and Gynecology, The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School. b Department of Obstetrics

Transcription

1 FERTILITY AND STERILITY VOL. 74, NO. 2, AUGUST 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Histochemical localization of endometrial insulin-like growth factor binding protein-1 and -3 during the luteal phase in controlled ovarian hyperstimulation cycles: a controlled study Samuel E. Brown, M.D., a Erik Mandelin, M.D., b Sergio Oehninger, M.D., a James P. Toner, M.D., Ph.D., a Markku Seppala, M.D., b and Howard W. Jones, Jr., M.D. a The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia; and Helsinki University Central Hospital, Helsinki, Finland Received November 12, 1999; revised and accepted January 31, Supported by The Jones Foundation (Norfolk, VA), The Cancer Society of Finland, the Academy of Finland, Finska Läkaresällskapet, and the Helsinki University Central Hospital Research Fund (Helsinki, Finland). Reprint requests: Samuel E. Brown, M.D., Florida Institute for Reproductive Medicine, 836 Prudential Dr. #902, Jacksonville, Florida (FAX: ; sambrown99@yahoo.com). a Department of Obstetrics and Gynecology, The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School. b Department of Obstetrics and Gynecology, Helsinki University Central Hospital /00/$20.00 PII S (00) Objective: To determine if controlled ovarian hyperstimulation (COH) affects the endometrial expression of IGFBP-1 and IGFBP-3. Design: Prospective, controlled study. Setting: Tertiary infertility clinic. Patient(s): Eighteen oocyte donors undergoing COH cycles and 17 natural cycle controls. Intervention(s): Controlled ovarian hyperstimulation, endometrial biopsies. Main Outcome Measure(s): Immunohistochemical scoring of endometrial IGFBP-1 and -3 expression, morphological endometrial dating, and serum estradiol (E 2 ), LH, and progesterone (P 4 ) concentrations. Result(s): No statistically significant difference was observed between natural and stimulated cycles in change in IGFBP-1 or -3 over standardized cycle days throughout the window of embryo implantation (days 17 24). The IGFBP-1 and -3 expression was zero or near zero for both the natural and COH cycles until day Both IGFBPs showed increased production throughout the secretory phase. Advanced endometrial histology ( 1 day) in glands and stroma was noted in COH cycles. Significant positive correlations of E 2 and P 4 were noted with IGFBP-1 and -3 but not with advanced endometrial morphology in the COH cycles. Conclusion(s): The COH cycles have no significantly increased endometrial IGFBP-1 or -3 expression throughout the implantation phase of the luteal cycle compared with normal menstrual cycles. Both IGFBPs were absent in the proliferative phase and increased throughout the secretory portion of the embryo implantation window. (Fertil Steril 2000;74: by American Society for Reproductive Medicine.) Key Words: Controlled ovarian hyperstimulation, natural menstrual cycle, insulin-like growth factor binding protein, implantation, endometria Human embryo endometrial implantation is a complex event. A symphony of histologic, biochemical, and physiologic endometrial changes prepare the endometrium for proper embryo attachment, invasion, and growth during early pregnancy. The insulin-like growth factor (IGF) autocrine-paracrine system is believed to play a role in endometrial differentiation and trophoblast growth. The IGFs bind with high affinity to a family of binding proteins (IGFBPs) that regulate the actions of the IGFs at their target cells. In the human endometrium, IGFBP messenger RNA (mrna) and proteins are expressed only in the endometrial stroma, and their expression is differentially regulated in secretory compared with proliferative phase endometrium (different steroidogenic environments) (1, 2). Further investigation using menopausal women showed a close relationship between progestins as the expression of IGFBP-1 (3). Controlled ovarian hyperstimulation (COH) advances endometrial development as evi- 338

2 denced by histologic changes, estrogen receptor, progesterone receptor, and pinopod expression (4, 5). Controversy exists about the impact of this advancement leading to a dyssynchrony between embryonic and endometrial events, reducing fecundity. Whether COH also advances insulin-like growth factor binding protein-1 and -3 expression was the subject of this study. MATERIALS AND METHODS Subjects This study was approved by the Institutional Review Board of Eastern Virginia Medical School. The subjects were fully informed and signed a study consent form. Oocyte donors and natural cycling subjects were recruited and studied at the Jones Institute (Norfolk, VA). Oocyte donors were recruited (n 18) for participation. Oocyte donors were treated with a long GnRH agonist protocol (Lupron; TAP Pharmaceuticals, Inc., Deerfield, IL) using 1 mg/d s.c. starting in the midluteal phase of the preceding cycle until the day of menses when it was reduced to 0.5 mg/d with a further reduction to 0.25 mg/d from the first day of gonadotropin injections through the day of hcg (Profasi; Serono, Norwell, MA) administration. Recombinant FSH (Gonal-F; Serono) IU/d for 9 12 days from the third day of menses was given, followed by 10,000 IU of hcg. Periodic transvaginal ultrasound examination and estradiol (E 2 ) measurement guided FSH dosing was used. Transvaginal oocyte retrieval was performed under light i.v. sedation and transvaginal ultrasound guidance hours after hcg injection. Serum determinations for LH, E 2, and progesterone (P 4 ) were performed throughout the follicular phase stimulation surveillance and with each luteal biopsy day. In addition, 17 healthy women aged years with normal menstrual cycles (i.e., 28 7 days) and no use of medications or hormonal contraception were studied (natural cycle controls). Subjects also consented to no over-thecounter medication or herbal remedy consumption throughout the study cycle. Standardization of Menstrual Cycle Days Natural cycle subjects (controls) underwent blood draws each morning (8 9 A.M.) and evening (8 9 P.M.) before LH surge. Serum determinations for LH, E 2, and P 4 were performed from each blood draw. The onset of the LH surge was defined as the day when the LH level exceeded 2 SDs above that patient s mean LH levels during the previous week and was called day 12 (6). The day of hcg injection during oocyte donor cycles was standardized to day 12 of the menstrual cycle. Endometrial Biopsy and Tissue Processing Endometrial biopsies were performed to maximize sampling throughout the embryo implantation period. Oocyte donors subjects were asked to return for endometrial biopsies every second or third day during the window of implantation cycle (days 17 through 24 to adequately evaluate each day during the implantation window [7 9]). Natural cycle controls had endometrial biopsies between cycle days 12 through 24. Each woman (donor or control) underwent a biopsy a minimum of two times (range, 2 4). Subjects had no luteal progesterone support, and endometrial biopsies were performed with use of a Pipelle endometrial suction curette (CooperSurgical, Shelton, CT). Biopsies were performed from all four uterine quadrants. Endometrial tissue was immediately placed into 10% formalin with subsequent embedding into paraffin block for routine sectioning. Insulin-Like Growth Factor Binding Protein-1 and -3 Determinations The IGFBP-1 and -3 determinations were performed by immunohistochemical staining at Helsinki University Central Hospital after being shipped from Norfolk, Virginia, in a coded fashion for blind reading. Formalin-fixed, paraffinembedded normal endometrial tissues were examined with use of the immunoglobulin (Ig)G fraction of polyclonal anti-igfbp-1 and monoclonalanti-igfbp-3 antibodies for immunoperoxidase method essentially as described before (10, 11). Staining of all specimens was performed on the same day. Staining of endometrial glands was subjectively scored by three investigators on a scale of 0 4. Subject information was unknown to the investigators by random slide numbering. Zero equated to no stain detection, 1 1% 25% cells staining, 2 26% 50%, 3 51% 75%, and 4 76% 100%. Intraassay and interassay reliability was 5% and 9%, respectively. Endometrial Dating Hematoxylin and eosin staining was performed to date the endometrium as described by the criteria of Noyes et al. (12). Statistical Analysis Comparisons of age, body mass index (BMI), gravidity, and parity between COH and natural cycle subjects were made with unpaired t-tests. A generalized estimating equation (GEE) approach to longitudinal data was used to compare change in glycodelin over adjusted cycle days for COH and natural cycles. A best-fit linear model with polynomial terms for nonlinear trends was found. Serum P 4 and E 2 levels in the natural and COH cycles were correlated with glycodelin results with use of analysis of covariance to account for multiple observations per subject. RESULTS A total of 35 subjects were included in the analysis. The average age (years SE) of all subjects was 29.9 ( 0.69) and the average BMI (kg/m 2 SE) was 24.3 ( 0.84). Demographic parameters were similar between the control and study patient groups (Table 1). The IGFBP-1 and -3 were visualized in the endometrial stroma in all subjects. No FERTILITY & STERILITY 339

3 TABLE 1 Summary of demographic data for 18 oocyte donors undergoing COH cycles and 17 controls with natural cycles. Variable COH cycles Natural cycles No. of subjects No. of biopsies Mean ( SE) age (y) Mean ( SE) BMI (kg/m 2 ) Mean ( SE) gravidity Mean ( SE) parity Note: All P values were not significant. IGFBP-1 or -3 were observed in the gland or surface epithelium of the biopsies. In addition, no histochemical localization of IGFBP-1 or -3 was found in the endometrial glands before day 12 of the natural and COH cycles. No statistically significant difference was observed between natural and stimulated cycles in change in IGFBP-1 or -3 over standardized cycle days throughout the window of embryo implantation (days 17 24). For the most part, IGFBP-1 was zero or near zero for both the natural and COH cycles until day The IGFBP-1 increased thereafter for both cycle types, but at a faster rate for stimulated cycles. Figure 1 plots the actual curves and 95% confidence intervals for IGFBP-1. Peak IGFBP-1 expression was noted at the end of the implantation window (day 24) with noted increase to a mean score of 2 (0 4 scale). For the most part, IGFBP-3 was zero or near zero for both the natural and COH cycles until days The IGFBP-3 increased thereafter for both cycle types, but at a faster rate for stimulated cycles. Figure 2 plots the predicted curves and 95% confidence intervals for IGFBP-3. Peak IGFBP-1 expression was noted at the end of the implantation window (day 24) with noted increase to a mean score of 1 (0 4 scale). Endometrial dating was performed independently on endometrial biopsy samples obtained with use of the criteria of Noyes et al. (12). The COH cycles had advanced endometrial histology ( 1 day) in 42% (13 of 31) of glands and 70% (22 of 31) of the stroma. In COH cycles, higher GdA scores were associated with endometrial biopsies having advanced glandular dating (r 0.63, P 0.01). Correlation analysis was performed to evaluate the association with serum P 4 and E 2 levels on the same days of the endometrial biopsies. Significant positive correlations of E 2 and P 4 were noted with IGFBP-1 and -3 between standardized days 10 through 24 in the natural or COH cycles (Table 2). DISCUSSION This is the first report describing endometrial IGFBP-1 and -3 expression in COH cycles compared with natural cycles. Previous reports on the absence of IGFBPs during the FIGURE 1 Endometrial IGFBP-1 scoring throughout the implantation window; IGFBP-1 scoring (0 4). Dash lines are 95th confidence intervals for the natural and COH cycles. 340 Brown et al. Endometrial expression of IGFBP-1 and -3 Vol. 74, No. 2, August 2000

4 FIGURE 2 Endometrial IGFBP-3 scoring throughout the implantation window; IGFBP-1 scores (0 4). Dash lines are 95th confidence intervals for the natural and COH cycles. proliferative endometrial phase and its presence during the secretory phase suggest that cytokine-binding proteins may play a role in the early stages of reproduction (1, 2). Because of the supraphysiologic elevation of serum sex steroids and the advancement of morphologic changes inherent with COH cycles, this investigation was performed to determine if changes in these two IGFBPs would similarly be altered in COH cycles. Numerous cytokines and growth factors have been found in endometrial secretory proteins that may serve as growthpromoting factors for embryos. In animal studies, exogenous IGFs were found to increase embryo cleavage, blastocyst formation, and outgrowth rates in vitro (13 15). Human embryos cocultured with endometrial stromal cells enhance the expression of IGFs and their receptors (16). The IGFs and IGFBPs have been implicated in the reproductive process, but their precise role is poorly understood. The IGF family is comprised of two polypeptide ligands (IGF-1 and IGF-2), their complementary receptors, and a group of binding proteins that regulate IGF bioactivity (17). Transcripts of IGFs and their receptors were detected in ovarian cells, fallopian tubes, and endometrial stromal cells (14, 18, 19). Thus, these cells may produce IGFs to nourish preembryos in the early stages of development (16). The precise role of IGFBPs in the reproductive process is poorly understood. It may be relevant for interaction at the fetomaternal interphase that the embryo and the trophoblast contain IGF-II, and decidual IGFBP-1 inhibits binding of trophoblastic IGFs to endometrial IGF receptors, thereby inhibiting trophoblastic invasion (20). On the other hand, IGFBP-1 also has non-igf related functions through its Arg-Gly-Asp (RGD) sequence that binds the 5 1 integrin (21). The invading blastocyst probably interacts through its 5 1 integrin with the RGD sequence in fibronectin on endometrial cells, inhibiting trophoblast attachment to endometrial fibronectin (22). In the current study, no difference in the endometrial tissue expression of IGFBP-1 or -3 was noted by comparing COH and natural menstrual cycles. Both cycle types showed an increased production of both IGFBPs in the secretory phase compared with no expression in the proliferative stage. Despite the advanced histologic morphology in the COH group, no similar earlier expression of the two IGFBPs was noted. The answer to why an earlier expression is not seen TABLE 2 Correlation analysis between COH and natural cycles. a Variable IGFBP-1 IGFBP-3 Natural cycle E COH cycle E Natural cycle P COH cycle P a All r values; P.05. FERTILITY & STERILITY 341

5 in COH cycles is unknown. It is possible that mechanisms other than increasing E 2 and P 4 secretion may direct IGFBP production. In conclusion, COH cycles have no significantly increased endometrial IGFBP-1 or -3 expression throughout the implantation phase of the luteal cycle compared with normal menstrual cycles. Both IGFBPs were absent in the proliferative phase and increased throughout the secretory portion of the embryo implantation window. The precise role of IGFBPs in the reproductive process are poorly understood. Acknowledgments: The authors thank Leif Andersson, M.D., and Riitta Koistinen, Ph.D., Helsinki University Central Hospital, for extensive work on the IGFBP assay reporting and the production and purification of the polyclonal anti-igfbp-1 IgG and the monoclonal anti-igfbp 3 antibodies, generated with use of recombinant IGFBP-3 from Celtrix Pharmaceuticals, California. We also thank Jeng G. Hsiu, M.D., Department of Pathology, DePaul Medical Center, Norfolk, Virginia, for morphological endometrial dating of specimens. References 1. Giudice LC, Milkowski DA, Lamson G, Rosenfeld RG, Irwin JC. Insulin-like growth factor binding proteins in human endometrium: steroid-dependent messenger ribonucleic acid expression and protein synthesis. J Clin Endocrinol Metab 1991;72: Julkunen M, Koistinen R, Suikkari AM, Seppala M, Janne OA. Identification by hybridization histochemistry of human endometrial cells expressing mrnas encoding a uterine beta-lactoglobulin homologue and insulin-like growth factor-binding protein-1. Mol Endocrinol 1990; 4: Suvanto-Luukkonen E, Sundstrom H, Penttinen J, Kauppila A, Rutanen EM. Insulin-like growth factor-binding protein-1: a biochemical marker of endometrial response to progestin during hormone replacement therapy. 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Growth factors and growth modulators in human uterine endometrium: their potential relevance to reproductive medicine. Fertil Steril 1994;61: Rutanen EM, Pekonen F, Makinen T. Soluble 34K binding protein inhibits the binding of insulin-like growth factor I to its cell receptors in human secretory phase endometrium: evidence for autocrine/paracrine regulation of growth factor action. J Clin Endocrinol Metab 1988;66: Jones JI, Gockerman A, Busby WH Jr, Wright G, Clemmons DR. Insulin-like growth factor binding protein 1 stimulates cell migration and binds to the alpha 5 beta 1 integrin by means of its Arg-Gly-Asp sequence. Proc Natl Acad Sci USA 1993;90: Martin KL CJ, Martin KL, Carver JG. The role of fibronectin in human implantation. In: 11th World Congress on In Vitro Fertilization and Human Reproductive Genetics; 1999 May 9 14; Sydney, Australia; 1999: Brown et al. Endometrial expression of IGFBP-1 and -3 Vol. 74, No. 2, August 2000