Departments of 1 Obstetrics and Gynecology and Reproductive Medicine, 2 Biology and Genetics of Reproduction, and 3 Biochemistry and Hormonology, Clamart, France
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology and Reproductive Medicine, Hôpital Antoine Béclère, 157, rue de la Porte de Trivaux, 92141, Clamart, France. Email: renato.fanchin{at}abc.ap-hop-paris.fr
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Abstract |
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Key words: anti-Müllerian hormone/controlled ovarian hyperstimulation/follicle size/hCG/luteal phase
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Introduction |
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Beyond the pre-ovulatory stage, the possible effects of granulosa cell luteinization and corpus luteum formation on AMH production are less well documented. Basic research studies conducted in rats indicated that isolated corpora lutea express much less AMH than small and large antral follicles (Baarends et al., 1995). Yet, AMH levels measured in follicular fluids from women undergoing controlled ovarian hyperstimulation (COH) for IVF and embryo transfer remained detectable 3234 h after hCG administration (Seifer et al., 1993
; Fallat et al., 1997
).
An interesting experimental model that could be helpful to clarify the effects of follicle luteinization and corpora lutea activity on peripheral AMH levels is that of COH. Indeed, contrary to the menstrual cycle in which only a single follicle ovulates and is converted into corpus luteum, COH is characterized by an extensive, hCG-driven transformation of maturing follicles into multiple corpora lutea. This supraphysiological process may be instrumental in amplifying the possible consequences of follicle luteinization and corpora lutea activity on peripheral AMH levels. Hence, the present study was conducted to clarify this issue by analysing peripheral AMH levels during the early to mid-luteal phase in pituitary-desensitized COH cycles.
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Materials and methods |
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COH protocol
All women received a time-release GnRH agonist, leuprolide acetate (1 mg/day s.c., Lucrin; Abott-France Pharmaceuticals, France) from cycle day 21 onwards. On day 3 of the subsequent cycle, complete pituitary desensitization was confirmed by the detection of low serum levels of E2 and gonadotrophins. Patients also underwent a conventional ultrasound examination to exclude ovarian cysts and to verify that endometrial thickness was <5 mm. Recombinant (r)FSH therapy (Gonal-F; Serono Pharmaceuticals, France) was then initiated at a dosage of 225 IU/day whereas daily GnRH agonist administration was continued until the day of hCG administration (Gonadotrophine Chorionique Endo, 10 000 IU i.m.; Organon Pharmaceuticals, France). Daily FSH doses and timing of hCG administration were adjusted according to the usual criteria of follicular maturation. Administration of hCG was performed when at least five follicles were >16 mm in diameter and E2 levels per mature follicle (>16 mm in diameter) were >200 pg/ml. Oocytes were retrieved 36 h after hCG administration by transvaginal ultrasound-guided aspiration. All embryo transfers were performed 2 days after oocyte retrieval using Frydman catheters (CCD Laboratories, France). Luteal phase was supported with micronized progesterone (Estima, 600 mg/day; Effik Pharmaceuticals, France) administered daily by vaginal route starting on the evening of embryo transfer.
Hormonal and follicular measurements
On the day of hCG administration (dhCG), 4 days later (hCG+4), and 7 days later (hCG+7), women underwent serum AMH, E2, progesterone and hCG measurements at 09:00. Blood samples drawn on hCG+4 and hCG+7 are usually performed to monitor the effectiveness of hCG administration and luteal progesterone support of IVF cycles in our centre. Serum AMH levels were determined using a second generation enzyme-linked immunosorbent assay (reference A16507; Immunotech Beckman Coulter Laboratories, France). Intra- and inter-assay coefficients of variation (CV) were <6 and <10% respectively, lower detection limit at 0.13 ng/ml, and linearity up to 21 ng/ml for AMH. Serum E2, progesterone and hCG levels were determined by an automated multi-analysis system using a chemiluminescence technique (Advia-Centaur; Bayer Diagnostics, France). For E2, lower detection limit was 15 pg/ml, linearity up to 1000 pg/ml, and intra- and inter-assay CV were 8 and 9% respectively. For progesterone, lower detection limit was 0.1 ng/ml, linearity
60 ng/ml, and intra- and inter-assay CV were 8 and 9% respectively. For hCG, lower detection limit was 2 mIU/ml, linearity
1000 mIU/ml, and intra- and inter-assay CV were 4 and 5% respectively.
On dhCG, ovarian ultrasound scans were performed using a 3.68.0 MHz multi-frequency transvaginal probe (EC9-4, Sonoline Antares; Siemens S.A.S., France) to evaluate the number and sizes of ovarian antral follicles. For the purposes of the present study, antral follicles were sorted into three size classes: small (311 mm in diameter), intermediate (1215 mm in diameter), and large (1622 mm in diameter) follicles. The choice of these thresholds to define follicle size classes was arbitrary and based on the fact that, in the menstrual cycle, the sizes of non-dominant follicles remain <12 mm (Pache et al., 1990) whereas follicle maturation is putatively achieved from 16 mm onwards (Dubey et al., 1995
).
Statistics
Measures of central tendency and variability used were, respectively, the mean and SEM when data distribution was normal, and the median and the ranges when normality could not be ascertained. Longitudinal changes in hormone levels from dhCG to hCG+7 were assessed by the Wilcoxon signed rank test. The association between two continuous variables was assessed by correlation when they were independent from each other and by simple regression when there was a dependency. The Fisher r to z test was used to determine if coefficients of correlation (r) were significantly different from zero. P<0.05 was considered statistically significant.
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Results |
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Discussion |
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The initial decrease in serum AMH levels is probably related to the putative adaptations that granulosa cells undergo during the follicle luteinization process. Indeed, the similarity between serum AMH and E2 level dynamics from dhCG to hCG+4 and the fact that AMH levels were positively correlated with E2 levels on hCG+4 support this hypothesis and suggests that both hormonal decreases shared the same origin. As in the menstrual cycle (Hoff et al., 1983) and in COH (Fanchin et al., 2000
), granulosa cell luteinization induces a remarkable decline in E2 levels due to the shift of follicular steroidogenesis from E2 to progesterone. In addition, the scarce expression of AMH and its type II receptor mRNA in isolated corpora lutea as compared to non-luteinized antral follicles from rats (Baarends et al., 1995
) further indicates that granulosa cell luteinization alters the follicular ability to produce AMH. The physiological mechanisms implicated in this striking phenomenon remain to be established. Also, if confirmed, these results suggest that AMH could be a potential marker of follicle luteinization after hCG in COH. Last, but not least, we observed a positive correlation between serum hCG and AMH levels on hCG+4, which may suggest that hCG administration stimulates early luteal AMH production. In contrast, hCG levels on hCG+4 failed to influence AMH levels on hCG+7, thereby suggesting that hCG does not exert a significant influence on AMH production by corpora lutea. Further studies are needed to elucidate the role of hCG on the follicular and luteal production of AMH.
During the interval from hCG+4 to hCG+7, serum AMH, progesterone and E2 levels increased simultaneously. The magnitudes of progesterone and E2 increases were positively correlated, despite the possible interference exerted by progesterone administration used for luteal support on endogenous progesterone levels. This suggests that the elevation in both hormone levels from hCG+4 to hCG+7 was triggered by a common phenomenon, i.e. the corpus luteum steroidogenesis (Hoff et al., 1983). The mechanisms involved in the subtle, yet significant, AMH increase observed from hCG+4 to hCG+7 are less evident. They are possibly linked to the hormonogenesis by corpora lutea and/or antral follicles during the luteal phase of COH. Yet, the first possibility is challenged by at least two reasons. First, corpora lutea have been shown to express negligible amounts of AMH (Baarends et al., 1995
). Second, the magnitude of AMH elevation was clearly dissociated from that of the putative markers of luteal activity, such as progesterone and E2.
Another possible explanation for the secondary increase in AMH levels is that it could result from the development of antral follicles during the luteal phase of COH. The positive relationship between the magnitude of AMH increase from hCG+4 to hCG+7 and the number of small follicles on dhCG is in agreement with this hypothesis. Recent well-designed studies have demonstrated that subtle waves of follicular growth sporadically occur during the luteal phase in the menstrual cycle despite the putative FSH suppression by luteal inhibin A and E2 secretions (Baerwald et al., 2003a,b
). It is conceivable that, during COH, the steadily high FSH doses administered have generated additional waves of follicle growth that might persist after hCG administration. The lack of relationship between the total dose of gonadotrophins used for COH and AMH levels on hCG+4 and hCG+7 observed in the present study does not disprove this hypothesis but suggests that such a phenomenon may be triggered even in the presence of low recombinant FSH doses. Unfortunately, the mass effect by multiple corpora lutea and their ability to produce E2 are factors that hamper the monitoring of possible follicle growth by ultrasound or E2 levels during the luteal phase of COH and prevented us from testing this hypothesis. Since AMH is probably scarcely expressed in the corpus luteum (Baarends et al., 1995
), luteal AMH measurements could theoretically serve as a valuable endocrine probe into the dynamics of follicle development during the luteal phase. The possible effects of exogenous FSH doses used for COH on the characteristics of non-dominant follicle growth during the luteal phase remain to be determined. Moreover, further studies aiming at monitoring serum AMH and follicular growth dynamics during the luteal phase in spontaneous menstrual cycles are needed to clarify this issue.
Furthermore, our observation that, on the day of hCG administration, serum AMH levels were strongly associated with the number of small but not large follicles confirms our own previous results (Fanchin et al., 2003a) and further supports the hypothesis that, with final follicle maturation, granulosa cells loose their ability to produce AMH (Baarends et al., 1995
; Fanchin et al., 2003a
). In addition, women whose serum AMH levels were high on dhCG showed an improved responsiveness to COH as indicated by a reduced gonadotrophin requirement, a large number of antral follicles and oocytes, and a more intense increase in progesterone levels from dhCG to hCG+7. These data further support the relationship between AMH and the ovarian follicular status (De Vet et al., 2002
; van Rooij et al., 2002
; Fanchin et al., 2003b
; Pigny et al., 2003
) and responsiveness to COH (Seifer et al., 2002
). Incidentally, on the day of hCG administration, we observed that serum AMH levels were higher in the present study than in an earlier report by our team (Fanchin et al., 2003a
). This difference presumably is related to the fact that, in the present investigation, serum AMH levels were determined using a second generation enzyme-linked immunosorbent assay (reference A16507; Immunotech Beckman Coulter Laboratories, France), which included several analytical modifications as compared to the original assay. These aimed at minimizing the background and matrix effects and improving AMH detection at low concentrations.
In conclusion, the present results represent the first documentation on the dynamics of serum AMH levels during the luteal phase of COH. They indicate that, after hCG, AMH levels initially decline, then increase during the mid-luteal phase. The mechanisms implicated in these remarkable hormonal changes are unclear. The initial decline may be attributed to adaptations of granulosa cells to the follicle luteinization process. The secondary increase may reflect luteal follicle development, as the magnitude of AMH increase was positively related to the number of small follicles at the end of COH. Further studies aiming at elucidating the mechanisms implicated in both phenomena will help to clarify the regulation of AMH secretion during the luteal phase and to determine the clinical usefulness of AMH measurements as a marker of follicle luteinization and possibly luteal follicle development.
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Submitted on October 6, 2004; resubmitted on November 3, 2004; accepted on November 22, 2004.