by inducing chemoattractants such as MCP-1 and endometrial cell growth-promoting activity. We provide evidence for the presence of oxidative milieu in the peritoneal cavity of women with endometriosis, the nonscavenging properties of macrophages that are nonadherent, and the synergistic interaction between macrophages, oxidative stress, and the endometrial cells. For example, the peritoneal fluid lipoproteins of subjects with endometriosis have increased the propensity to undergo oxidation as compared with plasma lipoproteins, and the subjects also have increased titer of autoantibodies to oxidatively modified proteins. If the oxidative proinflammatory nature of the peritoneal fluid is an important mediator of endometriosis growth, anti-inflammatory agents and antioxidants might afford protection against endometriosis.

Atherosclerosis, oxidation and endometriosis.

Endometriosis affects younger women of childbearing age. Atherosclerosis is considered as a disease of the old and increases with the ageing process. Both diseases are characterized by the increased presence of activated macrophages and associated increases in growth promoting activity and the production of inflammatory cytokines. In this review, we propose that oxidative stress and the presence of forms of oxidized low-density lipoprotein (LDL) might contribute to both Atherosclerosis and Endometriosis.