Timing of umbilical cord clamping during an elective caesarean delivery and its association with neonatal gut microbiome

During a caesarean delivery, umbilical cord clamping can be carried out before
or after providing the mother with a perioperative prophylactic antibiotic. If
the cord is clamped prior to giving the mother a prophylactic antibiotic, then
this antibiotic cannot pass on to the infant. However, if the cord is clamped
after giving the mother antibiotics these maternal antibiotics crosses the
placenta and reaches the circulation of the infant. In this study it is
hypothesized that such antibiotics alter the composition and functionality of
the neonatal gut microbiome. A modification of neonatal microbiome has been
associated with the development of asthma, allergies, type 1 diabetes and
obesity later in life. This small intervention thus could have important
consequences for the future health of the infant.

In the Netherlands, most of these elective CS are carried out primarily based
medical indications such as breech presentation. The most common complications
for the mother after C-sections include infectious morbidities like
endometritis and wound infection. The 2008 meta-analysis carried out by
Costantine et al. identified strong evidence that antibiotic prophylaxis for
caesarean delivery that is given before skin incision, rather than after
umbilical cord clamping, decreases the incidence of postpartum endometritis and
total infectious morbidities, without affecting neonatal outcomes. This
meta-analysis has been the basis for current protocols in the procedure for CS
in some hospitals in The Netherlands, including the UMCG. In 2014, results from
the systematic review of Mackeen et al. in The Cochrane Library, showed that
intravenous prophylactic antibiotics for all types of CS administered
preoperatively significantly decreases the incidence of composite maternal
postpartum infectious morbidity as compared with administration after cord
clamping. Such a review would indicate that, for the mother, antibiotics given
prior to skin incision would be a better choice in preventing post-operative
infections. An important point to note however is that, in all these studies,
the short and long term effects of antibiotic exposure on neonatal health has
been widely neglected. In their systematic review Mackeen et al. stressed the
need to elucidate short- and long-term adverse effects of antibiotics for
neonates. In 2015, in their randomized controlled trial and meta-analysis Zhang
et al. using the end points of postpartum endometritis and total infectious
morbidities, concluded that for an elective caesarean delivery, the effects of
antibiotic prophylaxis before skin incision and after umbilical cord clamping
were equal .

In studies that investigate the effect of giving antibiotics pre and post cord
clamping on mother and neonate, the effect of the antibiotics on neonatal gut
microbiome is ignored. The concerns regarding the administration of antibiotics
before cord clamping has traditionally been about unnecessary foetal exposure
that might mask foetal infections and increase need for sepsis work up in
infants. Recent studies, however, have focused more on the change in neonatal
microbiome as a result of pre cord clamping antibiotics. The antibiotics given
to the mother prior to skin incision rapidly cross the placenta and reach the
circulation of the neonate before birth, with an inevitable but not yet fully
characterized influence on newborn microbial colonization. Previous guidelines
advised cord clamping prior to giving mothers antibiotics to prevent such
collateral neonatal antibiotic exposure. There is increasing evidence for a
functional role of gut microbiota in driving immune development in the new-
born and the development of chronic conditions later in life.The human gut
microbiome plays an extremely important role in early neonatal development
(10), but also possible in later health and adult onset of disease. The
establishment of stable microbial communities within the gastrointestinal tract
closely parallels growth and immune development in early life. Antibiotic
exposure in children has been associated with increased risk of diabetes,
obesity, inflammatory bowel disease, asthma and allergies. Recent studies by
Yossour et al. and Bokulish et al. have noted the role of antibiotic exposure
in microbial community instability. Gut microbiome immaturity in infants has
been associated with malnutrition and reduced diversity has been observed in
infants who later develop type 1 diabetes. Furthermore, development of
childhood obesity has been linked with distinct microbial composition changes.
Disturbances in the intestinal microbiome in early life has been associated
with the development of immune-mediated disorders like allergy, asthma and
atopic eczema later in life. Thus, there is sufficient need to investigate this
possible source of microbiome disruption in early life that can result in grave
future consequences.

It is thus of extreme important that high quality evidence of immediate
benefits to the mother should be weighed against equally good evidence about
any potential risks of long-term harm to the infant. However, so far, no RCTs
have measured the effects on infants receiving intrapartum antibiotics. Such
studies are unlikely to be undertaken because of the long duration of follow up
required to measure health outcomes that might not present until years later.
Evidence suggesting an adverse effect of early antibiotic exposure on the
infant gut currently comes from observational studies but the limitations in
such studies mean they are less likely to be included in systematic reviews.
This RCT therefore seeks to explore the effect of antibiotics given before skin
incision on the composition and functionality of neonatal microbiome up to 1
year of life. Should this study reveal important changes in the neonatal
microbiome composition, this would be a strong indication to set up larger
studies, that follow infants for a longer period of time. Therefore, this study
is of crucial importance and seeks answer an important question regarding the
reproductive origins of adult disease.

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Primary Objective:
To compare the composition and functionality of neonatal microbiome arising
from mothers receiving antibiotic prophylaxis before skin incision with the
microbiome of neonates born when antibiotics are given after umbilical cord
clamping in caesarean delivery.

Secondary Objective(s):
To compare the rates of endometritis, wound infection and Urinary Tract
Infection (UTI) between the group of women receiving antibiotics prophylaxis
before skin incision and after umbilical cord clamping.

This randomized control trial will be carried out at the University Medical
Centrum Groningen (UMCG). All pregnant women undergoing elective CS at the UMCG
will be asked to participate when they come for their appointment at 36 weeks
of pregnancy. Participation will be noted only after asking a fully informed
consent.
Randomization will be carried out without involvement of investigator in trial
conducting. Fifty participants will be randomly assigned to receiving
antibiotics before skin incisions or after cord clamping in a 1:1 ratio.
Females scheduled for planned CS will be admitted to the hospital 4 hours prior
to their surgery. In case of the group having umbilical cord clamping cord
clamping after giving the antibiotic, the antibiotic (1 gram of Cefazolin) will
be administered 30 minutes prior to surgical incision by the anaesthesiologist.
In the group receiving the antibiotics after cord clamping, 1 gram of Cefazolin
(once, intravenous) will be administered by the anaesthesiologist as soon as
the umbilical cord has been clamped. Level of cephazolin in cord blood will be
measured by collecting 60 mL of cord blood.
One day before the caesarean section vaginal and blood samples will be
collected by obstetrician at the ward and the stool samples of the mother will
be collected on the day itself.
The stool of the neonate will be collected on days 1, 2 and 3 by the nurses at
the nursery ward. These samples will be stored at -80ºC. At the time of
discharge from the hospital after day 3, the parents of the infant will be
provided with 6 labelled stool collection kits for collection of stool samples.
The stool of the infant will be collected each week from week 1 to week 6. The
mother/father will be instructed to collect the samples and store them in her
own refrigerator in the freezer, after labelling them. On week 6 the parents
with the infant will return to the UMCG, bring the plastics bags with the
collected samples. These samples will be collected from the parents and stored
at -80ºC.
In addition to collecting the faecal samples, the parents will also be asked to
fill in a written questionnaire every week, answering questions related to the
diet, vaccinations, medication and general health of the infant during these
weeks.
The stool samples and questionnaires of the neonates at time points 6 months
and one year will be collected from the homes of the patients.

This study is a Phase 4 study with the intervention in this study being the
timing of umbilical cord clamping. It is either done 1) After giving the mother
the prophylactic antibiotic 2) Prior to giving the mother the prophylactic
antibiotic. Practically this will be done as follows:
One gram of Cefazolin (once, intravenous) will be administered 30 minutes
before CS by the anaesthesiologist in the group of women receiving antibiotics
before skin incision. In the other group, the same antibiotics will be
administered after umbilical cord clamping. Usage of 1g cefazolin as
prophylaxis in CS in line with the indications of the standard protocol.

The burden associated with the study can be divided into 2 parts 1) During the
length of stay in the UMCG and 2) After discharge from the UMCG. During the
length of the stay in the hospital the mother and the neonate face minimal
burden and no invasive tests. Stool samples from the mother will be collected
shortly before CS. The first stool of the neonate will be collected shortly
after the CS. After discharge of the mother and neonate, parents will be asked
to collect the stool of the infant in provided plastic bags, every week for a
period of 6 weeks and freeze this. The samples will be brought to the UMCG at
the regular check-up at 6 weeks. In addition to the stool sample, the parents
will have fill in a written questionnaire every week regarding diet,
medications and vaccinations of the neonates. The stool samples and
questionnaires will again be collected at 6 months and 1 year form the houses
of the mother and infant.


The risks associated with cord clamping before giving antibiotics to the mother
is the possible occurrence of post-operative infections like endometritis and
wound site infections. As mentioned previously, however, these studies have not
been limited women undergoing elective CS. Thus, the true increased risk of
these women, by exposing them to antibiotics post cord clamping cannot be truly
estimated based on these reviews. In the trail carried out by Zhang et al.,
taking into account only elective CS, the effects of antibiotic prophylaxis
before skin incision and after umbilical cord clamping were equal.Even though
the estimated risk is small we have kept in mind certain elements that can
minimize this risk further. In the UMCG, women undergoing elective CS stay in
the ward for 3 days after surgery and are under the supervision of nurses and
doctors, who can detect post-operative infections should they occur. These
women will immediately be treated, in case of signs of infection


The effects of the pre- incisional antibiotics on neonatal gut microbiome has
not been greatly studied. A number of studies implicate neonatal antibiotic
exposure in altering neonatal metabolism and the development of obesity in
later life. Furthermore, studies also associate early gut microbiome
disruptions with the development of obesity, allergy and insulin dependent
diabetes. Thus this study offers the tools to investigate this matter is of
great importance.