1 Obstetrics and Gynaecology, 2 Reproductive Endocrinology and 3 Pathology, Dokuz Eylul University School of Medicine, Izmir, Turkey
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Abstract |
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Key words: ageing/ovarian reserve tests/ovary
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Introduction |
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Basal serum follicle stimulating hormone (FSH) concentrations (Scott et al., 1989; Toner et al., 1991
; Farhi et al., 1997
), clomiphene citrate challenge tests (CCCT) (Navot et al., 1987
; Loumaye et al., 1990
; Scott et al., 1993
), gonadotrophin-releasing hormone agonist stimulation tests (GAST) (Winslow et al., 1991
), exogenous follicle stimulating hormone ovarian reserve test (EFORT) (Fanchin et al., 1994
), elevated day 10 serum progesterone levels (Hofmann et al., 1995
) and inhibin B (Hall et al., 1999
) have all been performed in order to assess response to gonadotrophins and chances of conception. Unfortunately, all the above tests are indirect measurements of ovarian reserve. Previous studies have assessed the role of these tests mainly in predicting ovarian follicular or oocyte response to gonadotrophins or chances of conception. To the best of our knowledge, the accuracy of these tests as a measure of the number of remaining primordial follicles within the ovaries has not been previously validated.
Attempts have been made to use follicular density in ovarian biopsy specimens as a direct means of assessing ovarian reserve (Lass et al., 1997). Although this may be a novel clinical tool for the evaluation of ovarian reserve, the limitation of this test is that follicular density within the biopsy specimen may not accurately represent the density of follicles within the whole ovary. The purpose of this study, therefore, was to examine the accuracy of the ovarian reserve tests by comparing the ability of basal serum FSH, CCCT, and GAST to predict the number of follicles within the ovaries as assessed by histology, in women over 35 years of age who underwent oophorectomy.
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Materials and methods |
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All three tests were performed in the same cycle but we were confident that there was no carry-over effect, especially between gonadotrophin releasing hormone (GnRH) agonist and clomiphene. Single s.c. administration of GnRH agonist (buserelin) and its half-life in normal women has been studied (Lemay et al., 1983). It was found that (after a 1 mg single shot of buserelin) at 24 h, serum concentrations of both FSH and LH returned to pre-treatment control values. Serum oestradiol concentrations rose progressively and reached their maximum (2.5-fold) at 11 h, then began to decrease steadily, but remained above pre-treatment control values (0.5-fold) at 24 h. In the present study there was an interval of at least 8 days between the two tests (GAST and CCCT).
FSH and LH were measured by a double antibody assay (coat-A count IRMA; DPC Diagnostics, Los Angeles, CA, USA). The intra- and interassay coefficients of variation (CV) for the FSH assay (for 24 mIU /ml) were 2.4 and 4.8% respectively. The intra- and interassay CV for the LH assay (for
23.8 mIU /ml) were 1 and 3.4% respectively. 17-ß oestradiol concentration was measured with commercially available radioimmunoassay kits (coat-A count; DPC Diagnostics). The intra- and interassay CV for oestradiol assay (for
50 pg /ml level) were 7 and 8.1% respectively.
Basal serum FSH concentration was considered abnormal if it exceeded 10 mIU/ml. This threshold was based on the upper limit of normal (95th centile) in our laboratory. Serum FSH >12 mIU/ml on day 10 was considered abnormal for CCCT (Tanbo et al., 1992). GnRH agonist stimulation test was considered normal for ovarian reserve if the serum oestradiol value on day 3 was double that of the baseline value (on day 2).
Of the 22 patients, 20 had operations for uterine myomata, one patient had uterine prolapse with urinary stress incontinence and the other had a stage 1 cervical carcinoma. Twenty of the patients had total abdominal hysterectomy (TAH) and two patients had myomectomy. In all women >39 years, at the time of the study it was routine to offer oophorectomy as prophylaxis for ovarian carcinoma. In the younger women, oophorectomy was offered as treatment for premenstrual syndrome or pelvic pain.
All the specimens were evaluated by the same pathologist (K.Y.). To assess the total number of follicles within the ovaries, all the ovaries were fixed for 24 h with buffered formaldehyde and cut into slices every 3 mm with a fractionater. All the tissues were processed in a routine manner and paraffin-embedded. From each 3 mm slice, a 5 µm section was cut and stained with haematoxylineosin (Gundersen et al., 1988). The sections were evaluated by light microscopy. The volume of each ovary (V(ov)) was calculated by the Cavalieri method (Gundersen et al., 1988
), by multiplying the parameters from parallel sections separated by a known distance t (3 mm), area associated with one point in the grid (a) (4 mm2), and sum of the number of points hitting the section of the ovary (
P).
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The sections were then evaluated for the number of follicles (primordial, primary, secondary, and Graffian follicles). Atretic follicles were not counted. The total follicle number (N(ov)) and follicle number per cubic centimetre (N(ov)/cc) of the tissue were calculated (Gundersen et al., 1988) using the formulae:
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At the beginning of the calculations, after the first 10 counts, variance of area, total variance of
P, Nugget effect (i.e. independent variance of each estimate), and Nug % was kept at 22.66 for point counting. The total variance of
P representing the error ratio was 7.1%. Uni- or bilateral ovarian follicle numbers and ovarian volume as well as oocyte numbers per centimetre were calculated and the number of follicles per unit tissue was determined.
Statistical Package for Social Sciences (SPSS, Corp., Chicago, IL, USA) was used for statistical analyses. The paired and unpaired t-test and simple linear regression analyses were used where appropriate. The cut-off value for the number of follicles per unit tissue was taken as the mean minus 2 SEM. The sensitivity and specificity of each test were calculated and receiver operator curve (ROC) analysis performed using this cut-off value. Values are given as mean ± SEM.
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Results |
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Table II shows the summary of results for each test. Ovarian reserve was considered as abnormal in a varying number of patients depending on the type of test performed. However, if the cut-off value for number of follicles was taken as 20 000/cc based on the mean 2 SEM, the ovarian reserve was abnormal in 11 patients. For this value, the sensitivity was 45.4% for CCCT whereas the specificity was 90.9%. Sensitivity and specificity for basal FSH were 36.3 and 81.8%, and for GAST were 72.7 and 45.4% respectively.
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Discussion |
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Traditionally, as a woman's age increases, her oestradiol response to ovulation induction and number of oocytes retrieved decreases. In fact her functional ovarian reserve plays the major role in this impaired response. This decrease in ovarian function has been attributed to an absolute reduction in the number of the follicles available for stimulation. To the best our knowledge this is the first study that directly compares ovarian reserve tests to the number of the follicles within the ovaries.
The chronological age of a woman is related to her chance of conceiving and the decline in fertility after the age of 35 years has been well documented, even in the assisted reproductive technologies (Tan et al., 1992). For this reason our study population was restricted to women over 35 years of age.
We are aware that a screening test should have a high sensitivity and low specificity in order not to underestimate the number of affected subjects. On the other hand, a test could only be diagnostic for a certain disease with a high specificity, although this would increase the cut-off value and allow individuals without disease to be included. It is therefore essential to perform receiver operator characteristic curves when comparing the accuracy of screening tests.
In the present study the basal FSH concentrations were abnormal in 27% (six out of 22) of our patients and did not accurately reflect the actual number of the follicles per unit tissue. The sensitivity of basal FSH was 36.3% and specificity 81.8%. In other words basal FSH has poor predictive value to estimate the follicles per unit tissue. However, the number of our patients who had basal FSH values <10 mIU/ml (i.e. normal) was greater than the number classified as abnormal, and this may have had an adverse effect on our results. Nevertheless, of the tests performed, basal serum FSH was the second best in providing accurate prognostic information according to ROC analysis (Figure 1).
The CCCT was first used by Navot et al. to assess the ovarian reserve (Navot et al., 1987) and this test appears to be more sensitive than basal FSH (Loumaye et al., 1990
; Tanbo et al., 1992
; Loumaye, 1995
). In our study, CCCT reflected a normal ovarian reserve in 73% (16 out of 22) of our patients. The mean number of follicles per unit tissue was significantly higher in patients with normal CCCT compared to those with an abnormal result. According to the ROC analysis, the CCCT was the most sensitive (area under the curve = 0.73, Table III
).
The prognostic value of GAST has been documented by many authors in different protocols (Muasher et al., 1988; Padilla et al., 1990
; Winslow et al., 1991
). Eight of our patients (36%) had normal ovarian reserve according to GAST. However, there was no statistically significant difference in the mean number of follicles per unit tissue between the groups with normal and abnormal GAST. Furthermore, GAST was the least sensitive test according to ROC analysis (area under the curve = 0.48, Table III
), and was relatively less accurate than the other two tests. In addition, we believe that the GAST is more invasive, expensive, and needs further standardization.
Some studies (Lass et al., 1997; Tomas et al., 1997
; Chang et al., 1998
) have assessed the use of ultrasound in evaluating ovarian reserve. Although ultrasound was performed in this study, ultrasonographic parameters were not an intended outcome measure.
We are aware of the limitations of the present study: The difficulty in recruiting patients who need an oophorectomy after 35 years of age and before they reach 50 years played a major role in defining the number of the study population. Secondly, since all of our patients were of proven fertility, the other possible factors that can affect the ovarian reserve in an infertile population cannot be eliminated in our study group. Finally, the number of the abnormal tests was small because of the limited number of the whole study group.
In conclusion, it seems unlikely that the sensitivity of the above tests can judge the ovarian reserve accurately. Nevertheless, according to ROC analysis, CCCT is more predictive of ovarian reserve compared with basal serum FSH and GAST.
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Acknowledgments |
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Notes |
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References |
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Submitted on March 29, 1999; accepted on August 16, 1999.