Evaluation of the Relationship Between Vaginal, Placental and Neonatal Mycobiota and Microbiome With Preterm Birth in Women With Short Cervical Length
Conditions: Preterm Birth, Microbial Colonization
Sponsor: Koç University

 
 
 
 
Evaluation of the Relationship 
Between Vaginal, Placental and 
Neonatal Mycobiota and Microbiome 
with Preterm Birth in Women 
 with  
Short Cervical Length 
      
EBRU CELIK 
KOC UNIVERSITY SCHOOL OF MEDICINE 
 
MARCH 1, 2019 

 
Ethic No: 2019.093.IRB2.030 
 
01.03.2019 
 
 
STUDY PROTOCOL 
 
Introduction 
 
The term microbiota is used to describe "ecological commensal, symbiotic and 
pathological communities" living on and / or within all multicellular microorganisms, from 
plants to animals. Microbiota contributes to the immunological, hormonal and metabolic 
homeostasis of the host. As in all natural orifices in the body, there is also a microbiota 
and mycobiota specific to the vagina. 
 
On the other hand, the sonographic short cervix in the second 3 months of pregnancy is 
associated with preterm delivery, which may be an important cause of mortality and 
morbidity in the neonatal period. American Society of Obstetricians and Gynecologists 
(ACOG), British Royal Society of Obstetricians and Gynecologists (RCOG) and the 
American Society of Maternal Fetal Medicine (SMFM) suggests the measurement of 
transvaginal sonographic cervical length at 20-24 gestational weeks for screening of 
preterm birth.  Aforementioned associations also recommend the use of progesterone in 
the treatment of women who diagnosed with short cervix by transvaginal ultrasonography 
due to the fact that progesterone is effective in prevention of preterm birth (Grade B) (1-
2). 
 
Previous vaginal microbiota studies have shown that some bacterial species, such as 
Lactobacillus insers, cause a predisposition to premature labor in patients with short cervix 
(3). However, the prominent deficiency in these studies is that the eukaryotic fungi in 
abundant vaginal flora have not been evaluated. 

 
Ethic No: 2019.093.IRB2.030 
 
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On the other hand, it is already known that progesterone treatment is not able to prevent 
all preterm birth in women with short cervical length. As a research group, our hypothesis 
is that the variety of microbiological and / or mycobiotal in pregnancies resulting in preterm 
birth are different than those who give birth at term although patients with sonographic 
short cervical length receive progesterone regularly from the second trimester. In this 
study, the aim of this study is to evaluate the patterns of microbiota and mycobiota from 
vaginal swabs of women who had preterm birth due to sonographic short cervical length 
and postpartum swabs of placenta and fetal oral cavity.  
In this study, it has already known that some species in vaginal flora present a greater risk 
for preterm delivery. Women with short cervical length tend to give preterm birth, 
especially in the second trimester of pregnancy (between 20-28 weeks of gestation). 
Babies born in this early period are exposed to high risk of morbidity, such as severe 
neurodevelopmental sequelae, even if they survive. Early diagnosis and appropriate 
treatment of cervical / vaginal mycobiota and microbiota changes in women with short 
cervical length might reduce the rate of preterm birth and the associated neonatal mortality 
and morbidity. 
 
Yet, measurements of cervical length are routinely performed in all pregnant women at 
11-14th and 18-22 th  gestational weeks by transvaginal ultrasonography. Of these 
patients, cervical swabs will be obtained just before the scans from women who agree to 
participate in the study will be kept in the -80 closet in our hospital until the samples are 
studied. Study group will include women who had cervical length 25 mm or less than 25 
mm at 20-24 th gestational week and underwent birth (preterm delivery) before 37 + 0 
weeks of gestation. The control group had pregnancies with a cervical length above 25 
mm and delivered at term (37 + 0 gestational week and above). During the follow-up of 
pregnancies, care will be taken not to use antibiotics. Plaques of placental tissue in sterile 

 
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conditions and oral swab samples from newborns will be taken from women whose follow-
up and delivery are performed in our hospital and  by our team. 
 
The inclusion criteria are: 
 
- Singleton pregnancies 
- The pregnant women who are willing to participitate sign the informed consent form  
 
The exclusion criteria are: 
 
- Multiple pregnancies 
- The presence of fetal anomaly 
- Finding the intrauterine mort de fetus 
- Antibiotic and / or antifungal use at any time of pregnancy 
- Pregnant women under 18 years of age 
- Women with previous cervical surgery 
- Women who do not accept to participate to be in the study 
- The presence of uterine anomaly 
- Women with vaginal bleeding at the time of cervical swabs taken 
 
Study Procedure 
 
The measurements of  cervical length will be performed by the perinatology team in two 
different periods of pregnancy (11-14 and 18-22 weeks of gestation) in the singleton 
pregnancies. Firstly, the vaginal swabs of women who accept to participate in the study  
will be collected at 11-14th weeks of gestation just before the measurement of cervical 
length. Also, the vaginal swabs through the aferomentioned procedure will be collected at 

 
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18-22 weeks of gestation. Micronized progesterone will be started in women with a 
cervical length equal or less than 25 mm (smaller than 3rd percentile) at 18-22 weeks of 
gestation. The medication (progesterone 200mg) will be administered intravaginally every 
night before bedtime and will continue until 36th gestational week unless the patient gives 
birth. Measurement of cervical length will be repeated transvaginally at 28th and 32nd  
gestational weeks, and samples will be taken with vaginal swab before these 
measurements. In order to evaluate the presence of placental mycobiota and microbiota 
after birth, 1 cm x 1 cm x 1 cm of tissue sample from placenta that is 3 cm lateral of the 
cord insertion will be taken under sterile conditions. In order to evaluate the amniotic 
compartment, buccal mucosal swabs will be taken from the neonates immediately after 
birth. All samples taken during pregnancy follow-up and after delivery will be delivered to 
our Research Laboratory immediately and will be kept and stored in -80 degrees cabinet 
until the evaluation.  
 
Study population 
 
The study will plan to take 2 years for recruitment of patients. The enrollment of 60 
participants with singleton pregnancies who have cervical length of 25 mm or less would 
give the study a power of 85% to show a treatment effect at a two-sided alpha level of 5%.  
The risk of preterm birth in a pregnant with a cervical length of 25 mm or less is 30%. The 
rate of preterm birth is 12% in the whole population. Thirty-two women who delivered at 
ter with cervical length of longer than 25 mm will be in the control group. For the 
recruitment of study group, 1429 women with  singleton pregnancies will need to be 
screened.  
The patients will be included in the study group; 
1) Pregnant women with cervical length of 25mm or less (n=60) 

 
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   A) Of 60 patients, 30% will be expected to give a birth before 37 gestational weeks 
(n=18) 
  B) Of 60 patients, almost 70% will be expected to give a birt at term (n=42) 
2) As a control group, women with a cervical length of more than 25 mm who give a birth 
at term will be included (n=32) 
A total of 1429 women will need to be screened to be able to recruit the above numbers 
of patients.   
 
Analysis of Microbiota  
Isolation of DNA  
 
Genomic DNA will be stored at 80 0 C until the analysis microbiota is performed by using 
the BIO PowerSoil DNA isolation kit. 
 
Bacterial microbiota 
 
The V3-V4 regions by the 16s ribosomal RNA sequencing method will be sequenced with 
the Illumina MiSeq device and the data will be analyzed according to the protocols 
standardized in the Human Microbiome Project. 
 
Fungal Microbiota 
 
DNA samples which will be ranked from the data to be sequenced in accordance with the 
ITS1 region metabiota protocol will be evaluated within the Human Microbiome Project. 
Pseudomonas, 
Escherichia, 
Neisseria, 
Streptococcus, 
Lactobacillus, 
Candida, 
Actinomyces will be used as positive controls and, V3-V4 and ITS regions will be matched 
for bacterial microbiota and Fungal microbiota. 
 

 
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Evaluation of Results 
 
After the "Operational taxonomic units" of the amplicons are configured with the 
VSEARCH program, the analyzes will be carried out with GENBANK microbiota and 
micobiota data. 
 
Recruitment and care of patients, collection of materials and management plan will be 
carried out by Perinatology team at Koç University Hospital Gynecology and Obstetrics 
Clinic during the study. The evaluation of materials will be carried out by the Department 
of Microbiology of Koç University, School of Medicine. 
 
Only the research team will have access to the identity information of the participants. 
Only physicians in the perinatology department of Koç University Hospital, Department of 
Obstetrics and Gynecology, have access to the GE Voluson E8 Ultrasonography device 
and the hospital-approved Viewpoint archiving system, which is already in place at the 
Perinatology Department. After the research is completed, the forms will be destroyed by 
paper milling machine after 5 years. 
 
 The project is supported by TUBITAK (prot no: 119S463).  
 
 
 
 

 
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References: 
 
[1] Aagaard, K., Ma, J., Antony, K.M., Ganu, R., Petrosino, J., ve Versalovic, J. 2014. “The placenta harbors a 
unique microbiome”, Sci Transl Med, 21(6),237. 
 
[2] DiGiulio, D.B., Romero, R., Amogan, H.P., Kusanovic, J.P., Bik, E.M., Gotsch, F., Kim, C.J., Erez, O., Edwin, 
S., ve Relman, D.A. 2008. “Microbial prevalence, diversity and abundance in amniotic fluid during preterm 
labor: A molecular and culture-based investigation”, PLoS One, 3(8),30-56. 
 
[3] DiGiulio, D.B., Romero, R., Kusanovic, J.P., Gómez, R., Kim, C.J., Seok, K.S., Gotsch, F., Mazaki-Tovi, S., 
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[5] Han, Y.W., Shen, T., Chung, P., Buhimschi, I.A., ve , C.S. 2009. “Uncultivated bacteria as etiologic agents of 
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[9] “Practice Bulletin No. 171: Management of Preterm Labor”, 2016. Obstet Gynecol, 128(4):155-64  
 

 
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[10] “Preterm labour and birth”, 2015. National Institute for Health and Care Excellence (NICE) guideline. National 
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https://www.ncbi.nlm.nih.gov/books/NBK327571/  Son erişim tarihi: 28 Mart 2019 
 
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Ethic No: 2019.093.IRB2.030 
 
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The first trimester scan 
(11-13 weeks of gestation 
• 
-Demographic data 
• 
-Vaginal swabs 
• 
- Measurement of cervical 
length 
The 
second 
trimester 
scan (18-24 weeks of 
gestation 
• 
-Demographic data 
• 
-Vaginal swabs 
• 
- Measurement of cervical 
CL≤ 25 mm 
CL>25 mm 
The 
second 
trimester 
scan (28-32 weeks of 
gestation 
• 
-Vaginal swabs 
• 
- Measurement of cervical 
length 
• 
Birth; 
• 
Collection 
of 
placental 
specimen 
• 
Neonatal buccal swabs 
 
Isolation fo 16s 
rRNA and 18s rRNA  
Analysis of 
bioinformatic data 
Administration of 
progesterone 
Recruitment of patients