The effect of oral contraceptive pills on markers of endometrial receptivity*t

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1 FERTILITY AND STERILITY Copyright ic 1996 American Society for Reproductive Medicine Vol. 65, No.3, March 1996 Printed on acid-free paper in U. s. A. The effect of oral contraceptive pills on markers of endometrial receptivity*t Stephen G. Somkuti, Ph.D., M.D. Jinghai Sun, M.D. Charles W. Yoell, B.S. Marc A. Fritz, M.D. Bruce A. Lessey, Ph.D., M.D.:!: Department of Obstetrics and Gynecology, University of North Carolina, Chapel Hill, North Carolina Objective: To determine if oral contraceptive (OC) usage alters expression ofintegrins associated ith endometrial receptivity. Design: Immunohistochemical staining intensity for integrins as compared in frozen sections of endometrial tissue specimens obtained from omen ith normal ovulatory cycles (n=3; cycle days to 4 based on LH surge) and from those taking oral contraceptives pills (n=3; cycle days 5 to 7). Setting: University-based outpatient infertility clinic. Results: Constitutive integrin expression in the endometrium ('131, '3131, '5131, '6131, and '6134) as similar in OC users and normally cycling individuals, except for an increase in epithelial '3131 in OC users. Oral contraceptive use as associated ith significant alterations in cycle-dependent integrin expression ('1131,'4131, and O'vj33). Specifically, e observed increased glandular '4131, and decreased O'vj33 expression. The P-regulated '1 subunit as present in both groups. Increased stromal O'V and 133 and decreased '3131 and '6131 staining as observed in OC users. Conclusions: Expression of those integrins most closely associated ith endometrial receptivity, O'vj33 and '4131, is altered in the glandular epithelium of omen taking OCs. Stromal integrin expression in OC users also differs from that in cycling omen. These alterations in epithelial and stromal integrin expression suggest that impaired uterine receptivity is one mechanism hereby OCs exert their contraceptive actions. Fertil Steril 1996;65:484-8 Key Words: Integrins, endometrium, oral contraceptives, endometrial receptivity, endometrial biopsy Received December, 1994; revised and accepted September,1995. * Supported by grants from the National Institutes of Health, Bethesda, Maryland, HD and HD , and the American College of Obstetricians and Gynecology Ciba-Geigy Felloship Aard for the Study of the Post-reproductive Woman, Washington, D.C. t Presented at the 5th Annual Meeting of The American Fertility Society, San Antonio, Texas, November 5 to 1, :j: Reprint requests: Bruce A. Lessey, Ph.D., M.D., University of North Carolina, Department of Obstetrics and Gynecology, Mac Nider Building, CB 757, Chapel Hill, North Carolina, (FAX: ). Integrins are a family of heterodimeric cell adhesion molecules composed of a and (3 subunits that have been implicated in a number of diverse physiological processes, including a role in fertilization and embryo implantation. An early study of hysterectomy specimens (1) suggests that implantation in humans occurs on approximately days 19 to ofthe menstrual cycle. More recently, those observations have been corroborated by experience gained through efforts to define the effective indo of transfer in donor oocyte-embryo recipiency programs (, 3). Three different integrins (a1(31, a4(31, and av(33) exhibit cycle-dependent patterns of expression in endometrial epithelium (4, 5) and e recently have shon that the putative implantation indo corresponds to the brief interval (cycle days through 4) during hich these specific integrins are coexpressed (6). In particular, expression of the av(33 integrin has emerged as a reliable marker of normal fertility, often conspicuously absent in the endometrium of patients ith luteal phase deficiency (4), endometriosis (7), and unexplained infertility (8). 484 Somkuti et al. Oral contraceptives and endometrial receptivity Fertility and Sterility

2 Based on these observations, e have suggested that integrins might prove useful as markers of normal endometrial receptivity (6, 9, 1). Contraceptive efficacy of oral contraceptives (OC) likely involves several complementary mechanisms. These include a central inhibition of ovulation, altered cervical mucus characteristics, and interference ith normal tubal motility (11). Clearly, pharmacologic levels of steroids in OC formulations also may affect endometrial development and function. Oral contraceptives induce characteristic morphological changes in the endometrium but the functional significance of these effects has not been investigated previously. Using integrins as markers of endometrial receptivity, e compared the patterns of expression of nine different integrin subunits in endometrial specimens obtained from OC users and cycling normal fertile controls. We hypothesized that significant differences, if found, ould support the notion that OC use renders the endometrium unreceptive and thereby provide direct evidence that this mechanism contributes to the contraceptive effectiveness of OCs. Tissue Specimens MATERIALS AND METHODS Endometrial tissue specimens ere obtained from 3 healthy normally cycling parous omen (aged 36 ± 6.1 years; mean ± SD) beteen cycle days and 4 ([6 to 1 days after detection of a urinary LH surge] Ovuquick; Quidel, San Diego, CA) and from 3 healthy omen (aged 7 ± 6.1 years) taking OCs (monophasic, 3 to 35 mg ethinyl E + 1 mg norethindrone acetate, n = 1; triphasic, 3 to 35 mg ethinyl E +.5 to 1 mg norethindrone acetate, n = 11) beteen cycle days 5 and 7. All tissue specimens ere obtained using the GynoSampler endometrial aspirator (GynoPharma Inc., Somerville, NJ) and ith the approval of the Committee for the Protection of Human Subjects at the University of North Carolina. Specimens ere snap frozen and stored in liquid nitrogen until processed. A portion of each specimen as fixed in Formalin and histologically dated according to the criteria established by Noyes et a1. (1). Photographs ere taken using ASA 1 Kodak film (Kodak, Rochester, NY). Immunohistochemistry and Monoclonal Antibodies Immunohistochemical staining of 5-/Lm cryostat sections as performed using a technique previously described in detail (4). Briefly, frozen tissue sections ere placed on poly-l-lysine-coated slides, lightly fixed in 3.7% formaldehyde, rinsed first in ice-cold methanol and acetone, and then in phosphate-buffered saline ph 7. (PBS). Sections ere incubated ith monoclonal antibodies directed against al-6, av, (33, or (34. P1H5 (a(31), P1B5 (a3(31), P1D6 (a5(31), and a6(34 ere purchased from GIBCO BRL (Grand Island, NY). The other antibodies, including TS17 (a1(31), B-5H1 (a4(31), GoH3 (a6(31), LM14 (av), and SSA6 ((33) ere gifts. All antibodies have been characterized previously (4). Monoclonal antibodies directed against individual a subunits also recognize those subunits hen paired ith their respective specific (3 subunits as intact integrins (e.g., a1(31). Exceptions include the a6 subunit, hich can pair ith (31 or (34, and av, hich can pair ith a number of (3 subunits. When recognition of more than one intact integrin as possible, antibodies against both subunits of interest ere used. Mter a I-hour incubation ith primary antibodies, sections ere blocked ith 1% bovine serum albumin in PBS and alloed to bind at room temperature for 1 hour. Sections then ere rinsed ith PBS, incubated for 3 minutes ith a biotinylated goat anti-mouse secondary antibody and, after rinsing in PBS,.3% H in absolute ethanol as added for 3 minutes, folloed by 3 minutes of rehydration in PBS. Sections then ere incubated for 3 minutes ith horseradish peroxidase coupled to avidin:biotin, folloed by diaminobenzadine for 3 minutes, rinsed again in PBS, and mounted. Immunohistochemical staining intensity and distribution in both endometrial glands and stroma ere assessed by to independent observers (B.A.L. and S.S.) using the semiquantitative HSCORE. HSCORE = L Pi (i + 1), here i = intensity of staining ith a value of 1,, or 3, (eak, moderate, or strong, respectively) and Pi represents the percentage of stained epithelial or stromal cells for each intensity, varying from % to 1% (4). Intraobserver (r =.983; P =.1) and interobserver (r =.994; P =.1) differences for HSCOREs in uterine tissue have been reported previously (13). All HSCOREs are reported as means ± SEM. A non parametric analysis of variance ith Scheffe's correction for multiple comparisons beteen groups as used to compare results in cycling omen and OC users. A Mann-Whitney Rank sum analysis as used for comparisons beteen monophasics and triphasics. A P value <.5 as considered significant. RESULTS With the exception of an increase in glandular a3(31 expression in OC users (Fig. 1A), the constitutive epithelial integrins (a(31, a3(31, a6(31, and a6(34) ere expressed similarly in OC users and normally cycling omen. As expected, the cycle-depen- Vol. 65, No.3, March 1996 Somkuti et al. Oral contraceptives and endometrial receptivity 485

3 4 3 II:: () I/) :J: 1 a a3 a6 134 expression (.3 ±.1 versus 1.1 ±.; P <.) also as observed in OC users (Fig. 3). We detected no differences in the patterns of integrin expression in monophasic versus triphasic OC users (P =.9). Moreover, results in tissues obtained from OC users ere consistent across the pill cycle. Of particular note, staining characteristics in five specimens obtained beteen cycle days and 4 (i.e., indo of implantation) in OC users did not differ from those in tissues obtained at other times in the cycle (P =.87). DISCUSSION 4 b 3 II:: () I/) :J: 1 al a4 133 Assessment of endometrial development and function ultimately relies on endometrial sampling. The histologic criteria established by Noyes et al. (1) for dating the endometrium have remained the gold standard despite their shortcomings (14, 15). The method cannot identify occult biochemical abnormalities unaccompanied by maturation delay (7, 16). Mindful of this limitation, e recently have described the use of integrins as an adjunct for endometrial dating (Lessey BA, Castelbaum AJ, Guzick D, Sun J, Fritz M, abstract). Figure 1 Immunohistochemical staining intensity and distribution (HSCORE) of endometrial epithelial cells in tissue specimens obtained from cycling controls (D) and OC users (_). (A), Constitutively expressed epithelial integrins (a,61, a3,61, a6,61, and a6,b4). (B), Cycle-dependent epithelial integrins (al,6l, a4,61, and av,63). Results are means ~ SEM. The HSCOREs ere calculated as described in the Materials and Methods section. *p <.5. dent epithelial integrins (a1,81, a4,81, and av,83) exhibited differing patterns of expression in OC users and cycling fertile controls (Fig. 1B). Staining intensity for the a4,81 integrin as increased significantly in tissues obtained from OC users hen compared ith cycling controls (3.3 ±.3 versus 1.1 ±.; P <.14), hereas that for av,83 as decreased (.8 ±. versus. ±.; P <.1). The P-regulated a1,81 integrin as expressed to a similar degree in the glandular elements of tissues obtained from both cycling controls and OC users (3. ±.3 versus.1 ±.3; P <.833). The immunohistochemical staining patterns of these three integrins are shon in Figure A through F. In stromal cells, the intensity of a1,81, a,81, a4,81, and a5,81 staining did not differ beteen OC users and controls (Fig. 3). Hoever, HSCOREs for stromal integrins in OC users ere increased significantly for the av (.1 ±. versus 1. ±.1; P <.46) and,83 (. ±. versus.8 ±.; P <.) integrin subunits (Figs. F and 3). A trend toard decreased a3,81 expression (.1 ±.1 versus.4 ±.1; P <.55) and a clear reduction in a6, Somkuti et al. Figure Light photomicrographs of endometrial sample cryosections from cycling controls (A, C, and E) and OC users (B, D, and F) stained immunohistochemically for the cycle-dependent integrins al,61 (A and B), a4,61 (C and D), and av,63 (E and F). (B), Arro points to vascular endothelium that serves as positive control for al,61 staining. (E), Arro points to av,63-positive staining gland; *lack of staining in the stromal epithelium. (F), Arro points to av,63 negative-staining gland; *positive-staining stromal epithelium. All magnifications at x. Oral contraceptives and endometrial receptivity Fertility and Sterility

4 3 a:: 1.5 UI :z:.5 *.5 a5 a6 ay In Figure 3 Immunohistochemical staining patterns (HSCOREs) of endometrial stromal integrins (al-6, av, and (33). Cryosections of endometrial tissues samples from cycling controls (D) and OC users (_) stained immunohistochemically. Results are means ± SEM. *p < 5. Normal implantation presumably requires that endometrial and embryonic development be synchronized precisely. Early observations by Hertig et al. (1) suggest that the "indo of implantation" opens approximately 5 to 6 days after ovulation, a deduction that is consistent ith more recent experience ith donor oocyte-embryo recipiency (, 3). Defects in uterine receptivity have long been suspected as the cause of the relatively lo cycle fecundity observed in IVF cycles and may be a contributing factor in other infertility states. Inevitably, attention has turned to finding reliable methods to define the crucial time interval and a number of potential markers of endometrial receptivity have emerged. These include cell surface lectins, mucins, cytokines, and groth factors; extracellular matrix molecules; and a variety of peptide hormones, including PRL, insulin-like groth factor (IGF) I and IGF-II and their binding proteins (9, 14). Fe studies have used such markers to examine the effects of OCs on the endometrium. Use of such markers to examine the effects of OCs on the endometrium (17) has been limited. We and others have described the characteristic patterns of epithelial integrin expression during the secretory phase of the endometrial cycle (4, 5). The al,81 collagen-laminin receptor, thought to be regulated by P (Tabibzadeh SS, Satyasaroop PG, abstract) is expressed throughout the luteal phase (4, 5, 17, 18) and to integrins not constitutively expressed in epithelial cells (av,83 and a4,81) are coexpressed conspicuously for a brief interval that corresponds ith the putative indo of maximal uterine receptivity (6). Because simultaneous expression of all three occurs only during this interval, these cycledependent integrins have emerged as reliable and reproducible markers for this critical stage of the menstrual cycle. Our understanding of the mechanisms that regulate these integrins is incomplete, but it appears likely that their expression is linked to P and its receptor (4,6,9, 19,). Altered expression of these integrins, mediated directly by sex steroids or indirectly through cytokines, may yield an unreceptive endometrium (4, 7) and predispose to implantation failure or early pregnancy loss. Oral contraceptives have a number of complementary actions at the hypothalamic, tubal, and cervical levels (11). They also have characteristic effects on endometrial morphology, including diminished thickness, narro, idely spaced glands, and predecidual changes in the stroma. The functional significance of these morphological effects is presumed to be an endometrium that is unreceptive to an implanting embryo. The changes in integrin expression e observed in the endometrium of OC users lend credence to this notion. Compared ith the patterns observed in fertile controls, expression of the a4,81 subunit is increased, hereas that of av,83 is diminished. These changes are consistent ith an unreceptive endometrium and likely reflect the influence of continuous exposure to pharmacologic levels of contraceptive steroids. Tissues obtained from OC users also exhibited elevated epithelial a3,81 expression, decreased stromal a3,81 and a6,81, and increased av,83 staining. Although such changes have been observed in stromal cells both in vitro (Grosskinsky CM, Yoell CW, Sun J, Parise LV, Lessey BA, abstract) and in vivo (1, ), there are no reports of regulation of a3,81 in endometrial epithelium. This finding arrants further study. During the normal menstrual cycle, Preceptor (PR) concentrations decline in the glandular epithelium around the time of implantation but are maintained in stromal and myometrial cells throughout the secretory phase (11). Don-regulation of epithelial PR is driven by rising serum P levels and corresponds ith the interval of av,83 expression. By contrast, PR levels remain elevated and av,83 expression is delayed or absent in luteal phase deficiency, a disorder in hich levels of P secretion are abnormally lo. Continuous exposure to contraceptive steroids also appears to disrupt normal patterns of endometrial integrin expression. The changes e observed ere consistent across the pill cycle and suggest a phenotype that is unreceptive to implantation. Interestingly, high-dose, short-term exposure to contraceptive steroids (morning-after pill) does not appear to induce any acute changes in endometrial integrin expression (17). These data suggest a different mechanism of action. Alternatively, the timing of endometrial sampling in this study may have obscured the ultimate effects of such treatment (1). The cycle-dependent epithelial integrins (al,81, a4,81, and av,83) have emerged as reliable markers for the indo of implantation in normal endome- Vol. 65, No.3, March 1996 Somkuti et at. Oral contraceptives and endometrial receptivity 487

5 trium (6) and their normal patterns of expression are altered grossly in OC users. These data suggest that the morphological changes observed in the endometrium of OC users have functional significance and provide evidence that reduced endometrial receptivity does indeed contribute to the contraceptive efficacy of OCs. Acknoledgments. The folloing individuals generously provided monoclonal antibodies: TS17 (a1,81) and B-5H1 (a4,81) by Martin Hemler, Ph.D. (Dana-Farber Cancer Institute, Boston, Massachusetts); GoH3 (a6,81) by Arnoud Sonnenberg, Ph.D. (Central Laboratory of The Netherlands Red Cross Transfusion Service, Amsterdam, The Netherlands); LM14 (av) by David Cheresh, Ph.D. (Scripps Clinic, LaJolla, California); SSA6 (,83) by Joel Bennett, M.D., and James Hoxie Ph.D. (University of Pennsylvania, Philadelphia, Pennsylvania). REFERENCES 1. Hertig AT, Rock J, Adams EC. A description of 34 human ova ithin the first 17 days of development. Am J Anat 1956; 98: Navot D, Bergh PA, Williams M, Garrisi GJ, Guzman I, Sandler B, et al. An insight into early reproductive processes through the in vitro model of ovum donation. J Clin Endocrinol Metab 1991;7: Bergh PA, Navot D. The impact of embryonic development and endometrial maturity on the timing of implantation. Fertil Steril 199; 58: Lessey BA, Damjanovich L, Coutifaris C, Castelbaum A, AIbeIda SM, Buck CA. Integrin adhesion molecules in the human endometrium. Correlation ith the normal and abnormal menstrual cycle. J Clin Invest 199;9: Tabibzadeh S. Patterns of expression of integrin molecules in human endometrium throughout the menstrual cycle. Hum Reprod 199; 7: Lessey BA, Castelbaum AJ, Buck CA, Lei Y, Yoell CW, Sun J. Further characterization of endometrial integrins during the menstrual cycle and in pregnancy. Fertil Steril 1994; 6: Lessey BA, Castelbaum AJ, Sain SJ, Buck CA, Schinnar R, Wilkins B, et al. Aberrant integrin expression in the endometrium of omen ith endometriosis. J Clin Endocrinol Metab 1994; 79: Lessey BA, Castelbaum AJ, Sain SW, Sun J. Integrins as markers of uterine receptivity in omen ith primary unexplained infertility. Fertil Steril 1995; 63: Ilesanmi AO, Hakins DA, Lessey BA. Immunohistochemical markers of uterine receptivity in the human endometrium. Microsc Res Technol 1993;5: Lessey BA. The use ofintegrins for the assessment of uterine receptivity. Fertil Steril 1994;61: Mishell DR. Oral steroid contraceptives. In: Mishell DR, Davajan V, Lobo RA, editors. Infertility, contraception, and reproductive endocrinology. Boston: Blackell Scientific, 1991: Noyes RW, Hertig AI, Rock J. Dating the endometrial biopsy. Fertil Steril 195; 1: Budit-Novotny DA, McCarty KS Sr, Cox EB, Soper JR, Mutch DG, Creasman WT, et al. Immunohistochemical analyses of estrogen receptor in endometrial adenocarcinoma using a monoclonal antibody. Cancer Res 1986;46: Somkuti SG, Appenzeller MF, Lessey BA. Advances in the assessment of endometrial function. Infert Reprod Med Clin North Am 1995;6: Gibson M. Clinical evaluation of luteal function. Semin Reprod Endocrinol 199;8: Isaacson KB, Galman M, Coutifaris C, Lyttle CR. Endometrial synthesis and secretion of complement component-3 by patients ith and ithout endometriosis. Fertil Steril 199; 53: Taskin, BronRW, Young DC, Poindexter AN, Wiehle RD. High doses of oral contraceptives do not alter endometrial a 1 and av,83 integrins in the late implantation indo. Fertil Steril 1994; 61: van der Linden PJQ, de Goeij AFPM, Dunselman GAJ, van der Linden EPM, Ramaekers FCS, Evers JLH. Expression of integrins and E-cadherin in cells from menstrual effiuent, endometrium, peritoneal fluid, peritoneum, and endometriosis. Fertil Steril 1994;61: Lessey BA, Killam AP, Metzger DA, Haney AF, Greene GL, McCarty KS Jr. Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle. J Clin Endocrinol Metab 1988;67: Lessey BA, Castelbaum AJ. Integrins in the endometrium. Reprod Med Rev 1995;4: Van der Linden PJQ, de Goeij AFPM, Dunselman GAJ, Erkens HWH, Evers JLH. Expression of cadherins and integrins in human endometrium throughout the menstrual cycle. Fertil Steril 1995; 63: Castelbaum AJ, Sain SW, Bellardo LJ, Lessey BA. Endometrial integrin expression in omen exposed to diethylstilbestrol in utero. Fertil Steril 63: Somkuti et al. Oral contraceptives and endometrial receptivity Fertility and Sterility