Home | Archive | AJA @ Nature | Online Submission | News & Events | Subscribe | APFA | Society | Links | Contact Us | 中文版 |
 |
Urinary
follicle stimulating hormone can be used as a biomarker to assess male reproductive
function
Xin-Ru
WANG1, James W Overstreet2, Heather Todd2,
Qing QIU1, Jiang-Hua YANG3, Shu-Yi WANG1,
Xi-Ping XU4, Bill L Lasley2
1School
of Public Health, Nanjing Medical University, Nanjing 210029, China
2Institute of Toxicology and Environmental Health, University of
California, Davis, California 95616, USA
3Anhui Meizhong Institute for Biomedical Science and Environmental
Health, Anhui, China
4School of Public Health, Harvard University, Boston, Massachusetts
02115, USA
Asian J Androl 1999 Jun; 1: 67-72
Keywords: follicle stimulating hormone; sperm count; reproduction; testis; spermatozoa; urine; urinalysis
Abstract
Aim: To develop an algorithm for use in population-based studies to assess testicular function by measurements of total urinary follicle stimulating hormone (FSH). Methods: Total concentrations of urinary FSH were measured in a group of 44 men at the University of California, Davis (UCD) and were compared to FSH measurements in serum. On the basis of these and other published data, a urinary FSH value of >2 ng/mg creatinine (Cr) was selected as the cutoff point to identify men with elevated serum FSH (>12 IU/L) or low sperm counts (<20 million/mL). Results: The sensitivity and specificity of this algorithm for detecting elevated serum FSH in a group of 58 agricultural workers in the People's Republic of China were 100% and 50%, respectively. The sensitivity and specificity of this algorithm for detecting low sperm counts in a population of 105 infertility patients at UCD were 58% and 76%, respectively. Conclusion: This test may have particular value in identifying populations with no evidence of testicular toxicity, and in which labor-intensive semen studies may not be feasible.1 Introduction
There
is growing concern that physical and chemical hazards in the environment
may be responsible for a general decline in male reproductive potential
during the past fifty years[1]. Although this interpretation
is controversial[2], there is no doubt that specific chemical
and physical agents can produce testicular injury and infertility in human
males[3].
The
laboratory evaluation of male fertility relies primarily on analysis of
semen[4], and a number of contemporary epidemiologic studies
have taken this approach for identifying reproductive
hazards to male fertility in the workplace or other environments[5].
Nevertheless, the use of semen tests to provide biomarkers for epidemiologic
studies presents substantial challenges for field workers. Many men will
not participate in such studies for cultural or personal reasons. An individual
who donates semen for a field study may be ambivalent about his participation
and may not be motivated to follow instructions. Even when thoroughly
instructed, such men may fail to understand the importance of the procedures
they must follow in obtaining the specimen, and they may not appreciate
the consequences of noncompliance with the instructions given. For example,
failure to observe the prescribed period of sexual abstinence may affect
the semen volume and sperm concentration, as may the technique of semen
collection (ie, there may be partial loss of the specimen)[6].
Endocrine
biomarkers have not been used exclusively in epidemiologic field studies
of male fertility although they have been used in some studies to provide
adjunctive data[7]. Follicle stimulating hormone (FSH) concentrations may be elevated in cases of testicular
damage because of adverse effects on the Sertoli cell and its production
of inhibin, which provides the primary negative feedback for FSH secretion[8].
Nevertheless, clinical studies have demonstrated that relatively normal
serum gonadal and pituitary hormone profies can be found in association
with significant defects in sperm production and infertility[9].
2 Materials and methods
2.1
Subjects
In
order to identify the concentration of urinary FSH metabolites which corresponds
to an elevated level of serum FSH, forty four male volunteers were recruited at
the University of California, Davis (UCD) to provide both serum and urine
samples. Ten of these subjects were fertile men who were semen donors
for a therapeutic artificial insemination program. All of these subjects
had normal serum levels of FSH. Thirty four subjects were infertility
patients who were selected on the basis of previous clinical evaluations
to provide a wide range of serum FSH values. The serum and urinary FSH
values from the 10 fertile men and 10 of the infertile men have been reported
previously[11]. Blood samples were collected at the clinic
by venipuncture, the samples were allowed to clot and the serum was removed
and stored frozen until analyzed. The subjects provided five consecutive
weekly urine samples, beginning in the week in which serum was obtained.
Early morning urine samples were collected at home and were stored immediately
without preservatives in the subject's refrigerator freezer (-10). The
samples were returned to the laboratory at the end of the five week collection
and were stored at -20. The samples were thawed and refrozen approximately
five times over an eight year period.
The
sensitivity of the urinary FSH assay for detecting men with abnormal serum
FSH values was tested in a population of 58 men recruited in the People's
Republic of China (PRC). These subjects were agricultural workers in Anhui
Province, who were not selected on the basis of previous medical history
or endocrine evaluations. These men are participants in an ongoing study
of the effects of environmental chemicals on male reproductive function.
Blood and urine samples were collected from the subjects in the PRC according
to the same basic protocol as used for the subjects at UCD, except that
three weekly blood and urine samples were collected from each man. The
samples were shipped frozen to the laboratory at UCD for analysis.
The
sensitivity of the urinary FSH assay for detecting men with abnormal sperm
numbers in semen was tested in a population of 105 men who were recruited
at UCD. These subjects were referred to the clinical andrology laboratory
for semen evaluation because they were members of infertile couples. They
were not selected on the basis of their medical history or any previous
laboratory evaluations. If they agreed to participate in the study, they
were asked to provide a single urine sample on the same day that they
collected a semen sample by masturbation for laboratory evaluation. Subjects
were instructed to abstain from ejaculation for forty eight hours prior
to semen collection.
2.2
Assays
Serum
FSH concentrations were measured using a commercial kit (Diagnostic Products
Corp, Los Angeles, CA). The assay for total urinary FSH, which is based on
measurement of the FSH beta subunit, was performed as previously described[11].
Briefly, microtiter plates (Nunc-Immuno Plate, Maxisorptm)
were coated with 200 L of 10 g/mL monoclonal anti-beta hFSH antibody
(FS2-4A10-G10, Scantibodies Lab Santee, CA) in 0.2 mol/L sodium bicarbonate
buffer, pH 9.6, and incubated for 6 h or overnight at room temperature
(RT, 27). Unbound sites in the wells were then blocked by overnight
incubation with 250 L of 1% casein, 0.05% Tween 20 and 0.1% sodium azide
in 10 mmol/L PBS, pH 7.5. On the next day, 100 L of 0.5 mol/L phosphate-buffer
saline (PBS) buffer containing 1% BSA and 0.05% sodium azide, pH 7.5,
were added to every well. Urine samples or internal controls were transferred
to polypropylene minitubes and placed into boiling water for 2 min to
dissociate the FSH subunits. Then, 100 L of treated urine samples, internal
controls or FSH beta subunit standard (provided by the National Hormone
and Pituitary Program) were added to each assigned well and incubated
overnight at RT. Two hundred microliters of polyclonal rabbit anti hFSH-
antisera were diluted 1:5000 in buffer B (0.1% beta gamma globulin,
1% polyethylene glycol 8000, 0.05% Tween 20 and 10 mmol/L ethylene-diamine-tetraacetic
acid (EDTA) in 10 mmol/L PBS) and added to each well before overnight
incubation at RT. The next day, 200 L of biotinylated goat anti rabbit
IgG (Bio-Rad 170-6401) diluted 1:64 K in buffer B were added
and incubated for 2 h at RT, followed by incubation with 200 L of alkaline
phosphatase streptavidin diluted 1:2000 in APS buffer (1.0 mol/L NaCl,
0.1% Tween 20 in 10 mmol/L Tris-HCl, pH 7.5) for 1 h at RT. Finally, 200 L of
1 mg/mL p-Nitrophenyl Phosphate (pNPP) in substrate buffer (1.0 mmol/L
MgCl2, 1.0 mol/L Diethanoamine-HCl, pH 9.0) were added to each well.
The plates were emptied and thoroughly washed with ELISA wash (0.15 mol/L
NaCl plus 0.05% Tween 20) between each step. Color development was expected
within 1 h, and each plate was read on a microtiterplate reader (Dynatec
Microplate Reader model MR600; Dynatech Laboratory Inc, Alexandria, VA)
using the dual wavelength mode, 405 nm minus reference 650 nm. The absorbances
were automatically transferred to an enzyme immunoassay data reduction
program for curve fitting and data analysis. Urinary concentrations of
FSH were indexed by the creatinine (Cr) concentration in the urine sample[10].
The intra-assay reliability was 2.9% and the inter-assay coefficient of
variation was 7.4%[11].
Semen evaluations were carried out in accordance with published methods[13]. Briefly, the semen volume was measured to the nearest 0.1 mL using a serological pipette. A -Cell slide (Spectrum Technologies) in combination with a reticle grid was used for determining the sperm concentration in millions per mL. The total number of sperm per ejaculate was calculated by multiplying the semen volume and the sperm concentration. Semen was classified as normal or abnormal according to the criteria of the World Health Organization[14]. Sperm numbers in semen were considered to be abnormal if the sperm concentration was less than 20 million sperm/mL, or the total number of sperm in the ejaculate was less than 40 million.
2.3
Data Analysis
3 Results
For
the 44 subjects recruited at UCD there was a highly significant correlation
between serum FSH and total urinary FSH (r=0.75, P<0.001,
Figure 1). The relationship between the FSH values in serum
and urine in this group of men was used to establish a cutoff value for
urinary FSH which could be tested for its utility in identifying men
with low sperm counts. The regression analysis of these data resulted
in a first order equation of Y=3.7X+6.44. The serum FSH values of 765
men with sperm concentrations >20 million/mL was reported to range
from undetectable to 11.7 mIU/mL[9]. On the basis of the data
of Morrow et al, and the regression analysis of the present data, the range of urinary FSH for normal
men was calculated to be 0 to 1.42 ng/mg Cr. Taking into account the report
from the same previous study[9] that men with normal testicular biopsies
had serum FSH levels as high as 15.4 mIU/mL (corresponding to 2.42 ng/mg
Cr in urine), we selected a total urinary FSH value of>2.0 ng/mg Cr
as the cutoff point for identifying men with abnormal testicular function.
Figure 1. The scattergram of individual serum FSH concentrations
and the average values of five total urinary FSH
concentrations from 44 men with a range of serum FSH values. The serum
samples were collected at the same time that the first urine sample was
collected and the remaining urine samples were collected at weekly intervals.
For
the 58 subjects recruited in the PRC the correlation between FSH values
in serum and in urine also was highly significant (r=0.72, P<0.001).
In this group of men 20 had serum FSH values>12 mIU/mL and all of these
subjects had a total urinary FSH>2.0 ng/mg Cr. Therefore, the sensitivity
of the urinary FSH assay to detect elevated serum FSH in this population
was 100% (Table 1). The predictive value of a negative urinary FSH test
(ie <2.0 ng/mg Cr) also was 100%. The specificity of the urinary assay
to detect elevated serum FSH was 50 % and the predictive value of a positive
urinary FSH assay was 51% (Table 1).
Table
1. Sensitivity and specificity of total urinary FSH to detect men with
elevated levels of serum FSH.
| Total Urinary FSH |
Serum FSH |
|
|
| 12 mIU/mL |
< 12 mIU/mL |
||
| 2 ng/mg Cr |
20 |
19 |
39 |
| < 2 ng/mg Cr |
0 |
19 |
19 |
| |
20 |
38 |
58 |
Sensitivity=100%
Specificity (95% CI)=50% (34-66)
Predictive value of a positive test (95%CI)=51%(35-67)
Predictive value of a negative test=100%
In
the group of 105 infertility patients at UCD, 37 men (35 %) had urinary
FSH levels that were >2.0 ng/mg Cr. When we evaluated the sensitivity
and specificity of the urinary FSH assay for detecting low sperm concentrations,
we found that the assay had a sensitivity of 58 % and a specificity of
76% to detect the thirty eight men with a sperm concentration <20 million
sperm/mL (Table 2). The predictive value of a negative test was 76% and
the predictive value of a positive test was 59% (Table 2). Similarly,
the sensitivity and specificity of the urinary FSH assay to detect the
32 men with total sperm numbers <40 million were 53% and 71%, respectively,
and the predictive values of a negative test and a positive test were
78% and 45%, respectively (Table 3).
Table
2. Sensitivity and specificity of total urinary FSH to detect men with
low sperm concentrations in semen Total Urinary FSH Sperm Count.
| Total Urinary FSH |
Sperm Count |
|
|
| < 20 million/mL |
20 million/mL |
||
| 2 ng/mg Cr |
22 |
15 |
37 |
| < 2 ng/mg Cr |
16 |
52 |
68 |
| |
38 |
67 |
105 |
Sensitivity
(95% CI)=58% (42-74)
Specificity (95% CI)=76% (66-86)
Predictive value of a positive test (95%CI)=59% (43-75)
Predictive value of a negative test (95%CI)=76% (66-86)
Table
3. Sensitivity and specificity of total urinary FSH to detect men with
low numbers of sperm in semen.
| Total Urinary FSH |
Total Sperm Per Ejaculate |
|
|
| < 40 million |
40 million |
||
| 2 ng/mg Cr |
17 |
21 |
38 |
| < 2 ng/mg Cr |
15 |
52 |
67 |
| |
32 |
73 |
105 |
Sensitivity (95% CI)=53% (36-70)
Specificity (95% CI)=71% (61-81)
Predictive value of a positive test (95% CI)=45% (29-51)
Predictive value of a negative test (95% CI)=78% (62-88)
4 Discussion
Measurements
of serum gonadotropins are considered essential for the clinical evaluation
of men with suspected infertility[8,16]. Although some studies
have demonstrated a relationship between serum gonadotropin levels and
sperm concentration in semen[17-19], other investigators found
that serum FSH levels were consistently increased only in cases of testicular
failure which resulted in azoospermia[20].
In clinical practice, an endocrine evaluation usually is obtained only
when the semen parameters are abnormal; and in these cases, elevated gonadotropins
usually indicate primary gonadal insufficiency, for which there is no
medical therapy[16]. Thus, the endocrine signals of male reproductive
dysfunction have been regarded as relatively late signs of toxicity, and
their utility as biomarkers of male reproductive function in population-based
studies has been questioned[21].
There
are few clinical data on the relationship between urinary FSH values and
testicular function. Previous studies utilized murine bioassays of FSH
activity, and showed no correlation between the histologic characteristics
of the testis and urinary FSH levels[22]. Before contemporary,
enzyme-based immunoassays could be applied for measurements of urinary
FSH, it was necessary to address the problem of dissociation of the FSH
heterodimer in urine samples. Such dissociation results from the freezing
and thawing of urine, and causes a decline in the concentration of immunoreactive,
intact FSH during storage[23]. As a consequence, subjects must
collect urine samples with some type of cyro-preservative such as glycerol.
This requires an additional effort in terms of sample kit preparation
and may confound the later use of urine samples if biomarkers for exposure
are also used. This problem was overcome by the development of an assay
procedure which dissociated all urinary FSH into its alpha and beta subunits
and then measured the total amount of beta subunit in the sample[11].
In
a previous study, we investigated the relationship between urinary concentrations
of FSH and serum concentrations of FSH[11]. Weekly urine samples
were collected and analyzed from a group of 10 fertile men and 10 infertile
men following the collection of paired blood and urine samples. The overall
correlation between FSH concentration in the weekly urine sample and FSH
concentration the initial blood sample remained strong for four weeks
following blood collection (r values ranged from 0.90 to 0.94). However,
for unknown reasons, the correlation fell to r=0.76 by the fifth
week after blood collection[11]. Our data from normal women,
who exhibit dynamic FSH production profiles, demonstrate that daily changes
in serum FSH are matched by similar changes in daily urine measurements
using this same assay[12]. Thus, it appears that in men FSH
production and excretion are closely linked and relatively stable over
prolonged periods of time.
The results of the present study demonstrate that this assay for total urinary FSH[11] is highly sensitive for identifying men with elevated serum FSH. The negative predictive value of the test also is very high for assessing elevated serum FSH levels. Those men in our sample who did not have elevated FSH in urine did not have high serum FSH levels. As might be predicted from the clinical literature, the urinary FSH assay lost sensitivity when it was applied for identification of men with low sperm production. This result can be explained because many men with low sperm counts do not have sufficiently advanced testicular failure to cause an elevation of pituitary gonadotropins. An additional complexity arises from the fact that some men can be expected to have low sperm counts for reasons other than gonadal failure, such as blockage of the reproductive tract. The present data also demonstrate that urinary FSH measurements have good negative predictive value for identifying men who do not have low sperm counts. This quality of the test could be expected since there are few situations other than testicular failure in which FSH production is likely to be increased.
Because urine collection is non-invasive, participation of subjects is likely to be enhanced in epidemiologic studies which use urine rather than blood or semen as the material for biological measurements. For this reason alone, urinary gonadotropin measurements may prove to be cost effective for screening populations of men with suspected reproductive impairment. These tests may be particularly valuable for identifying populations with no evidence of testicular toxicity, and in which labor-intensive semen studies may not be feasible.
References
[1]
Carlsen E, Giwercman A, Keiding N, Skakkebaek NE. Evidence for decreasing
quality of semen during past 50 years. Br Med J 1992; 305: 609-13.
[2] Olsen GW, Bodner KM, Ramlow JM, Ross CE, Lipshultz LI. Have sperm counts been reduced 50 percent
in 50 years? A statistical model revisited. Fertil Steril 1995; 63: 887-93.
[3] Gold EB, Lasley BL, Schenker MB. Introduction: rationale for an update.
Reproductive hazards. Occup Med 1994; 9: 363-72.
[4] Overstreet JW. Clinical approach to male reproductive problems. Occup
Med 1994; 9: 387-404.
[5] Schrader SM, Kanitz MH. Occupational hazards to male reproduction.
Occup Med 1994; 9: 405-14.
[6] Overstreet JW, Davis RO. Methods and interpretation of semen analysis.
In: Keye WR, Chang RJ, Rebar RW, Soules M, edtors. Infertility: Evaluation
and Treatment. Philadelphia: WB Saunders; 1995. p 580-91.
[7] Whorton MD, Milby TH, Krauss RM, Stubbs HA. Testicular function in
DBCP exposed pesticide workers. J Occup Med 1979; 21: 161-6.
[8] Santen RJ. Male hypogonadism. In: Yen SC, Jaffe RB, editors.
Reproductive Endocrinology. 3rd ed. Philadelphia: WB Saunders; 1991. p
739-94.
[9] Morrow AF, Gordon Baker HW, Burger HG. Different testosterone and
LH relationships in infertile men. J Androl 1986; 7: 310-5.
[10] Lasley BL, Lohstroh P, Kuo A, Gold EB, Eskenazi B, Samuels
SJ, et al. Laboratory methods for evaluating early pregnancy loss
in an industry-based population. Am J Ind Med 1995; 28: 771-81.
[11] Qiu Q, Kuo A, Todd H, Dias JA, Overstreet JW, Lasley BL. Enzyme immunoassay
method for the beta subunit of urinary follicle stimulating hormone (FSH)
and its application for measurement of total urinary FSH. Fertil Steril
1998; 69: 278-85.
[12] Qiu Q, Overstreet JW, Todd H, Nakajima ST, Stewart DR, Lasley
BL. Total urinary follicle stimulating hormone as a biomarker for detection
of early pregnancy and perimplantation spontaneous abortion. Environ Health
Perspect 1997; 105: 862-6.
[13] Overstreet JW, Brazil C. Semen Analysis. In: Lipshultz LI, Howards SS, editors. Infertility
in the Male. 3rd ed. St Louis: Mosby-Year Book, Inc; 1997. p 487-90.
[14] World Health Organization. WHO laboratory manual for the examination
of human semen and sperm-cervical mucus interaction. World Health Organization.
3rd ed. London: Cambridge University Press, 1992.
[15] Beyer WH, editor. CRC standard probability and statistics: tables
and formulae. Boca Raton, FL: CRC Press; 1991. p 151-69.
[16] Sokol RZ, Swerdloff RS. Endocrine evaluation. In: Lipshultz, LI,
Howards SS, eds. Infertility in the Male. 3rd ed. St Louis: Mosby-Year
Book, Inc; 1997. p 210-8.
[17] Franchimont P, Millet D, Vendrely E, Letawe J, Legros JJ, Netter
A. Relationship between spermatogenesis and serum gonadotropin levels
in azoospermia and oligospermia. J Clin Endocrinol Metab 1972; 34: 1003-8.
[18] Handelsman DJ, Conway AJ, Boylan LM, Turtle JR. Testicular function
in potential sperm donors: normal ranges and the effects of smoking and
varicocele. Int J Androl 1984; 7: 369-82.
[19] Mieusset R, Bujan L, Plantavid M, Grandjean H. Increased levels of
serum follicle-stimulating hormone and luteinizing hormone associated
with intrinsic testicular hyperthermia in oligospermic infertile men.
J Clin Endrocrinol Metab 1989; 68: 419-25.
[20] De Kretser DM, Burger HG, Fortune D, Hudson B, Long AR, Paulsen CA,
et al. Hormonal, histological
and chromosomal studies in adult males with testicular disorders. J Clin
Endocrinol Metab 1972; 35: 392-401.
[21] Overstreet JW. Assessment of disorders of spermatogenesis. In: Lockey
JE, editor. Reproduction: The New Frontier in Occupational and Environmental
Health Research. New York: Alan Liss; 1984. p 275-92.
[22] Leonard JM, Leach RB, Couture M, Paulsen CA. Plasma and urinary follicle-stimulating
hormone levels in oligospermia. J Clin Endrocrinol Metab 1972; 34: 209-14.
[23] Saketos M, Shartma N, Adel T, Raghuwanshi M, Santoro N. Time-resolved immunofluorometric assay
and specimen storage conditions for measuring urinary gonadotropins. Clin
Chem 1994; 40: 749-53.
Project
supported by National Institutes of Health Grants ES04699,ES06198 and
ES05707.
Correspondence to Dr James Overstreet, ITEH, University of California,
One Shields Avenue, Davis, CA 95616-8615 USA.
Tel: +1-530-752 3012 Fax: +1-530-752 5300
E-mail: jwoverstreet@ucdavis.edu
Received 1999-03-08 Accepted 1999-05-10
