新一代PGT可直接應用於偵測常見單基因疾病偵測, 不須事先set-up 或 pre-testing.

 J Assist Reprod Genet . 2024 Jan;41(1):185-192.

OneGene PGT: comprehensive preimplantation genetic testing method utilizing next-generation sequencing

Purpose: Preimplantation genetic testing for monogenic disorders (PGT-M) allows early diagnosis in embryos conceived in vitro. PGT-M helps to prevent known genetic disorders in affected families and ensures that pathogenic variants in the male or female partner are not passed on to offspring. The trend in genetic testing of embryos is to provide a comprehensive platform that enables robust and reliable testing for the causal pathogenic variant(s), as well as chromosomal abnormalities that commonly occur in embryos. In this study, we describe PGT protocol that allows direct mutation testing, haplotyping, and aneuploidy screening.

Methods: Described PGT protocol called OneGene PGT allows direct mutation testing, haplotyping, and aneuploidy screening using next-generation sequencing (NGS). Whole genome amplification product is combined with multiplex PCR used for SNP enrichment. Dedicated bioinformatic tool enables mapping, genotype calling, and haplotyping of informative SNP markers. A commercial software was used for aneuploidy calling.

Results: OneGenePGT has been implemented for seven of the most common monogenic disorders, representing approximately 30% of all PGT-M indications at our IVF centre. The technique has been thoroughly validated, focusing on direct pathogenic variant testing, haplotype identification, and chromosome abnormality detection. Validation results show full concordance with Sanger sequencing and karyomapping, which were used as reference methods.

Conclusion: OneGene PGT is a comprehensive, robust, and cost-effective method that can be established for any gene of interest. The technique is particularly suitable for common monogenic diseases, which can be performed based on a universal laboratory protocol without the need for set-up or pre-testing.

高達50%囊胚PGS染色體顯示鑲嵌化異常

鑲嵌化異常囊胚植入後仍可能產生正常胚胎嬰兒 

鑲嵌化異常囊胚植入後流產率較高 

Review

 

. 2020 Apr 15;26(3):313-334.

 doi: 10.1093/humupd/dmz050.

Chromosomal mosaicism in human blastocysts: the ultimate diagnostic dilemma

Background: Trophectoderm (TE) biopsy and next generation sequencing (NGS) are currently the preferred techniques for preimplantation genetic testing for aneuploidies (PGT-A). Although this approach delivered important improvements over previous testing strategies, increased sensitivity has also prompted a rise in diagnoses of uncertain clinical significance. This includes reports of chromosomal mosaicism, suggesting the presence of karyotypically distinct cells within a single TE biopsy. Given that PGT-A relies on the chromosomal constitution of the biopsied cells being representative of the entire embryo, the prevalence and clinical implications of blastocyst mosaicism continue to generate considerable controversy.

Objective and rationale: The objective of this review was to evaluate existing scientific evidence regarding the prevalence and impact of chromosomal mosaicism in human blastocysts. We discuss insights from a biological, technical and clinical perspective to examine the implications of this diagnostic dilemma for PGT-A.

Search methods: The PubMed and Google Scholar databases were used to search peer-reviewed publications using the following terms: 'chromosomal mosaicism', 'human', 'embryo', 'blastocyst', 'implantation', 'next generation sequencing' and 'clinical management' in combination with other keywords related to the subject area. Relevant articles in the English language, published until October 2019 were critically discussed.

Outcomes: Chromosomal mosaicism predominately results from errors in mitosis following fertilization. Although it appears to be less pervasive at later developmental stages, establishing the true prevalence of mosaicism in human blastocysts remains exceedingly challenging. In a clinical context, blastocyst mosaicism can only be reported based on a single TE biopsy and has been ascribed to 2-13% of embryos tested using NGS. Conversely, data from NGS studies disaggregating whole embryos suggests that mosaicism may be present in up to ~50% of blastocysts. However, differences in testing and reporting strategies, analysis platforms and the number of cells sampled inherently overshadow current data, while added uncertainties emanate from technical artefacts. Moreover, laboratory factors and aspects of in vitro culture generate further variability. Outcome data following the transfer of blastocysts diagnosed as mosaic remain limited. Current studies suggest that the transfer of putative mosaic embryos may lead to healthy live births, but also results in significantly reduced ongoing pregnancy rates compared to the transfer of euploid blastocysts. Observations that a subset of mosaic blastocysts has the capacity to develop normally have sparked discussions regarding the ability of embryos to self-correct. However, there is currently no direct evidence to support this assumption. Nevertheless, the exclusion of mosaic blastocysts results in fewer embryos available for transfer, which may inevitably compromise treatment outcomes.

常規使用PVP減低精蟲活動力，研究顯示不能暴露時間超過15分鐘，超過會對於精蟲DNA染色體有不量影響

Prolonged exposure of human spermatozoa in polyvinylpyrrolidone has detrimental effects on sperm biological characteristics

Polyvinylpyrrolidone (PVP) has been utilized in intracytoplasmic sperm injection (ICSI) for immobilization and manipulation of spermatozoa. This study aims to determine the suitable time that sperm cells could be safely exposed to PVP during ICSI procedure. Twenty-five normal semen samples were prepared using the swim-up method and then were exposed to 10% PVP at different time intervals (15, 30 and 60 min). The effect of PVP on sperm parameters (viability and morphology), DNA fragmentation index (sperm chromatin dispersion test), chromatin quality (aniline blue, toluidine blue and chromomycin A3 staining), acrosome reaction, mitochondrial membrane potential and sperm ultrastructure was assessed at different time intervals. Our results showed that prolonged sperm exposure in PVP for 15, 30 and 60 min significantly affects viability and morphology with a concomitant increase in DNA fragmentation and abnormal chromatin structure, while the percentage of acrosome-reacted spermatozoa was additionally increased. In addition, the spermatozoa with high mitochondrial membrane potential were significantly decreased compared to unexposed spermatozoa to PVP. In conclusion, the detrimental effects of PVP were increased significantly following sperm exposure in PVP after 15 min. Therefore, the sperm exposure to PVP should be limited to less than 15 min during ICSI procedure.

 PVP使用於精蟲顯微注射之精蟲遲滯

傳統PVP濃度為7%，研究顯示較低濃度之PVP (5%)可能有助於提高胚胎

Effects of different polyvinylpyrrolidone concentrations on intracytoplasmic sperm injection

Published online by Cambridge University Press:  14 January 2020

Summary

To explore whether different polyvinylpyrrolidone (PVP) concentrations affect the results of intracytoplasmic sperm injection (ICSI), a prospective study was conducted for 194 couples undergoing 210 ICSI therapy cycles. These cycles were divided into three groups (10, 7 and 5% groups) using the corresponding concentration of PVP for sperm immobilization. The main outcome measures were analyzed. Results indicated that, with a decrease in PVP concentrations, all of the main outcome measures increased. In particular, the high-quality cleavage embryo rate in the 7% group was significantly lower than in the 5% group (P < 0.01), and the cleavage, high-quality cleavage embryo, and high-quality blastocyst rates in the 5% group were significantly higher than those in the 10% group (all P < 0.001). For high-/intermediate-quality semen, all of the main outcome measures were significantly increased with 5% PVP. For the poor-quality semen, only the high-quality cleavage embryo and high-quality blastocyst rates were significantly higher in the 5% group. Therefore, lowering PVP concentrations greatly promoted the development of embryos in ICSI cycles, with an optimal concentration of 5% for ICSI.

是否D3更新培養液並無臨床差異

one step   vs two step 囊胚率: 68.3 vs. 66.8%)

Fertil Steril  2016 Mar;105(3):707-713.

 doi: 10.1016/j.fertnstert.2015.11.038. Epub 2015 Dec 12.

Blastocyst development in single medium with or without renewal on day 3: a prospective cohort study on sibling donor oocytes in a time-lapse incubator

Objective: To evaluate the efficiency of using a continuous (one-step) protocol with a single medium for the culture of human embryos in a time-lapse incubator (TLI).

Patient(s): Embryos from 59 patients.

Intervention(s): Culture in a TLI in a single medium with or without renewal of the medium on day-3.

Main outcome measure(s): Embryo morphology and morphokinetic parameters, clinical pregnancy, take-home baby rate, and perinatal outcomes.

Result(s): The blastocyst rates (68.3 vs. 66.8%) and the proportion of good-quality blastocysts (transferred plus frozen) obtained with the two-step (80.0%) protocol were statistically significantly similar to those obtained in the one-step protocol (72.2%). Similarly, morphokinetic events from early cleavage until late blastocyst stages were statistically significantly equivalent between both groups. No differences were found either in clinical pregnancy rates when comparing pure transfers performed with embryos selected from the two-step (75.0%), one-step (70.0%, respectively), and mixed (57.1%) groups. A total of 55 out of 91 embryos transferred implanted successfully (60.4%), resulting in a total of 37 newborns with a comparable birth weight mean among groups.

Conclusion(s): Our findings support the idea that in a TLI with a controlled air purification system, human embryos can be successfully cultured continuously from day 0 onward in single medium with no need to renew it on day-3. This strategy does not affect embryo morphokinetics or development to term and offers more stable culture conditions for embryos as well as practical advantages and reduced costs for the IVF laboratory.

傳統培養箱  vs  time lapse培養

懷孕率無差異

. 2018 Feb;109(2):302-309.e1.

 doi: 10.1016/j.fertnstert.2017.10.008. Epub 2017 Nov 23.

Randomized controlled trial comparing embryo culture in two incubator systems: G185 K-System versus EmbryoScope

Objective: To study whether the closed culture system, as compared with a benchtop incubator with similar culture conditions, has a positive impact on intracytoplasmic sperm injection (ICSI) outcomes.

Patient(s): A total of 386 patients undergoing ICSI cycles with at least six mature oocytes were randomized.

Intervention(s): Of these patients, 195 were assigned to the group with culture in a time-lapse imaging (TLI) system (EmbryoScope) and 191 to the group with culture in the G185 K-System (G185).

Main outcome measure(s): Rate of implantation (primary endpoint) and embryo morphology grade.

Result(s): No significant differences were found in the implantation rates. The proportion of high-grade embryos on day 2 was significantly higher in the TLI group compared with the G185 group (40.4% vs. 35.2%). The impact of the incubator on embryo morphology remained significant in multivariate analysis, which took into account the woman's age, the rank of attempt, and the smoking status (TLI vs. G185: odds ratio = 1.27; 95% confidence interval, [1.04-1.55]). No difference was found in the mean number of frozen embryos, even though the total proportion of frozen embryos was significantly higher in the TLI group than in the G185 group (29.5% vs. 24.8%).

Conclusion(s): No difference in implantation rate was found between the two incubators for fresh cycles. It remains to be determined whether the observed differences in embryo morphology and the total number of embryos cryopreserved would translate into higher cumulative outcomes with subsequent frozen embryo transfers.

 time-lapse(不取出觀察) vs傳統培養箱(定期取出觀察)

胚胎DNA甲基化程度並無差異(DNA甲基化程度越高代表外界干擾程度越大)

結論: 

胚胎有一定抵抗外界環境變動之能力  短暫取出觀察溫度環境變動對胚胎影響並不太大

\

. 2023 Jan;40(1):113-123.

 doi: 10.1007/s10815-022-02669-9. Epub 2022 Dec 2.

Comparison of DNA methylation profiles of human embryos cultured in either uninterrupted or interrupted incubators

Purpose: We aimed to compare the DNA methylation profiles of human embryos cultured in uninterrupted or interrupted incubators.

Methods: This study included 9 women, ≤ 30 years old (range: 20-30 years), without a history of genetic diseases or smoking, undergoing ICSI treatment, and each woman donated one oocyte. Embryos were randomly assigned to culture in either time-lapse imaging or standard incubators after ICSI. We compared the DNA methylation profiles of human eight-cell embryos cultured in uninterrupted condition using time-lapse imaging (TLI) incubator (EmbryoScope) to those cultured in interrupted culture model using standard incubators (SI, G185 K-System). Nine single-cell whole-genome bisulfite sequencing (WGBS) datasets were analyzed, including four SI-cultured embryos and five TLI-cultured embryos at the eight-cell stage.

Results: A total of 581,140,020 and 732,348,182 clean reads were generated from the TLI and SI groups, respectively. TLI-cultured embryos had similar genome-wide methylation patterns to SI-cultured embryos. There were no significant differences in the methylation and transcription levels of transposable elements and imprinted control regions. Although a total of 198 differentially methylated genes (DMGs) were identified, only five DMGs had significantly different transcription levels between the two groups.

Conclusions: This is the first study to compare the DNA methylation profiles of embryos cultured in TLI and SI and will provide a foundation for evaluating the safety of TLI application in assisted reproductive technologies. However, further study with a larger cohort of samples was needed for the data validation.

PGS是否提高懷孕率仍具爭議  

本篇指出年輕ㄉIVF施行PGS並無明顯提升累積懷孕率(甚至有下降疑慮)

. 2023 Jan;40(1):137-149.

 doi: 10.1007/s10815-022-02667-x. Epub 2022 Dec 1.

PGT-A is associated with reduced cumulative live birth rate in first reported IVF stimulation cycles age ≤ 40: an analysis of 133,494 autologous cycles reported to SART CORS

Purpose: To evaluate the impact of preimplantation genetic testing for aneuploidy (PGT-A) on cumulative live birth rate (CLBR) in IVF cycles.

Methods: Retrospective cohort study of the SART CORS database, comparing CLBR for patients using autologous oocytes, with or without PGT-A. The first reported autologous ovarian stimulation cycle per patient between January 1, 2014, and December 31, 2015, and all linked embryo transfer cycles between January 1, 2014, and December 31, 2016, were included in the study. Exclusion criteria were donor oocyte cycles, donor embryo cycles, gestational carrier cycles, cycles which included both a fresh embryo transfer (ET) combined with a thawed embryo previously frozen (ET plus FET), or cycles with a fresh ET after PGT-A.

Results: A total of 133,494 autologous IVF cycles were analyzed. Amongst patients who had blastocysts available for either ET or PGT-A, including those without transferrable embryos, decreased CLBR was noted in the PGT-A group at all ages, except ages > 40 (p < 0.01). A subgroup analysis of only those patients who had PGT-A and a subsequent FET, excluding those without transferrable embryos, demonstrated a very high CLBR, ranging from 71.2% at age < 35 to 50.2% at age > 42. Rates of multiple gestations, preterm birth, early pregnancy loss, and low birth weight were all greater in the non-PGT-A group.

Conclusions: PGT-A was associated with decreased CLBR amongst all patients who had blastocysts available for ET or PGT-A, except those aged > 40. The negative association of PGT-A use and CLBR per cycle start was especially pronounced at age < 35.

 取卵過程汙染造成卵泡液微生物會導致懷孕率下降  (19.7% vs. 32.2%)

Review

 

. 2023 Nov;40(11):2501-2511.

 doi: 10.1007/s10815-023-02912-x. Epub 2023 Sep 9.

Associations between microbial presence in follicular fluid with IVF outcomes: a systematic review and meta-analysis

Purpose: The aim of the study was to synthesize disparate studies to investigate potential impact of microbial presence in FF of infertile women on IVF outcomes.

Methods: Following preliminary searches to find medical subject heading (MeSH) terms plus free terms, a systematic search was performed in the PubMed, Cochrane Library, Embase, Web of Science, and Clinicaltrials.gov databases from January 10, 2022, to July 5, 2023. Data collected for each study were analyzed using RevMan 5.4 software available on the Cochrane website.

Results: After correcting for contamination from the vagina, the FFs of 289 women were detected positively by microbial culture and identification, ELISA, and IPA. The pregnancy rate of the FF-positive group was significantly lower than the FF-negative group (19.7% vs. 32.2%) and (OR: 0.57, 95% CI: 0.28-1.14, P=0.11; I2=56%) while the fertilization rate was almost equal (60.0% vs. 62.0%) and (OR: 1.03, 95% CI: 0.88-1.20, P=0.72; I2=0%). Evidence quality was very low.

Conclusions: The different species of microorganisms in FF of infertile women may have different effects on IVF outcomes. The Lactobacillus spp. may have a positive effect, while other microorganisms may have the opposite effect.

 Letrozole 使用於冷凍胚胎植入療程可能可下降胎兒生長過重& 懷孕高血壓

. 2023 Dec;40(12):2885-2894.

 doi: 10.1007/s10815-023-02956-z. Epub 2023 Oct 10.

Letrozole use in vitrified single-blastocyst transfer cycles is associated with lower risk of large for gestational age infants in patients with polycystic ovary syndrome

Purpose: To evaluate the obstetric and perinatal outcomes of three routine endometrial preparation protocols in women with PCOS who underwent frozen embryo transfer (FET).

Methods: This was a retrospective study in women with PCOS who underwent FET in an academic reproductive medical center. A total of 2710 cycles were enrolled and classified into three groups according to different endometrial preparation protocols; human menopausal gonadotropin (HMG), letrozole + HMG, or hormone replacement therapy (HRT).

Results: The stimulation groups had reduced risks of hypertensive disorders of pregnancy (HDP), large for gestational age (LGA) infants, and cesarean delivery than the HRT group. After adjustment for different confounder combinations in the two models, the frequencies of LGA and HDP in the letrozole + HMG group and the HMG group were still significantly lower than those in the HRT group. The letrozole + HMG group exhibited a reduced risk of LGA than HMG group after adjustment of confounders. A trend toward risk reductions in HDP and LGA was observe in turns of HRT, HMG, and letrozole + HMG groups, and the trends were statistically significant (Ptrend = 0.031 and 0.001).

Conclusion: In patients with PCOS, ovarian stimulation protocols for endometrial preparation are associated with reduced risks of HDP and LGA compared to HRT cycles. The use of letrozole could further reduce risk of LGA compared to HMG only protocol. We propose that ovarian stimulation protocols can be used widely for endometrial preparation in FET cycles in women with PCOS, especially with the use of letrozole.

 2段式胚胎植入( afterload catheters 先放外管後 再吸取胚胎植入內管)  

懷孕率優於

1段式胚胎植入(direct catheters 外管內管同時一起植入)

. 2023 Dec;40(12):2895-2902.

 doi: 10.1007/s10815-023-02957-y. Epub 2023 Oct 11.

Steps forward in embryo transfer technique: a retrospective study comparing direct versus afterload catheters at different time frames

Purpose: To assess whether embryo transfer (ET) technique can influence the clinical pregnancy rate (CPR) and its correlation with the embryo transfer difficulty.

Design: This single center retrospective cohort analysis of fresh and frozen single blastocyst transfers performed between January 2016 and December 2021 included fresh and frozen single blastocyst transfers performed during the study timeframe. Direct technique was the only one used from January 2016 to September 2017. From September 2017 to March 2019, the choice between the two techniques was given by randomization, due to a clinical trial recruitment. From April 2019, only the afterload technique was used. Preimplantation genetic testing cycles and gamete donation procedures and cycles performed with external gametes or embryos were excluded. CPR was the primary outcome, while difficult transfer rate the secondary one. Univariate and multivariate logistic regressions were performed.

Results: During the period, 8,189 transfers were performed. CPR of the afterload group resulted significantly higher compared to the direct group (44.69% versus 41.65%, OR 1.13, 95% CI 1.02-1.25, p = 0.017) and the rate of difficult transfers two-thirds lower (9.06% versus 26.85%, OR 0.27, 95% CI 0.24-0.31, p < 0.001).

Conclusion: Our study demonstrated that CPR is significantly affected by the ET technique. In particular, with the afterload protocol, both CPR and easy transfer rates increased.

雷射Laser輔助孵化可輕微提高卵裂期胚胎凍融胚胎移植週期的懷孕率

Laser vs Non-Laser 

臨床懷孕率40.7% vs. 38.3%

活產率34.9% vs. 31.4% 

Laser-assisted hatching improves pregnancy outcomes in frozen-thawed embryo transfer cycles of cleavage-stage embryos

J Assist Reprod Genet. 2023 Feb; 40(2): 417–427.

Published online 2023 Jan 7. doi: 10.1007/s10815-022-02711-w

PMCID: PMC9935798

PMID: 36609944

Laser-assisted hatching improves pregnancy outcomes in frozen-thawed embryo transfer cycles of cleavage-stage embryos: a large retrospective cohort study with propensity score matching

Introduction

Laser-assisted hatching (LAH) is a commonly used adjunct technique; however, its effectiveness has not been fully established.

Objective

We evaluated the effects of LAH on pregnancy outcomes in frozen-thawed embryo transfer (FET) cycles of cleavage-stage embryos.

Materials and methods

This retrospective study involved 5779 FET cycles performed at the Reproductive and Genetic Center in the Affiliated Hospital of Shandong University of Traditional Chinese Medicine between January 2016 and December 2020. After propensity score matching, 3535 FET cycles were included, out of which 1238 were subjected to LAH while the remaining 2297 cycles were non-LAH (NLAH). The primary outcomes were clinical pregnancy rate (CPR) and live birth rate (LBR) while secondary outcomes included implantation rate (IR), biochemical pregnancy rate (BPR), ectopic pregnancy rate (EPR), pregnancy loss rate (PLR), multiple pregnancy rate (MPL), and monozygotic twinning rate (MTR). Logistic regression analysis was conducted to adjust for possible confounders. Subgroup analysis was also performed based on the endometrial preparation regimen.

Results

The LAH group exhibited a higher LBR, compared to the NLAH group (34.9% vs. 31.4%, OR = 1.185, 95% CI = 1.023, 1.374, P = 0.024). Additionally, the LAH group showed a decreasing trend in PLR and EPR; however, differences were insignificant (P = 0.078, P = 0.063 respectively). Differences in IR (24.6% vs. 24.3%), BPR (41.8% vs. 40.4%), CPR (40.7% vs. 38.3%), MPR (14.1% vs. 17.3%), and MTR (1.4% vs. 1.1%) were insignificant. Subgroup analysis revealed that LAH may be more conducive for pregnancy outcomes in hormone replacement cycles.

Conclusions

In summary, LAH has an increased chance of achieving live births. However, further prospective studies should be performed to confirm our findings.