Department of Obstetrics and Gynaecology, Chang-Gung Memorial Hospital, Lin-Kou Medical Centre, Taipei, Taiwan, Republic of China
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Abstract |
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Key words: embryo score/estradiol/granulosa cell/inhibin B/oocyte maturation
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Introduction |
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In women with ovulatory cycles, the circulating inhibin B levels increase slowly and steadily in the early follicular phase, and reach a peak in the mid-follicular phase, then decrease progressively in the late follicular phase before ovulation. Shortly after the LH surge, there is a short-lived peak in the circulating inhibin B, which in turn declines to a low concentration for the remainder of the luteal phase (Groome et al., 1996). In contrast, the inhibin A levels are low in the early follicular phase, rise at the pre-ovulatory phase, and reach a peak in the mid-luteal phase (Groome et al., 1996
; Wang et al., 2000
).
Previous studies have shown that serum inhibin B is believed to be of predictive value in monitoring ovarian stimulation treatment for IVF (Hayes et al., 1998; Eldar-Geva et al., 2000
). In addition, serum inhibin A may also act as a useful marker for monitoring the effects of gonadotrophin stimulation (Lockwood et al., 1996
). Furthermore, concentrations of inhibin in FF, though not specified inhibin A or B, are greater in the fertilized group as compared with the unfertilized (Fowler et al., 1995
), and are suggested to be used as an index of follicular maturation (Franchimont et al., 1990
). However, the relationship between inhibin levels in follicular fluid (FF) and the quality of oocytes and embryos has not yet been explored.
In the work reported here, FF and matched oocytes were collected simultaneously to study further the correlation among the inhibin B and estradiol levels in FF as well as the embryo development. In addition, the association between oocyte quality and age was also investigated.
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Materials and methods |
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The protocol for controlled ovarian stimulation included the pituitary down-regulation with subcutaneous injection of GnRH agonist (1 mg/day, Lupron®; Abbott Laboratories, North Chicago, IL, USA) started on the 21st day of the previous menstrual cycle. Subcutaneous administration of recombinant human (r-hFSH) (150450 IU/day, Gonal-F®; Laboratories Serono SA, Aubonne, Switzerland) was initiated on the third day of the menstruation, based on the women's age and previous response of ovulation induction. HCG (10 000 IU, Pregnyl®; NV Organon, Oss, The Netherlands) was applied i.m. 3436 h before oocyte retrieval, and ovulation induction was monitored by transvaginal sonography.
During oocyte retrieval, FF without contamination of blood and matched oocyte from each single follicle were collected individually. Follicle aspirates which were not clear and contaminated with blood were excluded. Oocytes were evaluated, immediately after retrieval, for nuclear maturity and graded as metaphase II, metaphase I, or prophase I. FF was stored at -70°C until subsequent assays for inhibin B and estradiol.
Laboratory evaluation of embryos
Oocytes were cultured in Petri dishes in IVF-20® (Vitrolife, Göteborg, Sweden) at 37°C in a humidified 5% CO2/95% air environment. The semen was processed using 80% Percoll (Sigma Chemicals, St Louis, MO, USA) discontinuous gradient centrifugation at 800 g for 15 min. Insemination with 10 000 15 000 progressively motile sperm for each oocyte was performed 46 h after oocyte retrieval. Following IVF, the resulting embryos were cultured in IVF-20® at 37°C under 5% CO2 in air.
Quality of embryo was assessed by the embryo score described previously (Steer et al., 1992). Briefly, a quality score of each embryo on days 2 and 3 was produced by the morphological grade of the embryo multiplied by the number of blastomeres. Morphologically, embryos were graded as follows: grade 4, equal sized symmetrical blastomeres; grade 3, uneven blastomeres with <10% extracellular fragmentation; grade 2, uneven blastomeres with 1050% extracellular fragmentation; grade 1, >50% blastomeric fragmentation with unequal sized blastomeres.
Enzyme-linked immunosorbent assay (ELISA) for inhibin B
Concentrations of inhibin B in FF were determined by a commercial ELISA kit (Serotec, Oxford, UK). The assay employed the quantitative sandwich enzyme immunoassay technique. Samples of FF were diluted 1:500 in inhibin-free fetal calf serum prior to measurement. The minimum detection limit of assay was 15.6 pg/ml. The intra-assay coefficients of variation were 7.7% at 75.2 pg/ml, and 6.4% at 185 pg/ml (n = 8). The interassay coefficients of variation were 8.6% at 68.3 pg/ml and 7.3% at 196 pg/ml (n = 6).
Immunofluorometric assays (IFMA) for estradiol and FSH
Estradiol levels in FF were measured by immunofluorometric assays (Pharmacia, Turku, Finland). The detection limit of the estradiol assay was 13.6 pg/ml. The intra-assay coefficients of variation were 7.2% at 152 pg/ml and 5.3% at 446 pg/ml (n = 16). The interassay coefficients of variation were 9.1% at 163 pg/ml and 7.5% at 465 pg/ml (n = 12).
Serum levels of FSH on day 3 of the menstrual cycle were determined by immunofluorometric assays (Pharmacia, Turku, Finland). The detection limit of the estradiol assay was 0.5 mIU/ml. The intra-assay coefficients of variation were 6.3% at 2.4 mIU/ml and 7.1% at 6.6 mIU/ml (n = 8). The interassay coefficients of variation were 8.5% at 2.5 mIU/ml and 9.7% at 7.5 mIU/ml (n = 6).
Statistical analysis
In order to explore the relationship among inhibin B and estradiol in FF, embryo scores on days 2 and 3, as well as age, analysis using linear regression was employed. In all cases, a P value < 0.05 was considered statistically significant.
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Results |
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Inhibin B levels in FF were significantly related to embryo scores on days 2 and 3 (n = 179, P < 0.000 001 and n = 159, P < 0.000 1 respectively) (Figure 1). Notably, inhibin B levels in FF from which 54 oocytes failed to develop into embryos were relatively low [49.1 ng/ml (median), ranged from 10.9103.8 ng/ml], although they were overlapped with those from which developed into embryos. In FF, inhibin B showed a positive correlation with estradiol (P < 0.02) (Figure 2
). Nevertheless, no association was found between estradiol levels in FF and embryo scores on days 2 and 3 (Figure 3
).
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Discussion |
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A previous study failed to show the relationship between inhibin A and B levels in FF and the quality of oocytes and found little effect on oocyte quality of fertilization (Lau et al., 1999). In the present study, although FF inhibin B did not correlate with oocyte quality, it did so with the quality of embryos which were fertilized (Figure 1
). This might suggest that some other variable is dominating the actual fertilizability of the oocytes, but that oocytes made in a follicle with well-functioning granulosa cells, once fertilized, are better fitted to start embryonic development.
Reproductive capacity in women is believed to be age-related and declines dramatically beyond the fourth decade of life. In this respect, oocyte quality is most likely the primary determinant of reproductive potential (Klein and Sauer, 2001). Results from the present study showed that concentrations of both inhibin B and estradiol in FF were inversely associated with age (P < 0.02 and P < 0.05 respectively) (Figure 4a,b
). In addition, inhibin B levels in FF appeared to be an indicator of oocyte quality, since they were significantly correlated with embryo scores on days 2 and 3 (P < 0.000001 and P < 0.0001 respectively) (Figure 1
). Collectively, these findings demonstrate a notion that oocyte quality declines as the age of women progresses.
Serum FSH levels on day 3 of the menstrual cycle, but not inhibin B, are believed to be an indicator of ovarian reserve (Corson et al., 1999). It is also evident that age and day 3 FSH, rather than inhibin B, are the stronger predictors for successful pregnancy (Creus et al., 2000
). In addition, follicular phase inhibin B secretion is decreased in older ovulatory women (aged 4045 years) who demonstrate a monotrophic FSH rise, implying a diminished follicular pool in older women (Klein et al., 1996
). Moreover, decreased serum inhibin B throughout the menstrual cycle is suggested to be an early marker of the decline in follicle number across reproductive ageing (Welt et al., 1999
). In the present study, an inverse correlation was observed between day 3 serum FSH and inhibin B levels in FF (P < 0.0001) (Figure 5a
), further suggesting that the age-related decline in oocyte quality can be reflected by inhibin B levels in FF at the time of oocyte aspiration.
Ontogenically, inhibin is a product of granulosa cells and serves as a protein marker of granulosa-cell function (McLachlan et al., 1986). This is further confirmed by a previous study demonstrating that inhibins A and B levels in FF increase as follicles grow and develop (Magoffin and Jakimiuk, 1997
). In contrast, production of estradiol in the human ovary is through a co-operation of theca and granulosa cells (Erickson, 1996
). In clinical practice, serum or plasma estradiol is an established variable of follicular development. A close correspondence has been reported between circulating inhibin and estradiol concentrations in women undergoing ovulation induction in the IVF programme (McLachlan et al., 1986
), suggesting that both circulating inhibin and estradiol can be used as a monitoring indicator of ovarian response during ovulation induction.
Although serum estradiol has been used to monitor the follicular growth during controlled ovarian stimulation, estradiol levels in FF may not play a role in predicting the quality of oocytes and embryos. This is supported by the results from the present study that estradiol levels in FF showed no relation to embryo scores on both days 2 and 3 (Figure 3). In Figure 2
, there was a positive association between FF inhibin B levels and estradiol concentrations (P < 0.02), suggesting that both inhibin B and estradiol in FF reflect the follicular function. However, as described previously, inhibin is a product of granulosa cells and estradiol is predominantly a theca-cell product (McLachlan et al., 1986
) (Erickson, 1996
). Thus, unlike inhibin B, FF estradiol may not be a strong indicator for predicting the embryo quality.
In conclusion, inhibin B in FF may reflect the ovarian response and serve as an effective marker of follicular development and quality of oocyte/embryo during controlled ovulation stimulation. We also demonstrate, deduced from the present results, that quality of oocyte is age-related and declines as age increases.
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Acknowledgements |
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Notes |
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References |
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Submitted on January 9, 2002; accepted on March 11, 2002.