1. Intracytoplasmic morphologically
selected sperm injection
(IMSI)
M. Xenariou
Laboratory Andrologist

2. Sperm morphology & IVF outcome
• Head abnormalities mainly responsible for sperm
fertilization deficiency
• Flagellar abnormalities haven’t been shown to have
any impact on fertilization (Berkovitz et al., 2006; Chemes
et al., 2003)
• Sperm nuclear DNA damage is closely related to male-
derived repeated failed ICSI attempts (Tesarik et al., 2004;
Tesarik 2005)
• Presence of large nuclear vacuoles: negatively
associated with natural male fertility potential (Bartoov
et al., 1994; Mundi et al., 1994)

3. ICSI + MSOME = IMSI
• ICSI (Palermo et al., 1992): inverted microscope
magnification of x 400
• MSOME - motile sperm organellar morphology
examination (Bartoov et al., 2002)
The resulting magnification was based on 4 parameters:
1. Objective magnification x100
2. Magnification selector x1.5
3. Video coupler magnification x0.99
4. Calculated video magnification (ccd x monitor diagonal
dimension) x 44.45
TOTAL MAGNIFICATION: 100 x 1.5 x 0.99 x 44.45 = x 6600 !!!

4. IMSI equipment
IMSI can be achieved on a Nikon inverted microscope equipped with high
resolution Nomarski optics (100x or 60x oil Plan Apo VC) enhanced by a
videozoom and digital imaging. The final magnification of the system can
be calculated as follows:
(objective) X (magnification changer) X (videozoom) X (diagonal of image
camera CCD chip diagonal)

5. ICSI vs IMSI

6. IMSI Sperm assessment (1)
The MSOME criteria for the morphological normalcy of the
sperm nucleus were defined as below:
 Smooth
Symmetric
Oval Configuration
 Homogeneity of the nuclear chromatin mass (no more
than one vacuole / less than 4% of the nuclear area)
The average length and width limits in 100 spermatozoa
with a normally looking nucleus, are estimated as follow:
 Length: 4.75 ± 0.28 μm
 Width: 3.28 ± 0.20 μm

7. IMSI Sperm assessment (2)

8. Vacuoles (1)
• Vacuole: concavity extending from the surface of the sperm
head to the nucleus through the acrosome
• Initially: considered as degenerative structures with no
physiological significance – normal feature of sperm head
(Fawcett, 1958; Chrzanowski 1966; Bedford 1967; Pedersen 1969)

9. Vacuoles (2)
Post-IVF era:
• Mundi et al., 1994: sperm nuclei in
subfertile men contained more
intranuclear vacuoles than fertile
controls
• ICSI with sperm with vacuoles =
decreased PR, early miscarriage
(Bartoov et al., 2001, 2002, 2003; Berkovitz et
al., 2005, 2006; Franco et al., 2008; Antinori
et al., 2008)
Vacuoles are not just a polymorphism
but pose a risk for an abnormality
that is accompanied by DNA injury

10. Sperm grading: vacuoles
• Vanderzwalmen et al., 2008:
Grade I: absence of vacuoles
Grade II: max. of 2 small vacuoles
Grade III: >2 small vacuoles or at
least 1 large
Grade IV: abnormal head shapes or
other abnormalities with or
without vacuoles (identified even
at x400 magnification)

11. Vacuoles & DNA fragmentation
• Spermatozoa with large vacuoles should be avoided (Tanaka et al., 2012)
• Spermatozoa with large vacuoles (>50% of the sperm head) & other potential
abnormalities were described as having higher DNA fragmentation levels than
normal spermatozoa (Franco et al., 2008)
• Spermatozoa with multiples vacuoles (>4% of the head area) have higher DNA
fragmentation levels than normal spermatozoa (Wilding et al., 2011)
• Large vacuoles associated with DNA fragmentation (Oliveira et al., 2010; Hammoud
et al., 2012)
• No difference in DNA fragmentation between normal ones & morphometrically
normal ones with a single, large vacuole (15%-25% of head area) (Watanabe et al.,
2011; Boitrelle et al., 2011)
• No difference in DNA fragmentation between spermatozoa with 1 large vacuole
& non-selected ones (Perdrix et al., 2011)

12. Studies (1)
• Prospective randomized trials with unselected pt
- Antinori et al. , 2008
- Balaban et al., 2011
• Benefit of IMSI after ICSI implantation failure (without assessing influence of
sperm abnormalities)
- Berkovitz et al., 2006
- Bartoov et al., 2003
- Nadalini et al., 2009
- Oliveira et al., 2011  not significantly higher ongoing pregnancy, miscarriage
& LBR after IMSI
• Pt with isolated teratozoospermia
- Knez et al., 2012  higher PR in IMSI group

13. Studies (2)
• Comparable embryo development between
IMSI & ICSI on Day 2 (Mauri et al., 2010; Oliveira et al., 2011)
and Day 3 (Vanderzwalmen et al., 2008; Balaban et al., 2011; de
Almeida Ferreira Braga et al., 2011; Knez et al., 2011)

14. Meta-analysis
Setti et al., 2010
 IMSI outcomes (% of top quality embryos, IR, PR) significantly improved as
compared with ICSI cycles.
 Miscarriage rate significantly decreased as compared with ICSI.
 NO difference between ICSI & IMSI regarding the FR (late paternal effect??)

15. When to use IMSI
• Severe oligo-terato-asthenospermia
Antinori et al., 2008 (prospective randomized trial; 446 couples: ICSI (n=219) VS IMSI
(n=227)  higher IR & PR)
Setti et al., 2011: Sperm selection under high magnification results in improve
treatment outcomes in patients with OATS
• Previous conventional ICSI failures with significant % of DNA fragmented
spermatozoa in the ejaculate
Hazout et al., 2006:
- Improved pregnancy outcome in pt with repeated conventional ICSI failure
- Subgroup of pt (n=72): assessment of DNA integrity (TUNEL)improvement in
clinical outcomes (IR & LBR) in pt with >30% DNA fragmentation
• Couples with 1-3 yrs of infertility & semen characteristics 1-20mill/ml
Wilding et al., 2011:
- Improved embryo morphology together with significantly increased PR & IR
• De Vos et al., 2013: no benefit of IMSI in a non-selected population with fresh
ejaculation sperm containing >1mil/ml
• Klement et al., 2013: IMSI procedure is an option to be mentioned after the 1st
unsuccessful ICSI cycle

16. Sperm preparation techniques
• Both SUP & DGP efficient in recovering lower
percentage of sperm cells with vacuolization (Monqaut et
al., 2011; Borges et al., 2013)
• POOR SPERM MOTILITY :
temperature 37°C, no PVP and supplemented with 6% of human
serum albumin
• HIGH SPERM MOTILITY :
temperature 20°C and concentration of PVP about 8%.

17. Benefits
• Berkovitz et al., 2007
Sperm nucleus morphological normalcy assessed
at high magnification could decrease the
prevalence of major fetal malformations in ICSI
children

18. Concerns
• Prolonged exposure to 37°C during IMSI could
impair the morphological integrity of the
sperm nuclei (Peer et al., 2007)
• The No of spermatozoa without vacuoles in
humans is extremely small and trying to find
and then inject such cells is a very difficult &
time-consuming process (Tanaka et al., 2012)

19. References
• Antinori, M., Licata, E., Dani, G., et al., 2008. Intracytoplasmic morphologically selected sperm injection: a
prospective randomized trial. Reprod. Biomed. Online 16, 835–841.
• Bartoov, B., Berkovitz, A., Eltes, F., 2001. Selection of spermatozoa with normal nuclei to improve the pregnancy
rate with intracytoplasmic sperm injection. N. Engl. J. Med. 345, 1067–1068.
• Bartoov, B., Berkovitz, A., Eltes, F., et al., 2003. Pregnancy rates are higher with intracytoplasmic morphologically
selected sperm injection than with conventional intracytoplasmic injection. Fertil. Steril. 80, 1413–1419.
• Bartoov, B., Berkovitz, A., Eltes, F., et al., 2002. Real-time fine morphology of motile human sperm cells is
associated with IVF-ICSI outcome. J. Androl. 23, 1–8.
• Hazout, A., Dumont-Hassan, M., Junca, A.M., et al., 2006. High-magnification ICSI overcomes paternal effect
resistant to conventional ICSI. Reprod. Biomed. Online 12, 19–25.
• Vanderzwalmen, P., Hiemer, A., Rubner, P., et al., 2008. Blastocystmdevelopment after sperm selection at high
magnification is associated with size and number of nuclear vacuoles. Reprod. Biomed. Online 17, 617–627.
• Palermo, G., Joris, H., Devroey, P., et al., 1992. Pregnancies after intracytoplasmic injection of single spermatozoon
into an oocyte. Lancet 340, 17–18.
• Berkovitz, A., Eltes, F., Yaari, S., Katz, N., Barr, I., Fishman, A., Bartoov, B., 2005. The morphological normalcy of the
sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm. Hum.
Reprod. 20, 185–190.
• Berkovitz, A., Eltes, F., Lederman, H., Peer, S., Ellenbogen, A., Feldberg, B., Bartoov, B., 2006a. How to improve IVF-
ICSI outcome by sperm selection. Reprod. Biomed. Online 12, 634–638.
• Berkovitz, A., Eltes, F., Ellenbogen, A., Peer, S., Feldberg, D., Bartoov, B., 2006b. Does the presence of nuclear
vacuoles in human sperm selected for ICSI affect pregnancy outcome? Hum. Reprod. 21, 1787–1790.
• Knez, K., Zorn, B., Tomazevic, T., Vrtacnik-Bokal, E., Virant-Klun, I., 2011. The IMSI procedure improves poor
embryo development in the same infertile couples with poor semen quality: a comparative prospective
randomized study. Reprod. Biol. Endocrinol. 9, 123.

20. References
• Franco Jr., J.G., Baruffi, R.L., Mauri, A.L., Petersen, C.G., Oliveira, J.B., Vagnini, L., 2008. Significance
of large nuclear vacuoles in human spermatozoa: implications for ICSI. Reprod. Biomed. Online 17,
42–45.
• Souza Setti A, Ferreira RC, Paes de Almeida Ferreira Braga D, de Cassia Savio Figueira R, Iaconelli A
Jr, Borges E Jr. Intracytoplasmic sperm injection outcome versus intracytoplasmic morphologically
selected sperm injection outcome: a meta-analysis. Reprod Biomed Online 2010;21:450–5.
• Balaban B, Yakin K, Alatas C, Oktem O, Isiklar A, Urman B. Clinical outcome of intracytoplasmic
injection of spermatozoa morphologically selected under high magnification: a prospective
randomized study. Reprod Biomed Online 2011;15 [Epub ahead of print].
• Fawcett DW. The structure of the mammalian spermatozoon. Int Rev Cytol 1958;7:195–234.
• Chrzanowski S. Ultrastructure of human spermatozoon. Pol Med J 1966;5:
• 482–91.
• Bedford JM. Observation on the fine structure of spermatozoa of the Bush baby (Galago
senegalensis), the African green monkey (Cercopothecus aethiops) and man. Am J Anat
1967;121:443–60.
• Pedersen H. Ultrastructure of the ejaculated human sperm. Z Zellforsch 1969;94:542–54.
• Mundy AI, Ryder TA, Edmonds DK. A quantitative study of sperm head ultrastructure in subfertile
males with excess sperm precursors. Fertil Steril 1994;61:751–4.