Hum. Reprod.-2002-Barnes-88-91.pdf

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Human Reproduction Vol.17, No.1 pp. 8891, 2002

CASE REPORT

The role of LH and FSH in ovarian androgen secretion andovarian follicular development: Clinical studies in a patientwith isolated FSH deficiency and multicystic ovaries

Randall B.Barnes1,5, Anne B.Namnoum2, Robert L.Rosenfield3 and Lawrence C.Layman4

Departments of Obstetrics and Gynecology, 1University of Chicago, Chicago, IL 60637, 2Emory University, Atlanta, GA 30322,3Department of Pediatrics and Medicine, University of Chicago, Chicago, IL 60637, 4Department of Obstetrics and Gynecology,

Medical College of Georgia, Augusta, GA 30912, USA

5To whom correspondence should be addressed at: Dept of Obstetrics and Gynecology, University of Chicago, 5841 Maryland

Avenue, Chicago, IL 60637, USA. E-mail: [email protected]

Inactivating mutations have proven to be instructive in elucidating the role of FSH in human ovarian function. We

performed a detailed reproductive endocrine evaluation of a patient with inactivating mutations in the FSH

-subunit gene who was hypo-estrogenic and had LH excess. The patient underwent a pelvic ultrasound andovernight frequent blood sampling followed by a human chorionic gonadotrophin (HCG) stimulation test. One

month later she received human recombinant FSH, followed 24 h later by a second HCG stimulation test. Despite

a mean LH serum concentration and LH pulse characteristics typical for polycystic ovaries (PCOS), baseline and

dexamethasone-suppressed free testosterone were lownormal. The administration of HCG led to minimal stimulation

of 17-hydroxyprogesterone and androgens. The patient had multicystic ovaries containing follicles 35 mm in

diameter and responded to FSH with prompt increases in estradiol and inhibin B. There were no clinical or

laboratory consequences of LH excess in this FSH-deficient woman. These findings support the hypothesis that

excessive LH stimulation alone does not cause ovarian hyperandrogenism. We also found that follicular development

was present in the absence of FSH. These antral follicles had apparently developed normally, since estradiol and

inhibin B increased promptly after FSH administration.

Key words: androgens/FSH deficiency/LH excess/ovary

Introduction

Inactivating mutations have proven to be instructive in elucidat-

ing the role of FSH in human ovarian function (Layman and

McDonough, 2000; Themmen and Huhtaniemi, 2000). Our

studies of a patient with compound heterozygous mutations of

the FSH -subunit gene (Layman et al., 1997), undetectableserum FSH and elevated serum LH have elucidated two aspects

of FSH action which challenge common wisdom. We report

here evidence that FSH is not necessary for the development

of small, healthy antral follicles readily responsive to FSH,

which contrasts with studies in FSH-knockout mice (Kumar

et al., 1997; Dierich et al., 1998). However, FSH is necessary

for ovarian theca cell function, contrary to expectations from

the 2-gonadotrophin, 2-cell model of ovarian steroidogenesis

(Barnes et al., 2000). The latter finding has important

implications for theories about the role of LH excess in the

pathogenesis of polycystic ovary syndrome (PCOS).

88 European Society of Human Reproduction and Embryology

Case report

Subject

The patient presented at 16 years of age with primary

amenorrhoea and absent breast development. Her baseline

and gonadotrophin-releasing hormone (GnRH)-stimulatedFSH concentrations were undetectable by immunoassay, while

LH concentrations at baseline (30 mIU/ml) and post-GnRH

(150 mIU/ml) were elevated. The patient was subsequently

treated with estrogen and underwent normal breast develop-

ment. Analysis of the FSH -subunit gene demonstrated thatshe was a compound heterozygote for two different mutations,

a two base-pair deletion in exon three at codon 61 (Val61X)

inherited from her mother, and a missense mutation changing

a cysteine to a glycine at codon 51 (Cys51Gly) inherited from

her father. When these mutations were stably transfected into

Chinese hamster ovary cells, they demonstrated no measurable

FSH immuno- or bioactivity (Layman et al., 1997).


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Clinical studies in a patient with FSH deficiency

Figure 1. Outline of clinical research center protocols.Dexamethasone, 0.5 mg four times daily, was started on day 1 andcontinued until the blood sample was drawn 24 h after HCG.

Reproductive endocrine evaluation

The patient underwent a detailed reproductive endocrine

evaluation at 21 years of age in the University of Chicago

General Clinical Research Center (GCRC), after dis-

continuing the oral contraceptive pill for one month and giving

informed consent. The study protocols were approved by the

University of Chicago Institutional Review Board. Her heightwas 155 cm and her weight was 53.2 kg. There was no

hirsutism (Ferriman and Gallway score of 4; normal 8),

breasts and pubic hair were Tanner stage 5, and the pelvic

examination was normal. An initial blood sample was drawn for

immunoactive FSH, LH and free testosterone. The patient then

received dexamethasone 0.5 mg four times daily for 4 days

prior to and continuing throughout the endocrine evaluation

to suppress adrenal function. Following admission to the

GCRC, she underwent a transvaginal pelvic ultrasound fol-

lowed by blood sampling every 10 min from 7.00 p.m. to 6.00

a.m. Serum immunoactive FSH and LH were determined for

each sample; inhibin A and B, bioactive FSH and free -

subunit were determined in a pooled sample. The followingmorning, a human chorionic gonadotrophin (HCG) test was

performed. A blood sample was drawn for baseline testoster-

one, free testosterone and estradiol. The following steroid

intermediates were also determined: 17-hydroxyprogesterone

(17-Prog), androstenedione, 17-hydroxypregnenolone and

dehydroepiandrosterone. HCG 5000 IU was then administered

i.m. and blood was drawn 24 h later for repeat steroid

measurements.

The patient remained off any sex steroids for another month,

then returned to the GCRC for a second HCG study which

was like the first except she received 300 IU of recombinant

human FSH s.c. in the morning, 24 h prior to the HCG test.

A blood sample was drawn 24 h later for steroids and inhibin

A and B, and HCG 5000 IU was administered. She then

returned 24 h after HCG (48 h after FSH) for a final blood

sample for steroid measurements (Figure 1).

All samples for steroids were frozen at 20C and

measured in the same assay using previously published methods

(Barnes et al., 1989; Rosenfieldet al., 1994). Serum immuno-

active LH and FSH were measured using a -subunit-specificassay (Delfia, Wallach, Finland; lower limit of sensitivity

0.15 mIU/ml for LH and 0.2 mIU/ml for FSH). Bioactive

FSH, inhibin A, inhibin B and free -subunit were measuredusing previously published methods (Christin-Maitre et al.,

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Figure 2. Transvaginal ultrasound of the left ovary before FSHtreatment. The ovary is fusiform in shape, measuring ~61.5 cm.The stroma is scant but there are multiple follicles throughout theovary, the largest measuring 5 mm. The right ovary (not shown)was similar to the left.

1996; Lavoieet al., 1998; Weltet al., 1999). LH pulse analysis

was performed using the ULTRA program (Van Cauter andCopinschi, 1981).

Results

The ovarian ultrasound prior to FSH treatment is shown in

Figure 2. The ovaries were multicystic containing follicles

35 mm in diameter, but unlike typical polycystic ovaries

(Adams et al., 1986), there was no dense stroma and the

follicles were scattered throughout the ovary rather than being

predominately subcapsular.

Immunoactive LH was 46 mIU/ml and FSH was undetectable

(0.2 mIU/ml) in the single sample drawn before dexametha-

sone. There was no immunoactive FSH detected in the samplescollected overnight (Table I), and no bioactive FSH was

detected in the pooled sample. The mean immunoactive

LH in the overnight samples was 46 mIU/ml; mean pulse

amplitude was 15.8 mIU/ml and nine pulses were detected in

11 h (Figure 3). This LH profile is similar to that reported for

PCOS (Waldstreicher et al., 1988). Free -subunit measuredin the pooled 11 h sample was in the postmenopausal range

(3501740 pg/ml; Table I).

Although LH concentrations were elevated, the patient had

no laboratory evidence of ovarian hyperandrogenism. The

patients baseline free testosterone was 6 pg/ml (normal

range 310 pg/ml; ovarian hyperandrogenism 10 pg/ml)

(Rosenfield et al., 1994). After 4 days of dexamethasone

suppression the free testosterone fell to 2 pg/ml (normal range

8 pg/ml; ovarian hyperandrogenism 8 pg/ml; Rosenfield

et al., 1994). The 17-Prog concentration 24 h after HCG was

40 ng/dl which was 2.7 standard deviations (SD) below the

mean in women with ovarian hyperandrogenism (288 ng/dl)

(Levrantet al., 1997) and 1.5 SD below the mean of normal

follicular phase women (139 ng/dl: Levrant et al., 1997).

One month later, during the second GCRC study,

estradiol increased substantially 24 h after FSH administration

(2880 pg/mL). Inhibin B was near the lower limit of the

normal range at baseline and rose to above the range for


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R.B.Barnes et al.

Table I. Gonadotrophin and inhibin concentrations in an FSH-deficient patient

FAS LH FSH Bioactive FSH Inhibin A Inhibin B(pg/ml) (mIU/ml) (mIU/ml) (mIU/ml) (pg/ml) (pg/ml)

Baselinea 1247 46 0.2 4 0.6 25.324 h after FSH 8.4 0.8 283Normal women 103257 015 320 440 0.680 15220

aBaseline is mean of every 10 min samples over 11 h for LH and FSH. For all others, baseline is a value ofthe pooled 11 h sample. Normal values are from Christein-Maitre et al., 1996; Lavoie et al., 1998; Weltet al., 1999.FAS free alpha subunit.

Figure 3. LH pulse analysis. LH was measured at 10 min intervalsover 11 h. Significant pulses are indicated by an asterisk.

menstruating women 24 h after FSH injection (Table I). In

contrast, inhibin A was present at low concentrations.

The response of testosterone and the steroid intermediates

to the two HCG tests have been previously reported (Barnes

et al., 2000). In brief, all steroid concentrations 24 h after the

second HCG injection (48 h after FSH) were greater than at

24 h after the first HCG injection (HCG alone). The difference

between the two HCG tests was most dramatic for testosterone,

which was unaffected by the first HCG test (12 ng/dl before

and after HCG), but increased from 16 to 41 ng/dl after the

second, FSH-primed HCG test.

Discussion

This patient, with well characterized inactivating mutations in

the FSH -subunit gene, was hypo-estrogenic with secondaryLH excess. Two features were noteworthy: antral follicular

development was present in spite of the lack of FSH, and

there were no clinical or laboratory consequences of the

LH excess.

The point at which FSH is necessary for follicular develop-

ment in the human ovary is debated (Gougeon, 1996; McGee

and Hsueh, 2000). Despite having no detectable FSH, our

patient had multicystic ovaries with follicles up to 5 mm in

diameter. Similar sized follicles have been reported in some

women with FSH receptor mutations (Aittomaki et al., 1996;

Beau et al., 1998); however, those FSH receptor mutations

90

may not be completely inactivating. These findings are in

contrast to FSH- and FSH receptor-knockout mice, in whichantral follicles are not maintained (Kumar et al., 1997; Dierich

et al., 1998). In studies of human ovarian xenografts trans-

planted into the kidney capsule of immunodeficient and hypo-

gonadotrophic mice, FSH was required for the growth of

follicles beyond the two-layer granulosa cell stage (Oktay

et al., 1998), which is about the point at which the FSHreceptor gene isfirst expressed in human follicles (Oktay et al.,

1997). Although the xenograft data are in contrast to our

findings, it is likely that the endocrine and growth factor milieu

of the in-situ human ovary is very different than that in the

immunodeficient, hypogonadotrophic mouse.

The prompt estradiol and inhibin B responses 24 h after

exogenous FSH suggest that our patients antral follicles

contained granulosa cells that had developed normally in the

absence of FSH. Our findings are similar to those in two other

patients with isolated FSH deficiency who ovulated and had a

successful pregnancy after ~14 days of menotrophin therapy

(Rabinowitz et al., 1979; Matthews et al., 1993). Ovulation

after a short exposure to menotrophins implies that somehealthy follicles had reached the point of recruitability without

FSH exposure, since ~14 days are required for an ovulatory

follicle to develop from follicles a few millimeters in diameter,

in contrast to the 3 months required to develop from the pre-

antral stage (Gougeon, 1996). These earlier reports and our

findings suggest that in the complete absence of FSH stimula-

tion, human ovarian antral follicles can develop up to 5 mm

in diameter.

Our patient had no clinical or laboratory evidence of ovarian

hyperandrogenism despite a mean LH concentration, LH pulse

characteristics and ovarian follicular sizes typical for PCOS.

On ultrasound her multicystic ovaries lacked the excess stroma

of classic polycystic ovaries. Indeed, her ovaries produced

little, if any, androgen. Her baseline and dexamethasone-

suppressed free testosterone were lownormal. The administra-

tion of HCG led to minimal stimulation of 17-Prog or other

thecal cell steroids. However, we have previously shown that

exogenously administered FSH augmented her LH- and HCG-

stimulated production of testosterone and all steroids of thecal

cell origin (Barneset al., 2000). This suggests that FSH action,

probably via granulosa cell-produced paracrine intermediates,

is necessary for thecal cells to respond to LH. One of many

such paracrine factors is inhibin B, which increased markedly

with FSH administration in this patient (Barnes 1998).


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Clinical studies in a patient with FSH deficiency

These findings are relevant to the role of LH excess in the

pathogenesis of the ovarian hyperandrogenism of PCOS. It

has been reported that 75% of women with clinical evidence

of PCOS have an elevated LH concentration and 94% have

an increased LH/FSH ratio (Taylor et al., 1997). These

gonadotrophin secretory abnormalities have been thought to

play an important role in the development of the ovarian

hyperandrogenism characteristic of PCOS (Hall, 1993). In arelated study, we found that a woman with a constitutively

activating mutation of the LH receptor, identified because she

was the mother of two sons with gonadotrophin-independent

precocious puberty, had no clinical or laboratory evidence of

ovarian hyperandrogenism (Rosenthal et al., 1996). Thus,

increased LH stimulation, even in the presence of FSH, appears

to be insufficient to induce the hyperandrogenism and stromal

hyperplasia of PCOS. The findings in the previous, as well as

the current, case report support the hypothesis that thecal cell

androgen secretion in response to excessive LH stimulation is

strictly limited by intra-ovarian factors. Ovarian hyperandro-

genism is more likely a result of escape from down-regulation

by these intra-ovarian factors than a result of elevated LHconcentrations (Ehrmann et al., 1995). Taken together, our

studies support the hypotheses that normal ovarian androgen

production depends on both FSH and LH and that excessive

ovarian androgen production is a result of abnormal intra-

ovarian regulation, and not of excessive LH stimulation.

Acknowledgements

We are greatly indebted to Patrick Sluss,Reproductive Endocrine Unit,The Massachusetts General Hospital, Boston, MA, who performedthe FSH bioassay, FAS, inhibin A, and inhibin B assays; to ZubieSheikh, Department of Obstetrics and Gynecology, University ofChicago, for performing the transvaginal ultrasound; and to Jennifer

M.Cunningham, Department of Medicine, University of Chicago, forperforming the LH pulse analysis. We also appreciate the helpfuldiscussions of J.Larry Jameson from the Center for Endocrinology,Metabolism, and Molecular Medicine, Northwestern University,Chicago, IL and William F.Crowly Jr from the ReproductiveEndocrine Unit and the National Center for Infertility Research, TheMassachusetts General Hospital, Boston, MA.

L.C.L. was supported by NIH grant support PHS NICHD HD33004;R.L.R. was supported by NIH grant support RR-00055 (CRC).

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Received on February 28, 2001; accepted on August 21, 2001

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