The present study, for the first time, reported the efficiency of frozen/warmed blastocyst biopsy for PGS. Our data showed that frozen/warmed blastocyst biopsy can be done within an hour after completion of warming, and FET can be done within 24 h after warming if there are normal embryos. Because biopsy and aCGH take more than 12 h, FET was scheduled on the second day after embryo warming and biopsy. If the analysis could be done in a shorter time, FET could be scheduled on the same day for the patients. However, based on our experience, it would appear that embryo quality was not affected by extended culture up to 24 h. It has been reported that transfer of frozen/warmed blastocysts after overnight culture (less than 18 h) had similar implantation rates as compared with transfer of blastocysts after same day warming .
Usually blastocysts start to expand 2–3 h after completion of warming, and it may be easier to choose the location for biopsy if blastocysts re-expand. However, this would take more time from embryo warming to FET and it may be unfavorable to blastocyst implantation. From the results obtained in the present report, we found that our method is practical. It is still not known if biopsy of re-expanded blastocysts can further improve clinical outcomes.
For the warmed blastocyst biopsy, it is not necessary to warm all blastocysts if patients have many cryopreserved embryos in order to avoid re-freezing spare blastocysts after transfer. Although reports indicated that refreezing human blastocysts did not further impair embryo quality and implantation [19–22], repeated cryopreservation and warming may be not a good practice for human IVF, as long term effects of refreezing/warming on human embryos are not clear. Only limited case reports indicated that embryo survival was not affected by these procedures . Therefore, in order to avoid repeated freezing/warming, it may be necessary to warm a limited number of embryos for biopsy and analysis. The only concern of testing a limited number of blastocysts is that there may not be normal blastocysts for transfer after analysis, thus embryo transfer has to be cancelled. In the present study, some patients had all frozen blastocysts warmed and analyzed while some patients had a portion of blastocysts warmed and analyzed. As a result, only one patient did not have a euploid embryo, so her FET was cancelled, while all others had at least one normal embryo for FET.
Another concern is that there are limited number of good quality of normal blastocysts for transfer if a portion of embryos are warmed and analyzed. Because some euploid blastocysts may have fair or poor quality. It has been found that embryo implantation is related to the total available embryos for PGS, which would ensure that there is at least one good blastocyst to be euploid for transfer . Morphology and euploid status are the two most important factors affecting embryo implantation. Several studies have shown that morphology assessment alone to select embryos for transfer has limited prediction capacity for embryo implantation, as some high morphology score embryos may be aneuploid [7, 24, 25], especially in patients of advanced maternal age. Also, aneuploid embryos with good morphology may result in miscarriage. Therefore, high live birth rate should be obtained after transfer of embryos that have high morphology score and are euploid.
In the present study, all embryos were biopsied at the blastocyst stage. Many studies have proven that blastocyst stage is the optimal stage to perform biopsies for PGS, which produces higher implantation rates and provides more reliable aneuploidy results than cleavage stage embryos [13, 14, 26]. Biopsy at blastocyst stage does not result in a loss of inner cell mass and would not affect subsequent fetal development, so it is less harmful to embryo development [14, 15]. Furthermore, blastocyst biopsy can acquire multiple TE cells , thus the results are more accurate.
It has been repeatedly reported that more than half of all morphologically normal blastocysts in women >35 years old are aneuploid [7, 8, 12], and more than 50 % of first-trimester spontaneous abortions are chromosomal aneuploid . So by doing PGS, euploid embryos can be selected for transfer, thus implantation rates can be improved, and miscarriages and trisomy offspring loss can be reduced [11, 12].
As for the prevalence of aneuploidy and degree of chromosome anomalies in the cryopreserved/warmed blastocysts, it would appear that there was not much difference between fresh blastocysts and frozen blastocysts. In the present study, we found that the overall aneuploidy rate was 40.7 %, which was similar to that in our previous report with fresh blastocyst aCGH . However, direct comparison is difficult because patients’ populations are different between studies. Another similar result was the degree of chromosome anomalies in the aneuploid embryos. In the previous study with fresh blastocysts, it was found that 62.3 % of aneuploid blastocysts had single chromosome errors , while the rate was 65.6 % in the present study with frozen/warmed blastocysts. Also, it was found that chromosomes 21 and 22 were two of the most frequent chromosome abnormalities in the aneuploid blastocysts in the fresh blastocyst  as well as in the frozen/warmed blastocysts (current study). Again, although the comparison is difficult between studies, the results may suggest that the prevalence of aneuploidy in fresh blastocysts and frozen blastocysts would be similar and cryopreservation/warming may not increase chromosome abnormalities in human blastocysts.
In the present study, our goal was limited to examine the feasibility of biopsy of cryopreserved blastocyst for PGS. We did not compare the outcome between PGS and non-PGS cases, so the necessity of PGS on all previous IVF failures is still debatable as other factors (not only embryo ploidy) also affect embryo implantation, such as endometrium preparation and transfer itself. All IVF failures cannot be attributed to embryonic aneuploidy. As indicated in two recent review papers [29, 30], chromosomal mosaicism may cause incorrect embryo screening, especially diploid-aneuploid mosaicism, which is the most common chromosome mosaic in human embryos , thus some screened “abnormal” embryos may have normal inner cell mass [8, 31]. Transfer of these kinds of embryos may produce normal healthy babies. Indeed, recently, Greco et al. reported that transfer of mosaic aneuploid blastocysts can result in a high healthy live birth rate (33 %) . In this report, some of the embryos transferred had two chromosomal abnormalities . One of our patients also had a healthy baby after transfer of a screened embryo, but this pregnancy had a mosaic placenta although PGS showed it was a euploid . These results indicate that mosaic embryos can produce healthy babies. As suggested by Liu et al., if all embryos are aneuploidy in an IVF cycle, a second biopsy may be necessary to find a transferrable embryo . However, according to Greco et al.’s report , transfer of these “aneuploid’ embryos after additional consents were signed by the patients may be practical without a second biopsy that may cause more damages to embryos. Therefore, a careful discussion and decision with patients should be necessary if the patients do not have a normal embryo for transfer after PGS.
It has been reported that PGS can also improve clinical outcome in young patients, especially when eSET is applied . In our previous study, we did not find significant increase in either clinical pregnancy or embryo implantation when blastocysts from donated eggs were screened and transferred, especially when two blastocysts were transferred. As suggested by these authors, unrestricted clinical application of current PGS (either aCGH or NGS) should be avoided [30, 31, 34].
In the present study, we found that 65.6 % of aneuploid samples had single chromosomal error. If diploid-aneuploid mosaic rate is high in these embryos as indicated in the previous study (59 %) , it would be possible that transfer of these embryos can produce normal pregnancy and result in healthy live births as reported by Greco et al. . Thus it must be careful to make the decision to transfer, cryopreserve or discard these embryos. As hypothesized by Greco et al. , the extent and type of mosaicism may affect the IVF success rate, and additional studies with large sample size are necessary to test this hypothesis.