microRNA-based signatures obtained from endometrial fluid identify implantative endometrium

mic oRNA-based signa u es ob ained
om endome ial luid iden i y
implan a i e endome ium
Jone Iba~
nez-Pe ez
1,2,3,4
, Ma 
ıa D
ıaz-Nu~
nez
1,2
, Ma c Clos-Ga c
ıa
5
,
Luc
ıa Lainz
1,2
, Ma 
ıa Iglesias
1,2
, Mi en D
ıez-Zapi ain
1,2
,
Ain zane Rabanal
1,2
, Lau a Ba´ cena
6
, Monika Gonza´lez
6
,
Juan J. Lozano
7
, U ko M. Ma igo a
8,9
, Espe anza Gonza´lez
4
,
Fe´lix Royo
4,10
, Ana M. A ansay
6,10
, Ne ea Subi an
2,11
,
Robe o Ma o as
1,2,3,12,
*, and Juan Manuel Falco´n-Pe´ ez
4,9,10,13,
*
1
Human Rep oduc ion Uni , C uces Uni e si y Hospi al, Uni e si y o he Basque Coun y (UPV/EHU), Ba akaldo, Spain
2
Inno a ion in
Assis ed Rep oduc ion G oup, Bioc uces Bizkaia Heal h Resea ch Ins i u e, C uces Uni e si y Hospi al, Ba akaldo, Spain
3
Depa men o
Obs e ics and Gynecology, Uni e si y o he Basque Coun y (UPV/EHU), Leioa, Spain
4
Exosomes Labo a o y, CIC bioGUNE-BRTA,
De io, Spain
5
No o No disk Founda ion Cen e o Basic Me abolic Resea ch (CBMR), Facul y o Heal h and Medical Sciences, Uni e si y
o Copenhagen, Copenhagen, Denma k
6
Genome Analysis Pla o m, CIC bioGUNE-BRTA, De io, Spain
7
Bioin o ma ics Pla o m, Cen o
de In es igacio´n Biome´dica en Red de En e medades Hepa´ icas y Diges i as (CIBERehd), Mad id, Spain
8
In eg a i e Genomics Lab, CIC
bioGUNE-BRTA, De io, Spain
9
IKERBASQUE, Basque Founda ion o Science, Bilbao, Spain
10
Cen o de In es igacio´n Biome´dica en Red
en el A
´ ea ema´ ica de En e medades Hepa´ icas (CIBEReh), Mad id, Spain
11
Depa men o Physiology, Facul y o Medicine and Den is y,
Uni e si y o he Basque Coun y (UPV/EHU), Leioa, Spain
12
Ins i u o Valenciano de In e ilidad (IVI) Bilbao/IVIRMA, Leioa, Spain
13
Me abolomics Pla o m, CIC bioGUNE-BRTA, De io, Spain
*Co espondence add ess. Human Rep oduc ion Uni , C uces Uni e si y Hospi al, Uni e si y o he Basque Coun y (UPV/EHU),
Ba akaldo, Spain; E-mail: jose o[email p o ec ed] (R.M.) h ps://o cid.o g/0000-0002-4279-6823; Exosomes
Labo a o y, CIC bioGUNE-BRTA, De io, Spain; E-mail: [email p o ec ed] (J.M.F.-P.) h ps://o cid.o g/0000-0003-3133-0670
Submi ed on Sep embe 30, 2021; esubmi ed on Augus 2, 2022; edi o ial decision on Augus 9, 2022
STUDY QUESTION: Is i possible o use ee and ex acellula esicle-associa ed mic oRNAs (miRNAs) om human endome ial fluid
(EF) samples as non-in asi e bioma ke s o implan a i e endome ium?
SUMMARY ANSWER: The ee and ex acellula esicle-associa ed miRNAs can be used o de ec implan a i e endome ium in a non-
in asi e manne .
WHAT IS KNOWN ALREADY: miRNAs and ex acellula esicles (EVs) om EF ha e been desc ibed as media o s o he emb yo–en-
dome ium c oss alk. The e o e, he analysis o miRNA om his fluid could become a non-in asi e echnique o ecognizing implan a i e
endome ium. This analysis could po en ially help imp o e he implan a ion a es in ART.
STUDY DESIGN, SIZE, DURATION: In his p ospec i e s udy, we fi s op imized di e en p o ocols o EVs and miRNA analyses us-
ing he EF o a se up coho (n ¼72). Then, we examined di e en ially exp essed miRNAs in he EF o women wi h success ul emb yo im-
plan a ion (disco e y coho n ¼15/ alida ion coho n ¼30) in compa ison wi h hose o whom he implan a ion had ailed (disco e y
coho n ¼15/ alida ion coho n ¼30). Success ul emb yo implan a ion was conside ed when p egnancy was confi med by aginal ul a-
sound showing a ges a ional sac 4 weeks a e emb yo ans e (ET).
PARTICIPANTS/MATERIALS, SETTING, METHODS: The EF o he se up coho was ob ained be o e s a ing e ili y ea men
du ing he na u al cycle, 16–21 days a e he beginning o mens ua ion. Fo he disco e y and alida ion coho s, he EF was collec ed
om women unde going ozen ET on Day 5, and he samples we e collec ed immedia ely be o e ET. In his s udy, we compa ed fi e di -
e en me hods; wo o hem based on di ec ex ac ion o RNA and he o he h ee wi h an EV en ichmen s ep be o e he RNA ex ac-
ion. Small RNA sequencing was pe o med o de e mine he mos e ficien me hod and find a p edic i e model di e en ia ing be ween
implan a i e and non-implan a i e endome ium. The models we e confi med using quan i a i e PCR in wo se s o samples (disco e y and
alida ion coho s) wi h di e en implan a ion ou comes.
V
CThe Au ho (s) 2022. Published by Ox o d Uni e si y P ess on behal o Eu opean Socie y o Human Rep oduc ion and Emb yology.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-NonComme cial License (h ps://c ea i ecommons.o g/licenses/by-nc/4.0/), which
pe mi s non-comme cial e-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. Fo comme cial e-use, please con ac
jou nals.pe [email protected]
Human Rep oduc ion, Vol.37, No.10, pp. 2375–2391, 2022
Ad ance Access Publica ion on Augus 27, 2022 h ps://doi.o g/10.1093/hum ep/deac184
ORIGINAL ARTICLE In e ili y
Downloaded om h ps://academic.oup.com/hum ep/a icle/37/10/2375/6678065 by Uni e sidad del Pais Vasco use on 08 No embe 2022
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MAIN RESULTS AND THE ROLE OF CHANCE: The p o ocols using EV en ichmen de ec ed mo e miRNAs han he me hods based
on di ec RNA ex ac ion. The wo mos e ficien p o ocols (using polyme -based p ecipi a ion (PBP): PBP-M and PBP-N) we e used o
ob ain wo p edic i e models (based on h ee miRNAs) allowing us o dis inguish be ween an implan a i e and non-implan a i e endome-
ium. The fi s Model 1 (PBP-M) (disco e y: AUC ¼0.93; P- alue ¼0.003; alida ion: AUC ¼0.69; P- alue ¼0.019) used hsa-miR-200b-
3p, hsa-miR-24-3p and hsa-miR-148b-3p. Model 2 (PBP-N) (disco e y: AUC ¼0.92; P- alue ¼0.0002; alida ion: AUC ¼0.78;
P- alue ¼0.0002) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-99b-5p. Func ional analysis o hese miRNAs showed s ong associa-
ion wi h key implan a ion p ocesses such as in u e o emb yonic de elopmen o ans o ming g ow h ac o -be a signaling.
LARGE SCALE DATA: The FASTQ da a a e a ailable in he GEO da abase (access numbe GSE178917).
LIMITATIONS, REASONS FOR CAUTION: One impo an ac o o conside is he inhe en a iabili y among he women in ol ed in
he ial and among he ans e ed emb yos. The emb yos we e p e-selec ed based on mo phology, bu nei he gene ic no molecula
s udies we e conduc ed, which would ha e imp o ed he accu acy o ou es s. In addi ion, a limi a ion in miRNA lib a y cons uc ion is
he low amoun o inpu RNA.
WIDER IMPLICATIONS OF THE FINDINGS: We desc ibe new non-in asi e p o ocols o analyze miRNAs om small olumes o EF.
These p o ocols could be implemen ed in clinical p ac ice o assess he s a us o he endome ium be o e a emp ing ET. Such e alua ion
could help o a oid he loss o emb yos ans e ed o a non-implan a i e endome ium.
STUDY FUNDING/COMPETING INTEREST(S): J.I.-P. was suppo ed by a p edoc o al g an om he Basque Go e nmen
(PRE_2017_0204). This s udy was pa ially unded by he G an o Fe ili y Inno a ion (GFI, 2011) om Me ck (Da ms ad , Ge many). I
was also suppo ed by he Spanish Minis y o Economy and Compe i i eness MINECO wi hin he Na ional Plan RTI2018-094969-B-I00,
he Eu opean Union’s Ho izon 2020 esea ch and inno a ion p og am (860303), he Se e o Ochoa Cen e o Excellence Inno a i e
Resea ch G an (SEV-2016-0644) and he Ins i u o de Salud Ca los III (PI20/01131). The unding en i ies did no play any ole in he s udy
design, collec ion, analysis and in e p e a ion o da a, w i ing o he epo o he decision o submi he a icle o publica ion. The au ho s
decla e no compe ing in e es s.
Key wo ds: emb yo implan a ion / endome ial fluid / non-in asi e bioma ke s / ex acellula esicles / mic oRNAs / implan a i e endo-
me ium / non-implan a i e endome ium / implan a i e IVF cycles / non-implan a i e IVF cycle / IVF
In oduc ion
Inc easing emb yo implan a ion a es is one o he g ea es challenges
in ART, as only 35% o emb yo ans e s (ETs) esul in a clinical p eg-
nancy (Ma o as e al., 2002;De Gey e e al., 2020). Despi e nume -
ous s udies ocused on imp o ing implan a ion a es, a eliable me hod
o de e mining he compe ence o he endome ium, undamen al o
success ul implan a ion, is s ill lacking (S owi zki e al., 2006;C aciunas
e al.,2019). Cu en ly, he endome ial biopsy is used o es ablish
whe he he endome ium is eady o ET (Caspe , 2020). This is an
in asi e me hodology, and he ET is no pe o med in he same cycle
in which he sample is aken as i can ha e de imen al e ec s on im-
plan a ion ( an de Gaas e al.,2009). I he biopsy shows ha he en-
dome ium is ecep i e, he esul s will be ex apola ed o he nex
cycle. This assump ion is no ealis ic, since he endome ial cycle is a
dynamic p ocess in ol ing many ac o s a ec ing he ecep i i y o he
endome ium. The analysis o endome ial luid (EF) ob ained in a non-
in asi e manne , wi hou biopsy, is a p omising al e na i e ( an de
Gaas e al., 2003). I has been demons a ed ha he aspi a ion o EF
immedia ely be o e he ET does no a ec he implan a ion.
Mo eo e , he p omp analysis o EF composi ion migh allow he ET
in he same cycle ( an de Gaas e al.,2003;Azka go a e al.,2018;
Ma o as e al., 2018,2020). The EF can be ob ained se e al imes
du ing he cycle and i s analysis could e eal whe he he endome ium
is eady o implan a ion o he apeu ic in e en ion is necessa y o a
success ul p ocedu e.
The EF is a complex biological luid ha can modula e endome ial
homeos asis and ecep i i y, i can sus ain he p eimplan a ion emb yo
and ini ia e he implan a ion p ocess and i plays an impo an ole in
he emb yo–endome ium communica ion (Ng e al.,2013;Vilella
e al., 2015;Bhusane e al.,2016;Nguyen e al., 2016). mic oRNAs
(miRNAs) a e small non-coding RNA sequences (18–22 nucleo ides)
ha a e impo an egula o s o genes a he pos - ansc ip ional le el
(Bhaska an and Mohan, 2014). They a e essen ial du ing ea ly emb y-
onic de elopmen since hey egula e cell p oli e a ion and di e en ia-
ion (Bhaska an and Mohan, 2014). Some o hese miRNAs ha e been
associa ed wi h he ex acellula esicles (EVs), also p esen in he luid
ob ained om he u e ine ca i y (Vilella e al.,2015). EVs a e widely
known media o s o in e cellula communica ion, ansmi ing in o ma-
ion om one cell o a mul i ude o o he cells and loca ions (Han
e al., 2020). Mo eo e , analyses o miRNA con en o endome ium-
de i ed EVs show ha hey a e aken up by he emb yos, modi ying
hei ansc ip omic and adhesi e pheno ypes (Ng e al.,2013;Vilella
e al., 2015;G eening e al., 2016;Balague e al., 2018;Ma ina o
e al., 2019). Fo example, he EV-associa ed hsa-miR-30d is in e nal-
ized by mouse ophoec ode m and inc eases he emb yo adhesion
ia up egula ion o adhesi e molecules (Vilella e al., 2015).
One o he main challenges in ART is inding non-in asi e ools o
de ec ing he bes ime o pe o m he ET. He e, we de eloped a e-
p oducible, sensi i e, low-in asi e me hod o comp ehensi ely exam-
ine he miRNA landscape o he EF. Fi s , we op imized he EF sample
collec ion echnique. Then, we es ablished a obus me hod o analyz-
ing esicula and non- esicula miRNAs om EF ob ained in clinical
se ings, whe e sample size is limi ed and no sophis ica ed equipmen
is a ailable. Finally, we applied hese me hods o a se o EF samples
om women wi h di e en implan a ion ou comes. Ou aim was o
de ine a miRNA signa u e o iden i y he compe ence o he endome-
ium. I we could de e mine he s a e o he endome ium, i would
2376 Iba~
nez-Pe ez e al.
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hen be possible o change he ET s a egy when he esul s show an
un a o able implan a i e pa e n. Thus, he implan a ion a es could
po en ially be imp o ed and he loss o emb yos minimized by a oiding
hei ans e o non-implan a i e endome ium.
Ma e ials and me hods
E hical app o al
E hical app o al o he s udy was ob ained om he C uces
Uni e si y Hospi al E hics Commi ee and Ins i u ional Re iew Boa d
(CEIC 11/45) and all he pa icipan s ga e w i en consen o hei
pa icipa ion.
S udy popula ion
The popula ion unde s udy consis ed o a coho o 162 women who
a ended he Human Rep oduc ion Uni o C uces Uni e si y Hospi al
(Basque Coun y, Spain) om Janua y 2018 o Feb ua y 2021. Fo he
se up and op imiza ion o he echniques, he samples we e collec ed
be o e s a ing he e ili y ea men . The samples we e collec ed du -
ing he na u al cycle, 16–21 days a e he beginning o mens ua ion.
To es he selec ed me hod, he samples we e collec ed jus be o e
Day-5 ozen ETs, a p ac ice which is pe o med inc easingly o en
(Ma o as e al., 2021). Ou o 162 women (Supplemen a y Fig. S1),
72 pa icipa ed in he se up, 30 in he disco e y o he p edic ed mod-
els and 60 in he alida ion o he models. Fo y- i e women became
p egnan and we e included in he implan a i e endome ium g oup.
The o he 45, who did no achie e p egnancy, we e included in he
non-implan a i e endome ium g oup. The endome ium was consid-
e ed implan a i e when p egnancy was con i med by aginal ul asound
showing a ges a ional sac 4 weeks a e ET. Cases wi h a posi i e b-
hCG es whe e a ges a ional sac was no seen on aginal ul asound
(biochemical misca iages) we e no included in he s udy.
The inclusion c i e ia in he se up s udy we e: age be ween 18 and
37 yea s; cycle du a ion be ween 27 and 29 days; absence o o ula o y
diso de s, myomas, endome iosis, polyps, u e ine sca s o hyd osal-
pinges; no mal u e ine and o a ian ul asound; se um an i-Mu¨lle ian
ho mone >0.4 ng/ml; and no his o y o gynecological in ec ions, im-
mune diso de s o gynecological su ge y. The inclusion c i e ia o he
disco e y and alida ion coho s also included: ozen ET on Day 5
(good quali y emb yos; Types A and B o he Spanish Socie y o he
S udy o Rep oduc i e Biology (ASEBIR) classi ica ion (ASEBIR, 2015)
and ans e o 1–2 emb yos de i ed om he oocy es o he same
subjec .
The managemen o endome ial p epa a ion was always ca ied
ou using he same p o ocol. A aginal ul asound was pe o med on
Day 1 o 2 o con i m o a ian quiescence (absence o ollicles
>10 mm). An a i icial cycle was s a ed on Day 2 by adminis e ing
6 mg o es adiol daily (P ogyno a, Baye , Ba celona, Spain). The de el-
opmen o he endome ium was moni o ed using se ial aginal ul a-
sounds. When he endome ium became 7-mm hick, he ans e day
was scheduled. Vaginal p oges e one a a dose o 400mg/12 h
(U oges an, SEID, Ba celona, Spain) was s a ed he nex mo ning,
and he ET was pe o med on he 5 h day o p oges e one
adminis a ion. I p egnancy was achie ed, he es adiol and p oges e -
one ea men was main ained un il he 12 h week o ges a ion.
Emb yo i i ica ion was pe o med on Day 4 o 5 using a C yo op
de ice (Ki aza o BioPha ma Co., Shizuoka, Japan). The emb yos we e
c yop ese ed and wa med using he Ki aza o i i ica ion/wa ming ki
(Ki aza o BioPha ma Co.), acco ding o he manu ac u e ’s ins uc-
ions. F ozen Day-4 emb yos we e hawed and cul u ed o 24 h be-
o e he ET and Day-5 blas ocys s o 2 h be o e he ET.
Sample collec ion and s o age
The EF was aspi a ed wi h a ca he e used o ET (F ydman,
Ins umen os Me´dicos Es e´ iles SA, Spain) connec ed o a 10-ml sy-
inge unde abdominal ul asound guidance. Sample ex ac ion was
pe o med by gen ly applying a nega i e p essu e wi h he sy inge. The
aspi a ion was in e up ed a he in e nal ce ical os o p e en con-
amina ion wi h ce ical mucus. Special ca e was aken o a oid ouch-
ing he u e ine undus o inju ing he ce ix and minimize sample
con amina ion wi h blood and endome ial issue. In cases wi h exces-
si e aginal sec e ions, he agina was cleaned wi h saline solu ion be-
o e aspi a ion. Aspi a e olumes anged om 5 o 50ml. A e
aspi a ion, he 10-ml sy inge was eplaced wi h a 2-ml sy inge con ain-
ing 1.5 ml o 1Dulbecco’s PBS (DPBS) (Gibco, The mo Fishe
Scien i ic, # 14190250, MA, USA) o expel he EF. The aspi a es we e
mixed wi h he 1DPBS and expelled in o a c yogenic ube (5–50 ml
o EF þ1500 mlo 1DPBS). The mixed samples we e cen i uged o
emo e con aminan s a 2500g o 5 min a oom empe a u e, and
he supe na an s we e hen kep ozen a 80C un il p ocessed.
The dilu ion o he supe na an s was 1:30, wi h a inal olume be ween
400 and 1300 ml.
EV en ichmen me hods
Size-exclusion ch oma og aphy
A Poly-P ep ch oma og aphy column (BioRad, # 731-1550, He cules,
USA) was illed wi h 2.5 ml o Sepha ose CL-2B c oss-linked esin
(Sigma, # CL2B300-100ML) and le packing o e nigh a 4C. The
column was hen washed wice wi h 2.5 ml o 1DPBS. Th ee ali-
quo s o 400 ml om he se up coho sample pool we e used. Each
aliquo was applied o he column, and hen 4 ml o 1DPBS was
added. The size-exclusion ch oma og aphy (SEC) sepa a ed he sam-
ple in o 12 ac ions (F1–F12); he EVs we e elu ed mainly in F3 bu
also in F4 and F5 ac ions, as desc ibed by P ie o-Fe na´ndez e al.
(2019). F1 o F10 had a inal olume o 200 ml, and F11 and F12 o
1 ml. The 12 ac ions o one aliquo we e each used o RNA ex ac-
ion wi h mi Vana
TM
PARIS
TM
Ki (The mo Fishe Scien i ic,
# AM1556). The RNA ob ained om ac ions F3 and F4 was u he
analyzed by small RNA-sequencing (RNA-Seq). The 12 ac ions o he
o he wo aliquo s we e cha ac e ized using wes e n blo (WB).
Polyme -based p ecipi a ion me hod
Since he e was no published p o ocol o using he In i ogen To al
Exosome Isola ion Reagen wi h he EF, we compa ed he To al
Exosome Isola ion Reagen o he cell cul u e media (In i ogen by
The mo Fishe Scien i ic, # 4478359) wi h To al Exosome Isola ion
Reagen o o he body luids (In i ogen by The mo Fishe Scien i ic,
# 4484453). Al hough bo h wo ked well wi h he EF, we used he
# 4478359 because o i s be e cos -e ec i eness a io. The
miRNAs as bioma ke s o implan a i e endome ium 2377
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op imized p o ocol was as ollows: cen i uge he EF supe na an s a
3000g o 30 min a 4C; ans e he supe na an s o a esh ube and
add an equal olume o he To al Exosome Isola ion Reagen (1:1);
s i he mix u e by o exing un il he e is a homogeneous solu ion
and incuba e he sample o 30 min a oom empe a u e; a e he in-
cuba ion, cen i uge he samples a 10 000g o 1 h a 4C; aspi a e
he supe na an by pipe ing and disca d i ; he EVs a e con ained in
he pelle , which may no be isible a he bo om o he ube; and i-
nally, add 100 mlo 1DPBS o esuspend he pelle .
Ul acen i uga ion
Ul acen i uga ion (UC) was ca ied ou in a single s ep (100 000g o
75 min a 4C) using a Beckman-Coul e TLA 120.2 o o . The EV
pelle s we e esuspended in 100 mlo 1DPBS.
RNA ex ac ion me hods
We used wo RNA isola ion me hods; we ollowed he manu ac u e ’s
ins uc ions o he mi Vana
TM
PARIS
TM
Ki (The mo Fishe Scien i ic,
# AM1556) (DCT-M) and No gen Plasma/Se um RNA Pu i ica ion ki
(DCT-N). Two di e en No gen ki s we e used as needed; he midi ki
(No gen Bio ek Co p., # 56100, On a io, Canada) o he mini ki
(No gen Bio ek Co p., # 55000). The RNA was elu ed in nuclease-
ee wa e (Ambion, # AM9930 by The mo Fishe Scien i ic).
cDNA syn hesis and TaqMan miRNA assay
Following he manu ac u e ’s ecommenda ions, cDNA was syn he-
sized om 2 ml o RNA using he TaqMan Ad anced miRNA cDNA
Syn hesis ki (Applied Biosys ems, # A28007, by The mo Fishe
Scien i ic). The TaqMan eac ions used we e he TaqMan Fas
Ad anced Mas e Mix (The mo Fishe Scien i ic, # 4444557) and
TaqMan Ad ance miRNA assays (The mo Fishe Scien i ic,
# A25576). The quan i a i e PCR was pe o med using a Viia7 o
QS6 sys em, and he da a we e analyzed using he Quan S udio Real-
Time PCR Sys em e sion 1.3 (Applied Biosys ems, by The mo Fishe
Scien i ic). The exp ession p o iles o se en EV-associa ed miRNAs
we e used as e e ence (The mo Fishe Scien i ic): hsa-le -7-5p
(478579_mi ), hsa-miR-17-5p (478447_mi ), hsa-miR-200c-3p
(478351_mi ), hsa-miR-30c-5p (478008_mi ), hsa-miR-30d-5p
(478606_mi ), hsa-miR-451a (478107_mi ) and hsa-miR-92a-3p
(477827_mi ) (Supplemen a y Table SI). These miRNAs ha e been
epo ed as sec e ed by endome ial epi helial cell lines (Ng e al.,
2013), ound in he EF aspi a es (Vilella e al.,2015;Campoy e al.,
2016) and sec e ed in endome ial exosomes associa ed wi h ea ly
emb yo implan a ion (Vilella e al., 2015;Balague e al., 2018). Two
o he miRNAs we e selec ed a e he small RNA-Seq analysis using
he se up pool coho sample. These we e he hsa-miR-21-5p
(477975_mi ) and hsa-miR-155-5p (483064_mi ) miRNAs, which
we e among he mos and leas abundan miRNAs in he pool, e-
spec i ely (Supplemen a y Table SI). In addi ion, wo exogenous
miRNAs we e used as in e nal con ols. To examine he e iciency o
he RNA ex ac ion, 4 ml o cel-miR-39 (478293_mi , The mo Fishe
Scien i ic) o a 0.1 nM s ock we e added o he sample be o e each
RNA ex ac ion p ocedu e. To es he di e ences be ween he
cDNA syn hesis eac ions, 0.2 ml o a h-miR-159a (478411_mi ,
The mo Fishe Scien i ic) o a 0.001nM s ock was added a he begin-
ning o each cDNA syn hesis eac ion.
Compa ing he miRNA ex ac ion me hods
Fi e di e en me hods we e compa ed o de ine a simple and e ec i e
s a egy o de ec ing esicula and non- esicula miRNAs in small ol-
umes o EF (Fig. 1). Two o hese in ol ed di ec ex ac ion using
di e en RNA ex ac ion ki s, DCT-N (No gen ki , # 56100) and
DCT-M (mi Vana PARIS ki , # AM1556). The o he h ee equi ed en-
ichmen o EVs be o e RNA ex ac ion. In one case, he en ichmen
was ca ied ou by UC ollowed by RNA ex ac ion wi h mi Vana
PARIS ki (UC-M). In he emaining wo cases, he en ichmen was ca -
ied ou using he polyme -based p ecipi a ion (PBP) me hod and he
RNA was ex ac ed using he No gen (# 55000) (PBP-N) o mi Vana
PARIS ki (PBP-M). In pa allel, SEC was pe o med o cha ac e ize he
p o ein and miRNA con en o he EF (Fig. 1). The olumes o eco -
e ed EF samples a ied depending on many ac o s, such as he ope a-
o collec ing he sample and he EF olume o iscosi y. In gene al, he
olumes anged om 400 ml o 1.3 ml. The e o e, we op imized he
p o ocols o be used wi h he minimum olume a ailable (400 ml) in all
cases. All he es s we e pe o med in iplica e.
Technical ep oducibili y expe imen
A echnical ep oducibili y expe imen was conduc ed using he PBP-M
and PBP-N p o ocols. Two ope a o s (J.I.-P. and M.C.-G.) pe o med
he es s independen ly. The samples used in hese expe imen s came
om he se up pool coho , and each o he ope a o s es ed 10 ali-
quo s using each me hod. Quan i a i e PCR (qPCR) was used o ex-
amine ep oducibili y; nine miRNAs we e analyzed (se en e e ence
miRNAs and wo miRNAs ob ained om he small RNA-Seq analysis)
(Supplemen a y Table SI).
Di hio h ei ol ea men assay
Two aliquo s om he se up pool coho we e used o pe o m he
expe imen . One o he aliquo s was ea ed wi h a 1.4% di hio h ei ol
(DTT) solu ion in a 1:1 a io (Mille e al.,2012;Wang e al., 2017)
and he o he se ed as con ol; 1DPBS (1:1) was added. The sam-
ples we e mixed by o exing and incuba ed a oom empe a u e o
15 min. Then, 1DPBS was added un il he EF samples we e dilu ed
o he a io o 1:8, and he samples we e cen i uged a 3000g o
15mina 4
C. The supe na an s we e eco e ed, and 400-ml aliquo s
we e aken. The EV en ichmen was conduc ed using 400-ml aliquo s,
ollowing he PBP me hod, and he pelle was esuspended in 100 ml
o 1DPBS. F om his olume, 15 ml was ese ed o WB, 5 ml o
c yo-elec on mic oscopy, 5 ml o nanopa icle- acking analysis
(NTA), and he es o he suspension was used o RNA analysis wi h
No gen (# 55000). The isola ed RNA was elu ed in 100 mlo
nuclease- ee wa e . Two mic oli e s o he elua e was used o he
subsequen cDNA syn hesis, and he es was s o ed a 80C.
RNase p o ec ion assay
The samples used in his s ep came om he se up pool coho , and
each aliquo es ed had a inal olume o 400 ml. All he samples we e
i s EV-en iched using he PBP me hod (desc ibed abo e) and he EVs
we e esuspended in 200 ml o DPBS. In he RNase p o ec ion assay,
ou di e en p ocedu es we e compa ed. Samples we e ea ed
acco ding o ollowing p o ocols: RNase A (Sigma-Ald ich,
# 10109142001, MA, USA) (RNase); P o einase K (Sigma-Ald ich,
2378 Iba~
nez-Pe ez e al.
Downloaded om h ps://academic.oup.com/hum ep/a icle/37/10/2375/6678065 by Uni e sidad del Pais Vasco use on 08 No embe 2022
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# 03115879001) þRNase (PRT-K); T i on X-100 (Sigma-Ald ich,
# T8787) þRNase (TX-100); and TX-100 þP o einase K þRNase
(TX þPRT). An un ea ed sample was used as a con ol. Samples
we e ea ed wi h TX-100 o a inal concen a ion o 0.1%. P o einase
K (0.05 mg/ml concen a ion) was added and he mix u e was incu-
ba ed o 10 min a 37C. The eac ion was s opped by adding 5 mM
o phenylme hylsul onyl luo ide (Sigma-Ald ich, # 10837091001) and
hea ing a 90C o 5 min. The samples we e inally ea ed wi h
0.1 mg/ml RNase A (RNase) o 20 min a 37C. The con ol samples
we e kep a 4C un il RNA ex ac ion. Be o e ex ac ion, b-me cap-
oe hanol was used o inhibi RNases, as desc ibed by No gen (#
55000). The RNA was elu ed in 50 ml o nuclease- ee wa e . Two
mic oli e s we e used o he subsequen cDNA syn hesis, and he es
was s o ed a 80C. All he analyses we e pe o med in iplica e
wi h wo echnical duplica es, ending wi h six TaqMan qPCR eplica es.
Analysis and quan i ica ion o he EF
p o ein con en
WB analysis
Asampleo 15ml was mixed wi h 5 ml o NuPAGE LDS Sample Bu e
4(In i ogen # NP0007, by The mo Fishe Scien i ic). The ac ions
ob ained by SEC we e concen a ed using 99.5% ace one (Pan eac
Applichem, # 161007, Da ms ad , Ge many) and esuspended in
20 mlo 1LDS sample bu e . They we e hea ed o 5 min a 37C,
10 min a 65C and 15 min a 95C and cen i uged o 10 min a
13 000g. Each p o ein p epa a ion was loaded and sepa a ed unde
non- educing condi ions in 4–12% Bis–T is p ecas gels (In i ogen,
# NP0336BOX, by The mo Fishe Scien i ic) in MOPS SDS Running
Bu e 1(In i ogen, # NP0001, by The mo Fishe Scien i ic).
P ecision Plus P o ein Dual Colo S anda d (BioRad, # 161-0374) was
used as a ma ke o p o ein molecula weigh s. The p o eins we e
ans e ed o an Immobilon-P T ans e memb ane (Me ck Millipo e,
# IPVH00010, MA, USA) in NuPAGE T ans e Bu e 1(In i ogen,
# NP0006-1 by The mo Fishe Scien i ic) o 1 h a 100 V. The block-
ing was pe o med using 5% Blo ing-G ade Blocke (BioRad, # 170-
6404) and 0.2% Tween-20 (Sigma-Ald ich, # P2287, MA, USA) dilu ed
in 1DPBS, o 1 h . P ima y an ibodies we e incuba ed o e nigh
and he memb anes we e washed h ee imes o 10 min wi h 1
DPBS. Incuba ion wi h he seconda y ho se- adish pe oxidase-conju-
ga ed an ibody (1:6000) was pe o med a oom empe a u e o
30 min. The chemiluminescence was de ec ed using Pie ce ECL Plus
Wes e n Blo ing Subs a e (The mo Fishe Scien i ic, # 32132). The
bands we e isualized on high-pe o mance ilms (GE Heal hca e,
Figu e 1. Wo kflow summa izing he di e en me hods used o analyze mic oRNAs om he endome ial fluid o pa ien s un-
de going ART. We compa ed fi e di e en me hods, wo o which used he di ec ex ac ion o RNA om he endome ial fluid (EF) (DCT-N and
DCT-M). The o he h ee included he ex acellula esicle (EV) en ichmen (UC-M, PBP-N and PBP-M) be o e RNA ex ac ion. In pa allel, we ca -
ied ou a size-exclusion ch oma og aphy (SEC-M) o cha ac e ize he p o eins and miRNAs in he EF. The samples came om he se up pool coho ,
and each expe imen was pe o med in iplica e, using sample aliquo s o 400 ml. DCT-N: di ec RNA ex ac ion wi h No gen Plasma/Se um RNA
pu ifica ion ki . DCT-M: di ec ex ac ion o RNA wi h mi Vana PARIS ki . UC-M: EV en ichmen by ul acen i uga ion and RNA ex ac ion using
mi Vana PARIS ki . PBP-N: EV en ichmen wi h a polyme -based p ecipi a ion me hod and RNA ex ac ion wi h No gen Plasma/Se um RNA pu ifi-
ca ion ki . PBP-M: EV en ichmen using he polyme -based p ecipi a ion me hod and RNA ex ac ion wi h mi Vana PARIS ki . SEC-M: EV en ichmen
wi h SEC and RNA ex ac ion wi h mi Vana PARIS ki . miRNAs, mic oRNAs; PBP, polyme -based p ecipi a ion.
miRNAs as bioma ke s o implan a i e endome ium 2379
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# 28906844, IL, USA) employing he AGFA Cu ix-60 au oma ic p o-
cesso (Ag a, Cologne, Ge many). The p ima y an ibodies used in his
s udy we e mouse an i-CD63 (1:500; clone H5C6 om
De elopmen al S udies Hyb idoma Bank, IA, USA), mouse an i-CD9
(1:500; clone 209306, R&D Sys ems, Minneapolis, MN, USA), mouse
an i-CD81 (1:500, Clone JS-81, 555675, BD, NJ, USA), mouse an i-
CD133 (1:500 clone W6B3C1, Mil enyi Bio ec, No h Rhine-
Wes phalia, Ge many), mouse an i-Rab8 (1:1000; Clone 4, 610844,
BD, NJ, USA), mouse an i-Flo illin-1 (1:500; Clone 18 610820, BD, NJ,
USA), mouse an i-HSP90 (1:500; 610418, BD, NJ, USA) and abbi
an i-Limp II (1:500; ab16522, Abcam, Camb idge, UK). The in ensi y
o he bands was quan i ied by densi ome y using ImageJ so wa e .
1.52a (ImageJ so wa e, MD, USA).
Coomassie blue s aining
SimplyBlue
TM
Sa eS ain om In i ogen (Ca . # LC6060, The mo
Fishe Scien i ic) was used ollowing he manu ac u e ’s ecommenda-
ions. The in ensi y o he bands was quan i ied by densi ome y using
ImageJ so wa e ( . 1.52a).
Spec opho ome e
Spec opho ome ic measu emen s we e pe o med using a
NanoD op
TM
One Mic o olume UV–Vis Spec opho ome e (The mo
Fishe Scien i ic) in he wa eleng h ange o 230–576 nm.
Concen a ions o RNA and p o eins we e ob ained a e measu ing
he abso bance o 1 mlo hesample.
Nanopa icle- acking analysis
The size dis ibu ion o he EV p epa a ions was analyzed by measu ing
he a e o B ownian mo ion using a NanoSigh LM10 sys em
(NanoSigh , Amesbu y, UK), equipped wi h as ideo cap u e and
pa icle- acking so wa e. NTA acquisi ion se ings we e he same o
all samples, and each ideo was analyzed o ob ain he mean and
mode o esicle size and es ima e he pa icle concen a ion (D ago ic
e al., 2011).
C yo-elec on mic oscopy
EV p epa a ions we e di ec ly adso bed on o glow-discha ged holey
ca bon g ids (Quan i oil, G oßlo¨bichau, Ge many). The g ids we e
blo ed a 95% humidi y and apidly plunged in o liquid e hane wi h
he aid o Vi obo (Maas ich Ins umen s BV, Maas ich , The
Ne he lands). Vi i ied samples we e imaged a liquid-ni ogen empe -
a u e using a JEM-2200FS/CR ansmission c yo-elec on mic oscope
(JEOL, Tokyio, Japan) equipped wi h a ield emission gun and ope a ed
a an accele a ion ol age o 200 kV.
Real- ime qPCR assay
The ela i e exp ession le els o he miRNAs ob ained o he se up
pool coho we e no malized o a h-miR-159 exp ession and calcu-
la ed using he 2
DC (C miRNAC a h-miR-159a)
me hod. The ela i e ex-
p ession le els o he disco e ed and alida ed miRNAs we e
no malized o in e nal con ols; he di e ences be ween he g oups
we e calcula ed employing he 2
DC (C miRNAC mean in e nal con ols)
equa ion. Subsequen ly, he old changes we e ob ained using he
2
DDC
me hod (Rao e al., 2013). Endogenous con ols we e selec ed
om he e e ence miRNAs (Supplemen a y Table SI) using he
No mFinde so wa e (MOMA, Aa hus, Denma k). The No mFinde
is an algo i hm using a model-based app oach o calcula e he s abili y
o a e e ence ansc ip ; he calcula ion is based on he in e g oup
and in ag oup a ia ions. The s abili y sco e is a weigh ed measu e o
hese wo pa ame e s, and he mos s able e e ence ansc ip is he
one wi h he smalles s abili y alue (Ande sen e al., 2004).
Only he samples o which we could ind he in e nal con ols wi h
ewe han 30 C cycles (in qPCR) we e used in he eg ession s udy
o he disco e y coho (Supplemen a y Table SII) and o alida e he
models in he alida ion coho (Supplemen a y Table SIII).
Co ela ion analysis
The co plo package (Wei e al.,2017) o he R 3.6.2 p og am was
used o analyze he co ela ions be ween he p o eins (2019-12-12, R
Founda ion o S a is ical Compu ing, Vienna, Aus ia).
S a is ical analysis
G aphPad P ism .8.0 (G aphPad So wa e, Cali o nia, USA) was
employed o analyze he da a. The s a is ical signi icance o he expe i-
men s ca ied ou wi h he se up pool coho was de e mined using
pai ed S uden ’s - es s. Fo he esul s ob ained o he disco e y and
alida ion coho s, unpai ed S uden ’s - es s wi h Welch’s co ec ion
we e employed. S a is ical di e ences we e conside ed signi ican a a
P- alue smalle han 0.05 ( wo-sided). Sample sizes and P- alues a e all
shown in he igu es and igu e cap ions.
Small RNA-Seq
The quan i y and quali y o he RNA we e e alua ed using Agilen
RNA 6000 Pico Chips (Agilen Technologies, Ca . # 5067-1513, CA,
USA). Sequencing lib a ies we e p epa ed ollowing he p o ocol in-
cluded wi h he NEXT lex
TM
Small RNA-Seq Ki 3 (V
CBioo Scien i ic
Co p., Ca . # 5132-06, p o ocol V19.01, Aus in, TX, USA). B ie ly,
he o al RNA om each sample was incuba ed o 2 min a 70C.
Then, a 304 N adenyla ed adap e (adap e dilu ion 1/4) and ligase
enzyme we e added, and liga ion was ca ied ou by incuba ion o e -
nigh a 20C. A e emo ing he excessi e 30adap e , 5’ adap e was
added wi h he ligase enzyme and he mix u e was incuba ed a 20C
o 1 h . The liga ion p oduc was used o e e se ansc ip ion wi h
he M-MuLV e e se ansc ip ase in a he mocycle o 30 min a
42C and 10 min a 90C. Nex , he en ichmen o he cDNA was
pe o med using PCR cycling: 2 min a 95C; 20–27 cycles o 20 s a
95C, 30 s a 60Cand15sa 72
C, wi h he inal elonga ion o 2 min
a 72C and a pause a 4C. The PCR p oduc s we e esol ed on 8%
No ex TBE polyac ylamide gels (Ca . # EC6215BOX, The mo Fishe
Scien i ic), and a band be ween 150 and 400 bp was cu ou . Small
RNAs we e ex ac ed om he polyac ylamide gel using an adap ed
p o ocol in which he DNA om gel slices was dissol ed in ddH
2
O
o e nigh a oom empe a u e. A e wa ds, he lib a ies we e isual-
ized employing an Agilen 2100 Bioanalyze wi h an Agilen High
Sensi i i y DNA ki (Agilen Technologies, Ca . # 5067-4626) and
quan i ied using a Qubi dsDNA HS DNA Ki (The mo Fishe
Scien i ic, Ca . # Q32854). The amoun o cDNA in each lib a y ha
was sen o sequencing was 10 nM. Sequencing was ca ied ou in
pools o isomola lib a ies and all o hem we e sequenced in a
2380 Iba~
nez-Pe ez e al.
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HiSeq2500 (Illumina Inc) o achie e a leas 10 million 50-n single-
eads pe sample.
Alignmen
The FASTQs we e immed o he adap e s ollowing he ecommen-
da ions o he NEXT lex
TM
Small RNA-Seq Ki manu ac u e s. We
used he Bow ie p og am (Langmead e al., 2009) oalign he eads
agains he human genome (GRCh38), wi h a misma ch o 0 o a oid
alse posi i es. We chose miRBase 22 o quan i y he ma u e
miRNAs, employing he Pa ek Flow applica ion ( e sion 7.0).
Small RNA-Seq da a analysis
We pe o med di e en ial abundance analyses o iden i y miRNAs as-
socia ed wi h di e en implan a ion ou comes. To a oid a e mole-
cules, ollowing he T immed Mean o M- alues (TMM) no maliza ion,
we e ained miRNAs wi h coun s pe million >1, non-ze o coun s in
a leas 15 indi iduals, and a mos 10 ze o coun s in each o he wo
subg oups, i.e., he success ul (n ¼15) and unsuccess ul implan a ions
(n ¼15). Di e en ial exp ession was hen assessed employing he
edgeR (Robinson e al.,2010) using he SARTools R package (Va e
e al., 2016). The p og am i s a log-linea model o each miRNA ha
uses a g oup (implan a i e e sus non-implan a i e) as he ac o o
con as . Applying he edgeR de aul pa ame e s o no maliza ion and
sh inkage, his gi es a old change es ima e ha co esponds o he
mean exp ession le el in he implan a i e samples di ided by he mean
exp ession le el in he non-implan a i e g oup. Fo u he analysis, we
selec ed he miRNAs wi h logFC >1.5 o logFC <1.5 and he ad-
jus ed P- alue <0.05. The Benjamini–Hochbe g p ocedu e was used
o calcula e he alse disco e y a e o each compa ison and ob ain
he adjus ed P- alues (Supplemen a y Tables SIV and SV).
Reg ession s udy
A subse o miRNAs was used o gene a e wo linea eg ession mod-
els wi h k- old c oss- alida ions, one o each miRNA ex ac ion p o-
ocol assessed. Samples we e andomly di ided in o aining and
es ing da ase s (80–20%). Th ee miRNAs we e used pe modeling
p ocess. The hsa-miR-24-3p, hsa-miR-200b-3p and hsa-miR-148b-3p
we e selec ed o PBP-M and hsa-miR-24-3p, hsa-miR-200b-3p and
hsa-miR-99b-5p o PBP-N. The esul ing model ep oducibili y was
u he es ed by boo s ap co ec ion wi h 500 eplica ions. The
analysis was pe o med using R 4.0.0 so wa e (R De elopmen Co e
Team; h p://c an. -p ojec .o g) wi h ROCR (Sing e al.,2005)and
caTools packages.
Func ional analysis o he miRNAs
The a ge genes o he alida ed miRNAs we e ob ained om he
Ta Base da abase, 7.0. The biological p ocesses in which hese
miRNAs a e in ol ed we e analyzed using he Kyo o encyclopedia o
genes and genomes (KEGG) and gene on ology (GO) in e ms o bio-
logical p ocess ca ego ies employing Diana-miRPa h ools 3.0
(Vlachos e al., 2015). The Fishe ’s exac es and alse disco e y a e
co ec ion we e pe o med o selec en iched KEGG pa hways and
GO p ocesses. We selec ed only he pa hways and p ocesses wi h P-
alues <0.05. The esul s o he KEGG we e me ged by ‘pa hway
union’ and he esul s o GO by ‘ca ego y union’.
Resul s
Op imiza ion o EF sample p epa a ion
The ea men o he samples wi h 1.4% DTT (Mille e al., 2012;
Wang e al.,2017) was use ul o deg ading he mucus pelle o med
a e cen i uga ion; mos mucus disappea ed, as shown in
Supplemen a y Fig. S2A. The NTA and c yo-elec on mic oscopy anal-
yses (Supplemen a y Fig. S2B and C) e ealed he e ogeneous EV pop-
ula ions wi h diame e s be ween 100 and 800nm unde bo h
expe imen al condi ions (wi h and wi hou DTT). In he un ea ed
samples, he a e age concen a ion was 1.9 10
9
§7.8 10
7
pa -
icles/ml, wi h a mean size o 291.5 §0.1 nm and mode
196.4 §3.2 nm. In he DTT- ea ed samples, we de ec ed mo e pa -
icles (mean 2.7 10
9
§5.9 10
7
pa icles/ml), wi h la ge mean
size (mean 313.6 §2.5 nm and mode 248.5 §8.1 nm). The WB
showed di e en pa e ns o esicula ma ke s in he wo condi ions
(Supplemen a y Fig. S2D). The in ensi y o he Rab8 ma ke in he
DTT- ea ed samples was s onge han in he un ea ed samples, in
ag eemen wi h he numbe o pa icles de ec ed in he NTA. In con-
as , he in ensi ies o he Limp II, CD133 and CD63 ma ke s we e
s onge in he un ea ed samples. The se en e e ence miRNAs
(Supplemen a y Table SI) we e de ec ed in all he samples o bo h
condi ions. In he DTT- ea ed g oup, he le els o he ollowing
miRNAs we e signi ican ly educed compa ed o he un ea ed g oup:
hsa-le -7e-5p, hsa-miR-17-5p, hsa-miR-200c-3p, hsa-miR-30c-5p and
hsa-miR-451a (Supplemen a y Fig. S2E).
Cha ac e iza ion o he miRNAs in EF
The SEC me hod sepa a ed he EF in o se e al ac ions. The esul s
o WB analysis o he ac ions demons a e ha i was possible o de-
ec exosomal ma ke s in small- olume EF samples (Fig. 2A). The
CD63 and CD81 ma ke s we e de ec ed in he F3 and o a lesse ex-
en in F4 and F5 ac ions. Rab8 was also mainly de ec able in F3–F5
ac ions. Immunoglobulins we e also ound in ac ions F6 o F11. The
s udy o he dis ibu ion o he se en e e ence miRNAs
(Supplemen a y Table SI) showed ha he ela i e quan i y o miRNAs
inc eased in ac ions F3 o F11 (Fig. 2B). In F3, which co esponds o
he esicula ac ion, he mos abundan miRNAs we e hsa-miR-451a
and hsa-miR-92a-3p, and he leas abundan we e hsa-miR-30d-5p and
hsa-miR-200c-3p. This end was main ained in he es o he ac-
ions, excep o F7, whe e hsa-miR-200c-3p was he second mos
abundan miRNA.
The p o ec i e e ec o he EVs on he miRNAs was con i med by
he RNase assay. In he samples ea ed wi h RNase o T i on X-100
(TX-100) wi h RNase, only he hsa-miR-30c-5p was signi ican ly de-
g aded compa ed o he con ol (Fig. 2C). In he samples ea ed wi h
p o einase K (PRT-K) and RNase, he e was a signi ican dec ease in
he le els o all he analyzed miRNAs (al hough all he miRNAs we e
de ec able in all he eplica es). The miRNAs we e u he deg aded
when he samples we e ea ed wi h TX-100, PRT-K and RNase (TX
þPRT). In his case, we could only de ec hsa-miR-200c-3p and hsa-
miR-92a-3p in all he eplica es. Unde hese condi ions, hsa-miR-451a,
hsa-miR-17-5p and hsa-miR-30d-5p we e de ec ed in ou o six epli-
cas, hsa-le -7-5p in wo o six eplicas and hsa-miR-30c-5p was unde-
ec able. In he TX þPRT ea men , he de ec ion o all miRNAs was
signi ican ly educed compa ed o he p e ious combina ions.
miRNAs as bioma ke s o implan a i e endome ium 2381
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Iden i ica ion o an e icien me hod o
pe o m a comp ehensi e analysis o
miRNAs om EF
Al hough we de ec ed all he e e ence miRNAs (Supplemen a y
Table SI) in all he ex ac ion eplica es ob ained using he DCT-M,
DCT-N, PBP-M, PBP-N and UC-M p o ocols, di e ences in he abun-
dance o each miRNA we e ound among hem (Fig. 3A). The miRNA
analysis showed ha he me hods employing he EV en ichmen s ep
wi h PBP (PBP-N and PBP-M) pe o med be e han he o he s, wi h
PBP-N being he mos e icien me hod. The p o ocols using di ec
Figu e 2. Cha ac e iza ion o he mic oRNAs (miRNAs) in he endome ial fluid o pa ien s unde going ART. (A) Wes e n blo
shows di e en EV ma ke s (CD63, CD81 and RAB8) and soluble p o eins (Igs) in he ac ions o size-exclusion ch oma og aphy (SEC). The ac-
ions ob ained by SEC we e numbe ed om F1 o F12. (B) Dis ibu ion o he se en e e ence miRNAs among he ac ions o he SEC. No malized
ela i e quan ifica ion was used o de ec he miRNAs in he ac ions. To pe o m expe imen s A and B, a 400-ml sample aliquo om he se up
pool coho was added on o he column. The numbe o eplica es o each ac ion was six and he da a show he mean wi h SEM. (C) RNase p o-
ec ion assay. Sample analysis o examine he associa ion o miRNAs wi h p o eins and EVs. The g aphs show he C alues o he e e ence miRNAs
e alua ed using he qPCR. The numbe o eplica es o each condi ion was six and he da a show he mean wi h SEM. The numbe o eplica es in
which each miRNA was de ec ed is shown a he bo om o each column. Each aliquo (400 ml) came om he se up pool coho . S a is ical signifi-
cance was de e mined using he pai ed S uden ’s - es analysis. *
,$,&,#
P<0.05; **
,$$,&&,##
P<0.01; ***
,$$$,&&&,###
P<0.001. * e sus Con ol,
$
e -
sus RNase,
&
e sus TX-100,
#
e sus PRT-K. Con ol: con ol sample wi hou ea men . RNase: samples ea ed wi h RNase. TX-100: samples
ea ed fi s wi h T i on-X 100 (TX-100) ollowed by RNase ea men . PRT-K: samples ea ed fi s wi h p o einase K and hen wi h RNase. TX-
PRT: samples ea ed fi s wi h TX-100, hen wi h p o einase K and finally wi h RNase. EVs, ex acellula esicles; qPCR, quan i a i e PCR.
2382 Iba~
nez-Pe ez e al.
Downloaded om h ps://academic.oup.com/hum ep/a icle/37/10/2375/6678065 by Uni e sidad del Pais Vasco use on 08 No embe 2022
Figu e 3. Op imiza ion o di e en me hods o analyzing he miRNAs in endome ial fluid o pa ien s unde going ART. (A)
Resul s o he se en e e ence miRNAs analyzed by quan i a i e PCR o each o he compa ed echniques. No malized ela i e quan ifica ion
e ealed ha he mos e ficien me hod was he PBP-N, while he UC-M me hod was he leas e ficien . S a is ical significance was de e mined using
pai ed - es analysis. The numbe o eplica es o each case was 12 and he da a show he mean wi h SEM. * e sus PBP-N;
$
e sus PBP-M;
&
e sus
DCT-N;
#
e sus DCT-M. (B) The Venn diag am shows he numbe o unique miRNAs de ec ed using small RNA-Seq o each me hod and he
numbe o miRNAs common among hem. The numbe o unique miRNAs de ec ed by each echnique was 251 o PBP-M, 151 o PBP-N, 204 o
SEC F3 and 149 o SEC F4. The samples (400 ml) o expe imen s A and B came om he se up pool coho , and each expe imen was pe o med in
iplica e. (C) A echnical ep oducibili y expe imen was conduc ed o compa e he pe o mance o PBP-M and PBP-N me hods. The g aphs show
C alues o each miRNA, each ope a o (a, JIP; b, MCG) and me hod (PBP-M o PBP-N). Box plo s show he median, maximum and minimum al-
ues and all he poin s. The 400-ml samples came om he se up pool coho . Each ope a o analyzed 20 aliquo s, 10 by employing he PBP-M and 10
miRNAs as bioma ke s o implan a i e endome ium 2383
(con inued)
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Ins i u o de Salud Ca los III (PI20/01131). The unding en i ies did no
ha e any ole in s udy design, sample collec ion, analysis and in e p e a-
ion o da a, epo w i ing o decision o submi he a icle o
publica ion.
Con lic o in e es
The au ho s decla e no compe ing in e es s.
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