Serine inhibits granulosa cell ferroptosis to maintain ovarian function

Domain: neuroscience
Published: 2026-01-21
Editorial outcome: major_revision

Abstract
Cyclophosphamide(CTX)isaprimarymedicineforcuringbreastcancerwhich
oftencausesprematureovarianinsufficiency(POI).Ourrecentpublication
revealsthatCTXinducesPOIbypromotingtheexpressionofSLC1A4,a
transporterofserineefflux,inovariangranulosacells(GCs).Here,wereport
thatthereisaclosedconnectionbetweenthereductionofserumserineand
ovarianhypofunctioninthebreastcancerpatientstreatedwithCTXorwomen
ofchildbearingagewhoaresufferedfromthestaying-up-late.Additionally,we
observethatdietaryserinesupplementationprotectsmicefromCTX-induced
POIwithoutalteringitsanti-breastcancer.Furthermore,wedemonstratethat
theelevatedserinepromotesS1Psynthesis,andinturn,inhibitsthenuclear
translocationofNrf2andconsequentHO-1expression,tosuppressferroptosis
inGCs.Ourstudyrevealsthatthechemotherapy-inducedoridiopathicPOI
sharethesamemechanisms,indicatingthatserineisacriticalfactorfor
maintainingovarianfunction.

Introduction
Prematureovarianinsufficiency(POI)/Prematureovarianfailure(POF)
hasemergedasasignificantglobalconcern,adverselyaffectingfamily
well-being and social harmony1. According to the latest clinical
guidelines,POIcanbroadlyreplacePOF.Therefore,inthistext,wewill
usePOIinsteadofPOFtodemonstrateourrespectforthepatient.The
pathogenesis of POI remains unclear, leading to a lack of effective
clinical treatments to mitigate the ovarian hypofunction. Studies
indicatedthatchemotherapyledtoanincidenceofappropriate40%of
POIinthepatientswithbreastcancer,whichaccountsfor~5–10%of
totalpatientswithdiminishedovarianfunction2–5.However,thecauses
of the majority of patients with the idiopathic ovarian insufficiency
wereundetermined6–9.

Cyclophosphamide(CTX)isaprimarydrugusedinthetreatment
ofbreastcancer,favoredforitscost-effectivenessandpotentinhibi-
toryeffects.However,CTXisassociatedwithsignificantreproductive
toxicity,which adverselyaffectsthe reproductive health ofpatients
undergoing chemotherapy for breast cancer10–12. More than half of
breastcancerpatientsexperienceadversereactionssuchasmenstrual
delaysorevenamenorrheafollowingCTXtreatment,withsomefacing
severecomplications,includingdifficultiesinconception13–15.Notably,
withtheincreasingincidenceandthetrendofyoungeronsetofbreast
cancer,agrowingnumberofyoungpatientsweredevelopingthePOI
afterCTXchemotherapy16–18.Meanwhile,arecentsurveyrevealedthat
50%ofyoungfemalepatientswishtopreservetheirfertilitywithpost-
chemotherapy3,8,19,20.Therefore,itiscrucialtoprotectovarianfunction
duringCTXtreatmentinpatientswithbreastcancer21.Moreover,to
elucidatethemechanismandtheetiologyofPOIinducedbyCTXor
idiopathicPOIisanurgentneedfordevelopingeffectivepreventive
andtherapeuticstrategiestomaintainovarianfunction22.Ourrecent
study demonstrated that human urine stem cells (hUSC)-derived
miRNA-27-3p selectively suppressing CTX-induced elevated expres-
sions of SLC1A4, a serine transporter for excreting the intracellular
serine, in ovarian GCs to alleviate CTX-induced POI23. Inhibiting the
expressionofSLC1A4inovariangranulosacells(GCs)viasiRNAcan
directlysuppressCTX-inducedgermcelldeath.Thisfindingindicates
thattheelevatedexpressionofSLC1A4inducedbyCTXiscrucialfor
thedevelopmentofPOI.Buildingonthisfinding, we were eagerto
knowwhethertherewasaconnectionbetweenthereductionofserum
serineandtheovarianhypofunctioninbreastcancerpatientstreated
withCTXorwomenofchildbearingagewhosufferedfromidiopathic
POI, and whether the CTX-induced POI or idiopathic POI could be
preventedandcuredbydietaryserinesupplementation.

Serineisaconditionalnon-essentialaminoacidthatfunctionsasa
vital precursor in the synthesis of proteins, phospholipids, neuro-
transmitters,andnucleotides.Ithasbeenreportedthatserineplaysa
centralroleinvariousphysiologicalprocessesassociatedwithmultiple
disease24–28.Abnormalitiesofserinemetabolismhavebeenlinkedto
neurodegenerative diseases, cardiovascular disorders, immune dys-
function, and cancer29–34. However, the roles and the underlying
mechanismofserineinfemalereproductivefunctionhavenotbeen
explored35.

Inthepresentstudy,weobservedthatCTXsignificantlyreduced
thelevelsofserumEstrogen(E2),anti-Mullerianhormone(AMH),and
serineinpatientswithbreastcancer,andtherewasacloseconnection
betweenthereductionofserumserineandovarianhypofunctionin
thewomenofchildbearingagewhosufferedfromstayinguplate.In
addition, we observed that dietary serine supplementation could
effectively prevent CTX-induced POI in mice. Furthermore, we
demonstratedthattheelevatedserinepromotedS1Psynthesis,andin
turn,inhibitedthenucleartranslocationofNrf2andconsequentHO-1
expression,tosuppressferroptosisinGCs.Moreover,ourstudyfur-
ther revealed that the idiopathic and chemotherapy-induced POI
sharedthesamemechanism,suggestingthatserineisacriticalfactor
formaintainingovarianfunction.Recently,wehaveinitiatedarelevant
clinicaltrial(ChiCTR2400079348)toevaluatetheprotectiveeffectsof
serineagainstCTX-inducedPOI.Certainly,ourstudyshouldprovide
insightintoelucidatingthemechanismofchemotherapy-inducedPOI
oridiopathicPOI,andthedietaryserinesupplementationmightbea
simpletherapeuticstrategyforpreventingandcuringovarianhypo-
functionclinically.

Results
CTXreducedserumserineconcentrationinbreastcancer
patients related to the reduction of the intracellular serine23, L-serine was
administered to mice through gavage or drinking water supple-
mentation(Fig.3aandFig.S4a).Theresultsshowedthatregardlessof
theformsofserinesupplementation(intragastricalordrinkingwater
administration), serine effectively preserved ovarian morphology,
evidencedbyrestoringthesizeandorganrateofovariesduringCTX
chemotherapy(Fig.3b,candFigs.S3aandS4b–d).HEstainingresults
showedthatserinealsomarkedlypreventedCTX-induceddecreaseof
folliclesandtheincreaseinatresia.Inaddition,serinepreventedCTX-
inducedalterationofovarianstructureandthereductionofovarian
surfacearea(Fig.3dandFigs.S3b,candS4e–h),andcorrectedCTX-
induced abnormal secretions of E2, follicle-stimulating hormone
(FSH),andanti-Müllerianhormone(AMH)(Fig.3e–gandFig.S4i,j).In
theovary,twocrucialtypesoffunctionalcellsarepresent:GCsand
oocytes. GCs play a vital role in the growth and development of
oocytes by secreting associated hormones38–40. Our results showed
thatserinesupplementationeffectivelypreventedCTX-induceddeath
ofGCs,enhancedtheirproliferation,andincreasedtheexpressionof
AMH(Fig.3h,iandFig.S3d,e),indicatingthatserinesupplementation
protectedmicefromCTX-inducedPOIthroughpreventingthedeath
ofGCs,enhancingGCsproliferation,andmaintaininghomeostasisof
sexhormonelevels.

Next,weclarifiedwhethertheprotectiveeffectofdietaryserine
supplementation on CTX-induced POI was able to reduce its anti-
cancereffectinbreastcancer.Giventhatthemajorityofpatientswith
breastcancerpredominantlyexhibitedPOIduringchemotherapy,and
consideringthatthereweredifferencesintheinternalmicroenviron-
ment induced by breast cancer and the normal internal micro-
environment,wesimulatedthedosageanddurationofchemotherapy
toestablishabreastcancermousemodelofPOI41–43.Briefly,1×1064T1
cellsweretransplantedintothemousesecondbreastpadtogenerate
breastcancermodel,inwhichCTX(20mg/kg)wasadministratedby
injectionwithtwiceeachweekfromthirdto7thweek,andserine(50
and500mg/kg)wassupplementedbygavagewithonetimeeachweek
fromsecondweekto7thweek(Fig.3j),andthroughoutthestudy,we
closely tracked changes in tumor volume. Our results showed that
serinedidnotaffecttheefficacyofCTXinbreastcancer(Fig.3k–mand
Fig. S5a). In contrast, serine significantly improves CTX-induced
reduction in the size, organ rate, and surface area of the ovaries
(Fig.S5b–g).Inaddition,ourstudyalsorevealedthatserinesupple-
mentationremarkablyamelioratedthedysregulationofsexhormone
secretion and promoted the development of antral follicles in the
ovary,aswellastheproliferationofGCs(Fig.3n–pandFigS5h).Fur-
thermore, three different tumor cell lines were selected for further
validatingwhetherserineaffectstheanti-cancereffectofCTXinvitro,
andresultsshowedthatserinedidnotalterthetherapeuticeffectof
CTXonmultipletumorcells(Fig.S5i–k).Theseresultsdemonstrated
thattheprotectiveeffectofserinesupplementationonCTX-induced
POIwasnotrelatedtoitsanti-cancereffectinthemousemodel.

CTXinducedferroptosisinGCsbutnotinbreastcancercells
Next, we focused on the protective mechanism of serine in CTX-
inducedPOI.PreviousstudiessuggestedthatCTXmighttriggerPOIby
excessively activating primordial follicles8,44,45. Additionally, other
researchersobservedthatCTXinducedPOIbypromotingROSpro-
ductionorimpairingmitochondrialfunction46,47.Furthermore,some
studieshavereportedthatCTXinducesapoptosisinGCs,whichleads
toadeclineinovarianfunction48–50.OurrecentreportidentifiedGCsas
theprimarytargetcellsofCTXintheovary23.Subsequently,wecon-
ducted transcriptome sequencing on GCs treated with CTX. The
sequencing results showed that therewere significant alterations in
geneexpressioninGCsafterCTXtreatment(Fig.4aandFig.S6a).We
conductedaheatmapanalysisofthetop50upregulatedanddown-
regulatedgenesinGCsfollowingtreatmentwithCTX(Fig.4b,Fig.S6b,
and Supplementary Tables 3 and 4). GSEA and KEGG enrichment
analysesoftheupregulatedgenesrevealedsignificantenrichmentin
the ferroptosis signaling pathway (Fig. 4c, d and Fig. S6c). A Venn
diagramcomparisonbetweentheupregulateddifferentiallyexpressed
genesandthoseintheferroptosisdatabase(FerrDbV2)identifiedfour
overlappinggenesthatwerenotablyrankedamongtheupregulated
genes(Fig.4e).TheqPCRresultsandtheenrichmentmapofthefer-
roptosissignalingpathwayindicatedthatCTXprimarilyinducesthe
upregulation of HO-1, leading to the accumulation of ferrous ions
(Fig. 4f, g). Furthermore, CTX did not induce any changes in GPX4
expression(Fig.S6d).Wealsoobservedthattheelevatedexpression
levels of nuclear receptor coactivator 4 (NCOA4), zinc transporter 14
(ZIP14),andzinctransporter8(ZIP8)maycontributetoironaccumu-
lation,althoughtheirupregulationwasnotaspronouncedasthatof
Hmox1(Fig.4h).Therefore,weproposedthatCTX-inducedPOImight
notbedirectlyrelatedtotheapoptosisoroxidativestressofGCs,but
ratherthatitwascausedbytheaccumulationofferrousionsinGCs
mediatedbyHO-1,whichsubsequentlytriggersferroptosis.

Therefore,weinvestigatedwhetherserinesupplementationcould
inhibitHO-1-inducedferroptosisinthemousePOImodel.Ourresults
showedthatserinesuppressedCTX-inducedtheaccumulationofthe
intraovarianferricionandferrousion,lipidperoxidationinadose-
dependentmanner(Fig.5a–dandFig.S4l,m),andinhibitedtheCTX-
activatedKeap1/Nrf2/HO-1signalingpathway,whichinturn,reduced
the expression of HO-1 (Fig. 5e, f and Fig. S4m). Importantly, the
accumulation of MDA and ferrous ions induced by CTX was not
observed in the tumor tissues of the mouse POI model with breast
cancer (Fig. 5f–h). In addition, the expressions of the ferroptosis-
relatedgenesinbreastcancertissuesexhibitednosignificantchanges
following the administration of CTX and the oral gavage of serine
(Fig.5i).However,theaccumulationofferricionandferrousionswas
observedintheovariantissuesofbreastcancermodelmicewithPOI,
suggesting that CTX-induced POI could be significantly improved
uponsupplementationwithserine(Fig.S6e).Ourresultsindicatedthat
themechanismsthroughwhichCTXaffectsovarianandbreastcancer
tissuesweredifferent,andserinesupplementationshouldsafeguard
ovarianfunctionwithoutreducingtheeffectivenessofchemotherapy.

SerineelevatedthesynthesisofS1Ptoinhibitferroptosis
Tofurtherelucidatetheprecisemolecularmechanismbywhichserine
inhibitsferroptosisinGCs,mouseGCswereisolatedfollowingestab-
lished protocols. Our in vivo results showed that serine effectively
hinderedthebuildupofferrousions.Tovalidatethisobservation,we
employedtheferrousionchelatordeferoxamine(DFO)andtheago-
nistErastinasexperimentalcontrols(Fig.6a).Ourresultsshowedthat
DFO or serine had a notable inhibitory effect on CTX-induced cell
deathinGCs,whereasErastinexacerbatedthisalteration(Fig.6band
Fig.S6f,g).Furthermore,DFOorserineeffectivelyreducedthelevelof
lipidperoxidationinducedbyCTX,whileErastinenhancedlipidper-
oxidation in GC (Fig. 6c). In addition, DFO or serine inhibited the
accumulation of ferrous ions in GCs (Fig. 6d). Mitochondria play a
crucialroleintheprocessofferroptosis,promptingustoinvestigate
the morphological structure and membrane potential of the
mitochondria51–54. Our results revealed that CTX or Erastin induced
mitochondrialatrophy,increasedmembranearea,reducedridges,and
decreased membrane potential, whereas DFO or serine effectively
mitigatedthemitochondrialdamagecausedbyCTX(Fig.6e,f).Fer-
roptosiswascharacterizedbythegenerationofROS,andbothDFO
andserinenotablysuppressedROSproductioninGCs(Fig.6g).Fur-
thermore,ourresultsrevealedthatserinewasabletocounteractthe
elevatedexpressionsofNrf2andHO-1inducedbyCTX(Fig.6h,i),and
downregulated the expressions of NCOA4, ZIP14, and ZIP8,
consequentlydecreasedtheaccumulationofferrousions(Fig.S6h).
Overall,ourresultsdemonstratedthatserineattenuatedCTX-induced
ironoverloadandinhibitedferroptosisbyinhibitingtheKeap1/Nrf2/
HO-1signalingpathway.

Previous studies have demonstrated that serine reacted with
palmitoyl-CoA in cells, leading to the production of sphingosine
throughaseriesofreactions30,55.SphingosineisconvertedintoS1Pby
sphingosinekinase(Sphk),andS1Pmightbeinvolvedincrucialpro-
cesses such as ovarian growth, development, and maturation56,57.
Basedontheseobservations,wehypothesizedthatserineexertsits
effectsthroughtheactionofS1P.Totestthishypothesis,weemployed
theSphkinhibitorSKI178tosuppressS1Pproduction(Fig.6j),andthe
resultsshowedthatCTXorSKI178ledtoanotablereductioninS1P
levelsinGCscomparedwiththeuntreatedGCs(Fig.S6i).Ourstudy
furtherrevealedthatSKI178reversedtheinhibitoryeffectsofserineon
CTX-inducedcelldeathinGCs,whilepromotedlipidperoxidationand
theaccumulationofferrousions(Fig.6k,landFig.S6j).Inaddition,
SKI178alsoreversedtherestorativeeffectofserineonmitochondrial
morphology,structure,andmembranepotentialandROSgeneration
(Fig. 6m, m and Fig. S6k). These results demonstrated that serine
hinderedtheCTX-inducedbuildupofferrousionsinGCsviaS1P.Our
studiesfurtherdemonstratedthatwhenS1Psynthesiswasinhibited,
the expressions of Nrf2 and HO-1 were elevated, resulting in the
accumulationofferrousions.Moreover,theexpressionofSLC1A4was
alsosignificantlyincreased(Fig.6o–q).Notably,theconfocalmicro-
scopyresultsindicatedthatCTXpromotedthenucleartranslocation
ofNrf2,leadingtoanincreaseintheexpressionofSLC1A4andHO-1.
Conversely,serinesupplementationenhancedtheproductionofS1P,
which,inturn,reducedthenuclearimportofNrf2andsubsequently
decreasedtheexpressionofHO-1andSLC1A4(Fig.6randFig.S6l).To
furtherclarifythatserineinhibitsGCsferroptosisbyconvertingtoS1P,
weconductedanS1Prescueexperimentandmeasuredkeyindicators
offerroptosisinducedbyferrousionaccumulation.Theresultsindi-
cated that S1P significantly suppressed CTX-induced ferrous ion
accumulation,lipidperoxidation,andROSaccumulation,effectively
inhibitingGCsferroptosis(Fig.S6m–o).

Tovalidatethattheaccumulationofferrousionsisattributedto
HO-1 and to verify the regulation of the Keap1/Nrf2/HO-1 signaling
pathwayonferroptosis,weemployedtheHO-1activityinhibitorZnpp
toinhibitHO-1activity(Fig.6sandFig.S6p,q).Theresultsshowedthat
HO-1inhibitorsignificantlyreducedCTX-inducedGCdeath,aswell
as a decrease in lipid peroxidation and ferrous ion accumulation
(Fig.6t,uandFig.S6r).Inaddition,ourresultsalsoshowedthatZnpp
led to a significant improvement in abnormalities in mitochondrial
morphology, structure, and membrane potential induced by CTX
(Fig.6v,w),andsuppressedCTX-inducedproductionofROSinGCs
(Fig. 6x). Collectively, our data demonstrated that serine supple-
mentationalleviatedferroptosisinGCsbyinhibitingtheaccumulation
offerrousionsduringchemotherapy,sheddinglightontheunderlying
mechanismofCTX-inducedPOI.

Serinedeficiencyinducedbystayinguplatewasthemaincause
ofidiopathicPOIinwomen
Although chemotherapy-induced POI accounts for ~5–10% of total
patientswithdiminishedovarianfunction,thecausesofthemajority
ofthepatientswithidiopathicPOIwereundetermined58,59,indicating
thattheeffectivetreatmentsforthesepatientsarenotavailable60.Our
studyindicatedthattherewasacloserelationshipbetweenidiopathic
POIandthereductionofserumserinelevels.Consequently,weaimed
to investigate whether idiopathic POI shares the same pathological
mechanismsaschemotherapy-inducedPOI.Existingstudiesindicated
thatstayinguplatemightadverselyaffectthesecretionoffemalesex
hormones,ovariandevelopment,and the ovarian cycle61,62.Wecon-
ducted a questionnaire survey involving 124 enrolled patients, dis-
tributing124questionnairesandreceiving106completedresponses.
Theanalysisofthequestionnaireresultsrevealedthatstayinguplate
was the most significant factor contributing to idiopathic ovarian
insufficiency in women,which might significantlysurpassthe stress
anddieting(SupplementaryTable5).Furtheranalysisofthemetabo-
lomerevealedthatthereweresignificantdifferencesinserumamino
acidmetabolismlevelsbetweenwomenwithnormalovarianfunction
andthosewithdiminishedovarianfunction.Inaddition,serumlevels
of AMH and serine were significantly decreased in patients who
experiencedsleepdeprivation(Fig.7a,bandFig.S7a–d).Theresultsof
thequestionnairesurveyandmedicalexaminationsindicatedthattwo
primaryfactorscontributingtolate-nightactivitiesamongwomenare
the use of electronic devices and sleep disorders (Supplementary
Table 5). Among female patients experienced with sleep disorders,
their serum levels of AMH and serine were significantly decreased,
whichweresignificantlycorrelatedwithanincreasedfrequencyoflate-
nightactivitiesperweekinthisdemographic(Fig.7c–e).Ouranalysis
indicatedthattherewasanegativecorrelationbetweenserumAMH
and serine levels and the frequency of staying up late per week in
patientswithdiminishedovarianfunction.Theseresultsindicatedthat
stayinguplatemightcontributetoserinedeficiencyinfemalepatients,
subsequentlyleadingtodiminishedovarianfunction.Therefore,inour
study,astrongcorrelationhasbeenestablishedbetweenstayingup
late,thesubsequentdecreaseinserinelevels,andtheresultingdecline
inovarianfunctioninpatientswithidiopathicPOI(Fig.7f–h).

Duetotheinabilitytoobtainovariantissuefromthewomenwho
experienced sleep deprivation, we established a mouse model to
investigatetheaccumulationofferrousionsintheovaries(Fig.7i,j)63.
Followingextendedperiodsofsleepdeprivation,themicedisplayeda
markedreductioninbothsurvivalrateandbodyweight.Inaddition,
therewasasignificantincreaseintheproportionofwhitebloodcells,
neutrophils, and granulocytes, alongside heightened inflammatory
levelsobservedintheovaries(Fig.S8a–d).Furthermore,weobserved
thatthemouseovariesshowedasignificantshrinkagefollowingsleep
deprivation,accompaniedbydisorderedsecretionofsexhormones
andamarkedreductioninAMHlevelswithintheovaries(Fig.7k–r).
Interestingly,we alsoobserved an increasein SLC1A4 expressionin
GCs,andanaccumulationofironionsintheovariesofsleep-deprived
mice (Fig. 7s, t). These findings indicated that the mechanisms
underlyingsleepdeprivation-inducedidiopathicovarianhypofunction
mightlargelyoverlapwiththoseofCTX-inducedPOI,suggestingthat
serine might be a critical factor for maintaining ovarian func-
tion(Fig.8).

Methods
The study was conducted following the Declaration of Helsinki
(as
revisedin2013),andwrittenconsentfortissuedonationwasobtained
fromeachpatient.Thetwoclinicalprotocolsreceivedapprovalfrom
theInstitutionalReviewBoardoftheNanchangFirstPeople’sHospital
(approvalno.K-kt2025001;approveddateJanuary14,2025)andthe
FirstAffiliatedHospitalofNanchangUniversity(approvalno.IIT2024-
329;approveddateMay11,2024).Theprotocolswereapprovedbythe
AnimalResearchEthicsBoardofNanchangUniversity(approvalno.
NCULAE-20221228066; approved date July 2, 2022) following the
GuidelinesfortheCareandUseofAnimals.Thestudyinvolvingani-
malswasconductedfollowingtheBaselDeclaration.

Chemotherapyandserumcollectioninbreastcancerpatients
Twenty-sevenbreastcancerpatients(LuminalAorLuminalB)aged
between25and40wereenrolledinthestudy.Thesepatientsexhibited
normalovarianfunction,asevidencedbyregularmenstrualcyclesand
serumlevelsofAMHandE2priortoenrollment.Theyhadnotreceived
anyhormoneoraminoacid-basedmedicationswithinonemicemonthprior
to enrollment,and conditions thatcould potentially influence AMH
values were systematically excluded. All patients received che-
motherapy based on CTX (600mg/Body surface area), which con-
sisted of four cycles. In each cycle, CTX was administered once,
followedbya21-dayintervalbeforethesubsequentcycle.Noamino
acid drugs were administered before or during the chemotherapy
regimen. Serum samples were collected from 27 patients prior to
chemotherapytomeasureserumE2levelsandtoconductaminoacid-
targeted metabolomic sequencing, utilizing serum E2 levels as an
indicator of ovarian function. Following the first course of che-
motherapy,a21-daywaitingperiodwasobservedbeforeinitiatingthe
secondcourse.Serumsampleswereagaincollectedfromthesame27
patientsbeforethesecondcourseofchemotherapyforE2measure-
mentandaminoacid-targetedmetabolomicsequencing.Thepartici-
pantsofourstudyreceivedstandard-of-caretreatment.

POIstudyparticipantsandstudydesign
We recruited 124 infertile patients who provided written informed
consent,andthestudyreceivedapprovalfromthelocalinstitutional
reviewboard.Theclinicaldiagnoseswereconductedbyexperienced
clinicians in the Department of Reproduction at the First Affiliated
Hospital of Nanchang University. Most patients completed a ques-
tionnaireregardingtheirlate-nightactivitiesduringtheirinitialhos-
pital visit, and we collected ovarian B-ultrasound images and blood
samples.BasedontheAMHlevelsandthenumberofbilateralfollicles,
patientswerecategorizedintotwogroups:thosewithnormalovarian
function (Control, AMH >1.3ng/mL) and those with POI (POI, AMH
<1.3ng/mL). We compared sex hormone levels, bilateral follicle
counts, and blood amino acid levels between the two groups, and
analyzed the relationships among late-night activities, ovarian func-
tion, and amino acid levels. The participants of our study received
standard-of-caretreatment.

Clinicalexaminations
All participants underwent sex hormone screening, which included
measurements of AMH, E2, FSH, and Luteinizing hormone (LH).
Radionuclides were utilized to detect sex hormone levels. Bilateral
ovarian images of each participant were collected. The amino acid
levels in each patient were analyzed using LC-MS, a process that
involvesrelativelycomplexsteps.

Inclusioncriteria
Inclusioncriteriafortheexperimentalgroupcomprisedfemaleparti-
cipantsagedbetween20and35,withserumAMHlevelsbelow1.3ng/
mL. In addition, imaging data, including B-ultrasound, indicated
compromised ovarian function, characterized by a bilateral antral
folliclecountofsixorfewer.Participantswerealsorequiredtoexhibit
irregularmenstruation patterns, which could manifest as significant
delays,infrequentperiods,oramenorrhea.
Theinclusioncriteriaforthecontrolgroupconsistedoffemales
agedbetween20and35whoexhibitedregularmenstrualcycles.In
addition, their imaging data, including serum AMH levels and
B-ultrasoundresults,indicatednormalovarianfunction.

Clinicalstudyexclusioncriteria
PatientswithconditionsthataffectserumAMHlevels,suchaspoly-
cysticovarysyndrome(PCOS)anddiabetes,wereineligibletoparti-
cipateinthisclinicalstudy.Additionally,patientswhohaveundergone
hormonetherapyortakenaminoacid-basedmedicationswithinone
monthpriortoenrollment,aswellasthosewithphysicaloriatrogenic
ovarian function impairment, including prior ovarian surgery, were
alsoexcludedfromparticipation.Detailedpatientenrollmentcriteria
arepresentedintheSupplementaryTable2.

Aminoacidtargetedmetabolomesequencing
Sampleswereextractedwith400μLof10%formicacidinmethanol-
water(1:1,v/v),vortexedfor30s,andthencentrifugedat2000×g
and4°Cfor5min.Takeanappropriateamountofsupernatantand
add10%formicacidinmethanol-water(1:1,v/v)todilute20times
andvortexfor30s.Take100μLofsupernatantandadd100μLTrp-
d3(20ng/mL),vortexfor30s.Thesupernatantwasfilteredthrough
a 0.22-μm membrane, and the filtrate was added to the LC-MS
bottle.
The samples were injected into the ACQUITY UPLC® BEH C18
Column (2.1×100mm, 1.7μm, Waters, USA), the injection volume
was5μL,thecolumntemperaturewas40°C,andthemobilephaseA-
50%methanolinwater(containing0.1%formicacid),B-10%methanol
in water (containing 0.1% formic acid). The gradient elution
conditionswere0–6.5min,90–70%B;6.5–7min,70–0%B;7–14min,
0% B; 14–14.5min, 0–90% B; 14.5–17.5min, 90% B. Flow rate
0–8.0min, 0.3mL/min; 8.0–17.5min, 0.4mL/min (Supplementary
Table6).
Electrospray ionization (ESI) source, positive ionization mode.
Theionsourcetemperaturewas500°C,theionsourcevoltagewas
5500V,thecollisiongaswas6psi,thecurtaingaswas30psi,andthe
atomizing gas and auxiliary gas were both 50psi. Scans were per-
formedusingmultiplereactionmonitoring(MRM).

Dietaryserinesupplementationinmice
C57BL/6orBalb/cfemalemiceat8weeksofagewerepurchasedfrom
Jiangsu GemPharmatech Co., Ltd (Nanjing, China). The mice were
maintainedona12-hlight/darkcycleandhadadlibitumfreelyaccess
to food (1010083,jsxtsw,China)andwaterattheLaboratoryAnimal
Center of the Institute of Translational Medicine of Nanchang Uni-
versity.Allanimalproceduresdescribedinthisstudywerereviewed
andapprovedbytheAnimalCareandUseCommitteeofNanchang
University.Thestudyinvolvingani-
malswasconductedfollowingtheBaselDeclaration.
ToestablishPOImodels,atotalof50C57BL/6micewereintra-
peritoneallyinjectedwithCTX(50mg/kg;MCE,USA)for15days.For
establishingthebreastcancermousemodels,atotalof1×1064T1
cells (CBP60352,NanjingCobioerBioscienceCo.,Ltd.)weretransplanted
into the second breast pad of the mouse. The tumor volume was
measuredeverythreedaysfollowingtransplantation.Oncethetumor
volumeexceeded100mm3,themousereceivedintraperitonealinjec-
tionsofCTX(20mg/kg)everyMondayandThursdayforchemother-
apyfor4weeks.Exogenousserinewassupplementedintomiceintwo
ways: (1) Supplementation with water: L-serine (100g/L; Solarbio,
China) was dissolved in sterile water for free drinking; (2) Supple-
mentationbygavage:L-serine(50mg/kgor500mg/kg)wasadminis-
teredbygavageeverydayfor5weeks.Inaccordancewiththeanimal
ethicsandwelfarerequirementsestablishedbyNanchangUniversity,
mice were euthanized through cervical dislocation following anes-
thesiainducedbyanintraperitonealinjectionofAvertin.

Enzyme-linkedimmunosorbentassay(ELISA)
Serumwascollectedfrombloodandthenallowedtoclotfor4hat
room temperature and subsequently stored overnight at 4°C. The
nextday,thesampleswerecentrifugedat180×gfor20min,andthe
supernatantserumwascollected.SerumE ,FSH,andAMHlevelswere
2
measured using the corresponding ELISA kits (Elabscience, China)
accordingtothemanufacturer’sprotocol.Toestablishthestandard
curve, add 50μL of the serially diluted E2, FSH, and AMH standard
solutiontothestandardwells.Intheblankwells,add50μLofsample
diluentasanegativecontroltoeliminatereagentbackgroundinter-
ference.Subsequently,add50μLofthetestsamplesolutiontoeachof
theremainingwells.Aftercompletingthesampleaddition,immedi-
atelyintroduce50μLofthefreshlypreparedHRP-conjugatedworking
solutionintoeachreactionwelltoensureuniformitywithinthereac-
tionsystem.Finally,coverthe96-wellplatewithaspecializedsealing
film and incubated itin a 37°Cconstanttemperature incubator for
60min.Aftercompletingtheincubationstepoftheexperiment,add
350μLofwashingbuffertoeachwellofthemicroplateandallowedit
to stand for 1minute to ensure the thorough removal of unbound
impurities.Followingthesoakingperiod,invertedthemicroplateto
draintheliquidandblotteditdryusingabsorbentpaper.Thiswashing
process must be strictly repeated five times. After completing the
washingprocess,carefullyadded90μLofsubstratesolutiontoeach
well. Subsequently,covered the platewitha sterile sealing film and
transferthesealed96-wellplatetoaconstanttemperatureincubator
fora15minincubationperiod.Finally,added50μLofstopsolutionto
eachwelltoterminatethereactionandmeasuretheopticaldensity
(OD)valueusingamicroplatereader.

Histopathologyandfolliclecount
Ovariantissueswerecollectedwhentheexperimentswerecompleted,
fixedin4%paraformaldehydefor24h,subsequentlydehydratedand
embedded,andcutinto5-μm-thickslicesforhematoxylinandeosin
staining (H&E staining). Place the ovarian tissue sections under the
microscopeandsystematicallycountthenumberoffolliclesateach
developmentalstage,includingprimordialfollicles,primaryfollicles,
secondaryfollicles,andantralfollicles.Followingthecompletionof
theserinegavage,boththemouseovariantissueandthebreastcancer
tissue were collected. The collection steps of tumor tissues were
identicaltothosedescribedabove,withtheonlyvariationbeinginthe
dehydrationprocessforthetumortissue.

Immunofluorescencestaining
Ovarian tissues and GCs were fixed with 4% paraformaldehyde and
permeabilizedwith0.1%TritonX-100,followedbyincubationwiththe
correspondingantibodiesat4°Covernight.Theantibodiesusedinthe
study were as follows: anti-PCNA (1:200, rabbit monoclonal, CST,
13110) and anti-NRF2 (1:200, rabbit monoclonal, CST, 12721s). After
washingwithPBS,thecellswereincubatedat37°Cfor1hwithasec-
ondarybiotinylatedgoatanti-rabbitIgGantibody(dilution1:300).The
cellswerethenwashedwithPBSandincubatedat37°Cfor3minwith
DAPIdyeliquor.Thecorrespondingimmunofluorescencestainingwas
recordedwithalaserconfocalmicroscope(OlympusCKX41).

Ferrousionmeasurement
Theovariantissueswerelysedwiththecorrespondinglysisbuffer.The
appropriateamountofdetectionworkingsolutionandlysateswere
mixedaccordingtotheinstructions(Solarbio,BC5415,China)andthen
themixturewasincubatedat37°Cfor10min.Aftertheincubation,
100μL chloroformsolutionwasadded and vortexed for5min, and
then centrifuged at 12,000×g for 10min. The supernatant was col-
lected and detected with a microplate reader (ThermoFisher
Scientific,USA).

MDAassay
Theunilateralovariantissueswerecollected,and1mLofextraction
buffer was added for homogenization in an icebath. Subsequently,
centrifugedthemixtureat8000×gfor10minat4°C,andcollected
thesupernatantoniceforfurthertesting.Mixed100µLofthesample
with300µLoftheMDAworkingsolution,thenincubatedina100°C
waterbathfor60min,followedbycoolinginanicebath(Solarbio,
BC0025,China).Finally,centrifugedat10,000×gfor10minatroom
temperature,andtransferred200µLofthesupernatanttoa96-well
plate for detection using a microplate reader (ThermoFisher
Scientific,USA).

GCscultureandinvitroPOImodel
The 3-week-old female mice were injected intraperitoneally with
pregnant mare serum gonadotropin (PMSG; Solarbio, China) to sti-
mulatefolliclegrowth.Themiceweresacrificedanddissectedafter
48hofinjection.Theadiposetissueandconnectivetissueattachedto
theovarieswerepeeledoffinapre-chilledPBSsolution.Theovarian
GCswerereleasedbypuncturingthefollicleswitha1mLsyringeunder
abodyvisionmicroscope.ThecellswerewashedwithPBSthreetimes
andcentrifugedat180×gfor5min.ThecollectedGCswerecultured
withDMEM/F12(1:1)mediumcontaining15%FBS,1%Insulin-transfer-
rin-Se,and100U/mlpenicillinandstreptomycinat37°Cinahumi-
difiedatmospherewith5%CO .ThefirstpassageofGCswasusedinall
2
experiments, and the ovarian GCs were identified by detecting the
density of immunofluorescence for the expression of follicle-
stimulating hormone receptors (FSHR) since only ovarian GCs
expressFSHRinovariantissue.
InvitroPOImodelwasestablishedwithCTX(5mg/mL),andfer-
roptosisagonistsandinhibitorssuchasDFO(50μM;MCE,HY-B0988,
USA), Erastin (5μM; MCE, HY-15763, USA), and HO-1 inhibitor Zinc
protoporphyrin (5μM; Znpp, MCE, HY-101193, USA) were added to
inhibitorpromotetheprocessofferroptosisinGCs.Theconcentra-
tionoftheserinegroupininvitroexperimentswas800μM.

Transcriptomesequencing
Transcriptome Sequencing Library Preparation and Sequencing:
Granulosacells(GCs)weretreatedwith5mg/mLCTXfor24hpriorto
total RNA isolation with Trizol. An mRNA-seq library was prepared
fromthetotalRNA.Briefly,mRNAwasselectedusingpoly-Tmagnetic
beadsandfragmented.cDNAwassynthesizedintwosteps:first-strand
synthesisusedrandomhexamersandM-MuLVReverseTranscriptase
(RNaseH–),andsecond-strandsynthesisusedDNAPolymeraseIand
RNaseH.ThecDNAfragmentswereend-repaired,A-tailed,andligated
toadapters.Fragmentssized370–420bpwereselectedandpurified
(APureXPsystem,BeckmanCoulter).ThelibrarywasamplifiedbyPCR
with Phusion High-Fidelity DNA Polymerase using indexed primers,
followed by purification (AMPure XP system) and quality control
(AgilentBioanalyzer2100).
ClusterGenerationandSequencing:Indexedlibrarieswereclus-
teredonacBotsystem(Illumina)withtheTruSeqPEClusterKitv3-
cBot-HS.SequencingwasperformedonanIlluminaNovaSeqplatform
togenerate150bppaired-endreads.

AnnexinV-PIapoptosisassay
After completing each group treatment, the culture medium in the
plate was collected and transferred to a 15mL centrifuge tube. A
digestionsolutionwasthenaddedtodetachtheadherentGCs,which
were subsequentlyincluded in the same 15mLcentrifuge tube. Fol-
lowing centrifugation at 180×g for 5min, the supernatant was dis-
carded,andthecellpelletwasretained.Thepelletwasresuspendedin
pre-cooledPBSandsubjectedtoanothercentrifugationat180×gfor
5min,afterwhichthesupernatantwasagaindiscarded.Thepelletwas
thenresuspendedin100μLofAnnexinVbindingbuffer,containing
5μL of Annexin V-FITC and 5μL of propidium iodide (PI) solution
(Dojindo, AD10, Japan). After incubating at room temperature for
15min,thecellswerewashedwithPBS,centrifugedat180×gfor5min,
andsubsequentlyresuspendedin400μLofAnnexinVbindingbuffer.
ApoptosisdetectionwasperformedandanalyzedusingtheBDJazz
system.

Lipidperoxidationassay
To prepare the C11-BDP581/591 DMSO stock solution, add 20μL of
DMSOtothetubecontainingC11-BDP581/591andmixthoroughlyuntil
fullydissolved.Subsequently,dilutetheC11-BDP581/591DMSOstock
solution1000-foldwithcompleteGCsmediumtogeneratetheC11-
BDP581/591DMSOworkingsolution.Add200μLoftheC11-BDP581/
591 working solution to the cells and incubate at 37°C for 30min
(Dojindo,L267,Japan).Afterincubation,removetheworkingsolution
andwashthecellstwicewithHBSS.Finally,observethecellsusingthe
OlympusCKX41invertedfluorescencemicroscope.

Reactiveoxygenspecies(ROS)assay
TheDCFH-DAworkingsolution(10μmol/L;Solarbio,CA1410,China)
waspreparedfollowingthemanufacturer’sinstructions.DCFH-DAwas
dilutedwithserum-freeculturemediuminaratiorangingfrom1:1000
to 1:10,000 to achieve a final concentration of 1–10μmol/L. Subse-
quently,500μLoftheDCFH-DAworkingsolutionwasaddedtothe
cellsandincubatedat37°Cfor20min.TheGCswerethenwashed
threetimeswithserum-freeGCsmediumtothoroughlyremoveany
unincorporatedDCFH-DA.Finally,thegreenfluorescenceemittedby
theGCswasobservedunderafluorescencemicroscope.

S1Pcontentassay
GCsweredigestedwithtrypsin,centrifugedat360gfor5min,andthe
cellpelletswereresuspendedinpre-chilledPBSandquicklyfrozenin
liquid nitrogen, then thawed in water at 37°C. And this step was
repeated three times to disrupt the cells,and the lysates were cen-
trifugedat1500×gfor15min,thesupernatantwasdetectedaccording
totheinstructionsoftheS1Pcontentkit(mlbio,m1062988,China).

Mitochondrialmembranepotentialassay
The JC-1 working solution (Solarbio, M8650, China) was prepared
accordingtothemanufacturer’sinstructions.OnemLofJC-1working
solution was added to the cell culture and incubated at 37°C for
20min.Aftertheincubation,thesupernatantwasdiscarded,andthe
cells were washed twice with JC-1 staining buffer. Then, GCs were
coveredwithculturedmediumandmonitoredunderafluorescence
microscope.

Intracellularferrousionfluorescenceassay
The FerroOrange working solution (Dojindo, F374, Japan) was pre-
pared according to the manufacturer’s instructions. GCs culture
mediumwasthenremoved,andGCswerewashed3timeswithserum-
freeGCsculturemedium.FerroOrangeworkingsolution(2μmol/L)
wasaddedintotheGCsandincubatedat37°Cfor30mininanincu-
batorandthen,theGCswereobservedunderaconfocalmicroscope
(LeicaSP8,Germany).

Mitochondrialmorphologyobservation
TheGCssampleswerefixedwithprefixedelectronmicroscopyfixative
(Servicebio, China) and sent to Wuhan Servicebio Co., Ltd (Wuhan,
China)tocompletetheelectronmicroscopescan.

Westernblotanalysis
Total proteins were extracted from ovarian tissues, and the con-
centrationoftotalproteinswasdeterminedbythebicinchoninicacid
method (BCA; Solarbio, China). The lysates were separated by 10%
denaturing SDS-PAGE gels, then transferred to nitrocellulose mem-
branes(BioRad,USA),whichwereincubatedwithprimaryantibodies
anti-β-actin (1:5000, mouse monoclonal, CST, 3700), anti-FSHR
(1:1000, rabbit monoclonal, Protein technology, 22665-1-AP), anti-
AMH(1:1000,rabbitmonoclonal,Abclonal,A8535),anti-Nrf2(1:1000,
rabbitmonoclonal,CST,12721),anti-Keap1(1:500,rabbitmonoclonal,
Abclonal, A17062), anti-Nox4 (1:1000, rabbit monoclonal, Abclonal,
A22149),anti-HO-1(1:1000,rabbitmonoclonal,Wanleibio,WL02400)
andanti-SLC1A4(1:1000,rabbitmonoclonal,Abclonal,A12507)at4°C
overnight.After16h,themembranewasincubatedwithhorseradish
peroxidase (HRP)-conjugated goat anti-rabbit or rabbit anti-mouse
secondaryantibody(Invitrogen,USA)for1hatroomtemperature.The
blots were detected with enhanced chemiluminescence (ECL), and
imageswerequantifiedusingtheSuperSignalWestPicochemilumi-
nescencedetectionsystem.

Quantitativereverse-transcriptionpolymerasechainreaction
(qRT-PCR)
Total RNAs were isolated from ovarian tissues or GCs using Trizol
regent(ThermoFisher,USA)andreversetranscribedintocDNAusing
M-MLVreversetranscriptase(Promega,Shanghai,China).Theprimers
for the target genes were designed as in Supplementary Table 7.
Polymerasechainreactions(PCR)werecarriedoutinaPCRthermal
cycler(ThermoHybaid,USA).QuantitativePCRwasperformedusing
theABI-ViiA7PCRmachine.TheprimersforqPCRarelistedinSup-
plementaryTable7.

Ultrasoundimagingoftheovariesininfertilepatients
Ultrasoundimagesoftheovarieswereobtainedfrominfertilewomen
early in the follicular phase of the menstrual cycle (2–5 days). The
patientwassupinewithamoderatelyfullbladder.Transvaginalultra-
sound uses a high-frequency probe (frequency range: 5–9MHz) to
ensureoptimalresolution.Theovariesweresystematicallyscannedin
multiple planes to assess ovarian volume, follicle count (including
sinus follicle count, AFC), and the presence of ovarian cysts or
abnormalities.Allultrasoundexaminationsareperformedbyexperi-
encedsonographersaccordingtoastandardizedprotocoltoensure
dataconsistencyandreliability.

Sleepdeprivationmousemodel
Balb/cfemalemicewerepurchasedfromJiangsuGemPharmatechCo.,
Ltd(Nanjing,China).Themiceweremaintainedona12-hlight/dark
cycleandhadadlibitumaccesstofoodandwaterattheLaboratory
AnimalCenter oftheInstituteofTranslationalMedicine.Miceaged
6–8weekswererandomlydividedintoanexperimentalgroup(n=20)
and a control group (n=20). The experimental group of mice was
placedinasleepdeprivationdevice,stayup20haday,rest4h,for
4 days, then rest 2 days, according to this schedule for four con-
secutivetimes.Thecontrolmiceatethesamedietastheexperimental
group,exceptthattheywerenotdeprivedofsleep.Theirmortalityand
weightchangesweremeasured.

Statisticalanalysis
The results were presented asaverage value±standarderror ofthe
mean(SEM).Student’sttestwasusedforanalysisbetweenthetwo
groups.One-wayanalysisofvariance(ANOVA)wasusedtocompare
dataamongthreeormoregroups.DifferenceswithaPvalueof<0.05
wereconsideredstatisticallysignificant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study innovatively proposes the mechanism by which the \"serine-S1P-Nrf2 axis inhibits ferroptosis to protect ovarian function,\" with significant translational potential. However, several critical issues require attention.\nMain points:\n1. The clinical trial lacks descriptions of primary endpoints (e.g., AMH recovery rate, pregnancy rate) and the serine administration regimen, reducing its translational value. Additionally, patients’ baseline characteristics (such as CTX dosage, breast cancer stage) are not presented in the main text or supplementary tables. It is recommended to merge Figures 1 and 2, particularly Figure 1e.\n2. In Figure 3K, growth curves for all groups increase before Day 27 but drop sharply between Day 27 and Day 30. Is there an explanation for this phenomenon? Figure 3 omits a \"serine-only supplementation (without CTX)\" control group, making it impossible to rule out serine’s direct effects on the ovaries.\n3. Figure 4 relies solely on transcriptomic inferences and requires protein-level validation. Key markers such as GPX4 protein levels/activity and ACSL4 expression should be detected. Additionally, transmission electron microscopy (TEM) images of ultrastructural changes (e.g., mitochondrial shrinkage, reduced cristae) are needed to fully establish the ferroptosis evidence chain.\n4. Figure 5 does not use inhibitors like Ferrostatin-1 to directly block ferroptosis, weakening the specificity of conclusions. In Figure 6, the claim that serine promotes S1P synthesis is inferred indirectly only via the inhibitor SKI178, without measuring in vivo S1P level changes or performing exogenous S1P rescue experiments. It is recommended to add in vivo S1P detection and rescue experiments using HO-1-KO cells.\n5. Regarding Figure 7: First, the sudden shift to discussing staying up late and sleep deprivation is confusing, and the connection to the study’s core focus—serine inhibiting ovarian ferroptosis to maintain ovarian function—requires clarification. Second, evidence for the mechanism of idiopathic POI is weak, and the causal relationship of \"staying up late\" remains unproven: Sleep deprivation was assessed subjectively via questionnaires (106 responses) without objective monitoring (e.g., actigraphy), and confounding factors (stress, diet) were not controlled, making the assessment insufficient. While the sleep deprivation model shows iron accumulation, the animal model does not validate serine’s core role (e.g., verifying its rescue effect through supplementation), failing to demonstrate that \"serine deficiency is the primary cause.\"\nMinor points:\n1. Please revise the title to reflect that serine inhibits ovarian ferroptosis to maintain ovarian function.\n2. \"POI (premature ovarian insufficiency)\" and \"POF (premature ovarian failure)\" are used interchangeably. It is recommended to unify terminology as \"POI\" in line with the latest clinical guidelines.\n3. Figure 2e: Correlation coefficients (R² and p-values) between serum serine levels and AMH/E₂ need to be added.\n4. All tumor images, as well as immunofluorescence, H&E staining, and electron microscopy images, must include scale bars.\n5. Key literature in the field is missing, including studies on the dual roles of serine and SLC1A4 in tumors, and crosstalk mechanisms between ferroptosis, apoptosis, and senescence in POF.", "reviewer_2": "In this manuscript, the authors investigate how the breast cancer chemotherapeutic cyclophosphamide (CTX) induces premature ovarian failure (POF). They demonstrate that CTX upregulates the amino acid transporter SLC1A4, leading to reduced serum serine levels. Clinical correlations between serum serine and ovarian function markers (E2, AMH) support the idea that serine depletion is linked to ovarian insufficiency. Importantly, dietary serine supplementation in murine breast cancer models prevented CTX-induced POF without impairing CTX’s anti-tumor efficacy.\nMechanistically, the study shows that CTX induces ferroptosis in ovarian granulosa cells (GCs), but not in breast cancer cells. The ferroptosis regulator Hmox1/HO-1 is highlighted as a key mediator via iron overload, with upstream regulation through serine-dependent S1P synthesis and Nrf2/HO-1 signaling. Furthermore, data from women with idiopathic POF revealed similar reductions in serum serine, strengthening the translational relevance.\nThis work is timely, clinically meaningful, and novel. However, while the descriptive evidence is strong, the mechanistic claims remain underdeveloped. In particular, the causal involvement of SLC1A4, HO-1, and S1P signaling is not rigorously validated. With additional functional experiments, the manuscript would make a more compelling mechanistic and translational contribution.\nMajor Comments\nMechanistic validation is insufficient.\nThe central role of SLC1A4 is proposed but not directly tested. Knockdown or knockout approaches (even in vitro, e.g., siRNA in GCs) are critical to establish causality. The authors note plans for SLC1A4 knockout mice in the Discussion, but some functional SLC1A4 loss-of-function data should be included here.\nSimilarly, the roles of S1P synthesis and HO-1 are inferred from correlative data. Silencing/rescue experiments (e.g., Sphk inhibition + serine rescue; HO-1 inhibition or overexpression) would provide much stronger evidence for the proposed pathway.\nComparison of CTX effects in GCs vs. breast cancer cells is incomplete.\nCTX-induced ferroptosis is highlighted as GC-specific, yet the assays used are inconsistent between cell types. For example, ferroptosis in GCs is demonstrated via MDA assay and HO-1 protein, whereas in breast cancer cells it is measured by GPX4, Fth1, and Slc3a2 mRNA. Consistent assays across both systems are necessary for a rigorous comparison.\nIn Figures 5a–b vs. 5g, differences in microscopy magnification and the lack of quantification in 5g weaken the conclusion. Standardized imaging and quantification would strengthen this point.\nGenerality beyond CTX.\nBecause many alkylating agents are known to be gonadotoxic, testing whether other alkylators also reduce serine and promote POF would broaden the clinical impact and generalizability of the model.\nPathway cohesion (S1P → HO-1).\nThe manuscript proposes a serine–S1P–Nrf2/HO-1 axis, but the experimental evidence remains fragmented. Integration of up/downregulation studies (as noted in Major Comment 1) is needed to convincingly link these steps.\nMinor Comments\nSeveral figures are difficult to interpret. For example, the schematic in Figure 4f is overly complex, and some confocal images are too small or unclear. Redesigning these for clarity would help readers.\nThe incidence of ovarian dysfunction after just one CTX dose in patients (25/27 cases) appears strikingly high compared to prior reports. Some discussion of this discrepancy is warranted.\nTerminology should be standardized: “POF” and “POI” are used interchangeably; the authors should clarify and adopt one term consistently.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "This study investigates the protective role of serine against cyclophosphamide (CTX)-induced ovarian damage by inhibiting granulosa cell ferroptosis. The findings propose a novel mechanism involving the serine–S1P–Nrf2–HO-1 pathway, with potential relevance to both chemotherapy-induced and idiopathic ovarian insufficiency. While the topic is of significant interest, several major and minor concerns must be adequately addressed before the manuscript can be considered for publication.\nMajor Comments:\n1. The classification of infertile patients into normal and POI groups based solely on AMH levels requires further justification. It is unclear whether using infertile patients as a control group is appropriate without more comprehensive clinical and endocrine profiling.\n2. Claims regarding ovarian function and follicular reserve cannot be sufficiently supported solely by representative histological sections. Quantitative analysis of follicular counts at different developmental stages (e.g., primordial, primary, secondary, antral) across the entire ovary is essential in Figures 3D, 3O, S4E, 7P, and related panels.\n3. The statement that “granulosa cells are the most crucial functional cells in the ovary” overlooks potential oocyte-specific contributions. A systematic evaluation of oocyte quality—such as meiotic competence, maturation rate, fertilization ability following serine rescue—is needed to fully assess functional recovery.\n4. Many microscopic images (e.g., IF, HE staining in Figs. 3D, 3M, 3O, 3P, 5A, 5G, 6B–C, 6F–G, 6K, 6N, 6T, 6W–6X) lack sufficient magnification, resolution, and/or clear labeling of enlarged areas. Higher-quality images with scale bars and annotated regions of interest are required.\n5. The genes highlighted in Figure 4 appear without clear justification. A brief explanation of the screening strategy or bioinformatic methodology used to select these targets should be provided.\n6. To comprehensively assess CTX-induced alterations, proteomic analysis of granulosa cells following CTX exposure is recommended to complement transcript-level findings.\n7. Figure 5G should be supplemented with in vivo evidence of ferroptosis (e.g., immunohistochemistry or western blotting for key ferroptosis markers in granulosa cells.\n8. The proposed regulatory relationship between Nrf2, SLC1A4, and HO-1 remains unclear. Experimental evidence or cited literature supporting how Nrf2 activation modulates SLC1A4 and HO-1 expression is needed.\nMinor Comments:\n1. The manuscript lacks line numbers, which hinders precise feedback. For example, in the second paragraph of the introduction, “in the reproductive-aged women” should be corrected.\n2. Abbreviations such as E₂, AMH, FSH, and LH should be spelled out upon first use.\n3. The PCNA staining results are not clearly visible and require higher-magnification images.\n4. Ethical approval details and reference numbers for human studies must be explicitly provided.\n5. Axis labels in Figures S1B–S1G are illegible and should be enlarged.\n6. Figure S4F does not sufficiently support its conclusions; quantitative follicular staging counts are necessary.\n7. The high variability between replicates in Figure S4J should be discussed or addressed statistically.\n8. Several figures (e.g., S4E, 3D, 3O, 5A) do not indicate the specific regions from which magnifications are taken. These should be clearly marked.\n9. Cells in Figure S5I are too small to evaluate; a higher-magnification image is recommended.\n10. The signals in Figure S5J (red and green) are not defined in the figure legend. Their identity should be clearly indicated.\n11. Figure legends must consistently report statistical annotations (e.g., ns, *, **, ***, ****) across all panels. For instance, Fig. 1 includes all significance levels, while Fig. 2 omits the definition of “ns”.\n12. Font size in Figure 4F is too small to read, and scale bars appear duplicated or misaligned.\n13. The magnified regions in Fig. 3O and Fig. 6V do not match the corresponding low-magnification images, suggesting potential misrepresentation."}, "author_response": "The point-to-point responses to reviewers’ comments\nReviewer#1\nThis study innovatively proposes the mechanism by which the \"serine-S1P-Nrf2 axis\ninhibits ferroptosis to protect ovarian function,\" with significant translational\npotential. However, several critical issues require attention.\nResponse: We would like to express our sincere gratitude to your recognizing the\ninnovation and translational potential for our study, as well as for your valuable\ncomments.\nMain points:\n1.The clinical trial lacks descriptions of primary endpoints (e.g., AMH recovery rate,\npregnancy rate) and the serine administration regimen, reducing its translational\nvalue. Additionally, patients’ baseline characteristics (such as CTX dosage, breast\ncancer stage) are not presented in the main text or supplementary tables. It is\nrecommended to merge Figures 1 and 2, particularly Figure 1e.\nResponse: Thank you very much for your concern regarding the design of our clinical\ntrial. Both of our clinical study was related to determine the correlation between the\nserum serine levels and ovarian function in the breast cancer patients who were treated\nwith a single chemotherapy session, or in the patients with idiopathic ovarian\ninsufficiency, respectively. In fact, these two clinical studies were not involved in the\ndietary serine supplementation although these clinical studies were to examine similar\nparameters including the serum AMH, E2, and serine levels in patients, supplemented\nby bilateral ovarian ultrasound data. Currently, the most decisive clinical indicator for\nassessing ovarian insufficiency in patients is to examine the serum AMH level.\nThe basic characteristics of the breast cancer patients (Luminal A or Luminal B)\nhave been described in at the lines 702 to 706 of the page 20, and the inclusion and\nexclusion criteria for patients with idiopathic POI were listed at lines 740 to 759 of the\npage 21 in the revised manuscript. Additionally, the relevant supplementary tables have\nbeen appended to the supplementary materials to provide a more intuitive presentation.\nThe CTX dosage for breast cancer patients undergoing chemotherapy was 600 mg per\nbody surface area, as specified in the line 707 of the page 20 of the revised manuscript.\nRegarding your suggestion of merging Figure 1 and Figure 2, we would like to\npresent our perspective on this matter. Figure 1 and Figure 2 represented two distinct\nobservational clinical studies, which were characterized by significant differences of\nthe enrolled populations, despite the similar detection indicators have been examined.\nWe are concerned that merging them may lead to confusing the readers. Furthermore,\nthe overall framework of our article was based on our previous published article\n(DOI:10.1186/s11658-025-00701-1), which demonstrated that there was a close\ncorrelation between the CTX-induced POI and the reduced levels of serum serine.\nBased on the clinical observations, we extended our investigation to determine whether\nthere was also a close correlation between ovarian hypofunction and the reduced serum\nserine levels in the patients with idiopathic POI. These results suggested that CTX-\ninduced POI was related to the ferroptosis in GCs. Consequently, in the present study,\nwe initiated a research program to further explore the underlying mechanism regarding\nCTX-induced ferroptosis.\n2. In Figure 3K, growth curves for all groups increase before Day 27 but drop sharply\nbetween Day 27 and Day 30. Is there an explanation for this phenomenon? Figure 3\nomits a \"serine-only supplementation (without CTX)\" control group, making it\nimpossible to rule out serine’s direct effects on the ovaries.\nResponse: Thank you very much for your questions. In the present study, we\nestablished the mouse breast cancer models by injecting 4T1 cells into the second\nmammary fat pad of mice. Due to individual differences among the mice, there were\nsome variations in the initial tumor sizes. During the grouping process, we ensured that\nthe initial average tumor size was similar across groups, and however, we could not\nguarantee that the tumor growth rates were consistent for each mouse. Throughout the\nstudy, we observed that the tumor growths were relatively slowed in some mice. As the\nnumber of chemotherapy sessions increased, the mice with smaller tumors exhibited a\nsignificant reduction in tumor volume, while the mice with larger tumors experienced\nstagnation in tumor growth. As shown in Figure 3k, starting from day 13, we observed\nthat the average tumor growth rate was significantly slowed down in the mice receiving\nCTX chemotherapy, and reached a plateau phase by day 27, even the slightly declines\nof the tumor volumes in one group from day 27 to day 30 in some mice.\nRegarding the serine-only group was not established in our study, our explanation\nis as follows. Firstly, serine is a non-essential amino acid, and under physiological\nconditions, the concentration of the intracellular serine can be supplemented by the SSP\npathway-mediated serine synthesis if the dietary serine supplementation is not enough,\nindicating that the exogenous serine supplementation might be not necessary in the\nphysiological condition since excess serine may be not absorbed. However, under\npathological conditions, the endogenous serine synthesis pathway may be obstructed,\nand the exogenous serine supplementation may be necessary. Secondly, our study\ndemonstrated that serine can be converted into S1P, thereby inhibiting CTX-induced\nferroptosis in ovarian granulosa cells through suppressing the Keap1/Nrf2/HO-1\nsignaling pathway. Without CTX-induced ferroptosis, the supplemented serine cannot\nexert this function. Finally, from a safety perspective, serine is a non-essential amino\nacid, and is considered very safe with no toxic side effects. Based on these three points,\nwe did not consider to establish a separate serine group in our study.\n3. Figure 4 relies solely on transcriptomic inferences and requires protein-level\nvalidation. Key markers such as GPX4 protein levels/activity and ACSL4 expression\nshould be detected. Additionally, transmission electron microscopy (TEM) images of\nultrastructural changes (e.g., mitochondrial shrinkage, reduced cristae) are needed\nto fully establish the ferroptosis evidence chain.\nResponse: Thank you very much for your suggestion. Accordingly, we did the\nadditional experiments to validate the expressions of the proteins including GPX4. Our\nresults revealed that CTX significantly increased the expression of HO-1, but not GPX4,\nindicating that CTX caused ferroptosis by promoting ferrous ion accumulation in Fig\nS6d, and the relevant textual description has been incorporated into the lines 224 to 227\nof the page 7 of the revised manuscript.\nIn addition, we also examined the ultrastructure of mitochondria through\ntransmission electron microscopy (TEM) to comprehensively establish the evidence of\nferroptosis in GCs. As showed in Figure 6, the TEM image of mitochondria showed\nthat CTX induced mitochondrial atrophy and a reduction in mitochondrial cristae in\nGCs. Furthermore, the results from FerroOrange and mitochondrial membrane\npotential with JC-1 indicated that the ferrous ions were remarkably accumulated in GCs.\nWe believe these results sufficiently demonstrated that CTX induces ferroptosis in GCs.\nFig S6d\n4. Figure 5 does not use inhibitors like Ferrostatin-1 to directly block ferroptosis,\nweakening the specificity of conclusions. In Figure 6, the claim that serine promotes\nS1P synthesis is inferred indirectly only via the inhibitor SKI178, without measuring\nin vivo S1P level changes or performing exogenous S1P rescue experiments. It is\nrecommended to add in vivo S1P detection and rescue experiments using HO-1-KO\ncells.\nResponse: Based on the analysis presented in Figure 4, we hypothesized that CTX\ninduces ferroptosis in GCs, which was attributed to the accumulation of ferrous ions.\nConsequently, in Figure 5, we employed Prussian blue staining to assess the changes\nof ovarian iron ion levels, and the results showed that CTX led to the accumulation of\nferrous ions in GCs, while serine supplementation effectively mitigated the alterations.\nThese results combined with our transcriptomic findings indicated that ferrous ions play\na pivotal role in this process. In addition, our study also demonstrated that CTX-induced\novarian ferroptosis was not related with its antitumor effect. Furthermore, as showed in\nFigure 6, we demonstrated that DFO, a ferroptosis inhibitor, effectively inhibited CTX-\ninduced ferroptosis in GCs, indicating that CTX caused ferroptosis through increasing\nthe accumulation of ferrous ions in GCs.\nSerine serves as the primary precursor for the synthesis of S1P. In vivo, serine\nundergoes a series of enzymatic reactions, ultimately catalyzed by sphingosine kinase\nto produce S1P. SKI178, a sphingosine kinase inhibitor, effectively inhibits all four\nsubtypes of sphingosine kinase within cells, thereby preventing the conversion of serine\nto S1P. In our manuscript, we have cited the relative literature that indicated the crucial\nrole of S1P in ovarian growth, development, and maturation. Additionally, we\nmeasured S1P levels in GCs treated with CTX, and the results showed that CTX\nsignificantly reduced S1P levels in GCs. Therefore, we hypothesized that serine\nprimarily exerts its effects through its conversion to S1P. To validate our hypothesis,\nwe employed SKI178 to inhibit the activity of sphingosine kinase, and our results\nrevealed that the serine remarkably mitigated CTX-induced ferroptosis in GCs, whereas\nthe inhibitory effect on ferroptosis was completely abolished when SKI178 was\nadministered prior to serine, indicating that the added serine primarily was able to\nconvert to S1P. By combining these results with our previous findings, in which we\ndemonstrated that CTX increased the expression of SLC1A4 which led to a deficiency\nin intracellular serine levels in GCs, and therefore, we concluded that serine\nsupplementation compensated for the CTX-induced serine deficiency. Importantly, we\ndid not observe that CTX resulted in the inactivation of sphingosine kinase, suggesting\nthat the addition of serine would inhibit the ferroptosis by increasing the synthesis of\nS1P.\n5. Regarding Figure 7: First, the sudden shift to discussing staying up late and sleep\ndeprivation is confusing, and the connection to the study’s core focus—serine\ninhibiting ovarian ferroptosis to maintain ovarian function—requires clarification.\nSecond, evidence for the mechanism of idiopathic POI is weak, and the causal\nrelationship of \"staying up late\" remains unproven: Sleep deprivation was assessed\nsubjectively via questionnaires (106 responses) without objective monitoring (e.g.,\nactigraphy), and confounding factors (stress, diet) were not controlled, making the\nassessment insufficient. While the sleep deprivation model shows iron accumulation,\nthe animal model does not validate serine’s core role (e.g., verifying its rescue effect\nthrough supplementation), failing to demonstrate that \"serine deficiency is the\nprimary cause.\"\nResponse: We greatly appreciated very much for your question raised from our study\nregarding the idiopathic POI which might affect much more younger women. In our\nprevious and the present studies, we did demonstrate that there was a close correlation\nbetween ovarian hypofunction and the reduced serum serine levels in breast cancer\npatients who were treated with CTX chemotherapy. Later, we were eagerly to know\nwhether that the ovarian hypofunction in the patients with idiopathic POI was related\nto their serum serine levels. Therefore, we initiated the clinical observational\ninvestigation in the patients with the idiopathic POI. Subsequently, we performed a\nmore in-depth study presented in Figure 7, focusing on the patients depicted in Figure\n2, with the aim of determining whether this type of idiopathic POI shares the same\nmechanism of action as CTX-induced POF.\nAt present, the idiopathic POI remains unclear, resulting in a persistent lack of\nprimary treatment options in clinical practice. Long-term clinical analyses have shown\nthat most young patients with idiopathic POI have a history of staying up late.\nFurthermore, our review of the relevant literature suggested that staying up late might\ncontribute to idiopathic POI in women. A cross-sectional study involving 365 women\nfound a negative correlation between estradiol levels and sleep quality1. Additionally,\nanother cross-sectional study of 801 adolescents revealed that the incidence of self-\nreported irregular menstrual cycles was increased with reduced sleep duration2.\nHowever, a definitive explanation for how staying up late affects ovarian function is\nstill lacking. Certainly, a significant challenge in human sleep research is the lack of\nhighly objective assessment methods. Most studies depend on relatively subjective\nevaluations conducted through questionnaires. To minimize confounding factors such\nas diet, we included questions in the questionnaire regarding whether the enrolled\npatients have a long-term high-fat and high-oil diet. Furthermore, since we were not be\nable to obtain the ovarian tissue from women with POI, we established a mouse model\nof sleep deprivation. The horizontal platform method for sleep deprivation in mice was\ncontrollable, and the mice were unable to sleep on the horizontal platform, and this\nmethod was able to eliminate the interference from confounding factors such as stress\nand diet, thereby rendering the results more objective. In our study involving sleep-\ndeprived mice, the accumulation of ferrous ions and the reduced ovarian function were\nobserved. Furthermore, we did observe that there was a reduced serum serine level in\nthe women with idiopathic POI, indicating that the ovarian hypofunction was closely\nrelated to the reduced serum serine in patients with idiopathic POI although we did not\nmeasure the serine levels in the serum or ovaries of these mice. Moreover, in our\nanother ongoing study, we are primarily exploring the relationship between staying up\nlate, SLC1A4, serine, and ovarian function. Through the use of transgenic mice and\nserine detection, we aimed to elucidate in detail how staying up late contributes to\novarian hypofunction via serine.\nMinor points:\n1.Please revise the title to reflect that serine inhibits ovarian ferroptosis to maintain\novarian function.\nResponse: Thank you very much for your valuable suggestion. Accordingly, the\noriginal title of the manuscript was replaced with the “Serine inhibits granulosa cell\nferroptosis to maintain ovarian function” in the revise manuscript.\n2.\"POI (premature ovarian insufficiency)\" and \"POF (premature ovarian failure)\"\nare used interchangeably. It is recommended to unify terminology as \"POI\"\nin line with the latest clinical guidelines.\nResponse: Thank you very much for your suggestions. In response,all the ‘POF’ were\nreplaced with ‘POI’ throughout the manuscript and highlighted these changes in blue\nfont in the revised manuscript. We have also explained the reasons for using POI\ninstead of POF in lines 56-60 of the Main section.\n3.Figure 2e: Correlation coefficients (R² and p-values) between serum serine\nlevels and AMH/E₂ need to be added.\nResponse: Thank you very much for your suggestions. Accordingly, the R² value was\nincorporated into the Figure 2e. Furthermore, by given the fact that estrogen levels were\ninherently unstable and affected by various factors including the menstrual cycle, serum\nAMH levels were predominantly utilized for diagnosing POI in clinical practice.\nConsequently, we have established a linear relationship solely between serum AMH\nand serine.\nFig 2e\n4.All tumor images, as well as immunofluorescence, H&E staining, and electron\nmicroscopy images, must include scale bars.\nResponse: Thank you very much for suggestion. Accordingly, the clearer scale bars\nhave been added into the all images that were missing or unclear scale bars (Fig3, 5,\n6\nand Fig S4, S5, S6) in the revised manuscript.\n5.Key literature in the field is missing, including studies on the dual roles of serine\nand SLC1A4 in tumors, and crosstalk mechanisms between ferroptosis, apoptosis,\nand senescence in POF.\nResponse: We would like to express our gratitude to your insightful suggestions.\nAccordingly, we have supplemented the relevant references and discussions at line 365\non page 12, and at lines 396 to 399 on page 12 in the revised manuscript. Furthermore,\nthe relationship between apoptosis and POI has been thoroughly described and cited in\nthe manuscript at line 211 on page 7 in the revise manuscript.\nReviewer#2 and 3\nIn this manuscript, the authors investigate how the breast cancer chemotherapeutic\ncyclophosphamide (CTX) induces premature ovarian failure (POF). They\ndemonstrate that CTX upregulates the amino acid transporter SLC1A4, leading to\nreduced serum serine levels. Clinical correlations between serum serine and ovarian\nfunction markers (E2, AMH) support the idea that serine depletion is linked to\novarian insufficiency. Importantly, dietary serine supplementation in murine breast\ncancer models prevented CTX-induced POF without impairing CTX’s anti-tumor\nefficacy.\nMechanistically, the study shows that CTX induces ferroptosis in ovarian\ngranulosa cells (GCs), but not in breast cancer cells. The ferroptosis regulator\nHmox1/HO-1 is highlighted as a key mediator via iron overload, with upstream\nregulation through serine-dependent S1P synthesis and Nrf2/HO-1 signaling.\nFurthermore, data from women with idiopathic POF revealed similar reductions in\nserum serine, strengthening the translational relevance.\nThis work is timely, clinically meaningful, and novel. However, while the\ndescriptive evidence is strong, the mechanistic claims remain underdeveloped. In\nparticular, the causal involvement of SLC1A4, HO-1, and S1P signaling is not\nrigorously validated. With additional functional experiments, the manuscript would\nmake a more compelling mechanistic and translational contribution.\nResponse: We sincerely appreciated very much for your comments on our work, and\nwe will provide point-by-point responses to address all of your concerns.\nMajor Comments:\n1. Mechanistic validation is insufficient\nThe central role of SLC1A4 is proposed but not directly tested. Knockdown or\nknockout approaches (even in vitro, e.g., siRNA in GCs) are critical to establish\ncausality. The authors note plans for SLC1A4 knockout mice in the Discussion, but\nsome functional SLC1A4 loss-of-function data should be included here.\nResponse: Thank you very much for your comments and suggestions. SLC1A4 is a\ncrucial transporter for serine exclusion in cells. We are preparing the conventional and\novarian granulosa cell-specific SLC1A4 knockout mice to investigate the role of\nSLC1A4 in serine transport in primary ovarian insufficiency (POI). However, currently,\nthe relevant mice have not yet been obtained. Certainly, these knockout mice are critical\nfor establishing the role of SLC1A4 in serine transport in cells, especially in ovaries,\nsince currently, very few studies were involved in SLC1A4.\nIn our previous article, we utilized siRNA technology to knockdown the\nexpression of SLC1A4 in GCs and conducted subsequent validation. Our results\nshowed that the knockdown of SLC1A4 effectively inhibited CTX-induced death in\nGCs, and the serine supplementation also had a comparable protective effect. In the\npresent study, we further validated the observation that CTX promoted the expression\nof SLC1A4 by detecting its levels through Western blot analysis. Based on existing\nliterature, this enhancement may be attributed to the role of Nrf2 in promoting SLC1A4\nexpression. Furthermore, in our ongoing study, we will focus on the mechanisms of\nSLC1A4 in POI with the transgenic animal models.\nOnce again, we sincerely appreciated very much your suggestions. In the ongoing\nstudy, we will provide a comprehensive explanation for the central role and molecular\nmechanisms of SLC1A4 in POI.\nDOI:10.1186/s11658-025-00701-1\nD, E Detection of knockdown efficiency of Slc1a4 on GCs after siRNA transfection by qPCR and Western blotting.\nF The relative intensity of Slc1a4 in Western blotting. G Live–dead staining was used to detect death in the\nnormal+NC group, CTX+NC group, CTX+siRNA group, and CTX+serine group. H The apoptotic ratios of\nGCs in each group were detected by flow cytometry. I The apoptosis of GCs was quantitatively detected by\nflow cytometry\n2.Similarly, the roles of S1P synthesis and HO-1 are inferred from correlative data.\nSilencing/rescue experiments (e.g., Sphk inhibition + serine rescue; HO-1 inhibition\nor overexpression) would provide much stronger evidence for the proposed pathway.\nResponse: Thank you very much for your suggestion. The relevant experiments have\nbeen completed, and the synthesis of sphingosine-1-phosphate (S1P) is primarily\nfacilitated by serine as the raw material, leading to the formation of S1P through the\naction of sphingosine kinase. We specifically measured the intracellular sphingosine\nkinase after the application of SKI178, a sphingosine kinase inhibitor, using an S1P\nassay kit. Our results showed that through ROS detection, ferrous ion detection,\nmitochondrial electron microscopy, JC-1 assays, and lipid peroxidation assays, the\naddition of serine did not mitigate CTX-induced ferroptosis in GCs (Fig. 6j-r and S6i)\nwhen sphingosine kinase activity is inhibited by SKI178.\nFig S6i\nFig 6j-r\nIn addition, in the present study, we successfully inhibited HO-1 enzyme activity\nusing Znpp, a specific inhibitor of HO-1 through an HO-1 enzyme activity assay kit.\nUpon confirming the inhibition of HO-1 activity, we employed ROS assays, ferrous ion\nprobes, JC-1, lipid peroxidation probes, and mitochondrial electron microscopy to\ndemonstrate that the inhibition of HO-1 significantly suppresses CTX-induced\nferroptosis in GCs. These results indicated that the upregulation of CTX-induced HO-\n1 expression led to the accumulation of ferrous ions, suggesting that HO-1 was a\nprimary contributor to chemotherapy-induced POI (Fig. 6s-x and S6n).\nFig S6n\nFig 6s-x\n3.Comparison of CTX effects in GCs vs. breast cancer cells is incomplete.\nCTX-induced ferroptosis is highlighted as GC-specific, yet the assays used are\ninconsistent between cell types. For example, ferroptosis in GCs is demonstrated via\nMDA assay and HO-1 protein, whereas in breast cancer cells it is measured by GPX4,\nFth1, and Slc3a2 mRNA. Consistent assays across both systems are necessary for a\nrigorous comparison.\nResponse: Thank you very much for your question regarding the similar assays should\nbe used for determining CTX-induced ferroptosis in both GCs and tumor cells. In the\npresent study, our Prussian blue staining demonstrated that CTX led to a significant\naccumulation of ferrous ions in GCs, but not in tumor cells. Consequently, based on\nyour suggestion, we conducted the additional experiments for measuring the\nconcentrations of MDA and ferrous ions in the tumor tissue, and the results showed that\nCTX did not lead to the accumulation of ferrous ions or MDA formation in tumor\ntissues (Fig. 5g, h).\nFig. 5g and h\n4.In Figures 5a–b vs. 5g, differences in microscopy magnification and the lack of\nquantification in 5g weaken the conclusion. Standardized imaging and\nquantification would strengthen this point.\nResponse: Thank you very much for your suggestion. Accordingly, we have revised\nFigure 5f to align its magnification with that of Figure 5a, thereby facilitating a more\neffective comparison.\nPrussian blue staining is the most intuitive method for detecting ferrous ions in\nhistology. Our results revealed that there were the distinct blue-stained areas in the\novarian tissue, whereas no distinct blue-stained areas were observed in the tumor tissue,\nindicating that CTX caused a significant accumulation of ferrous ions in the ovary, but\nnot in the tumor tissues. Consequently, due to the absence of distinct blue-stained areas\nindicating ferrous ion accumulation in the tumor tissue, we were unable to conduct a\nquantitative analysis of the blue-stained areas. Furthermore, consistent with the results\nobtained from Prussian blue staining, the mRNA expressions of the key genes such as\nHmox 1 associated with ferroptosis were not altered.\nFig 5f\n5.Generality beyond CTX.\nBecause many alkylating agents are known to be gonadotoxic, testing whether other\nalkylators also reduce serine and promote POF would broaden the clinical impact\nand generalizability of the model.\nResponse: Thank you very much for your suggestion. We concur that expanding the\nmodel is essential for future clinical applications with enhancing its general adaptability.\nThe decision to utilize cyclophosphamide (CTX) in this study was grounded in several\nkey considerations. First, CTX is widely recognized as a classic agent for constructing\na mouse model of POI3, 4, 5. Second, CTX serves as a first-line agent for breast cancer,\ncharacterized by its cost-effectiveness and efficacy, which have contributed to its\nextensive clinical application6. However, CTX is associated with significant\nreproductive toxicity, increasing the risk of infertility in breast cancer patients\nundergoing treatment by 4 to 5 times. Furthermore, despite its prevalent use, the\nspecific underlying mechanisms of its reproductive toxicity remain poorly understood,\nresulting in a lack of effective protective strategies. Certainly, we totally agree with you\nto test whether serine protects against the reproductive toxicity by the other alkylators,\nand we are also eager to know whether there is an elevated expression of SLC1A4 in\nGCs by other alkylators. Looking ahead, we are planning to expand our research with\na variety of alkylating agents to determine the protective role of serine in the\nreproductive toxicity associated with these agents, as well as its potential impacts on\nother organs across different tumor types. We sincerely appreciated very much for your\nsuggestion, and we are committed to pursue the related research initiatives in the future.\n6.Pathway cohesion (S1P → HO-1).\nThe manuscript proposes a serine–S1P–Nrf2/HO-1 axis, but the experimental\nevidence remains fragmented. Integration of up/downregulation studies (as noted in\nMajor Comment 1) is needed to convincingly link these steps\nResponse: Thank you very much for your concern and insightful suggestions. We\nwould like to provide a detailed explanation for the specific molecular mechanisms of\nserine protecting CTX-induced ferroptosis in GCs. In our previous article, we\ndemonstrated that CTX significantly upregulated SLC1A4 in GCs, whereas the\nknockdown of SLC1A4 by siRNA or by adding serine remarkably inhibited CTX-\ninduced GC death7. In the present study, we aimed to elucidate the specific molecular\nmechanisms of CTX-induced POI and the protective effects of serine.\nIn the present study, we first established the correlation between the reduced serum\nserine levels and ovarian hypofunction in patients with chemotherapy-induced POI and\nidiopathic POI through two observational clinical studies. Furthermore, by dietary\nserine supplementation via gavage in mice, we observed that serine supplementation\neffectively mitigated symptoms of CTX-induced POI without compromising the\nchemotherapeutic efficacy of CTX against breast cancer. Subsequent transcriptomic\nanalysis suggested that the underlying mechanism might be involved in ferroptosis\ninduced by the accumulation of ferrous ions mediated by HO-1 in GCs. Furthermore,\nwe observed that CTX resulted in significant ferrous ion accumulation in the ovaries,\nbut not in breast cancer tissues. In our in vitro experiments, by utilizing the ferrous ion\nchelator DFO and the ferroptosis inducer Erastin, we further demonstrated that serine\ninhibited CTX-induced ferroptosis resulting from ferrous ion accumulation through\nmodulating the Keap1/Nrf2/HO-1 signaling pathway.\nIt has been reported that S1P is essential for the normal proliferation and\ndevelopment of ovaries, and serine serves as a significant precursor for S1P synthesis8,\n9, in which the conversion of serine to S1P necessitates the activation of sphingosine\nkinase. Therefore, by using SKI178, a sphingosine kinase-specific inhibitor, we\nobserved that the conversion of serine to S1P was obstructed, and the addition of serine\nno longer inhibited CTX-induced ferroptosis in GCs. Furthermore, through\nimmunofluorescence analysis, we found that the conversion of serine to S1P\nsignificantly inhibited the nuclear translocation of Nrf2, subsequently suppressed the\nexpression of HO-1. By employing Znpp. By employing Znpp, an inhibitor of HO-1, we further\ndemonstrated that CTX-induced the accumulation of ferrous ions/ferroptosis was\nalmost completely abolished by inhibition of HO-1. Taken together, our data\ndemonstrated that the serine–S1P–Nrf2/HO-1 axis played a pivotal role in CTX-\ninduced ferroptosis in GCs.\nIn addition, we are currently preparing SLC1A4 knockout mice. In the future\nresearch projects, we will utilize the transgenic mice to further investigate the specific\nmechanisms associated with SLC1A4.\nGraphical Abstract\nMinor Comments:\n1.Several figures are difficult to interpret. For example, the schematic in Figure 4f\nis overly complex, and some confocal images are too small or unclear. Redesigning\nthese for clarity would help readers.\nResponse: Thank you very much for your advice. Figure 4f presents a schematic\ndiagram of the ferroptosis-related signaling pathway derived from KEGG analysis. The\nred sections of the figure represent the pathways with high relevance and significant\ncorrelation. The results depicted in the figure suggested that ferroptosis induced by\nCTX was closely associated with the accumulation of ferrous ions mediated by HO-1.\nSubsequent experiments focused on the ferroptosis triggered by the accumulation of\nferrous ions. However, the clarity of Figure 4f was insufficient, and some confocal\nimages were too small, primarily due to the extensive experimental data and limited\nspace in the manuscript. The images included in the uploaded manuscript were\ncompressed, leading to reduced clarity. If our article will be accepted, we will provide\nthe original highest resolution images, which will maintain clarity when magnified.\nThank you for your understanding.\nFig 4g\n2.The incidence of ovarian dysfunction after just one CTX dose in patients (25/27\ncases) appears strikingly high compared to prior reports. Some discussion of this\ndiscrepancy is warranted.\nResponse: Thank you very much for your concern and suggestion. In our study, we\ndid observe that 25 out of 27 patients exhibited a downward trend in serum estrogen\nlevels which did not imply to develop the significant symptoms of POI in these patients.\nOur clinical investigation revealed that that nearly all breast cancer patients experienced\na decline in sex hormone levels following a single chemotherapy session of CTX.\nThroughout the course of four chemotherapy sessions, most patients encounter\namenorrhea, and some of them were spontaneously recovered after completing\nchemotherapy. Among these patients, 40%-50% develop POI. The corresponding\ndiscussion has been addressed in the discussion section of the revised manuscript.\n3.Terminology should be standardized: “POF” and “POI” are used\ninterchangeably; the authors should clarify and adopt one term consistently.\nResponse: Thank you very much for your suggestion. Accordingly, all the “POF”\nhave been replaced with “POI” in the revised manuscript.\nReviewer#4\nThis study investigates the protective role of serine against cyclophosphamide (CTX)-\ninduced ovarian damage by inhibiting granulosa cell ferroptosis. The findings\npropose a novel mechanism involving the serine–S1P–Nrf2–HO-1 pathway, with for\nrelevance to both chemotherapy-induced and idiopathic ovarian insufficiency. While\nthe topic is of significant interest, several major and minor concerns must be\nadequately addressed before the manuscript can be considered for publication.\nResponse: We greatly appreciated very much for your recognition to our study. We are\ncommitted to address your concerns raised from our manuscript in order to further\nrefine and enhance our study.\nMajor Comments:\n1.The classification of infertile patients into normal and POI groups based solely on\nAMH levels requires further justification. It is unclear whether using infertile\npatients as a control group is appropriate without more comprehensive clinical and\nendocrine profiling.\nResponse: Thank you very much for your concern. In clinical practice, physicians\nprimarily determine the POI based on their serum AMH levels, supplemented by the\nassessment of antral follicle counts in bilateral ovaries. The serum AMH level is\nconsidered the gold standard for diagnosing POI. Specifically, the patients with a serum\nAMH level less than 1.3 ng/mL are able to be diagnosed with POI.\nWe selected infertility patients as the enrollment population based on several\nconsiderations. Women with POI represent a specific subset of infertile patients,\ncharacterized by infertility due to diminished ovarian function. However, within the\nbroader infertile patients, there exists a group with normal ovarian function whose\ninability to conceive may be attributed to the factors related to their partners or other\nuterine conditions. Through the evaluation of physicians to these patients by\ndetermining their sex hormone levels and antral follicle count, the patients who exhibit\nnormal sex hormone secretion and a robust ovarian reserve might be suitable as a\ncontrol group for patients with POI.\n2.Claims regarding ovarian function and follicular reserve cannot be sufficiently\nsupported solely by representative histological sections. Quantitative analysis of\nfollicular counts at different developmental stages (e.g., primordial, primary,\nsecondary, antral) across the entire ovary is essential in Figures 3D, 3O, S4E, 7P,\nand related panels.\nResponse: Thank you very much for your suggestions. Accordingly, we have re-\nperform the statistical analyses of the number of follicles at various stages in the\nrevised manuscript.\nFig S3c\nFig S4h\nFig S5g\n3.The statement that “granulosa cells are the most crucial functional cells in the\novary” overlooks potential oocyte-specific contributions. A systematic evaluation of\noocyte quality—such as meiotic competence, maturation rate, fertilization ability\nfollowing serine rescue—is needed to fully assess functional recovery.\nResponse: We appreciated very much for your suggestions. Certainly, our previous\nstatement lacked sufficient rigor. Consequently, we have revised the wording on line\n174-176, page 6 of the revised manuscript to enhance clarity and precision.\nWe fully agree with your perspective that oocytes are of great importance to\nreproductive system diseases. Our study primarily investigated the POI caused by CTX-\ninduced ferroptosis in GCs. We aimed to elucidate the precise molecular mechanisms\nof CTX-induced POI and the protective role of serine through clinical analysis,\ntranscriptomic studies, and both in vivo and in vitro experiments. Following our initial\narticle, which revealed that CTX primarily damages GCs in the ovary, our current\nresearch mainly focused on GCs, yielding notable progress and discoveries.\nAdditionally, your suggestion has proven to be highly valuable and has inspired us.\nConsidering GCs and oocytes as two critical cell types within the ovary, we are\nparticularly interested in whether, under pathological conditions, GCs might secrete\nmicro-substances, including miRNAs, that could alter the communication dynamics\nbetween GCs and oocytes. In the future studies, we will explore the differences in\ncellular communication between GCs and oocytes under both normal and pathological\nconditions. Through reviewing the relevant literatures which indicated that serine\nmetabolism plays a significant role in oocytes10, 11, certainly, we should address the role\nof serine metabolism in oocytes in our future studies.\n4.Many microscopic images (e.g., IF, HE staining in Figs. 3D, 3M, 3O, 3P, 5A, 5G,\n6B–C, 6F–G, 6K, 6N, 6T, 6W–6X) lack sufficient magnification, resolution,\nand/or clear labeling of enlarged areas. Higher-quality images with scale bars and\nannotated regions of interest are required.\nResponse: Thank you very much for your constructive suggestions. In responses, we\nhave relabeled the previously unclear scale bars, providing detailed information within\nthe corresponding figure legends. Additionally, we have marked the magnified areas\nwith boxes on the images for clarity (Fig. 3d, 3o). For smaller images that were difficult\nto observe clearly, we have reorganized them by enlarging their size (Fig.5a).\nDue to the layout constraints imposed by the article's images and the substantial\nvolume of data, we had to reduce the size of certain images to maintain logical\ncoherence and aesthetic appeal. The current version of our upload system compresses\nimage clarity when generating PDFs. In the future, upon acceptance of the article, we\nwill provide the images with the highest resolution.\n5.The genes highlighted in Figure 4 appear without clear justification. A brief\nexplanation of the screening strategy or bioinformatic methodology used to select\nthese targets should be provided.\nResponse: Thank you very much for your insightful questions. To better address your\ninquiries, we have reformatted Figure 4 and added a Venn diagram (Page7, lines 217-\n231). Initially, we plotted the top 50 differentially expressed genes in GCs induced by\nCTX. We observed that among the upregulated genes, several were closely associated\nwith ferroptosis with a high rank. Subsequently, we conducted GSEA and KEGG\nanalyses on the upregulated genes, and found that the differentially expressed genes in\nthe top 50 were particularly enriched in the ferroptosis signaling pathway. Using Venn\nenrichment analysis, we compared genes from the ferroptosis database with the\nupregulated differentially expressed genes and identified four distinct genes. The\ndifferential expression of Hmox-1 was extremely significant, and the enrichment map\nof the ferroptosis pathway showed a notable association with the pathway of ferrous\nion accumulation induced by HO-1. We supplemented the results with Western blot\nanalysis, which clearly indicated a particularly significant increase in HO-1 expression\nfollowing the use of CTX. Based on this, we hypothesized that ferroptosis induced by\nHO-1 might be the primary cause, in which the subsequent experiments were performed\nfor validating our hypothesis.\nFig 4e Fig S6d\n6.To comprehensively assess CTX-induced alterations, proteomic analysis of\ngranulosa cells following CTX exposure is recommended to complement transcript-\nlevel findings.\nResponse: We would like to express our gratitude to your insightful suggestions.\nInitially, we selected transcriptomics for our experiments due to several compelling\nreasons. Firstly, transcriptomic technology has reached a higher level of maturity and\noffers superior sensitivity. Secondly, it enabled the detection of all transcripts, including\nboth mRNA and non-coding RNA, in a single run, thereby providing a more\ncomprehensive overview of gene expression. Furthermore, the alterations at the\ntranscriptional levels occur more rapidly than those at the protein level, making it a\nmore immediate reflection of cellular responses to external stimuli. Additionally,\ntranscriptomics is currently more cost-effective compared to proteomic methods.\nNonetheless, we recognize the importance of the proteomic analysis suggested by the\nreviewer. To address our validation being limited to mRNA levels through\ntranscriptomics, we have supplemented our study with an examination of the protein\nHO-1, which is regulated by the key gene Hmox1, both before and after the\nadministration of CTX. Our results showed that there was a significant upregulation of\nHO-1 expression following CTX treatment (Fig. S6d). In our subsequent in vitro\nexperiments, we have consistently validated the protein expression levels of HO-1\nalong with related ferroptosis assays, further confirming the accuracy of our\ntranscriptional findings (Fig. 6h, o). We greatly value your suggestions and plan to\ngradually incorporate proteomic analysis in our future study.\n7.Figure 5G should be supplemented with in vivo evidence of ferroptosis (e.g.,\nimmunohistochemistry or western blotting for key ferroptosis markers in granulosa\ncells.\nResponse: Thank you very much for your suggestions. Figure 5g illustrates the\nPrussian blue staining of tumor tissue, which was employed to detect the accumulation\nof ferrous ions within the tumor. The results indicated that CTX did not promote the\naccumulation of ferrous ions in the tumor tissue. We recognized that relying solely on\nPrussian blue staining and qPCR was insufficient to substantiate our conclusion.\nConsequently, we supplemented our analysis with measurements of MDA and ferrous\nion concentrations in the tumor tissue. The results further confirmed that CTX did not\nlead to an increase in MDA or ferrous ions. By incorporating these additional\nmeasurements, we demonstrated that CTX did not induce ferroptosis in the tumor tissue.\nFig. 5g and h\n8.The proposed regulatory relationship between Nrf2, SLC1A4, and HO-1 remains\nunclear. Experimental evidence or cited literature supporting how Nrf2 activation\nmodulates SLC1A4 and HO-1 expression is needed.\nResponse: Thanks. In the present study, we demonstrated that serine was converted\ninto S1P, which subsequently regulates the Keap1/Nrf2 signaling pathway. The\nincreased S1P inhibited CTX-induced increase in Nrf2 nuclear translocation and\nsuppressed the transcription and translation of HO-1, thereby reducing HO-1\nexpression. Previous literature has reported that Nrf2 promoted the expression of HO-\n1, and we cited the relevant studies to support our conclusion12, 13, 14. Additionally, our\nimmunofluorescence results also demonstrated that CTX enhanced Nrf2 nuclear\ntranslocation, while the addition of serine inhibited this translocation.\nIn the present study, we primarily focused on how serine inhibits CTX-induced\nferroptosis, and the role of SLC1A4 will be investigated in our future project. However,\nour Western blot analysis revealed that CTX promoted the expression and nuclear\ntranslocation of Nrf2, and also significantly increased the expression of SLC1A4. Upon\nthe addition of serine, the levels of both Nrf2 and SLC1A4 were decreased,\ndemonstrating a consistent change in their protein levels. Previously, due to limited\nresearch on SLC1A4, we lacked sufficient literature evidence to support our findings.\nHowever, a recent study has indicated that Nrf2 promoted the expression of SLC1A4\n(ASCT1), which aligns with our results15.\nIn the future, we will explore the specific molecular mechanisms associated with\nSLC1A4, aiming to address the existing gaps in the understanding of SLC1A4 across\nvarious disease fields. We appreciated very much for your valuable suggestions again.\nMinor Comments:\n1.The manuscript lacks line numbers, which hinders precise feedback. For example,\nin the second paragraph of the introduction, “in the reproductive-aged women”\nshould be corrected.\nResponse: Thank you very much for your valuable suggestions. Accordingly, the line\nnumbers of the manuscript were added in the revised manuscript. In response to the\ninappropriate terminology you highlighted, we have made the necessary revisions by\nchanging all instances of “in the reproductive-aged women” to the “women of\nchildbearing age” at the line 29 on page 1, line 42 on page 2, lines 85 and 98 on page\n3, line 409 on page 13, and line 1293 on page 45 in the revised manuscript.\n2.Abbreviations such as E₂, AMH, FSH, and LH should be spelled out upon first\nuse.\nResponse: Thank you very much for your suggestion. Accordingly, we have annotated\nthe full names of E2, AMH, FSH, and LH when they have been first appeared at line\n96 of page 3, line 149 of page 5, and line 735 of page 21 in the revised manuscript,\nrespectively.\n3.The PCNA staining results are not clearly visible and require higher-magnification\nimages.\nResponse: Thank you very much for your suggestion. We regretted that we were unable\nto upload higher magnification PCNA images due to the limitation of the size of\nuploading files. Therefore, should the manuscript be accepted, we will upload high-\nresolution images for publication. We sincerely apologize for the inconvenience. To\nensure better clarity for your viewing, we have provided this image to you separately.\nNormal CTX CTX+Serine 50 mg/kg CTX+Serine 500 mg/kg\n4.Ethical approval details and reference numbers for human studies must be\nexplicitly provided.\nResponse: Thank you very much for your suggestion. Both clinical studies were\nobservational, and the approval numbers were IIT2024-329 and K-kt2025001,\nrespectively. The ethical documentations were approved by the respective hospitals,\nand uploaded in the supplementary files of the revised manuscript.\n5.Axis labels in Figures S1B–S1G are illegible and should be enlarged.\nResponse: Thank you very much for your comments, and accordingly, we have\nredrawn the axes to make them clearer.\nFig S1B-1G\n6.Figure S4F does not sufficiently support its conclusions; quantitative follicular\nstaging counts are necessary.\nResponse: We sincerely appreciated your suggestion. Accordingly, we have conducted\nthe follicle counting on the HE images.\nFig S4h\n7.The high variability between replicates in Figure S4J should be discussed or\naddressed statistically.\nResponse: Thank you very much for your insightful question. The variations observed\namong the groups may be attributed to individual differences among the mice. However,\nwe also conducted a statistical analysis on the strips, and the results showed that the\noverall trend is clearly discernible although there were the significant variations within\ncertain groups.\n8.Several figures (e.g., S4E, 3D, 3O, 5A) do not indicate the specific regions from\nwhich magnifications are taken. These should be clearly marked.\nResponse: Thank you very much for your valuable suggestions. We have annotated the\nmagnified areas in the corresponding images in the revised manuscript.\nFig 3d\nFig 3o\nFig 5a\nFig S4e\n9.Cells in Figure S5I are too small to evaluate; a higher-magnification image is\nrecommended.\nResponse: Thank you very much your suggestion. Accordingly, we have revised the\nlayout of the Figure to enhance the clarity of the images which was enclosed, and if the\narticle is accepted, we will ensure to upload the high-resolution images for publication.\nFig S5i\n10.The signals in Figure S5J (red and green) are not defined in the figure legend.\nTheir identity should be clearly indicated.\nResponse: We have annotated the significance of the green and red fluorescent signals\nin Figure S5J according to your suggestions. Additionally, we have included\ncorresponding notes in the figure legends of the Extended Data (Lines 123 to 125) in\nthe revised manuscript.\nFig S5j\n11.Figure legends must consistently report statistical annotations (e.g., ns, *, **, ***,\n****) across all panels. For instance, Fig. 1 includes all significance levels, while\nFig. 2 omits the definition of “ns”.\nResponse: We appreciate very much for your reminder. We have thoroughly checked\nthe entire text and made the necessary corrections. Thank you very much.\n12.Font size in Figure 4F is too small to read, and scale bars appear duplicated or\nmisaligned.\nResponse: We appreciated very much for your reminder. We have reformatted Figure\n4 and corrected the misaligned scale bar in Figure 4F. The current resolution of Figure\n4F was not enough due to the limitation of uploading images, and the high-resolution\nimages were enclosed for your reference. If the manuscript is accepted the high-\nresolution images will be uploaded. Thank you.\nFig 4g\n13.The magnified regions in Fig. 3O and Fig. 6V do not match the corresponding\nlow-magnification images, suggesting potential misrepresentation.\nResponse: We appreciated very much for your careful observation. Upon verification,\nwe have corrected the error in Figure 3O, and we also confirmed that Figure 6V was\nnot misused.\nFig 3O\nReference\n1. Beroukhim G, Esencan E, Seifer DB. Impact of sleep patterns\nupon female neuroendocrinology and reproductive outcomes: a comprehensive\nreview. Reprod Biol Endocrinol 20, 16 (2022).\n2. Nam GE, Han K, Lee G. Association between sleep duration and menstrual cycle\nirregularity in Korean female adolescents. Sleep Med 35, 62-66 (2017).\n3. Qin X, et al. TrkB agonist antibody ameliorates fertility deficits in aged\nand cyclophosphamide-induced premature ovarian failure model mice. Nat Commun\n13, 914 (2022).\n4. Meng Y, et al. Therapeutic effects of curculigoside on cyclophosphamide-\ninduced premature ovarian failure in mice. Climacteric 27, 421-432 (2024).\n5. Karaoglan O, Kuyucu Y, Saker D, Daglioglu G, Tap O. An Investigation of the\nEffects of Melatonin and Vitamin D on the Ovaries of a Rat Model of Premature\nOvarian Failure Induced by Cyclophosphamide. Int J Mol Sci 26, (2025).\n6. Munzone E, Colleoni M. Clinical overview of metronomic chemotherapy in breast\ncancer. Nat Rev Clin Oncol 12, 631-644 (2015).\n7. Gu HC, et al. Human urine stem cells protect against cyclophosphamide-induced\npremature ovarian failure by inhibiting SLC1A4-mediated outflux of\nintracellular serine in ovarian granulosa cells. Cell Mol Biol Lett 30, 21\n(2025).\n8. Wang F, et al. Roles of follicle stimulating hormone and sphingosine 1-\nphosphate co-administered in the process in mouse ovarian vitrification and\ntransplantation. J Ovarian Res 16, 173 (2023).\n9. Di Paolo A, et al. Pathogenic Role of the Sphingosine 1-Phosphate (S1P)\nPathway in Common Gynecologic Disorders (GDs): A Possible Novel Therapeutic\nTarget. Int J Mol Sci 23, (2022).\n10. He H, et al. Autophagy dictates PHGDH-mediated serine metabolism in a timely\nmanner to support oocyte development. Autophagy, 1-20 (2025).\n11. Zhang H, McClatchie T, Baltz JM. l-Serine transport in growing and maturing\nmouse oocytes. J Cell Physiol 235, 8585-8600 (2020).\n12. Li H, Yuan J, Dong W, Yang C, Lu L, Wang Y. Bavachin stimulates ferroptosis\nand reduces malignant phenotype progression of hepatocellular carcinoma cells\nby inducing lipid peroxidation by modulation of the Nrf2/HO-1 signaling\npathway. Am J Transl Res 16, 6925-6934 (2024).\n13. Chen C, et al. Na-Cl Cotransporter (SLC12A3) Inhibition Exacerbates Age-\nRelated Macular Degeneration Via the Nrf2/HO-1 Ferroptosis Pathway. Invest\nOphthalmol Vis Sci 66, 24 (2025).\n14. Yu X, et al. C7ORF41 Alleviates Ferroptosis Via the Keap1/Nrf2/HO-1 Axis in\nEndotoxin-Associated Acute Kidney Injury. Inflammation, (2025).\n15. Kurniawan H, et al. Glutathione Restricts Serine Metabolism to Preserve\nRegulatory T Cell Function. Cell Metab 31, 920-936 e927 (2020)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have thoroughly addressed the previous concerns and made substantial revisions in response to all reviewer comments. These updates have significantly enhanced the quality, clarity, and scientific depth of the manuscript, which now meets the standards for minor revision in Nature Communications.\nIn addition, the following recent study might be relevant and worth citing:\ndoi: 10.1016/j.cmet.2020.03.004.\ndoi: 10.1016/j.cmet.2024.10.019.\ndoi: 10.1038/s42255-020-00329-9", "reviewer_2": "Thank you to the authors for their thoughtful revision. The additional experiments and clarifications provided are appreciated.\nPlease find below specific comments regarding the authors’ responses.\n1. Mechanistic Validation Remains Insufficient\nThe authors reference their previously published article, which demonstrates SLC1A4 silencing and its effect on CTX-induced granulosa cell death. This prior work is appreciated and provides supporting evidence that SLC1A4 contributes to CTX-induced granulosa-cell death. However, because this mechanistic validation lies outside the current dataset, it is recommended that the authors incorporate a more detailed discussion of their prior findings in the main text.\nIn addition to this, the experiments that the authors highlight from their Supplementary data, which examine S1P synthesis and HO-1 function are valuable; however, they do not yet establish causality, which was the central concern. These experiments rely exclusively on chemical inhibition without complementary genetic manipulation or rescue approaches. Specifically, for S1P synthesis, the proposed link between serine availability and S1P production remains unclear, as serine supports de novo sphingolipid synthesis rather than directly serving as an S1P precursor. Inclusion of a direct rescue experiment (e.g., exogenous S1P supplementation) would substantially strengthen the mechanistic argument.\nRegarding HO-1, while ZnPP inhibition is informative, ZnPP has known off-target effects; without supporting overexpression or genetic knockdown data, the conclusion that HO-1 is the primary driver of CTX-induced ferroptosis or POI remains speculative. The authors are therefore encouraged to temper causal statements and present these findings as supportive rather than definitive evidence of this pathway’s involvement.\n2. Standardized Imaging and Quantification\nThe rationale for not quantifying Prussian blue staining remains unconvincing. Prussian blue detects ferric (Fe³⁺), not ferrous (Fe²⁺) ions, the latter being directly involved in driving ferroptosis via the Fenton reaction. Accordingly, the interpretation of “ferrous ion accumulation” should be corrected. Moreover, the absence of visible staining in tumor sections does not preclude quantification; semi-quantitative image analysis could still reveal meaningful differences (e.g., near-zero signal relative to ovarian tissue).\nThe authors point to Hmox-1 mRNA expression changes as supporting evidence. However, Hmox-1 represents an indirect marker of labile iron accumulation. Therefore, at a minimum, the authors should either provide such image-based quantification or revise the discussion to frame these findings as qualitative observations rather than quantitative conclusions regarding iron accumulation and ferroptosis.\nOther concerns have been adequately addressed.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors have addressed most of my concerns, with the exception of the following issue:\nIn Figures S3C, S4H, and S5G, it appears that the authors quantified the number of follicles at different developmental stages based on a single ovarian section. This approach is not accurate, as a reliable quantification should involve counting follicles across the entire ovary."}, "author_response": "The point-to-point responses to reviewers’ comments\nReviewer#1\nThe authors have thoroughly addressed the previous concerns and made substantial\nrevisions in response to all reviewer comments. These updates have significantly\nenhanced the quality, clarity, and scientific depth of the manuscript, which now\nmeets the standards for minor revision in Nature Communications.\nIn addition, the following recent study might be relevant and worth citing:\ndoi: 10.1016/j.cmet.2020.03.004.\ndoi: 10.1016/j.cmet.2024.10.019.\ndoi: 10.1038/s42255-020-00329-9\nResponse: Thank you very much for your approving our revision and suggestion.\nAccordingly, we have cited the references you mentioned, which correspond to\nreferences 28, 75, and 76 in the revised manuscript.\nReviewer#2 and 3\n1. Mechanistic Validation Remains Insufficient\nThe authors reference their previously published article, which demonstrates\nSLC1A4 silencing and its effect on CTX-induced granulosa cell death. This prior\nwork is appreciated and provides supporting evidence that SLC1A4 contributes to\nCTX-induced granulosa-cell death. However, because this mechanistic validation\nlies outside the current dataset, it is recommended that the authors incorporate a\nmore detailed discussion of their prior findings in the main text.\nResponse: We sincerely appreciated very much for your suggestion. Our previous\nmanuscript contained extensive descriptions of our previously published findings in\nboth the Main and Discussion sections. Based on your suggestion, we have incorporated\nmore detailed descriptions regarding SLC1A4 silencing in the lines 83-86 and lines\n381-384 of the revised manuscript.\nIn addition to this, the experiments that the authors highlight from their\nSupplementary data, which examine S1P synthesis and HO-1 function are valuable;\nhowever, they do not yet establish causality, which was the central concern. These\nexperiments rely exclusively on chemical inhibition without complementary genetic\nmanipulation or rescue approaches. Specifically, for S1P synthesis, the proposed link\nbetween serine availability and S1P production remains unclear, as serine supports\nde novo sphingolipid synthesis rather than directly serving as an S1P precursor.\nInclusion of a direct rescue experiment (e.g., exogenous S1P supplementation) would\nsubstantially strengthen the mechanistic argument.\nResponse: Thank you very much for your suggestions. Serine can be converted to\nsphingosine in vivo, which is subsequently activated by sphingosine kinase to form S1P.\nTherefore, we hypothesized that serine exerts its effects through S1P. In the present\nstudy, we observed that the inhibitory effect of serine on GC ferroptosis was attenuated\nwhen the activity of sphingosine kinase was inhibited by SKI178, an inhibitor of\nSPKAs. However, it is important to note that serine is not the direct upstream precursor\nof S1P, as several intermediate steps are involved in the conversion from serine to S1P.\nTherefore, we are very grateful for your advice. To demonstrate the role of S1P, we\nconducted additional experiments with exogenous S1P (MCE, HY-108496) for\nexamining key ferroptosis markers. Our results showed that S1P supplementation\nsignificantly inhibited CTX-induced lipid peroxidation (Fig. S6n), ferrous iron\naccumulation (Fig. S6o), and ROS production (Fig. S6m) in GCs, exhibiting the effects\nsimilar to those of serine. These results further confirmed that serine primarily exerts\nits function by being converted into S1P, and the relevant description of the additional\nresults have been incorporated into the lines 298-303 of the results section in the revised\nmanuscript.\nNormal CTX CTX+S1P\nFig.S6 m\nNormal CTX CTX+S1P\nd\ne\nz\nid\nix\no\nn\no\nN\nd\ne\nz\nid\nix\no\nFig.S6 n.\nNormal CTX CTX+S1P\ne\ng\nn\na\nr\nO\no\nr\nr\ne\nF\nFig.S6 o.\nRegarding HO-1, while ZnPP inhibition is informative, ZnPP has known off-target\neffects; without supporting overexpression or genetic knockdown data, the\nconclusion that HO-1 is the primary driver of CTX-induced ferroptosis or POI\nremains speculative. The authors are therefore encouraged to temper causal\nstatements and present these findings as supportive rather than definitive evidence\nof this pathway’s involvement.\nResponse: We sincerely appreciated very much for your advice and suggestion. Znpp\nis known as a specific inhibitor of HO-1 enzyme activity, your comment prompted us\nto review the relevant literature, which revealed potential off-target effects of Znpp.\nAlthough our study demonstrated that Znpp significantly inhibits HO-1 enzymatic\nactivity and markedly alleviates CTX-induced iron accumulation, the possibility of off-\ntarget effects in our study remains unclear. Consequently, we have revised the relevant\ndescription accordingly (lines 304-306 in the manuscript). We acknowledged that we\nmight not be rigorous for concluding that HO-1 was a key driver for iron accumulation\nin our previous version. Therefore, considering the potential off-target effects of Znpp,\nwe have revised the description to emphasize the regulation of ferroptosis by the\nKeap1/Nrf2/HO-1 pathway, which is more accurate. In fact, our study primarily\nfocused on extensive validation studies of this signaling pathway, in which serine\nsupplementation has been approved to inhibit Nrf2 nuclear translocation.\n2. Standardized Imaging and Quantification\nThe rationale for not quantifying Prussian blue staining remains unconvincing.\nPrussian blue detects ferric (Fe³⁺), not ferrous (Fe²⁺) ions, the latter being directly\ninvolved in driving ferroptosis via the Fenton reaction. Accordingly, the\ninterpretation of “ferrous ion accumulation” should be corrected. Moreover, the\nabsence of visible staining in tumor sections does not preclude quantification; semi-\nquantitative image analysis could still reveal meaningful differences (e.g., near-zero\nsignal relative to ovarian tissue).\nResponse: Thank you very much for your advice and suggestion. In the present study,\nwe utilized Prussian blue staining and ferrous ion concentration assay kits to detect the\nalterations of the ferric and ferrous ions, respectively. Subsequently, we focused on the\nFenton reaction induced by ferrous ions. Therefore, we concluded that CTX induced\nferrous ion accumulation in GCs.\nIn addition, based on your suggestion, we have quantitatively re-analyzed the\nPrussian blue-stained sections of tumor tissues, and the results showed that there were\nno the differences between the statistical results and the staining images. In our previous\nrevision, we contacted a quantitative assessment of ferrous ion concentrations in tumor\ntissues, and the results showed that CTX significantly induced the accumulation of\nferrous ion in ovarian tissues, but not tumor tissues. Therefore, we can conclude that\nCTX did not induce ferrous ion accumulation in tumor tissues.\nFig. 5f-i\nThe authors point to Hmox-1 mRNA expression changes as supporting evidence.\nHowever, Hmox-1 represents an indirect marker of labile iron accumulation.\nTherefore, at a minimum, the authors should either provide such image-based\nquantification or revise the discussion to frame these findings as qualitative\nobservations rather than quantitative conclusions regarding iron accumulation and\nferroptosis.\nResponse: Thank you very much for your suggestions. In the present study, we\ndemonstrated that CTX-induced GC death was tightly associated with ferroptosis,\nin\nwhich we not only observed the elevated expression of Hmox-1 mRNA, but also clearly\nshowed the accumulations of the intracellular ferrous ions and MDA formation in GCs,\nproviding a direct quantitative evidence for the accumulations of the intracellular\nferrous ions and MDA. In addition, our KEGG enrichment analysis and qPCR\nvalidation revealed that Hmox1 was one of the most elevated differential genes.\nSubsequently, we demonstrated that there were the marked increases of Hmox-1\nexpression, ferrous ions accumulation and MDA formation following CTX\nadministration in animal models. Furthermore, in vitro experiments, we demonstrated\nthat CTX was able to induced the accumulation of the intracellular iron ion by applying\nan iron-specific probe FerroOrange to label the intracellular iron ions. Moreover,\nalthough Znpp may exhibit off-target effects, we did observe that Znpp significantly\ninhibited HO-1 enzyme activity, resulting in a substantial reduction of CTX-induced\nferrous iron accumulation in GCs. Therefore, our in vivo and in vitro experiments\nconclusively demonstrated the accumulation of ferrous irons of GCs treated with CTX.\nTo ensure the rigor of our description, we have revised the relevant discussion section\nfrom lines 399 to 404 in the revised manuscript.\nReviewer#4\nThe authors have addressed most of my concerns, with the exception of the following\nissue:\nIn Figures S3C, S4H, and S5G, it appears that the authors quantified the number of\nfollicles at different developmental stages based on a single ovarian section. This\napproach is not accurate, as a reliable quantification should involve counting\nfollicles across the entire ovary.\nResponse: Thank you very much for your suggestion. Accordingly, we have recounted\nthe follicles across the entire ovary by referencing the relevant literature, and the\ncorrected results have been presented in the relevant Supplementary Figures S3c, S4h\nand S5g. We acknowledged that our previous results were only involved in a statistical\nanalysis of a single HE section.\nFig. S3c\nFig. S4h\nFig. S5g"}, {"round": 3, "reviewer_comments": {"reviewer_2": "Thank you to the authors for their detailed responses and revisions. The additional mechanistic clarification, inclusion of exogenous S1P rescue data, tempering of the HO-1 interpretation, and quantitative reassessment of Prussian blue staining satisfactorily address the prior concerns.\nThe authors have also appropriately corrected the follicle quantification by recounting across the whole ovary, resolving the remaining issue raised by Reviewer #4. Overall, the manuscript has been substantially improved, and we are now supportive of publication."}, "author_response": ""}]

Decision letter