Placebo-Controlled Trial of Indole-3-Carbinol in the Treatment of CIN

Abstract

Most precancerous lesions of the cervix are treated with surgery or ablative therapy. Chemoprevention, using natural and synthetic compounds, may intervene in the early precancerous stages of carcinogenesis and prevent the development of invasive disease. Our trial used indole-3-carbinol (I-3-C) administered orally to treat women with CIN as a therapeutic for cervical CIN.
Thirty patients with biopsy proven CIN II-III were randomized to receive placebo or 200, or 400 mg/day I-3-C administered orally for 12 weeks. If persistent CIN was diagnosed by cervical biopsy at the end of the trial, loop electrocautery excision procedure of the transformation zone was performed. HPV status was assessed in all patients.
None (0 of 10) of the patients in the placebo group had complete regression of CIN. In contrast 4 of 8 patients in the 200 mg/day arm and 4 of 9 patients in the 400 mg/day arm had complete regression based on their 12-week biopsy. This protective effect of I-3-C is shown by a relative risk (RR) of 0.50 ((95% CI, 0. 25 to 0.99) P = 0.023) for the 200 mg/day group and a RR of 0.55 ((95% CI, 0.31 to 0.99) P = 0.032) for the 400 mg/day group. HPV was detected in 7 of 10 placebo patients, in 7 of 8 in the 200 mg/day group, and in 8 of 9 in the 400 mg/day group.
There was a statistically significant regression of CIN in patients treated with I-3-C orally compared with placebo. The 2/16 alpha-hydroxyestrone ratio changed in a dose-dependent fashion.

Full text

Placebo-Controlled Trial of Indole-3-Carbinol in the Treatment of CIN
Maria C. Bell,* Peg Crowley-Nowick,† H. Leon Bradlow,‡ Daniel W. Sepkovic,‡ Delf Schmidt-Grimminger,*
Patti Howell,* E. J. Mayeaux,* Angela Tucker,* Elba A. Turbat-Herrera,* and J. Michael Mathis*
*Department of Obstetrics and Gynecology, Louisiana State University Medical Center–Shreveport, 1501 Kings Highway, Shreveport,
Louisiana 71130–3932; †Department of Obstetrics and Gynecology, Harvard Medical School, Fearing Laboratory, P.O. Box 701, Boston,
Massachusetts 02081; and ‡Strang Cancer Research Laboratory, Murray Rayburn Laboratory for Biochemical Endocrinology,
Box 231, The Rockefeller University, 1239 York Avenue, New York, New York 10021
Received September 14, 1999
Objective. Most precancerous lesions of the cervix are treated
with surgery or ablative therapy. Chemoprevention, using natural
and synthetic compounds, may intervene in the early precancerous
stages of carcinogenesis and prevent the development of invasive
disease. Our trial used indole-3-carbinol (I-3-C) administered
orally to treat women with CIN as a therapeutic for cervical CIN.
Methods. Thirty patients with biopsy proven CIN II–III were
randomized to receive placebo or 200, or 400 mg/day I-3-C ad-
ministered orally for 12 weeks. If persistent CIN was diagnosed by
cervical biopsy at the end of the trial, loop electrocautery excision
procedure of the transformation zone was performed. HPV status
was assessed in all patients.
Results. None (0 of 10) of the patients in the placebo group had
complete regression of CIN. In contrast 4 of 8 patients in the 200
mg/day arm and 4 of 9 patients in the 400 mg/day arm had
complete regression based on their 12-week biopsy. This protective
effect of I-3-C is shown by a relative risk (RR) of 0.50 ((95% CI,
0.25 to 0.99) Pⴝ0.023) for the 200 mg/day group and a RR of 0.55
((95% CI, 0.31 to 0.99) Pⴝ0.032) for the 400 mg/day group. HPV
was detected in 7 of 10 placebo patients, in 7 of 8 in the 200 mg/day
group, and in 8 of 9 in the 400 mg/day group.
Conclusions. There was a statistically significant regression of
CIN in patients treated with I-3-C orally compared with placebo.
The 2/16
␣
-hydroxyestrone ratio changed in a dose-dependent
fashion. © 2000 Academic Press
Key Words: placebo; indoles; dysplasia.
BACKGROUND
Chemoprevention is the attempt to use natural and synthetic
compounds to intervene in the early precancerous stages of
carcinogenesis, before invasive disease begins [1–4]. Cervical
cancer is ideally suited to chemopreventive efforts given its
well-characterized preinvasive state, its multistep progression
to invasive disease, and the clinical ease with which the clini-
cian can follow the lesion without significantly invasive tech-
niques.
Many families of fruits and vegetables contain relatively
high concentrations of unique phytochemicals with potential
cancer-preventing properties. Diets high in cruciferous vege-
tables (e.g., broccoli, cabbage, cauliflower, bok choy, and
brussels sprouts) can retard cancer growth in animals and
increased consumption of these vegetables by humans is asso-
ciated with decreased cancers of the colon, lung, and breast [5].
A specific compound that has been identified in crucifers,
indole-3-carbinol (I-3-C), may be able to prevent or halt car-
cinogenesis [6].
Clinical success has been achieved in the treatment of hu-
man-papillomavirus-induced lesions of the larynx using in-
dole-3-carbinol [7, 8], and I-3-C has been shown to prevent the
development of cervical and vaginal cancers in HPV 16 trans-
genic mice [9]. Although extrapolation of data found in trans-
genic mice has its limitations, these data provide additional
information for the rationale of using I-3-C in the treatment of
cervical dysplasia. Indole-3-carbinol has been shown to
strongly induce the activity of an enzyme found in the liver in
humans and other mammals, called P450 1A1/1A2 (CYP1A1/
1A2) and, to a lesser extent, other p450s [10–12]. This group
of enzymes is known to detoxify and/or metabolize a variety of
chemical substances known to be carcinogenic, and this mech-
anism may explain its cancer-preventive activity in animals as
well as humans [13].
Indole-3-carbinol, through its action on cytochrome P-450
1A1/1A2, is known to alter the pathway of estrogen metabo-
lism in human males and females in a manner that decreases
the risk of certain tumors [13–15]. Metabolic degradation of
estradiol by liver cells results primarily in either 2-hy-
droxyestrone or 16
␣
-hydroxyestrone, and to a lesser extent
4-hydroxyestrone—a potent carcinogen. It is known that 16-
␣
-hydroxyestrone causes proliferation of some breast tumor
cell lines [12], while the alternative metabolite, 2-hy-
droxyestrone, is anti-estrogenic and anti-proliferative [13, 16].
Presented at the 30th Annual Meeting of the Society of Gynecologic
Oncologists, San Francisco, CA, March 20–24, 1999.
Gynecologic Oncology 78, 123–129 (2000)
doi:10.1006/gyno.2000.5847, available online at http://www.idealibrary.com on
123 0090-8258/00 $35.00
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.

Likewise, it has been shown that I-3-C abrogates the prolifer-
ative effect of estradiol on human laryngeal cells in culture and
decreases the development of papillomas in HPV-infected
grafts in immunocompromised mice [7]. Women with
CIN II/III have lower estrogen metabolite ratios than normal
women [17].
Estradiol is normally metabolized to 16
␣
-hydroxyestrone,
2-hydroxyestrone, and, to a lesser extent, 4-hydroxyestrone.
The decreased ratio of these metabolites (i.e., elevated 16
␣
-
hydroxyestrone) is thought to be tumorigenic and, importantly,
16
␣
-hydroxyestrone is elevated in breast cancer [12]. 16
␣
-
Hydroxyestrone also stimulates cell proliferation and anchor-
age-independent growth [13]. 2-Hydroxyestrone, however,
has less activity than estradiol in inducing anchorage-
independent growth and seems to have an anti-estrogenic
effect. 4-Hydroxyestrone is a known carcinogen of consid-
erable potency [16].
While tumor-associated viruses can profoundly affect the
2-hydroxyestrone to 16
␣
-hydroxyestrone ratio, attempts to di-
rectly decrease 16
␣
-hydroxylation have not proved effective.
However, by upregulating 2-hydroxylation, estradiol is metab-
olized to a benign product at the expense of 16
␣
-hydroxyla-
tion. Although several compounds are capable of upregulating
2-hydroxylation, Niwa et al. found that I-3-C was the most
potent of these compounds [18]. I-3-C also suppresses 4-hy-
droxylation [19].
Since laryngeal papillomatosis (an HPV-induced disease)
has been effectively treated with indole-3-carbinol, we hypoth-
esized that indole-3-carbinol may be effective in the treatment
of cervical dysplasia—another HPV-induced disease. In this
study, our goal was to use indole-3-carbinol in capsule form to
treat women with CIN, most of which are human-papilloma-
virus-induced.
MATERIALS AND METHODS
Patient Eligibility
Thirty subjects with histologically confirmed CIN (Table 1)
were enrolled. The study protocol was reviewed and approved
for human research subjects by the Institutional Research
Board for Human Research of LSU Medical Center–Shreve-
port. Because indole-3-carbinol was available in health food
stores prior to October 1994, a FDA IND number was not
required. Eligibility requirements were as follows: adequate
colposcopy, negative ECC, no suspicion of invasion, nonpreg-
nant, and HIV⫺. There was not a “size” requirment for the
dysplastic lesion prior to entry.
Evaluating colposcopists and patients were blinded to the
treatment. All residents and staff involved in the study were
instructed regarding the protocol. Typically second, third, and
fourth year ob/gyn residents staff the colposcopy clinic with
four different attending physicians supervising. The patients
were randomized to placebo or 200 or 400 mg/day treatment of
indole-3-carbinol administered orally for 12 weeks. Random-
ization was performed by successively assigning patients from
a computer-generated randomization table. Ten patients were
randomized to each group; however, 2 patients in the 200
mg/day treatment arm and 1 patient in the 400 mg/day treat-
ment arm did not complete the study. These patients were
randomized, but did not return for their appointment to receive
the medication and were excluded from analysis.
Patient Evaluation
All patients underwent colposcopy at the initial visit as well
as their 4-, 8-, and 12-week visits. Photodocumentation of each
colposcopic examination was obtained. At the end of the trial,
each patient was reexamined, and if the patient had persistent
CIN on 12-week biopsy or if the colposcopic impression was
persistent CIN, she underwent loop electrocautery excision
procedure of the transformation zone. Patients were asked at
each visit about side effects and compliance. Patients returned
remaining pills at the end of the trial for assessment of remain-
ing unused medication.
Pathology
The dysplasia was classified by the pathologists in the usual
manner and defined as follows: mild dysplasia meaning the
presence of immature (dysplastic) cells present in the lower
third of the squamous mucosa, moderate dysplasia in the lower
two-thirds, severe dysplasia the lower three-fourths of the
thickness of the mucosa, and carcinoma in situ full-thickness
immaturity. The cases were originally diagnosed by a sign-out
pathologist. The diagnosis of all cases was verified by the same
pathologist (ETH) for uniformity. In all cases, the pathologist
(ETH) was in agreement with the pathologist that had signed
out the case.
Indole-3-Carbinol Assay
In order to assess the purity of the indole-3-carbinol used in
this study, high-performance liquid chromatography was per-
formed on the bottles labeled “product X,” which was the
indole-3-carbinol, and on the bottles labeled “product Y,”
which was the placebo. Each of the product X capsules con-
tained slightly more than 100 mg of the indole-3-carbinol per
capsule, and no indole-3-carbinol was present in the product Y
capsule. Design Nutritional Products (Orem, UT), provided the
indole-3-carbinol capsules and placebo capsules.
HPV Analysis
HPV status was assessed in all patients by Hybrid capture
assay tests (Digene Diagnostics, Beltsville, MD) for low risk
(HPV types 6, 11, 42, 43, 44) and high risk (HPV types 16, 18,
124 BELL ET AL.

31, 33, 35, 45, 51, 52, 56). If the Hybrid capture assay was
negative, a follow-up polymerase chain reaction (PCR) analy-
sis was performed using consensus primers shown below for
the L-1 region of HPV to confirm HPV positivity.
Primer 1 (MY09) sequence: 5⬘-CGTCCMARRGGAWAC-
TGATC-3⬘
Primer 2 (MY11) sequence: 5⬘-GCMCAGGGWCATA-
AYAATGG-3⬘
The PCR amplification conditions were optimized using DNA
isolated from paraffin-embedded control HPV-positive cervi-
cal cancer specimens prior to analysis of the study patient’s
samples.
Estradiol Metabolism
Clean catch urine specimens were obtained from each pa-
tient at the initial visit and again at their 4-week visit. 2-Hy-
droxyestrone to 16-
␣
-hydrosytestrone ratios were assessed in
each urine sample. Ascorbic acid was added to the urine
specimens to aid in preservation for accurate analysis. ELISA
assays were performed to assess the quantity of 2-hy-
droxyestrone and 16-
␣
-hydroxyestrone in each urine sample
and were reported in nanograms per milliliter [20–22]. All
specimens were shipped to the Strang Cancer Research Labo-
ratory for analysis in the laboratory of Dr. H. Leon Bradlow.
Statistical Analysis
The placebo group was compared to each treatment group
using Fisher’s exact test with two-tailed Pvalues. Statistical
significance was defined as P⬍0.05. Taylor 95% confidence
intervals were computed for relative risks. The statistical pack-
age used was Trus Epistat (Richardson, TX).
TABLE 1
Dose, Diagnosis, and HPV Status
Patient (No.)
dose I-3-C
(mg/day) Diagnosisa
(week 1) Diagnosisa
(week 12) Low-riskbHPV High-riskbHPV HPV PCR
1. Placebo CIN II CIN I Pos Pos
2. Placebo CIN II CIN I Neg Neg Neg
3. Placebo CIN II CIN I Neg Pos
4. Placebo CIN III CIN I Neg Pos
5. Placebo CIN III CIN I Neg Neg Pos
6. Placebo CIN II CIN I Neg Neg
7. Placebo CIN II CIN I Neg Neg
8. Placebo CIN III CIN III Neg Pos
9. Placebo CIN III CIN I Neg Pos
10. Placebo CIN III CIN III Neg Pos
11. 400 CIN II CIN I Neg Neg Pos
12. 400 CIN II CIN I Neg Pos Pos
13. 400 CIN III CIN I Neg Pos
14. 400 CIN II CIN I Neg Neg Neg
15. 400 CIN II Normal Neg Neg Pos
16. 400 CIN III CIN I Neg Pos Pos
17. 400 CIN II Normal Neg Neg Pos
18. 400 CIN III Normal Neg Neg Pos
19. 400 CIN II Normal Neg Pos
20. 200 CIN III CIN I Pos Pos
21. 200 CIN II Normal Neg Pos
22. 200 CIN III CIN III Neg Neg Neg
23. 200 CIN II Normal Neg Pos
24. 200 CIN III Normal Pos Pos
25. 200 CIN II CIN II Pos Pos
26. 200 CIN II Normal Neg Neg Pos
27. 200 CIN II CIN II Neg Pos
Note. mg/day, milligrams per day; HPV, human papillomavirus; LEEP, loop electrocautery excision procedure; CIN, cervical intraepithelial neoplasia; neg,
negative; pos, positive; PCR, polymerase chain reaction.
aDiagnosis by biopsy.
bHPV types 6, 11, 42, 43, 44.
cHPV types 16, 18, 31, 33, 35, 45, 51, 52, 56.
125INDOLE-3-CARBINOL IN THE TREATMENT OF CIN

RESULTS
In this study, we evaluated the efficacy of indole-3-carbinol
in 27 histologically confirmed cervical dysplasia patients (Ta-
ble 1). Routine pathologic evaluation of the study patients
showed that none (0 of 10) of the patients in the placebo group
had complete regression of their dysplasia as summarized in Table 1. In contrast, 50.0% (4 of 8) of patients in the 200
mg/day treatment arm and 44.4% (4 of 9) of patients in the 400
mg/day treatment arm had complete regression based on their
12-week biopsy.
In order to evaluate the degree of regression between the
treatment groups, the mean grade of CIN regression was com-
pared as shown in Fig. 1. A linear dose–response relationship
was noted among the placebo and the 200 and 400 mg/day
treatment groups. The mean grade change of CIN in the pla-
cebo group was 1.22, in the 200 mg/day treatment group 1.38,
and in the 400 mg/day treatment group 1.78. Fisher’s exact test
was used to make comparisons between groups and the signif-
icance level was defined as P⬍0.05. The protective effect of
I-3-C is shown by a relative risk (RR) of 0.50 ((95% CI, 0.25
to 0.99) P⫽0.023) for the 200 mg/day group and a RR of
0.55 ((95% CI, 0.31 to 0.99) P⫽0.032) for the 400 mg/day
group.
The urinary 2-hydroxyestrone to 16-
␣
-hydroxyestrone ratios
were evaluated in each patient at the initiation of the trial and
at 4 weeks in order to assess the physiologic response to
indole-3-carbinol (Table 2, Fig. 2). Although there was some
variability in the individual ratios, a general trend in increase in
the ratios was found in the 200 and 400 mg/day treatment
groups before and after therapy (Table 2). In contrast, the
placebo treatment group showed a decrease in the ratio before
and after therapy (Fig. 3).
To determine whether HPV infection alters the response to
indole-3-carbinol, HPV status was determined on all patients
(Table 1). In the placebo group, 7 of 10 patients had HPV
detected. Of these 7 placebo patients, 6 had high-risk HPV
TABLE 2
2-Hydroxyestrone/16
␣
-Hydroxyestrone Ratio versus Dose
Patient (No.) and
dose (mg/day) Ratio
pretherapy Ratio after
I-3-C Ratio
% change
1. Placebo 0.83 1.56 88
2. Placebo 1.09 1.38 26
3. Placebo 1.26 1.91 52
4. Placebo 0.65 0.79 21
5. Placebo 0.67 0.55 ⫺18
6. Placebo 0.57 0.64 12
7. Placebo 1.18 1.43 21
8. Placebo 3.48 0.36 ⫺89
9. Placebo 2.80 1.58 ⫺44
10. Placebo ND ND ND
11. 400 1.52 1.42 ⫺0.07
12. 400 1.31 0.67 ⫺48
13. 400 0.48 0.87 81
14. 400 2.44 2.85 17
15. 400 0.61 1.10 80
16. 400 1.18 3.43 190
17. 400 1.42 1.45 0.02
18. 400 1.13 1.51 33
19. 400 0.89 1.06 19
20. 200 0.92 1.08 17
21. 200 0.69 0.69 0
22. 200 0.29 0.47 62
23. 200 1.08 0.66 ⫺39
24. 200 0.55 1.37 149
25. 200 0.70 0.91 30
26. 200 0.90 1.30 44
27. 200 1.32 1.66 26
FIG. 1. Regression of CIN by dose.
FIG. 2. Change in 2-hydroxyestrone/16
␣
-hydroxyestrone ratio.
126 BELL ET AL.

subtypes (HPV 16 or 18) and 1 patient had HPV present by
PCR, but was not typed. In the 200 mg/day treatment group, 7
of 8 patients had detectable HPV and, of these, 6 had high-risk
subtypes and 1 patient had HPV present by PCR, but was not
typed. In the 400 mg/day treatment group, 8 of 9 patients had
detectable HPV, of which 4 were high-risk subtypes and 4
patients had HPV present by PCR, but were not typed. These
results show that the incidence of HPV was similar between all
FIG. 3. H&E staining of cervical biopsies before and after therapy. Representative sections of cervical biopsies before and after treatment with
indole-3-carbinol are shown. Note the persistent dysplasia in the placebo section at 12 weeks, but regression of CIN in the 200 and 400 mg/day sections.
127INDOLE-3-CARBINOL IN THE TREATMENT OF CIN

treatment groups and historical control, and suggest that pres-
ence of HPV does not correlate with response to indole-3-
carbinol.
DISCUSSION
HPV infection of the lower genital tract is the most common
sexually transmitted viral disease in the United States [23].
Most of these viral infections remain dormant and never result
in clinically evident disease. However, in some cases, the virus
may propagate and cause clinically recognizable HPV-associ-
ated changes including condylomata (genital warts), precan-
cerous lesions of the cervix, and invasive cervical cancer. The
association between HPV infection and genital cancer is well
documented [24, 25]. The progression of HPV infection to
genital cancer is not absolute, and other factors (e.g., smoking,
diet, and immunosuppression) probably contribute to the pro-
gression. In our study we found that the majority of patients
had HPV present in their cervix. Typing of HPV also demon-
strated a similar proportion of high-risk HPV subtypes in each
of our three treatment groups. However, it is unclear from our
data whether the presence of high-risk HPV is more or less
likely to respond to I-3-C since we did not type all of the PCR⫹
patients.
The primary mechanism by which I-3-C is hypothesized to
work is by altering the 2-hydroxyestrone/16
␣
-hydroxyestrone
ratio. We were able to demonstrate an increasing change in this
ratio among placebo and the 200 and 400 mg/day groups in a
dose-response fashion. Indole-3-carbinol, normally present in
cruciferous vegetables, has been administered to healthy hu-
man volunteers in short courses (3 months) in doses of 5–7
mg/kg (350–500 mg), which is equivalent to approximately
300–500 g of raw cabbage or brussels sprouts per day [11, 26].
Bradlow et al. assessed the effects of I-3-C administered to
healthy subjects versus placebo. In this study, multiple organ
systems were monitored including hematologic, renal, and
hepatic, and no adverse effects were noted in any of these
subjects at this dosage. In this study, the highest dose (400
mg/day) was chosen because it is equivalent to 1/3 of a head of
cabbage, an amount thought to be what most that people would
reasonably consume daily [27]. Anecdotal reports about chil-
dren taking I-3-C for suppression of their laryngeal papillomas
indicate that I-3-C can be administered for several years with-
out any toxicity. In our study no toxicity was observed in either
the 200 or the 400 mg/day group.
When looking at individual ratios and determining whether
a positive change in ratio would predict whether the patient
would have regression of her lesion, our data showed that six
of the eight patients who had regression demonstrated a posi-
tive change in their urinary 2-hydroxyestrone to 16
␣
-hy-
droxyestrone ratio. In contrast, five of the six patients who had
a negative change in their urinary 2-hydroxyestrone/16
␣
-hy-
droxyestrone ratio demonstrated persistent CIN. The fact that
not all the responders had a significant (⬎50%) increase in
their 2/16 ratio may be explained by the other ways in which
I-3-C has been reported to work. These alternate pathways
include induction of apoptosis [28], inhibition of microtubule
assembly [29], and decreased effect of NNK—a known to-
bacco carcinogen [30, 31]. In light of the fact that five of six of
our responders had a positive shift in their urinary 2-hy-
droxyestrone/16
␣
-hydroxyestrone ratio, the urinary 2-hy-
droxyestrone/16
␣
-hydroxyestrone ratio was considered a fea-
sible and simple method for monitoring of potential responses.
However, further studies are necessary to confirm this result.
Indole-3-carbinol may not induce a shift in estrogen metab-
olism in a subset of the general population. Some patients have
a CYP 1A1 genotype (MSP-1 polymorph), in which case the
administration of I-3-C would not be expected to shift the
2/16-hydroxyestrone ratio. We did not assay for the CYP 1A1
genotype in our patients. The CYP 1A1 genotype is present in
1–16% of the population, depending on their ethnic back-
ground [32]. Further studies using I-3-C for cervical dysplasia
should incorporate CYP 1A1 genotyping to assess efficacy of
I-3-C in different CYP 1A1 genotypes.
Not all dysplastic lesions will go on to develop into cancer.
Mildly dysplastic lesions will spontaneously regress without
therapy in the majority of patients, and only a small subset of
cases actually posses cancerous potential. Less than one-half of
more severely dysplastic lesions in patients will eventually
progress to invasive disease [33]. There is currently no avail-
able test that can accurately predict the clinical behavior of a
cervical lesion. Therefore, most high-grade lesions are treated
with surgical excision. Chemoprevention is the attempt to use
natural and synthetic compounds to intervene in the early
precancerous stages of carcinogenesis, before invasive disease
begins. Cervical cancer is ideally suited to chemopreventive
efforts given its well-characterized preinvasive state and its
multistep progression to invasive disease and the clinical ease
with which the clinician can follow the lesion without signif-
icantly invasive techniques (i.e., colposcopy).
In conclusion, this placebo-controlled trial of indole-3-carbi-
nol in the treatment of CIN does show promise for I-3-C as a
therapeutic. We were able to demonstrate a physiologic effect
of the drug, which was seen in a dose-dependent fashion. The
strengths of this study are that there was placebo control, which
is important because of the high rate of spontaneous regression
of CIN, and that indole-3-carbinol was well tolerated with no
adverse events noted. Although the response rate in this study
is encouraging, these data need to be confirmed with a large
placebo-controlled multicenter trial. If such a large trial sup-
ports the efficacy of I-3-C for the treatment of cervical dys-
plasia, it would give patients and physicians a nonsurgical
option for the treatment of CIN.
128 BELL ET AL.

ACKNOWLEDGMENTS
The authors thank Dr. Steve London (Department of Obstetrics and Gyne-
cology, LSU Medical Center–Shreveport) for his generous support of this
project, as well as Murray and Isabella Rayburn.
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