Dynamic measurements of serum inhibin B and estradiol: a predictive evaluation of ovarian response to gonadotrophin stimulation in the early stage of IVF treatment

Dynamic measurements of serum inhibin B and estradiol: a predictive evaluation of ovarian response to gonadotrophin stimulation in the early stage of IVF treatment first-author †E-mail:mmf1966@sina.com.cnhuanghefg@hotmail.com †E-mail:mmf1966@sina.com.cnhuanghefg@hotmail.com mmf1966@sina.com.cn corresp ‡Corresponding Author ‡Corresponding Author †E-mail:mmf1966@sina.com.cnhuanghefg@hotmail.com †E-mail:mmf1966@sina.com.cnhuanghefg@hotmail.com mmf1966@sina.com.cn Department of Reproductive Endocrinology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China Department of Reproductive Endocrinology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China Objective: 2 We dynamically measured serum inhibin B and estradiol in the early stage of hormonal stimulation to predict the ovarian response in in vitro fertilization (IVF) treatment. Methods: 2 A total of 57 patients (<40 years of age) who underwent the first cycle of long protocol IVF or introcytoplasmic sperm injection (ICSI) treatment were included. Serum inhibin B, estradiol, follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels were measured four times: (1) on Day 3 of the menstrual cycle (basal); (2) on the day before the first administration of gonadotrophin (Gn) (Day 0); (3) on Day 1 of Gn therapy; and (4) on Day 5 of Gn therapy. Comparisons of these measurements with ovarian responses and pregnancy outcomes were made and analyzed statistically. Results: 2 (1) On Day 1 and Day 5 of recombinant FSH (rFSH) stimulation, ovarian response, i.e., numbers of follicles, oocytes, fertilized oocytes, and embryos, had a positive correlation ( r s =0.46~0.61, P =0.000) with raised inhibin B and estradiol concentrations, but a negative correlation ( r s =−0.67~−0.38, P =0.000 or P < 0.01) with total rFSH dose and total days of rFSH stimulation. (2) No significant variation ( P > 0.05) between the pregnant and non-pregnant groups on the basis of mean age or on all hormone concentrations at four times of the IVF cycle was observed. However, all the seven patients aged > 35 years did not reach pregnancy. Conclusions: 2 (1) Serum inhibin B and estradiol concentrations obtained shortly after Gn therapy may offer an accurate and early prediction of ovarian response; (2) Low levels of serum inhibin B and estradiol obtained shortly after Gn stimulation indicate the need for a longer period of Gn treatment and a higher daily dosage; (3) No obvious pregnancy difference among patients of age <35 years was found; however, IVF pregnancy outcome is significantly lower in women of age >35 years. Inhibin B Estradiol Ovarian response In vitro fertilization (IVF) Pregnancy INTRODUCTION 1 Since the advancement of in vitro fertilization-embryo transfer (IVF-ET) in assisted reproductive technologies (ART), controlled ovarian hyperstimulation (COH) has been widely utilized. However, women may react differently to the dosage of gonadotrophin (Gn), resulting in different ovarian responses and different IVF pregnancy outcomes. Poor ovarian response to Gn stimulation results in a small number of oocytes collected and thus a smaller number of embryos available for transfer, which therefore reduces the success rate of IVF (Bancsi et al., 2002 2002 ). On the other hand, an excessive ovarian response to Gn stimulation increases the risk for ovarian hyperstimulation syndrome (OHSS) (Navot et al., 1992 1992 ). A patient’s ovarian response to Gn stimulation is mainly determined by her ovarian reserve, which comprises the quantity and functional capacity of follicles and germ cells. Pre-evaluation of ovarian reserve and prediction of ovarian response would provide a valuable means of assisting clinicians in selecting appropriate Gn dose for each patient. This customized prescription of Gn dosage will not only reduce the possibility of either poor or excessive ovarian responses but also provide better chance to reach optimal ovarian response and ultimately improve the outcome of IVF pregnancy. Traditional criteria used to predict ovarian response to Gn stimulation include the patient’s age, baseline serum concentration of hormones such as follicle stimulating hormone (FSH), estradiol, FSH to luteinizing hormone (LH) ratio, and dynamic tests (such as the clomiphene citrate (CC) challenge test, the Gn-releasing hormone analogue (GnRHa) stimulation test, and the human menopausal Gn (HMG) stimulation test) (Navot et al., 1987 1987 ; Scott et al., 1989 1989 ; Scott and Hofmann, 1995 1995 ; Toner et al., 1991a 1991a ; Cahill et al., 1994 1994 ; Licciardi et al., 1995 1995 ; Mukherjee et al., 1996 1996 ; Kim et al., 1997 1997 ; Ravhon et al., 2000 2000 ). The effects of age, basal FSH and estradiol concentrations have been well documented (Scott et al., 1989 1989 ; Scott and Hofmann, 1995 1995 ; Toner et al., 1991b 1991b ; Cahill et al., 1994 1994 ; Licciardi et al., 1995 1995 ; Mukherjee et al., 1996 1996 ; Kim et al., 1997 1997 ). However, the most important aspect of diminished ovarian reserve and associated decline in reproductive potential is that its onset is highly variable (Mosher and Pratt, 1990 1990 ; 1991 1991 ; Maroulis, 1991 1991 ). This means that functional ovarian age as reflected by basal FSH and estradiol concentrations can be discordant with biological age (Scott et al., 1989 1989 ; Scott and Hofmann, 1995 1995 ; Toner et al., 1991b 1991b ; Cahill et al., 1994 1994 ). Patients who have normal FSH and estradiol levels or are younger than 35 years of age may still have a diminished ovarian reserve that leads to poor response to Gn stimulation (Farhi et al., 1997 1997 ). They may have a decreased Day 3 serum inhibin B concentration despite the unchanged Day 3 serum FSH and estradiol concentrations (Seifer et al., 1997 1997 ; 1999 1999 ). Inhibin B was suggested as an early sensitive predictor of ovarian reserve (Seifer et al., 1997 1997 ; 1999 1999 ). With better understanding of the control of synthesis and secretion of the inhibins and their potential endocrine role in the menstrual cycle, attention has turned to the possibility of this family of peptides to provide a more direct index of ovarian reserve and an improved predictor of ART outcome (Hughes et al., 1990 1990 ; Groome et al., 1994 1994 ; 1996 1996 ; Balasch et al., 1996 1996 ; Lockwood et al., 1996 1996 ; Seifer et al., 1997 1997 ; 1999 1999 ; Danforth et al., 1998 1998 ; Corson et al., 1999 1999 ; Hall et al., 1999 1999 ; Peñarrubia et al., 2000 2000 ; Laven and Fauser, 2004 2004 ; Shanthi et al., 2004 2004 ). Inhibins are dimeric polypeptides produced by granulosa cells and composed of an α-subunit along with a βA-subunit (inhibin A) or a βB-subunit (inhibin B). Inhibin B concentrations rise across the luteal-follicular transition and peak in the mid-follicular phase, suggesting that they are secreted by the developing cohort of follicles, and may mark the number or quality of developing follicles at the baseline (Groome et al., 1996 1996 ). In contrast, inhibin A does not begin to increase until just after the increase of estradiol in the late follicular and luteal phases, suggesting that it is secreted by the dominant follicle and may indicate follicle maturity (Groome et al., 1994 1994 ; Hall et al., 1999 1999 ). Because of being produced by granulosa cells, inhibin B has been suggested as a direct biochemical marker of ovarian reserve and may prove to be a useful adjunct in discriminating patients who are likely to respond to exogenous ovarian stimulation (Balasch et al., 1996 1996 ; Danforth et al., 1998 1998 ; Hall et al., 1999 1999 ; Seifer et al., 1999 1999 ). Danforth et al.( 1998 1998 ) found that women with normal menstrual cycle in the ages of 46~52 years had lower Day 3 serum inhibin B levels than those in the ages of 39~45 years. Seifer et al.( 1999 1999 ) considered inhibin B as a direct indicator of ovarian reserve and FSH as an indirect indicator. They discovered that the decrease of ovarian reserve might be demonstrated by a decrease in basal inhibin B concentration before a rise in FSH concentration. However, other studies do not support such a claim (Hughes et al., 1990 1990 ; Corson et al., 1999 1999 ; Hall et al., 1999 1999 ), and this discrepancy needs further clarification. Normal baseline hormonal values, such as inhibin B, estradiol and FSH, do not guarantee that an endocrine system is functioning normally. In fact, patients with baseline values in the normal range may still have a diminished ovarian reserve (Farhi et al., 1997 1997 ; Kim, 1998 1998 ; Fábregues et al., 2000 2000 ; Ranieri et al., 2001 2001 ). Thus, a number of provocative tests have been devised to indirectly assess ovarian reserve and identify patients who might not be defined by basal hormone screening alone (Navot et al., 1987 1987 ; Scott et al., 1989 1989 ; Hofmann et al., 1998 1998 ; Ravhon et al., 2000 2000 ). However, whether these indirect provocative tests are more informative of ovarian reserve than basal hormone remains controversial (Galtier-Dereure et al., 1996 1996 ; Ravhon et al., 2000 2000 ). Furthermore, neither basal hormonal measurements nor those dynamic tests are capable of providing direct manifestation concerning the responsiveness of the ovaries to the exogenous Gn used in ovarian stimulation for ART (Roest et al., 1996 1996 ; Farhi et al., 1997 1997 ; Peñarrubia et al., 2000 2000 ). Recently, Phelps et al.( 1998 1998 ) found that estradiol concentration obtained on Day 4 of Gn therapy was highly predictive of a successful ovulation induction and pregnancy outcome in IVF cycles. Neither observation of any variation of inhibin B nor measurement of baseline hormone values was mentioned in their study. This was explained on the basis that estradiol concentration obtained early after the initiation of ovarian stimulation directly reflects follicular activity and ovarian responsiveness to the ongoing regimen of Gn stimulation. Further studies are required to investigate whether in GnRHa-recombinant FSH (rFSH) treatment cycles there is any correlation between the increase in inhibin B concentration and the ovarian response and the IVF outcome. Since inhibin B may regulate the production of estradiol through increasing the level of substrates of estradiol, it would be of interest to delineate whether inhibin B is more sensitive than estradiol in predicting ovarian response to Gn stimulation (Porchet et al., 1994 1994 ). This study confirmed and expanded the findings from previous studies (Phelps et al., 1998 1998 ; Dzik et al., 2000 2000 ; Ravhon et al., 2000 2000 ). A total of 57 patents undergoing their first cycle of long protocol IVF or introcytoplasmic sperm injection (ICSI) treatment were studied, and serum inhibin B, estradiol, FSH, LH levels were detected four times in the IVF cycle: on Day 3 of the menstrual cycle, before the first administration of Gn (Day 0), on Day 1 after Gn therapy, and on Day 5 after Gn therapy. This study was designed to identify prospectively the values of basal and dynamic measurements of inhibin B, estradiol, FSH, and LH following Gn therapy for IVF cycles in predicting ovarian response to Gn stimulation and IVF pregnancy outcome. Comparisons of these basal and dynamic measurements with different ovarian responses enabled us to customize the dosage at the early stage of Gn and GnRHa treatment to improve the IVF outcome. MATERIALS AND METHODS 1 Patients 2 After approval from Hospital Ethics Committee and consents from all participants, the study was commenced in January 2004 and concluded in September 2004. Fifty-seven patients were selected from the regular patients admitted at the Reproductive Medicine Department, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China, for their first cycle of IVF/ICSI treatment. All patients accepted had regular menstrual cycles, as well as normal serum prolactin (PRL) level and without hormone treatment within 3 months. The patients’ ages ranged from 24 to 39 years with a mean age of (30.8±3.2) years (mean± SD ). Thirty-two patients belonged to tubal infertility, 19 to male infertility, and 6 to both tubal and male infertilities. Twenty-five patients were of primary infertility, and 32 of secondary infertility. Twenty-seven patients underwent IVF, and 30 underwent ICSI. Sample collection 2 In the morning, 8:00 a.m. to 10:00 a.m., on Day 3 of patient’s menstrual cycle, 3~5 ml venous blood sample was taken to be assayed. Concentrations of inhibin B, estradiol, FSH, LH, and PRL were measured. Ovarian stimulation was started using rFSH (Gonal-F®; Serono, Switzerland) under pituitary suppression with the daily subcutaneous injection of 0.05~0.10 mg of triptorelin acetate (Decapeptyl®; Ferring, Germany) in the midluteal phase, i.e., after basal temperature had risen for 6~7 d and the serum progesterone concentration was greater than 36 nmol/L. Starting on Day 3 of the next menstrual cycle, rFSH was injected daily with a dosage of 225~375 U/d for the first 2 d, and 150~300 U/d thereafter. More blood samples were collected and assayed for inhibin B, estradiol, FSH and LH in the mornings of Day 0 (before injection of rFSH), on Day 1 and Day 5 of rFSH injection, respectively. On Day 8 of the menstrual cycle (after 5 d of rFSH injection), patients took the first ultrasonographic scan and repeated the scan every 2 or 3 d as necessary. The dosage of rFSH was adjusted from Day 5 of stimulation according to the ovarian response. When at least 1 follicle ≥18 mm in diameter or at least 3 or more follicles ≥15 mm in diameter appeared, we stopped the injections of rFSH and triptorelin acetate for 36 h and then injected human chorionic gonadotrophin (HCG) (10 000 U) (Profasi®; Serono, Switzerland). Oocyte aspiration was performed with vaginal ultrasonography 34~36 h after HCG administration, and embryo ( n ≤4) transfer (ET) was done 2 to 3 d later. Then we started the injection of 80 mg progesterone daily. Serum HCG level was measured 2 weeks later, and if serum HCG level was more than or equal to normal level, we performed ultrasonography to detect the pulse of fetus to confirm pregnancy. Hormone assay 2 According to the manufacturer’s protocol, inhibin B was analyzed in duplicate using a double-antibody enzyme-linked immunosorbent assay (ELISA; Serotec, Kidlington, Oxford, Oxon, UK). Estradiol, FSH and LH were measured using the microparticle enzyme immuno-assay by the ECL2012 system (Rocer). Statistical analysis 2 Data were analyzed using SPSS 10.0 for Windows. Pearson’s correlation analysis or Spearman’s correlation analysis was performed to assess the correlation between different variables and ovarian response and the correlation between one variable and another as appropriate. Independent dual-sample test or Pearson’s square test was performed to assess the correlation between various variables and pregnancy outcome as appropriate. One-way analysis of variance (ANOVA) test or Kruskal-Wallis (K-W) test was used to analyze the difference of variables in poor, normal, and OHSS ovarian responses with changes in ovarian response as appropriate. Mann-Whitney (M-W) test was performed to analyze the difference between ovarian response and pregnancy outcome. RESULTS 1 All the 57 patients reached oocyte aspiration, of which 53 reached ET (3 ET failures due to vaginal bleeding and 1 failure due to fever). A total number of 162 embryos were transferred. Twenty-one women became pregnant with 3 cases of triplets and 2 cases of twins. The clinical pregnancy rate per ET was 39.6% (21/53), clinical pregnancy rate per cycle was 36.8% (21/57), and implantation rate was 17.3% (28/162). Five patients had poor response (criterion for poor response is oocytes ≤4), 5 patients developed OHSS, and other 47 patients had normal ovarian response. Table 1 1 shows patient’s characteristics, ovarian response, and dose of rFSH used. Mean numbers of follicles and eggs were 15.1±6.6 and 13.7±6.4, respectively. Median number of fertilized eggs, embryos, and transferred embryos were 9.0 (1~25), 8.0 (1~25) and 3.0 (1~4), respectively. Median days of rFSH stimulation, ampoules of rFSH and infertility years were 11.0 (8~23), 30.0 (20~82) and 5.0 (1~14), respectively. M ( Qr ): median (grade) Mean± SD M ( Qr ) * * Range Age (years) 30.8±3.2 24~39 Follicles 15.1±6.6 4~31 Eggs 13.7±6.4 3~31 Fertilized eggs 9.0 (7.5) 1~25 Embryos 8.0 (7.0) 1~25 Transferred embryos 3.0 (0.0) 1~4 Days of rFSH stimulation 11.0 (2.0) 8~23 Ampoules of rFSH 30.0 (11.0) 20~82 Infertility years 5.0 (4.0) 1~14 Table 2 2 shows median values of hormone concentrations at four different times of IVF cycle. Data given in the table were presented in median and grade. Inhibin B and estradiol concentrations rose across the increase of days of rFSH stimulation. Data are expressed as median (grade) Inhibin B (pg/ml) Estradiol (pmol/L) FSH (U/L) LH (U/L) Basal 36.5 (41.3) 127.4 (89.0) 6.9 (1.8) 3.7 (1.6) Day 0 16.7 (29.0) 66.3 (44.7) 3.2 (2.2) 2.0 (1.8) Day 1 53.4 (44.9) 157 (130.8) 8.3 (3.0) 2.0 (1.6) Day 5 696.2 (875.9) 1743.3 (2630.2) 8.8 (3.5) 1.5 (0.7) Correlations between hormone concentrations and age, ovarian response, dose of rFSH used 2 On Days 1 and 5 of rFSH stimulation, inhibin B and estradiol concentrations had a positive correlation with ovarian response (numbers of follicles, oocytes, fertilized oocytes and embryos) ( r s =0.46~0.61, P =0.000) (Table 3 3 ), but a negative correlation with total rFSH dose and days of rFSH administrated ( r s =−0.67~−0.38, P =0.000 or P < 0.01). Inhibin B concentrations on Days 1 and 5 and estradiol concentration on Day 5 had a negative correlation with age ( r s =−0.38~−0.28, P < 0.01). However, there was no correlation between age and estradiol concentration on Day 1 ( r s =−0.20, P > 0.05). This clearly indicated that higher total Gn dose and more days of Gn stimulation were needed in the case of lower serum inhibin B and estradiol concentrations shortly after Gn therapy. 2 2 Inhibin B on Day 1 2 Inhibin B on Day 5 2 Estradiol on Day 1 2 Estradiol on Day 5 r s P r s P r s P r s P Age −0.37 0.008 −0.38 0.004 −0.20 0.16 −0.28 0.040 Follicles 0.57 0.000 0.60 0.000 0.50 0.000 0.61 0.000 Eggs 0.57 0.000 0.61 0.000 0.47 0.000 0.58 0.000 Fertilized eggs 0.54 0.000 0.55 0.000 0.51 0.000 0.48 0.000 Embryos 0.57 0.000 0.55 0.000 0.52 0.000 0.46 0.000 Total rFSH dose −0.59 0.000 −0.38 0.000 −0.53 0.000 −0.67 0.000 Days of rFSH stimulation −0.47 0.001 −0.65 0.000 −0.45 0.001 −0.52 0.000 Basal hormone, Inhibin B, estradiol, FSH, and LH levels before rFSH stimulation had no correlation with age, ovarian response and dose of rFSH used ( P < 0.05). There was no correlation among basal inhibin B and estradiol concentrations and those on Day 1 and Day 5 of rFSH stimulation ( r s =−0.22~−0.17, P > 0.05). Negative correlation existed between age and the numbers of follicles and oocytes ( r s =−0.30~−0.27, P < 0.05). Inhibin B concentration had a positive correlation with estradiol concentration after rFSH stimulation ( r s =0.62~0.79, P =0.000) (Fig. 1 1 ). Correlations between all variables and pregnancy outcome 2 Table 4 4 shows mean values and standard deviation ( SD ) of age, follicles, eggs, and the independent samples test for the ampoules of rFSH, days of rFSH stimulation, fertilized eggs, embryos, transferred embryos, and infertility years in pregnancy group ( n =21) and non-pregnancy group ( n =36). Data are expressed as mean± SD or median (grade) Group Age (year) Follicles Eggs Ampoules of rFSH Days of rFSH stimulation Fertilized eggs Embryos Transferred embryos Infertility years Pregnancy ( n =21) 30.6±2.4 15.0±7.3 13.4±6.7 30 (11) 11 (2.5) 8.0 (8.5) 9.5 (8.0) 3.0 (0.5) 5.0 (4.5) Non-pregnancy ( n =36) 30.6±3.7 15.6±6.1 14.3±6.3 31 (11) 11 (2.0) 9.5 (8.2) 9.0 (7.5) 3.0 (0.0) 4.5 (3.8) 10 P >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 Table 5 5 shows independent samples test for hormone concentrations at four different times of IVF cycle in pregnancy group ( n =21) and non-pregnancy group ( n =36). Data are expressed as median (grade) 2 Group 4 Inhibin B (pg/ml) 4 Estradiol (pmol/L) 2 Basal FSH (U/L) 2 Basal LH (U/L) Basal Day 0 Day 1 Day 5 Basal Day 0 Day 1 Day 5 Pregnancy ( n =21) 26.4 (18.1) 8.8 (34.2) 46.2 (39.7) 603.3 (837.2) 114.5 (93.1) 72.9 (47.4) 157.5 (140.4) 1562.5 (3254.5) 7.0 (1.8) 3.7 (1.5) Non-pregnancy ( n =36) 38.5 (45.7) 18.6 (27.7) 57.2 (65.3) 762.5 (1059.3) 133.9 (94.2) 66.2 (39.8) 174.9 (155.1) 2133 (2533) 6.9 (1.9) 3.8 (2.1) 11 P >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 There was no significant difference between pregnancy and non-pregnancy groups in relation to different variables ( P > 0.05). In this study, all the 7 patients of age > 35 years failed to become pregnant. Correlations between pregnancy outcome and cause of infertility, sort of infertility, and IVF manner 2 Pearson’s square tests as shown in Table 6 6 show that pregnancy outcome had no correlation with cause of infertility, sort of infertility or IVF manner ( P > 0.05). χ 2 df P  Cause of infertility 0.53 2 0.77  Sort of infertility 0.50 1 0.70  IVF manner 0.91 1 0.34 Comparisons of variables in poor, normal, and OHSS ovarian responses with pregnancy outcome 2 For the benefit of easier and more direct comparisons of ovarian responses to different variables, we categorized the results into three different groups: poor response (5 patients whose oocytes were ≤4), normal response (47 cases), and OHSS (5 patients who developed OHSS). Comparisons of variables among the three groups are shown in Table 7 7 (results of one-way ANOVA test) and Table 8 8 (results of K-W test). Data are expressed as mean± SD Group Age (year) Follicles Eggs Fertilized eggs Embryos Days of rFSH stimulation Ampoules of rFSH Poor response ( n =5) 33.4±3.0 4.0±0.0 4.0±0.0 3.2±1.2 3.0±1.3 50.2±20.9 15.0±5.1 Normal response ( n =47) 30.5±3.3 15.4±5.7 13.9±5.5 9.5±5.1 9.0±4.8 30.2±6.8 11.0±1.6 OHSS ( n =5) 30.6±1.3 22.6±5.1 21.2±6.7 14.8±4.6 14.2±5.0 30.4±5.7 11.0±1.5 8 P 0.16 0.000 0.000 0.001 0.002 0.000 0.001 C 5 : concentration on Day 5; C b : basal concentration; C 0 : concentration on Day 0; C 1 : concentration on Day 1 2 Group 4 Inhibin B concentration (pg/ml) 4 Estradiol concentration (pmol/L) C 5 C 5 − C b C 5 − C 0 C 5 − C 1 C 5 C 5 − C b C 5 − C 0 C 5 − C 1 Poor response ( n =5) 164.7 (155.4) 132.2 (83.1) 153.6 (98.0) 180.3 (198.4) 303.2 (1241.2) 91.2 (144.7) 202.0 (566.9) 137.0 (1358.9) Normal response ( n =47) 696.2 (907.9) 659.7 (980.2) 640.2 (1000.2) 647.9 (917.9) 1709.5 (2252.0) 1582.1 (1934.5) 1920.0 (25.2.4) 1619.0 (2278.4) OHSS ( n =5) 1263.5 (792.9) 1237.1 (642.3) 1358.7 (743.5) 1027.3 (614.9) 4261.0 (1689.1) 4129.6 (1070.3) 3586.0 (2072.1) 3359.2 (1899.5) 9 P 0.001 0.001 0.007 0.006 0.002 0.001 0.005 0.009 Age was not an influential factor among the three different groups ( P > 0.05). The numbers of follicles, oocytes, fertilized oocytes and embryos were significantly different ( P < 0.01) among the three different groups. The total rFSH dose and days of rFSH stimulation in the poor ovarian response group were higher than those in the normal ovarian response and the OHSS groups ( P < 0.01). There was no significant difference in the levels of basal inhibin B, pre-rFSH inhibin B (Day 0) and post-rFSH inhibin B (Day 1) among the three groups ( P > 0.05). However, inhibin B concentration on Day 5 of rFSH stimulation was significantly different among the three groups ( P < 0.01). There was no significant difference ( P > 0.05) in the levels of basal estradiol, estradiol on Day 0 and Day 1 of rFSH stimulation among the three groups ( P > 0.05), but estradiol levels showed a significant difference on Day 5 of rFSH stimulation among the three groups ( P < 0.05). FSH and LH concentrations at four different times of IVF cycle were very similar among the three groups ( P > 0.05). Table 9 9 shows comparison of pregnancy outcome among the three groups. The pregnancy outcome was found similar by M-W test ( U =296, P > 0.05). Group Pregnancy Non-pregnancy Total Poor response ( n =5) 2 2 4 Normal response ( n =47) 15 29 44 OHSS ( n =5) 4 1 5 4 Total 21 32 53 DISCUSSION 1 Although there are many factors that affect the extent of COH and the result of IVF-ET, it is generally accepted that the ovarian response to the Gn stimulation plays a key role in an IVF outcome. In the present study, we demonstrated the predictive values of various ovarian reserve indicators that affect ovarian response as well as IVF pregnancy outcome. An accurate prediction of ovarian response could provide a valuable means in prescribing appropriate Gn and GnRHa dosage in the early days of treatment for a patient. This timely dosage adjustment may avoid the poor ovarian response and the risk of OHSS, while IVF-ET outcome was improved. Significance of inhibin B measurement after Gn stimulation in predicting the ovarian response and IVF pregnancy outcome 2 Dzik et al.( 2000 2000 ) found that the mean baseline inhibin B concentrations and the inhibin B concentration 24 h after exogenous follicle-stimulating hormone ovarian reserve test (EFORT) were significantly higher in good responders than that in poor responders. Thus, they concluded that inhibin B concentration 24 h after EFFORT may provide a method for predicting ovarian response to hyperstimulation in a subsequent IVF cycle. Ravhon et al.( 2000 2000 ) reported that ovarian response was defined in two ways: the number of oocytes/total rFSH dose and square root (the number of follicles/total rFSH dose). The following measurements were highly correlated with the ovarian response to stimulation: increase in estradiol (Day 3 estradiol concentration−Day 2 estradiol concentration) ( r s =0.68, P < 0.0001) and sum of inhibin B (Day 3 estradiol concentration+Day 2 inhibin B concentration) ( r s =0.60, P < 0.0001). Age and basal concentrations of FSH and inhibin B were inferior to the preceding measurements in terms of correlation with the ovarian response. Although it was concluded that dynamic measurements of inhibin B and estradiol following single administration of buserelin acetate were highly correlated with the ovarian response to stimulation for IVF treatment, the correlations between different variables and the pregnancy outcome remain unknown. Moreover, those reports did not provide direct information regarding the responsiveness of the ovaries to the exogenous Gn used in ovarian stimulation for ART. In fact, other studies proposed that the clinical response to Gn treatment would be a more direct way to assess ovarian reserve and a better predictor of fertility potential (Roest et al., 1996 1996 ; Farhi et al., 1997 1997 ; Peñarrubia et al., 2000 2000 ). Phelps et al.( 1998 1998 ) found that the estradiol concentration measured on Day 4 of Gn therapy was highly predictive of successful ovulation induction and pregnancy outcome in GnRHa-rFSH cycles. This study confirmed previous studies and expanded some of their findings (Phelps et al., 1998 1998 ; Ravhon et al., 2000 2000 ; Dzik et al., 2000 2000 ; Urbancsek et al., 2005b 2005b ). We performed dynamic assays of basal inhibin B, estradiol, FSH, and LH. These measurements were taken on Day 3 of the menstrual cycle, and at three different times in the rFSH stimulation cycle: before injection of rFSH (Day 0), Day 1 and Day 5 after injection of rFSH. These data were subjected to statistical analyses for correlations between ovarian responses and different sets of variables. Our findings 2 Our results suggest that serum inhibin B and estradiol concentrations obtained shortly after Gn therapy may offer an accurate and early prediction of ovarian response. Low levels of inhibin B and estradiol may indicate the possibility of low ovarian response; conversely, high levels of inhibin B and estradiol obtained on Day 5 indicate the occurrence of OHSS (Table 9 9 ). With early detection of low ovarian response or OHSS, a timely adjustment of dosage of rFSH and GnRHa can be considered in order to correct the physical conditions and to improve the ovarian response. A negative correlation between age and inhibin B existed on Day 1 after injection of rFSH, indicating that the older the age, the lower the inhibin B level after Gn stimulation. However, there was no correlation between age and estradiol level on Day 1 after injection of rFSH, indicating that inhibin B concentration may be a more sensitive and earlier index of ovarian response than serum estradiol concentration and age. The concentrations of inhibin B and estradiol had a higher correlation coefficient ( r s =0.47~0.61, P =0.000) with the numbers of follicles and oocytes than that with age ( r s =−0.27~−0.30, P < 0.05), indicating that the inhibin B and estradiol levels in the early days can better predict the ovarian response to Gn stimulation than age. We can conclude that the basal inhibin B and estradiol concentrations could not predict the change of inhibin B and estradiol after Gn stimulation. This may be due to the patients’ relatively young ages (mean value of (30.8±3.2) years). We can also conclude that it is less valuable to use concentrations of inhibin B, estradiol, FSH and LH on the early days after Gn stimulation as a predictive marker of IVF pregnancy outcome for younger women. These results agree with those of Hall et al.( 1999 1999 ) and Urbancsek et al.( 2005a 2005a ), but contradict with that reported by Seifer et al.( 1997 1997 ). Indeed, it is highly possible that uterine receptivity and a perfect dialogue between good-quality embryos and receptive endometrium may play a more important role in an IVF pregnancy outcome. Effects of basal hormone in predictions of ovarian response and IVF pregnancy outcome 2 This study shows that basal inhibin B, estradiol, FSH, and LH do not correlate with the numbers of follicles, oocytes, and IVF pregnancy. These results agree with previous reports (Hughes et al., 1990 1990 ; Rotsztejn and Asch, 1991 1991 ; Corson et al., 1999 1999 ; Hall et al., 1999 1999 ; Frattarelli et al., 2000 2000 ; Ravhon et al., 2000 2000 ). It is understandable that the basal inhibin B (36.5 ng/ml), estradiol (127.4 pmol/L) or FSH (6.9 U/L) levels had no correlation with ovarian response and pregnancy outcome, because patients’ ages in this study are relatively young [(30.8±3.2) years] and their basal hormone levels are in the normal range. There were 5 patients of low ovarian response and 5 patients developed OHSS, which confirmed that there is an uncertainty between basal hormone level and ovarian response; the normal baseline hormone levels cannot guarantee normal ovarian response to Gn stimulation. Effects of age on predictions of ovarian response and pregnancy outcome 2 Our results show that pregnancy outcome may be significantly lower in women of age > 35 years, but the pregnancy outcome varies little for those of age < 35 years. Our results agree with previous reports (Spandorfer et al., 2000 2000 ; Watt et al., 2000 2000 ). Watt et al. ( 2000 2000 ) reported that the ovarian response to Gn stimulation decreased with age as demonstrated by lower estradiol concentrations and that patients needed larger doses of Gn for stimulation. They also reported that age had a negative correlation with the numbers of follicles, oocytes, and pregnancy rate. Spandorfer et al.( 2000 2000 ) found that the implantation rate did not change in women of age < 35 years, but it reduced by 2.77% per year in those of age ≥35 years. The implantation rate was 36.8% in women of age < 33 and only 2.3% in those of age > 44. Correlation between ovarian response and pregnancy outcome 2 The success of IVF pregnancy depends on several factors such as patient’s age, embryo quality, uterine receptivity, and a perfect dialogue between good-quality embryos and receptive endometrium. However, the correlation between ovarian response and IVF pregnancy outcome remains controversial (Toner et al., 1991a 1991a ; Simon et al., 1995 1995 ; 1998 1998 ; Pellicer et al., 1996 1996 ; Sharara and McClamrock, 1999 1999 ; Kably et al., 2000 2000 ; Bancsi et al., 2002 2002 ). Previous reports indicated that low estradiol concentration may induce the decline in oocytes number and embryos quality which may lead to low fertilization rate and low pregnancy rate (Kably et al., 2000 2000 ). Different ovarian responses were considered to contribute to different IVF pregnancy rates (Kably et al., 2000 2000 ; Bancsi et al., 2002 2002 ). Poor ovarian responders had significantly lower clinical and ongoing pregnancy rates than did normal responders (Bancsi et al., 2002 2002 ). In contrast, Toner et al.( 1991a 1991a ) retrospectively reported that implantation and pregnancy rates were not different among the women retrieved 1~5, 6~10, or > 10 oocytes, despite a decrease in the numbers of retrieved oocytes and embryos in low responders. Low peak estradiol concentration may be in favor of embryos implantation. Although high responders had more retrieved oocytes and good-quality embryos, their pregnancy rates were similar to low and normal responders’ (Simon et al., 1998 1998 ; Sharara and McClamrock, 1999 1999 ). High estradiol concentration and altered estradiol/progesterone ratios, which also are associated with the impairment of endometrial receptivity, may be the main factors affecting endometrial receptivity in high responders (Simon et al., 1995 1995 ; Pellicer et al., 1996 1996 ). Our results show that although there were significant differences in the numbers of follicles, oocytes, fertilized oocytes, and embryos among poor, normal, and OHSS groups, the cycle pregnancy rates were not different. Our results agree with previous reports (Toner et al., 1991a 1991a ; Simon et al., 1995 1995 ; 1998 1998 ; Pellicer et al., 1996 1996 ; Sharara and McClamrock, 1999 1999 ). However, for the poor responder, the number of embryos obtained in the IVF cycle is only enough to have one chance of ET. An early, accurate identification of patients who tend to have poor response would help clinicians to make a prompt modification on the dosage of Gn and GnRHa for stimulation, so that ovarian response can be improved and more retrieved oocytes and better embryos can be gained. As a result, women have more chances to accept frozen-thawed ET. As to the OHSS patients, an appropriate dose of Gn and GnRHa will probably reduce or avoid the occurrence of OHSS. We, therefore, conclude that serum inhibin B and estradiol levels obtained shortly after Gn therapy offer an early and accurate prediction of ovarian response to Gn stimulation. They are superior indicators compared with age or basal hormonal levels in predicting ovarian response. Serum inhibin B and estradiol levels obtained shortly after Gn therapy predict poor ovarian response and OHSS. A higher total Gn dose and longer COH cycle would be warranted for the case of poor ovarian response. In predicting ovarian response of women in different ages, serum inhibin B may be a more sensitive index than serum estradiol. Normal basal hormonal levels could not guarantee normal ovarian response to Gn stimulation, and possess little predictive value for IVF pregnancy outcome in younger women. 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Serum CA-125 and inhibin B levels in the prediction of ovarian response to gonadotropin stimulation in in vitro fertilization cycles AH Watt , AT Legedza , ES Ginsburg , RL Barbieri , RN Clarke , MD Hornstein . The prognostic value of age and follicle-stimulating hormone levels in women over forty years of age undergoing in vitro fertilization . J Assist Reprod Genet , 2000 . 17 ( 5 ): 264 - 268 . 10976413 DOI: 10.1023/A:1009458332567 http://doi.org/10.1023/A:1009458332567 . The prognostic value of age and follicle-stimulating hormone levels in women over forty years of age undergoing in vitro fertilization 10.1631/jzus.B0820029 2008-01-22 2008-11-26 2008-09-23 2022-01-05 Copyright © 2009, Journal of Zhejiang University Science 2009 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 01 10