Management of Male Infertility - What the future holds

Sandro Esteves, MD., PhD.

Medical & Scientific Director, ANDROFERT

Campinas, Brazil

Management of Male

Infertility

What the future holds

Reproductive Andrology Surgery Workshop II

Reproductive Medicine Unit - Jahra Hospital - Kuwait 2014

Learning objectives

At the completion of this presentation, participants will have an overview of:

• Sperm biomarkers as diagnostic and treatment tools

• Prospects of male fertility preservation

• Stem cells as new agents for the treatment of male infertility

ANDROFERTandrofert.com.br

ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION

S ESTEVES, 2

2014 December

ANDROFERT

Irvin S, et al 1996, Auger J et al, 1995, Irvine DS 1994, Jørgensen N et al 2001,

Jørgensen N et al 2002, Swan SH 2003, Feki NC et al, 2009



S ESTEVES, 5

2014 December

ANDROFERT

Male reproductive relatively simple anatomy hides an overwhelmingly

complex system



S ESTEVES, 6

2014 December

ANDROFERT

Spermatogenesis Process



S ESTEVES, 7

2014 December

ANDROFERT

Genetic andepigenetic-regulated process

~2,000 genes, but only

30 in the Y

chromosomeHamada et al 2012



S ESTEVES, 8

2014 December

ANDROFERT

Extremely specialized cell type



S ESTEVES, 9

2014 December

ANDROFERT

Unexplained infertility affects up

to 40% of infertile men

1 in 100 men have no sperm in ejaculate (azoospermia)

Up to 50% aspermatogenic(absolute sterility)



S ESTEVES, 10

2014 December

ANDROFERT

Limitations of current management

Conventional semen analysis

Conventional surgeries

Empirical medical treatments

ART overuse



S ESTEVES, 11

2014 December

ANDROFERT

Recent Advancements

Sperm Function Testing

MicrosurgeryGenetic diagnosis



S ESTEVES, 12

2014 December

ANDROFERT

YCMD molecular

diagnosis by PCR

Genomics

Transcriptomics

Proteomics

Metabolomics

The Era of Biomarkers



S ESTEVES, 14

2014 December

ANDROFERT

Researchers first to determine entire

genetic sequence of individual sperm

We can look at a

particular individual,

make some calls about

what they would likely

contribute genetically to

an embryo and perhaps

even diagnose or detect

potential problems, and

identify healthy sperm

for use in IVF



S ESTEVES, 15

2014 December

ANDROFERT

Normalcy of Sperm Chromatin Content

DNA Micro-array Technology

• Glass or silicon slides

where thousands of

features are arrayed

• Gene expression is

measured hybridization

process

• Microarrays are read using

laser-based fluorescence

scanners

• Determine which genes are

active and at what levels

• Compare with controls, etc.



S ESTEVES, 16

2014 December

ANDROFERT



S ESTEVES, 17

2014 December

ANDROFERT

Potential Clinical Applications of Sperm Molecular Genetic

Fingerprinting

• Infertility diagnosis

• Assessment of treatment outcome (medical, surgical)

• ART outcome (IUI, IVF, ICSI)

• Sperm selection techniques



S ESTEVES, 18

2014 December

ANDROFERT



S ESTEVES, 19

2014 December

ANDROFERT

Seminal Fluid Molecular Genetic Fingerprinting



S ESTEVES, 20

2014 December

ANDROFERT

Proteins are also critical to understanding disease



S ESTEVES, 21

2014 December

ANDROFERT

Proteomics



S ESTEVES, 22

2014 December

ANDROFERT

Mass Spectroscopy

Raman Spectroscopy

Nuclear Magnetic

Resonance



S ESTEVES, 23

2014 December

ANDROFERT

Sperm & Seminal Plasma Proteomic Profiles



S ESTEVES, 24

2014 December

ANDROFERT

Sp

erm

P

rote

om

ics



S ESTEVES, 26

2014 December

ANDROFERTCamargo M et al. Hum. Reprod. 2013;28:33-46

Change in proteomic profiling in seminal plasma of adult men before and after

varicocelectomy

Sperm,

spermatids

Embryos

Oocytes

Ovarian tissue

?Male germ cells

?Testicular

tissue



S ESTEVES, 28

2014 December

ANDROFERT



S ESTEVES, 29

2014 December

ANDROFERT



S ESTEVES, 30

2014 December

ANDROFERT



S ESTEVES, 31

2014 December

ANDROFERT

Spermatogonial stem cell preservation and transplantation

• Boys facing gonadotropic treatment

• Klinefelter syndrome patientsCryopreservation before SSC loss

• Extraction: testicular biopsy

• Freezing : slow-freezing and storage of testicular

tissue or cell suspension in LN

• Cryoprotectant: DMSO

• Grafting: ectopic or homotopic (mouse)

• When to graft: unknown

• Fertilizing capacity in humans: unknown



S ESTEVES, 32

2014 December

ANDROFERT

Nonobstructive azoospermia irreversible condition

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)

Esteves et al. Clinics 2011; 66: 691-700



S ESTEVES, 34

2014 December

ANDROFERT

Current method for identification of sperm production sites in nonobstructive

azoospermia

Esteves et al Int Braz J Urol 2013; 37: 570-83; Deruyver et al Andrology 2014; 2: 20-4



S ESTEVES, 35

2014 December

ANDROFERT

Morphometric Evaluation of Seminiferous Tubules

Median 25%-75% 5%-95% Raw Data

yes No

Presence of Sperm

160

180

200

220

240

260

280

300

320

340

360

380

400

420

Max.

Tubule

Dia

mete

r

Verza Jr S, Esteves SC. Fertil Steril 2012; 98: S242

N=54; Tubule Diameter: KW-H (1;54) = 25.2; P<0.001



S ESTEVES, 36

2014 December

ANDROFERT

Novel methods for identification of sperm production sites in men with nonobstructive

azoospermia

Multi-photon microscopy(ex vivo; human model)

Confocal fluorescence microscopy

(in vivo; murine model)

Full-field optical coherence tomography

(ex vivo; rat model)

Najari et al, J Urol 2012; Smith et al J Urol 2012; Ramasamy et al., J Pathol Inform 2012



S ESTEVES, 37

2014 December

ANDROFERT



S ESTEVES, 38

2014 December

ANDROFERT

No residual sperm production in up to 50% of men with NOA

In mice, transplanted stem cell-derived male gametes resulted in

proper spermatogenesis



S ESTEVES, 39

2014 December

ANDROFERT

In mice, derived gametes generate viable offspring



S ESTEVES, 40

2014 December

ANDROFERTAponte et al, Clinics 2013

Biotechnological advancements in the treatment of aspermatogenic men



S ESTEVES, 41

2014 December

ANDROFERT

Conclusions



S ESTEVES, 42

2014 December

ANDROFERT

• Novel genomic and proteomic biomarkers could add to work-up and clinical treatment strategies

• Advances in immature germ cells cryopreservation/transplantation will expand fertility options for oncological boys

• Biotechnological approaches for generating male gametes last frontier to be accomplished

Why we should do all that …

Courtesy of E.N. & T.A.S. with permission



S ESTEVES, 43

2014 December

ANDROFERT

Health & Medicine

Management of Male Infertility - What the future holds