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Impact of Metformin on Male Reproduction

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Current Pharmaceutical Design, 2015, 21, 3621-3633 3621

Impact of Metformin on Male Reproduction

Carolina Ferreira1,2, Mário Sousa1,3, Ana Rabaça1,4, Pedro F. Oliveira1,5, Marco G. Alves5 and Rosália Sá1*

1
Department of Microscopy, Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS)
and Unit for Multidisciplinary Research in Biomedicine (UMIB), University of Porto, Rua Jorge Viterbo Ferreira
228, 4050-313 Porto, Portugal; 2University of Trás-os-Montes and Alto Douro (UTAD), Quinta de Prados, 5000-
801 Vila Real, Portugal; 3Centre for Reproductive Genetics Prof. Alberto Barros, Av. do Bessa 240, 1° Dto.
Frente, 4100-009 Porto, Portugal; 4Faculty of Medicine of University of Porto (FMUP), Al. Prof. Hernâni Mon-
teiro, 4200-319 Porto, Portugal; 5CICS-UBI, Health Sciences Research Centre, Faculty of Health Sciences, Uni-
versity of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal

Abstract: Male infertility has been increasing over the last decades being nowadays a pressing health problem.
Diabetes mellitus (DM) can contribute directly or indirectly to male infertility due to an abnormal spermatogenesis,
which results in a decreased sperm quality. Type 2 Diabetes mellitus (T2DM) is responsible for the vast majority of
DM cases, being frequently treated with oral antidiabetic drugs. Metformin is the most cost-effective therapy for the treatment of T2DM.
This biguanide is an oral insulin-sensitizing agent capable of increasing insulin sensitivity and decreasing plasma fasting insulin levels.
The main metabolic action of this drug occurs in the liver. However, it has been shown that metformin acts on a variety of organs includ-
ing the male reproductive system. With the rising numbers of diabetic individuals among younger populations, there is an increase in the
consumption of metformin in individuals of this age group. As a result, it is important to discuss the role of metformin in male fertility.
This review presents the most recent data available from studies on the effects of metformin on male reproductive system. Together with
the discussion of these effects, their significance to male fertility is also debated.

Keywords: Diabetes mellitus, metabolism, metformin, male fertility, Sertoli cell, spermatogenesis.

INTRODUCTION
Human infertility is a serious health problem and a social con-
cern that affects approximately 15% of couples of reproductive age
[1, 2]. On average, 50% of all infertility cases can be attributed to a
male factor [3]. Male reproductive health has been decreasing over
the last few decades with a high prevalence of infertile men below
their forties [4-7]. Male infertility is a multifactorial disorder clini-
cally characterized by a low or absent sperm count and/or the pres-
ence of nonfunctional sperm cells due to abnormal spermatogenesis
[8]. Spermatogenesis is a complex process both dependent and
controlled by different somatic cell types present in the testes.
Therefore, Sertoli cells (SCs), Leydig cells (LCs) and developing
germ cells are strictly associated in order to maintain a normal
spermatogenesis, with production of mature spermatozoa capable of
fertilizing an oocyte [9].
Systemic diseases, such as Diabetes mellitus (DM), may irre-
versibly affect spermatogenesis leading to a decrease in sperm qual-
ity, which can directly or indirectly contribute to male infertility
[10, 11]. DM is a metabolic disorder characterized by a defective
insulin production, insulin resistance, or both [12, 13]. Patients with
this disease exhibit reduced antioxidant activity [14] and dysfunc-
tion of the hypothalamic-pituitary-gonadal axis [15, 16]. Type 1 and
type 2 Diabetes mellitus (T1DM and T2DM, respectively), are the
most commonly found types of DM in the general population.
Nonetheless, T2DM is responsible for the vast majority of DM
cases. In the clinical practice, T2DM is regularly treated with a
combination of oral antidiabetic drugs, healthy food regimens and
physical activity [17]. T2DM has been proliferating in the younger
population [18]. As a result, there is an increase in the consumption
of oral antidiabetics by this age group. Consequently, there is a high
percentage of males in reproductive age exposed to the adverse
*Address correspondence to this author at Department of Microscopy,
Laboratory of Cell Biology, ICBAS-UP, UMIB, Rua Jorge Viterbo Ferreira
228, Building 1, Floor 2, Room 03, 4050-313 Porto, Portugal; Tel: +351-22
042 8000x5242; Fax: +351-22 042 80 90; E-mail: rmsa@icbas.up.pt
effects of this disease and to the eventual effects of its treatment
[19].
The first aim for DM management is the proper control of gly-
cemia, in order to minimize the patient’s risk of developing vascu-
lar and neurologic complications. Lifestyle interventions (including
diet, physical activity and weight control) are cost-effective means
to improve DM outcome. Still, for the majority of patients, a life-
style modification alone is not sufficient to maintain normal blood
glucose levels. Thereby, pharmaceutical interventions are required
to maintain these levels and reduce the risk of clinical complica-
tions. Oral antidiabetic agents that are currently in clinical use can
be categorized into insulin-sensitizing agents (e.g., metformin and
thiazolidinediones), enhancers of insulin secretion (e.g., sulfony-
lureas and meglitinides), agents that modify intestinal absorption of
carbohydrate or fat (e.g., a-glucosidase inhibitors, lipase inhibitors),
and exogenous insulin [20, 21]. Metformin (1, 1-dimethylbigua-
nide) belongs to the insulin sensitizers class and is a synthetic drug
implemented as the first-line oral treatment for individuals with
T2DM [17, 22]. This agent has been clinically used for over 50
years, and is now consumed daily by more than 100 million indi-
viduals worldwide [23]. Notably, in the last years several other
effects have been attributed to metformin. For instance, metformin
administration has been reported to inhibit the proliferation of neo-
plastic cells [24, 25] and, when given in combination with
clomifene, to improve the fertility of women with polycystic ovary
syndrome (PCOS) through the induction of ovulation [26]. These
reports showed that metformin, not only has anti-hyperglycaemic
capacity, but can also act in various body tissues, including the
reproductive system [27, 28].
DM impact on male fertility is widely known. Nevertheless,
DM is a chronic disease and more research is needed to identify the
effects of its continued therapy on male reproductive functions.
Hence, it is necessary to further investigate the effects of metformin
on this system, evaluating its potentially harmful or beneficial ef-
fects and thus establishing its safety. Herein, we aim to discuss the

1873-4286/15 $58.00+.00 © 2015 Bentham Science Publishers

3622 Current Pharmaceutical Design, 2015, Vol. 21, No. 25
recent literature available on the effects of this drug on male fertil-
ity.
SPERMATOGENESIS AND MAINTENANCE OF FUNC-
TIONAL SPERM PRODUCTION
The male reproductive system is of great complexity. This sys-
tem comprises the testes, the accessory glands and the penis. The
perfect coordination between physical and hormonal components is
necessary to the normal occurrence of spermatogenesis. In addition,
this process is dependent on a strict regulation by the brain [29].
Testes are the central reproductive organs, being responsible for
two essential functions: synthesis of sex steroid hormones (steroi-
dogenesis) and production of spermatozoa (spermatogenesis) [30,
31]. Steroidogenesis is mainly maintained through LCs’ steroid-
secreting activity, being highly important for the development of
spermatogenesis [32]. This process occurs within the seminiferous
tubules (ST) of the testes of pubertal men. ST basically consist of
an association of SCs with germ cells. SCs create tight junctions
between the neighboring cells giving rise to the Sertoli/blood-testis
barrier (BTB) [33]. This barrier physically divides the seminiferous
epithelium into basal and apical compartments, each containing
germ cells in different stages of development. The basal compart-
ment consists of type A and B spermatogonia and leptotene sperma-
tocytes I, while the apical region comprises pachytene spermato-
cytes I, spermatocytes II and spermatids [34]. The BTB also con-
trols the entry of some substances from the blood into the
seminiferous epithelium luminal environment, controlling their
levels, and immunologically isolating the germ cells [35-37]. Each
SC is responsible for the structural maintenance and nurturing of
multiple germ cells at various stages of maturation [38-40]. These
Basement membrane
Myoid cell
Spermatogonium A
BTB
Endoplasmic
reticulum
Spermatocyte II
Mitochondria
Fig. (1). Schematic illustration of spermatogenesis. Spermatogonia type A divide and develop into spermatogonia type B, which enter mei-
otic prophase and differentiate into primary spermatocytes that undergo meiosis I and form the haploid secondary spermatocytes. Meiosis II
yields four equalized spermatids that migrate toward the lumen where fully formed spermatozoa are finally released. BTB - Sertoli/blood-testis
barrier.
Ferreira et al.
somatic cells are also responsible for water transport from the inter-
stitial space into the lumen, facilitating the movement of sperm
from the testis to the epididymis [41]. SCs secrete multiple nutri-
ents, peptides and metabolic intermediates necessary for the effi-
cient development of germ cells during spermatogenesis [40].
Among these substances, lactate plays a key role, being the pre-
ferred fuel for germ cells development and survival [42]. Research
conducted on this subject by Courtens and peers, concluded that the
infusion of lactate in rat testes significantly improves the spermato-
genesis index [43]. In addition, other studies reported that lactate
exerts anti-apoptotic effects [44], stimulates RNA and protein syn-
thesis [45, 46], and increases the respiratory rate of male germ cells
[46].
Spermatogenesis is under strong hormonal regulation, particu-
larly by the pituitary hormones, follicle-stimulating hormone (FSH)
and luteinizing hormone (LH). These hormones promote the func-
tional connection between the brain and testis through the hypo-
thalamus-pituitary-testis axis [47]. FSH and LH are dependent on
the release of the gonadotropin-releasing hormone (GnRH) by the
hypothalamus. When FSH and LH are produced, they are responsi-
ble for the stimulation of SCs and LCs, respectively. However,
when the stimulation index is at its highest, a negative-feedback
mechanism takes place. Hormones produced by LCs and SCs, tes-
tosterone and inhibin, respectively, act on the hypothalamus and
pituitary inhibiting the production of GnRH, FSH and LH [48].
This complex regulatory mechanism is able to maintain the appro-
priate levels of hormones for the normal course of spermatogenesis.
Consequently, any disruption on these mechanisms may affect male
fertility.
Interstitial Space
Leydig
cell
Spermatogonium B
Leptotene
Spermatocyte I
Pachytene
Spermatocyte I
Sertoli cell
nucleus
Round
Spermatids
Spermatozoa
Sertoli cell
Metformin and Male Fertility
In mammals, spermatogenesis (Fig. 1) is a complex event of
cellular differentiation that features three main continuous steps: 1)
mitotic spermatogonia proliferation and differentiation; 2) meiotic
phase; and 3) metamorphosis of round spermatids [49]. In the first
phase, the male stem cell, spermatogonium (2n), undergoes mitosis.
At the end of mitosis, this cell originates two different cell types:
spermatogonium A (2n) and B (2n). While spermatogonium A re-
mains undifferentiated, ensuring the germ cell line maintenance,
spermatogonium B originates primary spermatocyte which under-
goes meiosis, continuing the spermatogenesis process [49, 50]. At
the end of meiosis I, the primary spermatocyte (2n) originates two
secondary spermatocytes (n). These go through the second meiotic
division and form four round spermatids (n). Spermatids undergo
maturation, becoming more elongated, compact and motile in a
mechanism known as spermiogenesis [49, 51]. Finally, spermiation
occurs, the release of immature spermatozoa into the lumen of ST.
Subsequently, the last vestiges of cytoplasm are removed as the
spermatozoon transits to the rete testis and epididymis where the
final maturation process occurs [49, 51].
RELEVANCE OF THE NUTRITIONAL SUPPORT OF DE-
VELOPING GERM CELLS AND SPERMATOZOA TO
SPERMATOGENESIS
During spermatogenesis, germ cells present very specific needs
for their development into mature spermatozoa. Both germ cells
and spermatozoa rely on specific nutrients for their development
and to obtain the necessary energy for several cellular functions.
Sugars (mainly hexoses like glucose and fructose) and other me-
tabolites (such as lactate and citrate) are the preferred substrate of
germ cells and spermatozoa for ATP production [52, 53]. In fact,
glucose is the main fuel for the normal maintenance of spermato-
genesis and, subsequently, a normal production of mature, motile
spermatozoa [30]. Furthermore, glucose metabolism is responsible
for the SCs production of lactate, which is then exported into the
intra-tubular fluid, where it is the main metabolite used by germ
cell during spermatogenesis [42, 54].
Glucose is a polar molecule, thus requiring the presence of
transport proteins to effectively cross the lipid bilayer. Glucose
transporters are divided into two groups, sodium-dependent glucose
transporters (SGLT) and glucose transporters (GLUTs) [55, 56].
The SGLT family is composed of six different active glucose trans-
porter proteins, SGLT 1 to 6, that spend energy to transfer glucose
across the plasma membrane [55]. On the contrary, GLUTs are
passive transporters that despite their name, may carry other mole-
cules in addition to glucose, such as fructose, mannitol, vitamins
[57] and glucosamine [52]. The GLUTs family encompasses 14
proteins that are grouped into 3 classes, I (GLUT1-4 and 14); II
(GLUT5, 7, 9 and 11); and III (GLUT6, 8, 10 and H+-coupled myo-
inositol transporter (HMIT)), varying on their tissue distribution,
hexose affinity and structure [52]. However, only some of these
passive transporters (GLUT 1-3,5,8 and 9) are present in mammal-
ian testicular cells and are responsible for the uptake of glucose
[52].
Being located in the basal compartment of ST, undifferentiated
spermatogonia are supplied with nutrients directly from the intersti-
tial fluid and use glucose as fuel for ATP production [58]. Mature
germ cells, particularly pachytene spermatocytes and round sper-
matids, express all enzymes of the glycolytic pathway. However
they are unable to use glucose and require lactate from the extracel-
lular environment for energy production and survival [42, 46, 54,
58]. SCs play a crucial role in lactate production for the mature
germ cells and are able to produce lactate via glycolysis at high
rates [46, 58-62]. Glucose is imported into the SCs by a number of
different GLUTs [59]. GLUT1, 2, 3 and 8 are reported as the
GLUTs responsible for the glucose transport on these cells, how-
ever, their contribution is not equal [63-65]. Since GLUT1-3 are
located in the plasma membrane, they are suggested as the main
Current Pharmaceutical Design, 2015, Vol. 21, No. 25 3623
GLUTs accountable for the glucose uptake in these cells. GLUT8 is
located in the membrane of intracellular organelles and is not ex-
pected to contribute for the glucose intake from the extracellular
milieu [63, 64, 66, 67]. In these cells, glucose intake is highly regu-
lated by a group of different substances (hormones, cytokines and
growth factors) that have the ability to modulate the mRNA and
protein expression of GLUTs, thereby ensuring an appropriate lac-
tate production [61, 62, 64]. In fact, it was shown that in SCs, when
the levels of glucose or insulin are decreased, GLUTs expression
may be altered [67, 68]. In human and rat SCs, insulin deprivation
[67] or glucose deprivation [68] increases GLUT1 and decreases
GLUT3 mRNA expression. Similarly in rats SCs, factors such as
FSH, interleukin-1
(IL1
) and the basic fibroblast growth factor
(bFGF), modulate the mRNA expression of GLUT1 during pubertal
development [64]. Once inside the SCs, the majority of glucose
follows the glycolytic pathway being metabolised into pyruvate
which is subsequently converted to lactate with the oxidation of
NADH to NAD+ [69]. Once produced, lactate is exported to the
extracellular environment, where it can be used by the developing
germ cells. This transport through the SCs plasma membrane to
germ cells is mediated by specific set of carriers called monocar-
boxylate transporters (MCTs), isoforms MCT1, MCT2 and MCT4
[58, 61, 67, 68, 70, 71]. While MCT4 exports lactate of SCs, MCT1
and MCT2 are responsible for lactate uptake by germ cells [70, 71].
Notably, SCs are also capable of producing this substrate in the
absence of glucose. Indeed, SCs are able to produce lactate through
the metabolism of lipids [72], amino acids or glycogen [68, 73] in
order to ensure the metabolic requirements of spermatogenesis.
After spermatogenesis is completed, the newly formed sper-
matozoa are stored in the epididymis for a final maturation process.
Hither and once required, it is important for spermatozoa to obtain
energy necessary to acquire and maintain motility [74]. The major-
ity of functional characteristics, like motility and fertilization capac-
ity, rely on a high-energy production [52]. Several studies have
been conducted on the existing GLUTs on spermatozoa. In fact,
several authors have reported that GLUT1-3 and GLUT5 are pre-
sent in spermatozoa, both in humans as in numerous other mam-
mals like rats, bulls, dogs, boars, stallions and donkeys [57, 75-78].
Additionally, mRNA and protein expression of GLUT8 was ob-
served in human mature spermatozoa [79], while both isoforms of
GLUT9 (GLUT9a and GLUT9b) were reported in mouse sper-
matozoa [80, 81]. Contrastingly, the presence of GLUT4 was not
reported in any of the above mentioned species [57, 75-78]. Con-
cerning cellular distribution, GLUTs can be found in different re-
gions along the spermatozoa. GLUT1 is present in the initial and
final portion of the tail and in the apical region of the spermatozoa
head. GLUT2 is mainly concentrated in the acrosomal region,
whereas GLUT3 and GLUT5 are located in the head and midpiece
of spermatozoa. Besides, GLUT8 can be found in the acrosomal
membrane, whereas GLUT9a and GLUT9b are localised on the tail
of spermatozoa [79-81].
Even when subjected to anaerobic conditions, mature spermato-
zoa have the ability to use distinct substrates to obtain energy, such
as glucose and fructose [82-84]. Indeed, two distinct mechanisms
can be used by spermatozoa to obtain energy: oxidative phosphory-
lation (midpiece) and glycolysis (principal piece) [85]. These
mechanisms can act together or independently, depending on the
energetic needs of the spermatozoa [84]. In spermatozoa, glucose
undergoes the glycolytic pathway and is converted to lactate by a
specific isoform of lactate dehydrogenase (LDH), present only in
the testes (LDHC) [86]. Lactate is then accumulated in these cells,
where it can further be used for energy production and for sper-
matozoa motility enhancement [83, 84, 87]. Consequently, glucose
metabolism in SCs and in mature spermatozoa are closely related
with male fertility, thus being essential processes for the regular
development of germ cells during spermatogenesis and for the final
maturation of spermatozoa.
3624 Current Pharmaceutical Design, 2015, Vol. 21, No. 25
BASIC ASPECTS OF DIABETES
Systemic diseases, especially DM, are highly prevalent disor-
ders that tremendously affect today’s world health. The number of
individuals with diabetes has been increasing over the last decades,
and it is expected that by the year 2030 more than 552 million peo-
ple will be affected by this disease [13]. There are numerous factors
that contribute to its high prevalence, particularly those closely
related to obesity and a generally unhealthy lifestyle [12, 13]. DM
is a metabolic disorder, characterized by an intensified oxidative
stress and a hyperglycaemic state, which induce structural and func-
tional alterations in cells, tissues and organs [88]. Individuals af-
fected by this disorder present a defective insulin production, insu-
lin resistance, or both factors combined [12, 13]. Moreover, DM is
accompanied with a reduced antioxidant activity [14] and dysfunc-
tion of the hypothalamic-pituitary-gonadal axis [15, 16]. Notably,
DM is associated with a wide range of other health issues, like heart
diseases, stroke, nerve damage, and kidney or eye problems [89].
Although there are several types of DM, T1DM and T2DM are the
most widespread.
T1DM is characterized by a complete absence of insulin due to
an autoimmune destruction of pancreatic
-cells, which are the
insulin-producing cells. Hence, patients with this disorder need
exogenous insulin to control their blood glucose levels. As a result,
T1DM patients are commonly referred as insulin-dependent [90-
92]. Although T1DM can affect a wide range of age groups, chil-
dren and young people are undoubtedly the most affected [93].
T2DM is responsible for the great majority of all DM cases. It
is characterised by insulin resistance and impaired insulin secretion
[89, 92], associated with a continued decline in
-cell function and
an increase of
-cell apoptosis [94]. The incomplete suppression of
hepatic glucose production associated with a decreased efficiency
of the liver and muscle glucose uptake promotes an increase in
blood glucose levels of patients with this disorder [95]. Cells are
unable to respond to the abnormal blood insulin levels, and pancre-
atic cells display a limited capacity to produce higher amounts of
insulin in response to insulin resistance. All these factors combined
lead to a failure in the control of the blood glucose levels. Patients
with T2DM do not require exogenous insulin to control their blood
glucose levels, thus being referred to as non-insulin dependent [89].
There are various individuals with pre-diabetes, an intermediate
state between a normal control of glucose levels and T2DM [96]
that have a higher predisposition to develop T2DM. Interestingly,
pre-diabetic patients exhibit important metabolic alterations, like
impaired fasting glucose, impaired glucose tolerance and/or dis-
turbed insulin secretion [97]. Although there is a high incidence of
pre-diabetic patients, age seems to be the crucial factor for T2DM
development, with most patients being from the adult and elder age
groups [89]. Nonetheless, the number of young patients with T2DM
has been increasing over the last years, mainly because of the high
prevalence of obesity that is now found in the younger population
[12, 13, 95, 98]. So, it is expected that the number of individuals
suffering from T2DM and its associated complications will con-
tinue to rise, affecting more individuals each year.
DIABETES: A THREAT FOR MALE FERTILITY
Diabetes is associated with a wide range of health complica-
tions. Male fertility is no exception. Males with DM are at high risk
for fertility issues, due to alterations induced on their reproductive
potential [15, 99]. In addition, depending on the patient’s age, his
control of blood glucose levels and the duration of the disease, DM
is reported to cause erectile dysfunction [100, 101]. Other compli-
cations associated with DM in males are impotence, retrograde
ejaculation and decreased sexual desire [101-104], as well as ab-
normalities in testicular functions and disruption of the spermato-
genesis endocrine control, leading to an abnormal sperm production
[105, 106].
Ferreira et al.
Several studies have reported that DM has a detrimental effect
on male reproductive system, commonly resulting in infertility or
subfertility by the induction of a number of alterations associated
with testicular dysfunction [107]. Male DM patients present numer-
ous alterations on their reproductive system, being that changes in
sperm parameters are the most noticeable. A decrease in testicular
weight, together with a reduction of testicular and epididymal
sperm content and a regression of the epididymis, have been consis-
tently observed in animals with DM [108-110]. The metabolic al-
terations induced by DM can instigate various abnormalities on
male fertility, which can ultimately lead to the production of ab-
normal spermatozoa. Sperm motility, count and volume were the
mainly altered properties, being significantly reduced on individu-
als with T1DM and T2DM [11, 111-114]. Clinical studies reported
alterations of sperm morphology and an increase of nuclear and
mitochondrial DNA fragmentation in human spermatozoa of dia-
betic individuals [11, 93]. Furthermore, there are multiple animal
studies reporting a decrease on sperm parameters quality on dia-
betic subjects, which are in clear agreement with most results ob-
tained in humans [108, 110, 115-117].
With the growth of T2DM among children and adolescents
[92], there is a definite need to fully understand the exact mecha-
nisms involved in the subfertility or infertility of these male dia-
betic patients, and the correlation between the two pathological
states. With the rising number of diabetic individuals there is also a
growth in the consumption of antidiabetics [118]. There are several
oral antidiabetics available, categorized into different groups ac-
cording to their means of action. Individuals suffering from T2DM
can be treated with different classes of oral antidiabetics, such as
insulin sensitizers, insulin secretagogues, and external insulin deliv-
ery [20]. Among these, metformin is one of the most frequently
prescribed drugs for T2DM and is usually recommended in combi-
nation with a healthy lifestyle [17]. This drug is the first line treat-
ment for this type of DM, being able to control the glycemic levels,
reduce the risk of micro- and macrovascular complications, and
significantly decrease mortality rates [119]. In spite of all the dis-
coveries made on the mechanisms of action of this agent, little is
known about its effects on the male reproductive system and spe-
cifically on germ cell development. Consequently, it is crucial to
further investigate the implications of metformin consumption on
male fertility.
METFORMIN AND ITS THERAPEUTIC RELEVANCE
Metformin (1, 1-dimethylbiguanide) is a synthetic drug origi-
nated from a medicinal herb used since the medieval Europe,
Galega officinalis [120]. This agent is the most cost-effective ther-
apy for the treatment of individuals with T2DM [17, 22] and is now
used by more than 100 million individuals [23]. In the clinical prac-
tice, metformin has been used for over 50 years, being characterised
as an oral biguanide insulin-sensitizing agent capable of increasing
insulin sensitivity and significantly decreasing plasma fasting insu-
lin levels [121]. This drug exerts its principal metabolic action, the
glucoregulatory, in the liver. However, it may act on a variety of
body tissues like skeletal muscles, adipose tissue, endothelium and
reproductive tissues [27]. Therefore, owing to its multiple action on
several body regions, which are affected by insulin resistance and
hyperinsulinemia, metformin has gained interest for its multiple
potential therapeutic uses [27, 122]. When administered, metformin
is not metabolised and is excreted in the urine and bile [123]. Thus,
this agent does not affect the action of other drugs prescribed to
patients.
The best-known effects mediated by metformin are associated
with its anti-hyperglycemic action. Its major effects are: 1) blocking
of liver gluconeogenesis; 2) increasing skeletal muscle uptake of
glucose [124]; 3) reduction of the absorption of glucose in intestinal
mucosa; and 4) decreasing lipogenesis in the liver, which can re-
duce lipotoxicity and improve insulin signaling reducing glu-
Metformin and Male Fertility
coneogenesis [125]. In addition, it has also been shown that met-
formin can be used as an antineoplastic agent. Indeed, metformin
restricts the growth and proliferation of various neoplastic cells
both in vitro and in vivo. These results were described in different
tumors, such as pulmonary adenocarcinoma [24], gastric [25], ovar-
ian [126-129], endometrial [130], prostate and colon cancer [131],
bladder neoplastic cells [132] and different types of breast cancer
[133-135]. Additionally, metformin increases the effect of the che-
motherapeutic agents in cultures of ovarian cancer cells, when co-
administered with cisplatin [126, 128, 129], carboplatin [136, 137],
phenethyl isothiocyanate [138], and in cultures of breast cancer
cells, when co-administered with doxorubicin [135]. Notably, it has
also been reported that this drug diminishes the incidence of breast
cancer in diabetic women [136]. Recently, Singhal and collabora-
tors established as well metformin as a possible adjuvant for anti-
tuberculosis therapy, by reduction of intracellular growth of Myco-
bacterium tuberculosis [139]. Metformin can also be used in the
treatment of anovulatory infertility in women with PCOS [140],
inducing ovulation and increasing pregnancy rates [26, 141]. Like-
wise, was proven that metformin modulates the reproductive axis,
affecting the release of GnRH and LH [142]. Moreover, reactive
oxygen species (ROS) can be reduced by metformin treatment
[143-145] and the expression of antioxidant genes (i.e. Nrf2) can be
increased [146].
Although this drug has been clinically available since the 50s,
little is known about its exact mechanisms of action. Even so, it is
known that metformin requires different isoforms of “Organic
cation transporters” (OCT1 and OCT2) or of “multidrug and toxic
compound extrusion type” transporters (MATE1 and MATE2), to
be transported into the various tissues. OCT1 is mainly responsible
for the transport of metformin in the liver and gut, whereas OCT2 is
primarily found in the kidney [147, 148]. Metformin is rapidly ab-
sorbed across the intestinal epithelium and transported to the liver,
where it accumulates [123]. In the hepatocytes, the predominant
expression of OCT1 facilitates cellular uptake of metformin into the
liver [148] and as a result, there is a higher accumulation of this
drug in the liver than in other tissues [149]. Once inside the cytoso-
lic compartment, mitochondria constitute the primary target of this
drug. At this point, metformin inhibits the complex I of the mito-
chondrial respiratory chain, without affecting any other steps of its
machinery [150, 151]. However, the exact mechanism by which
this drug promotes this inhibition remains unknown. After the spe-
cific inhibition of complex I, an increase in the intracellular
AMP/ATP ratio occurs leading to serine/threonine protein kinase
(AMPK) activation in the liver and in other tissues. This activation
promotes a decrease in lipid synthesis along with an increase of
fatty acid oxidation [152, 153]. Furthermore, it also inhibits glu-
coneogenesis [152-155], mitochondrial ROS production [156] and
cell proliferation [127, 157]. The decrease in ROS production re-
sults in an increase of glutathione (GSH), catalase (CAT), and su-
peroxide dismutase (SOD) levels [158]. As AMPK is present in
many tissues, metformin effects are designated as pleiotropic [28].
Therefore, in energy-rich environments, such as is the case of obe-
sity and insulin resistance, AMPK is frequently inactivated and
high levels of ROS can be found [156, 159, 160]. However met-
formin can also act independently from AMPK activation. Experi-
ments with AMPK and the upstream liver kinase K1 (LKB1)
knockout (KO) mice, as well as wild type mice, have shown that
serum glucose levels were reduced after metformin treatment. This
effect may be caused by alterations in AMP/ATP ratio, which can
modulate the hepatic glucose output upstream of AMPK, inhibiting
the AMPK pathway [161].
IMPACT OF METFORMIN ON MALE REPRODUCTIVE
SYSTEM
While the effects of metformin on several organs have been
broadly studied, little is known about its effects on the human male
reproductive system. The vast majority of the in vivo studies that
Current Pharmaceutical Design, 2015, Vol. 21, No. 25 3625
have been conducted on this subject took use of animal models.
Rodents were the most frequently used animal models, although
other animals have been exploited, as is the case of chickens and
rabbits. Furthermore, in vivo and in vitro approaches were also
employed in order to evaluate the effects of metformin on the male
reproductive tract. Data were obtained from several cell types, like
fetal testicular cells, adult germ cells, SCs or spermatozoa. How-
ever, the existing results remain controversial with some conflicting
results. We have summarized the different metformin-related ef-
fects on the male reproductive system in Table 1.
Tartarin and collaborators conducted an extensive study on the
effect of metformin in human and murine testes using in vitro ex-
perimental models [162]. The authors evaluated the impact of this
drug by exposing organotypic cultures of fetal testes to increasing
doses of metformin (50 μM to 5 mM) for 3 days. They reported that
both murine and human systems were affected, exhibiting de-
creased testosterone production and increased lactate secretion with
the supra-pharmacological (500
M) concentration, in the human
model, and a 500
M and 5 mM concentrations in mice. Further-
more, the mRNA expression levels of key players involved in ster-
oid secretion were also reduced after exposure to 5 mM of met-
formin. While for humans the lowest observed effect concentration
on testosterone secretion was with the pharmacological concentra-
tion of metformin (50
M), in mouse it was only verified at 500

M. With these concentrations, the production of testosterone was
reduced, which suggests that its pharmacological dose may cause
impaired steroidogenesis [162]. In mouse cultures, it was also ob-
served a significant increase in the mRNA levels of LDH at a con-
centration of 5 mM. A similar stimulation of the LDH mRNA ex-
pression has also been described in rat SCs after treatment with
aminoimidazole carboxamide ribonucleotide, another AMPK in-
ducer [9]. The increase of lactate production promoted by AMPK
activation on fetal murine testis in vitro, thus seems to be resultant
of augmented LDH levels. Due to this increased lactate production,
particularly at high concentrations of metformin, it has been hy-
pothesized that this drug may also have a protective effect on sper-
matogenesis, which could lead to a promotion of sperm production,
caused by an increase of the glycolysis index. In fact, to better
comprehend the conflicting results obtained in vitro, the authors
also conducted an in vivo study, where pregnant mice were exposed
to a daily dose of metformin (300 mg/kg the dose that is needed to
obtain a similar therapeutic effect in diabetic animals as in humans -
50
M). The treatment took place between embryonic day 0.5 and
13.5 or 16.5. The results obtained revealed that the pre-natal met-
formin exposure reduces the testes size and the ST diameter. A
reduction in the SC population, due to a substantial decrease in the
proliferation indexes of these cells, was also observed. This can
also be explained by the activation of the AMPK pathway since it
may lead to a decrease in cell proliferation. However, this decline in
the SCs number did not affect the total number of germ cells pre-
sent in the testes [162]. In contrast with the in vitro studies, met-
formin did not affect the intratesticular lactate concentration.
Hence, the authors concluded that some alterations in fetal testicu-
lar function due to metformin were obtained at concentrations equal
to or 10 times higher than the therapeutic doses used, since they
were able to promote a deleterious effect on several parameters
associated with the male reproductive function both in vivo and in
vitro.
An in vivo study where male rats were exposed to a metformin
dose equivalent to that used for the treatment of T2DM patients (30
mg/kg/day for 21 days by oral gavage), reported a significant de-
crease in sperm motility and sperm count [165]. The authors also
described an increase in testicular injury and testicular dysfunction,
concurrent with high testicular lipid peroxidation (LPO) levels.
Metformin administration resulted in a general decrease in the anti-
oxidant status of the rat testes due to a reduction in the activities of
testicular SOD, CAT and GSH. The histological study of the rat
3626 Current Pharmaceutical Design, 2015, Vol. 21, No. 25 Ferreira et al.

Table 1. Summary of main studies reporting metformin-associated effects on male reproductive system.

Biological Study Effects on Male Reproduction

Species Studied Reference
Material Type Metabolic Physiologic

lactate production
Mouse
In vitro  testosterone levels
Fetal
Human  steroidogenic factors expression
Testicular [162]
Cells  ST diameter
In vivo Mouse  number of SCs
 testes size

testosterone levels
 ST degeneration

LH levels

thickness of the germinal epithelium [163]

FSH levels

spermatogenesis index

insulin levels

sperm motility

testes antioxidant status [164]

sperm concentration
Testes In vivo Rat

 sperm motility
 sperm count
 testes antioxidant status
ST degeneration [165]

spermatocytes defoliation
 cells involved in spermatogenesis

lactate production
Sertoli Cells In vitro Rat  GLUT1, GLUT3, MCT4 expression [166]

antioxidant activity

sperm motility

lactate production
Chicken
sperm viability [167]

AMPK phosphorylation

acrosome reaction
In vitro
 sperm motility
Mouse
AMPK phosphorylation
sperm viability [156]

membrane integrity

sperm motility
Spermatozoa  oxidative stress
sperm count [168]
Rat
normal sperm morphology

sperm count
 oxidative stress [169]
In vivo
normal sperm morphology

 sperm motility
 testosterone levels  sperm count
Rabbit [170]
 normal sperm morphology
 testicular weight
ST- Seminiferous tubules, SCs - Sertoli cells, GLUT - glucose transporter, MCT - monocarboxylate transporter, LH - luteinizing hormone, FSH - follicle-stimulating hormone,

- increase,  - decrease.

testes, where the testicular dysfunction was accessed, revealed a
noticeable necrosis of the testicular tissue, with degeneration of the
ST and spermatocyte defoliation. Additionally, metformin admini-
stration caused ST atrophy and a decrease in the number of cells
involved in spermatogenesis, indicating that the production of ma-
ture spermatozoa was seriously affected in the metformin-treated
animals [165]. Thus, it can be concluded that the dose used may not
be able to activate AMPK pathway and thereby increase the con-
sumption of glucose and/or decrease ROS production in these ani-
mals. However, the metformin’s blood glucose lowering effect was
not taken into account in this study, which may explain (at least) the
results obtained. In addition, the previous effect should be further
evaluated and different concentrations of metformin tested since the
treated animals do not show blood glucose levels similar to those
found in patients with diabetes. Nevertheless, the results described
in these studies were obtained by exposing healthy animals with
Metformin and Male Fertility
normal (lower) levels of blood glucose to metformin, which may
explain (at least partly) the deleterious reproductive effects ob-
served. Therefore, several studies have been performed to deter-
mine the effect of this therapeutic drug on testicular cells or tissue
and on male reproductive function in diabetic conditions. In a re-
cent study, Alves and collaborators evaluated the effect of the expo-
sure to increasing doses (5, 50 and 500 M) of metformin, for 50
hours, on rat SCs metabolism, in a hyperglycemic medium [166].
These cells create the BTB and are targeted by the numerous sub-
stances present in the blood, as is the case of metformin. In this in
vitro study, the authors showed that 50 M of metformin reduces
the mRNA and protein expression of glucose (GLUT1 and GLUT3)
and lactate (MCT4) transporters. For the same concentration of
metformin, authors also found an increase in LDH activity accom-
panied by an increased production of lactate and alanine [166],
which indicates a possible AMPK pathway activation by the phar-
macological concentration of metformin (Fig. 2). In addition, even
on cells treated with the supra-pharmacological dose of metformin
(500 M), no cytotoxic effects were observed. These results illus-
trate that metformin decreases the number of SCs’ glycolysis-
related transporters, but increases SCs activity, also indicating that
the glycolytic pathway is enhanced. However, glucose consumption
was maintained in these cells. Furthermore, their data showed that
metformin has the capacity to induce the antioxidant activity, in
response to increased production of alanine, in rat SCs exposed to a
hyperglycemic condition. Then, the possible activation of AMPK
by metformin acts primarily by decreasing oxidative stress in rat
SCs and not by glucose consumption. Notably, germ cell develop-
ment is positively regulated by these factors (stimulated lactate
production and less oxidative stress) and so the authors suggested
that metformin may be used as an efficient drug for the treatment of
T2DM male individuals on reproductive age, improving their over-
all fertility status. However, although these results were based on an
in vitro model that attempts to mimic the in vivo conditions they
need to be further validated, as for instance, by the use of germ
cells-Sertoli cells co-cultures.
Still, data obtained in several in vivo studies were concomitant
with the results obtained in this in vitro approach. In fact, several
authors reported that metformin can improve the spermatogenic
index and the male reproductive functions of diabetic rats [163,
164]. A study conducted by Nasrolahi and collaborators evaluated
the effects of daily metformin in vivo administration (100 mg/kg),
for a 40 day period, on the testes of streptozotocin-induced diabetic
rats (
220 mg/dl of blood glucose concentrations) [163]. They
reported an increase in testosterone, LH and FSH levels, when
compared with non-treated rats. In the same study, the authors ob-
served that, when administered as a single agent or in a combina-
tion therapy with honey, metformin produced a significant increase
of the percentage of ST with a positive spermatogenesis index and
of the germinal epithelium thickness. Co-treatment also reduced the
ST degeneration caused by DM and increased its diameter. The
results obtained by these authors suggested that the administration
of metformin alone or in combination with honey improves the
male reproductive function of diabetic rats and has the ability of
reducing the detrimental changes induced by DM in testicular tissue
[163]. Fang and collaborators reported concurrent results when
evaluating the effect of metformin, for 12 weeks, on the epididymal
sperm quality and testicular antioxidant capacity in obese rodent
models [164], which is closely associated with the development of
DM. These authors reported that metformin-treated rats presented
an increase in sperm concentration and motility. Furthermore, met-
formin-treated obese rats displayed an improvement in the oxida-
tion status of the testes, evidenced by the reduction of LPO levels.
These results illustrate that a combination of metformin with an
appropriate diet enhances sperm quality and promotes the antioxi-
dant ability of the testes of obese rats, thereby attenuating the ef-
fects of obesity on the male reproductive function [164]. In fact, the
data obtained on these studies confirmed the protective role of met-
Current Pharmaceutical Design, 2015, Vol. 21, No. 25 3627
formin on male reproductive function in diabetic and obese rats, by
improving steroidogenesis and the antioxidant ability of the testes,
leading to an increase in the spermatogenesis index and increased
sperm concentration and motility [163, 164]. Consequently, these
data illustrates that metformin acts primarily by decreasing the oxi-
dative stress in the testis, and not by improving glucose consump-
tion.
Some studies have also been performed to evaluate the impact
of the in vitro exposure of spermatozoa to metformin. In a study
where mouse spermatozoa were incubated with different doses of
metformin (50, 500 and 5000 M) for 30 minutes, the authors re-
ported that this drug did not induce any deleterious effect on sper-
matozoa quality either sperm viability, membrane integrity or acro-
some reaction, except a slight reduction in sperm motility at 5000
M of metformin due to a reduction of active mitochondria [156].
These authors also investigated the effects of metformin incubation
(30 minutes) before the sperm cryopreservation. They reported that
viability and membrane integrity were increased in cryopreserved
sperm subjected to this pre-incubation. This study also evidenced
that this pre-incubation before cryopreservation has a positive im-
pact in the fertilization ability of cryopreserved mouse spermatozoa
due to a significantly increase in the percentage of fertilized oocytes
[156]. Furthermore, the pre-treatment with 5000 M of metformin
reduced the percentage of fragmented embryos and increased the
number of oocytes reaching the 4 cell-stage embryo. It was also
observed that the lower metformin concentrations (50 and 500 M)
did not modify the basal phosphorylation of AMPK. However,
frozen spermatozoa pre-treated with 5000 M of metformin pre-
sented a 5-fold increase in AMPK phosphorylation [156]. Accord-
ingly, an in vitro study using chicken spermatozoa (1 mM of met-
formin for 40 minutes) demonstrated that exposure to metformin
significantly improved AMPK phosphorylation, sperm motility,
viability, percentage of sperm able to undergo a successful acro-
some reaction and lactate production [167], evidencing a possible
beneficial effect of metformin in bird spermatozoa, at least during
in vitro storage. In fact, the use of metformin in cryopreservation
protocols seems to have beneficial effects on the quality of
(cryo)preserved sperm, improving its quality and increasing its
fertilization ability, in response to the activation of the AMPK
pathway.
Contradictory results were obtained in in vivo studies. In a
study conducted by Attia and collaborators, authors evaluated the
impact of single (100, 500 or 2500 mg/kg) and multiple oral ad-
ministration (100 or 500 mg/kg/day for 4 and 8 weeks) of met-
formin on sperm quality parameters on a streptozotocin-induced
diabetic rat model (blood glucose levels >350 mg/dL) [168]. In all
the doses tested, metformin was neither genotoxic nor cytotoxic for
the spermatozoa. No significant effects were observed with the
single administration. However, the two doses of metformin used in
multiple treatment attenuated the sperm oxidative stress, commonly
observed on diabetic rats caused by ROS overproduction, with in-
creased levels of GSH and decreased levels of LPO. In fact, the
multiple treatment significantly increased sperm motility, count and
the percentage of morphologically normal sperm, in a dose-
dependent manner, with significantly alterations observed with 500
mg/kg [168]. Similar data were described by Rabbani and collabo-
rators, who reported an increase on sperm count and on the general
antioxidant status, and a decrease on the percentage of sperm with
abnormal morphology, when metformin (50 mg/kg) is co-
administered orally daily for 4 weeks with pioglitazone (1 mg/kg)
to streptozotocin-induced diabetic rats (blood glucose levels >230
mg/kg) [169]. Although these authors have not evaluated the activa-
tion of AMPK pathway, they observed a reduction in the production
of ROS. However, these results demonstrate that metformin may
lead to the AMPK pathway activation preferentially leading to a
reduction of oxidative stress rather than changing glucose metabo-
lism. So, in response to this alteration, in the last two studies, met-
3628 Current Pharmaceutical Design, 2015, Vol. 21, No. 25 Ferreira et al.

Extracellular Space
Glucose
Glucose
T1
GLU GL
UT
3

Glycolysis
PFK LDH
Glucose Pyruvate Lactate

ALT Sertoli Cell

Alanine Lactate
Lactate
-oxidation Pyruvate

4
CT Lactate
Acetyl-CoA M MCT 2

Mitochondria Germ Cell

Lactate

Sertoli cell nucleus

ATP

Fig. (2). Schematic illustration of the main metabolic pathways described in Sertoli cells (SCs) and the metformin actions on the metabolism of these cells.
The extracellular glucose enters via glucose transporters (GLUT1 and GLUT3) and is converted into pyruvate by phosphofructokinase (PFK) action. Pyruvate
can either be transported into the mitochondrial matrix to form Acetyl-coA, or can be converted into lactate or alanine by lactate dehydrogenase (LDH) and
alanine transaminase (ALT), respectively. SCs actively consume glucose producing large quantities of lactate, for the developing germ cells, which is then
exported to the extracellular medium by proton-linked plasma membrane transporters, the monocarboxylate transporters (MCT2 and MCT4). SCs incubation
with metformin results in decreased mRNA and proteins levels of GLUT1, GLUT3 and MCT4, increased activity of LDH and improves the production of
lactate and alanine by these cells.
Dotted lines - metabolism steps affected by metformin action in SCs

formin was able to reduce the genomic alterations and the damages
induced by oxidative stress.
Dissonant results were however described in a study performed
on normal and alloxan-induced diabetic rabbits (blood glucose lev-
els >400 mg/kg) treated with a high dose (120 mg/kg) of met-
formin, once a day for 3 months [170]. Treated non-diabetic rabbits
displayed a significant reduction in testicular weight (which can be
explained by decreased cell proliferation triggered by AMPK acti-
vation), in sperm motility and count and serum testosterone level
with a significant increase in abnormal and dead sperm. However,
the greatest reduction in these parameters was observed in met-
formin-treated diabetic rabbits [170]. In face of their data, authors
suggested that this dose of metformin might potentiate the effect of
DM, impacting negatively the male reproductive function [170].
These results are not in agreement with the ones obtained in some
of the in vivo studies mentioned above using other animal models,
particularly using rodent models. Still, the method of induction of
DM in rabbits was distinct as that used in rodent. Although, in both
rodents and rabbits, metformin has been capable of lowering the
glucose plasma levels to control levels, in rabbits the dose used may
not have been enough to improve sperm parameters thus leading to
adverse effects on male fertility [170].
In vivo exposure to metformin only had beneficial effects in the
male reproductive function of diabetic animals, particularly in
mammals, except the case of alloxan-induced diabetic rabbits. On
the other hand, the male reproductive capacity of individuals with
normal glucose levels was negatively affected. As discussed, there
are some contradictory results concerning the safety and therapeutic
potential of metformin in male reproduction. The different out-
comes may be explained by the wide variety of species studied and
the diverse experimental conditions. Nonetheless, it was also possi-
ble to verify that AMPK phosphorylation appears to play important
functions in the regulation of sperm parameters and metabolism.
Nevertheless, further studies are needed to elucidate the exact influ-
ence of metformin on male reproduction. Even so, from the major-
ity of data available it seems evident that metformin improves the
antioxidant activity of male reproductive system, by reduction of
ROS production, due to the AMPK pathway activation. Less fre-
quently, the activation of AMPK pathway by metformin treatment
was also able to improve the glycolytic pathway, which increased
lactate production leading to an improvement of the spermatogenic
process. However, it has to be noted that not all studies have evalu-
ated simultaneously the AMPK activation with the glucose metabo-
lism and/or oxidative stress. The most promising results were ob-
tained with mouse spermatozoa, where metformin treatment acti-
vated the AMPK pathway, improving the quality of frozen sperm
and consequently the fertilizing capacity of cryopreserved sper-
matozoa. This data is of great importance and additional research is
needed for future application of metformin on human reproduction.
Metformin and Male Fertility
MOLECULAR MECHANISMS OF METFORMIN ACTION
IN THE TESTES
The molecular pathways by which metformin may employ its
potential actions on male reproductive system remain elusive. Cur-
rently, few studies are available reporting the effects of metformin
on male reproduction and, specifically, on human sperm quality
parameters. Nonetheless, some studies on animal models have
emerged in an attempt to identify the effect of this drug on male
fertility and reproductive health.
Several authors reported that metformin can indirectly influence
the activation of the AMPK pathway and an association between
the improvement of male reproductive functions and AMPK activa-
tion on testicular tissue was recently reported [152, 167]. Accord-
ingly, some authors have studied the activity of AMPK in different
testicular cell types, with and without metformin action, to better
clarify the mechanisms of this drug on testicular tissue. AMPK acts
as a sensor and regulator of cell metabolism [171] and is a hetero-
trimeric complex composed by one catalytic unit (
AMPK) and
two regulatory subunits ( and ) [172, 173]. Isoforms of all three
subunits (
1,
2, 1, 2, 1, 2 and 3) have been identified in
mammals [174]. A decline in cellular energy status, and increased
intracellular levels of AMP, lead to the binding of AMP to the regu-
latory  subunit of the AMPK complex. This association leads to a
conformational change of the AMPK complex that promotes its
activation via phosphorilation of the catalytic subunit in the
threonine 172 residue (Thr172) by an upstream LKB1 (100-fold
increase in activity) [173, 175, 176], and inhibition of its dephos-
phorilation by protein phosphatases [177, 178].
AMPK can be found in several mammalian tissues were it is
responsible for the activation of some metabolic pathways involved
in the activation of ATP producing pathways (glucose and lipid
catabolism), and inhibition of ATP consuming anabolic pathways
(protein, fatty acid and cholesterol synthesis) [178, 179]. It has been
shown that AMPK is present in reproductive cells like in boar
ejaculated sperm [171] and tissues of some mammals including the
rodent testis [180, 181]. Regardless of the fact that all the subunits
of AMPK are expressed in rat testis,
1AMPK is responsible for
most of the total AMPK activity [180]. Notably, it has been de-
scribed that rodent haploid spermatids have a specific isoform of
upstream LKB1, called LKB1s, responsible for AMPK activation,
illustrating the relevance of this pathway on male fertility. It was
also reported in mice that the lack of LKB1 can result in a decrease
of spermatozoa production leading to a reduction of mature sper-
matozoa in the epididymis, with an increase on the amount of mor-
phologically abnormal and non-motile spermatozoa [182].
The existence of high levels of
AMPK was noticed while
studying cultures of rat SCs. In these cells, activation of AMPK
pathway leads to an increase of lactate production, in response to an
increase in glucose uptake. The rise in glucose uptake and lactate
production results of an increase in the GLUT1 and MCT4 levels.
These data led to the hypothesis that the AMPK pathway is respon-
sible for the nutritional modulation of SCs function [9, 183]. A
different study revealed that AMPK is expressed in boar spermato-
zoa at higher levels than those found in somatic cells. Moreover, it
was reported that the inhibition of this enzyme in boar spermatozoa
resulted in a decrease of sperm motility, and fertilization capacity
[171]. More recently, the same authors reported that AMPK is also
involved in plasma membrane organization, since its inhibition
induced plasma membrane lipid disorganization and reduced the
outward translocation of phosphatidylserine at the plasma mem-
brane of spermatozoa [184]. Thus, it has been suggested that
AMPK is involved in the function of plasma membrane of mam-
malian spermatozoa at different levels, playing a key role on the
production of normal spermatozoa. Tartarin and collaborators stud-
ied the role of this kinase on male reproductive function using

1AMPK KO male mice [181]. They reported a significant reduc-
tion in fertility capacity by induction of asthenozoospermia and a
Current Pharmaceutical Design, 2015, Vol. 21, No. 25 3629
high percentage of morphological abnormalities. However, no al-
teration in testes morphology or sperm production rate was verified.
These animals also presented high levels of androgens, with in-
creased intra-testicular cholesterol and testosterone concentrations.
Furthermore, the LH and FSH levels were lower in KO mice due to
negative feedback induced by increased levels of testosterone.
Therefore, the lack of AMPK activation seems to result in an in-
crease of androgens levels, which can influence sperm quality, and
consequently male fertility [181]. As previously referred, a study
with male chickens reported that AMPK is also present in ejacu-
lated spermatozoa. This enzyme was located in the spermatozoon
acrosomal region, midpiece and flagellum. The AMPK active form,
phospho-Thr172-AMPK, was mainly observed in the flagellum and
acrosome region, while a reduced enzyme protein expression was
observed in the midpiece of chikens’ spermatozoa [167]. These
results seem to explain why ATP production is not restricted to the
spermatozoa midpiece (mitochondrial respiration), but can likewise
occur in other locations along spermatozoa through anaerobic gly-
colysis [185, 186]. The expression pattern of AMPK suggests that
this kinase may be closely related with the promotion of spermato-
zoa motility and acrosome reaction process [167].
Indeed, a mechanism of action involving AMPK has been pro-
posed for metformin on the chicken male reproductive tract. In this
model, metformin induced the inhibition of the mitochondrial respi-
ratory chain complex I and, in response to the decrease of ATP
production and the increase in AMP intracellular level, AMPK was
activated [151]. AMPK activation regulates ATP levels, improving
the metabolic functions needed to ensure high energy consuming
processes, such as sperm motility and acrosome reaction [167].
Furthermore, activation of the AMPK pathway leads to increased
sperm motility, as well as increased lactate production. Still, this
effect is not equivalent among species. Although metformin acts as
AMPK modulator, altering sperm ATP levels and function in sev-
eral animals, the kinetics and magnitude of these modifications is
variable. For instance, in the chickens these modifications occur
faster than in boars. Therefore, it has been suggested that the rele-
vance of AMPK pathway on male reproductive function differs
between mammals and birds. Yet, it is widelly accepted that this
pathway is susceptible of activation by metformin in the cells of the
male reproductive tract, particularly in spermatozoa.
In summary, activation of the AMPK pathway can improve
some sperm parameters, like motility and morphology, thereby
improving the overall male fertility status. This activation is also
able to stimulate the acrosome reaction and lactate production, and
maintain normal hormone levels, which conduct to an efficient
spermatogenesis.
CONCLUSION
Metformin is an oral antidiabetic with anti-hyperglicemic activ-
ity. Recently, it has been reported that this drug can act in different
organs and tissues including the male reproductive system. How-
ever, the existing literature presents some contradictory results on
the impact of metformin on this system. While several studies pro-
vide evidence that metformin improves sperm motility, others do
not confirm this observation. Analysis of the antioxidant status,
lactate production, LDH activity and sperm count, likewise re-
vealed opposing results. The differences found between all experi-
ments can justify the contraditory results observed. Nevertheless, it
is accepted that metformin improves the reproductive functions of
diabetic individuals, although the same was not always verified in
normal individuals. Additionally, the incubation of spermatozoa
with metformin improves their fertilization capacity after
cryopreservation. However, data from studies involving humans or
human material are scarse and it is important to investigate the
pharmacological and toxicological effects of metformin incubation
on human spermatozoa and reproductive function.
3630 Current Pharmaceutical Design, 2015, Vol. 21, No. 25
The metformin mechanism of action is not yet fully understood.
However, there are evidences that this drug acts via AMPK path-
way in numerous tissues, including the male reproductive system.
Activation of the AMPK pathway can improve some sperm pa-
rameters, as is the case of motility by activation of anaerobic path-
ways involved in ATP production. The AMPK pathway is also
involved in a reduction of morphologically abnormal sperm accom-
panied with increased lactate production [9, 167, 182]. These altera-
tions seem to be caused by a reduction in ROS production in re-
sponse to AMPK activation, which improves sperm quality. In most
cases of the reported studies, the action of metformin seems to be
responsible for the decrease of oxidative stress rather than for an
increase in glycolysis rate. Nonetheless, the studies conducted ob-
tained different results for the effects of metformin on male fertility.
Therefore, additional studies are needed in order to fully understand
the influence of metformin on the molecular mechanisms involved
on its effects on the male reproductive system and consequently on
male fertility and the role of the AMPK pathway on sperm func-
tions.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
ACKNOWLEDGEMENTS
UMIB (Pest-OE/SAU/UI0215/2014) and MG Alves (SFRH/
BPD/80451/2011) were funded by National Funds through FCT-
Foundation for Science and Technology and AR by the Master
Course in Molecular Medicine and Oncology, FMUP.
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