Association of Hexachlorobenzene (HCB), Dichlorodiphenyltrichloroethane (DDT), and Dichlorodiphenyldichloroethane (DDE) with In Vitro Fertilization (IVF) Outcomes

Hexachlorobenzene (HCB) and dichlorodiphenyltrichloroethane (DDT) are organochlorines (OC) used in fungus and pest control. These organochlorines have demonstrated estrogenic activity in in vitro assays and adverse reproductive effects in fish and wildlife (Tiemann 2008). Though HCB and DDT have been banned in the US, HCB is a byproduct of organic chemical production processes and is still produced outside the US (ATSDR 2002b). DDT continues to be used abroad for the control of mosquito-borne diseases (van den Berg 2009).
The main source of human exposure to these OCs is through dietary ingestion. Generally, HCB and DDT are found in meat, fish, and milk products (Brilhante and Franco 2006; Fontcuberta et al. 2008; Mawussi et al. 2009; Yu et al. 2009). DDT is in higher concentrations in foods imported from countries still using DDT (ATSDR 2002a). Due to their chemical stability, bioaccumulation up the food chain, and the ongoing production or use in some countries, HCB, DDT, and DDE, the primary metabolite of DDT, continue to be detected in human blood (ATSDR 2002a), breast milk (Tsang et al.), follicular fluid (Meeker et al. 2009), amniotic fluid (Foster et al. 2000; Luzardo et al. 2009), and human umbilical cord blood (Jimenez Torres et al. 2006).
There is conflicting data regarding the effect of exposure to DDT/DDE on early reproductive outcomes. A recent study described no association between oocyte, fertilization and implantation parameters in women undergoing IVF and exposure to DDT (Al-Saleh et al. 2009). Another study found an association between increased time to pregnancy and selfreport of exposure to agricultural and home pesticides (Harley et al. 2008). Some studies suggest an association of DDT/DDE with early clinically detected fetal loss (Longnecker, et al., 2005, Venners, et al., 2005). Other studies reported early pregnancy loss, early neonatal and childhood mortality after a widespread accidental exposure to HCB in Turkey (Jarrell et al. 1998; Peters et al. 1982). Due to concerns raised by these earlier studies, we explored the association of serum levels of these persistent organochlorines with early reproductive failure uniquely observable within the context of an in vitro fertilization (IVF) study.

Measurement of HCB, DDT, and DDE in serum
Blood samples were collected from women during the follicular phase of their first IVF/ICSI cycle and subsequent cycles in a non-fasting state immediately prior to human chorionic gonadotropin (HCG) administration. The serum fraction was separated for all blood samples by centrifugation and stored at -80°C. Measurement of the organochlorine pesticides hexachlorobenzene, p,p’-DDT, p,p’-DDE, o,p’-DDT, o,p’-DDE was conducted by the Organic Chemistry Analytical Laboratory, Harvard School of Public Health, using methods described previously (Korrick et al. 2000). Briefly, after liquid-liquid extraction and column chromatography, samples were analyzed by dual gas chromatography with electron capture detection (GC/ECD), on two capillary columns of different polarity using two internal standards.
Samples were accompanied by the following quality control samples: a procedural blank, matrix spike samples, and a laboratory control sample. Each sample was spiked with two surrogate compounds to monitor the efficiency of the extraction procedure. All final results were reported after subtracting the amount of the analyte measured in the procedural blank associated with the analytic batch. Method detection limits (MDL) were determined as recommended by the US Environmental Protection Agency (EPA 1984).

The wet-weight MDL values for HCB, and congeners of DDT and DDE were all <0.01 ng/g. The organochlorine concentrations were measured at part per billion (ppb) concentrations. The within-assay variation for all target pesticides ranged from 2% to 5%. The between-assay variation (%CV) for all target pesticides was below 10%. Serum total cholesterol and triglycerides were measured enzymatically. Total serum lipid content was calculated by Phillip’s equation 2 (out of 3) and then used for lipid-standardization of serum organochlorine concentration (Phillips et al. 1989).

Statistical analysis
Serum organochlorine concentrations in the study population of 765 women were grouped into quartiles. Total DDT was defined for this analysis as a sum of the 2 congeners each of DDT and DDE: p,p’-DDT, o,p’-DDT, p,p’-DDE, o,p’-DDE. The relation between IVF outcomes and quartiles of HCB, p,p’-DDE, and total DDT (tDDT) was explored with multivariable generalized linear regression models, with data structured to accommodate joint models for multiple outcomes and multiple cycles contributed per woman. Whenever a woman was at risk of a cycle failure due to implantation failure, chemical pregnancy, or spontaneous abortion in each cycle, a binary outcome (Y) (1=failure, 0=not a failure) was recorded. Thus, a woman provided up to three contributions to risk sets for the possible outcomes within each IVF cycle. A logistic regression model was then employed for each early pregnancy outcome, while adjusting for covariates. Interaction terms between organochlorine quartile and failure type were included in the model to allow for a distinct relation between the organochlorine of interest and reproductive failure type. Interaction terms between age and failure type were also included to account for the possibility of differing age effects on the various failure endpoints. A woman specific random effect term, assumed to be Gaussian, was included to account for the correlation among outcomes from different cycles for the same woman. Trend tests were conducted by
assigning ordinal integer values to HCB, DDE, and DDT quartiles (0=lowest quartile to 3=highest quartile).
Potential confounding variables that were considered in our analyses included maternal age (<35, 35-37, 38-40, >40), body-mass index (BMI), serum lipids, smoking status (never, past, or current), site (of the three participating Boston area clinics), study phase (1994-1998 or 1999- 2003), race (Caucasian or other), previous live birth (yes/no), ampules of gonadotropins, protocol (down regulation or other), ICSI (yes/no), number of embryos transferred, and primary infertility diagnosis. Primary infertility diagnoses were categorized as male factor, ovulatory dysfunction, and other/unexplained. The category entitled ‘other/unexplained’ included mullerian anomaly and uterine or tubal pathology. Cycles that were missing race, infertility diagnosis, or previous live birth, 6% of cycles, were assigned a missing indicator variable and retained in all analyses. If a cycle was missing any other covariate information, that cycle was excluded. The serum organochlorine concentrations were standardized by dividing by the calculated total serum lipid concentration. In addition, we also modeled IVF outcomes in relation to wet weight serum levels of the specific organochlorine and adjusted for serum lipids as a covariate in multivariable models, to reduce bias, as suggested by Schisterman, et al. (Schisterman et al. 2005).

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