FERTILITY AND STERILITY
Vol. 56, No. 3, September 1991
Copyright c 1991 The American Fertility Society
Printed on acid-free paper in U.S.A.
Suppression of corpus luteum function by the gonadotropinreleasing hormone antagonist Nai-Giu: effect of the dose and timing of human chorionic gonadotropin administration*
Sophie Dubourdieu, M.D.t:j: Bernard Charbonnel, M.D. t Maria Rebecca Massai, M.D.§
Julia Marraoui, M.D.§ Irving Spitz, M.D. II Philippe Bouchard, M.D.§
Clinique Endocrinologique, HOtel-Dieu, Nantes, and Service d'Endocrinologie Hopital Bicetre, Le Kremlin Bicetre, France, and The Population Counci~ Center for Biomedical Research, New York, New York
Objective: To assess the effect of gonadotropin-releasing hormone antagonist Nal-Glu administration in the luteal phase and the potential rescue by exogenous human chorionic gonadotropin (hCG) of corpus luteum (CL) after antagonist treatment. Design: We studied the dose of Nal-Glu required for luteolysis and subsequently we coadministered low doses of hCG for 3 consecutive days either simultaneously to Nal-Glu administration (n = 5), or 48 (n = 5), or 72 hours (n = 5) later. Six additional participants received pharmacological doses of hCG 48 hours after the luteolytic dose of Nal-Glu. Setting: Participants were studied in Clinique Endocrinologique, Nantes, and in Service d'Endocrinologie, Hopital Bidltre, Le Kremlin Bicetre, France. Participants: Twenty-nine normal young women (ages 20 to 35) were studied. Interventions: None. Main Outcome Measure: Measurements of follicle-stimulating hormone, luteinizing hormone (LH), estradiol, Progesterone (P) levels were performed by radioimmunoassay before, during, and after the various treatment regimens. Results: Complete luteolysis occurred in women who received 10 mg of Nal-Glu daily on days 4 and 5 after the LH surge. The coadministration of Nal-Glu and hCG overrode the effect of the antagonist (P = 48.8 ± 22.5 versus 60.8 ± 3.1 nmol/L in controls treated with hCG alone [NS]). When hCG treatment was started 48 hours after Nal-Glu, a partialluteolysis occurred (P = 33.8 ± 10.9 versus 117 ± 12.9 nmol/L, P < 0.01). When hCG was started 72 hours after Nal-Glu, a complete luteolysis occurred (P = 5.8 ± 2.05 versus 36.2 ± 0.6 nmol/L, P < 0.01). Higher doses of hCG (1,500 or 5,000 IU) administered 72 hours after Nal-Glu resulted in a significant rescue of CL function (P = 37.7 ± 4.8 and P = 43.8 ± 22.2 versus 74.5 ± 19.8 and 130.2 ± 14.3 nmol/L, P < 0.05), respectively. Conclusions: These results confirm the LH dependence of CL function. The suppression of CL LH support for 72 hours induced a compromise of the CL nonreversible by low doses of hCG Fertil Steril 56:440, 1991 mimicking early pregnancy but reversible with pharmacological doses.
Received December 12, 1990; revised and accepted May 7, 1991. * Supported through cooperative agreement from the United States Agency for International Development, as well as the George J. Hecht Fund, the Andrew W. Mellon, and The Rockefeller Foundations, New York, New York (P.B., B.C.). t Clinique Endocrinologique, Hotel-Dieu. :j: Reprint requests: Sophie Dubourdieu, M.D., Clinique Endocrinologique, Hotel-Dieu, Nantes, France. § Service d'Endocrinologie, Hopital Bidltre. II The Population Council, Center for Biomedical Research.
440
Dubourdieu et al.
Luteolytic effect of Nal-Glu
It is widely believed that endogenous luteinizing hormone (LH) secretion is required to sustain corpus luteum (CL) function in primates1- 4 as well as in women. 5•6 The interruption of pulsatile gonadotropin-releasing hormone (GnRH) administration during the luteal phase in monkeys with hypothalamic lesions results in a dramatic decline of serum progesterone (P) levels. 7 However, CL function can be rescued if pulsatile GnRH therapy is reinitiated Fertility and Sterility
within 3 days, suggesting that CL viability can be preserved without LH support for at least 72 hours. 8 Gonadotropin-releasing hormone antagonists are synthetic peptide analogs of GnRH that suppress pituitary gonadotropin secretion. 9 They therefore provide an important tool to study the LH dependency of CL function. A single injection of the GnRH antagonist detirelix ([N-Ac-D-Nal(2)\ DpCl-Phe2 , D-Trp 3 , DhArg (Et2 ) 6 , DAla10 ]GnRH; Syntex Research, Palo Alto, CA) to stumptailed macaques suppressed bioactive LH and P during all stages of the luteal phase. 3 When the antagonist was administered as a single dose in the early to midluteal phase, P levels recovered once the effect of the antagonist had worn off. But early luteal phase treatment with the same antagonist for 3 days suppressed serum P levels for the remainder ofthe cycle. Similar results have been shown in women by McLachlan et al. 5 using the GnRH antagonist Nal-Glu ([AcD2Nal\ D 4-ClPhe 2 , D3Pal3 , Ar~, DGlu6 (AA), D Ala10 ]GnRH). In their study, the duration of LH suppression was dose-related, and the induction of luteolysis required gonadotropin deprivation for >3 consecutive days. These results suggest that the immediate decrease in P, estradiol (E 2 ), and inhibin levels seen after GnRH antagonist administration during the luteal phase did not necessarily imply luteolysis; CL viability could be preserved for about 3 days without LH support. A contraceptive (postcoital) strategy, based only on GnRH antagonist treatment in the luteal phase, may be successful, but the presence of human chorionic gonadotropin (hCG) may produce rescue of CL function. The aim of this study was to assess the potential rescue of CL function by hCG after GnRH antagonist treatment, using different doses and various timings of hCG administration.
to induce luteolysis. On day 4 after the midcycle LH surge, four subjects received 10 mg of Nal-Glu, and four subjects received two consecutives injections (10 mg) of Nal-Glu on days 4 and 5 after the LH surge. All injections were given by the subcutaneous (SC) route into the abdomen. The timing of the midcycle LH surge (day 0) was determined by rapid analysis of plasma LH levels performed daily from day 12 of the cycle. Study 2: Administration of Nal-Glu and Low Doses ofHCG
An experimental paradigm was designed to study the effect of physiological doses ofhCG on CL function in women who had previously received antagonist to induce luteolysis. In this model, there was a variable duration of LH deprivation before hCG treatment. Each volunteer received the dose of N alGlu found in study 1 to induce luteolysis, i.e., 10 mg on days 4 and 5 after the LH surge. In addition, on 3 successive days intramuscular hCG (gonadotrophine chorionic "Endo"; Organon, Saint Denis, France) was given in increasing doses to mimic plasma hCG levels seen in early pregnancy6 (100 IU /m 2 on day 1,200 IU /m 2 on day 2, and 300 IU/m 2 on day 3). The subjects were divided into three groups depending on which day they received the first injection ofhCG. Group 1 (n = 5) received hCG along with Nal-Glu on days 4, 5, and 6 after the LH surge; group 2 (n = 5) received hCG on days 6, 7, and 8, i.e., 48 hours after the first injection of NalGlu, and group 3 (n = 5) received hCG on days 7, 8, and 9 after the LH surge, i.e., 72 hours after the first injection of Nal-Glu. Results were compared with three control groups each comprising three subjects who received hCG alone, on the same experimental days.
MATERIALS AND METHODS Subjects
Twenty-nine normal young women, 20 to 35 years of age, with regular menstrual cycles (mean length, 28 days; range, 26 to 32) and ovulatory basal body temperature charts were studied. None of the subjects were using a hormonal method of contraception or taking any medication. Each subject signed informed consent. Protocol Study 1: Single and Repeated Administration of Nal-Glu
Two groups of volunteers were studied to determine the dose and duration of treatment necessary Vol. 56, No.3, September 1991
Study 3: Administration of Nal-Glu and Pharmacological Doses of HCG
To evaluate the ability of pharmacological doses of hCG to rescue CL function after a 3-day period of LH suppression, each women received the dose of Nal-Glu found in study 1 to induce luteolysis, i.e., 10 mg on days 4 and 5 after the LH surge and hCG on days 7, 8, and 9 after the LH surge, i.e., 72 hours after the first injection of Nal-Glu. The hCG doses employed were 1,500 IU (n = 3) and5,000 IU (n = 3) daily. Results were compared with two control groups of two subjects each who received hCG alone on the same experimental days. Dubourdieu et al.
Luteolytic effect of Nal-Glu
441
Hormone Assays
To determine the day of the midcycle LH surge, LH was evaluated by a chemiluminescence system (Amerlite Estradiol-60 Assay; Amersham International plc, Amersham, United Kingdom). Measurement of gonadotropins was performed by radioimmunoassay (RIA, cotropin-F; Clinetics Corporation Laboratories, Tustin, CA). The intra-assay and interassay coefficients of variation for follicle-stimulating hormone (FSH) and LH were 3.7% and 3.5% and 3.1% and 3.7%, respectively. The lower limits of sensitivity of the assays were 2.5 mUI/mL for LH and 0.5 miU/mL for FSH. Cross-reactivity in the FSH assay was 0.2% for both LH and hCG. Human chorionic gonadotropin was measured by an immunoenzymatic method (IMX Abbott, North Chicago, IL). The sensitivity was 0.76 miU/mL. Intraassay and interassay CVs were 4.4% and 6.7%, for low levels ofhCG (6 miU/mL). Estradiol and P were also determined by RIA (Biomerieux Laboratories Marcy l'Etoile, France). The sensitivity of the Pas~ say was 0.17 nmol/L and 44 pmol/L for the E 2 assay. The intra-assay and interassay CVs for P were 3.5% and 4.5% for values on the order of 34 nmol/L. For E2, the intra-assay and interassay CVs variation were 4.8% and 3.9% for values in the normal preovulatory range. The duration of the luteal phase was calculated as being the period of time between the day after the preovulatory LH surge and the 1st day of the menstrual cycle. The results were expressed as means ± SEM. Statistical analysis of the results was performed using Student's t-test with a P value of significance assigned at 95% (from 25.6 ± 12.9 nmol/L to 1.0 ± 0.6 nmol/L, P < 0.01), both reaching their lowest point 72 hours after the initial administration of the drug (Fig. 1). Luteinizing hormone levels recovered their preinjection levels 5 days after the first injection of Nal-Glu, but E 2 and P hormone levels remained consistently low for the duration of the menstrual cycle, reflecting a compromise of the CL viability. Two of the four subjects who received 10 mg of Nal-Glu experienced varying delays in the occurrence of menstrual bleeding. In all women treated with 10 mg Nal-Glu antagonist for 2 consecutive days, premature menstrual bleeding occurred within 48 hours of the first dose of Nal-Glu antagonist and lasted' 2 to 5 days. The next menstrual period occurred at a minimum of 1 month after the initial injection of antagonist. Study 2: Administration of Nal-Glu and Low DosesofHCG
Plasma hCG levels rose in parallel with the increasing doses of hCG to 5.6 ± 2.7 miU/mL, 7.3 ± 2.7 miU/mL, and 9.6 ± 5.5 miU/mL 48, 72, and 92 hours, respectively, after the first injection of hCG, i.e., in the range of plasma levels usually seen around the time of implantation. 10 Group 1: Coadministration of Nal-Glu With HCG
Simultaneous administration ofhCG and Nal-Glu blocked the effect of the antagonist. Serum P
RESULTS Study 1: Single and Repeated Administration ofNal-Glu
Administration of Nal-Glu 4 to 5 days after the LH surge resulted in a decrease of LH, P, and E 2. Follicle-stimulating hormone levels, however, did not vary significantly. After 10 mg Nal-Glu, LH levels decreased by 20% to 72% (from 5.2 ± 1.4 to 2.5 ± 0.3 mUI/mL, P < 0.05) to reach a nadir 24 to 48 hours after drug administration. Estradiol fell by 71% to 76% (from 719.3 ± 75.5 to 339.3 ± 57.2 pmol/L, P < 0.05), and P fell by 48% to 97% (from 43.6 ± 19.2 to 5.0 ± 1.3 nmol/L) to reach a nadir 72 hours after treatment. Luteinizing hormone returned to baseline levels 4 days after injection, and E 2 and P returned by day 6. 442
Dubourdieu et al.
Luteolytic effect of Nal-Glu
p 100 nmoi/L 80
60
40
20
3
4
5
A A
6
7
8
9
10 11
12 13 14
DAYS AFTER THE LH SURGE
Nal Glu
Figure 1 Mean (±SE) serum P during the luteal phase in women treated with Nal-Glu (10 mg, SC) on days 4 and 5 after the LH surge (day 0) and during the control cycles (0-0).
Fertility and Sterility
rose progressively in both groups, Nal-Glu and control groups, to a maximum value of 48.8 ± 22.5 nmol/L in the Nal-Glu-treated group versus 60.8 ± 3.1 nmol/L in the control group (not significant) (Fig. 2). Because of a difference in P values between the two groups at day 10, the area under the curve (nmol/1- 1 per day) was calculated and was not statistically different: 66.7 ± 7.3 in the Nal-Glu treated group versus 100.1 ± 1.2 in the control group (not significant). The E 2 profile was also similar in both groups with a maximum of 484.1 ± 45.1 pmol/L in the Nal-Glu-treated group versus 477 ± 27.5 pmol/L in the controls (not significant). Group 2: Delay of 48 Hours Between 1st Day of Nal-Glu Treatment and HCG Administration
A complete luteolysis with premature menstruation was observed in one of the five women 48 hours after the first injection of Nal-Glu without any further increase of E 2 and P levels after hCG administration. Partial rescue of CL function occurred in four of the five subjects. Mean LH fell from 11.7 ± 1.8 miU/mL to 4.7 ± 0.5 miU/mL (P < 0.05) and 3.5 ± 0.2 miU/mL (P < 0.05) 24 and 48 hours after the first injection of Nal-Glu, respectively. Progesterone levels decreased from 23.2 ± 3. 7 nmoljL to 1.0 ± 2.4 nmol/L 48 hours after the first injection ofNal-Glu versus 67.7 ± 10.4 nmol/L in the control group on the corresponding day. After hCG treatment, P levels increased to reach a maximum level of33.8 ± 10.9 nmol/L in the Nal-Glu + hCG-treated group versus 117.6 ± 12.9 nmoljL in the control group (P < 0.01) (Fig. 3). Estradiol levels decreased
p nmoi/L 160 140
llO
.. ..
hCG
lO
tUIGIU+hCG
100
..
4
5
A
A
A
A
6
7
NeiGiu
I I I I DAYS AFI'ER THE LH SURGE
AhCG
Figure 2 Mean (±SE) serum P during the luteal phase, in women treated with Nal-Glu (10 mg, SC) on days 4 and 5 after the LH surge (day 0). Increasing daily doses of hCG were administered (100 IU/m2 , 200 IU/m2 , 300 IU/m2 ) for 3 successive days and started along with Nal-Glu. Control women were treated with hCG alone (0-0).
Vol. 56, No. 3, September 1991
Copyright c 1991 The American Fertility Society
Printed on acid-free paper in U.S.A.
Suppression of corpus luteum function by the gonadotropinreleasing hormone antagonist Nai-Giu: effect of the dose and timing of human chorionic gonadotropin administration*
Sophie Dubourdieu, M.D.t:j: Bernard Charbonnel, M.D. t Maria Rebecca Massai, M.D.§
Julia Marraoui, M.D.§ Irving Spitz, M.D. II Philippe Bouchard, M.D.§
Clinique Endocrinologique, HOtel-Dieu, Nantes, and Service d'Endocrinologie Hopital Bicetre, Le Kremlin Bicetre, France, and The Population Counci~ Center for Biomedical Research, New York, New York
Objective: To assess the effect of gonadotropin-releasing hormone antagonist Nal-Glu administration in the luteal phase and the potential rescue by exogenous human chorionic gonadotropin (hCG) of corpus luteum (CL) after antagonist treatment. Design: We studied the dose of Nal-Glu required for luteolysis and subsequently we coadministered low doses of hCG for 3 consecutive days either simultaneously to Nal-Glu administration (n = 5), or 48 (n = 5), or 72 hours (n = 5) later. Six additional participants received pharmacological doses of hCG 48 hours after the luteolytic dose of Nal-Glu. Setting: Participants were studied in Clinique Endocrinologique, Nantes, and in Service d'Endocrinologie, Hopital Bidltre, Le Kremlin Bicetre, France. Participants: Twenty-nine normal young women (ages 20 to 35) were studied. Interventions: None. Main Outcome Measure: Measurements of follicle-stimulating hormone, luteinizing hormone (LH), estradiol, Progesterone (P) levels were performed by radioimmunoassay before, during, and after the various treatment regimens. Results: Complete luteolysis occurred in women who received 10 mg of Nal-Glu daily on days 4 and 5 after the LH surge. The coadministration of Nal-Glu and hCG overrode the effect of the antagonist (P = 48.8 ± 22.5 versus 60.8 ± 3.1 nmol/L in controls treated with hCG alone [NS]). When hCG treatment was started 48 hours after Nal-Glu, a partialluteolysis occurred (P = 33.8 ± 10.9 versus 117 ± 12.9 nmol/L, P < 0.01). When hCG was started 72 hours after Nal-Glu, a complete luteolysis occurred (P = 5.8 ± 2.05 versus 36.2 ± 0.6 nmol/L, P < 0.01). Higher doses of hCG (1,500 or 5,000 IU) administered 72 hours after Nal-Glu resulted in a significant rescue of CL function (P = 37.7 ± 4.8 and P = 43.8 ± 22.2 versus 74.5 ± 19.8 and 130.2 ± 14.3 nmol/L, P < 0.05), respectively. Conclusions: These results confirm the LH dependence of CL function. The suppression of CL LH support for 72 hours induced a compromise of the CL nonreversible by low doses of hCG Fertil Steril 56:440, 1991 mimicking early pregnancy but reversible with pharmacological doses.
Received December 12, 1990; revised and accepted May 7, 1991. * Supported through cooperative agreement from the United States Agency for International Development, as well as the George J. Hecht Fund, the Andrew W. Mellon, and The Rockefeller Foundations, New York, New York (P.B., B.C.). t Clinique Endocrinologique, Hotel-Dieu. :j: Reprint requests: Sophie Dubourdieu, M.D., Clinique Endocrinologique, Hotel-Dieu, Nantes, France. § Service d'Endocrinologie, Hopital Bidltre. II The Population Council, Center for Biomedical Research.
440
Dubourdieu et al.
Luteolytic effect of Nal-Glu
It is widely believed that endogenous luteinizing hormone (LH) secretion is required to sustain corpus luteum (CL) function in primates1- 4 as well as in women. 5•6 The interruption of pulsatile gonadotropin-releasing hormone (GnRH) administration during the luteal phase in monkeys with hypothalamic lesions results in a dramatic decline of serum progesterone (P) levels. 7 However, CL function can be rescued if pulsatile GnRH therapy is reinitiated Fertility and Sterility
within 3 days, suggesting that CL viability can be preserved without LH support for at least 72 hours. 8 Gonadotropin-releasing hormone antagonists are synthetic peptide analogs of GnRH that suppress pituitary gonadotropin secretion. 9 They therefore provide an important tool to study the LH dependency of CL function. A single injection of the GnRH antagonist detirelix ([N-Ac-D-Nal(2)\ DpCl-Phe2 , D-Trp 3 , DhArg (Et2 ) 6 , DAla10 ]GnRH; Syntex Research, Palo Alto, CA) to stumptailed macaques suppressed bioactive LH and P during all stages of the luteal phase. 3 When the antagonist was administered as a single dose in the early to midluteal phase, P levels recovered once the effect of the antagonist had worn off. But early luteal phase treatment with the same antagonist for 3 days suppressed serum P levels for the remainder ofthe cycle. Similar results have been shown in women by McLachlan et al. 5 using the GnRH antagonist Nal-Glu ([AcD2Nal\ D 4-ClPhe 2 , D3Pal3 , Ar~, DGlu6 (AA), D Ala10 ]GnRH). In their study, the duration of LH suppression was dose-related, and the induction of luteolysis required gonadotropin deprivation for >3 consecutive days. These results suggest that the immediate decrease in P, estradiol (E 2 ), and inhibin levels seen after GnRH antagonist administration during the luteal phase did not necessarily imply luteolysis; CL viability could be preserved for about 3 days without LH support. A contraceptive (postcoital) strategy, based only on GnRH antagonist treatment in the luteal phase, may be successful, but the presence of human chorionic gonadotropin (hCG) may produce rescue of CL function. The aim of this study was to assess the potential rescue of CL function by hCG after GnRH antagonist treatment, using different doses and various timings of hCG administration.
to induce luteolysis. On day 4 after the midcycle LH surge, four subjects received 10 mg of Nal-Glu, and four subjects received two consecutives injections (10 mg) of Nal-Glu on days 4 and 5 after the LH surge. All injections were given by the subcutaneous (SC) route into the abdomen. The timing of the midcycle LH surge (day 0) was determined by rapid analysis of plasma LH levels performed daily from day 12 of the cycle. Study 2: Administration of Nal-Glu and Low Doses ofHCG
An experimental paradigm was designed to study the effect of physiological doses ofhCG on CL function in women who had previously received antagonist to induce luteolysis. In this model, there was a variable duration of LH deprivation before hCG treatment. Each volunteer received the dose of N alGlu found in study 1 to induce luteolysis, i.e., 10 mg on days 4 and 5 after the LH surge. In addition, on 3 successive days intramuscular hCG (gonadotrophine chorionic "Endo"; Organon, Saint Denis, France) was given in increasing doses to mimic plasma hCG levels seen in early pregnancy6 (100 IU /m 2 on day 1,200 IU /m 2 on day 2, and 300 IU/m 2 on day 3). The subjects were divided into three groups depending on which day they received the first injection ofhCG. Group 1 (n = 5) received hCG along with Nal-Glu on days 4, 5, and 6 after the LH surge; group 2 (n = 5) received hCG on days 6, 7, and 8, i.e., 48 hours after the first injection of NalGlu, and group 3 (n = 5) received hCG on days 7, 8, and 9 after the LH surge, i.e., 72 hours after the first injection of Nal-Glu. Results were compared with three control groups each comprising three subjects who received hCG alone, on the same experimental days.
MATERIALS AND METHODS Subjects
Twenty-nine normal young women, 20 to 35 years of age, with regular menstrual cycles (mean length, 28 days; range, 26 to 32) and ovulatory basal body temperature charts were studied. None of the subjects were using a hormonal method of contraception or taking any medication. Each subject signed informed consent. Protocol Study 1: Single and Repeated Administration of Nal-Glu
Two groups of volunteers were studied to determine the dose and duration of treatment necessary Vol. 56, No.3, September 1991
Study 3: Administration of Nal-Glu and Pharmacological Doses of HCG
To evaluate the ability of pharmacological doses of hCG to rescue CL function after a 3-day period of LH suppression, each women received the dose of Nal-Glu found in study 1 to induce luteolysis, i.e., 10 mg on days 4 and 5 after the LH surge and hCG on days 7, 8, and 9 after the LH surge, i.e., 72 hours after the first injection of Nal-Glu. The hCG doses employed were 1,500 IU (n = 3) and5,000 IU (n = 3) daily. Results were compared with two control groups of two subjects each who received hCG alone on the same experimental days. Dubourdieu et al.
Luteolytic effect of Nal-Glu
441
Hormone Assays
To determine the day of the midcycle LH surge, LH was evaluated by a chemiluminescence system (Amerlite Estradiol-60 Assay; Amersham International plc, Amersham, United Kingdom). Measurement of gonadotropins was performed by radioimmunoassay (RIA, cotropin-F; Clinetics Corporation Laboratories, Tustin, CA). The intra-assay and interassay coefficients of variation for follicle-stimulating hormone (FSH) and LH were 3.7% and 3.5% and 3.1% and 3.7%, respectively. The lower limits of sensitivity of the assays were 2.5 mUI/mL for LH and 0.5 miU/mL for FSH. Cross-reactivity in the FSH assay was 0.2% for both LH and hCG. Human chorionic gonadotropin was measured by an immunoenzymatic method (IMX Abbott, North Chicago, IL). The sensitivity was 0.76 miU/mL. Intraassay and interassay CVs were 4.4% and 6.7%, for low levels ofhCG (6 miU/mL). Estradiol and P were also determined by RIA (Biomerieux Laboratories Marcy l'Etoile, France). The sensitivity of the Pas~ say was 0.17 nmol/L and 44 pmol/L for the E 2 assay. The intra-assay and interassay CVs for P were 3.5% and 4.5% for values on the order of 34 nmol/L. For E2, the intra-assay and interassay CVs variation were 4.8% and 3.9% for values in the normal preovulatory range. The duration of the luteal phase was calculated as being the period of time between the day after the preovulatory LH surge and the 1st day of the menstrual cycle. The results were expressed as means ± SEM. Statistical analysis of the results was performed using Student's t-test with a P value of significance assigned at 95% (from 25.6 ± 12.9 nmol/L to 1.0 ± 0.6 nmol/L, P < 0.01), both reaching their lowest point 72 hours after the initial administration of the drug (Fig. 1). Luteinizing hormone levels recovered their preinjection levels 5 days after the first injection of Nal-Glu, but E 2 and P hormone levels remained consistently low for the duration of the menstrual cycle, reflecting a compromise of the CL viability. Two of the four subjects who received 10 mg of Nal-Glu experienced varying delays in the occurrence of menstrual bleeding. In all women treated with 10 mg Nal-Glu antagonist for 2 consecutive days, premature menstrual bleeding occurred within 48 hours of the first dose of Nal-Glu antagonist and lasted' 2 to 5 days. The next menstrual period occurred at a minimum of 1 month after the initial injection of antagonist. Study 2: Administration of Nal-Glu and Low DosesofHCG
Plasma hCG levels rose in parallel with the increasing doses of hCG to 5.6 ± 2.7 miU/mL, 7.3 ± 2.7 miU/mL, and 9.6 ± 5.5 miU/mL 48, 72, and 92 hours, respectively, after the first injection of hCG, i.e., in the range of plasma levels usually seen around the time of implantation. 10 Group 1: Coadministration of Nal-Glu With HCG
Simultaneous administration ofhCG and Nal-Glu blocked the effect of the antagonist. Serum P
RESULTS Study 1: Single and Repeated Administration ofNal-Glu
Administration of Nal-Glu 4 to 5 days after the LH surge resulted in a decrease of LH, P, and E 2. Follicle-stimulating hormone levels, however, did not vary significantly. After 10 mg Nal-Glu, LH levels decreased by 20% to 72% (from 5.2 ± 1.4 to 2.5 ± 0.3 mUI/mL, P < 0.05) to reach a nadir 24 to 48 hours after drug administration. Estradiol fell by 71% to 76% (from 719.3 ± 75.5 to 339.3 ± 57.2 pmol/L, P < 0.05), and P fell by 48% to 97% (from 43.6 ± 19.2 to 5.0 ± 1.3 nmol/L) to reach a nadir 72 hours after treatment. Luteinizing hormone returned to baseline levels 4 days after injection, and E 2 and P returned by day 6. 442
Dubourdieu et al.
Luteolytic effect of Nal-Glu
p 100 nmoi/L 80
60
40
20
3
4
5
A A
6
7
8
9
10 11
12 13 14
DAYS AFTER THE LH SURGE
Nal Glu
Figure 1 Mean (±SE) serum P during the luteal phase in women treated with Nal-Glu (10 mg, SC) on days 4 and 5 after the LH surge (day 0) and during the control cycles (0-0).
Fertility and Sterility
rose progressively in both groups, Nal-Glu and control groups, to a maximum value of 48.8 ± 22.5 nmol/L in the Nal-Glu-treated group versus 60.8 ± 3.1 nmol/L in the control group (not significant) (Fig. 2). Because of a difference in P values between the two groups at day 10, the area under the curve (nmol/1- 1 per day) was calculated and was not statistically different: 66.7 ± 7.3 in the Nal-Glu treated group versus 100.1 ± 1.2 in the control group (not significant). The E 2 profile was also similar in both groups with a maximum of 484.1 ± 45.1 pmol/L in the Nal-Glu-treated group versus 477 ± 27.5 pmol/L in the controls (not significant). Group 2: Delay of 48 Hours Between 1st Day of Nal-Glu Treatment and HCG Administration
A complete luteolysis with premature menstruation was observed in one of the five women 48 hours after the first injection of Nal-Glu without any further increase of E 2 and P levels after hCG administration. Partial rescue of CL function occurred in four of the five subjects. Mean LH fell from 11.7 ± 1.8 miU/mL to 4.7 ± 0.5 miU/mL (P < 0.05) and 3.5 ± 0.2 miU/mL (P < 0.05) 24 and 48 hours after the first injection of Nal-Glu, respectively. Progesterone levels decreased from 23.2 ± 3. 7 nmoljL to 1.0 ± 2.4 nmol/L 48 hours after the first injection ofNal-Glu versus 67.7 ± 10.4 nmol/L in the control group on the corresponding day. After hCG treatment, P levels increased to reach a maximum level of33.8 ± 10.9 nmol/L in the Nal-Glu + hCG-treated group versus 117.6 ± 12.9 nmoljL in the control group (P < 0.01) (Fig. 3). Estradiol levels decreased
p nmoi/L 160 140
llO
.. ..
hCG
lO
tUIGIU+hCG
100
..
4
5
A
A
A
A
6
7
NeiGiu
I I I I DAYS AFI'ER THE LH SURGE
AhCG
Figure 2 Mean (±SE) serum P during the luteal phase, in women treated with Nal-Glu (10 mg, SC) on days 4 and 5 after the LH surge (day 0). Increasing daily doses of hCG were administered (100 IU/m2 , 200 IU/m2 , 300 IU/m2 ) for 3 successive days and started along with Nal-Glu. Control women were treated with hCG alone (0-0).
