Cycle abnormalities in infertile women with regular menstrual cycles: effects of clomiphene citrate* treatmentt

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1 FERTILITY AND STERILITY Vol. 62, No.1, July 1994 Copyright 1994 The American Fertility Society Printed on acid-free paper in U. S. A. Cycle abnormalities in infertile women with regular menstrual cycles: effects of clomiphene citrate* treatmentt D. Andrew Rodin, M.D.:j: Aeneas M. Fisher, M.D. Richard N. Clayton, M.D. II Clinical Research Centre and Northwick Park Hospital, Harrow, United Kingdom Objective: To investigate the incidence and nature of cycle abnormalities and the effect of clomiphene citrate (CC) treatment in women with apparently ovulatory cycles and unexplained infertility. Design: Nonrandomized, open study of patients before and during treatment. Setting: The Reproductive Medicine Clinic of a District General Hospital. Patients: Thirty-five women with regular, apparently ovulatory menstrual cycles and unexplained infertility. Interventions: Detailed ultrasound and hormonal cycle tracking was performed before and during treatment with CC. Main Outcome Measures: Serial ultrasound scans and measurements of serum LH, FSH, E 2, andp. Results: Before treatment, 54% of cycles were uniovulatory, 40% were characterized by cyst formation, and 6% were characterized by poor follicular growth. Fifty-one percent of pretreatment cycles had normal hormone profiles, 31% had defective luteal phases, 14% had increased early follicular phase serum FSH levels, and 9% had increased early follicular phase serum LH levels. Treatment with CC reduced the incidence of cyst formation to 9% and the incidence of luteal phase defects to 3%. However, 28% of CC-treated cycles showed ultrasound features of overstimulation and 51 % had high follicular phase E2 peaks so that only 34% of CC-treated cycles had normal hormone profiles. Conclusions: Cycle abnormalities are common in unexplained infertility. The incidence of cyst formation and luteal phase defects, the most common abnormalities in this group, is reduced by CC treatment. Fertil Steril 1994;62:42-7 Key Words: Unexplained infertility, clomiphene, ultrasound, cyst formation, luteal phase defects, gonadotropins, sex steroids Received May 13, 1993; revised and accepted March 10, * Clomiphene citrate, Marion Merrell Dow Ltd., Uxbridge, United Kingdom. t Supported by the Medical Research Council, London, United Kingdom. :j: Present address: Division of Biochemical Medicine, St. George's Hospital Medical School, London, United Kingdom. Present address: Department of Endocrinology, North Staffordshire Royal Infirmary, Stoke-on-Trent, United Kingdom. II Reprint requests: Richard N. Clayton, M.D., Department of Endocrinology, North Staffordshire Royal Infirmary, Princes Road, Hartshill, Stoke-on-Trent ST4 7LN, United Kingdom (FAX: ). Routine investigations fail to establish the cause of infertility in about one quarter of infertile coupies (1, 2). In many such cases, disorders of follicular development, ovulation, or the luteal phase are suspected and these patients are often treated with clomiphene citrate (CC) empirically. Clinical studies in infertile women with apparently normal cycles have led to the description of several rather subtle cycle abnormalities, such as the abnormal luteal phase, transient hyperprolactinaemia, and elevated basal levels of LH (3). Laparoscopic observation of the ovaries combined with hormonal investigations demonstrated the occurrence ofthe luteinized unruptured follicle syndrome (4). Using ultrasound (US), the cyclical changes in ovarian morphology can be visualized. The accuracy of US measurements of preovulatory follicles 42 Rodin et al. CC in unexplained infertility Fertility and Sterility

2 has been confirmed by laparoscopic studies (5) and the US changes associated with ovulation can be demonstrated by daily scans in >90% of normal cycles (6). In the normal situation, serum E 2 levels correlate well with the mean follicular diameter as measured by US (7) but, in some circumstances, hormonal and US investigations may give apparently divergent results. For example, the luteinized unruptured follicle syndrome may be associated with either normal hormone profiles or luteal phase abnormalities (S, 9) and poor follicular growth may be associated with a normal hormone profile (10). In this study, we have used both US and hormonal cycle monitoring to define abnormalities of follicular growth and ovulation and luteal phase abnormalities in a group of women with unexplained infertility who had regular menstruation and apparent ovulation. We have evaluated the effectiveness of CC therapy in this subgroup of infertile women by examining its effects on both the US and hormonal parameters. MATERIALS AND METHODS The subjects were 35 women with unexplained infertility who attended the Reproductive Medicine Clinic at Northwick Park Hospital, Harrow, United Kingdom. The criteria used to assign patients to the unexplained infertility category were as follows: [1] a history of ~2 years of involuntary infertility; [2] a normal semen analysis as judged by the following parameters: sperm density> 20 X 10 6 /ml, motility> 40%, abnormal morphology < 40%; [3] regular menstrual cycles (23 to 34 days, but not varying from the mean by >2 days in any individual); [4] a midluteal serum P concentration of ~S ng/ml (25 nmoljl); and [5] a normal uterus and patent fallopian tubes as demonstrated by laparoscopy and dye test and/or hysterosalpingography (HSG). Two women had HSG alone. All were nonobese, none had clinical evidence of hyperandrogenism, and none had polycystic ovaries on US examination in the early follicular phase of the cycle (11). Normal cycle data were obtained from 10 female volunteers, recruited from the local community, who were similar to the study group in respect of age, body mass index, and average cycle length. These control cycles consisted of two conception cycles, five cycles in women with proven fertility, and three cycles in women in whom fertility was untested. These groups are compared in Table 1. Table 1 Clinical Charactersitics of Women With Unexplained Infertility and Normal Women* Age (y) Body mass index (kg/m 2 ) Cycle length (d) Duration of infertility (y) Women with unexplained infertili tyt 31.4 ± ± ± ± 1.78 Normal women (n = 10) 29.1 ± ± ± 2.35 * Values are means ± SD. t Primary infertility n = 24; secondary infertility n = 11. Each subject was studied first during an untreated cycle and then during a CC-treated cycle. Twenty-five subjects received treatment with 50 mg CC daily for 5 days starting from day 2 of the menstrual cycle and 10 subjects received treatment with 100 mg CC daily for 5 days. Ultrasound scans were performed with a Diasonics DRF250 real time sector scanner (Diasonics Sonotron Ltd., Bedford, United Kingdom) with 3.5 and 5 MHz transducers through the full bladder. The first scan was arranged within the first 5 days of the cycle to exclude the presence of residual cystic structures from the previous cycle. Scans were then performed every 2 to 3 days until a single follicle> 15 mm in diameter was observed. Scans were then performed daily until ovulation. Generally, scans were not continued after ovulation but, in those cycles in which US criteria for ovulation were not met, scans were performed throughout the cycle. Venous blood samples were obtained at the time of each scan and, after presumed ovulation, blood samples were collected every 3rd day until the onset of menstruation. Serum was stored at -20 C and subsequently LH, FSH, E 2, and P were measured in each of these samples by specific RIA as described previously (12). Reference ranges (geometric mean ± 2 SD) for serum LH, FSH, E 2, and P throughout the cycle were derived from the 10 normal control cycles. The serum LH peak was used as a reference point (day 0) and serum hormone concentrations from days ±1, ±2 to ±4, ±5 to ±7, ±S to ±1O, and ±11 to ±14 were grouped together for analysis. Values for serum hormone concentrations did not have a normal distribution and therefore were transformed logarithmically before statistical analysis. Differences in serum hormone concentrations between the study group and the normal control group were compared using the unpaired Student's t-test. Paired t-tests were used to compare serum Vol. 62, No.1, July 1994 Rodin et al. CC in unexplained infertility 43

3 ( ( j I ( Table 2 Ultrasound Findings in Women With Unexplained Infertility Before and During CC Treatment* Ultrasound Uniovulatory Poor follicular growth Cyst formation Overstimulation Pretreatment 19 (54.3)t 2 (5.7) 14 (40.0) 0(0.0) Clomiphene 21 (60.0) 1 (2.8) 3 (8.6)* 10 (28.6) * The absolute proportions of untreated and CC-treated subjects within each category were compared using the X 2 test. t Values in parentheses are percentages. *P < P < hormone concentrations in the study group before treatment and during CC treatment. The absolute proportions of subjects in the study group with each of the defined US and hormone findings before and during CC treatment were compared using the X 2 test with Yates' correction. A P value < 0.05 was considered significant. RESULTS Four cycle types were identified by US examination: ovulatory, poor follicular growth, cyst formation, and overstimulation. Uniovulatory cycles were characterized by progressive follicular maturation and formation of a preovulatory follicle (> 15 mm in diameter) that disappeared suddenly or reduced in size by 50%. In retrospect, this sudden disappearance or shrinkage ofthe preovulatory follicle was shown to occur within 48 hours of the serum LH peak. Poor follicular growth was demonstrated by the failure of any follicle to reach the dimensions of a preovulatory follicle. Cycles in which a preovulatory follicle developed but serial daily US scans failed to detect evidence of ovulation were termed cyst formation cycles. The resultant cyst either disappeared before the end of the cycle or persisted into the subsequent cycle. This ultrasonically defined abnormality is equivalent to the luteinized unruptured follicle syndrome. Overstimulation was defined as the development of more than two preovulatory follicles. Multiple ovulation and/or multiple cyst formation occurred in these cycles. The results of the US evaluation of the cycles in the study group are shown in Table 2. Before treatment, 54 % of cycles were uniovulatory and cyst formation occurred in 40% of cycles. In 13 of 14 cyst formation cycles there was a single cyst and in 1 of 14 cycles two cysts were observed. The median maximum diameter of these cysts was 22.8 mm (range 16.5 to 48.6 mm). With CC treatment, there was a significant reduction in the incidence of cyst formation but there was no increase in the proportion of cycles classified as uniovulatory because 28% of these cycles resulted in overstimulation. This included 6 of 25 (24%) cycles treated with 50 mg CC and 4 of 10 (40%) cycles treated with 100 mg CC. There was, however, US evidence of multiple ovulation in 7 of these 10 overstimulation cycles. The hormone profiles in the study group, both before and during CC treatment, are compared with those in the normal control group in Table 3. The following biochemical cycle abnormalities were defined as values for serum LH, FSH, E 2, or P that were outside the normal reference range for that hormone at the relevant stage in the cycle: [1] increased serum LH in the early follicular phase of the cycle (>6.0 mlu/ml [>6.0 IU/L]) between days -11 and -14); [2] increased serum FSH in the early follicular phase (>9.3 mlu/ml [>9.3 IU/L]) between days -11 and -14); [3] increased peak serum E2 concentration in the follicular phase (>432 pg/ml [>1585 pmoljl]); and [4] defective luteal phase (peak luteal phase serum P concentration <9.3 ng/ml [<29.5 nmoljl]). One half of the untreated cycles in the study Table 3 Hormone Results From Normal Women and Women With Unexplained Infertility Before and During CC Treatment* Early follicular phase LHt (miu/ml) FSHt (miu/ml) E2 max (pg/ml) LH peaktt (miu/ml) P maxh (ng/ml) Unexplained infertility Pretreatment 3.2 (2.8 to 3.7) 7.1 ( ) ( ) 22.4 ( ) ( ) CC 3.6 (3.0 to 4.3) 7.8 ( ) [** ( ) 17.8 ( ) ** ( ) Normal women (n = 10) 3.2 (2.6 to 3.8) 6.2 ( ) ( ) 24.6 ( ) 13.4 ( ) * Values are geometric means with 95% confidence intervals, in parentheses. t Serum LH concentration in the early follicular phase of the cycle (11 to 14 days before ovulation); conversion factor to SI units, 1.0. t Serum FSH concentration 11 to 14 days before ovulation; conversion factor to SI units, 1.0. Peak serum E2 concentration; conversion factor to SI units, II P < 0.05 versus normal controls. I[ P < 0.01 versus normal control. P < versus study group before treatment. tt Midcycle serum LH peak; conversion factor to SI units, 1.0. H Peak luteal phase serum P concentration; conversion factor to SI units, Rodin et al. CC in unexplained infertility Fertility and Sterility

4 Table 4 Hormone Profiles in Women With Unexplained Infertility Before and During CC Treatment* Hormones Normal High early follicular phase LH:I: High follicular phase FSH High E2 max II Luteal phase defects Pretreatment 18 (51.4)t 3 (8.6) 5 (14.3) 0(0.0) 11 (31.4) Clomiphene 12 (34.3) 5 (14.3) 9 (25.7) 18 (51.4)1f 1 (2.9)** * The x 2 test was used to compare the absolute proportions of untreated and CC-treated subjects in each category. t Values in parentheses are percentages. :I: Serum LH concentration in the early follicular phase of the cycle (11 to 14 days before ovulation); Serum FSH concentration 11 to 14 days before ovulation; II Increased peak serum E2 concentration in the follicular phase. 1f:l: P < **t P < group were normal according to these hormonal criteria and there was evidence of a defective luteal phase in almost one third of the cycles (Table 4). With CC treatment, there was a significant reduction in the incidence of luteal phase defects but an increased peak follicular phase serum E2 concentration occurred in half of the treated cycles (10 of 25 [40%) cycles treated with 50 mg CC from days 2 to 6 and 8 of 10 [80%) cycles treated with 100 mg CC from days 2 to 6). Nineteen untreated cycles and 21 CC-treated cycles in the study group were uniovulatory according to our US criteria. Only 63% of these untreated cycles and 48% of these CC-treated cycles had normal hormone profiles. Of the untreated uniovulatory cycles, 16% had increased early follicular phase serum LH concentrations, 5% had increased early follicular phase serum FSH concentrations, and 16% had defective luteal phases. Both LH and FSH concentrations in the early follicular phase were increased in 19% of the CC-treated uniovulatory cycles. None of the CC-treated uniovulatory cycles had defective luteal phases but 38% were characterized by increased peak follicular phase serum E2 concentrations (P < 0.01). Using hormonal criteria only, 18 untreated cycles and 10 CC-treated cycles in the study group were normal. Only 67% of these untreated cycles were uniovulatory: cyst formation occurred in 28%, and 5.5% were characterized by poor follicular growth. Eighty-three percent of the CC-treated cycles with normal hormone profiles were uniovulatory, 8% were characterized by cyst formation, and 8% showed poor follicular growth. There was no US evidence of overstimulation in any of the cycles with normal hormone profiles. Before treatment with CC, 14 cycles (30%) in the study group resulted in cyst formation. Five (36%) of these cycles had normal hormone profiles, but 7 cycles (50%) were characterized by biochemical evidence of a defective luteal phase. Three cyst formation cycles (21 %) were associated with increased levels of serum FSH in the early follicular phase. Of these 14 women, 10 had uniovulatory cycles on CC treatment, but only 5 of these had normal hormone profiles. These "CC-responders" included six of seven cyst formers with luteal phase defects and four of seven cyst formers with normal luteal phases. Ultrasound evidence of overstimulation was observed in four CC-treated cycles (one of seven [14%) treated with 50 mg CC from days 2 to 6 of the cycle and three of seven [43%) treated with 100 mg CC from days 2 to 6). Increased peak follicular phase serum E2 concentrations occurred in one of seven (14%) cyst formers treated with 50 mg CC but in five of seven (71 %) cyst formers treated with the higher dose of CC. DISCUSSION This study highlights the value of the combined use of serial US scanning and blood hormone measurements in the investigation of women with unexplained infertility who have regular menstrual cycles and hormonal evidence of ovulation. In keeping with previous observations, we identified cycle abnormalities in half of the women studied (10, 13). The reproducibility of cycle abnormalities in successive cycles was not addressed in this study but this has been reported by others (13, 14). The frequency with which these cycle abnormalities occur in an individual may be an important determinant of fertility because cyst formation (or luteinized unruptured follicle syndrome) and other subtle cycle abnormalities may also occur in women with apparently normal fertility (15). Cyst formation may be associated with either normal luteinization (5) or a poor P surge (3). Eissa et al. (13) differentiated luteinized unruptured follicle cycles in which there was a normal endocrine profile from cyst-forming cycles that were associated with a poor E2 surge and deficient luteinization, but the practical significance of this subclassification has not been demonstrated. Poor follicular growth was uncommon in the present study and was associated with either a normal hormone profile or a defective luteal phase. Presumably, a pre- Vol. 62, No.1, July 1994 Rodin et al. CC in unexplained infertility 45

5 mature LH surge is triggered in these cycles by E2 derived from multiple small follicles. Clearly, cyst formation or poor follicular growth cycles could remain undetected if hormone measurements alone were used to assess a cycle. Only a small proportion of the subjects in this study had cycles that were characterized by increased follicular-phase serum LH levels. None of these subjects had evidence of hyperandrogenism and none had polycystic ovaries on US examination. Although it has been reported that elevated serum LH levels may have a deleterious effect on fertility (16), this is unlikely to be the explanation in our patients. Although CC is used widely in the treatment of anovulatory infertility, its mechanism and site(s) of action remain only partly understood (17). In this study, treatment with low doses of CC resulted in a significant reduction in the incidence of cyst formation cycles, but this was not reflected in a corresponding increase in the number of normal ovulatory cycles because of the high incidence of overstimulation. Overall, however, in patients with unexplained infertility, CC treatment leads to an increase in fertility (18-20). In this respect, the increased follicular hormone production and the frequent occurrence of multiple follicular development might have a beneficial effect in increasing the chances of any pregnancy occurring. But, CC can disturb, rather than enhance, the process of follicular maturation in normal women and, when CC was administered in higher doses to women with unexplained infertility who had ovulatory cycles, there was a high incidence of luteinized unruptured follicle cycles (21). Follicular function often is impaired after treatment with CC (22) and there is no correlation between conception and the degree of rise in P with CC treatment (18). Furthermore, up to 45% of anovulatory women treated with clomiphene exhibit abnormal endometrial and corpus luteum development (23) and there is an increased incidence of subclinical pregnancy loss (24). The cause ofthese problems may, of course, be independent ofthe CC treatment and relate to the underlying pathology. However, these factors and the abnormal hormonal milieu in so many of the clomiphene-treated cycles may help to explain the discrepancy between the apparent ovulation rates and the pregnancy rates achieved in women treated with CC. In conclusion, we have identified a number of abnormalities in the cycles of women with regular menstruation, apparent ovulation, and unex- plained infertility. Cyst formation, with or without luteal phase defects, is the most common abnormality in these women and treatment with CC produces a significant reduction in the frequency of cyst formation cycles. The clinical significance of cyst formation in terms of its effect on fertility has not been addressed in this study and it follows that the clinical benefit of CC treatment of this disorder is not known. Overstimulation clearly is more common in women treated with the higher dose of CC, although its occurrence in at least one quarter of cycles treated with 50 mg CC from days 2 to 6 suggests that an even lower dose might be considered in these patients. Further studies are needed to determine the effectiveness of CC treatment in terms of pregnancy and birth rates and to optimize the pharmacologic treatment of these subtle cycle abnormalities. 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