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Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong Special Administrative Region, People’s Republic of China
Address all correspondence and requests for reprints to: Ernest H. Y. Ng, Department of Obstetrics and Gynaecology, The University of Hong Kong, 6/F, Professorial Block, Queen Mary Hospital, Pokfulam Road, Hong Kong. E-mail: nghye{at}hkucc.hku.hk.
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Abstract |
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Objective: The in vivo expression of T helper type 2 (Th2) cytokines in the periimplantation endometrium of infertile patients was compared between natural and stimulated cycles.
Interventions and Main Outcome Measures: Uterine flushings and endometrial biopsies were collected 7 d after the LH surge in natural cycles or after human chorionic gonadotropin injection in stimulated cycles. Th2 cytokines were determined by immunolocalization and by ELISA. Natural cycles were in group A, whereas stimulated cycles with peak serum E2 of no more than 20,000 pmol/liter (moderate responders) and more than 20,000 pmol/liter (excessive responders) were classified as group B and group C, respectively.
Results: Higher E2 had a negative effect on IL-11 and IL-6 expression in the endometrium and IL-11 concentration in the uterine flushing. In endometrial biopsies, a significantly lower immunostaining of stromal IL-11 (P < 0.001) and glandular IL-6 (P < 0.05) was detected in group C compared with that of groups A and B. IL-11 concentration by ELISA was significantly lower in group C (P < 0.05). Endometrial leukemia inhibitory factor and IL-4 expression was similar in the three groups. In uterine flushings, a significantly higher percentage of women in group C had undetectable IL-11 and a lower IL-11 concentration (P < 0.01) compared with group A, whereas no difference in IL-6 concentration was noted in the three groups.
Conclusion: Reduced expression of IL-11 and IL-6 in periimplantation endometrium may account for lower implantation in excessive responders.
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Introduction |
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Uterine flushing provides a reliable and noninvasive tool to evaluate endometrial function by measuring endometrial proteins, which may be important for the process of implantation. We have previously concluded that the adverse impact of high E2 concentration on endometrial function in the periimplantation period is not associated with alterations in the placental protein 14 and CA-125 concentrations performed in the midluteal-phase uterine flushings (8). However, many of the actions of steroids in controlling reproduction are mediated by locally acting factors such as growth factors and cytokines.
Cytokines are involved in the regulation of endometrial function, because they are expressed in the human endometrium throughout the menstrual cycle. The endometrial cells and the implanting embryo cross-talk via a series of cytokines that regulate the implantation process in a complex manner. The evidence for a dichotomous T helper (Th) cell response in reproduction originates from murine models that show that pregnancy rejection is mediated by Th type 1 (Th1) cytokines, whereas a Th2 cytokine response confers protection (9). The initial study showed that the functional dominance of Th2 cells is of primary importance in sustaining pregnancy (9). Subsequent human endometrial studies demonstrated increased Th2 cytokine expression during the secretory phase of the cycle at mRNA (10) and intrauterine protein levels (11). Th2 cytokine was predominantly present in the periimplantation endometrium of multiparous women (12) and deciduas during early pregnancy (13). Moreover, compared with women with normal gestation, women with unexplained recurrent abortions had decreased decidual T cells Th2 cytokines production (14), and anembryonic pregnancies had a higher Th1/Th2 ratio in peripheral blood and deciduas (15). These studies suggest that specific cytokines are necessary for successful implantation.
The aims of this study are to evaluate and compare the in vivo expression of Th2 cytokines, leukemia inhibitory factor (LIF), IL-11, IL-6, and IL-4 in the periimplantation endometrium of infertile patients between natural and stimulated cycles.
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Patients and Methods |
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Consecutive infertile patients undergoing IVF treatment at the Assisted Reproduction Unit of the Department of Obstetrics and Gynaecology of the University of Hong Kong were recruited when ET was not performed because of no spermatozoa in testicular sperm extraction, the absence of fertilization because of male factors, or serum E2 concentrations on the day of hCG greater than 20,000 pmol/liter. All fresh embryos were cryopreserved in these patients because of the associated risks of ovarian hyperstimulation syndrome and reduced pregnancy rate (2). Patients on the waiting list for IVF/ET with regular ovulatory cycles and no evident uterine pathology were recruited in natural cycles for comparison. The study was approved by the University Ethics Committee, and every patient gave a written informed consent before their participation. A total of 81 women were recruited, and each patient was evaluated only once during the study period.
A long protocol for ovarian stimulation is followed at our center (2). Briefly, women were pretreated with 150 µg Buserelin (Suprecur; Hoechst, Frankfurt, Germany) nasal spray four times a day from the midluteal phase of the cycle preceding the treatment cycle and received human menopausal gonadotropin (hMG) (Pergonal; Serono, Geneva, Switzerland). hCG (Profasi; Serono), 10,000 IU, was given im when the leading follicle diameter reached 18 mm and there appeared at least three follicles of greater than 15 mm. Serum E2 concentrations were measured on the day of hCG and 7 d after it.
Natural cycle patients did not receive any previous steroidal hormones for at least 2 months, and they used condoms for contraception during the study cycle. They were asked to attend the clinic daily from 18 d before the next expected period for serum E2 and LH concentrations until the day of the LH surge, which was defined as the day when the serum LH concentration was more than double compared with the mean of the preceding values.
Uterine flushing and endometrial biopsy
All uterine flushings were performed, as an outpatient procedure, 7 d after the LH surge in the natural cycles or hCG injection in stimulated cycles (8). Five milliliters of normal saline solution were used to carry out the flushing. One milliliter of fresh saline was flushed gradually each time into the uterine cavity through the opening of the inner lumen of a size 8 Foley catheter. Samples were pooled, centrifuged, and stored at –20 C for subsequent assays. After uterine flushing, endometrial biopsy was performed using a Pipelle sampler (Unimar, Neuilly-en-Thelle, France). The endometrial tissue was fixed in formalin and was paraffin embedded for sectioning.
Cytokine expression by immunohistochemistry (IHC)
The immunolocalization of LIF, IL-11, IL-6, and IL-4 was performed using commercially available mouse antihuman monoclonal antibodies from R&D Systems Inc. (Minneapolis, MN) on rehydrated 5-µm tissue sections. Nonspecific binding was blocked by 3% hydrogen peroxide and normal rabbit serum (Dako, Ely, UK). One section from the same tissue block was included in every staining run for quality control. A negative control was included with exclusion of the primary antibody and application of normal mouse IgG (Dako) at the same concentrations instead of the primary antibody. Primary antibodies were used in the following concentrations: LIF, 6.25 µg/ml; IL-11, 10 µg/ml; IL-6, 16.67 µg/ml; and IL-4, 33.3 µg/ml. All incubations were carried out in a humid chamber at room temperature, except for the incubation with primary antibodies, which was carried out at 4 C. Five-minute washes were carried out between incubations. Biotinylated rabbit antimouse IgG (Dako) was the secondary antibody. Color was developed using diaminobenzidine tetrahydrochloride substrate (Sigma Chemical Co., St. Louis, MO). Sections were counterstained, dehydrated, and mounted with hematoxylin (Vector Laboratories, Burlingame, CA).
The IHC slides were coded by a pathologist and read by G.M., and both were unaware of all the clinical information. The intraobserver variability was calculated as 6.8% for IL-11, 9.9% for IL-6, 8% for LIF, and 8.6% for IL-4 by reading a set of 20 slides two times (with a different set for each cytokine). The assessment of staining was done semiquantitatively by analyzing each of the 10 fields from epithelial and stromal fractions. The intensity of staining was graded on a five-point scale of 0–4, with each numeral representing the intensity as follows: 0, absent staining; 1, mild; 2, moderate; 3, strong; 4, very strong or intense staining.
Cytokine concentration by ELISA
Solid-phase, human quantitative sandwich enzyme immunoassay kits (R&D Systems) were used according to the manufacturer’s protocol. Samples were measured in duplicate for each ligand and were appropriately diluted (when necessary) to ensure that they were within the standard curve range. LIF (DLF00), IL-11 (D1100), and IL-4 (DY204) each had the lowest detectable limit of 8 pg/ml, with each measuring from 15.6–1000 pg/ml. IL-6 (DY206) had the lowest detectable limit of 4.7 pg/ml and was measured as 9.5–600 pg/ml. The interassay and intraassay coefficients of variations (percentage) for cytokines were as follows: 5.4 and 3.9 for LIF, 8.9 and 4.8 for IL-11, 6.9 and 3.7 for IL-4, and 5.7 and 3.4 for IL-6. The total protein concentration was measured by colorimetric method (Coomassie Plus-200 protein assay; Pierce Biotechnology Inc., Rockford, IL).
All Th2 cytokines from endometrial biopsies and IL-11 and IL-6 in the uterine flushings were quantitatively measured. To account for the differences in weight and the inconsistencies in protein extraction between endometrial specimens, the resultant final cytokine concentrations were standardized against total protein concentration measured from the sample. Protein extraction was done by homogenizing the biopsy in PBS with Protease Inhibitor Cocktail Set III (1:100; Calbiochem, Bad Soden, Germany).
Statistical analysis
Natural cycles were classified as group A, whereas stimulated cycles with peak serum E2 of no more than 20,000 pmol/liter (moderate responders) and peak serum E2 of more than 20,000 pmol/liter (excessive responders) were classified as group B and group C, respectively. The primary outcome measures were LIF, IL-11, IL-6, and IL-4 expression in endometrial biopsies and concentrations of IL-11 and IL-6 in uterine flushings. Cytokine levels by ELISA measurements were standardized against total protein concentrations of the sample and were expressed in picograms per milligram protein. Continuous variables are given as median (range), unless otherwise indicated. Statistical analysis was performed using Statistical Program for Social Sciences (SPSS Inc., Chicago, IL). Comparison was done using Kruskal-Wallis, Mann-Whitney U, and 
2 tests, where appropriate. The effect of serum E2 concentration on the expression of IL was examined by a linear regression model. Two-tailed P < 0.05 was considered statistically significant.
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Results |
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). The number of women with polycystic disease was similar in groups B and C. Likewise, the duration and dosage of hMG used were similar in group B and group C. Significantly higher serum E2 concentrations on the day of hCG were found in group C and group B compared with group A. Endometrial thickness was significantly higher in group B and group C than in group A.
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By IHC.
Figure 1 shows the representative staining of Th2 cytokines in endometrial biopsies from the three groups and a negative control. Table 2 summarizes the expression of Th2 cytokines in endometrial biopsies by IHC. Luminal epithelium was not observed in these sections from all biopsies. The expression of IL-11 in the stromal compartment was significantly lower in group C than in group A and group B. The glandular IL-11 expression was also lower in group C than in group A and group B, but the difference failed to reach statistical significance.
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By ELISA.
IL-11 concentration in group C was significantly lower by about 3-fold compared with that in group A and group B (P < 0.05), and no difference was noted between groups A and B (Table 3 and Fig. 2). IL-6 concentration was also lower in group C than in groups A and B, but the difference was not statistically significant (P > 0.05). The concentration of IL-4 was lower in group B compared with that in group A, whereas LIF was similar among the three groups. The protein concentrations of IL-11 and IL-6 were negatively affected by increasing E2 concentrations (ß = 0.355, P = 0.012 for IL-11; and ß = 0.354, P = 0.02 for IL-6).
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Only IL-11 and IL-6 were measured in the uterine flushings because there were no differences in LIF and IL-4 expression in the endometrial biopsies among the three groups. Group C had a significantly higher percentage of women having undetectable IL-11 in their uterine flushings and a lower IL-11 concentration than group A (Table 4). Increasing serum E2 had a negative effect on IL-11 concentrations (ß = 0.305, P = 0.005). No significant differences in the percentage of undetectable IL-6 and IL-6 concentration were noted among the three groups.
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Discussion |
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Our data demonstrated that IL-11 expression in endometrial biopsies and uterine flushings was negatively affected by serum E2 concentration. IL-11 is essential for murine endometrial function and embryo implantation. Female mice with either an inactive or null mutation for IL-11R
chain are fertile, and their blastocysts implant. However, uterine decidualization reaction is defective, leading to the formation of small decidua, which subsequently degrades and leads to embryo death (16, 17). In humans, IL-11 is important because IL-11 mRNA concentration, and not its receptor (as in mice), was higher in the decidua and chorionic villi in normal compared with anembryonic pregnancies (18). Moreover, plasma IL-11 concentrations were significantly decreased in women with spontaneous miscarriages compared with normal pregnancies (19). IL-11 plays a role in endometrial stromal cell decidualization (20) by enhancing cell survival and protecting against cell damage during embryo implantation and trophoblastic invasion (21).
Both ELISA- and IHC-determined IL-11 expression was decreased in excessive responders compared with moderate responders and natural cycles. This finding is consistent with our previous morphometric analysis of the periimplantation endometrium (5). Excessive responders had a significantly higher number of subnuclear vacuoles but a lower percentage of moderate to abundant filling of endometrial glands than moderate responders. In terms of uterine flushings, significantly more excessive responders had undetectable or lower IL-11 concentration. Previous studies had undetectable IL-11 in the plasma of nonpregnant (19) women and in the follicular fluid of women having IVF (22). However, it is more appropriate to measure cytokines in uterine flushings. Cytokines act in paracrine and/or autocrine fashion; hence, the measurement of their concentration at the site of production and action accurately reflects their physiological concentration rather than measurement from the circulation, which applies more to classical hormones. The undetectable IL-11 in uterine flushings may be a result of the very minute amount of locally secreted cytokines present in the flushings, which may not be easily detectable (23, 24).
IL-6 shares the glycoprotein gp130 signal transduction accessory signal protein with other members of the IL-6 family of cytokines including IL-11 and LIF. Recent findings support the role of IL-6 in the establishment of pregnancy because endometrial mRNA is suppressed in the midsecretory phase of patients with recurrent abortions (10, 25). Moreover, impaired embryo implantation in patients with tubal infertility was suggested to result from the lack of endometrial IL-6 (26). Animal experiments show the crucial role of IL-1 in implantation and IL-6, with the greatest bioactivity on d 5 and 6 of pregnancy in mice, which is a mediator of many actions of IL-1 at the endometrial-trophoblast interphase (27). In the human endometrium, IL-6 and LIF protein and mRNA are expressed at maximum concentrations in the mid- to late secretory phases (28, 29) and more in the epithelial cells than in the stromal cells, coinciding with the implantation window. Additionally, human trophoblasts express IL-6 receptor and produce IL-6, which induces the production of hCG in an autocrine manner, suggesting a role of IL-6 in early implantation and its continuation in early pregnancy.
Our data demonstrated that increasing E2 concentration had a negative effect on IL-6 expression in endometrial biopsies. The finding that glandular IL-6 expression was significantly reduced in biopsies from excessive responders is consistent with our previous report of a lower percentage of endometrial glands filling in excessive responders compared with moderate responders (5). ELISA-measured IL-6 concentration was similar among the three groups, but was negatively affected by E2.
A higher percentage of women had undetectable IL-6 and a lower IL-6 concentration in stimulated cycle uterine flushing compared with those with natural cycles. The lack of statistical significance may be due to wide variability and the small number of patients with detectable IL-6 concentration. Although all samples used in this study were from the midsecretory phase, when IL-6 protein concentration in uterine secretions was high, the reason for undetectable IL-6 in the majority of these samples was not known. The volume (5 ml) used to flush the uterine cavity may be too large, and perhaps concentrating the cytokines in a smaller volume would reduce the percentage of undetectable IL-6.
This study demonstrated similar LIF expression in endometrial biopsies by IHC and ELISA among the three groups. LIF is crucial for implantation in mice (30). In the human endometrium, LIF protein and mRNA are expressed maximally in the glandular epithelium during the luteal phase of the cycle. This spatiotemporal expression of LIF in the endometrium suggests a potential role of LIF in implantation and early pregnancy. However, varying results were found when fertile women were compared with women with unexplained infertility. Higher LIF concentrations were present in fertile women in uterine flushings (31), endometrial biopsy (32), and cultured explant tissues (33, 34). On the other hand, no differences were found in endometrial biopsy from the periimplantation period (29) and in the irrigated fluid of the uterine cavity (35). During IVF treatment, increased LIF and TNF- concentrations were pointed to as the negative prognostic factors for subsequent pregnancy (23), whereas no difference was found in LIF concentrations in uterine flushings at the time of egg retrieval between pregnant and nonpregnant women (24). The conflicting results from these studies may result from different timings in the measurement of LIF expression and the heterogeneous nature of the samples used.
Although LIF plays a strong role in mammalian implantation, LIF expression was not different in moderate and excessive responders. The LIF production in vivo and LIF action during embryo implantation may not be under the direct control of steroid hormones. In an endometrial cell culture model, there is a lack of direct stimulatory effect of E2 and progesterone on LIF mRNA expression or protein production (31, 36). Moreover, cytokines may directly control in vivo LIF production, because LIF in vitro was affected by other cytokines (36). Furthermore, LIF expression is inconsistent and has a larger variation in infertile women compared with fertile women (34) and hence may not add much credence to the Th2 cytokines determined in this study in a subset of infertile women.
The expression of IL-4 in endometrial biopsies by IHC and ELISA was comparable among women in natural cycles, moderate responders, and excessive responders. IL-4 is present in the periimplantation human endometrium (12), and its production is less in women with unexplained recurrent abortions (14). IL-4 favors normal pregnancy by the generation and maintenance of Th2-type (vs. Th1-type) responses by inducing the immune deviation shift to Th2 (from Th0) (37). Anembryonic pregnancies had fewer IL-4-secreting cells resulting in a higher Th1/Th2 ratio compared with normal pregnancies (15). IL-4 inhibits Th1 cell proliferation and Th1 cytokine production (38) and opposes their effect on natural killer cells (39). Because IL-4 enhances the production of other cytokines (40), it may be possible that IL-4 is controlled by other cytokines, and steroid hormones may not directly affect IL-4 secretion.
In conclusion, a high serum E2 concentration had a negative effect on endometrial IL-11 and IL-6 expression. Reduced IL-11 and IL-6 expression in the periimplantation endometrium may account for the lower implantation and pregnancy rates in the excessive responders compared with those in natural cycles and moderate responders. The effect of high E2 on IL-6 and IL-11 may result either from direct action or through other upstream molecules, e.g. IL-1. In excessive ovarian responders, reduced endometrial IL-6 can affect angiogenesis through altered vascular endothelial growth factor expression, which may subsequently influence the endometrial blood flow (unpublished data from our center). The results of the present study encourage additional research to explore the mechanisms by which high serum E2 concentration suppresses cytokines that are vital for successful implantation.
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Footnotes |
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First Published Online May 16, 2006
Abbreviations: E2, Estradiol; ET, embryo transfer; hCG, human chorionic gonadotropin; hMG, human menopausal gonadotropin; IHC, immunohistochemistry; IVF, in vitro fertilization; LIF, leukemia inhibitory factor; Th, T helper.
Received January 26, 2006.
Accepted May 4, 2006.
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References |
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function is required for normal decidua and fetoplacental development in mice. Genes Dev 12:2234–2243, ß, and 
. Cell Immunol 170:71–77[CrossRef][Medline] on cytokine production by endometrial stromal cells. Hum Reprod 13:2598–2601This article has been cited by other articles:
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