Sphingosine-1-phosphate inhibits H2O2-induced granulosa cell apoptosis via the PI3K/Akt signaling pathway.

OBJECTIVE: To investigate the protective effect of sphingosine-1-phosphate (S1P) against H(2)O(2)-induced apoptosis in human granulosa cell cultures with freshly harvested granulosa cells. DESIGN: Experimental study. SETTING: Academic medical center for reproductive medicine. PATIENT(S): Cultures of primary granulosa cells isolated from women undergoing in vitro fertilization (IVF). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Cell apoptosis and Western blot analysis of signaling pathway proteins. RESULT(S): We found that S1P (1 and 10 mM) statistically significantly decreased granulosa cell apoptosis after H(2)O(2) treatment. The decreased cell apoptosis induced by S1P was abolished after treatment with VPC23019, an inhibitor of S1P1 and S1P3 receptors, W146, an inhibitor of S1P1 receptors, and CAY10444, an inhibitor of S1P3 receptors. A Western blot analysis revealed that the level of phospho-Akt increased and peaked at 10 minutes after 10 mM S1P exposure. CONCLUSION(S): Treatment with S1P can inhibit the apoptosis of granulosa cells in response to oxidative stress induced by H(2)O(2). The protective effect of S1P is mediated by activating the PI3K/Akt pathway, and the antiapoptotic effect of S1P is mainly mediated through the S1P1 and S1P3 receptor.

Establishment of a human nonluteinized granulosa cell line that transitions from the gonadotropin-independent to the gonadotropin-dependent status.

The ovary is a complex endocrine organ responsible for steroidogenesis and folliculogenesis. Follicles consist of oocytes and two primary steroidogenic cell types, the granulosa cells, and the theca cells. Immortalized human granulosa cells are essential for researching the mechanism of steroidogenesis and folliculogenesis. We obtained granulosa cells from a 35-yr-old female and immortalized them by lentivirus-mediated transfer of several genes so as to establish a human nonluteinized granulosa cell line (HGrC1). We subsequently characterized HGrC1 and investigated its steroidogenic performance. HGrC1 expressed enzymes related to steroidogenesis, such as steroidogenic acute regulatory protein, CYP11A, aromatase, and gonadotropin receptors. Stimulation with FSH increased the mRNA levels of aromatase, which consequently induced the aromatization of androstenedione to estradiol. Activin A increased the mRNA levels of the FSH receptor, which were synergistically up-regulated with FSH stimulation. HGrC1 also expressed a series of ligands and receptors belonging to the TGF-ß superfamily. A Western blot analysis showed that bone morphogenetic protein (BMP)-4, BMP-6, and BMP-7 phosphorylated small mother against decapentaplegic (Smad)1/5/8, whereas growth differentiation factor-9 phosphorylated Smad2/3. BMP-15 and anti-Müllerian hormone phosphorylated Smad1/5/8 while also weakly phosphorylating Smad2/3. These results indicate that HGrC1 may possess the characteristics of granulosa cells belonging to follicles in the early stage. HGrC1 might also be capable of displaying the growth transition from a gonadotropin-independent status to gonadotropin-dependent one.

Expression and localization of CXCL16 and CXCR6 in ovarian endometriotic tissues.

PURPOSE: Inflammatory mediators, including chemokines, may play crucial roles in the development of endometriosis. Therefore, we investigated the expression and localization of CXCL16 and its receptor, CXCR6, in ovarian endometriotic tissues. We also examined whether CXCL16 induces IL-8 production in endometriotic stromal cells. METHODS: We performed immunohistochemical and Western blotting analyses of in vivo and in vitro samples. IL-8 production was assayed using an ELISA. RESULTS: Both CXCL16 and CXCR6 were expressed by endometriotic epithelial cells and stromal cells, but not normal ovarian stroma. A Western blotting analysis using primary cultured endometriotic stromal cells showed a constant expression of CXCL16 and CXCR6 in the proliferative phase, secretory phase and during gonadotropin-releasing hormone agonist therapy. CXCL16 induced IL-8 production in several endometriotic stromal cells in vitro. CONCLUSIONS: CXCL16 and CXCR6 might be involved in the pathophysiology of endometriosis through regulation of the inflammatory response.

Successful fertility management of a patient with factor V deficiency: planned transfusion of fresh frozen plasma under infertility treatment.

OBJECTIVE: To report the case of a patient with factor V deficiency who achieved pregnancy with the planned transfusion of fresh frozen plasma (FFP) while monitoring follicle development and ovulation induction using gonadotropin. DESIGN: Case report. SETTING: University hospital. PATIENT(S): A 28-year-old nulliparous female. INTERVENTION(S): Medical management including infertility treatment. MAIN OUTCOME MEASURE(S): Clinical follow-up. RESULT(S): A patient with factor V deficiency experienced repeated ovulation-related hemoperitoneum following the withdrawal of oral contraceptive pills. The monitoring of follicle development and ovulation induction using gonadotropin followed by FFP transfusion was useful to avoid hemoperitoneum. Pregnancy was achieved within a relatively short period using intrauterine insemination. CONCLUSION(S): Planned prophylactic FFP administration and intervention with infertility treatment might be useful to minimize the risk of ovulation-related hemoperitoneum in patients with factor V deficiency.

The post-operative decline in serum anti-Mullerian hormone correlates with the bilaterality and severity of endometriosis.

BACKGROUND To assess the impact of ovarian cystectomy for endometriomas on the ovarian reserve, we evaluated the pre- and post-operative levels of serum anti-Müllerian hormone (AMH). We also analyzed the correlations between factors related to endometriosis and surgery for endometriomas and the serum AMH levels to investigate which factors affect ovarian reserve. METHODS Thirty-eight patients who were undergoing ovarian cystectomy for unilateral endometrioma (n = 20) and bilateral endometriomas (n = 18) participated. Preoperative and post-operative serum samples were collected and assayed for AMH levels, and changes between the two samples were analyzed in association with parameters of endometriosis and surgery for endometriomas. RESULTS The mean AMH level was 3.9 ng/ml prior to surgery, and was reduced to 2.1 ng/ml at 1 month post-surgery. The rate of decline of the serum AMH level was significantly higher in the bilateral group than the unilateral group (62.8 ± 29.6 versus 24.7 ± 32.5%, P < 0.001). The rate of decline in the serum AMH levels showed a significant correlation to the revised American Society for Reproductive Medicine (rASRM) score (P = 0.003), but not age, cyst diameter, blood loss during the operation or the number of follicles removed in the specimens. CONCLUSIONS Our results suggest that the decrease in ovarian reserve should be taken into account in patients indicated for cystectomy for bilateral endometriomas or unilateral endometrioma with high rASRM scores.

Serum anti-Müllerian hormone level is a useful marker for evaluating the impact of laparoscopic cystectomy on ovarian reserve.

To assess the impact of laparoscopic surgery on ovarian reserve, we evaluated pre- and postoperative levels of serum anti-Müllerian hormone (AMH) in comparison with basal levels of FSH. The median AMH level was 2.98 ng/mL and 3.92 ng/mL before operation and was significantly reduced to a median level of 2.24 ng/mL and 3.29 ng/mL at 1 month after operation in the endometrioma group (n = 29) and the nonendometrioma group (n = 21), respectively, whereas postoperative basal FSH levels did not significantly change in comparison with preoperative levels.

Three-dimensional CT angiography is useful for diagnosis of postabortion uterine hemorrhage: 3 case reports and review of the literature.

Uterine hemorrhage is a major complication associated with abortion. There are various causes of postabortion uterine hemorrhage. The objective of this article is to estimate the efficacy of three-dimensional computed tomography (3D-CT) angiography in the diagnosis of this condition. We present 3 case reports of women with massive genital bleeding after abortion. 3D-CT angiography clearly demonstrated the 3-D features of the feeding artery, the draining vein, and the surrounding normal structures. The diagnosis in patient 1 was a uterine arteriovenous malformation, in patient 2 was a placental polyp mimicking a uterine arteriovenous malformation, and in patient 3 was a placental polyp. Patients were all successfully treated with uterine artery embolization or transcervical resection of the placental polyp. We conclude that 3D-CT angiography is useful for making a differential diagnosis and for preoperative planning in patients with postabortion uterine hemorrhage.

Evaluation of the safety of time-lapse observations for human embryos.

PURPOSE: To assess the effects of light from an integrated optical microscope and evaluate the safety of time-lapse observations using a built-in microscope incubator. METHODS: We prospectively compared the fertilization rate and embryonic morphology after intracytoplasmic sperm injection between embryos cultured with time-lapse observations every 15 min in an incubator with an integrated optical microscope and embryos with intermittent observations (once a day) in conventional incubators. RESULTS: No significant differences were observed in the fertilization rate (57.5% vs. 57.5%) or the rate of excellent-good cleavage embryos (36.0% vs. 36.0%). CONCLUSIONS: These results suggest that time-lapse observations using an incubator with an integrated optical microscope may therefore be safely utilized in clinical practice.

Assessment of the predictive value of follicular fluid insulin, leptin and adiponectin in assisted reproductive cycles.

PURPOSE: To assess the correlation of intrafollicular insulin, leptin and adiponectin levels with assisted reproductive technologies (ART) outcome. METHODS: This was a retrospective study of 46 patients undergoing in vitro fertilisation/intracytoplasmic sperm injection. Follicular fluid (FF) samples collected at oocyte retrieval were assayed for insulin, leptin and adiponectin levels using enzyme-linked immunosorbent assay, and correlations with ART outcome were analysed. RESULTS: There was no significant correlation between intrafollicular insulin, leptin and adiponectin levels. There was a significant difference in the concentration of insulin (P = 0.007), but not leptin or adiponectin, between pregnant (n = 20) and non-pregnant (n = 26) cycles. Only two pregnancies was observed in the 12 cycles in which the concentration of insulin was greater than 7 mU/l in FF, while 18 pregnancies was observed in the 34 cycles in which the concentration of insulin was less than 7 mU/l (P = 0.043). The significantly high concentration of insulin in FF was observed in non-pregnant cycles of patients with polycystic ovary syndrome (PCOS). CONCLUSIONS: Our results suggest the possible involvement of intrafollicular insulin in folliculogenesis. Insulin resistance-related substances may affect the reproductive process in patients with PCOS.

Insulin attenuates the insulin-like growth factor-I (IGF-I)-Akt pathway, not IGF-I-extracellularly regulated kinase pathway, in luteinized granulosa cells with an increase in PTEN.

CONTEXT: Insulin resistance is considered as part of the pathogenesis of polycystic ovary syndrome (PCOS), and PCOS patients often show hyperinsulinemia. The influence of insulin on folliculogenesis in women with PCOS has not been fully investigated. OBJECTIVE: Our objective was to assess the induction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression with insulin treatment and effects of PTEN on IGF-I-induced granulosa cell proliferation as well as the correlation of PTEN levels with the concentration of insulin in follicular fluid in PCOS and non-PCOS patients. DESIGN, SETTING, PATIENTS, AND MAIN OUTCOME MEASURES: A cell proliferation assay, real-time RT-PCR, and Western blotting for PTEN, Akt, and ERK1/2 were conducted in primary cultured granulosa cells under IGF-I stimulation with or without insulin pretreatment. Phosphorylation of Akt and ERK1/2 was also determined by Western blotting. We also measured the insulin concentration in follicular fluid and the levels of PTEN expression in granulosa cells collected at the time of oocyte retrieval of in vitro fertilization in PCOS (n = 13) and non-PCOS patients (n = 37). RESULTS: PTEN expression was induced by insulin. Pretreatment with insulin attenuated the IGF-I-induced Akt phosphorylation and cell proliferation but not ERK1/2 phosphorylation. A phosphatidylinositol 3-kinase inhibitor, LY294002, inhibited the IGF-I-induced cell proliferation. Suppression of insulin-induced PTEN expression using small interfering RNA recovered IGF-I-induced Akt phosphorylation. PTEN levels in granulosa cells, which tended to be higher in PCOS patients, were correlated with the insulin concentration in follicular fluid. CONCLUSIONS: PTEN may influence the proliferation of human granulosa cells as well as disturbance of follicular growth in PCOS patients.

IGF1-induced AKT phosphorylation and cell proliferation are suppressed with the increase in PTEN during luteinization in human granulosa cells.

Granulosa cells proliferate and then undergo differentiation; an inverse relationship between these processes is observed during terminal follicular growth. During terminal follicular growth and initial luteinization, there is a necessary transition of granulosa cells to a less proliferative and highly steroidogenic form in response to LH. Although the expression of several molecules has been reported to be up-regulated by LH, proliferation/differentiation transition is not fully understood. Here, we show that the expression of a tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was induced with human chorionic gonadotropin (hCG) treatment in human luteinized granulosa cells. Pretreatment with hCG attenuated insulin-like growth factor (IGF)-1-induced phosphorylation of AKT and cell proliferation, not phosphorylation of ERK1/2. Moreover, suppression of hCG-induced PTEN expression with siRNA increased AKT phosphorylation and cell proliferation in response to IGF1. We also demonstrate that a PI3K inhibitor, LY294002, not a MEK inhibitor, PD98059, inhibited IGF1-induced cell proliferation. In conclusion, PTEN induced to express by hCG in luteinized granulosa cells that inactivates AKT, not ERK, and attenuates IGF1-induced cell proliferation. PTEN expression may be a trigger for proliferation/differentiation transition in human granulosa cells.

Laparoscopic management of mature cystic teratoma of bilateral ovaries with adnexal torsion occurring in a 9-year-old premenarchal girl.

BACKGROUND: Laparoscopic surgery is a minimal-access procedure with many advantages. However, reports of young girls with adnexal disease treated by laparoscopic surgery are limited in the literature. CASE: A 9-yr-old premenarchal girl presenting with acute abdomen was treated by emergency laparoscopic surgery. Bilateral adnexal torsion was noted. After detorsion, bilateral cystectomy was successfully performed and ovarian tissues were preserved. Pathological diagnosis was mature cystic teratoma of bilateral ovaries. CONCLUSION: Although adnexal torsion occurring in a premenarchal girl is an extremely rare disorder and bilateral adnexal torsion is even more rare, gynecologists should possess sufficient knowledge about the manifestations of such disorder for immediate diagnosis and treatment to preserve future fertility and, if available, laparoscopic approach should be chosen for a young girl.

Spontaneous ectopic pregnancy occurring in the isthmic portion of the remnant tube after ipsilateral adnexectomy: report of two cases.

Two cases of spontaneous ectopic pregnancy occurring in the isthmic portion of the remnant tube after previous ipsilateral adnexectomy are presented. Laparoscopic observation and postoperative histopathological examination suggested intrauterine transmigration of the fertilized egg as the etiology. Laparoscopic excision of the remnant tube was performed and the postoperative course was uneventful in both cases. Attention should be paid to this unusual type of ectopic pregnancy while examining patients with previous history of adnexal surgery.

Management of patients with ectopic pregnancy with massive hemoperitoneum by laparoscopic surgery with intraoperative autologous blood transfusion.

STUDY OBJECTIVE: To evaluate the feasibility and safety of surgical laparoscopy with intraoperative autologous blood transfusion for ectopic pregnancy with massive hemoperitoneum. DESIGN: Retrospective analysis (Canadian Task Force classification II-1). SETTING: Department of gynecology at a general hospital. PATIENTS: Seventeen consecutive patients with ectopic pregnancy with massive hemoperitoneum. INTERVENTION: Laparoscopic surgery with salvage device-based intraoperative autologous blood transfusion. MEASUREMENTS AND MAIN RESULTS: From January 2000 through June 2005, one hundred and twelve women with ectopic pregnancy (interstitial/cornual: 4; isthmic: 18; ampullary: 86; and ovarian: 4) were treated by laparoscopic surgery. Seventeen patients who demonstrated more than 501 g of intraabdominal bleeding were classified as having massive hemoperitoneum and retrospectively analyzed. Site of pregnancy in these 17 patients was interstitial/cornual: 3; isthmic: 5; ampullary: 7; and ovarian: 2. Except for two women with tubal abortion of ampullary pregnancy, all other patients had rupture at the pregnancy site. During laparoscopic surgery, blood pooled in the abdominal cavity was collected by an irrigation and aspiration procedure, and sent to an autologous blood-salvage device to make concentrated red blood cell solution. Processed blood was immediately transfused back to the patient through a leukocyte reduction filter. The mean amount of estimated intraabdominal bleeding, which was calculated by the difference between the volumes of aspirated and irrigated fluids, was 1362.1 +/- 491.4 g, and the mean volume of reinfused processed blood was 680.6 +/- 209.5 g. No patient received banked blood at any time. The degree of hemoperitoneum was well correlated with the shock index calculated by dividing the heart rate by systolic blood pressure at triage (r = 0.72; 95% CI 0.37-0.89; p = .001). In all cases of massive hemoperitoneum, there was no need for laparotomic conversion, and homologous blood transfusion was avoided. CONCLUSIONS: Even in women with ectopic pregnancy with massive hemoperitoneum, laparoscopic surgery can be safely conducted by experienced laparoscopists with intraoperative autologous blood transfusion if hemodynamic stability is achieved by perioperative management.

Laparoscopic surgery in 12 cases of adnexal disease occurring in girls aged 15 years or younger.

STUDY OBJECTIVE: To evaluate the treatment by laparoscopic surgery of adnexal disease occurring in young girls. DESIGN: Retrospective analysis (Canadian Task Force classification II-1). SETTING: Department of gynecology at a general hospital. PATIENTS: Twelve consecutive girls aged 15 years or younger. INTERVENTIONS: Laparoscopic surgery. MEASUREMENTS AND MAIN RESULTS: Seven patients had dermoid cysts, and three of these were associated with adnexal torsion. Two patients had ruptured lutein cysts with ovarian bleeding, and one of them was pregnant. Torsion of the tube with paraovarian cyst, torsion of normal ovary, and serous cystadenoma were noted in one patient each. Although the underlying diseases varied, the chief symptom in each of these patients was lower abdominal pain. Because the symptom is nonspecific, the clinical features were confusing, especially in emergency cases; in two patients with adnexal torsion with dermoid cysts and one patient with adnexal torsion of a normal ovary, there was substantial delay in diagnosis, and salpingo-oophorectomy was required as a result. CONCLUSION: Even in young girls, laparoscopic surgery can be performed in an acceptable manner using regular instruments designed for adults.

A case of a mature cystic teratoma of the uterosacral ligament successfully treated by laparoscopic surgery.

An asymptomatic tumor was found in the pelvic cavity of a 49-year-old woman during a routine examination. With a diagnosis of mature cystic teratoma of the right ovary, laparoscopic surgery was performed. The intraoperative finding showed the presence of a cystic tumor firmly attached to the uterosacral ligament in a position distant from the bilateral ovaries. Laparoscopic excision was performed, and the diagnosis of mature cystic teratoma of the uterosacral ligament was made histopathologically. This is the first report in the literature of successful laparoscopic treatment for a mature cystic teratoma of the uterosacral ligament.

Preoperative evaluation of submucosal myoma by virtual hysteroscopy.

STUDY OBJECTIVE: To assess the utility of a new technique called virtual hysteroscopy in the evaluation of the size and location of submucosal myomas before hysteroscopic myomectomy. DESIGN: Retrospective analysis (Canadian Task Force classification II-1). SETTING: Department of gynecology at a general hospital. PATIENTS: Thirteen consecutive women. INTERVENTION: Sixteen-slice computed tomography (CT) scanner. MEASUREMENTS AND MAIN RESULTS: Thirteen women with submucosal myomas were examined by virtual hysteroscopy. The lesions were filmed by multislice CT scanner, immediately after CO2 injection into the uterine cavity with an intravenous dosage of iodide contrast media. The filmed image was subsequently reconstituted and analyzed by endoscopy mode and volume mode using three-dimensional computer graphics software. The size and depth of invasion of the submucosal myoma were clearly identified by the procedure. CONCLUSION: Accurate preoperative evaluation of the size and location of submucosal myomas before hysteroscopic myomectomy is important for a safe surgical procedure. Virtual hysteroscopy can provide such information with good reproducibility and is superior to previously described diagnostic procedures.