Research progress on the effects of phthalates on reproductive health of childbearing population and their offspring

LIN Cai-yun, YIN Fei-fei, WANG Hui-zi, YANG Hua✉

1.Women and Children’s Medical Center of Hainan Province, Haikou 570206, China

2.Key Laboratory of Tropical Translational Medicine of Ministry of Education, Hainan Medical University, Haikou 571199, China

3.Hainan Medical University-The University of Hong Kong Joint Laboratory of Tropical Infectious Diseases, Hainan Medical University, Haikou 571199,China

Keywords:

ABSTRACT The total fertility rate of women in childbearing age showed a downward trend in China.In addition to the age and genetic factors, environmental endocrine disruption can also impair fertility.The impact of increasing new environmental pollutants on the couples in childbearing age has become a research hotspot recently.Phthalate acid esters (PAEs) is a common plasticizer in plastic products, which is widely found in toys, food packaging, construction materials, electronic and medical components, personal care products, office and school supplies and other plastic packaging products, and is the main substance of environmental pollution.Multiple studies have shown that PAEs can not only cause environmental and water pollution, but also have a variety of toxic effects such as reproductive toxicity, genotoxicity,immunotoxicity, neurotoxicity, teratogenicity, and carcinogenesis.Therefore, its impact on human health, especially on reproductive health of people of reproductive age and their offspring, cannot be ignored.However, the current epidemiological study of PAEs and new alternatives in reproductive health population is still controversial, and the toxicity mechanism is still in the exploration stage.This article through to PAEs of parental generation, children(including embryo) of reproductive development and the influence of genetic toxicity research progress at home and abroad to do a review, aims to promote effective control measures for the establishment of PAEs pollutants rather than on reproductive health risk prediction, thus for PAEs of adverse reproductive outcomes of reproductive stage of people provide a scientific basis for precision control and guidance.

In recent years, with the rapid development of social economy,the total fertility rate of women of childbearing age shows the trend of decreasing continuously.According to the data of the seventh national census, the total fertility rate of Chinese women of childbearing age is only 1.3%[1], indicating that China has entered the ranks of countries with low fertility rate in the world.At the same time, the infertility rate of Chinese couples of childbearing age is as high as 15.6%[2], and the spontaneous abortion rate has reached 15%[3].Therefore, the identification of new interventionable factors affecting the reproductive health of people of childbearing age will significantly reduce the downward risk of fertility, which is of great socio-economic development and public health significance.In addition to age and genetic factors, there is now substantial evidence that synthetic and naturally occurring environmental chemicals in food, water, air and consumer products can also cause impaired fertility[4].Endocrine-disrupting chemicals (EDCs), disrupting the“third generation of environmental pollutants”, has become another serious global environmental hazard after “greenhouse effect” and“ozone layer destruction”.Multiple evidences have shown that EDCs destroy human endocrine function by interacting with hormone receptors, interfering with hormone action or changing hormone synthesis, release, transport or metabolism, and may directly or indirectly damage the development and function of reproductive system[5-7].Perfluorinated and polyfluoroalkylate (PFASs),bisphenol A (bisphenol A), organopesticides (OPs) and pyrethroids(PYRs) were found to be significantly associated with decreased fertility in women of childbearing age.However, studies on the effects of new environmental pollutants, such as new plasticizers -phthalates, phthalate acid esters, PAEs, etc.on fertility of couples of childbearing age are generally very limited.At present, the epidemiological studies of PAEs and new alternatives in reproductive health are still controversial, and the mechanism of toxicity is still in the stage of exploration.This paper summarizes the current research progress on the effects of PAEs on human population and various animal reproductive systems at home and abroad, in order to further explore the relationship between PAEs and reproductive health of reproductive age people in the future.

1.Introduction of phthalates and phthalate acid esters(PAEs)

PAEs, also called phthalates, is the general name for esters formed by phthalates.At room temperature, it is a colorless transparent oillike viscous liquid, which is not volatile.It is difficult to dissolve in water but easy to dissolve in organic solvents, and belongs to medium polar substances[8-9].PAEs is a plasticizer commonly used in plastic products, widely found in toys, food packaging, building materials, electronic and medical parts, personal care products,office and school supplies and other plastic packaging products[10].PAEs can be divided into two categories: low molecular weight phthalates, LMWP, PAEs 6 carbon chains and high molecular weight phthalates, HMWP, PAEs ＞ 6 carbon chains[11].Di-2(ethylhexyl)-phthalate (DEHP) is one of the most productive PAEs compounds and has been the focus of recent studies.Because PAEs are combined with plastic products in a non-covalent way,and their service life is long and they are susceptible to changes in ultraviolet light and other factors, they are easy to release or migrate to the surrounding environmental media[12], and thus widely exist in soil[13], water [14], indoor dust[15], food[16] and other ecological environments.Therefore, it is considered as a new type of environmental pollutant.A number of studies have shown that PAEs can not only cause environmental and water pollution, but also have a variety of toxic effects such as reproductive toxicity,genetic toxicity, immunotoxicity, neurotoxicity, teratogenesis and carcinogenesis [17], so the impact on human health, especially on the reproductive health of people of childbearing age and their offspring cannot be ignored.

2.Effects of PAEs on parental reproductive health

2.1 Effects on the female reproductive system

Exposure to PAEs can affect the reproductive organs of female animals in different germlines, which may damage the female reproductive organs and related reproductive cells, change the morphology of reproductive tissues and affect steroid hormone levels, lead to abnormal development and function of the female reproductive system, and ultimately affect the growth and development of offspring.

2.1.1 Effects on female reproductive organs

In terms of population studies, a case-control study of premature ovarian failure (POF) and female phthalate exposure in China [18]found a significant inverse relationship between urinary phthalate metabolite concentration and serum estradiol level in most women.follicle-stimulatinghormone (FSH) levels were in positive correlation, suggesting that exposure to certain phthalates may impair ovarian function and increase the probability of premature ovarian failure.Another overseas one based on primary ovarian insufficiency (POI) female insufficiency and mono-butyl phthalate,A cross-sectional case-control study of MBP or its metabolite concentration[19] found a positive correlation between serum levels of phthalate metabolites in women with POI, of which MBP is the most important and may be a risk factor for the development and development of POI.Although some scholars have proposed that phthalates can adversely affect ovarian function, epidemiological studies in the population are very limited.

In animal studies, there are many reports related to the influence of female reproductive organs, including quail, zebrafish, mice,rats and so on.Li et al.[20] studied the ovaries of quail exposed to DEHP and its major metabolite, mono- (2-ethylhexyl) phthalate(MEHP).It was found that the receptor mediated signaling pathway could further inhibit the production of estrogen.In addition,interference in the regulation of the hypothalamic- piitary -tuitaryovarian axis (HPOA) can inhibit sex hormone synthesis, resulting in sex hormone secretion disorders.Park et al.[21] studied 21-day female zebrafish exposed to different doses of MEHP (0, 2, 10 and 50 μg/mL) and found that the levels of estradiol and progesterone in all MEHP exposed groups were significantly increased, while the number of ovulation of female zebrafish exposed to 50μg/mL MEHP was significantly decreased.Liu et al.[22] found that the ovaries of lactating female mice exposed to DEHP (maternal and F1 generation) had significantly lower expression level of 17βhydroxysterosteroid dehydrogenase 1 (HSD17B), and further reduced serum and ovarian estrogen production.Moreover, the proportion of DNA damage marker γH2AX in ovary increased, and the level of ovarian apoptosis also increased, and the proliferation of ovarian cells was inhibited.Chen et al.[23] exposed adult female zebrafish to dibutyl phthalate (DBP), diisobutyl phthalate (DIBP),and a combination of both for 30 d.By studying the ovarian histology, sex hormone levels and ovarian transcriptomics of the above three groups, it was found that the level of estradiol was significantly decreased in DBP and DIBP exposure groups, and the percentage of advanced/mature oocytes was also significantly decreased.Interestingly, there was no significant effect under joint exposure; When sequenced in transcriptomics, differentially expressed genes (DEGs) in the ovaries of the combined exposure group exhibited potential reproductive toxicity at the molecular level,suggesting that thecombined exposure group may have mediated the transcription of key genes in therelevant signaling pathways, Thus disrupting the reproductive ability of the female zebrafish.Fu et al.[24] conducted a randomized controlled study of ICR mice exposed to DEHP for 30 consecutive days and found that exposure to DEHP could cause significant reproductive toxicity, mainly represented by significant reduction of ovarian organ coefficient and estrogen levels.Takai et al.[25] found histopathological abnormalities in ovary, uterus and vagina by conducting toxicity studies on female rats with oral dose levels of 0, 300, 1000 and 3000 mg/kg DEHP for 2 weeks and 4 weeks respectively.In the group exposed to 300 mg/kg or above,the ovarian stromal cells vacuolated.In the group exposed to 1000 mg/kg or more, the ovarian atresia follicles increased.During the 4-week observation, the 3000 mg/kg dose exposure group showed new luteinemia, uterine atrophy, thinning of vaginal epithelial cells and mucocliquefaction.In addition, in the observation of fertility, the average estrous cycle of each dose exposure group was prolonged after exposure to DEHP: irregular estrous cycle appeared in the 3000 mg/kg dose exposure group, while the estrous cycle of the other dose exposure groups was within the normal range.Chiang et al.[26] in breeding adult female CD-1 mice after oral administration of DEHP or diisononyl phthalate (DINP) found that breeding immediately after administration did not affect fertility; However,after 3 months of administration, both DEHP and DINP exposure decreased the fertility of female mice.In addition, DINP exposure disrupted the estrous cycle of female mice; Exposure to DINP 9 months after administration was still significantly disrupting the estrous cycle of female mice and shortening mating time with male mice.Somasundaram et al.[27] studied lactating female F1 generation rats by intragastric administration of DEHP (1, 10, 100 mg·kg-1·d-1),and found that the weight of ovaries and uterus of adult female rats decreased significantly.At the same time, it also affected the structure of ovary, which showed that the arrangement of ovarian granulosa cells was disorganized and the morphology of oocyte was changed.The number of luteal corpus cells decreased, interstitial space increased, and serum levels of estradiol, testosterone, and progesterone decreased significantly, suggesting that exposure to DEHP during lactation may impair the reproductive system of female rats.All the above studies have shown that exposure to PAEs can affect the reproductive organs of female animals in different species lines, resulting in varying degrees of damage to the reproductive system.

2.1.2 Effects on egg quality

In terms of population studies, existing studies have shown that PAEs accelerate ovarian aging by interfering with the synthesis and metabolism of endogenous steroid hormones, destroying the growth pattern of follicles, increasing oxidative stress and leading to follicle death[28].In a cross-sectional study of follicular fluid in 194 women[29], it was found that most PAEs metabolites were detected in all follicular samples, Further analysis showed that mono-butyl phthalate (MBP) and mono-2-ethyl-5-hydroxylhexyl phthalate, of the metabolites of PAEs.MEHHP was positively correlated with ovarian steroid hormones, suggesting that PAEs metabolites in follicular fluid were associated with changes in reproductive hormone levels in follicles.This is consistent with the current research results that PAEs accelerate ovarian aging by interfering with the synthesis and metabolism of endogenous steroid hormones.In women of reproductive age seeking pregnancy aid treatment, PAEs exposure is associated with a significant decrease in Antral Follicle Counting(AFC)[30-31] and oocyte production[32-34].However, a study by Du et al.[35] found no correlation between follicular fluid or urine metabolite concentration and oocyte production.Moreover, Du et al.[36] found in a retrospective analysis based on 415 infertile women of childbearing age that there was a negative correlation between the concentration of PAEs metabolites in urine and the level of serum inhibin-b (INH-b).There was no correlation with serum antimullerain hormone (AMH) levels.Sacha et al.[37] observed a negative correlation between urinary concentrations of PAEs metabolites and AMH concentrations in preovulatory follicular fluid in women receiving pregnancy-assisted treatment, which may influence sinus follicular recruitment and reproductive outcomes.Therefore,existing studies on the correlation between PAEs exposure and the development of follicles and reproductive endocrine hormone levels in women of reproductive age are still controversial.

In animal studies, a study on the offspring of female SD rats(exposed to 1 20, 50, or 300 mg DEHP·kg-1·d-1)[38] found that exposure to DEHP during the embryonic period resulted in a decrease in adult estradiol levels during estrus, Follicle-stimulating hormone levels increased significantly before and after estrus.When the dose of DEHP was 50 mg·kg-1·d-1or above, the thickness of follicular membrane cell layer decreased.In terms of fertility,the reproduction of F1 generation was not affected, but when the exposure dose was 20 mg DEHP·kg-1·d-1, the number of pups was increased and the weight of pups was decreased in F3 generation.Therefore, despite the above effects, it does not lead to the outcome of infertility in the offspring.Liu et al.[39] injected 21-day-old ICR mice with DEHP (0, 100, 400 and 1600 mg·kg-1·d-1) for 6 weeks by gavage, and the results showed that the expression of follicular development related factors (including c-kit, kitl, gdf9 and atm) was significantly reduced.However, the expression levels of ovarian miRNA (let-7b, miR-17-5p, miR-181a and miR-151)were significantly increased, indicating that PAEs could inhibit the proliferation of follicular granulosa cells.Xu et al.[40] found that the number of follicles in the ovary decreased and the atretic follicles increased significantly in the infected female rats through continuous gavage of 600 mg/kg DEHP for 60 d.Similarly, FU et al.[41], in a randomized controlled study of 4-week-old ICR female mice injected with DEHP for 30 consecutive days, found that the number of follicles in female mice exposed to DEHP was reduced (primary follicles, secondary and sinus follicles were reduced, and the number of primary oocytes was significantly reduced).This was consistent with the findings of Liu et al.[22], indicating that DEHP exposure would affect follicle development.

In conclusion, PAEs may alter the morphology of reproductive tissues by destroying female reproductive organs and related reproductive cells, affect steroid hormone levels, reduce the number of follicles and the quality of eggs, and lead to abnormal development and function of female reproductive system, and ultimately affect the growth and development of offspring.

2.2 Effects on the male reproductive system

PAEs exposure will adversely affect the development of male reproductive organs and their reproductive cells, not only lead to the loss of testis and epididymis weight, testicular tissue damage, but also lead to the reduction of semen quality, and ultimately lead to the decline of male reproductive ability.

2.2.1 Effects on male reproductive organs

Adolescence is an important period of reproductive system development and maturity, and it also plays a key role in male reproductive system development.In terms of population research,previous studies on reproductive toxicity produced by PEAs substances and male reproductive organ damage all suggest that there is a direct or indirect relationship between them.In a domestic study of PAEs metabolites in urine of boys aged 7-14, it was found that the level of mono-butyl phthalate (MBP) in urine was inversely correlated with the volume of testis.The level of mono (2-ethyl-5-oxohexyl) phthalate (MEOHP) was also inversely related to pubic hair development[42].A study on children showed that PAEs exposure was correlated with adolescent development in boys, and exposure of mono-n-butyl phthalate (MnBP) in urine samples might be related to delayed pubic hair development in boys[43].A study on the correlation between PAEs exposure and Constitutional Delay of Growth and Puberty (CDGP) found that the concentration of PAEs in the urine of children in the CDGP group was significantly higher than that in the control group.In addition, urinary MBP,monoethylphthalate (MEP) and total phthalate concentrations were negatively correlated with serum testosterone levels, suggesting that PAEs could indirectly affect CDGP by decreasing serum testosterone levels[44].

In animal experimental studies, PAEs exposure has obvious reproductive toxicity on male animals, which can affect spermatogenesis and reproductive endocrine of animals, but the related mechanism is still unclear.The process of spermatogenesis includes the differentiation of spermatogenic cells, the function of supporting cells and the regulation of androgens.In the study of oral exposure to DEHP (250 and 500 mg·kg-1·d-1) in prepubertal male rats, Zhao et al.[45] observed that exposure to DEHP could worsen testicular histology, decrease testosterone level and increase apoptosis of testicular cells, suggesting that DEHP exposure could cause testicular injury.Sun Liu Jia et al.[46] exposed 4-week-old ICR male mice to DEHP (0.5, 50 and 500 mg/kg) by gavage for 35 days,and found that testicular tissue was damaged in both the medium and high dose groups, and the wet weight of testis in the high dose group decreased significantly.However, this result is contrary to the study of Capela et al.[47], which may be caused by the difference between exposure doses.At the same time, OUDIR et al.[48] studied male Wistar rats exposed to low doses of DEHP (0.5, 50, and 5 000 μg·kg-1·d-1) at 21 to 120 d postnatal, and found that low dose of DEHP not only reduced testis and epididymal weight and sperm count,but also reduced the number of testicular sertoli cells.In addition,Abarikwu et al.[49] exposed di-butyl phthalate (DnBP) (1 mL/kg)for 15 d, and found that the diameter of sperm tubules, epithelial height, epithelial germ cell count and tubule length of rats decreased significantly.These results indicate that DnBP exposure can cause testicular tissue damage in male rats.All the above studies indicated that PAEs exposure would adversely affect the development of male reproductive organs and their germ cells.

2.2.2 Effect on semen quality

In terms of affecting semen quality in men, multiple studies and meta-analyses in the population have shown that exposure to phthalate has higher levels of decreased sperm density and motility as well as morphological abnormalities[50].Xie et al.[51] observed in vitro studies on DBP and its main metabolite MBP and human sperm function that high levels of DBP and MBP accumulation in semen may further damage male sperm function by inhibiting sperm tyrosine phosphorylation.Lidia et al.[52] found that semen volume was negatively correlated with mono-isononylphthalate(MINP) and sperm concentration was significantly negatively correlated with MEP in a study evaluating the correlation between seven phthalate metabolites and semen quality in 105 subjects.This has also been confirmed in animal studies.Mondal et al.[53] studied low dose Diethyl phthalate (DEP) exposure in 8-week-old mice for 3 months and found that testicular tissue structure was associated with disturbed antioxidant ratio and many structural abnormalities in sperm.Sperm function decreased, indicating that long-term exposure to low dose of DEP had adverse effects on sperm count,motility, viability and morphology.Goudarzi et al.[54] found that adult male mice treated with DEHP (2 g/kg) significantly decreased sperm motility and serum testosterone level, suggesting that DEHP exposure could lead to the destruction of testicular function in adult mice.This was in line with the results of a study by Sun Liu Jia et al.[46], which found that the spermatozoa count and spermatomotility level of the epididymis in mice in the 500 mg/kg group significantly decreased.In general, PAEs exposure will not only lead to the loss of testis and epididymis weight, testicular tissue damage, but also lead to reduced semen quality, which will affect the reproductive system and ultimately lead to the decline of male reproductive ability.

3.Effects of PAEs on reproductive development and genotoxicity of offspring (including embryos)

Multiple population studies have shown that exposure to P AEs and its metabolites can interfere with the normal growth and development of progeny, and the degree of influence varies with the dose and duration of exposure.In a study on the influence of maternal serum PAEs concentration during pregnancy on the long-term development of male offspring reproductive system, it was found that the higher the concentration of DEHP and DINP metabolites in maternal serum during pregnancy, the smaller the volume of male testis.Therefore,the concentration of maternal PAEs may affect the reproductive function of male offspring[55].In a prospective cohort study of 1059 maternal and infant pairs, higher maternal urine PAEs were associated with pubic hair development during early puberty in both sexes[56].

Similar effects have been observed in animal studies.Brehm et al.[57] compared the number of follicles, estrous cycle, and sex hormone levels in multiple generations (F1, F2, and F3 generations)of female CD-1 mice exposed to different doses of DEHP (20 and 200 μg·kg-1·d-1and 500 and 750 mg·kg-1·d-1), and found that prenatal exposure to DEHP changed the estrous cycle in F1 generations.The incidence rate of ovarian cyst increased and the total follicle number decreased.In F2 generation, not only the genital distance is shortened, but also the number of follicles and the estrous cycle are changed.Follicle production is reduced in F3 generation.In addition,prenatal exposure to DEHP resulted in increased serum estradiol levels (F1 and F3 generations), decreased serum progesterone levels (F2 generations), decreased serum testosterone levels (F1,F2 and F3 generations), altered serum gonadotropin levels (F1 and F3 generations), and decreased serum INH-b levels (F1 and F3 generations).This suggests that prenatal exposure to DEHP can produce reproductive toxicity in female offspring.Rattan et al.[58]studied the effect of prenatal DEHP exposure on the ovarian function of female mouse offspring, and found that prenatal exposure to DEHP in F1 generation would reduce ovarian weight and follicle production, and increase estrogen level.In F2 generation, not only ovarian weight decreased, but also follicular formation and serum progesterone level decreased.In F3 generation, the formation of primary follicles was accelerated.This study also suggested that prenatal exposure to DEHP could adversely affect ovarian function in offspring or even multiple generations.In addition, Barakat et al.[59] found that CD-1 mice during pregnancy were given PAEs mixture orally until the birth day of male mice of F1 generation,and their reproductive function indexes were evaluated.The gonads,prostates and seminal vesicles of normal male mice and male mice exposed to PAEs mixture before delivery were compared.In the prenatal exposure group, testosterone concentration and mRNA expression of testicle-associated steroid-producing genes were significantly decreased, and sperm concentration and motility were also decreased, suggesting that exposure to PAEs during pregnancy may have a long-term effect on male offspring.Dobrzy et al.[60]found that when adult male mice are exposed to DEHP for 8 weeks,the sperm count and quality of their F1 offspring are not significantly abnormal, but the fetal mortality of their F2 offspring is significantly increased.Wang et al.[61] conducted single-cell RNA sequencing on ovarian cells of offspring mice after maternal exposure to DEHP,and found that DEHP exposure not only hindered the formation of primorial follicles, but also interfered with the development of follicles, inhibited the proliferative activity of ovarian granulosa cells, and further activated the regulation of their death.Moreover,DEHP exposure also disrupted the interaction between ovarian cells mediated by transforming growth factor-β signaling, resulting in DNA damage and increased apoptosis in germ cells and/or somatic cells.Ozkemahli et al.[62] conducted A randomized controlled experimental study in which pregnant SD rats were lavaged with DEHP and bisphenol A (BPA) in the stomach until laculation, and found that after mixed exposure, the levels of plasma testosterone and estradiol in the female offspring were significantly reduced, as well as the number of corpus luteum and the number of primordial follicles.It is further suggested that early combined exposure to DEHP and BPA may have adverse effects on the reproductive system of their female offspring.Therefore, exposure to PAEs during pregnancy and lactation may cause different degrees of damage to reproductive metabolism, growth and development and reproductive toxicity of the offspring.

4.Prospect

At present, the total fertility rate of Chinese women of childbearing age continues to decline.In addition to age and genetic factors,a large amount of evidence shows that synthetic and natural environmental chemicals in food, water, air and consumer products can also lead to impaired fertility, and exposure to PAEs may cause damage to the reproductive system of parents and offspring or even multiple generations, resulting in adverse reproductive outcomes.In recent years, more and more studies have focused on the correlation between exposure to non-persistent endocrine disruptors PAEs and female reproductive health, but the research on female reproductive toxicity is still not comprehensive, the dose of PAEs exposure has different effects on female reproductive system, the minimum dose causing toxic reactions is still unclear, and the conclusion of the research is always controversial.Some studies have found no significant association between PFAS exposure and reproductive hormone levels in women of reproductive age[63-66], while other studies have pointed to endocrine disrupting effects of PFAS[67-69].Previous studies were mostly limited to considering the association between individual PAEs and reproductive health outcomes, and the effects of mixed exposure to multiple PAEs were unknown.The existing studies are mostly limited to the exposure of animal models,and most of them are single pollutant exposure, which cannot fully reflect the actual human exposure.The actual internal exposure of the population was the cumulative effect of long-term low concentration of multiple PAEs.In addition, the source, level, route and dose of exposure are unknown.Therefore, how to evaluate the exposure level of PAEs in the population, how to reflect the current situation of mixed exposure of multiple PAEs in the study, and how to determine the exposure dose required for its influence are the directions we need to explore in the future.In the future, we need to further design rigorous animal toxicological model experiments that consider the influence of various confounding factors to carry out research on the mechanism of fertility decline caused by PAEs exposure, so as to explore the correlation between PAEs exposure and reproductive outcome of people of reproductive age, so as to improve the fertility of people of reproductive age.

In conclusion, in-depth research on the impact of PAEs exposure on the reproductive health of people of reproductive age and determine the new factors that can be intervened in promoting the reproductive health of people of reproductive age and their offspring, so as to further precise prevention and control of its harm, protect the reproductive health and offspring health of people of reproductive age, which has important public health significance for promoting social and economic development and ensuring the healthy growth of newborn population.