Molecular Human Reproduction, Vol. 5, No. 2, 146-152,
February 1999
© 1999 European Society of Human Reproduction and Embryology
Tumour necrosis factor-
(TNF-) in human endometrium and uterine secretion: an evaluation by immunohistochemistry, ELISA and semiquantitative RT–PCR
1 Department of Anatomy and Reproductive Biology, RWTH University of Aachen, Wendlingweg 2, 52057 Aachen, and 2 Department of Gynecology and Obstetrics, St. Antonius Hospital, 52249 Eschweiler, Germany
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Tumour necrosis factor-
(TNF-) 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- 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- was investigated in homogenized endometrial tissue by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) (n = 18). An assessment of the cellular TNF- protein localization in the endometrial glands was performed immunohistochemically (n = 39). The concentrations of the secreted TNF- 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- mRNA and TNF- protein during the cycle. Concentrations of endometrial TNF- mRNA in tissue samples and TNF- 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- 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.
endometrium/menstrual cycle/tumour necrosis factor-/uterine secretion
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Tumour necrosis factor-
(TNF-) is known to be one of the most versatile cytokines. It serves as a normal mediator of tissue homeostasis, it has pathophysiological effects at high concentrations and it is expressed in various tissues. In the reproductive tract, TNF- was detected in the ovaries (Roby et al., 1990
), the oviduct (Hunt, 1993), in preimplantation embryos (Zolti et al., 1991; Sharkey et al., 1995) and the endometrium (Hunt et al., 1992; Philippeaux et al., 1993; Tabibzadeh et al., 1995).
The endometrium is characterized by a variety of cell types, e.g. fibroblasts, immunocompetent cells (Parr et al., 1995), glandular epithelial cells (Hunt et al., 1992) and vascular cells (Philippeaux et al., 1993), all of which have been found to express TNF-. The involvement of these cells in the regulation of proliferation, transformation and menstrual shedding as well as in implantation and trophoblast invasion is still a matter of research. Increasing evidence suggests that TNF- plays an important role in the cyclic changes of the endometrium, regulated by the modulation of the different cell types. However, increased expression of this cytokine can also cause pathophysiological effects reflected by its involvement in implantation failure (Hazout, 1995), immunologically-mediated abortion (Giacomucci et al., 1994) and endometriosis (Zhang et al., 1993).
As proliferation, transformation and endometrial shedding are controlled by oestradiol and progesterone, regulation of TNF- expression may logically follow these hormones. In fact, previous studies revealed a cycle-dependent expression of TNF- in the human endometrium. Hunt et al. (1992), using immunohistochemistry and in-situ hybridization, detected increasing values of TNF- and its mRNA in the endometrial glands of the proliferative phase, followed by a decreased expression in the early secretory phase and high levels in the mid- and late secretory phases. Philippeaux et al. (1993), described TNF- expression as being absent or weak in endometrial glands and arteries during the proliferative phase, and found a strong expression of TNF- (by immunohistochemistry) and its mRNA (by Northern blotting and in-situ hybridization) throughout the secretory phase. Tabibzadeh et al. (1995), measured by incubating endometrial biopsy samples in medium for 15 min, the release of TNF- by enzyme-linked immunosorbent assay (ELISA) and observed increasing concentrations throughout the cycle with a maximum in samples taken during menstruation.
Even though all three studies described a cycle-dependent expression of TNF-, the patterns of fluctuation were not uniform. It is difficult to postulate potential roles of TNF- in the regulation of endometrial biology. It was speculated that TNF- promotes DNA synthesis in the early proliferative phase (Hunt, 1993), that it participates in cell differentiation and tissue remodelling, which is required to support embryonic attachment (Terranova et al., 1995), and that it facilitates apoptosis and, therefore, initiates menstrual shedding (Tabibzadeh et al., 1995). It was postulated that TNF- ligands bind to TNF- receptors expressed in human embryos (Sharkey et al., 1995).
To get a better understanding of the role of TNF- in the regulation of endometrial tissue, we investigated the expression of this cytokine and its mRNA during the cycle in endometrial tissue and uterine secretion using three molecular and cell biological techniques.
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Materials and methods |
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Collection of material
Endometrial tissue, serum and plasma were collected from 39 premenopausal patients at different phases of the endometrial cycle and from seven post-menopausal patients after abdominal or vaginal hysterectomy for benign reasons. None of the patients was hormonally stimulated. For the collection of plasma, blood samples were immediately processed to avoid loss of TNF-
activity as described by Exley and Cohe (1990). Serum was collected after keeping the blood samples at room temperature for 2–3 h until the blood was fully clotted. Plasma and serum samples were stored at –20°C. For immunohistochemistry, samples of ~5 mm in diameter were snap frozen and stored at –20°C. For PCR studies, endometrium from 18 patients was scraped out of the uterus and stored in a solution of guanidium isothiocyanate (GTC) and mercaptoethanol at –70°C until processed.
Endometrial secretions were taken for ELISA from 30 patients just before hysterectomy to avoid changes in TNF- concentration caused by manipulation of the uterus during hysterectomy. Secretions were taken with an intraluminal device, manufactured for the collection of uterine surface cells for cytological investigations (Prevical; Nourypharma, Oberschleißheim, Germany). The secretions were flushed into a solution of phosphate-buffered saline (PBS) and stored at –20°C until the ELISA was performed.
The samples were dated according to the last menstrual period, the histological criteria of Noyes et al. (1950), and the serum concentrations of oestradiol, progesterone and luteinizing hormone.
The menstrual cycle was divided into six phases: the early proliferative phase (days 1–5), the mid proliferative phase (days 6–9), the late proliferative phase (days 10–14), the early secretory phase (days 15–19), the mid secretory phase (days 20–23) and the late secretory phase (days 24–28).
Immunohistochemistry
Sections were cut on a cryostat microtome (8 µm thick), placed on aminopropyl-triethoxy-silane (APES)-coated glass slides, air-dried and fixed in acetone (5°C) for 10 min. Blocking of endogenous peroxidase activity was achieved by a 10 min incubation in 3% H2O2. After incubating in 10% porcine serum, dissolved in PBS and 1.5% bovine serum albumin (BSA), sections were incubated with a polyclonal rabbit anti-human TNF- antibody, supplied as hyperimmune antiserum (Genzyme-Diagnostics, Cambridge, UK) at a concentration of 1:100 in PBS/BSA for 1 h. Immunostaining was performed using a multilink biotinylated secondary antibody purchased from Dako, Hamburg, Germany (1:150 in PBS/BSA, incubated for 30 min), followed by incubation with a streptavidin peroxidase conjugate (Zymed Laboratories, San Francisco, CA, USA) for 15 min and finally by use of an AEC staining-kit (Zymed). Red deposits indicated the sites of immunoreactive protein. Control experiments included staining without the primary antibody and substitution of the TNF- antibody by non-immune rabbit serum at a dilution of 1:100 in PBS/BSA. The intensity of the immunohistochemical staining was semiquantitatively assessed by two observers (M.v.W., I.C.-L.) as follows: weak staining (+), moderate staining (++) and strong staining (+++).
Polymerase chain reaction
The tissues were homogenized and the total RNA isolated according to Chomczynski and Sacchi (1987). The content of RNA was quantified by UV spectrophotometry at 260 and 280 nm. First strand cDNA was synthesized using a First-Strand cDNA Synthesis Kit (Pharmacia, Uppsala, Sweden) and 2 µg of total RNA. The reaction was incubated at 37°C for 1 h, heated to 99°C for 5 min and chilled on ice.
Aliquots of 2 µl of each cDNA was amplified in a total volume of 100 µl containing PCR buffer (10 mmol/l Tris, 50 mmol/l KCl, 1.5 mmol/l MgCl), 200 mmol/l dNTP, 50 pmol/l of oligonucleotide primers, 0.1 µl alkali-stable digoxigenin-11-dUTP (DIG dUTP) and 2.5 IU of Taq polymerase (Boehringer Mannheim). In order to quantify the PCR products comparatively and confirm the integrity of the RNA we co-amplified a housekeeping gene, cytochrome C oxidase subunit I (CO-I), in a companion tube. The TNF- primers were synthesized according to the human TNF- sequence (Shirai et al., 1985) and yielded a 254 bp product: sense (5'-CGAGTGACAAGCCTGTAGCC-3') and antisense (5'-GTTGACCTTGGTCTGGTAGG-3'). The CO-I primers were synthezised in accordance with the human CO-I sequence (Sanger et al., 1980) and yielded a 268 bp product: sense (5'-CGTCACAGCCCATGCATTTG-3') and antisense (5'-GGTTAGGTCTACGGAGGCTC-3'). The numbers of PCR cycles were within the linear logarithmic phase of the amplification curve. 30 cycles were chosen for TNF- and 22 cycles for CO-I and amplified as follows: 1 min at 92°C, 1 min at 59°C (TNF-) respectively 57°C (CO-I), 1 min at 72°C. The PCR products were separated electrophoretically in a 1.2 % high melting agarose gel, denatured and blotted to a Hybond-N membrane (Amersham, Buckinghamshire, UK).
The quantity of the PCR products was based on the detection of the incorporated DIG-dUTP during PCR. The membranes were incubated with anti-DIG alkaline phosphatase conjugate (Boehringer Mannheim), rinsed with the chemiluminescence substrate `CSPD' (Boehringer Mannheim) and exposed to an X-ray film. The luminescent signal was quantified using a laser densitomer (Pharmacia, Uppsala, Sweden) and the results were expressed as relative levels of TNF- mRNA normalized to CO-I mRNA.
Specificity of the amplification process was verified either by Southern Blot hybridization (TNF-) or by restriction mapping (CO-I): (i) following PCR (performed without dig-dUTP), electrophoresis and southern blotting the membranes were hybridized with a 3' DIG dUTP-tailed (Boehringer Mannheim) TNF- oligonucleotide probe (5'-ATTGACCTCAACTACATGGTTTACA-3') which was detected as described above for the PCR products; (ii) The PCR products generated with the CO-I primers were incubated with the restriction enzyme HindIII (Boehringer Mannheim). The PCR products were digested to fragments of 130 and 138 bp.
ELISA
TNF- was determined in the uterine secretions, serum and plasma using a commercially available ultra sensitive ELISA kit (Laboserv Diagnostica, Giessen, Germany). Following centrifugation the supernatants of the samples were analysed. The ELISA was performed according to the instructions of the manufacturer. The serum and plasma concentrations of TNF- were measured to estimate the contamination of the samples with TNF- from blood. The TNF- concentrations in the samples were expressed relative to the sample's protein content, determined according to Lowry et al. (1951).
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Immunohistochemistry
A polyclonal anti-TNF-
antibody was used to detect TNF- protein in frozen endometrial sections. The non-specific staining of the polyclonal antibody was evaluated by control experiments substituting the anti TNF- hyperimmune serum by non immune serum of the same species (Figure 1b,d,f).
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In the glandular cells TNF-
protein was detected in the cytoplasm, but nuclei were negative. The cells from an individual gland were similarily stained, but the staining intensity varied slightly from one gland to another. TNF- content in the glands was negative to weak in the early proliferative phase (Figure 1a
) and increased during the proliferative phase with a maximum in the late proliferative phase (Figure 1c) in which three of five samples were maximally stained on the apical and the basal part of the epithelial cells (Figure 2). The positive immunoreaction stayed high in the secretory phase (Figure 1e) with a staining intensity slightly lower than in the late proliferative phase. In the secretory phase staining was only pronounced on the apical part of the glandular epithelium.
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Analysis of the basal and functional layers of the endometrium revealed no striking differences in staining intensity. Staining of the stroma was weak to moderate throughout the whole cycle. The staining patterns from the seven post-menopausal patients were different from those collected premenopausally. In three of the samples, staining of the stroma and the epithelium were completely negative. The other samples revealed negative staining of the epithelium and interestingly very strong staining of the stroma (Figure 3
).
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PCR
Semiquantitative analysis of endometrial mRNA was achieved by normalizing the TNF-
mRNA to the mRNA of the co-amplified housekeeping gene `Cytochrome C oxidase subunit I' (COI) (Figure 4a). One of the main pitfalls in the semiquantitative PCR is the fluctuation in the expression of the housekeeping genes to which the analysed RNA values are normalized. COI is supposed to be expressed at almost constant levels throughout the cycle. In our experiments expression of COI was only slightly higher in the proliferative phase than in the secretory phase, which demonstrates that COI is suitable as a housekeeping gene for the experiments performed.
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TNF-
mRNA expression of the homogenized tissue appeared similar to TNF- protein concentration in the epithelial glands in the proliferative and early secretory phase: it was low at the beginning of the menstrual cycle, rose sharply in the mid to late proliferative phase and stayed high during the early secretory phase. Towards the end of the cycle, TNF- mRNA decreased whereas TNF- protein remained high in the endometrial glands. TNF- mRNA concentrations were about three times higher in the middle than at the beginning and the end of the menstrual cycle.
ELISA
A commercially available ultrasensitive ELISA kit was used to measure the TNF- concentration in uterine secretion samples, expressed in pg TNF-/mg protein. The limit of detection of TNF- was 2 pg TNF- per ml. The protein concentration in uterine secretions was on average 1.7 mg/ml which resulted in a limit of detection of TNF- per mg protein of about 1.2 pg. In serum and plasma, the limit of detection per mg protein was much lower due to the high protein content in serum and plasma.
The pattern of secreted TNF- protein concentration during the cycle revealed parallels with the results of the PCR studies. At the beginning and the end of the cycle, TNF- concentrations were very low (Figure 5). Although in about half of the samples concentrations were even too low to be detected by the applied ELISA, in the mid- to late proliferative and the early to mid-secretory phase, TNF- concentrations rose to levels three times higher than at the beginning and the end of the cycle. In addition, the TNF- concentration in plasma and serum was even lower: in plasma on average 0.01 pg/mg protein and in serum 0.003 pg/mg protein. The low concentrations in plasma and serum indicated that most of the TNF- in the uterine secretions originated from the endometrial cells and not from contamination by blood. The higher concentrations of TNF- in plasma in comparison to serum are presumably due to a loss of activity in serum samples during clotting of the blood at room temperature for 2–3 h (Exley and Cohe, 1990).
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Seven samples were collected from post-menopausal patients. Interestingly, three samples contained TNF-
concentrations too low to be detected and four showed surprisingly high concentration of TNF- (mean 4.8 pg/mg protein, data not shown), which corresponds with the high stromal immunoreactivity for TNF- as shown in Figure 2.
In summary, it may be concluded that the three different techniques used in this study revealed similar patterns of TNF- protein and TNF- mRNA expression during the menstrual cycle; levels were quite low at the beginning and the end of the cycle and peaked in the mid- to late proliferative phase and the early to mid-secretory phase. Towards the end of the cycle, TNF- mRNA in homogenized tissue and TNF- protein in uterine secretion decreased whereas TNF- protein in the endometrial glands, as detected by immunohistochemistry, remained high.
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Discussion |
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To study the synthesis of TNF-
and its mRNA in human endometrial tissue, we applied three cell biological techniques to evaluate the expression of protein biosynthesis and secretion: TNF- mRNA in homogenized endometrium by RT–PCR, TNF- protein in endometrial sections by immunohistochemistry and TNF- protein in uterine secretions by ELISA.
TNF- is known to be a very sensitive molecule, resulting in a significant loss of activity in tissues and biological fluids if not rapidly processed (Exley and Cohe, 1990). To avoid degradation of TNF-, uterine secretions were taken just before hysterectomy, endometrial tissue samples were snap-frozen immediately after hysterectomy, and plasma samples were collected in addition to serum as plasma can be separated from whole blood without any delay.
All three techniques applied in this study revealed a cycle-dependent expression of TNF- and its mRNA. Values were low at the beginning and the end of the cycle and peaked in the mid- to late proliferative phase and the early to mid-secretory phase. The cycle-dependent expression of TNF- suggests a regulation of its synthesis and secretion by ovarian steroid hormones. This concept corresponds with several previously published studies, involving in-vitro experiments on cultured endometrial cells and in-vivo experiments in mice stimulated by ovarian steroids.
Cell culture experiments by Laird et al. (1996), revealed maximal TNF- production by human endometrial epithelial cells taken from uteri in the late proliferative and the mid-secretory phase. Studies on ovariectomized and hormone-substituted mice showed that uterine TNF- mRNA expression is stimulated by oestradiol and progesterone (De et al., 1992; Roby and Hunt, 1994). However, other studies in mice demonstrated an unresponsiveness of TNF- genes to steroid hormones (Mc Master et al., 1992; Kover et al., 1995). Controversial results were also obtained by Laird et al. (1996). Progesterone and oestradiol caused an increase in TNF- production by cells prepared from proliferative endometrium. In contrast, both hormones caused a decrease in the production of TNF- by cells prepared from secretory endometrium.
A cycle-dependent expression of TNF- in human endometrial tissues has already been described in previous studies with some differences compared with our study. In-situ hybridization and immunohistochemical experiments by Hunt et al. (1992), demonstrated highest RNA concentrations in human endometrial epithelium and stroma in the late proliferative phase and the mid- to late secretory phase. Philippeaux et al. (1993), described an absent to weak TNF- expression in the endometrial glands during the proliferative phase and a strong expression of TNF- (by immunohistochemistry) and its mRNA (by Northern blotting and in-situ hybridization) throughout the secretory phase without obvious fluctuations. Tabibzadeh et al. (1995), measured in endometrial biopsy samples the release of TNF- by the ELISA-technique and observed increasing concentrations throughout the cycle with a maximum during menstruation. This led the authors to suggest that TNF- plays a role in cytolysis and menstruation. The most striking difference detected in our study, was the decrease of TNF- in the late secretory phase. This contrasts to the high concentration of TNF- in previously published studies.
However, an interpretation of these differences is difficult as different techniques were applied. Tabibzadeh et al. (1995), measured the release of TNF- in endometrial biopsy samples, after incubation in medium for 15 min. It cannot be excluded that the incubation of endometrial tissue led to a stimulation of immunocompetent cells resulting in the release of TNF- into the analysed medium. Another possible explanation of the differences might be the considerable individual variation in the expression of TNF-. In all published studies, tissue samples were collected from women undergoing hysterectomy for benign reasons. Patients whose uteri are removed are usually already pre- or perimenopausal or have bleeding disorders or other pathologies resulting in changes of endometrial physiology. Such an individual variation might also explain the unexpected low or high levels of TNF- mRNA and protein in some samples in our study. Methodical errors as a cause of this variation were unlikely as most reactions were performed twice.
Finally, if the results of different studies are compared, their significance should be carefully assessed. Any final evaluation would need far more samples than have been included in all those studies, including ours, to perform statistical analysis.
Immunohistochemical and ELISA studies on endometrial tissue samples and uterine secretions from post-menopausal patients revealed high TNF- concentrations in four of seven investigated samples. These results would not be expected if the expression of TNF- is only regulated by ovarian steroid hormones. The pathology of the uterus could also contribute to the unexpected high concentrations. High concentrations were found only in those postmenopausal patients who had suffered from a significant pathological variation of the whole uterus (e.g. prolapse), whereas patients with pathohistological changes (e.g. cervical dysplasia) showed only low concentrations. Prolapse of the uterus might cause a mechanical irritation, which may in turn attract immunocompetent cells into the endometrial stroma, resulting in increased stromal TNF- release.
To speculate about the role of TNF- in the regulation of the endometrium, the expression of TNF- receptors, the concentration of TNF- itself and the regional distribution of protein and receptor has to be taken into account. TNF- receptors are expressed on the surface of most cells. In the endometrium, TNF- receptor type I and type II are both maximally expressed in the mid- to late secretory phase. TNF- receptor type II shows an additional peak of expression in the mid- to late proliferative phase (Hunt et al., 1991). The fluctuation of receptor expression roughly corresponds with the concentration of TNF- and its mRNA described in our study.
The multifunctional character of TNF- and its expression at different phases of the menstrual cycle suggest a complex role in the function of the cycling endometrium. In the proliferative phase TNF- might enhance the proliferation of the endometrium by promoting DNA synthesis in endometrial and stromal cells (Hunt et al., 1992). Another mode of action could be the induction of receptors and cytokines with a high proliferative potential, as is seen with epidermal growth factor induction (Tomooka et al., 1986; Strowitzki et al., 1991) by TNF- as described in glioma cells (Adachi et al., 1992).
In the early and mid-secretory phase, TNF- might act not only on the endometrium but also on the preimplantation embryo, leading to a successful implantation. Chen et al. (1995), described the stimulation of prostaglandin release in cultured human luteal phase endometrial cells by TNF-. It can be speculated that the increase of endometrial vascular permeability by prostaglandins could have an effect on the implantation process (Peek et al., 1992). An effect of endometrial TNF- on the preimplantation embryo is only possible if the blastocysts have been proven to express TNF- receptors and if TNF- is secreted into the uterine secretion. The first requirement, the expression of TNF- receptors on human embryos, has been demonstrated by Sharkey et al. (1995), who found TNF- receptors Rp60 and Rp80 in the early preimplantation embryos by RT–PCR. The second requirement, the release of TNF- into uterine secretions, has been demonstrated in this study: high levels of TNF- were found in uterine secretions in the late proliferative phase and the early and mid-secretory phase.
The function of TNF- in the development of the preimplantation embryo however, is not clear. Based on the rate of morphological growth, no significant effect of TNF- on blastocyst formation (Wincek et al., 1991), and subsequent attachment (Haimovici et al., 1991) was detected. Pampfer et al. (1994), studied the selective effect of TNF- in mouse blastocysts and found even a reduction of the inner cell mass, suggesting a suppressive effect of TNF- on the preimplantation embryo.
In the late secretory phase, our study revealed low concentrations of TNF- which is compatible with studies demonstrating inhibition of decidualization of endometrial stromal cells by TNF- (Inoue et al., 1994; Jikihara and Handwerger, 1994; Terranova et al., 1995). Low concentrations of TNF- at the end of the cycle might be necessary for the important process of decidualization.
Tabibzadeh et al. (1995) and Tabibzadeh (1996), have suggested that TNF- might be a `menstruation inducing factor' by the induction of apoptosis and by acting on vasculature and cell–cell dissociation. Even though this suggestion is based on data showing maximal release of TNF- from endometrial tissue samples taken during menstruation, it is also compatible with our data showing strong TNF- expression in the mid-secretory phase and a decrease in the late secretory phase. Compromise of the integrity of the microvasculature system, stromal oedema and apoptosis (Tabibzadeh, 1996; Rango et al., 1998) are already noticeable in the mid-secretory phase. Bearing in mind the classical concepts of human chorionic gonadotrophin (HCG) rescue of the corpus luteum by an embryonic signal, one could easily compare the higher level of TNF- at the mid-luteal phase with this regulatory process. If an additional signal is lacking, ending of the luteal phase and consequent menstruation would necessarily occur.
In conclusion, we can summarize that TNF- is a cytokine synthesized in the endometrium, expressed in glandular epithelial cells and released into the uterine secretion in relation to the menstrual cycle, which suggests a regulation by ovarian steroid hormones. Most possibly, it has an interesting spectrum of functions, depending on the phase of the menstrual cycle and the regional distribution of its expression. More work remains to be carried out for a better understanding of the role of this cytokine as part of the complex network of cytokines in the endometrium.
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3 To whom correspondence should be addressed at Klinik und Poliklinik fuer Fraunheilkunde und Geburtshilfe, Klinikum Großhadern, Marchionistraße 15, D-81377, Muenchen, Germany
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Submitted on May 28, 1998; accepted on November 8, 1998.
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