BACKGROUND

Sperm DNA Fragmentation Testing is an important diagnostic and prognostic tool in determining the fertility potential of a Male. Of critical importance for the validation of any test is its precision and accuracy. The Sperm Chromatin Structure Assay (SCSA®) Test 1 and Sperm Chromatin Dispersion (SCD) Kits 2 are utilized in India to assess sperm DNA fragmentation levels and ascertain the course of clinical action. Despite the differences in methodology, both of these tests use similar threshold levels to determine the extent of sperm DNA damage and inform on further treatment management 3. Variations between assays and protocols can lead to ambiguity in the clinical setting.

High Variability between SCD-Kit 1 & SCSA® Test

Very high inter and intra-observer variation noted in 20 samples tested using SCD- Kit 1 and SCSA® Test methods.

Coefficient of Variation (CV) for SCD-Kit 1 was very high at 25%. Coefficient of Variation (CV) for SCSA® Test was 5.1%.

High Variability between SCD-Kit 2 & SCSA® Test

Very high inter and intra-observer variation noted in 10 samples tested using SCD- Kit 2 and SCSA® Test methods.

Coefficient of Variation (CV) for SCD-Kit 2 was very high at 14%. Coefficient of Variation (CV) for SCSA® Test was 6.3%.

COMPARISON AND VARIATION OF % DNA FRAGMENTATION INDEX (%DFI) BETWEEN SPERM CHROMATIN STRUCTURE ASSAY (SCSA®) AND SPERM CHROMATIN DISPERSION (SCD) TESTS

Tara Mahendran, Silviya S, Vidya Laxme B, Ramyashree D Andrology Center, Coimbatore, TamilNadu

%DFI Sperm DNA Integrity Treatment

Recommendation

≤15% Excellent - Good Natural, IUI

≥15% to ≤25% Good - Fair IUI

≥25% to ≤50% Fair - Poor IVF, ICSI, IMSI

≥50% Very Poor ICSI, TESE, TESA

Clinical threshold values for the SCSA® Test in relation to Natural and IUI conceptions.

PURPOSE

The aim of this preliminary study was to perform a comparative analysis to assess potential differences in Sperm DNA Fragmentation levels between the SCSA® and SCD methodologies in India and its clinical relevance.

METHODS

A cohort of 23 Indian patient samples was analysed using the SCSA® Test and SCD kit methods.

SCSA® Test 5000 sperm were measured twice independently on a FacsCalibur ™

Flow Cytometer to calculate the %DFI and %HDS (High DNA Stainability) with SCSA® Software as per standard protocol 1.

SCD Test 2 Indian commercially available kits, Kit 1 (CANfrag) and Kit 2 (Sperm

Processor) were used. 3 technicians analysed 100 sperm in duplicate on a bright-field

microscope; the average was used to calculate %DFI as per kit instructions.

Group A: 20 patient samples tested for Sperm DNA fragmentation (%DFI) using SCD-Kit 1 and SCSA®

Test. Group B: 10 patient samples tested for Sperm DNA fragmentation (%DFI) using SCD-Kit 2 and

SCSA® Test. A total of 7 samples from Groups A and B were compared for %DFI values between SCD-Kit 1, SCD-

Kit 2 and SCSA® Tests.

High Variability between SCD Kits 1 & 2 and SCSA® Tests

Results from 7 samples tested using SCD-Kit 1, SCD-Kit 2 and SCSA® Test methods showed high levels of variation.

Variations in %DFI values between the 2 SCD kits for the same patients were observed.

Group A Variation in Clinical Thresholds

Group B Variation in Clinical Thresholds

RESULTS

Patient Name SCD-Kit 1 (%DFI) Treatment Recommendation

SCSA® Test (%DFI)

Treatment Recommendation

Pt 4 (P - Frozen) 27 IVF 18 IUI

Pt 5 (M) 21 IUI 25 IVF

Pt 12 (SV) 24 IUI 33 ICSI

Pt 20 (R) 11 IUI 26 IVF

In 4 of the 20 samples (20%) tested from Group A, the %DFI values varied significantly, affecting further treatment recommendations by moving clinical thresholds.

In 4 of the 10 samples (40%) tested from Group B, the %DFI values varied significantly, affecting further treatment recommendations by moving clinical thresholds.

CONCLUSIONS

Despite differences in methodology the report values of the SCD tests are defined by the SCSA® test and small variations can have a huge impact in further clinical treatment management. The SCD test, being a human light microscope method, apparently leads to high intra and inter-observer variations. Such differences between technicians and the most widely used SCD kits in India question the accuracy and reliability of the SCD test, as it significantly affects the clinical result, interpretation and the treatment to

be recommended.

REFERENCES

1. Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 1980;210:1131-3. 2. Fernandez et al. The Sperm Chromatin Dispersion Test: A Simple Method for the Determination of Sperm DNA Fragmentation. Journal of Andrology, 2003. 24 (1): 59-66. 3. Evenson DP and Wixon R. Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay. Reprod. Biomed Online, 2006 (12): 466-472. 4. Evenson DP. Evaluation of sperm chromatin structure and DNA strand breaks is an important part of clinical male fertility assessment. Transl Androl Urol 2017. doi: 10.21037/tau.2017.07.20

Pt 1 (RK) %DFI report values varied from as

high as 73% to as low as 35% between SCD kits 1

and 2.

Pt 4 (RJ) %DFI report values varied from as

high as 33% to as low as 14% between SCD kits 1

and 2.

Patient Name SCD-Kit 1-

Tech 1 SCD-Kit 1-

Tech 2 SCD-Kit 1-

Tech 3 SCD-Kit 2-

Tech 1 SCD-Kit 2-

Tech 2 SCD-Kit 2-

Tech 3 SCSA®

Test

Pt 1 (RK) 56 73 59 35 35 42 63

Patient Name SCD-Kit 1-

Tech 1 SCD-Kit 1-

Tech 2 SCD-Kit 1-

Tech 3 SCD-Kit 2-

Tech 1 SCD-Kit 2-

Tech 2 SCD-Kit 2-

Tech 3 SCSA®

Test

Pt 4 (RJ) 16 16 14 33 27 27 21

Odds of in vivo/IUI pregnancy vs. %DFI 4

SCSA® Test

SCD Kit 2 SCD Kit 1

10 20

7

7

Patient Name SCD-Kit 2 (%DFI) Treatment Recommendation

SCSA® Test (%DFI)

Treatment Recommendation

Pt 2 (DK) 28 IVF 16 IUI

Pt 4 (MDN) 20 IUI 25 IVF

Pt 7 (RJ) 29 IVF 21 IUI

Pt 10 (R) 18 IUI 26 IVF

0

10

20

30

40

50

60

70

Pt 1 (RK) Pt 2 (DK) Pt 3 (U) Pt 4 (MDN) Pt 5 (VK) Pt 6 (SP) Pt 7 (RJ) Pt 8 (AK) Pt 9 (JP) Pt 10 (R)

% D

NA

Fra

gme

nta

tio

n In

de

x (

%D

FI)

Patient

SCD-Kit 2-Tech 1

SCD-Kit 2-Tech 2

SCD-Kit 2-Tech 3

SCSA® Test

0

10

20

30

40

50

60

70

80

90

% D

NA

Fra

gme

nta

tio

n In

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x (

%D

FI)

Patient

SCD-Kit 1-Tech 1

SCD-Kit 1-Tech 2

SCD-Kit 1-Tech 3

SCSA® Test

0

10

20

30

40

50

60

70

80

90

Pt 1 (RK) Pt 2 (DK) Pt 3 (SP) Pt 4 (RJ) Pt 5 (AK) Pt 6 (JP) Pt 7 (R)

% D

NA

Fra

gme

nta

tio

n In

de

x (%

DFI

)

Patient

SCD-Kit 1-Tech 1

SCD-Kit 1-Tech 2

SCD-Kit 1-Tech 3

SCD-Kit 2-Tech 1

SCD-Kit 2-Tech 2

SCD-Kit 2-Tech 3

SCSA® Test