Two waves of transcriptomic changes in periovulatory human granulosa cells.

STUDY QUESTION: How does the human granulosa cell (GC) transcriptome change during ovulation? SUMMARY ANSWER: Two transcriptional peaks were observed at 12 h and at 36 h after induction of ovulation, both dominated by genes and pathways known from the inflammatory system. WHAT IS KNOWN ALREADY: The crosstalk between GCs and the oocyte, which is essential for ovulation and oocyte maturation, can be assessed through transcriptomic profiling of GCs. Detailed transcriptional changes during ovulation have not previously been assessed in humans. STUDY DESIGN, SIZE, DURATION: This prospective cohort study comprised 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital-affiliated fertility clinic in 2016-2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: From each woman, one sample of GCs was collected by transvaginal ultrasound-guided follicle aspiration either before or 12 h, 17 h or 32 h after ovulation induction (OI). A second sample was collected at oocyte retrieval, 36 h after OI. Total RNA was isolated from GCs and analyzed by microarray. Gene expression differences between the five time points were assessed by ANOVA with a random factor accounting for the pairing of samples, and seven clusters of protein-coding genes representing distinct expression profiles were identified. These were used as input for subsequent bioinformatic analyses to identify enriched pathways and suggest upstream regulators. Subsets of genes were assessed to explore specific ovulatory functions. MAIN RESULTS AND THE ROLE OF CHANCE: We identified 13 345 differentially expressed transcripts across the five time points (false discovery rate, <0.01) of which 58% were protein-coding genes. Two clusters of mainly downregulated genes represented cell cycle pathways and DNA repair. Upregulated genes showed one peak at 12 h that resembled the initiation of an inflammatory response, and one peak at 36 h that resembled the effector functions of inflammation such as vasodilation, angiogenesis, coagulation, chemotaxis and tissue remodelling. Genes involved in cell-matrix interactions as a part of cytoskeletal rearrangement and cell motility were also upregulated at 36 h. Predicted activated upstream regulators of ovulation included FSH, LH, transforming growth factor B1, tumour necrosis factor, nuclear factor kappa-light-chain-enhancer of activated B cells, coagulation factor 2, fibroblast growth factor 2, interleukin 1 and cortisol, among others. The results confirmed early regulation of several previously described factors in a cascade inducing meiotic resumption and suggested new factors involved in cumulus expansion and follicle rupture through co-regulation with previously described factors. LARGE SCALE DATA: The microarray data were deposited to the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/gds/, accession number: GSE133868). LIMITATIONS, REASONS FOR CAUTION: The study included women undergoing ovarian stimulation and the findings may therefore differ from a natural cycle. However, the results confirm significant regulation of many well-established ovulatory genes from a series of previous studies such as amphiregulin, epiregulin, tumour necrosis factor alfa induced protein 6, tissue inhibitor of metallopeptidases 1 and plasminogen activator inhibitor 1, which support the relevance of the results. WIDER IMPLICATIONS OF THE FINDINGS: The study increases our understanding of human ovarian function during ovulation, and the publicly available dataset is a valuable resource for future investigations. Suggested upstream regulators and highly differentially expressed genes may be potential pharmaceutical targets in fertility treatment and gynaecology. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by EU Interreg ÔKS V through ReproUnion (www.reprounion.eu) and by a grant from the Region Zealand Research Foundation. None of the authors have any conflicts of interest to declare.

Abnormally invasive placenta-prevalence, risk factors and antenatal suspicion: results from a large population-based pregnancy cohort study in the Nordic countries.

OBJECTIVE: The objective was to investigate prevalence, estimate risk factors, and antenatal suspicion of abnormally invasive placenta (AIP) associated with laparotomy in women in the Nordic countries. DESIGN: Population-based cohort study. SETTING AND POPULATION: A 3-year Nordic collaboration among obstetricians to identify and report on uterine rupture, peripartum hysterectomy, excessive blood loss, and AIP from 2009 to 2012 The Nordic Obstetric Surveillance Study (NOSS). METHODS: In the NOSS study, clinicians reported AIP cases from maternity wards and the data were validated against National health registries. MAIN OUTCOME MEASURES: Prevalence, risk factors, antenatal suspicion, birth complications, and risk estimations using aggregated national data. RESULTS: A total of 205 cases of AIP in association with laparotomy were identified, representing 3.4 per 10 000 deliveries. The single most important risk factor, which was reported in 49% of all cases of AIP, was placenta praevia. The risk of AIP increased seven-fold after one prior caesarean section (CS) to 56-fold after three or more CS. Prior postpartum haemorrhage was associated with six-fold increased risk of AIP (95% confidence interval 3.7-10.9). Approximately 70% of all cases were not diagnosed antepartum. Of these, 39% had prior CS and 33% had placenta praevia. CONCLUSION: Our findings indicate that a lower CS rate in the population may be the most effective way to lower the incidence of AIP. Focused ultrasound assessment of women at high risk will likely strengthen antenatal suspicion. Prior PPH is a novel risk factor associated with an increased prevalence of AIP. TWEETABLE ABSTRACT: An ultrasound assessment in women with placenta praevia or prior CS may double the awareness for AIP.

Severe postpartum haemorrhage and mode of delivery: a retrospective cohort study.

OBJECTIVE: To examine the association between intended mode of delivery and severe postpartum haemorrhage. DESIGN: A retrospective cohort study. SETTING: Material from a nationwide study in Denmark. POPULATION: Danish women giving birth in 2001-08. METHODS: We compared use of red blood cell transfusion by intended mode of delivery in the total population (n = 382 266), in low-risk nulliparous women (n = 147 132) and in women with a previous caesarean delivery (n = 25 156). MAIN OUTCOME MEASURE: Red blood cell transfusion within 7 days of delivery. RESULTS: In the total population the crude transfusion rates for women with planned caesarean delivery and intended vaginal delivery were 2.24 and 1.75%. After adjustment for maternal age, body mass index, birthweight, smoking, parity, number of infants and previous caesarean delivery, the risk of red blood cell transfusion was significantly lower in women with planned caesarean delivery compared with intended vaginal delivery (odds ratio 0.82; 95% CI 0.73-0.92; P < 0.01). In low-risk nulliparous women and in women with a previous caesarean delivery the transfusion rates were lower for planned caesarean delivery compared with intended vaginal delivery before and after adjustment. CONCLUSION: Compared with intended vaginal delivery, planned caesarean delivery was associated with a reduced risk of severe postpartum haemorrhage indicated by use of red blood cell transfusion.

Preparation of bioconjugates by solid-phase conjugation to ion exchange matrix-adsorbed carrier proteins.

A solid-phase conjugation method utilizing carrier protein bound to an ion exchange matrix was developed. Ovalbumin was adsorbed to an anion exchange matrix using a batch procedure, and the immobilized protein was then derivatized with iodoacetic acid N-hydroxysuccinimid ester. The activated protein was conjugated with glutathione, the conjugation ratio determined by acid hydrolysis, and amino acid analysis performed with quantification of carboxymethyl cysteine. Elution of conjugates from the resin by a salt gradient revealed considerable heterogeneity in the degree of derivatization, and immunization experiments with the eluted conjugates showed that the more substituted conjugates gave rise to the highest titers of glutathione antibodies. Direct immunization with the conjugates adsorbed to the ion exchange matrix was possible and gave rise to high titers of glutathione antibodies. Conjugates of ovalbumin and various peptides were prepared in a similar manner and used for production of peptide antisera by direct immunization with the conjugates bound to the ion exchanger. Advantages of the method are its solid-phase nature, allowing fast and efficient reactions and intermediate washings, and the ability to release conjugates from the solid phase under mild conditions.

Application of fluorescence in situ hybridization techniques in clinical genetics: use of two alphoid repeat probes detecting the centromeres of chromosomes 13 and 21 or chromosomes 14 and 22, respectively.

Two cloned DNA fragments, one derived from an alpha satellite subfamily common to chromosomes 13 and 21, and the other derived from a similar subfamily common to chromosomes 14 and 22, have been used as biotinylated probes in in situ hybridization studies. Under high stringency conditions, chromosome specific centromeric labelling can be obtained. The applications of this technique in clinical situations are illustrated on metaphases from a fetus with trisomy 21, a fetus with trisomy 13, and a child with clinical features of cat-eye syndrome.

Prenatal diagnosis of alpha-1-antitrypsin deficiency using polymerase chain reaction (PCR). Comparison of conventional RFLP methods with PCR used in combination with allele specific oligonucleotides or RFLP analysis.

Prenatal diagnosis of alpha 1-antitrypsin (AAT) deficiency can be performed in the 1st trimester of pregnancy. These diagnoses have been based on DNA technology using either RFLP analysis or hybridization with allele specific oligonucleotides. Several RFLPs within and flanking the AAT gene have been found to render most families informative. The polymerase chain reaction allows specific DNA sequences to be amplified up to ten million fold. Both sequences containing a specific mutation or an RFLP can be amplified by this method. We have compared conventional RFLP methods with PCR used in combination with allele specific oligonucleotides or RFLP analysis, in a case of prenatal diagnosis of AAT deficiency of the ZZ type.

Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ.

It is demonstrated that either general staining of the centromeric regions of all primate chromosomes, or selective staining of the centromeric region of specific chromosomes, may be obtained in preparations of metaphase chromosomes by probing specifically for different regions within the alpha satellite DNA monomer. In order to exploit observed patterns of sequence variation within the monomer for this purpose, we have developed two new DNA analysis methods. In PRimed IN Situ labelling (PRINS), synthetic oligonucleotides derived from subsections of the monomer are hybridized to the chromosomes. The oligonucleotides then serve as primers for the in situ incorporation of biotin-labelled nucleotides catalysed by Klenow polymerase. Incorporated biotin is visualized with fluorescein isothiocyanate-labelled avidin (FITC-avidin). In Primed Amplification Labelling (PAL), biotin-labelled hybridization probes are produced in a polymerase chain reaction (PCR, Saiki et al. 1985), in which two synthetic oligonucleotide primers anneal within the same monomer. With the right choice of primers libraries of labelled probes derived from most monomers present as templates are produced. If DNA from a specific chromosome is used as template, then the resulting probe mixture gives stronger and more chromosome-specific signals in in situ hybridization experiments than does a cloned alpha satellite DNA probe derived from the same chromosome. The results obtained indicate that the alpha-repeat monomer is composed of regions with different degrees of chromosome specificity.

Detection of point mutations in amplified single copy genes by biotin-labelled oligonucleotides: diagnosis of variants of alpha-1-antitrypsin.

Specific analysis for point mutations in genomic DNA has until recently been a difficult and time-consuming task, using large amounts of unstable, hazardous and expensive 32P. By enzymatically amplifying the mutation-bearing sequence of the DNA the sensitivity of the analysis is increased several 100-fold, making the detection possible with stable, non-radioactive and inexpensive biotinylated oligonucleotides. We have applied this method (polymerase chain reaction (PCR] to the detection of the Z-mutation in the alpha-1-antitrypsin gene. After amplification, dot-blots of amplified DNA were subjected to hybridization with allele specific biotinylated oligonucleotide probes and washed at temperatures giving allele specificity. The bound biotin was visualized with avidin conjugated alkaline phosphatase using 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium as colour reagents. The detection can be performed on less than 1 microgram genomic DNA, and is therefore applicable on small amounts of blood, fibroblasts and chorionic villus biopsies.

Improved methods for the detection of unique sequences in Southern blots of mammalian DNA by non-radioactive biotinylated DNA hybridization probes.

Biotinylated DNA hybridization probes offers a stable, cheap and non-radioactive alternative to probes labelled with 32P. Insufficient sensitivity has, however, up till now, been prohibitive for the use of such probes in detecting unique sequences in Southern blots of human DNA. By optimizing the steps in the procedure we have improved the sensitivity enough for such use. We have showed (1) that long probes (greater than 500 nucleotides) perform unproportionally better than short probes; (2) that a simple affinity labelling with avidin alkaline phosphatase conjugate performs better than laborious immunochemical systems; (3) that use of 3% BSA as blocking agent at 37 degrees C and the presence of 0.5 mol/l NaCl together with 1% BSA during the affinity labelling nearly eliminate background staining; (4) that a dramatic gain in sensitivity is gained by affinity labelling at pH 9.0 instead of 7.5; (5) that biotin-labelling can be highly reproducibly performed on a preparative scale with cheap and easily synthesized bio-11-dUTP in a two step nick-translation and (6) that biotinylated probes and hybridization mixtures can be stored for months and reused. The study has resulted in the presentation of a fast procedure, which is generally applicable to routine DNA diagnostic work, also in parts of the world where it is difficult to get a regular supply of 32P.

A hotel fire.

A hotel fire in Copenhagen claimed 35 victims of eight different nationalities. An account is given of some major aspects of the dental identification work, including a few case reports. This disaster became professionally important because the dental expert team was allowed a completely free hand. Eight dental experts cooperated closely in recording, photographing and radiographing dental conditions in the victims and thereby became able to establish that, with adequate facilities at hand, complete dental registration of a single victim required an average of three man-hours. Three of the experts subsequently cooperated in establishing comparable antemortem data on the known missing persons, in comparing the ante- and postmortem data sets, and in completing the necessary paper work. Here, it could be shown that a further two man-hours were required per victim. In a final report to the police it was proposed that---in any future case---such number of dentists should be assigned which, allowing three man-hours per victim, would enable the dental team to finish the oral autopsy of the given number of victims within five full working days, i.e. a minimum of two dentists per 30 victims. Two dentists will accomplish complete recording in less than half the time it will take any dentist working single-handed to finish it, so that the question of cost can hardly become an obstacle. It remains for the profession to make sure that an adequate number of knowledgeable dental experts are always available.