In the first phase of FREIA, the “Discovery phase”, we looked for biological characteristics (biomarkers) that can be linked to female reproductive toxicity and test methods to measure this. For this, we use two well-understood EDCs: diethylstilbestrol (DES, a potent oestrogen receptor activator) and ketoconazole (a blocker of steroid hormone production).

Next, in the “Testing Phase”, we assessed how well our test methods and potential novel endpoints can identify EDCs that cause female reproductive toxicity, using the test compound propylparaben.

Here, we briefly describe the main findings from the first 54 months of the FREIA project, from January 2019 until June 2023. The full summaries of the periodic reports that were submitted to the European Commission can be downloaded below.


Exposure to ketoconazole diminishes the number of immature oocytes in cultivated human embryonic ovaries.
From women undergoing fertility treatment, fewer oocytes could be retrieved when levels of some EDCs were higher in the biological fluids surrounding the oocytes.

Female rats that were exposed to DES or ketoconazole in the womb started puberty at a later age than female rats that were not exposed. The hormone release in the brain of these animals was also delayed. Interestingly, fewer animals are needed to see this effect in the brain, which may reduce the need for test animals in the future.

Several cell models and computer models have been developed or improved. We focus on ovarian cells and targets that are involved in female reproductive health, such as the oestrogen receptor (target for oestrogen) and steroid hormone formation.

We have constructed sixteen possible pathways to explain how EDCs can cause female reproductive disorders. This will help us to pinpoint which test methods are needed to predict a chemical’s effect on female reproductive health.


The FREIA project aims to provide better test methods to identify human-made chemicals that disturb hormones and their actions on development and function of the reproductive system in women.

The FREIA findings point toward the following possible improvements:

  • The developing mammary gland is very sensitive for EDCs. Therefore, it may have added value to assess the mammary gland of rats after exposure to potential EDCs in the womb.
  • A quick screening of rat ovaries using ‘surface photo counting’ (SPC) methodology shows good predictive value in the assessment of ovulations and is simpler, faster, and more cost-effective than traditional histological assessment.
  • We found clear age-dependent changes in steroid hormones in rat blood. Exposure to EDCs did not changes these steroid hormone levels, which makes circulating steroid hormones a poor indicator of EDC exposure.
  • We did find many effects of EDCs on the formation of steroid hormones that could be linked to effects on reproductive cells directly:
    • We found that SCD (stearoyl-CoA desaturase) and DHCR7 (7-dehydrocholesterol Reductase), both involved in cholesterol biosynthesis, are potential biomarkers for effects of EDCs in human ovaries.
  • The KGN cell line may provide a valuable cell model to assess effects on ovary-specific endpoints.

Ultimately, we aim to integrate our newly identified biomarkers and sensitive endpoints with existing test systems from OECD to improve on future test methods and a strategy to determine the effect of an EDC on female reproductive development and health. 

We explored the association between the effects of EDCs on female fertility in women attending fertility clinics in Sweden and Estonia.

  • We provide additional evidence that the phthalate DEHP can negatively influence female fertility. In addition, several other chemicals, i.e. methylparaben and some PFAS, were identified that may harm ovarian function and contribute to female infertility.
  • We found that frequent use of perfumes was associated with higher phthalate levels. Henn’s egg consumption led to higher PFAS exposure. PFAS levels were also associated with certain fish consumption. We did not observe a correlation between the semi-persistent chemicals and use of plastics in microwave heating of food or flooring material.

We are currently collecting available scientific data on how humans, and women in particular, can be exposed to EDCs and what actions effectively can be taken to avoid exposure.