Hum. Reprod. Advance Access originally published online on June 21, 2006
Human Reproduction 2006 21(10):2521-2529; doi:10.1093/humrep/del215
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Ovarian stimulation with GnRH agonist, but not GnRH antagonist, partially restores the expression of endometrial integrin 
3 and leukaemia-inhibitory factor and improves uterine receptivity in mice
1 Department of Reproductive Endocrinology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China and 2 Department of Pharmacology & Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada
3 To whom correspondence should be addressed at: Department of Reproductive Endocrinology, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310006, China. E-mail: huanghefg{at}hotmail.com
4 To whom correspondence should be addressed at: Department of Pharmacology & Therapeutics, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. E-mail: jzsheng{at}ucalgary.ca
* These authors contributed equally to this work.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
BACKGROUND: The impact of different ovarian stimulation (OS) protocols on endometrial receptivity remains controversial. In this study, the effects of different OS on the expression of endometrial integrin
3 subunit and leukaemia-inhibitory factor (LIF) during the implantation window and the implantation rate in mice were investigated. METHODS: Three OS protocols were used, involving either pregnant mare’s serum gonadotrophin (PMSG) alone, PMSG plus GnRH agonist or PMSG plus GnRH antagonist. Uterus samples were collected at 48 h after OS or ovulation and were detected with immunohistochemistry, Western blot and RT–PCR analyses. Normal embryos at gestation day 4 were transferred into the uteri of mice in the control and OS groups. RESULTS: All OS groups showed a significant decrease in the expression of both the endometrial integrin 3 subunit and LIF during the implantation window and the implantation rate. Among the three OS groups, GnRH agonist-treated mice showed a higher endometrial integrin 3 subunit and LIF expression and a higher implantation rate. No significant difference was found in the measured indices between the GnRH antagonist and PMSG groups. CONCLUSIONS: OS may inhibit the expression of endometrial integrin 3 subunit and LIF and impair endometrial receptivity in mice. OS with GnRH agonist, but not GnRH antagonist, may partially restore the endometrial physiological secretion and improve uterine receptivity.
Key words: endometrial receptivity/GnRH antagonist/integrin/leukaemia-inhibitory factor/mouse/ovarian stimulation
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Ovarian stimulation (OS) with gonadotrophins is an important approach in IVF. However, the impact of different OS on endometrium receptivity remains controversial (Al-Inany and Aboulghar, 2002
; Fauser and Devroey, 2003; Coccia et al., 2004; Tarlatzis and Bili, 2004).
Many studies in human have shown that the periovulatory endometrial characteristics in OS cycles are considerably different compared with the natural cycle (Devroey et al., 2004; Fauser and Devroey, 2003; Saadat et al., 2004; Papanikolaou et al., 2005). This difference could affect luteal phase function and alter endometrial receptivity (Devroey et al., 2004; Kolibianakis et al., 2002), even to the extent of greatly compromising the likelihood of pregnancy (Al-Inany and Aboulghar, 2002; Beckers et al., 2003). An earlier meta-analysis (Al-Inany and Aboulghar, 2002) focusing on five multicentre comparative trials concluded that a small but significant reduction in pregnancy rate was observed per started cycle using a GnRH antagonist co-treatment protocol compared with the routine GnRH agonist co-treatment OS. Therefore, concerns have been raised regarding the possibility of direct effects of OS on endometrial receptivity and embryonic implantation, especially the possibility that adverse effects may arise from GnRH antagonist co-treatment (Devroey et al., 2004). Concordant evidence of this aspect is accumulating (Mirkin et al., 2004; Horcajadas et al., 2005; Vlahos et al., 2005), whereas the non-concordant results are also appearing (Bahceci et al., 2005; Saadat et al., 2004; Takahashi et al., 2004; Prapas et al., 2005; Simon et al., 2005). This means that a controversial state of this aspect exists and that further clarification is needed.
Regarding the effect of different OS strategies on endometrial receptivity and embryonic implantation in the human, the data shown in previous studies were mainly in the clinical and endometrial histologies (Beckers et al., 2003; Kolibianakis et al., 2002; Mirkin et al., 2004; Papanikolaou et al., 2005; Saadat et al., 2004; Simon et al., 2005; Tavaniotou et al., 2003; Thomas et al., 2002). Little direct data were available in the literature on human endometrial receptivity. Similar situations existed in related studies carried out in mice (Ertzeid and Storeng, 1992, 2001; Van der Auwera and D’Hooghe, 2001).
Integrin 3 subunit and leukaemia-inhibitory factor (LIF) are the two cellular factors which have been best characterized and largely accepted as the promising candidates of biomarkers of uterine receptivity in both human (Cavagna and Mantese, 2003; Hoozemans et al., 2004; Kondera-Anasz et al., 2004) and mice (Bhatt et al., 1991; Stewart et al., 1992; Illera et al., 2000a). The expression of these two cellular factors in both human and mouse endometrium is in a menstrual cycle-dependent manner, and the maximal expression is observed in the mid-secretary phase of the menstrual cycle, coinciding with the time of implantation (Arici et al., 1995; Lessey et al., 1992, 1994; Cullinan et al., 1996; Damario et al., 2001). It has been found that the expression of both alpha(v)beta(3) integrin and LIF was significantly reduced in endometrium from infertile women (Hambartsoumian, 1998; Lessey et al., 1995; Seli et al., 2005). Although these two cellular factors have not been established as obligatory uterine ones in human implantation (Creus et al., 2002, 2003; Thomas et al., 2003; Devroey et al., 2004), many studies have demonstrated that LIF is one of the obligatory implantation factors in mice (Cavagna and Mantese, 2003; Devroey et al., 2004; Kondera-Anasz et al., 2004).
The purpose of the present study was to investigate the effects of different classical OS protocols (gonadotrophins alone, GnRH agonist co-treatment or GnRH antagonist co-treatment) on endometrial receptivity and embryonic implantation in a human IVF-mimicked mouse model. The expression of integrin 3 subunit and LIF and embryonic implantation rate were used to evaluate endometrial receptivity and embryonic implantation. Our hypothesis was that different OS protocols would induce different biological and molecular profiles of endometrium that might lead to altered endometrial receptivity and implantation outcome.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Animals
Animal care and use procedures were in accordance with the institutional guidelines established by the Animal Care and Use Committee (ACUC) of the School of Medicine, Zhejiang University. Virgin 6- to 8-week-old female CD1 mice (Zhejiang University Animal Center, Hangzhou, China) were housed in 12/12 h light/dark cycle (light on from 0:00 to 12:00 hours) at 25 ± 0.5°C and 50–60% humidity. The mice were fed ad libitum with a standard pellet diet and water. Estrus was identified by daily vaginal discharge and smear samples. Only mice that had exhibited estrus for more than two consecutive periods of regular 4-day cycles were used in the present study. Suitable mice [8- to 12-week old and 18–22 g body weight (bw)] were randomly allocated to four groups, with 30 mice in each group. Within each group, 10 mice were either used for OS treatment or allowed to remain in their natural cycle, and the other 20 mice were used as recipients in the embryo-donation model described below.
Ovarian stimulation
The procedures for OS performed in different groups were: (i) GnRH agonist group: GnRH agonist (Decapetyl, Ferring GmbH, Germany) was i.p. injected at 1.5 µg/100 g bw/day from day 3–9 of estrus. At 9.00 a.m. of day 9, the pregnant mare’s serum gonadotrophin (PMSG) (Intervet, USA) was i.p. injected at 40 IU/100 g bw, followed by an injection (i.p.) of hCG (Pregnyl, Organon, The Netherlands) (100 IU/100 g bw) 28 h after the injection of PMSG; (ii) GnRH antagonist group: GnRH antagonist (Cetrotide, Serono, USA) was i.p. injected at 4 µg/100 g bw on day 3 of estrum. PMSG was i.p. injected at 40 IU/100 g bw at 9.00 a.m. of day 9, followed by an injection (i.p.) of hCG (100 IU/100 g bw) 28 h after the injection of PMSG; (iii) PMSG group: The mice were i.p. injected with saline at the same volume from day 3–9 of estrus and then treated with the same procedure as described for the GnRH agonist and GnRH antagonist groups; (iv) Control group: The mice were i.p. injected with saline only at the same volume from day 3 of estrus onwards, following the same injection schedule as described for the three groups above.
Tissue collection and application
Fresh whole uterus samples were collected from OS-treated or naturally ovulated mice at 48 h after OS (treated groups) or ovulation (control group). After quickly being rinsed with cold phosphate-buffered saline, each fresh whole uterus sample was divided into three parts. One part was fixed in 10% formalin and embedded in paraffin for a streptavidin peroxidase (SP)-conjugated immunohistochemical assay. The other two parts of each sample, including both endometrium and myometrium, were stored at –80°C until the extraction of protein and mRNA.
Immunohistochemical analysis
Formalin-fixed, paraffin-embedded tissues were sectioned to a thickness of 3 µm. Sections were then deparaffinized in xylene, dehydrated in a series of ethanol solutions and stained using standard immunohistochemistry procedures. Tissue sections were pretreated by boiling in 10 mmol/l citrate buffer (pH 6.0) for 10 min as recommended by the supplier. For immunohistochemical detection of integrin 3 and LIF, goat-anti-human polyclonal antibodies (Santa Cruz Biotechnology, USA) at dilution of 1 : 75 and 1 : 100 were used, respectively. The secondary antibodies were visualized using the avidin–biotin method recommended by the supplier (SP Kit) (Zymed, USA). The brown colour was developed with diaminobenzinine (DAB Kit) (Zymed). Staining intensity of tissue sections was evaluated and graded (0, absence; 1, weak; 2, moderate; 3, strong) in a blinded fashion by two examiners and was assessed by using the HSCORE. The HSCORE was calculated using the following equation:
 |
where i is the staining intensity and Pi the percentage of stained epithelial cells at each level of intensity. In order to confirm that the staining was specific, a human endometrium sample from a 29-year-old woman in her mid-secretary phase of menstrual cycle was used as positive control. The negative control was carried out, omitting the primary antibody in the same human endometrial sample.
Western blot analysis
Total proteins from the tissues were extracted by RIPA buffer containing 50 mmol/l Tris–HCl (pH 7.4), 150 mmol/l NaCl, 1% NP-40, 0.5% sodium deoxycholic acid, 0.1% SDS, 100 µg/ml PMSF and 100 µg/ml leupeptin. Samples were electrophoresed with sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) using 10% polyacrylamide gels and were transferred to nitrocellulose membranes (Bio-Rad, USA). Membranes were blocked at room temperature for 1 h with 5% fat-free powdered milk in TBS-T (10 mmol/l Tris, 150 mmol/l NaCl, 0.05% Tween-20, pH 8.0). Following three washes with TBS-T, the membranes were incubated overnight with primary antibody in 1% TBS-T at 4°C. For Western blot analysis of integrin 3 and LIF, goat-anti-human polyclonal antibodies (Santa Cruz Biotechnology) at dilution of 1 : 250 and 1 : 100 were used, respectively. After incubation, the membranes were washed three times with TBS-T and then incubated with the appropriate secondary antibody at dilution of 1 : 2000 conjugated to peroxidase (Santa Cruz Biotechnology) at room temperature for 2 h. Following three washes with TBS-T and three washes with distilled water, the bound antibodies were detected by enhanced chemiluminescence (ECL kit) (Santa Cruz Biotechnology). -Actin was used as an internal control to validate the amount of protein loaded onto the gels.
RT–PCR analysis
Poly(A)+ RNA was isolated from the mouse uterus samples using the Micro-Fast kit (Invitrogen, CA, USA), from which single-strand cDNA was synthesized using the cDNA cycle kit (Invitrogen). The purity of the extracted RNA was photometrically tested, and the ratio of the optical density (OD) at 260/280 nm was 1.8–2.0. A total of 20 µl reaction contained 0.5 µg random primer (Sigma-Genosys, Australia), 40 U RNase inhibitor (Promega Corporation, USA), 200 IU M-MLV reverse transcriptase, 2 µl dNTP at 25 pmol/l (Sigma), 4 µl reverse transcriptase buffer (x5) and 2 microgram RNA. Reactions were incubated at 70°C for 5 min, 37°C for 90 min, 95°C for 5 min and then rapidly cooled on ice.
For integrin 3, PCR was performed in a 50 µl volume containing 1 µl RT reaction product, 5 µl PCR buffer (x10), 4 µl MgCl2 at 2.5 mmol/l, 1 µl dNTP at 25 pmol/l, 1 µl integrin 3 sense and antisense primer, 0.5 µl -actin sense and antisense primer as control and 0.4 µl Taq DNA polymerase (Takara, Japan). LIF was performed as described above except that the dosage of the primers was 1.2 µl for the LIF and 0.4 µl for -actin.
The primers used were as follows: (i) integrin 3: sense: 5'-GCCTTCGTGGACAAGCCTGTA-3'; antisense: 5'-GGACAATGC CTGCCAGTCTTC-3', which yield a 337 bp PCR product. (ii) LIF: sense: 5'-CCTTACTGCTGCTGGTTCTG-3'; antisense: 5'-GCTCCA CCAACTTGGTCTTC-3, which yield a 288 bp PCR product. (iii) -actin: sense: 5'-TTCCAGCCTTCCTTCCTGG-3'; antisense: 5'-TTGCGCTCAGGAGGAGCAAT-3', which yield a 224 bp PCR product. All primers were obtained from Bio Basic (Shanghai, China). PCR was performed using the following conditions: for integrin 3, an initial denaturation at 94°C for 5 min, followed by 28 cycles of 30 s at 94°C, 30 s at 60°C and 30 min at 72°C; for LIF, an initial denaturation at 94°C for 5 min, followed by 30 cycles of 30 s at 94°C, 30 s at 65°C and 30 min at 72°C. All PCR was terminated with a final extension at 72°C for 10 min The PCR products were subjected to electrophoresis on 2% agarose gels and visualized by ethidium bromide staining and then verified with -actin as a control.
Embryo collection and transfer
Successfully mated female mice were identified with vaginal plugs. The day the vaginal plug presented was designated gestation day 1. Pregnant donor mice were killed by cervical dislocation on gestation day 2. The oviducts were excised and flushed with human tubal fluid (HTF) medium (Irvine Science, USA). Morphologically normal day 2 embryos (2- to 4-cell) were pooled after collection. The quality of embryos was evaluated based on cell numbers, size of blastomeres and cytoplasmic fragments. The embryos were cultured in HTF medium, supplemented with 10% synthetic serum substitute under oil at 37°C in a humidified atmosphere with 5% CO2. On day 4, normal embryos that reached the morula/blastocyst stage were randomly divided and transferred into the mice in control and OS groups. There were 20 mice in each group. Six embryos were transferred into the right uterine horn of each mouse in the estrus of natural cycle (control group) or at 48 h later after being given HCG (three OS groups) in a way of embryo-donation model. Mice were placed in a separate cage supplied with ad libitum food and water. The recipients were killed on day 10 of gestation, and the conceptuses were removed from the uteri. The number of implantation sites, moles (resorbing conceptuses) and live fetuses in the right uterine horn were recorded. The implantation rate was calculated as: total number of implantation sites, moles and live fetuses/number of embryos transferred.
Statistical analysis
Mann–Whitney U-test was used to compare the expression of endometrial integrin 3 and LIF in different OS groups and the control group. The effect of OS with different treatment on embryonic implantation rate was analysed by Chi-square test. All P-values are two-sided, and P < 0.05 was considered statistically significant. Data were analysed using the commercially available software package SPSS 11.0 (SPSS, Chicago, IL, USA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Immunohistochemical localization of endometrial integrin
3 subunit and LIF during the implantation window of miceImmunohistochemical detection of integrin
3 subunit and LIF was observed mainly in glandular epithelial cells and luminal epithelial cells of murine endometrium during the implantation window (Figures 1A and 2A). Compared with the control group (HSCORE: 2.44 ± 0.09), OS groups showed lower staining intensities of integrin 3 subunit (HSCORE: 2.31 ± 0.09 in GnRH agonist group; 2.09 ± 0.08 in GnRH antagonist group and 2.06 ± 0.10 in PMSG group) during the implantation window of the treated mice (P-values of 0.009, <0.001 and <0.001, respectively; Figure 1B). OS groups also showed lower staining intensities of LIF (HSCORE: 1.67 ± 0.10 in GnRH agonist group; 1.44 ± 0.05 in GnRH antagonist group and 1.41 ± 0.07 in PMSG group) than that of the control group (HSCORE: 1.84 ± 0.08) during the implantation window (P-values of 0.121, <0.001 and <0.001, respectively) (Figure 2B). Among OS groups, the staining intensities of integrin 3 subunit and LIF during the implantation window in the GnRH agonist group were significantly higher than that in GnRH antgonist group (P-values of <0.001 in integrin and <0.001 in LIF, respectively) or PMSG group (P-values of <0.001 in integrin and <0.001 in LIF, respectively) (Figures 1B and 2B).
|
|
Expression of endometrial integrin
3 subunit and LIF mRNA during the implantation windowBoth integrin
3 subunit and LIF mRNA were expressed in endometrium of mice during the implantation window in the natural control group and all the OS groups (Figures 3A and 4A). Compared with the control group, the integrin 3 subunit mRNA expression levels, normalized by -actin expression levels, in all three OS groups were significantly decreased (P-values of 0.049, 0.016 and 0.009, respectively; Figure 3B). LIF mRNA expression levels were also reduced in three OS groups (P-values of 0.023 in GnRH agonist versus control group, 0.009 in GnRH antagonist versus control group and <0.001 in PMSG versus control group, respectively) (Figure 4B). Among the three OS groups, integrin 3 subunit and LIF mRNA expression levels in GnRH agonist group were significantly higher than those in the other two OS groups (integrin 3 subunit: P-values of 0.037 in GnRH agonist versus GnRH antagonist group and 0.017 in GnRH agonist versus PMSG group, respectively; LIF: P-values of 0.049 in GnRH agonist versus GnRH antagonist group and 0.018 in GnRH agonist versus PMSG group, respectively) (Figures 3B and 4B).
|
|
Expression of endometrial integrin
3 and LIF proteins during the implantation windowThe protein expression of endometrial integrin
3 subunit and LIF during the implantation window of the mice was confirmed in all groups by Western blotting (Figures 5A and 6A). Consistent with the results of mRNA expression, the protein levels of both integrin 3 subunit and LIF, normalized with -actin expression level, in OS groups were lower than those in the control group (integrin 3 subunit: P-values of 0.028 in GnRH agonist versus control group, 0.009 in GnRH antagonist versus control group and <0.001 in PMSG versus control group, respectively; LIF: P-values of 0.016 in GnRH agonist versus control group, <0.001 in GnRH antagonist versus control group, and <0.001 in PMSG versus control group, respectively; Figures 5B and 6B). Among the three OS groups, both integrin 3 subunit and LIF expression levels in GnRH agonist group were significantly higher than those in the other two OS groups (integrin 3 subunit: P-values of 0.028 in GnRH agonist versus GnRH antagonist group and 0.019 in GnRH agonist versus PMSG group, respectively; LIF: P-values of 0.013 in GnRH agonist versus GnRH antagonist group, and <0.001 in GnRH agonist versus PMSG group, respectively; Figures 5B and 6B).
|
|
Embryonic implantation rate
In each group of the present study, 120 normal mouse embryos that reached the morula/blastocyst stage at day 4 were transferred to the corresponding mice. The number of implantation sites, moles (resorbing conceptuses) and live fetuses at gestation day 10 in each group was 58 in the control group, 44 in GnRH agonist group, 28 in GnRH antagonist group and 20 in PMSG group. The embryonic implantation rate in the control group (48.4%) was significantly higher than that in GnRH antagonist group (23.4%, P < 0.001) and PMSG group (16.7%, P<0.001). Among the three OS groups, the embryonic implantation rate in GnRH agonist group (36.7%) was also significantly higher than those in the other two OS groups (P-values of 0.018 in GnRH agonist versus GnRH antagonist group and <0.001 in GnRH agonist versus PMSG group, respectively).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
The present study is a pilot, prospective, randomized and controlled comparison study on the effects of different OS strategies on the endometrial receptivity and embryonic implantation in the human IVF-mimicked mouse models. Using immunohistochemical, Western blot and RT–PCR analyses, we demonstrated that integrin
3 subunit and LIF were expressed in the endometrium of mature female mice during the implantation windows. There were significant differences in the expression of endometrial integrin 3 subunit and LIF between OS groups and the control group during the implantation window of mice. By using an embryo-donation model, we also demonstrated that the embryonic implantation rates in all three OS groups were lower than that in the control group. Immunohistochemical analysis showed that the integrin 3 subunit and LIF were mainly located in endometrial glandular epithelial and luminal epithelial cells. The immunohistochemical location of both integrin 3 subunit and LIF in the mouse uterus at the time of implantation window was similar to that in the previous studies in mice (Bhatt et al., 1991; Illera et al., 2000b). Our results are not only consistent with the existing viewpoint that endometrial expression of integrin 3 subunit and LIF positively correlates with endometrial receptivity and embryonic implantation (Cavagna and Mantese, 2003; Hoozemans et al., 2004; Kondera-Anasz et al., 2004), but also infer that OS may impair endometrial receptivity at least in part by inhibiting the expression of endometrial integrin 3 subunit and LIF.
Previous studies on early mouse pregnancy have shown that both integrin 3 subunit and LIF appeared in the endometrium coinciding with the implantation window and might be used as markers of endometrial receptivity (Bhatt et al., 1991; Illera et al., 2000b). Targeted disruption of the LIF gene (Stewart et al., 1992) or blockade of the alpha(v)beta(3) integrin could adversely affect implantation in mice (Illera et al., 2000a). This likely explains why the lower expression of either endometrial integrin 3 subunit or LIF in OS groups, especially in the GnRH antagonist or PMSG alone group, is associated with a lower embryonic implantation rate.
The adverse effects of superovulation with gonadotrophins on implantation in mice have been documented in previous studies (Fossum et al., 1989; Ertzeid and Storeng, 2001; Van der Auwera and D’Hooghe, 2001). These studies showed that the implantation rate was significantly less in the superovulated mouse recipients by using a mouse embryo-donation model similar to the one used in the present study. A decreased implantation rate was supposed to be due to the changes in the uterine milieu, especially due to the change in endometrial receptivity. Unfortunately, all of these studies did not provide detailed information on the change in endometrial receptivity after OS treatment. The present study, for the first time, provides evidence that the endometrial expression of integrin 3 subunit and/or LIF is downregulated following OS and that this downregulation negatively impacts endometrial receptivity.
Up to now, the effects of different OS strategies, including OS with GnRH agonist or GnRH antagonist co-treatment, in human and other mammals on both endometrial receptivity and embryonic implantation are still unclear (Coccia et al., 2004; Ertzeid and Storeng, 2001; Devroey et al., 2004). In the present comparison study, we demonstrated for the first time that, among three OS groups in mice (GnRH agonist, GnRH antagonist and PMSG alone), differences arose both in the expression of endometrial integrin 3 subunit and LIF during the implantation window and in the embryonic implantation rate. Compared with the PMSG alone protocol, OS with GnRH agonist co-treatment showed elevated expression levels of endometrial integrin 3 subunit and LIF and a higher embryonic implantation rate, suggesting that OS with GnRH agonist co-treatment, but not with GnRH antagonist co-treatment, may potentially improve uterine receptivity, and this improvement of uterine receptivity in mice might be partially through the restoration of physiologically endometrial secretion changed in the OS cycle.
The mechanisms underlying the observed difference in endometrial receptivity among the different OS protocols, especially the modest endometrial receptivity in GnRH antagonist co-treatment OS, are unclear. In the human, a significantly lower serum estradiol concentration was observed on the day of HCG administration in the OS with GnRH antagonist co-treatment (Albano et al., 2000). This observation is in agreement with the results shown in the previous mouse studies, which reported that both integrin 3 subunit and LIF expression in the endometrium were under maternal control and were regulated by circulating estrogen levels (Bhatt et al., 1991; Yang et al., 1996; Illera et al., 2000b). A recent study reported that the profile of endometrial gene expression during the window of implantation was significantly different when comparing OS cycles using a GnRH agonist versus a GnRH antagonist (Mirkin et al., 2004). These pharmacological or pharmacodynamic properties in GnRH antagonist co-treatment cycles may be associated with the differences in aforementioned endometrial receptivity indices. However, definite conclusions about this issue cannot be drawn now (Ortmann et al., 2001; Coccia et al., 2004; Griesinger et al., 2004; Tarlatzis and Bili, 2004). Further studies are required to establish a clear causative relation.
In the human IVF clinic, either the commonly used OS with GnRH agonist co-treatment or the uncommonly used OS with GnRH antagonist co-treatment is intended to reduce cycle-cancellation rate and to improve the overall IVF outcome (Coccia et al., 2004; Hughes et al., 1992; Shapiro and Mitchell-Leef, 2003). By relying on competitive receptor blockage rather than receptor desensitization, OS with GnRH antagonist co-treatment is considered more advantageous in IVF over the OS with GnRH agonist co-treatment (Al-Inany and Aboulghar, 2002; Shapiro and Mitchell-Leef, 2003; Weiss et al., 2003; Coccia et al., 2004; Tarlatzis and Bili, 2004). However, an earlier meta-analysis concluded that a small but significant reduction in pregnancy rate was observed per started cycle using a GnRH antagonist co-treatment protocol compared with the routine GnRH agonist co-treatment OS (Al-Inany and Aboulghar, 2002). Furthermore, although some recent studies showed that no significant difference in either endometrial maturation (Saadat et al., 2004) or pregnancy outcomes (Takahashi et al., 2004; Bahceci et al., 2005; Prapas et al., 2005) was found between different OS protocols, the concurrent relevant studies (Horcajadas et al., 2005; Mirkin et al., 2004; Simon et al., 2005; Vlahos et al., 2005) still infer the possibility of adverse effects of OS on endometrial receptivity and embryonic implantation, including the possibility that arose specifically from GnRH antagonist co-treatment (Devroey et al., 2004). In the present study, the observed alteration in endometrial receptivity, as shown by significantly lower expression of both endometrial integrin 3 subunit and LIF and a significantly decreased embryonic implantation rate in GnRH antagonist co-treatment OS, supports the inference mentioned above and may provide some extra insights into this issue. Respecting the effect of GnRH antagonist co-treatment OS on endometrial receptivity, the results obtained in the present study were contrary to those from a recently reported study, in which it was shown that the endometrial development after GnRH antagonist co-treatment mimics the natural endometrium more closely than after GnRH agonist co-treatment (Simon et al., 2005). The conflict might be due to the differences between human and mice and/or the different indices measured between the two studies. Further studies are needed to establish their true effects on human IVF outcome and to optimize each of these two OS protocols (Felberbaum and Diedrich, 2003; Weiss et al., 2003; Prapas et al., 2005).
The present study used a human IVF-mimicked mouse model to investigate the effects of different classical OS protocols on the endometrial receptivity and embryonic implantation. Although there are considerable similarities amongst mammals in the early stage of development, it is difficult to extrapolate the information obtained from this mouse model directly to the human IVF clinic. Indeed, numerous differences exist in this aspect between the human and the mouse (Dey et al., 2004; Wimsatt, 1975; Lee and DeMayo, 2004). Hence, the extrapolation mentioned above should be done with caution. Moreover, because of a relatively small size of each group and the uncertain roles of both integrin 3 and LIF in endometrial receptivity and embryonic implantation in either humans or mice, further studies are needed to clarify the reality of all inferences and extrapolations on the basis of the present results.
In conclusion, our results indicate that routine OS protocol may have a negative influence on endometrial receptivity and embryonic implantation in mice. Conversely, OS with GnRH agonist, but not GnRH antagonist, appears to reverse the expression of endometrial integrin 3 and LIF and improves the uterine receptivity in mice. In the human IVF clinic, the low implantation rate observed with OS protocol in combination with GnRH antagonist might partly result from the impaired endometrial receptivity. Further studies in this aspect are needed to provide more definitive answers.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
The authors thank Dr Andrew P. Braun in Department of Pharmacology & Therapeutics, University of Calgary, for his helpful discussion and advice on the use of English. This work was supported by the grant to He-Feng Huang from Zhejiang Natural Science Foundation of China and Pao Yu-Kong and Pao Zhao-long Scholarship.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Albano C, Felberbaum RE, Smitz J, Riethmuller-Winzen H, Engel J, Diedrich K, Devroey P. (2000) Ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the luteinizing hormone-releasing hormone (LHRH)-antagonist cetrorelix and the LHRH-agonist buserelin. European Cetrorelix Study Group. Hum Reprod 15:526–531.
Al-Inany H and Aboulghar M. (2002) GnRH antagonist in assisted reproduction: a Cochrane review. Hum Reprod 17:874–885.[Abstract]
Arici A, Engin O, Attar E, Olive D. (1995) Modulation of leukemia inhibitory factor gene expression and protein biosynthesis in human endometrium. J Clin Endocrinol Metab 80:1908–1915.[Abstract]
Bahceci M, Ulug U, Ben-Shlomo I, Erden HF, Akman MA. (2005) Use of a GnRH antagonist in controlled ovarian hyperstimulation for assisted conception in women with polycystic ovary disease: a randomized, prospective, pilot study. J Reprod Med 50:84–90.[ISI][Medline]
Beckers NGM, Macklon NS, Eijkemans MJ, Ludwig M, Felberbaum RE, Diedrich K, Bustion S, Loumaye E, Fauser BCJM. (2003) Nonsupplemented luteal phase characteristics after the administration of recombinant human chorionic gonadotropin, recombinant luteinizing hormone, or gonadotropin-releasing hormone (GnRH) agonist to induce final oocyte maturation in in vitro fertilization patients after ovarian stimulation with recombinant follicle-stimulating hormone and GnRH antagonist cotreatment. J Clin Endocrinol Metab 88:4186–4192.
Bhatt H, Brunet LJ, Stewart CL. (1991) Uterine expression of leukemia inhibitory factor coincides with the onset of blastocyst implantation. Proc Natl Acad Sci USA 88:11408–11412.
Cavagna M and Mantese JC. (2003) Biomarkers of endometrial receptivity – a review. Placenta 24:Suppl B,, S39–S47.[Medline]
Coccia ME, Comparetto C, Bracco GL, Scarselli G. (2004) GnRH antagonists. Eur J Obstet Gynecol Reprod Biol. 115:Suppl 1,, S44–S56.
Creus M, Ordi J, Fabregues F, Casamitjana R, Carmona F, Cardesa A, Vanrell JA, Balasch J. (2003) The effect of different hormone therapies on integrin expression and pinopode formation in the human endometrium: a controlled study. Hum Reprod 18:683–693.
Creus M, Ordi J, Fabregues F, Casamitjana R, Ferrer B, Coll E, Vanrell JA, Balasch J. (2002) 
v3 integrin expression and pinopod formation in normal and out-of-phase endometria of fertile and infertile women. Hum Reprod 17:2279–2286.
Cullinan EB, Abbondanzo SJ, Anderson PS, Pollard JW, Lessey BA, Stewart 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.
Damario MA, Lesnick TG, Lessey BA, Kowalik A, Mandelin E, Seppala M, Rosenwaks Z. (2001) Endometrial markers of uterine receptivity utilizing the donor oocyte model. Hum Reprod 16:1893–1899.
Devroey P, Bourgain C, Macklon NS, Fauser BC. (2004) Reproductive biology and IVF: ovarian stimulation and endometrial receptivity. Trends Endocrinol Metab 15:84–90.[CrossRef][ISI][Medline]
Dey SK, Lim H, Das SK, Reese J, Paria BC, Daikoku T, Wang H. (2004) Molecular cues to implantation. Endocr Rev 25:341–373.
Ertzeid G and Storeng R. (1992) Adverse effects of gonadotrophin treatment on pre- and postimplantation development in mice. J Reprod Fertil 96:649–655.[Abstract]
Ertzeid G and Storeng R. (2001) The impact of ovarian stimulation on implantation and fetal development in mice. Hum Reprod 16:221–225.
Fauser BCJM and Devroey P. (2003) Reproductive biology and IVF: ovarian stimulation and luteal phase consequences. Trends Endocrinol Metabolism 14:236–242.[CrossRef][ISI][Medline]
Felberbaum RE and Diedrich K. (2003) Gonadotrophin-releasing hormone antagonists: will they replace the agonists? Reprod Biomed Online 6:43–53.[Medline]
Fossum GT, Davidson A, Paulson RJ. (1989) Ovarian hyperstimulation inhibits embryo implantation in the mouse. J In Vitro Fert Embryo Transf 6:7–10.[CrossRef][ISI][Medline]
Griesinger G, Felberbaum RE, Schultze-Mosgau A, Diedrich K. (2004) Gonadotropin-releasing hormone antagonists for assisted reproductive techniques: are there clinical differences between agents? Drugs 64:563–575.[CrossRef][ISI][Medline]
Hambartsoumian E. (1998) Endometrial leukemia inhibitory factor (LIF) as a possible cause of unexplained infertility and multiple failures of implantation. Am J Reprod Immunol 39:137–143.[ISI][Medline]
Hoozemans DA, Schats R, Lambalk CB, Homburg R, Hompes PG. (2004) Human embryo implantation: current knowledge and clinical implications in assisted reproductive technology. Reprod Biomed Online 9:692–715.[ISI][Medline]
Horcajadas JA, Riesewijk A, Polman J, van Os R, Pellicer A, Mosselman S, Simon C. (2005) Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles. Mol Hum Reprod 11:195–205.
Hughes EG, Fedorkow DM, Daya S, Sagle MA, Van de Koppel P, Collins JA. (1992) The routine use of gonadotropin-releasing hormone agonists prior to in vitro fertilization and gamete intrafallopian transfer: a meta-analysis of randomized controlled trials. Fertil Steril 58:888–896.[ISI][Medline]
Illera MJ, Cullinan E, Gui Y, Yuan L, Beyler SA, Lessey BA. (2000a) Blockade of the alpha(v)beta(3) integrin adversely affects implantation in the mouse. Biol Reprod 62:1285–1290.
Illera MJ, Juan L, Stewart CL, Cullinan E, Ruman J, Lessey BA. (2000b) Effect of peritoneal fluid from women with endometriosis on implantation in the mouse model. Fertil Steril 74:41–48.[CrossRef][ISI][Medline]
Kolibianakis E, Bourgain C, Albano C, Osmanagaoglu K, Smitz J, Van Steirteghem A, Devroey P. (2002) Effect of ovarian stimulation with recombinant follicle-stimulating hormone, gonadotropin releasing hormone antagonists, and human chorionic gonadotropin on endometrial maturation on the day of oocyte pick-up. Fertil Steril 78:1025–1029.[CrossRef][ISI][Medline]
Kondera-Anasz Z, Sikora J, Mielczarek-Palacz A. (2004) Leukemia inhibitory factor: an important regulator of endometrial function. Am J Reprod Immunol 52:97–105.[CrossRef][ISI][Medline]
Lee KY and DeMayo FJ. (2004) Animal models of implantation. Reproduction 128:679–695.
Lessey BA, Castelbaum AJ, Buck CA, Lei Y, Yowell CW, Sun J. (1994) Further characterization of endometrial integrins during the menstrual cycle and in pregnancy. Fertil Steril 62:497–506.[ISI][Medline]
Lessey BA, Castelbaum AJ, Sawin SW, Sun J. (1995) Integrins as markers of uterine receptivity in women with primary unexplained infertility. Fertil Steril 63:535–542.[ISI][Medline]
Lessey BA, Damjanovich L, Coutifaris C, Castelbaum A, Albelda SM, Buck CA. (1992) Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle. J Clin Invest 90:188–195.[ISI][Medline]
Mirkin S, Nikas G, Hsiu J-G, Diaz J, Oehninger S. (2004) Gene expression profiles and structural/functional features of the peri-implantation endometrium in natural and gonadotropin-stimulated cycles. J Clin Endocrinol Metab 89:5742–5752.
Ortmann O, Weiss JM, Diedrich K. (2001) Embryo implantation and GnRH antagonists: ovarian actions of GnRH antagonists. Hum Reprod 16:608–611.
Papanikolaou EG, Bourgain C, Kolibianakis E, Tournaye H, Devroey P. (2005) Steroid receptor expression in late follicular phase endometrium in GnRH antagonist IVF cycles is already altered, indicating initiation of early luteal phase transformation in the absence of secretory changes. Hum Reprod 20:1541–1547.
Prapas N, Prapas Y, Panagiotidis Y, Prapa S, Vanderzwalmen P, Schoysman R, Makedos G. (2005) GnRH agonist versus GnRH antagonist in oocyte donation cycles: a prospective randomized study. Hum Reprod 20:1516–1520.
Saadat P, Boostanfar R, Slater CC, Tourgeman DE, Stanczyk FZ, Paulson RJ. (2004) Accelerated endometrial maturation in the luteal phase of cycles utilizing controlled ovarian hyperstimulation: impact of gonadotropin-releasing hormone agonists versus antagonists. Fertil Steril 82:167–171.[CrossRef][ISI][Medline]
Seli E, Kayisli UA, Cakmak H, Bukulmez O, Bildirici I, Guzeloglu-Kayisli O, Arici A. (2005) Removal of hydrosalpinges increases endometrial leukaemia inhibitory factor (LIF) expression at the time of the implantation window. Hum Reprod 20:3012–3017.
Shapiro DB and Mitchell-Leef D. (2003) GnRH antagonist in in vitro fertilization: where we are now. Minerva Ginecol 55:373–388.[Medline]
Simon C, Oberye J, Bellver J, Vidal C, Bosch E, Horcajadas JA, Murphy C, Adams S, Riesewijk A, Mannaerts B, et al. (2005) Similar endometrial development in oocyte donors treated with either high- or standard-dose GnRH antagonist compared to treatment with a GnRH agonist or in natural cycles. Hum Reprod 20:3318–3327.
Stewart CL, Kaspar P, Brunet LJ, Bhatt H, Gadi I, Kontgen F, Abbondanzo SJ. (1992) Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature 359:76–79.[CrossRef][Medline]
Takahashi K, Mukaida T, Tomiyama T, Goto T, Oka C. (2004) GnRH antagonist improved blastocyst quality and pregnancy outcome after multiple failures of IVF/ICSI-ET with a GnRH agonist protocol. J Assist Reprod Genet 21:317–322.[CrossRef][ISI][Medline]
Tarlatzis BC and Bili H. (2004) Safety of GnRH agonists and antagonists. Expert Opin Drug Saf 3:39–46.[CrossRef][Medline]
Tavaniotou A, Bourgain C, Albano C, Platteau P, Smitz J, Devroey P. (2003) Endometrial integrin expression in the early luteal phase in natural and stimulated cycles for in vitro fertilization. Eur J Obstet Gynecol Reprod Biol 108:67–71.[CrossRef][ISI][Medline]
Thomas K, Thomson AJ, Sephton V, Cowan C, Wood S, Vince G, Kingsland CR, Lewis-Jones DI. (2002) The effect of gonadotrophic stimulation on integrin expression in the endometrium. Hum Reprod 17:63–68.
Thomas K, Thomson AJ, Wood SJ, Kingsland CR, Vince G, Lewis-Jones DI. (2003) Endometrial integrin expression in women undergoing IVF and ICSI: a comparison of the two groups and fertile controls. Hum Reprod 18:364–369.
Van der Auwera I and D’Hooghe T. (2001) Superovulation of female mice delays embryonic and fetal development. Hum Reprod 16:1237–1243.
Vlahos NF, Bankowski BJ, Zacur HA, Garcia JE, Wallach EE, Zhao Y. (2005) An oocyte donation protocol using the GnRH antagonist ganirelix acetate, does not compromise embryo quality and is associated with high pregnancy rates. Arch Gynecol Obstet 272:1–6.[CrossRef][Medline]
Weiss JM, Ludwig M, Ortmann O, Diedrich K. (2003) GnRH antagonists in the treatment of infertility. Ann Med 35:512–522.[CrossRef][ISI][Medline]
Wimsatt W. (1975) Some comparative aspects of implantation. Biol Reprod 12:1–40.[Medline]
Yang Z-M, Chen D-B, Le S-P, Harper MJK. (1996) Differential hormonal regulation of leukemia inhibitory factor (LIF) in rabbit and mouse uterus. Mol Reprod Dev 43:470–476.[CrossRef][ISI][Medline]
Submitted on January 9, 2006; resubmitted on March 22, 2006; resubmitted on May 7, 2006; accepted on May 11, 2006.
CiteULike 
Connotea 
Del.icio.us What's this?
| |||||||||||||||||||||||||||