Sonographic determination of a possible adverse effect of domiphene citrate on endometrial growth

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1 Human Reproduction vol.5 no.6 pp , 1990 Sonographic determination of a possible adverse effect of domiphene citrate on endometrial growth Yael Gonen 1 and Robert F.Casper Division of Reproductive Sciences, Department of Obstetrics and Gynaecology, University of Toronto, Toronto, Canada 'Present address: Department of Obstetrics and Gynaecology, Carmel Hospital, Haifa, Israel To whom correspondence should be addressed at: 6-40 EN, Toronto General Hospital, 00 Elizabeth Street, Toronto, Ontario, M5G C4, Canada We recently demonstrated, using transvaginaj sonography, that conception cycles in in-vitro fertilization (TVF) are associated with a significantly thicker endometrium at midcycle than non-conception cycles, suggesting that endometrial growth may influence implantation. In the present study, to examine whether the type of stimulation protocol affects endometrial development, we compared the sonographic appearance of the endometrium in patients randomized to receive domiphene citrate and human menopausal gonadotrophin () and in 19 who received alone. A significantly thicker endometrium was observed in the patients compared to the group (P < 0.005) throughout the folhcular phase of the cycle, although serum concentrations of oestradiol ( ) did not differ in the two groups. Twenty-three patients (13 in the group and 10 in the group) had previous IVF cycles with stimulation in which endometrial thickness was measured. A thin endometrium recurred with subsequent cycles while increased growth occurred with only compared to previous cycles. Therefore, ultrasound examination of the endometrium in this study demonstrated that CC results in a thinner endometrium than alone. We believe these findings may be of importance in improving pregnancy rates in IVF and possibly in other infertility therapy which involves the use of domiphene citrate. Key words: domiphene citrate/endometrium/ultrasound/lvf Introduction Routine sonographic examination of the pelvis during controlled ovarian stimulation has focused mainly on measurement of follicular development and, in the past, little attention has been directed toward endometrial growth. Vaginal sonography, with its high frequency and high resolution capability, enables very detailed measurement of the growth of the endometrium and visualization of subtle textural changes within the uterine cavity. Recently, we (Gonen et al., 1989; Gonen and Casper, 1990) and others (Glissant et al., 1985; Welker et al., 1989) have shown that the sonographic appearance of the endometrium at midcycle may be important in the prediction of implantation. We have found that implantation is unlikely to occur when endometrial thickness, measured from one side of the endometrial cavity to the other, is <6 mm on the day after HCG administration. Similarly, Welker et al. (1989) have shown that a 'triple line' endometrial pattern at midcycle, as described by Forrest et al. (1988), appears to favour implantation. We have speculated that unfavourable endometrial development may be a consequence of oestrogen receptor depletion in the endometrium by domiphene citrate (CC) (Gonen et al., 1989). If so, our findings may have important implications not only for IVF but also for any infertility treatment in which CC is used. The purpose of the present study was to compare the sonographic appearance of the endometrium during the follicular phase of the cycle, in randomized IVF patients in whom ovarian stimulation was performed using human menopausal gonadotrophin () with and without CC. We asked the following questions, (i) Does the addition of CC to the ovarian hyperstimulation regimen influence the thickness of the endometrium? (ii) Does an endometrial thickness of < 6 mm recur in subsequent cycles in the same patient with the same stimulation protocol? (iii) Can poor endometrial thickness be improved by alteration of the stimulation protocol? Materials and methods Forty-one consecutive patients enrolled in the University of Toronto IVF programme at the Toronto General Hospital were recruited to participate in the study. Serial sonographic examination of the endometrium was performed as part of the daily monitoring during controlled ovarian stimulation. The choice of stimulation protocol was randomly assigned. Twentytwo patients received CC (Serophene, Serono, Randolph, MA) 100 mg/day cycle days 5-9 and human menopausal gonadotrophin ( group only). The dose of was increased Table I. Indications for IVF treatment Tubal Endomelnosis Unexplained Male and tubal or endometriosis Oxford University Press

2 Endometrial effects of domipbene citrate Sonographic studies were initiated on cycle day 8 and were performed daily up to the day prior to oocyte retrieval (day after administration of HCG). The maximum thickness of endometrium on both sides of the midline was measured on the 'frozen' sonographic image in the plane through the central longitudinal axis of the uterine body as previously described (Gonen et ad., 1989). The endometrial pattern was determined by comparing the reflectivity of texture of the endometrium to that of the myometrium as previously described (Gonen and Casper, 1990; Welker etal, 1989; Forrest etal., 1988). The endometrial thickness on each cycle day was compared between the two groups. Thirteen patients in the only group and 10 in the group had had previous IVF cycles, in our programme, in which controlled ovarian stimulation with was performed and.endometrial thickness on the day after HCG was recorded. In those patients, the mean endometrial thickness on the day after HCG was compared between the Table II. Endometrial thickness and serum oestradiol (E) concentrations during ovarian stimulation with human menopausal gonadotrophin () alone or with the addition of clomiphene citrate (CC) Ej day 8 (pmol/1) End day 8 (cm) Ej day of HCG (pmol/l) End day of HCG (cm) Ej day after HCG (pmol/1) End day after HCG (cm) Significance P < P < P < End, endometrial thickness., not significant. 1 1 B 10 UJ 6- u. o ENDOMETRIAL THICKNESS (cm) Fig. 1. Endometrial thickness, on the day after human chorionic gonadotrophin (HCG) administration, in 41 IVF patients receiving either clomiphene citrate and human menopausal gonadotrophin (; open bars) or human menopausal gonadotrophin alone (; hatched bars). in both groups from cycle day 7 if needed, according to the changes in oestradiol (E) levels. Therefore, the main difference between the two stimulation protocols was the presence or absence of CC. Human chorionic gonadotrophin (HCG, Profasi, Serono) (5000 IU) was routinely used to trigger the final stage of follicular maturation in both groups. Oocyte retrieval, fertilization and embryo transfer were performed using standard techniques as previously described (Wood and Trounson, 1984). Age, gravidity and parity were similar in both groups. The indication for IVF was mainly tubal infertility as outlined in Table I. A male factor was involved as the cause of infertility, either as the sole problem or combined with tubal factor or endometriosis more often in patients stimulated with than in the alone group. Ultrasound scanning for the study was performed with a Bruel and Kjaer (type 1846, Naerum, Denmark) ultrasound equipped with a 7 MHz vaginal transducer (Bruel and Kjaer, type 8538).

3 Y.Gonen and R.F.Casper previous cycles and the present study cycles in both groups. The correlation between endometrial thickness and oestradiol (EJ levels, measured by radioimmunoassay (Gonen and Casper, 1989) on cycle day 8, the day of HCG and the day following HCG administration was determined. We also calculated the correlation between endometrial thickness on the day after HCG administration and the amount of required by regression analysis in both groups. Statistical analysis The data obtained were compared by Student's f-test for paired and unpaired results, the Chi square test and Fisher's Exact Test. Correlation was checked by linear regression analysis. The data are presented throughout this paper as a mean standard error of the mean ( SEM). Results The average cycle day for HCG administration did not differ between the and only groups ( and , respectively). On the day prior to oocyte retrieval, no difference was found in the mean number of follicles 1.5 cm between the two groups ( and 7.0, respectively). The amount of required to achieve an optimal E response and follicular development was significantly higher in the only group than in the group ( 1.9 and ampoules, respectively; P < 0.01). However, serum E concentrations were not different between the two groups on cycle day 8, on the day of HCG administration or on the day after HCG (Table II). Table II presents the mean endometrial thickness and serum E levels on cycle day 8, on the day of HCG and on the day following HCG administration. A significantly thicker endometrium was observed in the group of patients stimulated with without CC on each cycle day compared to the group (P < 0.005), although the serum concentrations of E did not differ between the two groups on any cycle day. As shown in Figure 1, none of the 19 patients in the stimulated group had an endometrial thickness <6 mm on the day following HCG, whereas seven out of patients in the group had endometrial thickness <6 mm (P < 0.05). Table III demonstrates endometrial thickness on the day after HCG administration in the present study and in a prior treatment cycle in our programme. The endometrial thickness in the previous cycles was not different from the thickness in the present study in 10 women in the group ( versus cm, respectively). Five of the 10 women had an endometrial thickness of 6 mm or less which was the same or thinner in the subsequent cycle. In contrast, a significantly greater endometrial thickness was seen in the 13 women in the only group ( 0.05 cm) compared to the previous cycles ( cm) in the same patients (P < ). Five of 13 patients had endometrial thickness < 6 mm in the first cycle of and all of these patients had endometrial thickness >6 mm in the subsequent only cycle. 67 A correlation between serum Ej levels and endometrial Table m. Comparison of endometnal thickness in consecutive IVF cycles in the same patients Mean Endometrial Endometrial thickness (cm) thickness (cm) day after HCG day after HCG in the study in the previous cycle cycle P < Endometnal Endometrial thrckiness (cm) thickness (cm) day after HCG day after HCG in the study in the previous cycle cycle P = In the group, the women received clomiphene citrate (CC) and human menopausal gonadotrophin () in the first (previous) cycle followed by alone in the study cycle. In the group, the women received stimulation protocols in both cycles. Table IV. Correlation between Ej levels (pmol/1) and endometrial thickness E^-end day 8 Ej-end day of HCG E-end day after HCG End, endometrial thickness (cm). *P < r = 0.7* 1 = 008 r = 0.01 r = = 0.03 r = 0.11 thickness was found with stimulation only on cycle day 8 (r = 0.7; P < 0.05). No such correlation was found in either group of patients on any of the other cycle days (Table IV). Assessment of the endometrial pattern in each cycle demonstrated that a 'triple line' pattern was observed in 16 of the 19 women in the only group compared to 13 of the women in the group treated with (84. versus 59.1%; P = 0.077; Fisher's Exact Test). Two pregnancies occurred in the group and two pregnancies occurred in the only group during this study. All four pregnancies were associated with an endometrial thickness >6 mm on the day after HCG. Discussion We (Gonen et al, 1989; Gonen and Casper, 1990) and others (Glissant et al., 1985) have recently demonstrated that midcycle endometrial thickness and the amount of endometrial growth during controlled ovarian stimulation for IVF, are significantly increased in conception compared with non-conception cycles.

4 Endometrial effects of domjphene citrate This finding may have potential predictive value with regard to implantation, especially when combined with the ultrasound pattern of endometrial development as previously described (Gonen and Asper, 1990; Welker et al., 1989). In the present study, we demonstrate that the use of CC in the stimulation protocol for IVF may result in a poor endometrial thickness and pattern in some patients. Endometrial thickness was significantly decreased in patients in whom was used for follicle stimulation compared to the use of alone. Furthermore, patients who underwent previous IVF cycles in our programme with a stimulation protocol, showed a significantly thicker endometrium during the present study when stimulated with alone. The anti-oestrogenic effect of clomiphene citrate is well known. In the area of anovulatory infertility, an adverse (anti-oestrogenic) effect on cervical mucus is thought to be one factor responsible for the discrepancy between the ovulation rate (70%) and the pregnancy rate (3%) of women receiving clomiphene citrate treatment (Speroff et al., 1989). Another possibility, that an antioestrogenic action of CC upon the endometrium may interfere with implantation, has also been considered by some authors (Wall et al., 1965; Sterzik et al., 1988). Studies of endometrial biopsies in IVF patients have demonstrated a high incidence of histological and ultrastructural abnormalities of the endometrium in some CC stimulated cycles when compared with natural cycles or cycles stimulated with alone (Dellenbach-Hellweg, 1981). Cohen et al. (1984) performed endometrial biopsies in the early luteal phase in FVF patients in whom no embryos were available for transfer and found an atrophic-appearing endometrium in almost half of the patients treated with a combination of CC and. This finding is consistent with the thin endometrium measured at midcycle in some patients stimulated with in the present study. Clomiphene citrate has been shown to deplete oestrogen receptors in oestrogen-sensitive tissues (Aksel et al., 1986; Clark et al., 1974; Katzenellenbogen and Ferguson, 1975) and it is likely that the level of oestrogen receptors in the endometrium can influence both endometrial growth and pattern. In some patients, endometrial oestrogen receptors may be depleted to a greater extent than others, resulting in less endometrial growth. Our finding of a significant correlation between serum E concentrations and endometrial thickness in the only cycles but not in the cycles is compatible with this hypothesis. Fleisher et al. (1984) using transabdominal ultrasound, also demonstrated relatively low endometrial thickening in women receiving CC alone compared to patients treated with alone. In the present study, we demonstrate a highly significant difference in endometrial growth in women stimulated with for IVF depending on the presence or absence of clomiphene citrate. The number of pregnancies in this study was small and did not differ between the two groups of women despite the significant findings in terms of endometrial thickness. A previous study of the sonographic appearance of the endometrium in the midluteal phase by Deichert et al. (1986) demonstrated a thicker endometrium in pregnancy cycles than in cycles not associated with conception. We believe that a difference in pregnancy rates was not seen in the present study because of the relatively small number of women treated and our lack of experience with an only stimulation protocol. The combination of clomiphene with is routinely used in many IVF programmes in order to reduce the amount of required, usually by ~ 50% (Blankstein et al., 1986). The results of the present study confirm that addition of clomiphene citrate to the stimulation protocol significantly reduces the requirement for. However, we propose that transvaginal ultrasound examination of the endometrial thickness and pattern should be performed in all infertility patients receiving clomiphene citrate to rule out poor endometrial development. Our data suggest that a thin endometrium with CC use is likely to recur from cycle to cycle and if detected on ultrasound, may be improved by switching the stimulation protocol to one which does not utilize CC. Acknowledgements The authors wish to thank Dr William Jacobson for his assistance with the statistical evaluation of the data and Ms Mary Coates for her help with the preparation of the manuscript. This work was supported by a research grant from the Medical Research Council of Canada and by a Fellowship Training Grant to Dr Y.GonenfromWyeth Pharmaceutical Company, Toronto, Canada. References Aksel.S., Saracoglu,O.F., Yeoman,R.R., Wiebe.R.H. (1986) Effects of clomiphene citrate on cytosolic estradiol and progesterone receptor concentrations in secretory endometrium. Am. J. Obstet. Gynecol., 155, Blankstein,;., Mashiach.S. and Lunenfeld.B. (eds) (1986) Ovulation Induction and In Vitro Fertilization. Year Book Medical Publisher, Inc., Chicago, London, 1 pp. ClarkJ.H., Peck,E.J.,Jr and AndersonJ.N. (1974) Oestrogen receptors and antagonism of steroid hormone action. Nature, 51, Cohen.J.J., Debache,C, Pigeau.F., MandelbaumJ., Plachot.M. and de Brux.J. (1984) Sequential use of clomiphene citrate, human menopausal gonadotropin and human chorionic gonadotropin in human in vitro fertilization. II. Study of luteal adequacy following aspiration of the preovulatory follicles. Fertil. 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5 Y.Gonen and R.F.Casper Gonen.Y., Casper.R.F., Jacobson.W. and Blankier.J. (1989) Endometria] thickness and growth during ovarian stimulation: a possible predictor of implantation of in vitro fertilization. Fertil. Steril, 5, Katzenellenbogen.B.S. and Ferguson,E.R. (1975) Antiestrogen action in the uterus: biological ineffectiveness of nuclear bound estradiol after antiestrogen. Endocrinology, 97, 1 1. Speroff.L., CHass.R.H. and Case,N.G. (eds) (1989) Clinical Gynecologic Endocrinology and Infertility. 4th edn. Williams and Wilkins, Baltimore, MD, 600 pp. Sterzik.K., Dallenbach.C, Schneider.V., Sasse.V. and Dallenbach- Hellweg.G. (1988) In vitro fertilization: the degree of endometrial insufficiency varies with the type of ovarian stimulation. Fertil. Steril., 50, WalU.A., Franklin.R.R., Kaufrnan.R.H. and Kaplan.A.L. (1965) The effects of clomiphene citrate on the endometrium. Am. J. Obstet. Gynecoi, 93, Welker.B.G., Gembruch,U., Diedrich.K., Al-Hasani,S. and Krebs.D. (1989) Transvaginal sonography of the endometrium during ovum pickup in stimulated cycles for in vitro fertilization. J. Ultrasound Med., 8, Wood.C. and Trounson.A. (1984) Clinical In Vitro Fertilization. Springer-Verlag, Berlin. Received on January, 1990; accepted on May 1,