1. 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

2. Declining conception rates among cohorts of younger women Jensen et al
Declining conception rates among cohorts of younger women Jensen et al. IJA, 2008

3. Declining conception rates among cohorts of younger women Jensen et al
Declining conception rates among cohorts of younger women Jensen et al. IJA, 2008

4. 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?

5. Structure of the Testis

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

7. Gene expression in CIS cells in relation to germ cell differentiation
PGC
ESC
Gonocyte
Infantile
sp-gonium
Birth
Puberty
Sp-gonium
CIS:
Spermatocytes
Spermatids
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:
Pluripotency, self-renewal, survival in undifferentiated stage
Regulation of cell cycle,
DNA repair (mitotic division)
Germ cell specific
e.g. RNA processing

8. Rates in testis cancer
During the many decades there have been a steadly increase in testicular cancer incidence in all the Nordic countries, with Denmark having the highest incidence followed by Norway, then Sweden and Finland.
However, recently there have been a change as the incidence have decreased for denmark in the period
In the search for causal factors irregularities from the general tendencies can often provide new clues as have been seen forexample with the war time effect, with men being born during the second world war had a lower risk of TC in many western countries.

9. Lessons from recent biological and epidemiological research on 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
Increasing incidence of testis cancer. Other male reproductive problems are also becomming more common!

10. Perinatal exposure of Rats to Phthalates (DEHP, DBP)
May cause a ”phthalate syndrome” in male offspring, including
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)

11. Phthalate Effects on Testis
Dysgenetic areas in the testes on d4 postnatal after in utero exposure to DBP (500 mg/kg/day) Fisher et al. Hum Reprod, 2003)
Control
DBP
Brown = smooth muscle actin

12. The Rat Phthalate Syndrome Resembles Human Problems

13. 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

14. 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

15. Collection of breast milk
Breast milk: post nataly month 1, n=130
Serum samples from the children at 3 months, n= 96
Btreats milk samples wree collected from 1 month after birth as additive aliquots over consecutive feedings, to obtain an everage exposure level during the first months of life. They were frozen at home and delivered to us at the 3 months exam of the child. In the 130 we selecetd for chemical analyseis, we were successful in obtaining a serum sample form 96 children at three months of age as well.

16. mEP, mBP: mBP: mMP, mEP, mBP: miNP:
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
- 15% free testosterone
+ 26, +19, +18 % LH /free testosterone ratio
These effects were seen also with some of the other metabolites, and I want to draw your attention especially to miNP, the phthalate foudn in highest concentration in milk, which was associaed with LH. Together, the effects seen fit with an anti-androgenic effect which triggers a higher LH drive.
+ 97% LH
Main KM et al. EHP 2006

17. 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

18. 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

19. Mono-butyl-phthalate in serum during 24 hours
Janjua NR, Mortensen GK, Skakkebæk NE, Wulf HC, Andersson AM, 2008.

20. It seems safe to conclude….
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.
Janjua NR, Mortensen GK, Skakkebæk NE, Wulf HC, Andersson AM, 2008.

21. Effects of Mixtures of Endocrine Disrupting Substances
Ulla Hass  
Dept. of Toxicology and Risk Assessment, National Food Institute, Danish Technical University, Denmark

22. Design of EDEN studies Anti-androgen alone or in mix
Young adults: Malformations, semen quality, behaviour
Via placenta
Via milk
Birth
Pups: AGD, nipple retention, reproductive organs, gene expression

23. Malformations of external reproductive organs in adult male offspring
1) Completely split penis and visible os penis, i.e. marked hypospadia 2) Blind vaginal opening

24. Something from Nothing
0% + 0% + 0% ~ 60%
Christiansen et al 2008, Int. J. Androl. 31

26. Differences in prevalence of genital abnormalities between Denmark and Finland (From Boisen et al. Lancet 2004;Boisen et al. JCEM 205)
Denmark
Finland

27. 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

28. 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?

29. Chemicals in breast milk from mothers of newborn boys
Danish
Finnish
*: p<0.05

30. 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

31. 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

32. Testicular Dysgenesis Syndrome
Environmental factors incl. endocrine disrupters
REDUCED SEMEN QUALITY
DISTURBED SERTOLI CELL FUNCTION
IMPAIRED GERM CELL DIFFEREN-TIATION
TESTICULAR CANCER
CIS
TESTICULAR DYSGENESIS
HYPOSPADIAS
DECREASED LEYDIG CELL FUNCTION
ANDROGEN INSUFFICIENCY
Genetic defects incl. 45,X/46,XY and point mutations
TESTICULAR MALDESCENT