Relationship: 394



irregularities, ovarian cycle leads to impaired, Fertility

Upstream event


irregularities, ovarian cycle

Downstream event


impaired, Fertility

Key Event Relationship Overview


AOPs Referencing Relationship


AOP Name Adjacency Weight of Evidence Quantitative Understanding
Aromatase (Cyp19a1) reduction leading to impaired fertility in adult female non-adjacent Moderate

Taxonomic Applicability


Sex Applicability


Life Stage Applicability


Key Event Relationship Description


The ovarian cycle irregularities impact on reproductive capacity of the females that may result in impaired fertility:

1. Irregular cycles may reflect impaired ovulation. Extended vaginal estrus usually indicates that the female cannot spontaneously achieve the ovulatory surge of LH (Huang and Meites, 1975). The persistence of regular vaginal cycles after treatment does not necessarily indicate that ovulation occurred, because luteal tissue may form in follicles that have not ruptured. However, that effect should be reflected in reduced fertility. Conversely, subtle alterations of cyclicity can occur at doses below those that alter fertility (Gray et al., 1989).

2. Persistent or constant vaginal cornification (or vaginal estrus) may result from one or several effects. Typically, in the adult, if the vaginal epithelium becomes cornified and remains so in response to toxicant exposure, it is the result of the agent’s estrogenic properties (i.e., DES or methoxychlor), or the ability of the agent to block ovulation. In the latter case, the follicle persists and endogenous estrogen levels bring about the persistent vaginal cornification. Histologically, the ovaries in persistent estrus will be atrophied following exposure to estrogenic substances. In contrast, the ovaries of females in which ovulation has been blocked because of altered gonadotropin secretion will contain several large follicles and no corpora lutea. Females in constant estrus may be sexually receptive regardless of the mechanism responsible for this altered ovarian condition. However, if ovulation has been blocked by the treatment, an LH surge may be induced by mating (Brown-Grant et al., 1973; Smith, E.R. and Davidson, 1974) and a pregnancy or pseudopregnancy may ensue. The fertility of such matings is reduced (Cooper et al., 1994).

3. Significant delays in ovulation can result in increased embryonic abnormalities and pregnancy loss (Fugo and Butcher, 1966; Cooper et al., 1994).

4. Persistent diestrus indicates temporary or permanent cessation of follicular development and ovulation, and thus at least temporary infertility.

5. Prolonged vaginal diestrus, or anestrus, may be indicative of agents (e.g., polyaromatic hydrocarbons) that interfere with follicular development or deplete the pool of primordial follicles (Mattison and Nightingale, 1980) or agents such as atrazine that interrupt gonadotropin support of the ovary (Cooper et al., 1996). Pseudopregnancy is another altered endocrine state reflected by persistent diestrus. The ovaries of anestrous females are atrophic, with few primary follicles and an unstimulated uterus (Huang and Meites, 1975). Serum estradiol and progesterone are abnormally low.

6. Lengthening of the cycle may be a result of increased duration of either estrus or diestrus.

Evidence Supporting this KER


Biological Plausibility


In females, normal reproductive function involves the appropriate interaction of central nervous system, anterior pituitary, oviducts, uterus, cervix and ovaries. During the reproductive years the ovary is the central organ in this axis. The functional unit within the ovary is the follicle which is composed of theca; granulosa cells and the oocyte. The somatic compartment synthesizes and secrets hormones (steroids and growth factors) necessary for the orchestration of the inter-relationship between the other parts of the reproductive tract and the central nervous system. Oestrus cycle is under strict hormonal control, therefore perturbations of hormonal balance lead to perturbations of normal cyclicity (change in number of cycles or duration of each phase) and/or ovulation problems leading to impaired female reproductive function. However, there are other mechanisms that might result in impaired fertility (e.g cellular maturation in ovary).

Empirical Evidence


Many chemicals are found to interfere with reproductive function in the female. This interference is commonly expressed as a change in normal morphology of the reproductive tract or in ovarian cycle irregularities (disturbance in the duration of particular phases of the estrous cycle and/or ovulation problems). Monitoring estrous cyclicity provides a means to identify alterations in reproductive functions which are mediated through nonestrogenic as well as estrogenic mechanisms (Blasberg, Langan, & Clark, 1997), (Clark, Blasberg, & Brandling-Bennett, 1998). Adverse alteration in the nonpregnant female reproductive system have been observed at dose levels below those that result in reduced fertility or produce other overt effects on pregnancy or pregnancy outcomes. A disruption of cycling caused by xenobiotic treatment can induce a persistent estrus, a persistent diestrus, an irregular pattern with cycles of extended duration and ovulation problems. Common classes of chemicals have been shown to cause cycle irregularities in rats, humans, and non-human primates. Examples include the polychlorinated biphenyls (PCBs) and dioxins, which are associated with such irregularities in rats and humans (e.g (Li, Johnson, & Rozman, 1995) (Meerts et al., 2004), (Chao, Wang, Lin, Lee, & Päpke, 2007) and various agricultural pesticides, including herbicides, fungicides, and fumigants for review see (Bhattacharya & Keating, 2012),(Bretveld, Thomas, Scheepers, Zielhuis, & Roeleveld, 2006).

Compound class


AO:ovarian cycle irregularities

AO:Impaired fertility


Phthalates (DEHP)


5-400 mg/kg/day females differed from the control in the relative amount of time spent in oestrous stages

number of live pups (P0) reduced (400 mg/kg/day)

(Blystone et al., 2010)

Phthalates (DEHP)


irregular estrous cycles (3,000 mg/kg/day)

slight decline in pregnancy rate (3,000 mg/kg/day)

(Takai et al., 2009)

Phthalates (DEHP)


dose-dependent decreases in fertility

(Lamb, Chapin, Teague, Lawton, & Reel, 1987)

Phthalates (DEHP)


No change

abortion rate of 100% in F0 dams (500-mg/kg/day)

(Schmidt, Schaedlich, Fiandanese, Pocar, & Fischer, 2012).

Phthalates (DEHP)


dose-dependent effect on the duration of the estrous cycles shortening of the ovulatory cycles due mainly to a reduction in the size and lifespan of CL

(Herreros, Gonzalez-Bulnes, et al., 2013)

Phthalates (DEHP)


No effect on ovulatory efficiency

(Herreros, Encinas, et al., 2013)

Phthalates (DEHP)


No changes in F0, increase of cycle by 0.4 day in F1 at 10,000ppm

18% and 21% decrease in live pups/litter F0 at 7500ppm and 10,000ppm respectively, no viable litters (F1 10,000 ppm ~643.95mg/kg/day)

(NTP, 2005)

Phthalates (DEHP)


Deficit in growing follicles and corpora lutea

4-fold increase in females with stillborn pups in F0 at 9000ppm 2.1-fold Postimplantation loss in F0 at 9000ppm

(Schilling, K., Deckardt. K., Gembardt, Chr., and Hildebrand, 1999)

Phthalates (DEHP)


prolong the estrous cycle, anovulation

(Davis, Maronpot, & Heindel, 1994)


Reduced fertility and fecundity

(Wolf et al., 1999)

Organochlorine (methoxychlor)


Decreased number of cycles, extended diestrus and estrus

(Laws, 2000)

Organotins tributyltin chloride (TBTCl)


At 125 ppm vaginal opening and impaired estrous cyclicity

(Ogata et al., 2001)

Table 1 Summary the empirical evidence supporting the KER.

It is known that exposure to 17-β-estradiol can disrupt the normal 4- to 5-day estrous cycle in adult female rats by inducing an extended period of diestrus consistent with pseudopregnancy within 5–7 days after the exposure (Gilmore & McDonald, 1969). This is due to the estrogen-dependent increase in prolactin that rescues ovarian corpora lutea and the subsequent synthesis and release of progesterone (Cooper, R. L., and Goldman, 1999). Significant evidence that the estrous cycle (or menstrual cycle in primates) has been disrupted should be considered an adverse effect (OECD, 2008).

Uncertainties and Inconsistencies


Chemicals may be found to interfere with reproductive function in the female. This interference is commonly expressed as a change in normal morphology of the reproductive tract or a disturbance in the duration of particular phases of the estrous cycle. However, menstrual cyclicity is affected by many parameters such as age, nutritional status, stress, exercise level, certain drugs, and the use of contraceptive measures that alter endocrine feedback. In nonpregnant females, repetitive occurrence of the four stages of the estrous cycle at regular, normal intervals suggests that neuroendocrine control of the cycle and ovarian responses to that control are normal. Even normal, control animals can show irregular cycles. However, a significant alteration compared with controls in the interval between occurrence of estrus for a treatment group is cause for concern. Generally, the cycle will be lengthened or the animals will become acyclic. Therefore changes in cyclicity should be interpreted with caution and not judged adverse without a comprehensive consideration of additional relevant endpoints in a weight-of-evidence approach.


Two generation studies by Tyl et al with Butyl benzyl phthalate (BBP) did not observe effects in F0 females on any parameters of estrous cycling, mating, or gestation. However, F1 females carrying F2 litters at and reduced number of total and live pups/litter at birth, with no effects on pre- or postnatal survival (Tyl et al., 2004).

Quantitative Understanding of the Linkage


Response-response Relationship




Known modulating factors


Known Feedforward/Feedback loops influencing this KER


Domain of Applicability


In many instances, human female reproductive toxicity of an agent is suspected based on studies performed in experimental animals. The neuroendocrinology, steroid biochemistry, and other physiologic events in the females of most small experimental species often used (mouse, rat, hamster) are similar in their susceptibility to disruption by toxicants (Massaro, 1997).

Although the assessment of the human ovarian cycle may have a variety of biomarkers distinct from those in rats, many of the underlying endocrine mechanisms associated with successful follicular development, ovulation, pregnancy, and parturition are homologous between the two (for review see (Bretveld et al., 2006). For this reason, a toxicant-induced perturbation of ovarian cycles in female rats suggest that a compound may function as a reproductive toxicant in human females.


  • environmental air pollution (Mohallem et al., 2005)
  • phthalates (DEHP)
  • abortion rate of 100% in F0 dams in the 500-mg/kg/day was observed, in F1 females found that the total number of F2 embryos (exposed to DEHP only as germ cells) was not impaired. However, in the 0.05- and 5-mg DEHP groups, 28% and 29%, respectively, of the blastocysts were degenerated, compared with 8% of controls (Schmidt et al., 2012).
  • Lamb et al. studied fertility effects of DEHP in mice (both sexes) and found that DEHP caused dose-dependent decreases in fertility. DBP exposure resulted in a reduction in the numbers of litters per pair and of live pups per litter and in the proportion of pups born alive at the 1.0% amount, but not at lower dose levels. A crossover mating trial demonstrated that female mice, but not males, were affected by DBP, as shown by significant decreases in the percentage of fertile pairs, the number of live pups per litter, the proportion of pups born alive, and live pup weight. DHP in the diet resulted in dose-related adverse effects on the numbers of litters per pair and of live pups per litter and proportion of pups born alive at 0.3, 0.6, and 1.2% DHP in the diet. A crossover mating study demonstrated that both sexes were affected. DEHP (at 0.1 and 0.3%) caused dose-dependent decreases in fertility and in the number and the proportion of pups born alive. A crossover mating trial showed that both sexes were affected by exposure to DEHP. These data demonstrate the ability of the continuous breeding protocol to discriminate the qualitative and quantitative reproductive effects of the more and less active congeners as well as the large differences in reproductive toxicity attributable to subtle changes in the alkyl substitution of phthalate esters (Lamb et al., 1987).

Rat phthalates (DEHP)

  • female rats exposed to a high dose of DEHP (3,000 mg/kg/day) had irregular estrous cycles and a slight decline in pregnancy rate (Takai et al., 2009). At 1,000 mg/kg bw/day over a period of 4 weeks did not disturb female fertility or early embryo development.
  • There was significant evidence that 5, 15, 50, and 400 mg /kg/day females differed from the control females in the relative amount of time spent in oestrous stages, however no changes were revealed in the number of females with regular cycles, cycle length, number of cycles, and in number of cycling females across the dose groups as compared to the control females The litter size (number of live pups) produced by the P0 generation was significantly reduced in the 400 mg/kg/day dose group (Blystone et al., 2010).


Studies showing a correlation between decreased fertility and;

  • professional activity (Olsen, 1994)
  • phthalates (DEHP) In occupationally exposed women to high concentration of phthalates exhibit hypoestrogenic anovulary cycles and was associated with decreased pregnancy rate and higher miscarriage rates (Aldyreva,M.V.,Klimove,T.S.,Iziumova,A.S.,Timofeevskaia,L.A., 1975).
  • smoking (Hull, North, Taylor, Farrow, & Ford, 2000)
  • the use of certain drugs or radiation exposure (Dobson & Felton, 1983)

For the taxonomic applicability see also the Table 1.



Aldyreva,M.V.,Klimove,T.S.,Iziumova,A.S.,Timofeevskaia,L.A. (1975). The effect of phthalate plasticizers on the generative function. Gig.Tr.Prof.Zabol., (19), 25–29.

Bhattacharya, P., & Keating, A. F. (2012). Impact of environmental exposures on ovarian function and role of xenobiotic metabolism during ovotoxicity. Toxicology and Applied Pharmacology, 261(3), 227–35. doi:10.1016/j.taap.2012.04.009

Blasberg, M. E., Langan, C. J., & Clark, A. S. (1997). The effects of 17 alpha-methyltestosterone, methandrostenolone, and nandrolone decanoate on the rat estrous cycle. Physiology & Behavior, 61(2), 265–72.

Blystone, C. R., Kissling, G. E., Bishop, J. B., Chapin, R. E., Wolfe, G. W., & Foster, P. M. D. (2010). Determination of the di-(2-ethylhexyl) phthalate NOAEL for reproductive development in the rat: importance of the retention of extra animals to adulthood. Toxicological Sciences : An Official Journal of the Society of Toxicology, 116(2), 640–6. doi:10.1093/toxsci/kfq147

Bretveld, R. W., Thomas, C. M. G., Scheepers, P. T. J., Zielhuis, G. A., & Roeleveld, N. (2006). Pesticide exposure: the hormonal function of the female reproductive system disrupted? Reproductive Biology and Endocrinology : RB&E, 4(1), 30. doi:10.1186/1477-7827-4-30

Chao, H.-R., Wang, S.-L., Lin, L.-Y., Lee, W.-J., & Päpke, O. (2007). Placental transfer of polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls in Taiwanese mothers in relation to menstrual cycle characteristics. Food and Chemical Toxicology : An International Journal Published for the British Industrial Biological Research Association, 45(2), 259–65. doi:10.1016/j.fct.2006.07.032

Clark, A. S., Blasberg, M. E., & Brandling-Bennett, E. M. (1998). Stanozolol, oxymetholone, and testosterone cypionate effects on the rat estrous cycle. Physiology & Behavior, 63(2), 287–95.

Cooper, R. L., and Goldman, J. M. (1999). Vaginal cytology. In An Evaluation and Interpretation of Reproductive Endpoints for Human Health Risk Assessment. Washington. Davis, B. J., Maronpot, R. R., & Heindel, J. J. (1994). Di-(2-ethylhexyl) phthalate suppresses estradiol and ovulation in cycling rats. Toxicology and Applied Pharmacology, 128(2), 216–23. doi:10.1006/taap.1994.1200

Dobson, R. L., & Felton, J. S. (1983). Female germ cell loss from radiation and chemical exposures. American Journal of Industrial Medicine, 4(1-2), 175–90.

Gilmore, D. P., & McDonald, P. G. (1969). Induction of prolonged diestrus in the rat by a low level of estrogen. Endocrinology, 85(5), 946–8. doi:10.1210/endo-85-5-946 Herreros, M. A., Encinas, T., Torres-Rovira, L., Garcia-Fernandez, R. A., Flores, J. M., Ros, J. M., & Gonzalez-Bulnes, A. (2013). Exposure to the endocrine disruptor di(2-ethylhexyl)phthalate affects female reproductive features by altering pulsatile LH secretion. Environmental Toxicology and Pharmacology, 36(3), 1141–9. doi:10.1016/j.etap.2013.09.020

Herreros, M. A., Gonzalez-Bulnes, A., Iñigo-Nuñez, S., Contreras-Solis, I., Ros, J. M., & Encinas, T. (2013). Toxicokinetics of di(2-ethylhexyl) phthalate (DEHP) and its effects on luteal function in sheep. Reproductive Biology, 13(1), 66–74. doi:10.1016/j.repbio.2013.01.177

Hull, M. G., North, K., Taylor, H., Farrow, A., & Ford, W. C. (2000). Delayed conception and active and passive smoking. The Avon Longitudinal Study of Pregnancy and Childhood Study Team. Fertility and Sterility, 74(4), 725–33.

Lamb, J. C., Chapin, R. E., Teague, J., Lawton, A. D., & Reel, J. R. (1987). Reproductive effects of four phthalic acid esters in the mouse. Toxicology and Applied Pharmacology, 88(2), 255–69.

Laws, S. C. (2000). Estrogenic Activity of Octylphenol, Nonylphenol, Bisphenol A and Methoxychlor in Rats. Toxicological Sciences, 54(1), 154–167. doi:10.1093/toxsci/54.1.154

Li, X., Johnson, D. C., & Rozman, K. K. (1995). Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on estrous cyclicity and ovulation in female Sprague-Dawley rats. Toxicology Letters, 78(3), 219–22.

Massaro, E. J. (Ed.). (1997). Handbook of Human Toxicology, Volume 236. Taylor & Francis.

Meerts, I. A. T. M., Hoving, S., van den Berg, J. H. J., Weijers, B. M., Swarts, H. J., van der Beek, E. M., … Brouwer, A. (2004). Effects of in utero exposure to 4-hydroxy-2,3,3’,4',5-pentachlorobiphenyl (4-OH-CB107) on developmental landmarks, steroid hormone levels, and female estrous cyclicity in rats. Toxicological Sciences : An Official Journal of the Society of Toxicology, 82(1), 259–67. doi:10.1093/toxsci/kfh251

Mohallem, S. V., de Araújo Lobo, D. J., Pesquero, C. R., Assunção, J. V., de Andre, P. A., Saldiva, P. H. N., & Dolhnikoff, M. (2005). Decreased fertility in mice exposed to environmental air pollution in the city of Sao Paulo. Environmental Research, 98(2), 196–202. doi:10.1016/j.envres.2004.08.007

NTP. (2005). Multigenerational Reproductive Assessment by Continuous Breeding when Diethylhexylphthalate (CAS 117-81-7).

OECD. (2008). No 43: Guidance document on mammalian reproductive toxicity testing and assessment.

Ogata, R., Omura, M., Shimasaki, Y., Kubo, K., Oshima, Y., Aou, S., & Inoue, N. (2001). Two-generation reproductive toxicity study of tributyltin chloride in female rats. Journal of Toxicology and Environmental Health. Part A, 63(2), 127–44. doi:10.1080/15287390151126469

Olsen, J. (1994). Is human fecundity declining--and does occupational exposures play a role in such a decline if it exists? Scandinavian Journal of Work, Environment & Health, 20 Spec No, 72–7.

Schilling, K., Deckardt. K., Gembardt, Chr., and Hildebrand, B. (1999). Di-2-ethylhexyl phthalate – two-generation reproduction toxicity range-finding study in Wistar rats. Continuos dietary administration.

Schmidt, J.-S., Schaedlich, K., Fiandanese, N., Pocar, P., & Fischer, B. (2012). Effects of di(2-ethylhexyl) phthalate (DEHP) on female fertility and adipogenesis in C3H/N mice. Environmental Health Perspectives, 120(8), 1123–9. doi:10.1289/ehp.1104016

Takai, R., Hayashi, S., Kiyokawa, J., Iwata, Y., Matsuo, S., Suzuki, M., … Deki, T. (2009). Collaborative work on evaluation of ovarian toxicity. 10) Two- or four-week repeated dose studies and fertility study of di-(2-ethylhexyl) phthalate (DEHP) in female rats. The Journal of Toxicological Sciences, 34 Suppl 1(I), SP111–9.

Tyl, R. W., Myers, C. B., Marr, M. C., Fail, P. a, Seely, J. C., Brine, D. R., … Butala, J. H. (2004). Reproductive toxicity evaluation of dietary butyl benzyl phthalate (BBP) in rats. Reproductive Toxicology (Elmsford, N.Y.), 18(2), 241–64. doi:10.1016/j.reprotox.2003.10.006

Wolf, C., Lambright, C., Mann, P., Price, M., Cooper, R. L., Ostby, J., & Gray, L. E. (1999). Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p’-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differen. Toxicology and Industrial Health, 15(1-2), 94–118. doi:10.1177/074823379901500109