Dangers of Control From the Cryogenic Liquid Nitrogen Storage Container and Cryopreservation Of Embryo and Sperm Dr . Mustafa ZAKARIA Deputy Executive Director and Administrative Coordinator of the Scientific Research Group R,B and ART, consultant Reproductive Biology in the IVF laboratory, fertility center IRIFIV Casablanca ,Morocco dr.zakaria@irifiv-aisrg.com

1. Dangers of Control From the Cryogenic
Liquid Nitrogen Storage Container and
Cryopreservation Of Embryo and
Sperm
22 July 2020
Casablanca
Dr . Mustafa ZAKARIA
Deputy Executive Director and
Administrative Coordinator of the
Scientific Research Group
R,B and ART, consultant
Reproductive Biology in the IVF
laboratory, fertility center IRIFIV
Casablanca ,Morocco
dr.zakaria@irifiv-aisrg.com

2. Continuing Education
#EN Casa Con LFC 2020
Wed, July 22, 2020
Dangers of Control From the Cryogenic LN2 Storage Container
and Cryopreservation Of Embryo and Sperm - IVF Laboratory
Deputy Executive Director and
Administrative Coordinator of the Scientific
Research Group
R,B andART, consultant Reproductive
Biology in the IVF laboratory, fertility center
IRIFIV,
Directly by registering on the Webinar website as well as on
the channel of Dr. Mustafa Zakaria on the live YouTube site

3. Ms Azhar Ismail
Clinical Embryologist Experience
more than 20 years
Worked at Michigan IVF
Lebanon
Moderator : Department of IVF Lab
Dr Ritu S,Santwani
FICOG – FIAOG AMRCOG –ART-
Singapore IVF specialist Director
Pune Test Tube Baby center &
shyam well Woman Clinic
India
Moderator ; M,D – Obst & Gynaec
Dr MUSTAFA ZAKARIA
Deputy Executive Director and
Administrative Coordinator of
the Scientific Research Group
R,B and ART, consultant
Reproductive Biology in the IVF
laboratory, fertility center IRIFIV,
Kingdom of Morocco
Speaker M,D Reproductive Biology
3
Speakers and moderators of a seminar

4. Introduction to
Cryopreservati
on
Temperature
Liquid Nitrogen
Freezin
g
Vitrification
Container
s
Cryopreservati
on Blastocyst
Day 5 – Day 6 – Day 7
Egg Freezing
Includes Presentation Content
Of the SIX Main Paragraphs
4
Cryogenic LN2
Storage Container
Introduction to liquid
nitrogen Storage Container
in show IVF lab
Of Embryo
Blastocyst

5. Introduction to
Cryopreservatio
n
Introduction to liquid
nitrogen Storage Container in
show IVF laboratory

6. Historical to Vitrification
6

7. Introduction to Cryopreservation
7
Cryogenic storage :
➢ 2 types of storage :
 Gas phase
■ No contact between liquid nitrogen and the sample
 Liquid phase
■ Discontinuous immersion: the sample will be in liquid nitrogen depending on:
● Nitrogen level
● From its position in the rack or canister
■ Continuous immersion: the sample will be continuously in liquid nitrogen
● Requires specific adjustment

8.  Liquid nitrogen is a cryogenic fluid essential for in vitro reproductive technologies widely used in
human (IVF), in the cattle industry (in vitro embryo production), and for livestock breeding
research purposes.
 LN2 is a liquid substance produced through an industrial process by means of a fractional
distillation method. Air is liquefied and then distilled in order to separate the nitrogen gas.
 Its main characteristic is the ability to maintain the ultralow temperature of −196°C, well below
the freezing point of the water (0°C), making it useful for several applications.
 One of them is the cell cryopreservation process, used ART to preserve gametes and embryos
for the treatment of human infertility and fertility preservation issues, as well as in cryobanking
of animal gametes and embryos in the cattle industry.
 The cryogenic temperature slows chemical and physical reactions of the biomolecules and stops
the samples from degrading for future
Introduction to liquid nitrogen Storage Container in
show IVF lab

9. liquid nitrogen Settlement transportation equipment
9
 What are the Hazards of Nitrogen?
Nitrogen is not toxic since about 78% of the
air we breathe contains this gas. However, it
is not harmless and it has NO SMELL.
 AS A GAS:
• It can cause suffocation by replacing the
oxygen in a confined area
• Its presence will give false readings when
using explosimeters or flammable gas detectors
• And, like other compressed gases, there are
the risks related to its pressurised containment
when it is stored in high pressure cylinders
 AS A LIQUID:
• The same as the gas, when it evaporates
• By creating an intense coldness (-196oC) that
can cause frostbite, crack steel equipment and
explode tyres

10. Open and Closed Cryopreservation Systems
Figure 1
Illustration showing the open vitrification and warming
system and the risk it offers for the germplasm samples.
(a) Cryopreservation straw with the vitrified embryo. (b)
Immersion of straw in contaminated LN2. (c) Straw stored
in LN2 container. (d) Warming of the straw with the
contaminated sample. (e) Contaminated germplasm.
Figure 2
Illustration showing the closed vitrification and warming
system and its low risk for germplasm samples. (a)
Cryopreservation straw with the vitrified embryo. (b) The
straw is covered and sealed prior to contact with LN2. (c)
Embryo vitrification in the closed system. (d) Straw
stored in LN2 container. (e) Straw cover is removed prior
to warming, avoiding contact of the germplasm sample
with the microorganisms. (f) Contaminant-free
germplasm.

11. Cryogenic LN2
Storage Container

12. Cryopelletization
LN provides refrigeration to vials of sample via a secondary heat-transfer fluid (HTF)
circuit. During vacuum drying, LIN also cools the condenser to create a low-
temperature cold trap for solvent removal.
Cryopreservation

13. Best practices for Cryopreservation
3-Structure directives and system for the manufacture of
refrigerated containers for small containers
1-control room
4-Structure of conveyor tubes
2-Distribution
center is a major
container
8-Embryo
containers
room
9-Container
temperature
monitor
7-Alarm system
6-Alarm
control
room
5-Quality
Control
Schedule

14. 14
 Guidance and risks of controlling the room of
refrigerated storage containers LN2 inside the
embryo laboratory and prevents the movement
of refrigerated storage containers from their place
in order to preserve the embryos.
 Check the liquid nitrogen every day when
entering a laboratory and when leaving a
laboratory.
 temperature control of refrigerated storage
containers, temperature controller.
 Place the implant in a container room in order to
be alerted in cases of danger from nitrogen
spreading into the air.
 Reviewing protection plates in the refrigerated
storage containers room for embryos and sperm
from nitrogen liquid diffusion chambers and lack
of oxygen.
Guidance and risks of controlling the room
of refrigerated storage containers LN2

15. Cryopreservation Equipment IVF labs
Part of cryogeniccontainers
A- Durable tamper-prooflid design
B-High strengh neck tube reduces liquid nitrogen lass
C-Strong lightweight aluminum can struction
D-Advanced chernial vacuum retention system
E-Hydrophobic liquid Nitrogen absorbent system

16. IVF Containers Liquid nitrogen
Container Single Embryos Container Embryos
Container Biopsy Testicular
Container Sperms

17. Cryogenic LN2 storage Containers
Operating a Liquid Withdrawal Device from Worthington Industries Video viewer

18. Labotect Cryo Unit
From Labotect
Programmable device for cryo preservation
consisting of:
(1) Cryo Controller
(2) Actuator
(3) Dewar vessel containing LN2
(4) Handling box
(5) Welding apparatus to close the straws
Open-vessel system
* Self-seeding
* Temperature measured in reference straw
* Economical consumption of liquid nitrogen
* Autocalibration of temperature sensor
* Documentation of freezing process via PC

19. Container Biopsy Testicular and SPERM BANK
Container Biopsy Testicular and
SPERM BANK Background
Testicular biopsies and ejaculated
spermatozoa are routinely
cryopreserved in many units but the
fate of these samples has
not provoked large interest. This
prompted us to review our data
accumulated during a period of 20
years (1997 to 2016).

20. Precautions during handling
Portable trolleys must be used for
moving large containers of cryogens.

21.  Container must be rated for liquid
nitrogen
 Dewar's must have handles or
wheels
 Person filling Dewar must be in
constant attendance during filling
(Never leave unattended)
 To prevent splashing, place filling
hose at or below mouth of the
receiving Dewar
Dispensing Liquid Nitrogen from Storage Tanks

22. The best way to use ofcryogeniccontainers
Use manual rice
Use electric rice
SAFETY PERSONAL

23. LIQUIDNITROGEN TRANSFER

24. Temperature Liquid
Nitrogen

25. LN2 Temperature
25
 Optimizing existing equipmen
 Reviewing current freezers and operating methods to
improve proper freezers and settings
 Storage vessels, protective devices, sensors in LN2
 Complete solutions:
 dewar vessels, , Nitrogen level and temperature meters
 Storage of cryogenic liquids
 is a critical issue in many cryogenic applications.
Subcooling of the liquid by bubbling a gas has been suggested to extend
the storage period by reducing the boil-off loss. Liquid evaporation into
the gas may cause liquid subcooling by extracting the latent heat of
vaporization from the liquid. The present study aims

26. Variation of Temperature LN2
26
Figure 3. Variation of LOX temperature at gas flow rates of 15, 20
and 25 g/s and gas injection temperature of 91 K.
West Bengal,
India CEC 2017

27. Control Company Liquid Nitrogen Traceable LIVE
Datalogger Thermometer
27
Condition: New product
WiFi Datalogging LN2 Monitoring Thermometer with
Remote Notification.
Wireless Digital Datalogger Thermometer connects
easily to wifi and provides continuous data
transmission to the cloud-based interface.
Specifications:
•Temperature Range:-200˚C to 105˚C
•Temperature Resolution: 0.01°C
•Temperature Accuracy:±0.25°C
•Cable Length: 10 feet
•Calibration Points:-196°C, -80°C, 0°C and 100°C

28. Cryopreservation Of
Embryo - Blastocyst

29.  Storage of gametes and embryos cryopreservation
• Gamete is sex cell containing the genetic material
necessary for reproduction
• Embryo is the early form of life in the uterus between the
stages of blastocyst and fetus
• cryopreservation is a process to store gametes or embryos
by freezing them at low temperatures
Positives and Negatives of cryopreservation in embryos

30. Major actor involved in the process of cryopreservation
LIQUID WATER: Major component of the cell
Cryopreservation is a process that preserves organelles, cells,
tissues, or any other biological constructs by cooling the
samples to very low temperatures. The responses of living
cells to ice formation are of theoretical interest and practical
relevance. Stem cells and other viable tissues, which have
great potential for use in basic research as well as for many
medical applications, cannot be stored with simple cooling or
freezing for a long time because ice crystal formation,
osmotic shock, and membrane damage during freezing and
thawing will cause cell death.

31. Problems of Embryo cryopreservation
Not all sperm and embryos can survive the freezing thawing
process
 Pregnancy rate using frozen sperm or embryo may have
lower chance compared to using fresh sperm or embryo
 No apparent birth defects from using frozen-thawed sperm
but it is not sure whether problems will not appear in the
future
 Chance of possible infection with a bacteria or virus

32.  Sperm cryopreservation embryo cryopreservation
 Collect semen from self-masturbation
 More portions of semen can be cryopreserved-this so called
test thaws will be warmed up to verify success
 Retrieve egg under ultrasound Guidance
 Embryo can be frozen at stage
 Both sperm and embryo are stored in liquid nitrogen around -
19 c
Pros and Negs of cryopreservation in embryos

33.  Embryo cryopreservation or embryo freezing is a method used to
Embryo preserve embryos, generally at embryogenesis stage by cooling
and storing them at low temperatures.
 Started in 1984.
 It is one of the most common and well established fertility preservation
treatments, with proven successful pregnancy rates.
 In addition, the duration of storage had no significant effect on clinical
pregnancy, miscarriage, implant, or live birth rate.
Cryopreservation Of Embryo

34.  It is commonly known as sperm banking .
 Sperm banking is a procedure to preserve sperm cells for
future use.
 The first successful cryopreservation of spermatozoa was
initiated over 50 years ago.
 For human sperm, the longest reported successful storage is
22 years.
 It is also useful for men suffering from azoosprmia (Lack of
motile sperm) & Gonadial cancer.
 These frozen sperms can be used in association with one of
the Assisted Reproductive Techniques ( ART) to induce
pregnancy.
Cryopreservation Of Sperm

35. T°
0°C
~-40°C
-137°C
20°C
Concentration of SALT
Heterogenous
Liquid H20
Thermal
hysteresis
-100°C
Solid Glass – vitreous- amorphe H20
Solid crystalline H20
Tm melting T°
Tg glass transition T°
Avoid intracellular ice crystal formation
the key of success
Cryopreservation
of Cells
Cryopreservation
protocols begin with
hypothermic
exposures which
persist through the
period of active
extracellular ice
growth until
equilibrium is
reached in the
glassy-state
(vitrified).

36. T°
0°C
~-40°C
-137°C
20°C
Concentration of SALT
Heterogenous
Liquid H20
Thermal
hysteresis
-100°C
Solid Glass – vitreous- amorphe H20
Solid crystalline H20
Tm melting T°
Tg glass transition T°
Avoid intracellular ice crystal formation
the key of success
The relevance of ice crystal formation
for the cryopreservation of tissues and
organs

37. Vitrification: Definition
T°T°
0°C
~-40°C
-120°C
20°C
Concentration of SALT
Heterogenous
Thermal
hysteresis
-100°C
Solid crystalline H20
Tm melting T°
Tg glass transition T°
Liquid H20
Solid Glass – vitreous- amorphe H20
SupercooledGlass
CPH2O L
vitrification consists of the solidification of a solution by extrême
increase in the viscosity of this supercooled solution Cryopreservation
protocols
begin with
hypothermic
exposures which
persist through
the period of
active
extracellular ice
growth until
equilibrium is
reached in the
glassy-state
(vitrified).

38. Freezing
Vitrification

39. Successfull vitrification?
The balance required in vitrification is between
(i) establishment of a safe system for maximal , reliable and
optimal cooling and warming rates while avoiding consequent
damage of the cells, and
(ii) to find, the minimal and optimal conditions of exposure
to high cryoprotectant concentrations (time and T°) needed to obtain
and maintain an intracellular non-toxic vitrified environment.
xCooling -
Rate
CPs
Concentration
Solution
Volume
T O X I C I T Y !!!!!!!
The lower the cooling rate, the higher the required
cryoprotectant concentration is, and vice versa.

40. 2 approaches to extend embryo culture
and vitrification of YBL and DCA
24 h
24 h
Day 5
Day 5 Day 6
3 h
Day 5 Artificial
Endometrium
Vitrification and warming The vitrification and warming procedures were done according
to standard protocols of vitrification and warming kits
(Kitazato, Japan).

41. • The procedure of Embryo freezing is done along with fertility treatment in an
IVF laboratory. It allows people to store gametes, reproductive tissue and
embryos for later use.
• many people have frozen embryos and used them later. If your doctor has given
option for Embryo freezing you must know the risks and advantages.
• they may wish to use them at once to become pregnant. Fertilisation of eggs
with sperm can be achieved either with in vitro fertilization (IVF) or
intracytoplasmic sperm injection (ICSI).
• Often with in vitro fertilisation (IVF) or Intracytoplasmic sperm injection (ICSI)
treatment, there may be good quality embryos left over after embryo transfer.
Embryo freezing
41

42. What are the risks associated with Embryo freezing?
42
 Risk Of Contamination
 Since viruses and bacteria can also survive at
cryogenic temperatures it needs special attention.
 The risk of cross-contamination, i.e., transfer of
bacteria or other microorganisms from liquid
nitrogen to stored samples, is should be taken care.
 Risk Of Reduced Or Lost Viability In Embryo
Freezing
 In Embryo freezing Long-term storage does not
impact viability/developmental potential of slow-
frozen embryos. Cryostorage appears to be safe for
slow-frozen oocytes as well.
 Risk Of Specimen Loss In Frozen Embryo
 In contrast to the risk of infection or time-related
decrease in viability, under current practices the
risk of loss of cryopreserved gametes and embryos
due to human error or equipment failure is
relatively high.
 It is the responsibility of the IVF laboratory to take
proper care of the procedure in vital procedures
like cryo-freezing.
 Risks Inherent In Shipment And Handling
 At times, frozen eggs or embryos need
transportation for further use according to patient
convenience. The shipping procedure carries risk of
vessels being exposed to elevated ambient
temperature and air pressure, vibration/other
physical shock, and horizontal storage.
 Embryo freezing is a type of fertility preservation.
 Embryo freezing may be useful for women with
cancer who want to have children after having
radiation therapy, chemotherapy, or certain types
of surgery, which can cause infertility. Also, called
embryo banking and embryo cryopreservation it is
widely used in IVF treatment

43. Vitrification: definition
Liquid Water Ice crystals Vitrified water
Vitrification consists of the solidification of a solution by
extreme increase in viscosity of this solution in
supercooling during cooling
Liquid water
contains a mix of short, straight
strong hydrogen bonds and
long, weak, bent hydrogen
bonds with many intermediate
forms between these extremes.

44. Water:
Major component of our cells
44
Freezing: crystal formation,
cell damage,
volume increase +++
Need to master +++
Luyet 1937

45. Vitrification: Steps
45
1- Exposure to non-vitrifying and vitrifying solutions of Cryo-Protectors
(minimum of 2 steps)
2- Dives in LN2 after loading the biological material on
an “open” support (CR:> 20,000 ° C / min) or an “closed” support (CR: ~ 1,500
° C / min)
3- Storage in LN2 or steam
4- Rapid heating (> 20,000 ° C / min)
5- Dilution of the CP by stages and embryo transfer

46. Vitrification: Basic principle
46
BeforeplungingthebiologicalmaterialinLN2:
cellsareexposedtodifferent hypertonicsolutionsofCP
NON vitrifying
Solutions (nVS1,2,3)
2.4 - 3.2 M
2700 - 3500 mosm
EG – DMSO
EG – PROH
EG – Glycerol
PROH – Glycerol
EG
First Step

47. Vitrification: Basic principle
47
NON vitrifying
Solutions (nVS1,2,3)
2.4 - 3.2 M
2700 - 3500 mosm
EG – DMSO
EG – PROH
EG – Glycerol
PROH – Glycerol
EG
First Step
solution hypertonique non vitrifiante
Objectives of this stage
 Prepare the intracellular
compartment for the optimal
concentration of cryoprotector
 Dehydration and permeation of
CP by osmotic equilibrium

48. EG – DMSO
EG – PROH
EG – Glycerol Sucrose
trehalose
PROH – Glycerol
EG
VITRIFYING
Solution (VS)
4.8 – 6.4 M
5600 - 7300 mosm
Final Step
Vitrification: Basic principle

49. VITRIFYING
Solution (VS)
4.8 – 6.4 M
5600 - 7300 mosm
Final Step
Vitrification: Basic principle
 Create a vitrifying intra and
extracellular environment
 Controlled dehydration and
concentration of the penetrating
cryo-protector during the first stage
Vitrifying solution
time
volume
EG – DMSO
EG – PROH
EG – Glycerol Sucrose
trehalose
PROH – Glycerol
EG

50. VITRIFIED
Slow freezing
1.6 M
1900 mosm
NON vitrifying
Solution (nVS)
2.4 - 3.2 M
2700 - 3500 mosm
VITRIFYING
Solution (VS)
4.8 – 6.4 M
5600 - 7300 mosm
Cryoprotectantconcentrations
worries and skepticisms

51. Vitrification
Vitrification Oocyte
Vitrification Embryos
Vitrification Biopys
Vitrification Sperm

52. Vitrification of Oocytes
52
Preservation of fertility
before medical treatment, for example before chemotherapy
IVF cycles
ethical or legal reasons
Oocyte donation cycles: oocyte banks
for societal reasons:
No partner,
Freezing of eggs to prevent age-related decline in fertility
risk of OHSS, endometrial problem
Avoid embryo storage
low-responders - (cryo-accumulation)
 Inability to receive sperm on time or a few sperm ++

53. Disadvantage of « open » carrier
• Possible risks of contamination and harm by
toxic compounds in LN2
Direct contact
with LN2
Drawback
CONTAMINATION

54. Are there data showing :
- direct contamination with LN2
- cross contamination during storage
- Toxic compounds present in LN2
NO contamination at all NO
YES
Bielanski, 2000, 2006,
2009
Charles 1971
Jones 1989
Schafer 1976
Tedder 1995
Morris 2005
Bielanski 2003
Piasecka 1972
THE RISK IS NOT ZERO
Precautionary Principle
The potentiality of contamination is not totally proven in the human application.
CONTAMINATION

55. To vitrify low quality embryos (morphology – delayed development)
YES
To vitrify expanded blastocysts without collapsing: YES
To vitrify in large volume: YES
In reduce the cooling rate: YES
To warm in 4°C Sucrose solutions: YES
Conclusions Is it possible :

56. Vitrification in media containing no HSA in the vitrification and warming solution ?
Storage for several days at –80°C before plunging in LN2 ?
Vitrification in presence of extracellular ice: YES
To vitrify in extremely low or even in absence of intracellular CP: YES NEW
CHALLENGE
THE FUTURE

57. Technical
1.Stimulation of the ovaries to promote
the growth of several follicles
2. Vaginal oocyte retrieval
OVARIAN RESPONSE
MONITORING
Blood test and ultrasound
3. Search for oocytes

58. Vitrification
Déshydratation
H2O
High electrolyte / CP concentration
Solution effect

59. Cryoloop (CL)
1999; Mukaida 2001, 2003; Liebermann &
ker, 2002; Reed et al., 2002;
Tucker, 2003)
Liebermann
EM grid
(Martino 1996,; Park 2000; Chung
2000; Wu 2001; Son 2003; Yoon
2003)
Cooling rate> 20,000 ° Cmin
Heating rate> 20,000 ° Cmin
(HS)Hemi-straw systemCryotop
(Vanderzwalmen 2000, 2003; Liebermann & Tuc
2002; Sugioka 2003, Zech 2005)
(Kuwayama & Kato, 2000, ; Liebermann &
Tucker, 2003 Al Hasani 2006)
Vitrification supports OPEN
Contact of biological material with LN2

60. Droplets of VS with embryos/oocytes < 1 µl)
Vitrification support

61.  380 embryo transfer cycles at the vitrified / warmed cleavage stage were included.
 S-R after vitrification and warming for the second day embryos and the third day embryos were 92.7% and 92.8%
respectively.
 cPR for heated embryos vitrified on the third day was significantly higher than that of heated embryos on the second
day (17.6 vs 4.0% per transfer cycle, P = 0.036
 cryopreservation of the entire cohort of embryos on day 3 gives better clinical results than cryopreservation on
day 2.
2014
Day 3 > Day 2

62. Results after transfers to D3 or D5 of vitrified
embryos
D3 good
qualities
Blastocysts from D3 of poor
quality
Systematic extended culture
supernumerary embryos
day3 of poor quality

63. Vitrification protocols :
Only if we use high concentration of CP
And very high cooling rates
We have to expose the embryos to the CP in
precise conditions of T° and time.
Otherwise !!!
Vanderzwalmen, Barcdelone , 2005
Conclusions

64. Blastocyst
Day 5 – Day 6 – Day 7
Egg Freezing

66. 2017
 Vitrified blastocyst transfer was significantly superior to vitrified cleavage-stage
embryo transfer regarding the implantation rate.

67. Early blastocyst: 1-2
blastocoel <half the volume of the embryo
Full blastocyst: 3-4
blastocoel Completely fills the embryo
Expanded blastocyst: 5
blastocoel volume is now larger than that of the
early embryo and the shingles is thinning
Hatching 6
Hatched
blastocyst

68. ICM Grading: well defined
A) Tightly packed, many cells
B) Loosely grouped, several cells
C) Very few cells
Size and shape of ICM
Trophectoderm Grading:
A) Many cells forming a cohesive epithelium
B) Few cells forming a loose epithelium
Scoring System for Human Blastocyst
Gardner 1999

69. Blastocyst score
Prediction of pregnancy rate by blastocyst
morphological score and age, based on
1,488 single frozen-thawed blastocyst
transfer cycles
Sakae Goto et al. Hum Reprod 2011
Significant correlation between blastocyst quality (according to the
Gardner Schoolcraft score) and pregnancy rate/ birth rate in addition
to the influence of age

70. 2017
 Vitrified blastocyst transfer was significantly superior to vitrified cleavage-stage embryo
transfer regarding the implantation rate.
 This meta-analysis shows that vitrification at any stage has no detrimental effect on clinical
outcome. Blastocyst transfer will still remain a favorable and promising option in ART.
Vitrified blastocyst transfer

71. ter 3- 24
Before vitrification
ET 128 ET 29
After Thawing
Survie 92 % Survie 96 %
Af hours
Ong Preg 38.4 % Ong Preg 26.6 %
Vitrification of Hatching and Hatched Blastocyst
Vanderzwalmen 2013

72. Factors influencing live birth outcomes of day 7
blastocysts following autologous single vitrified-
warmed blastocyst transfert:
A single-centre large cohort study
Vienna
June 2019Day 7

73. Results in terms of live births of blastocysts on day 7 after SVBT influenced by the
age of the mother, the time elapsed between insemination and blastulation,
the time of expansion of the blastocyst and the diameter of the blastocyst.
Rates of live births after Day 7-VBT are comparable to those after
J6-SVBT if the Day 7 blastocysts meet strict criteria.
Therefore, vitrified Day 7 blastocysts have potential clinical utility for FBT.
Results in terms

74. Oocytes or embryos are in
Direct contact with LN2
During Cooling and / or Storage
ULTRA-FAST GLAZING
LN2 can be a source of contamination
presence of bacteria, virus, fungus (sterile LN2)
by poor quality of LN2
by an accumulation of toxic substances in LN2
(heavy metals - Ozone)
(Tedder 1995, Fountain 1997, Bielanski 2000, Fountain 1997, Bielanski 2003)
European
directives
!!!!!!!

75. 2017
no statistically significant differences in survival rates, implantation, clinical
pregnancy rates ,and live birth rates between closed and open vitrification.

76. • A child if I want A child WHEN I want
 Woman without partner
 Woman or spouse with no short and medium term child project
 Woman wishing to privilege professional or couple life
 Woman looking for financial stability
 Often 35-38 year old woman with an intense professional life or having no
partner
1968: liberation from manners
Social indications: social freezing

77. Embryos with developmental delays on D5
What strategy?
82 cycles de vit (37.2 an) survie 92.3% 34 gross évol: 41.4%
Culture D 6
Vitrification J6
if blasto
Compact
Morulae
8 cell.
D5
Culture D 6
Transfer
blasto
if
Vitrification D 5
37 cycles de vit (36.4 an) survie 86.4% (J5) 74.2 % (J6) 10 gross évol: 27.0%

78. Conclusions 1
Vitrification of oocytes in case:
 of unexpected semen production or inability to produce the sample
according to a normal schedule post hCG is a valuable strategy.
 severe oligozoospermia is also an approach that permits to rescue oocytes.
 Of course such strategy implies the vitrification of zygotes
(orembryos) post fertilization of the warmed oocytes.
 Three cycles of vitrification (oocyte – zygote – blastocyst) seems to have no
adverse effect (but only one case)
 Kryo-accumulation of oocytes in case of poor responder may
be a good option. However a disparity in our results in term of embryo
development post warming is noticed, reflecting probably the intrinsic
problem linked with the oocyte quality.

79. VANDERZWALMEN P, ZECH NH, WIRLEITNER B
Blastocyst transfer after aseptic vitrification of zygotes: an approach to
overcome an impaired uterine environment.
Reprod Biomed Online. 2012
 We can expect that in the near future the vitrification cycles will increase at
the expense of fresh transfers in order to replace the embryos in a more
physiological uterine environment.
Future of vitrification
 Fresh transfer of second quality embryos and vitrification
of the best is a strategy not to be overlooked.

80. Conclusions 2
Similar rates of fresh
and thawed embryos
Great flexibility in the
management of
transfers and HSOs
Prevention of oocyte
aging in sperm
emission difficulties
Ensure compliance
with technical details
to prevent the toxicity
of cryo-protectors
Valuable contribution
to ovarian
insufficiency by
collecting eggs
Proven effectiveness
at all stages of
embryonic
development ++

81. Conclusions 3
Several very significant advances have recently been made in ultra rapid
cooling of human embryos, and as a result we may now be at the point
where a transition from slow to rapid cooling procedures will start to take
place also for the human
This inevitable that both freezing and vitrification methods will improve as a
result of directed research, and it is possible that vitrification will prove to be
a more favourable technique for the cryopreservation of cold-sensitive
oocytes and the more complex cellular system of the blastocyst.
The requirement for direct contact between the
cryo-medium and liquid nitrogen for successful vitrification introduces a
potential difficulty associated with the packaging for storage after
vitrification,

82. Conclusions 4
 To date, most results after cryopreservation of biopsied embryos are disappointing. However,
biopsied embryos surviving the freezing and thawing procedure can implant and develop to term.
 In contradiction to cryopreservation results after ICSI, the creation of a relatively large opening
in the ZP of human embryos has a significant impact on cryopreservation outcome.
In order to improve cryosurvival, further efforts have to be made. Several possibilities may be investigated:
- Evaluation of the possible protective role of macromolecules such as globulins as well as other non-physiological
macromolecules (Ficoll, polyvinyl alcohol…)
- Evaluate the use of alternative procedures to close the opening in the zona
- Limit swelling of blastomeres during thawing by modifying the cryoprotectant solution
- Reduce the impact of ice crystal formation by using ultra rapid cryopreservation procedures or vitrification.

83. Thank you for your
attention!
Dangers of Control From the
Cryogenic Liquid Nitrogen Storage
Container and Cryopreservation Of
Embryo and Sperm
Dr . Mustafa ZAKARIA
Deputy Executive Director and
Administrative Coordinator of the
Scientific Research Group
R,B andART, consultant
Reproductive Biology in the IVF
laboratory, fertility center IRIFIV
Casablanca ,Morocco
dr.zakaria@irifiv-aisrg.com

84. thank you for listening
to my presentation
Questio

85. 85
Titre de section 2
Sous-titre
85

86. Thank you for your
attention!
Dangers of Control From the
Cryogenic Liquid Nitrogen Storage
Container and Cryopreservation Of
Embryo and Sperm
Dr . Mustafa ZAKARIA
Deputy Executive Director and
Administrative Coordinator of the
Scientific Research Group
R,B andART, consultant
Reproductive Biology in the IVF
laboratory, fertility center IRIFIV
Casablanca ,Morocco
dr.zakaria@irifiv-aisrg.com