Antral follicle count and FSH concentration after clomiphene citrate challenge test in the prediction of ovarian response during IVF treatment

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1 Human Reproduction Vol.20, No.6 pp , 2005 Advance Access publication February 25, 2005 doi: /humrep/deh833 Antral follicle count and FSH concentration after clomiphene citrate challenge test in the prediction of ovarian response during IVF treatment Ernest Hung Yu Ng 1, Carina Chi Wai Chan, Oi Shan Tang and Pak Chung Ho Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong Special Administrative Region, People s Republic of China 1 To whom correspondence should be addressed. nghye@hkucc.hku.hk BACKGROUND: We compared: (i) antral follicle count (AFC) in the early follicular phase, after the clomiphene citrate challenge test (CCCT) and before ovarian stimulation following pituitary down-regulation; and (ii) age of women, body mass index, basal and stimulated serum FSH concentrations and AFC in predicting the ovarian response of infertile women aged <40 years with basal FSH <10 IU/l on recruitment in their first IVF cycle. METHODS: Two months prior to the treatment cycle, AFC and basal FSH concentration were determined on day 2 3 of a spontaneous period and on day 10 after CCCT. All women received a standard stimulation regimen. Ovarian response was represented by the number of oocytes, serum estradiol, the duration and dosage of gonadotrophins. RESULTS: There was no significant difference between basal, stimulated and down-regulated AFC. AFC achieved the best predictive value in relation to the number of oocytes, followed by combined FSH concentration (sum of the two FSH concentrations) and age of women. Both basal AFC and combined FSH concentration were predictive factors of serum estradiol concentration, whereas stimulated FSH concentration was predictive of the total dosage of gonadotrophins. CONCLUSION: Combined FSH concentration after CCCT provides additional information in predicting ovarian response. Key words: antral follicle count/clomiphene citrate challenge test/fsh/ovarian response Introduction Recruitment and development of multiple follicles in response to gonadotrophin stimulation are the key factors leading to a successful outcome of the IVF treatment. Poor ovarian response may be associated with poor pregnancy rates and many of these cycles are cancelled without proceeding to oocyte retrieval (Keay et al., 1997; Tarlatzis et al., 2003). On the other hand, exaggerated ovarian response leads to an increased risk of ovarian hyperstimulation syndrome (OHSS) (Aboulghar and Mansour, 2003) and the resulting high serum estradiol (E 2 ) concentrations may adversely affect the outcomes of the IVF treatment (Ng et al., 2000a). Prediction of ovarian responses prior to stimulation is useful in counselling patients and may be helpful in tailoring the dosage of gonadotrophin to individual patients. Different hormonal and ultrasound markers for ovarian reserve have been examined to predict the ovarian response to gonadotrophins, including early follicular serum FSH (Scott and Hofmann et al., 1995; Sharara et al., 1998), serum inhibin B (Seifer et al., 1997; Tinkanen et al., 1999; Dzik et al., 2000), serum anti-müllerian hormone (Seifer et al., 2002; van Rooij et al., 2002; Fanchin et al., 2003), ovarian volume (Syrop et al., 1995, 1999; Lass et al., 1997), antral follicle count (AFC) (Tomás et al., 1997; Chang et al., 1998; Frattarelli et al., 2000; Ng et al., 2000b; Hsieh et al., 2001; Nahum et al., 2001; Bancsi et al., 2002) and ovarian stromal blood flow (Zaidi et al., 1996; Engmann et al., 1999; Kupesic and Kurjak, 2002; Kupesic et al., 2003; Popovic-Todorovic et al., 2003). Early follicular FSH concentration is widely used in many IVF units to predict the ovarian response and is a better predictor of ovarian response than the age of women (Cahill et al., 1994; Sharif et al., 1998). In order to identify a greater number of women with decreased ovarian reserve, challenging the pituitary gland may be more likely to uncover an abnormality that would be missed by obtaining basal FSH concentration alone. Navot et al. (1987) first described the clomiphene citrate challenge test (CCCT), which consisted of measuring serum FSH concentrations on cycle day 3 (basal FSH) and then again on day 10 (stimulated FSH) after the administration of 100 mg of clomiphene citrate (CC) from day 5 to day 9. Subsequently, abnormal CCCT has been shown to be predictive of poor ovarian response, cycle cancellation and reduced pregnancy rate (Loumaye et al., 1990; q The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oupjournals.org 1647

2 E.H.Y.Ng et al. Tanbo et al., 1992; Hofmann et al., 1996; Kahraman et al., 1997; Gülekli et al., 1999; Van der Stege and Van der Linden, 2001; Csemiczky et al., 2002; Yanushpolsky et al., 2003; Hendriks et al., 2005). The significance of AFC in predicting ovarian response and pregnancy rate of IVF treatment has been demonstrated in many studies. We previously demonstrated that AFC achieved the best predictive value, followed by basal FSH concentration, body mass index (BMI) and age of women in a prospective study of 128 infertile patients undergoing the first IVF cycle using a standard regimen of ovarian stimulation (Ng et al., 2000b). In the majority of the above studies, AFC was determined after pituitary down-regulation and prior to ovarian stimulation in the treatment cycle. Bancsi et al. (2002) performed ultrasound assessment on day 3 of a spontaneous cycle prior to the IVF treatment. As the use of CC stimulates the growth of these antral follicles, perhaps it may be more accurate to determine AFC after CCCT. Moreover, there are very few studies in the literature comparing stimulated FSH concentration and AFC in relation to the prediction of ovarian responsiveness. The objectives of this prospective study were to: (i) compare AFC in the early follicular phase, after CCCT and before ovarian stimulation following pituitary downregulation; and (ii) compare age of women, BMI, basal and stimulated serum FSH concentrations and AFC in predicting the ovarian response of infertile women undergoing the first IVF cycle using a standard regimen of ovarian stimulation. Materials and methods Consecutive women attending the Department of Obstetrics and Gynaecology, University of Hong Kong between January 2002 and November 2003 for their first IVF treatment were recruited. Women who were aged.40 years at the time of treatment or whose basal FSH concentration on repeated testing on recruitment was $10 IU/l were advised against the IVF treatment according to the recruitment guideline issued by the Hospital Authority in Hong Kong. The current waiting time for IVF treatment was, 1 year after recruitment. The indications of conventional IVF treatment included tubal, male, endometriosis, unexplained and mixed factors. ICSI was performed for couples with severe semen abnormalities (, motile sperm recovered after sperm preparation) and surgically retrieved sperm from epididymis or testis in cases of obstructive azoospermia. Poor visualization of ovaries because of abdominal position, presence of an ovarian cyst of $20 mm in diameter and the presence of polycystic ovaries on scanning (Adams et al., 1986) were excluded. Every woman gave a written informed consent prior to participating in the study, which was approved by the Ethics Committee, Faculty of Medicine, University of Hong Kong. They received no monetary compensation for participation in the study. The results of CCCT did not affect their IVF treatment. Two months prior to the treatment cycle, they attended the clinic for a transvaginal ultrasound examination for AFC and a blood test for basal FSH concentration in the early follicular phase (days 2 4) of the cycle. They were asked to take clomiphene citrate (Clomid; Merrell, UK) 100 mg daily from day 5 to day 9. Transvaginal scanning for AFC and the blood test for FSH concentration were repeated on day The details of the long protocol of ovarian stimulation regimen at our centre have been previously published (Ng et al., 2000a). All women were pre-treated with buserelin (Suprecur; Hoechst, Germany) nasal spray 150 mg four times a day from the mid-luteal phase of the cycle preceding the treatment cycle. On the second day of the treatment cycle, transvaginal scanning was performed to determine AFC and blood was then taken for basal serum E 2 concentration. When the ultrasound scanning showed no ovarian cyst and serum E 2 concentrations were,200 pmol/l, hmg (Pergonal; Serono, Switzerland) injections were started at 300 IU daily for the first 2 days followed by 150 IU daily afterwards. The ovarian response was monitored by serial transvaginal scanning and the hmg dosage was increased if there was no follicle $10 mm after 7 days of stimulation. hcg (Profasi; Serono, Switzerland) was given i.m. when the leading follicle reached 18 mm in diameter and there were at least three follicles of $16 mm in diameter. Serum E 2 concentration was measured on the day of hcg administration. Cycles were cancelled when the follicles remained,10 mm after 14 days of stimulation. Oocyte retrieval would be performed even when there was only one dominant follicle and was scheduled 36 h after the hcg injection and any visible follicles were aspirated during the procedure. A maximum of three normally cleaved embryos was replaced into the uterine cavity 48 h after the retrieval and excess good quality embryos were frozen. All fresh embryos were cryopreserved if serum E 2 on the day of hcg injection was pmol/l in order to reduce the risks of ovarian hyperstimulation syndrome. Luteal phase was supported by two doses of hcg. A urine pregnancy test was done 16 days after embryo transfer; if positive, ultrasound examination was performed days later to confirm intrauterine pregnancy and to determine the number of gestation sacs present. Only clinical pregnancies were considered and were defined by the presence of one or more gestation sacs or the histological confirmation of gestational product in miscarriages. Ongoing pregnancies were those pregnancies beyond weeks of gestation, at which stage the patients were referred out for antenatal care. AFC was determined during transvaginal scanning performed at 08:00 10:00 by EHYN (Ng et al., 2000b) using a 6.5 MHz vaginal probe (Aloka, Model SSD-5500; Aloka Co. Ltd, Japan). The intraobserver coefficient of variation (CV) for AFC was 7%. AFC in the early follicular phase of a spontaneous cycle, day 10 after taking clomiphene citrate and before ovarian stimulation following pituitary down-regulation, were termed as basal, stimulated and downregulated respectively. Serum FSH and E 2 concentrations were measured using commercially available kits (Automated Chemiluminescence ACS-180 System; Bayer Corporation, USA). The FSH assay is standardized against the World Health Organization 2nd International Standard 94/632 reference material. The sensitivity of the FSH assay was 0.3 pmol/l and the intra- and inter-assay CV were 2.8 and 4.6% respectively. The sensitivity of the E 2 assay was 36.7 pmol/l and the intra- and inter-assay CV were 8.1 and 8.7% respectively. FSH concentration on day 2 and day 10 were added together to give combined FSH concentration. CCCT was considered to be abnormal when either basal or stimulated FSH concentration was. 10 IU/l. Statistical analysis The correlation coefficient between AFC and the number of oocytes obtained in the previous study (Ng et al., 2000b) was Assuming that AFC and combined FSH concentration had similar correlation coefficients, the sample size required would be 107 to give a test significance of 0.01 and a power of 0.9 (Sigmastat; Jandel Scientific, USA).

3 CCCT in predicting ovarian response The primary outcome measure was the number of oocytes obtained and the secondary outcome measures included the serum E 2 concentration on the day of hcg, the dosage and duration of hmg. Statistical tests were carried out by Friedman, Mann Whitney U-tests, x 2 and Fisher s exact tests, where appropriate. Multiple regression analysis with the least-squares regression was applied to evaluate the predictive values of different parameters on the ovarian response. Correlation was assessed by the Spearman rank method. Two-tailed P, 0.05 was taken as significant. Results A total of 300 eligible women undergoing the first IVF cycle during the study period was approached and 213 women agreed to participate in the study. After the first ultrasound examination, 68 women were excluded from the study: poor visualization of ovaries in 14 women, an ovarian cyst in 28 women and polycystic ovaries in 26 women. Five women did not return for the assessment on day 10 because they forgot to take clomiphene citrate. Two women became pregnant after taking clomiphene citrate and five women postponed their IVF treatment for personal reasons. Therefore, 131 women underwent ovarian stimulation and were included in the final analysis: 38 tubal factors; 15 endometriosis; 64 male infertility; eight unexplained and six mixed causes. Table I summarizes the demographic data and ovarian response. Four cycles did not proceed to oocyte retrieval because of premature luteinization in two cycles and absent follicular development in another two cycles. These patients were considered to have no oocyte obtained and oocytes were obtained in all planned retrievals. Failed fertilization was encountered in four cycles and in another three cycles Table I. Summary of demographic data and ovarian responses Parameters Median (inter-quartile range) Age (years) 35 (32 37) Duration of infertility (years) 4.0 (3 6) Primary/secondary infertility a 89/42 Body mass index (kg/m 2 ) 21.5 ( ) hmg dosage (IU) 1950 ( ) hmg duration (days) 11.0 (10 13) Total no. of oocytes obtained 8.0 (4 12) Estradiol on hcg day (pmol/l) 9,303 ( ) a Given as number of patients with primary/secondary infertility. embryos failed to cleave. Embryo transfer was postponed in three cycles because of the risk of OHSS. Embryo transfer was performed in 117 cycles and 31 clinical pregnancies resulted. The pregnancy rate was 23.7% per initiated cycle and 26.5% per transfer. A paired analysis revealed no significant difference between basal (median: 11.0; range: 1 21), stimulated (median: 12.0; range: 3 28) and down-regulated (median: 11.0; range: 3 29) AFC (Figure 1). Basal AFC was negatively correlated with age of women (r ¼ ; P, 0.001), basal FSH concentration (r ¼ ; P, 0.001) and combined FSH concentration (r ¼ ; P ¼ 0.004) but positively correlated with the number of oocytes aspirated (r ¼ 0.402; P, 0.001) and serum E 2 on hcg day (r ¼ 0.327; P, 0.001). Basal FSH concentration was positively correlated with age of women (r ¼ 0.202; P ¼ 0.021) and stimulated FSH concentration (r ¼ 0.348; P, 0.001) but was negatively correlated with the number of oocytes aspirated (r ¼ ; P, 0.001) and serum E 2 on Figure 1. Box plot of antral follicle count determined in the early follicular phase of a spontaneous period (basal), day 10 after the clomiphene citrate test (stimulated) and prior to ovarian stimulation following pituitary down-regulation (down-regulated). 1649

4 E.H.Y.Ng et al. hcg day (r ¼ ; P ¼ 0.001). Stimulated FSH concentration was positively correlated with hmg duration (r ¼ 0.3; P, 0.001) and hmg dosage (r ¼ 0.328; P, 0.001) but was negatively correlated with the number of oocytes obtained (r ¼ ; P, 0.001), the number of oocytes obtained (r ¼ ; P, 0.001) and serum E 2 on hcg day (r ¼ ; P ¼ 0.001). Combined FSH concentration was negatively correlated with basal AFC (r ¼ ; P ¼ 0.004), the number of oocytes obtained (r ¼ ; P, 0.001) and serum E 2 on hcg day (r ¼ ; P ¼ 0.001) but was positively correlated with hmg duration (r ¼ 0.313; P, 0.001) and hmg dosage (r ¼ 0.278; P, 0.001). Age of women, BMI, basal AFC, basal/stimulated and combined FSH concentrations were entered in a stepwise fashion in the multiple regression analysis using the number of oocytes obtained as the dependent variable with a constant included in the equation. Basal AFC had the largest R 2 change, which was followed by combined FSH concentration and age of women (Table II). When these parameters were entered in a stepwise fashion in the multiple regression analysis using serum E 2 concentration on the day of hcg as the dependent variable, basal AFC had the largest R 2 change, which was followed by combined FSH concentration (Table III). When these parameters were entered in a stepwise fashion in the multiple regression analysis with the dosage of hmg used as the dependent variable, stimulated FSH concentration was the only predictive parameter and all other parameters were excluded from the equation (Table IV). Abnormal CCCT was encountered in 16 patients: basal FSH concentration. 10 IU/l in eight patients and stimulated FSH concentration. 10 IU/l in 13 patients. Eight patients with basal FSH concentration, 10 IU/l had stimulated FSH concentration. 10 IU/l. There were no significant differences in age of women, primary/secondary infertility, cause of infertility, duration of infertility, BMI, dosage/duration of hmg between women with normal and abnormal CCCT (Table V). Those with abnormal CCCT had significantly lower basal AFC, serum E 2 concentration on the day of hcg and lower number of oocytes aspirated. Similar number of embryos transferred, pregnancy and implantation rates, multiple pregnancy rates and the pregnancy outcome were found in women with normal and abnormal CCCT (Table VI). Discussion The present study compared AFC in the early follicular phase, after CCCT and before ovarian stimulation following pituitary down-regulation. In the majority of relevant studies, AFC was determined after pituitary down-regulation and prior to ovarian stimulation in the treatment cycle. The effects of pituitary down-regulation on AFC are still controversial in the literature, despite extensive use of GnRHa for pituitary down-regulation in IVF cycles. Sharara et al. (1999) and Hansen et al. (2003) showed that AFC did not change after pituitary down-regulation, whereas a significant decrease in AFC was observed after pituitary down-regulation by Järvelä et al. (2003). In another group of patients, we have recently demonstrated that AFC, ovarian volume and ovarian power Doppler flow indices measured by threedimensional ultrasound did not significantly change after pituitary down-regulation, both in patients with normal ovaries and with polycystic ovaries (Ng et al., 2004). In the present study, we could not demonstrate any significant difference between basal and down-regulated AFC. Huang et al. (2001) demonstrated that AFC determined on day 6 or 7 after gonadotrophin stimulation was predictive of the ovarian response. Similarly, the combination of AFC on day 3 and day 7 had high positive and negative predictive values of ovarian response during IVF treatment (Durmusoglu et al., 2004). Both clomiphene citrate and gonadotrophins stimulate the growth of antral follicles and it is logical to postulate that CCCT might improve the assessment of AFC. To the best of our knowledge, this is the first Table II. Multiple regression analysis evaluating the values of different parameters in predicting the number of oocytes obtained B (95% CI) b R 2 change P Constant (13.319, ) Basal antral follicle count (0.134, 0.536) Combined FSH (20.570, ) Age (20.674, ) Body mass index, basal and stimulated FSH concentrations were excluded in the equation. Total number of oocytes ¼ þ basal AFC combined FSH concentration age. R ¼ Adjusted R 2 ¼ Table III. Multiple regression analysis evaluating the values of different parameters in predicting serum estradiol concentrations on the day of hcg B (95% CI) b R 2 change P Constant ( , ) Basal antral follicle count ( , ) Combined FSH ( , ) Age, body mass index, basal and stimulated FSH concentrations were excluded in the equation. Serum estradiol concentration ¼ þ basal AFC combined FSH concentration. R ¼ Adjusted R 2 ¼

5 CCCT in predicting ovarian response Table IV. Multiple regression analysis evaluating the values of different parameters in predicting the dosage of hmg used B (95% CI) b R 2 change P Constant (19.579, ) Stimulated FSH (0.133, 1.161) Age, body mass index, basal/stimulated and combined FSH concentrations were excluded in the equation. hmg dosage ¼ þ stimulated FSH concentration. R ¼ Adjusted R 2 ¼ study comparing AFC determined in the early follicular phase and after CCCT. Our result indicated that there was again no difference between basal and stimulated AFC. Taking these results together, it can be concluded that the assessment of AFC for the prediction of ovarian response can be performed in the follicular phase either before or after pituitary down-regulation, and there is no additional advantage to count AFC again after CCCT. Basal FSH concentration measured prior to the treatment cycle is widely used in many IVF programmes. A metaanalysis (Bancsi et al., 2003) showed that the performance of basal FSH concentration for predicting poor response was moderate and the performance for predicting no pregnancy was poor. Screening for elevated FSH concentrations is of no additional value in the prediction of fecundity in a general subfertility population with ovulatory menstrual cycles (van Montfrans et al., 2000). Therefore, a challenge test such as CCCT will be able to identify more women with impaired ovarian reserve than basal FSH screening alone. Women with abnormal CCCT were more likely to have poor ovarian response, higher cycle cancellation and reduced pregnancy rates (Loumaye et al., 1990; Tanbo et al., 1992; Hofmann et al., 1996; Kahraman et al., 1997; Gülekli et al., 1999; Van der Stege and Van der Linden, 2001; Csemiczky et al., 2002; Yanushpolsky et al., 2003; Hendriks et al., 2005). The utility of CCCT has also been validated in women among general infertile patients not receiving assisted reproduction methods (Scott et al., 1993, 1995). Many studies on the prediction of ovarian response are retrospective in nature and patients received different stimulation regimens and different starting dose of gonadotrophins. Moreover, various factors affecting ovarian response such as age of women, BMI and AFC were not considered at the same time. In order to avoid the confounding variables, the study group consisted of women undergoing their first IVF cycle and receiving the same starting dose of gonadotrophin after a standard protocol of pituitary down-regulation for ovarian stimulation. All patients receiving ovarian stimulation would be offered oocyte collection, unless there was no follicular development after 14 days of stimulation. A multiple regression analysis was also applied to compare the values of these factors in the prediction of ovarian response. Ovarian response was represented by the number of oocytes aspirated, serum E 2 on the day of hcg, and the duration and dosage of hmg, because we found that these measures are important in the counselling of patients undergoing IVF treatment. It is important to highlight that we examined a group of infertile women having relatively normal ovarian reserve as suggested by age,40 years at the time of treatment and basal FSH concentration, 10 IU/l. Those patients who did not satisfy these criteria could have self-funded treatment cycles in a private unit. In the present study, we showed that AFC achieved the best predictive value in relation to the number of oocytes aspirated, followed by combined FSH concentration and age of women. The result indicated that combined FSH concentration was a better predictor of the number of oocytes than either basal or stimulated FSH concentration. Unlike our previous finding (Ng et al., 2000b), BMI was not taken into the equation when combined FSH concentration was used. Both basal AFC and combined FSH concentration were predictive factors of serum E 2 concentration on the day of hcg, whereas stimulated FSH concentration was predictive of Table V. Comparison of demographic data and ovarian responses between women with normal and abnormal clomiphene citrate challenge test (CCCT) Normal CCCT Abnormal CCCT P (n ¼ 115) (n ¼ 16) Age of women (years) 35.0 ( ) 35.0 ( ) NS Primary/secondary infertility a 77/38 12/4 NS Causes of infertility a NS Tubal 38 0 Male 56 8 Endometriosis 11 4 Unexplained 5 3 Mixed 5 1 Infertility duration (years) 4.0 ( ) 4.0 ( ) NS Body mass index (kg/m 2 ) 21.5 ( ) 21.0 ( ) NS Basal antral follicle count 12.0 ( ) 7.0 ( ) hmg dosage (IU) 1950 ( ) 2025 ( ) NS hmg duration (days) 11.0 ( ) 11.5 ( ) NS Serum estradiol b (pmol/l) 9578 ( ) 5538 ( ) No. of oocytes obtained 8.0 ( ) 4.0 ( ) Data given as median (range). a No. of patients. b On the day of hcg. NS ¼ not significant. 1651

6 E.H.Y.Ng et al. Table VI. Comparison of treatment outcomes between women with normal and abnormal clomiphene citrate challenge test (CCCT) Normal CCCT Abnormal CCCT (n ¼ 115) (n ¼ 16) No. of embryos replaced Zero 9 (7.8) 5 (31.2%) One 12 (10.4) 2 (12.5) Two 88 (76.5) 7 (43.8) Three 6 (5.3) 2 (12.5) Pregnancy rate/cycle 27/115 (23.8) 4/16 (25.0) Pregnancy rate/transfer 27/106 (25.5) 4/11 (36.4) Implantation rate 33/206 (16.0) 4/22 (18.2) Multiple pregnancy rate 7/27 (25.9) 0/4 (0) Pregnancy outcome First trimester miscarriage 7 (25.9) 0 Ectopic 2 (7.4) 0 Ongoing pregnancy 18 (66.7) 4 (100) Data given as number (%). No differences were significant. the total dosage of hmg used. We could not find any predictive factor for the duration of hmg. Serum inhibin B concentration was not measured in this study. There is still controversy in the literature as to whether inhibin B is a useful marker for ovarian reserve. Seifer et al. (1997), Tinkanen et al. (1999) and Dzik et al. (2000) reported that women with inhibin B concentration, 45 pg/ml demonstrated a poor ovarian response during ART than those $45 pg/ml, whereas others (Hall et al., 1999; Corson et al., 1999; Creus et al., 2000) found no value in the prediction of ovarian response or pregnancy rate. Reduced production of inhibin B by granulosa cells of developing follicles during CCCT is responsible for the elevated FSH concentration in those with abnormal CCCT (Hofmann et al., 1998). Serum anti-müllerian hormone appears to be an early marker for ovarian reserve (Seifer et al., 2002; van Rooij et al., 2002; Fanchin et al., 2003) but unfortunately was not checked in the present study. There is still no consensus on threshold values for abnormal CCCT. We considered CCCT abnormal when either basal or stimulated FSH concentration was.10 IU/l. Such a definition was adopted by many groups (Kahraman et al., 1997; Gülekli et al., 1999; Van der Stege and Van der Linden, 2001; Csemiczky et al., 2002; Yanushpolsky et al., 2003) but other threshold values included stimulated FSH concentration. 26 IU/l (Navot et al., 1987) and. 12 IU/l (Tanbo et al., 1992) and combined FSH concentration. 26 IU/l (Loumaye et al., 1990),. 12 IU/l (Scott et al., 1995),.16 IU/l (Hofmann et al., 1996). In the present study, women with abnormal CCCT had significantly lower AFC, E 2 concentration on the day of hcg and the number of oocytes obtained but similar pregnancy and implantation rates, when compared with those with normal CCCT. Our results were contradictory to those of others (Navot et al., 1987; Hofmann et al., 1996; Csemiczky et al., 2002; Hicks et al., 2003; Yanushpolsky et al., 2003) and did not support the conclusion in a meta-analysis that an abnormal CCCT result virtually confirmed that pregnancy would not occur with IVF treatment (Jain et al., 2004). In this study, CCCT was applied as a secondary screening test to women who had 1652 relatively normal ovarian reserve, i.e., 40 years old and serum FSH concentration on recruitment, 10 IU/l. Women with normal CCCT had more oocytes obtained, and pregnancy rates from frozen thawed embryo transfer cycles were not taken into account. Moreover, implantation and pregnancy rates of patients with poor ovarian responses were similar to those with normal response (Lashen et al., 1999; Biljan et al., 2000). It is well known that basal FSH concentrations may vary from cycle to cycle (Scott and Hofmann, 1995). Significant cycle-to-cycle variability of the CCCT was also reported (Hannoun et al., 1998; Kwee et al., 2003). These may lead to variable results in the prediction of the ovarian response. On the other hand, there is only moderate intercycle variability in AFC and the pooled SD for intercycle variability up to a mean AFC of 15 was 3.0 (Hansen et al., 2003). In conclusion, there was no significant difference between basal, stimulated and down-regulated AFC. AFC achieved the best predictive value in relation to the number of oocytes aspirated, followed by combined FSH concentration and age of women. Both basal AFC and combined FSH concentration were predictive factors of serum E 2 concentration on the day of hcg while stimulated FSH concentration was predictive of the total dosage of hmg used. Women with abnormal CCCT had significantly lower AFC, E 2 concentration on the day of hcg and the number of oocytes obtained but comparable pregnancy and implantation rates, when compared with those with normal CCCT. Although CCCT may add some information on the prediction of ovarian response, it may not be justified to screen our patients by CCCT, as only eight patients (6.1%) with normal basal FSH concentration were found to have abnormal CCCT and the pregnancy rate was not affected by the abnormal CCCT result. Acknowledgements This study was funded by the Hong Kong Research Grant Council (HKU 7280/01M). References Aboulghar MA and Mansour RT (2003) Ovarian hyperstimulation syndrome: classifications and critical analysis of preventive measures. Hum Reprod Update 9, Adams J, Polson DW and Franks S (1986) Prevalence of polycystic ovaries in women with anovulation and idiopathic hirsutism. Br Med J 293, Bancsi LF, Broekmans FJ, Eijkemans MJ, de Jong FH, Habbema JD and te Velde ER (2002) Predictors of poor ovarian response in in vitro fertilization: a prospective study comparing basal markers of ovarian reserve. Fertil Steril 77, Bancsi LF, Broekmans FJ, Mol BW, Habbema JD and te Velde ER (2003) Performance of basal follicle-stimulating hormone in the prediction of poor ovarian response and failure to become pregnant after in vitro fertilization: a meta-analysis. Fertil Steril 79, Biljan MM, Buckett WM, Dean N, Phillips SJ and Tan SL (2000) The outcome of IVF-embryo transfer treatment in patients who develop three follicles or less. Hum Reprod 15, Cahill DJ, Prosser CJ, Wardle PG, Ford WC and Hull MG (1994) Relative influence of serum follicle stimulating hormone, age and other factors on ovarian response to gonadotrophin stimulation. Br J Obstet Gynaecol 101,

7 CCCT in predicting ovarian response Chang MY, Chiang CH, Hsieh TT, Soong YK and Hsu KH (1998) Use of the antral follicle count to predict the outcome of assisted reproductive technologies. Fertil Steril 69, Corson SL, Gutmann J, Batzer FR, Wallace H, Klein N and Soules MR (1999) Inhibin-B as a test of ovarian reserve for infertile women. Hum Reprod 14, Creus M, Peñarrubia J, Fábregues F, Vidal E, Carmona F, Casamitjana R, Vanrell JA and Balasch J (2000) Day 3 serum inhibin B and FSH and age as predictors of assisted reproduction treatment outcome. Hum Reprod 15, Csemiczky G, Harlin J and Fried G (2002) Predictive power of clomiphene citrate challenge test for failure of in vitro fertilization treatment. Acta Obstet Gynecol Scand 81, Durmusoglu F, Elter K, Yoruk P and Erenus M (2004) Combing cycle day 7 follicle count with the basal antral follicle count improves the prediction of ovarian response. Fertil Steril 81, Dzik A, Lambert-Messerlian G, Izzo VM, Soares JB, Pinotti JA and Seifer D (2000) Inhibin B response to EFORT is associated with the outcome of oocyte retrieval in the subsequent in vitro fertilization cycle. Fertil Steril 74, Engmann L, Sladkevicius P, Agrawal R, Bekir JS, Campbell S and Tan SL (1999) Value of ovarian stromal blood flow velocity measurement after pituitary suppression in the prediction of ovarian responsiveness and outcome of in vitro fertilization treatment. Fertil Steril 71, Fanchin R, Schonäuer LM, Righini C, Guibourdenche J, Frydman R and Taieb J (2003) Serum anti-müllerian hormone is more strongly related to ovarian follicular status than serum inhibin B, estradiol, FSH and LH on day 3. Hum Reprod 18, Frattarelli JL, Lauria-Costa DF, Miller B, Bergh PA and Scott RT Jr (2000) Basal anthral follicle number and mean ovarian diameter predict cycle cancellation and ovarian responsiveness in assisted reproductive technology cycles. Fertil Steril 74, Gülekli B, Bulbul Y, Onvural A, Yorukoglu K, Posaci C, Demir N and Erten O (1999) Accuracy of ovarian reserve tests. Hum Reprod 14, Hall JE, Welt CK and Cramer DW (1999) Inhibin A and inhibin B reflect ovarian function in assisted reproduction but are less useful at predicting outcome. Hum Reprod 14, Hannoun A, Abu Musa A, Awwad J, Kaspar H and Khalil A (1998) Clomiphene citrate challenge test: cycle to cycle variability of cycle day 10 follicle stimulating hormone level. Clin Exp Obstet Gynecol 25, Hansen KR, Morris JL, Thyer AC and Soules MR (2003) Reproductive ageing and variability in the ovarian antral follicle count: application in the clinical setting. Fertil Steril 80, Hendriks DJ, Broekmans FJ, Bancsi LF, de Jong FH, Looman CW and te Velde ER (2005) Repeated clomiphene citrate challenge testing in the prediction of outcome in IVF: a comparison with basal markers for ovarian reserve. Hum Reprod 20, Hicks ABT, Fox MD, Sanchez-Ramos L, Kaunitz A and Freeman MF (2003) Clinical characteristics of patients with an abnormal clomiphene citrate challenge test. Am J Obstet Gynecol 189, Hofmann GE, Sosnowski J, Scott RT Jr and Thie J (1996) Efficacy of selection criteria for ovarian reserve screening using the clomiphene citrate challenge test in a tertiary fertility center population. Fertil Steril 66, Hofmann GE, Danforth DR and Seifer DB (1998) Inhibin-B: the physiological basis of the clomiphene citrate challenge test for ovarian reserve screening. Fertil Steril 69, Hsieh YY, Chang CC and Tsai HD (2001) Antral follicle counting in predicting the retrieved oocyte number after ovarian hyperstimulation. J Assist Reprod Genet 18, Huang FJ, Chang SY, Tsai MY, Kung FT, Wu JF and Chang HW (2001) Determination of the efficiency of controlled ovarian hyperstimulation in the gonadotropin-releasing hormone agaonist-suppresion cycle using the initial follicle count during gonadotropin stimulation. J Assist Reprod Genet 18, Jain T, Soules MR and Collins JA (2004) Comparison of basal follicle-stimulating hormone versus the clomiphene citrate challenge test for ovarian reserve screening. Fertil Steril 82, Järvelä IY, Sladkevicius P, Kelly S, Ojha K, Campbell S and Nargund G (2003) Effect of pituitary down-regulation on the ovary before in vitro fertilization as measured using three-dimensional power Doppler ultrasound. Fertil Steril 79, Kahraman S, Vicdan K, Işik AZ, Őzgün OD, Alaybeyoğlu L, Polat G and Biberoğlu K (1997) Clomiphene citrate challenge test in the assessment of ovarian reserve before controlled ovarian hyperstimulation for intracytoplasmic sperm injection. Eur J Obstet Gynecol 73, Keay SD, Liversedge NH, Mathur RS and Jenkins JM (1997) Assisted conception following poor ovarian response to gonadotrophin stimulation. Br J Obstet Gynecol 104, Kupesic S and Kurjak A (2002) Predictors of IVF outcome by three-dimensional ultrasound. Hum Reprod 17, Kupesic S, Kurjak A, Bjelos D and Vujisic S (2003) Three-dimensional ultrasonographic ovarian measurements and in vitro fertilization outcome are related to age. Fertil Steril 79, Kwee J, Elthing MW, Schats R, Bezemer PD, Lambalk CB and Schoemaker J (2003) Comparison of endocrine tests with respect to their predictive value on the outcome of ovarian hyperstimulation in IVF treatment: results of a prospective randomized study. Hum Reprod 18, Lashen H, Ledger W, Lopez-Bernal A and Barlow D (1999) Poor responders to ovulation induction: is proceeding to in-vitro fertilization worthwhile? Hum Reprod 14, Lass A, Skull J, McVeigh E, Margara R and Winston RM (1997) Measurement of ovarian volume by transvaginal sonography before ovulation induction with human menopausal gonadotrophin for in-vitro fertilization can predict poor response. Hum Reprod 12, Loumaye E, Billion JM, Mine JM, Psalti I, Pensis M and Thomas K (1990) Prediction of individual response to controlled ovarian hyperstimulation by means of a clomiphene citrate challenge test. Fertil Steril 53, Nahum R, Shifren JL, Chang YC, Leykin L, Isaacson K and Toth T (2001) Antral follicle assessment as a tool for predicting outcome in IVF is it a better predictor than age and FSH? J Assist Reprod Genet 18, Navot D, Roisenwaks Z and Margalioth EJ (1987) Prognostic assessment of female fecundity. Lancet 2, Ng EHY, Yeung WSB, Lau EYL, So WWK and Ho PC (2000a) High serum oestradiol levels in fresh IVF cycles do not impair implantation and pregnancy rates in subsequent FET cycles. Hum Reprod 15, Ng EHY, Tang OS and Ho PC (2000b) The significance of the number of antral follicles prior to stimulation in predicting ovarian responses in an in-vitro fertilization programme. Hum Reprod 15, Ng EHY, Chan CCW, Tang OS, Yeung WSB and Ho PC (2004) Effect of pituitary downregulation on antral follicle count, ovarian volume and stromal blood flow measured by three-dimensional ultrasound with power Doppler prior to ovarian stimulation. Hum Reprod, in press. Popovic-Todorovic B, Loft A, Lindhard A, Bangsboll S, Andersson AM and Andersen AN (2003) A prospective study of predictive factors of ovarian response in standard IVF/ICSI patients treated with recombinant FSH. A suggestion for a recombinant FSH dosage normogram. Hum Reprod 18, Scott RT, Leonardi MR, Hofmann GE, Illions EH, Neal GS and Navot D (1993) A prospective evaluation of clomiphene citrate challenge test screening of the general infertility population. Obstet Gynecol 82, Scott RT Opsahl MS, Leonardi MR, Neall GS, Illions EH and Davot D (1995) Life table analysis of pregnancy rates in a general infertility population relative to ovarian reserve and patient age. Hum Reprod 10, Scott RT Jr and Hofmann GE (1995) Prognostic assessment of ovarian reserve. Fertil Steril 63,1 14. Seifer DB, Lambert-Messerlian G, Hogan JW, Gardiner AC, Blazar AS and Berk CA (1997) Day 3 serum inhibin-b is predictive of assisted reproductive technologies outcome. Fertil Steril 67, Seifer DB, MacLaughin DT, Christian BP, Feng B and Shelden RM (2002) Early follicular serum müllerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles. Fertil Steril 77, Sharara FI, Scott RT Jr and Seifer DB (1998) The detection of diminished ovarian reserve in infertile women. Am J Obstet Gynecol 179, Sharara FI, Lim J and McClamrock HD (1999) The effect of pituitary desensitization on ovarian volume measurements prior to in-vitro fertilization. Hum Reprod 14, Sharif K, Elgendy M, Lashen H and Afnan M (1998) Age and basal follicle stimulating hormone as predictors of in vitro fertilisation outcome. Br J Obstet Gynecol 105, Syrop CH, Willhoite A and Van Voorhis BJ (1995) Ovarian volume: a novel outcome predictor for assisted reproduction. Fertil Steril 64, Syrop CH, Dawson JD, Husman KJ, Sparks AE and Van Voorhis BJ (1999) Ovarian volume may predict assisted reproductive outcome better than 1653

8 E.H.Y.Ng et al. follicle stimulating hormone concentration on day 3. Hum Reprod 14, Tanbo T, Dale PO, Lunde O, Norman N and Åbyholm T (1992) Prediction of response to controlled ovarian hyperstimulation: a comparison of basal and clomiphene citrate-stimulated follicle-stimulating hormone levels. Fertil Steril 57, Tarlatzis BC, Zepiridis L, Grimbizis G and Bontis J (2003) Clinical management of low ovarian response to stimulation for IVF: a systematic review. Hum Reprod Update 9, Tinkanen H, Bläuer M, Laippala P, Tuohimaa P and Kujansuu E (1999) Prognostic factors in controlled ovarian hyperstimulation. Fertil Steril 72, Tomás C, Nuojua-Huttunen S and Martikainen H (1997) Pretreatment transvaginal ultrasound examination predicts ovarian responsiveness to gonadotrophins in in-vitro fertilization. Hum Reprod 12, Van der Stege JG and Van der Linden PJQ (2001) Useful predictors of ovarian stimulation response in women undergoing in vitro fertilization. Gynecol Obstet Invest 52, Van Montfrans JM, Hoek A, van Hooff MHA, de Koning CH, Tonch N and Lambalk CB (2000) Predictive value of basal follicle-stimulating hormone concentrations in a general subfertility population. Fertil Steril 74, Van Rooij IAJ, Broekmans FJM, te Velde ER, Fauser BCJM, Bancsi LFJMM, de Jong FH and Themmen APN (2002) Serum anti-müllerian hormone levels: a novel measure of ovarian reserve. Hum Reprod 17, Yanushpolsky EH, Hurwitz S, Tikh E and Racowsky C (2003) Predictive usefulness of cycle day 10 follicle-stimulating hormone level in a clomiphene citrate challenge test for in vitro fertilization outcome in women younger than 40 years of age. Fertil Steril 80, Zaidi J, Barber J, Kyei-mensah A, Bekir J, Campbell S and Tan SL (1996) Relationship of ovarian stromal blood flow at the baseline ultrasound scan to subsequent follicular response in an in vitro fertilization program. Obstet Gynecol 88, Submitted on December 6, 2004; resubmitted on January 31, 2005; accepted on February 10,