PCR primer employed in the detection of
Abstract
The objects of study is concerted to investigate the occurrence of Ureaplasma parvum in women with recurrent abortion and to determine the distribution of U. parvum serovars (1, 3, 6, 14) in women with recurrent abortion by conventional polymerase chain reaction (PCR) technique. In total, 130 samples included vaginal bleeding, vaginal swab, and urine, were collected from women with recurrent abortion and 40 samples included vaginal swab and urine from control women without recurrent abortion. Through the study, two types of media were used, Ureaplasma broth (IH Broth) and Ureaplasma agar (IH Agar). The positive isolates for Ureaplasma spp. were investigated by conventional PCR technique for identification of U. parvum and subtyping to their serovars (1, 3, 6, 14). The results revealed the U. parvum was identified in 29.6% from patient group and 11% from the control group. U. parvum isolates were further subtyped by using PCR, the results showed the serovar 3 was the most frequent isolate in proportion (42.8%), whereas serovar 1 (28.5%), serovar 6 (14.2%), and serovar 14 (14.2%) in patient group but in the control group only serovar 1 was isolated in rate (11%). These results evidently indicate that U. parvum may be an important etiologic agent for recurrent abortion.
Keywords
- IH medium
- PCR
- recurrent miscarriage
- serovars
- Ureaplasma parvum
1. Introduction
Recurrent miscarriage is the loss of three or more consecutive pregnancies ending of pregnancy by removing a fetus or embryo before it can survive outside the uterus [1, 2]. A miscarriage which occurs spontaneously is also known as a miscarriage and World Health Organization (WHO) explained the around 56 million recurrent miscarriage before the 24th week of gestation occur each year in the world unexplained [3].
Any sever infection that leads to bacteraemia or viraemia can cause sporadic miscarriage. The role of infection in recurrent miscarriage is unclear; the presence of bacterial vaginosis in the first trimester of pregnancy has been reported as a risk factor for second-trimester miscarriage and preterm delivery [4].
Although more than 30 years of study inside and outside of Iraq, many clinical importance of genital
2. Materials and methods
2.1. The bacterial isolates
In total, 130 samples included vaginal bleeding, vaginal swab, and urine, were collected from women with recurrent abortion and 40 samples included vaginal swab and urine from control women without recurrent abortion. All specimens were cultured in IH broth, which consists of PPLO broth, tryptone soya broth, yeast extract powder, distilled water, and supplements [22]. Then make a subculture to IH agar, which consists of PPLO agar, tryptone soya broth, MgSO4·H2O, yeast extract powder, agar-agar, distilled water, and supplements [22]. The
2.2. Molecular experiments
Molecular experiments included the extraction of
Organism | Primer (F)/(R) | Sequence (5′-3′) | Size of amplified product (bp) | Target gene |
---|---|---|---|---|
UMS-57 | F (TAA ATC TTA GTG TTC ATA TTT TTT AC -57) | 326 | 5′ Ends of MBA genes and upstream regions | |
UMA222 | R (GTA AGTGGA TTA AAT TCA ATG 222) |
Organism | Primer (F)/(R) | Sequence (5′-3′) | Size of amplified product (bp) | Target gene |
---|---|---|---|---|
Serovar 1 | UMS-83/UMA1A | F (TTACT GTA GAA ATT ATG TAA GAT TGC) R (TTT CTT TTG GTT CTT CAG TTT TTG AAG) | 578 | MBA |
Serovar 3 | UMS3S/UMA269 | F (TTA CTG TAG AAA TTA TGT AAG ATT ACC) R (AA CTA AAT GAC CTT TTT CAA GTG TAC) | 400 | MBA |
Serovar 6 | UMS-54/UMA269 | F (AAT CTT AGT GTT CAT ATT TTT TAC TAG) R (ACCA AAT GAC CTT TTG TAA CTA GAT) | 370 | MBA |
Serovar 14 | UMS14S/UMA314A | F (AAT TAC TGT AGA AAT TAT GTA AGA TTA AT) R (GTT GTT CTT TAC CTG GTT GTG TAG) | 572 | MBA |
2.3. PCR technique
The 20 ul amplification reaction mixtures contained 10 pmol of each primer, 5 ul of DNA template, and PCR water added to 20 ul for identification
3. Statistical analysis
The data were analyzed using SPSS statistic software version 20 (IBM, Armonk, USA) for comparison of qualitative variables using
4. Results and discussion
4.1. Laboratory identification of Ureaplasma spp. (colonial morphology)
In this study
4.2. Isolation of Ureaplasma spp. on culture media
Detection of
The medium contains urea and sensitive indicators of ammonia, Manganous sulfate which is firstly used by [27]. Sulfate salt of manganese was described to support the growth. Manganous sulfate was added in a final concentration of 0.03% was therefore, selected as the ammonia – detecting reagent of choice.
Moreover, putrescine – dihydrochloride was added to enhance the
Other study used IH medium for isolate
4.3. Comparison between age groups of miscarried women and percentage of isolation of Ureaplasma spp.
The results of this study showed that the age patients range 17–26 & 27–36 years old represented a high rate (46%), (40.7%) respectively compared with group (37–46) represented (13.3%) as shown in Figure 3 included in this study. The incidence of
This can be attributed to the sexual activity among this groups since there is an increased in estrogen hormone produced from female genital tract leading to change the vaginal environment which is regarded as a factor for infection [33]. because the estrogen is important hormone during pregnancy, it makes to adjust the level of the hormone progesterone is essential in the formation and development of the fetus, so pregnancy is one of the reason leading to the rise in the hormone estrogen [34]. For this reason, when the increased of estrogen, it affects the hormone progesterone is necessary in pregnancy, which affects the thickness of the lining of the uterus and the difficulty of adapt fetus with her. However it is generally difficult to determine whether these agents cause colonization or infection. Since the incidence of infection is affected by some factors, such as: menstrual cycle, bacterial and protozoan infection (co-infections), and socio-economic conditions like poverty. Also the age group 17–26 & 27–36 years are the most widely accepted for marriage and reproduction in our society for this reason the proportion of isolation the
4.4. Relationship between the isolation of genital Ureaplasma spp. and type of specimen
Figure 4 shows the distribution of bacterial isolate of genital
An explanation for these variations may be related to the type of specimens investigated for isolation, the methods used for transport and storage, and media used for primary isolation of
4.5. Molecular detection for diagnostic of Ureaplasma parvum by polymerase chain reaction (PCR)
The results showed the
Although
5. Conclusion
The results evidently indicate that
References
- 1.
Grimes DA, Stuart G. Abortion jabberwocky: The need for better terminology. Contraception. 2010; 81 (2):93-96 - 2.
Clark P, Walker ID, Langhorne P, Crichton L, Thomson A, Greaves M, et al.; Scottish Pregnancy Intervention Study (SPIN) collaborators. SPIN (Scottish Pregnancy Intervention) study: A multicenter, randomized controlled trial of low-molecular-weight heparin and low-dose aspirin in women with recurrent miscarriage. Blood. 2010; 115 :4162-4167 - 3.
Capoccia R, Greub G, Baud D. Ureaplasma urealyticum ,Mycoplasma hominis and adverse pregnancy outcomes. Current Opinion in Infectious Diseases. 2013;26 (3):231-240 - 4.
Zhang N, Wang R, Li X, et al. Are Ureaplasma spp. a cause of nongonococcal urethritis? A systematic review and meta-analysis. PLoS One. 2014;9 :e113771 - 5.
Waites MD. Mycoplasma and Ureaplasma infection [Ph.D. thesis]. Collage of Medicine, University of Alabama at Birmingham; 2015 - 6.
Huang C, Zhu HL, Xu KR, et al. Mycoplasma and ureaplasma infection and male infertility: A systematic review and meta-analysis. Andrology. 2015; 3 :809 - 7.
Wetmore CM, Manhart LE, Lowens MS, et al. Ureaplasma urealyticum is associated with nongonococcal urethritis among men with fewer lifetime sexual partners: A case-control study. The Journal of Infectious Diseases. 2011;204 :1274 - 8.
Redelinghuys MJ, Ehlers MM, Dreyer AW, et al. A cross-sectional study on the relationship of age, gestational age and HIV infection to bacterial vaginosis and genital mycoplasma infection. BMJ Open. 2015; 5 :e008530 - 9.
Kokkayil P, Dhawan B. Ureaplasma : Current perspectives. Indian Journal of Medical Microbiology. 2015;33 :205-214 - 10.
Urszula K, Joanna E, Marek E, Mączyńska B, Sobieszczańska BM. Colonization of the lower urogenital tract with U. parvum can cause asymptomatic infection of the upper reproductive system in women. International Journal of Gynecology & Obstetrics. 2014;289 :1129-1134 - 11.
Kacerovsky K, Pavlovsky M, Tosner J. Preterm premature rupture of the membranes and genital mycoplasmas. Acta Medica (Hradec Králové). 2009; 52 (3):117-120 - 12.
Waites K, Xiao L, Paralanov V, Viscardi RM, Glass JI. Molecular methods for the detection of mycoplasma and Ureaplasma infections in humans. The Journal of Molecular Diagnostics. 2012; 14 :437-450 - 13.
Larsen B, Hwang J. Mycoplasma, Ureaplasma, and adverse pregnancy outcomes. Infectious Diseases in Obstetrics and Gynecology. 2011; 23 :138-141 - 14.
Kataoka S, Yamada T, Chou K, Nishida R, Morikawa M, Minami M, et al. Association between preterm birth and vaginal colonization by mycoplasmas in early pregnancy. Journal of Clinical Microbiology. 2006; 44 :51-55 - 15.
Glass JI, Lefkowitz EJ, Glass JS, Heiner CR, Chen, Cassell GH. The complete sequence of the mucosal pathogen Ureaplasma urealyticum . Nature. 2000;407 :757-762 - 16.
Trembath A, Laughon M. Predictors of bronchopulmonary dysplasia. Clinics in Perinatology. 2013; 39 :585-601 - 17.
Agbakoba NR, Adetosoye AI, Adesina OA, Adewole IF. Isolation of vaginal pathogens along with genital mycoplasmas from asymptomatic gynaecology and antenatal clinic attendees. American-Eurasian Journal of Scientific Research. 2008; 3 (2):195-198 - 18.
Holly B, Danny J. Recurrent pregnancy loss: Etiology, diagnosis, and therapy. Obstetrics and Gynecology. 2009; 2 :76-83 - 19.
Goldenberg RL, Hauth JC, Andrews WW. Intrauterine infection and preterm delivery. The New England Journal of Medicine. 2000; 342 :1500-1507 - 20.
Miralles R, Hodge R, McParland PC, Field DJ, Bell SC, Taylor DJ, et al. Relationship between antenatal inflammation and antenatal infection identified by detection of microbial genes by polymerase chain reaction. Pediatric Research. 2005; 57 :570-577 - 21.
Waites KB, Katz B, Schelonka RL. Mycoplasma and Ureaplasma as neonatal pathogens. Clinical Microbiology Reviews. 2005; 18 :757-789 - 22.
Al-Azawiy IH. Cultural and molecular detection of mycoplasmal urogenital infection in woman. International Journal of Medical Sciences. 2013; 1 :25-29 - 23.
Kong F, Ma Z, James G, Gordon S, Gilbert GL. Species identification and subtyping of Ureaplasma parvum andUreaplasma urealyticum using PCR-based assays. Journal of Clinical Microbiology. 2000;38 :1175-1179 - 24.
Kong F, Zhu X, Wang W, Zhou X, Gordon S, Gilbert GL. Comparative analysis and serovar-specific identification of multiple banded antigen genes of Ureaplasma urealyticum . Journal of Clinical Microbiology. 1999;37 :538-548 - 25.
Ahmadi A. Association between U. urealyticum endocervical infection and spontaneous abortion [Ph.D thesis]. Department of Microbiology, College of Medicine, Kurdistan University of Medical Sciences; 2014 - 26.
Miles RJ, Nicholas RA. Method in Molecular Biology. Vol. 102. ToTowa, NJ: Human Press Inc.; 2010 - 27.
Sudolska MB, Zakrzewska DR, Lauterbach R. Assessment of various diagnostic methods of Ureaplasma respiratory tract infections in newborns. Acta Biochimica Polonica. 2006; 53 (3):609-612 - 28.
Al-Talqani IA, Al-Musawi HS. Using multiplex PCR assay for detection of genital mycoplasmas and Ureaplasma spp. in an infertility males. Kufa Journal for Nursing Sciences. 2015;5 (2):17-23 - 29.
Al-Nassri AR. Isolation and identification of Mycoplasma pneumoniae from tonsillitis patient by morphology and molecular characterization [M.Sc. thesis]. Iraq: College of Medicine, University of Al-Qadisyia; 2016 (in English) - 30.
AL-Karawy HA. Immunological and molecular evaluation of occurrence of mycoplasma with urogenital tract infections of infertile patients in Al-Qadissyia Province [Ph.D. thesis]. Iraq: Collage of Medicine, University of Babylon; 2015 (in English) - 31.
Gupta A, Mittal A, Chandra P, Gill AK. Correlation of Mycoplasma with explained infertility. Archives of Gynecology and Obstetrics. 2009; 280 :P981-P985 - 32.
Demba E, Morison L, Loeff MS, Awasana AA, Gooding E, Bailey R, Mayau DP, West B. Bacterial vaginosis, vaginal flora patterns and vaginal discharge syndrome in the Gambia, West Africa. BMC Infectious Diseases. 2005; 5 (12) - 33.
Zeighami H, Peerayeh SN, Yazdi RS, et al. Prevalence of Ureaplasma urealyticum andUreaplasma parvum in semen of infertile and healthy men. International Journal of STD & AIDS. 2009;20 :378-398 - 34.
Lee JS, Kim KT, Lee HS, Yang KM, Seo JT, Choe JH. Concordance of Ureaplasma urealyticum andMycoplasma hominis in infertile couples: Impact on semen parameters. Urology. 2013;81 (6):1219-1224 - 35.
Yang J, Savitz DA, Dole N, et al. Predictors of vaginal bleeding during the first two trimesters of pregnancy. Paediatric and Perinatal Epidemiology. 2005; 19 :276-283 - 36.
Gomez R, Romero R, Nien JK, et al. Idiopathic vaginal bleeding during pregnancy as the only clinical manifestation of intrauterine infection. The Journal of Maternal-Fetal & Neonatal Medicine. 2005; 18 :P31-P37 - 37.
Harville EW, Wilcox AJ, Baird DD, Weinberg CR. Vaginal bleeding in very early pregnancy. Human Reproduction. 2003; 18 :1944-1947 - 38.
Dhawan B, Malhotr N, Sreenivas V, Rawre J, Khanna N, Chaudhry R, Mittal S. Ureaplasma serovars & their antimicrobial susceptibility in patients of infertility & genital tract infections. The Indian Journal of Medical Research. 2012; 136 :991-996 - 39.
Aydin Y, Atis A, Ocer F, Isenkul R. Association of cervical infection of Chlamydia trachomatis U. urealyticum andM. hominis with peritoneum colonisation in pregnancy. The Obstetrician and Gynaecologist. 2010;30 (8):809-812 - 40.
Knox C, Allan A, Allan M, Edirisinghe WR, Stenzel D, Lawrence FA, et al. Ureaplasma parvum andUreaplasma urealyticum are detected in semen after washing before assisted reproductive technology procedures. Fertility and Sterility. 2003;80 :921-929 - 41.
Ning H, Nakura Y, Motooka D, Nakamura S, Nishiumi F, Ishino S, et al. Complete genome sequence of Ureaplasma parvum serovar 3 strain SV3F4, isolated in Japan. Genome Announcements. 2014;2 :254-256 - 42.
Breugelmans M, Vancutsem E, Naessens A, Laubach M, Foulon W. Association of abnormal vaginal flora and Ureaplasma species as risk factors for preterm birth. Acta Obstetricia et Gynecologica Scandinavica. 2010;89 :256-260 - 43.
Ekiel AM, Friedek DA, Romanik MK, Jozwiak J, Martirosian G. Occurrence of Ureaplasma parvum andUreaplasma urealyticum in women with cervical dysplasia in Katowice, Poland. Journal of Korean Medical Science. 2009;24 :1177-1181 - 44.
Fouad N, Assar E, Abdullah O. Detection of Ureaplasma spp. in endotracheal aspirates from preterm infants with respiratory distress syndrome. Egyptian Journal of Medical Laboratory Sciences. 2014;23 (3):9-17