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

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

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

In the routine ICSI procedure, sperm cells are selected from the sperm pool under a regular microscope that magnifies 200-400x 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.

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

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

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

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

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

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

vacuoles estrusion

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

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

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.

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

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

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

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.

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.

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.

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

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

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

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

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

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

“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

Study design: prospective randomized Original Group: 446 couples January 2006 - 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

Antinori et al. 2008

Antinori et al. 2008

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

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

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

Unselected infertile population 2011 Unselected infertile population

BARTOOV’S CLASSIFICATION

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

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

Cassuto & Barak SCORE SYSTEM

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

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

RAPRUI CLASSIFICATION Choice Specific nuclear malformations 1 st Oval-Symmetric-Smooth nucleus (even with a small vacuole in the middle : 0.78 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

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

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

IMSI OBSTETRIC OUTCOMES N° Deliveries 335 ( 291 singletons+ 38twins+6 triplets) N° Live Birth 380 (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

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

“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.

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?

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.

Thank you