1 Department of Endocrinology and Reproductive Medicine, 2 Department of Endocrinology, Gynecology and Pediatrics, 3 Department of Physiology Necker Hospital, 75015 Paris, 4 INSERM U584, Faculté Médecine Necker, 75015 Paris, 5 INSERM U407, 69921 Oullins, 6 Department of Hormonal Biochemistry, 7 INSERM E120, Bicêtre Hospital, 94275 Le Kremlin-Bicêtre, 8 Department of Pathology, 9 Department of Gynecology and Obstetrics, Centre Hospitalier Intercommunal de Créteil, 94000, Créteil, 10 Department of Pathology, 11 Department of Obstetrics, Cochin Hospital, 75014 Paris, France
12 To whom correspondence should be addressed: Department of Endocrinology and Reproductive Medicine, Necker Hospital, 149, rue de Sèvres, 75743 Paris Cedex 15, France. Email: philippe.touraine{at}nck.ap-hop-paris.fr
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Abstract |
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Key words: ovarian biopsy/ovarian follicle/ovarian histology/premature ovarian failure
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Introduction |
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Subjects and methods |
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All patients gave written informed consent for participating in the present study. On the first day of the investigation, blood samples for hormonal measurements and a pelvic ultrasonography were performed. The following day, patients underwent a surgical laparoscopy with an ovarian biopsy.
Hormone measurements
Plasma levels of FSH and LH were measured by conventional radioimmunoassay (Immunotech Beckman, France). Those of estradiol (E2) were determined (DiaSorin, Italy) after previous plasma extraction. Normal range was based on hormonal results obtained from women during a normal menstrual cycle and given by the laboratory of hormonal investigations from Necker Hospital. The intra-assay coefficient varied from 2.6 to 6.7% whereas the inter-assay coefficient varied from 3.7 to 6.3%. The inhibin B concentration was measured in duplicate in serum samples using a solid phase sandwich enzyme-linked immunosorbent assay. The inhibin B assay used a capture monoclonal antibody raised against a sequence from near the C-terminal of the human subunit, immobilized on a hydrazide plate, as described by Groome et al. (1996)
and a second monoclonal antibody specific for the
subunit of inhibin coupled to alkaline phosphatase. The assay detection limit was 5 pg/ml.
Pelvic ultrasonography
Pelvic ultrasonography was performed in all patients, using a Siemens Sonoline Elegra sonograph and a 6.5 MHz probe. The surface area of the ovaries was calculated as: S = L (length)xW (width)x0.8. The normal surface area of the ovary is between 2 and 6 cm2 (Halligan et al., 2000).
Morphological analysis of ovarian biopsies from POF patients
After informed consent, the 61 patients underwent ovarian biopsies per laparoscopy. For each patient, two biopsies of 35 mm, one on each ovary, were sampled and fixed in neutral formalin. After embedding in paraffin, biopsies were serially sectioned at 5 µm, then stained with haematoxylin/eosin/safran. One section out of every 20 was observed and the number of resting and growing follicles was counted. The sections were carefully examined to detect fragments of large follicles that could be present on the edges of the biopsy as well as atretic follicles in the ultimate stages of atresia and degenerated corpora lutea.
Morphological analysis of ovarian samples from normo-ovulating women
In an attempt to test the reliability of small-sized biopsies sampled at random, to reflect accurately the presence of follicles in a given ovary, follicular counts were performed on ovaries from 20 normo-ovulating patients (A.Gougeon). These patients were operated on between 1973 and 1975 for tubal ligation, tubal plasty, explorative laparotomy, hysteroplasty, fibromas, breast carcinoma and small benign teratoma. All these patients were either fertile, having between one and three children, or presented with apparently normal folliculogenesis, as proven by the presence of a preovulatory follicle. The age of these patients ranged from 17 to 31 years (26.4±0.9 years). The volume of the ovarian biopsies varied from 26 to 3200 mm3 (654±147 mm3). Some patients underwent several biopsies on both their ovaries, and the whole ovaries were sliced for pathological studies. The tissues were fixed in Bouin's fluid, embedded in paraffin, and serially sectioned at 10 µm, then stained with haematoxylin Masson Blue.
Sections were observed at x2.5 magnification. In one section out of every 100, a surface area of 10 mm2 (5.5 mm longx1.8 mm deep) was chosen at random in the cortical part of the sample. The surface area studied was equivalent to the mean size of sections in biopsies from POF patients. All non-growing and growing follicles, whether healthy or atretic, as well as follicular or luteal fragments, were counted.
Stastistical analysis
Analyses were processed with Statview version 4.5 (Abacus Concepts, USA). Descriptive statistics were performed for each variable; quantitative results are presented as median (range); qualitative results are presented as a distribution of a number of patients. Hormonal and ovarian parameters were compared using the MannWhitney test. Proportions for the two groups were compared using the 2-test. The concordance between histology and ultrasonography was analysed with the Kappa test. P<0.05 was accepted as significant.
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Results |
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Discussion |
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The population of 61 women studied in the present report, recruited either in our Department of Endocrinology for adult patients or in the Pediatric Gynecology Unit of Necker Hospital, showed a typical pattern of ovarian insufficiency with high FSH and low E2 plasma levels. Inhibin B levels were also low, confirming previous data (Kalantaridou et al., 1998; Petraglia et al., 1998
).
In this study, only 56% of patients with normal-sized ovaries and a presence of follicles >2 mm suggested at ultrasonography displayed follicles when histological examination of an ovarian biopsy was performed. Nevertheless, a parallel can be drawn between this proportion of 56% and that of 57.5% corresponding to histological sections in which no follicle theoretically detectable at US were seen. When women had no image of a follicle at ultrasonography, histological examination did not detect any follicle in 19 out of 29 patients, whereas follicles were observed in the 10 other patients. Consequently, it can be assumed that ultrasonography is not predictive of the presence of follicular structures within the ovary. We also wished to determine whether hormonal levels were sufficient to indicate the presence or absence of ovarian activity. Although E2 and inhibin B median levels were significantly higher in patients with the presence of follicles at histology, the overlap between the two groups appears considerable, pointing to the poor sensitivity of the hormonal markers to predict the presence or absence of a follicular activity; specifically, the predictive value of the presence of follicles appears very weak for low plasma levels of either estradiol or inhibin B.
To ensure that follicular content in ovarian biopsies from POF patients was representative of the whole ovarian follicular content, counts were randomly performed on ovaries from 20 normal patients, without POF, who were considered controls in this study. The present study provides useful data to determine whether ovarian biopsies sampled at random allow for reliable assessment of follicle presence and activity for a given patient. Ovarian biopsies are effective in discriminating between patients having or not having resting follicles, even if a very small biopsy (less than 5 x 2x2 mm) is less powerful in estimating the actual number of resting follicles. In our opinion, it can be assumed that the error in estimating the presence or absence of resting follicles in a given ovary is only 0.5%. Less accurate but still informative is the reliability of small ovarian biopsies to detect early follicular growth. Therefore, small biopsies appear very instructive for distinguishing ovarian phenotypes marked by the presence of a follicular reserve from ovaries deprived of such resting follicles. On the contrary, the determination of the stage of follicular growth, based on the observation of a few slides from an ovarian biopsy, appears less accurate and should be carefully considered.
The informative aspect of the ovarian biopsy has also been demonstrated in both healthy volunteers and patients with cancer, enabling the selection of women with numerous primordial follicles and for whom an ovarian conservation before chemotherapy may be suggested (Meirow et al., 1999). The follicular density measured after an ovarian sampling obtained by laparoscopy has also been shown to be correlated with age and appears to be a good marker of the ovarian reserve (Lass et al., 1997
), even if follicular density may be highly variable within the same ovary (Schmidt et al., 2003
). However, a prospective study comparing all methods available (hormonal and morphological) to appreciate the follicular reserve would probably be very interesting in the future for common medical practice.
To date, in our experience, the distinction between two different ovarian phenotypes, i.e. with or without follicles, appears useful in determining a strategy to search for genetic anomalies that could be responsible for the POF syndrome. In the past, this strategy has enabled us to identify patients with FSHR gene mutations, already described by other groups (Aittomaki et al., 1996; Allen et al., 2003
). The description of a partial follicular maturation was also given in the initial report of a patient bearing an FSH
gene mutation (Rabin et al., 1972
; Matthews et al., 1993
). However, these cases are probably rare and both the absence/presence of follicles and the stage at which follicular growth is impaired should prove helpful in orienting research and screening among other candidate genes. The comparison of the phenotype of POF patients with data obtained from mouse models presenting gene defects related to ovarian development and function suggests that mutations of those genes are possibly associated with POF syndrome. Various sets of genes are now identified and involved in the different steps of ovarian and follicular development. Genes involved in migration of germinal cells and meiosis such as Sporulation 11 (SPO 11), Disrupted Meiotic CDNA1 (DMC1), Mut S Homologue 4 (MSH4), Mat S Homologue 5 (MSHS) (Cotinot et al., 2002
) may be associated with defects leading to a complete absence of follicles. On the contrary, in mice bearing defects of genes normally involved in follicular maturation, such as Connexin 37 or GDF9 (Burns and Matzuk, 2002
), the ovarian histology confirms the presence of the resting follicles with a blockade of the follicular maturation at later stages. Finally, the recent description of the target disruption of genes encoding for transcription factors such as Foxl2 (Schmidt et al., 2004
) and Foxo3 (Castrillon et al., 2003
) confirms their role in the regulation of the follicular growth. It is highly probable that mutations of such genes exist in women and the search for them will depend on the ovarian phenotype observed. However, we cannot exclude that a heterogeneous syndrome such as POF might be secondary to multiple gene abnormalities.
Therefore, in the future, the identification of the ovarian transcriptome in those patients compared with normal young adults also appears necessary to better understand the role of different genes in follicular and ovarian development. This strategy using DNA chips would allow for the qualitative and quantitative analysis of gene expression and the understanding of gene function and detection of gene alterations (Ben-Shlomo et al., 2002), but would be based on the initial extraction of the ovarian mRNA, therefore justifying the ovarian biopsy. Finally, our knowledge of POF syndrome should be improved by the study of familial cases enabling a linkage analysis of such cases, with the determination of the gene defects associated with the syndrome.
In conclusion, the clinical presentation associated with the hormonal investigations, the ultrasonographic description and the histological ovarian phenotype constitute the global phenotyping of our POF patients and are mandatory in the identification of such new genetic mutations.
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Acknowledgements |
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References |
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Submitted on December 9, 2003; resubmitted on April 26, 2004; accepted on July 16, 2004.