Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter.
Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.
OCR for page 187
1 ~
Developing Assays of Biologic Markers
for Epidemiologic Studies:
Experience with a Marker of
Pregnancy and Early Loss
Human chorionic gonadotropin (hCG)
is a glycoprotein hormone secreted by
the syncytiotrophoblast; it appears to
enter the maternal circulation at the time
of endometrial implantation of a fertil-
ized ovum (Jaffe, 1986~. Its principal
known role is to act at the ovarian corpus
luteum to stimulate further secretion of
progestins to support endometrial growth.
Blood and urinary hCG concentrations rise
rapidly and peak approximately 12 weeks
after onset of the last menstrual period;
thereafter, they slowly decline until the
fetus and placenta are delivered. An addi-
tional function of hCG may involve stimulus
of steroid hormone production by the fetus.
The period of maximum testosterone produc-
tion by the fetal testis corresponds to
the peak period of placental hCG produc-
tion, and gaffers group has demonstrated
that hCG binds to receptors in the fetal
testis and stimulates the production of
testosterone (Huhtanemi et al., 1977~.
(Physiologic changes associated with the
initiation of pregnancy are discussed in
Chapter 20.)
This chapter describes the development
and application of an assay to assess uri-
nary hCG content as a biologic marker.
The purpose of describing the problems
encountered in laboratory development
and early use of inappropriate assays is
187
to make clear the need for complete devel-
opment in the laboratory and preliminary
studies in the field before assays are used
in large epidemiologic studies. It is
demonstrated that even with this widely
studied hormone, there remains a need to
improve the assays for use in field studies
and a need for more extensive studies of
normal populations.
ASSAYS OF hCG
In contrast with many other plasma pro-
teins, hCG is excreted in the urine at a
concentration approximately equal to that
in blood. Marshall and colleagues (1968)
demonstrated that to be the case throughout
the first trimester, and Armstrong et al.
( 1984) verified that, even when minute
quantities of serum hCG are present, the
urine specimen is a ready source for assay
and purification of hCG.
. . ~ . . . — . ~
However, it is
also Hey that urine specimens contain
partially degraded hormone fragments,
and measurement of a serum specimen might
be preferred for diagnostic interpreta-
tion.
Bioassays for hCG were developed by As-
cheim and Zondek (1928) (the A-Z test)
as a method to detect pregnancy several
weeks after the first missed menstrual
period. In the 1960s, immunologic assays
OCR for page 188
188
for hCG replaced the more cumbersome bio-
assays (Wide, 1962; Wide and Gemzell,
1960~; these added increased sensitivity
and reproducibility, which permitted
earlier diagnoses of pregnancy (Bell,
1969~. However, polyclonal (usually rab-
bit) antibodies raised against hCG always
cross-reacted with human luteinizing
hormone (hLH) in the maternal serum sample
or with hormone components in urine, owing
to the high degree of homology between hCG
and hLH. The sensitivity of these assays
was limited by the inevitable presence
of hLH immunoreactivity in maternal serum
and also by either intact hLH or its frag-
ments in urine.
The relationship of all the homologous
glycoprotein hormones became apparent
· .
with the discovery that each comprised
a single ax subunit and a single ~ subunit
(Pierce et al., 1971~. The amino acid se-
quences of the ~ subunits of hLH and hCG
were shown to be identical, but minor dif-
ferences were found in the structures
of the ,B subunits (Bellisario et al., 1973;
Morgan et al., 1973, 1975; Birken and Can-
field, 1977; Kessler et al., 1979; Fiddes
and Goodman, 1981; Pierce and Parsons,
1981~. When immunized with the purified
,6 subunit of hCG, rabbits occasionally
made antibodies that were directed predom-
inantly against this hormone and had lim-
ited cross-reactivity with human hLH (Vai-
tukaitis et al., 1972~; radioimmunoassays
were developed that routinely detected
hCG at 1 ng/ml in serum in the presence of
circulating hLH. Radioimmunoassays with
urine specimens were more complex, because
of interfering substances and the presence
FIGURE 15-1 Antibodies with specificity to ~ and B
subunits of hCG. Shaded areas indicate regions that
give rise to antibodies. Source: Canf~eld et al., 1987.
FEAL9LE REPRODUCTIVE TOXICOLOaY
of degradation products. Nonetheless,
this assay method was used to develop non-
radioisotopic home-testing kits for hCG
in urine specimens to detect pregnancy
with moderate reliability (Doshi, 1986)
during the first 9 days after the first
missed menstrual period.
Two developments led to new hog assays
with improved sensitivity and specifici-
ty. First, monoclonal antibodies against
the hCG ~ subunit could be produced, so that
urine could be extracted efficiently by
immunoaffinity adsorption. Second, pre-
parations of the unique ,B COOH-terminal
peptide region of hCG could be used as im-
munogens to make high-affinity polyclonal
antibodies (Birken et al., l 982~.
Several laboratories have made prepara-
tions of antibodies that recognize hCG
and hLH to varied degrees (c.f., Vaitukai-
tis et al., 1972; Birken et al., 1982; Thau
et al., 1983; Ehrlich et al., 1985~. Of
particular interest are antibodies that
exhibit a high degree of specificity for
hCG, that is, have very low reactivity with
hLH.
Four separate regions in hCG give rise
to antibodies that may be used in assays
with selectivity for hCG over hLH (Fig.
15- 1). Each region presumably reflects
a conformational change resulting from
structural differences.
Region IV represents an antigenic deter-
minant (epitope) that is not readily reac-
tive in native hCG, but is present on the
free ,B subunit and on forms of the degraded
hCG' subunit excreted in human urine. That
region has been most recently identified
and affords the opportunity to distinguish
B20 1
B202 \
,:5 J Bl03
/ a
A 109' /~°
A102 H 1 B107
B I 09
~[B2 06
( \ P3W80
tB108
OCR for page 189
MARKERS FOR EPIDEMIOLOGIC STUDIES
between degraded hCG ~ subunit fragments
and the intact hormone. The complete
structure of the degraded ~ core fragment
has been determined, and monoclonal an-
tibodies that bind to Region IV have been
developed (Birken et al., 1988; Krichevsky
etal., 1988~.
The importance of the four discrete re-
gions of the hCG molecule is demonstrated
by immunoradiometric assays (sandwich
assays) that use two antibodies selected
on the basis of binding to different hCG
epitope regions. An early study found that
one such assay exhibited an affinity far
beyond that expected (Ehrlich et al.,
1982~. A mathematical model was developed
that accounted for that observation by
predicting formation of a circular complex
composed of one molecule of each antibody
and two molecules of antigen (Moyle et al.,
1983 a,b) (Fig. 15-2~.
With those immunochemical reagents,
a sensitive assay for hCG in urine was de-
veloped that used an immunoradiometric
assay (Wilcox et al., 1985~. The f-specif-
ic monoclonal antibody B 101 is coupled
to Sepharose beads, suspended in urine,
spun down; the hCG binds to the antibody.
To measure the amount of hCG captured, a
Fc
F(ab~/\\ F(ab)
189
i26I-labeled antibody from the rabbit
(R525) is mixed with the resuspended Seph-
arose-B 101 -hCG mixture. When this is spun
down, amounts of radioactivity bound to
the solid phase indicate quantities of
hCG attached to the capture antibody. The
assay permits hCG detection at concentra-
tions approaching 0.01 ng/ml (highly puri-
fied reference preparations of hCG have
a bioassay value of approximately 13,000
IU/mg) and is 50-100 times more sensitive
than any other existing method (Armstrong
et al., 1984)(Fig. 15-3).
When a conventional radioimmunoassay
is performed on a urine specimen, contami-
nating proteolytic enzymes in the urine
can degrade the radiolabeled tracer
during its incubation. This proteolytic
artifact might lead to falsely increased
measurements of hCG (Maruo et al., 1979~.
In addition, other interfering urinary
substances can add to the background noise
and decrease the signal-to-noise ratio
(Ayala et al., 1978~. In the immunoradio-
metric assay, the radioactive tracer is
introduced after proteases and interfer-
ing substances have been washed away. SB-
6 radioimmunoassay in urine displays poor
signal-to-noise ratio with wide variation
FIGURE 15-2 High-af~mity circular complex that is formed in a
A n t i b 0 d y "sandwich" assay. Source: Moyle et al., 1983b.
B 1 02
h C G ~ ~) ()~
Im ~ ) 1 ~
~ a
F(ab) ~
1 a ~
\\//
~//
// F(ab)
~ ,—
Fc Antibody
_ B I O I
\ hCG
OCR for page 190
190
Figure 15-3 A. Schematic diagram of procedure
for ~mmunoradiometr~c assay of hCG In unne;
B. Sensitivity of hCG detection by ~mmunoradim
metric assay. Source: Canfield et al., 1987.
10,000 -
o
at
m 6000-
:E
between 1 and 10 ng/ml (Wilcox et al.,
1985~. This contrasts with data from im-
munoradiometric assay of same specimens,
which displays greater sensitivity for
hCG without widely fluctuating baseline.
Those factors might have contributed to
the differing results (8% to 57%) obtained
in prior studies of fetal loss (Miller et
al., 1980; Edmonds et al., 1982; Whittaker
etal., 1983~.
FIELD STUDIES OF EARLY FETAL
LOSS: TESTING THE UTILITY OF
THE hCG ASSAY
A small trial was conducted to ascertain
the feasibility of the study design and
whether new assays with improved sensitiv-
ity for hCG in urine would yield new data;
specimens also were analyzed with earlier
methods (Wehmann et al., 1981).
FEMALE REPRODUCTIVE TOXICOLOGY
A
~ ,
in,
I m '
Lo
CO
-I<
~ +
HOG
I ) Wash to remove / 2) Incubate with rediolabel.d
int~rtering substance: BCTP specific antibody
~.
Washing and
~ centritugation
ANTIGEN ANTIBODY
"SANDWICH" ~
B
2000
Ouantitation
of R525.
a/
it
HCG (ngJml)/ · Urine
,6/ ° But ~ c,
·'
to
HL~ (~g/ml )
Nonspeclf ic Blndlng
001 010 1 0
CONCENTRAT ION
Paid volunteers in the field study col-
lected approximately 1 oz of urine each
morning and stored the containers frozen
until the containers were picked up for
assay (Wilcox et al., 1985). Three studies
testing the utility of these assays have
been completed. In the first study, 30
women collected daily urine specimens
from the time they stopped contraception
until they became pregnant or for 6 months
if no pregnancy occurred (Wilcox et al.,
1985~. The study showed the feasibility
of the epidemiologic design and permitted
investigators to detect several cycles
with early fetal loss that would not have
been detected with other available meth-
ods. The second (control) group comprised
women who had undergone tubal ligation;
no patterns suggestive of pregnancy were
seen. The study was performed blindly.
OCR for page 191
MARKERS FOR EPIDEMIOLOGIC STUDIES
In the third study, women using intra-
uterine devices for contraception were
evaluated to determine whether this con-
traceptive technique prevented implanta-
tion of the fertilized ovum. Urinary hCG
was detected in only 1 of 107 menstrual
cycles in this study. Wilcox et al. (1987a)
concluded that implantation is infrequent
among IUD users; the single pregnancy loss
detected might have been a tubal implanta-
tion.
Figure 15-4 illustrates the types of
findings that can be obtained in field
studies. Three consecutive menstrual
cycles are illustrated in which daily
urine specimens were collected from one
person during the second half of each cy-
cle. During the first cycle, no conception
occurred, and all hCG values were less than
0.01 ng/ml. During the second cycle, 3
weeks after the onset of the prior menstru-
al period, urinary hCG rose for 6 days to
0.38 ng/ml. hCG steadily declined over
the next 6 days and became undetectable.
With the decline of hCG came the onset of
menses-27 days after the onset of the pre-
vious menses. The woman was unaware of her
pregnancy or the episode of early fetal
loss. However, the finding of 11 consecu-
tive increases in urinary hCG above the
normal background for that woman and the
pattern of rise and fall leave little doubt
10
HUMAN 1
CHORIONIC
GONADOTROPIN
(ng/ml) 0 1
191
that a loss occurred. Three weeks after
the apparent fetal loss, the woman again
exhibited increasing urinary hCG. This
time, she remained pregnant and delivered
a normal, full-term infant (Wilcox et al.,
1985~.
FUTURE ASSAY DEVELOPMENTS
Tests for urinary hCG have evolved
from relatively insensitive bioassays
to sensitive and specific immunoassays.
The ectopic secretion of hCG by some tumors
and its slight increase in postmenopausal
urine specimens (Armstrong et al., 1984;
Kuida et al., 1988) are unlikely to dimin-
ish its epidemiologic value as the
biologic marker of pregnancy in healthy
women of reproductive age because the char-
acter~st~c pregnancy-related changes
in hCG concentration are not observed from
tumor secretions. Other proteins that
appear to be pregnancy-specific have
shown less utility as markers in epidemio-
logic studies; furthermore, they are usu-
ally assayed in blood specimens, and their
fate in urine is less well known. (An ex-
tended discussion of markers of early preg-
nancy is given in the Chapter 20.)
Field trials involving regular hCG test-
ing in women attempting to conceive have
proved adaptable to large-scale epidemio-
. . . . . ~
. . .
. . .
. . .
. . .
. . .
..... ~
. . ...
.. ...
. . - .
. . . .
..... i.
..... _
..... _
..... r
. . . . I
. . .
. . .
. . . .
. . . . I
. . . .. I
..... 1
..... ~
..... ~
~222"'"' ~
l 2 V"".2 I
I ~ (~ by
1 :: \ ; 1
. . · . I
I
/ · · · ~ .
1~ 1 ~ ~ ~ ~ I I
5 6 7 8
WEEKS
(BARS INDICATE MENSES)
9 10 11
FIGURE 15~ hCG in urine during three consecutive menstrual cycles in human female unaware of pregnancy
or earn fetal loss. Source: Wilcox et al., 1987b.
OCR for page 192
192
logic studies. Within a few days of the
expected date of implantation of a fertil-
ized ovum, hCG can be detected, as it can
at least 2 days before the onset of the next
expected menstrual period (Wilcox et al.,
1985~. Early fetal loss has been detected
by urinary hCG testing; this requires an
hCG assay that is sensitive to a concentra-
tion lower than 0.05 ng/ml.
Much more needs to be done to advance
methods in this field of reproductive biol-
ogy, if large-scale epidemiologic studies
are to be undertaken to search for environ-
mental factors that alter the rate of early
fetal loss. Any change in the rate of clini-
cally apparent abortions potentially
caused by environmental factors can be
detected readily by routine hCG testing
and by the clinical events that surround
abortion.
Improved Methods for hCG Detection
To expand the present labor-intensive
and time-consuming research methods for
immunoradiometric measurement of hCG,
several advances would be desirable.
A simpler assay method is needed that
preserves sensitivity and specificity
for hCG while reducing the needs for labor,
large urine specimens, and large quanti-
ties of antibodies. These advances
should be made in the direction of a non-
radioactive-assay format. Ideally,
many specimens could be processed accu-
rately, rapidly, and inexpensively in the
laboratories shortly after urine collec-
tion. That would minimize the need for
extensive deep-freeze storage space,
because more than 90% of the specimens have
negative results and would be discarded.
Only urine specimens with positive screen-
ing tests that suggest an episode of fetal
loss would be shipped to a central assay
laboratory.
New Monoclonal Antibodies for hCG
Detection
In the quest for an improved assay for
the urinary products of hCG, new antibod-
ies with high affinities for the various
forms of urinary hCG will be important.
These should be monoclonal antibodies,
FEMALE REPRODUCTIVE TOXICOLOGY
to ensure a continuous source of complete-
ly characterized immunochemical rea-
gents. It is important to be able to char-
acterize and detect not only the entire
hormone, but also its degraded forms in
urine; to maximize sensitivity; and to
ensure that unusual biologic events, such
as a selective decrease in the secretion
of one of the subunits, do not lead to in-
complete or inaccurate results. New and
specific monoclonal antibody cell lines
might be required to accomplish this task.
Nonradioactive Methods for hCG
Detection
Many of the most sensitive research im-
munoassays use radioactive iodine, but
this approach has disadvantages, includ-
~ng:
· Radiolabeled reagents must be freshly
prepared and characterized every few weeks
and then shipped to the users.
· An ever-increasing problem with ra-
dioactive waste disposal is of concern.
· Use of radioactive methods requires
special training and surveillance in the
laboratory.
Epidemiologic research related to
early fetal loss would be advanced substan-
tially if a nonradioactive method to meas-
ure hCG were devised in which the reagents
had a shelf-life of 6 months to 1 year. The
most widely used nonradioactive method
is enzyme-linked immunosorbent assay
(ELISA), which requires only basic instru-
mentation (spectrophotometry) for quan-
tification. The sensitivity of the assay
depends on the enzyme amount and activity
that can be specifically linked to the
antigen or antibody measured.
Several recent advances have been intro-
duced to amplify the signal and increase
sensitivity, including enzyme-antibody
coupling through the avidin-biotin system
(Fuccillo, 1985), lectin-carbohydrate
coupling, and use of chimera antibodies
of double specificity (Guesdon et al.,
1983~. Those techniques involve noncova-
lent linkage and therefore avoid the loss
of enzyme catalytic activity due to chemi-
cal modification.
i
OCR for page 193
MARKERS FOR EPIDEMIOLOGIC STUDIES
Assays based on fluorescence or phos-
phorescence depend on light absorption
to provide the excitation energy to produce
the emission. Assays based on chemilumine-
scence depend on the energy from an oxida-
tive chemical reaction to produce mole-
cules in an electronically excited state;
return to the ground state is accompanied
by photon emission, which is detected pho-
tometrically. Low quantum yields and high
background interference limit the sen-
sitivity of chemiluminescent, fluore-
scent, and phosphorescent techniques.
To avoid those problems, time-resolved
fluorometric immunoassay has been devel-
oped. Lanthanide chelates have a high
quantum yield and a large Stokes shift
(340-nm excitation wavelength and 614-
nm emission wavelength). In addition
to a ion' decay time for europium chelates
( 103- 10 ns), these properties permit an
assay wherein a pulsed light of short dura-
tion (compared with the decay time of the
lanthanide chelates) is transmitted to
the sample, and the interfering rapid
decay fluorescence of other serum or uri-
nary constituents is discriminated
against by activating the detection system
after a delay sufficient to complete the
decay of naturally occurring fluoro-
phores.
Those techniques and others have the
advantage of a nonradioactive detection
system and increased reagent stability.
Further investigation is required to de-
termine which approaches would provide
the most satisfactory sensitivity and
specificity, which until the present have
been hallmarks of radioisotope - based
technology.
Requirement to Measure Other Biologic
Markers
Urinary hCG is a valuable biologic marker
of implantation of a fertilized ovum,
but it does not detect a fertilized ovum
that does not implant. New methods to test
for that should be developed. One approach
would be a technique to collect uterine
secretions and assay for the presence
of hCG secreted by the nonimplanted ovum.
Another approach involves developing
adequate methods for the assay of other
193
pregnancy markers that do not depend on
implantation. Although a wide variety
of pregnancy-associated proteins have
been described (Home and Nisbet, 1979),
most appear to be associated with the later
stages of pregnancy development. One ex-
ception is the pregnancy-specific protein
early pregnancy factor (EPF) (Morton et
al., 1977~. EPE properties have been stud-
ied in humans (Tinneberg et al., 1985),
sheep (Morton et al., 1979) and mice
(Morton et al., 1976), mainly by the ro-
sette-inhibition assay.
~ .,
Its appearance
can be detected in maternal serum within
6-48 hours after fertilization and does
not depend on implantation of the fertil-
ized ovum for detection (Sinosich et al.,
1985~. EPF persists in serum throughout
the first two trimesters of pregnancy (Ca-
vanagh et al., 1982) and rapidly disappears
after embryo death or surgical removal
(Nancarrow et al., 1979~.
In a study of 13 multiparous women
(Rolfe, 1982), EPF was detected within
48 hours of fertilization (ovulation
dated by progesterone determination).
Of cycles studied in which intercourse
occurred at the time of ovulation, EPF
was detected in 18 of 28 cycles, but con-
tinued to be produced beyond 2 weeks in 4
instances; of these 4 subjects, only 2
proceeded to full-term pregnancy. In
the remaining 14 of the 18 subjects, EPF
became undetectable before the onset of
the next menstrual cycle. The data suggest
that many fertilized ova are lost before
implantation. An assay less cumbersome
and more sensitive than the rosette-in-
hibition test should be investigated.
EPF should be thoroughly elucidated, and
the possible presence and assay of EPF or
its metabolites in urine investigated.
In epidemiologic studies of reproduc-
tive function, improved markers of ovula-
tion need to be developed in easily col-
lected biologic specimens—saliva or
urine. These improvements are needed to
develop better methods to detect the vari-
ous forms of the pituitary gonadotropins
and also to detect various estrogen and
progesterone derivatives throughout the
menstrual cycle, to estimate the time of
ovulation, and to document the formation
of a corpus luteum.
OCR for page 194
194
The requirement for new high-affinity
antisera directed against hLH and FSH is
based on the heterogeneity of the glycopro-
tein hormones from the pituitary (Franchi-
mont et al., 1972~. Isoelectric focusing
data show that hLH from regularly cycling
women is less acidic than that circulating
in men and postmenopausal women, pre-
sumably owing to a lower sialic acid con-
tent (Wide, 1981~. Differences in sialic
acid content are manifested not only in
physicochemical properties, but also in
immunoreactivity, receptor affinity,
and biologic activity, even to the extent
of having a variant of hLH that is immuno-
logically reactive but devoid of biologic
effect (Axelrod et al., 1979~. Multiple
forms of FSH are also observed in other
mammals, with as many as six immunoreactive
forms present in the hamster (Ullsa-
Aguirre and Chappel, 1982~. The different
forms have the same molecular weight, but
exhibit different affinities in lectin
binding and have different bioactivity-
to-immunoreactivity ratios when tested
by radioreceptor assay.
Those findings stress the importance
of developing assays that measure the per-
tinent forms of excreted pituitary gonado-
tropins. Assays that- use antisera raised
against antigens derived from postmeno-
pausal or male sources might not be optimal
for assays on normally cycling women and
might have contributed to the primary lack
of sensitivity in ovulation detection.
Recent work documented that urinary FSH
patterns closely resemble those found in
serum FSH patterns when measured by granu-
losa cell aromatase bioassay (Dahl et al.,
1987~. Future work in gonadotropin-assay
development should include assays for the
intact hormone and its subunits, as well
as determination of the correlation be-
tween the immunoreactivity and bioactivi-
ty of the substances being measured (e.g.,
gonadotropins isolated from normally
cycling women), to monitor the validity
of the assay.
Two objectives are met by measuring cir-
culating steroids or their metabolites.
The first is to signal the onset of ovula-
tion and thereby validate the accuracy
of gonadotropin assays as an ovulation
marker. The second is to assess the exis-
FEA~ll;"E REPRODUCTIVE TOXICOLC)GY
fence and adequacy of the corpus luteum.
A variety of urinary estrogen metabolites
have been assayed and their utility as
markers of ovulation evaluated. Baker
and colleagues ( 1979) assayed directly
El-3-glucuronide, E2-3-glucuronide, E2-
1 7,8-glucuronide, E5-3-glucuronide, and
Es- 1 6a-glucuronide in urine and found that
the E2-17,6-glucuronide assay was the most
sensitive predictor of ovulation, fol-
lowed closely by E,-3-glucuronide. The
authors recommended the latter, because
5 times more of it is excreted than of the
former and it can be detected in a 100-fold
diluted urine specimen, in which poten-
tially interfering substances would be
diluted virtually to nonexistence.
The Baker et al. study (1979) also indi-
cated that random urine collections can
be valid ovulation markers. Dividing
the mass of steroid metabolite by the exact
duration of collection gives the produc-
tion rate in nanomoles per hour, and com-
parison of this pattern for first-morning
voids and 24-hour collections showed ex-
cellent correlation. Results expressed
as the steroid:creatinine ratio also cor-
related highly with the 24-hour collection
results. Thus, either method may be used
in lieu of 24-hour collections. The E,-
3-glucuronide:pregnanediol 3a-glucuro-
nide ratio is independent of urine volume
and proved to be another valid ovulation
marker.
In a study of the urinary E~-3-glucuro-
nide, hLH, and pregnanediol 3~-glucuro-
nide as markers of ovarian function, Col-
lins et al. (1979) found a good correlation
between early-morning collection and 24-
hour collections and found that the ratio
of E~-3-glucuronide to pregnanediol 3a-
glucuronide could be used to demarcate
the duration of the fertile period.
A multicenter study (WHO, 1 980b)
evaluated the excretion pattern of E~-
3-glucuronide, E2- 1 7,8-glucuronide, E2-
3-glucuronide, E~- 1 6~-glucuronide, E~-
3-glucuronide, pregnanediol 3~-glucuron-
ide, and pregnanetriol 3~-glucuronide
throughout the menstrual cycle, to deter-
mine which best indicated the fertile peri-
od during the cycle; E~-3-glucuronide in
the follicular phase provided a marker
of ovulation 72 hours before ovulation,
OCR for page 195
MARKERS FOR EPIDEMIOLOGIC STUDIES
and assay of pregnanediol 3~-glucuronide
was the best indicator of whether ovulation
had occurred.
The WHO study (1980b) also confirmed
that first-morning voids yielded results
as reliable as those obtained from 24-
hour collections.
OTHER CLINICAL OPPORTUNITIES
If research is limited to young and
otherwise normal women, unusual patterns
might be missed. However, women with ab-
normal physiology enrolled in an epidemio-
logic study can lead to unexpected and
inexplicable findings. Thus a wide spec-
trum of clinical studies is important
for epidemiologic research.
Fecundity decreases with age in women.
The rate of early fetal loss might differ
between younger women and older women,
and enrollment of older women in a study
might affect the outcome; but no adequate
data exist on the rate of early fetal loss
in women older than 35 or 40 years.
Effects of different types of prior con-
traceptive use such as steroidal agents,
IUDs, and spermicides should be studied.
Furthermore, not all women have regular
menstrual periods, and those with short
and long luteal phases should be studied
to determine, for example, how this affects
the rate of fetal loss and timing of sneci-
men collection to detect implantation and
loss. The study of luteal phase charac-
teristics has become feasible with the
development of direct assays for urinary
estrogen and progestin metabolites using
nonradioisotopic assay systems.
Postmenopausal women have a strong stim-
ulus for pituitary gonadotropin secre-
tion; to some extent, this appears to stim-
ulate a minute degree of hCG synthesis and
195
secretion as well (Robertson et al., 1978;
Armstrong et al., 1984~. Important ques-
tions to study are whether this might be
a problem when studying perimenopausal
women who conceive, whether this is a pat-
tern of hCG, and whether the degree of ovar-
ian failure that leads to a slight amount
of hCG secretion occurs only when a woman
becomes infertile.
The National Center for Health Statis-
tics reports that 14% of couples in this
country have a problem with infertility.
Some men or women might have a genetic pre-
disposition to a high rate of early fetal
loss or failure of the fertilized ovum to
implant. Artificial insemination pro-
grams provide a fine opportunity to study
this problem. It is also important to de-
termine whether frozen and fresh semen
specimens lead to different rates of con-
ception and fetal loss. Although several
studies have indicated that the use of
frozen semen leads to diminished fecun-
dability, at least one study reports that
there is no difference in pregnancy rate
whether fresh or frozen semen is employed
(Trounson et al., 1980~.
Epidemiologic research provides an
opportunity to identify persons with un-
usual reproductive patterns that might
occur with low frequency in the population.
Protocols should be designed that test
the major epidemiologic hypotheses and
also are sensitive to the occurrence of
such unusual patterns.
The research described above will in-
crease the knowledge regarding the use
of hCG as a marker of pregnancy and early
fetal loss; that increased knowledge
will benefit not only epidemiologic stud-
ies, but also other research to detect
abnormalities of human fertility.
OCR for page 196
Representative terms from entire chapter:
urinary hcg
