spontaneus metritis to the presence of vaginal septum in pregnant

8/13/2019 Spontaneus Metritis to the Presence of Vaginal Septum in Pregnant ...

http://slidepdf.com/reader/full/spontaneus-metritis-to-the-presence-of-vaginal-septum-in-pregnant- 1/7

http://vet.sagepub.com/ Veterinary Pathology Online

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

Published by:

http://www.sagepublications.com

On behalf of:

Pathologists.American College of Veterinary Pathologists, European College of Veterinary Pathologists, & the Japanese College of Veteri

can be found at:Veterinary Pathology Online Additional services and information for

http://vet.sagepub.com/cgi/alertsEmail Alerts:

http://vet.sagepub.com/subscriptionsSubscriptions:

http://www.sagepub.com/journalsReprints.navReprints:

http://www.sagepub.com/journalsPermissions.navPermissions:

What is This?

- Dec 15, 2010OnlineFirst Version of Record

- Aug 24, 2011Version of Record>>

at UNAM IIMAS on November 26, 2013vet.sagepub.comDownloaded from at UNAM IIMAS on November 26, 2013vet.sagepub.comDownloaded from

http://vet.sagepub.com/



http://vet.sagepub.com/subscriptions

http://vet.sagepub.com/content/48/5/964


http://vet.sagepub.com/content/48/5/964.full.pdf



http://www.sagepub.com/journalsPermissions.nav

http://online.sagepub.com/site/sphelp/vorhelp.xhtml

http://www.sagepublications.com/

http://vet.sagepub.com/cgi/alerts



http://www.sagepub.com/journalsReprints.nav





http://vet.sagepub.com/content/early/2010/12/11/0300985810391113.full.pdf

























http://www.sagepublications.com/





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

Reprints and permission:

sagepub.com/journalsPermissions.nav

DOI: 10.1177/0300985810391113

http://vet.sagepub.com

at UNAM IIMAS on November 26, 2013vet.sagepub.comDownloaded from









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









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)










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










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

968 Veterinary Pathology 48(5)








spontaneus metritis to the presence of vaginal septum in pregnant

Documents