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Ovarian and peripheral androgen and oestrogen levels in post-menopausal women: correlations with ovarian histology
Muturitas, 8 (1986) 57-65 Elsevier
57
MAT 00388
Ovarian and peripheral androgen and oestrogen levels in post-menopausal women: correlations with ovarian histology A. Lucisano
I, N. Russo ‘, M.G. Acampora’, A. Fabian0 *, M. Fattibene E. Parlati ‘, E. Maniccia ’ and S. Dell’Acqua ’
‘,
Departments of I Obstetrics and GynaecologV and ’ Human Pathology, Sacro Cuore Catholic Unioersit_v, Rome, Italy (Received
22 January
1985; revision received 26 August
1985; accepted
13 November
1985)
Ovarian and peripheral plasma levels of oestrone (E,), oestradiol (E,), androstenedione (A) and testosterone (T) were assayed in 58 post-menopausal women who underwent hysterectomy and oophorectomy (35 for endometrial carcinoma and 23 for benign gynaecological diseases). No significant difference between the two groups was seen when they were matched for percentage of ideal weight. However, significant differences were found between the ovarian and peripheral levels of the four steroids investigated. To facilitate analysis of the data, 40 of these women were classified into three groups (1, 2 and 3) according to degree of stromal hyperplasia of the ovary. Group 1 comprised those with atrophic ovaries, group 2 those with slight stromal hyperplasia and group 3 those with moderate or marked stromal hyperplasia. Ovarian levels of A and T were significantly higher than peripheral levels in all three groups, but the ovarian/peripheral oestrogen differences were significant only in groups 1 and 2. The ovarian steroid levels in group 3 were significantly higher than those in groups 1 and 2 in the cases of E, (P < 0.01). E, (P < 0.001) and A (P < O.OOl),but not in that of T. It was concluded that ovaries showing marked stromal hyperplasia can produce significant amounts of the hormonal pattern in not only of androgens but also of E,, and hence that any evaluation post-menopausal women must also take into account the microscopic characteristics of the ovary. (Key words: Ovarian Peripheral steroids)
stromal
hyperplasia,
Post-menopausal,
Endometrial
carcinoma,
Ovarian
steroids,
Introduction Over the past few years
many
authors
have
clearly
demonstrated
that
the
[l-5]. However, its contribution to the circulating oestrogen pool is still open to debate. Some authors consider it irrelevant [2-81, while others have recently suggested that the ovary is still able to produce C-18 steroids in some subjects [9-121. Dennefors et
post-menopausal
ovary
continues
to secrete
significant
amounts
of androgens
Correspondence to: Antonino Lucisano, Department of Obstetrics and Gynaecology, de1 Sacro Cuore, Policlinico ‘A. Gemelli’, Largo Gemelli, 8, 00168 Rome, Italy
0378-5122/86/$03.50
0 1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
Universita
Cattolica
58
al. [ll] have shown by means of in vitro studies that the post-menopausal ovarian stroma is able to aromatise androgens to oestrogens and that this capacity is greater in ovaries with stromal hyperplasia. Moreover, in vivo studies have demonstrated the existence in some patients of a significant E, gradient between the ovarian artery and vein [9] and between ovarian and peripheral venous blood [12]. Many authors [9,10,13,14] have evidenced a certain discrepancy between oestrogen production rates and the androgen contribution to these. Such data would suggest that peripheral synthesis may not be the only source of circulating oestrogens. Histological examination has revealed that not all post-menopausal ovaries are atrophic, since they frequently exhibit varying degrees of cortical stromal hyperplasia [16]. A highly cellular tissue containing large ovoid cells with prominent nuclei and nodules composed of spindle cells arranged in whorls can be found in the cortical rim of such ovaries [11,15,16]. Luteinized stromal cells can be observed in the medulla; although absent in the atrophic ovary, these become more and more frequent as stromal hyperplasia increases [16]. There is histochemical evidence of steroidogenesis in the post-menopausal ovarian stroma and the main sites of enzymatic activity seem to be stromal and hilus cells [6]. However, the data merely indicate that biochemical activity occurs in the stromal cells and do constitute proof that the post-menopausal ovary is able to secrete steroids in vivo. This study was undertaken in order to determine whether or not the postmenopausal ovary continues to be a source of oestrogens, and whether histological appearance can be correlated with ovarian oestrogen production. An attempt was also made to find evidence of differences between women affected by endometrial carcinoma and control subjects. Plasma E,, E,, A and T concentrations were assayed in samples collected simultaneously during surgery from a peripheral vein and from veins draining both ovaries in a group of post-menopausal women with and without endometrial cancer, who were undergoing oophorectomy and hysterectomy. The patients were subsequently classified into three groups according to the degree of stromal hyperplasia as determined by histological examination of the ovaries.
Subjects and methods Fifty-eight women undergoing hysterectomy and oophorectomy were studied, 35 of whom were suffering from histologically confirmed adenocarcinoma of the endometrium and 23 from benign gynaecological diseases (uterine prolapse, fibromatosis). All had been post-menopausal for 2-3 yr. Mean age + SD was 59.9 k 7.6 yr and mean weight was 64.4 f 8.9 kg. Excess weight was calculated for each patient by dividing actual weight by ideal weight and multiplying by 100. Ideal weights were taken from the Metropolitan Life Insurance Co. tables; the mean percentage of ideal weight was 129.8 -t 21.4. None of the subjects had received hormone replacement therapy during the preceding 12 mth. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, evaluated by the double-antibody technique [17], were in the normal
59
ranges for post-menopausal women. None of the subjects were suffering from ovarian disease. A lo-ml blood sample was drawn from the cubital vein at 8 :00 a.m. on two consecutive days before operation, and two or more further samples were collected during surgery. At laparotomy, one or more blood samples were taken from veins draining both ovaries. These samples were immediately centrifuged and the plasma was frozen until analysed. Plasma E,, E,, T and A concentrations were assayed in all samples by previously published radioimmunoassay procedures [18-221. Before radioimmunoassay, chromatographic separation of all samples was performed on Sephadex LH 20 microcolumns. All samples from the same subject were assayed together. Our results were similar to those of other authors with regard to accuracy, precision, sensitivity and specificity of the methods used [18-221. Statistical analysis was performed using Student’s t-test. Immediately following operation, the ovaries of 40 of these women were sectioned along the main diameter through the hilus, fixed in a neutral solution of 10% buffered formalin and then embedded in paraffin. Five to seven sections taken at different levels from each ovary were prepared and stained with haematoxylin-eosin and Van Gieson’s acid fuchsin stain. These were subsequently examined under a light microscope. The 40 women were then classified into three groups, depending on the degree of stromal hyperplasia found in their ovaries according to the criteria of Boss et al. [16].
Results
Mean peripheral and ovarian plasma levels of total E,, E,, A and T in the 35 cancer subjects and the 23 control subjects are shown in Fig. 1. These values are in agreement with those reported by many other investigators [2,7,9,23,24]. Significant differences between the two groups were seen in the cases of E, and E, in peripheral plasma. These differences could have been due to the different mean percentages of ideal weight in the two groups (147 + 20 in the cancer patients and 125 f 17 in the control subjects). However, when each control subject was matched with a specific cancer patient for percentage of ideal weight, no differences were found between the two groups of 20 patients each (Fig. 2). These data constitute further evidence of the influence of body weight on circulating levels of E, and E,, as already demonstrated by other investigators [2,5,24]. In fact, analysis of the possible relationship between the peripheral and ovarian levels of the four steroids considered and the patients’ clinical characteristics revealed a significant positive pre-operative correlation between excess fat and peripheral levels of E, (r = 0.682, P < 0.01 for cancer patients; r = 0.730, P -c0.01 for control subjects) and of E, (r = 0.568, P -c0.05 for cancer patients; r = 0.626, P < 0.05 for control subjects). In addition, a significant correlation was found between the peripheral E,/A ratio and the percentage of ideal weight in both groups (r = 0.664, P < 0.001 for cancer patients; r = 0.632, P < 0.001 for control subjects) and also between E, and E, (r = 0.598, P < 0.05 for cancer patients; r = 0.643, P < 0.05 for control subjects. No correlations were found
60
PE”l.“(C”AL
O”.“l.”
P.“lP”.“.L
O”A”,A”
Fig. 1. Oestrone (E,), oestradiol (E,), testosterone (T) and androstenedione (A) (&SE) levels in plasma from peripheral and ovarian veins in 35 post-menopausal women with endometrial carcinoma (unshaded columns) and in 23 control subjects (shaded columns). 1, Before anaesthesia; 2, after anaesthesia; 3, at laparotomy.
tween clinical characteristics and androgens. The ovarian levels of the four steroids investigated did not correlate with each other, with peripheral levels or with clinical characteristics. In both groups the ovarian levels of the four steroids were significantly higher than those in peripheral plasma (third sample): conspicuously so for androgens (P < 0.01for A and P < 0.001 for T), but only minimally in the case of oestrogens (P < 0.01 for E, and P < 0.001 for E,). Since no differences were seen between cancer patients and control subjects, the patients were classified into three groups, depending on the degree of stromal hyperplasia of the ovary. Group 1 comprised 16 patients (10 with endometrial carcinoma and 6 without tumours) whose ovaries showed a thin cortex, little stroma and no sign of proliferation, corresponding to Grade I according to the criteria of Boss et al. [16]. Group 2 was made up of 17 patients (7 with and 10 without tumour) whose ovaries showed slight signs of stromal hyperplasia corresponding to Grade II [16]. Finally, group 3 numbered 7 patients (4 with and 3 without tumours) whose ovaries were characterised by moderate or marked stromal hyperplasia, corresponding to Grades III and IV [16]. No correlation was found either between the macroscopic appearance of the ovary and the degree of stromal hyperplasia, this being in accordance with the findings reported by other investigators [16], or between the degree of stromal
61
T
Fig. 2. Oestrone (Ei). oestradiol (E,), testosterone (T) and androstenedione (A) ( f SE) levels in plasma from peripheral and ovarian veins in 20 post-menopausal women with endometrial carcinoma (unshaded columns) and in 20 control subjects (shaded columns) matched for I ideal weigi;!s 1. Before anaesthesia; 2, after anaesthesia; 3, at laparotomy.
hyperplasia and the presence of endometrial carcinoma. The mean ovarian and peripheral values of the four steroids in groups 1-3 are shown in Fig. 3. The ovarian T and A values differed significantly from the peripheral values in all three groups, but the ovarian/peripheral oestrogen differences were significant only in groups 2
TABLE
1
PERCENTAGE DISTRIBUTION OF SIGNIFICANT OVARIAN/PERIPHERAL GRADIENTS 40 POST-MENOPAUSAL WOMEN CLASSIFIED INTO 3 GROUPS ACCORDING TO THE GREE OF STROMAL HYPERPLASIA Group Number of ovaries Oestrone Oestradiol Androstenedione Testosterone a 1, Atrophic
ovaries;
1
Group
32 6.2 18.7 18.7 78.1 2, slight stromal
2
34 11.7 23.5 41.1 82.3 hyperplasia;
3, moderate
Group
3
14 57.1 71.4 71.4 85.7 or marked
Total 80 17.5 30 37.5 81.2
stromal
hyperplasia
IN DE-
. i,.. :..:: :.. .._ ::::::::: _... _.... ./...... .::::::::: .. A ..i.._ dl
62
..A.... ,A...... P....... . .. . . . . ::,.:. .A...... P....... ::..:.
c....... c....,., . ...i... c....... . .. . . .._ . .. . . . .. . .:.:.:.:. :.:.:.:.: . . ..ii. . . .._..L
. . .. .. . .. . . . . .._ ._
P-J.01
pco,olI
nglml 0
4
A
.:..,..._ :.:.:.:.:. .,. I dl i
a
.::::::::: ...... . .?...._._ . J.3
y:::::.: >:+:.:. .:::::::.: ...._.. :. P....... ::::::::: :::::::::
2
G
R
J
p
p.a.01
P-w2
2 A
D
G
;:;:i:;:j ::::::::: ::::::::: .P..,..._ ::::::::: ........_ ......(..... ..._.... ......... ..... .‘ ...._....
3 R
A
D
E
Fig. 3. Oestrone (E,), oestradiol (El), testosterone (T) and androstenedione (A) ( f SE) levels in plasma from peripheral and ovarian veins in 40 post-menopausal women classified into 3 groups according to degree of ovarian stromal hyperplasia. Grade 1, atrophic ovaries: Grade 2, slight stromal hyperplasia; Grade 3, moderate or marked stromal hyperplasia.
and 3. Ovarian steroid levels in group 3 were significantly higher than in groups 1 and 2 in the cases of E, (P -C O.Ol), E, (P < 0.001) and A (P < O.OOl), but not in that of T. In order to facilitate analysis, we calculated the two ovarian peripheral gradients for each patient. Only gradients where the difference between ovarian and peripheral values was greater than the sum of each concentration multiplied by twice the maximal coefficient of variation for the methods used (12%) were deemed significant. Table I shows the percentages of significant gradients for each hormone found in the three groups of patients. Attention is drawn in the very low incidence of significant gradients for oestrogens and for A in the group of patients with atrophic ovaries (Group 1). However, the incidence is higher, especially as regards E, and A, among the patients in group 3. In the case of T, there are no marked variations between the groups.
Discussion Our results confirm once again that there are no differences peripheral levels of androgens and oestrogens in cancer patients
between ovarian or and control subjects
63
when they are matched according to body weight. They also confirm the existence of a strong positive correlation between levels of circulating oestrogens -and body weight in both groups, as previously reported by other authors [2,5,12,24], whereas this correlation is not seen in the case of ovarian steroid levels. These findings suggest that circulating oestrogens in post-menopausal women are produced mainly from A in peripheral tissue. Nevertheless, our study results indicate that the post-menopausal ovary is able to secrete androgens and strongly suggest that, at least in some subjects with ovarian stromal hyperplasia, the ovary can produce significant amounts of oestrogens. If we accept that peripheral steroid levels adequately represent steroid concentrations in arterial blood delivered to steroid-producing tissue, then the difference between ovarian and peripheral venous blood reflects the arteriovenous gradient through the ovary [25]. Accordingly, the significant differences between ovarian and peripheral E, and E, levels seen in groups 2 and 3, and the significant differences between ovarian and peripheral A and T levels in all three groups, could mean that all post-menopausal ovaries produce T and that ovaries with stromal hyperplasia are also able to produce E,, E, and A. This is confirmed by the fact that significant gradients for E,, E, and A occur more frequently in group 3 and that mean ovarian levels of E,, E, and A are significantly higher in group 3 than in group 1 (P -C0.01 for E,; P < 0.0001 for E,; P < 0.001 for A). Although the blood flow rate through the post-menopausal ovary is not known, we have arbitrarily taken it to be equal to 60 l/day, as calculated in pre-menopausal subjects by Baird and Fraser [26] and have estimated maximal ovarian secretion rates of 3.6, 5.5, 381 and 134 pg/24 h for E,, E,, A and T, respectively, for the women in group 3 with significant gradients. We consequently believe that an ovary with moderate or marked stromal hyperplasia can contribute not only to the pool of circulating T, but also to the pools of E, and A. Even though 21 women out of the 40 had endometrial carcinoma, this is not considered to have influenced our results. In fact, no difference was found between ovarian and peripheral levels in patients with or without endometrial carcinoma. Moreover, no correlation was established between the presence of stromal hyperplasia and endometrial carcinoma, in contrast with the findings of other investigators, who conducted a retrospective study in which only histological appearance was evaluated [16]. No significance differences in body weight were seen between patients in the three groups, which suggests that the presence or absence of a gradient does not seem to be influenced by the degree of obesity. Since the ovarian and the third peripheral blood samples were taken simultaneously during operation and would therefore be subject to the same variations as a consequence of surgery, the evaluation of the gradients ought not to have been affected by anaesthesia or surgical stress. During surgery only the peripheral A levels showed a rise, which could have resulted from an increased adrenal A contribution. There was a 17% incidence of moderate or marked stromal hyperplasia among the patients, which is lower than that found by Boss et al. [16] in autopsy specimens (28%) or that reported by Dennefors [ll] based on small pieces of tissue (47%). This is probably due to the fact that we included in group 3 only subjects with evident
64
stromal hyperplasia. The difficulty lies in distinguishing an ovary with slight stromal hyperplasia from a normal ovary. Our results are not in agreement with those of authors who studied ovarian secretion by indirect methods rather than by ovarian vein catheterisation [5] or who reported a minimal oestrogen gradient on the basis of only a few cases [2]. They are, however, in agreement with those of a number of other investigators [3,6,9] and indeed confirm what has already been demonstrated in vitro by Dennefors et al. [ll], namely that the ovarian stroma in post-menopausal women is able to aromatise androgens to oestrogens, and that ovaries affected by stromal hyperplasia can produce greater amounts not only of A but also of E, than atrophic ovaries. In conclusion, we believe that post-menopausal ovaries showing stromal hyperplasia contribute to the peripheral oestrogen pool, and that studies of hormonal patterns in post-menopausal women should always therefore take into account the histological characteristics of the ovary, which vary from individual to individual and cannot be detected by macroscopic examination alone.
References 1 Mattingly RF, Huang WY. Steroidogenesis of the menopausal and postmenopausal ovary. Am J Obstet Gynecol 1969; 103: 679-693. 2 Judd HL, Judd GE, Lucas WE. Yen SSC. Endocrine function of the postmenopausal ovary: concentration of androgens and oestrogens in ovarian and peripheral vein blood. J Clin Endocrinol Metab 1974; 39: 1020-1024. 3 Greenblatt RB, Colle ML, Mahesh VB. Ovarian and adrenal steroid production in the postmenopausal woman. Obstet Gynecol 1976; 47: 383-387. 4 Shenker JG, Polishuk WZ, Eckstein B. Pathways in the biosynthesis of androgens in the postmenopausal ovary in vitro. Acta Endocrinol 1971; 66: 325-332. 5 Vermeulen A. The hormonal activity of the postmenopausal ovary. J Clin Endocrinol Metab 1976; 42: 247-253. 6 Gronroos M, Klemi P, Sahni T, Rauramo L, Punnonen R, Ovarian production of oestrogens in postmenopausal women. Int J Gynecol Obstet 1980; 18: 93-98. 7 Judd HL, Lucas WE, Yen SSC. Serum 17/3-oestradiol and oestrone levels in postmenopausal women with and without endometrial cancer. J Clin Endocrinol Metab 1976; 43: 272-278. 8 Siiteri PK. Post-menopausal oestrogen production. In: Van Keep PA, Lauritzen C, eds. Oestrogens in the postmenopause. Frontiers in hormone Research. Vol. 3. Base]: Karger, 1975; 40-45. 9 Longcope C, Hunter R, Franz C. Steroid secretion by the postmenopausal ovary. Am J Obstet Gynecol 1980; 138: 564-568. 10 Kirshner MA, Cohen FB, Ryan C. Androgen-oestrogen production rates in post-menopausal women with breast cancer. Cancer Res 1978; 38: 4029-4035. 11 Dennefors’ BL, Janson PO, Knutson F, Hamberger L. Steroid production and responsiveness to gonadotropin in isolated stromal tissue of human post-menopausal ovaries. Am J Obstet Gynecol 1980; 136: 997-1002. 12 Lucisano A, Acampora MG, Russo N, Maniccia E, Montemurro A, Dell’Acqua S. Ovarian and peripheral plasma levels of progestogens, androgens and oestrogens in post-menopausal women. Maturitas 1984; 6: 45-53. 13 Judd HL, Shamonki IM, Frumar AM, Lagasse LD. Origin of serum oestradiol in post-menopausal women. Obstet Gynecol 1982; 59: 680-686. 14 Trevoux R. De Brux JA, Scholler R. Longitudinal histohormonal research on 220 peri- and postmenopausal women. In: Proc Xth World Congr Gynecol Obstet. San Francisco. CA, USA, 1982; 217.
65 15 Novak ER, Goldberg B, Jones GS, O’Toole RV. Enzyme histochemistry of the menopausal ovary associated with normal and abnormal endometrium. Am J Obstet Gynecol 1965; 93: 669-682. 16 Boss JH, Scully RE, Wagner KH. Cohen RB. Structural variations in the adult ovary: clinical significance. Obstet Gynecol 1965; 25: 747-764. 17 Serra GB, Caniglia R, Barile G. Some characteristics of the double antibody method for the assay of gonadotropins. In: Crosignani PG. James VHT, eds. Recent progress in reproductive endocrinology. London and New York: Academic Press, 1974; 65-82. 18 Emment Y, Collins WP, Sommerville IF. Radioimmunoassay of oestrone and oestradiol in human plasma. Steroid Biochem 1972: 3: 893-901. 19 Youssefnejadian E, Florensa E, Collins WP. Sommerville IF. Radioimmunoassay of plasma progesterone. J Steroid Biochem 1972; 3: 893-901. 20 Youssefnejadian E. Florensa E, Collins WP, Sommerville IF. Radioimmunoassay of 17-hydroxyprogesterone. Steroids 1972; 20: 773-788. 21 Collins WP. Mansfield MD, Alladina NS, Sommerville IF. Radioimmunoassay of plasma testosterone. Steroid B&hem 1972; 3: 333-348. 22 Montemurro A, Johnson MW, Barile G. Youssefnejadian E. A simple radioimmunoassay of androstenedione without column chromatography. J Obstet Gynecol 1981; 1: 247-250. 23 Meldrum DR, Davidson BJ, Tataryn IV. Judd HL, Changes in circulating steroids with aging in post-menopausal women. Obstet Gynecol 1981; 57: 624-628. 24 Vermeulen A, Verdonck L. Sex hormone concentrations in post-menopausal women. Clin Endocrinol 1978; 9: 59-66. 25 Chang RJ, Abraham GE. Peripheral arterial and venous concentrations of various androgens in patients with and without hirsutism. Obstet Gynecol 1975; 46: 549-550. 26 Baird DT. Fraser IS. Blood production and ovarian secretion rates of oestradiol-17P and oestrone in women throughout the menstrual cycle. J Clin Endocrinol Metab 38: 1009.
57
MAT 00388
Ovarian and peripheral androgen and oestrogen levels in post-menopausal women: correlations with ovarian histology A. Lucisano
I, N. Russo ‘, M.G. Acampora’, A. Fabian0 *, M. Fattibene E. Parlati ‘, E. Maniccia ’ and S. Dell’Acqua ’
‘,
Departments of I Obstetrics and GynaecologV and ’ Human Pathology, Sacro Cuore Catholic Unioersit_v, Rome, Italy (Received
22 January
1985; revision received 26 August
1985; accepted
13 November
1985)
Ovarian and peripheral plasma levels of oestrone (E,), oestradiol (E,), androstenedione (A) and testosterone (T) were assayed in 58 post-menopausal women who underwent hysterectomy and oophorectomy (35 for endometrial carcinoma and 23 for benign gynaecological diseases). No significant difference between the two groups was seen when they were matched for percentage of ideal weight. However, significant differences were found between the ovarian and peripheral levels of the four steroids investigated. To facilitate analysis of the data, 40 of these women were classified into three groups (1, 2 and 3) according to degree of stromal hyperplasia of the ovary. Group 1 comprised those with atrophic ovaries, group 2 those with slight stromal hyperplasia and group 3 those with moderate or marked stromal hyperplasia. Ovarian levels of A and T were significantly higher than peripheral levels in all three groups, but the ovarian/peripheral oestrogen differences were significant only in groups 1 and 2. The ovarian steroid levels in group 3 were significantly higher than those in groups 1 and 2 in the cases of E, (P < 0.01). E, (P < 0.001) and A (P < O.OOl),but not in that of T. It was concluded that ovaries showing marked stromal hyperplasia can produce significant amounts of the hormonal pattern in not only of androgens but also of E,, and hence that any evaluation post-menopausal women must also take into account the microscopic characteristics of the ovary. (Key words: Ovarian Peripheral steroids)
stromal
hyperplasia,
Post-menopausal,
Endometrial
carcinoma,
Ovarian
steroids,
Introduction Over the past few years
many
authors
have
clearly
demonstrated
that
the
[l-5]. However, its contribution to the circulating oestrogen pool is still open to debate. Some authors consider it irrelevant [2-81, while others have recently suggested that the ovary is still able to produce C-18 steroids in some subjects [9-121. Dennefors et
post-menopausal
ovary
continues
to secrete
significant
amounts
of androgens
Correspondence to: Antonino Lucisano, Department of Obstetrics and Gynaecology, de1 Sacro Cuore, Policlinico ‘A. Gemelli’, Largo Gemelli, 8, 00168 Rome, Italy
0378-5122/86/$03.50
0 1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
Universita
Cattolica
58
al. [ll] have shown by means of in vitro studies that the post-menopausal ovarian stroma is able to aromatise androgens to oestrogens and that this capacity is greater in ovaries with stromal hyperplasia. Moreover, in vivo studies have demonstrated the existence in some patients of a significant E, gradient between the ovarian artery and vein [9] and between ovarian and peripheral venous blood [12]. Many authors [9,10,13,14] have evidenced a certain discrepancy between oestrogen production rates and the androgen contribution to these. Such data would suggest that peripheral synthesis may not be the only source of circulating oestrogens. Histological examination has revealed that not all post-menopausal ovaries are atrophic, since they frequently exhibit varying degrees of cortical stromal hyperplasia [16]. A highly cellular tissue containing large ovoid cells with prominent nuclei and nodules composed of spindle cells arranged in whorls can be found in the cortical rim of such ovaries [11,15,16]. Luteinized stromal cells can be observed in the medulla; although absent in the atrophic ovary, these become more and more frequent as stromal hyperplasia increases [16]. There is histochemical evidence of steroidogenesis in the post-menopausal ovarian stroma and the main sites of enzymatic activity seem to be stromal and hilus cells [6]. However, the data merely indicate that biochemical activity occurs in the stromal cells and do constitute proof that the post-menopausal ovary is able to secrete steroids in vivo. This study was undertaken in order to determine whether or not the postmenopausal ovary continues to be a source of oestrogens, and whether histological appearance can be correlated with ovarian oestrogen production. An attempt was also made to find evidence of differences between women affected by endometrial carcinoma and control subjects. Plasma E,, E,, A and T concentrations were assayed in samples collected simultaneously during surgery from a peripheral vein and from veins draining both ovaries in a group of post-menopausal women with and without endometrial cancer, who were undergoing oophorectomy and hysterectomy. The patients were subsequently classified into three groups according to the degree of stromal hyperplasia as determined by histological examination of the ovaries.
Subjects and methods Fifty-eight women undergoing hysterectomy and oophorectomy were studied, 35 of whom were suffering from histologically confirmed adenocarcinoma of the endometrium and 23 from benign gynaecological diseases (uterine prolapse, fibromatosis). All had been post-menopausal for 2-3 yr. Mean age + SD was 59.9 k 7.6 yr and mean weight was 64.4 f 8.9 kg. Excess weight was calculated for each patient by dividing actual weight by ideal weight and multiplying by 100. Ideal weights were taken from the Metropolitan Life Insurance Co. tables; the mean percentage of ideal weight was 129.8 -t 21.4. None of the subjects had received hormone replacement therapy during the preceding 12 mth. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, evaluated by the double-antibody technique [17], were in the normal
59
ranges for post-menopausal women. None of the subjects were suffering from ovarian disease. A lo-ml blood sample was drawn from the cubital vein at 8 :00 a.m. on two consecutive days before operation, and two or more further samples were collected during surgery. At laparotomy, one or more blood samples were taken from veins draining both ovaries. These samples were immediately centrifuged and the plasma was frozen until analysed. Plasma E,, E,, T and A concentrations were assayed in all samples by previously published radioimmunoassay procedures [18-221. Before radioimmunoassay, chromatographic separation of all samples was performed on Sephadex LH 20 microcolumns. All samples from the same subject were assayed together. Our results were similar to those of other authors with regard to accuracy, precision, sensitivity and specificity of the methods used [18-221. Statistical analysis was performed using Student’s t-test. Immediately following operation, the ovaries of 40 of these women were sectioned along the main diameter through the hilus, fixed in a neutral solution of 10% buffered formalin and then embedded in paraffin. Five to seven sections taken at different levels from each ovary were prepared and stained with haematoxylin-eosin and Van Gieson’s acid fuchsin stain. These were subsequently examined under a light microscope. The 40 women were then classified into three groups, depending on the degree of stromal hyperplasia found in their ovaries according to the criteria of Boss et al. [16].
Results
Mean peripheral and ovarian plasma levels of total E,, E,, A and T in the 35 cancer subjects and the 23 control subjects are shown in Fig. 1. These values are in agreement with those reported by many other investigators [2,7,9,23,24]. Significant differences between the two groups were seen in the cases of E, and E, in peripheral plasma. These differences could have been due to the different mean percentages of ideal weight in the two groups (147 + 20 in the cancer patients and 125 f 17 in the control subjects). However, when each control subject was matched with a specific cancer patient for percentage of ideal weight, no differences were found between the two groups of 20 patients each (Fig. 2). These data constitute further evidence of the influence of body weight on circulating levels of E, and E,, as already demonstrated by other investigators [2,5,24]. In fact, analysis of the possible relationship between the peripheral and ovarian levels of the four steroids considered and the patients’ clinical characteristics revealed a significant positive pre-operative correlation between excess fat and peripheral levels of E, (r = 0.682, P < 0.01 for cancer patients; r = 0.730, P -c0.01 for control subjects) and of E, (r = 0.568, P -c0.05 for cancer patients; r = 0.626, P < 0.05 for control subjects). In addition, a significant correlation was found between the peripheral E,/A ratio and the percentage of ideal weight in both groups (r = 0.664, P < 0.001 for cancer patients; r = 0.632, P < 0.001 for control subjects) and also between E, and E, (r = 0.598, P < 0.05 for cancer patients; r = 0.643, P < 0.05 for control subjects. No correlations were found
60
PE”l.“(C”AL
O”.“l.”
P.“lP”.“.L
O”A”,A”
Fig. 1. Oestrone (E,), oestradiol (E,), testosterone (T) and androstenedione (A) (&SE) levels in plasma from peripheral and ovarian veins in 35 post-menopausal women with endometrial carcinoma (unshaded columns) and in 23 control subjects (shaded columns). 1, Before anaesthesia; 2, after anaesthesia; 3, at laparotomy.
tween clinical characteristics and androgens. The ovarian levels of the four steroids investigated did not correlate with each other, with peripheral levels or with clinical characteristics. In both groups the ovarian levels of the four steroids were significantly higher than those in peripheral plasma (third sample): conspicuously so for androgens (P < 0.01for A and P < 0.001 for T), but only minimally in the case of oestrogens (P < 0.01 for E, and P < 0.001 for E,). Since no differences were seen between cancer patients and control subjects, the patients were classified into three groups, depending on the degree of stromal hyperplasia of the ovary. Group 1 comprised 16 patients (10 with endometrial carcinoma and 6 without tumours) whose ovaries showed a thin cortex, little stroma and no sign of proliferation, corresponding to Grade I according to the criteria of Boss et al. [16]. Group 2 was made up of 17 patients (7 with and 10 without tumour) whose ovaries showed slight signs of stromal hyperplasia corresponding to Grade II [16]. Finally, group 3 numbered 7 patients (4 with and 3 without tumours) whose ovaries were characterised by moderate or marked stromal hyperplasia, corresponding to Grades III and IV [16]. No correlation was found either between the macroscopic appearance of the ovary and the degree of stromal hyperplasia, this being in accordance with the findings reported by other investigators [16], or between the degree of stromal
61
T
Fig. 2. Oestrone (Ei). oestradiol (E,), testosterone (T) and androstenedione (A) ( f SE) levels in plasma from peripheral and ovarian veins in 20 post-menopausal women with endometrial carcinoma (unshaded columns) and in 20 control subjects (shaded columns) matched for I ideal weigi;!s 1. Before anaesthesia; 2, after anaesthesia; 3, at laparotomy.
hyperplasia and the presence of endometrial carcinoma. The mean ovarian and peripheral values of the four steroids in groups 1-3 are shown in Fig. 3. The ovarian T and A values differed significantly from the peripheral values in all three groups, but the ovarian/peripheral oestrogen differences were significant only in groups 2
TABLE
1
PERCENTAGE DISTRIBUTION OF SIGNIFICANT OVARIAN/PERIPHERAL GRADIENTS 40 POST-MENOPAUSAL WOMEN CLASSIFIED INTO 3 GROUPS ACCORDING TO THE GREE OF STROMAL HYPERPLASIA Group Number of ovaries Oestrone Oestradiol Androstenedione Testosterone a 1, Atrophic
ovaries;
1
Group
32 6.2 18.7 18.7 78.1 2, slight stromal
2
34 11.7 23.5 41.1 82.3 hyperplasia;
3, moderate
Group
3
14 57.1 71.4 71.4 85.7 or marked
Total 80 17.5 30 37.5 81.2
stromal
hyperplasia
IN DE-
. i,.. :..:: :.. .._ ::::::::: _... _.... ./...... .::::::::: .. A ..i.._ dl
62
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. . .. .. . .. . . . . .._ ._
P-J.01
pco,olI
nglml 0
4
A
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a
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y:::::.: >:+:.:. .:::::::.: ...._.. :. P....... ::::::::: :::::::::
2
G
R
J
p
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P-w2
2 A
D
G
;:;:i:;:j ::::::::: ::::::::: .P..,..._ ::::::::: ........_ ......(..... ..._.... ......... ..... .‘ ...._....
3 R
A
D
E
Fig. 3. Oestrone (E,), oestradiol (El), testosterone (T) and androstenedione (A) ( f SE) levels in plasma from peripheral and ovarian veins in 40 post-menopausal women classified into 3 groups according to degree of ovarian stromal hyperplasia. Grade 1, atrophic ovaries: Grade 2, slight stromal hyperplasia; Grade 3, moderate or marked stromal hyperplasia.
and 3. Ovarian steroid levels in group 3 were significantly higher than in groups 1 and 2 in the cases of E, (P -C O.Ol), E, (P < 0.001) and A (P < O.OOl), but not in that of T. In order to facilitate analysis, we calculated the two ovarian peripheral gradients for each patient. Only gradients where the difference between ovarian and peripheral values was greater than the sum of each concentration multiplied by twice the maximal coefficient of variation for the methods used (12%) were deemed significant. Table I shows the percentages of significant gradients for each hormone found in the three groups of patients. Attention is drawn in the very low incidence of significant gradients for oestrogens and for A in the group of patients with atrophic ovaries (Group 1). However, the incidence is higher, especially as regards E, and A, among the patients in group 3. In the case of T, there are no marked variations between the groups.
Discussion Our results confirm once again that there are no differences peripheral levels of androgens and oestrogens in cancer patients
between ovarian or and control subjects
63
when they are matched according to body weight. They also confirm the existence of a strong positive correlation between levels of circulating oestrogens -and body weight in both groups, as previously reported by other authors [2,5,12,24], whereas this correlation is not seen in the case of ovarian steroid levels. These findings suggest that circulating oestrogens in post-menopausal women are produced mainly from A in peripheral tissue. Nevertheless, our study results indicate that the post-menopausal ovary is able to secrete androgens and strongly suggest that, at least in some subjects with ovarian stromal hyperplasia, the ovary can produce significant amounts of oestrogens. If we accept that peripheral steroid levels adequately represent steroid concentrations in arterial blood delivered to steroid-producing tissue, then the difference between ovarian and peripheral venous blood reflects the arteriovenous gradient through the ovary [25]. Accordingly, the significant differences between ovarian and peripheral E, and E, levels seen in groups 2 and 3, and the significant differences between ovarian and peripheral A and T levels in all three groups, could mean that all post-menopausal ovaries produce T and that ovaries with stromal hyperplasia are also able to produce E,, E, and A. This is confirmed by the fact that significant gradients for E,, E, and A occur more frequently in group 3 and that mean ovarian levels of E,, E, and A are significantly higher in group 3 than in group 1 (P -C0.01 for E,; P < 0.0001 for E,; P < 0.001 for A). Although the blood flow rate through the post-menopausal ovary is not known, we have arbitrarily taken it to be equal to 60 l/day, as calculated in pre-menopausal subjects by Baird and Fraser [26] and have estimated maximal ovarian secretion rates of 3.6, 5.5, 381 and 134 pg/24 h for E,, E,, A and T, respectively, for the women in group 3 with significant gradients. We consequently believe that an ovary with moderate or marked stromal hyperplasia can contribute not only to the pool of circulating T, but also to the pools of E, and A. Even though 21 women out of the 40 had endometrial carcinoma, this is not considered to have influenced our results. In fact, no difference was found between ovarian and peripheral levels in patients with or without endometrial carcinoma. Moreover, no correlation was established between the presence of stromal hyperplasia and endometrial carcinoma, in contrast with the findings of other investigators, who conducted a retrospective study in which only histological appearance was evaluated [16]. No significance differences in body weight were seen between patients in the three groups, which suggests that the presence or absence of a gradient does not seem to be influenced by the degree of obesity. Since the ovarian and the third peripheral blood samples were taken simultaneously during operation and would therefore be subject to the same variations as a consequence of surgery, the evaluation of the gradients ought not to have been affected by anaesthesia or surgical stress. During surgery only the peripheral A levels showed a rise, which could have resulted from an increased adrenal A contribution. There was a 17% incidence of moderate or marked stromal hyperplasia among the patients, which is lower than that found by Boss et al. [16] in autopsy specimens (28%) or that reported by Dennefors [ll] based on small pieces of tissue (47%). This is probably due to the fact that we included in group 3 only subjects with evident
64
stromal hyperplasia. The difficulty lies in distinguishing an ovary with slight stromal hyperplasia from a normal ovary. Our results are not in agreement with those of authors who studied ovarian secretion by indirect methods rather than by ovarian vein catheterisation [5] or who reported a minimal oestrogen gradient on the basis of only a few cases [2]. They are, however, in agreement with those of a number of other investigators [3,6,9] and indeed confirm what has already been demonstrated in vitro by Dennefors et al. [ll], namely that the ovarian stroma in post-menopausal women is able to aromatise androgens to oestrogens, and that ovaries affected by stromal hyperplasia can produce greater amounts not only of A but also of E, than atrophic ovaries. In conclusion, we believe that post-menopausal ovaries showing stromal hyperplasia contribute to the peripheral oestrogen pool, and that studies of hormonal patterns in post-menopausal women should always therefore take into account the histological characteristics of the ovary, which vary from individual to individual and cannot be detected by macroscopic examination alone.
References 1 Mattingly RF, Huang WY. Steroidogenesis of the menopausal and postmenopausal ovary. Am J Obstet Gynecol 1969; 103: 679-693. 2 Judd HL, Judd GE, Lucas WE. Yen SSC. Endocrine function of the postmenopausal ovary: concentration of androgens and oestrogens in ovarian and peripheral vein blood. J Clin Endocrinol Metab 1974; 39: 1020-1024. 3 Greenblatt RB, Colle ML, Mahesh VB. Ovarian and adrenal steroid production in the postmenopausal woman. Obstet Gynecol 1976; 47: 383-387. 4 Shenker JG, Polishuk WZ, Eckstein B. Pathways in the biosynthesis of androgens in the postmenopausal ovary in vitro. Acta Endocrinol 1971; 66: 325-332. 5 Vermeulen A. The hormonal activity of the postmenopausal ovary. J Clin Endocrinol Metab 1976; 42: 247-253. 6 Gronroos M, Klemi P, Sahni T, Rauramo L, Punnonen R, Ovarian production of oestrogens in postmenopausal women. Int J Gynecol Obstet 1980; 18: 93-98. 7 Judd HL, Lucas WE, Yen SSC. Serum 17/3-oestradiol and oestrone levels in postmenopausal women with and without endometrial cancer. J Clin Endocrinol Metab 1976; 43: 272-278. 8 Siiteri PK. Post-menopausal oestrogen production. In: Van Keep PA, Lauritzen C, eds. Oestrogens in the postmenopause. Frontiers in hormone Research. Vol. 3. Base]: Karger, 1975; 40-45. 9 Longcope C, Hunter R, Franz C. Steroid secretion by the postmenopausal ovary. Am J Obstet Gynecol 1980; 138: 564-568. 10 Kirshner MA, Cohen FB, Ryan C. Androgen-oestrogen production rates in post-menopausal women with breast cancer. Cancer Res 1978; 38: 4029-4035. 11 Dennefors’ BL, Janson PO, Knutson F, Hamberger L. Steroid production and responsiveness to gonadotropin in isolated stromal tissue of human post-menopausal ovaries. Am J Obstet Gynecol 1980; 136: 997-1002. 12 Lucisano A, Acampora MG, Russo N, Maniccia E, Montemurro A, Dell’Acqua S. Ovarian and peripheral plasma levels of progestogens, androgens and oestrogens in post-menopausal women. Maturitas 1984; 6: 45-53. 13 Judd HL, Shamonki IM, Frumar AM, Lagasse LD. Origin of serum oestradiol in post-menopausal women. Obstet Gynecol 1982; 59: 680-686. 14 Trevoux R. De Brux JA, Scholler R. Longitudinal histohormonal research on 220 peri- and postmenopausal women. In: Proc Xth World Congr Gynecol Obstet. San Francisco. CA, USA, 1982; 217.
65 15 Novak ER, Goldberg B, Jones GS, O’Toole RV. Enzyme histochemistry of the menopausal ovary associated with normal and abnormal endometrium. Am J Obstet Gynecol 1965; 93: 669-682. 16 Boss JH, Scully RE, Wagner KH. Cohen RB. Structural variations in the adult ovary: clinical significance. Obstet Gynecol 1965; 25: 747-764. 17 Serra GB, Caniglia R, Barile G. Some characteristics of the double antibody method for the assay of gonadotropins. In: Crosignani PG. James VHT, eds. Recent progress in reproductive endocrinology. London and New York: Academic Press, 1974; 65-82. 18 Emment Y, Collins WP, Sommerville IF. Radioimmunoassay of oestrone and oestradiol in human plasma. Steroid Biochem 1972: 3: 893-901. 19 Youssefnejadian E, Florensa E, Collins WP. Sommerville IF. Radioimmunoassay of plasma progesterone. J Steroid Biochem 1972; 3: 893-901. 20 Youssefnejadian E. Florensa E, Collins WP, Sommerville IF. Radioimmunoassay of 17-hydroxyprogesterone. Steroids 1972; 20: 773-788. 21 Collins WP. Mansfield MD, Alladina NS, Sommerville IF. Radioimmunoassay of plasma testosterone. Steroid B&hem 1972; 3: 333-348. 22 Montemurro A, Johnson MW, Barile G. Youssefnejadian E. A simple radioimmunoassay of androstenedione without column chromatography. J Obstet Gynecol 1981; 1: 247-250. 23 Meldrum DR, Davidson BJ, Tataryn IV. Judd HL, Changes in circulating steroids with aging in post-menopausal women. Obstet Gynecol 1981; 57: 624-628. 24 Vermeulen A, Verdonck L. Sex hormone concentrations in post-menopausal women. Clin Endocrinol 1978; 9: 59-66. 25 Chang RJ, Abraham GE. Peripheral arterial and venous concentrations of various androgens in patients with and without hirsutism. Obstet Gynecol 1975; 46: 549-550. 26 Baird DT. Fraser IS. Blood production and ovarian secretion rates of oestradiol-17P and oestrone in women throughout the menstrual cycle. J Clin Endocrinol Metab 38: 1009.