Study I of apoptotic markers in semen is to our knowledge the first paper in- vestigating demographic and hormonal characteristics in fertile men and the two markers. Few studies have investigated Fas positivity in human ejaculated sperm from infertility clients in relation to semen characteristics (19;26;27;29;30) or genetic variations of Fas or FasL (141;142).
Our results point toward an impaired semen quality when the men had low levels of Fas positivity, not corresponding with previous observations in infer- tility clients (19;26). However, high levels of Fas antigen in ejaculated bull sperm have been found in three fertile bulls (143). As observable in Figure 5, a tendency towards a U shaped curve occurs for Fas according to normal mor- phology and head defects. Though this is speculative, it might indicate that men with average percentage normal morphology have low levels of Fas posi- tive sperm cells, whereas men with low or high percentage normal morpholo- gy have high levels of Fas positive sperm cells. This is in line with two previ- ous studies in which they observed low normal morphology in men with high percentage Fas positivity (19;26). This did not seem to be the case for sperm concentration and count.
We did not observe associations between demographic characteristics and the two apoptotic markers, indicating that lifestyle factors like BMI, smoking sta- tus and alcohol or coffee consumption, as well as personal factors like disease and age did not affect the levels of the two markers like observed for semen quality (139).
Levels were highly differentiated in the three study countries with a doubling of Fas positivity in Polish men. Genetic factors might therefore be important. A Chinese study investigating genetic variants of Fas in men seeking infertility treatment showed that men carrying specific Fas polymorphisms were more likely to have lower sperm concentration and higher TUNEL positivity (141). A study by Wang et al. (2009) showed significantly different polymorphisms of FasL in infertile and fertile men, with infertile men carrying certain FasL genotypes which increased the risk of idiopathic azoospermia or severe oligo- zoospermia (142).
The median percentage Fas positivity was high in Poland (42%), with more moderate levels in Greenland (19%) and Ukraine (17%). Several studies using flow cytometry have not been able to measure Fas or only low percentage Fas positivity on ejaculated sperm (27;29;30), whereas studies analyzed using immunofluorescence have shown higher Fas levels (19;26). The discrepancy between our results and earlier studies might be due to our use of an immuno- fluorescence flow cytometry method or genetic differences in study popula- tions.
We did not observe any associations with Bcl-xL, except for a positive correla- tion with Fas as observed by Sakkas et al. (2002). Our results did not corre- spond to results of a study examining leftover semen submitted for semen analysis, which observed proportionally higher levels of Bcl-xL on immature sperm cells (31).
In Study III, we investigated four PFASs and sperm DNA integrity, apoptotic markers and reproductive hormones. To our knowledge this is the first study investigating DNA damage and apoptosis in humans. Animal studies suggest adverse effects of some PFASs on TUNEL positive and apoptotic germ cells, and structural damage of the testis, probably due to a down regulation in Bcl-2 and an up regulation of Fas (85;86). We observed a positive association be- tween PFOA and TUNEL positive spermatozoa in men from Greenland, though
contrary, point estimates in Poland and Ukraine were in a negative direction. Our result in Greenland is in line with an animal study of 7-9 weeks old rats fed PFOA at doses of 0.31, 1.25, 5 or 20 mg/kg bw/day (85).
The no observed adverse effect level (NOAEL) for developmental toxicity is 0.1 mg/kg bw/day in rats, comparable with a tolerable daily intake (TDI) of 0.0015 mg/kg bw/day in humans (144). Averagely the general population is dietary exposed to 0.000002 mg/kg bw/day, which is well below the TDI (144). The daily intake (DI) might increase a little when taking other exposure pathways, like inhalation, and other oral exposures than food and water into account, but will probably not exceed the TDI (145). Therefore observations in animal studies highly exposed to PFASs might not be predictive for humans. Six studies have investigated PFASs and reproductive hormones. Some studies have shown associations with hormones (96;97;99), though some have not (90;98;101). None observed associations between PFOA and SHBG like we did. We did, however, observe positive relationships in all the study countries. In Study IV of HCB and male reproductive biomarkers, the Ukrainian men had high levels of serum HCB compared to other study populations (108-110;146). We observed a positive association between HCB and SHBG, and a negative association for FAI, both in men from Ukraine. In men from Poland, who have serum HCB levels comparable to previous study populations, borderline sig- nificant associations between HCB and the two outcomes emerged. No associ- ations arose for the Greenlandic study population.
In animal models high levels of HCB increases hepatic weight, concentrations of hepatic microsomal protein, cytochrome P-450, cytochrome b5, and cyto- chrome c reductase activity which increases metabolism of testosterone and decreases the serum concentrations of testosterone (147). HCB has further been associated with thyroid hormones which increases hepatic SHBG pro- duction (146;148). Results in animal studies might therefore fit our results of SHBG. Of the four previous epidemiologic studies investigating HCB levels and reproductive hormones three analyzed SHBG and calculated FAI. Hagmar et al. (2001) and Ferguson et al. (2013) reported significantly lower FAI in the crude analysis, but the associations disappeared after control by confounders (109;111). Contrary to our results and observations in crude analysis of oth- ers, Dhooge et al. (2011) reported positive associations between HCB and FAI in a population of boys (median age of 14 years). Ferguson et al. (2013) re- ported negative relationship between HCB and SHBG only in crude analysis. SHBG was not positively associated with HCB in any study but ours. Biological- ly, free testosterone (FAI) decreases when SHBG increases because of its bind- ing of testosterone in plasma. Our result of relationships between high HCB and higher SHBG and lower FAI levels are thus biologically plausible.
The main conclusion in the literature review of DEHP and its metabolites and human male reproductive function was the relationships between DEHP and testosterone, although only four of the eleven studies showed statistically sig- nificant negative associations. Possibly, the decreased testosterone resulted in a tendency toward a reduction in estradiol, though insignificant.
The two semen outcomes, sperm concentration and motility, might be nega- tively associated with DEHP. Eight out of the 13 papers investigating sperm concentration and motility showed negative relationships, two or four signifi- cantly so, respectively. The two statistical significant studies of sperm concen- tration and two of the significant associations for motility were DEHP meas- ured in semen (123;149). During absorption, the diester phthalate (DEHP) is rapidly hydrolyzed into monoesters and further metabolized at different posi- tions in the ester chain by oxidation and hydroxylation into secondary metab- olites (43). Assessment of the mother compound raises serious concerns since DEHP is not stable in serum, and probably also in semen, because the lipase activity rapidly metabolizes the di-ester into mono-esters. Furthermore, sam- ples may be contaminated ex-vivo with DEHP during sampling, storage and handling in the laboratory, because DEHP may occur in many products used to perform these procedures (150). We are therefore cautious towards conclud- ing on the evidence of decreased sperm concentration and motility by DEHP exposure. Several studies showed significant relationship with semen quality, but often the results were inconsistent between studies, indicating that DEHP is not strong enough to impair semen quality.
We did, however, observe a higher proportion of damaged DNA in five out of six studies (84;117;118;120;122). A study reported high reactive oxygen spe- cies (ROS) generation in spermatozoa of occupationally DEHP exposed men compared to men not occupationally exposed (151). Evidence suggests in- creased generation of ROS by phthalates followed by induced oxidative stress which might be the mechanism behind the anti-androgenic action of phthalates on male reproductive function (35;152). DNA damage can originate from oxidative stress which corresponds well to the results observed in the review with more DNA fragmentation in highly DEHP exposed men.
The discrepancies between studies in results for several of the outcomes could be due to that not all metabolites were measured in all studies, methodological limitations, different study populations and methods used. The inconsistent results can also be suggestive of weak associations between DEHP and male reproductive function. With too weak effect to prolong TTP.
In our study of phthalate metabolites and male reproductive function (Study V) we did not investigate DNA damage, but we observed lower testosterone concentrations in men highly exposed to DEHP or DiNP metabolites. The re- sults fit observations from previous studies of DEHP metabolites in animal models (67;70;153) and humans (84;123;154), but not for DiNP (78;83;84). Evidence points towards reduced testosterone production by DEHP due to impaired steroidogenesis in the Leydig cell in animal models and in vitro (67;70;153;155). It is difficult to compare results from animal models with human studies since animals often are highly exposed to the chemical of inter- est, in controlled environments, e.g. Helal (2013) observed decreased testos- terone in rats exposed to 300 mg DEHP/kg bw/day for 6 weeks (68). Based on anti-androgenic evidence from animal models, the European Food Safety Au- thorities (EFSA) has established TDI values for the purpose of the public health (156). The TDI for DEHP is 0.05 mg/kg bw/day and is based on testicu-
lar toxicity in animal models with NOAEL at 5 mg/kg bw/day with an uncer- tainty factor of 100 (156), whereas the TDI for DiNP is 1.5 mg/kg bw/day. These levels are far below exposure levels in animal models of diminished testosterone production (68) and still relatively high compared to DI calculat- ed in adult humans (median DI of DEHP = 0.002 mg/kg/bw/day in an Belgian population (157) and median dietary DI of DEHP or DiNP= 0.0004 mg/kg/bw/day in a Norwegian population (158)). It is important to note that the DI does not balance the day-to-day variability in phthalate exposure, since it is based on single spot urine (157) or calculated from foods and beverages (158).
In the risk assessment of phthalates the DI and TDI of one phthalate might not be useful. Several other chemicals to which we are exposed daily might act anti-androgenic. A study has shown that MEHP neither had agonistic nor an- tagonistic effects on the androgen receptor (AR), though when analyzed in a mixture with other weak anti-androgenic chemicals at no-observed-effect concentrations an additive antagonistic effect on the AR was observed (159). The effects in humans might be a consequence of mixtures of correlated, anti- androgenic xenobiotics here among DEHP and DiNP metabolites.
In Study VI of phthalate serum levels and couple TTP, we observed shorter TTP in women and men from Greenland with high Proxy-MEHP serum levels, but without reaching statistical significance for the OR for infertility. Similar results were observed for women in the analysis of all study populations pooled. Point estimates for Proxy-MiNP were not associated with TTP or OR for infertility in women, but men from Greenland with high levels had a short- er TTP. This association was also significant for the men in the pooled analysis of Proxy-MiNP. These results were contrary to our hypothesis.
The two previous studies investigating phthalates and TTP did likewise not observe prolonged TTP with high DEHP or DiNP levels (79;80). However, nei- ther did they report shorter TTP like we did. The two studies were a prospec- tive environmental study and a retrospective occupational study. The retro- spective occupational study measured DEHP in air, both personally and sta- tionary at different work locations and for different tasks (80). The exposure was calculated from the men’s tasks and working stations over time, thus the measured DEHP levels might be more constant over the time they tried to get their wives pregnant than environmental exposure. Due to our retrospective data collection in an environmental study of short half-lived phthalates, we cannot exclude the risk of non-differential bias toward the null hypothesis. If our Study VI is biased due to timing of exposure measurements, our results would fit the two previous results, as well as epidemiologic studies of male reproductive biomarkers, where DEHP was not consistently associated with semen quality and only weakly affected reproductive hormones.