Roger G. Gosden, PhD, DSc

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Assisted conception  is evidently an inefficient technology and, overall, only ~3% of oocytes  collected result in live-births. Embryos are often inviable or abnormal  as a result of defects arising during oogenesis and/ or in culture.  Advancing maternal age further reduces the fertile potential of oocytes.  Cytoplasmic (ooplasmic) transfer aims to improve egg and embryo quality  by the donation of ooplasm from supposedly fertile oocytes to patients  whose cells are of poorer quality. Cytoplasm can be transferred by  either 1) electrofusion or 2) direct injection (Cohen et al, 1998). In  1), a small ooplast recovered from a donor egg is injected under the  zona pellucida of a recipient oocyte (after removing its polar body)  before fusing the membranes. The cybrid is then fertilized by ICSI. In  2), a small amount of ooplasm (~10%) is aspirated into a pipette and  injected into the recipient cell at the same time as the fertilizing  sperm. Transfer of anucleate cytoplasm has been used on a small scale in  clinical trials, and children have been born. An alternative strategy  is to transfer the germinal vesicle nucleus (together with shreds of  attached cytoplasm) to an enucleated recipient ooplast (Zhang et al,  1999). In either case, the children should be genetic offspring of the  patients, apart from the variable degree of cytoplasmic inheritance from  the donor egg. 

       Cytoplasmic transfer is a technique of last resort after repeated  IVF failure or at advanced maternal age: it is based on an assumption  that a vital molecule, such as ATP or cell cycle-related kinase, or an  organelle, such as mitochondria, is deficient at a critical stage in  early development. Hormone replacement therapy is an obvious parallel  but, until there is a better understanding of the molecular development  of oocytes, a deficiency state remains hypothetical. Indeed, the  opposite hypothesis is also plausible - that poor quality reflects  exposure of oocytes to environmental toxins, including drugs, diet and  smoking. These hypotheses are not mutually exclusive, but if either is  true it will be important to test whether recent evidence that the  mammalian egg is polarized carries implications for the site of  cytoplasmic “biopsy” and injection.

       Cytoplasmic transfer has had some impressive achievements in  experimental science. For example, in certain strains of mice or culture  conditions embryos halt in early cleavage, and cytoplasm from  non-blocking strains helps to overcome the problem (Muggleton-Harris et  al, 1982). Nevertheless, experiments also reveal potential pitfalls. In  the DDK strain, “cytoplasmic incompatibility” due to a maternal RNA  interaction with the paternal genome causes embryonic wastage (Renard et  al, 1994), and there is evidence of a replicative race between  mitochondria in somatic cells and after nuclear transfer in farm animal  oocytes. This is less surprising in view of the natural phenomenon of  paternal mitochondria exclusion, which creates homoplasmic embryos. It  is therefore unsafe to assume that mitochondrial transplants will be  proportionately represented in the tissues of the fetus and child.

       In addition to uncertainties about development, there are concerns  about the safety of cytoplasmic transfer for future children. There is  an obvious danger of transferring stray chromosomes from the donor cell  to produce a hyperploid embryo, especially in light of evidence showing  that the polar body is not an infallible landmark for avoiding the  spindle apparatus. Public health scares about prion disease in cattle  and humans and concerns about the risks of xenotransplantation force us  to take heed of the risks of disease transmission and complementation.  Viral-like particles can be observed in mouse oocytes and endogenous  retroviral proteins are detectable on the surface of human oocytes  (Nilsson et al 1999).

       Finally, it is questionable whether cytoplasmic transfer is  justified on the basis of evidence-based medicine, which has been  defined as “the process of systematically finding, appraising, and using  contemporaneous research findings as the basis for clinical decisions”  (Rosenberg & Donald, 1995). There is no firm evidence that it can  improve oocyte quality either from animal studies or randomized clinical  trials, and isolated reports of successful pregnancies cannot, by  definition, provide assurance about reproducibility. Admittedly, some of  the key breakthroughs in ART were made before studies of safety and  reliability had been performed, and there are major practical and  ethical problems designing and executing trials in ART to the same  exacting standards as, say, clinical pharmacology. Yet, the onus is on  practitioners of cytoplasmic transfer to offer a convincing  justification, or to produce a persuasive and ethically defensible case  for making reproductive medicine an exception to the principles of  evidence-based medicine. I am not aware that a case has been made.  Nevertheless, it is important to remember that the reproductive medicine  community must tread a cautious path between unproven technology, on  the one hand, and damping valuable innovation, on the other. 


COHEN J, SCOTT R, ALIKANI, M et al. Ooplasmic transfer in mature human oocytes. Mol Hum Reprod 4:269-280,1998

MUGGLETON-HARRIS A, WHITTINGHAM DG, SCOTT L. Cytoplasmic control of preimplantation development in vitro in the mouse. Nature 299:460-462,1982

NILSSON BO, JIN MS, ANDERSSON AC et al. Expression of envelope proteins of endogenous C-type retrovirus on the surface of mouse and human oocytes at fertilization. Virus Genes 18:115-120,1999

RENARD J-P, BALDACCI P, RICHOUX-DURANTHON V et al. A maternal factor affecting mouse blastocyst formation. Development 120:797-802,1994

ROSENBERG W, DONALD A. Evidence-based medicine: an approach to clinical problem-solving. Brit Med J 310:1122-1126,1995

ZHANG J, WANG C-W, KREY L et al. In vitro maturation of human preovulatory oocytes reconstructed by germinal vesicle transfer. Fertil Steril 71:726-731,1999

Articles

The First World Congress On: Controversies in Obstetrics, Gynecology & InfertilityPrague, Czech Republic - 1999

The Role of Cytoplasmic Transfer

Low temperature  banking of ovarian tissue was first attempted over 40 years ago, but  only recently have the potential benefits become obvious. With rising  numbers of young people surviving high-dose chemotherapy and radiation  there is now an urgent need to conserve germ cells to avoid iatrogenic  infertility (Byrne, 1999). Whilst frozen semen banking is available for  adult males, options for females are limited, and there are none for  prepubertal girls. Ovarian tissue banking provides a potential  opportunity for females to keep their reproductive options open.  Experimental studies in animals have demonstrated that slices of ovarian  tissue can be preserved in liquid nitrogen, thawed and returned as  isografts or autografts (Harp et al, 1994; Baird et al, 1999). This  technology is now poised for clinical application. 

       Ovarian tissue is suited to low temperature banking because  primordial follicles are abundant in young ovaries and confined to a  thin band of cortical tissue. Thin slices of tissue can be rapidly  equilibrated with permeating cryoprotectants, such as dimethyl  sulphoxide (DMSO) and 1,2-propanediol (PROH), and cooled in an automated  freezer. Vitrification with rapid cooling has some practical and  theoretical advantages, but it is too soon to say whether it is as  effective as conventional slow cooling protocols. Whilst storage of  whole organs is still impracticable, and will require preservation of  the vasculature so that vessels can be anastomosed for transplantation,  simple grafts of frozen-thawed tissue slices are surprisingly  successful. Another strategy is to isolate primordial follicles by  enzymatic disaggregation of the ovary, since small units are better for  cryopreservation. After thawing and transferring to sterile host ovaries  in mice, fertility was restored (Carroll & Gosden, 1993), but many  follicles were damaged or lost and this may not be a prudent use of  precious human germ cells. In any case, storage of follicles in situ is  already effective (Hovatta et al, 1996; Newton et al, 1996) and, if  isolated follicles are required, disaggregation can still be carried out  after thawing (Oktay et al, 1997). 

       Cryopreserved ovarian tissue from human donors has been  successfully xenografted to immunodeficient SCID mice (Newton et al,  1996), paving the way for techniques to restore fertility in patients by  autografting. DMSO and PROH were much more efficient than glycerol  because the latter penetrates slowly and creates a steep osmotic  gradient which damages cells. Over 70% of the primordial follicles  survived freezing, thawing and grafting. The pieces of tissue used were  smaller than clinically desirable, but if good results can be obtained  with xenografts prospects should be even brighter with autografts.  Ovarian tissue is now being stored in many centres and an announcement  of the results of the first graft in a patient are imminent. It is  already clear that heterotopic implants of fresh cortical tissue can be  effective: ~25% of follicles survived on the anterior uterus for at  least 3-4 months in volunteers for autografting who were undergoing  routine surgery (Rutherford et al., unpublished). 

       A wide range of patients might consider ovarian banking for  conserving their fertile potential. Women of reproductive age, and even  children, who are at imminent risk of sterilisation from sterilizing  chemotherapy/ abdominal radiation/ could be offered emergency ovarian  banking after laparoscopic recovery of tissue. The cost of the whole  procedure would be modest. There is a proviso that tissue should not be  replaced where there is a risk of returning the old disease to the  patient (Shaw et al, 1996). A safer course for them would be to grow  follicles in vitro, but this technology is still at a very early stage.  For other patients who are concerned about premature menopause or even  wish to postpone the age of natural menopause, such risks are  irrelevant, but the widening of applications in non-emergency cases  should await more data about the reliability of tissue storage.

BAIRD DT, WEBB R, CAMPBELL BK et al. Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at -1960C. Endocrinology 140:462-471,1999

BYRNE J. Infertility and premature menopause in childhood cancer survivors. Med Ped Oncol 33:24-28,1999

CARROLL J, GOSDEN RG. Transplantation of frozen-thawed mouse primordial follicles. Hum Reprod 8:1163-1167,1993

HARP R, LEIBACH J, BLACK J et al. Cryopreservation of murine ovarian tissue. Cryobiology 31:336-343,1994

HOVATTA O, SILYE R, KRANSZ T et al. Cryopreservation of human ovarian tissue using dimethysulphoxide and propanediol-sucrose as a cryoprotectant. Hum Reprod 11:1268-1272,1996

NEWTON H, AUBARD Y, RUTHERFORD A et al. Low temperature storage and grafting of human ovarian tissue. Hum Reprod 11:1487-1491,1996

OKTAY K, NUGENT D, NEWTON H et al. Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue. Fertil Steril 67:481-486,1997

SHAW JM, BOWLES J, KOOPMAN P et al. Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit the cancer to graft recipients. Hum Reprod 11:1668-1673,1996 

Cryopreservation of Ovarian Tissue in Cancer Patients

  I’m with Dr Roger Gosden, who is, at  present, Scientific Director of the Howard and Georgeanna Jones  Institute for Reproductive Medicine in Norfolk, USA, and who has given  very important presentations in the last two days concerning the role of  the egg in the follicle, the inter-relationship between the egg and the  granulosa cells, and also the consequences that has for the subsequent  development in the first days of division.  Now, Roger, one of the  issues you presented was about the influence of the quality of the egg  on the granulosa cell and vice versa.  Could you elaborate on this for  the audience?
 
 

 Certainly.  Well, the egg grows over a  long period of time, of course.  The follicle has a growth span in  humans lasting many months, and throughout that time, it is intimately  connected with those granulosa cells; you cannot grow an egg to full  size or competence to undergo meiosis or fertilisation without that  layer of granulosa cells around it. 
 
 The granulosa cells, of course, are also an endocrine organ, but they  are vitally important for the development of that egg; and they  determine the timescale of the development of the egg to some extent,  although the egg is not a passive passenger in this, so we have to look  at the other side of the story -- that they are actually working  together, that the egg also instructs the granulosa cells and is  required for them to undergo mitosis and also to express certain  molecules like the mucilaginous hyaluronic acid.
 
 

  So, what implications could that have for  the subsequent development?  For instance, at the moment, the egg is  ready to ovulate and has prepared itself for the subsequent development.   Could that have an impact in later life if something goes wrong or,  for instance, if we interfere with that process?
 
 

 If we interfere with the process at any  stage up until the arrival of the LH surge, we are likely to run into  big problems.  If we interfere with the connection between the granulosa  cells after that time, probably the implications are much less because  the interactions and instructions that have been sent to and from the  egg to the granulosa cells are largely complete; of course, in ICSI we  strip off those granulosa cells, although there are still some remaining  very fine processes crossing from the granulosa to the egg.  We don’t  know whether they’re important and probably not by that stage, so the  key events are going on much earlier in development. 
 
 And if we disrupt that communication at any stage during the follicle  development by breaking those processes or interfering with the signal  molecules (e.g. kit ligand from the granulosa cells, or GDF-9 from the  egg) -- then follicle development will be aborted.  The implications for  egg quality are another matter, of course, and that is something that I  think we all have to be very focused upon if we really want to improve  IVF technology.
 
 

  The two questions that directly pop up  are:  first, the implications, for instance, for in vitro maturation,  which we do not know enough of in order to be able to use it at present  as an acceptable treatment to create human offspring; the second, of  course, is the influence of ICSI and maybe the later influence in terms  of the development of the babies and the occurrence of rare aspects that  have nothing to do with the genetic but more the epigenetic aspects.   Could you comment on that?
 
 

 Well, as you know, the safety of IVF  and ICSI has been quite extensively studied already by investigating  birth defects, by investigating carrier types and by some longer-term  follow-ups of the children.  The results have been broadly pretty  reassuring, just one or two controversies exist, and the major concern I  think we all have is with the multiple pregnancies and their  implications. 
 
 So that has been the background of research over the last twenty years  in follow-up studies. But recently, there’s been much more focus  especially in the last year or so on epigenetic factors.  We published  in the June 7 volume of The Lancet a description of studies, which have  reported an association between epigenetic abnormalities and ARTs.  Now  these abnormalities vary in severity and   are rare like Angelman  Syndrome and Beckwith-Wiedemann syndrome. It is a matter of concern that  the registries for these birth defects show a three- to six-fold  increase in frequency over expectation. 
 
 Now, I don’t think that this should be regarded with huge alarm because,  in the big picture of IVF with a million babies born around the world,  we’re still talking very small numbers.  But even if there is a slight  increase, we have to be aware and, if it’s connected with any new  technologies, we must be very watchful. 
 
 So, what is needed now, I think, are two things:  one is that we need to  do prospective clinical studies to verify if there is an association,  and whether it is a causal relationship; so we need to follow-up large  numbers of IVF children probably on a multi-national basis for  statistical validity and to test for cluster effects. So that’s one part  of the equation. 
 
 The other part is that we need to investigate the developmental biology  of the egg.  So I come back to the egg and egg quality, because during  that developmental span of many months when the egg is growing, it is  accumulating mRNAs, which are either stored for later translation or  translated immediately.  Among those transcripts, and of course there  are thousands of them, there are some which are involved in this  imprinting phenomenon where one of the alleles of imprinted genes is  repressed by methylation or another process in the chromatin, such as   histone modification. 
 
 For Concerns have been expressed whether the imprinting process is  influenced by ART procedures, perhaps raising the risk of syndromes like  Beckwith-Wiedemann Syndrome .  One is, does the condition or specific  conditions of infertility in themselves predispose to imprinting  abnormalities in which the DNA is not appropriately methylated. In that  case, you get expression of imprinted genes from both the paternal and  the maternal sides (alleles) in the embryo of the child, leading to the  abnormal phenotype? 
 
 So we have to focus very much, I think, upon the expression of those  molecules, which were involved in the imprinting process.  There are  certain enzymes which are expressed during egg development, called DNA  methyltransferases, which seem to be key for this process and they have  to be expressed at the appropriate time. 
 
  And then the question of whether there are influences of other factors  -- in our culture conditions, in our hormone stimulation?  There is a  seeming excess of BWS cases associated with ART, but we simply don’t  know if they are related to the technology or the clinical history of  the patient and their infertility condition? 
 
 Therefore, we need the prospective clinical studies and to understand  whether culture conditions might perturb the imprinting process in human  eggs and embryos, and the expression of the enzymes responsible for it.
 
 

  Well, that’s quite a lot, I must say, and I  totally agree with you.  I would like to summarize this by saying that  there are a lot of these subtle changes in the oocyte and also in the  fertilised egg and the embryo in terms of the selective methylation and  demethylation and other processes that we still don’t understand.  Would  you agree with me that it is imperative that we gain more knowledge in  this aspect in order to especially look at the subtle and not very often  occurring changes that may have large consequences for the offspring?
 
 

 I absolutely agree.  There has, of  course, been a huge emphasis upon chromosomal anomalies in PGD with  aneuploidy screening, because we know that a lot of chromosomal  anomalies arise during oogenesis and so that has been the focus for some  years, very appropriately.  But I think in the next few years we will  be looking for these more subtle modifications, as you say.  It’s going  to be quite difficult to investigate them in human embryology because  the material is always very precious, but I think we do have the tools  for that now and I hope we may even have other ways of screening embryos  in the future, whereby we can avoid any risk of some of these  epigenetic phenomena, just as we screen at the moment for single gene  defects.
 
 

  Well, I’m really looking forward to  research in that area.  Are you going to participate in that yourself  actively in your centre?
 
 

 Yes, we are investigating this at the  cell biology level, looking at these enzymes and when the imprinting  process takes place in human eggs, because there’s very little data on  that.  We also are interested in embryo stem cells, and I raise that  because I think human embryo stem cells, can provide a potential model  for looking at factors that influence the blastocyst.  We can never get  enough blastocysts for all the studies we might want to do; for  instance, to look at how culture conditions might alter imprinting  processes, and we know that does happen in animal studies. 
 
 This is where the connections between reproductive medicine and stem  cell technology are very strong. Besides, there is evidence from animals  that this imprinting process is quite unstable in the mouse embryo stem  cell. 
 
 So we have to watch this in the human embryo stem cell too, because  there would be problems potentially with embryo stem cell technology if  the imprinting marks were lost. Biallelic expression could lead to  tumour formation in the transplant.  But, in the same way as we provide  information relevant to transplantation medicine through these studies  of imprinting in embryo stem cells, we may gain information about what  could potentially destabilise imprints in the blastocyst before it is  transferred.
 
 

  Well, really, we are all are looking  forward to this research and we have high expectations of your input on  this.  Thank you very much.

OBGYN.net Conference Coveragefrom the 19th Annual Meeting of ESHRE - Madrid, Spain

Egg quality and imprinting

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Author | Roger G. Gosden, PhD, DSc

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