1 Institute for Reproductive and Developmental Biology, Wolfson & Weston Research Centre for Family Health, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK, 2 FertiGen, Düsseldorf, Germany, 3 Leuven Institute for Fertility and Embryology, Leuven, Belgium, 4 Sismer, Bologna, Italy, 5 Volvat Medical Center, Oslo, Norway, 6 IVF-Kliniken Öresund, Malmö, Sweden, 7 Institute Pronatal, Prague, Czech Republique, 8 AVA-klinikka, Tampere, Finland and 9 Zentrum für Sterilitätsbetreuung, Innsbruck, Austria
10 To whom correspondence should be addressed. e-mail: j.brosens{at}imperial.ac.uk
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Abstract |
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Key words: aromatase/endometrium/IVF/mRNA/pregnancy outcome
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Introduction |
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The CYP19 gene encodes aromatase P450, a cytochrome P450 enzyme that catalyses three consecutive hydroxylation reactions converting C19 androgens to C18 estrogenic steroids (Sebastian et al., 2001; Simpson et al., 2002
; Chen et al., 2002
). Previous reports indicated that aromatase P450 mRNA is expressed in the endometrium of women with benign and malignant reproductive tract disorders, but not in endometria of disease-free controls (Noble et al., 1996
; Kitawaki et al., 1997
; 1999
; Dheenadayalu et al., 2002
). We postulated that the abundance of aromatase P450 transcripts in the endometrium could reflect the degree of biochemical impairment and serve as a prognostic marker of IVF outcome. To test this hypothesis, the relative aromatase P450 transcript levels were determined in endometrial biopsies, taken randomly in the cycle, from 150 women prior to starting IVF treatment.
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Materials and methods |
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Total RNA was isolated from tissues using STAT-60 (Tel-Test) and treated with DNaseI. Equal amounts of total RNA (2 µg) were reverse transcribed using the Superscript First-Strand Synthesis System for RTPCR (Invitrogen) and the resulting first-strand cDNA was diluted and used as template in the real-time quantitative (RTQ)-PCR analysis. Detection of aromatase P450 and GAPDH expression was performed with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems), using the relative standard curve method. GAPDH represents a non-regulated gene, and its expression served as internal control and was used to normalize for variances in input cDNA. All measurements were performed in triplicate. The following gene-specific primer pairs and probes were designed using the ABI Primer Express software: GAPDH forward primer (GAAGGTGAAGGTCGGAGT), GAPDH reverse primer (GAAGATGGTGATGGGATTTC), GAPDH probe (5'6FAM, 3'TAMRA) (ATTTGGTCGTATTGGGCGCCTGGTCACC), aromatase P450 forward primer (GGCATACCTCCTATGGGTTGTC), aromatase P450 reverse primer (GTAGCCTGGTTCTCTG TGTGAA) and aromatase P450 probe 3 (5'6FAM, 3'TAMRA) (CCAAGCTAGGTGCTATTGGTCATCTGCTCCT).
Statistical analysis was performed using Students t-test for parametric variables and the 2-test for categorical variables. A P-value <0.05 was considered statistically significant. The sample size was calculated on the assumption that 50% of the IVF/ICSI patients would express endometrial aromatase, and that in this group the pregnancy rate per transfer is reduced from 35% to (a) 10% or (b) 15%. With the level of significance of 0.05 and with 80% power, the number needed for (a) is 51 and for (b) 83.
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Results |
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Discussion |
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We hypothesized that the level of aromatase P450 mRNA expression could reflect the degree of biochemical perturbation in the endometrium. To test this, endometrial aromatase P450 transcripts were quantified by RTQ-PCR in a cohort of infertile patients and correlated with subsequent IVF outcome. Twenty-one of 122 (17%) patients had elevated transcript levels (8.3 a.u), and although IVF outcome was indeed very poor, two successful pregnancies did occur in this group. Our study also demonstrates that aromatase P450 transcripts are detectable in all biopsy samples and throughout the cycle, albeit at relative low level in the majority of cases. Moreover, the cause of infertility did not differ significantly between patients with high versus low endometrial aromatase P450 mRNA levels, but this is not entirely surprising as it is known that categorization of the causes of infertility in current IVF/ICSI practice often lack specificity (Guzick et al., 2001
).
The results of the present study indicate that the level of aromatase P450 expression represents one of a multitude of factors that determine IVF treatment outcome. However, our study is limited in its size, and the prognostic value of measuring endometrial aromatase P450 expression levels for predicting IVF treatment outcome requires further validation in a larger cohort of patients. A number of other potential confounding factors also require further investigation. For instance, endometrial biopsies were, for obvious reasons, not taken during the IVF treatment cycle but in a preceding cycle. At present it is not known whether elevated endometrial aromatase P450 expression fluctuates from cycle to cycle, or whether serial determinations in different cycles would yield a better prognostic value. Moreover, Ishihara et al. (2003) recently demonstrated that prolonged treatment with GnRH agonists can inhibit endometrial aromatase P450 expression in patients with fibroids and endometriosis, suggesting that the type of ovarian down-regulation and stimulation protocol used in IVF treatment may have a profound impact on subsequent pregnancy rates in affected patients.
This study shows that the endometrium can be an unique source of potential markers that could be exploited clinically to tailor infertility treatment to individual patients. This notion is further supported by recent microarray studies demonstrating an astonishing diversity of endometrial genes that are aberrantly expressed during the implantation window in women with endometriosis compared with healthy controls (Kao et al., 2002; 2003
). For clinical translation, however, timing of the biopsy sample should ideally not matter, and it appears very likely that powerful screening tools, such as microarrays and proteomics, will uncover additional genes that are aberrantly expressed throughout the cycle. Finally, the observation that a subgroup of infertile women express high levels of aromatase P450 in the endometrium also provides a rational basis to test the use of highly selective aromatase inhibitors, such as letrozole and anastrozole, in the treatment of infertility (de Ziegler, 2003
; Mitwally and Casper, 2003
). However, whether expression at mRNA level correlates with protein level or aromatase P450 enzymatic activity remains to be determined.
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Acknowledgements |
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References |
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Submitted on August 22, 2003; accepted on October 16, 2003.