試管嬰兒囊胚期胚胎切片以qPCR進行PGS胚胎著床前染色體診斷，不必冷凍，直接新鮮胚胎植入子宮:先驅性研究
2015.04.21
Using multiplex q-PCR in pre-implantation
genetic screening of common aneuploidies
followed by fresh embryo transfer: A pilot study
Application of q-PCR to preimp
試管嬰兒囊胚期胚胎切片以qPCR進行PGS胚胎著床前染色
Table 1. Clinical outcomes
|
qPCR + Fresh cycle ET verification series |
No. |
% |
|
No. of cycles |
13 |
|
|
No. of embryos analysed |
72 |
|
|
No. of embryos diagnosed(%) |
72 |
100.0% |
|
No. of embryos with diagnosis failure |
0 |
0.0% |
|
No. of aneuploid embryos |
28 |
38.9% |
|
No. of embryos transferred |
28 |
|
|
Mean embryos transferred |
2.15 |
|
|
No. of clinical pregnancies (% cycles) |
8 |
61.5% |
|
No. of early miscarriages(<12 weeks) |
1 |
12.5% |
|
No. of ongoing pregnancies (% cycles) |
7 |
53.8% |
|
No. of embryonic sacs |
11 |
|
|
No. of embryonic heart tone (FHT) |
11 |
|
|
Sustained implantation rate (% ET) |
11/28 |
39.3% |
蔡鋒博*1, 陳昭雯1, 林招彰1, 張舜評2,3, 馬國欽 2, ,張月嬌1,陳曉青1,徐慧鈴1,潘孟麗1,張琇媛1,
Feng-Po Tsai1, Chao-Wen Chen1, Chao-Chang Lin1, Shun-Ping Chang2,3, Gwo-Chin Ma2, Yueh-Chiao Chang1,Hsiao-Ching Chen1,Hui-Ling Hsu1,Meng-LI Pan1,Hsiu-Yuan Chang1,Li-Chun Shih1, Ming Chen2,4,5
Poyuan Women Clinic IVF Centre, Changhua Taiwan1
博元婦產科,不孕症試管嬰兒中心[1]
彰化基督教醫院 基因醫學部[2]
中興大學 生命科學系[3]
台灣大學醫學院婦產部[4]
東海大學 生命科學系[5]
Introduction
Only a small fraction of embryos that can successfully implant and progress to live-birth in spite of recent advances in morphology-based embryological methodologies, selection of competent embryos is therefore a crucial step in IVF treatment. Aneuploidy is regarded as a primary etiology of implantation failure and it is vitally important to develop an effective strategy to solve this deficiency. Various measures including FISH, array CGH (SNP or oligo chromosomal microarray), and q-PCR have been reported. Among them, q-PCR seems a very promising new modality in PGS by a small number of IVF centers abroad. This pilot study aimed at analyzing the impact on implantation rate and pregnancy rate in IVF cycles using fresh embryos transfer which embryos were selected via blastocyst biopsy with rapid qPCR-based partial aneuploidy screening in our setting. It has been shown that biopsy at blastocyst stage is less detrimental in implantation potential than biopsy at Day-3 cleavage stage embryos and thus in this study, we tested a simple strategy by using q-PCR on Day5/6 blastocyst stage embryos and see its outcome.
Patient(s) Material and method
15 infertile couples with at least one previous IVF failure came to our clinic were enrolled. PGS was conducted by q-PCR with selectively amplified markers situated at chromosome 13, 18, 21, and X. The blastocyst biopsy was performed on day 5/6 and the sample was analyzed on the same day. The results were immediately available to the embryologist and the clinician. The euploid embryo was subsequently transferred in the fresh cycle.
Result(s)
A total of 163 blastocysts were biopsied and 4.9% of embryos (n=8) with no results. The aneuploidy rate for blastocysts was 39.2% (n=64). 26 blastocysts were transferred and implantation rate was 53.8% (n=14). Clinical pregnancy rate was 73.3% (n=11).
Results
A total of 72 blastocysts were biopsied. Successful diagnoses were established in
all embryos and the rate of successful diagnosis is 100%. The aneuploidy rate for
blastocysts was 38.9% (28/72). 28 blastocysts were transferred (the number of
embryos transferred ranged from 1~4). The clinical pregnancy rate was 61.5%
(8/13) per cycle. Chemical pregnancy with early abortion was encountered in 1
and therefore the ongoing pregnancy rate (up to second- trimester) was 53.8%
(7/13). There are no difference between the women with advanced maternal age
(AMA, defined as age ≥35 y/o, n=3) and the women without AMA (n=10)
regarding the rates of aneuploidy, clinical pregnancy, and sustained
implantation.
Conclusion
Our pilot study showed a promising high pregnancy rate and implantation rate. Despite it is yet comprehensive chromosomescreening aiming at all 24 chromosomes, the strategy combines the traditional methodology by FISH and the biopsy of blastocyst followed by fresh embryo transfer, which gains an advantage of biopsy of Day-3 cleavage stage embryos. Expanding the chromosomes being investigated (from a limited set of chromosomes to all 24 chromosomes) is underway and a randomized study by comparing q-PCR and array CGH is needed in the future.
==============================
Using multiplex q-PCR in pre-implantation
genetic screening of common aneuploidies
followed by fresh embryo transfer: A pilot study
Feng-Po Tsai4,
Yu-Shih Yang1,#, Shun-Ping Chang2,#, Hsin-Fu Chen1,3,#, Gwo-Chin Ma2, Wen-Hsiang Lin2, Chi-Fang Lin1, Chen-Hsuan Wu5, Horng-Der Tsai5,
and Ming Chen1,2,5,6*
1Department of Obstetrics and Gynecology, College of Medicine, National Taiwan
University, Taipei, Taiwan
2Department of Genomic Medicine, and Center for Medical Genetics, Changhua
Christian Hospital, Changhua, Taiwan
3Graduate Institute of Medical Genomics and Proteomics, College of Medicine,
National Taiwan University, Taipei, Taiwan
4Poyuan Women Clinic, Changhua, Taiwan
5Department of Obstetrics and Gynecology, Changhua Christian Hospital,
Changhua, Taiwan
6Department of Life Sciences, Tunghai University, Taipei, Taiwan
#These authors contributed equally to this study
*Corresponding author to:
Ming Chen, MD, PhD. Department of Genomic Medicine, Changhua Christian
Hospital, Changhua, Taiwan. Email: mchen_cch@yahoo.com or
Short Title: Multiplex q-PCR PGS for common aneuploidies
Abstract
Background
Aneuploidy is a primary etiology of implantation failure and q-PCR seems a very
promising technology of PGS adopted by a small number of IVF centers abroad.
This pilot study aimed at analyzing the impact on implantation rate and
pregnancy rate in IVF cycles using fresh embryos transfer in which the embryos
were selected by blastocyst biopsy with rapid q-PCR-based common aneuploidy
screening (chromosome 13,18,21,X,Y) in our settings.
Patients and methods
Validation was performed as follows: 58 blastocysts were biopsied and
simultaneously sent for FISH (targeted at chromosome 13,18,21, X and Y) and q-
PCR (with selectively amplified markers situated at chromosome 13, 18, 21, X
and Y). The rate of successful diagnosis at the validation stage is 98.1% (57/58).
A total of 13 infertile couples with more than once failed in vitro fertilization
were enrolled during July to October of 2014. PGS was conducted by q-PCR with
selectively amplified markers to detect common aneuploidies. The design of the
q-PCR molecular markers was modified from the locked nucleic acid (LNA)
strategy. The blastocyst biopsy was performed on day 5/6 and the sample was
analyzed on the same day. The results were immediately available to the
embryologist and the clinician, which enabled fresh embryo transfer (FET).
Results
A total of 72 blastocysts were biopsied. Successful diagnoses were established in
all embryos and the rate of successful diagnosis is 100%. The aneuploidy rate for
blastocysts was 38.9% (28/72). 28 blastocysts were transferred (the number of
embryos transferred ranged from 1~4). The clinical pregnancy rate was 61.5%
(8/13) per cycle. Chemical pregnancy with early abortion was encountered in 1
and therefore the ongoing pregnancy rate (up to second- trimester) was 53.8%
(7/13). There are no difference between the women with advanced maternal age
(AMA, defined as age ≥35 y/o, n=3) and the women without AMA (n=10)
regarding the rates of aneuploidy, clinical pregnancy, and sustained
implantation.
Conclusion
Our pilot study showed a favorable reproductive outcome of adopting PGS with
q-PCR + FET in our own setting. Expanding the repertoire of aneuploidies being
investigated (from a limited set to all 24 chromosomes) is underway and a
randomized study by comparing q-PCR and other PGS technologies is warranted
in the future.
Key words: Aneuploidy, Blastocyst, Fresh embryo transfer,
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