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Reproductive Biology and Endocrinology... Oct 2019Morulas with delayed growth sometimes coexist with blastocysts. There is still limited evidence regarding the optimal disposal of surplus morulas. With the advancement... (Observational Study)
Observational Study
BACKGROUND
Morulas with delayed growth sometimes coexist with blastocysts. There is still limited evidence regarding the optimal disposal of surplus morulas. With the advancement of vitrification, the freezing-thawing technique has been widely applied to zygotes with 2 pronuclei, as well as embryos at the cleavage and blastocyst stages. The freezing of morulas, however, has rarely been discussed. The purpose of this study was to investigate whether these poor-quality and slow-growing morulas are worthy of cryopreservation.
METHODS
This is a retrospective, observational, proof-of-concept study. A total of 1033 day 5/6 surplus morulas were cryopreserved from January 2015 to December 2018. The study included 167 women undergoing 180 frozen embryo transfer cycles. After the morulas underwent freezing-thawing procedures, their development was monitored for an additional day. The primary outcome was the blastocyst formation rate. Secondary outcomes were clinical pregnancy rate, live birth rate and abortion rate.
RESULTS
A total of 347 surplus morulas were thawed. All studied morulas showed delayed compaction (day 5, n = 329; day 6, n = 18) and were graded as having low (M1, n = 54), medium (M2, n = 138) or high (M3, n = 155) fragmentation. The post-thaw survival rate was 79.3%. After 1 day in extended culture, the blastocyst formation rate was 66.6%, and the top-quality blastocyst formation rate was 23.6%. The day 5 morulas graded as M1, M2, and M3 had blastocyst formation rates of 88.9, 74.0, and 52.8% (p < 0.001), respectively, and the top-quality blastocyst formation rates were 64.8, 25.2, and 9.0% (p < 0.001), respectively. The clinical pregnancy rate was 33.6%.
CONCLUSIONS
The post-thaw blastocyst formation rate was satisfactory, with approximately one-half of heavily fragmented morulas (M3) developing into blastocysts. Most of the poor-quality morulas were worth to freeze, with the reasonable goal of obtaining pregnancy and live birth. This alternative strategy may be a feasible approach for coping with poor-quality surplus morulas in non-PGS (preimplantation genetic screening) cycles.
Topics: Adult; Birth Rate; Blastocyst; Cryopreservation; Embryo Implantation; Embryo Transfer; Embryonic Development; Female; Humans; Live Birth; Morula; Pregnancy; Pregnancy Rate; Retrospective Studies; Vitrification
PubMed: 31666062
DOI: 10.1186/s12958-019-0535-2 -
PloS One 2014Embryo implantation into the maternal uterus is a decisive step for successful mammalian pregnancy. Osteopontin (OPN) is a member of the small integrin-binding ligand...
Embryo implantation into the maternal uterus is a decisive step for successful mammalian pregnancy. Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein family and participates in cell adhesion and invasion. In this study, we showed that Opn mRNA levels are up-regulated in the mouse uterus on day 4 and at the implantation sites on days 5 and 8 of pregnancy. Immunohistochemistry localized the OPN protein to the glandular epithelium on day 4 and to the decidual zone on day 8 of pregnancy. OPN mRNA and proteins are induced by in vivo and in vitro decidualization. OPN expression in the endometrial stromal cells is regulated by progesterone, a key regulator during decidualization. As a secreted protein, the protein level of OPN in the uterine cavity is enriched on day 4, and in vitro embryo culturing has indicated that OPN can facilitate blastocyst hatching and adhesion. Knockdown of OPN attenuates the adhesion and invasion of blastocysts in mouse endometrial stromal cells by suppressing the expression and enzymatic activity of matrix metalloproteinase-9 in the trophoblast. Our data indicated that OPN expression in the mouse uterus during early pregnancy is essential for blastocyst hatching and adhesion and that the knockdown of OPN in mouse endometrial stroma cells could lead to a restrained in vitro trophoblast invasion.
Topics: Animals; Blastocyst; Blotting, Western; Cells, Cultured; Embryo Implantation; Female; Gene Expression Regulation; Immunohistochemistry; Mice; Osteopontin; Pregnancy; Real-Time Polymerase Chain Reaction; Trophoblasts; Uterus
PubMed: 25133541
DOI: 10.1371/journal.pone.0104955 -
Fertility and Sterility Feb 2011To assess correlation between blastocyst morphology and chromosomal status.
OBJECTIVE
To assess correlation between blastocyst morphology and chromosomal status.
DESIGN
Observational research study.
SETTING
An IVF clinic and a specialist preimplanation genetic diagnosis (PGD) laboratory.
PATIENT(S)
Ninety-three couples undergoing IVF treatment in combination with chromosome screening of embryos.
INTERVENTION(S)
Five hundred blastocysts underwent trophectoderm biopsy and comprehensive chromosome screening using comparative genomic hybridization (CGH). The morphology of the embryos was evaluated using standard methods.
MAIN OUTCOME MEASURE(S)
Association of aneuploidy and morphologic score.
RESULT(S)
A total of 56.7% of blastocysts were aneuploid. One-half of the grade 5/6 blastocysts were euploid, compared with only 37.5% of embryos graded 1/2, suggesting an effect of aneuploidy on blastocyst development. Aneuploidy also had a negative effect on inner cell mass and trophectoderm grades. Morphologically poor blastocysts had a higher incidence of monosomy and abnormalities affecting several chromosomes. The gender ratio was significantly skewed in relation to morphology. A total of 72% of blastocysts attaining the highest morphologic scores (5AA and 6AA) were found to be male, compared with only 40% of grade 3 embryos.
CONCLUSION(S)
Morphology and aneuploidy are linked at the blastocyst stage. However, the association is weak, and consequently, morphologic analysis cannot be relied on to ensure transfer of chromosomally normal embryos. A significant proportion of aneuploid embryos are capable of achieving the highest morphologic scores, and some euploid embryos are of poor morphology. Gender was associated with blastocyst grading, male embryos developing at a significantly faster rate than females.
Topics: Adult; Aneuploidy; Blastocyst; Chromosome Aberrations; Comparative Genomic Hybridization; Embryo, Mammalian; Embryonic Development; Female; Fertilization in Vitro; Humans; Male; Pregnancy; Preimplantation Diagnosis; Sex Characteristics; Sex Ratio
PubMed: 20537630
DOI: 10.1016/j.fertnstert.2010.04.003 -
Reproductive Biology and Endocrinology... Aug 2022To generate an effective embryo prediction model and identify a non-invasive evaluation method by analyzing microRNAs (miRNAs) in embryo culture medium.
OBJECTIVE
To generate an effective embryo prediction model and identify a non-invasive evaluation method by analyzing microRNAs (miRNAs) in embryo culture medium.
DESIGN
Analysis of microRNA profiles from spent culture medium of blastocysts with good morphology that did or did not result in pregnancy.
SETTING
Clinical and experimental research.
PATIENTS
Sixty patients who underwent thawed embryo transfer of blastocysts after intracytoplasmic sperm injection.
INTERVENTION(S)
None.
MAIN OUTCOME MEASURE(S)
The association of miRNA abundance levels secreted by blastocysts in culture medium and implantation success.
RESULTS
Our RNA sequencing analysis found a total of 53 differentially expressed miRNAs in the culture media of pregnancy and non-pregnancy groups. Twenty-one miRNAs were analyzed for their potential to predict implantation success. Eight miRNAs (hsa-miR-191-5p, hsa-miR-320a, hsa-miR-92a-3p, hsa-miR-509-3p, hsa-miR-378a-3p, hsa-miR-28-3p, hsa-miR-512-5p, and hsa-miR-181a-5p) were further extracted from the results of a logistic regression analysis of qPCR Ct values. A prediction model for high-quality blastocysts was generated using the eight miRNAs, with an average accuracy of 0.82 by 5-fold cross validation.
CONCLUSION
We isolated blastocyst miRNAs that may predict implantation success and created a model to predict viable embryos. Increasing the number of investigated cases and further studying the effect of each miRNA on embryonic development is needed to refine the miRNA-based predictive model.
Topics: Blastocyst; Embryo Implantation; Humans; Male; MicroRNAs; Sperm Injections, Intracytoplasmic
PubMed: 36042522
DOI: 10.1186/s12958-022-00989-0 -
Fertility and Sterility Aug 2021To quantify the percentage of monopronuclear-derived blastocysts (MNBs) that are potentially useful for reproductive purposes using classic and state-of-the-art...
OBJECTIVE
To quantify the percentage of monopronuclear-derived blastocysts (MNBs) that are potentially useful for reproductive purposes using classic and state-of-the-art chromosome analysis approaches, and to study chromosomal distribution in the inner cell mass (ICM) and trophectoderm (TE) for intertissue/intratissue concordance comparison.
DESIGN
Prospective experimental study.
SETTING
Single-center in vitro fertilization clinic and reproductive genetics laboratory.
PATIENT(S)
A total of 1,128 monopronuclear zygotes were obtained between June 2016 and December 2018.
INTERVENTION(S)
MNBs were whole-fixed or biopsied to obtain a portion of ICM and 2 TE portions (TE1 and TE2) and were subsequently analyzed by fluorescence in situ hybridization, new whole-genome sequencing, and fingerprinting by single-nucleotide polymorphism array-based techniques (a-SNP).
MAIN OUTCOME MEASURE(S)
We assessed MNB rate, ploidy rate, and chromosomal constitution by new whole-genome sequencing, and parental composition by comparative a-SNP, performed in a "trio"-format (embryo/parents). The 24-chromosome distribution was compared between the TE and the ICM and within the TE.
RESULT(S)
A total of 18.4% of monopronuclear zygotes progressed to blastocysts; 77.6% of MNBs were diploid; 20% of MNBs were male and euploid, which might be reproductively useful. Seventy-five percent of MNBs were biparental and half of them were euploid, indicating that 40% might be reproductively useful. Intratissue concordance (TE1/TE2) was established for 93.3% and 73.3% for chromosome matching. Intertissue concordance (TE/ICM) was established for 78.8%, but 57.6% for chromosome matching. When segmental aneuploidy was not considered, intratissue concordance and chromosome matching increased to 100% and 80%, respectively, and intertissue concordance and chromosome matching increased to 84.8% and 75.8%, respectively.
CONCLUSION(S)
The a-SNP-trio strategy provides information about ploidy, euploidy, and parental origin in a single biopsy. This approach enabled us to identify 40% of MNBs with reproductive potential, which can have a significant effect in the clinical setting. Additionally, segmental aneuploidy is relevant for mismatched preimplantation genetic testing of aneuploidies, both within and between MNB tissues. Repeat biopsy might clarify whether segmental aneuploidy is a prone genetic character.
Topics: Biopsy; Blastocyst; Blastocyst Inner Cell Mass; Chromosomes; DNA Fingerprinting; Female; High-Throughput Nucleotide Sequencing; Humans; In Situ Hybridization, Fluorescence; Ploidies; Polymorphism, Single Nucleotide; Prospective Studies
PubMed: 33926715
DOI: 10.1016/j.fertnstert.2021.03.038 -
Reproductive Biology and Endocrinology... Sep 2009With single blastocyst transfer practice becoming more common in ART, there is a greater demand for a convenient and reliable cryostorage of surplus blastocysts.... (Review)
Review
With single blastocyst transfer practice becoming more common in ART, there is a greater demand for a convenient and reliable cryostorage of surplus blastocysts. Vitrification has emerged in the last decade as an alternative promising substitute for slow freezing. Blastocysts represent a unique challenge in cryostorage due to their size, multicellular structure and presence of blastocoele. The continuous acquisition of experience and introduction of many different technological developments has led to the improvement of vitrification as a technology and improved the results of its application in blastocyst cryostorage. The current information concerning safety and efficacy of the vitrification of blastocysts will be reviewed along with the variables that can impact the outcome of the procedure.
Topics: Blastocyst; Cryopreservation; Embryo Transfer; Female; Humans; Male; Pregnancy; Pregnancy Outcome; Reproductive Techniques, Assisted; Time Factors
PubMed: 19758458
DOI: 10.1186/1477-7827-7-99 -
Journal of Assisted Reproduction and... Aug 2019Does blastocyst morphology following euploid elective single embryo transfer (eSET) after preimplantation genetic testing for aneuploidies (PGT-A) via next generation...
PURPOSE
Does blastocyst morphology following euploid elective single embryo transfer (eSET) after preimplantation genetic testing for aneuploidies (PGT-A) via next generation sequencing impact clinical outcome?
METHODS
Two hundred ninety-six patients underwent PGT-A. Of 1549 blastocysts, 1410 blastocysts had a conclusive result after PGT-A and were included for analysis. An eSET policy was followed in a frozen embryo replacement cycle. A total of 179 euploid blastocysts were thawed and transferred. Clinical outcomes were categorized in four different embryo quality groups: excellent, good, average and poor.
RESULTS
Euploidy rate was 19/36 (52.7%, 95% CI 37-68), 199/470 (42.3%, 95% CI 38-47), 156/676 (23.0%, 95% CI 20-26) and 39/228 (17.1%, 95% CI 13-23) in the excellent, good, average and poor quality blastocyst groups, respectively. Fitted logistic regression analysis taking into account the following covariables: female, age, embryo chromosomal status and day of blastocyst development/biopsy showed that morphology was predictive of the comprehensive chromosome screening result (p < 0.05). A logistic regression analysis was also performed on clinical outcomes taking into account the effect of blastocyst morphology and day of blastocyst development/biopsy. None of the parameters were shown to be significant, suggesting morphology and day of blastocyst development/biopsy do not reduce the competence of euploid embryos (p > 0.05).
CONCLUSIONS
After eSET, implantation rate was 80-86%; live birth rate per embryo transfer was 60-73% and clinical miscarriage rate was found to be < 10% and were not significantly affected by the embryo morphology. Results are concordant with those reported when using aCGH and highlights the competence of poor-quality euploid embryos.
Topics: Adult; Aneuploidy; Birth Rate; Blastocyst; Embryo Implantation; Embryo Transfer; Embryonic Development; Female; Fertilization in Vitro; Genetic Testing; High-Throughput Nucleotide Sequencing; Humans; Maternal Age; Ploidies; Pregnancy; Pregnancy Rate; Preimplantation Diagnosis
PubMed: 31165389
DOI: 10.1007/s10815-019-01496-9 -
PloS One 2016In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell...
In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.
Topics: Animals; Apoptosis; Blastocyst; Cellular Reprogramming; Cloning, Organism; DNA Fragmentation; Embryo Culture Techniques; Embryo Transfer; Embryo, Mammalian; Embryonic Development; Gene Expression; Gene Expression Profiling; Swine; Transcription Factors
PubMed: 26894831
DOI: 10.1371/journal.pone.0146390 -
Fertility and Sterility Mar 2015To determine the effects of Benzo(a)pyrene (BaP) on the development of early preimplantation embryo by exposure to physiologic concentrations of BaP based on a previous...
OBJECTIVE
To determine the effects of Benzo(a)pyrene (BaP) on the development of early preimplantation embryo by exposure to physiologic concentrations of BaP based on a previous report in human ovarian follicular fluid and serum.
DESIGN
Zygotes were cultured in 5 nM or 50 nM BaP and then examined for development efficiency, embryo quality, and DNA damage. In addition, embryonic stem cells (ESCs) were used as a model to test the toxic effects of BaP on inner cell mass (ICM) of blastocysts.
SETTING
Laboratory.
ANIMAL(S)
CD1 mice.
INTERVENTION(S)
Mouse zygotes and ESCs were cultured in medium with 5 nM or 50 nM BaP.
MAIN OUTCOME MEASURE(S)
The percentage (rate) of blastocyst development, reactive oxygen species level, and quality of embryos assessed by total cell number, cell apoptosis, Oct4- and Nanog-positive cell ratio, and DNA damage on genomic and telomeric DNA were compared between dimethyl sulfoxide control and BaP treatments.
RESULT(S)
The BaP-treated zygotes exhibited significantly higher reactive oxygen species activity, which might lead to more cell apoptosis, low ratio of Nanog- or Oct4-positive ICM cells, and increasing DNA damage in both genomic and telomeric DNA in blastocysts. By using mouse ESCs derived from ICM cells as a model, we showed that pluripotent cells might also show serious DNA damage after a brief exposure to BaP.
CONCLUSION(S)
Our data show that BaP could seriously disrupt cell growth and genomic DNA stability and increase cell apoptosis in mouse preimplantation embryo development.
Topics: Animals; Apoptosis; Benzo(a)pyrene; Blastocyst; Blastomeres; Cell Differentiation; Cells, Cultured; Embryonic Development; Embryonic Stem Cells; Female; Mice; Reactive Oxygen Species
PubMed: 25516082
DOI: 10.1016/j.fertnstert.2014.11.013 -
PloS One 2017Is the timing of vitrification after trophectoderm (TE) biopsy associated with successful implantation and pregnancy after the embryo transfer of blastocysts subjected...
UNLABELLED
Is the timing of vitrification after trophectoderm (TE) biopsy associated with successful implantation and pregnancy after the embryo transfer of blastocysts subjected to preimplantation genetic screening (PGS)? In this retrospective cohort study, 1329 blastocysts from 223 patients were subjected to TE biopsy for performing array comparative genomic hybridization (CGH) tests. The PGS and frozen blastocyst transfer (FET) cycles were performed from December 2012 to May 2015. Only the good quality and expanded blastocysts on day 5 or 6 were selected for biopsy. After TE biopsy, the re-expansion grades relative to the original blastocoel were (1) collapsed blastocysts (CB), (2) 3/4 re-expansion but not full expansion (RE), and (3) full re-expansion or hatching (FE). All biopsied blastocysts were subjected to vitrification within 0.5-6 h after biopsy; the time intervals between TE biopsy and vitrification and the expansion grades at the time of vitrification were recorded. By combining two factors, namely the expansion grades and culture intervals between biopsy and vitrification, the patients were further divided into four groups, namely CB with a < 3 h culture interval (n = 34 cycles, Group I), RE and FE blastocysts with a < 3 h culture interval (n = 10 cycles, Group II); CB blastocysts with a ≥ 3 h culture interval (n = 6 cycles, Group III); and RE or FE blastocysts with a ≥ 3 h culture interval (n = 173 cycles, Group IV). The implantation (63.7%, 179/281) and clinical pregnancy (74.0%, 128/173) rates in Group IV were significantly higher than those in Group I (45.3%, 24/53; 50.0%, 17/34; P = 0.012 and 0.005, respectively). According to our findings, optimal vitrification timing > 3 hours to enable blastocysts to reach RE or FE provides improved implantation and pregnancy rates after FET.
TRIAL REGISTRATION
ClinicalTrials.gov NCT03065114.
Topics: Blastocyst; Embryo Transfer; Female; Humans; Pregnancy; Preimplantation Diagnosis; Retrospective Studies; Vitrification
PubMed: 28982142
DOI: 10.1371/journal.pone.0185747