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Endometrial LGR7 expression during menstrual cycle

2011; Elsevier BV; Volume: 95; Issue: 8 Linguagem: Inglês

10.1016/j.fertnstert.2011.01.124

1556-5653

Maria Rosaria Campitiello, Pasquale De Franciscis, Daniela Mele, Gaia Izzo, Antonio Agostino Sinisi, G. Delrio, Nicola Colacurci,

Pregnancy-related medical research

In a prospective observational study, 50 healthy patients aged 18–39 years, with regular ovulatory cycle and normal hormone levels, underwent endometrial biopsy in the proliferative and secretory phase of the menstrual cycle for semiquantitative reverse-transcription polymerase chain reaction analysis of mRNA for LGR7, the classic relaxin receptor. LGR7 is constitutively expressed in human endometrium, and an increased LGR7 immunostaining is demonstrated in the secretory phase, confirming the involvement of relaxin in the physiology of endometrium and suggesting its role in implantation. In a prospective observational study, 50 healthy patients aged 18–39 years, with regular ovulatory cycle and normal hormone levels, underwent endometrial biopsy in the proliferative and secretory phase of the menstrual cycle for semiquantitative reverse-transcription polymerase chain reaction analysis of mRNA for LGR7, the classic relaxin receptor. LGR7 is constitutively expressed in human endometrium, and an increased LGR7 immunostaining is demonstrated in the secretory phase, confirming the involvement of relaxin in the physiology of endometrium and suggesting its role in implantation. Relaxin is a polypeptide hormone belonging to the family of the insulin-like peptides associated with a wide range of function on ovary, endometrium, myometrium and connective tissue. In humans and other primates there is increasing evidence supporting the hypothesis that relaxin is able to substain the endometrial preparation for implantation and decidualization (1Einspanier A. Nubbemeyer R. Schlote S. Schumacher M. Ivell R. Furhrmann K. et al.Relaxin in the marmoset monkey: secretion pattern in the ovarian cycle and early pregnancy.Biol Reprod. 1999; 61: 512-520Crossref Scopus (48) Google Scholar, 2Stewart D.R. Celniker A.C. Taylor Jr., C.A. Cragun J.R. Overstreet J.W. Lasley B.L. Relaxin in the peri-implantation period.J Clin Endocrinol Metab. 1990; 70: 1771-1773Crossref PubMed Scopus (137) Google Scholar, 3Hayes E.S. Biology of primate relaxin: a paracrine signal in early pregnancy?.Reprod Biol Endocrinol. 2004; 2: 36-47Crossref PubMed Scopus (27) Google Scholar). Indeed, relaxin has been associated with endometrial angiogenesis, thickening and bleeding through the modulation of hormones, growth factors and other substances involved in the early stage of implantation (4Goldsmith L.T. Weiss G. Palejwala S. Plant T.M. Wojtczuk A. Lambert W.C. et al.Relaxin regulation of endometrial structure and function in the rhesus monkey.Proc Natl Acad Sci U S A. 2004; 101: 4685-4689Crossref PubMed Scopus (77) Google Scholar, 5Einspanier A. Muller D. Lubberstedt J. Bartsch O. Jurdzinski A. Fuhrmann K. et al.Characterization of relaxin binding in the uterus of the marmoset monkey.Mol Hum Reprod. 2001; 7: 963-970Crossref PubMed Scopus (27) Google Scholar). To understand the functional role of relaxin in the human endometrium, it is necessary to study the specific temporal endometrial expression of its receptor. Two relaxin receptors are known: LGR7 and LGR8, both G-protein–coupled receptors with 7-transmemebrane spanning domains (6Hsu S.Y. Nakabayashi K. Nishi S. Kumagai J. Kudo M. Sherwood O.D. et al.Activation of orphan receptors by the hormone relaxin.Science. 2002; 295: 671-674Crossref PubMed Scopus (666) Google Scholar). LGR7 receptor has higher affinity with relaxin compared with LGR8 (7Sudo S. Kumagai J. Nishi S. Layfield S. Ferraro T. Bathgate R.A. et al.H3 relaxin is a specific ligand for LGR7 and activates the receptor by interacting with both the ectodomain and the exoloop 2.J Biol Chem. 2003; 278: 7855-7862Crossref Scopus (234) Google Scholar) and is expressed in many tissues, including human endometrium, decidua, and term placenta (8Bond C.P. Parry L.J. Samuel C.S. Gehring H.M. Lederman F.L. Rogers P.A. et al.Increased expression of the relaxin receptor (LGR7) in human endometrium during the secretory phase of the menstrual cycle.J Clin Endocrinol Metab. 2004; 89: 3477-3485Crossref PubMed Scopus (33) Google Scholar, 9Bond C.P. Parry L.J. Samuel C.S. Gehring H.M. Lederman F.L. Rogers P.A. et al.Increased expression of the relaxin receptor (LGR7) in human endometrium during the secretory phase of the menstrual cycle.Ann N Y Acad Sci. 2005; 1041: 136-143Crossref PubMed Scopus (8) Google Scholar, 10Luna J.J. Riesewijk A. Horcajadas J.A. van Os R.D.R. Dominguez F. Mosselman S. et al.Gene expression pattern and immunoreactive protein localization of LGR7 receptor in human endometrium throughout the menstrual cycle.Mol Hum Reprod. 2004; 10: 85-90Crossref PubMed Scopus (31) Google Scholar, 11Krusche C.A. Kroll T. Beier H.M. Classen-Linke I. Expression of leucine-rich repeat–containing G-protein–coupled receptors in the human cyclic endometrium.Fertil Steril. 2007; 87: 1428-1437Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 12Ivell R. Balvers M. Pohnke Y. Telgmann R. Bartsch O. Milde-Langosch K. et al.Immunoexpression of the relaxin receptor LGR7 in breast and uterine tissues of humans and primates.Reprod Biol Endocrinol. 2003; 1: 114Crossref PubMed Scopus (66) Google Scholar, 13Lowndes K. Amano A. Yamamoto S.Y. Bryant-Greenwood G.D. The human relaxin receptor (LGR7): expression in the fetal membranes and placenta.Placenta. 2006; 27: 610-618Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 14Horton J.S. Yamamoto S.Y. Bryant-Greenwood G.D. Identification of the relaxin-responsive cells in the human choriodecidua at term.Ann N Y Acad Sci. 2009; 1160: 136-137Crossref Scopus (1) Google Scholar). To date, there are controversial data in the reported endometrial LGR7 protein expression (8Bond C.P. Parry L.J. Samuel C.S. Gehring H.M. Lederman F.L. Rogers P.A. et al.Increased expression of the relaxin receptor (LGR7) in human endometrium during the secretory phase of the menstrual cycle.J Clin Endocrinol Metab. 2004; 89: 3477-3485Crossref PubMed Scopus (33) Google Scholar, 9Bond C.P. Parry L.J. Samuel C.S. Gehring H.M. Lederman F.L. Rogers P.A. et al.Increased expression of the relaxin receptor (LGR7) in human endometrium during the secretory phase of the menstrual cycle.Ann N Y Acad Sci. 2005; 1041: 136-143Crossref PubMed Scopus (8) Google Scholar, 10Luna J.J. Riesewijk A. Horcajadas J.A. van Os R.D.R. Dominguez F. Mosselman S. et al.Gene expression pattern and immunoreactive protein localization of LGR7 receptor in human endometrium throughout the menstrual cycle.Mol Hum Reprod. 2004; 10: 85-90Crossref PubMed Scopus (31) Google Scholar, 11Krusche C.A. Kroll T. Beier H.M. Classen-Linke I. Expression of leucine-rich repeat–containing G-protein–coupled receptors in the human cyclic endometrium.Fertil Steril. 2007; 87: 1428-1437Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The aim of the present study was to investigate in vivo physiologic variations of LGR7 expression in a large sample of human endometrium in different phases of menstrual cycle. Fifty patients were enrolled, 18–39 years of age, with regular ovulatory cycles, normal pelvic examinations, and normal karyotypes. Hormone determinations were normal on day 3 of the menstrual cycle at the time the study was performed: FSH ≤10 mIU/mL, LH ≤10 mIU/mL, E2 >50 pg/mL. Transvaginal ultrasound was performed to ensure ovarian normality. Exclusion criteria were uterine, ovarian, endocrine, or chronic disease, known or suspected endometriosis, recurrent spontaneous abortion, poor ovarian response in previous ART cycles, and previous uterine surgery. Institutional Review Board was obtained for this study by the local Ethics Committee. All the patients signed a written informed consent. An endometrial biopsy was planned to be obtained from all of the patients in the proliferative (days 10–12) and in the secretory phases (days 19–21) of the menstrual cycle. In the proliferative phase, an endometrial biopsy was performed on both anterior and posterior aspect of the uterine cavity by means of a 5-mm continuous-flow hysteroscope with an incorporated 5-F working channel to introduce the grasping forcep. In the secretory phase an endometrial biopsy was performed with a Pipelle de Cornier (Prodimed, Neuilly-en-Thelle, France) without dilatation of the cervix. Two endometrial specimens were obtained from all of the biopsies: one was fixed in formalin and subsequently paraffin embedded for pathologic analysis and one was immediately immersed in RNA-later and stored at −20° for polymerase chain reaction (PCR) analysis. All 100 biopsies were considered to be adequate for histologic analysis according to the routine criteria for adequacy of endometrial biopsies applied by the consultant pathologists in the routine biopsy service. Histologic examination was performed "blindly" for clinical and laboratory data, and histologic criteria (15Noyes R.W. Hertig A.T. Rock J. Dating the endometrial biopsy.Fertil Steril. 1950; 1: 3-25Crossref Google Scholar) were considered to differentiate the endometrial samples in relation to day of ovulation: 50 samples in proliferative phase and 50 samples in secretory phase. PCR analysis was performed using specific oligonucleotides raised against the human LGR7 sequence available in GenBank (accession no. NM_021634). The primer sequences were: hLGR7for, 5′-CAGGCCGATGAGGACAACTG-3′; hLGR7rev, 5′-CAAAAACACCCGGCTTCAGGA-3′). Specific oligonucleotides were used to amplify human GAPDH (accession no. NM_002046; hGAPDH for: 5′-GTGGATATTGTTGCCATCAATGA-3′; hGAPDHrev: 5′-TGACCACAGTCCATGCCATC-3′) as a control. The amplified cDNA bands were analyzed using a Geldoc 2000 (Biorad). The relative densitometry of each sample was calculated normalizing the values obtained for LGR7 to the GAPDH values. All analyses were performed with R software, version 2.9.1 (R Foundation for Statistical Computing). The data reported represent the median values and the interquartile (IQ) range. A Wilcoxon rank sum test was performed to compare proliferative and secretory phases. LGR7 transcript (418 bp) was detected in the human endometrium in both proliferative and secretory phases (Fig. 1, top). GAPDH products were used as control of quality and equal loading of the cDNA. The PCR analysis on mRNA untreated with reverse-transcriptase did not show any signal (data not shown). The relative densitometry of hLGR7 and hGAPDH band (462 bp) measured using Gel Doc (Fig. 1, bottom) showed a significant difference (P=.032) between proliferative (median 0.780, IQ range 0.645–1.005) and secretory (median 1.165, IQ range 0.900–1.657) groups. The role of relaxin and its receptor in the human endometrium has been neglected in recent years. Two studies (8Bond C.P. Parry L.J. Samuel C.S. Gehring H.M. Lederman F.L. Rogers P.A. et al.Increased expression of the relaxin receptor (LGR7) in human endometrium during the secretory phase of the menstrual cycle.J Clin Endocrinol Metab. 2004; 89: 3477-3485Crossref PubMed Scopus (33) Google Scholar, 10Luna J.J. Riesewijk A. Horcajadas J.A. van Os R.D.R. Dominguez F. Mosselman S. et al.Gene expression pattern and immunoreactive protein localization of LGR7 receptor in human endometrium throughout the menstrual cycle.Mol Hum Reprod. 2004; 10: 85-90Crossref PubMed Scopus (31) Google Scholar) demonstrated that LGR7 receptor is expressed in the human endometrium but showing conflicting results. Bond et al. (8Bond C.P. Parry L.J. Samuel C.S. Gehring H.M. Lederman F.L. Rogers P.A. et al.Increased expression of the relaxin receptor (LGR7) in human endometrium during the secretory phase of the menstrual cycle.J Clin Endocrinol Metab. 2004; 89: 3477-3485Crossref PubMed Scopus (33) Google Scholar, 9Bond C.P. Parry L.J. Samuel C.S. Gehring H.M. Lederman F.L. Rogers P.A. et al.Increased expression of the relaxin receptor (LGR7) in human endometrium during the secretory phase of the menstrual cycle.Ann N Y Acad Sci. 2005; 1041: 136-143Crossref PubMed Scopus (8) Google Scholar) demonstrated increased expression of LGR7 and relaxin binding in the secretory endometrial tissue, suggesting a specific role for the hormone in the physiology of the human uterus; on the other hand, Luna et al. (10Luna J.J. Riesewijk A. Horcajadas J.A. van Os R.D.R. Dominguez F. Mosselman S. et al.Gene expression pattern and immunoreactive protein localization of LGR7 receptor in human endometrium throughout the menstrual cycle.Mol Hum Reprod. 2004; 10: 85-90Crossref PubMed Scopus (31) Google Scholar) reported the highest intensity of epithelial LGR-7 immunostaining during the proliferative phase but a trend to decreased LGR7 expression during all of the secretory phase, failing to demonstrate any significant regulation of LGR7 mRNA during the menstrual cycle. Indeed, some criticisms could be raised: In one set of research (8Bond C.P. Parry L.J. Samuel C.S. Gehring H.M. Lederman F.L. Rogers P.A. et al.Increased expression of the relaxin receptor (LGR7) in human endometrium during the secretory phase of the menstrual cycle.J Clin Endocrinol Metab. 2004; 89: 3477-3485Crossref PubMed Scopus (33) Google Scholar, 9Bond C.P. Parry L.J. Samuel C.S. Gehring H.M. Lederman F.L. Rogers P.A. et al.Increased expression of the relaxin receptor (LGR7) in human endometrium during the secretory phase of the menstrual cycle.Ann N Y Acad Sci. 2005; 1041: 136-143Crossref PubMed Scopus (8) Google Scholar), endometrial tissues were obtained from uteri of women with advanced age undergoing hysterectomy for gynecologic disorders and therefore not adequate for the study of physiologic endometrial regulation by LGR7; in the other paper (10Luna J.J. Riesewijk A. Horcajadas J.A. van Os R.D.R. Dominguez F. Mosselman S. et al.Gene expression pattern and immunoreactive protein localization of LGR7 receptor in human endometrium throughout the menstrual cycle.Mol Hum Reprod. 2004; 10: 85-90Crossref PubMed Scopus (31) Google Scholar), the samples were small. In the present study, a large homogeneous sample of healthy women was included with regular ovulatory cycle and normal hormone levels. Our results show that LGR7 transcript is detectable in the human endometrium in all phases of menstrual cycle, that LGR7 expression changes in a cyclic manner with decreasing level during the proliferative phase, and a striking significant increase in the secretory phase. However, a potential bias of our study could be the analysis of LGR7 expression after normalizing to the GAPDH values which are known to be sex steroid dependent. More recently, a trend of LGR-7 immunostaining similar to our findings has been shown (11Krusche C.A. Kroll T. Beier H.M. Classen-Linke I. Expression of leucine-rich repeat–containing G-protein–coupled receptors in the human cyclic endometrium.Fertil Steril. 2007; 87: 1428-1437Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar): LGR7 was in fact constitutively expressed throughout the menstrual cycle in the endometrium, with a small but significant increase in the early secretory phase compared with the late proliferative phase, but after removal of the outlier in the early secretory phase the LGR7 mRNA expression no longer exhibited significant differences. Our data could be relevant for the implantation process: Because the late preimplantation reflects the secretory phase, it should be assumed that relaxin plays an important role in embryo-endometrial recognition and decidualization of endometrial stromal cells (3Hayes E.S. Biology of primate relaxin: a paracrine signal in early pregnancy?.Reprod Biol Endocrinol. 2004; 2: 36-47Crossref PubMed Scopus (27) Google Scholar). Support for a relaxin role in implantation was provided by Stewart et al. (16Stewart D.R. Overstreet J.W. Celniker A.C. Hess D.L. Cragun J.R. Boyers S.P. et al.The relationship between hCG and relaxin secretion in normal pregnancies vs peri-implantation spontaneous abortions.Clin Endocrinol. 1993; 38: 379-385Crossref PubMed Scopus (61) Google Scholar), who showed that relaxin levels were significantly impaired in women with early pregnancy loss and that granulosa cell production of relaxin was predictive of pregnancy outcome in IVF patients (17Stewart D.R. VandeVoort C.A. Relaxin secretion by human granulosa cell culture is predictive of in vitro fertilization-–embryo transfer success.Hum Reprod. 1999; 14: 338-344Crossref PubMed Scopus (30) Google Scholar). However, substantial experimental data have suggested that circulating levels of relaxin may not explain the full biologic impact of this hormone: Circulating relaxin levels in steroid hormone supplemented patients undergoing ovum donation cycles remain below the limits of detection despite successful pregnancy (18Emmi A.M. Skurnick J. Goldsmith L.T. Gagliardi C.L. Schmidt C.L. Kleinberg D. et al.Ovarian control of pituitary hormone secretion in early human pregnancy.J Clin Endocrinol Metab. 1991; 72: 1359-1363Crossref Scopus (41) Google Scholar) and nonhuman primates subjected to ovariectomy also maintain pregnancy when supplemented with steroid hormones (19Castracane V.D. Lessing J. Brenner S. Weiss G. Relaxin in the pregnant baboon: evidence for local production in reproductive tissues.J Clin Endocrinol Metab. 1985; 60: 133-136Crossref Scopus (15) Google Scholar). Taken together, these observations show that the primate ovary may be the major source of relaxin, but that high levels of circulating relaxin may not be necessary for embryo implantation in primates. Moreover, experimental data have suggested that endometrial production of relaxin may play a role in decidualization and embryo implantation (3Hayes E.S. Biology of primate relaxin: a paracrine signal in early pregnancy?.Reprod Biol Endocrinol. 2004; 2: 36-47Crossref PubMed Scopus (27) Google Scholar) through the modulation of hormones, growth factors, and other substances (4Goldsmith L.T. Weiss G. Palejwala S. Plant T.M. Wojtczuk A. Lambert W.C. et al.Relaxin regulation of endometrial structure and function in the rhesus monkey.Proc Natl Acad Sci U S A. 2004; 101: 4685-4689Crossref PubMed Scopus (77) Google Scholar, 5Einspanier A. Muller D. Lubberstedt J. Bartsch O. Jurdzinski A. Fuhrmann K. et al.Characterization of relaxin binding in the uterus of the marmoset monkey.Mol Hum Reprod. 2001; 7: 963-970Crossref PubMed Scopus (27) Google Scholar). Studies of relaxin transcripts in human endometrium in pregnant and nonpregnant cycles have clearly indicated that relaxin expression preceded the expression of IGFBP-1 and PRL (20Bryant-Greenwood G.D. Rutanen E.M. Partanen S. Coelho T.K. Yamamoto S.Y. Sequential appearance of relaxin, prolactin and IGFBP-1 during growth and differentiation of the human endometrium.Mol Cell Endocrinol. 1993; 95: 23-29Crossref PubMed Scopus (82) Google Scholar), two classic markers of decidualization, suggesting a paracrine role for relaxin during the periimplantation period. Other data have shown that relaxin and prostaglandin E2 drive decidualization via interleukin-11 production by human endometrial stromal cells in mid-late secretory-phase endometrium (21Dimitriadis E. Stoikos C. Baca M. Fairlie W.D. McCoubrie J.E. Salamonsen L.A. Relaxin and prostaglandin E2 regulate interleukin 11 during human endometrial stromal cell decidualization.J Clin Endocrinol Metab. 2005; 90: 3458-3465Crossref PubMed Scopus (65) Google Scholar). Relaxin contributes to the regulation of vascular tone and angiogenesis through the induction of VEGF and nitric oxide (NO), with an inhibitory effect in the proliferative phase and stimulatory effect in the secretory phase (22Palejwala S. Tseng L. Wojtczuk A. Weiss G. Goldsmith L.T. Relaxin gene and protein expression and its regulation of procollagenase and vascular endothelial growth factor in human endometrial cells.Biol Reprod. 2002; 66: 1743-1748Crossref PubMed Scopus (99) Google Scholar). Relaxin may also help to enhance embryonic access to the maternal vasculature through non–vascular endothelial growth factor–mediated effects, such as reduction of platelet release and aggregation (23Bani D. Bigazzi M. Masini E. Bani G. Sacchi T.B. Relaxin depresses platelet aggregation: in vitro studies on isolated human and rabbit platelets.Lab Invest. 1995; 73: 709-716Google Scholar), reduction of vascular fibrosis by stimulating the expression of tissue plasminogen activator (24Qin X. Garibay-Tupas J. Chua P.K. Cachola L. Bryant-Greenwood G.D. An autocrine/paracrine role of human decidual relaxin. I. Interstitial collagenase (matrix metalloproteinase-1) and tissue plasminogen activator.Biol Reprod. 1997; 56: 800-811Crossref PubMed Scopus (81) Google Scholar), increase of prorenin production (25Poisner A.M. Regulation of utero-placental prorenin.Adv Exp Med Biol. 1995; 377: 411-426Crossref Scopus (16) Google Scholar), and direct vasodilatation of the endometrial vessels (26Bani D. Relaxin: a pleiotropic hormone.Gen Pharmacol. 1997; 28: 13-22Crossref PubMed Scopus (200) Google Scholar). It has been demonstrated in endometrial stromal cells of mice that relaxin is able to stimulate production of laminin (27Bani G. Maurizi M. Bigazzi M. Bani S.T. Effects of relaxin on the endometrial stroma. Studies in mice.Biol Reprod. 1995; 53: 253-262Crossref PubMed Scopus (40) Google Scholar), needed for trophoblast adherence and its invasion into the endometrial stroma. Relaxin also up-regulates the expression of the glycoprotein glycodelin during the last week of the luteal phase and during the periimplantation phase (28Sunder S. Lenton E.A. Endocrinology of the peri-implantation period.Baillieres Best Pract Res Clin Obstet Gynaecol. 2000; 14: 789-800Abstract Full Text PDF PubMed Scopus (51) Google Scholar). In the light of such evidences, a direct relationship between relaxin, its receptor, and achievement of implantation could be hypothesized. Our data showing the increase of LGR7 expression in the secretory phase add new information and insight regarding this topic. However, further studies are necessary to correlate the expression of LGR7 with the success of implantation.