Anatomic double-bundle ACL reconstruction restricts knee extension in knees with hyperextension.

PURPOSE: Double-bundle ACL reconstruction has been demonstrated to be at least as effective as single-bundle reconstruction in terms of restoring knee rotational and translational stability. Until now, the influence on knees with hyperextension has not been evaluated. It was the purpose of this study to evaluate whether double-bundle ACL reconstruction restricts extension in hyperextendable knees. METHODS: Hamstring tendon reconstructions of 10 human cadaveric knees with the ability of hyperextension (age: 48 ± 14 years) were performed as single bundle (SB) on one side and double bundle (DB) on the other side. A surgical navigation system (BrainLab, Germany) was used to assess the kinematics of each knee at the intact and reconstructed state. A difference with regard to the anterior-to-posterior translation (AP) and rotational stability at 30° of knee flexion, 90° of flexion and the hyperextension capability of each specimen was analysed. RESULTS: The difference in AP translation before and after the reconstruction was not significantly different in 30° and 90° of flexion (n.s). Both single- and double-bundle reconstructions restored the preoperative kinematics at 30° and 90° of knee flexion (n.s). The knee extension was 4° ± 1.8° with the intact ACL and 4° ± 1.7° after reconstruction in the SB group (n.s). The knee extension was 5° of hyperextension ± 1.1° with the intact ACL and 0° ± 0.4° after reconstruction in the DB group; the limitation of the extension was significantly larger in this group (p = 0.013). CONCLUSION: Both single- and double-bundle ACL reconstruction techniques are capable of restoring knee anteroposterior and rotational stability. Double-bundle reconstructions significantly reduce knee extension in knees with hyperextension capability. Care must be taken when using double-bundle techniques in patients with knee hyperextension as this procedure may limit the knee extension after double-bundle ACL reconstruction.

The feasibility of a less invasive method to assess endometrial maturation--comparison of simultaneously obtained uterine secretion and tissue biopsy.

OBJECTIVE: To compare the assessment of endometrial maturation parameters in endometrial secretion samples obtained by a novel minimally invasive technique with those assessed in tissue biopsies. DESIGN: Prospective study. SETTING: University Hospital. POPULATION: Healthy female volunteers attending a gynaecological outpatient clinic. METHODS: Endometrial secretion fluid and tissue sampling 5 days after a spontaneous ovulation assessed with ultrasound. MAIN OUTCOME MEASURES: Progesterone (P) receptor, Ki-67 expression and the Noyes criteria were used to date endometrial biopsies. In the endometrial fluid samples, glycodelin A (GdA), leukaemia inhibitory factor (LIF) and P levels were analysed, and protein content and electrophoresis patterns were determined. RESULTS: All data were correlated to estradiol (E2) and P serum concentrations. The dating according to histology and immunohistochemical staining patterns correlated significantly with GdA levels (r=0.376, P=0.048) in endometrial fluid samples as well with serum levels of E2 (r=0.568, P=0.001) and P (r=0.408, P=0.023). No correlation was observed between tissue dating and LIF levels and protein content in endometrial fluid samples. CONCLUSIONS: The measurement of GdA in endometrial secretion samples may provide a less invasive method for assessing endometrial maturation in potential conception cycles without disrupting implantation.

Impact of ovarian stimulation on mid-luteal endometrial tissue and secretion markers of receptivity.

The objective of this study was to investigate the effect of ovarian stimulation for IVF on endometrial secretion and tissue markers of receptivity in the mid-luteal phase. In 10 oocyte donors, endometrial secretions and biopsies were sampled 5 days after spontaneous ovulation and oocyte retrieval in consecutive cycles. Four subjects received progesterone in the luteal phase of the stimulated cycles. Mid-luteal endometrial maturation in the stimulated cycle was compared with the spontaneous cycle, by histological dating, Ki-67, oestrogen receptor (ER) and progesterone receptor (PR) expression, secretion levels of leukaemia inhibitory factor (LIF), glycodelin A (GdA) and progesterone, and protein profile. No significant differences in histological markers, expression of Ki-67, PR, ER, secretion protein profiles or concentrations of LIF, GdA, or progesterone were observed when comparing natural with stimulated cycles. Progesterone supplementation of stimulated cycles was associated with significantly lower Ki-67 (P = 0.03) and ER (P = 0.04) expression compared with the non-supplemented stimulated cycle. In this pilot study, ovarian stimulation was not demonstrated to alter the studied markers of endometrial maturation in the mid-luteal phase.

[Interlaboratory trials for quality assurance of breast cancer biomarkers in Germany]. / Ringversuche zum Nachweis von therapeutischen Zielmolekülen beim Mammakarzinom in Deutschland.

In the age of personalized medicine, and in addition to typing and grading, breast cancer pathologists are now also involved in determining biomarkers such as steroid hormone receptors and Her-2, which are of the utmost importance in adjuvant therapy. In order to assure quality of these biomarker assays, external proficiency testing has been implemented in Germany. Since 2002 trials have been conducted annually, with up to 180 participating laboratories. More than 85% of all participants achieved good results in clearly negative and positive cases seen in daily practice. If at all, discordant results were observed in the rarer low steroid-hormone receptor expressing tumors and Her-2 borderline cases (2+). Regular participation in interlaboratory testing leads to significantly improved immunohistochemical results, particularly in these problematic cases. Tissue microarrays (TMA) with 20-24 different breast cancer samples including cell lines meant that a huge number of pathologists were challenged with identical samples, providing the prerequisite for comparability. Participation is recommended for pathology departments involved in the service for breast units. The organizational frame work of the trials is described here. The confidence of cooperating disciplines in breast cancer biomarkers assessed by pathologists will be fostered by external proficiency testing as presented here.

Indoleamine-dioxygenase is expressed in human decidua at the time maternal tolerance is established.

The semi-allogeneic fetus has to be tolerated by the maternal immune system. In mice, it has been shown that inhibiting indoleamine-dioxygenase (IDO) leads to fetal rejection, suggesting a central significance for IDO in establishing maternal tolerance. Consequently, we have analyzed IDO expression in human endometrium and decidua to determine whether it may be of significance in human reproduction. Endometrial (n=60) and decidual (n=68; first and second trimester) tissue samples and isolated cells were analyzed for IDO mRNA and protein expression by real-time PCR, Western blot and immunohistochemistry. IDO expression in the decidua of proven fertile women (n=34) was compared to women presenting with their first pregnancy (n=22) and women with a history of miscarriages (n=12). Expression of IDO was localized in glandular epithelial cells and scattered stromal leukocytes. Expression started at the mid-luteal phase in the menstrual cycle and was high until the second trimester of pregnancy. However, glandular expression of IDO decreased during the second trimester, whereas expression in villous trophoblast started at this time. There were no significant differences in decidual IDO expression between proven fertile women and women presenting with their first pregnancy or women with a history of miscarriages. From the expression pattern we conclude that IDO may play a central role in human pregnancies for the establishment of maternal tolerance of fetal antigens. Thereby, IDO expression may be needed in each pregnancy independently from prior pregnancies, and a history of miscarriage may not reflect a general deficiency in IDO expression.

Estradiol and medroxyprogesterone acetate regulated genes in T47D breast cancer cells.

Many mammary tumors express estrogen receptors (ER) and progesterone receptors (PR), and there is increasing evidence that progestins influence gene expression of breast tumor cells. To analyse the impact of progestins on breast cancer cells, we compared (a) the expression of two cytokines, involved in tumor progression, and searched (b) for differentially regulated genes by a microarray, containing 2400 genes, on T47D breast cancer cells cultured for 6 days with 17beta-estradiol (E2) or E2+medroxyprogesterone acetate (E2+MPA). Lower amounts of PDGF and TNFalpha were found in culture supernatants of E2+MPA treated T47D cells. MPA addition induced a 2.8-3.5-fold increase of the mRNA expression of (a) tristetraprolin, which is involved in the posttranscriptional regulation of cytokine biosynthesis, and (b) zinc-alpha2-glycoprotein and Na, K-ATPase alpha1-subunit, which both resemble differentiation markers of breast epithelium. In contrast, the mRNA expression of lipocalin 2, which promotes matrixmetalloproteinase-9 activity, was decreased five-fold in E2+MPA treated cells. Our data show that the expression of genes from various functional gene families is regulated differentially by E2 and E2+MPA treatment in T47D cells. This suggests that exogenous progestins applied for therapy and endogenous changes of the progesterone levels during the menstrual cycle both influence breast cancer pathophysiology.

Apoptosis of extravillous trophoblast cells limits the trophoblast invasion in uterine but not in tubal pregnancy during first trimester.

During the first trimester of pregnancy extravillous trophoblast cells (EVT) invade the maternal decidua. Invasion normally is reduced from the second trimester onwards and stops in the inner third of the myometrium. By contrast, in extrauterine tubal pregnancy, trophoblast invasion may even penetrate the tubal wall, which ultimately leads to the rupture of the fallopian tube. Induction of apoptosis of EVT cells, by maternal immune competent cells, may be an important mechanism to limit EVT invasion in uterine pregnancy. Tissue specimens from first and second trimester uterine pregnancy and first trimester tubal pregnancy were analyzed for apoptosis by TUNEL- and M30-staining. By immunohistochemical double labelling, maternal leukocyte subtypes were co-localized to apoptotic cells and in this context, the number of CD56(+)NK cells was analyzed. Our data show that apoptosis is confined to the decidua basalis. Most apoptotic cells are single cytokeratin-positive epithelial cells residing in the stromal compartment. Consequently these cells can only be EVT cells. Maternal leukocytes are not apoptotic. They are located in close contact to apoptotic cells. The number of apoptotic cells in the second trimester (1.8+/-0.7 per cent) is reduced compared to first trimester (5.6+/-0.7 per cent) of uterine pregnancy. In parallel, the number of NK cells declines from first (24.4+/-2.9) to second (12.4+/-1.8) trimester. Furthermore, apoptosis is significantly reduced in ectopic (0.9+/-0.3 per cent) compared to eutopic first trimester pregnancies. Consequently, we suggest that in first trimester uterine pregnancy, induction of EVT cell apoptosis by the maternal immune system is one mechanism to limit EVT invasion. During the second trimester, in parallel to declining numbers of NK cells, the mechanism changes. However, in tubal pregnancy due to differing immunological microenvironments at the ectopic implantation site, apoptosis induction fails, which deleteriously may result in uncontrolled invasion and penetration of the tubal wall.

Expression and regulation of 17beta-hydroxysteroid dehydrogenase 7 in the rabbit.

We cloned partial cDNA sequences of rabbit 17beta-hydroxysteroid dehydrogenase 1 (17beta-HSD1) and 17beta-HSD7. We analyzed the tissue distribution of 17beta-HSD7 as well as the expression in corpus luteum and endometrium during pseudopregnancy. The obtained cDNA sequence of 17beta-HSD7 coded for all functional regions of the protein and showed 86 and 81% similarity to human and rodent sequences, respectively. The partial sequence of rabbit 17beta-HSD1 was 76 and 82% similar to rodent and human sequences. By Northern analysis 17beta-HSD7 expression was predominantly found in reproductive organs like ovary, oviduct, endometrium, placenta and mammary gland. Substantial expression was also apparent in the heart, stomach and cerebellum. The 17beta-HSD1 could be detected in placenta by reverse transcriptase-polymerase chain reaction (RT-PCR), so far. During rabbit pseudopregnancy 17beta-HSD7 expression was found to be regulated in corpus luteum as well as in endometrium. In the corpus luteum, strongest expression occurred from d10 to d14 of pseudopregnancy (p. hCG) and was downregulated on d16 p. hCG. In endometrium strongest expression of 17beta-HSD7 was found on d6 p. hCG, when the endometrium was differentiated to its implantation permissive status.

The progesterone antagonist onapristone retards the advanced endometrial transformation after gonadotropin stimulation in rabbits.

Ovarian stimulation with gonadotropins (GN) during human in vitro fertilization and embryo transfer (IVF/ET) therapy alters the ovarian steroid output, especially that of progesterone. As a consequence, endometrial transformation is advanced, and embryo implantation is hampered. This study used the rabbit model to determine if the application of the progesterone antagonist (PA) onapristone (ONA) could retard endometrial development after GN-stimulation. Rabbits were GN-stimulated twice daily with 5 IU FSH and 5 IU LH on 3 consecutive days with a) hMG (n = 10) or b) with a mixture of recombinant FSH and LH (n = 10). The animals were then mated, and hCG was injected i.v. to ensure ovulation. This day is designated as day 0 post coitum (d 0 p.c.). On day 2 p.c., five animals of each group were treated with 20 mg ONA/kg body weight and five with vehicle for control. On d 5 p.c. endometrial transformation was analyzed by morphology, uteroglobin (Ugl)-mRNA expression, and proliferation. Embryos were flushed from the uteri. Their number and morphology was evaluated. The endometrium of GN-stimulated control animals demonstrated very long endometrial glands and narrow stromal septa. Ugl-mRNA expression was restricted to the cells at the bottom of the gland. 17.0 +/- 4.6% (mean +/- SD) of glandular cells and 6.0 +/- 5.3% of luminal epithelial cells proliferated. In ONA-treated animals, endometrial glands were significantly shorter, and the pattern of arborization was less pronounced. Endometrial gland cells and luminal epithelial cells expressed Ugl-mRNA. Furthermore, glandular and luminal cells proliferated with high intensity (38.6 +/- 6.8% and 36.4 +/- 9.3%, respectively). These results indicate that the status of endometrial differentiation was retarded after ONA-treatment. Nevertheless, the embryos of these ONA-treated animals were well developed. In conclusion, after GN-stimulation, ONA treatment retarded the advanced endometrial transformation in rabbits. Therefore, postovulatory administration of a PA might be a possible strategy to modulate the advanced endometrial development in IVF-cycles.

Progestins, progesterone receptor modulators, and progesterone antagonists change VEGF release of endometrial cells in culture.

The influences of the synthetic progestin, medroxyprogesterone acetate (MPA), the progesterone receptor modulator J867, and the antagonist ZK137316 were studied in vitro on isolated endometrial epithelial cells, as well as endometrial fibroblasts. We evaluated the expression of estrogen receptor alpha (ER) and the progesterone receptor (PR) by RT-PCR. ER and PR were strongly expressed in the fibroblasts and epithelial cells under treatment with 10(-8) M 17beta-estradiol (E(2)). Treatment with 10(-6) M J867 or ZK137316 upregulated the PR expression as did E(2), in contrast to treatment with 10(-6) M MPA, which caused a downregulation of PR in epithelial cells, but not in fibroblasts. In addition, the vascular endothelial growth factor (VEGF) release into the cell culture medium was analyzed by a VEGF-ELISA. VEGF which plays an important role in angiogenesis, is regulated by steroid hormones as well as hypoxia. E(2) stimulates VEGF release into the medium in both cell types. MPA reduces VEGF release significantly in the fibroblast cell culture, but increases it in the epithelial cell culture. ZK137316, in the presence or absence of E(2), reduces VEGF release in fibroblast cell culture. J867 increases the VEGF production in fibroblasts only in the presence of E(2). Both compounds show no significant effects, compared to E(2), in epithelial cell culture. The different results for the epithelial cells and fibroblasts indicate that the pharmacological effects of PR modulators (PRMs) and progesterone antagonists (PAs) may be cell specific and depend on the presence or absence of partial progestagenic agonistic activities. This observation opens up new perspectives for various clinical applications.

Localization of uteroglobin mRNA by nonradioactive in situ hybridization in the pregnant rabbit endometrium.

The distribution of uteroglobin mRNA has been investigated in the endometrial compartments of the rabbit uterus during early pregnancy (day 0.5 p.c.--day 12 p.c.) using nonradioactive in situ hybridization. Digoxigenin-dUTP labeled oligodesoxy-nucleotide-probes (24mer) and an anti-digoxigenin-antibody conjugated with alkaline phosphatase were developed and used. It could be shown, that uteroglobin mRNA localization is restricted to the endometrial epithelium. There are differences in the extent of uteroglobin mRNA expression within the epithelial cells, which is in accord with our interpretation on the existence of different epithelial cell populations. From day 0.5 p.c. to day 9 p.c. the cells of the basal glands express uteroglobin mRNA continuously, whereas the proliferating surface epithelium shows a remarkable fluctuating pattern of uteroglobin mRNA expression. On day 2 p.c. the whole surface epithelium starts to express the uteroglobin message, and up to day 5 p.c. all these cells show a high level of uteroglobin mRNA expression, which drops significantly on day 6 p.c., when significant changes in the cyto-morphology of the surface epithelium for implantation occur. On day 7 p.c., there is no more uteroglobin mRNA expression in the surface epithelium, however remaining expression in the basal glands. The latter is evident up to day 9 p.c. From day 10 p.c. onwards, neither the luminal nor the deep glandular epithelium express any uteroglobin mRNA. Our observations on the cellular level have been continued in parallel studies on endometrial homogenates by Northern Blot analysis of uteroglobin mRNA (600 bases). Finally, it is discussed whether Uteroglobin mRNA may be an useful marker for the differentiation of various endometrial epithelial cell populations.

Insulin and insulin-like growth factor-I promote rabbit blastocyst development and prevent apoptosis.

Insulin as well as insulin-like growth factor-I (IGF-I) promote early embryo development, and IGF-I binds to the coats of preimplantation rabbit embryos. As the IGF-I receptor is expressed from the morula stage onwards, the embryos are capable of responding to insulin and IGF-I, which is present in the oviductal and uterine secretions that surround them. The embryonic coats were removed to exclude any influence by IGF-I bound to the coats. The in vitro development of such embryos under classical conditions appears to be retarded. Addition of IGF-I (68 pM-6.8 nM) or insulin (68 nM-6.8 microM), however, promotes blastocyst formation. Embryo development under such conditions is not significantly different from that of embryos cultured with intact coats. In contrast, coat-free embryos cultured without IGF-I or insulin supplementation show apoptosis. Because IGF-I stimulates cell proliferation and prevents apoptosis, we investigated whether insulin or IGF-I may act as "survival factors" in preimplantation development. Therefore, apoptosis was induced by slight UV irradiation (254 nm wave length; 11.8 W/m2). Compared to the untreated controls, embryos displaying retarded development or degeneration were increased by 22% and 14%, respectively. Addition of IGF-I or insulin to the culture medium of UV-irradiated embryos improved [3H]thymidine incorporation and blastocyst formation significantly. By immunohistochemistry we could show that addition of insulin (0.68-68 nM) decreased apoptosis and increased cell proliferation in a dose-dependent manner, supporting blastocyst development significantly.

Haptoglobin expression and release by rabbit oviduct and endometrium, its localization in blastocyst extra-embryonic matrix and fluid during preimplantation time.

BACKGROUND: Evidence is emerging that haptoglobin, an acute phase protein with immunomodulatory properties, is expressed by the endometrium of various species. The present study describes an in-depth investigation of haptoglobin expression and release in the rabbit reproductive tract and in preimplantation embryos. METHODS: The full-length cDNA sequence of rabbit haptoglobin was determined by rapid amplification of cDNA ends PCR. Haptoglobin expression was studied in the oviductal ampull, and isthmus, endometrium and embryos from the time of ovulation up to adhesion. These results were completed by western blot analysis of reproductive tract secretions and embryonic tissues. RESULTS: cDNA sequencing showed a high homology between rabbit and human haptoglobin (84.1%). In oviductal tissues haptoglobin mRNA is clearly expressed from 6 h post-conception (p.c.) to day 3, and in the uterus on days 5 and 6. In the oviductal fluid highest haptoglobin protein content was found between 6 h p.c and day 2, and in the uterine fluid on days 5 and 6 p.c. Embryos do not express haptoglobin mRNA during preimplantation development. However, considerable amounts of maternal haptoglobin protein were detected in the blastocyst coverings and in blastocyst fluid. CONCLUSIONS: Already during periovulatory time and oviductal passage, high amounts of haptoglobin are present in the microenvironment surrounding the oocyte/embryo. Two days before implantation, again, high haptoglobin levels are detectable in the embryo's environment. The incorporation of haptoglobin into the extra-embryonic matrix may be of particular functional significance.

The receptive endometrium is characterized by apoptosis in the glands.

Apoptosis in the human endometrium up to now has been detected during the mid to late luteal phase and therefore connected to the onset of the menstrual shedding. However, there is increasing evidence that regulated apoptosis may be important during decidualization and implantation. To investigate a possible role for apoptosis in the human endometrium and its regulation, we correlated the immunolocalization of the apoptosis regulatory protein bcl-2 and the proliferation marker Ki67 to the in-situ nuclear DNA fragmentation - a key feature of apoptosis - detected by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling (TUNEL) method during the menstrual cycle. Whereas proliferation and bcl-2-expression were predominantly detected in the glandular compartment during the proliferative phase, only single apoptotic cells could be shown during this period. During the transformation of the endometrium (days 15-19) proliferation and bcl-2 expression decreased markedly and there was no sign of apoptosis. At the beginning of the implantation window (days 19-20) we could detect the first signs of apoptosis in the glandular epithelia in the basalis, which extended to the functionalis during the luteal phase. Proliferation and bcl-2 expression are limited to the stromal compartment comprising the large granular lymphocytes - during this time, and extend in parallel with apoptosis from the basal to the functional layers. Apoptosis therefore may be related to the loss of the protective effect of bcl-2 and may have significance for the establishment of an endometrium adequately prepared for successful implantation.

Marker molecules of human endometrial differentiation can be hormonally regulated under in-vitro conditions as in-vivo.

An established cell culture system of isolated human endometrial stromal and epithelial cells has been used to study the effects of oestrogen and progesterone, as well as their antagonists, upon endometrial cells. Normal hormonal regulation in vivo was investigated simultaneously in endometrial tissue samples taken at different phases of the menstrual cycle. Several marker molecules analysed by immunohistochemistry appeared to depend strongly on endocrine regulation and could be traced in culture. Immunohistochemically, basic parameters of cell biology were identified in vitro, e.g. cell proliferation (Ki-67), adhesion molecules (beta3 integrin) and paracrine factors (leukaemia inhibitory factor). The most reliable parameters to assess hormonal influences were oestrogen and progesterone receptor molecules. Immunohistochemical localization could be improved by molecular biological analysis using RT-PCR. In the presence of oestrogen, a significant expression of hormone receptors was also shown by RT-PCR, and withdrawal of oestrogens and addition of gestagen, i.e. medroxyprogesterone acetate, caused receptor downregulation. Addition of the anti-oestrogen ICI 182.780 to cell-culture medium significantly decreased the synthesis of progesterone receptors.

Modulation of endometrial transformation in gonadotrophin-stimulated and unstimulated pseudo-pregnant rabbits: studies with the progesterone receptor antagonist, onapristone.

Advanced endometrial transformation often occurs in IVF and embryo transfer therapy after ovarian stimulation with gonadotrophins. One reason is probably the early rise in peripheral progesterone concentration after ovulation induction. Consequently, we studied in a rabbit model, whether the post-ovulatory application of the progesterone receptor antagonist, onapristone, could prevent such an advancement of endometrial transformation after stimulation with different gonadotrophin preparations. The inhibitory effect of onapristone on the endometrium is dependent upon the strength of ovarian stimulation. In unstimulated animals or animals treated with recombinant LH (nine corpora lutea/animal in both groups), secretory differentiation and proliferation of the endometrium was strongly inhibited by onapristone. After weak ovarian stimulation with a 3:1 mixture of FSH and LH (22 corpora lutea/animal), secretory differentiation was strongly inhibited, while proliferation was enhanced. After strong stimulation with either a 1:1 mixture of FSH and LH, or human menopausal gonadotrophin (HMG; >40 corpora lutea/animal), only limited inhibitory effects of onapristone on secretory transformation or proliferation could be detected. In conclusion, these graded effects of onapristone after stimulation with gonadotrophins, resemble the basic observations from which a therapeutic strategy emerges, to modulate the advanced endometrial transformation which occurs in many IVF patients after ovarian stimulation.

Expression of uteroglobin in the human endometrium.

Uteroglobin is a progesterone binding protein, a member of the antiflammin gene family and possibly a novel cytokine. Initially, uteroglobin was identified as the major protein of rabbit uterine secretion during the phase of preimplantation. Counterparts of the rabbit uteroglobin or its gene are described in rat, mouse, hamster, hare, pig, horse and human. While uteroglobin appears as one of the most extensively studied proteins, particularly its physico-chemical properties, including its crystal structure and its gene, the true physiological role of this protein still remains to be unravelled. Essential to understanding the significance of human uteroglobin in reproductive organs, particularly in the endometrium, is a knowledge of the spatial and chronological expression of this secretory protein. Our studies on 115 volunteers combined reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry and quantitative assessment by an enzyme-linked immunosorbent assay for uteroglobin. The expression, localization and release of uteroglobulin in the human endometrium are presented. Secretory uteroglobin is found in endometrial tissue homogenates in highest levels of expression during the early luteal phase (days 15-19, 340 pg/mg total protein). In turn, uteroglobin is released into the uterine lumen in peak amounts during the receptive phase of the menstrual cycle (mid-luteal phase, days 20-23, secretion level 833.4 pg/mg total protein). Our immunohistochemical studies match with these results, as uteroglobin is located during the early and mid-luteal phase in the apical compartments of endometrial gland cells. These observations strongly suggest an involvement of uteroglobin in endometrial preparations for implantation.

Tumour necrosis factor-alpha (TNF-alpha) in human endometrium and uterine secretion: an evaluation by immunohistochemistry, ELISA and semiquantitative RT-PCR.

Tumour necrosis factor-alpha (TNF-alpha is a pleiotropic cytokine synthesized throughout the female reproductive tract. Even though evidence has accumulated that supports its role in autocrine and paracrine processes, its expression and function in the human endometrium are still not completely understood. To gain a better understanding of the synthesis and release of TNF-alpha in the endometrium and how this relates to concentrations in uterine secretion, its expression throughout the menstrual cycle was investigated by three different techniques. Samples of endometrial tissue and uterine secretions were collected from patients undergoing abdominal and vaginal hysterectomy for benign reasons. The mRNA expression of TNF-alpha was investigated in homogenized endometrial tissue by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) (n = 18). An assessment of the cellular TNF-alpha protein localization in the endometrial glands was performed immunohistochemically (n = 39). The concentrations of the secreted TNF-alpha protein in endometrial secretion were determined by enzyme-linked immunosorbent analysis (n = 30). All three methods gave similar results on the temporal expression of TNF-alpha mRNA and TNF-alpha protein during the cycle. Concentrations of endometrial TNF-alpha mRNA in tissue samples and TNF-alpha protein in uterine secretion were quite low at the beginning of the cycle, rose sharply in the mid- to late proliferative phase and decreased towards the end of the cycle. The concentrations of TNF-alpha protein in the endometrial glands, as shown by immunohistochemical investigation, stayed high throughout the secretory phase at values slightly below those of the late proliferative phase.