Supervisors info:
Σιμοπούλου Μαρία, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ, Επιβλέπουσα
Μαστοράκος Γεώργιος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Φιλίππου Αναστάσιος, Αναπληρωτής Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Summary:
Nowadays an increased trend of inability of achieving a
pregnancy has been observed, with the underlying etiology of infertility to be unclear.
This may hinder the optimal management of infertile couples. Albeit the great progress
that has been observed, the complex male physiology along with the functioning
mechanism of their reproductive system are complex and require intensive and
continuous research. Insulin-like growth factor I (IGF-I) has been suggested as a factor
that may play a pivotal role in male infertility due to its anabolic effect and differential
expression pattern of its three isoforms, as it has been observed in many other
pathological situations. The aim of the present study is to investigate the occurrence of
differential expression of the three IGF-I isoforms between men presenting with
abnormal semen parameters in semen analysis and normozoospermic males, as well as
a possible association between a different expression pattern of the IGF-I isoforms and
male infertility. Forty semen samples from men aged from 32 to 54 years old (43.9
± 4.6) were collected for the present study. Each sample was evaluated for certain
parameters according to the World Health Organization criteria. Written informed
consent was obtained from all participants. Four study groups were identified according
to the number of defects being identified in their semen analysis: “Normozoospermic”,
“Single defect”, “Double defect” and “Oligoasthenoteratozoospermic (OAT)”. First,
semen samples were diluted with phosphate-buffered saline (PBS) to 10x106
spermatozoa per mL and underwent osmotic shock to eliminate the non-gamete
component. Total RNA was extracted using TRItidy GTM according to the
manufacturer’s instructions and its concentration and purity was determined using a
BioSpec-nano Life Science spectrophotometer. Thereafter, complementary DNA
(cDNA) synthesis was performed for each sample using Protoscript II First Strand
cDNA synthesis kit. cDNA quantification was then conducted through real time
polymerase chain reaction using specific primers for the three IGF-I isoforms. The
mRNA level of each sample was normalized against 18s rRNA and expressed as fold
change. At the same time, proteins were also isolated from spermatozoa using TRItidy
GTM protocol and were used alongside with seminal plasma in western blot for detection
of the IGF-IEc isoform at protein level. Statistical analysis was conducted using the R
statistical programming language.
IGF-IEc mRNA was present in all 40 samples regardless of their
semen pathology, however, the Ec isoform was not detectable at protein level given the
amount of protein available. Moreover, IGF-IEa mRNA was not expressed except for
a single individual where it appeared to be related to the subject’s temperament. Two
out of 40 samples may have expressed the IGF-IEb isoform but only sequencing
analysis could confirm that. The mean mRNA levels of IGF-IEc in the
“Normozoospermic” group were statistically significant lower than those with “Double
defect” (5.1 ± 8.5, p-value=0.0117*) and “OAT” (2.9 ± 2.3, p-value=0.0072*) and with
a marginally non-significant difference with the “Single defect” group (4 ± 3.9, p-
8 -
value= 0.0254). Furthermore, a statistically negative correlation was found in the
mRNA levels of IGF-IEc with three of the semen parameters: total sperm count (r=-
0.3895225, p-value=0.01298), progressive motility (r =-0.4505146, p-value=
0.003532) and total motility (r =-0.3790446, p-value=0.01586). This study reported for the first time the distinct expression of the IGF-I isoforms in spermatozoa. The aforementioned results suggest a possible correlation between IGF-IEc mRNA levels and infertile men carrying two or more sperm defects and also that in spermatozoa the function of IGF-I is mediated mainly through the IGF-IEc isoform. Furthermore, the negative correlation of IGF-IEc mRNA levels with the aforementioned parameters indicates that high levels of IGF-IEc may negatively affect spermatogenesis and maturation process of spermatozoa. However, the conduction of larger, well-designed studies regarding the influence of the locally produced IGF-I isoforms on semen quality and spermatogenesis depending on their expression pattern, are needed in order to strengthen our findings.
Keywords:
IGF-1, Isoforms, Male infertility, Expression pattern
