Endometrial expression of the estrogen-sensitive genes MMP-26 and TIMP-4 is altered by a substitution protocol without down-regulation in IVF patients.

BACKGROUND: The aim of this study was to analyse the effects of an estradiol (E(2))-progesterone substitution protocol on the endometrial expression of estrogen-sensitive genes during the peri-implantation period. METHODS: Peripheral blood and endometrial biopsies were obtained from 13 infertile women both in a natural cycle (NC), on days 5 and 7 after ovulation (NC5, NC7), and in an artificial (substituted) cycle (AC), on days 5 and 7 of progesterone addition (AC5, AC7). Estrogen receptor-alpha (ERalpha) and progesterone receptor (PR) were assayed by immunohistochemistry. Matrix metalloproteinase-26 (MMP-26) mRNA and tissue inhibitor of metalloproteinase-4 (TIMP-4) mRNA were semiquantitatively assessed in tissue sections using in situ hybridization (ISH) and quantified in tissue extracts using real-time PCR. RESULTS: Levels of both E(2) and progesterone were higher in the peripheral blood in AC than in NC. Also on day AC5, expressions of ERalpha, PR and MMP-26 mRNA (focally) were increased in the epithelium and TIMP-4 mRNA in the stroma. Expression levels of these genes dropped significantly between AC5 and AC7, but not between NC5 and NC7. Abnormally high levels in AC5 samples suggest overstimulation with E(2), and the rapid decrease between AC5 and AC7 suggests overstimulation with progesterone. CONCLUSIONS: In ACs, increased levels of E(2) in the blood exaggerate the endometrial expression of estrogen-sensitive genes, whereas higher levels of progesterone in the blood in the secretory phase exaggerate the drop in expression of these genes. Dramatic variations in the gene expression may not be optimal for the implantation process.

[Protein bcl-2 expression in the secretory endometrium after oral estrogen-progesterone replacement]. / Exprese proteinu bcl-2 v sekrecním endometriu po perorální estrogen-progesteronové substituci.

OBJECTIVE: To compare the changes of the protein bcl-2 expression, regulatory mechanism in the process of apoptosis, in the secretory endometrium throughout natural and artificial (estrogen-progesterone substitution) cycles in the same group of patients. DESIGN: A prospective study. SETTING: Dept. of Obstetrics and Gynecology, University Hospital Olomouc, Dept. of Histology and Embryology, Faculty of Medicine, Palacký University in Olomouc, Dept. of Obstetrics and Gynecology, Bata Hospital, Zlín. METHODS: Endometrial samples (n=56) were obtained on days 5 and 7 after ovulation, progesterone addition, resp. Patients (n=14) included in the study had regular menstrual cycle, age under 40, BMI range 20-32 and basal FSH level range 5-9 IU/l. The collected samples were processed routinely and bcl-2 was estimated by indirect three level imunohistochemic method. RESULTS: We demonstrated the bcl-2 expression in all evaluated layers (epithelial surface, stroma, glands) of the mid-secretory endometrium in both cycles. No difference was found in bcl-2 expression between days 5 and 7 in the secretory phase of the natural cycle. In artificial cycles higher bcl-2 expression was found only in epithelial surface (p<0.05) between days 5 and 7. On day 7 higher bcl-2 expression was found in the artificial cycle in the endometrial epithelial surface (p<0.001) as well as in spongy layer (p<0.01) and compact layer (p<0.05) comparing to day 7 of the natural cycle. CONCLUSION: The bcl-2 expression in the mid-secretory endometrium is significantly higher in the cycle with estrogen-progesterone substitution comparing to the natural cycle. These changes were more significant in endometrial and glandular epithelium than in stromal cells.

Changes in the development of uterine pinopodes in steroid hormone supplemented cycles.

The endometrium acquires the ability to implant a hatched blastocyst only within a specific time termed the receptive phase. Ovarian steroid hormones are essential for structural and functional changes that prepare the endometrium to be receptive. Pinopodes have been suggested to be markers of uterine receptivity. The aim of this study was to compare the pinopode expression and serum levels of ovarian steroid hormones in the mid-luteal phase of the natural cycle and in a "mock" cycle in the same subject. Sequentional endometrial biopsies within 48 hours were obtained from women in the mid-luteal phase (ovulation +5, ovulation +7) of the natural cycle and in the "mock" cycle (progesterone supplementation +5 and +7). Biopsies were examined under a scanning electron microscope for pinopode detection. The expression of pinopodes was similar in both cycles, where pinopodes covered about 5 % of the endometrial surface. The developmental stages were also similar with a slight increase of fully developed pinopodes in both samples in the "mock" cycles. Our findings suggest that hormonal preparation of the endometrium do not change the timing of pinopode expression.

[Comparison of apoptosis expression in the secretory endometrium during a normal menstrual cycle]. / Porovnání exprese apoptózy v sekrecním endometriu prirozeného menstruacního cyklu.

OBJECTIVE: Comparison of level of apoptosis in the mid-secretory human endometrium. DESIGN: Clinical-laboratory trial. SETTING: Department of Gynecology and Obstetrics, Medical Faculty, Palacký University, Faculty Hospital, Olomouc, Department of Gynecology and Obstetrics, Bata Hospital, Zlín. METHODS: Samples of secretory endometrium were obtained from 14 women. The women included in the study had the next criteria: a history of infertility of more than 12 months, regular menstrual cycles, age below 40 and FSH basal level range 5-9 IU/L. Sampling of tissue was performed after confirmed ovulation by LH surge and repeated ultrasound examinations. Patterns were processed by routine methods and apoptosis was detected using TUNEL assay. RESULTS: The level of apoptosis in the endometrial epithelial surface was significantly higher on day +7 compared to day after ovulation (P < 0.02). Apoptotic cells were seen only sporadically in endometrial stromal cells. The level of apoptosis in endometrial stroma was also higher (but not statistically significant) on day +7 compared to day The level of apoptosis was markedly higher in spongy layer on day +7 (P < 0.01) compared to day There were no significant differences between day and +7 in compact layers of the glands.

Involvement of p53 and Bcl-2 family proteins in regulating programmed cell death and proliferation in human embryogenesis.

Homeostasis and development in vertebrates are regulated by cell proliferation, differentiation and death. Permeability of mitochondrial membranes, a decisive feature of apoptosis, is regulated by Bcl-2 family regulators. Protein p53 is able to reduce bcl-2 and promote bax expression. This study focused on the immunohistochemical detection of the expression levels of Bcl-2 family regulators (anti-apoptotic Bcl-2 and Bcl-XL, pro-apoptotic Bcl-Xs and Bax), p53, and PCNA as a marker of proliferation, together with the evaluation of the level of apoptosis in human embryos (anlage of limbs, axial skeleton, metanephros, and intestine). Expression of observed proteins was assessed by a three-step immunohistochemistry and evidenced by the double-staining technique. Apoptosis was detected by the TUNEL technique. This study provided circumstantial evidence of the exclusive role of Bcl-2 and Bcl-XL proteins in the inhibition of apoptosis - only rarely were the Bcl-2/ Bcl-XL positive cells stained by TUNEL. The role of pro-apoptotic members of Bcl-2 family remains ambiguous, as TUNEL positive cells are both Bax/Bcl-Xs positive and negative. This study provided substantial evidence that expression patterns of observed proteins are neither fully explainable by "rheostat" theory, nor are the findings obtained from animal model tissue or cell culture commonly applicable to human embryos.

[Histochemical analysis of enzymes in the rabbit cornea after LASIK surgery]. / Histochemické vysetrení aktivity nekterých enzymu v králicí rohovce po operaci LASIK.

The first experience with histochemistry of rabbit corneas after the operation LASIK is presented in this study. Activity of the following enzymes was assessed: alkalic phosphatase, acid phosphatase and dipeptidylpeptidase IV. No pathological activity of assessed enzymes was found in comparison with control corneas.

[LASIK--comparison of oscillating and rotating microkeratotomy using scanning electron microscopy]. / LASIK--srovnání oscilacní a rotacní mikrokeratotomie skeningovou elektronovou mikroskopií.

Scanning electron microscopy was used to evaluate the differences in the quality of the cut produced by oscilating microkeratome Flapmaker and Draeger rotating microkeratome. Higher quality of the edge of the cut was found with Flapmaker. Both microkeratomes produce uniform surface of the cut. Chatter is much less expressed with Flapmaker. No difference at the interface tissue was found with SEM three weeks postoperatively comparing the microkeratomes.

[Comparison of the diameter and thickness of the corneal lamella in LASIK surgery using the oscillating microkeratome]. / Porovnání prumeru a tloust'ky rohovkové lamely pro LASIK vytvorené oscilacním mikrokeratomem.

Corneal lamellas created by two different microkeratomes--oscillating and rotating are compared. Oscillating microkeratome Flapmaker (Refractive Technologies) and rotating Dreager microkeratome (Storz) were used for tis study. Diameter and thickness of the lamella were assessed. Rabbit lamella created by the same procedure as human differs significantly and this limits the interpretation of rabbit experiments to the human eyes. Oscillating microkeratome Flapmaker created more precise and standard lamella (standard deviation 4 times smaller) compared to rotating Storz on human eyes. Differences in lamella thickness in different topografical areas of the lamella were 3.3 times smaller with Flapmaker. We conclude that desired mean lamella thickness homogenity in different topografical areas of the lamella are much better with Flapmaker compared to Dreager microkeratome on human eyes.

[Comparison of the surface structure of the corneal stroma cut with a microkeratome using scanning and classical microscopy]. / Porovnání struktury povrchu rezu mikrokeratomem rohovkou ve skeningovém mikroskopu environmentálním a klasickém.

Surface of the corneal stroma after microkeratome cut was assessed by classical and environmental SEM. Environmental SEM allows magnification of tissue without it's previous drying. It is easy to prepare tissue for environmental SEM. Disadvantage of this leading technique is smaller scale of magnification and small angle of view. Classical SEM shows better the impact of LASIK on the cornea.

[Assessment of adhesion of the corneal lamella to the stroma in relation to the duration of the LASIK postoperative period]. / Merení adheze rohovkové lamely ke stromatu v závislosti na dobe od operace LASIK.

Adhesion of lamella created by two different microkeratomes was assessed during the healing period of 250 days. Oscillating microkeratome Flapmaker, Refractive Technologies and Draeger rotating microkeratome, Storz were used for this study. Increase of lamella adhesion during the healing period was found. The increase is little faster during the first weeks. The difference in adhesion between eyes operated by different microkeratome was not significant.

Expression of BCL-2 in the developing kidney of human embryos and fetuses qualitative and quantitative study.

Twelve human embryos and fetuses aged of 7-30 weeks of intrauterinal life were examined to determine the expression of bcl-2 gene in the developing kidney. Tissue samples were routinely processed and three-step indirect immunohistochemical method was used for the detection of Bcl-2 protein. End-point cytophotometry was performed with computer-controlled microscope photometer with a scanning table and the mean relative absorbance of the final product of peroxidase reaction was determined and taken as a measure of Bcl-2 expression. The morphometric evaluation was carried out from the TV display using Weibel s universal hexagonal raster and we determined the relative volume of Bcl-2 positive structures in the various zones of the embryonal kidney. The aim of our research was mapping of the Bcl-2 occurrence in the developing kidney of human embryos and fetuses. The Bcl-2 protein is involved in the regulation of apoptosis and its effect is antiapoptotic. The highest Bcl-2 expression was proved in the cells of metanephrogenic blastema. The lower occurrence of Bcl-2 positive cells was demonstrated in proximal tubules analges+ and it was almost on the borderline of detection in branches of ureteral bud. In the fetal period the marked Bcl-2 expression was maintained in the epithelial cells of proximal tubules analges.

Expression of apoptosis in the developing human cephalic region during the embryonic and early fetal period.

The aim of our work was to study the expression of apoptosis in the cephalic region in human embryonic and fetal material. Twelve human embryos and fetuses were processed by routine histological technique and then studied using the Boehringer-Mannheim Company kit for TUNNEL technique. Rich appearance of apoptotic cells was typical for mesenchyme and for tissues of the mesenchymal origin. Dispersed or focal occurrence of apoptotic cells was described in nervous system and some orofacial structures during the embryonic period. During the early fetal period occurrence of apoptosis decreased and only rare apoptotic nuclei were visible in studied areas. Often they failed at all.

[Measurement of adhesion of the corneal lamella to the stroma in the early postoperative period after microkeratotomy]. / Merení adheze rohovkové lamely ke stromatu po rezu mikrokeratomem v casné pooperacní fázi.

The early postoperative adhesion of corneal lamella after LASIK was measured. The interface was washed by hypo, iso and hypertonic saline solution and the influence on the lamella adhesion was assessed. Drying the interface and the excimer laser ablation of epithelial cells along the edge of the lamella were assessed in terms of the influence on the adhesion. The term "phototerapeutic epithelectomy (PTE)" was proposed for the excimer laser ablation of corneal epithelial cells. Small insignificant influence of PTE was found. The clinical application of PTE to increase the early adhesion of lamella is not recommended because of the epithelial destruction along the lamella edge. The adhesion was increased significantly by means of drying the interface which is recommended for the clinical application. High variability od measurements between different eyes was found.

Apoptosis and expression of proliferative proteins in the developing nervous system and orofacial region of human embryos.

We have studied expression of PCNA and Ki-67 in the developing nervous system, sensory organs and orofacial regions in human embryos and fetuses, using monoclonal antibodies PC-10 and MIB-1 in three-step immunohistochemical method and apoptosis performed by TUNEL technique. Expression of PCNA and Ki-67 increased with the age. Apoptosis was rare in above mentioned regions.

The role of the apoptosis and the genes regulating apoptosis in the early differentiation of human embryo.

Apoptosis (programmed cell death) is an important process participating in the formation of organs and tissues during embryogenesis. Our aim of the work is studying the role of the apoptosis during the human embryonic differentiation. We tend to give acquired findings into the correlation with expression of proteins Bcl-2 and Bax (products of genes regulating apoptosis). Detection of the apoptosis was carried out on 25 routinely processed human embryos by means of TUNEL technique. The level of expression of Bcl-2 and Bax was determined using standard three-step immunohistochemical procedure. Results were achieved by the comparison of apoptoic index and the level expression of Bcl-2 and Bax was semiquantitatively evaluated. The low value of apoptotic index was mostly accompanied by the high expression of Bcl-2 and the Bax expression was not proportionally related to the value of apoptic index.

Differences in p53 and Bcl-2 expression in relation to cell proliferation during the development of human embryos.

AIMS: To study the patterns of p53 and Bcl-2 expression in relation to cell proliferation during human embryogenesis in order to help elucidate their potential roles in the regulation of cell proliferation and apoptosis during morphogenesis. METHODS: Immunohistochemistry for p53, Bcl-2, and proliferating cell nuclear cell antigen (PCNA) proteins was performed, using a variety of monoclonal antibodies, on paraffin was embedded sections of tissues from 68 human embryos and fetuses of between 4 and 30 weeks gestation. RESULTS: Positive relations between sites of proliferative activity (as detected by PCNA expression) and p53 expression were found in the kidney, early developmental stages of intestine and lungs, liver, pancreas, heart, and in embryonic osteoblasts. On the other hand, positive relations between proliferative activity and Bcl-2 expression were found in the gonads, adrenal glands, in the cells of the dental lamina, hair follicles, syncytiotrophoblast, chondrocytes, and more advanced stages of intestinal development. In tissues of the central nervous system, p53 and Bcl-2 were co-expressed at the same sites but there was an inverse relation between p53/Bcl-2 expression and proliferative activity. CONCLUSIONS: These data suggest that p53 and Bcl-2 have tissue specific and stage specific functions during embryogenesis.

Enzyme histochemistry in fetal human hearts and kidneys during intrauterine development. Qualitative and quantitative results.

The activities of three enzymes, glycerol-3-phosphate dehydrogenase, succinate dehydrogenase and dipeptidylpeptidase IV, were histochemically demonstrated and cytophotometrically measured in fetal human hearts and kidneys. During the intrauterine development from the 7th to the 19th week 29 fetuses in 6 stages of age were investigated. Positive enzyme reaction was found in the myocardium of left and right ventricle and septum and in the different parts of the nephron. During the investigated period of development the enzyme activities changed in both heart and kidney. At the 16th week the activities of glycerol-3-phosphate dehydrogenase and succinate dehydrogenase showed the maximum in the heart, in contrast to kidney, where at this stage activity minima of succinate dehydrogenase and dipeptidylpeptidase IV were measured. The myocardium of the right ventricle showed higher metabolic activity than the right one, which could reflect the higher load of the right ventricle in the fetal circulation. Within the nephron differences of enzyme activities were observed. The highest enzyme activities were always measured in the proximal tubules, interpreted as sign of high degree of differentiation.