International Journal of

     Introduction
Assisted reproduction and development of in-vitro fertilization (IVF) techniques have revolutionized the treatment of infertility. Because IVF technique is expensive and not completly successful, many researches attempted to determine predictive factors for more successful outcome. Prognostic assessment of ovarian reserve has relied upon indirect markers of ovarian function, such as age (1-3), Follicle Stimulating Hormone (FSH) at baseline (4-7), and estradiol    concentrations (8,9). The  reduction  of  ovarian function or “reserve” is apparantly due to reduced number of ovarin premordial follicles, from over 250.000 at menarche  to  very few  at the  end  of reproductive age. Age and regularity of menses alone are unreliable predictors of ovarian reserve. Neither folliculaor phase FSH nor estradiol concontrations, could finally indicate that ovarian function is normal and unimpaired (1-3). Ovarian volume has also been proposed as a predictor of ovarian response but there were still a substantial number of pregnancies amoung the women with very small ovarian volumes (10,11). In theory, the direct products of granulosa cells might better reflect ovarian secretory capacity and follicle number. Inhibin-B is one of these products which regulates FSH secretion by negative feedback (11-17). The aging of ovary is accompanied by a decrease in inhibin-B secretion (18,19). Early follicular phase serum inhibin-B may be a suitable marker of ovarian follicle reserve and fertility potential (20-24). However, several studies found no or limited clinical value in measuring basal early follicular inhibin-B regarding to ART outcome (25-33).
     According to these contraversial data and in order to test the hypothesis that baseline inhibin-B concentration would serve as improved marker of IVF outcome, we examined baseline inhibin-B on day 3, prior to ovarian stimulation and compared it with standard markers of ART outcome.
 
Materials and Methods
 
     From April 2004 until June 2005, seventy one women undergoing IVF/ICSI treatment were included in this study in the Fatemieh Infertility Research Center. Our inclusion criteria were 1) age under 40 years old, 2) basal FSH level under 15 mIU/mL 3) presence of both ovaries, 4) no evidence of endocrine disorders (normal levels of thyroid-stimulating hormone, testostrone, androstendione, and prolactine), 5) no evidence of ovarian cyst bigger than 2cm in diameter, and 6) written informed consent. All patients received oral contraceptive pills (LD) from the 5th day of their previous menstrual cycle then administration of GnRH agonist (Suprefact, Hoechst, Germany), 500 g/day, was begun on the 19th day. After menstruation, ovarian volumes were calculated as the volume of an elliposoid (length× width× depth× /6) by transvaginal ultrasound (6.5 MHZ, Dynamic immaging) on day 3. If no follicular cysts larger than 12mm in diameter was detected, Busereline was reduced to 200 g/day and gonadotropin (Gonal F, Serono, Swiss) was started i.m. daily, depending on age (150 IU in >35 years old and 225 IU in <35 years old). Whenever follicular cysts bigger than 12mm were seen, estradiol was checked and if it was50 pg/ml, then the administration of gonadotropin was started. The dose of the gonadotropin was changed according to the folliclular growth. When more than 2 follicles bigger than or equal to 18mm were seen, HCG (Pregnyle, Organon, Germany) 10000 IU were injected to induce final oocyte maturation and 36 hours later, ovum was picked up. After 3 days if fertilization occured, embryo was transfered. Poor response was defined when fewer than four follicules at retrival were collected. Pregnancy was diagnosed by increasing concentrations of ß-HCG 2 weeks after embryo transfer and the subsequent demonstration of an intrauterine gestational sac by transvaginal scan 2 weeks later.
 
     Hormone analyses
     FSH and stradiol concentrations were measured at diagnostic laboratory of the Fatemieh Hospital using routine procedures. To detect Inhibin-B, all of collected sera immediately were freozen and stored at –80°C until assay. Inhibin-B concentration in serum was measured by Inhibin-B assay kit purchased from Serotec (Oxford, UK), using specific two-site enzyme immunoassay. Briefly, standards and samples were diluted as appropriate and mixed with a half volume of distilled water containing 10% sodium dodecyl sulphate (SDS). After 3 min at 100°C, tubes were cooled before adding freshly prepared hydrogen peroxide solution. After additional incubation at room temperature, duplicate aliquots of denatured and oxidized samples and standards were transferred to antibody-coated microtitre plates, that were incubated at room temperature, overnight. After washing with enzyme immunoassay (EIA) wash buffer [0.1 mol/l Tris–HCl, 0.15 mol/l NaCl, 10% (w/v) bovine serum albumin, 5% (v/v) Triton X-100, and 0.1% (w/v) sodium azide, pH 7.5], 50 µl alkaline phosphatase-conjugated mouse anti-human inhibin-α subunit antibody was used. The plates were then incubated for 3 hours. Plates were washed and bound alkaline phosphatase was quantified using a commercially available enzyme immunoassay amplification system (ELISA), which was used according to the supplier's instructions. The inhibin-B plates were read at 490 nm on an automated EIA plate reader (BRIO: Basic Radium Immunoassay Operator, Radim spa, Pomezia, Italy). The assay detection limit for inhibin-B was less than 10 pg/ml. Within and between plate coefficients of variation were <5.0 and 9.0% respectively. Cross-reactions for each assay with the various inhibin-related proteins were <0.5%.
 
     Statistical analysis
     Statistical analysis was performed using the commericially available software package SPSS version 11. Student’s t-test and ² were used for analysis and results were reported as mean  SD. p<0.05 was considered as significant level.
 
Results
 
     Amoung 71 patients, 11 cases were excluded from the study because of discontinuation of the cycle. The mean age of the women was 29 years (range 20-39). The casuses of infertility were: male factor infertility (40%), tubal infertility (13.3%), ovulatory infertility (18.3%), compound infertility (25%) and unexplained infertility (3.3%).
     According to the number of retrieved oocytes, the patients were divided into two groups; 12 poor responders (less than 4 oocyte),  and 48 normal responders (≥4 oocytes).
     Demographic characteristitcs, pretreatment hormonal profiles and results of two groups are shown on table I. Total pregnancy rate was 20%. There were no statistically significant differences in mean of age, mean of duration of infertility, type of infertility,  mean of  day 3 FSH, E₂ and  inhibin-B levels, total ovarian volume, number of ampoulses and duration of the ovarian stimulation in two groups. As expected, fertilization rate and clinical pregnancy rate are significantly reduced in poor responders. There was no significant differences in the mean of inhibin-B levels between two groups.
     According to inhibin-B levels, the patients were divided into two groups; inhibin-B concentrations <70 pg/ml (14 patients) and inhibin-B concentrations >70 pg/ml (46 patients) (11). Demographic characteristitcs, pretreatment hormonal profiles and results of  the two groups are shown on table II. There were no statistically significant differences in FSH and estradiol on day 3 between two groups. However, the number of retrieved oocytes in the patients with inhibin-B <70 pg/ml was lower than this number in the other group but this difference was not statistically significant. Correlation coefficients were determined between the number of oocytes retrieved and serum inhibin-B levels on day 3 administration, and significant correlations (r=0.358)  was found between them (fig.1).





Disscusion
 
     The most useful predictive information for an infertile couple should be obtained before beginning assisted reproductive techniques. Basal inhibin concentrations have been evaluated previously as predictive markers for pregnancy in IVF cycles. In two (12,24) of three (13) previous studies, higher inhibin-B on day 2–3 was associated with a greater number of oocytes retrieved in IVF cycles (24) and with subsequent pregnancy (12) which was better predictor of the response to exogenous gonadotrophins than age (24) and was equivalent to day 3 FSH.
     Seifer et al (20) demonstrated that, pregnancy rates were higher in patients with day 3 inhibin-B concentrations of over 45 pg/ml than this rate in patients with lower values. In 1999, they compared 109 normal responders with 47 poor responders and they did not find any significant difference in day 3 FSH leveles between the two groups, but poor responders had significantly lower day 3 inhibin-B leveles (21). The study of Hall et al (27) did not support the use of day 3 inhibin-B as a predictive marker of IVF outcome. In addition, base line FSH, E₂ and inhibin-B were not significantly different between pregnant and non pregnant patients. In the present study the mean inhibin-B in normal responders was 166.9141 pg/ml versus 115.887 pg/ml in poor responders, which the difference was not statistically significant (p= 0.24). In addition, there were no statistically significant differences in day 3 serum FSH, estradiol and ovarian volume between the two groups. A combination of FSH higher than the median value (6.51 mIU/ml) and inhibin-B lower than the median value (115.8 pg/ml) was not seen in poor responders. A cut off of serum inhibin-B conceantrations between normal and poor responders was calculated (cut- offs 45-70-100-120-150 pg/ml), but these cut-offs were not statistically significant.
     There was a positive correlation between inhibin-B level on day 3 and  retrived oocyte number but there was not any correlation between inhibin-B level on day 3 and ART outcome. There was tremendous overlap in baseline inhibin-B concentrations between pregnant and non-pregnant subjects, and inhibin-B alone failed to predict pregnancy.The results of this study confirm Hall et al findings (27) and are contraversal with Tsuchia et al (12)  and Seifer et al (20) results.
 
Conclusion
 
     The present  study shows that inhibin-B concentration on day 3 positevly correlate with the number of oocytes retrieved during ART. Our results do not support the use of day 3 inhibin-B as a predictive marker of IVF outcome.
 
Acknowledgment
 
     The authors wish to express thank to Mr. Mani Kashani, and other staff and personel of Fatemieh Infertility Research Center, specially Mrs. Ramazani for their kind assistance in this study.