Culture Media Flashcards
What conditions should culture media be evaluated for before use?
- Osmolality
- pH under working conditions
- endotoxin contamination
- biocompatibility
What are 3 main buffering systems used in IVF culture media?
- Bicarbonate buffers
- non-bicarbonate buffers
- zwitterion buffering
What is the pHi of embryos?
Between 7.1 and 7.3
What is the pH of the fallopian tube?
7.3
What are non-bicarbonate buffers?
- Designed for use with air
- phosphate with pH .7.2-7.43 and osmolality around 300mM was used in early days of IVF but less common now
- Should be used with calcium and magnesium salts and glucose
What are 2 zwitterion buffering systems?
- HEPES has working pH range of 6.8-8.2 and pKa 7.2-7.5
primarily used at mM from 20-25mM replacing much or equally molar concentrations of bicarbonate
can be used for sperm processing for IUI** because capacitation will proceed in vivo and lab can work without need for CO2 incubators - MOPS has pKa 7.15. used in sperm and oocyte handling media
Why must humidity be carefully controlled in the incubator?
- Prevent evaporation of water from medium
- incubators regulate CO2 injection with thermal sensors that depend on saturated humidity
What is the concentration of albumin in human plasma?
4mg/ml
conditions of culture attempt to mimic this
How much albumin is added to culture medium?
3-5mg/ml and sometimes 10mg/ml
What is the purpose of albumin in media?
- Undefined “vitality” promoting properties
- detoxifies culture medium
- Mask electrical charges on outer surface of cells and bottom of culture dish. Without protein or macromolecule like polyvinyl alcohol gametes and embryos stick
- secondary source of amino acids
What are the 4 types of proteins used in culture medium?
- Serum albumin commonly added between 3-5 up to 10 mg/ml. majority of commercial human protein supplements supplied in liquid dissolved in saline
- prepared homologous serum, follicular fluid, donor serum and fetal cord serum. routinely added in early days of human IVF before commercially prepared serum supplements became available. Must be prepared by centrifugation, filtration, heat inactivation and quarantined
- albumin and macroglobulin mixtures
- Recombinant human albumin.
note dilution effect with liquid protein supplements when added at higher percentage
protein free culture medium
capacitation involves removal of cholesterol from the membrane and albumin promotes cholesterol efflux via capacity to absorb steroid molecules
What are the 4 types of proteins used in culture medium?
- Serum albumin commonly added between 3-5 up to 10 mg/ml. majority of commercial human protein supplements supplied in liquid dissolved in saline
- prepared homologous serum, follicular fluid, donor serum and fetal cord serum. routinely added in early days of human IVF before commercially prepared serum supplements became available. Must be prepared by centrifugation, filtration, heat inactivation and quarantined
- albumin and macroglobulin mixtures
- Recombinant human albumin.
note dilution effect with liquid protein supplements when added at higher percentage
protein free culture medium
capacitation involves removal of cholesterol from the membrane and albumin promotes cholesterol efflux via capacity to absorb steroid molecules
Describe the benefits of mineral oil overlay?
- physical barrier
- dissolved gas sink
- prevent dehydration
4.
what is the best way to sterilize mineral oil?
membrane filtration. NOT heated or autoclaved in any fashion
Why is the function of heparin when added to oocyte retrieval media?
10,000 units per liter to decrease post-collection clotting of follicular fluid and blood
What is a damaging byproduct in culture medium taht is incubated for more than 48 to 72 hours?
Ammonia from breakdown of proteins/amino acids
glutamine is rapidly broek down
What are concerns with using phenol red?
mild estrogenic activity
What are the main antibiotics used in culture medium?
penicillin and streptomycin in the past.
allergy risk with penicillin and toxic effects of streptomycin
gentamicin is now used
some reports suggest that embryo development is inhibited to small dgree by presence of antibiotics
How many hours after administration of hCG is oocyte aspiration performed?
34-36 hours
What is the pressure used during oocyte aspiration?
100mmHg negative pressure
aspiration pressure should be maintained in narrow range of 100-105
must be applied at a constant level
What is the pH of culture media during retrieval?
pH 7.4
When does ovulation typically occur post-hCG
36 hours
How soon do sperm penetrate the zona in vitro?
as early as 3-4 hours post-insemination
sperm are penetrating the hyaluronate to the zona pellucida very soon after introduced
What is the range of concentration for sperm in conventional IVF?
50,000 sperm/oocyte/mL to 150,000 sperm/oocyte/mL
Why should sperm be washed from semen within one hour of collection?
- SPerm will die rapidly after 1-2 hours of exposure to seminal plasma
- after more than 30 minutes exposure the fertilizing capacity of sperm is reduced
- sperm placed in nutritive medium may live for over 72 hours
- semen processing removes seminal components such as decapacitation factors, hormones and pathogens
What are the benefits of sperm isolation from semen
- removing seminal fluid factors: ROS, inhibitors of capacitation, prostaglandins, anti-sperm antibodies, bacteria and viruses, other unknown inhibitors
- Addition of sperm culture medium: nutrition, pH buffers, electrolyte balance for extended culture, allows adjustment of sperm concentrations for insemination
List methods for separation of motile sperm from ejaculated semen, retrograde urine, cryopreserved semen, epididymal fluids
density gradient separation
pelleting
swim up
swim down
swim out
filtration columns (glass wool)
sedimentation/migration
transmembrane migration
What are some damaging consequences of centrifugation of semen samples?
Concentration of ROS (superoxide anions, hydroxyl radicals, hydrogen peroxide breakdown products) in sample that can damange sperm membrane
Not all sperm cells respond or tolerate centrifugal processing
What is the most common gradient material for sperm processing?
silane-coated silica
Do all sperm preparation techniques damage sperm?
All preparation techniques increase DNA fragmentation rate above that of raw semen samples
authors reported that pelleting followed by swim up or gradient separation followed by swim up had lowest levels of processing induced DNA fragmentation
What is the source of ROS in semen?
Leukocytes, less dense spermatozoa with excess cytoplasm
What is the typical centrifugation for gradient separation?
20 minutes at 300xg
How is capacitation effected by cryopreservation?
Cryopreservation alters the heterogeneous nature of the ejaculate
alters sperm membranes
causes earlier capacitation
cryopreserved semen sample can be thawed and incubated for shorter time before placing with oocytes
How should retrograde patients be prepared for ART?
Need to neutralize or alkalinize the bladder
take oral bicarbonate the night before and morning of procedure
acidic and hyperosmotic urine is detrimental to sperm
reduced motility is often observed
What is the order of importance for pre-wash semen parameters in IUI?
- Forward progressive motility
- concentration
- total motile count
- sperm morphology
What post-wash factors are correlated with succesfull IUI
- Motility and concentration (TMC)
- threshold minimum number needed for pregnancy is 1-2 million motile sperm
- too many sperm doesnt appear to be a concern
Where does capacitation occur in vivo?
Begins with paassage through cervical mucus and uterine environment
movement of sperm into female reproductive tract aids in removal of seminal decapactiation factors
exposes sperm to milieu of periovulatory duct that may include ovulated COC
What are the major events associated with capacitation?
- removal of surface proteins
- efflux of unesterified cholesterol from the sperm cell membrane (can be mediated by albumin)
- alteration of sperm proteins (tyrosine phosphorylation)
- modifies the acrosome membrane phospholipids that lead to membrane fenestration and increased ion channel availability
- increase in speed of sperms nonlineaer progression - hyperactivation
availability of extracellular ions, sodium, calcium, bicarbonate is essential for capacitation and subsequent AR
Timing of capacitation is sensitve to temperature, energy metabolism and pH
When does capacitation occur in vitro?
Spontaneously
initiated by washing the sperm to remove seminal proteins and decapacitation factors
incubation in bicarbonate buffered medium with albumin/serum proteins for several hours
What are capacitation enhancers?
promote likelihood that sperm undergo AR
include egg yolk buffers, pentoxyfylline and methylxanthine derivatives, homologous folliuclar fluid
they enhance motility
What is the frequency of azoospermia?
5-10% of men evaluated for infertility
What are the potential causes of azoospermia?
- Retrograde ejaculation
- obstructive azoospermia
- non-obstructive azoospermia (NOA) - result of impaired or non-existant sperm production
surgical recovery can be as low as 50% per patient
Are capacitation and AR required for successful fertilization by ICSI?
No. Acrosome is shed within the oocyte cytoplasm
What is TESE?
Testicular sperm extraction- seminiferous tubules are excised
What is TESA?
Testicular sperm aspiration - tissue removed by suction using wide-bore needle
can be perfomred as office procedure under local anesthesia
blind procedure- comes with greater risk of injury to the epididymis or testicular artery and failure to obtain a useful specimen
What types of movement do testicular sperm demonstrate after testicular extraction?
no motility, occasional tail movement twitching or vibrate
Why must ICSI be used from TESA/TESE?
The sperm are inherently immmature, limited mobility
incabable of achieving capacitation and penetration of the zona pellucida
What is ROSI? and when is it used?
round spermatid injection
round spermatids are haploid male germ cells
difficult to identify
nucleoli in nucleus
ASRM considers ROSI to be experimental
activation of oocyte rarely occurs after ROSI
artificial oocyte activation is required for fertilization
In what patients can Epididymal aspiration be used?
obstructive azospermia
testicular is needed for non-obstructive azoospermia
What is PESA?
Percutaneous aspiration of epididymal sperm through scrotum
done blindly
What is MESA?
Aspiration of epididymal sperm from single surgically exposed tubule of epididymis
Repeat at different site if no sperm are found
only requires a few microliters of epididymal fluid
Yield lower numbers of sperm relative to ejaculated semen
motility may be vigours
How can red blood cells be eliminated from surgically retrieved samples?
Hypotonic lysis buffer can be added then incubate for 5-10 minutes
solution changes from cloudy red to clear red
sample is mixed with culture medium and centrifuged to concetrate sperm
List sperm motility enhancers
- pentoxifylline - methylxanthine compount taht promotes motility via inhibition of phosphodiesterase eliciting rise in intracellular cAMP. 3-5mM up to 30 minutes exposure before dilution
- caffeine
- 2-deoxyadenosine
- homologous follicular fluid- can be collected at time of oocyte retrieval. centrifuged and filtered, heat inactivated. added at room temperature or 37 to washed sperm preparation incubated for 15 minutes. transient motility enhancement. should be used soon after for conventional insemination
What are main cellualr events that culminate in pronuclear development within oocyte cytoplasm?
- sperm penetration of the oolemma
- oocyte activation (calcium mobilization, alteration of oolemma eletrical potential, completion of metaphase II, signal transduction and exocytosis)
- sperm head detachment, decondensation and remodeling of sperm cell DNA and packing proteins, sperm centriole deposition, oocyte chromosomal mobilization and decondensation, pronuclear membrane coalescence, cytoskeletal alterations, microtubule formation to drive pronuclear migration
- Sperm cell DNA decondensation and remodeling begins within hours of sperm penetrating the oolemma
How many hours after insemination should fertilization check be performed?
14-18 hours
PN usually close in direct opposition not yet having initiated or udnergone syngamy
What does syngamy involve in human zygotes?
NOT fusion of pronuclear membranes
dissolution of closely opposed PN membranes and progression of maternal and paternal chromosomes into DNA replication for cell division
Are the human pronuclei identical?
male is slightly larger than female PN
PN morphology/ polar body orientation has been used to predict embryonic development
List morphological features that should be carefully evaluated at fertilization check time
Vacuoles - can mimic PN
PB1 may divide and may appear incorrectly as first and second PB
PB1 is usually larger than PB2
Sometimes small nculear membrane can be observed in PB1. First PB carries two haploid sets of maternal chromosomes.
Polar bodies may fragment or degenerate
Cytoplasmic halo
debris in PV space
refractile bodies in cytoplasm
What is the cytoplasmic halo?
Peripheral cytoplasmic translucency after fertilization. cytoplasm may retract following migration of cellular organelles towards the pronuclei, resulting in formation of a clear zone at the periphery of the zygote- can be large, small or nonexistant
What is an explanation for one PN and one PB at fertilization check?
PB2 retention
or parthenogenetic activation
What is an explanation for one PN and 2 PBs?
oocyte penetrated by sperm cell causing oocyte activation
but sperm nuclear condensation does not occur, is incomplete or delayed
single nucleus may be diploid
What are two methods incubators use to measure CO2 levels?
- Thermal conductivity - incubator must be fully humidified for accurate measurements
- Infrared sensors- allows CO2 to recover quickly without waiting for humidity to rise in the chamber
What is the relationship between altitude and CO2 concentration and pH of the medium?
Molar CO2 concentrations will vary based on atmospheric pressure and therefore altitude above sea level. The partial pressure of CO2 wil drop as we rise above sea level. Concentration of CO2 in the air will remain the same at 0.04% but air pressure falls.
if incubator is set to 6% CO2 at sea level and moved to higher altitude the display reading for CO2 will not change. However, amount of air in a given space will be less. Density of air will be less. 6% CO2 relative to other gases will remain the same but amount of CO2 will drop as air is thinner
How should the CO2 be adjusted for incubators at higher altitudes?
From sea level to 150m increase CO2 by 0.1%
For large altitude change at 3000m (10,000 ft) at 6% CO2 the effective molar equivlant is 4.1% CO2
Why is measuring pH of the culture medium superior to CO2?
pH is not directly affected by atmospheric pressure.
How is pH affected by higher elevations?
pH will increase at higher elevations
we need to increase CO2 percentage of the incubator to compensate
How does CO2 influence pH?
CO2 dissolves in culture medium to generate carbonic acid, then liberating one or two protons in successive reactions generating bicarbonate and then carbonate
CO2 + H2O = H2CO3
H2CO3 = H+ + HCO3-
HCO3- = H+ + CO32-
DECREASING media pH
equilibration is stable under constant conditions
How does temperature effect solubility of CO2?
Decrease in temperature will increase solubility of CO2. CO2 solubility is better at lower temperatures
For example, cold equilibrated medium placed directly from refrigerator into incubator at 37 and small bubbles of CO2 form on surface of medium
What is the henderson-hasselbach equation?
pH = pKa + Log10 ([A-]/[HA])
used to calculate the pH of a buffered solution. helps maintain pH under working conditions
Buffer solution contains an acid and a salt of the conjugate base of the acid
acid dissociation constant Ka
acid: HA
salt of the conjugate base of the acid: A-
What is the equation for pH?
pH = -log [H+]
0.0001 [H+] = pH 4
What is the pH of most cultured medium versus intracellular pH?
medium is 7.2-7.4
intracellular is 7.2
How much CO2 should be used in bicarbonate buffered medium to achieve desreable pH?
Gas phaes of at least 5.5% CO2 to achieve pH of 7.4 or below
increasingly media manufacturers are recomending incubators to be mainttained with >6% CO2 concentration to achieve the desired pH in their products
What is the relationship between temperature and pH?
lower temperatures will increase the pH
measurements should be taken at 37
pH of water at different temperatures- at 25 water is 7.0 At higher temperature (more energy) water molecules split into H+ and OH-. pH measurements will be lower because [H+] has increased.
How does serum affect the pH?
decrease the pH slightly
serum also helps stabilize the pH
How does the timing of pre-implantation embryonic development in vitro compare to in vivo?
Likely to be retarded in vitro like that of mouse.
Development of embryonic culture medium began in which two speices?
- mouse
- rabbit
What types of media did IVF pioneers use in the early 1980s?
Various balanced salt solutions modified to contain pyruvate, and 10-15% human serum
osmolarity adjusted to 280mOsmol/kg
pH 7.2
cultured 5% CO2 and 5% O2 and 90% N2
embryos often transferred to Hams F10 after fertilization before transfer
It was suggested that Earle’s medium could be used for entire process without effecting pregnancy rates. Just supplemented with bicarbonate, antibiotics, pyruvate and serum source.
Earle’s was commercially available
What was the first medium devised specifically for human embryo culture?
Quinn’s human tubal fluid (HTF)
similar to Earle’s and Whittingham’s but ionic composition based on that of human fallopian tube fluid
DID NOT demonstrate dramatic increase in success rates compard to Earle’s medium
Signified start of new era of media formulation for human embryonic culture
Widely accepted and used. basic and modified formulations still in use today
Based on premise that fairily simple medium can adequately support early embryo development has been established
most modern media evolved from HTF or based on fluid in the uterus or fallopian tube
What changed in the early 1990s with glucose composition in culture medium?
reports began to emerge suggesting presence of glucose was detrimental
deleteriuos effects in mouse, hasmter, bovine and sheep embryos
glucose not consumed in significant quantities in mouse and human embryos prior to 8 cell stage
However, glucose is actively used by embryos developing beyond the 8 cell stage and absolute requirement for blastocyst formation.
What is CZB medium?
first glucose-free medium devised for mouse embryos
human embryos later showed to benefit from media without glucose
What was observed with phopshate in culture medium in the early 1990s?
Phosphate had similar effects to glucose- detrimental.
Negative effects of gluocse and phosphate are likely related to composition of artificial media. Their negative effects are not seen in more complex media such as G1 (Vitrolife) containing EDTA amino acids and vitamins.
Glucose and phosphate-free media (Quinn’s Basal XI based on HTF and P1 from Irvine) were developed and widely used.
What change occured with media use in the mid 1990s?
Move away from in-house media production to commercial vendors.
Confidence in culture medium allowed embryos to be kept in culture for longer
performing transfers on Day 3 allowed better selection- reducing number transferred and decreasing unwanted high multiple pregnancy rates
lSequential media development. G1 and G2 or P1 and blastocyst medium. Alowes blastocyst transfers
What trend has occured over the last 10 years with culture medium?
Away from sequential medium to continuous culture with single medium
Media development history and developing embryo studies suggested that components in media should change over time.
Sequential media helped with this but required more manipulation of embryos, more resources.
What is a generally acceptable osmolarity and pH for culture media?
280-285 mOsm/kg
pH 7.2 or 7.3
What is simplex optimization and what was it used for?
Method to optimize the concentrations of several ingredients simultaneously. Culturing embryos in a number of different media each containing one of a group of common components at a high concentration. Ingredients found to be detrimental are lowered in concentration or removed completely.
High NaCl, pyruvate, KH2PO4 and glucose were detrimental
lead to development of KSOM for mouse embryo culture and human variant of Global IVF medium
What is the preferrred substrate in preimplantation human embryos?
Pyruvate
During early development glucose uptake is low. Following activation of embryonic genome at 4-8 cell stage in human, there is a surge in glucose uptake.
Removing glucose at specific times during culture enhances embryo development. However, glucose is present in fluid of fallopian tube
Inhibitory effects are likely induced by specific combinations of ingredients in media
Does complex medium yield superior growth of early human embryos?
No
Complex medium containing salts, amino acids, vitamins, energy substrates and other components have not dramatically improved early embryo culture when compared to use of balanced salt solutions supplemented with energy substrates, serum, antibiotics and pH buffer such as sodium bicarbonate
How does the concentration of nutrients in culture media compared with those present in vivo?
Nutrients supplied in media are mostly well above those present in vivo based on measurements of human plasma and follicular fluids
Excessive amounts of subtrates may be detrimental to embryo health, and erratic/abnormal patterns of development (eg effects of glucose and phosphate in some media formulations)
pyruvate is essential component but glucose appears to inhibit embryo development in simple media
Can human embryos develop in vitro without addition of serum or growth factors?
yes- develop for two days or more in simple chemically defined medium absence of serum and GFs without reduction in viability after transfer
What are thought to be the benefits of adding serum to culture medium?
due to cyclic adenosine monophosphate, vitamins, albumin and growth factors
may contribute additional energy substrates to medium also
What is the effect of adding growth factors to culture medium?
stimulates embryonic development
growth factors have not been measured in human fallopain tube fluid but evidence suggests that factors in the tube stimulate embryo development
What are the potential effects of insulin on development of embryos?
- stimulate protein synthesis in mouse
- double cleavage, compaction and blastocyst formation in mouse
- increased ICM cell numbers
in human? may have mitogenic or differentiative effects on human embryos
How can bioassay sensitivity be improved by adjusting serum amount and moust strain?
Protein-free medium
stripped-down and highly sensitive
more sensitive to toxins and good test for items embryos come in contact with
One-cell mouse embryos from outbred (wild type) mice further increases sensitivity of the assay
What are some additional additives for culture/handling medium?
- Chymotrypsin - breakdown viscous semen
- red blood cell lysis buffer - for testicular biopsies
- pentoxifylline/caffeine - stimulate motility
- digestive enzymes (collagenase, DNAse) - digesting testicular tissue
- Calcium ionophore/ionomycin - activating oocytes
- sodium citrate+fructose - HOS test of sperm viability (Hypoosmotic swelling)
- Growth factors - promoting embryo development but little research has been done on efficacy and concentrations in the reproductive tract are unknown
What is the preferred substrate for human embryos throughout preimplantation development?
Pyruvate
glucose consumption remains low until after 8 cell stage when there is a surge in uptake that cotinues to blastocyst stage
What is the proposed pattern of substrate utilization during early cleavage stages?
Pyruvate, lactate, amino acids and possibly fatty acids are utailized for AEROBIC RESPIRATION during early cleavage
Later glucose uptake, lactate production increase
May be in preparation for increasingly anoxic environment of uterus during implantation?
Glucose may be required for synthesis of macromolecules - ribose moieties for nucleic acids, glycerol phosphate for phospholipids, complex sugars for mucoproteins and mucopolysaccharides. Synthesis of these increase during blastocyst formation
Does pyruvate uptake change during preimplantation development for human embryos?
no unlike mouse
remains high at late preimplantation stages
How are the metabolite concentrations varied in reproductive tract based on location and cycle day?
all 3 metabolites (pyruvate, glucose and lactate) change from fallopian tube to uterus
cumulus cells also consume glucose and produce lactate
pyruvate in oviduct does not vary with cycle day. lactate and glucose varied with cycle day. lactate increased and glucose decreased.
Concentrations constant in uterine fluid throughout cycle
All metabolite concentrations in uterine fluid were significantly different from those in oviduct midcycle
What are the concentrations of pyruvate, glucose and lactate in the uterus and fallopian tube?
Uterus:
Pyruvate: 0.11 mM
Glucose: 3.15 mM
Lactate: 5.87 mM
Fallopian tube:
Pyrucate 0.24mM
Glucose: 3.11mM/follicular phase, 0.5mM/midcycle, 2.32mM/ luteal
Lactate: 4.87 mM/follicular, 10.5mM/ovulation
Do embryos require the addition of amino acids to medium?
In protein-supplemented medium embryos can develop to the blastocyst stage without exogenous amino acids
It is not common practice to supplement with amino acids
Only more recent and complex media such as G1/G2 (vitrolife) or blastocyst medium (Cook IVF and Irvine) include them
Inclusion of amino acids in human embryo culture media has been shown to enhance development to the blastocyst stage
most modern media used for blastocyst culture contain some combination of amino acids
What is the proposed purpose of the amino acids in culture medium?
amino acids are most likely to be utilized for synthesis of macromolecules, proteins and nucleic acids and oxidized to generate metabolic energy
also act as chelators, osmolytes, pHi buffers and antioxidants
What about the inclusion of glycogen in media?
While no evidence suggests that synthesis or metabolism of glycogen occurs in human preimplantation embryos, its inclusion in simple media appears to have a beneficial effect
Does volume or cellular mass of embryos change significantly during embryonic development?
in mouse there appears to be no growth
net decrease in protein content from 1 cell to morula which increases after blastocyst formation
whole protein is taken up by endocytosis
amino acid incorporation into proteins also occurs
might expect that protein content in human embryos does not have net increase in tissue mass during preimplantation development?
What is the function and use of EDTA in culture media?
Chelator of heavy metal cations taht contribute to free radical formation in culture media
proteins in media also offer similar protection
most commercially available media contain amino acids, EDTA or both
EDTA has been used more recently to prevent glycolysis in early cleavage stage embryos by inhibiting cytosolic kinases
Other media omit glucose to solve this issue.
For blastocyst development glucose must be added, and EDTA OMITTED
What is the role for lipids in preimplantation development?
likley contribute to embryo metabolism
embryos thought to contain fat droplets
* source of energy
* required for construction of cell membranes
What forms of transport are used for primary energy substrates, pyruvate and glucose uptake?
passive diffusion and carrier-mediated
simple and facilitated diffusion of pyruvate and glucose
GLUT1 and GLUT 2 likely responsible for glucose uptake
TE cells are thought to act as an epithelium transporting nutrients by facilitated diffusion into moust blastocyst cavity and inner cell mass
glucose levels were similar in blastocyst cavity to exetenal medium
What are the proposed benefits of embryo co-culture?
removing inhibitory substances from the medium (negative conditioning) such as hypoxanthine
secrete embryotrophic factors (positive conditioning) such as growth factors, glycoproteins and antioxidant taurine
