Molecular Human Reproduction, Vol. 6, No. 7, 627-634,
July 2000
© 2000 European Society of Human Reproduction and Embryology
Uterine physiology |
Regulated expression of cytokines in human endometrium throughout the menstrual cycle: dysregulation in habitual abortion
1 Department of Pathology, North Shore University Hospital, Biomedical Science Research Center, 350 Community Drive, Manhasset, NY 11030, USA, 2 Department of OB/GYN, Klinikum Grosshadern, Ludwig-Maximilians University, Marchioninistrasse 15, 81377 München, and 3 Department of OB/GYN, Tagesklinik München Nord, Ingolstaedterstrasse 166, 80939 München, Germany
Abstract
It is widely assumed that, after ovulation, the human endometrium undergoes specific changes and becomes receptive to the implantation of embryo during the mid-secretory phase. When implantation does not take place, further changes occur which eventually result in the shedding of human endometrium. The present study was carried out to examine whether there are changes in the cytokine gene expression in human endometrium which are correlated with endometrial function in various phases of the menstrual cycle. The RNase protection assay was performed on carefully dated endometria from normal subjects to characterize the expression of cytokines which potentially contribute to endometrial function. These included: tumour necrosis factor (TNF), interleukin (IL)-1ß, IL-6, IL-8, leukaemia inhibitory factor (LIF), transforming growth factor ß1 (TGF-ß1), macrophage colony stimulating factor (MCSF or colony stimulating factor-1), and vascular endothelial growth factor (VEGF) mRNAs. A low level of expression of these cytokine mRNAs was found during the proliferative and early secretory phase. Expression of cytokine mRNA increased during the mid-secretory phase and rose to a peak in the late secretory phase. The level of cytokine mRNA expression during gestation was most akin to that observed during the mid-secretory phase. Individuals with habitual abortion presented with an abnormal expression of IL-1ß and IL-6 mRNA in endometrium, during the mid-secretory phase. Taken together, these findings are consistent with a progressive rise in the expression of cytokines in human endometrium during the secretory phase in natural cycles. Furthermore, the findings show that habitual abortion is associated with the abnormal expression of IL-1ß and IL-6 in the mid-secretory phase.
cytokines/endometrium/implantation/interleukins/menstruation
Introduction
Human endometrium undergoes a series of carefully controlled changes, which include proliferation, secretion and menstrual shedding. Exquisite mechanisms control the processes that drive endometrium through these phases and prepare it for implantation (Tabibzadeh and Babaknia, 1995
). Implantation is a complex process which initially requires the interaction of the developing embryo and placenta with the endometrium. Studies show that a 100% pregnancy rate cannot be achieved by increasing the number of embryos placed in the uterine cavity (Medical Research International Society for Assisted Reproductive Technology, 1992). The requirement of a receptive human endometrium for embryo implantation has been suggested based on animal studies, and empirical clinical findings (Psychoyos, 1986, 1993). These studies show that endometrium can be maintained in various states which include neutral, receptive and non-receptive or refractory phases. It is postulated that in humans, an `implantation window' also exists, during which the endometrium becomes receptive to the implantation of the blastocyst (Navot et al., 1984, 1986, 1991; Paulson et al., 1990a,b; Psychoyos, 1993; Tabibzadeh and Babaknia, 1995). This phase is followed by a `non-receptive' phase during which the endometrium becomes refractory to the implantation process (Strauss and Gurpide, 1991; Psychoyos, 1993). If implantation does not occur or fails, the destiny of human endometrium is menstruation. We postulate that the menstrual shedding requires expression and activation of a set of molecules immediately prior to this phase. For example, expression of TNF and the new member of the TGFß superfamily, endometrial bleeding associated factor (ebaf), is primarily seen at the end of the menstrual cycle (Kothapalli et al., 1997). The expression of metalloproteinases also occurs immediately prior to and during the tissue shedding (Rodgers et al., 1994; Tabibzadeh, 1995, 1996). These findings suggest that human endometrium requires a specific molecular repertoire during the premenstrual period to ensure a successful tissue shedding in the absence of a healthy implanting blastocyst. The present study was carried out to simultaneously examine the expression of multiple cytokine mRNAs in human endometrium to gain an insight on their profile during the menstrual cycle. Then, the mRNA expression of these cytokines was examined in endometria of patients with infertility of unknown origin and habitual abortion to identify deviations from the normal pattern of cytokine mRNA expression.
Materials and methods
Collection and processing of endometrium
Consent of the internal review board of the institution was obtained before the samples used in this study were collected. Endometrial tissues were obtained from regularly menstruating and fertile women, undergoing hysterectomy for benign lesions of the cervix or myometrium. None of these women received any hormone. The age of the subjects ranged from 36–49 with an average of 43 years. Tissue samples of ~5 mm in diameter were obtained by curretting the endometrium and were snap-frozen and stored at –80°C.The samples were dated according to previously defined histological criteria (Noyes and Hertig, 1950) and the serum concentrations of oestradiol, progesterone and LH. Based on these criteria, the menstrual cycle was divided into four phases: the proliferative phase (days 7–14), the early secretory phase (days 15–19), the mid-secretory phase (days 20–22) and the late secretory phase (days 23–28). Decidual tissues were obtained in the first trimester from women between the ages of 19–36 years who underwent induced abortion by suction curettage for socio-economic reasons. Tissues were snap-frozen in liquid nitrogen and stored at –80°C until use. Endometrial biopsies were also obtained from women with a history of more than three IVF/intracytoplasmic sperm injection (ICSI) failures and women with more than two habitual abortions in the first 10 weeks of gestation (Table I). No anatomical, genetic, endocrine or immunological causes for infertility or abortions were found, therefore, allowing these cases to be classified as idiopathic in nature. Biopsies were obtained as curettings from infertile women in a natural cycle, 8–9 days after the LH surge (Table I). The LH surge was determined by daily urine analysis. The samples from patients with abortions were carefully examined to include only the decidua and not the embryonic or placental tissues for this study. Half of the tissue samples were analysed histologically to exclude endometrial retardation or other abnormalities in infertile patients and to exclude existence of placental and embryonic tissues in abortion samples. The other half was snap-frozen in liquid nitrogen and stored at –80°C until use.
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Isolation of RNA
RNA was extracted using the acid guanidinium thiocyanate–phenol–chloroform extraction method (Chomczynski and Sacchi, 1987
; Sambrook et al., 1989). Briefly, the tissues were homogenized in RNA STAT-60 (Tel-Test Inc, Friendswood, TX, USA). Each tissue was homogenized in 1 ml of RNA STAT-60 in a high-speed homogenizer at 30 000 rpm/min for 10 s. Each homogenate was stored for 5 min at room temperature to permit the complete dissociation of nucleoprotein complexes. Then 0.2 ml of chloroform was added for each ml of RNA STAT-60 used. Each sample was covered and shaken vigorously for 15 s and allowed to stand at room temperature for 2–3 min. Following centrifugation at 12 000 g for 15 min at 4°C, each homogenate was separated into a lower phenol/chloroform phase and an upper aqueous phase. RNA in the upper aqueous phase was transferred to fresh tubes and mixed with isopropanol to precipitate the total RNA. After centrifugation and drying, the precipitated RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water by vigorous pipetting. The amount of RNA in each sample was determined spectrophotometrically and its quality was evaluated by the integrity of ribosomal RNA by electrophoresis of 1 µg of total RNA in 1% formaldehyde–agarose gel in the presence of ethidium bromide.
RNase protection assay
The RNase protection assay was performed using the Riboquant kit (Pharmingen, San Diego, CA, USA), which allowed comparison and quantification of the expression of several mRNA species in a single RNA sample. By incorporating probes for the housekeeping genes GAPDH and the ribosomal RNA L32, the levels of individual mRNA species could be compared between samples. Two different sets of probes were used, one set containing probes for tumour necrosis factor 
(TNF), interleukin (IL)-1ß (IL-1ß), IL-6 and leukaemia inhibitory factor (LIF) and one set containing probes for macrophage colony stimulating factor (MCSF or colony stimulating factor-1), IL-8, transforming growth factor ß1 (TGFß1) and vascular endothelial growth factor (VEGF). First, the probes were internally labelled by in-vitro transcription with [-32P]-UTP (3000 Ci/mmol; 10 mCi/ml; Amersham Life Science Inc, IL, USA). In-vitro transcription was performed by incubating probes with T7 RNA polymerase, nucleotides and DTT for 1 h at 37°C. After terminating the reaction by addition of DNase for 30 min, probes were purified by phenol/chloroform extraction. Efficiency of the in-vitro transcription was quantified in a scintillation counter. A probe concentration of 6x105 cpm was used for each RNase protection assay. For the RNase protection assay, 2 µg (biopsies and mid-secretory controls) or 5 µg (hysterectomy specimen and decidua) of total RNA was dried in a vacuum evaporator centrifuge and diluted in hybridization buffer. RNA samples were mixed with the probe sets and hybridized for 14–16 h at 56°C. To reduce assay variability, 30 samples were analysed in one experiment. Non-hybridized RNA and free probes were digested by incubation with RNase A and T1 for 45 min at 30°C. Enzyme activity was stopped by addition of Proteinase K. The non-digested, RNase protected RNAs were purified by phenol/chloroform extraction and resolved on a denaturing 6% acrylamide sequencing gel for 2 h at 50 W. Unprotected probes were loaded as size markers. Gels were dried for 1 h at 80°C degrees and exposed to a film (Kodak X-AR, Kodak, Rochester, NY, USA) with an intensifying screen for 6 h to 20 days. The identity of the RNase protected bands was established by comparing their size with the size of the bands of the positive control samples and by comparing the migration distance of the bands with those of the probes, which were 23–29 nucleotides longer due to flanking sequences in the probes that did not hybridize to the target RNAs. For some of the genes, e.g. GAPDH, the protected fragments appeared as a doublet. This does not represent detection of splice variants or cross-hybridization of the probe. Rather, this is due to the fact that the protected fragments are often times not fully digested and there may be one or two bases hanging at either ends of the protected fragment. When analysing a mixture of genes, one cannot have the perfect digestion condition and therefore, an average time is used for digestion which, in some instances, may not be quite adequate leading to a slightly larger fragment as well as the completely digested and protected fragment. In the analysis of the data, the density of both bands is used to determine the frequency of the gene expressed in the tissue samples under study. Semi-quantification of the level of mRNA of various cytokines was achieved by normalizing the optical densities of the specific bands to the optical densities of the housekeeping genes, GAPDH and L32. The optical density of the specific protected bands was expressed as relative values. The expression of both housekeeping genes did not fluctuate throughout the menstrual cycle. A two-fold increase of the relative optical density values corresponded, on average, to a 1.8-fold increase of the specific RNA, as determined in several dilution series. The reproducibility of the RNase Protection Assay was determined by comparing the relative optical density values of the same samples, analysed in the same and in different assays. The intra-assay variation of the relative optical density values was ±8% and the inter-assay variation was ±12%. Samples from fertile control, infertile patients, from patients with recurrent abortions and the mid-secretory controls were analysed twice. Kruskal–Wallis and Mann–Whitney tests were used for statistical evaluation. P < 0.05 was considered to be statistically significant.
Results
The relative abundance of mRNA for various cytokines including TNF, IL-1ß, IL-6, LIF, MCSF (CSF1), IL-8, TGFß1 and VEGF were determined in a series of endometria from various phases of the menstrual cycle and during gestation (Figure 1). This was achieved by simultaneously determining the level of mRNA expression in the same tissue sample by using the ribonuclease protection assay. Various samples were not pooled, rather, specimens from different subjects were studied separately. The relative abundance of the mRNAs for all cytokines with the exception of TGFß1 was lowest during the proliferative and early secretory phase (Figure 1). However, IL-1ß and VEGF mRNAs were expressed at higher levels in the proliferative phase than the early secretory phase. Compared with the levels of cytokine mRNA during the early secretory phase, the relative abundance of mRNAs of all cytokines showed an increase during the mid-secretory phase (Figure 1). This increase was significant for LIF, IL-8 and VEGF (P < 0.05). With the exception of TGFß1, the cytokine mRNA was even higher during the late secretory phase than the levels seen during mid-secretory phase (Figure 1, P < 0.05). For some cytokines, e.g. TNF and IL-6, a high inter-individual variability was noted (Figure 1).
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comparison of weeks 7–8 of gestation with cycle days 20–22; Compared with the values obtained during the mid-secretory phase, for all cytokines, with the exception of TGFß1 and IL-1ß, the relative abundance of cytokine mRNA was not significantly increased during weeks 7–8 of gestation (Figure 1
). The relative abundance of the mRNA of TGFß1 and IL-1ß was significantly higher during the gestation, compared with the mid-secretory phase values (Figure 1). A significant decrease in the mRNA expression of some cytokines including TNF was seen during the 9–10th week of gestation (Figure 1). In contrast, the relative abundance of the MCSF, IL-8 and TGFß1 mRNAs showed an increase in the weeks 9–10 of gestation (Figure 1). These findings suggest that during the weeks 9–10 of gestation, only certain cytokines may have to be up-regulated to maintain the pregnancy.
One percent of women suffer from habitual abortion in spite of the lack of a recognizable anatomical, immunological or genetic abnormality (Stirrat et al., 1990). We postulated that habitual abortion may be due to an inability of human endometrium to establish a `dialogue' with the developing blastocyst and that such an abnormality might manifest itself as dysregulated cytokine mRNA expression in the endometrium as early as the mid-secretory phase. Therefore, endometrial tissues were obtained during the mid-secretory phase of the cycle from women who experienced habitual abortion (Table I). As an unrelated patient control group, the endometria of three patients with infertility was included. To maximize the possibility of finding a molecular lesion(s) in the endometria of these patients, we obtained tissue samples from infertile patients who failed to become pregnant despite multiple IVF and ICSI attempts (see Table I). In infertile patients, the RNase protection assay did not reveal a detectable change in the expression profile of the cytokine mRNAs during the mid-secretory phase (Figure 2), in comparison with the controls. In patients with habitual abortion, the expression of most cytokine mRNA also remained significantly unaltered as compared with the levels seen during the mid-secretory phase in normal fertile control subjects (Figure 2). However, the mRNA expression of IL-1ß and IL-6 was significantly lower during the mid-secretory phase in the endometria of patients with habitual abortion, in comparison with levels in the normal controls (Figure 2, P < 0.05). However, we cannot rule out the possibility that such a difference may be attributable to the age difference between the control (age range 19–36 years) and the experimental group (age range 36–49 years).
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Discussion
In recent years, several lines of evidence have suggested that cyokines are implicated in conferring receptivity to endometrium and in preparing it for menstrual shedding (Tabibzadeh, 1995, 1996). The cytokine mRNA profile shown here provides evidence that around the time of implantation, the expression of cytokines increases in human endometrium. Such a mid-secretory increase in the cytokine mRNA expression was not limited to few cytokines, rather, it was seen broadly for TNF, IL-1ß, IL-6, LIF, MCSF, IL-8, TGFß1 and VEGF. The relative values for the TNF, IL-1ß, IL-6, LIF, and IL-8 mRNAs in endometrium during weeks 7–8 of gestation are close to the values seen during the mid-secretory phase. An increased level of cytokine mRNA expression beyond that seen during the mid-secretory phase and during gestation tended to mark the pre-menstrual phase of the cycle. This finding shows a requirement for tight control over cytokine mRNA expression and suggests that only specific levels of cytokines are permissive to implantation and gestation.
The distinctly elevated expression of cytokine mRNA during the late secretory phase suggests that, in the absence of implantation, a second set of changes occur in the endometrium. It also suggests a requirement for a higher level of cytokine mRNA expression in human endometrium in the anticipation of menstruation. We postulate that a distinct premenstrual molecular repertoire exists in human endometrium. In pursuit of finding members of such a molecular repertoire, we carried out differential display analysis on human endometrium during the entire menstrual cycle. We identified an endometrial bleeding associated factor (ebaf), which is a new member of TGF-ß superfamily (Kothapalli et al., 1997). The maximal expression of ebaf mRNA in human endometrium is seen during the premenstrual/menstrual period (Kothapalli et al., 1997). The cytokines, TNF, IL-1ß, IL-6, LIF, MCSF, IL-8, TGFß1 and VEGF seem to be other members of this premenstrual molecular repertoire. These cytokines may be directly involved in the menstrual process. Endometrial bleeding and its regulation is a complex process which undoubtedly requires participation of a diverse group of local factors. We identified TNF as a cytokine which is synthesized and secreted by the human endometrium (Tabibzadeh, 1991; Hunt et al., 1992; Tabibzadeh et al., 1994, 1995b, Tabibzadeh et al., c). The known biological activities and menstrual cycle-dependent expression of the TNF in human endometrium suggests that it is implicated in menstrual bleeding and tissue shedding (Tabibzadeh et al., 1994, 1995b,c,d). Using in-situ hybridization and immunohistochemcial staining, we have shown that the TNF mRNA and protein are expressed, to a large extent, in the endometrial epithelial cells and, to a lesser extent, in the stromal and lymphoid cells (Tabibzadeh, 1991; Hunt et al., 1992). TNF induces haemorrhage in some tumours. However, the effect of this cytokine on the vasculature is not confined to tumours. In one study, ultrastructural examination showed that TNF was shown to impair the blood–retina barrier and induced retinal haemorrhage (Claudio et al., 1994). Administration of TNF to mice induced vascular damage as well as haemorrhage in endometrium that was indistinguishable from the bleeding which occurs during human menstruation (Shalaby et al., 1989). Furthermore, TNF induces apoptosis in endothelial cells (Robaye et al., 1991). Therefore, the disintegration of endometrial vasculature during menstruation may be attributable to the progressive rise in the endometrial tissue concentration of TNF during the secretory/menstrual phase.
With the exception of expression of MCSF mRNA in human endometrium throughout the menstrual cycle, the increase in mRNA of cytokines during the secretory phase reported here are consistent with the available published data (Kauma et al., 1990, 1991; Philippeaux and Piguet, 1993; Arici et al., 1995, 1998; Tabibzadeh et al., 1995a; Cullinan et al., 1996; Torry et al., 1996; Vandermolen and Gu, 1996; Vogiagis et al., 1996; Casslen et al., 1998). A previous publication showed that there is no significant increase in the MCSF mRNA during the secretory phase, compared with the proliferative phase (Kauma et al., 1991). However, a limited number of specimens (n = 5) was studied during the secretory phase and variations in the expression of the MCSF mRNA precluded reaching a definitive conclusion (Kauma et al., 1991). The limited published information regarding the comparison of the mRNA expression of cytokines during the secretory phase and gestation is also consistent with our findings. For example, an increase in the mRNA of IL-1ß, TGFß1 and MCSF has been reported in the first trimester decidua (Kauma et al., 1990, 1991; Daiter et al., 1992; Cullinan et al., 1996). A lack of T-helper (TH)-1 (IL-2 and IL-12 and interferon-
) and a predominant TH-2 (IL-4 and IL-6) cytokine response has been observed in the peri-implantation endometrium of 10 fertile, multiparous women on days LH+7 to LH+9 (Lim et al., 1998). Western blotting revealed that the immunoreactivity of IL-6 protein increases during the mid- and late secretory phases (Tabibzadeh et al., 1989). Other authors (Krasnow et al., 1996) have shown that the immunoreactivity of IL-6, both in the stroma and glands, increased significantly during the mid-secretory phase in comparison with the concentrations in the proliferative and early secretory phases. Therefore, an increase in the IL-6 concentrations in mid-secretory endometrium is a normal response. However, this response appears to be disturbed in individuals with habitual abortion. From all cytokines studied in this report, we found an abnormality in the expression of IL-1ß and IL-6. These findings corroborate with previous findings (Lim et al., 2000) reporting on the role of T-helper cytokines in human endometrium. Lim et al., studied the concentrations of TH-1 and TH-2 cytokines in patients with recurrent miscarriages by carrying out qualitative reverse transcription–polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbent assay (ELISA). Values of the TH-1 cytokines (interferon-, IL-2, IL-12, TNFß) were significantly greater in women with recurrent miscarriages as compared with controls. On the other hand, from the TH-2 cytokines (IL-4, IL-6, IL-10, IL-13), only the level of the IL-6 was found to be significantly lower in habitual aborters as compared with controls. Consistent with these findings, Dechaud et al. found lack of double staining for T cells and IL-6 immunoreactivity in the endometria of patients with tubal sterility (Dechaud et al., 1998). These findings imply that some cytokines may have a more important function in achieving or maintaining pregnancy and may be essential members of the `implantation window' whereas others may be supportive and/or redundant during this phase of the menstrual cycle. For example, animal experimentation shows a crucial role for IL-1 and LIF in implantation (Stewart et al., 1992; Simón et al., 1998; Smith et al., 1998). gp130 is the signal transducer for both LIF and IL-6 implying involvement of IL-6 in the implantation process (Tabibzadeh et al., 1995a). Taken together, these findings show abnormalities in expression of implantation specific cytokines in natural cycles of women with habitual abortion. However, the products of conception from women without recurrent spontaneous abortion had chromosomal abnormalities in 57% of cases, compared with 43% in the control group (Stern et al., 1996). This indicates that this population is not homogeneous and that abortion in these patients may be due to different causes. Further studies are needed to correlate the abnormal expression of IL-1ß or IL-6 in these patients with presence or absence of chromosomal abnormalities.
In addition to the endometrial glands and stroma, endometrial leukocytes are the source as well as targets for the action of cytokines in endometrium and are likely to be involved in implantation and/or menstruation. For example, it was recently reported that stress-triggered abortions are associated with a striking increase in the percentage of degranulated mast cells in the uteri of stressed animals, whereas the granularity of granulated metrial gland (GMG) cells was decreased by stress (Marker et al., 1997). The stress triggered abortion was associated with release of TNF (Arck et al., 1995) and soluble receptors neutralizing TNF- and IL-1 blocked such stress-triggered abortion (Arck et al., 1997). Furthermore, early loss of murine embryo was associated with infiltration of decidua by macrophages (Baines et al., 1997) and expression of macrophage activation markers in the decidua such as increased inducible nitric oxide synthase (iNOS) and TNF expression (Haddad et al., 1997). The importance of TNF in spontaneous resorption in rodents was recently reported (Lea et al., 1998). Two populations of V1.1(+)
6.3(+) T cells seems to exist in murine endometrium. An early population of TH1 T cells, present at the time of implantation, are abortogenic whereas a second TH2/3 T cell subset, present in the decidua later during pregnancy, protect the pregnancy (Arck et al., 1999). Patients who had miscarriages following endometrial biopsy had significantly more CD4+, CD8+, CD14+, CD16+, and CD56+ leukocytes in their endometrium than either those who had live births or women with proven fertility (Clifford et al., 1999; Quenby et al., 1999). Throughout the luteal phase, infertile women had fewer CD56+ cells than normal fertile controls (Klentzeris et al., 1994). Based on the available data, it seems that endometrial leukocytes actively participate both in the maintenance of normal pregnancy as well as in abortion and, some if not all of such functions, are likely to be mediated through cytokines. In the absence of data on cellular localization, we cannot tell whether the difference in the expression of IL-1ß and IL-6 in the endometria of patients with habitual abortion is related to changes in their expression in the endometrial stroma or epithelium or due to changes in the number of leukocytes.
The characteristic expression profile of mRNA of cytokines during the menstrual cycle in human endometrium suggests that these cytokines are under the control of steroid hormones. In fact, stimulation of cytokine mRNA in endometrial cells by steroid hormones has been reported for MCSF (Azuma et al., 1990), TGFß1 (Arici et al., 1996), and VEGF (Huang et al., 1998). However, suppression of cytokine expression in the endometrial stromal cells was reported by oestradiol for IL-6 (Tabibzadeh et al., 1989). The steroid hormone mediated regulation of cytokine mRNA expression is also consistent with the further up-regulation of cytokine mRNA at the end of the menstrual cycle and suggests that the cytokine mRNA may also be regulated by steroid hormone withdrawal. Consistent with these findings, we recently noted that the expression of the TNF mRNA is up-regulated by oestrogen withdrawal in endometrial epithelial cells (Tabibzadeh et al., 1999). A third control over the expression of cytokine mRNA is exerted at the autocrine and paracrine level by the endometrial cytokines. For example, the expression of IL-6 mRNA is stimulated by IL-1ß (Tabibzadeh et al., 1989) and the expression of IL-8 mRNA is enhanced by IL-1, TNF (Arici et al., 1993) and TGFß1 (Arici et al., 1996).
In summary, for most cytokines, a low level mRNA expression is seen during the proliferative and early secretory phases in human endometrium. The level of cytokine mRNA progressively increases during the secretory phase and reaches its maximum level prior to menstruation. During gestation, expression of cytokine mRNA is akin to that found during the secretory phase. The distinct and exquisite control over cytokine mRNA expression seems to be disturbed for IL-1ß and IL-6 in the endometrium of patients with habitual abortion.
Acknowledgments
This work was supported by a grant from German Academic Exchange Service (DAAD), Serono Pharma GmbH, Germany, and Baxter ImmunoGmbH, Germany, and by the NIH grant CA46866 to ST.
Notes
4 To whom correspondence should be addressed: Department of Pathology, North Shore University Hospital, Biomedical Science Research Center, 350 Community Drive, Manhasset, NY 11030, USA. E-mail: tabibzadeh{at}nshs.edu 
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Submitted on December 29, 1999; accepted on April 7, 2000.
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