[Show abstract][Hide abstract] ABSTRACT: Women with mutation in both alleles of the NLRP7 or C6orf221/KHDC3L genes are predisposed to diploid biparental moles, but it has also been suggested that mutation in these genes can predispose to diploid androgenetic or triploid moles and to other kinds of reproductive wastage. We have investigated the association between molar pregnancy and recurrent miscarriages regarding changes in the NLRP7 and C6orf221/KHDC3L genes. Our study group can be divided into three sub-cohorts: 1) women having had at least one molar pregnancy and at least two non-mole miscarriages, 2) women having had recurrent androgenetic hydatidiform moles and 3) women having had one diploid androgenetic hydatidiform mole and a relative having had a hydatidiform mole (familial hydatidiform moles).We observed a statistically non-significant tendency of non-synonymous variants in NLRP7 to be more frequent in women with familial hydatidiform mole and in women with female family members with hydatidiform mole or non-mole miscarriage compared to women with no family history of mole or miscarriage. However, we did not find any unequivocal pathogenic mutations (the term 'unequivocal pathogenic mutations' refers to mutations which indubitably have a pathogenic effect on the affected woman) in NLRP7 or C6orf221/KHDC3L in any of the women in the study group. This indicates that recurrent miscarriages plus hydatidiform mole, recurrent androgenetic hydatidiform moles and familial androgenetic hydatidiform moles in general do not have the same monogenetic etiology as familiar diploid biparental moles.
Molecular Human Reproduction 08/2013; · 4.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: When a triploid pregnancy is diagnosed prenatally, gynaecologists have traditionally relied on the histopathological examination of the tissue from the terminated pregnancy to determine if the pregnancy is molar. However, reproducibility is poor and variability is high when diagnosing hydatidiform moles. Triploid pregnancies can have either the chromosomal constitution of two maternal and one paternal set or two paternal and one maternal set but only the conceptuses with two paternal sets have the potential to cause maternal complications. Therefore it would be beneficial to introduce a method that gives the gynaecologist the parental origin of the genome of the triploid conceptus as early as possible, without delaying the process by first collecting parental samples.
Using Methylation specific Multiplex Ligation-dependent Probe Amplification we measured methylation levels at different imprinted sites.
We were able to correctly determine the parental origin of the genome in all 105 triploid pregnancies analysed.
We present Methylation specific Multiplex Ligation-dependent Probe Amplification as a method capable of determining the parental origin of the genome of triploid conceptuses within 24 hours; it is inexpensive, simple, and easy to use and parental samples are not needed. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: AIMS/HYPOTHESIS: Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. METHODS: The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 kg/m(2) and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p < 0.05) with case-control status, from four selected annotation categories or from loci reported to associate with metabolic traits. These variants were genotyped in 15,989 Danes to search for association with 12 metabolic phenotypes (stage 2). In stage 3, polymorphisms showing potential associations were genotyped in a further 63,896 Europeans. RESULTS: Exome sequencing identified 70,182 polymorphisms with MAF >1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, p = 8.5 × 10(-14)), COBLL1 (type 2 diabetes: MAF 12.5%, OR 0.88, p = 1.2 × 10(-11)) and MACF1 (type 2 diabetes: MAF 23.4%, OR 1.10, p = 8.2 × 10(-10)). CONCLUSIONS/INTERPRETATION: We applied exome sequencing as a basis for finding genetic determinants of metabolic traits and show the existence of low-frequency and common coding polymorphisms with impact on common metabolic traits. Based on our study, coding polymorphisms with MAF above 1% do not seem to have particularly high effect sizes on the measured metabolic traits.
[Show abstract][Hide abstract] ABSTRACT: Hydatidiform moles (HMs) most often occur sporadically and are either diploid androgenetic or triploid. The very rare familial recurrent HMs (FRHMs) have been related to NLRP7 and C6orf221 mutations in the mother. FRHMs are most often diploid with both maternal and paternal origin of the molar genome. We have screened a cohort of 11 women with diploid HMs with biparental contributions to the molar genome with regard to mutations in NLRP7, NLRP2, the NLRP gene most homologous to NLRP7, and C6orf221. This was done in order to reveal if mutations in the mentioned genes play a major role in development of non-recurrent biparental moles. Recently, we have shown that eight of these diploid moles consist of two different cell lines. Only one woman had a mutation in the coding DNA sequence of NLRP7, which most likely contributed to HM development. This woman had non-mosaic repeated moles, and she was the only woman in our cohort with FRHM. We found no unequivocal pathogenic mutations in NLRP2 or C6orf221. Our observations suggest that although NLRP7 and C6orf221 mutations are related to diploid biparental FRHMs, neither of these genes, nor NLRP2, are related to diploid HMs with biparental contributions to the molar genome, in general.
Molecular Human Reproduction 08/2012; · 4.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In 2004, the integrated European project GEHA (Genetics of Healthy Ageing) was initiated with the aim of identifying genes involved in healthy ageing and longevity. The first step in the project was the recruitment of more than 2500 pairs of siblings aged 90 years or more together with one younger control person from 15 areas in 11 European countries through a coordinated and standardised effort. A biological sample, preferably a blood sample, was collected from each participant, and basic physical and cognitive measures were obtained together with information about health, life style, and family composition. From 2004 to 2008 a total of 2535 families comprising 5319 nonagenarian siblings were identified and included in the project. In addition, 2548 younger control persons aged 50-75 years were recruited. A total of 2249 complete trios with blood samples from at least two old siblings and the younger control were formed and are available for genetic analyses (e.g. linkage studies and genome-wide association studies). Mortality follow-up improves the possibility of identifying families with the most extreme longevity phenotypes. With a mean follow-up time of 3.7 years the number of families with all participating siblings aged 95 years or more has increased by a factor of 5 to 750 families compared to when interviews were conducted. Thus, the GEHA project represents a unique source in the search for genes related to healthy ageing and longevity.
[Show abstract][Hide abstract] ABSTRACT: The purpose of our work was to establish an efficient-oriented enucleation method to produce transgenic embryos with handmade cloning (HMC). After 41-42 h oocytes maturation, the oocytes were further cultured with or without 0.4 microg/ml demecolcine for 45 min [chemically assisted handmade enucleation (CAHE) group vs polar body (PB) oriented handmade enucleation (OHE) group respectively]. After removal of the cumulus cells and partial digestion of the zona pellucida, oocytes with visible extrusion cones and/or polar bodies attached to the surface were subjected to oriented bisection. Putative cytoplasts without extrusion cones or PB were selected as recipients. Two cytoplasts were electrofused with one transgenic fibroblasts expressing green fluorescent protein (GFP), while non-transgenic fibroblasts were used as controls. Reconstructed embryos were cultured in Well of Wells (WOWs) with porcine zygote medium 3 (PZM-3) after activation. Cleavage and blastocyst rates were registered on day 2 and day 7 of in vitro culture respectively. Meanwhile, the total blastocyst cell number was counted on day 7. We found that the difference was only observed between blastocyst rates (38.6 +/- 2% vs 48.1 +/- 3%) of cloned embryos with GFP transgenic fibroblast cells after CAHE vs OHE. With adjusted time-lapse for zonae-free cloned embryos cultured in WOWs with PZM-3, it was obvious that in vitro developmental competence after CAHE was compromised when compared with the OHE method. OHE enucleation method seems to be a potential superior alternative method used for somatic cell nuclear transfer (SCNT) with transgenic fibroblast cells.
Reproduction in Domestic Animals 07/2008; 44(1):122-7. · 1.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abnormal epigenetic modification is supposed to be one of factors accounting for inefficient reprogramming of the donor cell nuclei in ooplasm after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone deacetylase, potentially enhancing cloning efficiency. The aim of our present study was to establish the optimal TSA treatment in order to improve the development of handmade cloned (HMC) porcine embryos and examine the effect of TSA on their development. The blastocyst percentage of HMC embryos treated with 37.5 nM TSA for 22-24 h after activation increased up to 80% (control group-54%; P<0.05). TSA mediated increase in histone acetylation was proved by immunofluorescence analysis of acH3K9 and acH4K16. 2-cell stage embryos derived from TSA treatment displayed significant increase in histone acetylation compared to control embryos, whereas no significant differences were observed at blastocyst stage. During time-lapse monitoring, no difference was observed in the kinetics of 2-cell stage embryos. Compact morula (CM) stage was reached 15 h later in TSA treated embryos compared to the control. Blastocysts (Day 5 and 6) from HMC embryos treated with TSA were transferred to 2 recipients resulting in one pregnancy and birth of one live and five dead piglets. Our data demonstrate that TSA treatment after HMC in pigs may affect reprogramming of the somatic genome resulting in higher in vitro embryo development, and enable full-term in vivo development.
[Show abstract][Hide abstract] ABSTRACT: The purpose of the present study was to improve cryotolerance using high hydrostatic pressure (HHP) pretreatment of porcine in vitro matured (IVM) oocytes, to facilitate their further developmental competence after parthenogenetic activation. A total of 1668 porcine IVM oocytes were used in our present study. The pressure tolerance and optimal duration of recovery after HHP treatment were determined. Oocytes were treated with either 20 or 40 MPa (200 and 400 times greater than atmospheric pressure) for 60 min, with an interval of 10, 70, and 130 min between pressure treatment and subsequent vitrification under each pressure parameter. Oocytes from all vitrification groups had much lower developmental competence than fresh oocytes (P<0.01) measured as cleavage and blastocyst rates. However, significantly higher blastocyst rates (P<0.01) were obtained in the groups of 20 MPa pressure, with either 70 (11.4+/-2.4%) or 130 (13.1+/-3.2%) min recovery, when compared with the vitrification control group without HHP treatment where no blastocysts were obtained. The influence of temperature at HHP treatment on further embryo development was also investigated. Treatments of 20 MPa with 70 min recovery were performed at 37 degrees C or 25 degrees C. Oocytes pressurized at 37 degrees C had a significantly higher blastocyst (14.1+/-1.4%) rate than those treated at 25 degrees C (5.3+/-1.1%; P<0.01). Our results demonstrate that HHP pretreatment could considerably improve the developmental competence of vitrified pig in vitro matured (IVM) oocytes. The HHP pretreatment will be tested as a means to improve survival and developmental competence at different developmental stages in different species including humans.
[Show abstract][Hide abstract] ABSTRACT: Somatic cell nuclear transfer (SCNT) offers the possibility of pig transgenesis. Importantly, specific genetic manipulations can be performed in donor cells before SCNT to derive pig models for specific human genetic diseases, including the neurodegenerative disorder Alzheimer's disease (AD). In the present study, we established pregnancies after transfer of SCNT blastocysts produced by the handmade cloning (HMC) technique. The blastocysts were transgenic for a human gene, amyloid precursor protein gene with the 'Swedish mutation' (APPsw), causing AD. For transgenesis, minipig fibroblasts were transfected by lipofection with a vector containing the APPsw gene under control of the platelet-derived growth factor β promoter (PDGF-APPsw) and a neomycin-resistance selection gene. Neomycin-resistant colonies were isolated, expanded, analyzed, and used for HMC. Cumulus–oocyte complexes were aspirated from ovaries of slaughtered sows and matured for 41 h. Subsequently, the cumulus cells were removed in hyaluronidase, and zonae pellucidae were partially digested by incubation in pronase. Oocytes with a visible polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm adjacent to the PB was removed with a microblade. The cytoplasts were used as recipients for embryo reconstruction. Reconstructed embryos were produced by a 2-step fusion procedure. At the first step, 1 cytoplast was fused with 1 fibroblast in the absence of Ca2+. After 1 h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously in the presence of Ca2+, incubated in cytochalasin B and cycloheximide for 4 h, and then cultured in PZM-3 medium. The development of reconstructed embryos to the blastocysts stage was determined after 5, 6, or 7 days of in vitro culture. To investigate the in vivo developmental capacity, blastocysts were transferred surgically to synchronized recipients. When using PDGF-APPsw-transgenic minipig fibroblasts, the rate of blastocyst formation (mean ± SEM) was 39 ± 3% (164/424). In comparison, non-transgenic fibroblasts resulted in a blastocyst development of 36 ± 7% (36/102). In 4 recipients that received an average of 54 Day 5, 6, and 7 PDGF-APPsw-transgenic blastocysts, 2 ongoing pregnancies were confirmed by ultrasonography, 1 pregnancy was lost, and 1 returned to estrus. The results show a high in vivo developmental competence of blastocysts produced after SCNT of PDGF-APPsw-transgenic minipig fibroblasts.
Reproduction Fertility and Development - REPROD FERT DEVELOP. 01/2008; 20(1).