A newly synthesized thiosemicarbazide derivative trigger apoptosis rather than necroptosis on HEPG2 cell line.

Thiosemicarbazide derivatives have been the focus of scientists owing to their broad biological activities such as anticancer, antimicrobial, and anti-inflammatory. Herein, we designed and synthesized a new thiosemicarbazide derivative (TS-1) and evaluated its antiproliferative potential against the human hepatocellular carcinoma cell line (HEPG2) and human umbilical vein endothelial cell line (ECV-304). Also, it was aimed to investigate the necroptotic and apoptotic cell death effects of TS-1 in HEPG2 cells, and these effects were supported by molecular docking. The new synthesized compound structure was characterized using various spectroscopic methods such as FT-IR, 1 H-NMR, 13 C-NMR, and elemental analysis. The cytotoxic activity of the tested compound was measured by the MTT assay. Apoptotic and necroptotic properties of the TS-1 were evaluated by indirect immunoperoxidase method using antibodies against Ki-67, Bax, Bcl-2, caspase-3, caspase-8, caspase-9, RIP3, and RIPK1. Apoptotic and necroptotic effects of TS-1 were supported by molecular docking. Compound TS-1 was synthesized as a pure compound with a high yield. The effective value of TS-1 was 10 µM in HEPG2 cells. TS-1 did not show any cytotoxic effect on ECV-304. Caspase-3 and RIPK1 immunoreactivities were significantly increased in HEPG2 cells after being treated with TS-1. As the results of the molecular docking studies, the molecular docking showed that the TS-1 exhibits H-bond interaction with various significant amino acid residues in the active site of both RIPK1. It could be concluded that TS-1 could be a promising novel therapeutic agent by inducing apoptosis rather than necroptosis in HEPG2 cells.

Boron-Doped Carbon Nanodots as a Theranostic Agent for Colon Cancer Stem Cells.

Carbon nanodots have drawn a great deal of attention due to their green and expedient opportunities in biological and chemical sciences. Their high fluorescence capabilities and low toxicity for living cells and tissues make them excellent imaging agents. In addition, they have a fluorimetric response against inorganic and organic species. Boron-doped carbon nanodots (B-CDs) with high fluorescence yield were produced from phenylboronic acid and glutamine as boron and carbon sources, respectively, by a hydrothermal method. First, the effects of the temperature on their fluorescence yield and the structural characteristics of B-CDs were investigated. Second, their cytotoxicity and cell death and proliferation behaviors were examined. The cytotoxicity was evaluated by the MTT assay. The cellular properties were evaluated with the distribution of caspase 3, Ki67, lamin B1, P16, and cytochrome c after the indirect immunoperoxidase technique. After the MTT assay, 1:1 dilution of all applicants for 24 h was used in the study. After immunohistochemical analyses, the application of B-CDs synthesized at 230 °C did not change control cell (Vero) proliferation, and also apoptosis was not triggered. Colo 320 CD133+ and CD133- cell-triggered apoptosis and cellular senescence were found to be synthesis temperature dependent. In addition, Colo 320 CD133- cells were affected relatively more than CD133+ cells from B-CDs. While B-CDs did not affect the control cells, the colon cancer stem cells (Colo 320 CD133+) were affected in a time-dependent manner. Therefore, the use of the synthesized B-CD product may be an alternative method for controlling or eliminating cancer stem cells in the tumor tissue.

Exosomes obtained from adipose mesenchymal stem cells prevent ischemia-reperfusion injury after torsion-detorsion in rat testes.

PURPOSE: To investigate the effect of exosomes obtained from adipose-derived mesenchymal stem cells (AD-MSCs) on testicular ischemia-reperfusion (I/R) injury. METHODS: AD-MSCs from rat adipose tissue were cultured. Characterization of cells was evaluated with CD44, CD90, CD34 and CD45 antibodies. Exosomes from AD-MSCs were obtained with the miRCURY exosome isolation kit. 21 rats were divided into 3 groups. The I/R model was created as 720° torsion for 4 h and reperfusion for 4 h. In the Sham group (SG), only scrotal incision was made. 100 µl of medium in the torsion-control group (T-CG) and 100 µl of exosome in the treatment group (TG) were injected into the testicular parenchyma after detorsion. Johnsen scores of testicles were determined. Apoptosis was evaluated by the TUNEL method. RESULTS: It was observed that the seminiferous tubule structures were partially disrupted in T-CG, but normal in SG and TG. Johnsen scores in SG, T-CG, and TG were 8.64 ± 0.39, 7.71 ± 0.37, and 8.57 ± 0.39, respectively. Apoptotic cell distribution was 11.28 ± 5.25%, 60.58% ± 1.68% and 17.71 ± 8.34% in SG, T-CG and TG, respectively. In both parameters, the difference between SG and TG was insignificant (p > 0.05), the difference between T-CG/TG and SG/T-CG was significant (p < 0.05). CONCLUSION: Exosomes obtained from AD-MSCs are effective in preventing testicular I/R injury. This effect appears to occur because of suppression of apoptotic activity.

Comparative phytochemical studies on the roots of Polygala azizsancarii and P. peshmenii and neuroprotective activities of the two xanthones.

Six known sucrose mono-, di- and triesters and five xanthone derivatives were isolated from the roots of Polygala peshmenii Eren, Parolly, Raus & Kürschner which is a narrow species endemic to Türkiye. Among the xanthones, 1,7-dihydroxy-2,3-methylenedioxy-5,6-dimethoxy-xanthone is an undescribed compound isolated for the first time from a natural source. The studies on the roots of P. azizsancarii Dönmez have resulted in the isolation of four known compounds including sucrose mono-, di- and triesters. The structures of the sucrose esters and xanthones isolated from P. azizsancarii and P. peshmenii were established by spectroscopic methods, including 1D-NMR (1H NMR, 13C NMR, DEPT-135), 2D-NMR (COSY, NOESY, HSQC, HMBC). Neuroprotective activities of two xanthones, 1,3,6-trihydroxy-2,5,7-trimethoxyxanthone and 3-O-ß-D-glucopyranosyloxy-1,6-dihydroxy-2,5,7-trimethoxyxanthone isolated from the roots of P. azizsancarii were evaluated in vitro using in a cellular model of Alzheimer's disease. SKNAS human neuroblastoma cells were used in the study and treated with different consecrations of Aß25₋35 oligomer for up to 48 h. Cell viability was evaluated using MTT assay. The distribution of ß-amyloid, α-synuclein, tau, JAK2, STAT3, caspase 3 and BMP-2 were investigated using indirect immunoperoxidase staining. Our results suggested that both xanthones control tau aggregation with no effect on ß-amyloid plaque formation. In addition, for neuronal pathophysiology in AD cell model, decreased distributions of JAK/STAT3 and BMP2 signaling pathways were demonstrated, therefore they play a role in the protective effect on neurons in neurodegenerative disease. A significant decrease in caspase 3 immunoreactivity was detected after the administration of both compounds in AD cells. Therefore, both compounds control neuronal pathophysiology and rescue cell death in AD disease.

Histologic and physiologic analysis of the relationship between the dorsal nerve of the penis and the corpus cavernosum on a rat model. A complementary pathway on the innervation of penile erection?

AIM: The dorsal nerve of the penis (DNP) is the terminal branch of the pudendal nerve which is responsible for the somatic innervation of the penis. This study aims to outline any direct role of the DNP in the hemodynamics of erection histologically and physiologically. MATERIALS AND METHODS: Fifteen Wistar albino rats were sorted into the electrical activity (n = 6), intracavernous pressure (n = 4), and control (n = 5) groups. The dorsal nerve was electrostimulated and the simultaneous changes in intracavernous pressure and smooth muscle activity were recorded. Penile tissues were collected, fixed, and sectioned, the slides were stained with either hematoxylin-eosin for morphological evaluation or using the indirect immunoperoxidase technique to analyze the distributions of eNOS, iNOS, and nNOS. RESULTS: During electrostimulation, there was a simultaneous statistically significant decrease in the electrical activity inside the corpora in electromyography and an increase in intracavernous pressure. eNOS and iNOS immunoreactivities were higher in the study group than in the control group. nNOS immunoreactivity was moderate in both study and control groups. CONCLUSION: Some fibers in the dorsal nerve of penis continue into the corpora cavernosa through the tunica albuginea and have an active, direct role in the hemodynamic process of erection, which may be complementary to the main route of innervation.

Age-related changes in the immunomodulatory effects of human dental pulp derived mesenchymal stem cells on the CD4+ T cell subsets.

Mesenchymal stem cells (MSCs) are powerful immunomodulatory cells. The effects of the aging on these abilities of MSCs have not been adequately clarified. In this study, alterations in immunomodulatory abilities of MSCs caused by aging were investigated. For this, dental pulp (DP) MSCs and peripheral blood mononuclear cells (PBMCs) of elderly and young donors were co-cultured age-matched and cross. We detected that the effects of DP-MSCs on Th1 and Th2 cells and their specific cytokines IFN-γ and IL-4 are not affected by aging. However, we observed that young and elderly DP-MSCs have different effects on Th17 and Treg cells. Th17 frequencies of young and elderly PBMCs were significantly increased only by young DP-MSCs, in contrast, Treg frequencies were significantly increased by elderly DP-MSCs. IL-6, IL-17a and HGF levels of both young and elderly PBMCs showed a significant increase only by young DP-MSCs, but TGF-ß levels were significantly increased only by elderly DP-MSCs. The oral cavity is home to a rich microflora. The interactions of dental tissues with this microflora can lead them to acquire different epigenetic modifications. Aging can affect the microflora composition of the oral cavity and change this process in different directions. According to our findings, DP-MSCs are effective cells in the regulation of CD4+ T cells, and their effects on Th1 and Th2 cells were not affected by aging. However, pleiotropic molecules IL-6 and HGF expressions, which are important in dental and bone tissue regeneration, decreased significantly in elderly DP-MSCs. This situation may have indirectly made a difference in the modulation effects of young and elderly DP-MSCs on the Th17 and Treg cells.

The effects of tramadol on cancer stem cells and metabolic changes in colon carcinoma cells lines.

Opioids are widely used in the treatment of cancer related pain. They mainly exert their effects on opioid receptors. The most common opioid in the treatment of pain is morphine. Previous studies show that they may have effects on cancer cell behavior. These may include apoptosis, angiogenesis, invasion, inflammation and immune reactions. Tramadol, also an opioid is widely used in the treatment of cancer pain and is not well studied in cancer behavior. We aimed to investigate the effects of tramadol on cancer stem cells and metabolic changes in colon carcinoma cells. We used Colo320 (ATCC, CCL-220), Colo741 (ECACC, 93052621) and HCT116 (ATCC, CCL-247) colon cancer cell lines. CD133 was considered colon cancer stem cell marker and used to sort CD133+ and CD133- cells by magnetic cell sorting. MTT (mitochondria-targeted therapeutics) technique was used to detect tramadol's cytotoxic effect on cells in the study groups. Cells were treated with 1 mg/kg, 1.5 mg/kg and 2 mg/kg tramadol for 24 h at 37 °C and 5% CO2.Caspase-3, Ki-67, Bcl-2 and VGEF distributions were performed using indirect immunoperoxidase staining for immunohistochemical analysis. The study showed that tramadol has triggering effect on apoptosis in Colo320 colon cancer stem cells.

Photostimulation of osteogenic differentiation on silk scaffolds by plasma arc light source.

Low-level laser therapy (LLLT) has been used for more than 30 years to heal wounds. In recent years, LLLT or photostimulation has been indicated as an effective tool for regenerative and dental medicine by using monochromatic light. The aim of this study is to indicate the usability of plasma arc light source for bone regeneration. This is why we used polychromatic light source providing effective wavelengths in the range of 590-1500 nm for cellular response and investigated photostimulation effects on osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on 3D silk scaffolds. Cellular responses were examined by using cell culture methods in terms of proliferation, differentiation, and morphological analyses. The results showed that photostimulation with a polychromatic light source (applied for 5 min from the 3rd day after seeding up to the 28th day in 2-day intervals with 92-mW/cm2 power from 10-cm distance to the cells) enhanced osteogenic differentiation of hMSCs according to higher alkaline phosphatase (ALP) activity, collagen and calcium content, osteogenic gene expressions, and matrix mineralization. In conclusion, we suggest that the plasma arc light source that was used here has a great potential for bone regeneration.

Effects of Keratinocytes Differentiated from Embryonic and Adipogenic Stem Cells on Wound Healing in a Diabetic Mouse Model.

OBJECTIVE: The current study aims to assess the molecular effects of keratinocytes derived from embryonic and adipose-derived stem cells (ADSCs) on wound healing in mice with diabetes mellitus. MATERIALS AND METHODS: Sixty BALB/c mice were randomly allocated into 6 groups of 10. Following diabetes mellitus induction by intraperitoneal injection of streptozocin, wounds were created and covered with gauze dipped in various solutions: isotonic saline, carrier and transfer medium-engineered dermal template, keratinocytes derived from embryonic stem cells (ESCs), keratinocytes differentiated from ADSCs, or ADSC medium alone. Histopathological changes and immunohistochemical alterations in the activities of cytokeratin 8, cytokeratin 14, epidermal growth factor (EGF), interleukin 8 (IL-8), fibroblast growth factor 2 (FGF-2), monocyte chemoattractant protein 1 (MCP-1), and collagen I were compared among the 6 groups. RESULTS: Histopathological analysis revealed that wound healing was accelerated by application of keratinocytes derived from ESCs. Such cells increased the activities of cytokeratin 8 and cytokeratin 14. No significant among-group differences were noted in terms of IL-8, FGF-2, MCP-1, or collagen I production. CONCLUSIONS: Keratinocytes derived from ESCs accelerated wound healing in mice with diabetes mellitus. The beneficial effects were evident both histomorphologically and immunohistochemically. Although keratinocytes derived from ADSCs are readily available, such cells did not accelerate wound healing.

Potential clinical use of differentiated cells from embryonic or Mesenchymal stem cells in orthopeadic problems.

Stem cells are classified by their tissue source. Embryonic stem cells that derived from inner cell mass of blastocyst stage embryos are highly proliferative in their undifferentiated state. A multipotent type of mesenchymal stem cells isolated from different type of tissues such as bone marrow, fat tissue etc. The dynamics of embryonic and adult stem cell cycles are profoundly dissimilar than the culture of stem cells. After improving the culture conditions and differentiation potentials, differentiated stem cells are the first cells to be preferred in modern regenerative medicine and tissue engineering. This review article focuses on the cell-based therapy on orthopedic problems. We explore the challenges associated with bone repair and regeneration using embryonic or mesenchymal stem cells that use undifferentiated or/and differentiated condition. This paper also discusses optimizing the best cell type, differentiation condition and using them on bone tissue engineering in future investigation.

Significance of apoptosis related proteins on malignant transformation of ovarian tumors: A comparison between Bcl-2/Bax ratio and p53 immunoreactivity.

In this study, we compared the immunoreactivities of Bcl-2, Bax and p53 proteins in ovarian tumors and related the immunohistochemical findings to the histological type of the tumors. Formalin-fixed, paraffin wax-embedded tissue sections from 40 patients who had serous-mucinous borderline tumors and serous-mucinous adenocarcinoma of the ovary (n=10 each) were stained with hematoxylin-eosin (H&E). After histopathological examination, serial sections were stained immunohistochemically with primary antibodies to Bcl-2, Bax and p53 using an avidin-biotin-peroxidase method. A semi-quantitative grading system was used to compare the immunohistochemical staining intensities. The nuclear DNA fragmentation of apoptosis was determined using TUNEL method. As a result of immunohistochemical staining, increased immunoreactivity of Bcl-2 was observed in adenocarcinomas when compared to borderline tumors (P<0.001). Strong immunoreactivity of Bcl-2 and mild immunoreactivities of Bax and p53 were detected in ovarian adenocarcinomas. There were no significant statistical differences in the immunoreactivity of Bax among the histological type of ovarian tumors. Whereas a balance was observed between the immunoreactivities of Bcl-2 and Bax in the borderline cases, and this balance was strongly changed toward the anti-apoptotic Bcl-2 protein in patients with adenocarcinoma. TUNEL staining of sections indicated apoptotic cells in the serous borderline tumors were about 8-fold higher than in the serous adenocarcinoma. The results of this study on apoptosis-related factors might help to develop novel protective and therapeutic approaches, such as isoflavonoids and isothiocyanates, which were associated with decreased Bcl-2/Bax ratio, against the malignant epithelial ovarian tumors.

Analysis of transferred keratinocyte-like cells derived from mouse embryonic stem cells on experimental surgical skin wounds of mouse.

Autologous/allogenic skin grafts constituted from differentiated adult or embryonic stem cells can be used in treatment of skin disorders. In our study we aimed to differentiate keratinocytes from mouse embryonic stem cells and the transfer of viable keratinocyte-like cells to a model of surgical skin wound of mouse. Embryoid bodies, derived from mouse embryonic stem cells, were cultured on basement membrane matrix with added BMP-4 for 10 days. The identification of differentiated keratinocyte-like cells was done by detection of cytokeratin-8 and cytokeratin-14 localization using an indirect immunoperoxidase technique and transmission electron microscopy evaluation. Distribution of BrdU, cytokeratin-8 and cytokeratin-14 were evaluated using an indirect immunoperoxidase technique from the experimental (dressing including BrdU labelled cells applied after the surgical wound was created on mouse), control (only the surgical wound was created on mouse) and sham (only the dressing applied after the surgical wound was created on mouse) in groups after 3, 5 and 7 days. Immunohistochemically and ultrastructurally, cells derived from mouse embryonic stem cells were similar to differentiated keratinocyte-like cells. Differentiated keratinocyte-like cells were demonstrated by positive BrdU, cytokeratin-8 and cytokeratin-14 staining after transfer to the wound area. In the experimental group wound healing was better after transferring differentiated keratinocytes when compared to the sham and control groups. In vivo continuity and usability of derived cells are very important issues. In wound repair mechanisms, keratinocyte-like cells could provide positive effects during the wound healing and could be used in clinical treatments of wound repair process.

Simultaneous folate intake may prevent adverse effect of valproic acid on neurulating nervous system.

PURPOSE: The aim of this study is to elucidate the preventive effect of folic acid (FA) on teratogenic effects of valporic acid (VA) in early stage chick embryos on neural tube development. MATERIALS AND METHODS: One hundred and fifty specific pathogen-free (SPF) chick eggs were used to investigate the neurulation in five groups. Group A was the control group. Group B was injected 0.02 ml of saline (0.9% NaCl) and was used for sham group. VA (0.72 mg) in 0.02 ml saline was injected in Group C, and 0.342 mcg of FA in 0.02 ml NaCl were administered to the embryos in Group D. VA (0.72 mg) + 0.342 mcg of FA in 0.02 ml saline were administered simultaneously to the eggs in Group E. At the end of 72 h, all embryos were extracted from eggs and were fixed, and for histological analyses hematoxylin and eosine was used, for detection of apoptotic cells terminal deoxyribonucleotide transferase-mediated dUTP-X nick end labeling (TUNEL) was used and for distribution of P53, bcl-2 and caspase-3, caspase-6, caspase-8 and caspase-9 immunoperoxidase techniques were used. RESULTS: While there were no neural tube defects in the embryos of groups A, B and D, eight embryos died in group C and there were 12 embryos with retarded embryological development. In contrast to that, no death was observed in group E, but only eight embryos were detected with maldevelopmental delay stage. CONCLUSION: These results suggested that VA may induce apoptotic mechanisms but not through the p53 pathway. In addition, FA effectively prevents the teratogenic influence of VA on chick embryo at neurulation stages by stopping cascade of apoptosis before caspase 3 expression.

Effect of lisinopril on renal tissue damage in unilateral ureteral obstruction in rats.

In this study, it was aimed to investigate apoptosis in renal injury and the effect of lisinopril in rat model, which constitute unilateral ureteral obstruction. The retroperitoneal ureter was ligated with a 4.0 silk for the experimental model of ureteral obstruction in Wistar albino rats. Untreated group (n = 20) received no treatment. For the lisinopril-treated group (n = 20), 20 mg/kg/day of drug was given orally. Ultrastructural differences were analyzed using electron microscopic technique; apoptotic distribution was analyzed using the TUNEL method. After electron microscopic evaluation, on the 4th and 14th day in the untreated group, edema in the glomeruli, loss of microvillus and apoptotic cells in proximal tubule cells and sclerosis in the glomeruli were detected. On the 4th day in the lisinopril-treated group, the kidney was ultrastructurally normal and a less number of apoptotic cells were only observed on the 14th day. On light microscopic examination on the 4th and 14th day in the untreated group, while the glomeruli were normal in structure, the boundary of the proximal tubule was disrupted and some picnotic cells in both the proximal and collecting tubules were observed. In both 4th and 14th day of the lisinopril-treated group, kidney showed normal structure, although in some places picnotic cells in the collecting tubules were observed. In conclusion, lisinopril was effective and it may prevent early renal damage in the direct obstruction model.

The effects of meloxicam on neural tube development in the early stage of chick embryos.

AIM: The aim of this study is to demonstrate the effect of meloxicam in early stage chick embryos on neural tube development. MATERIAL AND METHODS: One hundred specific pathogen-free (SPF) chicken eggs were used to investigate the neurulation. SPF eggs were invastigated in four groups (n:25). All of the groups were incubated at 37.2 +/- 0.1 degrees C and 60 +/- 5 % relative humidity for 30 hours, and an embryological development in the ninth stage as classified by Hamburger and Hamilton was obtained. In the end of the 30th hour, group A(control group) was administered 0.1 ml of saline (0.9% NaCl) in ovo and the other groups were administered meloxicam in increasing doses. At the end of 72 hours, all of the embryos were extracted from eggs and they underwent pathological examination with hematoxylin eosine and immunohistopathological examinations with CD138 and tubulin beta II. RESULTS: While the groups Aand B showed no neural tube defects, totally eight defective embryos were detected in the groups C and D (three in group C and five in group D. CONCLUSION: Our results suggested that meloxicam, a nonselective COX inhibitor, caused neural tube closure defects when injected at supratherapeutic doses. However, further studies with larger numbers of subjects are needed for its use in lower doses.

Changed Bcl:Bax ratio in endometrium of patients with unexplained infertility.

Apoptosis has been shown to be an important regulator of endometrial function during the menstrual cycle and implantation. Recently, some possible implantation defects were identified in patients with unexplained infertility. In this study, we investigated the role of spontaneous apoptosis, which is regulated by death regulatory genes, such as Bcl-2, Bax, p53, and isoenzymes of nitric oxide synthases; eNOS and iNOS during the implantation window in women with unexplained infertility. Endometrial samples were evaluated from fertile (n=15) and unexplained-infertile women (n=15) during post-ovulatory 7th or 8th day of their menstrual cycles. Apoptotic cells were detected using the dUTP nick-end labelling assay and Bcl-2, Bax, p53, iNOS and eNOS were assessed immunohistochemically. Reduced apoptotic cells, weak immunoreactivity of p53 and strong immunoreactivity of Bcl-2 were observed in the unexplained-infertile group compared with the fertile group (p<0.001). Bax intensity was similar in both groups. While weak iNOS immunoreactivity was detected in both groups, moderately increased eNOS immunoreactivity was observed in infertile cases. Spontaneous apoptosis is reduced in the endometrium of unexplained-infertile women, and is associated with the changed Bcl-2:Bax ratio. This finding may be a contributing factor to defective implantation causing infertility in this group of patients.

Effect of thermal energy produced by drilling on the facial nerve: histopathologic evaluation in guinea pigs.

The effect of thermal energy due to drilling around the facial nerve canal on the facial nerve was histopathologically evaluated in four guinea pigs. The bony canal of the facial nerve was drilled using a 3 mm diamond burr for one minute. The temperature changes on the facial nerve canal were noted before and after dissection. The temporal bones of the animals were histopathologically examined under light microscopy using haematoxylin & eosin (H&E) and solochrome cyanine staining for myelin, and immunohistochemical staining for neuronal nitric oxide synthase (nNOS). Compared to the control group, it was observed with H&E staining that there was oedema among the axonal fibres and with solochrome cyanine staining that the thickness of the myelin fibres was decreased, and that the severity and extent of nNOS activity was decreased in the axonal fibres. It was concluded that a temperature increase on the facial canal may potentially lead to inflammation of the nerve, and may also cause deterioration of nerve conduction to some extent.

Changes in distribution patterns of integrins in endometrium in copper T380 intrauterine device users.

Intrauterine contraception is the most cost-effective reversible method of contraception today, but its mechanism of action is not well understood. Our objective was to investigate immunohistochemical distribution patterns of alphav, alpha3, beta1 integrins in women using a copper T380 intrauterine device (IUD) for different periods of time to obtain insight into the role of integrins in intrauterine contraception. Endometrial biopsies were obtained from patients using T Cu380A IUD in follicular and luteal phases and in menopausal women grouped according to the period of time of IUD use (group 1: <3 year, and group 2:>or=3 years). Each group consisted of 10 patients, with a total number of 60 patients. Labelling intensity of all integrins, except for beta1 which increased in the follicular phase, were decreased in women who used IUD for>or=3 years when compared with group 1 in the follicular and luteal phases and in the menopause. We conclude that long-term use of IUD affects integrin expression in endometrium not only in follicular and luteal phases of premenopausal women but also in postmenopausal women. Copper IUD can inhibit binding of integrins to the extracellular matrix and it may cause inhibition of the implantation stage, which is crucial for pregnancy.

The aromatase inhibitor anastrozole is associated with favorable embryo development and implantation markers in mice ovarian stimulation cycles.

OBJECTIVE: To investigate the embryonic and endometrial effects of anastrozole in preimplantation and implantation phases in FSH-induced cycles in mice. DESIGN: Blind randomized study. SETTING: University research laboratory. ANIMAL(S): Twenty-seven mature female mice. INTERVENTION(S): Single-dose anastrozole (25 mg/kg [0.75 mg]), recombinant FSH (5 IU/mL), and hCG (5 IU/mL) (n = 9); recombinant FSH (5 IU/mL) and hCG (5 IU/mL) (n = 9); or sterile saline (1 mL) (n = 9). The morning of finding the vaginal plug was designated as day 1 of embryonic development (E1). Three mice from each group were sacrificed on E1 and embryos aspirated from uterine tubes. The rest of the mice were sacrificed on E2.5-3 and uteruses removed. MAIN OUTCOME MEASURE(S): Embryo quality, endometrial histologic evaluation, and immunohistochemical analysis of tumor necrosis factor-alpha, leukemia inhibitory factor, laminin, and collagen IV staining. RESULT(S): Anastrozole use in FSH-induced cycles not only caused an increase in preimplantation receptivity and implantation but also supported release of implantation markers. The enhanced embryo development seen in this study would explain the higher implantation because embryo development is synchronized with endometrial development. CONCLUSION(S): In mice, the use of anastrozole in FSH-induced cycles has a positive effect on embryo quality and implantation. This effect might be species dependent, and human studies are needed.

Immunolocalization of integrins and fibronectin in tubal pregnancy.

Integrins are a large family of cell adhesion molecules that serve as receptors involved in cell-to-cell and cell-to-matrix interactions during implantation. We studied immunohistochemical staining of integrins (alpha 3, alpha V, beta 1, and alpha 2 beta 1) and fibronectin in ectopic tubal pregnancy. Thirty fallopian tube samples with ectopic pregnancies and five normal tubal segments were obtained during ligation operations; the latter specimens served as controls in the study. Formalin-fixed paraffin-embedded tissue sections were stained with hematoxylin-eosin or primary antibodies against alpha 3, beta 1, alpha V, and alpha 2 beta 1 integrins and fibronectin, using the avidin-biotin-peroxidase method. A semi-quantitative grading system was used to compare staining intensities. In the control samples, immunostaining of all integrins was found in a single layer of tall columnar epithelial cells, the lamina propria (Lp) and the muscular layer. Fibronectin staining was detected in the Lp and the muscular layer. Staining intensities of alpha 3 and beta 1 integrins and fibronectin were increased in the normal part of fallopian tubes with ectopic pregnancies. Staining of beta 1 integrin was more intense than staining of alpha 3 and fibronectin, whereas there was no difference in alpha V and alpha 2 beta 1 integrin expression between normal tubal tissue in the ectopic pregnancy group and control tubal tissue. In the tubal pregnancy group at the site of implantation, staining intensity of alpha 3 and beta 1 integrins and fibronectin was strong in decidual cells, supporting tissue and placental villi, whereas alpha V and alpha 2 beta 1 staining was mild. We concluded that integrins, especially beta 1 and alpha 3, and fibronectin may play a role in progression of tubal implantation. Although the role of integrins has not yet been clearly defined, these molecules may function as markers of normal and abnormal states of receptivity. We like to suggest that integrins and fibronectin, which are needed in utero implantation, are expressed in tubal tissues during ectopic pregnancy and are involved in ectopic implantation.