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http:/ /vet.sagepub.com/content/48/5/964The online version of this article can be found at:
DOI: 10.1177/0300985810391113
2011 48: 964 originally published online 15 December 2010Vet Pathol
Clifford and I. Leconte. Lezmi, K. Thibault-Duprey, A. Bidaut, P. Hardy, M. Pino, G. Saint Macary, S. Barbellion, P. Brunel, O. Dorchies
Crl:CD(SD) Rats: Impact on Reproductive Toxicity Studies
Spontaneous Metritis Related to the Presence of Vaginal Septum in Pregnant Sprague Dawley
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Laboratory Animals
Spontaneous Metritis Related to thePresence of Vaginal Septum in PregnantSprague Dawley Crl:CD(SD) Rats:Impact on Reproductive Toxicity Studies
S. Lezmi1, K. Thibault-Duprey2, A. Bidaut2, P. Hardy2, M. Pino2,
G. Saint Macary1, S. Barbellion2, P. Brunel2, O. Dorchies2,
C. Clifford3, and I. Leconte1
Abstract
Recently, 6% of 1,176 Sprague Dawley rats examined in our reproductive toxicity studies presented with dark-red uterine
contents with or without fetuses demonstrating delayed development. Sometimes, a high proportion of the litter was found dead,
and dystocia with death or preterminal euthanasia of the dam occurred. Microscopic findings in the uterus consisted of necro-hemorrhagic and suppurative periplacentitis associated with the presence of bacterial colonies identified as Escherichia coli. In thevagina, similar findings were observed that were associated with mucus accumulation and the presence of a transverse occlusive
or partially occlusive thin membrane identified as a vaginal septum. Microscopically, this septum consisted of a thin band of con-nective tissue covered on both sides by a mucous epithelium that was continuous with vaginal epithelium. In some cases, there wasonly mucus accumulation retained by a septum in the vagina without evidence of bacterial infection. Serological and histological
examinations did not reveal any specific pathogenic agent. The presence of these septa in the vagina most likely favored mucusaccumulation, nonspecific ascending bacterial infection, and dystocia.
This colony of rats presented with an unusually high incidence of vaginal septa as it was described in different strains of mice and rats in the past. We hypothesized that the use of an impedance meter by the breeder—to determine the phase of the estrous cycle by introducing a probe in the vagina—likely facilitated gestation by perforating the vaginal septum in some
cases.
Keywords
malformation, metritis, rat, Sprague Dawley, septum, vagina
Vaginal malformations in humans (eg, atresia, persistent
hymen, hypoplasia, dysgenesis) are generally rare and can
result from genetic factors or prenatal exposure to a toxicant,
or they may have an unknown etiology.5 Transverse (occlusive
or partially occlusive) and longitudinal vaginal septa have been
described in various species, including different strains of
mice,10 Wistar rats,3 one Sprague Dawley rat,1 and in humans.9
The presence of transverse septa usually results in the accu-mulation of mucus and cellular debris in the anterior part of the
vagina and in the uterus (mucometra), which can lead to muco-
purulent vaginitis and endometritis in rats.3 In humans, the
absence of perforation could result in retention of menstrual
blood and cervical mucus, and hematocolpos may develop
(vagina filled with menstrual blood). Furthermore, in women
with a septum, dyspareunia (painful sexual intercourse) and
obstructed and painful labor can occur.9
Since 2006, in our facilities, an unusually high incidence
of transverse vaginal septum associated with metritis was
noted in mated Sprague Dawley Crl:CD(SD) female rats and
thereby interfered with the interpretation of our reproductive
toxicity studies (embryofetal and pre- and postnatal develop-
mental studies). The presence of the vaginal septa did not
result in a lower fertility rate, possibly because of the proce-
dure of identification of the estrous cycle in female rats (use
of an impedance meter) that resulted in perforation, thereby
enabling fertilization.
1 sanofi-aventis R&D, Drug Safety Evaluation, Porcheville, France2 sanofi-aventis R&D, Drug Safety Evaluation & Laboratory Animal Science and
Welfare, 94140 Alfortville, France3 Charles River Laboratories, Wilmington, MA, USA
Corresponding Author:
StephaneLezmi,2-8 routede Rouen, ZI de Limay Porcheville, 78440Porcheville,
France
Email: [email protected]
Veterinary Pathology
48(5) 964-969
ª The American College of
Veterinary Pathologists 2011
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DOI: 10.1177/0300985810391113
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Material and Methods
During the past few years (2006-2009), 1,176 Sprague Dawley
Crl:CD (SD) rats (10-12 weeks old) originating from the same
supplier were used in our laboratory for embryofetal and pre-
and postnatal developmental studies. In embryofetal toxicity
studies, dams were treated with test compound between gesta-
tion day (GD) 6 to GD17 (main organogenesis period). OnGD21, the dams were euthanized, and the uterus was examined
macroscopically for identification of implantation sites, resorp-
tions, and live or dead fetuses; live fetuses were weighed,
sexed, and examined for external, visceral, and skeletal defects.
In pre- and postnatal developmental studies, dams were treated
from GD6 throughout lactation period to weaning (21 days
after parturition).
In all of these studies, mated female rats were supplied by
Charles River Laboratories France. The supplier selected
females for pairing using an impedance meter to determine the
estrous cycle phase. A probe that was 1.5 cm long and 3 mm
in diameter was introduced into the vagina to measure the localelectrical resistance.8 Females in the estrous phase were then
placed with a male, and mating was confirmed by the presence
of the vaginal plug after a 4-hour pairing.
In one study, an in-house natural mating (without the use of
an impedance meter) was performed using approximately 200
mature females and 25 breeder males to obtain 96 Crl:CD
(SD) mated female rats (pairing ratio: 1 male for 3 females max-
imum overnight) to evaluate thepotential impactof theuse of an
impedance meter. The mating was evidenced by the presence of
sperm in the vagina using cytological examination.
At necropsy, macroscopic examination of the entire genital
tract was performed. In some studies, if abnormal uterine and/
or vaginal contents were observed, the ovaries, uterus, placen-tae, fetuses, and vagina were sampled and fixed in 10% buffered
formalin. The liver, heart, brain, spleen, kidney, and lung of
some affected pregnant females were also sampled and fixed
in formalin. These tissues were processed to paraffin block, sec-
tioned, stained with hematoxylin-eosin (H&E) or hematoxylin-
eosin-saffron (HES) and Periodic Acid-Schiff (to identify a pos-
siblefungal infection) and examined microscopically. Cytologi-
cal samples of the uterine and vaginal contents were stained
with May-Grunwald-Giemsa. Samples from selected uteri pre-
senting with abnormal content were taken for microbiological
examination, and serum samples from selected affected dams
and from sentinel animals were taken for serological evaluations
for rat coronavirus (RCV/Sendai), parvovirus (RPV 1&2,
PARVO NS1, TOOLAN H1, Kilham RV), pneumonia virus
of mice (PVM), reovirus (REO3), Hantaan, Lymphocytic chor-
iomeningitis virus (LCMV), Theiler, Clostridium piliforme,
Mycoplasma spp., Encephalitozoon cuniculi, and CAR Bacillus;
these samples were shipped to Charles River and Vebiotel
Laboratories, France. These analyses were performed in 3
different studies during the period 2006-2009. Each time, one
to three rats were analyzed.
All studies and the procedures of our laboratories complied
with the French regulation (Decree 87-848, revised 2001)
implementing European Directive 86/609 and European
Convention ETS123. Furthermore, the studies were performed
in accordance with the standards of the ILAR Guide4bis (1996)
and the sanofi-aventis Charter on the Humane Care and Use of
Laboratory Animals, including ethical review.
Results
Anatomic Pathology Observations
Macroscopic examination at GD21 of pregnant SD rats from
these studies revealed the presence of an abnormal number of
rats with red hemorrhagic or dark fluid in the uterus (Fig. 1) with
an abnormal development of fetuses (Fig. 2).When compared to
a normal fetus (Fig. 3), fetuses from affected pregnant females
were pale, with delayed development (smaller size), and with
a hemorrhagic fibrin clot often seen at the surface of the corre-
sponding placentae (Fig. 4, arrow). In one of the females found
dead before the end point of the study, the uterus was filled with
a dark liquid containing tissue debris, and only one macerated fetus was identifiable (Fig. 5).
When compared to a normal vagina(Fig. 6),the presence of a
transverse occlusive or partially occlusive membrane was iden-
tified as a vaginal septum of variable thickness (Figs. 7-9).
Rarely, the vaginal septum was associated only with the pres-
ence of a dense mucous plug in the anterior part of the vagina,
without evidence of uterine and fetal anomalies (Fig. 10).
(Cervix on the top for Figs. 6-10.)
Microscopic findings of all organs sampled from affected
dams and fetuses were limited to thefemale genital tract andpla-
centae. In all uteri examined, the lumen and glands were filled
with variable amount of cellular debris, mucus and neutrophils.
When compared to a normal placenta (Fig. 11), placentae fromaffected dams were covered by a dense accumulation of fibrin,
red blood cells mixed with degenerated neutrophils, and bacter-
ial colonies (Figs. 12 and 13). Large areas of ischemic necrosis
were observed in a few of the affected placentae. Cytological
examination of vaginal contents confirmed the presence of bac-
teria, mucus, and neutrophils (Fig. 14).
In the vagina of affected uteri, the septum consisted of a thin
band of vascular connective tissue sometimes associated with
some myofibers (Fig. 16, arrow), and covered by a mucous
and/or squamous epithelium that was continuous with vaginal
epithelium (Figs. 15 and 16).
Serological examinations did not demonstrate the presenceof any specific rat pathogen. The negative PAS stain ruled out
fungal infections. All bacterial cultures obtained from the
abnormal uterine contents identified the presence of a large
amount of Escherichia coli bacteria only.
Incidence
In all embryofetal toxicity studies evaluated, the incidence of
vaginal septum associated with metritis ranged from 0 to 10%
per study (Table 1: nos. 1 to 10). Approximately 6% of mated
females from all embryofetal toxicity studies (785 mated
Lezmi et al 965
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rats) had vaginal septum/mucus accumulation and/or
abnormal uterine content in both control and treated groups.
In pre- and postnatal developmental toxicity studies (Table 1:
nos. 11 to 13), 23 of 391 mated females presented with a vagi-
nal septum that was associated most often with dystocia,
representing an incidence of 5.9% of mated females received
from the supplier. For example, in one pre/postnatal study,
vaginal septa were observed in 10/100 rats; dystocia, death
of dams, and loss of the entire litter near the time of parturi-
tion were observed in 8 of the 10 affected rats.
Fig. 1. Uterus, rat, gestation day 21; abnormal hemorrhagic uterine contents. Fig. 2. Litter from an affected dam, rat; gestation day 21; fetuseswith different sizes (delayed development). Fig. 3. Normal fetus and placenta, rat, gestation day 21. Fig. 4. Pale rat fetus with delayed devel-opment from an affected dam, gestation day 21; hemorrhagic placenta (arrow). Fig. 5. Macerated rat fetus from an affected dam found dead atgestation day 19. Fig. 6. Normal vagina, rat. Fig. 7. Vagina, rat, vaginal septum with necro-hemorrhagic fluid accumulation. Fig. 8. Vagina, rat,thin vaginal septum. Fig. 9. Vagina, rat, thick vaginal septum. Fig. 10. Vagina, rat, mucus plug accumulation in the proximal part of the vagina.
966 Veterinary Pathology 48(5)
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In the embryofetal toxicity study in which an
impedance meter was not used (in-house natural matings),
an unusually high incidence of nonpregnant females (19%)
was observed. No macroscopic examination of the vagina
was performed at this time to check for the presence of a
septum; however, in the past, the background incidence
of nonpregnant female rats for this study type was
approximately 10%.
Fig. 11. Normal placenta, rat, hematoxylin-eosin (15). Fig. 12. Placenta, rat, hemorrhagic and suppurative periplacentitis, hematoxylin-eosin (15). Fig. 13. Placenta, rat, higher magnification of Fig. 12. Presence of bacterial colonies (arrow), hematoxylin-eosin (200).Fig. 14. Cytological smear of the vaginal contents, rat. Presence of mucus, bacteria, and neutrophils, May-Grunwald-Giemsa (400). Fig. 15.
Vagina, rat, vaginal septum, hematoxylin-eosin-saffron (15). Fig. 16. Vagina, rat, vaginal septum (higher magnification of Fig. 15), presence of myofiber (arrow), hematoxylin-eosin-saffron (100).
Lezmi et al 967
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Investigations into the origin of the mated female rats
(breeding room) in the supplier facilities revealed that most
of the females with genital tract anomalies were from a specific
breeding area.
Discussion
It was concluded that the high incidence of metritis that was
observed in pregnant females in our facilities was a conse-
quence of an abnormal number of female rats with a vaginal
septum (partial or complete) that possibly reflected a genetic
drift in the supplier facilities. In the literature, the incidence
of vaginal septa in Wistar rats is reported to be approximately
2%.3 In our studies with Sprague Dawley Crl:CD(SD) rats, the
overall incidence was 6%, with a maximum of 10% in some
reproductive toxicity studies.
A high incidence of metritis associated with the presence of
vaginal septum in reproductive toxicity studies may seriously
interfere with the evaluation of data. Indeed, vaginal septum can
lead to mucus accumulation in the vagina, vaginitis, metritis,
placentitis, reduced fetal weight, pale fetuses, growth retarda-
tion, and, in some cases, fetal death, as observed in our studies.
In pre- and postnatal developmental toxicity studies, dystocia
may occur that can be associated with the death or euthanasiaof the damand entire litter losses. As a consequence, the evalua-
tion of thedevelopmental toxicity of a compoundcould be influ-
enced. Vaginal septa and their sequelae may also have
consequences on fertility studies by having an impact on ferti-
lity, fecundity, and gestation indices. A systematic examination
of the vagina must be done to identify vaginal septa to cull
affected dams from reproductive toxicity studies or to take this
finding into account during interpretation of results.
It is hypothesized that the use of an impedance meter by the
breeder to determine the phase of the estrous cycle could cause
perforation of the vaginal septum, which may favor pregnancy
of females that would not become pregnant by natural mating.
Furthermore, in the study in which the female rats were mated
naturally, the incidence of nonpregnant females was unusually
high, which suggested that the use of an impedance meter likely
allowed pregnancy to occur in rats with a vaginal septum. The
hypothesis that vaginal trauma caused by the impedance meter
can induce the presence of vaginal septa is highly unlikely;indeed, these septa are very well organized structures with con-
nective tissue, myofibers and a well-defined epithelium on both
sides that do not fit with scar or healing tissue.
In humans, transverse and longitudinal vaginal septa have
been described. Transverse septa are approximately 1 cm thick
and may be complete or have a small central or off center per-
foration. They are usually isolated defects, but they are also a
component of the McKusick-Kaufman syndrome, which
includes congenital heart malformation and polydactyly.6 In
our studies, no other morphological anomalies were reported
in affected females, but careful examination of other organs
was not systematically performed, and we are not able to rule
out the presence of possible other congenital malformations.In different mouse strains (BALB/c, B6, DBA, etc.), the pres-
ence of vaginal septa was suspected to be inherited as a com-
plex recessive genetic defect;7 in a Balb/cJ mouse colony,
38% of females presented with longitudinal septa that were
likely induced by more than one recessive gene, the expression
of which was influenced by the genetic background.2
Morphologically, in humans, the transverse septum is distin-
guished from an imperforate hymen by the identification of a
hymen separated from the septum. In the rat, a membrane that
closes the distal part of the vagina and that is observable by the
naked eye is present during the prepubertal period.4 In Sprague
Dawley rats, this membrane degenerates between postnatal days
30 and 38, at the time of puberty. In the present studies, the pres-
ence of a septum in the middle of the vagina allowed us to rule
out a possible persistence of this prepubertal membrane; the
presence of a septum was likely related to an incomplete cana-
lization of the vaginal sinus during organogenesis.
The absence of perforation in humans results in retention of
menstrual blood andcervical mucus (hematocolpos) and, in rare
cases, obstructed labor may occur.9 In different strains of mice
(BALB/c, B6, DBA, etc.), imperforate vagina leads to a marked
accumulation of mucus or fluidin the uterus (mucometra, hydro-
metra). In one study, the authors isolated 3 types of bacteria in
10% of the affected mice that were considered as contaminants,
as none of the uteri had microscopic findings consistent with aninfection.10 In contrast, in Wistar rats3 as in our studies in Spra-
gue Dawley Crl:CD(SD) rats, a transverse septum (occlusive
and/or partially occlusive) can cause frequent mucopurulent
metritis and vaginitis. The presence of these septa most likely
caused mucus accumulation in the vagina followed by second-
ary nonspecific bacterial infection resulting from proliferation
of E. coli bacteria. With regard to our observations, it appeared
that mostbacterialproliferation associated with metritis and pla-
centitis likely occurred during the third part of gestation.
In an attempt to reduce theincidence of longitudinal septa in a
colony of Balb/cJ mice, the breeding of only nonseptate female
Table 1. Detailed Incidence of Vaginal/Uterine Findings and/orDystocia by Studies From Both Control and Treated Groups
Studynumber
Number of MatedFemale Rats
Number of Mated FemaleRats With Affected
Genital Tracta
Study 1 100 10
Study 2 96 4Study 3 96 0Study 4 96 7Study 5 60 5Study 6 106 6Study 7 16 2Study 8 24 0Study 9 96 8Study 10 95 5Study 11 100 10Study 12 159 5Study 13 132 8Total 1176 70 (6%)
a
Presence of a septum associated with mucus, metritis, and/or dystocia
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