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The Clinical Application of a Gas Chromatographic Method for the Routine Determination of Urinary Pregnanediol
The Clinical Application of a Gas Chromatographic Method for the Routine Determination of Urinary Pregnanediol ANTONIO SCOMMEGNA, M.D., SAT! C. CHATTORAI, PH.D., and HERBERT H. WOTIZ, PH.D.
EVER
Marrian23 first isolated pregnanediol from the urine of pregnant humans and identified it as a major metabolite of progesterone, the analysis of urinary pregnanediol has been regarded as a convenient way of estimating the progesterone production in the body. It is well known that pregnanediol excretion is increased in situations where progesterone production is augmented, as in pregnancy and the luteal phase of the cycle. Daily injections of progesterone in doses of 50 mg.jday for a 2-week period to men and postmenopausal women resulted in the recovery of urinary pregnanediol in the amount of 12.2% of the hormone administered. 24 Similar results were obtained with the use of radioactive tracers,29.30 and soon urinary pregnanediol was used to calculate secretory rates of progesterone after injection of the radioactive precursor by the specific activity of its urinary metabolite. 30 In fact, as late as 1965, Dorfman and Ungar stated that pregnanediol may be considered a unique catabolite of progesterone. However, with the application of more sophisticated double isotope technics, l it became evident that this simplified view of a progesteronepregnanediol relationship is not entirely correct. Several substances other than progesterone are known to be excreted as urinary pregnanediol, and in certain circumstances they may be secreted in sufficient quantity to invalidate the assumption that the assay of pregnanediol in the urine accurately reflects progesterone secretion. SINCE
From the Section on Gynecic Endocrinology, Department of Obstetrics and Gynecology, Michael Reese Hospital and Medical Center, Chicago, Ill., and the Department of Biochemistry, Boston University School of Medicine, Boston, Mass. We are grateful to Dr. Melvin Cohen of the Fertility Center, and to Dr. Richard Frank of the Planned Parenthood Federation, Chicago, for sending patients to us to participate in this study.
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Steroids which may possibly contribute in a quantitatively important manner to the pregnanediol excreted in the urine are shown in Fig. 1. Besides progesterone, pregnenolone1 and desoxycorticosterone1 3 are known to be secreted and to contribute to pregnanediol excretion. While desoxycorticosterone is probably of little importance, since its conversion to pregnanediol-as far as we know-is thought to occur only to a quite limited extent, the precursor role of pregnenolone might be more important, especially in man, where it may constitute a major pregnanediol precursor. Yet the exact extent of each is uncertain, since Arcos et al. have presented indirect evidence pointing toward the fact that pregnenolone sulfate might also be secreted and participate in the pregnanediol pool. Furthermore, the isolation of 20a-OHa 4 pregnene-3-one and its 20(3 isomer by Zander in biologic material makes the pregnanediol picture still further uncertain. In the rabbie 2 this steroid constitutes the major progestin secreted by the ovary, in quantities 10 times larger than progesterone. In the human it is not known whether it is derived from peripheral metabolism of progesterone or secreted as such by the ovary. Therefore, its role, if any, as a direct precursor to urinary pregnanediol is at present open to question. However, in spite of all these limitations and qualifications, it can be safely stated that for clinical purposes, urinary pregnanediol excretion in ~H3
c·o
o~ I
_
HO-S-O II
o
r
2
2
~H20H
H3
J~' ~ 3
3
Fig. 1. Secretory precursors of pregnanediol. At left, urinary metabolite, pregnanediol. At center, proved secretory products: (1) progesterone; (2) pregnenolone; (3) desoxycorticosterone. At right, possible secretory products: ( 1) pregnenolone sulfate; (2) 20a-hydroxYA4 pregnene-3-one; (3) 20,B-hydroxy pregnene-3-one.
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women, except under unusual circumstances, can still be used as a test of progesterone secretion. The knowledge of the progesterone production can be of significance in many clinical conditions, since the importance of this steroid spans the whole spectrum of reproductive physiology. Oftentimes this knowledge must be quickly acquired in order to be of practical value in the intelligent management of the patient. However, older methods of pregnanediol measurements were often inaccurate, or if precise, their elaborate and time-consuming nature sharply curtailed their clinical usefulness. The application of gas chromatography to the analysis of steroid hormones offered to the clinician the promise of a tool which older clinical technics could not match either in speed or sensitivity, and this new tool was soon applied to the analysis of pregnanedioP4, 35 However, only a few of the methods published after being developed in the research laboratory have been exposed to extensive routine use in the clinical laboratory. The procedure of Wotiz was modified by Chattoraj and Wotiz, who further explored in depth the validity of this method. Its specificity, sensitivity, precision, and accuracy favorably compare with the established technic described by KlopperY The unique advantage of this gas chromatographic technic lies in its rapidity, and for this reason we chose it for the routine determination of pregnanediol in our laboratory. A technician can comfortably perform 75 assays per week; however, if needed, the result of 1 assay can be obtained within 3 hr. after the urine reaches the laboratory. _" Pregnanediol excretion as an index of ovarian and placental function has been extensively evaluated by classic chemical methods, and its usefulness has been proved beyond doubt. 3 , 18,19, 22 Similar data on the applicability of a relatively modem technic such as gas chromatography in clinical cases are lacking in the literature. The purpose of the present communication is to report our recent experience in the performance of over 500 assays with this technic, and to show its usefulness and validity in different clinical conditions. MATERIALS AND METHODS
Urine specimens were obtained from normal volunteers, as well as from patients attending our clinic for endocrine and nonendocrine gynecologiC disorders. A large number of patients were also made available to us through other organizations. Most of the patients, as well as the normal volunteers, were ambulatory. During collection, 24-hr. urine specimens were refrigerated. The completeness of collection was checked by measurement of urinary creatinine, and
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the urine specimens were processed as soon as they were received in the laboratory. The method 5 used for the analysis, in brief, consists of an acid hydrolysis, toluene extraction, sodium hydroxide and water washes, acetylation of the organic extract, and introduction into a gas chromatograph. Most of the analyses were carried out on a glass column, 6 ft. X 4 mm. I.D., packed with 3% SE-30 on Diatoport S* (80-100 mesh size) equipped with a flame ionization detector. RESULTS AND DISCUSSION Investigation of Ovarian Function
During a normal menstrual cycle, progesterone is essentially secreted by the adrenal and by the corpus luteum, as a result of the transformation of the ruptured follicle following ovulation. While it seems that there is a slight production of progesterone before ovulation, its secretion does not become quantitatively Significant until about 36 hr. after the discharge of the ovum, and it ceases almost abruptly about 36 hr. before menstruation. In order to investigate ovarian function during this cycle, measurement of plasma progesterone would be the most desirable test; however, urinary pregnanediol determination is the most practical. In 98 menstrual cycles of 91 normal volunteers and patients with nonendocrine gynecologic complaints, the urinary pregnanediol values obtained were as follows: proliferative phase, 0.10-1.26 mg.j24 hr. (mean, 0.48 -+- 0.31 S.D.); secretory phase, 1.17-9.50 mg.j24 hr. (mean, 2.68 -+1.68 S.D.). The values obtained were scattered throughout the cycle. In all cases the occurrence of ovulation was documented by one or more of the following technics: culdoscopic visualization of the corpus luteum, secretory changes in the endometrial biopsy, biphasic basal body temperature, or the occurrence of pregnancy. After ovulation had been documented and menstruation occurred, ovulation was assumed to have taken place 14 days prior to the initiation of the menstrual flow. The ovulation date was used to divide the proliferative from the secretory phase. The date so established coincided with the shift in the basal body temperature when this information was available. The pregnanediol values were then grouped according to the phase of the cycle. During the proliferative phase of the cycle, pregnanediol excretion rarely exceeds 1 mg.J24 hr. The majority of the values are between 0.3 and 0.8 mg.j24 hr. During the luteal phase, the range is 1.2-9.0 mg.j24 hr. *F & M Scientific Corp., Avondale, Pa.
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The highest values were obtained around Days 21-23 of a 28-day cycle. However, the majority of the measurements randomly obtained during this phase of the cycle were between 1.8 to 3.5 mg.j24 hr. These data are quite similar to values reported by Klopper .19 One may readily appreciate that interference by adrenal pregnanediol, while diminishing the specificity of this assay as a test of luteal function, does not cause any difficulty in the recognition of a corpus luteum, once this structure is formed. The daily pregnanediol excretion pattern in 6 patients with ovulatory cycles is shown in Fig. 2. . Whenever a secretory endometrium was obtained, or a thermal shift in the basal body temperature occurred, or a fresh corpus luteum was seen at culdoscopy, pregnanediol values in the pro gravidic range were obtained. There were only 4 patients in whom pregnanediol in the second half of the cycle was below the secretory ranges (Table 1). Endometrial biopsy was available in 2()f the 4 patients, while basal body
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30
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-36
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Fig. 2. Pregnanediol excretion patterns in 6 patients with ovulatory cycles. X indicates beginning of menses.
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TABLE 1. Pregnanediol Values in Patients Probably Showing a Poor Luteal Phase
Patient
Length of cycle (days)
B.B.
31
B.C.
27 29
Pregnanediol value (mg./24 hr.)
Basal body temperature
25
.38 .44
Short biphasic
9 18 10 20
.34 .93 .28 1.78
Poor biphasic
Biphasic
Day of cycle
11
E.B.
28
18
1.04
B.B.
28
12 19 10 24
.28 .51 .46 .22
36
Biphasic
Poor bipbasic Biphasic
temperature was available in all cycles. In each of the 4 patients (except EB ), the thermal shift was atypical, showing one or more dips in the thermal plateau, and/or lasting less than 14 days. The histology of the endometrium in Patients EB and BB was not consistent with the day of the cycle during which the biopsy was obtained, showing merely quite early secretory changes in only a few glands, indicating irregular ripening. 'V'hether these biopsies were examples of poor luteal phase, and if so, what the significance is, we are not prepared to say. However, all 4 patients were complaining of infertility. In Patient B.B., low pregnanediol values in the progravidic half of the cycle were obtained in 2 consecutive cycles, while B.G., in another cycle, had a good biphasic temperature together with pregnanediol values in the secretory range. Even with this small amount of data, it might be tempting to postulate that urinary pregnanediol is an exceedingly useful test, not only in ascertaining the occurrence of ovulation, but in evaluating the functional status of the corpus luteum as well. It seems to us that more appropriate to the latter task would be the use of human chorionic gonadotropin stimulation of the corpus luteum, as reported by Jayle et al. They suggested that the administration of HCG in the second half of the cycle could be used to evaluate the dynamic capabilities of the corpus luteum to respond to the stimulus of pregnancy. The usefulness of this test is currently being investigated in our laboratory.
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The occurrence of pregnancy is accompanied by a marked rise in pregnanediol even prior to the date of the expected period. This is confirmed with our present technic in a patient who conceived during this study. Pregnanediol values on Days 10, 18, and 27 were, respectively, 0.24, 2.12, and 9.6 mg.j24 hr. A positive pregnancy test was obtained on Day 36 of the same cycle. The determination of urinary pregnanediol has also been used by us as a parameter of corpus luteum function after the administration of clomiphene citrate (Clomid*) in the induction of ovulation. In this group of patients, if spontaneous menstrual cycles were not present, vaginal bleeding was induced by medroxy progesterone acetate (Proverat), 20 mg.jday for 3 days. Although small elevations can be observed in patients without specific signs of ovulation16 (Table 2), pregnanediol levels in the secretory range are consistently accompanied by other signs of corpus luteum formation. Increased urinary pregnanediol excretion, however, is per se not a sufficient indication of ovulation and normal corpus luteum function, especially in patients treated with Clomid. Luteinization of follicles, with or without cyst formation, is a rather frequent occurrence. In these cases, though, pregnanediol excretion is quite frequently in the upper limit or above the normal secretory range. Recently we have been experimenting with smaller doses of Clomid (250 mg. per cycle) in order to decrease the side effects of this drug. With this reduced dosage we have not encountered any symptomatic cyst formation, and the urinary pregnanediol values reflect a more physiologic ovarian stimulation (Table 2). Whether this reduced dosage will result in an increased number of conceptions along with decreased side effects, we do not know. However, we feel that pregnanediol determination greatly supplements other parameters in the evaluation of the ovarian status after the administration of this drug. Another useful area for the use of urinary pregnanediol determination is its estimation in patients using oral contraceptives. While the exact mechanism of action of these drugs is not well understood, it is generally assumed that pituitary and gonadal inhibition with consequent suppression of ovulation offers the most satisfactory explanation of their mechanism of contraception. Their antiovulatory activity is, however, difficult to evaluate since the commonly employed tests of ovulation, such as basal body temperature and progestational endometrial changes, are affected by the thermogenic and progestational activities of the steroids administered. *William S. Merrell Co., Cincinnati, Ohio. tThe Upjohn Company, Kalama~oo, Mich.
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TABLE 2. Urinary Pregnanediol in Patients Receiving Clomid Pregnanediol Patient
Days of Clomid adminUit.*
Cycle dayt
Values (mg./24 hr.)
Basal body temp.
DiagnosUi and remarks
Anovulation, cause unknown; ovulated on treatment; had period on Day 45 of cycle Polycystic ovaries; period on Day 42 Polycystic ovaries; period on Day 37 Polycystic ovaries; period on Day 60 Polycystic ovaries; pregnancy test pos. Day 36
R.K.
16-20
11 22 38
0.30 0.30 1.20
Flat Flat Rise, Day 31
L.S.
20-24
D.M.
13-17
R.S.
37-41
L.S.
3-7
18 35 13 35 30 48 10 18 27 7 22 37 24 40
0.12 2.14 0.40 1.75 0.46 3.60 0.24 2.12 9.60 0.11 0.20 5.92 0.41 0.92
Flat Rise, Flat Rise, Flat Rise, Flat Rise,
Flat Flat Rise, Day 26 Flat Flat
M.Z.
8-12
D.C.
26--30
N.K.
13-17
13 37
0.22 0.53
Flat Flat
D.M.
30-34
30 44
0.39 0.96
Flat Flat
Day 28 Day 23 Day 45 Day 15
Polycystic ovaries; pregnancy test pos. Day 56 Secondary amenorrhea, cause unknown; no resp to treatment Secondary amenorrhea, cause unknown; no resp. to treatment Polycystic ovaries; period on Day 49
*Dosage of Clomid was 50 mg. except in Patients D.M. and D.C., given 100 mg. tThe first day of vaginal bleeding, spontaneous or induced, was considered Day I of the cycle.
Fortunately, none of the contraceptive compounds administered causes an increased excretion in urinary pregnanediol, which has proved to be a highly reliable test for the detection of the anovulatory status. 4 , 9, 26 Furthermore, suppression of pituitary 'gonadotropin might not occur in all cases, and changes in the uterine environment, such as thickening of cervical mucus and advancing of endometrial age, have also been implicated to explain the remarkable effectiveness of oral contraceptives.lO As a matter of fact, Goldzieher et al. have found elevated luteal-phase pregnanediol in 6.8% of users of Orthonovum* in the lO-mg. size, a finding which suggests the possible establishment of a ~orpus luteum. One might *Ortho Pharmaceutical Corporation, Raritan, N. J.
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TABLE 3. Pregnanediol Excretion in Patients Taking Ovulen (1 Mg.) for Birth Control
Patient
F.D. e.S. J.W. B.H. O.S. W.A. J.B.
S.P.
Length of treatment (mo.)
Pregnanediol (mg./21, hr.)*
1 4 4 8 8 8 8 9 9
0.35 0.31 0.40 0.09 0.14 0.05 0.26 0.11 0.51
,!,
*AU values shown are for second half of cycle; Patient F .D. showed an excretion of 0.27 mg.j24 hr. in the first half of cycle after 3 months of treatment.
think that the possibility of corpus luteum fonnation in the presence of effective oral contraception might be more likely to occttr with the use of smaller doses of medication. In fact, Rock reported that 2 of 11 patients taking 1 mg. of ethynodiol diacetate in combination with 0.1 mg. of mestranol (Ovulen*) for contraception had elevated pregnanediol iJ;! the second part of the cycle. It seemed interesting to us to investigafle urinary pregnanediol excretion in some women receiving Ovulen in the l~g. dose. Table 3 shows the values obtained in patients on this medication, while Table 4 shows values obtained in patients on sequential mestranol-chlormadinone (C-Quenst) contraceptive medication. It is apparent that if an increase in urinary pregnanediol occurs, it does not take place with any greater frequency than in patients using larger doses of oral contraceptives.l l The re-establishment of normal ovulatory function following cessation of treatment is also documented by urinary pregnanediol determination in 13 patients (Table 5). In 10, the first cycle proved to be ovulatory; however, 2 more patients might have ovulated also, but it could not be documented because specimens were not obtained during the second half of the cycle. Ovulation was proved to be established in subsequent cycles. In only 1 patient (C.P.) did ovulation seem not to occur until the third menstrual cycle after discontinuing the treatment. In this study, whenever a pregnanediol detennination was performed, endometrial biopsy was also obtained, and in each case a perfect correlation was established. *Brand of ethynodiol diacetate 1.0 mg. with mestranol 0.1 mg. G. D. Searle & CQ, tEli Lilly and Company, Indianapolis, Ind.
-,
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TABLE 4. Pregnanediol Values for Patients Taking C-Quens Pregnanediol values in luteal phase After treatment Patient No.
Before treatment (,..g./24 hr.)
13 1 8 12
3450 3000 1150 4396 1796
9
5429
10 5 3
915 2590 1617
2 7 6 4 11
6039 8700 3861 1302 5400
14
Mo. of treatment
1 2 3 3 3 6 3 7 4 4 5 11 7 7 10 11 11
p.g./24 hr.
280 360 438 975 372 330 68 97 37 107 737 43 197 270 676 160 360
When failure of corpus luteum development occurs, regardless of its causes, it is reasonable to assume that the urinary pregnanediol excretion would be in the proliferative range. It has been reported that in some cases of polycystic ovaries there was an increase in pregnanediol or in the "pregnane-complex."8. 33 These cases were supposed to be amenable to corticosteroid treatment. The findings apparently were a result of using methods of poor specificity, since pregnanediol per se is not elevated in patients with the classic Stein-Leventhal syndrome or in patients with the postpubescent variety of the adrenogenital syndrome, even though ovarian enlargement might be present.2 • 21. 31 This fact is evident in Table 6, where the pregnanediol values of all patients suffering from anovulation, regardless of cause, are indistinguishable from the values obtained in the proliferative phase of the cycle. The daily pregnanediol excretion pattern in 2 patients with anovulatory cycles are shown in Fig. 3. Investigation of Adrenal Function
The adrenal contribution to urinary pregnanediol, while small, is nevertheless real, and in men it might contribute a major part of it. 1 Pregnanediol values obtained in patients after bilateral oophorectomy are shown in
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Table 7. Small but definitely measurable amounts are present, and the implication is that the adrenal is responsible for the excretion of this compound in these patients. The adrenal origin of the pregnanediol is further confirmed by its response to ACTH stimulation (Table 8). Klopper et al. 20 first suggested that pregnanediol assay may be of value in evaluating adrenocortical function. No conclusions can be drawn from our data as to the value of pregnanediol in the diagnosis of adrenal diseases. However, in a recent report on TABLE 5. Pregnanediol Excretion Following Discontinuation of Treabnent with Ovulen
Patient
M.T.
E.W. A.R.
S.W. P.E. V.M. D.M.
c.P. B.H. F.D.
P.B. M.A.
C.B.
Length of treatment (mo.)
3 3 3 3 4 3 5 5 5 6 6 6 6
Pregnanediol ellJcretion (mg./24 hr.) after discontinuation of treatment First half of cycle
0.28, * 0.33t 0.15* 0.62t 1.00* 0.12*
0.64* 0.22,* 0.19,* 0.17* O.52t
Second half of cycle
0.37,t O.90,t 2.09:1: l.71t 1.86* 2.25* 2.90,* 1.43t 9.50* 2.05* 0.79,* 0.10,t 1.18:1: 7.80* 2.01* 3.84* 2.10* 1.64,* 1.64t
Most of the cycles were longer than 28 days; the second half of the cycle was arbitrarily assumed to begin 14 days prior to the following menstrual period. *First cycle. tSecond cycle. :::Third cycle.
TABLE 6. Pregnanediol Excretion in Patients with Anovulatory Cycles or Amenorrhea
Diagnosis
No. of cases
Secondary amenorrhea (other than polycystic ovaries) Anovulatory cycles (second half) Stein-Leventhal syndrome Postpubescent adrenogenital syndrome
23 6 12 2
Mean ellJcret., pregnanediol (mg./24 hr.)
0.39 ± 0.13 0.42 ± 0.09 0.45 ± 0.12 0.26 0.39
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20 cases of Cushing's syndrome, Martin and Hammer showed that pregnanediol excretion was elevated in all 4 patients with carcinoma, while it was normal in the rest of patients with hyperplasia or adenoma. Furthermore, in the latter group, after ACTH stimulation, all 5 patients with adenoma had a normal response, while 7 of 8 patients with hyperplasia had an elevated response in the pregnanediol output. The authors therefore concluded that urinary pregnanediol may be the most useful single test in determining the cause of Cushing's syndrome. With the ready availability of pregnanediol assay, the applicability of this test in the diagnosis of adrenal diseases should be further explored. ~
4.0
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"';
g
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_
A.
0 L.---'--::~=---_-=---='_~_-==O!II....
~ ~ 4.0
____x_x_x
>.
6c: .~
....o 0.
~
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Fig. 3. Pregnanediol excretion patterns in 2 patients with anovulatory cycles. X indicates beginning of menses. At bottom, Patient 1; at top, Patient 2 .
xxx OJ--T~.-~=r-.~r-~~-,-.-,-~-
5
7
9
II
13 15
17 19 21 23 25 27
Day of cycle
TABLE 7. Pregnanediol Excretion after Bilateral Oophorectomy in Patients with Carcinoma of the Breast Patient
Age
A.D.A.
40
A.L.P.
65
B.A.R. M.C.C.
35 47
T.R.A.
55
Z.A.G. O.L.E.G.
58 56
Pregnanediol ea:cretion (/LU./24 hr.)
201 182 148 103 200 152 193 125 223 102 129 172 450
All but Patient D.L.E.G., who received X-ray therapy, were treated by surgery. The meall excretion rate in the group on whom an operation was performed, was 161 ± 40 pg./24 hr.
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Investigation of Corpus Luteum of Pregnancy and Placental Function
Because of the large increase of urinary pregnanediol which accompanies the establishment of pregnancy, its measurement is relatively easy during this condition. Here again, gas chromatography, because of its speed, might prove t~ be an exceedingly useful tool in the management of pregnancy disturbances. While progestin treatment in patients suffering from habitual abortion might be a debatable issue, its use in patients in whom no progesterone deficiency is demonstrable is to be d~plored. The potential risks of progestational therapy in the mother as well as in the baby range from induced missed abortion through masculinization of the female fetus. It seems to us that the restriction of progestational therapy to cases where a progesterone deficiency, as evidenced by low pregnanediol values, can be demonstrated might result not only in less therapeutic meddling, but also in an apparent improvement of the results obtained, since the therapy will then be restricted to patients who are most likely to benefit from such management. The necessity for a means of accurately assessing fetoplacental function in the second and third trimester of pregnancy is self-evident. However, it is debatable whether pregnanediol assay is suitable for this purpose. Pregnanediol excretion does not seem to correlate with fetal outcome in diabetic pregnancies,7 although it might be of some usefulness in toxemia of pregnancy and preeclampsia. 15 • 27 However, urinary estriol determinations TABLE 8. Pregnanediol Excretion in Patients after ACTH Administration 17-0HCS (mg./24 hr.)
Patient
17-KS (mg./24 hr.)
Pregnanediol (mg./24 hr.)
[nit.
Sere
Control
ACTH
Control
ACTH
Control
ACTH
AP.U.
M
4.2
6.0
M M
5.4 6.5
M.O.O. P.E.A.
M F
8.6 3.6
M.A.R B.RE.
F F
5.4 8.4
18.0 22.7 23.5 15.7 27.7 46.8 24.2 24.9 14.1 18.3
0.54
RO.T. B.O.R
7.1 20.1 26.4 27.0 22.3 26.3 13.9 15.0 16.8 9.4
0.59 0.77 2.90 0.31 1.20 0.82 4.00 5.30 1.20 3.28
12.9 9.4 20.3 11.0 7.3 15.1
0.44 0.38 0.46 2.30 0.46 1.16
All male subjects were normal. Of the female patients, Patient P.E.A. was hirsute and was given ACTH in second half of cycle; M.A.R. had anovulatory cycles and was given ACTH in first half of cycle; B.R.E. had what was believed to be adrenal hyperplasia.
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TABLE 9. Pregnanediol and Estriol Excretion in Some Patients with Abnormal Gestation Estriol Pregnanediol (mg./24 hr.) (mg./24 hr.)
Gestation Patient
(wk.)
B.L.
J.P.
18 26 36
E.H.
38 14
J.T.
26
p.e.
Statm
Hydatidiform mole Intra-uterine fetal death Triplets
Therapeutic abortion Before intra-amniotic infusion After intra-amniotic infusion Rh sensitization; intra-uterine fetal death Mter intra-amniotic infusion
14.16 49.20 52.00 41.40 45.60 16.60 14.00 10.38 2.57 26.80 10.75 16.50
.34 .62 40.40 38.20 48.00 .89 .88 .36 .14 .80 .56 .80
are more helpful since decreases in estriol precede the reduced pregnanediol excretion. 32 We have not accumulated enough material to be in the position to confirm or refute these observations, yet some of the preliminary data agree with them. Some representative estimates obtained with our present technic are presented in Table 9. It can be seen that pregnanediol values in intra-uterine fetal death are still normal, while estriol values are markedly decreased (Patients T.}. and P.C.). After intraammiotic instillation of hypertonic saline for therapeutic abortion (Patients E.H. and }.T. ), estriol proves to be a better indicator of the fetal status, although a moderate pregnanediol drop is also evident. SUMMARY
Urinary pregnanediol, while not a unique metabolite of progesterone, is still a useful indication of progesterone secretion in women. It may be helpful in the management of many clinical conditions if assays of its excretion can be obtained with speed. A relatively new gas chromatographic technic has been applied to the investigation of normal cycles as well as of many clinical situations. Its usefulness in the measurement of pregnanediol favorably compares with established chemical methods. The speed with which it can be performed renders it highly suitable for routine use in the steroid laboratory. Department of Obstetrics and Gynecology Michael Reese Hospital and Medical Center 29 St. and Ellis Ave. Chicago, Ill. 60616
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REFERENCES 1. ARcos, M., GURPIDE, E., VAN DE WIELE, R L., and LIEBERMAN, S. J Clin Endocr 24:237, 1964. 2. BAULIEU, E. L., MAUVOIS-JARVIS, P., and CORPECHOT, L. J Clin Endocr 23:374, 1963. 3. BROWN, J. B., KLOPPER, A., and LORAINE, J. A. J Endocr 17:401,1958. 4. BUCHHOLZ, R, NOCKE, L., and NOCKE, W. Int J Fertil9:231, 1964. 5. CHATTORAJ, S. C., and WOTIZ, H. H. Fertil Steril In press. 6. DORFMAN, R I., and UNGAR, F. Metabolism of Steroid Hormones. Acad. Press, New York, 1965, p. 546. 7. EDDIE, D. A. C. J Obstet Cynaec Brit Comm 70:847, 1963 . 8. GOLD, J. J., and FRANK, RAmer J Obstet Cynec 75:1034,1958 . 9. GOLD, J. J., SMITH, L., SCOMMEGNA, A., and BORUSHEK, S. Int J Fertil 8:725, 1963. 10. GOLDFARB, A. Metabolism 14:339, 1965. 11. GOLDZIEHER, J. W., MOSES, L. E., and ELLIS, L. T. JAMA 180:359, 1962 . 12. HILLIARD, J., ARCIDBALD, D., and SAWGERS, C. H. Endocrinology 72:59, 1963. 13. HORWITT, B. N., DORFMAN, R I., SIDPLEY, R A., and FISH, W. R J Bioi Chem 155:213, 1944. 14. JAYLE, M. F., GELLER, S., SCHOLLER, R, and CHARMINE, H. Bull Fed Cynec Obstet Franc 13:594, 1961. 15. KANKAARINKA, T. Scand J Clin Lab Invest 15 (Supp. 74) :1,1963. 16. KISTNER, R. W. Obstet Cynec Survey 6:873, 1965. 17. KLOPPER, A., MICHIE, E. A., and BROWN, J. B. J Endocr 12:209,1955. 18. KLOPPER, A. J Endocr 13:291, 1956. 19. KLOPPER, A. J Obstet Cynaec Brit Comm 64:504,1957. 20. KLOPPER, A., STRONG, J. A., and COOK, L. R J Endocr 15:180,1957. 21. LANTIDER, A., and SANDOR, T. Acta Endocr 46:245, 1964. 22. LORAINE, J. A. The Clinical Application of Hormone Assay. Livingstone, London, 1958, p. 218. 23. MARRIAN, G. F. Biochem J 23:1090, 1929. 24. MARRIAN, G. F., RUSSELL, M. E., and ATHERDEN, S. M. J Endocr 10:351, 1954. 25. MARTIN, M. M., and HAMMER, B. L. J Clin Invest 44: 1073, 1965. 26. NEVINNy-STICKEL, J. Int J Fertil 9:57, 1964. 27. ROBERTSON, J. G., and MAXWELL, A. W. J Obstet Cynec 70:422, 1963. 28. ROCK, J. Clinical investigation data in files of Clinical Research Department, G. B. Searle & Co. 29. ROMANOFF, L. P. Excerpta Med. Int. Congr. Ser. 51, 1962, p. 194. 30. ROMANOFF, L. P., MORRIS, C. W., WELCH, P., GRACE, M.P., and PINCUS, G. J Clin Endocr 23:286, 1963. 31. SHERMAN, R P., and Cox, R I. Amer J Obstet Cynec 92:747,1965. 32. STRAND, A. Acta Obstet Cynec Scand 42 (Supp. 6) :96,1963. 33. VAN DE WIELE, R L. Bull Sloane Hosp Wom 6:82,1960. 34. WOTIZ, H. H. Biochim Biophys Acta 69:415, 1963. 35. WOTIZ, H. H., and MOZDEN, P. J. In Human Ovulation. Keefer, C. S., Ed. Little, Boston, 1965, p. 160. 36. ZANDER, J. Brook Lodge Symposium on Progesterone. Brook Lodge Press; Augusta, Mich., 1961, p. 77.
EVER
Marrian23 first isolated pregnanediol from the urine of pregnant humans and identified it as a major metabolite of progesterone, the analysis of urinary pregnanediol has been regarded as a convenient way of estimating the progesterone production in the body. It is well known that pregnanediol excretion is increased in situations where progesterone production is augmented, as in pregnancy and the luteal phase of the cycle. Daily injections of progesterone in doses of 50 mg.jday for a 2-week period to men and postmenopausal women resulted in the recovery of urinary pregnanediol in the amount of 12.2% of the hormone administered. 24 Similar results were obtained with the use of radioactive tracers,29.30 and soon urinary pregnanediol was used to calculate secretory rates of progesterone after injection of the radioactive precursor by the specific activity of its urinary metabolite. 30 In fact, as late as 1965, Dorfman and Ungar stated that pregnanediol may be considered a unique catabolite of progesterone. However, with the application of more sophisticated double isotope technics, l it became evident that this simplified view of a progesteronepregnanediol relationship is not entirely correct. Several substances other than progesterone are known to be excreted as urinary pregnanediol, and in certain circumstances they may be secreted in sufficient quantity to invalidate the assumption that the assay of pregnanediol in the urine accurately reflects progesterone secretion. SINCE
From the Section on Gynecic Endocrinology, Department of Obstetrics and Gynecology, Michael Reese Hospital and Medical Center, Chicago, Ill., and the Department of Biochemistry, Boston University School of Medicine, Boston, Mass. We are grateful to Dr. Melvin Cohen of the Fertility Center, and to Dr. Richard Frank of the Planned Parenthood Federation, Chicago, for sending patients to us to participate in this study.
257
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Steroids which may possibly contribute in a quantitatively important manner to the pregnanediol excreted in the urine are shown in Fig. 1. Besides progesterone, pregnenolone1 and desoxycorticosterone1 3 are known to be secreted and to contribute to pregnanediol excretion. While desoxycorticosterone is probably of little importance, since its conversion to pregnanediol-as far as we know-is thought to occur only to a quite limited extent, the precursor role of pregnenolone might be more important, especially in man, where it may constitute a major pregnanediol precursor. Yet the exact extent of each is uncertain, since Arcos et al. have presented indirect evidence pointing toward the fact that pregnenolone sulfate might also be secreted and participate in the pregnanediol pool. Furthermore, the isolation of 20a-OHa 4 pregnene-3-one and its 20(3 isomer by Zander in biologic material makes the pregnanediol picture still further uncertain. In the rabbie 2 this steroid constitutes the major progestin secreted by the ovary, in quantities 10 times larger than progesterone. In the human it is not known whether it is derived from peripheral metabolism of progesterone or secreted as such by the ovary. Therefore, its role, if any, as a direct precursor to urinary pregnanediol is at present open to question. However, in spite of all these limitations and qualifications, it can be safely stated that for clinical purposes, urinary pregnanediol excretion in ~H3
c·o
o~ I
_
HO-S-O II
o
r
2
2
~H20H
H3
J~' ~ 3
3
Fig. 1. Secretory precursors of pregnanediol. At left, urinary metabolite, pregnanediol. At center, proved secretory products: (1) progesterone; (2) pregnenolone; (3) desoxycorticosterone. At right, possible secretory products: ( 1) pregnenolone sulfate; (2) 20a-hydroxYA4 pregnene-3-one; (3) 20,B-hydroxy pregnene-3-one.
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women, except under unusual circumstances, can still be used as a test of progesterone secretion. The knowledge of the progesterone production can be of significance in many clinical conditions, since the importance of this steroid spans the whole spectrum of reproductive physiology. Oftentimes this knowledge must be quickly acquired in order to be of practical value in the intelligent management of the patient. However, older methods of pregnanediol measurements were often inaccurate, or if precise, their elaborate and time-consuming nature sharply curtailed their clinical usefulness. The application of gas chromatography to the analysis of steroid hormones offered to the clinician the promise of a tool which older clinical technics could not match either in speed or sensitivity, and this new tool was soon applied to the analysis of pregnanedioP4, 35 However, only a few of the methods published after being developed in the research laboratory have been exposed to extensive routine use in the clinical laboratory. The procedure of Wotiz was modified by Chattoraj and Wotiz, who further explored in depth the validity of this method. Its specificity, sensitivity, precision, and accuracy favorably compare with the established technic described by KlopperY The unique advantage of this gas chromatographic technic lies in its rapidity, and for this reason we chose it for the routine determination of pregnanediol in our laboratory. A technician can comfortably perform 75 assays per week; however, if needed, the result of 1 assay can be obtained within 3 hr. after the urine reaches the laboratory. _" Pregnanediol excretion as an index of ovarian and placental function has been extensively evaluated by classic chemical methods, and its usefulness has been proved beyond doubt. 3 , 18,19, 22 Similar data on the applicability of a relatively modem technic such as gas chromatography in clinical cases are lacking in the literature. The purpose of the present communication is to report our recent experience in the performance of over 500 assays with this technic, and to show its usefulness and validity in different clinical conditions. MATERIALS AND METHODS
Urine specimens were obtained from normal volunteers, as well as from patients attending our clinic for endocrine and nonendocrine gynecologiC disorders. A large number of patients were also made available to us through other organizations. Most of the patients, as well as the normal volunteers, were ambulatory. During collection, 24-hr. urine specimens were refrigerated. The completeness of collection was checked by measurement of urinary creatinine, and
260
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the urine specimens were processed as soon as they were received in the laboratory. The method 5 used for the analysis, in brief, consists of an acid hydrolysis, toluene extraction, sodium hydroxide and water washes, acetylation of the organic extract, and introduction into a gas chromatograph. Most of the analyses were carried out on a glass column, 6 ft. X 4 mm. I.D., packed with 3% SE-30 on Diatoport S* (80-100 mesh size) equipped with a flame ionization detector. RESULTS AND DISCUSSION Investigation of Ovarian Function
During a normal menstrual cycle, progesterone is essentially secreted by the adrenal and by the corpus luteum, as a result of the transformation of the ruptured follicle following ovulation. While it seems that there is a slight production of progesterone before ovulation, its secretion does not become quantitatively Significant until about 36 hr. after the discharge of the ovum, and it ceases almost abruptly about 36 hr. before menstruation. In order to investigate ovarian function during this cycle, measurement of plasma progesterone would be the most desirable test; however, urinary pregnanediol determination is the most practical. In 98 menstrual cycles of 91 normal volunteers and patients with nonendocrine gynecologic complaints, the urinary pregnanediol values obtained were as follows: proliferative phase, 0.10-1.26 mg.j24 hr. (mean, 0.48 -+- 0.31 S.D.); secretory phase, 1.17-9.50 mg.j24 hr. (mean, 2.68 -+1.68 S.D.). The values obtained were scattered throughout the cycle. In all cases the occurrence of ovulation was documented by one or more of the following technics: culdoscopic visualization of the corpus luteum, secretory changes in the endometrial biopsy, biphasic basal body temperature, or the occurrence of pregnancy. After ovulation had been documented and menstruation occurred, ovulation was assumed to have taken place 14 days prior to the initiation of the menstrual flow. The ovulation date was used to divide the proliferative from the secretory phase. The date so established coincided with the shift in the basal body temperature when this information was available. The pregnanediol values were then grouped according to the phase of the cycle. During the proliferative phase of the cycle, pregnanediol excretion rarely exceeds 1 mg.J24 hr. The majority of the values are between 0.3 and 0.8 mg.j24 hr. During the luteal phase, the range is 1.2-9.0 mg.j24 hr. *F & M Scientific Corp., Avondale, Pa.
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PREGNANEDIOL DETERMINATION
The highest values were obtained around Days 21-23 of a 28-day cycle. However, the majority of the measurements randomly obtained during this phase of the cycle were between 1.8 to 3.5 mg.j24 hr. These data are quite similar to values reported by Klopper .19 One may readily appreciate that interference by adrenal pregnanediol, while diminishing the specificity of this assay as a test of luteal function, does not cause any difficulty in the recognition of a corpus luteum, once this structure is formed. The daily pregnanediol excretion pattern in 6 patients with ovulatory cycles is shown in Fig. 2. . Whenever a secretory endometrium was obtained, or a thermal shift in the basal body temperature occurred, or a fresh corpus luteum was seen at culdoscopy, pregnanediol values in the pro gravidic range were obtained. There were only 4 patients in whom pregnanediol in the second half of the cycle was below the secretory ranges (Table 1). Endometrial biopsy was available in 2()f the 4 patients, while basal body
4.0 2.0
j
O~~~~~~~------~~__
6.0
4.0 .c
BAR5
2.0
~
,
x x
o~----------------------------
12'] ~ 4.0j
~xx
e
A.
.: 2.0
DELA 4
DEL
3
~ O.L-..:~,------",-
____ X_x_ _
co
~2·]~~xx
L);
IDI
4.0j 2~
o
SHD 2
.
7 i i i -r""r7t 6
10
14
18
22
26
30
34
~x
-36
Day af cycle
Fig. 2. Pregnanediol excretion patterns in 6 patients with ovulatory cycles. X indicates beginning of menses.
262
SCOMMEGNA ET AL.
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TABLE 1. Pregnanediol Values in Patients Probably Showing a Poor Luteal Phase
Patient
Length of cycle (days)
B.B.
31
B.C.
27 29
Pregnanediol value (mg./24 hr.)
Basal body temperature
25
.38 .44
Short biphasic
9 18 10 20
.34 .93 .28 1.78
Poor biphasic
Biphasic
Day of cycle
11
E.B.
28
18
1.04
B.B.
28
12 19 10 24
.28 .51 .46 .22
36
Biphasic
Poor bipbasic Biphasic
temperature was available in all cycles. In each of the 4 patients (except EB ), the thermal shift was atypical, showing one or more dips in the thermal plateau, and/or lasting less than 14 days. The histology of the endometrium in Patients EB and BB was not consistent with the day of the cycle during which the biopsy was obtained, showing merely quite early secretory changes in only a few glands, indicating irregular ripening. 'V'hether these biopsies were examples of poor luteal phase, and if so, what the significance is, we are not prepared to say. However, all 4 patients were complaining of infertility. In Patient B.B., low pregnanediol values in the progravidic half of the cycle were obtained in 2 consecutive cycles, while B.G., in another cycle, had a good biphasic temperature together with pregnanediol values in the secretory range. Even with this small amount of data, it might be tempting to postulate that urinary pregnanediol is an exceedingly useful test, not only in ascertaining the occurrence of ovulation, but in evaluating the functional status of the corpus luteum as well. It seems to us that more appropriate to the latter task would be the use of human chorionic gonadotropin stimulation of the corpus luteum, as reported by Jayle et al. They suggested that the administration of HCG in the second half of the cycle could be used to evaluate the dynamic capabilities of the corpus luteum to respond to the stimulus of pregnancy. The usefulness of this test is currently being investigated in our laboratory.
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The occurrence of pregnancy is accompanied by a marked rise in pregnanediol even prior to the date of the expected period. This is confirmed with our present technic in a patient who conceived during this study. Pregnanediol values on Days 10, 18, and 27 were, respectively, 0.24, 2.12, and 9.6 mg.j24 hr. A positive pregnancy test was obtained on Day 36 of the same cycle. The determination of urinary pregnanediol has also been used by us as a parameter of corpus luteum function after the administration of clomiphene citrate (Clomid*) in the induction of ovulation. In this group of patients, if spontaneous menstrual cycles were not present, vaginal bleeding was induced by medroxy progesterone acetate (Proverat), 20 mg.jday for 3 days. Although small elevations can be observed in patients without specific signs of ovulation16 (Table 2), pregnanediol levels in the secretory range are consistently accompanied by other signs of corpus luteum formation. Increased urinary pregnanediol excretion, however, is per se not a sufficient indication of ovulation and normal corpus luteum function, especially in patients treated with Clomid. Luteinization of follicles, with or without cyst formation, is a rather frequent occurrence. In these cases, though, pregnanediol excretion is quite frequently in the upper limit or above the normal secretory range. Recently we have been experimenting with smaller doses of Clomid (250 mg. per cycle) in order to decrease the side effects of this drug. With this reduced dosage we have not encountered any symptomatic cyst formation, and the urinary pregnanediol values reflect a more physiologic ovarian stimulation (Table 2). Whether this reduced dosage will result in an increased number of conceptions along with decreased side effects, we do not know. However, we feel that pregnanediol determination greatly supplements other parameters in the evaluation of the ovarian status after the administration of this drug. Another useful area for the use of urinary pregnanediol determination is its estimation in patients using oral contraceptives. While the exact mechanism of action of these drugs is not well understood, it is generally assumed that pituitary and gonadal inhibition with consequent suppression of ovulation offers the most satisfactory explanation of their mechanism of contraception. Their antiovulatory activity is, however, difficult to evaluate since the commonly employed tests of ovulation, such as basal body temperature and progestational endometrial changes, are affected by the thermogenic and progestational activities of the steroids administered. *William S. Merrell Co., Cincinnati, Ohio. tThe Upjohn Company, Kalama~oo, Mich.
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TABLE 2. Urinary Pregnanediol in Patients Receiving Clomid Pregnanediol Patient
Days of Clomid adminUit.*
Cycle dayt
Values (mg./24 hr.)
Basal body temp.
DiagnosUi and remarks
Anovulation, cause unknown; ovulated on treatment; had period on Day 45 of cycle Polycystic ovaries; period on Day 42 Polycystic ovaries; period on Day 37 Polycystic ovaries; period on Day 60 Polycystic ovaries; pregnancy test pos. Day 36
R.K.
16-20
11 22 38
0.30 0.30 1.20
Flat Flat Rise, Day 31
L.S.
20-24
D.M.
13-17
R.S.
37-41
L.S.
3-7
18 35 13 35 30 48 10 18 27 7 22 37 24 40
0.12 2.14 0.40 1.75 0.46 3.60 0.24 2.12 9.60 0.11 0.20 5.92 0.41 0.92
Flat Rise, Flat Rise, Flat Rise, Flat Rise,
Flat Flat Rise, Day 26 Flat Flat
M.Z.
8-12
D.C.
26--30
N.K.
13-17
13 37
0.22 0.53
Flat Flat
D.M.
30-34
30 44
0.39 0.96
Flat Flat
Day 28 Day 23 Day 45 Day 15
Polycystic ovaries; pregnancy test pos. Day 56 Secondary amenorrhea, cause unknown; no resp to treatment Secondary amenorrhea, cause unknown; no resp. to treatment Polycystic ovaries; period on Day 49
*Dosage of Clomid was 50 mg. except in Patients D.M. and D.C., given 100 mg. tThe first day of vaginal bleeding, spontaneous or induced, was considered Day I of the cycle.
Fortunately, none of the contraceptive compounds administered causes an increased excretion in urinary pregnanediol, which has proved to be a highly reliable test for the detection of the anovulatory status. 4 , 9, 26 Furthermore, suppression of pituitary 'gonadotropin might not occur in all cases, and changes in the uterine environment, such as thickening of cervical mucus and advancing of endometrial age, have also been implicated to explain the remarkable effectiveness of oral contraceptives.lO As a matter of fact, Goldzieher et al. have found elevated luteal-phase pregnanediol in 6.8% of users of Orthonovum* in the lO-mg. size, a finding which suggests the possible establishment of a ~orpus luteum. One might *Ortho Pharmaceutical Corporation, Raritan, N. J.
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TABLE 3. Pregnanediol Excretion in Patients Taking Ovulen (1 Mg.) for Birth Control
Patient
F.D. e.S. J.W. B.H. O.S. W.A. J.B.
S.P.
Length of treatment (mo.)
Pregnanediol (mg./21, hr.)*
1 4 4 8 8 8 8 9 9
0.35 0.31 0.40 0.09 0.14 0.05 0.26 0.11 0.51
,!,
*AU values shown are for second half of cycle; Patient F .D. showed an excretion of 0.27 mg.j24 hr. in the first half of cycle after 3 months of treatment.
think that the possibility of corpus luteum fonnation in the presence of effective oral contraception might be more likely to occttr with the use of smaller doses of medication. In fact, Rock reported that 2 of 11 patients taking 1 mg. of ethynodiol diacetate in combination with 0.1 mg. of mestranol (Ovulen*) for contraception had elevated pregnanediol iJ;! the second part of the cycle. It seemed interesting to us to investigafle urinary pregnanediol excretion in some women receiving Ovulen in the l~g. dose. Table 3 shows the values obtained in patients on this medication, while Table 4 shows values obtained in patients on sequential mestranol-chlormadinone (C-Quenst) contraceptive medication. It is apparent that if an increase in urinary pregnanediol occurs, it does not take place with any greater frequency than in patients using larger doses of oral contraceptives.l l The re-establishment of normal ovulatory function following cessation of treatment is also documented by urinary pregnanediol determination in 13 patients (Table 5). In 10, the first cycle proved to be ovulatory; however, 2 more patients might have ovulated also, but it could not be documented because specimens were not obtained during the second half of the cycle. Ovulation was proved to be established in subsequent cycles. In only 1 patient (C.P.) did ovulation seem not to occur until the third menstrual cycle after discontinuing the treatment. In this study, whenever a pregnanediol detennination was performed, endometrial biopsy was also obtained, and in each case a perfect correlation was established. *Brand of ethynodiol diacetate 1.0 mg. with mestranol 0.1 mg. G. D. Searle & CQ, tEli Lilly and Company, Indianapolis, Ind.
-,
266
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& STERILITY
TABLE 4. Pregnanediol Values for Patients Taking C-Quens Pregnanediol values in luteal phase After treatment Patient No.
Before treatment (,..g./24 hr.)
13 1 8 12
3450 3000 1150 4396 1796
9
5429
10 5 3
915 2590 1617
2 7 6 4 11
6039 8700 3861 1302 5400
14
Mo. of treatment
1 2 3 3 3 6 3 7 4 4 5 11 7 7 10 11 11
p.g./24 hr.
280 360 438 975 372 330 68 97 37 107 737 43 197 270 676 160 360
When failure of corpus luteum development occurs, regardless of its causes, it is reasonable to assume that the urinary pregnanediol excretion would be in the proliferative range. It has been reported that in some cases of polycystic ovaries there was an increase in pregnanediol or in the "pregnane-complex."8. 33 These cases were supposed to be amenable to corticosteroid treatment. The findings apparently were a result of using methods of poor specificity, since pregnanediol per se is not elevated in patients with the classic Stein-Leventhal syndrome or in patients with the postpubescent variety of the adrenogenital syndrome, even though ovarian enlargement might be present.2 • 21. 31 This fact is evident in Table 6, where the pregnanediol values of all patients suffering from anovulation, regardless of cause, are indistinguishable from the values obtained in the proliferative phase of the cycle. The daily pregnanediol excretion pattern in 2 patients with anovulatory cycles are shown in Fig. 3. Investigation of Adrenal Function
The adrenal contribution to urinary pregnanediol, while small, is nevertheless real, and in men it might contribute a major part of it. 1 Pregnanediol values obtained in patients after bilateral oophorectomy are shown in
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PREGNANEDIOL DETERMINATION
Table 7. Small but definitely measurable amounts are present, and the implication is that the adrenal is responsible for the excretion of this compound in these patients. The adrenal origin of the pregnanediol is further confirmed by its response to ACTH stimulation (Table 8). Klopper et al. 20 first suggested that pregnanediol assay may be of value in evaluating adrenocortical function. No conclusions can be drawn from our data as to the value of pregnanediol in the diagnosis of adrenal diseases. However, in a recent report on TABLE 5. Pregnanediol Excretion Following Discontinuation of Treabnent with Ovulen
Patient
M.T.
E.W. A.R.
S.W. P.E. V.M. D.M.
c.P. B.H. F.D.
P.B. M.A.
C.B.
Length of treatment (mo.)
3 3 3 3 4 3 5 5 5 6 6 6 6
Pregnanediol ellJcretion (mg./24 hr.) after discontinuation of treatment First half of cycle
0.28, * 0.33t 0.15* 0.62t 1.00* 0.12*
0.64* 0.22,* 0.19,* 0.17* O.52t
Second half of cycle
0.37,t O.90,t 2.09:1: l.71t 1.86* 2.25* 2.90,* 1.43t 9.50* 2.05* 0.79,* 0.10,t 1.18:1: 7.80* 2.01* 3.84* 2.10* 1.64,* 1.64t
Most of the cycles were longer than 28 days; the second half of the cycle was arbitrarily assumed to begin 14 days prior to the following menstrual period. *First cycle. tSecond cycle. :::Third cycle.
TABLE 6. Pregnanediol Excretion in Patients with Anovulatory Cycles or Amenorrhea
Diagnosis
No. of cases
Secondary amenorrhea (other than polycystic ovaries) Anovulatory cycles (second half) Stein-Leventhal syndrome Postpubescent adrenogenital syndrome
23 6 12 2
Mean ellJcret., pregnanediol (mg./24 hr.)
0.39 ± 0.13 0.42 ± 0.09 0.45 ± 0.12 0.26 0.39
268
SCOMMEGNA ET AL.
FERTILITY
& STERILITY
20 cases of Cushing's syndrome, Martin and Hammer showed that pregnanediol excretion was elevated in all 4 patients with carcinoma, while it was normal in the rest of patients with hyperplasia or adenoma. Furthermore, in the latter group, after ACTH stimulation, all 5 patients with adenoma had a normal response, while 7 of 8 patients with hyperplasia had an elevated response in the pregnanediol output. The authors therefore concluded that urinary pregnanediol may be the most useful single test in determining the cause of Cushing's syndrome. With the ready availability of pregnanediol assay, the applicability of this test in the diagnosis of adrenal diseases should be further explored. ~
4.0
1
~ 2.0
"';
g
A..
_
A.
0 L.---'--::~=---_-=---='_~_-==O!II....
~ ~ 4.0
____x_x_x
>.
6c: .~
....o 0.
~
2.0
Fig. 3. Pregnanediol excretion patterns in 2 patients with anovulatory cycles. X indicates beginning of menses. At bottom, Patient 1; at top, Patient 2 .
xxx OJ--T~.-~=r-.~r-~~-,-.-,-~-
5
7
9
II
13 15
17 19 21 23 25 27
Day of cycle
TABLE 7. Pregnanediol Excretion after Bilateral Oophorectomy in Patients with Carcinoma of the Breast Patient
Age
A.D.A.
40
A.L.P.
65
B.A.R. M.C.C.
35 47
T.R.A.
55
Z.A.G. O.L.E.G.
58 56
Pregnanediol ea:cretion (/LU./24 hr.)
201 182 148 103 200 152 193 125 223 102 129 172 450
All but Patient D.L.E.G., who received X-ray therapy, were treated by surgery. The meall excretion rate in the group on whom an operation was performed, was 161 ± 40 pg./24 hr.
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PREGNANEDIOL DETERMINATION
Investigation of Corpus Luteum of Pregnancy and Placental Function
Because of the large increase of urinary pregnanediol which accompanies the establishment of pregnancy, its measurement is relatively easy during this condition. Here again, gas chromatography, because of its speed, might prove t~ be an exceedingly useful tool in the management of pregnancy disturbances. While progestin treatment in patients suffering from habitual abortion might be a debatable issue, its use in patients in whom no progesterone deficiency is demonstrable is to be d~plored. The potential risks of progestational therapy in the mother as well as in the baby range from induced missed abortion through masculinization of the female fetus. It seems to us that the restriction of progestational therapy to cases where a progesterone deficiency, as evidenced by low pregnanediol values, can be demonstrated might result not only in less therapeutic meddling, but also in an apparent improvement of the results obtained, since the therapy will then be restricted to patients who are most likely to benefit from such management. The necessity for a means of accurately assessing fetoplacental function in the second and third trimester of pregnancy is self-evident. However, it is debatable whether pregnanediol assay is suitable for this purpose. Pregnanediol excretion does not seem to correlate with fetal outcome in diabetic pregnancies,7 although it might be of some usefulness in toxemia of pregnancy and preeclampsia. 15 • 27 However, urinary estriol determinations TABLE 8. Pregnanediol Excretion in Patients after ACTH Administration 17-0HCS (mg./24 hr.)
Patient
17-KS (mg./24 hr.)
Pregnanediol (mg./24 hr.)
[nit.
Sere
Control
ACTH
Control
ACTH
Control
ACTH
AP.U.
M
4.2
6.0
M M
5.4 6.5
M.O.O. P.E.A.
M F
8.6 3.6
M.A.R B.RE.
F F
5.4 8.4
18.0 22.7 23.5 15.7 27.7 46.8 24.2 24.9 14.1 18.3
0.54
RO.T. B.O.R
7.1 20.1 26.4 27.0 22.3 26.3 13.9 15.0 16.8 9.4
0.59 0.77 2.90 0.31 1.20 0.82 4.00 5.30 1.20 3.28
12.9 9.4 20.3 11.0 7.3 15.1
0.44 0.38 0.46 2.30 0.46 1.16
All male subjects were normal. Of the female patients, Patient P.E.A. was hirsute and was given ACTH in second half of cycle; M.A.R. had anovulatory cycles and was given ACTH in first half of cycle; B.R.E. had what was believed to be adrenal hyperplasia.
270
SCOMMEGNA ET AL.
FERTILITY
&
STERILITY
TABLE 9. Pregnanediol and Estriol Excretion in Some Patients with Abnormal Gestation Estriol Pregnanediol (mg./24 hr.) (mg./24 hr.)
Gestation Patient
(wk.)
B.L.
J.P.
18 26 36
E.H.
38 14
J.T.
26
p.e.
Statm
Hydatidiform mole Intra-uterine fetal death Triplets
Therapeutic abortion Before intra-amniotic infusion After intra-amniotic infusion Rh sensitization; intra-uterine fetal death Mter intra-amniotic infusion
14.16 49.20 52.00 41.40 45.60 16.60 14.00 10.38 2.57 26.80 10.75 16.50
.34 .62 40.40 38.20 48.00 .89 .88 .36 .14 .80 .56 .80
are more helpful since decreases in estriol precede the reduced pregnanediol excretion. 32 We have not accumulated enough material to be in the position to confirm or refute these observations, yet some of the preliminary data agree with them. Some representative estimates obtained with our present technic are presented in Table 9. It can be seen that pregnanediol values in intra-uterine fetal death are still normal, while estriol values are markedly decreased (Patients T.}. and P.C.). After intraammiotic instillation of hypertonic saline for therapeutic abortion (Patients E.H. and }.T. ), estriol proves to be a better indicator of the fetal status, although a moderate pregnanediol drop is also evident. SUMMARY
Urinary pregnanediol, while not a unique metabolite of progesterone, is still a useful indication of progesterone secretion in women. It may be helpful in the management of many clinical conditions if assays of its excretion can be obtained with speed. A relatively new gas chromatographic technic has been applied to the investigation of normal cycles as well as of many clinical situations. Its usefulness in the measurement of pregnanediol favorably compares with established chemical methods. The speed with which it can be performed renders it highly suitable for routine use in the steroid laboratory. Department of Obstetrics and Gynecology Michael Reese Hospital and Medical Center 29 St. and Ellis Ave. Chicago, Ill. 60616
VOL. 18, No.2, 1967
PREGNANEDIOL DETERMINATION
271
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