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Dose of GnRH agonist (nafarelin acetate) affects intrafollicular PAPP-A expression in controlled ovarian hyperstimulation cycle
European Journal of Obstetrics & Gynecology and Reproductive Biology 112 (2004) 65–68
Dose of GnRH agonist (nafarelin acetate) affects intrafollicular PAPP-A expression in controlled ovarian hyperstimulation cycle JiHyun Suha, EunYoung Leeb, SeongSoo Hwangb, Sarah Yoonc, Byung-Koo Yoona, DukSoo Baea, DooSeok Choia,* a
Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong Kangnam-Ku, Seoul 135-710, South Korea b Infertility Clinic, Seoul 135-710, South Korea c Center for Clinical Research, Samsung Biomedical Research Institute, Seoul 135-710, South Korea Received 6 February 2003; received in revised form 9 April 2003; accepted 13 May 2003
Abstract Objective: To determine the effect of dose of GnRH agonist on the follicular environment in controlled ovarian hyperstimulation (COH) cycles. Study design: Twenty-eight IVF patients with normal ovarian function were divided into three groups: group I received GnRHa (nafarelin acetate/Synarel) intranasally at 200 mg daily, group II received 400 mg daily until hCG injection, and group III was given 400 mg daily before the initiation of ovarian stimulation, then 200 mg daily before the day of hCG injection. Serum estradiol, progesterone, and leptin levels were measured on the day of hCG injection. After aspiration, expression of pregnancy-associated a-plasma protein (PAPP)-A in the follicular fluid of dominant follicles (>20 mm) was determined by Western blot analysis. Results: No significant difference was noted in serum estradiol, progesterone, and leptin levels. But intrafollicular PAPP-A expression was significantly higher in group II compared to other groups (P < 0:05). Conclusion: The dose of GnRHa may have an impact on the intrafollicular environment of dominant follicles in COH cycles. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Follicular environment; GnRH agonists (GnRHa); Pregnancy-associated a-plasma protein-A (PAPP-A); Controlled ovarian hyperstimulation (COH)
1. Introduction Ovarian follicular development is a critical factor determining the success of an assisted reproductive technology (ART). The quality, as well as the number, of follicles is known to be improved by GnRH agonist (GnRHa) [1,2], which is used in conjunction with gonadotropin in controlled ovarian hyperstimulation (COH) patients, primarily to prevent preovulatory premature luteinization [3,4]. To reduce the possible detrimental effects of GnRHa and the economic burden of patients, restriction on the dose of GnRHa has been considered. Feldberg and others have shown that employing reduced dose of GnRHa has resulted in beneficial in vitro fertilization (IVF) outcomes, in terms of increased serum estradiol level and number of oocytes harvested, and * Corresponding author. Tel.: þ82-2-3410-3514; fax: þ82-2-3410-0630. E-mail address: [email protected] (D. Choi).
decreased cancellation rate [5–8]. After appropriate pituitary desensitization, no evidence of premature LH surge was noted with low dose GnRHa [9,10]. Despite such clinical advantages, the impact of reduced dose of GnRHa on ovarian intrafollicular physiology remains unclear. Recently, much attention has been drawn on pregnancyassociated a-plasma protein-A (PAPP-A) known to be found highly in the serum of pregnant women. PAPP-A, a member of metalloproteinase family, has been shown to degrade IGFBP-4, an inhibitor of IGF-mediated FSH activity, thus indirectly enhancing FSH action on the ovary [11]. This identification of PAPP-A as the protease of IGFBP-4 [12] has compelled many scientists to believe PAPP-A to be a critical determinant of FSH-induced folliculogenesis and a potential marker reflecting a positive effect on the follicular environment [13–15]. The aim of our study was to determine whether different doses of GnRHa have any effects on the follicular
0301-2115/$ – see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0301-2115(03)00317-8
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J. Suh et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 112 (2004) 65–68
environment of dominant follicle, represented by the expression of PAPP-A, in COH cycles.
Belgium) and P4 level was measured by a double-antibody RIA kit (Coat-A-Count, Diagnostic Products Corp. CA, USA). Leptin was measured using a RIA kit (Linco Research Inc., MO, USA).
2. Materials and methods 2.4. Western blot 2.1. Patients Twenty-eight women with regular menstrual cycle and normal basal serum FSH, LH, E2 levels undergoing COH for IVF in the Infertility Clinic, Department of OB/GY, Samsung Medical Center, Seoul, Korea, from February to November of 2001 were recruited in the order of enrollment. All patients were aged 35 or below with body mass index (BMI) under 30 kg/m2. This study was approved by the Ethical Committee of Samsung Medical Center and informed consents were obtained from all patients. 2.2. Protocols All eligible 28 patients were given monophasic oral pill (Mercilon, Organon, Oss, The Netherlands) from menstrual day 1 or 2 for 2 weeks before commencing GnRHa. All patients received a daily intranasal spray of GnRHa (nafarelin acetate, Synarel, Searle & Co., Chicago) after completion of oral pill. GnRHa at a daily dose of 200 mg was administered intranasally in group I and 400 mg of GnRHa was given in group II daily until the day of human chorionic gonadotropin (hCG, Profasi, Serono, UK) injection. Group III patients received 400 mg of GnRHa daily before the starting day of FSH stimulation, then 200 mg daily until the day of hCG injection. Ovarian stimulation with recombinant FSH (rec-FSH, Puregon, Organon, Oss, The Netherlands) was started in all patients after confirming ovarian suppression (serum estradiol, E2 < 50 pg/ml). Rec-FSH at a daily dose of 200 IU was injected for 4–5 days, then the dose and duration of rec-FSH were adjusted according to individual’s response based on E2 level and transvaginal follicle monitoring. When one or more follicles reached a mean diameter of 18 mm, 10,000 IU of hCG was administered intramuscularly. On the day of hCG injection, serum E2, progesterone (P4), and leptin levels were measured. Oocyte retrieval under transvaginal ultrasound guidance was done 36 h after hCG administration. After isolation of oocytes, only follicular fluid aspirates of large follicles (>20mm) were collected and centrifuged for 10 min at 600 g to compare the dominant follicles of similar development stage [16]. Follicular fluids without red blood cells (RBCs) were frozen at 70 8C. 2.3. Hormone assays Serum E2 was measured using a commercially available radioimmunoassay (RIA) kit (E2-RIA-CT, Biosource,
Total protein in the follicular fluid was obtained by direct centricon (Amicon, YM-10). Equal amount of total protein (30 mg) were separated by SDS-PAGE and the transferred to supported Western PVDF (Schleicher & Schuell, New Hampshire, USA) by applying 100 V for 2 h with a plate electrode apparatus (Mini Trans-Blot Cell, USA). The blots were blocked for 2 h in Tris-buffered saline (TBST, 0.2 M NaCl, 0.1% Tween 20, 10 mM Tris, pH 7.4) containing 5% nonfat dry milk. Subsequently, the blots were incubated with antibody against PAPP-A (1:1000; Zymed, 71–7000) in TBST. Then the blots were also incubated with anti-rabbit IgG (1:2500; Amersham Pharmacia Biotech, Germany) or with anti-goat IgG (1:5000; Santa Cruz, USA) in TBST. The blots were washed several times with TBST after each step. The bound antibody was detected with an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech). The blots were stripped with stripping buffer (100 mM b-mercaptoethanol, 2% (w/v) sodium dodecyl sulphate, 62.5 mM Tris–HCl, pH 6.7) before applying b-actin antibody. The stripped blots were incubated with antibody against b-actin used for the internal standard (1:1000; Sigma, USA) in the TBST followed by anti-mouse IgG (1:2500; Amersham Pharmacia Biotech) in TBST. 2.5. Statistical Analysis The data was analyzed using the SAS system. The differences of all parameters were statistically analyzed by Kruskal–Wallis test. Multiple tests by the least significant difference test using ranks were applied to compare the levels of PAPP-A expression between three groups in pairs. P-value less than 0.05 was defined to be statistically significant.
3. Results As shown in Table 1, there were no significant differences in the age, BMI, basal hormonal profile, and the distribution of causes of infertility among three groups. Serum E2 level after GnRHa administration and on the day of hCG injection showed no significant difference among three groups. There were no differences in serum P4 and leptin levels on the day of hCG injection among three groups (Table 2). But intrafollicular PAPP-A level of dominant follicle was significantly higher in group II compared to that of the other groups (P < 0:05, Fig. 1).
J. Suh et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 112 (2004) 65–68 Table 1 Patient characteristics in three groups studies (mean standard error) Variable Age BMI (kg/m2) Basal FSH (mIU/ml) Basal LH (mIU/ml) Basal E2 (pg/ml) Infertility factor (n) Tubal factor Male factor Unexplained
Group I (n ¼ 8) 31 21.8 6.7 4.0 25.9 2 2 4
3.5 3.2 1.5 1.9 18.2
Group II (n ¼ 12) 30 20.6 6.3 4.4 27.4 3 4 5
3.3 1.9 2.9 1.3 12.1
Group III (n ¼ 8) 31.9 21.1 7.1 3.7 29.4
2.6 3.1 2.3 1.1 18.3
P
Table 2 Comparisons of clinical parameters among three groups (mean standard error) Variable
NS NS NS NS NS
3 3 2
Group I: 200 mg of GnRHa was administered intranasally. Group II: 400 mg of GnRHa was administered intranasally. Group III: 400 mg of GnRHa until the starting day of FSH stimulation then 200 mg until the day of hCG injection. BMI: body mass index. NS: not significant.
67
Group I (n ¼ 8)
Serum E2a (pg/ml) 34.8 9.6 Serum E2b (pg/ml) 1189 263.9 Serum P4b (ng/ml) 1.24 0.22 Serum leptinb (ng/ml) 12.03 2.3 Total doses of 1181 121.7 rec-FSH (IU)
Group II (n ¼ 12) 18.4 1224 1.21 7.2 1163
Group III (n ¼ 8)
P
3.0 18.0 3.6 NS 239.8 967.3 127 NS 0.14 1.21 0.12 NS 0.9 9.86 1.96 NS 55.8 1144 69.1 NS
Group I: 200 mg of GnRHa was administered intranasally. Group II: 400 mg of GnRHa was administered intranasally. Group III: 400 mg of GnRHa until the starting day of FSH stimulation then 200 mg until the day of hCG injection. a Serum level at the day of rec-FSH administration. b Serum level at the day of hCG administration. NS: not significant.
Fig. 1. Comparisons of pregnancy-associated a-plasma protein-A (PAPP-A) level among three groups in follicular fluids by Western blot (data are expressed as mean standard error). P < 0:05.
4. Discussion Our preliminary data suggest the possibility that the dose of GnRHa, specifically nafarelin acetate (Synarel), may have a significant effect on the expression of intrafollicular PAPPA. Since the discovery of PAPP-A as the inhibitor of IGFBP4 by Lawrence et al. [12], the role of PAPP-A in relation to folliculogenesis has widely been explored [13–15,17–19]. High expression of PAPP-A in healthy, estrogen-active, dominant follicles had strong implication on its active role in the selection process of dominant follicles. In this study, PAPP-A has been selected as a parameter to reflect the status of follicle health in COH cycles under different doses of GnRHa administration. Administering GnRHa in mid-luteal phase is commonly practiced in COH cycle mainly to down-regulate pituitary secretion of endogenous hormones, thus preventing premature luteinization and achieving follicular synchroniza-
tion [5,6]. According to the comparative trial by Yuzpe et al. [10], both 400 and 600 mg per day of GnRHa intranasal administrations have produced sufficient pituitary suppression and favorable reproductive outcomes. Other studies confer the efficiency of reduced dose of GnRHa on hypophyseal desensitization and retrieving greater number of oocytes [6–8]. Takeuchi et al. [9] have demonstrated that reducing GnRHa to half the standard dose to 200 mg results in better IVF outcomes, in terms of significantly more oocytes recovered, and significantly greater number of oocytes available for ET, and is more beneficial in reducing side effects and costs. After complete pituitary desensitization with standard dose of GnRHa, no evidence of premature LH surge or reduced quality of oocytes were noted after draw-back therapy of GnRHa. To date, the molecular biological changes of follicular environment depending on the doses of GnRHa have not
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been studied. According to our results, continuous administration of daily 400 mg of GnRHa showed significantly higher expression of intrafollicular PAPP-A of dominant follicles compared to that of the other dose groups. Despite many advantages on the clinical results of COH, the role of GnRHa on folliculogenesis and its effect on intrafollicular microenvironment remains controversial [20–22]. Limited in vivo models and currently available clinical data have precluded scientists to draw any firm conclusion on the effect of GnRHa on ovarian physiology. From the previous studies, reduced dose of GnRHa appears to improve IVF outcomes, but the status of follicular health seems to be most superior from continuous administration of the standard dose (400 mg per day) of GnRHa in regards to the expression of PAPP-A. Our study implies that the role of GnRHa in COH is not only limited to preventing premature luteinization but may influence the quality of follicle at the molecular biological level, and its dose may have a significant effect on the growth of healthy follicles. Therefore, further prospective clinical trials are warranted to seek the most optimal dose, which adapts to the intricate balance between inducing an appropriate pituitary down regulation with the most compatible follicular environment and at the same time, preventing deleterious effects of GnRHa. This preliminary study suggests that the dose of GnRHa may have a significant effect on the intrafollicular environment, reflected by the expression of PAPP-A. References [1] Parinaud J, Beaur A, Bourreau E, Vieitez G, Pontonnier G. Effect of a luteinizing hormone-releasing hormone agonist (buserelin) on steroidogenesis of cultured human preovulatory granulose cells. Fertil Steril 1988;50:597–602. [2] Tureck RW, Mastroianni Jr. L, Blasco L, Strauss III JF. Inhibition of human granulosa cell progesterone secretion by a gonadotrophinreleasing hormone agonist. J Clin Endocrinol Metab 1982;54:1078–80. [3] Hughes EG, Fedorkow DM, Daya S, Sagle MA, Van de Koppel P, Collins JA. The routine use of gonadotropin-releasing hormone agonists prior to in vitro fertilization and gamete intrafallopian transfer: a meta-analysis of randomized controlled trials. Fertil Steril 1992;58:888–96. [4] Meldrum DR, Wisot A, Hamilton F, Gutlay AL, Kempton WF, Huynh D. Routine pituitary suppression with leuprolide before ovarian stimulation for oocyte retrieval. Fertil Steril 1989;51:455–9. [5] Feldberg D, Farhi J, Ashkenazi J, Dicker D, Shalev J, Ben-Rafae Z. Minidose gonadotrophin-releasing hormone agonist is the treatment of choice in poor responders with high follicle-stimulating hormone levels. Fertil Steril 1994;62:343–6. [6] Pantos K, Meimeth-Damianaki T, Vaxevanoglou T, Kapetanakis E. Prospective study of a modified gonadotropin-releasing hormone agonist long protocol in an in vitro fertilizing program. Fertil Steril 1994;61:709–13. [7] Elgendy M, Afnan M, Holder R. Reducing the dose of gonadotropinreleasing hormone agonist on starting ovarian stimulation: effect on
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ovarian response and in-vitro fertilization outcome. Hum Reprod 1998;13:2382–5. Dal Prato L, Borini A, Trevisi MR, Bonu MA, Sereni E, Flamigni C. Effect of reduced dose of triptorelin at the start of ovarian stimulation on the outcome of IVF: a randomized study. Hum Reprod 2001;16: 1409–14. Takeuchi S, Minoura H, Shibahara T, Tsuiki Y, Noritaka F, Toyoda N. A prospective randomized comparison of routine buserelin acetate and a decreasing dosage of nafarelin acetate with a low-dose gonadotropin-releasing hormone agonist protocol for in vitro fertilization and intracytoplasmic sperm injection. Fertil Steril 2001;76: 532–7. Yuzpe AA, Nisker JA, Kaplan BR, Tummon IS, Auckland J. Nafarelin acetate for pituitary down-regulation in vitro fertilization; comparison of two dosages. J Reprod Med 1995;40:83–8. Erickson GF, Shimasaki S. The physiology of folliculogenesis: the role of novel growth factors. Fertil Steril 2001;76:943–9. Lawrence JB, Oxvig C, Overgaard MT, Sottrup-Jensen L, Gleich GJ, Hays LG, et al. The insulin-like growth factor (IGF)-dependent IGF binding protein-4 protease secreted by human fibroblasts is pregnancy-associated plasma protein-A. Proc Natl Acad Sci USA 1999;96:3149–53. Hourvitz A, Widger AE, Filho FL, Chang RJ, Adashi EY, Erickson GF. Pregnancy-associated plasma protein-A gene expression in human ovaries is restricted to healthy follicles and corpora lutea. J Clin Endocrinol Metab 2000;85:4916–9. Conover CA, Faessen GF, Ilg KE, et al. Pregnancy-associated plasma protein-A is the insulin-like growth factor binding protein-4 protease secreted by human ovarian granulosa cells and is a marker of dominant follicle selection and the corpus luteum. Endorinology 2001;142:2155–8. Rivera GM, Chandrasekger YA, Evans AC, Giudice LC, Fortune JE. A potential role for insulin-like growth factor binding-4 proteolysis in the establishment of ovarian follicular dominance in cattle. Biol Reprod 2001;65:102–11. Dirnfeld M, Goldman S, Gonen Y, Koifmen M, Calderon I, Abramovici H. A simplified co-culture system with luteinized granulose cells improves embryo quality and implantation rates: a controlled study. Fertil Steril 1997;67:120–2. Hourvitz A, Kuwahara A, Hennebold JD, et al. The regulated expression of the pregnancy-associated plasma protein-A in the rodent ovary: a proposed role in the development of dominant follicles and of corpora lutea. Endocrinology 2002;143:1833–44. Mazerbourg S, Overgaard MT, Oxvig C, et al. Pregnancy-associated plasma protein-A (PAPP-A) in ovine, bovine, porcine, and equine ovarian follicles: involvement in IGF binding protein-4 proteolytic degradation and mRNA expression during follicular development. Endocrinology 2001;142:5243–53. Giudice LC. Insulin-like growth factor family in Graafian follicle development and function. J Soc Gynecol Invest 2001(Suppl 1); 8:S26–9. Hugues JN, Cedrin-Durnerin IC. Revisting gonadotrophin-releasing hormone agonist protocols and management of poor ovarian responses to gonadotropins. Hum Reprod Update 1998;4:83–101. Racowsky C, Prather AL, Johnson MK, Olvera SP, Gelety TJ. Prematurely condensed chromosomes and meiotic abnormalities in unfertilized human oocytes after ovarian stimulation with and without gonadotropin-releasing hormone agonist. Fertil Steril 1997; 67:932–8. Selva J, Martin-Pont B, Hugues JN, et al. Cytogenetic study of human oocytes uncleaved after in-vitro fertilization. Hum Reprod 1991;6:709–13.
Dose of GnRH agonist (nafarelin acetate) affects intrafollicular PAPP-A expression in controlled ovarian hyperstimulation cycle JiHyun Suha, EunYoung Leeb, SeongSoo Hwangb, Sarah Yoonc, Byung-Koo Yoona, DukSoo Baea, DooSeok Choia,* a
Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong Kangnam-Ku, Seoul 135-710, South Korea b Infertility Clinic, Seoul 135-710, South Korea c Center for Clinical Research, Samsung Biomedical Research Institute, Seoul 135-710, South Korea Received 6 February 2003; received in revised form 9 April 2003; accepted 13 May 2003
Abstract Objective: To determine the effect of dose of GnRH agonist on the follicular environment in controlled ovarian hyperstimulation (COH) cycles. Study design: Twenty-eight IVF patients with normal ovarian function were divided into three groups: group I received GnRHa (nafarelin acetate/Synarel) intranasally at 200 mg daily, group II received 400 mg daily until hCG injection, and group III was given 400 mg daily before the initiation of ovarian stimulation, then 200 mg daily before the day of hCG injection. Serum estradiol, progesterone, and leptin levels were measured on the day of hCG injection. After aspiration, expression of pregnancy-associated a-plasma protein (PAPP)-A in the follicular fluid of dominant follicles (>20 mm) was determined by Western blot analysis. Results: No significant difference was noted in serum estradiol, progesterone, and leptin levels. But intrafollicular PAPP-A expression was significantly higher in group II compared to other groups (P < 0:05). Conclusion: The dose of GnRHa may have an impact on the intrafollicular environment of dominant follicles in COH cycles. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Follicular environment; GnRH agonists (GnRHa); Pregnancy-associated a-plasma protein-A (PAPP-A); Controlled ovarian hyperstimulation (COH)
1. Introduction Ovarian follicular development is a critical factor determining the success of an assisted reproductive technology (ART). The quality, as well as the number, of follicles is known to be improved by GnRH agonist (GnRHa) [1,2], which is used in conjunction with gonadotropin in controlled ovarian hyperstimulation (COH) patients, primarily to prevent preovulatory premature luteinization [3,4]. To reduce the possible detrimental effects of GnRHa and the economic burden of patients, restriction on the dose of GnRHa has been considered. Feldberg and others have shown that employing reduced dose of GnRHa has resulted in beneficial in vitro fertilization (IVF) outcomes, in terms of increased serum estradiol level and number of oocytes harvested, and * Corresponding author. Tel.: þ82-2-3410-3514; fax: þ82-2-3410-0630. E-mail address: [email protected] (D. Choi).
decreased cancellation rate [5–8]. After appropriate pituitary desensitization, no evidence of premature LH surge was noted with low dose GnRHa [9,10]. Despite such clinical advantages, the impact of reduced dose of GnRHa on ovarian intrafollicular physiology remains unclear. Recently, much attention has been drawn on pregnancyassociated a-plasma protein-A (PAPP-A) known to be found highly in the serum of pregnant women. PAPP-A, a member of metalloproteinase family, has been shown to degrade IGFBP-4, an inhibitor of IGF-mediated FSH activity, thus indirectly enhancing FSH action on the ovary [11]. This identification of PAPP-A as the protease of IGFBP-4 [12] has compelled many scientists to believe PAPP-A to be a critical determinant of FSH-induced folliculogenesis and a potential marker reflecting a positive effect on the follicular environment [13–15]. The aim of our study was to determine whether different doses of GnRHa have any effects on the follicular
0301-2115/$ – see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0301-2115(03)00317-8
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J. Suh et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 112 (2004) 65–68
environment of dominant follicle, represented by the expression of PAPP-A, in COH cycles.
Belgium) and P4 level was measured by a double-antibody RIA kit (Coat-A-Count, Diagnostic Products Corp. CA, USA). Leptin was measured using a RIA kit (Linco Research Inc., MO, USA).
2. Materials and methods 2.4. Western blot 2.1. Patients Twenty-eight women with regular menstrual cycle and normal basal serum FSH, LH, E2 levels undergoing COH for IVF in the Infertility Clinic, Department of OB/GY, Samsung Medical Center, Seoul, Korea, from February to November of 2001 were recruited in the order of enrollment. All patients were aged 35 or below with body mass index (BMI) under 30 kg/m2. This study was approved by the Ethical Committee of Samsung Medical Center and informed consents were obtained from all patients. 2.2. Protocols All eligible 28 patients were given monophasic oral pill (Mercilon, Organon, Oss, The Netherlands) from menstrual day 1 or 2 for 2 weeks before commencing GnRHa. All patients received a daily intranasal spray of GnRHa (nafarelin acetate, Synarel, Searle & Co., Chicago) after completion of oral pill. GnRHa at a daily dose of 200 mg was administered intranasally in group I and 400 mg of GnRHa was given in group II daily until the day of human chorionic gonadotropin (hCG, Profasi, Serono, UK) injection. Group III patients received 400 mg of GnRHa daily before the starting day of FSH stimulation, then 200 mg daily until the day of hCG injection. Ovarian stimulation with recombinant FSH (rec-FSH, Puregon, Organon, Oss, The Netherlands) was started in all patients after confirming ovarian suppression (serum estradiol, E2 < 50 pg/ml). Rec-FSH at a daily dose of 200 IU was injected for 4–5 days, then the dose and duration of rec-FSH were adjusted according to individual’s response based on E2 level and transvaginal follicle monitoring. When one or more follicles reached a mean diameter of 18 mm, 10,000 IU of hCG was administered intramuscularly. On the day of hCG injection, serum E2, progesterone (P4), and leptin levels were measured. Oocyte retrieval under transvaginal ultrasound guidance was done 36 h after hCG administration. After isolation of oocytes, only follicular fluid aspirates of large follicles (>20mm) were collected and centrifuged for 10 min at 600 g to compare the dominant follicles of similar development stage [16]. Follicular fluids without red blood cells (RBCs) were frozen at 70 8C. 2.3. Hormone assays Serum E2 was measured using a commercially available radioimmunoassay (RIA) kit (E2-RIA-CT, Biosource,
Total protein in the follicular fluid was obtained by direct centricon (Amicon, YM-10). Equal amount of total protein (30 mg) were separated by SDS-PAGE and the transferred to supported Western PVDF (Schleicher & Schuell, New Hampshire, USA) by applying 100 V for 2 h with a plate electrode apparatus (Mini Trans-Blot Cell, USA). The blots were blocked for 2 h in Tris-buffered saline (TBST, 0.2 M NaCl, 0.1% Tween 20, 10 mM Tris, pH 7.4) containing 5% nonfat dry milk. Subsequently, the blots were incubated with antibody against PAPP-A (1:1000; Zymed, 71–7000) in TBST. Then the blots were also incubated with anti-rabbit IgG (1:2500; Amersham Pharmacia Biotech, Germany) or with anti-goat IgG (1:5000; Santa Cruz, USA) in TBST. The blots were washed several times with TBST after each step. The bound antibody was detected with an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech). The blots were stripped with stripping buffer (100 mM b-mercaptoethanol, 2% (w/v) sodium dodecyl sulphate, 62.5 mM Tris–HCl, pH 6.7) before applying b-actin antibody. The stripped blots were incubated with antibody against b-actin used for the internal standard (1:1000; Sigma, USA) in the TBST followed by anti-mouse IgG (1:2500; Amersham Pharmacia Biotech) in TBST. 2.5. Statistical Analysis The data was analyzed using the SAS system. The differences of all parameters were statistically analyzed by Kruskal–Wallis test. Multiple tests by the least significant difference test using ranks were applied to compare the levels of PAPP-A expression between three groups in pairs. P-value less than 0.05 was defined to be statistically significant.
3. Results As shown in Table 1, there were no significant differences in the age, BMI, basal hormonal profile, and the distribution of causes of infertility among three groups. Serum E2 level after GnRHa administration and on the day of hCG injection showed no significant difference among three groups. There were no differences in serum P4 and leptin levels on the day of hCG injection among three groups (Table 2). But intrafollicular PAPP-A level of dominant follicle was significantly higher in group II compared to that of the other groups (P < 0:05, Fig. 1).
J. Suh et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 112 (2004) 65–68 Table 1 Patient characteristics in three groups studies (mean standard error) Variable Age BMI (kg/m2) Basal FSH (mIU/ml) Basal LH (mIU/ml) Basal E2 (pg/ml) Infertility factor (n) Tubal factor Male factor Unexplained
Group I (n ¼ 8) 31 21.8 6.7 4.0 25.9 2 2 4
3.5 3.2 1.5 1.9 18.2
Group II (n ¼ 12) 30 20.6 6.3 4.4 27.4 3 4 5
3.3 1.9 2.9 1.3 12.1
Group III (n ¼ 8) 31.9 21.1 7.1 3.7 29.4
2.6 3.1 2.3 1.1 18.3
P
Table 2 Comparisons of clinical parameters among three groups (mean standard error) Variable
NS NS NS NS NS
3 3 2
Group I: 200 mg of GnRHa was administered intranasally. Group II: 400 mg of GnRHa was administered intranasally. Group III: 400 mg of GnRHa until the starting day of FSH stimulation then 200 mg until the day of hCG injection. BMI: body mass index. NS: not significant.
67
Group I (n ¼ 8)
Serum E2a (pg/ml) 34.8 9.6 Serum E2b (pg/ml) 1189 263.9 Serum P4b (ng/ml) 1.24 0.22 Serum leptinb (ng/ml) 12.03 2.3 Total doses of 1181 121.7 rec-FSH (IU)
Group II (n ¼ 12) 18.4 1224 1.21 7.2 1163
Group III (n ¼ 8)
P
3.0 18.0 3.6 NS 239.8 967.3 127 NS 0.14 1.21 0.12 NS 0.9 9.86 1.96 NS 55.8 1144 69.1 NS
Group I: 200 mg of GnRHa was administered intranasally. Group II: 400 mg of GnRHa was administered intranasally. Group III: 400 mg of GnRHa until the starting day of FSH stimulation then 200 mg until the day of hCG injection. a Serum level at the day of rec-FSH administration. b Serum level at the day of hCG administration. NS: not significant.
Fig. 1. Comparisons of pregnancy-associated a-plasma protein-A (PAPP-A) level among three groups in follicular fluids by Western blot (data are expressed as mean standard error). P < 0:05.
4. Discussion Our preliminary data suggest the possibility that the dose of GnRHa, specifically nafarelin acetate (Synarel), may have a significant effect on the expression of intrafollicular PAPPA. Since the discovery of PAPP-A as the inhibitor of IGFBP4 by Lawrence et al. [12], the role of PAPP-A in relation to folliculogenesis has widely been explored [13–15,17–19]. High expression of PAPP-A in healthy, estrogen-active, dominant follicles had strong implication on its active role in the selection process of dominant follicles. In this study, PAPP-A has been selected as a parameter to reflect the status of follicle health in COH cycles under different doses of GnRHa administration. Administering GnRHa in mid-luteal phase is commonly practiced in COH cycle mainly to down-regulate pituitary secretion of endogenous hormones, thus preventing premature luteinization and achieving follicular synchroniza-
tion [5,6]. According to the comparative trial by Yuzpe et al. [10], both 400 and 600 mg per day of GnRHa intranasal administrations have produced sufficient pituitary suppression and favorable reproductive outcomes. Other studies confer the efficiency of reduced dose of GnRHa on hypophyseal desensitization and retrieving greater number of oocytes [6–8]. Takeuchi et al. [9] have demonstrated that reducing GnRHa to half the standard dose to 200 mg results in better IVF outcomes, in terms of significantly more oocytes recovered, and significantly greater number of oocytes available for ET, and is more beneficial in reducing side effects and costs. After complete pituitary desensitization with standard dose of GnRHa, no evidence of premature LH surge or reduced quality of oocytes were noted after draw-back therapy of GnRHa. To date, the molecular biological changes of follicular environment depending on the doses of GnRHa have not
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been studied. According to our results, continuous administration of daily 400 mg of GnRHa showed significantly higher expression of intrafollicular PAPP-A of dominant follicles compared to that of the other dose groups. Despite many advantages on the clinical results of COH, the role of GnRHa on folliculogenesis and its effect on intrafollicular microenvironment remains controversial [20–22]. Limited in vivo models and currently available clinical data have precluded scientists to draw any firm conclusion on the effect of GnRHa on ovarian physiology. From the previous studies, reduced dose of GnRHa appears to improve IVF outcomes, but the status of follicular health seems to be most superior from continuous administration of the standard dose (400 mg per day) of GnRHa in regards to the expression of PAPP-A. Our study implies that the role of GnRHa in COH is not only limited to preventing premature luteinization but may influence the quality of follicle at the molecular biological level, and its dose may have a significant effect on the growth of healthy follicles. Therefore, further prospective clinical trials are warranted to seek the most optimal dose, which adapts to the intricate balance between inducing an appropriate pituitary down regulation with the most compatible follicular environment and at the same time, preventing deleterious effects of GnRHa. This preliminary study suggests that the dose of GnRHa may have a significant effect on the intrafollicular environment, reflected by the expression of PAPP-A. References [1] Parinaud J, Beaur A, Bourreau E, Vieitez G, Pontonnier G. Effect of a luteinizing hormone-releasing hormone agonist (buserelin) on steroidogenesis of cultured human preovulatory granulose cells. Fertil Steril 1988;50:597–602. [2] Tureck RW, Mastroianni Jr. L, Blasco L, Strauss III JF. Inhibition of human granulosa cell progesterone secretion by a gonadotrophinreleasing hormone agonist. J Clin Endocrinol Metab 1982;54:1078–80. [3] Hughes EG, Fedorkow DM, Daya S, Sagle MA, Van de Koppel P, Collins JA. The routine use of gonadotropin-releasing hormone agonists prior to in vitro fertilization and gamete intrafallopian transfer: a meta-analysis of randomized controlled trials. Fertil Steril 1992;58:888–96. [4] Meldrum DR, Wisot A, Hamilton F, Gutlay AL, Kempton WF, Huynh D. Routine pituitary suppression with leuprolide before ovarian stimulation for oocyte retrieval. Fertil Steril 1989;51:455–9. [5] Feldberg D, Farhi J, Ashkenazi J, Dicker D, Shalev J, Ben-Rafae Z. Minidose gonadotrophin-releasing hormone agonist is the treatment of choice in poor responders with high follicle-stimulating hormone levels. Fertil Steril 1994;62:343–6. [6] Pantos K, Meimeth-Damianaki T, Vaxevanoglou T, Kapetanakis E. Prospective study of a modified gonadotropin-releasing hormone agonist long protocol in an in vitro fertilizing program. Fertil Steril 1994;61:709–13. [7] Elgendy M, Afnan M, Holder R. Reducing the dose of gonadotropinreleasing hormone agonist on starting ovarian stimulation: effect on
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