Interleukin-8 expression and modulation in human preovulatory follicles and ovarian cells.

Just before the time of ovulation, the number of neutrophils increases markedly in the thecal layer of the leading follicle. A preovulatory rise in chemotactic activity for neutrophils in human follicular fluid has also been detected. We hypothesized that interleukin-8 (IL-8), a neutrophil chemoattractant/activating factor and a potent angiogenic agent, may be an important modulator of leukocyte chemotaxis in ovulatory function. In this regard we investigated the expression and modulation of IL-8 in human follicular fluid samples from patients undergoing in vitro fertilization-embryo transfer therapy and in ovarian stromal and granulosa-lutein cell cultures. The concentration of IL-8 in pre-hCG follicular fluid samples (n = 4) was 16 +/- 12 (mean +/- SEM) pg/ml, and that in post-hCG samples (n = 101) was 262 +/- 45 pg/ml (P = 0.001). In post-hCG samples, the concentration of IL-8 in an individual follicle correlated with the size of that follicle (r = 0.61; P = 0.02). We also observed a correlation between serum IL-8 levels (22 +/- 3 pg/ml) and follicular fluid levels (303 +/- 143 pg/ml), with a 14-fold gradient (r = 0.71; P = 0.01) in 11 patients tested for both. IL-8 messenger RNA (mRNA) and the protein were expressed constitutively in ovarian stromal cell cultures, and the level was increased by IL-1 alpha and tumor necrosis factor-alpha in a time- and concentration-dependent manner. hCG and LH induced higher levels of IL-8 mRNA expression and protein production. Granulosalutein cells also expressed IL-8 mRNA and protein, and the levels were increased by IL-1 alpha and tumor necrosis factor-alpha. Importantly, progesterone suppressed both basal and IL-1 alpha-stimulated IL-8 expression in stromal and granulosa-lutein cell types. In summary, we found that IL-8 levels are elevated in periovulatory follicular fluid, and both granulosa-lutein and ovarian stromal cells express the mRNA and produce the protein. The modulation of IL-8 in these cell cultures by steroid and trophic hormones suggests that IL-8 may play an important role in the physiology of ovulation, such as timely follicular rupture and neovascularization of the corpus luteum.

Modulation of leukemia inhibitory factor gene expression and protein biosynthesis in human endometrium.

Implantation is the process by which the blastocyst becomes intimately connected with the maternal endometrium/decidua. The independently developing preimplantation blastocyst then becomes dependent on the maternal environment for its continued development. Although the factors involved in the regulation of blastocyst implantation are incompletely understood, recent studies strongly suggest a critical role for leukemia inhibitory factor (LIF) in mice. We showed previously that LIF acts on human trophoblasts to shift their differentiation toward the anchoring phenotype by increasing the synthesis of fibronectin. In the present study, we first evaluated the temporal expression of LIF in the human endometrium in order to gain further insights into the role of LIF in human implantation. We established the LIF is expressed in the endometrium in a menstrual cycle-dependent manner. The most abundant LIF messenger ribonucleic acid (mRNA) levels are observed in the mid- and late-luteal phase samples. LIF mRNA was also present in decidual tissues of first trimester of pregnancy, but levels were lower than those found in the midluteal endometrium. We then investigated the regulation of LIF expression in human endometrial cells in culture by cytokines, steroid hormones, and growth factors. We could not show any direct stimulatory evidence of steroid hormones (estradiol and progestins) on LIF mRNA expression or protein production by endometrial cells in culture. On the other hand, we showed that interleukin-1, tumor necrosis factor-alpha, platelet-derived growth factor, epidermal growth factor, and transforming growth factor-beta are potent inducers of LIF expression in endometrial stromal cells in culture in a concentration- and time-dependent manner. Interferon-gamma acted to inhibit LIF expression induced by these cytokines. In contrast, we found high constitutive and relatively less regulated levels of LIF mRNA in the endometrial gland cells. The timing of the expression of LIF in the endometrium suggests a potential role in the implantation. The regulation of the expression of LIF may have an important role in the physiological and pathological processes involving human implantation.

Sex steroids mediate HOXA11 expression in the human peri-implantation endometrium.

Under the influence of sex steroids, human endometrium undergoes sequential development in preparation for implantation. Hoxa11 is essential for implantation in the mouse. Here we describe a potential role for HOXA11 in human endometrial development and implantation. Northern analysis demonstrates that HOXA11 is expressed in a menstrual cycle phase-dependent fashion in adult human endometrium. HOXA11 messenger RNA levels dramatically increase at the time of implantation and remain increased in pregnancy. In vitro, HOXA11 expression is increased in response to estrogen or progesterone. There is a dose-responsive increase over the physiologic range of progesterone concentration. Pretreatment with Cyclohexamide does not decrease the response to estrogen. Steroids are novel regulators HOX gene expression. The spatial and temporal pattern of HOXA11 expression in the human endometrium suggests a role in endometrial development, implantation, and maintenance of pregnancy.

Serum progesterone levels predict success of in vitro fertilization/embryo transfer in patients stimulated with leuprolide acetate and human menopausal gonadotropins.

Serum progesterone (P4) levels greater than 2.86 nmol/L (0.9 ng/mL) on the day of hCG administration are reportedly associated with decreased pregnancy rates in in vitro fertilization/embryo transfer (IVF/ET) cycles. To further assess this phenomenon we measured serial serum P4, LH, and estradiol levels in 115 consecutive patients undergoing stimulation for IVF/ET with midluteal leuprolide acetate and human menopausal gonadotropins. IVF/ET cycle outcome was retrospectively correlated with P4 levels on the day of hCG administration. Two critical breakpoints were identified, 1.27 nmol/L (0.4 ng/mL) and 286 nmol/L (0.9 ng/mL). Clinical pregnancies occurred in 9 of 18 patients in group I (P4, less than 1.27 nmol/L) compared to 11 of 81 patients in group II (1.27 less than P4 less than 2.86 nmol/L; P = 0.001) and 0 of 14 patients in group III (P4, less than or equal to 2.86 nmol/L) (P = 0.001). Eleven patients in group III had cryopreservation of embryos during that cycle. Six subsequently underwent frozen embryo transfer, and clinical pregnancies occurred in 2, both of whom have delivered. These findings demonstrate that even modest increases in serum P4 levels (greater than 1.27 nmol/L) are associated with reduced pregnancy rates in IVF/ET cycles. In addition, it appears that the mechanism may not exclusively involve poor oocyte quality.

Interleukin-8 induces proliferation of endometrial stromal cells: a potential autocrine growth factor.

Proliferation of endometrium is dependent on sex steroid hormones, but specific growth factors are likely to play an important role in regulating this process. A number of cytokines and growth factors are synthesized in the endometrium in response to sex steroid hormones and act to regulate endometrial function. Endometrial cells produce interleukin-8 (IL-8) both in vivo and in vitro. We hypothesized that IL-8, a neutrophil chemoattractant/activating factor and a potent angiogenic agent that has been shown to stimulate growth in other cell types, may directly stimulate proliferation of endometrial cells. We first investigated the effect of IL-8 and mouse antihuman-IL-8 neutralizing antibody on endometrial stromal cell proliferation using both a colorimetric assay and thymidine uptake. We then investigated the modulation of endometrial stromal cell IL-8 production and proliferation by antisense oligonucleotides specific for IL-8. There was a concentration-dependent increase of cell proliferation with IL-8 (2-fold at 1 ng/mL; P < 0.01 between control and concentrations above 0.01 ng/mL) and a concentration-dependent inhibition of cell proliferation with anti-IL-8 antibody (to 30% of the control at 1 microg/mL; P < 0.01 between control and concentrations above 0.1 microg/mL). IL-8 antisense oligonucleotide treatment decreased IL-8 production by endometrial stromal cells in culture as well as cell proliferation when it is compared with scrambled (nonsense) oligonucleotide treatment (P < 0.01). Addition of IL-8 (1 ng/mL) reversed the proliferation inhibitory effect of IL-8 antisense oligonucleotides. We propose that IL-8 may act as an autocrine growth factor in the endometrium, and suggest that it may also play a role in the pathogenesis of endometriosis.

The effect of leukemia inhibitory factor (LIF) on trophoblast differentiation: a potential role in human implantation.

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein strongly associated with normal implantation in the mouse. We have recently determined that LIF is expressed in the human endometrium in a menstrual cycle dependent manner. Maximal expression is observed between days 19 and 25 of the menstrual cycle, coinciding with the time of human implantation. In this study we have utilized purified cultures of human cytotrophoblasts to examine the effects of LIF on several morphologic and biochemical markers of the trophoblastic differentiation. We purified human cytotrophoblasts from term placentae and cultured them with and without LIF (10 ng/mL). The secretion of human CG, oncofetal fibronectin, and progesterone were measured at 24, 48, 72, and 96 h. Northern blot analysis was used to assess messenger RNA (mRNA) expression of beta hCG and oncofetal fibronectin. We found that LIF markedly decreased trophoblast production of hCG protein at 72 and 96 h, as well as expression of beta hCG mRNA. LIF also significantly increased the expression of oncofetal fibronectin mRNA and secretion of the protein. LIF did not affect steroidogenic activity of cultured trophoblasts, as determined by progesterone production. These biochemical changes are characteristic of cytotrophoblast differentiation toward an anchoring extravillous phenotype. Thus, LIF appears to be an important regulator of human embryonic implantation by directly modulating trophoblast differentiation.

Review of the evidence for treatment of leiomyomata.

Leiomyomata of the uterus are common tumors of women, and a number of treatments are now available for symptoms of this disorder. Surgical treatments include hysterectomy, abdominal myomectomy, laparoscopic myomectomy, and myolysis. Nonsurgical treatments include uterine artery embolization and medical therapy. All have a small amount of accumulated data, but existing studies are generally small and of poor quality. There is a strong need for appropriately designed and analyzed randomized clinical trials, and surgical trials should preferably be multicenter/multisurgeon. In summary, although a large array of options exist, there are still little data upon which to base decisions regarding optimal treatment approaches.

Limited hormonal responsiveness of ectopic endometrium: histologic correlation with intrauterine endometrium.

In order to assess the hormonal responsiveness of ectopic endometrium, 438 unselected endometrial implants and corresponding intrauterine endometrium from 196 patients were evaluated and classified by standard endometrial dating criteria. Only 13% of the endometrial implants were histologically synchronous with the corresponding intrauterine endometrium. Both proliferative and secretory implants were present in relatively constant proportions throughout the menstrual cycle, demonstrating a lack of correlation with cyclic endogenous hormones. A significant percentage (range, 25% to 49%) of endometrial implants displayed some form of local hemorrhage irrespective of the menstrual cycle timing. Sixty percent of the patients had evidence of hemorrhage in at least one implant. In women receiving hormonal therapy at the time of surgery, the proportion of endometrial implants that were histologically in concert with the corresponding endometrium ranged from 0% to 33%. In early pregnancy and menopause, 50% and 31% of endometrial implants were histologically similar, respectively. These data indicate that the hormonal responsiveness of endometrial implants is unpredictable and inconsistent.

Endometriosis and mullerian anomalies.

Although numerous etiologies for endometriosis have been proposed, it is clear that retrograde menstruation and cell-mediated lymphocytotoxicity each play a significant role in the disease's development. A comprehensive theory of pathogenesis of endometriosis holds that development of the disorder depends upon amount of retrograde menstruation and the ability of the immune response to remove the debris. To test this theory, 64 women with mullerian anomalies and intra-abdominal surgery were evaluated for the presence or absence of endometriosis, patency of tubes, hematocolpos or hematometra, and outflow obstruction. Results demonstrated that endometriosis was present in ten of 13 women with functioning endometrium, patent tubes, and outflow obstruction, whereas it could be identified in only 16 of 43 women with no obstruction (77 versus 37%, P less than .01). Similarly, eight of nine women with hematocolpos or hematometra had endometriosis, while only 18 of 47 with functioning endometrium but no hematometra/hematocolpos had it (89 versus 38%, P less than .01). None of the eight women without endometrium had endometriosis. These data support the concept that an increase in retrograde menstruation will increase the likelihood of endometriosis.

Patient education in a women's hospital: attitudes of the medical and nursing staffs.

A survey was conducted among the medical and nursing staff of Prentice Women's Hospital in an attempt to examine attitudes toward patient education. Ninety-two percent of the obstetrician-gynecologists were surveyed, as were 81% of the nursing staff. Areas of evaluation included current practices at the hospital, attitudes toward patient education, principles of patient education, attitudes toward territorial imperative, perceptions of educational background, and perceptions of current role. Results indicate considerable disorganization in educational practices at the present time. Attitudes toward and understanding of principles of patient education did not differ between the two groups studied. However, there was strong disagreement in issues of patient ownership. Perceptions of educational training of, as well as current roles played by, each profession in educating the patient were highly polarized. The implications of such interprofessional conflict are discussed.

Endometriosis: pathogenetic implications of the anatomic distribution.

The authors have reassessed the anatomic distribution of ectopic endometrium by the laparoscopic study of the location of implants, adhesions, and uterine position in 182 consecutive patients with infertility and endometriosis. The ovary was the most common site of implants with 54.9% having either unilateral or bilateral involvement. This was followed, in order of frequency, by the posterior broad ligament (35.2%), the anterior cul-de-sac (34.6%), the posterior cul-de-sac (34.0%), and the uterosacral ligament (28.0%). Adhesion formation followed the same anatomic distribution. No patients were noted to have endometriosis of the cervix and vagina. Endometriosis of the anterior compartment (anterior cul-de-sac, anterior broad ligament, and anterior uterine serosa) was significantly more common in patients with anterior uteri (40.7%) versus patients with posterior uteri (11.8%, P less than .0005). Exclusive anterior compartment disease was found only in patients with anterior uteri, and significantly more commonly in patients with severely anteflexed uteri (P less than .005). These data suggest that factors influencing implantation of retrograde menstrual debris include: the dependent pooling of peritoneal fluid as affected by uterine position; epithelial cell type at the site of implantation; unique ovarian susceptibility; route of entry; and mobility of the pelvic structures. The data support the Sampson hypothesis of retrograde menstruation as the primary model of development of endometriosis.

Reevaluation of hysterosalpingography in infertility investigation.

The hysterosalpingogram has been extensively used in infertility investigations to assess tubal patency, however, the diagnostic reliability of this technique is not known. Two hundred thirty-one consecutive hysterosalpingograms were retrospectively evaluated. Sixty-two percent (143) of the patients subsequently underwent laparoscopy. Comparison of hysterosalpingogram and laparoscopic findings revealed a 15.9% false positive tubal patency rate and a 14.9% false negative tubal patency rate. Seventy-six percent of laparoscopies revealed previously undiagnosed intraperitoneal disease. Seventeen percent of hysterosalpingograms demonstrated intrauterine pathology. There was a 0.9% major complication rate with hysterosalpingograms due to two cases of acute pelvic inflammatory disease. No significant laparoscopic complications were noted. The results suggest that laparoscopy provides a more accurate assessment of tubal patency and peritoneal factors than hysterosalpingogram in the investigation of infertility.

Duplicate cervix and vagina associated with infertility, endometriosis, and chronic pelvic pain.

BACKGROUND: Müllerian anomalies are associated with several gynecologic complications including endometriosis, infertility, and pelvic pain. CASE: A woman with duplicate cervix and a non-communicating longitudinal vaginal septum, but no other uterine anomalies, presented with pelvic pain, secondary infertility, and a long history of endometriosis. She was treated with operative laparoscopy and excision of the vaginal septum. CONCLUSION: A thorough evaluation, including history, physical examination, and appropriate imaging techniques (hysterosalpingography and magnetic resonance imaging) facilitates accurate diagnosis of anatomical defects and any associated disease in cases of unusual müllerian anomalies. An accurate preoperative diagnosis allows a planned, efficient surgical approach.

A case of endometrial cancer following endometrial ablation for dysfunctional uterine bleeding.

BACKGROUND: A concern voiced by critics of endometrial ablation is the possibility that cryptic endometrial adenocarcinoma may develop after such ablation. To date, this concern has been theoretical. CASE: A patient presented with vaginal bleeding and was diagnosed with endometrial carcinoma 5 years after ablation of the superficial endometrium. CONCLUSION: The possibility exists for endometrial carcinoma to develop years after endometrial ablation. Careful patient selection, thorough evaluation of any post-procedure bleeding, careful postoperative surveillance, and continued evaluation of the procedure and its long-term efficacy are indicated.

Growth-regulated alpha expression in the peritoneal environment with endometriosis.

OBJECTIVE: To investigate the presence and modulation of growth-regulated alpha, a member of the chemokine family with neutrophil chemotactic activity, in the peritoneal fluid of women with or without endometriosis. METHODS: Peritoneal fluid samples were obtained at laparoscopy from 63 women with endometriosis and 19 fertile women without endometriosis. Endometrial tissue was obtained from uteri after hysterectomy for reasons other than endometrial disease or from endometrial biopsies of reproductive-age women. Cellular RNA was extracted and northern blots were hybridized with an oligonucleotide probe complementary to a specific sequence of growth-regulated alpha messenger RNA. Growth-regulated alpha in peritoneal fluid and culture supernatant was quantified using enzyme-linked immunosorbent assay. Statistical analyses were performed using Kruskal-Wallis and Mann-Whitney tests. RESULTS: The median (range) concentration of growth-regulated alpha in peritoneal fluid samples from 19 normal fertile women was 27 pg/mL (0-108), from 24 women with moderate endometriosis 34 pg/mL (8-150), and from seven with severe endometriosis was 73 pg/mL (10-221) (P = .04, P = .01, respectively). In the moderate and severe endometriosis groups, the levels of growth-regulated alpha were significantly higher in the peritoneal fluid of women with untreated endometriosis (73 pg/mL [10-221]) than in women with medically treated endometriosis (25 pg/mL [8-47]). In mesothelial and endometrial stromal cells in culture, growth-regulated alpha messenger RNA and protein were detectable constitutively; however, both interleukin-1 alpha and tumor necrosis factor-alpha induced higher levels of growth-regulated alpha messenger RNA and protein in a dose- and time-dependent manner. CONCLUSIONS: Growth-regulated alpha levels are elevated in the peritoneal fluid of women with moderate and severe endometriosis. This chemotactic factor, which acts via the interleukin-8 receptor, may play a role in the pathogenesis of endometriosis.

Method for the transportation of peritoneal macrophages.

This report outlines a method of PF transport that preserves the viability and activation level of peritoneal macrophages. Collection of macrophages in this manner will allow transport of PF from multiple centers for evaluation of macrophage function in a single laboratory. Such technology will make it possible to intensively study the relationship between peritoneal macrophages and infertility.

Treatment of endometriosis-associated infertility with CO2 laser laparoscopy: the use of one- and two-parameter exponential models.

Laparoscopic vaporization with the carbon dioxide laser has become a popular treatment modality for endometriosis-associated infertility, yet little data exist regarding the efficacy of such an approach. The authors have applied sophisticated modeling techniques to pregnancy rates in 129 endometriosis patients following laser laparoscopy. Patients were identified for stage of disease, danazol usage before and after surgery, and identifiable infertility factors. Fertility rates were analyzed with monthly fecundity rates and a two-parameter exponential model. Results indicate monthly fecundity rates comparable to other forms of therapy for stage I and stage II endometriosis, but substantially higher than more traditional modalities for stage III disease. The two-parameter model revealed "cure" rates comparable or lower than with major conservative surgery or danazol therapy, but a "monthly" probability of pregnancy" substantially higher than with other treatments. These results indicate that, in experienced hands, laser laparoscopic vaporization of endometriosis is as efficacious as other published forms of therapy, including use in moderate and severe disease. In addition, these results suggest an immediate, transient effect upon the patient with endometriosis-associated infertility.

Adverse effects of hydrosalpinx on pregnancy rates after in vitro fertilization-embryo transfer.

OBJECTIVE: To determine the effect of hydrosalpinx on the establishment of pregnancy after IVF-ET. DESIGN: Metaanalysis. SETTING: University medical center. PATIENT(S) AND INTERVENTION(S): All published reports (n=13) and abstracts (n=10) in English that examined the relation between hydrosalpinx and IVF-ET were included in the analysis. The metaanalysis was performed by first calculating the odds ratios for each trial and then combining them to obtain a pooled estimate of the odds ratio and a 95% confidence interval. MAIN OUTCOME MEASURE(S): Clinical pregnancy. RESULT(S): A total of 5,569 cycles was reviewed in the group without hydrosalpinx, and a total of 1,144 was reviewed in the group with hydrosalpinx. The clinical pregnancy rate was approximately 50% lower in patients who had hydrosalpinx. Similarly, the implantation rate was decreased by 50%. These effects were observed also in thawed ET cycles. The abortion rate was more than twofold higher in patients who had hydrosalpinx. CONCLUSION(S): This metaanalysis suggests that hydrosalpinx is associated with a reduced chance of implantation and an increased risk of pregnancy loss.

Expression and hormonal regulation of monocyte chemotactic protein-1 in myometrium and leiomyomata.

OBJECTIVE: The pathogenesis of leiomyoma likely involves interactions of sex steroids with paracrine growth factors or cytokines resulting in modulation of local immunity. Monocyte chemotactic protein-1 (MCP-1) is a chemotactic and activating factor for monocytes and is produced by multiple tumors and has antitumor effects. We investigated the expression of MCP-I in leiomyoma and myometrium as well as the regulatory role of steroid hormones and cytokines on MCP-1 expression and the effect of MCP-1 on the proliferation of leiomyoma cells. DESIGN: Prospective study. SETTING: University medical center. PATIENT(S): Women with (n = 20) or without (n = 11) leiomyoma. INTERVENTION(S): First. MCP-1 messenger RNA (mRNA) levels in myometrium and leiomyoma were measured, and then myometrial and leiomyoma cells in culture were treated with steroid hormones and cytokines. MAIN OUTCOME MEASURE(S): The MCP-1 mRNA was evaluated by Northern analysis. Immunoreactive MCP-1 in cell cultures was quantified by ELISA. Leiomyoma cell proliferation was assessed with [3H]thymidine incorporation. RESULT(S): The MCP-1 mRNA levels in myometrial samples were 4.7-fold higher than in the leiomyoma samples. Myometrial MCP-1 mRNA levels were 2.4-fold higher in secretory than in proliferative phase samples. The highest MCP-1 levels were observed in samples from women using GnRH analogues. Estradiol and progestins, alone or in combination, resulted in a decrease in MCP-1 protein production. There was an increase in the proliferation of leiomyoma cells treated with anti-MCP-1 neutralizing antibody. CONCLUSION(S): These findings suggest that MCP-1 may have antineoplastic activity in leiomyomata and that sex steroids may be exerting their growth stimulatory effect in leiomyomata through down-regulation of MCP-1.

The nature of the intraperitoneal exudate associated with infertility: peritoneal fluid and serum lysozyme activity.

An intraperitoneal inflammatory process has been associated with infertility in women without anatomic distortion of the pelvic viscera, particularly with endometriosis. This phenomenon was investigated by measuring peritoneal fluid (PF) and serum levels of a major secretory product of the macrophage, lysozyme, in fertile and infertile women undergoing laparoscopy. Serum lysozyme levels were in the normal range and did not correlate with total PF lysozyme. Regression analysis revealed a negative correlation between adnexal adhesions and PF volume (P less than 0.05), PF leukocyte number (P less than 0.05), and total PF lysozyme (P less than 0.05). Significant correlations were found between total PF lysozyme and PF volume (r = 0.72, P less than 0.0001) and leukocyte number (r = 0.67, P less than 0.0001). These results suggest that a localized intraperitoneal inflammatory reaction is associated with infertility in the absence of anatomic distortion of the pelvic viscera.