1 Medical department M (Endocrinology and Diabetes) and Medical Research Laboratories, Århus Kommunehospital, Århus University Hospital, DK-8000 Århus C, 2 Paediatric Department, Skejby Sygehus, Århus University Hospital, DK-8000 Århus C, 3 Department of Growth and Reproduction, Rigshospitalet, DK-2100 København, Denmark
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Abstract |
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Key words: gonadotrophins/inhibin A/inhibin B/puberty/spontaneous pregnancy/Turner's syndrome
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Introduction |
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During early puberty and late childhood in girls with TS the question arises whether or not there is any evidence of endocrine function of the ovaries. Often the levels of FSH and LH do not give a clear picture. Therefore, a definite marker of ovarian function would be valuable in daily clinical practice. Thus, hypothetically inhibin A and B could be candidate markers for evaluating the function of the ovaries in TS patients. Presently inhibins have not been evaluated in TS. We examined 24 females with TS without any signs of ovarian function and a control group of 60 normal prepubertal females. The levels of inhibin A and B were determined; the levels of FSH, LH, pubertal stage and body composition variables were also determined.
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Materials and methods |
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Design
A blood sample was drawn from all participants. From almost all TS patients (n = 23), 15 additional samples (median: 2 samples) were drawn with varying intervals (<14 years) and a detailed physical examination was performed with determination of Tanner stage; height and weight were measured and body mass index (BMI) calculated as weight (in kg) divided by height (in metres) squared.
Assays
Blood samples were drawn at random time points from an antecubital vein between 16.00 and 17.00 h in TS females, between 9.00 and 17.00 h in control females, and centrifuged. Although samples were obtained at different times of the day, there was no information on circadian periodicity of inhibin level in women and potential bias is difficult to assess. Serum was stored at 200C until analysis. Samples were stored for 46 years before analysis. Recent data indicate that inhibin B is relatively stable in samples during long-term storing (Andersson et al., 1998). Serum inhibin A was measured in duplicate in a double antibody enzyme immunometric assay using a monoclonal antibody raised against the inhibin ßA-subunit in combination with a labelled antibody raised against the inhibin
-subunit, as previously described (Groome et al., 1994
). Serum inhibin B was measured in duplicate in a double antibody enzyme immunometric assay using a monoclonal antibody raised against the inhibin ßB-subunit in combination with a labelled antibody raised against the inhibin
-subunit, previously described (Groome et al., 1996
). In the inhibin A assay the detection limit was 7 pg/ml, and the intra- and inter-assay coefficient of variation (CV) were 15 and 18% respectively; in the inhibin B assay the detection limit was 20 pg/ml, and the intra- and inter-assay coefficient of variation were 15 and 18% respectively. The given intra- and inter-assay CVs of 15 and 18% respectively, represent the highest CVs observed at the very low range of the assay (close to the detection limit). At higher levels both the intra- and inter-assay CVs were lower. Since this study involves samples at the very low range, intra- and inter-assay CVs at this very low range are reported. Serum FSH and LH were measured by time-resolved immunofluorometric assay (DELFIA, Wallac, Turku, Finland), with detection limits of 0.06 and 0.05 IU/l, respectively. Intra- and inter-assay CVs were both below 8% in the FHS and LH assays.
Statistics
All statistics were performed in SPSS Windows version 8.0. Data were examined by the MannWhitney test. Results are expressed as median and range. All inhibin A, inhibin B, and LH values less than assay sensitivity were assigned a value below assay sensitivity (inhibin A: 6 pg/ml; inhibin- B: 19 pg/ml; LH: 0.04 IU/l) for statistical computations. This approach to values below assay sensitivity was chosen, because it gives the most conservative estimate of a difference between any two groups. Statistical significance was assumed where P < 0.05.
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Results |
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Discussion |
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During the normal menstrual cycle inhibin B concentrations increase in the early follicular phase in response to the rising FSH concentrations, exerting negative feedback on the pituitary (Groome et al., 1996; Sehested et al., 2000
), and are primarily produced by the granulosa cells of the small developing follicles (Roberts et al., 1994
). However, inhibin A concentrations increase prior to ovulation in parallel with the late follicular phase rise in estradiol and subsequently increase further after ovulation and reach peak values in the mid-luteal phase, being primarily a product of the granulosa cells of the dominant follicle and the corpus luteum (Groome et al., 1996
; Muttukrishna et al., 1997
).
Reproductive ageing is associated with decreased levels of inhibin A and B in the face of increasing levels of FSH, with inhibin B possibly being the earliest marker of a decline in reproductive potential (Klein et al., 1996; Welt et al., 1999
). In older cycling women with a raised early follicular FSH, inhibin B is reduced (Muttukrishna et al., 2000
), and in post-menopausal women inhibin B is not detectable, while inhibin A is still present, suggesting extragonadal sources of inhibin A (Ala-Fossi et al., 1998
). Previously, it has been the general view that most females with TS never reproduce because of dysgenetic gonads as the germ cells degenerate during fetal life (Carr et al., 1968
). However, 1020% of females with TS do cycle regularly for a shorter or longer period (Pasquino et al., 1997
), and about 5% are able to become pregnant and give birth (Lippe, 1991
). Thus, a thorough evaluation of reproductive capacity is relevant in pubertal females and young adolescents with TS .
Recently data have been presented suggesting that viable follicles are present in females with TS and the `classical' karyotype, 45,X, even at the age of 1219 years. Hreinsson et al. took ovarian biopsies from five females with TS (45,X), and found 1.5128 follicles per mm3 of the ovarian cortical tissue (Hreinsson et al., 2001). The authors conclude that cryopreservation for later treatment of infertility might be an option in TS. The data from the latter study of and the present one suggest that there may indeed be viable follicles present in the ovaries of even females with classic TS (45,X), and this may explain why 30% or more of females with TS show at least some signs of puberty (Pasquino et al., 1997
). A future scenario could be to offer the possibility of cryopreserving ovarian tissue from young girls/adolescents with TS, as a part of an IVF programme that would therefore have to be integrated with paediatric departments to extend the offer before final degeneration of follicles. Technically, it should be possible to extend such an offer (Hovatta, 1999
), although ethical considerations may hinder such a development.
In the present study we used an assay for inhibin B with a detection limit of 20 pg/ml, and in the lower range of detection the CV was higher than in the mid-range. One must of course have to keep this level in mind when evaluating the results of the present study. The present detection level is clearly under the level for the entire follicular level of inhibin B in women. However, since we do not know where in a given cycle we sampled the cycling pubertal control females, this means that females that were actually sampled in the luteal phase, levels of circulating inhibin B would be undetectable in some. This may explain why some of the cycling females in the pubertal control group had undetectable levels of inhibin B (and inhibin A). In a recent study of more than 300 women (1845 years), we found that 4% had undetectable levels of both inhibin A and B, probably because they were sampled during the late luteal and early follicular phase (unpublished results). Furthermore, since inhibin B shows pulsatility in cycling women during the day, with pulses of 60 min, one measurement does not entirely reflect inhibin B secretion (in the mid-follicular phase at least) (Lockwood et al., 1998). However, since most of the females examined in the present were not cycling, pulsatility may be of only minor importance.
In conclusion, the present study showed that inhibin A and B can be detected in some females with TS, but without signs of ovarian function. Thus viable follicles may be present in more females with TS than previously expected. It may be suggested that a prospective study with systematic blood-sampling and ovarian biopsies or imaging will shed more light on ovarian function in apparently non-cycling adolescents with TS.
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Acknowledgements |
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Notes |
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4 To whom correspondence should be addressed. E-mail: ch.gravholt{at}dadlnet.dk
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References |
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Submitted on September 14, 1999; resubmitted on February 25, 2002; accepted on April 16, 2002.