Reproductive Genetics and the Aging Male

Paul J. Turek MD, FACS, FRSMPaul J. Turek MD, FACS, FRSMDirector, The Turek ClinicDirector, The Turek Clinic

Beverly Hills and San Francisco, CABeverly Hills and San Francisco, CA

Reproductive Genetics Reproductive Genetics andand the Aging Male the Aging Male

At the conclusion of this presentation, At the conclusion of this presentation, participants should be able to:participants should be able to:

Learning ObjectivesLearning Objectives

• Delineate 2 genetic abnormalities in sperm that are associated with advanced paternal age (APA).

• Explain APA associations with miscarriages, preterm birth and fetal death.

• Enumerate two adult diseases of offspring that occur with increased frequency in APA.

What’s Happening to Fathers? What’s Happening to Fathers?

% Men % Men Fathering Fathering ChildrenChildren

19701970 19931993 20032003

15%15%

25%25%

40%40%

Proportion of fathers over 35 years old in U.KProportion of fathers over 35 years old in U.K..

Bray I et al. J Epidemiol Comm Health 2006; 60: 851–3

7394

72

9999

When Does Paternity End? When Does Paternity End?

Courtesy: Dr. Saleh BinsalehCourtesy: Dr. Saleh Binsaleh

http://www.enterstageright.com/archive/articles/0604/062104trudeaupierre.jpg

Think of spermatogenesis as an engine.. Think of spermatogenesis as an engine..

By puberty: By puberty: 3030 spermatogonial divisions spermatogonial divisionsAfter puberty: After puberty: 2323 divisions/year divisions/yearBy age 70: By age 70: 12881288 divisions divisions (Women 23)(Women 23)

Crow J. Nat Rev Genet. 2000; 1: 41Crow J. Nat Rev Genet. 2000; 1: 41

What happens when engines get old? What happens when engines get old?

They donThey don’t run as smoothly, or at all. ’t run as smoothly, or at all.

What is the Relationship?What is the Relationship?

Genetic errorsGenetic errorsInfertilityInfertility

Genetic errorsGenetic errors AgingAging??

My interest in Male Reproductive GeneticsMy interest in Male Reproductive Genetics

1992: ICSI (Intracytoplasmic Sperm Injection)1992: ICSI (Intracytoplasmic Sperm Injection)

1993: TESE described (Testicular sperm extraction)1993: TESE described (Testicular sperm extraction)

1995: DAZ gene discovered on Y chromosome 1995: DAZ gene discovered on Y chromosome

My interest in Male Reproductive GeneticsMy interest in Male Reproductive Genetics

Response: Get Some Help!Response: Get Some Help!

• Hire Renee Reijo Pera PhDHire Renee Reijo Pera PhD• Recruited to 5 UCSF DeptsRecruited to 5 UCSF Depts• The rest is historyThe rest is history

Turek et al. UCNA. 2002; 29:767-92Turek et al. UCNA. 2002; 29:767-92

PROGENIPROGENI Program in the Genetics of Infertility Program in the Genetics of InfertilityPROGRAM LEADERSPROGRAM LEADERS Paul J. Turek M.D.Paul J. Turek M.D. Renee Reijo Pera Ph.DRenee Reijo Pera Ph.D

EXTERNAL ADVISORSEXTERNAL ADVISORS Andrew Wyrobek Ph.D.Andrew Wyrobek Ph.D.

INTERNAL ADVISORSINTERNAL ADVISORS Linda Giudice M.D, Ph.DLinda Giudice M.D, Ph.D

Mary Croughan Ph.DMary Croughan Ph.D

DIAGNOSTIC ANDDIAGNOSTIC AND

TREATMENT SERVICETREATMENT SERVICE

1. Cystic fibrosis mutations1. Cystic fibrosis mutationsFarid Chahab Ph.DFarid Chahab Ph.D

2. Karyotype analysis2. Karyotype analysisPhil Cotter M.DPhil Cotter M.D

3. Y chromosome deletion3. Y chromosome deletionRenee Reijo Pera Ph.DRenee Reijo Pera Ph.D

4. Preimplantation Diagnosis4. Preimplantation DiagnosisShehua Shen M.DShehua Shen M.D

RESEARCH PROGRAMSRESEARCH PROGRAMS

1. Genetic male infertility1. Genetic male infertilityRenee Reijo Pera Ph.DRenee Reijo Pera Ph.D

2. Epidemiology of2. Epidemiology of infertilityinfertility

Mary Croughan Ph.DMary Croughan Ph.D 3. Genetics of meiosis3. Genetics of meiosis

Renee Reijo Pera Ph.DRenee Reijo Pera Ph.D

Project CoordinatorProject CoordinatorGenetic CounselorsGenetic Counselors Lauri BlackLauri Black Kari DanzigerKari Danziger

Eugene Xu Ph.DEugene Xu Ph.D

Roger Pederson Ph.D.Roger Pederson Ph.D.

PROGENI®PROGENI®Clinical ResearchClinical Research

The The Genetic Genetic HistoryHistory

ConsanguinityConsanguinity

Translocation?Translocation?

Relevant Family History by Pedigree AnalysisRelevant Family History by Pedigree Analysis

OrOr

What is the Urologist Missing about Genetic What is the Urologist Missing about Genetic Infertility?Infertility?

Design: Design:

Reviewed pedigree analysis Reviewed pedigree analysis of n=215 of n=215 counseled couples. Both counseled couples. Both male and female family histories obtained. Exclude diagnoses male and female family histories obtained. Exclude diagnoses made by the urologist.made by the urologist.

Results:Results:

14 (14 (6.5%6.5%) of couples had significant and unique information ) of couples had significant and unique information revealed by the genetic counselor.revealed by the genetic counselor.

AUA 2001AUA 2001

PROGENI®PROGENI®Basic ResearchBasic Research

Infertility ClinicInfertility Clinic (Male and Female)(Male and Female)

Diagnosis & Diagnosis & TreatmentTreatment

DNA BankDNA Bank

TissueTissue

Genetic CounselorGenetic Counselor

BloodBloodRisk Risk CounselingCounseling

PROGENI®PROGENI®Clinical ResearchClinical Research

X

Gene Xu PhD “Fly guy”

Fred Moore MS “DAZ guy”

•Identified DAZ-binding proteins.Identified DAZ-binding proteins.

•Used a 2-hybrid screen.Used a 2-hybrid screen.

•Confirmed with co-precipitation.Confirmed with co-precipitation.

•Drosophila Drosophila bouleboule gene gene

•Compared sequences of Compared sequences of DAZDAZ interacting proteins with interacting proteins with boule.boule.

DAZLDAZL/human 1 /human 1 QGYILPEGKIMPNTVFVGGIDVRMDETEIRSFFARYGSVKEVKIITDRTGQGYILPEGKIMPNTVFVGGIDVRMDETEIRSFFARYGSVKEVKIITDRTGdazldazl/mouse 1 /mouse 1 QGYVLPEGKIMPNTVFVGGIDVRMDETEIRSFFARYGSVKEVKIITDRTGQGYVLPEGKIMPNTVFVGGIDVRMDETEIRSFFARYGSVKEVKIITDRTGHbouleHboule 1 1 PTSAPRYGTVIPNRIFVGGIDFKTNESDLRKFFSQYGSVKEVKIVNDRAGPTSAPRYGTVIPNRIFVGGIDFKTNESDLRKFFSQYGSVKEVKIVNDRAGboule/fly 1 boule/fly 1 PLAAPKYGTLIPNRIFVGGISGDTTEADLTRVFSAYGTVKSTKIIVDRAGPLAAPKYGTLIPNRIFVGGISGDTTEADLTRVFSAYGTVKSTKIIVDRAG * ..** .***** *... *. **.** **. **.** ..** .***** *... *. **.** **. **.*

RNP1RNP1DAZLDAZL/human 51 /human 51 VSKGYGFVSFFNDVDVQKIVES--QINFHGKKLKLGPAIRKQVSKGYGFVSFFNDVDVQKIVES--QINFHGKKLKLGPAIRKQ 9090dazldazl/mouse 51 /mouse 51 VSKGYGFVSFYNDVDVQKIVES--QINFHGKKLKLGPAIRKQVSKGYGFVSFYNDVDVQKIVES--QINFHGKKLKLGPAIRKQ 9090HbouleHboule 51 51 VSKGYGFVTFETQEDAQKILQEAEKLNYKDKKLNIGPAIRKQVSKGYGFVTFETQEDAQKILQEAEKLNYKDKKLNIGPAIRKQ 9292boule/fly 51 boule/fly 51 VSKGYGFVTFETEQEAQRLQADGECVVLRDRKLNIAPAIKKQVSKGYGFVTFETEQEAQRLQADGECVVLRDRKLNIAPAIKKQ 9292 ********.* . . *.. . . .** . ***.**********.* . . *.. . . .** . ***.**

=Shared by boules=Shared by boules

=Shared by hboule & DAZs=Shared by hboule & DAZs

RNPRNP22

Comparison of Comparison of BouleBoule and and DAZDAZ

An Autosomal Gene-BOULE-2q33

Xu E. et al. PNAS. 98: 7414, 2001Xu E. et al. PNAS. 98: 7414, 2001

Analysis of Human Analysis of Human Boule Boule MutationsMutations

•Examined DNA from n=164 infertile men in ProgeniExamined DNA from n=164 infertile men in Progeni

GeneGene RR eegiongion LL engengtthh PPoollyymmorphorphiicc SiSi ttees (s (FrFr eq.eq.))CodCodiingng rreegiongion 849849 bpbp 00NonNon--codcodiing ng rregegiionon 325325 bpbp 00II nnttronroniicc 872872 bpbp 33 (1(1//291291)) TT ototalal 20462046 33 (1(1//682682))

•Examined DNA from n=100 NIH Coriell reference Examined DNA from n=100 NIH Coriell reference panelpanel

Xu et al. Hum Mol Genet. 12: 169-175, 2003Xu et al. Hum Mol Genet. 12: 169-175, 2003

•Examined DNA from chimps, Old World monkeysExamined DNA from chimps, Old World monkeys

Xu et al. Hum Mol Genet. 12: 169-175, 2003Xu et al. Hum Mol Genet. 12: 169-175, 2003

The human The human BOULE BOULE gene rescues fertility gene rescues fertility in flies with a in flies with a Boule Boule mutationmutation

Wild-typeWild-type Boule Boule -/-knocko-/-knockoutut

Knockout Knockout with fly with fly

transgenetransgene

Knockout Knockout with human with human transgenetransgene

spermsperm No spermNo sperm sperm!sperm! sperm!!sperm!!

Primary spermatocytePrimary spermatocyte

Nodules withNodules withMMR proteinsMMR proteins

Meiotic RecombinationMeiotic Recombination

Gonsalves et al. Hum Mol Hum Genet. 2004; 13:2875Gonsalves et al. Hum Mol Hum Genet. 2004; 13:2875

Infertile men have reduced recombination frequenciesInfertile men have reduced recombination frequencies

spermatocytespermatocyte

Nodules withNodules withMMR proteinsMMR proteins

•There is decreased chromosomal pairing quality in azoospermic men.There is decreased chromosomal pairing quality in azoospermic men.•There is faulty progression through meiosis in infertile men.There is faulty progression through meiosis in infertile men.

Sun et al. Cytogenet Genome Res. 111: 366, 2005Sun et al. Cytogenet Genome Res. 111: 366, 2005

•Recombination errors have a distinct pattern of variation among Recombination errors have a distinct pattern of variation among various chromosomes. various chromosomes.

Centromere-specific Centromere-specific multi-color FISHmulti-color FISH

Sun et al. Cytogenet Hum Reprod. 15: 2376, 2006Sun et al. Cytogenet Hum Reprod. 15: 2376, 2006

•The localization patterns of 5 meiotic proteins described. The localization patterns of 5 meiotic proteins described. MSH4 appears to stabilize recombination foci.MSH4 appears to stabilize recombination foci.

Oliver Bonet et al. Mol Hum Reprod. 11: 517, 2005Oliver Bonet et al. Mol Hum Reprod. 11: 517, 2005

Recombination in Infertile Men

What Does This Mean?What Does This Mean?

• In animals and humans, faulty recombination is In animals and humans, faulty recombination is linked to chromosomal aneuploidy. linked to chromosomal aneuploidy.

• Smaller chromosomes at more risk (21, 22, X, Y). Smaller chromosomes at more risk (21, 22, X, Y). Faulty recombination could explain the “ICSI risk” Faulty recombination could explain the “ICSI risk” (sex chromosome anomalies) in offspring.(sex chromosome anomalies) in offspring.

• Recombination nodules harbor mismatch repair Recombination nodules harbor mismatch repair (MMR) genes. (MMR) genes.

• Do infertile men show defective MMR? Do infertile men show defective MMR?

Faulty Mismatch Repair & Cancer in MiceFaulty Mismatch Repair & Cancer in Mice

Mice Papers

Baker S et al. Cell. 82: 309, 1995Baker S et al. Cell. 82: 309, 1995

Edelmann et al. Cell. 85: 1125, 1996Edelmann et al. Cell. 85: 1125, 1996

Is There Evidence of Faulty DNA Repair Is There Evidence of Faulty DNA Repair in Infertile Men?in Infertile Men?

MouseMouse ManMan

•6 infertile men with 6 infertile men with azoospermia and azoospermia and maturation arrest.maturation arrest.

•5 infertile men with 5 infertile men with normal spermatogenesis.normal spermatogenesis.

•Examined blood and testis Examined blood and testis tissue for microsatellite tissue for microsatellite instability at polymorphic instability at polymorphic marker D19S49 on 19q12.marker D19S49 on 19q12.

Nudell D et al. Hum Reprod.15: 1289, 2000.

0

10

20

30

40%

Clo

nes

wit

h Po

int

Mut

atio

ns%

Clo

nes

wit

h Po

int

Mut

atio

ns

EARLY ARRESTEARLY ARREST LATE LATE ARREST ARREST

NORMAL NORMAL

22 1919 3131 55 3434 100100 500500 600600

4343 66 1111 1717

Infertile PatientsInfertile Patients

Nudell D. Hum Reprod.15: 1289, 2000.

Polymorphic Marker: D19S49

Human mean = 9% Mice Mlh1-/- =14%


• In animals and humans, meiotic recombination In animals and humans, meiotic recombination involves mismatch repair genes. involves mismatch repair genes.

• Faulty mismatch repair is associated with cancer Faulty mismatch repair is associated with cancer in animals and humans.in animals and humans.

• Infertile men have evidence of faulty mismatch Infertile men have evidence of faulty mismatch repair.repair.

• Are infertile men at higher risk of cancer? Are infertile men at higher risk of cancer?

An Epidemiologic Study An Epidemiologic Study Do Infertile Men Have Higher Rates of Cancer?Do Infertile Men Have Higher Rates of Cancer?

51,000+ infertile males

• 15 California centers

• 1965 to 1998

California Cancer Registry (CCR)

• 10 SEER Regions 1973 to 2003– Testis cancer– Prostate cancer– Colon cancer– Melanoma ??

Walsh et al. Arch Int Med. 169: 351, 2009Walsh et al. Arch Int Med. 169: 351, 2009Walsh et al. Cancer. Epub. Mar 2010Walsh et al. Cancer. Epub. Mar 2010

Standardized Incidence Ratios (SIR) Standardized Incidence Ratios (SIR) forfor Testicular Cancer Testicular Cancer in in Men Men withwith and and without without Male Factor InfertilityMale Factor Infertility

*P<0.05 *P<0.05

Standardized Incidence Ratios (SIR) Standardized Incidence Ratios (SIR) forfor Testicular Cancer Testicular Cancer inin Infertile Men Infertile Men

Walsh et al, Arch Int Med. 169: 351, 2009 Walsh et al, Arch Int Med. 169: 351, 2009

Walsh et al. Cancer. 116: 2140-7, 2010Walsh et al. Cancer. 116: 2140-7, 2010

SIRs SIRs forfor Prostate Cancer Prostate Cancer in in Infertile MenInfertile Men

Fertility Status # Men Fertility Status # Men # Cancers # Cancers SIR (95%CI) SIR (95%CI)

All cancersAll cancers Male Factor InfertilityMale Factor Infertility

NoNo 14,55714,557 6464 0.7 (0.6-0.9) 0.7 (0.6-0.9)YesYes 4,549 4,549 5656 1.1.33 (1.0-1.7) (1.0-1.7)

High-grade cancers (Gl 8-10)High-grade cancers (Gl 8-10) Male Factor InfertilityMale Factor Infertility

NoNo 14,55714,557 1616 0.8 (0.5-1.3) 0.8 (0.5-1.3)YesYes 4,549 4,549 1919 2.2.00 (1.2-3.0) (1.2-3.0)


• These infertility-cancer associations need These infertility-cancer associations need confirmation. confirmation.

• Other mechanisms exist that may explain infertility-Other mechanisms exist that may explain infertility-cancer relationships.cancer relationships.

• More recent literature supports the relationship.More recent literature supports the relationship.• Forms the basis for my interest in paternal age Forms the basis for my interest in paternal age

genetics!genetics!

What is the RelationshipWhat is the Relationship??

Genetic errorsGenetic errorsInfertilityInfertility

Genetic errorsGenetic errors AgingAging??

Paternal Age Issues May Differ…or Do They? Paternal Age Issues May Differ…or Do They?

Infertility

Aging

What Changes Occur with Paternal Age?What Changes Occur with Paternal Age?

Lots going on in Lots going on in young engines young engines

What changes occur What changes occur in older engines?in older engines?

Meiotic Recombination?Meiotic Recombination?DNA Mismatch Repair?DNA Mismatch Repair?Mitotic Replication?Mitotic Replication?

Paternal Age Effects: Sperm GeneticsPaternal Age Effects: Sperm Genetics

•Chromosomal issues: NumericalChromosomal issues: Numerical

Aneuploidy occurs in Aneuploidy occurs in 30-50% 30-50% of all pregnanciesof all pregnanciesMost are lethal (Most are lethal (0.3%0.3% of births are aneuploid) of births are aneuploid)Arise from non-disjunction during meiosis (I and II)Arise from non-disjunction during meiosis (I and II)Definite increase in aneuploidy in infertile vs. fertile sperm Definite increase in aneuploidy in infertile vs. fertile sperm Autosomal aneuploidy: No hard evidence of an increase Autosomal aneuploidy: No hard evidence of an increase

with paternal agewith paternal ageSex chromosomal aneuploidy and disomy: possible Sex chromosomal aneuploidy and disomy: possible increases with paternal age (1-2x)increases with paternal age (1-2x)

XY diploidy XY diploidy (Meiosis I) and (Meiosis I) and XX/YY diploidy XX/YY diploidy (Meiosis II)(Meiosis II)

Templado C. Cytogenet Genome Res 2005, 111:199-205. Templado C. Cytogenet Genome Res 2005, 111:199-205. Sloter et al. Fertil Steril. 2004; 81:925Sloter et al. Fertil Steril. 2004; 81:925Wyrobek et al. PNAS, 2006; 103:9601Wyrobek et al. PNAS, 2006; 103:9601

Paternal Age Effects: Sperm GeneticsPaternal Age Effects: Sperm Genetics•Chromosomal issues: StructuralChromosomal issues: Structural

0.25%0.25% of births of birthsChromosomal breaks & fragments Chromosomal breaks & fragments increase with age increase with age Pronounced Pronounced

relationshiprelationship: r=0.63: r=0.63Especially chromos 1Especially chromos 1

and acentricand acentricfragmentsfragments

Not evidentNot evident in in offspringoffspring

Sloter et al. Fertil Steril. 2007; 87: 1077Sloter et al. Fertil Steril. 2007; 87: 1077Martin and Rademaker. Am J Hum Genet. 1987, 41: 484Martin and Rademaker. Am J Hum Genet. 1987, 41: 484

Paternal Age Effects: Sperm GeneticsPaternal Age Effects: Sperm Genetics•Single Gene MutationsSingle Gene Mutations

•CommonCommon in male>>female germ cells; increase in male>>female germ cells; increase with age. Suggests with age. Suggests mitoticmitotic replication replication errorserrors•Postulated since 1912 Postulated since 1912 as causes of as causes of “sentinel “sentinel

mutations”mutations” in offspring (achondroplasia) in offspring (achondroplasia)•Now “Now “PAE Disorders.PAE Disorders.””•Wyrobek et al. n=88 healthy, Wyrobek et al. n=88 healthy,

non-smoking mennon-smoking men•Examined achondroplasia Examined achondroplasia

muatatonsmuatatons•r=.54; r=.54; Change: 2%/yrChange: 2%/yr

Goriely et al, Nat Genet. 2009, 41: 1247Goriely et al, Nat Genet. 2009, 41: 1247Wyrobek et al. PNAS, 2006; 103:9601Wyrobek et al. PNAS, 2006; 103:9601

20 yrs20 yrs 40 yrs 40 yrs 60 yrs 60 yrs 80 80

yrsyrs

AchondroplasiasAchondroplasias (FGFR3) (FGFR3) AniridiaAniridiaApert syndrome Apert syndrome (FGFR2(FGFR2) ) Bilateral retinoblastomaBilateral retinoblastomaCrouzon syndrome Crouzon syndrome (FGFR2) (FGFR2) Fibrodysplaisa ossificansFibrodysplaisa ossificansHemophilia AHemophilia A Lesch Nyhan syndromeLesch Nyhan syndromeMarfan syndromeMarfan syndrome (FGFR3) (FGFR3) MEN II MEN II (RET) (RET)

NeurofibromatosisNeurofibromatosis Oculodentodigital syndrome Oculodentodigital syndrome Osteogenesis Imperfecta Osteogenesis Imperfecta (FGFR3) (FGFR3) Polycystic kidney diseasePolycystic kidney diseasePfeiffer syndromePfeiffer syndrome (FGFR2) (FGFR2) ProgeriaProgeriaPolyposis coliPolyposis coli Thanatophoric dyspl. Thanatophoric dyspl. (FGFR3)(FGFR3)

Treacher-Collins syndromeTreacher-Collins syndrome Tuberous sclerosisTuberous sclerosisWardenburg syndromeWardenburg syndrome

Single Gene Mutations: Paternal Age Effect DisordersSingle Gene Mutations: Paternal Age Effect Disorders

More on Single Gene MutationsMore on Single Gene Mutations•Old: Old: Penrose Hypothesis (1955)Penrose Hypothesis (1955). It’s more likely paternal . It’s more likely paternal than maternally derived due to continuously dividing than maternally derived due to continuously dividing spermatogonia vs quiescent female germline.spermatogonia vs quiescent female germline.•OldOld: : Paternal contribution to Paternal contribution to de novo de novo single gene mutations single gene mutations

is is 6-9x greater 6-9x greater than maternal contribution.than maternal contribution.

Forster et al. Proc. Roy Soc B. 2015; Epub Mar 2015Forster et al. Proc. Roy Soc B. 2015; Epub Mar 2015Goriely and Wilkie, Am J Hum Genet. 2012, 90: 175Goriely and Wilkie, Am J Hum Genet. 2012, 90: 175

•NewNew: : These point mutations are These point mutations are not random not random (from (from generally poor quality control), but show generally poor quality control), but show “allelic “allelic skewing” skewing” and cluster in the receptor-RAS signalling and cluster in the receptor-RAS signalling pathway. pathway.

•New:New: Paternal age effect mutations follow a Paternal age effect mutations follow a "selfish "selfish selection” selection” pathway (e.g. oncogenesis) that favors clonal pathway (e.g. oncogenesis) that favors clonal proliferation of mutated spermatogonia, leading to testis proliferation of mutated spermatogonia, leading to testis cancer (spermatocytic seminoma) and specific cancer (spermatocytic seminoma) and specific phenotypes in offspring.phenotypes in offspring.

•New: New: PAE mutations occur up to 1000x more frequently PAE mutations occur up to 1000x more frequently than background nucleotide substitutions.than background nucleotide substitutions.

Even More on Single Gene MutationsEven More on Single Gene Mutations

Goriely and Wilkie, Am J Hum Genet. 2012, 90: 175Goriely and Wilkie, Am J Hum Genet. 2012, 90: 175

PAE Mutations: A Quality Control PAE Mutations: A Quality Control “Issue”“Issue”

Goriely and Wilkie, Am J Hum Genet. 2012, 90: 175Goriely and Wilkie, Am J Hum Genet. 2012, 90: 175Goriely et al, Nat Genet. 2009, 41: 1247Goriely et al, Nat Genet. 2009, 41: 1247

• Studied Studied spermatocytic seminomasspermatocytic seminomas..• Examined Examined PAE MutationsPAE Mutations; massive parallel sequencing; massive parallel sequencing• Found increased PAE mutations in tumors.Found increased PAE mutations in tumors.• Proposed that common Proposed that common “selfish” “selfish” mutations mutations “hijack” “hijack” the the

pathway and lead to: pathway and lead to: • tumorstumors• diseases in offspringdiseases in offspring

•Sperm produced are in Sperm produced are in “evolutionary conflict:”“evolutionary conflict:” great for great for maintaining production but bad for species.maintaining production but bad for species.

•““Houston, we have a problem…”Houston, we have a problem…”

Wyrobek et al. PNAS, 2006; 103:9601Wyrobek et al. PNAS, 2006; 103:9601

Paternal Age Effects: Sperm GeneticsPaternal Age Effects: Sperm Genetics•Sperm DNA Fragmentation:Sperm DNA Fragmentation:

Ji et al. BMC Med. 2012, 10: 49 Ji et al. BMC Med. 2012, 10: 49

•N=88 healthy non- N=88 healthy non- smokerssmokers

•r=0.72; p<0.001r=0.72; p<0.001

•Predicted change Predicted change of of 3.1%/year3.1%/year of age of age

•Associated with Associated with defective mismatch defective mismatch repair?repair? 20 yrs20 yrs 40 yrs 40 yrs 60 yrs 60 yrs 80 yrs 80 yrs

10% 25% 47% 88%

20 yrs 40 yrs 60 yrs20 yrs 40 yrs 60 yrs 80 yrs 80 yrs

Jenkins et al. PLOS Genet.2014, 10: e1004458 Jenkins et al. PLOS Genet.2014, 10: e1004458

Paternal Age Effects: Sperm EpigeneticsPaternal Age Effects: Sperm Epigenetics

• Ejaculates from 17 fertile sperm donorsEjaculates from 17 fertile sperm donors

1990s1990s9-19 years apart9-19 years apart

23-56 years old (mean 37.7 yrs) 23-56 years old (mean 37.7 yrs)

Mean 50.3 yrs Mean 50.3 yrs

• Global Methylation analysis (LINE)Global Methylation analysis (LINE)• High resolution (cGP) analysis (Illumina 450K)High resolution (cGP) analysis (Illumina 450K)• >10% change in methylation--significant>10% change in methylation--significant



• Age-associated global Age-associated global hyperhypermethylation. methylation. • Regional Regional hypohypomethylationmethylation• Altered regions are Altered regions are consistentconsistent among among

individuals and individuals and increase linearly increase linearly with age.with age.• Many altered regions occur in loci known to be Many altered regions occur in loci known to be

associated with associated with diseases diseases in offspring in offspring that are linked that are linked to advanced paternal age to advanced paternal age

Findings Findings



Diseases Diseases associated with associated with involved genes involved genes compared to compared to GAD-annoted GAD-annoted 11,326 disease-11,326 disease-associated associated genes.genes.

Mutations Mutations andand Methylation Changes Methylation Changes

• They are independent events.They are independent events.• Methylation changes are a consequence of mutationsMethylation changes are a consequence of mutations• Methylation changes influence mutational events Methylation changes influence mutational events

Are they related? 3 possibilities:Are they related? 3 possibilities:

Milekic et al. Nat Mol Psych.2014, 1-7Milekic et al. Nat Mol Psych.2014, 1-7


• Rapidly dividing spermatogonia are likely the Rapidly dividing spermatogonia are likely the source of DNA replication errors as men age.source of DNA replication errors as men age.

• Single gene mutations are the consequence of Single gene mutations are the consequence of these errors (unlike aneuploidy in women).these errors (unlike aneuploidy in women).

• Point mutations are rare, dominant and lead to Point mutations are rare, dominant and lead to significant disease in offspring.significant disease in offspring.

• Epigenetic changes to sperm genome also show Epigenetic changes to sperm genome also show correlations to disease in offspring.correlations to disease in offspring.

Changes in Testicles and Semen with AgeChanges in Testicles and Semen with Age

•Morphometric studies:Morphometric studies:Leydig cells: fall 80 million/testis/decadeLeydig cells: fall 80 million/testis/decadeAge-related decline in Sertoli cellsAge-related decline in Sertoli cellsDecreased germ cell proliferationDecreased germ cell proliferation

•Reduced semen qualityReduced semen quality

Hellstrom et al. J. Androl 2006Hellstrom et al. J. Androl 2006Wyrobek AJ. PNAS, 2006; 103:9601Wyrobek AJ. PNAS, 2006; 103:9601

Paternal Age Effects: Achieving PregnancyPaternal Age Effects: Achieving Pregnancy

•Non-clinical populations (Irish, Mormon, ALSPAC).Non-clinical populations (Irish, Mormon, ALSPAC).

•Demonstrate Demonstrate increased time to pregnancyincreased time to pregnancy..

•OR for fertility falls OR for fertility falls 2%/year of age2%/year of age

Kidd S. Fert Steril 2001 75:237-48 Kidd S. Fert Steril 2001 75:237-48 Ford W. Hum Reprod 2000 15:1703-Ford W. Hum Reprod 2000 15:1703-88

Paternal Age Effects: Achieving PregnancyPaternal Age Effects: Achieving Pregnancy

ConfoundersConfoundersFemale ageFemale ageErectile and sexual dysfunctionErectile and sexual dysfunctionCoital frequencyCoital frequencyComorbid conditionsComorbid conditions

Kidd S. Fert Steril 2001 75:237-48 Kidd S. Fert Steril 2001 75:237-48 Ford W. Hum Reprod 2000 15:1703-8Ford W. Hum Reprod 2000 15:1703-8

Paternal Age Effects: Pregnancy OutcomesPaternal Age Effects: Pregnancy Outcomes

•Miscarriages: Miscarriages: Fathers >50 yrs old associated Fathers >50 yrs old associated with with 2x increased 2x increased risk. risk.

•Preterm birth (<32 weeks): Preterm birth (<32 weeks):

•Fetal death: Fetal death: Fathers Fathers >>50 yrs 50 yrs HR 1.88 HR 1.88 (CI 0.93, 3.82)(CI 0.93, 3.82)

Lambert. World J Urol 2006; 24:611-617Lambert. World J Urol 2006; 24:611-617Kuhnert. Hum Reprod Upd 2004; 10:327-339Kuhnert. Hum Reprod Upd 2004; 10:327-339

CountryCountry YearsYears Maternal Maternal Findings Findings Ages (yrs)Ages (yrs)

ItalyItaly 1990-981990-98 20-29 20-29 OR 1.7 OR 1.7 (>45yrs)(>45yrs)DenmarkDenmark 1986-961986-96 20-29 20-29 OR 2.1 OR 2.1 (>50yrs)(>50yrs)USAUSA 1995-20001995-2000 20-35 20-35 No effectNo effect

Andersen et al. Am J Epi. 2004, 160: 1214.Andersen et al. Am J Epi. 2004, 160: 1214.

Paternal Age Effects: OffspringPaternal Age Effects: Offspring

•Congenital illness/birth defectsCongenital illness/birth defects•DiseasesDiseases


•Congenital illness/birth defectsCongenital illness/birth defectsChromosomalChromosomal

• General: no increase with paternal ageGeneral: no increase with paternal age• Exception: sex chromosomes Exception: sex chromosomes (47,XXY) (47,XXY)

55% 55% of sex chromosomal aneuploidies of sex chromosomal aneuploidies are paternal in origin are paternal in origin. . Increased risk Increased risk with paternal age? ( with paternal age? (RR 1.3-2.7)RR 1.3-2.7)

• Agrees with Agrees with spermsperm sex chromosomal sex chromosomal aneuploidy and disomyaneuploidy and disomy findings findings

• Trisomy 21: Trisomy 21: 7-9%7-9% of cases paternal of cases paternal

Toriello and Meck. Genet Med, 2008; 10-457Toriello and Meck. Genet Med, 2008; 10-457Zaragoza et al. Hum Genet, 1994, 94:411Zaragoza et al. Hum Genet, 1994, 94:411


•Congenital illness/birth defectsCongenital illness/birth defectsSingle Gene Mutations: Single Gene Mutations: “PAE Disorders”“PAE Disorders”

•4040 mutations; mutations; 4040 diseases. Selfish gene issue. diseases. Selfish gene issue.•Debilitating illnesses requiring lifelong careDebilitating illnesses requiring lifelong care•Rare, ranging from Rare, ranging from 1:10K to 1:1million1:10K to 1:1million•Fathers of affected children average Fathers of affected children average 6-7 years 6-7 years

olderolder than fathers of unaffected children than fathers of unaffected children•Diseases occur Diseases occur 10x more frequently 10x more frequently with fathers with fathers

>50 yrs old vs. 20-30 yrs old. >50 yrs old vs. 20-30 yrs old. •Overall prevalence is still Overall prevalence is still <1%<1%•Screening not recommendedScreening not recommended

AchondroplasiasAchondroplasias (FGFR3) (FGFR3) AniridiaAniridiaApert syndrome Apert syndrome (FGFR2(FGFR2) ) Bilateral retinoblastomaBilateral retinoblastomaCrouzon syndrome Crouzon syndrome (FGFR2) (FGFR2) Fibrodysplaisa ossificansFibrodysplaisa ossificansHemophilia AHemophilia A Lesch Nyhan syndromeLesch Nyhan syndromeMarfan syndromeMarfan syndrome (FGFR3) (FGFR3) MEN II MEN II (RET) (RET)

NeurofibromatosisNeurofibromatosis Oculodentodigital syndrome Oculodentodigital syndrome Osteogenesis Imperfecta Osteogenesis Imperfecta (FGFR3) (FGFR3) Polycystic kidney diseasePolycystic kidney diseasePfeiffer syndromePfeiffer syndrome (FGFR2) (FGFR2) ProgeriaProgeriaPolyposis coliPolyposis coli Thanatophoric dyspl. Thanatophoric dyspl. (FGFR3)(FGFR3)

Treacher-Collins syndromeTreacher-Collins syndrome Tuberous sclerosisTuberous sclerosisWardenburg syndromeWardenburg syndrome

Single Gene Mutations: Paternal Age Effect DisordersSingle Gene Mutations: Paternal Age Effect Disorders


•Congenital illness/Congenital illness/birth defectsbirth defects•DiseasesDiseases


•Birth defectsBirth defects

Paternal Age (Yrs)

Added Risk

30-35 4%

40-44 8%

45-49 8%

> 50 15%

Yang et al. Hum Reprod. 2007, 22: 696 Yang et al. Hum Reprod. 2007, 22: 696

•Pop. based, retrospective,Pop. based, retrospective, cohort studycohort study•5.2 million U.S. subjects5.2 million U.S. subjects•1999-2000 birth registry1999-2000 birth registry•Examined 22 serious birth Examined 22 serious birth

defect categoriesdefect categories•Overall rate Overall rate 1.5%1.5%

Comparison: Comparison: 220%220% increase with increase with maternal age >45 yrs maternal age >45 yrs

Green et al. Ann Epid. 2010, 20: 241Green et al. Ann Epid. 2010, 20: 241

•Pop. based, retrospective,Pop. based, retrospective, cohort studycohort study•U.S. Births from 1997-2004U.S. Births from 1997-2004•Overall rate Overall rate increases increases from 2% to 2.5%from 2% to 2.5%


•Birth defects-Birth defects-the usual suspectsthe usual suspects

Green et al. Ann Epid. 2010, 20: 241Green et al. Ann Epid. 2010, 20: 241

•Ventricular septal defectsVentricular septal defects•Atrial septal defectsAtrial septal defects•Pulmonary stenosisPulmonary stenosis•Situs inversusSitus inversus•Neural tube defects (spina bifida)Neural tube defects (spina bifida)•Cleft palateCleft palate•Diaphragmatic herniaDiaphragmatic hernia•Tracheoesophageal fistulaTracheoesophageal fistula

““Every reason to Every reason to believe that the believe that the patterns in the patterns in the origins of origins of spontaneous spontaneous mutations can be mutations can be extended to extended to complex traits.”complex traits.”J. Crow.2000J. Crow.2000

Crow J. Nat Rev Genet. 2000; 1: 41Crow J. Nat Rev Genet. 2000; 1: 41


•Congenital illness/birth defectsCongenital illness/birth defects•DiseasesDiseases


•Diseases-Diseases-Developmental,psychiatric conditionsDevelopmental,psychiatric conditions


•Diseases-Diseases-SchizophreniaSchizophrenia

Malaspina et al. Arch Gen Psych. 2001, 58: Malaspina et al. Arch Gen Psych. 2001, 58: 361361

0

1

2

3

20-24 25-29 30-34 35-39 40-44 45-49 50-54

Paternal Paternal AgeAge

1.01.0

2.962.96

•Israeli registryIsraeli registry

•n=87,907 birthsn=87,907 births

•Reproduced in Reproduced in 55 other countries other countries

Paternal Age Effects: DiseasesPaternal Age Effects: DiseasesAutism

DD’’Onofrio et al. JAMA Psych. Epub. Feb 2014 Onofrio et al. JAMA Psych. Epub. Feb 2014

•Sweden 1973-2001 Sweden 1973-2001 (n= 2,615,081, all births)(n= 2,615,081, all births)•Population-based, cohort studyPopulation-based, cohort study•Risk Risk of autism, ADHD, psychosis, bipolar disorder, suicide attempt, and low educational attainment.

Paternal Age Effects: Paternal Age Effects: “Psychiatric Morbidity”“Psychiatric Morbidity”

DD’’Onofrio et al. JAMA Psych. Epub. Feb 2014 Onofrio et al. JAMA Psych. Epub. Feb 2014

AutismAutism ADHDADHD PsychosisPsychosis

HR=3.45HR=3.45CI, 1.6-7.3CI, 1.6-7.3

HR=13.13HR=13.13CI, 6.8-25.2CI, 6.8-25.2

HR=2.07 HR=2.07 CI, 1.5-3.2CI, 1.5-3.2

A Perfect Storm in Basic and Population ScienceA Perfect Storm in Basic and Population Science

• DeCode Genetics, IcelandDeCode Genetics, Iceland• Examined whole genome sequences 78 trios of Examined whole genome sequences 78 trios of

mother, father and child. mother, father and child. • Child had schizophrenia or autism and parents did Child had schizophrenia or autism and parents did

not.not.• Searched for mutations in the child that were not Searched for mutations in the child that were not

present in parents.present in parents.

Kong et al. Nature. 2012, 488: 471 Kong et al. Nature. 2012, 488: 471

““Perfect Storm” of Basic and Population SciencePerfect Storm” of Basic and Population Science

• Fathers passed on Fathers passed on 4x4x as many new single gene as many new single gene mutations as mothers: (mutations as mothers: (55 versus 1455 versus 14). ).

• Paternal age accounted for Paternal age accounted for nearly all variation nearly all variation in new in new mutations in a child’s genomemutations in a child’s genome

• Number of new mutations being passed on Number of new mutations being passed on rose rose exponentiallyexponentially with paternal age. with paternal age.

20-yo passes on X mutations20-yo passes on X mutations 36-yo passes on 2X mutations36-yo passes on 2X mutations

70-yo passes on 8X mutations70-yo passes on 8X mutations


“…“…as a man ages, the number of de novo mutations in as a man ages, the number of de novo mutations in his sperm increases, and the chance that a child would his sperm increases, and the chance that a child would carry a deleterious mutation…that could lead to autism carry a deleterious mutation…that could lead to autism or schizophrenia increases proportionally. or schizophrenia increases proportionally.

Given that fully half of new mutations in Given that fully half of new mutations in offspring are neurodevelopmental in nature:offspring are neurodevelopmental in nature:

““Perfect Storm” of Basic and Population SciencePerfect Storm” of Basic and Population Science


Paternal Age Effects: SummaryPaternal Age Effects: Summary•Quality control Quality control in meiosis is compromised with in meiosis is compromised with both both

infertilityinfertility and and advanced paternal ageadvanced paternal age•Paternal age effects are characterized manly by Paternal age effects are characterized manly by de de

novo novo point mutations point mutations and not chromosomal and not chromosomal aneuploidy. aneuploidy. •Epigenetics and DNA fragmentation change with age.Epigenetics and DNA fragmentation change with age.•PAE on PAE on offspringoffspring::

Single gene mutationsSingle gene mutations 8-10x 8-10x Sex chromosome anomalies Sex chromosome anomalies 1.3-2.7x 1.3-2.7xMiscarriage, preterm birth, fetal death 1.5-2xMiscarriage, preterm birth, fetal death 1.5-2xBirth defectsBirth defects 1.25x 1.25xAdult diseasesAdult diseases 1.1- 13x 1.1- 13x

•Prevalence rates Prevalence rates remain lowremain low•No changes No changes to current genetic screening protocolsto current genetic screening protocols

Thanks to: Thanks to: The UCSF GroupThe UCSF Group

David Nudell MD David Nudell MD Uche Ezeh MD Uche Ezeh MD Shai Shefi MDShai Shefi MD Jeff Simko MD Jeff Simko MD Tom Walsh MD, MA Tom Walsh MD, MA James Smith MD, MPH James Smith MD, MPH Alan Shindel MD Alan Shindel MD Mary Croughan PhD Mary Croughan PhD Liza Jalalian BS Liza Jalalian BS Mike Abeyta BS Mike Abeyta BS Gina Davis MS, GCSGina Davis MS, GCS

Lance Armstrong FoundationLance Armstrong FoundationCalifornia Urology FoundationCalifornia Urology FoundationNIH K-12 MRHR NIH K-12 MRHR

The Reijo Pera LaboratoryThe Reijo Pera Laboratory

Amander Clark PhD Amander Clark PhD Dana Kostiner MD Dana Kostiner MD Joanna Gonsalves PhD Joanna Gonsalves PhD Fred Moore PhD Fred Moore PhD Mark Fox PhD Mark Fox PhD Nina Kossack MS Nina Kossack MS Ximena Ares PhD Ximena Ares PhD Renee Reijo Pera PhDRenee Reijo Pera PhD

CollaboratorsCollaborators

Fei Sun PhD Fei Sun PhD Renee Martin PhDRenee Martin PhD

Health & Medicine

Reproductive Genetics and the Aging Male