64 Impaired Post-Implantation Development Following Blastomere Biopsy is Associated with Placental Hypomethylation

2018 ◽  
Vol 30 (1) ◽  
pp. 171
Author(s):  
F. Zacchini ◽  
M. Ogluska ◽  
R. Arena ◽  
G. E. Ptak
Keyword(s):  
Congenital Anomalies ◽  
In Utero ◽  
Placental Weight ◽  
Fetal Weight ◽  
Postnatal Life ◽  
Developmental Potential ◽  
Blastomere Biopsy ◽  
Epigenetic Programming ◽  
Increased Risk

The removal of one cell from cleaving embryos (blastomere biopsy, BB) is the main component of pre-implantation genetic diagnosis (PGD), a diagnostic test aimed to select only healthy embryos before transfer in utero. Studies on animals have suggested BB as risk factor for impaired development during both pre- and postnatal life. However, we still have incomplete knowledge on the real side effects of BB and the mechanisms underlying them. The present study was designed to evaluate whether BB or miscellaneous factors (in vitro culture, IVC; and embryo transfer, ET) may affect developmental potential of conceptuses obtained following BB (i.e. pregnancy rate/loss, fetal/placental weight, feto:placenta ratio) and placental epigenetic programming. To this aim, 3-month-old C57 females, naturally mated without hormonal stimulation, were used as donors. At 2.5 days post-coitum (dpc), 8-cell stage embryos were collected and subjected to BB in PBS (without Ca2+/Mg2+) supplemented with 0.4% BSA and antibiotics. Embryos were cultured in KSOM medium in an incubator at 37.5°C and 5% CO2. Two in vitro control groups were used: (1) IVC, embryos cultured in vitro, not subjected to biopsy; (2) ET, blastocysts collected at 3.5 dpc and directly transferred into recipient females. At 3.5 dpc, 10 to 12 blastocysts were transferred into pseudo pregnant females (n ≥ 7/group). An additional control group consisted of naturally conceived pregnancy (n = 8, in vivo control, CTR). At 18.5 dpc, conceptuses were collected and subjected to gross morphological evaluations (n > 20 conceptuses/group). Placentae (n ≥ 4/group) were analysed for 5-methylation and hydroxymethylation content by ELISA assay. Decimal variables were analysed using the Mann-Whitney test, and percentages were analysed with the Fisher exact test. Assessment of development to term revealed reduced survival rate in BB, IVC, and ET v. CTR (45.23, 47.82, 60, and 98.15% respectively; P < 0.05) and increased number of stillborn/congenital anomalies in BB v. CTR fetuses (9.5 v. 1.5%; P < 0.05). Gross morphological observations displayed increased placental weight in BB, IVC, ET v. CTR (0.15, 0.17, 0.16 and 0.11 g, respectively; P < 0.0001) and reduced fetal weight in BB v. IVC, ET, and CTR groups (0.88, 1.09, 1.16, and 1.15 g, respectively; P < 0.05). Also, a significant reduction of feto:placenta ratio was detected in BB, IVC, ET v. CTR (5.9, 6.3, 8.1 v. 10.4; P < 0.05). The ELISA assays on placentae revealed significant reduction of 5-methylcytosine content in BB v. CTR (22.24 v. 33.55%; P < 0.05) but no differences in hydroxymethylcytosine. Altogether, our results showed that BB is associated with impaired development to term (reduced fetal weight, increased risk of perinatal mortality, and congenital anomalies) and hypomethylation in full-term placentae. Thus, our preliminary study suggests that BB, rather than IVC and ET, to be a factor of risk for proper development in utero through perturbation of developmental epigenetic programming. Further analysis will be necessary to better characterise the epigenetic profile in both fetuses and placentae obtained following BB.

1 INSULIN TREATMENT DURING IN VITRO OOCYTE MATURATION LEADS TO DIFFERENT GENE EXPRESSION AND METHYLATION PATTERNS OF KEY GENES ASSOCIATED WITH METABOLISM AND STEROID SYNTHESIS IN THE BOVINE BLASTOCYST

2017 ◽  
Vol 29 (1) ◽  
pp. 108
Author(s):  
D. Laskowski ◽  
P. Humblot ◽  
M. A. Sirard ◽  
Y. Sjunnesson ◽  
G. Andersson ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Lipid Metabolism ◽  
Oocyte Maturation ◽  
Steroid Synthesis ◽  
Developmental Potential ◽  
Increased Risk ◽  
In Vitro Oocyte Maturation ◽  
Methylation Patterns ◽  
And Oxidative Stress

Obesity and overfeeding are common causes for female infertility, leading to insulin resistance and hyperinsulinemia and associated with an increased risk for type 2 diabetes mellitus (Pasquali et al., http://dx.doi.org/10.1093/humupd/dmg024). We investigated here the effect of insulin during in vitro oocyte maturation on methylation changes in bovine Day 8 blastocysts (BC8) and focused on methylation patterns of candidate genes associated with metabolism and steroidogenesis (Day 0 = day of oocyte collection). Abattoir-derived oocytes (n = 882) were in vitro matured for 22 h with 2 different insulin concentrations, INS10 (10 µg mL−1) and INS0.1 (0.1 µg mL−1) or without insulin (INS0, control). Subsequently, IVF and IVC were performed to equal standardized conditions for all groups. Parallel genomic DNA and total RNA extraction (AllPrepDNA/RNA micro kit, cat no. 80284, Qiagen®, Valencia, CA, USA) from pools of 10 frozen (−80°C) BC8 was followed by transcriptome and epigenome analysis (Laskowski et al., http://dx.doi.org/10.1071/RD15315). An empirical Bayes moderated t-test and the ‘limma’ package in R (www.r-project.org) were used to search for differentially expressed genes between the control and the insulin groups. Analysis of the epigenome by using a specific pipeline, described by Shojaei Saadi et al. (2014 BMC Genomics 15, 451), showed that 7632 and 3914 regions were hypomethylated in the INS0.1 and INS10 v. INS0, whereas 6026 and 8504 regions were hypermethylated in INS0.1 and INS10 v. INS0. Combining epigenetic and transcriptomic data, we found that high methylation and low expression or the reverse (low methylation and high expression) were observed for a set of 14 and 11 genes for INS0.1 and INS10 respectively. Most of these genes are associated with lipid metabolism, steroid synthesis, and oxidative stress. Further investigation of the localization of differentially methylated regions (DMR) in genes showed that the conservation odds (methylation) was in general higher in coding regions and CpG islands than in noncoding regions. We observed a large overlap of DMR in the 2 insulin groups compared with controls (3233 common DMR). These numerous changes illustrate the potential unfavourable effects of elevated insulin during maturation leading to alteration of the methylation patterns of the early embryo. This model may help us better understand the mechanisms by which metabolic disorders observed pre-conception can affect embryonic development and subsequent health of the offspring. Our results based on changes in transcriptome or epigenome did show that insulin challenge during maturation leads to postponed effects associated with steroidogenesis, lipid metabolism and oxidative stress in the BC8. By this early stage, if persistent, specific changes in the expression and methylation patterns of genes associated to hyperinsulinemia may decrease the developmental potential of early embryos or could be responsible for subsequent pathologies. This study was funded by FORMAS.

scholarly journals A Genetic study in assisted reproduction and the risk of congenital anomalies

2021 ◽  
Vol 4 (4) ◽  
pp. 096-100
Author(s):  
Chrysoula Kaparelioti ◽  
Eleni Koniari ◽  
Vasiliki Efthymiou ◽  
Dimitrios Loutradis ◽  
George Chrousos ◽  
...  
Keyword(s):  
Birth Weight ◽  
In Vitro Fertilization ◽  
Assisted Reproduction ◽  
Congenital Anomalies ◽  
Clinical Genetics ◽  
Genetic Syndromes ◽  
Vitro Fertilization ◽  
Increased Risk ◽  
One Year

In vitro fertilization is one of the most common and effective procedure for thousands of couples worldwide who want to have a child and are unable to do so for various reasons. Diverse studies show that couples who conceive naturally after one year of trying had newborns with an increased risk of prematurity and low birth weight, compared with couples who conceived before completing one year of trying. Children from assisted reproduction (AR), have a 30% increased risk of prematurity and low birth weight, compared with children from infertile fathers. Regarding the conflicting results the present study aimed to record the frequency of genetic, congenital anomalies in children and adolescents who had examined in the last decade to the Clinical Genetics Clinic of the National and Kapodistrian University of Athens whose mothers had undergone assisted reproduction. The research process was conducted at the "Aghia Sofia" Children's Hospital based in Athens. However, the cases that were studied came from all over Greece. Initially, the researcher recorded the cases that came to the clinic of Clinical Genetics and whose conception occurred after technical assisted reproduction. After telephone communication and the consent of the parents, a live appointment was scheduled. In this meeting-interview all the provisions of the investigation and the protocol were asked and some elements of the medical history of the cases were confirmed. The total sample included 230 children and adolescents. The resulting data were recorded on a printed form/questionnaire. Then, they were registered electronically in the program SPSS 25.0 (Statistical Package for Social Sciences) with a specific unit code for each case/patient, followed by the processing and statistical analysis of the data as well as the recording of the results. The gender of the participants was male for 118 participants (51.3%) and 112 females (48.7%). Mean and standard deviation (SD) of maternal, paternal (at the time of delivery) age was equal to 36.38 (5.94) and 39.94 (6.58) respectively. The observed abdormalities were 35.53% psychomotor retardation, 23.68% facial abnormalities, 23.68% spinal cord abnormalities, 21.05% morphological abnormalities, 20.61% short stature, 19.74% developmental disorders, 19.30% heart disease, 16.67% neurological diseases, 14.47% genetic syndromes, 11.40% genital abnormalities, 8.33% limb abnormalities, 7.46% dermatological abnormalities, 6.14% eye abnormalities, 6.14% hypothyroidism, 5.70% endocrine disorders, 5.26%otolaryngology abnormalities, 2.63% disease of kidney, intestine, 2.19% vascular malformations. Regarding the karyotype chromosome analysis by G-banding technique, from the 230 children in: 24 (10.43%) a pathological result was found, in 158 children (68.70%) it was found normal (46, XX or 46, XY by case) without other findings, while in 48 children (20.87%) the test was not performed for various reasons. Regarding the results of molecular analysis (DNA) from the 230 children, in 50 (21.74%) a pathological finding was found, in 56 children (24.35%) no abnormalities were found and in 124 children (53.91%) no molecular analysis was performed for various reasons. In conclusion, the sample of this descriptive study is characterized as uniform in terms of the method of assisted reproduction since 96.24% had followed the classic IVF. Full-term pregnancy was associated with the appearance of malignancy and head morphological abnormalities (64.6%), normal pregnancy was associated with genetic syndromes (18.2%) and facial abnormalities (11.1%). It is recommended the screening oocyte and sperm donors in order to help protect the safety and health of donors, recipients, and future offspring. The present study confirms the association of the presence of congenital anomalies after in vitro fertilization (IVF). However, the absolute risk of developing severe dysplasias after an IVF procedure is limited.

scholarly journals Embryo biopsy and development: the known and the unknown

Reproduction ◽  
2017 ◽  
Vol 154 (5) ◽  
pp. R143-R148 ◽  
Author(s):  
Federica Zacchini ◽  
Roberta Arena ◽  
Adam Abramik ◽  
Grazyna E Ptak
Keyword(s):  
Clinical Practice ◽  
Preimplantation Genetic Diagnosis ◽  
Current Knowledge ◽  
Genetic Diagnosis ◽  
In Utero ◽  
Human Reproduction ◽  
Embryo Biopsy ◽  
Postnatal Life ◽  
Long Term Effects ◽  
Blastomere Biopsy

Preimplantation genetic diagnosis (PGD) has been introduced in clinical practice as a tool for selecting ‘healthy’ embryos before their transfer in utero. PGD protocols include biopsy of cleaving embryos (blastomere biopsy (BB)) or blastocysts (trophectoderm biopsy (TB)), followed by genetic analysis to select ‘healthy’ embryos for transfer in utero. Currently, TB is replacing the use of BB in the clinical practice. However, based on the European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis Consortium reports, BB has been used in >87% of PGD cycles for more than 10 years. An exhaustive evaluation of embryo biopsy (both BB and TB) risks and safety is still missing. The few epidemiological studies available are quite controversial and/or are limited to normalcy at birth or early childhood. On the other hand, studies on animals have shown that BB can be a risk factor for impaired development, during both pre- and postnatal life, while little is known on TB. Thus, there is an urgent need of focused researches on BB, as it has contributed to give birth to children for more than 10 years, and on TB, as its application is significantly growing in clinical practice. In this context, the aim of this review is to provide a complete overview of the current knowledge on the short-, medium- and long-term effects of embryo biopsy in the mouse model.

Do in vitro fertilization, intrauterine insemination or female infertility impact the risk of congenital anomalies in singletons? A longitudinal national French study

2020 ◽  
Author(s):  
Patricia Fauque ◽  
Jacques De Mouzon ◽  
Aviva Devaux ◽  
Sylvie Epelboin ◽  
Marie-José Gervoise-Boyer ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Birth Defects ◽  
National Health ◽  
Congenital Anomalies ◽  
Congenital Malformations ◽  
Female Infertility ◽  
Congenital Defects ◽  
Vitro Fertilization ◽  
Increased Risk

Abstract STUDY QUESTION Do IVF, IUI or female infertility (i.e. endometriosis, polycystic ovary syndrome [PCOS] and primary ovarian insufficiency [POI]) lead to an increased risk of congenital anomalies in singletons? SUMMARY ANSWER After multivariable adjustments, the increased risks of congenital defects associated with IUI were no longer significant, but the underlying maternal infertility presented a potential emental risk, in addition to the risk associated with IVF. WHAT IS KNOWN ALREADY Most epidemiological studies suggest that singletons born from ART have a higher risk of birth defects, specifically musculoskeletal, cardiovascular and urogenital disorders. However, most of these studies were established on data obtained at birth or in the neonatal period and from relatively small populations or several registries. Moreover, to our knowledge, female infertility, which is a potential confounder, has never been included in the risk assessment. STUDY DESIGN, SIZE, DURATION Using data from the French National Health System database, we conducted a comparative analysis of all singleton births (deliveries ≥22 weeks of gestation and/or &gt;500 g of birthweight) in France over a 5-year period (2013–2017) resulting from fresh embryo or frozen embryo transfer (fresh-ET or FET from IVF/ICSI cycles), IUI and natural conception (NC). Data were available for this cohort of children at least up to early childhood (2.5 years old). PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 3 501 495 singleton births were included (3 417 089 from NC, 20 218 from IUI, 45 303 from fresh-ET and 18 885 from FET). Data were extracted from national health databases and used to identify major birth defects. Malformations were classified according to the 10th revision of the International Classification of Disease. To analyse the effect of mode of conception, multivariable analyses were performed with multiple logistic regression models adjusted for maternal age, primiparity, obesity, smoking, history of high blood pressure or diabetes and female infertility. MAIN RESULTS AND THE ROLE OF CHANCE In our cohort of children, the overall prevalence of congenital malformations was 3.78% after NC, 4.53% after fresh-ET, 4.39% after FET and 3.91% after IUI (132 646 children with major malformations). Compared with infants conceived naturally, children born after fresh-ET and after FET had a significantly higher prevalence of malformations, with an adjusted odds ratio (aOR) of 1.15 [95% CI 1.10–1.20, P &lt; 0.0001] and aOR of 1.13 [95% CI 1.05–1.21, P = 0.001], respectively. Among the 15 relevant subgroups of malformations studied, we observed a significantly increased risk of eight malformations in the fresh-ET group compared with the NC group (i.e. musculoskeletal, cardiac, urinary, digestive, neurological, cleft lip and/or palate and respiratory). In the FET group, this increased risk was observed for digestive and facial malformations. The overall risk of congenital malformations, and the risk by subtype, was similar in the IUI group and the NC group (overall risk: aOR of 1.01 [95% CI 0.94–1.08, P = 0.81]). In addition, there was an overall independent increase in the risk of congenital defects when the mothers were diagnosed with endometriosis (1.16 aOR [95% CI 1.10–1.22], P &lt; 0.0001), PCOS (1.20 aOR [95% CI 1.08–1.34], P = 0.001) or POI (1.52 aOR [95% CI 1.23–1.88], P = 0.0001). Chromosomal, cardiac and neurological anomalies were more common in the three maternal infertility groups. LIMITATIONS, REASONS FOR CAUTION Male infertility, the in vitro fertilization method (i.e. in vitro fertilization without or with sperm injection: conventional IVF vs ICSI) and embryo stage at transfer could not be taken into account. Furthermore, residual confounding cannot be excluded as well as uncertainties regarding the diagnostic criteria used for the three female infertilities. Findings for specific malformations should be interpreted with caution because the number of cases was small in some sub-groups (potentially due to the Type I error or multiple testing). WIDER IMPLICATIONS OF THE FINDINGS In this large study, after multivariable maternal adjustments, a moderately increased risk of defects subsisted after IVF, while those associated with IUI were no longer significant. In addition, our results showed that underlying maternal infertility could contribute to the increased risk of defects associated with IVF. These novel findings highlight the importance of taking into account the ART treatment methods and the type of infertility. STUDY FUNDING/COMPETING INTEREST(s) This work was supported by the National Agency of Biomedicine. The authors have no competing interests to disclose. TRIAL REGISTRATION NUMBER NA.

scholarly journals Adipose Tissue Inflammation: Developmental Ontogeny and Consequences of Gestational Nutrient Restriction in Offspring

Endocrinology ◽  
2009 ◽  
Vol 150 (8) ◽  
pp. 3913-3920 ◽  
Author(s):  
Don Sharkey ◽  
Michael E. Symonds ◽  
Helen Budge
Keyword(s):  
Insulin Resistance ◽  
Endoplasmic Reticulum ◽  
Adipose Tissue ◽  
In Utero ◽  
Nutrient Restriction ◽  
Postnatal Life ◽  
The Metabolic Syndrome ◽  
Increased Risk ◽  
Pregnant Sheep ◽  
The Impact

Increasing adiposity predisposes to the development of the metabolic syndrome, in part, through adipose tissue dysregulation and inflammation. In addition, offspring nutrient-restricted (NR) in utero can exhibit an increased risk of early-onset insulin resistance and obesity, although the mechanisms remain unclear. We aimed to: 1) define adipose tissue ontogeny of key proinflammatory and endoplasmic reticulum stress gene expression from late fetal to early adult life and 2) examine the impact on these genes in gestational nutrient restriction. Pregnant sheep were fed 100% (control) or 50% (NR) of their nutritional requirements between early to mid (28–80 d, term ∼147 d) or late (110–147 d) gestation. In control offspring, toll-like receptor 4 (TLR4), and the macrophage marker CD68, peaked at 30 d of life before declining. IL-18 peaked at 6 months of age, whereas the endoplasmic reticulum chaperone glucose-regulated protein 78 peaked at birth and subsequently declined through postnatal life. TLR4 and CD68 positively correlated with relative adipose tissue mass and with each other. Early to midgestational NR offspring had decreased abundance of IL-18 at 6 months of age. In late gestational NR offspring, CD68 was significantly lower at birth, a pattern that reversed in juvenile offspring, coupled with increased TLR4 abundance. In conclusion, the in utero nutritional environment can alter the adipose tissue inflammatory profile in offspring. This may contribute to the increased risk of insulin resistance or obesity, dependent on the timing of nutrient restriction. Establishing the optimal maternal diet during pregnancy could reduce the burden of later adult disease in the offspring.

P–133 Monopronuclear (1PN) embryos can derive in healthy pregnancies

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Brinkmann ◽  
C Demmers. va. d. Werken ◽  
L Ramos
Keyword(s):  
Live Birth Rate ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
In Vitro Fertilisation ◽  
Developmental Potential ◽  
Increased Risk ◽  
Registration Number ◽  
The Right

Abstract Study question Should 1PN embryos be considered suitable for transfer when normal development is observed at day 3 or day 5? Summary answer In IVF/ICSI cycles, 1PN zygotes are encountered in 2.7% of inseminated oocytes. Transfer of 1PN-embryos should be considered in the absence of suitable 2PN embryos. What is known already During in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) zygotes containing only a single pronucleus (monopronuclear, 1PN) are encountered in 1–7.7% of cases, while the display of two pronuclei is expected in a normally fertilised oocyte. A 1PN zygote can be of gynogenetic or androgenetic origin, but it can also be biparental. Gynogenetic and androgenetic 1PN embryos can be haploid or diploid, so a diploid 1PN embryo is not guaranteed to be normally fertilised. Generally, 1PN are discarded, as they have an increased risk for aneuploidy. However, sporadically they can develop into healthyl babies. Study design, size, duration 1PN-zygotes (n = 1287, 2.7% from all inseminated oocytes) from 1–1–2016 up to 15–12–2020 were retrospectively evaluated. The development and fate (discarded/transferred/cryopreserved) of all embryos were recorded. Embryos were evaluated at day 2, 3 or 5 of development. The policy of our unit is that, in absence of 2PN embryos, normal developed 1PN-embryos can be transferred on day 3. Supernumerary 1PN embryos can be cryopreserved at blastocyst stage. Ongoing pregnancies from fresh embryo transfers (ET) were analysed. Participants/materials, setting, methods In 946 IVF/ICSI cycles, at least one 1PN zygote was observed (total 1287 embryos). ICSI with ejaculated, PESA or TESE sperm counted for a total of 795 embryos, IVF cycles for 494 embryos. Embryo evaluation was performed using a home-made numerical algorithm: A (top embryo; 150–200 points), B (regular embryo; 100–149 points) or C (poor embryo; 0–99 points). Monopronuclear embryos always scored lower than equal developed 2PN embryos. Blastocyst evaluation was according to Gardner score. Main results and the role of chance From the 795 ICSI embryos, 49 (6.1%) were used for fresh ET (26 scored quality A or B), and a total of 60 embryos developed to blastocyst and were cryopreserved. From these 49 ICSI transfers, 4 (8.1%) ongoing pregnancies were obtained, all 4 from DET (1PN+2PN embryo), from which one twin pregnancy was confirmed. From the 494 IVF embryos, 41 (8.3%) were used for fresh ET (24 scored A or B), and 62 blastocysts were cryopreserved. A total of 9/41 (22%) ongoing pregnancies were obtained: 5 from SET (1PN) and 4 from DET (1PN+ 2PN embryo). Therefore, in only five IVF cycles a confirmed pregnancy was observed from a 1PN embryo (all A-quality embryos). Considering six ongoing pregnancies with complete certainty of monopronuclear origin from fresh tranfers could be confirmed from our retrospective data, we can conclude that although the live birth rate of these embryos is very low (around 0,5- 1.0%), they should not be discarded when development is normal and no dipronuclear embryos are present. Limitations, reasons for caution Cryo-thawing data is missing as these embryos were not differentially marked at freezing. Therefore, the cumulative pregnancies from monopronuclear embryos could be higher. Embryos were not evaluated in a time lapse system, so asynchronicity of PN formation could explain missing the right moment for evaluation, while normal fertilized. Wider implications of the findings: Notably, IVF monopronuclear embryos display a higher developmental potential than those derived from ICSI. We suggest that, in absence of dipronuclear embryos, culture to blastocyst stage before considering fresh ET or cryopreservation will help differentiate viable 1PN embryos, reducing the higher chance of genetic anomalies and miscarriages. Trial registration number N.A.

scholarly journals In vitro cellular and proteome assays identify Wnt pathway and CDKN2A-regulated senescence affected in mesenchymal stem cells from mice after a chronic LD gamma irradiation in utero

2021 ◽  
Author(s):  
Martina Schuster ◽  
Gargi Tewary ◽  
Xuanwen Bao ◽  
Prabal Subedi ◽  
Stefanie M. Hauck ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Ionizing Radiation ◽  
Gamma Irradiation ◽  
Wnt Signaling Pathway ◽  
Genotoxic Stress ◽  
In Utero ◽  
Postnatal Life ◽  
Radiation Induced

AbstractReliable data on the effects of chronic prenatal exposure to low dose (LD) of ionizing radiation in humans are missing. There are concerns about adverse long-term effects that may persist throughout postnatal life of the offspring. Due to their slow cell cycle kinetics and life-long residence time in the organism, mesenchymal stem cells (MSCs) are more susceptible to low level genotoxic stress caused by extrinsic multiple LD events. The aim of this study was to investigate the effect of chronic, prenatal LD gamma irradiation to the biology of MSCs later in life. C3H mice were exposed in utero to chronic prenatal irradiation of 10 mGy/day over a period of 3 weeks. Two years later, MSCs were isolated from the bone marrow and analyzed in vitro for their radiosensitivity, for cellular senescence and for DNA double-strand break recognition after a second acute gamma-irradiation. In addition to these cellular assays, changes in protein expression were measured using HPLC–MS/MS and dysregulated molecular signaling pathways identified using bioinformatics. We observed radiation-induced proteomic changes in MSCs from the offspring of in utero irradiated mice (leading to ~ 9.4% of all detected proteins being either up- or downregulated) as compared to non-irradiated controls. The proteomic changes map to regulation pathways involved in the extracellular matrix, the response to oxidative stress, and the Wnt signaling pathway. In addition, chronic prenatal LD irradiation lead to an increased rate of in vitro radiation-induced senescence later in life and to an increased number of residual DNA double-strand breaks after 4 Gy irradiation, indicating a remarkable interaction of in vivo radiation in combination with a second acute dose of in vitro radiation. This study provides the first insight into a molecular mechanism of persistent MSC damage response by ionizing radiation exposure during prenatal time and will help to predict therapeutic safety and efficacy with respect to a clinical application of stem cells.

Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

1991 ◽  
Vol 66 (04) ◽  
pp. 453-458 ◽  
Author(s):  
John T Brandt
Keyword(s):  
Specific Activity ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Clinical Importance ◽  
Potential Method ◽  
Quantitative Expression ◽  
Increased Risk ◽  
Apparent Association

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

Thrombin Generation Measured Ex Vivo Following Microvasculor Injury Is Increased in SLE Patients with Antiphospholipid-protein Antibodies

1997 ◽  
Vol 78 (04) ◽  
pp. 1173-1177 ◽  
Author(s):  
Jacek Musiał ◽  
Jakub Swadźba ◽  
Miłosz Jankowski ◽  
Marek Grzywacz ◽  
Stanisława Bazan-Socha ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Skin Incision ◽  
Anticardiolipin Antibodies ◽  
Small Blood Vessel ◽  
Anionic Phospholipids ◽  
Increased Risk ◽  
High Concentrations

SummaryAntiphospholipid-protein antibodies (APA) include lupus-type anticoagulant (LA) and antibodies recognizing complexes of anionic phospholipids (e.g. cardiolipin) and proteins (e.g. prothrombin and (β2-glycoprotein I). The presence of APA is associated with an increased risk of both arterial and venous thrombosis. However, the pathogenic mechanism leading to thrombosis in patients with APA remains unclear. We studied 32 patients with systemic lupus erythematosus (SLE) who were divided into two groups depending on the presence (n = 19) or absence (n = 13) of APA. Healthy volunteers (n = 12) matched by age and sex served as controls. In all subjects LA and IgG class anticardiolipin antibodies (ACA) were determined. Thrombin generation was monitored ex vivo measuring fibrinopeptide A (FPA) and prothrombin fragment F1 + 2 (F1 + 2) in blood emerging from a skin microvasculature injury, collected at 30 second intervals. In subjects with antiphospholipid antibodies mean FPA and F1 + 2 concentrations were signiF1cantly higher at most blood sampling times than in controls. In some SLE patients with APA the process of thrombin generation was clearly disturbed and very high concentrations of F1brinopeptide A were detected already in the F1rst samples collected. Two minutes after skin incision SLE patients without APA produced slightly more FPA, but not F1 + 2, as compared to healthy subjects. Mathematical model applied to analyze the thrombin generation kinetics revealed that APA patients generated signiF1cantly greater amounts of thrombin than healthy controls (p = 0.02 for either marker). In contrast, in the same patients generation of thrombin in recalciF1ed plasma in vitro was delayed pointing to the role of endothelium in the phenomenon studied. In summary, these data show for the F1rst time that in SLE patients with antiphospholipid-protein antibodies thrombin generation after small blood vessel injury is markedly increased. Enhanced thrombin generation might explain thrombotic tendency observed in these patients.

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