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Gonadotropins in women with amenorrhea
Gonadotropins in women with amenorrhea The use of plasma follicle~stimulating hormone to differentiate women with and without ovarian follicles
R. L. GOLDENBERG JAY M. GRODIN D. RODBARD GRIFF T. ROSS Bethesda, Maryland Total urinary gonadotropins, plasma luteinizing hormone ( LH), and plasma follicle-stimulating hormone (FSH) were measured in normal women and in 234 Patients with primary and secondary amenorrhea and retrospectively correlated with the presence or absence of ovarian follicles. Total urinary gonadotropin levels and plasma LH values were greater in patients without follicles than in patients with follicles and normal women, but the degree of overlap prohibited reliable diagnosis in any individual patient. However, plasma FSH values reliably predicted the presence or absence of ovarian follicles in women with either primary or secondary amenorrhea.
IN THE EVA L U AT I 0 N and treatment of amenorrhea, knowledge of the presence or absence of ovarian follicles is of major importance, and ovarian biopsy either by culdoscopy, laparoscopy, or laparotomy is an accepted procedure for making this determination. Attempts have been made to associate biological parameters such as urinary gonadotropins and plasma luteinizing hormone (LH) levels with the presence of follicles, but correlation has often not been sufcient to obviate a diagnostic surgical procedure with its associated risks. 1 - 5 We have re-examined the possibility of
obtaining the necessary diagnostic information indirectly by correlating the presence or absence of ovarian follicles with measurements of urinary gonadotropins and plasma LH and follicle-stimulating hormone (FSH) values in 234 women with primary and secondary amenorrhea. Although the distributions of urinary gonadotropin and plasma LH values were clearly different in women with and without follicles, the degree of overlap prohibited reliable diagnosis in individual patients. However, a single plasma FSH value was found sufficient to diagnose the presence or absence of follicles in every woman in our series.
From the Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health. Received for publication December 28, 1972. Accepted February 2, 1973. Reprint requests: R. L. Goldenberg, Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar St., New Haven, Connecticut 06510.
Methods In the diagnostic evaluation of 234 women with primary (58) or secondary ( 176) amenorrhea, we have obtained an ovarian biopsy by either laparoscopy, culdoscopy, or laparotomy. Specimens were fixed, sectioned, and stained by standard methods and evaluated microscopically for presence or absence of follicles. Total urinary gonadotropins were 1003
1004
Goldenberg et al.
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Augml I. 197:l Obstet. Gynecol.
Table I. Distribution of women with primary and secondary amenorrhea with and without follicles according to their probable diagnosis Amenorrhea Primary
With follicles
With follicles Sclerocystic ovaries Karyotypic mosaic Unknown etiology Subtotal
25
Without follicles
Sclerocystic ovaries Pituitary adenoma Post oral contraceptives Post Depo-Provera Ahumada, Del Castillo, Argonz Chiari-Frommel Unknown etiology Subtotal
38
7 17 ')
4 7 69 144
Without follicles
Turner's syndrome 20 Gonadal dysgenesis with normal karyotype 13 Subtotal All patients with follicles All patients without follicles
176
Secondary
SBI
33
Premature menopause Castrate Subtotal
10 ')')
32
169
measured by bioassay." Plasma LH and FSH were measured by specific radioimmunoassay.'· ' The Semnd International Reference Preparation (IRP) of Human Menopausal Gonadotropin was used for dose interpolation in both LH and FSH assays. In our laboratory, 1 ml.U. of LH activity of the 2nd IRP is equivalent to 3.9 ng. of LER 907, a pituitary gonadotropin preparation, and 1 miT. of FSH activity equivalent to 30.0 ng. of LER 907. Plasma and urine specimens collected from a group of 40 women, judged to be normal on the basis of a history of regular menses associated with biphasic basal body tempt:>raturt> curves, served as controls for the study of similar specimens from the women with amenorrht:>a. In Table I the patients are grouped on the basis of primary or secondary amenorrhea, pre,enn· or absence of follicles, and the etiologic diagnosis of the disorder. Of those patients with primary amenorrhea. 20 had either the stigmata of Turner's syndrome or a karyotypf' 45,X or both. These patients and 1:) others without m·arian follicles had no eyidcncc of spontaneous sexual dewlopmf'nt. One patient with primary amenorrhea with follicles had :wkrocystic ovaries, con>istent with ~~ diagnosis of Stein-Leventhal syndrome. Another patient with a mosaic
65
karyotype of 45,X, 46,XX, and 47,XXX in cells from peripheral blood had follicles and well-developed secondary sexual characteristics. The remainder of the patients whose ovaries contained follicles were assumed to have amenorrhea secondary to hypothalamohypophyseal dysfunction. There were 176 patients with secondary amenorrhea. Again, these patients were grouped according to presence or absence of follicles and separated into categories on the basis of their probable diagnosis. Ten of the patients with secondary amenorrhea had symptoms suggestive of premature menopause, and no follicles were found in ovarian biopsy from any of these women. Since specimens from these women and from 22 surgically castrated patients did not differ significantly, the two groups were combined. The distributions of total urinary gonadotropins, plasma LH values, and plasma FSH values for each group of women are presented. In addition, a median plasma LH and plasma FSH value has been calculated for each group. Results
Normal volunteers. Fig. 1 presents the distribution of 321 total urinary gonadotropins (from 17 women) , 805 plasma LH samples, and 882 plasma FSH samples from 40 normal
Gonadotropins and amenorrhea
Volume 116 Number 7
1005
60 40
n' 321
20
0'--------
<10
>10<50 >50<200 >200 <500
>500
URINARY GONADOTROPINS (muu)
40
'" u.J ~
.
n ,805
20
'">-
:z
t5 0
a:
u.J 0..
0-10
10-20
20-30
30-40
40-50
50-75
75-100
>100
75-100
>100
LH mlU/m l
60
n '882
40
20
o
0-10
10-20
20-30
30-40
40-50
50-75
FSH mlU I ml
Fig. 1. Distribution of total gonadotropic activity (bioassay) in 24 hour urine collections and of immunoreactive FSH and LH (radioimmunoassay) in plasma samples from normal adult women. n Number of samples.
=
cycling women. The total gonadotropic activities measured in random 24 hour urine specimens were grouped as either < 10 mouse uterine units (MUU), > 10 < 50 MUU, > 50 < 200 MUU, > 200 < 500 MUU, and > 500 MUU. Although a majority of the values were less than 50 MUU, with 34 per cent having gonadotropins below the limits of detection, 10 per cent of the samples were greater than 50 MUU and 2 per cent were greater than 200 MUU. In our normal population, the median plasma LH value was 17 mI.U. per milliliter, and 50 per cent of the samples were in the range from 10 to 20 mI.u. per milliliter. Plasma LH values greater than 100 mI.U. per milliliter were found in one per cent of samples from normal patients, almost always at the time of the midcycle peak. In this population, the median FSH value was 9 mI.u. per milliliter. Sixty-eight per
cent of plasma FSH values were less than 10 mI.U. per milliliter, and not one sample gave a value exceeding 44 mI.U. per milliliter. Primary amenorrhea. Figs. 2, 3, and 4 present urinary gonadotropins, plasma LH, and plasma FSH in women with primary amenorrhea classified according to the presence or absence of ovarian follicles. When the distribution of total urinary gonadotropin activities were examined (Fig. 2), it was immediately clear that most values in women with follicles were different from most of those in women without follicles; but considerable overlap between groups was found. This precluded the possibility of using this measurement for diagnostic purposes for individual patients. Examination of LH concentrations ,· in plasma from patients with primary amenorrhea (Fig. 3) revealed that women with
1006 Goldenberg et 01.
August 1, 1973 Am.
60 •
J. Obstet. Gynecol.
o Without follicles n'160
With follicles
n' 153 50
VI
w --'
40
Cl.
::0 <[
VI I-
z
30
w
u
a:: w
Cl.
20
10
0
<10
>10<50 >50<200
-
>200<500
n
>500
URINARY GONADOTROPINS (muu)
Fig. 2. Distribution of total gonadotropic activity (bioassay ) in 24 hour urine collections from women with primary amenorrhea with and without follicles. n Number of samples.
=
60 •
With follicles
n'275
0
Without follicles
n'247
50
::3 40 --'
Cl.
2; <[
~ 30
:z w
u
a::
UJ Cl.
20
10
OL-~~
0-10
__
~~
10-20
__
.u~~~~
20-30
30-40
__
~L-~~
40-50
50-75
__.uLL__-l~
75-100
> 100
PLASMA LH mlU/ml
Fig. 3. Distribution of plasma LH (radioimmunoassay ) in samples from women with primary amenorrhea with and without follicles. n Number of samples.
=
ovarian follicles had a median value of 18 mI.U . per milliliter, while women without follicles had a median LH value of 75 mI.U . per milliliter. However, considerable overlap between groups was again found. The two groups (pa tients with and without follicles ) were separated completely on the basis of FSH levels (Fig. 4). Median FSH in patients with primary amenorrhea and follicles was 6 mI.u. per milliliter, and no patient had a plasma FSH value greater
than 33 mI.u. per milliliter. Women without follicles had a median plasma FSH of 104 mI.U. per milliliter, and none had an FSH value less than 50 mr.u. per milliliter. Thus, in this series, a single plasma FSH value could be used for diagnosis of the presence or absence of follicles. Secondary amenorrhea. Figs. 5, 6, and 7 present total urinary gonadotropins, plasma LH, and plasma FSH concentrations in women with secondary amenorrhea. The
Volume 116 Number 7
Gonadotropins and amenorrhea
60
•
0
With follicles
n·272
1007
Without follicles
n·382
50
t:l 40
...J Cl.
:::E
«
lZ
30
lj a::
UJ Cl.
20
10
OL-~--~--~·----~---~~~~~-=~~~20-30 30-40 40-50 50-75 75-100 >100 0-10 15-20 PLASMA FSH mlU/ml
Fig. 4. Distribution of plasma FSH (radioimmunoassay) in samples from women with primary amenorrhea with and without follicles . n Number of samples .
=
60
•
With follicles
n· 452
Without follicles
O n· 191
50
'j 40 Cl.
:::E
r
«
I-
~ 30 a::
u
UJ Cl.
20
10
<10
> 10<50
>50<200
>200<500
>500
URINARY GONADOTROPINS (muu)
Fig. 5. Distribution of total gonadotropin activity (bioassay) in 24 hour urine collections from women with secondary amenorrhea with and without follicles. n Number of samples.
=
median plasma values for LH and FSH in women with follicles was 17 and 12 mI.U. per milliliter, respectively, and 86 mI.u. per milliliter and 97 mI.u. per milliliter in women without follicles. Once again, however, because of the degree of overlap, neither urinary gonadotropins nor plasma LH values completely separated women with follicles from those without ovarian follicles. Plasma FSH clearly made this differentiation (Fig. 7) .
Comment
There is increasing evidence for both positive and negative feedback control of pituitary gonadotropin secretion mediated by ovarian sex steroid hormones in women. 9 For the most part, these steroid hormones are products of the follicular apparatus. Results of our studies suggest that the absence of follicles is reflected more accurately by changes in secretion of FSH than LH. Recent studies of Van Thiel and associates 10
1008 Goldenberg et 01.
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August I, 1973 GynecoJ.
J. Obstet.
60 •
Wilh follicles
n '785
0
Wilhout follicles
n ' 314
50 Vl
~ 40
Cl. ~
~ 30
u
a:
UJ Cl.
20
10
0L-~0·_-10--~I·OL _2-0~--20~_~301-~3· 0_~4~0---4·0U_L50L-~aL~~7· 5_uI0~0--~>~IO~0L-PLASMA LH mlU/ml
Fig. 6. Distribution of plasma LH (radioimmunoassay) in samples from women with secondary amenorrhea with and without follicles. n Number of samples.
=
60 •
With follicles
n' 713
o n'Without follicles 397
50
CJ')
40
UJ ....J Cl. ~
I-
30
:z UJ
1E UJ Cl.
20
I
10
OL-__L-__~L-__~____~______~CL__~~~~~__~~
0-10
10-20
20-30
30-40
40-50
50-75
75-100
> 100
PLASMA FSH mlU/ml
Fig. 7. Distribution of plasma }ISH (radioimmunoassay ) in samples from women with secondary amenorrhea with and without follicles. n Number of samples.
=
demonstrated that absence of germinal epithelium in the male is correlated with significant elevation of FSH but little or no elevation of LH. lO This suggests the possibility that negative feedback control of FSH in both the male and female may operate by a similar mechanism. We have demonstra ted that a single specific plasma FSH determination based upon a reliable radioimmunoassay was diagnostic of the presence or absence of follicles in
women with either primary or secondary amenorrhea. The distribution of plasma FSH levels in women with amenorrhea with oocytes was the same as that for normal women and never exceeded the maximum normal value. A single plasma FSH value above the normal range of values for this determination in our laboratory was indicative of the absence of follicles. Conversely, when no exogenous steroid hormones had been given, and there was no evidence of
Volume 116 Number 7
pituitary disease, a single FSH value within the normal range was indicative of the presence of ovarian follicles. A large interlaboratory variation in plasma FSH values has been reported.U Further, the normal range for FSH values appears to depend on the antiserum used.U Thus, it is necessary to establish the normal range of values from a specific laboratory before using FSH values for diagnostic purposes and to use appropriate statistical quality control procedures. 14 To our knowledge there are only a limited number of patients for whom such FSH determinations might not provide correct classification. Several patients with follicles reported by Jones and Moraes-Ruehsen 12 and Starup and co-workers 13 were stated to have high urinary and plasma gonadotropins, although the exact values were not reported. It is possible that plasma FSH values from similar patients would fall outside the normal range in our laboratory and make for exceptions to our conclusion. In addition, a thorough statistical analysis of the present data suggests that the diagnostic rule presented here may fail in a small percentage of cases. To date, however, we have never
REFERENCES
1. Taymor, M. L.: In Collins, C. J., editor: Management of Amenorrhea, Springfield, Ill., 1966, Charles C Thomas, Publisher. 2. Reschini, E., Giustina, G., D' Alberton, A., and Chierichetti, G.: AM. J. OnsTET. GYNEcoL. 111: 173, 1971. 3. Penny, R., Guyda, H. ]., Baghdassarian, A., Johanson, A. ]., and Blizzard, R. M.: J. Cliri. Invest. 49: 1847, 1970. 4. Ryan, R. J., Cloutier, M. D., Hayles, A. B., Paris, J., and Randall, R. V.: Med. Clin. North Am. 54: 1079, 1970. 5. Schalch, D. S., Parlow, A. F., Boon, R. C., and Reichlin, S.: J. Clin. Invest. 47: 665, 1968. 6. Ross, G. T., Brice, J., and Reid, R.: In Sunderman, F. W., and Sunderman, F. W., Jr., editors: Laboratory Diagnosis of Endocrine Diseases, St. Louis, 1971, Warren H. Green, Inc., p. 148. 7. Odell, W. D., Ross, G. T., and Rayford, P.: J. Clin. Invest. 46: 248, 1967.
Gonadotropins and amenorrhea
1009
seen a patient with follicles who had plasma FSH concentrations outside the normal range. Nevertheless, to rule out the possibility of laboratory error and to reduce the probability of misdiagnosis by a random sample outside predicted ranges for a given laboratory, we suggest that at least two plasma samples be evaluated. The treatment of anovulatory infertility has improved markedly over the last decade because of the introduction of clomiphene and human menopausal gonadotropins-two potent drugs for induction of ovulation. Because of the expense and side effects of these drugs, treatment without chance of success is contraindicated. Identification of patients with amenorrhea who have follicles and therefore can respond to one or the other of these drugs is of major importance. For the purpose of identifying patients with the potential to respond, ovarian biopsy, regardless of how it is performed, is costly and associated with the morbidity of an anesthetic and operative procedure. Because plasma FSH values predict the presence or absence of ovarian follicles, a surgical procedure to obtain this information should rarely be necessary.
8. Cargille, C. M., Ross, G. T., and Yoshimi, T.: J. Clin. Endocrinol. 29: 12, 1969. 9. Ross, G. T., Cargille, C. M., Lipsett, M. B., Rayford, P. L., Marshall, J. R., Strott, C. A., and Rodbard, D.: Recent Progr. Hormone Research 26: 1, 1970. 10. Van Thiel, D. H., Sherins, R. J., Myers, G. H., and DeVita, V. T.: J. Clin. Invest. 51: 1009, 1972. 11. Cargille, C. M., Rodbard, D., and Ross, G. T.: J. Clin. Endocrinol. Metab. 28: 1276, 1968. 12. Jones, G. S., and Moraes-Ruehsen, M.: AM. J. OnsTET. GYNECOL. 104: 597, 1969. 13. Starup, ]., Sele, V., and Henriksen, B.: Acta Endocrinol. 66: 248, 1971. 14. Rodbard, D.: In Odell, W. D., and Daughaday, W. H., editors: Principles of Competitive Protein Binding Assays, Philadelphia, 1971, J. B. Lippincott Co., p. 204. 15. Harris, E. K., and De Mets, D. L.: Clin. Chern. 18: 244, 1972.
August 1, 1973
1010 Goldenberg et al.
Statistical appendix 1. These data were collected in a retrospective study; accordingly, the interpretation of the results is subject to the limitations inherent in any clinical study of this type. Ideally, these results should be confirmed in a prospective study. 2. The nature of the clinical "population" studied at the NIH as shown in Table I is not necessarily representative of the clinical "population" at other medical centers. No formal method of "random sampling" was used. However, all patients referred to an infertility clinic were included and no apparent preselection of patients were used. Ideally, these findings should be confirmed on an independent patient population. 3. The data presented here (Fig. 1 to 7) were obtained in many different assays over the period of several years. Further, the standards, antisera, second antibody, and minor details of the radioimmunoassays (RIA's) for FSH and LH changed on at least two occasions during the course of these studies. This introduces extraneous sources of variation which, if anything, should reduce (rather than create or accentuate) the observed differences between diagnostic groups. Accordingly, we infer that the diagnostic accuracy of the plasma FSH value as an indicator of the presence or absence of follicles might be even better if all measurements are made within the same assay. When measurements obtained in different RIA's (or bioassays) are to be compared, it is essential to include a rigorous quality control system. 14 4. The distributions shown in Figs. 1 to 7 are "composite"; i.e., they include variation within individuals (between days), as well as a component of variation between individuals. The number of observations per subject is quite variable (from one to 30). Further, measurement error (both within and between assays) is superimposed on the genuine physiologic variation. 5. The shape of the distributions is asymmetrical, generally with "skewing to the right." Use of a logarithmic transformation results in an approach to Gaussian distributions (i.e., the measurements have a "log-normal" distribution as a first approximation). Also, the use of a log transform markedly reduces the severity of nonuniformity of variance and provides "additivity." Accordingly. a subset of the data were studied by analysis of variance ( ANOVA) after the log transformation, using a "one-way, nested design with subsampling." 15 The results are shown in Table ITA. These results must be in-
Am.
J. Obstet. Gynecol.
terpreted, however, keeping in mind the limitations of the data (items 1 to 4, above) . These results indicate, that for log (plasma FSH), the means of the distributions for patients with and without follicles are approximately 4 standard deviations apart (using the standard deviation obtained if we had only one measurement per subject). Thus, using a "cut point" midway between the two means (on a log scale), we might expect a 2.5 per cent chance of misdiagnosis if the distributions were truly "log-normal." By increasing the number of measurements per subject, we reduce the measurement error, but do not reduce the variability between subjects (the latter may be reduced, if all appropriate comparisons are made within the same assay). It should be emphasized, that in the present study, there was no overlap observed. Also, by appropriate selection of the "cut point," one can control the relative magnitude of the two types of possible misdiagnosis. Also, this ANOVA shows that the difference in the means (for groups with and without follicles) for log LH is smaller, and the variation within diagnostic groups is greater, than for the log FSH measurements, such that the means are only separated 2.5 standard deviations. Thus, using a cut point midway between the two means, one would expect at least a 10 per cent rate of misdiagnosis. This is the case for the secondary amenorrhea patient population. The overlap is even greater for patients with primary amenorrhea, where a bimodal distribution is observed (see Table liB, Fig. 3, and Fig. 6). 6. If data are analyzed without the log transform, then variance (or u) for patients without follicles is enormous compared to that for patients with follicles. However, after the log transform, the standard deviation for patients with follicles (and normal gonadotrophins) is now larger (i.e., the large absolute values have a larger standard deviation but a smaller coefficient of variation). By use of transforms such as log (X + C) , or
y
X + C, one could probably obtain better uniformity of variance. However, in view of the problems with these data, further analysis was deemed unwarranted. 7. Urinary gonadotropins (Table IIC). Mean values for patients with secondary amenorrhea are significantly greater than values for patients with primary amenorrhea for patients with follicles. A significant difference is also seen in patients without follicles, although the difference
Volume 116 Number 7
Gonadotropins and amenorrhea
1011
Table IIA With follicles ]•
Log FSH
No. of observations Mean Standard deviation
155 0.955
660 1.01
0.330 0.225 0.271 0.241
(!'1
u, Ira
u.
Without follicles
zo
2"
0.285 0.192 0.313 0.256
164 2.03 0.183 O.o75 0.143 0.167
2• 180 1.93 0.212 0.074 0.169 0.108
following footnotes refer to Tables IIA and liB: 1. All calculations use logto of the LH (or FSH) values. 2. Number of degrees of freedom not sbown. 3. Some of these values were calculated on a subset of the data. 4. Even after log transform, there is heterogeneity of variance, in that the u for patients without follicles is less than the corresponding u for patients with follicles.
=
=
5. "' Standard deviation, between patients within diagnostic groups, assuming one measurement per subject. 112 Standard deviation (pooled) of all measurements within any given patient. us Standard deviation for all u measurements within a diagnostic group; this is a composite, and represents an "average" of "' and '"· depending on the number of rep· licates per subject, and the number of subjects which is variable. "' "Component of variance" attributable to differences between subjects, after subtracting the variance within subjects. 6. The linear additive model states that the variance between subjects (within diagnostic groups), expressed for a mean of r replicates per subject, is given by:
=
=
a2
=
a~/r
+ ai
i.e., the total error can be reduced by increasing the number of replicates, r; (to reduce day-to·day fluctuations and measurement error). However, it cannot be reduced below the intrinsic variation between subjects, U<. 7. a, May be biased upward, i.e., an overconservative estimate, since it includes between-assay variance, whereas U> usually contains only within-assay variance.
Table IIB With follicles Log LH
No. of observations Mean Standard deviation
2"
1"
2"
179 1.27
674 1.24
252 1.86
119 1.78
0.374 0.130 0.388 0.351
tr,
u, tr,
u,
Without follicles
1"
0.339 0.191 0.376 0.280
0.242 0.117 0.200 0.212
0.239 0.106 0.215 0.214
Table IIC
Urinary gonadotropins
1"
No. of patients No. of samples Mean Standard deviation Mean* Standard deviation*
17 321 38.8 88.9 1.21 0.58
35 153 25.6 39 0.98 0.60
*Calculation after log transform.
Without follicles
With follicles
Normal subjects
2'
]•
2'
132 452 51.1 60 1.42 0.57
34 160 215 254 2.09 0.50
19 91 240 241 2.23 0.35
1012 Goldenberg et al.
is smaller (on a log scale) . This indicates significant heterogeneity in the data, and prevents pooling of data from 1o and 2° amenorrhea. Also, patients with follicles, with 1o amenorrhea have significantly less urinary gonadotropins than do normal subjects, whereas patients with 2° amenorrhea have significantly more. Although these differences are statistically significant ( P < 0.05), they do not carry diagnostic significance. However, they may well reflect genuine physiologic differences among the various groups. 8. LH. Here the differences between 1o and 2 o amenorrhea are virtually zero (Table liB), whether one considers patients with or without follicles. The values for patients with either 1o or 2° amenorrhea with oocytes are indistinguishable from the normal range. 9. FSH. Again, there is no signicant difference
August I, 1973 Am. J. Obstet. Gynecol.
between patients with 1o or 2o amenorrhea, either for patients with or without oocytes. Values for patients with 2° amenorrhea with oocytes are higher than values for patients with 1o amenorrhea with oocytes. This difference is in the same direction as seen in the urinary gonadotropin data. 10. Ideally, one should examine the correlations between FSH, LH, and urinary gonadotropins. It is possible that using all 3 tests together (as in discriminant analysis or cluster analysis) , one could obtain better diagnostic accuracy than with any one test alone. Also, it would be worthwhile to study the urinary gonadotropin activity as it relates to plasma FSH and LH. Unfortunately, the data for this study were not collected in such a way as to make this type of analysis possible.
R. L. GOLDENBERG JAY M. GRODIN D. RODBARD GRIFF T. ROSS Bethesda, Maryland Total urinary gonadotropins, plasma luteinizing hormone ( LH), and plasma follicle-stimulating hormone (FSH) were measured in normal women and in 234 Patients with primary and secondary amenorrhea and retrospectively correlated with the presence or absence of ovarian follicles. Total urinary gonadotropin levels and plasma LH values were greater in patients without follicles than in patients with follicles and normal women, but the degree of overlap prohibited reliable diagnosis in any individual patient. However, plasma FSH values reliably predicted the presence or absence of ovarian follicles in women with either primary or secondary amenorrhea.
IN THE EVA L U AT I 0 N and treatment of amenorrhea, knowledge of the presence or absence of ovarian follicles is of major importance, and ovarian biopsy either by culdoscopy, laparoscopy, or laparotomy is an accepted procedure for making this determination. Attempts have been made to associate biological parameters such as urinary gonadotropins and plasma luteinizing hormone (LH) levels with the presence of follicles, but correlation has often not been sufcient to obviate a diagnostic surgical procedure with its associated risks. 1 - 5 We have re-examined the possibility of
obtaining the necessary diagnostic information indirectly by correlating the presence or absence of ovarian follicles with measurements of urinary gonadotropins and plasma LH and follicle-stimulating hormone (FSH) values in 234 women with primary and secondary amenorrhea. Although the distributions of urinary gonadotropin and plasma LH values were clearly different in women with and without follicles, the degree of overlap prohibited reliable diagnosis in individual patients. However, a single plasma FSH value was found sufficient to diagnose the presence or absence of follicles in every woman in our series.
From the Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health. Received for publication December 28, 1972. Accepted February 2, 1973. Reprint requests: R. L. Goldenberg, Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar St., New Haven, Connecticut 06510.
Methods In the diagnostic evaluation of 234 women with primary (58) or secondary ( 176) amenorrhea, we have obtained an ovarian biopsy by either laparoscopy, culdoscopy, or laparotomy. Specimens were fixed, sectioned, and stained by standard methods and evaluated microscopically for presence or absence of follicles. Total urinary gonadotropins were 1003
1004
Goldenberg et al.
Am.
J.
Augml I. 197:l Obstet. Gynecol.
Table I. Distribution of women with primary and secondary amenorrhea with and without follicles according to their probable diagnosis Amenorrhea Primary
With follicles
With follicles Sclerocystic ovaries Karyotypic mosaic Unknown etiology Subtotal
25
Without follicles
Sclerocystic ovaries Pituitary adenoma Post oral contraceptives Post Depo-Provera Ahumada, Del Castillo, Argonz Chiari-Frommel Unknown etiology Subtotal
38
7 17 ')
4 7 69 144
Without follicles
Turner's syndrome 20 Gonadal dysgenesis with normal karyotype 13 Subtotal All patients with follicles All patients without follicles
176
Secondary
SBI
33
Premature menopause Castrate Subtotal
10 ')')
32
169
measured by bioassay." Plasma LH and FSH were measured by specific radioimmunoassay.'· ' The Semnd International Reference Preparation (IRP) of Human Menopausal Gonadotropin was used for dose interpolation in both LH and FSH assays. In our laboratory, 1 ml.U. of LH activity of the 2nd IRP is equivalent to 3.9 ng. of LER 907, a pituitary gonadotropin preparation, and 1 miT. of FSH activity equivalent to 30.0 ng. of LER 907. Plasma and urine specimens collected from a group of 40 women, judged to be normal on the basis of a history of regular menses associated with biphasic basal body tempt:>raturt> curves, served as controls for the study of similar specimens from the women with amenorrht:>a. In Table I the patients are grouped on the basis of primary or secondary amenorrhea, pre,enn· or absence of follicles, and the etiologic diagnosis of the disorder. Of those patients with primary amenorrhea. 20 had either the stigmata of Turner's syndrome or a karyotypf' 45,X or both. These patients and 1:) others without m·arian follicles had no eyidcncc of spontaneous sexual dewlopmf'nt. One patient with primary amenorrhea with follicles had :wkrocystic ovaries, con>istent with ~~ diagnosis of Stein-Leventhal syndrome. Another patient with a mosaic
65
karyotype of 45,X, 46,XX, and 47,XXX in cells from peripheral blood had follicles and well-developed secondary sexual characteristics. The remainder of the patients whose ovaries contained follicles were assumed to have amenorrhea secondary to hypothalamohypophyseal dysfunction. There were 176 patients with secondary amenorrhea. Again, these patients were grouped according to presence or absence of follicles and separated into categories on the basis of their probable diagnosis. Ten of the patients with secondary amenorrhea had symptoms suggestive of premature menopause, and no follicles were found in ovarian biopsy from any of these women. Since specimens from these women and from 22 surgically castrated patients did not differ significantly, the two groups were combined. The distributions of total urinary gonadotropins, plasma LH values, and plasma FSH values for each group of women are presented. In addition, a median plasma LH and plasma FSH value has been calculated for each group. Results
Normal volunteers. Fig. 1 presents the distribution of 321 total urinary gonadotropins (from 17 women) , 805 plasma LH samples, and 882 plasma FSH samples from 40 normal
Gonadotropins and amenorrhea
Volume 116 Number 7
1005
60 40
n' 321
20
0'--------
<10
>10<50 >50<200 >200 <500
>500
URINARY GONADOTROPINS (muu)
40
'" u.J ~
.
n ,805
20
'">-
:z
t5 0
a:
u.J 0..
0-10
10-20
20-30
30-40
40-50
50-75
75-100
>100
75-100
>100
LH mlU/m l
60
n '882
40
20
o
0-10
10-20
20-30
30-40
40-50
50-75
FSH mlU I ml
Fig. 1. Distribution of total gonadotropic activity (bioassay) in 24 hour urine collections and of immunoreactive FSH and LH (radioimmunoassay) in plasma samples from normal adult women. n Number of samples.
=
cycling women. The total gonadotropic activities measured in random 24 hour urine specimens were grouped as either < 10 mouse uterine units (MUU), > 10 < 50 MUU, > 50 < 200 MUU, > 200 < 500 MUU, and > 500 MUU. Although a majority of the values were less than 50 MUU, with 34 per cent having gonadotropins below the limits of detection, 10 per cent of the samples were greater than 50 MUU and 2 per cent were greater than 200 MUU. In our normal population, the median plasma LH value was 17 mI.U. per milliliter, and 50 per cent of the samples were in the range from 10 to 20 mI.u. per milliliter. Plasma LH values greater than 100 mI.U. per milliliter were found in one per cent of samples from normal patients, almost always at the time of the midcycle peak. In this population, the median FSH value was 9 mI.u. per milliliter. Sixty-eight per
cent of plasma FSH values were less than 10 mI.U. per milliliter, and not one sample gave a value exceeding 44 mI.U. per milliliter. Primary amenorrhea. Figs. 2, 3, and 4 present urinary gonadotropins, plasma LH, and plasma FSH in women with primary amenorrhea classified according to the presence or absence of ovarian follicles. When the distribution of total urinary gonadotropin activities were examined (Fig. 2), it was immediately clear that most values in women with follicles were different from most of those in women without follicles; but considerable overlap between groups was found. This precluded the possibility of using this measurement for diagnostic purposes for individual patients. Examination of LH concentrations ,· in plasma from patients with primary amenorrhea (Fig. 3) revealed that women with
1006 Goldenberg et 01.
August 1, 1973 Am.
60 •
J. Obstet. Gynecol.
o Without follicles n'160
With follicles
n' 153 50
VI
w --'
40
Cl.
::0 <[
VI I-
z
30
w
u
a:: w
Cl.
20
10
0
<10
>10<50 >50<200
-
>200<500
n
>500
URINARY GONADOTROPINS (muu)
Fig. 2. Distribution of total gonadotropic activity (bioassay ) in 24 hour urine collections from women with primary amenorrhea with and without follicles. n Number of samples.
=
60 •
With follicles
n'275
0
Without follicles
n'247
50
::3 40 --'
Cl.
2; <[
~ 30
:z w
u
a::
UJ Cl.
20
10
OL-~~
0-10
__
~~
10-20
__
.u~~~~
20-30
30-40
__
~L-~~
40-50
50-75
__.uLL__-l~
75-100
> 100
PLASMA LH mlU/ml
Fig. 3. Distribution of plasma LH (radioimmunoassay ) in samples from women with primary amenorrhea with and without follicles. n Number of samples.
=
ovarian follicles had a median value of 18 mI.U . per milliliter, while women without follicles had a median LH value of 75 mI.U . per milliliter. However, considerable overlap between groups was again found. The two groups (pa tients with and without follicles ) were separated completely on the basis of FSH levels (Fig. 4). Median FSH in patients with primary amenorrhea and follicles was 6 mI.u. per milliliter, and no patient had a plasma FSH value greater
than 33 mI.u. per milliliter. Women without follicles had a median plasma FSH of 104 mI.U. per milliliter, and none had an FSH value less than 50 mr.u. per milliliter. Thus, in this series, a single plasma FSH value could be used for diagnosis of the presence or absence of follicles. Secondary amenorrhea. Figs. 5, 6, and 7 present total urinary gonadotropins, plasma LH, and plasma FSH concentrations in women with secondary amenorrhea. The
Volume 116 Number 7
Gonadotropins and amenorrhea
60
•
0
With follicles
n·272
1007
Without follicles
n·382
50
t:l 40
...J Cl.
:::E
«
lZ
30
lj a::
UJ Cl.
20
10
OL-~--~--~·----~---~~~~~-=~~~20-30 30-40 40-50 50-75 75-100 >100 0-10 15-20 PLASMA FSH mlU/ml
Fig. 4. Distribution of plasma FSH (radioimmunoassay) in samples from women with primary amenorrhea with and without follicles . n Number of samples .
=
60
•
With follicles
n· 452
Without follicles
O n· 191
50
'j 40 Cl.
:::E
r
«
I-
~ 30 a::
u
UJ Cl.
20
10
<10
> 10<50
>50<200
>200<500
>500
URINARY GONADOTROPINS (muu)
Fig. 5. Distribution of total gonadotropin activity (bioassay) in 24 hour urine collections from women with secondary amenorrhea with and without follicles. n Number of samples.
=
median plasma values for LH and FSH in women with follicles was 17 and 12 mI.U. per milliliter, respectively, and 86 mI.u. per milliliter and 97 mI.u. per milliliter in women without follicles. Once again, however, because of the degree of overlap, neither urinary gonadotropins nor plasma LH values completely separated women with follicles from those without ovarian follicles. Plasma FSH clearly made this differentiation (Fig. 7) .
Comment
There is increasing evidence for both positive and negative feedback control of pituitary gonadotropin secretion mediated by ovarian sex steroid hormones in women. 9 For the most part, these steroid hormones are products of the follicular apparatus. Results of our studies suggest that the absence of follicles is reflected more accurately by changes in secretion of FSH than LH. Recent studies of Van Thiel and associates 10
1008 Goldenberg et 01.
Am.
August I, 1973 GynecoJ.
J. Obstet.
60 •
Wilh follicles
n '785
0
Wilhout follicles
n ' 314
50 Vl
~ 40
Cl. ~
~ 30
u
a:
UJ Cl.
20
10
0L-~0·_-10--~I·OL _2-0~--20~_~301-~3· 0_~4~0---4·0U_L50L-~aL~~7· 5_uI0~0--~>~IO~0L-PLASMA LH mlU/ml
Fig. 6. Distribution of plasma LH (radioimmunoassay) in samples from women with secondary amenorrhea with and without follicles. n Number of samples.
=
60 •
With follicles
n' 713
o n'Without follicles 397
50
CJ')
40
UJ ....J Cl. ~
I-
30
:z UJ
1E UJ Cl.
20
I
10
OL-__L-__~L-__~____~______~CL__~~~~~__~~
0-10
10-20
20-30
30-40
40-50
50-75
75-100
> 100
PLASMA FSH mlU/ml
Fig. 7. Distribution of plasma }ISH (radioimmunoassay ) in samples from women with secondary amenorrhea with and without follicles. n Number of samples.
=
demonstrated that absence of germinal epithelium in the male is correlated with significant elevation of FSH but little or no elevation of LH. lO This suggests the possibility that negative feedback control of FSH in both the male and female may operate by a similar mechanism. We have demonstra ted that a single specific plasma FSH determination based upon a reliable radioimmunoassay was diagnostic of the presence or absence of follicles in
women with either primary or secondary amenorrhea. The distribution of plasma FSH levels in women with amenorrhea with oocytes was the same as that for normal women and never exceeded the maximum normal value. A single plasma FSH value above the normal range of values for this determination in our laboratory was indicative of the absence of follicles. Conversely, when no exogenous steroid hormones had been given, and there was no evidence of
Volume 116 Number 7
pituitary disease, a single FSH value within the normal range was indicative of the presence of ovarian follicles. A large interlaboratory variation in plasma FSH values has been reported.U Further, the normal range for FSH values appears to depend on the antiserum used.U Thus, it is necessary to establish the normal range of values from a specific laboratory before using FSH values for diagnostic purposes and to use appropriate statistical quality control procedures. 14 To our knowledge there are only a limited number of patients for whom such FSH determinations might not provide correct classification. Several patients with follicles reported by Jones and Moraes-Ruehsen 12 and Starup and co-workers 13 were stated to have high urinary and plasma gonadotropins, although the exact values were not reported. It is possible that plasma FSH values from similar patients would fall outside the normal range in our laboratory and make for exceptions to our conclusion. In addition, a thorough statistical analysis of the present data suggests that the diagnostic rule presented here may fail in a small percentage of cases. To date, however, we have never
REFERENCES
1. Taymor, M. L.: In Collins, C. J., editor: Management of Amenorrhea, Springfield, Ill., 1966, Charles C Thomas, Publisher. 2. Reschini, E., Giustina, G., D' Alberton, A., and Chierichetti, G.: AM. J. OnsTET. GYNEcoL. 111: 173, 1971. 3. Penny, R., Guyda, H. ]., Baghdassarian, A., Johanson, A. ]., and Blizzard, R. M.: J. Cliri. Invest. 49: 1847, 1970. 4. Ryan, R. J., Cloutier, M. D., Hayles, A. B., Paris, J., and Randall, R. V.: Med. Clin. North Am. 54: 1079, 1970. 5. Schalch, D. S., Parlow, A. F., Boon, R. C., and Reichlin, S.: J. Clin. Invest. 47: 665, 1968. 6. Ross, G. T., Brice, J., and Reid, R.: In Sunderman, F. W., and Sunderman, F. W., Jr., editors: Laboratory Diagnosis of Endocrine Diseases, St. Louis, 1971, Warren H. Green, Inc., p. 148. 7. Odell, W. D., Ross, G. T., and Rayford, P.: J. Clin. Invest. 46: 248, 1967.
Gonadotropins and amenorrhea
1009
seen a patient with follicles who had plasma FSH concentrations outside the normal range. Nevertheless, to rule out the possibility of laboratory error and to reduce the probability of misdiagnosis by a random sample outside predicted ranges for a given laboratory, we suggest that at least two plasma samples be evaluated. The treatment of anovulatory infertility has improved markedly over the last decade because of the introduction of clomiphene and human menopausal gonadotropins-two potent drugs for induction of ovulation. Because of the expense and side effects of these drugs, treatment without chance of success is contraindicated. Identification of patients with amenorrhea who have follicles and therefore can respond to one or the other of these drugs is of major importance. For the purpose of identifying patients with the potential to respond, ovarian biopsy, regardless of how it is performed, is costly and associated with the morbidity of an anesthetic and operative procedure. Because plasma FSH values predict the presence or absence of ovarian follicles, a surgical procedure to obtain this information should rarely be necessary.
8. Cargille, C. M., Ross, G. T., and Yoshimi, T.: J. Clin. Endocrinol. 29: 12, 1969. 9. Ross, G. T., Cargille, C. M., Lipsett, M. B., Rayford, P. L., Marshall, J. R., Strott, C. A., and Rodbard, D.: Recent Progr. Hormone Research 26: 1, 1970. 10. Van Thiel, D. H., Sherins, R. J., Myers, G. H., and DeVita, V. T.: J. Clin. Invest. 51: 1009, 1972. 11. Cargille, C. M., Rodbard, D., and Ross, G. T.: J. Clin. Endocrinol. Metab. 28: 1276, 1968. 12. Jones, G. S., and Moraes-Ruehsen, M.: AM. J. OnsTET. GYNECOL. 104: 597, 1969. 13. Starup, ]., Sele, V., and Henriksen, B.: Acta Endocrinol. 66: 248, 1971. 14. Rodbard, D.: In Odell, W. D., and Daughaday, W. H., editors: Principles of Competitive Protein Binding Assays, Philadelphia, 1971, J. B. Lippincott Co., p. 204. 15. Harris, E. K., and De Mets, D. L.: Clin. Chern. 18: 244, 1972.
August 1, 1973
1010 Goldenberg et al.
Statistical appendix 1. These data were collected in a retrospective study; accordingly, the interpretation of the results is subject to the limitations inherent in any clinical study of this type. Ideally, these results should be confirmed in a prospective study. 2. The nature of the clinical "population" studied at the NIH as shown in Table I is not necessarily representative of the clinical "population" at other medical centers. No formal method of "random sampling" was used. However, all patients referred to an infertility clinic were included and no apparent preselection of patients were used. Ideally, these findings should be confirmed on an independent patient population. 3. The data presented here (Fig. 1 to 7) were obtained in many different assays over the period of several years. Further, the standards, antisera, second antibody, and minor details of the radioimmunoassays (RIA's) for FSH and LH changed on at least two occasions during the course of these studies. This introduces extraneous sources of variation which, if anything, should reduce (rather than create or accentuate) the observed differences between diagnostic groups. Accordingly, we infer that the diagnostic accuracy of the plasma FSH value as an indicator of the presence or absence of follicles might be even better if all measurements are made within the same assay. When measurements obtained in different RIA's (or bioassays) are to be compared, it is essential to include a rigorous quality control system. 14 4. The distributions shown in Figs. 1 to 7 are "composite"; i.e., they include variation within individuals (between days), as well as a component of variation between individuals. The number of observations per subject is quite variable (from one to 30). Further, measurement error (both within and between assays) is superimposed on the genuine physiologic variation. 5. The shape of the distributions is asymmetrical, generally with "skewing to the right." Use of a logarithmic transformation results in an approach to Gaussian distributions (i.e., the measurements have a "log-normal" distribution as a first approximation). Also, the use of a log transform markedly reduces the severity of nonuniformity of variance and provides "additivity." Accordingly. a subset of the data were studied by analysis of variance ( ANOVA) after the log transformation, using a "one-way, nested design with subsampling." 15 The results are shown in Table ITA. These results must be in-
Am.
J. Obstet. Gynecol.
terpreted, however, keeping in mind the limitations of the data (items 1 to 4, above) . These results indicate, that for log (plasma FSH), the means of the distributions for patients with and without follicles are approximately 4 standard deviations apart (using the standard deviation obtained if we had only one measurement per subject). Thus, using a "cut point" midway between the two means (on a log scale), we might expect a 2.5 per cent chance of misdiagnosis if the distributions were truly "log-normal." By increasing the number of measurements per subject, we reduce the measurement error, but do not reduce the variability between subjects (the latter may be reduced, if all appropriate comparisons are made within the same assay). It should be emphasized, that in the present study, there was no overlap observed. Also, by appropriate selection of the "cut point," one can control the relative magnitude of the two types of possible misdiagnosis. Also, this ANOVA shows that the difference in the means (for groups with and without follicles) for log LH is smaller, and the variation within diagnostic groups is greater, than for the log FSH measurements, such that the means are only separated 2.5 standard deviations. Thus, using a cut point midway between the two means, one would expect at least a 10 per cent rate of misdiagnosis. This is the case for the secondary amenorrhea patient population. The overlap is even greater for patients with primary amenorrhea, where a bimodal distribution is observed (see Table liB, Fig. 3, and Fig. 6). 6. If data are analyzed without the log transform, then variance (or u) for patients without follicles is enormous compared to that for patients with follicles. However, after the log transform, the standard deviation for patients with follicles (and normal gonadotrophins) is now larger (i.e., the large absolute values have a larger standard deviation but a smaller coefficient of variation). By use of transforms such as log (X + C) , or
y
X + C, one could probably obtain better uniformity of variance. However, in view of the problems with these data, further analysis was deemed unwarranted. 7. Urinary gonadotropins (Table IIC). Mean values for patients with secondary amenorrhea are significantly greater than values for patients with primary amenorrhea for patients with follicles. A significant difference is also seen in patients without follicles, although the difference
Volume 116 Number 7
Gonadotropins and amenorrhea
1011
Table IIA With follicles ]•
Log FSH
No. of observations Mean Standard deviation
155 0.955
660 1.01
0.330 0.225 0.271 0.241
(!'1
u, Ira
u.
Without follicles
zo
2"
0.285 0.192 0.313 0.256
164 2.03 0.183 O.o75 0.143 0.167
2• 180 1.93 0.212 0.074 0.169 0.108
following footnotes refer to Tables IIA and liB: 1. All calculations use logto of the LH (or FSH) values. 2. Number of degrees of freedom not sbown. 3. Some of these values were calculated on a subset of the data. 4. Even after log transform, there is heterogeneity of variance, in that the u for patients without follicles is less than the corresponding u for patients with follicles.
=
=
5. "' Standard deviation, between patients within diagnostic groups, assuming one measurement per subject. 112 Standard deviation (pooled) of all measurements within any given patient. us Standard deviation for all u measurements within a diagnostic group; this is a composite, and represents an "average" of "' and '"· depending on the number of rep· licates per subject, and the number of subjects which is variable. "' "Component of variance" attributable to differences between subjects, after subtracting the variance within subjects. 6. The linear additive model states that the variance between subjects (within diagnostic groups), expressed for a mean of r replicates per subject, is given by:
=
=
a2
=
a~/r
+ ai
i.e., the total error can be reduced by increasing the number of replicates, r; (to reduce day-to·day fluctuations and measurement error). However, it cannot be reduced below the intrinsic variation between subjects, U<. 7. a, May be biased upward, i.e., an overconservative estimate, since it includes between-assay variance, whereas U> usually contains only within-assay variance.
Table IIB With follicles Log LH
No. of observations Mean Standard deviation
2"
1"
2"
179 1.27
674 1.24
252 1.86
119 1.78
0.374 0.130 0.388 0.351
tr,
u, tr,
u,
Without follicles
1"
0.339 0.191 0.376 0.280
0.242 0.117 0.200 0.212
0.239 0.106 0.215 0.214
Table IIC
Urinary gonadotropins
1"
No. of patients No. of samples Mean Standard deviation Mean* Standard deviation*
17 321 38.8 88.9 1.21 0.58
35 153 25.6 39 0.98 0.60
*Calculation after log transform.
Without follicles
With follicles
Normal subjects
2'
]•
2'
132 452 51.1 60 1.42 0.57
34 160 215 254 2.09 0.50
19 91 240 241 2.23 0.35
1012 Goldenberg et al.
is smaller (on a log scale) . This indicates significant heterogeneity in the data, and prevents pooling of data from 1o and 2° amenorrhea. Also, patients with follicles, with 1o amenorrhea have significantly less urinary gonadotropins than do normal subjects, whereas patients with 2° amenorrhea have significantly more. Although these differences are statistically significant ( P < 0.05), they do not carry diagnostic significance. However, they may well reflect genuine physiologic differences among the various groups. 8. LH. Here the differences between 1o and 2 o amenorrhea are virtually zero (Table liB), whether one considers patients with or without follicles. The values for patients with either 1o or 2° amenorrhea with oocytes are indistinguishable from the normal range. 9. FSH. Again, there is no signicant difference
August I, 1973 Am. J. Obstet. Gynecol.
between patients with 1o or 2o amenorrhea, either for patients with or without oocytes. Values for patients with 2° amenorrhea with oocytes are higher than values for patients with 1o amenorrhea with oocytes. This difference is in the same direction as seen in the urinary gonadotropin data. 10. Ideally, one should examine the correlations between FSH, LH, and urinary gonadotropins. It is possible that using all 3 tests together (as in discriminant analysis or cluster analysis) , one could obtain better diagnostic accuracy than with any one test alone. Also, it would be worthwhile to study the urinary gonadotropin activity as it relates to plasma FSH and LH. Unfortunately, the data for this study were not collected in such a way as to make this type of analysis possible.