I motivi del fallimento nella fecondazione. Laura Rienzi

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1 I motivi del fallimento nella fecondazione Laura Rienzi

2 Fertilization process Cumulus cell penetration Oocyte activation CG reaction completion of meiosis II Sperm/oocyte binding and penetration Sperm processing Sperm/oocyte fusion PN formation Swain and Pool, Human Reproduction Update 2008

3 Definition of fertilization failure Nagy et al., Hum Reprod PN (1 PB) 1PN (1 or 2 PB) 3PN (1 or 2 PB) Oocyte activation failure Abnormal fertilization Other

4 ICSI does not ensure fertilization Following ICSI, human oocytes still fail to fertilize 30% of the time. Complete fertilization failure occurs at an estimated rate of 2 3%. Payne et al., 1994; Flaherty et al., 1995; Mahutte and Arici, 2003

5 Fertilization failure: prevalence

6 Fertilization Failures: potential etiologies (oocyte-borne defects)

7 Successful fertilization is heavily dependent upon inherent qualities of the oocytes PERINATAL Erasure of imprints during Primordial Germ Cell Formation Pairing and Recombination and Genetic Chiasma Formation and Sister Chromatid Cohesion POSTNATAL Follicle formation and Follicular Dynamics Chronological ageing and depletion of pool PERIOVULATORY Follicular development and Oocyte growth - Extensive crosstalk in regulation and gene expression - Aquisition of maturity Oocyte maturation Chromosome segregation and Preparation for Fertilization/Support of Embryonal Development

8 Oocyte Maturation Blocks Maturation-promoting factor is the cyclin-cdk complex that was discovered first in frog eggs. It stimulates the mitotic and meiotic phases of the cell cycle. MPF promotes the entrance into mitosis (the M phase) from the G2 phase by phosphorylating multiple proteins needed during mitosis. MPF is activated at the end of G2 by a phosphatase, which removes an inhibitory phosphate group added earlier.

9 Failed fertilization: oocyte deficiency Bypassed by ICSI

10 Failed fertilization: oocyte deficiency

11 Oocyte Activation Failure: oocyte responsive system defect Even when sperm provides the oocyte activation factor, any of the multiple elements of the oocyte responsive system (SFKs, PIP2, 1P3 receptor, or PKC) could be aberrant, resulting in failure to resume meiosis. Barroso et al., 2008

12 Failures in Post-Activation Events

13 Paternal Chromatin remodeling activity resides in the oocyte After fertilization, the protamines are replaced by oocyte-supplied histones. After the protamine histone exchange, 5-methylcytosine becomes undetectable on the paternal DNA. Other oocyte proteins further modify the chromatin and DNA replication begins The activity responsible for the early events of remodeling becomes functional during oocyte meiotic maturation McLay and Clarke, 2002, 2003

14 Failed fertilization: altered oocyte gene expression profiles CHX Maternal mrnas Maternal proteins Permanent embryo arrest Transcriptional analysis of unfertilized MII oocytes revealed an altered gene expression profile associated with fertilization failure. Meiosis, cell growth and apoptosis controlling genes are mis-expressed with important fold changes. Hamatani et al., 2004, Gasca et al., 2008

15 Fertilization Failures: potential etiologies (sperm-borne defects)

16 Fertilization Failures: potential etiologies (sperm-borne defects) Lack of oocyte activation factor Sperm aster dysfunction Centrosomal defects Ultrastructural defects of the sperm flagellum

17 Failed fertilization: Spermiogenesis Defect Globozoospermia is associated with a significant reduction in, or the absence of, the sperm factor phospholipase C z (PLCz)

18 Failed fertilization: Spermiogenesis Defect Globozoospermia is a very rare type of Teratozoospermia (0.1% of infertile males) characterized by: 100% round-headed spermatozoa with an absent acrosome aberrant nuclear membrane midpiece defects Three genes with potential involvement in globozoospermia have now been identified : SPATA16 (spermatogenesis associated 16) PICK1 (protein interacting with PRKCA 1) DPY19L2 (Transmembrane gene family) Singh, 1992; Dam et al., 2007

19 Failures in Post-Activation Events: Sperm Structural Defects After penetration in the oocyte cytoplasm, the sperm centrosome forms an aster of microtubules which are necessary for male nucleus migration. During pronuclear formation the sperm derived centriole duplicates and recruits the retained oocyte centrosomal proteins to become a functional embryonic centrosome. Van Blerkom and Davis, 1995; Sathananthan et al., 1996; Manandhar et al., 2005; Carrell 2008

20 Failures in Post-Activation Events: Centrosme dysfunction Sperm Structural Defects Failed syngamy and cleavage

21 Absolute astenozoospermia Absolute asthenozoospermia, i.e. 100% immotile spermatozoa, is reported at frequency of 1 of 5000 men Eliasson et al., 1977, Ortega et al., 2011

22 Fertilization Failures: treatment strategies

23 Fertilization Failures: Treatment Strategies AOA Artificial oocyte activation Mechanical method (vigorous cytoplasm aspiration) Tesarik et al., 2002; Ebner et al., 2004 Physical method (electrical activation ) Yanagida et al., 1999 Chemical method (Ca2+ ionophore, Strontium chloride) Borges et al., 2009; Kyono et al., 2012 Most popular

24 Chemical Activation: calcium ionophores Calcium ionophores are lipid-soluble molecules that transport calcium ions across the oocyte cell membrane, inducing a single transient surge in intracellular calcium concentration, Ionomycin Calcimycin (A23187), including a commercially available calcimycin solution: GM508 Cult-Active (Gynemed, Germany) Moaz et al., 2006; Heindryckx et al., 2008; Nasr-Esfahani et al., 2008; Razavi et al., 2012 Borges et al., 2009, Montag et al., 2012; Vanden Meerschaut et al., 2012,Ebner et al., 2012, 2015

25 Chemical Activation: calcium ionophores Chemical and electrical methods of AOA, used in human assisted reproductive technology induce an aberrant calcium rise that includes a single surge without subsequent oscillations This raises concerns regarding the safety and physiological relevance of AOA Swann and Ozil, 1994; Vanden Meerschaut et al., 2014

26 Clinical pathway for the management of patients diagnosed with globozoospermia Kuentz et al. Hum Reprod. 2016

27 Fertilization Failures: Treatment Strategies

28 Fertilization Failures: Treatment Strategies

29 Fertilization Failures: Treatment Strategies 1,521 ICSI cycles

30 Does the use of calcium ionophore during artificial oocyte activation demonstrate an effect on pregnancy rate? Fertilization Live birth

31 AOA: no difference in birth defects N=595 N=83

32

33 AOA: potential genetic effects

34 AOA: potential genetic effects For all activated oocytes, polar bodies were removed, and the oocyte cytoplasm was successfully isolated and tubed for chromosome analysis

35 AOA: potential genetic effects The incidence of aneuploidy was comparable to those in a small sample of normally fertilized embryo

36 How to manage the case of Absolute Asthenozoospermia Invasive viability test TESE Ultrastructural analysis Non invasive viability test, Sperm activation Ortega et al., 2011

37 How to prevent fertilization failure in case of Absolute Asthenozoospermia Selection methods currently used to choose a viable sperm for ICSI are based on chemical substances added to the sample before the procedure Pentoxifylline treatment Higher fertilization rate compared The with sperm cycles tail using unselected flexibility immotile testsperm. (Kovacic et al., 2006) Theophilline Pentoxifylline is a methylxanthine treatment derivative that causes a nonselective inhibition of phosphodiesterase (PDE) leading to augmented generation of cyclic nucleotides such as camp and cyclic guanosine monophosphate. On sperm the drug induce sperm tail protein phosphorylation, thus stimulating motility Yovich, 1993 HOS (Hypoosmotic condition) Embryotoxicity??? Further studies are nedeed! Rubino et al., 2016

38 How to prevent fertilization failure in case of Absolute Asthenozoospermia Selection methods currently used to choose a viable sperm for ICSI are based on chemical substances added to the sample before the procedure Pentoxifylline treatment Significant improvement in searching time, increased fertilization and blastulation rates and higher The implantation sperm tail and clinical pregnancy flexibility ratestest (Ebner et al., 2011) Theophilline treatment Theophylline is a weak non-selective inhibitor of PDE isoenzymes leading to increased intracellular concentrations of camp and cyclic 3,5 guanosine monophosphate HOS (Hypoosmotic condition) Teratogenic effect reported in rodents Further studies are recommended

39 How to prevent fertilization failure in case of Absolute Asthenozoospermia Selection methods currently used to choose a viable sperm for ICSI are based on chemical substances added to the sample before the procedure Pentoxifylline treatment The sperm tail flexibility test Theophilline treatment HOS (Hypoosmotic condition) Limitations : Immotile viable sperms have a flexible tail Staff with a high level of experience and skills is nedeed. Sperm rigidity and incapacity to recover to the initial tail position is Only one retrospective study has investigated the clinical usefulness considereda sign of non-viability No significant differences in fertilization and pregnancy rates The benefits of the method remain to be proved Soares et al., 2003

40 How to manage Absolute Asthenozoospermia Selection methods currently used to choose a viable sperm for ICSI are based on chemical substances added to the sample before the procedure Pentoxifylline treatment The sperm tail flexibility test HOS (Hypoosmotic condition) The Hypo-osmotic swelling Theophilline test (HOS) is a simple and safe method treatment allowing the identification of live and intact spermatozoa. exposure to hypo-osmotic conditions (75 mm fructose, 25 mmsodiumcitrate dehydrate) induces in cells different tail swelling patterns, due to water influx, which are classified from a- sperm to g-sperm Casper et al., 1996;WHO, 2010

41 Fertilization Failures: abnormally fertilized oocytes 1PN 2.1PN

42 Fertilization Failures: abnormally fertilized oocytes About 10% of inseminated oocytes fertilize abnormally 1PN Parthenogenetic oocyte activation Abnormal formation of the nuclear envelope combination of the two genomes into a single PN or failure to organize a nuclear envelope around one of the parental genome Normal chromosomal constitution? Asynchrony in PN formation 2.1PN Two evenly sized PN plus one smaller PN Non-extrusion of the second polar body? Non inclusion of all chromosomes in the PN

43 Fertilization failures: the case of abnormally fertilized oocytes rescue

44 Fertilization failures: the case of abnormally fertilized oocytes rescue

45 Conclusions The failure of fertilization and subsequent embryo development have a complex aetiology, mainly related to oocyte borne defects; Specific sperm characteristics are associated with fertilization failure; Limited treatment options are available but AOA is possible; New technologies incorporating reliable ploidy assessment can provide an effective tool to rescue AFO-derived blastocysts for clinical use.

46 Thank you for your attention! >7.000

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