Akira Iwase

The post-operative decline in serum anti-Müllerian 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.

Mesenchymal stromal cells of human umbilical cord Wharton's jelly accelerate wound healing by paracrine mechanisms

BACKGROUND AIMS Mesenchymal stromal cells (MSC) can be isolated from the perivascular connective tissue of umbilical cords, called Whartons jelly. These human umbilical cord perivascular cells (HUCPVC) might provide therapeutic benefits when treating skeletal or cutaneous malformations in neonatal patients. METHODS HUCPVC were isolated, and their proliferation rate, marker expression and multilineage differentiation potential determined. HUCPVC or their conditioned medium (HUCPVC-CM) was injected into the excisional wound of a mouse splinted-wound model. The effects of the treatment on wound closure were examined by morphohistochemical and gene expression analyses. RESULTS HUCPVC expressed typical MSC markers and could differentiate into osteoblastic and adipogenic lineages. HUCPVC transplanted into the mouse wound accelerated wound closure. Immunohistologic analysis showed that the HUCPVC accelerated wound healing by enhancing collagen deposition and angiogenesis via paracrine mechanisms. Furthermore, treatment with HUCPVC-CM alone significantly enhanced wound closure. HUCPVC-CM increased the number of anti-inflammatory M2 macrophages expressing resistin-like molecule (RELM)-α/CD11b and promoted neovessel maturation. Quantitative polymerase chain reaction (PCR) analysis showed that HUCPVC-CM increased the expression of tissue-repairing cytokines interleukin (IL)-10, transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF)-1 and angiopoietin-1 at the healing wound. CONCLUSIONS Our results show that HUCPVC promotes wound healing via multifaceted paracrine mechanisms. Together with their ability to differentiate into the osteogenic linage, HUCPVC may provide significant therapeutic benefits for treating wounds in neonatal patients.

One-year follow-up of serum antimüllerian hormone levels in patients with cystectomy : are different sequential changes due to different mechanisms causing damage to the ovarian reserve?

OBJECTIVE To investigate whether the serum antimüllerian hormone (AMH) levels recover within 1 year after cystectomy for endometriomas, and to analyze the pattern of sequential changes in the serum AMH levels. DESIGN Prospective study. SETTING University hospital. PATIENT(S) Thirty-nine patients undergoing cystectomy for unilateral endometrioma (n = 22) and bilateral endometriomas (n = 17). INTERVENTION(S) Serum samples collected 2 weeks before, and 1 month and 1 year after surgery were assayed for AMH levels. MAIN OUTCOME MEASURE(S) Assessment of the ovarian reserve damage based on alterations in the serum AMH levels and the association with parameters of endometriosis and surgery for endometriomas. RESULT(S) The median AMH levels were 3.56, 1.90, and 2.10 ng/mL before, 1 month after, and 1 year after surgery, respectively. Twenty patients showed higher AMH levels 1 year after surgery than 1 month after surgery (increase group); 19 patients showed lower AMH levels (decrease group). We found a statistically significant difference in the number of follicles removed by surgery between the two groups. CONCLUSION(S) The decrease in the serum AMH levels caused by cystectomy can recover. Our results suggest that removal of ovarian cortex might be involved in the decrease of the ovarian reserve just after surgery, and that a continuous decrease of the ovarian reserve after cystectomy might be attributed to other mechanisms.

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.

PTEN and Akt expression during growth of human ovarian follicles

PurposeTo assess the expression of PTEN and total and phosphorylated Akt in human ovarian follicles during follicular growth.MethodsImmunohistochemistry of ovarian tissues and Western blotting and immunofluorescence of primary cultured luteinized granulosa cells for PTEN and Akt.ResultsImmunoreactivity 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.ConclusionsPTEN 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.

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.

The Loss of Endoglin Promotes the Invasion of Extravillous Trophoblasts

Endoglin is a coreceptor for TGF-β, which is expressed in syncytiotrophoblasts. The soluble form of endoglin (sEng) has been observed to increase in the serum of preeclamptic patients. Several studies have shown that endoglin is involved in cancer invasion. However, the role of endoglin in extravillous trophoblasts (EVT), which have an invasive phenotype, remains unknown. The present study was designed to investigate the expression and role of endoglin in human EVT. We found that endoglin was mainly expressed on cytotrophoblasts within the cell column during the first trimester and its expression decreased in the EVT by immunohistochemistry and immunocytochemistry. The expression of endoglin significantly increased after treatment with TGF-β1 and TGF-β3 in the human EVT cell line, HTR-8/SVneo, as detected by semiquantitative RT-PCR. To investigate the role of endoglin in EVT, the stable knockdown of endoglin was performed by lentiviral short hairpin RNA transfection into the HTR-8/SVneo cells. Although proliferation was not affected, the motility and invasiveness of the HTR-8/SVneo cells significantly increased by the knockdown of endoglin. Both the mRNA expression and secretion of urokinase-type plasminogen activator significantly increased in endoglin knockdown cells. The secretion of sEng was very low in HTR-8/SVneo, and the treatment of endoglin knockdown cells with 10 ng/ml sEng had no effect on their invasiveness. Therefore, the suppression of sEng was not involved in the increased invasiveness of endoglin knockdown cells. These results suggested that EVT increased their invasive function as a result of decreasing expression of transmembrane endoglin.

Chronic Peripheral Administration of Kappa-Opioid Receptor Antagonist Advances Puberty Onset Associated with Acceleration of Pulsatile Luteinizing Hormone Secretion in Female Rats

Puberty in mammals is timed by an increase in gonadotropin-releasing hormone (GnRH) secretion. Previous studies have shown involvement of the two neuropeptides, kisspeptin and neurokinin B (NKB), in controlling puberty onset. Little is known about the role of the other key neuropeptide, dynorphin, in controlling puberty onset, although these three neuropeptides colocalize in the arcuate kisspeptin neurons. The arcuate kisspeptin neuron, which is also referred to as the KNDy neuron, has recently been considered to play a role as an intrinsic source of the GnRH pulse generator. The present study aimed to determine if attenuation of inhibitory dynorphin-kappa-opioid receptor (KOR) signaling triggers the initiation of puberty in normal developing female rats. The present study also determined if stimulatory NKB-neurokinin 3 receptor (NK3R) signaling advances puberty onset. Female Wistar-Imamichi rats were weaned and intraperitoneally implanted with osmotic minipumps filled with nor-binaltorphimine (nor-BNI), a KOR antagonist, or senktide, a NK3R agonist, at 20 days of age. Fourteen days of intraperitoneal infusion of nor-BNI or senktide advanced puberty onset, manifested as vaginal opening and the first vaginal estrus in female rats. Frequent blood sampling showed that nor-BNI significantly increased luteinizing hormone (LH) pulse frequency at 29 days of age compared with vehicle-treated controls. Senktide tended to increase this frequency, but its effect was not statistically significant. The present results suggest that the inhibitory input of dynorphin-KOR signaling plays a role in the prepubertal restraint of GnRH/LH secretion in normal developing female rats and that attenuation of dynorphin-KOR signaling and increase in NKB-NK3R signaling trigger the onset of puberty in female rats.

N-Acetylglucosaminyltransferase V Regulates Extravillous Trophoblast Invasion through Glycosylation of α5β1 Integrin

For successful human placentation, invasion of trophoblast cells into the uterus and its associated vasculature is essential, and the regulation of this process is controlled by many factors at the fetal-maternal interface. N-acetylglucosaminyltransferase V (GnT-V) is a key enzyme that catalyzes beta1, 6-N-acetylglucosamine (beta1-6GlcNAc) branching on asparagine-linked oligosaccharides of cell proteins. GnT-V and its product, beta1-6GlcNAc, are known to regulate cellular transformation and correlate with the metastatic potential of various cancer cells. The aim of the present study was to determine whether extravillous trophoblast (EVT) expressed this molecule and examine the role of GnT-V in the regulation of human trophoblast invasion. Immunohistochemistry showed that GnT-V was strongly expressed within the cytoplasm of EVT in the anchoring villi; this expression was down-regulated in EVTs invading the decidua. Suppression of beta1-6GlcNAc glycosylation by swainsonine enhanced the migratory potential and invasive capability of both primary EVTs and the EVT cell line, HTR-8/SVneo. Down-regulation of GnT-V expression by small interfering RNA in the choriocarcinoma cell line Jar consistently enhanced the migration and invasive capacity of these cells and elevated cellular adhesion to extracellular matrix proteins, such as fibronectin and collagen type I/IV. The extent of beta1-6 branching of alpha5beta1 integrin was significantly reduced in small interfering GnT-V-transfected Jar cells compared with mock transfectants, although the expression of alpha1, alpha5, alpha6, and beta1 integrin on the cell surface was not changed. These results suggest that GnT-V is expressed in human EVT and is involved in regulating trophoblast invasion through modifications of the oligosaccharide chains of alpha5beta1 integrin.