JBRA Assist. Reprod 2007;11(2):9-13
ARTIGO ORIGINAL
doi: 10.5935/1518-0557.2007.11.2.02
Center: Departments of Obstetrics and Gynecology and Reproductive Medicine ( J.B.S., R.F., D.H.M.L, R.F.), Clamart, France
ABSTRACT
During the luteal-follicular transition, FSH is required for survival and further growth of a fraction of ovarian follicles. Basic research data have suggested the relationship between serum AMH levels and antral follicle responsiveness to exogenous FSH.
OBJECTIVE: To assess the possible relationship between serum AMH levels and antral follicle responsiveness to exogenous FSH.
DESIGN: PROSPECTIVE STUDY. Patients and Methods: We studied 116 IVF-ET candidates undergoing controlled ovarian hyperstimulation (COH) with GnRH agonist and FSH. After the achievement of pituitary suppression and before FSH administration (baseline), serum AMH levels were measured. The number of antral follicles was determined by ultrasound at baseline (small antral follicles; 3-10 mm) and on the day of hCG administration (dhCG; preovulatory follicles; ≥16 mm).
RESULTS: At baseline, median (ranges) serum AMH, E2, and P4 levels were 3.53 (0.71-8.59) ng/mL, 31 (30-50) pg/mL, and 0.25 (0.1-1.6) ng/mL, and median number of early antral follicles were 17±0.25. Serum AMH levels showed a positive correlation with preovulatory follicles on dhCG (r=0.23; P <0.01), and oocytes obtained (r=0.38; P <0.0001).
CONCLUSIONS: These data demonstrate a strong association between day 3 serum AMH level and IVF outcome. AMH may offer greater prognostic value than other currently available serum markers of assisted reproductive technology ART outcome.
Palavras-chave: Hormônio anti-mulleriano /AMH /FSH /Estimulação ovariana controlada.
RESUMO
Durante a transição da fase luteal a folicular, FSH é necessitado para a sobrevida e crescimento de uma fração de folículos ovarianos. Pesquisas têm sugerido a relação do nível sérico do AMH com a resposta folicular ao FSH.
OBJETIVO: Avaliar a possível relação entre o nível sérico do AMH e a resposta do folículo antral ao FSH exógeno.
DESENHO: ESTUDO PROSPECTIVO. Pacientes e Métodos: Nós estudamos 116 candidatas a fertilização in vitro e transferência embrionária (FIV-TE) submetidas à estimulação ovariana controlada com agonista de GnRH e FSH. Depois de atingir a supressão da hipófise e antes da administração do FSH (baseline), o nível sérico do AMH foi avaliado. O número de folículos antrais foi determinado pelo ultrasom (pequenos folículos antrais; 3-10 mm) e no dia da administração do hCG (dhCG; folículos préovulatórios; ≥16 mm).
RESULTADOS: Baseline, o nível sérico mediano de AMH, E2, e P4 foram 3.53 (0.71-8.59) ng/mL, 31 (30-50) pg/mL, e 0.25 (0.1-1.6) ng/mL, e o número mediano de folículos antrais precoce foi 17±0.25. O nível sérico do AMH mostrou uma correlação positiva com o número de folículos préovulatórios no dia do hCG (r=0.23; P <0.01), e oócitos obtidos (r=0.38; P <0.0001).
CONCLUSÃO: Esses dados demonstram a forte associação entre o nível sérico do AMH (d3) e as consequências da FIV-ET. AMH pode proporcionar um maior valor prognóstico do que outros marcadores séricos disponíveis atualmente das consequências do tratamento de reprodução assistida.
Key words: Anti-Müllerian hormone /AMH /FSH /Controlled ovarian hyperstimulation.
INTRODUCTION
In women undergoing treatment for infertility, the predictive value of serum hormone parameters such as FSH, E2, and inhibin B remains somewhat controversial, and disappointing.
Chronological age still appears to be the most reliable predictive factor (Chuang et al., 2003).
A serum marker that could be indicative of the pool of nongrowing, FSH-independent follicles before cyclic recruitment might better predict the ovarian response to ovulation induction for ART than current available serum markers.
In adult women, the physiological role of anti-Müllerian hormone (AMH), a peptide that exclusively is produced by the granulosa cells of ovarian follicles (Vigier et al., 1984), remains ill established. Basic research experiments suggest the involvement of AMH in the control of the primordial to primary follicle growth in mice (Durlinger et al., 2002). Moreover, growing evidence indicates that AMH is also implicated in the regulation of the responsiveness of early antral follicles to FSH. Indeed, Durlinger et al., 1999 have demonstrated that ovaries from 4-month-old, AMH-deficient mice contain more growing follicles as compared to those from wild-type matching controls. Later experiments conducted by the same investigators have shown that FSH administration increases the number of follicles and their pace of growth in AMH- deficient mice as compared with their wild-type littermates (Durlinger et al., 2001).
In these women, serum AMH levels measured on day 3 of the menstrual cycle have been positively associated to the strength of a subsequent ovarian response to controlled ovarian hyperstimulation (COH) (Seifer et al., 2002; Hazout et al., 2004; Fanchin et al., 2005). The quantitative relationship between AMH and ovarian response to COH may merely result from the positive correlation that exists between peripheral AMH levels and the number of early antral follicles on day 3 (De Vet et al., 2002; van Rooij et al., 2002; Pignyet al., 2003; Fanchin et al., 2003; 2005).
Hence, the aim of this study was to test the hypothesis that day 3 serum AMH may be a more useful prognostic marker of assisted reproductive technology ART outcome
MATERIAL AND METHODS
Subjects
We prospectively studied 116 infertile women, 21 to 41 years of age. All of them met the following inclusion criteria: 1) both ovaries present, deprived of morphological abnormalities, and adequately visualized in transvaginal ultrasound scans; 2) regular menstrual cycle lengths ranging between 25 and 35 days; 3) no current or past diseases affecting ovaries or gonadotropin or sex steroid secretion, clearance, or excretion; 4) no clinical signs of hyperandrogenism; 5) body mass indexes (BMI) ranging from 18-25 kg/m2; 6) number of early antral follicles (3-10 mm in diameter) <24; 7) no endometriosis. Infertility was due to sperm abnormalities (54%), tubal abnormalities (29%), or was unexplained (17%). An informed consent was obtained from all women and this investigation received the approval of our internal Institutional Review Board.
Controlled ovarian hyperstimulation protocol
All women received a time-release gonadotropin releasinghormone (GnRH) agonist, triptorelin (3 mg/day IM, Decapeptyl, Beaufour Ipsen Pharma, Paris, France) on cycle day 1. Three weeks later, complete pituitary desensitization was confirmed by the detection of low serum levels of E2 and gonadotropins. Patients also underwent a conventional ultrasound examination to exclude ovarian cysts and to verify that endometrial thickness was <5 mm. Recombinant FSH therapy (Gonal-F, Serono Pharmaceuticals, Boulogne, France) was then initiated at a dosage of 225 IU/day and continued until the day of hCG administration (Gonadotrophine Chorionique “Endo”, Organon Pharmaceuticals, Saint-Denis, France, 10,000 IU, IM). After the 6th day of recombinant FSH therapy, daily FSH doses were adjusted according to the number of growing follicles. Administration of hCG was performed when at least four follicles exceeded 16 mm in diameter and E2 levels per mature follicle (=16 mm in diameter) were >200 pg/mL. Women who did not meet these criteria had their cycles cancelled. Oocytes were retrieved 36 hours after hCG administration by transvaginal ultrasound-guided aspiration. All ETs were performed 2 days after oocyte retrieval using Frydman catheters (CCD Laboratories, Paris, France). Luteal phase was supported with micronized progesterone (P4) (Estima, Effik Pharmaceuticals, Bièvres, France, 600 mg/day) administered daily by vaginal route starting on the evening of ET.
Hormonal and follicular measurements
For the purposes of the present study, we considered blood samples obtained on the day in which pituitary desensitization was confirmed (baseline) and on the day of hCG administration (dhCG). All blood samples were obtained by venipuncture and serum was separated and frozen in aliquots at -20 C for subsequent centralized analysis. At baseline, serum AMH levels were determined using a “second generation” enzymelinked immunosorbent assay (reference A16507; Immunotech Beckman Coulter Laboratories, Villepinte, France). Intraand interassay coefficients of variation were <6% and <10%, respectively, lower detection limit at 0.13 ng/mL, and linearity up to 21 ng/mL for AMH. At baseline and dhCG, serum E2 and P4 levels were determined by an automated multi-analysis system using a chemiluminescence technique (Advia-Centaur, Bayer Diagnostics, Puteaux, France). For E2, lower detection limit was 15 pg/mL, linearity up to 1,000 pg/mL, and intraand interassay coefficients of variation were 8% and 9%, respectively. For P4, lower detection limit was 0.1 ng/mL, linearity up to 60 ng/mL, and intraand interassay coefficients of variation were 8% and 9%, respectively.At baseline and dhCG, ovarian ultrasound scans were performed using a 5.0-9.0 MHz multi-frequency transvaginal probe (Voluson 730 Expert, General Electric Medical Systems, Paris, France) to evaluate the number and sizes of ovarian antral follicles. We determined, at baseline, the number and mean diameters of all follicles measuring 3-10 mm for both ovaries and, on dhCG, the number and mean diameters of all follicles measuring from 12-22 mm. On dhCG, follicles measuring 16-22 mm in diameter were considered mature follicles (Dubey et al., 1995).
STATISTICS
Measures of central tendency and variability used were, respectively, the mean and standard error of the mean when data distribution was normal, and the median and the ranges when normality could not be ascertained. For menstrual cycle duration, we used the mode and the ranges. Relationship between two continuous variables was assessed by correlation when they were independent from each other and by simple regression when there was a dependency relationship. The Spearman’s test was used to determine if coefficients of correlation (r) were significantly different from zero. A P value <0.05 was considered statistically significant.
RESULTS
Hormonal and follicular relationships
At baseline, median (ranges) serum AMH, E2, and P4 levels were 3.53 (0.71-8.59) ng/mL, 31 (30-50) pg/mL, and 0.25 (0.1-1.6) ng/mL, and median number of early antral follicles were 17±0.25. Serum AMH levels showed positive correlation with preovulatory follicles on dhCG (r=0.23; P <0.01), and oocytes obtained (r=0.38; P <0.0001). On dhCG, the mean number of matured follicles (16-22mm in diameter) was 8±0.25.
Overall population characteristics and COH results
Mean women’s ages and menstrual cycle lengths were 33.0±0.37 years 28.7±0.17 days. Mean body mass index was 22.7±0.37 kg/m2 .Controlled ovarian hyperstimulation lasted 12±0.13 days and required 2,981±74 IU of recombinant FSH. We observed a negative relationship between recombinant FSH requirement for COH and serum AMH levels at baseline (r=-0.29; P <0.007). In addition, serum AMH levels at baseline were comparable in patients whose infertility was due to sperm or tubal abnormalities, or it was unexplained. The mean number of total oocytes and transferred embryos were 11±0.4, and 2.2±0.05, respectively.
DISCUSSION
Previous studies have examined the value of AMH and other markers, using as endpoints oocyte number (Seifer et al., 2002; Van Rooij et al., 2002) or follicular count (Fanchin et al., 2003). Van Rooij et al., 2002 examined ongoing pregnancy in a subset of his patients as a secondary endpoint but did not find a significant association with any serum marker including FSH, E2, or inhibin B. All these studies concluded that AMH may be a more useful serum marker than any of the known serum markers for assessment of ovarian reserve.
In cultured rat granulosa cells, exogenous AMH inhibits aromatase synthesis (di Clemente et al., 1992, 1994) and inhibits the proliferation of granulosa luteal cells (Seifer et al., 1993). AMH and its receptor mRNA are highly expressed in granulosa cells of mainly preantral and small antral follicles (Hirobe et al., 1992; Baarends et al., 1995). From previous mouse and rat studies (Durlinger et al., 1999), it became apparent that AMH plays an important role during both initial and cyclic recruitment of ovarian follicles (Weenen et al., 2004). AMH, produced by the pool of growing follicles, acts as a feedback signal by inhibiting the initial recruitment of primordial follicles (Durlinger et al., 2002). Moreover, AMH expression negatively correlates with future atresia of follicles that undergo selection, suggesting that AMH may be involved in the process of cyclic recruitment (Durlinger et al., 1999). In addition, FSH- dependent growth of mouse follicles in vitro is attenuated by the addition of AMH to the culture, indicating that AMH is one of the factors determining the sensitivity of ovarian follicles to FSH (Durlinger et al., 2001).
The mechanisms underlying this relationship remain unclear. They could involve an effect of AMH on FSH receptor expression. A reduction in the expression of the FSH receptor may change the sensitivity of a follicle to FSH, as it was demonstrated by studies in bovine (Bao et al., 1997). In line with this, several studies have shown that, in addition to FSH effects on the ovary, AMH can also inhibit the role of cAMP as a second messenger of FSH (Teixeira et al., 2001). This would suggest that the molecular target site of AMH action is downstream of the FSH receptor.
Assisted reproduction is expensive, time-consuming and stressful for patients. Evaluations of IVF/ICSI performance rarely consider cancelled cycles, which usually result from an inadequate ovarian response to the stimulation treatment. The cycle cancellations further increase the cost and duration of therapy. Therefore, a major challenge to the IVF teams is to predict prospective patients who will be low responders and to appropriately counsel women who are potential candidates for assisted reproduction. Traditional methodology used to assess ovarian reserve has consisted of baseline serum levels of hormones such as FSH, estradiol and inhibins, and chronological age. Also, a number of provocative tests have been devised to indirectly assess ovarian reserve and identify patients who might not be detected by basal hormone screening alone (Scott & Hofmann, 1995; Bukman & Heineman, 2001). However, neither basal hormone measurements nor such dynamic tests provide direct information concerning the responsiveness of the ovaries to exogenous gonadotropins used in ovarian stimulation for assisted reproductive treatment. Thus, despite the validity of all these tests, there still remain patients who respond poorly to stimulation despite having normal tests of ovarian reserve. Therefore, an early marker of ovarian responsiveness after the initiation of gonadotropin therapy would assist in deciding whether to proceed with an ongoing cycle. Ultimately, this will decrease the cost of continued monitoring and medication for patients in whom therapy will most likely fail. Assessment of the ovarian reserve is particularly important in the IVF clinic, where AMH may be useful as a predictor of poor response. However, in order to determine whether serum AMH level has prognostic value, additional prospective studies in a normal population are necessary to provide definite proof for this concept.
In conclusion, these data demonstrate a strong association between day 3 serum AMH level and IVF outcome. AMH may offer greater prognostic value than other currently available serum markers of assisted reproductive technology ART outcome.