GENERA Centres for Reproductive Medicine Rome, Marostica, Umbertide, Italy www.generaroma.it
Best of ASRM and ESHRE: where we are and where we are going?
Who is the most important for a successful outcome in ART? The clinician or the embryologist?
Cortina D’Ampezzo, 1‐3 March 2012
Laura Rienzi Senior Clinical Embryologist
I declare no commercial relationships or other activities that might be perceived as a potential conflict of interest.
Learning objectives
Embryologist vs. Clinician:
1. Evidently, none of them can exist without the other
2. Early achievements: resresult lt of close collaboration and ininventive enti e approach on both sides
3. Later: more innovative approach from the Embryologist side
4. Present state: the dominance of the Laboratory
5. Future: how to reach the level of the 21th century?
1978 1992 2000 2012 1. None can exist without the other 1978 1992 2000 2012 2. Early achievements
Embryologist Clinician
1. Hormonal stimulation 1. Oocyte recovery 2. Evaluation induction 2. In vitro insemination 3. Transvaginal ultrasound 3. Early embryo culture ovum pick‐up 4. Sperm and embryo cryopreservation 4. Non‐surgical embryo transfer
The IVF chain
Stimulation protocol Oocyte Embryo transfer & quality PATIENT LABORATORY luteal support OUTCOME Sperm Diet quality
# of Incubators # of Embryologists & trainig Air quality level
Oil overlay
CULTURE SYSTEM Tissue culture ware/ contact Gas phase supplies
QC & QA Culture media # of Embryo/drop
Oocyte/embryo handling outside the incubator
Adapted from Gardner and Lane, 2007
Outcome measures in ART
Fertilization rate / Ongoing implantation rate patients with TFF (fresh/cryo cycles)
Live birth rate/ITT Cleavage rate
Good quality embryos
Blastocyst rate/ ICM and THC cells Obstetric, neonatal and long term follow‐up !
Survival rate after cryopreservation MOTHER •AGE PERINATAL • GENETIC BACKGROUND CLINICAL APPROACH • Erasure of imprints during Primordial Germ Cell Formation •EXPOSURES/LIFE STYLES/ •Ovarian hyperstimulation NUTRITION • Pairing and Recombination and • Oocyte retrieval Genetic Chiasma Formation and •HORMONAL HOMEOSTASIS •Sperm retrieval Sister Chromatid Cohesion •Embryo transfer
POSTNATAL • Endometrial preparation • Follicle formation and Follicular Dynamics – Chronological ageing and depletion of pool LABORATORY •Culture media PERIOVULATORY •Gas conditions • Follicular development and Oocyte growth ‐ Extensive cross‐ • Maintenance of temperature talk in regulation and gene • Cells Manipulation (denudation, expression ‐ Aquisition of maturity FATHER IVF, ICSI, biopsy) • Oocyte maturation – • SPERM QUALITY •Cell evaluation and Selection Chromosome segregation and (morphology, metabolism, gene Preparation for • GENETIC BACKGROUND expression, proteomic, genetical Fertilization/Support of Embryonal •EXPOSURES/LIFE STYLES/ constitution) Development NUTRITION •HORMONAL HOMEOSTASIS
1978 1992 2000 2012 3. Innovative approaches
Clinician Embryologist
‐ICSI ‐Surgical sperm retrieval ‐Oocyte and embryo biopsy ‐Recombinant FSH ‐PGD/PGS ‐Use of antagonist ‐Blastocyst culture ‐Concept of mild stimulation ‐Revival of the natural cycles
1978 1992 2000 2012 3.1. Innovative approaches: ICSI
Palermo G, Joris H, Devroey P, Van Steirteghem AC Lancet, 1992: ICSI changes the figure of ART Immediately adopted as the treatment of choice
New possibilities for the all categories of male infertility, new opportunities for oocyte evaluation, necessary for PGD 1978 1992 2000 2012 3.2. Innovative approaches: PGD
Handyside et al., Nature 1990
Mechanisms for inheritance of disease:
‐Chromosomal (numerical or structural) ‐Mendelian (single gene defects) ‐Mitochondrial ‐Complex genetic inheritance
PGD can rule out a specific genetic disease known to exist in one or both parents before pregnancy has been established. The benefit is self‐evident!
“Understanding of the methods used for genetic diagnosis and research is becoming a standard requirement for the clinical practice of reproductive medicine” Crosignani, The ESHRE Capri Workshop Group, Human Reproduction Update, 2008
1978 1992 2000 2012 3.3. Innovative approaches: blastocyst culture
New approaches to improve embryo culture “Empirical optimisation” (John Biggers) vs. “Back to the Nature” (Henry Leese) vs. “Nutrition consumption” (David Gardner)
1978 1992 2000 2012 3.3. Innovative approaches: blastocyst culture
GOAL: Reduce number of embryos transferred ‐ Maintain high pregnancy rates per cycle
“The best available evidence suggests that the probability of live birth after fresh IVF is significantly higher after blastocyst‐stage embryo transfer as compared to cleavage‐stage embryo transfer when equal number of embryos are transferred in the two groups compared” Papanikolaou et al., 2008 “There is emerging evidence to suggest that in selected patients, blastocyst culture maybe applicable for single embryo transfer.” Blake et al., 2007 1978 1992 2000 2012 4. Present state: the dominance of the laboratory
Clinician Embryologist
1. Non invasive embryo evaluation 1. Sustained follicle stimulants 2. Trophectoderm biopsy and 2. Endometrial evaluation and advancement in genomics preparation 3. Oocytes and blastocyst vitrification 4. In Vitro Maturation 5. Ovarian tissue cryopreservation 3. Ovarian tissue transplantation
1978 1992 2000 2012 4.1. Present state: non invasive oocyte/embryo evaluation
1. Morphology ‐ Birefringence ‐ Continuous Monitoring System ‐ IMSI
2. Metabolic Activity ‐ Pyruvate/Glucose uptake ‐ AiAmino acids ‐ Oxygen consumption (Respirometry)
3. Constituents ‐ Genome ‐ Transcriptome (cumulus cells) ‐ Proteome ‐ Metabolome
4. Secreted Factors ‐ PAF ‐ HLAg ‐ “Secretome”
1978 1992 2000 2012 4.1.1 Present state: birenfringence
“most studies show that polarization microscopy may help us to further increase our knowledge on gametes and meiosis.” Montag et al., 2011 1978 1992 2000 2012 4.1.1. Present state: morphodynamic
1978 1992 2000 2012 4.1.1. Present state: morphodynamic
video
1978 1992 2000 2012 4.1.1. Present state: IMSI
Letter to New England Journal of Medicine:
“Selection of spermatozoa with normal nuclei to improve the pregnancy rate with intracytoplasmic sperm injection”
Bartoov et al. (()2001) 1978 1992 2000 2012 4.1.2. Present state: metabolic activity
1978 1992 2000 2012 4.1.3. Present state: ‐omic
Assou et al., 2003
1978 1992 2000 2012 4.2. Present state: new PGS approach
I and II polar bodies Blastocyst TE genetic screening Blastomere genetic genetic screening screening
•Meiotic and mitotic aneuploidies • Male and mitotic errors not detected •More robust genetic diagnosis •Amplification efficiency of both PBs • Reduced biopsy damage to the about 85% embryo •Non complementary aneuploidies • Reduced number of embryos •Balanced PD •Low mosaicism? 1978 1992 2000 2012 4.2. Present state: new PGS approach
Day 5 PGS
qPCR and Microarray Approaches FISH: 9 chromosomes on 1 aCGH ‐ 23 chromosomes on blastomere 5‐10 cells
1978 1992 2000 2012 4.3. Present state: vitrification
PubMed search for “vitrification” or “vitrified”
Full papers between 2006 and 2010 917 Dealing with human embryos or oocytes 80
1978 1992 2000 2012 4.3. Present state: oocyte vitrification
Oocyte cryopreservation is an emerging discipline that has already a key place for different applications:
1. Fertility preservation for medical reasons
2. Fert ility preservation for sociilal reasons
3. Use of cryo‐banked oocytes for egg donation
4. Avoids the production of supernumerary embryos in IVF
5. Accumulation of excess oocytes in IUI cycles
Gook et al., 1995; Porcu et al., 1997, 2000; Kuleshova, et al.1999; Borini et al., 2004; Kuwayama et al., 2005, 2007; Oktay et al., 2005; Oehninger 2005; Patrizio et al., 2005; Lee et al., 2006; Lucena et l., 2006; Norwak et al., 2007; Vajta and Nagy, 2007; Savulescu et al., 2008; Tao et al., 2008; Homburg et al., 2009; Dondorp et al., 2009; Nagy et al., 2009; Cobo et al., 2008, 2010; Ubaldi et al., 2010; Stoop et al., 2010; Garcia et al., 2011; Lee et al., 2011 1978 1992 2000 2012 5. Future: how to reach the 21° century
??? Clinician Embryologist
‐Validation of the embryo selection procedures ‐Optimization and automation of the handmade procedures= STANDARDIZATION
1978 1992 2000 2012 Preimplantation environment today
In vivo O2 In vitro CO2
Maternal tissues O2 CO2 Effectors
O2 CO2 Nutrients ↓Nutrients CO2 Waste CO \ 2 Effectors ↑ Waste Fluid Waste Ions film Ions Zona Ions Epithelium Ions Lumen Medium Adapted from Klaus Wiemer
1978 1992 2000 2012 5. Future: how to reach the 21° century
Does the present efficiency of in vitro embryo culture approach the natural limits?
1. We do not know where these limits are.
2. There are possibilities outside the present frames, that may result in considerable increase in efficiency.
3. A lot of things to consider, answer, resolve, improve, and create...
In vitro embryo culture is not an imperfect copy, is an artificial process, with its own frames, limitations, and possibilities. The embryologist task is to modify the frames, eliminate the limitations, exploit fully the possibilities Vajta, 2011 1978 1992 2000 2012 5. Future: how to reach the 21° century
New platforms: Dynamic platforms: Specialized surfaces: . Microdrops •Agarose . Ultramicrodrops •Shaking/rotation •Tilting •Matrigel . Submicroliter platforms •Vibration •Hyaluronic acid . Microwells •Controlled fluid flow •Co‐culture . Micro channels •3‐Dimensional matrix
Vajta et al., 2000, 2008 Swain and Smith, 2011
Heo et al., 2010
1978 1992 2000 2012 5. Future: how to reach the 21° century
™ Marksman ROBOTIC ICSI System: standardization
1978 1992 2000 2012 5. Future: how to reach the 21° century
Silicone based nano barcodes: safety
Magnified images of the barcodes Novo et al., 2011 1978 1992 2000 2012 5. Future: how to reach the 21° century
Media sampling: Vision of a complex automated embryo Metabolome, Secretome, production system Respiration, Amino acid and Sugar uptake, …..
Denudation Robotic ICSI Robotic Camera biopsy Dinamic morphological evaluation (light and polarized microscopy) Trophoblast genomic Cumulus corona cells transcriptome Rienzi et al., 2011
1978 1992 2000 2012 5. Future: how to reach the 21° century
“IVF on a Chip” Nanosensors
• pH • Temperature • O2 • Osmolality • Toxins/metabolites
Ability to monitor environmental parameters and potential to adjust/correct to desired settings Pool, 2011
Conclusions
Laboratory innovations have allow to increased the number of patients that can be successfully treated in IVF clinics. Revolutionary techniques (ICSI, PGD, blastocyst culture, cryopreservation) are today routinely used. Novel potentially useful procedures have been recently proposed and may revolutionize the near future. The past, present, and future is in the Laboratory technologies!!
All these involvements require high‐ laboratory standards with highly qualified personnel, defined quality management systems as well as investments. www.generaroma.it
CLINICA VALLE GIULIA, Roma SALUS – ASI MEDICAL, Marostica GENERA UMBERTIDE, Perugia
Gynecology: Embryology: Laura Rienzi Filippo Ubaldi Stefania Romano Elena Baroni Laura Albricci Silvia Colamaria Antonio Capalbo Maddalena Giuliani Roberta Maggiulli Fabrizio Fiorini Benedetta Iussig Antonio Ciconte Nicoletta Barnocchi Silvia Venanzi Sara Fusco Antonio Lore Federica Sanges Fabio Sapienza Catello Scarica Elena Ievoli