1 / 45

Roberta Maggiulli

www.generaroma.it. CLINICA VALLE GIULIA, Rome. Roberta Maggiulli. Can sperm and oocyte selection improve pregnancy rate?. June 4-6 2010, Istanbul. International symposium on Reproductive Medicine “PCOS, Ovulation induction and Fertility Preservation: New Trend in 2010”.

verda
Télécharger la présentation

Roberta Maggiulli

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. www.generaroma.it CLINICA VALLE GIULIA, Rome Roberta Maggiulli Can sperm and oocyte selection improve pregnancy rate? June 4-6 2010, Istanbul International symposium on Reproductive Medicine “PCOS, Ovulation induction and Fertility Preservation: New Trend in 2010”

  2. Can sperm selection improve pregnancy rate?

  3. Sperm morphology and ICSI 1995 Success ratesofintracytoplasmaticsperminjectionisindipendentofbasicspermparameters. HumanReproductionvol.10 no.5 pp.1123-1125, 1995 The resultofintracytoplasmicsperminjectionisnotrelatedtoanyof the threebasicspermparameters. Nagy ZP, Liu J, Joris H, Verheyen G, Tournaye H, Camus M, Derde MC, Devroey P, Van Steirteghem AC. HumanReproductionvol.11 no.5 pp.1019-1022, 1996 The outcomeofintracytoplasmicsperminjectionisunrelated to‘strictcriteria’ spermmorphology Peter Svalander1, Ann-HeleneJakobsson, Ann-SofieForsberg, Anna-CarinBengtsson and MattsWikland

  4. Sperm morphology and ICSI • The establishment of a pregnancy even with spermatozoa that are dysfunctional and with abnormal DNA may be attributed to the corrective role of selecting a single spermatozoon for ICSI. Virro, Larson-Cook et al. 2004 FERTILITY AND STERILITY VOL. 79, N°1, JANUARY 2003 Influenceofindividualspermmorphology on fertilization, embryomorphology, and pregnancyoutcomeofintracytoplasmicsperminjection. De Vos A, Van De VeldeH, JorisH, VerheyenG, DevroeyP, Van Steirteghem A. CentreforReproductive Medicine, University Hospital, Dutch-speakingBrussels Free University (VrijeUniversiteitBrussel), Belgium.

  5. Sperm morphology and ICSI Retrospectivestudy De Vos et al., 2003 * Significantly different

  6. REAL TIME FINE SPERM MORPHOLOGY ASSESSMENT

  7. IMSI LettertoNew England Journal of Medicine: “Selectionofspermatozoawithnormal nuclei toimprove the pregnancy rate withintracytoplasmicsperminjection” Benjamin Bartoovetal. (2001) Introduction of a new concept to observe spermatozoa called ‘motile-sperm organelle-morphology examination’ (MSOME) and to evaluate the fine nuclear morphology of motile spermatozoa in real time. IntracytoplasmicMorphologically Selected Sperm Injection (IMSI)

  8. Optical resolution • Image contrast • Maximal optical magnification • Magnification of the video system NOMARSKY contrast DIC FINAL DIGITALLY ENHANCED MAGNIFICATION: 100 X 1.5 X 0.99 X (355.6MM/8MM) = 6600 x 100X Objective Monitor Video coupler magnification 0.99 Camera 8mm

  9. IMSI: Spermpreparation Bartoovet al., 2002 • - Useof a density gradient in the preparationpriortoselection • - Useof PVP (differentconcentration) • low temperature (accordingtospermmotility) • glass-bottomdishover the top ofan100x objectivelenscoveredby a dropletof immersion oil - Examinationofindividualspermathigh magnificationby the inverted microscope equippedwithhigh-powernomarskiopticsenhancedbydigitalimaging - sperm selection according to MSOME criteria 100x

  10. IMSI: Sperm assessment MotileSpermOrganellarMorphologyExamination CRITERIA toselectSPERMATOZOA SUITABLE for IMSI The MSOME criteriaforthemorphologicalnormalcyof the spermnucleusweredefinedas: • SMOOTH • SYMMETRIC • OVAL CONFIGURATION • HOMOGENEITY OF THE NUCLEAR CHROMATIN MASS • (no more thanonevacuole/ lessthan4% of the nuclear area) The averagelenght and widthlimits in 100 spermatozoawith a normallylookingnucleus, are estimatedasfollow: • LENGHT: 4.75  0.28 µm • WIDTH: 3.28  0.20 µm Bartoovet al., 2003

  11. Lenght 4.68 mm Width 3.48 mm IMSI: Sperm assessment LENGHT: 4.75  0.28 µm WIDTH: 3.28  0.20 µm NUCLEUS: SMOOTH, SYMMETRIC, OVAL, HOMOGENEOUS Abnormal Not homogeneous Vacuole (>4%)

  12. IMSI: Clinical results Several publications report that the selection of spermatozoa with normal nuclear shapes at high magnification is positively associated with pregnancy rates: • Couples with previous and repeated implantation failure Bartoov et al., 2002, 2003; Berkovitz et al. 2006 • Patients with high degree of sperm DNA fragmentation rate Hazout et al., 2006

  13. IMSI: Clinical results First attempt Antinori et al., 2008

  14. IMSI: Clinical results First attempt, with ≥ 6 oocytes, sibling oocytes Takeuchi and Yoshida, 2009 ESHRE Amsterdam Kiba Park Clinic, Tokyo, Japan

  15. IMSI: Clinical results Repeated failures, with ≥ 6 oocytes, sibling oocytes Takeuchi and Yoshida, 2009 ESHRE Amsterdam Kiba Park Clinic, Tokyo, Japan

  16. Prospective randomized study: standard ICSI vs IMSI in OAT patients • Inclusion criteria: • female age < = 42 years • basal serum FSH (< = 10 mIU/ml) • severe OAT (<=4% according to Kruger) • Exclusion criteria: • Less than 3 MII obtained

  17. IMSI vs ICSI in OAT: Material and methods • Patients randomization performed on the day of hCG administration • Control group = sperm selection accurately performed at 400x magnification (standard ICSI) • Study group = sperm selection performed at high magnification (IMSI, according to Bartoov et al., 2003)

  18. PreliminaryResults

  19. Lessonfrom IMSI approach Sperm quality may affect ICSI results in terms of embryo development and clinical outcome. No clear evidence have been published yet (evidence-based medicine, prospective randomized studies, enough power, identification of a specific category of patients) about the real efficacy of IMSI approach. Time consuming technique, need for additional highly trained man power, additional cost of the procedure

  20. PERINATAL • ErasureofimprintsduringPrimordialGermCellFormation • Pairing and Recombination and Genetic Chiasma Formation and SisterChromatidCohesion • MOTHER • AGE • GENETIC BACKGROUND • EXPOSURES/LIFE STYLES/ NUTRITION • HORMONAL HOMEOSTASIS • POSTNATAL • Follicle formation and Follicular Dynamics – Chronological ageing and depletion of pool Involve the cohort • PERIOVULATORY • Folliculardevelopment and Oocytegrowth - Extensive cross-talk in regulation and gene expression - Aquisitionofmaturity • Oocytematuration–Chromosomesegregation and PreparationforFertilization/SupportofEmbryonalDevelopment • LABORATORY • HANDLING • CULTURE CONDITIONS Can oocyte selection improve pregnancy rate? Ursula Eichenlaub-Ritter

  21. Oocytes and embryos potential assessment • NON-INVASIVE EVALUATION • Microscopic observation Polarized light microscopy Metabolomic assessment Gene expression profiles • INVASIVE EVALUATION Genetic evaluation

  22. ASYNCHRONY Cumulus corona cell morphology not predictive Lauferet al., 1984 ICSI

  23. Cytoplasmic maturation Nuclearmaturation E2 LH FSH ENTRY IN MEIOSIS PRIMARY OOCYTE ARRESTED IN PI ARRESTED MII COMPLETION OF MEIOSIS ASYNCHRONY Oocyte morphological abnormalities OVULATION/OPU

  24. Morphologyasassessmenttool Human Reproduction Update in press Predictive value of oocyte morphology in human IVF: a systematic review of the literature Laura Rienzi,1,2 Gábor Vajta, 3 Filippo Ubaldi2 2G.EN.E.R.ACentreforReproductive Medicine, Clinica Valle Giulia, Via G. De Notaris 2, 00197 Rome, Italy 3Beijing Genomics Institute, Shenzhen, 518083 China. PURPOSE: to investigate if any noninvasive morphological feature or group of features of MII phase human oocytes has a strong predictive value for further development according to the literature between 1996 and 2009

  25. Systematic review: methods Literature search Medline, ISI Web of Knowledge Science Citation Index, Cochrane Controlled Trials Register and Ovid. The search was performed in December, 2009 for all available papers written in English and published in or after January, 1996 Studies with lack of any relevance: dealing exclusively with animal oocytes, sperm morphology, in vitro matured, cumulus- or zona-free oocytes as well as with cryopreservedoocytes were excluded according to the title.

  26. Systematic review: results Investigated features Meiotic spindle (15) Vacuoles or refractile bodies (14) Zonapellucida (12) The shape of the polar body (12) The shape of oocytes (10) Dark cytoplasm or diffuse granulation (12) Central granulation (8) Perivitelline space (10) Cumulus-oocyte complex (6) In average, each publication investigated 2.2 morphological features

  27. Systematic review: results Investigated outcomes Fertilization (37) Embryo quality (22) Clinical pregnancy rate (19) Cleavage rate (12) Development to blastocysts (9) Implantation rate (7) Embryo development (5) Aneuploidy (4) Ongoing pregnancy (2) and take home babies (2) In average each publication investigated 2.7 outcome parameters

  28. Systematic review: conclusions The analysis of 46 papers of the past 15 years about predictive value of non-invasive morphological parameters of MII phaseoocyteshas produced contradicting results No support of the average opinion about the features of ‘good’ and ‘bad’ quality and respective developmental competence More intensive and coordinated research is needed to reach a consensus on the predictive potentials of oocyte morphological examination

  29. Oocytes and embryos potential assessment • NON-INVASIVE EVALUATION • Microscopic observation Polarized light microscopy Metabolomic assessment Gene expression profiles • INVASIVE EVALUATION Genetic evaluation

  30. ~4% Oocyte nuclear status Metaphase II Telophase I 60-90 minutes (Montaget al., 2006) ~4% ~10%

  31. Meiotic Spindle: meta-analysis • Fertilization rate • Embryo development • Implantation rate • Clinical pregnancy rate per transfer

  32. Meiotic Spindle: Fertilization Rate 75.6% 61.5% P < 0.0001 Fertilization rate was significantly higher in group of oocytes where meiotic spindle was viewed than in the group of oocytes that did not show the meiotic spindle. Petersenet al., 2009

  33. Meiotic Spindle: PN morphology and cleavage rate 25.1% 12.7% 43.9% 63.1% P < 0.0001 Petersenet al., 2009

  34. Meiotic Spindle: Embryo quality 37.3% 28.0% 50.7% 28.6% P < 0.0001 Peterssenet al., 2009

  35. Meiotic Spindle: Clinical outcome Despite the tendency in favor of oocytes with meiotic spindle, this meta-analysis failed to show any statistically significant difference in the most relevant end points in IVF. Petersenet al., 2009

  36. 35% Comparative analysisof the MII MS ofhumanoocytesthroughpolarized light and confocalmicroscopy • Retardance measurements have limited predictive value of the degree of spindle fiber order and chromosome position • Polscope may still be an inefficient method for assessing the metaphase II spindle and, as a result, for noninvasive oocyte selection. Coticchioet al., 2009

  37. Zonapellucida Birefringence OuterLayer Middle Layer InnerLayer

  38. Zonapellucida Inner Layer retardance automatic user-independent polarization microscopy imaging system Retardance magnitude > 3 nm is predictive of CC, while retardance < 2 nm of the inner layer of the zonapellucida appears to be associated with reduced developmental potential and pregnancy rate. NCC NCC IL retardance NCC CC CC CC Shenet al.2005 Montaget al.,2007;Ebneret al.,2009

  39. Oocytes and embryos potential assessment • NON-INVASIVE EVALUATION • Microscopic observation Polarized light microscopy Metabolomic assessment Gene expression profiles • INVASIVE EVALUATION Genetic evaluation

  40. Metabolomic assesment www.generaroma.it “Exometabolomics”: the systematic analysis of the inventory ofmetabolites (small-molecule biomarkers) that represent thefunctional phenotype at the cellular level Metabolomicprofiling from spent culture medium of the oocyte is related to nuclear maturity 93% specificity and 100% sensitivity between MI and MII oocytes Nagyet al.,2009

  41. Metabolomic assesment Metabolomic profiling from spent culture medium of the oocyte is able to predict embryo development at day 3 and day 5 stages, and relates to embryo viability. Day 3 Day 5 Near-infrared (NIR) spectroscopy data obtained 412 oocyte culture samples collected from 43 patient cycles; Nagy et al.,2009

  42. Gene expression profiles of cumulus cells www.generaroma.it Future approaches analyzing the expression of relevant genes in cumulus cells may enable us to monitor the consequences of different stimulation protocols and identify the underlying molecular mechanisms by which they influence oocyte/embryo quality. Assouet al., 2003; McKenzieet al., 2004; Feuerstein et al., 2007

  43. Oocyte chromosomal status Meiosis I errors Meiosis II errors Wells et al., 2002

  44. Conclusion Oocyte morphology is probably not an indicator of oocyte developmental competence Embryologists are, thus, unable to select the best oocyte by simply looking at it (on the other hand bad oocytes can be discared) Novelapproaches (proteomic and metabolomic analysis, array-CGH of PB) shouldbecombinedtooocytemorphologyevaluationbutmulticenter randomized trials are needed to confirm the efficacy of these technologies.

  45. www.generaroma.it CLINICA VALLE GIULIA, Roma SALUS – ASI MEDICAL, Marostica Embriologia: Laura Rienzi Stefania Romano Laura Albricci Antonio Capalbo Benedetta Iussig Roberta Maggiulli Nicoletta Barnocchi Ginecologia: Filippo M. Ubaldi Elena Baroni Antonio Ciconte Silvia Colamaria Madda Giuliani Fabio Sapienza Silvia Venanzi

More Related