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Iranian Journal of Reproductive Medicine, Vol. 4, No.1, May 2006, pp. 1-5 Day 3 serum inhibin-B level is not predictive of ovarian assisted reproductive technologies outcome Marzieh Farimani1, M.D., Iraj Amiri2,Ph.D., Sedigheh Hoseini1, M.D.1 Department of
Gynecology & Obstetrics, Facculty of Medicine, Hamedan University of Medical Sciences, Hamedan, Iran. Correspondence Author:Dr. Iraj amiri, The Fatemieh Infertility Research Center, Fatemieh Hospital, Kermanshah Av., Hamedan, Iran. Received: 23, November, 2005; Code Number: rm06001 Abstract Background: The ability of the ovary to respond to exogenous
gonadotrophin stimulation and development of several follicles is essential in
assisted reproductive technology. Neither age and regularity of menses nor
follicular phase FSH and estradiol concentrations are reliable predictors of
ovarian response. Day 3 serum inhibin-B level, during induction ovulation, has
beenproposed as a predictor of ovarian response. Key words: Inhibin-B, IVF, Ovarian reserve, Poor responder Introduction Assisted reproduction and development ofin-vitro fertilization (IVF) techniques have revolutionizedthe treatment of infertility. Because IVF technique is expensive and notcompletly successful, many researches attempted to determine predictive factors for more successful outcome. Prognostic assessmentof ovarian reserve has relied upon indirect markers of ovarianfunction, such as age (1-3), FollicleStimulating 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 improvedmarker 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 μg/day, was begun on the 19th day. After menstruation, ovarian volumes were calculated as the volume of an elliposoid (length× width× depth× π/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 μg/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 was ≤50 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 wasdiagnosed by increasing concentrations of ß-HCG 2 weeks after embryo transfer and the subsequent demonstration ofan intrauterine gestational sac by transvaginal scan 2 weekslater. 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 serumwas measured by Inhibin-B assay kitpurchased from Serotec (Oxford, UK), using specific two-site enzyme immunoassay. Briefly, standardsand samples were diluted as appropriate and mixed with a halfvolume of distilled water containing 10% sodiumdodecyl sulphate (SDS). After 3 min at 100°C, tubes werecooled before adding freshly prepared hydrogen peroxide solution.After additional incubation at room temperature, duplicate aliquotsof denatured and oxidized samples and standards were transferredto antibody-coated microtitre plates, that were incubatedat room temperature, overnight. After washing with enzyme immunoassay (EIA) wash buffer[0.1 mol/l TrisHCl, 0.15 mol/l NaCl, 10% (w/v) bovineserum albumin, 5% (v/v) Triton X-100, and 0.1% (w/v) sodiumazide, pH 7.5], 50 µl alkaline phosphatase-conjugatedmouse anti-human inhibin-α subunit antibody was used. The plateswere then incubated for 3 hours. Plates were washed and bound alkaline phosphatase was quantifiedusing a commercially available enzyme immunoassay amplificationsystem (ELISA), which was used according to the supplier's instructions.The inhibin-B plates were read at 490 nm on an automatedEIA plate reader (BRIO: Basic Radium Immunoassay Operator, Radimspa, Pomezia, Italy). The assay detection limit for inhibin-B was less than 10 pg/ml. Withinand between plate coefficients of variation were <5.0 and9.0% respectively. Cross-reactions for each assay with the variousinhibin-related proteins were <0.5%. Statistical analysis Statistical analysis was performed using the commericially available software package SPSS version 11. Students t-test and χ2 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 numberof oocytes retrieved and serum inhibin-B levels on day 3 administration, and significant correlations (r=0.358) was found between them (fig.1). Table I.Demographic characteristics, pretreatment hormonal profiles and results in two groups of normal responders and poor responders
Table II. Demographic characteristics, pretreatment hormonal profiles and results in two groups, subdivided by day 3 inhibin-B serum concentrations.
Disscusion The most useful predictive information for an infertile coupleshould be obtained before beginning assisted reproductivetechniques. Basal inhibin concentrations have been evaluatedpreviously as predictive markers for pregnancy in IVF cycles.In two (12,24) of three(13) previous studies, higher inhibin-B on day23 was associated with a greater number of oocytes retrievedin IVF cycles (24) and with subsequent pregnancy(12) which was better predictorof the response to exogenous gonadotrophins than age (24) and was equivalent to day 3 FSH. Seifer et al (20) demonstrated that, pregnancy rates werehigher in patients with day 3 inhibin-B concentrations of over 45 pg/mlthan 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 166.9± 141 pg/ml versus 115.8± 87 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 tremendousoverlap in baseline inhibin-B concentrations between pregnantand non-pregnant subjects, and inhibin-B alone failed to predictpregnancy.The results of this study confirm Hall et alfindings (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 numberof oocytes retrieved during ART. Our results do not support the use of day 3inhibin-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. References
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