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• Authors’ response to Skafte-Holm et al. (668 words)
  Nicola Low, Marinjho Jonduo, Emma L Sweeney, Lisa M Vallely, Handan Wand, Dianne Egli-Gany, John M Kaldor and Andrew J Vallely
  Published on: 19 July 2023
• Mollicutes and adverse pregnancy outcomes: A challenging research area to navigate
  Axel Skafte-Holm, Thomas Roland Pedersen and Jørgen Skov Jensen
  Published on: 21 March 2023

• Published on: 19 July 2023

  Authors’ response to Skafte-Holm et al. (668 words)

  • Nicola Low, Professor of Epidemiology and Public Health University of Bern
  • Other Contributors:
    • Marinjho Jonduo, PhD student
    • Emma L Sweeney, Postdoctoral Research Fellow
    • Lisa M Vallely, Senior Research Fellow
    • Handan Wand, Associate Professor (Biostatistics and Databases)
    • Dianne Egli-Gany, Project Manager
    • John M Kaldor, Scientia Professor
    • Andrew J Vallely, Professor of Clinical Epidemiology

  To the editor,

  We are grateful to Axel Skafte-Holm et al. for their careful reading of our systematic review and for highlighting challenges in the classification of Ureaplasma species. We aimed to include studies that identified U. urealyticum or U. parvum using appropriate microbiological methods to examine associations with adverse pregnancy outcomes. For the 14 articles highlighted by Skafte-Holm et al. [1-14], we acknowledge that we used data that the authors of the included studies reported, but which were not consistent with the actual microbiological methods used.
  There is inconsistent reporting of Ureaplasma species owing to changes made to their taxonomy; the resulting misclassification of these species compounds the shortcomings of epidemiological methods of studies in this systematic review. As such, it reinforces our conclusion, that “The currently available literature does not allow conclusions about the role of mycoplasmas in adverse pregnancy and birth outcomes”.
  We updated our analysis of associations between the two Ureaplasma spp. and adverse pregnancy outcomes, following Skafte-Holm and colleagues’ investigations. We removed data from meta-analyses if identification of U. urealyticum was ambiguous and we amended data from studies that identified biovars of Ureaplasma spp. but reported them incorrectly.

  The original systematic review included 57 articles. Of the 14 articles highlighted by Skafte-Holm et al., 10 would remain; 2...

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  To the editor,

  We are grateful to Axel Skafte-Holm et al. for their careful reading of our systematic review and for highlighting challenges in the classification of Ureaplasma species. We aimed to include studies that identified U. urealyticum or U. parvum using appropriate microbiological methods to examine associations with adverse pregnancy outcomes. For the 14 articles highlighted by Skafte-Holm et al. [1-14], we acknowledge that we used data that the authors of the included studies reported, but which were not consistent with the actual microbiological methods used.
  There is inconsistent reporting of Ureaplasma species owing to changes made to their taxonomy; the resulting misclassification of these species compounds the shortcomings of epidemiological methods of studies in this systematic review. As such, it reinforces our conclusion, that “The currently available literature does not allow conclusions about the role of mycoplasmas in adverse pregnancy and birth outcomes”.
  We updated our analysis of associations between the two Ureaplasma spp. and adverse pregnancy outcomes, following Skafte-Holm and colleagues’ investigations. We removed data from meta-analyses if identification of U. urealyticum was ambiguous and we amended data from studies that identified biovars of Ureaplasma spp. but reported them incorrectly.

  The original systematic review included 57 articles. Of the 14 articles highlighted by Skafte-Holm et al., 10 would remain; 2 with revised data [1,13]; and 8 with data removed from some meta-analyses, but which also reported on Mycoplasma hominis [5-11,14]. Four articles would not be eligible for the review because the methods reported did not distinguish between Ureaplasma spp. [2-4,12].
  We repeated the random effects meta-analysis on revised datasets to estimate the summary odds ratio (OR) and 95% confidence intervals (CI) for 4 of 10 organism-outcome pairs involving Ureaplasma species. In 2 other pairs, only 1 study remained. We also removed data for one study, with no positive samples, from the meta-analysis of U. urealyticum and preterm birth.
  The direction of results did not change for new analyses of either U. urealyticum or U. parvum; the OR increased for 4 organism-outcome pairs and decreased for 2, with overlapping CIs for original and revised analyses in each pair. For U. urealyticum, the new analyses included fewer studies, which reduces the precision of summary ORs.

  Here we report the new findings, expressed as OR, 95% CI, and number of included studies (corresponding findings from the original version in brackets). The primary outcome was preterm birth. For U. urealyticum, the new summary was OR 1.96, 95% CI 1.14-3.39, 16 studies (original, 1.84, 1.34-2.55, 27 studies). For U. parvum, the new summary OR was 1.79, 95% CI 1.28-2.52, 13 studies (original, 1.60, 1.12-2.30, 11 studies).

  For secondary outcomes, we found the following associations between U. urealyticum and: premature rupture of membranes, summary OR 9.87, 95% CI 1.81-53.72, 4 studies (original, 4.27, 1.83-9.98, 11 studies); low birth weight, OR 1.08, 95% CI 0.08-14.41, 1 study (original, 2.24, 95% CI 1.16-4.33, 2 studies); spontaneous abortion, summary OR 2.43, 95% CI 1.21-4.86, 3 studies (original 1.74, 1.02-2.95, 4 studies); perinatal death, OR 3.52, 95% CI 0. 14-87.08, 1 study (original 9.50, 2.99-30.13, 2 studies). Secondary outcomes for associations with U. parvum were unaffected by the revised data.

  The revised results in this rapid response address Skafte-Holm et al.’s concern about how misclassification of Ureaplasma spp. Might have affected our systematic review findings. We will replace the full manuscript with a corrected version, in which we will replace text, tables, figures and supplemental material with the revised results. The authors of primary studies should also be alerted to errors in their articles. The causal role of genital mycoplasmas in adverse pregnancy outcomes still needs to be understood. The increasing number of commercially available PCR tests that detect multiple genital organisms, some of uncertain clinical relevance, will result in antimicrobial treatment that may be unnecessary. If, however, these organisms are important causes of adverse pregnancy outcomes, further research is needed to determine whether antenatal screening interventions are warranted. We propose multidisciplinary research to design definitive studies with state-of-the-art microbiological, clinical, epidemiological and statistical methods.

  References
  1. Mitsunari M, Yoshida S, Deura I, Horie S, Tsukihara S, Harada T, et al. Cervical Ureaplasma Urealyticum Colonization Might Be Associated with Increased Incidence of Preterm Delivery in Pregnant Women without Prophlogistic Microorganisms on Routine Examination. J Obstet Gynaecol Res. 2005;31(1):16-21. Epub 2005/01/27. doi: 10.1111/j.1447-0756.2005.00246.x. PubMed PMID: 15669986.
  2. Kafetzis DA, Skevaki CL, Skouteri V, Gavrili S, Peppa K, Kostalos C, et al. Maternal Genital Colonization with Ureaplasma Urealyticum Promotes Preterm Delivery: Association of the Respiratory Colonization of Premature Infants with Chronic Lung Disease and Increased Mortality. Clin Infect Dis. 2004;39(8):1113-22. Epub 2004/10/16. doi: 10.1086/424505. PubMed PMID: 15486833.
  3. Aaltone R, Jalava J, Laurikainen E, Karkkainen U, Alanen A. Cervical Ureaplasma Urealyticum Colonization: Comparison of Pcr and Culture for Its Detection and Association with Preterm Birth. Scand J Infect Dis. 2002;34(1):35-40. Epub 2002/03/05. doi: 10.1080/00365540110077074. PubMed PMID: 11874162.
  4. Povlsen K, Thorsen P, Lind I. Relationship of Ureaplasma Urealyticum Biovars to the Presence or Absence of Bacterial Vaginosis in Pregnant Women and to the Time of Delivery. Eur J Clin Microbiol Infect Dis. 2001;20(1):65-7. Epub 2001/03/14. doi: 10.1007/pl00011237. PubMed PMID: 11245329.
  5. Usui R, Ohkuchi A, Matsubara S, Izumi A, Watanabe T, Suzuki M, et al. Vaginal Lactobacilli and Preterm Birth. J Perinat Med. 2002;30(6):458-66. Epub 2003/01/18. doi: 10.1515/JPM.2002.072. PubMed PMID: 12530101.
  6. Kwak DW, Hwang HS, Kwon JY, Park YW, Kim YH. Co-Infection with Vaginal Ureaplasma Urealyticum and Mycoplasma Hominis Increases Adverse Pregnancy Outcomes in Patients with Preterm Labor or Preterm Premature Rupture of Membranes. J Matern Fetal Neonatal Med. 2014;27(4):333-7. Epub 2013/06/26. doi: 10.3109/14767058.2013.818124. PubMed PMID: 23796000.
  7. Kacerovsky M, Pavlovsky M, Tosner J. Preterm Premature Rupture of the Membranes and Genital Mycoplasmas. Acta Medica (Hradec Kralove). 2009;52(3):117-20. Epub 2010/01/16. doi: 10.14712/18059694.2016.115. PubMed PMID: 20073423.
  8. Montenegro DA, Borda LF, Neuta Y, Gomez LA, Castillo DM, Loyo D, et al. Oral and Uro-Vaginal Intra-Amniotic Infection in Women with Preterm Delivery: A Case-Control Study. J Investig Clin Dent. 2019;10(2):e12396. Epub 2019/01/22. doi: 10.1111/jicd.12396. PubMed PMID: 30663264.
  9. Lee MY, Kim MH, Lee WI, Kang SY, Jeon YL. Prevalence and Antibiotic Susceptibility of Mycoplasma Hominis and Ureaplasma Urealyticum in Pregnant Women. Yonsei Med J. 2016;57(5):1271-5. Epub 2016/07/13. doi: 10.3349/ymj.2016.57.5.1271. PubMed PMID: 27401661; PubMed Central PMCID: PMC4960396.
  10. Nasution TA, Cheong SF, Lim CT, Leong EW, Ngeow YF. Multiplex Pcr for the Detection of Urogenital Pathogens in Mothers and Newborns. Malays J Pathol. 2007;29(1):19-24. Epub 2007/06/01. PubMed PMID: 19105324.
  11. Daskalakis G, Thomakos N, Papapanagiotou A, Papantoniou N, Mesogitis S, Antsaklis A. Amniotic Fluid Interleukin-18 at Mid-Trimester Genetic Amniocentesis: Relationship to Intraamniotic Microbial Invasion and Preterm Delivery. BJOG. 2009;116(13):1743-8. Epub 2009/11/13. doi: 10.1111/j.1471-0528.2009.02364.x. PubMed PMID: 19906019.
  12. Abele-Horn M, Scholz M, Wolff C, Kolben M. High-Density Vaginal Ureaplasma Urealyticum Colonization as a Risk Factor for Chorioamnionitis and Preterm Delivery. Acta Obstet Gynecol Scand. 2000;79(11):973-8. Epub 2000/11/18. PubMed PMID: 11081683.
  13. Harada K, Tanaka H, Komori S, Tsuji Y, Nagata K, Tsutsui H, et al. Vaginal Infection with Ureaplasma Urealyticum Accounts for Preterm Delivery Via Induction of Inflammatory Responses. Microbiol Immunol. 2008;52(6):297-304. Epub 2008/06/26. doi: 10.1111/j.1348-0421.2008.00039.x. PubMed PMID: 18577163.
  14. Schwab FD, Zettler EK, Moh A, Schotzau A, Gross U, Gunthert AR. Predictive Factors for Preterm Delivery under Rural Conditions in Post-Tsunami Banda Aceh. J Perinat Med. 2016;44(5):511-5. Epub 2015/05/20. doi: 10.1515/jpm-2015-0004. PubMed PMID: 25980381.

  Authors
  Nicola Low, Professor of Epidemiology and Public Health, University of Bern (corresponding author)
  Marinjho Jonduo, PhD student, Papua New Guinea Institute of Medical Research
  Emma L Sweeney, Postdoctoral Research Fellow, University of Queensland
  Lisa M Vallely, Senior Research Fellow, University of New South Wales
  Handan Wand, Associate Professor (Biostatistics and Databases), University of New South Wales
  Dianne Egli-Gany, Project Manager, University of Bern
  John M Kaldor, Scientia Professor, University of New South Wales
  Andrew J Vallely, Professor of Clinical Epidemiology, University of New South Wales

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  Conflict of Interest:
  None declared.

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• Published on: 21 March 2023

  Mollicutes and adverse pregnancy outcomes: A challenging research area to navigate

  • Axel Skafte-Holm, MD Statens Serum Institut, Copenhagen, Denmark
  • Other Contributors:
    • Thomas Roland Pedersen, Cand. Scient. Biology
    • Jørgen Skov Jensen, Consultant physician, MD, PhD, DMedSci.

  Axel Skafte-Holm 1, Thomas Roland Pedersen 1 and Jørgen Skov Jensen 1*
  1 Department of Bacteria, Parasites and Fungi, Research Unit for Reproductive Microbiology, Statens Serum Institut, Copenhagen, Denmark

  *Corresponding author
  Jørgen Skov Jensen
  Consultant physician, MD, PhD, DMedSci.
  Statens Serum Institute
  5 Artillerivej
  DK-2300 Copenhagen S
  Denmark
  Research Unit for Reproductive Microbiology.
  Division of Diagnostic Infectious Disease Preparedness
  Telephone +45 3268 3636
  jsj@ssi.dk

  To the Editor,

  We read with interest the systematic review and meta-analysis by Jonduo et al. [1], investigating the association between Mycoplasma (M.) hominis, Ureaplasma (U.) urealyticum and U. parvum colonisation of the genital tract of pregnant women and adverse pregnancy outcomes. This is a highly relevant analysis, as this subject is a matter of ongoing debate, due to the complex interactions with other microbial and non-microbial factors. It is also a subject where enormous taxonomic confusion exists due to the re-classification of the ureaplasmas. We were, consequently, interested in the comprehensive analysis on the relative importance of the two Ureaplasma spp. Unfortunately, we detected some shortcomings, which need to be discussed.
  (I) Culture-based studies. The review excluded articles published before the year 2000 if unspeciated U. urealyticum were re...

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  Axel Skafte-Holm 1, Thomas Roland Pedersen 1 and Jørgen Skov Jensen 1*
  1 Department of Bacteria, Parasites and Fungi, Research Unit for Reproductive Microbiology, Statens Serum Institut, Copenhagen, Denmark

  *Corresponding author
  Jørgen Skov Jensen
  Consultant physician, MD, PhD, DMedSci.
  Statens Serum Institute
  5 Artillerivej
  DK-2300 Copenhagen S
  Denmark
  Research Unit for Reproductive Microbiology.
  Division of Diagnostic Infectious Disease Preparedness
  Telephone +45 3268 3636
  jsj@ssi.dk

  To the Editor,

  We read with interest the systematic review and meta-analysis by Jonduo et al. [1], investigating the association between Mycoplasma (M.) hominis, Ureaplasma (U.) urealyticum and U. parvum colonisation of the genital tract of pregnant women and adverse pregnancy outcomes. This is a highly relevant analysis, as this subject is a matter of ongoing debate, due to the complex interactions with other microbial and non-microbial factors. It is also a subject where enormous taxonomic confusion exists due to the re-classification of the ureaplasmas. We were, consequently, interested in the comprehensive analysis on the relative importance of the two Ureaplasma spp. Unfortunately, we detected some shortcomings, which need to be discussed.
  (I) Culture-based studies. The review excluded articles published before the year 2000 if unspeciated U. urealyticum were reported alone, as Ureaplasma spp. detected by culture previously were reported as U. urealyticum. In this matter, it is surprising that seven of the included articles [2-8] on U. urealyticum were culture-based. Although bacteriological culture is considered the gold standard it only identifies bacteria at the genus level, preventing differentiation between U. urealyticum (formerly biovar 2, T980, T960T or A) and U. parvum (formerly biovar 1, parvo or B). In the review, all results from culture-based articles were reported as U. urealyticum, probably because primary authors wrongly reported cultured ureaplasmas as U. urealyticum. Unless additional typing of isolates is performed, cultures should always be reported as Ureaplasma spp.
  (II) Confusion on nomenclature. Harada et al. determined the biovar of colonies using PCR-based typing where they reported all U. urealyticum (i.e. Ureaplasma spp.) colonies as biovar type 1 i.e. U. parvum among 23 women experiencing preterm birth, but the systematic review reported findings as U. urealyticum [9]. Two other articles [10, 11] reported on biovar 1 and biovar 2, but were cited only as U. urealyticum and the review failed to include these results in the U. parvum analysis.
  (III) Shortcomings in methodology. Most articles were based on real-time PCR and two articles [12, 13] used multiplex PCR to identify sexually transmitted infections. Some articles used conventional PCR, but one [14] failed to report information on PCR method and consequently evaluation of the species differentiation was not possible. In order to evaluate primers and probes in the studies included in the meta-analysis, we aligned sequences and queried them using the Basic Local Alignment Search Tool (BLAST) [15] against U. urealyticum serovar 8 (NCBI Reference Sequence: NZ_AAYN02000002.1) and U. parvum serovar 3 (NCBI Reference Sequence: NC_002162.1). Most studies correctly distinguished ureaplasmas at the species level. Unfortunately, we found methodological shortcomings in one paper [16], as primer/probe design did not allow discrimination of Ureaplasma spp. Two studies [17, 18] reported U. urealyticum, but alignment results indicated that the primers were 100% specific for U. parvum and contained several mismatches with the U. urealyticum sequence, thus, at best detecting Ureaplasma spp.
  In general, Mollicutes are complicated pathogens and our assessment of the literature demonstrates a laissez-faire approach to nomenclature and methods applied. The diagnostic methodologies applied should be carefully evaluated when assessing studies to allow conclusions about the relation between Ureaplasma spp. and clinical outcomes. The taxonomic and methodological confusion in the literature is concerning, so an in-depth analysis of the area is warranted and we suggest that the review authors update their analysis of associations between the two Ureaplasma spp. and adverse pregnancy outcomes. The review conclusions may not change because the majority of the studies suffered from lack of control for major confounding factors, but at least the combined effect estimates will be more correct.

  References

  1. Jonduo ME, Vallely LM, Wand H, Sweeney EL, Egli-Gany D, Kaldor J, et al. Adverse pregnancy and birth outcomes associated with Mycoplasma hominis, Ureaplasma urealyticum and Ureaplasma parvum: a systematic review and meta-analysis. BMJ Open. 2022;12(8):e062990. DOI: 10.1136/bmjopen-2022-062990.
  2. Lee MY, Kim MH, Lee WI, Kang SY, Jeon YL. Prevalence and Antibiotic Susceptibility of Mycoplasma hominis and Ureaplasma urealyticum in Pregnant Women. Yonsei Med J. 2016;57(5):1271-5. DOI: 10.3349/ymj.2016.57.5.1271.
  3. Daskalakis G, Thomakos N, Papapanagiotou A, Papantoniou N, Mesogitis S, Antsaklis A. Amniotic fluid interleukin-18 at mid-trimester genetic amniocentesis: relationship to intraamniotic microbial invasion and preterm delivery. Bjog. 2009;116(13):1743-8. DOI: 10.1111/j.1471-0528.2009.02364.x.
  4. Usui R, Ohkuchi A, Matsubara S, Izumi A, Watanabe T, Suzuki M, et al. Vaginal lactobacilli and preterm birth. J Perinat Med. 2002;30(6):458-66. DOI: 10.1515/jpm.2002.072.
  5. Kwak DW, Hwang HS, Kwon JY, Park YW, Kim YH. Co-infection with vaginal Ureaplasma urealyticum and Mycoplasma hominis increases adverse pregnancy outcomes in patients with preterm labor or preterm premature rupture of membranes. J Matern Fetal Neonatal Med. 2014;27(4):333-7. DOI: 10.3109/14767058.2013.818124.
  6. Kafetzis DA, Skevaki CL, Skouteri V, Gavrili S, Peppa K, Kostalos C, et al. Maternal genital colonization with Ureaplasma urealyticum promotes preterm delivery: association of the respiratory colonization of premature infants with chronic lung disease and increased mortality. Clin Infect Dis. 2004;39(8):1113-22. DOI: 10.1086/424505.
  7. Abele-Horn M, Scholz M, Wolff C, Kolben M. High-density vaginal Ureaplasma urealyticum colonization as a risk factor for chorioamnionitis and preterm delivery. Acta Obstet Gynecol Scand. 2000;79(11):973-8. DOI.
  8. Kacerovský M, Pavlovský M, Tosner J. Preterm premature rupture of the membranes and genital mycoplasmas. Acta Medica (Hradec Kralove). 2009;52(3):117-20. DOI: 10.14712/18059694.2016.115.
  9. Harada K, Tanaka H, Komori S, Tsuji Y, Nagata K, Tsutsui H, et al. Vaginal infection with Ureaplasma urealyticum accounts for preterm delivery via induction of inflammatory responses. Microbiol Immunol. 2008;52(6):297-304. DOI: 10.1111/j.1348-0421.2008.00039.x.
  10. Mitsunari M, Yoshida S, Deura I, Horie S, Tsukihara S, Harada T, et al. Cervical Ureaplasma urealyticum colonization might be associated with increased incidence of preterm delivery in pregnant women without prophlogistic microorganisms on routine examination. J Obstet Gynaecol Res. 2005;31(1):16-21. DOI: 10.1111/j.1447-0756.2005.00246.x.
  11. Povlsen K, Thorsen P, Lind I. Relationship of Ureaplasma urealyticum biovars to the presence or absence of bacterial vaginosis in pregnant women and to the time of delivery. Eur J Clin Microbiol Infect Dis. 2001;20(1):65-7. DOI: 10.1007/pl00011237.
  12. Koucký M, Malíčková K, Cindrová-Davies T, Smíšek J, Vráblíková H, Černý A, et al. Prolonged progesterone administration is associated with less frequent cervicovaginal colonization by Ureaplasma urealyticum during pregnancy - Results of a pilot study. J Reprod Immunol. 2016;116:35-41. DOI: 10.1016/j.jri.2016.04.285.
  13. Peretz A, Tameri O, Azrad M, Barak S, Perlitz Y, Dahoud WA, et al. Mycoplasma and Ureaplasma carriage in pregnant women: the prevalence of transmission from mother to newborn. BMC Pregnancy Childbirth. 2020;20(1):456. DOI: 10.1186/s12884-020-03147-9.
  14. Schwab FD, Zettler EK, Moh A, Schötzau A, Gross U, Günthert AR. Predictive factors for preterm delivery under rural conditions in post-tsunami Banda Aceh. J Perinat Med. 2016;44(5):511-5. DOI: 10.1515/jpm-2015-0004.
  15. Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol. 2000;7(1-2):203-14. DOI: 10.1089/10665270050081478.
  16. Aaltone R, Jalava J, Laurikainen E, Kärkkäinen U, Alanen A. Cervical ureaplasma urealyticum colonization: comparison of PCR and culture for its detection and association with preterm birth. Scand J Infect Dis. 2002;34(1):35-40. DOI: 10.1080/00365540110077074.
  17. Montenegro DA, Borda LF, Neuta Y, Gómez LA, Castillo DM, Loyo D, et al. Oral and uro-vaginal intra-amniotic infection in women with preterm delivery: A case-control study. J Investig Clin Dent. 2019;10(2):e12396. DOI: 10.1111/jicd.12396.
  18. Nasution TA, Cheong SF, Lim CT, Leong EW, Ngeow YF. Multiplex PCR for the detection of urogenital pathogens in mothers and newborns. Malays J Pathol. 2007;29(1):19-24. DOI.

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  Conflict of Interest:
  Jørgen Skov Jensen reports grants, personal fees, and non-financial support from Hologic, personal fees from Roche, grants and personal fees from SpeeDx, grants and personal fees from Nabriva, grants and personal fees from Cepheid, grants and personal fees from Abbott, and grants and personal fees from GSK all outside the submitted work.
  The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
  

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