1 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital and 2 School of Biological and Molecular Sciences, Oxford Brookes University, Oxford, UK
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Abstract |
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Key words: activin A/follicular fluids/inhibin A/inhibin B/oocytes
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Introduction |
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Serum inhibin A concentrations peak prior to the pre-ovulatory luteinizing hormone (LH) surge and in the mid-luteal phase suggesting that inhibin A is a product of the mature dominant follicle and the corpus luteum (Groome et al., 1994; Muttukrishna et al., 1994). Inhibin B concentrations peak in early follicular phase (day 4/day 5) and then remain low throughout the rest of the cycle suggesting it is mainly a product of pre-antral follicles (Groome et al., 1996). During the menstrual cycle, mean serum activin A concentrations vary in a biphasic manner, with highest concentrations around mid-cycle and the late luteal/early follicular phases and nadirs in both mid-follicular and mid-luteal phases (Muttukrishna et al., 1996
).
Classically, inhibin has an endocrine role by inhibiting pituitary production of FSH. Although activin was also initially purified as a substance that increases pituitary FSH secretion, there is increasing evidence for diversified biological activity of activin in various tissues (Vale et al., 1994). In-vitro studies show that both inhibin and activin can act directly to modulate androgen synthesis in the theca interna (Hsueh et al., 1987
). Treatment of cultured human theca cells with recombinant activin A potently inhibits stimulation of androgen production by LH and insulin-like growth factor 1 (IGF-1) (Hillier et al., 1991b
). Conversely, picomolar amounts of recombinant inhibin markedly augment LH/IGF-stimulated androgen production. Potent augmentation by activin of FSH-inducible aromatase activity has been convincingly demonstrated in granulosa cells in vitro in the rat (Hutchison et al., 1987
; Miro et al., 1991
) and in the non-human primate (Miro and Hillier, 1992
).
Studies in cattle suggest that inhibin A and activin A may play important roles during the final stages of oogenesis and that addition of recombinant inhibins and activins improves the performance of serum-free culture medium for in-vitro maturation of oocytes from cattle and possibly other mammalian species (Stock et al., 1997). Recently, follicular fluid concentrations of inhibin A, inhibin B and activin A from individual follicles from regularly cycling women have been measured using specific two-site enzyme-linked immunosorbent assays (Magoffin and Jakimiuk, 1997
). This study reported a linear relationship between follicular fluid concentrations of inhibin A and follicle size and maturation. A similar study has also been reported using a mouse follicle culture system (Smitz and Cortvrindt, 1998
). The aim of the present study was to investigate the association between follicular fluid and oocytecumulus complex culture medium concentrations of inhibin A, inhibin B and activin A and the quality and the fertilizing capacity of the oocyte. The relationship between the concentrations of these hormones in the follicular fluid and oocyte culture medium, and the quality, fertilizing capacity of the oocyte and follicle size were studied.
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Materials and methods |
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Hormone assays
Cross-reaction of the culture medium was checked in each assay. The absorbance values of the culture medium alone were similar to the diluent blanks in all three enzyme-linked immunosorbent assays (ELISA).
Inhibin A
All follicular fluid samples were diluted 100 times in Tris HCl + 10% bovine serum albumin (BSA) buffer. Concentrations of inhibin A in the follicular fluid and oocyte culture medium were assayed in duplicate using a specific ELISA reported elsewhere (Muttukrishna et al., 1997). The minimum detection limit of the inhibin assay was 2 pg/ml. The inter- and intraplate variations for the inhibin A ELISA were <10%.
Inhibin B
All follicular fluid samples were diluted 100 times in sample buffer. Follicular fluids and oocyte culture media were assayed in duplicate for inhibin B using a specific ELISA (Muttukrishna et al., 1997) with some modifications. The sample preparations involved a single oxidation step with no SDS treatment. The sensitivity of the assay was 6 pg/ml, and the mean intra- and interplate variations were <10% respectively.
`Total' activin A
All follicular fluid samples were diluted 10 times in phosphate-buffered saline (PBS) + 1% BSA buffer. Follicular fluids and oocyte culture media were assayed in duplicate for `total' activin A using the recently reported activin A ELISA (Muttukrishna et al., 1997). The sensitivity of the assay was 50 pg/ml, and the mean intra- and interplate variations were <12%.
Data obtained from patients' files and classification of oocyte
Information on follicular size, matching oocyte quality and fertilizing capacity (fertilized or unfertilized) was obtained from patients' case records. Follicular diameter assessed by transvaginal ultrasound was recorded in mm (average of three perpendicular planes was taken) and aspirated FF volume was noted in ml. Oocytes were graded by the clinical embryologists according to the amount of cumulus and maturity as reported elsewhere (Saith et al., 1998). Briefly, Grade 1 describes oocytes with a fully mature complex, normal amount of cumulus, and slightly dark corona and cumulus. Grade 2 describes oocytes with a fully mature complex, normal amount of cumulus, and signs of post-mature cumulus. Grade 3 describes oocytes with a borderline fully mature complex, normal amount of cumulus, and dark corona or cumulus. Grades 4 and 5 describe immature oocytes. In general, grade 1 is considered to be of good quality, grades 2 and 3 are average qualities, and grades 4 and 5 are unlikely to fertilize. The limitations of this oocyte quality grading system in terms of repeatability and relationship to oocyte maturity were recognized but, as it was the method used in our IVF unit, we had no alternative option.
Statistical analysis
Hormone concentrations in follicular fluid and oocyte culture media were compared between different oocyte qualities and fertilizing capacity. Hormone measurements did not vary significantly from normal distribution (normality tests, Prism statistical software; GraphPad Inc., San Diego, CA, USA) and parametric methods were used for analysis. One-way analysis of variance were carried out with BonferroniDunn post hoc tests to compare the concentrations of hormones in FF and culture medium and different grades of oocytes.
The relationships between the concentrations of inhibin A, inhibin B and activin A in follicular fluids and oocyte culture media and three parameters, follicular size, oocyte grade and fertilizing capacity, were determined by Pearson correlation analysis using the statistics package for social sciences (SPSS) software programme. Mean concentrations of hormones between the fertilized and non fertilized groups were compared by the unpaired Student's t-test using the same programme.
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Results |
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Inhibin A and inhibin B
High concentrations of inhibin A and B (~4050 ng/ml) were present in the FF. Oocytecumulus complex also produced measurable amounts of inhibin A and B in culture. Mean inhibin A concentrations in FF were not different in follicles yielding different grades of oocytes (Figure 1a). Inhibin B concentrations in the FF were significantly higher in grade 1 oocyte-containing follicles compared with grade 2 oocyte-containing follicles (Figure 1b
). Oocyte culture medium concentrations of inhibin A and B did not vary with different grades of oocytes (Figure 2
). Inhibin B concentrations in the oocytecumulus complex media were negatively correlated with fertilizing capacity of the oocytes (r = 0.3, P < 0.01). There were no significant correlations between the concentrations of inhibin A and B in both FF (Figure 3
) and oocyte culture medium (Figure 4
) with oocyte fertilizing capacity.
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Discussion |
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In this study, the concentration of inhibin A in the follicular fluid increases with follicle size and FF volume, whereas the concentration of inhibin B in the follicular fluid decreases when follicle size and FF volume increases. This is consistent with a previous report showing that smaller follicles express more ßB subunit mRNA (Hillier, 1991c) and would appear to agree with the changes in concentrations of inhibin A and B in serum seen as the pre-ovular dominant follicle matures (Muttukrishna et al., 1994; Groome et al., 1996). Magoffin and Jakimiuk (1997) in their natural cycle study found a significant increase in follicular fluid inhibin B with increasing follicle size (414 mm) and a decrease in inhibin B in follicles >14 mm diameter. Our patients all underwent gonadotrophin-releasing hormone (GnRH) agonist and gonadotrophin treatment as part of their IVF cycle, with possible effects on intrafollicular autocrine/paracrine events, so the studies are not directly comparable. However, almost all follicles in the IVF patients in this study were >14 mm diameter and the relationship between inhibin B and follicle size is consistent with the natural follicle data in the above reported study. The relationship between follicle size and the amount of inhibin A (r = 0.28, P = 0.03) and inhibin B (r = 0.3, P < 0.01) in the oocytecumulus complex culture medium is consistent with the relationship between FF concentrations of inhibin A (r = 0.4, P < 0.001) and inhibin B (r = 0.33, P = 0.02) with follicle size.
The concentrations of activin A in the oocyte culture medium appeared to be higher in the case of good maturity of cumulusoocyte complexes, while no relationship was found with inhibin A, inhibin B and activin A in the FF. There was an increase in activin A concentration in oocyte culture medium (60%) in the case of good quality cumulusoocyte complexes suggesting that activin A may be involved in the maturation of the oocyte. The measurement carried out in this study is `total' activin A (follistatin bound activin A + `free' activin A) and total follistatin concentrations have to be measured in the future in similar studies to evaluate the availability of `free' activin A that is biologically active.
It has been demonstrated (Dyson and Gurdon, 1997) that activin signalling is necessary in the early embryonic development of Xenopus. Stock et al. (1997) have demonstrated that both inhibin A and activin A play important roles during the final stages of oogenesis in cattle. Inhibin A and inhibin B were not found to be associated with oocyte quality in this study, possibly identifying a difference between species. Activin receptor mRNA (ActRII) has been shown to be strongly expressed by rat oocytes (Sadatsuki et al., 1993
; Cameron et al., 1994
) and human oocytes (Sidis et al., 1998
), supporting a putative action of activin on this cell type. As activins are structurally related to the transforming growth factor ß peptide family, it is not surprising to find that activins have growth and differentiation promoting effects on oocytes. Furthermore, Alak et al. (1998) have shown that activin A stimulates meiotic maturation of human oocytes in vitro. This study also indirectly supports the hypothesis that increased concentrations of activin A in the oocytecumulus culture medium may promote maturation of the oocyte. However, further studies have to be carried out with larger numbers to confirm that activin A promotes human oocyte maturity and/or embryo development.
The lack of correlation between the follicular fluid concentrations of inhibin A, inhibin B and activin A and the chance of fertilization (Figure 3c) suggests that these factors do not influence the chance of fertilization. In this study only cases with excellent sperm parameters were included to minimize one variable but it is not possible to control for all extrinsic factors and/or for the intrinsic genetic potential of the oocyte.
The availability of human recombinant inhibins and activins would allow further in-vitro studies to investigate their effect(s) on oocyte development. Activin receptor expression on human oocytes can also be studied to determine the mechanisms controlling the responsiveness of human oocytes to these intrafollicular proteins.
In summary, this study confirms previous reports on inhibin A and inhibin B as markers of follicle maturation. Complete overlap in data between FF and oocytecumulus culture medium concentrations of inhibin A, inhibin B and activin A and the quality of oocytes suggests that these hormones could not be biochemical predictors of oocyte quality. Future in-vitro studies are needed to assess the ability of activin A in promoting human oocyte maturation that could be applicable in IVF laboratories.
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Acknowledgments |
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S. Muttukrishna is funded by the Wellcome trust. This research was supported by the Oxford Fertility Unit, John Radcliffe Hospital, Oxford, UK.
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Notes |
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References |
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Submitted on January 20, 1999; accepted on June 21, 1999.