Objective: Hepatocyte growth factor is a pleiotropic cytokine with
potent mitogenic activity in many organs and tissues. The receptor for hepatocyte
growth factor is c-Met, a transmembrane glycoprotein with tyrosine kinase
activity. We investigated the relationships between hepatocyte growth factor and
c-Met expression levels on sperm motility and male infertility. Methods:
The study was designed as a prospective cohort, single-center clinical trial and
was conducted between March 2015 and June 2016. The control group consisted of 31
healthy male volunteers with children, while the study group consisted of 61 men
who were diagnosed with male infertility after presenting to the In
Vitro Fertilization Unit of our Institute. All participants in the study were
aged 18–60 years. Sperm samples were taken from each participant and divided in
two. The fresh sample was examined immediately for expression of c-Met, while the
other was stored frozen and evaluated later for hepatocyte growth factor. Primary
outcome measures were the levels of hepatocyte growth factor and c-Met
expression, while secondary outcome measures were the correlation of these levels
with sperm count, morphology and motility. Results: The c-Met expression
level in the control group (26.53
Hepatocyte growth factor (HGF) is a pleiotropic cytokine previously identified
as a ‘scatter factor’ and potent mitogen for hepatocytes that provides a
connection between different target cells [1, 2, 3, 4]. HGF is expressed in various
immunoreactive organs in the male reproductive system, including the seminiferous
tubules, epididymis, ductus deferens, epidermis, prostate and seminal vesicle
epithelium [5, 6]. It can act as a mitogen (stimulator of cell growth), a motogen
(stimulator of cell motility) and a morphogen (inducer of a structure similar to
multicellular tissue). It also has features that allow it to play a role in the
functional regulation of many systems involved in movement, such as the
spermatogenic epithelium [7, 8, 9]. HGF has a unique receptor called met, with the
encoding oncogene referred to as c-Met. The c-Met receptor has tyrosine kinase
activity and a heterodimer structure composed of an
The aim of this study was therefore to evaluate the levels of c-Met oncogene and its ligand HGF in relation to the sperm count and to sperm morphology and motility parameters in infertile males and healthy volunteers.
The study design was a prospective cohort study and was conducted between March 2015 and June 2016 at a single center following approval from the institutional ethics committee.
A control group and a study (infertile) group were created. The control group consisted of healthy males aged 18-60 years who had children, had no known chronic disease, had not undergone any urological surgery, had normal sperm count, motility and morphology as revealed by sperm analysis, and had volunteered to participate in the study. The infertile group consisted of males aged 18–60 years who were admitted to the Institute’s IVF Andrology Laboratory, had at least one value for sperm count, motility and morphology that was below the reference range from the WHO 2010 Semen Analysis Parameters on sperm analysis (Table 1) , and had volunteered to participate in the study.
|Parameter||Lowest reference value|
|Semen volume (mL)||1.5 (1.4–1.7)|
|Total sperm count (millions)||39 (33–46)|
|Sperm concentration (million/mL)||15 (12–16)|
|Total motility (%)||40 (38–42)|
|Progressive motility (%)||32 (31–34)|
|Sperm morphology (normal forms, %)||4 (3.0–4.0)|
All participants signed an Informed Volunteer Consent Form after being provided with detailed verbal and written information on the study.
A sperm sample was taken from all study participants under appropriate conditions at the Institute’s IVF Andrology Laboratory after 2–5 days of sexual abstinence. Individuals with a sperm sample under volume of 3 mL were excluded from the study.
Samples were subjected to a spermiogram following a liquefaction process lasting
30 minutes at 37 °C. The spermiogram procedure was performed by an
embryologist at the IVF Andrology Laboratory after placing a 10
Following the spermiogram procedure, the first sample of one milliliter was placed into a Falcon 2003 tube without any treatment and sent to the Institute’s Genetics Laboratory for the evaluation of c-Met expression on the same day. The second one milliliter sample was stored frozen at –10 °C in a Falcon 2003 tube for later HGF measurement.
Total RNA was extracted from 1 ml of fresh ejaculate using a tissue total RNA
Isolation Kit (HibriGen, Turkey). It was then transcribed in vitro to
cDNA using MuLV reverse transcriptase (High Capacity cDNA Reverse Transcription
Kit, Applied Biosystems, USA) and random hexamers as primers, according to the
manufacturer’s instructions. The presence of transcripts for the target gene
c-Met and the reference gene HPRT1 was shown by Real Time PCR
under the experimental conditions recommended by the manufacturer and using the
Human c-Met Expression Kit (HibriGen, Turkey) and LightCycler 2.0 device (Roche
Diagnostics, Germany). The amplification mixture for each Q-PCR consisted of PCR
mix and 50 ng of template cDNA in a final volume of 20
HGF levels were measured with a commercial kit (Human HGF Instant ELISA,
Affymetrix eBioscience) that had been stored at –20 °C. The previously
stored frozen samples were thawed at 24 °C and sent to the Institute’s
Biochemistry Laboratory for measurement of HGF. Each plate from the kit contained
96 small compartments, including14 standard and 2 empty compartments. Antibodies
coated with anti-human HGF had been absorbed into these compartments. Distilled
Statistical analysis: The NCSS (Number Cruncher Statistical System) 2007
(Kaysville, Utah, USA) program was used to perform the statistical analysis.
Descriptive statistical methods (mean, standard deviation, median, frequency,
percentage, minimum, maximum) were used to evaluate study data. Pairwise group
comparisons of quantitative data were conducted with the Student’s
t-test when the variables showed a normal distribution, and the
Mann-Whitney U test otherwise. The Wilcoxon Signed Rank test was used for
intragroup comparison of variables that did not show a normal distribution.
Spearman correlation analysis was used to evaluate the relationships between
variables. Significance was accepted at the p
The study was conducted on a total of 92 male patients (61 infertile and 31 fertile) who presented to the Florence Nightingale Hospitals Group’s IVF department between March 2015 and June 2016.
The sperm count, sperm motility percentage and sperm morphology percentage reported in the WHO 2010 Semen Analysis Parameters (Table 1) were considered when assigning participants to either the control or infertile groups. Participants with values below the reference range for any of the three parameters (sperm count, motility percentage and morphology percentage) were assigned to the infertile group.
The sperm count and sperm morphology percentage were significantly higher in the control group (Mann-Whitney U test). The sperm motility percentage was also higher in the control group but did not reach statistical significance, possibly because only one of the three semen analysis parameters had to be lower than the reference range in order to assign an individual to the infertile group.
Basal sperm count was 66.84
Sperm morphology percentage was 4.45
The c-Met expression level was 26.53
HGF levels were 4.87
|Total (n = 92)||Infertile (n = 61)||Control (n = 31)||p|
|Basal Sperm Count (x million)||Mean
|Min–Max (Median)||2–116 (33.5)||2–110 (21)||18–116 (64)|
|Sperm Motility (%)||Mean
|Min–Max (Median)||15–86 (45)||15–85 (45)||40–86 (45)|
|Sperm Morphology (%)||Mean
|Min–Max (Median)||0–6 (3)||0–5 (2)||4–6 (4)|
|Min–Max (Median)||19.43–33.07 (28.35)||21.58–33.07 (28.76)||19.43–32.73 (27.89)|
|Min–Max (Median)||0.83–21.58 (3.10)||0.83–8.25 (2.98)||1.01–21.58 (3.63)|
No statistically significant correlations were found between c-Met expression
and HGF levels in the total cohort, the infertile group or the control group
|c-Met - HGF|
|Total (n = 92)||0.130||0.219|
|Infertile (n = 61)||0.146||0.263|
|Control (n = 31)||0.287||0.117|
|r = Spearman’s Correlation Coefficient.|
|Sperm count (millions)|
|Total (n = 92)||Infertile (n = 61)||Control (n = 31)|
|r = Spearman’s Correlation Coefficient.
However, a significant negative correlation was found between the basal sperm count and c-Met expression level in the total cohort (Table 4). The correlation between these factors did not reach statistical significance in either the infertile or control groups. Fig. 1 shows the correlation between sperm count and c-Met expression level in the total cohort.
Correlation between basal sperm count and c-Met expression level in the total cohort (r = –0.309; p = 0.003).
No significant correlations were found between the sperm motility percentage and
HGF level in the total cohort, the infertile group or the control group (each
|Sperm motility percentage|
|Total (n = 92)||Infertile (n = 61)||Control (n = 31)|
|r = Spearman’s Correlation Coefficient.
However, significant positive correlations were observed between the sperm motility percentage and the c-Met expression level in the total cohort (Fig. 2) and in the infertile group (Fig. 3) (Table 5).
Positive correlation between sperm motility percentage and c-Met expression level in the total cohort (r = 0.421, p = 0.001).
Positive correlation between sperm motility percentage and c-Met expression level in the infertile group (r = 0.488, p = 0.001).
No statistically significant correlations were found between sperm morphology
and HGF level in the overall cohort or in the infertile group (each p
|Sperm morphology percentage|
|Total (n = 92)||Infertile (n = 61)||Control (n = 31)|
|r = Spearman’s Correlation Coefficient.
Significant negative correlations were also found between the sperm morphology
percentage and c-Met expression level in the total cohort (Fig. 4) and in the
infertile group (Fig. 5), but not in the control group (p
Negative correlation between sperm morphology percentage and c-Met expression level in the total cohort (r = –0.441, p = 0.001).
Negative correlation between sperm morphology percentage and c-Met expression level in the Infertile group (r = –0.422, p = 0.001).
Male infertility is responsible for 25–30% of all cases of infertility. It can be caused by many factors, but changes in sperm count, motility and morphology are the most common problems.
HGF regulates many different motile systems, including the spermatogenic epithelium. It is a pleiotropic cytokine known to be a potent mitogen for hepatocytes, and is also thought to be a ‘scatter factor’ that connects target cells [1, 2, 3, 4]. HGF binds with high affinity to c-Met, a transmembrane receptor with tyrosine kinase activity that is only expressed in mesenchymal cells.
HGF acts as both a paracrine and an autocrine modulator to support testicular functions. In this study we analyzed the sperm of infertile and healthy males for the parameters of basal sperm count, sperm morphology percentage and sperm motility percentage. We then analysed the relationships between these parameters and the levels of c-Met expression and HGF.
Depuydt et al.  reported the presence of significant amounts of HGF in human seminal plasma. The detection of HGF in vasectomized men indicates that it is not testicular in origin, with the main source in humans found to be the epididymis, prostate and seminal vesicle [11, 12].
Catizone et al.  was the first to investigate the role of HGF in male fertility by evaluating the in vitro motility of spermatozoa isolated from the caput of the epididymis. They found that motility in culture medium was significantly lower in the absence of HGF, and that sperm motility could be preserved for long periods in the presence of HGF. The HGF/c-Met key-lock system was therefore shown to have a positive role in preserving sperm motility during transition of the sperm from the epididymis to an in vitro environment.
However, another study did not find a positive correlation between the HGF level and sperm motility. Kitamura et al.  added recombinant HGF to washed spermatozoa from infertile males and measured sperm motility, as well as the HGF concentration in seminal plasma. The results were compared with the hormone profile and with various semen analysis parameters. Recombinant HGF had no effect on the linear motility or motility frequency of the sperm that would cause hyperactivation in the spermatozoa. Moreover, no correlation was observed between the HGF concentration in the seminal plasma and any of the clinical parameters investigated.
Wiltshire et al.  evaluated HGF and semen analysis parameters in three different patient groups: those with normozoospermia, subfertility or azoospermia. They found no significant correlations between serum HGF concentration and sperm concentration, sperm motility percentage, total sperm count or total motile sperm count. There was also no significant difference in the HGF level between the three patient groups.
Catizone et al.  reported that c-Met in rat spermatogonia, pachytene spermatocytes and round spermatids could increase germ cell proliferation in conjunction with HGF. C-Met also influences male germ cell homeostasis and consequently male fertility by decreasing the number of apoptotic cells in prepubertal experimental rats .
In the present study, c-Met expression in the infertile group was significantly higher than in the control group (Table 2). This suggests a role for c-Met in the regulation of spermatogenesis and sperm quality, either by directly affecting sperm germ cells or through tubular and interstitial somatic cells in the testis. HGF is a growth factor that induces sperm motility . The effect of HGF on spermatozoa increases during passage through the epididymis and this is directly correlated with high c-Met receptor expression .
Catizone et al. and others [14, 19, 20] have reported that c-Met is expressed in both human and rat germ cells and that in rats it is always present during the spermatogenetic process from spermatogonia to spermatozoa.
Using ex vivo organ culture of testicles from 8–30 day old rats, it has also been shown that HGF controls germ cell mitotic activity by increasing spermatogonial cell proliferation . Mitotic germ cells need this HGF signal, but germ cells must also be inhibited to allow the orderly entry of germ cells at the beginning of the meiotic process.
The above findings support our notion that the factors responsible for low HGF levels in the infertile group could be increased c-Met expression. C-Met upregulation is a response to the low HGF level. Elevated c-Met expression affect the HGF/c-Met key-lock system and could attempt to increase intracellular signaling.
The HGF level in the control group was significantly higher than in the infertile group (Table 2). The presence of sufficient ligand in the control group allows an appropriate amount of HGF/c-Met complex to form and therefore satisfactory intracellular signaling can occur. This finding may also explain the higher sperm motility percentage observed in the control group compared to the infertile group, despite the lack of statistical significance. Increased HGF levels appear to downregulate the c-Met receptor, to physiological levels of expression. Spearman’s correlation coefficient analysis did not reveal any significant correlations between c-Met expression and HGF levels in the total cohort, the infertile group or the control group (Table 3).
A positive correlation was found between the c-Met expression level and sperm motility percentage in the overall cohort and in the infertile group, thus supporting a positive effect of c-Met expression on sperm motility. However, no such correlation was observed in the control group where all parameters for the WHO 2010 Semen Analysis reference ranges were met. In the presence of healthy spermatozoa, sperm maturation and differentiation are consisted with the seminal plasma. If the sperm motility sufficient, there is no need to increase the c-Met expression.
Goda et al.  reported that adenovirus-mediated HGF gene transfer into the testis of experimental cryptorchid rat models stimulated the overexpression of HGF and significantly decreased the number of apoptotic germ cells by regulating spermatogenesis and testicular weight.
Naz et al.  reported that the highest specific localization of HGF in the rat epididymal tract where the sperm becomes motile was in the distal corpus and cauda. HGF was found to slightly induce cell motility in immotile sperm, to have a protective effect on epididymal sperm motility in vitro, but not to improve the motile sperm percentage [23, 24]. However, the absence of this motogenic effect in human sperm, although previously reported in rodents , means the role of HGF in human sperm physiology is still controversial.
We found significant negative correlations between the c-Met expression level and sperm morphology percentage in the total cohort and in the infertile group (Table 6). We believe that better sperm morphology indicates higher quality sperm within the fertile range, thus eliminating the need to increase intracellular signaling and resulting in decreased c-Met expression.
A negative correlation was found between c-Met expression and sperm concentration in the overall cohort (Table 4). Increased c-Met expression to stimulate sperm and increase its activation is therefore not required in the presence of a sufficient sperm concentration. This suggests there is a need to consider the function of the epididymis during evaluation. Since HGF expression and the regulation of c-Met expression occur in this region, it is possible that a “main control area” is active here to correct the sperm quality.
We have demonstrated in this study that HGF levels in healthy males are high and support sperm motility during passage of the ejaculate through the epididymis. The necessary stimulation is therefore provided by HGF without the requirement for c-Met upregulation.
The high c-Met levels observed in the infertile group suggest this receptor may be overexpressed in response to low HGF in order to stimulate sperm. This could be an attempt to compensate for functional defects by upregulating c-Met receptor expression when HGF-related functions in the cell are inadequate, particularly those concerning motility.
The observed increase in c-Met expression as sperm motility increases demonstrates the association between these two factors and is consistent with the results of earlier studies. Since we do not have a normogram for the ligand-receptor relationship between HGF and c-Met in healthy males and because this is variable even during spermatogenesis, it may be more useful to consider these two factors together in future work.
The negative correlations observed here between sperm count and c-Met expression (Table 4) and between sperm morphology percentage and c-Met expression (Table 6) suggest that upregulation of c-Met occurs in order to increase intracellular signaling. When the cause of infertility is other parameter than sperm motility, the relationship between the HGF ligand and the c-Met receptor is regulate with the activity of various processes trying to activate or increase it within the cell.
When the cause of infertility is a parameter other than sperm motility percentage (e.g., decreased sperm count or sperm morphology percentage), we believe the c-Met receptor controls the key-lock mechanism throughout the life of the sperm and starting in the epididymis in order to compensate for the defect. It is already well known that c-Met is upregulated in many other tissues, and especially in neoplastic tissue. As factors other than the sperm motility percentage deteriorate, the c-Met receptor is overexpressed in order to increase intracellular signaling. This improves the functional properties of sperm and leads to fertilization as an end result. It will be interesting to further clarify the relationship of various Semen Analysis Parameters such as sperm count and sperm morphology with c-Met and HGF levels. In future work, we plan to evaluate c-Met expression and HGF levels in cases where asthenospermia is suspected as the cause of male infertility and to monitor the effect of recombinant HGF.
Further studies in this area should advance our understanding of the pathogenesis and treatment of male infertility.
BOK, VSH and UG designed the research study. CO performed the research. FA provided help and advice on the ELISA experiments. CMB analyzed the data. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.
This study was conducted in accordance with the Declaration of Helsinki. The protocol was approved by the Ethics Committee of T.C. Istanbul Bilim University (approval number: 44140529-2015, 15/04).
We would like to express our gratitude to all those who helped us during the writing of this manuscript.
This research received no external funding.
The authors declare no conflict of interest.
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