Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress
Clinical Collections
Advances in Alzheimer’s

At a glance: The STEP trials

A round-up of the STEP phase 3 clinical trials evaluating semaglutide for weight loss in people with overweight or obesity.
ADVANCED SEARCH
Log in
Top
Reproductive Biology and Endocrinology
Published in: Reproductive Biology and Endocrinology 1/2020

Open Access 01-12-2020 | Infertility | Research

The complex microbiome from native semen to embryo culture environment in human in vitro fertilization procedure

Authors: Jelena Štšepetova, Juliana Baranova, Jaak Simm, Ülle Parm, Tiiu Rööp, Sandra Sokmann, Paul Korrovits, Madis Jaagura, Karin Rosenstein, Andres Salumets, Reet Mändar

Published in: Reproductive Biology and Endocrinology | Issue 1/2020

Login to get access

Abstract

Background

Only a few microbial studies have conducted in IVF (in vitro fertilization), showing the high-variety bacterial contamination of IVF culture media to cause damage to or even loss of cultured oocytes and embryos. We aimed to determine the prevalence and counts of bacteria in IVF samples, and to associate them with clinical outcome.

Methods

The studied samples from 50 infertile couples included: raw (n = 48), processed (n = 49) and incubated (n = 50) sperm samples, and IVF culture media (n = 50). The full microbiome was analyzed by 454 pyrosequencing and quantitative analysis by real-time quantitative PCR. Descriptive statistics, t-, Mann-Whitney tests and Spearman’s correlation were used for comparison of studied groups.

Results

The study involved normozoospermic men. Normal vaginal microbiota was present in 72.0% of female partners, while intermediate microbiota and bacterial vaginosis were diagnosed in 12.0 and 16.0%, respectively. The decreasing bacterial loads were found in raw (35.5%), processed (12.0%) and sperm samples used for oocyte insemination (4.0%), and in 8.0% of IVF culture media. The most abundant genera of bacteria in native semen and IVF culture media were Lactobacillus, while in other samples Alphaproteobacteria prevailed. Staphylococcus sp. was found only in semen from patients with inflammation. Phylum Bacteroidetes was in negative correlation with sperm motility and Alphaproteobacteria with high-quality IVF embryos.

Conclusion

Our study demonstrates that IVF does not occur in a sterile environment. The prevalent bacteria include classes Bacilli in raw semen and IVF culture media, Clostridia in processed and Bacteroidia in sperm samples used for insemination. The presence of Staphylococcus sp. and Alphaproteobacteria associated with clinical outcomes, like sperm and embryo quality.
Appendix
Available only for authorised users
Literature
1.
Shu Y, Prokai D, Berga S, Taylor R, Johnston-MacAnanny E. Transfer of IVF-contaminated blastocysts with removal of the zona pellucida resulted in live births. J Assist Reprod Genet. 2016;33(10):1385–8.PubMedPubMedCentralCrossRef
2.
Mandar R, Punab M, Korrovits P, Turk S, Ausmees K, Lapp E, et al. Seminal microbiome in men with and without prostatitis. Int J Urol. 2017;24(3):211–6.PubMedCrossRef
3.
Palini S, Primiterra M, De Stefani S, Pedna MF, Sparacino M, Farabegoli P, et al. A new micro swim-up procedure for sperm preparation in ICSI treatments: preliminary microbiological testing. JBRA Assist Reprod. 2016;20(3):94–8.PubMedPubMedCentral
4.
5.
Kastrop PM, de Graaf-Miltenburg LA, Gutknecht DR, Weima SM. Microbial contamination of embryo cultures in an ART laboratory: sources and management. Hum Reprod. 2007;22(8):2243–8.PubMedCrossRef
6.
Selman H, Mariani M, Barnocchi N, Mencacci A, Bistoni F, Arena S, et al. Examination of bacterial contamination at the time of embryo transfer, and its impact on the IVF/pregnancy outcome. J Assist Reprod Genet. 2007;24(9):395–9.PubMedPubMedCentralCrossRef
7.
Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol. 1991;29(2):297–301.PubMedPubMedCentralCrossRef
8.
Punab M, Loivukene K, Kermes K, Mandar R. The limit of leucocytospermia from the microbiological viewpoint. Andrologia. 2003;35(5):271–8.PubMedCrossRef
9.
WHO. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction (4th Edition). Cambridge: Cambridge University Press; 2010.
10.
Nasiri N, Eftekhari-Yazdi P. An overview of the available methods for morphological scoring of pre-implantation embryos in in vitro fertilization. Cell J. 2015;16(4):392–405.PubMedPubMedCentral
11.
Fabrice AD, R. Exploring microbial diversity using 16S rRNA high-throughput methods. J Comp Sci Syst Biol. 2009;2:074–92.
12.
Ott SJ, Musfeldt M, Ullmann U, Hampe J, Schreiber S. Quantification of intestinal bacterial populations by real-time PCR with a universal primer set and minor groove binder probes: a global approach to the enteric flora. J Clin Microbiol. 2004;42(6):2566–72.PubMedPubMedCentralCrossRef
13.
Bartosch S, Fite A, Macfarlane GT, McMurdo ME. Characterization of bacterial communities in feces from healthy elderly volunteers and hospitalized elderly patients by using real-time PCR and effects of antibiotic treatment on the fecal microbiota. Appl Environ Microbiol. 2004;70(6):3575–81.PubMedPubMedCentralCrossRef
14.
Martineau F, Picard FJ, Ke D, Paradis S, Roy PH, Ouellette M, et al. Development of a PCR assay for identification of staphylococci at genus and species levels. J Clin Microbiol. 2001;39(7):2541–7.PubMedPubMedCentralCrossRef
15.
Adderson EE, Boudreaux JW, Cummings JR, Pounds S, Wilson DA, Procop GW, et al. Identification of clinical coryneform bacterial isolates: comparison of biochemical methods and sequence analysis of 16S rRNA and rpoB genes. J Clin Microbiol. 2008;46(3):921–7.PubMedCrossRef
16.
Fite A, Macfarlane GT, Cummings JH, Hopkins MJ, Kong SC, Furrie E, et al. Identification and quantitation of mucosal and faecal desulfovibrios using real time polymerase chain reaction. Gut. 2004;53(4):523–9.PubMedPubMedCentralCrossRef
17.
Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, et al. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 2007;35(21):7188–96.PubMedPubMedCentralCrossRef
18.
Wang QG, Garrity M, Tiedje JM, Cole JR. Naıve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol. 2007;73(16):5261–7.PubMedPubMedCentralCrossRef
19.
WHO. Laboratory manual for the examination and processing of human semen. 5th ed. Geneva: WHO; 2011.
20.
Abeysundara PK, Dissanayake D, Wijesinghe PS, Perera R, Nishad A. Efficacy of two sperm preparation techniques in reducing non-specific bacterial species from human semen. J Hum Reprod Sci. 2013;6(2):152–7.PubMedPubMedCentralCrossRef
21.
Weng SL, Chiu CM, Lin FM, Huang WC, Liang C, Yang T, et al. Bacterial communities in semen from men of infertile couples: metagenomic sequencing reveals relationships of seminal microbiota to semen quality. PLoS One. 2014;9(10):e110152.PubMedPubMedCentralCrossRef
22.
Elder K, Baker D, Ribes J. Infections, infertility and assisted reproduction. Part II: infections in reproductive medicine & part III: infections and the assisted reproductive laboratory. Cambridge: Cambridge University Press; 2005.
23.
Pomeroy KO. Contamination of human IVF cultures by microorganisms: a review. J Clin Embryo. 2010;13:11–30.
24.
Cottell E, Lennon B, McMorrow J, Barry-Kinsella C, Harrison RF. Processing of semen in an antibiotic-rich culture medium to minimize microbial presence during in vitro fertilization. Fertil Steril. 1997;67(1):98–103.PubMedCrossRef
25.
Pelzer ES, Allan JA, Cunningham K, Mengersen K, Allan JM, Launchbury T, et al. Microbial colonization of follicular fluid: alterations in cytokine expression and adverse assisted reproduction technology outcomes. Hum Reprod. 2011;26(7):1799–812.PubMedCrossRef
26.
Ben-Chetrit A, Shen O, Haran E, Brooks B, Geva-Eldar T, Margalioth EJ. Transfer of embryos from yeast-colonized dishes. Fertil Steril. 1996;66(2):335–7.PubMedCrossRef
27.
Al-Mously N, Cross NA, Eley A, Pacey AA. Real-time polymerase chain reaction shows that density centrifugation does not always remove chlamydia trachomatis from human semen. Fertil Steril. 2009;92(5):1606–15.PubMedCrossRef
28.
Moretti E, Capitani S, Figura N, Pammolli A, Federico MG, Giannerini V, et al. The presence of bacteria species in semen and sperm quality. J Assist Reprod Genet. 2009;26(1):47–56.PubMedCrossRef
29.
Mandar R, Punab M, Borovkova N, Lapp E, Kiiker R, Korrovits P, et al. Complementary seminovaginal microbiome in couples. Res Microbiol. 2015;166(5):440–7.PubMedCrossRef
30.
Monteiro C, Marques PI, Cavadas B, Damiao I, Almeida V, Barros N, et al. Characterization of microbiota in male infertility cases uncovers differences in seminal hyperviscosity and oligoasthenoteratozoospermia possibly correlated with increased prevalence of infectious bacteria. Am J Reprod Immunol. 2018;79(6):e12838.PubMedCrossRef
31.
Hou D, Zhou X, Zhong X, Settles ML, Herring J, Wang L, et al. Microbiota of the seminal fluid from healthy and infertile men. Fertil Steril. 2013;100(5):1261–9.PubMedPubMedCentralCrossRef
32.
Shah HN, Collins DM. Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides. Int J Syst Bacteriol. 1990;40(2):205–8.PubMedCrossRef
33.
Donders G. Diagnosis and management of bacterial vaginosis and other types of abnormal vaginal bacterial flora: a review. Obstet Gynecol Surv. 2010;65(7):462–73.PubMedCrossRef
34.
Kiessling AA, Desmarais BM, Yin HZ, Loverde J, Eyre RC. Detection and identification of bacterial DNA in semen. Fertil Steril. 2008;90(5):1744–56.PubMedCrossRef
35.
O'Doherty AM, Di Fenza M, Kölle S. Lipopolysaccharide (LPS) disrupts particle transport, cilia function and sperm motility in an ex vivo oviduct model. Sci Rep. 2016;6:24583.PubMedPubMedCentralCrossRef
36.
Barrett E, Guinane CM, Ryan CA, Dempsey EM, Murphy BP, O'Toole PW, et al. Microbiota diversity and stability of the preterm neonatal ileum and colon of two infants. Microbiologyopen. 2013;2(2):215–25.PubMedPubMedCentralCrossRef
37.
Lambert JA, Kalra A, Dodge CT, John S, Sobel JD, Akins RA. Novel PCR-based methods enhance characterization of vaginal microbiota in a bacterial vaginosis patient before and after treatment. Appl Environ Microbiol. 2013;79(13):4181–5.PubMedPubMedCentralCrossRef
38.
Hugon P, Ramasamy D, Robert C, Couderc C, Raoult D, Fournier PE. Non-contiguous finished genome sequence and description of Kallipyga massiliensis gen. Nov., sp. nov., a new member of the family Clostridiales Incertae Sedis XI. Stand Genomic Sci. 2013;8(3):500–15.PubMedPubMedCentralCrossRef
39.
Moreno I, Codoner FM, Vilella F, Valbuena D, Martinez-Blanch JF, Jimenez-Almazan J, et al. Evidence that the endometrial microbiota has an effect on implantation success or failure. Am J Obstet Gynecol. 2016;215(6):684–703.PubMedCrossRef
40.
Pelzer ES, Allan JA, Waterhouse MA, Ross T, Beagley KW, Knox CL. Microorganisms within human follicular fluid: effects on IVF. PLoS One. 2013;8(3):e59062.PubMedPubMedCentralCrossRef
41.
Sluss PM, Reichert LE Jr. Secretion of an inhibitor of follicle-stimulating hormone binding to receptor by the bacteria Serratia, including a strain isolated from porcine follicular fluid. Biol Reprod. 1984;31(3):520–30.PubMedCrossRef
42.
Sluss PM, Fletcher PW, Reichert LE Jr. Inhibition of 125I-human follicle-stimulating hormone binding to receptor by a low molecular weight fraction of bovine follicular fluid: inhibitor concentration is related to biochemical parameters of follicular development. Biol Reprod. 1983;29(5):1105–13.PubMedCrossRef
43.
Dissanayake DM, Amaranath KA, Perera RR, Wijesinghe PS. Antibiotics supplemented culture media can eliminate non-specific bacteria from human semen during sperm preparation for intra uterine insemination. J Hum Reprod Sci. 2014;7(1):58–62.PubMedPubMedCentralCrossRef
44.
Kala S, Singh A, Prabha V, Singh R, Sharma P. Escherichia coli attaches to spermatozoa: affecting sperm parameters. Arh Appl Sci Res. 2011;3(5):618–23.
45.
Malik S, Agarwal A. Understanding male infertility global practices & Indian perspectives. Gurgaon: Elsevier India; 2014.
46.
Lemeire K, Van Merris V, Cortvrindt R. The antibiotic streptomycin assessed in a battery of in vitro tests for reproductive toxicology. Toxicol in Vitro. 2007;21(7):1348–53.PubMedCrossRef
47.
48.
Brook N, Khalaf Y, Coomarasamy A, Edgeworth J, Braude P. A randomized controlled trial of prophylactic antibiotics (co-amoxiclav) prior to embryo transfer. Hum Reprod. 2006;21(11):2911–5.PubMedCrossRef
Metadata
Title
The complex microbiome from native semen to embryo culture environment in human in vitro fertilization procedure
Authors
Jelena Štšepetova
Juliana Baranova
Jaak Simm
Ülle Parm
Tiiu Rööp
Sandra Sokmann
Paul Korrovits
Madis Jaagura
Karin Rosenstein
Andres Salumets
Reet Mändar
Publication date
01-12-2020
Publisher
BioMed Central
Published in
Reproductive Biology and Endocrinology / Issue 1/2020
Electronic ISSN: 1477-7827
DOI
https://doi.org/10.1186/s12958-019-0562-z

Other articles of this Issue 1/2020

Reproductive Biology and Endocrinology 1/2020 Go to the issue

Research

Establishment of a WHO Reference Reagent for anti-Mullerian hormone

Debate

The 2019 PGDIS position statement on transfer of mosaic embryos within a context of new information on PGT-A

Research

Regulation of [Ca2+]i oscillations and mitochondrial activity by various calcium transporters in mouse oocytes

Research

Cyclosporin A protects JEG-3 cells against oxidative stress-induced apoptosis by inhibiting the p53 and JNK/p38 signaling pathways

Correction

Correction to: Status of racial disparities between black and white women undergoing assisted reproductive technology in the US

Research

Attenuation of sleep deprivation dependent deterioration in male fertility parameters by vitamin C