用囊胚培養液偵測囊胚染色體狀況優點

1. 無侵犯性
2. 可以同時偵測ICM & TE
3. 主要針對鑲嵌狀況染色體  (以50%比率, 區分高 or 低染色體異常)
4. 可同時併用傳統TE biopsy 尤其針對 TE biopsy無法偵測出染色體之狀況(0.6%)

https://academic.oup.com/humrep/article/36/7/2020/6273656?login=false

• With the improvement and optimization of whole genome amplification protocols, the amplification rate of cell-free DNA has increased from 35% to 82% (when using blastocoele fluid, BF) to 100% (with the combined use of BF and SCM) 
• Cell-free DNA is more reliable than TE biopsy for diagnosing mosaic embryos because the genetic material is released into culture media from both ICM and TE cell lineages (Huang et al., 2019a). 
• During the period of using aCGH, we reported a result with 20–50% aneuploid cells as ‘normal embryo (possible mosaic)’ (low degree) and with 50–80% aneuploid cells as ‘abnormal embryo (possible mosaic)’ (high degree). 
• The raw concordance rates of SCM and TE re-biopsies compared with BE were 74.4% and 82% in terms of overall ploidy and 96.2% and 97.9% per single chromosome, respectively. 
• In our study, niPGT of putative whole chromosomal mosaic blastocysts showed reliable concordance rates with the gold standard of whole blastocyst chromosomal constitution. 
• cell-free DNA in SCM is leaked primarily from the ICM, which seems more representative of the fetal lineage.
• cell-free DNA in the SCM showed powerful detectability at the level of both a single chromosome and overall ploidy.

Non-invasive preimplantation genetic testing for putative mosaic blastocysts: a pilot study

Study question: What is the potential of applying non-invasive preimplantation genetic testing (niPGT) for chromosome abnormalities in blastocysts reported with a mosaic trophectoderm (TE) biopsy?

Summary answer: niPGT of cell-free DNA in blastocyst culture medium exhibited a good diagnostic performance in putative mosaic blastocysts.

What is known already: Advances in niPGT have demonstrated the potential reliability of cell-free DNA as a resource for genetic assessment, but information on mosaic embryos is scarce because the mosaicism may interfere with niPGT. In addition, the high incidence of mosaicism reported in the context of PGT and the viability of mosaic blastocysts raise questions about whether mosaicism really exists.

Study design, size, duration: The study was performed between May 2020 and July 2020. First, clinical data collected by a single-center over a 6-year period on PGT for chromosome aneuploidies (PGT-A) or chromosomal structural rearrangements (PGT-SR) were analyzed. After confirming the reliability of niPGT, 41 blastocysts classified as mosaics by trophectoderm (TE) biopsy were re-cultured. The chromosomal copy number of the blastocyst embryo (BE, the gold standard), TE re-biopsy, and corresponding cell-free DNA in the culture medium was assessed.

Participants/materials, setting, methods: Data on patients enrolled for PGT at a single center from 2014 to 2019 were collected and the cycles with available putative mosaic blastocysts were evaluated. To verify the diagnostic validity of niPGT, eight aneuploid blastocysts were thawed and re-cultured for 14-18 h. The concordance of the niPGT diagnosis results and the whole blastocyst testing results was analyzed. Forty-one blastocysts reported as mosaics from 22 patients were included and re-cultured for 14-18 h. The genetic material of the BE, TE re-biopsy, and corresponding cell-free DNA in the culture medium was amplified using multiple annealing and looping-based amplification cycles. The karyotype data from niPGT and TE re-biopsy were compared with that from the whole blastocyst, and the efficiency of niPGT was assessed.

Main results and the role of chance: Data on 3738 blastocysts from 785 PGT-A or PGT-SR cycles of 677 patients were collected. According to the TE biopsy report, of the 3662 (98%) successfully amplified samples, 24 (0.6%) yielded no results, 849 (23.2%) were euploid, 2245 (61.3%) were aneuploid, and 544 (14.9%) were mosaic. Sixty patients without euploid blastocysts opted for a single mosaic blastocyst transfer, and 30 (50%) of them obtained a clinical pregnancy. With the BE chromosome copy number as the gold standard, niPGT and TE re-biopsy showed reliable detection ability and diagnostic efficiency in eight putative aneuploid blastocysts. Of the 41 putative mosaic blastocysts re-cultured and re-tested, 35 (85.4%) showed euploid BE results. All but two of the blastocysts previously diagnosed with segmental chromosomal mosaic were actually euploid. In addition, all blastocysts previously classified as low degree (20-50%) mosaics were identified as euploid by BE PGT, whereas four of the six putative high degree (50-80%) mosaic blastocysts showed chromosomal abnormalities. The raw concordance rates of spent culture medium (SCM) and TE re-biopsies compared with BE were 74.4% and 82%, respectively, in terms of overall ploidy and 96.2% and 97.6%, respectively, per single chromosome when considering all degree mosaic results as true positives. However, when we set a mosaicism identification threshold of 50%, the concordance rates of SCM and TE re-biopsies compared with BE were 87.2% and 85% at the overall ploidy level and 98.8% and 98.3% at the chromosomal level, respectively. At the full ploidy level, the sensitivity and false negative rates for niPGT were 100% and 0, respectively. After adjustment of the threshold for mosaicism, the specificity of niPGT increased from 69.7% to 84.8% in terms of overall ploidy and from 96.1% to 98.9% at the chromosomal level.

 https://www.mdpi.com/1422-0067/22/5/2513

目前偵測胚胎之新興技術包括

Time lapse即時監測+搭配電腦AI綜合評估胚胎等級

以胚胎培養液體偵測

DNA(cell free DNA, 染色體狀況)(類似孕婦nifty技術)

mRNA(miRNA, miR-142-3p)

蛋白質體(mass spectrometry)

代謝產物 

(hCG, IL, stem cell factor (SCF), interferon (IFN), sHLA-G (soluble human leukocyte antigen,l etc)

Current Advancements in Noninvasive Profiling of the Embryo Culture Media Secretome

There have been over 8 million babies born through in vitro fertilization (IVF) and this number continues to grow. There is a global trend to perform elective single embryo transfers, avoiding risks associated with multiple pregnancies. It is therefore important to understand where current research of noninvasive testing for embryos stands, and what are the most promising techniques currently used. Furthermore, it is important to identify the potential to translate research and development into clinically applicable methods that ultimately improve live birth and reduce time to pregnancy. The current focus in the field of human reproductive medicine is to develop a more rapid, quantitative, and noninvasive test. Some of the most promising fields of research for noninvasive assays comprise cell-free DNA analysis, microscopy techniques coupled with artificial intelligence (AI) and omics analysis of the spent blastocyst media. High-throughput proteomics and metabolomics technologies are valuable tools for noninvasive embryo analysis. The biggest advantages of such technology are that it can differentiate between the embryos that appear morphologically identical and has the potential to identify the ploidy status noninvasively prior to transfer in a fresh cycle or before vitrification for a later frozen embryo transfer.

 Day4桑葚期胚胎

融合比率(>50%, 10-50%, <10%)&

脆片比率(<10% 10-50%, >50%)

---可用於評估胚胎未來囊胚比率&懷孕率

Morphological assessment on day 4 and its prognostic power in selecting viable embryos for transfer 

Zygote , Volume 24 , Issue 4 , August 2016 , pp. 477 - 484

DOI: https://doi.org/10.1017/S0967199415000404

The aim of this study was to describe a system for embryo morphology scoring at the morula stage and to determine the efficiency of this model in selecting viable embryos for transfer. In total, 519 embryos from 122 patients undergoing intracytoplasmic sperm injection (ICSI) were scored retrospectively on day 4 according to the grading system proposed in this article. Two separate quality scores were assigned to each embryo in relation to the grade of compaction and fragmentation and their developmental fate was then observed on days 5 and 6. Secondly, the prediction value of this scoring system was compared with the prediction value of the traditional scoring system adopted on day 3. Morulas classified as grade A showed a significant higher blastocyst formation rate (87.2%) compared with grades B, C and D (63.8, 41.3 and 15.0%, respectively), (P < 0.001). Furthermore, the ability to form top quality blastocysts was significantly higher for grade A morulas with respect to grades B, and C and D (37.8% vs. 22.4% vs. 11.1%), (P < 0.001). Finally, the morula scoring system showed more prediction power with respect to the embryo scoring a value of 1 [Akaike information criterion (AIC) index 16.4 vs. 635.3 and Bayesian information criterion (BIC) index −68.8 vs. −30.0 for morulas and embryos respectively]. In conclusion, results demonstrated that the presented scoring system allows for the evaluation of eligible embryos for transfer as a significant correlation between the grade of morula, blastulation rate and blastocyst quality was observed. Furthermore, the morula scoring system was shown to be the best predictive model when compared with the traditional scoring system performed on day 3.

https://www.youtube.com/watch?v=SPLHjFis_4M

胚胎等級從

D1 28小時 

D2 44-48小時  

D3 66-72小時

D4 88-96小時

D5110-120小時

D6-130-140小時

有不同之分類&評估

https://www.researchgate.net/figure/Sample-model-of-a-quality-grading-system-for-oocytes-and-embryos-A-Oocyte-grading_fig1_275098392

https://www.youtube.com/watch?v=SPLHjFis_4M

卵子細胞質內有不規則顆粒狀細胞質通常預後不佳

若呈現halo天使圈般細胞質  則預後不受影響  甚至更好

. 2000 Nov;15(11):2390-3.

 doi: 10.1093/humrep/15.11.2390.

Relationship between granular cytoplasm of oocytes and pregnancy outcome following intracytoplasmic sperm injection

Couples undergoing intracytoplasmic sperm injection (ICSI) for male infertility using oocytes with centrally located granular cytoplasm (CLCG) were evaluated for fertilization, embryo development, implantation and pregnancy rate. CLCG is a rare morphological feature of the oocyte, that is diagnosed as a larger, dark, spongy granular area in the cytoplasm. Severity is based on both the diameter of granular area and the depth of the lesion. Twenty-seven couples with 39 cycles presenting CLCG in >50% of retrieved oocytes were evaluated. A total of 489 oocytes was retrieved, out of which 392 were at MII. CLCG was observed in 258 of the MII oocytes (65. 8%); 66.7% of these oocytes had slight and 33.3% had severe CLCG. The overall fertilization rate was 72.2% and no statistical significant difference was found between normal and CLCG oocytes and between the oocytes representing slight and severe CLCG. The development and quality of embryos was the same in normal and CLCG oocytes. In nine cycles, preimplantation genetic diagnosis was executed to evaluate a possible accompanying chromosomal abnormality. Out of 44 blastomeres biopsied, 23 had chromosomal abnormality (52. 3%). Eleven pregnancies were achieved in 39 cycles (28.2%), six pregnancies resulted in abortion (54.5%). The implantation rate was found to be 4.2%. Only five ongoing pregnancies were achieved in 39 cycles (12.8%). Couples with CLCG oocytes should be informed about poor on-going pregnancy rates even if fertilization, embryo quality and total pregnancy rates are normal. Furthermore, a high aneuploidy rate may be linked to a high abortion rate.

. 2019 May 10;9(1):7215.

 doi: 10.1038/s41598-019-43757-8.

Relationship between the positions of cytoplasmic granulation and the oocytes developmental potential in human

To evaluate the relationship between the positions of cytoplasmic granulation and the oocytes developmental potential in human, we detected the developmental potentials of oocytes with centrally located cytoplasmic granulation (CLCG). The patients' age, body mass index (BMI), Infertility duration, follicle stimulation hormone (FSH) levels, average stimulate ovulation days, gonadotropin (GN) total dose, fertilization rate, cleavage rate, high quality embryo rate, embryo utilization rate and pregnancy rate were analyzed. The results showed that there were no significant difference on patients' age, BMI, infertility duration, FSH levels, average stimulate ovulation days, GN total dose, pregnancy rate and birth rate between CLCG group and control group in patients with BMI < 24 (P > 0.05). However, there was no significant difference in fertilization rate, cleavage rate, and high quality embryo rate in patients with BMI < 24 (P > 0.05). The pregnancy rate was low in both groups, but 35 and 15 healthy fetuses were born in each group. We also found that the central granulated area size did not affect fertilization rate, cleavage rate, embryo utilization rate, and high quality embryo rate (P > 0.05). These results suggested CLCG might be a normal morphology of oocyte. The oocytes from patients with or without CLCG had no significant difference in their developmental potentials. The patients who transferred CLCG embryos had successful delivery. The developmental potentials of oocytes with different CLCG grades had no obvious differences.

https://www.youtube.com/watch?v=SPLHjFis_4M

第一次分裂大多發生在受精後28小時左右

若提前到25小時內則稱之為早期分裂

通常早期分裂之胚胎懷孕率明顯較高(2倍)

若受精後32小時仍未分裂  通常胚胎預後不佳

https://www.researchgate.net/figure/Sample-model-of-a-quality-grading-system-for-oocytes-and-embryos-A-Oocyte-grading_fig1_275098392

This occurrence is checked 23-27 hours after insemination. At this moment, embryos with two blastomeres are classified as early cleavage (EC) embryos and those that do not reach this stage with intact nuclear membranes are classified as no early cleavage (NEC) embryos.  EC embryos show more than two times the pregnancy rate and three times the implantation rate compared with non EC (NEC) embryos. 

內質體ICM等級----是評估囊胚懷孕率最敏感指標  https://studio.youtube.com/video/LV_g3HGJga4/edit

Front. Endocrinol., 24 February 2021

Sec. Reproduction
Volume 12 - 2021 | https://doi.org/10.3389/fendo.2021.621221

The Morphology of Inner Cell Mass Is the Strongest Predictor of Live Birth After a Frozen-Thawed Single Embryo Transfer

Background: The scoring system for human blastocysts is traditionally based on morphology; however, there are controversies on the effect of morphology parameters on pregnancy outcomes. The aim of this study is to evaluate the predicting value of each morphology parameter on pregnancy outcomes in a setting of single embryo transfer.

Methods: This is a retrospective cohort study on patients undergoing frozen-thawed single blastocyst transfer at our center, between Jan. 2009 and Dec. 2018. A total of 10,482 cycles were analyzed. The blastocysts were scored according to the expansion and hatching status, morphology of inner cell mass (ICM), and cells of trophectoderm (TE). The primary outcome measure was live birth rate. One-way analysis of variance, chi-square test, and multiple logistic regression were used for statistical analysis.

Results: The clinical pregnancy rate was lower in the blastocysts of stage 3 (48.15%), compared with those of stage 4 (56.15%), stage 5 (54.91%), and stage 6 (53.37%). The live birth rate was lower in the blastocysts of stage 3 (37.07%), compared with those of stage 4 (44.21%) and stage 5 (41.67%). The rates of clinical pregnancy (A: 66.60%, B: 53.25%, C: 39.33%) and live birth (A: 54.62%, B: 41.29%, C: 28.45%) were both decreased with decreasing grade of ICM morphology, and these differences were pairwise significant. The miscarriage rate of blastocysts with ICM grade A was lower, compared with ICM grade C (17.53 vs. 27.66%). Blastocysts with TE morphology of C had lower rates of clinical pregnancy (43.53%) and live birth (32.57%), compared with those with TE morphology of A and B (clinical pregnancy rate: 64.26% for A, 58.11% for B; live birth rate: 52.74% for A, 45.64% for B). There were no significant differences in rates of clinical pregnancy, live birth, and miscarriage between the blastocysts with TE grade A and B.

Conclusions: The blastocyst expansion stage, ICM grade, and TE grade are all associated with pregnancy outcomes. ICM grade is the strongest predictor of live birth. A blastocyst with stage 4–5, ICM grade A, and TE grade A/B should be given priority for single embryo transfer.

Blastocyst Stage	Quality Grade	Description
Full blastocyst	3	The blastocyst cavity completely fills the embryo
Blastocyst	2	The blastocyst cavity is greater than or equal to half of the volume of the embryo
Early blastocyst	1	The blastocyst cavity is less than half the volume of the embryo
Expanded blastocyst	4	The blastocyst cavity volume is larger than that of the early embryo and the surrounding membrane is thinning
Hatched blastocyst	6	The blastocyst has completely escaped from the surrounding membrane
Hatching blastocyst	5	The outer layer of cells has started to herniate through the surrounding membrane

 

Blastocyst Structure	Grade	Description
Inner Cell Mass	A	Tightly packed, many cells
Inner cell mass	B	Loosely grouped, several cells
Inner cell mass	C	Very few cells

 

Blastocyst Structure	Grade	Description
Trophectoderm	A	Many cells forming a tightly knit epithelium
Trophectoderm	B	Few cells
Trophectoderm	C	Very few cells forming a loose epithelium.