Reproductive Andrology Surgery Workshop III17-21 January 2016 - Reproductive Medicine Unit - Jahra Hospital, Ministry of Health - KUWAIT

Dr. Sandro EstevesMedical and Scientific Director

www.androfert.com.br

ANDROFERT - Andrology & Human Reproduction Clinic Campinas / SP - Brazil

Stem Cell and Male Infertility

The testis

The

Sper

mat

ozoo

n

Sperm production involves complex Genetic and Epigenetic regulation

s

Disorders causing male

infertility

One every 100 men have what is called 'azoospermia' (absence of sperm in the semen)

Testicular torsion; Trauma Post-inflammatory (eg. Mumps orchitis) Exogenous factors (eg. Cytotoxic drugs, irradiation) Testicular cancer Systemic diseases (eg. Liver cirrhosis, renal failure)

Congenital Testicular dysgenesis/cryptorchidism Genetic abnormalities (Klinefelter syndrome, Yq microdeletions, etc.)

Acquired

Idiopathic (Unknown etiology) Revised in Esteves et al. Clinics 2011; 66: 691-700

In up to 50% it is possible to identify tiny areas of sperm production within the testis

using microsurgery

Esteves Asian J Androl 2015; 17: 459-70

Things to ponder to enhance Male Reproductive Health

On the contrary, there is NO residual sperm production in the other half of these men

the only hope

An undifferentiated cell of a multicellular organism that is capable of giving rise (by mitosis) to indefinitely more cells of the same type, and from which certain other kinds of cell arise by differentiation.

Stem cell

Two basic properties are self-renewal (ability to undergo multiple cycles of cell division while maintaining the undifferentiated state) and plasticity (capacity to differentiate into specialised cell types)

In mammals, two broad types:• Embryonic stem cells: from the inner cell mass of blastocysts• Adult stem cells (main sources: bone marrow, adipose tissue, blood)

Stem cell

Stem cells can also be taken from the umbilical cord blood. Induced pluripotent stem cells (iPSC): adult cells (eg. epithelial cells) reprogrammed to give rise to pluripotent capabilities.

Embryonic stem cell(Pluripotent)

Limitations of Embryonic and induced-Pluripotent Stem Cells

Ethical issuesTumorigenicityImmune rejectionViral transductionLow efficiency

Chen Z, Li Z, He Z. Plasticity of male germline stem cells and their applications in reproductive and regenerative medicine. Asian J Androl 2015;17:367-72

Responsible for male fertility (origin of spermatogenesis)

Ability of self-renewal and generation of large number of haploid cells

In humans, SSCs comprise a sub-fraction of undifferentiated type A spermatogonia (~0.002% of total testis cells; Nagano et al 1999)

Spermatogonial Stem Cell (SSC)

Chen et al Asian J Androl 2015; 17: 367-72

Spermatogonial Stem Cell (SSC)Ability to convert into pluripotent cells without gene manipulation under certain culture conditions (Kanatsu-Shinohara et al. 2004 )

Many important molecules regulating SSC self-renewal and differentiation have been identified, but the exact mechanism largely unknown (Oatley et al. 2010)

For men who harbor SSCs despite having azoospermia, a testis biopsy combined with in vitro proliferation and transplantation may solve the problem.

Spermatogonial stem cell (SCC) & Male Infertility

Culture medium consisting of HEPES-buffered DMEM/F-12 (Gibco) supplemented with 10 μg/ml insulin transferring-selenium solution (Gibco), 10–4mol/l vitamin C (Sigma), 10 μg/ml vitamin E (Sigma), 3.3×10–7 mol/l retinoic acid (Sigma), 3.3×10–7 mol/l retinol (Sigma), 1 mmol/l pyruvate (Sigma), 2.5×10–5 IU recombinant human FSH (Gonal-F; MerckSerono), 10–7 mol/l testosterone (Sigma), 1×antibiotic–antimycotic (ABAM, containing penicillin, streptomycin and amphotericin B; Gibco), and 10% bovine calf serum (Hyclone) (modified from Weiss et al., 1997).

Human SSC culture enabling SSC amplification. However, lack of an effective differentiation culture system hinders its clinical application.

An organ culture system that supported the whole spermatogenesis process has been created for the mouse (Sato et al 2013).

What has been achieved (and replicated) so far

Sato et al. Nature Protocols 2013; 8: 2098-2104

Process of stem cell cell injection

The system produced functional sperm in about

2 months

Testis tissue fragment transplanted with SSCs showing colonization

Sato et al. Nature Protocols 2013; 8: 2098-2104

Not yet validated in humansDemand for organ fragments for those patients with defective spermatogenesis inhibit clinical application. Xenotransplantation of human SSCs to primates may solve this problem.

Spermatogonial stem cells (SSCs) represent a novel cell source for both reproductive and regenerative medicine (unlimited potentials including pluripotency, self-renewal, differentiation, and transdifferentiation)

Future Perspectives and Concluding Remarks

Generation of mature and functional cells from SSCs have advantages over ES cells and iPS cells, including no ethical concern, lower frequency of tumorgenesis, and no immune rejection. Many issues await to be clarified, e.g. the dedifferentiation and transdifferentiation mechanisms, optimal induction protocols, and origin of the ES-like cells. Given the rapid progress in SSC research, SSCs would be eventually utilized from the bench to bedside to cure male sterility in the near future.

THANK YOU

شكراOBRIGADO