A comparison of CCRM's publication about blastocyst tranfer and blastocyst transfer after Comprehensive Chromosome Screening - are they biased?

I found two different publications of CCRM, one from the year 2000 and it talks about the importance of blastocyst transfer over day 3 embryo transfer. The other publication is a very recent one (2010) where they talk about the supremacy of comprehensive chromosome screening (CCS). Their published implantation rate caught my attention. They showed that when blastocyst transfer was performed they obtained an implantation rate of 70% (in 2000) in their patients and when blastocyst transfer was performed after CCS testing (in 2010) (that is transferring only euploid embryos) they obtained an implantation rate of 68.9%. But in control group where no CCS testing was performed they got only an implantation rate of 44.8% (in 2010). This means CCRM is able to get the same high implantation rate using blastocyst transfer (without performing CCS on them, that is, without selecting for euploid embryos) almost 10 years ago. But in 2010 when they performed blastocyst transfer without CCS (control group) they got only an implantation rate of 44.8%! I hope people get my point!!! So I decided to compare patient selection criteria used in both the studies and found not much difference.
 

2000, CCRM,  PMID: 10856474 ( publication on blastocyst transfer)         Patient selection criteria     FSH ≤ 15 mIU/ml, age ≤ 45 years atleast  10 follicles ≥ 12 mm on the day of HCG administration   Mean age     34 years   Age range     25-43   Mean number of blastocysts on day 5     8.6   Implantation Rate     70%   No of blastocysts transferred     2

2010, CCRM,  PMID: 19939370 ( publication on CCS)
Patient selection criteria			FSH  7.3-7.6 mIU/ml , patients with  AMA, RPL and RIA
Mean age			37.7 years
Age range			30-43
Mean number of blastocysts on day 5 (CCS group)			6.3
Implantation Rate (control group) Implantation Rate (CCS group)			44.8% 68.9%
No of blastocysts transferred			2 .7 (control group) 2 (CCS group)

In their 2000 publication they used blastocysts scored using morphological appearance (no selection for chromosomally normal embryos). There is not much information about the patient characteristics (like whether there are patients who underwent recurrent pregnancy loss (RPL) and implantation failure). But definitely there are patients with Advanced Maternal Age (AMA) as evidenced by the age range of patients given in the publication. But nowhere had they mentioned that the above said patients are first time IVFers! So naturally there would have been patients with previous IVF failures. 68 patients underwent 2 blastocyst transfers (top scoring blastocyst).

In 2010 publication where they applied CCS using aCGH to screen embryos, they say they have included patients with AMA, patients who underwent RPL and patients with recurrent IVF failure (RIF) (more than 2 failed IVF cycles!). They had 48 patients in CCS group and their control group consisted of 113 patients undergoing blastocyst transfer in the same center. They say that the patients were matched for age, day 3 FSH, previous unsuccessful attempt etc. But there is no mention whether there are patients with RPL in control group! It must be noted that the day 3 FSH in two groups is very less when compared to day 3 FSH of patients from 2000 paper. They said the selection criteria for including patients for blastocyst transfer (2000 publication) is day 3 FSH ≤ 15 mIU/ml. It does imply that they used patients who had FSH upto 15 mIU/ml.

If this is the case how come they got an implantation rate (with fetal heart tone) of 70% in 2000 (the same group and the same author too!) with the transfer of 2 good quality blastocyst and when they performed 3 blastocyst transfer (2.7 mean) in control group in 2010 they are able to achieve only an implantation rate (with fetal heart tone) of only 44.8%. In the CCS group they got an implantation rate of 68.9%. 

Even if they argue that they have included only the difficult patients (as per AMA, RPL and RIF) – the FSH level in patients and the blastocyst formation rate do not show much difference between the patients selected in 2000 and in 2010. Actually in 2010 paper the day 3 FSH level of patients seems to be lower than in 2000! 

The only question in my mind after comparing both their publications is - ARE THEY BIASED? 

Comprehensive Chromosome Screening (CCS) - panacea or pipe dream? - Part III

Trophectoderm biopsy

You can read the first part here and  the second part here.

Which biopsy is the best for detecting genetic errors in an embryo?

The removal of polar bodies from oocyctes or removal of one or more cells from pre-implantation embryo provides the material for the genetic screening of an embryo. Usually an embryo is biopsied on day 3 or day 5 of development. Day 3 embryo biopsy is called early cleavage stage biopsy and day 5 embryo biopsy is called trophectoderm biopsy or blastocyst biopsy.

Polar bodies are meiotic by products of an embryo. Polar body biopsy is considered to be less invasive and hence protects embryo against extensive mechanical damage. It gives information about the genetic status of the oocyte and not of the sperm, but this is acceptable in clinical practice because 90% of the genetic abnormality in an embryo arises from meiotic non-disjunction in the oocyte. Also, polar body biopsy does not give information about embryo mosaicism ( which arises due to mitotic cell divisions in an embryo).

Day 3 embryo biopsy is performed on one or two blastomeres. Because of embryo mosaicism arising due to mitotic non-disjunction,some cells on day 3 might have a normal karyotype and some may not. So when a single blastomere is biopsied on day 3 , there are chances that the result of CCS comes from the cell carrying a normal chromosomal make-up. This will lead to the labeling of entire embryo as euploid while it can be a mosaic embryo (diploid-aneuploid mosaic). Also, not all day 3 embryo develop into blastocysts , even if it carries a normal karyotype. Biopsy itself can be detrimental to its further development into a blastocyst, so there are chances of having no genetically normal embryo for transfer on day 5 when a day 3 biopsy is performed. On the other hand, when a biopsy is performed on day 3 and when the embryo is grown until day 5 before transfer, then it allows for a kind of double selection, because you get to transfer CCS normal day 3 embryos which had the potential to develop into blasts , giving a much higher chance of transferring the best embryo.

Day 5 biopsy is performed on a group of cells (2-10 cells) obtained from trophectoderm. Since more cells are obtained for biopsy , the chance of detecting chromosomal abnormality with precision is higher. But the trophectoderm is the layer which develops into the placenta, while the embryo develops from the inner cell mass . This means that it’s possible that the inner cell mass could have euploid cells , while the and the cells from the trophectoderm are aneuploid. This phenomenon is called as confined placental mosaicism, as a result of which trophectoderm biopsy can lead to the labeling of a chromosomally normal embryo as chromosomally abnormal.

Also, an embryo which seems to be genetically defective on day 3 may ‘self-correct’ itself on day 5. So doing a day 5 biopsy might help in obtaining accurate results. Trophoectoderm biopsy is considered to be less traumatic to the embryo as only a few cells out of over 100 cells are removed ).

Are we over-testing, just because we have the technology to do so?

As a biologist, I do have a critical view of all the new technologies and its effectiveness. Reading the scientific literature regarding embryo aneuploidy and comprehensive chromosome screening have raised a number of unanswered questions. It is true that this technology is a blessing for patients who carry genetic abnormalities , because it will prevent the genetic disorder from being transferred to their offspring. But, will this extensive genetic screening of embryos do any good for patients who undergo IVF for other reasons? Can older women who undergo repeated implantation failure or recurrent pregnancy loss benefit from it?

It was shown that greater than 50% of the embryos (even from younger women) created via IVF carry genetic abnormalities. It was hypothesized that, if we could select embryos which are genetically normal then the chance of implantation and pregnancy rate per embryo transfer will increase significantly. Since most miscarriages are the result of embryo aneuploidy, the rate of miscarriage is thought to decrease dramatically with aneuploidy screening. This seems to be logical but the data from PGS testing failed to show any clear benefit. A very nice review of literature which includes the biggest and best scientific study can be found here. But the genetic screening technique used in those studies is FISH (which cannot screen abnormalities in all 23 chromosomes) and not CCS and this might explain in-part the lack of improvement in pregnancy rate even after PGS testing. A recent (2011) meta-analysis of randomized controlled trials failed to show improved live birth rates after PGS. Paradoxically, it showed that for women of advanced maternal age PGS significantly lowers the live birth rate (PMID: 21531751).

Can we say that all genetically abnormal embryos are doomed to perish? There is no definite answer for this question. Since more than 60% of miscarriages show some form of chromosomal abnormality it is logical to think that aneuploidy arising either in the egg or embryo has to be blamed. But we should not also forget that an embryo has the ability to ‘self-correct’. There is lots of evidence in favor of thus ‘self-correction’ hypothesis and just 20% of euploid cells present in the inner cell mass of a mouse blastocyst can give rise to a genetically normal mouse. Embryonic stem cells created from genetically abnormal embryos (found to be genetically defective via PGS) produced stem cell lines which have a normal karyotype. There is proof that tetraploid blastocysts (genetically abnormal blastocyst) transferred into uterus gave rise to a genetically normal baby. Does this say something? This shows that an embryo which is designated as genetically defective might actually have the potential to develop into a normal baby. Then does it mean we are discarding embryos which could develop into much desired babies just because of technology over use?

What if faulty earlier cell division in embryo is just a physiological mechanism and not really a pathological mechanism? An embryo in its initial stages may be designed by evolution to divide very rapidly , so that it reaches the uterus at the appropriate stage to implant itself into the endometrium(it might be that slow and steady embryos fail in this situation!). We should not forget that our uterus is receptive to the embryo only for a short period of time. If the embryo is under the scrutiny of strict cell cycle control , it is possible that the time taken for cell division would be too long, and result in a failure to implant . So nature might inherently allow replication errors (by relaxing the cell cycle control), and the errors introduced in the genome can be ‘self-corrected’ later when cell differentiation starts. Should higher miscarriage rates in women of advanced maternal age be linked to higher aneuploidy rates due to replication errors? Isn’t there a possibility of ‘self-correction’ mechanisms being defective in their embryos because of aged oocytes? If this is true , are we really doing any good by screening and discarding the genetically abnormal embryos? We can check the embryo for its competency upto the blastocyst stage, but an embryo implanting and developing in the uterus also undergoes rapid cell division for the next few weeks during which organogenesis takes place. If there are genetic errors introduced at that time point and if the ‘self-correction’ machinery is defective will the embryo survive? Doesn’t this explain the lack of improvement in miscarriage rates in older women even with PGS? All these are questions which still need to be answered !

Should I opt for comprehensive chromosome screening?

It depends for what reason you are doing it. CCS is extremely useful for couples who are carriers of rare genetic disorders. Selecting embryos which do not carry that particular genetic defect will guarantee them a normal pregnancy and a genetically normal baby. Until now , this is the most reasonable indication for doing CCS on an oocyte or an embryo. All other conditions for which CCS is indicated are still not proved to be effective with larger, well-controlled studies. This technique is extremely costly, time consuming (when there are no appropriate labs nearby for doing CCS) and can be very emotionally draining too. So it is advisable to undergo such genetic screening tests only when absolutely necessary.