Assessment of serum essential/non-essential elements, biochemical variables and its association with semen parameters and the risk of male infertility

doi:10.21203/rs.3.rs-3872704/v1

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Assessment of serum essential/non-essential elements, biochemical variables and its association with semen parameters and the risk of male infertility

https://doi.org/10.21203/rs.3.rs-3872704/v1

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Essential metals are involved in various cellular processes of the body and non-essential metals are toxic even in small amounts and have no biological role. The deficiency or over-exposure of these elements perturbs the reproductive health of males. A cross-sectional study has been conducted to investigate the association of a) essential (Cr, Co, Ni, Fe, Mn, Cu, Zn, Se) and non-essential elements (Pb, Cd, As, Ba, Bi, U, V); b) biochemical variables (glucose, triglycerides, cholesterol, HDL, LDL, albumin, and total protein) with risk of male infertility. A total of 46 males from infertile couples have been selected for the study. Serum element concentrations were estimated using ICP-MS and biochemical parameters were estimated by colorimetric methods. Statistical analysis was performed with IBM SPSS and R package. A significant difference was observed for Cr and LDL at p < 0.10 among study groups. Significant difference was found for spermatozoa volume, count and motility between study groups. Serum albumin levels are positively associated with sperm motility. Cr is negatively correlated with triglyceride levels (p = 0.03). Fe and Co are also significantly but negatively correlated with cholesterol levels (p = 0.05). Mn, Cu, Pd, Cd, and Ba are significantly positively correlated with semen volume. Cr, Co, Ni, Mn, Cu, Zn, Pd, Cd, As, Ba, U, and V are significantly positively correlated with liquefaction time (p < 0.05). The investigation of metals may help in the screening of male infertility as some metals may have therapeutic potential or toxic nature that can be used in the management of condition.

Blood serum

Cross-sectional study

Essential elements

Male infertility

Non-essential elements

Reproductive health is significantly influenced by high exposure to different metal pollutants. The toxicity effects of these metals depend on the dose, duration of exposure and condition of workplace. Metal pollutants can be classified as either essential elements (required for biological functions) or non-essential elements (have no biological function and are toxic even in trace amounts) [1]. Consumption of essential elements above a critical concentration and non-essential elements even in trace amounts may lead to dysregulation of various cellular events including, proliferation, differentiation, DNA repair pathways, reduced antioxidant defense, and apoptosis [2]. Populations exposed to toxic non-essential elements are at higher risk of reproductive disorders both in males and females [3],[4]. Although, males are generally more predispose to the harmful effects of environmental and occupational exposures as compared to females [5],[6]. Essential elements are the basic components of biological enzymes required for various other functions including, sperm production and reproduction [7]. Essential elements (Zn, Se, Fe, Mn, Cu and Co) are although beneficial, an excess can cause alteration in male reproductive functions [8].

Presence of non-essential elements such as, lead (Pb), cadmium (Cd), arsenic (As), mercury (Hg), uranium (U), barium (Ba) have been reported in various body tissues of humans who had been reported to be adversely affected[9]. Chronic exposure to these elements may cause alterations in the structure of seminiferous tubules, sertoli cells and germ cells [10], which may cause infertility in males. Infertility affects about 17.5% of adult population (approximately 1 in 6 people) worldwide [11] and about 40–60% cases are due to male factor infertility [12]. Varicocele, erectile dysfunction, oligospermia, asthenospermia and azoospermia are the most common causes of male infertility [13]. Various factors contribute to decline in male fertility which may be anatomical, genetic, immunological and idiopathic. A number of studies have investigated the exposure to essential and non-essential elements among males with infertility, but the results are inconsistent and controversial and inadequate as limited number of essential elements has been analyzed in few human subpopulations so far. Hence, the study have been carried out to investigate the association of a) essential (Cr, Co, Ni, Fe, Mn, Cu, Zn, Se) and non-essential elements (Pb, Cd, As, Ba, Bi, U, V); b) biochemical variables [glucose (Glu), triglycerides (TG), cholesterol (Cho), HDL, LDL, albumin (Alb), and total protein (TP)] with risk of male infertility and to find the correlation of these elements with abnormal semen parameters among the population of Punjab, India.

Study population

The cross-sectional study was conducted on 46 males from infertile couples attending infertility clinic in Punjab, India, who had regular unprotected intercourse for at least 12 months without conception with their partners. The participants are in the age of 24 to 55 years. The cases were categorized according to the World Health Organization (WHO) standard recommendations. Subjects with diabetes mellitus, endocrine disease, varicocele, history of injury or testicular surgery were excluded from consideration in the study. Each patient completed a pre-designed questionnaire concerning his anthropometric data (height, weight, waist, and hip circumference), lifestyle habits (alcohol consumption, smoking history and dietary habits), physical activity and education. The study is approved by institutional ethics committee (IEC) (105/IEC-2018) and all the subjects participating in the study signed a consent form before their enrollment in the study. This study was in compliance with the Declaration of Helsinki.

Study design and sample collection

Participants were categorized into four groups: Normospermic (males with sperm count greater than 15 million/ml, n = 20), oligospermic (males with sperm count less than 15 million/ml, n = 7), asthenospermic (males with sperm motility less than 40%, n = 8) and azoospermic (males with no spermatozoa in the semen, n = 11), according to the WHO reference range for semen quality. A sample of 3 ml venous blood was collected by trained health care worker from all subjects into anticoagulant free sample collection tubes. Then serum was isolated and then stored at -20^◦ C for element analysis. The patient’s anthropometric data was measured using inch tape. Other lifestyle variables including, intake of tea, coffee, fruits, junk food, dairy products, vegetables, smoking history and alcohol consumption, were self-reported by the participants.

Instrumentation and sample preparation

Evaluation of serum biochemical parameters

Biochemical parameters namely glucose, triglycerides, cholesterol, HDL, LDL, albumin, and total protein were analyzed using automated clinical chemistry analyzer EM 200, Trans Asia. These parameters were estimated from serum samples using standard kits (ERBA). The quality control values for analysis were within ± 2 SD (standard deviation) of their respective means. Quality control standards Erba paths, Erba norms, Erba calibrator multical, and HDL calibrator were used for calibration during serum analysis. Inter-Assay Coefficient of variability (CV) was less than 10% and the intra-assay coefficient of variability was less than 5%.

Evaluation of serum essential and non-essential elements

Standard preparation

Multi-element standards were prepared from the stock solution of Pb, Cd, As, Ba, Cr, Bi, U, V, Co, Ni, Zn, Fe, Mn, Cu, Se (VHG labs PN.: VHG-SM68-1–100, LGC). For Pb, Cd, As, Ba, Cr, Bi, U, V, Co, and Ni multi-element calibration standard solutions were prepared at the concentration of 0.1 ppb, 1 ppb, 5 ppb, 10 ppb, and 25 ppb. In the case of Zn, Fe, Mn, Cu, and Se calibration standard solutions were prepared at the concentration of 5 ppb, 10 ppb, 25 ppb, 50 ppb and 100 ppb. All the glassware used for sample preparation and standard preparation was washed with 1% HNO₃ and then rinsed with deionized water to avoid any risk of contamination. The sample probe was washed after each sample with Milli-Q water (for 30 sec) followed by 5% HNO₃ (for 30 sec), then again with Milli-Q water (for 30 sec).

Sample preparation and experimental conditions

Serum samples were thawed and digested with 65% suprapur nitric acid (HNO₃) and hydrogen peroxide (H₂O₂) using microwave digestion system (MDS) (Anton Paar, Multiwave Go) under fume hood. Then samples were filtered and diluted to 5 ml. Double distilled purified deionized water was used for dilution before analysis of elements. Selected elements (Pb, Cd, As, Ba, Cr, Bi, U, V, Co, Ni, Zn, Fe, Mn, Cu and Se) were investigated using inductively coupled plasma mass spectrometry (iCAP Q ICP-MS, Thermoscientific). One blank tube containing deionized water was added to each batch of samples and levels of elements in blank samples were lower than the limit of detection (LOD). To check the instrument stability, the standard reference solution was run after each 10 samples. The readings of each samples were taken three times with RSD value < 15%. Operational conditions for the instrument were as follows: helium (He) flow at 0.1 MPa, argon (Ar) flow at 0.6 MPa with micro mist nebulizer type, and mass spectrometer detector. Readings of standards were repeated five times to obtain calibration curves.

Statistical analysis

Statistical analysis was carried out with software IBM SPSS statistic version 26 (SPSS, Chicago, IL, USA) and R package version 4.1.3. Summarization of data was done using descriptive statistics (mean, standard deviation, and median). Kruskal-Wallis test was performed to observe the normality between data set. The relationship between metal concentrations, biochemical variables, and semen parameters was analyzed with multiple linear regression. Correlations between various quantitative variables were analyzed by Pearson correlation. A significance level of p < 0.05 was kept for analysis and marginally significant if p < 0.10.

Demographic characteristics of the participants

Table 1 shows the socio-demographic factors, lifestyle characteristics, and semen parameters of the participants in the study. There were no significant differences for the age of participants, BMI, WHR, marriage duration, smoking, and alcohol consumption. A significant difference in the case of education status (p = 0.03) and physical activity (p = 0.002) was observed between study groups.

Table 1

Demographic and semen characteristics of subjects participating in study *p value < 0.05
Variables	Normospermic (n = 20)	Oligospermic (n = 7)	Asthenospermic (n = 8)	Azoopermic (n = 11)	p value
Demographic characteristics
Age (yrs) Mean ± SD (Median)	37.05 ± 7.65 (35.5)	33.85 ± 5.17 (35)	40.87 ± 9.28 (36.5)	35.72 ± 5.04 (35)	0.533 (0.807)
BMI (kg/m²)	26.9 ± 2.65 (26.35)	26.65 ± 4.11 (25)	26.3 ± 3.91 (24.45)	26.67 ± 3.08 (25.9)	0.814 (0.425)
WHR (Mean ± SD) (Median)	0.912 ± 0.03 (0.91)	0.90 ± 0.05 (0.91)	0.91 ± 0.02 (0.90)	0.90 ± 0.067 (0.88)	0.822 (0.481)
Duration of marriage (yrs) Mean ± SD (Median)	13 ± 6.11 (12)	9 ± 4.08 (11)	11.25 ± 8.37 (9.5)	12.27 ± 5.06 (11)	0.463 (0.291)
Residence
Rural (n %)	14 (70%)	4 (57.14%)	3 (37.5%)	9 (81.82%)	0.217
Urban (n %)	6 (30%)	3 (42.86%)	5 (62.5%)	2 (18.18%)	0.217
Education status
Un-educated (n %)	2 (10%)	0 (0%)	0 (0%)	1 (9.1%)	0.035*
Elementary and intermediate (n %)	9 (45%)	2 (28.6%)	5 (62.5%)	2 (18.18%)
High school (n %)	6 (30%)	1 (14.3%)	2 (25%)	8 (72.72%)
College and university (n %)	3 (15%)	4 (57.1%)	1 (12.5%)	0 (0%)
Physical activity
Sedentary (n %)	2 (10%)	0 (0%)	0 (0%)	2 (18.18%)	0.002*
Moderate (n %)	17 (85%)	3 (42.86%)	8 (100%)	9 (81.82%)
Vigorous (n %)	1 (5%)	4 (57.14%)	0 (0%)	0 (0%)
Drinking water
RO filtered (n %)	9 (45%)	5 (71.4%)	4 (50%)	5 (45.5%)	0.665
Tap water (n %)	11 (55%)	2 (28.6%)	4 (50%)	6 (54.5%)	0.665
Alcohol consumption
No (n %)	5 (25%)	3 (42.86%)	3 (37.5%)	4 (36.4%)	0.797
Yes (n %)	15 (75%)	4 (57.14%)	5 (62.5%)	7 (63.6%)	0.797
Daily (n %)	5 (33.3%)	2 (50%)	3 (60%)	4 (57.14%)	0.535
Occasionally (n %)	10 (66.7%)	2 (50%)	2 (40%)	3 (42.86%)	0.535
Smoking history
No (n %)	14 (70%)	6 (85.7%)	6 (75%)	9 (81.8%)	0.809
Yes (n %)	6 (30%)	1 (14.3%)	2 (25%)	2 (18.2%)	0.809
Daily (n %)	2 (33.3%)	1 (100%)	2 (100%)	1 (50%)	0.598
Occasionally (n %)	4 (66.7%)	0 (0%)	0 (0%)	1 (50%)	0.598
Lifestyle characteristics
Eating habits
Skipped (n %)	4 (20%)	0 (0%)	0 (0%)	1 (9.1%)	NS
Regular (n %)	16 (80%)	7 (100%)	8 (100%)	10 (90.9%)	NS
Dietary habits
Tea
Daily (n %)	20 (100%)	7 (100%)	8 (100%)	11 (100%)
Weekly (n %)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0.478
Coffee
Daily (n %)	1 (5%)	0 (0%)	0 (0%)	0 (0%)	0.874
Weekly (n %)	1 (5%)	0 (0%)	1 (12.5%)	1 (9.1%)
Occasionally (n %)	18 (90%)	7 (100%)	7 (87.5%)	10 (90.9%)
Dairy products
Daily (n %)	18 (90%)	6 (85.7%)	7 (87.5%)	11 (100%)	0.331
Weekly (n %)	2 (10%)	1 (14.3%)	1 (12.5%)	0 (0%)	0.331
Junk food
Daily (n %)	2 (10%)	1 (14.3%)	0 (0%)	0 (0%)	0.611
Weekly (n %)	6 (30%)	3 (42.85%)	5 (62.5%)	5 (45.5%)
Occasionally (n %)	12 (60%)	3 (42.85%)	3 (37.5%)	6 (54.5%)
Canned food
Daily (n %)	1 (5%)	1 (14.3%)	0 (0%)	0 (0%)	0.616
Weekly (n %)	7 (35%)	3 (42.85%)	5 (62.5%)	4 (36.4%)
Occasionally (n %)	12 (60%)	3 (42.85%)	3 (37.5%)	7 (63.6%)
Vegetarian/Non-vegetarian
Vegetarian (n %)	2 (10%)	2 (28.6%)	2 (25%)	3 (27.3%)	0.310
Non-vegetarian (egg/meat) (n %)	18 (90%)	5 (71.4%)	6 (75%)	8 (72.7%)	0.310
Mobile phone type
Keypad (n %)	7 (35%)	0 (0%)	1 (12.5%)	2 (18.18%)	0.213
Smartphone (n %)	13 (65%)	7 (100%)	7 (87.5%)	9 (81.81%)	0.213
Duration of mobile/laptop use
< 3 hrs/day (n %)	16 (80%)	4 (57.14%)	5 (62.5%)	7 (63.63%)	0.687
3–6 hrs/day (n %)	3 (15%)	3 (42.86%)	3 (37.5%)	3 (27.27%)
> 6 hrs/day (n %)	1 (5%)	0 (0%)	0 (0%)	1 (9.1%)
Semen parameters
Liquefaction time (minutes) Mean ± SD (Median)	26.8 ± 8.98 (30)	32.85 ± 12.19 (30)	29.37 ± 11.47 (25)	23.18 ± 6.19 (20	0.217 (0.982)
Volume (ml) Mean ± SD (Median)	2.7 ± 0.93 (2.5)	3.14 ± 1.34 (3)	1.56 ± 0.49 (1.5)	1.59 ± 0.86 (1)	0.001* (0.007)
Sperm count (mill mL-1) Mean ± SD (Median)	67.15 ± 38.86 (60)	4.28 ± 5.18 (1)	(36.62 ± 20.06 30)	-	0.000* (0.000)
Pus cells (hpf) Mean ± SD (Median)	9.72 ± 13.15 (4)	5.85 ± 8.96 (2.5)	3.93 ± 1.23 (4.5)	2.68 ± 3.05 (1.5)	0.334 (0.317)
Motility (%) Mean ± SD (Median)	59.8 ± 12.28 (60)	23.57 ± 15.46 (15)	17.25 ± 9.66 (17)	-	0.000* (0.000)

Association between semen parameters and study groups

No significant difference was observed for liquefaction time and pus cells, although significant association was observed for spermatozoa volume, count, and motility among the study groups. The spermatozoa volume was 3.14 ± 1.34, 1.56 ± 0.49, 1.59 ± 0.86, 2.7 ± 0.93 (p = 0.001), sperm count was 4.28 ± 5.18, 36.62 ± 20.06, Nil, 67.15 ± 38.86 (p = 0.000), motility was 23.57 ± 15.46, 17.25 ± 9.66, Nil, 59.8 ± 12.28 (p = 0.000), in oligospermic, asthenospermic, azoospermic and normospermic group respectively.

Association between essential elements and study groups

Mean concentration of essential, non-essential elements and biochemical parameters are summarized in Table 2. Out of 8 studied essential elements, only chromium (Cr) is significantly different among the study groups at p < 0.10, indicating its role in male infertility.

Table 2

Concentrations (ppb) of essential, non-essential elements and biochemical variables in the investigated groups Note: Vanadium concentration is obtained from 5 samples in asthenospermia group and from 8 samples in the case of azoospermia group (*p value < 0.10)
Variables	Normospermic (Mean ± SD) (Median) (n = 20)	Oligospermic (Mean ± SD) (Median) (n = 7)	Asthenospermia (Mean ± SD) (Median) (n = 8)	Azoospermic (Mean ± SD) (Median) (n = 11)	p value
Essential elements
Cu	14775.25 ± 4063.84 (14676.83)	20077.93 ± 13959.35 (14372.52)	14946.78 ± 3377.27 (16167.41)	13980.091 ± 4046.6 (14232.77)	0.594 (0.965)
Zn	8520.44 ± 5988.67 (6994.06)	10361.72 ± 6872.72 (8272.7)	6637.9 ± 2428.81 (5883.86)	6987.29 ± 2617.53 (7177.6)	0.255 (0.674)
Mn	364.344 ± 139.112 (355.79)	629.32 ± 889.522 (314.3)	332.63 ± 144.83 (348.83)	370.80 ± 204.74 (272.87)	0.826 (0.425)
Se	32507.99 ± 7325.65 (36810.97)	34212.24 ± 6535.63 (36004.7)	31416.97 ± 9201.01 (32473.92)	31794.88 ± 7082.20 (30102.07)	0.993 (0.790)
Fe	63652.721 ± 48660.17 (56384.1)	74370.81 ± 61312.39 (55966.26)	60809.31 ± 17554.41 (59115.58)	80315.62 ± 43725.99 (58643.2)	0.530 (0.258)
Co	3.308 ± 1.965 (3.025)	5.657 ± 7.581 (2.67)	3.565 ± 2.326 (2.875)	3.727 ± 1.82 (2.97)	0.821 (0.833)
Ni	70.486 ± 32.691 (63.987)	70.8 ± 36.16 (61.27)	63.718 ± 26.741 (54.66)	75.14 ± 40.0 (59.85)	0.812 (0.912)
Cr	152.373 ± 56.141 (132.387)	163.214 ± 47.424 (137.22)	181.953 ± 77.34 (146.22)	131.393 ± 52.446 (122.22)	0.082* (0.991)
Non-essential elements
Pd	15.148 ± 20.344 (9.775)	90.778 ± 218.55 (9.0)	8.665 ± 3.099 (7.87)	9.854 ± 3.849 (8.4)	0.720 (0.330)
Cd	0.778 ± 0.445 (0.575)	1.176 ± 1.790 (0.525)	0.703 ± 0.194 (0.66)	0.686 ± 0.178 (0.65)	0.521 (0.885)
As	5.27 ± 1.957 (4.6)	6.564 ± 2.308 (6.35)	4.912 ± 0.812 (4.76)	4.74 ± 1.917 (4.65)	0.244 (0.929)
Ba	203.868 ± 94.943 (170.687)	331.425 ± 234.61 (284.92)	217.268 ± 65.272 (231.72)	187.50 ± 69.179 (186.17)	0.274 (0.412)
U	5.428 ± 3.949 (3.887)	7.875 ± 5.466 (7.5)	7.103 ± 3.309 (7.712)	5.056 ± 3.707 (3.92)	0.309 (0.358)
V	3.44 ± 3.98 (1.93)	5.91 ± 7.05 (2.95)	6.603 ± 7.16 (4.11)	4.114 ± 4.578 (3.775)	0.970 (0.894)
Biochemical variables
Glu (mg/dl)	99.54 ± 38.71 (97.95)	73.17 ± 37 (61.9)	84.45 ± 45.16 (64.85)	86.327 ± 32.484 (93.9)	0.342 (0.088)
TG (mg/dl)	288.6 ± 189.2 (247)	200.14 ± 126.18 (167)	150.37 ± 60.728 (158.5)	272.818 ± 231.811 (189)	0.128 (0.068)
Cho (mg/dl)	218.65 ± 75.88 (198)	196.571 ± 79.60 (189)	154.75 ± 38.38 (147.5)	220.727 ± 93.31 (190)	0.182 (0.195)
HDL (mg/dl)	51.995 ± 20.38 (47.75)	56.742 ± 33.95 (55.2)	54.412 ± 19.09 (49.85)	62.509 ± 28.461 (57.8)	0.780 (0.471)
LDL (mg/dl)	140.545 ± 55.026 (123.68)	130.434 ± 66.68 (106.08)	90.083 ± 25.711 (79.06)	138.71 ± 74.421 (117.6)	0.083* (0.116)
Alb (g/dl)	4.947 ± 1.329 (4.585)	5.094 ± 1.90 (4.18)	4.606 ± 0.728 (4.385)	4.278 ± 0.807 (3.97)	0.308 (0.163)
TP (g/dl)	7.83 ± 1.69 (7.45)	8.41 ± 2.87 (7.18)	8.078 ± 2.738 (7.275)	8.56 ± 3.064 (7.76)	0.982 (0.912)

Association between non-essential elements and study groups

None of the 7 studied non-essential elements show significant difference among the study groups. This may suggest that these element concentrations in the serum of study population did not lower the fertility. However, when compared the concentrations between four groups, oligospermic group has higher concentrations of these elements in comparison to asthenospermic, azoospermic and normospermic group. The concentration of Bismuth was below limits of detection in most of the samples; hence no further statistical evaluation could be performed for its role.

Association between biochemical parameters and study groups

Out of seven (glucose, triglycerides, cholesterol, HDL, LDL, albumin and total protein) biochemical parameters, only low-density lipoprotein (LDL) at p < 0.10 showed significant association between study groups, suggesting its role in male infertility. Male reproductive function is highly dependent on cholesterol homeostasis as cholesterol is the main precursor of steroid synthesis [14]. High LDL and Oxidized LDL (OxLDL) blood levels may decrease sperm counts, viability, hormonal imbalance, and lower weights of testis, epididymis and seminal vesicle as suggested in a study performed on male rats [15].

Multiple linear regression analysis of studied elements, biochemical parameters and semen parameters

Table 3 shows the multivariate analysis for element concentrations, biochemical parameters and semen parameters. Cobalt levels were found to be negatively correlated with pus cells (p = 0.04). A cross-sectional study has found that pyospermia is associated with reduced motility and altered morphology in sperms [16]. Albumin levels are positively correlated with sperm motility (p = 0.01). Albumin protein helps in capacitation of spermatozoa during fertilization process [17]. Elzanaty et al. [18] found positive relationship between albumin levels in seminal plasma and sperm morphology, but no association with sperm motility. There are limited studies which investigate the association of albumin and semen motility.

Table 3

Multivariate analysis for element concentrations, biochemical parameters and semen parameters *p value < 0.05, β = Regression coefficient, CI = confidence interval
	Liquefaction time		Volume		Sperm count		Motility		Pus cells
	β	95% CI	β	95% CI	Β	95% CI	β	95% CI	β	95% CI
Essential elements
Cr	0.189	-0.033,0.095	0.00	-0.009,0.007	0.09	-0.222,0.403	0.041	-0.176,0.258	0.045	-0.024,0.114
Co	0.002	-1.445,1.456	-0.03	-0.215,0.147	-4.39	-11.432,2.655	-2.380	-7.280,2.520	-1.558	-3.105,-0.012*
Ni	0.014	-0.127,0.135	0.01	-0.011,0.022	0.02	-0.621,0.652	-0.078	-0.521,0.364	-0.021	-0.161,0.118
Fe	-0.196	0.000,0.000	0.00	0.000,0.000	0.00	0.000,0.000	0.000	0.000,0.000	0.000	0.000,0.000
Mn	0.549	-0.004,0.033	0.00	-0.001,0.004	0.00	-0.085,0.093	0.009	-0.053,0.070	0.005	-0.015,0.024
Cu	0.066	-0.001,0.001	0.00	0.000,0.000	0.00	-0.002,0.005	0.000	-0.002,0.002	0.000	-0.001,0.001
Zn	-0.107	-0.001,0.000	0.00	0.000,0.000	0.00	-0.003,0.003	0.001	-0.001,0.003	0.000	0.000,0.001
Se	-0.030	0.000,0.000	0.00	0.000,0.000	0.00	-0.002,0.002	0.000	-0.001,0.001	0.000	-0.001,0.000
Non-essential elements
Pd	0.023	-0.075,0.121	0.005	-0.007,0.017	-0.410	-0.851,0.031	-0.182	-0.502,0.138	-0.084	-0.185,0.018
Cd	1.964	-8.429,12.358	-0.117	-1.414,1.179	45.781	-0.834,92.396	20.572	-13.245,54.389	3.541	-7.179,14.260
As	0.716	-1.004,2.436	0.021	-0.194,0.235	5.909	-1.805,13.623	1.869	-3.727,7.465	-0.920	-2.694,0.854
Ba	0.009	-0.042,0.061	0.000	-0.006,0.007	-0.086	-0.319,0.147	0.014	-0.156, 0.183	0.038	-0.016,0.091
U	-0.034	-1.156,1.089	-0.001	-0.141,0.139	0.844	-4.189,5.877	-1.040	-4.692,2.611	0.694	-0.463,1.851
V	0.071	-0.493,0.635	0.009	-0.062,0.079	0.306	-2.222,2.835	-0.471	-2.305,1.364	-0.004	-0.586,0.577
Biochemical parameters
Glu	-0.001	-0.105,0.103	-0.001	-0.013,0.011	0.228	-0.189,0.644	0.114	-0.161,0.388	-0.053	-0.157,0.051
TG	-0.006	-0.027,0.016	-0.001	-0.003,0.002	0.032	-0.054,0.119	-0.004	-0.061,0.053	-0.010	-0.032,0.011
Cho	-0.107	-0.266,0.051	-0.002	-0.021,0.017	0.143	-0.493,0.779	-0.108	-0.527,0.311	0.016	-0.143,0.174
HDL	-0.013	-0.218,0.192	-0.006	-0.031,0.018	-0.297	-1.119,0.525	-0.398	-0.940,0.144	-0.148	-0.353,0.057
LDL	0.118	-0.059,0.294	0.005	-0.016,0.026	-0.158	-0.865,0.549	0.244	-0.222,0.710	0.061	-0.116,0.237
Alb	1.559	-1.546,4.664	0.169	-0.199,0.537	10.423	-2.014,22.860	10.062	1.863,18.261*	0.841	-2.261,3.944
TP	-0.256	-2.130,1.618	-0.073	-0.295,0.149	-4.674	-12.181,2.833	-4.370	-9.319,0.579	0.087	-1.785,1.960

Pearson correlation among studied elements, biochemical parameters and semen quality parameters

Table 4 summarizes the Pearson correlation between semen quality parameters, element concentrations and biochemical parameters. Mn, Cu, Pd, Cd, and Ba are positively correlated with semen volume. Cr, Co, Ni, Mn, Cu, Zn, Pd, Cd, As, Ba, U, and V are significantly correlated with liquefaction time. Liquefaction time is longer in oligospermic and asthenospermic men as compared to other groups. Prolonged liquefaction time can cause delay in conception and also significantly associated with abnormal semen parameters in hypofertile men [19]. Correlation between element-element concentrations and biochemical parameters is summarized in Table 5. Positive correlations between following pairs are observed: (Pd-Co, Cd-Co, As-Co, Ba-Co, U-Co, Pd-Ni, Cd-Ni, Pb-Fe, Cd-Fe, As-Fe, Ba-Fe, Pb-Mn, Cd-Mn, As-Mn, Ba-Mn, U-Mn, V-Mn, Pb-Cu, Cd-Cu, As-Cu, Ba-Cu, U-Cu, V-Cu, Pd-Zn, Cd-Zn, As-Zn, Ba-Zn, U-Zn, U-Se, Cd-Pd, As-Pd, Ba-Pd, U-Pd, V-Pd, As-Cd, Ba-Cd, U-Cd, V-Cd, U-As, V-As, As-Ba, U-Ba, V-Ba, V-U, Cr-Co, Ni-Co, Fe-Co, Mn-Co, Zn-Co, Se-Co, Cr-Ni, Fe-Ni, Mn-Ni, Mn-Fe, Cu-Fe, Cr-Mn, Zn-Mn, Se-Mn, Co-Cu, Mn-Cu, Zn-Cu, Se-Cu, Cr-Cd, Cr-U). In the case of biochemical parameters, there is significant negative linear relationship between chromium (Cr) and triglycerides, suggesting negative correlation between two. Iron (Fe) and cobalt (Co) are also significantly but negatively correlated with cholesterol levels. None of the other biochemical parameters (glucose, HDL, LDL, albumin, total protein) shows any correlation between studied elements.

Table 4

Correlation between semen quality parameters, element concentrations and biochemical parameters (*p value < 0.05, r = correlation coefficient)
	Volume	Sperm count	Motility	Liquefaction time	Pus cells
Essential elements
Cr	r = 0.148 p = 0.162	r = 0.078 p = 0.304	r = -0.007 p = 0.482	r = 0.333 p = 0.012*	r = 0.148 p = 0.164
Co	r = 0.231 p = 0.061	r = -0.115 p = 0.224	r = -0.130 p = 0.195	r = 0.440 p = 0.001*	r = -0.151 p = 0.159
Ni	r = 0.203 p = 0.088	r = -0.005 p = 0.487	r = -0.038 p = 0.400	r = 0.279 p = 0.030*	r = -0.033 p = 0.414
Fe	r = 0.020 p = 0.448	r = -0.117 p = 0.219	r = -0.054 p = 0.361	r = 0.143 p = 0.171	r = -0.204 p = 0.087
Mn	r = 0.342 p = 0.010*	r = -0.045 p = 0.383	r = -0.040 p = 0.396	r = 0.500 p = 0.000*	r = -0.033 p = 0.413
Cu	r = 0.315 p = 0.017*	r = 0.057 p = 0.354	r = -0.050 p = 0.370	r = 0.450 p = 0.001*	r = 0.021 p = 0.445
Zn	r = 0.223 p = 0.069	r = -0.028 p = 0.426	r = 0.089 p = 0.279	r = 0.245 p = 0.051*	r = 0.173 p = 0.126
Se	r = 0.022 p = 0.442	r = -0.027 p = 0.430	r = 0.043 p = 0.389	r = -0.169 p = 0.130	r = -0.186 p = 0.108
Non-essential elements
Pd	r = 0.379 p = 0.005*	r = -0.124 p = 0.206	r = -0.058 p = 0.350	r = 0.492 p = 0.000*	r = -0.069 p = 0.325
Cd	r = 0.345 p = 0.009*	r = -0.009 p = 0.477	r = 0.012 p = 0.469	r = 0.481 p = 0.000*	r = -0.020 p = 0.447
As	r = 0.183 p = 0.112	r = 0.163 p = 0.139	r = 0.055 p = 0.357	r = 0.342 p = 0.010*	r = 0.061 p = 0.344
Ba	r = 0.310 p = 0.018*	r = -0.074 p = 0.314	r = -0.057 p = 0.353	r = 0.462 p = 0.001*	r = 0.247 p = 0.049
U	r = 0.200 p = 0.092	r = 0.017 p = 0.455	r = -0.075 p = 0.309	r = 0.322 p = 0.014*	r = 0.371 p = 0.006*
V	r = 0.182 p = 0.113	r = -0.013 p = 0.467	r = -0.107 p = 0.240	r = 0.246 p = 0.049*	r = 0.072 p = 0.317
Biochemical parameters
Glu	r = -0.084 p = 0.289	r = 0.212 p = 0.079	r = 0.080 p = 0.298	r = -0.137 p = 0.182	r = -0.083 p = 0.291
TG	r = -0.064 p = 0.335	r = 0.242 p = 0.053	r = 0.099 p = 0.256	r = -0.199 p = 0.092	r = -0.015 p = 0.460
Cho	r = -0.016 p = 0.457	r = 0.085 p = 0.287	r = 0.037 p = 0.405	r = -0.188 p = 0.106	r = 0.169 p = 0.130
HDL	r = -0.046 p = 0.380	r = -0.101 p = 0.253	r = -0.189 p = 0.104	r = -0.064 p = 0.335	r = -0.040 p = 0.397
LDL	r = 0.016 p = 0.459	r = 0.042 p = 0.391	r = 0.079 p = 0.301	r = -0.117 p = 0.219	r = 0.222 p = 0.069
Alb	r = 0.054 p = 0.360	r = 0.176 p = 0.121	r = 0.146 p = 0.166	r = 0.024 p = 0.437	r = 0.024 p = 0.437
TP	r = -0.114 p = 0.225	r = -0.037 p = 0.402	r = -0.173 p = 0.126	r = -0.129 p = 0.196	r = -0.004 p = 0.489

Table 5

Correlation between element-element concentrations and biochemical parameters
	Cr	Co	Ni	Fe	Mn	Cu	Zn	Se	Pb	Cd	As	Ba	U	V
Essential elements
Cr	r = 1.000	r = 0.342 p = 0.020*	r = 0.627 p = 0.000*	r = 0.108 p = 0.476	r = 0.290 p = 0.051	r = 0.235 p = 0.116	r= 0.197 p = 0.190	r= -0.040 p = 0.791	r = 0.277 p = 0.063	r = 0.317 p = 0.032*	r = 0.144 p = 0.339	r = 0.151 p = 0.318	r = 0.293 p = 0.048*	r = 0.121 p = 0.423
Co	r = 0.342 p = 0.020*	r = 1.000	r = 0.349 p = 0.017*	r = 0.460 p = 0.001*	r = 0.809 p = 0.000*	r = 0.726 p = 0.000*	r = 0.423 p = 0.003*	r= -0.372 p = 0.011*	r = 0.829 p = 0.000*	r = 0.755 p = 0.000*	r = 0.291 p = 0.049*	r = 0.601 p = 0.000*	r = 0.375 p = 0.010*	r = 0.243 p = 0.104
Ni	r = 0.627 p = 0.000*	r = 0.349 p = 0.017*	r = 1.000	r = 0.489 p = 0.001*	r = 0.467 p = 0.001*	r = 0.227 p = 0.129	r = 0.276 p = 0.064	r = 0.036 p = 0.814	r = 0.338 p = 0.021*	r = 0.357 p = 0.015*	r = 0.227 p = 0.129	r = 0.267 p = 0.073	r = 0.132 p = 0.383	r = 0.161 p = 0.285
Fe	r = 0.108 p = 0.476	r = 0.460 p = 0.001*	r = 0.489 p = 0.001*	r = 1.000	r = 0.564 p = 0.000*	r = 0.314 p = 0.034*	r= 0.245 p = 0.101	r= -0.215 p = 0.151	r = 0.425 p = 0.003*	r = 0.382 p = 0.009*	r = 0.369 p = 0.012*	r = 0.374 p = 0.010*	r = 0.115 p = 0.446	r = 0.149 p = 0.322
Mn	r = 0.290 p = 0.051	r = 0.809 p = 0.000*	r = 0.467 p = 0.001*	r = 0.564 p = 0.000*	r = 1.000	r = 0.782 p = 0.000*	r = 0.592 p = 0.000*	r= -0.305 p = 0.039*	r = 0.918 p = 0.00*	r = 0.890 p = 0.000*	r = 0.344 p = 0.019*	r = 0.744 p = 0.000*	r = 0.448 p = 0.002*	r = 0.346 p = 0.019
Cu	r = 0.235 p = 0.116	r = 0.726 p = 0.000*	r = 0.227 p = 0.129	r = 0.314 p = 0.034*	r = 0.782 p = 0.000*	r = 1.000	r = 0.415 p = 0.004*	r=-0.353 p = 0.016*	r = 0.817 p = 0.000*	r = 0.749 p = 0.000*	r = 0.488 p = 0.001*	r = 0.650 p = 0.000*	r = 0.515 p = 0.000*	r = 0.340 p = 0.021*
Zn	r = 0.197 p = 0.190	r = 0.423 p = 0.003*	r = 0.276 p = 0.064	r = 0.245 p = 0.101	r = 0.592 p = 0.000*	r = 0.415 p = 0.004*	r = 1.000	r= -0.164 p = 0.276	r = 0.546 p = 0.000*	r = 0.629 p = 0.000*	r = 0.343 p = 0.019*	r = 0.667 p = 0.000*	r = 0.524 p = 0.000*	r = 0.185 p = 0.218
Se	r= -0.040 p = 0.791	r= -0.372 p = 0.011*	r = 0.036 p = 0.814	r=-0.215 p = 0.151	r= -0.305 p = 0.039*	r=-0.353 p = 0.016*	r=-0.164 p = 0.276	r = 1.000	r =-0.232 p = 0.121	r= -0.180 p = 0.232	r = 0.069 p = 0.648	r= -0.218 p = 0.146	r= -0.332 p = 0.024*	r= -0.218 p = 0.146
Non-essential elements
Pb	r = 0.277 p = 0.063	r = 0.829 p = 0.000*	r = 0.338 p = 0.021*	r = 0.425 p = 0.003*	r = 0.918 p = 0.000*	r = 0.817 p = 0.000*	r = 0.546 p = 0.000*	r=-0.232 p = 0.121	r = 1.000	r = 0.937 p = 0.000*	r = 0.358 p = 0.014*	r = 0.733 p = 0.000*	r = 0.452 p = 0.002*	r = 0.347 p = 0.018*
Cd	r = 0.317 p = 0.032	r = 0.755 p = 0.000*	r = 0.357 p = 0.015*	r = 0.382 p = 0.009*	r = 0.890 p = 0.000*	r = 0.749 p = 0.000*	r = 0.629 p = 0.000*	r =-0.180 p = 0.232	r = 0.937 p = 0.000*	r = 1.00	r = 0.351 p = 0.017*	r = 0.694 p = 0.000*	r = 0.462 p = 0.001*	r = 0.299 p = 0.044*
As	r = 0.144 p = 0.339	r = 0.291 p = 0.049*	r = 0.227 p = 0.129	r = 0.369 p = 0.012*	r = 0.344 p = 0.019*	r = 0.483 p = 0.001*	r = 0.343 p = 0.019*	r = 0.069 p = 0.648	r = 0.358 p = 0.014*	r = 0.351 p = 0.017*	r = 1.000	r = 0.575 p = 0.000*	r = 0.469 p = 0.001*	r = 0.308 p = 0.037*
Ba	r = 0.151 p = 0.318	r = 0.601 p = 0.000*	r = 0.267 p = 0.073	r = 0.374 p = 0.010*	r = 0.744 p = 0.000*	r = 0.650 p = 0.000*	r = 0.667 p = 0.000*	r=-0.218 p = 0.146	r = 0.733 p = 0.000*	r = 0.694 p = 0.000*	r = 0.575 p = 0.000*	r = 1.000	r = 0.788 p = 0.000*	r = 0.438 p = 0.002*
U	r = 0.293 p = 0.048	r = 0.375 p = 0.010*	r = 0.132 p = 0.383	r = 0.115 p = 0.446	r = 0.448 p = 0.002*	r = 0.515 p = 0.000*	r = 0.524 p = 0.000*	r= -0.332 p = 0.024*	r = 0.452 p = 0.002*	r = 0.462 p = 0.001*	r = 0.469 p = 0.001*	r = 0.788 p = 0.000*	r = 1.000	r = 0.332 p = 0.024*
V	r = 0.121 p = 0.423	r = 0.243 p = 0.104	r = 0.161 p = 0.285	r = 0.149 p = 0.322	r = 0.346 p = 0.019*	r = 0.340 p = 0.021*	r = 0.185 p = 0.218	r=-0.218 p = 0.146	r = 0.347 p = 0.018*	r = 0.299 p = 0.044*	r = 0.308 p = 0.037*	r = 0.438 p = 0.002*	r = 0.332 p = 0.024*	r = 1.000
Biochemical parameters
Glu	r= -0.125 p = 0.408	r=-0.200 p = 0.183	r = 0.034 p = 0.824	r =-0.233 p = 0.119	r= -0.093 p = 0.541	r= -0.130 p = 0.387	r = 0.018 p = 0.905	r = 0.121 p = 0.422	r =-0.052 p = 0.730	r = 0.052 p = 0.731	r =-0.076 p = 0.616	r= -0.085 p = 0.574	r =-0.043 p = 0.778	r= -0.060 p = 0.693
TG	r= -0.311 p = 0.035*	r= -0.028 p = 0.855	r =-0.243 p = 0.104	r= -0.134 p = 0.376	r =-0.019 p = 0.899	r = 0.122 p = 0.421	r =-0.017 p = 0.908	r=-0.083 p = 0.585	r= -0.096 p = 0.526	r =-0.048 p = 0.753	r = 0.009 p = 0.951	r= -0.027 p = 0.860	r = 0.005 p = 0.973	r= -0.108 p = 0.475
Cho	r= -0.276 p = 0.064	r= -0.285 p = 0.05*	r =-0.232 p = 0.121	r =-0.288 p = 0.05*	r=-0.207 p = 0.167	r =-0.133 p = 0.379	r = 0.008 p = 0.957	r=-0.011 p = 0.941	r =-0.165 p = 0.274	r =-0.147 p = 0.331	r = 0.023 p = 0.881	r= -0.055 p = 0.716	r = 0.097 p = 0.521	r= -0.143 p = 0.344
HDL	r =-0.190 p = 0.207	r= -0.175 p = 0.245	r =-0.052 p = 0.732	r= -0.195 p = 0.195	r= -0.176 p = 0.241	r= -0.134 p = 0.373	r=-0.080 p = 0.597	r= -0.027 p = 0.858	r =-0.159 p = 0.291	r= -0.194 p = 0.196	r= -0.028 p = 0.851	r =-0.020 p = 0.897	r = 0.130 p = 0.390	r= -0.116 p = 0.441
LDL	r=-0.234 p = 0.118	r= -0.230 p = 0.124	r =-0.208 p = 0.166	r =-0.250 p = 0.093	r= -0.163 p = 0.278	r= -0.108 p = 0.474	r = 0.057 p = 0.709	r= -0.040 p = 0.791	r= -0.114 p = 0.450	r= -0.098 p = 0.517	r = 0.072 p = 0.634	r = 0.030 p = 0.845	r = 0.157 p = 0.298	r= -0.077 p = 0.611
Alb	r=-0.048 p = 0.751	r= -0.143 p = 0.342	r = 0.013 p = 0.931	r=-0.012 p = 0.937	r=-0.050 p = 0.740	r= -0.054 p = 0.721	r = 0.025 p = 0.870	r = 0.102 p = 0.500	r= -0.063 p = 0.680	r= -0.077 p = 0.612	r = 0.242 p = 0.106	r = 0.043 p = 0.777	r = 0.091 p = 0.547	r= -0.087 p = 0.564
TP	r= -0.177 p = 0.240	r= -0.130 p = 0.389	r =-0.035 p = 0.819	r= -0.035 p = 0.818	r= -0.052 p = 0.733	r= -0.132 p = 0.383	r = 0.054 p = 0.722	r= -0.136 p = 0.368	r= -0.084 p = 0.577	r= -0.071 p = 0.640	r= -0.104 p = 0.493	r = 0.102 p = 0.501	r = 0.155 p = 0.304	r= -0.032 p = 0.834
*p value < 0.05, r = correlation coefficient

Essential elements

Chromium (Cr)

Significant difference was observed in chromium concentrations among all the four groups at p < 0.10. Chromium could induce reproductive toxicity and is known for its detrimental effects as toxic, and carcinogenic [20]. Higher levels of chromium concentration are found in oligospermic (163.214 ± 47.424) and asthenospermic (181.953 ± 77.34) group as compared to azoospermic (131.393 ± 52.446) and normospermic group (152.373 ± 56.141). Sperm concentration and motility of exposed workers was significantly lower as compared to non-exposed workers [21].

Cobalt (Co)

Cobalt is an essential element, but its deficiency or over-exposure perturbs the normal morphology and progressive motility of sperms [22]. No significant difference was observed in cobalt concentration among study groups in present study although higher cobalt levels are observed in oligospermic (5.657 ± 7.581), asthenospermic (3.565 ± 2.326) and azoospermic (3.727 ± 1.82) group in comparison to normospermic group (3.308 ± 1.965).

Nickel (Ni)

No significant difference in the nickel levels among the study groups was observed. Higher nickel concentration is observed in azoospermic group (75.14 ± 40.0) in comparison to other three groups. The experimental higher exposure to nickel resulted in abnormal semen parameters, reduced antioxidant levels, decreased testosterone levels as well as alterations in the hypothalamus and pituitary axis [23].

Iron (Fe)

Iron is an essential element and cofactor in various biological molecules. Its overload cause testicular atrophy, impaired spermatogenesis, and epididymal lesions, which results in impaired reproductive health [24]. However, no significant difference was observed in the current study.

Manganese (Mn)

Manganese is considered an essential element, but its impact on quality of spermatozoa remains unclear. We found increased concentrations of manganese in oligospermic (629.32 ± 889.522) and azoospermic men (370.80 ± 204.74) in comparison to athenospermic (332.63 ± 144.83) and normospermic men (364.344 ± 139.112). Li et al [25] found the negative impacts of higher manganese concentration on sperm morphology and quality.

Copper (Cu)

Negative correlations between high copper concentrations and spermatozoa quality parameters were found among older men. Increased frequency of sperm defects and poor semen quality were observed [26]. High concentrations of copper are observed in oligospermic (20077.93 ± 13959.35) and asthenospermic (14946.78 ± 3377.27) group in comparison to azoospermic (13980.091 ± 4046.6) and normospermic group (14775.25 ± 4063.84). Similarly Akinloye et al [27] also found the higher levels of serum copper µmol/l in oligospermic men (7.37 ± 0.44) as compared to azoopermic (5.70 ± 0.39) and normospermic men (6.18 ± 0.61).

Zinc (Zn)

Inconsistent results are observed in literature between zinc levels and male infertility. In present study, zinc levels are higher in oligospermic men (10361.72 ± 6872.72) as compared to asthenospermic (6637.9 ± 2428.81), azoopermic (6987.29 ± 2617.53) and normospermic men (8520.44 ± 5988.67). Similar to our findings, Akinloye and colleagues [27] found increased levels of zinc in serum of oligospermic men (25.43 ± 1.42) in comparison to azoospermic (9.79 ± 0.26) and normospermic men (11.31 ± 0.65).

Selenium (Se)

We found higher levels of selenium in oligospermic men (34212.24 ± 6535.63) as compared to other three goups. Similarly, Akinloye et al., 2005 [28] found higher levels of serum selenium (µmol/l) in oligospermic men 4.19 (93.93–4.45) as compared to azoopermic 1.62 (1.51–1.79) and normospermic men 1.75 (1.52–1.97).

Non-essential elements

Lead (Pd)

Lead is a non-essential element found in the environment as a result of human activities. Mean blood concentration of lead in oligospermic men was (90.778 ± 218.55), which was significantly higher as compared to mean concentration in the asthenospermic (8.665 ± 3.099), azoospermic men (9.854 ± 3.849) and normospermic men (15.148 ± 20.344). Our findings are consistent with Nsonwu-Anyanwu et al., 2019 [29], who found increased levels of lead (µg/L) (Mean ± SD) in oligospermic men (0.53 ± 0.17) in comparison to azoospermic (0.43 ± 0.15) and normospermic men (0.47 ± 0.17).

Cadmium (Cd)

Cadmium is one of the most detrimental non-essential element which induces oxidative toxicity, apoptosis, autophagy and represents a high risk factor for infertility. Mean cadmium concentrations in oligospermic men (1.176 ± 1.790) were also higher than its concentration in normospermic group (0.778 ± 0.445). The results were consistent with the findings of Nsonwu-Anyanwu and colleagues [29], which reported higher levels of cadmium (µg/L)( Mean ± SD) in oligospermic men (0.47 ± 0.16) than in azoospermic (0.37 ± 0.13) and normospermic men (0.41 ± 0.15). Cadmium induced toxicity causes structural alterations in the blood-testis barrier, sertoli cells, and seminiferous tubules, and, which results in reduced male fertility [30].

Arsenic (As)

Arsenic toxicity perturbs the normal reproductive function through generation of free radicals and oxidative stress, which further causes alterations in steroidogenesis and spermatogenesis [31]. Increased levels of arsenic are found in oligospermic men (6.564 ± 2.308) in comparison to normospermic men (5.27 ± 1.957). Our results are similar with Calogero and colleagues, who found higher arsenic levels in low quality semen 6.14 (3.06–9.52)[(Median (IQR)] as compared to normal quality semen 4.08 (2.35–8.55) [32].

Barium (Bi)

Mean barium concentrations in oligospermic (331.425 ± 234.61) and asthenospermic men (217.268 ± 65.272) were found to be higher as compared to normospermic men (203.868 ± 94.943). Likewise, barium levels are higher in cases (low quality semen) (134.78 ± 52.74) as compared to normal quality semen (63.40 ± 22.82) in a study conducted in Labanon [3].

Uranium (U)

Only few studies have been conducted which reported the exposure of uranium concentrations and its correlation with male reproductive function. High levels of uranium were found in oligospermic (7.875 ± 5.466) and asthenospermic men (7.103 ± 3.309) than in case of normospermic men (5.428 ± 3.949). Sukhn et al found uranium concentrations in seminal plasma of male partners in heterosexual couples and concluded that U concentrations are related with decline in semen quality parameters [3].

Vanadium (V)

Vanadium is among the main environmental toxicants. Mean concentrations of vanadium were higher in oligospermic (5.91 ± 7.05), asthenospermic (6.603 ± 7.16) and azoospermic men (4.114 ± 4.578) as compared to normospermic men (3.44 ± 3.98). Vanadium poisoning can cause increase in lipid peroxidation, free radical production, and inhibition of antioxidant defense system in adult male rats, which makes testis more prone to oxidative damage which further leads to loss in their function [33].

Bismuth (Bi)

Bismuth levels are below detection limit in all the four study groups, so that statistical analysis has not been done for bismuth. Although bismuth is considered to be non-toxic, but high level exposure along with other non-essential elements may inhibit sperm creatine kinase, which further reduces normal sperm metabolism [34].

Both essential and non-essential elements are associated with human fertility. The decline in sperm volume, count, motility, and normal morphology of spermatozoa might be due to adverse environmental factors, occupational pollution [35]. Although, humans are exposed to both essential and toxic elements but there is limited research investigating the impact of both types of elements on male infertility. In this study, the elemental concentrations of both essential (Cr, Co, Ni, Fe, Mn, Cu, Zn, Se) and non-essential elements (Pb, Cd, As, Ba, Bi, U, V) in blood serum have been assessed. Finally, blood serum may not be the most appropriate sample type in assessing male infertility, but other sample types (semen, seminal plasma) may provide better estimation of elemental concentrations. Furthermore, additional epidemiological studies are needed to explore the impact of these metals on reproductive health of males.

The higher concentration of chromium is observed among oligospermic and asthenospermic men that may cause alteration of male reproductive function. Albumin levels are positively associated with sperm motility. The levels of serum chromium significantly reduce the triglyceride levels. An increased Fe and cobalt concentration significantly lowers the cholesterol levels in infertile men. Increased levels of LDL have adverse impacts on semen quality, which further increase the risk of infertility in males.

Funding

The study is funded by Indian Council for Medical Research (ICMR), New Delhi, proposal ID: 2020–9200.

Conflict of interest

The authors declare no conflict of interest.

Funding

The study is funded by Indian Council for Medical Research (ICMR), New Delhi, proposal ID: 2020–9200.

Author Contribution

RK and PK conceived and designed the analysis; MK and KC collected the data; MK and HK performed the initial analysis and wrote the first draft; RK, HK, and PK checked and finalized the final draft. Each author has read and approved the manuscript.

Acknowledgements

We are sincerely thankful to the Central Instrumentation Laboratory (CIL), Central University of Punjab for providing infrastructure and facility support. Internet facility provided by Computer Department of Central University of Punjab, Bathinda and Punjabi University, Patiala is also acknowledged. Authors are thankful to all the participants of the study.

Data availability statement

Supporting data for the findings of this study are available from the corresponding author upon request.

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Assessment of serum essential/non-essential elements, biochemical variables and its association with semen parameters and the risk of male infertility

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Abstract

Introduction

Materials and methods

Study population

Study design and sample collection

Instrumentation and sample preparation

Evaluation of serum biochemical parameters

Evaluation of serum essential and non-essential elements

Standard preparation

Sample preparation and experimental conditions

Statistical analysis

Results and Discussion

Demographic characteristics of the participants

Association between semen parameters and study groups

Association between essential elements and study groups

Association between non-essential elements and study groups

Association between biochemical parameters and study groups

Multiple linear regression analysis of studied elements, biochemical parameters and semen parameters

Pearson correlation among studied elements, biochemical parameters and semen quality parameters

Essential elements

Chromium (Cr)

Cobalt (Co)

Nickel (Ni)

Iron (Fe)

Manganese (Mn)

Copper (Cu)

Zinc (Zn)

Selenium (Se)

Non-essential elements

Lead (Pd)

Cadmium (Cd)

Arsenic (As)

Barium (Bi)

Uranium (U)

Vanadium (V)

Bismuth (Bi)

Conclusion

Declarations

Funding

Conflict of interest

Funding

Author Contribution

Acknowledgements

Data availability statement

References

Additional Declarations

Status:

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