Long-term suppression of ovarian function by a luteinizing-hormone releasing hormone agonist implant in patients with endometriosis

FERTILITY AND STERILITY

Vol. 53, No.1, January 1990

Printed on acid-free paper in U.S.A.

Copyright ID 1990 The American Fertility Society

Long-term suppression of ovarian function by a luteinizing-hormone releasing hormone agonist implant in patients with endometriosis Hamish M. Fraser, D.Sc. *t Jurgen Sandow, M.D.:!: Gwen M. Cowen, B.Sc.t

Mary Ann Lumsden, M.D.§ Rosie Raining, MB.tll~ Stephen K. Smith, M.D.t~

Centre for Reproductive Biology, Edinburgh, United Kingdom, and Hoechst AG, Frankfurt, Federal Republic of Germany

Ten endometriosis patients received luteinizing hormone releasing hormone (LH-RH) agonist (buserelin) implant injections (6.6 mg subcutaneously) at days 0, 42, 84 and 126. Serum LH and follicle-stimulating hormone (FSH) were lowered by day 14. Luteinizing hormone remained at basal concentrations while FSH returned to values in the low-normal range of the menstrual cycle by day 35. At the end of the luteal phase during which treatment commenced, estrone and pregnanediol declined and remained at postmenopausal or early follicular phase values until days 305 to 460. Time to first ovulation ranged from 321 to 481 days after starting treatment. After the initial menstruation, only three instances of bleeding occurred during treatment. Pelvic pain was relieved or markedly reduced by day 42 and remained absent throughout the period of ovarian suppression. These results indicate the potential of a long-acting LH-RH agonist implant to form the basis for the treatment of symptomatic endometriosis. Fertil Steril53:61, 1990

The ability of chrbnic administration of luteinizing hormone-releasing hormone (LH- RH) agonists to desensitise pulsatile gonadotropin secretion and suppress ovarian steroidogenesis is beneficial for the treatment of a number of estrogen-dependent gynecological conditions including endometriosis.1-7 Current therapies, e.g., danazol have a high incidence of unacceptable androgenic side effects and use of LH-RH agonists that lack these side effects should have an important therapeutic role Received May 24, 1989; revised and accepted September 5, 1989. *Reprint requests: Hamish M. Fraser, D.Sc., MRC Reproductive Biology Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, United Kingdom. t Medical Research Council (MRC) Reproductive Biology Unit. :j: Hoechst AG. § Department of Obstetrics and Gynaecology. II Supported by a research fellowship from Hoechst AG. 11 Present address: Department of Obstetrics and Gynaecology, University of Cambridge Clinical School, Rosie Maternity Hospital, Cambridge, United Kingdom. Vol. 53, No.1, January 1990

in relieving symptoms associated with endometriosis. 3 - 5 The LH-RH agonist buserelin administered via intranasal spray has been used extensively in these clinical trials. In the treatment of endometriosis a defined period of ovarian suppression is required to induce regression of lesions and it is assumed that the degree of regression of endometriosis is proportional to the degree of suppression of estrogen because endometriosis resolves after ovariectomy or the menopause. To ensure a sustained level of suppression constant exposure of the pituitary to the agonist is more effective, and delivery of buserelin by programmable infusion pumps or osmotic minipumps has been described in women. 7 To facilitate patient compliance the use of injectable implants should be a further advantage. In the current study we evaluated a long-lasting buserelin implant that previously was found suitable for ovarian suppression lasting 3 months after a single injection in female macaques. 8 Patients with endometriosis were treated with the implant Fraser eta!.

LH-RH agonist implant in endometriosis

61

Table 1 Clinical Findings Before, During, and at First PostTreatment Recovery Cycle in 10 Patients Treated With Buserelin Implants Severity of symptoms" time (wk)

Patient

1 2 3 4

5 6 7

8 9 10

AFS score

5 90 50 22 50 11

12 59 4

16

0

6

12

36

Recovery (mean 58)

0+0 2+2 2+1 3+3 0+2 3+2 0+2 3+1 3+3 3+3

0+0 0+0 0+0 1+3 0+2 0+0 0+0 0+0 1+0 1+3

0+0 0+0 0+0 0+0 0+0 0+0 0+0 0+0 0+0 1+0

0+0 0+0 0+0 0+0 0+0 0+0 0+0 0+0 0+0 0+0

0+0 1+1 3+2 1+1 0+0 1+0 0+0 0+0 0+0 3+2

" Symptom severity score derived by grading and assigning a numerical value to the symptoms of pelvic pain (shown first) and dysmenorrhea: absent = 0; mild = 1; moderate = 2; severe = 3. Data from date of first implant treatment (week 0) and are represented by scores at 6, 12, and 36 weeks, and during the first recovery cycle at 58 weeks (mean) after first implant.

on four occasions at intervals of 6 weeks and effects on pituitary-ovarian function, symptoms of endometriosis, and side effects were monitored. The dose interval was based on a preliminary dose estimate of daily buserelin absorption during nasal spray treatment. The bioequivalent dose to the nasal spray formulation (900 JLg) used in endometriosis is 30 JLg subcutaneously (SC). To monitor buserelin absorption, a specific radioimmunoassay for serum and urinary concentrations was used validated by infusion studies in patients with endometriosis.7·9 Approximately 30% of the dose administered is excreted in the urine as immunoreactive buserelin.9 We postulated that a dose interval of 6 to 8 weeks would be required, and that ovulation suppression would be maintained until the release rate had decreased to 30 JLg per day, corresponding ·to a urinary excretion of 10 JLg/g creatinine.

Society (AFS) classification at time of laparoscopy.10 Two patients had mild disease, two moderate disease, and 6 had severe disease. All patients had a normal cervical smear. Nine of the 10 had dysmenorrhea at time of recruitment, 7 had pelvic pain (Table 1), and 3 had dyspareunia (2 severe, 1 mild). Five patients had abnormal findings on vaginal examination, one subject (no. 2) had an adnexal mass thought to be an endometrioma measuring 4 X 4 em. Only one patient had achieved a full-term pregnancy whereas 3 others had had spontaneous abortions, the remainder being nulliparous. Eight of the women had experienced infertility at some time although only 5 wished to conceive at the time of entering the study. All patients had regular menstrual cycles before treatment (26.6 ± 0.8 days; mean ± standard error of the mean [SEM]). Five patients had had previous me~ical treatment: three were given multiple courses of danazol, and two continuous oral contraceptives (OCs). None had had endocrine therapy or had taken OCs for at least 4 months before the onset of the study. Treatment by SC implant injection commenced within 3 months of the laparoscopy. The implants were administered during the luteal phase of the cycle (days 21 to 25) as assessed from day of onset of menstruation. The implants of slowly biodegradable polylactide/glycolide (molar ratio 75:25) were sterilized by ')'-radiation. Each implant contained 3.3 mg buserelin, [D-Ser (But) 6 , LH-RH (1-9) nonapeptide-ethylamide in a rod 1 em long and 0.13 em diameter. Each patient received 4 doses of 6.6 mg buserelin (2 implants per dose) at intervals of 6 weeks injected SC in the lateral region of the abdominal wall. Sample Collection and Monitoring of PituitaryOvarian Activity

Ten patients (aged 21 to 38 years) who had endometriosis confirmed at ·laparoscopy were recruited from the gynecological unit of the Royal Infirmary of Edinburgh. Permission for the study was obtained from the local ethical committee and written informed consent obtained from all participants. Clinical details of each patient are shown in Table 1 together with The American Fertility

Early morning urine samples were collected three times per week during the cycle before starting treatment. After the first implant, samples were collected daily ·until the return of two menstrual cycles. Urine samples were stored in the domestic freezer until being taken to the laboratory where they were stored at -2o•c until required for radioimmunoassay (RIA). Urine samples were assayed for estrone glucuronide, pregnanediol glucuronide, and LH using established RIAs.U- 13 The limits of sensitivity of the assays w~re 1 mg/mL pregnanediol, 6.8 JLg/mL estrone, and 1. 7 miU/mL

LH-RH agonist implant in endometriosis

Fertility and Sterility

MATERIALS AND METHODS Endometriosis Patients

62

Fraser et al.

LH. Interassay and intra-assay coefficients of variation were 14.4% and5.4% for pregnanediol, 13.8% and 2.2% for estrone, and 13.8% and 2.5% for LH. Creatinine (Cr) was measured by an automated enzymatic method. Normal ranges for urinary hormones were established from daily samples collected during cycles from six healthy volunteers. To compare urinary estrone concentrations during treatment with those of women with inactive ovaries, early morning urine samples were collected from 9 postmenopausal women. Blood samples were collected at time of first implant injection and at weekly intervals until day 253 and at monthly intervals thereafter until return of normal cycles. Serum concentrations of LH, follicle-stimulating hormone (FSH), estradiol17{3 (E 2 ), and progesterone (P) were determined using RIAs described previously.8 •14•15 Sensitivities of the assays were 10 pg/mL E 2 , 0.2 ng/mL P, 0.56 miU/mL LH, and 0.2 miU /L FSH. Luteinizing hormone and FSH results were expressed in terms of the appropriate standard obtained from the N ationa! Institute of Biological Standards and Control, London (LH:68/40, 77 U/ampule; FSH:69/ 104,10 U/ampule). Data for hormone concentrations were subjected to one factor analyses of variance for repeated measures. The individual means were further examined using the Newman-Keuls test for pairwise comparisons. Pharmacokinetics of the Buserelin Implants

Buserelin levels in urine and in serum were measured by RIA. 7•9 In validation of the analytical procedure, the effects of incubating buserelin (250, 2500, or 5000 pg/mL) in human serum or urine for up to 24 hours at 4 ·c, 22"C, and 37"C were determined. The concentration of immunoreactive buserelin in urine was not changed during incubation. In serum, buserelin was degraded slowly at 4 ·c and more rapidly at 22"C and 37"C. This serum degradation was inhibited by bacitracin 10-3 M. 16 In the present study, blood samples (5 mL) were collected into tubes containing bacitracin to give a final concentration of 10-3 M. Serum wa!! separated in a refrigerated centrifuge and stored at -20"C until required for RIA. Immunoreactive buserelin in serum was measured in aliquots of 100 JLL giving a sensitivity of 30 pg/mL. Normal serum with bacitracin in the standard curve was without significant effect when compared with a standard curve containing assay buffer. VoL 53, No. 1, January 1990

At the end of treatment, serum from each woman was tested for antibodies to buserelin as described previously _I? No specific binding oflabelled buserelin was present. Menstrual Bleeding and Side Effects

Using daily diaries, patients recorded details of menstrual bleeding, dysmenorrhea, dyspareunia, pelvic pain, and side effects attributable to therapy, e.g., hot flushes, night sweats, and vaginal dryness. These were reviewed and discussed during the patients weekly attendance at the out-patient clinic. On each visit, injection sites were inspected and routine hematologic and biochemical parameters checked. After treatment, patients were monitored until two regular menses had been established. RESULTS Pharmacokinetics of the Buserelin Implants

After each implant injection, there was a rapid rise of the urinary buserelin excretion to an average of 136.9 ± 28 Jtg/g Cr, followed by an early fast phase ofbuserelin release (halflife (t/2) = 10 days), a second phase representing a plateau of release (t/ 2 = 60 days), and a final slow exponential decrease of buserelin release with a terminal half life of buserelin release of t/2 = 45 to 50 days. The release rates of implants in individual patients were highly reproducible, e.g., lowest concentrations on day 42 after each implant treatment were 14 to 23 Jtg/g Cr. The serum concentrations ofbuserelin were closely correlated with the urinary excretion rates; correlation coefficient r = 0.583 for 51 observations, 95% significance limit 0.4473. Serum buserelin concentrations reached the limit of detection around day 134 after the last implant, whereas the urinary concentrations of buserelin remained detectable for at least 210 days after the last implant injection. Effect on Follicular Development and Ovulation

Figure 1 shows the urinary estrone, pregnanediol and LH concentrations during days 0 to 200 from first implant injection and permits comparison of the subsequent response with hormonal changes during the normal cycle. The first implant injection induced an immediate marked rise in urinary LH, peak values being observed the following day (day 1) in all women. Luteinizing hormone levels were significantly greater than pretreatment (P < 0.01) on Fraser et al.

LH-RH agonist implant in endometriosis

63

PLG - Buserelin Implants

Control

t

•

•

eo

10 OJ~~~~~~~~~~~~~~~~~~~

14

2 0 90

eo -70

~eo 2E

-

:I:

-'

50

and levels were in the range recorded in postmenopausal women (5.5 ± 0.6 J.Lgfg Cr; mean ± SEM) with transitory elevations to early follicular phase levels. The subsequent implant administrations were virtually devoid of stimulatory effects (Fig. 1). These effects on urinary steroid excretion were confirmed by data from serum E 2 and P that changed in a similar pattern (data not shown). Serum concentrations of LH and FSH for days 0 to 200 are shown in Figure 2. Since the first blood sample was collected on day 7 after the first implant, the stimulatory effect of the agonist was not detected and serum concentrations of LH and FSH were already declining by this time. LH was significantly suppressed (P < 0.01) by 7 days, then continued to fall markedly and remained below normal concentrations until the recovery period. By day 14, FSH concentrations had also declined below pretreatment values (P < 0.05), reached a nadir at day 21 (P < 0.01lower than pretreatment), then gradually began to rise. By day 35, FSH had returned to pretreatment luteal phase values and thereafter remained in the low-normal range of the menstrual cycle. Recovery Period

40

30 20

10 0 -20-10 0 10 20

Day after LH surge

0 10 20 3D 40 50 60 70 10 80 100110120130140150160170180190200

Day After First Implant

Figure 1 Urinary estrone glucuronide, LH, and pregnanediol glucuronide during cycles from 6 control women and during days 0 to 200 in 10 patients receiving implants containing 6.6 mg buserelin at days 0, 42, 84, and 126 starting during the midluteal phase. Values are means ± SEM.

There were no marked alterations in hormonal parameters after the 200 day period shown in Figures 1 and 2 until the recovery period. This is illustrated in the individual examples shown in Figures 3 and 4. The duration of suppression varied considerably between individuals. The time to a rise in urinary estrone secretion indicative of a normal follicular phase ranged from 179 to 334 days after

6

.days 1 and 2, but by day 7, LH had returned to pretreatment values. By day 12 LH levels were significantly lower (P < 0.01) than pretreatment after which they remained in this suppressed state until the recovery period. Urinary pregnanediol levels were stimulated during the first few days after starting treatment, then fell as luteal regression occurred and remained at.follicular phase values until the recovery period in all women. Urinary estrone levels were not significantly altered during the initial stimulation period. After regression -of the corpus luteum, urinary estrone declined to remain at or below early follicular phase levels in all women until the recovery period. No evidence of sustained rises in estrone were observed during this period 64

Fraser et al. LH-RH agonist implant in endometriosis

PLG - Buserelin lmplams

f

20

40

f

60

80

'

100

120

140

160

180

200

Day After First Implant

Figure 2 Serum concentrations of LH (•) and FSH (e) during days 0 to 200 in 10 patients receiving implants containing 6.6 mg buserelin at days 0, 42, 84, and 126. Values are means ±SEM.

Fertility and Sterility

90

1000

100

"' c:

I

5"

10

~

20 10

tion lasting approximately 10 to 15 days, similar to a normal follicular phase, but not followed by an LH surge or rise in pregnanediol (e.g., Fig. 4). In three patients, the rise in estrone was accompanied by the expected rise in urinary LH, but in two of the women the LH secretion remained suppressed. These anovulatory cycles were then followed by another rise in urinary estrogen excretion followed by a normal pregnanediol elevation although the magnitude of the surges in urinary LH tended to be lower than pretreatment. Ovulation was confirmed by corresponding increments in serum progesterone. Bleeding Patterns

-40

0

40

80

120

160

200

240

280

320

360

400

440

480

Day After First Implant

Figure 3 Urinary LH (•--•), buserelin (e---e) (top panel) and estrone glucuronide (0 - -- 0) and pregnanediol glucuronide (• - - •) during the entire study period in a patient receiving implants containing 6.6 mg buserelin at days 0, 42, 84, and 126. Note normal cycles during recovery.

the last implant injection. The resumption of estrogen secretion was related to the urinary buserelin excretion, suppression being maintained at very low buserelin release rates and returning when buserelin excretion fell to 0.4 to 0.6 f.Lg/g Cr (Figs. 3 and 4). Time to first ovulation after the last implant was 275 ± 15 days (mean ± SEM), range 195 to 355 days, i.e., 321 to 481 days after starting treatment. In 4 of the 10 patients the recovery period after the last implant injection was associated with regular ovulatory cycles-a follicular rise in urinary estrone, followed by a normal luteal rise in pregnanediol (e.g., Fig. 3). Beginning about 20 days before the preovulatory LH surge a small and sustained rise in urinary LH was observed associated with an abrupt rise in urinary estrone similar to the normal follicular phase. This was followed by an LH surge and rise in pregnanediol within the normal range (e.g., Fig. 3). The ovulatory cycles were confirmed by similar increases in serum E 2 and P. In one patient, T3, who required the longest time to recovery, the urinary estrone rise was associated with a minor LH peak and a short-lasting pregnanediol elevation suggesting a luteinized follicle or an inadequate corpus luteum. The subsequent two cycles were normal in this patient. In the remaining five patients the first indication of ovarian recovery was a period of estrone excreVol. 53, No.1, January 1990

The length of the menses at the end of the cycle after the first implant was prolonged in six of the women, resulting in a significant increase in the group (P < 0.01l;>ypaired t-test) from 5.5 ± 0.4 days in the pretreatment cycle to 10.2 ± 1.4 days (means ± SEM). One patient was admitted after 1 week of treatment with exacerbation of pelvic pain that resolved within 2 days. Apart from three instances of painless light breakthrough bleeding, all women were amenorrheic during the period of ovarian suppression. Dysmenorrhea was absent during this period. Two of the five women with anovulatory

90

1000

80

_I .. 10

~50 ::>

;s

10

E 40

:s

~

30

•

•

20 10

35

3

10 10

~

5 -

0 -40

0

40

80

120

160

200

240

280

320

360

400

440

480

520

Day After First Implant

Figure 4 Urinary LH (•--•), buserelin (e--- e) (top panel) and estrone glucuronide (0-- - 0) and pregnanediol glucuronide (• - - •) during the entire study period in a patient receiving implants containing 6.6 mg buserelin at days 0, 42, 84, and 126. Note anovular cycle during recovery, followed by ovular cycles associated with low LH surges. Fraser et al.

LH-RH agonist implant in endometriosis

65

cycles had light breakthrough bleeds before reestablishing regular ovulatory cycles. The remaining eight women had an ovulatory cycle preceding the first menses. Dysmenorrhea returned with resumption of ovulatory cycles. Clinical Results

Pelvic pain resolved in all seven affected women by the time of the second implant, three of the five with severe pain noting complete remission (Table 1). Six of the seven were pain free through 36 weeks, and remained pain free until after the second recovery cycle. Two patients noted occasional episodes of pain during the recovery period. The abnormal findings on vaginal examination were improved during treatment, although in two of the five patients with local tenderness and pain this was not apparent until the completion of treatment. The adnexal mass present in one patient before implant treatment was not palpable at 36 weeks after treatment but became palpable at 40 weeks and continued to increase in size during the remainder of the study period. Of the three women with dyspareunia, two experienced complete relief and one partial relief by the time of the second implant. Of the five women who initially presented with infertility, two (nos. 1 and 2) became pregnant within four months of returning to cyclical ovarian activity after 3 and 8 years of infertility respectively. During the 6 months after resumption of cycles, five of the patients presented at the clinic complaining of return of symptoms. Side Effects

Hot flashes were experienced by 8 of 10 patients (median onset was day 8, range 1 to 42). These were mild and of occasional occurrence in 6 subjects but were severe in two women. Intermittent headaches occurred in 2 subjects and one complained of nausea. Two women complained of mild superficial dyspareunia (vaginal dryness) at 6 weeks. Of the 40 implant treatments administered, one injection caused local swelling, another injection caused local pain, while after one injection a small section of the implant material protruded from the injection site and was removed at the time of the next injection. DISCUSSION

Taken together with previous studies using LHRH agonists delivered by nasal spray or by daily 66

Fraser et al.

LH-RH agonist implant in endometriosis

injection 1- 5 the present results demonstrate the potential of treating patients with symptomatic endometriosis by sustained release of an LH-RH agonist from a biodegradable implant. By giving the first implant injection during the luteal phase the initial stimulatory phase of the LH-RH agonist was restricted and rises in E 2 seen when agonist treatment begins during the follicular phase were avoided. It appears that pituitary desensitisation is more rapid when treatment begins during the luteal phase, avoiding stimulation of early recruited follicles. 7•15•18•19 The advantages of injectable implants to ensure a constant level of gonadal suppression and facilitate patient compliance were clearly demonstrated in this study. The sustained decline in serum and urinary LH levels confirms the desensitisation of the pituitary gonadotrope with resultant decreased LH release as the primary action of the agonist-mediated suppressio~} of ovarian function. This finding contrasts with results in which immunoreactive LH levels did not fall after chronic agonist therapy, 4 •7 because of use ofLH RIAs that detect the increased output of a-subunit during agonist treatment. The lack of acute LH increase after subsequent implants that had high initial release of buserelin confirms that the pituitaries of these women were desensitised during the treatment period. The effects of the treatment on FSH secretion are more difficult to explain. After the marked initial fall in serum FSH concentrations the secretion began to rise again after 21 days. Because the rise in FSH was associated with suppressive release rates of buserelin and with low levels of estrogen production, it is tempting to suggest that the initial decrease in FSH secretion is attributable to the negative feedback effect of increased secretion of ovarian factors during the initial stimulatory phase. However, a preferential decrease in FSH secretion during the initial period of LH-RH agonist-induced pituitary desensitisation was also observed in post-menopausal women20 and after LH-RH infusion in ovariectomized women. 21 This suggests that the initial fall in FSH secretion is due principally to the desensitisation mechanism. The subsequent rise in FSH was gradual and of low magnitude; nevertheless, it occurred during a period of maximal suppression of LH. A rise in FSH has been described in a number of other studies in women during infusion with LH-RH agonist and treatment with depot formulations. 6 •7 •19•22 This secretion could be either autonomous because of the withdrawal of negative feedback or because of a Fertility and Sterility

separate FSH-RH that operates independently of the LH-RH receptor. More likely, the FSH rise after prolonged treatment could be because of differential sensitivity to the LH-RH agonist, its continuous presence in high amounts desensitising FSH synthesis and secretion to a lesser extent than that of LH. Despite the gradual rise, FSH is not secreted in sufficient quantity to induce follicular development. The effective release rate of buserelin was much lower than expected. We had anticipated that using a dose interval of 6 to 8 weeks ovarian suppression would be maintained until the release rate had decreased to 30 !J,g per day, corresponding to an average urinary excretion of 10 JLg/g creatinine that is found during nasal spray treatment of 3 X 300 JLg buserelin per day. Unexpectedly, suppression of ovarian activity was maintained at much lower release rates, until the urinary buserelin excretion had declined to 0.4 to 0.6 JLgfg Cr and this resulted in the wide individual variation in return to pituitary function. From animal experiments it is known that progressive depletion of gonadotropin content and decreased synthesis occurs during long-term infusions of LH-RH and its agonists as part ofthe desensitisation process. 23 It is likely that in this study that prolonged inhibition of appropriate LH synthesis was also a major contributing factor to the suppression observed in the presence of low amounts of buserelin. The findings may also indicate that the dose requirement for pituitary inhibition decreases in the hypoestrogenic state. In other clinical trials with LH-RH agonists at lower daily doses the majority of women resume normal cycles shortly after nasal administration is stopped. 1- 5 This is also the case when implants of more rapidly degrading polylactide/glycolide are administered. 22 With the present treatment regimen the recovery of pituitary function seemed to be gradual. A source of individual differences may stem from the individual variation that exists for the amount of FSH required to initiate follicular development. 24 In the five patients with follicular development not followed by an immediate ovulatory rise in pregnanediol there was also no evidence of a LH surge suggesting that at this time pituitary gonadotropin output was sufficient to induce follicular development but not yet adequate for an ovulatory LH increase. Previous large scale clinical trials using LH-RH agonist delivered by nasal spray or daily injection have established a decrease in The American Fertility Society scores in 80% of patients after 6 Vol. 53, No.1, January 1990

months treatment. 1- 7 A second laparoscopy was not performed in the present study because suppression of estrogen was reliably obtained and it was considered that the absence of the repeat laparoscopy was compensated for by the detail of the endocrinological assessment and clinical examination. The buserelin implants proved effective in alleviating the symptoms of endometriosis during the period of ovarian suppression in all of these patients. Although 8 to 10 patients experienced hot flushes and minor side effects associated with the treatment, these were tolerable and no patient withdrew from the trial because of side effects. Injection ofbuserelin implants was well tolerated except for minor problems at the injection sites that might be avoided by improved injection technique. The implants ensured good patient compliance, and subjectively were more acceptable to the 10 patients than daily formulations. Some patients with endometriosis require prolonged treatment for recurrent symptoms of pain and LH- RH agonist implants offer an effective long term management of endometriosis-related pain. The most important side effect of the hypoestrogenism associated with the treatment is the risk of accelerated bone mineral loss. 25 While loss of bone mineral is likely to be reversed by the return to normal cycles, long-term studies are required to establish the risk group and the permissible suppression period more clearly. Because symptoms recurred in some patients even after the deep and prolonged suppression achieved in this study, the approach may require surgical intervention after down-staging of the endometriosis lesions or be used with another agent given in combination or sequentially. Acknowledgments. We thank Vicky Reid-Thomas R.G.N. and the staff of the Research Area, Simpson Memorial Maternity Pavilion for their help with this study, Stephen G. Hillier Ph.D. and the technical staff of the Regional Endocrine Laboratory for assay of creatinine and Heidrun Trabant M.D. (Hoechst AG, Frankfurt, Germany) and Patrick Magill Ph.D. (Hoechst UK) for their interest.

REFERENCES 1. Shaw RW, Fraser HM, Boyle H: Intranasal LHRH in the

treatment of women with endometriosis. Br Med J 287: 1667, 1983 2. Lemay A, Maheux R, Faure N, Jean C, Fazekas ATA: Reversible hypogonadism induced by a luteinizing hormonereleasing hormone (LH-RH) agonist (Buserelin) as a new therapeutic approach for endometriosis. Fertil Steril 41: 863,1984

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'

''

3. Schriock E, Monroe SE, Henzl M, Jaffe RB: Treatment of endometriosis with a potent agonist of gonadotropin-releasing hormone (nafarelin). Fertil Steril44:583, 1985 4. Steingold KA, Cedars M, Lu JKH, Randle D, Judd HL, Meldrum DR: Treatment of endometriosis with a long-acting gonadotropin-releasing hormone agonist. Obstet Gynecol69:403, 1987 5. Henzel MR, Carson SL, Moghissi KS, Buttram VC, Bergquist C, Jacobson J: Administration of nasal nafarelin as compared with oral danazol for endometriosis. N Engl J Med 318:485, 1988 6. Zorn JR, Tanger Ch, Roger M, Grenier J, Comaru-Schally AM, Schally AV: Therapeutic hypogonadism induced by a delayed-release preparation of microcapsules of D-Trp-6 luteinizing hormone-releasing hormone: a preliminary study in eight women with endometriosis. Int J Fertil 31: 11,1986 7. Lemay A, Sandow J, Bureau M, Maheux R, Fontaine J-Y, Merat P: Prevention offollicular maturation in endometriosis by subcutaneous infusion of luteinizing hormone-releasing hormone agonist started in the luteal phase. Fertil Steril49:410, 1988 8. Fraser HM, Sandow J, Seidel H, von Rechenberg W: An implant of a gonadotropin releasing hormone agonist (buserelin) which suppresses ovarian function in the macaque for 3-5 months. Acta Endocrinol (Copenh) 115:521, 1987 9. Kiesel L, Sandow J, Bertges K, Jerabek-Sandow G, Trabant H, Runnebaum K: Serum concentration and urinary excretion of the LHRH agonist buserelin in patients with endometriosis. J Clin Endocr Metab 68:1167, 1989 10. The American Fertility Society: Revised American Fertility classification of endometriosis: 1985. Fertil Steril 43:351, 1985 11. Samarajeewa P, Cooley G, Kellie A: The radioimmunoassay ofpregnanediol-3-a glucuronide. J Steroid Biochem 11: 1165,1979 12. Samarajeewa P, Kellie A: The radioimmunoassay of steroid glucuronides. Biochem J 151:369, 1975 13. Djahanbakhch 0, McNeilly AS, Hobson BM, Tempelton AA: A rapid luteinizing hormone radioimmunoassay for the prediction of ovulation. Br J Obstet Gynaecol88:1016, 1981 14. Vaughan-Williams CA, McNeilly AS, Baird DT: Comparison of a single and repeated applications of a long-acting synthetic analogue of LHRH [D-SER(TBU) 6 EA10LHRH] in the assessment of pituitary gonadotrophin secretory capacity. Clin Endocrinol (Oxf) 13:51, 1980

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