Resea ch A icle
An ibac e ial and An i ungal Ac i i y o he Human Endome ial
Fluid du ing he Na u al Cycle
Ma a B egón-Villahoz ,
1
Ma ia-Dolo es Mo agues ,
1
Inés A ie a-Agui e ,
1
Mikel Azka go a ,
2
Lucía Lainz,
3
Mi en Diez-Zapi ain,
3
Ma ia Iglesias,
3
Ma ia-Begoña P ie o,
3
Ana Ma o as,
4
An onia Exposi o,
3
Felix Elo za ,
2
and Robe o Ma o as
3,5
1
Depa men o Nu sing, Facul y o Medicine and Nu sing, Uni e si y o he Basque Coun y UPV/EHU, Ba io Sa iena s/n,
48940-Leioa, Spain
2
P o eomics Pla o m, CIC bioGUNE, CIBERehd, P o eoRed-ISCIII, Bizkaia Science and Technology Pa k, De io, Spain
3
Human Rep oduc ion Uni , C uces Uni e si y Hospi al, Uni e si y o he Basque Coun y, Ba akaldo, Spain
4
Facul ad de Medicina, Uni e sidad Eu opea de Mad id, Villa iciosa de Odon, Mad id, Spain
5
IVIRMAI, IVI Bilbao, Spain
Co espondence should be add essed o Ma ia-Dolo es Mo agues; [email p o ec ed]
Recei ed 22 Sep embe 2020; Re ised 26 Ap il 2021; Accep ed 2 June 2021; Published 16 June 2021
Academic Edi o : B yan La sen
Copy igh © 2021 Ma a B egón-Villahoz e al. This is an open access a icle dis ibu ed unde he C ea i e Commons A ibu ion
License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is
p ope ly ci ed.
Pu pose. Some mic obio a pa e ns ha e been associa ed wi h a o able IVF p ognosis and o he s wi h pa hological condi ions. The
endome ial fluid aspi a e (EFA) con ains an ibac e ial p o eins ha a e en iched in implan a i e IVF cycles, bu he an imic obial
effec o EFA has no been add essed. We aimed o e alua e he an imic obial ac i i y o he human endome ial fluid du ing he
na u al cycle. Me hods. EFA was ob ained h ough an emb yo ans e ca he e in 38 women, aged 18-40 yea s, wi h egula cycles
a ending o a e ili y clinic. The an imic obial ac i i y o EFAs was es ed agains wo s ains o S aphylococcus au eus; one s ain
each o S ep ococcus agalac iae,En e ococcus aecalis,Esche ichia coli, and Klebsiella pneumoniae; and h ee yeas s (Candida
albicans,Candida glab a a, and Candida k usei). Resul s. All samples exhibi ed an ibac e ial ac i i y agains S. au eus.In
addi ion, 32.4% o EFAs we e ac i e agains one o he o he mic oo ganisms assayed, 16.2% agains wo, and 5.4% agains ou
o hem. In con as , none exhibi ed an ibac e ial ac i i y agains E. coli o K. pneumoniae. The an imic obial ac i i y diffe s
conside ably be ween EFA samples, and we ailed o obse e a cycle- ela ed pa e n. Conclusions. EFA p esen ed wo
an imic obial ac i i y pa e ns: (a) one common o all he samples, exhibi ing ac i i y agains S. au eus and lack o ac i i y
agains E. coli and K. pneumoniae, and (b) an indi idualized pa e n, showing ac i i y agains some o he o he mic oo ganisms
es ed. The in ensi y o an ibac e ial ac i i y diffe s be ween EFA samples. Ou da a sugges ha he u e ine mic obio a is
con olled by means o endome ial fluid componen s.
1. In oduc ion
Emb yo implan a ion is one o he mos inefficien s eps o
assis ed ep oduc ion [1]. Implan a ion a es a e usually
<70% e en i euploid emb yos a e ans e ed [2]. Gi en his,
he e is a g owing in e es in in es iga ing he u e ine ole in
implan a ion. Since he ea ly wo ks o Noyes e al. [3], i is
well known ha he endome ium unde goes his ological
changes which a e pi o al o emb yo implan a ion.
In ecen yea s, a huge numbe o diffe en mic oo gan-
isms ha e been epo ed in diffe en o gans, such as he gu ,
skin, lungs [4], u ina y bladde [5], and agina [6]. Some
epo s ha e ocused on endome ial mic obio a. I has been
epo ed ha endome ial and aginal mic obio a can diffe
Hindawi
In ec ious Diseases in Obs e ics and Gynecology
Volume 2021, A icle ID 8849664, 9 pages
h ps://doi.o g/10.1155/2021/8849664
in s uc u e and composi ion [7, 8]. Indeed, a mic obio a pa -
e n a o able o emb yo implan a ion in in i o e iliza-
ion (IVF) cycles has been desc ibed [8].
Mo eo e , he sec e ions o cul u ed human endome ial
cells ha e shown an imic obial ac i i y [9–11], and an imi-
c obial pep ides ha e been epo ed in hem [12–14]. Ne e -
heless, he in i o implica ion o his ac i i y emains
unknown.
We ha e p e iously shown ha endome ial fluid can be
easily aspi a ed [15] wi hou impai ing p egnancy a es, e en
i pe o med a he same ime as emb yo ans e [16].
Depending on he analy ical app oach and he applied c i-
e ia, somewha mo e han 800 [17] o e en o e 2,200 p o-
eins ha e been de ec ed in he endome ial fluid aspi a e
(EFA) and some endogenous pep ides o he EFA ha e
shown an ibac e ial ac i i y [18]. On he o he hand, we ha e
shown ha in IVF he concen a ion o an ibac e ial pep ides
in he EFA is highe in cycles whe e implan a ion occu s han
hose whe e implan a ion ails [19].
Howe e , ac ual an ibac e ial ac i i y would p esumably
be he esul o in e ac ion be ween diffe en an ibac e ial
p o eins wi h endogenous pep ides o e en wi h o he mole-
cules p esen in he endome ial fluid. Thus, we ha e sough
o analyze he o e all an ibac e ial ac i i y o he EFA
ob ained du ing na u al o a ian cycles.
2. Ma e ial and Me hods
2.1. Popula ion. The popula ion unde s udy consis ed o 38
women o Caucasian e hnici y a ending he Assis ed Rep o-
duc ion Uni om he C uces Uni e si y Hospi al. The inclu-
sion c i e ia we e as ollows: (i) age be ween 18 and 40 yea s;
(ii) no mal u e ine ul asound; (iii) cycle leng h o 28-30
days; (i ) p e ious no mal ce ical cy ology; ( ) absence o
ubal condi ions; ( i) no his o y o ce ical in ec ions, sexu-
ally ansmi ed diseases, pel ic inflamma o y disease, endo-
me iosis, o polycys ic o a ian disease; ii) no p e ious
misca iages; ( iii) 0-2 p e ious IVF cycles; (ix) no an ibio ic
o ho monal he apy in he las 4 mon hs; (x) no mal o a ian
ese e; (xi) day o endome ial cycle 5-28; and (xii) body
mass index ðBMIÞ< 30 kg/m2.
Pa ien s cha ac e is ics we e as ollows: mean age o
35:4±3:8yea s, du a ion o in e ili y o 2:4±1:7yea s,
BMI o 26:9±3:8kg/m
2
, 94.4% ha ing no p e ious child en,
66.7% ha ing no p e ious IVF cycles, and 22.2% smoke s. In
42% o cases, he indica ion o IVF was he male ac o .
The s udy was app o ed by ou Ins i u ional E hical and
In es iga ion Boa d (CEIC code 11/45).
2.2. Endome ial Fluid Collec ion. Pa ien s we e asked o pa -
icipa e in he s udy by dona ing an EFA sample du ing he
mock emb yo ans e , which is usually pe o med as a pa
o he p e-IVF p o ocol. W i en in o med consen was
ob ained.
A s e ile disposable aginal speculum was placed wi hou
employing aginal lub ican s. In cases whe e leuco hea p e-
cluded access o he ce ical ex e nal os, he discha ge was
gen ly emo ed wi h a s e ile gauze. The EFA collec ion
me hod has been ecen ly epo ed [16, 19]. An “emb yo
ans e ”ca he e (F ydman ca he e , Ins umen os Médicos
Es é iles, SA, Spain) was inse ed h ough he ce ical canal,
in asep ic condi ions, wi hou i ouching he aginal walls
o he ec oce ix. Abdominal ul asound guidance was used
in o de o acili a e he passing h ough he ce ix [1] and
o p e en he ca he e om ouching he u e ine undus.
The sample was aspi a ed wi h gen le nega i e p essu e
applied wi h a 10 mL sy inge connec ed o he ca he e and
50-200 μL o EFA we e ob ained [16, 19]. To p e en con-
amina ion wi h ce ical fluid, aspi a ion was in e up ed a
he in e nal ce ical os, and special ca e was aken o a oid
auma izing he u e ine undus o he ce ix and con ami-
na ing he EFA sample wi h endome ial issue o blood.
Hea ily blood-s ained samples we e disca ded.
Since he e is no a s anda dized echnique o ob ain EFA
o an ibac e ial ac i i y analysis, we used wo me hods. Du -
ing he fi s pa o he s udy, he samples (n=20; EF-01 o
EF-19 and EF-26) we e expelled in o s anda d c yogenic
ubes, and he ca he e ip was cu and placed in o he same
ube. In he second pa o he s udy, a e he expulsion o
EFA, he inside o he ca he e was insed wi h 1 mL o s e ile
saline solu ion. The samples we e immedia ely ozen a
−80
°
C un il p ocessed. P io o be p ocessed in he labo a-
o y, he samples o he fi s g oup we e filled up o 1 mL final
olume wi h s e ile saline and he ca he e ip was emo ed
a e o exing o 15 seconds. The fi s me hod ca ied he
isk o d agging some ge ms om he ce ical canal ha
could p oduce some an imic obial subs ances. In he second
case, he in e io washing o he ca he e would a oid his
po en ial con amina ion o he sample.
EFA samples we e no collec ed by bo h me hods in he
same pa ien because EFA is e y sca ce and he e was a isk
ha he manipula ions o ob aining he fi s sample would
affec he quali y o he second one.
2.3. Assessmen o Mic obial G ow h om EFA Samples and
Iden ifica ion o Isola es. Ten μL o each dilu ed sample, om
bo h he “cu ip”sample g oup and he “flushed ou ca he e ”
g oup, was inocula ed on o Columbia aga supplemen ed
wi h 5% sheep blood (BD, USA) and incuba ed a 36 ± 1
°
C.
Ano he 10 μL aliquo o each sample was inocula ed on o
Man, Rogosa, and Sha pe aga (MRS; Sigma-Ald ich, USA)
and incuba ed a 36 ± 1
°
C in a 5% CO
2
a mosphe e.
Bac e ial DNA om he mos equen colony ypes iso-
la ed om samples was subjec ed o PCR amplifica ion o
he 16S RNA gene using he bac e ial uni e sal p ime s
27F and 1522R as desc ibed by Kang e al. [20]. The amplicon
sequences we e compa ed wi h hose s o ed in he NCBI
da abase o iden i y he isola ed bac e ia.
2.4. An imic obial Ac i i y o EFA Samples: B o h
Mic odilu ion Assay. EFA samples we e gen ly homogenized
in a o a ing wheel o 30 min a 4
°
C and hen cen i uged a
2,500 g o 15 minu es o elimina e he cellula deb is.
The cla ified EFA samples we e assayed agains h ee
Candida s ains (Candida albicans SC 5314, Candida glab-
a a ATCC 90030, and Candida k usei ATCC 6258) and six
bac e ial s ains (Esche ichia coli CECT 434, Klebsiella pneu-
moniae CECT 144, S ep ococcus agalac iae CECT 183T, and
2 In ec ious Diseases in Obs e ics and Gynecology
En e ococcus aecalis CECT 481 and wo S aphylococcus
au eus s ains— he CECT 435 e e ence s ain and a
me hicillin- esis an clinical isola e). ATCC s ands o Ame -
ican Type Cul u e Collec ion (Manassas, VA, USA) and
CECT o Colección Española de Cul i os Tipo (Pa e na,
Valencia, Spain). Candida spp. we e ou inely g own o
main enance in Sabou aud aga (BD) a 36 ± 1
°
C o 24 h,
while bac e ia we e g own in Nu ien Aga (Sigma-Ald ich)
unde he same condi ions.
The an imic obial ac i i y o EFA was es ima ed in 96-
well pla es, ollowing he guidelines o he Clinical and Labo-
a o y S anda ds Ins i u e (CLSI) p o ocols M-27-A3 o
yeas s [21], and M07-A9 o bac e ia [22], adap ed o ou
condi ions. B iefly, Candida s ains we e inocula ed in 100
μL o RPMI-MOPS, while bac e ia we e g own in Muelle -
Hin on b o h. Each well was supplemen ed wi h 25 μLo
he co esponding cla ified EFA sample. Pla es also included
a posi i e g ow h con ol o each mic oo ganism (wi h no
EFA sample) and a nega i e cul u e medium con ol (no
EFA sample and no mic oo ganism), as well as g ow h con-
ols o EFA samples wi h no added mic oo ganisms. The
pla es we e incuba ed a 36 ± 1
°
C, and g ow h inhibi ion wi h
e e ence o he co esponding g ow h posi i e con ols was
assessed isually a 24 h (48 h o S. au eus) using he ollow-
ing scale: -, no inhibi ion; +, sligh inhibi ion (less han 50%
educ ion o he cell pelle in he well); ++, mode a e o high
inhibi ion (≥50%); and +++, ull g ow h inhibi ion. G ow h
was always blindly assessed by he same in es iga o (MB-V).
2.5. S a is ical Analysis. The s a is ical associa ion o ca ego -
ical a iables was analyzed wi h he chi-squa e o he Fishe ’s
exac es , and P alues <0.05 we e conside ed s a is ically
significan (IBM® SPSS® S a is ics .24).
3. Resul s
3.1. Dis ibu ion o EFA Sampling o e he Mens ual Cycle.
Mos EFA samples (28/38) we e collec ed in he middle o
he cycle be ween days 13 and 19 (EF-12, sampled on day
16, was excluded om he subsequen s udy o an imic obial
ac i i y), while only fi e we e collec ed be ween days 5 and 12
and ano he fi e in he la e lu eal phase, be ween days 24 and
28 (Table 1).
Twen y samples included he ip o he ca he e , whe eas
he emaining 18 endome ial fluids we e flushed ou om
he ca he e . In bo h cases, EFA samples we e made up o 1
mL final olume wi h s e ile saline be o e being p ocessed
in he labo a o y.
3.2. Mic obial G ow h o EFA Samples. A ound 50% o he
EFA samples showed no mic obial g ow h on Columbia aga
(17/38) o MRS aga (21/38) (Table 2). The p opo ion o
cases wi h mic obiological g ow h was significan ly highe
o EFA samples s o ed wi h he ca he e ip compa ed wi h
hose which we e flushed ou (80% s. 27.8% on Columbia
aga (χ2=8:44;p<0:05) and 60% s. 27.8% on MRS aga
(χ2=3:98;p<0:05)). The analysis o he 16S RNA gene
sequences o colonies g own om 15 EFA samples showed
ha he mos abundan and equen ly isola ed mic obes
we e G am-posi i e od-shaped bac e ia (Lac obacillus spp.,
mos ly Lac obacillus gasse i), ollowed by colonies o G am-
posi i e coagulase-nega i e s aphylococci (S aphylococcus
epide midis)infi e samples (EF-04, 06, 10, 12, and 26). In
con as , o he colonies om EF-02, 10, and 30 ha only
g ew on Columbia aga we e iden ified as Ac inomyces u o-
geni alis and Co ynebac e ium spp.
The equency o posi i e mic obial g ow h was 2-3 imes
highe o samples ha had e ained he ca he e ip han o
hose ha only con ained he in e nal washing fluid o he
ca he e . In addi ion, he inoculum om h ee ou o he fi e
fluids wi h no ca he e bu posi i e g ow h on MRS aga only
yielded 1-2 colonies. In con as , one o he samples ha
included he ca he e (EF-12, collec ed on day 16 o he men-
s ual cycle) yielded uncoun able colonies; since excessi e
mic oo ganisms in e e ed wi h he an imic obial ac i i y
assays, his sample was disca ded o u he analyses.
3.3. An imic obial Ac i i y o EFA Samples. The da a o an i-
mic obial ac i i y displayed in Table 1 a e summa ized in
Figu e 1. Almos all he EFA samples s udied (34/37;
91.9%) inhibi ed he g ow h o he wo S. au eus s ains
es ed, while he emaining h ee samples only inhibi ed
one o hem; none o he EFA samples exhibi ed ull g ow h
inhibi ion o S. au eus (Table 1). In addi ion o inhibi ing S.
au eus, 32.4% (12/37) o samples we e ac i e agains only
one o he o he mic oo ganisms es ed, 16.2% (6/37) agains
wo mic oo ganisms, and 5.4% (2/37) agains ou mic oo -
ganisms (Table 1).
O e all, 40.5% (15/37) o EFA samples educed he
g ow h o a leas one o he h ee species o Candida, and fi e
o hem achie ed ull g ow h inhibi ion agains hem. Some
o he EFAs we e ac i e agains S. agalac iae (13.5%) o E. ae-
calis (13.5%). In con as , none o he dilu ed EFAs showed
ac i i y agains E. coli o K. pneumoniae (Table 1; Figu e 1).
Rega ding he day o EFA collec ion du ing he men-
s ual cycle, all samples exhibi ed a ce ain deg ee o ac i i y
agains S. au eus independen ly o he day o collec ion
(Table 1). Fou ou o fi e samples ha educed he g ow h
o E. aecalis we e ob ained in he cen al pe iod o he cycle
(days 14-17) and he fi h one on day 25 (Table 1). While C.
glab a a and/o C. k usei we e inhibi ed by se e al fluids col-
lec ed a diffe en poin s du ing he cycle (Table 1), C. albi-
cans was inhibi ed only by ou EFA samples om days 13
and 14; hus, he ac i i y agains C. albicans was s a is ically
associa ed (p<0:05) wi h EFA samples om he fi s pa
o he cycle (days 1-14) compa ed o he second pa (days
14-28). Th ee EFA samples—EF04, EF06, and EF26—we e
ac i e agains wo species o Candida (C. glab a a and C. k u-
sei) and wo—EF01 and EF27—agains all h ee species
es ed (C. albicans,C. glab a a, and C. k usei).
Al hough ca e was aken o a oid epi helial bleeding du -
ing he sample collec ion, an ibac e ial ac i i y agains S. aga-
lac iae was only egis e ed in fi e ou o 20 samples ha
con ained aces o blood (EF-01, 05, 11, 14, and 25; Table 1).
As o he an imic obial ac i i y o EFA depending on he
p esence o cul u able mic oo ganisms in he samples, we
ound no diffe ences in an i-S. au eus ac i i y: all samples
inhibi ed he g ow h o a leas one o he wo s ains es ed
3In ec ious Diseases in Obs e ics and Gynecology
(Table 1). Howe e , an imic obial ac i i y agains he es o
mic oo ganisms es ed was somewha mo e equen in cases
whe e he mic obial cul u e o EFA on MRS and/o Colum-
bia aga was posi i e (15/23; 65.22%) han hose wi h no
cul u able bac e ia (5/14; 35.71%) (χ2=3:05;p=0:08)
(Table 1). In ega d o he collec ion me hod and s o age,
Table 1: Time dis ibu ion, sample cha ac e is ics and an imic obial ac i i y o EFA samples.
Cycle
day
Sample Ac i i y agains mic oo ganisms
a
Iden ifica ion Ca he e
ip
b
Mic obial
g ow h
E.
coli
K.
pneumoniae
E.
aecalis
S.
agalac iae
S.
au eus MRSA C.
albicans
C.
glab a a
C.
k usei
5 EF-05 Yes Yes - - - + - + - - +
10 EF-07 Yes Yes - - - - ++ ++ - - -
EF-22 No No - - - - + + - - -
11 EF-24 No Yes - - - - + - - - +++
12 EF-23 No No - - - - + ++ - - -
13
EF-01 Yes Yes - - - ++ ++ ++ +++ +++ +++
EF-02 Yes Yes - - - - + ++ - - -
EF-10 Yes Yes - - - - ++ + - ++ -
EF-28 No No - - - - - + ++ - -
EF-29 No No - - - - ++ + - - -
14
EF-15 Yes Yes - - - - + + - - -
EF-20 No No - - - - ++ + - - -
EF-21 No Yes - - - - + ++ +++ - -
EF-25 No No - - - + ++ ++ - - -
EF-27 No No - - + - + ++ +++ +++ +++
15
EF-04 Yes Yes - - - - + ++ - ++ +
EF-19 Yes Yes - - - - + + - ++ -
EF-30 No Yes - - - - ++ ++ - - -
EF-37 No No - - - - ++ + - - -
16
EF-03 Yes Yes - - - - + ++ - - +
EF-13 Yes Yes - - + - + + - - -
EF-16 Yes No - - + - ++ ++ - - -
EF-26 Yes Yes - - - - + ++ - + ++
EF-31 No Yes - - - - ++ + - - -
EF-35 No Yes - - - - ++ ++ - - -
EF-36 No Yes - - - - ++ ++ - - -
17 EF-33 No No - - + - ++ + - - -
EF-34 No No - - - - + + - - -
18 EF-06 Yes Yes - - - - + + - +++ +
EF-14 Yes Yes - - - +++ + + - - -
19 EF-18 Yes Yes - - - - + + - - -
EF-32 No No - - - - + + - - -
24 EF-17 Yes No - - - - + + - - -
25 EF-11 Yes Yes - - - + ++ ++ - ++ -
26 EF-08 Yes Yes - - - - + ++ - - +
27 EF-09 Yes Yes - - + - ++ + - ++ -
28 EF-38 No No - - - - ++ + - - -
a
An imic obial ac i i y wi h e e ence o he cell pelle o he posi i e g ow h con ol well. -: no inhibi ion; +: sligh inhibi ion (<50% educ ion); ++: mode a e o
high inhibi ion (≥50%); +++: ull g ow h inhibi ion.
b
Ca he e : “yes”s ands o samples s o ed wi h he ca he e ip p io o p ocessing; “no”s ands o samples
flushed ou om he ca he e be o e being s o ed. Mic oo ganisms: Esche ichia coli,Klebsiella pneumoniae,En e ococcus aecalis,S ep ococcus agalac iae,
S aphylococcus au eus, MRSA (Me hicillin- esis an S. au eus), Candida albicans,Candida glab a a, and Candida k usei.
4 In ec ious Diseases in Obs e ics and Gynecology
samples ha included he ca he e ip we e mo e equen ly
associa ed wi h ac i i y agains mic oo ganisms o he han
S. au eus (14/19; 73.68%) han hose ha con ained only
he flushing fluid om inside he ca he e (6/18; 33.33%),
and his associa ion was s a is ically significan (χ2=6:06;
p<0:05).
4. Discussion
Mic obial popula ions o a ied composi ion ha e been
desc ibed in associa ion wi h diffe en ana omical loca ions
o he human body [23]. The in e ac ion be ween he hos
and mic obio a has been shown o play an essen ial ole in
many aspec s o human physiology [24–26].
Much less is known abou he u e ine side o implan a-
ion han he emb yonic side. The challenges o in es iga ing
he u e ine side o implan a ion include endome ial cyclic
changes, in e cycle a iabili y, and he influence o o a ian
s imula ion on endome ial changes, as well as he po en ial
ad e se effec o endome ial biopsy on implan a ion, i pe -
o med close o he ime o emb yo ans e [16]. In his con-
ex , we ha e ocused ou s udy on EFA, which can be
ob ained a he same ime as emb yo ans e , wi hou
impai ing implan a ion a es [16].
Mo eno e al. [8] iden ified a a o able pa e n in he
endome ial mic obio a o he success ul implan a ion o
he emb yo in IVF, cha ac e ized by a high p opo ion o
Lac obacillus. In a ecen s udy ocusing on he p o ein com-
posi ion o EFA ob ained a he same ime as he emb yo
ans e , we showed ha EFA om IVF cycles whe e implan-
a ion ook place (“implan a i e”cycles) was iche in an i-
bac e ial p o eins han EFA om “nonimplan a i e”cycles
[19]. This is in ag eemen wi h e y ecen da a om an
endogenous pep idomics- ocused mass spec ome y analy-
sis o EFA desc ibing he p esence o a numbe o pep ides
wi h po en ial an ibac e ial ac i i y in his fluid [18]. In line
wi h his, selec ed in silico p edic ed an ibac e ial pep ides
we e syn hesized and es ed in i o o an imic obial ac i -
i y. P elimina y esul s showed ha , indeed, some o hese
pep ides p esen an ibac e ial ac i i y [18]. In he same way,
i has ecen ly been sugges ed ha he inna e immune sys em
senses pa hogen-associa ed molecula pa e ns and his could
induce he elease o an imic obial pep ides in o he u e ine
ca i y [27].
In he p esen s udy, we ha e e alua ed he an imic obial
ac i i y o EFA, which con ains many diffe en an ibac e ial
p o eins and pep ides, whose syne gisms o e en an ago-
nisms a e s ill unknown. Indeed, some mic oo ganisms p es-
en in EFA could also play an an ibac e ial ole. Fo ins ance,
he Lac obacillus genus p oduces lac ic acid and sho -chain
a y acids, acidi ying he en i onmen o pH ≤4.5 in he
agina and p ohibi ing he g ow h o o he pa hogenic o
dysbio ic bac e ia in heal hy women [28, 29]. Howe e , con-
ce ning he endome ium, no co ela ion has been obse ed
be ween he pH alue and he endome ial mic obio a [8].
The possibili y o sample con amina ion is a significan
hu dle o in es iga e u e ine mic obio a [30]. The e is no
s anda dized me hodology o he s udy o human u e ine
mic obio a [30]. In mos o he s udies, he samples we e col-
lec ed h ough he ce ix [30]. Some au ho s used endome-
ial biopsies [31], o he s used ansce ical aspi a ion
h ough an emb yo ans e ca he e [8, 32–34] and hen
emp ied he con en (wi hou flushing ou he ca he e ),
o he s used a double lumen ca he e , wi hou aspi a ion,
and analyzed he dis al po ion o he ans e ca he e [7,
35], and some combined a double lumen ET ca he e wi h
la age and endome ial biopsy [36]. Aspi a ion o EF unde
asep ic condi ions has been shown o be a sa e and effec i e
Table 2: Mic obial g ow h o endome ial fluid aspi a e (EFA) samples om 38 women on Columbia aga and MRS aga . The a io o EFA
samples displaying mic obial g ow h in bo h media was significan ly highe o hose s o ed wi h he ca he e ip (∗χ2=8:44,p<0:05;∗∗
χ2=3:98,p<0:05) compa ed o EFA samples flushed ou om he ca he e .
Mic obial g ow h o EFA samples on
Columbia aga MRS aga
Posi i e Nega i e Posi i e Nega i e
EFA samples NN %N%N%N%
Wi h ca he e ip 20 16 80∗42012
60∗∗ 840
Flushed ou om ca he e 18 5 27.8 13 72.2 5 27.8 13 72.2
To al 38 21 55.3 17 44.7 17 44.7 21 55.3
13.5 8.1 2.7
13.5
51.4 54.1
2.7
2.7
13.5 2.7
43.2 43.2
2.7
8.1
8.1 8.1
0
E. coli
K. pneumoniae
E. aecalis
S. agalac iae
C. albicans
C. glab a a
C. k usei
S. au eus
MRSA
10
20
30
40
50
60
70
80
90
100
Pe cen age o EFA samples
Mic oo ganisms
Figu e 1: Pe cen age o EFA samples wi h an imic obial ac i i y
agains diffe en mic oo ganisms. Colo code e e s o he
in ensi y o inhibi ion as desc ibed in Ma e ial and Me hods (blue:
+; yellow: ++; salmon: +++).
5In ec ious Diseases in Obs e ics and Gynecology
me hod o e alua e he endome ial mic obio a [8, 37]. We
ha e unde aken his wo k in o de o e alua e he po en ial
an imic obial ac i i y o he endome ial fluid o 38 women
in ep oduc i e age. The EFA samples we e collec ed h ough
aspi a ion wi h an emb yo ans e ca he e passed h ough
he ce ical os, aking p ecau ions o a oid con amina ion
wi h he ce ix mic obio a. Two diffe en collec ion me hods
we e used: (i) emp ying he ca he e con en and s o ing he
sample wi h he ca he e ip and (ii) flushing he inside o
he ca he e wi h saline solu ion. In ou wo k, app oxima ely
hal o he EFA samples con ained cul u able mic oo gan-
isms, in ag eemen wi h da a s a ing ha he endome ial
ca i y is no s e ile in mos heal hy women [38].
Twice as many posi i e cul u es we e ob ained om sam-
ples s o ed wi h he ca he e ip compa ed o samples wi hou
i . Colonies o S aphylococcus epide midis we e only iden i-
fied in hose samples s o ed wi h he ca he e . I could be
specula ed ha he ex e nal su ace o he ca he e e ained
a numbe o mic oo ganisms om he ce ical canal, and
hence, o mic obiological analyses, i seems ad isable o col-
lec only he in e nal fluid o he ca he e . Ne e heless, he
iable bac e ia iden ified in bo h sample ypes consis ed
mainly o Lac obacillus spp. and o a much lesse ex en Ac i-
nomyces u ogeni alis and Co ynebac e ium spp. Lac obacillus
spp. we e he mos equen ly isola ed bac e ia om he EFA
samples, in ag eemen wi h p e ious epo s o Wee e al.
[39] and Mo eno and Simon [37]. Al hough significan di -
e ences ha e been epo ed in aginal and u e ine mic obi-
o a [8, 33, 34, 40], as a as we know, ce ical and u e ine
mic obio a ha e no been compa ed. Selman e al. s udied
endoce ical and ec oce ical samples as well as he in e nal
ip o he ca he e , bu hey did no aspi a e samples and
he esul s o each localiza ion we e no compa ed [35].
The e is also a con o e sy abou he me hod o analysis
o he u e ine mic obiome. Rega ding he cul u e o iable
mic obes, less han 1% o he bac e ia p esen in a sample
g ow and o m colonies; u he mo e, some human samples
may con ain a limi ed amoun o mic oo ganisms [41]. On
he o he hand, he nex -gene a ion sequencing (NGS)
analysis o EFA samples con aining a small numbe o
mic oo ganisms may be dis o ed by abundan con amina -
ing aginal mic obio a [30]. Mo eo e , NGS eadings do
no diffe en ia e be ween li e and dead mic oo ganisms,
and consequen ly, he u e ine mic obial popula ion may
be o e es ima ed.
The u e ine mic obio a ha e been epo ed o fluc ua e
wi h mens ual cycle iming [42], o a ian s imula ion [43],
e hnic g oup [44], ce ain pa hological condi ions [42], and
IVF p ognosis [37]. In ou s udy, pe o med in na u al cycles
o Caucasian women in ep oduc i e age, we ha e no been
able o es ablish a clea associa ion be ween he iable mic o-
bio a o EFA samples and he ime o sampling du ing he
mens ual cycle. Howe e , we acknowledge ha only a small
numbe o he samples s udied we e ou side he cen al pa
o he cycle.
Rega ding he an imic obial ac i i y, all he EFA samples
in he p esen wo k inhibi ed he in i o g ow h o S. au eus
o some ex en , and he e we e no significan diffe ences
be ween cul u able and noncul u able samples, o e en
be ween samples collec ed a diffe en imes o e he men-
s ual cycle. A numbe o po en ial an ibac e ial pep ides
ha e been epo ed om human endome ial issue cul u es
[12–14]. Ou esul s a e consis en wi h hose ob ained wi h
cul u es o u e ine epi helial cells [45], showing he p oduc-
ion o an ibac e ial ac o s ha effec i ely killed S. au eus,
and co ela ion wi h sec e o y leukocy e p o ease inhibi o
concen a ions. Ne e heless, in he same s udy on u e ine
epi helial cell cul u e [45], an ibac e ial ac i i y agains E. coli
was also obse ed, a ype o ac i i y ha was no obse ed in
any pa ien s om ou s udy. We we e also unable o find any
ac i i y agains K. pneumoniae in any o ou pa ien s’EFA
samples. On he o he hand, mo e han hal o he EFA sam-
ples o he s udied women (54.1%) exhibi ed an imic obial
ac i i y agains o he mic oo ganisms including C. albicans,
C. glab a a,C. k usei,S. agalac iae, and/o E. aecalis. Mo e-
o e , ou findings ega ding C. albicans a e in ag eemen
wi h hose o Wi a e al. [46], who epo ed ha sec e ions
o cul u es om he uppe emale ep oduc i e ac cells
inhibi yeas and hyphal o ms o C. albicans.
I has o be s essed ha he e was a ema kable he e oge-
nei y in an imic obial ac i i y pa e ns: (i) he in ensi y o he
an imic obial ac i i y diffe s no ably be ween he diffe en
specimens, and (ii) apa om S. au eus which was always
inhibi ed, he p opo ion o EFA inhibi ing some o he
emaining mic oo ganisms assayed anged om 10.8 o
24.3%. Fu he mo e, in addi ion o S. au eus, 32.4% o
EFA samples we e ac i e agains only one o he emaining
mic oo ganisms assayed, 16.2% agains wo mic oo gan-
isms, and only 5.4% agains ou mic oo ganisms. On he
o he hand, i mus be highligh ed ha a numbe o mic o-
o ganisms, especially anae obic bac e ia, we e no e alua ed
in ou s udy.
Conce ning he sou ce o an ibac e ial compounds, in
addi ion o endome ial issue, as sugges ed by issue cul u e
s udies, one canno ule ou an associa ed effec coming om
he mic obio a o he endome ial ca i y. In ag eemen wi h
his, apa om he an i-S. au eus ac i i y which was p esen
in e e y sample, we ound ha an imic obial ac i i y agains
he o he es ed mic oo ganisms was mo e equen in he
samples whe e mic obiological cul u es we e posi i e. Wang
e al. [47] a ibu ed an imic obial ac i i y o Lac obacillus
spp. ha p oduces lac ic acid and hyd ogen pe oxide, among
o he compounds, which can inhibi he g ow h o po en-
ially pa hogenic bac e ia, as well as he filamen a ion o C.
albicans. None heless, he anae obic en i onmen o he
endome ium does no acili a e hyd ogen pe oxide p oduc-
ion by Lac obacillus. Indeed, we ha e documen ed an i-Can-
dida ac i i y in mo e han one- hi d o he EFA samples,
e en in cases when no g ow h o Lac obacillus on MRS
medium was eco ded. Rega ding he me hod o collec-
ion and s o age, he samples con aining only he fluid
flushed ou om he ca he e we e associa ed wi h he
leas equen ly posi i e cul u es and an imic obial ac i i y.
Al hough ou sample numbe s we e limi ed and we we e
unable o assess a ange o con ounding ac o s, he la e
me hod is p obably he one ha can p o ide he mos eliable
in o ma ion on he u e ine mic obio a and i s an imic obial
ac i i y.
6 In ec ious Diseases in Obs e ics and Gynecology
In his s udy, we ha e shown ha EFA samples om
women o ep oduc i e age exhibi an imic obial ac i i y
agains some mic oo ganisms ha could impai IVF esul s.
All he EFA samples es ed in i o educed he g ow h o
S. au eus, and many o hem inhibi ed Candida spp., E. aeca-
lis, and/o S. agalac iae as well, bu no E. coli o K. pneumo-
niae. To he bes o ou knowledge, his is he fi s epo on
he di ec an imic obial ac i i y o endome ial fluid aspi a e
samples in humans.
Fu he s udies a e wa an ed o cha ac e ize he endo-
me ial fluid o women unde going IVF p ocedu es in o de
o assess whe he he e is a ela ionship be ween he an imi-
c obial ac i i y o aw EFA and implan a ion success.
Da a A ailabili y
The da a used o suppo he findings o his s udy a e a ail-
able om he co esponding au ho upon eques .
E hical App o al
The s udy was app o ed by ou Ins i u ional E hical and
In es iga ion Boa d (CEIC code 11/45).
Consen
Pa ien s we e asked o pa icipa e in he s udy by dona ing an
EFA sample du ing he mock emb yo ans e , which is usu-
ally pe o med as a pa o he p e-IVF p o ocol. W i en
in o med consen was ob ained.
Con lic s o In e es
The au ho s epo no conflic o in e es .
Au ho s’Con ibu ions
M B egón-Villahoz is esponsible o he sample analyses,
da a collec ion, and manusc ip w i ing. MD Mo agues is
assigned o he p o ocol de elopmen , da a analysis, and
manusc ip w i ing/edi ing. I A ie a-Agui e is esponsible
o he samples and da a analysis. L Lainz, M Diez-Zapi ain,
M Iglesias, A Exposi o, and MB P ie o a e assigned o pa ien
selec ion and sample collec ion. A Ma o as, F Elo za, and M
Azka go a did he da a analysis. R Ma o as is esponsible
o he p ojec de elopmen , pa ien selec ion, sample collec-
ion, da a managemen and analysis, and manusc ip
w i ing/edi ing.
Acknowledgmen s
This s udy was pa ially suppo ed by a G an o Fe ili y
Inno a ion (GFI, 2011) om Me ck, Da ms ad , Ge many.
M. B egón-Villahoz is ecipien o a p edoc o al g an om
he Uni e sidad del País Vasco-Euskal He iko Unibe si a-
ea (UPV/EHU) (PIF19/316). The au ho s hank he echni-
cal and human suppo p o ided by DNA Bank Se ice
(SGIke ) o he Uni e si y o he Basque Coun y
(UPV/EHU) and Eu opean unding (ERDF and ESF). CIC
bioGUNE is acc edi ed wi h he Se e o Ochoa Excellence
awa d by he Spanish Minis e io de Economía y Compe i i i-
dad, MINECO (SEV-2016-0644).
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