以囊胚培養液偵測囊胚DNA仍不可靠  最大問題為母體DNA之汙染干擾胚胎針測之準確性

Fertil Steril. 2018 Oct;110(5):870-879.e5. doi: 10.1016/j.fertnstert.2018.05.031.

Diagnostic efficacy of blastocoel fluid and spent media as sources of DNA for preimplantation genetic testing in standard clinical conditions.

Capalbo A1, Romanelli V2, Patassini C2, Poli M3, Girardi L2, Giancani A4, Stoppa M4, Cimadomo D4, Ubaldi FM4, Rienzi L4.

Author information

Erratum in

• Erratum. [Fertil Steril. 2019]

Abstract

OBJECTIVE:

To determine whether blastocoel fluid (BF) or spent blastocyst medium (SBM) is a suitable template for genotype and/or karyotype assessment of in vitro fertilization-generated embryos.

DESIGN:

Prospective blinded study.

SETTING:

Genetic laboratory.

PATIENT(S):

From 26 patients undergoing preimplantation genetic testing (PGT) treatments, 103 trophectoderms (TE), 92 BF samples, and 72 SBM samples.

INTERVENTION(S):

The BF and SBM were retrieved at the time of TE biopsy. Two DNA extraction strategies were evaluated on independent BF and SBM samples. Further enrolled samples were processed using next-generation sequencing and quantitative polymerase chain reaction for assessment of monogenic disorders (PGT-M) or aneuploidy (PGT-A).

MAIN OUTCOME MEASURE(S):

DNA amplification and concordance rates across BF, SBM, and TE to assess diagnostic efficiency.

RESULT(S):

No differences were detected among the DNA extraction methods tested. In PGT-M tests, for BF and SBM, 2.9% and 20.8% of all samples, respectively, produced a diagnosis concordant with the corresponding TE (n = 2 of 69 and 15 of 72, respectively). The SBM samples were associated with higher discordance rates and higher artifacts/contamination detection compared with BF. In multiple occasions, the maternal mutated variant was detected in the SBM of homozygous wild-type embryos, showing evidence of maternal DNA persistence in culture medium. In PGT-A tests, BF analysis showed high amplification failure rates (65.2%) and an overall concordance rate of 37.5% among amplified samples.

CONCLUSION(S):

Based on current methodologies, BF and SBM genetic analyses do not provide sufficiently reliable results to be employed clinically. Until the risk of maternal contamination can be properly prevented, SBM should not be used for PGT-M purposes.

AZF基因缺損除了造成精蟲品質異常外  還可能造成胚胎品質下降  神精精神系統異常   癌症發生

J Assist Reprod Genet. 2019 Jun 18. doi: 10.1007/s10815-019-01492-z. [Epub ahead of print]

Consequences of Y chromosome microdeletions beyond male infertility.

Colaco S1, Modi D2.

Author information

Abstract

PURPOSE:

The human Y chromosome plays a central role in sex determination and spermatogenesis. The azoospermia factor (AZF) loci on the Y chromosome contain genes that were thought to be testis specific with their deletions leading to spermatogenic failure. However, beyond the testis, the AZF genes (mainly those in AZFa and AZFb loci) are widely expressed in multiple tissues. Further, these genes are predicted to play roles in processes such as gene regulation and protein synthesis. These observations suggest that the AZF genes may have functions beyond regulation of fertility.

RESULTS:

Three major areas have emerged where alternations in AZF genes have effects beyond infertility. (1) Poor-quality embryos are generated in assisted reproduction when sperm from men harboring Y chromosome microdeletions are used, (2) a higher preponderance of neuropsychiatry disorders is observed in men with deletions in AZF genes, and (3) copy number variations and altered expression of AZF genes are found in several cancers.

CONCLUSION:

While our data is preliminary and observational in nature, systematic studies are required to address how genetic alterations in the Y chromosome can affect the health of men beyond infertility. This information will provide a different perspective in the area of androgenetics and have implications in devising strategies for maintaining the overall well-being of infertile males.

PGS ---切片TE 染色體異常假陽性率 7.5%
D3 胚胎切片染色體異常假陽性率30%

The clinicians´ dilemma with mosaicism—an insight from inner cell mass biopsies

B Lawrenz I El Khatib A Liñán A Bayram A Arnanz R Chopra N De MunckH M Fatemi

Human Reproduction, Volume 34, Issue 6, June 2019, Pages 998–1010,https://doi.org/10.1093/humrep/dez055

Published:

 

22 May 2019

 

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Abstract

STUDY QUESTION

How reliable are cleavage stage and trophectoderm (TE) biopsies compared to inner cell mass (ICM) biopsies?

SUMMARY ANSWER

The reliability of TE biopsy compared to ICM biopsy is almost perfect, but only substantial between cleavage stage biopsy and ICM biopsy.

WHAT IS KNOWN ALREADY

One of the prevailing reasons for implantation failure is presumed to be chromosomal aneuploidy in human preimplantation embryos. Preimplantation genetic testing for aneuploidies (PGT-A) has been introduced into assisted reproduction in an effort to increase pregnancy rates. Increasing evidence indicates that genetic results obtained following blastomere or TEbiopsy may not accurately reflect the true genetic status of the embryo due to the presence of embryonic mosaicism, and therefore the reliability of PGT is highly controversial.

STUDY DESIGN, SIZE, DURATION

This was an observational descriptive study, performed in a private infertility centre from August 2016 to January 2017.

PARTICIPANTS/MATERIALS, SETTING, METHODS

The mean female age was 33.9 years, ranging from 24 to 46 years, and the mean number of biopsied embryos per couple was 2.2 (range 1–7 embryos). Blastomere biopsies had been performed at cleavage stage on Day 3 (D3) due to the turnover time of genetic testing and the inability to cryopreserve embryos in accordance with the local law governing ART. To confirm the genetic results in embryos not chosen for transfer, additional biopsies of the TE at blastocyst stage (BLASTO–TE) as well as of the ICM (BLASTO–ICM) were performed on D5. Only surplus blastocysts, which had not been selected for transfer and were not cryopreserved in accordance with the law governing ART, had been included.

MAIN RESULTS AND THE ROLE OF CHANCE

Comparison of all biopsies (D3/BLASTO–ICM/BLASTO–TE) per embryo demonstrated that 50 (59.5%) out of 84 embryos showed concordance in all three results (= full concordance). Thirty-four (40.4%) embryos had at least two discordant results between the three biopsies, regardless of whether the embryo diagnosis (aneuploid/euploid) was discordant or not, or in aneuploid embryos, whether the chromosomal patterns were inconsistent. Nine (= 10.7%) embryos had complete discordance between all three biopsies. False positive results between D3/BLASTO–TE, D3/BLASTO–ICM and BLASTO–TE/BLASTO–ICM were 26.4%/30.2% and 7.5%, respectively, while the Kappa agreement between the different approaches was 0.647, 0.553 and 0.857, respectively. Therefore the reliability of D3/BLASTO–TE, D3/BLASTO–ICM and BLASTO–TE/BLASTO–ICM can be interpreted as substantial, as moderate and as almost perfect.

捐贈卵子年齡    <25歲  vs  25-30歲   
懷孕率無明顯差異

J Assist Reprod Genet. 2019 Jun 10. doi: 10.1007/s10815-019-01494-x. [Epub ahead of print]

Is younger better? Donor age less than 25 does not predict more favorable outcomes after in vitro fertilization.

Humphries LA1,2, Dodge LE1,2,3, Kennedy EB1, Humm KC1,2,4,5, Hacker MR1,2,3, Sakkas D6.

Author information

Abstract

OBJECTIVE:

To determine whether younger oocyte donor age is associated with better outcomes after in vitro fertilization (IVF) compared with older oocyte donor age.

DESIGN:

A retrospective cohort study.

SETTING:

Large academically affiliated infertility treatment center.

PATIENTS:

We included all women ≥ 18 years who started their first fresh cycle using donor oocytes at our center from January 2002 through October 2017; only the first oocyte recipient cycle was analyzed.

INTERVENTION:

Log-binomial regression was used to compare the incidence of clinical pregnancy and live birth among the following donor age groups: < 25 years, 25 to < 30 years, and 30 to <35 years.

MAIN OUTCOME MEASURE:

Incidence of clinical pregnancy and live birth among donor age groups.

RESULTS:

We included 774 donor cycles; 269 (34.8%) used donors < 25 years, 399 (51.6%) used donors 25 to < 30 years, and 106 (13.7%) used donors 30 to < 35 years. Median donor age was 26 years (range 18-34.5), and median recipient age and partner age were both 42 years. Per cycle start, after adjusting for recipient age, cycles using donors < 25 years were not associated with a higher incidence of clinical pregnancy (RR 0.90; 95% CI 0.77-1.06) or live birth (RR 0.87; 95% CI 0.72-1.04) compared with donors age 25-< 30 years.

CONCLUSIONS:

Donor age < 25 was not associated with better outcomes after IVF. Under the age of 30, the prioritization of <25 year old donors may not be recommended given the lack of evidence for superior pregnancy or live birth outcomes.

未來PGSㄉ趨勢可能是蒐集培養液內之DNA 用以取代囊胚切片  如同母血胎兒基因篩檢取帶羊水穿刺

Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications

Carmen Rubio, Ph.D.a,∗,Correspondence information about the author Ph.D. Carmen RubioEmail the author Ph.D. Carmen Rubio

, 

Laura Rienzi, M.Sc.b

, 

Luis Navarro-Sánchez, Ph.D.a

, 

Danilo Cimadomo, Ph.D.b

, 

Carmen María García-Pascual, Ph.D.a

, 

Laura Albricci, Ph.D.b

, 

Daria Soscia, M.Sc.b

, 

Diana Valbuena, M.D., Ph.D.c

, 

Antonio Capalbo, Ph.D.d

, 

Filippo Ubaldi, M.D., Ph.D.b

, 

Carlos Simón, M.D., Ph.D.a,e,f,g,h

DOI: https://doi.org/10.1016/j.fertnstert.2019.04.038

Article Info

• Abstract

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Objective

To study whether embryonic cell-free DNA (cfDNA) in spent blastocyst media is representative of the chromosomal constitution of a blastocyst.

Design

Pilot prospective blinded study.

Setting

In vitro fertilization center and genetics laboratory.

Patient(s)

A total of 115 trophectoderm (TE) biopsies and spent blastocyst media (SBM) from 46 patients with ages ranging from 32 to 46 years, whose indications for preimplantation genetic testing of aneuploidy (PGT-A) were advanced maternal age, recurrent miscarriage, or recurrent implantation failure.

Interventions(s)

Embryo collection after TE biopsy.

Main Outcome Measure(s)

Concordance rates, sensitivity, and specificity between TE biopsies and SBM. Clinical outcomes in cases with euploid TE biopsies and euploid SBM compared with cases with euploid TE and aneuploid SBM.

Result(s)

In general, the total concordance rate for ploidy and sex was 78.7%, and sensitivity and specificity were 94.5% and 71.7%, respectively. A significant increase for all parameters was observed for day 6/7 samples compared with day 5 samples, with day 6/7 samples showing total concordance for ploidy and sex of 84%, and sensitivity and specificity of 95.2% and 82.1%, respectively. Ongoing implantation rates in euploid TE/euploid SBM showed a threefold increase compared with euploid TE/aneuploid SBM (52.9% vs. 16.7%, respectively), without reaching significant differences. Interestingly, no miscarriages were observed when TE and SBM were euploidy concordant.

Conclusion(s)

These results offer a better understanding of the dynamics of cfDNA during embryo development and despite more basic research being needed, they are reassuring to consider in the future this noninvasive approach as an alternative to TE biopsy for PGT-A.

IVF受孕之囊胚約25%有基因mosaicism
ICSI受孕之囊胚約20%有基因mosaicism

J Assist Reprod Genet. 2019 Jan;36(1):153-157. doi: 10.1007/s10815-018-1347-6. Epub 2018 Oct 25.

Minimizing mosaicism: assessing the impact of fertilization method on rate of mosaicism after next-generation sequencing (NGS) preimplantation genetic testing for aneuploidy (PGT-A).

Palmerola KL1,2, Vitez SF3, Amrane S4,3, Fischer CP5, Forman EJ4.

Author information

Abstract

PURPOSE:

Advances in preimplantation genetic testing (PGT) have led to practice changes in assisted reproductive technologies (ART), enabling fertility centers to transfer single embryos while maintaining excellent ongoing pregnancy rates, reducing miscarriage rates, and dramatically reducing ART-associated multiple pregnancies. The introduction of next-generation sequencing (NGS) allows PGT laboratories to assess for embryo mosaicism-although the true incidence and reproductive potential of predicted mosaic embryos are controversial. Due to concern for genetic contamination from other spermatozoa, most reference laboratories require use of intracytoplasmic sperm injection (ICSI) for single gene preimplantation genetic diagnosis (PGT-M). However, in PGT for aneuploidy (PGT-A), conventional insemination (IVF) is typically permissible. The purpose of this study was to evaluate rates of euploid, aneuploid, and mosaic in trophectoderm biopsy samples from embryos in IVF versus ICSI PGT-A cycles. Secondary aims were to assess sex ratio, and subtypes of aneuploidy and mosaicism in IVF versus ICSI PGT-A cycles.

METHODS:

We performed a retrospective review of women undergoing PGT-A at a single academic fertility center from July 1, 2015, to September 1, 2017. In all cycles, PGT-A was performed via trophectoderm biopsy on day 5 or 6 and analyzed using NGS at a single reference lab. We collected and compared patient demographics, fertility testing, cycle characteristics, and PGT-A outcomes between IVF and ICSI cycles.

RESULTS:

Three hundred two PGT-A cycles were included for analysis: 75 IVF and 227 ICSI cycles, resulting in 251 IVF and 724 ICSI biopsied blastocysts. Mean oocyte age of included cycles was 38.6 years (IVF) and 38.5 years (ICSI), p = 0.85. Baseline characteristics of IVF and ICSI PGT-A cycles were similar with the exception of semen parameters: IVF cycles had higher sperm concentration and total motility compared to ICSI cycles. PGT-A outcomes did not differ between IVF and ICSI cycles: euploid 27.9% (IVF) versus 30% (ICSI); aneuploid 45.4% (IVF) versus 43.1% (ICSI); no result 4.4% (IVF) versus 6.2% (ICSI). Though not significant, we identified a trend toward higher rate of mosaicism in IVF (25.9%) versus ICSI (20.9%). Among mosaic embryos, a lower percentage of simple mosaic embryos resulted from IVF (53.8%) versus ICSI (70.2%). Among aneuploid embryos, a non-significant higher percentage of complex aneuploidy resulted from IVF (16.3%) versus ICSI (9%). IVF resulted in a non-significant higher proportion of cycles with no transferrable embryos (42.7%) versus ICSI (36.6%). Numerical and sex chromosome involvement in mosaicism and aneuploidy were similar between IVF and ICSI cycles.

CONCLUSION:

IVF and ICSI NGS PGT-A have similar rates of euploid, aneuploid, and no result embryos, though IVF may result in higher rates of mosaicism and demonstrates differences in proportions of mosaic and aneuploid subtypes compared to ICSI. ICSI may be preferable to conventional insemination to minimize the rate of mosaic results in NGS PGT-A cycles.

子宮腺肌症病患施行胚胎植入前有必要做endometrialreceptivity array (ERA)確定是否為適當之胚胎著床時機window of implantation (WOI) 

J Hum Reprod Sci. 2018 Oct-Dec;11(4):353-358. doi: 10.4103/jhrs.JHRS_52_18.

Window of Implantation is Significantly Displaced in Patients with Adenomyosis with Previous Implantation Failure as Determined by Endometrial Receptivity Assay.

Mahajan N1, Kaur S2, Alonso MR3.

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Abstract

BACKGROUND:

Adenomyosis is associated with implantation failure and poor reproductive performance in IVF/ICSI cycles.

AIMS:

To compare if window of implantation (WOI) is displaced in patients having adenomyosis compared to controls using endometrialreceptivity array (ERA) test.

SETTINGS AND DESIGN:

Retrospective Case control study. 374 patients with previous one or more IVF failures who underwent ERA test between 2013-2016 at our centre were enrolled. Patients were divided into two groups; Group A-36 patients with adenomyosis (study group) and Group B- 338 patients without adenomyosis (controls).

STATISTICAL ANALYSIS:

Normality assumptions for continuous variables were tested using Kolmogorov Smirnov test. Mean values of two groups were compared using Student's t-independent test. Frequency data by categories were compared using Chi-square/Fisher's exact test. Risk ratio and 95% confidence limits were calculated. P < 0.05 was considered for statistical significance.

RESULTS:

WOI was displaced (Non Receptive ERA) significantly in adenomyosis 47.2% (17/36) compared to controls 21.6% (73/338) (P < 0.001, CI-8.7%-42.5%) making risk ratio of displaced WOI in adenomyosis versus controls to be 2:1. The incidence of RIF was 66.6% in adenomyosis compared to 34.9% in controls (P < 0.001, CI- 15.5%-47.9%). Pregnancy rate after personalized embryo transfer in adenomyosis group was 62.5%, signifying displaced WOI as a cause of implantation failure in adenomyosis patients with previous implantation failure.

CONCLUSIONS:

Our study suggests it is prudent to evaluate Endometrial receptivity before embryo transfer in patients with adenomyosis to avoid wastage of good embryos.

endometrial receptivity array (ERA)對於囊胚著床失敗之病患可能有相當助益

J Assist Reprod Genet. 2018 Apr;35(4):683-692. doi: 10.1007/s10815-017-1112-2. Epub 2018 Jan 11.

The role of the endometrial receptivity array (ERA) in patients who have failed euploid embryo transfers.

Tan J1, Kan A1, Hitkari J1,2, Taylor B1,2, Tallon N1,2, Warraich G1,2, Yuzpe A1,2, Nakhuda G3,4.

Author information

Abstract

PURPOSE:

Endometrial receptivity issues represent a potential source of implantation failure. The aim of this study was to document our experience with the endometrial receptivity array (ERA) among patients with a history of euploid blastocyst implantation failure. We investigated whether the contribution of the endometrial factor could be identified with the ERA test and if actionable results can lead to improved outcomes.

METHODS:

A retrospective review was performed for 88 patients who underwent ERA testing between 2014 and 2017. Reproductive outcomes were compared for patients undergoing frozen embryo transfer (FET) using a standard progesterone protocol versus those with non-receptive results by ERA and subsequent FET according to a personalized embryo transfer (pET) protocol.

RESULTS:

Of patients with at least one previously failed euploid FET, 22.5% had a displaced WOI diagnosed by ERA and qualified for pET. After pET, we found that implantation and ongoing pregnancy rates were higher (73.7 vs. 54.2% and 63.2 vs. 41.7%, respectively) compared to patients without pET, although differences were not statistically significant.

CONCLUSIONS:

Our experience demonstrates that a significant proportion of patients with a history of implantation failure of a euploid embryo have a displaced WOI as detected by the ERA. For these patients, pET using a modified progesterone protocol may improve the outcomes of subsequent euploid FET. Larger randomized studies are required to validate these results.

口服黃體素Duphaston (30mg/d) vs 陰道塞劑黃體素(crinone)
對於IVF-ET無統計差異

Hum Reprod. 2018 Dec 1;33(12):2212-2221. doi: 10.1093/humrep/dey306.

Oral dydrogesterone versus intravaginal micronized progesterone gel for luteal phase support in IVF: a randomized clinical trial.

Griesinger G1, Blockeel C2, Sukhikh GT3, Patki A4, Dhorepatil B5, Yang DZ6, Chen ZJ7, Kahler E8, Pexman-Fieth C9, Tournaye H2.

Author information

Abstract

STUDY QUESTION:

Is oral dydrogesterone 30 mg daily non-inferior to 8% micronized vaginal progesterone (MVP) gel 90 mg daily for luteal phase support in IVF?

SUMMARY ANSWER:

Oral dydrogesterone demonstrated non-inferiority to MVP gel for the presence of fetal heartbeats at 12 weeks of gestation (non-inferiority margin 10%).

WHAT IS KNOWN ALREADY:

The standard of care for luteal phase support in IVF is the use of MVP; however, it is associated with vaginal irritation, discharge and poor patient compliance. Oral dydrogesterone may replace MVP as the standard of care if it is found to be efficacious with an acceptable safety profile.

STUDY DESIGN, SIZE, DURATION:

Lotus II was a randomized, open-label, multicenter, Phase III, non-inferiority study conducted at 37 IVFcenters in 10 countries worldwide, from August 2015 until May 2017. In total, 1034 premenopausal women (>18 to <42 years of age) undergoing IVF were randomized 1:1 (stratified by country and age group), using an Interactive Web Response System, to receive oraldydrogesterone 30 mg or 8% MVP gel 90 mg daily.

PARTICIPANTS/MATERIALS, SETTING, METHODS:

Subjects received either oral dydrogesterone (n = 520) or MVP gel (n = 514) on the day of oocyte retrieval, and luteal phase support continued until 12 weeks of gestation. The primary outcome measure was the presence of fetal heartbeats at 12 weeks of gestation, as determined by transvaginal ultrasound.

MAIN RESULTS AND THE ROLE OF CHANCE:

Non-inferiority of oral dydrogesterone was demonstrated, with pregnancy rates in the full analysis sample (FAS) at 12 weeks of gestation of 38.7% (191/494) and 35.0% (171/489) in the oral dydrogesterone and MVP gel groups, respectively (adjusted difference, 3.7%; 95% CI: -2.3 to 9.7). Live birth rates in the FAS of 34.4% (170/494) and 32.5% (159/489) were obtained for the oral dydrogesterone and MVP gel groups, respectively (adjusted difference 1.9%; 95% CI: -4.0 to 7.8). Oral dydrogesteronewas well tolerated and had a similar safety profile to MVP gel.