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Original Studies |
Monash IVF (T.E.-G., M.P.G., V.M., D.L.H) and the Department of Obstetrics and Gynecology, Monash University (T.E.-G., D.L.H), Prince Henry’s Institute of Medical Research (D.M.R., N.C.), Clayton, Victoria 3168, Australia; and Oxford Brookes University (N.G.), Oxford, United Kingdom
Address all correspondence and requests for reprints to: Dr. T. Eldar-Geva, Monash IVF and the Department of Obstetrics and Gynecology, Monash University, Clayton, Victoria 3168, Australia.
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The opportunity to explore in detail the relationship between serum inhibin A and B and ovarian follicular development arose as a side study to a multicenter trial aimed at the investigation of the efficacy of a fixed daily dose regimen of 100 or 200 IU recombinant human FSH (Puregon, Organon, Lane Cove, Australia) in promoting ovarian hyperstimulation in pituitary-suppressed infertile women undergoing IVF. In the work reported here, the relationships among circulating levels of inhibin A, inhibin B, FSH, and E2 at different times during FSH treatment; the number and size of ovarian follicles; and the number of oocytes retrieved after treatment were examined. The results suggest that serum inhibin B determined early during FSH treatment provides an index of the number of recruited follicles that will ultimately reach maturity and may provide a useful early marker in the clinical management of controlled ovarian hyperstimulation.
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Subjects and Methods |
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Thirty-eight women, aged 28–39 yr (mean, 32.7 yr), undergoing IVF treatment were included in the study. The inclusion criteria used were 1) subjects should have normal ovulatory cycles with a mean length of between 24–35 days, a body mass index between 18–29 kg/m2, and be in good physical and mental health; and 2) the cause of infertility must be treatable by IVF or intracytoplasmic sperm injection (ICSI) procedures.
The exclusion criteria used were 1) infertility attributed to endocrine abnormalities such as hyperprolactinemia, polycystic ovarian syndrome, and absence of ovarian function; 2) previous IVF, gamete intrafallopian transfer or zygote intrafallopian transfer attempts in which less than three oocytes were retrieved; 3) chronic cardiovascular, hepatic, renal, or pulmonary disease; 4) history of current abuse or abuse within the last 12 months of alcohol or drugs; 5) treatment with investigational drugs within 3 months before screening; and 6) presence of ovarian cysts more than 2 cm. The study protocol was approved by the Epworth Hospital research and ethics committee (Melbourne, Australia). Informed consent was obtained from all participants.
Treatment protocol
This study consists of a subset of a larger, randomized, double-blind clinical trial (14) examining the efficacy of a daily fixed dose regimen of 100 or 200 IU recombinant human FSH in pituitary-suppressed infertile women undergoing controlled ovarian hyperstimulation for IVF. After taking a blood sample for determination of basal hormone levels, patients were initially treated with a GnRH agonist, nafarelin (0.5 mg/day; Synarel, Searle, Sydney, Australia), by intranasal administration for 14 days, starting in the midluteal phase of the ovarian cycle. When E2 levels were below 150 pmol/L and any ovarian cysts were excluded by vaginal ultrasound, 100 or 200 IU recombinant human FSH (Puregon, Organon) were administered daily sc. The ovarian response was monitored by serum E2 and transvaginal ultrasonography every 1–3 days from the fourth day of FSH treatment. The ultrasonography detection limit was 1 mm, and its reproducibility in detecting follicles, as assessed from the coefficient of variation of repeated measurements, was 6%. The number and size of follicles with diameter greater than 2 mm were recorded. Serum samples were collected before FSH administration and every 1–3 days from days 4 to days 9–14 of treatment. Serum was stored at -20 C until assayed for progesterone, FSH, LH, inhibin A, and inhibin B. Treatment was continued for a maximum of 3 weeks until at least three follicles larger than 17 mm developed. hCG (5000 IU, im; Profasi, Serono, Melbourne, Australia) was given to promote ovulation. Oocytes were retrieved transvaginally 36 h later under general anesthesia. Routine IVF or ICSI and embryo culture were used as indicated, and a maximum of three embryos were transferred 2–6 days after oocyte retrieval.
Eighteen patients were randomized to receive 100 IU FSH/day, and 20
patients received 200 IU FSH/day. Both groups were comparable in
demographic and infertility characteristics (Table 1
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In patients with severe male factor infertility, the ICSI procedure was used (11 cases each were treated with 100 and 200 IU/day FSH). After hyaluronidase treatment, oocyte maturation were assessed according to the criteria described previously (15).
Hormone assays
Serum concentrations of E2, progesterone,
LH, FSH, and PRL were measured using a chemiluminescent immunoassay
(Diagnostic Products, Los Angeles, CA). The inter- and
intraassay coefficients of variation were, respectively, 9.2% and
4.9% for E2, 12.3% and 9.2% for progesterone,
7.8% and 1.9% for LH, 4.3% and 3.6% for FSH, and 3.7% and 1.7%
for PRL. Serum inhibin A was measured using an
/ßA-subunit dimeric enzyme-linked
immunosorbent assay (16), with the WHO inhibin A preparation (91/624)
used as standard. The assay sensitivity was 2 pg/mL, and the
between-assay variation was 18%. Serum inhibin B concentrations were
measured by the method of Groome et al. (10), using a human
inhibin B preparation isolated from human follicular fluid by N.
Groome. The assay sensitivity was 15 pg/mL, and the between-assay
variation was 18%.
Statistical analyses
Samples with hormone values below the assay detection limit were
assigned values equal to the detection limit of that assay. Testing for
differences between treatment groups was performed using both the
Mann-Whitney test (for differences between medians) and the two-sample
t test (for differences between means). Similar statistical
results were found with the use of both tests in this study. The
2 test was used to test for differences
between infertility groups. One-way ANOVA was used to analyze serial
changes in serum hormones concentrations and number of follicles.
Results are expressed as the mean ± SD
unless otherwise indicated. Differences were considered statistically
significant at P < 0.05.
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Results |
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Daily administration of either 100 or 200 IU FSH produced a
significant (P < 0.05) dose-related increase in serum
FSH, E2, and inhibin A (Fig. 1). By contrast, serum inhibin B showed a
maximum response with either FSH dose. The time course of response to
FSH treatment differed among the various hormones. After 4–6 days of
treatment, serum FSH values showed stable levels for the duration of
FSH administration at both FSH doses, whereas inhibin A and
E2 showed a continuous significant
(P < 0.05) increase from days 4–6 to 9–10 days of
FSH treatment. By contrast, inhibin B was significantly
(P < 0.05) increased by 4–6 days of treatment to
reach a maximum by days 7–8 and remained unchanged thereafter for the
duration of FSH treatment.
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). Serum progesterone levels measured 12–36
h before hCG administration were comparable in the two groups(Table 1). Follicle number and size
The follicles were divided into three groups according to
diameter: less than 11, 11–14, and 15 or more mm, respectively. The
number of 11- to 14-mm and 15-mm or more follicles significantly
(P < 0.05) increased with FSH treatment, although no
change was observed in the number of less than 11-mm follicles (Fig. 2). A significant FSH dose-related increase
was observed in the numbers of oocytes and mature oocytes collected and
the total number of embryos per patient transferred and frozen (Fig. 2).
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), the serum hormone levels and follicle count were also
expressed in terms of the last day of treatment (day of hCG
administration). As shown in Figs. 1 and 2 and Table 2, a similar FSH dose-response effect
with these parameters was observed compared to FSH responses during the
latter days of treatment.
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No significant correlations were observed between serum inhibin A (r = 0.16; n = 19) and inhibin B (r = 0.21; n = 19) on day 21 (midluteal phase) of the previous menstrual cycle and the number of eggs collected during the subsequent treatment cycle.
To investigate the ability of serum hormone levels or follicular number
during early FSH treatment to predict treatment outcome, correlation
coefficients were determined between the number of oocytes retrieved
and serum inhibin A, inhibin B, and E2 determined
on days 4–6, 7–8, and 9–10 and the last day (12–36 h before hCG
administration; Table 2 and Fig. 3). Highly
significant correlations (r = 0.72–0.89) were found between serum
inhibin B levels and the number of oocytes on all days of treatment.
Serum inhibin A and E2 showed significant, but
lower, correlations (r = < 0.78) with oocyte number later in the
treatment period. The number of 11- to 14-mm and more than 15-mm
follicles correlated highly (r = > 0.63) with oocyte number in
the middle to late stages of treatment (Table 2).
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The relationship between serum ovarian hormone levels, and
follicle number and size at various stages of FSH treatment was
examined (Table 3). Correlations of
limited (P < 0.05) or no significance were observed on
any day of treatment between the number of less than 11-mm follicles
and serum FSH, E2, and inhibin A and B.
Significant correlations (P < 0.05–0.001) were
observed between the number of 11- to 14-mm follicles and serum
E2 and inhibin B levels during days 7–12 of FSH
treatment. The highest correlations (r = 0.76–0.80) were observed
between the more than 15-mm follicles and both serum
E2 and inhibin A on days 9–10 of treatment.
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As shown in Table 4, a highly
significant correlation (P < 0.01–0.001) was observed
between serum inhibin A and B on days 4–8 of treatment, with a less
significant correlation (P = NS to P <
0.05) observed toward the end of the treatment period (days 9–10, day
of hCG administration). Serum E2 correlated
closely with inhibin A (P < 0.01–0.001) during the
early stages (days 4–10) and with inhibin B (P <
0.001) during the later stages (days 9–10, day of hCG administration)
of the treatment cycle.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The temporal relationship between follicle development and serum
hormone levels after FSH treatment provides a means to delineate the
contribution of each follicle group to the overall serum hormone
levels. On days 4–6 of FSH treatment, when the small (<11 mm)
follicles dominate, inhibin B provides the largest response, suggesting
that these small follicles are largely producing inhibin B. A similar
finding has been observed previously by Anderson and colleagues (17)
after FSH treatment of women with polycystic ovarian syndrome. On days
7–8 of treatment, although the numbers of medium-sized (11–14 cm)
follicles are increasing, the numbers of large (
15 mm) follicles are
still low. Serum inhibin A and B and E2 correlate
strongly with the number of medium-sized follicles, suggesting that
these follicles readily produce these hormones, with inhibin B showing
the most marked response. The late rise (days 9–12) in serum
E2 and inhibin A correlates closely with the
number of 15-mm or more follicles, whereas serum inhibin B, which does
not show a late rise, is less strongly correlated. These data support
the proposition that large follicles produce large amounts of both
E2 and inhibin A compared with earlier time
intervals.
Although these findings are consistent with earlier studies, the origin of inhibin B and its responsivity to FSH is still unclear. The high correlation between inhibin A and B during the early and middle stages of FSH treatment suggests that both small and medium sized follicles are producing these inhibins (8, 13, 18). However, during the late stages of treatment, serum inhibin A and B correlate significantly with E2, but less so with each other. On the other hand, it has been shown previously, based on immunocytochemical (6, 7) and in situ hybridization (4, 5) studies, that the ßA-subunit, but not the ßB-subunit protein and ribonucleic acid message are localized to the dominant human follicle. It is not clear how to reconcile these different observations. One explanation may relate in part to the continued production of inhibin A, inhibin B, and E2 by the medium-sized follicles developing in parallel with the large follicles and by the changing patterns of inhibin A, inhibin B, and E2 production as the large follicle develops.
A highly significant correlation was observed between the serum levels
of inhibin B during FSH treatment and the number of oocytes collected
up to 8 days later (Table 2). Based on the observation that inhibin B
is produced by immature follicles (4, 5, 6, 7), these data suggest that
inhibin B is being produced by immature follicles receptive to FSH
stimulation and destined for subsequent maturation. The size of this
FSH-responsive follicle pool probably represents a small proportion of
the total follicle number, as no or limited correlations were observed
between serum inhibin B levels and the number of small (<11-mm)
follicles at any stage of treatment. It is presumed that the remainder
is already programmed for apoptotic death.
This high correlation (r > 0.79) between serum inhibin B levels observed early in FSH treatment and subsequent number of oocytes collected suggests that the measurement of inhibin B may be useful as a marker of ovarian response during early FSH treatment. In fact, the correlation coefficient is higher than that noted between the number of less than 11-cm follicles detected on day 0 (r = 0.46) and the subsequent number of oocytes collected. As serum inhibin B levels at the 200-IU FSH dose are already elevated by days 4–6 of treatment, examination of inhibin B levels before day 4 may provide an even earlier marker of treatment outcome.
Although previous studies have proposed that inhibin B may be a good marker of ovarian reserve (10, 11, 12, 13), there have been few reported studies that have explored the relationship of serum inhibins, ovarian follicle number, and treatment outcome early in the FSH treatment period. Burger et al. (19) reported that serum inhibin B levels showed a larger incremental increase compared to inhibin A after a single injection of FSH early in the follicular phase. Lockwood and co-workers (13) found that the levels of inhibin A and B rose markedly after 8 days of FSH stimulation, with serum inhibin B increasing more than serum inhibin A in women undergoing ovarian stimulation. However, in contrast to the present study, they did not determine serial changes in serum inhibins earlier during treatment.
Siefer and colleagues (12) also proposed that inhibin B was a good
marker of treatment outcome based on serum inhibin B levels determined
in the early follicular phase of the previous spontaneous menstrual
cycle. Earlier studies (20, 21, 22, 23, 24, 25) using the Monash inhibin RIA (20) to
determine serum inhibin levels in FSH hyperstimulation cycles, showed
that E2 and inhibin levels increased in parallel
during ovarian hyperstimulation. However, this RIA detected both free
-subunit and inhibin dimers and thus was unable to identify changes
in the specific dimeric forms. Porchet et al. (18), using an
assay recognizing both heterodimeric inhibin and free -subunit
together, found that serum inhibin levels were the first
pharmacodynamic markers to increase after FSH treatment in pituitary
down-regulated women. Results from this study would suggest that the
primary inhibin form responsible is inhibin B.
In conclusion, these studies have further clarified the relationship between ovarian hormone production and follicle number and development after FSH stimulation. During the early stages of treatment, the elevated serum inhibin B levels are attributed to production by small follicles. In contrast, the late increase in serum E2 and inhibin A is associated with production by large follicles. Medium-sized follicles appear to be capable of producing inhibin A, inhibin B, and E2. Serum inhibin B levels determined during the early stages (e.g. days 4–6) of fixed dose FSH treatment provide an early indicator of the number of recruited follicles that are destined to form mature oocytes. The marked FSH stimulation of inhibin B early in treatment suggests that inhibin B may be a useful predictor in monitoring ovarian hyperstimulation treatment for IVF.
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Acknowledgments |
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Footnotes |
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Received November 3, 1998.
Revised July 30, 1999.
Accepted October 25, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, ßA and ßB
messenger ribonucleic acids in the normal human ovary and in polycystic
ovarian syndrome. J Endocrinol. 143:127–137.[Abstract]
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