Division of Animal Sciences.
Department of Veterinary Pathobiology, University of Missouri, Columbia 65211; University of Missouri Metagenomics Center, University of Missouri, Columbia 65201.
J Dairy Sci. 2017 Jun;100(6):4953-4960. doi: 10.3168/jds.2017-12592. Epub 2017 Apr 21.
We tested the hypothesis that the uterus of virgin heifers and pregnant cows possessed a resident microbiome by 16S rRNA gene sequencing of the virgin and pregnant bovine uterus. The endometrium of 10 virgin heifers in estrus and the amniotic fluid, placentome, intercotyledonary placenta, cervical lumen, and external cervix surface (control) of 5 pregnant cows were sampled using aseptic techniques. The DNA was extracted, the V4 hypervariable region of the 16S rRNA gene was amplified, and amplicons were sequenced using Illumina MiSeq technology (Illumina Inc., San Diego, CA). Operational taxonomic units (OTU) were generated from the sequences using Qiime v1.8 software, and taxonomy was assigned using the Greengenes database. The effect of tissue on the microbial composition within the pregnant uterus was tested using univariate (mixed model) and multivariate (permutational multivariate ANOVA) procedures. Amplicons of 16S rRNA gene were generated in all samples, supporting the contention that the uterus of virgin heifers and pregnant cows contained a microbiome. On average, 53, 199, 380, 382, 525, and 13,589 reads annotated as 16, 35, 43, 63, 48, and 176 OTU in the placentome, virgin endometrium, amniotic fluid, cervical lumen, intercotyledonary placenta, and external surface of the cervix, respectively, were generated. The 3 most abundant phyla in the uterus of the virgin heifers and pregnant cows were Firmicutes, Bacteroidetes, and Proteobacteria, and they accounted for approximately 40, 35, and 10% of the sequences, respectively. Phyla abundance was similar between the tissues of the pregnant uterus. Principal component analysis, one-way PERMANOVA analysis of the Bray-Curtis similarity index, and mixed model analysis of the Shannon diversity index and Chao1 index demonstrated that the microbiome of the control tissue (external surface of the cervix) was significantly different from that of the amniotic fluid, intercotyledonary placenta, and placentome tissues. Interestingly, many bacterial species associated with postpartum uterine disease (i.e., Trueperella spp., Acinetobacter spp., Fusobacteria spp., Proteus spp., Prevotella spp., and Peptostreptococcus spp.) were also present in the uterus of virgin heifers and of pregnant cows. The presence of 16S rRNA gene sequence reads in the samples from the current study suggests that the uterine microbiome is established by the time a female reaches reproductive maturity, and that pregnancies are established and maintained in the presence of a uterine microbiome.
我们通过对处女牛和妊娠牛子宫的 16S rRNA 基因测序来检验处女牛和妊娠牛子宫存在固有微生物组的假设。使用无菌技术从 10 头发情期处女牛的子宫内膜和 5 头妊娠牛的羊水、胎盘中、胎盘中、宫颈管内腔和宫颈外表面（对照）取样。提取 DNA，扩增 16S rRNA 基因的 V4 高变区，使用 Illumina MiSeq 技术（Illumina Inc.，圣地亚哥，CA）对扩增子进行测序。使用 Qiime v1.8 软件从序列中生成操作分类单元（OTU），并使用 Greengenes 数据库分配分类。使用单变量（混合模型）和多变量（置换多元方差分析）程序测试组织对妊娠子宫内微生物组成的影响。所有样品中均生成了 16S rRNA 基因的扩增子，支持处女牛和妊娠牛子宫内存在微生物组的论点。平均而言，胎盘中分别生成了 53、199、380、382、525 和 13589 个序列，注释为 16、35、43、63、48 和 176 OTU，在处女子宫内膜、羊水、宫颈管内腔、胎盘中，以及宫颈外表面。在处女牛和妊娠牛的子宫中，最丰富的 3 个门是厚壁菌门、拟杆菌门和变形菌门，它们分别占序列的约 40%、35%和 10%。妊娠子宫组织中门的丰度相似。主成分分析、Bray-Curtis 相似性指数的单向 PERMANOVA 分析以及香农多样性指数和 Chao1 指数的混合模型分析表明，对照组织（宫颈外表面）的微生物组与羊水、胎盘中的组织明显不同胎盘和胎盘组织。有趣的是，许多与产后子宫疾病相关的细菌物种（即 Trueperella spp.、不动杆菌 spp.、梭菌 spp.、变形杆菌 spp.、普雷沃氏菌 spp.和消化链球菌 spp.）也存在于处女牛和妊娠牛的子宫中。当前研究样本中存在 16S rRNA 基因序列读长表明，子宫微生物组在女性达到生殖成熟时就已建立，并且妊娠是在子宫微生物组存在的情况下建立和维持的。
