Journal of
Clinical Medicine
Article
Maternal Baseline Risk Factors for Abnormal Vaginal
Colonisation among High-Risk Pregnant Women and the
Association with Adverse Pregnancy Outcomes: A
Retrospective Cohort Study
Junesoo Jeon 1 , Yun-sun Choi 2 , Yejin Kim 2 , Siryeon Hong 2 , Ji-Hee Sung 2 , Suk-Joo Choi 2 , Soo-young Oh 2, *
and Cheong-Rae Roh 2
Citation: Jeon, J.; Choi, Y.-s.; Kim, Y.;
Hong, S.; Sung, J.-H.; Choi, S.-J.; Oh,
S.-y.; Roh, C.-R. Maternal Baseline
Risk Factors for Abnormal Vaginal
Colonisation among High-Risk
Pregnant Women and the Association
with Adverse Pregnancy Outcomes:
A Retrospective Cohort Study. J. Clin.
Med. 2023, 12, 40. https://doi.org/
10.3390/jcm12010040
Academic Editors: Gabriele Tonni
and Yariv Yogev
Received: 25 October 2022
Revised: 15 November 2022
Accepted: 18 December 2022
Published: 21 December 2022
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
J. Clin. Med. 2023, 12, 40. https://doi.org/10.3390/jcm12010040
1 School of Medicine, Sungkyunkwan University, Seoul 06355, Republic of Korea
2 Department of Obstetrics and Gynecology, Samsung Medical Centre, School of Medicine,
Sungkyunkwan University, Seoul 06351, Republic of Korea
* Correspondence: ohsymd@skku.edu; Tel.: +82-2-3410-3517; Fax: +82-2-3410-0630
Abstract: Abnormal vaginal colonisation can lead to adverse pregnancy outcomes such as preterm
birth through intra-amniotic inflammation. Despite the concern, little is known about its risk factors
and impact in pregnant women at high-risk for spontaneous preterm birth. Thus, we conducted this
single-centre retrospective cohort study including 1381 consecutive women who were admitted to
the high-risk pregnancy unit. The results of vaginal culture at admission were categorised according
to the colonising organism: bacteria (Gram-negative or -positive) and genital mycoplasmas. Maternal
baseline socioeconomic, and clinical characteristics, as well as pregnancy, delivery, and neonatal
outcomes were compared according to the category. Maternal risk factors for Gram-negative coloni-
sation included advanced maternal age, increased pre-pregnancy BMI, a greater number of past
spontaneous abortions, earlier gestational age at admission, and IVF. Gram-positive colonisation was
likewise associated with earlier gestational age at admission. Genital mycoplasmal colonisation was
associated with a greater number of past induced abortions, a lower level of education completed,
and a lower rate of multifetal pregnancy and IVF. The neonates from mothers with Gram-negative
colonisation had a greater risk of NICU admission, proven early onset neonatal sepsis, and mortality.
However, not Gram-positive bacteria or genital mycoplasma was directly associated with adverse
pregnancy outcomes.
Keywords: high-risk pregnancy; abnormal vaginal colonisation; maternal risk factor; preterm birth;
adverse pregnancy outcome; Gram-negative bacteria; Gram-positive bacteria; Ureaplasma urealyticum
1. Introduction
About 40–50% of preterm births are associated with intrauterine infection [1], most of
which occur through ascending vaginal infection [2]. It is well recognised that abnormal
vaginal flora (microbiota/microbiome), characterised by a decrease in lactobacilli and
overgrowth of aerobic or anaerobic microorganisms, is a contributing factor for ascending
infection [3]. Previous studies have indicated that the presence of abnormal vaginal flora
was associated with spontaneous preterm birth caused by preterm labour (PTL), preterm
premature rupture of membranes (pPROM), or cervical incompetency (CI) [4–7]. Moreover,
several clinical studies have assessed whether antibiotic treatment of abnormal vaginal flora
(as in bacterial vaginosis) reduces preterm birth rates in high-risk and even low-risk women:
however, antibiotics were found to be ineffective, contrary to many expectations [8].
There have been many investigations of the risk factors for abnormal vaginal flora, but
these mainly targeted the non-pregnant population. Previously identified risk factors for
bacterial vaginosis among women of reproductive age included low socioeconomic status,
https://www.mdpi.com/journal/jcm
J. Clin. Med. 2023, 12, 40 2 of 13

black ethnicity, and early sexual experience [9,10]. Use of intrauterine devices, history
of past spontaneous abortion, and menstrual cycle over 35 days were also identified as
risk factors for bacterial vaginosis in non-pregnant women [11]. With regard to aerobic
vaginitis, which has emerged as a relatively new disease entity [12], unmarried status,
use of intrauterine devices, long-term use of antibiotics (≥10 days in the past month),
and frequent vaginal douching were found to be independent risk factors in women of
reproductive age [13]. For genital mycoplasmas including Ureaplasma urealyticum, younger
age, sexual behaviour, and low socioeconomic status are known risk factors [14–16].
Previously, we found that few comprehensive studies regarding maternal risk factors
for abnormal vaginal flora during pregnancy have been published and have significant
limitations. These studies have been largely focused on bacterial vaginosis rather than
aerobic bacterial colonisation or genital mycoplasmas. In addition, the study designs
included low-risk pregnant women, making it unclear whether the presence of abnormal
vaginal flora adversely influences high-risk pregnancies among women who already had
signs and symptoms indicative of spontaneous preterm birth.
With this background, we aimed to identify the maternal risk factors for abnormal
vaginal flora, caused by colonisation of aerobic microorganisms or genital mycoplasmas, in
pregnant women at high risk of spontaneous preterm birth. We also examined whether
abnormal vaginal flora is associated with adverse outcomes in these women.

2. Materials and Methods

This retrospective cohort study included consecutive parturient women who were
admitted to our institution’s high-risk care unit and underwent vaginal culture at admission
between January 2010 and July 2020. The inclusion criteria were patients whose gestational
ages were 14 0/7–34 6/7 weeks at admission (n = 1381) and the presence of one of the
following indications: PTL, pPROM, CI, and short cervical length (CL). The diagnoses
were mutually exclusive. Women diagnosed with PTL required admission due to regular
uterine contraction and progressive cervical change. Women with pPROM were diagnosed
based on gross pooling of amniotic fluid in the vagina and an alkaline vaginal fluid pH
determined by a nitrazine test. Women with CI were those with painless cervical dilatation
(>1 cm) with visible membranes through the cervical os. Short CL was defined when the
cervix is closed on a sterile speculum, and the cervical length is less than 25 mm when
measured through transvaginal ultrasound.
In accordance with our institutional protocols, we routinely perform vaginal culture of
every patient at high risk of spontaneous preterm birth at admission under these indications.
Vaginal sampling for culture was performed by swabbing the posterior fornix using a sterile
cotton swab and a sterile non-lubricated speculum. The samples were transported for
analysis by Copan Venturi Transystem collection device (Copan innovation, Corona, CA,
USA), which is composed of Liquid Stuart transport medium. The Gram-staining and
culture were carried out according to the standard laboratory procedures of our institution.
The samples were directly inoculated on a blood agar plate, MacConkey agar plate, and
Thayer–Martin agar plate and were incubated for up to 72 h at 35 ◦ C in a 5% CO2 incubator.
Results of vaginal culture were categorised by the types of colonised microorganism;
bacteria (Gram-negative and -positive) and genital mycoplasmas (Mycoplasma hominis and
Ureaplasma urealyticum). Abnormal vaginal flora was defined as isolation of any of the
above-mentioned microorganisms. Colonisation with either Gram-negative or -positive
bacteria was defined as abnormal bacterial colonisation.
Data were collected from the review of the medical records and were compared
according to the category of vaginal flora. Maternal baseline characteristics collected
included maternal age and pre-pregnancy body mass index (BMI). Parity was reviewed in
detail, including the number of past spontaneous and induced abortions. With regard to
socioeconomic factors, marital status, smoking history, drinking history after pregnancy,
and highest level of education completed were recorded. Marital status was categorised
as either single or married/common-law. The highest level of education completed was
J. Clin. Med. 2023, 12, 40 3 of 13

categorised as lower than high-school graduate, high-school graduate, some college/college

graduate, or higher than college graduate. Clinical characteristics collected included
gestational age at admission, diagnosis at admission (PTL, pPROM, CI, or short CL),
multifetal pregnancy, and infertility treatment (intrauterine insemination [IUI] or in vitro
fertilisation [IVF]) for the index pregnancy.
We collected the pregnancy outcome of every foetus of the study population that
was alive at the time of admission (n = 1648). Pregnancy outcome was categorised as
abortion, stillbirth/foetal death in uterus (FDIU), or livebirth. Abortion included all foetal
loss in utero occurring before 20 weeks of gestation, while stillbirth/FDIU included foetal
loss occurring beyond 20 weeks of gestation with 5 min Apgar scores of 0. Delivery out-
comes included gestational age at delivery, mode of delivery, and diagnosis of histological
chorioamnionitis and were analysed for the 1381 mothers. Neonatal outcome was assessed
for the 1519 liveborn neonates of the study population. The variables included sex, birth
weight, Apgar score, neonatal intensive care unit (NICU) admission, diagnosis of neonatal
sepsis, and neonatal mortality. Neonates with birth weights below the 10th percentile
for gestational age were defined as small for gestational age (SGA). Neonatal sepsis was
specified as early onset neonatal sepsis (EONS) when diagnosed within the first week of
life, the subsequent diagnosis was classified as late-onset neonatal sepsis (LONS). Sus-
pected EONS was diagnosed clinically by individual paediatricians, whereas proven EONS
was diagnosed upon the isolation of microorganisms from blood or cerebrospinal fluid of
the neonates.
All statistical analyses were completed using SPSS® ver. 27.0 (IBM Corp., Armonk,
NY, USA) and the results were considered statistically significant when the p value was
<0.05. Categorical variables were analysed using Pearson’s chi-square test or Fisher’s exact
test, and continuous data were tested using the Mann–Whitney U test when appropriate.
Linear-by-linear association was used to evaluate the statistical significance of trends in the
number of past abortions and level of education, according to abnormal vaginal colonisa-
tion. To determine whether abnormal vaginal colonisation is a meaningful factor even after
adjusting factors which are generally known to directly affect neonatal outcomes, multivari-
ate analysis was conducted using a logistic regression model. We selected gestational age
at delivery and history of preterm delivery as the most influential factors and used them
for analysis. Moreover, treatment for vaginal colonisation, namely the use of antepartum
antibiotics, would affect the neonatal outcomes, so we also adjusted it for analysis. The use
of antepartum antibiotics is defined as use of antibiotics of any kind for 2 or more days after
admission, since using antibiotics one day or less is less likely to affect neonatal outcomes.
The history of antibiotics was unclear in 56 patients (65 foetuses) and were excluded from
the analysis.

3. Results
A total of 1381 parturient women were included in this study, and abnormal vaginal
bacterial flora was identified in 283 cases (20.5%). A total of 178 (12.9%) and 124 (9.0%)
cases had at least one of Gram negative and positive microbes isolated, respectively. The
most frequently isolated Gram-negative bacterium was E. coli (n = 122, 8.8%), followed by
Klebsiella (n = 29, 2.1%), Enterobacter (n = 14, 1.0%), Citrobacter (n = 10, 0.7%), Pseudomonas
(n = 6, 0.4%), and Proteus (n = 6, 0.4%). Gram-positive bacterial colonisation was mostly due
to group B Streptococcus (GBS) (n = 61, 4.4%), followed by coagulase-negative Staphylococcus
(n = 18, 1.3%), and S. anginosus (n = 4, 0.3%). Results of mycoplasma culture were available
for 1309 patients. Among these, the colonisation by genital mycoplasmas occurred in 43.6%
(n = 571), comprising 4.4% (n = 58) M. hominis and 42.4% (n = 555) Ureaplasma species.
Table 1 presents the maternal baseline characteristics, socioeconomic factors, and clini-
cal characteristics in the Gram-negative or -positive bacterial colonisation groups compared
to the group with no bacterial colonisation. Vaginal Gram-negative bacterial colonisation
was associated with advanced maternal age, increased pre-pregnancy BMI, greater numbers
of past spontaneous abortions, and pregnancy through IVF as compared with the group
J. Clin. Med. 2023, 12, 40 4 of 13

with no bacterial colonisation. In contrast, we identified no baseline or socioeconomic risk

factors for Gram-positive bacterial colonisation. Both Gram-negative and -positive bacterial
colonisation was associated with an earlier gestational age at admission as compared with
no bacterial colonisation. With regard to the diagnosis of admission, mothers with Gram-
negative bacteria colonisation were more likely to be admitted for CI (15.1% vs. 33.7%,
p < 0.001) and less likely to be admitted for PTL (36.4% vs. 28.7%, p = 0.044), but there was
no difference in the diagnosis of admission according to the presence of Gram-positive
bacteria colonisation.
Table 2 summarises the maternal baseline characteristics, socioeconomic factors, and
clinical characteristics in the group with genital mycoplasmal colonisation and the group
with no genital mycoplasma colonisation. Most of the factors were not significantly different
between the two groups. Of note, a greater number of past induced abortions, lower level
of education completed, and lower rates of multifetal pregnancy and IVF were found in
the genital mycoplasmal colonisation group. The genital mycoplasmal colonisation group
was more likely to be admitted for pPROM compared to those without colonisation (42.4%
vs. 35.6%, p = 0.013).
Table 3 presents the pregnancy, delivery, and neonatal outcomes in the Gram-negative
or -positive bacterial colonisation groups and the group with no bacterial colonisation.
The Gram-negative bacterial colonisation group had a greater number of stillbirths/FDIU
(6.5% vs. 3.1%, p = 0.014), and delivered babies of an earlier gestational age (27 4/7 weeks
[16 1/7 to 37 6/7] vs. 30 4/7 weeks [16 0/7 to 38 4/7], p < 0.001) compared with the
group with no bacterial colonisation. The Gram-positive bacterial colonisation group
showed a similar tendency: higher rate of stillbirth/FDIU (8.3% vs. 3.1%, p = 0.002) and
an earlier gestational age at delivery (27 6/7 weeks [16 0/7 to 37 4/7] vs. 30 4/7 weeks
[16 0/7 to 38 4/7], p = 0.002). Association with histological chorioamnionitis was only
evident in the Gram-negative colonisation group (57.4% vs. 43.6%, p < 0.001). In addition,
associations with neonatal morbidity and mortality were found only in the Gram-negative
colonisation group: specifically, the group’s infants had a higher risk of NICU admission
(97.4% vs. 89.4%, p < 0.001) and proven EONS (3.1% vs. 1.1%, p = 0.042). There was no
significant difference in neonatal outcomes between the Gram-positive and no bacterial
colonisation groups. After adjustment of gestational age at delivery, history of preterm
birth, and use of antibiotics two or more days after admission, only risk of NICU admission
remained significantly higher in the Gram-negative colonisation group (OR [95% CI]: 4.179
[1.471–11.873], p = 0.007), and other pregnancy, delivery, and neonatal outcomes did not.
After adjustment, there were still no factors related to the Gram-positive colonisation group.
Table 4 presents the pregnancy, delivery, and neonatal outcomes in the genital my-
coplasmal colonisation group compared and the group with no genital mycoplasma coloni-
sation. Genital mycoplasmal colonisation was associated with histological chorioamnionitis
(50.4% vs. 42.9%, p = 0.008), but was not associated with any of the other adverse preg-
nancy outcomes except for a greater percentage of 5 min Apgar score < 7. The association
with histological chorioamnionitis was still statistically significant after adjustment (OR
[95% CI]: 1.281 [1.005–1.633], p = 0.045), while other outcome factors was not.
J. Clin. Med. 2023, 12, 40 5 of 13

Table 1. Maternal baseline characteristics, socioeconomic factors, and clinical characteristics in the
groups with vaginal colonisation by Gram-negative or -positive bacteria compared with the group
with no bacterial colonisation.

Maternal Vaginal Colonisation at Admission *

No Bacterial Gram (−) Bacterial Gram (+) Bacterial
Colonisation Colonisation p Value Colonisation p Value
(n = 1098) (n = 178) (n = 124)
Maternal baseline characteristics
Age [years] 33 (18~45) 34 (20~42) 0.007 33 (19~43) 0.057
20.86
Pre-pregnancy BMI [kg/m2 ] † (14.04~43.41) 21.45 (15.55~47.34) 0.006 21.23 (14.90~35.52) 0.159
Parity
Nulliparity 60.4% (663/1098) 65.2% (116/178) 0.224 59.7% (74/124) 0.879
History of preterm delivery ‡ 25.5% (111/435) 29.0% (18/62) 0.555 24.0% (12/50) 0.815
Past spontaneous abortion [count] 0.010 0.184
none 77.1% (847/1098) 68.5% (122/178) 82.3% (102/124)
1 15.1% (166/1098) 19.7% (35/178) 13.7% (17/124)
2 5.5% (60/1098) 7.3% (13/178) 1.6% (2/124)
≥3 2.3% (25/1098) 4.5% (8/178) 2.4% (3/124)
Past induced abortion [count] 0.577 0.192
none 92.2% 89.9% (160/178) 87.9% (109/124)
(1012/1098)
1 5.4% (59/1098) 7.3% (13/178) 8.1% (10/124)
2 1.5% (17/1098) 2.2% (4/178) 3.2% (4/124)
≥3 0.9% (10/1098) 0.6% (1/178) 0.8% (1/124)
Socioeconomic factors
Marital status 0.073 1.000
Single 0.7% (8/1098) 2.2% (4/178) 0.8% (1/124)
99.3%
Married/Common-law (1090/1098) 97.8% (174/178) 99.2% (123/124)
Smoking history
Any before or after pregnancy † 2.0% (15/742) 1.3% (2/155) 0.751 0.0% (0/109) 0.239
Drinking history 1.000 1.000
Any after pregnancy 0.5% (5/1098) 0.0% (0/178) 0.0% (0/124)
Highest level of education † 0.167 0.479
Lower than high-school graduate 1.3% (13/1014) 2.9% (4/138) 0.0% (0/87)
High-school graduate 14.9% (151/1014) 13.8% (19/138) 12.6% (11/87)
Some college/college graduate 72.4% (734/1014) 76.8% (106/138) 75.9% (66/87)
Higher than college graduate 11.4% (116/1014) 6.5% (9/138) 11.5% (10/87)
Clinical characteristics of index pregnancy
Gestational age at admission [weeks] 28 6/7 (15 6/7 to 25 4/7 (14 4/7 to 34 <0.001 26 2.5/7 (15 6/7 to 0.005
34 6/7) 6/7) 34 6/7)
Diagnosis at admission
PTL 36.4% (400/1098) 28.7% (51/178) 0.044 29.0% (36/124) 0.103
pPROM 37.9% (416/1098) 31.5% (56/178) 0.099 40.3% (50/124) 0.597
CI 15.1% (166/1098) 33.7% (60/178) <0.001 21.0% (26/124) 0.090
short CL 4.1% (45/1098) 2.2% (4/178) 0.233 4.8% (6/124) 0.696
Multifetal pregnancy § 20.5% (225/1098) 22.5% (40/178) 0.546 18.5% (23/124) 0.610
Twin § 19.0% (205/1078) 20.2% (35/173) 0.706 17.2% (21/122) 0.629
Triplet § 2.2% (20/893) 3.5% (5/143) 0.374 1.9% (2/103) 1.000
Infertility treatment¶ 18.7% (205/1098) 31.5% (56/178) <0.001 19.4% (24/124) 0.853
IUI ¶ 2.2% (20/913) 3.2% (4/126) 0.521 4.8% (5/105) 0.169
IVF ¶ 17.2% (185/1078) 29.9% (52/174) <0.001 16.0% (19/119) 0.742
BMI: body mass index; PTL: preterm labour; pPROM: preterm premature rupture of membranes; CI: cervical
incompetence; short CL: short cervical length; IUI: intrauterine insemination; IVF: in vitro fertilisation. Data are
presented as the median (range) or percentage (number). * 1381 high-risk parturient women who underwent
vaginal culture at admission were included in analysis. † Data regarding pre-pregnancy BMI, history of smoking
before pregnancy, and final education were not available for some patients (Data existing in n = 1079, 987, 1231
each). ‡ Analysed for multiparous women only. § Compared with the singleton pregnancy group. ¶ Compared to
the group that did not receive any infertility treatment.
J. Clin. Med. 2023, 12, 40 6 of 13

Table 2. Maternal baseline characteristics, socioeconomic factors, and clinical characteristics in

the group with vaginal colonisation by genital mycoplasmas compared with the group with no
mycoplasmal colonisation.

Maternal Vaginal Colonisation at Admission *

No Genital Mycoplasmal Genital Mycoplasmal p Value

Colonisation Colonisation
(n = 738) (n = 571)
Maternal baseline characteristics
Age [years] 33 (18~45) 33 (18~45) 0.659
Pre-pregnancy BMI [kg/m2 ] † 20.93 (14.90~43.41) 21.23 (14.04~47.34) 0.052
Parity
Nulliparity 61.2% (452/738) 60.6% (346/571) 0.811
History of preterm delivery ‡ 23.4% (67/286) 29.8% (67/225) 0.105
Past spontaneous abortion [count] 0.635
none 75.7% (559/738) 76.5% (437/571)
1 16.0% (118/738) 15.4% (88/571)
2 5.3% (39/738) 6.0% (34/571)
≥3 3.0% (22/738) 2.1% (12/571)
Past induced abortion [count] 0.021
none 92.8% (685/738) 89.7% (512/571)
1 5.3% (39/738) 6.3% (36/571)
2 1.2% (9/738) 2.8% (16/571)
≥3 0.7% (5/738) 1.2% (7/571)
Socioeconomic factors
Marital status 0.455
Single 0.8% (6/738) 1.2% (7/571)
Married/Common-law 99.2% (732/738) 98.8% (564/571)
Smoking history
Any before or after pregnancy † 0.9% (4/457) 2.5% (12/477) 0.053
Drinking history 1.000
Any after pregnancy 0.4% (3/738) 0.4% (2/571)
Highest level of education † 0.022
Lower than high-school graduate 0.9% (6/675) 2.0% (10/497)
High-school graduate 14.2% (96/675) 15.3% (76/497)
Some college/college graduate 72.1% (487/675) 74.2% (369/497)
Higher than college graduate 12.7% (86/675) 8.5% (42/497)
Clinical characteristics of index pregnancy
Gestational age at admission [weeks] 28 4/7 (14 4/7 to 34 6/7) 28 0/7 (15 6/7 to 34 6/7) 0.922
Diagnosis at admission
PTL 36.7% (271/738) 32.9% (188/571) 0.153
pPROM 35.6% (263/738) 42.4% (242/571) 0.013
CI 18.6% (137/738) 17.0% (97/571) 0.461
short CL 3.8% (28/738) 4.2% (24/571) 0.707
J. Clin. Med. 2023, 12, 40 7 of 13

Table 2. Cont.

Maternal Vaginal Colonisation at Admission *

No Genital Mycoplasmal Genital Mycoplasmal p Value

Colonisation Colonisation
(n = 738) (n = 571)
Multifetal pregnancy § 24.7% (182/738) 15.2% (87/571) <0.001
Twin § 23.0% (166/722) 14.0% (79/563) <0.001
Triplet § 2.8% (16/572) 1.6% (8/492) 0.200
Infertility treatment ¶ 24.1% (178/738) 15.4% (88/571) <0.001
IUI ¶ 1.8% (10/570) 3.2% (16/499) 0.124
IVF ¶ 23.1% (168/728) 13.0% (72/555) <0.001
BMI: body mass index; PTL: preterm labour; pPROM: preterm premature rupture of membranes; CI: cervical
incompetence; short CL: short cervical length; IUI: intrauterine insemination; IVF: in vitro fertilisation. Data
are presented as median (range) or percentage (number). * 1309 high-risk parturient women who underwent
vaginal culture at admission were included in the analyses. † Data for pre-pregnancy BMI, history of smoking
before pregnancy, education data were not available for some patients. (Data existing in n = 1017, 934, 1172 each).
‡ Analysed for multiparous women only. § Compared with the singleton pregnancy group. ¶ Compared with the

group that did not receive any infertility treatment.

Table 3. Pregnancy, delivery, and neonatal outcomes in groups with vaginal colonisation by Gram-
negative or -positive bacteria compared with the group with no bacterial colonisation.

Maternal Vaginal Colonisation at Admission

No Bacterial Gram (−) Bacterial Gram (+) Bacterial

p Value p Value
Colonisation Colonisation Colonisation
Pregnancy outcome (n = 1648) * n = 1311 n = 216 n = 145
Abortion 3.5% (46/1311) 5.1% (11/216) 0.255 6.2% (9/145) 0.106
Stillbirth/FDIU 3.1% (41/1311) 6.5% (14/216) 0.014 8.3% (12/145) 0.002
93.4%
Livebirth 88.4% (191/216) 0.010 85.5% (124/145) <0.001
(1224/1311)
Delivery outcome (n = 1381) † n = 1098 n = 178 n = 124
30 4/7 (16 0/7 to 27 4/7 (16 1/7 to 37 27 6/7 (16 0/7 to
Gestational age at delivery [weeks] <0.001 0.002
38 4/7) 6/7) 37 4/7)
Caesarean section 53.9% (592/1098) 56.2% (100/178) 0.574 44.4% (55/124) 0.043
Histological chorioamnionitis § 43.6% (470/1077) 57.4% (101/176) <0.001 50.8% (61/120) 0.132
Neonatal outcome (n = 1519) ‡ n = 1224 n = 191 n = 124
Sex [male] 55.2% (676/1224) 53.4% (102/191) 0.637 60.5% (75/124) 0.262
Birth weight [g] 1570 (84~4110) 1140 (400~3130) <0.001 1300 (380~2870) 0.068
SGA 8.3% (102/1224) 11.0% (21/191) 0.225 4.8% (6/124) 0.172
1 min Apgar score <4 10.0% (122/1224) 12.6% (24/191) 0.272 9.7% (12/124) 0.918
5 min Apgar score <7 11.8% (144/1224) 12.6% (24/191) 0.750 14.5% (18/124) 0.369
89.4%
NICU admission 97.4% (186/191) <0.001 90.3% (112/124) 0.744
(1094/1224)
Neonatal sepsis 7.0% (86/1224) 9.9% (19/191) 0.152 10.5% (13/124) 0.160
LONS 4.2% (52/1224) 4.7% (9/191) 0.769 5.6% (7/124) 0.469
J. Clin. Med. 2023, 12, 40 8 of 13

Table 3. Cont.

Maternal Vaginal Colonisation at Admission

No Bacterial Gram (−) Bacterial Gram (+) Bacterial

p Value p Value
Colonisation Colonisation Colonisation
EONS 2.8% (34/1224) 5.2% (10/191) 0.069 4.8% (6/124) 0.258
proven 1.1% (14/1224) 3.1% (6/191) 0.042 3.2% (4/124) 0.076
suspected 1.6% (20/1224) 2.1% (4/191) 0.554 1.6% (2/124) 1.000
Neonatal mortality 6.2% (76/1224) 9.9% (19/191) 0.055 7.3% (9/124) 0.647
FDIU: foetal death in uterus; SGA: small for gestational age; NICU: neonatal intensive care unit; LONS: late-onset
neonatal sepsis; EONS: early onset neonatal sepsis. Data are presented as the median (range) or percentage
(number). * 1648 foetuses whose mothers were admitted to the high-risk pregnancy centre were included for
analysis. Vanishing or demised twins and foetuses that underwent selective foetal reduction were excluded
from the study population. † 1381 high-risk parturient women who underwent vaginal culture at admission and
delivered at the centre were included in the analyses. ‡ 1519 liveborn neonates whose mothers were admitted
to the high-risk pregnancy centre were included in the analyses. § Histological examination results were not
available for 27 patients.

Table 4. Pregnancy, delivery, and neonatal outcomes in the group with vaginal colonisation by genital
mycoplasmas compared with the group with no mycoplasmal colonisation.

Maternal Vaginal Colonisation at Admission

p Value
No Genital Mycoplasmal Genital Mycoplasmal
Colonisation Colonisation
Pregnancy outcome (n = 1564) * n = 911 n = 653
Abortion † 4.1% (37/911) 3.4% (22/653) 0.478
Stillbirth/FDIU † 3.3% (30/911) 4.4% (29/653) 0.240
Livebirth † 92.6% (844/911) 92.2% (602/653) 0.737
Delivery outcome (n = 1309) † n = 738 n = 571
Gestational age at delivery [weeks] 30 1/7 (16 0/7 to 38 4/7) 30 2/7 (16 0/7 to 37 6/7) 0.745
Caesarean section 55.8% (412/738) 50.3% (287/571) 0.045
Histological chorioamnionitis § 42.9% (308/718) 50.4% (284/564) 0.008
Neonatal outcome (n = 1446) ‡ n = 844 n = 602
Sex [male] 56.3% (475/844) 54.0% (325/602) 0.387
Birth weight [g] 1490 (84~3230) 1565 (300~4110) 0.097
SGA 8.4% (71/844) 7.8% (47/602) 0.679
1 min Apgar score <4 9.2% (78/844) 11.8% (71/602) 0.116
5 min Apgar score <7 10.8% (91/844) 14.3% (86/602) 0.045
NICU admission 90.4% (763/844) 91.0% (548/602) 0.686
Neonatal sepsis 6.6% (56/844) 8.1% (49/602) 0.277
LONS 3.9% (33/844) 4.8% (29/602) 0.401
EONS 2.7% (23/844) 3.3% (20/602) 0.510
proven 1.3% (11/844) 1.5% (9/602) 0.758
suspected 1.4% (12/844) 1.8% (11/602) 0.544
Neonatal mortality 6.0% (51/844) 7.1% (43/602) 0.403
FDIU: foetal death in uterus; SGA: small for gestational age; NICU: neonatal intensive care unit; LONS: late-onset
neonatal sepsis; EONS: early onset neonatal sepsis. Data are presented as median (range) or percentage (number).
* 1564 foetuses whose mothers were admitted to the high-risk pregnancy centre were included in the analyses.
Vanishing or demised twins and foetuses that underwent selective foetal reduction were excluded from the study
population. † 1309 high-risk parturient women who underwent vaginal culture at admission and delivered at the centre
were included in the analyses. ‡ 1446 liveborn neonates whose mothers were admitted to the high-risk pregnancy
centre were included in the analyses. § Results of histological examination were not available in 27 patients.
J. Clin. Med. 2023, 12, 40 9 of 13

4. Discussion
Abnormal bacterial colonisation by aerobic microorganisms during pregnancy has
been reported previously. According to a Belgian study that included non-symptomatic
pregnant women, the prevalence of abnormal vaginal flora during the first trimester was
9.35% [17]. In studies including high-risk pregnant women with PTL or pPROM, the rate
of aerobic vaginitis ranged from 11.3% to 18% [18,19]. In our study population including
high-risk pregnant women (PTL, pPROM, CI, and short CL), aerobic bacterial colonisation
was detected in 20.5% of the women, and the most frequently observed microbes were
E. coli (8.8%) and GBS (4.4%). These two microbes were reported to be among the most
common causative organisms of aerobic vaginitis in previous studies [12,20].
In this study, we found that risk factors for aerobic bacterial colonisation during
pregnancy manifested distinct patterns from those of bacterial vaginosis. In general,
maternal baseline risk factors for bacterial vaginosis in pregnancy have been reported
to be smoking at conception, high alcohol consumption, being single, and lower level
of education [21,22], none of which were found to be significant risk factors for either
Gram-negative or -positive bacterial colonisation in our analyses. In contrast, maternal
age, which was not previously reported to be associated with bacterial vaginosis [21,22],
was found to be a significant risk factor for Gram-negative colonisation in this study. In
addition, the history of past spontaneous abortion, which was the risk factor of bacterial
vaginosis in reproductive women [11], was common. Specifically, we found a linear
association between the number of past spontaneous abortions and Gram-negative bacteria
colonisation. Our finding that IVF pregnancies are risk factors for abnormal vaginal
colonisation is an extension of the results of our previous study, which showed that the
prevalence of vaginal colonisation by Gram-negative bacteria, especially Escherichia coli,
was significantly greater in pregnancies resulting from infertility treatment after adjustment
for confounding variables [23].
It is noteworthy that neonates from both the Gram-negative and -positive colonisation
groups had earlier gestational age at delivery in our study, but only the Gram-negative
group had significantly higher rate of histological chorioamnionitis. This result is partly
consistent with the results of a previous study showing that Gram-negative rods (but not
Gram-positive cocci) were the only chorioamniotic isolate to be significantly correlated
with histological chorioamnionitis [24]. In addition, our study showed that only the Gram-
negative colonisation group was associated with adverse neonatal outcomes, such as NICU
admission and proven EONS. The risk of NICU admission was significantly higher in
the Gram-negative group even after the adjustment of other risk factors. Bacteria from
the vaginal flora can migrate directly to foetal membranes, causing inflammation and
consequent neonatal morbidity and mortality [25]. Our results suggest that Gram-negative
bacteria are better able to produce ascending infection than Gram-positive bacteria and
produce greater impact on neonatal outcomes. In fact, a recent in vitro study demonstrated
that lipopolysaccharides in Gram negative bacteria induce time-dependent and cell-type-
specific pro-inflammatory cytokines, which contribute to propagation though decidua,
chorion, and amnion [26].
Genital mycoplasmas are sometimes considered part of the normal vaginal microbiota
but can also be associated with adverse pregnancy outcomes, especially where bacterial
loads are increased or in combination with other abnormal vaginal flora such as bacte-
rial vaginosis in preterm gestation [15,27]. In general, the risk factors for mycoplasmal
infection in non-pregnant women are younger age, risky sexual behaviour, and a lower
socioeconomic status [15]. However, it is not clear that these risk factors also applied
during pregnancy when the vaginal microbiome changes physiologically. In our study, we
confirmed that an increased number of artificial abortions and lower education level tended
to be associated with genital mycoplasmal colonisation in high-risk pregnant women. Un-
expectedly, we found that genital mycoplasmas were associated with reduced multifetal
pregnancies and infertility treatment. We presume that infertility treatment protocols in-
J. Clin. Med. 2023, 12, 40 10 of 13

cluding doxycycline administration before hysterosalpingography may reduce colonisation

by genital mycoplasmas, as previously noted [23,28].
Unlike the robust intra-amniotic infection or inflammation caused by Ureaplasma in
amniotic fluid [29], the impact of maternal vaginal colonisation by genital mycoplasmas
on pregnancy outcomes is less overt [27,30,31]. It was suggested in a meta-analysis that
genital mycoplasma presence in the vagina is insufficient to promote preterm birth [32]. For
example, for Ureaplasma urealyticum, the odds ratio for preterm labour was 1.94 [0.77–4.90]
for maternal samples, but 2.68 [1.18–6.09] for foetal samples [32]. In our study including
high-risk pregnant women, associations between vaginal genital mycoplasmas and adverse
pregnancy outcomes were not demonstrated except for histological chorioamnionitis. Such
associations between vaginal Ureaplasma species and histologic chorioamnionitis were
previously demonstrated in preterm births at less than 37 weeks [33] and in very preterm
infants with pPROM [34]. According to a Japanese study, isolation of Ureaplasma spp. in
the placenta from preterm births was associated with the severity of histologic chorioam-
nionitis [35]. In addition, the urease structural gene and protein of Ureaplasma spp. were
detected in the amnion, indicating direct infection of Ureaplasma spp. [35].
The clinical significance of antibiotic treatment for adverse vaginal colonisation in
pregnant women is uncertain. However, there are studies that suggest antibiotic therapy
may be more helpful earlier in pregnancy, since the irreversible negative effects of abnormal
vaginal microorganism are assumed to happen during that period [36,37]. Our finding
that patients in both the Gram-negative and -positive bacterial colonisation groups were
admitted for symptoms of preterm birth at earlier gestational age is also consistent with
this assertion.
There are some limitations of our study. Most of the former studies assessing abnormal
vaginal flora classified the concept as bacterial vaginosis, aerobic vaginitis, mixed abnormal
flora, or intermediate flora. For this classification, wet-mount microscopy, whiff test, and
pH measurement of the vaginal discharge are required in addition to the identification of
bacteria [7,38,39]. However, due to limited resources to perform such tests at our institution,
only the identification of the microorganism by culture was completed; this precluded
us from analysing the maternal inflammatory response to the colonised bacteria. Among
genital mycoplasmas, only Mycoplasma hominis and Ureaplasma urealyticum were checked,
so it could not be analyzed for other genital mycoplasmas, including Mycoplasma genitalium,
infection of which is known to be associated with preterm birth [40]. Additionally, there is
a possibility that treatments for preterm birth or other medical conditions before admission
might have altered the maternal vaginal flora [41]. The representativeness of vaginal culture
at admission might be a matter of debate.
Nonetheless, we consider our study to be clinically meaningful since there have been
relatively few studies addressing the risk factors and effects of abnormal vaginal flora on
parturient women who have already shown manifestations of preterm delivery. In addition,
having gathered data from a relatively large number of consecutive patients (n = 1381)
added strength to the results. While current guidelines indicate screening for bacterial
vaginosis in low-risk pregnant women is not beneficial, routine screening and treatment
of abnormal vaginal flora in high-risk pregnant women is still a matter of debate [42]. A
prospective study of antibiotic therapy focusing on Gram-negative microbes should also be
considered within the larger goal of reducing preterm births. With the prospective study, it
may be justified to conduct vaginal cultures to detect Gram-negative colonisation, at least
among women with high-risk pregnancies.
J. Clin. Med. 2023, 12, 40 11 of 13

Author Contributions: For Conceptualization: S.-y.O.; data curation: J.J., Y.-s.C., Y.K. and S.H.; formal
analysis: J.J.; supervision: J.-H.S., S.-J.C., S.-y.O. and C.-R.R.; writing—original draft preparation: J.J.
and S.-y.O.; writing—review and editing: J.J., Y.-s.C., Y.K., S.H., J.-H.S., S.-J.C., S.-y.O. and C.-R.R. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by a grant from the Patient-Centered Clinical Research.
Coordinating Center (PACEN) funded by the Ministry of Health and Welfare, Republic of Korea
(grant no. HC21C0123).
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki and approved by Institutional Review Board of Samsung Medical Center,
Sungkyunkwan University School of Medicine (IRB FILE No. 2020-08-006, and date of approval:
6 August 2020).
Informed Consent Statement: Patient consent was waived due to retrospective study design.
Data Availability Statement: The datasets analyzed during the current study are available from
the corresponding author on reasonable request, under approval of Institutional Review Board of
our institution.
Acknowledgments: We thank Jin-ha Kim for their invaluable contributions in data acquisition for
this study.
Conflicts of Interest: The authors declare no conflict of interest.

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