Leuprolide acetate lowers circulating bioactive luteinizing hormone and testosterone concentrations during ovarian stimulation for oocyte retrieval

Vol.

FERTILITY AND STERILITY Copyright c

1990 The American Fertility Society

53, No.4, April 1990

Printed on acid·free paper in U.S.A.

Leuprolide acetate lowers circulating bioactive luteinizing hormone and testosterone concentrations during ovarian stimulation for oocyte retrieval

Marcelle I. Cedars, M.D.*t Eric Surey, M.D.* Fredesminda Hamilton, B.S.E.:j:

Phillip Lapolt, Ph.D.* David R. Meldrum, M.D.*:j:

University of California, Los Angeles, and AMI South Bay Hospital IVF Center, Redondo Beach, California

Levels of immunoreactive luteinizing hormone (LH), bioactive LH, and testosterone (T) were determined in 52 women receiving human menopausal gonadotropins (hMG). In 26 women receiving leuprolide acetate (LA) preceding hMG, there was a significant suppression of immunoreactive LH and bioactive LH. The characteristic increase in serum levels ofbioactive LH and T were absent. Follicular fluid estradiol and T concentrations, and serum progesterone were not different. The lower circulating levels of T may reflect reduced LH -stimulated androgen accumulation in smaller nonaspirated follicles and may account for the enhanced follicle recruitment observed during LA. The lack of premature luteinization despite marked rises of bioactive LH in the absence of LA is consistent with normal events during the menstrual cycle and was due to the early termination ofhMG stimulation. Fertil Steril53:627, 1990

Two major problems occur during controlled ovarian hyperstimulation (COH) for oocyte retrieval. First, sustained growth of secondary follicles may not occur, resulting in follicle atresia and development of a single dominant follicle. Second, before full follicle maturation, the pituitary may respond to the rising level of estrogen (E), accompanying the growth of multiple follicles, resulting in raised basal secretion1 or a full surge 2 of luteinizing hormone (LH). Atresia of secondary follicles is associated with accumulation of intrafollicular androgen,3 an LH-dependent process. 4 Long-acting agonists of gonadotropin -releasing hormone Received June 26, 1989; revised and accepted December 7, 1989. * Department of Obstetrics and Gynecology, University of California. t Reprint requests and present address: Marcelle I. Cedars, M.D., Department of Obstetrics and Gynecology, University of Cincinnati Medical Center, 231 Bethesda Avenue (ML526), Cincinnati, Ohio 45267. :j: AMI South Bay Hospital IVF Center.

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(GnRH-a) suppress bioactive LH in normally menstruating women,5 increase recruitment of secondary follicles and prevent premature LH surges in prior poor responders,6-9 and suppress the basal level of immunoreactive LH during COH.9 The associated increase in the pregnancy rate with concommitant use of GnRH-a during COH 1o,n and the lower rate of pregnancy in non-GnRH-a cycles with raised basal secretion of LH1 have suggested beneficial effects of GnRH -a on oocyte quality by preventing premature luteinization of follicles and senescence of oocytes. Alternatively, the observed increased rate of pregnancy could be because of an improved ratio of androgen to E in aspirated follicles' or to other variables not controlled in these studies. To further investigate the effects of GnRH-a during COH, we have examined the levels of immunoreactive LH and bioactive LH, and circulating and intrafollicular testosterone (T) concentrations during COH in GnRH-a and non-GnRHa cycles. The peripheral levels of progesterone (P) were used to reflect the occurrence of premature luteinization.

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MATERIALS AND METHODS

Fifty-two women underwent COH for oocyte retrieval using daily injections of three to five ampules of human menopausal gonadotropins (hMG, 75 IU of follicle-stimulating hormone [FSH] and 75 U of LH per ampule, Pergonal; Serono Laboratories, Inc., Randolph, MA). In 26 women, ovarian stimulation was preceded by pituitary suppression with subcutaneous injections of leuprolide acetate (LA, Lupron; TAP Pharmaceuticals, North Chicago, IL), 1.0 mg/d for 10 days until serum estradiol (E 2) level was <35 pg/mL, and then accompanied by LA, 0.5 mg/d until the injection of human chorionic gonadotropin (hCG) as previously reportedP Leuprolide acetate was begun in the midluteal phase as determined by basal body temperature chart or P level. Human chorionic gonadotropin was injected when the leading follicle had a longest diameter of at least 1.5 cm in the hMG only group and 2.0 cm in the women receiving LA. Patients were matched for the circulating concentration of E2 on the day of hCG injection. Blood samples were drawn before starting hMG on day 2 of the menstrual cycle in the non-GnRH-a cycles, after LA suppression and before hMG administration in the GnRH -a cycles, and on the day ofhCG injection in both groups of women. Follicular fluid (FF) from nine women in each group was separated from two to six clear aspirates of mature preovulatory follicles by centrifugation. The supernatants and sera were stored at -20 DC until assay. Testosterone concentrations were analyzed using extraction, celite chromatographic separation, and radioimmunoassay (RIA) as previously described. 13 Levels of E 2, immunoreactive LH, and P were analyzed by 1125 RIA kits (Pantex, Santa Monica, CA; Serono-Baker Diagnostics, Allentown, PA; and Diagnostic Products Corp., Los Angeles, CA; respectively). To assure the lack of cross-reaction of the T antiserum with the numerous steroids and other substances in FF, the chromatographic profile of immunoreactive T extracted from pooled FF was compared with that of the authentic radiolabeled standard as previously described. 14,15 Serum bioactive LH was quantified in triplicate using a dispersed mouse Leydig cell bioassay adapted from several methods. 16,17 Testes from 4to 5-week-old mice were decapsulated and cut into small pieces in a Petri dish on ice. The pieces were added to 25 nM M199 containing 25 mM HEPES

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and 0.2% bovine serum albumin (BSA, Fraction V; Sigma Chemical Co., St. Louis, MO) and stirred gently with a magnetic stirrer for 30 minutes at room temperature. The remaining tissue clumps were drawn repeatedly through fire-polished pasteur pipettes until the entire mixture was homogenous. The mixture then was filtered through fine mesh gauze. The filtrate was preincubated for 1 hour at 34 DC with shaking at 60 cycles/minutes. After preincubation, the cell suspension was centrifuged for 15 minutes at 130 X g at 4 DC. The supernatants were discarded and the cells were suspended in M199 containing no BSA at a final concentration of 200,000 cells per 100 JLL (6 to 8 mL/ testis). Cell viability assessed after staining with 0.1 % trypan blue was routinely >90%. The standard used was LER-907 (National Pituitary Agency, National Institute of Diabetes, Digestive Diseases, and Kidney, Bethesda, MD). The standards (1.4 mlU /mL to 750 mlU /mL) and serum samples (5 JLL or 1:20 dilution) were added to 13 X 100 mm culture tubes in 0.1 mL M199 containing 0.2% BSA. Each standard tube contained serum (diluted 1:20) from a woman receiving an oral contraceptive. The cells were added in 100 JLL M199 (no BSA) with continuous stirring and the mixture incubated for 4 hours at 34 DC with shaking (60 cycles/min). At the end of the incubation period, the incubation mixtures were snap frozen and stored at -20 DC until assayed. At that time, they were thawed and extracted with ether. Testosterone was then quantitated by RIA without prior chromatographic purification. All samples were run in the same assay. To determine if GnRH-a had any direct effects on measurement ofbioactive LH activity, aliquots of GnRH-a were added to standards. There was no effect noted of various concentrations of GnRH-a on the resultant standard curves. For each woman, the FF levels of E2 and T were averaged. Hormone levels were compared between GnRH-a and non-GnRH-a cycles by group Student's t-tests and within each group by paired t-tests. When variances were not homogenous, the t-test was applied assuming unequal variances. RESULTS

Shown in Table 1 are the means ± standard error serum levels of E 2, immunoreactive LH, bioactive LH, T, and P before hMG stimulation and on the day of hCG in the hMG-only and LA/hMG cycles. Compared with day 2 of the menstrual cycle, the

Fertility and Sterility

Table 1 Levels ofE 2, Immunoreactive LH, Bioactive LH, T, and P Before HMG and on the Day ofhCG in Women Receiving Only HMG and in Women Also Receiving LA HMG BeforehMG"

DayofhCG"

LA/hMG Probability

Before hMG"

Probability

1299 ± 92

1304 ± 91

E2 (pg/mL) Immunoreactive LH (mID/mL) Bioactive LH (mID/mL) T(ng/mL) P (ng/mL)

DayofhCG"

3.8 ± 0.3 b

5.8 ± 0.9 c

P<0.05

2.4 ± 0.3

1.5 ± 0.1

P<0.05

76±6 0.40 ± 0.05

160 ± 17 d 0.65 ± 0.05 1 0.59 ± 0.03

P
69 ± 10 0.34 ± 0.03

51 ± 7 0.45 ± 0.04 0.58 ± 0.03

Nse P<0.05

" Values are means ± SE. b HMG-only patients significantly different from LA/hMG patients before hMG (P < 0.005). c HMG-only patients significantly different from LA/hMG patients on day ofhCG (P < 0.001).

d HMG-only patients significantly different from LA/hMG patients on day of hCG (P < 0.001). e Not significant. 1 HMG-only patients significantly different from LA/hMG patients on day of hCG (P < 0.01).

levels of bioactive LH and T after 10 days of LA were only mildly decreased. After an average of 10 further days of LA, on the day of hCG, the level of bioactive LH was significantly reduced (51 ± 7 mIU/mL) compared with the day before hMG in the women who were not receiving LA (76 ± 6 mIU/mL). In contrast, immunoreactive LH and bioactive LH concentrations were significantly increased on the day ofhCG in the non-GnRH-a cycles (5.8 ± 0.9 and 160 ± 17 mIU/mL) compared with day 2 of menses and compared with the values measured during LA administration. The ratio of bioactive LH to immunoreactive LH was significantly increased (P < 0.02) on the day of hCG in the absence of LA. Serum T increased in both groups but was significantly higher in the hMGonly group. The serum concentrations of P were not different in the GnRH-a versus non-GnRH-a groups (0.58 ± 0.03 and 0.59 ± 0.03 ng/mL, respectively). To characterize our assay system for T, we evaluated the chromatographic profile of FF T versus radiolabeled T standard. Figure 1 indicates that the chromatographic profiles of immunoreactive and radiolabeled T were superimposable when adjusted for peak concentrations. Shown in Table 2 are the FF E2 and T concentrations and the E2:T ratio in the two groups. No significant differences were noted in the absolute concentrations or the ratios between these two steroids.

reduced rate of fertilization 18 and a lower rate of pregnancy.1 The concommitant use of GnRH -a has been shown to reduce immunoreactive LH9 and has been associated with an increased rate of pregnancy,10,n although this remains to be proven by a prospective, randomized study in which COH is accomplished with hMG in both GnRH-a and nonGnRH-a groups. The present study confirms that bioactive LH is also suppressed during GnRH -a, with a significant reduction being achieved between 10 and 20 days of LA administration. We are aware that there is variability in the bioactivity of the LH in different lots of hMG. 19,2o However, the magnitude of the difference between the two groups cannot be accounted for by this variability. The use of GnRH -a has been shown to increase recruitment of secondary follicles. 6 - 9 Since atresia is associated with the accumulation of intrafollicu250

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DISCUSSION

During COH, an increased basal concentration of immunoreactive LH has been associated with a

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Figure 1 Chromatographic profile of immunoreactive testosterone in follicular fluid (open circles) compared with labeled standard (closed circles).

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Table 2 Levels of FF E 2, T, and the E2:T Ratio in Women Receiving Only HMG and in Women Also Receiving LA a

E2 (pg/mL) T (ng/mL) E2:T a

HMG

LA/hMG

505 ±68 0.63±0.1 833 ± 156

619 ± 154 0.76 ± 0.1 889 ± 130

Values are means ± SE.

lar androgen,3 which is an LH-dependent process,4 this enhancement of the growth of secondary follicles during GnRH -a may be due to suppression of bioactive LH and the intraovarian production of T. The significant reduction of circulating T without a corresponding reduction of FF T in mature preovulatory follicles in the present study suggests that LA reduced androgen secretion in the smaller follicles that were not aspirated. These findings are consistent with the hypothesis that GnRH-a enhances follicle recruitment by reducing androgen accumulation in secondary follicles. In the absence of LA, the concentration ofbioactive LH was markedly increased by the day of hCG injection, in spite of only modest elevations of immunoreactive LH. During the menstrual cycle, bioactive LH increases well in advance of immunoreactive LH,21 presumably due to an effect of rising concentrations of E on the processing of LH within the gonadotropes. This increase was not seen in the present study during LA, in spite of matched levels of E 2, suggesting that this effect of E on pituitary production of bioactive LH is not expressed in the presence of GnRH-a suppression. In spite of much higher bioactive LH levels in the non-GnRH-a group on the day of hCG injection, the mean level of serum P was not increased. During the menstrual cycle, bioactive LH increases well in advance of immunoreactive LH and P. 21 Clearly, for luteinization to occur, both increased bioactive LH and a sufficiently mature follicle are necessary. In the non-GnRH-a group, it appears that hCG was administered before the leading follicles were sufficiently mature to respond to these levels ofbioactive LH. During COH, premature release of LH has been examined by measuring immunoreactive LH in all studies to date. The results of the present study indicate that premature increases ofbioactive LH could be responsible for adverse changes in the follicle and would not be detected by examining immunoreactive LH concentrations alone. The intrafollicular level ofT was not measurably

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reduced by GnRH-a, in spite of a markedly lower level ofbioactive LH. In the presence of supraphysiologicallevels of FSH and presumably of aromatase, androgen precursor may be rapidly converted to E. If so, a lower production of intrafollicular T in the GnRH-a group might be reflected in a lower production of E 2. This is consistent with the observation in the present study that FF E2 was similar with GnRH-a in spite of the follicles being larger, with hCG being administered about 2 days later.10 It is also consistent with the finding of lower E2 levels with GnRH-a when follicle size was similar, with or without the agonist, on the day of hCG. 7 Measurement of FF T may also be affected by thecal androgen secretion occurring preferentially toward the circulation rather than leading to accumulation in the FF, 22 particularly in mature follicles' which have a well-vascularized theca. 23 In the present study, the FF levels of T were 10to 100-fold lower than in prior studies of spontaneOUS3,24 and hMG-stimulated cycles. 25 Our analysis of the chromatographic system used in these experiments indicate that FF, T, and T standard have superimposable profiles (Fig. 1), suggesting that our assay is measuring T specifically. This discrepancy warrants further investigation and stresses the need for specific assays to be used in any future studies of FF androgens. Obviously for complete validation of an assay system in new biological fluids, gas chromatography would be a more conclusive method.

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