Endometrial expression of HCG/LH receptor in infertile women with repeated implantation failure

Transcription

1 Original Article J Womens Med 2011;4(1):6-10 doi: /jwm pissn eissn Endometrial expression of HCG/LH receptor in infertile women with repeated implantation failure Byung Chul Jee, MD, PhD 1,2,3, Chang Suk Suh, MD, PhD 1,2,3, and Seok Hyun Kim, MD, PhD 1,3 1 Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam; 2 Department of Obstetrics and Gynecology, Seoul National University College of Medicine; 3 Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul, Korea Objective: To investigate endometrial expression of human chorionic gonadotropin (HCG)/luteinizing hormone (LH) receptor in infertile women who experienced repeated implantation failure. Methods: Sixty-one women participating in vitro fertilization due to unexplained infertility (n=37) or male factor (n=24) were included. Endometrial biopsies were taken during luteal phase in a spontaneous unmedicated cycle. The streptavidin-biotin-peroxidase complex technique was used for immunohistochemistry. The signal intensity was assigned semiquantitatively and then compared between the repeated implantation failure group (three or more failures, n=33) and controls (two or less failures, n=28). Results: Luteal expressions of HCG/LH receptor at all three compartments were not different statistically between the two groups. However, late luteal expressions of HCG/LH receptor tended to be consistently low in repeated implantation failure group. In repeated implantation failure group, HCG/LH receptor expression continuously decreased throughout the entire luteal phase. Conclusion: Low expression of HCG/LH receptor in late luteal endometrium might contribute to repeated implantation failure. Key words: Endometrium; HCG/LH receptor; In vitro fertilization; Implantation Introduction Human embryo implantation can only take place during the window of implantation, a period of endometrial receptivity spanning cycle day (CD) of the luteal endometrium. 1 Implantation involves a complex sequence Received: Revised : Accepted: Corresponding author: Seok Hyun Kim, MD, PhD Department of Obstetrics and Gynecology, Seoul National University Hospital, 28 Yeongeon-dong, Jongno-gu, Seoul , Korea Tel: , Fax: seokhyun@snu.ac.kr Copyright c Korean Society of Obstetrics and Gynecology of signaling events, consisting of a large number of molecular mediators such as ovarian hormones, cytokines, growth factors, adhesion molecules, and others. 2 Endometrial samples can be used to identify molecules associated with uterine receptivity to obtain a better insight into human implantation, but there has been no consensus as to what constitutes a marker of receptive endometrium. 3 Human chorionic gonadotropin (HCG) is one of the most specific molecules produced by the embryo, even before its implantation. Recently, the specific interaction of blastocyst-derived HCG and its endometrial receptor (HCG/ LH-R) has been emerged to be a fundamental component of the molecular dialogue at the materno-fetal interface. 4 From previous studies, HCG was shown to play a significant role in implantation and tolerance of the embryo, decidual differentiation and remodeling, as well as in placentation. 5,6 The profile pattern of HCG/LH-R expression by endometrial ep- 6

2 Byung Chul Jee, et al. Endometrial HCG/LH receptor ithelium correlates with the theoretical timing of the implantation window. 7 Although several studies have demonstrated the expressions and possible functions of endometrial HCG/LH-R in fertile women, clinical implications on the presence of its molecule in infertile women are sparse. In the present study, we examined whether expression of HCG/LH-R in a luteal endometrium contributes to repeated implantation failure. Materials and Methods Thirty-three infertile women who experienced repeated in vitro fertilization (IVF) failures (three or more) were included in this study. Twenty-eight women who experienced two or less failures of IVF served as control. All of the subjects had regular menstrual cycles and received IVF treatment due to unexplained infertility (n=37) or male factor (n=24). Women with irregular menstruation, such as women with polycystic ovary syndrome, and with apparent endometrial pathology were entirely excluded. Unexplained infertility was defined as those who had normal hysterosalpingogram, normal semen analysis, and normal hormonal profiles. Informed consent was obtained from all study participants. The study was approved by the institutional review board of Seoul National University Bundang Hospital. All patients underwent a hysteroscopic procedure under local and mild sedative anesthesia with pretreatment by insertion of laminaria. After careful hysteroscopic inspection, endometrial samplings were performed by silastic suction curette in the luteal phase in a spontaneous unmedicated cycle. The obtained endometrial samples were sent to one pathologist for routine histologic dating and pathologic examination. The remaining endometrial tissue samples were immediately stored in a paraffin block. When pathologic conditions were found in endometrium at the time of hysteroscopy or after microscopic pathologic examination (i.e., polyps, leiomyomas, inflammation, hyperplasia, or inactive endometrium), we excluded these women from the final analysis. The patients were grouped according to histologic endometrial dating by the Noyes criteria: early (CD 16-19), mid (CD 20-24), and late luteal phase (CD 25-27). The streptavidin-biotin-peroxidase complex technique was used for immunohistochemistry, as described previously. 8 All incubations were performed at room temperature unless otherwise stated. The sections were deparaffinized in xylol and then rehydrated in a graded series of ethanol according to standard procedures. Antigen retrieval was done by microwave treatment in sodium citrate buffer (10 mm/l, ph 6.0; Zymed Laboratories, San Francisco, CA, USA) for 20 minutes. Endogenous peroxidase activity was quenched with 1% H 2O 2 in phospate buffered solution (PBS) for 10 minutes. After the sections were washed in PBS (3-5 minutes), endogenous avidin and biotin binding sites were blocked by incubation with avidin and biotin blocking solution (Blocking Kit; Vector Laboratories, Burlingame, CA, USA) for 30 minutes. Nonspecific antibody binding was reduced by diluting the primary and secondary antibodies in antibody diluent solution (Zymed Laboratories) containing bovine serum albumin. The sections were incubated with rabbit polyclonal antibodies for HCG/LH-R (acris antibodies, SP4593P, 3.3 μg /ml) overnight at 4 o C. The sections were then incubated with the biotinylated secondary antibody diluted 1:200 for 45 minutes before incubation with horseradish peroxidase-streptavidin (1:200, SA-5004; Vector Laboratories) for 45 minutes. The antigen-antibody reaction was visualized with 3,3 -diaminobenzidine as chromogen (DAB, S ; DakoCytomation, Glostrup, Denmark). After washing, the sections were counterstained with Mayer s hematoxylin, dehydrated, and mounted in mounting medium (Pertex; Histolab Products AB, Göteburg, Sweden). Positive controls were performed for all antibodies by staining human placenta tissues with known expression of the corresponding epitopes. Negative controls were performed by replacing the primary antibodies with normal goat serum (X0907; DakoCytomation). Interassay variability in immunoreactivity was assessed by staining a section from the same paraffin block in every assay. The immunohistochemical staining was estimated by one pathologist in stained areas of the epithelial cells, stromal compartment and endothelial cells, separately. A signal intensity was assigned semiquantitatively on a 4-point scale from 0 to 3, where 0=no staining, 1=weak, 2=moderate, and 3=strong. An area was considered as strong when the staining intensity was similar to the corresponding positive control. Then, each intensity score was multiplied by their distribution score from 1 to 4, where 1=less than 5% of the area stained, 2=5-25%, 3=25-50%, and 4=>50%. At least five randomly chosen areas of the endometrium were evaluated and then averaged for each compartment. Statistical analysis was performed using MedCalc 4.15 (MedCalc Software, Mariakerke, Belgium). The Chi-square test was used to compare proportions. If data showed a normal distribution, the Student s t test was used to compare two means. If the data did not show a normal distribution, 7

3 J Womens Med Vol. 4, No. 1, 2011 the Wilcoxon rank test was used to compare two medians. A P-value of <0.05 (two-tailed) was considered statistically significant. Results The age of women, distribution of infertility factors and CD of endometrial biopsy did not differ between repeated implantation failure group and control (Table 1). HCG/LH-R was expressed evenly at all three compartments (i.e. more than 50% of the area stained) (Fig. 1). At glandular epithelium, the signals were mainly located in the subnuclear and cytoplasmic area. Luteal expressions of HCG/LH-R at all three compartments were not different statistically between the two groups (Fig. 2). However, late luteal expressions of HCG/LH-R tended to be consistently low in repeated implantation failure group at all three compartments (2.9±4.5 vs. 8.0±2.8 in glands, 6.9±5.0 vs. 12.0±0.0 in stroma, 7.4±4.9 vs. 11.2±1.8 in endothelium). In repeated implantation failure group, HCG/LH-R expression continuously decreased throughout the entire luteal phase at all three compartments. This expression pattern was not observed in the control. The present study demonstrated that late luteal expression of HCG/LH-R was consistently low in women who experienced repeated implantation failure although statistically not significant when compared with control. In addition, repeated implantation failure group showed decreasing tendency of HCG/LH-R expression throughout the luteal phase. Thus this peculiar expression pattern might contribute to repeated implantation failure. However, the late luteal endometrium could be obtained from only twelve women thus further large-scaled study will be needed to confirm our finding. The biological actions of LH and HCG are mediated by A C B D Discussion E Fig. 1. Representative photomicrographs showing human chorionic gonadotropin (HCG)/luteinizing hormone (LH) receptor expressions in endometrial glands (A, B, C) and stroma (D, E, F) (stained by rabbit polyclonal anti-hcg/lh-r antibody, 400). F Table 1. Clinical characteristics in the study population Repeated implantation failure (n=33) Control (n=28) P Age of woman (yr) 34.0± ±3.8 NS Duration of infertility (yr) 6.2± ± Number of previous IVF cycles 4.6± ± Infertility factor NS Unexplained Male Cycle day of endometrial biopsy 21.2± ±3.4 NS Number of patients according to luteal phase NS Early Mid 14 8 Late 7 5 Values are presented as mean±standard deviation or number. IVF, in vitro fertilization; NS, not significant. 8

4 Byung Chul Jee, et al. Endometrial HCG/LH receptor RIF Control Fig. 2. The mean intensity score of human chorionic gonadotropin/luteinizing hormone receptor expression at endometrial glands (upper), stroma (middle), and endothelium (lower) according to luteal phase (upper limit bar represents mean+ mean±standard deviation). By using the Wilcoxon rank test, each score was not statistically different between repeated implantation failure (RIF) group (three or more failures) and control (two or less failures) at all three compartments. binding to the same G-protein-coupled membrane receptor primarily expressed in gonadal cells. Several studies have also confirmed the presence of HCG/LH-R in non-gonadal tissues, such as endometrium, myometrium, placenta/decidua and fallopian tubes It is well known that HCG stimulates ovarian steroidogenesis and maintain corpus luteum during pregnancy. In addition, extraovarian receptors appear to be functional and have been shown to mediate various effects. 14 Earlier report indicated that full-length HCG/LH-R mrna was detected both in proliferative as well as early and mid-secretory phase. 12 A subsequent study through real-time PCR demonstrated an increased expression of HCG/LH-R in the mid-luteal phase, thus suggesting that HCG/LH-R could constitute a possible marker of endometrial receptivity. 13 In that study, HCG/LH-R mrna by reverse transcription-polymerase chain reaction was detected both in epithelial and stromal cells isolated from endometrial biopsies of fertile women. Several studies demonstrated that in vivo or in vitro injection of HCG could induce increase of uterine blood flow via vasodilation and angiogenesis, 15 decidual differentiation and remodeling, 5,6,10 and up-regulation of cyclooxygenase-2, SerpinA3, matrix metalloproteinase (MMP), LIF, IL-6 and/or C3. 16 HCG increased LIF but reduced IL-6 secretion by cultured human endometrial epithelial cells. 13 HCG administration during the secretory phase significantly modulated several endometrial paracrine parameters that correlate with endometrial marker for differentiation and decidualization (IGFBP-1), angiogenesis (VEGF), implantation (LIF, M-CSF) and tissue remodeling (MMP-9) assessed by intrauterine microdialysis. 17 Hence HCG appears to directly modulate endometrial differentiation and function in humans. Until now, there have been no studies with regards the clinical significance of HCG/LH-R expression as a new biomarker of uterine receptivity for embryo implantation. 4 Acknowledging the fact that HCG might have local and systemic effects on the embryo-endometrial microenvironment, Tesarik et al. 18 demonstrated an increased implantation rate after the administration of HCG in the early secretory phase of patients undergoing egg donation cycle. However, no advantage was found concerning pregnancy and implantation rate by supplementing HCG in patients undergoing transfer of frozen-thawed embryo in hormonally modulated cycles. 19 The assumed etiologies for repeated implantation failure after IVF are reduced endometrial receptivity, embryonic defects, or multifactorial effectors such as endometriosis and hydrosalpinx. 20 We excluded all women with apparent endometrial pathology, such as uterine cavity abnormalities and thin endometrium. We confirmed the normalcy of endometrium by both hysteroscopic and histologic inspection. All study subjects produced an adequate number of embryos and had no hydrosalpinx or endometriosis at the time of study. The women with irregular menstruation were excluded because endometrial dating was hard to determine in those women. Implantation failure remains an unsolved 9

5 J Womens Med Vol. 4, No. 1, 2011 problem in reproductive medicine. 21 Inadequate uterine receptivity is known to be responsible for approximately two thirds of implantation failures, whereas the embryo itself is responsible for only one third of these failures. 22 Although several studies suggested the fundamental role of endometrial HCG/LH-R on implantation and decidualization, clinical implications of HCG/LH-R in infertile women are sparse. Our present results are not conclusive for verifying the role of HCG/LH-R in repeated implantation failure because this was a small study. Further large-scaled study should be warranted to determine whether aberrant expression of HCG/LH-R contributes to repeated implantation failure. Acknowledgements This work was supported by grant no from the Seoul National University Bundang Hospital Research Fund. References 1. Revel A. Multitasking human endometrium: a review of endometrial biopsy as a diagnostic tool, therapeutic applications, and a source of adult stem cells. Obstet Gynecol Surv 2009; 64: Achache H, Revel A. Endometrial receptivity markers, the journey to successful embryo implantation. Hum Reprod Update 2006;12: Diedrich K, Fauser BC, Devroey P, Griesinger G; Evian Annual Reproduction (EVAR) Workshop Group. The role of the endometrium and embryo in human implantation. Hum Reprod Update 2007;13: Perrier d'hauterive S, Berndt S, Tsampalas M, et al. Dialogue between blastocyst hcg and endometrial LH/hCG receptor: which role in implantation? Gynecol Obstet Invest 2007;64: Cameo P, Srisuparp S, Strakova Z, Fazleabas AT. Chorionic gonadotropin and uterine dialogue in the primate. Reprod Biol Endocrinol 2004;2: Filicori M, Fazleabas AT, Huhtaniemi I, et al. Novel concepts of human chorionic gonadotropin: reproductive system interactions and potential in the management of infertility. Fertil Steril 2005;84: Licht P, Russu V, Lehmeyer S, Wissentheit T, Siebzehnrubl E, Wildt L. Cycle dependency of intrauterine vascular endothelial growth factor levels is correlated with decidualization and corpus luteum function. Fertil Steril 2003;80: Jee BC, Suh CS, Kim KC, et al. Expression of vascular endothelial growth factor-a and its receptor-1 in a luteal endometrium in patients with repeated in vitro fertilization failure. Fertil Steril 2009;91: Hudelist G, Huber A, Knoefler M, et al. beta-hcg/lh receptor (beta-hcg/lh-r) expression in eutopic endometrium and endometriotic implants: evidence for beta-hcg sensitivity of endometriosis. Reprod Sci 2008;15: Zhou XL, Lei ZM, Rao CV. Treatment of human endometrial gland epithelial cells with chorionic gonadotropin/luteinizing hormone increases the expression of the cyclooxygenase-2 gene. J Clin Endocrinol Metab 1999;84: Shemesh M, Mizrachi D, Gurevich M, et al. Expression of functional luteinizing hormone (LH) receptor and its messenger ribonucleic acid in bovine endometrium: LH augmentation of camp and inositol phosphate in vitro and human chorionic gonadotropin (hcg) augmentation of peripheral prostaglandin in vivo. Reprod Biol 2001;1: Licht P, von Wolff M, Berkholz A, Wildt L. Evidence for cycle-dependent expression of full-length human chorionic gonadotropin/luteinizing hormone receptor mrna in human endometrium and decidua. Fertil Steril 2003;79 Suppl 1: Perrier d'hauterive S, Charlet-Renard C, Berndt S, et al. Human chorionic gonadotropin and growth factors at the embryonic-endometrial interface control leukemia inhibitory factor (LIF) and interleukin 6 (IL-6) secretion by human endometrial epithelium. Hum Reprod 2004;19: Ziecik AJ, Bodek G, Blitek A, Kaczmarek M, Waclawik A. Nongonadal LH receptors, their involvement in female reproductive function and a new applicable approach. Vet J 2005;169: Toth P, Lukacs H, Gimes G, et al. Clinical importance of vascular LH/hCG receptors: a review. Reprod Biol 2001;1: Sherwin JR, Sharkey AM, Cameo P, et al. Identification of novel genes regulated by chorionic gonadotropin in baboon endometrium during the window of implantation. Endocrinology 2007;148: Licht P, Russu V, Lehmeyer S, Moll J, Siebzehnrubl E, Wildt L. Intrauterine microdialysis reveals cycle-dependent regulation of endometrial insulin-like growth factor binding protein-1 secretion by human chorionic gonadotropin. Fertil Steril 2002;78: Tesarik J, Hazout A, Mendoza C. Luteinizing hormone affects uterine receptivity independently of ovarian function. Reprod Biomed Online 2003;7: Ben-Meir A, Aboo-Dia M, Revel A, Eizenman E, Laufer N, Simon A. The benefit of human chorionic gonadotropin supplementation throughout the secretory phase of frozen-thawed embryo transfer cycles. Fertil Steril 2010;93: Margalioth EJ, Ben-Chetrit A, Gal M, Eldar-Geva T. Investigation and treatment of repeated implantation failure following IVF-ET. Hum Reprod 2006;21: Krussel JS, Bielfeld P, Polan ML, Simon C. Regulation of embryonic implantation. Eur J Obstet Gynecol Reprod Biol 2003;110 Suppl 1:S Christiansen OB, Nielsen HS, Kolte AM. Future directions of failed implantation and recurrent miscarriage research. Reprod Biomed Online 2006;13: