1. The role of IMSI in sperm selection
Monica Antinori
R.A.P. R.U.I
International Associated Research Institute for Human Reproduction
Rome, Italy.

2. INTRODUCTION
A different prognosis can be assigned on the basis of different normal morphology thresholds (poor prognosis: ≤4%; good prognosis: 5-14%; normal: >14%) in order to choose an adequate infertility management .
Kruger et al., 1988; Grow et al., 1994
Predictive value of sperm morphology for fertilization and pregnancy outcomes in IVF treatments.
Kruger et al., 1986; 1987; Parinaud et al., 1993; Ombelet et al., 1997; Eilish T. et al., 1998
Correct selection of spermatozoa improves ICSI outcome
Kahraman et al., 1999; Miller and Smith, 2001; De Vos et al., 2003
Sperm morphology demonstrated to be useful tool to assign to assign a prognosis and predict the outcomes of both IVF and ICSI

3. INTRODUCTION II
According to some authors, ICSI outcome is not related to strict morphology of the sperm used for microinjection
Oehninger et al., 1995; Kupker et al., 1998; Host et al., 2001; Celik-Ozenci et al.,2004
No differences in terms of fertilization and clinical pregnancy rates have been shown when samples with poor morphology (<5% normal cells) were used
Gomez 2000
Fertilization, embryo development and pregnancy seem to be achievable even if normal spermatozoa are not available (100% of terato-zoospermia)
Nagy et al., 1995; Tasdemir et al.,1997; Mckenzie et al., 2004
Nevertheless some authors didn’t find a strict correlation between this parameter and the chances of pregnancy even in those cases of extremely severe teratozoospermia

4. In the routine ICSI procedure, sperm cells are selected from the sperm pool under a regular microscope that magnifies x
Recently new devices to achieve high magnification levels (6600x) have been proposed in order to detect subtle ultra-structural alterations that would be impossible to identify with conventional methods.
Since it’s introduction, ICSI has consented the selection of a good looking spermatozoon under a magnification of maximum 400 times.
This limit was recently overcome by the introduction of new devices that have been proposed in order to detect subtle ultra-structural alterations undetectable with conventional methods.

5. Sperm sub-cellular organelles.
Sperm sub-cellular organelles
Bartoov in 1999,with the use of electron microscopy, could identify in the sperm structure, 6 main subcellular organelles, sites of different anomalies
Bartoov , et al.,1999 5

6. Specific Morphological Malformations of the Sperm Cell Subcellular Organelles (other than nucleus) Observed by MSOME
SPERM CELL SUBCELLULAR ORGANELLES
SPECIFIC MALFORMATIONS
Acrosome
Partial; Vesiculated; Lack
Post- Acrosomal Lamina
Vesiculated; Lack
Neck
Abaxial; Disorder; Cytoplasmic droplet
Tail
Coiled; Broken; Multi; Short; Lack
Mitochondria
Partial; Disorganization; Lack
Here you have displayed the specific malformation that can be detected for each of this organelles excluding the nucleus. 6

7. VACUOLES ACROSOMAL LACK Bartoov , et al,1999
And here some examples of anomalies visible by electron microscopy
Bartoov , et al,1999

8. Criteria for morphologically normal nucleus
-Oval shape
-Longitudinal symmetry
-Smooth content
And now focusing on the nucleus which is the most important structure to evaluate ,as you’ll see in the following slides, we can say that
it can be considered normal when shows an oval shape, a longitudinal symmetry and a smooth content.
To clarify this peculiarity

9. Oval Shape LARGE OVAL WIDE NORMAL SHORT LONG NARROW SMALL OVAL
Normal shape
WIDE
4.75μm (±0.28)
NORMAL
SHORT
LONG
3.28μm
(±0.20)
You have this example of oval shape, Width, Length and size have to be taken into consideration
SMALL
OVAL
NARROW

10. Smooth Content
No vacuoles/only one vacuole with a diameter greater than 0.78±0.18µm
Vacuolated spermatozoa
No extrusion or invagination of the nuclear chromatin mass
Regional disorder
A normal nucleus doesn’t show any vacuole out of one with a diameter smaller that 1 micron, that can be considered physiological . There shouldn’t be any extrusion or invagination of the nuclear chromatin mass, kind of anomalies also called regional disorders I E

11. vacuoles
estrusion

12. Sperm Functional Morphology is based on:
-High power light microscopy
-Single cell examination
-Real time observation
-Examination of only motile sperm cells
-Fine organellar morphology
Motile Sperm Organellar Morphology Examination
The sperm organellar morphological characteristics, which were established using electron microscopy, were "translated" to Sperm Functional
Morphology Criteria based on leggi:
MSOME 12

13. 97 men from an unselected group of couples undergoing infertility investigation
This new kind of evaluation, compared to Tygerberg Criteria, showed a positive correlation regarding the percentage of normal sperm forms present in the samples of 97 men from an unselected group of couples undergoing infertility investigation

14. But, despite the high positive correlation, MSOME seems a much stricter criterion of sperm morphology classification, since it identifies vacuoles and chromatin abnormalities that are not evaluated with the same precision by the analysis of Tygerberg criteria.

15. Moreover, according to more recent data by Oliveira, Msome evaluation doesn’t show fluctuations when assessed twice, in the same subject.
as far as normal and vacuolated spermatozoa are concerned
Diapo intra individual variant
Oliveira 2010

16. large/small spermatozoa 1.5 1.7 wide/narrow 3.1 2.8 regional disorder
HA bound
spermatozoa
HA unbound
P value
normal spermatozoa
2.7
2.6 ns
large/small spermatozoa
1.5
1.7
wide/narrow
3.1
2.8
regional disorder
4.7
4.3
Vacuoles 4-50%
72.5 72
Vacuoles > 50%
15.6
16.5
And finally Msome selection doesn’t seem to be replaceable by other methods like the use of Hyaluronic acid.
In fact when spermatozoa are collected from a PICSI dish neither those bounded nor those unbounded to Hyaluronic acid showed a correlation with a subsequent evaluation at high magnification
Petersen 2010

17. DNA status Clinical outcomes
System
Set Up
DNA status
Fragmentation
Aneuploidies
Clinical outcomes
Pregnancy
Abortion
Embryo quality
Classifications
Bartoov
Cassuto & Barak
Vanderzwalmen
Our proposal
Let’s move now to the major issues involved in this topic, that is nowadays one of the most debated
Equipment
Chablon design

18. Hazout et al. 2006
On the basis of the study published by Hazout in 2006,IMSI resulted to be more beneficial than ICSI at any level of sperm DNA fragmentation and particularly in those patients with more than 40% of fragmented spermatozoa. A link was then postulated between MSOME abnormalities and damaged DNA.
The improvement of clinical ICSI outcomes was evident both in patients with an elevated degree of sperm DNA fragmentation and in those with normal DNA status. It is concluded that high magnification ICSI improves clinical outcomes in couples with
previous repeated conventional ICSI failures.

19. 30 patients in an unselected group of couples undergoing infertility investigation and treatment
Franco et al. 2008
to determine the presence or absence of DNA damage in spermatozoa with large nuclear vacuoles selected by high magnification. These spermatozoa were submitted to DNA fragmentation analysis by Tunel test As you can see nearly 30% of those sperms with large vacuoles showed fragmented DNA compared to the 16% in the normal nucleus pool .The result was statistically significant Obtained results support an association between spermatozoa with large nuclear vacuoles and DNA damage and the routine use of MSOME
Obtained results support an association between spermatozoa with large nuclear vacuoles and DNA damage and the routine use of MSOME. This adverse effect (DNA fragmentation or denaturation) leads to concern, particularly about the possibility of iatrogenic transmission of genetic abnormalities.

20. Mitosensor Acridine orange Tunel Aneuploidies
Those results were confirmed by the study of Garolla where different tests were applied in order to analyse the mitocondrial activity and the DNA integrity of samples coming from patients affected by partial obstruction and testicular damage.

21. Garolla et al. 2008
Is evident that the worst results were obtained testing the whole sperm samples of the testicular damage group. But when single normal and vacuolated spermatozoa from the testicular damage group are collected at high magnification and tested again with the methods previously used, a strong relationship between high-magnification morphology and the status of spermatozoa was found.
. Thus the better results of ICSI obtained using spermatozoa selected by high-magnification microscopy may be explained

22. Oliveira 2010 5- 50% vacuolized > 50% vacuolized
The question now is: is DNA fragmentation mainly correlated with vacuolization? The results of the present study show that both normal and abnormal nuclear forms, under high-magnification analysis, appear to be equally favorable from a DNA fragmentation point of view. The only sperm type that correlates with a high rate of DNA fragmentation is the category of sperm with >50% vacuolated nucleus.
5- 50% vacuolized
> 50% vacuolized

23. Progressive motility: 43± 18% Vitality:79 ± 7.8%
RBM Online2010
Msome and acrosomal status assessment were simultaneously performed on 3237 spermatozoa of 30 man with the following sperm parameter:
concentration 65.6±0.2 x 106/ml
Progressive motility: 43± 18%
Vitality:79 ± 7.8%
Normal morphology (David’s criteria):29 ± 4.6%
Then we can say that these controversial results can indicate a different origin of some of the vacuoles visualized at high magnification.
In order to investigate the nature of vacuoles mainly located in the anterior region of the head Kacem analised the acrosomal status of single spermatozoa previously examined at high magnification

24. Kacem et al. 2010
Among the acrosome reacted the majority resulted vacuole free spermatozoa, whereas when the acrosome reaction was incomplete or intact most of them were vacuoleted

25. The same correlation arises when she compares the presence of vacuoles before and after the induction of the acrosomal reaction
Kacem et al. 2010

26. MSOME on acrosome reacting spermatozoa
A:Some protruding blebs are visible in the anterior part of the head
D:In the following picture the corresponding area shows a “vacuole-like” image
Dice che facciamo confusione tra vacuoli e regional disorders ma lei aveva analizzati spermatozoi immobili,quindi I risultati hanno questo limite visto che la valutazione MSOME va fatto proprio per quseta ragione su spermatozoi mobili
Kacem et al. 2010

27. “Crater” characterization for IMSI
“Thoughts on IMSI”
Giampiero Palermo et al.
In : "Biennial Review of Infertility, Volume 2" New York Inc. Springer-Verlag; June 2011
“Crater” characterization for IMSI
N°of (%)
Large
Small
None
ICSI
Oocytes injected 23 63 20
256
Fertilization
14(60.9)
54(85.7)
16(80.0)
167 (70.8)
Blastocyst Development
7(50.0)
28 (54.9)
4(25.0)
85 (51.0)
The structures commonly defined at the MSOME evaluation as vacuoles are craters that do not reflect abnormalities of the sperm head genome but common phisiological variations occurring during human spermnatogenesis

28. Study design: prospective randomized Original Group: 446 couples
January June 2007
Study design: prospective randomized
Original Group: 446 couples
Inclusion criteria : 1) at least 2 previous diagnoses of severe
oligo-astheno-terato-zoospermia
2) at least 3 years of primary infertility
3) the woman being 35 or younger
4) an undetected female factor
COH: GnRH Antagonist regimen (ganirelix acetate) + rFSH
OPU 35-36h after HCG
Transfer D3
Speaking about clinical effectiveness of IMSI procedure,our paper published in 2008 remain up to now the largest randomised trial present in literature

29. Antinori et al. 2008

30. Antinori et al. 2008

31. CONCLUSION
IMSI resulted in a significantly higher pregnancy rate than ICSI in all treated cases (P = 0.004) and notably in patients with ≥ 2 failures for whom the success rate increased by over 100% (P = 0.017), which confirms the data already published in the literature

33. Number of fertiized eggs Number of top quality embryos
Setti et al. 2010

34. Number of gestational sacs Number of Pregnancies
Setti et al. 2010

35. Unselected infertile population
2011
Unselected infertile population

37. BARTOOV’S CLASSIFICATION

39. Specific nuclear malformations
Bartoov’s retrieval hierarchy of morphologically evaluated "second best" sperm cells with minimally impaired nuclei.
Choice
Specific nuclear malformations 1
Large Oval
Small Oval 2
Wide forms (> 3.7 μm width)
Narrow forms (< 2.9 μm width) 3
Regional disorder 4
Large vacuoles +Normal Shape/Size 5
Abnormal forms + Large vacuoles

41. Cassuto & Barak SCORE SYSTEM
Score of spermatozoa: 2 Head Vacuole + Base
CLASS I (4-6) = 6 POINTS
CLASS II (1-3) = 2 POINTS
CLASS III (0) = 0 POINTS

42. Cassuto & Barak SCORE SYSTEM

43. VANDERLZWALMEN CLASSIFICATION
Grade
Specific nuclear malformations I
Normal form and no vacuoles II
Normal form and ≤ 2 small vacuoles
III
Normal form >2 small vacuoles or at least one large vacuole IV
Large vacuole and abnormal head shapes or other abnormalities

44. VANDERLZWALMEN CLASSIFICATION
Spermatozoi di I° e II°
Spermatozoi di III° e IV°
non mostrano significative differenze in quanto a sviluppo di blastocisti
Migliore lo spermatozoo selezionato maggiore la possibilità di formazione di blastocisti
VANDERLZWALMEN 2008

45. RAPRUI CLASSIFICATION
Choice
Specific nuclear malformations
1 st
Oval-Symmetric-Smooth nucleus
(even with a small vacuole in the middle : m)
2nd
Vacuolization < 15% (only small anterior vacuoles)
3rd
Vacuolization 15-30% (only small anterior vacuoles)
Size and shape anomalies, no vacuoles
(Large/Small; Wide/Narrow )
Normal nucleus with neck cytoplasmic droplet

47. Il contributo percentuale nella formazione di embrioni d I grado appare maggiore da parte di spermatozoi di I e II scelta
Gli embrioni di terzo grado sembrano derivare in maggior percentuale da spermatozoi di III scelta

48. IMSI at RAPRUI January 2005 and Dec 2010: 2082 IMSI cycles
Mean age: 36.6 yrs
Couples with >previous ICSI 2 failures
46.4%
Mean n° failures/couple in this group
3.87

49. IMSI OBSTETRIC OUTCOMES
N° Deliveries
335
( 291 singletons+ 38twins+6 triplets)
N° Live Birth
(211 females;169 males)
Cesarean section
326 (85.7%)
Gestational age (weeks,mean±SD)
37.4 ± 2.6
Birth weight (gr, mean±SD)
2980 ± 652
Obstetric complication
13 ( 3.8%)
7 PROM; 1gestational diabetes;
5 hypertensive desease
Congenital anomalies
(genetic deseases,malformations)
5 (1.4%)
1big vessels trasposition
1ipot. left ventricle*
1body stalk sindrome*
1Down sindrome*
1trysomy 18*
*terapeutical abortion

50. MSOME evaluation in daily IVF routine
ADDITIONAL CRITICAL POINTS
Expensive equipment to reach the necessary magnification (microscope, camcorder, composite system of lenses)
Experienced embriologists (hard training)
Work in pairs cold be usefull to increase accuracy of the evaluation
Time consuming

51. “IMSI as a Valuable Tool for Sperm Selection During ART ”
Monica Antinori, Pierre Vanderzwalmen and Yona Barak
In : "Biennial Review of Infertility, Volume 2" New York Inc. Springer-Verlag; June 2011
The introduction of IMSI has fostered a deeper understanding of those mechanisms that interfere with male fertility potential in both natural and assisted reproduction.
The lack of standardization in terms of basic techniques and morphological evaluation criteria, its routine application available in only a few ART units due to man-hours and high costs involved ,all these factors create skepticism regarding IMSI’s cost-effectiveness.

52. All things considered, the most important question is : is it ethically acceptable, according to the current literature, to not provide the infertile couple with spermatozoa of the best quality available when the technology gives you the opportunity to do so, even with the knowledge that this could compromise the ART success rate?

53. In order to fully answer this question, it’s important to first change the pervasive mind-set which is limiting the full potential that could be gained by employing the most technologically advanced procedures like IMSI.
ART treatments can no longer be considered mere “shots in the dark” they must become a decisive therapy, with much more weight being given to the first attempt.

57. Thank you