Serum FSH and LH measurements in the evaluation of menstrual disorders

Serum FSH and LH measurements in the evaluation of menstrual disorders WILLIAM ALBERT THOMAS Los Angeles

J. F.

DIGNAM,

M.D.

PARLOW,

A.

PH.D.*

DAANE,

and Torrance,

COMMANDER

(MC)

USN**

California

Serum FSH and LH levels were measured serially, for 30 days, in primary, oligo-, secondary amenorrheas, and amenorrheas associated with galactorrhea or obesity, Comparison with normal subjects revealed a lack of the characteristic midcycle FSH and LH hypersecretion in amenorrheic patients. Comparison among the amenorrheic patients, most of whom exhibited normal levels of FSH and LH, revealed differences in absolute levels and ratios of FSH and LH. Striking differences in the day-to-day patterns of serum FSH and LH were also noted. A significant correlation existed between the nature of these gonadotropin patterns and responsiveness of gonadotropins to clomiphene citrate. Thus, amenorrheic patients with either low or normal but unfluctuating gonadotropin levels responded poorly to clomiphene citrate, while those with normal levels which did fluctuate from day to day exhibited an ovulatory-type of gonadotropic response to clomiphene citrate. A hypothesis relating serum qonadotropin patterns to the intensity of hypothalamic regulation of gonadotropins t!s presented.

P R 0 G R E S s in resolution of the conflict in characterizations of the pattern of secretion of pituitary gonadotropic hormones during the normal menstrual cycle has been achieved since the application of radioimmunoassay methods. Regarding the particularly controversial area of FSH secretion, several laboratories,1-5 including our own, now concur in a pattern consisting of (a) an early follicular rise, (b) a midcycle peak, From the Department of Obstetrics and Gynecology, UCLA School of Medicine, Los Angeles, and Harbor General Hospital, Torrance. Supported Foundation,

by a grant 67-514

from

the Ford

Presented (by invitation) at the Ninetysecond Annual Meeting of the American Gynecological Society, New Orleans, Louisiana, May 5-7, 1969. *Supported and USPHS Development **Supported the United

by NIH Grant AM Research Career Award AM 7735. by a Fellowship States Navy.

11214

Award

of

and (c) luteal phase values lower than those of the follicular phase.3y 4 Agreement on the outstanding features of the serum LH pattern, namely, higher follicular phase than luteal phase levels, and a midcycle elevation, is virtually universal. However, the precise physiologic role of these characteristics of the variations in serum FSH and LH levels during the normal menstrual cycle is not known. The importance or essentiality of any of these characteristics of the patterns of serum FSH and LH for the secretion of estrogens, the induction of ovulation, and the secretion of progesterone remains to be rigidly delineated. Therefore, it was our aim to study the variations in serum levels of FSH and LH daily, for prolonged periods of time, in a variety of disorders of the menstrual cycle, for a threefold purpose: (1) to compare serum FSH and LH patterns in menstrual disorders with those of normal cycles; (2) to attempt to characterize menstrual dis-

680

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orders in terms of particular patterns of gonadotropin secretion; (3) to attempt to differentiate subjects with menstrual disorders in terms of the nature of their gonadotropic response to treatment with clomiphene citrate. Material

and

methods

Radioimmunoassays for FSH and LH in human serum. Double-antibody radioimmunoassays for LH and FSH, similar to those previously reported,“, ‘i were employed. Highly purified human pituitary FSH8 and human pituitary LH (DEAE-2-2)” were iodinated with 1311. Anti-LH serum, prepared in rabbits immunized with pituitary LH, was used in a final dilution in the incubation tubes of 1:200,000. Anti-FSH serum, prepared in rabbits immunized (in our laboratory) with pituitary FSH, was used in a final dilution of 19,000. Absorption of FSH antiserum prior to use was accomplished with the serum of a hypophysectomized human. In addition, 10 I.U. of HCG was added to each incubation tube, in order to remove cross-reactivity with serum LH. Human serum samples being tested were added in volumes of 200 to 400 ~1 per tube. One hundred microliters of 1311-labeled antigen, in 0.15M NaCl-O.OlM phosphate pH 7.8 buffer containing 0.1 per cent bovine serum albumin was added per tube. Two hundred microliters of antiserum, diluted in the same buffer plus 0.05M EDTA, containing 3 per cent normal rabbit serum was added per tube. A final volume of 800 ,LJ was achieved with addition of buffer containing 1 per cent BSA. After incubation at 4“ C. for 5 days, sufficient sheep antirabbit gamma globulin serum was added to produce maximal precipitation of antibody bound radioactivity, after additional incubation for 72 hours at 4%. Estimates of FSH potency of pituitary fractions obtained with our radioimmunoassay method are similar to those previously rep0rted.l” Collection of blood samples. From all subjects with menstrual disorders, blood samples were obtained daily for a period of approximately 30 days. In numerous cases additional blood was collected during

an ensuing period of trcatrnent with cloiuiphene citrate and for approximately 2 weeks following clomiphene citrate administration. Blood was drawn at a constant time of day for exh subject. The blood was allowed to clot for a maximum of 48 hours, centrifuged, and the serum stored at -20° C. Blood samples were obtained daily throughout a menstrual cycle, in the case of normal subjects. All samples for a particular subject were tested simultaneously in one radioimmunoassay, as well as in a replicate assay. Final estimate of the concentration of serum FSH and LH (expressed in terms of the Second International Reference Preparation for human menopausal gonadotropins as milli-International Units per milliliter serum [mI.U. /ml.]) was based on the mean of the two independent determinations.

Physical and laboratory examinations of patients with menstrual disorders. All patients studied had a complete general physical and pelvic examination. Vaginal smears were taken for an estimation of estrogen effect. The endocrine work-ups included thyroid and adrenal function tests. Where a skull x-ray for visualization appropriate. of the sella turcica and visual field examinations were done. Unless otherwise stated in the brief clinical description of each case, these parameters were reported to be normal. Results

Illustration of the pattern of serum FSH and LH in normal menstrual cycles. The pattern of serum FSH changes during normal ovulatory cycles of three women is illustrated in Fig. 1. The early follicular rise, midcycle elevation, and luteal depression in serum FSH levels is clearly seen. These examples are very similar to those presented previously by other investigators.l-” The temporal relationships between serum FSH and LH changes during the normal menstrual cycles of two women are presented in Fig. 2. Once again, the general patterns are similar to those observed by others.lm5 A closer scrutiny of the serum FSH and LH changes of greatest magnitude, those

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30 i 2 .z I if

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330. I J

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G 0. Ovulatory

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Fig. 1. Serum FSH levels during normal ovulatory cycles in 3 women. Results expressed in terms of the Second International Reference preparation for Human Menopausal Gonadotropins (IRP-HMG) as milli-Internationl Units per milliliter serum.

0

10

20

30

DAYS

NORMAL

Fig. 3. Serum FSH and LH levels every 8 hours at midcycle in a normally menstruating woman.

MENS.

G.G.

ence of the LH than the FSH peak are apparent in this subject. Patterns of serum FSH and LH in subjects with menstrual disorders. Primary amenorrhea. Two subjects with primary amenorrhea were studied. They differed markedly in respect to their serum gonadotropin profiles, as well as in their clinical features.

,,,,*,*, 10

20

+. 30

lo

20

30

DAYS

Fig. 2. Temporal relationships between serum FSH and LH changes during normal menstrual cycles in 2 women.

at midcycle, was obtained by sampling one subject every 8 hours at that period (Fig 3). Diurnal variation in serum FSH and LH levels, the greater magnitude of LH than FSH changes, and the longer persist-

SUBJECT 1 (K. w.). This 23-year-old subject had no secondary sexual development. Vaginal smears were atrophic. A chromatin-positive buccal smear was obtained. A small cervix and uterus were present. Ovarian biopsy showed numerous primordial follicles.

In this subject serum FSH was undetectable and serum LH barely detectable (Fig. 4, lower portion). However, administration of clomiphene citrate* (50 mg. per day) did succeed in stimulating a significant elevation of serum FSH and LH levels, al*AU indicated

subjects treated with clomiphene citrate dose daily for 5 consecutive days.

received

the

682

Dignam,

Parlow,

and Daane

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.:a...

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--___--

t

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z-

----,.

;-------lo----

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DAYS -5

10

0 DAYS

1~

0

2b

lb

3b

DAYS

Fig. 4. Serum FSH and LH levels prior to (bottom portion), during and after clomiphene citrate (upper portion) in a subject (K. W.) with primary amenorrhea.

though not to the range ordinarily with- the induction of ovuIation upper portion).

associated (Fig. 4,

SUBJECT 2 (L. c.). This 20-year-old subject did have normal secondary sexual development. Vaginal smears indicated moderate estrogenic activity. Withdrawal bleeding was produced with progesterone.

Both serum FSH and LH (Fig. 5, lower portion) were within the range of norma levels. However, the pattern of serum FSH and LH changes lacked the cyclicity characteristic of a normal ovulatory cycle, although some degree of variation from day to day, especially for LH levels, was observed. Subsequent to this series of serum collections the patient did experience a spontaneous period. Several months thereafter

Fig. 5. Serum FSH and LH levels in subject (L. C.) with primary amenorrhea (lower portion). Upper portion of the figure depicts serum gonadotropins several months after a sponfaneous period, prior to and after clomiphene citrate.

a second collection of serum samples (Fig. 5, upper portion) again revealed serum FSH and LH levels within the range of normal, but lacking the cyclicity of normal cycles. FSH levels were higher than those previously recorded for this subject. Clomiphene citrate (50 mg. per day) administration resulted in an elevation of serum LH levels, particularly, although the pattern seen was not that usually associated with a normal ovulation (Fig. 5, upper portion). Oligomenorrhea. Three subjects with oligomenorrhea were studied. Each subject experienced irregular menstrual cycles, varying substantially in length (in one case as long as 6 months). The first of these oligomenorrheic subjects (L. M.) was studied during an apparently normal, spontaneous ovulatory cycle. The other two subjects were studied during a period of amenorrhea.

Volume

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OLIGO.

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683

- OVUL 117 I

I, Lb-3

I. OLIGOMEN. z 34o.tt

).

z I J 20.

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20

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0

10

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SA F

i\ 260. E

OLIGOMEN

Clomld -

I 2w Ln 0, -50

-40

-30

-20

-10

0

10

20

DAYS

Fig. 6. Oligomenorrhea. Ufifier, left (L. M.) : serum gonadotropins during a spontaneous ovulatory cycle. Bottom (S. A. F.) : serum gonadotropins prior to and during an induced ovulatory cycle. Upper, right (C. G.) : serum gonadotropins prior to and during an induced ovulatory cycle.

SUBJECT 1 (L. M.). This 21-year-old nulligravida had experienced normal menarche at the age of 12 years. Her menstrual cycle had always been irregular with periods of amenorrhea as long as 6 months.

The cycle in which this patient was studied was a spontaneous ovulatory cycle. The levels and the patterns of serum FSH and LH were normal (Fig. 6, upper, left). SUBJECT 2 (s. A. F.). This 25-year-old, married, nulligravida gave a history of irregular menses following an appendectomy at the age of 18 years. I.,aparotomy 3 years later for per-

sistent pelvic pain revealed multiple abdominopelvic adhesions. Subsequent culdoscopy revealed a left ovary of normal contour; the right ovary was obscured by adhesions. This subject was studied during a prolonged period of amenorrhea. Levels of serum FSH were within the range of normal and relatively constant from day to day. Serum LH levels were within the “high normal” range and varied considerably from day to day (Fig. 6, bottom). Administration of clomiphene citrate (100 mg. per day) stimulated an elevation of

684

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Parlow,

and Daane

2’ AMEN &f-J 3

.

5’ I

CT

-I 20. JJJQJf%Al 0, -30

Clomid -

-20

-10

0

10

20

Clomid 5 k20. f El In

J&\ 0,

*b

-30

-a

-20

0

-10

10

.

20

DAYS

Fig. 7. Secondary ovulatory cycle.

amenorrhea

(C.

T.):

serum

gonadotropins

prior

to and

during

an induced

both FSH and LH during treatment. Following the treatment FSH and LH levels both dipped and then rose again to heights ordinarily associated with a normal ovulation.

subjects had been amenorrheic for periods ranging from one year to 5 years. Significant differences in the serum FSH and LH patterns can readily be seen among these subjects.

SUBJECT 3 (c. G.). This 23-year-old married nulligravida experienced menarche at the age of 13 years. Her cycles were characterized by 3 month intervals, until she began contraceptive pill treatment the first year of her marriage. On discontinuing the pills, after one year, she experienced more irregular menses, with amenorrhea of as long as 7 months’ duration.

SUBJECT 1 (c. T.). This l&year-old single nulligravida had spontaneous amenorrhea of 2 years’ duration. Approximately one year prior to our study she underwent a right oophorectomy for serous cystadenoma. Vaginal smears revealed a mild estrogenic effect.

This subject was also studied during a period of amenorrhea. The serum FSH and LH levels were within the range of normal, although lacking the cyclicity of normal ovulatory cycles. However, in contrast to the pattern seen in the second subject (S. A. F.) the third subject demonstrated relatively constant levels of FSH and, to a large degree, LH also (Fig. 6, upper: right ) . Once again, clomiphene citrate treatment (100 mg. per day) resulted in a type of serum FSH and LH response characteristic of an ovulatory cycle (Fig. 6, upper, right). Secondary amenorrhea. Five subjects with secondary amenorrhea were studied. These

Subject 1 displayed values for serum FSH and LH which were entirely within the range of normal, although the cyclicity characteristic of normal ovulatory cycles was not apparent (Fig. 7). Despite the lack of a cyclic gonadotropin pattern, both serum FSH and LH evinced a high degree of rhythmicity, in that these levels varied substantially from day to day. Clomiphene citrate treatment (50 mg. per day) stimulated an ovulatory-type of response in serum FSH and LH levels (Fig. 7) . SUBJECT 2 (N. H.). This 20-year-old single nulligravida had 3 years of spontaneous amenorrhea. Vagina1 smears showed a low estrogenic effect.

In

striking

contrast

to

the

first

subject

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2” AMEN.

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Clomid -

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io

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Fig. 8. Secondary amenorrhea gonadotropin levels.

(C. T.), Subject 2 (N. H.) undetectable levels of FSH serum (Fig. 8) . These low able levels of FSH and LH the lack of estrogenic activity the vaginal smear record. Yet another variation in the patterns seen in this category is presented.

(N. H.) : serum

had IOW or and LH in or undetectcorrelate well indicated in

DAYS

Fig. 9. Secondary amenorrhea (C. B.): serum gonadotropin levels, indicating differential hyposecretion of LH, and the response to clomiphene citrate.

I

2” AMEN.

gonadotropin of patients

h

SUBJECT 3 (c. B.). This 27-year-old single nulligravida had a 5 year history of spontaneous amenorrhea. Menarche occurred at age 11 and menstrual cycles were regular until age 21. After a year of irregular cycles, complete amenorrhea developed. Multiple vaginal smears showed minimal estrogen activity. No withdrawal bleeding occurred after treatment with Provera (10 mg. daily for 5 days).

Unlike the two previous subjects in the category of secondary amenorrhea. Subject 3 presented the unusual serum gonadotropins profile of normal FSH levels with undetectable 1eveIs of LH (Fig. 9). This differential hyposecretion of LH may have been responsible for the low estrogenic activity apparent in the vaginal smear. Note, also, the failure of clomiphene citrate treatment (50 mg. per day) to have induced a hypersecretion of gonadotropins in this subject, although a slight elevation in serum LH did occur.

i/

Clomid -

‘Fig. 10. Secondary amenorrhea (S. H.) : serum gonadotropin levels prior to and during an induced ovulatory cycle.

SUBJECT 4 (s. H.). This 20-year-old single nulligravida experienced menarche at the age of 15 years. Regular cyclical menses had occurred to the age of 18 years, when irregularity began. Following a period of undefined medication, she became amenorrheic, for one year prior to the current study.

This

subject

demonstrated

normal

but

686

Dignam,

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and Daane

acyclical levels of both serum FSH and LI-I (Fig. 10). Both FSH and LH appeared to undergo a small degree of variation from day to day. This subject’s serum gonadotropin patterns were similar to those of Subject 1 of this series (C. T.), although the degree of variation from day to day was less marked for Subject S. H. The response of Subject

S. H. to c-lomiphene citrate (50 tug. /XI day) was of the ovulatory type ( E’ic. lo’:. SZJBJECT

5

N(II.

B.).

This

1%year-old

single

nulligravida had a history of normal menarchc at age 14. She experienced a sudden spnntaneous amenorrhea at age 17j/, years.

Normal but acyclic levels of FSH and LH were observed in this subject (Fig. 1 I ) LH levels were relatively unvaried, although a small but definite degree of rhythmicity was present for FSH. The response of gonadotropins to clomiphene citrate treatment (100 mg. per day) was of the ovulatory type (Fig. 11) . Anorexia rhea. SUBJECT

nervosa

associated

with

1 (M. c.). The one patient

amenorstudied

(M. C.)

is a 29-year-old nulligravida who developed amenorrhea 6 years prior to the study, coincidental with a weight loss of 30 pounds. Vaginal smears revealed a mild to moderate estrogenic effect.

-20

-10

0

10

This subject secreted normal amounts of both FSH and LH (Fig. 12). The response of her gonadotropins to clomiphene citrate treatment (50 mg. per day) was significant. However, the lack of one serum sample, 4 days after cessation of clomiphene citrate

20

DAYS

Fig. 11. Secondary amenorrhea gonadotropin levels prior to duced ovulatory cycle.

and

(H. B.): during

serum an in-

ANOREX

NERV CEid

Clomld -

DAYS

Fig. 12. citrate.

Anorexia

nervosa

(M.

C.):

serum

gonadotropins

and

the

response

to clomiphene

Volume Number

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Serum

FSH

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GALACT

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20

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40

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:,

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LH in menstrual

-AMEN

0 ClEid

lL 20 I 2 30

-+--&A -10

0 DAYS

10

-20

-10

D

la

20

-10

DAYS

0 DAYS

lo

Fig. 13. Galactorrhea. upper, left (C. I.) : serum gonadotropins in a subject with galactorrhea and normal menstrual cycles. The response to norethynodrel plus mestranol (5 mg. per day for 20 days). Lower, right (G. E.) : serum gonadotropin levels in a woman with amenorrhea; the response to clomiphene citrate. Botlom, center (M. C. V.) : serum gonadotropins prior to and following clomiphene citrate in a woman with amenorrhea. Lower, left (J. S.) : serum gonadotropins and the response to clomiphene citrate in an amenorrheic woman. Upper, right (A. C.) : serum gonadotropins in an amenorrheic woman prior to and during clomiphene citrate and one day during the ensuing pregnancy.

treatment, prevents a critical evaluation of the gonadotropic response as the ovulatory type. Galuctorrhea. Five subjects with galactorrhea have been studied. One of these subjects experienced normal menstrual cycles despite the galactorrhea. Four of the subjects were amenorrbeic. SUBJECT 1 (c. I.). This 33-year-old para 3 developed postpartum galactorrhea which continued after resumption of cyclical menses. The galactorrhea was copious with a thick milky color and consistency. A basal body temperature curve during the study cycle showed the charac-

teristic rise of an ovulatory pattern. Endometrial biopsy on the first day of menses following the study cycle revealed secretory endometrium.

Serum FSH and LH showed characteristic patterns for a normal ovulatory cycle, especially with regard to midcycle and luteal phase values (Fig. 13, upper, left). Administration of norethynodrel with mestranol (5 mg. daily for 20 days) in the following cycle had some effect in lowering serum gonadotropin levels. SUBJECT

patient

2 (c. had noted

This 30-year-old para 3 galactorrhea since her last

E.).

688

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pregnancy. She had spontaneous resumption ot menses, but developed amenorrhea together with headaches and visual difficulties 2 years later. A large pituitary chromophohe adenoma was removed and the seila area irradiated. Postoperatively she was treated with thyroid extract and cortisone. Galactorrhea did not subside, suggesting that some pituitary function remained. Vaginal smears indicated minimal estrogenic effect. No change in the radiologic evaluation of the enlarged sella turcica had been noted since treatment. Visual field examinations Wvealed a slight improvement. Serum FSH and LH levels were usually undetectable in this subject (Fig. 13, lower, right), although a slight elevation did appear to occur after clomiphene citrate treatment ( 100 mg. per day). SUBJECT 3 (M. c. v.). This gravida developed amenorrhea 12 months prior to our study, prednisone therapy (20 mg. asthma. Her initial complaint infertility of 2 years’ duration.

27-year-old nulliand galactorrhea coincidental with daily) for severe was involuntary

Serum FSH levels were within the range of normal (Fig. 13, bottom, center), showing little variation from day to day. “High normal”. LH levels, with substantial day-today variation, were apparent. Clomiphene citrate treatment (100 mg. per day) stimulated hypersecretion of gonadotropins, as judged by serum levels, although the fullscale response was somewhat slower than usual and not exactly of the ovulatory

type. SUBJECT 4 (J. s.). This 26-year-old para 2 patient nursed her last child and started on contraceptive pills. Galactorrhea stopped during contraceptive therapy but resumed along with amenorrhea after contraceptives were discontinued 2 years later. Vaginal smears, during the period of study, showed moderate to substantial cstrogenic effect.

The gonadotropin profile (Fig. 13, lower, left) again presented normal levels of both hormones with a nearly unvarying pattern for FSH and little variation for LH from day to day. An effect on gonadotrophins following clomiphene citrate treatment (50 mg. per day) was not marked.

i I,\. C.). ‘I’his 26-year-old para T nursed postpartum for 6 months, thert contraceptive pills. Galactorrhea LOTIduring contraceptive therapy. She expel inormal withdrawal menses during this On discontinuing contraceptives, after 2 she experienced amenorrhea with pergalactorrhea. Vaginal smears showed estrogenic effect.

SUBJECT

patient started tinued enced time. years, sistent marked

Both FSH and LH levels in serum were normal (Fig. 13, upper, right), and somewhat rhythmic, but lacked evidence of an clomiphene “ovulatory surge.” However, citrate treatment (100 mg. per day) provided the needed stimulus for the “ovulaFollowing clomiphene citrate tory surge.” treatment the subject became pregnant. Srrum LH was highly elevated approximately 45 days after the clomiphene citrate-induced ovulation. Amenorrhea associated with obesity. Of the 5 patients studied in this category all had a history of significant weight gain associated with the onset of amenorrhea. SUBJECT 1 (s. w.). This 2-l-year-old nullipara had a history of menarche at the age of 13 years and regular cycles to 18 years of age. Coincidental with a steady weight gain she developed amenorrhea, of 6 years’ duration. Vaginal smears showed a moderate estrogenic effect and endometrial biopsy showed endometrial hyperplasia.

Serum FSH was in the low normal range, while LH levels were normal (Fig. 14, lower, right). Both gonadotropins showed little day-to-day variation and only a slight elevation in response to clomiphene citrate treatment (100 mg. per day). SUBJECT 2 (A. T.). This 25-year-old para 1 patient was delivered via cesarean section 7 years prior to the study. She had a progressive weight gain following the pregnancy with increasingly irregular cycles. She was amenorrheic for one year prior to the study. Vaginal smears showed moderate to marked estrogen effect and endometrial biopsy revealed endometrial hyperplasia. Ovaries were normal in appearancc on culdoscopy.

The subject had completely normal but unvarying levels of both serum FSH and LH (Fig. 14, lower, left), which responded

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citrate

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(S. W.) : serum gonadotropins and the response to clomiphene woman. Lower, left (A. T.) : serum gonadotropins and the in an obese amenorrheic woman. Upper, right (S. S.) : serum response to clomiphene citrate in an obese amenorrheic : serum gonadotropins and the response to clomiphene citrate Upper, left (B. A.) : serum gonadotropins and the response clomiphene citrate in an obese amenorrheic woman.

(50

mg.

per day). A subsequent source of treatment with 100 mg. per day produced the same effect. (s. s. ) This 17-year-old nulligravida had been arnenorrheic for 18 months during a 50 pound weight gain. Her history included laparotomy at the age of 13 years with a left oophoretcomy for removal of a “tumorcyst.” SUBJECT

20

Clb&d

Fig. 14. Obesity. Lower, right citrate in an obese amenorrheic response to clomiphene citrate gonadotropins and an ovulatory woman. Bottom, center (B. H.) in an obese amenorrheic woman. to stilbestrol (3 mg./day) and

slightly

10

B*

Clomld

only

- AMEN.

3

In contrast to the 2 preceding subjects of this series, Subject 3 displayed normal but varying levels of LH associated with normal but constant levels of FSH (Fig. 14, upper, right). An ovulatory-type of response of the gonadotropins to clomiphene citrate

treatment SUBJECT

nulligravida years There

with was

(100 mg. per day) 4

(B.

had

This

H.).

was observed.

21-year-old

been amenorrheic

an associated no significant

steady endocrine

single

for several weight disease

gain. other

than borderline low thyroid function, for which she had been treated with 2 to 3 grains of thyroid daily. Daily vaginal smears showed mild to moderate estrogenic effect.

Serum range

FSH

of

normal,

and

LH

varied

levels, little

within from

thi

day

to

day and sesponded poorly to clomiphene citrate (100 mg. per day) (Fig. 14, bottom, center). SUBJECT

gravida

had

5 (B. A.). amenorrhea

This 25-year-old of 18 months’

nulliduration

690

Dignam,

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and Daane

associated with progressive obesity. Base-line vaginal smears showed a low estrogenic effect.

features ot gonadotropin secretion in normal ryclrs also appear to be lacking in amenorrheic women, namely, the sequence of early follicular elevation, premidcycl(* depression, midcycle elevation. and lutcal depression of FSH secretion. From thvsc obser\,ations it is clrar that proper assessment and characttkation oi pituitary yonadotropic acti\.it) in wonders with menstrual disorders CXI~ he ‘1c1‘0111plished only with prolonged serial collections of blood samples. Among the subjects with metlstrual di+ orders studied. diffetenctbs NY:W appart:nt in respect Lo rhc following parameters: Absolute level of serum FSH and LH. These 1 arit:d ronsidcrably fern subject tr, subject (see ‘I’able I). For FSH. mean vah~s computed for each subject’> [)I’:--clomiphcr!c citrate serirs of collections ran,qed from less than 2.0 m1.l:. per milliliter tu 19.4. For LH. mean values computed for each suhject’s pre--clomiphene citrate srricts oi’ CC)Ilections ranFcd frorn 3.4 to 23.0 mI.IT. ~VI milliliter. Mean serum FSH/LH ratio. ‘l’his ratio. computed from the mpan FSH vahlc ic;r

iNormal and somewhat rhythmic patterns of serum FSH and LH responded fully to clomiphene citrate treatment (100 mg. pet day) with an ovulatory type of response ‘Fig. 14, upper, left). The effect of stilbestrol administration (3 mg. per day) can also be observed. Comment Our data indicate that a number of disorders of the menstrual cycle are compatihlc with serum FSH and LH levels within the range of normal, as determined by radioimmunoassay methods. Therefore, assay of a single randomly selected serum specimen for FSH and LH in subjects with such disorders would not permit differentiation from women with normal menstrual cycles. The characteristic which most clearI>. differentiates amenorrheic women from those with normal cycles is the lack, in the former group, of the typical midcyck hypersecretion of LH and FSH usually obser\:ed in normal women. However, othel,

Table I. Mean

serum

FSH and LH Mean

Fig. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

/ K. L. I,. S. c:. i:. N. C!. S. H. M. G. M. J. A. s. A. s.

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serum

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Serum FSH and LH in menstrual

each subject’s pre-clomiphene citrate series of collections and the mean LH value for each subject’s pre-clomiphene citrate series of collections (Table I) ranged from less than 0.33 to 2.22. One subject (C. B.) presented a differentia1 hyposecretion of LH.

Patterns of serum FSH and LH levels. Among subjects with normal levels of serum FSH and LH, different patterns were discernible. For example, some subjects displayed levels of either FSH or LH or both which varied considerably from day to day. Thus, dynamic changes in serum FSH and LH, albeit of the acyclic kind, were evident in amenorrheic women. Other women displayed levels of gonadotropins which were unvarying with respect to either FSH or LH or both, Some of these different patterns are illustrated in Fig. 15.

disorders

Responseof serum FSH and LH to cloof clomiphene citrate. Administration miphene citrate to amenorrheic women resulted in two principal patterns of response: (a) a full-scale, ovulatory-type of response, characterized generahy by an elevation of serum gonadotropins during the 5 day period of treatment, a slight, transient depression of FSH and LH levels, and, finally, a in FSH and LH secretion, within “surge” 5 to 9 days after cessation of clomiphene citrate treatment, comparable to “midcycle surge” seen in normal, ovulatory cycles; and (b) the lack of a full-scale “ovulatory surge” type of response, but a slight elevation in either or both serum FSH and LH levels.

Responsiveness of serum gonadotropins of subjects with menstrual disorders to clo-

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Fig. 15. Patterns ences in patterns

of FSH and among them.

LH

in the serum

691

of amenorrheic

women,

illustrating

the differ-

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miphene citrate treatment. Among subjects with menstrual disorders, an absolute predictability of the response to clomiphene citrate treatment is not yet possible. However, a general differentiation is suggested, in these terms. Subjects with levels of either or both gonadotropins which are below the limit of normal fail to display a full ovulatory type of response to clomiphene citrate. In addition, most subjects whose serum gonadotropin levels are normal, but relatively unvarying from day to day, also fail to present a full ovulatory response to clomiphene citrate. If the degree of variation of serum gonadotropins from day to day (rhythmicity) in amenorrheic women is a function of the stimulatory activity of the hypothalamus, then it might be reasoned that those women with greater hypothalamic activity are more

likely to respond fully and completely to clomiphene citrate therapy. Of special interest is the observation that the syndrome of galactorrhea was compatible with serum gonadotropin levels which were either low or normal, and even serum gonadotropin patterns which are cyclic, of the normal ovulatory type. It was not possible to correlate consistent serum gonadotropin patterns with the various major subdivisions of the classification scheme used for presentation of subjects with menstrual disorders, namely, primary amenorrhea, oligomenorrhea, secondary amenorrhea, amenorrhea associated with galactorrhea, and amenorrhea associated with obesity. However, it is possible that certain of the serum gonadotropin patterns of subjects with oligomenorrhea or secondary amenorrhea, discerned in this study,

Fig. 16. Patterns of FSH and LH in the sertefn of m+ic sequence of changes associated with decret&xg hypothalamic tropin secretion.

women, ji~~~~ a pos&le cDntro1 of pituitary gondo-

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represent a sequence of changes in the intensity of hypothalamic regulation of pituitary gonadotropin secretion. Thus, the cyclical pattern of gonadotropin secretion of normal ovulatory cycles undoubtedly represents the most intimate degree of hypothalamic regulation of pituitary gonadotropin secretion. A first step in the loss of this intimate control of the pituitary by the hypothalamus may be illustrated in Subject C. T. (Fig. 16, upper, center), whose serum FSH and LH patterns are acyclic, but highly rhythmic. A second possible phase in the decline of the hypothalamic-pituitary interrelationship may be illustrated in Subject S. A. F., whose serum LH pattern is highly rhythmic, while the serum FSH pattern is relatively unvarying or tonic (Fig. 16, upper, right) . In the case of both these subjects, clomiphene citrate treatment evoked a full ovulatory response. (Figs. 7 and 6, bottom.) As a possible example of the third phase, amenorrheic Subject A. T. has patterns of both FSH and LH which are relatively unvarying or tonic, perhaps representing a further decrease in the degree of intensity of hypothalamic control of pituitary gonadotropins (Fig. 16, lower, left). As additional evidence of decreased hypothalamic activity, an apparent Ioss of full-scale responsiveness

Serum

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and

LH

in

menstrual

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to clomiphene citrate is observed in this subject (Fig. 14, lower, left). A possible next phase is represented by amenorrheic Subject C. B. (Fig. 16, lower, center), in whom the level of one of the gonadotropins has fallen below normal. Clomiphene citrate therapy failed to evoke a major response in this subject (Fig. 9). A final phase might by amenorrheic Subject be represented N. H., whose serum levels of both FSH and LH are below normal (Fig. 16, lower, right), and in whom clomiphene citrate fails to stimulate a major response (Fig. 8). It is not suggested that each amenorrheic subject necessarily undergoes a series of changes in her serum FSH and LH patterns exactly in accordance with the sequence described above. However, such a proposed sequence of changes may serve as a useful framework for further study of amenorrheas and other disorders of the menstrual cycle. It is hoped that such further studies would comprehend not only serum FSH and LH, but serum levels of estrogens and progesterone as well, so that the entire hypothalamic-pituitary-gonadal axis can be fully explored. The authors thank Mrs. Betty Hayakawa, Mr. Louis Lizardi, Mr. Francis Martinson, Mrs. Rae Taylor, and William Meyer for skilled technical assistance.

REFERENCES

Faiman, C., and Ryan, R. J.: J. Clin. Endocrinol. 27: 1711, 1967. 2. Franchimont, P.: Le dosage des hormones hypophysaires somatotropes et gonadotropes et son application en clinique, Bruxelles, 1966, Arscia S. A., p. 231. Cargille, C. M., Ross, G. T., and Yoshimi, T.: J. Clin. Endocrinol. 29: 12, 1969. Midgley, A. R.: and Jaffe, R. B.: J. Clin. Endocrinol. 28: 1699, 1968. Taymor, M. L., A&no, T., and Pheteplace, C.: Acta endocrinol. 59: 298, 1968. Schalch, D. S., Parlow, A. F., Boone, R. C., 1.

Discussion FUCHS, New York, New York. There can be no doubt that the development of radioimmunoassays for pituitary gonadotropins has enormously increased our ability to assess DR.

FRITZ

7. 8. 9. 10.

and Reichlin, S.: J. Clin. Invest. 47: 665, 1968. Odell, W. D., Parlow, A. F., Cargille, C. M., and Ross, G. T.: J. Clin. Invest. 47: 2551, 1968. Peckham, W. D., and Parlow, A. F.: Endocrinology 84: 1953, 1969. Shome, B., Parlow, A. F., Ramirez, V. D., Elrick, H., and Pierce, J. G.: Arch. Biochem. 103: 444, 1968. Albert, A., Rosemberg, E., Ross, G. T., Paulsen, C. A. and Ryan, R. J.: J. Clin. Endocrinol. 28: 1214, 1968.

the release of the hormones controlling gonadal function. Although separate bioassays were available for follicle-stimulating hormone and luteinizing hormones, one such even developed by your co-author, we usually had to make do with the

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very poor bioassay for total gonadotropins based on the increase in weight of the immature mouse uterus. However, before we can utilize these vastly improved assays, we must know some details about the method used. I would like to know why there is such a low sensitivity of the assay? The assay we use and which was developed by Saxena and associates1 is sensitive to 0.05 m1.U. per milliliter. From your figures, I would have to conclude that your assays are considerably less sensitive, particularly the assay for FSH. I imagine that you get zero values in hypophysectomized individuals. I must compliment you for the fact that you have been able to obtain daily blood samples from your patients, in some as long as a month or more. We all know how difficult daily sample collection is. How much blood is needed for your assay? The purpose of the paper is to demonstrate the value of the radioimmunoassay in the evaluation of menstrual disorders and the material is quite convincing in this regard. But it would have been extremely useful to have for comparison more than just a single parameter of gonadal function apart from menstruation, namely, the vaginal smear. Were you able to assay the steroid levels in the blood or urine? I shall not have time to comment on your individual curves which obviously represent an enormous amount of work. But I would like to know more about your values for the “normal” menstrual cycle. On how many cases were they based and what were the criteria for the individuals being normal? I ask because, as you know, there is no general agreement about the follicular and luteal phase levels. All we can agree upon is the midcycle peak. Is there a higher level around day 10 or is there not? I would question whether the FSH pattern could really be called “normal” in the three patients with oligomenorrhea. The radioimmunoassays will permit a much more detailed study of the pituitary-gonadal axis than was possible before, as you have pointed out. But any new tool which provides better answers also raises new questions. I have asked a lot of questions, too many perhaps, but I would like to stress that your work and that of the other members of the “Gonadotropin Club”2 is very important and has already provided us with a much better understanding of the menstrual cycle, normal as well as ahnormal.

REFERRNCES

1. Saxena, B. B., Demura, H., Gandy, H. M., and Peterson, R. E.: J. Clin. Endocrinol. 28: 519, 1968. 2. Rosemberg, Eugenia, editor: Gonadotropins 1968, Proc. Workshop Conference, Vista Hermosa, Mexico, 1968, Los Altos, California, 1968, Geron-X. DR. S. LECIN ISRAEL, Philadelphia, Pennsylvania. I rise merely to add more questions to those of Dr. Fuchs. This important paper of Dignam and Parlow may be recognized by all of us as an important contribution to our fund of knowledge. It is also easy to ask of Dr. Dignam information regarding not only the level of elevation of the luteal hormone rise, but also whether or not he had noted functional differences in the corpus luteum that fohows. It is recognized by some that it is not only important that ovulation occur, but it is of perhaps equal weight in certain patients who have reproductive difficulties to concern ourselves with the degree of luteal function that follows. While I appreciate the fact that Dr. Dignam could not possibly say in 15 minutes all that he knows concerning his work, can he perhaps now say a word or two concerning observations that he may have made regarding duration and quality of the function of the corpus luteum? DR. DIGNAM (Closing). I wish we did have some of the answers that you asked about. We don’t have them all, of course. I think gynecologic endocrinologists today would love to have daily everything on their patients, including, at least, temperatures, vaginal smears, circulating estrogens, circulating progesterone, circulating gonadotropins, hopefully, the measurement of releasing factors, and so on. I think these things are not unrealistic over the long term. I believe that we will be able to measure all of them simultaneously. To date, we are just starting. We do have the measurements of gonadotropins. We are beginning to measure both estrogens and progesterone, the several progestins, in the circulation, but we do not have adequate data to state facts. The sensitivity of our method is not as good as we would like to see it. In the early parts of our study, it was approximately 4 milli-International Units per milliliter of FSH and 2 of LH. What we must do is to obtain better antiserum. As those of you who work in the area know, once one finds a rabbit that produces good antiserum, you surround the rabbit with four

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armed guards and you bleed him very carefully. As we have found better and better antiserum, our sensitivity has been improved, but it is not in the range that Dr. Fuchs mentioned. The sensitivity of the method of Saxena and associates is astonishingly high for a method which is claimed to be specific for FSH. The fact that the FSH peak was low in some of the patients who did ovulate is, I think, probably due to the variation within the day. I think that we probably did not pick up the maximum level of FSH in those patients, but the LH peaks usually occurred at the same time and the usual parameters of ovulation that you asked about were present. We, in common with so many people who are working in the area, are repeatedly more and more impressed with the thought that luteinized follicles produce all of the features which we formerly associated with ovulation and that, in truth, the old saw that only pregnancy or a biopsy of the ovary is absolutely confirmatory of ovulation is quite valid, particularly as one administers gonadotropin or gonadotropin-releasing products one does stimulate the production of progesterone and the signs that it causes. We have not come up with any significant ratio between gonadotropins that does characterize ovulation. Once again, we wouldn’t expect to, based on measurements that occur once a day. We have come to think of this process as an extremely dynamic one, one that changes very rapidly within a period of hours. The ratio of the gonadotropins might be different at a different hour in the day of study.

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You ask how much blood is necessary for the assay, One can get along with a very small amount; a milliliter of serum is certainly enough to measure both gonadotropins. We do obtain more so that we can repeat the measurements. Each of the measurements that we have shown here today is a mean of two independent determinations. If there is any question, they certainly are repeated more often than that. You asked about how many normal patients we have studied. To date, we have studied 35 normal menstrual cycles. I do not have a composite curve of those with me, but many do show the pattern that I illustrated, admittedly one of the best examples. Dr. Israel asked about the functional differences in the corpus luteum. Again, we certainly do not have the final word to say about this. I would say only that from the patients whom we have been able to study with the usual parameters, we do feel that there are two different corpora lutea which can be formed. That is, there is a certain amount of evidence that the corpus luteum once formed goes aIong through pregnancy without further support in the form of gonadotropins or certainly any supplementation with ovarian steroids to protect the pregnancy, and this may be true of the normal corpus luteum which results from ovulation, However, there no doubt are some corpora lutea which are not completely normal. That is, the patient does ovulate, but the corpus luteum does not function normally. There has been insufficient study of circulating progesterone, though, to say anything more about such a corpus luteum.