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IVF Başarısında Kültür Medyumlarının Rolü; Rivayetler ve Gerçekler

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IVF Başarısında Kültür Medyumlarının Rolü;

Rivayetler ve Gerçekler

Prof.Dr.Lale Karakoç Sökmensüer Hacettepe Üniversitesi Tıp Fakültesi

Kadın Hastalıkları ve Doğum A.D. Tüp Bebek Üniversitesi

 

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sub-optimal culture conditions  

impaired embryo development  

loss of viability  

(-­‐)  ART  outcome  

(4)

more physiological and effective culture media   decrease in

the number of pregnancies

lost  

increase in implantation

rates  

increase in the overall success rates of ART  

embryos more able to survive cryopreservation  

(5)
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human embryo culture

•  mammalian embryo culture in vitro should not be regarded as

an imperfect copy of the in-vivo procedures

•  an artificial process

•  standard embryo culture procedures use somatic cell cultures as templates

•  situation, morphology, function and regulation of somatic cells in vivo are basically different from those of

preimplantation mammalian embryos

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differences and their consequence

•  preimplanta+on  embryos  are  not  part  of  the  maternal  body:  they  exist   physically  and  biologically  as  largely  independent  living    

•  while  soma+c  cells  of  mammals  are  surrounded  by  the  extracellular  fluid   that  is  rich  in  nutrients,  proteins  and  other  factors,  preimplanta+on  

embryos  are  surrounded  by  oviductal  and  uterine  fluid  

•   soma+c  cells  are  unable  to  proliferate  without  external  trophic  ligands  ,   mammalian  zygotes  are  intrinsically  programmed  to  develop  to  the  

hatched  blastocyst  stage  without  any  external  s+muli.    

(  in  vivo  these  embryos  are  exposed  and    respond  to  a  number  of   interrelated  endocrine,  paracrine  and  autocrine  factors)    

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differences and their consequence

•   oviductal  and  uterine  fluid  is  not  just  the  product  of  the  epithelial  cells.  It   contains  factors  derived  from  the  maternal  body,  and  may  have  profound   consequences  on  the  future  of  the  fetus  and  offspring    

 

•   role  of  oviductal  and  uterine  epithelial  cells  includes  a  passive  filtra+on  or  ac+ve   transport  of  these  factors  as  well;  this  role  cannot  be  reconstructed  by  a  simple   monolayer  of  a  co-­‐culture  system  

 

•  establishment  and  maintenance  of  the  microenvironment  plays  a  crucial  role  in   embryo  development  both  in  vivo  and  in  vitro.  Autocrine  factors  seem  to  be  

decisive  cons+tuents  of  this  microenvironment.  With  the  current  methods,  tools   and  dishes  (established  originally  for  soma+c  cells),  this  microenvironment  

cannot  be  properly  maintained  in  vitro.    

(9)

•  first successes of embryo culture in the mouse ……Krebs–

Ringer bicarbonate medium (Tyrode’s medium) supplemented

with lactate

(McLaren and Biggers, 1958; Whitten, 1957)

•  human ……..complex media (e.g. Earle’s balanced salt solution or Ham’s F-10)

(Edwards, 1981)

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two major approaches

•  ‘empirical  op+miza+on’    or  ‘let  the  embryos  choose’  (KSOM-­‐Global   family)                                                                                                                                                              

                 (Biggers,  2001)  

 

•   ‘back  to  nature’  principle,  (make  media  with  composi+on  similar  to   the  oviductal  fluid)    

         

                                                                                                   (Mor+mer,  1986;  Quinn,  1995;  2000;  Quinn  et  al.,  1985)    

 

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Lane  and  Gardner,  2007    

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Lane  and  Gardner,  2007    

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sequential or single medium?

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Renew medium or not?

•  embryos  are  le\  undisturbed;  

•  accumulated  endogenous  growth  factors  are  le\  in  place;  

•   the  rela+ve  environmental  stress  is  ‘low’    

•  labour  intensity  is  lower;    

•  cost  is  lower;  

•  less  quality  control  is  required    disadvantages  :    

Ø essen+al  nutrients  are  not  replaced     Ø toxins  may  accumulate    

(20)

Renew medium or not?

•  only  material  that  was  supposed  to  have  detrimental  effect  is  

ammonium:  by  replacing  glutamine  with  stable  dipep+des  (Biggers  et   al.,  2004),  this  danger  can  be  safely  eliminated.    

•   a  single  medium  without  renewal  may  be  a  realis+c  alterna+ve  of  the   present  culture  systems.    

•  It  may  offer  many  prac+cal  benefits  for  embryologists  and  it  may  also   help  embryos  to  build  up  and  maintain  their  microenvironment.    

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alternative possibilities for embryo culture

 

Ø Tubes  

Ø Microchannel  microfluidic  system  (con+nuous  or  stepwise  medium   exchange)  

Ø Glass  oviduct  system     Ø Well  of  the  Well  system      

         

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COMPOSITION OF EMBRYO CULTURE MEDIA

•  Carbohydrates  

•  An+bio+cs  

•  Nucleic  Acid  Precursors  

•  Amino  Acids  

•  Water  

•  Ions  

•  Chelators  

•   An+oxidants  

•  Protein/macromolecules  

•   Hormones  and  growth  factors  

•   Buffer  system  

•  Vitamins  

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Lane  and  Gardner,  2007    

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embryo culture medium: which is the best ???

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ü first  week  of  mammalian  embryo  development  consists  of  crucial   events  including  the  first  cleavage,  ac-va-on  of  the  maternal  

genome,  compac-on  of  morula  and  differen-a-on  with  blastocyst   development  ……..  programmed  and  orchestrated  events.    

ü adapta+on  to  an  inappropriate  environment  may  cause  changes  in   epigene-cs,  transcrip-on,  metabolism  and  cell  alloca-on  

ü components  of  media  can  produce  serious  altera-ons  in  the  gene   expression  pa<ern  of  preimplanta-on  embryos  with  poten-al  long-­‐

term  consequences  

(40)

ü 

 culture  media  is  only  one  part  of  the  culture  system,  op+mal  

performance  of  the  medium  is  dependent  on  the  quality  of  other   aspects  such  as  contact  supplies  and  oil  

ü 

 importance  of  the  quality  of  the  en+re  system  increases  with  

extended  culture,  high  levels  of  quality  control  and  assurance  exist   in  the  laboratory  to  enable  any  culture  medium  to  perform  to  its   maximum  

 

ü 

not  only  examine  the  ability  of  a  culture  system  to  produce  a  

pregnancy  with  the  one  or  two  highest-­‐grade  embryos,  but  also  to   determine  how  many  embryos  from  the  en+re  cohort  (both  fresh   and  frozen  embryos)  are  capable  of  producing  a  live  birth  

   

 

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ü 

understand the role of each medium component and to

identify possible sources of cellular stress to the embryo that will ultimately affect the function and viability of the

conceptus

optimized and standardized culture system

(42)

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