Why is Infertility Growing in Europe? Is there a link between different disorders of male reproductive organs and exposure to chemical compounds? Henrik.

Why is Infertility Growing in Europe?

Is there a link between different disorders of male reproductive organs and exposure to

chemical compounds?

Henrik Leffers University Department of Growth &

Reproduction, Rigshospitalet, Copenhagen

Declining conception rates among cohorts of younger womenJensen et al. IJA, 2008

1,00

1,20

1,40

1,60

1,80

2,00

2,20

2,40

Year of birth, pregnant woman

Nu

mb

er

of

pre

gn

an

cie

s

Total fertility rate without ART

Declining conception rates among cohorts of younger womenJensen et al. IJA, 2008

1,00

1,20

1,40

1,60

1,80

2,00

2,20

2,40

Year of birth, pregnant woman

Nu

mb

er

of

pre

gn

an

cie

s

Total fertility rate without ART

ART

Reproductive health problems

• Human reproductive health problems are very common, some are increasing

• The man sitting next to you has probably more than 80% morphologically abnormal sperms (>90 %, if he is a young Dane)

• High demand for ART: almost 8 % of all Danish children were in 2007 born after IVF, ICSI, homolog or donor insemination

• In most cases the etiology is really not known, although we use terms as ”oligozoospermia” and ”ovarian failure”

• Do endocrine disrupters play a role?

Structure of the Testis

Oct-4 expression in CIS cells (adult, subfertile male)

Spermatocytes

Spermatids

Sp-gonium

PGCESCGonocyte Infantile

sp-gonium

NANOG:OCT-4:AP-2 TRA-1-60:PLAP:KIT:p53: Cyclin D2: CHK-2 : TSPY:MAGE A4: NY-ESO-1: DAZL 1: Hiwi: VASA:IMP-1:

CIS:

PubertyBirth

Pluripotency, self-renewal, survival in undifferentiated stage

Regulation of cell cycle, DNA repair (mitotic division)

Germ cell specifice.g. RNA processing

Gene expression in CIS cells in relation to germ cell differentiation

Rates in testis cancer

• Testis cancer is a sensor for poor reproductive health (incl. cryptorchidism, poor semen quality)

• Testis cancer is of fetal origin. Therefore, a search for causal factors should be directed towards adverse effects on the fetal testis

Lessons from recent biological and epidemiological research on testis cancer

•Increasing incidence of testis cancer. Other male reproductive problems are also becomming more common!

Perinatal exposure of Rats to Phthalates (DEHP, DBP)

• Leydig cell abnormalities• Dysgenesis of Leydig cells and seminiferous

tubules• Hypospadias• Undescended testis • Spermatogenic failure and infertility

(Several papers from Paul Foster’s, Earl Gray’s, Rao Veeramachaneni’s and Richard Sharpe’s groups)

May cause a ”phthalate syndrome” in male offspring, including

ControlDBPBrown = smooth muscle

actin

Dysgenetic areas in the testes on d4 postnatal after in utero exposure to DBP (500 mg/kg/day) Fisher et al. Hum Reprod, 2003)

Phthalate Effects on Testis

The Rat Phthalate Syndrome Resembles Human Problems

Does Chemical Exposure Affect the Human Fetus?

• Association between excretion of phthalates in pregnant women’s urine and ano-genital distance in newborn boys, Swan et al. 2005

Does Chemical Exposure Affect the Fetus?

• Association between excretion of phthalates in pregnant women’s urine and ano-genital distance in newborn boys, Swan et al. 2005

• Association between Phthalate levels in breats milk and subtle changes in hormone levels in 3 month old boys, Main et al. 2006

Collection of breast milk

• Breast milk: post nataly month 1, n=130• Serum samples from the children at 3 months,

n= 96

Changes in boys hormone levels associated with 10 fold higher phthalate levels in the milk

• mEP, mBP:

• mBP:

• mMP, mEP, mBP:

• miNP:

+ 15,+8 % SHBG

+ 26, +19, +18 % LH /free testosterone ratio

+ 97% LH

- 15% free testosterone

Main KM et al. EHP 2006

Does Chemical Exposure Affect the Fetus?

• Association between excretion of phthalates in pregnant women’s urine and ano-genital distance in newborn boys, Swan et al. 2005

• Association between Phthalate levels in breats milk and subtle changes in hormone levels in 3 month old boys, Main et al. 2006

• Persistent pesticides in breast milk and cryptorchidism in the sons, Damgaard et al. 2006

• Flameretardants in breast milk and chryptorchidism in the sons, Main et al. 2007

Ongoing project: Uptake of phthalates and parabens through skin

• Comparisons between applications of ”basis lotion” with and without 2% phthalates and 2% parabens

• Bloodsampling during 24 hours

Janjua NR, Mortensen GK, Skakkebæk NE, Wulf HC, Andersson AM, 2008.

mBP

0

10

20

30

40

50

60

70

T=0 T=1 T=2 T=3 T=4 T=24

µg/L

control week exposure week

Mono-butyl-phthalate in serum during 24 hours

• The phthalates DEP, DBP, and BuP are rapidly taken up through the skin after topical application.

• Although these compounds have a high clearance rate some accumulation by every day use can occur.

• Normal use of cosmetics and skin care products containing these compounds is likely to result in recurrent serum spikes of the parent compounds and their metabolites.

It seems safe to conclude….

Janjua NR, Mortensen GK, Skakkebæk NE, Wulf HC, Andersson AM, 2008.

Effects of Mixtures of Endocrine Disrupting

Substances

Ulla Hass  

Dept. of Toxicology and Risk Assessment, National Food Institute, Danish Technical University, Denmark

Design of EDEN studies

Anti-androgen alone or in mix

Young adults: Malformations, semen quality, behaviour

Pups: AGD, nipple retention, reproductive organs, gene expression

Via placenta Via milk

Birth

1) Completely split penis and visible os penis, i.e. marked hypospadia 2) Blind vaginal opening

Malformations of external reproductive organs in adult male offspring

0% + 0% + 0% ~ 60% Christiansen et al 2008, Int. J. Androl. 31

Something from Nothing

0,001,002,003,004,005,006,007,008,009,00

10,00

cryptorchidismat birth

cryptorchidismat 3 months

hypospadias

Denmark

Finland

Differences in prevalence of genital abnormalities between Denmark and

Finland (From Boisen et al. Lancet 2004;Boisen et al. JCEM 205)

Geographic association between abnormalities in male reproductive health

Denmark

• High incidence of testicular cancer

• High prevalence of cryptorchidism

• High prevalence of hypospadias

• Low sperm counts• Smaller testes as

newborns and lower inhibin-B levels

• Finland

• Low incidence of testicular cancer

• Low prevalence of cryptorchidism

• Low prevalence of hypospadias

• High sperm counts• Bigger testes as

newborns and higher inhibin-B levels

Can Different Environmental Exposures between Denmark and Finland explain the Different Reproductive Health Patterns?

• More Danish women smoke and drink during pregnancy (Jensen et al. Am J Epidemiol. 2004)

• Associations to different exposures to EDs?

Chemicals in breast milk from mothers of newborn boys

p,p'-DDE

0

50

100

150

200

p,p'-DDE *

ng

/g

other persistent pesticides

0

10

20

30

DDT * HCH HCB * endosulfan-I oxychlordane dieldrin * cis-HE

ng

/g

brominated flame retardents

0

1

2

3

BDE 47 BDE 153 *

ng

/g

phthalate monoesters

0

50

100

150

mEHP * miNP

ug

/L

*: p<0.05

Danish

Finnish

Conclusions

• We are all exposed to a mixture of endocrine disrupters• ”Antiandrogenic” agents may be as important as chemicals

with estrogenic actions• Evidence that testicular cancer, cryptorchidism, hypospadias

and poor semen quality can be linked through a fetal mechanism causing testicular dysgenesis (TDS syndrome)

• We are beginning to see associations between fetal exposures of humans to some endocrine disrupters and symptoms of TDS

• Causal relationships between EDs and reproductive symptoms have been established in experimental animals, but not in humans

• Current ED research is focussed on the issue of possible additive effects of mixtures of different chemicals with different actions

Thanks to

• Niels E. Skakkebæk, Niels Jørgensen, Katharina Main, Anna-Maria Andersson, Ewa Rajpert-De Meyts, Anders Juul and several of their Ph.D. students

• Jorma Toppari, Finland• Other European collaborators• And many others

Testicular Dysgenesis Syndrome

TESTICULAR DYSGENESIS

DISTURBEDSERTOLI CELLFUNCTION

DECREASEDLEYDIG CELLFUNCTION

IMPAIREDGERM CELLDIFFEREN-TIATION

ANDROGENINSUFFICIENCY

REDUCED SEMEN QUALITY

TESTICULAR

CANCER

HYPOSPADIAS

TESTICULAR MALDESCENT

Environmental factorsincl. endocrine disrupters

Genetic defectsincl. 45,X/46,XY and point mutations

CIS