Why is Infertility Growing in Europe? Is there a link between different disorders of male reproductive organs and expo

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 womenJensen et al. IJA, 2008

3. Declining conception rates among cohorts of younger womenJensen 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 Puberty Birth CIS: Infantile sp-gonium ESC PGC Gonocyte Sp-gonium 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

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

16. 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 + 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 milk Via placenta 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 • 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 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

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 factorsincl. endocrine disrupters REDUCED SEMEN QUALITY DISTURBEDSERTOLI CELLFUNCTION IMPAIREDGERM CELLDIFFEREN-TIATION TESTICULAR CANCER CIS TESTICULAR DYSGENESIS HYPOSPADIAS DECREASEDLEYDIG CELLFUNCTION ANDROGENINSUFFICIENCY Genetic defectsincl. 45,X/46,XY and point mutations TESTICULAR MALDESCENT