1. SPERM SELECTION AND PGD
L. Gianaroli, M.C. Magli, F.P. Fiorentino, A.P. Ferraretti SISMER, Reproductive Medicine Unit, Bologna, Italy 1

2. SPERM QUALITY AND CLINICAL OUTCOME
5250 ICSI cycles %
Donor sperm
20 5 - < 1 - <5 1 TESE
106/ml
SISMER

3. CLINICAL OUTCOME ACCORDING TO SPERM INDICES
No. cycles at oocyte retrieval / transfer
105 / 81
134 / 104
29 / 23
27 / 21
Magli MC, Gianaroli L, Ferraretti AP, Gordts S, Fredericks V, Crippa A. Paternal contribution to aneuploidy in preimplantation embryos. Reproductive Biomedicine Online 2009, 18:536-42

4. INCIDENCE OF EMBRYONIC ANEUPLOIDIES ACCORDING TO SPERM INDICES
a, P<0.025
b, P<0.005 b a
a,b
Embryos diagnosed
594
695
127
133
Magli MC, Gianaroli L, Ferraretti AP, Gordts S, Fredericks V, Crippa A. Paternal contribution to aneuploidy in preimplantation embryos. Reproductive Biomedicine Online 2009, 18:536-42

5. EMBRYONIC ANEUPLOIDIES ACCORDING TO SPERM INDICES
ab, P<0.05
c, P<0.01
d, P<0.001
e, P<0.005
f, P<0.025
d,e,f e f a d c b
a,b,c
Aneuploid embryos
328
431 80 92
Magli MC, Gianaroli L, Ferraretti AP, Gordts S, Fredericks V, Crippa A. Paternal contribution to aneuploidy in preimplantation embryos. Reproductive Biomedicine Online 2009, 18:536-42

6. RE-ANALYSIS OF BLASTOMERES IN NON-TRANSFERRED EMBRYOS
X Y
Magli MC, Gianaroli L, Ferraretti AP, Gordts S, Fredericks V, Crippa A. Paternal contribution to aneuploidy in preimplantation embryos. Reproductive Biomedicine Online 2009, 18:536-42

7. MOSAICISM RATE IN NON-TRANSFERRED EMBRYOS ACCORDING TO SPERM INDICES
abc, P<0.001
a,b,c c b a
Non-transferred embryos
217
184 37 55
Magli MC, Gianaroli L, Ferraretti AP, Gordts S, Fredericks V, Crippa A. Paternal contribution to aneuploidy in preimplantation embryos. Reproductive Biomedicine Online 2009, 18:536-42

8. The proportion of chromosomally abnormal embryos increases with the severity of the male factor condition, with the highest incidence in those generated by non-obstructive azoospermia (NOA) patients.
This can be consequence of both the generation of aneuploid gametes during spermatogenesis, which eventually leads to monosomic and trisomic embryos, and the defective arrangement of the mitotic plate at first embryo division, which results in complex abnormalities.
We confirm that post-zygotic errors are the predominant cause of abnormal development in NOA patients.
Magli MC, Gianaroli L, Ferraretti AP, Gordts S, Fredericks V, Crippa A. Paternal contribution to aneuploidy in preimplantation embryos. Reproductive Biomedicine Online 2009, 18:536-42

9. CHROMOSOMALLY ABNORMAL CELLS
FISH ON SPERM
CHROMOSOMALLY ABNORMAL CELLS
IN FERTILE SAMPLES
Frequency of aneuploidy for
each autosome %
each gonosome %
Frequency of diploidy %
Frequency of total aneuploidy for 23 chromosomes 6.66%
Frequency of total aneuploidy for
X Y %

10. MALE CONTRIBUTION TO ANEUPLOIDY
FISH ON SPERM - CHROMOSOMALLY NORMAL SAMPLES (n=959)
Number of Diagnosed cells: 2’746’804
abcd a % b c d
Normospermia OAT MESA sev. OAT TESE
abcdP<0.001
SISMER

11. CHROMOSOMALLY ABNORMAL CELLS
FISH ON SPERM
CHROMOSOMALLY ABNORMAL CELLS
No. diagnosed cells 2’746’804 % d c b a ab
Normospermia OAT MESA sev. OAT TESE
abcdP<0.001
SISMER

12. CHROMOSOMALLY ABNORMAL CELLS
FISH ON SPERM – SAMPLES WITH SIGNIFICANTLY
HIGHER INCIDENCE OF ANEUPLOIDY
CHROMOSOMALLY ABNORMAL CELLS
No. diagnosed cells 248’262 / 188 samples d c b a %
abcd
Normospermia OAT MESA sev. OAT TESE
abcdP<0.005
SISMER

13. SPERM QUALITY AND EMBRYO DEVELOPMENT
“Given the shortage of cytoplasm, and the lack of any detectable protein synthesis in mature sperm heads, biologists had long assumed that sperm contributes little to an embryo but the father´s genes”
Ainsworth
“The idea was that the oocyte is supplying everything (protein and RNAs) and spermatozoa were just tagging along with his DNA” Krawetz
Ainsworth C. Cell biology: the secret life of sperm. Nature 2005, 436(7052):
Krawetz SA. Paternal contribution: new insights and future challenges. Nature Reviews. Genetics 2005, 6:633–642.

14. SPERM QUALITY AND EMBRYO DEVELOPMENT
Spermatozoa contain almost 3000 different mRNA, required for synthesis of development-related proteins or whose function is still unknown.
Moreover, human centrioles in pronuclear stage embryos are paternally derived, thus supporting the hypothesis that defective spermatozoa cause aberrant embryonic development based on centriolar defects.
Ainsworth C. Cell biology: the secret life of sperm. Nature 2005, 436(7052):
Krawetz SA. Paternal contribution: new insights and future challenges. Nature Reviews. Genetics 2005, 6:633–642.
Sathanathan H, Ratnam SS, Ng SC, Tarin JJ, Gianaroli L, Trounson A. The sperm centriole: its inheritance, replication and perpetuation in early human embryos. Hum Reprod 1996, 11:34-356

15. BIREFRINGENCE EFFECT
Light beam
Emerging ray
When crossing an anisotropic structure, the incident light beam is refracted into two rays traveling at different velocity. The retardance of the slow ray relative to the fast ray generates the birefringence effect.
Light beam
Emerging fast ray
Emerging slow ray
Retardance
BIREFRINGENCE

16. SPERM HEAD BIREFRINGENCE
In order to select normal spermatozoa, the analysis of birefringence, expression of an organized and very compact texture, is an indicator of their structural quality.
Spermatozoa possess characteristics of birefringence due to the anisotropy of their protoplasmic texture.
mature acrosomal complex
mature sperm nucleus
midpiece

17. SPERM HEAD BIREFRINGENCE
Total
Partial
Abnormal

18. BIREFRINGENT SPERMATOZOA AFTER SPERM PREPARATION
abcde, P<0.0001
a,b,c
a,d,e
b,d
c,e
No. of samples 9 46 43 14
Gianaroli L, Magli MC, Collodel G, Moretti E, Ferraretti AP, Baccetti B. Sperm head’s birefringence: a new criterion for sperm selection. Fertility and Sterility 2008, 90:104-12

19. PREGNANCY RATE PER TRANSFERRED CYCLE
Significantly higher ongoing-pregnancy rate after ICSI with birefringent spermatozoa
a, P<0,05
b, P=0,02 b a a b
Cycles 9 9 46 53 43 44 14 13
Gianaroli L, Magli MC, Collodel G, Moretti E, Ferraretti AP, Baccetti B. Sperm head’s birefringence: a new criterion for sperm selection. Fertility and Sterility 2008, 90:104-12

20. ACROSOME REACTION
Intact
Acrosome
Sperm cells are classified as acrosome intact when exhibiting uniform green fluorescence following PSA (pisum sativum agglutinin)-FITC immunofluorescence staining or acrosome reacted in cases of no fluorescence or when fluorescence is localized in an equatorial green band.
Reacted Acrosome

21. HEAD BIREFRINGENCE CORRELATES WITH ACROSOME REACTION
ab, P<0,001 b a b a
No. of sperm cells
988
974
Magli MC, Crippa A, Muzii L, Boudjema E, Capoti A, Scaravelli G, Ferraretti AP, Gianaroli L. Head birefringence properties correlate with acrosome reaction, sperm motility and morphology. Reprod Biomed Online In press.

22. BIREFRINGENCE PROPERTIES ACCORDING TO SPERM INDICES
ab, P<0.001
a,b b a
1601
677
668
Magli MC, Crippa A, Muzii L, Boudjema E, Capoti A, Scaravelli G, Ferraretti AP, Gianaroli L. Head birefringence properties correlate with acrosome reaction, sperm motility and morphology. Reprod Biomed Online In press.

23. BIREFRINGENCE IN SPERM HEAD
Reacted
Non-Reacted
Gianaroli L, Magli MC, Ferraretti AP, Crippa A, Lappi M, Capitani S, Baccetti B. Birefringence characteristics in sperm heads allow for the selection of reacted spermatozoa for intracytoplasmic sperm injection. Fertility and Sterility 2010, 93:807-13

24. 45 severe OAT 71 ICSI cycles 35.64.5 yrs 26 TESE
During ICSI, the type of birefringence of the injected spermatozoa was analyzed to distinguish between reacted and non-reacted spermatozoa.
Up to three oocyte per patient were inseminated.
45 severe OAT
71 ICSI cycles
35.64.5 yrs
26 TESE
Gianaroli L, Magli MC, Ferraretti AP, Crippa A, Lappi M, Capitani S, Baccetti B. Birefringence characteristics in sperm heads allow for the selection of reacted spermatozoa for intracytoplasmic sperm injection. Fertility and Sterility 2010, 93:807-13

25. PREGNANCY RATE PER TRANSFERRED CYCLE
Significantly higher delivery rate after ICSI with reacted spermatozoa
a, P=0,002
b, P=0,048
c, P=0,004
d, P=0,033 c a d b
a,b
c,d
Cycles 23 26 22
Gianaroli L, Magli MC, Ferraretti AP, Crippa A, Lappi M, Capitani S, Baccetti B. Birefringence characteristics in sperm heads allow for the selection of reacted spermatozoa for intracytoplasmic sperm injection. Fertility and Sterility 2010, 93:807-13

26. BIREFRINGENCE AND CHROMOSOMAL STATUS
X Y

27. SPERM’S HEAD BIREFRINGENCE AND ANEUPLOIDY
Total Aneuploidy / Total Birefringence
The proportion of birefringent-head spermatozoa was inversely correlated with the frequency of total aneuploidy (P= 9.59E-7).
2.56% 10 20 40 50 60 70 80 90
100
42 samples
Total
Birefringence
(%)
OAT Severe
+ OAT Moderate
X Normal spermic R= 5 15
Total Aneuploidy (%)

28. SPERM’S HEAD BIREFRINGENCE AND ANEUPLOIDY
Total Aneuploidy / Motile Sperm Birefringence
The proportion of birefringent-head motile spermatozoa was inversely correlated with the frequency of total aneuploidy (P= 9.04E-7).
2.56% 10 20
-40
-30
-20
-10 30 40 50 60 70 80
42 samples
Total
Birefringence
(%)
OAT Severe
+ OAT Moderate
X Normal spermic 5 15
Total aneuploidy (%)

29. SPERM’S HEAD BIREFRINGENCE + HIGH MAGNIFICATION
63X with high magnification
63X
Morphology
Dimentions
40X

30. SPERM SELECTION AT ICSI Priority is given to motile spermatozoa
58% total head birefringence
29% partial head birefringence
12% no head birefringence
Motile Normal morphology
Priority is given to motile spermatozoa
26% total head birefringence
27% partial head birefringence
47% no head birefringence
Motile Abnormal morphology
Magli MC, Crippa A, Muzii L, Boudjema E, Capoti A, Scaravelli G, Ferraretti AP, Gianaroli L. Head birefringence properties correlate with acrosome reaction, sperm motility and morphology. Reprod Biomed Online In press.

31. SPERM SELECTION AT ICSI Priority is given to motile spermatozoa
45% total head birefringence
23% partial head birefringence
32% no head birefringence
Immotile Normal morphology
Priority is given to motile spermatozoa
22% total head birefringence
19% partial head birefringence
59% no head birefringence
Immotile Abnormal morphology
Magli MC, Crippa A, Muzii L, Boudjema E, Capoti A, Scaravelli G, Ferraretti AP, Gianaroli L. Head birefringence properties correlate with acrosome reaction, sperm motility and morphology. Reprod Biomed Online In press.

32. BIREFRINGENCE spermatozoa Abnormal morphology abcdefghijk, P<0,001
g,j
spermatozoa
h,k f d b e a c
d,e,f
g,h,i
Abnormal morphology
Magli MC, Crippa A, Muzii L, Boudjema E, Capoti A, Scaravelli G, Ferraretti AP, Gianaroli L. Head birefringence properties correlate with acrosome reaction, sperm motility and morphology. Reprod Biomed Online In press.

33. BIREFRINGENCE No. of spermatozoa 742 107 1576 521 Normal morphology
b, P<0,01
acdfghij, P<0,001
e, P<0,005
a,b,c
h,j
b,d
f,g,j
f,h,i e a
c,d e
f,g,h,i
No. of spermatozoa
742
107
1576
521
Normal morphology
Abnormal morphology
Magli MC, Crippa A, Muzii L, Boudjema E, Capoti A, Scaravelli G, Ferraretti AP, Gianaroli L. Head birefringence properties correlate with acrosome reaction, sperm motility and morphology. Reprod Biomed Online In press.

34. CONCLUSIONS
Our results show a correlation between birefringence properties, sperm quality (morphology, motility and functionality) and an inverse relation with frequency of aneuploidy in sperm.
It is still unknown if sperm selection by birefringence affects the incidence of mosaic embryos in non-obstructive azoospermia patients, whose development to blastocyst can still occurs.
PGD for aneuploidy screening could be employed in order to detect euploid embryos in NOA patients.
Further studies will clarify the incidence of complex abnormalities, unrelated to sperm aneuploidy, in embryo after sperm selection by birefringence.

35. CONCLUSIONS
The data from our studies suggest that a severe male infertility condition contributes to high incidence of mosaicism and complex abnormalities in the resulting embryos, in particular for what concerns non-obstructive azoospermic patients.
Sperm selection by birefringence and PGD for aneuploidy screening can be an extremely valuable complementary technique to ICSI to reduce incidence of aneuploidy and mosaic abnormalities.

36. SPERM SELECTION AND CHROMOSOMAL TRASLOCATIONS
a, P<0.001 a a
No. of spermatozoa
10082
4275
No. of patients 16 23
SISMER

37. PGD FOR TRASLOCATIONS Robertsonian Reciprocal N° patients N° cycles
Age
N° diagnosed embryos
FISH normal (%)
FISH abnormal (%)
N° transferred cycles (%)
N° transferred embryos
N° clinical pregnancies (% ET)
(% PU)
N° abortions
Implantation rate (%)
Implantation rate/pregnant patient (%) 35 56
35.2±3.6
262
67 (26)
195 (74)
39 (68)
1.4±0.7
17 (44)
(30) 5*
35.7
66.7 47 68
35.3±4.8
366
47 (13)
319 (87)
26 (38)
1.3±0.5
7 (27)
(10) 2
22.9
66.7
<0.001
<0.005
<0.025
*1 after amniocentesis, normal karyotype; 1 t(13;14) was 47XY,21; 1 t(13;14) was 47XY,22
Modified from: Gianaroli et al. (2002) Possible interchromosomal effect in embryos generated by gemete from translocation carriers. Hum Reprod.

38. Array CGH – PGD for Translocations + PGS
Patient with a reciprocal translocation: 46XX,t(5q;8p)
Gain 19p
Loss 11q
Gain 8p
Loss 16
Loss 5q

39. PGD FOR TRASLOCATIONS Robertsonian Reciprocal Female carriers
N° cycles
N° diagnosed embryos
FISH normal (%)
FISH abnormal (%)
N° clinical pregnancies (% ET) 11 53
12 (23)
41 (77)
3/7 (43) 28
142
24 (17)
118 (83)
1/10 (10)
Male carriers
N° cycles
N° diagnosed embryos
FISH normal (%)
FISH abnormal (%)
N° clinical pregnancies (% ET) 45
209
55 (26)
154 (74)
14/32 (44) 40
224
23 (10)
201 (90)
6/16 (37,5)

40. Proportion of normal / balanced embryos
Female carriers
Pregnancy rate / PU
n=4
Pregnancy rate after PGD in translocation carriers is proportional to the % of normal / balanced embryos.
n=21
n=5
n=4
n=3
n=2
n=21
Proportion of normal / balanced embryos
Male carriers
Pregnancy rate / PU
n=19
n=28
n=15
n=9
n=11
n=4

41. Proportion of normal / balanced embryos
Male + Female carriers
Pregnancy rate / PU
Pregnancy rate after PGD in translocation carriers is proportional to the % of normal / balanced embryos.
Proportion of normal / balanced embryos
The analysis of gametes could have a prognostic value on the chances of pregnancy after PGD

42. FREQUENCY OF UNBALANCED SPERMATOZOA PER SINGLE CHROMOSOME%
cases
SISMER

43. ROBERTSONIAN TRANSLOCATIONS
FISH ON SPERM IN MALE TRANSLOCATION CARRIERS
ROBERTSONIAN TRANSLOCATIONS
Translocation Unbalanced Unbalanced Aneuploid
sperm (%) embryos (%) embryos (%)
45,XY,der(13;14)(q10;q10)
45,XY,der(13;14)(q10;q10)
45,XY,der(14;21)(q10;q10)
45,XY,der(13;14)(p11;q11)
45,XY,der(13;14)(q10;q10)
45,XY,der(13;21)(p11;p11)
45,XY,der(14;22)(q10;q10)
45,XY,der(13;14)(q10;q10)
45,XY,der(13;14)(q10;q10)
45,XY,der(13;14)(q11;p11)
45,XY,der(13;14)(q10;q10)
45,XY,der(13;14)(q10;q10)
45,XY,der(14;22)(q11;p11)
45,XY,der(13;14)(q10;q10)
45,XY,der(13;14)(q10;q10)
45,XY,der(14;21)(p11;p11)
45,XY,der(13;13)(q10;q10)

44. ROBERTSONIAN TRANSLOCATIONS
FISH ON SPERM IN MALE TRANSLOCATION CARRIERS
ROBERTSONIAN TRANSLOCATIONS
FISH on sperm: 3.3% % sperm unbalanced, mean 21%
FISH on embryos: mean 31% unbalanced
Prenatal diagnosis: 1% - 2% unbalanced

45. RECIPROCAL TRANSLOCATIONS
FISH ON SPERM IN MALE TRANSLOCATION CARRIERS
RECIPROCAL TRANSLOCATIONS
Translocation Unbalanced Unbalanced Aneuploid
sperm (%) embryos (%) embryos (%)
46,XY,t(4;14)(p14;q12) ,9
46,XY,t(1;15)(p31.2; q25.3)
46,XY,t(14;20)(p11.2; q11.2)
46,XY,t(14;20)(p11.2; q11.2)
46,XY,t(1;9)(q21; q32)
46,XY,t(14;20)(p11.2; q11.2)
46,XY,t(8;10)(q13;q14) ,4
46,XY,t(3;14)(3pter→3q23::14p11.2→14pter) ,2
46,XY,t(7;18)(p22;q21) ,3
46,XY,t(9q;15p)(q12;p12)
46,XY,t(4;17)(p14;q21.33)
46,XY,t(9;14)(q32;q32)
46,XY,t(7;17)(q11.2;q12)
46,XY,t(10;12)(q22;q24.1)
46,,XY,t(7;10)(p21;q22.3)
46,XY,t(10;13)(p24.3;q12.1)
46,XY,t(7;13)(q36;q21.2)
46,XY,t(12;14)(q15;q21)
46,XY,t(2;19)(p24;q12)
46,XY,t(3;7)(q25;q32)
46,X,t(Y;15)(q11.23;p11.2)
46,XY,t(9;15)(q34;q13)

46. RECIPROCAL TRANSLOCATIONS
FISH ON SPERM IN MALE TRANSLOCATION CARRIERS
RECIPROCAL TRANSLOCATIONS
FISH on sperm: 9.9% % sperm unbalanced, mean 55%
FISH on embryos: mean 81% unbalanced
Prenatal diagnosis: 12% unbalanced

47. SPERM SELECTION AND PGD
L. Gianaroli, M.C. Magli, F.P. Fiorentino, A.P. Ferraretti SISMER, Reproductive Medicine Unit, Bologna, Italy 47

48. Best Practices of ASRM and ESHRE