www.generaroma.it




                            CLINICA VALLE GIULIA, Rome



Why should we use the IVF chambers
(L323IV...
Introduction


Preimplantation embryos are known to be sensitive
  to environmental conditions that can affect future
  gr...
Introduction


Whatever incubation system is chosen, a key to
successful embryo culture is to minimize
perturbations in th...
Preimplantation embryo is a free-living organism

The early embryo represents a unique stage of development where environm...
Short-term effects of embryo environmetal

Working in vitro the embryos are exposed to several stress that may compromise
...
Culture conditions

With the appropriate media and laboratory set-up, it is possible to obtain
blastocysts form on Day 4, ...
Culture conditions

Stimulation
 protocol
                       Oocyte
                                                  ...
OXIDATIVE STRESS


Potential sources of oxidative stress:

• Exposure to light

• Presence of transitional metals in the c...
Culture system: Gas Phase

Human in vitro embryo culture is reported to be performed using a gas
phase containing either a...
Effect of Oxygen concentration on mammalian
            embryonic development

                             90
           ...
Oxygen concentration

Prospective randomized trial: six hundred couples undergoing IVF
Main Outcome Measure(s):      live ...
Oxygen concentration




           Meintjes et al., Human Reproduction 2008
Oxygen concentration

Under atmospheric oxygen conditions the main contributor to poor embryo
development is supposed to b...
REDOX state

 REDOX state is considered to be an important mechanism that regulates
 several cell functions, particularly ...
Hypoxia-inducible factors

Members of the hypoxia inducible factor family of transcription factors are
influenced directly...
Effect of Oxygen concentration on gene and
        protein expression (mammalian embryos)

A recent proteomic analysis of ...
Oxygen is a physiological signal

            Embryos encounter a decreasing O2 concentration gradient as they
           ...
Effect of Oxygen concentration on human embryo
                        development

Oxygen concentration in the oviduct of...
Effect of Oxygen concentration on mammalian embryonic
                         development



Even transient exposure to h...
Culture system: pH and temperature

Most embryo culture media utilize a bicarbonate/CO2 buffer
system to maintain physiolo...
Buffer System and Hydrogen Ion Concentration (pH)


pHi in somatic cells
is implicated in:




   The pH and hence the [H+...
Recommended pH Ranges for Commercially
         Available IVF Media


                    7.1           7.3
Quinn’s Advant...
pHo ≠ pHi
                                 This elevation in pHi observed as development proceeds may reflect the very
   ...
Embryos ability to recover from a pHi stress


It has been established that even relatively small fluctuations in pHi can
...
Cellular buffer systems




• At cytoplasmatic level, locally and rapidly by intrinsic
          buffers such as the zwitt...
Cellular buffer systems


                             Relieves alkalosis, with set point
                                ...
Cellular buffer system

It has been determined that the oocyte lacks any functional transport systems
to regulate pHi in e...
Standard micro-environment for embryo culture




           Temperature: ~37°C

                CO2: 5-6%

              ...
Choosing the right incubator
          Incubators


     •   Capacity
     •   Temperature distribution
     •   Heated do...
Out side the incubator?
Out side the incubator?


FOR HANDLING: chambers           FOR INVERTED MICROSCOPE:
                                 stage...
Quality




 People forget how fast you did a job
but they remember how well you did it

                        Howard W ...
www.generaroma.it


                     CLINICA VALLE GIULIA, Roma
                   SALUS – ASI MEDICAL, Marostica




...
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L323 Semi Closed Incuchamber Laura R. Ksystem Eshre 2009

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Dr. Laura Rienzi lecture on L323 semi-closed incubator and importance of Oxygen on cells.

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L323 Semi Closed Incuchamber Laura R. Ksystem Eshre 2009

  1. 1. www.generaroma.it CLINICA VALLE GIULIA, Rome Why should we use the IVF chambers (L323IVF) during our procedures??? Laura Rienzi Senior Embryologist, Laboratory Director
  2. 2. Introduction Preimplantation embryos are known to be sensitive to environmental conditions that can affect future growth and developmental potential Embryologists have the difficult task to select culture media, supplies, instrumentations and methods that better meet with the physiological requirements of the developing embryo in the Lab.
  3. 3. Introduction Whatever incubation system is chosen, a key to successful embryo culture is to minimize perturbations in the atmosphere around the embryo. Temperature Air quality Media composition Gas composition
  4. 4. Preimplantation embryo is a free-living organism The early embryo represents a unique stage of development where environmental interactions must take place to set homeostatic mechanisms for the developmental program Stress tollerance Critical period determining oocyte competence Oocyte preparation Critical period determining blastocyst quality for “the great awakeness” first cleavage embryonic division genome activation morula compaction blastocyst formation Rizos et al., 2002
  5. 5. Short-term effects of embryo environmetal Working in vitro the embryos are exposed to several stress that may compromise their physiology, gene expression and development transcriptome metabolome proteome Suboptimal in vitro conditions may lead to irreversibly long-term alterations in the characteristics of foetal and postnatal growth and development
  6. 6. Culture conditions With the appropriate media and laboratory set-up, it is possible to obtain blastocysts form on Day 4, such as occurs in vivo (in the mouse model) and establish 80% pregnancy rates in humans (oocyte donor model). 5% CO2 5% O2 new medium in air 6% CO2 formulation Gardner and Lane, 2004
  7. 7. Culture conditions Stimulation protocol Oocyte Embryo PATIENT quality LABORATORY OUTCOME transfer & Sperm luteal support Diet quality # of Incubators # of Embryologists Air quality & trainig level Oil overlay CULTURE SYSTEM Tissue culture ware/ Gas phase contact supplies QC & QA Culture media # of Embryo/drop Oocyte/embryo handling outside the incubator Gardner and Lane, 2007
  8. 8. OXIDATIVE STRESS Potential sources of oxidative stress: • Exposure to light • Presence of transitional metals in the culture media • High oxygen tension (i.e., 20%)
  9. 9. Culture system: Gas Phase Human in vitro embryo culture is reported to be performed using a gas phase containing either atmospheric (~20%) or reduced (~5%) oxygen concentrations. High oxygen concentration has been shown to adversely affect embryonic development in several animal species. Quinn & Harlow 1978, Batt et al., 1991, Fujitani et al., 1997; Gardner et al., 1996; Karagenic et al., 2004; Booth et al., 2005 No beneficial (or too marginal) effect of low O2 concentrations has been reported in IVF programs. Dumoulin et al., 1995, 1999 More recent data have, however, underlined the importance of the Gas composition for human embryo culture.
  10. 10. Effect of Oxygen concentration on mammalian embryonic development 90 ** % Blastocyst / Cell number 80 70 * Blastocyst (%) 60 50 Cell Number 40 30 *, P<0.05 20 **, P<0.01 10 0 ~20% 5% Oxygen Concentration Gardner et al. personal comunication
  11. 11. Oxygen concentration Prospective randomized trial: six hundred couples undergoing IVF Main Outcome Measure(s): live birth rate Blastocyst culture with low-oxygen (5%) versus high-oxygen (~20%) concentration yielded a better blastocyst outcome and a marked improvement in birth rate. Waldenstròm et at., Fertility and Sterility 2008
  12. 12. Oxygen concentration Meintjes et al., Human Reproduction 2008
  13. 13. Oxygen concentration Under atmospheric oxygen conditions the main contributor to poor embryo development is supposed to be ROS production. However a ‘cause and effect’ mechanism is yet to be demonstrated. • There are some specific events in reproductive system that are positively associated with ROS production • Increase in antioxidant gene expression under 20% oxygen has not been observed • Reducing O2 tension is more effective in promoting embryo development in vitro than is treatment with detoxifying enzymes (superoxide dismutase and catalase) Harvey et al., 2002; Thomas et al., 1997
  14. 14. REDOX state REDOX state is considered to be an important mechanism that regulates several cell functions, particularly in the preimplantation embryo. Oxygen is an essential key energy substrate for oxidative Other cellular functions, phosphorylation such as apoptosis and cell cycle control, are also significantly influenced by oxygen availability and REDOX state, via transcription state a key modulator factors such as NFkB of metabolic pathways (OXPHOS and glycolysis) Harvey et al., 2004
  15. 15. Hypoxia-inducible factors Members of the hypoxia inducible factor family of transcription factors are influenced directly by the intracellular oxygen concentration, and are important for embryonic development within the hypoxic reproductive tract Core consensus sequence (A/G)CGTG in the Under normoxic conditions, HIF- 1a protein is degraded rapidly by hypoxia response element (HRE) regulatory region the ubiquitin–proteasome system Under hypoxic conditions, the HIF-1a protein is not targeted for degradation and can translocate to the nucleus to form the active DNA-binding complex Correct [O2] gene expression Semenza et al., 2000
  16. 16. Effect of Oxygen concentration on gene and protein expression (mammalian embryos) A recent proteomic analysis of mammalian preimplantation embryonic development revealed that specific pattern of 10 biomarkers effectively discriminated in vitro embryos cultured at low or high oxygen concentration High O2 In-vivo Low O2 Katz-Jaffe et al., 2005
  17. 17. Oxygen is a physiological signal Embryos encounter a decreasing O2 concentration gradient as they progress down the reproductive tract O2 from a 5% atmosphere [O2] ≈ 7% post-compaction development would be able to sustain is associated with a significant OXPHOS throughout shift in the REDOX state to a preimplantation more reduced state development glycolytic enzyme activity glucose uptake [O2] ≈ 2% Shift from glycolysis Oxigen gradient provide spatial information to cells within the embryo ICM TEC Harvey et al., 2002; Thomas et al., 1997
  18. 18. Effect of Oxygen concentration on human embryo development Oxygen concentration in the oviduct of hamster, rabbit and rhesus monkey is similar to 8%. Oxygen concentration in the uterus is significantly lower: 1.5% to 5% In humans? 5% O2 10% O2 P Patients (n) 27 27 NS Oocytes inseminated (n) 371 422 NS Fertilization rate 76% 74% NS Clinical pregnancy rate 56% 70% NS Implantation rate 27.2 39.5 0.09 Halicigil et al., Oral presentation ESHRE 2007
  19. 19. Effect of Oxygen concentration on mammalian embryonic development Even transient exposure to high O2 concentration reduce embryo development in vitro Culture dishes equilibrated at 20% oxygen concentration for five hours prior culture in low O2 concentration decreases mouse zygote development to the blastocyst stage and resultant blastocyst cell number IT TAKES MORE THAN 5 HOURS FOR THE OXYGEN CONCENTRATION TO FALL TO EMBRYO SAFE LEVELS!! Gardner, personal comunication
  20. 20. Culture system: pH and temperature Most embryo culture media utilize a bicarbonate/CO2 buffer system to maintain physiological pH of between 7.2 and 7.4 in the medium. The inclusion of sodium bicarbonate in the media requires the use of a CO2 incubator to maintain a 5-7% CO2 atmosphere. Drawback - rapid pH increase when exposed to air - impaired embryo development To reduce gas exchange when culture dishes are taken out of the incubator it is advisable to use oil overlay. For prolonged manipulation the use of HEPES/MOPS is required. Buffering capacity is temperature dependent.
  21. 21. Buffer System and Hydrogen Ion Concentration (pH) pHi in somatic cells is implicated in: The pH and hence the [H+] can be approximately estimated using the Henderson-Hasselbach equation: pH = 6.1 + log [[NaHCO3]/(PaCO2)] HCO3- + H+ ⇋ H2CO3 ⇋ CO2 + H2O
  22. 22. Recommended pH Ranges for Commercially Available IVF Media 7.1 7.3 Quinn’s Advantage 7.2 7.35 Global 7.30 ± 0,10 G1.3 7.40 ± 0,10 HTF 7.30 7.35 COOK 7.2 7.4 Medicult
  23. 23. pHo ≠ pHi This elevation in pHi observed as development proceeds may reflect the very 7,17 different physiology of the early embryo in comparison to its later counterpart 7,19 7,21 7,24 DAY 1 DAY 2 DAY 3 DAY 5-6 5- Rather than pHo affecting pHi directly, it appears that the presence of weak acids or bases in the culture medium markedly affect pHi zigote zigote Morula / blastocyst 60 min Edwards et al., 1998
  24. 24. Embryos ability to recover from a pHi stress It has been established that even relatively small fluctuations in pHi can significantly retard subsequent developmental competence Altering pHi disrupts the organization of mitochondria Bavister et al 2001, Edwards et al,1998
  25. 25. Cellular buffer systems • At cytoplasmatic level, locally and rapidly by intrinsic buffers such as the zwitterionic amino acids • Ionic membrane transport mechanisms
  26. 26. Cellular buffer systems Relieves alkalosis, with set point 7.3 pHi (AE gene family) Na+/ Cl- HCO3- with set point pH<7 25mM Cl- Na+ HCO3- CO2 X Relieves acidosis, with set- HEPES point 6.8 pHi (NHE gene family) 5 mM [NaHCO3] Baltz et al., 2005
  27. 27. Cellular buffer system It has been determined that the oocyte lacks any functional transport systems to regulate pHi in either the acid or the alkaline ranges around 6 h following fertilisation Oviduct pH 7,7 Uterin pH 7,1 Following the denudation procedure oocytes and early embryos cannot regulate their ionic homeostasis Phillips and Baltz, 1999
  28. 28. Standard micro-environment for embryo culture Temperature: ~37°C CO2: 5-6% O2: ~5% N2: 89-90%
  29. 29. Choosing the right incubator Incubators • Capacity • Temperature distribution • Heated door • Fast recovery • Gass tight split doors • Ergonomic: all shelves easily accessible • Reliable • Failure Alarm • Possibility to use independent probes • Remote alarm system • Easy to clean • Easy to desinfect • VOC filters
  30. 30. Out side the incubator?
  31. 31. Out side the incubator? FOR HANDLING: chambers FOR INVERTED MICROSCOPE: stage top incubators CCOCs screening, denudation, Oocyte evaluation, oocyte dishes change, embryo transfer micromanipulation, embryo evaluation
  32. 32. Quality People forget how fast you did a job but they remember how well you did it Howard W Newton
  33. 33. www.generaroma.it CLINICA VALLE GIULIA, Roma SALUS – ASI MEDICAL, Marostica Ginecology: Embriology: Filippo Ubaldi Laura Rienzi Elena Baroni Laura Albricci Antonio Ciconte Antonio Capalbo Silvia Colamaria Roberta Maggiulli Antonio Lo Re Stefania Romano Fabio Sapienza

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