novel concepts in male infertility: clinical and laboratory aspects

Novel concepts in male infertility: clinical and

laboratory aspects

Life Conference 2015 – TSRM – Taipei, Taiwan

Sandro C. Esteves, MD., PhD.Medical & Scientific Director, ANDROFERTAndrology & Human Reproduction Clinic

Campinas, BRAZIL

Learning Objectives1. Understand the WHO 2010 reference

values for semen analysis and the role of sperm DNA fragmentation testing

2. Appraise what’s new regarding clinical interventions to infertile men candidates to ART

3. Learn the novel concepts in the management of infertile males with azoospermia

ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION

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Semen analysis is still the most widely used biomarker to assess

male fertility


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1980 1987 1992 1999

Volume (mL) ND ≥2 ≥2 ≥2

Count (106/mL) 20-200 ≥20 ≥20 ≥20

Total count (106) ND ≥40 ≥40 ≥40

Motility (%) ≥ 60 ≥50 ≥50 ≥50

Progressive (%) ≥ 2 ≥25% ≥25% (a) ≥25% (a)

Vitality (%) ND ≥50 ≥75 ≥75

Morphology (%) 80.5 ≥50 ≥30 (14)*

Leukocytes (106/mL) <4.7 <1.0 <1.0 <1.0

*Strict criteria (Tygerberg); Esteves et al. Urology 2012

WHO reference values have changed

2010≥1.5 ≥15 ≥39

≥40

≥32%

≥58

≥4*

1.0


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~2,000 semen specimens;recent fathers

Percentile

5% 50% 95%

Volume (mL) 1.5 3.7 6.8

Count (x106/mL) 15.0 73.0 213.0

Total count (x106) 39.0 255.0 802.0

% Motile 40 61 78

% Progressive motility 32 55 72

% Normal (Kruger) 4 15 44

% Alive 58 79 91


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Urology 2012; 79(1):16-22


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Proposal for a new report template

Esteves, Int Braz J Urol 2014; 40:443-53


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History taking, physical examination, endocrine profile and laboratory sperm function testing are minimum standards

Esteves Int Braz J Urol 2014

Male infertility evaluation must go beyond a simple semen analysis


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Conventional semen analysis is not enough

single-strand

break mis-match

damaged

base double-strand

break inter-strand

crosslink

intra-strand

crosslink


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DNA Damage

Environmental factorsPhtalate exposure,

radiation, temperature

DiseasesVaricocele, GTI, fever

Life-styleObesity, smoking, medication

Aging

Factors associated with sperm DNA fragmentation

Rubes et al 2007; Esteves & Agarwal 2011


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Fertility and Sterility 2014; 101(1):58-63


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Frequency of elevated SDF in men with unexplained infertility

Elevated

SDF

(27%)

N=987

Elevated SDF (27%)


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19%

1.5%

Normal Elevated

Live birth rates with IUI

N=387; OR = 0.07 [95% CI: 0.01-0.48]

Bungum et al. Hum Reprod 2007

IUI outcome is negatively affected by elevated SDF


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26%

42%

IVF ICSI

Pregnancy in cases of elevated sperm DNA fragmentation

IVF outcome is negatively affected by elevated SDF

Robinson et al. Hum Reprod 2012

Meta-analysis of 16 studies; 2,969

couples:

Increased miscarriage in IVF/ICSI associated

to high SDF; RR = 2.16 95% CI: 1.54-3.03; p<0.00001

Bungum et al. Hum Reprod 2007


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Andrologia 2014; 46(6): 602–9

Patients with varicocele have higher proportion of sperm with massive DNA damage


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SDF is part of routine work-up of male infertility at Androfert

Does the patient have high SDF?

Semen analysis

including SDF testing (SCD

assay)

High SDF if results >30%

What does the doctor need

to know?

Determine test and internal

validation

Lab SOP with post-analytical info for clinical

decision


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Role of interventions to infertile men

candidates to ART


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Outcome Effect size (OR; 95% CI)

Live birth 4.85 [1.92, 12.24]

DNA fragmentation -13.80 [-17.50, -10.10]

Oral antioxidants decrease SDF and improve ART outcomes

Showell et al. Cochrane Database Syst Rev 2011


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Oral antioxidants to infertile malesPrescription:

Vitamin C 500mg; Vitamin E 400 mgFolic acid 2 mg, Zinc 25 mgSelenium 26 mcg

Duration: minimum 2 months

Misell et al. J Urol 2006; Esteves & Agarwal Int Braz J Urol 2011

Old concept ~90 days

New concept ~60 days


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Wang YJ et al. Reprod Biomed Online 2012;25:307-14

Meta-analysis of 7 studies including 336 pts. indicated that SDF is significantly decreased after varicocele repair (MD=3.4%; 95% CI -4.1 to -2.6; p<0.0001)

Varicocele repair alleviates SDF


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Microsurgical subinguinal varicocele repair with aid of intraoperative doppler


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• Microsurgical varicocele repair prior to ICSI (N=80)

• ICSI with untreated varicocele(N=162)


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ANDROFERTJ Urol 2010;184:1442-6


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SDF markedly lower in testicular versus ejaculated specimens

40.7%

8.3%

EjaculateTestis

P<0.001

Sánchez-Martín, Esteves & Gosálvez, in preparation

*Modified SCD test: dual fluorescent cocktail probe


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Management of azoospermia and the role of gonadotropin

therapy


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Azoospermia: the complete lack of sperm in ejaculate after centrifugation

10-15% infertile males

1-3% male population

Cooper et al. Hum Reprod Update 2009; Esteves & Agarwal, Clinics 2013


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Prognosis and management differentially affected by type of azoospermia

Obstructive

Non-obstructive

Hypo-hypo

Spermatogenicfailure

Clinical picture

FSH/LH: or nlTT: low or nL

Testes: small or nl

Normal testes & endocrine profile;

Mechanical blockage

FSH/LH <1.2 mUI/mL,

Low TT, small testis, poor virilization

Disrupted

Normal

Spermatogenesis

Esteves et al, Clinics 2011


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Rec-hCG for male hypo-hypo

Esteves & Papanikolaou Fertil Steril 2011;96:S230

• Series of men with adult-onset hypo-hypo • Recombinant hCG (250 mcg qw for 12 weeks)

Baseline Posttreatment


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Frequency of azoospermia among 2,383 patients attending an Infertility Clinic

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

Azoospermia

35%

61%

36%

3%Hypo-hypo

OA

SF


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Testicular torsion; traumaPost-inflammatory (eg. Mumps orchitis)

Exogenous factors (eg. Cytotoxic drugs, irradiation)

Testicular cancerSystemic diseases (eg. Liver cirrhosis, renal failure)

CongenitalTesticular dysgenesis/cryptorchidism

Genetic abnormalities (Klinefelter syndrome, Yq microdeletions, etc.)

Acquired

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

NOA due to spermatogenic failure: an irreversible condition


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Challenges faced by health professionals providing care for men with SF

Counseling about the chances of finding testicular sperm

Usefulness of any medical intervention before sperm retrieval

Which sperm retrieval method to apply

Reproductive potential of retrieved gametes in ICSI treatment

Health of offspring


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Complete AZFa, AZFb or AZFa+bmicrodeletions unfavorable prognosis

YCMD SRsuccess

AZFa nilAZFb nilAZFc 50-70%

Krausz et al. 2014; Esteves et al. 2013; Esteves 2015


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Interventions to infertile males men with SF prior to a sperm retrieval attempt

Medication

Hypogonadism (TT<300 ng/dl) in up to 50% men with SF

High ITT levels essential for regulating spermatogenesis in combination with Sertoli cell stimulation by FSH

Paradoxically weak stimulation of Leydig and Sertoli cells by endogenous gonadotropins

Due to high baseline FSH and LH levels the relative amplitudes are low

Shiraishi et al Hum Reprod 2012;27:331-9; Sussman et al Urol Clin N Am 2008;35:147-55


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ITT levels increase after hCG; stimulatory effect on residual spermatogenic areas

Shinjo E et al Andrology 2013;1:929-35; Shiraishi et al Hum Reprod 2012;27:331-9

273

1348

Before After

ITT (ng/dl)

ITT levels increased after hCG-based therapy

Spermatogonial DNA synthesis increased


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Testosterone and estradiol levels

<300

ng/dL(10.4 nmol/L)

Hypogonadismcategory

Pure

Medication algorithm at Androfert

Tx aimed at boosting T

Aromatase inhibitor (anastrozole 1mg orally

qid)

Rec-hCG(250 mcg SC qw);

rec-FSH added(75 IU SC biw) if FSH levels <1.5 mIU/ml

T/E ratio <10

Aromatase hyperactivity

T/E ratio >10 (nl)


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ANDROFERTEsteves Asian J Androl 2015;17:1-12

1Shiraishi et al Hum Reprod 2012;27:331-9; Esteves Int Braz J Urol 2013;39:440

Medical therapy may increase SR success in men with SF

Microdissection TESE

Rescue ~15% of patients with previous failed SR attempts1


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Options for sperm retrieval in spermatogenic failure

Technique Acronym Success

Testicular sperm aspiration TESA 15-50%

Testicular sperm extraction TESE 20-60%

Microdissectiontesticular sperm extraction

Micro-TESE 40-67%

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


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http://androfert.com.br/videos

Esteves SC Int Braz J Urol 2013; 39(3):440

Micro-TESE more effective than

conventional TESE

45%

93%

64%

20%25%

64%

9% 6%

Overall Hypospermatogenesis Maturation Arrest Sertoli-cell Only

Sperm Retrieval Success Rates

Micro-TESE single-biopsy TESE

Controlled series (N=60)

Histology categories

pairwise comparisons:

p<0.0001

Method

P=0.0005

Verza Jr & Esteves. Fertil Steril 2011; 96 (Suppl.): S53


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Seminiferous tubule diameter associated with presence of sperm

Verza Jr S, Esteves SC. Fertil Steril 2012; 98: S242; Esteves & Varghese J Reprod Sci 2012; 5(3):233-43

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

Ma

x.

Tu

bu

le D

iam

ete

r

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


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Tissue removed (mg)

Conventional TESE Micro-TESE P-value

65 ± 25 8.9 ± 2.5 <0.01Conventional TESE Micro-TESE

Verza Jr & Esteves Fertil Steril 2011; Esteves & Varghese J Reprod Sci 2013


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Micro-TESE more efficient than conventional TESE

• Optimize sperm retrieval• Mechanical mincing • Enzymatic tissue digestion• Avoid iatrogenic damage


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Clean Room IVF Lab


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CPRMiscarriage

rate

36.9%

23.0%

47.1%

15.0%

Severe Male Factor (N=399)

Conventional IVF lab Cleanroom IVF lab

P=0.03

Esteves et al. Fertil Steril 2006

2.3

3.2

Average No. Top Quality Embryos ET

Conventional lab Cleanroom lab

N=2,315

Esteves & Bento. Reprod Biomed Online 2013;26:9-21

P=0.01

Cleanroom IVF lab positively impact cycle outcome in severe male factor infertility

ANDROFERTANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION

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41.4 47 43.320

10064 61

34.2

Spermretrieval (%)

2PNFertilization

(%)

Top QualityEmbryos (%)

Live Birth (%)

Non-obstructive (N=365) Obstructive (N=146)

P<0.01


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3,412 cycles; Androfert

Individualized COS strategies to retrieve10 to 15 oocytes per treatment cycle

0%

10%

20%

30%

40%

50%

60%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 20 25

Number of oocytes retrieved

Clinical pregnancy

Live birth


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COS in cycles involving NOA


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• Main goal: effectiveness

• Clinical quality indicator: number oocytes

• Protocol of choice:

Antagonist + tailored recFSH dose according to patient subgroup

cetrorelix (flexible); 150-300 IU/d pen injector

>35yr and DOR: Antagonist + recFSH/recLH

cetrorelix (flexible); follitropin alfa + lutropin alfa2:1 ratio (1-2 vials/d); from stimulation D1

COS in poor responders involving NOA


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• Goal: minimum of 8 MII oocytes

• Strategy: Oocyte banking + fresh cycle and micro-TESE (day prior OPU)

- Antagonist + recFSH/recLH (2:1 ratio; 2 vials/d from Sd1)

- Minimal IVF stimulation

Empirical treatments

Reproductive AndrologyEmpowering clinical decisions & treatment efficiency

Conventional semen

analysis

Conventional surgeries

Sperm Function

Testing

Microsurgery

Genetic diagnosis

YCMD molecu

lar diagno

sis

ANDROFERT

Targeted therapy

Conclusions1. Conventional semen analysis limited as surrogate

for fertility assessment; SDF testing valuable laboratory tool for allowing better clinical decision making

2. Antioxidant therapy, microsurgical varicocelerepair and TESA-ICSI can improve ART outcome in selected individuals

3. Best management of azoospermia-related infertility includes proper diagnosis, interventions to optimize sperm production, microsurgical SR, state-of-art laboratory care and tailored COS


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Thank you 謝謝 Obrigado

This presentation is available at http://www.slideshare.net/sand

roesteves

novel concepts in male infertility: clinical and laboratory aspects

Health & Medicine

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male infertility

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clinical interventions

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management of infertile

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