Metformin Use Potentially Affects Sperm Count

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Metformin Use Potentially Affects Sperm Count

SUMMARY: DOES METFORMIN AFFECT SPERM

In this study, prepuberty Metformin treatment decreases the total number of mature Sertoli cells in the testis, which negatively effects sperm production and decreases sperm count later in adulthood, however no effect on sperm motility or morphology was observed.

doi.org/10.1111/andr.12957

Background

Sertoli cells are essential for the formation of male testicles formation and spermatogenesis. They provide the necessary structure and nutritional support for germ cells to develop into sperm. However, a single Sertoli cell can only sustain a limited number of germ cells. Therefore, the amount of sperm cells produced daily is directly related to the number of Sertoli cells in the testis.

In most species, immature Sertoli cells proliferate during specific periods (foetal, neonatal and start of puberty) before maturing to establish the final number of Sertoli cells just prior to adulthood. As these cells are essential for spermatogenesis, it goes without saying that any external factor which affect their proliferation, will also directly impact sperm cell production later in life.

One such external agent that has got health experts worried is metformin (1,1 -dimethylbiguanide hydrochloride). Metformin is currently the only oral agent approved by the US Food and Drug Administration for the treatment of paediatric diabetes.

Recent in vitro studies have reported that metformin has an antiproliferative effect on foetal and postnatal Sertoli cells. However, the impact of postnatal metformin treatment on in vivo mammalian Sertoli cell proliferation and its mechanism remains unknown.

Aim

To examine the effect of metformin treatment on Sertoli cell proliferation during the postnatal period of the rat and to analyze its potential impact on spermatogenic capacity during adulthood.

Methodology

Pregnant Sprague Dawley rats from the Central Animal Facility of the University of Veterinary (Universidad de Buenos Aires, Argentina) were housed in individual cages under temperature and humidity control, with 12-hour light/dark cycles and unrestricted access to water and commercial pellet laboratory chow.

The day the offspring was born was termed as Postnatal day 0 (Pnd-0). At Pnd-3, the animals were weighed and the male pups randomly categorised into 2 groups, a control group (control) and a metformin group (MET).

From Pnd-3 to Pnd-7, animals in the control group were injected daily with sterile saline solution, while those in the metformin group were given a daily dose of metformin (200mg/kg).

This dose of metformin was chosen according to previous studies, where it achieved similar plasma concentrations to those in humans following metformin treatment.

On Pnd-8, blood and histological analysis was carried out in 14 rats (7 metformin, 7 control), with spermatid head counts and sperm motility analysis performed later on the other 10 rats (5 metformin, 5 control) at Pnd-90.

Results

Initial analysis at Pnd-8 revealed no significant differences in mean weight or serum glucose level between the 2 groups.

ControlMET
Body weight on Pnd-3 (g)8.78.8
Body weight on Pnd-8 (g)15.315.1
Blood glucose (mg/dL)108117
Body weight and blood glucose levels of Control and MET groups

BrdU staining at Pnd-8 showed that MET treatment did not affect the quantity of germ cells but did significantly decrease the percentage of proliferating Sertoli cells (18% vs. 21%).

Histological analysis at Pnd-8 revealed both groups had similar testicular characteristics including testis weight and tubule diameter.

ControlMET
Testis weight (mg)13.414.2
Tubule diameter (μm)49.750.3
Testis weight and tubule diameter of Control and MET groups

TUNEL assay confirmed both groups had similar quantities of seminiferous tubule cells, meaning the same amount of cell apoptosis occurred in both groups.

Analysis of cell cycle regulators and inhibitors, suggests metformin negatively affects Sertoli cell proliferation, via these proteins with a significant decrease in the levels of Ccnd1, Ccne2 and Cdkn1b mRNA, and a significant increase in Cdkn1a mRNA.

Pathway analysis also revealed AMP-activated protein kinase (AMPK) was activated, 2 hours post metformin injection, as per previous in vitro study findings. At the same time, Phosphorylated AKT (P-AKT), which plays a role in the stimulation of Sertoli cell proliferation, was significantly decreased (-45%) in the MET group testis compared to controls.

Analysis of a variety of Sertoli cell maturation markers only revealed lower androgen receptor (AR) mRNA levels in the MET group, while mRNA levels of anti-Mullerian hormone (AMH), claudin 11 (Cldn11), connexin 43 (Gja1), FSH receptor (FSHR), along with hormones thyroxine and triiodothyronine were all similar in concentration. This suggests the negative effect of metformin on Sertoli cells is via an anti-proliferative action and not accelerated maturation.

Finally, further examination of the testis observed a significant decrease in the number of spermatids (step 19 shape) per testis and daily sperm production of rats treated with metformin compared to controls. However, adult testis weight, seminiferous tubule architecture, sperm morphology and motility were not affected by metformin treatment.

The authors noted that humans have a lower sperm production rate efficiency compared to rats. Therefore, the excess quantity of sperm cells per ejaculate as a ratio is much less, meaning the potential impact of metformin on male fertility is potentially more severe.

Limitations

  1. Animal only study.

Funding

This study was funded by the Agencia Nacional de Promoción Científica y Tecnológica and the Consejo Nacional de Investigaciones Científicas y Técnicas.

Glossary

Apoptosis
Programmed cell death.

Assay
A laboratory test.

BrdU
Bromodeoxyuridine (BrdU) is commonly used for the detection of proliferating cells.

Histological
The study of the structure of cells and tissue under a microscope.

Germ cell
Precursor cells which differentiate into an egg or sperm.

mRNA
A single-stranded RNA molecule that is complementary to one of the DNA strands of a gene.

Neonate
Newborn child.

Sertoli cell
Somatic cells essential for testis formation and spermatogenesis.

Seminiferous tubules
The site where sperm is produced within the testes.

Spermatid
A haploid cell produced by division of a spermatocyte.

Spermatogenesis
The process by which a complex, interdependent population of germ cells produces spermatozoa (sperm).

Thyroxine
A hormone produced by the thyroid gland, also known as T4.

Triiodothyronine
A hormone produced by the thyroid gland, also known as T3.

Similar studies

Rindone G M, et al. (2018). Metformin counteracts the effects of FSH on rat Sertoli cell proliferation. https://doi.org/10.1530/rep-18-0233

Faure M, et al. (2016). The insulin sensitiser metformin regulates chicken Sertoli and germ cell populations. https://doi.org/10.1530/rep-15-0565

Tartarin P, et al. (2012). Metformin exposure affects human and mouse fetal testicular cells. https://doi.org/10.1093/humrep/des264


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