Expression of leukemia inhibitory factor and its receptor is not altered in the decidua and chorionic villi of human anembryonic pregnancy

Hsin-Fu Chen1, Kuang-Han Chao1, Jin-Yuh Shew2, Yu-Shih Yang1 and Hong-Nerng Ho1,3

1 Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, College of Medicine and the Hospital, National Taiwan University, 2 Department of Biochemistry, College of Medicine, National Taiwan University, 3 Graduate Institute of Immunology, College of Medicine, National Taiwan University

Corresponding author addressed at: National Taiwan University Hospital, Department of Obstetrics and Gynecology, No.7 Chung-Shan S. Road, Taipei 100, Taiwan. Tel: 886 2 23123456 extension 5161; Fax: 886 2 23418557; e-mail: hnho{at}ha.mc.ntu.edu.tw


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Uterine expression of leukemia inhibitory factor (LIF) is absolutely essential for mouse, and critical for human, embryo implantation. However LIF is not required for post-implantation development of mouse embryo. The objective of this study was to examine the role of LIF system in post-implantation stage of human pregnancy. METHODS: Tissues from 25 patients with anembryonic pregnancy (AP; blighted ovum) and 25 matched patients with normal pregnancy (NP) were collected. LIF and its receptor {beta} (LIF-R{beta}) expression in the decidua and chorionic villi were analyzed by semi-quantitative reverse transcription and polymerase chain reaction (RT–PCR), real-time quantitative PCR and immunohistochemical study. RESULTS: LIF mRNA levels were not different either between different tissues (decidua vs chorionic villi) or between different patients (NP vs AP). LIF-R{beta} mRNA levels were significantly higher in chorionic villi than in decidua but were not different between NP and AP. Immunohistochemical staining supported these findings and showed a predominate expression of LIF-R{beta} in the trophoblast cells. CONCLUSIONS: This study concluded that at early human post-implantation stage, LIF is produced from both decidua and chorionic villi and may exert its major action on trophoblasts. A baseline expression of LIF and LIF-R{beta} is probably needed for early pregnancy, but AP cannot be accounted for by the defective expression of either LIF or LIF-R{beta} in most circumstances.

Key words: anembryonic pregnancy/blighted ovum/chorionic villi/decidua/leukemia inhibitory factor


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Anembryonic pregnancy (AP; blighted ovum in a clinical term) is a frequent presentation of first-trimester abortion and it accounts for ~15% of all spontaneous pregnancies. Excluding those with chromosome abnormality, there remains a high percentage of abortion occurring without discernible mechanisms. Recently defective expression of some growth factors and/or cytokines is suggested to be attributable to these reproductive failures. These include colony-stimulating factor (CSF), leukemia inhibitory factor (LIF) and interleukin-11 (IL-11) (Bhatt et al., 1991Go; Pollard et al., 1991Go; Stewart et al., 1992Go; Bilinski et al., 1998Go; Robb et al., 1998Go). LIF and IL-11 are related cytokines but exert different reproductive functions. They however share a common signal transduction component, gp130, in the formation of high-affinity receptor complexes (Yin et al., 1993Go). Recently, we reported that there is a defective production of IL-11 by decidua and chorionic villi in human anembryonic pregnancy (Chen et al., 2002aGo), indicating a significant role of IL-11 in maintaining early pregnancy after embryo implantation.

It was found that expression of LIF in the endometrium is absolutely essential for mouse embryo to implant (Bhatt et al., 1991Go; Stewart et al., 1992Go). Subsequent studies identified the peak expression of LIF and its receptor (LIF-R) in the mid-luteal phase of human endometrium. Therefore it is suggested that LIF plays a role in regulating human embryo development at the peri-implantation stage (Cullinan et al., 1996Go). In addition, LIF probably also exerts some function before embryo implantation, since LIF and LIF-R transcripts were detectable in early mouse and human embryos (Chen et al., 1999Go). However the role of LIF in the fetal–maternal interface after embryos have implanted has not been clarified. The expression of LIF and LIF-R genes has been detected in the decidua and chorionic villi of first-trimester and term placentas (Kojima et al., 1994Go; Kojima et al., 1995Go; Sawai et al., 1995a,b,Go 1997; Sharkey et al., 1999Go), and LIF regulates the growth and differentiation of trophoblasts (Kojima et al., 1995Go; Ren et al., 1997Go). These reports suggest that LIF may constitute one mechanism for local control of trophoblast and endometrial proliferation and therefore is likely associated with the continuation of pregnancy. However, an animal study has basically excluded the role of LIF in the maintenance of pregnancy in mice (Chen et al., 2000Go). These discrepant results apparently needs further clarification.

In addition, decidual NK cells and T cells produce a variety of cytokines including LIF (Jokhi et al., 1994Go), and the production of LIF and type 2 T-helper cytokines are both defective by decidual T cell clones in unexplained recurrent abortions (Piccinni et al., 1998Go). However, the clinical significance of LIF involvement in the evolution and formation of anembryonic pregnancy (and thus abortion) has not been established. Therefore, the present study was designed to examine the expression of LIF and its receptor in early human pregnancy, in order that the role of LIF system in the formation of anembryonic pregnancy might be clarified. To test this, we examined and compared the LIF and LIF-R gene expression and protein products in normal pregnancy (NP) and AP.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects and materials
In total, 25 patients with sonographically confirmed AP (at 7–10 weeks of gestation) were recruited into the study group (AP group). Twenty-five age-matched patients with NP (viable pregnancies at 7–10 weeks of gestation, which were artificially terminated due to multi-parity) were recruited as control group (NP group). The gestational age (mean ± SEM, 8.5 ± 0.4 vs 8.0 ± 0.5 weeks for AP and NP, respectively) and maternal age (34.5 ± 2.1 vs 32.4 ± 1.9 years) were comparable between study and control groups. For the confirmation of pregnancy status, the presence or absence of fetal heart motion was documented by either trans-abdominal or trans-vaginal ultrasound. Samples of decidua and chorionic villi were obtained during dilatation and curettage. Informed consent was obtained from each patient who participated in this study. This study was approved by the Ethical Committee of the National Taiwan University Hospital in 1998.

Tissue processing
The gestational tissues (decidua and chorionic villi) were collected aseptically, put into test tubes containing normal saline, and immediately taken to the laboratory for processing. The preparation of decidual and villus tissues was performed according to the previous report (Chen et al., 2002aGo). Briefly, tissue mixtures were grossly separated into decidua and chorionic villi, washed twice with Hank’s balanced salt solution (HBSS), and divided into portions for further RNA extraction or formalin fixation. Small pieces of each sample from each patient were also stained with hematoxylin and eosin (H&E) and examined by a pathologist to exclude any pathology.

Semi-quantitation of LIF and LIF-R{beta} mRNA in decidua and chorionic villi by standard RT–PCR
The RNA from decidua or chorionic villi was obtained using the protocol reported previously (Chadderton et al., 1997Go; Chen et al., 2002aGo). Briefly, tissue samples were cut into pieces, washed with PBS (diethyl pyrocarbonate-treated), and then homogenized in liquid nitrogen. Subsequently, RNA was extracted using the Trizol reagent (Life Technologies, Inc., Grand Island, NY), chloroform–isopropanol–ethanol method (Chadderton et al., 1997Go; Chen et al., 2002aGo). The purity and concentration of RNA were analyzed first by UV spectrophotometer at absorbance wavelengths of 260 nm and 280 nm (OD260 and OD280). An OD260/OD280 ratio of higher than 1.6 was considered adequate. Furthermore the integrity of the RNA sample was verified both by agarose gel electrophoresis and examination of the RT–PCR product of S26, a housekeeping gene (Vincent et al., 1993Go). Five micrograms of RNA samples were subsequently subjected to first strand cDNA synthesis using oligo(dT) primers and Superscript II reverse transcriptase following the manufacturer’s manual (First Strand cDNA Synthesis Kit, Pharmacia Biotech). The resultant cDNA template was further subjected to PCR amplification with oligonucleotide primers designed according to the published human LIF and LIF-R{beta} cDNA sequences (Stahl et al., 1990Go; Gearing et al., 1991Go). For LIF mRNA amplification, the following primers were used: sense, 5'-CAGCATCACTGAATCACAGAGC-3'; and antisense, 5'-AGTATG AAACATCCCCACAGGG-3'. The size of the PCR product was 560 base pairs (bp). For LIF-R{beta} mRNA amplification, the following primers were used: sense, 5'-CAAAAGAGTGTGTCTGTGAG-3'; and antisense, 5'-CCATGTATTTACATTGGC-3'. The predicted size of the product was 459 bp. For the purpose of semi-quantitation, the S26 gene transcript was also co-amplified in the same reaction to control for the mRNA amount in the same sample. The primer set used was designed according to the published cDNA sequence (Vincent et al., 1993Go), and were: sense, 5'-CCGTGCCTCCAAGAT GACAAAG-3'; and antisense, 5'-ACTCAGCTCCTTACATG GGCTT-3'. The predicted size of the PCR product was 371 bp. All the above primer sets were designed to span at least two exons, in order to exclude the possibility of amplifying contaminating genomic DNA.

In a 50 µl reaction mixture, PCR was performed in the presence of 25 µl Taq Master Mix (QIAGEN, Valencia, USA), 3 µl first strand cDNA template, 200 ng/µl 5'-primer, 200 ng/µl 3'-primer, and 20 µl d.d.H2O. A number of preliminary experiments were performed to determine the PCR conditions and the cycle numbers that ensured the PCR production in an exponential phase. The PCR was carried out in a thermal cycler (GeneAmp PCR System 9700; PERKIN-ELMER, Norwalk, CT) in the following sequences: denaturation at 94°C for 30–60 s, annealing at 55–65°C for 60–90 s, extension at 72°C for 30 s, and a final extension at 72°C for 15–30 min. The PCR was repeated for 28–38 cycles according to the gene of interest and the product was examined using 1.5% agarose gel electrophoresis and stained with SYBR green stain (SYBR Green I; Roche Molecular Biochemicals, Indianapolis, IN). The signal intensity of each product was measured by scanning the SYBR green luminescence using a phosphoimage scanner (Storm 840; Molecular Dynamics, Sunnyvale, CA). A reverse transcription reaction was carried out without the addition of reverse transcriptase, and the resulting product was subjected to PCR to exclude the possibility of genomic DNA contamination. PCR was also performed without the presence of template DNA to test for cross-contamination of samples. All the samples were examined in duplicate by two researchers, and the data were compared. It was found that the data obtained by the two researchers were in most instances comparable.

In each sample, the relative mRNA levels of LIF and LIF-R{beta} were corrected by the mRNA amount, as reflected by the S26 level, using the following formulae: LIF/S26 and LIF-R{beta}/S26, to represent LIF mRNA and LIF-R{beta} mRNA levels, respectively.

Real-time quantitative PCR
Due to the inherently limited sensitivity of the semi-quantiation used above, we further conducted a quantitative PCR to measure these gene expressions. A real-time quantitative PCR system (ABI PRISM 7700 Sequence Detection System; PE Applied Biosystems) was used as previously described (Gibson et al., 1996Go; Chen et al., 2002aGo). Briefly, RNA was prepared and subsequently cDNA was obtained by random hexamer priming. The primers and probes used in the PCR were designed according to the TaqMan primer and probe design system (PE Applied Biosystems) (Table I). Based on the manufacturer’s protocol, the FAM (6-carboxyfluorescein) and VIC were used as the reporter dyes and TAMRA (6-carboxy-tetramethyl-rhodamine) as the quencher dye. The probes were labeled with both reporter dye and quencher dye on the 5'- and 3'-ends, respectively. During PCR, the reporter dye was released and the resultant fluorescence was detected and could be quantified. Before examinations on the study samples, a number of preliminary experiments were done to determine the PCR conditions. The PCR was carried out in a thermal cycler (ABI PRISM 7700 Sequence Detection System) in the following sequence: reaction at 50°C for 2 min, at 95°C for 10 min, and subsequently the PCR was repeated for 45 cycles at denaturation at 95°C for 15 s and annealing and extension at 60°C for 1 min. The authenticity of PCR products was verified by 2% agarose gel electrophoresis and by direct sequencing. Each sample was examined in triplicate and a mean value was obtained. The relative concentration of each mRNA was subsequently calculated according to the Manufacturer’s User Manual. Briefly, the threshold cycle (CT) values of the target gene (LIF or LIF-R{beta}) and the internal control gene ({beta}-actin) mRNA in the studied sample were first measured. The {Delta}CT value of the studied sample was calculated by the following formula: {Delta}CT = CT of target gene – CT of {beta}-actin (this {Delta}CT is designated as ‘sample {Delta}CT’). Similarly, the CT values of the target gene and its respective {beta}-actin of a positive control were also obtained and the {Delta}CT was calculated (designated as ‘calibrator {Delta}CT’). {Delta}{Delta}CT was then calculated using the following formula: {Delta}{Delta}CT = {Delta}CT (sample) – {Delta}CT (calibrator) and finally the relative value of each mRNA was calculated by the formula: 2{Delta}{Delta}CT. PCR without template was used as a negative control (called no template control) to verify experimental results.


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Table I. Sequences of TaqMan primers and probes
 
Immunohistochemistry
The immunohistochemistry protocol was modified from previous reports (Hsu et al., 1981Go; Lessey et al., 1994aGo,b; Chen et al., 2002aGo) and a Streptavidin/Biotin Universal Detection System (IMMUNOTECH, Marseille, France) was used. Briefly, formalin-fixed, paraffin-embedded tissue sections were deparaffinized with xylene, and hydrated with step-down concentrations of ethanol. The sections were then incubated with 30% H2O2/70% methanol solution at RT for 5 min, treated with protein blocking agent at RT for 5 min to reduce non-specific binding of antibodies, and incubated overnight at 4°C with primary antibodies (for LIF detection, goat anti-human LIF antibody, 1:25 dilution; R&D Systems Inc., Minneapolis, MN, USA; for anti-LIF-R detection, goat anti-human LIFR antibody, 1:40–80 dilution; R&D Systems). On the next day, the sections were washed with PBS, treated with polyvalent biotinylated secondary antibody, which bound to primary antibody at RT for 10 min, and then treated with streptavidin–peroxidase reagent, which bound to the secondary antibody, at RT for 10 min. The colour was developed using DAB chromogen at RT for 15 min and counterstained with hematoxylin for 1 min. An independent pathologist who was blind to the origins of the samples reviewed these stained slides and graded their staining intensity. Experiments without primary or secondary antibodies were used as negative controls.

Statistical analysis
Since a nonparametric testing was a more appropriate statistical method due to the smaller case number, the Wilcoxon Rank-Sum Test (the Mann–Whitney U test) was used to analyze the data. A P-value of <0.05 was considered statistically significant.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
The expression of LIF and LIF-R{beta} genes in decidua and chorionic villi
PCR revealed that all the samples expressed LIF and LIF-R{beta} mRNA. These included decidua and chorionic villi either from NP or AP (Figure 1). In addition, S26, a housekeeping gene, was detectable in all samples (Figure 1). Direct DNA sequencing for the PCR products was done and it was found that their sequences were identical to the published human LIF and LIF-R{beta} cDNA sequences, respectively (data not shown).



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Figure 1. Expression of LIF (560 bp), LIF-R{beta} (459 bp) and S26 (371 bp) mRNA in the decidua and chorionic villi of normal and anembryonic pregnancy by standard RT–PCR using the primer sets described in Materials and methods. The PCR product was resolved in 1.5% agarose gel electrophoresis and stained with SYBR green stain. S26 was used as an internal control. M, DNA marker; NP, normal pregnancy; AP, anembryonic pregnancy.

 
Semi-quantitation of LIF and LIF-R{beta} mRNA
Representative photographs of the semi-quantitation of the LIF and LIF-R{beta} mRNA are shown (Figure 2). Due to the lack of sufficient quantities of samples for study in some subjects, there were in total 24 samples in the NP group and 23 samples in the AP group available for analysis. For LIF mRNA, the relative transcript levels were 2.3 ± 0.5 (mean ± SEM) and 2.5 ± 0.3, respectively, in the decidua and chorionic villi of NP (Figure 3A). The levels were 3.1 ± 0.9 and 3.5 ± 1.0, respectively, in AP (Figure 3A). For LIF-R{beta} mRNA, the relative transcript levels were 0.7 ± 0.1 and 3.8 ± 0.8, respectively, in the decidua and chorionic villi of NP (Figure 3B). The levels were 1.2 ± 0.2 and 3.1 ± 0.6, respectively, in AP (Figure 3B). These data indicate that the LIF-R{beta} mRNA level was higher in chorionic villi compared to that in decidua, in either NP (0.7 ± 0.1 vs 3.8 ± 0.8, respectively, for decidua and villi, P = 0.002) or AP (1.2 ± 0.2 vs 3.1 ± 0.6, P = 0.008). However, the levels of LIF mRNA were not different within the same group (NP, 2.3 ± 0.5 vs 2.5 ± 0.3, P > 0.05 respectively in decidua and; AP, 3.1 ± 0.9 vs 3.5 ± 1.0, in villi P > 0.05) (Figure 3A). The comparison between different groups (NP vs AP) neither showed any difference of LIF mRNA levels, when either decidua (2.3 ± 0.5 vs 3.1 ± 0.9, P > 0.05) or chorionic villi (2.5 ± 0.3 vs 3.5 ± 1.0, P > 0.05) were concerned (Figure 3A). Similarly, the LIF-R{beta} mRNA levels were not different between groups (NP vs AP) in either decidua (0.7 ± 0.1 vs 1.2 ± 0.2, P = 0.18) or chorionic villi (3.8 ± 0.8 vs 3.1 ± 0.6, P = 0.50) (Figure 3B).



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Figure 2. Representative photographs of semi-quantitation of LIF (560 bp) and LIF-R{beta} (459 bp) mRNA levels in the decidua and chorionic villi. S26 (371 bp) was co-amplified as an internal control. Samples were subjected to RT–PCR using the primer sets described in the Materials and methods. Different cycle numbers were done (e.g. 26 and 28 cycles in these images) for the purpose of semi-quantitation. The relative levels of each mRNA of interest (LIF and LIF-R{beta}) were quantified by the formulae: LIF/S26 and LIF-R{beta}/S26, respectively. (A) LIF and S26 co-amplification; (B) LIF-R{beta} and S26 co-amplification; M, DNA marker.

 


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Figure 3. Comparison of LIF and LIF-R{beta} mRNA levels in the decidua and chorionic villi by semi-quantitative RT–PCR, using S26 as an internal control. Longitudinal axis indicates the relative levels of LIF (A) and LIF-R{beta} (B) mRNA, which were calculated by the formulae: LIF/S26 and LIF-R{beta}/S26, respectively. NP, normal pregnancy; AP, anembryonic pregnancy. *P = 0.002 (comparison between decidua and chorionic villi of NP for LIF-R{beta}). **P = 0.008 (comparison between decidua and chorionic villi of AP for LIF-R{beta}). The bars represent the mean ± SEM.

 
Quantitation of LIF and LIF-R{beta} mRNA by real-time quantitative PCR
By real-time quantitative PCR, it was found that the relative levels of LIF mRNA were 162.1 ± 59.5 (mean ± SEM) and 226.4 ± 105.5, respectively, in the decidua and chorionic villi of NP (n = 21) (Figure 4A). The levels were 926.3 ± 487.0 and 401.8 ± 210.7, respectively, in AP (n = 22) (Figure 4A). For LIF-R{beta}, the relative transcript levels were 15.1 ± 14.0 and 7333.2 ± 3198.0, respectively, in the decidua and chorionic villi of NP (Figure 4B). The levels were 0.3 ± 0.2 and 1053.4 ± 535.3, respectively, in AP (Figure 4B).



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Figure 4. Comparison of LIF and LIF-R{beta} mRNA levels in the decidua and chorionic villi by real-time quantitative PCR, using {beta}–actin as an internal control. Longitudinal axis indicates the relative values of LIF (A) and LIF-R{beta} (B) mRNA, which were calculated by the formula: 2{Delta}{Delta}CT, as described in the Materials and methods. NP, normal pregnancy; AP, anembryonic pregnancy. *P = 0.006 (comparison between decidua and chorionic villi of NP for LIF-R{beta}). **P = 0.03 (comparison between decidua and chorionic villi of AP for LIF-R{beta}). The bars represent the mean ± SEM.

 
These data indicate again that the levels of LIF mRNA were not different within the same group (NP, 162.1 ± 59.5 vs 226.4 ± 105.5, P = 0.60, respectively in decidua; AP, 926.3 ± 487.0 vs 401.8 ± 210.7, P = 0.35 in villi). The comparison between different groups (NP vs AP) neither showed any difference of LIF mRNA levels, when either decidua (162.1 ± 59.5 vs 926.3 ± 487.0, P = 0.13) or chorionic villi (226.4 ± 105.5 vs 401.8 ± 210.7, P = 0.44) were concerned (Figure 4A). Similar to the trend found by semi-quantitation in the previous section, the LIF-R{beta} mRNA level was statistically lower in decidua than that in chorionic villi, in either NP (15.1 ± 14.0 vs 7333.2 ± 3198.0, P = 0.006) or AP (0.3 ± 0.2 vs 1053.4 ± 535.3, P = 0.03) (Figure 4B). However, comparison between different groups of patients did not show difference in LIF-R{beta} mRNA levels in decidua (15.1 ± 14.0 vs 0.3 ± 0.2, respectively in NP and AP, P = 0.20) (Figure 4B). Though there was a trend towards lower level of LIF-R{beta} mRNA in chorionic villi of AP (Figure 4B), the difference did not reach statistical significance (7333.2 ± 3198.0 vs 1053.4 ± 535.3, P = 0.07) (Figure 4B).

Immunohistochemical study for LIF and LIF-R{beta} proteins in decidua and chorionic villi
Immunohistochemical study identified that the immunoreactive LIF was found in both decidua and chorionic villi of both NP and AP (Figure 5A–D). In decidua, the LIF staining was detectable in both the stroma cells and the luminal and glandular epithelium but the vascular endothelium did not show significant immunoreactivity (Figure 5A and C). In chorionic villi, the LIF protein was mostly localized to the trophoblasts but the stroma cells and endothelium also showed faint staining (Figure 5B and D). It was not microscopically possible to identify any difference in LIF staining intensity between NP and AP, in either decidua or chorionic villi (Figure 5A–D) (Table II).



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Figure 5. Representative photomicrographs of immunohistochemical study of LIF and LIF-R{beta} in the decidua and chorionic villi from normal pregnancy (NP) and anembryonic pregnancy (AP). Formalin-fixed, paraffin-embedded tissue samples were treated with either anti-LIF antibody or anti-LIF-R antibody, respectively, using the Streptavidin–Biotin Universal Detection System (Immunotech) as described in the Materials and methods. The color was developed using DAB chromogen and counterstained by hematoxylin. (AD) LIF examination. (EH) LIF-R{beta} examination. (IJ) Representative negative controls. A, C, E, G and I were decidua tissues. B, D, F, H and J were chorionic villi tissues. A, B, E and F were from NP. C, D, G and H were from AP. Brown color indicates positive staining (arrow). Magnification, x200. GL, gland; ST, stroma; TR, trophoblast; EN, vascular endothelium. Bar = 50 µm.

 

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Table II. Results of immunohistochemical study (LIF and LIF-R{beta} proteins)
 
LIF-R{beta} protein was also detectable in the decidua and chorionic villi of both NP and AP (Figure 5E–H). However, in striking contrast to that of LIF protein, the LIF-R{beta} staining was much less intense in decidua compared to that in chorionic villi (Figure 5E–H). In the chorionic villi, the intense staining was mostly localized to the trophoblasts. However, when tissues from NP and AP were compared, the staining intensity was comparable in most instances, when either decidua (Figure 5E and G) or chorionic villi (Figure 5F and H) was concerned (Table II). As expected, the negative controls (Figure 5I and J) did not show any immunoreactive staining.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
LIF is transiently expressed in the glandular epithelium of mice at ovulation and again on the fourth day of pregnancy, when the embryo is prepared to implant (Bhatt et al., 1991Go; Stewart et al., 1992Go). LIF-deficient female mice (LIF–/–) are fertile, but their blastocysts cannot implant unless they are treated with LIF (Stewart et al., 1992Go). In the human, LIF expression in the endometrium is up-regulated at the peri-implantation stage, suggesting that LIF may also be important for the human embryo to implant (Charnock-Jones et al., 1994Go; Cullinan et al., 1996Go). However, it has not yet been established whether continuous expression of LIF during the entire pregnancy is required for embryonic development. It is neither clear whether AP has a causal relationship with the defective production of LIF-related genes. The present study identified the expression of LIF and LIF-R{beta} genes in human decidua and chorionic villi of early gestation. However, LIF mRNA levels were not different either in different tissues (decidua vs chorionic villi) or between different groups of patients (NP vs AP). In contrast, a differential spatial distribution of LIF-R{beta} mRNA level was found, showing a significantly lower level in decidua than in chorionic villi. One of our previous studies (Wu et al., 2001Go) also demonstrated that serum levels of LIF neither changed before and after embryo implantation nor were related to the occurrence of abortion in an in vitro fertilization (IVF) program. All these data suggest a role but do not define the function of LIF system genes in post-implantation stage. While the major target of LIF is likely to be on the trophoblasts, the absence of differences in LIF or LIF-R{beta} levels between NP and AP suggests that in human, AP cannot be accounted for by the defective expression of either LIF or LIF-R{beta} in the maternal–fetal interface in most circumstances.

Discrepant results have been reported in a mouse model, which suggested that after implantation, LIF is neither required by the mouse embryo for further development nor for the maintenance of pregnancy (Chen et al., 2000Go). That specific study (Chen et al., 2000Go) basically excludes the active role of LIF in the continuation of pregnancy. However, in view of the significant expression and possible function of LIF-related genes in the placenta and decidua during pregnancy reported in this study and others (Jokhi et al., 1994Go; Kojima et al., 1994Go; Arici et al., 1995Go; King et al., 1995Go; Sawai et al., 1997Go; Sharkey et al., 1999Go; Modric et al., 2000Go), it remains premature to conclude that LIF action is totally unnecessary after implantation in other species. For example, greatest steady-state LIF mRNA levels were evident in the porcine placenta at post-implantation stages, suggesting its important function (Modric et al., 2000Go). In human, LIF-R mRNA was localized in the villous and extravillous trophoblast throughout pregnancy and strong expression of LIF mRNA was detected in decidual leukocytes, especially at the implantation sites, suggesting that LIF may mediate interactions between maternal decidual leukocytes and invading trophoblast (Sharkey et al., 1999Go). Piccinni and coauthors showed that progesterone-mediated production of both IL-4 and LIF might contribute to the development and maintenance of successful human pregnancy (Piccinni et al., 1998Go). Therefore, in the consideration of the discrepant results from different studies, species-related factor should be one to be explored. Actually there are good evidences that LIF action differs in different species. For example, LIF alone can support the self-renewal without differentiation of a mouse embryonic stem cell line (which is derived from pre-implantation embryo) in a feeder-free culture condition. In contrast, feeder cells are generally indispensable for a human embryonic stem cell line to proliferate without differentiation (Reubinoff et al., 2000Go). As yet we have not encountered a case with extreme down-regulation or alteration of either LIF or LIF-R. Therefore we cannot predict what will happen if baseline levels of LIF and LIF-R are not expressed in early human gestation. However, according to the present study and those previous reports (Jokhi et al., 1994Go; Kojima et al., 1994Go; Arici et al., 1995Go; Sawai et al., 1997Go; Piccinni et al., 1998Go; Sharkey et al., 1999Go; Chen et al., 2002bGo), it is probably reasonable to suggest that a baseline expression of LIF and LIF-R{beta} is important for the continuation of human pregnancy. Further study will be necessary to identify the implication of the difference of LIF action on human and mouse.

A number of known or unknown factors including certain cytokines may prove to be the mechanism leading to human AP or abortion. However, we did not find a significant difference in LIF or LIF-R{beta} expression between NP and AP in this study. It is reasoned that though LIF action may play an active role in the maintenance of human pregnancy, the occurrence of AP is not usually associated with a defective production of LIF or LIF-R{beta}. As far as our knowledge is concerned, this is one of the rare human reports regarding the investigation of LIF role in the evolution of AP or abortion. However, the results from this study are not completely agreeable with those of a previous human study which showed a decreased production of LIF by decidual T cell clones from women with unexplained recurrent abortion (Piccinni et al., 1998Go). Several mechanisms probably can explain these contradictory findings. The first is the difference in patient populations (first trimester abortion in this study vs unexplained recurrent abortion in the Piccinni study). We cannot exclude the possibility that heterogeneity of the patient population in first trimester abortion will compromise the analysis. However it is not always practical to identify a pure population of unexplained recurrent aborters, especially when the subject number needs to be beyond a threshold level for analysis. Secondly, the study by Piccinni et al. examined the in vitro function using clones of T cells. Their approach was different from that of the present study, which directly used an in vivo sample (decidua and chorionic villi) for examination. In addition, it is quite likely that decidual T cell may not be the only cell type that produces LIF in the human endometrium or placenta (Croy et al., 1991Go; Jokhi et al., 1994Go; Laird et al., 1997Go). For example, in situ hybridization and immunohistochemical studies identify most LIF production in human endometrium to the glandular epithelium (Laird et al., 1997Go). In this study, we also showed the production of LIF by both the stroma and epithelial cells in decidua and by trophoblast cells in chorionic villi, by immunohistochemical study. The possibility therefore cannot be excluded that the decreased LIF production by decidual T cell in recurrent aborters (Piccinni et al., 1998Go) may in some circumstances be overcome by exaggerated or supplemental production from other cell types. Since en bloc tissues (decidua and chorionic villi) were examined in this study, it is conceivable that if the rescue mechanism (i.e. supplemental production from other cells) actually exists, real-time quantitative PCR will not detect the difference in LIF gene expression between NP and AP. In a logical consideration based on clinical conditions, one has good reason to speculate that this type of rescue mechanism would be present if LIF-producing cell types other than T cells remained functionally intact. Therefore further study to identify the presentation in a more purified patient population (for example, unexplained recurrent abortion) and a detailed comparison between localized and general environment would be needed to clarify this issue.

In addition, it is known that both in mice and human (Cullinan et al., 1996Go), there are two isoforms of LIF, diffusible and matrix-associated, which may exert different biological effects. The present study did not specifically differentiate them and therefore we cannot exclude the possibility that expression and production of respective isoforms might be different in the study subjects. Therefore future detailed analyses of the LIF isoforms will probably provide further information about this issue. In addition, gp130, an important part of the LIF receptor system, should also be studied.

In this study, we used two experimental methods to quantify the concentration of target mRNA, one by conventional semi-quantitation and the other by real-time quantitative PCR. Some scientists argue that there are limitations about the sensitivity and specificity of semi-quantitation method. The present study did show some essential differences between these two methods, but the semi-quantitation method still identifies a trend in mRNA level which was reflected by real-time quantitative PCR. Therefore, the validity of the data obtained in this study was strengthened by the concomitant use of the two methods. However, due to the potentially higher sensitivity of real-time quantitative PCR, this method would probably provide a clearer insight into the significance of the data in some circumstances.

In conclusion, the present study suggests that LIF-related genes may play a functional role in the maintenance of human pregnancy, at least in the first trimester. At this stage, the LIF action may be predominantly on the trophoblasts through binding to LIF-R. However, there is no absolute indication that defective production of LIF or LIF-R is a major cause of abortion in early human pregnancy.


    Acknowledgements
 
This work was supported by grants from the National Science Council of the Republic of China (NSC90-2314-B-002-147 and NSC91-2314-B-002-374) and National Taiwan University Hospital (91A10-6).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Arici A, Engin O, Attar E and Olive DL (1995) Modulation of leukemia inhibitory factor gene expression and protein biosynthesis in human endometrium. J Clin Endocrinol Metab 80,1908–1915.[Abstract]

Bhatt H, Brunet LJ and Stewart CL (1991) Uterine expression of leukemia inhibitory factor coincides with the onset of blastocyst implantation. Proc Natl Acad Sci USA 88,11408–11412.[Abstract]

Bilinski P, Roopenian D and Gossler A (1998) Maternal IL-11Ralpha function is required for normal decidua and fetoplacental development in mice. Gene Dev 12,2234–2243.[Abstract/Free Full Text]

Chadderton T, Wilson C, Bewick M and Gluck S (1997) Evaluation of three rapid RNA extraction reagents: relevance for use in RT-PCRs and measurement of low level gene expression in clinical samples. Cell Mol Biol 43,1227–1234.

Charnock-Jones DS, Sharkey AM, Fenwick P and Smith SK (1994) Leukaemia inhibitory factor mRNA concentration peaks in human endometrium at the time of implantation and the blastocyst contains mRNA for the receptor at this time. J Reprod Fertil 101,421–426.[Abstract]

Chen HF, Shew JY, Ho HN, Hsu WL and Yang YS (1999) Expression of leukemia inhibitory factor and its receptor in preimplantation embryos. Fertil Steril 72,713–719.[CrossRef][ISI][Medline]

Chen HF, Lin CY, Chao KH, Wu MY, Yang YS and Ho HN (2002a) Defective production of interleukin-11 by decidua and chorionic villi in human anembryonic pregnancy. J Clin Endocrinol Metab 87,2320–2328.[Abstract/Free Full Text]

Chen HW, Chen JJ, Tzeng CR, Li HN, Chang SJ, Cheng YF, Chang CW, Wang RS, Yang PC and Lee YT (2002b) Global analysis of differentially expressed genes in early gestational decidua and chorionic villi using a 9600 human cDNA microarray. Mol Hum Reprod 8,475–484.[Abstract/Free Full Text]

Chen JR, Cheng JG, Shatzer T, Sewell L, Hernandez L and Stewart CL (2000) Leukemia inhibitory factor can substitute for nidatory estrogen and is essential to inducing a receptive uterus for implantation but is not essential for subsequent embryogenesis. Endocrinology 141,4365–4372.[Abstract/Free Full Text]

Croy BA, Guilbert LJ, Browne MA, Gough NM, Stinchcomb DT, Reed N and Wegmann TG (1991) Characterization of cytokine production by the metrial gland and granulated metrial gland cells. J Reprod Immunol 19,149–166.[CrossRef][Medline]

Cullinan EB, Abbondanzo SJ, Anderson PS, Pollard JW, Lessey BA, Stewart and CL. (1996) Leukemia inhibitory factor (LIF) and LIF receptor expression in human endometrium suggests a potential autocrine/paracrine function in regulating embryo implantation. Proc Natl Acad Sci USA 93,3115–3120.[Abstract/Free Full Text]

Gearing DP, Thut CJ, VandeBos T, Gimpel SD, Delaney PB, King J, Price V, Cosman D and Beckmann MP (1991) Leukemia inhibitory factor receptor is structurally related to the IL-6 signal transducer, gp130. EMBO J 10,2839–2848.[Abstract]

Gibson UE, Heid CA and Williams PM (1996) A novel method for real time quantitative RT–PCR. Genome Res 6,995–1001.[Abstract]

Hsu SM, Raine L and Fanger H (1981) A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol 75,734–738.[ISI][Medline]

Jokhi PP, King A, Sharkey AM, Smith SK and Loke YW (1994) Screening for cytokine messenger ribonucleic acids in purified human decidual lymphocyte populations by the reverse-transcriptase polymerase chain reaction. J Immunol 153,4427–4435.[Abstract/Free Full Text]

King A, Jokhi PP, Smith SK, Sharkey AM and Loke YW (1995) Screening for cytokine mRNA in human villous and extravillous trophoblasts using the reverse-transcriptase polymerase chain reaction (RT–PCR). Cytokine 7,364–371.[CrossRef][ISI][Medline]

Kojima K, Kanzaki H, Iwai M, Hatayama H, Fujimoto M, Inoue T, Horie K, Nakayama H, Fujita J and Mori T (1994) Expression of leukemia inhibitory factor in human endometrium and placenta. Biol Reprod 50,882–887.[Abstract]

Kojima K, Kanzaki H, Iwai M, Hatayama H, Fujimoto M, Narukawa S, Higuchi T, Kaneko Y, Mori T and Fujita J (1995) Expression of leukaemia inhibitory factor (LIF) receptor in human placenta: a possible role for LIF in the growth and differentiation of trophoblasts. Hum Reprod 10,1907–1911.[Abstract]

Laird SM, Tuckerman EM, Dalton CF, Dunphy BC, Li TC and Zhang X (1997) The production of leukaemia inhibitory factor by human endometrium: presence in uterine flushings and production by cells in culture. Hum Reprod 12,569–574.[ISI][Medline]

Lessey BA, Castelbaum AJ, Buck CA, Lei Y, Yowell CW and Sun J (1994a) Further characterization of endometrial integrins during the menstrual cycle and in pregnancy. [See comments]. Fertil Steril 62,497–506.[ISI][Medline]

Lessey BA, Castelbaum AJ, Buck CA, Lei Y, Yowell CW and Sun J (1994b) Further characterization of endometrial integrins during the menstrual cycle and in pregnancy. [See comments]. Fertil Steril 62,497–506.[ISI][Medline]

Modric T, Kowalski AA, Green ML, Simmen RC and Simmen FA (2000) Pregnancy-dependent expression of leukaemia inhibitory factor (LIF), LIF receptor-beta and interleukin-6 (IL-6) messenger ribonucleic acids in the porcine female reproductive tract. Placenta 21,345–353.[CrossRef][Medline]

Piccinni MP, Beloni L, Livi C, Maggi E, Scarselli G and Romagnani S (1998) Defective production of both leukemia inhibitory factor and type 2 T-helper cytokines by decidual T cells in unexplained recurrent abortions. Nat Med 4,1020–1024.[CrossRef][ISI][Medline]

Pollard JW, Hunt JS, Wiktor-Jedrzejczak W and Stanley ER (1991) A pregnancy defect in the osteopetrotic (op/op) mouse demonstrates the requirement for CSF-1 in female fertility. Dev Biol 148,273–283.[ISI][Medline]

Ren SG, Melmed S and Braunstein GD (1997) Decidual leukemia inhibitory factor production and action on human chorionic gonadotropin secretion at different stages of gestation in vitro. Early Pregnancy 3,102–108.[Medline]

Reubinoff BE, Pera MF, Fong CY, Trounson A and Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. [See comments]. [Erratum appears in Nat Biotechnol 2000; 18:559]. Nat Biotechnol 18,399–404.[CrossRef][ISI][Medline]

Robb L, Li R, Hartley L, Nandurkar HH, Koentgen F and Begley CG (1998) Infertility in female mice lacking the receptor for interleukin 11 is due to a defective uterine response to implantation. Nat Med 4,303–308.[ISI][Medline]

Sawai K, Matsuzaki N, Kameda T, Hashimoto K, Okada T, Shimoya K, Nobunaga T, Taga T, Kishimoto T and Saji F (1995a) Leukemia inhibitory factor produced at the fetomaternal interface stimulates chorionic gonadotropin production: its possible implication during pregnancy, including implantation period. J Clin Endocrinol Metab 80,1449–1456.[Abstract]

Sawai K, Azuma C, Koyama M, Ito S, Hashimoto K, Kimura T, Samejima Y, Nobunaga T and Saji F (1995b) Leukemia inhibitory factor (LIF) enhances trophoblast differentiation mediated by human chorionic gonadotropin (hCG). Biochem Biophys Res Commun 211,137–143.[CrossRef][ISI][Medline]

Sawai K, Matsuzaki N, Okada T, Shimoya K, Koyama M, Azuma C, Saji F and Murata Y (1997) Human decidual cell biosynthesis of leukemia inhibitory factor: regulation by decidual cytokines and steroid hormones. Biol Reprod 56,1274–1280.[Abstract]

Sharkey AM, King A, Clark DE, Burrows TD, Jokhi PP, Charnock-Jones DS, Loke YW and Smith SK (1999) Localization of leukemia inhibitory factor and its receptor in human placenta throughout pregnancy. Biol Reprod 60,355–364.[Abstract/Free Full Text]

Stahl J, Gearing DP, Willson TA, Brown MA, King JA and Gough NM (1990) Structural organization of the genes for murine and human leukemia inhibitory factor. Evolutionary conservation of coding and non-coding regions. J Biol Chem 265,8833–8841.[Abstract/Free Full Text]

Stewart CL, Kaspar P, Brunet LJ, Bhatt H, Gadi I, Kontgen F and Abbondanzo SJ (1992) Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. [See comments]. Nature 359,76–79.[CrossRef][ISI][Medline]

Vincent S, Marty L and Fort P (1993) S26 ribosomal protein RNA: an invariant control for gene regulation experiments in eucaryotic cells and tissues. Nucleic Acids Res 21,1498.[ISI][Medline]

Wu MY, Chen HF, Chen SU, Chao KH, Yang YS and Ho HN (2001) Increase in the production of interleukin-10 early after implantation is related to the success of pregnancy. Am J Reprod Immunol 46,386–392.[CrossRef][Medline]

Yin T, Taga T, Tsang ML, Yasukawa K, Kishimoto T and Yang YC (1993) Involvement of IL-6 signal transducer gp130 in IL-11-mediated signal transduction. J Immunol 151,2555–2561.[Abstract/Free Full Text]

Submitted on November 7, 2003; accepted on February 2, 2004.