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Is there a role for Serratia marcescens in male infertility: An experimental study?
Accepted Manuscript Is there a role for Serratia marcescens in male infertility: An experimental study? Kalpana Rana, Deepali Thaper, Vijay Prabha PII:
S0882-4010(16)30085-7
DOI:
10.1016/j.micpath.2017.01.061
Reference:
YMPAT 2108
To appear in:
Microbial Pathogenesis
Received Date: 15 February 2016 Revised Date:
27 January 2017
Accepted Date: 31 January 2017
Please cite this article as: Rana K, Thaper D, Prabha V, Is there a role for Serratia marcescens in male infertility: An experimental study?, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.01.061. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Is there a role for Serratia marcescens in male infertility: An experimental study?
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Kalpana Rana, Deepali Thaper and Vijay Prabha*
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Department of Microbiology, Panjab University, Chandigarh-160014, India.
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Running head: Bacteria and male infertility
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*Correspondence:
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Dr. (Mrs) Vijay Prabha
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Professor
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Department of Microbiology
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Panjab University
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Chandigarh -160014. India
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Telephone: 91-9417065675
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E mail: [email protected]
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Keywords: Serratia marcescens, male infertility, immobilization, decapitation, sperms
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Abbreviations and Acronyms
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PBS: phosphate buffered saline; TSI: Tissue Somatic Index
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Abstract
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Objective: Establishment of a male BALB/c mouse model to study the role of sperm impairing S.
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marcescens on mouse reproductive potential. The current study can add to use of reliable animal
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models to provide a noteworthy evidence for the microbial cause of infertility. in our environment.
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Methods: The mice in the experimental test groups II, III, IV were intraperitoneally administered
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with different doses (104, 106 or 108 cfu) of S. marcescens whereas, group I serving as control,
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received PBS, for 10 consecutive days. The groups were evaluated for any change in body weight,
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tissue somatic index (%), seminal parameters and histology. Confirmation of S. marcescens from
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reproductive organs was done by reisolating the same by cultural characteristics and biochemical
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tests. microorganism from tissue homogenates of reproductive organs giving a knowhow of its
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spread and distribution.
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Results: The results showed that weight gain was evident only in mice receiving PBS (group I),
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whereas a decrease was recorded in the test groups (group II, III and IV). Only testes of test groups
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showed significant changes in TSI values whereas, no change in TSI was observed in any
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reproductive organ of any test group. The testes of all the test groups showed significant change in
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tissue somatic index, whereas, no change was observed in TSI value of all the other reproductive
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organs of all the groups. Seminal parameters viz. sperm count, motility and viability were found to
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decrease in test groups II, III and IV as compared to control group I. Interestingly, the number of
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pus cells and percent decapitation was more prominent in test groups which received higher doses
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(i.e. group III and group IV). With the increase in dose, there were more regressive changes (eg.
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oligo- astheno-teratotospermia) in seminal parameters. The histopathological examination revealed
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hypospermatogenesis alongwith mild to dense inflammation in vas deferens and caudal epididymis
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in all test groups except hypospermatogenesis which was observed only in test group III and IV.
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However, in group I, neither adverse changes nor any sign of inflammation were observed.
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Conclusion: Intraperitoneal inoculation of S. marcescens could lead to alteration of semen
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parameters, induction of decapitation in spermatozoa and histopathological changes, thereby
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decreasing the reproductive potential of male mice.
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ACCEPTED MANUSCRIPT Introduction
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Microbial infections in the male genitourinary tract account for 15% of male infertility cases [1].
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The major classes of such pathogens include bacteria, viruses, fungi and parasites. Some of the
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most commonly isolated bacteria are Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria
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gonorrhoeae, Staphlyococcus sp., members of the family Enterobacteriaceae viz., Escherichia
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coli, Klebsiella sp. and Proteus sp. [2]. These pathogens can attack different sites of male
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reproductive tract such as testis, epididymis, vas deferens and male accessory sex glands, thereby,
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causing reproductive disturbances and hence, leading to infertility. This form of infertility, which
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arises as a result of infectious agents, is considered as a potentially correctable type of infertility
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and hence, can be treated by antibiotics [3]. However, a large number of patients remain untreated,
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because of lack of diagnosis of the infectious agent. The asymptomatic course of the disease and
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unspecific clinical signs allow the infection to remain unnoticed, thereby resulting into chronic
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illness [4]. This has brought into focus, the role of these asymptomatic colonizers in male
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infertility since, to date, there has been little knowledge about their absolute or relative
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contribution to male infertility. In this regard, So far, most of the work has been accredited to
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organisms such as C. trachomatis, Ureaplasma sp., N. gonorrhoeae and Herpes simplex virus for
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their role in male infertility [5-8]. However, the data concerning the effect of other asymptomatic
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microorganisms on male infertility is negligible.
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In an earlier work done in our laboratory, infertility in female BALB/c mice was observed
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upon vaginal colonization with sperm-impairing Serratia marcescens and Candida albicans [9].
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So, with an aim to assess the role of S. marcescens in male infertility, intraperitoneal infection in
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mouse model was established and the effects on the seminal parameters were studied.
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Material and Methods
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Ethical note
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All the experiments have been conducted in compliance with the protocols approved by
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Institutional Animal Ethics Committee, Panjab University (vide letter no. IAEC/504 dated 2 April
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2014). All experiments were completed in agreement with the guidelines of the Committee for the
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Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
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Animals
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Adult 5-6 weeks old male (25±2g) BALB/c mice were used in the present study. Animals were
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individually housed in polypropylene cages in the animal room of Department of Microbiology,
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Panjab University, Chandigarh, India. The animals were fed with standard pellet food and water ad libitum and maintained in laboratory conditions (12:12, dark:light cycle).
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Microorganism
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The standard strain of S. marcescens (MTCC 7641), used in the present study, was procured from
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the Microbial Type Culture Collection, Institute of Microbial Technology, Sector 39, Chandigarh,
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India. The strain was grown in Brain Heart Infusion (BHI) broth and maintained as glycerol stocks
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at -80oC. The strain was found to cause sperm impairment by agglutination in vitro.
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Inoculum
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Serratia marcescens strain was cultivated in BHI broth, incubated under shaking conditions (150
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rpm) for 24h at 37ºC and then centrifuged at 10,000 rpm for 20 min. The pellet obtained was
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washed twice with PBS (50mM, pH 7.2). The cells were resuspended in the same buffer to get a
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cell count of 104, 106, and 108cfu/20µl.
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Inoculation procedure
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Male BALB/c mice were divided into 4 groups with 3 mice in each group. Group I, inoculated
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intraperitoneally with 20µl of PBS alone for 10 consecutive days, served as a control. The
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remaining three test groups (Group II-IV) were intraperitoneally administered with 104, 106 or
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108cfu of S. marcescens per mouse in 20µl PBS respectively for the same time period. Weight
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profile of all the mice groups was evaluated. The mice were sacrificed on day 12 and the
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parameters which were evaluated include TSI (%), seminal parameters and histopathological
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changes. The experiment was repeated twice for consistency of the results.
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Weight responses and Tissue Somatic Indices TSI (%)
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Initial body weight of mice from each group was taken on the 1st day of experiment and final
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weight on the last day of experiment. On day 12, mice from each group were sacrificed by cervical
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dislocation and the various reproductive organs (viz. testis, caudal epididymis and vas deferens)
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were removed aseptically. The organs were grossly examined and weighed. The TSI (percent
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tissue/organ weight in relation to body weight) was evaluated [10].
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Determination of Seminal parameters
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Sperm count and sperm morphology
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Mice from each group were sacrificed by cervical dislocation on day 12 and were dissected. The
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vas deferens was pulled out and placed in freshly prepared 500 µl of PBS buffer (50 mM, pH 7.2).
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fixed volume of 10µl of the sample was placed on a glass slide and examined under light
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microscope at 400X magnification. Around six to eight fields were scanned and mean number of
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spermatozoa in all the fields was multiplied by 106. The slides were also assessed for the
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morphology of spermatozoa in each field to evaluate the respective abnormalities [11].
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Sperm motility assay
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Motility of the sperms extracted from the sacrificed mice was determined by the method of
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Emmens [12].
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Sperm viability assay
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In order to estimate the percentage of viable sperms, an equal volume of mouse spermatozoa was
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mixed with 0.5% eosin and examined under the light microscope at 400X magnification.
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Reisolation of S. marcescens from the various reproductive organs on day 12
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The various reproductive organs were homogenized manually in sterile PBS (50 mM, pH 7.2) to
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form a uniform homogenate. 100µl of serially diluted tissue homogenate was plated on BHI agar
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plates and incubated overnight at 37ºC. The bacterial load in terms cfu/mouse/g of tissue was
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calculated. The reisolated bacteria from various organs were confirmed as S. marcescens by
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culture characteristics and biochemical tests. Further the isolates were also checked for
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spermagglutinating activity in vitro.
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Histopathological Studies
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Histopathological analyses of the reproductive organs (testis, caudal epididymis and vas deferens)
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of mice from each group sacrificed on day 12 were carried out. The various reproductive organs
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were harvested, fixed in 10% formaldehyde for 24h and then embedded in paraffin according to
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standard histological methods. Serial paraffin sections of 4 mm were stained with hematoxylin-
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eosin and observed at 400X magnification for any significant changes in reproductive organs.
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Statistical analysis of data
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The results of all the experiments in this study were analysed using Microsoft Word Excel
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software. The statistical significance of differences between control and test groups was evaluated
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by Student’s t-test. Results are expressed as mean± standard deviation. Differences were
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considered to be statistically significant when p value < 0.05.
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The impact of S. marcescens on body weight, TSI (%) of various reproductive organs, seminal
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parameters and histological changes in mice belonging to each group was evaluated.
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Weight profile
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When the weight profile study of different groups of mice was conducted, the results showed that
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there was 21% increase in weight of the control group which received PBS alone, whereas 15%,
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18% and 21% decrease in body weight was recorded in test groups (II, III and IV) which
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received 104cfu, 106cfu and 108cfu of S. marcescens respectively (Figure: 1).
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Tissue somatic indices TSI (%)
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Tissue somatic indices of the reproductive organs excised from all test groups of mice were
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determined on day 12. In comparison with control group I, the significant changes in TSI values
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were observed in testes of all test groups. As compared to group I, there was decrease of approx.
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24%, 39%, and 50% in TSI values of testis of test group II, III, IV respectively. However, no
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significant alterations were observed in vas deferens and caudal epididymis of group II, III and IV
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when compared with group I (Figure: 2).
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Evaluation of Seminal parameters
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On day 12, all the animals were sacrificed and dissected for assessment of seminal parameters viz.
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sperm count, motility, viability and morphology. In the test groups, an alteration in seminal
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parameters was observed. The sperm count of test group II, III and IV group II, group III and
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group IV reduced significantly to 9x106 / ml, 7x106 /ml, 4x106 /ml respectively when compared to
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group I (28x106 / ml). Similarly, motility and viability percentage also declined drastically for the
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test groups (group II, III and IV) as compared to control group I. There was decrease in sperm
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count (68%, 75%, 86%), percent motility (86%, 93%, 93%), percent viability (66%, 76%, 83%) in
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test group II, III, IV respectively, as compared to control group I. . Interestingly, the number of pus
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cells and decapitation of mouse spermatozoa was observed in all the test groups, group II (14%),
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group III (24%) and group IV (61%). However, the This teratospermic effect was observed in all
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test groups, but was more pronounced in group IV. the number of pus cells and percentage of
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decapitation was more prominent in test groups which received higher doses (i.e. group III, IV).
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With the increase in dose, more regressive changes (i.e. oligo- astheno-teratotospermia) in the
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seminal parameters could be observed (Table 1, Figure: 3).
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S. marcescens could not be recovered from any of the tested reproductive organs of the mice in
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control group (i.e. group I). However, the organism could be efficiently reisolated from the
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homogenates of testis, caudal epididymis and vas deferens of mice belonging to test group II, III
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and IV (Table 2, Figure: 4). According to the biochemical tests viz. Oxidase – ve, MR – ve, VP +
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ve, Citrate + ve, and glucose- fermenting, the Gram –ve organism so isolated was confirmed to be
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S. marcescens. Moreover, it was capable of causing 100% spermagglutination in vitro.
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Histopathological examination of various organs of groups of male BALB/c mice
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administered with different doses of S. marcescens on day 12
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To check any adverse affect of S. marcescens on tissue morphology of various reproductive organs
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viz. testis, epididymis and vas deferens, histopathological analysis was carried out. The control
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group revealed normal tissue histology in all the organs. The testes showed regular seminiferous
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tubules and germinal cell morphology. Epididymis displayed the well-vascularized loose
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connective tissue present around the epididymal duct. Further, normal columnar epithelium was
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seen in vas deferens. However, the test group II mice showed that testis had nearly normal
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spermatogenesis whereas mild interstitial inflammation was observed in vas deferens and caudal
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epididymis. Interestingly, in case of test group III mice, testis showed hypospermatogenesis along
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with no excess of sertoli cells. Inflammation was observed both in epididymis (i.e. epididymitis)
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and vas deferens. Similarly, in case of test group IV mice, there were only few- matured
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spermatocytes leading to hypospermatogenesis. Also, there was dense inflammation in caudal
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epididymis (epididymitis) and vas deferens (F- Funiculitis) (Figure: 5).
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Discussion
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Infertility is a condition in which conception does not occur even after one year of regular
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unprotected sexual intercourse [13]. Out of the 15% of the affected couples, the male factor is
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involved in 40-50% of the infertility cases [14-16]. Of the various reasons cited for male infertility,
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the microbial infections have not been given their due because of their asymptomatic nature. Even
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though the microorganisms could be isolated from semen of both fertile as well as infertile men,
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yet several reports have surfaced up regarding their greater frequency in semen samples from
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asymptomatic infertile patients than in those from fertile men. The microorganisms which are most
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prevalent in semen belong to Gram positive viz. S. aureus, Coagulase negative staphylococci
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species, Enterococcus faecalis, Candida species and Gram negative microorganisms viz. E. coli,
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Klebsiella sp., Proteus sp., Pseudomonas sp. and Serratia sp. [17, 18]. The presence of these
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ACCEPTED MANUSCRIPT microorganisms in semen has been confirmed by various studies but their role in male fertility is
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still controversial. In this regard, several investigators have also reported the in vitro detrimental
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effect of microorganisms on sperms. For instance, Berktas et al. [19] has have reported that
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incubation of spermatozoa with E. coli resulted in reduced sperm motility in vitro. Similarly, U.
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urealyticum could cause loss of motility via multi-sperm agglutination [6] whereas S. aureus has
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been known to impede sperm motility in vitro by secreting the factor extracellularly [20]. The
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majority of these observations are derived from experimental in vitro studies, but their significance
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for in vivo infections has been questioned.
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The standard strain of S. marcescens, an opportunistic pathogen from the Enterobacteriaciae
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family which tends to colonize the genitourinary tract, was found to impair sperm motility in vitro
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by causing agglutination of sperms and hence, leading to infertility in female BALB/c mice upon
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intravaginal administration [9]. These results prompted us to extrapolate the same in male BALB/c
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mice. Therefore, the present study was undertaken to discover the impact of intraperitoneal
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administration of S. marcescens on seminal parameters of male BALB/c mice. For this, different
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doses of S. marcescens i.e. 104, 106, 108 cfu (20µl/mouse) were intraperitoneally administered in
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male BALB/c mice for 10 consecutive days. On day 12, the mice were sacrificed and evaluated for
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weight profile, TSI (%), seminal parameters and histopathological changes.
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The weight profile study showed decrease in body weight of mice in all the test groups (receiving
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104, 106, 108 cfu of S. marcescens) as compared to the control group which received PBS where an
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increase in weight was observed. These results are in concordance with the studies of Heeckeren et
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al. [21] wherein they have studied the role of Pseudomonas infection and have reported a drastic
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weight loss after 3 days of infection. Further, the relevance of body condition and somatic indices
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(testis, vas deferens and cauda epididymis) as indicators of health status of the animals was
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investigated. The results showed that there was significant reduction in TSI values of testes of all
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test groups which is indicative of diminished reproductive vigor of the mice as a result of S.
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marcescens infection. These results are in contrast to the study conducted by Kaur and Prabha [22,
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23] wherein no difference was found in TSI of all the reproductive organs of female mice
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receiving intravaginal sperm agglutinating S. aureus and E. coli as compared to control group
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which received PBS only.
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Some of the major determinants of fertilization potential of spermatozoa are based on their
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microscopic assessment of concentration, motility, viability, morphology and presence of pus cells.
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In the present study, an adverse effect was observed on all the seminal parameters in all test groups 8
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and viability could be very well correlated with increase in dose of S. marcescens indicating that
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the microorganism under study had detrimental effects on seminal parameters. To support this, a
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study conducted by Sellami et al. [5] has shown that the inoculation of male Swiss mice in meatus
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urethra with 106 C. trachomatis IFU resulted in decreased reproductive performance of male mice
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as observed by seminal parameter alterations. Furthermore, from our results, it was observed that
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an increase in the dose of S. marcescens lead to a rise in number of percent decapitation and pus
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cells which further indicated that the response is purely dose-dependent. In the light of present
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finding, it could be speculated that S. marcescens infection could be responsible for the separation
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of heads from tails by inducing specific morphogenetic defects at different stages of spermatid
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differentiation. This is supported supports the findings by the findings of Grewal et al. [24] who
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showed that the culture supernatants of Bacillus sp. and Pseudomonas sp. not only decreased the
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motility of spermatozoa but also decapitated a few spermatozoa. Also, while studying the
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relationship between semen quality and pyospermia, Khan et al. [25] showed that pyospermia is
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associated either with compromised sperm motility or altered morphology.
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The next experiment was aimed at validating the proposal that the changes in weight profile, TSI
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(%) and seminal parameters in the male BALB/c mice were only due to the pathogen studied.
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Therefore, reisolation of S. marcescens and histopathological examination of reproductive tissues
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of mice was conducted. With an aim to know about the spread and distribution of the organism to
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reproductive organs, homogenates of all these the reproductive organs were plated on BHI agar
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and the results showed that S. marcescens could be efficiently recovered from the reproductive
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organs of all the mice in test groups II, III and IV. These findings correspond to those of previous
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study of Kruszewska et al. [26], wherein they were able to successfully culture Coxiella burnetii
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from various reproductive and non-reproductive organs of male mice from day 7 till day 49 p. i.
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when challenged intraperitoneally. Similarly, intraperitoneal challenge of mice with Shigella strain
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lead to a systemic infection at very early time points and the organism could be efficiently
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recovered from intestinal tissues [27].
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It is already a known fact that successful intraperitoneal bacterial invasion and colonization of the
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host can lead to multiple histological changes that are produced by the intrinsic host defense
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system in response to the infection. On the similar lines, the histopathological evaluation of the
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present study shows mild to dense inflammation in vas deferens and caudal epididymis of mice in
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the test groups II, III and IV in comparison to the control (group I) where normal tissue histology 9
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inflammation was more prominent in the testes of group of mice receiving higher doses of S.
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marcescens. From these results, it could be inferred that inflammation infection could be the
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underlying reason behind disruption of spermatogenesis, which has earlier also been reported by
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Lu et al. where uropathogenic E. coli was inoculated in male rats and after seven days p. i., it was
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seen that bacterial infection resulted in severe impairment of spermatogenesis with significantly
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decrease in sperm number and damage to germ and testicular cells [30]. which in itself is a major
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determinant of fertility potential of males. In another study Similar histopathologic findings have
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been carried out by Jantos et al. [28, 29] reported in two different studies, where the testes of rats
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were characterized by inflammation and moderate to severe loss of spermatogenesis on day 14
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post inoculation with Chlamydia psittaci and C. trachomatis. These results indicate that
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reproductive tract infections as well as inflammations have profoundly negative effects on
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testicular function resulting in reduced androgen production, lowered sperm counts and temporary
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loss of fertility.
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In conclusion, our data showed that intraperitoneal inoculation of S. marcescens an asymptomatic
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colonizer could lead to regression in sperm concentration, motility, morphology and finally, the
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overall fertility potential of male mice. However, further studies in this direction need to be carried
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out, and that too with greater number of strains, so as to obtain a statistically significant data and
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gather conclusive evidence in support of this hypothesis.
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Conflicts of interest
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The authors report no potential conflicts of interest.
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Acknowledgement
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The support of the Department of Science and Technology, New Delhi, India is gratefully
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acknowledged.
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Nigeria. World J Med Sci 2008; 3: 28-33. 19.
Berktas M, Aydin S, Yilmaz Y, Cecen K, Bozkurt H. Sperm motility changes after
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coincubation with various uropathogenic microorganisms: an in vitro experimental study.
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Int Urol Nephrol 2008; 40:383-389. 20.
species in semen and sperm quality. J Assist Reprod Genet 2009; 26: 47-56.
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Moretti E, Capitani S, Figura N, Pammolli A, Federico MG, et al. The presence of bacteria
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aureus colonization in genital tract of female mice. PLoS One 2012; 7, e52325.
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Kaur K, Prabha V. Spermagglutinating Escherichia coli and its role in infertility: in vivo study. Microb Pathog 2014; 69-70:33-38.
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Kaur S, Prabha V. Infertility as a consequence of spermagglutinating Staphylococcus
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Grewal RK, Prabha V, Malhotra SK, Gupta KG. Decapitation of spermatozoa by
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endocervical isolates from cases of unexplained infertility. J Obstet Gynaecol India 2007;
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Immun 1993; 61:4188-4195.
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Yang JY, Lee SN, Chang SY, Jeong Ko H, Ryu S, et al. A mouse model of Shigellosis by
intraperitoneal infection. J Infect Dis 2014; 209:203-215.
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Kruszewska D, Tylewska-Wierzbanowska S. Coxiella burnetii penetration into the reproductive system of male mice, promoting sexual transmission of infection. Infect
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parameters in infertile Pakistani males. Sultan Qaboos Univ Med J 2012; 12:479-484.
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Khan MS, Mohammad SH, Deepa F, Tahir F. Association between pus cells and semen
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28.
Jantos CA, Augustin J, Durchfeld-Meyer B, Baumgartner W, Schiefer HG. Experimental
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genital tract infection with Chlamydia psittaci (GPIC agent) in male rats. Infection 1998;
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due to Chlaymydia trachomatis in rats. Infect Immun 1992; 60: 2324-2328.
392 393 394
Jantos CA, Baumgartner W, Durchfeld-Meyer B, Schiefer HG. Experimental epididymitis
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Lu Y, Bhushan S, Tchatalbachev S, Marconi M, Bergmann M, et al. Necrosis is the dominant cell death pathway in uropathogenic Escherichia coli elicited epididymo-orchitis 12
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and is responsible for damage of rat testis. PLoS ONE 2013 8: e52919.
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doi:10.1371/journal.pone.0052919.
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Table 1: Effect of different doses of S. marcescens on sperm parameters on day 12
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Seminal parameters Groups Sperm count (x 106 /ml)
Motility %
-
28
57
Group II
+
9
Group III
++
7
Group IV
++
402 403 404
8
20
14% Decapitation
4
14
24% Decapitation
4
10
61%
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Normal
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Morphology
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Viability %
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Pus cells
405 406 407 408 13
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Table 2: Reisolation of S. marcescens on day 12 from various organs after intraperitoneal
413
inoculation. Organs Testis
Caudal epididymis
-
-
+
Group III
+
Group IV
+
+
416
-
417 418 419
+
+
+
+
+
+
S. marcescens recovered S. marcescens not recovered
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Vas deferens
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Figure legends
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Figure 1: Weight profile of male BALB/c mice administered with different doses of S.
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marcescens for 10 consecutive days. Values represent mean values ±SD.
433
Figure 2: Tissue somatic indices (%) of various reproductive and non reproductive organs
434
after 10 days intraperitoneal administration of S. marcescens. Data represent mean values
435
±SD. * represents p < 0.05 which indicate significant value when compared to control PBS
436
group.
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Figure 3: (a) Light micrograph of spermatozoa of control group receiving PBS (b)
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Representative light micrograph of decapitated spermatozoa from mouse administered
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intraperitoneally with S. marcescens (c) Representative light micrograph of nonspecific
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aggregation showing large number of pus cells due to the S. marcescens infection in group
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IV. Arrows are showing decapitated heads.
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Figure 4: Representative photograph of reisolation of S. marcescens from homogenates of
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testis, caudal epididymis and vas deferens of (a, b, c) group I, (d, e, f) group IV.
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Figure 5: Histopathological examination of reproductive organs vas deferens, caudal
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epididymis and testis of mice of group I (a, b, c), representative photomicrograph of
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reproductive organs vas deferens, caudal epididymis and testis of mice of group IV (d, e, f) at
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400 X magnification, bar 50 µm. Arrows are showing infiltration of leukocytes, * represents
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empty lumen of testis with few mature spermatozoa.
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The role of uropathogens in male infertility since, to date, there has been little knowledge about their absolute or relative contribution to male infertility. Serratia marcescens is an opportunistic pathogen involved in urogenital tract infections. The mice were intraperitoneally administered with different doses of S. marcescens for 10 consecutive days. The groups were evaluated for any change in body weight, tissue somatic index (%), seminal parameters, reisolation of the microorganism from reproductive organs and histopathology. Results showed regression in sperm concentration, motility, morphology and finally, the overall fertility potential of male mice. The histopathological examination revealed hypospermatogenesis in testes alongwith mild to dense inflammation in vas deferens and caudal epididymis in test groups.
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DOI:
10.1016/j.micpath.2017.01.061
Reference:
YMPAT 2108
To appear in:
Microbial Pathogenesis
Received Date: 15 February 2016 Revised Date:
27 January 2017
Accepted Date: 31 January 2017
Please cite this article as: Rana K, Thaper D, Prabha V, Is there a role for Serratia marcescens in male infertility: An experimental study?, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.01.061. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Is there a role for Serratia marcescens in male infertility: An experimental study?
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Kalpana Rana, Deepali Thaper and Vijay Prabha*
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Department of Microbiology, Panjab University, Chandigarh-160014, India.
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Running head: Bacteria and male infertility
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*Correspondence:
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Dr. (Mrs) Vijay Prabha
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Professor
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Department of Microbiology
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Panjab University
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Chandigarh -160014. India
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Telephone: 91-9417065675
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E mail: [email protected]
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Keywords: Serratia marcescens, male infertility, immobilization, decapitation, sperms
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Abbreviations and Acronyms
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PBS: phosphate buffered saline; TSI: Tissue Somatic Index
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Abstract
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Objective: Establishment of a male BALB/c mouse model to study the role of sperm impairing S.
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marcescens on mouse reproductive potential. The current study can add to use of reliable animal
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models to provide a noteworthy evidence for the microbial cause of infertility. in our environment.
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Methods: The mice in the experimental test groups II, III, IV were intraperitoneally administered
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with different doses (104, 106 or 108 cfu) of S. marcescens whereas, group I serving as control,
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received PBS, for 10 consecutive days. The groups were evaluated for any change in body weight,
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tissue somatic index (%), seminal parameters and histology. Confirmation of S. marcescens from
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reproductive organs was done by reisolating the same by cultural characteristics and biochemical
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tests. microorganism from tissue homogenates of reproductive organs giving a knowhow of its
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spread and distribution.
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Results: The results showed that weight gain was evident only in mice receiving PBS (group I),
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whereas a decrease was recorded in the test groups (group II, III and IV). Only testes of test groups
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showed significant changes in TSI values whereas, no change in TSI was observed in any
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reproductive organ of any test group. The testes of all the test groups showed significant change in
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tissue somatic index, whereas, no change was observed in TSI value of all the other reproductive
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organs of all the groups. Seminal parameters viz. sperm count, motility and viability were found to
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decrease in test groups II, III and IV as compared to control group I. Interestingly, the number of
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pus cells and percent decapitation was more prominent in test groups which received higher doses
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(i.e. group III and group IV). With the increase in dose, there were more regressive changes (eg.
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oligo- astheno-teratotospermia) in seminal parameters. The histopathological examination revealed
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hypospermatogenesis alongwith mild to dense inflammation in vas deferens and caudal epididymis
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in all test groups except hypospermatogenesis which was observed only in test group III and IV.
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However, in group I, neither adverse changes nor any sign of inflammation were observed.
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Conclusion: Intraperitoneal inoculation of S. marcescens could lead to alteration of semen
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parameters, induction of decapitation in spermatozoa and histopathological changes, thereby
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decreasing the reproductive potential of male mice.
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Microbial infections in the male genitourinary tract account for 15% of male infertility cases [1].
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The major classes of such pathogens include bacteria, viruses, fungi and parasites. Some of the
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most commonly isolated bacteria are Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria
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gonorrhoeae, Staphlyococcus sp., members of the family Enterobacteriaceae viz., Escherichia
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coli, Klebsiella sp. and Proteus sp. [2]. These pathogens can attack different sites of male
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reproductive tract such as testis, epididymis, vas deferens and male accessory sex glands, thereby,
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causing reproductive disturbances and hence, leading to infertility. This form of infertility, which
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arises as a result of infectious agents, is considered as a potentially correctable type of infertility
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and hence, can be treated by antibiotics [3]. However, a large number of patients remain untreated,
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because of lack of diagnosis of the infectious agent. The asymptomatic course of the disease and
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unspecific clinical signs allow the infection to remain unnoticed, thereby resulting into chronic
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illness [4]. This has brought into focus, the role of these asymptomatic colonizers in male
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infertility since, to date, there has been little knowledge about their absolute or relative
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contribution to male infertility. In this regard, So far, most of the work has been accredited to
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organisms such as C. trachomatis, Ureaplasma sp., N. gonorrhoeae and Herpes simplex virus for
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their role in male infertility [5-8]. However, the data concerning the effect of other asymptomatic
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microorganisms on male infertility is negligible.
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In an earlier work done in our laboratory, infertility in female BALB/c mice was observed
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upon vaginal colonization with sperm-impairing Serratia marcescens and Candida albicans [9].
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So, with an aim to assess the role of S. marcescens in male infertility, intraperitoneal infection in
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mouse model was established and the effects on the seminal parameters were studied.
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Material and Methods
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Ethical note
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All the experiments have been conducted in compliance with the protocols approved by
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Institutional Animal Ethics Committee, Panjab University (vide letter no. IAEC/504 dated 2 April
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2014). All experiments were completed in agreement with the guidelines of the Committee for the
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Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
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Animals
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Adult 5-6 weeks old male (25±2g) BALB/c mice were used in the present study. Animals were
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individually housed in polypropylene cages in the animal room of Department of Microbiology,
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Panjab University, Chandigarh, India. The animals were fed with standard pellet food and water ad libitum and maintained in laboratory conditions (12:12, dark:light cycle).
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Microorganism
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The standard strain of S. marcescens (MTCC 7641), used in the present study, was procured from
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the Microbial Type Culture Collection, Institute of Microbial Technology, Sector 39, Chandigarh,
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India. The strain was grown in Brain Heart Infusion (BHI) broth and maintained as glycerol stocks
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at -80oC. The strain was found to cause sperm impairment by agglutination in vitro.
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Inoculum
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Serratia marcescens strain was cultivated in BHI broth, incubated under shaking conditions (150
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rpm) for 24h at 37ºC and then centrifuged at 10,000 rpm for 20 min. The pellet obtained was
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washed twice with PBS (50mM, pH 7.2). The cells were resuspended in the same buffer to get a
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cell count of 104, 106, and 108cfu/20µl.
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Inoculation procedure
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Male BALB/c mice were divided into 4 groups with 3 mice in each group. Group I, inoculated
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intraperitoneally with 20µl of PBS alone for 10 consecutive days, served as a control. The
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remaining three test groups (Group II-IV) were intraperitoneally administered with 104, 106 or
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108cfu of S. marcescens per mouse in 20µl PBS respectively for the same time period. Weight
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profile of all the mice groups was evaluated. The mice were sacrificed on day 12 and the
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parameters which were evaluated include TSI (%), seminal parameters and histopathological
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changes. The experiment was repeated twice for consistency of the results.
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Weight responses and Tissue Somatic Indices TSI (%)
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Initial body weight of mice from each group was taken on the 1st day of experiment and final
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weight on the last day of experiment. On day 12, mice from each group were sacrificed by cervical
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dislocation and the various reproductive organs (viz. testis, caudal epididymis and vas deferens)
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were removed aseptically. The organs were grossly examined and weighed. The TSI (percent
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tissue/organ weight in relation to body weight) was evaluated [10].
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Determination of Seminal parameters
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Sperm count and sperm morphology
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Mice from each group were sacrificed by cervical dislocation on day 12 and were dissected. The
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vas deferens was pulled out and placed in freshly prepared 500 µl of PBS buffer (50 mM, pH 7.2).
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fixed volume of 10µl of the sample was placed on a glass slide and examined under light
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microscope at 400X magnification. Around six to eight fields were scanned and mean number of
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spermatozoa in all the fields was multiplied by 106. The slides were also assessed for the
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morphology of spermatozoa in each field to evaluate the respective abnormalities [11].
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Sperm motility assay
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Motility of the sperms extracted from the sacrificed mice was determined by the method of
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Emmens [12].
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Sperm viability assay
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In order to estimate the percentage of viable sperms, an equal volume of mouse spermatozoa was
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mixed with 0.5% eosin and examined under the light microscope at 400X magnification.
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Reisolation of S. marcescens from the various reproductive organs on day 12
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The various reproductive organs were homogenized manually in sterile PBS (50 mM, pH 7.2) to
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form a uniform homogenate. 100µl of serially diluted tissue homogenate was plated on BHI agar
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plates and incubated overnight at 37ºC. The bacterial load in terms cfu/mouse/g of tissue was
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calculated. The reisolated bacteria from various organs were confirmed as S. marcescens by
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culture characteristics and biochemical tests. Further the isolates were also checked for
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spermagglutinating activity in vitro.
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Histopathological Studies
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Histopathological analyses of the reproductive organs (testis, caudal epididymis and vas deferens)
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of mice from each group sacrificed on day 12 were carried out. The various reproductive organs
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were harvested, fixed in 10% formaldehyde for 24h and then embedded in paraffin according to
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standard histological methods. Serial paraffin sections of 4 mm were stained with hematoxylin-
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eosin and observed at 400X magnification for any significant changes in reproductive organs.
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Statistical analysis of data
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The results of all the experiments in this study were analysed using Microsoft Word Excel
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software. The statistical significance of differences between control and test groups was evaluated
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by Student’s t-test. Results are expressed as mean± standard deviation. Differences were
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considered to be statistically significant when p value < 0.05.
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The impact of S. marcescens on body weight, TSI (%) of various reproductive organs, seminal
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parameters and histological changes in mice belonging to each group was evaluated.
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Weight profile
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When the weight profile study of different groups of mice was conducted, the results showed that
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there was 21% increase in weight of the control group which received PBS alone, whereas 15%,
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18% and 21% decrease in body weight was recorded in test groups (II, III and IV) which
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received 104cfu, 106cfu and 108cfu of S. marcescens respectively (Figure: 1).
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Tissue somatic indices TSI (%)
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Tissue somatic indices of the reproductive organs excised from all test groups of mice were
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determined on day 12. In comparison with control group I, the significant changes in TSI values
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were observed in testes of all test groups. As compared to group I, there was decrease of approx.
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24%, 39%, and 50% in TSI values of testis of test group II, III, IV respectively. However, no
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significant alterations were observed in vas deferens and caudal epididymis of group II, III and IV
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when compared with group I (Figure: 2).
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Evaluation of Seminal parameters
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On day 12, all the animals were sacrificed and dissected for assessment of seminal parameters viz.
180
sperm count, motility, viability and morphology. In the test groups, an alteration in seminal
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parameters was observed. The sperm count of test group II, III and IV group II, group III and
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group IV reduced significantly to 9x106 / ml, 7x106 /ml, 4x106 /ml respectively when compared to
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group I (28x106 / ml). Similarly, motility and viability percentage also declined drastically for the
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test groups (group II, III and IV) as compared to control group I. There was decrease in sperm
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count (68%, 75%, 86%), percent motility (86%, 93%, 93%), percent viability (66%, 76%, 83%) in
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test group II, III, IV respectively, as compared to control group I. . Interestingly, the number of pus
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cells and decapitation of mouse spermatozoa was observed in all the test groups, group II (14%),
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group III (24%) and group IV (61%). However, the This teratospermic effect was observed in all
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test groups, but was more pronounced in group IV. the number of pus cells and percentage of
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decapitation was more prominent in test groups which received higher doses (i.e. group III, IV).
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With the increase in dose, more regressive changes (i.e. oligo- astheno-teratotospermia) in the
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seminal parameters could be observed (Table 1, Figure: 3).
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S. marcescens could not be recovered from any of the tested reproductive organs of the mice in
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control group (i.e. group I). However, the organism could be efficiently reisolated from the
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homogenates of testis, caudal epididymis and vas deferens of mice belonging to test group II, III
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and IV (Table 2, Figure: 4). According to the biochemical tests viz. Oxidase – ve, MR – ve, VP +
199
ve, Citrate + ve, and glucose- fermenting, the Gram –ve organism so isolated was confirmed to be
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S. marcescens. Moreover, it was capable of causing 100% spermagglutination in vitro.
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Histopathological examination of various organs of groups of male BALB/c mice
202
administered with different doses of S. marcescens on day 12
203
To check any adverse affect of S. marcescens on tissue morphology of various reproductive organs
204
viz. testis, epididymis and vas deferens, histopathological analysis was carried out. The control
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group revealed normal tissue histology in all the organs. The testes showed regular seminiferous
206
tubules and germinal cell morphology. Epididymis displayed the well-vascularized loose
207
connective tissue present around the epididymal duct. Further, normal columnar epithelium was
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seen in vas deferens. However, the test group II mice showed that testis had nearly normal
209
spermatogenesis whereas mild interstitial inflammation was observed in vas deferens and caudal
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epididymis. Interestingly, in case of test group III mice, testis showed hypospermatogenesis along
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with no excess of sertoli cells. Inflammation was observed both in epididymis (i.e. epididymitis)
212
and vas deferens. Similarly, in case of test group IV mice, there were only few- matured
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spermatocytes leading to hypospermatogenesis. Also, there was dense inflammation in caudal
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epididymis (epididymitis) and vas deferens (F- Funiculitis) (Figure: 5).
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Discussion
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Infertility is a condition in which conception does not occur even after one year of regular
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unprotected sexual intercourse [13]. Out of the 15% of the affected couples, the male factor is
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involved in 40-50% of the infertility cases [14-16]. Of the various reasons cited for male infertility,
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the microbial infections have not been given their due because of their asymptomatic nature. Even
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though the microorganisms could be isolated from semen of both fertile as well as infertile men,
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yet several reports have surfaced up regarding their greater frequency in semen samples from
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asymptomatic infertile patients than in those from fertile men. The microorganisms which are most
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prevalent in semen belong to Gram positive viz. S. aureus, Coagulase negative staphylococci
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species, Enterococcus faecalis, Candida species and Gram negative microorganisms viz. E. coli,
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Klebsiella sp., Proteus sp., Pseudomonas sp. and Serratia sp. [17, 18]. The presence of these
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still controversial. In this regard, several investigators have also reported the in vitro detrimental
229
effect of microorganisms on sperms. For instance, Berktas et al. [19] has have reported that
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incubation of spermatozoa with E. coli resulted in reduced sperm motility in vitro. Similarly, U.
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urealyticum could cause loss of motility via multi-sperm agglutination [6] whereas S. aureus has
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been known to impede sperm motility in vitro by secreting the factor extracellularly [20]. The
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majority of these observations are derived from experimental in vitro studies, but their significance
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for in vivo infections has been questioned.
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The standard strain of S. marcescens, an opportunistic pathogen from the Enterobacteriaciae
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family which tends to colonize the genitourinary tract, was found to impair sperm motility in vitro
237
by causing agglutination of sperms and hence, leading to infertility in female BALB/c mice upon
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intravaginal administration [9]. These results prompted us to extrapolate the same in male BALB/c
239
mice. Therefore, the present study was undertaken to discover the impact of intraperitoneal
240
administration of S. marcescens on seminal parameters of male BALB/c mice. For this, different
241
doses of S. marcescens i.e. 104, 106, 108 cfu (20µl/mouse) were intraperitoneally administered in
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male BALB/c mice for 10 consecutive days. On day 12, the mice were sacrificed and evaluated for
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weight profile, TSI (%), seminal parameters and histopathological changes.
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The weight profile study showed decrease in body weight of mice in all the test groups (receiving
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104, 106, 108 cfu of S. marcescens) as compared to the control group which received PBS where an
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increase in weight was observed. These results are in concordance with the studies of Heeckeren et
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al. [21] wherein they have studied the role of Pseudomonas infection and have reported a drastic
249
weight loss after 3 days of infection. Further, the relevance of body condition and somatic indices
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(testis, vas deferens and cauda epididymis) as indicators of health status of the animals was
251
investigated. The results showed that there was significant reduction in TSI values of testes of all
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test groups which is indicative of diminished reproductive vigor of the mice as a result of S.
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marcescens infection. These results are in contrast to the study conducted by Kaur and Prabha [22,
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23] wherein no difference was found in TSI of all the reproductive organs of female mice
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receiving intravaginal sperm agglutinating S. aureus and E. coli as compared to control group
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which received PBS only.
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Some of the major determinants of fertilization potential of spermatozoa are based on their
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microscopic assessment of concentration, motility, viability, morphology and presence of pus cells.
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In the present study, an adverse effect was observed on all the seminal parameters in all test groups 8
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and viability could be very well correlated with increase in dose of S. marcescens indicating that
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the microorganism under study had detrimental effects on seminal parameters. To support this, a
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study conducted by Sellami et al. [5] has shown that the inoculation of male Swiss mice in meatus
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urethra with 106 C. trachomatis IFU resulted in decreased reproductive performance of male mice
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as observed by seminal parameter alterations. Furthermore, from our results, it was observed that
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an increase in the dose of S. marcescens lead to a rise in number of percent decapitation and pus
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cells which further indicated that the response is purely dose-dependent. In the light of present
269
finding, it could be speculated that S. marcescens infection could be responsible for the separation
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of heads from tails by inducing specific morphogenetic defects at different stages of spermatid
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differentiation. This is supported supports the findings by the findings of Grewal et al. [24] who
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showed that the culture supernatants of Bacillus sp. and Pseudomonas sp. not only decreased the
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motility of spermatozoa but also decapitated a few spermatozoa. Also, while studying the
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relationship between semen quality and pyospermia, Khan et al. [25] showed that pyospermia is
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associated either with compromised sperm motility or altered morphology.
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The next experiment was aimed at validating the proposal that the changes in weight profile, TSI
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(%) and seminal parameters in the male BALB/c mice were only due to the pathogen studied.
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Therefore, reisolation of S. marcescens and histopathological examination of reproductive tissues
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of mice was conducted. With an aim to know about the spread and distribution of the organism to
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reproductive organs, homogenates of all these the reproductive organs were plated on BHI agar
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and the results showed that S. marcescens could be efficiently recovered from the reproductive
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organs of all the mice in test groups II, III and IV. These findings correspond to those of previous
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study of Kruszewska et al. [26], wherein they were able to successfully culture Coxiella burnetii
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from various reproductive and non-reproductive organs of male mice from day 7 till day 49 p. i.
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when challenged intraperitoneally. Similarly, intraperitoneal challenge of mice with Shigella strain
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lead to a systemic infection at very early time points and the organism could be efficiently
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recovered from intestinal tissues [27].
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It is already a known fact that successful intraperitoneal bacterial invasion and colonization of the
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host can lead to multiple histological changes that are produced by the intrinsic host defense
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system in response to the infection. On the similar lines, the histopathological evaluation of the
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present study shows mild to dense inflammation in vas deferens and caudal epididymis of mice in
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the test groups II, III and IV in comparison to the control (group I) where normal tissue histology 9
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inflammation was more prominent in the testes of group of mice receiving higher doses of S.
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marcescens. From these results, it could be inferred that inflammation infection could be the
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underlying reason behind disruption of spermatogenesis, which has earlier also been reported by
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Lu et al. where uropathogenic E. coli was inoculated in male rats and after seven days p. i., it was
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seen that bacterial infection resulted in severe impairment of spermatogenesis with significantly
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decrease in sperm number and damage to germ and testicular cells [30]. which in itself is a major
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determinant of fertility potential of males. In another study Similar histopathologic findings have
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been carried out by Jantos et al. [28, 29] reported in two different studies, where the testes of rats
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were characterized by inflammation and moderate to severe loss of spermatogenesis on day 14
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post inoculation with Chlamydia psittaci and C. trachomatis. These results indicate that
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reproductive tract infections as well as inflammations have profoundly negative effects on
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testicular function resulting in reduced androgen production, lowered sperm counts and temporary
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loss of fertility.
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In conclusion, our data showed that intraperitoneal inoculation of S. marcescens an asymptomatic
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colonizer could lead to regression in sperm concentration, motility, morphology and finally, the
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overall fertility potential of male mice. However, further studies in this direction need to be carried
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out, and that too with greater number of strains, so as to obtain a statistically significant data and
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gather conclusive evidence in support of this hypothesis.
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Conflicts of interest
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The authors report no potential conflicts of interest.
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Acknowledgement
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The support of the Department of Science and Technology, New Delhi, India is gratefully
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acknowledged.
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Table 1: Effect of different doses of S. marcescens on sperm parameters on day 12
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Seminal parameters Groups Sperm count (x 106 /ml)
Motility %
-
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Group II
+
9
Group III
++
7
Group IV
++
402 403 404
8
20
14% Decapitation
4
14
24% Decapitation
4
10
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Normal
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4
Morphology
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Viability %
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Pus cells
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Table 2: Reisolation of S. marcescens on day 12 from various organs after intraperitoneal
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inoculation. Organs Testis
Caudal epididymis
-
-
+
Group III
+
Group IV
+
+
416
-
417 418 419
+
+
+
+
+
+
S. marcescens recovered S. marcescens not recovered
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Vas deferens
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Figure legends
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Figure 1: Weight profile of male BALB/c mice administered with different doses of S.
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marcescens for 10 consecutive days. Values represent mean values ±SD.
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Figure 2: Tissue somatic indices (%) of various reproductive and non reproductive organs
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after 10 days intraperitoneal administration of S. marcescens. Data represent mean values
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±SD. * represents p < 0.05 which indicate significant value when compared to control PBS
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group.
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Figure 3: (a) Light micrograph of spermatozoa of control group receiving PBS (b)
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Representative light micrograph of decapitated spermatozoa from mouse administered
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intraperitoneally with S. marcescens (c) Representative light micrograph of nonspecific
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aggregation showing large number of pus cells due to the S. marcescens infection in group
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IV. Arrows are showing decapitated heads.
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Figure 4: Representative photograph of reisolation of S. marcescens from homogenates of
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testis, caudal epididymis and vas deferens of (a, b, c) group I, (d, e, f) group IV.
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Figure 5: Histopathological examination of reproductive organs vas deferens, caudal
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epididymis and testis of mice of group I (a, b, c), representative photomicrograph of
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reproductive organs vas deferens, caudal epididymis and testis of mice of group IV (d, e, f) at
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400 X magnification, bar 50 µm. Arrows are showing infiltration of leukocytes, * represents
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empty lumen of testis with few mature spermatozoa.
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Highlights
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The role of uropathogens in male infertility since, to date, there has been little knowledge about their absolute or relative contribution to male infertility. Serratia marcescens is an opportunistic pathogen involved in urogenital tract infections. The mice were intraperitoneally administered with different doses of S. marcescens for 10 consecutive days. The groups were evaluated for any change in body weight, tissue somatic index (%), seminal parameters, reisolation of the microorganism from reproductive organs and histopathology. Results showed regression in sperm concentration, motility, morphology and finally, the overall fertility potential of male mice. The histopathological examination revealed hypospermatogenesis in testes alongwith mild to dense inflammation in vas deferens and caudal epididymis in test groups.
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