J Infertil Reprod Biol, 2020, Volume 8, Issue 3, Pages: 49-52. https://doi.org/10.47277/JIRB/8(3)/49  
Association of Copper-to Zinc Ratio with Sperm  
Concentration among Males Investigated for  
Infertility  
Mathias Abiodun Emokpae* and Muyiwa Adeleye Moronkeji  
Department of Medical Laboratory Science, School of Basic Medical Sciences, College of Medical Sciences, University of Benin, Benin City, Nigeria  
Received: 28/07/2020  
Accepted: 13/09/2020  
Published: 20/09/2020  
Abstract  
Background: The importance of copper and zinc in the fertility potential of males are well understood. The close relationship and the  
antagonistic interactions between copper and zinc requires further investigation especially because of conflicting reports in literature  
on the association of the trace elements with sperm quantity and quality. This study evaluates seminal plasma copper and zinc  
concentrations, copper-to-zinc ratio and their associations with sperm concentration in infertile males.  
Methods: This is a cross-sectional study of 400 males investigated for infertility and 100 male control subjects. Seminal plasma  
copper and zinc were determined using atomic absorption spectrophotometry technique (Buck Scientific Model VGP-210, Germany).  
6
6
The subjects were grouped based on sperm count; normozoospermia(>15 x 10 cells/mL), oligozoospermia (<15x10 cells/mL) and  
azoospermia (no sperm cell). The levels of copper, zinc and Cu/Zn ratio were compared using unpaired Students-t-test and analysis of  
variance while Pearson correlation coefficient was used to assess the correlation between measured variables and sperm count.  
Results: Seminal plasma zinc was significantly lower (p<0.001) while copper and Cu/Zn ratio were significantly higher (p<0.001) in  
infertile than fertile males. The Cu/Zn ratio was significantly higher (p<0.001) among infertile males than controls. Seminal plasma  
levels of copper and Cu/Zn ratio increased with decreasing concentrations of sperm cells while zinc levels increase with increasing  
concentration of sperm cells.  
Conclusion: Copper-to-zinc ratio correlated with sperm concentration in infertile males. The interaction between copper and zinc may  
be routinely considered in the clinical evaluation of the infertile men.  
Keywords: Copper, Zinc, Sperm concentration, Male infertility  
deficiencies are high. The seminal plasma contains several  
1
Introduction1  
elements such as copper, zinc, and Iron that protect the  
spermatozoa at the time of ejaculation (8). Zinc deficiency  
may be an important risk factor for poor quality and quantity  
of sperm and idiopathic male infertility (9). Zinc is required  
for adequate physiological function of the body such as  
growth, reproduction, DNA synthesis and repair, cell division  
and gene expression, wound healing and immune function  
Copper-to-zinc (Cu/Zn) ratio may be a good indicator of  
male fertility potential. The evaluation of the relationship  
between Cu/Zn ratio and sperm indices in seminal plasma  
may give  
a better understanding of the mechanisms  
underlying male infertility. Deficiency of Zn in the  
reproductive system can lead to hypogonadism and gonadal  
hypofunction (1). Several authors have shown the physiologic  
role zinc plays in the development of testicles, prostate and  
sperm motility (2, 3). Zinc and copper have very close  
relationship and their interactions are majorly antagonistic (4).  
Appropriate concentrations are needed for adequate  
physiological functions (5). Any variation from normal of  
either of the trace element will disrupt the equilibrium and  
affect the Cu/Zn ratio which may ultimately impact the  
fertility potential of the male. Some authors have indicated a  
range for the ratio of Cu/Zn 0.9 to 1.27 and this may be  
different with different semen abnormalities (6).  
Zinc is the second most abundant trace element in human  
body, it cannot be stored. It therefore needs to be regularly  
replenished via the diet. The importance of Zn in human  
reproductive health and its potential in sperm quality and  
quantity requires regular evaluation especially in the sub-  
Saharan Africa where the rates of infertility and micronutrient  
(10). In humans, the level of zinc is high in the prostate  
compared to the other tissues and body fluids. Zinc is a  
marker of prostatic function and it regulates the functions of  
spermatozoa, acts as a co-factor for several enzymatic  
reactions, and helps in the preservation of sperm motility (11).  
It has been reported that most of the copper present in seminal  
plasma comes from the prostate, but unlike zinc, copper is  
also released by other structures of the reproductive tract such  
as epididymis and seminal vesicles (11). Copper is an  
important trace element for several metalloenzymes and  
metalloproteins that are involved in energy or antioxidant  
metabolism. Unfortunately, the ionic form of copper rapidly  
becomes harmful to many cells at high levels, including  
human spermatozoa. Copper can promote the formation of  
free radicals and induce the oxidation of polyunsaturated fatty  
acids of membrane leading to the inactivation of enzymes and  
structural proteins abnormalities (12). There are however  
conflicting reports in literature as to the impact of low or high  
levels of copper or zinc on semen quantity and quality. Some  
have shown that oligozoospermic men with sperm counts <20  
million per mililiter had slightly lower seminal plasma zinc  
level than normozoospermia, while others observed that  
*
Correspondence author: Prof. M. A. Emokpae, Department  
of Medical Laboratory Science, School of Basic Medical  
Sciences, College of Medical Sciences, University of Benin,  
Benin Email:  
City,  
Edo  
State,  
Nigeria.  
mathias.emokpae@uniben.edu, Cell Phone: +2348034511182  
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J Infertil Reprod Biol, 2020, Volume 8, Issue 3, Pages: 49-52. https://doi.org/10.47277/JIRB/8(3)/49  
normozoospermic and oligoasthenozoospermic males had  
visits within two months for analysis and mean value of the  
determinations was used.  
similar seminal plasma zinc levels (13-15). A low Zn levels in  
cells was reported to be a contributing factor to poor sperm  
quality and testosterone in infertile males (16), while  
Danscher et al (17) reported a high concentration of Zn was  
associated with poor sperm motility. Experimental study has  
shown that low copper level may be lead to the production of  
ejaculates of lower volume, lower sperm concentration and  
poor sperm motility and morphology (6). On the other hand,  
some authors have reported that high Cu levels were harmful  
to reproductive function and Zn had an opposite influence on  
the semen quality (19). Despite the numerous studies of the  
levels of Cu and Zn only very few had associated Cu/Zn ratio  
with sperm indices among infertile males. This study seeks to  
determine the levels of Cu and Zn concentrations in seminal  
plasma of infertile men and correlates Cu/Zn ratio with sperm  
concentrations.  
2.5 Laboratory Analysis  
2.5.1 Routine Semen Analysis  
After liquefaction the semen specimens were assessed for  
volume, appearance, pH, and viscosity. Routine semen  
analysis was performed microscopically with special interest  
in the sperm concentration, percentage motility and  
percentage  
morphology.  
Based  
on  
the  
sperm  
concentration/count according to WHO criteria (19), the  
overall samples were therefore grouped into the following  
6
categories:  
oligozoospermia; 1-14.9  
normospermia;  
≥15  
x10  
cells/mL,  
6
x
10 cells/mL,and azoospermia;  
absence of sperm cells in the ejaculate. After analysis, the  
semen specimen was centrifuged at 3000rpm for 10 minutes  
and the supernatant was separated and used for Cu and Zn  
determination.  
2
Materials and Methods  
2
.5.2 Determination of Cu and Zn concentrations  
Total seminal plasma copper concentration was measured  
2
.1 Study Design  
This is a cross-sectional study of males investigated for  
by atomic absorption spectrometry (Buck Scientific Model  
VGP-210, Germany). Samples were digested by adding nitric  
acid diluted with deionized water (1:4). This was aspirated  
into the instrument after adequate calibration with series of  
infertility and the study participants were between the ages of  
3-60 years with mean age of 40.4±5.6 years. The control  
group were males of proven fertility without chronic clinical  
illnesses with mean age of 40.2±5.5 years.  
2
2 2  
CuCl .H O standards at wavelength of 324.8 nm.  
2
.2 Ethical Consideration  
The study protocol was approved by the Health Research  
2
.5.3 Determination of zinc  
Zinc concentration in seminal plasma was measured by  
Ethics Committee of Osun State Ministry of Health, Abere,  
Osogbo, Osun State (Ref. OSHREC/PRS/569/149) dated 30  
November, 2017. All study participants gave informed  
consent before specimens were collected.  
th  
atomic absorption spectrophotometry (Buck Scientific Model  
VGP-210, Germany) at a wavelength of 213 nm . Before each  
assay, specimen was diluted 1:4 and mixed thoroughly. The  
instrument was calibrated with Zn standard solutions. The  
spectrophotometer was calibrated after each series of assays.  
2
.3.1 Inclusion Criteria  
All male subjects evaluated for infertility and consented to  
2
.6 Statistical Analyses  
The data were compared between the groups using  
be enrolled without physical abnormalities or chronic illnesses  
were included in the study. Subjects without chronic clinical  
illnesses and had their babies within the last one year, whose  
semen counts were over 15 million sperm cells per millilitre  
semen according to World Health Organization (WHO)  
criteria (19) were included and used as controls.  
unpaired Students-t-test and One way analysis of variance  
ANOVA) as appropriate by statistical software SPSS version  
1 (SPSS Inc, Chicago, IL, USA). A p-value ≤ 0.05 was  
(
2
considered statistically significant.  
2
.3.2 Exclusion Criteria  
Individuals with known pathological or congenital  
3 Results  
Table 1 shows the comparison of seminal plasma levels of  
copper, zinc and Cu/Zn ratio among infertile males than fertile  
males. Seminal plasma zinc was significantly lower (p<0.001)  
while copper and Cu/Zn ratio were significantly higher  
(p<0.001) in infertile than fertile males. In the same vein,  
Cu/Zn ratio was significantly higher (p<0.001) among  
infertile males than controls. Seminal plasma levels of copper  
and Cu/Zn ratio increased with decreasing concentrations of  
sperm cells while zinc levels increase with increasing  
concentration of sperm cells among infertile males (table 2).  
Table 3 indicates that copper and Cu/Zn ratio correlated  
negatively while zinc correlated positively with sperm  
concentrations.  
conditions such as hypertension, diabetes mellitus, sexually  
transmitted diseases, testicular varicocele and genital warts  
were excluded. In addition, individuals currently on  
antioxidant supplementation, smokers and alcoholics were  
also excluded.  
2
.4 Sample Collection  
Semen samples were collected in a sterile container by  
self or assisted masturbation after at least 72 hours of sexual  
abstinence (without the use of spermicidal lubricants). The  
specimens were delivered to the laboratory within 30 minutes  
of ejaculation. Two specimens were collected at different  
Table 1: Comparison of seminal plasma levels of copper, zinc and copper-to-zinc ratio in infertile males  
Parameters  
Age (Years)  
Infertile males (n=400)  
40.4±5.61  
Fertile males (Controls)(n=100)  
40.2±5.60  
P-value  
0.91  
Zinc (µg/mL)  
Copper (µg/mL)  
Copper-to-zinc ratio  
0.81±0.01  
1.41±0.08  
1.73±0.01  
2.06±0.08  
1.93±0.05  
0.92±0.02  
0.001  
0.001  
0.001  
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J Infertil Reprod Biol, 2020, Volume 8, Issue 3, Pages: 49-52. https://doi.org/10.47277/JIRB/8(3)/49  
Table 2: Comparison of copper, zinc and copper-to-zinc ratio among subtypes of infertile males  
Seminal plasma  
copper(µg/mL)  
Seminal plasma  
zinc(µg/mL)  
Copper-to-zinc  
ratio  
Subtypes of infertility  
N
b
b
b
Azoospermia(Absence of sperm cells)  
Oligozoospermia(<15 x 10 /mL)  
Normozoospermia(>15 x 10 /mL)  
P-value  
41  
2.73±1.48  
1.97±1.62  
0.92±0.61  
1.26±0.81  
2.95±0.02  
1.56±0.01  
6
a
b
b
168  
191  
400  
6
1.95±1.39  
0.001  
3.08±0.59  
0.01  
0.62±0.01  
0.001  
a= p>0.05; b=p<0.001  
Table 3: Correlation of seminal plasma copper, zinc and copper-to-zinc ratio with sperm concentrations  
Copper  
R-value P-value  
-0.160  
-0.59  
-0.359  
Zinc  
Cu/Zn Ratio  
R-value  
-0.155  
Correlation  
R-value P-value  
P-value  
0.01  
Normozoospermia  
Oligozoospermia  
Azoospermia  
0.05  
0.05  
0.02  
0.187  
0.221  
0.310  
0.01  
0.005  
0.02  
-0.244  
0.002  
0.001  
-0.492  
5
α-dihydro-testosterone (7). The finding of significantly  
5
Discussion  
higher copper levels in seminal plasma of infertile men is  
consistent with previous studies (27-29). The authors  
observed that copper chelation was implicated in the  
suppression of spermatogenesis suggesting that higher levels  
are harmful to reproductive potential. Experimental study has  
indicated that implantation of copper in the epididymis, vas  
deferens and scrotum of mammals adversely affected fertility,  
hence copper was considered as a highly toxic element for  
sperm (30). High levels of copper in seminal plasma were  
associated with decreased sperm motility. An action that was  
attributed to induction of free radicals generation by copper  
ions,that ultimately resulted in lipid peroxidation of  
spermatozoa membrane (27). Copper-to-zinc ratio correlated  
with sperm concentration, finding that is consistent with  
previous study (6). Significantly higher Cu/Zn ratio was  
reported among subjects with abnormal progressive motility  
than that of the healthy control group. Higher CU/Zn ratio  
was also reported among subjects with poor sperm quality  
than normozoospermia. Low serum zinc, higher copper levels  
and higher Cu/Zn ratio were reported among infertile men  
than fertile control subjects (30, 31).  
The conflicting reports in literature coupled with the  
importance of biological functions of copper and zinc in  
reproductive health, it is important to assess the Cu/Zn ratio in  
the assessment of their association with sperm concentration.  
The Cu/Zn ratio is a more important assessment than the  
concentration of individual element in clinical practice. In  
healthy individuals, the body has the ability to manage and  
regulate the amount of essential trace elements in the blood.  
The dietary essential metals are included into blood if their  
blood concentrations are low, incorporated into the cells when  
their cellular levels are depleted or eliminated when blood and  
cellular levels are adequate or excess (20). When this  
regulatory system fails to function optimally, abnormal levels  
and imbalance in their ratios occur (21).  
In this study, we observed that seminal plasma copper and  
Cu/Zn ratio were significantly higher while zinc was  
significantly lower in infertile males than fertile control  
subjects. This observation is consistent with previous studies  
(9, 21-24). Zhao and Xiong reported a lower mean level of  
zinc and a positive association with poor spermatogenesis and  
poor sperm quality among infertile men (22). Also, Chia et al  
reported that seminal plasma zinc levels correlated directly  
with sperm density and motility (23). A significantly low level  
of seminal plasma zinc was reported in oligozoospermia and  
azoospermia and that poor zinc in diet is a risk factor for low  
sperm quality of sperm and idiopathic infertility (9).  
Conversely, other authors observed that no significant  
correlation exist between zinc concentration and semen  
indices (25, 26).  
6 Conclusion  
Significantly higher seminal plasma copper and Cu/Zn  
ratio coupled with lower zinc was observed among infertile  
males than control. Copper-to-zinc ratio correlated with sperm  
concentration in infertile males. The interaction between  
copper and zinc may be considered in the clinical evaluation  
of the infertile men.  
Zinc plays important roles in the physiological functions  
of spermatozoa. The zinc finger motif Cys2/His2 of protamine  
plays vital role in the prevention of transcription via sperm  
chromatin stabilization and in the prevention of oxidative  
damage. Also, zinc is a cofactor for antioxidant enzymes (7).  
Zinc protects the testes from injury by stressors such as toxic  
metals, fluoride and heat (7). It is accumulated in the testes  
during early spermatogenesis and probably plays a role in the  
modulation of spermatogonial proliferation and differentiation  
Acknowledgments  
The authors appreciate the contributions of all medical,  
Nursing and Medical laboratory services Departments towards  
the completion of the study.  
Source of funding  
Nil  
(
7). The impact of low seminal plasma zinc on fertility also  
Ethical issue  
stem from its effect on the hypothalamus-pituitary-gonadal  
axis. Low zinc has adverse effect on serum testosterone  
secretion. Adult males with low zinc levels had inadequate  
testosterone synthesis in the leydiq cell, because zinc is co-  
factor for 5α-reductase enzyme that is necessary for the  
transcription of testosterone to the biologically active form,  
Authors are aware of, and comply with, best practice in  
publication ethics specifically with regard to authorship  
(avoidance of guest authorship), dual submission,  
manipulation of figures, competing interests and compliance  
with policies on research ethics. Authors adhere to publication  
5
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J Infertil Reprod Biol, 2020, Volume 8, Issue 3, Pages: 49-52. https://doi.org/10.47277/JIRB/8(3)/49  
requirements that submitted work is original and has not been  
published elsewhere in any language.  
17. Danscher G, Hammen R, Fjerdingstad E, Rebbe H. Zinc content  
of human ejaculate and the motility of sperm cells. Int J Androl.  
1
978;1(1-6):576-581.  
1
8. Peizhi H, ShijuW, Xu’e L. General chemistry. Beijing: Science  
Publish House, 2001:372379.  
Competing interests  
The authors declare that there is no conflict of interest that  
would prejudice the impartiality of this scientific work.  
19. WHO laboratory manual for the examination and processing of  
human semen. 5th ed. WHO Press, World Health Organization,  
2
0 Avenue Appia, 1211 Geneva 27, Switzerland; 2010. p. 7-113.  
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0. Osredkar, J.; Sustar, N.J. Copper and zinc, Biological role and  
significance of copper/zinc imbalance. J. Clin. Toxicol. 2011;  
S3:001.  
Authors’ contribution  
All authors of this study have a complete contribution for  
data collection, data analyses and manuscript writing.  
21. Emokpae MA, Fatimehin EB. Copper-to-Zinc Ratio Correlates  
with an Inflammatory  
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2. Marker in Patients with Sickle Cell Disease Sci 2019; 1: 55.  
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