Effects of clomiphene citrate on the endometrium of regularly cycling women

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1 FERTILITY AND STERILITY VOL. 73, NO. 2, FEBRUARY 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Effects of clomiphene citrate on the endometrium of regularly cycling women Wisan Sereepapong, M.D.,* Somchai Suwajanakorn, M.D.,* Surang Triratanachat, M.D.,* Pichet Sampatanukul, M.D., Kamthorn Pruksananonda, M.D.,* Wisut Boonkasemsanti, M.D.,* and Damrong Reinprayoon, M.D.* Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand Received May 10, 1999; revised and accepted August 23, Supported by the Ratchadapiseksompotch Fund, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (Grant No ). Reprint requests: Wisan Sereepapong, M.D., Department of Obstetrics and Gynaecology, Faculty of Medicine, Chulalongkorn University, Rama IV Road, Bangkok 10330, Thailand (FAX: ; E- mail: * Department of Obstetrics and Gynaecology. Department of Pathology /00/$20.00 PII S (99) Objective: To study the effects of clomiphene citrate (CC) on the endometrium of regularly cycling women. Design: Prospective, controlled study. Setting: Department of Obstetrics and Gynaecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. Patient(s): Thirty healthy, regularly cycling, female volunteers. Intervention(s): All volunteers were studied for two consecutive, one control cycle and one CCtreated cycle. Clomiphene citrate (100 mg/d) was given on days 3 7 of the. Ultrasonography was performed daily to assess ovulation. Ultrasonography and endometrial biopsy were performed, and blood samples were obtained for determination of E 2 and progesterone levels 7 days after ovulation in both the control and. Main Outcome Measure(s): Histologic dating, morphometric analysis, and ultrasonographic appearance and thickness of the endometrium. Result(s): Histologic dating and ultrasonographic appearance and thickness of the endometrium were similar in the control and, but morphometric parameters were different. The number of glands per square millimeter and the mean diameter of the glands were lower in the than in the control, but the number of vacuolated cells per 1,000 glandular cells was higher. Conclusion(s): Clomiphene citrate has effects on the endometrium of regularly cycling women, as demonstrated by a reduction in glandular density and an increase in the number of vacuolated cells. (Fertil Steril 2000;73: by American Society for Reproductive Medicine.) Key Words: Clomiphene citrate, endometrium, morphometric analysis Clomiphene citrate (CC) is a triphenylethylene derivative. It was first synthesized in 1956 and was approved for clinical use in the United States by the Food and Drug Administration in 1967 (1). Thereafter, CC became the most common agent in clinical use for ovulation induction (2). Clomiphene citrate recently has been widely used in ovulatory women to stimulate multiple follicular development and ovulation. Clomiphene citrate binds to the estrogen receptor at numerous sites throughout the reproductive system, including the hypothalamus, pituitary, ovary, endocervix, and endometrium. Clomiphene citrate can act as either an estrogen agonist or antagonist on estrogen-sensitive tissue, depending on the endogenous hormonal environment (1). Its mechanism of action in ovulation induction appears to be its capacity to act as an antiestrogen at the hypothalamic site. Clomiphene citrate is thought to displace endogenous estrogen from hypothalamic estrogen receptor sites, alleviating the putative negative feedback effect exerted by endogenous estrogen and causing a central misinterpretation of the circulating estrogen concentration as a hypogonadal state (1, 2). Although the ovulation rate with the use of CC is 57% 91%, the pregnancy rate is only 27% 40%. The discrepancy between the ovulation and pregnancy rates is explained partly by the antiestrogenic effect of CC on the cervical mucus and endometrium (3 5). Successful implantation requires a receptive 287

2 endometrium, with synchronous development of glands and stroma. Abnormal development of the endometrium results in pregnancy failure (6). Several studies have reported conflicting results about the effect of CC on the endometrium (2, 4, 7 11). For analysis, some studies used only ultrasonographic findings of the endometrium, which do not correlate directly with histologic findings (12, 13). Some studies used histologic dating of the endometrium, which is a subjective criterion that results in intraobserver and interobserver variations (14). Morphometric analysis of the endometrium is a quantitative and objective technique (15). It reduces the unreliability of histologic dating caused by subjective criteria. Therefore, this prospective study was undertaken to investigate the effect of CC on the endometrium of regularly cycling women using morphometric analysis. MATERIALS AND METHODS Subjects Thirty healthy, female volunteers were recruited for this study. They ranged in age from years and had regular menstrual of days duration and biphasic basal body temperature patterns. Volunteers who had used hormonal drugs or intrauterine devices or who had undergone uterine curettage within the previous 2 months were excluded from the study. All volunteers provided their written informed consent. The study was approved by the Ethical Committee of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. Study Design All subjects were studied for two consecutive menstrual, one control cycle, and one cycle. In the control, the subjects underwent daily transvaginal ultrasonographic examination (Aloka, Model SSD-1700; Aloka Co., Ltd., Tokyo, Japan) with a 5-MHz transvaginal transducer, starting 18 days before the next menstruation, to determine ovulation. The ultrasonographic examinations were performed by a single investigator (W.S.). Ovulation was confirmed by documenting at least two of the following parameters: a decrease in follicular diameter, blurring of the follicular border, the presence of internal echoes, and free fluid in the cul-de-sac (16). Follicular diameter was determined by calculating the mean of two perpendicular diameters measured at the largest plane of the follicle. On day 7 after ovulation, endometrial thickness and appearance were observed, endometrial biopsy was performed, and blood samples were obtained for determination of E 2 and progesterone levels. In the next cycle, the cycle, the subjects were treated with CC (100 mg/d, Clomid; Merrell Pharmaceuticals Inc., Kansas City, KS) from days 3 7 of the cycle. The subjects underwent ultrasonographic examination daily, starting on day 10 of the cycle, to determine ovulation. Then ultrasonographic examination, blood sampling, and endometrial biopsy were carried out as in the control cycle. Endometrial Thickness and Appearance The endometrial thickness and appearance were assessed by aligning the uterus along the central longitudinal axis. The endometrial thickness was measured from the echogenic interface between the endometrium and myometrium to the opposite interface at the point of maximum thickness. The measurement was taken three times by a single investigator (W.S.) who was blinded to the experimental conditions, and a mean value was calculated for analysis. The endometrial appearance was classified into four grades. Grade 1 was a hypoechoic functionalis with a minimal hyperechoic basalis and a distinct lumen. Grade 2 was an approximately 50% hypoechoic and 50% hyperechoic pattern with a distinct lumen. Grade 3 was a predominantly hyperechoic endometrial pattern with a minimal hypoechoic area centrally and a distinct lumen. Grade 4 was a totally hyperechoic pattern. Grades 1, 2, and 3 were considered an unsatisfactory endometrial appearance, and grade 4 was considered satisfactory (12). Histologic Dating and Morphometric Analysis of the Endometrium Each subject underwent an outpatient endometrial biopsy 7 days after ovulation with a Novak curette. Specimens were taken from the anterior and posterior walls of the uterine fundus to ensure assessment of an area of maximal physiologic development (15). The specimens were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for microscopic evaluation. Histologic dating was evaluated by two pathologists (S.T. and P.S.) who were blinded to the experimental conditions using the histologic criteria of Noyes et al. (17). When their findings were dissimilar, the specimens were reevaluated by both pathologists. Histologic dating that was 2 days out of phase (before day 19 or after day 23 of the cycle) was considered abnormal. Morphometric analysis was performed using an image analysis system (Quantimet 500, Leica; Cambridge Ltd., Cambridge, Cambridgeshire, United Kingdom). Morphometric parameters shown to be specifically sensitive to tissue differentiation were chosen. These were the number of glands per square millimeter, the diameter of the glands (in micrometers), the height of the glandular epithelium (in micrometers), and the number of vacuolated cells per 1,000 glandular cells (15). The number of glands per square millimeter was determined by counting the glands in five areas, each of which measured 1 mm 2, and calculating the mean value. The diameter of the glands was determined by randomly selecting 10 cross-sectional glands, measuring two perpendicular diameters, and calculating a mean value. The height of the glandular epithelium was determined by measuring 20 glan- 288 Sereepapong et al. Effects of CC on the endometrium Vol. 73, No. 2, February 2000

3 dular epithelial cells of typical columnar shape and calculating a mean value. The number of vacuolated cells per 1,000 glandular cells was determined by randomly selecting 20 glands and counting the vacuolated cells and the glandular cells. Serum E 2 and Progesterone Concentrations Venous blood samples were allowed to clot and the separated serum samples were stored at 70 C until assayed for E 2 and progesterone levels. All serum samples were assayed simultaneously for each hormone, using a fluoroimmunoassay (DELFIA; Wallac Oy, Turku, Finland). The intraassay and interassay coefficients of variation were 3.8% and 5.1% for the E 2 level and 5.5% and 8.1% for the progesterone level, respectively. Accuracy was 94.4% for the E 2 level and 96.8% for the progesterone level. Limits of detection were 13.6 pg/ml for the E 2 level and 0.25 ng/ml for the progesterone level. Data Analysis Data analysis was performed using the Statistical Package for the Social Sciences for Windows, Release (SPSS Inc., Chicago, IL). Histologic dating and endometrial appearance in the control and were compared using McNemar s test. Morphometric analysis of the endometrium, endometrial thickness, and E 2 and progesterone levels in the control and were compared using the paired t test. P.05 was considered statistically significant. RESULTS A total of 30 volunteers were included in this study. Their mean ( SD) age was years (range, years). Their mean ( SD) body mass index was kg/m 2 (range, kg/m 2 ). Their mean ( SD) parity was (range, 0 4). Two volunteers had severe pain during the endometrial biopsy and no tissue could be obtained. One volunteer was found to have insufficient endometrial tissue for analysis. Therefore, 54 endometrial specimens obtained from 27 volunteers were available for histologic analysis. The cycle lengths, numbers of follicles, and E 2 and progesterone levels for the control and are shown in Table 1. The cycle lengths were not different between groups (paired t test, P.05). The mean numbers of follicles in the control and were 1.0 and 2.4, respectively. The E 2 and progesterone levels in the were higher than those in the control (paired t test, P.001), but the E 2 and progesterone levels per follicle and the progesterone-to-e 2 ratio of the were not different (paired t test, P.05). The endometrial thickness on day 7 after ovulation in the control was not different from that in the (paired t test, P.05), as shown in Table 1. The endometrial appearance on day 7 after ovulation in the TABLE 1 Cycle length, E 2 and progesterone levels, and endometrial thickness in the control and. Variable Control P value Cycle length (d) NS No. of follicles E 2 level (pg/ml) Progesterone level (ng/ml) E 2 level per follicle (pg/ml) NS Progesterone level per follicle (ng/ml) NS Progesterone-to-E 2 ratio NS Endometrial thickness (mm) NS Note: Values are means SD. NS not significant. control also was not different from that in the CCtreated (McNemar s test, P.05). Grade 4 endometrium was found in 23.3% (7/30) of both control and CCtreated. Grades 1, 2, and 3 endometria were found in 16.7% (5/30), 6.7% (2/30), and 53.3% (16/30) of the control and in 16.7% (5/30), 10% (3/30), and 50% (15/30) of the, respectively. The endometrial thickness and appearance also were not different between the in-phase and out-of-phase endometria (t test and 2 test, P.05). The results of the histologic dating of the endometria are shown in Table 2. In-phase endometrium (days of the cycle) was found in 55.6% (15/27) of the control and 40.7% (11/27) of the. Out-of-phase endometrium (before day 19 or after day 23) represented either delayed or accelerated growth. Delayed growth of the endometrium was found in 40.7% (11/27) of the control and 55.6% (15/27) of the. Accelerated growth of the endometrium was found in 3.7% (1/27) of both. The percentages of in-phase and out-of-phase endometria were not different between the control and (McNemar s test, P.05). The degree of endometrial TABLE 2 Histologic dating of the endometrium in the control and. Histologic dating Control In phase (days 19 23) Delayed growth (before day 19) Accelerated growth (after day 23) 1 1 Total FERTILITY & STERILITY 289

4 TABLE 3 Morphometric analysis of the endometrium in the control and. Morphometric analysis Control stromal edema in the control and also was not different (McNemar s test, P.05). Focal edema and diffuse endometrial stromal edema were found in 33.3% (9/27) and 44.4% (12/27) of the control and in 22.2% (6/27) and 70.4% (19/27) of the, respectively. The results of the morphometric analyses of the endometria are shown in Table 3. The number of glands per square millimeter and the diameter of the glands in the were lower than in the control (paired t test, P.001 and P.05, respectively). There were more vacuolated cells per 1,000 glandular cells in the than in the control (paired t test, P.01). The height of the glandular epithelium was not different between (paired t test, P.05). DISCUSSION P value No. of glands per mm Diameter of glands ( m) Height of glandular epithelium ( m) NS No. of vacuolated cells per 1,000 glandular cells Note: Values are means SD. NS not significant. In humans, the implantation process occurs 4 8 days after ovulation (6). During this period, the endometrium undergoes many morphologic and biochemical changes in preparation for embryo implantation. Any abnormal changes in the endometrium during this period may adversely affect implantation. Therefore, we performed endometrial biopsy for histologic evaluation 7 days after ovulation to evaluate the effect of CC on the endometrium during this period. We did not include irregularly cycling women in the study because their endometrium might already have shown abnormal development. We used the natural cycle before the cycle as a self-control cycle because numerous confounding factors may affect endometrial development in each woman. This study demonstrated that CC did not have any effect on endometrial thickness or appearance as demonstrated by ultrasonography. Our results are in agreement with those of Li et al. (13), but differ from those of Randal and Templeton (18) and those of Nakamura et al. (11). However, endometrial thickness and appearance on ultrasonographic examination cannot predict accurately the endometrial histologic findings (12, 13). This study demonstrated that CC did not affect the midluteal endometrial development based on the histologic criteria of Noyes et al. (17). Our results are in agreement with those of Thatcher et al. (7) and Li et al. (4), but differ from those of Cook et al. (19), Yeko et al. (8), Massai et al. (9), and Bonhoff et al. (10, 20). The percentage of out-of-phase endometria in the control was 40.7%. This may imply that 40.7% of the subjects already had abnormal endometrial development before treatment with CC, and the number of subjects who had in-phase endometria before treatment with CC may not be large enough for statistical comparison. Although we recruited only regularly cycling women, these women might have had abnormal endometrial development. The histologic dating of the endometrium was evaluated by two pathologists who were blinded to the experimental conditions. When their findings were dissimilar, the specimens were reevaluated by both pathologists. Therefore, bias on the part of the pathologists is unlikely. However, histologic dating of the endometrium, which is a subjective criterion, has high intraobserver and interobserver variation, as demonstrated in a previous study (14). It can be used to predict accurately dating of the endometrium in only 60% of cases (14). Therefore, histologic dating is not a reliable method for evaluating the endometrium. Based on the morphometric analysis, our results demonstrated that CC affects endometrial development. We found that the number of glands per square millimeter and the diameter of the glands were lower in the than in the control and the number of vacuolated cells per 1,000 glandular cells was higher in the than in the control, but the height of the glandular epithelium and the degree of stromal edema were not different between the control and. This implies that CC reduces the glandular density of the endometrium. Because the vacuoles normally appear in the cytoplasm of the epithelial cells between day 16 and day 19 of the cycle (17) and we performed endometrial biopsy on day 21 of the cycle, the larger number of vacuolated cells per 1,000 glandular cells in the compared with the control may indicate that there was delayed endometrial development or impairment of the secretory activity leading to persistence of the subnuclear vacuoles (21). Our results are in agreement with those of Bonhoff et al. (10), but differ from those of Li et al. (4). Li et al. (4) reported that CC did not have a major adverse effect on endometrial morphology, but their study had a small sample size and no control group. However, there is no clear evidence to suggest that changes in morphometric parameters would interfere with embryo implantation. The increase in midluteal E 2 and progesterone levels observed in the probably reflects the re- 290 Sereepapong et al. Effects of CC on the endometrium Vol. 73, No. 2, February 2000

5 cruitment of additional follicles rather than increased synthesis from the dominant follicle itself, as shown by the equal levels of these hormones per follicle in the control and demonstrated in this and a previous study (22). Our results showed that the progesterone-to-e 2 ratio was similar in both and was close to 100:1. A progesterone-to-e 2 ratio of close to 100:1 has been postulated to represent the optimal conditions for successful conception (20). Therefore, CC does not alter the optimal progesteroneto-e 2 ratio produced by the corpus luteum. Because normal secretory endometrial maturation requires the combined actions of estrogen and progesterone, and because both steroids exert their effects through specific estrogen-induced endometrial receptors, direct interference with estrogen induction of effective endometrial estrogen and progesterone receptor populations is one mechanism that might explain the increased incidence of abnormal midluteal endometrial growth associated with the use of CC (19). This hypothesis has been both supported and refuted in previous studies (2, 23). Direct adverse endometrial effects of CC may operate through other mechanisms. The occasionally marked elevation of E 2 in the luteal phase that follows CC-induced ovulation may interfere directly with endometrial decidualization (24). Inadequate folliculogenesis and aberrations in gonadotropin secretion during the cycle may be one mechanism (25). 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