Usefulness of epidural anesthesia in gynecologic laparoscopic surgery for infertility in comparison to general anesthesia.

BACKGROUND: Although the advantages of epidural anesthesia in open surgery have been established, its usefulness in the setting of laparoscopic surgery remains to be studied. METHODS: Patients undergoing laparoscopic surgery for infertility were randomly administered epidural anesthesia (group A, n = 11) or general anesthesia (group B, n = 9). The operation was performed under 4 mmHg pneumoperitoneum and in the 20 degrees Trendelenburg position. Respiratory function tests using a spirometer and blood gas analysis were performed during the intra- or perioperative period. Pain status was evaluated with visual analog scale scoring. The number of postoperative recovery days needed to resume daily activities was obtained by a questionnaire. RESULTS: Respiratory rate, minute volume, P(a)CO2, % vital capacity (VC), and forced expiratory volume in 1 s (FEV1) % were virtually constant throughout the study period in group A, whereas %VC was decreased immediately after operation in group B (p < 0.05). Minute volume immediately after operation was significantly increased in group B compared with group A (p < 0.01), suggesting shallow respiration in women undergoing general anesthesia. Observed pain scores on abdominal pain, shoulder pain, and dyspnea were very low during operation in group A. Pain scores immediately and 3 h after operation were also minimal in group A, whereas abdominal pain scores at these points were significantly higher in group B than those in group A (both p < 0.01). The number of days required for a half reduction in wound pain, trotting, and full recuperation for group A were less than those for group B (p < 0.05). CONCLUSIONS: Epidural anesthesia, when used in laparoscopic surgery for infertility treatment, has advantages over general anesthesia in terms of analgesic effects, postoperative respiratory function, and a return to preoperative daily activities.

A primary cell culture system for human cytotrophoblasts of proximal cytotrophoblast cell columns enabling in vitro acquisition of the extra-villous phenotype.

Cytotrophoblast (CT) differentiation into the extra-villous phenotype is a crucial process in initiating their invasion into the decidua and thereby developing the placenta. However, how CTs differentiate into extra-villous CTs (EVCTs) is not fully elucidated. To address this, a suitable culture model for CTs has been long-sought. But this has been hampered by annoying problems such as; cell aggregation, in vitro syncytialization, low plating efficiency, etc. The aim of this study is to develop a culture system in which CTs differentiate into EVCTs. CTs were isolated from the first trimester placenta using density gradient separation and immuno-depletion using anti-CD9 antibody to remove contaminating fibroblasts and EVCTs. The resultant isolated CTs were found to have the character similar to poorly differentiated CTs comprising proximal cytotrophoblastic cell columns as confirmed by immunocytochemical and flowcytometric analyses. When cultured on type 4 collagen-coated plates in culture media containing low calcium concentration, CTs neither aggregated nor syncytialized, remaining mononuclear and monolayer state. Interestingly, cultured CTs gradually upregulated integrin alpha1, CD9, and human leukocyte antigen (HLA)-G; the known markers specific for EVCTs invading into the decidua diffusely. Hence, the CT culture system provides a sophisticated experimental model in which highly purified CTs acquire the extra-villous phenotype without syncytialization.

Demonstration of angiogenin in human endometrium and its enhanced expression in endometrial tissues in the secretory phase and the decidua.

Angiogenesis is thought to be crucial for normal physiology of the endometrium, where dynamic vascular remodeling occurs during the menstrual cycle and pregnancy. We investigated the presence of angiogenin, a potent inducer of angiogenesis, and the regulatory mechanisms of its production in the human endometrium. Western blot analysis demonstrated that angiogenin protein expression increased by 3- to 4-fold in the endometrium in the mid and late secretory phases and in early gestation relative to that during the proliferative phase. Quantitative mRNA analysis showed the similar tendency in the expression of angiogenin mRNA in the endometrium, with the highest levels observed in the mid and late secretory phases and early gestation. An immunohistochemical study showed that angiogenin was expressed in both stromal cells and epithelial cells, with indistinguishable intensity between these cells regardless of phases of the menstrual cycle. In support of the Western blot analysis, the intensity of staining appeared to be highest in the mid to late secretory phases relative to other phases. Consistent with these in vivo results, decidualized cultured stromal cells, after treatment with progesterone or progesterone plus E2, exhibited the capacity to secrete significantly increased amounts of angiogenin compared with untreated or E2 alone-treated control group. Both the treatment with (Bu)2cAMP and hypoxic conditions stimulated angiogenin secretion by stromal cells. For isolated epithelial cells, hypoxia stimulated angiogenin secretion, whereas (Bu)2cAMP had no appreciable effect. In summary, we demonstrated the presence of angiogenin in human endometrium and its possible local regulatory factors, such as progesterone, cAMP, and hypoxia. These findings along with its enhanced expression in the endometrium in the secretory phase and in decidual tissues raise the possibility that angiogenin may play a role in establishing pregnancy.

Evidence for the presence of keratinocyte growth factor (KGF) in human ovarian follicles.

The presence of keratinocyte growth factor (KGF) in human follicular fluid (FF) was investigated in a total of 145 FFs obtained during oocyte retrieval for in vitro fertilization (IVF) from 29 patients with no apparent endocrine disorders. The concentrations of KGF, estradiol, progesterone, testosterone and human chorionic gonadotropin (hCG) in FF were measured by enzyme-linked immunosorbent assay. FF samples contained relatively higher amounts of KGF (2194+/-87 pg/ml), whereas its concentrations in serum were below assay limit (<31.2 pg/ml). Concentrations of KGF in FF were positively correlated with both progesterone (r=0.311, p<0.0005) and testosterone (r=0.230, p<0.01) concentrations in FF. However, KGF concentrations were not significantly correlated with estradiol and hCG concentrations. KGF in FF was detected as a broad band (26-29 kD) by immunoblotting, the size being reduced by 7kD after N-glycosidase treatment. In an in vitro experiment, KGF suppressed the basal and hCG-stimulated progesterone production by cultured human luteinized granulosa cells. summary, we demonstrated the presence of KGF in human ovarian follicles, suggesting its possible role as a local factor in regulating human ovarian functions.

Evidence for the expression of interleukin (IL)-18, IL-18 receptor and IL-18 binding protein in the human endometrium.

To see whether the interleukin (IL)-18 system is operative in the endometrium, we examined the expression of IL-18, IL-18 receptor (IL-18R) and IL-18 binding protein (IL-18BP), the substance known to neutralize IL-18 activity, in this tissue. Reverse transcription-polymerase chain reaction analyses showed that IL-18, IL-18R and IL-18BP mRNA were constitutively expressed without significant fluctuation throughout the menstrual cycle. When epithelial cells and stromal cells were cultured separately, the expression levels of IL-18 mRNA in epithelial cells were about 18-fold higher compared to those in stromal cells. Furthermore, the IL-18 precursor protein was detected by Western blot analysis in cultured epithelial cells but not in stromal cells. Recombinant human IL-18 stimulated the secretion of interferon (IFN)-gamma by resident bone marrow-derived cells in the endometrium. On the other hand, IFN-gamma up-regulated the IL-18BP expression both in cultured epithelial cells and stromal cells. Thus, we have presented evidence for the presence of the IL-18 system in the human endometrium. In light of its immunomodulatory roles in a variety of tissues, this system may afford protection against pathogenic micro-organisms and provide a regulatory mechanism for controlled trophoblast invasion by modulating a local cytokine network.

Molecular cloning and characterization of mouse EBAG9, homolog of a human cancer associated surface antigen: expression and regulation by estrogen.

We previously identified a human estrogen-responsive gene, EBAG9 (ER-binding fragment-associated antigen9) (Watanabe, T. et al., Mol. Cell. Biol. 18, 442-449, 1998). It was later reported as RCAS1 (receptor-binding cancer antigen expressed on SiSo cells) that induced apoptosis and suppressed the growth of several cells such as activated T cells (Nakashima, M. et al., Nat. Med. 5, 938-942, 1999). Here, we have isolated both cDNA and genomic DNA of mouse EBAG9/RCAS1. Mouse EBAG9 gene spans about 30 kb in genomic DNA and consists of 7 exons. Mouse EBAG9 cDNA encodes a protein that contains the transmenbrane segment and coiled-coil domain. An alignment between the predicted mouse and human EBAG9 shows a high degree of homology at the amino acid level (98%). Northern and Western blot analyses demonstrate that EBAG9 is expressed in several tissues including the heart, brain, spleen, liver, kidney, and testis, and also in developing embryo. In the uterus, a target organ for estrogen, the EBAG9 was shown to be upregulated in vivo by 17beta-estradiol. To determine the biological action of mouse EBAG9, NIH3T3 fibroblastic cells were incubated with recombinant EBAG9 protein, resulting in suppression of cell growth. These findings suggest that EBAG9 is an in vivo estrogen-responsive gene that inhibits the cell growth.

Is an elevation in basal follicle-stimulating hormone levels in unexplained infertility predictive of fecundity regardless of age?

An elevation in follicle-stimulating hormone (FSH) levels is considered to reflect lowered ovarian function, resulting in poor fecundity in infertile women. However, it remains to be clarified whether or not the significance of FSH levels applies equally to all women irrespective of age. The objective of the present study is to compare basal FSH levels in infertile women who conceived or not after stratification by age. A total of 144 infertile women between ages 25 and 45 who underwent infertility treatment due to unexplained infertility in the University of Tokyo Hospital were included in the retrospective study. Subjects were divided by age into two groups, < 38 (n=98) vs > or = 38 (n=46) years, with ages ranging from 25 to 37, and from 38 to 45, respectively. Blood samples were collected in early follicular phase (day 4-6) for assessment of basal levels of LH, FSH, and PRL. In the older group, pregnant cases had significantly lower FSH levels (6.07 +/- 2.83 mIU/ml) than nonpregnant cases (9.60 +/- 3.67 mIU/ml), whereas no difference in basal FSH levels was observed between pregnant and nonpregnant cases in the younger group. Basal FSH levels of pregnant cases in the older group were significantly lower than those of pregnant cases in the younger group (8.26 +/- 2.95 mIU/ml). Basal LH and PRL levels were not related to fecundity in either group. Thus, an increase in basal FSH levels as a predictor of fecundity should be considered in the context of age.

A movable gestational sac in association with a myometrial defect.

An ultrasound examination in a woman at 9 weeks' gestation demonstrated a gestational sac with a live fetus in the left uterine horn which was surrounded by an extremely thin outer lining. An interstitial pregnancy was suspected and laparoscopy was performed. During laparoscopy the purple bulge in the left horn gradually reduced in size and eventually disappeared following the external pressure by the tip of the forceps. We believe this pressure caused contraction of the superficial myometrial fibers resulting in a shift in the location of the gestational sac to the central area of the uterus. The pregnancy continued and on Cesarean section at 34 weeks' gestation, a membranous protrusion of the uterine wall in the left horn was noticed. A myometrial defect caused by an intrauterine intervention in the patient's previous pregnancy was suspected to be the cause of this "movable gestational sac" phenomenon.

Stem cell factor (SCF) concentrations in peritoneal fluid of women with or without endometriosis.

PROBLEM: In the quest for possible involvement of stem cell factor (SCF), a cytokine known to have multiple effects, in the pathogenesis of endometriosis, we evaluated concentrations of SCF in peritoneal fluid (PF) of women with or without endometriosis. METHOD OF STUDY: SCF concentrations in PF collected from women undergoing laparoscopy were measured, using a specific enzyme-linked immunosorbent assay (ELISA). Reverse transcription-polymerase chain reaction (RT-PCR) analysis to detect gene expression of c-kit, the receptor for SCF, was performed using the endometriotic tissue and the eutopic endometrium collected during the operation. RESULTS: SCF concentrations in PF of women with endometriosis were significantly higher compared to women without endometriosis. Looking at SCF concentrations in PF of women with endometriosis stratified by disease stage, women with stage I and II exhibited relatively higher SCF levels in PF, whereas SCF levels in PF with stage III and IV were comparable with those without endometriosis. The expression of mRNA for c-kit was detected in both the endometriotic tissue and the eutopic endometrium. CONCLUSION: We demonstrated an elevation in SCF levels in PF associated with endometriosis and the presence of its receptor in endometriotic tissues. Given the known pleiotropic properties of SCF, the present results suggest that SCF might play a role in the pathogenesis of endometriosis.

A case of XY pure gonadal dysgenesis with 46,XYp-/47,XXYp- karyotype whose gonadoblastoma was removed laparoscopically.

A case of pure gonadal dysgenesis was investigated. The patient was an 18-year-old Japanese woman with a history of primary amenorrhea. She had poorly developed breasts, a hypoplastic uterus, a normal vagina and infantile genitalia. The patient's karyotype was 46,XYp-/ 47,XXYp-. Microsatellite analysis revealed that the X chromosomes of this patient originated from one of the two maternal X chromosomes. DNA analysis of the Y chromosome revealed that she had a deletion of SRY (the sex-determining region on the Y chromosome). She underwent laparoscopic gonadectomies with a final pathology consistent with gonadoblastoma. Laparoscopic surgery is recommended as it is much less invasive and associated with rapid postoperative recovery.

Increased concentrations of soluble tumour necrosis factor receptor (sTNFR) I and II in peritoneal fluid from women with endometriosis.

Tumour necrosis factor alpha (TNFalpha), a proapoptotic cytokine, is known to be present in peritoneal fluid from women with endometriosis. An emerging view is that soluble TNF receptors (sTNFR) can modulate the effects of TNFalpha by acting as TNFalpha antagonists. To assess the relevance of sTNFRs in the pathophysiology of endometriosis, concentrations of sTNFR I, sTNFR II and TNFalpha in peritoneal fluid from women with endometriosis (n = 53) and without endometriosis (n = 40) were measured. Concentrations of both sTNFR I and sTNFR II in peritoneal fluid from women with endometriosis were significantly higher than in peritoneal fluid from women without endometriosis, both in the follicular and the luteal phases. TNFalpha concentrations did not differ in patients with and without endometriosis in both phases. When stratified by the stage of the disease, women with both stages I/II and stages III/IV exhibited significantly higher concentrations of sTNFR I and sTNFR II in peritoneal fluid, compared with women without endometriosis, whereas no appreciable difference in the concentrations was detected between stages I/II and stages III/IV. A significant correlation was found between the concentrations of sTNFR I and sTNFR II; while the correlations between TNFalpha and sTNFR I or sTNFR II, were either not significant or were very weak. Furthermore, mRNA for the membrane-associated TNF receptor type 1 and TNF receptor type 2, both of which convey the effects of TNFalpha, were shown to be expressed in endometriotic tissues as well as eutopic endometrium. Together, these findings suggest a possible involvement of sTNFRs in the pathophysiology of endometriosis.

Evidence for the presence of angiogenin in human follicular fluid and the up-regulation of its production by human chorionic gonadotropin and hypoxia.

Angiogenesis is an essential event during the development of the ovarian follicle and ensuing formation of the corpus luteum. We investigated the presence of angiogenin, a potent inducer of angiogenesis, and the regulatory mechanisms of its production in the human ovary. Follicular fluid (FF) and granulosa cells (GCs) were collected from women undergoing in vitro fertilization and embryo transfer. The presence of angiogenin in FF and GCs was demonstrated by Western blot analysis. The production of angiogenin by cultured GCs was stimulated with the addition of human CG or cAMP or under the hypoxic milieu. Concentrations of angiogenin in FF from an individual follicle were positively correlated with those of progesterone, but not estradiol and testosterone. Given the presence of angiogenin in FF and up-regulation of its production by human CG and hypoxia, it seems logical to assume that angiogenin may play a role as a local angiogenic factor in the human ovary.

Schnurri interacts with Mad in a Dpp-dependent manner.

BACKGROUND: Decapentaplegic (Dpp) is a member of the transforming growth factor-beta superfamily. Dpp governs various developmental processes in Drosophila through the transcriptional regulation of a variety of genes. Signals of Dpp are transmitted from the cell membrane to the nucleus by Medea and Mad, both belonging to the Smad protein family. Mad was shown to bind to the Dpp-responsive element in genes such as vestigial, labial, and Ultrabithorax. The DNA binding affinity of Smad proteins is relatively low, and requires other nuclear factor(s) to form stable DNA binding complexes. schnurri (shn) was identified as a candidate gene acting downstream of Dpp receptors, but its relevance to Mad has remained unknown. RESULTS: We characterized the biochemical functions of Shn. Shn forms homo-oligomers. Shn is localized in the nucleus, and is likely to have multiple nuclear localizing signals. Finally, we found that Shn interacts with Mad in a Dpp-dependent manner. CONCLUSIONS: The present results argue that Shn may act as a nuclear component of the Dpp signalling pathway through direct interaction with Mad.

An earlier menopause as clinical manifestation of granulosa-cell tumor: a case report.

We present a case of a granulosa-cell tumor, which can cause menopause at an earlier than normal age. The hormonal profiles were characterized by undetectable FSH levels associated with an estradiol level compatible with the level seen in perimenopausal women and by a significant increase in the inhibin level.

Estrogen receptor-mediated effects of a xenoestrogen, bisphenol A, on preimplantation mouse embryos.

The effects of bisphenol A, a xenoestrogen widely used in industry and dentistry, were studied in early preimplantation mouse embryos. Two-cell mouse embryos were cultured with 100 pM to 100 microM bisphenol A with or without 100 nM tamoxifen and evaluated at 24-h intervals for their development to eight-cell and blastocyst stages. At 72 h, blastocysts were cultured for another 48 h without bisphenol A, and surface areas of trophoblast spread were measured. At 24 h, more embryos exposed to 3 nM bisphenol A than to controls had reached the eight-cell stage. At 48 h, more embryos exposed to 1 nM and 3 nM bisphenol A than to controls had become blastocysts. At 100 microM, bisphenol A decreased frequency of development to blastocysts. Tamoxifen counteracted both stimulatory and inhibitory effects of bisphenol A on blastocyst formation. Although bisphenol A did not alter blastocyst morphology or cell number, early exposure to 100 microM bisphenol A increased subsequent trophoblast areas. These findings suggest that bisphenol A may not only effect early embryonic development via estrogen receptors even at low, environmentally relevant doses, but also exert some late effects on subsequent development of these embryos.

Pregnancy rates after laparoscopic treatment. Differences related to tubal status and presence of endometriosis.

OBJECTIVE: To examine how preexisting tubal adhesions and endometriosis affect pregnancy outcome after laparoscopic treatment in infertile women with no apparent causes of infertility other than tubal factors. STUDY DESIGN: Pregnancy outcomes in 186 infertile women for a follow-up period of 18 months after laparoscopy were analyzed. Laparoscopic manipulations consisted of adhesiolysis of tubes and removal of endometriotic lesions. RESULTS: The patients were classified into three groups, those with no tubal adhesions (group A, n = 83), unilateral tubal adhesions (group B, n = 46) and bilateral tubal adhesions with at least one tube patent (group C, n = 57). The cumulative pregnancy rate in group C (13.2%) was lower than in groups A (41.8%) and B (45.7%) 18 months after laparoscopy. The average time to conception in group A (6.7 +/- 0.8 months) tended to be shorter than that in group B (10.6 +/- 1.2 months). In group A, pregnancy rates were essentially the same between minimal/mild endometriosis and moderate/severe endometriosis. Regarding group B, women with minimal/mild endometriosis exhibited significantly higher pregnancy rates than those with moderate/severe endometriosis, while pregnancy rates in women without endometriosis fell in between. CONCLUSION: Pregnancy rates after laparoscopic treatment are different in relation to tubal status and the presence of endometriosis.