1 Introduction

Several studies have reported a decline in semen quality over time^[1-3]. Circumstantial evidence is accumulating that environmental xenobiotics may disrupt reproductive processes in human males. These include disorders of development and function of the male reproductive tract such as testicular cancer^[4], maldescent (cryptorchidism)^[5], urethral abnormalities (hypospadias)^[6], and a striking drop in semen volume and sperm counts^[7]. These abnormalities have occurred during periods of increased exposure to and body burdens of oestrogenic chemicals^[8].

PCBs are halogenated aromatic hydrocarbons consisting of complex lipophilic mixtures of 200 or more congeners with oestrogenic and anti-oestrogenic effects^[9]. Several adverse have been documented in animals on different organs and at different metabolic levels such as the thyroid hormone metabolism^[10], gonadal steroidogenesis and semen quality^[11], enzyme metabolism^[12], and immune system^[13]. In man, levels of some PCB congeners have been inversely correlated to sperm motility in semen samples with sperm concentrations <20 million/mL^[14]. Boys exposed to PCBs in utero were shown to have significantly smaller penis^[15].

PCBs have been used extensively as insulators in electrical equipment such as transformers, ballasts in fluorescent lighting, circuit breakers and switchgear, as plasticisers in PVC products, in carbonless copy paper, as de-inking solvents for recycling of newspaper and as waterproofing agents. They are resistant to degradation, hence widespread and persistent in the environment. Different routes of exposure include dermal exposure, ingestion of contaminated water and food, and inhalation of ambient air contaminated with PCBs. They tend to accumulate in marine and terrestrial food chains and due to the low biodegradation and excretion in humans, these substances accumulate in the body fat. Humans are the potential endpoint reservoirs for PCB contamination in the aquatic ecosystem. The current major sources of concern are their entry into the environment from unauthorized disposal practices, landfill leachate, leaks in transformers and hydraulic and heat transfer systems, and from transformer fires and transportation spills. Rigorous placebo controlled trials have revealed many of the supposed treatments for male infertility to be ineffective^[16]. Intracytoplasmic sperm injection (ICSI), the most significant advance in the treatment of men who will not respond to specific treatment, may carry a substantial recurrence risk for infertility in the offspring of treated couples^[17] as it allows fertilization with defective sperm without knowing the cause of the infertility. Identifying and eliminating toxic factors in the environment that result in this condition is an urgent need of the day.

2 Materials and methods

2.1 Subjects

Five hundred and fifty-seven infertile couples were screened at The Assisted Conception Services Unit, Mahavir Hospital and Research Centre, a referral centre that receives cases from all over Andhra Pradesh. A complete case history evaluation including the nature and duration of infertility, residential area, habits and addictions, history of illness/disease, occupation, life style, and diet was made. Laboratory investigations included a routine semen analysis after a proscribed three-day abstinence^[18]. Seminal volume, sperm count, the number and quality of sperm motility, sperm morphology, sperm hypo-osmotic swelling (HOS) test, sperm nuclear chromatin stability, and sperm DNA normality were recorded. The HOS was assessed in a sodium citrate fructose medium^[19]. Sperm nuclear chromatin decondensation was analysed following treatment of spermatozoa with 1% SDS+6 mmol/L EDTA, stained according to the Shorr method and classified according to the scheme of Rodriguez et al^[20].

Individuals with sperm counts <20 million/mL,  grade A motility 25%, and/or 30% normal forms, and/or hypo-osmotic swelling test scores 50% were considered to have a male factor problem. In 300 individuals with a male factor problem, an obvious cause for the infertility could be found in 44.52% of the men, while no cause could be identified in 9.34%. These men were diagnozed Unexplained Male Factor infertility. PCBs were estimated in the seminal plasma of 21 infertile men with unexplained male factor and 32 controls. Male partners of women with a previous conception were selected as controls. Criteria for the inclusion of infertile men included i) no history of associated clinical pathology eg, varicocoele, retractile testis, cryptorchidism, hormonal disorders, ii) no history of febrile illness in the six months prior to analysis, iii) no history of smoking or tobacco consumption, iv) no evidence of semen infection. On the basis of the possible source of PCBs, men were further subcategorized into i) fish-eating urban dwellers, ii) fish-eating rural dwellers, iii) non fish-eating urban dwellers with an exclusively vegetarian diet, and iv) non fish-eating rural dwellers with an exclusively vegetarian diet. The total motile sperm count was taken as a measure of semen quality. It was calculated as the product of grade 2+3 motility and the sperm concentration. For the sake of comparison of semen quality between these sub-categories of men, two control groups of fertile men were considered: i) Control 1: urban men with a mixed diet and no reported consumption of fish, and ii) Control 2: rural men with a mixed diet and no reported consumption of fish. Urban was used to denote areas within the control of the Muncipal Health Corporation whereas 'Rural' was used to denote areas not within Muncipal control.

2.2 Assays

The extraction of xenoestrogens was carried out by the method described by Burse et al^[21]. The extraction procedure was divided into five phases.

2.2.1 Phase 1: Isolation of seminal plasma

Semen was collected as a part of the treatment protocol and after analysis of the semen parameters, seminal plasma was isolated by centrifugation at 2000g for 20 min.

2.2.2 Phase 2: Removal of sex steroids by Charcoal-Dextran treatment.

Charcoal (Norit A, acid washed, Sigma) was washed with cold sterile water immediately before use. A 5% charcoal and 0.5% dextran T-70 (D-4876, Sigma) suspension was prepared. Charcoal-Dextran (CD) suspension aliquots of a volume similar to that of seminal plasma aliquots to be processed were centrifuged at 100g for 10 min. Supernatants were aspirated and seminal plasma aliquots mixed with charcoal pellets. The charcoal seminal plasma mixture was maintained by rolling at 4 cycles/min at 37 for 1 hour. The suspension was centrifuged at 2000g for 20 min. The supernatant was then filtered through a Nalgene filter with a pore size of 0.45 m.

2.2.3 Phase 3: Extraction of xenoestrogens

Extraction of xenoestrogens was performed by the method described by Burse et al^[21] with modifications. CD-treated seminal plasma was divided into two parts. One part was used as a blank and a known quantity of standard PCB (Polychlorinated biphenyl mix, Supelco) was added to the other part to establish a comparison with the blank and so as to monitor the recovery of the added chemical. The two aliquots were allowed to equilibrate at room temperature. The extracts from the blank preparation are devoid of absorbance at 280 nm, and therefore do not interfere with the quantification of synthetic xenoestrogens. CD-treated seminal plasma lacks estrogenic activity.

Sample extraction: 1 mL of methanol was added to each of 2 mL aliquots of seminal plasma, mixed by vortexing, and then 3 mL of hexane:ethyl ether (1:1 by vol) (HPLC grade, Spectrochem) was added to extract the mixture. The mixture was agitated on a rotary mixer for 15 min, and then centrifuged at 2000g for 5 min. The organic phase was collected and the aqueous phase extracted twice more. The organic phases were pooled and subsequently concentrated to 1  mL by evaporation under nitrogen steam.

2.2.4 Phase 4: Acid clean-up of the organic phase prior to high performance liquid chromatography (HPLC)

Concentrated sulphuric acid 0.5 mL was added to the concentrated 1 mL of sample of organic phase. The organic phase was seperated, and the aqueous phase extracted twice more with 1 mL hexane. The organic phases were pooled and dried completely under nitrogen. The sample was resuspended in hexane and then injected into HPLC.

2.2.5 Phase 5: HPLC analysis

Seperation was performed by the method of Medina and Sherman^[22] modified by Sonnenschein et al^[23] in a Shimadzu LC6A Liquid Chromatograph Solvent Delivery System; Shimadzu SCL-6A System Controller; Shimadzu SPD-6AV UV-vis Spectrophotometric detector equipped with a C-R6A Chromatopac.

Aliquots of 500 L were injected into a 4220 Partisil 5 silica column (Whatman) equilibrated with 100% n-hexane (Phase A), and n-hexane: methanol: isopropanol (40:45:15 by vol) (Phase B) at a flow rate of 1.5 mL/min and a pressure of 510^-6 Pa. All the organic solvents were of HPLC grade, filtered with a 0.22 m filter and degassed prior to use. The gradient was developed as follows: 100% n-hexane was allowed to pass through the column for 2 min, following which the concentration of solvent B was increased to 10% in 10 min, 20% in the next 15 min, 50% in the next 5 min, and 100% in the next 5 min. Solvent B 100%  was maintained in the column for 5 min before the concentration of solvent B was reduced to zero. The elution profile was monitored on a Shimadzu SPD-6AV UV-Vis Spectrophotometric detector at 280 nm with an absorbance range of 0.001 to 2.56. The chromatogram was recorded on a C-R6A Chromatopac (Shimadzu).

Prior to standardisation with the commercially available standards and the sample runs, a hexane blank was injected. For each patient, 2 samples were run: a seminal plasma blank and a seminal plasma to sample  which a known amount of PCB was added. The peak HPLC fractions were collected in aliquots and the spectrum of each fraction confirmed spectrophotometrically with a UV 1601 UV Visible Spectrophotometer using a working wavelength of 210-320 nm. Fractions with peak absorbances at the suggested wavelength were recorded to differentiate compound peaks from solvent peaks. The peak retention times of the blank sample were compared with those of the PCB-added sample and standard PCB retention times. Following spectrophotometric estimation, the fractions with peak absorbances at 280 nm were pooled, evaporated under liquid nitrogen, resuspended in hexane and reinjected into the HPLC unit to purify the compound and verify the peak retention times of the eluted compounds. 

Estrogenic pesticides, PCBs, hydroxylated PCBs, phenolic antioxidants and plasticizers elute from the HPLC column during the first 10 min, whereas ovarian estrogens, phytoestrogens, DES, and mycoestrogens are retained for more than 12 min. The concentration of the eluted compound was calculated from the area percentage under each peak. Individual PCB fractions were pooled and expressed as total PCBs in g/mL. Individual PCB congener levels are however presented as a separate publication. The PCB concentrations and semen parameters namely sperm count, sperm motility, sperm morphology, sperm osmoregulatory capacity and measures of sperm function such as sperm nuclear chromatin stability and DNA normality were compared between infertile men and controls. PCB levels and total motile counts were compared among infertile men from different areas and with different diets. The detection limits of PCBs ranged from 0.00323 to 14.97 g/mL with a percentage recovery of 80.75%.

2.3 Ethics

Evaluation of semen analysis parameters was executed as a part of the treatment protocol, thus obviating the need for an informed consent.

2.4 Statistical analysis

3 Results

It was observed  that the ejaculate volume, sperm concentration, grade 3, grade 2+3 motility, the hypo-osmotic swelling test, and sperm nuclear chromatin stability were significantly lower in the infertile men compared to controls. Head defects and percentage of single,  stranded DNA were significantly higher in the infertile men. PCBs were detected in the seminal plasma of infertile patients but absent in fertile controls (Table 1).

Table 1. Semen values and xenoestrogen concentrations (meanSD) in infertile men and controls.

4 Discussion

This study carried out on infertile men is the first report from the Indian sub-continent demonstrating the presence of PCBs in semen samples. The presence of PCBs in the seminal plasma of occupationally unexposed infertile men, their absence from fertile controls, and the significantly poor semen parameters in this group is a clue to the possible role of PCBs in the deterioration of sperm quantity and quality. These chemicals are ubiquitous in the environment.

The significantly lower ejaculate volume in infertile men with PCBs provides evidence for the effect of these chemicals on accessory gland function. Organs that appear to be at particular risk for estrogenic effects are those with receptors for gonadal hormones in the male- the prostate, seminal vesicles, epididymides and testes^[24]. Xenoestrogens may modulate the hormonal milieu within the prostate and seminal vesicles by possibly interfering with androgen binding to the androgen receptors. They may interfere with hormone mediated events in the testis and epididymis by either binding to sex-hormone binding globulin (SHBG) and androgen binding protein (ABP), or blocking the cell-surface receptors^[25] for these proteins, thus inhibiting normal signal transduction.

Acknowledgements

References

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