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.

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.

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.

Distribution of endothelin-converting enzyme-1 and neutral endopeptidase in human endometrium.

The expression of endothelin-1 (ET-1), which has been proposed to have a potential autocrine/paracrine role, varies during the menstrual cycle, and therefore, ET-1 may be involved in the cyclic change of the human endometrium. However, neither the synthesis nor the degradation of ET-1 in the endometrium has been determined in detail. We investigated endothelin-converting enzyme-1 (ECE-1), which converts big-ET-1 to active ET-1, and neutral endopeptidase (NEP), which cleaves and inactivates ET-1 in human endometrium in vivo and in vitro. Western blot analysis demonstrated that the change in the expression of ECE-1 during the menstrual cycle differed from that of NEP in the endometrium. ECE-1 was expressed by endometrial epithelial cells, whereas NEP was predominantly expressed by stromal cells in vivo and in vitro. In conclusion, our results suggest that spacio-temporal expression of two endopeptidases, ECE-1 and NEP, involved in the synthesis and degradation of ET-1, might regulate ET-1 action in human endometrium.

Neutral endopeptidase expressed by decidualized stromal cells suppresses akt phosphorylation and deoxyribonucleic acid synthesis induced by endothelin-1 in human endometrium.

Endothelin-1 (ET-1) in human endometrium has been proposed to have a potential paracrine role, for its receptors are also present within this tissue. In addition, the expression of ET-1 varies during the menstrual cycle, and therefore, ET-1 may be involved in the cyclic change of the human endometrium, such as proliferation and decidualization. However, neither the inactivation of ET-1 in the endometrium nor the paracrine effect of ET-1 on endometrial cells has been determined. We investigated the production of ET-1 and the presence of neutral endopeptidase (NEP), which cleaves and inactivates ET-1, in primary cultured human endometrial cells. We found primary cultured endometrial epithelial cells, not stromal cells, to be the major source of ET-1. Western blot analysis and RT-PCR demonstrated that NEP was predominantly expressed by endometrial stromal cells. We also demonstrated that ET-1 stimulated the phosphorylation of Akt and DNA synthesis in endometrial stromal cells via the ET(A) receptor and phospahtidylinositol-3 kinase signaling pathways. The effect of ET-1 was regulated by NEP expressed by stromal cells. We also found that conditioned medium containing ET-1 from endometrial epithelial cell culture stimulated phosphorylation of Akt via the ET(A) receptor. In conclusion, ET-1 has a paracrine effect of Akt phosphorylation and cell proliferation on endometrial stromal cells, which occurs via the ET(A) receptor and phospahtidylinositol-3 kinase signaling pathways, and is regulated by cell-surface NEP.

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.

Successful management of a massive hemorrhage due to rupture of cystic cervical endometriosis by a loop electrosurgical excision procedure.

OBJECTIVE: To report a case of cystic cervical endometriosis that caused a massive hemorrhage due to rupture of a cyst and successful management with a loop electrosurgical excision procedure (LEEP). DESIGN: Case report. SETTING: University Hospital. PATIENT: A 37-year-old nulliparous woman. INTERVENTION(S): Medical management including surgical treatment. MAIN OUTCOME MEASURE(S): Clinical follow-up and pathologic diagnosis. RESULT(S): A hemorrhagic cystic cervical mass was excised with emergent LEEP. The mass was found to be cervical endometriosis. There was no evidence of recurrence until 1 year after excision. CONCLUSION(S): Cystic formation of cervical endometriosis--like ovarian endometrioma, which causes a massive hemorrhage due to rupture--is extremely rare, although cervical endometriosis is generally asymptomatic. Hysterectomy is considered in such cases but can be avoided via LEEP that incorporates partial excision of the uterine cervix.

Oral progestogen versus intramuscular progesterone for luteal support after assisted reproductive technology treatment: a prospective randomized study.

OBJECTIVES: To evaluate the efficacy of oral progestogen, chlormadinone acetate, and intramuscular (IM) progesterone for luteal support in patients, undergoing assisted reproductive technology (ART) treatment, who were treated with a gonadotropin-releasing hormone agonist (GnRHa). METHODS: This was a prospective randomized study of 40 patients with normal and high response (serum estradiol > 2,000 pg/ml) in GnRHa down-regulation. Patients were randomized to receive either oral chlormadinone acetate or IM progesterone. The outcomes of ART treatment, including pregnancy and embryo implantation rates, were analyzed. RESULTS: There were no significant differences in the clinical pregnancy rates (25 vs. 20%) and in the implantation rates (12.7 vs. 9.1%) of patients who received IM progesterone and oral chlormadinone acetate. Endometrial thickness was also comparable between oral chlormadinone acetate and IM progesterone. CONCLUSION: Oral progestogen, chlormadinone acetate showed a comparable pregnancy rate and live birth rate with IM progesterone as luteal support for the high responders. The optimal methods for luteal support may be dependent on responses to stimulation with gonadotropin, although it is not concluded that oral chlormadinone acetate is recommended as an option for luteal support in high responders.

PTEN and Akt expression during growth of human ovarian follicles.

PURPOSE: To assess the expression of PTEN and total and phosphorylated Akt in human ovarian follicles during follicular growth. METHODS: Immunohistochemistry of ovarian tissues and Western blotting and immunofluorescence of primary cultured luteinized granulosa cells for PTEN and Akt. RESULTS: Immunoreactivity of Akt was found in the oocytes, granulosa cells and theca cells in primordial follicles, follicles at each growing stage and luteal cells. As the follicles grew, staining for PTEN became intense in the granulosa cells, whereas the intensity of phospho-Akt became weak. Western blotting and immunofluorescence analysis using primary cultured granulosa-lutein cells showed Akt and PTEN expression, and phosphorylation of Akt in vitro. CONCLUSIONS: PTEN and Akt are present in the granulosa cells during folliculogenesis. An increase in PTEN may lead to changes in proliferation and/or differentiation of granulosa cells during follicular growth via regulation of Akt phosphorylation.

Localization of angiotensin II, the AT1 receptor, angiotensin-converting enzyme, aminopeptidase A, adipocyte-derived leucine aminopeptidase, and vascular endothelial growth factor in the human ovary throughout the menstrual cycle.

OBJECTIVE: To assess the expression and cellular distribution of angiotensin II (Ang II), angiotensin type 1 receptor (AT1R), angiotensin-converting enzyme (ACE), aminopeptidase A (APA), adipocyte-derived leucine aminopeptidase (A-LAP), and vascular endothelial growth factor (VEGF) in human ovarian tissue during the menstrual cycle. DESIGN: Ovarian tissues (n = 52) and corpora lutea (n = 34) were obtained from patients undergoing hysterectomy/oophorectomy, and tissue sections were immunostained for each antigen. SETTING: University hospital. PATIENT(S): Patients undergoing hysterectomy or oophorectomy for benign conditions. INTERVENTION(S): Immunostaining of tissue sections using antibodies to each antigen. MAIN OUTCOME MEASURE(S): Microscopic evaluation to assess the presence, distribution, and cellular localization. RESULT(S): The luteal tissue is the major site of Ang II, ACE, AT1R, and VEGF, with highest staining intensity found during the midluteal phase and at pregnancy. The AT1R was found in theca cells. The APA was strongly immunolocalized in pericytes. Immunolocalization of AT1R was almost similar to that of VEGF including oocytes in the primordial and intermediate follicles. CONCLUSION(S): The expression and distinct pattern of the cellular localization of Ang II and its related proteins in human ovarian tissue during folliculogenesis and in the luteal tissue suggest their roles in the growth and differentiation of theca, granulose, and luteal cells.

Dual renin-angiotensin blockade therapy in patients at high risk of early ovarian hyperstimulation syndrome receiving IVF and elective embryo cryopreservation: a case series.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is an important and dangerous aspect of assisted reproduction techniques. Although elective cryopreservation of all embryos can prevent pregnancy-induced late OHSS, it cannot prevent early OHSS, which is induced by hCG administration. METHODS: We undertook this trial to assess the efficacy with which the combined oral administration of angiotensin-converting enzyme inhibitor (ACEI) and angiotensin II receptor blocker (ARB) could prevent early OHSS in IVF patients at very high risk for this syndrome. Four women, who had serum estradiol concentration > or =8000 pg/ml on the day of hCG injection, were treated with the combination of the ACEI alacepril and the ARB candesartan cilexetil for 8 days starting the day after oocyte retrieval. Embryos were cryopreserved and embryo transfer was postponed until later cycles. RESULTS: Despite the extremely enlarged ovaries, no ascites was accumulated in any of the cases. Haematocrit (34.1 +/- 1.0) and serum albumin concentration (4.1 +/- 0.2 g/dl) were normal throughout the treatment period. These patients showed elevated plasma renin and angiotensin II concentration before the treatment. CONCLUSIONS: The dual renin-angiotensin blockade therapy used here would be worth exploring further in a study with more patients and a prospective, randomized design.