Development of a hematocrit-insensitive device to collect accurate volumes of dried blood spots without specialized skills for measuring clozapine and its metabolites as model analytes.

Dried blood spots have been used as alternatives to traditional plasma and serum samples. We have now developed new devices, named volumetric absorptive paper disc (VAPD) and mini-disc (VAPDmini), to collect accurate volumes of dried blood spots in a simple manner and without the need for additional instruments. VAPD consists of a filter paper disc and a filter paper sheet with holes slightly larger than the disc. The disc is fixed in one such hole without direct contact with the filter sheet. VAPDmini is a scaled-down version of the same device. When several drops of whole blood are applied, the disc becomes saturated and any excess sample is absorbed by the surrounding filter sheet. Accuracy and precision of sampling were assessed by determining the levels of clozapine and its metabolites as target analytes by liquid-liquid extraction and high-performance liquid chromatography with coulometric detection. In addition, differences in analyte recovery were within ±15% for all analytes in samples with 30-60% hematocrit, suggesting that VAPD and VAPDmini are insensitive to hematocrit for the analytes tested. The devices were also validated for analyte concentrations in the range 50-1000 ng/mL, and the limit of detection and lower limit of quantification were 5-17 ng/mL and 15-51 ng/mL, respectively. Intra- and inter-day precision ranged from 3% to 13%, whereas accuracy ranged from a -14% to 12% bias. Analytes were stable in the devices for at least 2 weeks at room temperature. Collectively, these results indicate that sampling using VAPD and VAPDmini is comparable to conventional hole punch sampling of entire dried blood spots, even for samples obtained from patients treated with clozapine. Importantly, the devices were also found to be suitable for sample self-collection.

Glycolipids from spinach suppress LPS-induced vascular inflammation through eNOS and NK-κB signaling.

Glycolipids are the major constituent of the thylakoid membrane of higher plants and have a variety of biological and pharmacological activities. However, anti-inflammatory effects of glycolipids on vascular endothelial cells have not been elucidated. Here, we investigated the effect of glycolipids extracted from spinach on lipopolysaccharides (LPS)-induced endothelial inflammation and evaluated the underlying molecular mechanisms. Treatment with glycolipids from spinach had no cytotoxic effects on cultured human umbilical vein endothelial cells (HUVECs) and significantly blocked the expression of LPS-induced interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), and intracellular adhesion molecule-1 (ICAM-1) in them. Glycolipids treatment also effectively suppressed monocyte adhesion to HUVECs. Treatment with glycolipids inhibited LPS-induced NF-κB phosphorylation and nuclear translocation. In addition, glycolipids treatment significantly promoted endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) production in HUVECs. Furthermore, glycolipids treatment blocked LPS-induced inducible NOS (iNOS) expression in HUVECs. Pretreatment with a NOS inhibitor attenuated glycolipids-induced suppression of NF-κB activation and adhesion molecule expression, and abolished the glycolipids-mediated suppression of monocyte adhesion to HUVECs. These results indicate that glycolipids suppress LPS-induced vascular inflammation through attenuation of the NF-κB pathway by increasing NO production in endothelial cells. These findings suggest that glycolipids from spinach may have a potential therapeutic use for inflammatory vascular diseases.

Astilbin from Engelhardtia chrysolepis enhances intestinal barrier functions in Caco-2 cell monolayers.

Astilbin, which is one of polyphenolic compounds isolated from the leaves of Engelhardtia chrysolepis HANCE (Chinese name, huang-qui), is available as the effective component in food and cosmetics because of its anti-oxidant and anti-inflammatory effects. The tight junction (TJ) proteins, which protect the body from foreign substances, are related to adhesion between a cell and a cell. Previously, the enhancement of TJ's functions induced by aglycones of flavonoids has been demonstrated, but the effects of the glycosides such as astilbin have not been observed yet. In this study, we investigated the effects of astilbin on the TJ's functions, and human colon carcinoma Caco-2 cell monolayers were used to evaluate the effects of astilbin on transepithelial electrical resistance (TER) value and the mRNA and proteins expressions of TJ-related molecules. Astilbin increased the TER value, mRNA expression levels of claudin-1 and ZO-2, and protein expression levels of occludin and ZO-2 in Caco-2 cells. Astilbin also increased the TER value in Caco-2 cells co-stimulated with TNF-α plus IFN-γ, and moreover upregulated the protein expression of TJ-related molecules in Caco-2 cells co-treated with TNF-α plus IFN-γ. These results suggest that astilbin can enhance the expressions of TJ-related molecules, leading to upregulation of the barrier functions in the intestinal cells.

Kisspeptin in the Hypothalamus of 2 Rat Models of Polycystic Ovary Syndrome.

Hyperandrogenism, disturbance of the hypothalamus-pituitary-ovary axis followed by elevated serum luteinizing hormone (LH) levels, and insulin resistance are involved in the complicated pathophysiology of polycystic ovary syndrome (PCOS). Kisspeptin is coexpressed with neurokinin B (NKB) in the arcuate nucleus (ARC), the center of the gonadotropin-releasing hormone pulse generator that is responsible for pulsatile LH secretion. We compared 2 androgenized rat models of PCOS to evaluate the estrous cycle, hormonal profiles, and expression of kisspeptin and NKB in the ARC. Rats in our postnatal dihydrotestosterone (DHT)-treatment model exhibited weight gain and persistent diestrus with normal LH levels. In contrast, irregular cycles, with elevated LH serum levels and normal body weight, were found in the prenatally DHT-treated rats. We also found increased signals of kisspeptin and NKB in the ARC of the prenatally DHT-treated rats, and not in the postnatally DHT-treated rats. Our results suggest that prenatal exposure to androgens may result in higher kisspeptin and NKB levels in the ARC, which could be associated with 1 phenotype of PCOS that is characterized by normal body weight and higher LH secretion, whereas in postnatally DHT-treated rats, characteristics such as weight gain and normal LH levels are seen in the obese PCOS phenotype.

Choosing the optimal therapeutic strategy for placental polyps using power Doppler color scoring: Transarterial embolization followed by hysteroscopic resection or expectant management?

OBJECTIVE: To evaluate a protocol for selection of placental polyp management, including expectant management and hysteroscopic resection with or without transarterial embolization (TAE), using power Doppler color score (PDCS) as the vascularity parameter. MATERIALS AND METHODS: This retrospective case-control study included 25 patients who were diagnosed with placental polyps. We evaluated the vascularity of placental polyps with PDCS measured by transvaginal ultrasonography as follows: PDCS 1, no blood flow; PDCS 2, minimal flow; PDCS 3, moderate flow; and PDCS 4, marked blood flow. We then selected expectant management or hysteroscopic resection with or without TAE. RESULTS: Three of 17 patients with PDCS 1 or 2 underwent surgical intervention, and expectant management was successful in 14. Seven of eight patients with PDCS 3 or 4 underwent surgical intervention, while expectant management was successful in only one patient. CONCLUSION: PDCS is a simple examination for evaluating the vascularity of placental polyps. PDCS might be useful for selecting the optimal treatment for placental polyps, such as expectant management or surgical intervention, according to their vascularity.

The Establishment of an Assay to Measure DNA Polymerase-Catalyzed Repair of UVB-Induced DNA Damage in Skin Cells and Screening of DNA Polymerase Enhancers from Medicinal Plants.

An in vitro assay method was established to measure the activity of cellular DNA polymerases (Pols) in cultured normal human epidermal keratinocytes (NHEKs) by modifying Pol inhibitor activity. Ultraviolet (UV) irradiation enhanced the activity of Pols, especially DNA repair-related Pols, in the cell extracts of NHEKs. The optimal ultraviolet B (UVB) exposure dose and culture time to upregulate Pols activity was 100 mJ/cm² and 4-h incubation, respectively. We screened eight extracts of medicinal plants for enhancement of UVB-exposed cellular Pols activity using NHEKs, and found that rose myrtle was the strongest Pols enhancer. A Pols' enhancement compound was purified from an 80% ethanol extract of rose myrtle, and piceatannol was isolated by spectroscopic analysis. Induction of Pol activity involved synergy between UVB irradiation and rose myrtle extract and/or piceatannol. Both the extract and piceatannol reduced UVB-induced cyclobutane pyrimidine dimer production, and prevented UVB-induced cytotoxicity. These results indicate that rose myrtle extract and piceatannol, its component, are potential photo-protective candidates for UV-induced skin damage.

Increase of kisspeptin-positive cells in the hypothalamus of a rat model of polycystic ovary syndrome.

Kisspeptin, a hypothalamic neuropeptide, is expressed in the arcuate nucleus (ARC) that is considered as the center of the gonadotropin-releasing hormone (GnRH)-pulse generator. We hypothesized that kisspeptin expressed in the ARC is implicated in the disturbance of the hypothalamus-pituitary-ovary axis observed in polycystic ovary syndrome (PCOS). To test this hypothesis, we evaluated the hormonal profiles, luteinizing hormone (LH) pulse, and ARC kisspeptin immunoreactivity in a PCOS rat model using the anti-progestin RU486. We found an alteration of the LH pulse, including a trend towards an increased mean LH concentration and area under the curve, and a significant upregulation of the mean LH pulse amplitude. Additionally, a higher number of kisspeptin-positive cells was observed in the ARC of RU486-treated rats than in the ARC of intact rats. These results suggest the possible involvement of hypothalamic kisspeptin in the hypothalamus-pituitary-ovary axis and therefore, in PCOS pathophysiology.

ß-Amyrin induces angiogenesis in vascular endothelial cells through the Akt/endothelial nitric oxide synthase signaling pathway.

ß-Amyrin is a pentacyclic triterpene found in various plants and has a variety of biological and pharmacological activities. However, the angiogenic effects of ß-amyrin in vascular endothelial cells have not been elucidated. Herein, we investigated the effects of ß-amyrin on angiogenesis and evaluated the underlying molecular mechanisms. ß-Amyrin treatment had no cytotoxic effect on cultured human umbilical vein endothelial cells (HUVECs). It promoted the formation of tube-like structures and enhanced HUVEC migration and the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) in HUVECs. Pre-treatment with a PI3 kinase or NOS inhibitor blocked ß-amyrin-induced phosphorylation of Akt and eNOS. ß-Amyrin treatment significantly induced nitric oxide (NO) production in HUVECs. Furthermore, pre-treatment with a PI3 kinase or NOS inhibitor significantly inhibited ß-amyrin-induced tube-like structures formation of vascular endothelial cells and HUVEC migration. These data indicate that ß-amyrin-induced angiogenesis in vascular endothelial cells may be mediated by Akt-eNOS signaling-dependent mechanisms. These findings suggest that ß-amyrin could be a novel therapeutic agent for ischemic vascular diseases.

Focal Ischemic Injury with Complex Middle Cerebral Artery in Stroke-Prone Spontaneously Hypertensive Rats with Loss-Of-Function in NADPH Oxidases.

By means of introgressing a loss-of-function mutation in the p22phox gene from the Matsumoto Eosinophilia Shinshu (MES) rat to stroke-prone spontaneously hypertensive rats (SHRSP), we constructed the SHRSP-based congenic strain lacking the P22PHOX expression (i.e., lacking NADPH oxidases [NOX] activities) (SHRSP.MES-Cyba(mes)/Izm; hereafter referred to as SP.MES). To examine the effects of Nox activities on the focal ischemic injury or stroke, we performed middle cerebral artery (MCA) occlusion in this new congenic strain; the distal MCA was occluded by 561-nm laser-driven photothrombosis. Resting mean arterial blood pressure was significantly lower in SP.MES when compared with the control PM0/SHRSP (150±11 mmHg vs. 166±11 mmHg). Cerebral blood flow decreased to 37±13% in SP.MES and 35±17% in PM0/SHRSP at 10 min after MCA occlusion (not significant). Infarct volume determined at 24 h after MCA occlusion in SP.MES was 89±39 mm3, which was not significantly different from 83±35 mm3 in PM0/SHRSP. The distal MCA pattern was more complex in SP.MES (median 3, IQR 3-5) than PM0/SHRSP (median 2, IQR 1-3) (p = 0.001). Because more complex distal MCA is known to produce larger infarction after distal MCA occlusion in SHR, we adjusted for the branching pattern in an ANCOVA. The adjusted mean of infarct volume was significantly smaller in SP.MES compared with that in PM0/SHRSP (67 [95% CI 46 to 87] mm3 vs. 100 [95% CI 82 to 118] mm3, p = 0.032). Elimination of the P22PHOX expression induced complex distal MCA, which would suggest the presence of 'loss of complexity' induced by enhanced oxidative stress in SHRSP; infarct size in SP.MES--when adjusted for distal MCA complexity--was significantly attenuated compared with that in PM0/SHRSP. Therefore, the present results suggest that Nox is harmful for ischemic brain tissue.

Rose myrtle (Rhodomyrtus tomentosa) extract and its component, piceatannol, enhance the activity of DNA polymerase and suppress the inflammatory response elicited by UVB­induced DNA damage in skin cells.

A number of naturally occurring agents are hypothesized to protect against ultraviolet (UV)­induced skin damage. The present study screened >50 plant extracts for inhibitors of UVB­induced cytotoxicity, using cultured normal human epidermal keratinocytes (NHEK), and identified that the fruit of rose myrtle (Rhodomyrtus tomentosa) was the most marked inhibitor of cell death. The protective effect of rose myrtle extract and the two key components, piceatannol and piceatannol­4'­O­ß­D­glucopyranoside, on UVB­induced damage and inflammation in cultured NHEK was investigated. The 80% ethanol extract from rose myrtle fruit with piceatannol exhibited protection of UVB­induced cytotoxicity in NHEK; however, piceatannol­4'­O­ß­D­glucopyranoside exhibited no protection, as determined by a 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide assay. This extract and piceatannol reduced the production of UVB­induced cyclobutane pyrimidine dimers and enhanced the cellular enzyme activity of the DNA polymerases in UVB­irradiated NHEK, suggesting that UVB­stimulated DNA damage was repaired by the polymerases. In addition, the secretion of prostaglandin E2, which is an inflammatory mediator, was decreased. These results indicated that rose myrtle fruit extract and its key constituent, piceatannol, are potential photoprotective candidates for UV­induced skin damage.

Anti-Müllerian hormone and assessment of ovarian reserve after ovarian toxic treatment: a systematic narrative review.

Since serum anti-Müllerian hormone (AMH) levels enable quantitative evaluation of ovarian damage, we conducted a computer-based search, using key words, of all articles published in English through the PubMed database from inception until September 2013 to summarize available studies evaluating ovarian reserve after ovarian toxic interventions to discuss the usefulness of serum AMH levels. We found that most of the studies demonstrated a decline in serum AMH levels when compared to control or pretreatment levels, with levels dependent on the type of treatment modality. Measurement of serum AMH levels enables quantitative evaluation of ovarian damage caused by ovarian toxic interventions, such as chemotherapy and radiotherapy, instead of qualitative evaluation using menstrual condition or basal follicle-stimulating hormone levels. Serum AMH levels are becoming indispensable to assess the ovarian reserve of patients who desire preservation of ovarian function for fertility and endogenous sex steroid hormones.

CYP51A1 induced by growth differentiation factor 9 and follicle-stimulating hormone in granulosa cells is a possible predictor for unfertilization.

Growth differentiation factor 9 (GDF9), an oocyte-secreted factor, whose receptors exist in granulosa cells, is involved in follicle progression. Therefore, GDF9 is considered to potentially mediate signals necessary for follicular growth. However, the effect of GDF9 on human granulosa cells is not fully understood. Human immortalized nonluteinized granulosa cell line (HGrC1) which we have previously reported was stimulated with GDF9 and/or follicle-stimulating hormone (FSH). Granulosa cells obtained from in vitro fertilization (IVF) patients were also evaluated with quantitative reverse transcription polymerase chain reaction (RT-PCR). Real-time RT-PCR showed that GDF9 increased messenger RNA (mRNA) levels of enzymes required for cholesterol biosynthesis, such as 3-hydroxy-3-methylglutanyl-CoA synthase 1 (HMGCS1), farnesyl-diphosphate farnesyltransferase 1, squalene epoxidase, lanosterol synthase, and cytochrome P450, family 51, subfamily A, polypeptide 1 (CYP51A1). A greater increase in mRNA levels of HMGCS1 and CYP51A1 was observed by combined treatment with GDF9 and FSH. Clinical samples showed a significant increase in CYP51A1 mRNA in the group of granulosa cells connected with unfertilized oocytes. Our results suggest that GDF9, possibly with FSH, may play significant roles in the regulation of cholesterol biosynthesis and the expression of CYP51A1 which might be a predictor for unfertilization.

Anti-Müllerian hormone as a possible predictor of fecundability in subfertile women over 38 years: a retrospective cohort study.

Anti-Müllerian hormone (AMH) is a relatively novel method for examining the ovarian reserve that reflects female reproductive function. In the era in which the number of women delaying attempts to conceive has increased, a good predictor for long-term fecundability has been explored. We performed the retrospective cohort study to investigate whether initial serum AMH levels are useful for predicting long-term fertility during infertility treatments. We recruited 149 women in the retrospective cohort, and 52 women were gravid during the follow-up period. According to the multiple logistic analyses, only age was found to have a significant correlation with pregnancy success in all women. In women ≥38 years, significantly higher serum AMH levels were detected in the pregnant group (median = 2.83 ng/mL, range = 1.11-6.29 ng/mL) than the non-pregnant group (median = 1.22 ng/mL, range = 0-9.46 ng/mL; p = 0.015). None of the women with serum AMH levels <0.7 ng/mL were pregnant during treatment. AMH may be used to identify poor pregnancy prospects in women who are above 38 years.

Assessment of ovarian reserve using anti-Müllerian hormone levels in benign gynecologic conditions and surgical interventions: a systematic narrative review.

The usefulness of anti-Müllerian hormone (AMH) for the quantitative evaluation of ovarian reserve has been established. Therefore, serum AMH has been recently applied to the assessment of ovarian reserve outside infertility treatment. We conducted a computer-based search, using keywords, through the PubMed database from inception until May 2014 and summarized available studies evaluating ovarian damage caused by gynecologic diseases, such as endometriosis and ovarian tumor, as well as surgical interventions, such as cystectomy and uterine artery embolization (UAE), to discuss the usefulness of serum AMH. Most of the studies demonstrated a decline of serum AMH levels after cystectomy for endometriomas. It is not conclusive whether electrocoagulation or suturing is preferable. The effects of other gynecologic diseases and interventions, such as hysterectomy and UAE, on ovarian reserve are controversial. Serum AMH levels should be considered in determining the indication and selection of operative methods for benign gynecologic conditions.

Pelvic tuberculous granuloma successfully treated with laparoscopy to preserve fertility: a case report and review of the published work.

Tuberculous granuloma must be considered in the differential diagnosis of pelvic masses in women of reproductive age because the major sequela of pelvic tuberculosis is infertility; however, currently there is very little information about its fertility-preserving treatment. We report the case of a woman with a history of tuberculous peritonitis who referred to our hospital for evaluation of an adnexal mass and primary infertility. The patient underwent excision of pelvic tuberculous granuloma with fertility-preserving laparoscopic surgery. We resected as much of the tuberculous granuloma as possible using the laparoscopic technique without causing damage to the uterus or ovaries. In particular, we report for the first time in the published work the laparoscopic removal of tuberculous granuloma without causing damage to the uterus or ovaries. Our experience from this case suggests that laparoscopic diagnosis and treatment of tuberculous granuloma is a feasible procedure in a patient who wants to conceive.

Time-lapse observations to analyze the effects of assisted hatching.

PURPOSE: Assisted hatching (AH) is an artificial disruption of the zona pellucida with the aim of facilitating embryo implantation. We used time-lapse observations of mouse embryos to examine the effect of AH in mouse blastocysts. METHODS: AH techniques were performed with acid Tyrode's solution. We compared the rates of blastocyst formation and blastocyst attachment to Ishikawa cells between the control (n = 28) and the AH group (n = 24). To analyze the effects of AH, 8-cell mice embryos were cultured under time-lapse observations (every 15 min). The time required for hatching, the hatching rates, the frequency of contraction, and the contraction rates in the blastocysts were analyzed. RESULTS: There were no significant differences between the two groups in hatching rate or attachment rate. The times required for hatching were 286 ± 22 min in the AH group and 990 ± 437 min in the control group (P = 0.018). The contraction frequencies in blastocysts were 3.5 ± 0.7 times in the AH group and 7.5 ± 2.5 times in the control group (P = 0.020). CONCLUSIONS: From the time-lapse observations we found that the time required for hatching and the frequency of contraction in blastocysts were both reduced by AH, although blastocyst formation and attachment were not affected.

Possible involvement of CD10 in the development of endometriosis due to its inhibitory effects on CD44-dependent cell adhesion.

A reduced response to progesterone in the eutopic endometrium with endometriosis and in endometriotic tissues is considered to be the underlying factor for endometriosis. CD10 is known to be expressed by endometrial and endometriotic stromal cells and may be induced by progestins, although the function of CD10 is not fully revealed in endometrial or endometriotic tissues. In the current study, the expression of CD10 was significantly increased by treatment of the cells with progesterone, 17ß-estradiol, and dibutyryl cyclic adenosine monophosphate (cAMP) in the endometrial stromal cells. On the other hand, the expression of CD10 following treatment with progesterone, 17ß-estradiol, and dibutyryl cAMP was not significantly increased in endometriotic stromal cells. The adhesion assay for endometrial and endometriotic stromal cells to hyaluronan using 5- or 6-(N-succinimidyloxycarbonyl)-fluorescein 3', 6'-diacetate-labeled cells demonstrated that the CD44-dependent adhesion of stromal cells was inhibited by CD10. As far as the induction of CD10 is concerned, the effect of progesterone was different between endometrial stromal cells and endometriotic stromal cells. CD10 might be involved in the development of endometriosis due to its influence on CD44-dependent cell adhesion.

[Serum of transthyretin as a treatment-responsive biomarker for schizophrenia].

We investigated the changes in protein profiles of treatment responsive biomarkers in three subjects with first-onset schizophrenia using the surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology. Our results showed that eight protein peaks were down-regulated or up-regulated during the acute phase, and returned toward the control values during the recovery phase. In particular, a 13,761Da protein peak was markedly down-regulated during the acute phase, and returned during the recovery phase. This protein was identified as unmodified transthyretin using two-dimensional electrophoresis followed by peptide mass fingerprinting based on matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). However, we failed to show transthyretin changes with the treatment using enzyme-linked immunosorbent assay (ELISA) and two-dimensional electrophoresis. The distinction between unmodified and modified subtypes of transthyretin was difficult with these methods because of similar molecular weights and isoelectric points in these subtypes. The present study showed that the application of SELDI-TOF-MS technology has higher precision for the distinction of detailed molecular weight than conventional proteomics techniques such as ELISA and two-dimensional electrophoresis. The dynamic changes in transthyretin have been reported to be associated with acute-psychosis condition; the present study suggested that unmodified transthyretin has the potential to be a treatment responsive biomarker for schizophrenia.

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.

A proteomic analysis of human follicular fluid: comparison between fertilized oocytes and non-fertilized oocytes in the same patient.

PURPOSE: Human follicular fluid constitutes the microenvironment of follicles and includes various biological active proteins that can affect follicle growth and oocyte fertilization. Conducting proteomic evaluations of human follicular fluid may be helpful for identifying potential biomarkers possibly possessing a predictive value for oocyte quality and the success of in vitro fertilization. METHOD: We performed proteomic profiling of human follicular fluids containing oocytes that were fertilized and resulted in pregnancy and follicular fluids containing oocytes that were not fertilized in the same patients undergoing intracytoplasmic sperm injection using the LTQ Orbitrap coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) analyses. RESULTS: We identified a total of 503 proteins in human follicular fluids containing fertilized and non-fertilized oocytes obtained from 12 patients. We also found that 53 proteins exhibited significantly different spectral counts between the two groups, including heparan sulfate proteoglycan perlecan, which showed significant upregulation in the follicular fluids containing fertilized oocytes in comparison with that observed in the follicular fluids containing non-fertilized oocytes. CONCLUSION: Our results suggest a possibility that proteins identified by LC/MS/MS in follicular fluid might not only be involved in folliculogenesis, but also function as biomarkers possessing predictive potential for oocyte maturation and the success of IVF when their expression levels are significantly different between fertilized and non-fertilized oocytes, although no distinctive biomarkers were identified in the current study.