NUTRITIONAL SUPPORT FOR INFERTILE MALES

(1)

NUTRITIONAL SUPPORT FOR INFERTILE MALES

Maurizio Dattilo, MD

Parthenogen, Lugano, Switzerland

MSRM

Izmir Apr 22, 2016

(2)

Conflict of interest

Maurizio Dattilo is manager and shareholder of PARTHENOGEN SAGL, Switzerland

Parthenogen is involved in the development and

marketing of dietary supplements

(3)

Causes of oxidative stress

Environmental factors

Genetic substrate

e.g. MTHFR, CBS and MTRR genes

(4)

Sperms are passive in oxy-redox balancing

The oxidation-sensitive cells

Differenziation

Compacted nucleus &

transcription silencing No chances to react

« Exposed »

epididimal transit Long time

(4 months)

Cumulative damages

(5)

The obvious assumption

It is an oxidative damage, therefore …

(6)

Cochrane review, 2014

Antioxidants for male subfertility

^.

Showell MG, Mackenzie-Proctor R, Brown J, Yazdani A, Stankiewicz MT, Hart RJ Cochrane Database Syst Rev. 2014;12:CD007411

Note:

Data based on 44 live births in a total of 277 couples from 4 small studies

AUTHORS' CONCLUSIONS:

There is low quality evidence from only four small randomised controlled trials suggesting that antioxidant supplementation in subfertile males may improve live birth rates for couples

attending fertility clinics. Low quality evidence suggests that clinical pregnancy rates may increase. There is no evidence of increased risk of miscarriage but this is uncertain as the evidence is of very low quality. Data were lacking on other adverse effects. Further large well- designed randomised placebo-controlled trials are needed to clarify these results.

(7)

Reductive stress from oral antioxidants

Interference with protamination and DNA synthesis

Strong direct antioxidants are predicted to cause a fall of NAD+

Protamination depends on Spermatid DNA relaxation by TOP2B and PARP

Meyer-Ficca et al. 2011

NAD

⁺

NADH

PARP

PARP activity is NAD+ dependent

(8)

Antioxidants to reduce sperm DNA fragmentation: An unexpected adverse effect

Menezo Y, RBMonline 2007; 14(4): 418-421

Reductive stress in the clinical setting

Defective protamination

Decrease of fragmentation (positive effect)

Increase of decondensation (negative effect)

Treatment:

-  Vit. C 400 mg -  Vit E 400 mg

-  Beta-carotene 18 mg -  Zinc 500 microM -  Selenium 1 microM

(9)

Oral antioxidants are powerful tools with strong pharmaceutical effects and a misuse is easy to occur.

They are to be prescribed only in front of a confirmed damage, under medical control and for limited duration

Oral antioxidants

(10)

Oral antioxidants: Mode of action

From Silvestris E. et al, Womens Health Gynecol 2016; 2(4): 030

(11)

The antioxidant cascade

(12)

GSH is the primary cellular antioxidant

GSH

GS-SG

E .

Direct ROS scavenging, NADH, NADPH Regeneration of hydrophilic

antioxidants e.g. vitamin C and uric acid (blood, cytoplasm and body fluids)

Regeneration of hydrophobic antioxidants e.g. carotenes, tocopferoles and ubiquinone (lipids, cell membrane)

Antioxidant Enzymes, e.g.

-  Catalase -  SOD

Glutathione (GSH) acts as a redox buffer feeding E .

into the

whole antioxidant cascade

(13)

Where does GSH come from?

Cysteine Cystathionine

γ-glutamylcysteine

Glutathione (GSH) Homocysteine

Serine

Glutamate

Glycine

Transsulfuration pathway

-  Homocysteine (Hcy) as the starting substrate

•  transsulfurated with serine to form cystathionine

-  The reducing –SH group is provided by Hcy

- Ubiquitary (all cells) - Massive cycling

- Mandatory for cell survival

No dietary sources/adsorption: MUST be synthetized into the cell !

CBS enzyme

(14)

Where does homocysteine come from?

S-adenosylmethionine (SAMe)

S-adenosylhomocysteine

Dimethyl- glycine

Betaine

Choline Tetrahydrofolate

5-methyl- tetrahydrofolate

Homocysteine Mehtionine

Methylated acceptor

Adenosine

Homocyseine re-methylated from betaine Homocyseine

re-methylated from folates

One carbon cycle (1CC)

A molecule of Hcy is formed any time a carbon unit is added to a molecule

(15)

Interplay between transmethylations and GSH production

One carbon cycle:

The methyl group is released to an acceptor, Hcy is formed and is re- methylated to cycle again

Transsulfurations:

Hcy binds serine to form cistathionine toward the synthesis of GSH

CBS Hcy

regulatory step

SAMe

(16)

The key enzyme for GSH synthesis:

Cystathionine Beta Synthase (CBS)

A unique mechanism of activation

Full efficiency of GSH synthesis will only occur after the requirement for activated methyl groups (SAMe) from the one carbon cycle is satisfied

Activation from oxidative stress

Amplification from 1CC activity

(17)

Antioxidant homeostasis in simple terms

GSH synthesis Oxidative

aggression

1CC CBS activity

Cell growth, DNA methylation

The system is well equipped to manage any oxidative aggressions ... At least as long as properly feeded

Activation Amplification

Protection

(18)

Substances without pharmacological activity that feed the production of reducing equivalents (GSH) and their permissive pathway (1CC).

Full correction

No rebound effect

Can be given to everybody with no limits of dose or duration

Indirect antioxidants

Regulated by cellular

homeostasis

(19)

Indirect antioxidants: Which substances?

B vitamins + Zinc + cysteine donor

Carbon cycle

GSH

synthesis

(20)

The importance of the one carbon cycle nutritional support in human male fertility: a preliminary clinical report.

Maurizio Dattilo, Dominique Cornet, Edouard Amar, Marc Cohen, Yves Menezo Reprod. Biol & Endocrin., 2014; 12:71

Non comparative study

Patients Female partners Mean age, years (range, SD) 37 (25-63, 5.5) 35 (25-44, 4.4)

Normospermic, n (%) 19 (23%)

Asthenospermic, n (%) 14 (17%)

Oligoasthenospermic, n (%) 51 (61%)

Female factor, n (%) 28 (33%)

Previous ART cycles (mean n, range) 2.4 (2-6)

84 male partners of couples with at least 2 previous ART failures and willing for a new attempt.

DFI > 20% or SDI > 20%, irrespective of spermiogram.

(21)

Methods

Intervention

Condensyl^TM, 1 or two tablets per day (mean 1.33 tabs/day).

Target duration 4 months, eventually adapted to the patients willingness and to the actual planning of the following ART cycle (mean 130 days; range 59-365 days).

ART was performed by IUI, IVF or ICSI

balancing the clinical indication with the couples willingness

Measurements

Sperm DNA Fragmentation Index (DFI) by TUNEL

Sperm chromatin Decondensation Index (SDI) by aniline blue staining

Nutri&onal characteris&cs Per day (1 tab) % of NRV

N-‐acetyl L-‐cysteine 250 mg

Extract of oputn;a fruit pulp With betalaine With querece;ne

100 mg 0.05 mg 0.001 mg Zinc bisglicinate

Containing zinc

66.49 mg 12.5 mg

125%

Vitamine B3 16 mg 100%

Vitamine E 12 mg 100%

Vitamine B6 1.4 mg 100%

Vitamine B2 1.4 mg 100%

Vitamine B9 400 µg 200%

Vitamine B12 2.5 µg 100%

Condensyl^TM, Parthenogen SAGL – Lugano, Switzerland

(22)

Responder rates and their DFI and SDI values before and after the treatment

* p < 0.001, Wilcoxon test

Groups n (%)

DFI SDI

Pre Post %

change Pre Post %

change All Patients 84 (100) 29.7% 23.1% -6.6%* 40.1% 36.3% -3.8%*

DFI responders 60(71%) 33.0% 20.0% -13.0% 38.5% 34.6% 3.9%

SDI Responders 54 (64%) 28.5% 21.4% -7.0% 42.0% 30.2% -11.8%

DFI and SDI responders 40 (48%) 31.8% 18.8% -13.1% 40.2% 28.9% -11.4%

First time ever both DFI and SDI

significantly decreased

(23)

Pregnancy outcomes

Categories n Clinical pregnancies Live b irths

n Rate p n Rate p

All patients 84 40 47.6% 33 39.3%

Pts improvin g both DFI and SDI 40 28 70.0% 0.000 23 57.5% 0.001

Pts improvin g SDI 54 35 64.8% 0.000 30 55.6% 0.000

Pts improvin g DFI 60 33 55.0% 0.032 26 43.3% 0.23

Pts not improving DFI nor SDI 10 0 0.0% 0.001 0 0.0% 0.007

Independence CHI² test

18 pregnancies and births from spontaneous pregnancies (occuring before performing the planned ART cycle)

Pregnancies strongly related to the decrease of the indexes

SDI decrease plays a stronger effect compared to DFI decrease

(24)

DFI and SDI response according to the occurrence of a clinical pregnancy

Groups n (%)

DFI SDI

Pre Post %

change p Pre Post %

change p Any pregnancy

YES 40 (47 .6) 29.4% 20.1 % -9.3% 0.168 40.6% 29.3% -11.3% 0.000

NO 44 (52 .4) 30.1% 25.9 % -4.2% 39.6% 42.6% 3.0%

Spontaneous pregnancy

YES 18 (21 .5) 23.2% 18.4 % -4.8% 0.571 44.8% 29.8% -15.0% 0.000

NO 66 (78 .5) 31.5% 24.4 % -7.2% 38.8% 38.0% -0.7%

ART pregnancy

YES 22 (33) 34.4% 21.4 % -13.0% 0.046 37.2% 29.0% -8.2% 0.001

NO 44 (67) 30.1% 25.9 % -4.2% 39.6% 42.6% 3.0%

Mann -Whitney test

Spontaneous pregnancies related to SDI decrease

ART pregnancies related to SDI decrease and, to a minor extent, to DFI

decrease

(25)

A: Transcriptionally active DNA has an open chromatin structure with widely spaced

nucleosomes (light blue circles), unmethylated CpG residues (white circles) and specific histone modifications. The resultant DNA is readily accessible to enzymes important in gene transcription.

B: Chromatin in transcriptionally repressed conformation, with methylated CpGs (red circles), compacted nucleosomes (dark blue circles), and methylation of histones (red ovals). This repressive conformation renders the DNA inaccessible to the transcriptional machinery

DNA methylation trıggers chromatin compaction and transcription

silencing

(26)

Comparative study 1

Amar E et al., Austin J. of Reprod. Med. 2015; 2(1):1006

Methods

Patients

- Couples: primary infertility with at least 2 previous ART failures - Female partners: no female factor, age < 43

- Male partners: spermiogram >WHO threshold for infertility, high DFI and/or SDI Groups

-  Active treatment: Condensyl, 2 tabs per day for 4 months

-  Controls: No treatment (matched patients not willing to take supplements) Analyses

-  Blinded analyst -  DFI by TUNEL

-  SDI by aniline blue staining

Treatment for High Levels of Sperm DNA Fragmentationand Nuclear De condensation:

Sequential Treatmen twith a Potent Antioxidant Followed by Stimulation of theOne-

Carbon Cycle vs One-Carbon Cycle Back-up Alone

(27)

Amar E et al., Austin J. of Reprod. Med. 2015; 2(1):1006

Outcomes

N Age DFI SDI Pregnancies

M F Pre Post Δ Pre Post Δ CPR LBR Condensyl 69 37.6

(5.9)

36.1 (4.1)

24.6 (12.4)

20.0

(12.7) -‐19% 41.7 (9.7)

35.0

(15.0) -‐16% 50.7%

(35)

42.0%

(29) Controls 84 37.8

(4.5)

34.6 (5.4)

30.9 (34.4)

31.2

(34.3) +1% 30.5 (6.4)

31.1

(3.5) +2% 21.4%

(18)

21.4%

(18) p* < 0.01 <0.01

P** 0.003 0.003

P* = Wilcoxon tests; p** = Chi² test

Spontaneous pregnancies (i.e. before the planned ART):

- Condensyl = 9 (28%) - Controls = 0 (0%)

Warning: the study was NOT truly randomized

(28)

Comparative study 2

Cornet D et al., Austin J. of Reprod. Med. 2015; 2(2):1011

Methods

Same as previous studies, 2 arms

Clinical Evidence for the Importance of 1-Carbon Cycle Support in Subfertile Couples

Male study

Male partners of ART-resistant couples, female partner normal

Treated Controls n = 95 n = 84 Female study

Female partners of ART-resistant couples, male partner normal

Treated Controls n = 100 n = 73

Nutri;onal characteris;cs Per day (2 tabs) % of RDA

N-‐acethyl L-‐cysteine 300 mg

Extract of oputn;a ﬁgue With betalaine With querece;ne

200 mg 0.10 mg 0.002 mg

Magnesium 100 mg 26.6%

Extract from green tea leaves

Titrated in polyphenols

68 mg 20.40 mg Extract from dunaliella salina

Titrated in carotenoids

48 mg 1.20 mg Extract from sweet orange fruits

Titrated in hexperidine

34 mg 20.40 mg

Vitamin B3 16 mg 100%

Zinc bisglicinate

Containing zinc

66.49 mg 12.50 mg

125%

Vitamin E 10.30 mg 85.8%

Vitamin B6 1.4 mg 100%

Vitamin B2 1.4 mg 100%

Vitamin B9 200 µg 100%

Vitamin B12 2.4 µg 100%

Procrelia^TM, Parthenogen SAGL – Lugano, Switzerland

(29)

Cornet D et al., Austin J. of Reprod. Med. 2015; 2(2)

Outcomes - Men

Table 4 DFI and SDI before and after the treatment, only male partners treated, female partner "normal", treatment vs controls

Again!

High rate of

spontaneous

pregnancies

(30)

Cornet D et al., Austin J. of Reprod. Med. 2015; 2(2)

Outcomes - Ladies

N of pts Pregnancies Miscarriages Deliveries Treated 100 45

¹

(45%) 5 (11%) 40 (40%) Controls 73 10

²

(14%) 2 (20%) 8 (10.9%)

p 0.0001

Statistics: Chi square test

Table 3: Pregnancies and deliveries: women treated only, male partner

“normal”, treatment vs. Control.

Spontaneous pregnancies = 66% (30 out of 45 CP)

Discussion

“This type of nutritional support seems at least as important in women as in men”

(31)

Chromatin condensation in (bovine) oocytes

Lodde et al. 2007

Bright field and fluorescent images after Hoechst 33342 labeling of bovine oocytes with GV0 (A, A1), GV1 (B, B1), GV2 (C, C1), and GV3 (D, D1) configuration. Arrows in the bright fields indicate the nuclear envelope. Scale bar: 50 mm.

GV0

nonsurrounded nucleolus (NSN)

surrounded nucleolus (SN)

GV3

Ooc yte matur ation

(32)

Clinical studies: Take home messages

¨ 

A proper structure of sperm chromatin is of paramount importance for reproduction

¤ 

It is susceptible to environmental factors, pollution and pro-oxidants on one side and dietary manipulations on the other side

¨ 

A significant rate of ART-resistant infertility seems strongly linked to metabolic imbalances

¤ 

An adequate nutritional care offered since the first referral might decrease the time to pregnancy

¨ 

Interventions to take into account the complexity of cellular homeostasis

¤ 

Caution with aggressive direct antioxidants

¤ 

Indirect antioxidants (Condensyl) improve pregnancy outcomes

(33)

Spontaneous pregnancies

¨ 

Males

¤ 

Linked to the positive effect on chromatin

maturation (overcomes the main barrier to natural fertilization)

¤ 

Vouches for a pronounced effect on natural conception, high gain in IUI is predicted

¨ 

Females

¤ 

Also oocyte maturation includes a phase of tight chromatin compaction, benefit from DNA

methylation support

¤ 

Oocyte sensitivity to oxidative damage may be higher than previously supposed

Selection bias?

ART-resistance may have

selected couples with a

main metabolic problem

(34)

Homocysteine: the epicentre of the problem

¨ 

Blood Hcy is widely available, non-invasive and cheap

¤ 

Normal = 4-12 µM/liter (optimal 6-10)

¤ 

Borderline = 12-20 µM/liter

¤ 

High = > 20 µM/liter

¨ 

Highly predictive for metabolic problems

If Hcy is high THERE IS A METABOLIC PROBLEM

Test your patients !!

(35)

THANK YOU FOR YOUR

QUESTIONS

Mail to: maurizio.dattilo@parthenogen.ch

(36)

Events in (bovine) oocyte maturation

Luciano et al. 2014

(37)

Chromatin condensation in sperms

90% volume reduction

- Hydrodynamic shape

- Easier pack delivery at fertilization

- Gene and genomic stability

(38)

The key role of histone to protamine

transition in sperm nuclear condensation

Sperm DNA organised in histone/

nucleosomes just like any other cell

Histone/nucleosomes largely substituded by protamine/toroids: 90% reduction of

chromatine volume.

A few histones nucleosomes are retained Post-fertilization the paternal protamines substituted by maternal histones. Paternal histone/nucleosomes (retained) are the

first transcribed to activate the embryo

Ward 2010

Histone to protamine transition is triggered by DNA and H3K79 methylation

followed by H3 and H4 methylation and hyperacetylation (Dottermusch-Heidel 2014)

(39)

Gametes nuclear condensation in summary

Nuclear condensa tion

Transcription silencing

Protamine transition DNA and Histone methylation

Histone hyperacetylation

DNA and Histone methylation

Embryo activation Embryo

feeding Massive transcription

Selective transcription

Keeps compaction

Reverts to «open»

(40)

Oxidative damage affects protamination

Decreased protamin content, decreased P2 and increased P1/P2 ratio correlate with the level of oxidative stress

Hammadeh et al. - Human Reproduction, Vol.25, No.11 pp. 2708–2720, 2010

(41)

Oxidative damage may affect GENOMIC STABILITY

Wu et al., J Neurosci. 2008 January 2; 28(1): 3–9

Methylated CpG islands may be oxidised on both the C and the G side

*OH-mC may trigger extensive DNA de-methylation (seen in stem cells and cerebral tissues)

G and/or mC oxidations nullifies the effect of C methylation and allow the transcription factors to bind and read the gene

8-oxo-guanosine (8OG) Oxidation of methyl cytosine Oxidation of guanosine

Hydroxymethylcytosine* (OH- mC)

Loss of regulation on gene transcription

O

^-

OH

^-

(42)

Sperm oxidative damage in summary

Oxidative Aggression

Molecular damages:

DNA, Proteins, Lipids

Structural damages:

Protamination, genomic stability

Repair Apoptosis

Fertilization DNA fragmentation,

infertility

(43)

DNA fragmentation contributes to abortive apoptosis:

Natural selection of good sperms

Oxidative stress DNA damages

DNA repair Apoptosis

Mitochondria committed to the oxidative burst (H₂O₂ release)

Fragmented sperms: massive DNA damages aimed at

removal of the damaged cell DNA damages from ROS

(44)

About DFI

¨ 

Good marker of poor reproductive prognosis

¨ 

Poor marker of response to antioxidant treatments

¤ 

« Cosmetic » reduction of the index due to the blockade of abortive apoptosis

¤ 

The savage of damaged cells could disturb selection for ICSI

¨ 

It is not an indipendent predictor of male reproductive outcome

¤ 

Residual sperm DNA damage is repaired into the zygote

¤ 

Chances of pregnancy: balance between DNA repair capacity of the

oocyte vs the quality of the sperm

(45)

About SDI

¨ 

Strong marker of poor reproductive prognosis

¨ 

Good marker of the response to the treatment

¤ 

« Cosmetic » reduction of the index unlikely to occur

¨ 

It is a strong indipendent predictor of male reproductive outcome

¤ 

The zygote is not known to be able to repair sperm chromosome structure

¨ 

The test with blue aniline is simple and cheap

¤ 

NUTRITIONAL SUPPORT FOR INFERTILE MALES