Changes of β3 Integrins and Extracellular Matrix Proteins in the Endometrium of Unexplained Infertility

The purpose of this study was to investigate changes of β3 integrins and extracellular matrix proteins including fibronectin (FN) , laminin (LN) and collagen type Ⅳ (CL type Ⅳ) on the endometrium of secretory phase from 31 fertile women (fertility group)and 34 women with unexplained infertility (infertility group) by a histochemical method. The results were as follows : In glandular epithelium, β3 integrin appeared in the mid secretory phase and continued to late secretory phase in the fertility group, but was not expressed during the secretory phase in the infertility group.Extracellular matrix proteins from the fertility group were expressed more strongly in mid secretory phase than that in the early secretory phase, and were weakest in the late secretory phase. Compared with the fertility group, the levels of extracellular matrix proteins in the infertility group were elevated in the secretory phase. In conclusion: our current study demonstrate that fie integrin and extracellular matrix proteins are expressed at different levels in the endometrium during the menstrual cycle. They are involved in endometrial changes during the menstrual cycle and during the implantation of the blastocyst. Their unusual expression result in the failure of implantation.     

Expressions of β_3 Integrin and Fibronectin in Salpingitis

Objective To investigate the association between β3 integrin-fibronectin signaling and salpingitis. Methods Thirteen chronically inflamed tubal samples were included in the case group, while 29 normal fallopian tube samples were set as the controls. Immunohistochemistry was used to determine the distribution patterns and expressions of β3 integrin and fibronectin in the fallopian tube samples. Results β3 integrin was presented in the ciliated cells within the whole length of the normal fallopian tube, but no staining was detected in the tubal epithelium of salpingitis. A similar distribution pattern of fibronectin was revealed between the cases and the controls, with exclusive staining in the tubal mucosal matrix and the basement membranes of the tubal epithelium and mucosal vessels, whereas no staining was detected in the tubal epithelial cells. Fibronectin in the samples of salpingitis showed a remarkably stronger-staining than that of the normal controls (P<0.01). Conclusion Salpingitis could reduce β3 integrin expression and raise fibronectin expression in the fallopian tube epithelium, concomitantly changed concentrations of β3 integrin and fibronectin seem to have impacts on the onset of salpingitis.     

Expression of nucleophosmin in glandular epithelium of non-pregnant human endometrium during the menstrual cycle

Background  Nucleophosmin plays a critical role in embryonic development. This study aimed to examine the expression pattern of nucleophosmin in glandular epithelium of human endometrium during the menstrual cycle.

Methods  Endometrial tissues used for this study were obtained from 46 non-pregnant patients who underwent hysterectomy which had been performed to treat benign diseases. Nucleophosmin expression was assessed by in situ hybridization and immunohistochemistry.

Results  At the early-, mid- and late-proliferative phase, nucleophosmin mRNA was highly expressed in glandular epithelium of human endometrium. At the secretory phase, the expression of nucleophosmin mRNA was reduced in glandular epithelium in early-secretory phase, and the expression in mid- and late-secretory phases was not detected. Similarly, nucleophosmin protein was strongly expressed in endometrial glands throughout the proliferative phase, but was gradually reduced during secretory phase.

Conclusion  Nucleophosmin mRNA and protein are expressed in glandular epithelium of human endometrium throughout the menstrual cycle.

     

Genetic imprinted gene PEG10 expression in deciduas of normal early pregnant women

Objective： To investigate the role and significance of paternally expressed gene 10 （PEG! 0） in deciduas of normal pregnant women. Methods： Sixty deciduas tissue from pregnant 6 to 11 weeks were divided into six groups and in each group ten samples were done every gestational week. The expression and distribution of PEG10 in deciduas were examined by RT-PCR, hybridization in situ, Western Blot and laser scanning confocal microscopy. Results： The PEG10 mRNA and protein were expressed in deciduas tissue from pregnant 6 to 11 weeks. Among them, the expression of PEG10 showed a gradual increase as the pregnancy weeks increased. In RT-PCR, the PEG10 expression was lower at pregnant 6th week （0.6743±0.114）, from pregnant 7 to 8 weeks, the expression was increased gradually （7th week 0.7349±0.0083） and reached the pinnacle at 8th week （0.7354±0.0074）. And then the pinnacle began to descend （9th week 0.6340±0.0084, 10th week 0.5901±0.0089 and llth week 0.5261±0.0112）. There was a significant difference in the expression of PEG10 from pregnant 6 to 11 weeks except 7th week and 8th week. This expression characteristic was demonstrated by hybridization in situ. The similar conclusion was proved by Western Blot and laser scanning confocal microscopy. Conclusion： Expression of PEG 10 may play an important role in early pregnancy.     

Clinial Implication of tThe Expression of Aurora B in Normal Endometrium and Endometrial Carcinoma

The expression of Aurora B in normal endometria and endometrial carcinomas and its relation with clinicopathologic parameters of endometrial carcinomas were investigated.Streptavidin-biotin peroxidase(SP) immunohistochemical technique was used to detect the expression of Aurora B in 10 cases of normal proliferative phase endometria,10 cases of normal secretory phase endometria and 72 cases of endometrial carcinomas respectively.According to the 1988 International Federation of Gynecology and Obstetrics(FIGO) grade,there were 37 patients in grade 1,23 in grade 2 and 12 in grade 3 respectively.According to the FIGO stage,there were 59 patients in stage Ⅰ-Ⅱ and 13 patients in stage Ⅲ-Ⅳ.Aurora B was expressed in both normal proliferative phase endometria,secretory phase endometria and endometrial carcinomas,but its positive labeling index(PLI) in proliferative phase endometria was significantly higher than that in secretory phase endo-metria(P<0.01) and endometrial carcinomas(P<0.01).The PLI of Aurora B was lower in tumors with well differentiation(G1),low surgical staging(Ⅰ-Ⅱ),and ≤1/2 myometrial invasion than that in tumors with moderate and low differentiation(G2-G3),higher surgical staging(Ⅲ-Ⅳ),and >1/2 myometrial invasion(all P<0.01).Aurora B exerts its functions in the replication of normal endometrial glandular cells;Expression of Aurora B is significantly correlated with biologic behavior of endo-metrial carcinoma,indicating that Aurora B may be a promising prognostic factor in endometrial carcinoma.     

Regulation and Expression of Tagln2 in Early Rabbit Pregnant Uterus

Objective To examine the expression and regulation of Tagln2 gene in rabbit uterus during early pregnancy. Methods In situ hybridization was used to detect the expression of Tagln2 mRNA in rabbit uterus during early pregnancy and their regulation under pseudopregnancy and hormonal treatment. Results In situ hybridization analysis revealed Tagln2 mRNA expression in a spatiotemporally regulated pattern in early pregnant uterus. Tagln2 mRNA was highly expressed in the uterine epithelium from days 2 to 4. A low expression level was detected in the glandular epithelium near the myometrium on days 5 and 6. On days 7.0 and 7.25, Tagln2 expression was upregulated at implantation sites and mainly expressed in luminal epithelium and blastocyst at the anti-mesometrial and lateral sides. On day 8, Tagln2 expression was mainly detected in fusion area and glandular epithelium at anti-mesometrial sides, and became stronger in trophectoderm and luminal epithelium at mesometrial side. Tagln2 expression was not detected in inter-implantation sites from days 6 to 8 of pregnancy and from days 6 to 8 of peudopregnancy. In the ovariectomized rabbit uteri, Tagln2 expression was upregulated by either estrogen or progesterone. Conclusion Tagln2 was highly expressed in the endometrium at implantation sites and need actived embryo, upregulated by steroid hormones.     

Effect of Timing and Number of Intrauterine Insemination （IUI） on the Cycle Clinical Outcome

Objective To study the effect of timing and number of intrauterine insemination （IUI） per cycle on the outcome of artificial insemination by husband （AIH）. Methods A total of 195 infertile couples underwent 379 cycles of lUI with husband＇s sperm. They were divided into 4 groups according to the ovulation and the number of IUIs per cycle： single IUI was performed 24 h after hCG injection, including single IUI before ovulation （group A） and single IUI after ovulation （group B）; double IUI was performed 24 h and 48 h after hCG injection, including double IUI before and after ovulation （group C） and double IUI before ovulation （group D）. The relation-ship between IUI pregnancy rate and the factors like processed total motile sperm （PTMS）, timing and number of lUIs per cycle was analyzed. Results When PTMS〈5 × 10^6, only one case in group B got pregnant, while no pregnancy was observed in other groups. When PTMS ≥ 5 × 10^6, pregnancy rates in all group were improved significantly. The pregnancy rate in group B reached 32.22%, which was significantly higher than that in group A （14.12%）, group C （20.00%） and group D （17.39%）, respectively （P〈0.05）. Conclusion IUI treatment is recommended to be performed when PTMS ≥ 5 × 10^6. An ideal pregnancy rate can be achieved by single IUI which is performed 24 h after hCG injection, and double IUI performed without ovulation could not result in significant improvement of cycle pregnancy rate.     

TCM TREATMENT OF LUTEAL PHASE DEFECT—An Analysis of 60 Cases

Sixty cases of infertility due to luteal phase defect were treated with herbs to tonify the kid-ney and regulate the menstrual cycle.After the treatment,the hyperthermal phase score ofbasal body temperature(BBT)was markedly increased(P<0.05),the hyperthermal phase7-8 days after ovulation improved(P<0.001),the transitional period of BBT remarkeblyshortened,and the pregnancy rate in 32 uncomplicated cases of luteal phase defect was 56%.The close relationship between luteal phase defect and the kidney deficiency syndrome inTCM was discussed.The key points of the treatment included coordination of yin and yang,regulation of qi and blood,and combination of tonification with reduction.     

Endometrial Collagen Fibril Hyperplasia is Associated with Implantation Failure in Women Undergoing IVF-ET

Objective To analyse the effects of controlled ovarian hyperstimulation （ COH） on the endometrial expression of collagen fibril （CF） during the peri-implantation period in patients undergoing IVF, and its relation to endometrial receptivity （ER） in repeated implantation failure（RIF）. Methods Peripheral blood and endometrial biopsies were obtained from 45 infertile women on days 5, 7 or 9 after oocytes retrieval or ovulation in a stimulated cycle （SC） and natural cycle （NC） respectively. CF was assayed by transmission electron microscope and quantified by modified Masson dyeing. The outcome of subsequent embryo transfer（ET） was observed. Results Levels of both E2 and progesterone were higher in the peripheral blood in SC than in NC. Also the expression of CF in the stroma in each secretory phase was increased significantly in SC （P 〈O.05）. After embryo transferring, expression levels of CF in the pregnancy group dropped between the mid- and late-secretory phase, but no change in the non-pregnancy group. In the same term, all patients undergone endometrial curettage had higher pregnancy rate than those without. Conclusion Imbalance of production and degradation of endometrial CF in the secretory phase resulting from COH may be the cause of defective ER and implantation failure in some RIF patients. Endometrial curettage may improve implantation rate by inducing appropriate CF hyperplasia and degradation.     

CHANGES OF FIBRINOLYTIC ACTIVITY IN ENDOMETRIUM OF INTRAUTERINE DEVICE USERS

The levels of endometrial plasminogen activator were determined with Todd's histochemical method in 85 women using IUD and 76 women without IUD as controls. It was found that the levels in all cases were bimodal, being high in the proliferative phase, lower in the secretory phase and highest in the premenstrual phase. The concentrations of plasminogen activator in the endometrium of IUD users complicated with menorrhagia were much higher than those in the IUD users with normal menstrual blood loss and in the controls during the proliferative, secretory and premenstrual phases. There was no difference in the concentrations of plasminogen activator between the IUD users with normal menstrual blood loss and the controls. The fluctuation of endometrial plasminogen activator levels during the different phases, and the uterine bleeding related to IUD are disscused.     

Antifertility Activity of Hydroalcoholic Extract of Ocimum Basilicum Linn. Leaves on Female Wistar Rats

Objective To determine the effect of hydroalcoholic extract of Ocimum basilicum Linn. leaves on ovulation, implantation and maintenance of pregnancy in adult female cyclic Wistar rats. Methods Ocimum basilicum leaves extract (364 mg/kg and 624 mg/kg) was screened for its antiovulatory, antiimplantation and abortifacient activities. Vaginal smear of each rat was monitored daily during the 15-day treatment of the first experiment. Estimation of cholesterol in ovarian tissue and its histology was carried out to confirm its antiovulatory activity. In the second and third experiments, animals received treatment from 1st to 7th and 8th to 16th day of pregnancy, respectively. The number of implantations and litter size were determined through laparotomy on 10th and 20th day of pregnancy, respectively in two tests. The treated groups were compared with pain control. Results A significant increase in duration of estrus cycle and diestrus phase was observed (P<0.001). A significant decrease in ovarian weight and notable increase in ovarian tissue cholesterol level was also observed (P<0.001). Weight of uterus in treated group was found to be decreased. Histological report showed presence of large corpora lutea in ovarian parenchyma. Neither antiimplantation nor abortifacient effect was observed in rats treated with both the doses of extract. Conclusion Hydroalcoholic extract of Ocimum basilicum interferes normal ovulation by disrupting the estrus cycle and prolonging diestrus phase thus, has a potential of being developed into a female contraceptive.     

Implication of Expression of Osteopontin and Its Receptor Integrin αvβ3 in the Placenta in the Development of Preeclampsia

To investigate the expression of osteopontin （OPN） and its receptor integrin αvβ3 in the placental tissue from pregnant women complicated with preeclampsia, the expression of OPN and αvβ3 in the placenta of the pregnant women with preeclampsia and healthy pregnant women was detected by immunohistochemistry, Western blotting and RT-PCR. Our results showed that OPN and αvβ3 protein were expressed in the placenta from normal pregnant woman and those with preeclampsia. OPN was located in the placental syncytiotrophoblasts and the cytoplasm of capillary endothelial cells and integrin αvβ3 was mainly expressed on the surface of trophoblast cells. Expression of OPN and integrin αvβ3 in the placental tissue from preeclampsia subjects was significantly lower than that from the control group （P〈0.05）. Compared with the control group, expression of OPN in the placental tissue from preeclampsia group was significantly lower （P〈0.05） but there was no significant difference in the expression of αv and β3 between the preeclampsia group and the controls. It is concluded that OPN and its receptor integrin αvβ3 may be involved in the pathogenesis of preeclampsia.     

IL-24 Expression at Maternal-fetal Interface and Its Roles in Trophoblast Invasion

In this study, the expression of IL-24 at maternal-fetal interface and the roles in extravillous trophoblast （the TEV-1 cell line） invasion were examined. Immunohistochemistry was used to detect the expression of IL-24 in villi and decidual tissue. The proliferation of TEV-1 cells under the effect of IL-24 was measured by MTT assay. The invasiveness of TEV-1 cells under the effect of recombinant IL-24 （rhIL-24） was examined by transwell system. Immunohistochemical detection showed that IL-24 was expressed in the villi and decidual tissue, and distributed in villous column, trophoblasts, stroma and blood vessels. The proliferation of TEV-1 cells was not inhibited by rhIL-24 of various concentrations. The examination of invasion in vitro showed that rhIL-24 could inhibit the invasion of TEV-1 cells in a concentration-dependent manner. The results suggested IL-24 could inhibit the invasion of TEV-1 cells. Therefore, IL-24 produced by maternal-fetal interface in human first trimester pregnancy may influence the invasion of trophoblasts and is involved in normal pregnancy.     

Expression of TWEAK on the Ectopic and Eutopic Endometrium from Women with Endometriosis

Objective To investigate the relationship between Tumor necrosis factor （TNF）-like weak inducer of apoptosis（TWEAK） and endometriosis.
Methods TWEAK mRNA and protein concentrations in paired samples of eutopic endometrial tissue from women with and without endometriosis, and also of ectopic endometrial tissue from women with endometriosis were measured by Real-time PCR, immunohistochemistry and Western blotting, respectively.
Results Compared with control endometrium from women without endometriosis and eutopic endometrium from women with endometriosis, TWEAK expressions were reduced on the ectopic endometrium （P〈O.05）. Moreover, the expressions of TWEAK mRNA on eutopic endometrium and control endometrium in proliferative phase were much lower than those in secretory phase. TWEAK protein was expressed in the cytoplasm of glandular cells and stromal cells of endometrium.
Conclusion TWEAK is expressed on the endometrium of reproductive women, and its concentration rises in secretory phase, compared with that in proliferative phase. Patients with endometriosis had a lower expression of TWEAK on ectopic endometrium, which may lead to a decreased level of apoptosis on endometrial cells. Consequently, endometrial cells were able to survive outside uterus helping the development of endometriosis.     

The Expression of PLF in Placenta in Pregnancy-Induced Hypertension and the Relationship between the PLF and Serum VCAM-1

Summary:To study the expression of placental isoferritin(PLF)in placental tissue of pregnancy-in-duced hypertension(PIH)and the relationship between the level of expression of PLF and theamount of vascular cell adhesion molecule-1(VCAM-1)in serum,immunohistochemical techniquewas used to detect the expression of PLF in placenta tissue in 45 PIH patients(PIH group)and 15normal pregnant women(normal group).High resolution pathological image analysis system(HPIAS-100)was employed to determine the quantity of PLF.The VCAM-1 in serum was exam-ined by enzyme linked immunoabsorbent assay(ELISA).The results showed that the levels of PLFexpressions in moderate and severe PIH patients were significantly lower than that of normal group(P<0.01).The serum VCAM-1 was significantly decreased in PIH group(1310±177ρ/mg/ml)than that of normal group(609±72ρ/ng/ml,P<0.01).The significant negative correlation exist-ed betweene the expression of PLF in placental tissue and the serum VACM-1(r=0.58,P<0.01).It was concluded     

The Expression of hTER and hTERT mRNA in Human Tumors with Tissue Microarray

To explore expression-pattems of hTER and hTERT mRNA in human tumors by tissue chip with in situ hybridization （ISH）, simultaneously, evaluate the applicability and value of tissue chip in ISH. Tissue chips consisted of 230 specimens of human tissues （10 cases normal tissues, 13 cases benign tumors, 207 cases malignant tumors） were prepared through the patent tissue arrayer. The expression ofhTER and hTERT mRNA in specimens were determined by ISH. The expression of hTER and hTERT mRNA in normal tissues, benign and malignant tumors were related to characterization of tumor （p〈0.005）. The distribution of hTER and hTERT positive cells was regulated from limited in basal layer to diffused throughout most layers of squamous epithelium is correlated with increasing severity of histopathologic changes. That means the expression-patterns of tissue chip provides a rapid and powerful histological method that would be used in ISH and play an important role in tumor biology. And, the expression-patterns of up-regulation and dysregulation of hTER and hTERT of is an important histology sign could be used as a useful molecular marker for diagnosis and identification human tumor.